Protocols STroke imAging pRevention and treatment (START): A longitudinal stroke cohort study: Clinical trials protocol Leeanne M. Carey1,2*, Sheila Crewther1,3, Olivier Salvado4, Thomas Lindén1,5, Alan Connelly6, William Wilson7, David W. Howells1, Leonid Churilov1, Henry Ma1,8, Tamara Tse1,2, Stephen Rose4, Susan Palmer1, Pierrick Bougeat4, Bruce C. V. Campbell9, Soren Christensen9, S. Lance Macaulay10, Jenny Favaloro1, Victoria O’ Collins1, Simon McBride4, Susan Bates11, Elise Cowley11, Helen Dewey12, Tissa Wijeratne13, Richard Gerraty14, Thanh G. Phan8, Bernard Yan9, Mark W. Parsons15, Chris Bladin16, P. Alan Barber17, Stephen Read18, Andrew Wong18, Andrew Lee19, Tim Kleinig20, Graeme J. Hankey21,22, David Blacker22, Romesh Markus23, James Leyden24, Martin Krause25, Rohan Grimley26, Neil Mahant27, Jim Jannes28, Jonathan Sturm29, Stephen M. Davis9**, Geoffrey A. Donnan1**, and the START Research Team (http://www.START.csiro.au) Correspondence: Leeanne M. Carey*, National Stroke Research Institute, Florey Institute of Neuroscience and Mental Health, Melbourne Brain Centre-Austin Campus, 245 Burgundy Street, Heidelberg, Vic. 3084, Australia. E-mail: [email protected] 1 National Stroke Research Institute, Florey Institute of Neuroscience and Mental Health, Heidelberg, Vic., Australia) 2 Department of Occupational Therapy, School of Allied Health, La Trobe University, Bundoora, Vic., Australia 3 School of Psychological Sciences, La Trobe University, Bundoora, Vic., Australia 4 Preventative Health National Research Flagship, The Australian e-Health Research Centre, Commonwealth Scientific and Industrial Research Organisation (CSIRO), Herston, Qld, Australia 5 Institute of Neuroscience and Physiology, The Sahlgrenska Academy, Gothenburg University, Gothenburg, Sweden 6 Brain Research Institute, Florey Institute of Neuroscience and Mental Health, Heidelberg, Vic., Australia 7 Preventative Health National Research Flagship, Neurodegenerative Diseases, Mental Disorders and Brain Health, CSIRO, North Ryde, NSW, Australia 8 Stroke Unit, Monash Medical Centre, Department of Medicine, Monash University, Clayton, Vic., Australia 9 Department of Medicine, Melbourne Brain Centre, Royal Melbourne Hospital, University of Melbourne, Parkville, Victoria, Australia 10 Preventative Health National Research Flagship, Neurodegenerative Diseases, Mental Disorders and Brain Health, CSIRO, Parkville, Vic. Australia 11 Neuroscience Trials Australia, Melbourne Brain Centre – Austin Campus, Heidelberg, Vic., Australia 12 Department of Neurology, Austin Health, Heidelberg, Vic., Australia 13 Department of Neurology, Western Hospital, Western Health, Melbourne, Vic., Australia 14 Epworth Healthcare, Melbourne, Vic., Australia 15 Department of Neurology, John Hunter Hospital, University of Newcastle, Newcastle, NSW, Australia 16 Department of Neurology, Box Hill Hospital, Eastern Health, Melbourne, Vic., Australia 17 Department of Neurology, Auckland City Hospital, Auckland, New Zealand 18 Department of Neurology, Royal Brisbane and Women’s Hospital, Brisbane, Qld, Australia 19 Flinders Comprehensive Stroke Centre, Flinders Medical Centre and University, Adelaide, SA 20 Department of Neurology, Royal Adelaide Hospital, Adelaide, SA, Australia

