A MOUSE MODEL FOR STUDYING STROKE INDUCED 
IMPAIRMENTS, RECOVERY, AND COMPENSATION IN 
THE MOTOR CORTEX. 
Tracy Deanne Farr 
BSc: Biology, University of Lethbridge, 2001. 
A thesis submitted to the University of Lethbridge School of Graduate 
Studies in partial fulfillment of the requirements for the degree 
MASTER OF SCIENCE 
Department of Psychology and Neuroscience 
University of Lethbridge 
LETHBRIDGE, ALBERTA, CANADA 
May 2003 
? Tracy Farr, 2003 
"A mouse is an animal that, if killed in sufficiently many and 
creative ways, will generate a PhD." unknown 
Thesis Abstract 
Stroke is the third leading cause of death and survivors suffer motor impairments. 
The rodent sensorimotor system is similar to the human's, making rodents a good model 
to study the effects of stroke. Transgenic technology makes the mouse a desirable stroke 
model, however, there are few behavioural tests to assess behavioural outcome. This 
thesis evaluates mice subjected to permanent or temporary occlusion focal motor cortex 
strokes in a skilled reaching task. The first experiment documents changes in skilled 
movements in mice with a permanent occlusion focal motor cortex stroke. The second 
experiment is identical but uses a temporary occlusion focal motor cortex stroke. The 
third experiment compares the two strokes. The results indicate permanent occlusion 
mice suffer greater impairments, and a larger injury, than temporarily occluded animals. 
The mice with the largest insults were most impaired. Mice make an excellent 
behavioural and genetic model for studying motor system stroke. 
iv 
Acknowledgements 
There are so many people who deserve recognition because I could never have 
done this without them. I hope I don't forget anyone. 
Reed Kindt for the picture of the mouse single pellet reaching apparatus, and 
Robbin Gibb for advice regarding immunochemistry. I am not at all statistically inclined, 
but Doug and Patty Wallace still helped me make sense of my data. 
Dionne Piecharka, Felicia Drever, Joanna Gorny, and Greg Silasi also contributed 
a portion of their lives to the training of mouse subjects. Without them I surely would 
have gone mad alone with so many mice. I need to thank Nafisa Jadavji for volunteering 
so much of her time testing mice, slicing tissue and scoring video. 
Thank you Rob Sutherland for helping me pursue "the GAP-43 Saga" and for 
being so generous. 
Ying Wu has spent literally hours upon hours with me in the surgical suite 
struggling, in vain, to get a valid waveform during the LTP experiments. She is 
absolutely amazing. 
Carmen Hanson, for her exceptional care of the mice. 
Bogdan Gorny and Paul Whishaw for helping prepare all the beautiful footage of 
the mice. Notice the difference between the first and second experiments! 
Thank you to Gerlinde Metz, Lisa Thomson, and Af'ra Foroud for your always 
present listening ears and being so kind to me. 
I owe so much to the Canadian Stroke Network for not only funding me, but 
giving me so much publicity and exposure. 
Mom, for her mother's pride. 
Colin, for being my shoulders and for loving me enough to spend your weekends 
with the mice! 
Thank you Peter Dibble and Theresa Jones, for helpful comments and sacrifice of 
time to read this monster. 
Glen Prusky for honest advice, abuse, friendship, and for believing in me, even 
when I am having trouble believing in myself. 
And, finally I need to thank Ian Whishaw for guiding me, challenging me, and 
taking this chance on me. 
v 
Table of Contents 
Title page 
Signature page 
Quotation 
Thesis Abstract 
Acknowledgments 
Table of Contents 
List of Tables and Figures 
List of Abbreviations 
1. Introduction 
1.1. Introduction to the thesis. 
1.2. Introduction to stroke. 
1.3. Introduction to the motor system. 
1.4. The phenomenon of plasticity. 
1.5. Modeling stroke in the rodent. 
1.6. Molecular mechanisms of cell death. 
1.7. Testing behaviour in the rodent. 
1.8. Why use mice? 
1.9. Introduction to the experiments. 
2. Experiment 1: Skilled movements in a mouse are impaired after a focal 
motor cortex stroke. 
3. Experiment 2: The effects of a new stroke model on mouse skilled reaching. 
4. Experiment 3: The effects of a permanent and temporary occlusion stroke 
on skilled reaching in the mouse. 
5. Discussion 
Outline of the discussion. 
A review of stroke. 
Mice as a model for stroke. 
An introduction to transgenics. 
The contribution of transgenics to stroke. 
A thesis summary. 
6. References 
vi 
List of Tables and Figures 
Figure 1.21. The cerebrovasculature of the human brain. 
Figure 1.31. A spinal cord cross section. 
Figure 1.32. The layers of the sensory and motor cortices. 
Figure 1.33. The cortical organization of the human brain. 
Figure 1.34. The cortical organization of the rat brain. 
Figure 1.35. The corticospinal tract. 
Figure 1.41. A map of the rat motor cortex. 
Figure 1.42. The cortical piano theory. 
Figure 1.51. The cortical surface vasculature of the rat brain. 
Figure 1.61. The excitotoxicity hypothesis. 
Figure 2.11. The single pellet reaching apparatus. 
Figure 2.12. The pial strip histology. 
Figure 2.13. A quantitative measurement of percent success. 
Figure 2.14. A qualitative measurement of the ten movement components. 
Figure 2.15. Movements 1-3: Digits to the Midline and Aim. 
Figure 2.16. Movements 4-5: Advance and Digits Extend. 
Figure 2.17. Movement 6: Pronation. 
Figure 2.18. Movements 7-8: Grasp and Supination I. 
Figure 2.19. Movement 9-10: Supination II and Release. 
Figure 3.11. The modified single pellet reaching apparatus. 
Figure 3.12. Representative photographs of the endothelin-1 stroke. 
Figure 3.13. Coronal sections and cellular morphology of the endothelin-1 stroke. 
Figure 3.14. The quantitative measurement of the endothelin animals' success. 
Figure 3.15. The qualitative measurement of the endothelin animals' movements. 
Figure 3.16. Endothelin animals' movements: Digits to the Midline- Aim. 
Figure 3.17. Endothelin animals' movements: Advance- Pronation. 
Figure 3.18. Endothelin animals' movements: Grasp and Supination I. 
Figure 3.19. Endothelin animals' movements: Supination II and Release. 
Figure 4.11. A histological comparison of the pial strip and endothelin-1 stroke. 
Table 4.11. A table of the estimated stroke volume in the pial strip and endothelin 
animals. 
Figure 4.12. The quantitative success rates of the endothelin-1 and pial strip stroke 
animals. 
Figure 4.13. The qualitative analysis of the endothelin-1 and pial strip stroke animals' 
movements. 
Table 4.12. A table of the correlation statistics, and probabilities, for lesion volumes and 
the ten different movement components. 
vn 
List of Abbreviations 
ADP- adenosine diphosphate 
AMPA- alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate 
Apo A-l- apolipoprotein A-1 
Ca2+- calcium 
CFA- caudal forelimb area 
DAG- diacylglycerol 
DNA- deoxyribonucleic acid 
ER- endoplasmic reticulum 
ET- endothelin 
fMRI- functional magnetic resonance imaging 
HDL- high density lipoproteins 
HLA- hindlimb area 
IP3- inositol triphosphate 
K+- potassium 
MCA- middle cerebral artery 
MCAO-middle cerebral artery occlusion 
MRI- magnetic resonance imaging 
Na+- sodium 
NAD- nicotinamide adenine dinucleotide 
NGF- nerve growth factor 
NMDA- N-methyl-D-aspartate 
NO- nitric oxide 
NOS- nitrous oxide synthase 
PARP- poly ADP-ribose polymerase 
PIP2- phosphatidyl inositol 
PLC- phospholipase C 
PKC- protein kinase C 
RFA- rostral forelimb area 
ROS- reactive oxygen species 
SI- primary somatosensory cortex 
SII- secondary somatosensory cortex 
viii 
1. Introduction 
1.1. Introduction to the thesis. 
"In forty years I plan to have a decent job. I will have a nice house with 
furnishings and will drive a quality car to work every day." 
Isn't this how many people picture their future? What if one day, in my future, I 
had a headache that developed into nausea? Would I stay home, thinking I have the flu? 
What if that headache doesn't go away and I suddenly have trouble speaking, my vision 
becomes blurry, and I can't move one side of my body. By the time I would make it to 
the emergency room I would have already suffered neurological damage from a stroke. 
What if I had to stay in the hospital because I required assistance dressing and eating? 
What would my family have to sacrifice to help me live? This isn't how I picture my 
future; 1 don't think anyone does. Every year approximately 50 000 Canadians live this 
future. Experimental research into stroke is essential because it may help reduce the 
impact of the disease on the lives and economy of Canadians. 
It is not ethical to conduct invasive procedures in humans for the purpose of 
experimental research. Therefore most stroke research is conducted in animals. Rodents 
make a good model for studying the pathology of stroke for a variety of reasons (for a 
review see Cenci, et ah, 2002). One reason is that it is possible to control many 
extraneous factors in the rodent environment that may influence experiments; these 
include atmosphere, temperature, activity, diet, experience and mating. In addition, the 
rodent motor system is remarkably similar to humans. Studying the motor system has 
important clinical implications because impaired motor function is one of the most 
common and frustrating symptoms exhibited by stroke patients. 
This thesis describes a focal model of stroke to the motor cortex of mice. The 
motor cortex stroke will be described to have drastic effects on the movements of these 
animals. The following eight sections comprise the introduction portion of this thesis. 
1 
The first section of the introduction defines and describes stroke. The second section 
emphasizes the differences that exist between the human and rodent motor system. 
Because the second section portrays a fixed view of the motor system, the third section 
will emphasize the phenomenon of plasticity. Plasticity is a general term used to describe 
experience induced changes in the brain, including the motor system. The fourth section 
discusses the various rodent models of stroke, and highlights some considerations that 
should be made when choosing a model of stroke. Since the pathology of stroke is an 
important factor when choosing a model, the fourth section leads into the fifth section, 
which is a discussion about the mechanisms of cell death associated with stroke. The 
sixth section describes behavioural testing in the rodent. The seventh section briefly 
describes the use of transgenic mice to study stroke. The final section of the introduction 
outlines the experiments. 
1.2. Introduction to stroke. 
Stroke is a cardiovascular disease that affects the vessels that supply the brain 
with blood. A stroke occurs when a vessel bursts or is clogged. Stroke is the fourth 
leading cause of death in Canada, claiming 16 000 lives each year. There are 
approximately 50 000 strokes annually in Canada, making stroke the leading cause of 
disability for those who survive. Stroke survivors have a twenty percent chance of 
suffering a second stroke within two years of the initial event. Stroke is primarily a 
disease of the elderly as the risk of stroke doubles after the age of fifty-five. Out of every 
hundred stroke victims twenty will die, fifty will return home, ten will undergo hospital 
rehabilitation and fifteen will require long term care. It is estimated that stroke costs the 
Canadian economy 2.7 billion dollars each year (Heart and Stroke Foundation of Canada; 
The Canadian Stroke Network). 
There are two types of strokes: twenty percent are hemorrhagic and eighty percent 
are ischemic. A hemorrhage is uncontrolled bleeding in the brain. A subarachnoid 
hemorrhage is bleeding between the brain surface and the skull. An intracerebral 
hemorrhage occurs when a vessel within the brain ruptures. Hemorrhages are often 
2 
caused by structural defects in the walls of the brains blood vessels. Hemorrhages can 
occur at an aneurysm, which is a small weak section of the vessel wall, or at an 
arteriovenous malformation, which are areas of the brain where there are many thin-
walled blood vessels. 
Ischemic stroke refers to a reduction in blood supply to the brain. The reduction is 
typically produced by a blood clot or the buildup of plaques formed from atherosclerosis 
(hardening of the arteries). The best treatment for an ischemic stroke is immediate 
administration of anticoagulants to break up the clots. Clots can be either thrombotic, 
formed in an artery of the brain, or embolic, formed in the body and carried through the 
bloodstream to the brain. Depending on their size, clots can enter any of the numerous 
vessels of the brain (Netter and Dalley, 1998). Clots are particularly dangerous for a 
patient when they block one of the major artery systems that supply a large area of the 
brain. 
One of the main brain blood vessels is the middle cerebral artery, depicted in 
Figure 1.21. 
3 
Middle 
Cerebral 
Artery 
External 
Carotid 
Internal 
Carotid 
Basilar 
Artery 
Vertebral 
Artery 
Figure 1.21. A diagram depicting the middle cerebral artery in the brain. The carotid 
artery branches into the middle and anterior cerebral arteries while the vertebral artery 
bifurcates into the posterior cerebral and basilar arteries. This Figure was modified from 
Blumenfeld, 2002. 
4 
Humans have four main arteries that supply the brain with blood. The two vertebral 
arteries supply the hindbrain by bifurcating into the posterior cerebral artery, which 
supplies the occipital and temporal lobes, and the basilar artery, which extends into the 
cerebellum and brainstem. The other two arteries entering the brain, the carotids, 
originate in the aorta and are located on each side of the neck. They bifurcate below the 
jaw line into the internal and external carotids. The external carotid supplies the facial 
muscles. The internal carotid also bifurcates near the eye into the anterior cerebral artery 
and the middle cerebral artery (MCA) both of which supply the forebrain. The MCA is a 
frequent destination for clots in stroke patients (Hossmann, 1998). Because it supplies the 
majority of the lateral cortex, thalamic structures, and the basal ganglia, ischemia to the 
forebrain can result in many types of motor impairments (for a review see Slater and 
Johns, 2003). 
The nature of the impairment a patient suffers depends on the location and 
severity of the stroke. People with cognitive impairments can lose the ability to speak or 
understand speech or become confused about their environment (Kalaria and Ballard, 
2001). Motor impairments affect a patient's ability to move and perform every day tasks 
such as dressing or eating. These impairments are often unilateral, meaning one side is 
more affected. This is usually the side opposite or contralateral to the location of the 
insult in the brain (for a review see Hendricks, et al., 2002). Often a patient will fail to 
recognize the disabled side of the body; this phenomenon is referred to as neglect (Swan, 
2001). In order to understand the process of movement, and impairments that arise from 
injury, it is helpful to have an appreciation of the structure of the motor system. 
