Injectable Chemokine-Releasing Gelatin Matrices for
Enhancing Endogenous Regenerative Responses in the Injured Rat Brain
by
MASSAdHUsETTs INSTITUTE
OF TECH2CLO Y
Teck Chuan Lim
B.S. Biomedical Engineering
Johns Hopkins University, 2007
Li BRAR ES
SUBMITTED TO THE HARVARD-MIT PROGRAM IN HEALTH SCIENCES AND
TECHNOLOGY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF
DOCTOR OF PHILOSOPHY IN MEDICAL ENGINEERING AND MEDICAL PHYSICS
AT THE
MASSACHUSETTS INSTITTUTE OF TECHNOLOGY
SEPTEMBER 2014
C Teck Chuan Lim. All rights reserved.
The author hereby grants to MIT permission to reproduce and distribute publicly paper and
electronic copies of this thesis document in whole or in part in any medium now known or
hereafter created.
Signature redacted
Signature of Author:
Harvard-MIT PAogram in Health Sciences & Technology
Aug 1, 2014
Signature redacted
Certified by:
Myron Spector, Ph.D.
Professor of Orthopedic
rgery (Bio aterials), Harvard Medical School
Senior Lec
r, Department of Mechanical Engineering, MIT
Thesis Supervisor
Signature redacted
Accepted by:
Emery N. Brown, MD, Ph.D.
Director, Harvard-MIT Program in Health Sciences & Technology
Professor of Computational Neuroscience and Health Sciences and Technology
1
2
Injectable Chemokine-Releasing Gelatin Matrices for
Enhancing Endogenous Regenerative Responses in the Injured Rat Brain
By
Teck Chuan Lim
Submitted to the Harvard-MIT program in Health Sciences and Technology on Aug
Partial Fulfillment of the Requirements for the Degree of
Doctor of Philosophy in Medical Engineering and Medical Physics
1
t, 2014 in
ABSTRACT
Brain injuries acquired from hemorrhage, ischemic strokes and trauma affect millions
worldwide each year and often cause irreversible loss of neural tissue that disrupts vital
neurological functions. Cell transplantation has traditionally been the strategy for achieving
neuroregeneration but endogenous regenerative responses such as neurogenesis and
neovascularization are increasingly recognized as an elegant alternative. These responses are
directed toward injured tissues via chemokine signaling such as the stromal cell-derived factor-I a
(SDF-1 a)/CXCR4 pathway and offer the potential to replace lost neurovascular elements.
Endogenous regenerative responses are, however, not fully effective in the injured brain.
Two prominent barriers are the loss of chemokine expression and disappearance of stroma
following tissue loss in the brain lesion, which lead to sub-optimal engagement of endogenous
regenerative responses and inability of recruited cells to infiltrate the lesion. The overall goal of
this thesis was therefore to develop an injectable lesion-filling matrix that could re-establish
chemokine release and stroma, thereby enhancing endogenous regenerative responses. Toward this
goal, we demonstrated injectable gelatin-hydroxylphenylpropionic acid (Gtn-HPA) hydrogels as an
appropriate scaffolding material that was permissive for proliferation, migration and differentiation
of adult neural progenitor cells (aNPCs). We also synthesized dextran sulfate/chitosan
polyelectrolyte complex nanoparticles (PCN), which could encapsulate SDF- 1 a efficiently and
sustain its release for 4 weeks. When used in an in vitro migration assay to fill a core region that
was surrounded by an aNPC-laden hydrogel construct, the resulting Gtn-HPA/SDF- 1 a-PCN matrix
recruited aNPCs to accumulate around and migrate into the core region.
When injected into the brain lesion in a rat model of intracerebral hemorrhage, GtnHPA/SDF- 1 a-PCN matrix successfully increased the migration of endogenous neuronal precursors
into the injured striatum and amplified neovascularization. Gtn-HPA/SDF-la-PCN matrix also led
to a newly formed vasculature within the lesion and supported infiltration by endogenous cells that
included neutrophils expressing CXCR4 and VEGF. The neutrophil infiltrate did not spread to
surrounding tissue or induce necrosis and compelled further investigation for their role in the
injured brain. Importantly, Gtn-HPA/SDF-la-PCN matrix reduced brain tissue loss and improved
behavioral recovery. Overall, Gtn-HPA/SDF-la-PCN matrix offered an opportunity to enhance
endogenous regenerative responses and confer benefits to the injured rat brain.
Thesis Supervisor: Myron Spector
Title: Professor of Orthopedic Surgery (Biomaterials), Harvard Medical School.
Senior Lecturer, Department of Mechanical Engineering, MIT
3
Acknowledgements
This work would not have been possible without my thesis supervisor, Dr. Myron Spector.
It really began with Dr. Spector placing his trust on a relatively young and inexperienced graduate
student when I first broached the research idea with him five years ago. Along the way, his passion
to advance treatment for challenging tissue injuries had been the source of motivation to press
ahead and his unwavering faith and optimism in this project the source of resolve at times when
straying to another research direction seemed easier. My appreciation also goes out to his care and
concern over the years. Never have I seen someone take so much personal interests in the wellbeing and career development of his students/employees. I will truly miss my days under the
mentorship of a wise and caring leader.
Many heartfelt thanks to my collaborators, Dr. Eng H. Lo and Dr. Emiri Mandeville, from
the Massachusetts General Hospital Neuroprotection Laboratory and Dr. Motoichi Kurisawa and
Dr. Li-Shan Wang from Institute of Bioengineering and Nanotechnology, Singapore. To Dr. Lo,
thank you for the opportunity to participate in this multi-disciplinary project. It is always a pleasure
to learn from someone who is so knowledgeable and yet, so unassuming. To Dr. Mandeville, thank
you for imparting me the surgical skills and the know-how to run behavior tests, especially when I
had near to none to begin with. To Dr. Kurisawa and Dr. Wang, thank you for all the fruitful
discussions and the preparation of materials needed to pursue this work.
A huge thank you to my thesis committee members, Dr. Jeffrey Karp and Dr. David
Mooney for their guidance and all the tremendously helpful feedback.
My sincere thanks to all the friends and co-workers who have made my life easier and
more interesting. To all members and alumni of the Spector Tissue Engineering Lab, thank you for
being part of an enriching experience. Especially to Dr. Swetha Rokkappanavar, Dr. Wanting Niu,
Dr. Ravikumar Rajappa, Dr. Ding Weng and Dr. Wei Seong Toh, thank you for providing the extra
pair of hands when 24 hours seemed insufficient for a day. To Dr. Kimberly Leite-Morris, Angela
Young and Marsha Guy, thank you for guiding me through the art of cryosectioning the brain and
for the interesting conversations about the life of a researcher. To Dr. NurgUl Aytan, thank you for
being comrade-in-arms in trying to get finicky antibodies to work. To Ying Kai Chan, one of my
best friends since high school, thank you for your infectious passion in science and being the
biology guru to dish out tips and advice on experimental methods.
My deepest thanks to my family whom I owe everything to. To my Dad and Mom, thank
you for molding me to who I am today. I can only strive to be as good a parent as you have been to
me. To my parents-in-law, thank you for all the care and loving encouragement. To my brother
Teck Yong, thank you for always being supportive and taking care of everything during my long
period of absence from Singapore. To my son Wen Shuo, thank you for being such a precious joy.
You have taught me as much as I have taught you (so far). To Chyan Ying, my beloved wife, my
best friend and my fellow classmate for the past 9 years, "thank you for everything" is an
understatement. You understand my every thought, sense my every emotion and care for my every
need. It is truly my fortune to experience life with a soul mate like you.
Finally, my utmost appreciation goes to all the rats involved in this work. They went under
the knife for the sake of science, had the most vested interests in this project and had truly
4
sacrificed their lives for this work. Without their lives, my idea will always remain as just an idea
on paper.
The funding for this work was provided by Agency for Science, Technology and Research
(A*STAR), Singapore as well as the U.S. Department of Veteran Affairs, Veterans Health
Administration, Rehabilitation Research and Development Service.
5
Table of Contents
Acknowledgm ents.....................................................................................
4
Chapter 1: Introduction
10
........ - -
-..--.............................
..........................................
1.1
Major Forms of Brain Injury......................................12
1.1.1
Hemorrhagic Stroke................................................................................................12
1.1.2. Ischemic Stroke ...................................................................................................
1.1.3. Traumatic Brain Injury (TBI)...............................................................................
1.2 Post-Injury Sequelae......................................................................................................17
1.2.1 Excitotoxicity and Ionic Imbalance .....................................................................
1.2.2 Oxidative Stress ...................................................................................................
1.2.3 Necrosis and Apoptosis............................................................................................18
1.2.4 Inflammatory mechanisms...................................................................................
1.2.5
Tissue loss: Atrophy and Liquefactive Necrosis...................................................
1.2.6 Glial Scar Formation.............................................................................................
1.2.7 Growth Inhibitory M olecules...............................................................................
1.3
17
17
19
20
21
21
Neuroregeneration..............................................................................................22
1.3.1 Goals .....................................................................
...............................................
1.3.2 Tools: M olecular Agents.........................................................................................23
1.3.3 Tools: Cell transplantation........................................................................................24
1.3.4 Tools: Biomaterials...............................................................................................
1.4 Endogenous Regenerative Responses......................................................................
1.5
1.6
14
15
1.4.1
Neurogenesis............................................................................................................26
1.4.2
1.4.3
Neovascularization...............................................................................................
Tools to Manipulate Endogenous Stem and Progenitor Cells
...............
22
25
26
28
28
Outline of research.........---.....
.-........................................................................
....... 30
References..... .............
........... .......................................................................
33
Chapter 2: Characterization of Injectable Gtn-HPA Hydrogels as Supporting
M atrices for Neural Progenitors............................................................................................46
2.1
Introduction...............
....... .............................................................................
2.2 M aterials and M ethods.............................................................................................
2.2.1y
g
.....................................................................
2.2.2 Rheological Measurement.........................................................................................48
2.2.3 In vitro Degradation Assay for Gtn-HPA Hydrogels............................................
2.2.4 Neural Progenitor Cell Culture ............................................................................
2.2.5
Adhesion Assay....................................................................................................
2.2.6 Evaluation of Viability..........................................................................................
2.2.7 Oxidative Stress Resistance Assay .......................................................................
2.2.8
Evaluate of proliferation and migration .................................................................
2.2.9 Evaluation of differentiation.................................................................................
2.2.10 Immunofluorescent Staining...............................................................................
2.2.11
Quantitative Real-Time Polymerase Chain Reaction
....................
2.2.12
Statistical Analysis..................................................................................................52
2.3 Results..................................
. --................................................................................
6
46
48
48
49
49
49
50
50
51
51
51
52
53
2.3.1
2.3.2
2.3.3
2.3.4
2.3.5
Rheological Measurements of Gtn-HPA Hydrogels............................................
In vitro Degradation Profiles of Gtn-HPA Hydrogels .........................................
Adhesion of Neural Progenitor Cells on Gtn-HPA Hydrogels .............................
Viability of Neural Progenitor Cells within Gtn-HPA Hydrogels........................
Oxidative Stress Resistance of Neural Progenitor Cells
within Gtn-HPA Hydrogels ................................................................................
2.3.6 Proliferation of Neural Progenitor Cells within Gtn-HPA Hydrogels..................
2.3.7 Migration of Neural Progenitor Cells within Gtn-HPA Hydrogels ......................
2.3.8 Differentiation of Neural Progenitor Cells within Gtn-HPA Hydrogels ..............
2.3.9 Survival of Progenitor-derived Neurons and Astrocytes
within Gtn-HPA Hydrogels ................................................................................
2.4.
Discussion......................................................................................................................66
2.5 References .......................................................................................................................
53
54
55
56
56
59
59
62
65
70
Chapter 3: Development of Gtn-HPA/SDF-la-PCN Matrices for Recruitment of
Neural Progenitor Cells.........................................................................................................75
3.1 Introduction................................................................................................................
75
3.2 Materials and Methods..............................................................................................
77
3.2.1 Preparation of SDF-la PCNs...............................................................................
77
3.2.2 Characterization of SDF-1a PCNs........................................................................
77
3.2.3 Preparation of Gtn-HPA conjugate .....................................................................
78
3.2.4 Neural Progenitor Cell Culture ............................................................................
78
3.2.5 Characterization of Gtn-HPA/ PCN Matrices.....................................................
78
3.2.6 Three-Dimensional Migration Assay...................................................................
79
3.2.7 Three-Dimensional Proliferation Assay...............................................................
81
3.2.8 Fluorescent Immunohistochemical Analysis .......................................................
82
3.2.9 Statistical analysis .................................................................................................
83
3.3
Results .............................................................................................................................
84
3.3.1 Characterization of SDF-Ia PCNs........................................................................
84
3.3.2 Characterization of Gtn-HPA/PCN matrices .......................................................
85
3.3.3 Neural Progenitor Cell Recruitment: Accumulation around
Gtn-HPA/SDF-Ia-PCN matrices .......................................................................
86
3.3.4 Neural Progenitor Cell Recruitment: Migration into
Gtn-HPA/SDF-lIa-PCN matrices .......................................................................
87
3.3.5
Chemotactic nature of Neural Progenitor Cell Recruitment by Gtn-HPA/SDF-laPCN matrices......................................................................................................
90
3.3.6 Secondary Effects of Gtn-HPA/ SDF- I-PCN matrices.....................................
91
3.3.7 Effect of crosslinking degree on migration..........................................................
94
3.3.8 Profile of matrix metalloproteinases used to mediate migration of Neural Progenitor
Cells into Gtn-HPA/ SDF-Ia-PCN matrices........................................................
95
3.3.9 Phenotypic fate of Recruited Neural Progenitors ................................................
97
3.4 Discussion......................................................................................................................101
3.5 References .....................................................................................................................
107
7
Chapter 4: Implantation and Optimization of Gtn-HPA/SDF-la-PCN matrices in a
Rat Model of Intracerebral Hemorrhagic Stroke
........................
111
4.1gIntroduction..............................................
4.1 Introduction
.................................................................................................................. Ill
4.2 Materials and Methods................................................................................................113
4.2.1
4.2.2
4.2.3
4.2.4
4.2.5
4.2.6
4 .2.7
4.3
Preparation of Gtn-HPA conjugate and SDF- 1 a PCNs .......................................... 113
Rheological Measurements of Gtn-HPA Hydrogels...............................................113
Design of Animal Experiment ................................................................................
113
Collagenase-Induced Model of Intracerebral Hemorrhage.....................................114
Stereotaxic Injection of Gtn-HPA/SDF-la-PCN Matrices.....................................115
Animal Sacrifice and Specimen Processing............................................................116
Histology .................................................................................................................
117
Results...........................................................................................................................118
4.3.1 Storage Moduli of Gtn-HPA Hydrogels ...........................................................
118
4.3.2 Intracerebral Hemorrhagic Lesion 1 day and 2 weeks post-ICH ............................ 119
4.3.3 Gtn-HPA/SDF-1 a-PCN matrices 1 hour after Injection.........................................120
4.3.4 Optimization of Gtn-HPA/SDF-l a-PCN matrices .................................................
120
4.4 Discussion.................................................................................................................122
4.5 References ..............................................................................................................
124
Chapter 5: Evaluation of Gtn-HPA/SDF-la-PCN matrices in a Rat Model of
Intracerebral Hemorrhagic Stroke .....................................................................................
5.1
Introduction..................................................................................................................126
5.2
Materials and Methods................................................................................................130
126
5.2.1 Experimental Design ...............................................................................................
130
5.2.2 Preparation of Gtn-HPA conjugate and SDF- 1 a PCNs .......................................... 130
5.2.3 Collagenase-Induced Model of Intracerebral Hemorrhage.....................................131
5.2.4 Stereotaxic Injection of aCSF and Matrices ...........................................................
132
5.2.5 B ehavioral Testing ..................................................................................................
132
5.2.6 Animal Sacrifice and Specimen Processing............................................................134
5.2.7 H istology .................................................................................................................
134
5.2.8 Fluorescent Immunohistochemical Analysis ..........................................................
135
5.2.9 Statistical A nalysis..................................................................................................137
5.3 Results...........................................................................................................................138
5.3.1 Behavioral Outcomes ..............................................................................................
138
5.3.2 Gross Histological Outcomes..................................................................................140
5.3.3 Endogenous Neurogenesis in Host Tissue ..............................................................
144
5.3.4 Neovascularization in Host Tissue..........................................................................149
5.3.5 Astrocytic Response in Host Tissue...................................................................
152
5.3.6 Neutrophils and Activated Macrophages/Microglia in Host Tissue.......................153
5.3.7 Endogenous Neuronal Precursors in Lesion/Implanted Matrices...........................156
5.3.8 Blood vessels in Lesion/Implanted Matrices ....................................................
158
5.3.9 Astrocytes in Lesion/Implanted Matrices ...............................................................
160
5.3.10 Neutrophils and Activated Macrophages/Microglia
in Lesion/Implanted Matrices ............................................................................
161
5.4 Discussion......................................................................................................................167
5.5 References................................................................................................................178
8
Chapter 6: Conclusions and Future Work ........................................................................
9
185
Chapter 1
Introduction
The adult brain of mammals in general is an organ that is highly sophisticated and
specialized to handle bioelectrical processing and control various life-sustaining processes as well
as voluntary thoughts, emotions and actions that largely determine the quality of life for the
being. Amidst this high degree of sophistication and specialization lies an inherent susceptibility
to injury arising from various
conditions. High metabolic demand with little or no
oxygen/nutrient reserves creates liability for hypoxic or ischemic injury in the event of
compromised systemic blood flow or blocked artery. Strong dependence on tightly regulated
transport of molecules and cells across the blood-brain-barrier sets up vulnerability to toxicity
upon exposure to blood and immune cells in a hemorrhagic event. Lack of substantial mechanical
strength further opens room for traumatic injury once excessive forces are transmitted to the brain
in the event of a sudden impact or a compromised skull. Given the critical roles of the organ,
there is no shortage of hope and ambition to heal the injured brain. Conventional wisdom has,
however, pitted against the possibility of brain healing for a very long time. This conventional
wisdom is best expressed by Spanish neuroscientist and Nobel laureate, Santiago Ram6n y Cajal
when he made the famous statement about the central nervous system: "Once the development
was ended, the sources of growth and regeneration of the axons and dendrites dried up
irrevocably. In the adult centers, the nerve paths are something fixed, ended, and immutable.
Everything may die, nothing may be regenerated. It is for the science of the future to change, if
possible, this harsh decree."
True to his keen observation, there is still not a single treatment available a century later
to reverse the damage inflicted by any form of brain injury. However, the science around the
injured brain has grown tremendously since then. Much has been revealed about the molecular
and cellular mechanisms driving the pathophysiology of brain injuries. The toolbox available to
attempt protection, repair and regeneration in the injured brain has also undergone vast expansion
to include tailored molecules, genetic manipulation, instructive biomaterials and multi-potent
10
stem and progenitor cells. Most importantly, seminal discoveries of endogenous neural stem and
progenitor cells in the adult brain have sparked a new paradigm that the regenerative prospect of
the injured brain is not be as absolutely bleak as the "harsh decree" illustrated by Cajal. Overall,
these advancements have given rise to a vast array of experimental therapies and immensely
increase the possibility that a fruitful treatment can be materialized for the injured brain.
The key subject of this thesis is about enhancing endogenous regenerative responses in
the wake of a brain injury. Compared to other strategies such as neuroprotection or
transplantation of stem and progenitor cells, endogenous regenerative responses are much less
commonly involved in experimental therapies. Many of the tools that are available in other
strategies have also not been attempted to exploit the full benefit of endogenous regenerative
responses. This is possibly because the discovery of the endogenous regenerative responses is
relatively recent and existing knowledge about them is limited. Also, the scale of endogenous
regenerative responses often pales in comparison to the extent of tissue loss and damage after
brain injury. However, these current limitations should not be viewed as reasons to disregard the
valuable resources that the injured brain is endowed with but as urging challenges to be resolved.
Moreover, the notion of promoting the injured brain to heal itself is too appealing to completely
disregard endogenous regenerative responses. Even if they prove to be insufficient to ultimately
achieve complete healing, they can also be reasonably expected to increase the robustness of
other forms of therapies. Simply put, the promise of endogenous regenerative responses is so
great and yet, so little has been done to enable the injured brain to fully exploit them.
In this chapter, we provide an overview of several pertinent brain injuries, their
pathophysiology and the state of the art in neuroregeneration in the brain. We also highlight the
promises and limitations of endogenous regenerative responses in the injured brain. Finally, we
outline the specific aspects of endogenous regenerative responses that we aim to target, the design
criteria for tools that can achieve our aim and the approach we have taken to develop an
appropriate injectable matrix to enhance endogenous regenerative responses in the injured brain.
11
1.1
Major Forms of Brain Injury
1.1.1
Hemorrhagic Stroke
Each year, stroke inflicts brain injuries on 15 million people worldwide, of whom around
5 million progress to become permanently disabled and another 5 million to death [1]. This makes
stroke the leading cause of serious, long-term disability and 3rd leading cause of deaths (close
after coronary heart diseases and cancer). Stroke is broadly classified into hemorrhagic and
ischemic strokes. Of the these two subtypes, hemorrhagic stroke is associated with significantly
higher morbidity and mortality and generally accounts for 8 - 18% of all strokes [2], although
the figure can be substantially higher in certain ethnicities [3] and age groups [4]. Hemorrhagic
stroke is an event where a blood vessel in the brain ruptures and leads to spontaneous bleeding.
Depending on the location of the ruptured vessel and bleeding, hemorrhagic strokes can occur in
the form of subarachnoid hemorrhage (SAH) or intracerebral hemorrhage (ICH). SAH describes
the bleeding within the meninges. On the other hand, ICH refers to bleeding within the brain
parenchyma, which can include the cerebral lobes, basal ganglia (striatum, globus pallidus and
subthalamic nucleus), thalamus, cerebellum and the pons.
ICH is typically the more concerning type of hemorrhagic strokes, given that ICH is more
common and affects many important structures within the brain.
Hypertension is the primary
cause of ICH. Chronic hypertension has been known to induce degenerative changes and
reduction of compliance of the wall of blood vessels, thereby setting the stage for spontaneous
rupture, especially near bifurcation of arteries where microaneurysms are prone to occur. Other
causes also involve atypical blood vessels and include brain tumors, amyloid angiopathy,
arteriovenous malformations, cavernous angiomas and arteriovenous fistulae. ICH may also occur
non-spontaneously as: 1) a secondary injury event during hemorrhagic transformation of ischemic
strokes when cerebral blood flow is restored to the affected brain tissues in thrombolytic therapies
or; 2) a consequence of mechanical damage during traumatic brain injury.
The key concern in ICH is the mass effect of the hematoma or in other words, the
pathological effects due to the hematoma exerting pressure or causing displacements to the
surrounding. Management of ICH has therefore been centered on reducing the mass effect or the
12
hematoma itself. The use of ventricular catheters to drain cerebrospinal fluid and reduce
intracranial pressure provides improvements to the neurological status of the affected individual
[5]. American Heart Association guidelines also recommend anti-hypertensive medication to
control elevated blood pressure. Surgical removal of the hematoma can also be an option,
especially in the cases of large lobar hemorrhages (> 50 cm3 ) in young patients and cerebellar
hemorrhages (>3cm) that are accompanied with neurological deterioration [6]. Although
craniotomy is conventionally performed to approach the hematoma, the surgical practice, for the
obvious reason of reducing morbidity, appears geared toward the use of minimally invasive
stereotactic aspiration of hematoma under the guidance of imaging modalities such as computed
tomography [7]. To liquefy the hematoma and facilitate its complete removal, thrombolytic
agents such as urokinase can also be infused into the hematoma [8]. Pilot studies on such
techniques have revealed a 40% reduction in hematoma volume after surgery and a 40% in death
rate [9].
Several pre-clinical models have been developed to facilitate experimental work on ICH.
One model consists of inserting a microballoon into the brain and keeping it inflated for a period
of time before its eventual removal [10]. This microballoon insertion model can recapitulate the
physical mass effect of hematoma in ICH but does not reproduce chemical and biological aspects
such as toxicity of hematoma and disruption of blood-brain-barrier. Another model involves the
injection of blood into the brain to mimic the bleeding event in ICH. Several blood injection
procedures have been attempted over the years and they include: single [11] or multiple [12]
injections of isolated blood, use of a catheter to bridge the femoral artery to the brain [13] and
allow bleeding into brain and the use of microinfusion pump to assist with the reproducibility of
the rate and volume of blood injected [14]. Each of the procedures is an improvement over others
in some ways but usually retains some disadvantages such as the inability to create bleeding
under arterial pressure or the lack of reproducibility in the hematoma formed. In addition, they
generally do not mimic the disruption of blood-brain-barrier, the spontaneity of intraparenchymal
bleeding and the expansion of hematoma over time. A third model involves the injection of
bacterial collagenase into the brain parenchyma and was discovered during a study to study the
effects of proteolytic enzymes on the extracellular matrix in rats [15]. The injection of
collagenase disrupts the basal lamina of blood vessels and results in spontaneous extravasation of
blood into the surrounding brain tissue. Compared to the blood injection model, this model is
13
attractive for its simplicity as well as the ability to recapitulate hematoma expansion and
continuous tissue loss over time [16]. Concerns have regularly been raised over the potential of
the injected collagenase causing neurotoxicity, inflammation and therefore artifacts in the injury
produced, but are proven to be unfounded by studies showing that the concentration of
collagenase used does not cause neuronal toxicity [17] or inflammation [18].
1.1.2. Ischemic Stroke
Ischemic stroke is the dominant subtype of all strokes and affects as many as 800 000
people in the United States alone [19]. Although not as critically morbid and fatal as ICH,
ischemic stroke still poses devastating repercussions on the neurological functions of the affected
individual. The event underlying all ischemic strokes is the occlusion of blood vessels supplying
the brain. This creates a disruption in the supply of oxygen and nutrients to meet the metabolic
demands of the brain and often leads to severe distress or death for the affected brain tissue.
Depending on the cause of the vessel occlusion, ischemic strokes can be grouped into thrombotic
or embolic strokes. In thrombotic stroke, occlusion is caused by the formation of a thrombus (or
blood clot) in the vessel. The formation of blood clots is generally induced by existing
pathological
states of the vessels such as atherosclerosis, vasoconstriction
as well as
inflammatory diseases. In embolic stroke, occlusion is caused by an embolus that travels into the
brain after originating from somewhere else. The embolus is often a thrombus, especially in
patients with cardiovascular complications, but may also be any sizeable bodies such as air,
tumor, fat and bacteria clumps [20].
The only FDA-approved treatment for ischemic stroke is the use of tissue plasminogen
activator (tPA), which lyses the blood clot occluding blood vessels in the brain and restores blood
flow to the brain. Since its approval, only 1 - 6 % of ischemic stroke patients are treated with tPA
[21]. Part of the reason lies in the fact that tPA administration is only approved for a tight 3-hour
window after the onset of stroke and 4.5-hour window for a smaller selection of patients meeting
stricter requirements. Another part lies in the reluctance of physicians [22], which may arise from
the possible risks of tPA. One principal risk is that tPA may exacerbate hemorrhagic
transformation, which may arise during reperfusion when blood flow is restored to the damaged
14
vasculature of the ischemic region. Such hemorrhagic transformation typically increases the
morbidity and mortality and is of grave concern to the affected patients.
Several models have been developed to recapitulate the pathophysiology of ischemic
stroke. In a model for thrombotic stroke, photochemicals are injected systematically followed by
irradiation of the target cortical region through the intact skull [23]. The irradiation leads to
platelet aggregation and subsequently, vessel occlusion in the target region but is faulted with
prominent vascular injury that is usually not seen in acute ischemic strokes [24]. In another model
that serves to mimic embolic stroke, blood clots are injected into the intracarotid arteries [25].
The model is attractive with its high degree of resemblance with actual embolic strokes and
permits further investigation on the use of thrombolytic agents to lyse the blood clots. For
modeling strokes arising from vasoconstriction, endothelin- 1, a natural peptide that potently
induces vasoconstriction, is injected into the brain next to a targeted artery [26]. The
vasoconstriction causes a decline in blood flow to the territory of the targeted artery, thereby
inducing a focal ischemic stroke. While the model is straightforward in terms of surgical
procedures, the ischemia may not be sufficiently consistent due to high variability in the
vasoconstriction response to endothelial and the return of blood flow after the effect wears off
[27]. Among all models, the most widely used one is the intraluminal middle cerebral artery
(MCA) occlusion model. The model involves threading a filament into internal carotid artery to
occlude the MCA [28]. On top of being relatively simple and non-invasive, it results in a
reproducible infarct with sizeable penumbra, enables precise control of the occlusion duration and
allows for the investigation of transient and permanent occlusions.
1.1.3. Traumatic Brain Injury (TBI)
As the worldwide leading cause of death and disability for children and adults under the
age of 45 years, TBI is another form of brain injury that draws significant attention, especially in
sports and the military. TBI is a broad term to describe numerous events where physical
disturbances are imposed on the brain. TBI can be classified into: 1) closed injury if the physical
damage is incurred without the cranial space being compromised or; 2) penetrating injury if a
foreign object physically invades the cranial space. It can also be classified into: A) focal injury if
15
the damage occurs in a specific location or; B) diffuse injury if the damage spreads over a wide
area.
The cause of penetrating injury is usually clear; a foreign object (e.g. knife, bullet and
shrapnel) pierces through the skull and into the brain matter. Such an event typically causes
compression of the brain tissue as the foreign object competes for the constrained space within
the skull, laceration of the brain tissue as well as hemorrhage when the blood vessels are
mechanically ruptured. Closed injury can arise when an impact on the head produces contact
and/or inertial forces on the brain [29]. Contact forces directly damage the impacted area and
often lead to focal injuries. On the other hand, inertial forces result from sudden acceleration (and
deceleration) of the head. Translation acceleration typically causes focal injuries (e.g. contusion
and hemorrhage) due to differential movement of the brain relative to the skull while angular
acceleration can cause either focal injury (e.g. laceration) or diffuse axonal injury due to
differential movements of different brain structures.
As with ICH, treatment for TBI is limited to supportive measures. Intracranial pressure is
monitored and if necessary, reduced by the placement of a drainage catheter into the ventricles or
the use of diuretics to reduce fluid in the system [30]. Seizures may be prevented by the
administration of benzodiazepines. Surgery may also be performed to remove foreign matter in
the case of penetrating brain injury and to evacuate the hematoma in the event of a hemorrhage.
Preclinical models for the various types of TBI include: 1) fluid percussion model [31];
2) controlled cortical impact model [32]; 3) weight drop model [33]; 4) penetrating ballistic injury
[34] and; 5) shock tube model [35]. In the first three models, a physical impact is delivered, either
with a fluid pressure pulse, a rigid impactor or a free falling guided weight, to exposed intact dura
to cause a contusion. Of these, the controlled cortical impact model offers better control over the
traumatic injury by allowing for easy adjustment of the time, velocity and depth of impact.
Penetrating ballistic injury, on the other hand, is modeled with the use of an inflatable probe. To
simulate the energy dissipation from a penetrating bullet round, the probe is inserted into the
brain parenchyma, inflated and deflated rapidly and eventually withdrawn. Finally, to simulate
TBI that typically results from bomb blasts, a compression-driven shock tube is used to replicate
16
blast waves and direct them to the brain. The model is shown to replicate diffuse axonal injury
that is commonly observed with non-contact blast injury.
1.2
Post-Injury Sequelae
Many of the molecular and cellular processes in the complex environment of the brain are
tightly regulated in a homeostatic state. In this regard, although the nature of the injury may vary
substantially from hemorrhage, ischemia to trauma, the resulting disturbance to the homeostatic
state triggers a similar chain of events. Furthermore, each form of injury usually develops to
incorporate elements of other forms e.g. ischemia in ICH and hemorrhage in ischemic stroke and
trauma. All in all, these cause the post-injury sequelae to share many common features.
1.2.1
Excitotoxicity and Ionic imbalance
With metabolic dysfunction (resulting from ischemia or impaired blood flow in
hemorrhage or traumatic injury) and loss of energy stores, neurotransmitters are released while
their reuptake is inhibited. As a result, excitatory amino acid neurotransmitters such as glutamate
become excessively abundant in the extracellular environment [36].
ionotropic
N-methyl-D-aspartate
(NMDA)
and
Glutamate can bind to
a-amino-3-hydroxy-5-methyl-4-isoxazole
(AMPA) receptors to induce calcium influx, which in turn activates lipid peroxidases,
phospholipases and proteases and triggers the onset of various catabolic processes. It can also
bind to: 1) ionotrophic glutamate receptors to mediate sodium influx and cell swelling or; 2)
metabotropic glutamate receptors to trigger cell death [37]. Depolarization of excitatory neurons
may also trigger the release of zinc, which is neurotoxic and mediates neuronal death [38].
1.2.2
Oxidative Stress
In addition to activating enzymes that drive the degradation of membranes and essential
proteins, excessive glutamate stimulation and ionic imbalance can potently increase the
production of reactive oxygen species (ROS). Specifically, more nitric oxide is produced with the
heightened activity of nitric oxide synthase and more superoxide is generated by NADPH oxide
synthase, xanthine oxidase, cyclo-oxygenase and leakage from the mitochondrial electron
17
transport chain. In the case of ICH, breakdown of heme may also give rise to iron, which
catalyzes the Fenton reaction to produce highly reactive hydroxyl radicals [39].
The high production of ROS is poorly handled by the brain where endogenous levels of
antioxidant enzymes (such as superoxide dismutase, catalase and glutathione) are low and easily
exhausted. Also, the brain contains a high concentration of peroxidizable lipids which can be
readily targeted by the ROS [40]. In addition to lipids, ROS also damages a variety of proteins,
carbohydrates and nucleic acids. Their effects on DNA have particular serious consequences due
to resultant over-activation of poly(ADP-ribose) polymerase-1 (PARP-1), which depletes ATP
stores and leads to eventual cell death [41].
1.2.3
Necrosis and Apoptosis
Necrosis occurs when the primary injury (from the initial mechanical or ischemic insult)
or the secondary damage is too severe or when ATP is too depleted to allow for apoptosis. In this
mode of cell death, the lipid peroxidases, phosolipases and proteases activated through
mechanisms mentioned above catalyze the autolysis of membranes and proteins in an unregulated
fashion.
