Recombinant IL-4 injection into the brain alters the inflammatory response and grey matter injury in a rat model of

ischemic stroke

by

Sarah Hutchings

A thesis submitted in conformity with the requirements for the degree of Master of Science

Department of Physiology University of Toronto

© Copyright by Sarah Hutchings 2014

ii

Recombinant IL-4 injection into the brain alters the inflammatory

response and grey matter injury in a rat model of ischemic stroke

Sarah Hutchings

Master of Science

Department of Physiology University of Toronto

2014

Abstract

Following stroke, there is an inflammatory response involving activation of CNS-resident

microglia, and infiltration of peripheral immune cells. This response can last for hours to days,

making it more amenable to treatment. CNS-resident microglia can exist in several activation

states, and in vitro, their alternative activation can be induced by the cytokine, IL-4. Here, I

modeled transient focal ischemia by injecting the vasoconstrictor peptide, endothelin-1 (ET-1),

into the rat striatum, either alone (untreated) or with 500 ng of recombinant IL-4 (treated). Gene

expression analysis revealed a significant increase in the alternative activation markers, IL-4r!,

ARG1, CD163 and CCL22; and in the IL-4 signaling molecules, STAT6 and PPAR". IL-4

treatment increased the number of phagocytic neutrophils and degenerating neurons in the

ischemic core, but did not alter the number of microglia/macrophages or the extent of white

matter injury. Thus, IL-4 might be harmful when administered at the onset of ischemia.

iii

Acknowledgments

First and foremost I would like to thank my supervisor Dr. Lyanne Schlichter. Dr.

Schlichter is a meticulous and honest scientist. Without trying, Dr. Schlichter motivates and

inspires her students. Her passion for science is undeniably contagious. I truly could not have

asked for a more supportive supervisor and feel very lucky to have been trained by such an

incredible scientist. Thank you.

Secondly, I would like to thank my committee members Dr. Jim Eubanks and Dr. Isabelle

Aubert. Both Dr. Eubanks and Dr. Aubert went above and beyond as committee members,

making themselves available for questions whenever needed and provided additional support

while I was trouble shooting difficult experiments. I cannot thank them enough.

I would also like to thank all the members of the Schlichter lab: XiaoPing, Starlee, Roger,

Raymond, Doris, Michael, Tamjeed, Laurel and Mary. Working along side all of you has been

such a blessing. You have provided me with stimulating intellectual conversations and very

special friendships. I would especially like to thank Starlee for training me and Laurel, for all of

her help with the never-ending brain sectioning.

I would like to thank my amazingly supportive friends and family, many of whom do not

share the same passion for science as I do. Nevertheless this has not prevented any of them from

providing me with an incredible support system for the duration of my degree. I would especially

like to thank my parents and brother for believing in me when I have needed it most.

Finally, I would like to dedicate the work in this thesis to one of the smartest people I

know, my grandfather, Gerald Roger. From a very young age my grandpa has taught me the

importance of honesty. I’ve learned to model his honesty in my role as a scientist, as I conduct

experiments and present my results with utmost integrity. My grandpa never ceases to amaze me

with his widespread knowledge, constantly sharing with me facts that are never restricted to one

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genre but range from history to science to sports. Although a brilliant man, my grandpa is

humble. He acknowledges what he does not know and is always eager to learn more. Throughout

the years, I have always felt that I can turn to him, regardless of how large or small the problem,

and that has been such a blessing while I was completing my MSc and always. Thank you so

very much, I am so lucky to have you. !

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Table of Contents

Abstract.………………………………………………………………………………………… ii Acknowledgements.………………………………………………………….………………… iii Table of contents.……………………………………………………………………………….. v List of tables……………………………………………………………………………………. ix List of figures………………………………………………………………………………….… x List of abbreviations…………………………………………………………………………… xi List of appendices………………………………………………………………………..…… xiii

1. Introduction…………………………………………………………………………………... 1

1.1 Ischemic Stroke ………………………………………………………………..…… 1

1.2 Damage after stroke.…………………………………………………………...…… 3

1.2.1 White vs Grey matter ……………………………………………………… 3

1.3 Animal models used to study white and grey matter injury after ischemia ….… 3

1.3.1 White matter locations in the brain ……………………….……………… 4

1.3.2 Vessel Occlusion ……..…………………………………………………… 5

1.3.2.1 MCAo…...………………...……………………………………… 5

1.3.2.2 AchAo…..………………………………………………………… 7

1.3.3 Vasoconstriction…………………………………………………………… 7

1.3.3.1 Endothelin-1……………………………………………………… 7

1.4 Inflammation of the CNS...………………………………………………………… 9

1.4.1 General introduction…….………………………………………………… 9

1.4.2 Microglia……….………………………………………………………… 10

1.4.3 Neutrophils……..………………………………………………………… 11

1.4.4 Astrocytes……….………………………………………………………… 12

1.5 Activation of microglia and macrophages……..………………………………… 13

1.6 Injection of cytokines into the brain…………………………………………...…. 14

1.7 Monitoring changes in gene expression, grey and white matter injury and inflammation after injection of IL-4 into the ischemic rat striatum……..………… 15

vi

1.7.1 Downstream IL-4 signaling molecules and alternative activation/anti-inflammatory genes….………………………………………… 15

1.7.2 Pro-inflammatory genes…………………………………………………. 16

1.7.3 Neuron-glial interaction markers: CD200-CD200R………………...….. 17

1.7.4 Phagocytic cells……..……………………………………………………. 17

1.7.5 Grey matter injury…..……………………………………………………. 18

1.7.6 White matter injury………………………………………………………. 18

1.8 Hypotheses…………………………………………………………………………. 19

1.8.1 Gene expression analyses……...………………………………………… 19

1.8.2 Immunohistochemistry analyses………………………………………… 19

2. Materials and Methods.…………………………………………………………………….. 20

2.1 Induction of transient focal ischemia..…………………………………………… 20

2.2 Enhancing the alternative microglia/macrophage activation state in the striatum...……………………………………………………….……………………… 21

2.3 mRNA analysis……………….……………………………………………………. 21

2.4 Immunohistochemistry……………………………………………………………. 23

2.5 Fluoro-Jade B histochemistry.……………………………………………………. 26

2.6 Sampling procedure and quantification….……………………………………… 26

2.7 Statistical Analysis…..…………………………………………………………….. 27

3. Results….………………………………………………….………………………………… 28

3.1 Evaluating the effects of IL-4 treatment on expression of inflammation-related and alternative activation-related genes in ischemic animals………………………………………………………………….……………… 28

3.1.1 Recombinant IL-4 treatment increased expression of signaling molecules downstream of the IL-4 receptor…………………………………… 28

3.1.2 IL-4 treatment increased expression of alternative activation markers……………………………………………………………………….… 30

3.1.3 IL-4 treatment did not significantly alter expression of pro-inflammatory markers…..………………………………….……………… 30

vii

3.1.4 IL-4 treatment altered expression of CD200 but not CD200R, which are markers of neuron-glial interactions…..………………………………..… 33

3.2 Monitoring changes in tissue...……………………………………………………. 36

3.2.1 ET-1+ saline and IL-4 treated animals showed BBB leakiness………… 36

3.2.2 IL-4 treatment did not alter the presence of the glial scar or its thickness.............................................................................................................. 36

3.2.3 IL-4 treatment increased phagocytosis and the number of neutrophils, but not microglia/macrophages, in the ischemic core………………………… 39

3.2.4 IL-4 treatment increased grey matter injury after ischemia….………… 43

3.2.5 IL-4 treatment did not alter the extent of white matter injury..………… 43

4. Discussion………………………………………………………….………………………… 48

4.1 Overview…...…………………………………………………….………………… 48

4.2 Endothelin-1 stroke model..…………………………………………………….… 48

4.3 Salient Findings.…...………………………………………………….…………… 49

4.3.1 Recombinant IL-4 treatment increased expression of downstream signaling molecules of IL-4 activation……...……………….………………… 49

4.3.2 IL-4 treatment increased expression of alternative activation genes.…... 51

4.3.2.1 IL-4r!...…………………………………………………….…… 51

4.3.2.2 ARG1.……………………...………………………………….… 52

4.3.2.3 CD163.……………….……………………………………….… 54

4.3.2.4 CCL22.…………….………………………………………….… 56

4.3.3 IL-4 treatment did not significantly alter expression of pro-inflammatory genes……..………………………………….……………… 58

4.3.4 IL-4 treatment altered expression of CD200 but not CD200R, markers of neuron-glial interactions ……………………...………………………….… 58

4.3.4.1 CD200........................................................................................... 58

4.3.4.2 CD200R………………………………………………………… 60

4.3.5 ET-1+ saline and IL-4 treated animals showed signs of BBB leakiness.. 61

4.3.6 IL-4 treatment did not alter presence of the glial scar or its thickness…. 62

viii

4.3.7 IL-4 treatment did not alter the number of activated microglia/macrophages in the ischemic core.……………………………….… 63

4.3.8 IL-4 treatment increased phagocytic neutrophils in the ischemic core… 64

4.3.9 IL-4 treatment increased grey, but not white matter injury after ischemia…..…………………………………………………….………………. 67

4.4 Limitations..…………………………………………………….………………..… 70

4.5 Conclusions……………………………………………………………………….... 72

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List of Tables

Table 2.1 Antibodies used to identify cells, their state and white and grey matter injury……... 25

x

List of Figures

Figure 1.1. A generally accepted time course of damaging events occurring after stroke in humans…………………………………………………………………................ 2

Figure 2.1. Endothelin-1 stroke model and sampling regions for quantification……………. 6 Figure 2.2. Summary of experimental design…..…………………………………………… 22

Figure 3.1. Recombinant IL-4 treatment increased expression of signaling molecules

downstream of the IL-4 receptor. …………………………………………….… 29 Figure 3.2. Recombinant IL-4 treatment increased expression of the alternative activation

or anti-inflammatory markers, IL-4r! and ARG1. ……………………..………. 31 Figure 3.3. Recombinant IL-4 treatment increased expression of alternative activation

markers CD163 and CCL22. …………………………………………………… 32 Figure 3.4. Recombinant IL-4 treatment did not significantly alter the expression of pro-

inflammatory markers…………………………………………………………... 34 Figure 3.5. Recombinant IL-4 treatment altered the expression of CD200, but not its

receptor…………………………………………………………………………. 35 Figure 3.6. ET-1+ saline and IL-4 treated animals showed signs of BBB leakiness……….. 37 Figure 3.7. Recombinant IL-4 treatment did not alter the presence or thickness of the

glial scar………………………………………………………………………… 38 Figure 3.8. Recombinant IL-4 treatment did not alter the number of phagocytic

microglia/macrophages in the lesion core………………………………………. 40 Figure 3.9. Recombinant IL-4 treatment increased the phagocytic activity in cells other

than microglia/macrophages at 1 day…………………………………………... 42 Figure 3.10. Recombinant IL-4 treatment increased the number of phagocytic neutrophils

in the ischemic core at 1 day. …………………………………………………... 44 Figure 3.11. Recombinant IL-4 treatment increased grey matter injury, judged by neuronal

degeneration after ischemia. …………………………………………………… 45 Figure 3.12. Recombinant IL-4 treatment did not alter white matter injury, judged by myelin

staining in the ischemic core. …………………………………………………... 47

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List of Abbreviations

AchAo Anterior choroidal artery occlusion

ARG1 Arginase-1

BBB Blood-brain barrier

CCR2 Chemokine receptor 2

CD163 Haptoglobin-hemoglobin scavenger receptor

CD200 Cluster of Differentiation 200

CD200R Cluster of Differentiation 200 receptor

CNS Central nervous system

CX3CR1 Fracktalkine receptor

DAPI 4’-6-diamidino-2-phenylindole

dMBP Degraded myelin basic protein

ET-1 Endothelin-1

EAE Experimental autoimmune encephalomyelitis

FACS Fluorescence-activated cell sorting

GFAP Glial fibrillary acidic protein

GP Globus pallidus

HO-1 heme-oxygenase-1

HPRT1 Hypoxanthine-guanine phosphoribosyltransferase

IB4 Griffonia simplicifolia B4 isolectin

Iba1 Ionized calcium binding adapter molecule

ICH Intracerebral hemorrhage

IFN-! Interferon gamma

IgG Immunoglobulin G

IL Interleukin

IL1ra IL-1 receptor antagonist

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iNOS Inducible nitric oxide synthanse

IP Intraperitoneal injection

LPS Lipopolysaccharide

MBP Myelin basic protein

MDC Macrophage derived chemokine

MCA Middle cerebral artery

MCAo Middle cerebral arter occlusion

MMP Matrix metalloproteinases

MPO Myeloperoxidase

NeuN Neuronal nuclear antigen

NO Nitric oxide

PBS Phosphate buffered saline

PBT Phosphate buffered triton

PMN Polymorphonuclear leukoctye

SDHA Succinate dehydrogenase complex subunit A

SN Substantia nigra

TNF" Tumor necrosis alpha

TTC 2,3,5-triphenyl tetrazolium chloride

tPA Tissue plasminogen activator

YWHAZ Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide

xiii

List of Appendices

A1. Inflammation-related genes evaluated using NanoString technology after recombinant IL-4 treatment in the striatum of ischemic rats…………………………………………. 92

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1. Introduction

1.1 Ischemic Stroke

Stroke is the leading cause of long-term disability in the USA and second leading cause

of death worldwide (Kelly-Hayes et al., 1998, Lopez and Mathers, 2006). There are two types of

stroke, hemorrhagic and ischemic. Hemorrhagic stroke is caused by weakening, thinning and

rupture of a blood vessel or artery in the brain. The majority of strokes are ischemic, caused by

transient or permanent occlusion to an artery or deep blood vessel (American Heart Association,

2012). There is currently only one available treatment for ischemic stroke; administration of the

clot buster Tissue Plasminogen Activator (tPA). If delivered within 3-4.5 hours of stroke onset,

tPA can break down blood clots that are preventing normal blood flow in the brain, and can

produce a remarkable reperfusion to the infarct area (Wang et al., 2004). Unfortunately, very few

stroke patients are in hospital and treated within the narrow window of time after stroke onset

needed to receive tPA. This translates to fewer than 5% of ischemic stroke patients receiving tPA

treatment (del Zoppo, 1998), and has spawned a huge and ongoing effort to identify new

therapeutic targets and drugs. Hundreds of chemicals that target the primary injury phase after

stroke, namely, early neurotoxicity have been identified. Dozens have proven promising in pre-

clinical studies in rodents but have been ineffective in improving survival or functional outcomes

in clinical trials (Brott and Bogousslavsky, 2000, Ginsberg, 2009, Lakhan et al., 2009).

In the hours to days following initial injury after stroke, there is a secondary injury phase, which

is characterized by a prominent inflammatory response and damage to white matter (Fig. 1.1). In

the past, a great deal of stroke research has focused on the initial injury phase. However,

targeting damaging events that occur in the secondary injury phase might be more amenable to

treatment, given the larger time window. Identifying a treatment that targets the secondary injury

phase would benefit a much larger proportion of ischemic stroke patients.

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Figure 1.1

Figure 1.1. A generally accepted time course of damaging events occurring after stroke in humans.

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1.2 Damage after stroke

1.2.1 White vs Grey matter

In 1543, the Belgian anatomist, Andreas Veslius was the first to identify two distinct

neuroanatomical divisions of the brain (Filley, 2012). At the macroscopic level, these regions are

distinguishable by colour, giving rise to the nomenclature, white and grey matter. Much more

recently, scientists have delineated discrete microscopic features that account for these

differences in colour. White matter consists of nerve fibers, axons and glia, with few neuronal

cell bodies or synapses, while grey matter is comprised of neuronal cell bodies and synapses

(Petty and Wettstein, 1999). Historically, white matter was thought to be less susceptible to

stroke than grey matter (Marcoux et al., 1982). More recent advances in imaging technology

have led to a paradigm shift. Magnetic Resonance Imaging and Diffuse Tensor Imaging have

provided a means to map the networks of white matter in the human brain. Clinical observations

have shown that ischemic stroke is rarely confined to grey matter (Goldberg and Ransom, 2003)

and primary white matter injury occurs in about 25% of human strokes (Matute et al., 2013). At

the microscopic level, more recent research has demonstrated the sensitivity of the major cellular

components of white matter to ischemia. Several animal models have shown the susceptibility to

ischemic damage of oligodendrocytes, the myelin forming cells of the central nervous system

(CNS; reviewed by Arai and Lo, 2009), and of myelin and axons (Pantoni et al., 1996, Petty and

Wettstein, 1999, Irving et al., 2001, Hughes et al., 2003, Medana and Esiri, 2003, Moxon-Emre

and Schlichter, 2010, Lively and Schlichter, 2012). Thus, white matter damage is now

considered an important contributor to stroke outcome, and a tractable therapeutic target.