1.3. Introduction to the motor system. 
The motor system is a complex system that includes many regions of the brain 
and areas of the body, all of which contribute to the production of movement. Because 
the motor system is closely connected to the sensory system, the two are often 
collectively referred to as the sensorimotor system. This section is designed to provide a 
brief description of the sensorimotor system by describing, in sequence, the sensory input 
5 
to the cortex, the layers of the cortex, cortical topography, and the motor output pathway. 
Throughout this section the similarities and differences between the rodent and human 
will be emphasized. 
Movement begins with sensation. The epidermis, or outer layer of the skin, 
contains many receptors, whose neurons carry sensory information into the spinal cord. 
These neurons are called the dorsal root ganglion neurons because their cell bodies lie 
beside the spinal cord (Kolb and Whishaw, 2001). The dorsal root ganglion neurons 
extend upwards into the spinal cord, forming the dorsal column (Nieuwenhuys, et al., 
1981). The dorsal column is depicted in a cross section of the spinal cord in Figure 1.31. 
6 
Dorsal column 
(sensory) 
(motor) 
Figure 1.31. A cross section of the spinal cord indicating the gray and white matter, as 
well as the column organization. The cross section background was modified from 
Kandel, et al, 2000. 
7 
The fibres of the dorsal column enter the brain and cross over to the other hemisphere to 
form the medial lemniscus. The medial lemniscus synapses with the ventrolateral 
thalamus and the thalamus projects to Layer IV of the primary somatosensory cortex. 
This system is similar in humans and rats (Paxinos, 1994; Burstein and Giesler, 1989). 
The neo cortex of both humans and rats is composed of six layers (Hepp-
Reymond, 1988; Kandel, et al, 2000; Kolb and Whishaw, 2001). The six layers of the 
motor and sensory cortex are depicted respectively in Figure 1.32. 
8 
i 
I 
[in 
j 
Integrative 
functions 
J v 
T\/VInput of sensory/ iiSiiS^f ifc 
1 V information lIVl^KS?? 
y v Output to brain < 
Figure 1.32. The six layers of the motor and sensory cortices respectively. This figure 
was modified from Kolb and Whishaw, 2001. 
9 
n 
Layer I contains few projections and many glial cells that have primarily a supportive 
role. Layers II and III contain some pyramidal neurons, named such because they have 
pyramid shaped cell bodies (Hepp-Reymond, 1988) that are involved primarily in 
information processing. Layer IV is dense in the sensory cortex (Figure 1.32) as it 
contains many non-pyramidal, stellate-shaped, neuron cell bodies that receive sensory 
input. A large concentration of pyramidal cell bodies exists in Layer V of the motor 
cortex (Figure 1.32). These cells form the output pathway of the cortex. 
The divisions of the cortex are illustrated in a lateral view of the human brain in 
Figure 1.33 (Kolb and Whishaw, 2001). 
10 
Figure 1.33. The cortical organization of the human brain. The prefrontal cortex is 
indicated in green, the premotor in purple, the motor in blue, the primary somatosensory 
in red and the secondary somatosensory in yellow. The arrows indicate the flow of 
information. This figure is adapted from Kolb and Whishaw, 2001. 
11 
After information is received in Layer IV of the primary somatosensory cortex it travels 
to the secondary somatosensory cortex. The secondary somatosensory cortex, in turn, 
sends information forward to the prefrontal cortex, which is involved in planning 
movements. Subsequently, information is received by the premotor area, which is 
involved in coordinating and sequencing movements, and then sent to the motor cortex to 
begin the execution of movement. 
The cortical division in a rat is illustrated in a dorsal photograph of a rat brain in 
Figure 1.34. Though the rat brain may look different from that of the human, there are 
many similarities. 
12 
Figure 1.34. The cortical organization of the rat brain. The big structure at the back of the 
brain is the cerebellum and the two tiny structures on the front of the brain are the 
olfactory bulbs. The motor cortex is divided into the rostral forelimb area (RFA) 
represented in green, the caudal forelimb area (CFA) represented in blue, and the 
hindlimb area (HLA) is represented in red. The purple region indicates the sensory 
cortex. 
13 
As in the human, sensory information from the body and face is received in Layer IV of 
the rat primary sensory cortex (SI). SI stretches down the side of the rat brain and 
overlaps extensively with the motor cortex. As in humans, sensory information is 
transferred from the sensory to the motor cortex. The rat motor cortex, similar to that of 
the human, is composed of many sub areas, each of which can elicit movement from 
different body parts. Specifically the rat has three motor regions that overlap, the most 
anterior being the rostral forelimb area (RFA), the middle being the caudal forelimb area 
(CFA) and the most posterior being the hindlimb area (TELA). Despite the topographical 
differences, Layer V of the motor cortex is responsible for the initiation of movement 
from all three regions. 
In the rat, as in humans, Layer V of the motor cortex consists of many pyramidal 
cells whose axons project downwards into the spinal cord. These descending fibres form 
the corticospinal tract, which later divides into the lateral and ventromedial systems 
(Kandel, et ah, 2000; Miller, 1987; Nieuwenhuys, et al., 1981). These tracts are depicted 
in Figure 1.35. 
14 
Figure 1.35. The corticospinal tract originates in the motor cortex and projects to the 
pyramidal decussation where it becomes the lateral and ventromedial tracts. This figure 
was modified from Kandel, et al., 2000. 
15 
The corticospinal tract regroups in the medullary pyramid where some of the projections 
exit to terminate in the brain stem nuclei and the others travel down to the pyramidal 
decussation. It is here that seventy five percent of the corticospinal fibres cross over to 
form the lateral system, which innervates the contralateral side of the body. The lateral 
system controls distal limb, or hand and foot movements. The uncrossed fibres that 
continue to innervate the ipsilateral side of the spinal cord are called the ventromedial 
system. The ventromedial neurons control the proximal musculature near the midline 
such as the shoulders and pelvis. (For a depiction of the lateral and ventral columns of the 
spinal cord, see Figure 1.31). 
Movement is not solely dependent on somatosensory information. Other sensory 
systems including the vestibular, visual, and auditory systems, as well as brain stem tracts 
and nuclei, and other brain structures like the cerebellum (Thach, et al., 1991), and basal 
ganglia (Alexander, et al., 1990) are also important in producing movement. The 
diversity of structures emphasizes the complexity that goes into producing movement. 
The complexity is made worse by the fact that the system is subject to change, a 
phenomenon called plasticity. 
1.4. The phenomenon of plasticity. 
Plasticity refers to the ability of the nervous system to reorganize and change in 
response to environmental influences or injury. This thesis is particularly concerned with 
motor cortex plasticity that results from injury. Historically, it has been believed that the 
areas of the motor cortex were very specific and discrete and that the adult brain was 
incapable of change. The idea that a different cortical area represented each part of the 
body was first hypothesized in the late seventeenth century (Fritsch and Hiitzig, 1870). 
Studies conducted in dogs (Ferrier, 1875) and primates (Leyton and Sherrington, 1917) 
indicated that stimulation of discrete brain regions elicited movements of one area of the 
body. Similar research in humans (Jackson, 1958) led to the coining of the term 
homunculus (Penfield and Boldrey, 1937). The homunculus is a size-specific pictorial 
representation of the body parts associated with each area of the cortex. Today it is 
16 
believed, not only that the motor cortex is not so discretely organized, but also that the 
cortical representations for each body part are constantly changing (Strick, 1988; Nudo, 
et al., 2001; Schieber, 2001). The following is an example of motor cortex plasticity in 
the rat. 
The rat motor cortex is organized into the two forelimb areas, rostral and caudal, 
and a hindlimb area. Electrostimulation of the motor cortex results in the generation of 
movements in limbs contralateral to the side of the brain being stimulated. It is possible 
to assess the total cortical area that elicits movement from each body part in each new 
animal. As it turns out, the RFA and the CFA are often indistinguishable in that they are 
both able to produce proximal (elbow and shoulder) and distal (wrist and digit) 
movements. A control rat would have an approximately equal distribution of proximal 
and distal musculature in the motor cortex. A rat trained to perform skilled movements 
will develop more distal movement representation in the forelimb areas. This change in 
relation between areas committed to proximal and distal movement is an example of 
plasticity (Kleim, et al., 1998). A representative surface map of the motor cortex of a 
trained rat is indicated in Figure 1.41. 
17 
Figure 1.41. A motor cortex electrostimulation map of a control rat trained in the single 
pellet reaching task. Green dots represent the distal movements, blue the proximal, 
yellow the head and whiskers, pink the neck, and black is non responsive. This figure was 
donated by Dionne Piecharka at the University of Lethbridge. 
18 
There are many green dots and each represents a site that elicits distal wrist and digit 
movements. The green dots outnumber the blue dots that represent proximal elbow and 
shoulder movements. The yellow and red dots respectively indicate head, vibrissae, and 
neck movements. The black dots indicate non-responsive areas where no movements 
were elicited. 
Schieber (2001) describes the underlying complexity of the cortex that allows the 
process of plasticity and subsequently movement to occur. He proposes that there are 
many different areas of cortex that elicit movement from one muscle, thus the territories 
for each muscle experience substantial dispersal across the cortex. In addition, the motor 
output of one corticospinal neuron diverges and innervates many other corticospinal 
neurons. Multiple collateral projections between cortical neurons allow for horizontal 
triggering of action potentials between motor neurons. Schieber (2001) uses a piano 
keyboard in Figure 1.42 to illustrate his more modern view of how the complex and 
plastic motor cortex produces movement. 
19 
Figure 1.42. A: The 42 white keys on a standard piano keyboard were colored in order 
to distinguish them from one another. Each note can be located by its placement in the 
standard keyboard. It is impossible to play certain combinations of notes at the same 
time, such as the keys indicated by the black dots, because they are so far apart. B: A 
larger, nonstandard keyboard was created by placing each note at many possible 
locations, thus each note is represented over many larger areas that overlap with other 
notes. It is easy to see that there are many more possible note combinations than what is 
available on the standard keyboard, including the impossible to combine black dotted 
notes on the standard keyboard in A. This is Figure 10 from Schieber, 2001. 
20 
Each key in a standard piano keyboard is colored in Figure 1.42 A. It is impossible to 
play the combination of notes indicated by the black dots because they are distant. Figure 
1.42 B shows a complex keyboard composed of many different combinations of the 
original keys. With the new arrangement there are many more possible combinations of 
notes. One of these combinations allows the notes indicated in Figure 1.42 A to be 
played. Thus, by analogy, motor movements can be produced by simultaneous activation 
of various combinations of neurons that do not always lie in sequential relation to one 
another. 
Because movement is so complex, it is necessary to choose an appropriate model 
in order to study the effects of stroke on the motor system. The primate would be an ideal 
model, because their motor system most closely resembles ours. Rodents make a more 
cost effective alternative, while sharing many similarities to the human motor system. 
The following section describes the most popular rodent models in stroke research, along 
with justification for choosing the mouse. 
1.5. Modeling stroke in the rodent. 
When modeling stroke it is important to choose a model that will be best suited to 
the types of questions being asked. Rodents are a good model system in which to study 
stroke because they are relatively inexpensive, they have a short lifespan, and are similar 
to humans. There are essentially two general methods of modeling stroke in rodents (for a 
review see Hossmann, 1998 and Nedergaard, 1988). The first is a global ischemia model, 
which will temporarily impair blood flow to the entire brain. It is accomplished by 
occluding various combinations of the four arteries that service the brain. This method is 
most commonly used to mimic the clinical condition of cardiac arrest and will result in 
more extensive damage in the periphery of the brain where the vasculature is branched 
and more diffuse (Hossmann, 1998). The second method of modeling stroke is a focal 
ischemia model that impairs blood flow to a smaller region of the brain damaging 
primarily that area. This can be accomplished using a middle cerebral artery occlusion 
21 
(MCAO)(Tamura, et al., 1981). The middle cerebral artery in the rat is indicated in a 
dorsal photograph of the rat brain in Figure 1.51. 
22 
lIllBlI 
Anterior Cerebral 
Figure 1.51. The surface cerebrovasculature of the rat. The divisions in the corner 
represent 1 mm. This photograph is courtesy of Richard Dyck at the University of 
Calgary. 
23 
There are two main variations of the MCAO method. The first requires feeding a thread 
through the internal carotid until it reaches the base of the MCA; the thread can either 
permanently or temporarily occlude the MCA. In the second variation, the MCA itself 
can be accessed via a craniotomy and permanently occluded via electro-coagulation or 
temporarily occluded with a knot or a clip. Both methods have two primary 
disadvantages. The first is that infarct sizes are variable because of collateral blood flow. 
Both the Circle of Willis and the pial network, which connects the anterior, posterior and 
middle cerebral arteries (Hossmann, 1998), can maintain blood flow to some degree 
when the MCA is occluded. Figure 1.51 depicts the numerous tiny vessels that 
interconnect the main vessels in the cortex. The second disadvantage is that an MCAO 
produces massive damage to numerous brain structures: the white matter, striatum, 
hippocampus and the motor cortex. 
Because this thesis is concerned primarily with the motor system, an alternative 
stroke that affects primarily the motor cortex was chosen. This alternative model 
devascularizes the motor cortex and it is known as the pial strip. A pial strip removes the 
pial vasculature over a designated surface of the motor cortex, ensuring that the stroke is 
localized primarily to that area. The cerebrovasculature on the surface of the motor cortex 
is also illustrated Figure 1.51. 
When choosing a model, it is also important to take into account the unique 
cellular pathology stroke produces. The next section will describe the affects of a stroke 
at a cellular level. 