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Rationale Stroke and poststroke depression are common and have a profound and ongoing impact on an individual’s quality of life. However, reliable biological correlates of poststroke depression and functional outcome have not been well established in humans. Aims Our aim is to identify biological factors, molecular and imaging, associated with poststroke depression and recovery that may be used to guide more targeted interventions. Design In a longitudinal cohort study of 200 stroke survivors, the START – STroke imAging pRevention and Treatment cohort, we will examine the relationship between gene expression, regulator proteins, depression, and functional outcome. Stroke survivors will be investigated at baseline, 24 h, three-days, three-months, and 12 months poststroke for blood-based biological associates and at days 3–7, three-months, and 12 months for depression and functional outcomes. A sub-group (n = 100), the PrePARE: Prediction and Prevention to Achieve optimal Recovery Endpoints after stroke cohort, will also be investigated for functional and structural changes in putative depression-related brain networks and for additional cognition and activity participation outcomes. Stroke severity, diet, and lifestyle factors that may influence depression will be monitored. The impact of depression on stroke outcomes and participation in previous life activities will be quantified. 21 School of Medicine and Pharmacology, The University of Western Australia, Perth, WA, Australia 22 Department of Neurology, Sir Charles Gairdner Hospital, Perth, WA, Australia 23 Department of Neurology, St.Vincent’s Hospital, Sydney, NSW, Australia 24 Department of Neurology, Lyell McEwin Hospital, Adelaide, SA, Australia 25 Department of Neurology, Royal North Shore Hospital, Sydney, NSW, Australia 26 Department of Neurology, Nambour General Hospital, Nambour, Qld, Australia 27 Department of Neurology, Westmead Hospital, Sydney, NSW, Australia 28 Department of Neurology, The Queen Elizabeth Hospital, SA 29 Department of Neurology, Gosford Hospital, Gosford, NSW, Australia

Received: 31 March 2013; Accepted: 5 August 2013; Published online 10 November 2013 **Co-chair of START collaborative study group. Conflicts of interest: None DOI: 10.1111/ijs.12190 © 2013 The Authors. International Journal of Stroke © 2013 World Stroke Organization

L. M. Carey et al. Study Outcomes Clinical significance lies in the identification of biological factors associated with functional outcome to guide prevention and inform personalized and targeted treatments. Evidence of associations between depression, gene expression and regulator proteins, functional and structural brain changes, lifestyle and functional outcome will provide new insights for mechanism-based models of poststroke depression. Key words: cohort, cortical thickness, depression, functional neuroimaging, gene expression, stroke

Introduction Stroke is a leading cause of ‘global burden of disease’, with this burden projected to rise substantially (1). Depression is a common sequela of stroke, ranging from 30% to 60%, with a pooled estimate of 33% (2), compared with 13% in age-matched population controls (3). Despite evidence that depression and stroke are epidemiologically linked, reliable correlates of poststroke depression (PSD) and the mechanisms underlying this association are still relatively unknown. Recognition and diagnosis of PSD are critical, as PSD is an independent determinant of handicap (4) and is associated with worse outcome (3), recurrent stroke, and greater risk of early death (5). Depressed stroke patients have more days in hospital, utilize more care, have poorer rehabilitation outcome compared with nondepressed stroke patients, and are often institutionalized (3). Even those with relatively mild stroke return to fewer household, social, leisure, and work activities (6). Knowledge of return to previous activities is important, as activity participation poststroke is associated with health-related quality of life (6,7) and is a modifiable factor. Despite the high prevalence and negative impact of depression, only a minority of those affected are diagnosed in clinical settings and even fewer are treated in common clinical practice (8). Thus, we need to better highlight the theoretical likelihood of depression with clinicians and differentiate and monitor patients ‘at risk’ of developing PSD. Of the large number of clinical and social associates investigated, meta-analysis revealed that more severe stroke was the only common variable associated with depression (2). Biological associates of depression, derived from gene expression and blood protein analyses, which could differentiate ‘at risk’ patients early as part of the clinical care pathway for stroke, currently do not exist. Biological markers of depression Investigation of biomarkers linked with underlying mechanisms may not only be predictive but is also a likely guide for the targeting of therapy, both prevention and treatment. Depression is increasingly recognized as a stress-associated disorder involving interactions between neural and immune systems within the brain and periphery (9,10). Major depression is often associated with a prolonged proinflammatory response (11). Ischemic brain injury is also associated with acute oxidative stress and cell death that leads to an increased inflammatory response of immune-related chemicals (cytokines) both from within the brain and outside from the peripheral immune system (12). Systematic review has identified a common pattern of excitotoxicity and consequent © 2013 The Authors. International Journal of Stroke © 2013 World Stroke Organization

