The World Health Organization (WHO) definition of stroke is rapidly developing clinical signs of focal (or global) disturbance of cerebral function, with symptoms lasting 24 hours or longer or leading to death, with no apparent cause other than of vascular origin [1,2]. Ischaemic strokes accounts for majority of strokes which occur due to interrupted blood supply to a brain area [3].
The NO is a gaseous molecule that can easily cross biological membranes and plays an important role in the pathogenesis of AIS (<24 hour). Cerebral ischaemia leads to multiple changes in NO signaling as well as NO content of brain [4]. NO may have some positive (neuroprotective) or, negative (neurodestructive) effects depending on the stage of stroke and isoform of NO produced during the stroke [5,6].
The NO derived from iNOS appears to contribute to neurotoxicity after ischaemic stroke, as animal knockout models have smaller infarcts than their wild type counterparts [7]. An increase in NO above basal levels secondary to iNOS occurs 12-24 hour after Middle Cerebral Artery (MCA) occlusion and contributes to neurotoxicity, with iNOS inhibition leading to reduced infarct volume [8].
Apart from NO, haematological parameters such as polymorphs and other types of WBCs had also been implicated in pathogenesis of stroke [9-12]. Many studies had also highlighted that various haematological and biochemical parameters like blood glucose, White Blood Cell (WBC), Erythrocyte Sedimentation Rate (ESR) alteration had prognostic significance in AIS [13,14]. Some of the studies had also shown that elevated WBC, ESR, creatinine was independent predictor of 30 days case fatality in patient with first time AIS [15,16].
The present study attempts to see the interaction of the above parameters in the stroke cohort.
Materials and Methods
This hospital based, cross-sectional, observational study was conducted in Himalayan Institute of Medical Sciences (HIMS), Dehradun, India, over a period of 12 months (August 2009 to July 2010). Fifty patients of AIS, attending the indoor and outpatient departments of Medicine at HIMS were enrolled in this study as cases and 25 were healthy individuals who acted as controls. Age matched healthy controls were enrolled from subjects coming for routine health checkup and healthy blood donors.
Considering the limited study period and limited financial budget, to ensure adequate strength of study and its outcome a case to healthy comparative control ratio of 2:1 was considered. Based on previous studies and considering a mean difference of serum NO of 4.50 and the pooled Standard Deviation (SD) of 10.6, a sample size of 43 AIS cases was calculated [6,7]. With the 10% attrition the figure was rounded up to 50. With the case and control ratio of 2:1, 25 healthy controls were recruited for the study.
All patients included in the study had a confirmed clinical diagnosis of ischaemic stroke supported by immediate neuroimaging (CT/MRI). Stroke was defined according to WHO guidelines [1]. The time of onset of the stroke was defined as the time when the patient or observer first became aware of the symptoms.
All adult patients (55-70 years of age) suffering from ischaemic stroke, which is less than 24 hours duration were included in this study. The exclusion criteria were as follows: i) Stroke associated with myocardial infarction; ii) Stroke associated with Central Nervous System (CNS) infection; iii) Stroke in patients using nitrate drug; iv) Previous history of stroke.
After inclusion, each participant was subjected to thorough history taking and detailed clinical examinations {physical and systemic examination with emphasis on neurological examination and evaluation of Glasgow Coma Scale (GCS) score. After taking written informed consent, serum NO, complete haemogram including ESR, complete lipid profile, serum BUN, creatinine, and RBS were evaluated from all participants.
Collection of Samples
Blood samples were collected at the time of admission. Three milliliters (mL) of blood was drawn from the antecubital vein in a plain vacutainer for the estimation of serum NO, RBS, BUN, urea and creatinine and 2 mL of blood was taken in Ethylenediaminetetraacetic Acid (EDTA) vacutainer for haemogram. After 12 hours of overnight fasting, 2 mL of blood was collected in a plain vacutainer for the estimation of lipid profile.
Estimation of serum NO was done by Enzyme-Linked Immunosorbent Assay (ELISA) technique with NO colorimetric assay kit using Griess reagent (BioVision Research Products, USA). Estimation of serum total cholesterol, HDL cholesterol, triglycerides, glucose, BUN and creatinine were done on SYNCHRON CX9, automated chemistry analyser (Beckman Coulter, USA). Serum LDL cholesterol and Very Low-Density Lipoprotein (VLDL) cholesterol were calculated by using Friedewalds formula [19]. Although, previous reports suggest that Friedewald’s formula underestimates LDL-C only when value of triglyceride is very high, the equation remains one of the best in routine use [20]. Estimation of haemoglobin, total leukocyte count and differential leukocyte count were done on the automated haematology cell counter “MS-9”. All the haematological and biochemical investigations were carried out in the laboratories of Himalayan Institute of Medical Sciences Hospital, Dehradun.
