PLASMA ALDOSTERONE LEVELS INCREASE AFTER CEREBRAL ISCHEMIA,
AND MINERALOCORTICOID RECEPTOR ANTAGONISM AT THE TIME OF
REPERFUSION REDUCES INFARCT SIZE; THE ROLE OF
MINERALOCORTICOID RECEPTOR ANTAGONISM ON THE INFLAMMATORY
RESPONSE POST-STROKE

By

Carla Maria Dams Ramos

A THESIS

Submitted to
Michigan State University
In partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

NEUROSCIENCE

2012

ABSTRACT

Stroke is a leading cause of death and disability, and the available
therapies are limited. Stroke stimulates inflammatory mechanisms that
exacerbate the damage caused by cerebral ischemia. Interestingly, inflammation
is linked to the effects of aldosterone and mineralocorticoid receptor (MR)
activation in different organ systems. Previous studies show that chronic MR
antagonism before cerebral ischemia improves the outcome of stroke, and MR
activation worsens it. The aim of this study was investigate the hypothesis that
plasma aldosterone levels increase after cerebral ischemia, and MR antagonists
administered post-stroke, will reduce the infarct size by mediating a shift in the
phenotype of the immune cells in the brain from pro- to anti-inflammatory. I
induced transient focal cerebral ischemia using the middle cerebral artery
occlusion technique, and the MR antagonist spironolactone was administered at
the time of reperfusion. Plasma aldosterone levels were measured by ELISA,
and inflammatory marker expression was analyzed by qRT-PCR. My results
indicate that plasma aldosterone levels increase rapidly after the induction of
cerebral ischemia in Wistar Kyoto (WKY) rats but not in stroke-prone
spontaneously hypertensive rats (SHRSP). MR antagonism post-stroke reduced
infarct size in both strains. In SHRSP, but not in WKY rats, MR antagonism
increased the expression of anti-inflammatory microglia/macrophages (MG/M)
and cytokines, which may be positive for stroke outcome. These results suggest
that aldosterone post-stroke exacerbates the injury caused by cerebral ischemia
and that MR antagonists are a potential drug for the treatment of stroke.

To my loving and supportive husband Alex,
who brought meaning and joy
to this chapter of my life.

iii

ACKNOWLEDGEMENTS

I would like to thank Dr. Anne Dorrance for her infinite support and
guidance. Your mentorship in and out of the lab is one of the greatest gifts I have
received and I will treasure every single lesson you taught me, and every word of
wisdom you gave me. I have never admired anyone else as I admire you, and
although it is a tough challenge to measure up to you in any aspect of life, I am
grateful that I have you as a role model. I will never be able to say thank you
enough for your unlimited support, kindness, and encouragement during this
important chapter of my life.
I would also like to thank my lab-mate Nusrat, who quickly became a true
friend and brought a breeze of fresh air to the lab. Your daily company almost
makes me not want to graduate and leave the lab! Thank you for the
unconditional support. Similarly, I want to thank all past and presents
undergraduate students, specially Saavia, Kellen, Stephanie, Kaye, and Curt. I
learned so much from each of you and working alongside was a pleasure.
I would also like to gratefully acknowledge my committee members, Drs.
James Galligan, Susan Barman, and William Jackson. I truly appreciate all your
time and dedication to my research and professional development. I was blessed
with the most intellectual, engaged, kind, and supportive team of advisors.
I need to thank the members of the administrative staff in the
Neuroscience Program Shari and Jim Stockmeyer for always going beyond
expectations to help me and care for me.

iv

Finally, I want to thank all my family and friends, specially my husband
and best friend, Alex Anders, for unconditionally cheering me up every step along
the way.

v

TABLE OF CONTENTS

List of Figures………………………………………………………………………….ix

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

Statement of the Problem………………………………………….......1

II.

Cerebral Ischemia……………………………………………………….2

III.

The Ischemic Cascade…………………………………………………4

IV.

Inflammatory Response to Ischemia………………………………….7

V.

Aldosterone and the Mineralocorticoid Receptor (MR)……………11

VI.

Aldosterone Regulation……………………………………………….13

VII.

Stroke Risk is Increased by the Effects of Aldosterone……………15

VIII.

MR Activation and Inflammation……………………………………..17

IX.

Choice of Models………………………………………………………19

Experimental Design………………………………………………………………...21
I.

Aim 1…………………………………………………………………….21

II.

Aim 2…………………………………………………………………….21

Methodology…………………………………………………………………………..21
I.

Animals………………………………………………………………….21

II.

Transient Middle Cerebral Artery Occlusion (tMCAO)…………….23

III.

Infarct Size Quantification…………………………………………….24

vi

IV.

Measurement of Plasma Aldosterone Levels………………………24

V.

Analysis of Inflammatory Markers mRNA Expression……………..25

VI.

Statistical Analysis……………………………………………………..26

Results………………………………………………………………………………....27
I.

Analysis of plasma aldosterone levels in WKY rats with femoral
artery catheters that underwent either tMCAO or sham
surgery………………………………………………………………….30

II.

Analysis of plasma aldosterone levels from blood samples
collected by cardiac puncture in SHRSP and WKY rats that
underwent tMCAO compared to sham rats…………………………30

III.

Infarct size quantification in SHRSP and WKY rats………………..32

IV.

Analysis of inflammatory markers in SHRSP and WKY rats……...35

Discussion…………………………………………………………………………….40
I.

Ischemia Increases Plasma Aldosterone Levels…………………..40

II.

Effect of MR Antagonism at the Time of Reperfusion on Infarct
Size……………………………………………………………………...43

III.

Effect of MR Antagonism Post-stroke on the Inflammatory
Response……………………………………………………………….44

Limitations.……………………………………………………………………………47

vii

Conclusions…………………………………………………………………..……….48

Literature Cited……………………………………………………………………… 50

viii

LIST OF FIGURES

Figure 1.

Plasma Aldosterone Concentration Analysis in WKY Rats
at Multiple Time Points Post-tMCAO………………………………...29

Figure 2.

Plasma Aldosterone Concentration Analysis Pre- and PosttMCAO………………………………………………………………..…31

Figure 3.

Analysis of the Effect of Spironolactone Treatment Post-Stroke
on Infarct Size in SHRSP…………………………………...…………33

Figure 4.

Analysis of the Effect of Spironolactone Treatment Post-Stroke
on Infarct Size in WKY Rats…………………………………...……...34

Figure 5.

Analysis of mRNA Expression of General Markers of
Inflammation in SHRSP and WKY Rats Treated with
Spironolactone Post Stroke…………………………………………...37

Figure 6.

Analysis of TNF- mRNA Expression in SHRSP and WKY
Rats Treated with Spironolactone Post-Stroke……………………..38

Figure 7.