Protocols upregulation in proinflammatory cytokines that mediate depression-related apoptotic cell death in limbic regions in rodents (12). However, investigation of biological factors associated with PSD is currently limited. A recent meta-analysis identified high cortisol levels, at approximately three months poststroke, and lower levels of brain-derived neurotrophic factor levels, as associates (13). Gene expression and PSD The potential now exists to explore patterns of gene expression in peripheral blood for their capacity to biologically differentiate persons who have PSD compared with those that do not show the behavioral phenotype. Altered gene expression is an integral feature of ischemic cerebral injury in animals and humans (14). However, few studies have examined gene expression in patients soon after stroke and followed their recovery over 12 months. In the STroke imAging pRevention and Treatment (START) study, we will examine the genomic changes in whole blood at 24 h, three-days, three–months, and 12 months poststroke to identify a genomic signature associated with PSD. Analyses will be complemented with measurements of blood proteins to give a better indication of, for example, cytokine responses. The least number of genes that best differentiates the groups may be used as a biomarker profile for PSD. The analysis will also reveal a larger number of pathway genes that may be investigated for networks they impact, thus providing insight to mechanisms (14). Structure and function changes in brain networks Strong evidence links (nonstroke) depression and alterations in brain morphology and function. Meta-analyses provide evidence of gross morphological cerebral changes, including atrophy of prefrontal cortex and smaller hippocampal and amygdala volumes (15). A systems-level model of clinical depression highlights failure of ‘limbic-cortical pathways’ (16) and is supported more recently by evidence of decreased functional connectivity (FC) in limbic-cortical regions (17). Disruption of FC to central nodes of brain networks, such as hippocampus and caudate nucleus, is evident with first episode major depressive disorder (18). Baseline severity of white matter changes on Magnetic Resonance Imaging (MRI) (‘cerebrovascular burden’) may independently predict future depression in healthy elderly (19). While limbic network and cortical changes are well established in clinical depression, they have only been investigated to a limited extent after stroke. Evidence for a relationship between lesion location and PSD is unresolved (20), although relationships have been identified with more advanced imaging protocols (20). White matter changes and cerebral microbleeds predict PSD (21). An association with smaller amygdala volumes and reduction in overall brain perfusion poststroke is reported based on metaanalysis (13). Based on these findings we will investigate functional and structural brain changes in putative depression networks using advanced imaging protocols. Multimodal approach A profile of biochemical changes and functional and structural brain alterations that interact with each other and depression is likely but to our knowledge has not been systematically investigated poststroke. Convergent functional and structural changes in Vol 10, June 2015, 636–644

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Protocols limbic-cortical brain circuit are reported with clinical depression (17). Distinctive RNA expression profiles in blood have been associated with white matter hyperintensities and with oxidative stress and inflammation (22). These findings combine to suggest that inflammatory changes, gene expression, functional brain connectivity, volume of putative brain regions, and white matter hyperintensities have potential as associates for risk profiles in PSD. Knowledge of changes across modalities will advance our understanding of the mechanism and provide insight into targets for prevention and intervention. The proposed study Using a multimodal approach we will investigate blood-based correlates, functional and structural brain alterations, depression, and functional outcomes in a prospective, observational, longitudinal stroke cohort over a 12-month period with the aim to: • discover biological correlates that differentiate persons with depression after stroke compared with those that do not develop depression; • derive gene lists associated with PSD, to be used for pathway and gene ontology analyses, to assess pathways, networks, and mechanisms of PSD; • characterize associations between PSD and functional and structural connectivity in limbic-cortical networks (threemonths poststroke), as well as late morphological changes in hippocampus, amygdala, and prefrontal regions (12 months poststroke); and • quantify the impact of PSD on participation in household, social, and leisure activities and return to productive occupations in an Australian cohort. Key to the study is the multimodal and longitudinal investigation of neurobiological associates of PSD.