1.3 Animal models used to study white and grey matter injury after ischemia

It is clear that no animal model can fully recapitulate all components of human stroke

(Durukan and Tatlisumak, 2007, Howells et al., 2010). Most pre-clinical studies are conducted in

4

small animals, especially rodents (Durukan and Tatlisumak, 2007). While mouse models

facilitate using transgenic animals, which are of limited availability in rats (Durukan and

Tatlisumak, 2007), rats are commonly used for several reasons. The cerebrovascular anatomy

and physiology is reasonably similar between rats and humans (Macrae, 1992), and the infarct

size is more consistent than in mice (Carmichael, 2005). In the most commonly used mouse

model of ischemia (middle cerebral artery occlusion (MCAo), discussed below), the infarct size

is highly variable and shows strain-dependence that is less pronounced in rats (Carmichael,

2005). The positive correlation between brain size and amount of white matter (Zhang and

Sejnowski, 2000) and larger brain size of rats yields more material for analysis. The larger size is

especially useful when one seeks to analyze several parameters or stains, and to quantify

differences across brain regions.

1.3.1 White matter locations in the brain

It is often noted that the ratio of white matter to grey matter in the human brain is 60:40,

but much lower in the rat (14:86) and mouse (10:90) (Krafft et al., 2012). While this might be

considered an impediment to studying white matter injury, rodents have several brain regions

with high densities of white matter (Johnson et al., 2012). Many of these regions are commonly

used to study white matter injury in rodents. The corpus callosum is comprised of white matter

tracts (nerve fibers) that connect the two cerebral hemispheres of the brain, transferring and

integrating information from the left and right hemispheres (Bruni and Montemurro, 2009). The

internal capsule is a compact band of fibers that lies deep in the brain, separating the caudate

nucleus from the putamen. It consists of projection fibers that relay information from the cortex

and spinal cord, brain stem and subcortical structures (Bruni and Montemurro, 2009). Other

white matter-dense brain regions include, but are not limited to, the optic nerve and anterior

commissure (Bruni and Montemurro, 2009). Some brain regions, such as the striatum, provide an

5

excellent template for studying white and grey matter injury side-by-side. The striatum contains

a large number of myelinated axon bundles that are surrounded by neuronal cell bodies (Figure

2.1B).

I have exploited the striatum in rats to simultaneously analyze damage to both white and grey matter.

1.3.2 Vessel Occlusion

1.3.2.1 MCAo

Most ischemic strokes in humans occur in the area surrounding the middle cerebral artery

(MCA). A rodent model of MCAo was developed early and has since been refined (Robinson et

al., 1975, Koizumi et al., 1986). Robinson and colleagues (1975) used craniotomy to expose the

MCA and distally ligated the artery, which produced an ischemic lesion that extended into the

cortex. Now, a more commonly used method for occluding blood flow is to advance an

intraluminal suture through the internal carotid artery until it reaches the origin of the MCA (del

Zoppo et al., 1992). With this method, ischemia is made transient by removing the suture to

allow reperfusion. The duration of MCAo needed to produce a significant non-lethal lesion is

variable. The common duration for rat is 60, 90 or 120 minutes (Carmichael, 2005), and usually

results in neuron death throughout the striatum and into the dorsolateral cortex, but not in

contralateral brain regions(Garcia et al., 1995). Based on the distribution of injury, the MCAo

model is useful for simultaneously analyzing damage that is isolated to grey matter (in the

cortex), white matter (in the corpus callosum), or occurring in both (in the striatum). Despite its

popularity, an important limitation of the MCAo model is the variable size and location of the

infarct (Liu and McCullough, 2011). Some of the variability seen in the literature can be

attributed to differences in size and quality of the suture/filament used (Kuge et al., 1995);

silicone-coated thread induces a larger lesion than uncoated thread (Laing et al., 1993).

6

Figure 2.1

Figure 2.1. Endothelin-1 stroke model and sampling regions for quantification. (A) Representative coronal section at 3 days after stroke induction is stained with 2% TTC (2,3,5-triphenyl-2H-trazolium chloride). The white area delineates the ischemic infarct. Six 200 # 200 µm sampling areas (red boxes) across the infarct core were used for quantification of immune cell infiltration (microglia/macrophages and neutrophils), phagocytic activity and grey matter injury, judged by the number of degenerating neurons in ET-1+ saline and ET-1+ IL-4 treated animals at 1, 3 and 7 days post injection. As a control, four 300 # 300 µm sampling areas were imaged across the contralateral striatum. (B) Grey and white matter can be clearly distinguished in the naive rat striatum. White matter tracts were identified using an antibody against myelin basic protein (MBP, rabbit polyclonal; green) and grey matter (neurons) was labeled with an antibody against neuronal nuclei (NeuN, mouse monoclonal, red). Scale bars = 100 µm.

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When using mice, some strains, such as C57/Bl6 demonstrate substantially larger infarcts after

MCAo than others (129J mice; Connolly et al., 1996). Other limitations of the MCAo model are

the risk of subarachnoid hemorrhage (Carmichael, 2005), and the added difficulty in aged

animals, which have less flexible blood vessels and higher mortality (Liu and McCullough,

2011).

1.3.2.2 AchAo

Occlusion of the anterior choroidal artery (AchAo) can be obtained by advancing an

intraluminal suture through the ICA to a region proximal to the MCA (He et al., 1999). As

illustrated in detail in their Figure 4, the infarct location is similar in MCAo and AchAo (He et

al., 2000). The main differences are that after AchAo, the infarct is generally smaller but nearly

always encompasses the internal capsule. Thus, AchAo reliably produces ischemia in a white

matter-dense region, and facilitates studies of deep lacunar strokes with a less distributed lesion

than MCAo.

1.3.3 Vasoconstriction

1.3.3.1 Endothelin-1

An increasingly popular model uses injection of the potent vasoconstrictor, endothelin-1

(ET-1). ET-1 is a naturally occurring, 21 amino acid peptide produced by endothelial cells,

which binds to the endothelial receptors, ETA and ETB (Verhaar et al., 1998). There are several

advantages of this model over vessel occlusion models. Provided the potency of each ET-1 batch

is properly established, the injected dose can be titrated to obtain different degrees of transient

ischemia, and lesion sizes that are relatively reproducible. For instance, with moderate amounts

of ET-1 injected directly into the brain parenchyma, a reduction of cerebral blood flow of ~60%

for up to 3 hours has been reported (Hughes et al., 2003). ET-1 is sometimes applied to the MCA

8

to cause constriction, instead of ligating the artery in rats (Robinson et al., 1990, Gresle et al.,

2006). Importantly, stereotaxic ET-1 injection can be used to produce a focal infarct in brain

regions that are selected to focus on white matter (e.g., corpus callosum, internal capsule), grey

matter (e.g., cortex) or both (e.g., striatum; Sozmen et al., 2012). When injected into white

matter-rich regions, ET-1 produces hallmarks of white matter injury seen in humans, including

axonal damage, demyelination, inflammation and glial scar formation (Hughes et al., 2003,

Moxon-Emre and Schlichter, 2010, Lively and Schlichter, 2012).

There are few mouse studies using ET-1 injection to evoke transient ischemia, and the

results have been inconsistent. In one study, injecting ET-1 into the striatum failed to induce

ischemia in multiple strains of mice, and increasing the dose increased mortality without

inducing a lesion (Horie et al., 2008). Another study showed a dose-dependent increase in infarct

size in mouse brain subjected to multiple focal injections of ET-1 (Sozmen et al., 2009).

Differences in expression of endothelin receptor subtypes might be a confounding factor. Mouse

brain expresses a lower proportion of ETA receptors, which evoke the vasoconstriction needed to

produce ischemia, than ETB receptors, which generally cause vasodilation (but see below; Wiley

and Davenport, 2004, Sozmen et al., 2012). The possibility was raised that the larger proportion

of ETB receptors interferes with induction of ischemia in the mouse brain (Sozmen et al., 2012).

Could differences in ET-1 affinities be a factor? The Kd for ET-1 binding is 0.12 nM for ETB

and 0.6 nM for ETA receptors (Haynes et al., 1995). A further complication is that in humans,

ETB receptors can mediate both vasodilation and vasoconstriction (Clozel et al., 1992, Seo et al.,

1994, Haynes et al., 1995). This might explain the finding that although the rat striatum contains

mainly ETB receptors (Tayag et al., 1996), a focal ET-1 injection reliably produces an ischemic

infarct (described above). Further research is needed to clarify the regional and cell-specific

expression, and roles of the ET-1 receptor subtypes in rodents. Nevertheless, focal ET-1 injection

9

into the rat CNS is a widely accepted model of transient ischemia that can create reproducible

lesions restricted to a desired region of the brain (Sozmen et al., 2012).

In order to study transient focal ischemia in a restricted region of the brain and induce reproducible lesions, I used the ET-1 model in rats.

1.4 Inflammation of the CNS

1.4.1 General introduction

Although the CNS was originally thought to be immune-privileged, it has since been

demonstrated that neurons, microglia, astrocytes and neutrophils can contribute to the

inflammatory response in the CNS (Jin et al., 2010). In the healthy adult brain, the blood-brain

barrier (BBB) provides a protective shield between the CNS and blood and lymphatic systems,

creating a stable environment for proper neural function and preventing harmful molecules and

substances from entering into the CNS. The BBB is comprised of tight junctions between

endothelial cells which function with what is described as the “neurovascular unit”, comprised of

astrocytes, glia, pericytes, neurons and the basement membrane (Persidsky et al., 2006). After

damage (such as ischemia) or if the brain is in a diseased state the BBB is compromised (often

described as leakiness of the BBB). Leakiness allows infiltration of peripheral immune cells and

potentially harmful molecules from the blood. Using the ET-1 model in rats, within a week of

ischemia induction, activation of innate immune cells in the CNS and infiltration of blood-borne

immune cells has been found (Souza-Rodrigues et al., 2008, Moxon-Emre and Schlichter, 2010).

In contrast, others have found no breakdown in the BBB or infiltration of neutrophils after ET-1

injection into the striatum (Hughes et al., 2003). The Schlichter lab and others have used

extravasation of the blood protein, immunoglobulin G (IgG), as an indicator of BBB dysfunction

(Moxon-Emre and Schlichter, 2011).

I used an antibody against IgG to evaluate BBB integrity.

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1.4.2 Microglia

Microglia are the innate immune cells of the CNS. They act as sensors, searching for signs

of damage. In the resting state, microglia have long processes that extend and retract as they

survey their environment (Nimmerjahn et al., 2005). In the activated state, microglia pull in their

processes and appear rounded in shape, indistinguishable from invading macrophages; however,

there are several different modes of microglial activation (see section 1.5). Currently, there is no

microglia-specific marker. There are, however, several markers for microglia/macrophages,

including ‘integrin alpha M’16 subunit (ITGAM or CD11b; Robinson et al., 1986, Akiyama and

McGeer, 1990), complement component receptor 3 alpha (CR3A; Robinson et al., 1986) and

‘ionized calcium binding adapter molecule 1’ (Iba1; Ito et al., 1998). It is important to note that

CD11b is also found on neutrophils and thus is not solely a microglia/macrophage marker.

Microglia/macrophages can also be labeled with the stains, tomato lectin and Griffonia

simplicifolia B4 isolectin (IB4) but they are less specific, as they also stain blood vessels (Streit

and Kreutzberg, 1987, Acarin et al., 1994).

Many investigators have exploited fluorescence-activated cell sorting (FACS) in an attempt

to discriminate microglia from invading macrophages. Resting microglia are labeled with CD11b

and low levels of the antigen, CD45 (hematopoietic cell surface marker), and are thus referred to

as CD11b+CD45low; whereas, peripheral macrophages are CD11b+CD45high (Sedgwick et al.,

1991). When using FACS, tissue samples are homogenized, and thus all information regarding

the location of cells in the CNS is lost. Additionally, activated microglia (in pathological states

or after damage) have increased CD45, making it more difficult to distinguish between

CD11b+CD45low and CD11b+CD45high cells (Ford et al., 1995).

Recently, a substantial improvement in differentiating microglia from macrophages has

been made through the use of fluorescent transgenic knock-in mice. A cross between a

11

fractalkine receptor-green fluorescent protein (CX3CR1-GFP) labeled mouse and a chemokine

(C-C Motif) receptor 2 red fluorescent protein (CCR2-RFP) labeled mouse appears to be the

most promising tool for discriminating microglia from macrophages to date. CCR2 is specific to

peripheral macrophages and the fractalkine receptor (CX3CR1) is specific to microglia (Mizutani

et al., 2012). Thus, monitoring green versus red cells allows discrimination of microglia from

invading macrophages. Furthermore, CCR2 (unlike CD45) is not up-regulated in pathological

states (Saederup et al., 2010).

As early as 24 hr after stroke onset, some microglia demonstrated an “activated”

morphology (rounded; Moxon-Emre and Schlichter, 2010, Lively et al., 2011). Our lab (2010)

and others have demonstrated that by 3 days after ischemic stroke induction there is an

infiltration of microglia/macrophages to the lesion, and by 7 days, the lesion is completely

infiltrated (Souza-Rodrigues et al., 2008). Microglia are the primary phagocytes in the brain, and

thus engulf debris after an insult in the CNS, which might be beneficial after ischemia (Patel et

al., 2013). However, microglia release both pro and anti-inflammatory mediators (see section

1.5) and thus their exact role after stroke remains elusive.

For the purposes of this project, I used an antibody against Iba1 to evaluate microglia/macrophage infiltration into the ischemic infarct. I did not attempt to discriminate between microglia and macrophages.

1.4.3 Neutrophils In a healthy state, the CNS is devoid of neutrophils. However, neutrophil infiltration into

the CNS has been reported as early as 12 hours post-ischemia induction (Matsuo et al., 1994). In

animal models of stroke, neutrophil infiltration is routinely monitored via a myeloperoxidase

(MPO) activity assay (Barone et al., 1991, Weston et al., 2007) or by staining with antibodies

against MPO (anti-MPO; Moxon-Emre and Schlichter, 2010) or against polymorphonuclear

12

leukocytes (anti-PMN; Weston et al., 2007). MPO is an enzyme found abundantly in the

granules of neutrophils (Amulic et al., 2012), and an increase in number of neutrophils is

associated with an increase in MPO activity (Matsuo et al., 1994).

One widely received hypothesis is that neutrophils are harmful following ischemia. Most

of the evidence supporting this belief is based on neutrophil depletion studies (where depletion

before ischemia induction attenuated post-ischemic injury; Matsuo et al., 1994, Hartl et al.,

1996). Neutrophils release harmful cytokines, such as tumor necrosis alpha (TNF!; Nguyen et

al., 2007), as well as matrix metalloproteinases (MMPs), such as MMP-9 (Romanic et al., 1998,

Justicia et al., 2003), which can contribute to breakdown of the BBB further exacerbating

damage after ischemia. Furthermore, neutrophil infiltration after ischemia is correlated with

enhanced MMP-9 expression, which was attenuated in neutrophil-depleted animals (Justicia et

al., 2003). In contrast, some studies have found no clear positive or negative correlation between

neutrophil infiltration and ischemic damage (Emerich et al., 2002, Harris et al., 2005). In fact,

Hughes and colleagues (2003) were unable to detect the presence of neutrophils after ischemia

(induced by ET-1 injection), while others have reported neutrophil infiltration that peaks at 1 day

(Souza-Rodrigues et al., 2008, Moxon-Emre and Schlichter, 2010).

To evaluate neutrophil presence in the CNS after ischemia without losing important spatial information (i.e., closeness to the lesion), I used an antibody against PMN.

1.4.4 Astrocytes

Astrocytes are the most abundant type of glial cells in the brain (Tower and Young,

1973). In the healthy CNS, astrocytes are known to provide basic support functions, allowing for

optimal CNS functioning (Kimelberg and Nedergaard, 2010). After stroke, astrocytes become

hypertrophic, and aggregate around the infarct, forming what is known as the glial scar

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(Anderson and Nedergaard, 2003). There are several antibodies that allow visualization of

astrocytes and the glial scar, such as glial fibrillary acidic protein (GFAP), vimentin (Sancho-

Tello et al., 1995, Anderson and Nedergaard, 2003) and nestin (Duggal et al., 1997). The glial

scar provides a barrier between damaged/necrotic tissue and healthy tissue. While the barrier

could prevent harmful substances (released from damaged tissue) from reaching unharmed

tissue, it might also prevent axonal regeneration.

The presence of the glial scar (accumulation of reactive astrocytes) was evaluated using an antibody against nestin.

1.5 Activation of microglia and macrophages

There are several proposed modes of activation of microglia/macrophages; however,

classical and alternative activation are especially relevant to acute damage of the CNS (Colton,

2009). Identification of activation states comes mostly from in vitro studies, in which treatment

with certain activators selectively induced expression of unique markers. Classical activation of

microglia/macrophages is associated with an increase in expression and protein synthesis of pro-

inflammatory mediators, such as interleukin-1 beta (IL-1$) and TNF!. Furthermore, classical

activation induces production of reactive oxygen and nitrogen species, as well as MMPs, which

as previously noted, contribute to BBB breakdown. In general, classically activated

microglia/macrophages are thought to be involved in tissue defense and production of pro-

inflammatory cytokines, as well as nitric oxide (NO; Colton, 2009). In vitro, microglia cells

treated with the endotoxin, lipopolysaccharide (LPS) or interferon gamma (IFN-") increase their

production of markers characteristic of the “classical state” (Colton, 2009).