Analysis of mRNA Expression of M2 Markers in SHRSP and
WKY Rats Treated with Spironolactone Post-Stroke………………39

ix

INTRODUCTION

Statement of the Problem

Stroke is a cerebrovascular accident and the second leading cause of death
in the world. According to the World Health Organization, annually 15 million
people suffer a stroke, and 5.5 million stroke victims die. Stroke is also the
leading cause of disability in the U.S. [1]. The vast majority of stroke survivors
face life-long disability and devastating consequences to their quality of life that
include impairment of language, memory, and movement. Approximately 50% of
stroke survivors will have permanent disability, placing a burden on their families
and caregivers. To make this problem worse, despite the billions of dollars
invested to research stroke therapies, treatment options are still very limited. The
only drug approved to treat stroke is the recombinant human tissue plasminogen
activator, also known as rtPA. This is a serine protease that causes thrombolysis
leading to reperfusion, which is the reinstatement of the blood supply to the
affected area. However, because rtPA conveys a risk of hemorrhagic
transformation, this treatment can only be administered to patients within 4.5
hours of the onset of ischemia [2]. This limitation means that only 4.5% of
patients receive this therapy [3]. Some patients with large vessel occlusions that
are not eligible for rtPA can be treated by mechanical thrombectomy, which is the
recannulation of the occluded artery by physical removal of the clot. This involves
insertion of an arterial catheter to the site of occlusion. Mechanical clot extraction

1

with retriever (e.g. MERCI retriever) or aspiration methods (e.g. Penumbra
Aspiration Systems), have higher rates of successful reperfusion than
thrombolysis [4]. However, high risk of complications like hemorrhage and the
difficulty of the procedures make them a last resort intervention. Therefore, there
is a great need for new and improved therapies for the treatment of ischemic
strokes.

Substantial research has been conducted to develop neuroprotective
strategies,

but

despite

their

promising

results

in

pre-clinical

studies,

neuroprotective agents have not been effective in the human population.
Subsequently, the stroke field has devoted greater efforts to stroke prevention,
and reperfusion strategies post-stroke [5]. However, reperfusion itself can worsen
the outcome of a stroke by exacerbating the effects of ischemia, leading to blood
brain barrier (BBB) disruption [6], and further damage known as reperfusion
injury [7]. Most stroke patients ultimately require stroke rehabilitation therapy for
successful neurorecovery [1]. The human brain has a remarkable ability to adapt
and recover functionality after injury. Therefore, rehabilitation helps patients with
disabilities relearn physical and cognitive skills impaired by the stroke. Physical,
occupational, and speech therapies are the foundations of stroke rehabilitation.

Cerebral Ischemia
Stroke is a cerebrovascular condition that is diverse in nature. Strokes are
classified as ischemic or hemorrhagic depending on the underlying cause.

2

Ischemic strokes are the most common type with an incidence of 87%, and they
are the type of stroke of interest in this study. An ischemic stroke occurs when a
cerebral blood vessel is occluded, interrupting the supply of oxygen and glucose
to the affected area of the brain. Cerebral arteries are most commonly occluded;
although venous strokes occur, these are rare [8, 9]. Cerebral blood vessels can
be occluded by an embolus (traveling particle), or a thrombus, which is usually a
platelet deposit (blood clot) that forms in the vessel. Thrombi often originate from
atherosclerotic lesions, which are accumulations of lipids, leukocytes, and
smooth muscle cells on the vessel wall [10]. On the other hand, most emboli are
dissociated thrombus material from cerebral and peripheral vessels. However,
cardiogenic blood clots, air, cholesterol crystals, and cell lumps are all potential
emboli that can occlude cerebral vessels [11, 12].

About 50% of ischemic

strokes are attributed to large-artery atherothrombotic disease, 25% are lacunar
strokes which occur in small intracranial arteries, 20% are caused by cardiac
emboli, and 5% are attributed to various rare causes [13].

The brain is particularly vulnerable to ischemia because of its high
metabolic rate and dependence on aerobic respiration. In addition, the brain does
not have the ability to store energy as in other tissues, therefore even a
momentary interruption of the blood supply can lead to permanent tissue
damage, this is known as infarcted tissue and it is the direct result of necrosis,
which is cell death due to exogenous factors or insults such as ischemia [14].
These cells die as a direct result of the lack of oxygen and glucose supply,

3

forming the ischemic core. Surrounding the core, there is a layer of affected cells
that are severely impaired, but could be rescued under the right conditions. This
layer is known as the penumbra; these cells may die or survive affecting the
overall outcome of stroke. Cell death in the penumbra is the result of both
necrotic and programmed cell death processes (apoptosis). Some post-stroke
therapeutic interventions aim to rescue the cells in the penumbra and minimize
the size of infarcted tissue. Minimizing the damage in the ischemic penumbra is
imperative because final infarct volume impacts neurological outcome [15].
Although, progression of the ischemic penumbra and infarct maturation are
influenced by multiple factors, some studies using immunohistochemical and
imaging techniques report that completed infarct maturation can take up to 4
weeks after stroke [16]. Therefore, there is potentially a large window for poststroke therapeutic intervention.

The Ischemic Cascade

Ischemic conditions and signaling molecules from affected cells trigger a
complex series of biochemical reactions identified as the ischemic cascade,
which leads to inflammation and neuronal cell death [14]. The term cascade is a
misnomer, as the order of events after ischemia does not follow a linear
sequence of reactions; instead they form a network of processes involved in cell
dysfunction. A major component of this network is the complex inflammatory
response observed after cerebral ischemia, which is the main focus of this thesis

4

and will be discussed in detail later.

After onset of ischemia, the high metabolic rate of the brain causes rapid
depletion of adenosine triphosphate (ATP) from cells. The brain is composed of
aerobic tissue that generates ATP using the mitochondrial electron transport
chain; therefore, seconds after the onset of ischemia, ATP depletion causes
failure of diverse cellular functions, including ATP-dependent ion pumps [17].
This leads to collapse of the membrane electrochemical gradient and
depolarization. Depolarization leads Ca2+ influx and release of excitatory
neurotransmitters, including glutamate, which is cytotoxic at high concentrations
[18]. Studies have shown that oligodendrocytes, and the myelin they provide, are
damaged by glutamate [19], while axonal processes are sensitive to Ca2+
accumulation [20]. Ca2+ influx also acts as a second messenger to activate an
array of transcription factors, phospholipases, endonucleases, and proteases
which compromise cell function and integrity [21] [22]. In addition, the influx of
Na+, Ca2+, and Cl- into the cell leads to brain edema [23].

Apoptosis also contributes to cell death; this is a cell-driven series of
biochemical events and morphological changes that lead to the cells selfdestruction. The morphological changes observed during apoptosis include the
formation of spherical masses of chromatin within the nucleus, and the
appearance of membrane–bound structures containing dark chromatin [24]. The
main biochemical changes observed during apoptosis include the breakdown of

5

DNA into nucleosomal segments of about 200 base pairs [25], and the activation
of the caspase system [26] which is a complex system of cysteine proteases
essential for the execution of apoptosis. These enzymes are activated by second
messenger molecules such as cytochrome c, which are released from the
mitochondria and play a significant role in the activation of caspase-3 [27], a
ubiquitous enzyme of apoptosis. Activation of caspase-3 by cytochrome c is
necessary for the execution of apoptosis [14].

Several of the signaling mechanisms involved in the ischemic cascade
contribute to an overproduction of reactive oxygen species (ROS). Complex I and
III of the mitochondrial electron transport chain are major sources of ROS [28],
including detrimental molecules such as superoxide anion (O 2-), hydrogen
peroxide (H2O2), and hydroxyl radical (OH-). However, these free radicals are
also generated by a number of cytosolic reactions mediated by the enzymes
cyclo-oxygenase, lipo-oxygenase, and xanthine oxidase [29, 30]. ROS cause
oxidative stress by damaging cellular structures, nucleic acids, and lipid
membranes. Peroxidation, a biochemical indicator of tissue injury by ROS [31,
32], leads to apoptosis through activation of mitochondrial-derived products [33]
and activation of the JNK 1/2 pathway via NMDA receptor-mediated extracellular
Ca2+ influx [34]. Lipid peroxidation causes neurotoxicity by activating map
kinases, PKC isoforms, and activating the JNK-C-jun/AP-1 pathway which
induces caspase activity [33, 35-37]. Other ROS, like peroxynitrile, interfere with
dehydrogenase activity in the Krebs cycle reducing pyruvate dehydrogenase and

6

alpha-ketoglutarate dehydrogenase activity [38], this decreases the mitochondrial
transmembrane potential [38].