Statistical Analysis
Demographic data was stratified by gender to assess its association with AIS by Chi-square test. Difference between the case and control subjects were analysed by the unpaired t-test for demographic, biochemical parameters, haematological parameters and NO. Finally, the Pearson’s correlation coefficients were analysed to look for the relationship between the NO and biochemical and haematological parameters among the acute ischaemic stroke patients.
The p-value was considered significant if p<0.05, highly significant if p<0.01 and very highly significant if p<0.001. All the data were expressed as mean±standard deviation (SD).
Results
Number of cases and controls, mean age and sex are shown in [Table/Fig-1]. The difference between the mean age of controls and cases was statistically insignificant.
Comparison of baseline characteristics of controls and cases.
| Parameter | Controls (n=25) | Cases (n=50) | p-value |
|---|
| Age in years (mean±SD) | 53.64±5.62 | 57.18±10.64 | 0.064# |
| Sex (male/female) | 18/7 | 32/18 | 0.07* |
| Hypertension | - | 20 (40%) | - |
| Diabetes mellitus | - | 17 (34%) | - |
| Smoking | 7 (28%) | 24 (48%) | - |
#unpaired “t” test, *chi-square test, p>0.05-non-significant (ns), p<0.05-significant
Among controls, the mean value and range of NO was 24.41 μmol/L and 14.94-32.31 μmol/L respectively. Among cases, the mean value and range of NO was 32.06 μmol/L and 17.55-43.70 μmol/L respectively. On comparison, statistically very highly significant elevation in mean serum NO level was observed in cases as compared to controls (p<0.001).
Comparison of serum biochemical profile among controls and cases are given in [Table/Fig-2]. Pearson’s correlation matrix composed showed levels of NO were positively correlated with TAG, Total Cholesterol (T-Chol), LDL-C, VLDL-C, RBS and negatively correlated with HDL-C, BUN and serum creatinine, but none of the correlation significantcoefficient was found to be statistically significant in the cohort. On the other hand, a statistically highly significant positive correlation of BUN with the serum creatinine (r=0.541, p<0.01) was found. The levels of blood glucose were correlated positively with the T-Chol and LDL-C levels and the relation was statistically very highly significant [Table/Fig-3].
Comparison of biochemical parameters (Mean±SD) among controls and cases.
| Parameter | Status | Mean±SD | n | Mean Difference | p-value |
|---|
| TAG (mg/dL) | Controls | 128.68±22.05 | 25 | 24.32 | 0.001 |
| Cases | 153±36.41 | 50 |
| T-Chol (mg/dL) | Controls | 185.88±32.87 | 25 | 31.58 | 0.001 |
| Cases | 217.46±44.57 | 50 |
| LDL-C (mg/dL) | Controls | 115.14±32.14 | 25 | 31.53 | 0.001 |
| Cases | 146.68±42.08 | 50 |
| VLDL-C (mg/dL) | Controls | 25.73±4.41 | 25 | 4.86 | 0.001 |
| Cases | 30.60±7.28 | 50 |
| HDL-C (mg/dL) | Controls | 45.00±7.70 | 25 | 4.82 | 0.012 |
| Cases | 40.18±6.45 | 50 |
| RBS (mg/dL) | Controls | 118.32±21.12 | 25 | 24.70 | 0.001 |
| Cases | 143.03±37.81 | 50 |
| BUN (mg/dL) | Controls | 15.8±3.58 | 25 | 4.24 | 0.001 |
| Cases | 20.04±6.60 | 50 |
| Creatinine (mg/dL) | Controls | 0.89±0.17 | 25 | 0.20 | 0.014 |
| Cases | 1.10±0.50 | 50 |
unpaired “t” test, p>0.05-non significant, p<0.05-significant, p<0.01-highly significant, p<0.001-very highly significant.