In vitro, alternative activation of microglia/macrophages is induced by treatment with the

cytokine, interleukin-4 (IL-4) and to a lesser extent, by interleukin-13 (IL-13; Colton, 2009).

14

Administration of IL-4 produces a profile of gene expression and protein synthesis that differs

from the classical state. This “alternative” activation state is thought to contribute to tissue repair

(as opposed to tissue defense). The alternative state is associated with up-regulation of anti-

inflammatory mediators, such as arginase-1 (ARG1; Colton, 2009). In some ways, alternative

activation could be described as an activation state that counteracts classical activation, because

anti-inflammatory functions could offset classically activated pro-inflammatory functions.

In vivo, microglia/macrophage activation states are much more difficult to study, because

the number of factors contributing to the activation state is large. Furthermore, there is an on-

going debate about whether the phenotype of activated microglia is committed or reversible. For

example, are classically activated microglia/macrophages committed to a certain molecular

profile (e.g., production of pro-inflammatory cytokines) or can exposure to an alternative

stimulus reverse or alter the activation phenotype. In vitro studies demonstrate that the order and

duration in which stimuli are applied to microglia influences whether they are committed to a set

phenotype or not (Schwartz et al., 2006). If microglia/macrophage activation states are

reversible, this would increase the difficulty of differentiating between classically versus

alternatively activated microglia/macrophages in vivo.

The focus of my research was to identify whether or not enhancing alternative activation, via recombinant IL-4 administration is beneficial or harmful in the ET-1 model of ischemia in rats.

1.6 Injection of cytokines into the brain

This project will not be the first study to inject a cytokine into the brain. In 2002,

Sibson and colleagues injected TNF" into the rat striatum and found a marked decreased in

cerebral blood flow and a greater breakdown of the BBB compared to controls, demonstrating

the harmful effects TNF". An injection of IL-10 into the lateral ventricles of the rat brain

15

decreased abnormal social behaviour after an intraperitoneal (IP) injection of LPS (Bluthe et al.,

1999). Damage (as judged by TTC stain (2,3,5-triphenyl tetrazolium chloride) see section 4.3.9

for discussion) produced by injection of the excitotoxin, glutamate, decreased when co-injected

with IL-1 receptor antagonist (IL-1ra; Lawrence et al., 1998); suggesting that inhibiting the

effects of IL-1# is beneficial after excitotoxic brain damage. One study evaluated the effect of

intracerebroventricular administration of IL1ra after MCAo induction in the rat and found a

decrease in the lesion size (judged by TTC; Loddick and Rothwell, 1996). This suggests that

decreasing activity of IL-1# after stroke is beneficial. Another study, intracerebroventricaularly

injected the cytokine of interest for this project, IL-4, in an attempt to counteract the sickness

behaviour in rats induced by systemic administration of LPS. If IL-4 was administered prior to

LPS, animals showed decreased sickness behaviour (Bluthé et al., 2002) .

1.7 Monitoring changes in gene expression, grey and white matter injury and inflammation after injection of IL-4 into the ischemic rat striatum

1.7.1 Downstream IL-4 signaling molecules and alternative activation/anti-inflammatory genes

As previously stated, alternatively activated microglia/macrophages have been

characterized by induction of cell surface receptors and other genes after IL-4 exposure in vitro.

Some markers of alternative activation have been induced by stimuli other than IL-4; thus, it is

important to examine several markers in combination (Van Dyken and Locksley, 2013). Several

markers and reasons for their use are described next.

Haptoglobin-hemoglobin scavenger receptor (CD163) is expressed by macrophages

during the wound-healing phase of injury (Zwadlo et al., 1987). CD163 contributes to clearance

of pro-inflammatory haptoglobin after injury, thus demonstrating its anti-inflammatory role

(Akila et al., 2012). Furthermore, expression of CD163 is up-regulated in human and rat

macrophages treated with IL-4 (Gordon, 2003, Polfliet et al., 2006) and decreased by a pro-

16

inflammatory stimulus (LPS; Colton, 2009). ARG1 expression is increased in both microglia and

macrophages after IL-4 treatment in vitro (Colton et al., 2006). ARG1 competes with inducible

nitric oxide synthase (iNOS) for the substrate, L-arginine; thus, an increase in ARG1 could lead

to a subsequent decrease in the level of nitric oxide produced (Colton, 2009). IL-4 binds to the

IL-4r! chain of the IL-4 receptor complex to induce several intracellular signaling events, up-

regulating downstream signaling molecules such as signal transducer and activator of

transcription 6 (STAT6) and peroxisome proliferator-activated receptor gamma (PPAR "; Nelms

et al., 1999). Additional markers of alternative activation include certain chemokines, which are

regulatory polypeptides that play an important role in recruiting immune cells to sites of damage.

In vitro treatment with IL-4 increased expression of chemokine (C-C motif) ligand 22 (CCL22)

in human and rat macrophages, and has since been used as a marker for alternative activation

(Jaguin et al., 2013).

I monitored changes in expression of signaling molecules downstream of the IL-4 receptor and alternative activation markers in the rat striatum at 1, 3 and 7 days following injection of [Saline alone], [ET-1+ Saline] or [ET-1 +Recombinant IL-4].

1.7.2 Pro-inflammatory genes

Several pro-inflammatory genes are typically associated with classically activated

microglia/macrophages. LPS stimulated microglia and mixed glial cultures show increased

expression of IL1$, IL6, TNF!, and iNOS, which are suppressed by treatment with IL4

(Kitamura et al., 2000, Colton et al., 2006, Colton, 2009). After ischemia, it is reported that

several pro-inflammatory markers are up-regulated (Liu et al., 1994). Furthermore, inhibition of

pro-inflammatory cytokines, such as TNF!, can reduce ischemic injury (Yang et al., 1998).

I monitored changes in expression of pro-inflammatory markers in the rat striatum at 1, 3 and 7 days after injecting [Saline alone], [ET-1+ Saline] or [ET-1 +Recombinant IL-4].

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1.7.3 Neuron-glial interaction markers: CD200-CD200R

Binding of the ligand, Cluster of Differentiation 200 (CD200), to its receptor, CD200R,

suppresses immune cell activity (Koning et al., 2009), and this interaction is thought to maintain

microglia in a resting state (Yi et al., 2012). CD200R is normally found on microglia, and its

ligand, CD200, on neurons (Neumann, 2001). CD200 expression increases when microglial cells

are treated with IL-4 in vitro (Yi et al., 2012). In an excitotoxic lesion in vivo, a decrease in

CD200 expression was associated with an increase in microglial activation (Yi et al., 2012).

Experimentally inhibiting binding of CD200 to CD200R (by injecting a CD200R antibody)

exacerbated microglial activation in a rat model of Parkinson’s disease (Zhang et al., 2011).

Interestingly, after ischemia, an increase in CD200 was found in microglia/macrophages in the

core of the lesion (Matsumoto et al., 2007a). Together, these data suggest that a dysfunction of

the interaction of CD200 and CD200R could increase microglial activation.

I monitored changes in expression of CD200 and the CD200 receptor in the rat striatum at 1, 3 and 7 days following injection of [Saline alone], [ET-1+ Saline] or [ET-1 +Recombinant IL-4].

1.7.4 Phagocytic cells

Phagocytosis of debris by microglia/macrophages or neutrophils after damage is

described as a “double edged sword” because it can attenuate inflammation, but can also lead to

engulfment of potentially viable cells (Ramprasad et al., 1996). CD68, which is also known as

ED1, is a lysosomal marker whose expression is associated with increased phagocytic activity

(Damoiseaux et al., 1994). Microglia/macrophages and neutrophils have been found to be ED1

positive after injury (Hansen et al., 2001, Wasserman et al., 2008, Moxon-Emre and Schlichter,

2010, 2011).

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To evaluate phagocytic activity of microglia/macrophages or neutrophils after IL-4 treatment, I double stained tissue with antibodies against ED1(phagocytic marker) and either Iba1 (microglia/macrophage marker), and or PMN (neutrophil marker) after injection of [ET-1+ Saline] or [ET-1 +Recombinant IL-4]

1.7.5 Grey matter injury

As previously mentioned, damage to grey matter following ischemia has been studied

more extensively than white matter damage. In the most commonly used rodent model of focal

ischemia, MCAo, neuron death has been characterized using several staining methods. Neuronal

nuclear antigen (NeuN) is expressed in the nucleus and soma of neurons; and its disappearance

can be used as an indicator of neuron death. Also commonly used is Fluoro-Jade B, a dye that

labels the cell body of degenerating neurons (Schmued and Hopkins, 2000). Temporal loss of

NeuN and the appearance of Fluoro-Jade B display similar patterns following ischemia induction

by MCAo(Liu et al., 2009). Neuron loss has been reported as early as 1.5 hr after ischemia

induction and peaks at 1 day (in the MCAo model; Liu et al., 2009). By 7 days Fluoro-Jade B

staining and NeuN staining in rats subjected to MCAo is comparable to saline-injected controls.

I monitored the effect of IL-4 treatment on grey matter injury at 1,3, and 7 days following injection of either [ET-1+ Saline] or [ET-1 +Recombinant IL-4] using Fluoro-Jade B staining.

1.7.6 White matter injury

There are several ways to evaluate white matter injury following ischemia. Death of

oligodendrocytes has been studied by staining with antibodies against oligodendrocyte markers,

such as adenomatous 13 polyposis coli (Bhat et al., 1996). After ischemia, loss of

oligodendrocytes suggests that re-myelination cannot occur because the myelin producing cells

of the CNS are no longer present. Thus, lack of oligodendrocytes could be used to estimate the

extent of white matter damage, but also as an indication of the potential for re-myelination and

recovery.

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One of the main components of myelin is myelin basic protein (MBP). Our lab (2010) and

others (Hughes et al., 2003; Souza-Rodriguez et al., 2008) have used an antibody against MBP to

evaluate white matter injury after in the ET-1 model of ischemia. MBP staining 24 hr after

ischemia induction is comparable to MBP staining in naïve animals. However, by 3 days there is

a significant loss of MBP in the infarct core. By 7 days, the lesion centre is completely devoid of

MBP staining (Souza-Rodrigues et al., 2008, Moxon-Emre and Schlichter, 2010). To further

support using MBP loss as a marker of white matter injury, it is possible to double-stain with an

antibody against degraded myelin basic protein (dMBP; Moxon-Emre and Schlichter, 2010).

dMBP staining parallels the loss of MBP staining. Thus, as the level of healthy myelin (indicated

by MBP staining) increases, the level of unhealthy myelin (dMBP) increases, so that by day 7 the

ischemic infarct is strongly labeled with dMBP (Moxon-Emre and Schlichter, 2010).

I monitored the effect of IL-4 treatment on white matter injury using antibodies against MBP and dMBP at 1, 3 and 7 days following injection of either [ET-1+ Saline] or [ET-1 +Recombinant IL-4].

1.8 Hypotheses

1.8.1 Gene expression analyses

Recombinant IL-4 treatment at stroke induction will increase expression of downstream

IL-4 signaling molecules, alternative activation markers, and the neuron-glial interaction

markers, CD200 and CD200R in the rat striatum. IL-4 treatment will decrease expression of pro-

inflammatory markers.

1.8.2 Immunohistochemistry analyses

Recombinant IL-4 treatment will have beneficial effects after ischemia, decreasing grey

and white matter injury, immune cell infiltration into the lesion core, and glial scar formation.

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2. Materials and Methods

2.1 Induction of transient focal ischemia

All experimental procedures were performed in accordance with the University Health

Network animal care committee, with guidelines established by the Canadian Council on Animal

Care. Adult male Sprague-Dawley rats (Charles River; 3-4 months, 250-300 g) were

anaesthetized using 5% isoflurane. Animals were placed in a stereotaxic instrument (David Kopf

Instruments, Tujunga, CA) in which their heart rate, breathing rate and temperature were

monitored for duration of the surgery, using MouseOx® system (Starr Life Sciences, St. Laurent,

QC) and an electric heating pad. A 2 cm long incision was made to expose the skull.

Subsequently, a 1 mm in diameter burr hole was drilled (0.2 mm anterior and 3.5 mm lateral to

bregma) and a 27-gauge needle was lowered into the center of the right striatum (0.6 mm

ventral). The striatum was injected with 400 picomoles of the vasoconstrictor peptide,

endothelin-1 (ET-1; Calbiochem, EMD Biosciences, Gibbstown, NJ; 2 µL, in physiological

saline) using an UltraMicroPump II (World Precision Instruments, Sarasota, FL) at a rate of 500

nL/min. Control animals were injected with saline (final volume of 2 µL). ET-1 injection into the

brain reduces local blood flow by ~60% for about 3 hr (Hughes et al., 2003). Upon completion of

injection, the needle was left in place for 5 min to prevent backflow. In accordance with previous

work in the Schlichter lab (Moxon-Emre and Schlichter, 2010, Lively et al., 2011, Moxon-Emre

and Schlichter, 2011, Lively and Schlichter, 2012), the 400 pmol ET-1 injection created

reproducible lesions that were restricted to the striatum. Prior to each set of surgeries, a new

batch of ET-1 was tested for efficacy, as judged by the lesion present, monitored with TTC stain

(2,3,5-triphenyl tetrazolium chloride). TTC labels metabolically active tissue; thus, its absence

clearly identifies the stroke lesion (Figure 2.1A).

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2.2 Enhancing the alternative microglia/macrophage activation state in the striatum

To enhance alternative microglia/macrophage activation, the rat striatum was co-injected

with ET-1 (400 pmol) and 500 ng/rat of rat recombinant IL-4 (R&D Systems, Burlington, ON).

ET-1 and IL-4 were mixed together in physiological saline and injected at a final volume of 2

µL. The stroke control was 2 µL of ET-1 + saline. The 500 ng/rat dose of recombinant IL-4 was

selected based on a series of doses tested in a pilot study. For mRNA analysis, the striata of ET-

1+ saline and ET-1 + IL-4 treated rats were isolated, while the whole brain was isolated for

immunohistochemical analysis. For mRNA analysis, 6 animals were used for each time point

following stroke induction (1, 3, 7 days) for each treatment group. For immunohistochemistry

analysis, 3-4 animals were used for each time point (1, 3, 7 days) for each treatment group. See

Figure 2.2 for a summary of the experimental design.

2.3 mRNA analysis

Rats were administered a lethal dose of 100% isoflurane at 1, 3 or 7 days following

stroke induction. When there was no toe pinch reflex, the rat was transcardially perfused with

120 mL of phosphate buffered saline. The striatum was isolated and stored at -40°c until. For

multiplexed gene expression analysis, striata were removed from the -40°C freezer and

homogenized in TRIzol (Invitrogen, Life Technologies, Burlington, ON). Total RNA was

extracted and purified using the RNeasy Mini Kit (Qiagen, Mississauga, ON) following the

manufacturer’s instructions. RNA samples (200 ng/rat per time point, per treatment group) were

sent to be analyzed using Nanostring nCounterTM System, at the Princess Margaret Genomics

Centre (Toronto, ON). This assay uses sequence-specific probes to measure target mRNAs

within a sample, combining the high-throughput of microarrays while maintaining sensitivity

similar to RT-PCR (Geiss et al., 2008). mRNA hybridization, detection and scanning were

completed as per Nanostring nCounterTM instructions. Prior to submitting the samples, we

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Figure 2.2

Figure 2.2. Summary of experimental design. For gene expression analyses, animals received an injection of either saline only (A, I), 400 picomoles of endothelin-1+ saline (A, II) or 400 picomoles of endothelin-1 + 500 ng/rat of recombinant IL-4 (A, III). Animals were sacrificed at 1, 3 or 7 days post-injection; number of rats indicated. Ipsilateral and contralateral striata of animals from each treatment group were isolated, RNA extracted and subsequently used for Nanostring analysis. For immunohistochemistry analyses, animals were treated with either 400 picomoles of endothelin-1+ saline (B, I) or 400 picomoles of endothelin-1 + 500 ng/rat of recombinant IL-4 (B, II) and sacrificed at 1, 3 or 7 days post-injection for the number of rats indicated. Whole brains were isolated, frozen, and cryo-sectioned.

23

selected 28 inflammation-related genes (See Table A1), and a CodeSet was created by

NanoString nCounterTM Technologies to identify them. The CodeSet contains capture and

reporter probes that recognize complimentary sequences (35!50 base pairs) in mRNAs for the

specified genes of interest. Raw gene counts underwent both technical and biological

normalizations, as follows. First, raw counts were normalized to positive control probes, and

then to three housekeeping genes, (hypoxanthine-guanine phosphoribosyltransferase (HPRT1),

succinate dehydrogenase complex subunit A (SDHA) and tyrosine 3-monooxygenase/tryptophan

5-monooxygenase activation protein, zeta polypeptide (YWHAZ). These genes have been

described as good housekeeping genes for stroke studies (Gubern et al., 2009). Expression levels

of the selected genes were compared between treatment groups (ET-1+ saline versus ET-1+ IL-

4) and across time (1, 3, 7 days) within each treatment group. In every case, at every time point,

treatment groups were normalized to saline-only injected animals.