1.6. Molecular mechanisms of cell death. 
Stroke produces a distinctive pattern of tissue damage. The site of the stroke 
leaves an area of complete cell death that is called the infarct. Typically the tissue 
surrounding the infarct will also experience some degree of damage due to the impaired 
blood flow. This area is referred to as the penumbra. The penumbra is of specific interest 
because cell death is often delayed and thus the penumbral tissue may be salvageable. 
24 
The pathology associated with a focal and global model of rodent ischemia is reviewed 
elsewhere (Lipton, 1999; Hossmann, 1994; Nedergaard, 1988). 
There are many mechanisms that may contribute to cell death. Because the 
primary focus of this thesis is with motor impairments and not cellular pathology, this 
section will briefly outline the complex process of cell death. The prevailing idea related 
to stroke induced cell death is called the Excitotoxicity or Excessive Neuronal Excitation 
Hypothesis. Cell death begins when the reduction of oxygen and glucose results in a 
decrease in energy production in the affected area. The cell needs energy to maintain ion 
gradients. Sodium (Na+) and toxic calcium (Ca2+) ions (Fleckenslein, et al., 1974; for a 
review see Kristian and Siesjo, 1998) are present in large quantities outside the cell 
primarily due to pumps that ensure these ions are expelled (for a review see Carafoli, 
1987). If ion gradients are disrupted K+ flows out, voltage gated ion channels open, and 
Na+ and Ca2+ flow in (Nicholson, et al, 1977). 
Calcium entry results in release of the primary excitatory neurotransmitter 
glutamate; it had been hypothesized that glutamate release is toxic to the cell (Olney, et 
al., 1971). This is confirmed by studies that show glutamate causes a Ca2+ mediated cell 
death (for a review see Choi, 1987). Glutamate activates the N-methyl-D-aspartate 
(NMD A) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (A MP A) 
receptors on the cell surface (Pelligrino-Giampietro. et al., 1992). Activation of the 
AMP A and NMDA receptors will allow Na+ translocation into the cell, which will 
depolarize the postsynaptic membrane. Excessive glutamate will cause excessive 
depolarization, which will then displace the magnesium (Mg2+) ion inside the NMDA 
Ca2+ channel. Additional Ca2+ will then flow into the cell via the NMDA receptor. This 
hypothesis is attractive because the CA1 and CA4 neurons of the hippocampus have high 
concentrations of NMDA receptors and they are particularly vulnerable to ischemic 
damage. 
25 
Calcium is also released from the intracellular endoplasmic reticulum (ER). Cell 
surface receptors activate a membrane bound G protein, which in turn activates the 
intramembrane phospholipase C. Phospholipase C contributes to membrane destruction 
and also cleaves the intramembrane phosphatidyl inositol (PIP2) into the two secondary 
messengers: inositol triphosphate (IP3) and diacylglycerol (DAG). IP3 can translocate 
into the cytoplasm where it acts on the ER promoting Ca2+ release (for a review Berridge, 
1993). 
Ca2+ also activates a number of cellular cascades and products including: 
phospholipases, endonucleases, proteases, lipases, and protein kinases involved in 
phosphorylation. Ca2+ also promotes the formation of enzymes that produce free radicals 
such as nitric oxide (NO) and reactive oxygen species (ROS) (Choi, 1987; Kristian and 
Siesjo, 1998). ROS, also called free radicals, are groups of atoms that have unpaired 
electrons. The unpaired electron makes the radicals unstable so they react quickly with 
other molecules to obtain an additional electron, often destroying them in the process. 
Common cellular targets are cell membranes because they have many exposed hydrogen 
atoms with available electrons. Ischemic cells experiencing an energy crisis will also 
exhibit enhanced ROS production that contributes to cell death. Figure 1.61 summarizes 
the excitotoxicity hypothesis. 
26 
ISCHEMIA 
Pump Failure 
Depolarization 
Glutamate Release / 
AMPA 
receptors Metabotropic 
receptors I 
Intracellular 
calcium release 
from stores 
\ 
Calcium channels 
"N^ open 
NMDA 
receptors 
CALCIUM 
INFLUX 
Total 
intracellular 
calcium 
CELL DEATH 
Figure 1.61. A summary of the processes that are hypothesized to result in cell death. 
This figure is adapted from the Lyden and Wahlgren, 2000 review. 
27 
Understanding the complex process of cell death may lead to the ability to salvage 
the injured tissue. This is because there are many possible mechanisms involved in the 
complex cascade that can potentially be manipulated. These manipulations that are 
thought to be neuroprotective can be tested for functional outcome using behavioural 
studies with measurable parameters in standardized tests. The following section will 
describe some aspects of behavioural testing useful for assessing motor function. 
1.6. Testing behaviour in the rodent. 
There are a variety of behavioural tests available for testing sensorimotor function 
in rats, but there are very few available for mice. Immediately following a motor system 
injury, a rat's ability to walk may be affected and these deficits can be measured by 
performance on the rotarod, a rotating cylinder, or a rung-walking test (Metz and 
Whishaw, 2002). Because the sensorimotor deficits a stroke animal suffers are often 
unilateral (Yamamoto, et al., 1988), most tests measure forelimb asymmetry. For 
example, if an animal is lowered towards a surface it will extend its forelimb in 
anticipation of contact; this response is impaired in the injured forelimb (Hua, et al., 
2002; Schallert, et al., 2000). The injured forelimb is also relatively inactive during 
spontaneous vertical exploration (Schallert, et al., 1997; Schallert, et al., 2000). In 
addition, control rats do not use their forelimbs while swimming, except to turn, but this 
forelimb inhibition may be lost following an ischemic insult causing the rat to paddle 
with all four limbs (Stoltz, et al., 1999). Most of these tests provide a quantitative score 
of performance. The animal often appears to recover quickly on these measures after the 
insult. It is therefore necessary to have a detailed qualitative assessment of impairments 
to distinguish between recovery and compensation. 
The single pellet reaching task has the advantage of providing both a quantitative 
and a qualitative measure of performance (Whishaw, 2000; Whishaw, et al., 1992; 
Whishaw, et al., 1992a). It has been standardized for both rats and mice (Farr and 
Whishaw, 2002, Whishaw, 1996). When a laboratory mouse is reaching for a single piece 
of food in the single pellet reaching task, the entire sequence can be subdivided into ten 
28 
stereotypical movements (Farr and Whishaw, 2002). These movements can be evaluated 
using a scale adapted from a movement notation that expresses the limbs as axis and 
documents movement by observing the changes in the limb and body axis (Eshkol and 
Wachmann, 1958). A reach occurs as follows, the paw is lifted so that the semiflexed 
digits are aligned with the body's midline. Then the elbow is brought to the midline. This 
is called the "aiming" position. During the advance the digits extend as the paw is moved 
through the slot in anticipation of contact with the food. The digits then pronate over the 
food item so that each digit contacts the surface of the shelf in succession. This 
movement is called the arpeggio. The digits close around the food item during the grasp. 
The paw is supinated for the first time as it is withdrawn through the slot and supinated 
further to present the food to the mouth. Aiming, pronation and supination are performed 
mainly by upper arm movements. 
Mice, like rats and humans, are also impaired at skilled reaching following injury 
to the motor system (for a review see Whishaw, 2000; Whishaw and Coles, 1996; 
Whishaw and Gorny, 1994, Whishaw, et al, 1992a; Whishaw, et al., 1992b; Whishaw. et 
al., 1991; Castro, 1972). Injury to the motor cortex affects various aspects of the reach, 
specifically the aiming and rotatory movements of the forelimb (Whishaw, 2000; 
Whishaw and Coles, 1996; Whishaw, et al., 1992a; Whishaw, et al., 1992b; Whishaw, et 
al., 1991) and often recovery is minimal and the animals learn to compensate for the 
deficits (Whishaw, 2000; Friel and Nudo, 1998; Whishaw, et al, 1991). 
The most commonly used mouse behavioural examinations are neurological 
rating scales (Rogers, et al, 1997; Hunter, et al, 2000; Sampei, et al, 2000). Cognitive 
and sensory tests, such as passive avoidance, that incorporate learning and memory are 
also commonly used (Hattori, et al, 2001; Zhang, et al, 2002). They are not specific for 
the motor system, however. Tasks that are commonly used for testing the motor system in 
mice are the rotarod, in which mice are required to walk on a rotating cylinder (Jones and 
Roberts, 1968) or the raised beam where the footsteps are counted. These tests do not 
often reflect any long term deficit because mice recover the ability to walk quite quickly. 
The Montoya staircase task (Montoya, et al, 1991) places the animal in an enclosure 
29 
with a divided staircase, ideally the impaired limb will be unable to retrieve the food 
source placed on that side of the divided staircase; this task has been adapted for mice 
(Baird, et al, 2001). Nevertheless, the mice exhibit unusual strategies when retrieving the 
food, making it difficult to assess the degree of impairment. 
Because there are few ways to assess functional outcome in mice, one might ask, 
why use mice at all? There is one strong advantage to using mice and it will be described 
in the following section. 
1.6. Why use mice? 
The recent development of transgenic technology has made it possible to create 
many strains of mice, which is revolutionizing genetic research (for an excellent review 
see Wagner, et al, 1995 or Jaenisch, 1988). Transgenic technology involves specific 
manipulations of the genome, making it possible to assess the function of individual 
genes to the animal. Because transgenics have been developed in the mouse, it is possible 
to remove or insert genes from any living organism including humans, as well as 
promoters and reporters into the transgenic genome. Stroke research has benefited from 
transgenic technology by using genetic manipulations to answer some molecular concepts 
(for relevant papers see Sampei, et al, 2000; Guegan, et al, 1998; Eliasson, et al, 1997; 
Chan, et al, 1995). The contributions of transgenics to stroke research will be discussed 
later. For the present it is worth making note of the most important transgenic 
contributions to stroke research. Knocking out the gene that codes for nitrous oxide 
synthase (NOS) in mice prevented the production of nitric oxide, which is known to be a 
powerful and disastrous free radical (Chan, et al, 1995). Thus, these "knockouts" 
suffered minimal tissue damage from a stroke when compared to the wild type controls. 
This advantage of using mice makes it necessary to develop mouse appropriate 
behavioural tests. Therefore, the first goal of the thesis was to adapt the single pellet 
skilled reaching task to mice with a stroke in order to obtain a quantitative and qualitative 
functional measure of behavioural outcome, recovery and compensation. 
30 
1.7. Introduction to the Experiments. 
Because the single pellet reaching task is fairly specific to the sensorimotor 
system, the focal model, pial stripping, was chosen to localize the stroke to the motor 
cortex. The first experiment documents the single pellet reaching task in C57/Black/6 
mice that have received a stroke. The C57/Black/6 mice are one of the most common 
strains of mice (Fujii, et al., 2003). Testing took place both before and after a focal motor 
cortex stroke produced by pial stripping. Post stroke, the animals are severely impaired 
both quantitatively in the number of pellets they were able to retrieve, and qualitatively, 
in the way the pellets were retrieved. 
The second experiment was designed in much the same way. It was apparent that 
the mice were not able to easily grasp 20mg food pellets, so a smaller food item, dehulled 
millet seeds, was obtained. In addition, the method of producing stroke was altered. The 
focal motor cortex stroke was produced by applying the potent vasoconstrictor 
Endothelin-1 (ET-1) (Yanagishawa, et al., 1988) to the motor cortex; this compound has 
been used to produce reproducible temporary occlusions (Sharkey, et al., 1993). An 
endothelin application does not damage the pial vasculature and it's affects are 
temporary. The endothelin-type stroke did not result in a large quantitative deficit, but the 
animals were impaired qualitatively. 
The third experiment compared the effects of the two stroke types. Post surgery, 
the ET-1 group was not significantly different from the control group in their quantitative 
success rates; the mice that received the pial strip were impaired and gradually recovered 
to the level of the endothelin mice over the remainder of the testing period. The detailed 
qualitative analysis revealed that both groups of mice suffered impairments in the same 
types of movements, though the pial strip animals were more severely impaired. A 
correlation analysis indicated that the larger the stroke insult the more impaired the mice 
were at many of the movements. The brain tissue of the endothelin group showed little 
infarct damage but microscopic observation indicated that the underlying cell 
31 
morphology was abnormal. Thus, it seems possible to titrate the endothelin model to 
produce varying degrees of damage. 
32 
2. Experiment 1; 
Skilled movements in a mouse are impaired after a focal motor cortex 
stroke. 
Abstract 
Background and Purpose - Skilled reaching movements are an important aspect of 
human motor behavior, but are impaired following motor system stroke. The purpose of 
this study was to document skilled movements in mice before and after a focal motor 
cortex stroke for the purpose of developing a mouse model of human stroke. 
Methods - Male C57/BL6 mice were trained to reach with a forelimb for food pellets and 
then given a motor cortex stroke, induced by pial stripping, contralateral to their preferred 
reaching limb. Reaching success and the movements used in reaching were analyzed by 
frame-by-frame inspection of presurgical and postsurgical video records. 
Results - Reaching success was severely impaired following the stroke. Improvement in 
success over two postsurgical weeks was moderate. Analysis of ten movement 
components, comprising reaches pre- and postsurgically, indicated that most of the 
rotatory movements of the limb used for aiming, advancing, pronating, and supinating the 
paw were impaired. When successful reaches did occur, body movements that 
compensated for the impairments in limb rotatory movements aided them. 
Conclusions - The results indicate that skilled reaching in the mouse is impaired by focal 
motor cortex stroke and they suggest that the mouse, and the skilled reaching task, 
provides an excellent model for studying impairments, compensation, and recovery after 
motor system stroke. 
33 
Introduction 
Comparative analyses of food handling and skilled reaching by laboratory rats 
(Rattus norvegicus) and mice (Mus musculus) show that the forelimb movements used by 
the two species are similar (Whishaw, 2000; iwaniuk and Whishaw, 2000; Whishaw, et 
al, 1998; Whishaw, 1996; Whishaw and Coles, 1996). To eat they use five movements in 
sequence. They identify the food via olfaction, pick up the food using the mouth, sit back 
on their haunches, grasp the food with the forepaws by bringing the elbows inward, and 
manipulate the food with the digit tips. 