Hypotheses Primary hypothesis (Total START cohort) Based on changes in gene expression at three-days and threemonths poststroke, it will be possible to identify a profile of best-

L. M. Carey et al. classifier genes (14) that differentiates those with PSD compared with those without PSD (at 3 and/or 12 months), when adjusted for age, gender, and stroke severity. It is anticipated that these best-classifier genes will be associated with neuroinflammatory processes and oxidative stress. Secondary hypotheses [START_ Prediction and Prevention to Achieve Optimal Recovery Endpoints after stroke (PrePARE) cohort] Depressive symptoms will be associated with: • differences in functional and/or structural connectivity patterns in the putative limbic-cortical network at three-months; and • reduced hippocampal and amygdala volumes and/or atrophy in prefrontal and cingulate regions at 12 months. Survivors of stroke with more depressive symptoms will participate in fewer household, social, leisure, and work activities, relative to prestroke, after adjusting for stroke severity and age.

Methods Study design We will conduct a prospective, observational, longitudinal study of a cohort of 200 stroke survivors. Patients will be recruited via the START collaborative research program. The START study has two arms: an intervention arm (START_EXTEND, NTA 0901) and a cohort arm that recruits from START_EXTEND (NTA 0901), and START_PrePARE (NTA 0902) (see Fig. 1). The intervention arm, START_EXTEND (EXtending the time for Thrombolysis in Emergency Neurological Deficits), is a randomized, multicentre, double-blinded, placebo-controlled phase 3 trial for thrombolysis with tissue plasminogen activator (tPA) (23). START_PrePARE is a longitudinal cohort study with advanced imaging and clinical outcomes. It is a standalone study with independent recruitment as well as a sub-study of START_EXTEND. The Stroke Cohort: Blood-based markers and diet and lifestyle factors will be investigated in relation to PSD and functional

Fig. 1 START cohort: overview and recruitment path. START = STroke imAging pRevention and Treatment; EXTEND = EXtending the time for Thrombolysis in Emergency Neurological Deficits; PrePARE = Prediction and Prevention to Achieve Optimal Recovery Endpoints; tPA = tissue plasminogen activator.

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© 2013 The Authors. International Journal of Stroke © 2013 World Stroke Organization

L. M. Carey et al. outcome in the total START cohort (n = 200). The sample will comprise: (i) stroke survivors randomized in the START_EXTEND trial; (ii) stroke survivors recruited via START_EXTEND who did not achieve penumbral criteria for randomization but are monitored via the cohort study, and (iii) patients recruited to the START_PrePARE study (this study includes broader inclusion criteria). Patients recruited to START_PrePARE (n = 100) will have additional imaging, cognition, and activity participation outcome data collected to permit investigation of secondary hypotheses. Consistent with current clinical care and enrolment in START_EXTEND, a proportion of the patients will have received thrombolysis with tPA. Investigators and patients will remain blinded to START_EXTEND treatment designation. Approximately one-third of patients will be recruited via the EXTEND clinical trial arm, one-third via the cohort only arm, and one-third via the PrePARE standalone arm that has a broader inclusion criteria. This will facilitate generalizability to the broader stroke population. Stroke patients will be assessed within the first week and at 3 and 12 months poststroke for gene expression analysis, core clinical status, diet and lifestyle factors, and depression. Advanced imaging and clinical testing will also be obtained at 3 and 12 months poststroke in the PrePARE participants. Advanced imaging, involving intrinsic FC, cortical thickness, and fiber tract connectivity, will be undertaken at a dedicated site. Advanced clinical measures of cognition, sensorimotor function, and activity participation will also be collected at these time points by an experienced therapist. Patient population Patients who meet eligibility criteria will be recruited consecutively from participating hospitals. Patients presenting with acute ischemic stroke, aged ≥18 years, and with clinical signs of hemispheric infarction will be recruited. The patient, family member, or legally responsible person will be required to consent for START_PrePARE. Patients recruited to START_EXTEND have the additional inclusion criteria that they are recruited 4·5–9 h after stroke onset, to permit onset of thrombolysis treatment if randomized, and have a National Institute of Health Stroke Scale (NIHSS) score of ≥4 to 26. It is anticipated that at least one-third will develop clinical depression poststroke. Exclusion criteria for START_PrePARE are: contraindication to imaging with MRI; prestroke modified Rankin Score (mRS) score of ≥2 (indicating previous disability); any terminal illness such that the patient would not be expected to survive more than one-year; clinically evident pregnancy; and non-English speaking. Patients will not be excluded on the basis of prior depression. Our focus is to identify biological factors associated with the presence of depression in stroke survivors at a particular point in time, irrespective of whether depression was present prior to or post stroke. Patients recruited via START_EXTEND will also meet the additional exclusion criteria of that study (23). Schedule of study assessments A schedule of study assessments is provided in Table 1 and briefly described below. © 2013 The Authors. International Journal of Stroke © 2013 World Stroke Organization

