INTRODUCTION

The pathophysiological consequences of acute ischemic stroke are still not fully understood. There is much evidence, largely derived from animal models, to suggest that neuroinflammatory mechanisms play an important role in ischemic injury, and that interruption of these processes can result in improved neurological outcomes¹. In experimental animal models of stroke, peripheral blood leukocytes migrate into the brain parenchyma within the first 12 hours after ischemia. Migration of peripheral blood leukocytes requires prior adhesion to cerebral endothelial cells, which is mediated by adhesion molecules on the surface of cerebral endothelial cells and peripheral blood leukocytes². 

The leukocyte-endothelial adhesion process consists of several steps, in which three families of leukocyte-endothelial adhesion molecules have been identified: the selectins, the immunoglobulin gene superfamily, and the integrins. An initial transient contact of the circulating leukocytes with vascular endothelium generally mediated by adhesion molecules of the selectin family and their respective carbohydrate ligands, slows down the leukocyte in the blood stream. Subsequently, the leukocyte rolls along the vascular  wall with greatly reduced velocity.  Firm adhesion of leukocytes to the endothelial cells as well as leukocyte activation and transmigration across the endothelium is mediated by receptors of the immunoglobulin gene superfamily mainly: intercellular  adhesion molecule-1(ICAM-1)and vascular cell adhesion molecule-1 (VCAM-1)³. VCAM -1 was first described as a cytokine-inducible endothelial adhesion molecule. It can bind to leukocyte integrin VLA-4 (very late antigen-4) to recruit leucocytes to sites of inflammation⁴. Because most adhesion molecules are not only expressed on cell surfaces but are also released into the circulation, they can easily be quantified  by ELISA in peripheral blood².

Aim of The Work

The aim of this work is to assess the levels of serum soluble vascular cell adhesion molecule-1 (sVCAM-1) in acute ischemic stroke and  their correlation to the extent of  neurological deficits  and short term  clinical outcome .

SUBJECTS AND METHODS

Forty four patients admitted with first ever acute ischemic stroke within 72 hours of stroke onset. All   patients were admitted in stroke unit in Ain Shams Specialized hospital .

Inclusion criteria:  First ever acute ischemic stroke .

Exclusion criteria: 1- Transient ischemic attack. 2- Past history of stroke. 3- Hemorrhagic stroke. 4- Presence of infections. 5- Other inflammatory or malignant disease. 6- Condition with tissue injury (myocardial infarction, or major surgical procedures) within the last year or immunosuppressive treatment.

All patients were subjected to full  neurological history taking on admission, and clinical examination  on admission and after 14 days. At each point scores were assessed  according to the National Institutes of Health stroke Scale (NIHSS) (no disability=0), and the Barthel Index (BI) (no disability =100) . Both scales of neurological deficits and disability have been used in many stroke  trials and found to be reliable and valid measures^5,6. All patients were  subjected to  laboratory investigation in the form of  fasting and two hours post prandial blood glucose level, complete lipid profile, renal and liver profile, complete blood picture and others according to the suspected etiology of the stroke. Vascular studies: ECG and Echocardiography: transthoracic (TTE) and/or transoesophageal (TOE). Magnetic resonance imaging (MRI) of the brain: Cerebral MRI and MR angiography (MRA) were performed with a Siemens Magnetom 1.5 –T imager, according to MRI protocol of stroke in Ain Shams Specialized Hospital, which include Axial T1,T2,T2*, FLAIR, diffusion, and MRA of the circle of Willis. Stroke   mechanisms were determined according to strict criteria elaborated by Caplan⁷: large artery occlusive disease required  the demonstration of occlusion or severe stenosis of large artery. Branch penetrator (small vessel) disease was demonstrated by infarction limited to the territory of penetrating branch(es)  without large artery or cardiac disease.

Eighteen normal controls and twelve patients with vascular risk factors (hypertension, diabetes mellitus and hyperlipidaemia) were matched with age and sex to the patients group and were subjected to the measurement of the level  of sVCAM-1 in their serum.

Blood sampling and quantification of sVCAM:

Blood  samples were   obtained from the patients at the third day of stroke onset  and from the control groups. After collection by venipuncture, blood was allowed to clot at  room temperature for one hour, and after centrifugation  the serum was stored at -80°C until it was analysed. Determination of sVCAM-1 was done by ELISA technique suing a kit supplied by BioSource (BioSource International Inc.California USA).

Statistical analysis:

The data were collected  and processed  to a personal computer. Data analysis was done using SPSS program version 11.0.1. The quantitative data were compared using student t-test, and Pearson correlation Coefficient test.

RESULTS

The study was conducted on 74 subjects, of whom 44 (group I) were patients of first ever acute ischemic stroke, eighteen normal controls (group II) and twelve subjects with vascular  risk factors (group III) (hypertensives, diabetics and hyperlipidaemics).