The components of the ischemic cascade that have been discussed are
interrelated and can influence each other; they are also exacerbated by
reperfusion, which is the reinstatement of blood supply to the affected area.
Reperfusion increases the production of second messengers and prostaglandins,
inflammation, and mitochondrial dysfunction as indicated by elevated ROS and
opening of the mitochondrial permeability transition pore (MPTP) [39].
Reperfusion also provides oxygen influx for ROS generation, and contributes to
the effects of the ischemic cascade leading to BBB disruption [6]. This leaves the
central nervous system vulnerable to infection.

Inflammatory Response to Ischemia

Inflammation is a complex reaction by the immune system to noxious
stimuli such as pathogens, irritants, or ischemia. It is necessary for combating
infection and wound healing. However, chronic inflammation can be harmful and
even deadly. For this reason, the inflammatory response is tightly regulated. The
inflammatory response is initiated when leukocytes are mobilized from the blood
to the site of injury. In the brain, the inflammatory response to ischemia mediates
a rapid activation of microglia (MG), the resident immune cells. These are
myeloid cells that differentiate from monocytes and migrate from the bone

7

marrow to the brain where they reside and monitor the environment. MG act as
the first line of immune defense in the brain by destroying foreign matter using
phagocytic and cytotoxic mechanisms [40].

MG respond to changes in homeostasis and signaling molecules such as
cytokines, ATP, glutamate, prostaglandins, and ROS [40]. These molecules can
induce activation, migration, and proliferation of MG. Studies indicate that MG
are activated within minutes of stroke onset [41, 42], leading to the release of
cytokines and inflammatory mediators [41, 43]. Hours to days later, there is
infiltration of inflammatory monocytes from the periphery [44], homologous to
MG, which are known as macrophages (M). In experimental models of stroke,
M start infiltrating 24-48 hours after ischemia and reach a peak 7 days later [45].
Because MG and M are morphologically and functionally similar, after M
infiltrate in the brain, they cannot be differentiated from MG [46, 47]. For this
reason, in the brain MG/M are analyzed and referred to as the same type of
inflammatory cells. Other inflammatory cells such as neutrophils and T
lymphocytes infiltrate the brain. Neutrophils are the first leukocytes to enter the
brain, beginning after 30 minutes [48], T lymphocytes infiltrate to a lesser extent
and play a role in the later stages of ischemic injury [49].

Inhibiting the inflammatory response by immunoblockade or genetic
deletion of adhesion molecules and cytokines necessary for the activation and
recruiting of inflammatory cells reduces the damage caused by ischemia [48].

8

Although the inflammatory response observed after stroke can significantly
exacerbate the damage caused by cerebral ischemia [46] [50], inflammatory cells
seem to play a dual role after stroke [51, 52]. MG/M are not a uniform
population of cells, instead, they express multiple phenotypes depending on their
environment. The expression of different phenotypes depends on signaling
molecules and cytokines that can induce classical or alternative activation of
MG/M [12]. Classically activated MG/M, also known as M1 are considered to
be pro-inflammatory, while alternatively activated MG/M or M2 play an antiinflammatory role and are involved in wound healing and tissue repair [52]. The
M1 phenotype is induced by cytokines such as TNF- and interlukin (IL)-1 [53].
M1 MG/M can be cytotoxic and exacerbate tissue damage. They mediate
neurotoxicity by producing NADPH oxidase derived ROS, [54] and

pro-

inflammatory cytokines [55] which damage nearby cells and potentiate leukocyte
infiltration, BBB disruption and apoptosis [56]. The cytokines associated with the
M2 phenotype include transforming growth factor 1 (TGF-1), IL-4 and IL-10
[53, 55]. This phenotype is characterized as a debris scavenger; M2 MG/M
promote angiogenesis and extracellular matrix remodeling for cell growth [57].
M2 MG/M cannot carryout antigen presentation or promote inflammation. M2
MG/M are unable to activate NF-, a transcription factor that responds to
noxious stimuli [58] and is necessary to induce the pro-inflammatory response
mediated by M1 MG/M.

9

One recent study showed that polarization of MG/M post-stroke is highly
dynamic. Hu et al. conducted a temporal characterization of MG/M phenotype
by M1 and M2 marker analysis with quantitative real-time polymerase chain
reaction (qRT-PCR) and immunohistochemistry (IHC) [52]. The results revealed
that in the early stages of cerebral ischemia, MG/M predominantly express the
M2 phenotype, but gradually convert to M1 phenotype in and around the
penumbra. They found that mRNA expression of all the tested M2 markers,
which included TGF-1 and Arginase (Arg) 1, were predominant soon after
stroke and peaked by day 3 post-stroke. However, levels of M2 markers begin to
decline around day 7 post-stroke until they are minimal compared to M1 markers.
In contrast, mRNA of common M1 markers such as the glycoproteins Cluster of
Differentiation (CD) 16, 32, 86, and 11b, as well as inducible nitric oxide synthase
(iNOS) gradually increased from day 3 post-stroke and remained elevated for 2
weeks. mRNA expression changes were supported by IHC and in vitro
experiments. Using an in vitro model of ischemia by oxygen-glucose deprivation
of cultured neurons, the authors showed that M1 MG/M or conditioned media
from these cells intensifies induced neuronal death. In contrast, M2 microglia
protected neurons against ischemia. These results suggest that suppressing the
shift from M2 to M1 is a potential therapeutic strategy to reduce the damage
caused by ischemia [52]. This thesis aims to investigate if MG/M polarization is
a target of the effects of the hormone aldosterone.

10

Aldosterone and the Mineralocorticoid Receptor (MR)

Aldosterone is a steroid hormone with high affinity for the MR. It is
synthesized in the zona glomerulosa (ZG; outer layer) of the adrenal cortex [59].
Aldosterone primarily acts in the collecting duct of the kidney to regulate blood
pressure through water and sodium reabsorption. Activation of the MR in
epithelial tissues leads to up-regulation of epithelial sodium channel expression,
causing Na+ and water retention [59]. As a consequence the increase in
extracellular volume leads to an increase in blood pressure. The MR plays a
major role in the regulation of renal physiological functions, but recent studies
have revealed that MR activation and high levels of its ligand, aldosterone can
contribute to the pathology of other organ systems. In the vasculature, for
example, MR activation leads to inflammation and remodeling, increasing the risk
of stroke [60, 61].

The chemical structure of aldosterone was identified in 1953, but it was
not until the 1990’s when researchers began to elucidate the hormone’s
pathophysiological effects in cardiovascular disease [59, 62]. Since then,
different roles of aldosterone, including effects on the cerebral vasculature and
risk of stroke keep emerging. The main objective of this thesis is to further this
research and investigate if blocking the effects of aldosterone by antagonizing
the MR after a stroke reduces the damage caused by cerebral ischemia.