In situations where the cells are less severely injured, cell death may occur via apoptosis.
The family of caspases plays a central role in mediating apoptosis. In caspase-mediated apoptosis,
signaling may commence with members of the TNF family, namely tumor necrosis factor-alpha
(TNF-a) and first apoptotic signal (FAS). These cytokines lead to the activation of initiator
caspase 8, which in turn activates other caspases or pro-apoptotic factors (e.g. Bcl-2 associated X
protein, BH3 interacting-domain
death agonist, Bc2 homologous antagonist killer, Bcl2
associated death promoter) to begin an amplificatory feedback loop of caspase activation.
Following activation, effector caspases 3, 7 and 6 serve the role of proteases to degrade
intracellular proteins and pave the way for cell death. Apoptosis may also occur via a caspaseindependent pathway where activation of NMDA receptor leads to activation of PARP-1, which
in turn results in the release of apoptosis-inducing factor from mitochondria [42].
18
1.2.4
Inflammatory mechanisms
Under normal physiologic conditions, the brain is largely isolated from systemic
inflammatory cells but contains resident macrophages known as microglia which carry out similar
functions such as immune surveillance, phagocytosis and productions of various cytokines to
drive or regulate inflammation [43]. In the event of an injury, microglia are the first among the
inflammatory cells to respond to the damaged tissue. Their production of ROS, pro-inflammatory
cytokines and MMP-9, together with the initial insult to the cerebral vasculature and blood-brainbarrier, aids in the elevated expression of leukocyte adhesion molecules (e.g. intercellular
adhesion molecule-1, E/P-selection) on the brain endothelial cells and the subsequent influx of
circulating leukocytes into the brain [44].
Neutrophils arrive within a few hours from the brain injury and are among the first to
infiltrate the brain [45]. They typically accumulate rapidly and may disappear within days via
either apoptosis and phagocytosis by microglia/macrophages or active or reverse migration into
the circulation [46]. Infiltration and accumulation of neutrophils in the acute setting after injury
serve the critical role of clearing cellular debris in the damaged tissue but have also been
generally viewed as a principal propagator of secondary damage. The neutrophil-mediated
mechanisms underlying the observed secondary damage include the release of proteolytic
enzymes such as elastase and MMPs and the production of pro-inflammatory cytokines to drive
further cascades of inflammation. Neutrophils have been classically thought to induce bystander
tissue damage with their oxidative burst (or rapid release of ROS), although a recent study now
shows that brain injury such as stroke results in significant reduction in the oxidative burst of
neutrophils, which in turn explains the susceptibility of stroke patients toward infections [47].
Instead of being completely pro-inflammatory and detreimental, neutrophils have now been
shown to play diverse roles and can also mediate regenerative processes. In one study, neutrophils
are found to mediate VEGF-induced focal angiogenesis [48] while in another, neutrophils are
observed to express oncomodulin and essential for retinal ganglion cells to regenerate axons
through the injured optic nerve [49].
Activated microglia/macrophages are the predominant cell types after the neutrophilic
response. Similar to neutrophils, they serve the role of clearing debris (and neutrophils), which is
critical to reduce additional damage and to prepare the environment for any plausible repair
19
and/or regeneration. They may also produce either secondary damage through the usual
mechanisms of ROS, pro-inflammatory cytokines and proteases or neuroprotective effects with
their secretion of neurotrophins such as FGF and TGF-pl. Current understanding organizes these
opposing roles of microglia/macrophages into a simplified model where microglia/macrophages
exist either as pro-inflammatory M1 state or the pro-regeneration/immunoregulatory M2 state
[50, 51]. In one study looking at ischemic injury after MCAO, the microglia/macrophages
appeared in the injury site mostly in the M2 state, but switched to the M1 state over time [52].
Such polarization dynamics may have implications on the brain injury and be possible
intervention targets in the future.
T lymphocytes also infiltrate into the injured brain and contribute to the inflammatory
processes. Different subtypes play substantially different roles. For example, CD4+ THi cells
release pro-inflammatory cytokines and may contribute to the pathogenesis of brain injuries while
CD4+ TH2 cells secrete anti-inflammatory cytokines and may aid in the resolution of
inflammation [53]. Also, regulatory T cells modulate the activation, infiltration and cytokine
production of inflammatory cells and are found to be neuroprotective in acute experimental stroke
in mice [54].
1.2.5
Tissue loss: Atrophy and Liquefactive Necrosis
Loss of tissue volume is a salient feature of the injured brain. As mentioned above,
numerous catabolic enzymes are produced by the dying neural cells and inflammatory cells and
all of them contribute to process of liquefactive necrosis where cells and their associated
extracellular matrix (ECM) are degraded and phagocytosed. In the case of diffuse injury and cell
death, the affected region of the brain undergoes marked atrophy. In the case of a focal injury, the
lesion is typically transformed into a fluid-filled cavity upon completion of the liquefactive
process. In a significant departure from other non-CNS tissues, a collagenous scar tissue does not
develop in the place the original neural tissue, unless the lesion opens up to the meninges and be
invaded by meningeal fibroblasts that deposit a collagen-rich ECM to form a fibrotic scar.
20
1.2.6
Glial Scar Formation
In response to injury, astrocytes upregulate their expression of glial fibrillary acidic
protein, S100 protein, vimentin and the intermediate filament protein plectin, undergo significant
hypertrophy and transform into the reactive phenotype [55-58]. Reactive astrocytes amass around
the lesion and have their processes stacked against the lesion boundary. The astrocytes then
couple to one another via tight and gap junctions to form a barrier around the lesion [59].
The glial scar plays the critical role of sealing the site of injury from the rest of the brain.
It does so with scavenging capabilities for glutamate and ions, thereby stemming excitotoxicity at
the injury site [60]. It also aids in free radical scavenging and reduces oxidative stress in the
environment [61]. On top of these, reactive astrocytes have also been known to produce
chondriotin sulfate proteoglycans (CSPG) to create a diffusion barrier and prevent the spread of
neurotoxic molecules [62].
1.2.7
Growth Inhibitory Molecules
A plethora of growth inhibitory molecules is expressed or secreted after brain injury.
Inhibitory molecules derived from astrocytes include CSPGs [63] and ephrins [64]. Those derived
from oligodendrocytes and myelin include Nogo [65], myelin-associated glycoprotein [66],
oligodendrocyte myelin glycoprotein [67] as well as ephrin-B3 [68]. In the case of open lesions
that are invaded by meningeal fibroblasts, sema IIIA may also be produced. Acting largely
through the Rho GTPase signaling pathway that regulates the actin cyotskeleton [69], these
inhibitory molecules robustly stall neurite outgrowth and mediate the collapse or repulsion of
growth cones. The jarring of axonal regeneration in the injured brain has been mainly attributed
to the hostile environment established by these growth inhibitory molecules.
21
1.3
Neuroregeneration
1.3.1
Goals
The ultimate goal of neuroregeneration is to restore the brain to its original state. Given
the enormous complexity of the brain and thus the daunting nature of this goal, a realistic
interpretation, based on the current state of the art, is that neuroregeneration aims to restore or
initiate the path toward restoration for specific facets of the injured brain. The hope is for the
advancement for each and every facet to be put together in a collective strategy to reverse the
effects of injury on the brain.
Neuroregeneration can be considered on the axonal or cellular level. At the axonal level,
neuroregeneration works on the premise that numerous neurons survive the brain injury but have
suffered impairments to their axons. Specifically, affected neurons can lose their projections and
synaptic connections if their axons are damaged or severed. In this regard, neuroregeneration
strives to restore projections or synaptic connections to the appropriate target neurons. The target
neurons can be the original synaptic partners if they have also survived the injury. They can also
include new synaptic partners to exploit redundancy in brain circuitry and achieve the same
nervous functions with new signal transmission pathways. While most neurons seem to retain the
intrinsic ability to extend their axons and form synapses, they generally fail to do so due to the
presence of various inhibitory molecules. The key challenge is therefore to derive strategies to
overcome the highly inhibitory environment.
At the cellular level, neuroregeneration recognizes the loss of certain indispensable neural
cells that have to be replaced in order for the original nervous functions to be restored. The focus
here lies predominantly on neurons, which lack the same replicative ability as the two other
principal neural cell types astrocytes and microglia. Loss of neurons is therefore deemed
irreversible without any intervention. Multipotent stem and progenitor cells have generally been
regarded as a promising resource to enable the replacement of loss neurons. However,
reproducible in vivo differentiation of the stem and progenitor cells into neurons in general is
non-trivial and the difficulty is compounded by the existence of neurons in numerous subtypes.
Both axonal and cellular regeneration are inextricably linked. At times, successful projection and
22
synaptic connections may require replacement of neurons, especially when no or few target
neurons remain after a severe brain injury. On the other hand, replacement of neurons will only
be meaningful if the new neurons are integrated into the brain circuitry.
Understandably, neuroregeneration is an approach that complements and synergizes with
neuroprotection. While neuroprotection aims to minimize the consequences of brain injury by
salvaging any non-dying neural cells and tissues, its purpose is usually limited to a relatively
narrow time window right after the onset of the brain injury. Also, it does not address
irremediable damage. Neuroregeneration therefore complements neuroprotection by serving as an
option in the chronic phase of the injury when neuroprotection becomes increasingly ineffective
and by addressing the irremediable damage directly for the overall benefit of the injured brain.
The synergy between neuroprotection and neuroregeneration arises from the fact that their
efficacies are intimately tied to the state of the injured brain. In this regard, any benefit by one
approach improves the tissue environment for the other to be more effective. For instance,
transplantation of neural stem cells to replace lost cells may lead of suppression of post-injury
immune responses, which reduce secondary damage to surrounding neurons and in turn produce a
environment that is more conducive for the transplanted cells [70].
1.3.2
Tools: Molecular Agents
The role of molecular agents in neuroregeneration is generally focused on inducing
axonal regeneration. Molecular agents can achieve this by enhancing the intrinsic ability of
neurons to extend their axons and form synapses. For this, the pathway involving brain derived
neutrotrophic factor (BDNF)/tropomyosin-related kinase B (Trk B) is often implicated. BDNF
has been widely known to induce axonal growth after injury [71]. It has also been found to
increase markers of synaptogenesis [72] and result in structural instability in dendrites and spines
so as to aid in morphological changes in accordance with synaptic activity [73]. To exploit this
pathway, molecular agents, other than taking the form of BDNF itself, can include: 1)
pharmaceutical drugs (e.g. niacin) that increase the endogenous expression of BDNF [74]; 2)
small molecule agonist of the TrkB receptor [75] and; 3) antibodies that bind to and activate
TrKB receptor [76]. Outside the BDNF/Trk B pathway, FGF-2 [77], NGF [78] and neurotrophin3 [79] have also been used to promote axonal regeneration. In addition to using these molecular
23
agents one at a time, a combination may be used to achieve synergistic effects. For instance, the
combined use of FGF-2, BDNF and NT-3 produced axon growth more than the sum of each
neurotrophic factor alone [80].
Axonal regeneration may also be induced if growth inhibitors in the injured environment
are overcome. Molecular agents can achieve this by taking the form of enzymes and dismantling
the inhibitory signaling. Examples for this include phospholipase C that releases the Nogo
receptor from neurons [81] or chondriotinase ABC to remove CSPG from the extracellular matrix
[82]. They may also take the form of soluble receptors [83] and antibodies [84] to block the
inhibitory signaling.
1.3.3
Tools: Cell transplantation
Given that mature neurons lack the ability to replicate and that the adult brain is only
found relatively recently to contain resident neural stem and progenitor cells, neuroregeneration,
at the level of cell/tissue reconstitution, has relied heavily on cell transplantation. Over the years,
numerous types of stem cells have been isolated, processed' and transplanted via intravenous
administration or direct injection into the injured brain. They include neurons [85], embryonic
stem cells [86], neural stem cells [87], bone marrow-derived mesenchymal stem cells [88],
adipose-derived stem cells [89] and umbilical cord blood-derived stem cells [90]. Since the
discovery of induced pluripotent stem cells (iPSCs) that can be generated by reprogramming
somatic cells, neural stem cells have also been derived from iPSCs and transplanted into the brain
[91]. Each type of cells has been shown to provide benefits along the lines of: 1) replacing
neurons, astrocytes, oligodendrocytes and endothelial cells; 2) increased angiogenesis; 3)
improvements in neurological functions and; 4) regulating post-injury immune responses [70].
However, particular types of cells are expectedly more attractive in terms of their source and
lineage restriction. Specifically, iPSCs are immensely appealing given that they can be isolated
without donor site morbidity or ethical concerns and that they are autologous cells that do not
evoke immune responses upon transplantation. On the other hand, neural stem cells are attractive
for having a lineage that is appropriately restricted to the three principal neural cell types found in
the brain.
24
Cell transplantation is a highly promising strategy to induce neuroregeneration, but can
be associated with several downsides. For instance, preparations for cell transplantation, which
may include expanding the number of cells and differentiating them to the desired phenotype, can
be highly costly, laborious and technically challenging.
Many cell types also pose the risk of
detrimental immune complications after their transplantation. Furthermore, control over the cells
is promptly
lost after their transplantation.
The transplanted
cells may differentiate
inappropriately into undesirable cell types or fail to survive over time due to their sudden
introduction in large numbers into the harsh environment in the post-injury brain tissue [92] and
anoikis (dissociation-induced programmed cell death) in the structureless cavitary lesions.
1.3.4
Tools: Biomaterials
Biomaterials are non-viable materials intended to interact with biological systems. One
application of biomaterials has been to re-establish structure in injury sites where liquefactive
necrosis characteristically leads to cavitary lesions. To date, various scaffolds and hydrogels,
derived either from natural polymers such as hyaluronan [93] and gelatin [94], or synthetic
polymers such as PLGA [95] and polycaprolactone [96] have been developed for this purpose. Of
particular interest is the class of injectable hydrogels that can: 1) be implanted (injected) in a
minimally invasive fashion; 2) conform effectively to a irregularly shaped lesion; and 3) undergo
safe cross-linking in situ after injection to impart resistance to in vivo degradative processes.
Injectable hydrogels, which have been successfully injected to induce regeneration in the injured
brain, include peptide nanofiber scaffold [97], xyloglucan [98], matrigel [99] and alginate [100].
These scaffolds and hydrogels have been shown to ameliorate injuries such as distortion of
cerebral cortex around the defect as well as support axonal regeneration across the injury gap.
However, most of them can be relatively biologically inert such that they are often used with
biological factors or cells to improve interactions with the native tissue.
In addition to serving as a physical structure in the injured brain, biomaterials have been
particularly useful in steering molecular agents through the complex aspects of the injured brain,
ensuring that they preserve their integrity and function as well as meet spatial and temporal
requisites. For example, nogo-66 receptor antibody had been conjugated to hyaluronic acid
hydrogels that were implanted within both brain ischemic lesions. Through degradation,
25
the hydrogels helped to gradually release the bioactive antibodies to block inhibitory
signaling via the nogo-66 receptor and enhance axonal growth [101].
Biomaterials have also been particularly useful in improving the survival of transplanted
cells, presumably by serving as a support to reduce anoikis and protecting the cells from the harsh
environment in the injured brain. In a rat model where focal cerebral ischemia led to a cystic
human neural precursor cells to survive 8 weeks after transplantation and mediate a -50
-
cavity in the cerebral cortex, matrigel made the critical difference in allowing transplanted
60% reduction in infarct cavity volume [102]. In another example, VEGF-releasing PLGA
microspheres provided physical support for the transplanted neural stem cells and the in
growth of blood vessels, ultimately leading to the formation of a de novo tissue within the
stroke cavitary lesion [103]. The value of scaffolding biomaterials is also convincingly
illustrated in a study where polyglycolic acid scaffolds were implanted into cystic lesions
resulting from a unilateral hypoxia-ischemic brain injury [104]. In this instance, only cystic
lesions implanted with scaffolds enabled neurons derived from neural stem cells grafted
into the lesion to innervate the contralateral hemisphere as well as host neurons from
contralateral hemisphere to innervate the lesion. Given these successful applications, it is
reasonable to expect biomaterials to become regular features of cell-mediated neuroregenration.
1.4
Endogenous Regenerative Responses
1.4.1
Neurogenesis
The mammalian brain has long been believed to stop producing any nascent neurons soon
after birth until the paradigm was overturned by the discovery that neurogenesis persists into
adulthood [105, 106]. The adult mammalian brain is now widely known to contain NSPCs in both
the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of the
dentate gyrus. These endogenous NSPCs can: 1) generate neurons throughout life; 2) produce
neuroblasts or immature neurons in the SVZ and SGZ that respectively migrate considerable
distances into the olfactory bulb (OB) or a short distance into the granule cell layer of the
hippocampus; and 3) contribute to new neurons that functionally integrate into local neural
26
circuitry. The endogenous NSPCs are regulated by their local neurogenic niches which
encompass i) cellular components; ii) biological molecules; and iii) extracellular matrix. This
dependence on environmental signals allow endogenous NSPCs to behave accordingly when redirected to a different environment (as exemplified by SGZ progenitors which, upon being
grafted into SVZ, behave like SVZ progenitors[107]) and even more importantly, respond to
perturbations to their local environments.
Since their discovery in the adult brain, endogenous NSPCs have been extensively
reported to respond to various forms of brain injury such as hemorrhagic stroke [108], ischemic
stroke [109] and traumatic brain injury [110]. Their ability to respond has moreover been
observed across many species ranging from rodents [111, 112] to humans [113, 114] and in both
the young and the old [115-117]. The responses of endogenous NSPCs proceed as follows. First,
brain injury potently increases the proliferation of endogenous NSPCs through various pathways
such as FGF [118], Notch [119], Wnt and erythropoietin [120]. Second, it strikingly induces cells
(e.g. astrocytes, microglia [121], and endothelial cells [122]) in the injury site to express
chemoattractive signals (e.g. SDF-la [123], angiopoietin 1 [122] and 2 [124], osteopontin [125]
and monocyte chemoattractant protein-l
[126]) which direct NSPCs to migrate towards the
damaged tissue. Third, the NSCs differentiate into the region-specific types of neurons (e.g
striatal medium-sized spiny neurons [109]) destroyed by the injury, form synapses with
neighboring neurons and integrate functionally into the local circuit [127]. Notably, these
responses can last for as long as 16 weeks after the injury [121, 125].
These responses undertaken by endogenous NSPCs have not only proven to offer cell
replacement for the purpose of neuroregeneration, but have also been associated with
improvements in functional behavior after brain injury [128, 129]. This association has further
been scrutinized in a transgenic model where endogenous neuronal progenitors derived from
NSPCs in the SVZ could be specifically ablated after stroke injury [130]. In this study, the
removal of endogenous neuronal progenitors that would typically migrate out of the SVZ into the
injured striatum led to both increased lesion size and aggravated behavioral deficits, thus
demonstrating the importance of endogenous NSPCs in ameliorating the detrimental effects of
brain injury. All in all, the entirety of the endogenous regenerative responses (from proliferation,
migration, differentiation to integration into brain circuitry), their persistence as well as their
27
definitive role in determining the outcomes of brain injury strongly motivate their use in novel
therapies to promote neuroregeneration.
1.4.2
Neovascularization
In the aftermath of brain injury, a highly parallel set of events is also known to occur with
endothelial progenitor cells (EPCs). In response to brain injury (as well as injury in numerous
other tissues), EPCs mobilize from the bone marrow into the circulation and travel into the injury
site. The mediators for this mobilization and recruitment of EPCs overlap significantly with those
for the post-injury neurogenic response and include SDF-la, erythropoietin and VEGF [131]. At
the same time, elevated expression of pro-angiogenic factors stimulate the proliferation and
migration of endothelial cells in existing vessels. The vasculogenesis resulting from recruited
EPCs and angiogenesis mediated by newly generated endothelial cells collectively contribute to
neovascularization in the injured brain [132].
There are several postulated roles for neovascularization in the injured brain. One
plausible role is to increase blood flow and thereby, the delivery of oxygen and nutrients to the
compromised brain [133]. Another possible role is to provide for the passage of phagocytotic
cells and facilitate the removal of debris after brain injury. Recently, there is also an emerging
concept that considers neuronal and vascular elements as a neurovascular unit. This has largely
been informed by observations where the development of neural tube and embryonic blood
vessels is highly integrated [134] and neuronal progenitors migrate in close association with
blood vessels [129]. As such, blood vessels may play the essential role as a guiding cellular
scaffold as well as a signaling partner in neuroregeneration. It is also highly plausible that
neovascularization contributes to functional recovery after brain injury. By far, several reports
have indicated that treatments which increase neovascularization improve behavioral outcomes
[132].
1.4.3
Tools to Manipulate Endogenous Stem and Progenitor Cells
Endogenous regenerative responses constitute an elegant and appealing resource for
neuroregeneration. Unlike non-autologous cells, the endogenous cells involved do not trigger host
28
immune reactions that may lead to catastrophic consequences in the brain or suppress stem cell
differentiation and compromise the efficacy of the treatment. Even when compared to autologous
cells or iPSCs, endogenous cells avoid the complications and significant costs typically
associated with isolating/purifying/processing cells for transplantation.
However, there exist several limitations that prevent these endogenous regenerative
responses from mediating appreciable neuroregeneration after brain injury. First, the endogenous
regenerative responses are largely driven by the expression of various cytokines in the damaged
tissue. In certain injuries, cytokine expression may be limited in duration [135]. As the damaged
tissues die and disappear, cytokine expression is limited in both scale and amount to the surviving
tissue in the periphery of the lesion [136]. Overall, these limitations are likely to cause the
endogenous regenerative responses to proceed in a sub-optimal fashion. Second, the liquefaction
of dying tissue often results in a cavitary lesion and limits the migration of endogenous cells up to
the peri-lesion [137]. Third, the number of endogenous NSPCs resulting from injury-induced
neurogenesis, albeit substantially higher than basal level, is inadequate relative to the massive
loss of neuronal cells after ischemic injury. Lastly, as much as 80 - 90% of the neurons derived
from the endogenous NSPCs recruited to the damaged tissue die in the long term [109, 122]. The
poor survival has been attributed to caspase-mediated apoptosis [121].
Given the huge potential of the injury-induced endogenous regenerative responses,
several tools have been developed to overcome the limitations or stimulate the responses.
Molecular agents have successfully been utilized to target the various aspects of endogenous
regenerative responses, e.g. 1) increasing proliferation of endogenous NSPCs through the use of
TGF-a [138, 139], Notch ligand [140], epidermal growth factor [141]; 2) enhancing the
recruitment of NSPCs towards the damaged tissue by SDF-lp [122], angiopoietin [122] and EPO
[142]; 3) enhancing neovascularization with SDF-la [143]; and 3) inhibiting apoptotic death with
caspase inhibitors [121].
Biomaterials have also contributed to the effort to manipulate the
endogenous cells. The polymer, polyethylene glycol, has been used to facilitate intranasal
delivery of TGF-a, enabling the mitogen to increase the pool of endogenous NSPCs via a noninvasive route [144].
Hyaluronan/methyl cellulose hydrogels have also been applied on the
cortex to provide sustained delivery of EGF [145] and EPO [146] to stimulate the proliferation of
the endogenous NSPCs after stroke injury. Many of the tools above have also been shown to
29
result in functional improvements. Overall, it is clear that the development of tools to enhance
endogenous regenerative responses is a compelling strategy to benefit the injured brain.
1.5
Outline of research
Brain injury typically leads to the liquefaction of affected neural tissue, which in turn
results in the loss of chemokine expression and disappearance of stroma within the lesion. These
two commonly observed deficiencies limit the robustness of the endogenous regenerative
responses and the ability of the involved endogenous cells to migrate into the lesion. The overall
goal of this research is to address these two deficiencies and thereby enhance endogenous
regenerative responses. Our approach toward this goal is to develop a matrix to fill the lesion and
concurrently replaces both elements of chemokine release and stromal support. The overall
hypotheses are that such a lesion-filling matrix can: i) influence or enhance neurogenesis and
neovascularization in the surrounding brain tissue and; ii) re-establish a physical framework to
enable recruited endogenous cells to migrate into the otherwise structureless brain lesion. The
design criteria guiding the development of the matrix include: 1) the need for injectability to
enable the matrix to be delivered via a minimally invasive route and therefore be applicable to
deeply seated brain lesions and; 2) achieving both elements of chemokine release and stromal
support by modular design so that each element can be optimized individually.
The
first
stage
of
the
research
involves
in
vitro
evaluation
of
gelatin
hydroxylphenylpropionic acid (Gtn-HPA) hydrogel as a candidate biomaterial to formulate the
desired matrix for filling brain lesions. This recently developed biomaterial is selected for
investigation because: i) Gtn-HPA is poised to fulfill the criterion for injectability given its ability
to transition from a pre-gel solution into a semi-solid hydrogel; 2) Gtn-HPA permits independent
tuning of gelation rate and crosslinking degree, a flexibility that allows control of the material
properties and the injection procedure without causing constraints to each other; 3) Gtn-HPA
offer additional benefit of undergoing covalent crosslinking which may be useful to appropriately
resist liquefactive processes in brain lesions. The evaluation takes into consideration of the
eventual need for Gtn-HPA hydrogels to provide a permissive environment within the lesion for
endogenous cells to mediate neuroregeneration and is geared toward assessing the ability of Gtn-
30
HPA hydrogels support proliferation, migration and differentiation of adult neural progenitor
cells.
In the second stage, we investigate the use of dextran sulfate/chitosan (DS/CS)
polyelectrolyte complex nanoparticles (PCN) as independent vehicles to encapsulate and deliver
SDF-la, a chemokine that is widely implicated in driving the endogenous regenerative responses
after brain injury. DS/CS PCNs are chosen for their facile preparation and their similarities with
heparan sulfate-based growth factor sequestration in natural extracellular matrices. We assess
whether the encapsulation and loading efficiencies are in line with reported dosage of SDF-la for
in vivo applications and examine their release kinetics. Both the hydrogel and nanoparticle
modules are combined to formulate Gtn-HPA/SDF-la-PCN matrix. Prior to its in vivo
application, we exploit the well-controlled environment of an in vitro three-dimensional
migration assay to examine the ability of Gtn-HPA/SDF-la-PCN matrix to recruit adult neural
progenitors, the chemotactic nature of the recruitment as well as the profile of MMPs involved in
migration into Gtn-HPA/SDF-l a-PCN matrix.
In the third stage, we conduct a pilot study to explore the stereotactic injection of GtnHPA/SDF-la-PCN matrix into a rat brain lesion resulting from ICH injury, as part of the
transition from in vitro experimentation to in vivo application. The collagenase-induced ICH
model is chosen for its ease of implementation, the reproducibility of injury and more
importantly, for the overall relevance of ICH in the sense that this form of brain injury is
clinically managed by stereotactic surgerical procedures which can be readily adopted for
implanting injectable matrices. We specifically seek to validate the delivery of Gtn-HPA/SDF1a-PCN matrix into the injured brain via the needle-based injection procedure and the location of
the implanted matrix relative to the lesion and surrounding host tissue. We also evaluate GtnHPA/SDF-la-PCN matrices composed to varying wt% Gtn-HPA to identify the optimal
formulation for further investigation.
Success criteria include persistence of the matrix,
maintenance of interface with surrounding host tissue and permissiveness toward infiltration by
endogenous cells.
In the final stage, we investigate the use of Gtn-HPA/SDF-l a-PCN matrices as a form of
treatment for brain injury in a rat ICH model. Gtn-HPA/SDF-la-PCN matrices are compared
against aCSF as well as Gtn-HPA and Gtn-HPA/PCN matrices. We assess the effects on
31
behavioral outcome and overall tissue loss, which are two fundamental metrics used to determine
the beneficial value of a treatment for brain injury. We specifically seek to verify our hypothesis
by examining post-injury neurogenesis and neovascularization as well as the infiltration of
matrix-filled lesions by endogenous cells. Alongside, we also examine common injury-induced
responses such as gliosis and neutrophil/macrphage/microglia-mediated inflammation. Overall,
the study is structured to provide a conclusion on the two overall hypotheses of this thesis and
provide a comprehensive picture on the effects of Gtn-HPA/SDF-l a-PCN matrices on the injured
brain.
32
1.6
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45
Chapter 2
Characterization of Injectable Gtn-HPA
Hydrogels as Supporting Matrices for Neural
Progenitors
2.1
Introduction
Neural stem and progenitor cells (NSPCs) can self-renew or differentiate into the three
principal central nervous system (CNS) cell types (neurons, astrocytes and oligodendrocytes).
When generated endogenously or transplanted into the brain, they can potentially mitigate tissue
loss after injuries such as hemorrhage, ischemic stroke or trauma. However, the environment at
injury sites in the brain is highly unfavorable for the introduction of these cells. The progression
of a CNS injury dictates the dissolution and clearance of necrotic tissue resulting in a cavitary
lesion. Without structural support within the lesion, transplanted NSPCs lack cell-matrix
interactions and may suffer from anoikis [1]. Of the cells that survive, most lack a matrix to be
spatially retained and organized within the lesion and typically leave the lesion for the
surrounding viable host tissue [2]. For similar reasons, endogenous NSPCs migrating towards the
lesion usually fail to infiltrate the lesion core [3]. Within the lesions, transplanted or endogenous
NSPCs are also exposed to hostile factors [4] such as oxidative stress, hypoxia and inflammatory
responses and tend to survive poorly.
Collectively, these conditions reduce the potential of
NSPCs to mediate meaningful regeneration in the brain lesions.
For these reasons, injectable hydrogels are increasingly important in therapeutic
approaches focusing on NSPC-mediated regeneration in cavitary lesions after brain injuries.
Injectable hydrogels provide the potential means to reestablish structure in the cavitary lesions
and modulate the hostile environment. Their injectable nature enables their introduction with
minimal disruption of surrounding tissue and upon gelling in situ, optimal conformation to the
46
cavity. Notable benefits (e.g. increased survival of transplanted NSCs and reduction in lesion
cavity [5]) have also been demonstrated with the use of injectable hydrogels in brain lesions.
Injectable hydrogels which crosslink non-covalently upon thermal or .ionic stimuli (e.g. Matrigel
[5] and self-assembling peptide hydrogels [6]) have attracted much attention over the years due to
their cytocompatibility and availability. However, such hydrogels may suffer from potential
mechanical instability or premature dissolution and are often passive matrices for the transplanted
cells. Therefore, there is growing interest in hydrogels with cytocompatible covalent crosslinking
chemistries [7, 8] as well as those with features that can actively influence NSPCs in aspects
relevant for neural tissue regeneration [9, 10].
Recently, a class of biopolymers [11-14], including gelatin-hydroxyl-phenylpropionic
acid (Gtn-HPA) [15], was developed in order to enable safe in vivo covalent crosslinking via
enzyme-mediated oxidative coupling of phenol moieties conjugated along the polymers. During
crosslinking, the concentrations of the enzyme (horseradish peroxidase, HRP) and the oxidant
(hydrogen peroxide, H 2 02 ) independently control the gelation rate and the degree of crosslinking
respectively. An injectable formulation of these biopolymers can potentially be fine-tuned to: i)
meet procedural requirements; ii) optimize conformity to irregularly-shaped cavitary defects; and
iii) produce a matrix which matches the mechanical behavior of surrounding neural tissues.
Among them, Gtn-HPA is of particular interest for neural applications given the role of its
tunable mechanical stiffness in promoting neurogenic differentiation of mesenchymal stem cells
(MSCs) [16].
Towards the ultimate goal of using injectable Gtn-HPA hydrogels in brain injury sites to
support endogenous and transplanted NSPCs in brain injuries, the objective of this study was to
investigate whether Gtn-HPA hydrogels would serve as appropriate scaffolding materials
for adult neural progenitor cells (aNPCs). Specifically, we evaluated cytocompatibility of GtnHPA and examined the effects of the hydrogels on the oxidative stress resistance of the aNPCs.
We further investigated if Gtn-HPA hydrogels would provide a stromal framework that was
permissive for the proliferation, migration and differentiation of aNPCs, given the importance of
these processes in tissue regeneration. Finally, we tracked the survival of aNPC-derived neurons
and astrocytes within Gtn-HPA hydrogels.
47
2.2
Materials and Methods
2.2.1
Preparation of Gtn-HPA Hydrogels
As previously described,
Gtn-HPA conjugate was synthesized
via a general
carbodiimide/active ester-mediated coupling reaction [15]. Specifically, 3.32g of HPA (Sigma
Aldrich) was dissolved in 250ml of 40 vol% NN-dimethylformamide (in deionized water),
followed by addition of 3.20g
of N-hydroxysuccinimide
and 3.82g
of 1-ethyl-3-(3-
dimethylaminopropyl)-carbodiimide hydrochloride. The solution was stirred for 5 hr at room
temperature. 150ml of 6.25 wt% Gelatin (MW = 80 -140 kDa; pI =5; Wako Japan) solution was
then added to the solution, which stirred overnight at room temperature. Using dialysis tubes with
molecular cut-off of 1 kDa, the solution was dialyzed against 0. 1M sodium chloride solution for 2
days, 25 vol% ethanol (in deionized water) for 1 day and deionized water for 1 day. The purified
solution was eventually lyophilized and stored at 4"C until use. 90% of the amine groups in
gelatin were conjugated with HPA. To prepare Gtn-HPA hydrogels (GH-0.85, GH-1.0, GH-1.2
and GH- 1.7), 2 wt% solution of Gtn-HPA, either alone or added with cells at the required density,
was sequentially mixed with 0.1 U/ml HRP (Wako USA), unless specified otherwise, and 0.85,
1.0, 1.2 or 1.7 mM H 20 2 (Sigma) respectively.
2.2.2
Rheological Measurement
Measurements were performed with a TA instruments AR-G2 rheometer using cone and
plate geometry of 40mm diameter and 20 angle. For each measurement, 750ptl of Gtn-HPA
containing 0.03 U/ml HRP and varying concentrations of H 20 2 was applied to the bottom plate
immediately after mixing. The upper cone was lowered to a measurement gap of 54ptm. A layer
of silicone oil was also applied during the experiment to prevent evaporation. All measurements
were taken at 370C in the oscillation mode with a constant strain of 1% and frequency of 1 Hz. To
estimate the gelation rate, the time at which gel point (as defined by the crossover between
storage modulus, G' and loss modulus, G") occurred was measured.
