INTRODUCTION

 

               Atherosclerosis, one of the major causes of ischemic cerebrovascular disease is considered to involve the inflammatory system. C-reactive protein (CRP) widely known to be an inflammatory marker was detected in atherosclerotic plaques and was recently reported to be involved in the development of atherosclerosis^1,2,3.

Elevated plasma CRP level may be an atherothrombotic biomarker that provides additive prognostic information about not only coronary heart disease⁴ but also cerebral and peripheral arterial disease⁵. Moreover, elevated CRP level could be a marker that predicts the risk of ischemic stroke in elderly⁶.

The realization that CRP genetic polymorphism does exists and that it directly and predictably influences steady-state blood CRP level could be of substantial clinical importance⁷.

This work aimed at studying the high sensitivity-CRP (hs-CRP) assay as an inflammatory marker of stroke risk and valuable additional diagnostic and prognostic tool. Also, to study genetic association between CRP gene polymorphism and ischemic stroke.

 

SUBJECTS AND METHODS

 

Subjects

The participants were recruited from Neurology Department, Beni-Sueif University Hospital and Cairo University Hospital during the period from April 2006 to December 2008.

               Forty-one patients (group I) (24 males and 17 females) with acute first ever ischemic stroke (within first 48 hours of stroke onset) diagnosed clinically and by computed tomography (CT) were included in the study. Patients' age ranged from 45 to 76 years with a mean age of 57.9±9.1 years.

Excluded from the study were patients with hemorrhagic stroke diagnosed by CT, and patients with cerebral embolism of cardiac origin diagnosed clinically and by ECG, and echocardiography. Also, patients with cancer and autoimmune disease were excluded. No participants had a history of peripheral arterial occlusive disease, myocardial infarction, recurrent stroke or any acute or chronic infection at the time of sampling.

Forty healthy subjects (group II) (21 males and 19 females) were enrolled as control subjects, their age ranged from 45 to 71 years (mean age of 55.5±6.6 years). Excluded from this group, subjects with history of stroke or myocardial infarction, rheumatic heart diseases, autoimmune diseases. Diabetic and hypertensive persons or persons with any acute or chronic infection at the time of sampling were also excluded.

 

METHODS

 

Patients were evaluated by clinical examination and were clinically classified using Oxfordshire Community Stroke Project Classification (OCSP)⁸ into lacunar infarction syndrome (LACI), total anterior circulation infarction syndrome (TACI), partial anterior circulation infarction syndrome (PACI) and posterior circulation infarction syndrome (POCI).

Assessment of stroke severity and level of impairment using the National Institute of Health Stroke Scale (NIHSS)⁹, which is a standardized neurologic stroke severity scale developed to quantify stroke patients' deficits in clinical trials. A score of 1-4= minor stroke, 5-15 = moderate stroke, 15-20 = moderate /sever stroke and 21-42=sever stroke.

Assessment of functional outcome using different stroke scales: Modified Rankin Scale (MRS)⁽¹⁰⁾, which is an efficient global functional outcome index after stroke. It is a 7 grade scoring system with 0 corresponding to persons having no symptoms and 6 being dead.

Glasgow Outcome Scale (GOS)¹¹, a 5 point scale that reflects disability and handicap. A score of 1-2 is considered good outcome, whereas a score of 3-6 is considered poor one.

The outcome functional scales were done for patients one month after stroke onset.

Laboratory investigations were done and they included routine laboratory profile and specific laboratory investigations including: (a) hs-CRP serum level using automated chemiluminescence using Immulite analyzer (immunometric assay). Normal values of serum hs-CRP are 0.14 mg/dl to 1.1 mg/dl, serum hs-CRP was done in the first 48 hours of stroke onset. (b) Genetic profile: mutation detection of CRP gene was done using RFLP (Restriction Fragment Length Polymerase) technique. The human CRP gene was mapped to chromosome 1q21. The CRP 1059 G/C polymorphism could be detected by digestion of endonuclease Mae III¹².

Computed tomography of the brain was done using the Toshiba Express/XS System, initially and follow-up after 72 hours if needed. The scans were conducted in the axial imaging planes. The axial cuts were normally at 10 mm. Size of infarction was detected as follows: small infarction: <1 cm, moderate sized infarction: 1-3 cm and large sized infarction: >3 cm.