11

The MR is a nuclear receptor that has equal affinity for mineralocorticoids
and glucocorticoids such as cortisol (humans) or corticosterone (rodents) [63].
The MR is expressed in many different tissues including the kidney, heart, and
brain regions such as the hypothalamus and hippocampus [64, 65]. However,
tissues where the MR is specific to mineralocorticoids, including vascular smooth
muscle cells, contain the enzyme 11--hydroxysteroid dehydrogenase type 2
(11-HSD2) [65-67]. This enzyme metabolizes cortisol and corticosterone to
cortisone, which is unable to bind to and activate the MR. This protects the MR
from

being

constantly

activated

because

physiological

glucocorticoid

concentrations are 100 to 1000-fold higher than circulating aldosterone [68].
Therefore, deficiencies in 11-HSD2 lead to arterial hypertension and
hypokalemia, which normally reflect excessive MR activation due to elevated
levels of mineralocorticoids, however patients with 11-HSD2 deficiency present
low levels of mineralocorticoid and high levels of glucocorticoids [69].

The MR belongs to the group of 3-ketosteroid receptors in the estrogen
receptor-like subfamily. The NR3C2 gene encodes the MR protein, which
consists of three regions: the N-terminal domain, the C-terminal domain, which is
also the ligand-binding domain, and the DNA binding domain, which serves as a
transcription factor [63]. Similar to other nuclear receptors, when the MR is
activated by a ligand, it undergoes conformational changes, leading to its
dissociation from the heat shock protein 90, and homodimerization. Upon
translocation to the nucleus, the MR complex interacts with coactivators and

12

hormone response elements present in the promoter of some genes. This results
in transcription of target genes [65]. Although the genomic effects of the MR are
better characterized, there is evidence of non-genomic effects of the MR that
lead to activation of second messenger pathways [65].

Spironolactone and eplerenone are MR antagonists that block the MR with
high affinity. Spironolactone is a synthetic compound that acts predominantly as
a competitive antagonist, and has been used clinically as a K +-sparing diuretic
mostly for patients with cardiovascular conditions like heart failure and resistant
hypertension [62, 70].

Aldosterone Regulation

It is unknown what happens to aldosterone levels post-stroke. Aldosterone
production is mainly regulated by the renin-angiotensin system in response to
elevated [K+] or low blood pressure. High [K+] stimulates aldosterone production
by depolarizing cells in the ZG and activating voltage-dependent Ca2+ channels
[59]. On the other hand, the hormones involved in the renin-angiotensin system
stimulate aldosterone synthesis when the juxtaglomerular cells in the kidney
release the enzyme renin upon sensing low blood pressure [71]. Renin catalyzes
the conversion of angiotensinogen released by the liver to angiotensin (Ang) I,
which is metabolized into AngII. This is a potent vasoconstrictor that can also act
on the cells of the ZG to stimulate aldosterone production [59, 71].

13

Although studies indicate that MR antagonism is beneficial, particularly in
cardiovascular disease, even when aldosterone levels are not elevated [72], the
literature suggests that cerebral ischemia has the potential to increase
aldosterone production. There is evidence that the sympathetic nervous system
(SNS) could mediate increased aldosterone production via the increase in nerve
activity that occurs post-stroke [73]. The ZG in the adrenal cortex is innervated by
SNS nerves that come from the adrenal medulla and outside the adrenal gland
[74, 75]. Some of the nerves that reach the adrenal cortex are post-ganglionic
and express tyrosine hydroxylase, the enzyme necessary for NE production [76].
In addition, in vitro studies have shown that electrical stimulation of the capsule
and ZG causes NE release [76] and NE, by activating 1-adrenoreceptors,
stimulates aldosterone secretion [77, 78], and enhances AngII-stimulated
aldosterone production [79]. Furthermore, studies show the steroidgenic acute
regulatory (StAR) protein, which is required to transfer cholesterol to the
mitochondria for steroid biosynthesis [79] increases 24 hours after MCAO [80],
suggesting steroidgenic activity may be elevated. All this evidence suggests that
after stroke, sympathetic nerve activity may increase plasma aldosterone levels.

In models of congestive heart failure, aldosterone augments the activity of
the paraventricular nucleus of the hypothalamus, which is critical for regulation of
sympathetic drive and contributes to the progression of the disease [81]. This
study also showed that MR blockade decreases sympathetic nerve activity in rats

14

through direct actions in the brain [81]. In addition, patients with heart failure
treated with MR antagonists have improved NE uptake and heart-rate variability
[82]. This evidence raises the question of whether cerebral ischemia induces a
system of positive-feedback regulation between aldosterone and the SNS, similar
to other positive-feedback mechanisms observed in states of disease.
Understanding the mechanism for the increase in aldosterone production could
open up alternative therapeutic targets for the treatment of stroke.

Stroke Risk is Increased by the Effects of Aldosterone

Elevated plasma aldosterone is associated with stroke independently of its
link with blood pressure. Patients with elevated plasma aldosterone have a
higher risk of suffering a stroke [83]. Comparison studies between patients with
primary aldosteronism and essential hypertension showed that patients with
primary aldosteronism suffer more strokes despite having lower blood pressure
[84]. In addition, cerebral ischemia induces upregulation of MR expression in the
hippocampus and cerebral cortex where the MR is normally expressed, but
ischemia also induces MR expression in brain regions that do not express the
MR under normal conditions such as the striatum [85]. In this study using mice,
the authors showed that a short duration of ischemia (only 20 minutes of tMCAO)
remarkably increases MR expression in the ischemic brain and that the majority
of MR-positive cells are astrocytes that migrate towards the ischemic area [85].

15

Previous studies published by the Dorrance laboratory have shown that
activation of the MR aggravates the outcome of cerebral ischemia. In one study,
inhibiting the effects of aldosterone by blocking the MR prior to stroke decreased
damage caused by cerebral ischemia by 50% without affecting blood pressure
[84]. Similar results have been obtained in studies conducted with mice; one
study showed that long term MR antagonism prior to cerebral ischemia reduces
infarct size. The authors argued this results from elevated levels of
neuroprotective and angionenic agents, growth factors such as vascular
endothelial growth factor (VEGF), and a reduction in ROS production around the
infarcted region [85]. However, it is important to point out that increasing levels of
VEGF has been shown to be detrimental because it causes an increase in BBB
permeability [86]. Another study using mice showed that MR inhibition for 2 days
prior to the induction of cerebral ischemia with the antagonist eplerenone
reduces the infarct size and neurological impairments by improving cerebral
blood flow in the penumbra and reducing oxidative stress [87]. This study
suggests that beneficial effects of MR antagonism are linked to improving
vascular function because treatment for 48 hours with spironolactone is unlikely
to have an effect on slow processes such as vascular remodeling and
angiogenesis. So far, all the beneficial effects of MR antagonism seem to be
blood pressure independent. In addition, chronic MR activation by two different
mechanisms corroborated that MR activation increases the size of infarct after
cerebral ischemia [60, 88]. These studies reported a significant, but small
increase in blood pressure. However, the MR antagonists were administered

16

long-term before onset of ischemia, limiting the clinical relevance of these
studies. At this point, there is enough evidence suggesting that MR antagonism
could be a strategy for the treatment of stroke to warrant further studies with
clinically appropriate treatment regimes. Importantly, several of the components
of the ischemic cascade like oxidative stress, inflammation, and apoptosis have
been linked to the effects of aldosterone and MR activation in different organ
systems like the heart and kidney [89, 90].

MR activation and Inflammation

There is evidence suggesting that cerebral ischemia induces an increase
in plasma aldosterone levels soon after stroke [73, 91]. If this is the case, it is
possible that MR activation after ischemia exacerbates the outcome of stroke by
altering the immune response and ROS generation. MR activation and
aldosterone are linked to inflammation; MR activation induces activation of
transcription factor NF.

