Ischemic Stroke Programme

A target of 1000 patients will be recruited in a hospital-based (NUHS), single centre, prospective study with 3-year recruitment period and maximum 4-year follow-up (minimum 2 years) which will be conducted in patients admitted to the inpatient Stroke Program or referred to the Outpatient Stroke Clinics with a recent (within preceding 2-4 months) ischemic stroke or transient ischemic attack. All patients would have been investigated and managed as per existing Clinical Practice Guidelines; which include a thorough series of investigations for vascular risk factors, demographic information, brief neuropsychological assessment, stroke subtype, as well as neurovascular imaging. Use of all medications will be documented. Eligible patients will be identified from a registry of patients admitted to the Inpatient Stroke Service or referred to the Outpatient Stroke Clinics of the National University Hospital and invited to participate in this study. After informed consent is obtained, patients will undergo neurovascular screening for the presence or absence of ICLAD. While catheter angiography is the gold standard for ICLAD assessment, Transcranial Doppler (TCD), Transcranial Color Coded Duplex (TCCD), CT Angiography and MR Angiography are viable alternatives for screening and repeated assessments with high negative predictive values and without the potential complications of catheter angiography.

Study follow-up will be arranged every 6 months up to a maximum of 4 years. Assessments will be performed by investigators blinded to the baseline data. Data will be collected on the control of vascular risk factors, outcome events, secondary stroke prevention measures, cardiovascular surgery and occurrence of major vascular events (e.g. ischemic stroke, myocardial infarction, peripheral vascular disease, other thrombotic events, and vascular death). Functional status of patients will be assessed on the modified Rankin score. Blood samples will be collected (into SST tubes) on enrolment into the study and subsequently every 12 months for biomarker studies.

Inclusion criteria

• Patients with recent (2-4 months) ischemic stroke or transient ischemic attack
• Aged 45 years and above
• mRS < 5 at enrolment
• Adequate TCD examination available to determine presence or absence of ICLAD
• Informed consent obtained from patient or legal representative
• Able to return for follow-up visits
• No other major illness which may reduce life expectancy within study period

Novel imaging methodology will be utilized to study the presence, severity and nature of intracranial stenosis and plaque characteristics. Moreover, the consequence of ICLAD in terms of hypoperfusion, metabolic changes and structural lesions will be examined. Magnetic resonance imaging will be performed on all patients without contraindications to such imaging at baseline (2-4 months post-stroke/TIA) and repeated at 2 years, on a 3T state-of-the-art MRI scanner to investigate the following according to a standardized protocol.

For Neurocognitive studies, we aim to determine if the presence and severity of ICLAD, baseline MRI features (such as periventricular & deep white matter hyperintensities, infarction, hippocampal and entorhinal atrophy),pro- and anti- inflammatory biomarkers and genetic variability in selected candidate genes involved in the inflammatory pathway are associated with baseline cognitive classification and risk of progression to dementia, in a cohort of Singaporean stroke patients with clinically diagnosed Mild Cognitive Impairment (MCI). We hypothesize that patients with multiple-domain MCI and memory impairment at baseline will be associated with: ICLAD, increased cerebrovascular disease load, atrophy, elevated "pro-inflammatory" and reduced "anti-inflammatory" biomarkers and relevant genetic polymorphisms. Finding genes and other biomarkers that underlie susceptibility to a complex disease like cognitive impairment and dementia after stroke is important for several reasons. In patient management, such discoveries may enhance decision-making algorithms for diagnosis, prognosis, prediction of susceptibility, or may enable patient stratification for therapy and counseling. Our findings may identify groups of patients at higher risk and may also identify new molecular targets for classical pharmacological and/or novel interventions and thus lead to the development of rational therapeutic strategies for treatment or prevention. A weakness of this proposal is that only a limited number of inflammation related candidate genes will be investigated. Other technologies, insights gained from the Human Genome Project and collaborations with other groups may lead to future major research projects which will require larger cohorts and resources.

For Biomarker Studies, we propose to apply established and new biomarkers to improve the diagnosis of ICLAD, risk stratification, selection of therapy and as surrogate end-points in clinical trials. This program allows us to seamlessly translate state-of-the-art novel biomarkers to clinical practice, to aid the management of patients with ICLAD, develop evidence-based diagnostic algorithms and critical pathways for triage and therapy. We will establish a database consisting matched clinical and human samples to complement research using animal models in functional genomics, molecular mechanisms and stem cell studies.

Our group has recently shown that markers of oxidative stress, inflammation, and cysteine and its metabolic products are significantly altered in the hyperacute and acute stages following ischemic stroke. To expand on these findings, we propose to study the changes in these markers in the subacute and chronic phases of stroke recovery (up to two years) to elucidate their roles in the development and progression of ICLAD and their prognostic use in ischemic stroke.

Both homocysteine and cysteine have also recently being recognized as important risk factors for cerebrovascular diseases. Hence their use as prognostic markers for the clinical outcome of acute stroke will be investigated.

