Neuropathology of Post-Stroke Depression:

Possible Role of Inflammatory Molecules and Indoleamine 2,3-dioxygenase

by

Amy Wong

A thesis submitted in conformity with the requirements for

the degree of Master of Science (MSc.)

Graduate Department of Pharmacology and Toxicology, University of Toronto

© Copyright by Amy Wong 2010

ii

Neuropathology of Post-Stroke Depression: Possible Role of Inflammatory Molecules and

Indoleamine 2,3 dioxygenase

Amy Wong, MSc Candidate, 2010

Graduate Department of Pharmacology, University of Toronto

ABSTRACT

The study evaluated whether the activity of the indoleamine 2,3 dioxygenase (IDO)

enzyme is increased post-stroke and contributes to the development of post-stroke depression

(PSD) via tryptophan (TRP) depletion and neurotoxic kynurenine (KYN) metabolite production.

The activity of IDO was measured using the KYN/TRP ratio. Participants were assessed for

depression severity using the Center for Epidemiological Studies Depression Scale (CES-D).

Blood TRP, KYN, large neutral amino acids and cytokines were measured and compared.

Fifty-four (mean age=69.9±15.2, male=52.7%, mean NIHSS=7.3±4.6) patients within

28.9±40.3 days of stroke were separated into two groups: non-depressed (n=38, CES-

D=6.1±4.9) and those with significant depressive symptoms (n=16, CES-D=26.8±10.8). Higher

mean KYN/TRP ratios were demonstrated in stroke patients with depressive symptoms (non-

depressed=69.3±36.9 vs. depressive symptoms=78.3±42.0, F3,50=4.61, p=0.006) after

controlling for LNAA (p=0.026) and hypertension (p=0.039). As the KYN/TRP ratio reflects

decreased TRP and increased neurotoxic KYN metabolites, both mechanisms may play an

etiological role in PSD.

iii

ACKNOWLEDGEMENTS

I would like to thank Dr. Krista Lanctôt for her continued support, encouragement and

motivation throughout the course of my studies. Her loving maternal nature coupled with her

outstanding commitment to her work has made my experience as a graduate student an

unforgettable journey. She is a true inspiration to myself and others around me.

I would like to thank Dr. Nathan Herrmann for his continued support, encouragement

and enormous knowledge in the field of psychiatry. He possesses a genuine thirst for excellence

in medicine without limiting himself to any boundaries--a rare trait that I respect and admire

wholeheartedly.

Thank you to both Drs. Krista Lanctôt and Nathan Herrmann for the opportunity to

broaden my horizons in the field of neuropsyhopharmacology research. They have always

made themselves available to my needs and have been patient during my times of struggle.

Their dedication to their work and kindness to others is a great example to their pupils and

motivates me to continually strive for my passions, never ceasing to reach beyond my wildest

dreams or highest expectations.

Thank you to Abby Li, Allison Gold, Jaclyn Cappell, Karen Levy, Kelly Smart,

Mahwesh Saleem, Philip Francis, Robert Mitchell, and Walter Swardfager for their

friendship, blessings, and encouragements. I came in as the slightly crazy one, and I am leaving

as the extraordinarily insane one. I am certain that we will meet once again long after this is all

over—though hopefully not as one of your patients (not for my benefit, but for yours).

Finally, I am most indebted to my Father and my family who have showered me with

their love and support since before I was even born. You have sacrificed so much for me—

some without me knowing, I’m sure—and I am humbly honoured to call myself your daughter,

and your sister. You are my rock, my beacon, my guidepost to life; but most importantly, you

are my best friend. I would not have survived some of my toughest obstacles and fears had it not

been for your strength and belief in my capabilities to succeed. I love you, always.

The skills and lessons I have learned during the past two years are forever internalized

within me. It was at Sunnybrook that I discovered an amount courage I never even knew I

possessed; I am no longer afraid to take the risks I always talk about but never have been able to

realize. I know that in the future, after reaching what then seemed like an impossible dream, I

will look back to this day with both corners of my mouth slightly curled upwards, my eyes

gleaming with understanding, gratitude, and merriment.

iv

TABLE OF CONTENTS

ABSTRACT ..................................................................................................................... 1

1 INTRODUCTION ......................................................................................................... 1

1.1 Statement of the Problem ..................................................................................... 1

1.2 Purpose of the Study and Objectives .................................................................. 2

1.3 Statement of Research Hypotheses and Rationale for Hypotheses ..................... 3

1.3.1 Primary Hypothesis ......................................................................................... 3

1.3.2 Secondary Hypothesis .................................................................................... 4

1.4 Review of the Literature ........................................................................................ 5

1.4.1 Post-stroke Depression ................................................................................... 5

1.4.2 Inflammatory Processes and Stroke ............................................................. 18

1.4.3 Inflammatory Processes and Depression ...................................................... 21

1.4.4 Current Hypotheses on the Etiology of Post-Stroke Depression .................. 25

1.4.5 Neurological Consequences of Reduced Serotonin Synthesis and the Formation of Neurotoxic Metabolites ...................................................................... 29

1.4.6 Rationale for This Study ................................................................................ 38

2 METHODS ................................................................................................................. 43

2.1 Study Design ...................................................................................................... 43

2.2 Subjects .............................................................................................................. 44

2.2.1 Inclusion and Exclusion Criteria ................................................................... 44

2.2.2 Demographics and Medical History ............................................................... 45

2.3 Clinical Assessments .......................................................................................... 47

2.3.1 Depression Scales ........................................................................................ 47

2.3.2 Conition Scales ............................................................................................. 49

2.3.3 Stroke Severity and Functional Disability ...................................................... 50

2.3.4 Blood Draw .................................................................................................... 51

2.4 Laboratory Analyses ........................................................................................... 51

2.4.1 Cytokine Assays ............................................................................................ 51

2.4.2 KYN Assay .................................................................................................... 53

2.4.3 Tryptophan and LNAA Assays ...................................................................... 54

2.5 CT Scan Analysis ............................................................................................... 54

2.6 Statistical Analyses............................................................................................. 55

2.6.1 Preliminary Data Transformations ................................................................. 55

2.6.2 Planned Analyses ......................................................................................... 56

2.7 Sample Size Calculations ................................................................................... 58

2.8 Control of Confounders ...................................................................................... 59

3 RESULTS ................................................................................................................... 59

3.1 Demographic and Clinical Characteristics .......................................................... 61

3.2 Assay Results ..................................................................................................... 64

v

3.3 Biological Correlates of Post-Stroke Depression ................................................ 65

3.3.1 Hypothesis I: Inflammatory Markers of Depression ...................................... 65

3.3.2 Hypothesis II: Kynurenine Activation and Depression ................................... 69

4 DISCUSSION ............................................................................................................ 72

4.1 The Role of IDO Activation in Post-stroke Depression ...................................... 72

4.2 The Role of Cytokines in Post-Stroke Depression .............................................. 76

4.3 The Role of Hypertension in Post-Stroke Depression ........................................ 81

4.4 Limitations .......................................................................................................... 86

4.5 Recommendations for Future Research ............................................................. 88

4.6 Clinical Implications ............................................................................................ 91

5 REFERENCES .......................................................................................................... 96

APPENDIX .................................................................................................................. 122

Appendix A: Post-stroke Depression Study Patient Consent Form ......................... 122

Appendix B: Sunnybrook REB Approval Letter ....................................................... 126

Appendix C: York Central REB Approval Letter ...................................................... 129

Appendix D: Baycrese REB Approval Letter ........................................................... 130

Appendix E: St. John's REB Approval Letter ........................................................... 131

Appendix F: Toronto Research Institute Approval Letter ......................................... 132

Appendix G: Study Roles ........................................................................................ 133

Appendix H: Presentations and Publications ........................................................... 134

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LIST OF TABLES

Table 1: Mean KYN/TRP Levels in Various Depression, Neurodegenerative Diseases, and Inflammatory Illnesses ............................................................................................ 40

Table 2: Cyotkine Assay Sensitivities ............................................................................ 53

Table 3: Participant Ineligibility ...................................................................................... 61

Table 4: Participant Demographics and Assessment Scores by Depression Group ..... 62

Table 5: Stroke Lesion Characteristics by Depression Group ....................................... 63

Table 6: Cerebrovascular Risk Factors and Concomitant Medication Use by Depression Group.......................................................................................................... 64

Table 7: Distribution of Hypertensive Patients on Anti-Hypertensive Medication ........... 69

Table 8: Measured and Imputed Cytokine Plasma Levels by Depression Group .......... 70

Table 9: Cytokine Detectability by Depression Group.................................................... 70

LIST OF FIGURES

Figure 1. The KYN Pathway: TRP Metabolism and its Products .................................. 35

Figure 2. Patient Recruitment ........................................................................................ 60

Figure 3. Mean KYN/TRP ratios in stroke patients with and without depressive symptoms ..................................................................................................................... 65

Figure 4. Box-plot of unadjusted KYN/TRP ratios in stroke patients with and without depression ..................................................................................................................... 66

Figure 5. Correlation between CES-D total scores and KYN/TRP ratio ......................... 66

Figure 6. Correlation between LNAA concentrations and KYN/TRP ratio ...................... 67

Figure 7. Mean CES-D Scores by Hypertension Group ................................................. 68

Figure 8. Correlation between unadjusted IL-6 concentrations and KYN/TRP ratio ...... 71

1

1. INTRODUCTION

1.1 Statement of the Problem

Stroke is the third leading cause of death in Canada, with more than 50,000 strokes

occurring annually1. This figure amounts to approximately one stroke for every 10 minutes of

time that has elapsed. About 30% of stroke victims do not survive the acute attack1. For the

survivors, rehabilitation involves addressing the physical, cognitive, psychological and

psychosocial impairments that are common post-stroke sequelae. One of the major

psychological changes observed in post-stroke patients is depression.

Depression is a significant health problem among the elderly population leading to

diminished quality of life2, impaired daily functioning

3-6, greater health service use

7, poorer

perceived health7 and increased mortality risk

8. With regards to stroke, depression occurs in

approximately one-third of all reported cases9-14

and is the strongest predictor of quality of life

(QoL)15-18

. Consequentially, post-stroke depression (PSD) patients have increased functional

disabilities19-23

, increased health service utilization24, 25

, increased cognitive impairment26

,

higher mortality rates27-31

even after controlling for other factors such as associated illnesses and

prior stroke27, 28

and poorer rehabilitation outcomes32-34

compared to non-PSD patients.

Moreover, stroke survivors are at elevated risk for clinically significant depressive symptoms

even two years after stroke onset35

.

Although antidepressant medications can be prescribed, the drugs are not efficacious in

all depressed survivors despite the availability of different formulas acting upon distinct

neurological pathways. Even when effective for depressive symptoms, post-stroke cognitive

impairments frequently persist36

. Additionally, response rates to antidepressant treatments are

inconsistent between studies partly due to variable definitions of response. The high prevalence

of depression post-stroke (about three times greater than the general population) combined with

2

the impact of these symptoms on antidepressant response highlights the urgency to discover

possible pharmacological targets for disease management. The generation of reliable data

regarding the neurological, physical and social correlates impacting PSD is a crucial factor in

the field of stroke recovery. Ultimately, a sound understanding of PSD etiology may lead to

novel therapeutic approaches that can propagate into successful management of this stroke

outcome.

1.2 Purpose of the Study and Objectives

PSD remains profoundly problematic due to its high prevalence, negative impact, and

lack of effective treatments. Several hypotheses regarding the etiology of PSD have circulated

since the early 1980s, beginning with Robinson’s lesion location theory37

. It was published that

stroke victims suffering from left anterior lesions were more prone to PSD than those of right or

posterior lesions. His hypothesis not only stimulated several debates, but also generated the

formation of subsequent hypotheses by other groups exploring other biological and psychosocial

components relevant to the disease. Despite such increasing interest in PSD research, no

conclusive data have been collected to date. The primary objective of this study was to assess

novel biological components that may contribute to the etiology PSD. In particular,

concentration changes of specific inflammatory markers and their ability to modulate tryptophan

(TRP) metabolism post-stroke was investigated as a possible cause of PSD. Tryptophan was of

special interest since it acts as a precursor to both the mood modulatory neurotransmitter

serotonin and neurotoxic kynurenine (KYN) metabolites.

KYN is formed by metabolism of TRP by indoleamine 2,3-dioxygenase (IDO);

therefore, an increase in IDO activity as stimulated by increases in pro-inflammatory cytokines

post-stroke is thought to initiate the development of PSD. Here, it is hypothesized that a

decrease in serotonin synthesis accompanied by an accumulation of neurotoxic KYN

3

metabolites act as a possible mechanism of the disease. This study measured the KYN/TRP ratio

as a marker of IDO activity in order to elucidate the hypothesis. Additionally, measurements of

pro- and anti-inflammatory cytokines were taken and analyzed to examine the relationship

between cytokines, IDO, and PSD. Finally, demographic and clinical factors were characterized

between depressed and non-depressed stroke patients in order to elucidate any markers that may

contribute to post-stroke inflammatory response mechanisms.

1.3 Statement of Research Hypotheses and Rationale for Hypotheses

1.3.1 Primary Hypothesis

Hypothesis 1: Increased IDO enzyme activity will be found in stroke patients experiencing

depressive symptoms compared to non-depressed stroke patients as evidenced by an increased

kynurenine/tryptophan (KYN/TRP) ratio.

Rationale: Initially, an increased KYN/TRP ratio was documented among depressed

immunotherapy patients38

, in post-partum depression39

, and in major depression40

. Most

recently, our group demonstrated that an elevated KYN/TRP ratio was associated with

depression among coronary artery disease patients, a group similar with respect to age and a

high prevalence of cerebrovascular risk factors41

. It is hypothesized that post-stroke stimulation

of IDO activity increases the production of KYN from its TRP precursor. Since TRP also acts

as a precursor to serotonin synthesis42

, decreased levels of serotonin are left available for

synapses between mood regulatory neurons. In addition, KYN can be metabolized into

neurotoxic metabolites43-45

that may cause further damages to areas of the brain responsible for

processing emotions. The effect of decreased serotonin synthesis accompanied by an escalation

of neuronal injuries is hypothesized to play a major role in the etiology of PSD. Thus, it is of

interest to measure IDO activity as a potential marker of disease progression. Since IDO has an

extremely short half life, an indirect measurement of enzyme activity (KYN/TRP ratio) will be

4

utilized instead. The KYN/TRP ratio has been used as a sensitive estimate of IDO activity and

cellular immunity both in vivo and in vitro since the late 1980s

46, 47.

1.3.2 Secondary Hypothesis

Hypothesis 2: Increased levels of pro-inflammatory cytokines (IL-1, IL-6, TNF-, IFN-) and

decreased levels of anti-inflammatory cytokines (IL-10) will be found in stroke patients

experiencing depressive symptoms compared to non-depressed stroke patients.

Rationale: Early pre-clinical studies have documented several cases of behavioural disturbances

in animals administered a variety of pro-inflammatory cytokines48, 49

. These behavioural

changes were collectively termed ―sickness behaviors‖ and are thought to mimic those of

depressive symptoms observed in humans50, 51

. More recent experimental and epidemiological

studies have reported higher levels of inflammatory markers within the nervous and circulatory

systems of depressed people52-58

. Marked increases in peripheral pro-inflammatory cytokines

have been documented in several post-stroke cases59-64

. By linking the two findings together,

the development of depression in immunocompromised patients is thought to be cytokine-

mediated. This theory has been supported by reported cases of depression after cytokine

immunotherapy65-68

where pro- and anti-inflammatory cytokines are thought to have opposite

effects. Although data regarding the role of pro- and anti-inflammatory cytokines remain

controversial, it has been hypothesized that a balance between both inflammatory markers is

crucial for maintaining mental fitness69-71

. According to the mechanism proposed for the

primary hypothesis, elevations in pro-inflammatory cytokines and reductions in anti-

inflammatory cytokines contribute to IDO activation and may contribute to PSD.

5

1.4 Review of the Literature

1.4.1 Post-stroke Depression

1.4.1.1 Prevalence

One systemic review of observational studies reported a 33% prevalence of PSD at any

time after stroke10

. Although this number concurs with most PSD studies, it is difficult to define

the true prevalence of PSD due to methodological (evaluation time, study setting,

inclusion/exclusion criteria) and diagnostic (different cut-off scores in rating scales, non-

consistent use of structured-clinical interviews, clinical findings only) variability present

amongst studies. PSD may also be over-diagnosed due to similar somatic symptoms produced

by other medical illnesses, or under-diagnosed particularly in patients with co-morbid cognitive

impairment. The correct attribution of somatic symptoms is important since it is relevant to

accurate scoring of depression severity scales, namely the Hamilton Depression Scale (HAM-

D), Montgomery Asberg Depression Rating Scale (MADRS) and Beck Depression Inventory

(BDI). Furthermore, the frequency of PSD may increase in prevalence over time since

symptoms may even begin to appear one year post-stroke irrespective of the level of handicap13,

72, 73. Conversely, some patients with early onset PSD (0-3 months post-stroke) are not

necessarily affected at one year follow-up74, 75

. Ultimately, quantification of PSD prevalence

proves to be challenging due to the dynamic nature of the disease in both its early and late

stages.

1.4.1.2 Risk Factors

Although the prevalence of PSD remains unpredictable, the disorder has been

documented to affect a high percentage of stroke victims (at least 30%). Thus, it is important to

investigate predictive demographic factors early after stroke onset in order to determine those at

highest risk for the disease. One study has shown that stroke severity, non-lacunar stroke

6

subtype and the melancholy index of the HAM-D (which includes depressed mood, feelings of

guilt, work inhibition, psychic anxiety and general somatic symptoms) were significant risk

factors for PSD at 3 months follow-up76

, and adjustment for stroke severity increased the

predictive value of the melancholy index at the same follow-up. Other studies have

demonstrated that prior personal or family history of major depression75

, poor social support74

,

greater impairment in activities of daily living (ADLs)74, 77, 78

, cognitive impairment79

and one or

more negative life events during the six months prior to stroke75

increases risk for PSD. Time

needed for recovery may also play an important role as it has been documented that those who

have not yet recovered from the disease at the one year mark have a high risk of developing

chronic depression74

. Despite these separate findings a more recent systematic review reported

that only the degree of physical disability, stroke severity and cognitive impairment are

consistently associated with the disease80

.

Because stroke often occurs in discrete lesion locations, researchers have examined the

neuroanatomical correlates of depression to identify regions that would put one at most risk of

PSD. Currently, literature surrounding lesion location remains controversial. There is some

evidence that PSD is accompanied by left anterior and left basal ganglia lesions and proximity

to the frontal pole has been positively correlated with depressive symptoms37, 81-85

. However,

these results have not been consistency replicated23, 86

and the reasons for these discrepancies

are not known. Thus, ready identification of patients at risk for PSD remains complicated.

Further studies are necessary to support, add to, or even contrast the current evidence available

before a sound pattern of PSD risk factors can be elucidated.

1.4.1.3 Demographics

To date, little is understood on the demographic determinants related to PSD onset.

Whether age increases the likelihood of developing PSD remains controversial. A couple of

7

studies support a correlative relationship between the two variables16, 87

while others report no

association between them9, 19, 88, 89

. With gender, there have been reports that women are more

likely to suffer from depression in the general population; however many studies have found

minimal to no effect between gender and PSD16, 22, 87, 89, 90

. Despite these negative findings, a

more recent systemic review evaluating sex differences in the prevalence of PSD reported the

disease to be slightly more common among women than men after combining post-stroke cases

ranging from less than two weeks to 15 years91

. The same factors explaining the greater

prevalence of depression in women among cardiac patients may also be applicable to post-

stroke cases, including elements of genetics, psychosocial inequities, issues related to recovery,

differential support and access to rehabilitation92

. Interestingly, women93, 94

and stroke patients

less than 59 years of age94

have been found to be more susceptible to post-stroke anxiety (PSA),

a disease that often manifests with PSD.

More recently, racial and ethnic disparities have been examined as potential

demographic correlates for PSD. In one study, white, non-Hispanic acute stroke patients from a

national cohort of Department of Veterans Affairs were more likely to be diagnosed with PSD

even after adjusting for sociodemographic and clinical characteristics95

. Another study

examining the relationship between ethnicity and risk for post-stroke mortality reported that

Non-Hispanic Black veterans with a history of stroke had a higher risk for mortality than did

Non-Hispanic White veterans96

. Because PSD patients have been shown to have a higher

mortality risk than non-depressed stroke patients27-31

racial and ethnic factors may be important

predictors in the development of the disease. At this time, it remains unclear whether racial and

ethnic backgrounds can be utilized as correlates for PSD.

Whether the stroke victim was an inpatient or an outpatient may be predictive of PSD. A

systematic review reported that inpatients with left anterior lesions were significantly more

8

likely to have MDD than outpatients97

. Note however, that this finding was only applicable to

the acute stroke period as no difference in mood was found between in- and outpatients during

the chronic stroke period.