This enhances the expression of inflammatory

cytokines such as tumor necrosis factor alpha (TNF-), and monocyte
chemoattractant protein- 1 (MCP-1) [92, 93] which are involved in activation of
M1 MG/M. An in vitro study using cultured human peripheral mononuclear cells
(M precursor) showed that incubation with aldosterone increases TNF-
expression [94]. In addition, both in vitro and in vivo studies have shown that
spironolactone treatment blocks TNF- release through a serum- and

17

glucocorticoid-mediated kinase-1-dependant pathway (sgk1), which is the main
mediator of MR genomic effects in epithelial and smooth muscle cells [95, 96].

Recently, studies using M specific MR knockout mice suggest that the
MG/M phenotype is partly controlled by the MR. Usher et al, 2010
demonstrated that efficient classical activation of MG/M requires a functional
myeloid MR both in vitro and in vivo [97]. This means that myeloid MR deficiency
promotes inducers of alternative activation such as IL-4 to shift the inflammatory
response towards a M2 phenotype. In vivo myeloid MR knockout mice showed
increased mRNA expression of anti-inflammatory cytokines such as TGF-, IL-4,
and Arg1 in heart tissue, and a reduction in cardiac hypertrophy and vascular
damage caused by nitric oxide synthase inhibitor, NG-nitro-l-arginine methyl
ester (L-NAME) and AngII administration [97]. Importantly, studies of cerebral
ischemia using the myeloid MR knockout mice show that MG/M in the infarct
are phenotypically similar to alternatively activated MG/M and infarct size is
reduced [98, 99]. These suggest that the MR plays a role in the immune
response observed post-stroke and spironolactone treatment at the time of
stroke could potentially have the same effect that MR knockout on shifting the
inflammatory phenotype. My study aims to investigate if MR inhibition with the
antagonist spironolactone after the onset of ischemia has the potential to reduce
infarct size by shifting the inflammatory response from pro- to anti-inflammatory
phenotype. For this purpose the main hypothesis of this thesis is that: Plasma
aldosterone levels increase after cerebral ischemia, and MR antagonist,

18

administered post-stroke, will reduce the infarct size by mediating a shift in the
phenotype of the immune cells in the brain from pro- to anti-inflammatory.

Choice of Models

Stroke-prone spontaneously hypertensive rats (SHRSP) have been widely
used to study spontaneously occurring hemorrhagic strokes and induced
ischemic strokes. This strain was developed by the selective breeding of
spontaneously hypertensive rats (SHR), which originated from Wistar Kyoto rats
(WKY). The WKY is considered a normotensive control for SHR and SHRSP.
The SHRSP shows elevated blood pressure from 6-weeks of age, malignant
hypertension by 12 weeks, and as they age they can show stroke symptoms like
circling and limb weakness [100, 101]. The SHRSP have been used to study
spontaneous strokes, often, as the result of salt-loading and hypertension [102].
This high salt or stroke-prone diet induces cerebral hemorrhages, cortical
infarcts, and small subcortical infarcts [101]. The latter resemble lacunar infarcts
(micro-bleeds in brain white matter) in humans [103]. The subcortical infarcts
suffered by the SHRSP are not only the consequence of elevated blood
pressure. By developing consomic strains, Carswell et al. demonstrated that the
incidence and magnitude of infarcts are independent of blood pressure and
dependent on the genotype [104]. In addition, immunohistochemistry analysis of
the progressive pathological changes that occur in the neurovascular unit of
SHRSP rats showed that endothelial dysfunction, BBB impairment, and chronic

19

inflammation play contributing roles in the development of hypertension and
stroke [105]. Although, SHRSP have proven to be an efficient model of
spontaneous stroke, most commonly SHRSP are used to produce large middle
cerebral artery (MCA) territory infarcts by MCA occlusion (MCAO). SHRSP have
larger infarcts than WKY rats when ischemia is induced experimentally. In this
study, I chose to study SHRSP to better understand the role of inflammation in
the pathology of cerebral ischemia. In addition, because hypertension is a risk
factor for stroke, the SHRSP provides a model with a co-morbidity, and
comparing SHRSP to WKY rats allowed me to validate that MR antagonism poststroke has beneficial effects on normotensive rats. The SHRSP used in this
study were housed and fed identically to WKY rats and the SHRSP did not
receive a high salt diet. However, the SHRSP and WKY rats used in this study
were 12 weeks old, which is an age when SHRSP rats have developed
hypertension.

20

EXPERIMENTAL DESIGN

This thesis consists of two specific aims that address the question
presented in the central hypothesis: Plasma aldosterone levels increase after
cerebral ischemia, and MR antagonist, administered post-stroke, will reduce the
infarct size by mediating a shift in the phenotype of the immune cells in the brain
from pro- to anti-inflammatory.

Aim 1: To test the hypothesis that plasma aldosterone levels increase after
ischemic stroke and that MR antagonism at the time of reperfusion reduces the
infarct size.

Aim 2: To test the hypothesis that MR antagonism improves stroke outcome by
increasing the expression of anti-inflammatory cytokines as measured by qRTPCR.

METHODOLOGY

Animals

All animal procedures were followed in accordance with Michigan State
University animal use guidelines. A total of 53 SHRSP and WKY rats were used
in this study.

Eighteen SHRSP were obtained from the breeding colony

21

maintained by our laboratory at Michigan State University. Thirty-eight WKY rats
were purchased from Harlan (Indianapolis, IN) at 11 weeks old, to allow one
week of transport recovery before surgery. Aldosterone was measured in male
WKY rats with femoral artery catheters that underwent 3 hours of tMCAO
(Stroke; n=8) or sham surgery (SHAM; n=4). Aldosterone was also measured in
SHRSP and WKY rats where blood was collected by cardiac puncture. This
included, a group of intact and untreated SHRSP (SHRSP_SHAM; n=6) and
WKY rats (WKY_SHAM; n=6) used as controls, a group of SHRSP
(SHRSP_tMCAO+Control; n=6) and WKY rats (WKY_tMCAO+Control; n=6) that
were only administered 200 ul of DMSO intraperitoneally (i.p.) as vehicle, and
finally a group of SHRSP (SHRSP_tMCAO+Spir; n=6) and one group of WKY
rats (WKY_tMCAO+Spir; n=6) were administered spironolactone at the time of
reperfusion.

For infarct size quantification and inflammatory marker analysis rats were
randomly assigned to the following groups: WKY: (WKY_MCAO+Spir; n = 11) or
vehicle (WKY_MCAO+Control; n = 11). SHRSP: (SHRSP_MCAO+Spir; n = 5) or
vehicle (SHRSP_MCAO+Control; n = 6).

The MR antagonist spironolactone (25 mg/kg b.w.) was diluted in 200 ul of
the organic solvent dimethyl sulfoxide (DMSO; used as vehicle) immediately prior
to use. Spironolactone was administered i.p. to the treatment groups of SHRSP
and WKY rats right before removal of the monofilament to facilitate reperfusion.

22

Transient Middle Cerebral Artery Occlusion (tMCAO)

This method was previously described in Longa et al [106]. Rats were
anesthetized with isoflurane and body temperature monitored with a rectal
temperature probe to be maintained at 37°C with a heating pad. To achieve MCA
occlusion, the carotid artery was exposed, the thyroid and lingual arteries were
cauterized, and the external carotid and pterygopalatine arteries were ligated.
The MCA was occluded by placing a 3.0 monofilament (Doccol, Sharon, MA.)
through the internal carotid artery, and advancing it into the MCA. Occlusion of
the MCA was verified by a drop in blood flow in the region supplied by the MCA
as measured by pin-point laser Doppler. After MCAO, isoflurane administration
was stopped to allow the rats to wake up. In WKY rats, MCAO was maintained
for 3 hours, the occlusion time was limited to 1 hour in SHRSP. The rats were
checked for neurodeficit during this time. The rats were then re-anesthetized, and
the monofilament was removed. The rats were euthanized either 48 or 144 hours
after the initial induction of ischemia.