2.4 Immunohistochemistry

For immunohistochemistry, rats were transcardially perfused with 120 mL of phosphate

buffered saline followed by 120 mL of 4% paraformaldehyde. The whole brain was isolated,

placed in 4% PFA solution for 24 hr and cryoprotected by sequential incubation in 10% sucrose

solution for 6 hr and 30% sucrose until the brain sank in the sucrose solution. Brains were

embedded in freezing medium (Dako-Canada, Mississauga, ON), flash frozen in a mixture of dry

ice and 95% ethanol, and stored at -40°c until sectioning. Coronal sections of 16 µm thickness

were cut using a cryostat (Model CM350S, Leica, Richmond Hill, ON), placed onto gelatin-

coated slides (1% gelatin, 0.5% chromium potassium sulfate), and stored at -40°C until

immunohistochemistry analysis. Slides were removed from -40°C, allowed to reach room

temperature and rehydrated in phosphate buffered Triton X-100 solution (PBT; 0.1 mol/L PBS,

24

pH 7.5, 0.1% bovine serum albumin, 0.2% Triton X-100). Following a 2 hr incubation in 10%

donkey serum to prevent non-specific binding, primary antibodies were added to sections

outlined with a Pap pen (Ted Pella, Inc. Redding, CA) and incubated for 24 hr. Antibodies, their

sources and dilutions used are listed in Table 2.1.

Healthy myelin was labeled with a mouse monoclonal antibody against myelin-basic

protein (anti-MBP; 1:100; Sigma Aldrich, Oakville, ON; Hughes et al., 2003, Souza-Rodrigues

et al., 2008, Moxon-Emre and Schlichter, 2010) and damaged myelin basic protein was labeled

with a rabbit polyclonal antibody against degraded myelin basic protein (dMBP; 1:250;

Chemicon, Temecula, CA; Moxon-Emre and Schlichter, 2010). Reactive microglia were

detected using rabbit polyclonal anti-ionized calcium-binding adapter-1 (anti-Iba1; 1:1000;

Wako, Japan; Imai and Kohsaka, 2002) and neutrophils were labeled with an antibody against

rabbit polyclonal polymorphonuclear leukocyte (anti-PMN; 1:10,000; Cedarlane, Burlington,

ON; Weston et al., 2007). Glial scar formation was monitored using mouse monoclonal anti-

nestin (1:250; Chemicon, Temecula, CA; Duggal et al., 1997). Phagocytic activity was assessed

with mouse monoclonal anti-ED1 (1:200; Serotec, Raleigh, NC; Damoiseaux et al., 1994). BBB

breakdown was detected using a mouse monoclonal antibody against IgG (Chemicon, Temecula,

CA), and blood vessels were labeled with a mouse monoclonal antibody against collagen (CgIV,

1:250; San Diego, CA; Moxon-Emre and Schlichter, 2010).

After an overnight incubation in primary antibodies and 3# 15 min washes in PBT,

Dylight™-conjugated secondary antibodies were applied for 2 hr: donkey anti- mouse-488 and

donkey anti-rabbit-594 (1:400; Jackson Immunoresearch, Baltimore, PA). Sections were then

incubated for 10 min in DAPI (4’-6-diamidino-2-phenylindole; 1:5000 Sigma-Aldrich, St. Louis,

MO), followed by washing 3# for 15 min each in PBS. Slides were cover slipped using

mounting medium (Dako-Canada, Mississauga, ON) and stored at -4°C until imaged.

25

Table 2.1. Antibodies used to identify cells, their state and white and grey matter injury.

Antibody Used to Label Concentration Manufactor Iba1 Microglia/macrophages 1:1000 Wako, Japan

nestin Glial scar 1:250 Chemicon, Temecula, CA

IgG BBB breakdown 1:150 Chemicon, Temecula, CA

MBP Myelin 1:100 Sigma Aldrich, Oakville, ON

dMBP Damaged myelin 1:250 Chemicon, Temecula, CA

PMN Neutrophils 1:10,000 Cedarlane, Burlington, ON

ED1 Phagocytic cells 1:200 Serotec, Raleigh, NC

CgIV Blood vessels 1:250 Abbiotec, San Diego, CA

DAPI Nuclei of all cells 1:5000 Sigma Aldrich, Oakville, ON Iba1, ionized calcium-binding adapter-1; IgG, immunoglobulin G; MBP, myelin basic protein; dMBP, damaged myelin basic protein; PMN, polymorphonuclear leukocytke;CgIV, collagen; DAPI, 4'-6-diamidino-2-phenylindole

26

2.5 Fluoro-Jade B histochemistry

To label degenerating neurons in ET-1+ IL-4 treated animals versus ET-1+ saline only

treated animals, tissue sections were stained with a polyanionic fluorescein derivative, Fluoro-

Jade B (Histochem Inc, Jefferson, AR), a commonly used marker for degenerating neurons

(Schmued and Hopkins, 2000). As for immunohistochemistry, frozen sections from 3-4 animals

were selected for each time point (1, 3, 7 days) for each treatment group (ET-1+ saline, ET-1+

IL-4). Slides were immersed in 100% ethanol for 3 min, followed by 1 min immersion in 70%

ethanol and a 1 min rinse in distilled H2O. Then, the slides were immersed in a 0.06% solution of

potassium permanganate, shaken gently for 15 min, followed by a 1 min rinse in distilled H2O.

Slides were placed in a 0.001% Fluoro-Jade solution for 30 min with gentle shaking, and then

rinsed 3# for 1 min each in distilled H2O. Slides were left to dry in a fume hood for 30 min.

After dying, slides immersed in 100% xylene 3# for 2 min each, cover-slipped with DPX

mounting medium (Sigma-Aldrich, St. Louis, MO), and stored at -4°C.

2.6 Sampling procedure and quantification

All labeled tissue (immunohistochemistry and Fluoro-jade B histochemistry) was

examined with either a confocal microscope (Zeiss LSM 700 META, Oberkocken, Germany) or

epi-fluorescent microscope (Axioplan 2, Zeiss, Toronto, ON) with deconvolution capability, and

later quantified using Image J software (Version 1.33K, NIH). To reduce variability, all images

for each antibody were made on the same day, and the contralateral hemisphere brain images

were captured as an additional control. Pinhole and exposure times were determined at the start

of each imaging session and kept constant throughout. Images were captured at 10#, 20# or 40#

magnification depending on the marker examined.

For MBP and dMBP antibodies, the area fraction of staining was computed and averaged

from 5 sampling boxes (300 # 300 µm) in the lesion core of ET-1+ saline and ET-1+ IL-4 treated

27

rats. Where the core of the lesion was not clearly defined (e.g., MBP staining at 1 day after

stroke induction) the approximate location of the lesion was estimated, based on where it was

seen in all rats at 3 days. Using Image J, the same threshold was set across all treatment groups

and time points for all captured images and the area fraction of MBP and dMBP was recorded

for each sample box.

All cell counts were made by an observer blinded to the treatment. Six sampling boxes

(200 # 200 µm) were selected in the lesion core for all stains [microglia/macrophages (Iba1+ve

cells), neutrophils (PMN+ve cells), phagocytic cells (ED1+ve), degenerating neurons (Fluoro-

Jade B+ve cells)] (Fig. 2.1A). Counts for microglia/macrophages and neutrophils were assessed

by identifying cells with the nuclear stain, DAPI. To evaluate the phagocytic nature of

microglia/macrophages or neutrophils, tissue was triple stained with Iba1, ED1 and DAPI or

with PMN, ED1 and DAPI, respectively (see Results, Fig. 3.8A and 3.10A). For all cell-

counting, images were imported into Image J and manually counted.

The glial scar length, labeled with an antibody against nestin, was measured at the

dorsal-, ventral-, lateral- and medial-most portions of the scar. The overall scar length was

determined by averaging lengths from each of the 4 sections of the scar (see Results, Fig. 3.7A).

No scar was detected in the ipsilateral striatum at 1 day post-stroke or in the contralateral

striatum at 1, 3 or 7 days.

2.7 Statistical Analysis

All analyses were carried out using GraphPad Prism 6 statistics software (Version 6.0,

San Diego, CA). All results are reported as the mean ± SEM. Statistical differences between ET-

1+ IL-4 treated rats and ET-1 stroke controls were assessed using a 2-way ANOVA, with time

(1, 3, 7 days) and treatment (ET-1+ saline, ET-1+IL-4) as the independent factors, or using

Student’s t-test, where appropriate. Post-hoc analyses were performed using Bonferoni’s test,

28

with p<0.05 taken as significant.

3. Results

3.1 Evaluating the effects of IL-4 treatment on expression of inflammation-related and alternative activation-related genes in ischemic animals.

The effect of IL-4 treatment on several inflammation-related and alternative activation-related genes was evaluated in ischemic animals. As a baseline, I first examined the effect of ischemia alone on the genes of interest. To study the effect of ischemia, comparisons were made between saline-injected animals (control) and ischemic animals (ET-1+ saline injected). To study the effect of IL-4 treatment in ischemic animals, comparisons were made between ischemic animals (ET-1+ saline injected; ischemic control) and IL-4 treated animals (co-injected with ET-1+ IL-4).

3.1.1 Recombinant IL-4 treatment increased expression of signaling molecules downstream of the IL-4 receptor

There are several signaling molecules involved in the IL-4 signaling (discussed in section

4.3.1). Signal transducer and activator of transcription-6 (STAT6) is the main STAT activated in

the IL-4 pathway and provides a connection between the IL-4 receptor and the cell’s

transcription machinery (Nelms et al., 1999). I first examined the effect of ischemia on STAT6

expression and found a trend toward elevated transcript levels in the ipsilateral ET-1 treated

striatum at 3 and 7 days (Fig. 3.1A). Next, I examined the effect of IL-4 treatment on STAT6

expression. IL-4 treated animals showed a significant increase in STAT6 expression in the

ipsilateral striatum compared to ischemic controls (ET-1+saline injected) at the 7 day time point

(Fig. 3.1B). An additional molecule involved in the IL-4 pathway is PPAR" (Van Dyken and

Locksley, 2013). PPAR" transcripts were elevated at 3 days post-ischemia compared to saline-

injected animals (Fig. 3.1C). Although IL-4 treatment did not significantly alter PPAR"

expression, there was a trend towards increased expression at 1 day (Fig. 3.1D).

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Figure 3.1

Figure 3.1. Recombinant IL-4 treatment increased expression of signaling molecules downstream of the IL-4 receptor. Rats were injected with saline alone, 400 picomoles of endothelin-1 and saline or 500 ng/rat of rat recombinant IL-4, and sacrificed at 1, 3 or 7 days post-stroke. mRNA transcripts from the ipsilateral striatum were measured using Nanostring technology for signal transducer and activator of transcription-6 (STAT6) after ischemia (A) and IL-4 treatment (B), and for peroxisome proliferator-activated receptor gamma (PPAR-") after ischemia (C) and IL-4 treatment (D). Results were normalized to saline-injected animals. n= 6 per time point (1, 3, 7 days) per treatment group (ET-1+ saline versus ET-1+ IL-4), except for saline only treated animals (n=4 per time point). %p<0.05, %%p<0.05, %%% p<0.001 for comparisons between ischemic and saline-injected animals. # p<0.05, ## p<0.01, ### p<0.001 for comparisons between IL-4 treated (ET-1+IL-4) and ischemic controls (ET-1 + saline injected). * p<0.05, ** p<0.01, *** p<0.001 denotes time-dependent changes within a treatment group.

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3.1.2 IL-4 treatment increased expression of alternative activation markers

I assessed the expression levels of several alternative activation related genes (for a more

detailed list, see Table A1). After ischemia, there is a progressive increase in IL-4 expression

from 1-7 days; recombinant IL-4 treatment did not alter IL-4 (data not shown). IL-4 binds to IL-

4r! and stimulates downstream signaling molecules that have anti-inflammatory roles (Nelms et

al., 1999). Ischemia induction significantly increased IL-4R! expression compared to saline-

injected controls at 3 days (Fig. 3.2A). Although not reaching significance, stroke apparently

increased ARG1 expression at 3 days, compared to saline injected animals (Fig. 3.2C). ARG1 is

an enzyme that competes with the pro-inflammatory enzyme, iNOS, for the substrate, L-arginine,

thus decreasing the level of NO (Colton, 2009). An additional marker of alternative activation is

the haptoglobin-hemoglobin scavenger receptor, CD163, which contributes to removal of

hemoglobin from the blood (Akila et al., 2012). CD163 transcripts were apparently increased

after stroke at 1 and 3 days but did not reach significance for the small number of animals used

(Fig. 3.3A). Expression levels of the chemokine, CCL22, were significantly elevated 3 days after

stroke (Fig. 3.3C). CCL22 is up-regulated in rat macrophages after IL-4 treatment in vitro

(Jaguin et al., 2013). IL-4 treatment significantly increased IL-4r! and ARG1 expression

(compared to ischemic controls) at 7 and 3 days, respectively (Fig. 3.2B, D). Most striking were

the dramatic increases in CD163 and CCL22 transcript levels at 1 day (Fig. 3.3B, D).

3.1.3 IL-4 treatment did not significantly alter expression of pro-inflammatory markers

IL-4 treatment decreases expression of IFN"-induced pro-inflammatory mediators in

vitro; one of the ways it is postulated to exert anti-inflammatory effects (Donnelly et al., 1990).

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Figure 3.2

Figure 3.2. Recombinant IL-4 treatment increased expression of the alternative activation or anti-inflammatory markers, IL-4r" and ARG1. Rats were injected with saline alone, 400 picomoles of endothelin-1 and saline or 500 ng/rat of rat recombinant IL-4, and sacrificed at 1, 3 or 7 days post-stroke. mRNA transcripts from the ipsilateral striatum were measured using Nanostring technology for arginase 1 (ARG1) after ischemia (A) and IL-4treatment (B), and for the interleukin-4 receptor alpha chain (IL-4r!) after ischemia (C) and IL-4 treatment (D). Results were normalized to saline-injected animals. n= 6 per time point (1, 3, 7 days), per treatment group (ET-1+ saline versus ET-1+ IL-4), except for saline only treated animals (n=4 per time point). %p<0.05, %%p<0.05, %%% p<0.001 for comparisons between ischemic and saline-injected animals. # p<0.05, ## p<0.01, ### p<0.001 for comparisons between IL-4 treated (ET-1+IL-4) and ischemic controls (ET-1 + saline injected). * p<0.05, ** p<0.01, *** p<0.001 denotes time-dependent changes within a treatment group.

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Figure 3.3

Figure 3.3. Recombinant IL-4 treatment increased expression of alternative activation markers CD163 and CCL22. Rats were injected with saline alone, 400 picomoles of endothelin-1 and saline or 500 ng/rat of rat recombinant IL-4 and sacrificed at 1, 3 or 7 days post-stroke. mRNA transcripts from the ipsilateral striatum were measured using Nanostring technology for the haptoglobin-hemoglobin scavenger receptor (CD163) after ischemia (A) and IL-4treatment (B), and for the chemokine (C-C motif) ligand 22 (CCL22) after ischemia (C) and IL-4 treatment (D). Results were normalized to saline-injected animals. n= 6 per time point (1, 3, 7 days), per treatment group (ET-1+ saline versus ET-1+ IL-4), except for saline only treated animals (n=4 per time point). %p<0.05, %%p<0.05, %%% p<0.001 for comparisons between ischemic and saline-injected animals. # p<0.05, ## p<0.01, ### p<0.001 for comparisons between IL-4 treated (ET-1+IL-4) and ischemic controls (ET-1 + saline injected). * p<0.05, ** p<0.01, *** p<0.001 denotes time-dependent changes within a treatment group.

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I examined the expression of three classic pro-inflammatory molecules: IL-1$), iNOS (Fig.

3.4C), and TNF! (Fig. 3.4E) at 1, 3 and 7 days following transient ischemia. While expression

levels of IL-1$, iNOS and TNF! were not significantly increased after ischemia, there was a

trend toward increased expression at each time point compared to saline-injected controls (Fig.

3.4A, C, E). In addition, IL-4 treatment did not significantly affect expression of IL-1$, iNOS or

TNF! at any time (Fig. 3.4B, D, F). However, a trend worth noting was the level of IL-1$, iNOS

and TNF! expression in IL-4 treated animals at 1 day, which was apparently closer to saline-

injected animals than ischemic controls (Fig. 3.4B, D, F). Additionally, over time there was a

trend towards elevated IL-1$, iNOS and TNF! levels in IL-4 treated animals (Fig. 3.4B, D, F).

This suggests that IL-4 treatment at stroke induction might affect expression of pro-inflammatory

molecules at later times. Unfortunately, the study was limited by the small number of animals

used at each time point for the very costly NanoString analysis.

