5-6 September 2014
The 16th Spinal Research Network Meeting
ABSTRACTS
ABSTRACTS
Speakers’ abstracts appear in presentation order, followed by poster
abstracts in alphabetical order
POSTER PRESENTATIONS
Poster session is scheduled from 5pm at the end of the first day,
immediately after the main meeting, on Friday, 5th September. The
posters are also available to view during the coffee and lunch breaks
on Friday and Saturday.
SCIENTIFIC ORGANIZING COMMITTEE
Professor Susan Barnett BSc PhD FSB
University of Glasgow
Professor James Fawcett PhD FRCP
University of Cambridge
Professor Stephen B. McMahon PhD, FMedSci
King's College, London
Professor Wolfram Tetzlaff MD DrMed PhD
University of British Columbia
Professor Joost Verhaagen PhD
Netherlands Institute for Neuroscience
Table of Contents
Presentation Abstracts
Friday
………………………………………………………………………… 3
Saturday………………………………………………………………….13
Poster Abstracts…………………………………………………………...26
Delegate list………………………………………………………………..67
Table of Contents – Presentation Abstracts
5th September 2014
Mechanisms of intermittent hypoxia-induced functional recovery after spinal cord injury
Gordon S. Mitchell ........................................................................................................................................ 3
Diaphragm pacing: early utilization to decrease invasive mechanical ventilation and optimize functional
respiratory recovery after spinal cord injury
Raymond P. Onders ...................................................................................................................................... 4
Enhancing respiratory plasticity following cervical SCI
Michael Lane ................................................................................................................................................. 5
Restoring bladder function
Graham Creasey ............................................................................................................................................ 6
Promoting regeneration with electrical stimulation
Thomas M. Brushart ..................................................................................................................................... 7
Autonomic control and sport performance in Paralympic athletes with spinal cord injury: What we
learned from London 2012 Games?
Andrei Krassioukov ....................................................................................................................................... 8
Pharmacological management of autonomic dysreflexia: Effects on intraspinal plasticity and
inflammation after complete spinal cord injury
Alexander G. Rabchevsky .............................................................................................................................. 9
Regulation of autonomic control of bladder voiding after a complete spinal cord injury
Parag Gad .................................................................................................................................................... 10
Infections as ‘outcome modifying riskfactor’ after spinal cord injury (SCI) – phenotype and underlying
mechanisms: a bed-side to bench approach
Jan M. Schwab ............................................................................................................................................ 11
Promoting neuroplasticity after spinal cord injury by over-expressing polysialic acid
Louise Adams .............................................................................................................................................. 12
1
6th September 2014
Recent progress with a high-performance brain-computer interface
Andrew Schwartz ........................................................................................................................................ 13
Improvements in nerve-electrode integration for closed-loop control of neuroprostheses
Daniel Chew ................................................................................................................................................ 14
Brain machine interface controlled functional electrical stimulation therapy
Milos R. Popovic .......................................................................................................................................... 15
Systemic administration of Epothilone B promotes axon regeneration and functional recovery after
spinal cord injury
Frank Bradke ............................................................................................................................................... 16
Successful spinal cord regeneration in the zebrafish
Catherina G. Becker .................................................................................................................................... 17
Visualization of neuronal networks in the mouse brain, spinal cord and mouse embryos by
ultramicroscopy
Hans-Ulrich Dodt ......................................................................................................................................... 18
A transgenic approach to permanently labeling stressed or damaged neurons
Matt S. Ramer ............................................................................................................................................. 19
Transplantation of corticospinal motor neurons derived from human iPS to repair spinal cord cervical
injuries
Giles W. Plant .............................................................................................................................................. 20
Intravenous multipotent adult progenitor cell treatment for acute spinal cord injury: promoting
recovery through immune modulation
Sarah A. Busch............................................................................................................................................. 21
Role of endogenous neural stem cells in spinal cord injury
Moa Stenudd............................................................................................................................................... 22
Inclusive SCI clinical trials: Predicting homogeneous trial participants and modeling outcome measures
for incomplete SCI participants
John D. Steeves ........................................................................................................................................... 23
The SCIentinel study - prospective multicenter study to define the spinal cord injury-induced immune
depression syndrome (SCI-IDS)': aiming at protection of the endogenous recovery potential after SCI
Marcel A. Kopp............................................................................................................................................ 24
Cell transplantations for SCI – will we really need them?
Sue Barnett , Frank Bradke, Armin Blesch, Simone Di Giovanni, Karim Fouad, James Guest,
Dana McTigue, Adina Michael-Titus, Giles Plant , Phil Popovich, Jerry Silver, Wolfram Tetzlaff ........... 25
2
Day 1
Friday, 5th September 2014
Session I: RESPIRATION AND SCI
Chair: Professor Joost Verhaagen
Mechanisms of intermittent hypoxia-induced functional recovery after spinal cord injury
Gordon S. Mitchell
Department of Comparative Biosciences, University of Wisconsin - Madison, Madison, USA
After December, 2014: McNight Brain Institute and Physical Therapy, University of Florida, Gainesville, Florida, USA
Mitchell@vetmed.wisc.edu
Spinal plasticity is essential for even limited spontaneous functional recovery of respiratory and
non-respiratory motor function after spinal cord injury. Strategies that induce spinal motor plasticity have
considerable potential to further improve function after incomplete spinal injuries. An important model of
spinal motor plasticity that has guided us in our development of new strategies to treat chronic spinal
injury is phrenic long-term facilitation, a long-lasting increase in phrenic motor induced by exposure to
modest protocols of acute intermittent hypoxia (AIH). Preconditioning with repetitive AIH enhances
subsequent expression of AIH-induced phrenic long-term facilitation, demonstrating that meta-plasticity
amplifies its functional effect (and that repetitive AIH may amplify therapeutic benefits). We have identified
multiple, distinct cellular cascades, each capable of giving rise to similar long-lasting phrenic motor
facilitation in different circumstances. Two important pathways are initiated and orchestrated by spinal Gprotein coupled metabotropic receptors activated during hypoxic episodes, including serotonin 2 and
adenosine 2A receptors; these receptors, in turn, differentially up-regulate BDNF or TrkB function in the
phrenic motor nucleus, leading to amplification of phrenic motor output. AIH-induced respiratory motor
plasticity can be harnessed to improve respiratory function in rodent models of clinical disorders that
cause severe respiratory insufficiency, including cervical spinal injury and motor neuron disease (ALS).
However, we have also come to realize that AIH and AIH pre-conditioning elicit similar mechanisms in
non-respiratory motor nuclei, improving limb/leg function after incomplete chronic spinal injuries in both
rodent models and humans with chronic, incomplete spinal injuries. Intermittent hypoxia induced spinal
motor plasticity may be a general feature of motor systems, reflecting an evolutionary coupling of hypoxia,
breathing and movement (swimming) in aquatic vertebrates. We continue progress towards an
understanding of cellular mechanisms giving rise to intermittent hypoxia induced motor plasticity, factors
that amplify or constrain this plasticity, its biological significance, and its clinical application. Although
research on intermittent hypoxia induced motor plasticity is still in its infancy, progress has been rapid,
and there is considerable promise that it will lead to novel, safe and effective therapeutic approaches to
treat devastating clinical disorders that compromise respiratory and non-respiratory motor function,
particularly chronic, incomplete spinal injury.
References
Dale, E.A., F. Ben Mabrouk and G.S. Mitchell (2014). Unexpected benefits of intermittent hypoxia: enhanced respiratory and nonrespiratory motor function. Physiology 29: 39-48.
Devinney, M.J., A.G. Huxtable, N.L.Nichols and G.S. Mitchell (2013). Hypoxia-induced phrenic long-term facilitation: emergent
properties. Ann. N.Y. Acad. Sci. 1279:143-53.
Lovett-Barr, M.R.*, I. Satriotomo*, G. Muir*, J.E.R. Wilkerson, M.S. Hoffman and G.S. Mitchell (2012). Repetitive intermittent hypoxia
induces respiratory and somatic motor recovery following chronic cervical spinal injury. J. Neuroscience. 32: 3591-3600.
Nichols, N.L., G. Gowing, I. Satriotomo, L.J. Nashold, E.A. Dale, M. Suzuki, P. Avalos, P. Mulcrone, J. McHugh, C.N. Svendsen and
G.S. Mitchell (2013). Intermittent hypoxia and stem cell implants preserve breathing capacity in a rat model of ALS. Am. J. Resp.
Crit. Care Med. 187(5): 535-42.
Hayes, H.B., A. Jayaraman, A., M. Herrmann, G.S. Mitchell, W.Z. Rymer and R.D. Trumbower (2014). Daily intermittent hypoxia
enhances walking after chronic spinal cord injury: a randomized trial. Neurology 82: 104-13.
Supported by NHLBI R3769064, 080209 and 111598; and DoD CDMRP SC090355, SC120226 and SC130298.
3
Diaphragm pacing: early utilization to decrease invasive mechanical ventilation and
optimize functional respiratory recovery after spinal cord injury
Raymond P. Onders
Department of Surgery, University Hospitals Case Medical Center, Cleveland, Ohio, USA
Raymond.onders@uhhospitals.org
High cervical tetraplegics with intact phrenic nerves can be removed from mechanical ventilation
with diaphragm pacing (DP). Being removed from tracheostomy mechanical ventilation has been shown
to decrease pneumonia rates, decrease secretions, decrease cost of care and improve multiple aspects
of quality of life. The long term benefits of DP have shown that it is durable and can be used to maintain
natural negative pressure ventilation. There are no significant adverse effects of early implantation or long
term use of DP. Recent reports have shown that early use of DP, within days and weeks, after the initial
trauma, will allow more rapid weaning from ventilators. Up to 36% of patients implanted early can have
recovery of respiration and removal of the implanted electrodes. Control of respiration is incompletely
understood, but utilizing the therapeutically implanted electrodes to analyze diaphragm electromyographic
activity(dEMG) has allowed a greater understanding of what is occurring to the respiratory system after
SCI injury in humans.
Functional electrical stimulation has been shown to have neuroplasticity effects. There is growing
evidence in compensatory plasticity of the respiratory system. Recent reports of DP in amyotrophic lateral
sclerosis have shown not only improvement in maintaining ventilation but improvement in the central
control of the phrenic motor neurons. Mechanical ventilation rapidly causes atrophy of the diaphragm
converting Type I muscle fibers to the less functional glycolytic fast fatigable Type IIb muscle fibers. DP
converts the entire diaphragm to Type I muscle fibers with subsequent changes to the involved motor
neurons. So not only will early use of DP after SCI decrease atrophy of the diaphragm muscle and
decrease pneumonia, but it will help in recovery of respiration. This presentation will outline how DP can
have the maximum effect with early use in SCI and should become a primary intervention in the intensive
care units treating these patients acutely.
References
Posluszny JA, Onders R, Kerwin AJ, Weinstein MS, Stein DM, Knight J, Lottenberg L, Cheatham ML, Khansarinia S, Dayal S,
Byeno PM. Multicenter Review of Diaphragm Pacing in Spinal Cord Injury: Successful not only in weaning from ventilators but also
in bridging to independent respiration. J Trauma Acute Care Surg 2014;76:303-310.
Onders R, Elmo MJ, Kaplan C, Katirji B, Schilz R. Extended Use of Diaphragm Pacing in Patients with Unilateral of Bilateral
Diaphragm Dysfunction: A New Therapeutic Option. Accepted for Surgery 2014
Onders R, Elmo MJ, Kaplan C, Katirji B, Schilz R. Identification of Unexpected Respiratory Abnormalities in Patients with
Amyotrophic Lateral Sclerosis through Electromyographic Analysis Using Intramuscular Electrodes Implanted for Therapeutic
Diaphragmatic Pacing. Accepted for American Journal
Onders RP. Functional Electrical Stimulation: Restoration of Respiratory Function. Handbook Clinical Neurol. 2012;109:275-82
4
Enhancing respiratory plasticity following cervical SCI
Michael Lane
Department of Neurobiology, Drexel University College of Medicine, PA, USA
mlane.neuro@gmail.com
Impaired breathing is a devastating consequence of cervical spinal cord injury (SCI), representing
a significant burden to injured people and increasing the risk of mortality. While there is mounting
evidence for spontaneous improvements in respiration, the extent of recovery – or functional plasticity –
remains limited. Elucidating the mechanisms of respiratory plasticity and enhancing functional recovery
after cervical SCI is becoming an important experimental and clinical goal. Many of the anatomical and
functional changes contributing to post-injury respiratory plasticity remain a subject of ongoing
investigation. The results from experimental studies by our research team and others, however, suggests
that spinal interneurons may contribute to spontaneous diaphragm recovery and represent a therapeutic
target for enhancing functional improvement following SCI.
Our ongoing research aims to improve our understanding of spinal and supraspinal changes that
may influence respiration following cervical SCI, and assess whether therapeutic strategies can harness
ongoing neuroplastic changes to improve function post-injury. The primary goal of the present work is to
test whether transplantation of neural precursor cells can restore anatomical continuity, contribute to
formation of novel interneuronal relays, and enhance diaphragm recovery. Adult, female Sprague Dawley
rats receive lateralized C3/4 contusion injuries were allowed to recover for 1 week. At that time, the injury
site is re-exposed and allogeneic donor tissues obtained from fetal spinal cord (E13.5) were transplanted
directly into the lesion epicenter. Ventilatory patterns were assessed using whole-body plethysmography
weekly pre- and post-injury for 4 weeks. Transneuronal tracing with pseudorabies virus was then used to
examine the extent of synaptic integration between host and donor neurons, and between transplanted
cells and host phrenic circuitry. One month post-transplantation, terminal neurophysiological studies were
used to assess diaphragm activity or phrenic motor output, and record multiunit activity from transplanted
neurons.
These experiments have revealed that transplanted neural precursor cells survive, proliferate and
become integrated with host phrenic circuitry ipsilateral to injury. Host neurons also become integrated
with donor cells. Furthermore, terminal electrophysiology has shown improvement in diaphragm function
in transplant recipients. Multiunit recordings made from within transplanted tissue have revealed phasic
patterns of activity consistent with inspiration. These results suggest that transplantation of neural
progenitor tissue from the developing spinal cord may contribute to an interneuronal relay capable of
improving diaphragm recovery following cervical SCI.
5
Session II: ELECTRICAL STIMULATION AND SCI
Chair: Professor Robin Franklin
Restoring bladder function
Graham Creasey
Department of Neurosurgery, Stanford University, California
gcreasey@stanford.edu
The essential functions of the bladder are storage of urine at safe pressures for convenient
intervals, and nearly complete emptying of urine at safe pressures when desired. The majority of methods
of managing the bladder after spinal cord injury restore one but not both of these functions. Restoring
these functions fully requires methods of dealing with both paralysis and hyper-reflexia of the bladder and
sphincter muscles.
Voiding of the paralyzed bladder can be restored by intermittent electrical stimulation of the sacral
preganglionic parasympathetic neurons, provided these are still intact, to produce bladder contraction.
Although this stimulation by the Finetech-Brindley Bladder Controller also directly causes intermittent
contraction of the sphincter, urine can be effectively voided in a pattern of post-stimulus voiding.
Implantation of this stimulator has usually been combined with cutting the sacral sensory roots to
reduce hyper-reflexia of the bladder and restore storage of urine at safe pressures. This posterior
rhizotomy has the added benefits of reducing hyper-reflexia of the sphincter, which improves voiding, and
reducing autonomic dysreflexia originating from the bladder and bowel. However, it also abolishes reflex
erection and reflex ejaculation, and although erection and ejaculation can be restored by other methods
this has limited acceptance of the technique.
It is possible that neuromodulation may be able to inhibit hyper-reflexia of the bladder sufficiently
to restore storage of urine at safe pressures for convenient intervals. It is also possible that electrical
block may be able to reduce hyper-reflexia of the sphincter sufficiently to restore voiding of urine at safe
pressures when desired. It has been shown that conduction of action potentials can be blocked rapidly
and reversibly, and probably safely, by application of high frequency stimuli to nerves. This has been
used to prevent sphincter contraction during electrically stimulated voiding in animals with chronic spinal
cord injury. We are now starting a clinical trial to test the feasibility and efficacy in human subjects of
restoring both continence and voiding by electrical stimulation without posterior rhizotomy.
References
Neuromodulation through sacral nerve roots 2 to 4 with a Finetech-Brindley sacral posterior and anterior root stimulator. Kirkham
AP, Knight SL, Craggs MD, Casey AT, Shah PJ. Spinal Cord 40(6):272-81, 2002
High frequency sacral root nerve block allows bladder voiding. Boger AS, Bhadra N, Gustafson KJ. Neurourol Urodyn. 31(5):677-82,
2012
Reversible nerve conduction block using kilohertz frequency alternating current. Kilgore KL, Bhadra N. Neuromodulation 17(3):24254, 2014
Supported by US Department of Veterans Affairs, US Department of Defense and National Institutes of Health
6
Promoting regeneration with electrical stimulation
Thomas M. Brushart
Department of Orthopaedics, Johns Hopkins University, Baltimore Maryland, USA
tbrusha@jhmi.edu
Electrical stimulation has been used for over a century in attempts to promote neural
regeneration. In 2000 the Brushart and Gordon laboratories began a systematic evaluation of electrical
stimulation at the time of peripheral nerve repair. Experiments were performed in the rat femoral nerve
model. Proximally, at the site of transection and repair, sensory and motor axons intermingle. Distally, the
nerve bifurcates into the saphenous (cutaneous) nerve and the motor branch to the quadriceps muscle.
We found that one hour of 20 Hz stimulation at the time of nerve repair nearly doubled the number of
motoneurons projecting axons correctly to the muscle branch 3 weeks later. Subsequent investigations
revealed that the stimulation effect was mediated by the neuron and not the peripheral pathway, and that
it involved enhanced promotion of motor axons across the repair site rather than increased regeneration
speed in the distal pathway. Stimulation without axotomy did not serve as a conditioning lesion as
determined by radiotracer transport after nerve crush or retrograde labeling after subsequent nerve
transection and repair.
Examination of the neuronal consequences of stimulation revealed accelerated and enhanced
upregulation of BDNF and its receptor TrkB, GAP-43, and T1 tubulin. Expression of the motor-specific
HNK-1 carbohydrate epitope was enhanced in the femoral muscle branch after stimulation; this could be
a secondary consequence of promoting motor axons into the motor pathway rather than a direct effect of
stimulation on Schwann cells.
Clinically, electrical stimulation at the time of carpal tunnel decompression was found to enhance
the electrophysiologic parameters of regeneration without affecting outcome. Potential challenges to the
clinical use of stimulation as an adjunct to nerve repair include the interval between injury and
repair/stimulation and the dramatic difference in regeneration distance between the rodent and man.
References
Brushart, TM. Nerve Repair. Oxford University Press, 2012
Brushart lab supported by NIH RO1 NS034484
7
Session III: REHAB AND AUTONOMIC FUNCTION
Chair: Professor Stephen McMahon
Autonomic control and sport performance in Paralympic athletes with spinal cord injury:
What we learned from London 2012 Games?
Andrei Krassioukov
ICORD; University of British Columbia; Vancouver, BC, Canada
krassioukov@icord.org
The field of autonomic function and exercise performance in athletes with spinal cord injury (SCI)
is still in its infancy. For individuals with SCI who partake in competitive sport, the cardiovascular and
autonomic consequences of SCI, such as blood pressure, heart rate, and temperature dysregulation are
of critical importance for athletic performance[4]. For example, it is well known that cervical or highthoracic SCI is associated with life-long abnormalities in systemic arterial pressure control, whereby
resting arterial pressure is lower than that of individuals with mid-to-low thoracic injuries or uninjured
controls[6]. Further, high level SCI is commonly accompanied by persistent orthostatic intolerance[2, 3],
along with transient episodes of hypertension, known as ‘autonomic dysreflexia’, which are often
accompanied by disturbances in heart rate and rhythm[5]. Recent evidence suggests that autonomic
completeness of injury, that is the degree of disruption to the descending spinal autonomic pathways, also
plays a critical role in resting cardiovascular control, whereby those with an autonomic complete injuries
exhibit the most severe disruption in resting cardiovascular function independent of injury level[7].
Athletes with SCI have also been documented to self-induce autonomic dysreflexia prior to competition
with a view of increasing blood pressure and improving their performance, a technique known as
‘boosting’[1]. For health safety reasons, boosting is officially banned by the International Paralympics
Committee. The purpose of this chapter is to review the present literature available on the complex
relationship between autonomic function and aerobic exercise performance/exercise capacity in athletes
with SCI, specifically focusing on the cardiovascular response to exercise.
We should recognize that exercise performance in individuals with SCI is an extremely complex
issue and multiple additional factors should also be considered. The unique cardio-autonomic profiles of
athletes with SCI present a variety of challenges to coaches, medical practitioners, classifiers and the
athletes themselves. Despite a growing body of literature demonstrating the importance of autonomic
integrity on athletic performance no sporting classification currently accounts for between-athlete
differences in autonomic function. Such an addition would provide a valuable opportunity for collaboration
between athletes, scientists, clinicians and governing bodies with the overall goal to level the sporting
playing field and ensure athletes with a similar degree of cardio-autonomic dysfunction can compete fairly
against one another.
References
1. Blauwet CA, Benjamin-Laing H, Stomphorst J, Van d, V, Pit-Grosheide P, Willick SE. Testing for boosting at the Paralympic
games: policies, results and future directions. Br.J.Sports Med. 2013; 47:832-837
2. Claydon VE, Krassioukov AV. Orthostatic hypotension and autonomic pathways after spinal cord injury. J Neurotrauma 2006;
23:1713-1725
3. Illman A, Stiller K, Williams M. The prevalence of orthostatic hypotension during physiotherapy treatment in patients with an acute
spinal cord injury. Spinal Cord 2000; 38:741-747
4. Mills PB, Krassioukov A. Autonomic function as a missing piece of the classification of paralympic athletes with spinal cord injury.
Spinal Cord. 2011; 49:768-776
5. Wan D, Krassioukov AV. Life-threatening outcomes associated with autonomic dysreflexia: a clinical review. J.Spinal Cord.Med.
2014; 37:2-10
6. West CR, Mills P, Krassioukov AV. Influence of the neurological level of spinal cord injury on cardiovascular outcomes in humans:
a meta-analysis. Spinal Cord. 2012; 50:484-492
7. West CR, Romer LM, Krassioukov A. Autonomic function and exercise performance in elite athletes with cervical spinal cord
injury. Med.Sci.Sports Exerc. 2013; 45:261-267
Supported by CIHR; C. Nielsen Foundation; Rich Hansen Institute
8
Pharmacological management of autonomic dysreflexia: Effects on intraspinal plasticity
and inflammation after complete spinal cord injury
Alexander G. Rabchevsky
Department of Physiology and Spinal Cord & Brain Injury Research Center (SCoBIRC), University of Kentucky,
Lexington, KY USA
AGRab@uky.edu
Autonomic dysreflexia (AD) is an abnormal hypertensive syndrome that affects the majority of
individuals who have a spinal cord injury (SCI) above the sixth thoracic (T6) level. It is typically elicited by
unperceived noxious somatosensory or visceral stimuli below the injury level which then activate
uninhibited spinal sympathetic neurons to elicit widespread vasoconstriction that rapidly elevates blood
pressure creating a potential life-threatening syndrome. Importantly, AD events often occur on a daily
basis because full bladders and constipated bowels are common triggers of this aberrant viscero-spinalsympathetic reflex after SCI. Remarkably, there is currently no prophylactic treatment to prevent the onset
of AD in susceptible individuals, only anti-hypertensive agents to relieve the cardiovascular crises, but
which do not target the causative factor(s). In our search for palliative therapeutics, we first documented
post-traumatic maladaptive intraspinal plasticity of nociceptive afferent fibers and subsequent activation
and sprouting of ascending propriospinal neuron pathways that potentiate uninhibited sympathetic
preganglionic neuron responses (i.e., hypertension) to noxious colorectal distension (CRD) (Cameron,
Smith et al. 2006; Hou, Duale et al. 2008; Hou, Duale et al. 2009). Subsequently, employing blood
pressure telemetry we found that the neuropathic pain medication, gabapentin (GBP), reduces the
incidence of spontaneous AD (sAD) when given once daily and significantly diminishes the severity of
CRD-induced AD (iAD) when given acutely (Rabchevsky, Patel et al. 2011; Rabchevsky, Patel et al.