Infarct Size Quantification

SHRSP and WKY rats underwent tMCAO (1 and 3 hours respectively).
They were treated with either spironolactone or vehicle at the time of reperfusion
as described above. The rats were euthanized, 48 hours after the onset of
ischemia, under anesthesia and the brains were collected. Brains were coronally

23

sectioned in five 2 mm thick slices. The 4th slice from the frontal pole was snapfrozen in liquid nitrogen for mRNA expression analysis. The rest of the slices
were stained with 2,3,5-triphenyltetrazolium chloride (TCC). The infarct volume
was obtained using the formula of Swanson et al [107] (shown below), and is
expressed as the percentage of the infarcted hemisphere to take cerebral edema
into account. The slices were scanned and measurements were taken digitally
using ImageJ (version 10.2). The 3rd slice was scanned front and back to account
for the loss of the 4th slice from the equation.

The Swanson equation for measuring brain infarct volume:
% I = 100 x ((Vc– VL) / Vc)
Where,
% I = Percent of hemisphere infarcted
Vc = Volume of normal tissue in the structure of the control hemisphere
VL = Volume of normal tissue in the structure of the infarcted hemisphere

Measurement of Plasma Aldosterone Levels

Catheter Implantation

Femoral artery catheters (SAI, Lake Ville, IL) were implanted 2 days prior
to tMCAO or sham surgery using the method described in Jespersen et al. [108].
Catheters were flushed with 200 ul of heparanized saline (100 U/ml) daily.

24

ELISA
To measure plasma aldosterone in SHRSP and WKY rats, an ELISA
assay (Cayman Chemical, Ann Arbor, MI.) was used. Blood was collected either
by cardiac puncture at the time of sacrifice or in conscious rats through a femoral
artery catheter. Blood was centrifuged at 1000 rpm for 10 minutes to isolate
plasma. Blood samples were collected before surgery and at the following poststroke time points: 6, 24, 48, and 144 hours.

Analysis of Inflammatory Markers mRNA Expression

Real-Time PCR (qRT-PCR)

Total RNA was extracted from the ischemic hemisphere of SHRSP and
WKY rats that underwent tMCAO and were treated with either spironolactone or
vehicle. RNA was extracted using the Ambion® mirVanaTM PARIS Kit
(Invitrogen, Grand island, NY), and reverse transcribed with either qScript™
cDNA SuperMix (Quanta Biosciences, Gaithersburg, MD) or SuperScript®
VILO™ (Life Technologies, Grand Island, NY) Specific TAQMAN primers were
obtained from Applied Biosystems (Life Technologies, Grand Island, NY). The
following inflammatory markers were analyzed: General MG/M markers; CD68
and intracellular adhesion molecule 1 (ICAM-1); M1 MG/M markers and proinflammatory cytokines; TNF-, and MCP-1; M2 MG/M markers and M2
phenotype inducers; TGF-1, Arg1. mRNA expression of each of the

25

inflammatory markers was normalized to expression of the housekeeping gene
-2-microglobulin

(2M).

The

2-CT

method was

used

to

analyze

the relative changes in gene expression from the qRT-PCR experiments [109].

Statistical Analysis

All data are presented as means ± standard error of the mean (SEM). For
the comparison of two groups the appropriate Student’s t test was used. In all
cases, a p value of 0.05 or less was considered significant.

Plasma aldosterone concentration was quantified using a standard curve,
and reported as concentration in picograms per milliliters (pg/ml).

For

aldosterone concentration analysis over time, a two-way repeated measures
ANOVA was performed, and post-hoc testing using Bonferroni’s procedure at
each time point was performed to correct for multiple comparisons (Graph Pad
Prism 5). For data analysis of aldosterone concentration in groups subjected to
cardiac puncture, a One-way ANOVA with post-hoc testing using the Bonferroni’s
procedure was performed to analyze differences within treatment of SHRSP
groups. For WKY data analysis within treatments, a One-Way ANOVA KruskalWallis Test was performed with Dunn’s post-hoc testing to correct for multiple
comparisons with unequal variances.

26

RESULTS

Aim 1: To test the hypothesis that plasma aldosterone levels increase after
stroke and MR inhibition at the time of reperfusion reduces infarct size.

Experiment 1: Plasma aldosterone concentration analysis after cerebral
ischemia.

Experiment 2: Effect of MR antagonism at the time of reperfusion on infarct size.

Results of Experiment 1:

Analysis of plasma aldosterone levels in WKY rats with femoral artery catheters
that underwent either tMCAO or sham surgery

Plasma aldosterone increased after cerebral ischemia in WKY rats with
femoral artery catheters. Plasma aldosterone was significantly elevated 6 and 48
hours after induction of cerebral ischemia compared to shams. At 48 hours poststroke plasma aldosterone was double that in shams (Figure 1). At 24 and 144
hours post-stroke aldosterone was also elevated, but this was not statistically
different from the sham group, possibly due to the slight increase in aldosterone
observed in shams. This increase could be due to the catheterization surgery
itself. However, when plasma aldosterone levels at 24 and 144 hours were

27

compared to basal levels in tMCAO rats, they were both significantly increased
(24 hours post-tMCAO: p value<0.0001; 144 hours post-tMCAO: p value<0.05;
compared to Basal tMCAO). In contrast, plasma aldosterone levels did not
increase in sham rats at any time compared to basal levels.

28

29

Analysis of plasma aldosterone levels from blood samples collected by cardiac
puncture in SHRSP and WKY rats that underwent MCAO compared to sham rats

SHRSP
tMCAO did not change plasma aldosterone concentration in SHRSP rats
compared to SHRSP-SHAM (Figure 2). However, spironolactone treatment at the
time of reperfusion did increase aldosterone levels compared to vehicle treated
SHRSP and SHRSP sham groups. Plasma aldosterone levels were not statically
different between SHRSP and WKY sham rats.

WKY
Plasma aldosterone was dramatically increased at WKY 48 hours post
tMCAO (Figure 2, p value < 0.001). Plasma aldosterone levels increased about
15-fold after 3 hours of tMCAO followed by 45 hours of reperfusion in WKY rats.
In addition, treatment with spironolactone at the time of reperfusion
(WKY_MCAO+Spir) did not change plasma aldosterone levels when compared
to WKY_MCAO rats.

30

31

Results of Experiment 2:

Infarct size quantification in SHRSP and WKY rats

MR antagonism with spironolactone post-stroke reduces the size of the
infarct caused by cerebral ischemia in SHRSP (Figure 3) and WKY (Figure 4)
rats. Spironolactone reduced infarct size (average percentage of infarcted
hemisphere) in SHRSP_tMCAO+Spir (n=5) by more than 30% compared to
SHRSP_MCAO+Control (n=6) (p value < 0.01). In the WKY groups, similar
results were observed; spironolactone treatment post-stroke significantly reduced
infarct size in WKY_MCAO+Spir rats (n=11) compared to WKY_MCAO+Control;
n = 11 (p value < 0.05).

32

33

34

Aim 2: To test the hypothesis that MR inhibition improves stroke outcome by
increasing the expression of anti-inflammatory cytokines as measured by qRTPCR.