3.1.4 IL-4 treatment altered expression of CD200 but not CD200R, which are markers of neuron-glial interactions

Interaction between the ligand CD200, found on neurons, and its receptor CD200R, found

on microglia, is thought to exert immunosuppressive effects (Koning et al., 2009). CD200

expression levels did not change after ischemia (ET-1 injection), compared to saline-injected

animals (Fig. 3.5A). However, CD200R expression increased at 3 days post-ischemia (Fig.

3.5C). IL-4 treatment significantly decreased CD200 expression at 1 day compared to ischemic

controls (Fig.3.5C), but did not alter CD200R transcript levels at any time point (Fig. 3.5D).

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Figure 3.4

Figure 3.4. Recombinant IL-4 treatment did not significantly alter expression of pro-inflammatory markers. Rats were injected with saline alone, 400 picomoles of endothelin-1 and saline or 500 ng/rat of rat recombinant IL-4, and sacrificed at 1, 3 or 7 days post-stroke. mRNA transcripts from the ipsilateral striatum were measured using Nanostring technology for interleukin-1 beta (IL-1$) after ischemia (A) and IL-4 treatment (B), tumor necrosis factor alpha (TNF!) after ischemia (C) and IL-4 treatment (D) and for inducible nitric oxide synthase (iNOS) after ischemia (E) and IL-4 treatment (F). Results were normalized to saline-injected animals. n= 6 per time point (1, 3 and 7 days), per treatment group (ET-1+ saline versus ET-1+ IL-4) with the exception of saline only treated animals (n=4 per time point). %p<0.05, %%p<0.05, %%% p<0.001 for comparisons between ischemic and saline-injected animals. # p<0.05, ## p<0.01, ### p<0.001 for comparisons between IL-4 treated (ET-1+IL-4) and ischemic controls (ET-1 + saline injected). * p<0.05, ** p<0.01, *** p<0.001 denotes time-dependent changes within a treatment group

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Figure 3.5

Figure 3.5. Recombinant IL-4 treatment altered the expression of CD200, but not its receptor. Rats were injected with saline alone, 400 picomoles of endothelin-1 and saline or 500 ng/rat of rat recombinant IL-4 and sacrificed at 1, 3 or 7 days post-stroke. mRNA transcripts from the ipsilateral striatum were measured using Nanostring technology for CD200 after ischemia (A) and IL-4treatment (B) and for CD200 receptor (CD200R) after ischemia (C) and IL-4 treatment (D). Results were normalized to saline-injected animals. n= 6 per time point (1, 3 and 7 days), per treatment group (ET-1+ saline versus ET-1+ IL-4) with the exception of saline only treated animals (n=4 per time point). %p<0.05, %%p<.05, %%% p<0.001 for comparisons between ischemic and saline-injected animals. # p<0.05, ## p<0.01, ### p<0.001 for comparisons between IL-4 treated (ET-1+IL-4) and ischemic controls (ET-1 + saline injected). * p<0.05, ** p<0.01, *** p<0.001 denotes time-dependent changes within a treatment group.

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3.2 Monitoring changes in tissue

For immunohistochemistry analyses, I evaluated only the effect of IL-4 treatment (co-injection of ET-1+ IL-4) after ischemia compared to ischemia alone (ET-1+saline injected/ischemic controls). Saline-injected animals were omitted from this portion of the analysis because our lab has already shown that the contralateral brain region is a sufficient control (Moxon-Emre and Schlichter, 2010).

3.2.1 ET-1+ saline and IL-4 treated animals showed BBB leakiness

A recent report stated that the BBB is not breached after ET-1-induced stroke in rats

(Sozmen et al., 2012). To evaluate BBB leakiness, I stained for IgG, which is normally confined

to circulating plasma, but is found in the brain parenchyma when the BBB is compromised (Ruth

and Feinerman, 1988). As shown in Fig. 3.6, both ET-1+ saline and ET-1+ IL-4 treated animals

showed pronounced staining for IgG (green) in blood vessels (stained for collagen IV, red) but

also in cells and in the extracellular space outside blood vessels at, 1 day. There was no obvious

difference between ET-1+saline and ET-1+IL-4 treated animals. In both treatment groups, IgG

staining (green) was restricted to the ipsilateral striatum, and absent in the corresponding

contralateral striatum (Fig. 3.6).

3.2.2 IL-4 treatment did not alter the presence of the glial scar or its thickness

Glial scar formation after stroke is argued to be beneficial by some and detrimental by

others. Although the glial scar is thought to act as a barrier protecting healthy tissue from

harmful substances released from damaged cells (Anderson and Nedergaard, 2003), it can also

prevent axonal regrowth (Yiu and He, 2006). I found that a glial scar, identified using an

antibody against nestin (red; Fig. 3.7) develops with time around the ET-1 lesion core, consistent

with our lab’s earlier study (Moxon-Emre and Schlichter, 2010). Not surprisingly, no scar

formation was seen in the contralateral striatum at 1, 3 or 7 days post-stroke (results not shown).

The scar was not yet formed in the ipsilateral striatum at 1 day; however, by 3 days, a glial scar

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Figure 3.6

Figure 3.6. ET-1+ saline and IL-4 treated animals showed signs of BBB leakiness. Animals were injected with 400 picomoles of endothelin-1 and either saline or 500 ng/rat of rat recombinant IL-4. Brains were isolated, sectioned and examined using immunohistochemistry. BBB leakiness was evaluated using an antibody against the blood protein, IgG (mouse monoclonal anti-IgG, green), which is not found in the healthy CNS. A rabbit polyclonal antibody against collagen IV (red) was used to label microvessels. Representative images of IgG in the ET-1+ saline treated animals (left panel) and ET-1+ IL-4 treated animals (right panel). IgG staining was restricted to the ipsilateral brain regions in both treatment groups. Scale bar = 100 µm.

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Figure 3.7

Figure 3.7. Recombinant IL-4 treatment did not alter the presence or thickness of the glial scar. Animals were injected with 400 picomoles of endothelin-1 and either saline or 500 ng/rat of rat recombinant IL-4. Brains were isolated, frozen, cryo-sectioned, and examined using immunohistochemistry. The glial scar was visualized using a mouse monoclonal antibody against nestin (red). Scar length (µm) was measured at the dorsal-, medial-, lateral- and ventral-most portions of the scar (marked a, b, c, d) at 3 and 7 days post-stroke (A). Overall summary of the average scar length in ET-+ IL-4 treated and ischemic controls at 3 and 7 days post-stroke, at the dorsal- (C), medial- (D) lateral- (E) or ventral-(F) most portions of the scar. n=3-4 per time point, per treatment group (ET-1+ saline vs. ET-1+ IL-4). Scale bar = 100 µm.

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clearly defined the edge of the lesion (Fig. 3.7A), and was even more pronounced at 7 days. I

noticed that the scar thickness varied depending on what portion was analyzed, and the shape

varied between animals within the same treatment group. Therefore, I measured the thickness at

the dorsal, ventral, lateral and medial portions of the scar in IL-4 treated and ischemic animals.

Recombinant IL-4 treatment did not affect the timing of appearance of the glial scar or its

thickness at each portion measured (Fig. 3.7B-E).

3.2.3 IL-4 treatment increased phagocytosis and the number of neutrophils, but not microglia/macrophages, in the ischemic core

Following stroke, circulating neutrophils and monocyte-derived macrophages enter the

CNS where, together with the brain’s innate immune cells, they respond to injury. Although

microglia/macrophages exert potentially harmful effects (Lai and Todd, 2006), they also have

potentially beneficial roles, such as phagocytosing cellular debris (Patel et al., 2013). Previously,

our lab (2010) and others (Hughes et al., 2003, Souza-Rodrigues et al., 2008), evaluated the

number of microglia/macrophages that infiltrated the lesion core. The number of

microglia/macrophages (Iba1+ve cells) progressively increased after ET-1 induced stroke,

completely infiltrating the lesion core by 7 days (Moxon-Emre and Schlichter, 2010). I evaluated

the effect of IL-4 treatment on microglia/macrophage infiltration into the ischemic core at 1,3

and 7 days. The number of Iba1+ve/DAPI+ve cells (Fig. 3.8A; examples shown by solid arrows)

was counted from 6 different sample boxes selected from regions spanning the lesion core

(Figure 2.1A). Compared to ischemic controls, IL-4 treatment did not significantly alter the

number of microglia/macrophages in the lesion core at any time examined (Fig. 3.8B, C).

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Figure 3.8

Figure 3.8. Recombinant IL-4 treatment did not alter the number of phagocytic microglia/macrophages in the lesion core. Animals were injected with 400 picomoles of endothelin-1 and either saline or 500 ng/rat of rat recombinant IL-4. Brains were isolated, frozen, cryo-sectioned, and examined using immunohistochemistry. Microglia/macrophages were labeled with a rabbit polyclonal antibody against Iba1 (red). Phagocytic activity was evaluated using a mouse monoclonal antibody against the lysosomal marker, ED1 (green). The number of microglia/macrophages in the lesion core was evaluated by counting Iba1+ve cells in the lesion core (solid arrow), while blinded to the treatment. The phagocytic activity of microglia/macrophages was examined by double staining for microglia/macrophages (Iba1) and ED1 (hollow arrows)(A). Representative images of phagocytic microglia/macrophages in the lesion core of ET-1+ saline (left panel) and ET-1 and IL-4 treated (right panel) animals at 1, 3 and 7 days post-stroke (B). Quantitative analysis of Iba1+ve microglia/macrophages (C) and phagocytic Iba1+/ED1+ve microglia/macrophages (D) counted from 6 sampling regions (200 # 200 µm) per animal spanning the lesion core. n=3-4 animals/per time point (1, 3, 7 days), per treatment group . # p<0.05, ## p<0.01, ### p<0.001 for comparisons between IL-4 treated (ET-1+IL-4) and ischemic controls (ET-1 + saline injected). * p<0.05, ** p<0.01, *** p<0.001 denotes time-dependent changes within a treatment group. Scale bars = 20 µm (A) and 100 µm (B).

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2.1A). Compared to ischemic controls, IL-4 treatment did not significantly alter the number of

microglia/macrophages in the lesion core at any time examined (Fig. 3.8B, C).

Next, to evaluate the phagocytic state of microglia/macrophages, I triple stained tissue

with DAPI (to identify individual cells), Iba1 (to identify microglia/macrophages), and the

lysosomal marker, ED1. ED1 has been extensively used as a marker of phagocytosis, and is up-

regulated in phagocytic macrophages (Damoiseaux et al., 1994). Thus, I counted the number of

Iba1+ve/ED1+ve/DAPI+ve cells as an indicator of the number of phagocytic

microglia/macrophages (Fig. 3.8A; examples shown by hollow arrows). In both the IL-4 treated

animals and ischemic controls, the number of Iba1+ve/ED1+ve cells increased progressively

from 1 to 7 days, but did not significantly differ between the two groups at any time (Fig. 3.8B,

D). Interestingly, I noticed a large number of ED1+ve cells at 1 day that were Iba1-ve (Fig. 3.9A;

solid arrows), especially in IL-4 treated animals. In ischemic controls there were apparently

fewer ED1+ve/Iba1-ve cells, and most ED1+ve cells were microglia/macrophages (Iba1+ve)

(Fig. 3.9A; examples shown by hollow arrows). Quantitative analysis revealed significantly more

phagocytic cells (ED1+ve/DAPI+ve; Fig3.9A; hollow arrows) that were not

microglia/macrophages (i.e., Iba1-ve) in IL-4 treated animals compared to ischemic controls

(Fig. 3.9B). Because they were not microglia/macrophages, I next asked whether the

ED1+ve/DAPI+ve/Iba1-ve cells were neutrophils.

Neutrophils have been found to infiltrate the CNS by 1 day after stroke in the ET-1

model, and to decrease substantially by 3 days. A similar time course was found for

ED1+ve/Iba1-ve cells; thus, I predicted these cells might be neutrophils. To begin, I examined

the effect of IL-4 on the number of neutrophils in the lesion core by double staining with DAPI

and an antibody against polymorphonuclear leukocytes (anti-PMN). IL-4 treatment significantly

increased the number of neutrophils (PMN+ve/DAPI+ve cells) compared to ischemic

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Figure 3.9

Figure 3.9. Recombinant IL-4 treatment increased the phagocytic activity in cells other than microglia/macrophages at 1 day. Animals were injected with 400 picomoles of endothelin-1 and either saline or 500 ng/rat of rat recombinant IL-4. Brains were isolated, frozen, cryo-sectioned, and examined using immunohistochemistry. Phagocytic cells were labeled with a mouse monoclonal antibody against the lysosomal marker, ED1 (green) and microglia/macrophages were labeled with a rabbit polyclonal antibody against Iba1 (red). To evaluate differences in phagocytic activity of cells other than microglia/macrophages, the number of ED1+ve/Iba1-ve cells was counted (solid arrows), while blinded to the treatment. Representative ipsilateral images demonstrating that most ED1+ve cells were also Iba1+ve in ischemic controls (A; top panel, hollow arrows) compared to IL-4 treated animals, in which the number of ED1+/Iba1-ve cells was greater (A; bottom panel, solid arrows). Quantitative analyses of ED1+ve/Iba1-ve cells counted from 6 sampling regions (200 # 200 µm) per animal spanning the lesion core of ET-1+saline and ET-1+IL-4 treated animals (B). n=3-4 animals/per time point (1, 3, 7 days), per treatment group. # p<0.05, ## p<0.01, ### p<0.001 for comparisons between IL-4 treated (ET-1+IL-4) and ischemic controls (ET-1 + saline injected). * p<0.05, ** p<0.01, *** p<0.001 denotes time-dependent changes within a treatment group. Scale bar = 20 µm.

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controls at 1 day after stroke (Fig. 3.10A, B). Next, I triple stained tissue for PMN, ED1 and

DAPI. I considered cells labeled with all three markers (PMN+ve/ED1+ve/DAPI+ve) to be

phagocytic neutrophils. Most of the PMN+ve cells in IL-4 treated animals were also ED1+ve,

suggesting that most neutrophils were phagocytic, and the number of phagocytic neutrophils

(PMN+ve/ED1+ve/DAPI+ve) was significantly higher than in ischemic controls (Fig. 3.10C).

3.2.4 IL-4 treatment increased grey matter injury after ischemia

Grey matter injury after stroke has been extensively characterized, especially when

compared to the number of studies in which white matter damage is the main focus. Fluoro-Jade

B is a stain extensively used to evaluate grey matter injury, specifically by labeling the number

of degenerating neurons (Schmued and Hopkins, 2000, Schmuck and Kahl, 2009). Neuronal

damage after stroke has been reported to be highest at 1 day after MCAo (Liu et al., 2009). Using

Fluoro-Jade B staining, I evaluated the effect of IL-4 treatment on neuronal degeneration.

Fluoro-Jade B positive cells were counted in the ipsilateral striata only because none were

detected in the contralateral striatum of either treatment group (Fig. 3.11). After ischemia,

Fluoro-Jade B staining was highest at 1 day and progressively decreased thereafter, with few, if

any, stained cells remaining by 7 days (Fig. 3.11; left panel). A similar time course of

neurodegeneration occurred in the IL-4 treated animals (Fig. 3.11; right panel); however, a

25.6% increase in the number of degenerating neurons was detected at 1 day (Fig. 3.11B). Thus,

IL-4 treatment apparently increased grey matter injury.

3.2.5 IL-4 treatment did not alter the extent of white matter injury

Although white matter was traditionally thought to be less susceptible to ischemia

(Marcoux et al., 1982), it is now widely accepted that both white and grey matter are damaged

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Figure 3.10

Figure 3.10. Recombinant IL-4 treatment increased the number of phagocytic neutrophils in the ischemic core at 1 day. Animals were injected with 400 picomoles of endothelin-1 and either saline or 500 ng/rat of rat recombinant IL-4. Brains were isolated, frozen, cryo-sectioned, and examined using immunohistochemistry. Neutrophils were labeled with a rabbit polyclonal antibody against polymorphonuclear leukocytes (PMN; red; solid arrow) and phagocytic cells were labeled with a mouse monoclonal antibody against the lysosomal marker, ED1 (green). Phagocytic activity of neutrophils was evaluated by counting PMN+ve/ED1+ve (hollow arrow). Representative images of phagocytic neutrophils (PMN+ve/ED1+ve cells) in ET-1+saline treated (top panel) and ET-1+ IL-4 treated (bottom panel) animals, 1 day after ischemia induction (A). Quantitative analyses of PMN+ve neutrophils (B) and phagocytic PMN+ve/ED1+ve neutrophils (C) counted from 6 sampling regions (200 # 200 µm) per animal spanning the lesion core. n=3-4 animals per treatment group (ET-1+saline vs. ET-1+IL-4). ** p<0.01 compared to ET-1 + saline ischemic controls. Scale bar = 20 µm.

