The Norwegian Stroke in the Young Study (NOR-SYS): Rationale and design

Background Ischemic stroke in young adults is a major health problem being associated with a higher vascular morbidity and mortality compared to controls, and a stroke recurrence rate of 25% during the first decade. The assumed cause of infarction and the detected risk factors determine the early- and long-term treatment. However, for many patients the cause of stroke remains unknown. Risk factor profile and etiology differ in young and elderly ischemic stroke patients, and atherosclerosis is the determined underlying condition in 10 to 15%. However, subclinical atherosclerosis is probably more prevalent and may go unrecognized. Ultrasound imaging is a sensitive method for the detection of arterial disease and for measurement of adipose tissue. The relationship between intima-media thickness (IMT), plaques, cardiovascular risk factors including visceral adipose tissue (VAT) and ischemic events has repeatedly been shown. We have established The Norwegian Stroke in the Young Study (NOR-SYS) as a three-generation research program with the goal to increase our knowledge on heredity and the development of arterial disease and ischemic stroke. Extended standardized ultrasound examinations are done in order to find subclinical vessel disease for early and better prophylaxis. Methods/Design NOR-SYS is a prospective long-term research program. Standardized methods are used for anamnestic, clinical, laboratory, imaging, and ultrasound data collection in ischemic stroke patients aged ≤60 years, their partners and joint adult offspring. The ultrasound protocol includes the assessment of intracranial, carotid and femoral arteries, abdominal aorta, and the estimation of VAT. To date, the study is a single centre study with approximately 400 patients, 250 partners and 350 adult offspring expected to be recruited at our site. Discussion NOR-SYS aims to increase our knowledge about heredity and the development of arterial vascular disease in young patients with ischemic stroke and their families. Moreover, optimization of diagnostics, prophylaxis and early intervention are major targets with the intention to reduce stroke recurrence and other clinical arterial events, physical disability, cognitive impairment and death. NOR-SYS is reviewed and approved by the Regional Committee for Medical and Health Research Ethics, Western-Norway (REK-Vest 2010/74), and registered in ClinicalTrials.gov: NCT01597453.


Background
Cerebrovascular and coronary artery disease are the main causes of disability and death in the western world [1]. According to observational studies where TOAST criteria have been used, atherosclerosis is the underlying condition in 10 to 15% of patients with ischemic cerebrovascular events of determined etiology [2]. However, in 30-40% of cases the cause of stroke remains unknown [3]. Risk factor profile and etiology differ in young ischemic stroke patients compared to the elderly [3][4][5]. In addition, young patients have a higher vascular morbidity and mortality compared to healthy controls [6][7][8][9], and recurrent ischemic events are common [10,11]. Further, a significant portion of ischemic stroke patients have unrecognized atherosclerosis not only located to cervical arteries, but as well to intracranial arteries [12], to coronary arteries [13], to the aortic arch [14] and to femoral arteries [15]. As therapeutic options are limited, primary and secondary prophylaxis of atherosclerosis and generalized arterial disease should be a major target with the purpose to reduce long-term disability and death among young stroke patients.
Ultrasound imaging is a sensitive, non-invasive, and low-cost method for the detection of arterial vessel disease in major arteries [16,17]. The measurement of carotid intima-media thickness (cIMT) and plaques in B-mode ultrasound has become a tool for vascular risk prediction, as the relationship between IMT, plaques, cardiovascular risk factors and future ischemic events has consequently been shown in several longitudinal studies, predominantly in older individuals [17][18][19][20][21][22][23][24][25][26][27]. However, the value of IMT measurements in all carotid artery segments compared with measurements in the distal CCA alone is disputable [28], and a recent meta-analysis concluded that cIMT measurements in the CCA alone adds little to the improvement of a 10-year risk prediction [29].
NOR-SYS is a concept for the standardized gathering of anamnestic, clinical and biological data in young ischemic stroke patients, their partners, and their family members. The intention is to estimate the presence of arterial vessel disease, to determine the individual's vascular risk profile, and to offer optimal prevention.
Inclusion of the patients' partners and joint adult offspring is providing a platform for primary vascular prevention and early intervention. Stroke is a result of multifactorial causes with genetic, environmental and life-style components [30]. The combination of a standardized case-history, standardized ultrasound protocols, and a prospective long-term follow-up schedule is expected to give knowledge regarding heredity and vascular co-morbidity. The optimal goal and the major purpose of the study is to reduce vascular morbidity, disability, cognitive impairment and mortality in young ischemic stroke patients.