1.4.1.4 Clinical Diagnosis of PSD

It is well-known that both major and minor depression are possible consequences of

stroke, though the true prevalence of each remains less certain. Major depression can be

accurately diagnosed in those with stroke98, 99

using the Diagnostic and Statistical Manual

Version of Mental Disorders, Fourth Edition (DSM-IV)100

. According to the manual, a patient

is diagnosed with major depression if at least five of the following nine symptoms are met:

depressed mood, anhedonia, appetite/weight change, insomnia or hypersomnia, psychomotor

agitation or retardation, fatigue or loss of energy, feelings of worthlessness or guilt, diminished

concentration, or recurrent thoughts of death. The five symptoms must include either depressed

mood or anhedonia and must persist for at least two weeks prior to evaluation. In contrast,

minor depression must only meet three of the previously mentioned nine symptoms with either

depressed mood or anhedonia listed as one of the three symptoms. Although studies have

documented major depression to occur in approximately 25%89, 98

of all stroke patients, the

occurrence of minor depression remains more elusive, with a reported prevalence ranging from

14-31%89, 98

.

Currently, there is no consensus on how to define "early" and "late" onset PSD.

However, Robinson demonstrated that 30% of stroke survivors who are non-depressed at initial

evaluation in an acute hospital setting will be diagnosed with depression 6 months later101

.

Following that criterion, one study examined the individual characteristics of early and late-

onset PSD, where early and late-onset were described as 3-6 months and 24 months post-stroke

respectively. It was found that the frequency of melancholic, vegetative, and psychological

9

symptoms and morning depression were significantly greater in patients with early-onset post-

stroke major or minor depression than in patients with late-onset major or minor depression102

.

Furthermore, early-onset post-stroke major depression was associated with larger lesion

volumes, and late-onset post-stroke major and minor depressions were associated with poorer

social functioning. Another study reported that early-onset PSD is characterized by anxiety,

loss of libido and feelings of guilt, whereas later-onset PSD is more frequently associated with

diurnal variation of mood and social isolations103

. Still, the prognostic differences and clinical

patterns between early-onset and late-onset PSD remain quite vague and are suspected to result

from differences in the biological or psychological processes acquired from the stroke.

Recently, diagnosis of PSD has shifted the emphasis from expressed mood and feelings

to the patient’s affect and behavior104

. This is especially true in cases where PSD is complicated

by significant neurological and functional impairment. Dominant hemispheric strokes are often

associated with aphasia making it difficult for patients to articulate thoughts and emotions of

sadness, hopelessness and guilt. On the other hand, patients with non-dominant hemisphere

stroke may still have intact language but be stricken with speech prosody, thus affecting their

ability to comprehend the importance voice tone and attitude expression in their speech (e.g. the

ability to imply sarcasm through tone of voice).

Patients with language impairments are not the only group of individuals who require

special attention. Several somatic and vegetative symptoms that are important clinical features

of PSD may be misattributed to a simple distaste for hospital environment. For example, weight

loss due to post-stroke dysphagia may be ascribed to a disliking for hospital food. Insomnia

may be noted as difficulty sleeping in a hospital setting rather than a symptom of depression. In

all cases, the opposite effect may also be true where patients are misdiagnosed with depression

due to misinterpretation of the aforementioned symptoms. Note, that vegetative symptoms are

10

significantly more common in depressed patients over the first two years post-stroke103

and that

certain somatic symptoms, specifically loss of appetite, psychomotor retardation and fatigue

have been found to be predictive of depression99

. Thus, it remains crucial for physicians be

attentive to such symptoms despite their unspecific nature.

1.4.1.5 Hypertension and the Vascular Hypothesis of Depression

Cerebral blood flow (CBF) and vasomotor reactivity are specific hemodynamic changes

that play a major role in stroke. Once CBF falls below threshold, delivery of vital nutrients

through the brain's microvasculature becomes compromised. This subsequently leads to

detrimental effects on neuronal functioning. If further decline in regional CBF occurs, cellular

mechanisms governing ionic equilibrium are also jeopardized105

. Furthermore, patients with

stroke or transient ischemic attacks have reduced vasomotor reactivity, indicating that cerebral

arterioles are unable to dilate in order to compensate for increased blood demand106

.

Hypertension is well-known to the public by its simplest definition--a condition

characterized by consistently elevated arterial blood pressure. However, hypertension entails

much more complicated hemodynamic changes than the previous definition describes. With

respect to blood vessels, it is characterized by two main factors: (1) the structural remodeling of

cerebral arteries via increasing wall thickness/lumen ratio107

and (2) impaired endothelium-

mediated vasodilatation as a result of collagen build-up108

. Ultimately, the effects of decreased

lumen size and decreased vessel distensibility may reduce both CBF and cerebrovascular

reactivity. Abnormal changes in regional CBF have been documented in the past where

hypertensive subjects had decreased CBF to the subcortical regions of the brain as well as to the

limbic and paralimbic structures109

. Thus, not only may hypertension create a vulnerability state

for the development of neurodegenerative disorders via oxidative stress mechanisms, but it may

also be relevant to the development of depression since limbic structures of the brain are widely

11

known to regulate emotions and behaviour. Indeed, both of the aforementioned hemodynamic

properties have previously been found to be impaired in subjects with depressive symptoms110,

111 where a reduction in blood flow velocity was especially prevalent in subjects suffering from

a DSM-IV depressive disorder110

. Furthermore cerebrovascular reactivity (CVR) is

significantly improved after antidepressant treatment at 21 months follow-up111

. Interestingly,

CVR has also been found to be significantly reduced in a group of depressed patients free of

vascular risk factors112

. This suggests that major depression may be the actual cause of CVR

function rather than its consequence. A combination of results from both studies suggests that

the link between hypertension and depression may be bidirectional with both leading to

increased risk for stroke.

Because depressive syndromes have been shown to be comorbid with cerebrovascular

lesions and cardiovascular risk factors, a vascular hypothesis to depression was introduced by

Alexopoulos and colleagues in the late 1990s. Here, it was stated that ―cerebrovascular disease

may predispose, precipitate, or perpetuate some geriatric depressive symptoms‖, and are

uniquely attributed to cases of late-onset depression113

. Further research has described the

symptomatology of the disease as distinct from non-vascular depression with more cognitive

impairment, prominent psychomotor retardation, apathy, lassitude and pronounced disability114

.

However, not all studies report a specific symptom profile of vascular depression115

.

Nevertheless, the vascular depression hypothesis remains a topic of interest as positive

associations between cardiovascular risk factors (i.e. hypertension114

, hyperlipidemia116

),

cerebro-117

and cardiovascular diseases118

, diabetes119

and depression have been documented in

the past.

In terms of size and location of the stroke lesion, Alexopoulos and his team postulated

that the neurocircuitry responsible for processing emotions can be affected by not only one large

12

infarct but by several macrovascular or microvascular lesions. Here, neuroimaging data

consisted of acute infarctions and white matter changes confined to three frontal-subcortical

loops of the brain that were later described to be key features of vascular depression. In support

of the multi-lesion hypothesis, one study found no relationship between focal vascular

pathology and PSD in post-mortem elderly subjects31

. This finding suggests that the vascular

burden from chronic accumulation of multiple lesions may be a crucial determinant to the

etiology of PSD rather than a single large infarct from stroke120

. Two years prior, Brodaty and

colleagues proposed the same neuroanatomical change of focus for PSD stating ―depression

after stroke may be related to cumulative vascular brain pathology rather than side and severity

of single stroke‖121

.

1.4.1.6 Treatment of PSD

Currently, antidepressants are prescribed to either prevent the occurrence of PSD or as

treatment of a newly diagnosed case. In 2005, a Cochrane review was published examining the

efficacies of both indications for therapy within a post-stroke population122

. The results,

however, were discouraging as no evidence of improved mood or cognitive function was found

in either the treatment or prevention group. Even at best, there were only some documented

effects of reduced depression severity within the treatment group. These negative findings may

reflect heterogeneity between studies, including differences in patient characteristics, methods

of diagnosis, assessment tools of depression and cut-off scores used in those forms of

assessments. Furthermore, patient recruitment varied from a few days to two years after stroke

onset. This likely complicated the analysis since the balance of risks and benefits of

pharmacotherapy may change over various stages of stroke recovery. Finally, the primary goals

of therapy in many trials were unclear which made it difficult to define important outcomes such

as remission and recovery. As a result of the methodological limitations encountered during the

13

review process, no successful recommendations regarding the applicability of antidepressants to

post-stroke conditions were made.

In contrast to the Cochrane review, more recent data supports the use of antidepressants

for the prevention of the disease. One RCT examining the SSRI escitalopram found the drug to

be significantly superior to placebo in preventing the development of PSD123

. The same study

also reported that problem-solving therapy, a manual-based intervention that originated from

England for treating medical patients with depression124

, was significantly superior to placebo.

Other classes of antidepressants have also been effective in preventing PSD, though the study

designs were less rigorous. In one study, 5.7% of patients receiving the tetracyclic acid (TeCA)

mirtazapine developed PSD compared to 40% of non-treated patients125

. While those

proportions were significantly different, the study was not placebo controlled and participants

were not blinded to treatment, which likely introduced a bias in favour of mirtazapine. As well,

more than 90% of people screened for recruitment were not enrolled as participants.

Milnacipran, which can be classified as a serotonin and norepinephrine reuptake inhibitor

(SNRI), was given open label to 11 patients and compared to historical controls and found to be

associated with improved PSD in rehabilitative in-patients126

. Finally, a 2007 meta-analysis

exploring prophylactic uses of antidepressants significantly favoured the treatment group

outcome. The study examined 10 randomized clinical trials where a total of 703 non-depressed

post-stroke patients were identified and reported a difference in PSD occurrence rate of about

17% between treated and non-treated groups (PSD occurrence of 29.17% and 12.54% in non-

treated and treated groups, respectively)127

. The authors also noted that the prophylactic effects

of antidepressants were not related to duration of use. However, the previously quoted studies

were the basis of that meta-analysis, thus study quality was a problem.

14

Additional publications suggest that antidepressants may reduce other post-stroke

sequelae. Several studies have suggested their positive effect on abating symptoms of cognitive

impairment that are often found co-morbid with PSD128-130

, and these improvements are likely to

remain stable over the next 2 years in the absence of subsequent reinjury to the central nervous

system131

. It has been suggested that post-stroke recovery from impaired activities of daily

living (ADLs)132

and cognition133

, and reductions in aggression and irritability134

may also be

enhanced by antidepressants if treatment were to begin within the first few months of stroke

onset. Some physicians opt to prescribe antidepressants to stroke patients before a formal

diagnosis of depression is made in order to prevent such disabilities. Although the prophylactic

use of these drugs has been reported to significantly reduce the occurrence of PSD127

, it does not

completely eradicate the disease and may manifest into different disease characteristics of PSD

compared to patients without treatment. For example, one study randomly assigned non-

depressed patients to receive nortriptyline, fluoxetine, or placebo for three months and examined

the occurrence of depression thereafter135

. In patients treated with antidepressants, lesion

volume and degree of social impairment were associated with late-onset PSD. In contrast, those

who were on placebo developed PSD associated with the severity of ADL impairments. The

differences in the clinical and pathological correlates between groups suggest that the

pathophysiology of the disease changes with the use of antidepressants, and that these subtle

differences may be important to post-stroke recovery.

Physicians are often reluctant to prescribe antidepressants to older stroke patients co-

morbid for other medical illnesses due to the perceived risk of side effects (e.g. drug-drug

interactions) leading to morbidity. Therefore, the selection of the antidepressant class with the

lowest risk/benefit ratio is of great importance. Both selective serotonin reuptake inhibitors

(SSRIs)136-138

and tricyclic antidepressants (TCAs)139

have been suggested to significantly

15

reduce depressive symptoms in some PSD patients. However, the use of TCAs in elderly

populations with vascular disease remains a safety concern among the medical community. A

systematic review comparing SSRIs and TCAs in patients vulnerable to stroke and

cardiovascular disease found that those receiving TCAs were at significantly greater odds of

developing non-serious cardiovascular adverse events (AEs) than those on SSRIs140

. These

non-serious events include the following: heart palpitations, chest pain, angina, arrhythmia,

hypertension and hypotension-syncope. Note, however, that serious cardiovascular AEs such as

death, cerebrovascular disease, heart failure, TIA, stroke and MI were not associated with the

use of TCA.

Interestingly, a couple of studies have documented potential benefits to SSRI use in

cardiovascular disease patients. Most notably, congestive heart failure (CHF) patients on active

aspirin medication yielded additional anti-platelet benefits during concomitant therapy with

SSRIs141

. Prior data from the same research group found that the SSRI sertraline and its

neurologically inactive metabolite N-desmethylsertraline (NDMS) exhibited potent, dose-

dependent antiplatelet activity in both in vitro

142 and ex vivo

143 clinical scenarios in patients

undergoing coronary stenting. These findings suggest that the use of SSRIs after ischemic

stroke may translate into improvement of depressive symptoms and a decreased rate of mortality

that would previously have been caused by unwanted cardiovascular AEs.

The findings above are likely the reason why SSRIs remain the most recommended

treatment for PSD due to their believed favourable tolerability (e.g. less serious cardiovascular

side effects, lack of anticholinergic effects)144, 145

. However, one must not discount the negative

side effects of SSRIs, as they are known to elicit sexual dysfunction, weight gain, and sleep

disturbances during long-term therapy146

. A recent study in post-menopausal women reported

that those using SSRIs had a 45% increased relative risk of incident stroke and 32% increased

16

risk of death compared to those with no antidepressant use

147. When analyzed by stroke type,

SSRI use was associated with incident hemorrhagic stroke and fatal stroke. The results are

consistent with prior evidence suggesting SSRIs may increase the risk of abnormal bleeding due

to their antiplatelet effects when combined with aspirin141

. Although the risk of death was

similarly increased among TCA users, no significant differences in risk between SSRI and TCA

users were found. However, it remains difficult to determine whether the non-significant results

can be attributed to drug-type exposure alone or unaccounted differences in other cardiovascular

risk factors. For example, depression has been an established risk factor for cardiovascular

morbidity and mortality148

and has been associated with an increased risk of stroke149

. This

raises the possibility that any ―excess‖ risk to stroke may be caused by depression and not solely

on drug use. Thus, it is necessary to assess the effect of different antidepressants specifically on

the PSD population in order to clarify their side effects. The optimal length of treatment is

unclear, though one research group recommends carrying on treatment for 4-6 months followed

by slow withdrawal of the drug150

. Conversely, one meta-analysis reported that the prophylactic

effects of antidepressants were not related to the duration of drug intake127

. Since there has been

several documented cases of robust placebo responses during antidepressant trials151

, it would

be necessary to examine the significance of placebo-effects before determining the best

treatment time-course for PSD patients.

1.4.1.7 Co-morbid Depression and Cognitive Decline Post-stroke

Cognitive symptoms are hypothesized to be indicators of chronic depression in the

elderly and have been found to persist in major depressive disorder (MDD) outpatients in

remission152-156

. Although the appearance of cognitive deficits remains under scrutiny, several

studies suggest that it may be immune-mediated. For example, higher levels of inflammation

during baseline assessments have been shown to predict cognitive symptoms at follow-up in

17

depressed patients157

. Thus, inflammation may act as an initiator and contributor to cognitive

symptoms of depression in the overall progression of the disease.

It has been reported that cognitive function and depressive symptoms are independently

associated with risk of mortality within the elderly population158

. Here, the study participants

were stratified into best, middle and worst groups based on scores collected from various

depression and cognition scales. It was found that older participants in the worst cognitive

function or worst depressive symptoms tertile at baseline had higher rates of mortality than

those in the middle and best tertiles. Furthermore, for each grouping of cognitive function,

greater depressive symptoms were associated with higher mortality rates, and for each grouping

of depressive symptoms, worse cognitive function was associated with higher mortality rates.

Thus, the combination of poor cognitive function and depressive symptoms appear to increase

the likelihood of mortality in an additive fashion, suggesting that the risk for poor outcome is

best explained by the cumulative effects of two or more risk factors.

With relation to post-stroke periods, one study related cognitive functioning to

depression and anxiety whereby verbal learning and reduced cognitive speed were correlated

with increased depression and anxiety scores159

. Furthermore, stroke patients with both

depression and cognitive impairment have been shown to present with longer durations of

depression than depressed patients without cognitive impairment160

. This finding was most

pronounced during the initial evaluation period of the study and highly associated with left

hemispheric lesions. Whether the acute post-stroke period and left hemispheric lesions are

absolutely related to co-morbid depression and cognitive decline requires further research.

However, it is apparent that cognition and mood post-stroke are linked and neither should be

ignored during the rehabilitative process.

_____________________________________

18

1.4.2 Inflammatory Processes and Stroke

1.4.2.1 Time course of inflammatory molecules post-stroke

Experiments inducing focal ischemia in rats via medial cerebral artery occlusion

(MCAO) have given insight to the inflammatory events that occur post-stroke. Circulating

monocytes have been detected within the capillaries and venules after 4 to 6 hours of induced

ischemia161

. The study also detected accumulation of polymorphonuclear neutrophils (PMNs)

12 hours after the same injury161

, though PMN infiltration may occur within 6 hours and with

greater severity in reperfused tissues than in permanently occluded tissues162

. Mechanistically,

it has been reported that leukoctyes and PMNs exacerbate tissue damage via edema formation,

physical obstruction through adherence to the vessels, and the release of oxygen radicals, pro-

inflammatory cytokines, and cytolytic enzymes leading to cellular necrosis162-164

. In support of

these findings, administration of inflammatory markers into rat models has been found to

augment increases in brain water content and tended to correlate with neutrophilic infiltration

into the parenchyma165

.

1.4.2.2 Cytokines

Cytokines are small, soluble glycoproteins (~15-25kd) that function locally or

systemically to orchestrate immune responses. They are produced by a variety of activated cell

types including endothelial cells, platelets, leukocytes, and fibroblasts166

. Additionally,

autocrine, paracrine, or endocrine effects are possible, depending on the particular cytokine105

.

Once released into the environment in response to an activating stimulus (e.g. stroke), they

control the growth and differentiation of different cell types through specific membrane

receptors105

. Ultimately, intracellular signaling pathways are activated and alter gene

transcription patterns within the cell, one of which modulates the expression of their own

specific target cell receptor.

19

In addition to being mediators of inflammation, cytokines also induce the expression of

cell adhesion molecules on endothelial cells that propagates cerebral ischemia and contribute to

tissue damage via alterations in levels of endogenous mediators such as tissue-plasminogen-

activator (tPa)105

. One post-mortem study of elderly patients showed that intercellular adhesion

molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were increased in the

cerebral endothelium in late life depression167

. Combining these two findings suggests a

possible causal relationship between inflammatory cytokines and MDD.

1.4.2.3 Cytokines and Stroke: Pro- Versus Anti-inflammatory Cytokines

Cytokines are not stationary inflammatory markers and thus, are able to coordinate

immune responses between several physiological systems in the body. Post-stroke plasma

elevations of different cytokines168-172

(including IL-1, IL-6, IL-8, and TNF-) and their

mRNA expression in mononuclear cells168-172

have been previously reported and are

significantly correlated with brain infarct volume171, 173

, functional disability173

, risk of recurrent

stroke172

, and less favourable prognosis171

. Thus, not only is the inflammatory response related

to stroke severity, but clinical outcome as well. Two major categories of cytokines are the pro-

and anti-inflammatory cytokines which play opposite roles in regulating stroke outcome.

Pro-inflammatory cytokines (IL-1, TNF-α, IL-6, IL-8 and IL-18) function to amplify

immunological responses during reperfusion injury after acute cerebral ischemia. However,

they may also induce the depressogenic effects in depressed patients174, 175

. For example,

increased levels of IL-1β, TNF-α and IL-6 have been observed in the cerebrospinal fluid

(CSF)176, 177

and plasma178-183

of patients suffering from mood disorders. In addition, inhibition

of these cytokines and their respective signaling pathways can improve depressed mood184

.

Furthermore, both acute and chronic infectious and non-infectious diseases that primarily affect

immune regulation are often reported to be accompanied by depression185-188

. Note, however,

20

that the opposite is also true where immune dysregulation is found to be common in major

depression disorder, suggesting that depression by nature may be an autoimmune disease189

.

In contrast to pro-inflammatory cytokines, anti-inflammatory cytokines (IL-10, IL-4 and

insulin-like growth factors (IGF)) are proposed to have beneficial effect to the injured brain. In

one animal model, IL-10 administration to MCAO treated rats reduced infarct size compared to

vehicle treated controls190

. The opposite is also true where low levels of IL-10 have been linked

to larger infarct size191

and early neurological deterioration192

. Additionally, both IL-10 and

IGF have been shown to block lipopolysaccharide (LPS) and TNF- induced behavioural

changes in rats193, 194

. Although these results implicate neuroprotective properties of anti-

inflammatory cytokines to depression, their exact roles as immune modulators are clouded by

studies that have associated both increased192

and decreased195

IL-10 levels with poor outcome

and neurological worsening. Still, the balance between pro- and anti-inflammatory cytokines

must also be considered, highlighted by an elevated IL-6/IL-10 ratio in major depression69

.

Although the unfortunate prognosis of PSD has been well-documented, there is limited

research available explaining why certain post-stroke fates occur and the underlying

mechanisms involved in their development. With regards to mortality, the relationship between

pro- and anti-inflammatory cytokines may provide clues as to why the rate of mortality is

significantly higher in the PSD population compared to healthy aging individuals. Here, robust

evidence has been gathered from experiments supporting the development of an

―immunodepression syndrome‖, as summarized by two recent publications196, 197

. It is

hypothesized that the fate of injured brain tissue is dependent on the intricate balance between

pro- and anti-inflammatory cytokines where divergence from their naturally occurring

concentrations is thought to induce an immune response. Such prominent changes in

21

inflammatory markers are believed to facilitate systemic infection, thus resulting in a higher risk

for mortality post-stroke.