The control group underwent the same routine and specific laboratory tests (hs-CRP and genetic testing).

 

Statistical Methods

Quantitative data were summarized as means and standard deviations. Categorical data were summarized as percentages. Qualitative data were compared by Chi-square or Fisher' exact tests according to the expected frequencies. Persons Correlation Coefficient, which is a test to measure the strength of the linear relationship between two variables was used. A 5% probability level (p<0.05) was considered statistically significant.

 

RESULTS

 

Hypertension was found in 18 patients (43.9%), twenty-two patients were cigarette smokers (53.7%), TIAs in 2 patients (4.9%) and family history of stroke in 6 patients (14.6%). Hypercholesterolemia was found in 15 patients (36.6%), elevated blood sugar in 15 patients (36.6%) and hyperuriceamia in 11 patients (26.8%).

Clinically LACI syndrome was diagnosed in 6 patients (14.6%), TACI in 2 patients (4.9%), PACI in 33 patients (80.5%) and no patients had POCI syndrome.

Assessment of stroke severity by NIHSS revealed mean of 8.6±3.7 with 36 patients (87.8%) having minor and moderate stroke and 5 patients (12.2%) having moderate/sever and sever stroke.

               Clinical functional outcome scales showed mean MRS of 3.1±1.2 with 23 patients (56.1%) with good outcome, and GOS mean of 2.3±0.8 with 24 patients (58.5%) with good outcome.

CT results showed 6 patients (14.64%) had small sized infarction, 18 patients (43.9%) had medium sized infarction and 17 patients (41.46%) had large sized infarction.

 

Specific Laboratory Results:

(a)    Serum high sensitivity CRP levels (hs-CRP):

Elevated serum hs-CRP was found in 22 patients (53.7%) while normal serum hs-CRP was found in 19 patients (46.3%). Whereas all subjects in control group had normal serum hs-CRP levels. There was a highly significant difference between acute stroke patients and control subjects regarding the mean level hs-CRP (p-value <0.01) (Table 1).

 

(b)    CRP genotyping:

Human CRP G1059C polymorphism genotype results revealed that (GG allele) (the common allele) was found in 39 patients (95%) and all persons of control group, while (GC allele) (the less common allele) was found in 2 patients only and absent in control group. The third rare allele (CC) was absent in both groups. In patients having the homozygous allele GG, the mean CRP level was 1.9±1.0 mg/dl whereas the mean level of CRP in patients having GC allele was 0.52±0.4 mg/dl (Table 2).

 

Relation of hs-CRP level and risk factors and CT findings:

1.      Male patients had significantly higher mean level of hs-CRP (3.1 ± 2.0 mg/dl) than female patients (1.7 ± 1.1 mg/dl).

2.      Hypertensive patients had significantly higher mean level of hs-CRP (3.7 ± 3.7mg/dl) than normotensive patients (P-value = 0.02). Also patients with hypercholesterolemia and high blood sugar were significantly associated with elevated hs-CRP (2.4±2.2 mg/dl), (4.3±1.3 mg/dl) respectively.

3.      However, smoking, TIAs, +ve family history of stroke, hyperuricemia, elevated WBCs count did not reach a significant level.

4.      According to OCSP: patients with TACI had significantly higher serum level of hs-CRP (3.5±0.5 mg/dl) than patients with either PACI or LACI (2.3±0.5 mg/dl) (0.5±0.4 mg/dl) respectively.

5.      Patients with large infarction had highly significant mean level of CRP (4.5±2.9 mg/dl) than patients with small and medium sized infarction (0.5±0.4 mg/dl) (Table 3).

 

Correlation study between hs-CRP serum level and different laboratory risk factors and outcome scales (Table 4):

1.      There was positive correlation between hs-CRP and different laboratory risk factors, which reached statistical significance regarding cholesterol level only.

2.      Negative correlation between hs-CRP and HDL serum level (did not reach significant value).

3.      Also positive correlation was present between hs-CRP level and different assessment and outcome scales (NIHSS, GOS and MRS). Correlation with GOS reached significant value and that with MRS reached a highly significant value.