Methods and design
NOR-SYS is intended to be a national multicenter study, performed by co-operating neurological departments in Norway. The study was initiated at Haukeland University Hospital, Bergen, in September 2010. The inclusion period will be 5 years. NOR-SYS is designed as a three-generation study with prospective long-term follow-up design. In addition to a routine cerebro-cardiovascular work-up including clinical examination, neuroimaging, cardiac investigations, and laboratory analyses, all participating patients and relatives are investigated according to the NOR-SYS protocol ( Figure 1). This includes questionnaires regarding vascular disease burden in the family, the patient's medical history and life styles. In addition, all patients are examined by transcranial, extracranial, abdominal and peripheral ultrasound, arterial stiffness measurements, and 24 hour blood pressure monitoring. Participants with undocumented but suspected coronary and/or peripheral arterial disease are referred to the Department of Cardiology and the Department of Vascular Surgery, respectively, for further appropriate investigations, including cardiac computertomography-angiography (CCTA) and CT of the thoracic aorta.
Anthropometric variables, such as height, weight, and waist-hip ratio are measured and EDTA-blood and serum samples are collected to a biobank. The patients' partners and biological offspring aged ≥18 years are being offered investigations as shown in Figure 1. Data on medical history and life styles are collected from the patients' biological parents, partners, and offspring by standardized questionnaires. For deceased first-degree relatives, the patient will be asked about their cardio-vascular clinical events and the achieved information will be verified by medical records and data from the Norwegian Cause of Death Registry.
NOR-SYS will be carried out in two phases. In the first, cross-sectional phase, a comprehensive stroke data base on vascular risk factors, arterial ischemic events, and clinical and subclinical atherosclerotic disease burden in the study population is being established.
The second, longitudinal phase will constitute long-term follow-ups, at 5, 10 and 15 years from the time of inclusion for patients and their partners, and at 10 and 20 years from time of inclusion for offspring. The purpose of the follow-up is to observe the biological development of atherosclerosis and vascular disease over time, and to optimize primary and secondary medical prophylaxis. The complete work-up is shown in Figure 1.

Subject selection
Study participation is offered to all patients with Norwegian residency aged 15 to 60 years, with radiologically documented acute cerebral infarction. All study participation is based on informed written consent. Patients of non-western European ethnicity are investigated in agreement with the NOR-SYS protocol, but are not included in statistical study analyses. Patients with ischemic stroke due to a traumatic cause or subarachnoidal bleeding are excluded from study participation. Spouses and partners of included patients are offered participation as control persons and as reference persons to participating joint offspring. Parents of patients and partners are invited to return standardized questionnaires. All participants are asked for permission to review their relevant medical records from hospitals, specialists or general practitioners for verification.  Infrarenal diameter >30 mm is suspect for aneurysm and considered for additional vascular surgical investigations [37]. Hemodynamically significant stenosis is  ABI is measured after a resting period of at least 5 to 10 minutes by Ultrasonic Doppler Flow Detector, Model 811-BTS, Parks Medical Electronics, Inc., Aloha, OR, USA. Bloodpressure measurements are performed bilaterally in the radial, the dorsalis pedis, and the posterior tibial artery. ABI ≤ 0.9 at rest is defined as the cut-off point for peripheral artery disease (PAD). ABI 0.7-0.9 is considered as mild, 0.4-0.7 as moderate and <0.4 as severe arterial disease. ABI >1.4 may be explained by medial sclerosis or other conditions leading to arterial incompressibility [38]. Suspect subjects and participants with known diabetes mellitus are reported to the respective departments of vascular surgery for further investigation.   Samples of serum and EDTA-plasma are collected, processed, coded and stored at −80°C until analyzation for each participating subject. Analyses are scheduled after completion of the 5-years inclusion period. Biomarkers to be investigated will be determined at the time of analyses according to the most relevant biomarkers known at that time point. GWAS, exone sequencing or any newer technology, relevant and feasible at the time of genetic analyses, will be applied.

