INTRODUCTION

 

Pentraxin 3 (PTX3) is a member of the pentraxin family that includes the traditional C-reactive protein(CRP) but unlike it ,is expressed locally at atherosclerosotic patches and have been considered a biomarker of atherosclerotic cardiovascular diseases and correlated with its severities¹. PTX-3, as an acute phase reactant, is induced by pro-inflammatory cytokines (inteleukin1 beta & tumor necrosis factor) and has anti-inflammatory effect through binding and activation of complement system². Intracranial and extracranial atherosclerosis is not only a major cause for ischemic strokes but also for hemmorhagic ones due to rupture of unstable atherosclerotic plaques³. Although PTX-3 was reported to be expressed in atherosclerotic plaques and high levels were seen in patients with carotid artery atherosclerosis⁴, its pathological role in acute stroke situation is still not fully studied or understood. The aim of this study was to evaluate PTX-3 in acute stroke patients and its prognostic value.

PATIENTS AND METHODS

 

This prospective study was conducted on patients with acute stroke admitted to neuro-critical care unit, Mansoura University Hospital within the first 24 hours of symptom onset. Thirty two, age and sex matched, healthy subjects, not having any evidence of stroke or previous history of transient ischemic attacks, were served as a control.

Stroke was ascertained and classified into ischemic or hemorrhagic by brain computed tomography and/ or magnetic resonance imaging done on hospital admission.

Ischemic stroke cases were further classified into 5 subtypes according to the Trial of Org 10172 in Acute Stroke Treatment (TOAST): cardioembolic infarct (CEI), large-artery atherosclerosis (LAAS), lacunar infarct (LAC), stroke of undetermined etiology (UDE), and stroke of other determined etiology (ODE)⁵.

Infarct volumes were measured by brain CT according to the formula 0.5*A*B*C where (A) is the largest diameter of the infarct and (B) is its largest perpendicular diameter, while (C) was determined by summing the thicknesses of the slices in which the lesion was visible.  The infarcts were classified as large when the volume > 1.5 cm and small when the volume was < 1.5 cm. ⁶

On admission, stroke severity was assessed by the National Institute of Health Stroke Scale (NIHSS) and stroke was categorized as mild (NIHSS 0–7), moderate (NIHSS 8–14), or severe (NIHSS >14) ⁷.

Seven days following the onset of stroke, we evaluated the outcome of patients using modified Rankin Scale (MRS). Poor outcome was considered to be >2 by MRS score⁸.

The presence of baseline vascular risk factors, including hypertension, diabetes mellitus, hypercholesterolemia, and smoking, was determined. Hypertension was defined as blood pressure ≥ 140/90 mmHg; or current antihypertensive therapy. Diabetes mellitus was defined as fasting blood glucose level ≥ 126 mg/dl or a history of treatment with antidiabetics. Dyslipidemia was defined as a total cholesterol level ≥ 200 mg/dl.

Subjects admitted more than 24h after symptoms onset, or with recent inflammatory conditions, such as major trauma, surgery, or obvious acute infectious disease, acute coronary syndrome, cerebrovascular events, autoimmune disease, liver cell failure, and chronic renal failure were excluded from this study.

 

Blood sampling & laboratory measurements:

For PTX-3 measurement: 2 ml of venous blood were collected within 24 - 48 hours of hospitalization into tubes containing ethylene diamine tetraacetic acid (EDTA). Within 30 minutes of collection, tubes were centrifuged at 1500×g  for 10 min. Plasma samples were stored frozen at – 20^o C until the analysis were performed.

For blood glucose & cholesterol measurements: after an overnight fasting, 2ml of venous blood were withdrawn into dry plastic tubes and allowed to clot then the serum was separated by centrifugation at 3000 r/min for 10 min and immediately analyzed.

Plasma PTX-3 levels were determined using enzyme-linked immunosorbent assay (ELISA) according to the manufacture's procedure (R&D Systems, Inc, USA) ⁹.