 

 

Table 1. Serum high sensitivity CRP levels in group I (patients with acute stroke) and group II (Healthy controls)

 

CRP	Group I	Group II	P-value
Minimum	0.01	0.01	0.001*
Maximum	15.7	0.67
Mean±SD	2.1±0.9	0.3±0.2

CRP C-reactive protein, SD standard deviation

* Significant at p <0.01

 

 

Table 2. hs-CRP levels according to CRP polymorphism in group I (patients with acute stroke)

 

CRP polymorphism	Serum hs-CRP (mean±SD) mg/dl
1059GG	1.9±1.0
1059 GC	0.52±0.4
1059CC	Absent

CRP C-reactive protein, SD standard deviation

 

 

Table 3. hs-CRP levels and size of infarction in group I (Patients with acute ischemic strokes)

 

Infarction size	Large infarction	Small and medium infarction	P-value
hs-CRP (mean±SD)	4.5±2.9	0.5±0.4	0.006**

CRP C-reactive protein, SD standard deviation

* significant at p <0.01

 

Table 4. Correlation between hs-CRP level and laboratory risk factors and clinical assessment and outcome scales in patients with acute ischemic strokes

 

Laboratory risk factors and different assessment and outcome scales	“r” value	P-value
Cholesterol level	0.312	0.04*
Triglycerides level	0.039	0.806
Fasting blood sugar	0.146	0.361
HDL	-0.175	0.274
LDL	0.104	0.517
Uric acid	0.113	0.482
ESR	0.090	0.575
WBCs count	0.004	0.982
NIHSS	0.157	0.328
GOS	0.337	0.031*
MRS	0.441	0.004**

ESR erythrocyte sedimentation rate, GOS Glasgow Outcome Scale, HDL high density lipoprotein, LDL low density lipoprotein, MRS Modified Rankin Scale,  NIHSS national institute of health stroke scale, WBC white blood cells

* significant at p<0.05 ** significant at p<0.01

 

 

DISCUSSION

 

There is much evidence to suggest that neuro-inflammatory mechanisms play an important role in ischemic injury, and that interruption of these processes can result in improved neurological outcomes¹³.

In this study there was a highly significant increase in the hs-CRP serum level in the first 48 hours after stroke onset and this was in accordance with some preceding studies^14,15. A finding which can be explained by the fact that, inflammation plays a central role in all phases of atherosclerosis, from the initial recruitment of circulating leukocytes to the arterial wall to the rupture of unstable plaques, which results in the clinical manifestations of the disease. Hs-CRP may be involved in each of these stages by direct influencing processes like complement activation, apoptosis, vascular cell activation, lipid accumulation and thrombosis¹⁶.

On the other hand, DiNapoli et al.¹⁷ concluded that, there is no sufficient evidence to recommend measurement of hs-CRP in the routine evaluation of cerebrovascular disease risk in primary prevention. As hs-CRP is simply a marker reflecting inflammatory state, any factor that alters the relationship between its concentration and inflammation may distort interpretation.

Roquer et al.³ recently demonstrated that many biomarkers of inflammation are elevated years in advance of first ever thrombotic stroke and that these same biomarkers are highly predictive of recurrent stroke.

               With regards to cerebral vessels increased biomarkers of inflammation, including hs-CRP have been associated with increased stroke risk as well as an increased rate of atherosclerosis progression in the carotid vessels^4,6.

               Serum hs-CRP level was higher in acute ischemic stroke men than women. This was supported by the study of Devaraj et al.¹⁸ who stated that elevation of hs-CRP level was an independent risk factor for future ischemic stroke in men not in women. On the other hand, clinical evidence has shown that endogenous estrogen protects the development of atherosclerosis¹⁹, and has anti-inflammatory action in premenopausal women²⁰.

The significantly higher mean levels of serum hs-CRP in hypertensive patients observed in this study can be explained on the basis of the relation between atherosclerosis as a chronic inflammatory process, and increased CRP level as an indicator of inflammatory process. Moreover, Sesso et al.²¹ reported that increased CRP precedes blood pressure elevation, implying an active involvement of inflammation in the development of hypertension.