TAG: Triacylglycerol; T-Chol: Total cholesterol; LDL-C: Low density lipoprotein cholesterol; VLDL-C: Very low density lipoprotein cholesterol; HDL-C: High density lipoprotein cholesterol; RBS: Random blood sugar; BUN: Blood urea nitrog
Pearson’s correlation among biochemical parameters in cerebral ischaemic patients.
| NO | TAG | T-Chol | LDL-C | VLDL-C | HDL-C | RBS | BUN | CREA |
|---|
| NO | 1 | 0.118 | 0.247 | 0.253 | 0.118 | -0.096 | 0.208 | -0.091 | -0.032 |
| TAG | | 1 | 0.218 | 0.139 | 1.00*** | - 0.453* | 0.218 | 0.109 | 0.042 |
| T-Chol | | | 1 | 0.992*** | 0.218 | 0.214 | 0.624*** | - 0.146 | - 0.005 |
| LDL-C | | | | 1 | 0.138 | 0.175 | 0.645*** | - 0.167 | - 0.016 |
| VLDL-C | | | | | 1 | - 0.453* | - 0.125 | 0.109 | 0.042 |
| HDL-C | | | | | | 1 | 0.240 | - 0.019 | 0.041 |
| RBS | | | | | | | 1 | - 0.013 | 0.111 |
| BUN | | | | | | | | 1 | 0.541** |
| CREA | | | | | | | | | 1 |
*p<0.05-Significant, **p<0.01-Highly significant, ***p<0.001-Very highly significant
NO: Nitric oxide; TAG: Triacylglycerol; T-Chol: Total cholesterol; LDL-C: Low density lipoprotein cholesterol; VLDL-C: Very low density lipoprotein cholesterol; HDL-C: High density lipoprotein cholesterol; RBS: Random blood sugar; BUN: Blood urea nitrogen; CREA: Creatinine
Comparison of different haematological parameters between cases and controls has been shown in [Table/Fig-4]. Pearson’s correlation matrix showed levels of NO were negatively correlated with the Hb, TLC, platelet and ESR; however none of the correlation coefficient was found to be statistically significant in the cohort. TLC was found to be positively correlated with the ESR and the relation was statistically very highly significant (r=0.655, p<0.001) [Table/Fig-5].
Comparison of haematological parameters (Mean±SD) among controls and cases.
| Parameter | Status | Mean±SD | n | Mean difference | p-value |
|---|
| Hb (mg/dL) | Controls | 13.42±1.17 | 25 | 0.59 | 0.07 |
| Cases | 12.83±1.62 | 50 |
| TLC (/mm3) | Controls | 8492.56±1178.47 | 25 | 1547.64 | 0.001 |
| Cases | 10040.20±2717.04 | 50 |
| Neutrophil (%) | Controls | 72.68±4.99 | 25 | 4.8 | 0.001 |
| Cases | 77.48±6.71 | 50 |
| Lymphocyte (%) | Controls | 25.72±5.35 | 25 | 4.32 | 0.003 |
| Cases | 21.40±6.39 | 50 |
| Eosinophil (%) | Controls | 1.56±0.91 | 25 | 0.06 | 0.814 |
| Cases | 1.62±1.24 | 50 |
| Platelet (105/ mm3) | Controls | 2.55±0.57 | 25 | 0.278 | 0.057 |
| Cases | 2.27±0.59 | 50 |
| ESR (mm first hour) | Controls | 10.36±2.36 | 25 | 19.64 | 0.000 |
| Cases | 30.00±14.46 | 50 |
unpaired “t” test, p>0.05-non significant, p<0.05-significant, p<0.01-highly significant, p<0.001-very highly significant.
Hb: Haemoglobin; TLC: Total leukocyte count; ESR: Erythrocyte sedimentation rate
Pearson’s correlation of NO with GCS and haematological parameters in cerebral ischaemic patients.
| NO | Hb | TLC | Platelet | ESR |
|---|
| NO | 1 | -0.108 | -0.269 | -0.085 | -0.004 |
| Hb | | 1 | -0.028 | -0.444* | -0.409* |
| TLC | | | 1 | 0.035 | 0.655*** |
| Platelet | | | | 1 | 0.208 |
| ESR | | | | | 1 |
*p<0.05-significant, **p<0.01-highly significant, ***p<0.001-very highly significant
NO: Nitric oxide; Hb: Haemoglobin; TLC: Total leukocyte count; ESR: Erythrocyte sedimentation rate
Discussion
Cerebrovascular disease or stroke is one of the major cause of mortality and disability worldwide [21,22]. Several studies have established cellular and molecular pathophysiological processes in acute ischaemic stroke, which are complex [23,24]. Among the many players of stroke, NO and various haematological parameters were implicated in pathogenesis of stroke [5,10,11,25].
This study was planned to evaluate the serum levels of NO and other biochemical and haematological parameters in patients of ischaemic stroke of less than 24 hours duration and to find out a correlation between them.
The mean age of our stroke patients was 57.18±10.64 years which was relatively younger than those seen in the Western studies. In the Indian subcontinent, stroke happens nearly a decade earlier than West [26]. A total of 35 (70%) of the patients were in age group 41-60 years, 14 (28%) of the patients were aged more than 60 years and only 1 (2%) patient was less than 40 years of age. The gender distribution showed an M:F ratio of 1.78:1. Present study was comparable to other Indian studies on stroke patients where greater preponderance was seen among males [26,27].