Protocols Clinical status and stroke outcome Core clinical assessments These will be administered by a stroke specialist or health-care professional trained in their administration at three- to sevendays, three–months, and 12 months poststroke to all patients. Depression Depressive symptoms and disorder will be assessed using the Montgomery-Åsberg Depression Rating Scale (MADRS) structured interview format (SIGMA): a standardized and validated observer-rating of depression at a point in time (24). The measure comprises 10 structured interview questions covering areas such as reported sadness, reduced appetite, and pessimistic thoughts. Nonverbal supports (25) will be used for administration with patients who have aphasia and/or poor language and cognitive skills. These have been operationalized within our protocol. Depressive symptoms are scored on a scale of 0–38; higher scores indicating more depressive symptoms and 18 being the standard cutoff point for major depression (24). Inter-rater reliability is high (intraclass correlation coefficient = 0·93), with sensitivity of 87% and specificity of 61% for differentiating major depression relative to the Diagnostic and Statistical Manual of Mental Disorders-Edition 4 (DSM-IV) (24). Prior history of depression or mood disorders will be obtained from medical history and patient/carer interview at each time point using a set of structured interview questions (26). Neurological status Neurological status will be assessed using the NIHSS (27). Global cognitive impairment Global cognitive impairment will be screened using the Montreal Cognitive Assessment (MoCA) (28). Functional outcome Functional disability will be assessed using the modified Rankin Scale (29) and functional independence in activities of daily living assessed using the Barthel index (30). Return to previous activities Return to previous activities will be assessed using the Work and Social Adjustment Scale (31). Stroke-specific quality Stroke-specific quality of life will be assessed using the Stroke Impact Scale (SIS) Version 3 (32). Diet and Lifestyle Demographic, physical risk factor, and socioeconomic histories will be obtained from semi-structured interview. Dietary habits will be assessed using the Cancer Council of Victoria Diet Questionnaire (33) and physical activity using the Rapid Assessment of Physical Activity (34). Body mass index will be determined from physical examination. Recurrent stroke Recurrent stroke will be monitored throughout. Clinical history and medications Clinical history and medications, including information on antidepressants, will be taken at each time point using a structured Vol 10, June 2015, 636–644

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Table 1 Study schedule of assessments

Study outcome Informed consent Inclusion/exclusion criteria Patient details/demographics, medical history/radiological scans Medications Clinical examinations

Depression

Diet and lifestyle questionnaires

Routine laboratory blood tests Research bloods Advanced imaging (MRI) (START_PrePARE only)

Cognition (advanced) (START_PrePARE only)

Sensorimotor function (START_PrePARE only) Participation (START_PrePARE only) Safety Stroke recurrence

Investigation

Baseline

Including stroke history

X X X

National Institute of Health Stroke Scale (NIHSS) Modified Rankin Scale (mRS) Physical examination Demographics Barthel Index Physical risk factors interview Montreal Cognitive Assessment (MoCA) Quality of life – Stroke Impact Scale (SIS) Depression – Montgomery Asberg Depression Rating Scale (MADRS) Depression history and interview questions Cancer Council of Victoria (CCV) diet questionnaire (part) Rapid Assessment of Physical Activity (RAPA) questionnaire Work and Social Adjustment questionnaire (WSAS) Hematology, lipid profile Vascular, inflammatory, dietary Biomarker investigations Lesion characterization Cortical thickness Functional resting state Fractional anisotropy Tractography Microhemorrhages Mini Mental State Examination Trail Making Test – Part B Digit Span Raven’s Coloured Progressive Matrices Shape cancellation task Stroop Test Action Research Arm Test Tactile Discrimination Test Timed up and go test Activity Card Sort

protocol. History of comorbidities will be recorded. Treatments for depression prior to or during the study will be recorded on the basis of a structured interview (26). Advanced clinical assessments These will be administered at 3 and 12 months to patients recruited to START_PrePARE. Cognition In addition to the MoCA, the following measures will assess the corresponding functions: Shape Cancellation Task (35) – hemispatial neglect; auditory Digit Span (36) – working memory; Trail Making Test – Part B (TMT-B) (37) and Stroop test (38) – execu-