Similarly, while performing in a formal laboratory skilled reaching task, requiring 
extension of a forelimb through an opening in a cage in order to obtain a food pellet, rat 
and mouse movements are similar (Whishaw, 1996; Whishaw, et al., 1992). The paw is 
lifted so that the digits, semiflexed, are aligned with the midline of the body, and then the 
elbow is also brought to the midline, to an "aiming" position. As the limb is advanced 
through the slot, the digits are extended. As the limb is pronated, the digits are opened 
thus placing the paw over the food. The food item is grasped by digit closing, the wrist is 
extended to lift the paw, and the paw is withdrawn through the slot. The paw is then 
supinated further so that the food can be presented to the mouth. Aiming, pronation and 
supination are performed mainly by upper arm movements (Whishaw and Pellis, 1990; 
Castro, 1972). Pronation consists of rotating the paw so that digits 5 (outermost) through 
2 (innermost) contact the surface on which the food pellet is placed, and this "arpeggio" 
movement is assisted by movements around the wrist (Whishaw and Gorny, 1994). 
Studies using rats show both spontaneous and skilled limb use are impaired 
following damage to the motor system (Stein, 2001; Whishaw, 2000; Hunter, et al, 2000; 
Kolb, et al, 2000; Zhang, et al, 2000; Modo, et al, 2000; McKenna and Whishaw, 1999; 
Petullo, et al, 1999; Whishaw, 1996; Whishaw. et al, 1992a; Whishaw, et al, 1992b; 
Montoya, et al, 1991; Whishaw, et al, 1991; Gonzalez, et al, 1986; Castro, 1972). 
Impairments produced by damage to different motor regions are also distinctive. For 
example, following a motor cortex injury, a rat still retrieves food by reaching with the 
34 
affected forelimb but success is reduced (Biernaskie and Corbett, 2001; Whishaw, 2000; 
Kolb, et al, 2000). In addition, most of the movement components of the reach are 
abnormal. Compensatory body movements provide the rotatory movements to assist 
pronation and supination (Whishaw, 2000). Thus, the species has provided a primate 
substitute for studying the neural basis of skilled movements. 
Given recent interest in using transgenic mice for stroke research (Hattori, et al, 
2001; Sampei, et al, 2000; Guegan, et al, 1998; Rogers, et al, 1997; Eliasson, et al, 
1997; Chan, et al, 1995) the present study investigated the effects of motor cortex stroke 
on skilled reaching in mice. The mice were trained to reach through a slot for food and 
their performance was video recorded before and after a cortex stroke produced by pial 
stripping. 
Materials and Methods 
Subjects 
Eight five-month old male C57/Black 6 mice (Mus rnusculus) weighing between 
25-30g were raised in the University of Lethbridge animal colony from animals originally 
obtained at Jackson Laboratories, Bar Harbor, ME. Animals were housed individually in 
standard plastic mouse cages (lights on a 12:12 hour cycle beginning at 08.00; room 
temperature 22?C ) and given one piece of Lab Chow (4g) after the testing period each 
day. Two of the eight animals consistently had less than half of a gram of food remaining 
in the morning that was removed at 8:00. Because testing began at 1:00, even the mice 
with remaining food had to wait five hours before regaining access to food. Each mouse 
was thus sufficiently restricted and motivated to perforin the task. The experiment was 
conducted according to the Canadian Council on Animal Care code. 
Surgery 
After adaptation to reaching and six days of training the animals were 
anaesthetized using isoflurane (BIMEDA-MTC Animal Health Inc.). Eight mice, 5 left, 
35 
and 3 right paw preference, received a pial strip stroke to the motor cortex contralateral to 
the preferred forelimb. Using stereotactic surgery, a 3mm by 3mm region of the skull was 
removed, 0.5mm behind bregma to 3mm anterior to bregma, and from 0.5mm lateral to 
the midline to 3.5mm lateral to the midline. The dura was removed within the 
trephination and the underlying pia, arachnoid, and vasculature was wiped with a cotton 
swab until no vasculature was visible. Shams were not performed because the removal of 
the skull and dura did not result in any motor deficits in similar experiments in rats 
(Kleim, et al, 1998). Vetropolycin (Janssen) gel was applied to the eyes and the incisions 
were closed. The animals were returned to their home cages and postsurgical testing 
began the following day. 
Reaching Task 
The Plexiglas reaching box (Whishaw, 1996) was 19.5cm long, 8cm wide, and 
20cm high (Figure 2.11). 
36 
Figure. 2.11. The single pellet reaching apparatus. The shelf in front of the box contains 
two divots for food. 
37 
A 1cm wide vertical slit ran up the front of the box. A 0.2cm thick plastic shelf (8.3cm 
long and 3.8cm wide) was mounted 1.1cm from the floor on the front of the box. Twenty-
milligram food pellets (Bioserve Inc.) were placed in indentations spaced 1cm away from 
the slit and centered on its edges. Animals were habituated for one week by placing them 
in the cages for ten minutes. Pellets were initially available on the cage floor and within 
tongue distance on the shelf. Pellets were gradually removed from the floor and placed 
farther away on the shelf until the mice were forced to reach to retrieve the food. The 
pellets were placed in both indentations allowing the mice to display which paw they 
preferred to use after which the food was placed in the indentation contralateral to the 
preferred paw. As the animal pronates the paw medially, this placement allows the mouse 
to obtain the pellet with a paw and not with the tongue (Whishaw, et al., 1991). 
Videorecording 
Filming was done on the last two pre- and postsurgical days of training with a 
Sony DSRPDIOO digital camcorder (30 frames/sec; shutter speed of 1000). Illumination 
was provided with a 2 arm Nikon MKII150W fiber optic light. The animals were filmed 
from a frontal and ventral view (by placing the apparatus over an inclined mirror table). 
The tapes were viewed on a Sony DV cam DSR-20 player and Trinitron monitor. 
Representative movements were captured using Final Cut Pro frame grabbing software 
on a Macintosh G3 computer. 
Reaching Success 
Each animal reached for twenty pellets each day during the testing period. All 
animals were more than sufficiently motivated to attempt to obtain 20 pellets. If an 
animal reached through the slot and obtained a food pellet, the reach was scored as a 
success. If an animal knocked the food away, or dropped the food after grasping it, the 
reach was scored as a miss. Performance was defined by Percent Success = (number of 
successful retrievals/ 20)* 100 
38 
Qualitative Analysis 
Qualitative movement scoring was derived from a conceptual framework adapted 
from Eshkol-Wachmann Movement Notation (Eshkol and Wachmann, 1958). EWMN is 
designed to express relations and changes of relation between the parts of the body. The 
body is treated as a system of articulated axes (i.e., body and limb segments). A limb is 
any part of the body that either lies between two joints or has a joint and a free extremity. 
These are imagined as straight lines (axes), of a constant length, which move with one 
end fixed to the center of a sphere. On the basis of descriptions obtained from EWMN, 
rating scales of movements were derived (Whishaw and Pellis, 1990). 
Five presurgical and five postsurgical reaches from each mouse were rated for 
qualitative features of the movement (Whishaw, et al., 1991). Ten components of a reach 
were rated: 
1) Digits to the midline. Using mainly the upper arm, the reaching limb is lifted 
from the floor so that the tips of the digits are aligned with the midline of the body. 
2) Digits semiflexed. As the limb is lifted, the digits are flexed and the paw is 
supinated so that the palm of the paw is aligned almost vertically. 
3) Aim. Using an upper arm movement, the elbow is adducted to the midline 
while the tips of the digits remain aligned with the midline. 
4) Advance. The limb is advanced directly through the slot toward the food target 
using an upper arm movement and during advancement the snout is raised to allow 
passage of the paw into the slot. 
5) Digits extend. The digits extend during the advance. 
6) Pronation. When the paw is over the target, the paw pronates and digit 5 (the 
outer digit) through to digit 2 touches the surface in succession, mainly by abduction of 
the elbow and also by a rotational movement around the wrist. During pronation, the 
digits open. 
7) Grasp. The digits flex over the food and close around it. The paw remains in 
place and the wrist is slightly extended to lift the food. 
8) Supination I. As the paw is withdrawn, it supinates by almost 90 degrees. 
39 
9) Supination II. Once the paw is withdrawn from the slot the paw further 
supinates by 45 degrees to present the food to the mouth. 
(10) Release. The mouth contacts the paw and the digits open to release the food. 
Each movement was rated on a 3-point scale. If the movement was normal, a 
score of 0 was given. In cases where there was some ambiguity concerning the 
occurrence of a movement, or the movement was present but incomplete, a score of 1 
was given. If the movement was absent, a score of 2 was given. 
Histology 
The mice were deeply anaesthetized and perfused through the heart with 0.9% 
saline followed by 4% para-formaldehyde/0.9% saline. Brains were removed and cryo-
protected in 30% sucrose/4% para-formaldehyde for one week. The brains were sectioned 
(50um) on a 2800 Frigocut E cryostat (Reichert-Jung), mounted on 1% gelatin and 0.2% 
chromalin dipped slides, stained with Cresyl violet, and cover slipped using Permount 
(Fisher-Scientific). 
Results 
Histology 
All of the brains had damage in the motor cortex. Figure 2.12 indicates 
representative dorsal (Figure 2.12A-B) and coronal views (Figure 2.12C-E) of the lesion. 
40 
9> t.m 
E 
Figure 2.12. (A-B) Dorsal view of a typical motor cortex stroke. (C-E) Photomicrographs 
(Cresyl violet) of a representation of the motor cortex stroke at three coronal levels. The 
arrows in the right hemisphere indicate the location of the lesion in the motor cortex. The 
intact left hemisphere indicates the brain regions present (MFL- Motor Forelimb Area, 
Cgl and Cg2- Cingulate Cortices 1 and 2, SFL- Sensory Forelimb Area, SI- Primary 
Sensory Area, S2- Secondary Sensory Area, S1BF- Primary Sensory Barrel Forelimb 
Area). 
41 
Pial stripping produced a conical cavity beneath the area of pial removal, and the cortex 
adjacent to the lesion evaginated and filled in the cavity produced by the lesion, making 
quantification of the actual lesion size difficult. Nevertheless, based on a comparison of 
the lesion location relative to the large layer V cells in the contralateral hemisphere, the 
lesions appeared restricted to the caudal and rostral forelimb areas as defined by 
electrophysiological stimulation studies in the rat (Kleim, et al., 1998). 
Quantitative changes in reaching following stroke 
Reaching Success 
The ANOVA on success revealed a significant Treatment effect, F(l, 15)= 
38.962, P< 0.001, indicating that the mice achieved higher reaching scores before the 
motor cortex lesion. There was a significant effect of Days, F(5, 75)=3.48, P< 0.007, but 
there was no significant Group by Days interaction, F(5, 75)= 2.114, P= 0.0729. The 
mice achieved an average percent success of 40? 10 prior to the lesion and the average 
percent success dropped to 10?10 following the lesion (Figure 2.13). An ANOVA on 
postsurgical performance was not significant, although the animals displayed a trend 
toward improving. 
42 
U-j 1 , , , , , , 1 , 1 , 1 1 
1 3 5 1 6 12 
Days 
Figure 2.13. Reaching success (means ? SEM). The dotted line represents the 
administration of a motor cortex stroke. The single point post surgery averages the 
control performance. (*** P < 0.001). 
43 
Movement Components 
The ANOVA of five presurgical and postsurgical reaches revealed a significant 
main effect of Group, F(l,16)= 65.01, P < 0.001, indicating that the mice accumulated 
higher scores for the ten movement components following cortical injury. There was also 
a main effect of Movement, F(9, 144)= 14.68, P < 0.001, indicating that some movements 
were more impaired than others. More importantly, there was a significant Group by 
Movement interaction for the Postsurgery stroke group, F(9, 144)= 8.232, P < 0.001, as 
some movements in the stroke group were more impaired relative to control scores. 
Follow-up students unpaired t-tests indicated significant group differences for the aim, 
advance, pronation, supination II and release components of the reach, but not for the 
digits to the midline, digits semiflexed, digits extend, grasp and supination I (Figure 
2.14). 
44 
0) 
Release 
Supination 2 
Supination 1 
Grasp 
Pronation 
Digits Extend 
Advance 
Aim 
Digits Semiflexed 
Digits to Midline 
? Control 
? Stroke 
0 
Score 
Figure 2.14. Scores (means ? SEM) for each of the ten movement components of the 
reach. Control white bars, stroke solid bars. A score of 0 indicates a normal movement 
while a score of 2 indicates complete absence of the movement. (*** P < 0.001)(** P < 
0.01)(* P < 0.05). 
45 
o I 
o U 
a; S 
QJ 
> 
O 
Qualitative changes in movement following stroke 
The following descriptions and ratings were all taken from reaches that were 
successful. Nevertheless, it is likely that the abnormalities displayed by the stroke mice 
on successful reaches did contribute to the lower incidence of successful reaches. 
Digits to the Midline and Aim 
Control mice lifted the reaching limb from the surface, semiflexed the digits, and 
aligned the digit tips with the midline of the body with a single movement (Figure 
2.15A). 
46 
Control Stroke 
Figure 2.15. Digits to Midline and Aim. A, B: Control. The control mouse lifts the 
reaching limb and aligns its digit tips with the midline of the body (arrow in A) and then 
aims the limb by bringing the elbow to the midline (double sided arrow in B). C, D: 
Stroke. The stroke mouse also aligns the digit tips with the body but must shift the 
shoulders to achieve this (arrows in C). Note: The non-reaching forelimb is supporting 
the unusual posture. Note: The double-sided arrow in B and D indicates the angle of the 
paw-elbow segment relative to the angle of the body. 
