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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)
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
Stroke Center, University Hospital
Ischemic stroke
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
Coronal view
WebMD
• Infarct • 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
Intact cortex
Stroke
Middle cerebral territory
Lateral view
Primary and secondary motor cortices in primates
Preuss et al., 1996; Dum and Strick, 2002; Dancause et al., 2005; owl monkey
Primary motor cortex
Premotor cortex
Neurons that are interconnected to the infarct will undergo molecular responses immediately following infarct induction
To sensory hand
Primary motor cortex hand representation
Premotor cortex hand
digit
wrist
face
proximal
no response
medial
rostral
Previous work in the squirrel monkey model has highlighted neuronal changes following an infarct
medial
rostral
An infarct was induced in 30% of the M1 hand representation (Nudo and Milliken, 1996)
Infarct
medial
rostral
Following spontaneous recovery, there is a further loss of hand representation (Nudo and Milliken, 1996)
Infarct
medial
rostral
Following rehabilitative motor skill training, there was an actual increase in M1 hand representation (Nudo et al., 1996)
Infarct
medial
rostral
Infarct
PMv hand neurons undergo axonal sprouting to novel targets in primary somatosensory cortex (Dancause et al., 2005)
To sensory hand Dancause et al,
J Neurosci, 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
University of Glasgow Glasgow Experimental MRI Centre
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.
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
Pardridge, 1997
• High-resistance tight junctions
• Capillaries are 40m apart
• No transcellular pathways
Pardridge, 1997
• Share basement membrane with EC
• Antigen-presenting properties
• May regulate blood vessel growth and EC proliferation in quiescent cortex
Blood-Brain Barrier - Pericytes
Pardridge, 1997
•Foot processes cover more than 99% of brain capillary surface
•Site of p-glycoprotein, product of the multi-drug resistance gene
•Effective efflux system
Blood-Brain Barrier - Astrocytes
• 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
The BBB is physically uncoupled in areas of ischemic injury
Post-Ischemic Inflammation: Leukocyte diapedesis occurs in the post-
capillary 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 Contralateral WT WT
B cell-depleted 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
Magnitude of Stress
Tiss
ue o
r C
ellu
lar
Res
pons
e
none
tolerance
apoptosis
necrosis
* *
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
3 week exercise period 2 week sedentary period Flow cytometry on brain and spleen
Flow cytometry on brain and spleen
3 week exercise period
3 or 5 week sedentary period Flow cytometry on brain and spleen
stroke
stroke
stroke
3 days
3 days
3 days
SEDENTARY (SED)
EXERCISE (EX)
DETRAINING (DET)
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
EXER
CIS
E D
ETR
AIN
ING
All leukocytes in the brain
W1W
2W
3W
1W
2W
3W
1W
2W
3W
1W
2W
3W
1W
2W
3W
1W
2W
3W
1W
2W
3W
1W
2W
3W
1W
2W
3W
1W
2W
3
0
10000
20000
30000
40000
50000
60000
Av
era
ge
nu
mb
er
of
rota
tio
ns/w
ee
k
Ex27 Ex28 Ex29 Ex30 Ex31 Ex32 Ex33 Ex34Ex25 Ex26
**
******
*** *
EXERCISE
W1W
2W
3W
1W
2W
3W
1W
2W
3W
1W
2W
3W
1W
2W
3W
1W
2W
3W
1W
2W
3W
1W
2W
3W
1W
2W
3W
1W
2W
3
0
10000
20000
30000
40000
50000
60000
Av
era
ge
nu
mb
er
of
rota
tio
ns
/we
ek
Det13 Det14 Det15 Det16 Det17 Det18 Det19 Det20Det11 Det12
**
*****
****
*** **
***
DETRAINING
0 10000 20000 30000 40000 500000
1000000
2000000
3000000
Average Wheel Rotations
# c
ells
/he
mis
ph
ere
(me
an
/SD
)
All leukocytes
R2 = 0.8423
0 10000 20000 30000 40000 500000
500000
1000000
1500000
2000000
Average Wheel Rotations
# c
ells
/he
mis
ph
ere
(me
an
/SD
)
All leukocytes
R2 = 0.02860
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/chapter-7-opinion-about-the-use-of-animals-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
AN D T H AN K S !
Questions?