_____________________________________

1.4.3 Inflammatory Processes and Depression

1.4.3.1 Sickness Behaviour

Sickness behaviour is a collective term describing a constellation of behavioural

disturbances in animal models that mimic the depressive symptoms of humans. These

characteristics are comprised of malaise, decreased motivation (fatigue, lethargy, adipsia and

anorexia), decreased pleasure (anhedonia), psychomotor retardation, affective and cognitive

changes, hyposomnia or hypersomnia, decreased physical and social activities, poor

concentration and confusion198-202

. Current research suggests that inflammatory response

mechanisms may play a role in such behavioural disturbances. For example, administration of

the bacterial endotoxin LPS or inflammatory cytokines in animals produces some of the

behavioural characteristics encompassed in sickness behaviour48, 49, 203, 204

. Furthermore, the

absence of pro-inflammatory genes may inhibit the appearance of these depressive

characteristics. In support of this, one study found that mice deficient for the IL-6 gene are less

likely to exhibit the depressiogenic effects induced by LPS administration compared to mice

that do express the IL-6 cytokine205

.

1.4.3.2 Animal models of stroke

Behavioural correlates of depressive-like symptoms in animal models mainly include: 1)

reduced preference for sucrose solution when both the sweetened liquid and water are made

available206, 207

and 2) reduced locomotor and exploratory activity198

. The former observation is

an indicator of anhedonia and the latter suggests changes in incentive and motivation. A recent

study demonstrated both these symptoms in a novel animal model of stroke where MCAO

22

treated rats exhibited decreased locomotor activity and reduced sucrose solution preference208

.

Moreover, the observed behavioural abnormalities were ameliorated after the administration of

citalopram. These results are congruent with another study that also reported reduced sucrose

intake in MCAO-treated rats compared to control rats. However, the researchers took a

different approach to restore sucrose consumption. Instead, they administered the IL-1 receptor

antagonist (IL-1Ra) to MCAO-treated rats and found that it produced the same alleviation of

behavioural disturbances as antidepressants in the previously mentioned study209

. Results from

both findings not only confirm the presence of depressive symptoms post-stroke, but also

suggest that the disease may be immune-mediated.

1.4.3.3 Clinical Cases of Immunotherapy or Immune Challenges

Clinically, cytokines have been shown to be effective in the treatment of various medical

conditions including hepatitis C, leukemia, multiple sclerosis, Kaposi’s sarcoma, melanoma,

myeloma, renal carcinoma and other forms of cancer. However, cytokine immunotherapy may

also lead to pro-inflammatory molecule release causing depression210-217

, and may even

exacerbate preexisting psychotic symptoms218

. For example, the therapeutic use of cytokines

(e.g. IL-2, IFN-a) in the treatment of malignancies and chronic viral infections is frequently

complicated by cognitive, emotional, and behavioural disturbances219

. Moreover, antidepressant

therapy can prevent or attenuate cytokine induced depression220-223

and may even possess a

cytokine-linked anti-inflammatory effects222, 224-228

. These findings have opened up new

avenues for alternative treatments to depression, since pro-inflammatory cytokine inhibitors and

anti-inflammatory drugs may be able to prevent or attenuate depressive symptoms in humans.

In cases of immune challenges, one study found that mild stimulation of the primary host

defense in humans (using a safe and well-tolerated dose of endotoxin) produces negative effects

on both emotional and cognitive function229

. Elevations in both anxiety and depression levels

23

have been reported in other human cases of immune challenges186, 230

with those of lower

socioeconomic status most affected231

. All together, these findings suggest that pro-

inflammatory cytokines may play a crucial role in the pathogenesis of mood disorders in

humans, with implications for novel cytokine targeting therapies in neuropsychopharmalogical

research.

1.4.3.4 Cytokines in Clinical Depression and Cognitive Decline

The involvement of cytokines in the etiology of psychiatric disorders has been

extensively reviewed in the past by various authors174, 175, 184, 232, 233

. Under physiological

conditions, cytokine concentrations remain low in the body but are up-regulated during various

disease states, particularly those involving inflammation. With regards to depression, both

experimental and epidemiological research has found that depressed people have higher levels

of inflammatory markers52-58, 234

. For example, one study examined the levels of inflammatory

cytokines in a community-based sample of already depressed elderly subjects and found that

persons with depressed mood had higher median plasma levels of IL-6 and TNF-α57

. These

results became more significant after adjustments for health and demographic variables,

suggesting that depressed mood is either causing or caused by systemic inflammation.

Clinical studies have associated depression with increased levels of systemic cytokines

and acute phase proteins52, 58, 235-238

although such results were not always reproducible239, 240

.

Note, however, that the complex balance between pro- and anti-inflammatory cytokines may

play a crucial role in the development of mood disturbances, highlighted by reports of elevated

IFN- to IL-470, 241

and IL-6 to IL-1069

ratios in major depression. This may explain why a few

studies did not find a positive correlation between pro-inflammatory cytokines and depression

since none of them explored the significance of this balance. In addition, one of the authors

based their concentrations on lipopolysaccharide (LPS) induced peripheral blood mononuclear

24

cell (PBMC) cytokine production, which may be drastically different from the natural biological

concentrations of interest.

Cytokines are believed to affect cognitive function via active transport into the CNS

through afferent neurons that centrally activate cognitive responses, or upon local release by

activated microglia causing neurodegeneration242

. As previously mentioned, the

neuropsychiatric effects of cytokine therapy on cancer and HIV patients encompasses cognitive

changes that may involve a decline in verbal memory, cognitive speed and executive function216,

243, 244. Other forms of neurological disease associated with cognitive impairment have been

well documented in the past. Most notably, the severity of dementia in Alzheimer’s disease

patients is often accompanied with higher levels of circulating cytokines than controls, including

IL-1245-248

, IL-6247, 249, 250

, IL-18251

, and TNF-248

, although conflicting reports exist251, 252

. This

is not a disease related phenomenon since healthy aging individuals who experience cognitive

decline also exhibit higher levels of inflammatory markers253-257

independent of demographic

status and social status, and may also be at higher risk for mortality258

. Although the mechanism

of cytokine-induced cognitive changes has yet to be elucidated, it has been suggested to involve

damages to the fronto-subcortical circuitry. For example, one study found that stroke lesions of

the frontal lobe were associated with amusia (the absence of music perception), and that the

disorder also entailed general cognitive deficits in working memory, learning, semantic fluency,

executive functioning, and visuospatial coordination259

. Therefore, post-stroke elevations in

cytokines may contribute to post-stroke cognitive deterioration alongside depression.

The precise mechanisms behind cytokines and their ability to induce radical changes in mood

states remain unanswered. Generally, it is believed that increased peripheral cytokine levels

enter the brain and alter neurotransmitter metabolism, neuroendorcrine function, and neural

plasticity mechanisms responsible for mood regulation184

. Additionally, cytokine-mediated

25

interactions between neurons and glial cells may contribute to cognitive impairment (especially

in cases of memory and learning) via alterations in cholinergic and dopaminergic pathways242,

260. The next step would be to determine the cytokine concentrations and their time of activation

post-stroke to create a more detailed definition of their pathological outcome. Nevertheless,

elevations in cytokine levels may be useful prognostic markers for PSD despite their detrimental

effect to neuronal functioning.

_____________________________________

1.4.4 Current Hypotheses on the Etiology of Post-Stroke Depression

The etiology of PSD is thought to be multifactorial involving both psychosocial

vulnerability and biological mechanisms. In addition, several scientists believe that location of

the infarct plays a crucial role in understanding the mechanisms behind the disease. Thus, three

major hypotheses currently exist in the etiology of PSD: lesion location, psychosocial factors,

and biological factors261

.

1.4.4.1 Lesion Location Hypothesis

Those who support the lesion location hypothesis believe that PSD is directly caused by

focal damage to brain regions involved in the mood regulatory system. An early pioneer of the

lesion location hypothesis is Robinson and his team who were the first to report that left-

hemisphere lesions located to the vicinity of the frontal pole were more frequently associated

with PSD than lesions elsewhere in the brain82

. Since then a couple of other studies have

reported the same phenomenon74, 159, 262, 263

. These findings were encouraging to the lesion

location hypothesis since they support theories regarding: 1) frontal structures in the regulation

of emotional behavior and 2) lateralized differentiation in the organization of emotion, where

the left side of the brain is more often activated than the right side during emotional

processing264, 265

. In support of this, a MRI study found that left lesions of the frontal-subcortical

26

circuits (i.e. pallidum and caudate) predisposed stroke patients to depression with size of infarcts

being larger in depressed patients12

. Furthermore, the severity of depression has been associated

with left frontal lobe damage, with the exception of the basal ganglia266

. However, two more

recent systematic reviews267, 268

and several other studies76, 89, 269-273

failed to confirm the

association between left-sided strokes and depression, while one review stated such an

association varied depending on whether patients were sampled as inpatients or from the

community97

. Post-stroke cognitive impairment may also be related to lesion location as

comorbid MDD and cognitive impairment has been associated with left-sided lesions274

.

To counter the unsuccessful replication of their findings, Robinson and colleagues

carried out a meta-analysis275

and claimed to have found an interaction between lesion location

and time of PSD onset. Here, left-hemisphere lesions would increase the risk of PSD especially

in the first couple of months after stroke, whereas in the subacute to chronic course,

psychosocial factors would play a more important role. In addition, it was suggested that the

proximity of the left hemispheric lesion to the frontal pole was correlated with depression

severity, but not in cases of right-hemisphere stroke276

. However, prior to such claims, Gainotti

and his team had tested this same assumption and found that symptom profiles and the

anatomical and clinical correlates of major PSD were no different in the acute and chronic

stages of stroke270

. Currently, confirmation or rejection of Robinson and colleagues’ findings

has yet to be elucidated. However, many researchers agree that the etiology of PSD does not

and cannot rely on the lesion location theory alone.

1.4.4.2 Psychosocial Factors Hypothesis

The psychosocial hypothesis states that PSD is a psychological reaction to the physical

devastation caused by stroke whereby disability is the major predictor to disease development.

Several studies have supported this hypothesis22, 270, 277, 278

even after controlling for other stroke

27

characteristics including size and location of infarct85, 89, 279

. Lack of social companionship272, 273,

280 and absence of family support

74 have also been associated with the development of PSD and

are predictive of depression severity at six months post-stroke272

. Additionally, the development

of functional disabilities as a result of the stroke may lead to greater depressive symptoms which

would further complicate functional outcome20, 33, 281

. In support of this, improvement of

depressive symptoms post-stroke has been also associated with better functional recovery88

.

Although the severity of functional disability has been shown to be a strong predictor of

depression at 3 months post-stroke74, 85

the association disappears after around 12 to 24 months

post-stroke74, 85, 279

. Instead, few social contacts outside the immediate family and diagnosis of

the disorder at an in-hospital setting contributes most to depression at those times. Thus,

disability may only be a risk factor for depression in the acute and subacute post-stroke period,

where it does not determine the onset of depression per se, but interacts with it to limit the

results of long-term rehabilitation280

. These suggestive findings have led to the hypothesis that

PSD is at least, in part, a consequence of the direct biological consequence (e.g. immune

response) of the stroke.

1.4.4.3 Other Biological Factors

It has been hypothesized that during acute brain infarction, there is decreased

monoamine synthesis leading to decreased serotonin levels in the brain. In blood, serotonin is

present in high concentrations in platelets and released into the plasma when platelets aggregate

at the site of tissue damage. It is then subsequently catabolized by monoamine oxidase enzyme

activity in the liver and lungs. In the CNS, serotonin is present in high concentrations in

localized regions of the midbrain, serving as a neurotransmitter. The level of CSF or brain

serotonin remains controversial—some studies associate MDD with reduced levels of CSF

serotonin and increased levels of serotonin turnover282, 283

whereas other studies find no

28

differences in CSF concentrations of serotonin metabolite (5-hydroxyindoleacetic acid) between

depressed and healthy subjects284, 285

.

It has been proposed that PSD is caused by ischemic brain lesions that directly disrupt

neural circuits involved in mood regulation286

. In particular, it has been postulated that stroke

lesion interrupts the biogenic amine containing axons ascending from the brainstem to the

cerebral cortex, thereby leading to a decreased production of serotonin and norepinephrine in

uninjured limbic structures of frontal and temporal lobes as well as basal ganglia287

. Thus,

deficits in important mood regulatory neurotransmitters or failure of the body to up-regulate

their respective receptors after stroke may trigger PSD. This hypothesis has been supported by

studies demonstrating significantly lower CSF and plasma concentrations of serotonin288

and

CSF concentrations of its metabolite, 5-hydroxy-indoleacetic acid289

, in PSD patients compared

to non-depressed stroke survivors. Moreover, one study reported an inverse correlation between

serotonin receptor binding in the left temporal cortex and HAM-D scores of depressed

patients290

.

In further support of the biological hypothesis, one study found that the most commonly

reported symptoms in a group of post-stroke patients were discouragement/hopelessness, self-

criticalness, tiredness and feelings of being punished159

, all of which describe non-physical

symptoms. Furthermore, a community-based post-stroke cohort was found to have an elevated

risk for clinically significant depressive symptoms that was not mediated by functional

disability35

. Lastly, one study reported an association between the presence of small silent

strokes and late-onset depression, suggesting that patients who are unaware of their existing

cerebral lesions develop depression independently of psychological mechanisms related to

functional debilitation291

.

_____________________________________

29

1.4.5 Neurological Consequences of Reduced Serotonin Synthesis and the Formation of Neurotoxic Metabolites 1.4.5.1 Serotonergic Hypothesis of Depression

The serotonergic hypothesis of depression has been in existence for over 40 years292

and

proposes that diminished activity of 5-HT pathways is responsible for the pathopysiology of

depression. The hypothesis was evidenced by countless observations of patient response to

treatment with tricyclic antidepressants in which inhibition of 5-HT and noradrenaline reuptake

was presumed to enhance 5-HT-mediated neuroendrocrine responses. Moreover, the

development of the more efficacious SSRIs suggested that depressive symptoms can sufficiently

be attenuated as a result of increased synaptic 5-HT concentration293

. Today, 5-HT is

recognized to influence many symptoms of depression, including mood, appetite and sleep

patterns, physical activity, sexual interest and cognitive dysfunction.

Imaging investigations of the 5-HT receptors via ligand detection in conjunction with

positron emission tomography (PET) or single photon emission computed tomography (SPECT)

have provided consistent and convincing evidence that the binding density of 5-HT1A294, 295

and

5-HT2296

receptors is significantly decreased in MDD patients. The reduction in receptor

binding density was particularly prominent in areas of the brain involving the amygdala297

,

midbrain297, 298

, and brainstem299

. Age-related effects may also be of significant importance in

which reduced receptor binding density occurs with increasing age298, 300, 301

. Similar results

have been reported for the serotonin transporter (SERT), where drug-free unipolar depressed

patients exhibited reduced SERT availability in the brain stem299

. However, these findings are

not necessarily reproducible as no difference in SERT availability between depressed and

healthy volunteers has also been documented302

. Similarly, no significant difference in 5-HT

binding potential was found between recovered depressed patients and healthy controls in one

study303

while another study documented widespread persistent reductions in 5-HT receptor

30

binding among patients who have already recovered from the disease. Taken together, these

results suggest that reduced binding density may represent a genetic abnormality that confers

vulnerability to recurrent major depression rather than an observation made only during the

course of the disease303

.

In relation to post-stroke cases, the presence of an acute infarct is hypothesized to reduce

monoamine synthesis (e.g. 5-HT) within the brain. In the past, PSD patients have been

consistently documented to have lower CSF concentrations of the serotonin metabolite 5-

hydroxyindoleacetic acid compared non-PSD patients289

. More recently, one study measured

the CSF levels of serotonin in PSD patients288

and compared these levels to plasma

concentrations of serotonin. It was reported that serotonin concentrations in the CSF and

plasma of PSD patients were considerably lower than non-depressed patients and that a greater

number of PSD patients had lower than normal serotonin concentrations than the control group.

Furthermore, a sound correlation was found between the plasma and CSF serotonin

concentrations in both PSD and control patients, suggesting that plasma concentrations of

serotonin may be useful in predicting the central concentrations in patients with stroke or

depression.

1.4.5.2 Tryptophan and Central Serotonin Synthesis

Tryptophan is an essential amino acid that can only be obtained through external

sources. Once absorbed by the body, tryptophan travels around the peripheral circulation in

equilibrium between its albumin-bound and free form, the former comprising of 90% of the

body’s tryptophan level42

. Tryptophan can only be transported across the BBB in its free form

by the L-type amino acid transporter304

and once in the CNS, tryptophan acts as a precursor in

various metabolic pathways. The end products of these pathways are comprised of various

proteins, kynurenine, and serotonin42

. Note that brain TRP levels can be considered a reflection

31

of its plasma levels since TRP transport across the BBB directly affects the amount of

metabolites synthesized.

Tryptophan hydroxylase (TPH) is located in the cytoplasm of neurons of the raphe

nuclei and acts as the rate limiting enzyme of 5-HT synthesis. Using molecular oxygen and the

cofactor tetrahydobiopterin, it catalyzes the hydroxylation of TRP into 5-hydryoxytryptophan

(5-HTP). A subsequent decarboxylase enzyme then rapidly converts 5-HTP into 5-HT using

pyridoxal-5’-phosphate as co-factor42

. Serotonin is then either: 1) bound by the carrier protein

serotonin-binding protein (SBP) and released into the extracellular fluid (synapse) via calcium-

dependent exocytosis; or 2) metabolized into 5-hydroxyindoleacetic acid (5-HIAA) by

monoamine oxidase enzymes and excreted from the cell via energy dependent mechanisms.

Following release into the synapse, 5-HT can be recycled back into the neuron through SERT

reuptake located on the pre-synaptic membrane or metabolized into 5-HIAA in the post-synaptic

neuron.

Several researchers have examined the role of TRP and 5-HT in depression and the

clinical and neuropsychological consequences of abnormal 5-HT activity in the brain. For

example, one of the most reliable pieces of evidences linking 5-HT abnormalities to depression

was first documented in the mid-1980s, where total plasma tryptophan was significantly lower

in depressed patients compared to controls305

. More recently, researchers have constructed a

technique to induce a brief tryptophan depleted state in healthy subjects in order to assess the

mood effects of reducing central 5-HT function. This is simply accomplished by administering

an amino acid mixture free of TRP; thus, lowering both plasma and brain TRP concentrations.

Because the rate limiting step of 5-HT synthesis is determined by TPH activity, and because

TPH is only 50% saturated with TRP under normal physiological conditions306

, tryptophan

depletion is predicted to produce a transient lowering of central 5-HT activity.

32

The return of depressive symptomatology as a result of TRP depletion has been

documented in recovered depressed patients withdrawn from medication307, 308

. However, TRP

depletion appears insufficient to cause clinically defined depressive symptoms in healthy

volunteers308

. Although the difference in response to tryptophan depletion between the two

populations has yet to be elucidated, it has been postulated that depression may be the result of

persistent neurobiological changes that produce clinically significant mood abnormalities when

affected individuals are exposed to low 5-HT activity309

. The types of neurobiological changes

remain undefined but are hypothesized to involve altered or impaired innervations in

neurocircuitry that consistently leads to negative biases in emotional perception. Whatever the

mechanism, it can be said that tryptophan depletion studies generally support a casual

relationship between decreased central 5-HT and depression, and that those with risk factors for

depression or a family history of depression are most affected.

1.4.5.3 Central Tryptophan Availability

Knowledge underlying the specificities of tryptophan uptake into the serotonergic

neurons remains scarce. In its most simple definition, it is generally agreed upon that a

dynamic, regulatory barrier exists between plasma and intraneuronal tryptophan concentrations.

Although it is known that the L-type amino acid transporter is responsible for tryptophan

transport into the brain, two important factors surrounding this transport system must be

considered in central tryptophan availability: 1) the ratio between free tryptophan to its albumin-

bound form since only the free fraction crosses the BBB; and 2) the competitive nature of other

large neutral amino acids (LNAA = tyrosine, valine, leucine, isoleucine and phenylalanine) for

entry into the brain via the same transporter.

Several studies have reported lower tryptophan and tryptophan to LNAA ratio

(TRP/LNAA) in MDD patients compared to healthy controls305, 310-312

or subjects with obsessive

33

compulsive disorder (OCD)311

. The ratio appears to be most correlated with depressed mood313

,

retardation313

, and melancholic features314

. While some studies demonstrated reduction by using

total tryptophan, others have found reductions in only free tryptophan. Depression severity has

also been related to lower TRP/LNAA ratio among the depressed population315

, though the

opposite finding has also been documented312

. TRP/LNAA analysis may also successfully

predict treatment response to antidepressants, with a lower baseline TRP/LNAA ratio indicative

of greater improvements in depressive symptoms316-318

. Additionally, one study reported that

good responders exhibited a steady increase in TRP/LNAA ratio as the length of treatment

proceeded for six weeks319

. All in all, there is substantial evidence to support the relationship

between reduced central tryptophan availability and the presence of depression. Indeed,

increased brain tryptophan availability can increase brain 5HT synthesis320

. However, these

findings have yet to be tested among the PSD population.