47 
They then adducted the elbow to the midline of the body so that the forelimb was aligned 
with the body midline in an "aiming" position (Figure 2.15B). Stroke mice also lifted the 
limb and semiflexed the digits, but the digit tips were displaced more laterally. Then, 
rather than adducting the elbow to the midline, to bring the paw to a position from which 
it could enter the slot, the animals made an ipsiversive rotatory movement of the body to 
bring the limb to an aiming position. Thus, the comparison in Figure 2.15A (control) and 
Figure 2.15C (stroke) indicate that although the digits seem aligned with the body in both 
animals, the stroke animal has achieved alignment in part by using body rotation. The end 
result was that the control mice were able to advance the paw from an "aiming" position 
directly through the slot toward the food, whereas the stroke mice directed the paw 
through the slot diagonally. 
Advance and Digits Extend 
The control mice advanced the paw directly forward over the food item, while at 
the same time extending the digits (Figure 2.16A), leaving the paw in a position to 
pronate over the food pellet (Figure 2.16B). 
48 
Advance and Digits Extend 
Figure 2.16. Advance and Digits Extend. A, B: Control. The control animal can advance 
the reaching forelimb directly over the food pellet (A) and extend the digits while 
beginning to rotate the paw over the food pellet (arrow in B). C, D: Stroke. The stroke 
animal lifts the reaching forelimb with the shoulders during the advance (arrow in C) and 
slaps laterally to contact the pellet while extending the digits (arrow in D). 
49 
The stroke mice also opened the digits as the paw advanced, but the paw entered the slot 
diagonally so that it was located dorsally and medially relative to the food pellet (Figure 
2.16C.D). 
Pronation 
Once the limb is advanced and the digits are extended, the paw is pronated over 
the food pellets as the digits are opened from digit 5 through digit 2. The digits also 
contact the shelf in the sequence digit 5 through digit 2. This arpeggio movement is 
illustrated for a control mouse reaching in an instance in which no food pellet was located 
in the slot (Figure 2.17A-D top). 
50 
Figure 2.17. Pronation. A-D, top: Control. The digits contact the surface of the shelf in 
succession from the most medial (pinky) to the most lateral (pointer). The digits extend 
and open. A-D, bottom: Stroke. The paw comes in from the side, or slaps laterally, and 
digits do not contact the surface in succession. 
51 
Typically, when the paw and digits contact a food pellet, grasping occurs. If the paw fails 
to contact a food pellet, grasping does not occur. The stroke mice slapped the paw 
laterally, instead of using a rotatory wrist movement, and grasped whether or not a food 
pellet was contacted (Figure 2.17A-D bottom). 
Grasp and Supination I 
Once the food was contacted, the control mice closed the digits around the pellet 
and supinated the closed paw so that the palm of the paw was oriented vertically (Figure 
2.18A), from which position the paw was withdrawn through the slot (Figure 2.18B). 
52 
sp SEC; S^piBatie 
Figure 2.18. Grasp and Supination I. A, B: Control. The control animal closes the digits 
around the food item (A) and exhibits more supination during the withdrawal (arrow in 
B). C, D: Stroke: The stroke animal also closes the digits around the food item (C) and 
degree of supination is less than that of the control, using a dragging motion to obtain the 
food (arrow in D). 
53 
Stroke animals displayed little supination after grasping the food (Figure 2.18C), and 
whatever supination they did achieve occurred by leaning on the pellet with the forelimb 
as they withdrew the paw from the slot (Figure 2.18D). 
Supination II and Release 
Once the paw with food was withdrawn, the control mice further supinated the 
paw, so that the palm faced upward to present the food to the mouth (Figure 2.19A). 
54 
Supination II and Release 
Control Stroke 
Figure 2.19. Supination II and Release. A, B: Control. A control animal supinates the 
paw to present the food to the mouth (arrow in A) and opens the digits to release it (arrow 
in B). C, D: Stroke. A stroke animal chases the paw downward (arrow in C) and the other 
paw provides assistance while releasing the pellet (arrow in D). 
55 
Supination II and release of the food was frequently accomplished without assistance 
from the non-reaching paw (Figure 2.19B). Stroke mice typically used the non-reaching 
forepaw to grasp the reaching forepaw and assist in Supination II and Release (Figure 
2.19C,D). 
Discussion 
This study examined the effect of motor cortex stroke on quantitative and 
qualitative measures of skilled reaching in mice. Following a focal motor cortex stroke, 
reaching success by the paw contralateral to the lesion was impaired and did not recover 
over two postsurgical weeks of testing. At this time many component movements used 
for reaching remained abnormal, especially the rotatory movements of the limb 
associated with aiming, pronating, and supinating the paw. When reaches were 
successful, they were achieved using compensatory body movements. This is the first 
study to describe qualitative impairments in skilled reaching in the mouse and the results 
suggest that this species provides a good model for analysis of motor skills, plasticity, 
and recovery processes. 
The mice were trained and tested pre-and postsurgically in an apparatus in which 
they reached through a slot in a test box for single pieces of food located on a shelf just 
outside the slot. Prior to the lesion, the animals were able to orient to the slot, advance a 
paw through the slot, grasp and retrieve the food pellets for eating. Performance 
presurgicaliy was reasonably accurate but following the lesion the mice were extremely 
impaired, consistent with findings in mice subjected to prefrontal damage on another 
motor task (Baird, et al., 2001). 
Prior to surgery, the mice used a distinctive pattern of movement when reaching 
for food. They located the food by sniffing for the food pellet, oriented their body, and 
then made a characteristic series of limb movements in order to retrieve a food pellet. The 
paw was lifted so that the slightly flexed digits were aligned with the tips to the midline 
of the body. The elbow was then adducted to the midline of the body so that the limb was 
56 
aimed to enter the slot. As the paw was advanced through the slot to the food the digits 
were extended, and then the paw was pronated while opening the digits to grasp the food. 
The paw was then supinated to withdraw the food through the slot, and then once 
through, was supinated further to place the food in the mouth. 
Following the stroke reaching movements were altered, even when the animals 
successfully obtained food. The paw and elbow were not aligned to the aiming position 
along the midline of the body, but rather proceeded toward the food diagonally. Some 
aiming was obtained by an ipsiversive rotation of the fore portion of the trunk. Pronation 
was incomplete as the animals swiped at the food pellet with a sideways motion of the 
paw. Finally, the movements of supination during withdrawal and release were abnormal 
as the paw was incompletely supinated. Again, food retrieval was assisted by rotatory 
movements of the trunk and through the assistance of the non-reaching paw. 
Some of the stroke animals' movements were not impaired. Stroke animals 
remained able to semiflex and extend the digits as well as grasp the food. Again, these 
findings are consistent with the impairments described for the rat following a focal motor 
cortex injury (Whishaw, 2000; Whishaw, et al., 1992a; Whishaw, et al., 1992b; 
Whishaw, et al., 1991; Gonzalez, et al., 1986). 
These changes in movements post stroke suggest that the function of the motor 
cortex is in part to produce rotatory movements of the limb (Evarts, 1979). This 
conclusion is consistent with other findings of impaired limb use (Palmer, et al., 2001; 
Nudo, et al., 2001; Biernaskie and Corbett, 2001; Whishaw, 2000; Borlongan, 2000; Friel 
and Nudo, 1998) with recovery likely due to compensatory movements. 
In conclusion, this is the first study to document a detailed qualitative impairment 
in skilled movements following a motor cortex stroke in mice. The main findings are that 
mice perform skilled reaching movements quite well and the skilled reaching test is 
sensitive to damage to the motor cortex. A strength of the model is that it is able to 
57 
provide a quantitative score and a qualitative assessment of deficits, compensation and 
recovery. 
58 
3. Experiment 2: 
The effects of a new stroke model on mouse skilled reaching. 
Abstract 
Background and Purpose - Focal ischemia via permanent occlusion of the motor cortex 
vasculature results in massive motor impairments in the mouse, similar to the rat and 
comparable to what is seen in humans. There are many methods of modeling stroke in 
rodents and each is capable of producing different pathological outcomes that may 
correlate with behavioural outcome. The purpose of this study was to document skilled 
movements in mice subjected to a temporary reduction in blood flow in the motor cortex 
followed by tissue reperfusion. 
Methods - Male C57/BL6 mice were trained in the single pellet reaching task to obtain 
seeds with their preferred forelimb. Following baseline training the mice received a focal 
motor cortex stroke, induced by topical application of the vasoconstrictor Endothelin-1, 
in the hemisphere contralateral to their reaching limb. Video recordings were made pre 
and post-surgically to examine the movements. 
Results - Reaching success was not affected following the stroke, but an analysis of the 
ten movement components of the reach indicated that most of the rotatory movements of 
the limb used for aligning, aiming, and supinating the paw were abnormal. The histology 
showed no evidence of infarct or swelling although irregular cellular morphology directly 
below the endothelin application site was evident. 
Conclusions - The results indicate that certain aspects of skilled reaching in the mouse are 
impaired by a temporary focal motor cortex stroke, in contrast to the large degree of 
impairment that is seen following a permanent focal motor cortex stroke. 
59 
Introduction 
The popularity and availability of transgenic mice for stroke research (Hattori, et 
al, 2001; Baird, et al, 2001; Hunter, et al, 2000; Sampei, et al, 2000; Rogers, et al, 
1997) has created a demand for behavioural assessment in mice. A detailed analysis of 
laboratory mice (Mus musculus) indicates that they perform skilled reaching in a manner 
similar to laboratory rats (Rattus norvegicus) (Farr and Whishaw, 2002; Iwaniuk and 
Whishaw, 2000; Whishaw, 1996; Whishaw and Coles, 1996; Whishaw, et al, 1992; 
Whishaw and Pellis, 1990). Mice also experience the same deficits following a 
permanent focal motor cortex stroke (Farr and Whishaw, 2002). 
There are many methods of producing a stroke, each resulting in complex cellular 
and behavioural changes. Therefore, it is attractive to hypothesize that a different model 
of stroke can result in different behavioural impairments in the mouse. Endothelin-1 is a 
potent vasoconstrictor peptide (Yanagisawa, et al, 1988) that is synthesized in 
endothelial cells (Russell and Davenport, 1999). It can be used to create a temporary 
reduction of blood flow in rats both globally via intracarotid injection (Willette, et al, 
1990), and focally via intracerebral injection (Sharkey, et al, 1993; Macrae, et al, 1993; 
Sharkey, et al, 1992). MRI imaging confirmed a focal injection of endothelin adjacent to 
the MCA can reduce striatal and cortical blood flow by 30-50%, which persists for 7-16 
hours respectively (Biernaskie, et al, 2001). Other studies with a slightly larger dose of 
endothelin result in a 70-96% reduction in cerebral blood flow (CBF) with effects lasting 
up to four hours (Macrae, et al, 1993). 
There are three types of endothelin: ET-1, ET-2 and ET-3. Endothelin-1 is found 
primarily in vascular endothelial cells while 2 and 3 are located primarily in the kidneys, 
lungs and adrenal glands (Sakurai, et al, 1992). There are two identified types of 
endothelin receptors, ETA and ETB (for a review see Sakurai, et al., 1992). The receptors 
have different affinities for the endothelins. ETB, located primarily in endothelial cells, 
binds the three endothelin subtypes equally. ETB induces a vasodilatory response via the 
60 
release of endothelium derived relaxing factor (EDRF) from endothelial cells. ETA binds 
ET-1 and 2 preferentially to ET-3 and results in vasoconstriction. The ETA receptors are 
located primarily on smooth muscles, as well as vascular tissue, neurons and glia 
(MacCumber, et al, 1990). Both receptors are coupled via a G protein to phospholipase 
C (PLC) (Kawai, et al, 1997; Stanimirovic, et al, 1994). Endothelin binding to the 
receptor releases the G protein, which in turn activates the membrane bound PLC. 
Activated PLC hydrolyses the major membrane component phosphatidylinositol 4,5 
bisphosphate (P1P2) into diacylglycerol (DAG) and inositol triphosphate (IP,) (for a 
review see Berridge, 1996). DAG will bind to protein kinase C (PKC). PKC 
phosphorylates many proteins, some of which are responsible for the vasoconstriction 
(He, et al, 2000). In addition to inducing a vasoconstrictive response ET-1 has been 
shown to increase intracellular Ca (Koizumi, et al, 1994) via the L Type Ca~ channel 
that is activated by PKC phosphorylation (He, et al, 2000). This elevated calcium may be 
linked to enhanced glutamate release that contributes to neurotoxicity (Shihara, et al, 
1998; Koizumi, et al, 1994). Regardless, endothelin has the advantage of producing a 
temporary reduction in blood flow that results in reperfusion. 
Motor impairments have been documented in mice subjected to a permanent focal 
motor cortex stroke produced by pial stripping. The present study investigated the effects 
of a temporary, perfusion-reperfusion focal motor cortex stroke, produced by topical 
application of ET-1, on skilled reaching in mice. The mice were trained to perform 
skilled reaching through a slot for food and their performance was video recorded before 
and after the focal motor cortex stroke. 
Materials and Methods 
Subjects 
Sixteen four-month old male C57/BL6 mice (Charles River, Montreal. Canada) 
weighing between 20-30g were housed individually. The 12:12 hour light cycle began at 
61 
08:00 and room temperature was constant at 22?C. Testing was conducted at 1:00pm and 
the animals were given one piece of Lab Chow (4g) after the testing period each day. The 
experiment was conducted according to the Canadian Council on Animal Care code. 
Surgery 
After habituation the mice were given ten days of baseline training. On day eleven 
they were anaesthetized with isoflurane (BIMEDA-MTC Animal Health Inc.). 
Vetropolycin (Janssen) ointment was applied to the eyes to prevent drying. The mice 
received a stroke to the motor cortex contralateral to the preferred forelimb. Using 
stereotactic surgery the following area of skull was removed. From 0.5mm anterior to 
bregma to 2.5mm posterior to bregma, and from 0.5mm lateral to the midline to 2.5mm 
lateral to the midline. The dura was removed within the trephination and 5uL of 
40pmol/uL Endothelin-1 (Calbiochem) was applied to the surface of the brain. Five 
minutes later any remaining endothelin was wicked away and the incision was closed. 