48
2.2.3
In vitro Degradation Assay for Gtn-HPA Hydrogels
100pl gels were incubated in 2ml of phosphate-buffered saline (PBS) containing 10U/ml
type I collagenase (Invitrogen) and incubated at 37C on an orbital shaker at 150rpm. Samples
were collected at every hour for six hours and analyzed for degradation products from Gtn-HPA
hydrogels using the bicinchoninic assay (Thermo Fisher Scientific). Degradation rate constants
were derived by fitting the data for mass loss into an inverse exponential model.
2.2.4
Neural Progenitor Cell Culture
aNPCs were kindly provided by the University of California, San Diego and had been
isolated from the hippocampi of adult female Fischer 344 rats as previously described [17]. Cells
were grown on poly-ornithine/laminin (P/L)-coated flasks in N2 media [DMEM/F12 media with
1% N2 supplement (Invitrogen) and 1% Penicillin-Streptomycin (Sigma)] containing 20ng/ml
recombinant human fibroblast growth factor (FGF)-2 (R&D Systems) as a monolayer culture to
achieve a high degree of population homogeneity as compared to neurosphere culture [18]. At
approximately 80% confluency, cells were subcultured using accutase (Sigma).
2.2.5
Adhesion Assay
Gtn-HPA conjugate and hyaluronic acid-tyramine (HA-Tyr) conjugate were dissolved in
PBS and mixed at different ratios to a final concentration of 2wt%. 200pl of the mixture was
added into each well in a 24-well plate and crosslinked with 0.03U/ml HRP and 1700pM H 0
2
2
for 30 min. aNPCs were seeded onto hydrogel- or P/L-coated 24-well plates at 75,000/cm 2 and
incubated for 4 hours to allow for cell attachment. Each well was then aspirated and washed once
with PBS to remove any unattached cells. Cells attached on hydrogels were then harvested using
an enzyme cocktail containing 1000U/ml collagenase and 400U/ml hyaluronidase (Sigma) while
those attached on P/L-coated wells were harvested using accutase. The picogreen DNA
quantification assay (Invitrogen) was used to determine the number of attached cells.
49
2.2.6
Evaluation of Viability
aNPCs [(5,000 cells/cm 2 on P/L-coated 4-well chamber slides or 4x 10 5/ml within 100pl
of Gtn-HPA hydrogels in transwell cell culture inserts (6.4mm diameter, 3.0im pore,
polyethylene terephthalate membrane, BD Biosciences)] were incubated with 2pM calcein AM
and 4pM ethidium homodimer-1 in N2 media for 1 hour at 37*C, 5% CO 2. They were further
incubated with PBS for 30 min at room temperature before imaging with epifluorescence
microscope (Olympus BX60). Cells in three randomly selected fields of view under 20x objective
lens magnification were counted.
2.2.7
Oxidative Stress Resistance Assay
aNPCs were encapsulated at 4x 105/ml within 100pl of 0.lwt% collagen type I (BD
Biosciences), 2 wt% alginate (FMC BioPolymer; crosslinked with 25mM CaCl 2) or 2 wt% GtnHPA (crosslinked at 1mM H 2 0 2 ). Collagen and alginate were chosen as control hydrogels to
encapsulate aNSCs because they are commonly used, well-characterized biomaterials with highly
cytocompatible crosslinking [19, 20] that would not pose detrimental effects on the aNPCs prior
to the oxidative stress challenge, and could each partially mimic Gtn-HPA in aspects such as
mass density and cell adhesiveness. Following 24 hours of culture, aNPCs were exposed to media
containing H 2 0 2 for 4 hours to induce oxidative stress and measured for survival. The
concentration range of H 202 was rationalized at 50 - 500pM to impose sufficient oxidative stress
on the aNPCs even in the presence of phenol red in DMEM/F12 media and possible shielding by
the three-dimensional matrices around the cells. To investigate the effect of HRP/H 20 2
crosslinking on the bioreduction capacity of aNPCs, cells encapsulated within Gtn-HPA were
either compared directly with cells encapsulated within collagen hydrogels or extracted from GtnHPA hydrogels with 500U/ml collagenase type I and plated on P/L surfaces to be compared with
control monolayer cultures. To investigate if soluble HPA could prevent the cytotoxic effects of
H 2 0 2 , aNPCs were fed with fresh media containing HPA before the addition of H202. Survival or
bioreduction capacity of aNPCs was measured using the MTS assay (Promega) according to the
manufacturer's instructions. For relevant measurements, background absorbance determined from
cell-free hydrogels was subtracted prior to analysis.
50
2.2.8
Evaluate of proliferation and migration
aNPCs (5000/cm 2 on P/L-coated tissue cultureware or Ix 10 5/ml within Gtn-HPA
hydrogels) were maintained in N2 media containing lOng/ml FGF-2. At 4 and 7 days postplating, accutase or 1000U/ml collagenase was added to harvest monolayer and hydrogelencapsulated aNPCs respectively. The number of cells harvested was quantified using the
Picogreen Assay.
2.2.9
Evaluation of differentiation
aNPCs (5,000 cells/cm 2 onto P/L-coated 4-well chamber slides or 4x 10 5/ml within Gtn-
HPA hydrogels in transwell cell culture inserts) were treated with differentiation media for 6
days. The differentiation conditions included: 1) 1PM retinoic acid (RA; Sigma) and 5piM
forskolin (For; Sigma) for neuronal differentiation; 2) 50ng/ml leukemia inhibitory factor (LIF;
Millipore) and 50ng/ml bone morphogenetic protein (BMP)-4 (Peprotech) for astrocytic
differentiation; and 3) lpM RA and 1% fetal bovine serum (FBS; Invitrogen) for mixed
differentiation.
2.2.10 Immunofluorescent Staining
aNPCs
in
monolayer
culture
or
Gtn-HPA
hydrogels
were
fixed
with
4%
paraformaldehyde at room temperature for 15 min and 1 hour respectively. The Gtn-HPA
hydrogels were further cryoprotected in 25% sucrose solution, flash-frozen in isobutane (-30*C),
embedded in Tissue Tek O.C.T compound (Sankura Finetek) and cryosectioned (50pm). All
staining procedures were carried out at room temperature unless mentioned otherwise. Samples
were blocked and permeabilized with 10% donkey serum (Sigma) and 0.3% Triton X-100 in PBS
for 2 hours. Samples were then incubated with primary antibodies for 2 hours at room
temperature or overnight at 40C. After washing in PBS/0.3% Triton X-100, samples were
incubated with secondary antibodies for 2 hours and eventually with DAPI (1:50,000, Invitrogen)
to counterstain cell nuclei. The primary antibodies used were mouse monoclonal anti-p-tubulin III
(TuJI, 1:5000, Covance) to identify neurons and rabbit polyclonal anti-glial fibrillary acidic
protein (GFAP, 1:2000, Dako) to identify astrocytes. The secondary antibodies used were Dylight
488-conjugated donkey anti-mouse and anti-rabbit IgG (1:500, Jackson ImmunoResearch).
51
Images were collected using epifluorescence microscope (Olympus BX-60). Controls, in which
undifferentiated aNPCs were stained or primary antibodies were replaced with relevant IgG
(Dako), resulted in no detectable staining. Cells were counted in three randomly selected fields of
view for each well of a chamber slide (monolayer culture) or each of three cryosections (culture
within Gtn-HPA hydrogel). Cells with pyknotic nuclei were excluded from counting.
2.2.11 Quantitative Real-Time Polymerase Chain Reaction
Total RNA was isolated using RNeasy Mini Kit (Qiagen, USA) and measured with
NanoDrop ND-1000 to determine concentration and purity. RNA was then reverse transcribed
into complementary DNA using iScript cDNA Synthesis Kit (Bio-Rad, USA) according to the
manufacturer's protocol. For quantitative real-time polymerase chain reaction (qRT-PCR),
reactions were prepared with 10pl Fast SYBR Green Master Mix, 250nM of each forward and
reverse primer, 500ng cDNA and sufficient nuclease-free water to achieve 20pl in total and were
performed in duplicates on Applied Biosystems StepOnePlus PCR System. Samples were
analyzed for the genes TuJl and GFAP with normalization to the reference gene glyceraldehyde3-phosphate dehydrogenase (GAPDH). Expression level of each target gene was compared to day
0 controls and the relative fold change was calculated as 2 -sAct [21]. Forward and reverse primers
for GAPDH, TuJI and GFAP were summarized in Table 1.
2.2.12 Statistical Analysis
A sample size of 3 - 6 was used in all experiments. Values are expressed as mean
standard error mean (SEM). Analyses of variance (ANOVA) and Student's t-test were used for
statistical analysis. Statistical significance was set at p<0.05.
52
2.3
Results
2.3.1
Rheological Measurements of Gtn-HPA Hydrogels
-
All Gtn-HPA hydrogels (crosslinked to varying degrees with 0.85, 1.0, 1.2 and 1.7mM
H 20 2 and termed as GH-0.85, GH-1.0, GH-1.2 and GH-1.7 respectively) reached gel point in 40
45 seconds when crosslinked with the same HRP concentration of 0.03U/ml. On the other hand,
the crosslinking degree, which was controlled by H 20 2 concentration, significantly influenced the
storage moduli of Gtn-HPA hydrogels (one-factor ANOVA, p<0.0001). With increasing
crosslinking degrees, G' was found to increase from 449 Pa to 1717 Pa (Figure 2.1).
-
2000
-
1600
-
400
0-
El
K]z
GG
.
GH-0.85 GH-1.0
5
-
P 80 0
-1-
-
1200
-
'%
GH-1.2
GH-1.7
Figure 2.1: Storage moduli of Gtn-HPA hydrogels changed significantly with the crosslinking degree
controlled by H 20 2 concentration (n=3, p<0.0001, ANOVA).
53
2.3.2
In vitro Degradation Profiles of Gtn-HPA Hydrogels
Varying crosslinking degrees also resulted in concomitant changes in the degradation
rates of Gtn-HPA hydrogels. When degraded with type I collagenase, Gtn-HPA hydrogels with
higher crosslinking degrees underwent a slower rate of mass loss (Figure 2.2). The effect of
crosslinking degree on degradation rate constant was significant (one-factor ANOVA, p<0.01),
with the rate constant decreasing from 94 to 38 %/hr over the selected crosslinking degrees
(Figure 2.2 insert).
100
S80
Degradation
60
60 -
k 40
0
--
-
rate constant (%/hr)
so
100
150
GH-0.85
Al-GH41.7~]
0
0
1
2
3
4
Time (hr)
5
6
Figure 2.2: Mass loss for Gtn-HPA hydrogels upon enzymatic degradation by lOU/ml collagenase I (n=3).
(Insert) Degradation rate constants were derived from an inverse exponential model and changed inversely
with the crosslinking degree (p=0.0003, ANOVA).
54
2.3.3
Adhesion of Neural Progenitor Cells on Gtn-HPA Hydrogels
Gtn-HPA hydrogels served as adhesive matrices for aNPCs, yielding an attachment of
83.1% that was as high as well-established substratum coatings such as P/L (Figure 2.3). We
further examined the efficacy of Gtn-HPA in promoting aNPC adhesion at lower densities by
preparing a "titrated" series of 2wt% composite hydrogels with varying weight ratios of Gtn-HPA
: HA-Tyr (Hyaluronic acid-Tyramine, a minimally adhesive polymer which could be crosslinked
via the same mechanism as Gtn-HPA). Hydrogels comprising of HA-Tyr alone resulted in a low
level of aNPC adhesion (6.2%). Proportions of Gtn-HPA at 10% or 20% produced little or no
enhancement to aNPC adhesion. However, further increments of Gtn-HPA to 30% and 40% gave
rise to steep increases in aNPC adhesion (Fisher's post-hoc test, p<0.001). Once the proportion of
Gtn-HPA in the composite hydrogels was increased to 40% or higher, aNPC adhesion reached the
optimal level seen with P/L or 100% Gtn-HPA hydrogels.
100
80
60
40
20
0
60
80
100
%of Gtn-HPA in hydrogel coating
0
20
40
P/L
Figure 2.3: aNPC adhesion on 2wt/o composite hydrogels with varying ratios of Gtn-HPA : HA-Tyr (n=6).
55
2.3.4
Viability of Neural Progenitor Cells within Gtn-HPA Hydrogels
The viability of aNPCs encapsulated within Gtn-HPA hydrogels was measured to assess
the cytocompatibility of Gtn-HPA. Despite exposure to H 2 02 during the enzyme-mediated
oxidative crosslinking process, the viability of encapsulated aNPCs remained as high as the
control cultures that were spared of any hostile handling (-95-97% for cultures on P/L and within
GH-0.85, GH-1.0 and GH-1.2). Only GH-1.7 resulted in a slight decrease in cell viability to
-93%.
2.3.5
Oxidative Stress Resistance of Neural Progenitor Cells
within Gtn-HPA Hydrogels
The high tolerance of encapsulated aNPCs towards H 20 2 used in the crosslinking process
prompted further evaluation of their oxidative stress resistance. Using GH-1.0 as representative
Gtn-HPA hydrogels, aNPCs were challenged with H 2 02 one day after their encapsulation and
compared with similar aNPC cultures within collagen and alginate hydrogels. Two-factor
ANOVA revealed a significant dependence of metabolic viability on H 0 concentration
2
2
(p<0.0001) and more importantly, the type of hydrogel (p<0.0001) (Figure 2.4).
Specifically,
aNPCs encapsulated within collagen and alginate hydrogels were vulnerable to the oxidative
stress induced by H 2 0 2 and suffered massive cell death, as evidenced by MTS assay and
OCollagen
120
S100
U Alginate
GGtn-HPA
M*
S80
60
0
0
40
20
0
0
50
100
200
500
Oxidative stress [H202] (pM)
Figure 2.4: Metabolic viability of aNPCs upon challenge with cytotoxic levels of oxidative stress (n=3).
aNPCs encapsulated within Gtn-HPA hydrogels, as represented by GH-1.0, maintained a significantly
higher level of viability when compared to those in collagen and alginate hydrogels (** p<0.01, Fisher's
post-hoc test)
56
increased appearance of necrotic cells, especially towards the higher H 2 0
2
concentrations. In
contrast, the metabolic viability of aNPCs encapsulated within Gtn-HPA hydrogels remained
unchanged by 11202 concentrations up to 200pM, with cell death becoming more apparent only at
500pM. The viability of aNPCs within Gtn-HPA hydrogels was therefore higher than those
within collagen and alginate at nearly every level of oxidative stress investigated (Fisher's posthoc test, p<0.01), particularly at 500pM H202 where metabolic viability within Gtn-HPA
hydrogels persisted at -84% as opposed to -8% and -15% in collagen and alginate hydrogels
respectively.
The increased resistance of aNPCs against oxidative stress could stem from either an
"extracellular" effect mediated by the presence of protective agents such as free radical
scavengers or an "intracellular" effect where NPCs were preconditioned by prior exposure to subcytotoxic levels of oxidative stress [22]. Since aNPCs would have been transiently exposed to
H202 during their encapsulation within Gtn-HPA hydrogels, we determined if the higher
oxidative stress resistance was due to an altered state in the aNPCs arising from preconditioning.
In the MTS assay which examined the deydrogenase-dependent bioreduction of the tetrazolium
salt, aNPCs encapsulated within GH-1.0 (and therefore had been exposed to oxidative
crosslinking)
exhibited 27.4% higher bioreduction capacity than aNPCs that had been
encapsulated within collagen hydrogels and thus not been exposed to HRP/H 20 2 crosslinking (ttest, p<0.01, Figure 2.5).
ID 0.4
Figure 2.5: Bioreduction capacity of aNPCs
measured
by
dehydrogenase-dependent
0 Hydrogel culture
culture
-0Monolayer
0.3
bioreduction of tetrazolium salts when the cells
U
were in either i) Gtn-HPA (+) or collagen
0
hydrogels (-) (n=3); or ii) monolayer culture (n=6).
*M 0.1
Prior exposure to HRP/H 20 2 crosslinking led to a
0
significant increase in bioreduction capacity (**
p<0.01, * p<0.001, Student's t test).
0
+
Z
Exposure to
HRP/H2O2 crosslinking
57
To confirm that the higher bioreductive capacity was not due to differences in the culture
microenvironment (i.e., Gtn-HPA versus collagen) during assay, aNPCs within Gtn-HPA
hydrogels were also extracted via enzymatic digestion of the hydrogels and plated on P/L to be
compared with control cultures. Again, aNPCs with prior exposure to HRP/ H 2 0 2 crosslinking
displayed 32.4% higher bioreductive capacity (t-test, p<0.0001).
A fair amount of HPA moieties on Gtn-HPA might remain uncoupled after crosslinking
and be responsible for protecting encapsulated aNPCs from oxidative stress by scavenging
radicals generated from H 2 0 2 or by increasing the activity of cellular enzymes that aids in
breaking down H 2 02 [23]. We tested this alternative hypothesis by challenging aNPC cultures on
P/L with H 202 in the presence of soluble HPA to determine if the cytotoxicity effects of oxidative
stress could be ameliorated. 50RM H202 produced a low level of cytotoxicity that decreased
,
aNPC viability to 77%. The presence of soluble HPA, even up to 10-fold in excess of H2 0 2
failed to produce any recovery in aNPC viability (Figure 2.6). Similarly, soluble Gtn-HPA
conjugates, providing approximately -279RM HPA immobilized on the soluble gelatin polymers,
did not significantly reduce the cytotoxic effects arising from 50ptM H2 0 2 (data not shown). It
was therefore likely that HPA did not directly mediate the increased oxidative stress resistance
observed in aNPCs encapsulated within Gtn-HPA hydrogels. Collectively, the above findings
suggested that exposure
to the HRP/H 202 crosslinking of Gtn-HPA could provide a
preconditioning effect on aNPCs and increase their oxidative stress resistance.
100
-
-
120
80T
:560
40
20
-
+
+
+
0
50
200
+
H
HPA (pM)
0
500
Figure 2.6: Effects of soluble HPA on the viability of aNPCs that were challenged with oxidative stress
induced by 50pM H20 2 (n=3-5).
58
2.3.6
Proliferation of Neural Progenitor Cells within Gtn-HPA Hydrogels
aNPCs encapsulated within Gtn-HPA hydrogels increased significantly in number by day
4 and 7 (two-factor ANOVA, p<0.0001) in response to mitogen FGF-2 (Figure 2.7). The
proliferation rate within the Gtn-HPA hydrogels was, however, generally lower compared to
monolayer culture on P/L (Fisher's post-hoc test, p<0.0001). The crosslinking degree of GtnHPA also exerted a significant influence on the rate of proliferation (two-factor ANOVA,
p<0.0001). By day 4, proliferation of aNPCs was evidently in an inverse trend with the
crosslinking degree i.e. each higher crosslinking degree resulted in a significantly lower rate of
proliferation. By day 7, proliferation within the hydrogel with the highest crosslinking degree
(i.e., GH-1.7) continued to be the lowest (Fisher's post-hoc test, p<0.0001) while those in the
other three crosslinking degrees exhibited no significant difference.
60
3JP/L
E 50
0 GH-0.85
= 40
O GH-1.0
0 GH-1.2
30
* GH-1.7
20
2
0
10
U-
0
Day 4
Day 7
Figure 2.7: Proliferation of aNPCs cultured on P/L surfaces or within Gtn-HPA hydrogels by day 4 and 7
(n=4). aNPCs in Gtn-HPA hydrogels proliferated more slowly as compared to monolayer cultures
(p<0.0001, Fisher's post-hoc test). Among Gtn-HPA hydrogels, higher crosslinking degrees significantly
decreased the rate of proliferation (p<0.0001, ANOVA).
2.3.7
Migration of Neural Progenitor Cells within Gtn-HPA Hydrogels
When cultured in the mixed differentiation condition, aNPCs encapsulated in GH-1.0,
GH- 1.2 and GH- 1.7 were noted to form neurospheres with processes extending outwards (Figure
59
2.8A). (aNPCs in GH-0.85 mostly remained as individual cells and were excluded from the
following analysis). Immunofluorescence staining for immature neuronal marker TuJl revealed
that the processes in all hydrogels were TuJl' and therefore originated from cells of neuronal
lineage. DAPI nuclei staining further revealed that the nature of these processes changed as the
crosslinking increased. In moderately crosslinked Gtn-HPA hydrogels (i.e. GH-1.0 and GH-1.2),
these processes contained multiple nuclei along their length and were likely to be migratory
chains of young neurons (Figure 2.8B & C). On the other hand, processes in the heavily
crosslinked Gtn-HPA hydrogels (i.e., GH-1.7) did not contain any nucleus and were neurites
extending from the neurospheres (Figure 2.8D).
B
Figure 2.8: Chain migration from neurospheres. (A) Phase contrast of neurospheres formed after 6 days
in
mixed differentiation condition. Representative immunofluorescence images showing neuronal marker
TuJI (green) and DAPI nuclei (red pseudocolor) staining in neurospheres within (B) GH-1.0, (C) GH-1.2
and (D) GH-1.7. Bar=50pm.
60
This was also reflected in our measurements of process length and particularly, process
width, which gave a strong indication if entire cells were present in the process. One-factor
ANOVA revealed significant effects of crosslinking degree on both process length (p<0.0001)
and width (p<0.001) (Figure 2.9 A & B). Between GH-1.0 and GH-1.2, the increment in
crosslinking did not affect the process width but caused a reduction in process length (Fisher's
post-hoc test, p<0.05). This agreed with the immunostaining findings that the increment in
crosslinking from GH-1.0 to GH-1.2 did not prevent chain migration from occurring but did
impede the extent of migration of the immature neurons along these chains. On the other hand,
between GH-1.2 and GH-1.7, both process length and width were significantly reduced (Fisher's
post-hoc test, p<0.0001 and p<0.001 respectively). This was also in line with the immunostaining
findings that further increment in crosslinking from GH- 1.2 to GH- 1.7 eliminated chain migration
and only permitted the formation of neurites.
B
20 - NeurosphereI
absent I
15
Migratory
chains
-
INeurites
I
200
1
100
150
NeurosphereI
absentl
Migratory
chains
I Neurites
*
A
0T
10
05
so
CL
0
1
GH-0.85 GH-1.0 GH-1.2 GH-1.7
GH-0.85 GH-1.0 GH-1.2 GH-1.7
Figure 2.9: (A) Width of processes extending from neurospheres (n=4), showing that increases in the
crosslinking degree only produced a significant reduction at GH-1.7 (*** p < 0.001, Fisher's post-hoc test).
(B) Length of processes extending from neurospheres (n=4), which decreased significantly at each
increment in the crosslinking degree (* p<0.05, *** p<0.001, Fisher's post-hoc test).
61
2.3.8
Differentiation of Neural Progenitor Cells within Gtn-HPA Hydrogels
We first examined the influence of Gtn-HPA hydrogels on gene expression for neuronal
marker TuJ1 and astrocytic marker GFAP when aNPCs were cultured in various differentiation
conditions. Using GH-1.0 as representative Gtn-HPA hydrogels, encapsulated aNPCs were noted
to exhibit higher gene expression for the lineage-specific markers when compared to those
cultured on P/L [i.e. enhancements in gene expression for i) TuJ1 in neuronal differentiation
condition (Figure 2.1 OA); ii) GFAP in astrocytic differentiation condition (Figure 2.1 OB); and iii)
both markers in mixed differentiation condition (Figure 2.1 OC)].
We further performed immunofluorescence staining for TuJ1 and GFAP and quantified
the proportion of aNPCs that differentiated into neurons and astrocytes. In general, Gtn-HPA
hydrogels constituted a permissive environment for aNPCs to differentiate specifically in
response to the given stimuli. aNPCs encapsulated in Gtn-HPA hydrogels differentiated mostly
into neurons (60% vs 3% astrocytes) in neuronal differentiation condition (Figure 2.1 lA-B),
mostly astrocytes (57% vs 12% neurons) in astrocytic differention condition (Figure 2.11C-D)
and a mixture of neurons and astrocytes (43% and 18% respectively) in mixed differentiation
A
8
B
TuJ1--6-GFAPr 8
-
-
TuJ1-'&-GFAP
8
786
2
0
6 rn
V
R.
ai
E
0-
4
C,
I,
CL
2
0
8
GTu1
4
-VL
x2-
Gtn-HPA
--
2
W,
0
0
P/L
C
3
G) M2
V
-4
0
P/L
Figure 2.10:
GFAP 78
Gtn-HPA
QRT-PCR measurements
of gene
expression (fold change from Day 0) for neuronal
S6
ID
marker TuJI and astrocytic marker GFAP in aNPCs
'C
a'
cultured on P/L surfaces and within GH-1.0 (n=3-4)
60
Ew
4
3
-2
P/L
Gtn-HPA
mixed differentiation conditions (** p<0.01,
p<0.001, Student's t test).
62
*
after 6 days in (A) neuronal, (B) astrocytic and (C)
0
A
._.
Gtn-HPAB
P/L
-
100
OTuJ1+
* GFAP+
80
40
-
60 60
20
C
P/L
D
Gtn-HPA
-U
P/L
100
OTuJi1+
P/L
*GFAP+
80
60
P/L
E
GH-0.85 GH-1.0 GH-1.2 GH-1.7
GH-0.85 GH-1.0 GH-1.2 GH-1.7
F
Gtn-HPA
100
0TuJ 1+
M GFAP+
80
60
*
..
40
20
P/L
GH-0.85 GH-1.0 GH-1.2 GH-1.7
Figure 2.11: Immunofluorescence staining for TuJI and GFAP after after 6 days in (A-B) neuronal, (C-D)
astrocytic and (E-F) mixed differentiation conditions. (A, C, E) Representative immunofluorescence
images of aNPCs cultured on P/L surfaces and within GH-1.0 (Bar=20jm). (B, D, F) Quantification of
aNPCs stained positive for TuJI and GFAP (n=3) (** p<0.01, Fisher's post-hoc test).
63
condition (Figure 2.11E-F). The proportions of neurons and astrocytes derived from aNPCs
resulting from neuronal and astrocytic differentiation conditions were similar between P/L and
Gtn-HPA hydrogels. In mixed differentiation condition, aNSCs within Gtn-HPA hydrogels
produced 2.0-fold more neurons (Fisher's post-hoc test, p<0.01) and 1.5-fold more astrocytes
(Fisher's post-hoc test, p<0.01) than those on P/L. Finally, we determined if the crosslinking
degree of Gtn-HPA hydrogels would affect the differentiation of aNPCs. In all differentiation
conditions and across all four selected crosslinking degrees, one-factor ANOVA did not reveal a
significant effect in the crosslinking degree of Gtn-HPA hydrogels on neuronal or astrocytic
differentiation.
64
2.3.9 Survival of Progenitor-derived Neurons and Astrocytes within Gtn-HPA
Hydrogels
In addition to the direct influence on the lineage commitment of aNPCs, we examined if
Gtn-HPA hydrogels might exert selective effects over the course of differentiation. We
specifically applied neuronal or astrocytic differentiation conditions and tracked the cell number
and cell death to determine if Gtn-HPA hydrogels would preferentially promote proliferation or
survival in progenitors of a particular lineage. In measuring cell number over the 6 days of culture
(Figure 2.12A), we observed significantly smaller increases in cell number in Gtn-HPA hydrogels
as compared to P/L in both neuronal and astrocytic differentiation conditions. These resembled
the trend for proliferation during expansion culture with FGF-2. Two-factor ANOVA analysis on
the increases in cell number within Gtn-HPA hydrogels over 6 days did not demonstrate any
significant effect in the type of differentiation condition, suggesting that Gtn-HPA did not favor
proliferation of progenitors of one lineage over the other. In terms of cell survival (Figure 2.12B),
a modest increase in cell death was observed within Gtn-HPA hydrogels when compared to P/L.
Interestingly, two-factor ANOVA analysis on cell death within Gtn-HPA hydrogels further
revealed a significant effect in the type of differentiation condition (p<0.0001). While cell death
was minimal in neuronal differentiation condition and remained below 10% throughout all 6
days, a slight but significant increase in cell death was observed in astrocytic differentiation
condition at day 4 and 6 (t test, p<0.01).
A
B
Gtn-HPA:
E
,--Gtn-HPA:
20
Neure
P/L Neuro
6
-*-Gtn-HPA:
16
ti..
It.~tro
--
Neuro
-0-
Gtn-HPA: Astro
P/L Neuro
Day
0
-*
Astro
T
A- P/L: Astro
2~1
-
0
Day
0
Day 2
Day 4
Day 6
Day 2
Day 4
Day
6
Figure 2.12: Selective effects of Gtn-HPA hydrogels on aNPC proliferation and survival in neuronal and
astrocytic differentiation conditions. (A) Fold increase (relative to day 0) in cell number and (B) cell death
over the course of differentiation on P/L surfaces and within GH-1.0 (n=4, ** p<O.01, Student's t test).
65
2.4. Discussion
Gtn-HPA hydrogels, with their independent tuning of gelation rate and crosslinking
degree, have been shown to be promising biomaterials for delivering and influencing stem cells
[16, 24, 25].
Moreover, other data in our lab (not shown) have revealed that the hydrogels
display minimal swelling characteristics (a mere 7% increase in the diameter of MSC-laden GH0.85 hydrogels and an 8% decrease in the diameter of MSC-laden GH-1.7 hydrogels after 7 days
of culture) that are appropriate for applications in the CNS where fluctuations in interstitial
pressure should be avoided. Our study further revealed several physical and chemical aspects of
Gtn-HPA hydrogels that were notable for their potential role as injectable matrices to support
endogenous or transplanted aNPCs. First and foremost, the storage modulus of Gtn-HPA
hydrogels changed sensitively with the crosslinking degree and could be tuned to levels that were
appropriately close to that of neural tissue in the adult brain (400 - 100OPa [26, 27]). Studies on
biomaterial-based implants in a variety of tissue types (e.g. heart [28, 29] and spinal cord [30]) or
on the extreme case of silicon-based neural electrodes implanted in the brain [31] have suggested
that mechanical mismatch between implant and surrounding tissue could result in strain along the
implant-tissue interface, inflammatory responses or even tissue laceration. With the capability to
match the storage modulus of surrounding neural tissue in the adult brain, Gtn-HPA can
constitute appropriate implants that avoid complications arising from mechanical mismatch.
The enzyme-mediated covalent crosslinking of Gtn-HPA, which involved the use of
H202, was shown to be highly cytocompatible with aNPCs. Our supposition is that such high
cytocompatibility could arise from the rapid consumption of H 2 0 2 during the oxidative
crosslinking process. This might be verified in the future with probes such as dihydrorhodamine
or 3'-(p-Aminophenyl) fluorescein [32] to track the concentration of H 0 and free radicals,
2
2
although the probes, which would consume H2 0 2 to provide fluorescent signals, would need to be
employed in an appropriate fashion that would not severely interfere the HRP/ H 202 crosslinking
of Gtn-HPA. In addition to the high cytocompability, the encapsulated aNSCs unexpectedly
displayed a dramatically increased resistance against oxidative stress. In the lesion environment
after brain injury, resistance against oxidative stress can be beneficial. During CNS injury,
endogenous antioxidant mechanisms, such as superoxide dismutases, dehydrogenases and
glutathione peroxidases, are perturbed, leading to insufficient clearance of oxygen radicals [33].
66
Such excessive oxidative stress has been postulated to contribute to the poor survival of
transplanted neural stem and progenitor cells [34]. Two approaches exist to tackle the issue of
low cell survival due to oxidative stress. One is to modulate the oxidative environment with
antioxidants or free radical scavengers. Biomaterials, which can be functionalized [35] or loaded
[36] with these protective agents, generally adopt this approach. The other is to induce the
transplanted cells to become more resistant by preconditioning them with sub-cytotoxic levels of
oxidative stress [22] or by overexpressing pro-survival signals [37]. Our study using Gtn-HPA is
the first to demonstrate the role of a biomaterial and its associated crosslinking chemistry in
preconditioning aNPCs and increasing their resistance against oxidative stress to attain better cell
survival. Gtn-HPA and other biomaterials utilizing similar enzyme-mediated oxidative coupling
may therefore present a one-step approach for preconditioning and transplantation of NSCs as
well as a variety of other cell types (e.g. MSCs [38], cardiomyocytes [39] and retinal pigment
epithelium cells [40]) that could be preconditioned to achieve increased oxidative stress
resistance. Importantly, our findings emphasize the need to look beyond the conventional role of
crosslinking and examine additional biological benefits that crosslinking may plausibly provide.
We also found Gtn-HPA to exemplify the strategy of employing cytocompatible
crosslinking chemistries on appropriate extracellular matrix (ECM) derivatives such as gelatin to
yield an injectable matrix which can crosslink covalently in situ and mimic natural ECM in its
interactions with aNPCs. While cell adhesive molecules have to be added to certain biomaterials
to promote adhesion of neural cells and prevent anoikis [9, 41], Gtn-HPA is inherently abundant
in arginine-glycine-aspartic acid (RGD) sequences and in our study, strongly promoted aNPC
adhesion even when diluted to lower densities with minimally adhesive materials. Therefore, it is
likely that Gtn-HPA can readily prevent anoikis when used alone or in mixtures with other
biomaterials to support aNPCs in CNS injury sites. Likewise, Gtn-HPA inherently carries
cleavage sites for matrix metalloproteinases (MMPs) and is amenable to remodeling by cells. In
our observations, aNPCs, which are known to express MMPs [42], were able to proliferate as
well as to give rise to neuronal cells that migrated into the surrounding Gtn-HPA matrix in
chains. Chain migration is a vital mechanism by which neural progenitors from neurogenic niches
such as the subventricular zone may deviate from stereotypical migratory routes (e.g. rostral
migratory stream towards the olfactory bulb) and instead, home towards brain injury sites [3, 43,
44]. By being permissive for chain migration of aNPCs and their differentiated progeny, Gtn-
67
HPA hydrogels may be well poised as injectable matrices for use in brain cavitary defects to
allow the endogenous migratory mechanisms to proceed in a similar manner as the brain ECM.