Experiment 1: Analysis of inflammatory markers in SHRSP and WKY rats that
underwent tMCAO and MR antagonism at the time of reperfusion.

Results of Experiment 1:

Analysis of Inflammatory marker mRNA expression in SHRSP and WKY rats

Figure 5 shows the fold increase in mRNA expression for general MG/M
markers CD68 and MG/M cytokine ICAM-1. Groups treated with spironolactone
or vehicle were compared for both strains to identify the effect of MR antagonist
treatment post-stroke in the expression of inflammatory markers. SHRSP and
WKY rats showed different patterns of marker expression in response to
spironolactone treatment. CD68 was unchanged by spironolactone treatment in
both

SHRSP

and

WKY

rats.

ICAM-1

remained

unchanged

in

the

WKY_MCAO+Spir group compared to the control group. In contrast, in
SHRSP_MCAO+Spir rats, expression of ICAM-1 mRNA is increased 3-fold
compared to SHRSP_MCAO+Control (p value < 0.001). Figure 6 shows fold
increase of pro-inflammatory cytokine, and M1 phenotype inducer, TNF-.
Spironolactone treatment did not change the mRNA expression of TNF- in

35

SHRSP or WKY rats. Finally, mRNA expression of M2 MG/M phenotype and
anti-inflammatory cytokines (TGF-1, Arg1, and MRC-1) are shown in Figure 7.
SHRSP and WKY rats exhibited different patterns of M2 marker mRNA
expression; spironolactone treatment in WKY rats did not change the mRNA
expression of any of the M2 markers. In contrast, mRNA expression of M2
phenotype marker Arg1 was significantly increased in SHRSP_MCAO+Spir rats
compared to the SHRSP_MCAO+Control group (p value < 0.05). mRNA
expression of M2 markers TGF-1 and MRC-1 were not significantly changed.

36

37

38

39

DISCUSSION

The results obtained in this study support parts, but not all, of my central
hypothesis. Plasma aldosterone levels are increased by cerebral ischemia in
WKY rats but not in SHRSP. However, MR antagonism at the time of reperfusion
reduces infarct size in both strains studied. Finally, MR antagonism improves the
outcome of stroke by shifting the phenotype of MG/M from pro- to antiinflammatory in SHRSP, but not in WKY rats. This indicates that the mechanism
of action for the beneficial effects of MR antagonism is different in the two strains
and could depend on pre-existing or genetic factors.

Ischemia Increases Plasma Aldosterone Levels

My study showed that plasma aldosterone levels increase remarkably
after the onset of ischemia. WKY rats with femoral artery catheters had elevated
plasma aldosterone concentrations 6 hours after the onset of ischemia compared
to sham operated rats. Aldosterone levels remained elevated up to 48 hours
post-stroke. Surprisingly, aldosterone levels were not significantly higher at 24
hours post-stroke.

This could be due to the slight increase in aldosterone

observed in the sham-operated rats at 24 and 144 hours post-stroke. Although
we do not know the exact mechanism, it is possible that stress and inflammation
associated with surgery could mediate the increase in aldosterone levels after
catheterization in the sham rats. For this purpose we wanted to investigate if

40

MCAO increased plasma aldosterone levels in the same manner in rats without
femoral artery catheters. These blood samples were collected while the rats were
under isoflurane anesthesia; however, this anesthetic has little effect on
aldosterone levels [114]. We analyzed data from both SHRSP and WKY rats to
determine if the increase in aldosterone levels was strain dependent. We
previously used Sprague-Dawley rats and found that aldosterone levels
increases post-stroke. This suggests that the increase in aldosterone levels is
blood pressure instead of strain dependent. Moreover, it is possible that plasma
aldosterone levels in SHRSP do not have the ability to increase any further if
SNS activity is already elevated. In addition, we analyzed data from groups
treated with MR antagonist spironolactone to determine if spironolactone
treatment could increase aldosterone levels further. The results were very
interesting; first of all, catheterization seems to increase aldosterone levels, WKY
rats that did not undergo any treatment or surgery had much lower basal levels of
aldosterone (about an 8-fold difference). This finding is potentially important
when one considers that catheters are generally accepted as one of the best
ways to collect blood for aldosterone analysis. Second, MCAO alone did not
increase plasma aldosterone levels in SHRSP as observed in WKY rats.
However, MR antagonism significantly increases plasma aldosterone levels in
SHRSP. Finally, in contrast to SHRSP, MR antagonism in this group significantly
increased plasma aldosterone levels. This was an interesting finding, and could
indicate that aldosterone production increases to its maximal capacity after
cerebral ischemia in WKY rats; therefore, MR antagonism cannot further

41

increase aldosterone levels. One way MR antagonism could increase the
production of aldosterone is by mimicking a positive-feedback mechanism to the
HPA axis. Differences in plasma aldosterone levels between the strains were
surprising because the Dorrance laboratory had reported no differences between
the strains [84]. It is important to mention that in this study the rats were given a
fatal dose of pentobarbital, which could potentially increase aldosterone levels.
Nevertheless, this is a controversial area. A different study reported that SHRSP
have higher levels of plasma aldosterone than WKY rats [115]. These could be
due to different diets, and blood sample collection methods, including methods of
anesthesia.

Importantly, aldosterone levels increase dramatically during a critical
period of cerebral ischemia resolution. Within 48 hours post-stroke onset,
peripheral M infiltrate the brain and there is a widespread opening of the BBB
[6, 14], which is highly detrimental for the outcome of ischemia and central
nervous system health [116]. Therefore, MR activation could have negative
effects on the outcome of cerebral ischemia through multiple pathways.

Although the mechanism for increased aldosterone production post-stroke
remains unclear, some studies suggest ischemia can lead to increased
aldosterone levels by stimulating sympathetic drive [117, 118]. One particular
study showed that ischemic conditions in the heart have the potential to
permanently activate central nervous system (CNS) nuclei that control

42

sympathetic outflow and increase norepinephrine release [118]. Similarly, there
are other mechanisms that could lead to increased plasma aldosterone levels
post-stroke,

for example,

the

renin-angiotensin-system,

which

regulates

aldosterone synthesis under normal circumstances. In addition, the HPA axis
could also induce aldosterone production post-stroke via ACTH; since this
hormone is also increased by ischemia [119]. Regardless of the responsible
mechanism, finding that plasma aldosterone concentrations increase after
cerebral ischemia raises questions and opens an opportunity to better
understand the pathology of stroke. To the best of my knowledge, these are the
first studies to show a marked increase in aldosterone with ischemia reperfusion
injury. Increased aldosterone levels post-stroke could potentially affect other
organ systems as well, particularly, the myocardium, where MR activation leads
to structural remodeling [92].

Effect of MR Antagonism at the Time of Reperfusion on Infarct Size

To better understand the role of the MR in the outcome of cerebral
ischemia I administered the MR antagonist spironolactone at the time of
reperfusion. Previous studies have shown that chronic treatment with MR
antagonists before stroke reduces infarct size, however, in this study I used a
clinically relevant treatment paradigm to investigate the therapeutic potential of
spironolactone. Interestingly, we found that MR antagonism post-stroke
significantly reduces infarct size in both SHRSP and WKY rats compared to their

43

respective vehicle-treated controls. The duration of ischemia was increased to 3
hours in the WKY rats to produce a similar infarct size to the SHRSP. The
differences in infarct size between the spironolactone-treated and control groups
are not due to differences in cerebral blood flow; scanning laser Doppler (PIM3;
Perimed, Stockholm, Sweden) measurements of pial artery blood flow showed
no difference between the groups in SHRSP rats that underwent 1 hour of
tMCAO followed by 23 hours of reperfusion (data not shown). However, since the
scanning laser Doppler only measure flow in the pial arteries we do not know if
there is a difference in blood flow in the sub-cortical regions or in the ischemic
penumbra. The results from these experiments indicate that MR antagonism
post-stroke is beneficial by intervening in pathways that aggravate stroke
outcome, for example the inflammatory response, and the effects are not limited
to the hypertensive population.