1.4.5.4 Kynurenine Biosynthesis and Metabolism

Tryptophan is a ubiquitous molecule that is not only essential for protein synthesis but

plays a crucial role in central 5-HT metabolism. Additionally, the amino acid is a precursor for

the kynurenine pathway of metabolism in astrocytes, infiltrating macrophages, microglia and

dendritic cells321, 322

. The end products include kynurenine and several of its metabolites, some

of which are neurotoxic and others which are neuroprotectant. Under this pathway, the indole

ring of tryptophan is first metabolized by the rate limiting enzymes tryptophan 2,3-dioxygenase

(TDO), found highly concentrated in hepatic cells323

, or by indoleamine 2,3-dioxygenase (IDO),

most prominently expressed by cells in the CNS and infiltrating macrophages321, 324

. It can then

be inferred that TDO regulates homeostatic plasma tryptophan concentration in non-

inflammatory conditions while the extra-hepatic IDO predominantly maintains brain tryptophan

levels and is thus, of special interest in to PSD.

34

IDO is up-regulated in response to infection and tissue inflammation by certain

cytokines325-330

and can be induced in vitro by other inflammatory molecules such as LPS331

and

amyloid peptides332

. Once IDO metabolizes tryptophan into kynurenine (KYN) it proceeds

along the pathway until nicotinamide adenosine dinuleotide (NAD) is achieved as the final

product. Several neuroactive intermediates are generated during this process and comprising of:

1) 3-hydroxykynurenine333, 334

and 3-hydroxyanthranilic acid45

, both free-radical generators; 2)

quinolinic acid335

, the excitotoxin and selective N-methyl-D-aspartic acid (NMDA) receptor

agonist; and 3) kynurenic acid (KA)336

, a neuroprotective NMDA antagonist. A detailed

diagram of TRP metabolism and its products are displayed in Figure 1. Since the kynurenine

pathway produces both neurotoxic and neuroprotective metabolites, the ratio between these

products are of great importance when correlating their concentrations to the pathological

consequences of PSD. For example, the KA/KYN ratio has been show to be decreased in

depressed hepatitis C patients undergoing IFN- therapy337

and MDD patients40

.

Several pro-inflammatory cytokines possess the ability to induce IDO activity, including

IFN-338

, IFN-339

, IFN-338, 340

, IL-2341

, IL-6342

, IL-18343

, and TNF-331

. Among this list, IFN-

has consistently been reported to be the most potent regulator of the enzyme. Conversely, the

anti-inflammatory cytokine IL-4, IL-10 and TGf- act to inhibit IDO activity344

. Interestingly,

one study found that IL-10 is able to suppress IFN- induced IDO expression in cells derived

from the hypothalamic-pituitary-adrenal axis (HPA)329

. It cannot be stressed enough that the

balance between pro- and anti-inflammatory cytokines is crucial for homeostatic maintenance of

the human body. Therefore, an excess of pro-versus anti-inflammatory cytokines have the

potential to cause mood dysfunction in several neurological diseases if IDO activity exceeds its

normal boundaries of TRP metabolism.

35

Figure 1. The KYN Pathway: TRP Metabolism and its Products345

36

1.4.5.6 Linking Cytokines and Tryptophan Metabolism to the KYN/TRP ratio

A newer hypothesis to the etiology of depression involving the metabolic pathways of

tryptophan was recently described336

. Increased cytokine levels (such as in the case of stroke)

upregulates IDO activity, an enzyme that is widely distributed in the intestinal tissues, lungs,

placenta and brain291, 343, 346-349

. Because it is in direct competition with TPH for the metabolism

of tryptophan into 5-HT, heightened IDO activity as a result of elevated in cytokine levels may

reduce tryptophan availability for 5-HT production. Consequently, central serotonin

concentrations become sparse, leading to mood complications and behavioural disturbances in

the affected individual. In support of this hypothesis, several studies have shown a significant

decrease in serum TRP concentrations in patients treated with cytokines350-352

. In addition, TRP

levels have been found to be decreased in depressed patients314

while the administration of TRP

produces an antidepressant effect340

. Based on such findings, one is inclined to postulate that

clinical conditions associated with increases in pro-inflammatory cytokines lead to activation of

the IDO enzyme, subsequently resulting in decreased 5-HT synthesis and thus, increased risk of

depression.

We postulate that the mechanism above may have a significant effect on the etiology of

PSD. To reiterate, elevations in post-stroke cytokine levels shunts tryptophan metabolism from

the serotonin pathway into the kynurenine pathway via upregulation of the IDO enzyme. Under

the kynurenine pathway, tryptophan is catabolized into kynurenine; thus reducing the

availability of tryptophan to be converted into the mood regulatory neurotransmitter 5-HT.

Kynurenine is subsequently broken down into several neuroactive intermediates, including three

potent neurotoxins; the hydrogen peroxide generators, 3-hydroxykynurenine and 3-

hydroxyanthranilic acid223

and the NMDA agonist, quinolinic acid (QUIN)335

. Indeed, one

study reported IFN-α immunotherapy treatment in hepatitis C patients significantly increased

37

peripheral blood KYN353

. This was accompanied by marked increases in CSF KYN and QUIN

and correlated with the severity of depressive symptoms.

Several studies have examined the effects of the neurotoxic kynurenine metabolites. 3-

hydroxykynurenine levels are elevated in Huntington’s disease patients354, 355

and has been

shown to induce neuronal cell death in cortical and striatal cell cultures44

. Intracerebral

injection of 3-hydroxyanthranilic acid can decrease choline acetyltransferase activity356

, thus,

decreasing acetylcholine synthesis. QUIN is produced by infiltrating macrophages, microglia

and dendritic cells under inflammatory conditions and acts as a potent agonist of the neuronal

NMDA subtype of glutamate receptors335

. Moreover, several neurodegenerative conditions

have been linked to QUIN-induced apoptosis, including Huntington’s disease357

and HIV-

associated dementia358

. Most recently, it was suggested that KYN preferentially metabolized

along the QUIN pathway at the subcortical level (amygdala/striatum) in mice models359

. This

has its importance since a reduction in 5-HT receptor binding density has been associated with

MDD and is most pronounced in the amygdala. Altogether, it is possible that these metabolites

inflict further damage to the post-stroke brain by destroying essential neuronal pathways

responsible for regulating mood and cognition.

Because measurement of IDO activity has not been thoroughly explored in stroke and is

difficult to measure, the kynurenine to tryptophan ratio (KYN/TRP) has been proposed as a

marker for its activity instead. The KYN/TRP is a reliable marker of IDO activity based on its

reproducibility in past studies examining depression and related neurologic diseases. For

example, enhanced TRP degradation and higher KYN/TRP ratios in Alzheimer's disease,

Parkinson's disease, Huntington's disease, cancer, malaria, and rheumatoid arthritis have been

associated with advanced stages of the diseases and more severe symptoms and fatal outcomes

360-367. Furthermore, it has been reported that the inflammatory marker neopterin, is correlated

38

with both KYN/TRP ratio and kynurenine concentrations and inversely correlated with

tryptophan in cases of immune activation368, 369

. Elevations in the KYN/TRP ratio has not only

been associated with the severity of well-known inflammatory diseases but has also been linked

to cases of depression. Individuals undergoing cytokine therapy for hepatitis C and cancer

developed depressive symptoms that were specifically linked to increases in plasma KYN and

KYN/TRP ratio38, 337

. Similar increases in the KYN/TRP ratio have also been found in women

with post-partum depression39, 370

. Most recently, our group demonstrated that a higher

KYN/TRP ratio was significantly associated with greater depression scores in coronary artery

disease (CAD) patients, a relatable population to the stroke population41

. Taken all together,

these findings support the possibility of a biological contribution to PSD (alongside the current

psychosocial models) through IDO activation, whereby upregulation of IDO activity stimulates

the KYN pathway; thus depleting the amount of TRP available for 5-HT synthesis and

increasing the levels of KYN neurotoxins. A summary of KYN/TRP levels in psychiatric,

neurodegenerative, and inflammatory illnesses is displayed in Table 1.

_____________________________________

1.4.6 Rationale for this Study

Currently, no definite relationship has been established between pro-inflammatory

cytokine levels and PSD in humans. This study attempts to determine the relationship between

such and expects to find a positive causal relationship between plasma pro-inflammatory

cytokine concentrations and depression after stroke. In addition, this study also aims to examine

the counterintuitive reports of elevated anti-inflammatory cytokine levels192, 191

in post-stroke

patients. Instead, it is proposed that anti-inflammatory cytokines are involved in an innate

protective mechanism initiated by the body to counterbalance the depressiogenic effects induced

by pro-inflammatory cytokines. .

39

Finally, the study will examine IDO activity as measured by the KYN/TRP ratio. Since

TRP can only be obtained through diet, and since KYN371

and TRP compete with LNAAs for

entry through the BBB, peripheral measurements of both molecules should reflect the

concentrations circulating within the CSF. As summarized in the literature review, an elevated

KYN/TRP ratio will act as a marker for both possible neurotoxicity and decreased central TRP

availability. The additive affects of the neurotoxic KYN metabolites and reduced 5-HT

synthesis is predicted to play a significant role in the development of PSD. Indeed, increased

KYN/TRP ratio has recently been associated with depressive symptoms of other diseases (e.g.

post-partum depression, CAD); however, this will be the first study to examine its relationship

to PSD.

40

Table 1: Mean KYN/TRP Levels in Various Depression, Neurodegenerative Diseases, and Inflammatory Illnesses

Author Condition KYN/TRP Controls*

KYN/TRP Patients*

p-value Comments

Depression

Kohl39

Post-partum depression

29.4 ± 44.7 (n=86)

35.1 ± 47.5 (n=9)

0.067

KYN/TRP measured 4 days after birth; Edinburgh Postnatal Depression Scale (EPDS) score ≥ 12 positive for depression

Myint40 Major depression disorder

25.0 ± 11.0 (n=58) 17.0 ± 14.0 (n=189) 0.036 DSM-IV used for diagnosis of MDD; fasting blood levels drawn

Neurodegenerative Diseases

Forrest367

Huntington's

disease

33.8 ± 8.3 (n=11)

45.0 ± 7.9 (n=40)

<0.05

KYN/TRP significantly higher in only patients with the severest form of the disease (≥37 CAG triplet expansions on the gene)

Widner365

Alzheimer's

disease

34.1 ± 9.91 (n=20)

46.1 ± 19.8 (n=21)

0.018

AD patients with cognitive score lower than the median (<6; as measured by the Mini Mental State Examination (MMSE)) had significantly higher KYN/TRP (p=0.02) than those with MMSE≥6

Widner366

Parkinson's

disease

36.1 ± 8.2 (n=11)

58.5 ± 34.2 (n=7)

<0.05

Means displayed only compare controls to patients with the severest form of PD. Mean difference between controls and mild-moderate PD patients was non-significant

Inflammatory Illnesses

Bonaccorso38

Hepatitis C virus (Before and after IFN-α therapy)

39.0 ± 14.0

61.0 ± 31.0

<0.001

Patient group with IFN-α therapy, control group without IFN-α therapy. Treatment resulted in significantly higher depression scores at 4-6 months (p=0.03) as measured by the Hamilton Depression Scale (HAM-D)

*Reported as mean ± SD unless otherwise indicated

41

Author Condition KYN/TRP Controls*

KYN/TRP Patients*

p-value Comments

Inflammatory Illnesses

Lögters372

Major trauma (with or without

sepsis)

5.6 ± 0.4 (n=42)

21.9 ± 1.1 (n=17)

<0.01

KYN/TRPx100 reported here. Plasma levels measured 6 days after trauma; patient group is with sepsis, control group without sepsis

Huengsberg373

HIV

34.9 (32.4, 36.9)ɸ (n=72)

50.5 (44.9, 54.5)ɸ (n=82)

<0.01

Most patients were on treatment during the course of the study (53 patients took zidovudine (AZT); 23 took didanosine or zalcitabine)

Pertovaara374

Sjögren's syndrome

Females (n=139): 24.3 (21.0–28.9)

---------------- Males (n=7):

27.0 (23.6–32.1)

Females (n=170): 34.0 (25.1–44.3)

--------------- Males (n=170):

34.5 (29.3–38.7)

<0.0001

-------- 0.014

Patients were not age-matched; mean years control = 45 ± 11 years vs. mean years patients = 60 ± 12 years

Schroecksnadel363

Rheumatoid

arthritis

26.9 ± 8.10 (n=49)

37.9 ± 42.4 (n=22)

N/A

KYN/TRP tended to be higher in men than women, strongly associated with age (p<0.01), and significantly correlated with neopterin (p<0.05)

Swardfager41

CAD

(depressed vs. non-depressed)

38.5 ± 15.7 (n=24)

45.6 ± 20.0 (n=71)

0.055

All patients had CAD; control group represents those who were non-depressed, patient group represents those who were depressed

Wirleitner375

Coronary heart

disease

28.1 ± 5.25 (n=35)

36.3 ± 13.0 (n=35)

<0.01

KYN/TRP positively correlated with neopterin levels. Neopterin concentrations also positively correlated with older age

Zangerle376

HIV

30.7 ± 8.7 (n=40)

79.2 ± 60.3 (n=45)

<0.001

KYN/TRP significantly decreased (p<0.001) in patient group during anti-retroviral therapy (ART)

*Reported as mean ± SD unless otherwise indicated

ɸReported as median (95% confidence intervals) Reported as median (interquartile ranges)

42

Author Condition KYN/TRP Controls*

KYN/TRP Patients*

p-value Comments

Cancer

Schroecksnadel369 Gynecological cancer

35.1(19.2-59.0) (n=19)

41.4 (not reported) (n=20)

N/A

Patients with ovarian cancer had higher KYN/TRP trending on significance compared to controls

Suzuki360

Lung cancer

32.9 ± 9.10 (n=45)

47.1 ± 21.3 (n=123)

<0.0001

Higher KYN/TRP ratio was associated with advanced stages of the disease

Weinlich377

Malignant melanoma

26.9 ± 8.10 (n=49)

46.3 ± 20.7 (n=87)

<0.001

KYN/TRP and neopterin levels were positively correlated. Patients with KYN/TRP above the median (=41.2μmol/mmol) had a significantly decreased survival time compared to those above the median (p=0.0025)

*Reported as mean ± SD unless otherwise indicated Reported as median (interquartile ranges)

43

2. METHODS

2.1 Study Design

This was a cross-sectional observational study examining the role of several

neurochemical factors in the etiology of PSD. Patients meeting the World Health Organization

Multinational Monitoring of Trends and Determinants in Cardiovascular Disease (WHO-

MONICA) Project378

and National Institute of Neurological Disorders and Stroke (WHO-

NINCDS)379

criteria for stroke with CT or MRI evidence of acute ischemic infarcts were invited

to participate into the study. Based on the WHO-NINCDS guidelines, stroke was defined as ―a

sudden, nonconvulsive, focal neurologic deficit persisting for greater than 24 hours‖ and

excluded cases of transient ischemic attacks (TIAs). Five different health and rehabilitation

centres were utilized for recruitment: (1) Sunnybrook Health Sciences Centre, (2) Baycrest

Rehabilitation Centre, (3) St. John’s Rehabilitation Centre, (4) York Central Hospital and (5)

Toronto Rehabilitation Centre. Only patients who had an ischemic stroke within 12 weeks of

the initial assessment who spoke and understood English were considered for enrolment into the

study. If the patient fully agreed to the voluntary terms outlined for the study, a written

informed consent was completed (Appendix A). The individual then remained an active

participant until all consent terms were met unless the patient chose to terminate participation

prematurely.

At baseline interview, patients were considered depressed if they met the Diagnostic and

Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV) criteria for depression, either

major or minor. Once screening tools and standardized mood questionnaires were completed, all

participants had blood samples drawn for cytokine, KYN, TRP, and LNAA analyses. In

addition, general cognitive function and stroke severity was examined in all consenting

participants.

44

2.2 Subjects

2.2.1 Inclusion and Exclusion Criteria

In all of the five listed recruitment sites, the study was approved by their respective

Research Ethics Boards (REBs) as shown by the most recent approval letters in Appendixes B

through F. The eligibility criteria for the study were carefully considered before approaching

patients and are described below:

Inclusion Criteria:

Age ≥18 years

Speaks and understands English

Clinical diagnosis of stroke according to WHO-MONICA378

and NINCDS379

criteria

Evidence of acute infarction on either CT or MRI

Maximum time since stroke onset <12 weeks

Written, informed consent

Exclusion Criteria:

Subarachnoid or intracranial hemorrhage

Severe aphasia or dysarthria that would interfere with the assessor’s ability to understand

patient response

Imminently suicidal or, in the opinion of the affiliated clinician, has inadequate family

monitoring for suicidality

Significant acute medical illness, including:

• Infection

Drug overdose

Alcohol abuse

Untreated hypothyroidism

Uncontrolled diabetes

Uncontrolled anemia

Severely disturbed liver,

kidney, or lung function

Significant acute neurologic illness, including:

Decreased Level of

Consciousness (LOC)

Active brain tumor

Parkinson’s disease

Huntington’s disease

Multiple sclerosis

Binswanger’s disease

Hydrocephalus

Subdural hematoma

Progressive supranuclear

paralysis

Severe aphasia

Presence of a premorbid Axis I psychiatric diagnosis, including:

Major depressive disorder

Schizophrenia

Bipolar disorder

Dementia

Concomitant use of psychotropics except for short acting benzodiazepines

47

2.2.2 Demographics and Medical History

Patient demographics including age, body mass index (BMI), level of education,

employment status, living status, marital status, history of depression, and number of vascular

risk factors (including hypertension, cigarette smoking, obesity, hyperlipidemia, and diabetes)

were either collected through patient interviews or medical histories stored on hospital

electronic databases. Current or past medical illnesses were determined through a review of

patient history, physical examinations, and/or routine laboratory test results by a licensed

practitioner as indicated in the patient charts. Finally, the time since stroke onset and

concomitant medications were recorded based on information gathered by the attending

physician upon admission.

_____________________________________

2.3 Clinical Assessments

2.3.1 Depression Scales

Centre of Epidemiological Studies Depression Scale (CES-D)

The Centre for Epidemiological Studies Depression Scale (CES-D)380

is a 20-item self-

rated questionnaire that was originally designed for use in community surveys as a means of

determining one’s depression quotient. Today, it is clinically employed as a screening tool for

depression and examines various aspects of the respondent’s perceived mood, anxiety and

physical functioning within the past seven days. However, because certain somatic symptoms

listed on the CES-D were originally intended to elicit symptoms of depression in otherwise

healthy individuals, the appropriateness of the scale requires validation among the stroke

population. Indeed, the scale has previously been validated in post-stroke patients using a

structured psychiatric interview as an established criterion. Here, a cut-off score of 16 was

found to be highly predictive of clinical depression, with a specificity of 90%, a sensitivity of

48

86%, and a positive predictive value of 80%381

. Thus, a score of 16 or greater was used as an

indicator of depressive symptoms in this study but may not have necessarily reflected definite

clinical depression. The CES-D has also been shown to exhibit high inter-observer reliability

and concurrent construct validity with several depression measures in the geriatric stroke

population (e.g. Geriatric Depression Scale (GSD), Zung Scale), as well as high discriminant

validity with measures of other factors including social functioning, cognition, and

disability382, 383

. As such, it has consistently been used as a screening instrument for

depression among the post-stroke population, including the NINCDS Stroke Data Bank384

and

other various PSD studies22, 385-387

.

Structured Clinical Interview for the DSM-IV: Depression Module (SCID)

In cases where patients scored 16 or greater on the CES-D, the attending psychiatrist

was called to further assess their depressive symptoms to ensure that they were not evoked by

the physical impairments suffered from stroke. The psychiatrist then confirmed a clinical

diagnosis of depression according to the DSM-IV criteria for a Major Depressive Episode

(MDE)388

, using the depression module of the Structured Clinical Interview for the DSM-IV

(SCID). The use of the SCID has previously been validated among the PSD population98, 99

.

Patients were considered to be suffering from major depression if at least five of the following

nine modules were met in the past two weeks: depressed mood, anhedonia, changes in weight

or appetite, insomnia or hypersomnia, psychomotor agitation or retardation, fatigue or loss of

energy, feelings of worthlessness or guilt, difficulties in concentration and decision-making, or

recurrent thoughts of death; with at least depressed mood or anhedonia present among their

symptoms. Patients were considered to be suffering from minor depression if they met three of

the nine modules, with at least depressed mood or anhedonia present among their symptoms.

Those who scored 16 or higher on the CES-D but failed to meet a formal diagnosis of

49

depression were placed in a "depressive symptoms only‖ group. However, the clinically

depressed group and the depressive symptoms only group were eventually combined for

analytical purposes due to the limitations of a small sample size. Thus, the patient population

used for analyses were not necessarily diagnosed with clinical depression. The CES-D has

consistently been used as a screening instrument for depression among the post-stroke

population, including the NINCDS Stroke Data Bank384

and other various PSD studies22, 385-

387. Even a briefer version of the CES-D that only included half of the original questions was

found to be a positive predictor of depression when compared to the DSM-IV criteria, with a

sensitivity of 97%, a specificity of 84%, and a positive predictive value of 85%

389.