In addition to providing a permissive environment for proliferation and migration, GtnHPA hydrogels offers control over these cellular behaviors via its tunable crosslinking. Our study
showed that while proliferation persisted in all Gtn-HPA hydrogels, those with higher
crosslinking degrees were able to decrease the extent of proliferation. Similar effects have
previously been reported [20] and attributed to increased mechanical stiffness. Given the
degradability of Gtn-HPA, the concomitant increases in the resistance to degradation could have
also played a contributing role. The tunable crosslinking of Gtn-HPA hydrogels also influenced
the migration of aNPCs. In hydrogels of the lowest crosslinking degree (GH-0.85), it was
possible that aNPCs migrated and dispersed efficiently, therefore failing to form neurospheres
that our study used as reference points to track aNPC movement. In the other Gtn-HPA hydrogels
examined in the study, neurosphere formation was observed and increasing levels of crosslinking
were seen to impede and even completely arrest chain migration from the neurospheres. Again,
crosslinking of matrices affects both mechanical stiffness and degradation rate, which can in turn
regulate cell migration [45] and thus explain the influence of tunable Gtn-HPA hydrogels on the
migration of aNPCs.
Gtn-HPA hydrogels
influenced
aNPCs
in several
ways
over their course
of
differentiation. Compared to P/L, Gtn-HPA hydrogels enhanced gene expression for TuJ1 and/or
GFAP for all the differentiation conditions examined, with the enhancements being specific to the
type of differentiation condition. As seen in immunofluorescence staining, Gtn-HPA hydrogels
also induced encapsulated aNPCs to produce a higher proportion of neurons and astrocytes in
mixed differentiation condition, although such increases were not observed for neuronal and
astrocytic differentiation conditions. It appears plausible that in the presence of strong, specific
stimuli (e.g., RA/For in neuronal differentiation condition or LIF/BMP-4 in astrocytic
differentiation condition), the majority of aNPCs was already directed towards the relevant
lineages and was expressing relatively high levels of the relevant genes. Gtn-HPA hydrogels
mainly acted to further increase the gene expression for these committed cells, thus explaining
why enhanced gene expression did not translate into a higher proportion of neurons or astrocytes.
On the other hand, in the presence of mild, mixed stimuli, aNPCs were driven less strongly
68
towards a particular lineage commitment. In this context, Gtn-HPA-induced enhancement in gene
expression was able to serve as an additional factor to promote lineage commitment in aNPCs
and therefore, resulted in higher proportions of neurons and astrocytes.
Regarding the higher efficiencies of differentiation for aNPCs within Gtn-HPA hydrogels
in mixed differentiation condition, we had noted that the increase in the resulting proportion of
neurons was greater than that for astrocytes. This was consistently observed across all the
examined crosslinking degrees, which had been selected in this study to tune Gtn-HPA hydrogels
to be soft matrices (449 - 1717 Pa) approximating adult brain tissue. At the same time, we also
observed that when compared to P/L-coated polystyrene
(-109 Pa [46]), Gtn-HPA hydrogels
exerted a selective effect where aNPCs driven towards neuronal lineage exhibited a higher
survival than those towards astrocytic lineage. These observations on the bias that Gtn-HPA
hydrogels had for neuronal cells were in line with similar studies where soft substrates promoted
neuronal differentiation [20, 47, 48] or induced poor survival among aNPCs over astrocytic
differentiation [49]. Further investigation will be necessary to verify if the bias in Gtn-HPA
hydrogels is driven by substrate modulus. Finally, it should be noted that these effects of GtnHPA hydrogels on aNPCs over their differentiation might be extended into animal models given
the injectable nature of Gtn-HPA. It is of great interest to examine these effects in vivo and more
importantly, to exploit them in actively directing NSPCs to mediate brain tissue repair and
regeneration.
In conclusion, brain lesions may benefit from implanted matrices to support endogenous
or transplanted NSPCs. Towards the aim of using Gtn-HPA hydrogels for such purposes, this
work demonstrates that Gtn-HPA, which is an injectable hydrogel that undergoes covalent
crosslinking via an enzyme-mediated process, features high cytocompatibility and adhesive
support for aNPCs. We also show that beyond the role as a supporting matrix, Gtn-HPA presents
an approach to augment the oxidative stress resistance of encapsulated aNPCs for enhanced cell
survival. Gtn-HPA hydrogels are capable of modulating proliferation and migration of aNPCs via
their tunable crosslinking. Furthermore, Gtn-HPA hydrogels can exert bias for neuronal cells by
promoting neuronal differentiation and maintaining a higher level of cell survival over neuronal
differentiation.
69
2.5
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neural electrodes. J Neurosci Methods. 2005;148:1-18.
[32] Soh N. Recent advances in fluorescent probes for the detection of reactive oxygen species.
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[33] Chan PH. Reactive oxygen radicals in signaling and damage in the ischemic brain. J Cereb
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[37] Lee HJ, Kim MK, Kim HJ, Kim SU. Human neural stem cells genetically modified to
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IKVAV peptides for tissue repair and axonal regeneration in an injured rat brain. Biomed Mater.
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74
Chapter 3
Development of Gtn-HPA/SDF-la-PCN
Matrices for Recruitment of Neural
Progenitor Cells
3.1
Introduction
With the discovery of endogenous stem cells and the factors influencing their behaviors,
tissue regeneration using endogenous stem cells has become a viable approach for tissue
engineering. By recruiting endogenous stem cells into compromised tissues and directing them to
replace lost cells, provide paracrine signals or modulate the microenvironment, this approach is
an attractive alternative to conventional cell transplantation therapies in terms of the potential to
avoid exogenous cells and their associated costs/complications. Depending on the persistence of
cell generation and homing, endogenous stem cells can also be continuously, albeit gradually,
introduced into the compromised tissues. In cases where the blood supply is impaired, this may
orchestrate regenerative processes that are more congruent with the physiological provisions than
the sudden introduction of cells in large numbers by transplantation [1]. So far, such an approach
has been successfully attempted in tissues such as cartilage [2] and muscle [3].
For the adult brain that has long seemed to lack regenerative capacities, evidence has now
surfaced to underscore the possibility of tissue regeneration using endogenous stem cells. After
brain injury, endogenous neural progenitor cells (NPCs) from stem cell niches in the lateral
ventricles [4] and hippocampus [5] as well as from local resident pools found throughout the
brain parenchyma [6] become activated. Injured neural tissues release chemotactic cues for
recruiting endogenous NPCs [7] and retain an adequately intact stroma where the recruited NPCs
reside and generate both glia [8] and region-specific neurons [9]. The newly differentiated
neurons may further integrate into existing brain circuitry [10]. Notable advances have been made
towards exploiting the endogenous NPCs for repairing/regenerating injured neural tissues. These
75
include the use of caspase inhibitors to improve the survival of newly formed neurons [11] and
mitogens to amplify the number of progenitors [12].
However, making similar advances in cavitary brain lesions has remained highly
challenging. Formed after the death and clearance of neural tissues, cavitary brain lesions are
characteristically depleted of most cells and stroma and constitute a predominantly structureless
environment [13]. As a result, cavitary brain lesions, unlike injured neural tissue, lack an inherent
ability to recruit endogenous NPCs, even though the NPCs typically accumulate within close
proximity around the cavity [14]. It is therefore imperative to establish chemotactic cues within
the cavity to recruit the NPCs as well as fill the cavity with a structural support that is permissive
to the migration of recruited NPCs. This will essentially require multi-faceted scaffolding
materials that can concurrently fulfill both aspects and recruit NPCs into themselves.
In this study, we develop matrices from gelatin-hydroxylphenylpropionic acid (Gtn-HPA)
hydrogels
and
stromal
cell-derived
factor-1
alpha
(SDF-1a)
polyelectrolyte
complex
nanoparticles (PCNs). Gtn-HPA hydrogels are injectable scaffolds that cater for tunable gelation
rate and crosslinking degree [15]. The tunable gelation rate facilitates minimally invasive
injection of Gtn-HPA solutions into target locations within the brain prior to their covalent
crosslinking in situ to form hydrogels. The tunable crosslinking degree, as shown in our recent
study [16], tailors Gtn-HPA hydrogels to match the storage modulus of adult brain tissues and be
permissive matrices to the proliferation, migration and differentiation of adult rat hippocampal
NPCs (aNPCs). On the other hand, SDF-la is known to mediate homing of endogenous NPCs to
sites of brain injury [7]. The use of PCNs, which can entrap cationic proteins facilely without
affecting their bioactivity and serve as biocompatible delivery vehicles for the proteins [17],
further offers control over the release of SDF-la. We hypothesize that by simultaneously
providing NPC-compatible structural support and sustained release of SDF-la, Gtn-HPA/SDF1 a-PCN matrices can recruit NPCs into themselves. These matrices may ultimately be valuable in
enabling brain cavitary lesions to mobilize endogenous NPCs. We investigated this in vitro by
formulating Gtn-HPA/SDF-la-PCN matrices as cylindrical cores that interfaced with annular
tissue simulant containing aNPCs and their neuronal progeny. We then examined the migration of
cells towards and into the matrices as well as the underlying mechanisms.
76
3.2
Materials and Methods
3.2.1
Preparation of SDF-la PCNs
Dextran sulfate (DS; Mw 500kDa; Fisher Scientific, Pittsburgh, PA) was dissolved in
deionized water. Chitosan (CS; Mw lOkDa; Polysciences, Warrington, PA) was dissolved in
0.175% acetic acid and the solution was adjusted to pH 5.5 using IM NaOH. 5% Trehalose and
15% Mannitol were prepared using deionized water. All solutions were sterilized by filtration
prior to use. Lyophilized recombinant rat SDF-la (Peprotech, Rocky Hill, NJ) was reconstituted
in 5% Trehalose and stored at -20C until use. Preparation of SDF-la PCNs was performed on a
magnetic stirrer placed within the sterile environment of a tissue culture hood. Mixing was done
in a sterile cryogenic vial using a micro flea stir bar. To prepare SDF-la PCNs with a
DS:CS:SDF-la ratio of 1:0.33:0.2, 25pl 0.5mg/ml recombinant rat SDF-la was mixed
sequentially with 25pl 2.5mg/ml DS and 20.8pl lmg/ml CS for 30 min each. Finally, 35.4pl 15%
mannitol (Sigma Aldrich) was added and mixed for 10 min. The PCNs were centrifuged at
15,000g for 20min at 4C. The PCNs were washed and centrifuged twice in 5% mannitol prior to
lyophilization. To vary DS:CS or DS:SDF-la ratios, the concentration of SDF-la or the volume
of CS was adjusted accordingly. Blank PCNs were similarly prepared without SDF- la.
3.2.2
Characterization of SDF-1a PCNs
Mean particle size and polydispersity index (PDI) were determined by dynamic light
scattering using a 90Plus/ZetaPALS particle analyzer (Brookhaven Instruments, Holtsville, NY).
The amount of encapsulated SDF-la and DS was determined by analyzing the supernatant from
each centrifugation step using bicinchoninic acid (BCA) protein assay (Fisher Scientific) and
toluidine blue colorimetric assay (EMD Millipore, Billerica, MA) respectively. Toluidine blue
colorimetric assay was performed by mixing 40pl of the collected supernatants with lml 35pg/ml
toluidine blue (in deionized water) and measuring its absorbance at 595nm. Mass of PCNs was
estimated from the mass of encapsulated SDF-la and DS together with the added mass of CS
(assumed to be fully reacted given that DS was in excess relative to CS). Encapsulation efficiency
and loading efficiencies were calculated using Equation 3.1 and 3.2.
77
Encapsulation efficiency (%) = Mass of SDF - la encapsulated X 100
Total mass of SDF - la added
Equation 3.1
Loading efficiency (%) = Mass of SDF - la encapsulated X 100
Mass of PCNs
Equation 3.2
To measure release kinetics, approximately 50pg of lyophilized SDF- 1 a PCNs was suspended in
400 l of a release buffer that comprised of 50% PBS and 0.02% sodium azide. The suspension
was transferred to a Protein Lobind tube (Eppendorf, Hauppuage, NY) and incubated at 37"C
with shaking at 100 rpm. At each time point, the suspension was centrifuged at 15,000g for
20min at 4"C. Following this, 120pl of supernatant was collected and replaced and the suspension
was returned to incubation at at 370C. All collected supernatant was stored in -20 "C and analyzed
together using BCA protein quantification assay.
3.2.3
Preparation of Gtn-HPA conjugate
Gtn-HPA conjugate was synthesized from gelatin (Wako Pure Chemical Industries,
Japan) and HPA (Sigma Aldrich) as previously described in Section 2.2.1.
3.2.4
Neural Progenitor Cell Culture
NPCs were cultured as previously described in Section 2.2.4.
3.2.5
Characterization of Gtn-HPA/ PCN Matrices
For all Gtn-HPA hydrogels in this study, 2% Gtn-HPA solution was crosslinked using
0. 1U/ml horseradish peroxidase (HRP; Wako Chemical USA, Richmond, VA, USA) and targeted
concentrations of H2 0 2 (Sigma Aldrich). To incorporate PCNs within Gtn-HPA hydrogels, PCNs
were mixed into Gtn-HPA solutions prior to gelation. To investigate degradation rate, 50pl GtnHPA solution incorporating PCNs (0 - lmg/ml) was crosslinked with HRP and 1mM H 2 0 2 in
78
cylindrical molds and incubated with 1OU/ml type IV collagenase (Life Technologies) at 37'C.
Samples collected every hr were measured for degradation products from Gtn-HPA/PCN
matrices using the BCA assay. The data for mass loss was fitted into an inverse exponential
model to determine degradation rate constants as previously described [16]. To investigate
-
cytocompatibility of PCNs, I00gl Gtn-HPA solution containing NPCs (4x10 5/ml) and PCNs (0
Img/ml) was crosslinked with HRP and
1mM
H 2 02 in 96-well tissue culture plates. After
1 or 3
days, metabolic viability was assessed using the CellTiter 96@ AQueous One Solution Cell
Proliferation Assay (Promega, Madison, WI).
3.2.6
Three-Dimensional Migration Assay
Assembly of Annular + Core (A+C) Construct
A three-dimensional construct consisting of a NPC-laden annulus and an acellular core
was assembled to evaluate the migration of NPCs toward and into the core. To create the core,
236pil Gtn-HPA solution was crosslinked with HRP and 0.95mM H 2 02 in a cylindrical mold and
incubated for lhr at 37'C. The Gtn-HPA core was transferred to a 24-well tissue culture plate
(Figure 3.1A & B). To form the annulus, 141p l of 0.8mg/ml type I collagen solution and 423pl of
0.8mg/ml type I collagen solution containing 10ng/ml FGF-2 and 1x106 NPCs/ml were
sequentially added around the core and incubated for 30min and 2hr respectively at 37'C. Iml
neurogenic medium i.e N2 medium containing 1pM retinoic acid and 5[tM forskolin (Sigma
Aldrich) was finally added, followed by an 80% change every other day.
B
A 1i
-
1
8.66mm
15.96mm
iii
I
* Gtn-HPA Q Collagen
13mm
m
pm aNPC
U Media
Figure 3.1: (A) Schematic illustrating the procedure to assemble A+C constructs. (B) Gross appearance of
A+C construct in a 24- well plate.
79
Characterization of cell accumulation at the interface
After 7 days, A+C constructs were fixed overnight with 4% paraformaldehyde at 40C.
The constructs were incubated with 20ng/ml DAPI for lhr at room temperature to stain for cell
nuclei and washed thoroughly with PBS prior to imaging using an epifluorescence microscope
(Olympus BX-60). Micrographs were computed into intensity plots using ImageJ software. For
each sample, four regions in the annulus, spanning 1mm along the interface and 0.5mm from the
interface, were randomly selected from the micrographs. DAPI fluorescence in the selected
regions was integrated using ImageJ software and averaged to quantify cell accumulation at the
interface.
Characterization of cell migration across the interface
At day 4 and 7, ten fields of view along the annulus/core interface were imaged using a
Phase Contrast Microscope (Olympus IX5 1). For each sample, the total number of migrated cells
(i.e cells that traversed the interface into the core and/or their daughter cells after proliferation)
was counted and normalized by the length of interface imaged. To determine migration distance
into the core, the perpendicular distance of each cell body from the interface was measured.
Study on effects of SDF-la PCNs
Gtn-HPA core containing SDF-la PCNs that equated to 5pg/ml SDF-la or -25pg/ml
PCNs were compared with control Gtn-HPA cores that were blank or contained soluble SDF-la
or PCNs at matching concentrations. To ascertain the specific involvement of SDF- 1 a in cell
accumulation and migration, the medium bath for the A+C constructs was supplemented with
25pg/ml AMD3100 (Sigma Aldrich). Dose responsiveness was examined with a range of SDFla-PCN doses that equated to 0 - 20pg/ml SDF-la or 0 - 100g/ml PCNs and with matching
doses of soluble SDF-la or PCNs.
To distinguish chemotactic recruitment due to SDF-la
concentration gradients from chemokinetic effects, medium bath for the A+C constructs was
supplemented with 1.31 g/ml soluble SDF-la.
Study on secondary effects of PCNs
DS, CS, low molecular weight DS (DSL; Mw 9 - 20 kDa; Sigma Aldrich) and heparin
(Mw 9 - 2 1kDa; Sigma Aldrich)] were either incorporated within the Gtn-HPA core or added to
the medium bath. In each experiment, the doses of DS, DSL and heparin were matched by mass
80
to the DS component within the PCN group in comparison and the dose of CS to the CS
component.
Study on crosslinking degree of Gtn-HPA/SDF-la-PCN matrix cores
The influence of crosslinking degree on migration of NPCs was examined in the A+C
constructs using Gtn-HPA/SDF-la-PCN matrix cores crosslinked with 0.85, 0.95, 1.05 or 1.20
2
.
mM H 20
Study on MMPs used for cell migration into Gtn-HPA/SDF-la-PCN matrix cores
For broad-spectrum MMP inhibition, medium bath was supplemented with lOM
GM6001 (EMD Millipore). To inhibit individual MMPs, specific MMP inhibitors with high
selectivity were used at a concentration five-fold of their known half maximal inhibitory
concentration. The specific MMP inhibitors (and their working concentrations) were: i) MMP-2
inhibitor (60nM; Santa Cruz Biotech, Santa Cruz, CA, USA); ii) MMP-3 inhibitor (29.5nM;
Sigma Aldrich); iii) MMP-8 inhibitor I (20nM; EMD Millipore); iv) MMP-9 inhibitor I (25nM;
EMD Millipore); and v) MMP-13 inhibitor (40nM; EMD Millipore). Matching volumes of
DMSO were added to culture medium for controls.
3.2.7
Three-Dimensional Proliferation Assay
A bilayer construct was assembled to determine how Gtn-HPA matrices of varying
compositions influenced the proliferation of NPCs in an overlying collagen matrix. To assemble
this construct, 100pl of Gtn-HPA solution was crosslinked with HRP and 0.95mM H2 0
2
in a 48-
0
well tissue culture plate and incubated for lhr at 37 C. 100pl of 0.8mg/ml type I collagen solution
containing lOng/ml FGF-2 and lx106 NPCs/ml was then added on top of the Gtn-HPA matrix
and incubated for 2hr at 37*C (Figure 3.2). 250gl of neurogenic medium was finally added,
followed by an 80% change every other day.
81
Gtn-HPA ( Collagen
.3.33mm
$1.33mm
i aNPC
W Media
9.77mm
Figure 3.2. Schematic of bilayer constructs
At day 0, 4 or 7, 200pl of the medium was replaced with 1000U/ml type I collagenase to
extract the encapsulated cells. The number of cells was determined using the Picogreen DNA
quantification assay (Invitrogen). To study the effects of SDF-la PCNs, Gtn-HPA core
containing SDF-Ia PCNs that equated to 5pg/ml SDF-la or -25pgg/ml PCNs were compared with
control Gtn-HPA cores that were blank or contained soluble SDF-la or PCNs at matching
concentrations. To study secondary effects of PCNs, DS, CS, low molecular weight DS (DSL;
Mw 9 - 20 kDa; Sigma Aldrich) and heparin (Mw 9 - 21kDa; Sigma Aldrich)] were either
incorporated within the Gtn-HPA cores or added to the medium bath.
3.2.8
Fluorescence Immunohistochemical Analysis
After
1 or 7 days of culture, A+C constructs were fixed overnight in 4%
paraformaldehyde at 40C. The constructs were further cryoprotected via overnight incubation
with 12.5% and 25% sucrose solutions. The constructs were then detached from the culture well,
flash-frozen in isobutane (-30'C) and embedded in Tissue Tek O.C.T compund (Sankura
Finetek). The embedded constructs were further cut into 50ptm cryosections and mounted on
Superfrost Plus Slides (Fisher Scientific). All staining procedures were performed at room
temperature unless specified otherwise. Sections were blocked and permeabilized with 5%
donkey serum/0.3% Triton X-100/PBS for 2hr and then incubated with primary antibodies
overnight at 4'C. After washing with 0.3% Triton X-100/PBS, sections were incubated with
secondary antibodies for 2hr and eventually with 1Ong/ml DAPI (Invitrogen) for 5 min to
counterstain cell nuclei. All antibodies were diluted in 5% donkey serum/PBS. The primary and
secondary antibodies used are listed in Table 3.1. Sections were subsequently imaged using
epifluorescence microscope (Olympus BX-60). Controls performed with relevant IgG (Dako) in
place of the primary antibodies resulted in no detectable staining. To quantify the percentage of
82
cells positive for each marker, three randomly selected fields of views from cryosections of four
independent hydrogel constructs were analyzed.
Dilution
Secondary Antibody
1:2500
Dylight 488 Donkey Anti-Mouse IgG
(Jackson Immunoresearch 715-485-151)
Mouse Monoclonal IgG2a
Anti-f-tubulin III
(TUJ1;
Covance MMS-435P)
Rabbit Polyclonal AntiGlial Fibrillary Acidic
Protein
(Dako Z0334)
Mouse monoclonal IgGi
Anti-CNPase
(Abcam ab6319)
1:2000
Dylight 488 Donkey Anti-Mouse IgG
(Jackson Immunoresearch 715-485-151)
Neurons
1:2000
Dylight 488 Donkey Anti-Rabbit IgG
(Jackson Immunoresearch 711-485-152)
Astrocytes
1:1000
Dylight 488 Donkey Anti-Mouse IgG
(Jackson Immunoresearch 715-485-151)
Oligodendrocytes
Rabbit polyclonal.
Anti-Ki67
(Abcam ab15580)
1:100
Dylight 488 Donkey Anti-Rabbit IgG
(Jackson Immunoresearch 711-485-152)
Proliferating cells
Primary Antibody
Mouse Monoclonal IgGi
Anti-Nestin
(Millipore MAB353)
Cells Identified
Neural
Progenitors
Table 3.1 List of Primary and Secondary Antibodies used in Fluorescence Immunohistochemical Analysis
3.2.9
Statistical analysis
A sample size of 3-8 was used in all experiments. Values are expressed as mean
standard error mean (SEM). One-Factor Analysis of Variance (ANOVA) followed by Fisher's
protected least square difference post-hoc test and Student's t test were performed accordingly.
Statistical significance was set atp < 0.05.
83
3.3
Results
3.3.1
Characterization of SDF-la PCNs
PCNs formulated with DS and CS displayed a mean particle size of 466
6nm (PDI:
0.03). To achieve sustained release of SDF-la, the chemokine was entrapped within
0.28
PCNs. Within the range investigated, both the entrapment and loading efficiencies were
minimally influenced by the proportion of CS relative to DS but increased significantly with the
proportion of SDF-la relative to DS (p < 0.001, Fisher's post-hoc test; Figure 3.3A & B).
Overall, high entrapment efficiency of 75 - 91% and loading efficiency of 7 - 31wt% were
achieved. SDF-la PCNs formulated with the DS:CS:SDF-la ratio of 1:0.33:0.2 released 44.7%
of their SDF- 1 a load in a sustained linear fashion over the 28-day evaluation period (Figure 3.3C)
and were used in all subsequent studies.
A
B
*L4-1
*-5o
DDS:SDF-1a
080
1:0.1
60 -
0 DS:SDF-la
40 -20
r:
43
20 -04
DDS:SDF-1a
30 -
1:0.2
Z
DS:SDF-la
1:.4
t
0 DS:SDF-la
1:0.2
0
C10
0
DS:SDF-la
1:0.4
ll0
0
1:0.33
1:0.5
DS:CS Ratio
1:0.33
1:0.5
DS:CS Ratio
C
Figure 3.3: (A) Entrapment efficiency and (B)
loading efficiency of SDF-la PCNs formulated
50 -
using different DS:CS and DS:SDF-la ratios
(n=3). (C) Cumulative SDF-la release from
SDF-la PCNs formulated with the DS:CS:SDF-
40 U30
3
>1:0.1
-
100
-
75 w
E cc 20V 20
a6 10
1 -P
0
|
la ratio of 1:0.33:0.2 (n=3). PCNs were
incorporated within Gtn-HPA hydrogels at
'
0
5
'
concentrations
ranging from 0 ***p < 0.001, Fisher's post-hoc test.
10 15 20 25 30
Time (Days)
84
lmg/ml.
3.3.2
Characterization of Gtn-HPA/PCN matrices
To determine if PCNs posed any interference with hydrogel crosslinking, the degradation
profiles of Gtn-HPA/PCN matrices incorporating PCNs at mass percentage ranging from 0 - 5%
were examined. Within the entire range, incorporated PCNs did not alter the hydrogels to induce
any significant changes in the degradation rate of the hydrogels (Figure 3.4A & B). Incorporated
PCNs also posed minimal cytotoxicity to NPCs encapsulated within the same hydrogels, which
remained 91% and 97% viable on day 1 and 3 respectively (Figure 3.4C).
A
B
120
Hna
nn
-
100
a
100
.U)
-C
80
-
-
4-I
tn
E
Cu
60
40
Mass %of PCN
00
AO.5
x 1.25
20
I-
+2.5
0
1
I
I
I
1
2
3
4
5
60
40
0U 20
. 5
0
.- I
-
E ta
E 0
80
0
6
Time (hr)
0.5 1.25 2.5
5
Mass % of PCN in Gtn-HPA/PCN matrix
C
CL
'4-
0
(U
Figure 3.4: (A) Cumulative mass loss of
120
100
**80
u
<60- 60
ODay 1
ODay3
7T--H
Gtn-HPA/PCN hydrogels degraded with
1OU/ml type IV collagenase and (B)
their
associated
constants
derived
degradation
from
an
rate
inverse
exponential model (n=5). (C) Viability
20
,.I',.''I'1
0
I'
0.5
1.25
2.5
5
Mass % of PC N in Gtn-HPAIPCN ma trix
85
of NPCs
encapsulated
HPA/PCN
matrices relative to those
within
Gtn-
within blank Gtn-HPA matrices (n=6).
3.3.3 Neural Progenitor Cell Recruitment: Accumulation around Gtn-HPA/SDFla-PCN matrices
After 7 days in the A+C constructs, cells in the annulus accumulated at the interface with
the core. Compared to blank Gtn-HPA cores, Gtn-HPA/SDF-la-PCN cores significantly
increased cell accumulation in terms of the area of increased cellularity (Figure 3.5A) and the
number of cells as reflected by DAPI fluorescence integrated along the interface (p < 0.001,
Fisher's post-hoc test; Figure 3.5B). In contrast, Gtn-HPA/SDF-la cores did not influence cell
accumulation while Gtn-HPA/PCN cores only produced slight increases. The CXCR4 antagonist
AMD3100 blocked the increase between Gtn-HPA/PCN and Gtn-HPA/SDF-la-PCN cores but
did not affect the slight increase induced by Gtn-HPA/PCN cores.
A
Annulus
B
Core
Intensity
ISO
125
2
T
L.
Blank SDF-Th
AMD3100
-
-
PCN
-
PCN SDF-lc SDF-lcc
PCN
PCN
+
-
+
-
75
-
y
Figure 3.5: Cell accumulation along the interface
Intensity
Z
between annulus and core. After 7 days, cell
nuclei in the A+C constructs were stained with
DAPI. (A) Regions of the annulus directly
adjacent to the interface were imaged
and
converted into intensity plots to visualize
I.
Wy
ntensity
distribution and intensity of DAPI fluorescence.
Scale bars = 100sm. (B) DAPI intensities from
four randomly selected regions spanning 1mm
along the interface and 0.5mm from the interface
were integrated and averaged for each sample
(n=5). * p < 0.05 vs. blank and SDF-la,
*** p < 0.001 vs. others, Fisher'spost-hoc test.
86
3.3.4 Neural Progenitor Cell Recruitment: Migration into Gtn-HPA/SDF-la-PCN
matrices
Upon assembly of A+C constructs, cores were completely cell free (Figure 3.6). At day 7,
relatively few cells were found with Gtn-HPA, Gtn-HPA/soluble-SDF-la and Gtn-HPA/PCN
cores. In contrast, a significantly higher number of cells had migrated into the Gtn-HPA/SDF- 1 aPCN cores. Migration into Gtn-HPA/PCN hydrogels occurred in the form of either individual
bipolar cells or chains (Figure 3.7). In the presence of AMD3 100, the number of migrated cells
was not affected in Gtn-HPA/PCN cores but was substantially decreased that for Gtn-HPA/SDFla-PCN cores.
Day 7
Collagen
4 Gtn-HPA
Figure 3.6: All cells were positioned in the annulus at day 0. Varying numbers of cells migrated into the
hydrogel core by day 7. Scale bars = 100pm.
A -B_
Figure 3.7(A) Cells migrated individually through the Gtn-HPA/SDF-la-PCN matrix core. Many cells
exhibited a distinct bipolar morphology that was reflective of leading and trailing processes. (B) Cells were
also observed to participate in chain migration where cells putatively migrated one following the other (20)
or along one another (7). Scale bars = 50ptm.
87
Quantification of the cells that migrated into the cores revealed that only -1
and -8
migrated cells per cm interface were observed within blank Gtn-HPA cores on days 4 and 7
respectively (Figure 3.8). The number of migrated cells in Gtn-HPA/SDF-la cores reached -3
and -14 cells per cm interface on days 4 and 7 respectively but was not significantly higher than
that in blank Gtn-HPA cores. Despite the absence of SDF-la, Gtn-HPA/PCN cores significantly
increased the number of migrated cells by -40-fold and -16-fold over the blank Gtn-HPA cores
on days 4 and 7 respectively (p < 0.05, Fisher's post-hoc test). Gtn-HPA/SDF-la-PCN cores
achieved the highest number of migrated cells which amounted to a -81-fold and -45-fold
increase over the blank Gtn-HPA cores on days 4 and 7 respectively (p < 0.00 1, Fisher's post-hoc
test) and surpassed even the combined number from both Gtn-HPA/SDF-la and Gtn-HPA/PCN
cores (p < 0.01, Student's t test). Similar to its effects on cell accumulation, AMD3100
obliterated the increase between Gtn-HPA/PCN and Gtn-HPA/SDF-la-PCN cores but failed to
completely reduce the number of migrated cells to the level seen with blank Gtn-HPA cores.
o
40 Day 4 O Day 7
400
(U
00
SE 21w'
00
AME3200
%tU.
-
-
-
Blank SDF-lcc PCN
+0
PN
C
PCN SDF-lcx SDF-lcx
Figure 3.8. Number of migrated cells in the core by day 4 and 7, summed over ten fields of views and
normalized to the length of interface imaged (n=5). 0 p < 0.05 vs. blank and SDF- 1a at the same time
point,
* p < 0.001 vs.
others at the same time point
Measurement of the distance each cell migrated into the hydrogel core on day 7 further
revealed that the migrated cells were distributed furthest into Gtn-HPA/SDF-l1a-PCN cores
(Figure 3.9). Cell migration changed significantly with the SDF-la PCN dose on both days 4 and
88
7 (p < 0.001, ANOVA; Figure 3.10A & B). Specifically, cell migration increased significantly
with the SDF-la PCN dose in the range of 1.25 - 5gg/ml SDF-la but remained relatively
unchanged across the higher dose range of 5 - 20gg/ml SDF- 1a.
'A
50
45
40
35
30
25
0
20
0
15
10
5
0
* SDF-la
U PCN
N=3S
N=4S3
N=944
47.8
27.4
213.4
141.3
128.0
399.4
176.7
159.5
490.9
Mean
Median
Max
I-
t4
V4
4 N
i
N N N
E SDF-1a PCN
MneMn
Mn Mt
n*n
Migration distance (jam)
Figure 3.9: Percentage of migrated cells at various distances from the interface on day 7.
A
100
u 80
60
B
0 SDF-lca
U'
n" PCN
M
SDF-1a PCN
500
u 400
300
*0
a' E 40
-c
U E 200
a'
a 20
CL100
(U
0
SDF-la
-
PCN
-E
SDF-la PCN
**#
0
0
1.25 2.5
5
10
20
SDF-1a concentration (pg/ml)
0
0 1.25 2.5 5
10
20
SDF-1a concentration (pg/ml)
Figure 3.10: Number of migrated cells in the core on (A) day 4 and (B) 7 with varying doses of soluble
SDF-la, PCNs and SDF-la PCNs (n=4). # p < 0.05 vs. SDF-la and PCN at the same dose, *** p < 0.00 1
vs. the immediate lower dose of SDF-la PCNs, Fisher's post-hoc test.
89
3.3.5 Chemotactic nature of Neural Progenitor Cell Recruitment by GtnHPA/SDF-la-PCN matrices
To examine whether Gtn-HPA/SDF-la-PCN matrices had increased cell accumulation
and migration by enhancing proliferation, we measured changes in cell number in the bilayer
constructs (Figure 3.11). Cell number was found to increase by days 4 and 7 for all groups. At
both time points, significantly higher increases in cell number were observed in the GtnHPA/PCN and Gtn-HPA/SDF-la-PCN matrices as compared to the PCN-free matrices (p < 0.05,
Fisher's post-hoc test). However, no significant differences were observed between GtnHPA/PCN and Gtn-HPA/SDF-la-PCN matrices, as well as between blank Gtn-HPA and GtnHPA/SDF- 1 a matrices.
CJDay 4 C Day 7
(U
**
S'4
0T
Blank SDF-1a PCN SDF-1a
PCN
Figure 3.11: Fold increase in cell number by day 4 and 7 (n=6). * p < 0.05, *** p < 0.001, Fisher'spost-hoc
test.
To further differentiate between chemokinetic and chemotactic effects, we introduced
soluble SDF-la into the medium bath for the A+C constructs to disrupt chemotactic
concentration gradients of SDF-la while preserving any chemokinetic effect that might be
present. In the SDF-la bath, the number of migrated cells in Gtn-HPA/SDF-la-PCN cores
decreased substantially on both days 4 and 7 (p < 0.01, Student's t test; Figure 3.12A) to levels
90
typically seen with Gtn-HPA/PCN corees. A similar trend was also observed with the integrated
DAPI fluorescence that was used to quantify cell accumulation at the interface (p < 0.05,
Student's t test; Figure 3.12B).
