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 measures5,6. 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.
sVCAM |
Mean |
SD |
T |
P |
Normal control (group II) N=18 |
541.6 |
120.3 |
1.3 |
>0.05 NS |
Control with RF
(group III) N=12 |
594.2 |
103.6 |
P>0.05 not significant
Table 2. Comparison between ischemic stroke patients and normal control as regards the mean sVCAM-1
sVCAM |
Mean |
SD |
T |
P |
Stroke patients N=44 (group I) |
1032 |
708.7 |
4.4 |
<0.01 HS |
Normal Control
N=18 (group II) |
541.6 |
120.3 |
P<0.01 Highly significant
Table 3. Comparison between ischemic stroke patients and controls with risk factors (RF) as regards the mean sVCAM-1.
sVCAM |
Mean |
SD |
T |
P |
Stroke patients N=44 (group I) |
1032 |
708.7 |
3.94 |
<0.05 S |
Control with RF
N=12 (group III) |
594.2 |
103.6 |
P<0.05 significant
Table 4. Comparison between stroke patients with small and large vessel disease as regards the mean sVCAM-1.
sVCAM |
Mean |
SD |
T |
P |
Stroke patients with small vessel disease (N=14) |
494.6 |
177.6 |
5.6 |
<0.01 HS |
Stroke patients with large vessel disease (N=30) |
1282.8 |
725.4 |
P<0.01 Highly significant
Table 5. Comparison between males and female patients with ischemic stroke as regards the mean sVCAM-1.
sVCAM |
Mean |
SD |
T |
P |
Male patients (N=28) |
1013.3 |
638.7 |
0.2 |
>0.05 NS |
Female patients (N=16) |
1064.6 |
839.0 |
P>0.05 not significant
Table 6. Comparison between young and old patients with ischemic stroke as regards the mean sVCAM-1.
sVCAM |
Mean |
SD |
T |
P |
Patients <60 years (N=24) |
1011.4 |
761.6 |
0.2 |
>0.05 NS |
Patients >= 60 Years (N=20) |
1056.7 |
658.4 |
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.
sVCAM |
Mean |
SD |
t |
P |
Anterior circulation N=25 |
1143.4 |
781.8 |
1.2 |
>0.05 NS |
Posterior circulation N=19 |
885.5 |
587.5 |
P>0.05 not significant
Table 8. Comparison between patients with ischemic stroke with different risk factors as regards the mean sVCAM-1.
sVCAM |
Mean |
SD |
t |
P |
Patients with HTN n=31 |
1101.6 |
784.4 |
1.2 |
>0.05 NS |
Patients without HTN . N=13 |
866.1 |
468.3 |
Patients with DM N= |
985.7 |
632.1 |
-0.5 |
>0.05 NS |
Patients without DM. N= |
1113.1 |
842.5 |
Patients with dyslipid. N= |
1167.1 |
827.6 |
0.9 |
>0.05 NS |
Patients without dyslipid.. N= |
954.8 |
634.3 |
NS : not significant
Table 9. Correlation coefficient between sVCAM-1 and NIHSS, Barthel Index Scores and age.
|
sVCAM-1 |
NIHSS |
r =0.689
P<0.01 HS |
Barthel Index |
r = - 0.301
P<0.05 significant |
Age |
r=0.016
P>0.05 non significant |
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 ischemia8,9. 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 humans12-16. 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 injury2,18. 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 level2,17. 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.
REEFRENCES
1. Price C, Warburton E and Menon D (2003): Human cellular inflammation in the pathology of acute cerebral ischaemia. Journal of Neurology Neurosurgery and Psychiatry; 74:1476-1484.
2. Bitsch A, Klene W, Murtada L (1998): A Longitudinal Prospective Study of Soluble Adhesion Molecules in Acute Stroke. Stroke; 29:2129-2135.
3. Dirnagel U and Elger B (2004): Neuroinflammation in stroke. Springer-Verlag Berlin Heidelberg New York. pp: 25-26.
4. Lee Y, Kuhn H, Hennig B (2001): IL-4-induced oxidative stress upregulates VCAM-1 gene expression in human endothelial cells. . Mol Cell Cardiol 33: 83-94 (abstract).
5. The National Institute of Neurological Disorders and Stroke rt-PA Stroke Study Group (1995). Tissue plasminogen activator for acute ischemic stroke. N Engl J Med.;333:1581–1587.
6. Hacke W, Kaste M, Fieschi C, Toni D, Lesaffre E, von Kummer R, Boysen G, Bluhmki E, Hoxter G, Mahagne MH (1995): Intravenous thrombolysis with recombinant tissue plasminogen activator for acute hemispheric stroke: the European Cooperative Acute Stroke Study (ECASS). JAMA.;274: 1017–1025.
7. Caplan LR. Vertebrobasilar territory ischaemia: an overview (1996) . In: Caplan LR, ed. Posterior circulation diseases: clinical findings, diagnosis and management. Cambridge, Mass: Blackwell ;pp:179-197.
8. Kim JS (1996): Cytokines and adhesion molecules in stroke and related diseases. J Neurol Sci.;137: 69–78
9. Stanimirovic DB, Wong J, Shapiro A, Durkin JP (1997): Increase in surface expression of ICAM-1, VCAM-1 and E-selectin in human cerebromicrovascular endothelial cells subjected to ischemia-like insults. Acta Neurochir Suppl; 70:12–16.