2.3.2 Cognition Scales

Mini Mental State Examination (MMSE)

In addition to depression measurements, all patients were evaluated for general cognitive

function post-stroke. The Mini-Mental State Examination (MMSE)390

is the most is a popular

screening tool for this purpose and was administered to all recruited participants. It consists of

a brief, 30-point questionnaire for cognitive ability and evaluates various mental functions

including orientation, registration, short-term memory, attention, calculation, and visuo-spatial

skills. A patient who scored less than 24 was considered to be cognitively impaired and a

patient who scored 24 or more was considered non-cognitively impaired. Previously, the

MMSE has detected differences in concentration and memory function between depressed and

non-depressed stroke patients391

, as well as worse global cognitive function in PSD patients

with significant executive dysfunction392

. The MMSE has also been used to test the efficacy

of different antidepressants on reducing cognitive impairment in the PSD population128-130

.

50

2.3.3 Stroke Severity and Functional Disability

National Institutes of Health Stroke Scale (NIHSS)

Since the study was interested in examining the biological correlates related to PSD, it

was important to evaluate stroke severity and functional disability as possible confounding

factors to the results. To fulfill this task, The National Institute of Health Stroke Scale

(NIHSS)393

was administered to all stroke patients by the treating medical team or by a NIHSS

certified research associate. The NIHSS is a systematic assessment tool designed to

quantitatively evaluate the neurological deficits and degree of recovery of stroke patients. The

scale examines the following outcomes: level of consciousness, extraocular movements, visual

field defects, facial muscle function, extremity strength, sensory function, coordination

(ataxia), language (aphasia), speech (dysarthria), and hemi-inattention (neglect)394

. In this

study, the level of stroke severity was scored as follows: 0 = no stroke; 1-4 = minor stroke; 5-

15 = moderate stroke; 15-20 = moderate/severe stroke; 21-42 = severe stroke.

Previously, The NIHSS has demonstrated high intra- and inter-observer reliability to

non-neurologists and showed that study coordinators can be rapidly and reliably trained via

certification videos/DVDs to administer the NIHSS395, 396

. Furthermore, these certification

videos are reliable across multiple venues396

. NIHSS scores are also robust predictors of

stroke outcome and their values at baseline correlate strongly with the Trail of Org 10172 in

Acute Stroke Treatment (TOAST) stroke subtype diagnosis397

. In general, lacunar strokes are

related to lower NIHSS scores and thus, forecast better patient outcome. NIHSS scores are

also well correlated with infarct volumes, indicating strong concurrent validity of the scale398

.

_____________________________________

51

2.3.4 Blood Draw

Blood was drawn from inpatients by IV technicians at the time of their routine clinical

blood draws. Although blood orders were usually carried out in the morning (approximately

9:00 a.m.), there was no guarantee of a standardized collection time or that the desired blood

fasting levels were obtained. Serum levels of various pro- and anti-inflammatory cytokines

(IL-1β, IL-6, IL-10, IL-18, TNF-a, IFN-g) and kynurenine were analyzed, as well as plasma

levels of tryptophan and LNAA. Blood samples used for cytokine and kynurenine analysis

were collected in SST gel vacutainer tubes, while TRP and LNAA were collected in EDTA

vacutainer tubes.

Immediately after collection, blood samples were centrifuged at 1000g for 10 minutes.

Plasma or serum samples were transferred into labeled cryovials after centrifugation and

stored at -70°C until assayed. Because only the free fraction of plasma TRP reflects its

concentration in the brain, TRP samples required an additional ultrafiltration process to

separate its free form from its protein-bound form. Here, 1.0 cc of plasma was transferred into

an ultrafiltration device (Centrifree 4104®, Millipore) and centrifuged at 1000g for an

additional 15 minutes. Mechanistically, the device retained all protein-bound TRP while

allowing for the smaller free TRP to pass through the 30,000 kDa membrane into ultrafiltrate

reservoir cups. The cups containing the free TRP were then capped and stored with the other

samples at –70˚C until ready for batched analysis.

_____________________________________

2.4 Laboratory Analyses

2.4.1 Cytokines Assay

This study focuses on peripheral cytokine measurements. Several cytokines,

including IL-1α, IL-1β, IL-6, and TNF-α, are capable of crossing the blood brain barrier399

.

52

Although it cannot be positively confirmed that plasma cytokine levels are a direct reflection

of CSF levels, peripheral and central cytokine concentrations have been correlated in the

past400

. Finally, reproducibility of several past studies have indicated plasma cytokines to be

reliable markers of depression175, 401

and stroke outcome171, 191, 192, 400, 402

.

Cytokine measurements of IL-6, IFN-γ, and I-10 were performed by Dr. Angela

Panoskaltsis-Mortari who heads the Cytokine Reference Laboratory at the University of

Minnesota. The technique employed for all three cytokines was multiplex immunobead-based

assay using the Human Fluorokine Multianalyte Profiling (MAP) Base Kit A (R&D Systems,

Minneapolis, MN). This bead-based assay only requires a small sample size, allows the user

to define specific molecules of interest, and is known to be highly efficient and flexible for the

simultaneous detection of process-related molecules via Luminex xMAP® technology. Each

sample has been evaluated for cytokine sensitivity, precision, recovery, sample linearity, and

specificity by the company panel. A brief mechanistic summary of the technique is provided

as follows from the company website:

"Detection is achieved through a bead-based antibody-antigen sandwich

method. Briefly, samples are incubated with color-coded beads that are pre-

coated with analyte-specific capture antibodies for the molecule of interest.

Expression levels are determined following incubation with a biotinylated

detection antibody and streptavidin-conjugated phycoerythrin (PE). Using a

Luminex® analyzer, independent lasers determine the color of each bead

and the magnitude of the PE-derived signal, which is directly proportional to

the levels of bound analyte."

The procedure is similar to that described by Djoba Siawaya et al403

, who evaluated

and compared several commercial bead-based luminex cytokine assays. The group reported

that R&D Systems Fluorokine-MAP assays were one of the most accurate for the

measurement of cytokine concentrations in whole blood culture supernatant and achieved

good recovery ranges and reproducibility for most cytokines. According to the data provided

53

by R&D Systems, assay sensitivities were 1.11 pg/mL for IL-6, 0.3 pg/mL for IL-10, and 1.27

pg/mL for IFN-γ. A more detail description of assay sensitivities as described as the minimum

detectable dose is shown in Table 2 below:

Table 2: Cytokine Assay Sensitivities

Cytokine Number of Assays MDD range MDD mean

IL-6 43 0.10 - 1.11 pg/mL 0.36 pg/mL

IL-10 43 0.07 - 0.30 pg/mL 0.13pg/mL

IFN-γ 42 0.09 - 1.27 pg/mL 0.31 pg/mL

2.4.2 KYN Assay

The KYN assay was performed by the Pharmacy Quality Control and Research Group at

Sunnybrook Health Sciences Centre. The technology utilized was reverse phase high performance

liquid chromatography (HPLC) at UV detection of 258nm. The exact steps taken for the KYN assay

were provided by Scott Walker in a personal communication. Briefly, the method for KYN

involves protein precipitation with an equal volume of 3% perchloric acid. The sample was

centrifuged for 10 minutes and 0.1 mL of the supernate was injected directly into the high

liquid chromatographic system using an autoinjector (715 Ultra WISP, Waters Canada). The

mobile phase consists of 9% acetonitrile in 0.05 M potassium phosphate mono basic, which

was pumped through a reverse phase 5 µm ODS column , 250 mm x 4.6 mm (Symmetry;

Waters Corp) at 1.0 mL/min using a chromatographic pump (P4000; ThermoSeparation

Systems, San Jose CA). KYN was detected using an ultraviolet detector (UV 6000;

ThermoSeparation Systems, San Jose CA) at a wavelength of 258nm. Chromatograms were

recorded on a computer using the ChromQuest software (ThermoQuest, San Jose CA).

The standard curve was constructed by creating 9 standards from a common serum

stock sample. To approximately 10 mL of serum, additional KYN was added to the KYN

present in the sample to create standards with 0, 0.125, 0.250, 0.3125, 0.50, 0.75, 1.0, 1.5 and

2 mg/L of additional KYN added. Analysis of these standards in duplicate resulted in linear

54

relationship between the KYN peak area and concentration with a slope and intercept (due to

the endogenous kynurenine in the common serum stock). Unknown concentrations of KYN in

plasma were determined by dividing the KYN peak area in the unknown sample by the slope.

Replicate error, as measured by the coefficient of variation (CV(%) = 100 x standard

deviation/mean), based on duplicate analysis of standards between 0 mg/L of kynurenine

added to 2 mg/L added ranged from 1.54% to 0.5% with deviations from the known or

expected concentrations averaging less than 7%.

2.4.3 TRP and LNAA Assay

The TRP and LNAA assays were performed by Dr. Simon Young in the Department of

Psychiatry at McGill University who used the same procedure as described by Anderson et

al404

. Details of the TRP and LNAA assays were provided by Simon Young via personal

communication:

―Plasma was deproteinized by adding one part of 1 M perchloric acid to

three parts of plasma, mixing and centrifuging. Tryptophan in plasma

was measured by high-performance liquid chromatography (HPLC) on

a Waters Bondapack C18 reverse phase column (Phenomenex,

Torrance, California) with fluorometric detection (Anderson and Purdy,

1979). The other large neutral amino acids (LNAA) (tyrosine,

phenylalanine, leucine, isoleucine, valine) were measured on a

Beckman System Gold amino acid analyzer using precolumn

derivatization with o-phthalaldehyde and gradient reverse phase HPLC

with fluorometric detection. Aminoadipic acid was used as an internal

standard.‖

_____________________________________

2.5 CT Scan Analysis

The majority of patients admitted to Sunnybrook for acute stroke had CT scans

performed within the first 24 hours of admission. Lesion characteristics were determined from

CT scans obtained without a contrast agent on a General Electric LightSpeed VCT series

scanner (General Electric Healthcare, Waukesha, WI). Ischemic lesions were manually traced

55

on these images using Medical Image Processing, Analysis, and Visualization (MIPAV;

National Institutes of Health, Bethesda, MD). Lesion area was measured with the use of a

digitizing tablet (Sigma Scan, version 3.0, Jandel Scientific) from tracings of the infarct on

each slice of the CT scan on which the lesion appeared. The area of the lesion on each slice

was multiplied by the slice thickness (1 mm3) and summed to obtain the total lesion volume.

The criteria for anterior lesion was that its anterior most border must extend in a rostral

manner past the 40% anterior-posterior (A-P) distance. The criterion for posterior lesion was

that its posterior border must extend in a caudal manner past the 60% A-P distance. Lesions

that fell between the 40% and 60% A-P distance were classified as intermediate. Lesions that

eclipsed both the 40% and 60% A-P distances was classified as extending. Infarct side (left

vs. right) was determined by examining which side the lesion laid to the longitudinal fissure

on the CT scans.

_____________________________________

2.6 Statistical Analyses

The Statistical Package for the Social Sciences (SPSS), version 17.0 was used for all

statistical analyses.

2.6.1 Preliminary Data Transformations

NIHSS scores are supposed to be collected upon patient admission and entered into the

chart. However, 23 scores were missing from the charts. The NIHSS score was imputed at the

group mean for both the control and depression group (since the stroke scale was not

performed on all patients) in order to keep a consistent sample size for analyses. NIHSS

scores were collected based on forms that were filled out by the attending medical staff upon

patient admission. Thus, about half of all scores were missing as some of the NIHSS forms

were not contained within the patient charts. In total, 23 NIHSS scores were imputed—18

56

from the control group and 5 from the depressed group. In the case of the MMSE, a percent

score was calculated for all patients who could not complete the entire examination due to

physical impairments from stroke (e.g. motor injuries would void the individual form drawing

and writing activities), where the correct number of responses was divided by the largest

possible score that can be obtained during assessment. The percent was then multiplied by 30

(the standard MMSE total score) to maintain an invariable range of scores and the method has

been validated among the elderly population405, 406

. Additionally clinical measurements of

cytokine assays were sometimes returned with undetectable levels or below the lowest level of

detectability of the assay. When this occurred, the lowest level of detectability for each

cytokine (1.11 pg/mL for IL-6, 0.30 pg/mL for IL-10, and 1.27 pg/mL for IFN-), was used as

the imputed value.

Although this method would generate cytokine values that deviate from the true mean,

the main goal of this study was not to capture low concentrations of pro-inflammatory

cytokines but their elevated levels. Thus, the approach should not affect our ultimate intent of

finding clinically significant elevated cytokine concentrations. However, data pertaining to

anti-inflammatory cytokines may be affected since our hypothesis predicts the presence of

decreased concentrations. This limitation will be explored further in the discussion.

2.6.2 Planned Analyses

Clinically depressed patients and those who only exhibited depressive symptoms

(CES-D≥16) were combined into one group and collectively labeled the "depressed group".

As mentioned previously, the number of patients in the study with major versus minor

depression made subanalysis by depression subtype unfeasible. Thus, throughout this paper,

the term "depressed group" will be used to represent the combined depressed and depressive

symptoms group. Those who did not exhibit any clinically significant signs of depressive

57

symptoms (CESD≤15) were labeled the "non-depressed group". Complete demographic data

were collected during recruitment and compared between the two groups using independent

samples t-tests for continuous data (e.g. age, BMI) and chi-square or Fisher's exact tests for

categorical data (e.g. marital status, employment status, living situation). Variables that were

significantly different between groups were included as covariates in subsequent analyses.

2.6.2.1 Primary Analysis

Hypothesis 1: Increased idoleamine 2,3-dioxygenase (IDO) enzyme activity will be found in

stroke patients experiencing depressive symptoms compared to non-depressed stroke patients

as evidenced by an increased kynurenine/tryptophan (KYN/TRP) ratio.

Demographic and clinical characteristics were compared between depressed and non-

depressed groups using independent samples t-test for continuous variables and Pearson's chi-

square analysis for categorical variables. ANCOVA was used to compare differences in mean

KYN/TRP ratio between groups with any between-group demographic or clinical differences

entered as covariates.. Depression was also analyzed as a continuous variable by looking at the

relationship between CES-D scores and KYN/TRP using a partial correlation.

2.6.2.2 Secondary Analyses

Hypothesis 2: Increased levels of pro-inflammatory cytokines (IL-6, IFN-) and decreased

levels of anti-inflammatory cytokines (IL-10) will be found in stroke patients experiencing

depressive symptoms compared to non-depressed stroke patients.

Skewness and kurtosis were examined for each cytokine variable. If the data presented

itself as unskewed, ANCOVA was used to compare differences in mean cytokine levels

between depressed and non-depressed patients, with the appropriate demographic variables

acting as covariates as determined during preliminary analysis. If the data presented itself as

being skewed, a non-parametric Mann-Whitney test was employed instead to determine the

58

mean cytokine difference between patient groups. Additionally, Spearman's rank rho were

performed between all cytokines and the KYN/TRP in order to determine the relationship

between the two variables.

2.7 Sample Size Calculations

This is the first study to evaluate the relationship between KYN/TRP ratio and its

predictive value of depression post-stroke. Thus, estimates of effect size were based on

previously published data examining the relationship between KYN/TRP ratio and depression

in CAD patients41

. Since the primary analysis of this study was based on ANCOVA statistics,

sample size calculations for the multivariate data were not possible as no supportive data was

available for this method407

. Thus, all effect estimates were based on the procedure for

independent samples t-tests. Based on the CAD study, mean KYN/TRP ratios and the

weighted standard deviation were used in the equation described as follows. Effect size (d)

were calculated according to the equation d = (mA – mB) / σ; where mA = the population mean

for group A (MDD patients), mB = the population mean for group B (non-depressed controls),

and σ = the population standard deviation. Required sample size was calculated according to

the equation n = 2 [ (zα + zβ) σ / Δ ]2 or n = 2 [ (zα + zβ) / d ]

2 where Δ = (mA – mB), the

difference between the two population means, zα = the z-value that corresponds to the

accepted α level (probability of type I error), and zβ = the z-value that corresponds to the

accepted β level (probability of type II error). For α = 0.05, zα = 1.96 and for β = 0.20, zβ =

0.84. It was determined that for α = 0.05, 88 patients per group would be necessary to obtain

an observed power of 0.80.

_____________________________________

59

2.8 Control of Confounders

Both depression and stroke have been individually and collectively linked to several

clinical and cardiovascular risk factors in the past research studies. For this reason, potential

confounders were compared between depressed and non-depressed patients. Based on the

statistical analyses, variables found to be significantly different between groups were

considered as covariates in the primary analyses. Only hypertension and levels of LNAA

were found statistically significant among all measured demographic and clinical factors. As

previously mentioned in 1.4.1.5 hypertension has previously been found in subjects with

depressive symptoms110

and thus may be pertinent to the development of PSD.

3. RESULTS

Patient recruitment began at the inpatient stroke unit at Sunnybrook Health Sciences

Centre in July 2007. A list of possible study participants was provided weekdays by a nurse

responsible for keeping track of admitted stroke patients. This list of potential research

subjects is generated as part of the Heart and Stroke Foundation Centre for Stroke Recovery

research initiative. Potential recruits were subsequently approached by the study coordinator

to ask if they were willing to participate in a research study. The coordinator described the

premise of the study, what was required of the patient if he or she chose to participate, and the

risks and benefits of those requirements. However, because the research is complex in its

variability, the ability for each individual to understand the research also varies. Thus, the

capacity for consent was verified by independent physicians for all patients as part of standard

care. As a further precaution, the assessor had the patients repeat the purpose and details of

the study to ensure that they were fully aware of their responsibilities with participation.

Finally, the study requires consenting to questionnaires and a blood sample, which are not

60

complicated tasks to understand in themselves. A flowchart of patient recruitment progress

can be viewed in Figure 2 along with a table of ineligibility in Table 3.

Figure 2. Patient Recruitment

Ischemic Stroke Patients n=489

Minimum Length of Stay ≥5 days n=346

Screened n=303

Non-Depressed n=38

Depressive Symptoms n=16

Eligible n=125

Not Eligible n=178

Recruited n=61

Completed n=54

No Consent n=64

61

Table 3: Participant Ineligibility

Reason for Ineligibility Number of Patients

History of Depression 34

Non-English Speaking 29

Severe Aphasia/Dysarthria 19

Decreased LOC 15

Neurological Disorder

Dementia 18

Parkinson’s Disease 5

Other 9

Medical Illness

Infection 13

Rheumatoid Arthritis 3

Other 6

Other 27

Total 178

At the time of this paper, 61 patients have agreed to participate in the study with

recruitment ongoing. All individuals who were selected as participants met the eligibility

criteria, signed a written informed consent and provided a blood sample for biomarker

analyses. However, due to loss of follow-up, only 54 (38 non-depressed, 16 depressive

symptoms) were used for analyses. Based on these numbers, the study group consists of 30%

of stroke patients experiencing significant depressive symptoms, which is an agreement with

previously published results regarding the prevalence of PSD10

.

_____________________________________

3.1 Demographic and Clinical Characteristics

All pertinent demographic and clinical characteristics are reported in Table 4 via

independent t-test and chi-square analyses. It is uncertain how long a patient was depressed

because the diagnosis of depression was made by our team. However, patients with a prior

history of depression or who were on anti-depressants at the time of approach were excluded

62

from the study. Therefore, it is unlikely that the depression evaluated by our team was a result

of a previous history of depression rather than the events prompted by stroke.

Table 4: Participant Demographics and Assessment Scores by Depression Group

Non-depressed (n=38)

Depressive Symptoms (n=16)

p-value

Demographics

Sex 52.6% 50.0% 0.860

Age 71.3 ± 17.7 69.4 ± 14.3 0.674

BMI (kg/m2) 28.0 ± 4.8 26.1 ± 5.4 0.228

Time between date of stroke and assessment (days)

30.2 ± 42.0 25.6 ± 36.8 0.713

Marital status (% married) 39.5% 68.8% 0.233

Living situation (% living with others)

68.4% 75.0% 0.751

Employment (% retired) 76.3% 75.0% 0.286

Education (% with Bachelor’s degree or greater)

36.8% 18.8% 0.514

Assessment Scores

CES-D score 6.2 ± 5.0 26.8 ± 10.8 <0.0001

MMSE imputed score 26.6 ± 4.3 25.55 ± 4.7 0.090

NIHSS score 7.6 ± 5.6 11.4 ± 5.4 0.097

Blood Markers

LNAA (µg/mL) 578.8 ± 147.0 538.6 ±141.2 0.022

KYN (µmol/L) 2.8 ± 1.6 3.3 ± 1.6 0.582

TRP (µg/mL) 8.8 ± 2.5 9.3 ± 3.7 0.120 For continuous variables, data is represented as mean ± SD

No significant differences were found between patient groups except for hypertension

(p=0.043). Although PSD is often found comorbid with cognitive impairments, the current

patient population did not display such a relationship as no significant difference was found

between mean MMSE scores (non-depressed group=26.6±4.3, depressed group=25.6±4.7,

F2,49=2.53, p=0.090). Plasma markers were examined using ANCOVA analysis with

hypertension acting as the covariate. Mean LNAA concentrations were found to be

significantly lower in the depressed patient group (F2,51=4.10, p=0.022), while other plasma

markers including TRP and KYN were found to be non-significant.