The mice were injected with a 0.2mg/kg dose of Metacam (Boehringer Ingelheim) to 
control post operative pain and inflammation. The animals were returned to their home 
cages and ten days of postsurgical testing began the following day. 
Reaching Task 
A Plexiglas reaching box (Farr and Whishaw, 2002) was modified to be 20cm 
long. 9cm wide, and 20cm high (Figure 3.11). 
62 
V' .tv ' % -'fit' , 
Figure. 3.11. The modified mouse reaching apparatus. The shelf in front of the box 
contains two indentations for food. 
63 
A 0.2cm thick plastic shelf (8cm long and 4cm wide) was mounted on the front of the 
box 1.5cm from the floor. Dehulled millet seeds, obtainable at any grocery store, were 
placed in indentations (depth < 1mm) spaced 1cm away from the slit and centered on its 
edges. A 1cm wide vertical slit ran up the front of the box to allow access to the food. 
Mice were habituated for one week by placing them in the cages for ten minutes each 
day. Seeds were initially available within tongue distance, and in both indentations. They 
were gradually moved away until the mice were forced to use their paws. The mice 
displayed the paw they preferred to use after which the food was placed in the indentation 
contralateral to the preferred paw. 
Videorecording 
Filming occurred on the last day of baseline training as well as two weeks post 
surgery for each animal. The camera was a Sony DSRPD100 digital camcorder (30 
frames/sec; shutter speed of 1000). Illumination was provided with a 2 arm Nikon MKII 
150W fiber optic light and a cold source Caselite (Lowel). The tapes were analyzed on a 
Sony DV cam DSR-20 player and Trinitron monitor. 
Reaching Success 
Only successful reaches were counted from a daily total of twenty seeds. If the 
mouse knocked the food away, dropped it, or required more than one attempt to obtain 
the food the reach was scored as a miss. Performance was defined by the formula: 
Percent Success = (number of successful retrievals/ 20)* 100 
Qualitative Analysis 
The ten movement components were evaluated using a variation of the Eshkol-
Wachmann movement notation, as described previously. Each movement was rated on a 
3-point scale. If the movement was normal, a score of 0 was given. In cases where there 
was some ambiguity concerning the occurrence of a movement, or the movement was 
64 
present but incomplete, a score of 1 was given. If the movement was absent, a score of 2 
was given. 
Histology 
The mice were deeply anaesthetized and perfused through the heart with 
phosphate buffered 0.9% saline followed by 4% para-formaldehyde. Brains were cryo-
protected in 30% sucrose/4% para-formaldehyde for three days and cut into 40 um 
sections on a 2800 Frigocut E cryostat (Reichert-Jung). The sections were mounted on 
1% gelatin and 0.2% chromalin dipped slides, stained with Cresyl violet, and cover 
slipped using Permount (Fisher-Scientific). 
Results 
Histology 
Figure 3.12 depicts representative photographs of stroke insults. 
65 
Figure 3.12. (A-D) Dorsal views of typical motor cortex strokes. (A-B) An example of 
the smallest stroke, the lesion site is clearly indicated in between the arrows in B. (C-D) 
An example of the largest stroke, the lesion site is indicated in D. 
66 
The visible damage on the surface of the cortex produced very small infarcts that were 
not easily visible in the sections. Figures 3.13 A and B depict representative sections 
through the lesion site. C and E depict cells within the intact hemisphere while D and F 
illustrate cells in the stroke hemisphere. 
67 
i.lO-x.t.;. 
Figure 3.13. (A-B) Coronal photomicrographs of a representation of a motor cortex 
stroke through the lesion site (Cresyl violet). The intact hemisphere contains labeling for 
the appropriate areas: SI-sensorimotor cortex, SFL- sensoriforelimb area, MFL-Motor 
forelimb area and Cgl-cingulate cortex). The stroke site is indicated within the arrows. 
(C-D) Representations of the cellular morphology of the control (C) and stroke (D) motor 
cortex hemispheres at 100X magnification. (E-F) Representations of the cellular 
morphology of the control (E) and stroke (F) hemispheres at 400X magnification. Note: 
The abnormal cells present in the stroke hemisphere in the area immediately below the 
site of endothelin application. 
68 
Endothelin-1 did not produce a large infarct below the area of application. Instead there is 
a conical area below the lesion site with abnormal cell morphology. There are fewer cells 
and the ones that are present exhibit abnormal structure. The nucleus is not clearly visible 
and the cell staining is much darker, possibly indicating ruptured nuclei (Figure 3.13D,F). 
In addition, the tissue below the endothelin application did not swell. The damage 
appears to be restricted to the motor forelimb area. 
Quantitative changes in reaching following stroke 
Reaching Success 
The percent success ANOVA, with days as a repeated measure, indicated that 
there was no treatment effect (Figure 3.14). 
69 
Baseline Post-surgery 
fiO-i 
0^ 20- ; 
U .
?H ! 
g_|?r_1?j i?i?j?,?i?i ii i?i?i i?i i?i?i r 
Days 
Figure 3.14. Reaching success (means ? SEM). The dotted line represents the 
administration of a motor cortex stroke. 
70 
The lack of statistical difference indicated that the animals' performance was not 
significantly affected by the stroke. 
Movement Components 
The qualitative ANOVA of five presurgical and postsurgical reaches, evaluated 
for the 10 different movement components, revealed a significant main effect of Group, 
F(l,29)= 17.883, P < 0.001. This indicated that the mice accumulated higher scores for 
the ten movement components following the stroke. There was also a main effect of 
Movement, F(9, 261)=46.787, P < 0.001, indicating that some movements were more 
affected than others. Finally, there was a significant Group by Movement interaction for 
the stroke group, F(9, 261)= 8.739, P < 0.001. This indicated that some movements in the 
stroke group were more impaired than the baseline components. Follow-up students 
unpaired t-tests were performed and indicated significant group differences for the digits 
to the midline, aim, and supination II components of the reach, but not for the digits 
semiflexed, advance, digits extend, pronation, grasp, supination I and release (Figure 
3.15). 
71 
Score 
Figure 3.15. Scores for each of the ten movement components of the reach (means ? 
SEM). Clear bars indicate baseline performance and solid bars indicate post surgical 
performance. A score of 0 indicates a normal movement while a score of 2 indicates 
complete absence of the movement. (*** P < 0.001)(** P < 0.01)(* P < 0.05). 
72 
Qualitative changes in movement following stroke 
The following descriptions were all taken from successful reaches. 
Digits to the Midline and Aim 
During baseline testing, mice lifted the semiflexed digits of the reaching paw and 
quickly aligned them with the midline of the body (Figure 3.16A). 
73 
Digits to the Midline and Aim 
Control Stroke 
Figure 3.16. Digits to Midline and Aim. A, B: Baseline. During presurgical testing the 
mouse lifted the semiflexed digits to align them with the bodies midline (arrow in A). 
The limb was then aimed by bringing the elbow to the midline, this is visible by the 
position of the digits (arrow in B) forcing the elbow to be beneath them in the same axis 
as the paw. C, D: Stroke. The same mouse following stroke surgery shifted the shoulders 
to align the digits with the midline (arrows in C) and the elbow is not brought into the 
aim, indicated by the misplaced, diagonal paw and additional shoulder shifting. 
74 
The elbow was then aligned bringing the reaching limb into an "aiming" position (Figure 
3.16B). Following surgery the mice lifted the limb and semiflexed the digits to a lesser 
extent (Figure 3.16C). The animals shifted their shoulders to bring the elbow into an 
aiming position, indicated by the position of the paw and angle of the trunk (Figure 
3.16D). 
Advance and Pronation 
Prior to surgery the mice were able to advance the paw and extend the digits 
directly over the food item (Figure 3.17A) and begin pronation (Figure 3.17B). 
75 
Advance and Pronation 
Figure 3.17. Advance and Pronation. A, B: Baseline. The baseline animals advanced the 
reaching forelimb directly over the food pellet and extend the digits (A) while beginning 
to rotate the paw over the food pellet (arrow in B). C, D: Stroke. After surgery the mice 
were still able to advance and extend the digits (arrow in C), though to a lesser extent, but 
can not fully pronate indicated by the paw (D) coming straight down, rather than rotating 
over. 
76 
Post surgery the mice were still somewhat able to advance and opened the digits (Figure 
3.17C) but slapped down rather than rotating over the seed (Figure 3.17D). 
Grasp and Supination I 
Once the food was contacted, baseline mice closed the digits around the pellet 
(Figure 3.18A) and supinated the closed paw in order to begin withdrawl (Figure 3.18B). 
77 
Grasp and Supination I 
Control Strok 
Figure 3.18. Grasp and Supination I. A, B: Baseline. The presurgical animal can close the 
digits around the food item (A) and supinate the paw during withdrawl (arrow in B). C, 
D: Stroke: The post surgical animal is not able to fully close the digits (C) and the degree 
of supination is less than that of the control, using a dragging motion to obtain the food 
(arrow in D). 
78 
Stroke animals displayed less grasping when compared to the their baseline scores 
(Figure3.18C) and food retrieval was begun by dragging the item backward (Figure 
3.18D). In addition, the stroke animals would continue body rotation in order to rotate 
the limb. 
Supination II and Release 
Once the food was withdrawn, the baseline mice further supinated the paw to 
present the food to the mouth (Figure 3.19A). 
79 
Supination II and Release 
Control Stroke 
Figure 3.19. Supination II and Release. A, B: Baseline. A presurgical animal supinated 
the paw to present the food to the mouth (arrow in A), opened the digits to release it, and 
brought the non-reaching limb up to aid in food manipulation (arrows in B). C, D: Stroke. 
A post surgical animal chased the paw downward, unable to complete the second 
supination (arrow in C) and forced the paw to the ground to release the food (D). 
80 
Supination II and release of the food was frequently accomplished without assistance 
from the non-reaching paw (Figure 3.19B). Following surgery the mice typically 
exhibited difficulty with the second supination and chased the paw down to force release 
with the help of the floor (Figure 3.19 C,D). 
Discussion 
This study documented skilled reaching in mice subjected to a focal motor cortex 
stroke produced by a temporary occlusion of the cortical blood vessels. Surprisingly, the 
stroke did not produce a quantitative deficit in reaching success, though a qualitative 
analysis revealed the mice exhibited difficulty with some of the movements in the reach. 
The findings are particularly important especially when considering the method 
employed to produce the insult. This is the first study to describe a subtle lesion that has 
no effect on the quantitative performance but effects the qualitative performance in mice. 
The mice were trained to perform skilled movements by reaching through a slot 
to obtain single seeds rewards. Following the focal motor cortex stroke the mice were 
still able to retrieve a comparable number of seeds. This finding contrasts what is 
normally seen in rats and mice subjected to a permanent focal motor cortex stroke. 
Animals with such a lesion suffer a large quantitative impairment that improves with 
training but does not completely return to baseline level (Whishaw, 1996; Whishaw, 
2000; Whishaw, et al, 1992; Castro, 1972; McKenna, et al, 1999; Modo, et al, 2000; 
Petullo. et al, 1999; Whishaw, et al, 1992; Whishaw, et al, 1991). 
The stroke insult caused the mice to exhibit abnormalities in the different 
components used during a reach. Specifically, the mice had difficulty aligning the digit 
tips to the body midline and subsequently with bringing the elbow into midline position. 
The mice were able to partially achieve the aim by shifting the shoulders and rotating the 
body. Due to the misplaced aim the paw advanced diagonally through the slot and often 
minor adjustments were required to get the paw over the food. The animals also had 
difficulty with the second supination that requires another rotation of the paw to present 
81 
the food to the mouth. Rotatory movements of the trunk assisted food retrieval. The 
stroke mice also exhibited subtle impairments in the ability to extend the digits and grasp. 
These findings are consistent with the impairments described for the rat following a focal 
motor cortex injury (Whishaw, 1996; Whishaw, 2000; Whishaw, et al, 1992; Castro, 
1972; McKenna, et al, 1999; Whishaw, et al, 1992; Whishaw, et al, 1991). 
These findings suggest that the small damage produced by the endothelin 
application may only produce subtle impairments with rotatory movements of the limb. 
This impairment may contribute only to deficits in qualitative assessments; quantitatively 
the mice are able to compensate quickly, it is possible that a stronger concentration of 
endothelin would result in increased damage that may mimic what is commonly seen in 
permanent focal stroke models. 
In conclusion, this is the first study to document the impairments in skilled 
reaching in mice subjected to a temporary perfusion-reperfusion stroke. The main 
findings are that mice exhibit qualitative impairments but not quantitative impairments 
following the endothelin produced lesion. The method of producing the stroke may be 
influential on the types of impairments exhibited. 
82 
4. Experiment 3: 
The effects of a permanent and temporary occlusion stroke on skilled 
reaching in the mouse. 
Abstract 
Background and Purpose - Stroke can be modeled in rodents using a variety of methods 
and each is capable of producing different histological damage. The previous experiments 
revealed that permanent removal of the cortical vasculature in mice results in massive 
quantitative and qualitative impairment whereas a temporary constriction of the same 
vessels results in only a subtle qualitative impairment. Thus, the purpose of this study 
was to compare the effects of the two models of stroke on skilled movements and 
histological outcome in the mouse. 
Methods - Male C57/BL6 mice were trained to reach for seeds with their preferred limb. 
They were subdivided into three groups. One group remained a control. The other two 
groups received a focal motor cortex stroke in the hemisphere contralateral to their 
preferred limb, either via a pial strip or the topical application of Endothelin-1. Reaching 
success and the movements used in reaching were analyzed by pre and post-surgical 
video records. 
Results - Reaching success indicated that the pial strip animals were significantly affected 
by the stroke, whereas the endothelin animals were not. An analysis of the ten movement 
components of the reach indicated that both groups were impaired with many of the same 
rotatory movements but the pial strip animals experienced a greater degree of 
impairment. A histological comparison indicated that the pial strip strokes were 
significantly larger than the endothelin strokes and the degree of impairment for certain 
movements an animal exhibited was positively correlated with the size of the lesion. 