10. del Zoppo GJ, Schmid-Schonbein GW, Mori E, Copeland BR, Chang CM (1991): Polymorphonuclear leukocytes occlude capillaries following middle cerebral artery occlusion and reperfusion in baboons. Stroke. ; 22:1276–1283
11. Ward PA (1991): Mechanisms of endothelial cell killing by H2O2 or products of activated neutrophils. Am J Med.;91:89-94
12. Connolly ES Jr, Winfree CJ, Springer TA, Naka Y, Liao H, Yan SD, Stern DM, Solomon RA, Gutierrez-Ramos JC, Pinsky DJ (1996): Cerebral protection in homozygous null ICAM-1 mice after middle cerebral artery occlusion: role of neutrophil adhesion in the pathogenesis of stroke. J Clin Invest.; 97:209–216.
13. Blann A, Ridker P , Lip G (2002): Inflammation, cell adhesion molecules, and stroke :tools in pathophysiology and epidemiology? Stroke; 33: 2141-2145.
14. Hayward R, Campbell B, Shin YK, Scalia R, Lefer AM (1999): Recombinant soluble P-selectin ligand-1 protects against myocardial ischemic reperfusion in cats. Cardiovasc Res.; 41: 65–76
15. Zhang RL, Chopp M, Jiang N, Tang WX, Prostak J, Manning AM, Anderson DC (1995): Anti-intercellular adhesion molecule-1 antibody reduces ischemic cell damage after transient but not permanent middle cerebral artery occlusion in the Wistar rat. Stroke.; 26: 1438–1443
16. Tsukamoto K, Yokono K, Amano K, Nagata M, Yagi N, Tominaga Y, Moriyama H, Miki M, Okamoto N, Yoneda R (1995): Administration of monoclonal antibodies against vascular cell adhesion molecule-1/very late antigen-4 abrogates predisposing autoimmune diabetes in NOD mice. Cell Immunol.; 165: 193–201.
17. Fassbender K, Mossner R, Motsch L, Kischka U, Grau A ,and Hennerici M (1995):Criculating Selectin-and immunoglobulin – type adhesion molecules in acute ischemic stroke. Srtoke.; 26: 1361-1364
18. Calabresi L, Gomaraschi M, Villa B (2002): Elevated Soluble Cellular Adhesion Molecules in Subjects With Low HDL-Cholesterol. Arteriosclerosis, Thrombosis, and Vascular Biology; 22: 656-659.
19. Frijns CJ and Kappelle LJ (2002): Inflammatory Cell Adhesion Molecules in Ischemic Cerebrovascular Disease. Stroke; 33:2115-2119.
20. Danton G and Dietrich W (2003): Inflammatory mechanisms after ischemia and stroke. J Neuropathol Exp Neurol.; 62: 127–136.
21. Becker K, Kindrick D, Relton J (2001): Antibody to the 4 integrin decreases infarct size in transient focal cerebral ischemia in rats. Stroke; 32:206-210.
22. Vemuganti R, Dempsey R, Bowen K (2004): Inhibition of intercellular adhesion molecule-1 protein expression by antisense oligonucleotides is neuroprotective after transient middle cerebral artery occlusion in rat. Stroke; 35:179-184.
الملخص العربى
الجزيئات الذائبة اللاصقة لخلايا الأوعية الدموية – رقم ( 1) الموجودة بالمصل فى حالات الجلطة الدماغية الحادة
يلعب ميكانيزم الالتهاب العصبى دور مهم فى الإصابة الناتجة عن جلطات الدماغ والتدخل فى هذه العملية قد يؤدى إلى تحسن مآل الحالة العصبية للمرضى.
فالالتصاق المتين لكرات الدم البيضاء بالخلايا المبطنة للوعاء الدموى بالإضافة إلى تنشيط وهجرة هذه الخلايا عبر الأوعية الدموية إلى خلايا المخ يعتمد على الجزيئات اللاصقة.
وقد تم هذا البحث بهدف تقييم مستوى الجزيئات اللاصقة فى الدم (المصل) فى حالات الجلطة الدماغية الحادة وعلاقة هذه الجزيئات بمدى شدة الإصابة الإكلينيكية وعلاقتها بمآل المرض على المدى القصير.
تمت هذه الدراسة على 44 مريض بالجلطة الدماغية الحادة فى أول 3 أيام من بداية الجلطة وتكونت العينة الضابطة من 18 شخص سليم و 12 شخص يعانون من عوامل خطورة لحدوث الجلطات ولكن بدون سكتة دماغية. وتم تقييم مستوى الجزيئات اللاصقة للأوعية الدموية رقم – 1 فى المرضى فى اليوم الثالث لحدوث الجلطة الدماغية وفى العينة الضابطة.
وأظهرت النتائج ارتفاع متوسط مستوى هذه الجزيئات مقارنة بالعينة الضابطة وكان مستوى هذه الجزيئات فى مرضى الأوعية الدموية الكبيرة مرتفع مقارنة بمرض الأوعية الدموية الصغيرة. وقد وجدت علاقة إيجابية بين مستوى هذه الجزيئات ومدى شدة الإصابة الإكلينيكية بواسطة مقياس الجلطة الدماغية للمعهد القومى للصحة، وقد وجدنا أيضاً علاقة سلبية بين مستوى هذه الجزيئات مع مقياس بارثيل لمزاولة النشاط اليومى لتقييم مدى الإعاقة.
وقد أكدت هذه الدراسة على أهمية دور الجزيئات اللاصقة فى حدوث الجلطات الدماغية ودورها فى مدى شدة الإصابة والإعاقة العصبية.
وأشارت إلى أهمية الدراسات العلاجية التى تعوق دور هذه الجزيئات فى علاج الجلطة الدماغية.