How we use animal studies to understand
recovery from brain injury
Ann M. Stowe, PhD
Assistant Professor
Neurology & Neurotherapeutics
Overview
 Introduction to clinical stroke
 Post-stroke plasticity in non-human primates
 Stroke models in rodents
 Methods to promote recovery after stroke
 Educational resources for the use of animals in
biomedical research
General considerations
 The brain is highly aerobic tissue
 Dependent upon a steady supply of well-oxygenated
blood

650 – 750 ml of arterial blood /minute




15% of total cardiac output
20% of body’s total O2 consumption
Global interruption in blood flow results in loss of
consciousness within 10 seconds!
Irreversible CNS injury occurs of blood flow drops to less than
15 ml / 100 gm tissue / minute
 Blood flow to the brain is maintained at a constant rate
over a wide range of blood pressure (autoregulation)
Stroke
Global mortality, all
causes: 65 million people
Global mortality, stroke:
6.5 million people – 10%
In the US, stroke #1 cause
of long-term adult
disability
800,000 U.S. strokes/yr
$73 billion annually (US)
to provide long-term care
Stroke Center, University Hospital
Coronal view
Ischemic stroke
WebMD
 88% of all strokes are ischemic
 Thrombotic – blood clots formed in the artery (50%)
 Embolic – blood clots dislodged from the body and
trapped in arteries in brain
Stroke
• Infarct
•
Intact cortex
Tissue necrosis, neuronal
death, possible loss of
pre-lesion function
• Peri-infarct
•
CBF is 20-50% of normal
values
•
Cells are at risk of apoptosis
or necrosis
Infarct
Peri-infarct
Middle cerebral territory
Lateral view
Primary and secondary motor cortices in primates
Primary motor cortex
Premotor cortex
Preuss et al., 1996; Dum and Strick, 2002; Dancause et al., 2005; owl monkey
Neurons that are interconnected to the infarct will undergo
molecular responses immediately following infarct induction
medial
rostral
Primary motor cortex
hand representation
To
sensory
hand
Premotor
cortex
hand
digit
wrist
face
proximal
no
response
Previous work in the squirrel monkey model has highlighted
neuronal changes following an infarct
medial
rostral
Infarct
An infarct was induced in 30% of the M1 hand representation
(Nudo and Milliken, 1996)
medial
rostral
Infarct
Following spontaneous recovery, there is a further loss of hand
representation (Nudo and Milliken, 1996)
medial
rostral
Infarct
Following rehabilitative motor skill training, there was an actual
increase in M1 hand representation (Nudo et al., 1996)
medial
rostral
Infarct
Dancause et al,
J Neurosci, 2005
To
sensory
hand
PMv hand neurons undergo axonal sprouting to novel targets in
primary somatosensory cortex (Dancause et al., 2005)
Findings:
 Disuse of the hand can decrease the number of
neurons that directly control hand movement
after brain injury
 Rehabilitation after stroke directly affects
neuronal plasticity during recovery
 Recovery after brain injury takes months to
complete in larger brains, especially during new
connections
Stroke models in rodents – why?
 Much more readily available
 Behavioral recovery can be monitored in rats