Primary and secondary prevention strategies
Additionally to stroke treatment and secondary prevention in the patient population, all subjects are being evaluated concerning the presence and severity of established clinical and sub-clinical cardiovascular disease and modifiable vascular risk factors. An evaluation sheet is given to all patients at discharge, issues where improvement is recommended are pointed out and intervention is initiated as soon as possible during hospital stay. For family members, a short report is being sent to their respective general practitioner, in which clinical and anamnestic results are discussed and recommendations for intervention or further investigation are given. A modified Essen Stroke Risk Scale is applied for all participants [39].

Prospective follow-up
During a standardized telephone interview one week after discharge performed by a study nurse, patients are asked to evaluate the information they received concerning their stroke, investigation results, and individual vascular risk factors, as well as their hospital stay in general. Three months follow-up is performed at the out-patient clinic and includes standardized questionnaires concerning recurrent ischemic events, seizures, pain, cognitive function, psychological disorders, tolerability of medication, quality of life, employment/ education after the stroke, sick leave, as well as changes with respect to life styles and modifiable risk factors after discharge. Clinical and functional scoring by NIHSS, mRS, and Barthel index are performed, and weight and blood pressure measurements are repeated.
One-year follow-up is performed by telephone interview as short standardized questionnaire update on changes concerning modifiable risk factors. For long-term follow-up, examinations C. a-h and D. will be repeated after 5, 10 and 15 years or after 10 and 20 years from inclusion regarding patients and partners or their offspring, respectively.

Study endpoints
Primary endpoints are death and documented cerebral, coronary and/or peripheral arterial events. Secondary endpoints are the long-term development or progression of atherosclerosis and the failure of therapeutic goal achievement (tobacco cessation, well-regulated bloodpressure, dyslipidemia and diabetes mellitus, and normal weight or slight overweight). Data validation will be done by medical record information.

Statistics
All obtained data are registered in the NOR-SYS Research Registry. Statistical analyses are performed by' STATA/SE for Windows'and 'R' in cooperation with a biostatistician.