2012). Since acute GBP treatment effectively reduces the severity of AD, likely by impeding excitatory
neurotransmission, we have begun investigating whether it abrogates the expression of sprouting
transcription factors and inflammatory cytokines in dorsal root ganglia (DRG) and spinal cord that
contribute to maladaptive plasticity underlying the temporal development of AD. We have preliminary
evidence that prolonged repeated iAD is correlated with increased expression of inflammation/sprouting
genes (e.g. ATF-3/IL-1b) in DRGs, their corresponding spinal cord segments, and also visceral organs
they innervate compared to SCI alone. Moreover, such elevated gene expression profiles in central and
peripheral tissues were partially reversed by a single acute GBP administration that effectively mitigated
repeated iAD. Accordingly, ongoing studies are testing whether continuous prophylactic GBP treatment
beginning after complete T4 SCI will prevent the development of sAD by abrogating maladaptive plasticity
as measured by 1) sprouting/synaptogenesis of primary afferent and ascending propriospinal fibers and
2) attenuation of inflammatory cytokine and sprouting transcription factor expression in the spinal cord
and DRG.
References
1. Cameron, A.A., Smith, G.M., Randall, D.C., Brown, D.R. & Rabchevsky, A.G. Genetic manipulation of intraspinal plasticity after
spinal cord injury alters the severity of autonomic dysreflexia. J Neurosci 26, 2923-2932 (2006).
2. Hou, S., et al. Plasticity of lumbosacral propriospinal neurons is associated with the development of autonomic dysreflexia after
thoracic spinal cord transection. J Comp Neurol 509, 382-399 (2008).
3. Hou, S., Duale, H. & Rabchevsky, A.G. Intraspinal sprouting of unmyelinated pelvic afferents after complete spinal cord injury is
correlated with autonomic dysreflexia induced by visceral pain. Neuroscience 159, 369-379 (2009).
4. Rabchevsky, A.G., et al. Gabapentin for spasticity and autonomic dysreflexia after severe spinal cord injury. Spinal cord 49, 99105 (2011).
5. Rabchevsky, A.G., et al. Effects of gabapentin on muscle spasticity and both induced as well as spontaneous autonomic
dysreflexia after complete spinal cord injury. Front Physiology 3, 329 (2012).
Supported by Kentucky Spinal Cord and Head Injury Research Trust (KSCHIRT), NIH/NINDS R01NS049901; 2P30NS051220
9
Regulation of autonomic control of bladder voiding after a complete spinal cord injury
Parag Gad
Terasaki Life Sciences Building, UCLA, Los Angeles, CA, USA
paraggad@gmail.com
The inability to control bladder emptying after a complete spinal cord injury is one of the most
dangerous functional deficits that occurs after complete paralysis. Maintenance of bladder health and
avoiding retro-flow of urine to the kidneys and urinary tract infections are of utmost importance. The
inability to hold urine (incontinence) and to void when desired is extremely socially humiliating. Having
demonstrated that electrodes placed epidurally on the dorsum of the spinal cord can be used in animals
and humans to recover postural and locomotor function after complete paralysis, we hypothesized that a
similar approach could be used to recover bladder function after paralysis. Also knowing that posture and
locomotion can be initiated immediately with a specific frequency-dependent stimulation pattern and that
with repeated stimulation-training sessions these functions can improve even further, we reasoned that
the same two strategies could be used to regain bladder function. Herein, we show that both of these
mechanisms. First, we have observed that daily, chronic stimulation leads to spontaneously improved
bladder function over a period of a few weeks even without the presence of stimulation. Secondly
immediate voiding can be initiated and completed with seconds of the onset of a specific pattern of
stimulation in awake, completely paralyzed rats. The clinical implications of these results are substantial
in that this intervention has the potential to have significant impact on the quality of life and longevity of
patients, while simultaneously reducing ongoing health maintenance after a spinal cord injury.
References
Edgerton et al., 2001, Courtine et al.. 2009, Harkema et al.,2011,Angeli et al., 2014
This research was supported by the National Institute of Biomedical Imaging and Bioengineering (NIBIB) R01EB007615, the
National Institute of Health (NIH) R01NS062009, Christopher and Dana Reeve Foundation, the Walkabout Foundation, the Russian
Foundation for Basic Research Grants 13-04-0109a, 13-04-01091 and 13-0412030 ofi-m. The work is performed according to the
Russian Government Program of Competitive Growth of Kazan Federal University.
10
Infections as ‘outcome modifying riskfactor’ after spinal cord injury (SCI) – phenotype
and underlying mechanisms: a bed-side to bench approach
a
a
a
a
b
c
c
a
M.A. Kopp , B. Brommer , H. Prüss , V. Failli , T. Liebscher , M.J. DeVivo , Y. Chen , Meisel , U.,
a
a,b
Dirnagl , Jan M. Schwab
a
Department of Neurology and Experimental Neurology, Clinical and Experimental Spinal Cord Injury Research
b
(Neuroparaplegiology), Charite Universitaetsmedizin Berlin, D-10117 Berlin, Germany, Spinal Cord Injury Center,
c
Trauma Hospital Berlin, D-12683 Berlin, Germany, NSCISC, University of Alabama, AL 35233, USA
jan.schwab@charite.de
Spinal cord injury (SCI) causes a systemic immunodeficiency, which increases mortality and
worsens outcome by increasing the susceptibility to infections, such as pneumonia and sepsis. It is
known that immunosuppression is differentially modulated by lesion localization, suggesting a critical role
for central and peripheral neuronal circuits as well as of immune organ innervation. SCI induces a
disturbance of the normally well-balanced interplay between the immune system and the CNS (SCIinduced immunodeficiency syndrome, SCI-IDS). We hypothesized that infections operate as disease
modifying factors after SCI in human. In order to address this we investigated the association of infections
(i.e. pneumonia and/or postoperative wound infections) with functional neurological outcome after acute
severe traumatic spinal cord injury.
We screened data sets of 24 762 patients enrolled in a prospective cohort study (National Spinal
Cord Injury Database, Birmingham, AL, USA). The group with pneumonia and/or postoperative wound
infections (n = 855) revealed significantly less ASIA impairment scale upward conversions after 1 year
than the control group (n = 855). ASIA motor score gain [median (interquartile range)] was significantly
lower in patients with infections.
Infections associated with spinal cord injury, such as pneumonia and/or postoperative wound
infections, qualify as independent risk factors for poor neurological outcome after motor complete human
spinal cord injury. Within a bed-to-benchside approach we established an experimental model of
controlled pneumonia in order to investigate the neurogenic origin of SCI-IDS and the underlying pathoneurobiology of infections as prevalent co-morbidity, disease modifying factor and neurobiological
rehabilitation confounder.
 Infections constitute a clinically relevant target for protecting the limited endogenous functional
regeneration capacity. Upcoming interventional trials might gain in efficacy with improved patient
stratification and might benefit from complementary protection of the intrinsic recovery potential after
spinal cord injury.
 SCI-IDS might serve as a model to study the mechanisms and mediators of CNS control over
immunity. More importantly, understanding SCI-IDS will allow us to work on developing effective
therapeutic strategies to protect the outcome ‘at risk’ after SCI by eliminating a significant negative
denominator of poor motor recovery.
References
Failli V et al. Functional neurological recovery after spinal cord injury is impaired in patients with infections. Brain. 2012 135:3238-50
Glass CK, et al. Mechanisms underlying inflammation in neurodegeneration. Cell. 2010 140:918-34
Lucin KM, Sanders VM, Popovich PG. Stress hormones collaborate to induce lymphocyte apoptosis after high level spinal cord
injury. J Neurochem. 2009 110:1409-21
Meisel C et al., Central nervous system injury-induced immune deficiency syndrome. Nat Rev Neurosci. 2005 10:775-86
Moreno B et al. Systemic inflammation induces axon injury during brain inflammation. Ann Neurol. 2011 70:932-42
Schwab JM et al., The paradox of chronic neuroinflammation, systemic immune suppression, autoimmunity after traumatic chronic
spinal cord injury. Exp Neurol. 2014 258C:121-129
Zhang et al., Autonomic dysreflexia causes chronic immune suppression after spinal cord injury. J Neurosci. 2013 33:12970-81
Supported by the German Research Council (DFG; Cluster of Excellence NeuroCure), the Berlin-Brandenburg Center for
Regenerative Therapies (BCRT; # 81717034) and the Wings for Life Spinal Cord Research Foundation (# WfL-DE-006/1)
11
Session IV: NATHALIE ROSE BARR PHD STUDENTSHIP PRESENTATION
Chair: Professor Ann Logan
Promoting neuroplasticity after spinal cord injury by over-expressing polysialic acid
Louise Adams, Yi Zhang, Ping Yip, Adina Michael-Titus, John Priestley, Xuenong Bo
Centre for Neuroscience & Trauma, Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen
Mary University of London, UK
L.Adams@qmul.ac.uk
Polysialic acid (PSA) is a linear homopolymer of alpha 2,8-linked sialic acid. It is abundantly
expressed in the CNS during development, where it is found closely associated with axon growth and
synapse formation. Previous results from our laboratory noted the appearance of perineuronal nets
(PNN) in the spinal cord was correlated with diminished expression of PSA. We postulate that overexpression of PSA in neurons around the spinal cord lesion site may modify the PNN structure and
promote neuroplasticity and behavioural improvement in a rat cervical spinal cord injury model. To
engineer expression of PSA in the spinal cord, we used a lentiviral vector to express polysialyltransferase
under the control of the neuronal specific promoter human synapsin-1 (LV/PST). Lentiviral vector
expressing GFP controlled by the same promoter (LV/GFP) was used as a control. Adult rats received a
mid-cervical lateral hemisection, which was immediately followed by four injections of either LV/PST or
LV/GFP rostral and caudal to the injury site. Motor recovery was assessed for six weeks using the
Montoya staircase test, open field, grid exploration test and the Catwalk XT system. Anterograde tracing
of the corticospinal tract was performed by injection of BDA into the motor cortex. LV/PST injection
resulted in strong PSA expression on the surface of transduced neurons. Behavioural tests showed a
tendency towards improved function in the LV/PST group compared with LV/GFP; however this did not
reach significance due to the large variance among the animals. Whether PSA over-expression promotes
sprouting of the corticospinal tract and the formation of new synaptic connections with the neurons on the
lesioned side is still being examined.
Supported by the International Spinal Research Trust (ISRT)
12
Day 2
Saturday, 6th September 2014
Session V: ELECTRONIC INTERFACES WITH THE NERVOUS SYSTEM
Chair: Professor Peter Ellaway
Recent progress with a high-performance brain-computer interface
Andrew Schwartz
Systems Neuroscience Institute, University of Pittsburgh, Pittsburgh, PA, USA
abs21@pitt.edu
A better understanding neural population function would be an important advance in systems
neuroscience. Neurons encode many parameters simultaneously, but the fidelity of encoding at the level
of individual neurons is weak. However, because encoding is redundant and consistent across the
population, extraction methods based on multiple neurons are capable of generating a faithful
representation of intended movement. The realization that useful information is embedded in the
population has spawned the current success of brain-controlled interfaces. Since multiple movement
parameters are encoded simultaneously in the same population of neurons, we have been gradually
increasing the degrees of freedom (DOF) that a subject can control through the interface. Our early work
showed that 3-dimensions could be controlled in a virtual reality task. We then demonstrated control of an
anthropomorphic physical device with 4 DOF in a self-feeding task.
Currently, monkeys in our laboratory are using this interface to control a very realistic, prosthetic
arm with a wrist and hand to grasp objects in different locations and orientations. Our recent data show
that we can extract 10-DOF to add hand shape and dexterity to our control set. This technology has now
been extended has been extended to a paralyzed patient who cannot move any part of her body below
her neck. Based on our laboratory work and using a high-performance “modular prosthetic limb” she has
been able to control 10 degrees-of-freedom simultaneously. The control of this artificial limb is intuitive
and the movements are coordinated and graceful, closely resembling natural arm and hand movement.
This subject has been able to perform tasks of daily living-- reaching to, grasping and manipulating
objects, as well as performing spontaneous acts such as self-feeding. Current work is progressing toward
making this technology more robust and extending the control with tactile feedback to sensory cortex.
Supported by DARPA (972352, N66001-12-C-4027) and NIH (R01NS0256-05S1)
13
Improvements in nerve-electrode integration for closed-loop control of neuroprostheses
Daniel Chew
John Van Geest Centre for Brain Repair, University of Cambridge, Cambridge, UK
Dc501@cam.ac.uk
The applications of neuroprostheses have currently a modest integrative capacity with the
nervous system. This is in part due to biological incompatibility leading to foreign body reactions and scar
tissue formation. Many implantable devices also suffer from physiological incompatibility, in that they are
not able to adequately deconstruct and translate recorded neurological activity from the milieu of
compounded activity and electrical interference. Neural activity can be stimulated robustly; but to
electrically inhibit erroneous neural activity in cases of neuropathic pain, neurogenic bladder (hyper
reflexia and over-activity), and other neurological disorders is a challenging task.
Here we present data that describes a surgical manipulation of peripheral nerves and dorsal roots
to provide robust improvements in recordable extracellular signal through a neuroprosthetic device.
These ‘rootlets’ or ‘nervelets’ are small enough in diameter to grow into, or be surgically placed into,
electrically insulated micro-cuffs (microchannels). These microchannels are fabricated to include
nanometer thin gold electrode contact sites for optimal recording.
In the case of the peripheral nerve, sciatic ‘nervelets’ of 100um in diameter can grow after
transection into a 3 dimensional microchannel network. This can occur as early as 3 days post
implantation, and the axons survive anatomically and functionally up to 9 months. Recordings from the
device enable individual action potential spikes to be detected, providing a potential robust recording
system for use in a closed loop neuroprosthesis in amputee patients.
Additionally dorsal roots can be surgical micro-dissected into 100um diameter ‘rootlets’, enabling
direct implantation into a linear array of microchannels. These rootlets remain anatomically and
functionally viable up to 3 months post implantation. This technique could have direct application as a
bladder fullness indicator, in combination to the clinically marketed bladder nerve stimulators, such as the
Medtronic Interstim or Finetech Brindley devices, used as treatment for overactive or neurogenic bladder
respectively.
Further; refining stimulation parameters, such as applying low frequency (10Hz) stimulation of the
dorsal roots or high frequency (20KHz) stimulation of the ventral roots, one can neuromodulate the local
bladder circuitry, to retain bladder continence. Combined with ‘traditional’ stimulation parameters (30Hz)
of the ventral roots in cases of bladder areflexia, this neuromodulation and improved recording dynamics
provides the basis for the development of a reactive and intelligent neuroprosthesis.
References:
Chew DJ, Zhu L, Delivopoulos E, Minev IR, Musick KM, Mosse CA, Craggs M, Donaldson N, Lacour SP, McMahon SB, Fawcett JW
(2013). A microchannel neuroprosthesis for bladder control after spinal cord injury in rat. Sci Trans Med 6;5(210)
Delivopoulos E, Chew DJ, Minev IR, , Fawcett JW, Lacour SP (2012). Concurrent recording of bladder afferents from multiple
nerves using a microfabricated PDMS microchannel electrode array. Lab Chip 21;12(14);2540-51
Minev IR, Chew DJ, Delivopoulos E, Fawcett JW, Lacour SP (2012). High sensitivity recording of afferent nerve activity using ultracompliant microchannel electrodes: an acute in vivo validation. J Neural Eng. 9(2):026005
FitzGerald JJ, Lago N, Benmerah S, Serra J, Watling CP, Cameron RE, Tarte E, Lacour SP, McMahon SB, Fawcett JW (2012). A
regenerative microchannel neural interface for recording from and stimulating peripheral axons in vivo. J Neural Eng 9(1):016010
Supported by a grant from the EPSRC
14
Brain machine interface controlled functional electrical stimulation therapy
2
1,2
1,2
Cesar Marquez-Chin , Kathryn Atwell , Steve McGie , Milos R. Popovic
1,2
(1) Institute of Biomaterials and Biomedical Engineering, University of Toronto, Canada
(2) Toronto Rehabilitation Institute, University Health Network, Toronto, Canada
milos.popovic@utoronto.ca
In recent years, through a number of clinical trials, our team has demonstrated that functional
electrical stimulation (FES) therapy can be used to improve upper limb function in C4 to C7 spinal cord
injured (SCI) individuals. We have demonstrated that the therapy is effective in both sub-acute and
chronic patients. This therapy essentially promotes neuroplasticity in the spinal cord and the brain, that
ultimately results in improved upper limb function. In parallel with these activities we have carried out a
series of experiments evaluating which control strategy is more effective in further promoting
neuroplasticity when it is coupled with the FES therapy. The outcome of this study was that out of 3
different control strategies applied, the brain machine interface controlled FES therapy produced the most
profound neuroplastic changes. For the purpose of that experiment we have used fairly ineffective brain
machine interface, and yet the results achieved surpassed electromyography and push button controlled
methods for delivering FES therapy. This further motivated us to develop a real-time
electroencephalography-based (EEG-based) brain machine interface that is able to reliably and robustly
detect different grasps, such as palmar, pinch, and lumbrical grasps, from EEG recordings alone. In this
lecture, we will present the results of these three independent activities, and we will present the road map
for development of the brain machine interface controlled FES therapy for improving upper limb function
in SCI individuals.
15
Session VI: NOVEL TECHNIQUES FOR SCI RESEARCH
Chair: Professor Jerry Silver
Systemic administration of Epothilone B promotes axon regeneration and functional
recovery after spinal cord injury
1
1
1†
1‡
1
2
Frank Bradke , Jörg Ruschel , Farida Hellal , Kevin C Flynn , Sebastian Dupraz , Margaret Bates ,
3
4
4
4
3
3
Christopher Sliwinski , Kristina Dobrint , Michael Peitz , Oliver Brüstle , Armin Blesch , Norbert Weidner ,
2
2
Mary Bartlett Bunge , John L. Bixby
1) Axonal Growth and Regeneration, German Center for Neurodegenerative Diseases, Bonn, Germany
2) The Miami Project to Cure Paralysis, University of Miami Miller School of Medicine, Miami, USA
3) Spinal Cord Injury Center, Heidelberg University Hospital, Heidelberg, Germany
4) Institute of Reconstructive Neurobiology, Life&Brain Center, University of Bonn and Hertie Foundation, Bonn,
Germany
† Current address: Institute for Stroke and Vascular Dementia Research, University of Munich Medical Center,
Munich, Germany
‡ Current address: Department of Molecular Medicine, Max Planck Institute of Biochemistry, Martinsried, Germany
Frank.Bradke@dzne.de
After central nervous system (CNS) injury, axon regeneration is prevented by growth inhibitory
factors in the lesion scar as well as by poor intrinsic axon growth potential. Microtubule stabilization
controls scar formation and axon growth. However, the action of microtubule stabilization on these
processes has remained unclear. Here, systemic and post-injury administration of a blood-brain barrier
permeable microtubule stabilizing drug, epothilone B, decreased scarring in spinal cord injured rodents by
disrupting cell polarity of meningeal fibroblasts, which abrogated directed cell migration. Further,
epothilone B propelled axon growth through an inhibitory environment by enabling microtubule protrusion
into the axon tip. Finally, epothilone B treatment improved walking disabilities after spinal cord injury. As
epothilones received recently clinical approval, they hold promise for clinical translation in enabling axon
regeneration and functional recovery after CNS injury.
This work was supported by NIH, IRP, WfL and DFG
16
Successful spinal cord regeneration in the zebrafish
Catherina G. Becker, Thomas Becker, Gianna Maurer, Jochen Ohnmacht, Antón Barreiro-Iglesias, Yujie
Yang
Centre for Neuroregeneration, The University of Edinburgh, Edinburgh, UK
catherina.becker@ed.ac.uk
In contrast to mammals, the adult zebrafish can successfully repair injury to the spinal cord
leading to full functional recovery (1,2). During repair, axons recross the lesion site and motor- and
interneurons are generated from progenitor cells at the central canal (3). We have used the embryonic
fish to screen for compounds enhancing this neurogenesis and have found that during development,
dopamine and serotonin augment the number of motor neurons. This mechanism is also active during
regeneration of the spinal cord in adult fish. Furthermore, application of dopamine to human ES cell
derived neural stem cells enhances the number of motor neurons generated in vitro, underscoring the
relevance of fish findings for mammalian spinal cord development, and potentially, repair (4).
We have now begun to use mechanical lesion of the larval spinal cord in zebrafish (Ohnmacht et
al., unpublished). Lesions as early as 3 days post fertilisation lead to a macrophage/microglial response
and regeneration of motor neurons in the proximity of the lesion site. This can be enhanced by application
of dopaminergic drugs, similar to adults. We are now further refining mechanic and genetic lesions
paradigms in the post-embryonic/larval zebrafish to investigate signalling mechanisms directing
neurogenesis and to allow compound screens on the regenerating spinal cord.
References
1. Becker T, Becker CG (2014) Axonal regeneration in zebrafish. Curr Opin Neurobiol. 27C:186-191
2. Becker T, Wullimann MF, Becker CG, Bernhardt RR, and Schachner M (1997) Axonal regrowth after spinal cord transection in
adult zebrafish. J Comp Neurol 377:577-595
3. Reimer, MM, Sörensen I, Frank RE, Liu C, Becker CG*, Becker T* (2008) Motor neuron regeneration in adult zebrafish, J Neurosci
28: 8510-8516
4. Reimer MM, Norris A, Ohnmacht J, Patani R, Zhong Z, Dias TB, Kuscha V, Scott AL, Chen Y, Rozov S, Frazer SL, Wyatt C,
Higashijima S, Patton EE, Panula P, Chandran S, Becker T*, Becker CG* (2013) Dopamine signaling from the brain augments
spinal motor neuron generation during development and adult regeneration via hedgehog pathway activation, Dev Cell 25(5): 478491
Supported by the BBSRC, the National Centre for the 3Rs and the Motor Neurone Disease Association
17
Visualization of neuronal networks in the mouse brain, spinal cord and mouse embryos
by ultramicroscopy
Hans-Ulrich Dodt, C. Hahn, N. Jährling, S. Saghafi, K. Becker
Department of Bioelectronics, Institute of Solid State Electronics, TU Vienna, Vienna, Austria
Section of Bioelectronics, Center for Brain Research, Medical University Vienna, Vienna, Austria
dodt@tuwien.ac.at
It would be very helpful for the analysis of neuronal networks of the brain, if one could visualize
these networks in 3 dimensions. Up to now this was only possible with limited resolution by sequential
slicing and reconstruction of the brain. This time consuming attempt is easily hampered by artifacts as
shrinkage and distortion induced by standard histological procedures.
To overcome these problems we used a microscopy based on extreme darkfield illumination with a
light sheet, once called ultramicroscopy. This microscopy allows optical sectioning of whole mouse brains
and was combined with an approach to clear fixed neuronal tissue: Mouse brains were made completely
transparent by immersion in oil of the same refractive index as protein. By illuminating the brains with blue
light (λ = 488 nm), neurons labeled with GFP were visualized by fluorescence. This way we could detect
single neurons in hippocampi inside whole brains.
By surface rendering the shape and position of hippocampi relative to the brain surface could be
depicted. In complete excised hippocampi subcellular resolution was obtained by 3D reconstruction from
several hundred optical sections. The dendritic trees of CA1 hippocampal neurons with dendrites and
dendritic spines could be visualized.
Many proteins can be labelled in transgenic mice with genetically encoded fluorescent markers.
Using these markers our approach will represent a high-throughput screening method for protein
expression in 3 D. This expression can be monitored with µm resolution and should allow the elucidation
of complex neuronal networks in the brain and spinal cord.
We show that ultramicroscopy allows also optical sectioning and detailed 3D reconstruction of
whole mouse embryos by imaging autofluorescent structures. Especially the circulatory system in the
body and brain became apparent as blood remaining in the preparation showed strong fluorescence.
Also other applications like e.g. visualization of nerve bundles in whole embryos and visualization of
plaques in brains of mice with Alzheimers disease will be shown. In general the method is well suited for
high-throughput phenotype screening of transgenic mice and thus will benefit the investigation of disease
models.