A
B
3 300 -
S200
0
60
11EJSDF-1a
PCN
*
125
-
%M0 400 -
150
**
0 SDF-1a PCN
+ SDF-1a
4100
7
Bath
s0
S
-0
10
CL 10
25
0-
SDF-lct PCN SDF-1a PCN
SDF-1a Bath
+
0
Day 4
Day 7
Figure 3.12: Soluble SDF-la was added into the medium bath to disrupt any SDF-la concentration
gradient arising from SDF-la PCNs. (A) The number of migrated cells in the core (n=4) and (B) cell
accumulation as reflected by integrated DAPI intensity along the interface (n=4) decreased significantly
with the SDF-la bath, evidencing the chemotactic role of SDF-la PCNs. * p < 0.05, ** p < 0.01, Fisher's
post-hoc test.
3.3.6
Secondary Effects of Gtn-HPA/ SDF-la-PCN matrices
Even without SDF- I a, PCNs mediated appreciable increases in cell migration (Figure
3.8) and proliferation (Figure 3.11). To understand this, we first used the bilayer hydrogel
constructs to examine if the proliferative effects could be traced to the polyelectrolytes used to
prepare PCNs. DS mediated similar increases in proliferation as PCNs while CS did not induce
any significant effect relative to blank controls (Figure 3.13A). To determine if DS had achieved
this effect through its heparin-like properties, we also evaluated heparin, which reproduced an
increase in proliferation similar to DS and PCNs. To test the possibility that the FGF-2
accompanying the aNPCs during their incorporation into the tissue simulant was implicated in
these secondary effects of PCNs, we omitted FGF-2 during the assembly of bilayer and A+C
hydrogel constructs. In the absence of FGF-2, the proliferative effects of PCNs, DS and heparin
disappeared (Figure 3.13B) and the number of migrated cells in Gtn-HPA/PCN core reduced
91
drastically by -81% on day 7 (Figure 3.14).
Interestingly, the diminished PCN-mediated
migration was expected to decrease the number of migrated cells in Gtn-HPA/SDF-la-PCN
A
aEs
U0
=2
U. 0
M Day 4
Blank
B
S6
5
S4
-E
W&.
C 0
r
I
PCN
DS
rfl rflP
0Day7
CS
Heparin
U Day 4
O Day 7
mFlrflrHrI
Blank
PCN
DS
CS
Heparin
Figure 3.13: (A) In the presence of FGF-2, PCNs as well as DS and heparin increased
proliferation when compared to blank control and CS (n=4). (B) Proliferation was minimal for all
groups when FGF-2 was excluded (n=4). ** p < 0.01, *** p < 0.001 vs. control, Fisher's post-hoc
test.
92
2"
500
400
ra
W
U+FGF
0-FGF
300
E E zo00
20
1100
PCN
SDF-1a
PCN
Figure 3.14: Number of migrated cells under the influence of blank and SDF-la PCNs was in the
absence of FGF-2 (n=4). **p < 0.01, ***p < 0.001 vs. -FGF, Student's ttest
hydrogels by -32% but we observed an overall decrease by -77% instead. (Notwithstanding this,
the number of cells recruited by Gtn-HPA/SDF- 1 a-PCN hydrogels remained 6.6-fold higher than
that for Gtn-HPA/SDF-la hydrogels.)
To verify that the FGF-dependent secondary effects of PCNs on cell migration could be
similarly traced to the relevant polyelectrolytes, we incorporated individual polyelectrolytes
within the Gtn-HPA cores of A+C constructs assembled with FGF-2 (Figure 3.15). Compared to
controls, both CS and DS failed to produce any effect on the number of migrated cells even
though DS had previously been observed to increase proliferation (Figure 3.13A). On the other
hand, heparin and DSL, which has a low molecular weight akin to heparin, mediated increased
number of migrated cells in the hydrogel core. Given the instance of DS where enhanced
proliferation did not mediate an increased level of cell migration, we focused on the role of
chemokinesis and/or chemotaxis by examining additional A+C hydrogel constructs that contained
the individual polyelectrolytes in their medium bath. For both DSL and heparin, increases in the
number of migrated cells were similar regardless of the location to which the polyelectrolytes
were added. On average, these increases with DSL and heparin reached -88% and -60% of that
with PCNs by day 4 and 7 respectively.
93
-
200
ODay4
kA
ODay7
U150
(U
E EU
6
z
***
100-
~.50
mmr
0
Blank
PCN
F
r-n*
***
IT-
I*
t
DS
DS
DSL
DSL
CS
CS
Bath
Core
Bath
Core
Bath
Core
j
Heparin Heparin
Core
Bath
Figure 3.15: Number of migrated cells in the presence of FGF-2 when DS, CS, DSL and heparin
were either loaded into the core or added to the medium bath (n=4). ***p <0.001 vs. control,
Fisher's post-hoc test
3.3.7
Effect of crosslinking degree on migration
Cell migration into Gtn-HPA/SDF-la-PCN cores was significantly affected by their
crosslinking degree (p < 0.001, ANOVA; Fig. 3.16). Relative to the crosslinking degree
examined thus far using 0.95mM H 2 0 2 , a decrease in crosslinking degree to 0.85mM H 0
2
2
resulted in a 34% and 65% increase in the number of migrated cells on day 4 and 7 respectively.
On the other hand, an increase in crosslinking degree to 1.05mM H 2 0 2 sharply decreased the
number of migrated cells by 80% and 75% on day 4 and 7 respectively. Very few migrated cells
2
.
were observed when the crosslinking degree was further increased to 1.20mM H 0
2
94
-
800
)
w W
T
0Day
4 O Day 7
-
600
Z 400
b
2000.=dI
0.85
0.95
1.05
1.20
H 20 2 crosslinking (mM)
Figure 3.16: The number of migrated cells in the core on day 4 and 7 in relation to the crosslinking degree
of Gtn-HPA hydrogels (n=5)
3.3.8 Profile of Matrix Metalloproteinases Used to Mediate Migration of Neural
Progenitor Cells into Gtn-HPA/ SDF-la-PCN matrices
We examined the effects of a broad-spectrum MMP inhibitor, GM6001, to establish the
role of MMPs in cell migration into Gtn-HPA/SDF-la-PCN matrices. Compared to controls, the
number of migrated cells in the presence of GM6001 was greatly diminished to 5.5% and 6.2%
on days 4 and 7 respectively (Figure 3.17). The material nature of Gtn-HPA hydrogels might
dictate particular types of MMPs to play more important roles than others during cell migration,
prompting further examination with specific MMP inhibitors. For the two inhibitors of
gelatinases, MMP-2i reduced cell migration by 41.8% while MMP-9i resulted in an even larger
reduction by 54.7% (Figure 3.18). Cell migration also decreased by 30.1% when MMP-3, a type
of stromelysins, was inhibited. Between the two inhibitors for collagenases, MMP8i decreased
cell migration significantly by 79.6% while MMP13i exhibited no significant effect.
95
500
U
a,U
(U
'4I."
E DMSO
400
a,
C
300
to
a,
E EU 200
***
O GM6001
4.'
1~
0 a,
0.
z
100
***
0
Day 4
Day 7
Figure 3.17: The number of migrated cells in the core on day 4 and 7 in relation to the presence of broadspectrum MMP inhibitor GM6001 (n=6). *** P<0.001 vs. control, Student's t test
120
100
***
-
0
80
0
0
OR/
60
40
20
n
F]
****
Control MMP2i MMP3i MMP8i MMP9i MMP13i
Figure 3.18: Level of cell migration at day 7, expressed as a percentage of control, when specific
inhibitors for MMP-2, 3, 8, 9 and 13 were applied (n=8). *** P<0.001 vs. control, Fisher's posthoc test.
96
3.3.9
Phenotypic Fate of Recruited Neural Progenitors
On day 1, the cell population in the annular tissue simulant consisted mostly of
proliferating NPCs, as seen from the fact that -91% and -88% of the cells expressed NPC marker
nestin and proliferation marker Ki67 (Figure 3.19 & 20). After 7 days in neurogenic medium, this
cell population evolved into a mixture of NPCs (-35%) and TUJ1 neurons (-45%) (Figure 3.21
& 22). The proportion of the Ki67+ cells also decreased to -34%. Within the Gtn-HPA/SDF- 1 aPCN matrix core on day 7, nestin+ and Ki67' cells constituted -82% and -73% of the recruited
&
cells which were relatively larger proportions compared to those in tissue simulant (Figure 3.23
24). Also, only -15% of the recruited cells expressed TUJI. Very few cells in either the tissue
simulant or the Gtn-HPA/SDF-la-PCN hydrogel core were GFAP+ or CNPase+.
97
(0
I
(U
0
Figure 3.19: Immunostaining for neural progenitor marker (Nestin), neural differentiation markers (TUJi,
GFAP and CNPase) and proliferation marker (Ki67) to identify the cells in the (A) annulus on day 1. Scale
bars = 50m.
c1 0 0
'
80
0 60
a.
ue40
S20
S0
1
1
, ,
limminimma
Nestin TUJ1
I
-
I
v
A-.---L-
GFAP CNPase Ki67
Figure 3.20: Quantification of Nestin+, TUJ1, GFAP+, CNPase+ and Ki67+ cells in the annulus on day 1.
98
Figure 3.21: Immunostaining for neural progenitor marker (Nestin), neural differentiation markers (TUJ 1,
GFAP and CNPase) and proliferation marker (Ki67) to identify the cells in the annulus on day 7. Scale bars
=50pm.
W1 0
0
. 80
0
60
a4.
H
.4
-
U
.I
.I
Nestin TUJ1
.
20
,,,,I
i,
GFAP CNPase K167
Figure 3.22: Quantification of Nestin+, TUJ1+, GFAP+, CNPase+ and Ki67+ cells in the annulus on day 7.
99
O
0
I-
Figure 3.23: Immunostaining for neural progenitor marker (Nestin), neural differentiation markers (TUJ 1,
GFAP and CNPase) and proliferation marker (Ki67) to identify the cells in the core on day 7. Scale bars =
50pm.
W1 0 0
z80
o 60
a.
40
S20
m
.ELLm
I
Nestin TUJ1
I
I
LL
i
GFAP CNPase Ki67
Figure 3.24: Quantification of Nestin+, TUJ1+, GFAP+, CNPase+ and Ki67+ cells in the core on day 7.
100
3.4
Discussion
A crucial step in the current work to develop NPC-recruiting biomaterial scaffolds had
been to integrate injectable Gtn-HPA hydrogels with the functionality of sustained SDF-la
release. To allow independent optimization of SDF-la release and structural support for NPCs,
we adopted the strategy of devising delivery vehicles that could provide the desired release
kinetics and serve as a modular addition to Gtn-HPA hydrogels. The PCNs formulated from
biodegradable polymers, DS and CS, were found to fulfill the aforementioned requirements.
When loaded with SDF-la, they released 44.7% of their load gradually over a 28-day period,
conceivably retaining the remaining load for release over a further prolonged period. The PCNs
could also be incorporated into Gtn-HPA hydrogels without affecting the hydrogel covalent
crosslinking and without being cytotoxic to aNPCs. Furthermore, SDF-la PCNs met the practical
requirements relating to their preparation and use. They offered high entrapment efficiencies (75
- 91%) that addressed the pragmatic need to minimize the loss of costly proteins. They also
offered high loading efficiencies (7 - 31 wt%) that were critical in avoiding excessively high
concentrations of delivery vehicles when introducing therapeutically relevant doses of SDF-la
into the small volumes associated with cavitary brain lesions.
The resulting Gtn-HPA/SDF-la-PCN matrices were evaluated in specially developed
standardized migration assays based on A+C constructs. Unlike conventional migration assays in
Boyden, Dunn and Zigmond chambers, the A+C constructs did not only quantitatively assess the
bioactivity and chemotactic potency of test substances, but also concurrently evaluated the
compatibility of test matrices with cell migration. Cells in the A+C constructs were also
maintained in three-dimensional culture, which mimic in vivo environments more closely than the
monolayer cell cultures typically used in conventional migration assays [18]. Moreover, the A+C
constructs were devised to distill the complexity of cavitary brain lesions into elements that were
pertinent to the recruitment of NPCs. They featured a core-annular geometry to represent the
hydrogel-filled cavity and the peri-lesion tissue. NPCs were encapsulated within the annular
tissue simulant to correspond to endogenous NPCs within peri-lesion tissues. A one-time dose of
FGF-2, which was added into the annular tissue simulant together with the NPCs to stabilize their
encapsulation, served to reflect the transiently increased FGF-2 expression in peri-lesion tissues
after injury [19]. Similar to implanting materials into the cavitary brain lesions where they contact
101
and interact with peri-lesion tissues, materials could be placed in the core where they interface
with the annular tissue simulant to influence or recruit the NPCs. Finally, neurogenic cues were
provided in the medium to allow NPCs to undergo neuronal differentiation, as would the
endogenous NPCs in vivo during their homing towards injured tissues [9]. The A+C constructs
therefore allowed us to systematically evaluate candidate materials in a rapid, yet physiologically
relevant manner, as well as perform characterization/mechanistic studies (e.g. those to distinguish
between chemotaxis and chemokinesis) that were otherwise difficult to implement in vivo.
In the A+C constructs, Gtn-HPA/SDF-la-PCN matrices produced significantly larger
enhancements in cell accumulation at the annulus/core interface and in the number of migrated
cells in the core when compared to the effects of Gtn-HPA/SDF-la matrices, Gtn-HPA/PCN
matrices or even the sum of both. Most recruited cells migrated into Gtn-HPA/SDF-la-PCN
matrices either as individual cells exhibiting leading and trailing processes or as chains where
cells were known to follow preceding cells [20] or use one another as guiding structures [7]. Both
forms were highly reminiscent of the migratory behavior of endogenous NPCs observed in the
injured brain [20]. The use of AMD3100 to antagonize SDF-la receptor CXCR4 demonstrated
that the enhancements by Gtn-HPA/SDF-la-PCN matrices were largely mediated through the
SDF- la/CXCR4 pathway. The effects of Gtn-HPA/SDF- la-PCN matrices also displayed a dosedependent behavior, which further confirmed the involvement of SDF-la. Apart from being a
well-known chemotactic cue, SDF- 1 a may also stimulate proliferation [7] and chemokinesis [21].
However, SDF-la did not increase proliferation of NPCs in our study, thus dismissing the
possibility that Gtn-HPA/SDF-la-PCN matrices drove more cells towards the interface and into
the core by causing overcrowding in the tissue simulant. SDF-la from Gtn-HPA/SDF-la-PCN
matrices also did not enhance cell accumulation and migration relative to Gtn-HPA/PCN matrices
when we disrupted chemotactic SDF-la concentration gradients and examined chemokinetic
effects in isolation. This excluded the possibility that Gtn-HPA/SDF-la-PCN matrices enhanced
cell accumulation and migration simply by increasing the motility of NPCs and expediting their
random dispersal from the tissue simulant. Therefore, Gtn-HPA/SDF-la-PCN matrices had
enhanced cell accumulation and migration relative to Gtn-HPA/PCN hydrogels via chemotactic
recruitment of cells towards the interface and into the core. The ability of Gtn-HPA/SDF-la-PCN
hydrogels to chemotactically recruit aNPCs clearly highlighted the instrumental role of SDF-la
102
PCNs in sustaining the release of SDF-l a sufficiently for concentration gradients to be
established.
Along with releasing
SDF-la,
Gtn-HPA/SDF-la-PCN matrices also provided a
compatible structural support for cell migration during chemotactic recruitment. This was
subsequently found to be contingent on utilizing the tunable crosslinking of Gtn-HPA matrices to
maintain crosslinking degrees in the range of around 0.85 - 0.95mM H 2 02 . Above this range, the
higher crosslinking degrees led to a drastic reduction or even a complete arrest in cell migration,
which was a likely consequence of cells being unable to remodel/degrade highly crosslinked
polymer networks. As expected from the abundance of MMP cleavage sites along gelatin
polymers, migration into the Gtn-HPA matrices was almost fully dependent on MMPs and was
severely impeded by broad-spectrum MMP inhibitor GM6001. It was of further interest to
identify the specific MMPs involved since endogenous NPCs express MMP-2, 3 and 9 to mediate
their migration through brain extracellular matrices (ECM) after brain injuries [22, 23] and will
reasonably interact better with structural support that can respond readily to the MMPs they
secrete. By using specific MMP inhibitors, we first noticed MMP-8 to be critically essential. This
might be attributed to MMP-8 being a potent type I collagenolytic enzyme and heavily utilized to
transverse the tissue simulant that was composed of type I collagen. The specific MMP inhibitors
additionally revealed the involvement of MMP-2, 3 and 9. Unlike MMP-8, neither MMP-2, 3 nor
9 played a particularly dominant role relative to one another, suggesting that all three of these
MMPs were used collaboratively. The MMP profile employed during cell migration through GtnHPA/SDF-la-PCN matrices was therefore highly similar to that used by endogenous NPCs to
migrate through the brain ECM after brain injuries. This strongly reflects the compliance of GtnHPA/SDF-la-PCN matrices towards endogenous NPCs when employed within cavitary brain
lesions.
Importantly, our findings demonstrated the promise of injectable Gtn-HPA/SDF-la-PCN
matrices for treating cavitary brain lesions. In the A+C hydrogel constructs, in addition to
providing chemotactic cues and structural support in the otherwise structureless core, GtnHPA/SDF-la-PCN matrices showcased the proof-of-concept for properly interfacing with
apposing cell-laden matrices in order to achieve the crucial continuity in structural support for
recruited cells to migrate across the interface. In addition, Gtn-HPA/SDF-1 c-PCN matrices did
103
not only recruit the highest number of cells but also induced recruited cells to migrate the furthest
into the core. With supportive levels of angiogenesis which could presumably be stimulated by
Gtn-HPA/SDF-l a-PCN matrices given the ability of SDF- I a to recruit endothelial cells [24] and
hematopoietic stem cells [25], the matrices might offer the potential to maximize the volume of
the cavitary brain lesions that would be populated by recruited neurovascular cells. Furthermore,
the ability of Gtn-HPA/SDF-la-PCN matrices to enhance cell accumulation around themselves
would be of value in vivo to similarly recruit and accumulate greater numbers of endogenous
neural cells in the peri-lesion tissues. This could benefit the peri-lesion tissues and provide more
cells for eventual recruitment into the cavitary lesion itself. Finally, Gtn-HPA/SDF-la-PCN
matrices preferentially recruited proliferative NPCs as opposed to neurons and offered the
possible advantage of directing NPCs, which hold greater regenerative potential due to their
proliferative capacity and multipotency, into the cavitary lesions while leaving the newly
differentiated neurons and their reparative roles in the peri-lesion tissues minimally perturbed.
Consistently throughout our investigations, PCNs themselves increased proliferation and
the number of migrated cells in the hydrogel core, the latter of which was unaffected by
AMD3 100 and occurred independently of the SDF- 1 a/CXCR4 pathway. These effects were
subsequently found to involve PCNs inheriting heparin-like properties from DS and utilizing
FGF-2 from the tissue simulant. Related polyelectrolytes, DSL and heparin, could also utilize
FGF-2 and enhance cell migration, albeit to a lower extent than PCNs, through stimulating
chemokinesis. It thus appeared that at least a large part of the PCN-mediated effect on cell
migration might be driven by similar increases in chemokinesis. We further observed that when
FGF-2 was omitted, cell migration into Gtn-HPA/SDF-la-PCN matrices decreased substantially
more than the amount expected from cell migration with Gtn-HPA/PCN matrices. This indicated
that when utilizing FGF-2, PCNs did not only constitute a stand-alone mechanism that increased
the chemokinetic migration of NPCs but also likely displayed synergistic effects with SDF-la by
stimulating the chemotactic responses
of NPCs towards SDF-la and/or promoting the
proliferation of recruited NPCs (Figure 3.25).
104
Co
(
0
*
0
Id
Chen okinesi
s
Proliferation
-
0
SDF-1a Chemotactic
Recruitment
PCN *
Delivered
SDF-1a
FGF-2 from
(:i# NPCs
annulus
Figure 3.25: Proposed model for SDF-la PCNs. The released SDF-la induced aNPCs to migrate into the
core primarily via chemotactic recruitment. The PCNs utilized FGF-2 from the annular tissue simulant
(dotted box) to enhance chemokinesis of aNPCs, potentiate chemotactic recruitment by SDF-la and
promote proliferation of migrated aNPCs, orchestrating an overall higher number of migrated aNPCs in the
core.
Given the known effects of FGF-2 in stimulating proliferation [26], chemokinesis [27] as
well as chemotaxis towards other chemokines [28], the process of utilizing FGF-2 in the above
findings likely entailed PCNs stabilizing FGF-2 and/or potentiating its activity. These secondary
effects of PCNs, although a serendipitous finding, were not entirely surprising because the
property of PCNs that enabled the entrapment of SDF-la had been the heparin-like property of
DS. Heparin, DS and similar glycosaminoglycans have been demonstrated to bind and stabilize
FGF-2 [29]. These glycosaminoglycans can further participate in the presentation of FGF-2 to its
receptors and modulate the activity of the FGF-2 on cell behaviors such as mitosis and motility
[30]. The secondary roles of PCNs were, however, remarkable in the following way. Unlike the
DS-bearing PCNs and low molecular weight counterparts such as DSL and heparin, DS itself
failed to stimulate chemokinetic migration of NPCs in the presence of FGF-2. Since the activities
of growth factors can vary with the structure of binding glycosaminoglycans [31], we conjectured
105
that the chitosan moieties within PCNs bound to DS and altered its structural properties to
approximate DSL and heparin. Nevertheless, it was evident that other than delivering SDF-la,
PCNs themselves conferred beneficial properties to Gtn-HPA/SDF-la-PCN matrices. By being
molecular aggregates that were less prone to diffuse, PCNs served as an anchored form of DS
within the matrices and stably imparted the matrices with heparin-like properties, allowing them
to emulate brain ECM in the aspects of sequestering and modulating the activities of heparinbinding growth factors such as FGF-2 [32]. More importantly, PCNs enabled Gtn-HPA/SDF-laPCN matrices to utilize FGF-2 found in situ in the tissue simulant to "self-enhance" their efficacy
to draw in NPCs and in this sense, bear similar potential as biomaterials that took advantage of
endogenous growth factors expressed in situ [33]. When employed within cavitary brain lesions,
Gtn-HPA/SDF-la-PCN matrices present an appealing possibility of harnessing the FGF-2 or
other heparin-binding growth factors expressed in the peri-lesion tissues to synergize the
recruitment of endogenous NPCs by the SDF-la they release. This will maximize the number of
endogenous NPCs recruited into the lesion and facilitate the ultimate goal of neural tissue
repair/regeneration.
106
3.5
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110
Chapter 4
Implantation and Optimization of GtnHPA/SDF-la-PCN matrices in a Rat Model
of Intracerebral Hemorrhagic Stroke
4.1
Introduction
Implantation of matrices can play several roles in brain lesions. They can act repository
materials to deliver drugs/cytokines to drive targeted cell/tissue responses [1], maintain the
structure of surrounding brain tissues [2], provide stroma for in growth of axons [3, 4] and neural
cells [5] and support transplanted cells in the injury environment [6, 7]. Nevertheless, implanting
matrices into brain lesions can often be a challenging endeavor. Unlike many other organs, the
brain is securely housed in the rigid skull where the close fit between the brain and the skull as
well as the connection of the brain to the spinal cord allows little room to manipulate the position
or orientation of the brain during surgical procedures. The brain is also usually accessible only
from its dorsal surface due to the presence of numerous anatomical structures around its ventral
space. At the same time, much of the brain, especially the cortical region near the dorsal surface,
is densely packed with axonal projections and cannot be temporarily removed or push aside lest
there be injury due to stretching or shearing of the axons. On top of these, brain lesions tend to be
irregularly shaped and can vary significantly from one injury to the next. When compounded
together as in the case of deep-seated lesions, these circumstances pose a huge challenge for
implanting matrices in brain lesions.
Numerous matrices have been developed for application in the injured brain. Several of
them have displayed promising benefits for the injured brain, but their applications have been
largely restricted to exposed cortical lesions [2, 5, 8] because the matrices are fabricated to a
predetermined shape and size and are difficult to be implanted into deep-seated lesions for the
reasons above. This has spurred the interest in identifying or developing matrices that are
injectable. The property of injectability involves having the matrices be introduced into the lesion
only through a syringe needle and is an attractive strategy to reach deep-seated lesions without
111
causing extensive damage to the overlying tissue. A few materials, such as PLGA microspheres
[7], matrigel [9], platelet-rich plasma scaffolds [10] and self-assembling peptide hydrogels [3]
have been employed with injectable matrices thus far, each displaying certain strengths and
weaknesses. Microspheres represent miniaturized pre-fabricated matrices and allow for their
material properties to be verified prior to application but may not interface seamlessly with the
surrounding brain tissue if they only loosely occupy the lesion. Naturally-derived materials such
as matrigel and platelet-rich plasma scaffolds are relatively easy to obtain and offer in situ
gelation to conform to the irregular shape of the lesion but may have varying composition or
material properties depending on their sources and collection. Self-assembling peptide hydrogels
offer both well-defined chemical composition and enable robust regeneration of severed optic
nerve but do not undergo covalent crosslinking and may not be adequately persistent in brain
lesions that are relatively more severe.
Prior chapters in this thesis have detailed the development and characterization of
matrices that comprise of Gtn-HPA hydrogels and SDF-la-loaded polyelectrolyte complex
nanoparticles (PCNs). Gtn-HPA hydrogels belong to a class of enzymatically crosslinked
matrices whose crosslinking rate and degree can be tuned by varying concentration of the
crosslinking reagents, horseradish peroxidase (HRP) and H 2 0 2 [11]. By appropriate control of the
crosslinking rate, Gtn-HPA hydrogels can be employed as an injectable matrix to circumvent the
difficulties associated with implanting matrices into deep-seated brain lesions. Relative to other
injectable materials that have been gainfully employed in the injured brain, Gtn-HPA hydrogels
also provide for in situ gelation which is critical in ensuring conformity to irregularly shaped
lesions [12] and give the additional benefit of covalent crosslinking to allow for adjustment of
matrix persistence against the multitude of degradative processes in the lesion. SDF-la PCNs
were utilized for their easy incorporation into Gtn-HPA hydrogels and their ability to sustain (for
up to 4 weeks) the release of SDF-la, a well-established chemoattractant for various cell types
including neural progenitors and endothelial cells. It is therefore compelling to examine the
implantation of Gtn-HPA/SDF-la-PCN matrices in brain lesions and optimize the matrices for
future application in the injured brain.
In this study, we employ a rat model of intracerebral hemorrhage (ICH) to examine the
implantation of Gtn-HPA/SDF-la-PCN matrices into deep-seated lesions. In this model, ICH is
induced by the injection of collagenase to cause the disruption of blood vessels, spontaneous
extravasation of blood and hemotoma expansion in the rat brain. The severity of the injury, and
112
the subsequent size of the brain lesion, can be consistently manipulated by the amount of
collagenase injected, thus avoiding huge variations that may complicate comparative analyses.
More importantly, the location of the hemorrhage is also easily controlled by the injection site of
collagenase to induce injury well below the dorsal surface of the brain. This allows the creation of
deeply seated brain lesions to realistically evaluate the implantation of Gtn-HPA/SDF-la-PCN
matrices.
4.2
Materials and Methods
4.2.1
Preparation of Gtn-HPA conjugate and SDF-la PCNs
Gtn-HPA conjugate and SDF-la PCNs were prepared as previously described in Section
2.2.1 and 3.2.1.
4.2.2
Rheological Measurements of Gtn-HPA Hydrogels
Rheological measurements of Gtn-HPA hydrogels were performed with a TA instruments
AR-G2 rheometer using cone geometry of 40mm diameter and 20 angle. For each measurement,
600pl of Gtn-HPA containing 0.03U/ml HRP and varying concentrations of H 20 2 was applied to
the bottom plate immediately after mixing.
Solvent trap cover was placed to prevent
evaporation. All measurements were taken at 370 C in the oscillation mode with a constant strain
of 1% and frequency of 1 Hz.
4.2.3
Design of Animal Experiment
All experiments were performed under protocols approved by the Massachusetts General
Hospital Institutional Animal Care and Use Committees (IACUC, protocol 2012N000165)
following the NIH Guide for Use and Care of Laboratory Animals. A total of 14 male SpragueDawley Rats (250 - 300g) were used in this study and were subjected to intrastriatal injections of
collagenase to induce ICH. Of these animals, a) 2 was sacrificed 1 day post-ICH to reveal
characteristics of the hemorrhage; b) 2 was sacrificed 2 weeks post-ICH to reveal characteristics
of the lesion; 3) 2 received an injection of Gtn-HPA/SDF-la-PCN matrix at 2 weeks post-ICH
113
and sacrificed immediately after to reveal characteristics of the injected matrix and; 4) 8 received
injections of Gtn-HPA/SDF-la-PCN matrices (formulated with 4, 8, 12 and 16 wt% Gtn-HPA
(n=2 for each type) and SDF-la-PCNs carrying containing 6gg of SDF-la) at 2 weeks post-ICH
and sacrificed 4 weeks post-ICH to identify the optimal formulation for further investigation
(Figure 4.1).
No treatment
L_
1-day survival
Day 0
1
0
4
2-week survival
Day 0
14
Treatment with
Gtn-HPA/SDF-la-PCN matrix
4wt% Gtn-HPA
8wt% Gtn-HPA
12wt% Gtn-HPA
28
14
Day 0
16wt% Gtn-HPA
ICH
11, Matrix Injection
4
Sacrifice
Figure 4.1: Time course for ICH, matrix injection and animal sacrifice for various experimental
groups (n=2).
4.2.4
Collagenase-Induced Model of Intracerebral Hemorrhage
Rats were anesthetized using isofluorane (induction 3.5%, maintenance 1.5% to 2% in
70% N 2 0 and 30% 02) and placed in a stereotaxic frame (Kopf, Tujunga, CA). Body temperature
was monitored and maintained around 37'C during surgery using a rectal probe and a heating
pad. A midline scalp incision was made to expose the skull. A small cranial burr hole was then
drilled 0.5mm anterior and 3mm lateral to the bregma. The 25-Gauge needle of a 2d Hamilton
syringe was inserted 5mm ventral to the dural surface. 5 min after the insertion of the needle, 0.5
114
U Collagenase Type VII (Sigma Aldrich) in 2pl of 0.9% saline was injected into the striatum over
5 min. The needle was then left in place for 5 min and slowly withdrawn over 5 min. The burr
hole was sealed with bone wax and the incision was subsequently sutured. The rats were allowed
to recover from surgery in cages warmed under an incandescent bulb.
4.2.5
Stereotaxic Injection of Gtn-HPA/SDF-la-PCN Matrices
Rats were placed in a stereotaxic frame using the same procedures for anesthesia and
temperature monitoring/maintenance. A midline scalp incision was made to expose the cranial
burr hole created in the previous surgery and bone wax removed to unseal the burr hole. 25pl of
pre-gel solution of Gtn-HPA/SDF-la-PCN matrix (containing 6pg of SDF-la) was loaded into a
50pl Hamilton syringe. Pre-gel solution of Gtn-HPA/SDF-la-PCN matrix was prepared
immediately prior to loading by mixing Gtn-HPA solution with SDF- 1 a PCNs and solutions of its
cross-linking reagents, horseradish peroxidase (HRP) and H 2 0 2 . The final composition of the
various matrices is shown in Table 4.1. After loading of pre-gel solutions of matrices, the 22-G
needle of the Hamilton syringe was promptly lowered 5mm ventral to the dural surface. The
loaded pre-gel solution was injected into the ICH lesion over 3 min (Figure 4.2). After injection,
the needle was left in place for 5 min and slowly withdrawn over 5 min. Similar to the previous
surgery, the burr hole was sealed with bone wax, the incision sutured and the rats returned to
cages warmed under an incandescent bulb.
Matrix
Gtn-HPA
SDF-la
Horseradish
PCN
peroxidase
H 20 2
(HRP)
4wt% Gtn-HPA/SDF-Ia-PCN matrix
40mg/ml
1.3mg/ml*
55.0 mU/ml
3.4mM
8wt% Gtn-HPA/SDF-la-PCN matrix
80mg/ml
1.3mg/ml*
70.0 mU/mi
6.8mM
12wt% Gtn-HPA/SDF-la-PCN matrix
120mg/ml
1.3mg/ml*
115.0 mU/ml
10.2mM
16wt% Gtn-HPA/SDF-la-PCN matrix
160mg/ml
1.3mg/ml*
167.5 mU/ml
13.6
mM
*
2
corresponds to a SDF-la concentration of 0.24 mg/ml that yields 6pg of SDF-la in 5pl of injected matrices
Table 4.1: Final composition of Gtn-HPA/SDF-la-PCN matrices injected into ICH lesion
115
Figure 4.2: Photograph showing stereotaxic injection of Gtn-HPA/SDF-la-PCN matrices into the brain
lesion 2 weeks after ICH.
4.2.6
Animal Sacrifice and Specimen Processing
Rats were deeply anesthetized using 5% isofluorane and sacrificed via transcardial
perfusion with 120ml of 0.9% saline followed by 120ml 4% paraformaldehyde solution. Brains
were extracted and submerged into Tissue Tek O.C.T compound contained in a tinfoil mold. The
brains were then flash-frozen using isopentane cooled on liquid nitrogen and cryosectioned
coronally at 30m. The tissue sections were mounted on treated glass slides, air-dried and stored
at -200 C. Prior to histological staining, the tissue sections were baked at 500C for 1 hr to increase
adhesion to the glass slides, rehydrated in PBS, permeabilized in ice-cold methanol for 10 min
and washed with PBS.