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X X Prestroke X

12–24 h

Day 3–7 ± 1 day

Three-months ± 7 days

12 months ± 7 days

X

X X

X X X X

X X X X

X

X X X X X

X X X X X

X X

X X

X X

X

X

X

X

X

X

X

X

X X

X X X X X X X X X

X X X X X X X X X

X

X

X

X

X X

X X

X X X

X X X

X X

X

tive function; Raven’s Coloured Progressive Matrix Test (39) – nonverbal, abstract reasoning; Mini Mental State Examination (40) will assess global cognition and facilitate comparison with other studies. Sensorimotor function Upper limb function will be assessed using the Action Research Arm Test (41) and brief version of the Tactile Discrimination Test (42). Mobility will be assessed using the Timed Up and Go test (43). Activity participation Actual participation in activities across household, leisure, and social domains will be assessed using the Activity Card © 2013 The Authors. International Journal of Stroke © 2013 World Stroke Organization

Protocols

L. M. Carey et al. Sort (ACS) – Australian version (44).The ACS is a pictorial interview-based measure of participation relative to prestroke activity profile. The percentage of retained activities, relative to prestroke, will be the outcome measure. The ACS-Aus is a valid measure of participation in the Australian population aged 60–95 years (44). Blood samples In addition to blood taken for standard care blood tests, 30 ml of venous blood will be collected from patients for gene expression analyses. Whole blood will be collected at baseline, at 12–24 h, three-days, three–months, and 12 months poststroke and stored following standardized protocols. The number of blood samples collected at early time points within the first three days post stroke onset will depend upon the time that has elapsed between stroke onset and enrolment for each individual patient. Blood for RNA preparation will be collected into QIAGEN PAXgene tubes and stored at −80°C. RNA will be prepared using a QIAGEN PAXgene Blood RNA Kit (Qiagen, Venlo, Limburg, Netherlands). Results from standard care blood tests that may have been performed will be recorded (i.e. routine hematology, biochemistry, and coagulation screening tests). Imaging protocols Advanced imaging data will be acquired for START_PrePARE patients at 3 and 12 months on a 3 Tesla scanner at a single site to maximize reliability. Scanning time is 30–40 mins. Morphological brain alterations High-resolution T1-weighted anatomical 3D MPRage images (1 × 1 × 1 mm) will be acquired to quantify cortical thickness and volume of relevant brain structures. Intrinsic FC Resting-state data will be acquired over seven-minutes of continuous functional MRI while subjects do not perform any particular task (Repetition Time, TR = 3·0; 3 × 3 × 3 mm isotropic voxels; 72 × 72 matrix; 44 slices; 216 mm Field of View, FOV; Echo Time, TE = 30 ms). White matter tracts Diffusion-weighted imaging protocols will acquire images for measurement of fiber tract integrity [fractional anisotropy; fourminutes, 25 directions, b = 1000] and for white matter tract estimation (45). The fiber tracking protocol takes nine-minutes with 60 diffusion-weighted directions and b = 3000 s/mm2 (TE/TR: 110/8500 ms, 54 contiguous slices, 96 × 96 matrix, 240 mm FOV, 2·5 × 2·5 × 2·5 mm isotropic voxels). Lesion characterization and white matter hyperintensities 2D and/or 3D FLAIR (fluid attenuation inverse recovery sequence; 1 × 1 × 1 mm) images will be acquired for delineation of the lesion and white matter hyperintensities. Susceptibility weighted imaging (SWI) SWI images will permit assessment of microhemorrhages and bleeding. © 2013 The Authors. International Journal of Stroke © 2013 World Stroke Organization