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Figure 3.11

Figure 3.11. Recombinant IL-4 treatment increased grey matter injury, judged by neuronal degeneration after ischemia. Animals were injected with 400 picomoles of endothelin-1 and either saline or 500 ng/rat of rat recombinant IL-4. Brains were isolated, sectioned and examined using immunohistochemistry. Fluoro-Jade B was used to identify degenerating neurons. Representative image of Fluoro-Jade B+ve cells in the ET-1+saline treated animals (left panel) and ET-1+ IL-4 treated animals (right panel) 1, 3, and 7 days after ischemia induction (A). Quantitative analysis demonstrating a significant increase in the number of Fluoro-Jade B +ve cells in ET-1+IL-4 counted from 6 sampling regions (200 # 200 µm) per animal spanning the lesion core (B). n=3-4 animals/per time point (1, 3, 7 days), per treatment group. # p<0.05, ## p<0.01, ### p<0.001 for comparisons between IL-4 treated (ET-1+IL-4) and ischemic controls (ET-1 + saline injected). * p<0.05, ** p<0.01, *** p<0.001 denotes time-dependent changes within a treatment group. Scale bar = 100 µm.

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after stroke (Goldberg and Ransom, 2003). Damage to white matter in the ET-1 stroke model can

be characterized by the loss of normal myelin, indicated by decreased myelin basic protein

(MBP) staining or by increased degraded MBP (dMBP) staining (Moxon-Emre and Schlichter,

2010). Area fraction measurements (% of area staining positively for MBP or dMBP) were made

in frozen sections from ET-1+ saline and ET-1+IL-4 treated animals at 1, 3 and 7 days. Under

both conditions, MBP staining (green) in the lesion core was prominent at 1 day, began to

decline by 3 days, and was undetectable by 7 days (Fig. 3.12A). Concurrent with the decline in

MBP, an increase in dMBP (red) occurred over time, filling the lesion core by 7 days in both

treatment groups (Fig. 3.12A). Therefore, by these criteria, recombinant IL-4 treatment did not

alter the extent of myelin damage that occurs during the first week after transient ischemia.

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Figure 3.12

Figure 3.12. Recombinant IL-4 treatment did not alter white matter injury, judged by myelin staining in the ischemic core. Animals were injected with 400 picomoles of endothelin-1 and either saline or 500 ng/rat of rat recombinant IL-4. Brains were isolated, sectioned and examined using immunohistochemistry. White matter injury was evaluated using a mouse monoclonal antibody against healthy myelin basic protein (green) and a rabbit polyclonal antibody against degraded MBP (red). A. Representative image of MBP and dMBP staining in ET-1+ saline (left panel) and ET-1 + IL-4 treated (right panel) animals at 1, 3 and 7 days post-stroke. Quantitative analysis of MBP staining (B) and dMBP staining (C) evaluated by measuring area fraction of positive staining from 5 sampling boxes (300 # 300 µm) per animal. n=3-4 per time point (1, 3, 7 days), per treatment group (ET-1+ saline vs. ET-1+ IL-4). * p<0.05, ** p<0.01, *** p<0.001 denotes changes within a treatment group across time. Scale bar = 100 µm.

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4. Discussion

4.1 Overview

This study aimed to evaluate white and grey matter injury in ischemic rats after altering

the activation state of microglia/macrophages. First, I examined changes in expression of 28

alternative activation or inflammation related genes after stroke induction with or without a co-

injection of recombinant IL-4. Subsequently, I evaluated the degree of neuronal degeneration and

damage to white matter, glial scar formation, immune cell infiltration and phagocytic in IL-4

treated animals. The immune response following stroke is complex. This project begins to

delineate changes in outcomes post-stroke, after attempting to alter the inflammatory state of the

brain.

4.2 Endothelin-1 stroke model

Few studies have explored the intersection between inflammation, white and grey matter

damage, and transient ischemia. When selecting an animal model for studying inflammation

attention must be given to sources of unnecessary inflammation due to surgical procedures (such

as those that accompany cannula implantation or a craniectomy). Furthermore, a needle poke

into the brain is enough to induce an inflammatory response, characterized by activation of

microglia/macrophages (Moxon-Emre and Schlichter, 2010). Thus, for the purposes of the

current study, selecting a minimally invasive model better allows for examining changes in

inflammation not induced by the model itself. The ET-1 model does not require craniectomy or

cannula implantation, and produces reproducible lesions (Sozmen et al., 2012). Whereas the

commonly used MCAo model produces lesions that often vary in size and location (Liu and

McCullough, 2011). There is evidence that the ET-1 model does disrupt the BBB (Hughes et al.,

2003) and also evidence that it does not (Souza-Rodrigues et al., 2008). Furthermore there has

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been controversy over whether or not neutrophils infiltrate the CNS after ET-1 stroke induction,

some studies reported their presence (Moxon-Emre and Schlichter, 2010) and others their

absence (Hughes et al., 2003). To address these parameters, I examined both BBB leakiness

(discussed in section 4.3.5) and neutrophil infiltration (discussed in section 4.3.8) after ET-1

injection into the striatum; and found the BBB is comprised and neutrophils do infiltrate. The

ET-1 model in mice has not been nearly as promising as in rat (see section 1.3.3.1), which limits

the possibility for future experiments using transgenic mice.

4.3 Salient Findings

4.3.1 Recombinant IL-4 treatment increased expression of downstream signaling molecules of IL-4 activation

Cytokines are polypeptides that act on several different cell types in the brain (Planas et

al., 2006). Most often, cytokines act on membrane receptors, activating a myriad of intracellular

signaling pathways and inducing changes in gene transcription. In a healthy physiological state,

cytokine expression is typically low in the brain, increasing after injury, such as stroke (Planas et

al., 2006). The cytokine I used in an attempt to increase the alternative activation state of

microglia/macrophages in vivo, is IL-4. IL-4 binds to the IL-4r! chain of the IL-4 receptor

complex, causing heterodimerization with either the "c chain or IL-13R! (Nelms et al., 1999).

The heterodimerization recruits tyrosine kinases, which then go on to phosphorylate substrates

and initiate further signaling cascades. Signal Transducer and Activator of Transcription 6

(STAT6) is the primary STAT that binds to the phosphorylated IL-4 receptor, later translocating

to the nucleus to induce changes in gene expression (Nelms et al., 1999).

STAT6 expression was increased after MCAo in rats (Sun et al., 2007) and after IL-4

treatment in microglia/macrophages in vitro (Sica and Mantovani, 2012). Based on these

50

findings, I hypothesized STAT6 expression would be elevated after ischemia. Consistent with

my hypothesis and a previous MCAo study (Sun et al., 2007), STAT6 expression (although not

reaching the level of significance) was apparently increased in ischemic animals at 3 and 7 days.

This is not a surprising finding as IL-4 expression is elevated at later time points after stroke

(Lively and Schlichter, 2012), thus I would predict at later time points (3 or 7 days), downstream

STAT6 would demonstrate a similar pattern of increased expression.

Given STAT6 is downstream of IL-4, I predicted that IL-4 treated animals would have

enhanced STAT6 expression compared to ischemic controls. In accordance with my hypothesis,

IL-4 treated animals showed increased expression of STAT6 at 7 days. Thus, suggesting, the

injected recombinant IL-4 protein was sufficient to stimulate IL-4 pathways, therefore,

enhancing downstream signaling molecules. This finding was further supported by exploring

expression of an additional downstream molecule in the IL-4 pathway, peroxisome proliferator-

activated receptor gamma (PPAR").

PPAR" is a nuclear receptor that heterodimerizes with retinoid X receptors, which can

then modulate transcription of several genes (Van Dyken and Locksley, 2013). IL-4 treatment in

vitro strongly increases PPAR" expression in rat macrophages (Huang et al., 1999). PPAR" has

been a large focus of research in Multiple Sclerosis (MS), after Niino and colleagues (2001)

demonstrated administration of a PPAR" agonist hindered the development of experimental

autoimmune encephalomyelitis (EAE). In the non-ischemic rat brain, PPAR" levels are found to

be very low (Victor et al., 2006), however, in a rat MCAo model of ischemia, PPAR" expression

increased significantly at 1 day (Victor et al., 2006). In accordance with the literature, I

hypothesized that ischemia would increase PPAR" expression. I found a significant increase in

PPAR" expression 3 days after ischemia. As predicted with the increase in STAT6 expression

51

after ischemia, elevated PPAR" at 3 days is likely a downstream effect of enhanced endogenous

IL-4 (seen typically at later time points after stroke (Lively and Schlichter, 2012).

Based on the in vitro studies in which IL-4 stimulation increased PPAR" expression

(Huang et al., 1999); I predicted that in vivo administration of IL-4 would increase PPAR"

expression in ischemic animals. Although not reaching the level of significance for the small

number of animals used, I found a trend towards increased PPAR" expression in IL-4 treated

animals at 1 day. One possibility is that the increase in PPAR" expression is restricted to the 1-

day time point because the injected dose of recombinant IL-4 was not sufficient to maintain

stimulation of the IL-4 pathway, and thus, elevation of downstream molecules such as PPAR", at

later time points. This however, is unlikely as changes in STAT6 expression were found 7 days

after ischemia in IL-4 treated animals. It is not known whether the changes found at 7 days (e.g.,

increased STAT6 expression and IL-4r! expression (discussed in section 4.3.2.1)) are the result

of recombinant IL-4 that is still present in the brain at 7 days, or are the result of recombinant IL-

4 acting at the time of ischemia induction, and producing a cascade of changes in the brain with

prolonged effects (seen at later time points).

4.3.2 IL-4 treatment increased expression of alternative activation genes

4.3.2.1 IL-4r!

IL-4r! is a chain of the IL-4 receptor complex whose expression is up-regulated after

stroke induction in a rat model of intracerebral hemorrhage (Lively and Schlichter, 2012). I

predicted that IL-4r! expression would increase in the ET-1 stroke model (compared to saline

controls) and that recombinant IL-4 treatment would further enhance IL-4r! expression.

Consistent with my hypotheses, IL-4r! expression was elevated in ET-1 injected animals

compared to saline controls (3 and 7 days). Similar to the downstream signaling molecules,

52

STAT6 and PPAR", IL-4r! expression was increased at later time points, which coincides with

increased endogenous IL-4 expression after stroke. I found a significant up-regulation in IL-4r!

mRNA at 7 days in IL-4 treated ischemic rats. As IL-4r! is used as a marker of alternative

activation (Colton, 2009, Liu et al., 2012), this finding provides evidence that recombinant IL-4

treatment enhanced alternative activation.

IL-4r! is found not only on microglia/macrophages in the rat brain, but also on astrocytes

(Brodie et al., 1998) and on human neutrophils (Girard et al., 1997). Thus, we cannot attribute all

findings associated with IL-4 treatment solely to the effect of IL-4 on microglia/macrophages; as

some effects might be mediated by astrocytes and/or neutrophils. In the future, it would be of

interest to determine which cells are responsible for the increase in IL-4r! expression. Future

studies should exploit immunohistochemistry analyses, staining for IL-4r!. If an effective

antibody is found, double stained tissue with an antibody against IL-4r! co-localized with either

an antibody against microglia/macrophages (Iba1), or astrocytes (nestin or GFAP), or neutrophils

(PMN) would allow one to attribute the increase in IL4r! expression to a certain cell type.

4.3.2.2 ARG1

The enzyme, arginase, has been shown to play a role in the immune response, certain

pathological infections and tumor suppression (Munder, 2009). There are two arginase isoforms,

arginase 1 (ARG1) and arginase 2 (ARG2). ARG2 is expressed in several peripheral tissues and

at lower levels in the brain; whereas, ARG1 is robustly expressed in the brain (Colton, 2009).

Arginase (1 and 2, varying in subcellular locations) convert L-arginine to L-ornithine.

Downstream effects of L-ornithine production include enhanced cell proliferation and decreased

cytotoxicity (Munder, 2009). Importantly, arginase competes with iNOS for the substrate, L-

arginine; thus, ARG1 decreases the availability of L-arginine for NO production (Munder, 2009).

53

A microarray study showed a dramatic increase in ARG1 expression following induction of ICH

in rats (Tang et al., 2002). Similar results were found using qRT-PCR analysis after ICH in rats

(Lively and Schlichter, 2012). Patients having suffered from ischemic stroke also demonstrated

increased levels of ARG1 expression in the blood (Tang et al., 2006, Barr et al., 2010). Based on

findings in the literature, I hypothesized that ARG1 expression would increase after ischemia. I

found a trend towards elevated expression of ARG1 at the 3-day time point. An increase in

ARG1 expression after stroke is likely to counteract the prominent increase in iNOS also found

at 3 days after ischemia (discussed in section 4.3.3).

ARG1 is a widely accepted marker of alternative activation (Colton, 2009). In vitro, IL-4

stimulation increased ARG1 expression in microglia and macrophages (Gordon, 2003, Colton,

2009, Martinez et al., 2009). I predicted that IL-4 treatment would increase ARG1 expression in

ischemic animals. In support of my hypothesis, I found a dramatic increase in ARG1 expression

in IL-4 treated animals at 3 days. IL-4 treated animals also showed a trend toward increased

expression of iNOS (compared to ischemic controls; discussed in section 4.3.3). Thus, the

increase in ARG1 at 3 days could be an attempt to dampen the pro-inflammatory NO production.

ARG1 is found in activated microglia/macrophages and astrocytes (Quirie et al., 2013). It is

unlikely that the increase in ARG1 expression in IL-4 treated animals is due solely to an increase

in the number of activated microglia/macrophages or astrocytes. That is, my analyses show that

IL-4 treatment did not alter the number of microglia/macrophages in the ischemic core or the

appearance of the glial scar (comprised of reactive astrocytes; discussed in section 4.3.7 and

4.3.6). ARG1 is found in human neutrophils (Rotondo et al., 2011) and my results show an

increase in number of neutrophils after IL-4 treatment (discussed in section 4.3.8). Thus, the

increase in ARG1 expression could be due to increased neutrophil infiltration. A recent study

examining ARG1 staining in an ischemic lesion, showed an increase in expression in 30% of

54

activated rat microglia/macrophages (Quirie et al., 2013). One explanation for their findings is

that 30% of the cells (ie., ARG1+ve) were alternatively activated. Similarly, in the current study,

it is possible that IL-4 treated animals have an increased number of microglia/macrophages

expressing ARG1 in the lesion core. If so, this could be the result of an increase in the number of

alternatively activated microglia/macrophages (but not the overall number of

microglia/macrophages present in the lesion core). To explore this possibility in the future, one

could examine the number of activated microglia/macrophages (Iba1+vecells) that are also

ARG1+ve (for further discussion of double-immunostaining for alternative activation markers;

see section 4.3.2.4).

4.3.2.3 CD163

Hemoglobin is the oxygen carrier protein that is preferentially cleared from the body via

extravascular hemolysis, a process in which red blood cells are phagocytosed by macrophages

(Akila et al., 2012). During pathological conditions such as stroke, hemoglobin is released into

the circulation and binds with a serum glycoprotein, haptoglobin (Akila et al., 2012). The

hemoglobin-haptoglobin complex then binds with high affinity to the CD163 receptor, also

known as hemoglobin scavenger receptor. CD163 is found on microglia and macrophages (and

other cells of the monocyte lineage; Guillemin and Brew, 2004); however, most of the literature

focuses on macrophages. In vitro studies provide strong evidence for an anti-inflammatory role

for CD163. One study, examining the effect of pro-inflammatory stimuli, such as IFN-", TNF!

and LPS on macrophages demonstrated decreased CD163 expression (Akila et al., 2012);

whereas, anti-inflammatory stimuli induced its expression (Buechler et al., 2000). Furthermore,

macrophages treated with a hemoglobin-haptoglobin complex showed increased CD163

expression, and suppression of the LPS-induced phenotype (Zhang et al., 2012). CD163 is highly

55

expressed on macrophages during the wound-healing phase (Zwadlo et al., 1987) and acts as a

receptor for TNF-like weak inducer of apoptosis (TWEAK). Binding of TWEAK to CD163 on

macrophages leads to internalization of TWEAK and its degradation (Bover et al., 2007). In

studies staining healthy human tissue, CD163+ cells were absent, however a strong accumulation

of CD163+ cells was found in MS lesions. Interestingly, CD163+ve cells were not found in all

MS lesions, but were restricted to active lesions and absent in inactive lesions. The authors

speculate that CD163+ve microglia/macrophages might play an anti-inflammatory/resolution

role in the more severe stages of MS (Zhang et al., 2012). Additional evidence for an anti-

inflammatory role of CD163 comes from studies on traumatic brain injury, in which CD163+ve

cells were found days later in the lesioned area (Zhang et al., 2012).

In vitro, IL-4 treatment up-regulated CD163 expression in human and rat macrophages

(Polfliet et al., 2006), although others have reported no change (Van den Heuvel et al., 1999). I

predicted that CD163 expression would exhibit a similar pattern of expression as other

alternative activation/anti-inflammatory markers after ischemia, showing increased levels of

expression at later times after stroke. In contrast to my predictions, I found that CD163

expression was comparable to saline-injected animals at 7 days, and there a trend towards

increased expression at 1 and 3 days, although the variability in the ischemic animals was high.