The possible use of miRNA and stem cell as biomarkers will also be investigated as one of the aims of this project, as described in sections II (b) and II (c).

a) Animal Models

Animal modelling is a precious tool in studying any disease. There is currently no established animal model for the study of intracranial stenosis. We propose to employ a multi-prong approach by using a combination of two or three (if necessary) well-established stroke risk factors in rabbits, rats and mice. Development of atherosclerotic stenosis in both intracranial and extracranial blood vessels would be monitored by histological and immunohistochemical techniques. In the presence of cross-sectional luminal stenosis, the following parameters will be evaluated: (1) degree of stenosis of the artery, (2) thickness of the fibrous cap, (3) extent of lipid area including necrotic core, (4) presence of intra-plaque haemorrhage, (5) presence of neovasculature in the plaques, (6) presence of thrombus in the artery, (7) presence of calcification, and (8) macrophage and T-lymphocyte infiltration.

Overall, we believe that using multiple risk factors in several species provide the best chance of success in establishing useful animal models for ICLAD. These models would be invaluable in the study of changes in the brain after cerebral artery occlusion, changes in blood and brain biomarkers for disease progression, and response of these markers to therapeutic interventions.

b) Functional Genomics and Molecular Mechanisms

Besides studying the relationships between the genes and proteins involved in intracranial stenosis and associated processes such as inflammation, apoptosis, edema etc., we will also be examining the expression of riboregulators (microRNAs) that control the expression of various genes both at the transcriptional and translational levels. MicroRNAs (miRNA) are highly conserved, small, RNA molecules encoded in the genomes of plants and animals and they regulate the expression of many genes either by RNA interference (RNAi) or RNA activation (RNAa). miRNAs have been found to regulate multiple genes and seem to be crucial factors in many cellular pathways, including development, cell differentiation, proliferation and apoptosis. The molecular studies will be carried out on the following lines:

• Profiling mRNA, miRNA and proteins in both animal model of intracranial stenosis and human patient blood samples
• Identification of specific molecules (mRNA/miRNA and proteins) that can be used as biomarkers and therapeutic targets
• Identification of specific targets (for miRNA)
• Identification of specific genes by in vitro assays
• Verification of the involvement of selected miRNAs in intracranial stenosis, endothelial and neuronal repair using animal models

c) Stem Cell Studies

Stem cells, which are capable of self-renewal and differentiation, hold the key to regenerative medicine. Researchers have identified stem cells from various sources, including bone marrow and the brain subventricular zone, which had been considered to be incapable of regeneration. Bone marrow stem cells (BMSCs) are a heterogeneous population comprising of hematopoietic stem cells and mesenchymal stem cells (MSCs). Because of the potential of autologous cell transplantation and hypoimmunogenic allogenic transplantation, the adult BMSCs are of particular interest for cell therapy to the aging population. In developing a stem cell therapy for aging patients with intracranial stenosis, we propose to use the subpopulations of BMSCs-endothelial progenitor cells (EPCs) and MSCs for cerebrovascular repair and the level of circulating EPC as a biomarker for preliminary diagnostic screening.

Cerebrovascular repair for patients diagnosed with and stroke will be as important as, if not more than, the repair of damaged neural tissue, because:

• For the intracranial stenosis patients, the most urgent task will be to repair their ICLAD-occluded vasculature, to prevent the occurrence of stroke.
• For patients who have already suffered ischemic stroke either in acute or chronic phase, it is also important to repair the occluded vasculature to prevent stroke recurrence.
• Repaired cerebral vasculature will also provide a more favorable environment for neurogenesis.

The presence of adult bone marrow-derived EPCs present in the peripheral blood circulation has been demonstrated to be essential for revascularization of damaged vessels via vasculogenesis. As the EPCs are recruited to damaged vascular endothelium for repair, the overall circulating population of these EPCs is postulated to decrease as a result. The level of peripheral EPCs present in an individual was also found to be correlated with various risk factors, such as hypertension, diabetes, peripheral vascular disease, smoking, myocardial infarction and age. Intracranial stenosis arises from the formation of atherosclerosis, thus measurement of the CD34+ cell population in blood circulation can serve as an initial screening.

Even though bone marrow stem cells and EPCs have the potential in providing a promising therapy for intracranial stenosis, several challenges have yet to be addressed:

• Isolation and expansion. Aged ICLAD patients may have low EPC level, thus an effective EPC isolation method and ex vivo expansion strategy will be required for a successful autologous therapy.
• Alternative cell sources. The EPCs in patients with multiple risk factors could be impaired, limiting the potential of autologous transplantation. An alternative allogenic cell source will be needed.
• Delivery and therapeutic use. The cell delivery method, therapeutic efficacy, repairing mechanism of EPCs in ICLAD will be assessed in an ICLAD animal model.
• Low level of EPCs in the aged ICLAD patients.

Aged ICLAD patients may have multiple risk factors and other complications, thus their circulating EPC level could be lower than a healthy individual. The EPC level assessment will not only serve as a potential biomarker, it will also provide the information to identify the type of stem cell therapy to be administered for cerebrovascular repair in different groups of patients at different stages of recovery.