116
4.2.7
Histology
Tissue sections were stained with hematoxylin and eosin for visualization of brain tissue
and implanted matrices. The procedure included staining in Gill's hematoxylin (10 min), rinsing
in water (5 min), differentiation in acid alcohol (1 s), rinsing in water (5 min), staining in eosin (1
min), dehydration in 100% alcohol (2
x
2 min), processing in xylene (2
x
3 min) and mounting of
coverslip using cytoseal. Brightfield imaging was performed on the stained cryosections using an
Olympus BX51 microscope. For each rat, histomorphometric analysis was performed on 7
cryosections which were taken at 1mm intervals from -3mm to 3mm anterior to bregma to span
over the lesion and/or implanted matrices. Areas of both hemispheres, both ventricles, both
cavitary and non-cavitary lesions resulting from ICH as well as implanted matrix were measured
using ImageJ. Area of intact brain tissue in each hemisphere was calculated using Equation 4.1.
Area of brain tissue loss for each hemisphere was calculated using Equation 4.2 and linearly
integrated using Equation 4.3 to determine the volume of brain tissue loss.
Area of intact brain tissue = Area of hemisphere - Area of ventricle Area of lesion - Area of implanted matrix
Equation 4.1
Area of brain tissue loss = (Area of intact brain tissue)conmaeE
(Area of intact brain tissue)lr
i
Equation 4.2
Volume of brain tissue loss = Y,(Area of brain tissue loss), x 1
Equation 4.3
i=-3
117
4.3
Results
4.3.1
Storage Moduli of Gtn-HPA Hydrogels
In preparation of their use in vivo, Gtn-HPA hydrogels of varying wt% were subjected to
rheological measurements to determine their storage moduli, G'. The measurements revealed that
G' of Gtn-HPA hydrogels increased significantly with increasing wt% (one-factor ANOVA,
p < 0.001) and spanned from 8591 to 54353 Pa (Table 4.2).
Matrix
G' (Pa)
4wt% Gtn-HPA
8591
8wt% Gtn-HPA
22930
1205
12% Gtn-HPA
35160
8188
16wt% Gtn-HP A
54353
13115
894
Table 4.2: G' of Gtn-HPA hydrogels
118
4.3.2
Intracerebral Hemorrhagic Lesion 1 day and 2 weeks post-ICH
1 day after the injection of collagenase, a hemorrhagic lesion was observed, spanning
over significant area of the striatum and the globus pallidus (Figure 4.3A). Within the lesion,
numerous extravasated erythrocytes dissected the brain parenchyma and aggregated at multiple
sites. Cell nuclei staining by hematoxylin appeared more diffuse in the lesion than in the rest of
the brain. Relative to the contralateral side, the injured hemisphere underwent significant
enlargement, a characteristic commonly observed to occur acutely after ICH due to the presence
of extravasated blood and brain edema. Two weeks after the onset of ICH, the lesion became a
well-demarcated region characterized by the presence of a hematoma, degenerating parenchyma
and by the appearance of small fluid-filled cavities (Figure 4.3B). The injured hemisphere
evolved to be smaller in size compared to the contralateral hemisphere and featured a
considerably enlarged ventricle. Histomorphometric analysis indicated tissue loss to be 21.5 6.8
.
mm 3
Figure 4.3: Micrographs showing the brain lesion at (A)
119
1 day and (B) 2 weeks after the onset of ICH.
4.3.3
Gtn-HPA/SDF-la-PCN matrices 1 hour after Injection
25mm 3 of Gtn-HPA/SDF-la-PCN matrix, whose volume was chosen to approximately
match the volume of tissue loss, was injected into the lesion in the form of a pre-gel solution
shortly before the completion of its cross-linking process and its transition into a gel-based
matrix. Gtn-HPA/SDF-la-PCN matrix was successfully deposited as a continuous piece of
implant around the injection site in the lesion (Figure 4.4). The implanted matrix conformed to
the boundary of the lesion, enveloped the hematoma and was tightly interfaced with the host brain
tissue or the hematoma. Notably, the presence of the Gtn-HPA/SDF-
la-PCN matrix appeared to
compensate for the structural effects of tissue loss after ICH. Specifically, the matrix reversed the
enlargement of the ipsilateral ventricle, restored the size of the injured hemisphere and reestablished overall structural asymmetry between both hemispheres.
Figure 4.4: Micrograph showing Gtn-HPA/SDF- 1 a-PCN matrix at
1 hour after injection into a 2-week old
ICH lesion.
4.3.4
Optimization of Gtn-HPA/SDF-la-PCN matrices
4 formulations of Gtn-HPA/SDF-la-PCN matrices, prepared with SDF-la-PCNs and
either 4, 8, 12 or 16wt% Gtn-HPA, were injected in the injured brain at 2 weeks post-ICH and
evaluated 2 weeks after. At the time of evaluation, a substantial proportion of the 4wt% GtnHPA/SDF-la-PCN matrix had been degraded (Figure 4.5A). Any remaining proportion of the
matrix had evolved into loose cell-filled fragments that were disconnected from the surrounding
host brain tissue, resulting in the emergence of small cavitary spaces within the lesion. The
injured hemisphere also reverted to having an enlarged ventricle and a reduced size relative to the
contralateral hemisphere. In contrast, 8wt% Gtn-HPA/SDF-la-PCN matrix was better preserved
120
at 2 weeks after implantation (Figure 4.5B). The matrix continued to exist as a single piece of
implant in the striatum without any cavitary spaces. The matrix retained its tight interface with
the surrounding brain tissue and was heavily infiltrated with host cells and vessel-like structures.
Overall,
the injured hemisphere retained its structural symmetry with the contralateral
hemisphere. 12 and 16 wt% Gtn-HPA/SDF- 1 a-PCN matrices were also well preserved at 4 weeks
post-ICH (Figure 4.5C & D). However, the interface with the host brain tissue was split in several
locations around the implanted matrices, resulting in the loss of physical continuity between the
host brain tissue and the matrices. Compared with 8wt% Gtn-HPA/SDF-la-PCN matrix, the
denser 12 and 16 wt% Gtn-HPA/SDF-la-PCN matrices permitted little or no cell infiltration.
Structural symmetry between both hemispheres was also distorted as both of these matrices
dissected into the striatum and ventricular wall and extended substantially into the ipsilateral
ventricle. 8wt% Gtn-HPA was therefore selected to formulate all matrices in subsequent
experiments.
f I
Figure 4.5: Micrographs showing (A) 4wt%, (B) 8wt%, (C) 12wt% and (D) 16wt% Gtn-HPA/SDF-laPCN matrices at 2 weeks after injection into a 2-week old ICH lesion.
121
4.4
Discussion
The first part of the study focused on the implantation of Gtn-HPA/SDF-la-PCN
matrices into deeply seated brain lesions after ICH injury. The physical space needed to house the
Gtn-HPA/SDF- 1 a-PCN matrices could be created in the acute setting by the use of procedures to
aspirate the hematoma or be allowed to gradually appear in the chronic setting with focal
cavitation or atrophy of the injured brain tissue. The latter scenario was chosen with the intent for
this study to be informative toward other forms of brain injuries that undergo similar tissue
cavitation/atrophy. The value of injectability and in situ crosslinking was clearly displayed in the
implantation of Gtn-HPA/SDF-la matrices into a lesion 5mm below the dorsal surface of the
brain. By being injectable, a Gtn-HPA/SDF-la matrix that was eventually as wide as -3mm
could be delivered to its target location via a needle whose diameter measured to be 0.7mm. In
other words, compared to a similarly sized preformed matrix which might have to affect -35mm
3
of overlying cortical tissue' for its passage into the brain lesion, the implantation of GtnHPA/SDF-la-PCN matrix only had a volumetric footprint of -2mm
3
on the overlying cortical
tissue." Examination of the brain right after implantation showed that the matrix had optimally
filled the lesion and the overlying cortical tissue was well preserved except for the slight
mechanical disruption along the needle tract. On the other hand, the ability to undergo in situ
crosslinking evidently gave Gtn-HPA/SDF-la matrix the benefit of being transiently fluid right
after injection into the lesion. This transient fluidity persisted sufficiently long for the injected
matrix fill the irregularly shaped lesion and wrap around any residual hematoma but ended soon
enough for the matrix to undergo sol-gel transition into a semi-solid material that remained
securely located within the lesion. In situ crosslinking also enabled the matrix to undergo the solgel transition while in direct contact with the surrounding tissue. This was presumably a key
factor in establishing the seamless physical continuity observed between the matrix and host
tissue, which would be critical for the inward migration of any endogenous cells.
Gtn-HPA/SDF- 1 a-PCN matrices are a form of biomaterial that is primarily composed of
denatured collagen and can be enzymatically degraded by matrix metalloproteinases, which are
known to be expressed at elevated levels in the injured brain. A crucial component of this pilot
study was to identify an optimal formulation of Gtn-HPA/SDF-la matrix for implantation into
estimated with a matrix (with a circular cross-section of diameter 1.5mm) moving through 5mm of cortical
tissue
" estimated with a needle (with a circular cross-section of diameter 0.35mm) moving through 5mm of
cortical tissue
122
brain lesions after ICH injury. The need to identify an optimal formulation was furthermore
pertinent given the opposing design criteria that stemmed from the roles that Gtn-HPA/SDF-l a
matrices were expected to serve in the brain lesions. On one hand, the expectation to reside in the
brain lesion and release SDF-la over time demanded Gtn-HPA/SDF-la matrices to have
adequate macroscopic persistence against degradation after being implanted. On the other hand,
the expectation to serve as a stromal framework for endogenous cells demanded Gtn-HPA/SDF1 a-PCN matrices to be amenable to cell migration by possessing sufficient susceptibility toward
degradation at the microscopic scale. Given the isotropic nature of Gtn-HPA/SDF-la-PCN
matrices, one way to satisfy both the lower and upper constraints placed by the opposing design
criteria was to tune the overall degradation rate of the matrices. To achieve this, we varied the
wt% of Gtn-HPA (and amount of crosslinking reagents to maintain the same crosslinking degree
per wt% Gtn-HPA). When we evaluated for matrix persistence and cell infiltration at 2 weeks
after being implanted in the brain lesion, we successfully identified 8wt% Gtn-HPA/SDF-la
matrix as an optimal formulation satisfying both constraints.
A cross reference showed that the storage modulus of the optimal formation, 8wt% GtnHPA/SDF-la matrix, was 22930
1205 Pa and was considerably higher than the reported range
of 400 - 3100 Pa for the brain. Given the material nature of Gtn-HPA, this high storage modulus
of the matrix was a concomitant of the increase in wt% of Gtn-HPA to achieve sufficient matrix
persistence in the injured brain. In our observation with 8wt% Gtn-HPA/SDF-la matrix, we did
not observe any tissue laceration, which a severe mechanical mismatch would pose a risk for.
This might partly be because the storage modulus of the implanted 8wt% Gtn-HPA/SDF-la
matrix would drop significantly with degradation to approach that of the surrounding brain tissue.
Although mechanical matching of matrix to host tissues has almost become a dogma in the field
of regenerative medicine, our experience here argues for the cautious consideration of mechanical
matching with other design criteria, especially for a degradable matrix whose mechanical
properties will evolve over time. Blind pursuit of mechanical matching would have disregarded
8wt% Gtn-HPA/SDF-la matrix as a potentially valuable formulation to investigate in an in vivo
model. Nevertheless, mechanical mismatch beyond a certain point does result in detrimental
outcomes. In our case, implantation of 12 and 16wt% Gtn-HPA/SDF-la matrices, which had
even higher storage moduli, lacerated the adjacent striatum and ruptured the lateral ventricle.
123
4.5
References
[1] Elias PZ, Spector M. Treatment of penetrating brain injury in a rat model using collagen
scaffolds incorporating soluble Nogo receptor. Journal of tissue engineering and regenerative
medicine. 2012.
[2] Deguchi K, Tsuru K, Hayashi T, Takaishi M, Nagahara M, Nagotani S, et al. Implantation of
a new porous gelatin-siloxane hybrid into a brain lesion as a potential scaffold for tissue
regeneration. J Cerebr Blood F Met. 2006;26:1263-73.
[3] Ellis-Behnke RG, Liang YX, You SW, Tay DK, Zhang S, So KF, et al. Nano neuro knitting:
peptide nanofiber scaffold for brain repair and axon regeneration with functional return of vision.
Proceedings of the National Academy of Sciences of the United States of America.
2006; 103:5054-9.
[4] Ma J, Tian WM, Hou SP, Xu QY, Spector M, Cui FZ. An experimental test of stroke recovery
by implanting a hyaluronic acid hydrogel carrying a Nogo receptor antibody in a rat model.
Biomed Mater. 2007;2:233-40.
[5] Huang KF, Hsu WC, Chiu WT, Wang JY. Functional improvement and neurogenesis after
collagen-GAG matrix implantation into surgical brain trauma. Biomaterials. 2012;33:2067-75.
[6] Park KI, Teng YD, Snyder EY. The injured brain interacts reciprocally with neural stem cells
supported by scaffolds to reconstitute lost tissue. Nature biotechnology. 2002;20:1111-7.
[7] Bible E, Chau DY, Alexander MR, Price J, Shakesheff KM, Modo M. The support of neural
stem cells transplanted into stroke-induced brain cavities by PLGA particles. Biomaterials.
2009;30:2985-94.
[8] Zhang H, Kamiya T, Hayashi T, Tsuru K, Deguchi K, Lukic V, et al. Gelatin-siloxane hybrid
scaffolds with vascular endothelial growth factor induces brain tissue regeneration. Current
neurovascular research. 2008;5:112-7.
[9] Jin K, Mao X, Xie L, Galvan V, Lai B, Wang Y, et al. Transplantation of human neural
precursor cells in Matrigel scaffolding improves outcome from focal cerebral ischemia after
delayed postischemic treatment in rats. Journal of cerebral blood flow and metabolism : official
journal of the International Society of Cerebral Blood Flow and Metabolism. 2010;30:534-44.
[10] Vaquero J, Otero L, Bonilla C, Aguayo C, Rico MA, Rodriguez A, et al. Cell therapy with
bone marrow stromal cells after intracerebral hemorrhage: impact of platelet-rich plasma
scaffolds. Cytotherapy. 2013;15:33-43.
124
[11] Wang LS, Chung JE, Chan PP, Kurisawa M. Injectable biodegradable hydrogels with
tunable mechanical properties for the stimulation of neurogenesic differentiation of human
mesenchymal stem cells in 3D culture. Biomaterials.31:1148-57.
[12] Stabenfeldt SE, Garcia AJ, LaPlaca MC. Thermoreversible laminin-functionalized hydrogel
for neural tissue engineering. Journal of biomedical materials research Part A. 2006;77:718-25.
125
Chapter 5
Evaluation of Gtn-HPA/SDF-la-PCN
matrices in a Rat Model of Intracerebral
Hemorrhagic Stroke
5.1
Introduction
Brain injuries such as ischemic strokes, intracerebral hemorrhage and trauma often cause
irreversible impairments to brain parenchyma that disrupts vital neurological functions and
severely impact quality of life. Depending on the severity of the injury, the mature brain with its
remarkable degree of cellular specialization requires cell/tissue reconstitution for the recovery of
affected neurological functions. The strategy of cell/tissue reconstitution has generally relied on
the transplantation of stem cells. Over the years, numerous types of stem cells have been isolated,
processed and transplanted via intravenous administration or direct injection into the injured
brain. They include embryonic stem cells [1], neural stem cells [2], bone marrow-derived
mesenchymal stem cells [3], adipose-derived stem cells [4] and umbilical cord blood-derived
stem cells [5]. Since the discovery of induced pluripotent stem cells (iPSCs) that can be generated
by reprogramming somatic cells, neural stem cells have also been derived from iPSCs and
transplanted into the brain after ICH [6].
Common outcomes
following the transplantation
of stem cells have
included:
1)
differentiation of the transplanted stem cells into useful cell types such as neurons, astrocytes,
oligodendrocytes and endothelial cells; 2) increased angiogenesis and; 3) improvements in
neurological functions. Scaffolding matrices (e.g. polyglycolic acid scaffolds [7], poly-lactic-coglycolic acid microspheres [8] and matrigel [9]) have further been used to improve the survival of
the transplanted cells and enabling the donor and host cells to participate in reciprocal interactions
which are absent otherwise. Despite all this promise demonstrated in pre-clinical models,
transplantation of exogenous cells into the injured brain remains highly challenging. Isolating and
126
preparing the cells for transplantation are typically associated with costly processing. The number
of transplanted cells has to be carefully titrated to avoid sudden introduction of cells in large
numbers into the harsh environment in the injured brain [10]. Any cells from non-autologous
sources need to be further weighed against possible detrimental immune complications.
Over the years, endogenous regenerative responses in the injured brain have become
increasingly recognized as an alternative way to achieve cell/tissue reconstitution in the injured
brain. One prominent regenerative response is injury-induced neurogenesis, which involves
endogenous neural stem and progenitor cells in well-defined niches such as the subventricular
zone. In the wake of a brain injury, the endogenous neural stem and progenitor cells increase their
proliferation [11, 12], migrate toward the lesion persistently for several weeks after the injury [13,
14] and differentiate into neural cells that include region-specific neurons [15] and neurons that
integrate successfully
into
the local
circuitry
[16].
Another regenerative
response is
neovascularization, which has been observed consistently across ischemic [17], hemorrhagic [18],
traumatic [19] as well as surgical brain injury [20]. Neovascularization can occur in the
angiogenic fashion where endothelial cells in the peri-lesion tissues proliferate, migrate and
engage in tube formation to sprout new blood vessels from existing ones [21]. Circulating bone
marrow-derived cells (e.g. endothelial progenitor cells) may also be recruited into the injured
brain where they differentiate into endothelial cells and participate in the formation of new blood
vessels [19, 20].
Both injury-induced neurogenesis and neovascularization have further been
correlated with functional recovery in several pre-clinical models, demonstrating their potential to
compose meaningful treatments for the injured brain.
Nevertheless, the injured brain remains far from being an adequate environment for
endogenous regenerative responses to proceed favorably. One of the key inadequacies lies in the
physical tissue loss commonly observed to beleaguer the injured brain. Such tissue loss can arise
from: 1) clearance of necrotic tissue without any accompanying mechanism to produce a tissue
substitute; 2) atrophy of localized regions due to diffuse loss of neural cells and their associated
extracellular matrix or; 3) surgical removal of compromised neural tissue. The implications of
tissue loss on endogenous regenerative responses are twofold. First, expression of cytokines to
engage the endogenous regenerative responses is lost with the physical neural tissue. Any
remaining expression is limited to a far smaller volume of tissue in the peri-lesion area and fails
127
to drive endogenous regenerative responses optimally, spurring several efforts to perform
intracerebral infusion of cytokines to augment the responses. Second, the loss of tissue typically
creates a cavity that lacks stroma to support the migration of endogenous progenitors or in-growth
of blood vessels. Even with infused cytokines to augment recruitment, progenitors and blood
vessels can only migrate/grow up to the surrounding tissue and fail to invade the cavitary space to
initiate any form of cell/tissue reconstitution.
Toward resolving these inadequacies in the injured brain to enhance endogenous
regenerative responses, we have sought to develop a matrix that can: 1) be implanted into brain
lesion to occupy the physical space resulting from brain tissue loss; 2) provide sustained release
of an appropriate cytokine that can engage endogenous regenerative responses and; 3) provide
structural support for the infiltration of endogenous cells. Gtn-HPA/SDF-la-PCN matrices have
demonstrated their efficacy in recruiting and supporting the migration of neural progenitors in
vitro. We hypothesize that upon implantation into brain lesion to occupy the physical space
resulting from brain tissue loss, Gtn-HPA/SDF-la-PCN matrix can: i) release SDF-la to
influence or enhance neurogenesis and neovascularization in the surrounding brain tissue and; ii)
re-establish a physical framework to enable recruited endogenous cells to repopulate the
otherwise structureless brain lesion.
These hypotheses are tested in an animal model of intracerebral hemorrhage (ICH),
which is a debilitating subtype of strokes that results from extravasation of blood into the brain
parenchyma From the clinical point of view, ICH is a significant brain injury that confronts
numerous people suffering from hypertension, aneurysm and brain trauma and urgently needs the
development of effective treatment approaches to improve outcomes after injury. Furthermore,
current clinical management of ICH already includes stereotactic needle-based surgery to aspirate
hemotoma. The existence of clinical procedures to physically access the brain lesion makes ICH a
highly relevant type of brain injury that can readily benefit from the current work on injecting
purposefully designed matrices into brain lesions.
From the experimental point of view,
modeling ICH in animals can be relatively straightforward with injection of either autologous
blood or collagenase. The collagenase injection model is chosen here to better mimic the
disruption of blood vessels, spontaneous extravasation of blood and hemotoma expansion. The
location of the hemorrhage is easily controlled by the injection site of collagenase to induce
128
injury well below the dorsal surface of the brain, allowing the creation of deeply seated brain
lesions to realistically evaluate the implantation of Gtn-HPA/SDF-la-PCN matrices via
stereotactic injection. The severity of the injury, and the subsequent size of the brain lesion, can
also be consistently manipulated by the amount of collagenase injected, thus avoiding huge
variations that may complicate comparative analysis among different treatment groups. Most
importantly, numerous studies have already been performed on animal models of ICH. Details of
injury-induced functional behavioral deficits, macroscopic changes (e.g. tissue loss and cavity
formation)
as
well
as
endogenous
regenerative
responses
(e.g.
neurogenesis
and
neovascularization) are well characterized and available to inform our study on the value on
implanting Gtn-HPA/SDF-la-PCN matrices in the injured brain.
129
5.2
Materials and Methods
5.2.1
Experimental Design
All experiments were performed under protocols approved by the Massachusetts
General
Hospital
Institutional
Animal
Care
and
Use
Committees
(IACUC,
protocol
2012N000165) following the NIH Guide for Use and Care of Laboratory Animals. A total of 30
male Sprague-Dawley Rats (250 - 300g) were subjected to intrastriatal injections of collagenase
to induce ICH. They were then randomly selected to receive injections of artificial cerebrospinal
fluid (aCSF; n=8), Gtn-HPA matrices (n=7); Gtn-HPA/PCN matrices (n=7) or Gtn-HPA/SDF-lcaPCN matrices (n=8) at 2 weeks post-ICH and sacrificed at 4 weeks post-ICH (Figure 5.1).
Behavior tests were performed before ICH as well as at 3 days, 1, 2, 3 and 4 weeks after ICH. 5Bromo-2'-Deoxyuridine (BrdU) was administered every other day for 2 weeks between ICH and
aCSF/matrix injection.
L0
ICH
BrdU
Saciifice
I
I
I
I
I
I
Day0
3
7
14
21
28
0
0
0
B
B
0
Pre-ICH
+
+
+
+
Pre-Injection
+
+
*
Behavior Test
aCSF / Matrix
Injection
Figure 5.1: Time course for ICH, aCSF/matrix injection, behavior test, BrdU administration and animal
sacrifice for rats treated with aCSF (n=8), Gtn-HPA matrix (n=7), Gtn-HPA/PCN (n=7) and GtnHPA/SDF-la-PCN matrix (n=8).
5.2.2
Preparation of Gtn-HPA conjugate and SDF-1a PCNs
Gtn-HPA conjugate and SDF-la PCNs were prepared as described in Section 2.2.1 and
3.2.1.
130
5.2.3
Collagenase-Induced Model of Intracerebral Hemorrhage
Rats were anesthetized using isofluorane (induction 3.5%, maintenance 1.5% to 2% in
70% N 2 0 and 30% 02) and placed in a stereotaxic frame (Kopf, Tujunga, CA). Body temperature
was monitored and maintained around 37"C during surgery using a rectal probe and a heating
pad. A midline scalp incision was made to expose the skull. A small cranial burr hole was then
drilled 0.5mm anterior and 3mm lateral to the bregma. The 25-Gauge needle of a 2pl Hamilton
syringe was inserted 5mm ventral to the dural surface. 5 min after the insertion of the needle, 0.5
U Collagenase Type VII (Sigma Aldrich) in 2pl of 0.9% saline was injected into the striatum over
5 min. The needle was then left in place for 5 min and slowly withdrawn over 5 min. The burr
hole was sealed with bone wax and the incision was subsequently sutured. The rats were allowed
to recover from surgery in cages warmed under an incandescent bulb. Between ICH and
aCSF/matrix injection, BrdU (50mg/kg) was administered via intraperitoneal injections every
other day for a total of 7 times.
131
5.2.4
Stereotaxic Injection of aCSF and Matrices
Rats were placed in a stereotaxic frame using the same procedures for anesthesia and
temperature monitoring/maintenance. A midline scalp incision was made to expose the cranial
burr hole created in the previous surgery and bone wax removed to unseal the burr hole. 251 of
aCSF, pre-gel solution of Gtn-HPA matrix, pre-gel solution of Gtn-HPA/PCN matrix or pre-gel
solution of Gtn-HPA/SDF-la-PCN matrix (containing 6gg of SDF-1a) was loaded into a 50l
Hamilton syringe. Pre-gel solution of Gtn-HPA matrix was prepared immediately prior to loading
by mixing Gtn-HPA solution with solutions of its cross-linking reagents, horseradish peroxidase
(HRP) and H 2 0 2 . Pre-gel solutions of Gtn-HPA/PCN and Gtn-HPA/SDF-la-PCN matrices were
prepared with the addition of blank PCNs and SDF-la PCNs respectively to the Gtn-HPA
solutions prior to the mixing with the cross-linking reagents. The final composition of the
matrices used is shown in Table 5.1. After loading of aCSF or pre-gel solutions of matrices, the
22-G needle of the Hamilton syringe was promptly lowered 5mm ventral to the dural surface. The
loaded aCSF or pre-gel solution was injected into the ICH lesion over 3 min. After injection, the
needle was left in place for 5 min and slowly withdrawn over 5 min. Similar to the previous
surgery, the burr hole was sealed with bone wax, the incision sutured and the rats returned to
cages warmed under an incandescent bulb.
Matrix
Gtn-HPA
SDF-la
Horseradish
PCN
peroxidase
H20 2
(HRP)
Gtn-HPA matrix
80mg/ml
-
70.0 mU/ml
6.8mM
Gtn-HPA/PCN matrix
80mg/ml
1.3mg/ml
70.0 mU/ml
6.8mM
Gtn-HPA/SDF-I a-PCN matrix
80mg/ml
1.3mg/ml*
70.0 mU/ml
6.8mM
Table 5.1: Final composition of matrices injected into 2-week-old ICH lesions. (*) corresponds to a SDFla concentration of 0.24 mg/ml that yields 6pg of SDF-la in 25.d of injected matrices
5.2.5
Behavioral Testing
Behavioral testing was performed 1 day before ICH as well as 3 days, 1, 2, 3 and 4
weeks after ICH. Behavioral testing at 2 weeks after ICH was performed prior to the injection of
132
aCSF or matrices. All behavior tests were conducted in an isolated room with dim lighting and by
personnel blinded to the experiment design.
Modified Neurological Severity Scores
Each rat was evaluated in a composite of motor, sensory, reflex and balance tests
(Table 5.2) [22] where points were awarded for particular test outcomes, the inability to perform
the test or the absence of reflexes. The overall neurological function was reflected on a scale of 0
to 18 where higher scores would denote higher severity of neurological impairment.
Motor Test
Raising rat by tail (Normal = 0, Maximum = 3)
Flexion of Forelimb
Flexion of Hindlimb
Head moved >100 within 30s
Placing rat on floor (Normal = 0, Maximum =3)
Normal walk
Inability to walk straight
Circling toward the paretic side
Fall down to the paretic side
Sensory Test (Normal= 0, Maximum = 2
Placing test (visual and tactile test)
Proprioceptive test (deep sensation, pushing the paw against the table edge to stimulate limb
Beam Balance Test (Normal= 0, Maximum =6)
Balances with steady posture
Grasps side of beam
Hugs the beam and one limb falls down from the beam
Hugs the beam and two limbs fall down from the beam or spins on beam (> 60s)
Attempts to balance on the beam but falls off (> 40s)
Attempts to balance on the beam but falls off (> 20s)
Falls off: No attempt to balance or hang on to the beam (<20s)
Reflex absence and abnormal movements (Normal = 0, Maximum = 4)
Pinna reflex (head shake when touching the auditory meatus)
Corneal reflex (eye blink when lightly touching the cornea with cotton))
Startle reflex (motor response to a brief noise from snapping a clipboard paper)
Seizures, myoclonus, myodystony
Maximum Points
Table 5.2: Modified Neurological Severity Scores.
133
1
1
1
0
1
2
3
1
1
0
1
2
3
4
5
6
1
1
1
1
18
Forelimb Placing Test
Each rat was carried with the forelimb under evaluation hanging freely and brought to
approach a tabletop sideways via a maneuver that brushed the ipsilateral vibrissae. Intact rats
spontaneously place the forelimb onto the tabletop while rats with a brain injury contralateral to
the stimulated vibrissae may, depending on the nature and extent of the injury, fail to do so. Each
rat was tested 10 times for each forelimb. The forelimb placing score reflected the percentage of
trials in which the rat placed the forelimb on the tabletop upon stimulation of the vibrissae.
Corner Turn Test
Two boards (each measuring 30cm
x
30cm
x
lcm) were attached along their shorter
edge to form a 300 corner. Each rat was placed halfway into the corner. Upon entering the corner,
both sides of the vibrissae were stimulated simultaneously, prompting the rat to rear (i.e. stand on
rear limbs) and turn to face the open end of the corner. Uninjured rats turn randomly to either
direction while injured rats turn to the direction ipsilateral to the brain injury. For each test, 10
trials, spaced at least 30s apart from one another, were performed and the percentage of ipsilateral
turns was calculated.
5.2.6
Animal Sacrifice and Specimen Processing
Rats were deeply anesthetized using 5% isofluorane and sacrificed via transcardial
perfusion with 120ml of 0.9% saline followed by 120ml 4% paraformaldehyde solution. Brains
were extracted and submerged into Tissue Tek O.C.T compound contained in a tinfoil mold. The
brains were then flash-frozen using isopentane cooled on liquid nitrogen and cryosectioned
coronally at 30[im. The tissue sections were mounted on treated glass slides, air-dried and stored
at -200C. Prior to histological and immunofluorescent staining, the tissue sections were baked at
500C for 1 hr to increase adhesion to the glass slides, rehydrated in PBS, permeabilized in icecold methanol for 10 min and washed with PBS.
5.2.7
Histology
Tissue sections were stained with hematoxylin and eosin (H&E) for visualization of
brain tissue and implanted matrices. The procedure included staining in Gill's hematoxylin (10
134
min), rinsing in water (5 min), differentiation in acid alcohol (1 s), rinsing in water (5 min),
staining in eosin (1 min), dehydration in 100% alcohol (2
x
2 min), processing in xylene (2
x
3
min) and mounting of coverslip using cytoseal. Brightfield imaging was performed on the stained
cryosections using an Olympus BX51 microscope. For each rat, histomorphometric analysis was
performed on 7 cryosections which were taken at 1mm intervals from -3mm to 3mm anterior to
bregma to span over the lesion and/or implanted matrices. Areas of both hemispheres, both
ventricles, both cavitary and non-cavitary lesions resulting from ICH as well as implanted matrix
were measured using ImageJ. Volume of brain tissue loss for each hemisphere was calculated
using Equation 4.1 - 4.3 from the previous chapter. The area of stroma in each hemisphere, which
is defined with the physical framework in both brain tissue and implanted matrices, was
calculated using Equation 5.1 and linearly integrated using Equation 5.2 to determine the stromal
volume.
Stromal area = Area of hemisphere - Area of ventricle
- Area of cavitary lesion
Equation 5.1
3
Stromal volume=
(Stroma area)i
x
1
Equation 5.2
i=-3
H&E-stained sections were also analyzed for the presence of neutrophils. Neutrophils
were identified as cells with multi-lobulated nuclei. The presence of neutrophils was scored using
a rating scale of 0-3 where 0 denotes an absence of neutrophils while 1, 2 and 3 denote mild,
moderate and significant presence respectively.
5.2.8
Fluorescence Immunohistochemical Analysis
When necessary, antigen retrieval was performed by: 1) heating tissue sections in a
pressure cooker (Biocare Medical, Concord, CA); 2) cooling to room temperature over 30 min
and; 3) washing with PBS. All subsequent staining procedures were performed at room
temperature and all washes with PBS unless otherwise mentioned. Tissue sections were blocked
with 5% donkey serum/0.3% triton-X100 in PBS for 1 hr and incubated overnight with primary
antibodies at 4 0C. Following 3 washes, tissue sections were incubated with secondary antibodies
(dilution 1:400) for 2 hrs. Tissue sections were then washed twice, incubated with 100ng/ml 4',6-
135
diamidino-2-phenylindole (DAPI) (in PBS) for 5 min and washed once. Sections subjected to
antigen retrieval were further incubated in 0.1% Sudan Black B (in 70% ethanol) for 30 min to
reduce tissue autofluroescence and washed thoroughly. All tissue sections were eventually
mounted with coverslips using Fluorescence Mounting Medium (Dako). In the particular case of
detecting surface expression of CXCR4, tissue sections were not permeabilized with methanol or
triton-X100 and blocked with 5% donkey serum in PBS. Details of the antibodies used and their
associated antigen retrieval procedure are listed in Table 5.3. The stained sections were imaged
using an epifluorescence microscope (Olympus BX60) or confocal laser scanning microscope
(Zeiss LSM 510) and analyzed using ImageJ.