References
1. H.U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C.P. Mauch, K. Deininger, J.M. Deussing , M. Eder, W. Zieglgänsberger, and
K. Becker, Nat. Meth., 2007, 4, 331-336
2. A. Ertürk, C.P. Mauch, F. Hellal, F. Förstner, T. Keck, K. Becker, N. Jährling, H. Steffens, M. Richter, M. Hübener, E. Kramer, F.
Kirchhoff, H.U. Dodt, and F. Bradke, Nat. Med., 2012, 18, 166-171
3. A. Ertürk, K. Becker, N. Jährling, C.P. Mauch, C.D. Hojer, J.G. Egen, F. Hellal, F. Bradke, M. Sheng, and H.U. Dodt, Nat. Protoc.,
2012, 7, 1993-95
4. C. Schönbauer, J. Distler, N. Jährling, M. Radolf, H.U. Dodt, M. Frasch, F. Schnorrer, Nature, 2011, 479, 406-409
18
A transgenic approach to permanently labeling stressed or damaged neurons
1,2,
2
2
3
4
Matt S. Ramer Franziska Denk , Leanne M. Ramer , Mohammed A. Nassar , Yury Bogdanov ,
5
4
2
Massimo Signore , John N. Wood , Stephen B McMahon
1. International Collaboration on Repair Discoveries, the University of British Columbia, Vancouver, Canada
2. Wolfson Centre for Age-Related Diseases, King’s College London, UK
3. Department of Biomedical Science, University of Sheffield, UK
4. Molecular Pain Laboratory, University College London, UK
5. Institute of Child Health, London, UK
ramer@icord.org
A major challenge in spinal cord injury research, particularly in clinically-relevant contusive
injuries, is in distinguishing injured axons from spared fibres. This is an important issue since
experimental therapies targeting injured axons may in fact be better suited to harnessing spared systems
(and vice versa).
Here we describe a new transgenic mouse knock-in which was designed to exploit upregulation
of the injury-induced transcription factor Activating in Transcription Factor 3 (ATF3) in order to effect
genomic recombination. An ATF3 - Cre-ERT2 construct, flanked by an ATG start codon and the ATF3
3’UTR and SV40 stop signal was inserted into exon 2 of the native ATF3 gene. Mice carrying the knockin mutation were then bred with lox-stop-lox Rosa26-tdtomato mice. The expectation was that
upregulation of ATF3 gene expression in the offspring, along with tamoxifen treatment to allow Cre
translocation to the nucleus, would cause excision of the floxed stop signal and induction of reporter
expression. In naïve adult mice, occasional neurons underwent recombination in the olfactory bulb (OB)
granular layer, in the dentate gyrus and in trigeminal and dorsal root ganglion (DRG) neurons. This is in
line with the known expression patterns of ATF3 in the adult mouse. In the OB and dentate gyrus, the
few neurons labeled had Golgi-like morphology. The central projections of primary afferent axons were
likewise completely filled.
Peripheral nerve injury induced recombination in approximately 50% of DRG neurons and in a
smaller fraction of sympathetic neurons by four days post-lesion, indicating that the Cre construct is
“leaky”. Lateral spinal hemisection induced reporter expression in DRG neurons close to the lesion, in
midline neurons below the injury (in area X) which send ascending axons into the lateral funiculus, in
magnocellular reticular neurons, in vestibular nuclei (lateral and spinal), in the red nucleus, and in the
descending part of the paraventricular hypothalamic nucleus. Treatment of uninjured animals with
tamoxifen induced extensive recombination (i.e. ATF3 expression) in the OB, in the dentate gyrus, in
vascular endothelium and arteriolar smooth muscle, and in primary sensory (and sympathetic) neurons.
We show that tamoxifen-induced ATF3 induction in the brain occurs via the antiestrogen binding site
(rather than via estrogen receptors), and can be blocked by alpha-tocopherol (vitamin E). In the DRG,
tamoxifen induces ATF3 via anti-estrogenic and non-estrogenic mechanisms. These mice may be useful
for labeling specific neuronal populations following injury in the absence of tamoxifen treatment, or
knocking out floxed constructs with or without tamoxifen in addition to injury.
This work was carried out by the M.S. Ramer while on sabbatical in the laboratory of Prof. SB McMahon. Dr. Franziska Denk
designed the mouse.
19
Session VII: CELL THERAPIES FOR SCI
Chair: Professor Sue Barnett
Transplantation of corticospinal motor neurons derived from human iPS to repair spinal
cord cervical injuries
Giles W. Plant
Department of Neurosurgery and Stanford Institute for Neuroscience, Stanford University, Stanford, CA, USA
gplant@stanford.edu
Regeneration of corticospinal tract (CST) axons is a major goal in the successful repair of the
adult spinal cord following an injury. Determining how severed axons that regrow can produce functional
and anatomical benefits is vital to creating future treatments for patients. Following injury to the spinal
cord CST axons fail to regenerate, and retract rostrally from the original lesion site. Therefore an effective
therapeutic intervention needs to provide both a positive growth milieu, and meaningful connections to the
limbs once innervated. Many current stem cell therapies have not been defined well making any direct
correlation of repair with a specific cell type difficult to assess.
Stem cell strategies can also involve the use of undifferentiated or differentiated embryonic and
adult neural stem cells, which cannot be obtained from the injured patient. Our development of human
induced pluripotent stem cells (iPSC) differentiated into neuronal lineages gives us the potential to
immunologically match grafts without the need for immunosuppression and avoids the ethical issues
arising from embryonic tissue use. We employ a rat cervical unilateral cut or contusion model of spinal
cord injury at the C5 level to measure anatomical and functional outcomes after transplanting human iPS
derived corticospinal motor neurons (CSMN). In addition we have electrophysiologically mapped host
CST neurons, which infer specific forelimb movements. Previous studies have indicated that rehabilitative
strategies have additive benefits when combined with therapeutic interventions. For this reason, our
animals are undergoing rehabilitation and functional testing paradigms following CSMN transplantation.
We hypothesize that our treatment with CSMN transplants will improve functional and anatomical
outcomes after a cervical spinal cord injury. We propose that any functional improvement observed will
arise from long tract growth of the transplanted CSMN neurons forming synaptic connections to host
spinal cord circuitry. In addition we propose that host CST axons will form synaptic connections to those
neurons from the CSMN transplants via the injured ipsilateral or uninjured contralateral CST. Functional
tests encouraging the use of the injured forelimb and assessment will also be undertaken in groups to
ascertain improved growth and target specificity.
Supported by the International Spinal Research Trust (ISRT), Klein Family Research Fund, and Wings for Life
20
Intravenous multipotent adult progenitor cell treatment for acute spinal cord injury:
promoting recovery through immune modulation
1
2
2
2
1
2
Marc DePaul , Marc Palmer , Rochelle Cutrone , Jason A. Hamilton , Bradley T. Lang , Robert J. Deans ,
2
1
2
Robert W. Mays , Jerry Silver , Sarah A. Busch
1
Dept. of Neurosciences, Case Western Reserve Univ., Cleveland, OH, USA
Athersys, Inc. Regenerative Medicine, Cleveland, OH, USA
2
sbusch@athersys.com
Adult bone marrow-derived stem cells are known to have immunomodulatory capabilities, but
their potential to alter inflammatory processes and promote regeneration after spinal cord injury (SCI) has
not been thoroughly studied. We have previously demonstrated that multipotent adult progenitor cells (or
MAPCs) prevent axonal dieback and promote re-growth of injured axons in vivo in a dorsal column crush
model (Busch et al., 2011). In the current study, we sought to determine the optimal window of
administration, dosing, and the biodistribution of human multipotent adult progenitor cells (MAPCs) in a
spinal cord contusion model (250 kdyn, Infinite Horizon Impactor injury at T8). A clinical grade variant of
®
MAPC therapy, known as MultiStem , is currently in clinical evaluation for treatment of ischemic stroke.
6
Rats received saline or 4x10 cells via intravenous (iv) injection immediately following or 1 day post injury
(DPI). We performed locomotor testing through 10 weeks post injury (WPI) and found significant and
sustained improvements in BBB scores, subscores, and the Catwalk regularity index. We monitored
urination every two weeks using metabolic cages and found the void volume significantly reduced 10
weeks post injury in cell treated animals. At the study endpoint, rats underwent urodynamic assessment.
Bursting activity of the external urethral sphincter was seen in correlation with a void in several treated
animals, whereas in untreated animals bursting was rare, sporadic, and uncoordinated. Treated animals
urinated at a smaller bladder volume, had less residual volume, showed improved return to baseline
6
pressure following a void, and had a decrease in bladder weight. Increasing the dose to 8x10 cells did
not lead to additional improvements in locomotor recovery, but did improve bladder function. Cell
distribution was determined using CryoViz technology by iv infusing Qdot-labeled MAPCs into either SCI
or laminectomy control animals 1 DPI. Lungs, liver, spleen, and spine were collected 24 or 48 hours after
treatment. MAPCs were found in the lungs, liver, and spleen at 24 hours, amounting to <5% of
administered cells, and cell numbers decreased at 48 hours. Normalizing cell counts to tissue weight
showed a preferential homing to the spleen, while few cells were found in the spinal column. Microarray
analysis of the lesion, blood, and spleen suggests MAPCs administered 1 day post injury alter many
injury-induced pathways including those involved in recruitment, activation and migration of immune cells,
+
which we confirmed using qPCR. In support of this data, we found a decrease of ED1 macrophages at
the lesion site 4 DPI in treated animals. These data suggest that MAPCs, when administered iv in an
acute model of SCI, are more likely to exert benefit through peripheral organ systems than via homing
and direct interaction with the site of injury.
References
S.A. Busch, J.A. Hamilton, K.P. Horn, F.X. Cuascut, N. Lehman, A.E. Ting, R.J. Deans, R.W. Mays, J. Silver. Multipotent Adult
Progenitor Cells Prevent Macrophage-Mediated Axonal Dieback and Promote Regrowth after Spinal Cord Injury. Journal of
Neuroscience. 2011 31: 944-953
Supported by a grant from the Ohio Third Frontier to J. Silver / R.W. Mays through the National Center for Regenerative Medicine
21
Role of endogenous neural stem cells in spinal cord injury
Moa Stenudd
Jonas Frisén lab, Department of Cell and Molecular Biology, Karolinska Institutet, Sweden
moa.stenudd@ki.se
Spinal cord injury is followed by the formation of a glial scar, which has both positive and negative
effects on recovery from the injury (1). The astrocytes in the glial scar are generated from both selfduplicating resident astrocytes and neural stem cells. Ependymal cells are the neural stem cells in the
adult spinal cord, and after spinal cord injury they proliferate vigorously and generate both
oligodendrocytes and more than half of the astrocytes in the glial scar (2).
A good understanding of the separate functions of astrocyte-derived and neural stem cell-derived
astrocytes is important to develop therapies modulating the contribution to the glial scar. By blocking the
neural stem cells’ contribution to the scar, we have shown that neural stem cell progeny is necessary to
maintain tissue integrity and prevent neuronal death after spinal cord injury (3). These results identify
neural stem cells as key players in protective mechanisms following spinal cord injury, making them an
interesting target for future non-invasive treatments for spinal cord injury.
References
(1) Burda JE, Sofroniew M V. Reactive gliosis and the multicellular response to CNS damage and disease. Neuron.
2014;81(2):229–48
(2) Barnabé-Heider F, Göritz C, Sabelström H, et al. Origin of new glial cells in intact and injured adult spinal cord. Cell Stem Cell.
2010;7(4):470–82
(3) Sabelström H, Stenudd M, Réu P, et al. Resident neural stem cells restrict tissue damage and neuronal loss after spinal cord
injury in mice. Science. 2013;342(6158):637–40
Work in the lab of Dr. Jonas Frisén is supported by Swedish Research Council, the Swedish Cancer Society, the Karolinska
Institute, Tobias Stiftelsen, AFA Försäkringar, the Strategic Research Programme in Stem Cells and Regenerative Medicine at
Karolinska Institutet (StratRegen), Torsten Söderbergs Stiftelse and Knut och Alice Wallenbergs Stiftelse
22
Session VIII: CLINICAL SESSION
Chair: Professor James Guest
Inclusive SCI clinical trials: Predicting homogeneous trial participants and modeling
outcome measures for incomplete SCI participants
1
2
1
John D. Steeves , Lorenzo Tanadini , Dirk Haupt , Armin Curt
1
2
2
ICORD, University of British Columbia, Vancouver, Canada
Uniklinik Balgrist, University of Zurich, Zurich, Switzerland
steeves@icord.org
Never before have there been so many clinical trials and human studies examining a wide range
of therapeutic approaches for the treatment of spinal cord injury (SCI). It is desirable to recruit and enroll
as many participants as possible to a trial. However, we know this is burdened by ethical and safety
concerns. In order to minimize possible harm, many Phase I SCI trials begin by enrolling volunteers with a
thoracic sensorimotor complete (AIS A) injury. If there are no adverse events and risks seem minimal, the
trial program often advances to a Phase II trial involving participants with cervical sensorimotor complete
(AIS A) SCI. This sequential recruitment approach is safe, but slow and an inefficient utilization of
equipment, staff and participants. After safety has been preliminarily established in a small Phase I study,
an alternative approach would recruit a broader range of participants with either complete or incomplete
SCI (iSCI) to a Phase II trial. Such a strategy has some justification since many of the experimental
treatments were developed using animals models with iSCI (AIS B-D).
Nevertheless, people living with iSCI exhibit highly variable patterns of functional recovery. Such
heterogeneity in trial participants makes it difficult to detect statistically valid treatment effects or clinical
benefits. Some trials have recruited iSCI subjects and used a common clinical endpoint for all SCI types,
hoping the therapeutic would have a large enough effect size to overcome any participant variability in the
patterns of recovery. But, this approach has not been successful and will likely miss detecting subtle, but
significant treatment effects. Thus, how can we create more homogeneous study cohorts in an objective
and unbiased manner, while identifying those participants who should be excluded from participation
because they may recover to extensively to detect a therapeutic benefit (e.g. ceiling effect)? Finally, what
outcome measures (trial endpoints) should we use for different cohorts in a phase II study?
We require predictive data-driven algorithms that will accurately stratify participants based on
early neurological and functional values. In addition, we need to model (test) reasonable cohort-specific
outcomes for iSCI participants. We have been employing the EMSCI database and a recently developed
multivariate statistical method, unbiased recursive partitioning, to identify homogeneous cohorts at an
early time point after SCI, as well as to model different cohort-specific outcome measures. If such a
recruitment strategy could be implemented, it would mean that trials could be completed faster and more
efficiently, as well as identify which types of SCI respond best to the therapeutic being examined.
23
The SCIentinel study - prospective multicenter study to define the spinal cord injuryinduced immune depression syndrome (SCI-IDS)': aiming at protection of the
endogenous recovery potential after SCI
1,2
2,3
4,5,6
7
7
1,2
Marcel A. Kopp , Claudia Druschel , Christian Meisel , Thomas Liebscher , Erik Prilipp , Ralf Watzlawick ,
8
7
3
8
9
9
Paolo Cinelli , Andreas Niedeggen , Klaus-Dieter Schaser , Guido A. Wanner , Armin Curt , Gertraut Lindemann ,
10
10
11
11
11
12
Natalia Nugeva , Michael G. Fehlings , Peter Vajkoczy , Mario Cabraja , Julius Dengler , Wolfgang Ertel , Axel
13
14
4,5
5
5
1,2
Ekkernkamp , Peter Martus , Hans-Dieter Volk , Nadine Unterwalder , Uwe Kölsch , Benedikt Brommer , Rick C.
1,2
1,2
1,2
1,15
1,2
1,16
Hellmann , Ramin Raul Ossami Saidi , Ines Laginha , Harald Prüss , Vieri Failli , Ulrich Dirnagl , Jan M.
1,2,7
Schwab
1
Dept. of Neurology and Experimental Neurology, 2 Spinal Cord Injury Research (Neuroparaplegiology), 3Dept. of Musculoskeletal
Surgery, 4Institute of Medical Immunology, 5 Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Charité Universitätsmedizin Berlin, Germany; 6Dept. of Immunology, Labor Berlin – Charité Vivantes GmbH, Berlin, Germany; 7Treatment
Centre for Spinal Cord Injuries, Trauma Hospital Berlin, Germany; 8Division of Trauma Surgery, University Hospital of Zürich, 9Spinal
Cord Injury Center, University Hospital Balgrist, Zurich, Switzerland; 10Dept. of Neurosurgery, University of Toronto, Canada; 11Dept.
of Neurosurgery, 12Centre for Trauma- and Reconstructive Surgery, Charité - Universitätsmedizin Berlin, Germany; 13Trauma
Surgery and Orthopedics Clinic, Trauma Hospital Berlin, Germany; 14Dept. of Clinical Epidemiology and Applied Biostatistics,
Eberhard Karls Universität Tübingen, Germany; 15German Center for Neurodegenerative Diseases (DZNE), 16Center for Stroke
Research Berlin, Charité - Universitätsmedizin Berlin, Germany
marcel.kopp@charite.de or jan.schwab@charite.de
Infections are the leading cause of death following acute spinal cord injury (SCI). Furthermore,
infections represent a ‘disease modifying factor’ independently associated with poor neurological
1
outcome . The increased susceptibility for infections is not sufficiently explained by the risk of aspiration,
e.g.2,3,4
bladder dysfunction, or high-dose methylprednisolone treatment. Experimental and clinical pilot data
suggest that SCI disturbs the signaling pathways between the central nervous system and the immune
system and shifts homeostasis towards an acute and chronic state of suppressed immune functions.
The objectives of the SCIentinel trial are to characterize the dysfunction of the innate and adaptive
immune system after SCI and to explore its proposed ‘neurogenic’ origin by analyzing its correlation with
lesion height and severity. Decreased HLA-DR (MHC II) expression on monocytes serves as a key
surrogate parameter. Secondary hypotheses are that the Spinal Cord Injury-induced Immune Depression
5
Syndrome (SCI-IDS) causes transient lymphopenia and triggers qualitative functional leukocyte deficits,
which may complicate the post-acute phase after SCI.
SCIentinel is a prospective, international, multicenter study aiming to recruit about 120 patients
with acute SCI or control patients with acute vertebral fracture without neurological deficits scheduled for
spinal surgery. The assessment points are: <31 hours, 31-55 hours, 7 days, 14 days, and 10 weeks posttrauma. Neurological classification includes American Spinal Injury Association impairment scale and
neurological level. Laboratory analyses comprise haematological profiling, immunophenotyping, cytokines
and gene expression of immune modulators. Preliminary results reveal a SCI severity dependent immune
depression in terms of reduced HLA-DR molecule numbers on monocytes occurring within the first days
after SCI and extending into the sub-acute phase in the group of complete SCI. Better definition of the
SCI-IDS provides a basis to prevent infectious complications in order to attenuate the impact of ‘disease
modifying factors’ on neurological outcome. This putatively results in improved SCI care.
References
1 Failli V, Kopp MA, Gericke C, Martus P, Klingbeil S, Brommer B, Laginha I, Chen Y, DeVivo MJ, Dirnagl U, Schwab JM:
Functional neurological recovery after spinal cord injury is impaired in patients with infections. Brain 2012, 135(Pt 11):3238-3250
2 Cruse JM, Lewis RE, Jr., Bishop GR, Kliesch WF, Gaitan E, Britt R: Decreased immune reactivity and neuroendocrine alterations
related to chronic stress in spinal cord injury and stroke patients. Pathobiology 1993, 61(3-4):183-192
3 Campagnolo DI, Keller SE, DeLisa JA, Glick TJ, Sipski ML, Schleifer SJ: Alteration of immune system function in tetraplegics. A
pilot study. Am J Phys Med Rehabil 1994, 73(6):387-393
4 Riegger T, Conrad S, Schluesener HJ, Kaps HP, Badke A, Baron C, Gerstein J, Dietz K, Abdizahdeh M, Schwab JM:
Immunedepression syndrome following human spinal cord injury (SCI): a pilot study. Neuroscience 2009, 158(3):1194-1199
5 Meisel C, Schwab JM, Prass K, Meisel A, Dirnagl U: Central nervous system injury-induced immune deficiency syndrome. Nat
Rev Neurosci 2005, 6(10):775-786
Supported by the German Research Council (DFG; Cluster of Excellence NeuroCure), the Berlin-Brandenburg Center for
Regenerative Therapies (BCRT; # 81717034) and the Wings for Life Spinal Cord Research Foundation (# WfL-DE-006/1)
24
Session IX: DISCUSSION FORUM / DEBATE
Chair: Professor Wolfram Tetzlaff
Cell transplantations for SCI – will we really need them?
Panel members:
Sue Barnett
Susan.Barnett@glasgow.ac.uk
Frank Bradke
frank.bradke@dzne.de
Armin Blesch
Armin.Blesch@med.uni-heidelberg.de
Simone Di Giovanni
s.di-giovanni@imperial.ac.uk
Karim Fouad
karim.fouad@ualberta.ca
Jim Guest
jguest@med.miami.edu
Dana McTigue
dana.mctigue@osumc.edu
Adina Michael-Titus
a.t.michael-titus@qmul.ac.uk
Giles Plant
gplant@stanford.edu
Phil Popovich
Phillip.Popovich@osumc.edu
Jerry Silver
jxs10@po.cwru.edu
Wolfram Tetzlaff
tetzlaff@icord.org
25
Table of Contents – Poster Abstracts
Promoting neuroplasticity after spinal cord injury by over-expressing polysialic acid
Louise Adams ........................................................................................................................................ 29
Regaining over-ground locomotor function following severe contusion injury with epidural
stimulation and treadmill training
Yazi D. Al’joboori ................................................................................................................................... 30
Remyelination of spinal axons by Schwann cells following spinal cord injury is a Neuregulin-1
dependent endogenous repair mechanism
Katalin Bartus ........................................................................................................................................ 31
Assessing functional recovery in the injured corticospinal tract: an optogenetic approach
Murray G. Blackmore ............................................................................................................................ 32
Investigating functional plasticity and synaptogenesis following experimental spinal cord injury
Emily R. Burnside .................................................................................................................................. 33
Alpha9 integrin activation in neurite outgrowth and axon regeneration
Menghon Cheah .................................................................................................................................... 34
Olfactory ensheathing cell transplants improve vertical climbing in rats after cervical level dorsal root
rhizotomy
Andrew Collins ...................................................................................................................................... 35
Transcriptomic changes evoked by culturing Dorsal Root Ganglion neurons overwhelm those evoked
by axonal injury
Matthew C. Danzi.................................................................................................................................. 36
Nanotechnology in neuroregeneration
Suradip Das ........................................................................................................................................... 37
Transduction of cells with a lentiviral vector encoding mammalian chondroitinase ABC in an in vitro
model of neurite outgrowth: enhanced neurite outgrowth is accompanied by a drop in PTEN levels
Priscilla Day ........................................................................................................................................... 38
Regulation of IL10 by chondroitinase ABC promotes a distinct immune response following spinal cord
injury
Athanasios Didangelos .......................................................................................................................... 39
The role of PKA in translating rehabilitative training after SCI into neuroplasticity
Karim Fouad .......................................................................................................................................... 40
MicroRNA-155 deletion restricts inflammatory signaling in macrophages and enhances axon growth
capacity: implications for spinal cord repair
Andrew D. Gaudet................................................................................................................................. 41
26
Artificial Intelligence in Medical Systems Neurobiology: Finding a Treatment for Paralysis
Barbara Grimpe ..................................................................................................................................... 42
Does overexpression of Fibroblast Growth Factor Receptor 1 (fgfr1) in CNS neurons enhance axon
regeneration and recovery after spinal cord injury in rats?
Barbara Haenzi ...................................................................................................................................... 43
AAV9-IL4 exacerbates a pathogenic systemic immune response that impairs functional recovery after
contusive spinal cord injury
Jodie C.E. Hall ........................................................................................................................................ 44
Human neural progenitors transplanted to the deaffarented murine spinal cord promote
regeneration of functional sensory fibers
Jan Hoeber ............................................................................................................................................ 45
Do severity and duration of compression impact on recovery after severe acute spinal cord injury in
dogs?