63

Lesion characteristics of stroke patients with and without depression can be viewed in

Table 5. The mean stroke lesion volume among all stroke patients was 21.89 ± 50.8 cm3 but

was not significantly different among non-depressed and depressed stroke patients (non-

depressed=23.6 ± 58.3, depressed=18.3 ± 31.8, p=0.763). The majority of stroke patients

(38.5%) had lesions located in the intermediate region (between anterior and posterior

lesions). The rest of the patient population had lesions in the following locations: 35.9% of in

the anterior circulation, 23.1% in the posterior circulation, and 2.6% extended from the

anterior circulation to the posterior circulation. There was no significant difference in lesion

locations between groups (2=3.74, p=0.291). Among all patients, 54.9% had right-sided

lesions, 43.1% had left-sided lesions, and 2.0% had lesions spanning both the left and right

sides. Finally, there was no significant difference in infarct side between depressed and non-

depressed patients (2=0.49, p=0.781).

Table 5: Stroke Lesion Characteristics by Depression Group

Non-depressed (n=27)

Depressive Symptoms (n=13)

p-value

Volume (cm3) 23.6 ± 58.3 18.3 ± 31.8 0.763

Location • %Anterior • %Posterior • %Intermediate • %Extending

34.6% 30.8% 30.8% 3.8%

38.5% 7.7% 53.8%

0%

0.291

Laterality (% Right) 59.3% 61.5% 0.781 For continuous variables, data is represented as mean ± SD

Almost all patients had at least one cardiovascular risk factor based on the entire study

population, including hypertension (71.4%), hypercholesterolemia (37.0%), diabetes (18.5%)

or cigarette smoking (18.5%). All recorded cardiovascular risk factors were found to be

higher in the depressed patient population as displayed in Table 5; however, only the

proportion of hypertensive patients in the depressed groups was significantly greater than the

non-depressed group (93.8% vs. 65.8%, χ2=4.48, p=0.04).

64

Based on different medical histories, all patients were treated with a variety of

medications including anti-hypertensives (57.4%), aspirin (66.7%), beta-blockers (37.0%),

calcium channel blockers (29.6%) and diuretics (16.7%). The proportion of patients taking a

certain medications did not differ between patient groups. Although the regular use of

psychotropics was counted as an exclusion criterion during recruitment, short acting

benzodiazepines (e.g. lorazepam) were permitted in this study to sedate or to aid patients in

their sleep. Medications consumed by both patient groups by proportion are displayed in

Table 6 via chi-square analysis.

Table 6: Cerebrovascular Risk Factors and Concomitant Medication Use by Depression Group

Non-depressed (n=38)

Depressive symptoms (n=16)

p-value

Cerebrovascular Risk Factors

Hypertension 65.8% 93.8% 0.043

Hypercholesterolemia 34.2% 43.8 0.507

Obesity (BMI ≥ 30) 31.6% 25.0% 0.629

Diabetes 15.8% 25.0% 0.426

Cigarette smoking 18.4% 18.8% 0.977

Total number of CVRFs 1.7 ± 1.2 2.1 ± 1.1 0.198

Concomitant Medications

Aspirin 68.4% 62.5% 0.673

-blockers 36.8% 37.5% 0.964

Ca+2 channel blockers 23.7% 43.8% 0.140

Diuretics 15.8% 18.8% 0.790

Insulin 15.8% 25.0% 0.426

Benzodiazepines 13.2% 6.3% 0.461 For continuous variables, data is represented as mean ± SD

_____________________________________

3.2 Assay Results

A total of 54 plasma samples (38 non-depressed, 16 depressive symptoms) were

collected and sent for analysis. Plasma markers of interest were KYN, TRP, LNAA and the

cytokines IL-6, IL-10, and IFN-. However, several cytokine samples were below the lower

limit of the assay’s detectability. As previously mentioned, these limits were 1.11 pg/mL for

65

IL-6, 0.30 pg/mL for IL-10, and 1.27 pg/mL for IFN-. Thus, imputed scores were also used

for cytokine statistical analyses, where the lowest level of detectability was used as the

imputed values.

3.3 Biological Correlates of Post-stroke Depression

3.3.1 Hypothesis 1: KYN/TRP ratio as a marker of depression

As displayed in bar graph form in Figure 3, ANCOVA analysis revealed a significant

difference in mean KYN/TRP ratio between depressed and non-depressed patient groups with

hypertension and LNAA concentrations as covariates (non-depressed group=69.32±36.90,

depressed group=78.33±42.03, F3,50=4.61, p=0.006) and an observed power of 86.4%.

Figure 3. Mean KYN/TRP ratios in stroke patients with and without depressive symptoms

* F3,50=4.61, p=0.006; non-depressed=69.3±36.9; depressive symptoms = 78.3±42.0 Values reported as mean ± SD.

A box-plot displaying further differences in the range, median, and interquartile ranges

of the KYN/TRP ratios can be viewed in Figure 4. However, partial correlation analysis

revealed a non-significant relationship between CES-D scores and KYN/TRP while

66

controlling for hypertension and disparities in LNAA concentrations (r50=-0.06, p=0.67). A

scatter plot outlining the relationship between CES-D scores and KYN/TRP ratio is displayed

in Figure 5.

Figure 4. Box-plot of unadjusted KYN/TRP ratios in stroke patients with and without depression

Figure 5. Correlation between CES-D total scores and KYN/TRP ratio

The same ANCOVA analysis was conducted after separating the combined depression

group into ―depressed‖ and ―depressive symptoms only‖ groups such that a total of three

67

groups existed. The results remained significant between groups (F4,49=4.027, p=0.007),

where those in the depressive symptoms category had a much higher KYN/TRP that the other

two groups. These results suggest that KYN/TRP may act as a red-flag for those most prone to

depressive symptoms post-stroke and possibly developing clinical depression thereafter. No

comment can be made about the depressed group due to the small sample size.

Other exploratory analysis found a significant correlation between KYN/TRP levels

and LNAA concentrations using bivariate correlation analysis (r2=0.21, p<0.001). A scatter-

plot of the relationship can be viewed in Figure 6.

Figure 6. Correlation between LNAA concentrations and KYN/TRP ratio

The presence of hypertension between depressed and non-depressed patients was

explored further since chi-square analysis found a significantly greater number of hypertensive

patients in the depressed than the non-depressed population via chi-square analysis (93.8% vs.

65.8%, χ2=4.48, p=0.03). Unfortunately, the production of a correlative relationship between

the two variables was not possible since systolic pressure over diastolic pressure values were

not collected during recruitment. To observe the reverse relationship where groups were

68

separated by the presence of hypertension rather than depression, ANOVA analysis revealed a

significant difference in mean CES-D scores between hypertensive and non-hypertensive

patients (non-hypertensive=7.0 ± 5.7, hypertensive=14.2 ± 12.9, F1,52=3.9, p=0.05). A graph of

this relationship can be viewed below in Figure 7.

Figure 7. Mean CES-D Scores by Hypertension Group

Further exploratory analysis was conducted to determine the relationship between

hypertension and the KYN/TRP, the KYN/TRP values were first separated into two groups,

―low‖ vs. ―high‖ (based on the median), proceeded by chi-square analysis with hypertension

acting as the other variable. However, results were non-significant (percent non-hypertensive

above the median KYN/TRP ratio=50% vs. percent hypertensive above the median KYN/TRP

ratio=73%, χ2=2.36, p=0.12). When ANOVA was conducted using the same variables, the

results were no different in significance between hypertension and mean KYN/TRP ratio

(mean KYN/TRP non-hypertensive=59.5±41.7, hypertensive=76.3±36.6, F1,52=2.3, p=0.16).

Although the relationship between hypertension and KYN/TRP was not significant,

hypertension acted as a significant co-variate in our analysis of KYN/TRP and depression

69

group. Thus, the effect of anti-hypertensive medication on the results of this primary

hypothesis was also examined. Hypertension was diagnosed based on reviewing the past

medical history in the patient chart. Out of all 54 patients, 40 were hypertensive and 31

patients were on anti-hypertensive medication. The distribution of hypertensive/non-

hypertensive patients on anti-hypertensive medication is listed in Table 7 as follows:

Table 7: Distribution of Hypertensive Patients on Anti-Hypertensive Medication

Hypertensive Non-hypertensive

On anti-hypertensives 27 4

Not on anti-hypertensives 13 10

Those who did not have a history of hypertension but were currently placed on anti-

hypertensives as an inpatient may have acquired hypertensive characteristics because of

complications from stroke. Likewise, those who had a history of hypertension but were not

give anti-hypertensives as an inpatients may have been taken off the medication due to

medical complications as seen fit by the attending doctor. When both hypertension and the

use of anti-hypertensive were controlled for in analysis of the primary hypothesis, The

KYN/TRP ratio remained significantly higher in the depressed group (F4,49=4.0, p=0.007).

Thus, the presence or absence of anti-hypertensive medication did not significantly alter our

results.

3.3.2 Hypothesis 2: Inflammatory markers of depression

Because preliminary analysis found the cytokine data to be highly skewed, a Mann-

Whitney test was employed to compare the mean cytokine levels between depressed and non-

depressed stroke patients with hypertension acting as the covariate. All three measured

cytokines (IL-6, IL-10, IFN-) revealed non-significant results (p=0.32, p=0.19, p=0.21)

respectively. When the imputed values were used for analysis, results remained non-

significant (p=0.627, p=0.216, p=0.297) respectively. However, about half or more of the data

70

required imputation creating a skewed set of data that would be hard to detect significance

even with non-paramatric tests. Nevertheless, the values remained numerically in the

direction of expectation, where pro-inflammatory cytokine levels were greater in depressed

group and the anti-inflammatory cytokine levels were lower in the depressed group. A more

detailed summary of the results can be viewed in Table 8.

Table 8: Measured and Imputed Cytokine Plasma Levels by Depression Group

N Non-depressed Depressed p-value*

Unadjusted values

IL-6 (pg/mL) 30 7.9 ± 5.0 9.3 ± 3.7 0.329

IL-10 (pg/mL) 13 1.9 ± 1.5 1.0 ± 0.4 0.192

IFN- (pg/mL) 27 5.1 ± 3.7 5.3 ± 1.7 0.205

Imputed values

IL-6 (pg/mL) 54 4.9 ± 5.0 5.7 ± 5.0 0.627

IL-10 (pg/mL) 54 0.59 ± 0.86 0.56 ± 0.42 0.216

IFN- (pg/mL) 54 3.1 ± 3.2 3.5 ± 2.4 0.297

*Based on Mann-Whitney statistics

Data is represented as mean values ± SD

Due to the high number of samples lost as a result of undetectability, chi-square

analysis was conducted to compare if there was a significant difference in percentage of

cytokine detectability between groups depression group. This was not the case as displayed in

the table below. Thus, detectability of cytokines should not have affected our results and was

not treated as a covariate during any analyses.

Table 9: Cytokine Assay Detectability by Depression Group

Non-depressed Depressed 2 p-value*

IL-6 56.8% 56.3% 0.001 0.973

IL-10 18.4% 37.5% 2.242 0.134

IFN- 47.4% 56.3% 0.355 0.551

Spearman's rank rho analysis revealed a relationship trending toward significance

between measured unadjusted IL-6 concentrations and KYN/TRP ratio (rho=0.33, p=0.08) and

is exhibited in scatter-plot form in Figure 8. However no other cytokine resulted in a similar

relationship with the KYN/TRP ratio. In addition, non-significant correlations were found

71

between all cytokines and total CES-D score when both the raw and imputed data were used

for analysis.

Figure 8. Correlation between unadjusted IL-6 concentrations and KYN/TRP ratio

Finally, the magnitude of variability imposed by combining the depression groups was

observed. When the combined depression group was separated into ―depressed‖ and

―depressive symptoms only‖ such that a total of three groups existed rather than two, the

results did not significantly differ from the originally reported results after running the same

analysis. The time of blood collection with respect to stroke onset was also evaluated for its

variability. Only 10 of the total 54 patients had blood drawn within greater than 1 month post-

stroke, 2 of which were in the depressed group. However, our preliminary analysis showed

that time since stroke and enrollment into the study did not significantly differ between patient

groups, thus, the inclusion of patients >3 months post-stroke should not significantly affect our

results. Nevertheless, when the 10 patients were removed from the subject population and the

cytokine analysis were rerun, they did not significantly differ from our initially reported

results.

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4. DISCUSSION

This study is the first to support the role of IDO activation in the etiology of PSD via

the KYN/TRP ratio marker. Mean KYN/TRP ratio was significantly higher in depressed than

non-depressed stroke patients. However, the severity of depressive symptoms, as measured by

total CES-D score, did not significantly correlate with KYN/TRP ratio or plasma cytokine

levels. Additionally, cytokine levels did not significantly differ between patient groups, but a

correlation trending on significance was found between KYN/TRP and IL-6 plasma levels.

Finally, the percent of hypertensive patients was significantly higher in depressed stroke

patients, a finding supported by a higher mean CES-D score among hypertensive patients than

non-hypertensive patients. No significant difference in mean KYN/TRP was found between

hypertensive and non-hypertensive patients.

_____________________________________

4.1 The Role of IDO Activation in Post-stroke Depression

Several studies examining neurological illnesses associated with unstable levels of

inflammatory markers have reported elevations in KYN/TRP ratio, including in Alzheimer’s

disease365

, Parkinson’s disease366

, HIV infection408

, rheumatoid arthritis363

, coronary heart

disease41, 375

and healthy aging409, 410

. Additionally, an increase KYN/TRP ratio has also been

associated with states of depression including women with post-partum depression39, 370

and

depressed hepatitis C and cancer immunotherapy patients38, 337

. However, despite several

speculations of a causal relationship between activation of the KYN pathway and the

development of PSD by various research groups, no association has been made to date. This

study is the first to report a significant difference in mean KYN/TRP ratio between depressed

and non-depressed stroke patients with a high observed power of 86.4%. Although this result

73

was relatively surprising since the sample size calculation predicted that 88 patients per group

was required to fulfill an observed power of 80%, we were still able to demonstrate

significantly elevated KYN/TRP ratios among depressed patients.

The results presented in this study agree with our hypothesis that IDO is activated upon

inflammatory burden post-stroke. Because the IDO enzyme has an extremely short half-life,

direct measurement of its plasma concentrations was not feasible. Thus, the ratio of its

primary metabolite KYN to TRP was used as a marker of its activity instead. An increase in

IDO activity may reduce 5-HT production since the enzyme is in direct competition with TPH

for the metabolism of TRP into 5-HT. Consequently, central 5-HT levels are decreased below

the level for normal mood and behavioural regulations. For example, TRP levels have been

found to be decreased in depressed patients314

while the administration of TRP produces an

antidepressant effect340

. Thus, depletion of TRP as a result of increased IDO activity may

explain our finding that the depressed patient population had a significantly higher KYN/TRP

than non-depressed subjects.

Increases in KYN levels may also affect mood since it can be further metabolized into

several neurotoxic metabolites including three potent neurotoxins; the hydrogen peroxide

generators, 3-hydroxykynurenine and 3-hydroxyanthranilic acid223

and the NMDA agonist,

quinolinic acid (QUIN)335

. Because these intermediates are neuroactive, central elevations in

their concentrations may destroy essential neurons responsible for controlling mood and

behaviour within the limbic structures of the brain. For example, 3-hydroxykynurenine levels

have been found to be elevated in other neurological disorders such as Huntington’s disease

patients354, 355

and has been shown to induce neuronal cell death in cortical and striatal cell

cultures44

. Intracerebral injection of 3-hydroxyanthranilic acid can decrease choline

acetyltransferase activity225

thus, decreasing acetylcholine synthesis. Finally, several

74

neurodegenerative conditions have been linked to QUIN-induced apoptosis, including

Huntington’s disease357

and HIV-associated dementia358

. Furthermore, laboratory studies

have demonstrated the production of QUIN to be immune mediated (in this case, stroke) and

acts as a potent agonist of the neuronal NMDA subtype of glutamate receptors335

. It is thus no

surprise that a significantly elevated KYN/TRP ratio was detected in the depressed patient

population as a combination of both decreased serotonin synthesis and increase neurotoxic

KYN metabolites can result in the clinical manifestations of PSD. Also note that stroke

severity was not significantly different between groups, suggesting that the described

biological pathway may be partially responsible for PSD rather than the sole influence of

post-stroke physical impairments.

An effort was made to control the potential demographic confounders collected during

the recruitment period. The presence of both hypertension and low LNAA concentrations

significantly differed between patient groups and were thus used as covariates in the analyses.

Properties of hypertension including decreased lumen size and decreased vessel distensibility

have previously been found to be prominent in patients with depressive symptoms and those

who also suffer from reduced blood flow velocity were diagnosed with ad DSM-IV depressive

disorder110

. LNAA directly competes with TRP for transport into the brain and because the

direction of TRP metabolism affects synthesis of 5-HT and KYN, significantly different levels

of LNAA would be expected from depressed patients.

In this study, plasma levels of LNAA were significantly lower in depressed patients

while TRP levels (although not significant) were numerically higher. This counters past

studies that have reported lower TRP and TRP/LNAA ratio in MDD patients compared to

healthy controls305, 310-312

. Our contrasting results may include one of the following three

explanations, a combination of all, or other factors outside our control. First, it is common for

75

clinical studies to measure plasma TRP in the morning after overnight fast since TRP can only

be obtained exclusively through dietary sources. Although our study attempted to ensure all

blood samples were collected in the morning (approximately 8 a.m.) during the IV

technologist’s regular rounds, patients were known to have breakfast as early at 7 a.m. in some

cases, while blood draw times were delayed to approximately 11 a.m. in other cases. Thus,

variability in collection time and diet may have contributed to the unexpected increase in TRP

availability among stroke patients. Secondly, the amount of dietary tryptophan and LNAAs

(e.g. tyrosine) consumed may differ between patient groups depending on their willingness to

eat. Since one of the most defining symptoms of depression includes the loss of appetite,

variations in TRP and LNAA consumption between depressed and not depressed patients may

have confounded our results. Finally, peripheral levels of TRP, KYN, and LNAA may not

parallel with its central availability. It would be necessary to devise experiments looking at

both the central and peripheral availability of these molecules in order to validate if an

increased TRP/LNAA ratio or its opposite is representative of the observed depressive

symptoms in PSD patients.

Interestingly, a significant positive correlation could not be found between total CES-D

scores and the KYN/TRP. Therefore, it would be necessary to test this hypothesis on a greater

sample population of depressed patients as our study was limited to a small group of subjects.

This was mostly due to a decrease in sample availability in order to prevent the confounding

effect of plasma analyses from two different laboratories. Furthermore, because research

regarding the clinical and neuropsychiatric correlates of KYN/TRP are still in its primitive

phase, other demographic or clinical factors that have not yet been documented may be

confounding the analysis. For example, it has been documented that post-stroke major and

minor depressions have different prognoses. The prognosis of MDD has been shown to be

76

more favourable than that of minor depression, with resolution of depressive symptoms in

75% of the MDD patient population compared to only 30% of the minor depression cohort411

.

In addition, those with minor eventually developed major depression. By increasing sample

size, we would be able to segregate post-stroke MDD and minor depression groups along with

other demographic variables that each cohort may entail. Determination of these factors may

lead to more significant results when controlling for them during partial correlations.

Whether elevations of the KYN/TRP ratio is a phenomenon directly related to PSD

and other neurological illness or a marker that can be employed in otherwise healthy

individuals has yet to be elucidated. It would be necessary to conduct the same study by also

enrolling healthy, non-stroke depressed and non-depressed individuals. Although the

relationship between depression and inflammation has been well-documented in the past, there

is still uncertainty whether the former induced the latter or vice versa. Through the inclusion

of healthy individuals in a longitudinal study design one may be able to decipher if the

relationship between the two variables is uni- or bidirectional depending on the individuals

neurological state and also assess the role of the KYN/TRP ratio in both subgroups.

Moreover, the addition of follow-up visits would allow for the time-course evaluation of the

KYN/TRP ratio and changes in inflammatory states of each participant. Even so, current

animal models suggest that IDO is most active following cerebral ischemia412, 413

and may be

necessary to induce sickness behaviours208

, suggesting that basal cytokine levels are rarely

sufficient enough to alter mood states via IDO induction.

_____________________________________

4.2 The Role of Cytokines in Post-stroke Depression

Since the inflammatory pathways are triggered under post-stroke conditions and are

77

the primary inducers of IDO activity, elevated pro-inflammatory cytokines (IL-6, IFN-) and

diminished anti-inflammatory cytokines (IL-10) were expected among the depressed patient

population. Although the numerical mean values equate to the anticipated direction of

movement, none of the measured levels significantly differed between patient groups.

However, a positive correlation trending on significance was found between IL-6 levels and

KYN/TRP ratio as demonstrated in Figure 5, supporting the role of pro-inflammatory

cytokines in IDO activation.