Effect of MR Antagonism Post-stroke on the Inflammatory Response

As previously discussed, several components of the ischemic cascade,
including inflammation, have been linked to the effects of aldosterone and MR
activation in different organ systems and conditions. The effect of spironolactone
treatment on the post-stroke inflammatory response in SHRSP and WKY rats
present interesting differences. MR antagonism at the time of reperfusion does
not change the inflammatory response in WKY rats. There is a small but
insignificant reduction in TNF-α with spironolactone treatment. It is possible that

44

the inflammatory analysis in WKY rats was underpowered and increasing the
number of subjects could have led to the identification of a significant reduction in
TNF-α expression. On the other hand, when spironolactone treatment is
administered in SHRSP at the time of reperfusion, the expression of general
MG/MΦ marker CD68 remains unchanged while ICAM-1 is significantly
increased. ICAM-1 is required for MΦ to infiltrate into the tissue; therefore, this
increase in ICAM-1 mRNA expression could indicate increased MΦ infiltration in
the brain. This idea is supported by mRNA expression analysis of TNF-α, which
is a pro-inflammatory cytokine that induces classical activation, and is unchanged
by spironolactone treatment. In addition, the marker of M2 phenotype Arg1 is
significantly increased in SHRSP treated with spironolactone. TGF-1, an
inducer of alternative activation shows a small increase after MR blockade,
however, this was not significant different from SHRSP controls (p value =
0.0583). Similarly, although the M2 marker MRC-1 seems to be slightly increased
by spironolactone treatment (p value = 0.09), this is not significant. It is possible
that in these studies, statistical variability is high because we analyzed samples
that contained normal and infarcted tissue; our findings might have been more
conclusive if we had only analyzed tissue punches from the ischemic penumbra.

The inflammatory response has normally been considered to have
detrimental effects in the resolution of ischemia. It leads to increased ROS
production and apoptosis [46]. However, more recent evidence has shown that
the inflammatory response can be beneficial and lead to tissue repair depending

45

on the phenotype exhibited by MG/M [97-99]. This study demonstrates that in
SHRSP MR antagonism post-stroke has the potential to improve the outcome of
cerebral ischemia by shifting the polarization of MG/M toward an alternative
phenotype that leads to an anti-inflammatory response. In WKY rats, the
beneficial effects of MR antagonism seem to be independent from the
inflammatory response. The difference between strains can possibly be
explained by the differences in duration of ischemia and timing of the treatment.
Because we treated the rats at the time of reperfusion spironolactone was
administered to WKY rats 2 hours later than in the SHRSP. Therefore, it is
possible that spironolactone can only effectively modulate the inflammatory
response within that one-hour window. Another possible explanation for the
difference between strains is the evidence that SHRSP rats develop chronic
inflammation in the absence of an ischemic insult, as evidenced by increased
inflammatory

cytokines

and

vascular

lesions

associated

with

chronic

inflammation. Therefore, it is possible that the acute inflammatory response
caused by ischemic conditions in WKY rats does not respond to MR blockade
because there are other factors signaling the urgent state of injury.

The study on the temporal characterization of MG/M post-stroke recently
published by Hu et al. indicates that the initial ischemia induces a “healthy”
inflammatory response in the early stages of injury. However, within 48 hours
after ischemic onset MG/M transitions to a “sick” phenotype which can worsen
the outcome of the stroke [52]. It is possible that this transition is partly mediated

46

by the increase in plasma aldosterone observed after cerebral ischemia,
Therefore, blocking the MR with spironolactone could be beneficial by prolonging
the prevalence of M2 phenotype, increasing the potential for tissue repair and
improving stroke outcome. Hu et al. subjected the mice to 1 hour of tMCAO; the
same duration than the SHRSP in my study. This could explain my results in
SHRSP, but not in WKY rats, are in accordance with those reported Hu et al.
Further research is necessary to elucidate these gaps in our knowledge.
However, the studies in this thesis are the first to demonstrate it is possible to
use a clinically relevant treatment regime to alter the inflammatory response
observed post-stroke in a rat model with a common co-morbidity for stroke such
as hypertension.

LIMITATIONS

There were several limitations in the experimental design of this study.
First of all, only one spironolactone dose, administration route, and treatment
time was used for the study. It is possible that a different dose or time of
administration would be more efficient in reducing infarct size and/or increasing
the

expression

of

anti-inflammatory

markers

and

cytokines.

Similarly,

administration of the antagonist intravenously would be more precise and
potentially more effective. In addition, this study only investigated the effects of
MR antagonism in a model of ischemia/reperfusion injury. Therefore, we do not
know if the same response would be observed in a model of permanent MCAO.

47

Another limitation is that the decrease in infarct size observed post-stroke is
relatively small. Therefore, we do not know if spironolactone treatment would
significantly improve neurological function. For this reason, neurological testing of
the subjects before euthanasia would be insightful and imperative to assess the
therapeutic potential of MR antagonism post-stroke. I did not investigate the
mechanism by which MR antagonism mediates a shift in the inflammatory
response. Finally, spironolactone, although it is an effective MR antagonist, is not
MR specific. Spironolactone has been shown to also block the androgen receptor
[120], which could potentially have an effect on the infarct size and the
inflammatory response. Future studies should address these issues.

CONCLUSION

The studies conducted in this thesis highlight the therapeutic potential of
the MR antagonist spironolactone for the treatment of cerebral ischemia. These
studies indicate that the MR is involved in the pathology of cerebral ischemia and
that increased plasma aldosterone levels post-stroke aggravate the injury caused
by stroke. For this reason, a better understanding of the mechanisms involved in
MR activation post-stroke is imperative. The complex nature of a stroke makes it
unlikely that one therapeutic strategy will be a magic bullet for the treatment of
stroke. MR blockade is a promising strategy because it might globally target
multiple components of the ischemic cascade. Particularly interesting is the effect
of MR antagonism on the inflammatory response observed post-stroke. A better

48

characterization of inflammatory cell phenotypes and function has suggested that
the best strategy is not to suppress the inflammatory response, but instead to
shift it towards a beneficial phenotype, which will contribute to tissue repair and
healing. Spironolactone treatment post-stroke seems to have the potential to
accomplish this. However, the complexity of the inflammatory response demands
a better understanding of the pathways involved in MR mediation of
inflammation, and we hope this study has helped spark interest in these
questions.

49

LITERATURE CITED

50

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Donnan, G.A., et al., Stroke. Lancet, 2008. 371(9624): p. 1612-23.

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Adams, H., et al., Guidelines for the early management of patients with ischemic
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36(4): p. 916-23.

3.

Weinberger, J.M., Evolving therapeutic approaches to treating acute ischemic
stroke. J Neurol Sci, 2006. 249(2): p. 101-9.

4.

Smith, W.S., Safety of mechanical thrombectomy and intravenous tissue
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Embolus Removal in Cerebral Ischemia (MERCI) trial, part I. AJNR Am J
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