Hilary Hu................................................................................................................................................ 46
Sympathetic and sensory sprouting after spinal cord injury: peripheral consequences of central
injuries
Diana V. Hunter ..................................................................................................................................... 47
Chondroitinase gene therapy as a treatment for spinal cord injury
Nicholas D. James.................................................................................................................................. 48
Combinatorial treatment with GSK3β inhibitors and chondroitinase ABC to regulate glial scar
formation and promote axon regeneration in the spinal cord
Ashik Kalam ........................................................................................................................................... 49
Optimizing and understanding the use of intracellular sigma peptide as a translatable therapeutic for
spinal cord injury
Bradley T. Lang ...................................................................................................................................... 50
Early intravenous delivery of mesenchymal progenitor cells modulates the secondary inflammatory
response after cervical spinal cord injury leading to behavioral and pathological amelioration
Seok Voon Lee ....................................................................................................................................... 51
Facilitating reproducibility and data integration for SCI research with MIASCI and RegenBase
Vance P. Lemmon ................................................................................................................................. 52
Expression of a hyperactive transcription factor increases axon growth and regeneration
Saloni T. Mehta ..................................................................................................................................... 53
Daily acute intermittent hypoxia following cervical spinal cord injury
Kristiina Negron .................................................................................................................................... 54
A novel role for Wnt signalling in regulating astrogliosis in adult white matter
Andrea Rivera........................................................................................................................................ 55
27
Wnts: more than an axonal growth inhibitor in the adult spinal cord
F. Javier Rodríguez. ............................................................................................................................... 56
Characterization of a novel axon growth repellent and its role in spinal cord injury
Julia Schaeffer ....................................................................................................................................... 57
Transplantation of neural progenitors to improve respiration following spinal cord injury
Victoria M. Spruance............................................................................................................................. 58
Investigating neuroprotection by carbon nanotubes following spinal cord injury
Merrick Strotton ................................................................................................................................... 59
Intramuscular Tibialis Anterior coherence and subacute spinal cord injury: mechanisms of
neuroplasticity underlying SCI
Julian Taylor .......................................................................................................................................... 60
Oral administration of the p38α MAPK inhibitor, UR13870, inhibits anterior cingulate microglial
expression and affective pain behaviour following spinal cord injury
Julian Taylor .......................................................................................................................................... 61
The fate of boundary cap neural crest stem cells following transplantation to the surface of avulsed
or uninjured spinal cord
Carl Trolle .............................................................................................................................................. 62
Early transplantation of mesenchymal stem cells after spinal cord injury relieves pain
hypersensitivity through suppression of pain-related signaling cascades and reduced inflammatory
cell recruitment
Kenzo Uchida…………………………………………………………………………………………………………………………………..63
Effects of pudendal and cortical paired associative stimulation on reflex and cortico-spinal control of
anal sphincter responses in patients with incomplete spinal cord injury: a feasibility study
Natalia Vásquez..................................................................................................................................... 64
Chondroitinase ABC rescues complete respiratory motor activity following cervical contusion injury
Philippa M. Warren ............................................................................................................................... 65
Extensive recovery of respiratory motor function at chronic and super-chronic time points following
cervical spinal cord injury
Philippa M. Warren ............................................................................................................................... 66
28
Promoting neuroplasticity after spinal cord injury by over-expressing polysialic acid
Louise Adams, Yi Zhang, Ping Yip, Adina Michael-Titus, John Priestley, Xuenong Bo
Centre for Neuroscience & Trauma, Blizard Institute, Barts and the London School of Medicine and Dentistry,
Queen Mary University of London, UK
L.Adams@qmul.ac.uk
Polysialic acid (PSA) is a linear homopolymer of alpha 2,8-linked sialic acid. It is abundantly
expressed in the CNS during development, where it is found closely associated with axon growth and
synapse formation. Previous results from our laboratory noted the appearance of perineuronal nets
(PNN) in the spinal cord was correlated with diminished expression of PSA. We postulate that overexpression of PSA in neurons around the spinal cord lesion site may modify the PNN structure and
promote neuroplasticity and behavioural improvement in a rat cervical spinal cord injury model. To
engineer expression of PSA in the spinal cord, we used a lentiviral vector to express
polysialyltransferase under the control of the neuronal specific promoter human synapsin-1 (LV/PST).
Lentiviral vector expressing GFP controlled by the same promoter (LV/GFP) was used as a control.
Adult rats received a mid-cervical lateral hemisection, which was immediately followed by four
injections of either LV/PST or LV/GFP rostral and caudal to the injury site. Motor recovery was
assessed for six weeks using the Montoya staircase test, open field, grid exploration test and the
Catwalk XT system. Anterograde tracing of the corticospinal tract was performed by injection of BDA
into the motor cortex. LV/PST injection resulted in strong PSA expression on the surface of
transduced neurons. Behavioural tests showed a tendency towards improved function in the LV/PST
group compared with LV/GFP; however this did not reach significance due to the large variance
among the animals. Whether PSA over-expression promotes sprouting of the corticospinal tract and
the formation of new synaptic connections with the neurons on the lesioned side is still being
examined.
Supported by the International Spinal Research Trust (ISRT)
29
Regaining over-ground locomotor function following severe contusion injury with
epidural stimulation and treadmill training
Yazi D. Al’joboori, Ronaldo M. Ichiyama
School of Biomedical Sciences, University of Leeds, Leeds, UK
bsydaj@leeds.ac.uk
Spinal contusion injuries result in a loss of motor, sensory and autonomic function. Electrical
epidural stimulation (ES) of the lumbar spinal cord (L2 to S1) has previously been shown to improve
locomotor function in complete transection models of rat spinal cord injury in conjunction with
monoaminergic and serotonergic agonists and bipedal locomotor training; however, ES has never
been assessed in incomplete contusion lesions where some descending and ascending pathways
remain. Here we demonstrate that the use of epidural stimulation (40 Hz; L2) and locomotor training
following severe spinal contusion injury (T9/10) leads to improved locomotor function.
Adult Sprague-Dawley rats received a severe spinal contusion injury (T9/10) and epidural
implantation at segmental levels L2 and S1 and were randomly assigned to one of four groups: cage
control, training only, ES only or ES+training. Rats in either trained group stepped bipedally on a body
weight supported treadmill (5-16 cm/s) (5 days/week, 20 mins/day) for 8 weeks. By the end of the 8week period rats in the ES+training group showed improvements not only in supported treadmill
stepping ability but also in open field locomotion (BBB), with combination treated animals achieving
the highest overall increase in mean BBB score (12.5±1.5), significantly different from the
improvement seen in cage control animals (8.2±0.6; *p-value 0.016). Therefore these results suggest
that a combination of step training and epidural stimulation in an incomplete model of SCI
successfully improved locomotor function further than either therapy administered alone; with animals
not only improving in treadmill step performance but were also able to transfer this skill to an open
field task which has not previously been observed in complete transection models.
Support provided by the International Spinal Research Trust (ISRT) and Medical Research Council (MRC)
30
Remyelination of spinal axons by Schwann cells following spinal cord injury is a
Neuregulin-1 dependent endogenous repair mechanism
1
2
2
1
2
Katalin Bartus , Jorge Galino , John M. Dawes , Nicholas D. James , Florence R. Fricker , Andrew
3,4
5
3,4
2
1
N. Garratt , Matt S. Ramer , Carmen Birchmeier , David L. H. Bennett & Elizabeth J. Bradbury
1
The Wolfson Centre for Age-Related Diseases, Regeneration Group, King’s College London, Guy’s Campus,
2
London Bridge, London, UK; Nuffield Department of Clinical Neurosciences, West Wing John Radcliffe Hospital,
3
4
Oxford, UK; Max Delbrück Center for Molecular Medicine, Berlin, Germany; Charité Universitätsmedizin Berlin,
5
Charitéplatz, Berlin, Germany; International Collaboration on Repair Discoveries, The University of British
Columbia, Vancouver, Canada
katalin.bartus@kcl.ac.uk
One of the spontaneous intrinsic regenerative responses following traumatic spinal contusion,
which is the most common form of human spinal cold injury (SCI), is remyelination that is triggered by
acute demyelination of spinal axons. However, this endogenous repair response is suboptimal and
may account for the persistently compromised function of some surviving axons. This remyelination is
largely mediated by Schwann cells, a phenomenon commonly observed in the chronically injured
human spinal cord, where injured demyelinated axons become associated with peripheral myelin.
This phenomenon is particularly prominent in the dorsal columns which contain long tracts of
ascending myelinated axons that are known to undergo severe demyelination after SCI, followed by a
degree of remyelination. It remains unknown what governs this Schwann cell-mediated remyelination
of injured spinal axons. The growth factor neuregulin-1 (Nrg1) is known to play a key role of in virtually
every phase of Schwann cell development and myelination within the peripheral nervous system
(PNS), via signaling through ErbB tyrosine kinase receptors. Here we used a tamoxifen inducible
Nrg1mutant mouse to determine the mechanisms controlling remyelination after spinal contusion
injury. We examined whether Nrg1 is required for Schwann cell-mediated remyelination of dorsal
column axons after spinal contusion injury, and whether Nrg1 ablation influences the degree of
spontaneous remyelination and functional recovery. Conditional ablation of Nrg1 was associated with
a complete absence of Schwann cells within the spinal cord and profound demyelination of spinal
axons as determined by immunohistochemistry and electronmicroscopy. This was associated with a
significant deficit in spontaneous locomotor recovery following spinal contusion as assessed by using
the Basso Mouse Scale for hindlimb locomotor function. There was no compensatory oligodendrocyte
remyelination and the majority of remyelinating Schwann cells appeared to originate from within the
spinal cord. These findings provide novel mechanistic insight into the molecular signalling that
governs spontaneous Schwann cell remyelination and repair after SCI.
Supported by the International Spinal Research Trust (ISRT), Wings for Life, Medical Research Council, Wellcome Trust
31
Assessing functional recovery in the injured corticospinal tract: an optogenetic
approach
1
1
1
Naveen Jayaprakash , Brian Hoeynck , Zimei Wang , Murray G. Blackmore
1
1
Department of Biomedical Sciences, Marquette University, Milwaukee, WI, USA
murray.blackmore@marquette.edu
Spinal Cord Injury (SCI) impairs motor function as a consequence of axon transection and
lost synaptic connections. A major goal in SCI research is to increase axon growth and restore
synaptic connectivity. Axons in the spinal cord possess some endogenous capacity for compensatory
plasticity, particularly in situations of partial injury, and various therapeutics have succeeded in
evoking additional regenerative axon growth. For instance, we have shown that forced
overexpression of two genes, Sox11 and KLF7, can increase corticospinal tract (CST) sprouting into
contralateral spinal cord denervated by pyramidotomy, as well as regenerative growth after cervical
hemisection. Behavioral changes, both positive and negative, have been correlated with this
regenerative growth, but remain difficult to interpret. For instance, what are the contributions of direct
CST connectivity by regenerated axons in the spinal cord, as opposed to alterations in upstream
relays? A promising diagnostic tool in this regard is the use of optogenetics, which allows highly
specific activation of neuronal populations that express light-sensitive Channelrhodopsin proteins.
Accordingly, we are exploring optogenetic stimulation of murine CST neurons transduced with viral
Channelrhodopsin (rAAV9/CaG-ChR2-EYFP). Electrophysiology has confirmed light-evoked cell
firing in sensorimotor cortex that emerges one week after viral injection. By two weeks post-injection,
direct stimulation of Channelrhodopsin-expressing terminals in the spinal cord evokes post-synaptic
activity in spinal neurons. Importantly, because CST fibers are the only terminals in the spinal cord to
express light-sensitive channels, this approach distinguishes direct cortical drive from potential relays.
As expected, spinal responses to cortical stimulation are abolished acutely after dorsal hemisection of
the spinal cord, which severs descending cortical axons. Moreover, spinal responses occur
predominantly in tissue located contralateral to the treated cortex, and this laterality is maintained
after a pyramidotomy injury. We are now assessing whether sprouting CST axons in KLF7- or Sox11treated animals acts to restore direct synaptic connectivity in cervical hemisection or pyramidotomy
injury models. Our preliminary data suggest that optogenetics may be a powerful approach to monitor
the restoration of synaptic connections from specific neuronal populations after spinal injury.
Supported by grants from the International Spinal Research Trust (ISRT), the Craig H. Neilsen Foundation, the Bryon Riesch
Paralysis Foundation, and NINDS R01NS083983
32
Investigating functional plasticity and synaptogenesis following experimental spinal
cord injury
1
2
1
1
3
Emily R. Burnside , Federico Grillo , Karen D. Bosch , Stephen B. McMahon , Jonathon S. Carp ,
2
1
Juan Burrone , Elizabeth J. Bradbury
1
Neurorestoration Group, Wolfson Centre for Age-Related Diseases, King’s College London, UK
Medical Research Council Centre for Developmental Neurobiology, Kings College London, UK
3
Wadsworth Center, New York State Department of Health, Albany, New York, USA
2
emily.burnside@kcl.ac.uk
The CNS has a poor intrinsic capacity for regeneration, although some functional recovery
does occur. This is mainly in the form of sprouting, dendritic remodeling and changes in neuronal
coding, firing and synaptic properties; elements collectively known as plasticity. Following spinal cord
injury (SCI), a fundamental approach to repair the injured CNS is therefore to harness, promote and
refine plasticity. This is partly limited by some components of the extracellular matrix, important
inhibitory molecules which may be manipulated by therapeutics such as chondroitinase ABC.
The corticospinal tract (CST) is an important descending motor pathway involved in
locomotion, posture and voluntary skilled movements. Therefore regeneration and anatomical
reorganisation of this projection is often examined in studies of experimental SCI. Techniques such as
anterograde tracing have been combined with immunolabeling for synaptic proteins or electron
microscopy to elucidate connectivity; however whether active synaptogenesis occurs following SCI
and potential therapeutic manipulations has not been studied. Genetically encoded reporters of
presynaptic activity represent novel tools to assess synaptogenesis and gain insight into the
anatomical and functional status of new connections. Here we use an adeno-associated viral (AAV)
vector encoding SynaptopHluorin (SpH), as a tracing tool before exploring its potential for measuring
vesicular release and functional synaptogenesis. We present an ex-vivo acute cervical spinal slice
preparation from adult rats, in which the CST has been transduced using AAV vectors encoding
fluorescent probes reporting presynaptic activity or neurotransmitter release.
We
electrophysiologically stimulate the CST and use two-photon microscopy to real-time image
fluorescence indicative of activity. We aim to perform this ex vivo preparation on rats which have
undergone a unilateral pyramidotomy lesion, and in which the intact contralateral CST is transduced
with the functional reporter. This will allow us to investigate functional synaptogenesis resulting from
plasticity of a known, spared population of fibres following injury and furthermore how this may
change following therapeutic delivery of plasticity-inducing therapeutics such as chondroitinase gene
therapy.
This work is supported by the MRC
33
Alpha9 integrin activation in neurite outgrowth and axon regeneration
1
2
1
3
4
Menghon Cheah , Melissa Andrews , Daniel Chew , Joost Verhaagen , Reinhard Fässler , Andreas
5
1
Faissner , James Fawcett
1
John van Geest Centre for Brain Repair, University of Cambridge, Cambridge, UK
School of Medicine, University of St Andrews, North Haugh, St Andrews, UK
3
Netherlands Institute for Neuroscience, Amsterdam, NL
4
Department of Molecular Medicine, Max Planck Institute of Biochemistry, Martinsried, DE
5
Department of Cell Morphology and Molecular Neurobiology, Ruhr University Bochum, Bochum, DE
2
mc747@cam.ac.uk
Spinal cord injury is a debilitating condition which results in serious neurological
consequences due to the failure of axon regeneration in the central nervous system. Two recent
separate studies in our laboratory have shown that α9 integrin (a cell adhesion receptor subunit)
promotes neurite outgrowth on growth inhibitory tenascin-C, and kindlin-1 (a cytoplasmic integrin
activator) overcomes the inhibition of chondroitin sulfate proteoglycans (CSPG) such as aggrecan by
activating integrins to enhance axon growth. This study aims to combine the beneficial effects of both
α9 integrin and kindlin-1 to promote better neurite outgrowth and axon regeneration. Adult rat dorsal
root ganglion (DRG) was used as a model of study for both in vitro and in vivo. Significant neurite
outgrowth was observed from neurons co-expressing both α9 integrin and kindlin-1 in cell culture. For
the in vivo study investigating sensory axon regeneration, the virus AAV5-fGFP, AAV5-α9-V5 and
AAV5-kindlin1-GFP were used to transduce DRGs in the lower cervical region after a
hemilaminectomy and dorsal root crush. This was followed by ladder-walking, Randall-Siletto and
Hargreave’s behavioural testing for 12 weeks post-injury. Electrophysiological recording showed
significant recovery of axonal activity for the group of animals receiving both α9 integrin and kindlin-1
treatment for 12 weeks. Anatomical analysis also confirmed axon regeneration into dorsal root entry
zone, dorsal horn and dorsal column; this was coupled with recovery in behavioural testing. Alpha9
integrin activation results in better neurite outgrowth and axon regeneration in the presence of both
tenascin-C and CSPG after an axonal injury as α9 integrin is a receptor for tenascin-C and kindlin-1
overcomes the inhibition of CSPG.
References
Andrews et al, 2009. Alpha9 integrin promotes neurite outgrowth on tenascin-C and enhances sensory axon regeneration. J
Neurosci. 2009 Apr 29;29(17):5546-57
Tan et al, 2012. Kindlin-1 enhances axon growth on inhibitory chondroitin sulfate proteoglycans and promotes sensory axon
regeneration. J Neurosci. 2012 May 23;32(21):7325-35
Supported by Christopher and Dane Reeve Foundation, Cambridge Trusts, Yousef Jameel Academic Program
34
Olfactory ensheathing cell transplants improve vertical climbing in rats after cervical
level dorsal root rhizotomy
Andrew Collins, Sara Bowie, Daqing Li, Ying Li
Spinal Repair Unit, Institute of Neurology, University College London, London, UK
Andrew.Collins@ucl.ac.uk
A brachial plexus injury (BPI) involves damage to spinal roots at the cervical level of the
spinal cord. Such injuries most often result from road traffic accidents and can lead to sensory or
motor impairment. Up to 90% of BPI patients also face permanent pain, described by some as a
1
“burning and crushing” sensation on their arm . A lack of relevant preclinical models is one factor
behind the lack of effective treatments.
Transplants of olfactory ensheathing cells (OECs) have evoked long distance axon
2,3
regeneration in thoracic level lesions and restored breathing in a high cervical injury model . A matrix
method of transplantation was developed to ensure retention of the OEC transplant at the dorsal root
4
entry zone . We sought to establish a rat model of dorsal root injury (DRI) which mimics both the
sensory impairment and pain aspect of a BPI. The effect of OEC transplants on these parameters
would then be assessed.
Unilateral transection of C6, C7, C8 and T1 dorsal roots at the dorsal root entry zone (DREZ)
led to a long-term forelimb sensory deficit. Proprioceptive function and forepaw tactile sensitivity were
measured using vertical cage climbing and adhesive tape tests, respectively. Those rats which
received an acute transplant of GFP-labelled OECs climbed better than controls at 7- and 8-weeks
post injury, despite maintaining a deficit in forepaw tactile sensitivity. In terms of pain, a separate
cohort of (control) rats showed no increased sensitivity to mechanical, thermal or cold stimuli after C6T1 rhizotomy.
Immunohistochemical staining of the DREZ and dorsal horn revealed histological differences
between ipsilateral and contralateral sides after C6-T1 rhizotomy. Markers for GFAP, laminin, CGRP,
neurofilament and VGLUT were identified on transverse and longitudinal sections at various time
points up to 8 weeks post-injury. Clear differences were apparent between intact and injured regions
but further quantitative analysis is required to determine any potential effect of OECs.
A C7C8 dorsal root avulsion study is ongoing which is more likely to induce forepaw
sensitivity to cold, mechanical or thermal stimuli due to greater vascular damage and cell loss at the
5
dorsal horn . Specific preclinical models of cervical injury tailored to study either sensory impairment
or pain should allow us to assess the efficacy of OECs and optimize their use in patients.
References
1. Waikakul S. et al. 2000 J Med Assoc
2 .Ramon-Cueto A. et al. 1992 J Neurosci
3. Li Y et al. 2003 J Neurosci
4. Li et al. 2004 Exp Neur
5. Chew DJ et. al 2008 Neurosci Lett
Supported by Medical Research Council
35
Transcriptomic changes evoked by culturing Dorsal Root Ganglion neurons
overwhelm those evoked by axonal injury
1
1
Matthew C. Danzi , Dario Motti , John L. Bixby
1
1,2,3
, and Vance P. Lemmon
2
1,3
3
The Miami Project to Cure Paralysis, Departments of Molecular and Cellular Pharmacology, Neurological
Surgery, Miller School of Medicine, University of Miami, Miami, FL, USA
m.danzi@med.miami.edu
A necessary step toward functional recovery after spinal cord injury is axon regeneration.
Unfortunately, Central Nervous System (CNS) axons regenerate poorly. In contrast, injured axons of
the Peripheral Nervous System (PNS), such as those of Dorsal Root Ganglia (DRG), often succeed in
regenerating long distances. For this reason, the regenerative capabilities of DRG neurons are
commonly studied both in vitro and in vivo. We performed RNA-Seq on DRG neurons isolated from
mice that had undergone a sciatic nerve crush, DRG neurons from mice that had undergone a sham
surgery, and DRG neurons grown in culture, and characterized the transcriptomic similarities and
differences among these three DRG sample groups. This characterization included ontological
analysis of the pathways and biological processes overrepresented by gene isoforms differentially
expressed by each of the samples relative to the others, analysis of the diversity of transcription start
sites and examination of isoform expression frequency distributions. Additionally, we predicted the
relative overrepresentation of different transcription factor binding sites among the isoforms
differentially expressed in pairwise comparisons of each of the sample groups. Our analysis revealed
that placing DRG neurons in culture had a significantly greater effect on their transcriptome than did a
nerve crush carried out in vivo. Since DRGs in culture bear so little resemblance to their regenerationcompetent or actively regenerating counterparts in vivo, our findings call into question the validity of
using cultured DRG neurons as a model for gene expression by regeneration-competent neurons.
This work was supported by the National Institutes of Health grants HD057521 (to V.P.L and J.L.B.), and NS059866 (to
J.L.B.), DOD grant W81XWH-05-1-0061 (to V.P.L. and J.L.B.), State of Florida Specific Appropriation 538, the Buoniconti Fund
and the Walter G. Ross Distinguished Chair in Developmental Neuroscience (to V.P.L)
36
Nanotechnology in neuroregeneration
1,
1
2
3
Suradip Das , Manav Sharma , Dhiren Saharia , Kushal Konwar Sharma , Utpal Bora
1
1,4*
2
Department of Biotechnology, Indian Institute of Technology Guwahati; Saharia’s Path Lab & Blood Bank;
4
Department of Surgery and Radiology, College of Veterinary Sciences, Khanapara; Mugagen Laboratories
Private Limited, Technology Incubation Centre, IIT Guwahati, Guwahati, Assam, India
3
*Corresponding author – ubora@iitg.ernet.in, ubora@rediffmail.com
The application of nanotechnology for the development of advanced functional scaffolds for
neural regeneration has been predominantly restricted to the fabrication of nanofiber based scaffolds.
Although forming nano-dimensional fibers through electrospinning remains the preferred fabrication
method, some notable work has also been reported using self assembly of peptides to form
nanofibers. Self assempbled peptide nanofibers (SAPNs) having sequence arginine, alanine,
aspartate, and alanine (RADA)16 have been shown to support adhesion of neuronal cells (PC12) in
vitro, enhancing neurite outgrowth as well as facilitating functional synapse formation (Holmes et al,
2000). In vivo administration of this peptide has shown success in brain lesion repair by promoting
axonal regeneration and knitting the brain tissues together by forming nanofibers (Ellis-Behnke et al,
2006). The injectable sol-gel nature of SAPNs make them ideal for CNS interventions whereas
electrospun nanofibrous scaffolds have been mostly used in fabricating implantable nerve growth
conduits to facilitate peripheral nerve regeneration. Several natural materials like chitosan, laminin,
silk fibroin, as well as synthetic polymers like PGA, PLA and composites like laminin-PLLA, collagenPCL etc have been used as biomaterials to form nano-scaffolds by electrospinning technique.