136
Primary Antibody
Dilution
Rabbit Polyclonal
Anti-Doublecortin
1:2000
(Abcam ab18723)
Antigen
Retrieval
Citrate Buffer
(pH 6)
Secondary Antibody
100*C 20min
Immunoresearch 711-485-152)
Citrate Buffer
Dylight 488 Donkey AntiMouse IgG (Jackson
Immunoresearch 715-485-151)
Mature
Neurons
(pH 6)
Cy3 Donkey Anti-Sheep IgG
(Jackson Immunoresearch
110C 30min
713-165-147)
Cells that
proliferated
during BrdU
administration
Dylight 488 Donkey AntiRabbit IgG (Jackson
Immunoresearch 711-485-152)
Endothelial
cells
Endothelial
cells
Dylight 488 Donkey AntiRabbit IgG (Jackson
Cells
Identified
Neuronal
Precursors
Mouse Monoclonal
Anti-NeuN
(Millipore MAB377)
1:250
Sheep Polyclonal
Anti-BrdU
(Abcam ab1893)
1:250
Rabbit Polyclonal
Anti-von Williebrand
Factor (Dako A0082)
1:100
Tris-EDTA
Buffer
(pH 9)
Goat Polyclonal
Anti-CD34
(R&D Systems
AF4117)
Rabbit Polyclonal AntiGlial Fibrillary Acidic
Protein
(Dako Z0334)
Mouse Polyclonal
Anti-CD68
(Abcam ab31630)
1:40
Tris-EDTA
Buffer
Dylight 488 Donkey Anti-Goat
IgG (Jackson Immunoresearch
705-485-003)
1:1000
(pH 9)
100*C 20min
None
Dylight 488 Donkey AntiRabbit IgG (Jackson
Immunoresearch 711-485-152)
Astrocytes
1:400
None
Dylight 488 Donkey Anti-
Activated
macrophages
and microglia
(pH 6)
1 10C 30min
Citrate Buffer
100'C 20min
Mouse IgG (Jackson
Immunoresearch 715-485-151)
Rabbit Polyclonal
Anti-CXCR4
(Abcam ab2074)
1:250
Mouse Monoclonal
Anti-VEGF
(Abcam abl316)
1:100
Tris-EDTA
Buffer
(pH 9)
1 10"C 20min
Citrate Buffer
(pH 6)
1 10"C 20min
Dylight 488 Donkey AntiRabbit IgG (Jackson
Immunoresearch 711-485-152)
Various
types
express
CXCR4
cell
that
Dylight 488 Donkey AntiMouse IgG (Jackson
Immunoresearch 715-485-151)
Various
types
express
VEGF
cell
that
Table 5.3: List of antibodies and their associated antigen retrieval procedures for fluorescent
immunohistochemical analysis.
5.2.9
Statistical Analysis
Values are expressed as mean
standard error mean (SEM). Analyses of variance
(ANOVA) and Fisher's post-hoc test were performed accordingly. Statistical significance was set
atp < 0.05.
137
5.3
Results
5.3.1
Behavioral Outcomes
To evaluate the effects of Gtn-HPA/SDF- 1 a-PCN matrices on outcomes after ICH, rats
were subjected to ICH and randomly grouped to receive injections of aCSF, Gtn-HPA matrix,
Gtn-HPA/PCN matrix or Gtn-HPA/SDF-la-PCN matrix. Functional behavioral outcomes of the
rats were assessed by mNSS, forelimb placing test and corner turn test.
For mNSS, the scores of rats in all groups were nearly 0 pre-ICH and spiked to around
6 after ICH, indicating the appearance of neurological impairment upon the onset of injury to the
brain (Figure 5.2). Over week 2 - 4 after ICH, scores were significantly different over time and
across different treatments (2-factor ANOVA, p < 0.001 for both). Specifically, scores remained
similarly elevated across all groups at week 2 post-ICH prior to treatment injection. In the
subsequent 2 weeks, scores for the aCSF, Gtn-HPA matrix and Gtn-HPA/PCN matrix groups
declined slightly in a similar fashion while scores for the Gtn-HPA/SDF-la-PCN matrix group
declined more rapidly to be significantly lower at week 3 and 4 after ICH (Fisher's post-hoc test,
p < 0.01 andp<0.05 compared to all other groups).
8
Treatment
-G- aCSF
6
4
z
E
-a- Gtn-HPA matrix
2
0
-X- Gtn-HPA/PCN matrix
---
Be
Gtn-HPA/
SDF-la-PCN matrix
_---_-------A
Figure 5.2: Modified Neurological Severity Scores (mNSS). A decrease in the score suggests behavioral
recovery. * p<O.05, ** p < 0.01, Fisher's post-hoc test.
138
A similar trend was observed with the forelimb placing test. Before ICH, all rats
successfully placed their contralateral forelimb with every stimulus, thus giving a score of 100%
(Figure 5.3). Forelimb placing reduced drastically to below 20% after ICH. Over week 2 - 4 after
ICH, scores were significantly dependent on time and the type of treatment (2-factor ANOVA, p
< 0.05 for both). Specifically, scores remained similarly low across all groups at week 2 post-ICH
prior to treatment injection but improved subsequent to treatment injections. However, scores for
the Gtn-HPA/SDF-la-PCN matrix group improved more rapidly and reached the highest among
all groups and time points at week 4 after ICH (Fisher's post-hoc test, p < 0.05 compared to
aCSF).
Baseline
u
S
100
80-
(0
60
60
0
40
Cl)
20
Gtn-HPA/
SDF-la-PCN matrix
-X-
Treatrnent
Gtn-HPA/PCN matrix
+ Gtn-HPA matrix
-e- aCSF
0'
Figure 5.3: Forelimb placing score. An increase in the score suggests behavioral recovery.
*
p < 0.05, Fisher's post-hoc test.
In the corner turn test, rats randomly turned to their ipsilateral side approximately 50%
of the time before ICH (Figure 5.4). Turns became primarily directed toward the ipsilateral side
after ICH. Over week 2 - 4 after ICH, the percentage of turns toward the ipsilateral side was
significantly dependent on time and the type of treatment (2-factor ANOVA, p < 0.01 and p <
0.05 respectively). Specifically, rats in all groups continued to turn primarily to the ipsilateral
side. Rats treated with aCSF, Gtn-HPA matrix and Gtn-HPA/PCN matrix failed to recover in the
subsequent weeks while rats treated with Gtn-HPA/SDF-la-PCN matrix showed a modest
recovery at week 4 after ICH (Fisher's post-hoc test, p < 0.01 compared to both aCSF and GtnHPA matrix).
139
Gtn-HPA/
100
SDF-la-PCN matrix
80
40
-E
o-
-X- Gtn-HPA/PCN matrix
Baseline
60
Treatment
-0- Gtn-HPA matrix
20
________
_--
aCSF
Figure 5.4: Percentage of turns made towards the impaired side in the corner turn test. An increase
in the percentage toward 50% suggests behavioral recovery. ** p < 0.01, Fisher's post-hoc test.
5.3.2
Gross Histological Outcomes
At 4 weeks after ICH, the lesion in rats treated with aCSF presented as either a cavity
or a condensed region of tissue that was intensely stained by hematoxylin (referred hereafter as
cavitary and non-cavitary lesion respectively; Figure 5.5A). The cavitary lesion was completely
void of physical matter under histological staining but was presumably filled with fluid prior to
histological processing based on magnetic resonance images in other studies [23, 24] (Figure
5.6A). On the other hand, the non-cavitary lesion consisted of hypercellular tissue with fine
trabeculae and hemosiderin-filled cells (Figure 5.6B). In both cases, the hematoma had largely
resolved and the injured hemisphere shared several characteristics already observed at 2 week
post-ICH, namely: 1) enlargement of the ipsilateral ventricle; 2) reduction in hemispheric size
and; 3) overall asymmetry with the contralateral side.
140
A
C
CU
-
D
cc
E
z
CI
07
0LL
Figure 5.5: Micrographs showing H&E-stained sections with (A) aCSF-treated (left) cavitary and (right)
non-cavitary lesions, (B) Gtn-HPA matrix-treated lesions, (C) Gtn-HPA/PCN matrix-treated lesions and
(D) Gtn-HPA/SDF-la-PCN matrix-treated lesions. Bar:
141
1mm.
A
C
aCSF:
Cavitary Lesion
Gtn-HPA matrix
B
aCSF:
Non-cavitary Lesion
D
Gtn-HPAI
PCN matrii
E
Gtn-HPA/
SDF-la-PCN matrix
Figure 5.6: Micrographs showing brain tissue interfacing with (A) aCSF-treated cavitary lesion, (B) aCSFtreated non-cavitary lesion, (C) Gtn-HPA matrix-treated lesion, (D) Gtn-HPA/PCN matrix-treated lesion
and (E) Gtn-HPA/SDF-la-PCN matrix-treated lesion. Bar: 50gm.
Hematoma had also mostly resolved in the injured hemispheres of rats treated with
Gtn-HPA, Gtn-HPA/PCN and Gtn-HPA/SDF-la-PCN matrices (Figure 5.5B-D). The injured
hemispheres were, however, notably distinct from those of rats treated with aCSF in several other
aspects. Instead of empty space or necrotic-looking tissue, the lesion was substantially filled with
an implant material that was distinguishable from the surrounding host tissue. In addition, the
injured hemispheres mostly maintained the overall structural symmetry that had been achieved
with the injection of matrices at 2 week post-ICH and did not suffer the same ventricular
enlargement or hemispheric size reduction as those treated with aCSF. All the implanted GtnHPA-based matrices shared similar characteristics of being a physical structure weakly stained by
eosin and featuring a porous morphology that had likely resulted from histological processing
(Figure 5.6C-E). The matrices were also physically continuous with apposing host tissues and had
142
evidently maintained the tight interface achieved right after injection of matrix at 2 week postICH. In several locations within the striatum, the implanted matrices could be seen to directly
appose host tissues with distinct white and gray matter structures. Implanted Gtn-HPA matrices
were mostly void of cells. In contrast, the implanted Gtn-HPA/PCN and Gtn-HPA/SDF-la-PCN
matrices contained numerous host cells. The latter was also found to contain several vessel-like
structures.
The volume of stroma was measured to assess the physical framework present in the
host brain tissue (minus empty spaces such as ventricles and cavitary lesion) and implanted
matrices. When treated with aCSF. alone, the volume of stroma in the injured hemispheres of rats
was reduced to 92.0
HPA,
0.5 % as compared to the contralateral side (Figure 5.7A). Injection of Gtn-
Gtn-HPA/PCN and Gtn-HPA/SDF-la-PCN
matrices
reduction and significantly increased the volume of stroma to 96.2
substantially diminished this
0.6 %, 95.8
0.8% and 98.2
0.9 % respectively relative to the contralateral side (Fisher's post-hoc test, p < 0.0 1).
The volume of intact brain tissue (defined to exclude empty spaces, necrotic-looking
tissue or implanted matrices) was also measured to determine brain tissue loss, which has been
known to occur progressively over time after ICH [23]. Brain tissue loss in rats treated with aCSF
was found to reach 32.7
2.7 mm 3 at 4 weeks post-ICH (Figure 5.7B). Injection of Gtn-HPA and
Gtn-HPA/PCN matrices did not produce any significant effect on brain tissue loss. In contrast,
injection of Gtn-HPA/SDF-la-PCN matrix significantly reduced brain tissue loss by 37.7% to
20.4
3.7 mm 3 (Fisher's post-hoc test, p < 0.05).
143
so
100**
95-
0
E 12
_2 G
90-
d
85-
--
80
80-
E
40'
30-
0
,
.
2010
0
0
,9
AICI
4P
la
4.*
4P
I0;
Q
C., IQ
IQ
19
e
?P
,V14+
'
(D%1-
50-
E
*
E -c
B
-
I*
-
A
CIO
N
C,
Figure 5.7: (A) Relative volume of stroma as a percentage of contralateral hemisphere. (B) Tissue loss in
the injured hemisphere. * p < 0.05, **p<0.01, *** p < 0.001, Fisher's post hoc test.
5.3.3
Endogenous Neurogenesis in Host Tissue
Doublecortin was used as the marker to identify neuronal precursors (i.e. neuronal-
restricted progenitors and immature neurons). In the contralateral (i.e. uninjured) hemispheres of
rats across all groups, the DCXI cells in the subventricular zone (SVZ) existed as a thin lining
along most of the lateral border of the ventricle and as a small wedge-shaped aggregation of
contiguous cells at the dorsal tip of the ventricle (Figure 5.8). Very few DCX+ cells were
observed outside the subventricular zone. ICH injury resulted in marked changes to the
population of DCXI in the ipsilateral hemisphere. As seen in rats treated with aCSF, the overall
population of DCX+ cells had increased substantially.
144
A
Contralateral
Co*"ra'a-e'a
Ipsilateral
'psat-r'
B
Contralateral
Ipsilateral
Contralateral
Ipsilateral
X
E
LL
CO
0.
0
C
Contralateral
D
Ipsilateral
z
U
CL
E
z
LL 25
LMc
E.
Figure 5.8: Fluorescence micrographs showing DCX immunoreactivity in the contralateral and ipsilateral
hemispheres of 2 representative rats in the (A) aCSF, (B) Gtn-HPA matrix, (C) Gtn-HPA/PCN matrix and
(D) Gtn-HPA/SDF-la-PCN matrix. v: ventricle. Bar: 500 [m.
This increase manifested as a thicker lining of DCX+ cells in certain regions of SVZ and a
significantly larger aggregation at the dorsal tip of the ventricle. Several more DCXI cells were in
the striatum although the majority of the DCX* cells still remained in the SVZ. Such injuryinduced changes to the population of DCX+ cells were also observed in rats treated with Gtn-HPA
and Gtn-HPA/PCN matrices. An outstanding difference was, however, noted in rats injected with
Gtn-HPA/SDF-la-PCN matrices. In the aCSF, Gtn-HPA matrix and Gtn-HPA/PCN groups, most
DCXI cells remained as relatively rounded cells in a contiguous lining or aggregration in the SVZ
145
(Figure 5.8A-C, 5.9A-C). However, the majority of the DCX+ cell population in ipsilateral
hemispheres injected with the Gtn-HPA/SDF-la-PCN matrix was observed as discrete cells
scattered across the injured striatum (Figure 5.8D). Unlike the contiguous cells within the SVZ,
these discrete cells were mostly elongated with long processes (Figure 5.9D).
Figure 5.9: Fluorescence micrographs showing DCX+ neuronal precursors as contiguous cells in the SVZ
of the (A) aCSF, (B) Gtn-HPA matrix, (C) Gtn-HPA/PCN matrix groups and as discrete elongated cells in
the striatum of the (D) Gtn-HPA/SDF-la-PCN matrix. Bar: 50gm.
The area and distribution of the DCXI cell population in the SVZ and striatum were
analyzed as illustrated by the schematic in Figure 5.1 OA. The area occupied by DCX+ cells in the
contralateral hemispheres was similar across all four treatment groups at
-
0.17 mm 2 (Figure
5.10B). In comparison, the total area occupied by DCX+ cells in the ipsilateral hemispheres of
rats was also similar across all treatment groups but had increased significantly by -5-fold to ~
0.78 mm 2 (Fisher's post-hoc test, p < 0.01). In terms of distribution, the proportion of DCX+ area
in the contralateral striatum was measured to be
-
9 -11% and was similar across all treatment
groups (Figure 5. 1C). In contrast, the proportion of DCX+ area in the ipsilateral striatum had
increased significantly in all four treatment groups.
The increase in both the aCSF, Gtn-HPA and Gtn-HPA/PCN matrix groups was
similar, bringing the proportion of DCXI area in the ipsilateral striatum to
-
36 - 45%. On the
other hand, the increase in Gtn-HPA/SDF- 1 a-PCN matrix group was notably larger. Proportion
of DCX+ area in the ipsilateral striatum of the Gtn-HPA/SDF- 1 a-PCN matrix group reached 64%,
which was significantly higher than that for the other treatment groups (Fisher's post-hoc test, p <
0.05). Despite the distribution shift of DCX+ neuronal progenitors into the striatum, the vast
majority of them were observed up to approximately the midpoint between the SVZ and the
lesion/implanted matrix in all treatment groups.
146
A
. Subventricular zone
11. Striatum
B,-
C
E
E
Contra
0
Ipsi
E
U)
Ipsi
80
1.M
60-
V
0
Contra
.100
X
0
III.Lesion/Gel
0.520
0.0
0CC
VC
V,~
e9
0q
(9
N
4'
C,
Figure 5.10:
(A)
C,
'14
Schematic depicting regions in the SVZ and striatum where
areas of DCX
2
immunoreactivity are analyzed. (B) Total area covered by DCXI cells (expressed in mm ). (C) Percentage
of DCX+ area observed in the striatum. * p < 0.05, ** p <0.01, * * * p < 0.001, Fisher's post hoc test.
147
Double immunohistochemical staining for BrdU and NeuN was performed to identify
newly generated mature neurons in the striatum. The number of BrdU+/NeuN+ cells was relatively
few and only observed in striatal regions near the SVZ (Figure 5.11). Quantification showed the
density to be around 10 BrdU+/NeuN+ cells per mm 2 across all groups (Figure 5.12).
A
B
CD
Figure 5.11: Micrographs showing NeuN (green) and BrdU (red) immunoreactivity in the striatum of rats
in the (A) aCSF, (B) Gtn-HPA, (C) Gtn-HPA/PCN matrix and (D) Gtn-HPA/SDF-la-PCN matrix groups.
Arrows indicates cells that are immunoreactive for both BrdU and NeuN. Bar: 50ptm.
148
3
15
z
ZO
10
caa
0
,
z+
Figure 5.12. Number of BrdU+/NeuN+ cells per mm 2 in the striatum near the SVZ.
5.3.4
Neovascularization in Host Tissue
Regardless of the treatment group, all the contralateral hemispheres were generally
marked with thin blood vessels that were uniformly all scattered throughout the brain parenchyma
(Figure 5.13). In contrast, blood vessels in the ipsilateral hemispheres were thicker and appeared
to be of greater numbers, especially in areas near the lesion or implanted matrix. Quantitative
analysis revealed that the length density of blood vessels in the contralateral hemispheres were
~ 2 mm per mm2 across all the treatment groups (Figure 5.14). Relative to this, the length
densities of blood vessels in the ipsilateral hemispheres across the various treatment groups were
all significantly higher (Fisher's post-hoc test, p < 0.05 or p < 0.001). Specifically, the length
densities of blood vessels in the ipsilateral hemispheres of the aCSF, Gtn-HPA matrix and GtnHPA/PCN matrix groups had increased significantly to 3.5, 3.7 and 3.3 mm per mm2 respectively
(Fisher's post-hoc test, p < 0.05 and p < 0.001 respectively relative to contralateral hemispheres)
but were statistically indistinct from each other. On the other hand, the length density of blood
vessels in the ipsilateral hemispheres of the Gtn-HPA/SDF- 1 a-PCN matrix increased to reach 4.9
2
mm per mm (Fisher's post-hoc test, p < 0.001 relative to contralateral hemispheres), which was
also significantly higher than length density of blood vessels in the ipsilateral hemispheres in the
other groups (Fisher's post-hoc test, p < 0.01 respectively).
149
A
B
Contralateral
I silateral
C
Contralateral
Ipsilateral
D
Contralateral
Ipsilateral
Figure 5.13. Micrographs showing vWF+ vessels around (*) lesion/implanted matrices in the ipsilateral
hemisphere and corresponding regions in the contralateral hemisphere of rats treated with (A) aCSF, (B)
Gtn-HPA matrix, (C) Gtn-HPA/PCN matrix and (D) Gtn-HPA/SDF-la-PCN matrix. Dotted line denotes
interface between host tissue and lesion/implanted matrices. Bar: 100pm.
150
**
a
I
6I
ContralatE ral
Ipsilateral
*
0
+
0'
Cu
0~
IU.
~.1
I Cj
0
19
411
Figure 5.14. Length density of vWF+ blood vessels in brain tissue around the lesion/implanted matrices.
* p < 0.05, ** p <0.01, *** p < 0.001, Fisher's post hoc test.
151
5.3.5
Astrocytic Response in Host Tissue
Numerous GFAP+ astrocytes were observed around all lesions/implanted matrices (Figure 5.15).
Within approximately 200-300 pm of the lesion/matrix boundary, density of GFAP+ astrocytes
was so high that GFAP+ reactivity was largely continuous and individual astrocytes could not be
easily demarcated. In the host tissue outside this dense band, GFAP+ astrocytes mostly existed as
scattered cells. Some were in the form of enlarged, rounded cells with short, thick processes while
others appeared as elongated cells with long, thin processes. Quantitative analysis of the area
covered by GFAP+ astrocytes indicated no significant difference among the different treatment
groups (Figure 5.16).
AB
Figure 5.15: Micrographs showing GFAP immunoreactivity around (*) lesion/implanted matrices in the
ipsilateral hemisphere of rats treated with (A) aCSF, (B) Gtn-HPA matrix, (C) Gtn-HPA/PCN matrix and
(D) Gtn-HPA/SDF- 1 a-PCN matrix. Dotted line denotes interface between host tissue and lesion/implanted
matrices. Bar: 200pm.
152
U,
+
U
0.
60-
U-
0
.0
0
L.
0
40-
20-
0
U
(U
0
1..
0C,
+'
I9V~
&
V
Figure 5.16: Percentage of area covered by GFAP+ cells in brain tissue around the lesion/implanted
matrices.
5.3.6
Neutrophils and Activated Macrophages/Microglia in Host Tissue
The host tissue was assessed any acute and chronic inflammatory cells in response to the ICH
injury and/or the implanted matrices. Across all treatment groups, the presence of neutrophils
around the lesion and implanted matrices was very mild (Figure 5.17). Specifically, -0-2
neutrophils were observed for the aCSF and Gtn-HPA matrix groups in a typical field of view
(using a 40x objective) while ~3-5 neutrophils were observed for the Gtn-HPA/SDF-la-PCN
matrix group. These were consistent with the absence of necrotic cells/tissues in the host tissue,
which are common triggers of acute inflammation. The identified neutrophils were largely found
within 50 pm of the lesion/matrix boundary. Overall, the various groups were scored as either 0
or 1 (Figure 5.18)
153
C
Figure 5.17: Micrographs of H&E-stained sections showing the brain tissue in contact with (A) aCSFtreated lesion, (B) Gtn-HPA matrix, (C) Gtn-HPA/PCN matrix and (D) Gtn-HPA/SDF-la-PCN matrix.
Dotted line denotes interface between host tissue and lesion/implanted matrices. Arrows denote the
neutrophils identified. Bar: 50ptm.
0)
0
3.
AA
2-
J0
0
U
*
I-
z
~I~~
I
C4
C+
I'
Figure 5.18: Rating for the presence of neutrophils in brain tissue around the
154
lesion/implanted matrices.
On the other hand, immunostaining for CD68 showed a strong presence of activated
macrophages/microglia around the lesion and/or implanted matrices (Figure 5.19). Around both
cavitary and non-cavitary lesions in the aCSF group, the activated macrophages/microglia were
predominantly
enlarged,
spherical/spheroid
cells
without
any
processes.
Numerous
macrophages/microglia were found to contain hemosiderin or to localize around extracellular
deposits of hemosiderin, which was easily visualized with its intense autofluorescence. In
contrast, activated macrophages/microglia in the host tissue around implanted Gtn-HPA, GtnHPA/PCN and Gtn-HPA/SDF-la-PCN matrices possessed a small soma with a few ramified
processes.
Quantitative
analysis
showed
that the
area fraction occupied
by activated
macrophages/microglia was similar across all the treatment groups (Figure 5.20).
Figure
5.19: Micrographs showing CD68 immunoreactivity around (*) lesion/implanted matrices in the
ipsilateral hemisphere of rats treated with (A) aCSF, (B) Gtn-HPA matrix, (C) Gtn-HPA/PCN matrix and
(D) Gtn-HPA/SDF-la-PCN matrix. Dotted line denotes interface between host tissue and lesion/implanted
matrices. Bar: 200 m.
155
U)
co 25
20
15
0
0
5
0
0
C,
Figure 5.20. Percentage of area covered by CD68+ cells in brain tissue around the lesion/implanted
matrices.
5.3.7
Endogenous Neuronal Precursors in Lesion/Implanted Matrices
Both cavitary and non-cavitary lesions in the aCSF group as well as the implanted matrices in the
Gtn-HPA, Gtn-HPA/PCN and Gtn-HPA/SDF-la-PCN matrix groups were examined for any
infiltration by DCXI cells. Cavitary lesions were void of any physical framework and did not
contain any cells (Figure 5.21A). No DCX+ cell was also observed in the non-cavitary
lesion,
Gtn-HPA matrix and Gtn-HPA/PCN matrix (Figure 5.21B-D). On the other hand, DCX+ cells
could be found in the Gtn-HPA/SDF-la-PCN matrices near the interface with the host brain
tissue for 4 out of 8 rats in the group (Figure 5.21D). Their presence was generally
meager,
consisting of only 1 - 5 cells per brain tissue section from each rat.
156
A
C
C)
)Uh
(U
0
_
Ca
0>
CU
z
E
E
Matrix
MMtrix
Matrix
aCUs
zE
cz
C-
(U
CLz
LL
Figure 5.21: Fluorescent micrographs showing (green) DCX immunoreactivity, (blue) DAPI nuclei staining
and their merger for (A) aCSF-treated cavitary lesion, (B) aCSF-treated non-cavitary lesion, (C) lesion
treated with Gtn-HPA matrix, (D) lesion treated with Gtn-HPA/PCN matrix and (E) lesion treated with
Gtn-HPA/SDF-la-PCN matrix. Dotted line denotes interface between host tissue and lesion/implanted
matrices. Arrows denote DCXI cells that had successfully migrated into the Gtn-HPA/SDF-la-PCN matrix.
Bar: 100pm.
157
5.3.8
Blood vessels in Lesion/Implanted Matrices
Cavitary lesions did not contain any blood vessel (Figure 5.22A) while several blood
vessels were found to persist in the tissue within non-cavitary lesions (Figure 5.22B). For the
implanted matrices, Gtn-HPA and Gtn-HPA/PCN matrices remained void of blood vessels
(Figure 5.22C) while an appreciable number of blood vessels had grown into Gtn-HPA/SDF-laPCN matrices (Figure 5.22D). Quantitative analysis revealed that the vessel length of blood
vessels per mm 2 were statistically comparable between the non-cavitary lesions and the GtnHPA/SDF- 1 a-PCN matrices (Figure 5.23).
C
Gtn-HPA matrix
D
Figure 5.22: Micrographs showing vWF+ blood vessels within (A) aCSF-treated cavitary lesion, (B) aCSFtreated non-cavitary lesion, (C) lesion treated with Gtn-HPA matrix, (D) lesion treated with Gtn-HPA/PCN
matrix and (E) lesion treated with Gtn-HPA/SDF-la-PCN matrix. Dotted line denotes interface between
host tissue and lesion/implanted matrices. Bar: 200pm.
158
2.5
E
iii
2.0
15
M.E
SE
0
0.0-
LUL
3:
~T
I
I
Ig~
>,
I
I
P,
et
.0N
P-
+
+
4)
0'.~*
Figure 5.23: Length density of vWF+ blood vessels within lesions/implanted matrices.
159
5.3.9
Astrocytes in Lesion/Implanted Matrices
Immunostaining for GFAP revealed the absence of astrocytes within the lesion or implanted
matrices (Figure 5.24).
A
aCSF: Cavitarv lesion
B
C
Gtn-HPA matrix
D
aCSF: Non-cavitary lesion
Gtn-HPA/PCN matrix
Figure 5.24: Micrographs showing absence of GFAP+ immunoreactivity within (A) aCSF-treated cavitary
lesion, (B) aCSF-treated non-cavitary lesion, (C) lesion treated with Gtn-HPA matrix, (D) lesion treated
with Gtn-HPA/PCN matrix and (E) lesion treated with Gtn-HPA/SDF-l a-PCN matrix. Dotted line denotes
interface between host tissue and lesion/implanted matrices. Bar: 200pm.
160
5.3.10 Neutrophils and Activated Macrophages/Microglia in Lesion/Implanted
Matrices
No neutrophil was observed in the cavitary lesion (Figure 5.25A) while a trace number of
them was found throughout the non-cavitary lesion in the midst of hemosiderin-laden
macrophages (Figure 5.25B). A trace number of neutrophils was also noted within implanted
Gtn-HPA matrices but was mostly restricted within 50 gm of the interface between the host tissue
and matrix (Figure 5.25C). In contrast, significant numbers of neutrophils were observed
throughout the implanted and Gtn-HPA/PCN and Gtn-HPA/SDF-la-PCN matrices (Figure 5.25D
& E). They occurred as either scattered cells near the tissue/matrix interface, clusters that were
closely associated with in-grown blood vessels (Figure 5.26A) or dense pools of cells that were
typically located away from the tissue matrix interface (Figure 5.26B). When immunostaining for
CXCR4, the receptor for SDF-la, was performed without methanol- or detergent-mediated
permeabilization in order to evaluate surface expression, many neutrophils were stained
positively for CXCR4 (Figure 5.27A). CXCR4 staining was also observed to be punctate and
localized to the boundary of the cells (Figure 5.27B). Many neutrophils were also stained positive
for vascular endothelial growth factor (VEGF; Figure 5.28). Overall, the presence of neutrophils
in lesions treated with aCSF and Gtn-HPA matrix was rated as 0-1 while that in Gtn-HPA/SDF1 a-PCN matrix was mostly rated as 3 (Figure 5.32).
161
A
BC
0
cc
C
Cn
C
CO)
z0
Cu
C)
0D
D
Ex
E
z
E
z
E
a.
LI.
a
U)
Figure 5.25: Micrographs of H&E-stained sections showing (A) aCSF-treated cavitary lesion, (B) aCSFtreated cavitary lesion, (C) lesion treated with Gtn-HPA matrix, (D) lesion treated with Gtn-HPA/PCN
matrix and (E) lesion treated Gtn-HPA/SDF-la-PCN matrix. Neutrophils with multi-lobulated nuclei were
found to be a common cell type within Gtn-HPA/PCN and Gtn-HPA/SDF-la-PCN matrices. Bar: 50tm.
A
B
Figure 5.26: Micrograph showing neutrophils (A) closely associated with newly formed vasculature and
(B) in clusters within Gtn-HPA/SDF-la-PCN matrix. Bar: 50pm
162
B
Figure 5.27: Immunostaining for CXCR4 was performed without cell permeabilization. (A) Micrograph
showing CXCR4 immunoreactivity on a cluster of neutrophils within Gtn-HPA/SDF-la-PCN matrix.
Bar: 20pm.
(B) Confocal 3D reconstruction of neutrophils
showing
CXCR4 immunoreactivity.
Reconstructed orthogonal images are presented as viewed in the x-z (top) and y-z (right) planes.
Figure 5.28: Micrograph showing VEGF immunoreactivity on a cluster of neutrophils within
GtnHPA/SDF-la-PCN matrix. Bar: I00pm.
163
3-
2-
z
I-
M
IB
____W_ ,+
AP
C,
_AiMdAdA_
I
P
V
+
(0
I
MA.+
pr
Figure 5.29. Rating for the presence of neutrophils in aCSF-treated lesions and implanted matrices.
Activated macrophages/microglia were present in non-cavitary lesions and Gtn-HPA/SDF-laPCN matrices (Figure 5.30). Activated macrophages/microglia were noticeably more prevalent in
non-cavitary lesions than in Gtn-HPA/SDF-la-PCN matrices. Quantitative analysis revealed that
area fraction occupied by the activated macrophages/microglia was 30.5
cavitary lesions but was significantly lower at 4.2
+
8.9 % in the non-
1.0 % in the Gtn-HPA/SDF- 1 a-PCN matrices
(p <0.001, Fisher's post-hoc test; Figure 5.31). Within the Gtn-HPA/SDF-la-PCN matrices,
activated macrophages/microglia were observed to be scattered cells (Figure 5.30E) or closely
associated with neutrophil clusters (Figure 5.32).
164
A
aCSF: Cavitarv lesion
R
aCSF: Non-cavitarv lesion
D
Gtn-HPA/PCN matrix
I
F
Gtn-HPA/SDF-la-PCN matrix
Figure 5.30: Micrographs showing absence of CD68+ immunoreactivity within (A) aCSF-treated cavitary
lesion, (B) aCSF-treated non-cavitary lesion, (C) lesion treated with Gtn-HPA matrix, (D) lesion treated
with Gtn-HPA/PCN matrix and (E) lesion treated with Gtn-HPA/SDF-la-PCN matrix. Dotted line denotes
interface between host tissue and lesion/implanted matrices. CD68+ macrophages/microglia were mostly
observed in aCSF-treated non-cavitary lesion as well as both Gtn-HPA/PCN and Gtn-HPA/SDF-la-PCN
matrices. Bar: 200pm.
165
60Co
C)
0
0
I
40-
+
0
(a
0
C.)
20***
_
( aCSF
***
0*
-
0
.~e
FL:j:'
Figure 5.31: Percentage of area covered by CD68+ macrophages/microglia within the lesions/implanted
matrices. *** p < 0.001, Fisher's post-hoc test.
Figure 5.32: Micrograph showing some CD68+ macrophages/microglia to be closely associated with cell
clusters that were previously identified to be neutrophils. Bar: 200pm
166
5.4
Discussion
In this chapter, we implanted Gtn-HPA, Gtn-HPA/PCN and Gtn-HPA/SDF-la-PCN
matrices into brain lesions resulting from ICH injury and evaluated their effects alongside with
lesions that were only treated with aCSF. The foremost assessment we conducted was the
evaluation of behavior recovery, given that the most tangible and important goal of any strategy
to treat brain injury is to reduce neurological impairments and their devastating effects on quality
of life. We had selected a battery of three tests, namely mNSS, forelimb placing test and corner
turn test for various reasons. mNSS covers a spectrum of sensory functions, motor skills, balance
and reflexes and provides the means to obtain a perspective of the overall neurological well being
of the animal. Forelimb placing test is a relatively more focused evaluation that requires the
animal to display its ability to integrate sensory and motor functions to complete the task of
sensing a table edge and placing its limb on the surface. Corner turn test requires the animal to
approach and sense the corner, execute a turn based on its postural symmetry and emerge from
the corner. By examining the ability of the animal to perform a multi-step task, it is highly
sensitive and can detect long-term impairment [25]. In our study, animals with the control
treatment of aCSF were on the trajectory of behavior recovery after ICH injury in terms of the
tasks in mNSS and forelimb placing test. Such spontaneous behavior recovery is commonly
observed across various forms of brain injury and has been attributed to the return of functions to
neural cells that suffer but survive the initial injurious event and to neuro-plasticity that allows
certain functional behaviors to be re-wired to alternative neural transmission pathways. We also
found that the impairment determined by corner turn test failed to resolve to any degree,
suggesting that within a certain time frame, spontaneous recovery might be either inadequate or
incapable of providing noticeable improvements for relatively more challenging behaviors. Our
data showed that implantation of Gtn-HPA/SDF-la-PCN matrices accelerated the spontaneous
recovery observed in mNSS and forelimb placing test and initiated improvements in the corner
turn test. SDF-la PCNs had appeared to have an integral role in these effects, given that GtnHPA and Gtn-HPA/PCN matrices failed to elicit the same benefits. These findings are a
definitive statement that Gtn-HPA/SDF-l a-PCN matrices are beneficial to the injured brain after
ICH. They also add to gathering evidence that appropriate intervention can produce functional
improvements even when applied in a delayed setting after brain injury. The observed
enhancement in behavioral recovery might have been orchestrated by the multiple effects of the
167
Gtn-HPA/SDF- 1 a-PCN matrices, some of which we have observed in our study and will discuss
below. However, the exact mechanism by which Gtn-HPA/SDF-la-PCN matrices enhanced
behavioral recovery is difficult to pinpoint, given the highly sophisticated and largely unknown
processes underlying functional behavior.