In the present study, hypertensive and diabetic population was higher as compared to a similar study done by Aygul R et al., which may be due to increasing number of diabetic and hypertensive population in India [28].
Serum NO levels were very highly elevated in cases as compared to controls in present study. Experimental evidence by Malinski T et al., had shown that AIS induced by occlusion of Middle Cerebral Artery (MCA) of rats resulted in increased brain NO production [29]. Concomitant with changes in brain NO levels, serum and CSF NO levels were also found to be significantly higher in various studies [6,28,30]. Contrary to the above studies, Shibata M et al., showed a decrease brain NO level, after production of global ischaemia by occluding the common carotid artery of rat [31]. Similarly blood NO level was found to be low in various other studies also [25,32]. In ischaemic stroke, hypoxia and hypercapnia may be the triggering factors for NOS enzyme as evidenced by cell culture study [33].
Because of this it is now widely believed that NO production is enhanced at all stages of cerebral ischaemia [34].
We had found out statistically highly significant level of TAG, T-Chol, LDL-C while low level of HDL-C was present in cases as compared to controls. Present findings were in agreement with other study on Indian population, where significant increase in the LDL-C level and a significant decrease in the HDL-C level were observed [35]. Contrary to present findings, a study had also shown lipid lowering effect of acute ischaemic stroke [36], the mechanism of lipid lowering effect is unclear. Consistent with many other studies [13,37,38], significant increase in blood glucose level in the present study may be due to stress response after AIS as suggested by Murros K et al., [39].
Analysis of haematological parameters showed a statistically significant increase in TLC and ESR in AIS patients than controls. These findings were in agreement with previous studies where alterations of WBC, ESR, polymorphs were reported [40,41]. Increase in ESR values observed in their study was, at least in part, a consequence of the acute phase response to the ischaemic stroke event as suggested previously [42]. A high WBC count could be pinpointing to a vast immune system complexity in strokes patients. In addition, previous studies reported that WBC particularly polymorphs participate in the acute phase of ischaemic brain injury [10,43]. The probable effects of WBC and polymorph in the pathogenesis of ischaemic stroke patients include restriction of cerebral blood flow due to vessels plugging, exacerbation of parenchymal injury via hydrolytic enzymes, free oxygen radical production, and initiation of thrombosis [14].
Limitation
The present study is a single center, hospital-based study on a smaller sample hence the strength of relation of NO and other factors for AIS cannot be expanded to the regional population. A larger population-based study is needed to establish the relation of NO to covariates of AIS in the regional community.
Conclusion
Elevation in serum levels of RBS, TAG, T-Chol, LDL-C and VLDL-C while low levels of HDL-C were observed in patients of AIS. These changes constitute important risk factors for cerebral ischaemia.
Significant elevation in mean serum NO levels in patients of AIS was most probably due to the induction of NOS by the ischaemic condition which contributes to subsequent neuronal damage.
The present study findings suggest that haematological derangement and oxidative stress in AIS may be the result of imbalance in oxidant/antioxidant homeostasis.
#unpaired “t” test, *chi-square test, p>0.05-non-significant (ns), p<0.05-significantunpaired “t” test, p>0.05-non significant, p<0.05-significant, p<0.01-highly significant, p<0.001-very highly significant.TAG: Triacylglycerol; T-Chol: Total cholesterol; LDL-C: Low density lipoprotein cholesterol; VLDL-C: Very low density lipoprotein cholesterol; HDL-C: High density lipoprotein cholesterol; RBS: Random blood sugar; BUN: Blood urea nitrog*p<0.05-Significant, **p<0.01-Highly significant, ***p<0.001-Very highly significantNO: Nitric oxide; TAG: Triacylglycerol; T-Chol: Total cholesterol; LDL-C: Low density lipoprotein cholesterol; VLDL-C: Very low density lipoprotein cholesterol; HDL-C: High density lipoprotein cholesterol; RBS: Random blood sugar; BUN: Blood urea nitrogen; CREA: Creatinineunpaired “t” test, p>0.05-non significant, p<0.05-significant, p<0.01-highly significant, p<0.001-very highly significant.Hb: Haemoglobin; TLC: Total leukocyte count; ESR: Erythrocyte sedimentation rate*p<0.05-significant, **p<0.01-highly significant, ***p<0.001-very highly significantNO: Nitric oxide; Hb: Haemoglobin; TLC: Total leukocyte count; ESR: Erythrocyte sedimentation rate