Electrically conductive nanomaterials like grapheme films and foams have been reported to support
growth of mouse hippocampal cells as well as promote differentiaition of neural stem cells (NSCs)
towards astrocytes and neurons (nf4, nn6). Carbon nanotube based scaffolds have also emerged as
potential platforms promoting neural regeneration due to their ability to interface with neuronal circuits,
synapses and facilitating conduction of nerve impulse. On the other hand, the use of nanoparticles in
neurology has been largely limited to applications in drug delivery and diagnostics. There are very few
reports on the use of nanoparticles for enhancing nerve regeneration.
In the present poster, we attempt to review the extent of nanotechnology interventions in
solving neuro-regeneration problems as well as indicate the untapped potential of the field where
future research could be directed.
37
Transduction of cells with a lentiviral vector encoding mammalian chondroitinase
ABC in an in vitro model of neurite outgrowth: enhanced neurite outgrowth is
accompanied by a drop in PTEN levels
1
2
2
1
Priscilla Day , Joao Alves , James Fawcett , Roger Keynes , Elizabeth Muir
1
2
1
Department of Physiology Development and Neuroscience, University of Cambridge, Cambridge, UK
John Geest Centre for Brain Repair, University of Cambridge, Cambridge UK
It is known that chondroitin sulphate proteoglycans (CSPGs) bind to some receptors also
bound by myelin inhibitory proteins, and it is likely that they use some of the same signalling pathways
to bring about inhibition of neurite outgrowth. Using SH-SY5Y cells differentiated with retinoic acid and
DMEM containing 1% FCS and CSA at 75µg/ml, we show here that, in common with myelinassociated glycoprotein, digestion of chondroitin-4-sulphate (CSA) with chondroitinase via
transduction of the cells with a recombinant lentivirus (LVChABC) encoding the modified
chondroitinase transgene, results in a drop in cellular Phosphatase and tensin homolog (PTEN)
mRNA and protein levels. This is accompanied by enhanced neurite outgrowth on an inhibitory
substrate (CSA) compared to untransduced controls. Addition of the PTEN-specific inhibitor
VO(OH)pic resulted in a similar increase in neurite outgrowth, an effect that was not further enhanced
by transducing the cells with the ChABC vector, consistent with a role for PTEN in CSPG-based
inhibition of neurite outgrowth. Staining of the cells for beta1 integrin showed that it is up-regulated in
cells containing the ChABC transgene, suggesting that CSPGs, like myelin inhibitors, may negatively
remodel integrins at the cells surface in a PTEN-dependent manner.
38
Regulation of IL10 by chondroitinase ABC promotes a distinct immune response
following spinal cord injury
Athanasios Didangelos, Michaela Iberl, Elin Vinsland, Katalin Bartus and Elizabeth J. Bradbury
Wolfson Centre for Age Related Diseases, Guy’s Campus, King’s College London, London, UK
athanasios.didangelos@kcl.ac.uk
Abstract Chondroitinase ABC (ChABC) has striking effects on neuronal plasticity after spinal
cord injury (SCI) but little is known about its involvement in other pathological mechanisms. Recent
work from our lab showed that ChABC might also modulate the immune response by promoting M2
macrophage polarization (Bartus et al. 2014, Journal of Neuroscience, 34: 4822-36). Here we
investigate in detail the immunoregulatory effects of ChABC following clinically-relevant SCI in rats.
Initially, we examined the gene expression profile of 16 M1/M2 macrophage polarization markers at 3
hours and 7 days post-injury. ChABC treatment had a clear effect on the immune signature after SCI.
More specifically, ChABC increased the expression of the anti-inflammatory cytokine IL10,
accompanied by a reduction in the pro-inflammatory cytokine IL12B in injured spinal tissue. These
effects were associated with a distinct, IL10-mediated anti-inflammatory response in ChABC-treated
spinal cords. Mechanistically, we show that IL10 expression is driven by tissue injury and macrophage
infiltration, while the p38 MAP kinase is the central regulator of IL10 expression in vivo. These
findings provide novel insights into the effects of ChABC in the injured spinal cord and explain its
immunoregulatory activity.
Supported by Medical Research Council UK and Rosetrees Trust
39
The role of PKA in translating rehabilitative training after SCI into neuroplasticity
Karim Fouad, C Hurd, D Wei, D Galleguillos, S Sipione
University of Alberta, Edmonton, Alberta, Canada
Karim.fouad@ualberta.ca
It is well accepted that rehabilitative training promotes neuroplasticity and functional recovery
following injuries of the central nervous system. One mechanism that could be involved in translating
training and the subsequent neuronal activity into plasticity and recovery of function is the frequently
reported up-regulation of BDNF. Another possible mechanism is via an activity induced up-regulation
of cAMP levels, which would promote neurite outgrowth possibly via the activation of PKA. Earlier
studies supported this idea by showing that electrical stimulation of a peripheral nerve increased
intracellular cAMP levels and promoted neurite outgrowth in sensory fibers. We also reported that
rehabilitative training can counteract (at least partially) the drop in PKA activation in cortical neurons
following spinal cord injury. In our quest to show that PKA is a key player in translating training into
neuroplasticity we intended to inhibit PKA activation in the motor cortex during a rehabilitative phase
(i.e., single pellet reaching) in rats with a cervical spinal cord injury. However, as so often, the
hypothesis was quickly disproven when we found increased CST plasticity and improved training
induced functional recovery. It is noteworthy that we were able to repeat this result. Currently we are
attempting to confirm that the PKA inhibitor rp-cAMP actually did inhibit PKA activation. This however
is proofing to be a greater challenge than anticipated. If anything we are finding an increase of CREB
phosphorylation, which is the opposite of the predicted effect. In conclusion, the current view on how
neuronal activity translates into recovery is likely over simplified and other components of the pathway
have to be explored. The promoted recovery may occur via other pathways that get strengthened by
PKA inhibition or a potentially pro-inflammatory effect of the treatment.
This study was supported by a grant of the Canadian Health Research Council
40
MicroRNA-155 deletion restricts inflammatory signaling in macrophages and
enhances axon growth capacity: implications for spinal cord repair
1,2,
1,2
1,2
1,2
1,2
Andrew D. Gaudet Philipp J. Schmitt , Xinyang Xu , Amelia Hargrove , David R. Sweet ,
1,2
2,3
1,2
Zhen Guan , Mireia Guerau-de-Arellano , Phillip G. Popovich
1
2
3
Center for Brain and Spinal Cord Repair, Department of Neuroscience, Health and Rehab Sciences,
Wexner Medical Center, The Ohio State University, Columbus, OH, USA
andrew.gaudet@osumc.edu
MicroRNAs (miRs) bind to various target mRNAs limiting their expression, and consequently
their ability to encode proteins. Modifying expression of relevant miRs could elicit improved repair
after spinal cord injury (SCI) by altering responses extrinsic and intrinsic to the neuron. Our lab
showed that an intense and prolonged macrophage-dominant inflammatory response contributes to
1
SCI pathology ; therefore, discovering miRs that regulate macrophage phenotype could alter tissue
repair. In macrophages, miR-155 is a switch that initiates inflammatory cascades. miR-155 is
upregulated by inflammatory signals (e.g., toll-like receptor activation) and can regulate translation of
multiple target mRNAs to amplify inflammation.
We hypothesized that miR-155 deletion would improve macrophage-elicited axon growth and
limit pathology. In addition, we explored whether miR-155 deletion from neurons would impact
intrinsic neurite outgrowth capacity. First, we characterized the phenotype of cultured WT and miR155 knockout (KO) mouse macrophages. miR-155 KO macrophages expressed lower inflammationinduced iNOS and higher levels of the anti-inflammatory marker Ym1. When WT or miR-155 KO
macrophages were co-cultured with WT dorsal root ganglion (DRG) neurons, miR-155 KO
macrophages enhanced neurite extension and the percent of neurons bearing neurites. miR-155 also
had a neuron-intrinsic role; miR-155 deletion improved growth potential of DRG neurons. After
contusive SCI, smaller lesions in the rostral extension of the contusion sites of miR155 KO mice
correlated with reduced inflammatory macrophage density, although functional recovery was not
improved in these mice.
These data indicate that miR-155 deletion could improve tissue repair through both neuronextrinsic (macrophage phenotype) and –intrinsic (neuron growth capacity) mechanisms. Ongoing
studies will determine whether miR-155 deletion impacts axon plasticity in vivo after SCI.
References
1. Kigerl KA, Gensel JC, Ankeny DP, Alexander JK, Donnelly DJ, Popovich PG (2009) Identification of two distinct macrophage
subsets with divergent effects causing either neurotoxicity or regeneration in the injured mouse spinal cord. J Neurosci 29:
13435-13444
Supported by a Canadian Institutes of Health Research (CIHR) Postdoctoral Fellowship (ADG), The Ray W. Poppleton
Endowment (PGP), National Institutes of Health Grant R21NS081413 (PGP/MG), and International Foundation for Research in
Paraplegia (IRP) Grant #P129 (PGP/MG).
41
Artificial Intelligence in Medical Systems Neurobiology: Finding a Treatment for
Paralysis
1,
2
3
1
1
1
Baumgartner W. Jr.* Waldera-Lupa D.M.* , Pape D. , Georgiev I. , Grichtchenko I. , Hunter L. ,
#2
#1
#3
Stühler K. , Cohen K. , Grimpe B.
1
Computational Bioscience Program, School of Medicine, University of Colorado at Denver, USA
Molecular Proteomics Laboratory, Biological-Medical Research Center, Heinrich Heine University Düsseldorf,
Germany
3
Applied Neurobiology, Medical Center of the Heinrich Heine University Düsseldorf, Germany
2
* = equal experimental performance
# = equally supervising students
William.Baumgartner@ucdenver.edu,
Daniel.Waldera@uni-duesseldorf.de,
Daniel.Pape@med.uni-duesseldorf.de,
ivogeorg@gmail.com, irina.grichtchenko@gmail.de, Larry.Hunter@ucdenver.edu, Kai.Stuehler@uni-duesseldorf.de,
Kevin.Cohen@gmail.com, Barbara.Grimpe@med.uni-duesseldorf.de
Spinal cord injury (SCI) affects approximately three million people worldwide, and in spite of
over a century of research, no treatment is available. A potential solution to this problem lies in
capturing the complexity of the underlying processes that lead to regeneration failure.
To address this problem we modify an existing systems biology tool, called Hanalyzer, to work
within the SCI domain. We use this tool in conjunction with quantitative proteomics data obtained from
contused rat spinal cords tissue collected at different time points after trauma in comparison to sham
or unlesioned animals. The contusion injury model is justified because it is the most common trauma
to the spine leading to paralysis in humans. The results are consolidated in a data network and
associated with the knowledge network build within the Hanalyzer. This knowledge network is derived
from a wide variety of biological data sources including natural language text. To facilitate text mining
we defined a controlled vocabulary in order to extract the information contained in the SCI literature.
Here, a carefully selected list of words and phrases, which consists of basic scientific as well as
clinical vocabulary from the area of SCI and regeneration are used to tag units of information so that
they are more easily retrieved by a computer. The next step is the generation of the first SCI ontology
with the aim to annotate publications and databases from that domain.
The Hanalyzer is an example of a novel software tool, which deals with the problems of high
throughput data analysis, as it combines reading, reasoning, and reporting (3R) methods to facilitate
knowledge-based analysis of experimental data. The goal of the 3R system is to assist scientists in
forming explanations of the phenomena in genome and proteome-scale data, and to generate
significant hypotheses that can influence the design of future experiments.
Supported by the German Federal Ministry of Education and Research (BMBF, 01GQ1206, BG), the National Science
Foundation (NSF, LH) and National Institute of Health (NIH, 2T15LM009451, LH)
42
Does overexpression of Fibroblast Growth Factor Receptor 1 (fgfr1) in CNS neurons
enhance axon regeneration and recovery after spinal cord injury in rats?
Barbara Haenzi, Thomas Hutson, Sean Menezes, Claudia Kathe, Denise Duricki, Kat Gers-Barlag,
Mary Bartlett Bunge, Lawrence Moon
King’s College London, University of London, London, UK
barbara.haenzi@kcl.ac.uk
We have identified 500 genes which were up-regulated in adult spinal neurons that had
regenerated an axon into a Schwann cell transplant placed in the injured rat spinal cord. These genes
were screened to identify those which promote neurite outgrowth when overexpressed in CNS
neurons cultured on inhibitory substrates. Fifteen regeneration associated genes (RAGs) were
identified.
We are currently overexpressing one of the identified RAGs, fgfr1, in corticospinal axons in a
model of rat spinal cord injury. Adeno-associated viral vectors encoding fgfr1 (or a control gene)
linked via 2A to EGFP were injected into cortex and axonal regeneration and sensorimotor recovery
will be measured after unilateral pyramidotomy. In vitro: I will overexpress fgfr1 in postnatal CNS
neurons and investigate the cell signaling pathway in these cells in detail. I hope to identify candidates
for co-overexpression to increase regenerative performance of spinal axons in vitro and in vivo. I will
stimulate the pathway with different ligands and identify the ligand with the best effect. The most
potent ligands will be used for in vivo stimulation of CNS axon regeneration. Furthermore, we have
mutated important tyrosine residues of the fgfr1 receptor to dissect the downstream pathway most
important for axon regeneration.
In summary, we aim to improve repair after spinal cord injury by overexpression of the fgfr1
receptor in corticospinal neurons.
Supported by the Swiss National Foundation (BH), the Christopher and Dana Reeve Foundation (MBB) the Henry Smith
Charity (TH and LM), the International Spinal Research Trust (ISRT) (CK and LM), and Wings For Life (LM)
43
AAV9-IL4 exacerbates a pathogenic systemic immune response that impairs
functional recovery after contusive spinal cord injury
1, 2
Jodie C.E. Hall ; Kristina A. Kigerl
3
1, 2
, Phillip G. Popovich
1
1, 2
; Kevin D. Foust
1, 2
1
; Alexander D. Roszman ; Brian K. Kaspar
2,
2
Center for Brain and Spinal Cord Repair, Department of Neuroscience, Wexner Medical Center, The Ohio State
University, Columbus, Ohio, USA
3
Center for Gene Therapy, The Research Institute at Nationwide Children’s Hospital, Columbus, Ohio, USA
Jodie.Hall@osumc.edu
Spinal cord injury (SCI) elicits both a local and systemic immune response, which results in
macrophage accumulation at the injury site. The composition of activation stimuli at sites of
inflammation determines their phenotype and function. Classically activated ‘M1’ macrophages
predominate after SCI and have neurotoxic effector functions. Conversely, alternatively activated ‘M2’
macrophages are not toxic and augment regenerative growth in adult sensory axons, but these cells
1
occupy the lesion site only transiently . Interleukin-4 (IL4) is a cytokine that causes newly activated
1
macrophages to differentiate into M2 cells . We have created a viral vector engineered to produce
IL4, a non-invasive tool that reprograms macrophages at the injury site. Our hypothesis is that AAV9IL4 will promote differentiation of intraspinal macrophages toward an alternatively activated
phenotype, limiting intraspinal pathology and promoting functional recovery.
Two hours after a moderate (75 kDyn) or mild (60kDyn) mid-thoracic contusion SCI, female
C57BL/6 mice were injected (i.v.) with an adeno-associated viral (AAV9) vector engineered to
produce IL4 (AAV9-IL4) or GFP (AAV-GFP) under the control of a cytomegalovirus (CMV) promoter
9-11
(1 x 10
vg/mouse). Open field locomotor function was assessed regularly, blood was collected and
mice were perfused and tissues processed for western blot or histology.
Our data show that AAV9-IL4 causes widespread intraspinal transduction and a significant
shift in macrophage phenotype at the injury site (measured using M1 and M2 markers). However,
counter to our hypothesis, lesion pathology is exacerbated and functional recovery is impaired relative
to mice injected with AAV9-GFP. Similar results were obtained after titrating the dose or severity of
SCI. We suspected that systemic complications caused by SCI were synergizing with the immunemodulatory effects of IL4. Indeed, innate and adaptive immune responses elicited by SCI arise in
secondary lymphoid tissues (outside the CNS) and the effects of i.v. AAV9 will manifest throughout
the body (non-specific tropism). New data show that AAV9-IL4 stimulates IL4R expression on
circulating leukocytes and causes leukocytosis. Previously, we found that contusive SCI activates
2
pathogenic autoreactive B cells . Since IL4 is a B cell growth factor, AAV9-IL4 could exacerbate a
systemic autoimmune response elicited by SCI. Our data show that IL4 dependent signaling via
AAV9-IL4 is enhanced after SCI and exacerbates SCI-induced splenomegaly. These changes in the
periphery correlate with increased numbers of intraspinal B cells and anti-CNS antibodies found in
AAV9-IL4 SCI mice. Thus, post-injury intravenous injection of AAV9-IL4 redirects the intraspinal
inflammatory environment to an alternative macrophage phenotype, but it also enhances pathogenic
autoantibody synthesis and exacerbates functional recovery.
Future work will explore intraspinal IL4 injection, use of a second-generation AAV9 vector in
which IL4 production is controlled by a GFAP promoter (limit synthesis to CNS) and evaluate
axon/glial interactions within the M2 dominant lesion foci of AAV9-IL4 injected mice.
References
1. Kigerl KA, Gensel JC, Ankeny DP, Alexander JK, Donnelly DJ, Popovich PG (2009) Identification of Two Distinct
Macrophage Subsets with Divergent Effects Causing either Neurotoxicity or Regeneration in the Injured Mouse Spinal
Cord. J Neurosci 29: 13435-13444
2. Ankeny DP, Guan Z, Popovich PG (2009) B cells produce pathogenic antibodies and impair recovery after spinal cord
injury in mice. J Clin Invest 119(10): 2990-9
Supported by the International Spinal Research Trust (ISRT) and the Craig. H Neilsen Foundation
44
Human neural progenitors transplanted to the deaffarented murine spinal cord
promote regeneration of functional sensory fibers
1
1
1
2
2
Jan Hoeber , Carl Trolle , Niclas König , Allessandro Gallo , Emmanuel Hermans , Ronald
2
1
Deumens , Elena Kozlova
1
Regenerative Neurobiology, Department of Neuroscience, Biomedical Centre, Uppsala University, Sweden
Neuropharmacology, Pole Cellular and Molecular, Institute of Neuroscience, Université Catholique de Louvain,
Belgium
2
jan.hoeber@neuro.uu.se
Dorsal root avulsion (DRA) leads to a loss of sensorimotor functions due to deaffarentation
and direct injury of the dorsal horn (Carlstedt 2008). Therapies for the recovery of sensorimotor
functions in DRA need to address two major issues, first avulsed dorsal root axons fail to regenerate
across the CNS-PNS border and second to prevent the loss of second-order neurons in the dorsal
horn (Chew et al. 2008). Human embryonic cell derived neuronal progenitors (hNPE) have been
shown to promote regeneration of axons when transplanted into the transected spinal cord (Erceg et
al. 2010). Recently, we showed that murine neural progenitors are able to survive, migrate and
differentiate into different neuronal subtypes when transplanted to the junction between avulsed
dorsal root and injured dorsal root transitional zone (DRTZ) (König et al. 2014). By combining our
stem cell transplantation approach in a model of DRA of lumbar spinal roots 3-5 (L3-L5 DRA) with the
regenerative abilities of hNPE, we aimed to test the ability of hNPE to support regeneration of sensory
fibers across the CNS-PNS border and their potential for the recovery of sensorimotor functions.
hNPE were generated from human embryonic stem cells and expressed markers typical for a
spinal neuronal lineage of neuronal progenitors. 3 month after transplantation to the site of spinal cord
injury caused by L3-L5 DRA, hNPE were found to differentiate into interneurons and inhibitory nerve
cells. Further, transplantation of hNPE led to the ingrowth of myelinated sensory axons. Sensory
axons regenerated across the region of hNPE transplantation and grew back into the dorsal horn. In
long term behavioral experiments, L3-L5 DRA animals showed a severe loss of hind limb sensitivity
and grip strength. In contrast, hNPE treated animals showed both improved sensitivity and strength.
Rhizotomy peripheral to the region of hNPE transplantation completely abolished the observed
improvement.
Taken together, hNPE transplantation to the site of L3-L5 DRA resulted in good survival and
differentiation of transplanted cells and induced regeneration of functional sensory axons, resulting in
the recovery of sensorimotor functions in the dorsal root avulsion model.
References
Carlstedt, T. (2008). Root repair review: basic science background and clinical outcome. Restorative neurology and
neuroscience, 26(2), 225-241
Chew, D. J., Leinster, V. H., Sakthithasan, M., Robson, L. G., Carlstedt, T., & Shortland, P. J. (2008). Cell death after dorsal
root injury. Neuroscience letters, 433(3), 231-234
Konig, N., Trolle, C., Kapuralin, K., Adameyko, I., Mitrecic, D., Aldskogius, H., Shortland P. J. & Kozlova, E. N. (2014). Murine
neural crest stem cells and embryonic stem cell‐derived neuron precursors survive and differentiate after transplantation in a
model of dorsal root avulsion. Journal of tissue engineering and regenerative medicine. [Epub ahead of print]
Erceg, S., Ronaghi, M., Oria, M., Roselló, M. G., Aragó, M. A. P., Lopez, M. G., Radojevic, I., Moreno-Manzano, V., RodríguezJiménez, F. J., Bhattacharya, S. S., Cordoba, J. & Stojkovic, M. (2010). Transplanted oligodendrocytes and motoneuron
progenitors generated from human embryonic stem cells promote locomotor recovery after spinal cord transection. Stem Cells,
28(9), 1541-1549
This work was supported by the Swedish Research Council, proj no. 20716, Stiftelsen Olle Engkvist Byggmastare, Signhild
Engkvist’s Stiftelse and the Swedish Institute’s Visby program Dnr 00613/2011
45
Do severity and duration of compression impact on recovery after severe acute spinal
cord injury in dogs?
Hilary Hu, Nick Jeffery
College of Veterinary Medicine, Iowa State University, Ames, USA
hilaryhu@iastate.edu
Evidence from laboratory experiments suggests that recovery after spinal cord injury (SCI) is
1
adversely affected by more prolonged and severe compression. This suggests that it would be
beneficial for human SCI patients to undergo decompressive surgery as early as possible, but this
2,3
suggestion remains controversial. SCI is very common in domestic pet dogs (because intervertebral
disc herniation occurs with high prevalence), providing an ideal model in which effects of various
variables that impact on SCI in human patients can be investigated.
In this study we prospectively recruited dogs that had incurred thoracolumbar SCI that had
abolished all motor and sensory function to the hindquarters (i.e. equivalent to ASIA ‘A’ patients). We
recorded historical data that indicated the duration of cord injury and the dogs then underwent routine
imaging and decompressive surgery (which in dogs is routinely carried out as soon as possible for
this category of SCI). Maximal severity of cord compression in each dog was measured on cross
sectional MR and CT images Dogs were followed up until they recovered independent ambulation or
for three months from surgery (which in dogs is equivalent to ~12 months in humans in terms of
functional recovery). Logistic regression was used to evaluate the relationship of recovery with
duration and severity of compression and Cox regression used to analyze variable relationships with
rapidity of recovery.
78 cases were available for full analysis. Our data revealed that the delay from onset of
paraplegia till surgical intervention had no detectable effect on the likelihood of recovery. There was
weak evidence that more severe compression was associated with greater likelihood of recovery –
and even weaker evidence that slower onset of clinical signs prior to onset of paraplegia also had the
same effect. There was no evidence for an interaction between severity of compression and delay
between onset of paraplegia and surgery. In the second series of analyses we determined that, in
animals that recovered after surgery, more severe compression and longer duration of clinical signs
prior to onset of paraplegia were associated with more rapid recovery.
We conclude it is likely that, in this type of SCI in which an impact injury is followed by
prolonged spinal cord compression of varying severity, the prognosis appears to be largely defined by
the magnitude of the initial mechanical injury and that early decompressive surgery has no detectable
benefit. However, for cases in which the initial injury has not caused cross sectionally complete
destruction of the spinal cord, decompressive surgery within the first few days after injury may play a
role in reversing the clinical signs and accelerating functional recovery.