Given the increased expression of CD163 after IL-4 treatment in vitro, I hypothesized that IL-4

injection into the striatum of ET-1 treated rats would enhance CD163 expression compared to

ischemic controls. At 1 day, IL-4 treatment induced a substantial increase in CD163 expression.

Interestingly, CD163 expression is increased up to sixfold in phagocytic macrophages in vitro

(Buechler et al., 2000). It is possible that the increased CD163 expression after IL-4 treatment is

an indicator of phagocytosis. This agrees with my finding (discussed in section 4.3.8) that IL-4

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treatment in increased markers of phagocytosis at 1 day.

4.3.2.4 CCL22

Chemokines are regulatory polypeptides, involved in cellular communication as well as

immune cell recruitment (Stamatovic et al., 2005). Following stroke, expression of certain

chemokines is thought to be deleterious (Emsley and Tyrrell, 2002). Furthermore, inhibition of

chemokines after transient ischemia has been correlated with a decrease in MPO activity and

tissue damage (Garau et al., 2005). Some chemokines affect BBB integrity. For example, in an in

vitro BBB model treatment with the chemokine MCP-1 lead to a 17-fold increase in BBB

permeability (Stamatovic et al., 2005). Chemokine (C-C motif) ligand 22 (CCL22), previously

known as Macrophage Derived Chemokine (MDC), has been linked to alternative activation of

microglia/macrophages. This link comes from studies demonstrating that CCL22 is a

chemoattractant for polarized type II T cells. Studying the different polarization profiles of T

cells provided the theoretical basis for M1 vs. M2 (also described as classical vs. alternative)

microglia/macrophage activation (Bonecchi et al., 1998b). M2 (alternatively activated

microglia/macrophages) are proposed to act in a similar way as polarized type II T cells

(Bonecchi et al., 1998b). Furthermore, a high level of CCL22 expression is reported in Type II T

cell-skewed diseases (Mantovani et al., 2000). IL-4, a potent inducer of alternative activation,

has been found to increase CCL22 expression in human and rat macrophages (Jaguin et al.,

2013), monocytes (Bonecchi et al., 1998a) and mouse microglia (Columba-Cabezas et al., 2002).

Furthermore, treatment with pro-inflammatory mediators, LPS, IL-1$ and TNF!, attenuated

CCL22 expression in human macrophages (Rodenburg et al., 1998). I predicted an increase in

CCL22 expression after stroke (compared to saline injected). I also hypothesized an increase in

CCL22 expression in animals that received IL-4 treatment at ischemia induction, as IL-4 treated

57

increases its expression. My results revealed a significant increase in CCL22 after stroke at 3

days and a 25-fold increase in IL-4 treated animals (compared to ischemic controls). My results

are in accordance with the in vitro based literature, showing that IL-4 treatment up-regulates

CCL22 expression. Additionally, the increased expression of CCL22 (and CD163) in IL-4

treated animals, provides further support that recombinant IL-4 treatment stimulated the IL-4

pathway in vivo.

Previous work exploring the chemoattractant properties of CCL22 has shown increased

migration of polarized type II (but not I) T cells in response to CCL22 (Bonecchi et al., 1998b).

If recombinant IL-4 treatment is increasing CCL22, this might increase the number of

alternatively activated microglia/macrophages (M2- similar to type II T cells) recruited to the

lesion core. However, I found no difference in the number of microglia/macrophages (discussed

in section 4.3.7). It is possible that a larger percentage of the microglia/macrophages recruited to

the lesion core were alternatively activated (responding to the chemoattractant properties of

increased CCL22 expression) rather than classically activated. However, differentiating

activation states of microglia/macrophages in vivo is difficult. In the future, double

immunostaining of microglia/macrophage markers with CCL22 antibodies might shed light on

whether the IL-4 mediated increase in CCL22 expression (seen in the current study)

preferentially recruits alternatively activated microglia/macrophages to the lesion site. It is

important to note that in rats there is currently no widely accepted method to accurately

differentiate between alternative and classically activated microglia/macrophages in vivo.

However, studies (such as the previously proposed) using double immunostaining for antibodies

against alternative activation markers could begin to discriminate between classical vs.

alternative activation in vivo.

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4.3.3 IL-4 treatment did not significantly alter expression of pro-inflammatory genes

There is a prominent increase in pro-inflammatory genes after ischemia (reviewed in

Sharma and Kumar, 1998). Thus, I predicted an increase in the level of pro-inflammatory

cytokines, IL-1$, TNF! and iNOS, after ischemia. Supporting findings in the literature, I saw a

trend towards increased expression of all 3 pro-inflammatory genes. Because IL-4 treatment in

vitro decreases LPS- (Hart et al., 1989) and IFN"- (in (Donnelly et al., 1990) induced expression

of pro-inflammatory cytokines, I predicted that IL-4 treatment in vivo would also decrease levels

of IL-1$, TNF! and iNOS. At 1 day, there was a trend toward IL-4 treatment decreasing these

pro-inflammatory genes, suggesting that recombinant IL-4 treatment reduces the strong pro-

inflammatory response after ischemia. Interestingly, IL-4 treated animals showed a trend toward

increased expression of pro-inflammatory markers at later time points (3 and 7 days). If

prolonged, the pro-inflammatory response could be harmful. This suggests, that although IL-4

treatment appears to dampen the pro-inflammatory response early after ischemia (1 day) and was

predicted to be beneficial (but was later shown to increase grey matter injury; discussed in

section 4.3.9), the later enhancement of pro-inflammatory genes could cause more damage at

later times. White matter injury (discussed in section 4.3.9) was not altered after IL-4 treatment;

however, it is possible that the increased pro-inflammatory response at 3 and 7 days could induce

white matter damage at later times that were not evaluated in the current study. Future studies

should extend the study period to 14 or 28 days.

4.3.4 IL-4 treatment altered expression of CD200 but not CD200R, markers of neuron-glial interactions

4.3.4.1 CD200

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CD200 is a glycoprotein known for inducing immunosuppression through its receptor

CD200R (Koning et al., 2009). In the healthy brain, CD200 is found on neurons, but not

astrocytes or microglia (Yi et al., 2012), whereas CD200R is found on microglia (Koning et al.,

2010). In contrast, in the injured or diseased brain CD200 is reportedly expressed in

microglia/macrophages (Matsumoto et al., 2007a, Yi et al., 2012) and astrocytes (Koning et al.,

2009).

In general, interaction between CD200 and its receptor, CD200R, is thought to maintain

microglia in their quiescent state (Patel et al., 2013). Most evidence for an immunosuppressive

role for CD200/CD200R is based on studies of CD200 knockout mice, which have a heightened

neuroinflammatory response to harmful stimuli (such as LPS) compared to wild type mice

(Lyons et al., 2009). CD200 expression was increased at 1 day in a mouse model of cytotoxic

injury (Yi et al., 2012) and was found in the ischemic core of rats (Matsumoto et al., 2007a).

Based on these findings, I predicted increased CD200 expression in ischemic rats compared to

saline injected controls. Surprisingly, I found no dramatic increase in CD200. This could be

explained by Matsumoto and colleagues (2007a) findings that a subset of microglia/macrophage

cells expresses CD200 protein in the ischemic core. Microglia/macrophages (Iba1+ve) cells co-

localized with CD200+ve and also CD200-ve cells (Matsumoto et al., 2007a). A difference in

morphology between the Iba1+ve/CD200+ve (more spherical) and Iba1+ve/CD200-ve (more

irregular) cells was also reported. The difference in morphology and CD200 expression could be

an indication of the cells’ activation state. It is possible that in the current study there was a

significant increase in microglia/macrophages expressing CD200 mRNA in the lesion core

(compared to the surrounding striatum). However, this might have been missed, given that I

isolated the whole striatum, which would include cells with and without CD200 message. Future

studies could use a CD200 antibody and Iba1 antibody to examine whether the number of

60

microglia/macrophages containing CD200 protein is altered in the ischemic core of IL-4 treated

rats.

In vitro treatment of microglia cells with IL-4 increased CD200 expression (Lyons et al.,

2007). Additionally, In vivo intracerebroventricular injection of IL-4 into the rat increased

CD200 immunofluorescence (Lyons et al., 2007). Thus, I predicted that CD200 expression

would increase in ischemic animals treated with IL-4. Interestingly, IL-4 treatment slightly, but

significantly decreased CD200 expression at 1 day (compared to ischemic controls). One

possibility is that there was an increase in the death of CD200 expressing cells in IL-4 treated

ischemic animals. This idea is further supported by my findings that IL-4 treatment significantly

increased the number of degenerating neurons (discussed in section 4.3.9). Given that CD200 is

expressed in neurons (and suggested to increase in other cell types after damage), at 1 day, when

most of the damage occurring is restricted to grey matter, I predict that most of the CD200

expressing cells are neurons. If so, then increased neuronal death would coincide with decreased

CD200 expression.

4.3.4.2 CD200R

Blocking CD200R worsens the clinical course in an animal model of MS (EAE) (Meuth

et al., 2008) and neurodegeneration in an animal model of Parkinson’s disease (Zhang et al.,

2011). Such studies provide additional evidence the theory that CD200-CD200R interaction is

protective. Furthermore, the later time points after stroke induction are characterized by up-

regulation in anti-inflammatory genes (Hu et al., 2012); thus, I predicted elevated levels of

CD200R at later times. My results showed a pattern in expression in which CD200R appeared to

be higher in ischemic animals compared to saline injected animals at 3 and 7 days, this provides

support for a role of CD200R during the anti-inflammatory phase of injury after stroke.

61

In vitro, CD200R expression is induced in human macrophages treated with IL-4

(Koning et al., 2010). Thus, I hypothesized that IL-4 treated rats would exhibit an enhanced up-

regulation of CD200R expression compared to ischemic controls. Although not significant, there

was a trend toward an increase in CD200R in IL-4 treated animals at 7 days. At 7 days,

microglia/macrophages had completely infiltrated the lesion and exhibited an activated rounded

morphology. It is possible that an increase in CD200R expression increases after 7 days, when

the lesion is resolving and the microglia are in a resting/quiescent state. In the future, it would be

of interest to evaluate CD200R (and CD200) expression at 14 and 28 days.

4.3.5 ET-1+ saline and IL-4 treated animals showed signs of BBB leakiness

The CNS is protected from harmful molecules and substances found in the blood or

lymphatic systems by the blood brain barrier (BBB). When damage occurs in the CNS or the

brain becomes diseased, permeability across the BBB is increased, allowing harmful substances

to reach the brain (Persidsky et al., 2006). It has been debated whether or not the BBB is

breached after ischemia induction using ET-1 (Sozmen et al., 2012). To monitor leakiness of the

BBB, I evaluated extravasion of the blood protein, IgG, in the brain of animals treated with ET-1

and ET-1+ IL-4. Consistent with the literature reporting a compromised BBB after ET-1

injection (Souza-Rodrigues et al., 2008, Moxon-Emre and Schlichter, 2010), I found increased

IgG staining in the ipsilateral brain regions of animals injected with ET-1+ saline and ET-1+ IL-

4, suggesting that the BBB is compromised. A leaky BBB is often accompanied by the presence

of neutrophils in the CNS after stroke. Staining for IgG in the ischemic brain was variable and it

was difficult to quantitatively measure differences between treatment groups (ET-1+ saline and

ET-1+ IL-4). Overall, there appeared to be no drastic differences in the level of IgG staining

found in the brains of IL-4 treated versus ischemic controls. However, I found a difference in the

62

number of neutrophils in the ischemic core of IL-4 treated animals, which might be an indication

that the BBB is compromised to a greater extent. The increased neutrophil infiltration seen in IL-

4 treated animals could also be due to increased expression of chemokines attracting neutrophils,

as opposed to an increased leakiness of the BBB. In the future it would be interesting to

quantitatively evaluate differences in BBB permeability between IL-4 treated and stroke control

animals. This might involve a different means of examining BBB integrity, such as the use of

Evan’s Blue dye.

4.3.6 IL-4 treatment did not alter presence of the glial scar or its thickness

Astrocytes are found throughout the CNS. They are complex cells that play several roles

in the brain (Sofroniew, 2009). In response to injury, astrocytes undergo a process called reactive

astrogliosis (Sofroniew, 2009). In cases of extreme injury, reactive astrogliosis involves

formation of a glial scar. Whether or not scar formation is beneficial or detrimental remains

controversial. For example, scar formation inhibits axon regeneration, but ablation of

proliferating astrocytes (and in turn disruption of scar formation) can increase immune cell

infiltration, BBB permeability, neuronal loss, lesion size and demyelination (Bush et al., 1999,

Sofroniew, 2009). After ischemia, astrocytes secrete harmful molecules (e.g., NO) as well

neutrophic and angiogenic factors. I found that scar formation began at 3 days and was

pronounced by 7 days in ET-1 injected animals. This is consistent with previous characterization

of the glial scar formation by our lab (Moxon-Emre and Schlichter, 2010). Having predicted a

beneficial role for IL-4, I hypothesized that scar formation would not be as extensive in IL-4

treated animals. After qualitatively noting the presence of the scar in both IL-4 treated animals

and stroke controls at 3 and 7 days, the thickness of the scar was measured. I hypothesized that

although the scar formed in IL-4 treated animals, IL-4 treatment might have altered scar

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thickness. The shape of the scar itself was variable within each treatment group and

measurements showed there was no significant difference between IL-4 treated animals and

corresponding ischemic controls. As previously mentioned, functional IL-4 receptors are present

on astrocytes (Brodie et al., 1998). Therefore, in vivo, astrocytes could be responding to

numerous stimuli released from damaged neurons, neutrophils and microglia/macrophages,

including IL-4. Thus, perhaps the recombinant IL-4 did bind to and induce an anti-inflammatory

pathway in astrocytes. However, this effect might have been masked by the numerous other

stimuli (released from damaged neurons, and microglia/macrophages) acting on astrocytes after

stroke, which might have had more pronounced effects on their response.

4.3.7 IL-4 treatment did not alter the number of activated microglia/macrophages in the ischemic core

Microglia have been implicated in mediating several aspects of ischemia-induced damage

(Patel et al., 2013). When activated, microglia become essentially indistinguishable from

infiltrating macrophages. Activation of microglia/macrophages has been described as a “double

edge sword”, having both harmful and beneficial effects (Patel et al., 2013). Infiltration of

activated microglia/macrophages into the ischemic lesion has been well characterized by our lab

in the ET-1 model (Moxon-Emre and Schlichter, 2010). As early as 1 day after stroke,

microglia/macrophages begin to react to the damage, pulling in their processes and taking on an

amoeboid shape. By 7 days, the ischemic core is filled with activated microglia/macrophages

(Moxon-Emre and Schlichter, 2010).

Studies have begun to explore the possible use of anti-inflammatory agents to decrease

the extent of microglia/macrophage activation. In vitro, administration of the anti-inflammatory

agent, minocycline, selectively inhibited markers of the classical phenotype, but not the

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alternative activation markers (Kobayashi et al., 2013). In vivo administration of minocycline

after MCAo in mice, decreased microglia activation (as judged by Iba1 expression), which was

accompanied by an improved neurological score (Hayakawa et al., 2008). Similarly, melatonin

(thought to have anti-inflammatory effects) administered after MCAo in mice decreased grey and

white matter damage as well as microglia/macrophage activation (Lee et al., 2005). Given the

anti-inflammatory role of IL-4 in vitro, I predicted that IL-4 treatment in vivo would have

beneficial effects and and decrease the number of activated microglia/macrophages in the

ischemic core. However, I found there was no significant difference in the number of Iba1

positive cells in IL-4 treated animals compared to ischemic controls at 1, 3 or 7 days following

stroke. Both treatment groups (ET-1+ saline and ET-1+ IL-4) demonstrated the previously

characterized pattern, in which Iba1+ve cells began to accumulate in the lesion at 3 days and

fully infiltrated the core by 7 days. Given that the number of microglia/macrophages in the lesion

core was not altered by IL-4 treatment, it is of great interest to determine in the future, whether

injection of recombinant IL-4 altered their activation state. Increased phagocytic activity in

microglia/macrophages is suggested as a marker of alternative activation in rat macrophages

(Varin and Gordon, 2009). Thus, I next evaluated the presence of the phagocytic marker, ED1, in

microglia/macrophages in an attempt to identify their activation state.

4.3.8 IL-4 treatment increased phagocytic neutrophils in the ischemic core

Microglia are the primary phagocytes of the brain. After CNS injury, phagocytic

microglia are thought to play a role in re-establishing homeostasis (Patel et al., 2013). Our lab

and others have demonstrated that there is a prominent increase in ED1 (phagocytosis marker;

see section 1.7.4.) co-localized with the microglia/macrophage marker, Iba1, after stroke

(Hansen et al., 2001, Moxon-Emre and Schlichter, 2010). Although not well understood, it is

65

widely believed that alternative activation is associated with increased phagocytosis (Varin and

Gordon, 2009). For example, macrophages treated with IL-4 exhibit increased phagocytosis of

the parasite, Trypanosome cruzi (Wirth et al., 1989). I hypothesized that if I successfully

enhanced the alternative activation phenotype of microglia/macrophages in vivo via treatment

with recombinant IL-4, the animals would show an increase in number of

microglia/macrophages co-stained with the phagocytosis marker, ED1. Surprisingly, there was

no difference in the number of Iba1 +ve/ED1+ve cells in IL-4 treated vs. ischemic control

animals at any time point. It should be noted that phagocytosis is not the only marker of

alternative activation. Thus, the possibility that IL-4 treatment enhanced alternative activation of

microglia/macrophages should not be ruled out. In future studies immunostaining using

antibodies against other alternative activation markers (such as ARG1, CD163 and CCL22) as

previously suggested (see section 4.3.2) should still be examined.