Discussion
Long-term follow-up studies of young stroke patients have shown high mortality and vascular morbidity compared to healthy controls [6][7][8][9][10]40]. Hence, a prospective cohort follow-up based on thorough investigation of clinical and sub-clinical vascular disease and risk factors is necessary in order to achieve a better long-term outcome.
Ultrasound imaging has been proved to be a sensitive and cost-effective method for the detection of arterial vessel disease in major arteries [16], as well as for the evaluation of adipose tissue [35,41]. For this reason, ultrasound was chosen as the predominating tool for the investigations in the NOR-SYS protocol. IMT increases are dependent on age, sex and cardiovascular risk [42]. However, the increase and prevalence of atherosclerotic lesions vary among different anatomical segments. Moreover, increased IMT has repeatedly been associated with cardiovascular risk factors and the incidence of cardiovascular events [19,43], and has been validated as a surrogate marker of atherosclerosis [44,45]. Atherosclerotic lesions are not distributed circumferentially, but develop asymmetrically [46], and their prevalence varies in the different artery segments [47]. In our study, Meijer's Carotid Arc® is used for standardized imaging at defined angles [47,48], and cIMT and plaque measurements are aquired bilaterally in three carotid segments: the distal CCA, the bifurcation and the proximal ICA [47]. We suppose that this approach will improve the individual risk classification, as recently suggested [49]. It has also been suggested that the presence of carotid artery plaques may be even more representative for CVD prediction than increased cIMT itself [50]. Hence, plaque measurements are performed in addition to the standardized IMT measurements at all three carotid sites, if present.
Atherosclerosis is a systemic disease, and lesions are often to be found in several locations of the vasculature, such as in the peripheral arteries. Intermittent claudication is a frequent condition in western European populations [51,52] and associated with symptomatic CAD and cerebrovascular events [53,54]. Acute death due to PAD has been shown in 9% [40], compared to 45% and 42% due to cerebrovascular and coronary death, respectively [55]. The CFA has been reported as the segment most prone to IMT increase and plaque formation [42] compared to the SFA and the carotids. CFA IMT has beyond that been related to coronary angiographic [56] and echocardiographic parameters [57]. It is considered suitable for long-term observations concerning the natural development of atherosclerosis in healthy participants, and for the observation of treatment effects in a participant group requiring intervention [42]. For these reasons, IMT measurements are additionally performed bilaterally in the distal CFA and the proximal SFA segment, and included in study analyses. Atherosclerosis in the abdominal aorta is leading to aortic stenoses and PAD. Abdominal aortic aneurysms are also considered to be a manifestation of advanced atherosclerosis [58], and are frequently observed in patients with carotid stenoses, cardiovascular events and PAD [59]. Therefore, in NOR-SYS the abdominal aorta is evaluated with respect to atherosclerotic lesions, stenoses, and aneurysms. The ABI is performed in all participants as it is a wellestablished tool in investigation for peripheral artery disease and adds valuable information to vascular risk prediction [60,61].
Standard screening for a cardiac embolic source, including 24 hour heart rhythm registration and echocardiography is carried out in order to diagnose left ventricular hypertrophy, abnormal left ventricular geometry, and dilated left atrium as they are well-known predictors of stroke, both in the general as well as in the hypertensive population [62]. Blood pressure is measured after hospital discharge as an ambulatory 24-hour measurement as it has been proven to be closer associated with cardiovascular target organ damage and incident cardiovascular events than clinic pressure [63]. Ambulatory blood pressure measurements identify hypertension more accurately than clinic blood pressure measured during an acute stroke. Measurement of arterial stiffness by carotid-femoral pulse wave velocity by aplanation tonometry may be useful in identifying arterial disease which is not captured by routine carotid ultrasound visualization [64].
NOR-SYS includes CCTA and CT of the thoracic aorta because of the well-known association between peripheral and coronary disease [65]. In addition, aortic arch atheroma or other wall disease of the ascending aorta or the aortic arch might cause the index-stroke or recurrent stroke [66].
Obesity is an increasingly prevalent disorder [67] which is associated with atherosclerosis and cardiovascular disease. Particularily abdominal obesity has been associated with metabolic syndrome [68], pre-clinical atherosclerosis [69], cardiovascular events [70] and mortality [70].
Epicardial adipose tissue (EAT) has its embryologic origin in common with mesenteric and omental fat, and all these are accordingly classified as visceral adipose tissue (VAT) [71,72]. Associations between VAT and cIMT [73], metabolic syndrome [74,75] and cardiovascular disease [76,77] have been reported in several studies. Release of free fatty acids due to the proximity to the portal circulation leading to direct lipotoxicity [78,79], and release of pro-inflammatory and pro-atherogenic cytokines and hormones with impact on endothelial function [80,81] are related issues. The accumulation of VAT has therefore been found to be an independent vascular risk factor, even within the normal range of BMI [82]. Accordingly, the anatomical relationship of EAT to the heart is providing local interaction with modulation of the coronary arteries and the myocardium, which may subsequently affect cardiac function and morphology [83][84][85]. On the other hand, subcutaneous adipose tissue, which is a non-portal fat type with less metabolic activity [86], has previously shown only a weak relationship to increased cIMT [73]. Its evaluation related to the amount of VAT and anthropometric parameters is assumed to be relevant for risk prediction and for that reason included in NOR-SYS. Anthropometric parameters such as BMI and WHR are simply applicable clinical tools and widely used in obesity evaluation. They are as well associated with ultrasonographic visceral adipose tissue measurements [41,87], and applied in NOR-SYS.
In conclusion, the major objective of NOR-SYS is the standardized gathering of anamnestic, clinical, and biological data concerning life styles, medical history, and clinical and subclinical vascular disease at several sites of the vasculature including body fat composition and anthropometric measurements in young ischemic stroke patients and their families. Standardized questionnaires and standardized ultrasound examinations combined with detailed clinical data are assumed to increase the precision in diagnostics and risk estimation, and generate a solid basis of decision-making concerning secondary prophylaxis after acute ischemic stroke.
Further investigation and evaluation of vascular risk factors and sub-clinical artery wall disease in young ischemic stroke patients' family members provide a platform for primary prophylaxis and early intervention.
NOR-SYS aims to reduce co-morbidity, disability, recurrent stroke, cognitive impairment and mortality in young patients with acute ischemic stroke. We expect that a comprehensive work-up and long-term observation, combined with biological, genetical and clinical information gathered from three family generations, will give the opportunity to improve our basic knowledge concerning preclinical atherosclerosis in families with a vascular disease burden.
NOR-SYS is reviewed and approved by the Regional Committee for Medical and Health Research Ethics, Western-Norway (REK-Vest 2010/74), and registered in ClinicalTrials.gov: NCT01597453.