References
1. Dimar JR 2nd, Glassman SD, Raque GH, et al. The influence of spinal canal narrowing and timing of decompression on
neurolgic recovery after spinal cord contusion in a rat model. Spine 1999;24:1623-33
2. Fehlings MG, Vaccaro A, Wilson JR, et al. Early versus delayed decompression for traumatic cervical spinal cord injury:
results of the Surgical Timing in Acute Spinal Cord Injury Study (STASCIS). PLoS One 2012;7:e32037
3. van Middendorp JJ. Letter to the editor regarding: "Early versus delayed decompression for traumatic cervical spinal cord
injury: results of the Surgical Timing in Acute Spinal Cord Injury Study (STASCIS)”. Spine J 2012;12:540
Supported by
Hilary Hu is a graduate student funded through a grant from the International Spinal Research Trust (ISRT) to
investigate the effect of intralesional chondroitinase ABC on functional outcome after severe SCI in dogs.
46
Sympathetic and sensory sprouting after spinal cord injury: peripheral consequences
of central injuries
Diana V. Hunter, L.M. Ramer, M.A. Crawford, P.A. van Stolk, S.B. McMahon, M.S. Ramer
ICORD, University of British Columbia, Vancouver, Canada and Wolfson Centre for Age-Related Diseases,
King’s College London, London, UK
dianavhunter@gmail.com
Though motor system deficits may be the most outwardly apparent challenge after spinal cord
injury (SCI), dysfunction of the sensory and autonomic systems can result in an equal if not greater
impact on quality of life. Numerous studies have described injury-induced changes in sympathetic and
sensory pathways within the spinal cord itself, however less is known about how the peripheral
components of these pathways change and impact function after SCI. This work characterizes the
peripheral changes that occur within sensory (dorsal root ganglia; DRGs) and sympathetic ganglia,
with specific interest in the peripheral sprouting of these two neuronal populations. To examine the
changes in these two systems after SCI, a complete transection at the third thoracic (T3) level was
performed in adult male Wistar rats and both sensory and sympathetic ganglia were collected and
examined after one, two or four weeks. The density of sympathetic fibers within the DRGs rostral (T1),
caudal (T5, T10), and far distal (L1-S1) to the injury was analyzed immunohistochemically using
antibodies against tyrosine hydroxlase (TH). Sympathetic ganglionic axons invaded the cell layer of
the DRG within one month of SCI. The density of the TH-expressing axons in DRGs was significantly
increased in the SCI animals at one month when compared to sham-injured controls. Interestingly,
sympathetic sprouting was most pronounced in DRGs far distal to SCI. To examine sensory sprouting
in sympathetic ganglia, major pelvic ganglia, mixed sympathetic/parasympathetic ganglia of the pelvic
regions, were also collected and processed for markers of sensory axons. The changes occurring
within these two types of peripheral ganglia indicate that dysfunction after SCI may be mediated by
factors outside of spinal plasticity and the central nervous system. Further study into the crossing of
the sympathetic and sensory systems may shed light into both sensory and autonomic dysfunction
such as neuropathic pain and autonomic dysreflexia.
Supported by the International Spinal Research Trust (ISRT), ICORD and NSERC
47
Chondroitinase gene therapy as a treatment for spinal cord injury
Nicholas D. James, Katalin Bartus, Karen D. Bosch, John H. Rogers, Bernard L. Schneider, Joost
Verhaagen, Elizabeth M. Muir, Elizabeth J. Bradbury
King’s College London, Regeneration Group, The Wolfson Centre for Age-Related Diseases, London, UK
nicholas.d.james@kcl.ac.uk
Spinal cord extracellular matrix is densely packed with growth inhibitory chondroitin sulphate
proteoglycans (CSPGs), which become more abundant after injury. Thus, matrix modification has
become a leading experimental strategy for promoting repair following spinal cord injury. Despite the
beneficial effects that have been achieved by digesting CSPGs with the bacterial enzyme
chondroitinase ABC (ChABC), the potential for achieving long term efficacy in traumatic injuries that
mimic a human spinal cord injury has been limited, due to suboptimal delivery methods and issues of
enzyme instability. However, we have recently demonstrated that gene therapy, using a mammalian
compatible ChABC gene, offers a route to achieving stable and continuous delivery of ChABC,
resulting in a dramatic reduction in pathology and significant improvements in functional recovery
1
when used to treat a clinically relevant spinal contusion injury model in adult rats . Following on from
these findings we now demonstrate the efficacy of chondroitinase gene therapy in contusion injury
models at differing spinal levels (cervical and thoracic) and of differing severities. When used to treat
a contusion injury at either C5 or T10 spinal level, ChABC gene therapy resulted in increased spinal
conduction through the injury epicenter, improved functional performance in skilled locomotion,
significant neuroprotection and enhanced plasticity of intact spinal circuitry. Further to this, we present
findings from recent experiments in which we have assessed the efficacy of different viral vectors
(both adeno-associated viral vectors and lentiviral vectors) containing the modified ChABC gene in
order to determine the optimal vector structure to be used for ChABC gene therapy. We find that the
use of different promoters results in differing patterns of ChABC expression, due to which cell types
are transduced. Furthermore, the timing and pattern of expression affects the efficacy of ChABC gene
therapy. The use of a PGK promoter primarily leads to transduction of neuronal cells and axons,
resulting in widespread CSPG degradation throughout the spinal cord and the most dramatic
improvements in functional and anatomical outcome measures. Thus, we demonstrate the therapeutic
potential of ChABC gene therapy to treat clinically relevant injury models at different spinal levels and
present findings on optimizing the delivery of chondroitinase gene therapy.
References
Bartus, K, James, ND, Didangelos, A, et al. Large-Scale Chondroitin Sulfate Proteoglycan Digestion with Chondroitinase Gene
Therapy Leads to Reduced Pathology and Modulates Macrophage Phenotype following Spinal Cord Contusion Injury. J
Neurosci. 2014; 34:4822-4836
Supported by the U.K. Medical Research Council, the International Spinal Research Trust (ISRT), the Henry Smith Charity
and the International Foundation for Research in Paraplegia
48
Combinatorial treatment with GSK3β inhibitors and chondroitinase ABC to regulate
glial scar formation and promote axon regeneration in the spinal cord
1,2
2
2
1
2
Ashik Kalam , Athanasios Didangelos , Katalin Bartus , Andrea Rivera , Nicholas James ,
2
1
Elizabeth Bradbury , Arthur Butt
1
2
Institute of Biomedical and Biomolecular Sciences, University of Portsmouth, Portsmouth, UK
Wolfson CARD, Guy’s Campus, King’s College London, London, UK
ashik.kalam@myport.ac.uk
The glial scar is the key reason for the failure of axonal regeneration in the adult CNS and
1
acts as a physical and biochemical barrier for growing axons. Functional regeneration in the spinal
cord is thus limited by the glial scar inhibiting axonal growth. Therefore, modification of the glial scar
to a more growth permissive one is necessary for any regeneration to occur.
Lithium and small molecule inhibitors of GSK3β has been shown to trigger profound changes
in the astroglial cytoarchitecture in the optic nerve and promote neuronal survival and neurite
2,3
outgrowth in vitro and axonal sprouting following spinal cord injury (SCI). Hence, GSK3β is a
potential target for regulating astrogliosis and stimulating the formation of a potential scaffold for
regenerating axons. Moreover, the glial scar contains chondroitin sulphate proteoglycans (CSPGs)
that inhibit axon growth following SCI. Enzymatic digestion CSPGs with chondroitinase ABC (ChABC)
has been shown to disrupt the glial scar and promote axon sprouting and partial recovery of
4,5
function. Therefore, the gliomorphic effects of GSK3β inhibitors combined with enzymatic removal
of CSPGs could have a synergistic effect on promoting regrowth of axons, reformation of connections
and recovery of function in SCI.
The aim of this collaborative project is to determine the effects of a combinatorial therapy of
GSK3β inhibition and ChABC on glial scar formation and axon regeneration, using multiple
techniques and models, including an in vitro scratch assay as a model for astroglial scar formation, ex
vivo organotypic cultures of spinal cord and optic nerve, established rodent models of SCI, and
genomic, biochemical, histological, electrophysiological and behavioural analyses.
References
1. Sandvig et al. (2004) Glia. 46(3):225-51
2. Azim K and Butt AM. (2011) Glia. 59(4):540-53
3. Dill J et al (2008). J Neurosci. 28(36):8914-28
4. Bradbury EJ et al. (2002) Nature. 416(6881):636-40
5. Bartus K et al. (2014) J Neurosci. 34(14):4822-36
Supported by the Nathalie Rose Barr studentship award from the International Spinal Research Trust (ISRT)
49
Optimizing and understanding the use of intracellular sigma peptide as a translatable
therapeutic for spinal cord injury
1
1
1
1-2
Bradley T. Lang , Jared M. Cregg , Marc DePaul , Amanda Tran, Benjamin Brown , Sarah A.
1
3
1
Busch , Yingjie Shen , Jerry Silver
1
Case Western Reserve University, Department of Neurosciences, Cleveland, OH, USA
Baldwin Wallace University, Berea, OH, USA
3
The Ohio State University, Center for Brain and Spinal Cord Repair, Department of Neuroscience, Columbus,
OH, USA
2
BTL21@case.edu
Regeneration and sprouting following spinal cord injury is curtailed by several processes, with
the inhibitory chondroitin-sulfate proteoglycan (CSPG)-rich glial scar and perineuronal net being major
impediments. We designed a small membrane-permeable peptide modulator of the CSPG receptor
PTPσ (Intracellular Sigma Peptide, ISP), which was capable of blocking CSPG-mediated inhibition in
1-3
vitro . Delivered systemically over several weeks, ISP treatment restored coordinated walking and
urinary function following contusive SCI in a large percentage of animals. We sought to further
characterize the mechanism by which ISP regulates PTPσ function in order to devise strategies
toward optimizing treatment. We previously determined that ISP binds to the intracellular domain of
PTPσ. Using in silico techniques, we identified a potential binding pocket for ISP near the wedge
domain of PTPσ. ISP truncation and alanine substitution analysis using an in vitro CSPG gradient
assay provided experimental evidence for this in silico finding. We are currently performing the
necessary point mutation studies to confirm this binding site.
Interestingly, in addition to binding rat and mouse PTPσ, ISP also bound human recombinant
PTPσ. Therefore, we tested whether ISP treatment could overcome CSPG inhibition in more clinically
relevant human neurons. Human induced pluripotent neurons had severely diminished outgrowth on a
CSPG rich substrate vs a laminin only control substrate. Importantly, treatment with Ch’ABC as well
as ISP restored outgrowth of human neurons to that of laminin levels.
Finally, we sought to optimize the dose of ISP in vivo. Following a T8 contusion injury (Infinite
Horizon Impactor, 250kDyne), we treated animals daily with increasing doses of ISP, from 3.3μg to
44μg, for 7 weeks (n=5/dose). Escalating ISP to 44μg/day (4x of previous dose) led to a dramatic
improvement in urinary function, with all animals recovering function. This suggests that increasing
ISP efficacy, either through increased concentration or possibly via more direct delivery paradigms,
may further enhance urinary recovery. Interestingly, locomotor recovery, as measured by open field
BBB and gridwalk tests, was not dose dependent in our small sample of animals. A larger number of
animals may be required to show a dose response in these locomotor behaviors. We hypothesize
that combinatorial therapies in conjunction with ISP, including a variety of neuroprotective and
rehabilitation strategies, will be necessary to promote maximal recovery. Our data provide strong
verification of the role of CSPGs and PTPσ in regeneration/sprouting failure following neurological
trauma.
References
1. Shen, Y., A. P. Tenney, et al. (2009). "PTPsigma is a receptor for chondroitin sulfate proteoglycan, an inhibitor of neural
regeneration." Science 326(5952): 592-6
2. Lang, B.T., Cregg, J.M, et al (2014). “Systemic modulation of the proteoglycan receptor PTPσ promotes functional recovery
after spinal cord injury” Under Review
3:.Gardner, R. T., L. Wang, et al. (2014). "Targeting protein tyrosine phosphatase σ after myocardial infarction restores cardiac
sympathetic innervation and prevents arrhythmias." Under Review
Supported by NINDS NS25713 (JS); Case Western Reserve University Council to Advance Human Health (CAHH), Unite to
Fight Paralysis, The Brumagin Memorial Fund, Spinal Cord Injury Sucks (SCIS), and United Paralysis Found
50
Early intravenous delivery of mesenchymal progenitor cells modulates the secondary
inflammatory response after cervical spinal cord injury leading to behavioral and
pathological amelioration
1,3
1
2
2
3
Seok Voon Lee , Chris Czisch , Yingxiang Huang , May H. Han , Alan R. Harvey , Giles W. Plant
1
1
Department of Neurosurgery, Stanford University, Stanford, CA, USA
Department of Neurology and Neurological Sciences, Stanford University, Stanford, CA, USA
3
School of Anatomy, Physiology and Human Biology, The University of Western Australia, Perth, Australia
2
svlee@stanford.edu
Cellular transplantation strategies utilizing mesenchymal progenitor cells (MPCs) have
previously been reported to be beneficial in spinal cord injury (SCI). Methods of transplantation
include intraspinal, intrathecal and intravenous (IV) injection. Our previous data have shown efficacy
1, 2
of intraspinal injection of human MPCs into rat thoracic SCI . However, intraspinal injection is
technically challenging, especially in an unstable environment after an injury, thus we have
investigated the feasibility and benefits of IV injection of MPCs in a cervical contusion SCI injury
3
model. Compact bone MPCs were isolated from GFP-luciferase transgenic mice . Co-culture data of
the MPCs with splenocytes indicate that MPCs enhance anti-inflammatory and decrease proinflammatory cytokine expression. SCI (unilateral C5, 30kdy, 3s dwell; IH impactor, NY) was
6
performed on adult female FVB mice and 1x10 MPCs in 300μL HBSS or 300μL HBSS were IV
injected via tail vein at D1, D3, D7, D10 or D14 after injury (n=5 per group). After injection, MPCs
were tracked using bioluminescence. Live in vivo imaging data showed that 24hrs after IV injection,
MPCs tracked to the lungs and remained there. After 72hrs, minimal luciferase signal was detected in
the lungs and did not appear elsewhere. This was irrespective of when the MPCs were injected
following injury. Terminal bioluminescence tracking of the MPCs confirmed that cells tracked to the
lungs and no other major organ, and were cleared within 7 days. Comparison of naïve versus injured
mice showed that MPCs were cleared more rapidly within 24hrs in the injured animals. Behavioral
testing by cylinder test recorded at D7, D21, D35 and D49 showed that animals receiving IV injection
of MPCs at D1 and D3 after injury had significant amelioration compared to their control counterparts
(p<0.05). No improvement was seen at the other injection time points. Mice were sacrificed at 8
weeks after injury. Tissue analysis showed that both D1 and D3 IV injection of MPCs resulted in
smaller lesion size compared to their controls but no difference was observed at the other time points.
Immunohistochemical analysis revealed less scarring and vascularization only at the D1 and D3 IV
MPC injection time points. An additional study where SCI mice received IV injection of MPCs or
HBSS at D1 after injury and sacrificed at 6hrs following IV injection showed that the beneficial effect
of the IV MPC injection occurred almost immediately. These findings suggest positive modulation of
secondary inflammatory responses after the initial mechanical injury. In summary, IV injection of
MPCs is neuroprotective in terms of clinical amelioration shown by behavioral testing and
histopathological changes in the damaged spinal cord. The timing of MPC delivery is crucial to
achieve this benefit.
References
1
Hodgetts SI, Simmons PJ, Plant GW (2013). Exp Neurol 248:343-59
2
Hodgetts SI, Simmons PJ, Plant GW (2013). Cell Transplant 22:393-413
3
Short BJ, Brouard N, Simmons PJ (2009). Methods Mol Biol 482:259-82
This work is supported by the Saunders Family Neuroscience Fund, James Doty Neurosurgery Fund, Stanford Neuroscience
Institute, and an International Postgraduate Research Scholarship from The University of Western Australia
51
Facilitating reproducibility and data integration for SCI research with MIASCI and
RegenBase
1
2
1
3
Vance P. Lemmon , Alison Callahan , Kunie Sakurai , Saminda W. Abeyruwan , Adam R.
4
5
3
1
Ferguson , Phillip G. Popovich , Ubbo Visser , John L. Bixby ,
1
Miami Project to Cure Paralysis, University of Miami School of Medicine, Miami, FL, USA
Stanford Center for Biomedical Informatics Research, Stanford University, USA
3
Department of Computer Science, University of Miami, Coral Gables, FL, USA
4
Brain and Spinal Injury Center (BASIC), Department of Neurological Surgery, University of California, San
Francisco, San Francisco, CA, USA
5
Center for Brain and Spinal Cord Repair and the Department of Neuroscience, The Ohio State University,
Columbus, OH, USA
2
VLemmon@med.miami.edu
The lack of reproducibility in many areas of science, including spinal cord injury (SCI)
research, is due in part to the lack of common reporting standards. Over the past three years an ad
hoc consortium of scientists has developed a minimum information reporting standard for SCI, called
Minimum Information About an SCI Experiment (MIASCI, J Neurotrauma. 2014 Jul 11. PMID:
24870067).
Version 1.0 of the MIASCI contains 11 sections: investigator, organism, surgery,
perturbagen, cell transplantation, biomaterials, histology, immunohistochemistry, imaging, behavior,
and data analysis and statistics. Each section has a number of data elements to be filled in that detail
essential metadata about the project, materials and methods. Depending on a particular study, not all
sections will apply. The purpose of MIASCI is to improve transparency of reporting and to encourage
the use of best practices. A secondary benefit is to facilitate the aggregation and automated
interrogation of diverse datasets using a formal standard language. Thus, a parallel effort is
underway to develop an ontology about SCI: the RegenBase ontology. Expanding RegenBase by
incorporating MIASCI concepts facilitates paper curation and knowledge creation. We present
MIASCI concepts, show integration with the RegenBase Ontology and present different approaches
to paper annotation. Querying the RegenBase knowledgebase using the integrated ontology will also
be illustrated.
Acknowledgments NINDS NS080145 and NICHD HD057632
52
Expression of a hyperactive transcription factor increases axon growth and
regeneration
1
1
1
1,3
1,2,3
Saloni T. Mehta , Xueting Luo , Tatiana I. Slepak , Kevin K. Park , John L. Bixby
1,3
Lemmon
1
2
, Vance P.
3
The Miami Project to Cure Paralysis, Departments of Molecular and Cellular Pharmacology, Neurological
Surgery, University of Miami Miller School of Medicine, University of Miami, Miami, FL, USA
s.mehta8@med.miami.edu
Axonal regeneration after spinal cord injury (SCI) is intrinsically and extrinsically inhibited by
multiple factors. One major form of intrinsic inhibition of axon regeneration is the altered expression of
regeneration-associated transcription factors in mature neurons of the central nervous system (CNS);
these factors fail to be activated post-injury. A gene expression study of regeneration-capable
1
peripheral nervous system (PNS) neurons identified candidate transcription factors that could
potentially, if expressed in CNS neurons, promote axon growth and regeneration. Of these, signal
transducer and activator of transcription 3 (Stat3) showed a significant upregulation in PNS neurons
compared to CNS neurons.
To maximize gene transcription and, potentially, neurite outgrowth and axon regeneration, a
constitutively active variant of Stat3 (Stat3CA) was fused with a viral transcriptional activation domain
(VP16). VP16 “hyper-activates” transcription factors by recruiting transcriptional co-factors to the DNA
binding domain. This VP16-Stat3CA chimera significantly increased neurite outgrowth when
expressed in rat cortical neurons in vitro. Furthermore, when virally transduced into retinal ganglion
cells (RGCs) in vivo, VP16-Stat3CA led to increased axon regeneration in the optic nerve. These
findings indicate that hyperactivation and overexpression of transcription factor Stat3, which is
downregulated in the adult CNS, can promote axon regeneration after injury.
References
1
Smith RP, Lerch-Haner JK, Pardinas JR, Buchser WJ, Bixby JL, Lemmon VP. Mol Cell Neurosci. 2011;46:32–44
This work was supported by the National Institutes of Health grants HD057521 (to V.P.L), and NS059866 (to J.L.B.), DOD
grant W81XWH-05-1-0061 (to V.P.L. and J.L.B.), State of Florida Specific Appropriation 538, the Buoniconti Fund and the
Walter G. Ross Distinguished Chair in Developmental Neuroscience (to V.P.L)
53
Daily acute intermittent hypoxia following cervical spinal cord injury
Kristiina Negron, Tanya Bezdudnaya, Victoria Spruance, Timothy Whelan, Michael Lane
Department of Neurobiology, Drexel University College of Medicine, PA, USA
Kmn67@drexel.edu
Among the wide range of motor and sensory deficits that arise following cervical spinal cord
injuries (SCIs), impaired breathing remains one of the most devastating. Cervical SCIs typically result
in ventilator-dependence, thus increasing the risk of life-threatening secondary complications and
greatly impairing quality of life. Respiratory dysfunction primarily results from a compromise to the
phrenic motor system which controls the diaphragm – the primary respiratory muscle. While there is
some spontaneous functional neuroplasticity, the extent of recovery is limited and significant
respiratory deficits persist. The goal of the present research is to develop rehabilitative strategies
capable of harnessing respiratory plasticity and improving long-term functional recovery.
Previous work has identified that phrenic motor activity and respiratory plasticity can be
enhanced by repeated, intermittent exposures to hypoxia. While hypoxia stimulates increases in
respiratory activity, repetitive exposures to hypoxia can elicit phrenic motor facilitation. Several studies
have shown that intermittent hypoxia can be used to promote recovery of phrenic motor function
following high cervical hemisection. Whether such increases in respiratory activity can be used to
therapeutically enhance phrenic recovery following cervical contusion injury is not well defined. The
present work begins to address this gap in knowledge by testing whether daily acute intermittent
hypoxia (dAIH) can amplify respiratory plasticity and enhance recovery one week following
experimental contusion injury.
Adult female Srague Dawley rats received a lateralized mid-cervical (C3-4) contusion injury
(Infinite Horizon pneumatic impactor; intended impact force of 200 kilodynes). These injuries disrupt
descending bulbospinal respiratory pathways and result in loss of spinal phrenic inter- and
motoneurons, leading to diaphragm paresis. In addition, the diaphragmatic response to increased
respiratory demands – which can be stimulated with hypoxia or hypercapnia - is attenuated postcontusion. One week post-SCI animals underwent five days of respiratory challenge via dAIH, which
consisted of a total of 50, 5 minute hypoxic exposures over the course of five days (10 per day).
Implanted telemetric EMG electrodes were used to assess diaphragm function pre- and post- injury,
and daily during and following IH treatment. At the end of the 5-day IH therapy, retrograde
neuroanatomical tracers (pseudorabies virus or cholera toxin beta subunit) were used to trace the
phrenic circuit ipsilateral to injury. Terminal phrenic nerve recordings were used to assess phrenic
motor output. Animals were then perfuse-fixed with paraformaldehyde for histological analyses.
Transverse spinal cord sections (40 micron, frozen sections) were immunolabeled for the presence of
5HT, c-fos and neuronal tracers. Preliminary results from these ongoing studies have suggested that
IH may elicit only a very modest effect on respiratory plasticity and phrenic function following cervical
contusion injury.
54
A novel role for Wnt signalling in regulating astrogliosis in adult white matter
Andrea Rivera, Arthur M. Butt
University of Portsmouth, Southsea, UK
andrea.rivera@port.ac.uk ; arthur.butt@port.ac.uk
Astrocytes perform multiple functions that are essential for CNS function. Following a CNS
insult, astrocytes undergo a characteristic injury response, termed ‘reactive astrogliosis’ and form a
chemo-physical barrier called the glial scar, which is inhibitory to axonal regeneration. The
mechanisms regulating the astrocyte injury response are unresolved, but Wnt signalling is implicated
in a number of different pathologies, including injury, Parkinson Disease, Amyotrophic Lateral
Sclerosis and Alzheimer’s Disease. Wnt acts through the “canonical pathway”, or GSK3b/β-catenin
pathway, and the “non-canonical” pathway, which is independent of GSK3b. Here, we have examined
canonical Wnt/β-catenin signalling in adult white matter astrocytes, using a specific Wnt3a activator.