It is possible that a significant difference in cytokine levels between groups could not

be detected due to a broad separation between time of study assessment and time when

inflammation is most pronounced post-stroke. As displayed in Table 3 the average time

between stroke and recruitment averaged 25 to 30 days in the depressed and non-depressed

groups respectively, with some patients reaching almost 3 months post-stroke before the initial

evaluation. However, animal studies have detected circulating monocytes within the

capillaries and venules 4 to 6 hours after induced ischemia, as well as an accumulation of

PMNs 12 hours after the same injury161

. At this time inflammatory molecules initiate their

activity and exacerbate tissue damage via edema formation, release of oxygen radicals,

cytokines, and cytolytic enzymes leading to cellular necrosis162-164

. Because our cytokine

concentration measurements occurred as late as 3 months post-stroke for both depressed and

non-depressed patients, it was difficult to determine an accurate reflection of cytokine level

differences between patient groups. Furthermore, cytokines have previously been reported to

increase the risk of recurrent stroke172

. Since a few of our participants were not first-time

stroke patients, elevated cytokine levels may have already been present prior to their most

recent ischemic attack regardless of their mood state. This would also confound our ability to

precisely define significant differences in mean cytokine levels between depressed and non-

78

depressed subjects. However, because only four of our patients were previously diagnosed

with stroke, it is unlikely that recurrent stroke played a major role in cytokine variability in our

study. Nevertheless, previous history of stroke was treated as a covariate in further analysis

and did not the significantly change the results. Once sample size is increased and thereby,

also increasing the likelihood of recruiting patients with previous stroke, the same statistics

should be repeated while controlling for this variability. Taking everything into account, the

optimal recruitment time would be 5 days to 1 month post-stroke such that undesired early

spikes in cytokine concentrations or late undetectable differences between groups can be

eliminated.

The time of blood collection relative to stroke onset may have also hindered cytokine

detectability in this study which decreased the power of our analyses. Currently, the majority

of the data regarding the time course of cytokines post-stroke are available for IL-6. While it

has been suggested that its activity may remain elevated up to one year post-stroke414

, another

study has reported peak IL-6 response to occur 4 days post-stroke62

. The latter study also

found the detection of IL-6 to be significantly higher only up to 1 month post-stroke in stroke

patients compared to healthy controls. Although this numerical value was still 127% of

controls after 3 months, the difference was no longer statistically significant. Similarly, the

cytokine IL-10 has also been reported to peak in response 3 days after stroke415

. Thus,

assaying plasma samples collected from our patients 5 days to 3 months post-stroke may have

altered detection levels, as some may have had their cytokine concentrations documented at its

peak response while others may have had their concentrations recorded during the tail-end of

response. Based on past research, it would be necessary to narrow cytokine analysis to only

those patients recruited up to 1 month post-stroke. However, because our analysis was limited

by low detectability, patients recruited up to 3 months post-stroke were included to increase

79

yield. As recruitment continues and assay procedures become stabilized, our ability to assess

stroke survivors only up to 1 month post-ischemic attack will increase in power. However, it

is important to note that, only 10 of the total 54 patients had blood drawn within greater than 1

month post-stroke, 2 of which were in the depressed group. Furthermore, our preliminary

analysis showed that time since stroke did not significantly differ between patient groups

(Table 3). Thus, the inclusion of patients greater than 3 months post-stroke should not have

significantly affected our results. As suspected, when the 10 patients were removed from the

subject population and the cytokine analyses were rerun, they did not significantly differ from

our initially reported results.

Blood collection time relative to morning or night periods may also have an effect on

our study. Fasting blood levels were collected from patients in the mornings (around 9:00 am)

based on the IV technician's regular rounds in the wards. However, this was not always the

case, as the IV technician may have made rounds with ±2 hours of the study's 9:00 am

standard. This is a limiting factor to our study since cytokines have been found to be regulated

in accordance with the circadian rhythm. Such diurnal rhythms are believed to follow

consistent and remarkable patterns as demonstrated across a wide range of experiments.

These studies reported production of pro-inflammatory cytokines (IL-2, IL-6, IL-12, TNF-α,

IFN-γ) to be maximal during nocturnal sleep, peaking between 1–4 a.m.416-419

. Although these

findings suggest it would be most appropriate to draw blood from patients at night, a change in

procedure remains implausible since laboratory workers are not present at that time to collect

and centrifuge the blood for further analyses. Thus, our ability to detect levels of pro-

inflammatory cytokines may have been hindered by their natural fluctuations in accordance

with circadian rhythms. It should be noted nonetheless that all samples were taken in the

morning, and none were taken in the evening, or between 1 and 4 am. This should minimize

80

the impact of sample time variability. Finally, identifiers of possible mediators that enhance or

suppress actions of nocturnal sleep on pro- and anti-inflammatory cytokines include high

levels of growth hormone and prolactin and low levels of cortisol and catecholamines,

respectively420-423

. Thus, further research should explore how these molecules vary with

changing levels of cytokines and the possible role they may play in PSD progression.

The importance of pro- versus anti-inflammatory response during post-stroke

conditions was previously discussed. Two recent publications have described what is believed

to be an ―immunodepression syndrome‖, where the fate of the injured brain tissue is

dependent on the intricate balance between pro- and anti-inflammatory cytokines196, 197

.

Moreover, divergence from their naturally occurring concentrations is thought to induce an

immune response. A couple of studies have since then reported increased pro- to anti-

inflammatory cytokine ratios in major depression, including the IL-6/IL-1069

and the IFN-

γ/IL-4 ratio70, 71

. Unfortunately, low cytokine detectability hampered our ability to evaluate

the ―immunodepression syndrome‖ theory in our study as only a few patients had well

detected pro- and anti-inflammatory cytokines to generate such a ratio. This was a major

drawback since a balance between pro- and anti-inflammatory cytokine stimulation may be

particularly important with regards to IDO activation. In fact, cytokine ratios may even hold a

predictive value in the development of PSD when tied to the KYN hypothesis. Hopefully our

finding that IL-6 concentrations are trending towards significance with the KYN/TRP ratio

will generate greater interest in this field of study.

In summary, the numeric value of both raw and imputed cytokine levels were

directionally in line to our prediction, but not in full agreement with past studies reporting

significant differences in cytokine levels between depressed and non-depressed healthy

individuals52, 58, 235-238

. This was surprising since our hypothesis anticipated stroke to

81

significantly elevate inflammatory marker concentrations such that those suffering from the

highest level of inflammation would begin to manifest depressive symptoms. Thus, it would

be worthwhile to replicate our assays to improve the reliability of our findings. Furthermore,

measurements of LPS stimulated cytokine levels have been conducted in previous studies and

may mimic altered states of immune activation representative of post-stroke periods424

.

Evaluation of unstimulated and stimulated cytokine levels may also be necessary to further

elucidate the relationship between inflammation, stroke and depression.

_____________________________________

4.3 The Role of Hypertension in Post-stroke Depression

Our study reports a greater percent of hypertensive patients among the depressed

population. With respect to blood vessels, hypertension is characterized by two main factors:

(1) the structural remodeling of cerebral arteries via increasing wall thickness/lumen ratio107

and (2) impaired endothelium-mediated vasodilatation as a result of collagen build-up108

.

Ultimately, the effects of decreased lumen size and decreased vessel distensibility may reduce

both CBF and cerebrovascular reactivity. CBF have been found to be decreased in

hypertensive subjects compared to healthy controls109

. The limbic and paralimbic structures

of the brain, which are known to house the major areas responsible for emotional processing,

were found most altered due to this decrease. Both our prediction and results agree with these

concepts and findings and match other clinical studies that report impaired blood flow velocity

in subjects suffering from a DSM-IV depressive disorder 110

.

However, similarly to the relationship between depression and inflammation, no

conclusive statements can be made about the order of their appearance. Although research

surrounding this topic remains scarce, one study has reported CVR to be significantly reduced

in a group of depressed patients free of vascular risk factors112

. This suggests that major

82

depression may be the actual cause CVR malfunction rather than its consequence. Although

there is no definitive evidence linking depression to the development of hypertension, a couple

of studies have found that depression can impair the management and prognosis of

hypertension425, 426

. Since our study focused solely on stroke patients, it would be necessary to

include otherwise healthy depressed and non-depressed groups into the study to further

evaluate the possible relationship between depression and hypertension and whether this

phenomenon is only applicable to stroke conditions. It would also be necessary to include raw

blood pressure values to be able to analyze the data from a numerical standpoint.

Because depression was found to be significantly related to hypertension and

KYN/TRP, an analysis was conducted to compare the levels of KYN/TRP between

hypertensive and non-hypertensive patients. Although the numeric value was higher in

hypertensive compared to non-hypertensive patients, the results were non-significant (p=0.12

for chi-square analysis, p=0.16 for ANOVA analysis). Currently, this is the first study to

examine the relationship between KYN/TRP and hypertension. Furthermore, no other study

has previously examined the triple relationship between stroke, hypertension and the

KYN/TRP. As our results indicate a significantly higher KYN/TRP ratio in depressed

subjects while controlling for hypertension, the next step would be combine the depressed and

hypertensive variables to generate four patient groups (non-depressed and non-hypertensive,

non-depressed and hypertensive, depressed and non-hypertensive, and depressed and

hypertensive) and evaluate their association to KYN/TRP ratio. Due to the small sample size

of depressed patients in our study, we were unable to carry out the analyses ourselves as only

two subjects qualified for the depressed and non-hypertensive group. Thus, active recruitment

would allow for a greater number of individuals in order to evaluate the proposed

relationships.

83

In support of the triple relationship between depression, KYN/TRP ratio and

hypertension, previous studies have produced results that make it worthwhile to explore this

concept further. Most recently, it was reported that kynurenine formation by IDO-expressing

endothelial cells contribute to arterial vessel relaxation and regulation of blood pressure in

systemic inflammation427

. Although the effects and significance of this finding have yet to be

elucidated, the regulation of vascular tone by IDO may substantiate a link between

hypertension and PSD. However, the results described from that study appear to favour an

opposite outcome (hypotension) then the one hypothesized by this study (hypertension).

Conversely, animal models may favour an outcome more in line the predictions of this study.

As previously mentioned, KA is a NMDA antagonist that counters the neurotoxic effects of

the QUIN metabolite. It is synthesized in the brain by the enzyme kynurenine

aminotransferase-1 (KAT-1) which concentrations have been reported to be significantly

reduced in spontaneously hypertensive rats (SHR) compared to normotensive Wistar Kyoto

rats (WKY)428

. The same research group further reported that all examined strains of SHR

possessed a missense mutation in the KAT-1 gene but not in any of the WKY outbred

strains429

. These results suggest that a mutation in the KAT-1 gene leading to deficient

amounts of KA production may be associated with increased effects of the neurotoxic KYN

metabolites in hypertensive subjects. In keeping with stroke, further investigations searching

for a similar KAT-1 mutation in the human genome may relate hypertension to an elevated

KYN/TRP ratio among the PSD population.

The KA levels are also affected by the availability of the kynureninase enzyme. As

depicted in Figure 1, kynureninase is responsible for converting KYN into anthranilic acid

which is subsequently metabolized into 3-hydroxyanthranilic acid. It has been shown that

inhibition of kynureninase as achieved by the intraperitoneal administration of m-

84

benzoylalanine results in large accumulation of KA in the brain. Furthermore, one animal

study documented reduced basal blood pressure after KA injection into the rostral

ventrolateral medulla (RVLM) in SHR but not in normotensive WKY430

. Since the RVLM is

pertinent to the tonic and reflexive regulation of arterial blood pressure

431, kynureninase is

speculated to play a role in blood pressure regulation. Along this line of logic, those with a

genetic predisposition to increased kynureninase activity will experience higher concentrations

of the neurotoxic KYN metabolites under states of inflammatory stress (e.g. stroke) than those

without the genetic predisposition, and that this observation will be most pronounced in

hypertensive patients.

Finally, a reduction of TRP and simultaneous increase of KYN and QUIN

concentrations have been observed in patients with chronic renal insufficiency and correlated

with the severity of the disease432, 433

. Similar results are exhibited in renal insufficient rats

where QUIN and L-kynurenine levels in serum, brain, and CSF are increased parallel to the

severity of renal incompetence433

. Since the kidney is well-known for its homeostatic control

of blood volume via renin-angiotensin-aldosterone system and anti-diuretic hormone (ADH)

release from the adrenal cortex and posterior pituitary, damage to the organ may lead to

abnormal blood pressures (e.g. hypertension). Indeed, rat models with induced uremia (a

kidney disease where urea and other waste products normally excreted into the urine are

retained in the blood) display significantly decreased TRP plasma levels and augmented

concentrations of the KYN metabolites 3-hydorxykynurenine and QUIN434

. This suggests that

an elevated KYN/TRP ratio may be responsible for the severity of uremic symptoms such as

neuropathy, and increases one's susceptibility to infections, anemia and hypertension. Thus,

future PSD studies may also want to look into kidney function and how it may be related to

KYN/TRP and hypertension.

85

Several other risk factors relating depression to hypertension have been documented in

the past that were not included in this study. For example, a combination of depression and

anxiety symptoms as measured by the General Well-Being Schedule and DSM-IV diagnostic

criteria, have been associated with elevated risk of incident hypertension in a couple of

studies435, 436

. One of these studies also reported that race and gender may also play a role

since the effect of anxiety was more pronounced in black women compared to white women

or men435

. Certain aspects of depressive symptomatology may also be related to hypertension.

In a 4-year follow-up prospective study, hopelessness was associated with increased incidence

of hypertension in initially normotensive men. Here, men reporting high levels of

hopelessness at baseline were 3 times more likely to become hypertensive than men who were

not hopeless437

. Finally, it is widely accepted that blood pressure and susceptibility to

hypertension are influenced by genetic factors438

. Furthermore, twin, adoption and family

studies have linked genetics to the etiology of mood disorders. A combination of the two

findings suggests that a shared genetic vulnerability may play a role in the relationship

between depression and hypertension. Indeed, one study found that individuals with positive

family histories of hypertension exhibit more depressive-like behavior when exposed to

mental stress tasks439

. Results from this study also suggest that a combination of genetic and

psychological factors (i.e. stressors) may be involved with cardiovascular hyperreactivity.

Thus, inclusion of all the aforementioned risk factors (anxiety, race, specific aspects of

depressive symptomatology, genetics, and psychological well-being) should be executed in

future studies in order to achieve a well-rounded understanding of hypertension and its effect

on mood disorders.

Finally, pre-clinical studies suggest that a mutation in the KAT-1 gene leading to

deficient amounts of KA production may be associated with increased effects of the

86

neurotoxic KYN metabolites in hypertensive subjects428

. Thus, future studies may choose to

measure the KA/KYN ratio as an indicator of KAT-1 activity, where individuals with high

blood pressure would be expected to have a lower KA/KYN ratio than normotensive subjects.

This finding would also suggest the presence of higher KYN levels in hypertensive subjects

such that an elevated KYN/TRP should be observed. Recently, the KA/KYN ratio has been

speculated to be a biomarker for neuroprotection as one study reported reduced levels to be

correlated with depression severity in hepatitis C immunotherapy patients337

. Using this logic,

a lower KA/KYN ratio may be indicative of PSD severity, with hypertensive PSD patients

displaying the lowest KA/KYN ratios.

_____________________________________

4.4 Limitations

This study was primarily limited by the small sample size, particularly with respect to

the depressed group. Initial sample size calculations predicted statistical significance to be

achieved at 88 patients per group. However, this study only represents a post-stroke

population of 38 non-depressed and 16 depressed patients. Although results of the primary

hypothesis regarding differences in KYN/TRP ratios was significant under ANCOVA analysis

with a high observed power of 86.4%, the results from the secondary hypothesis examining

differences in cytokine levels were not significant. Furthermore, when the imputed values

were used for analysis, results remained non-significant. However, about half or more of the

samples required imputation creating a very skewed set of data that would be hard to detect for

significance even with the Mann-Whitney Test. Thus, sample size of the depressed patient

population should be increased to alleviate the impact of this limitation.

The secondary limitation to this study was the high level of undetectable cytokine

samples. To control for this loss, we opted to create imputed values for the missing data by

87

substituting them with the lower limit of the assay’s detectability. Although this method

generated cytokine values that deviated from their true means, the main goal of this study was

not to capture low concentrations of pro-inflammatory cytokines but their elevated levels.

Thus, the approach should not affect our ultimate intent of finding clinically significant

elevated cytokine concentrations. This is supported by our analysis examining differences in

undetectability of cytokines between patient groups which did not yield significant results.

From a more conservative perspective, it is likely that the technique overestimated all

data since it was not possible to generate discrete values for cytokine levels below the outlined

assay sensitivities. Even so, we felt that this method was the most applicable to our data and

provided us with the most conservative estimates. Unfortunately, incorporating the imputed

values to our analysis did not yield any significant results since such a large majority of our

patient population had cytokine levels below the detection limit of the assay. For example,

mean levels of pro-inflammatory cytokines could in truth, be significantly higher in the

depressed group even though the individual values may be lower than the assay sensitivity.

The reverse is also true where imputation would over-estimate plasma pro-inflammatory

cytokine levels in the non-depressed group if they were indeed lower as predicted in

hypothesis 2. Data pertaining to anti-inflammatory cytokines were much more affected since

our hypothesis predicts the presence of decreased concentrations. Imputation of a depressed

patient's anti-inflammatory cytokine level would overestimate the true lowest level of interest.

Taken all together, imputation would still make it very difficult to accurately define

differences in cytokine concentrations between patient groups.

Due to the small sample size, we were unable to categorize patients according to the

DSM diagnosis of major depression. Thus, patients who were clinically depressed and

displayed milder forms of depressive symptoms were combined and analyzed as one group.

88

Although differences in mean KYN/TRP ratio was significant under ANCOVA analysis, it is

possible that separation between clinically depressed and those who only exhibit depressive

symptoms (such that three groups would be employed in the analyses) would have yielded

different results. For example, patients who only display depressive symptoms may have

lowered the actual mean KYN/TRP ratio and pro-inflammatory cytokines among the clinically

depressed patient population. Correlative analysis also revealed that a possible, positive

correlation may exist between patients with the severest cases of depressive symptoms and

KYN/TRP ratio. Separation of the current "depressed group" into its major and minor

components would aid in this investigation. Furthermore, many studies examining the

biological correlates mentioned in this study categorize patients explicitly based on the DSM

diagnosis criteria. On-going recruitment would increase the sample size such that a more in

depth analysis can be carried out between non-depressed, depressive symptoms only, and

depressed patients. Moreover comparisons with other studies would carry greater legitimacy

by eliminating heterogeneity between different methods of study design.

In summary, the major limitations of this study were sample size, low levels of

cytokine detectability, and the shortcoming of having to combine milder depressive symptoms

patients with clinically depressed patients into one group due to small sample size.

_____________________________________

4.5 Recommendations for Future Research

The primary recommendation would be to increase the sample size of the study

population with greater emphasis on recruitment of depressed patients. The addition of more

patients would increase the observed power to detect differences in mean cytokine levels

between patient groups. Furthermore, it would no longer be a necessity to create a

heterogeneous group consisting of those with milder forms of depressive symptoms with those

89

who are clinically depressed. This would allow for analysis of the individual depression

groups according to the DSM diagnosis criteria as well as more appreciable comparisons with

other study populations by decreasing heterogeneity between studies. Since it has been

previously reported that cytokine levels are associated with recurrent stroke, it would be useful

to longitudinally track the depressive outcomes of these patients and assess their relationship

to cytokine concentrations and the KYN/TRP ratio. Indeed, the study is still actively

recruiting patients and has included two follow-up visits at 6 and 12 weeks post-baseline

assessment to track the time-course of these biological correlates, as well as demographic

influences on depression severity. Recently, low level of education, low income, severity of

stroke, worse functional outcome, and self-reported problems with ADLs have been shown to

be predictive of depression symptoms at 3 months post-stroke440

. Moreover, low social

support at three months and loneliness and low satisfaction with social network at six months

has been reported to be predictive of post-stroke psychological distress441

.

It would be interesting to examine the triple relationship between depression,

KYN/TRP ratio and hypertension in greater depth as our study indicates that 93.8% of the

depressed patient population was also hypertensive. Several demographic risk factors relating

depression to hypertension that have been documented in the past were not included in this

study, including race and level of anxiety. In addition, the relationship between hypertension

and KYN/TRP ratio was close to trending on significance. We hope that by collecting patient

demographics and clinical history in greater depth, we will be able to control for these factors

during analyses and generate significant results between hypertension, depression and

KYN/TRP ratio. It would also be interesting to include a psychophysiological aspect to the

study by submitting patients to certain mental tasks (or stressors) while measuring their

cardiovascular response. Previously, it has been found that both men and women with a

90

positive family history of hypertension exhibit higher tonic levels of blood pressure and heart

rate during such stressors439

. The next step would be linking the relationship between

hypertension and stress to depression, the KYN/TRP ratio, and other biological correlates of

mood disorders. Also, it would be worthwhile to investigate whether this observation applies

to otherwise healthy but hypertensive and depressed individuals, or if it has a greater effect on

those suffering from neurological conditions such as stroke, since stroke itself is can also be

considered a stressor.