Interestingly, there were a large number of ED1+ve cells at 1 day in the IL-4 treated

animals, which were not co-localized with microglia/macrophage marker, Iba1. This suggests

phagoctyic activity could not be attributed to microglia/macrophages only. Later statistical

analysis revealed the number of ED1+ve/Iba1-ve cells was significantly higher at 1 day in IL-4

treated animals compared to stroke controls.

Microglia are not the only cells that have phagocytic capacity. A previous study

demonstrated ED1 in neutrophils after stroke (Matsumoto et al., 2007b). ED1 was reported to

localize to neutrophils more so than microglia (Matsumoto et al., 2007b). Given the prominent

increase in phagocytic cells (ED1+ve/Iba1-ve cells), I hypothesized that at 1 day, IL-4 treated

animals would exhibit an increased number of neutrophils in the ischemic core. In support of my

hypothesis, I found a significant increase in the number of PMN +ve cells (antibody against

neutrophils) compared to ischemic controls. To attribute the significant increase in ED1+ve/Iba1-

66

ve cells to neutrophils, I double stained with antibodies against PMN and ED1. As predicted, IL-

4 treatment increased the number of PMN+ve/ED1+ve cells in the ischemic core of IL-4 treated

animals compared to stroke controls. Thus, one may attribute the increase in phagocytic activity

to the increased number of phagocytic neutrophils.

The contribution of neutrophils to CNS injury after stroke has been extensively studied

(Easton, 2013). Several researchers have evaluated the effects depleting neutrophils in ischemia

animal, most of which provided evidence for a harmful role for neutrophils after stroke. For

example, a correlation between a decrease in neutrophils and decreased lesion size as well as a

better functional recovery has been reported (Jin et al., 2010). Additionally, depleting neutrophils

reduced BBB breakdown and axonal damage in a model of intracerebral hemorrhage (ICH;

Moxon-Emre and Schlichter, 2011). A newly emerging concept is the existence of N1 and N2

polarized neutrophils, analogous to M1 and M2 macrophages (also refereed to as classical- and

alternative-activated macrophages; see section 1.5; Fridlender et al., 2009). Most studies

evaluating the N1 versus N2 phenotype examined tumor development. A N2 phenoytpe,

characterized by increased expression of ARG1, certain chemokines and angiogenic growth

factors, promotes tumor growth (Piccard et al., 2012). Interestingly, over 10 years ago, functional

IL-4 receptors were found on human neutrophils (Girard et al., 1997); however, the effect of IL-4

stimulation on neutrophils has not been reported. It will be important to evaluate to what extent

IL-4 treatment of neutrophils mimics the polarization of classically vs. alternatively activated

microglia/macrophages. Suppose N2 neutrophils demonstrate the phagocytic activity

characteristic of M2/alternatively activated microglia/macrophages; if so, the recombinant IL-4

treatment administered in the present study, might have driven the activation profile of

neutrophils towards an anti-inflammatory state.

67

A recent study evaluated the effect of administering the PPAR" agonist, rosiglitazone

(RSG), after stroke induction (permanent MCAo model) in mice. They found an increase in

neutrophil number and an increase in N2- like neutrophils, co-stained for the alternative

activation marker, YM1, and the phagocytic alternative activation marker, CD206. This finding

is in accord with the increased number of neutrophils (PMN +ve cells) and phagocytic

neutrophils (ED1+ve/PMN+ve cells) I found in IL-4 treated animals after ischemia. Both studies

suggest that enhancing IL-4 pathways (either by recombinant IL-4 treatment or by administrating

an agonist acting on a transcription factor downstream of IL-4, PPAR") increases the neutrophil

infiltration into the ischemic core, and the presence of N2-type neutrophils. As previously

discussed (see section 4.3.2.4), IL-4 treated animals showed increased expression of the

chemokine, CCL22, which acts as a chemoattractant for type II T cells but not type I (Bonecchi

et al., 1998a). Thus, perhaps the increase in N2-type neutrophils in the lesion is the result of

increased CCL22 expression, attracting only the alternatively activated/N2 type neutrophils.

4.3.9 IL-4 treatment increased grey, but not white matter injury after ischemia

Previous studies have demonstrated a protective role for IL-4 in vivo. IL-4 knockout

animals subjected to MCAo showed an increase in lesion size, which was accompanied by a

significantly worse neurological score (compared to wild type mice; Xiong et al., 2010). IL-4

knockout mice also had an exacerbated progression of EAE (Ponomarev et al., 2007). In a spinal

cord injury study, blocking IL-4 produced a more extensive contusion (Lee et al., 2010). Based

on these findings, I predicted that driving the inflammation profile in ischemic rats towards an

anti-inflammatory state (by recombinant IL-4 injection) would have beneficial effects, and

decrease white and grey matter damage. Gene expression analyses suggested that recombinant

IL-4 treatment did increase the anti-inflammatory state of the striatum (discussed in section

68

4.3.2). However, immunohistochemistry analysis showed that the changes in gene expression

were accompanied by un-predicted changes in the tissue, in which grey matter injury was

increased in IL-4 treated animals.

Prior to this study, the progression of grey matter injury in the ET-1 model had not been

extensively studied. Several groups have evaluated infarct size in the ET-1 model, using TTC

stain (Robinson et al., 1990, Gresle et al., 2006). However, TTC distinguishes metabolically

active versus less active cells, and does not indicate the cell type (Liu et al., 2009). Therefore, to

our knowledge, this is the first study to demonstrate the time course of neurodegeneration by

Fluoro-Jade B staining in the rat model of ET-1. Similar to the MCAo model (Liu et al., 2009),

neurodegeneration peaked at 1 day (number of Fluoro-Jade B+ cells) and then progressively

decreased.

Fluoro-Jade B staining was also evaluated across the full time course in IL-4 treated

animals. At 1 day, I found that IL-4 treatment increased neurodegeneration. At this time, we do

not know the type of neurons that were injured in the striatum. There are two main classes of

striatal neurons: spiny projection neurons and interneurons. Spiny projection neurons, which are

also known as medium spiny neurons, are GABAergic (neurons that primarily release

neurostransmitter gamma-aminobutyric acid (GABA)) and project to the globus pallidus (GP) or

substantia nigra (SN) (Kreitze, 2009). 95% of the neurons in the striatum are medium spiny

neurons, which receive input from the cerebral cortex and thalamus (Kemp and Powell, 1971).

The interneurons, which account for the remaining 5% of striatal neurons, are further classified

into medium GABAergic interneurons and large cholinergic interneurons. Interneurons also

receive glutamatergic input from the cortex and thalamus, but their output is local, forming

synapses onto medium spiny neurons and other striatal interneurons (Tepper et al., 1995). It is

likely that most of the neuronal degeneration was of the medium spiny neurons (given they are

69

more abundant). It would however, be of interest to evaluate whether certain striatal cells (i.e.,

projecting to the GP or SN, or GABAergic vs. cholinergic) were more susceptible to effects of

IL-4 treatment. For instance, different subtypes of striatal neurons are selectively

lost/dysfunctional in Parkinson’s Disease and Huntington’s Disease (Kreitzer, 2009).

As noted earlier the striatum provides an excellent location to evaluate both grey matter

damage and white matter damage (i.e., of neuronal cell bodies and white matter bundles; see Fig.

2.1). Our lab previously characterized the evolution of white matter damage in ischemia, using

the ET-1 model (Moxon-Emre and Schlichter, 2010). My results are consist with several

previous findings, demonstrating a marked loss of MBP expression (progressing from 1-7 days)

accompanied by a progressive increase in dMBP expression after ischemia. Again, having

predicted a beneficial role for IL-4, I hypothesized that IL-4 treated animals would show

decreased white matter injury. Interestingly, I found no difference in MBP or dMBP staining at

any time point between IL-4 treated animals and ischemic controls.

It was originally believed that white matter was less susceptible to stroke-related injury

than grey matter (Marcoux et al., 1982). At first glance it might appear my results support this

view, however, it is possible that the timing of IL-4 injection is responsible for the damage

having been restricted to grey matter, rather than a difference in susceptibility to stroke-induced

injury. Neuronal death occurs within hours of stroke onset. The recombinant IL-4 was co-

injected with ET-1 at the time of stroke induction. Thus, I assume that IL-4 is acting on

surrounding microglia/macrophages and astrocytes at a time when neuronal damage is underway.

Counteracting the harmful effects of pro-inflammatory molecules via enhancement of an anti-

inflammatory pathway after stroke was predicted to be beneficial. However, my results suggest

that evoking this shift too early after stroke induction is damaging. Pro-inflammatory mediators

released after initial stroke injury are thought to recruit inflammatory cells to the lesion site

70

(Konsman et al., 2007). This process is thought to increase phagocytosis of damaged or dying

cells. If so, then hindering this arguably beneficial process too early after stroke onset could lead

to increased damage, as I have seen in IL-4 treated animals. In the future, the effect of injecting

recombinant IL-4 at varying intervals after stroke onset should be explored. Ideally, an

experiment that evaluates the effect of IL-4 treatment within hours, days and weeks of stroke

onset, would allow researchers to identify time windows in which a heightened anti-

inflammatory milieu might be protective.

4.4 Limitations

I found several prominent changes in gene expression after IL-4 treatment (see section

4.3.2). These results were limited by using the whole striatum for analysis. Thus, all spatially

relevant information was lost, i.e., where in the striatum changes occurred, in what cell type

changes were induced, and finally, the proximity to the lesioned area in which changes were

found. If a change in expression could be attributed to a single cell type, it is likely robust, given

that all cells in the striatum were included in the gene expression analysis and many significant

changes were still found. As is common in most animal studies, I chose to use the lowest number

of animals possible. The Nanostring analysis was done on 4-6 rats per treatment and cost

$25,000. Although in most cases variability was reasonable, increasing the sample size could

have produced more statistically significant changes in gene expression (and tissue analyses).

This is especially true with ET-1+ saline animals, which demonstrated the highest variability in

gene expression.

This study was carefully designed with the goal to study inflammation after stroke. Thus,

we decided to use a minimally invasive surgery to induce stroke, and opted to inject recombinant

IL-4 at the same time to limit needle wounds to the brain. For this reason, we did not use the

71

cannula method for delivering recombinant IL-4. One advantage of the cannula method is the

ability to deliver recombinant IL-4 in different doses at varying times after stroke induction.

However, we have found that the cannula implantation itself produces a robust inflammatory

response (unpublished observations). Time constraints did not allow me to examine additional

time points after treatment. In the future, it would be of interest to compare changes in gene

expression and tissue in IL-4 treated and ET-1 control animals within hours and also at 14 and 28

days after stroke induction.

As with many in vivo studies, I was limited in my ability to attribute gene changes to a

certain cell (ie., microglia/macrophages, neutrophils or astrocytes) after introducing an

exogenous agent (in this case IL-4) into the CNS. With our findings, comes the realization of

how intricate and complex the CNS is in response to injury. There are several types of cells that

could have been responding to IL-4 treatment or responding to mediators released from cells that

were directly affected by recombinant IL-4. Furthermore, although it might appear that classical

and alternative microglia/macrophage activation differences are clear-cut, recent studies suggest

otherwise. In vitro, stimulation with classical vs. alternative stumuli has produced phenotypes

believed to be distinctly different from one another. For instance, LPS stimulation up-regulates

pro-inflammatory cytokines, and IL-4 increases anti-inflammatory cytokines. However, the order

in which stimuli are delivered, or the combining the stimuli (IL-4 with LPS or before LPS etc)

can produce dramatically different changes in gene expression (Porcheray et al., 2005, Cao et al.,

2007). This has raised the question as to whether microglia/macrophages are irreversibly

committed to a particular phenotype (Schwartz et al., 2006). If microglia/macrophages are not

committed to a particular phenotype, the way in which the cells respond to an in vivo injection of

IL-4 could be very different. For example, if microglia/macrophages can transition between

activation states, a dose of IL-4 might first drive all surrounding microglia/macrophages towards

72

an alternative phenotype. This response could then be short lived, as pro-inflammatory stimuli

from damaged neurons are released and convert those cells back to a classical activation state. It

is difficult to study such changes in vivo.

4.5 Conclusion

Stroke, as the leading cause of long-term disability in the US, is a debilitating and

increasingly prevalent disease (American Heart Association, 2012) . Unfortunately, there is

currently only one treatment for ischemic stroke (tPA injection) and it must be delivered within a

short time frame (Wang et al., 2004). For this reason, researchers are exploring treatment options

targeted at the secondary injury phase that is characterized by a strong inflammatory response. In

the current study, I attempted to alter the activation state of microglia/macrophages in the brain,

in attempt to induce the anti-inflammatory state, that is characteristic of microglia/macrophages

treated with IL-4 in vitro. I had predicted that the anti-inflammatory effects of recombinant IL-4

injection would be beneficial, reducing damage and immune cell activation/recruitment.

However, my results showed that IL-4 treatment increased neurodegeneration, and this was

accompanied by an increase in neutrophil infiltration and phagocytosis in the ischemic core.

Together, these results suggest that IL-4 treatment at stroke induction is harmful. However, a

protective role for IL-4 should not be dismissed. I now speculate that the time at which IL-4

treatment is administered after stroke induction influences whether the outcome is beneficial or

harmful. This thesis has begun to determine the effects of IL-4 treatment at the time of stroke

induction. In doing so, I provide a substantial start for future research to further characterize IL-4

treatment at different time points after stroke.

73

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Appendices

Table A1. Inflammation-related genes evaluated using NanoString technology after recombinant IL-4 or function-blocking anti-IL-4 treatment in the striatum of ischemic rats.

Gene name Sequence accession number Direction of change after recombinant IL-4 trt iNOS NM_012611 & TNF" NM_012675 & IL-1# NM_031512 & TACE NM_020306 & ICE NM_012762 & IL-4 NM_201270 & IL-13 NM_053828 & IL-10 NM_012854 & IL-1RA NM_022194 & IL-4R" NM_133380 '* MRC1 NM_001106123 & ARG1 NM_017134 '** CD163 NM_001107887 '*** CCL22 NM_057203 '*** CD200 NM_031518 (* CD200R NM_023953 & YM1 NM_001191712 & FIZZ 1, 2 NM_053333 & GFAP NM_017009 '* MMP2 NM_031054 & MMP9 NM_031055 & MMP12 NM_053963 & MMP14 NM_031056 & TIMP1 NM_053819 & Vimentin NM_031140 & BDNF NM_012513 & STAT6 NM_001044250 '* PPAR! NM_013124 &

Trt, treatment; IL, interleukin; ; iNOS, inducible nitric oxide synthase; TNF", tumour necrosis alpha; TACE, Tumor necrosis factor-alpha-converting enzyme; ICE, IL-1 beta-converting enzyme; MRC1, mannose receptor C type 1; ARG1, arginase-1; CD163, haptoglobin-hemoglobin scavenger receptor; CCL22, Chemokine (C-C motif) ligand 22; CD200, Cluster of Differentiation 200; CD200R, Cluster of Differentiation 200 receptor; FIZZ1,2, found in inflammatory zone-1,2; GFAP, glial fibrillary acidic protein; MMP, Matrix metalloproteinase; TIMP1, tissue inhibitor of metalloproteinase 1; BDNF, brain-derived neurotrophic factor; STAT6, Signal transducer and activator of transcription 6 ; PPAR!, Peroxisome proliferator-activated receptor gamma & no change,*p<.05, ** p<.01, ** p<.001, *** p<.0001, compared to ET-1 and Saline injected stroke controls. All expression levels were normalized to 3 housekeeping genes, succinate dehydrogenase complex subunit A (SDHA) and Tyrosine 3-Monooxygenase/Tryptophan 5-Monooxygenase Activation Protein, Zeta Polypeptide (YWHAZ) and Hypoxanthine-Guanine Phosphoribosyltransferase (HPRT1).

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Copyright Acknowledgements

The written portion of pages 3-8 of this thesis were taken verbatim from a portion of the chapter, “Inflammation and white matter injury in animal models of ischemic stroke”, in the book, “White Matter Injury in Stroke and CNS Disease”, with kind permission from Springer Science and Business Media.

Schlichter, L.C., Hutchings, S., Lively, S. (2014). Inflammation and white matter injury in animal models of ischemic stroke. In White Matter Injury in Stroke and CNS Disease (pp.461-504). New York, New York: Springer.