All procedures were in accordance with the Animal Scientific Procedures Act (1986). Optic nerves
(ONs) from adult mice were isolated intact and maintained in organotypic culture for 3 days in vitro.
Wnt3a significantly increased the number of astrocytes compared to controls (p<0.05, unrelated ttests) and induced the generation of novel astrocytes with a “simple” stellate morphology. To
characterise the genotype of these novel astrocytes we compared the transcriptome of Wnt3a treated
optic nerves with control and acutely isolated tissue. We identified a number of genes specifically
altered by Wnt3a which are involved in regulation of planar cell polarity (Ctnnd1 and Cpne1). Pathway
analysis (IOA, Ingenuity Systems) identified the Axon-Guidance and NGF pathways amongst the top
pathways regulated by Wnt3a. The results identify a novel role for Wnt signalling in regulating
astrogliosis and indicate that stimulation of Wnt/β-catenin in astrocytes may provide an environment
that supports axonal growth.
Supported by the Anatomical Society and IBBS
55
Wnts: more than an axonal growth inhibitor in the adult spinal cord
1
1
1-2
Pau H. González , Carlos González-Fernández , Carmen María Fernández-Martos , Ernest
3
1
Arenas , F. Javier Rodríguez .
1
2
Laboratory of Molecular Neurology, Hospital Nacional de Parapléjicos (HNP), Toledo, SPAIN Actual address:
3
Wicking Dementia Research and Education Centre, University of Tasmania Hobart, TAS, Australia Molecular
Neurobiology Unit, MBB, Karolinska Institute, Stockholm, Sweden
fjrodriguez@sescam.jccm.es
Wnt proteins are a large family of molecules that are critically involved in CNS development.
Interestingly, although initially reported a lack of Wnt expression in the adult spinal cord of mice with
an acute reinduction after injury of few Wnt ligands and receptors [1], subsequent reports have shown
that most Wnt ligands, modulators and receptors are constitutively expressed in mice and rats with
altered expression patterns after injury [2-5]. Furthermore, Wnts have been involved in a variety of
physiological (angiogenesis, adult neurogenesis, myelination, sensory function or activity-induced
synapse formation) and pathophysiological processes (neuroinflammation, cancer and
neurodegeneration). This is driven by a vast array of Wnt signalling effects, which are elicited by a
tightly regulated pattern of expression of Wnt ligands, modulators and receptors at each cell type
involved in a particular physiological/ pathophysiological mechanism. In this regard, SCI is a
multifaceted pathophysiology with different spatio-temporal actors and requirements post-injury, and
by extension Wnt patterns and signalling events. However, little is known regarding their specific
expression patterns at cellular level in the healthy and injured adult spinal cord, which will be essential
to understand their roles in the physiology and pathophysiology of CNS and, eventually, allow for the
development of novel Wnt-targeted therapies for SCI without secondary adverse effects. Therefore,
we decided to analyse the spatio-temporal mRNA and protein expression patterns of Wnt ligands,
modulators and receptors in the spinal cord of adult rats and mice after SCI using quantitative RTPCR and single and double IHQ. In brief, our results show that:
 The mRNA encoding most Wnt ligands, soluble inhibitors and Frizzled receptors are
constitutively expressed in the healthy spinal cord of adult rats. Strikingly, contusion spinal
cord injury induced a time-dependent increase in Wnt mRNA expression from 6 hours until 28
days post-injury, and a narrow peak in the expression of soluble Wnt inhibitors between 1 and
3 days post-injury.[2, 3].
 Analysis of cellular Frizzled 5 expression pattern showed that, in the uninjured rat spinal cord,
this receptor was expressed in neurons, oligodendrocytes, astrocytes, microglia and NG2+
glial precursors. After injury, Frizzled 5 was also found in axons at all evaluated time points
and in reactive microglia/macrophages from 3 to 14 days post-injury [3].
 In uninjured adult rats, Ryk is expressed in neurons, astrocytes, and blood vessels. Following
SCI, we observed an increase in Ryk expression from 24h until 14 dpi in the damaged tissue,
where it was observed in reactive astrocytes and microglia/macrophages, NG2+ glial
precursors, fibronectin+ cells, oligodendrocytes, and axons [4].
 The mRNAs of most Wnt proteins are constitutively expressed in the uninjured spinal cord of
adult mice, where IHC revealed a differential expression of Fz1 by neurons and
oligodendrocytes and Fz4 by astrocytes. After dorsal hemisection, we found significant timedependent variations with a prominent Wnt downregulation besides an upregulation of Wif1
[5].
Our results provide compelling evidences of constitutive expression of Wnts in both rats and
mice, as well as that SCI induces dramatic changes in their spatio-temporal profiles with striking
differences between the two rodent species analyzed. Remarkably, our results also show cell-specific
expression patterns of Wnt receptors and thus suggestive for different physiological and
pathophysiological functions. Future research interests include further characterization of the roles of
each Wnt receptor in SCI with a special focus in inflammation and the development of specific
peptides for their modulation.
References [1] Liu et al. J Neurosci 2008, 28: 8376-8382; [2] Fernandez-Martos et al. PLoS One 2011; 6(11):e27000; [3]
Gonzalez et al. PLoS One 2012; 7(12): e50793; [4] Gonzalez et al. J Neurotrauma 2013, 30(10):806-17; [5] GonzálezFernández et al. J Neurotrauma 2014, 31(6):565-81
Financial support FISCAM (Grant PI2008-39) and FIS (Grants PI08/1475 and PI12/2895 with FEDER co-funding)
56
Characterization of a novel axon growth repellent and its role in spinal cord injury
Julia Schaeffer, Geoffrey Cook, Roger Keynes
Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, UK
js2064@cam.ac.uk
During the development of the peripheral nervous system, migrating neural crest cells and
outgrowing motor and sensory axons follow a segmented pattern, the mesodermal somites. Growth
cone repulsion is an important mechanism controlling axon growth, and ensuring that the peripheral
nervous system develops without obstruction by the developing vertebral column. During
1
development it guides axons by excluding them from “no-go” areas in the embryo . Following injury to
the adult brain and spinal cord it may also block regeneration, with serious clinical consequences.
Among the candidate molecules, PNA-binding glycoproteins, chondroitin sulphate proteoglycans
(CSPGs) and semaphorin 3A, a member of the semaphorin family, have been proposed as
repellent molecules acting on different receptor systems expressed by primary sensory axon growth
2
cones .
Recent work in the lab has identified a protein in embryonic somites that generates spinal
nerve segmentation by contact-repulsive axon guidance. The protein is expressed selectively on the
surface of somite cells, and cause collapse of axon growth cones when applied to cultured sensory
neurons. It has also been shown previously that detergent extracts of mammalian (rat) grey matter
3
and of a cultured line of human astrocytes possess growth cone collapse-inducing activity . Further
experiments have indicated that this CNS-derived activity has molecular properties closely similar to
that in somites, so it is possible that this contact-repulsive system has been co-opted in the CNS to
play an important role in regulating connectivity and plasticity. The overall aim of the project is to
examine this novel CNS-derived system in more detail to confirm its molecular identity, elucidate how
it is related to the somite-derived axon repellent, and assess its role in spinal cord injury.
To date, I have shown that the protein is expressed at the cell surface of a human astrocyte
cell line by live cell immunostaining and 2D gel electrophoresis and immunoblotting. Detergent
extracts of these astrocytes have been shown to cause growth cone collapse, and a lectin, jacalin, is
currently being used as an affinity reagent to further analyse this molecular system.
The experimental objectives are:
1) to test whether inhibition of the somite protein blocks growth cone collapse induced by
detergent extracts of cultured human astrocytes;
2) to identify the protein responsible for this activity in human astrocytes extracts, using
biochemical techniques (2D gel electrophoresis, Western blotting and proteomics) and
molecular cloning;
3) to assess the sites and level of expression of the protein in normal and injured adult rat CNS;
4) to test the most effective in vitro inhibitor of the biologically active protein for its ability to
promote axon regeneration and functional recovery using an in vivo rat model of spinal cord
injury (in collaboration with Prof James Fawcett, Cambridge Centre for Brain Repair).
References
1. Keynes, R. et al. Surround repulsion of spinal sensory axons in higher vertebrate embryos. Neuron 18, 889–897 (1997)
2. Kuan, C.-Y. K., Tannahill, D., Cook, G. M. W. & Keynes, R. J. Somite polarity and segmental patterning of the peripheral
nervous system. Mech. Dev. 121, 1055–1068 (2004)
3. Fok-Seang, J. et al. An analysis of astrocytic cell lines with different abilities to promote axon growth. Brain Res. 689, 207–
223 (1995)
Supported by the International Spinal Research Trust (ISRT), Nathalie Rose Barr Studentship, and the Rosetrees Trust
57
Transplantation of neural progenitors to improve respiration following spinal cord
injury
Spruance V.M., Sanchez D.E., Bezdudnaya T., Negron K.M., Whelan T.J., Reier P.J., Lane M.A.
Drexel University, Department of Neurobiology & Anatomy, Philadelphia, PA, USA
Victoria.spruance@gmail.com
Spinal cord injury (SCI) at nearly any level can result in some degree of respiratory deficiency,
and impaired breathing remains one of the leading causes of morbidity and mortality following cervical
injury. Over 50% of cervical SCI patients require assisted ventilation at some point during their care,
incurring high costs of healthcare and significantly affecting quality of life. Given the majority of SCIs
occur at the cervical level, there is an urgent need for improved therapeutic treatments targeting
respiratory function following spinal injury. In recent years, there has been a growing appreciation for
the endogenous, spontaneous neuroplasticity that can occur following injury contributing to limited
functional recovery of respiratory systems. Recent experimental studies have suggested that
interneurons may modulate phrenic motor function and diaphragm activity following a cervical SCI.
The central hypothesis of the following work is that transplantation of fetal spinal cord (FSC),
inherently rich in interneuronal progenitors, will promote the establishment of a novel circuitry capable
of enhancing respiratory recovery following injury.
Adult, female Sprague-Dawley rats received lateralized C3/4 contusions using the Infinite
Horizons Impactor Device at a preset force of 200 kilodynes. One week later, mechanically
dissociated fetal spinal cord tissue (obtained from age E13.5 Sprague-Dawley or Fischer 344-GFP
rats) was injected directly into the lesion cavity. Animals were allowed to recover for one month, at
which time a retrograde, transynaptic tracer (pseudorabies virus, PRV) was applied to the ipsilateral
hemi diaphragm or injected into the transplant site. Tracing studies have revealed anatomical
connectivity between the transplanted cells and host phrenic neurons, and innervation of donor tissue
from neurons throughout the brainstem, cervical and thoracic spinal cord. Immunohistochemical
analysis has also revealed of a variety of neuronal phenotypes within transplanted tissue, including
GABAergic, glutamatergic, cholinergic and catecholaminergic neurons. Furthermore, serotonergic
and catecholaminergic projections were seen within outermost regions of donor tissue. Extensive cfos immunoreactivity was observed throughout donor neurons, suggesting that transplanted cells
were active. This was confirmed with multiunit recordings from within transplanted tissue, which
revealed neurons with phasic respiratory and non-respiratory activity. Terminal electrophysiological
recording of diaphragm and phrenic nerve revealed improved function during normal (eupneic)
breathing and in response to respiratory challenge (hypoxia), suggesting that respiratory recovery
was enhanced in transplant recipients. The results from these ongoing studies suggest that
transplantation of neural progenitors can facilitate improved respiratory function following cervical
spinal cord injury.
Supported by National Institute of Health, NINDS; R01 NS081112
58
Investigating neuroprotection by carbon nanotubes following spinal cord injury
1
2
2
1
Merrick Strotton , Noelia Rubio Carrero , Khuloud Al-Jamal , Elizabeth Bradbury
1
2
King’s College London, Regeneration Group, The Wolfson Centre for Age-Related Diseases, London, UK
King’s College London, Drug delivery group, Institute of Pharmaceutical Science, London, UK
merrick.strotton@kcl.ac.uk
In spinal cord injury (SCI) research, biomaterials are often thought of as growth permissive
scaffolds. However, a class of particulate nanomaterials also exists, which have the advantage of
being injectable. Carbon nanotubes (CNTs) are an example of such a material, but they have a
number of additional unique properties that could be exploited for studying SCI, such as a large
surface area for protein/genetic species delivery, infra-red absorption properties enabling in vivo
imaging, and ultrahigh conductivity which may modify neuronal properties by lowering action potential
firing thresholds (Cellot et al., 2009). Furthermore, functionalising (solubilising) carbon nanotubes
through the covalent addition of positively charged amine groups to CNT side walls (forming α-CNTs)
has been demonstrated to create an agent that when delivered to various stroke models leads to
reduced levels of cell death (Al-Jamal et al., 2011, Lee et al., 2011, Moon et al., 2012).
We explored the therapeutic potential of α-CNTs as a neuroprotective agent in a rat model of
SCI with bilateral intraspinal injections of α-CNTs rostral and caudal to a dorsal column crush injury at
the cervical level. Tissue sparing at the injury epicentre and the survival and spread of CNTs were
examined at various post injury time points. In a separate group, positively charged α-CNTs were coinjected with chondroitinase ABC to evaluate if cleaving negatively charged glycosaminoglycans in
the extracellular matrix potentiated the spread of α-CNTs through the lesion. Using a battery of
forelimb dependent behaviours, electrophysiological assessments of the descending corticospinal
tract and histological analyses of the lesion epicenter, we demonstrate a slightly reduced lesion
volume in the presence of α-CNTs, but no functional improvement (or deficit). Further analysis of
behaviour and electrophysiological data is ongoing, and electron microscopy studies are currently
underway to evaluate those cell types which uptake α-CNTs. This will be important information for
future studies where we aim to explore the use of α-CNTs as a non-viral vector for siRNA mediated
knockdown of targets of interest.
References
Al-Jamal, K. T., Gherardini, L., Bardi, G., Nunes, A., Guo, C., Bussy, C., Herrero, M. A., Bianco, A., Prato, M., Kostarelos, K. &
Pizzorusso, T. 2011. Functional motor recovery from brain ischemic insult by carbon nanotube-mediated siRNA silencing. Proc
Natl Acad Sci U S A, 108, 10952-7
Cellot, G., Cilia, E., Cipollone, S., Rancic, V., Sucapane, A., Giordani, S., Gambazzi, L., Markram, H., Grandolfo, M., Scaini, D.,
Gelain, F., Casalis, L., Prato, M., Giugliano, M. & Ballerini, L. 2009. Carbon nanotubes might improve neuronal performance by
favouring electrical shortcuts. Nat Nanotechnol, 4, 126-33
Lee, H. J., Park, J., Yoon, O. J., Kim, H. W., Lee Do, Y., Kim Do, H., Lee, W. B., Lee, N. E., Bonventre, J. V. & Kim, S. S. 2011.
Amine-modified single-walled carbon nanotubes protect neurons from injury in a rat stroke model. Nat Nanotechnol, 6, 121-5
Moon, S. U., Kim, J., Bokara, K. K., Kim, J. Y., Khang, D., Webster, T. J. & Lee, J. E. 2012. Carbon nanotubes impregnated
with subventricular zone neural progenitor cells promotes recovery from stroke. Int J Nanomedicine, 7, 2751-65
This work was supported by a studentship funded by Guy’s and St Thomas hospital through the King’s Bioscience Initiative
59
Intramuscular Tibialis Anterior coherence and subacute spinal cord injury:
mechanisms of neuroplasticity underlying SCI
1,2
1
2
1
2
2
Bravo-Esteban E. Taylor J. Aleixandre M. Simon-Martínez C. Torricelli D. Pons J.L. Avila-Martin
1,3
G., Galan-Arriero I., Gómez-Soriano J.
elisabethb@sescam.jccm.es
1
Sensorimotor Function Group, Hospital Nacional de Parapléjicos, Toledo, Spain
Spanish National Research Council (CSIC), Madrid, Spain
3
Nursing and Physiotherapy School, Castilla La Mancha University, Toledo, Spain
2
Tibialis Anterior (TA) coherence estimation is assumed to reflect common supraspinal
descending input spinal motoneurons, related to corticospinal tract activity (1-4). This study
documented residual voluntary motor recovery at 2 week intervals during subacute spinal cord injury
(SCI) with intramuscular TA coherence estimation within the 10-60Hz bandwidth, assessed during
controlled maximal isometric and isokinetic dorsiflexion. Several clinical and functional lower limb
measures (muscular testing, dorsiflexion maximal voluntary torque and gait function measured with
the WISCI II) and neurophysiological measures (TA motor evoked potentials, MEPs) were also
recorded.
Total and TA muscle strength, voluntary torque generation and gait function improved during
subacute SCI, in addition to 40-60Hz, but not 15-30Hz intramuscular TA coherence. TA MEPs failed
to reflect significant recovery of function in this cohort. The SCI spasticity syndrome non-specifically
reduced 15-30Hz TA coherence and was detected as high TA coherence values during fast isokinetic
movement in all frequency bands.
To conclude, longitudinal assessment of adaptive and maladaptive motor plasticity during
subacute SCI can be detected with TA EMG coherence estimation during controlled movement,
providing orientative diagnostic information during neurorehabilitation.
References
1. Bravo-Esteban E, Taylor J, Aleixandre M, Albu S,Cristina Simon ,Torricelli D, J. L. Pons, Gómez-Soriano J. Muscle
coherence during controlled voluntary movement in healthy subjects and patients with spinal cord injury: contraction and
velocity dependence. Journal of NeuroEngineering and Rehabilitation (In press)
2. Farina D, Negro F, and Jiang N: Identification of common synaptic inputs to motor neurons from the rectified
electromyogram. J Physiol 2013, 591(10): 2403-18
3. Gómez-Soriano, J, Bravo-Esteban E, Aleixandre M, Taylor J, Pons JL. Method for the measurement of muscle coherence
for neurological disorders.PATENT. January 12, 2014 with application nº EP14150864
4. Halliday DM, Conway BA, Christensen LO, Hansen NL, Petersen NP, and Nielsen JB: Functional coupling of motor units
is modulated during walking in human subjects.J Neurophysiol 2003, 89: 960-8
This project is funded by the Spanish Ministry of Science and Innovation CONSOLIDER-INGENIO, project HYPER (Hybrid
NeuroProsthetic and NeuroRobotic Devices for Functional Compensation and Rehabilitation of Motor Disorders, CSD200900067)
60
Oral administration of the p38α MAPK inhibitor, UR13870, inhibits anterior cingulate
microglial expression and affective pain behaviour following spinal cord injury
1
1
1
1,2
Iriana Galan-Arriero , Gerardo Avila-Martin , Agueda Ferrer-Donato , Julio Gomez-Soriano ,
1,3
1
Elisabeth Bravo-Esteban , Julian Taylor
igalan@jccm.es
1
Sensorimotor Function Group, Hospital Nacional de Parapléjicos, SESCAM, Toledo, Spain
E.U. E. Fisioterapia de Toledo, Universidad de Castilla la Mancha, Toledo, Spain
3
IAI, Consejo Superior de Investigaciones Científicas (CSIC), Arganda del Rey, Spain
2
The p38α MAPK cell signalling pathway is a key mechanism of microglia activation and has
been intensively studied as a potential target for neuropathic pain (NP) control (2). The effect of
UR13870 (4), a demonstrated p38α MAPK inhibitor, on the modulation of anterior cingulate cortex
(ACC) and spinal dorsal horn microglia reactivity was addressed in this study following T9 contusion
spinal cord injury (SCI) in the rat, in addition to behavioural testing of pain-related aversion and
anxiety.
Administration of UR13870 (1mg/kg i.v.) and Pregabalin (30mg/kg i.v.) reduced place escape
avoidance paradigm (PEAP) behaviour during chronic SCI at 42 days after injury. Animals treated
with UR13870, administered daily (10mg/kg p.o.) demonstrated reduced PEAP, but not anxiety
behaviour, at 28 days after SCI. Although administration of UR13870 (10mg/kg p.o.) failed to reduce
either microglia or astrocyte reactivity within the spinal dorsal horn following SCI, a reduction in the
total volume of damaged tissue was identified at 28 days after injury. In the ACC, an increase in
microglia reactivity, in addition to an upregulation of the metabotropic glutamate type 5 receptor
(mGluR5) expression, were identified after SCI. While UR13870 (10mg/kg p.o.) treatment significantly
reduced OX-42 expression, mGluR5 and NR2B in the ACC, no change in astrocyte reactivity was
observed.
To conclude, oral treatment with a p38α MAPK inhibitor reduces the affective cognitive
component of pain component following SCI, mediated preferentially by the inhibition of microglia
reactivity within the ACC remote from the injury site (1). This study supports a central role of ACC glia
reactivity for affective pain behaviour after SCI (3, 5, 6).
References
1. Detloff MR, Fisher LC, McGaughy V, Longbrake EE, Popovich PG, Basso DM. Remote activation of microglia and proinflammatory cytokines predict the onset and severity of below-level neuropathic pain after spinal cord injury in rats. Exp
Neurol 2008;212(2):337-347
2. Ji RR, Suter MR. p38 MAPK, microglial signaling, and neuropathic pain. Mol Pain 2007;3:33
3. Lu Y, Zhu L, Gao YJ. Pain-related aversion induces astrocytic reaction and proinflammatory cytokine expression in the
anterior cingulate cortex in rats. Brain Res Bull 2010;84(2):178-182
4. Mihara K, Almansa C, Smeets RL, Loomans EE, Dulos J, Vink PM, Rooseboom M, Kreutzer H, Cavalcanti F, Boots AM,
Nelissen RL. A potent and selective p38 inhibitor protects against bone damage in murine collagen-induced arthritis: a
comparison with neutralization of mouse TNFalpha. Br J Pharmacol 2008;154(1):153-164
5. Widerstrom-Noga E, Pattany PM, Cruz-Almeida Y, Felix ER, Perez S, Cardenas DD, Martinez-Arizala A. Metabolite
concentrations in the anterior cingulate cortex predict high neuropathic pain impact after spinal cord injury. Pain
2012;154(2):204-212
6. Zhao P, Waxman SG, Hains BC. Modulation of thalamic nociceptive processing after spinal cord injury through remote
activation of thalamic microglia by cysteine cysteine chemokine ligand 21. J Neurosci 2007;27(33):8893-8902
This work has been supported by the following founding sources: Fundación Mutua Madrileña, 2013, INNPACTO (Ministerio
de Ciencia e Innovación, IPT-010000-2010-016), Consorcio “Dendria-Draconis Pharma S.L.” (Centro para el Desarrollo
Tecnológico Industrial), Instituto de Salud Carlos III PI11/00592. UR13870, was manafactured and kindly donated by Heidi
Sisniega of PalauPharma, S.A. (Barcelona, Spain)
61
The fate of boundary cap neural crest stem cells following transplantation to the
surface of avulsed or uninjured spinal cord
Carl Trolle*, Niclas König*, Ninnie Abrahamsson, Svitlana Vasylovska, Elena N. Kozlova
*Equal contribution
Regenerative Neurobiology, Dept of Neuroscience, Biomedical Center, Uppsala University, Uppsala, Sweden
Carl.Trolle@neuro.uu.se, Niclas.Konig@neuro.uu.se
Spinal root avulsion results in loss of motor and sensory function as well as often chronic
intractable neuropathic pain. Although surgical restoration of the ventral root may improve motor
function, methods to improve sensory function still needs to be developed.
We have previously described the ability of boundary cap neural crest stem cells (BCs) to
form elongated bands, associated with regenerating sensory fibers, after transplantation to the
1
avulsed dorsal roots of rodents . Our previous findings also show that BCs remaining outside the
spinal cord express the glial marker GFAP whereas the BCs that migrate into the spinal cord as single
2
cells may adopt a neuronal phenotype . Here we investigate the fate of transplanted BCs in different
injury models (durectomy, dorsal root rhizotomy and dorsal root avulsion). Our results indicate that the
BCs survive in all the above mentioned conditions but at a lower extent following durectomy when the
nervous system is left intact. While the BCs form band-like structures when transplanted to either cut
or avulsed dorsal roots, they tend to remain in small clusters when transplanted to intact dorsal roots.