and mice
We know their genetics…
…which allows for genetic manipulation
Develop various models to ask different
questions
Myriad ways we can quantify injury and repair –
genetic, molecular, behavioral, imaging
Variety of rodent models of stroke:
University of Glasgow
Glasgow Experimental MRI Centre
Luo et al., JCBFM (2008) 28, 973–983
Middle Cerebral Artery
Occlusion (tMCAo)
Procedure
Intraluminal filament is threaded to
the origin of the MCA, with retraction
comes brain reperfusion.
Infarct Volume
2,3,5-triphenyltetrazolium chloride
(TTC)
Transient: Excellent for looking at
post-stroke inflammation
Permanent: Much larger infarct
volumes
Imaging techniques to measure infarct volumes
T2 weighted MRI 24h after permanent MCAo to measure edema
University of Glasgow
Glasgow Experimental MRI Centre
Diffusion tensor imaging after
permanent MCAo to measure
white matter tracks
Water moves along the axons
of neurons faster than in
cerebral cortex.
This allows for quantification
of direction based on the rate
of diffusion.
University of Glasgow
Glasgow Experimental MRI Centre
This means we can
track the in vivo
progression of the
infarct, along with
behavioral recovery, to
assess the efficacy of
drug or behavioral
interventions
60-min tMCAo
PBS (n=14), WT B cell
(n=12), RHP B cell (n=11)
Unpublished data
Motor recovery can
be measured as a
secondary outcome
Human CD20 transgenic mice
B cells depleted with Rituximab
8-12 week males
60-min tMCAo
(n=11 WT, n=13 B cell-depleted)
Unpublished data
Blood-Brain Barrier - Endothelial Cells
• High-resistance tight
junctions
• Capillaries are 40m
apart
• No transcellular
pathways
Pardridge, 1997
Blood-Brain Barrier - Pericytes
• Share basement
membrane with EC
• Antigen-presenting
properties
• May regulate blood
vessel growth and EC
proliferation in
quiescent cortex
Pardridge, 1997
Blood-Brain Barrier - Astrocytes
•Foot processes cover
more than 99% of brain
capillary surface
•Site of p-glycoprotein,
product of the multidrug resistance gene
•Effective efflux system
Pardridge, 1997
The BBB is physically uncoupled in areas of
ischemic injury
• Angiogenic factors facilitate
endothelial/pericyte dissociation
and disruption of tight junctions
• Astrocyte end-feet withdraw from
the vasculature
• Increase in vascular permeability
into peri-infarct tissue
• Cerebral edema
Post-Ischemic Inflammation:
Leukocyte diapedesis occurs in the postcapillary venules
modified from Eltzschig and Collard, 2004
• Selectins mediate rolling along the vessel wall
• Integrins mediate firm adherence to the vessel wall
Flow cytometry can be used to
quantify leukocyte
populations within the injured
(i.e. ischemic) hemisphere,
spleen, or blood
Abcam.com
B cells support post-stroke neurogenesis
Ipsilateral WT
B cell-depleted
WT
Contralateral
B cell-depleted
Scale bar = 20µm
hCD20Tg mice, WT littermate
controls
All receive Rituximab
Bottom border- subgranular zone
Dendrites extending into the molecular layer
Unpublished data
Recap:
 Several models for inducing stroke, can ask different
questions
 Concurrent quantification of outcomes
 Use of genetic manipulation to generate new mouse
strains
 Look at important mechanisms that can not be studied
in the clinical population
 Help to understand the contribution of genetic,
environmental, and physiological factors to stroke
outcome in the individual
“Preconditioning”
The presentation of a non-injurious stimulus that
promotes adaptive responses at the level of the cell,
tissue, organ, and/or whole animal to afford protection
against an injurious or lethal intervention.
“Tolerance”
The state of relative resistance to a normally injurious or
lethal intervention.
(Dirnagl et al., Trends Neurosci., 2003)
In Vivo Preconditioning Stimuli
Local
• brief ischemia
• mild trauma
Systemic
• hypoxia and hypoxia-mimetic drugs
• hyperoxia, hypoglycemia, caloric restriction
• heat shock
• cytokines, LPS, anesthetics,
metabolic inhibitors, antibiotics
• distant tissue ischemia (“remote” PC)
• exercise
Tissue or Cellular Response
necrosis
apoptosis
tolerance
none
*
*
Magnitude of Stress
Sustained exercise – but not the magnitude of exercise
– creates a unique B cell phenotype in the blood
Unpublished data
Hypothesis: Exercise-mediated changes in adaptive
immunity are lost with detraining
stroke
SEDENTARY
(SED)
3 or 5 week sedentary period
3 days
Flow cytometry on
brain and spleen
stroke
3 week exercise period
EXERCISE
(EX)
3 days
Flow cytometry on
brain and spleen
stroke
DETRAINING
(DET)
3 week exercise period
2 week sedentary period
3 days
Flow cytometry on
brain and spleen
Detrained animals exhibit increased infarct volumes
SED
EX
DET
Unpublished data
Exercise intensity induces a non-linear, dose-dependent
increase of immune cells in the ischemic brain that is lost
after detraining
All leukocytes in the brain
All leukocytes
40000
30000
20000
10000
2000000
1000000
0
*
Ex26
Ex28
Ex29
Ex30
Ex31
Ex32
Ex33
W
1
W
2
W
3
W
1
W
2
W
3
W
1
W
2
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3
W
1
W
2
W
3
W
1
W
2
W
3
W
1
W
2
W
3
W
1
W
2
W
3
W
1
W
2
W
3
Ex27
Ex34
**
***
**
30000
*
*
***
20000
10000
0
Det17
Det18
Det19
W
1
W
2
W
3
Det16
W
1
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2
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3
Det15
W
1
W
2
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3
W
1
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2
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3
Det14
W
1
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3
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1
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Det13
W
1
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2
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3
W
1
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3
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1
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3
Det12
30000
40000
50000
Det20
R2 = 0.02860
1500000
1000000
500000
0
Det11
20000
2000000
*** **
40000
10000
All leukocytes
DETRAINING
*
0
Average Wheel Rotations
# cells/hemisphere
(mean/SD)
60000
W
1
W
2
W
3
0
W
1
W
2
W
3
DETRAINING
*
**
Ex25
Average number of rotations/week
**
*
***
# cells/hemisphere
(mean/SD)
***
50000
50000
R2 = 0.8423
3000000
60000
W
1
W
2
W
3
EXERCISE
Average number of rotations/week
EXERCISE
0
10000
20000
30000
40000
Average Wheel Rotations
50000
Unpublished data
Recap:
 Spontaneously hypertensive rodents
 Obese and aged rodents
 Other environmental factors- exercise vs. sedentary
lifestyle
 We can use these interventions to determine the
mechanisms by which lifestyle and genetics can
contribute to injury and recovery after stroke.
http://www.pewinternet.org/2015/07/01/c
hapter-7-opinion-about-the-use-ofanimals-in-research/
Bringing up the concept of animals in
biomedical research
 Pew research into the public opinion of animal research
 American Physiological Society Advocacy and
Outreach


Presentations available online
http://www.the-aps.org/mm/SciencePolicy/Advocacy/Research-Benefits
 NIH RePORT website- search by any disease

http://report.nih.gov/NIHfactsheets/Default.aspx?key=S#S
Questions?
AND THANKS !