At the gross histological level, lesions resulting from ICH injury were found to present
either a cavitary or non-cavitary appearance. The non-cavitary appearance could be due to
incomplete liquefaction of the dying brain tissue, structural collapse of surrounding brain tissue
into a cavitary lesion or a combination of both. In either case, the benefit of implanting Gtn-HPAbased matrices was apparent; such matrices served as a persistent physical framework in the
injured brain and restored its stromal volume. Importantly, the matrices remained tightly
interfaced with the host brain tissue, where the absence of necrotic cells/tissues as well as the
preservation of distinct white and gray matter structures demonstrated that the matrices could
reside in the brain without causing any deleterious effect. The value of implanting matrices to
provide stroma is significant given that: 1) the mature brain typically undergoes loss of neural
tissue and its associated stroma after injury and; 2) a compensatory physical framework usually
does not materialize spontaneously or in response to stimulation by drugs/growth factors (unless
the lesion is physically connected with the meninges and can be readily infiltrated with matrixproducing fibroblasts). While this restoration of stromal volume does not necessarily equate
recovery of lost tissues, the provision of a physical framework is a pre-requisite for enabling
spontaneous or stimulated tissue remodeling and is of potential benefit to the injured brain. On
top of restoring stromal volume, Gtn-HPA/SDF-la-PCN matrices, but not Gtn-HPA ot GtnHPA/PCN matrices, were found to have the additional benefit of dampening the progressive loss
of tissue after the initial ICH injury and resulting in a reduced volume of tissue loss by the time of
our evaluation at 4 weeks after ICH. Their effects on tissue loss could have occurred through
reported neuroprotective effects of SDF-la and/or the spectrum of regenerative effects to be
discussed below.
One of our hypotheses in this study was that Gtn-HPA/SDF-la-PCN matrix, when
implanted into brain lesions,
could enhance the endogenous neurogenic response
and
neovascularization in the surrounding brain tissue. Similar to numerous reports on neurogenesis
in the injured brain, ICH triggered an endogenous neurogenic response in our study. Using DCX+
168
area as indicator for the presence of endogenous neuronal precursors [26], the endogenous
-
neurogenic response in the injured hemisphere of aCSF-treated animals was found to mount a
5-fold increase in the presence of neuronal progenitors over the baseline detected in the
contralateral hemisphere. This robust increase was similar to numerous reports on endogenous
neurogenesis in the injured brain and was likely mediated through injury-induced expression of a
plethora of mitogens such as FGF [27], SCF [28] and erythropoietin [29]. The endogenous
neurogenic response also included a second dimension where a higher proportion of DCX+
neuronal precursors exited the SVZ and migrated into the injured striatum, although the majority
of the newly generated DCX+ neuronal precursors still remained as contiguous cells in the SVZ.
Gtn-HPA, Gtn-HPA/PCN and Gtn-HPA/SDF-la-PCN matrices did not increase the total pool of
DCX+ neuronal precursors. In the case of Gtn-HPA/SDF-la-PCN matrices, this observation was
in line with the reportedly weak mitogenic effect of SDF-la [30] as well as our in vitro finding
that SDF-la did not increase the proliferation of adult neural progenitor cells beyond the level
achieved with a strong mitogen such as FGF-2 (Section 3.3.5). On the other hand, Gtn-HPA/SDFla-PCN matrices profoundly influenced the endogenous neurogenic response in terms of the
distribution of DCX+ neuronal precursors between the SVZ and striatum. In the presence of GtnHPA/SDF- 1 a-PCN matrices, a substantially higher proportion of DCX+ neuronal precursors were
dispersed over a wide area in the striatum with stereotypic migratory morphology [31], strongly
suggesting that they had been actively recruited out of the SVZ. Such a distribution shift was
absent with the implantation of Gtn-HPA and Gtn-HPA/PCN matrices. This ruled out the
possibility that the physical presence of Gtn-HPA matrices and PCNs in the striatum might
elevate endogenous expression of chemokines that could attract neuronal precursors and pointed
to SDF- 1 a PCNs as the primary driver of the observed effect.
Our observation that Gtn-HPA/SDF-la matrices induced a distribution shift of DCX+
neuronal precursors into the striatum is in accordance with numerous reports on SDF-la being
one of the key mediators for recruiting endogenous neuronal progenitors toward the lesion. Our
finding also relates particularly well with one study where intraventricular infusion of AMD3 100,
the small molecular antagonist of SDF- 1 a receptor CXCR4, was performed after ischemic stroke
injury [13]. In this event of disrupted SDF-la/CXCR4 signaling, more DCX+ neuronal precursors
stayed in the SVZ and less of them migrated into the striatum. The distribution shift of DCX+
neuronal progenitors toward the striatum has previously been achieved by other strategies. One
169
strategy involved the systemic administration of SDF- 1 P with the notion that SDF- 1 P would
permeate through the leaky blood-brain-barrier of newly formed vessels in the peri-lesion tissue
and recruit DCXI neuronal progenitors [32]. Another strategy involved the injection of
hepatocyte growth factor-loaded gelatin microspheres into the peri-lesion tissue [33]. Our study
demonstrates that using Gtn-HPA/SDF-la-PCN matrices to fill brain lesions can also be a
comparably effective strategy to shift the distribution of DCXI neuronal precursors toward the
striatum.
By far, findings from multiple studies have been strongly indicative of DCX+ neuronal
precursors being beneficial after brain injury. Their differentiation can be specific to the type of
neurons found in the striatum. The newly generated neurons can further form functional synapses
with neighboring neurons and integrate functionally into local circuit. They have also been
associated repeatedly with improvements in functional behavior. Highly specific ablation of
DCX+ neuronal precursors via transgenic manipulation is further shown to reduce spontaneous
behavioral recovery after ischemic stroke [34] and traumatic brain injury [35]. Channeling a
higher proportion of DCX+ neuronal precursors into the injured striatum with the use of GtnHPA/SDF-la matrices may therefore be a way to optimize the benefits of neurogenic responses
after every brain injury.
Despite the observed distribution shift, the presence of the DCX+ neuronal precursors in
the striatum did not extend significantly within close proximity of the implanted Gtn-HPA/SDF1 a-PCN matrices. We also found very few BrdU+/NeuN+ mature neurons in the striatum near the
SVZ and within the implanted Gtn-HPA/SDF-la-PCN matrices. Collectively, these observations
suggest that even with Gtn-HPA/SDF-la-PCN matrices to augment SDF-la release, the
endogenous neurogenic response remains constrained by several other limitations.
These
limitations may include inefficient differentiation of the DCXI neuronal progenitors into NeuNexpressing mature neurons, high death rate and limited motility within the injured brain.
Gtn-HPA/SDF-la-PCN matrices were also found to exert a prominent effect on
neovascularization around lesion. In our study, the vasculature in all the uninjured contralateral
hemispheres measured to be
-
2 mm per mm 2, which put every cell to be within 250pm from a
blood vessel and was reasonably consistent with the oxygen diffusion limit of 200gm in living
170
tissues [36]. In agreement with previous reports on injury-triggered neovascularization in the
brain, we found vessel length density to 3.4 mm per mm 2 around the lesion after ICH injury. GtnHPA/SDF-la-PCN matrices significantly amplified this injury-triggered neovascularization, the
vessel length density around the lesion further to 4.9 mm per mm 2. Gtn-HPA and Gtn-HPA/PCN
matrices did not produce such an amplificatory effect, attesting that the observed amplification of
injury-triggered neovascularization with Gtn-HPA/SDF-la-PCN matrices was primarily driven
by SDF-la PCNs and not due to the physical effect of an implanted matrix or the bioactivity of
Gtn-HPA leading to expression of angiogenic or vasculogenic factors in the peri-lesion tissue.
The ability of SDF-la PCNs to potently stimulate neovascularization in our study was likely
derived from the known effects of SDF-l a on angiogenesis and vasculogenesis. SDF-la has been
reported to boost the proliferation of endothelial cells [37], increase their survival, guide their
migration via chemoattraction [38] and regulate their ECM-dependent branching morphogenesis
[39], thus orchestrating an overall enhancement in angiogenesis. On the other hand, SDF- Ia also
actively recruits endothelial progenitor cells [40] that has been known to circulate in the
peripheral blood in greater numbers after brain injuries [41], attenuates their apoptosis [42] and
helps to drive vasculogenesis in the injured brain [43]. Neovascularization has been regarded to
be critically important in injured brain tissues. In ischemic injuries, neovascularization plays the
vital role of restoring perfusion to match physiological requirements and salvaging underperfused
brain tissues. In the non-ischemic context as was the case in the current study, neovascularization
has been postulated to serve the role of improving metabolite delivery to injured neurons [19] as
well as follow the "clean-up" hypothesis of facilitating macrophage infiltration for the removal of
necrotic brain tissue.
The above findings validate our hypotheses that implanted Gtn-HPA/SDF-la-PCN
matrices can enhance endogenous regenerative responses in the brain tissue surrounding the
lesion. A recurring theme has been that endogenous regenerative responses (along with
behavioral recovery) occurred spontaneously after brain injury but intensified/accelerated with in
the presence of Gtn-HPA/SDF-la-PCN matrices. This verifies the presumption underlying our
hypotheses that endogenous regenerative processes usually unfold in a sub-optimal manner due to
various barriers and typically fail to reach their full potential without intervention. The
implication of this is that more emphasis should be placed to identify barriers that curtail
endogenous regenerative responses and to develop tools to address these barriers as part of an
171
overall strategy to maximize the self-healing capacity of the brain. Several other plausible barriers
have already been noted over our assessment of the endogenous neurogenic responses with GtnHPA/SDF-la-PCN matrices. They include: 1) failure of endogenous neuronal precursors to give
rise to significant numbers of new mature neurons as a possible consequence of inefficient
differentiation or high death rate and; 2) failure of endogenous neuronal precursors to migrate
sufficiently far into the injured striatum prior to their disappearance. To further optimize the
endogenous neurogenic response, it is of great interest to retrofit Gtn-HPA/SDF- 1 a-PCN matrices
with additional proteins/drugs to address these deficiencies. The proteins/drugs may include: 1)
mitogens such as transforming growth factor-alpha (TGF-a) to enlarge the pool of endogenous
neuronal progenitors [44, 45]; 2) caspase inhibitors to reduce their apoptosis and increase their
survival [13] and; 3) neurotrophins such as brain-derived neutrophohic factor (BDNF) to enhance
their differentiation [46] and motility [47].
Another recurring theme is that although Gtn-HPA and Gtn-HPA/PCN matrices were on
par with Gtn-HPA/SDF- 1 a-PCN matrices in serving the role as a physical structure that
interfaced tightly with the surrounding host tissue and restored the stromal volume of the injured
brain, they did not yield any appreciable improvement in all other measured outcomes. This is a
significant departure from the experience in other tissues (e.g. skin and bone) where implanted
matrices themselves can induce appreciable regenerative effects, and is expectedly so given that
all processes are more strictly regulated in the brain. This is also in line with reports where
matrices without instructive cues remained as relatively inert physical structures in the injured
brain [48]. Instructive cues, such as SDF-la PCNs in this case, are therefore indispensable and
should be appropriately included in the design of matrices for the injured brain.
The other hypothesis of this study is that Gtn-HPA/SDF-la-PCN matrices, when
implanted into brain lesions, can re-establish a physical framework to enable recruited
endogenous cells to migrate into the brain lesion. To associate with our findings on endogenous
regenerative responses in the surrounding brain tissue, we first examined for the presence of
DCX+ neuronal precursors within the lesion/implanted matrices. In our observation, a very small
number of DCX+ neuronal pecursors successfully migrated into the implanted Gtn-HPA/SDF- IaPCN matrices when none did in the case of cavitary lesion, non-cavitary lesion as well as GtnHPA and Gtn-HPA/PCN matrices, presumably because of the absence of structure, the presence
172
of an unfavorable environment and lack of instructive cues respectively. Although this is a
promising proof-of-concept that the lesion can be restructured to promote the infiltration of
neuronal progenitors, it is clear that Gtn-HPA/SDF-la-PCN matrices must be combined with
other strategies to sufficiently amplify this migration in order to achieve meaningful repopulation
of brain lesions with endogenous neuronal precursors.
In addition to DCX+ neuronal progenitors, we observed the presence of vasculature into
brain lesions filled with Gtn-HPA/SDF-la-PCN matrices. The newly constructed vasculature was
an evident improvement over the absence of blood vessels in lesions that were cavitary in nature
or filled with Gtn-HPA and Gtn-HPA/PCN matrices. Despite being less developed than
vasculature in non-cavitary lesion in terms of vessel length density, Gtn-HPA/SDF-la-PCN
matrices were completely acellular and avascularized at the point of implantation and were
clearly engaging in an active process of vascular construction
over time. In general,
revascularization does not occur spontaneously in brain lesions (except those which are
physically connected to the meninges and can be filled with a fibrotic scar tissue after meningeal
fibroblasts intrude the lesion and secrete ECM [49]) after the clearance of necrotic tissues and the
associated blood vessels. Yet, blood vessels constitute a fundamental pre-requisite for
regeneration of neural tissue in the lesions, since the oxygen and metabolic demands of
endogenous or transplanted cells have to be met for them to survive and amount to any
regenerative effect. Blood vessels may also be necessary promoters of neuroregeneration given
their roles as conduits for blood-borne progenitors to enter the lesion and cellular scaffolds to
guide migration of neural cells [32, 50-52]. With these in mind, the use of Gtn-HPA/SDF-laPCN matrices to reconstruct vasculature of brain lesion is therefore a significant advancement
toward cell/tissue reconstitution in brain lesions. The reconstruction of vasculature in a
biomaterial-filled brain lesion has previously been demonstrated with a preformed collagen
scaffold carrying soluble Nogo receptor [53] and in unpublished work by a laboratory in Clemson
University and. The trauma-induced lesions in these contexts were either open wounds or cortical
surface wounds that remained in close contact with the meninges whose fibroblasts and
vasculature might facilitate the growth of blood vessels into the implanted biomaterial. In this
study, we demonstrated that vasculature could also be constructed in a deeply seated brain lesion
completely surrounded by neural tissues with the use of Gtn-HPA/SDF-Ia-PCN matrices.
173
One major finding from our examination of the implanted Gtn-HPA/SDF-la-PCN
matrices is the significant presence of neutrophils. As short-lived cells that last 5 - 6 days,
neutrophils typically participate a rapid inflammatory response toward any injury or introduction
of foreign material and undergo spontaneous apoptosis [54]. Their existence in the GtnHPA/SDF-la-PCN matrices 2 weeks after matrix implantation or 4 weeks after the ICH injury,
along with their absence in aCSF-treated lesions, therefore indicated the presence of an
underlying stimulus that continuously recruited neutrophils. One possible stimulus could be
persisting necrosis in the host tissue as a result of deleterious effects arising from the implanted
Gtn-HPA/SDF-la-PCN matrices. Our examination, however, revealed that the surrounding host
tissue remained tightly interfaced with the implanted matrices and showed no signs of cell/tissue
necrosis. The presence of neutrophils was also predominantly localized within the GtnHPA/SDF-la-PCN matrices instead of spreading into the surrounding host tissue. On this note,
the stimulus was likely to be PCNs given that: 1) implanted Gtn-HPA matrices in our study did
not elicit similar neutrophil response and; 2) Gtn-HPA/PCN elicited a highly similar neutrophilic
response as Gtn-HPA/SDF-la-PCN matrices. In our study, the infiltrated neutrophils were
stained positively for CXCR4 even in the absence of cell permeabilization. The CXCR4 staining
also displayed a punctate pattern around the perimeter of each positively stained cell and was
characteristic of receptors expressed on cell surface. In addition, they were vastly positive for the
angiogenic factor, VEGF.
The presence of neutrophils has been classically understood to be detrimental for the
injured brain. Upon being recruited into a site of inflammation, neutrophils release reactive
oxygen species, proteases and pro-inflammatory cytokines, all of which either adversely affect
the neural cells and blood-brain-barrier or further intensify the inflammatory process [55].
Several studies also show that neutrophil depletion after brain injury can be beneficial in terms of
reducing tissue loss [56] and behavioral impairments [57]. On the other hand, there has emerged
data that shows neutrophils to exhibit different phenotypes and functions instead of being a
homogenous population devoted only to driving deleterious acute inflammation. For instance,
neutrophils are the only cell type to secrete MMP-9 without its endogenous inhibitor, tissue
inhibitor of metalloproteinases-1 [58]. Through the use of MMP-9 to remodel ECM and release
sequestered angiogenic factors, neutrophils have been noted to mediate VEGF-induced
angiogenesis in the injured brain [59]. In another example, CDllb+/Gr-1+/CXCR4hi neutrophils
174
played the critical role of secreting MMP-9 to enable proper revascularization of transplanted
pancreatic islets in a mouse model [60]. Our study, together with several others [61, 62], also
found neutrophils to express VEGF. Such VEGF expression has been found to be vital in
orchestrating physiological angiogenesis in endometrial tissue [63], pathological angiogenesis in
tumors [64] as well as nerve regeneration in the cornea [65]. Finally, neutrophils infiltrating the
eye after optic nerve injury expressed high levels of oncomodulin and were found to represent an
essential innate immune response to reverse axonal growth inhibition in the CNS and allow
lengthy axons to regenerate through the optic nerve [66].
The observed neutrophil accumulation has several implications on the use of GtnHPA/SDF-la-PCN matrices in the injured brain. It is of interest to investigate the neutrophils as
an endogenous response that may contribute toward neuroregeneration and neovascularization,
akin to what has been observed in other studies [63, 66]. In our study, the neutrophil
accumulation alone in rats treated with Gtn-HPA/PCN matrices did not appear to bear any
positive influence on the injured brain at 2 weeks after treatment but should be monitored over a
longer period of time to fully determine the effects of the neutrophils.
It is also pertinent to
characterize the cytokine profile of the neutrophils. If the expression of pro-inflammatory
cytokines such as TNF-a and IFN-y remains persistently high, Gtn-HPA/SDF-la-PCN matrices
should be retooled to deliver SDF- Ia without the use of PCNs.
Apart from those discussed above, Gtn-HPA/SDF-la-PCN matrices do not address other
aspects of the complex environment in the injured brain by nature of their intent and design. One
such aspect is the presence of growth inhibitors such as Nogo-A, myelin-associated glycoprotein,
oligodendrocyte myelin glycoprotein, ephrin B3 and semaphorin 4D. Although not specifically
measured in this study, these growth inhibitors might be presumed to be prevalent around the
lesion given their robust production after brain injury [67]. The growth inhibitors might attribute
to the absence of axonal projections from host neurons into the implanted Gtn-HPA/SDF-la-PCN
matrices. They might also adversely affect the survival and functions of the recruited neuronal
progenitors and the newly differentiated neurons.
Gtn-HPA/SDF- 1 a matrices also do not address astrogliosis around the brain lesion. As a defense
mechanism against brain injury, astrogliosis involves massive proliferation of astrocytes followed
175
by their activation into the reactive state. The reactive astrocytes then participate in the formation
of a glial scar where their endfeet is stacked against the edge of the lesion and tightly woven with
gap and tight junctions to form a highly impermeable barrier around the lesion [68]. This cellular
barrier serves the beneficial role of reducing the spread of excitotoxicity, oxidative stress and
inflammation [69] but is inhibitory in terms of axonal regeneration [67]. In our study, GtnHPA/SDF- 1 a-PCN matrices did not intensify or dampen the presence of reactive astrocytes in the
peri-lesion tissues. Neither did Gtn-HPA/SDF-la-PCN matrices induce the migration of the
reactive astrocytes into the lesion to result in some disruption or disentanglement of the tightly
woven cellular barrier. Although migration of cells across the glial scar is not completely ruled
out (given reports of transplanted cells crossing the boundary of a chronic lesion into the host
tissue [70, 71] and our observation of a few endogenous neuronal progenitors crossing the lesion
boundary and migrating into the implanted Gtn-HPA/SDF-la-PCN matrices), it is reasonable to
expect that the glial scar is highly obstructive toward migrating cells and has to be appropriately
tackled to facilitate substantial in-growth of neural cells into the lesion.
The third aspect is the accumulation of macrophages/microglia. In the event of brain
injury, blood-borne monocytes infiltrate the lesion and differentiate into macrophages while
microglia, the resident macrophages of the brain, migrate into the lesion. Being highly
phagocytotic cells, they serve the critical role of clearing cell/tissue debris to reduce secondary
damage
and
prime
the
injured
brain
for
repair/regeneration.
Given
that
monocytes/macrophages/microglia share the SDF-la/CXCR4 signaling pathway [72, 73], GtnHPA/SDF-la-PCN matrices were poised to chemoattract them despite it not being the primary
intent of the matrices in this study. In our observations, Gtn-HPA/SDF-la-PCN matrices did not
increase the accumulation of macrophages/microglia in the peri-lesion tissues beyond the level
induced by ICH injury, presumably because the injury-induced accumulation was already
sufficiently robust. Lesions filled with Gtn-HPA/SDF-la-PCN matrices did, however, contain
more macrophages/microglia than cavitary lesions and lesions filled with Gtn-HPA matrices,
although they did not approach the level seen in the non-cavitary lesions where the necrotic-like
tissue was densely packed with macrophages/microglia.
To appreciate the significance of these
cells, the key issue, as recent studies of the role of macrophage/microglia in the CNS have
pointed out, is less about the number but more about the polarization state of these cells.
Macrophages/microglia of the MI state are pro-inflammatory and drive inflammation with their
176
secretion of TNF-a and IL- 12 while those of the M2 state are immunoregulatory or proregenerative and can secrete beneficial cytokines such as IL-10, VEGF and FGF [74]. With a
study showing effects of SDF-1 a of M2 polarization [75], it is of interest to examine in the future
if Gtn-HPA/SDF- Ia-PCN matrices, in their current form or with additional cytokines/agents, can
be a tool to drive M2 polarization of macrophages/microglia to achieve additional regenerative
effects after brain injury.
In summation, implantation of Gtn-HPA/SDF- 1 a-PCN matrices within brain lesions was
an effective strategy to increase migration of endogenous neuronal progenitors into the injured
striatum and boost neovascularization in the surrounding tissues. The use of Gtn-HPA/SDF-laPCN matrices also led to the reconstruction of vasculature within brain lesions and heavy
infiltration of neutrophils. While the newly constructed vasculature was valuable for potential
cell/tissue reconstitution of the brain lesion, the neutrophils did not seem to impose detrimental
inflammatory effects and compelled further investigation to verify their role in neuroregeneration.
Most importantly, Gtn-HPA/SDF-la-PCN matrices proved to be a tangibly useful treatment for
the injured brain in terms of reducing tissue loss and enhancing recovery in functional behaviors
that has relevance to quality of life after brain injury.
177
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differentiation and RUNX3
Chapter 6
Conclusions and Future Work
In this thesis, we developed a lesion-filling matrix to address the loss of chemokine
expression and the disappearance of stroma within brain lesions. The underlying hypotheses were
that when implanted within brain lesions, the matrix would: 1) provide chemokine release and
enhance endogenous regenerative responses in the surrounding brain tissues and; 2) re-establish a
physical framework to enable recruited endogenous cells to migrate into the brain lesions. The
developed matrix was eventually evaluated in a deep-seated brain lesion in a rat model of
collagenase-induced ICH.
Development of the matrix began with the in vitro characterization of Gtn-HPA
hydrogels. These hydrogels were selected for their unique crosslinking chemistry that allowed
them to be injectable, covalently crosslinked in situ and thus excellent candidate materials for
implantation into brain lesions. Adult neural progenitor cells (NPCs) were cultured within the
Gtn-HPA hydrogels as an approximation of the context where endogenous neural stem and
progenitor cells entered the Gtn-HPA hydrogels that were used to fill the brain lesions. In our
findings, the adult NPCs within the hydrogels retained their ability to proliferate in response to
the mitogen FGF-2, undergo migration to different positions and differentiate into neurons and
astrocytes when stimulated with the differentiation cues. The importance of proliferation,
migration and differentiation in regeneration and the demonstrated ability of Gtn-HPA hydrogels
to provide favorable environments for these processes collectively supported the use of Gtn-HPA
hydrogels as the building block for providing appropriate stroma.
The next phase
of matrix development
centered
on the
synthesis of DS/CS
polyelectrolyte complex nanoparticles (PCNs) to achieve the functionality of SDF-la release.
SDF-la was the chemokine of choice due to its widely reported involvement in driving numerous
endogenous regenerative responses. PCNs displayed great compatibility with Gtn-HPA hydrogels
and could be incorporated readily without compromising the integrity of the hydrogels. At the
DS:CS:SDF-la ratio of 1:0.33:0.2, encapsulation efficiency was over 80%, demonstrating PCNs
to be a practical delivery vehicle that minimized the loss of the costly chemokine. Loading
efficiency also reached ~ 20 wt%, indicating that the mass of PCNs needed to deliver a
185
therapeutic dose of SDF-la could remain within 5 times of the mass of SDF-la. Most
importantly, SDF- la PCNs sustained the release of SDF-1 a over 4 weeks and proved effective as
the second building block for providing chemokine release.
The resulting Gtn-HPA/SDF- 1 a-PCN matrix was evaluated in an in vitro threedimensional migration assay where it was used to fill a core region that was surrounded by a
NPC-laden hydrogel construct (akin to its eventual in vivo application of filling a brain lesion that
was surrounded by brain tissue containing endogenous cells). In this assay, Gtn-HPA/SDF- 1 aPCN matrix significantly enhanced accumulation of NPCs around the core region as well as
migration of NPCs into the core region when compared to Gtn-HPA and Gtn-HPA/PCN matrices,
therefore demonstrating its ability to achieve the objectives of providing: i) release of bioactive
SDF-la to recruit cells and; ii) appropriate stroma to support migration of recruited cells.
Specifically for the latter, Gtn-HPA/SDF-la-PCN matrix displayed the capacity to maintain
physical continuity with the apposing hydrogel construct and enabled recruited cells to
successfully migrate across the interface. Gtn-HPA/SDF-la-PCN matrix also greatly enhanced
the accumulation and migration of NPCs relative to Gtn-HPA/soluble SDF-1 a, thus proving to be
the necessary formulation for releasing SDF-la. The use of CXCR4 inhibitor and soluble SDF-la
to disrupt concentration gradients showed the Gtn-HPA/SDF-la-PCN matrix to act specifically
on the SDF- 1 a/CXCR4 pathway and to actively recruit NPCs through the chemotactic effects of
SDF- 1 a as opposed to merely increasing the chemokinesis of the surrounding NPCs. On the other
hand, the use of specific MMP inhibitors established that Gtn-HPA/SDF-la-PCN matrix
supported MMP-2 and MMP-9-mediated migration and was poised to be a highly compatible
stroma in the injured brain where endogenous cells commonly employ MMP-2 and MMP-9 to
migrate.
In the transition to in vivo experimentation, the emphasis for the developed matrix to be
injectable had proven to be particularly valuable. Gtn-HPA/SDF-la-PCN matrix was successfully
implanted into a deep-seated brain lesion in an ICH model with the use of a needle-based
injection procedure that caused minimal morbidity on the overlying cortical tissue. The in situ
crosslinking was also useful in enabling Gtn-HPA/SDF-la-PCN matrix to conform to the
irregularly shaped lesion and establish tight interface with the surrounding brain tissue. When
optimized to contain 8wt% Gtn-HPA, the lesion-filling Gtn-HPA/SDF-la-PCN matrix persisted
for at least 2 weeks after implantation, maintained the tight interface with surrounding host tissue
and was capable of support infiltration of endogenous cells.
186
In the in vivo study where our hypotheses were evaluated in a rat ICH model, GtnHPA/SDF-la-PCN matrix did not increase the overall scale of injury-induced neurogenesis as
compared to aCSF and Gtn-HPA matrix, but enhanced the response by shifting the distribution of
endogenous neuronal progenitors/immature neurons toward the injured striatum. Gtn-HPA/SDF1 a-PCN matrix also amplified injury-induced neovascularization around the lesion and led to the
construction of a new vasculature within the lesion. These verified our hypotheses that by reestablishing chemokine release and stroma within the lesion, the lesion-filling matrix could
enhance endogenous regenerative responses and enable migration of endogenous cells into the
lesion. Interestingly, the endogenous cells in Gtn-HPA/SDF-la-PCN matrix also included a
sizeable population of neutrophils. If found to have similar angiogenic roles as neutrophils in
other tissues, they could represent a novel endogenous response that would be useful for
reconstructing vasculature in matrix-filled brain lesions. Amidst these significant effects, GtnHPA/SDF-la-PCN matrix did not have any influence on the number of newly generated mature
neurons
in
the
striatum,
macrophages/microglia.
the
Finally,
injury-induced
gliosis
Gtn-HPA/SDF-la-PCN
and
accumulation
matrix
resulted
of
in
activated
behavioral
improvements and reduced tissue loss. This marked Gtn-HPA/SDF-la-PCN matrix as a
potentially beneficial treatment for the injured brain after ICH injury.
Overall, this thesis has followed the development of an injectable brain lesion-filling
matrix, the in vitro demonstration of its capability to provide chemokine release and stromal
support and the in vivo validation of its ability to enhance endogenous regenerative responses.
Even with these advancements, the work on developing highly effective lesion-filling matrices,
maximizing the potential of endogenous regenerative responses and inducing neuroregeneration
in general in the injured brain is clearly far from over. The course of pursuing this thesis has
revealed several specific directions that may be considered in the future.
187
Future work related to the development and use of lesion-filling matrices
*
The ideal brain lesion-filling matrix is expected to be macroscopically persistent to prevent
collapse of the surrounding brain tissue, stably anchor delivery vehicles
(such as
nanoparticles) and importantly, remain as a physical framework until new stroma is
produced. It is also expected to permit microscopic cell-mediated degradation that usually
precedes cell migration. For a homogeneous material such as Gtn-HPA/SDF-la-PCN matrix,
these requirements
create a sandwiching constraint where only a narrow range of
formulations may be applicable. This constraint may be removed if the modular design
approach extends to different roles of the lesion-filling matrix i.e. developing a heterogeneous
matrix where different components
address the macroscopic and microscopic
roles
separately. Gtn-HPA hydrogels may be employed with injectable macroscopic structures such
as cryogels [1] or short electrospun fibers [2] to achieve the desirable scenario where GtnHPA hydrogels provides the modality to conform to irregularly shaped lesion and establish
tight interface with surrounding brain tissue while the macroscopic structures provide the
macroscopic persistence.
-
The volume and location of lesions are consistent in the collagenase-induced induced model
and allow for the injection of Gtn-HPA/SDF-la-PCN matrix to be sufficiently guided by
previous histological findings and the stereotaxic coordinates of the prior collagenase
injection. For other forms of brain injury (e.g. ischemic strokes in a sub-cortical region)
where the lesion size and location are relatively more variable, an imaging modality such as
magnetic resonance imaging (MRI) will be necessary to ensure an appropriate volume of
matrix and accurate deposition of matrix within the lesion.
-
For greater relevance to current clinical management of ICH, it is pertinent to combine the
implantation of Gtn-HPA/SDF-la-PCN matrix with a preceding procedure to aspirate the
hematoma. The aspiration of hematoma can be performed with or without clot-lysing drugs
such as streptokinase [3, 4] and can enable Gtn-HPA/SDF-la-PCN matrix to be implanted
acutely after the ICH injury.
188
Future work related to maximizing endogenous regenerative responses in the injured brain
-
The number, motility, differentiation and survival of neuronal progenitors and immature
neurons are other aspects of injury-induced neurogenesis that need to be dealt with. As
mentioned previously, Gtn-HPA/SDF-la-PCN matrix can be further exploited to deliver
potent mitogens (e.g. EGF, EPO and TGF-a), motility-enhancing factors (e.g. BDNF [5]),
differentiating cues (ephrin-B2 [6] and retinoic acid [7]) and pro-survival factors (e.g. [8]) to
increase the robustness of injury-induced neurogenesis.
e
The presence of neutrophils within the implanted Gtn-HPA/SDF-la-PCN matrix compels
further investigation. First, neutrophil depletion should be performed to examine if the
infiltrating neutrophils contribute to any enhancements in endogenous regenerative responses
in the surrounding brain tissue. Second, the implanted Gtn-HPA/SDF-la-PCN matrix can be
retrieved and digested to isolate the neutrophil infiltrates and analyze their cytokine
expression profiles using RT-PCR and FACS. This will determine whether the neutrophils
display a pro-inflammatory or pro-regenerative phenotype. Third, the Gtn-HPA/SDF- 1 a-PCN
matrix should be evaluated at a later time point (e.g. 4 and 6 weeks) to examine if the
presence of neutrophil is transient or persistent. These future experiments will elucidate the
roles and consequences of the neutrophil infiltrates and clarify whether the neutrophil
infiltrate should be pursued as another beneficial endogenous regenerative response.
189
Future work related to inducing neuroregeneration in the injured brain
*
Along the course of characterizing Gtn-HPA hydrogels as an appropriate matrix for adult
neural progenitors, Gtn-HPA hydrogels were found to increase the oxidative stress resistance
of encapsulated neural progenitors. Further investigation is needed to determine the exact
pathway/defense mechanism involved in the heightened oxidative stress resistance, and to
verify if gene and protein expression profiles of the encapsulated neural progenitors remain
comparable to their native profiles.
*
Gtn-HPA hydrogels exhibit high cytocompatibility with neural progenitors and are appealing
injectable matrices that can be implanted into brain lesions in a minimally invasive manner. It
is of interest to employ Gtn-HPA hydrogels as a carrier matrix for transplanting neural
progenitors into brain lesions. Investigation should focus on whether the Gtn-HPA hydrogels
provide a supporting framework for the transplanted neural progenitors to occupy the lesion
and if they increase the survival of the transplanted cells, either by preventing anoikis or by
boosting their resistance against the highly oxidative environment in the injured brain.
190
I
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