Primary Outcome Measure The primary measure will be clinical evidence of depression at 3 and/or 12 months poststroke, as assessed using the MADRS (SIGMA) and diagnostic criterion score on this test. Secondary Outcome Measures There are two secondary measures: (i) depressive symptom score, assessed using the MADRS, and (ii) percentage of retained activities on the ACS-Aus, each assessed at 3 and 12 months. Specialized data analysis Gene expression and protein analysis Microarray analyses will be conducted for gene-signature analysis and investigation of mechanisms associated with PSD and functional outcome. Using a discovery approach and the strategy for identification of biological markers (14), we will derive the best classifier gene-signature for PSD with optimal specificity and sensitivity, based on three-day and three-month poststroke blood samples. Validation of classifiers identified from the genomic experiments will be performed using Reverse transcription polymerase chain reaction (RT-PCR) and Western blotting techniques. Initial gene expression analysis will be conducted using Affymetrix® Human Exon (Affymetrix, Santa Clara, CA, USA) microarray and protein expression using, for example, Luminex xMAP cytokine/chemokine panel (Merck KGaA, Darmstadt, Germany). RNA and blood plasma samples will be randomized before microarray and protein analysis. Gene expression and bioinformatic analysis will also be conducted. Dedicated bioinformatic algorithms will be used to analyze gene expression microarray data followed by ontological pathway analyses. Advanced imaging analysis Morphological brain changes Thickness and volume of cortical and deep brain structures will be quantified using automated 3D-volume analysis (46). Hippocampal and amygdala volume and cortical thickness of prefrontal regions will be quantified in absolute, normalized units. Intrinsic FC FC analysis will quantify strength and location of coherence of spontaneous activity in brain networks. Connectivity will be assessed at a whole-brain level, using independent component analysis (47), and from ‘seeded’ regions of interest. Seed regions of interest will be defined based on meta-analysis of the literature. White matter tracts Diffusion data will first be preprocessed. Fiber tracking will be performed by seeding each voxel of the brain (‘whole-brain’ tracking) and within regions of interest in the hippocampus, amygdala, cingulated, and prefrontal regions bilaterally (for ‘targeted’ tracking) using constrained spherical deconvolution and MRtrix software (48; Brain Research Institute, Melbourne, Australia, http://www.brain.org.au/software/). Super-resolution track-weighted imaging methodology will be used together with intrinsic FC analysis to derive track-weighted FC maps of FC Vol 10, June 2015, 636–644

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Protocols networks (49). The lesion will be masked out in the analysis, and the region-of-interest template will be warped into native space for each individual (50). Lesion characterization Lesions will be defined on the three-month 3D FLAIR images by a neurologist experienced in reading MRI images. Lesions will also be characterized relative to gray and white matter using image segmentation software and the 3D T1 images (51). A probabilistic map of stroke-related injury will be developed based on infarct location to determine the extent to which the lesion impacts putative brain networks and hubs. Infarct volume will be quantified. An index of cerebrovascular burden, i.e. white matter hyperintensity volume, will also be derived (52). Data monitoring Data will be captured electronically via an electronic Case Report Form linked to a database. The data transfer will be encrypted, and access to the database will be secure and password protected, and managed by data managers. The study has a data-safety monitoring committee. Sample size Primary hypothesis Investigation of the primary hypothesis will involve an exploratory analysis of gene expression data from 200 stroke survivors (total START cohort) at two time points (three days and three months). Based on a conservative assumption of medium predictive capacity (R2 being 0·5), using six alternative heuristic methods and six alternative values for shrinkage parameter with Precision Power method (53), the sample size sufficient for building a model with up to seven independent variables is approximately 200 patients. Secondary hypotheses The accepted standard, based on a critical analysis of sample size needed for reliable, group-based functional MRI analyses, indicates a minimum of 20 participants in a group (54). We will have a sample size of approximately 75 participants for our correlation analyses of FC maps (one per individual) at three-months and depressive symptom score. This relatively large sample size allows for expected missing data associated with eligibility for MRI while retaining adequate power and generalizability. Changes in brain morphology between 3 and 12 months will be correlated with depression score and covaried for age. Based on an estimated correlation effect size of r = 0·47 (pilot data; n = 36) between hippocampal volume and cognitive outcome in a neurologically impaired group, a sample of 34 stroke survivors will yield a power of 0·8 at a two-tailed significance of 0·05 to reject the hypothesis of no association. To correct for multiple comparisons, the corrected alpha of 0·01 will be used for each correlation, thus requiring a total sample of n = 48. Finally, we will explore the contribution of depressive symptoms on activity participation outcomes in the sample of 100 START_PrePARE participants. This sample size will be sufficient to detect an effect size f 2 = 0·1; a relatively small effect within the range of small (0·02) to medium (0·15). This is based on a regression model including depressive symptoms, stroke severity and