83 
Conclusions - The results indicate that certain aspects of skilled reaching in the mouse are 
impaired following focal motor cortex, stroke and that larger permanent occlusion lesions 
produce a greater degree of impairment. This may have implications for the choice of a 
stroke model. 
Introduction 
There are many methods of modeling stroke in rodents and the resulting 
pathology from each can be very different. Most focal motor cortex models result in 
striatal and cortical damage and animals exhibit a behavioural deficit in the contralateral 
limb. The previous experiments have indicated that variations in stroke methodology 
produce different histological and behavioural outcomes. The purpose of this study is to 
compare the permanent pial strip to the temporary endothelin application. 
The first experiment disrupted skilled reaching movements in the mice by using a 
permanent occlusion model of focal motor cortex stroke. The vasculature over the motor 
cortex was removed, eliminating the possibility of tissue reperfusion. Histological 
analysis revealed that the tissue surrounding the motor cortex swelled up into the area of 
the infarct. Behavioural analysis revealed that the mice were impaired quantitatively in 
the ability to obtain the food and qualitatively, meaning the movements used to obtain the 
food were altered. This is consistent with what is seen in rats (Whishaw, 1996; Whishaw, 
2000; Whishaw, et al, 1992; Castro, 1972; McKenna, et al, 1999; Whishaw, et al, 1992; 
Whishaw, et al, 1991). The second experiment made use of a temporary model of focal 
motor cortex stroke. A potent vasoconstrictor, ET-1 (Yanagisawa, et al, 1988), was 
applied to the surface of the motor cortex to reduce blood flow for a period of time 
(Biernaskie, et al, 2001; Sharkey, et al., 1993; Macrae, et al, 1993; Sharkey, et al, 1992; 
Willette. et al, 1990). Histological analysis of the ET-1 tissue revealed that there was a 
conical area of abnormal cellular morphology directly beneath the application site, 
though there was no visible infarct. Behavioural analysis indicated that the mice were not 
impaired quantitatively, though they did exhibit abnormal movements in order to retrieve 
the food. 
84 
It is possible that the extent of tissue damage is directly correlated with the degree 
of impairment. The purpose of this experiment was to increase the dose of the 
Endothelin-1 application and compare it's effects to that of the permanent pial strip. 
Materials and Methods 
Subjects 
Nine four-month old male C57/BL6 mice (Charles River, Montreal, Canada) 
weighing between 20-30g were housed individually in a 22?C room where the lights were 
on a 12:12 hour cycle beginning at 08:00. Testing was conducted at 9:00am and the 
animals were given one piece of Lab Chow (4g) after the testing period each day. The 
experiment was conducted according to the Canadian Council on Animal Care code. 
Surgery 
The mice were habituated to the testers and testing apparatus for three days and 
tested for ten baseline days prior to surgery. Six mice were anaesthetized with isoflurane 
(BIMEDA-MTC Animal Health Inc.), and Vetropolycin gel (Janssen) was applied to the 
eyes to prevent drying. The skull over the motor cortex was removed, from 0.5mm 
anterior to bregma to 2.5mm posterior to bregma, and from 0.5mm lateral to the midline 
to 2.5mm lateral to the midline. The dura was removed within the trephination. Three 
mice, all with right paw dexterity, received a pial strip to the motor cortex contralateral to 
the preferred forelimb, essentially the pial vasculature was wiped away with a sterilized 
cotton swab. The other three mice, all left paw preference, received a 5 minute topical 
endothelin application of 5uL of SOpmol/uL Endothelin-1 (Calbiochem), the pial 
vasculature was left intact. After the incision was closed the mice received a 0.2mg/kg 
dose of Metacam (Boehringer Ingelheim) to aid with post operative pain and 
inflammation. The post surgical testing began the following day. 
85 
Reaching Task 
The same Plexiglas reaching box described in the previous experiment was 
utilized. Briefly, the mice reached through a 1cm vertical slit to obtain millet seeds that 
were placed in divots 1cm away from the front of the box. Animals were habituated for 
three days as seeds were gradually moved further away until the mice indicated their 
preferred paw. 
Videorecording 
Filming occurred on the tenth day of baseline training as well as the tenth day of 
post surgical training. The filming set up from the previous experiment was duplicated. 
Briefly, the camera was a Sony DSRPD100 digital (30 frames/sec; shutter speed of 
1000), the lights were a 2 arm Nikon MKII150W fiber optic, and a cold source Caselite 
(Lowel). The tapes were analyzed on a Sony DV cam DSR-20 player and Trinitron 
monitor. 
Reaching Success 
Daily scores for percent success were calculated from twenty seeds with the 
following formula: 
Percent Success = (number of successful retrievals/ 20)* 100 
Qualitative Analysis 
The ten movement components were evaluated using a variation of the Eshkol-
Wachmann movement notation, as described previously. Briefly, a score of 0 indicated 
normal whereas a score of 2 indicated complete movement absence. 
Histology 
86 
The mice were deeply anaesthetized and perfused through the heart with 
phosphate buffered 0.9% saline followed by 4% para-formaldehyde. Brains were 
photographed with a Kodak DCS 410 digital camera, cryo-protected in 30% sucrose/4% 
para-formaldehyde solution and sectioned (40[im) on a 2800 Frigocut E cryostat 
(Reichert-Jung). The sections were mounted on 1% gelatin and 0.2% chromalin dipped 
slides, stained with Cresyl violet, and cover slipped using Permount (Fisher-Scientific). 
Four sections from each animal were digitized with a Polaroid DMC-3 live feed digital 
camera and analyzed using NIH image software (Bethesda, MD., U.S.A.). Lesion 
volumes were estimated by measuring the stroke area in each of the four sections, and 
multiplying it by the area between each of the sections. 
Results 
Histology 
All of the brains had damage in the motor cortex (Figure 4.11). The first two 
panels (A-F) represent a pial strip stroke and the second two panels (G-L) represent an 
endothelin induced stroke. 
87 
Figure 4.11. Pial strip. (A-B) Photomicrographs of a representative pial strip stroke, the 
infarct is indicated between the arrows. (C-D) Representative coronal sections through 
the stroke site (Cresyl violet). The intact hemisphere contains labeling for the appropriate 
areas: SI-sensorimotor cortex, SFL- sensoriforelimb area, MFL-Motor forelimb area. (E-
F) Representations of the cellular morphology in the motor cortex of the control (E) and 
stroke (F) hemispheres at 400X magnification. Endothelin. (G-FJ) Photomicrographs of a 
representative endothelin stroke. (I-J) Representative coronal sections through the stroke 
site (Cresyl violet). (K-L) Representations of the cellular morphology in the motor cortex 
of the control (K) and stroke (L) hemispheres at 400X magnification. Note: the swelling 
of the stroke site in the pial strip sections (C-D) and the presence of abnormal cells in the 
stroke hemispheres of both stroke models (F,L). 
88 
The pial strip stroke produced a noticeable infarct (Figure 4.11A-B) and tissue swelling 
around the infarct site (Figure 4.11 C-D). Detailed examination of the motor cortex cells 
indicated that the tissue in the intact hemisphere is normal, whereas the cells in the stroke 
hemisphere exhibit abnormal morphology. Abnormal cellular morphology includes cells 
with abnormal shape, and darker colors, as well as a reduction in the number of cells in 
general. Endothelin-1 did not produce a large infarct below the area of application. When 
superficially studying the sections it is often difficult to determine which hemisphere 
suffered the stroke (Figure 4.11I-J). Cellular examination reveals that there is a conical 
area below the endothelin application site with abnormal cell morphology (Figure 4.11L). 
Lesion volumes for each group are indicated in Table 4.11. 
89 
Table 4.11. The stroke volumes for each mouse in millimeters cubed. Average? SEM is 
indicated for each group. 
Group Stroke Volume (mm3) 
Pial Strip 
Mouse 1 10.03 
Mouse 2 10.73 
Mouse 3 12.36 
Average 11.04+ 0.69 
Endothelin 
Mouse 4 5.42 
Mouse 6 6.02 
Mouse 9 7.53 
Average 6.32? 0.63 
90 
The pial strip animals' average lesion volume was almost double the size of the 
endothelin animals. An unpaired students t-test revealed a t statistic of 5.056 and a 
corresponding probability of 0.0072, indicating that this difference in size is not simply 
due to chance. 
Quantitative changes in reaching following stroke 
Reaching Success 
A repeated measures ANOVA for the last five pre-surgical days and the first five 
postsurgical days indicated that there was a significant effect of treatment (F(l,6)= 
11.172, P < 0.05) and a significant group effect (F(2,6)= 6.137, P < 0.05) (Figure 4.12). 
91 
Baseline Po st-surge rj 
m 60-
U 50-
1357913579 
Days 
Figure 4.12. Reaching success (means ? SEM) of the three groups: Control, ET-1 and 
Pial Strip. The dotted line represents the administration of a focal motor cortex stroke. (* 
P < 0.05). 
92 
The first five post surgical days of the mice in the pial strip group were outside of the 
confidence interval when pairwise compared to the final five days of the control baseline 
performance. This indicates that the pial strip group's performance was statistically 
different from that of the controls during this time period. The pial strip mice had begun 
recovery by the eighth post surgical day and there is no statistical difference from the 
control group at this time. There was no statistical difference in performance between the 
endothelin and control groups, though the endothelin animals scored slightly lower than 
the controls following the stroke. 
Movement Components 
Five pre and post surgical reaches from each animal were used to provide one 
score for each of the ten movement components. There were no significant differences 
between any of the groups prior to surgery. The post surgical scores for the stroke treated 
groups and average for the control group are plotted in Figure 4.13. 
93 
Score 
Figure 4.13. Scores for each of the ten movement components of the reach (means ? 
SEM) for the two stroke groups, Pial Strip and Endothelin-1. A score of 0 indicates a 
normal movement while a score of 2 indicates complete absence of the movement. The 
dotted lines represent an average score for all of the ten movement components in the 
control group. (*** P < 0.001 )(** P < 0.01)(* P < 0.05). 
94 
A repeated measures ANOVA evaluated for the 10 different movement components post 
surgery, revealed a significant main effect of Group, F(2,9)= 20.295, P < 0.001. This 
indicates that the stroke mice accumulated significantly higher scores than the control 
group. There was also an effect of Movement, F(9, 18)=24.126, P < 0.001, indicating that 
some movements were more affected than others. Finally, there was a significant Group 
by Movement interaction F(18, 81)= 4.388, P < 0.001, indicating that there were 
differences between groups for certain movements. Follow-up students unpaired t-tests 
indicated that the pial strip group was significantly impaired when compared to the 
controls for digits to the midline, aim, advance, supination II, and release components of 
the reach. The Endothelin mice were only impaired at digits to the midline, aim and 
supination II. 
Qualitative changes in movement following stroke 
Both stroke groups exhibited difficulties aligning the digit tips and elbow with the 
bodies midline, as well as the second supination. In addition, the pial strip mice 
experienced difficulties with the advance and release. Because the pial strip animals 
exhibited a larger lesion volume than the endothelin group (Table 4.11) it is attractive to 
hypothesize that a larger degree of damage will impair a larger variety of movements. A 
correlation analysis between lesion volume and degree of impairment was performed for 
each of the ten movement components. A positive correlation was found for some of the 
movement components, meaning the larger the lesion volume the greater the impairment. 
Table 4.12 reports the corresponding r statistic and probability value for the 10 different 
movement components. 
95 
Table 4.12. The r correlation statistic value and corresponding probability for the Fisher's 
r to z test for the ten different movement components of the reach versus lesion volume. 
Movement Components r value P value 
Digits to the Midline 0.758 0.085 
Digits Semiflexed 0.9 0.011 
Aim 0.625 0.204 
Advance 0.654 0.175 
Digits Extend 0.620 0.210 
Pronation 0.379 0.490 
Grasp 0.329 0.554 
Supination I 0.424 0.432 
Supination II 0.959 0.0008 
Release 0.978 0.0001 
96 
An r statistic of greater than 0.5 but less than 0.8 is indicative of a moderate positive 
correlation while a value greater than 0.8 indicates a strong correlation. Thus, digits to the 
midline, aim, advance, and digits extend exhibit a moderate correlation with lesion 
volume. Digits semiflexed, supination II and release exhibit a strong correlation with 
lesion volume. The corresponding probabilities indicate that only digits semiflexed, 
supination II, and release were statistically correlated. Stroke animals with a larger lesion 
did receive larger scores on most of the ten components. The two mice with the largest 
lesion volume also exhibited impairments with digits semiflexed, advance and digits 
extend. 
Discussion 
This study compared the effects of two different models of focal motor cortex 
stroke. One was a permanent removal of the pial vasculature (pial strip) and the other a 
temporary constriction of the same vessels (Endothelin-1 application). The results 
indicate that the pial strip has drastic affects on the ability of the mice to obtain food, as 
well as impairing the way in which they obtain the food during skilled reaching. The 
endothelin model of stroke did not significantly impair the mice quantitatively, though 
the animals did exhibit some abnormal movements in the qualitative analysis. Both types 
of stroke produce impairments in the same rotatory types of movements such as: digits to 
the midline, aiming, and supinating the paw. This indicates that a stroke to the motor 
cortex always results in certain movement abnormalities. Because mice with a larger 
lesion volume, such as the pial strip group, exhibited a larger degree of impairment, it is 
attractive to hypothesize that the differences in impairments are a result of the extent of 
the injury. The findings are particularly relevant when considering the method employed 
to produce the insult. 
There are three main differences in the stroke models that could account for the 
differences in behavioural outcome. The first is that the pial strip permanently removes 
97 
the pia vasculature, irreversibly impairing blood flow in the motor cortex. Endothelin-1 is 
a vasoconstrictor that will eventually result in reperfusion of the intact motor cortex 
vessels. The second is that the removal of the pia disrupts the meninges of the brain 
during the pial strip. This membrane is left intact during a topical endothelin application. 