Our patients group included 28 males (63.6%), and 16 females (36.4%) with mean age 59.8±10.1 (range 39-80 years), of whom 31 (70.5%) patients were hypertensives, 28  were diabetics (63.5%) and 16 (36.4%) with dyslipidaemia. Patients having large vessel disease were 30  patients (68.2%) compared to 14 (31.8%) with small vessel disease. Patients with anterior circulation stroke were 25(56.8%)   compared to 19 (43.2%) with posterior circulation stroke.  Mean NIHSS score was 7.8±4.0 (range 1-18) and mean BI score was 61.1±25.9 (range 10-100). Mean level of  sVCAM-1in our patients was  1032±708.7 ng/ml, (range  225- 3750). 

Data regarding sVCAM-1 revealed higher mean sVCAM-1 among  control with vascular risk factors (group III) compared to normal control (group II) but the difference is not significant statistically p>0.05 (Table 1).

Also, there is a higher mean sVCAM-1 among ischemic stroke patients compared to normal controls and the difference is highly significant statistically p<0.01 (Table 2) (Fig .1).

Again, there is a higher mean sVCAM-1 among patients with ischemic stroke  compared to controls with risk factors  and the difference is significant statistically p<0.05 (Table 3).

The mean sVCAM-1 among female patients with ischemic stroke was comparable to that of the  male patients p>0.05 (Table 5).

There is no  statistically  significant difference of mean sVCAM-1 between older patients >60 years and  young patients p>0.05 (Table 6).

Regarding type of stroke, there is no significant statistical difference between  levels of sVCAM-1 among patients with anterior circulation compared to patients with posterior circulation stroke, p>0.0 5 (Table 7 and Fig. 3).

Regarding correlation of sVCAM-1 to risk factors, there is no statistical difference as regards  sVCAM-1 levels in patients with different risk factors (hypertensives vs normotensives 1101.6±784.4 and 866.1±468.3 respectively), (diabetics vs euglycemics  985.7±632.1 and 1113.1±842.5 respectively), (presence vs absence of dyslipidaemia 1167.1±827.6 and 954.8±634.3 respectively) p>0.05 (Table 8).

As  regards correlation of sVCAM to neurological and functional status among patients group, there is highly significant positive correlation between sVCAM and NIHSS score p<0.01 (Table 9 and Fig. 4) and significant negative correlation between Barthel Index  Score and sVCAM  p<0.05 (Table 9). 

There is no significant correlation between age and sVCAM among the studied stroke patients.

There is a higher mean sVCAM-1 among patients with large vessel disease compared to patients with small vessel disease and the difference is highly significant statistically p<0.01 (Table 4 and Fig. 2).

Table 1. Comparison between group II (normal controls) and group III (controls with risk factors) as regards the mean sVCAM-1.

P>0.05 not significant

Table 2. Comparison between ischemic stroke patients and normal control as regards the mean sVCAM-1

P<0.01 Highly significant

Table 3. Comparison between ischemic stroke patients and controls with risk factors (RF) as regards the mean sVCAM-1.

P<0.05  significant

Table 4. Comparison between stroke patients with small and large vessel disease as regards the mean sVCAM-1.

P<0.01 Highly significant

Table 5. Comparison between males and female patients with ischemic stroke as regards the mean sVCAM-1.

Table 6. Comparison between young and old patients with ischemic stroke  as regards the mean sVCAM-1.

P>0.05 not significant

Table 7. Comparison between cases of ischemic stroke  with anterior versus posterior circulation stroke as regards the mean sVCAM-1.

P>0.05 not significant

Table 8. Comparison between patients with  ischemic stroke  with different risk factors  as regards the mean sVCAM-1.

NS : not significant

Table 9. Correlation coefficient between sVCAM-1 and NIHSS, Barthel Index Scores and age.

P>0.05 not significant   P<0.05 Significant     P<0.01 highly significant

Fig. (1): Comparison between ischemic stroke patients  and normal controls as regards the mean sVCAM-1.

Fig. (2): Comparison between patients  with large and small vessel disease as regards the mean sVCAM-1.

Fig. (3): Comparison between cases with anterior and posterior circulation as regards the mean sVCAM-1.