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L. M. Carey et al. age as independent variables, a two-tailed alpha of 0·025 (two time points), and power of 0·8 [GPower 3·1.3 (55)]. Statistical Analyses Primary hypothesis Multivariate statistical techniques will be used to conduct analysis of microarray data to differentiate patients with and without depression, with covariates of age, gender, and stroke severity. Secondary hypotheses Functional Connectivity FC maps will be correlated with depressive symptoms using simple regression in Statistical Parametric Mapping (SPM software, University College London, UK http://www.fil.ion.ucl.ac .uk/spm/) and corrected for multiple comparisons. Morphological brain changes Regression models will be used to determine relationships between depressive symptoms and (i) hippocampal volume, (ii) amygdala volume, and/or (iii) atrophy in cingulate and/or prefrontal regions at 12 months poststroke. Participation outcomes Multiple regression methods will be used to determine the association between depressive symptoms and the percentage of activities retained. Factors such as severity of neurological impairment (NIHSS) and age will be controlled. Analyses will be conducted separately at three- and 12-month times. Exploratory analyses Diet and lifestyle factors and lesion location will be explored for a relationship with depression, recurrent stroke, and functional outcome using correlation analyses (56). Thrombolysis will be included as a covariate in all analyses. Associations between blood based measurements and imaging parameters will be explored. Study organization and funding The study is managed by the management committee, steering committee, and executive sub-committees. It is financially supported by Commonwealth Scientific and Industrial Research Organisation (CSIRO) of Australia and its Preventative Health Flagship program, Florey Institute of Neuroscience and Mental Health, and National Health and Medical Research Council (NHMRC) of Australia. The National Stroke Research Institute, a member of the Florey, is the sponsor of the study. Ethical considerations This study will be carried out according to the Declaration of Helsinki, the NHMRC National Statement on Ethical Conduct in Research Involving Humans (57), and the Notes for Guidance on Good Clinical Practice as adopted by the Australian Therapeutic Goods Administration and the ICH GCP Guidelines.

Summary Clinical significance of findings from this longitudinal stroke cohort lies in the identification of biological factors that help differentiate those who do and do not go on to develop PSD. This study has taken the approach of an in-depth, longitudinal analysis © 2013 The Authors. International Journal of Stroke © 2013 World Stroke Organization

L. M. Carey et al. of factors that may impact on depression and recovery over time. Targeted monitoring is crucial so that the one in three stroke survivors likely to develop depression can benefit from preventative interventions, achieve earlier access to effective treatments, and participate more optimally in rehabilitation and previous life activities. Novel evidence of associations between depression, genetic and protein markers, and functional and structural brain changes will advance our understanding of PSD from a multimodal perspective. New insights into underlying mechanisms may lead to improvements in prevention, early intervention, and targeted therapeutic interventions. Evidence of late morphological brain changes associated with depression will provide confirmatory evidence of change within brain regions implicated in the etiology of clinical depression in a stroke cohort. Identification of patterns of change in depression and functional recovery longitudinally will promote better understanding of the needs of depressed stroke survivors over time and provide quantitative evidence of the impact of depression on return to previous life activities and occupations following stroke.

Acknowledgements The START program of research which comprised START_EXTEND (NTA 0901) and START_PrePARE (NTA 0902) is supported in part by the CSIRO of Australia through the Preventative Health Flagship Cluster. The National Stroke Research Institute, Florey Institute of Neuroscience and Mental Health acknowledges the support from the Operational Infrastructure Support Grant. We would particularly like to acknowledge the participants, clinician nurses, radiologists, study manager Ms Sue Bates, study coordinator Ms Elise Cowley, and START researchers (see http:// www.START.csiro.au) who are contributing time and effort to the study. LMC is supported by an Australian Research Council Future Fellowship [number FT0992299]. The funding sources had no role in conduct of the study or writing of the report.

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