Furthermore, following dorsal root rhizotomy as well as dorsal root avulsion, single BCs migrate into
the superficial laminae of the spinal cord. In all the three different conditions, the BCs outside the
spinal cord tend to be positive for the glial marker GFAP. We believe that the inflammatory processes
initiated after nerve injury are beneficial for the survival of BCs and that the BCs are able to respond
to the injury by organizing into band-like structures possibly supporting regenerating fibers.
References
1. Konig N, Trolle C, Kapuralin K, Adameyko I, Mitrecic D, Aldskogius H, Shortland PJ, Kozlova EN (2014) Murine neural crest
stem cells and embryonic stem cell-derived neuron precursors survive and differentiate after transplantation in a model of
dorsal root avulsion. J Tissue Eng Regen Med (Epub ahead of print)
2.Trolle C, Konig N, Abrahamsson N, Vasylovska S, Kozlova EN (2014) Boundary cap neural crest stem cells homotopically
implanted to the injured dorsal root transitional zone give rise to different types of neurons and glia in adult rodents BMC
Neurosci 5;15:60
62
Early transplantation of mesenchymal stem cells after spinal cord injury relieves pain
hypersensitivity through suppression of pain-related signaling cascades and reduced
inflammatory cell recruitment
1
1
1
1
3
Kenzo Uchida , Hideaki Nakajima , Shuji Watanabe MD , Kazuya Honjoh , William E.B. Johnson ,
1
Hisatoshi Baba
1
Department of Orthopaedics and Rehabilitation Medicine, Faculty of Medical Sciences, University of Fukui,
Eiheiji, Fukui, Japan
2
Life & Health Sciences, Aston University, Aston Triangle, Birmingham, UK
kuchida@u-fukui.ac.jp
Bone marrow-derived mesenchymal stem cells (BMSC) modulate inflammatory/immune
1) 2)
responses and promote motor functional recovery after spinal cord injury (SCI).
However, the
effects of BMSC transplantation on central neuropathic pain and neuronal hyperexcitability after SCI
remain elusive. This is of importance because BMSC-based therapies have been proposed for clinical
treatment. We investigated the effects of BMSC transplantation on pain hypersensitivity in GFPpositive bone marrow-chimeric mice subjected to a contusion SCI, and the mechanisms of such
effects. BMSC transplantation at day 3 post-SCI improved motor function and relieved SCI-induced
hypersensitivities to mechanical and thermal stimulation. The pain improvements were mediated by
suppression of PKC-γ and p-CREB expression in dorsal horn neurons. BMSC transplants significantly
reduced levels of p-p38 MAPK and p-ERK1/2 in both hematogenous macrophages and resident
microglia, and significantly reduced the infiltration of CD11b and GFP double-positive hematogenous
macrophages without decreasing the CD11b-positive and GFP-negative activated spinal-microglia
population. BMSC transplants prevented hematogenous macrophages recruitment by restoration of
the blood-spinal cord barrier, which was associated with decreased levels of (i) inflammatory
cytokines (TNF-α, IL-6); (ii) mediators of early secondary vascular pathogenesis (MMP-9); (iii)
macrophage recruiting factors (CCL2, CCL5, CXCL10), but increased levels of a microglial stimulating
factor (GM-CSF). These findings support the use of BMSC transplants for SCI treatment; further, they
suggest that BMSC reduce neuropathic pain through a variety of related mechanisms that include
neuronal sparing and restoration of the disturbed blood-spinal cord barrier, mediated through
modulation of the activity of spinal-resident microglia and the activity and recruitment of
hematogenous macrophages.
References
1. Nakajima H, Uchida K, Guerrero AR, et al. Transplantation of mesenchymal stem cells promotes an alternative pathway of
macrophage activation and functional recovery after spinal cord injury. J Neurotrauma 2012;29:1614-25
2. Tan Y, Uchida K, Nakajima H, et al. Blockade of interleukin 6 signaling improves the survival rate of transplanted bone
marrow stromal cells and increases locomotor function in mice with spinal cord injury. J Neuropathol Exp Neurol 2013;72:98093
63
Effects of pudendal and cortical paired associative stimulation on reflex and corticospinal control of anal sphincter responses in patients with incomplete spinal cord
injury: a feasibility study
1
1
1
1
1,3,
Vásquez N. , Knight S. , Susser J. , Gall, A. Ellaway P.H.
1
3
1,2
Craggs M.D.
2
The London Spinal Cord Injury Centre, Royal National Orthopaedic Hospital, University College London,
Imperial College, London, UK
Natalia.Vasquez@rnoh.nhs.uk
Incomplete spinal cord injury (iSCI) frequently impacts on spino-bulbo-spinal pathways
causing major disruption to the control of the pelvic organs and sphincter muscles. Restoration of
bladder and bowel control are top priorities for those with iSCI, especially in cases of paraplegia
(Anderson, 2004). In iSCI subjects who have retained a pudendal anal reflex (PAR), a surrogate
marker for the bladder guarding reflex, the reflex can be facilitated by conditioning transcranial
magnetic stimulation of the motor cortex in approximately 50% of cases (Vásquez et al, 2014). This
raised the question as to whether the ability of paired associative stimulation (PAS) to induce plasticity
in neural circuits, based on paired pudendal nerve and cortical stimulation, could be shown to induce
plasticity in the cortico-spinal circuits controlling the PAR. A long-term aim would be to see whether
development of the technique might lead to changes in neural circuitry accompanied by functional
restoration of continence.
Aims: To assess whether repetitive, associative paired stimulation of the dorsal penile nerve
and the motor cortex produces changes in cortico-spinal circuitry controlling anal sphincter muscle
responses, in iSCI.
Methods: Eighteen male subjects with incomplete, supra-sacral spinal cord injuries and
symptoms of a neuropathic bladder were recruited. Incontinence was assessed using the
International Consultation on Incontinence Modular Questionnaire (ICIQ). Electromyographic activity
of the external anal sphincter was recorded. The PAR was elicited by electrical stimulation of the
dorsal penile nerve (DPN). Motor cortical excitation was achieved using transcranial magnetic
stimulation (TMS). A PAS protocol (DPN and TMS, interval 40ms) was applied for 8 min at 0.25Hz
using either real or sham TMS (randomised order) of the motor cortex. Pudendal (DPN)
somatosensory evoked potentials (pSSEPs) were recorded.
Results: A PAR could be recorded in all subjects and an anal sphincter MEP in 12 of the 18
subjects. In all but one subject the PAR could be facilitated by prior (30 ms) conditioning TMS. Group
mean amplitudes of the PAR, the conditioned PAR (cPAR) and MEP showed no significant change
immediately after or 20’ minutes after either real or sham PAS. There was no change in the group
mean ICIQ scores. 13 subjects individually showed significant changes (10 increases, 11 decreases)
in one or more anal sphincter responses to either real or sham PAS. These individual responses were
not correlated with the presence or latency of either pSSEPs or the anal sphincter MEP.
Conclusions: Paired associative stimulation has the potential to alter the excitability of
cortico-spinal and reflex circuitry controlling the anal sphincter in certain iSCI individuals. The basis for
the variable nature of the responses to this particular PAS protocol is not known but could not be
explained by differences in afferent or efferent spinal conduction pathways.
References
Anderson KD. J Neurotrauma 2004;21:1371–83
Vasquez N, Balasubramaniam V, Kuppuswamy A, Knight S, Susser J, Gall, A Ellaway PH, Craggs MD. Neurourology and
Urodynamics 2014 E-Pub
Supported by the INSPIRE Foundation (Registered Charity No. 296284 UK) and the RNOH Charity (Registered Charity No.
226955 UK)
64
Chondroitinase ABC rescues complete respiratory motor activity following cervical
contusion injury
1
1,2
P.M. Warren ; B.I. Awad ; W.J. Alilain
1
2
1
MetroHealth Medical Centre, Case Western Reserve University, Cleveland, OH, USA
Dept. of Neurological Surgery, Mansoura University School of Medicine, Mansoura, Egypt
pmw45@case.edu
Chondroitinase ABC mediates functional recovery of damaged respiratory motor pathways
following both acute and chronic C3 cervical contusion (C3C). This auspicious treatment evokes
functional improvement in multiple muscle groups associated with inspiratory activity (diaphragm,
external intercostals, genioglossus) and suggests that the modification of the extracellular matrix
(ECM) causes plasticity at multiple levels and motoneuron pathways throughout the spinal cord.
Relatively few studies have been conducted studying the treatment of respiratory motor paralysis and
dysfunction following cervical spinal cord contusion. One of the major impairments to functional
respiratory motor recovery following C3C is the development of the astro-glial scar. This CSPG
(chondroitin sulphate proteoglycan) rich matrix acts as a barrier to plasticity and regeneration.
Application of chrondroitiase ABC (ChABC) catabolises the CSPGs within the ECM, increasing
functional plasticity. We have assessed the therapeutic efficacy of delayed ChABC application (4
concurrent injections totaling 0.015 U) upon multiple respiratory motor systems at both acute and
chronic stages following severe lateral cervical (C3) contusion (LC3C). Diaphragmatic, intercostal,
and genioglossus electromyography were used to assess functional restoration of spared and
damaged tracts following LC3C while anatomical alterations were investigated through
immunohistochemistry. Control animals showed severely compromised respiratory motor activity
ipsilateral to the injury at 3 and 6 weeks post contusion with minimal endogenous recovery. Indeed,
the damaged/contused pathways were unable to maintain respiratory motor activity alone. However,
application of ChABC at both 1 and 4 weeks post LC3C caused significant restoration in respiratory
motor function and repair of these damaged pathways which, alone, were able to maintain complete
inspiratory activity. These data suggest that ChABC treatment, at both acute or chronic time points, is
sufficient to evoke recovery of respiratory function. This possibly occurs through plastic mechanisms
including anatomical reorganization and an increase in serotonergic signaling. Further, we
demonstrate that the treatment can positively affect multiple systems that collectively act to mediate
inspiratory activity. However, this functional change in respiratory motor activity is neither absolute nor
completely robust when applied at acute stages suggesting an optimal time course for treatment
application must be determined to mediate sustained recovery. These data support the use of ECM
modification to mediate total respiratory motor system recovery following acute and chronic cervical
contusion.
Supported by the International Spinal Research Trust (ISRT), the Craig H. Neilson Foundation, Wings for Life, and the
Egyptian Governmental Scholarship
65
Extensive recovery of respiratory motor function at chronic and super-chronic time
points following cervical spinal cord injury
1
2
3
P.M. Warren ; P.M. MacFarlane ; J. Silver ; W.J. Alilain
1
1
MetroHealth Medical Centre, Case Western Reserve University, Cleveland, OH, USA
Dept. of Pediatrics, Case Western Reserve University, Rainbow Babies & Children’s Hospital, Cleveland, USA
3
Dept. of Neurosciences, Case Western Reserve University, Cleveland, OH, USA
2
pmw45@case.edu
Treatments to restore respiratory function following chronic cervical spinal cord injury (SCI)
have not been extensively studied. We demonstrate that a pharmacological agent and rehabilitative
training may provide the key for recovery of diaphragm activity following chronic trauma. The ablation
of respiratory function is caused by disruption of motoneuron pathways, formation of the chondroitin
sulphate proteoglycan (CSPG) rich astroglial scar, and a reduction in interneuron, motoneuron and
synaptic density. Following acute cervical SCI, CSPG breakdown by application of chrondroitiase
ABC (ChABC) can restore functional diaphragm activity while intermittent hypoxia (IH) training
increases respiratory drive and synaptic strength. We now provide evidence for the recovery of robust
functional respiratory motor activity at both chronic (3 month) and super-chronic (1.5 year) time points
following LC2H through a combination of IH training and ChABC. We used diaphragmatic
electromyography (diaEMG) and phrenic nerve recordings to demonstrate that a single application of
ChABC (0.005 U) can recover extensive respiratory motor function following chronic and superchronic cervical SCI. Control treated animals showed no endogenous recovery of diaphragm function.
While having limited effect upon diaEMG patterns, IH training alone was shown to enhance maximal
phrenic nerve activity. However, the combined treatment of IH and ChABC was shown to substantially
enhance diaEMG and maximal phrenic nerve activity beyond that demonstrated by either group
alone. Interestingly, in a subpopulation of animals the muscle activity in this combination group can
become unstructured, demonstrated by degraded patterned activity on the lesioned side. This
tonic/chaotic activity is governed by a serotonergic (5-HT) mechanism and suggests considerable
remodeling of spinal cord circuitry below the level of the lesion at chronic stages. Indeed, ChABC and
IH treated animals which recover normal breathing patterns following treatment can be made chaotic
by giving exogenous 5-HT, while those that are already chaotic can be normalized by blocking certain
5-HT receptors. These data demonstrate the significant restoration of diaphragm function and nerve
activity at chronic and super-chronic time points following cervical SCI due to matrix modification,
induction of plasticity and facilitation of drive. Yet, the potential emergence of chaos is indicative of the
complications inherent in repairing the chronically injured spinal cord and suggests the need for tight
mechanistic and environmental control.
Supported by the International Spinal Research Trust (ISRT) and the Craig H. Neilson Foundation
66
Delegate list
First Name
Last Name
Company
Louise
Adams
Queen Mary University of London
louise_adams24@hotmail.co.uk
Hakan
Aldskogius
Uppsala University
Hakan.Aldskogius@neuro.uu.se
Alilain
Case Western Reserve University
wja4@case.edu
Al'Joboori
University of Leeds
bsydaj@leeds.ac.uk
Allan
National Spinal Injuries Unit for Scotland
davidballan@btinternet.com
Warren
Yazi
David
Email Address
Mark
Bacon
ISRT
research@spinal-research.org
Sue
Barnett
University of Glasgow
Susan.Barnett@glasgow.ac.uk
Katalin
Bartus
King's College London
katalin.bartus@kcl.ac.uk
Catherina
Becker
University of Edinburgh
catherina.becker@ed.ac.uk
Murray
Armin
Xuenong
Sara
Liz
Frank
Tom
Srinivasa
Blackmore
Marquette University
murray.blackmore@marquette.edu
Blesch
UniversitätsKlinikum Heidelberg
Armin.Blesch@med.uni-heidelberg.de
Bo
Queen Mary University of London
x.bo@qmul.ac.uk
Bowie
UCL Institute of Neurology
sara.bowie@ucl.ac.uk
Bradbury
King's College London
elizabeth.bradbury@kcl.ac.uk
Bradke
DZNE Bonn
frank.bradke@dzne.de
Brushart
Johns Hopkins
tbrusha@jhmi.edu
Budithi
Midlands Centre for Spinal Injuries, Oswestry
Srinivasa.budithi@rjah.nhs.uk
Emily
Burnside
King's College London
emily.burnside@kcl.ac.uk
Sarah
Busch
Athersys, Inc.
sab37@case.edu
Arthur
Butt
University of Portsmouth
arthur.butt@port.ac.uk
Cheah
University of Cambridge
mc747@cam.ac.uk
Menghon
Daniel
Chew
University of Cambridge
dc501@cam.ac.uk
David
Choi
UCL Institute of Neurology
d.choi@ucl.ac.uk
Collins
UCL Institute of Neurology
Andrew.Collins@ucl.ac.uk
Coupaud
National Spinal Injuries Unit for Scotland
sylvie.coupaud@strath.ac.uk
Andrew
Sylvie
Michael
Craggs
UCL Institute of Neurology
michael.craggs@ucl.ac.uk
Graham
Creasey
Stanford University
gcreasey@stanford.edu
Danzi
University of Miami
m.danzi@med.miami.edu
Das
Indian Institute of Technology Guwahati
suradip@iitg.ernet.in
Matt
Suradip
Priscilla
Day
University of Cambridge
pd380@cam.ac.uk
Simone
Di Giovanni
Imperial College London
s.di-giovanni@imperial.ac.uk
Athanasios
Didangelos
King's College London
athanasios.didangelos@kcl.ac.uk
Hans-Ulrich
Dodt
University of Technology Vienna
hans-ulrich.dodt@meduniwien.ac.at
Wagih
El Masry
Midlands Centre for Spinal Injuries, Oswestry
bellstonehse@btinternet.com
Peter
Ellaway
Imperial College London
p.ellaway@imperial.ac.uk
Karim
Fouad
University of Alberta
karim.fouad@ualberta.ca
Robin
Franklin
University of Cambridge
rjf1000@cam.ac.uk
Matthew
Fraser
National Spinal Injuries Unit for Scotland
matthewfraser@nhs.net
Gad
University of California, Los Angeles
paraggad@gmail.com
Andrew
Gaudet
The Ohio State University
Andrew.Gaudet@osumc.edu
Isabella
Gavazzi
King's College London
isabella.gavazzi@kcl.ac.uk
Marieta
Georgieva
University of Aberdeen
marieta.georgieva.10@aberdeen.ac.uk
Goganau
Heidelberg University Hospital
Ioana.Goganau@med.uni-heidelberg.de
Parag
Ioana
67
Alfredo
Andy
Barbara
James
Barbara
Jodie
Gorio
University of Milan
alfredo.gorio@unimi.it
Greenhalgh
McGill University
andrew.greenhalgh@mail.mcgill.ca
Grimpe
Heinrich Heine University Duesseldorf
Barbara.Grimpe@med.uni-duesseldorf.de
Guest
University of Miami
jguest@med.miami.edu
Haenzi
King's College London
barbara.haenzi@kcl.ac.uk
Hall
The Ohio State University
jodie.hall@osumc.edu
Jan
Hoeber
Uppsala University
jan.hoeber@neuro.uu.se
Kazuya
Honjoh
University of Fukui
kazuya@u-fukui.ac.jp
Hu
Iowa State University
hilaryhu@iastate.edu
Hilary
Wenlong
Huang
University of Aberdeen
w.huang@abdn.ac.uk
Diana
Hunter
University of British Columbia ICORD
dianavhunter@gmail.com
Ronaldo
Ichiyama
University of Leeds
R.M.Ichiyama@leeds.ac.uk
Nicholas
James
Kings College London
nicholas.d.james@kcl.ac.uk
Nick
Eustace
Linda
Ksenija
Ashik
Jeffery
Iowa State University
njeffery@iastate.edu
Johnson
Aston University
w.e.johnson@aston.ac.uk
Jones
Craig H. Neilsen Foundation
linda@chnfoundation.org
Jovanovic
National Hospital for Paraplegics
kjovanovic@sescam.jccm.es
Kalam
University of Portsmouth
ashik.kalam@myport.ac.uk
Naomi
Kleitman
Craig H. Neilsen Foundation
naomi@chnfoundation.org
Niclas
König
Uppsala University
niclas.konig@neuro.uu.se
Timea
Konya
ISRT
timea@spinal-research.org
Marcel
Kopp
Charité-Universitätsmedizin Berlin
marcel.kopp@charite.de
Kozlova
Uppsala University
elena.kozlova@neuro.uu.se
Krassioukov
University of British Columbia ICORD
krassioukov@icord.org
Michael
Lane
Drexel University
mlane.neuro@gmail.com
Bradley
Elena
Andrei
Lang
Case Western Reserve University
btl21@case.edu
Kaythi
Latt
RNOH, Southport and Ormskirk
kaythi.latt@nhs.net
Stuart
Law
UCL Institute of Neurology
stuart.law@ucl.ac.uk
Lederer
Wings for Life
rosi.lederer@wingsforlife.com
Lee
Stanford University
svlee@stanford.edu
Lemmon
University of Miami
vlemmon@med.miami.edu
Daqing
Li
UCL Institute of Neurology
daqing.li@ucl.ac.uk
Ying
Li
UCL Institute of Neurology
ying.li@ucl.ac.uk
Ann
Logan
University of Birmingham
a.logan@bham.ac.uk
Lopez-Dolado
National Hospital for Paraplegics
lamidolado@gmail.com
McMahon
King's College London
stephen.mcmahon@kcl.ac.uk
Dana
McTigue
The Ohio State University
dana.mctigue@osumc.edu
Rosi
Seok Voon
Vance
Elisa
Stephen
Claire
Meehan
Copenhagen University
claire@sund.ku.dk
Saloni
Mehta
University of Miami
s.mehta8@med.miami.edu
Madge
Miah
UCL Institute of Neurology
m.miah@ucl.ac.uk
Michael-Titus
Queen Mary University of London
a.t.michael-titus@qmul.ac.uk
Adina
Gordon
Lawrence
Liz
Hans Werner
Tiina
Mitchell
University of Wisconsin - Madison
Mitchell@svm.vetmed.wisc.edu
Moon
King's College London
lawrence.moon@kcl.ac.uk
Muir
University of Cambridge
emm1@mole.bio.cam.ac.uk
Müller
University of Düsseldorf
hanswerner.mueller@uni-duesseldorf.de
Negron
Drexel University
TiinaM.Negron@gmail.com
68
Raymond
Onders
University Hospitals Case Medical Center
Raymond.onders@uhhospitals.org
Karen
Oprych
UCL Institute of Neurology
k.gladwin@ucl.ac.uk
Aheed
Osman
Midlands Centre for Spinal Injuries, Oswestry
aheed.osman@rjah.nhs.uk
James
Phillips
UCL Institute of Neurology
jb.phillips@ucl.ac.uk
Plant
Stanford University
gplant@stanford.edu
Giles
Milos
Popovic
University of Toronto
milos.popovic@utoronto.ca
Phil
Popovich
The Ohio State University
Phillip.Popovich@osumc.edu
John
Priestley
Queen Mary University of London
J.V.Priestley@qmul.ac.uk
Purcell
National Spinal Injuries Unit for Scotland
margaret.purcell@ggc.scot.nhs.uk
Mariel
Alexander
Rabchevsky
University of Kentucky
agrab@uky.edu
Ramer
University of British Columbia ICORD
ramer@icord.org
Peter
Richardson
Queen Mary University of London
p.richardson@qmul.ac.uk
John
Riddell
University of Glasgow
j.s.riddell@bio.gla.ac.uk
Andrea
Rivera
University of Portsmouth
andrea.rivera@port.ac.uk
Rodriguez
National Hospital for Paraplegics
fjrodriguez@sescam.jccm.es
Savic
RNOH, Stoke Mandeville Hospital
Gordana.Savic@buckshealthcare.nhs.uk
Sayenko
University of Louisville
DimitrySayenko@KentuckyOneHealth.org
Matthew
Javier
Gordana
Dimitry
Julia
Schaeffer
University of Cambridge
julia.schaeffer13@gmail.com
Schwab
Charité-Universitätsmedizin Berlin
jan.schwab@charite.de
Andrew
Schwartz
University of Pittsburgh
abs21@pitt.edu
Derryck
Shewan
University of Aberdeen
d.shewan@abdn.ac.uk
Silver
Case Western Reserve University
jxs10@po.cwru.edu
Spruance
Drexel University
Victoria.spruance@gmail.com
John
Steeves
University of British Columbia ICORD
steeves@icord.org
Moa
Stenudd
Karolinska Institutet
moa.stenudd@ki.se
Jan
Jerry
Victoria
Merrick
Julian
Veronica
Carl
Strotton
King's College London
merrick.strotton@kcl.ac.uk
Taylor
National Hospital for Paraplegics
juliantaylorgreen@yahoo.es
Tom
Drexel University
veronica.tom@drexelmed.edu
Trolle
Uppsala University
carl.trolle@neuro.uu.se
Kenzo
Uchida
University of Fukui
kuchida@u-fukui.ac.jp
Anna
Varone
University of Aberdeen
varoneanna@gmail.com
Vasquez
RNOH, Stanmore
nataliavasquezp@yahoo.com
Verhaagen
Netherlands Institute for Neuroscience
J.Verhaagen@nin.knaw.nl
Natalia
Joost
Laurent
Vinay
CNRS & Aix Marseille Université
laurent.vinay@univ-amu.fr
Philippa
Warren
Case Western Reserve University
pmw45@case.edu
Claudia
Wheeler-Kingshott
UCL Institute of Neurology
c.wheeler-kingshott@ion.ucl.ac.uk
Edward
Wirth
Asterias Biotherapeutics, Inc.
ewirth@asteriasbio.com
Yip
Queen Mary University of London
p.yip@qmul.ac.uk
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69