The third is that the histology indicated the pial strip caused the tissue surrounding the 
stroke to swell and fill in the infarct, whereas there was little displacement of the tissue in 
the hemisphere of the stroke after the endothelin insult. It is likely these differences 
contribute to the complexities observed in the behaviour. 
The mice in both groups exhibited abnormalities in some of the same components 
used during a reach. Following the stroke all animals had difficulty with the initial 
alignment of the digits and the aim, they used body rotation to achieve these goals. All 
animals were also unable to perform the second supination often forcing the limb down to 
the ground to present the food to the mouth. All of these findings are consistent with the 
impairments from a focal motor cortex stroke described in the rat (Whishaw, 1996; 
Whishaw, 2000; Whishaw, etal, 1992; Castro, 1972; McKenna, etal, 1999; Whishaw, 
et al., 1992; Whishaw, et al., 1991). This suggests that the motor cortex has substantial 
contribution to the rotatory movements involved in a reach. In addition, the pial strip 
group exhibited lesion volumes that were significantly larger than that of the endothelin 
group. In addition, the pial strip animals experienced difficulties with other movements 
such as the advance and release. In extreme cases the mice with the largest pial strip 
lesion also exhibited subtle impairments in the ability to flex and extend the digits. This 
could mean that larger injuries can result in a greater degree of impairment. This is 
supported by the fact that there were moderate to strong positive correlations between 
lesion volume and degree of impairment for some of the ten movement components such 
as: digits to the midline, digits semiflexed, aim, advance, digits extend, supination II and 
release. 
Interestingly, the larger dose of endothelin used in this experiment did produce a 
slightly lower quantitative percent success score in the stroke animals when compared to 
the controls, though it was not statistically different. The fact that endothelin has the 
98 
ability to make subtle lesions that affect only certain types of movements and do not 
affect reaching performance makes it an attractive model because it can be titrated to 
produce strokes of various sizes. It may be possible, to provide an application of 
endothelin strong enough to mimic what is seen in the pial strip animals. 
In conclusion, this is the first study to document the impairments in skilled 
reaching in mice subjected to a permanent and temporary focal motor cortex stroke. The 
main finding is that the permanent model results in a larger degree of behavioural 
impairment and histological damage than the temporary model. 
99 
5. Discussion 
5.1. Outline of the discussion. 
This thesis described skilled motor movements in mice and the alterations in these 
movements following both a permanent removal, and a temporary occlusion of the blood 
vessels in the motor cortex. The conclusions were that mice perform skilled motor 
movements and that these movements are impaired following a stroke to the motor 
cortex. Different stroke models can result in different types of behavioural impairments, 
and the degree of impairment is correlated with the size of the insult. 
This thesis encourages the use of mice as an animal model for stroke research 
because of the current availability of transgenic mouse technology. Transgenic mouse 
technology can help evaluate the contribution of genetics to brain injury, specifically 
stroke. The discussion is therefore not intended to provide a summary of the results of the 
experiments but rather offer a rationale for, as well as a review of, the use of mice as an 
animal model for stroke research. The discussion is composed of five sections. The first 
section reviews the definition and description of a focal stroke. It will emphasize the most 
widely used method of modeling focal stroke in rodents, the MCAO. The second section 
will describe the current methods of modeling MCAO stroke in mice and some 
considerations that should be accounted for when choosing a mouse model. The third 
section describes the creation of transgenic mice, which will lead into a discussion of the 
contributions of transgenic technology to stroke research in the fourth section. The final 
section will summarize the thesis. 
5.2. A review of stroke. 
Stroke is a cardiovascular disease that occurs when there is an obstruction in the 
blood flow of the brain. Most strokes occur when a blood clot or atherosclerotic plaque 
forms in the brain, or breaks off from a buildup in the body, and travels through the blood 
100 
stream to lodge itself in a brain vessel. Once the supply of oxygenated blood is reduced 
the tissue that is irrigated by the blocked vessel often dies or suffers injury that can lead 
to death. The most commonly occluded vessel in human stroke patients is the MCA, 
which services a large portion of the forebrain. 
An MCA stroke can result in a variety of clinical disabilities for a patient (for a 
review see Slater and Johns, 2003). The loss of motor function is one of the most 
frustrating impairments a stroke patient can suffer. The sudden inability to use the upper 
and lower contralateral limbs can mean that the patient will be unable to dress, eat, or 
even move. Skilled motor movements such as writing or picking up small objects can 
also be disrupted. An MCAO in the mouse mimics the clinical situation by causing motor 
impairments. 
5.3. Mice as a model for stroke. 
There are not many models of MCAO in the mouse. The most common method 
involves the insertion of a thin nylon coated filament into the internal carotid until it 
reaches the origin of the MCA, there-bye preventing blood flow through the MCA. This 
technique results in a 50% reduction in cortical blood flow (Clark, et al., 1997). The 
thread can be left in place to create a permanent occlusion, which often results in an 
average infarct volume of 80mm3(Clark, et al., 1997). The MCA can also be temporarily 
occluded. Removal of the thread produces a smaller average infarct size of 55mm (Clark, 
et al., 1997). The murine model of MCA focal ischemia in the mouse is very similar. In 
addition to the filament insertion, the external carotid artery is cauterized (Sander 
Connolly, et al, 1996). This technique is able to produce extremely reproducible infarct 
volumes that are approximately 30% of the ipsilateral hemispheric volume (Sander 
Connolly, et al., 1996). Both MCAO techniques offer the advantage of being 
reproducible and they do not require a craniotomy to access the small MCA of the mouse. 
Despite the advantages, the main drawback of the thread insertion models is that there is 
a large degree of damage to many subthalamic brain structures as well as the cortex and 
striatum. Functional motor assessment of these animals would be difficult because of the 
101 
variety of impairments produced by the MCAO. A focal motor cortex stroke model, such 
as the pial strip or endothelin application, affects only the sensorimotor cortices, which 
makes it possible to study the contribution of the motor cortex to skilled movements. 
There is another factor that needs to be considered when choosing the appropriate 
mouse model. Because genetic background can influence stroke outcome, the choice of 
mouse strain should be evaluated carefully. The C57/BL6 mouse is one of the most 
characterized and widely used strains for stroke research; they are often used as 
background strains for the development of transgenic lines. Recent evidence indicates 
that the C57/BL6 strain is more susceptible to ischemic injury than other commonly used 
strains of mice (Fujii, et al., 1997). Detailed studies of vascular anatomy indicated that 
C57/BL6 mice have an incomplete Circle of Willis. The Circle of Willis is a circular 
structure attached to the base of the brain that encircles the brainstem. Both basilar and 
carotid arteries terminate in the circle, thus the loop gives off the anterior, middle and 
posterior cerebral arteries. Since blood flow can go in either direction, the Circle of Willis 
can provide collateral blood flow when one of the protruding arteries becomes occluded. 
Other sources of collateral blood flow in the brain are the pial anastomoses, which 
are tiny vessels that interconnect the major cerebral arteries. The anastomoses that 
connect the MCA to the anterior cerebral artery in C57/BL6 mice are located close to the 
midline. This means that the distal branches of the MCA must stretch further to reach the 
anastomoses, thus the MCA in C57/BL6 animals supplies a larger area than it would in 
other strains (Maeda, et al, 1998). Despite the variables in vascular anatomy that must be 
taken into consideration, mice are still a good model because transgenic technology-
allows the examination of genetic contributions to stroke. 
5.4. An introduction to transgenics. 
A literature search of the keywords "transgenic mice" in Pub Med results in over 
20 000 matches, most of which have been published in the last 20 years. Transgenics 
began in the early 1970's when it was found that injection if viral deoxyribonucleic acid 
102 
(DNA) into a mouse embryo produced an adult animal that exhibited viral DNA 
expression in many types of somatic tissue (Jaenisch and Mintz, 1974). Less than a 
decade later microinjection of foreign DNA into the pronucleus of a single celled embryo 
resulted in germline expression (Gordon, et al., 1980). Germline transmission refers to 
incorporation of the foreign DNA into reproductive cells so that it can be passed on to 
progeny. The first transgenic mouse with confirmed overexpression of a rat transgene 
encoding a growth protein was unveiled a short time later (Palmiter, et al., 1982). 
Pronucleus injection is used to obtain widespread transgene expression, however, the 
disadvantage is that it is hard to determine the incorporation site of the transgene in the 
genome of the generated animals. It is possible to target specific DNA regions of the 
genome with constructs that recognize specific DNA sequences. The constructs can be 
injected into embryonic stem cells (ES). ES are derived from blastocysts and have the 
ability to become any cell type, and they can be returned to the embryo following DNA 
injection. Both the adult mouse and any progeny must be screened for homozygous 
expression of the transgene. Homozygotes can then be used to produce a true breeding 
line. Transgene insertion is often accompanied by the insertion of promoters to drive 
transcription in specific tissue types, or reporters to imply transcription of other DNA 
sequences. The addition or removal of a gene in a mouse embryo is thought to produce an 
adult animal that displays the phenotype produced by that gene, making it possible to 
assess the contributions of various genes. For a review of these techniques see Jaenisch, 
1988 
Transgenic technology has had immense impact on the study of genetic 
contributions toward human disease (for a review see Wagner, et al., 1995 and Koretsky 
1992) particularly with regards to stroke research. 
5.5. The contribution of transgenics to stroke. 
Stroke research has benefited from transgenic insights into molecular pathology 
(for a review see Ahmed, et al., 2000). The contribution of various genes and their 
products towards cardiovascular hypertension is reviewed in Bader, et al, 2000. This 
103 
section will cover only a few important examples from the available literature. For 
example, high density lipoproteins (HDL) bind to cholesterol to transport it to the liver 
for metabolization. Lecithin-cholesterol acyltransferase is an enzyme that induces HDL 
and cholesterol binding by causing the two compounds to form an ester. Apolipoprotein 
A 1 (Apo A-l) is one protein that activates lecithin-cholesterol acyltransferase. Apo A-l 
knockout mice are no longer able to produce Apo A-l because they lack the gene that 
codes for it. As a result, the animals exhibit reduced cholesterol HDL complex levels in 
the blood plasma; cholesterol is more available. The knockouts have more atherosclerotic 
plaques and are hypertensive. Understanding the genetic contributions to hypertension 
can lead to a therapy that may help reduce the risk of stroke. 
A large number of transgenic models are dedicated to reducing the number of 
reactive oxygen species present in stroke injured tissue (for a review see Chan, et al., 
1995). For example, nitrous oxide synthase (NOS) is an enzyme that produces the free 
radical NO, and NOS is activated during an ischemic insult. Knocking out the gene that 
codes for NOS resulted in reduced NO production in the mice and they exhibited a 
reduction in infarct size when compared to the wild type reference strain (Huang, et al., 
1993). Sampei, et al., 2000 reported that mice overexpressing a copper superoxide 
dismutase (CuSOD) (SOD's are free radical scavengers or antioxidants) exhibited a 
reduced infarct volume that was up to 30% smaller in the transgenics when compared to 
the wild type (for a review see Chan, et al., 1995). This can have important clinical 
implications because antioxidants can easily be administered orally or via injection. 
Neurite growth factors are often produced in response to a stroke and many 
studies have looked at growth factor application as a potential treatment for ischemic 
injury. Guegan, et al, 1998 used a nerve growth factor (NGF) transgene to test the effects 
of endogenous NGF synthesis on infarct volume. They inserted the NGF transgene 
downstream of the c-fos promoter, which activates many early response genes 
immediately following an ischemic insult. The resulting adult animals were subjected to a 
focal ischemic insult via electrocoagulation of the MCA. The results indicated that both 
the promoter and the transgene exhibited peak expression 6 hours post ischemia and the 
104 
transgenic mice experienced a 40% reduction in infarct volume when compared to the 
wild type. 
Other research looked into the minimization of cell death post stroke. The poly 
ADP-ribose polymerase gene (PARP) is a nuclear enzyme that transfers adenosine 
diphosphate (ADP) to a variety of ribose groups by using the substrate nicotinamide 
adenine dinucleotide (NAD). NAD is an important energy source for the cell. During 
times of stress PARP is overactivated and NAD is rapidly depleted. PARP is also thought 
to be involved in the production of nitric oxide synthase. Transgenic mice, in which the 
PARP gene is disrupted and can no longer produce a viable protein, experience an 80% 
reduction in infarct volume as compared to the wild type (Eliasson, et al., 1997). 
This molecular research has contributed to the knowledge of the pathology of 
stroke and may in fact have clinical implications for treatments. There are many other 
genes whose effects can be examined with this type of technology. 
5.6. A thesis summary. 
Despite the fact that transgenic technology can provide insight into the genetic 
contributions to stroke, a reduction in infarct size does not necessarily mean an animal 
will exhibit improved functional outcome. Behavioural testing in stroke research is 
essential in order to evaluate the efficacy of any treatment. The purpose of this thesis was 
not only to demonstrate the usefulness of mice, but also to provide a detailed analysis of 
mouse motor behaviour that can be readily applied to any mouse strain in any 
experimental setting. The results of the experiments indicate that the single pellet 
reaching task is applicable to mice and that a focal motor cortex insult produces 
quantifiable impairments. There are also implications from the experiments for the choice 
of a stroke model. A larger focal motor cortex stroke produced by permanent removal of 
the cortical vasculature results in massive quantitative and qualitative impairments. Mice 
subjected to a stroke produced by temporary occlusion of the same vasculature only 
exhibit qualitative deficits and the area of tissue damage is much smaller. The fact that 
105 
both groups of mice exhibited impairments in the same rotatory limb movements 
indicates that even small lesions disrupt certain functions. The positive correlation 
between infarct size and degree of impairment for certain movement components 
indicates that as the lesion gets larger more of the movements become affected. This is 
particularly true for movements that are relatively stable. For example, mice rarely 
exhibit impairments in the movements that require fine digit manipulation such as digits 
semiflexed and digits extend, but animals with the largest insult did indicate impairments 
with these movements. In conclusion, the mouse makes an excellent model for studying 
stroke induced motor impairments, recovery and compensation. 
106 
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