Genetics of depression may play a role in the etiology of PSD based on findings from

past studies. Polymorphisms of the SERT have been repeatedly associated with several

neurological illnesses. Coded by the SLC64A gene in the SERTLPR, the presence or absence

of 43 kb yields a long (l) variant or short (s) variant of the gene442

. Current research suggests a

significant association between the s genotype and PSD among all ethnicities443

, 444

. It has

also been demonstrated that individuals with the SERTLPR s/s genotype have a 3-fold higher

odds of PSD compared with l/l type carriers. Additionally, another SLC64A polymorphism

known as the intron 2 VNTR (STin2 VNTR) has been implicated in the etiology of PSD.

STin2 VNTR is located in intron 2 and consists of a variable number (usually 9, 10, or 12) of

nearly identical 17-bp segments, with the 9-repeat allele (STin2.9) conferring increased odds

of major depression and bipolar disorder445

. In fact, one research group reported that stroke

patients with the STin2 9/a2 or 12/12 genotype had 4-fold higher odds of PSD compared with

STin2 10/10 genotypes444

.

There are also several known mammalian 5-HT receptor genes, including 5-

HT1A/B/D/E/F, 5-HT2A/B/C, 5-HT3A/B/C/D/E, 5-HT4, 5-HT5A/B, 5-HT6, 5-HT7. Moreover, some of

these receptor genes may encode additional receptor variants446

. Within this group,

polymorphisms in the 5-HT1A gene have been the most investigated for its role in psychiatric

91

disorders and treatment responses. The 5-HT1A receptor is encoded by an intronless gene

located on human chromosome 5q11.2–q13 and it spans about 1200 bp and was first cloned by

was first cloned by Fargin and Albert447, 448

. Both chronically stressed animal models and

subjects with MDD have been reported to have a significantly decreased dorsolateral pre-

frontal cortex and hippocampal expression of 5-HT1A mRNA449, 450

. Conversely, enriched

expression of the gene carrying the G allele of the C(-1019)G polymorphism has been

associated with higher risk of depressive episodes and greater resistance to antidepressant

therapy451-457

. Moreover, the polymorphism may be sex-linked as one study reported

significantly higher binding potential of the receptor in females compared to males, especially

in regions of the dorsal raphe, amygdala, anterior cingulate, cingulate body, and the medial

and orbital pre-frontal cortexes454

. Thus, further exploration into the 5-HT1a polymorphism

and its role in PSD should be implemented in future studies.

Finally polymorphisms within cytokine coding genes may also be related to PSD.

Several studies have reported that polymorphisms in IL-1a, IL-1Ra and TNF-a genes458-461

and

polymorphisms within the promoter region of the IL-6 gene are associated with increased risk

of stroke462, 463

. However, whether these polymorphisms are linked to both increased risk of

stroke and the development of depression thereafter requires further research. Thus, it would

be worthwhile to study both SERT and cytokine genetic components in our study population

and examine their roles in the etiology of PSD.

_____________________________________

4.6 Clinical Implications

Detection of elevated KYN/TRP ratio early post-stroke would aid clinicians in

determining which patients are most prone to PSD. In these cases, immediate preventative

measures can be taken to inhibit disease manifestation. Since the prevalence of depression

92

post-stroke is documented to be as high as 30%, some clinicians opt to prescribe

antidepressants to their patients immediately following stroke even before a formal diagnosis

of the disease is made. However, research regarding prompt pharmaceutical interventions

post-stroke is scarce.

Previously, a Cochrane systemic review was conducted examining methods for

preventing depression post-stroke464

. The group investigated randomized controlled trials

comparing pharmaceutical agents with placebo or psychotherapy against standard care as

means of intervention. After reviewing 10 pharmaceutical trials and 4 psychotherapy trials,

the authors concluded that there was no clear effect of pharmacological therapy on the

prevention of depression. Conversely, there was a significant improvement in mood for

psychotherapy patients. However, although the prevention of depression was evident, the

treatment effects were reported to be small. Overall, the general consensus is that more

reliable data is required before recommendations can be made about either technique.

Additionally, the results from our study indicating a significantly higher mean KYN/TRP

among those displaying depressive symptoms should also be explored alongside future

pharmacotherapy studies, and may provide an improved selection criterion for patients who

should undergo antidepressant treatment immediately following stroke.

The ability to predict treatment response has always been of great interest to clinicians

since it would allow them to determine the best available medical intervention for their

patients on an individual basis. One method currently being explored for the individualization

of pharmacotherapy is the use of biomarkers as a predictor to treatment response. According

to the Biomarkers Definitions Working Group, a biomarker can be described as ―a

characteristic that is objectively measured and evaluated as an indicator of normal biological

processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention465

‖.

93

For example, results from earlier research proposed that SSRIs would be most beneficial to

those who exhibit low TRP/LNAA ratios, while TCAs would generate a better response in

patients with a high TRP/LNAA ratio466

. This hypothesis has since then been refuted by other

studies that reported no significant difference between antidepressant response and

TRP/LNAA ratio467, 468

. Thus, a rational biomarker that is predictive of antidepressant

response specific to PSD patients has yet to be identified.

The KYN/TRP ratio has previously been hypothesized to be a potential biomarker for

stroke volume, where an elevated KYN/TRP ratio may be indicative of a larger infarct size469

.

Results from our study suggest that the KYN/TRP ratio may be utilized as a probable

biomarker to predict antidepressant response in PSD. For example, a higher KYN/TRP ratio

would indicate elevated levels of the neurotoxic KYN metabolites and lower TRP availability

for 5-HT synthesis. If this is the case, the 5-HT levels may already be so depleted that

inhibiting 5-HT uptake from the synapse via SSRI treatment will prove to be ineffective in

preventing depression. In addition, activation of the NMDA receptors and oxidative stress

mechanisms via QUIN and 3-hydroxykynurenine production may degenerate the SERT-

containing neurons at the site of SSRI action. Because our results already indicate a

significant difference in KYN/TRP ratio between depressed and non-depressed patients, the

next step would be to examine whether this biomarker is a suitable predictor of antidepressant

response in a RCT study.

As outlined in the review of literature, activation of the KYN pathway via pro-

inflammatory cytokines contribute to post-stroke glutamate excitotoxicity and subsequent

calcium accumulation through the production of QUIN. Since QUIN is an endogenous

NMDA receptor agonist, it may be advantageous to treat PSD patients exhibiting high

KYN/TRP ratio with drugs that inhibit the NMDA receptor. In fact, one study reported that

94

the NMDA antagonist memantine was able to block oligodendrocyte NMDA receptors at

clinically significant therapeutic concentrations after induced ischemia, as well as improve the

recovery of action potentials in myelinated axons470

. Clinically, memantine has been

demonstrated to significantly improve aphasia severity in chronic post-stroke aphasic patients,

with its beneficial effects persisting during long-term follow-ups. However, the best outcomes

were achieved when the drug was combined with constraint-induced aphasia therapy (CIAT),

a form of therapy that forces the patient to communicate verbally without using gestures, non-

word sounds or writing471

. Thus, the same concept may be applied to PSD, where a

therapeutic regimen combining a NMDA receptor antagonist with an antidepressant and

psychotherapy may aid in preventing disease manifestation. Furthermore, if response to

antidepressant alone is hindered by the neurodegeneration of the SERT containing neurons

due to KYN toxicity, an add-on of a NMDA receptor antagonist may facilitate recovery.

It is important to note that activation of the KYN pathway not only produces

neurotoxic metabolites, but also the neuroprotective kynurenic acid (KA). KA acts as a

NMDA antagonist and opposes the negative effects of QUIN. However, studies examining

the measure of its action compared to other KYN metabolites have been limited. The

KA/KYN ratio has been proposed to be a measure of neuroprotection since reduced levels of

this ratio has been associated with MDD patients40

, and correlated with depression severity in

hepatitis C patients undergoing immunotherapy337

. Although these findings suggest potential

treatment opportunities with KA to help restore mood balances in post-stroke patients, it does

not readily cross the BBB371

and required large doses to be effective in preclinical studies472

.

A more plausible route would be to inhibit the kynureninase and kynurenine-3-

monooxygenase (KMO) enzymes responsible for converting KYN into its neurotoxic

metabolites, such that KYN would be shunted into the kynurenine aminotransferase pathway

95

to form KA. Nicotinylalanine was the first compound that was reported to act in this manner;

it significantly increased the brain content of KA while also preventing the induction of

seizures473, 474

. The more recently developed KMO inhibitors, m-nitrobenzoyl-alanine

(mNBA) and 3,4-dimethoxy-[-N-4-(nitrophenyl)thiazol-2yl]-benzenesulfonamide (Ro 61-

8048) have also been reported to increase brain KA synthesis475, 476

as well as significantly

reduce infarct volume in MCAO treated rats477

, reduce post-ischemic neuronal death in gerbil

hippocampal slice cultures476

, and decrease extracellular basal ganglia concentrations of

glutamate that would have otherwise accumulated from overproduction of QUIN475

. Finally,

KMO is also inhibited by the compound PNU156561 (formerly FCE28833A), which has been

documented to increase levels of KYN by tenfold and KA by 80-fold in rat hippocampal

dialysates for 24 hours after a single dose478.

Most recently, orally active hydroxyamidine small molecule inhibitors (INCB023843

or INCB024360) have been shown to suppress IDO activity in the plasma of mice and dogs479

.

For example, they were reported to decrease plasma KYN concentrations in vivo in wild-type

mice to those seen in Ido-deficient mice, suggesting these compounds can completely block

IDO function. The same study also documented decreased tumour size in tumour-bearing

mice after application of the inhibitor. Although the results are promising, the study only

measured KYN concentrations as a marker of IDO activity rather than the more appropriate

KYN/TRP ratio. Thus, it would be worthwhile to replicate the study using the KYN/TRP as

marker for IDO activity before making premature implications on the efficacy of the

molecules.

To conclude, the KYN/TRP ratio was increased in those with PSD. The potential role

of this ratio as a biomarker for response to antidepressant treatments should be explored.

Furthermore, sound knowledge of the role of KYN pathway activation in PSD can lead to the

96

development of novel pharmacotherapies (e.g. kynureninase and KMO inhibitors,

hydroxyamidine inhibitors) to prevent or prohibit disease manifestation and other debilitating

stroke outcomes. As research continues to reveal the mechanisms involved in PSD etiology

and safe mediums of inhibiting its development, individualization of pharmacotherapies for

the disease is a reasonable goal for the future.

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APPENDIX A: Post-stroke Depression Patient Consent Form

Stroke and Depression: The Role of Cytokine - Serotonin Interactions

Patient Information and Consent

Investigators: Dr. K.L. Lanctôt Sunnybrook Health Sciences Centre

Dr. N. Herrmann Sunnybrook Health Sciences Centre

Dr. S.E. Black Sunnybrook Health Sciences Centre

Dr. D. Gladstone Sunnybrook Health Sciences Centre

Dr. J. Ween Baycrest

Dr. W. Goldstein York Central Hospital

Dr. M. Waldman St. John’s Rehab

1. Information for subject:

You are being asked to participate in a study conducted at Sunnybrook Health Sciences Centre

under the supervision of the above investigators. Participation is voluntary and will involve

the following:

2. Description and purpose of the trial:

The purpose of this study is to evaluate determinants of the development of depressive and

cognitive (memory and thinking) symptoms after a stroke. Both symptoms are common post-

stroke, and may be related to levels of serotonin (an important brain chemical involved in

regulating mood and thinking). We are interested in assessing the relationship between

different types of cytokines (naturally produced inflammatory chemicals) and serotonin, and

the role they play in any depressive or cognitive symptoms that you may or may not have. In

addition, we are also interested in the impact of cytokines and other chemicals related to

Sunnybrook Health Sciences Centre

2075 Bayview Ave., Suite FG-05 Toronto, ON M4N 3M5

University of Toronto Faculty of Medicine 1 King’s College Circle, Toronto, ON M5S 1A8

123

serotonin production on the size of the hippocampus (an important brain structure involved in

regulating mood and thinking).

3. Study Details:

If you agree to participate in this study, you will be asked to undergo an assessment with a

trained research assistant. The process of assessment will involve the following:

a) Assessments:

The study coordinator will first meet with you and review your medical chart in order to

assess your eligibility for the study. If you are eligible to participate, the study coordinator

will then interview you using standard questionnaires that assess your mood, cognition and

physical functioning. Certain details (e.g. medical history, demographic characteristics,

current medications and details of your stroke) will be copied from your medical chart.

Any CT and MRI scans conducted clinically during your hospital stay will also be

analyzed to determine the characteristics of your stroke. Lastly, if you report experiencing

significant depressive symptoms, the study physician will meet with you for further

assessment and treatment, if necessary. This study will not interfere with your selection of

treatment choice. However, information will be collected regarding your response to

treatment through the questionnaires described above.

b) Blood Draw:

A sample of blood will be drawn in order to measure levels of certain signaling molecules

related to the serotonin and inflammatory systems (called cytokines, kynurenines and

tryptophan). A total of 31 mL (2 tablespoons) of blood will be drawn.

c) Cheek Swab:

A sample of skin cells from the inside of your cheek will be taken using a sterile cotton-

tipped swab. The DNA inside these cells will be used for us to determine which forms of

certain genes (―polymorphisms‖) you have that are related to the cytokine, kynurenine and

serotonin pathways. Your sample will be identified only by a unique number and will be

destroyed once the genetic tests are complete.

4. Benefits:

You will not benefit directly from participation in this study.

5. Risks:

When your blood is drawn, there may be some discomfort and/or bruising, however these are

expected to be very mild. The mouth swab is simple and painless.

6. Alternative Treatments:

You are eligible to receive treatment for your stroke and any depressive symptoms you may

have even if you choose not to participate in this study. Participation in this study will not

affect your treatment in any way.

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7. Costs:

You will be given a $20.00 honorarium each time you visit Sunnybrook for the purposes of

this study. If you participate in the MRI substudy, you will be given a $40.00 honorarium for

your third visit. You will incur no costs as a result of participation in this study.

8. Participation/Termination:

Your participation in this study is voluntary. Thus, if you do not wish to take part in this study

or wish to withdraw at any time after commencing the study, your care will not be affected in

any way.

You may be withdrawn from the study, at any point, if the investigator of this study considers

it to be in your best interest. You may withdraw your consent at any point during the study.

9. Confidentiality:

Your identity in this study will be treated as confidential. Certain Sunnybrook research staff,

the Sunnybrook Research Ethics Board, and other agencies as required by law, may need to

review your medical chart. We will have access to your medical chart for information on:

blood pressure, heart rate, prescribed drugs, depressive symptoms and health status for 1 year.

On all data collected for this study, you will be identified only by a unique number. If you

disclose the intention to harm yourself or others, this information may not be kept confidential,

as required by law.

10. Contacts:

If you or your substitute decision maker have any questions about this study or for more

information, you may contact the Study Co-ordinators: Philip Francis or Amy Wong (416-

480-6100 x3185), Dr. Krista L. Lanctôt (416-480-6100 x2241) or Dr. Nathan Herrmann (416-

480-6100 x6133).

If you have any questions about your rights as a research subject, you may contact Dr. Philip

Hébert, the Chair of the Sunnybrook Research Ethics Board, at 416-480-4276.

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Stroke and Depression Study

Consent to Participate in this Study:

I, (patient’s name) _________________ _______ have read the above information and

fully understand the nature and the purpose of the study in which I have been asked to take

part. The explanation I have been given has mentioned both the possible risks and benefits of

the study. I understand that I will be free to withdraw from the study at any time without

affecting my subsequent treatment by my doctor in any way. I voluntarily consent to

participate in this study.

_________________________________

Name of Patient (typed or printed)

_________________________________ _____________________

Signature of Patient Date

_________________________________

Name of Investigator (typed or printed)

_________________________________ _____________________

Signature of the Investigator Date

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APPENDIX B: Sunnybrook REB Approval Letter

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128

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APPENDIX C: York Central REB Approval Letter

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APPENDIX D: Baycrest REB Approval Letter

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APPENDIX E: St. John's REB Approval Letter

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APPENDIX F: Toronto Research Institute REB Approval Letter

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APPENDIX G: Study Roles

Study Concept and Design: Drs. Krista Lanctôt and Nathan Herrmann

Data Acquisition: Amy Wong

Consisted of participant recruitment, completion of all study assessments, collection of

medical histories through chart review, venipuncture and database management

Cytokine Assays: Dr. Angela Panoskaltsis-Mortari, University of Minnesota

Kynurenine Assay: Mr. Scott Walker, Sunnybrook Health Sciences Centre

Tryptophan Assay: Dr. Simon Young, McGill University

CT Analysis: Dr. Richard Aviv, Sunnybrook Health Sciences Centre

Data Analysis and Interpretation: Amy Wong, Drs. Krista Lanctôt, Nathan Herrmann

Funding: A Heart and Stroke Foundation grant to: Drs. Krista Lanctôt (PI), Nathan Herrmann

(Co-PI), Sandra Black, Demetrios Sahlas, David Gladstone

Study Supervision: Drs. Krista Lanctôt, Nathan Herrmann

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APPENDIX H: Presentations and Publications

Presentations

Wong A, Herrmann N, Black SE, Tennen G, Gladstone DJ, Snaiderman A, Gao F, Aviv RI,

Albert PR, Lanctôt K. Activation of indoleamine 2,3-dioxygenase in post-stroke depression

patients. Presented at the 36th Annual Harvey Stancer Research Day, Department of

Psychiatry, University of Toronto, Canada, June 2010.

Wong A, Herrmann N, Black SE, Tennen G, Gladstone DJ, Snaiderman A, Gao F, Aviv RI,

Albert PR, Lanctôt K. Activation of indoleamine 2,3-dioxygenase in post-stroke depression

patients. Presented at Visions of Pharmacology Research Day, Department of Pharmacology,

University of Toronto, Canada, June 2010.

Wong A, Herrmann N, Black SE, Tennen G, Gladstone DJ, Snaiderman A, Gao F, Aviv RI,

Albert PR, Lanctôt K. Activation of indoleamine 2,3-dioxygenase in post-stroke depression

patients. Presented at the 1st Canadian Stroke Congress. Quebec City, Canada, June 2010.

Wong A, Herrmann N, Black SE, Tennen G, Gladstone DJ, Snaiderman A, Gao F, Aviv RI,

Albert PR, Lanctôt K. Activation of indoleamine 2,3-dioxygenase in post-stroke depression

patients. Presented at the 8th Annual Scientific Meeting of the Heart and Stroke Centre for

Stroke Recovery. Ottawa, Canada, June 2010.

Wong A, Herrmann N, Black SE, Tennen G, Gladstone DJ, Snaiderman A, Gao F, Aviv RI,

Albert PR, Lanctôt K. Activation of indoleamine 2,3-dioxygenase in post-stroke depression

patients. Presented at the 62nd Annual American Academy of Neurology. Toronto, Canada,

April 2010.

Wong A. Cytokine and serotonin interactions in post-stroke depression. Presented at Brain

Sciences Rounds. Department of Neurology, Sunnybrook Health Sciences Centre Toronto,

Canada, November 2009.

Wong A, Herrmann N, Black S, Tennen G, Francis P, Li A, Gold A, Lanctôt K. Role of

Inflammatory Cytokines in the Etiology of Post-stroke Depression. Presented at the 5th

Annual Toronto Rehabilitation Centre Research Day. Toronto, Canada, November 2009.

Wong A, Herrmann N, Black S, Tennen G, Francis P, Li A, Gold A, Lanctôt K. Role of

Inflammatory Cytokines in the Etiology of Post-stroke Depression. Presented at the 35th

Annual Harvey Stancer Research Day, Department of Psychiatry, University of Toronto,

Canada, June 2009.

Wong A, Herrmann N, Black S, Tennen G, Francis P, Li A, Gold A, Lanctôt K. Role of

Inflammatory Cytokines in the Etiology of Post-stroke Depression. Presented at Visions of

Pharmacology Research Day, Department of Pharmacology, University of Toronto, Canada,

May 2009.

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Wong A, Herrmann N, Black S, Tennen G, Francis P, Li A, Gold A, Lanctôt K. Role of

Inflammatory Cytokines in the Etiology of Post-stroke Depression. Presented at the

Sunnybrook Neuroscience Research Day, Department of Neurology, Sunnybrook Health

Sciences Centre, Toronto, Canada, January 2009.

Other Publications

Walter Swardfager, Krista Lanctôt, Lana Rothenburg, Amy Wong, Jaclyn Cappell, and Nathan

Herrmann. A Meta-Analysis of Cytokines in Alzheimer’s Disease. Biological Psychiatry.

2010 Aug 6.

Wong A, Herrmann N, Tennen G, Li A, Gold A, Francis P, Mitchell R, Lanctôt K. The Role

of Indoleamine 2,3-Dioxygenase Activation in the Etiology of Post-Stroke Depression.

Presented at the 62nd

American Academy of Neurology Annual Meeting, Toronto, April 2010.

Supplement to Neurology 2010; 74(9): Suppl 2.

Tennen G, Herrmann N, Li A, Francis P, Wong A, Lanctôt K. Do vascular risk factors impact

poststroke depressive symptoms? Presented at the 14th

International Congress of the

International Psychogeriatric Association, Montreal, September 2009. Int Psychogeriatr 2009;

21(2): S202.

Wong A, Kusznireckyi M. Red Yeast Rice (Professional Review). CAMLINE: An evidence-

based complementary and alternative medicine website for healthcare professionals. <

http://camline.ca/professionalreview/pr.php?NHPID=62>. Dec 2006.