It's interesting to know the effects of different antihypertensive medications on CRP level as it might be of relevance to treatment choices. Patients using beta blockers, angiotensin converting enzyme inhibitors had lower CRP levels than patients using other medications as diuretics²¹.

Contrary to expectations, mean level of hs-CRP in smoking patients was significantly lower than non-smokers. Other studies have found an association between hs-CRP levels and smoking in disease-free individuals⁽²²⁾, but few studies have evaluated this association in patients after stroke. Arenillas et al.²³ found no association, on the other hand, El-Kind et al.²⁴ found an elevated post-stroke levels of hs-CRP with smoking.

Patients with hypercholesterolemia and high blood sugar had significantly higher levels of hs-CRP pointing to a correlation between lipids, diabetes and inflammation. This was in accordance with previous studies^18,25.

Nakanishi et al.⁽²⁶⁾ added that, there is a growing body of evidence implicating low grade inflammation as a potential dynamic in the pathogenesis of type-2 diabetes. Moreover, they explained the association between CRP and diabetes through oxidative stress as oxidative stress generated hyperglycemia, and might be implicated in promoting a state of low-grade inflammation indicated by markers such as CRP with elderly type-2 diabetes.

In this study, a positive correlation was observed between hs-CRP and the NIHSS as indicator of stroke severity. Also a significant relation was found between increased hs-CRP and bad outcome in GOS and MRS scales as indicators of functional outcome. These data were in agreement with others^15,29. Moreover Mizrahi et al.⁽²⁸⁾ results reached a significant value as regards stroke severity.

Patients with large infarction had a highly significant higher CRP level than those with small and medium sized infarction. Others^15,16 concluded that increase hs-CRP was significantly associated with large territory infarction than lacunar infarction. This was in accordance with our results as regards a significant higher level of hs-CRP in patients presented with total anterior circulation infarction than its level in patients with either partial anterior circulation infarction or lacunar infarction syndrome.

Necrosis in the brain produces a strong inflammatory reaction, therefore, the association between lesion size and elevated inflammatory parameters (e.g. hs-CRP) is a sufficient explanation of previous observation²⁹.

This study revealed that the mean CRP level was higher in 1059GG allele polymorphism than that in 1059GC allele in stroke patients. On the other hand, all our control group had GG allele genotyping, so the significant difference between the CRP level in patient group and control group cannot be explained on the genetic basis as both groups had the GG allele (95% of patients and 100% of control).

This was supported by other previous studies^7,12, who found that, there was no significant difference of hs-CRP serum concentration regarding CRP gene G1059C polymorphism. Moreover, Ladenvall et al.¹⁵ failed to demonstrate that poor outcome of acute stroke patients which is associated with high CRP level is because the studied polymorphism (G1059C).

               On the other hand, Eklund et al.³¹ revealed that the carriers of the CRP 1059 C-allele had significantly lower CRP values than GG homozygotes in men. Whereas, Morita et al.³² indicated that the risk of ischemic stroke could be increased in subjects with C allele of 1059 polymorphism.

               Some other polymorphisms are reported to affect the basal and stimulated CRP level³³. Zee et al.³⁰ proved that polymorphisms in CRP gene are associated with marked increase in CRP level and thus with a theoretically predicted increase in the risk of ischemic vascular disease. However, the polymorphisms are not in themselves associated with an increased risk of ischemic vascular disease.

Kikuchi et al.³³ found no significant association of serum CRP levels with the genotypes of CRP C1444T. Also CRP 1009 A>G genotypes and associated haplotypes were associated with lower fasting serum hs-CRP in a group of elderly men³⁴.

These conflicting data suggests that, genetic and environmental determinants, each importantly contribute to the vascular risk associated with inflammation. Although the mechanism for a direct effect is unclear, so, it remains possible that this polymorphism is in strong linkage disequilibrium with a polymorphism in a distal regulatory element not within the region scanned for polymorphisms. Conclusion:

Elevated hs-CRP level is significant in acute ischemic stroke and associated with multiple risk and prognostic factors however, its genetic polymorphism did not influence its level.

 

 

[Disclosure: Authors report no conflict of interest]

 

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