Fig. (4): Correlation between NIHSS score and sVCAM-1 level among ischemic stroke patients

DISCUSSION

There is increasing evidence that inflammatory processes are involved in acute cerebral ischemia and adhesion molecules are involved in the pathogenesis of cerebral ischemia^8,9. Also, leukocytes are now considered to potentiate ischemic neuronal damage by microvasculature obstruction ¹⁰ and generation of neurotoxic substances such as reactive oxygen metabolites, enzymes or toxic cytokines¹¹. Since the discovery of their role approximately a decade ago, cell adhesion molecules have been attracting interest for a number of reasons. For example, the blockade of the interaction between leukocytes and the endothelium by agents that mimic or inhibit these adhesion molecules may provide the basis for a new class of therapeutic agents, although promising studies in animals, have yet to be translated into products proven to be effective in humans^12-16. These findings strongly suggest a pathogenetic role of leukocyte adhesion and migration in acute cerebral ischemia. However, the role of adhesion molecules in acute stroke in humans thus far is only incompletely understood. Our  study  showed that there is significant  elevation in the level of the sVCAM-1  in  patients with acute ischemic stroke in the  third day after the onset of stroke with statistically significant difference in comparison to the normal control and control people with multiple risk factors for atherosclerosis  without stroke (P<0.05) .Our result goes with the finding of Bitsch², who found  characteristic and  significant changes in the level of sVCAM-1 after completed stroke and not  after transient ischemic attacks. Our  result    is also similar to the finding of Fassbender et al.¹⁷, who  did not  find difference between the level of sVCAM-1 in patients with risk  factors for atherosclerosis  and  normal control subjects.  On the other hand, other studies  found significant difference  between other type of adhesion molecules, namely sICAM, in patients with risk factors of atherosclerosis in comparison to normal control group, which provide further evidence of involvement of sVCAM-1 in the pathogenesis of ischemic stroke and possible involvement of the inflammatory mediators  in the occurrence of secondary neuronal injury and cell  damage and also in the reperfusion injury^2,18. Leukocytes may adhere to the activated endothelium, plug capillaries, migrate into brain tissue and release proinflammatory mediators. This inflammatory reaction could lead to secondary injury of potentially salvageable neurons in the penumbra around the infarction¹⁹. High level of sVCAM-1 in the third day of ischemic stroke goes with the finding of many studies which found persistence elevation of its level in the 5 days  following ischemic stroke and not like other adhesion molecules, as serum endothelial leukocyte adhesion molecule-1(sELAM-1) which showed initial transient elevation of its level^2,17. The wide range of variation of levels  of the sVCAM- 1 in our study (225-3750 ng/ml ,mean  1032 is comparable to the wide range found in Bitsch study² (was 296-1770, with mean  708).

Regarding the correlation of sVCAM-1 level with the severity of neurological deficit  we found positive correlation between the raised serum level of the adhesion molecule and the NIHSS scores which may  support the role of  leukocytes  migration  and inflammatory mediators  in the neuronal injury and cell damage which lead to more severe neurological deficit. This result  goes with the  finding of Bitsch et al.², who found correlation between initial sVCAM-1 and  initial Scandinavian Stroke Scale (SSS) scores, although was not statistically significant.  Another study¹ has also shown enhanced astrocyte expression of VCAM-1 in samples of brain tissue of ischemic stroke patients nine to ten days after clinical onset (patients who died as a results of their stroke)  which support our finding of  positive correlation between the raised serum level of the sVCAM -1 and the NIHSS scores.  

On the other hand, regarding the disability , we found negative correlation between the level of the sVCAM-1 and the Barthel Index (BI) which mean good functional outcome (high level of BI score) with low serum level  of the adhesion molecule and vise versa. Other studies²  failed to find correlation between sVCAM-1 and  functional outcome, however, the same study found correlation between the initial rise of other adhesion molecules (s E-selectin) and BI scores.

Also, our finding revealed no correlation between level of adhesion molecules and the age of the patients, a finding that is similar to others² who found that the level of sVCAM-1 were independent neither to age  nor to the  different vascular territory (in our study, anterior versus posterior  circulation).

Our results demonstrated statistically significant higher mean sVCAM-1 in large vessels versus small lacunar infarction (p<0.01), a finding that can be explained by  the fact that wide area of injury may lead to more adhesion of leukocytes with more involvement of adhesion molecules compared to small lacunar infarction²⁰. Our results revealed statistically insignificant difference of sVCAM in stroke patients with different risk factors, a finding that highlights its role in occurrence of stroke whatever the risk factor is. Clinical implication of the role of sVCAM in acute ischemic stroke was translated in experimental studies by the work of Becker etal²¹  and Vemuganti and coworkers²², who suggested that blocking the action of adhesion molecules (e.g.; by anti-adhesion molecules antibodies) can reduce the size of infarction in experimental animals.

In conclusion, serum VCAM-1 could be reliable marker for severity of neuronal damage  in acute cerebral ischemic stroke after exclusion of stroke independent risk factors, like infection, fever, and other inflammatory conditions. Therapeutic trials that attempt to interfere with the function of adhesion molecules may help to reduce the infarction size  and  may  be beneficial in prolongation of therapeutic window for thrombolytic therapy and thus be of benefit in acute stroke management.

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