Blood glucose and cholesterol concentrations were measured on a Cobas Integra 400 chemistry autoanalyzer (Roche Diagnostics, Basel, Switzerland) using commercially available reagents and an enzyme-based kit.

 

Statistical Analysis

Statistical analyses were performed in the SPSS statistical package programme (version 16, SPSS, Chicago, IL). Normality was measured with Kolmogorov Smirmov test. The results were expressed as percentages for categorical variables and as mean ± SD in normal distributed variables or median (range) in non-normal distributed variables. The two-tailed Mann–Whitney U test was used to compare the median values of parameters in patient and control groups. Spearman & Pearson correlation coefficient test (r) was used to test a positive or negative relationship between two variables. Results were considered significant if P values ≤ 0.05. A receiver operating characteristic (ROC) analysis was performed to define a cut-off value of plasma PTX3 for ischemic, hemorrhagic stroke and the associated specificity and sensitivity levels. To test how much of the total variation in the stroke severity can be explained by the plasma PTX3 level we calculated R square by Simple linear regression analysis.

 

RESULTS

 

A hundred and forty acute stroke patients were enrolled. Their demographic, risk factors, & clinical variables as well as PTX-3 levels are presented in table 1. Plasma pentraxin-3 level were significantly higher in stroke patients when compared to control (P=0.000). Although, both ischemic and hemorrhagic groups showed significantly higher levels than control subjects, the PTX3 level was significantly higher among the ischemic group (P=0.000).

There were no significant gender differences of plasma PTX-3 level in each stroke type. Significantly higher PTX-3 levels were noticed in patients with comorbidity of hypertension (P=0.000), diabetes mellitus (P=0.000) or hypercholesterolemia (P=0.001) in both stroke types. On the other hand, patients with ischemic stroke who smoke have significantly higher PTX3 level when compared to non-smokers, a finding that was not seen among patients with hemorrhagic stroke.

Stroke severity among both types of strokes are shown in table 2 while ischemic stroke classification and infarcts volumes are shown in table 3. PTX-3 was significantly higher among cardioembolic (P=0.000) and atherosclerotic ischemia (P=0.002) but not in lacunar ischemia and those with undetermined etiology.

The level of PTX3 was significantly higher in patients with severe stroke both clinically (in both stroke types) and radiologically by infarct volume in the ischemic group as shown in Figures (1) and (2).  Also, Correlation between plasma PTX3 level and stroke severity (as measured by NIHSS) was significantly strong positive in ischemic stroke patients (r=0.91, P=0.000) and significantly moderate positive in hemorrhagic stroke patients (r=0.60, p=0.000) as shown in Figures (3) and (4)..

Simple linear regression analysis showed that plasma PTX3 level can explain 83.4% & 63.1% of ischemic & hemorrhagic stroke severity as measured clinically by NIHSS. Moreover, PTX3, hypertension and smoking were significant predictors of severe ischemic stroke type as measured by NIHSS on multivariate regression linear analysis while only PTX-3 was the only significant predictor for hemorrhagic stroke on the same analysis (Beta=1.59, SE=0.422 & P=0.000).

Our study showed that at a cutoff value of 3.18 ng/ml, PTX3 has specificity, sensitivity, negative and positive predictive values of  78.13%, 98.6%, 92.6% & 95.17% respectively for detecting patients with acute cerebral stroke (AUC, 0.96; 95%  CI,  0.93 – 0.99) as done by Receiver operating characteristic (ROC) curve (Figure 5).

 

Table 1. Clinical results of the studied stroke patients and control subjects.

 

♦ median (range)

 

Table 2. Stroke clinical severity assessment.

 

	Hemorrhagic stroke N(%)	Ischemic stroke N (%)
NIHSS-mild	27(38.57)	22(31.43)
NIHSS-moderate	0	6(8.57)
NIHSS-severe	43(61.43)	42(60)
MR scale-mild	26(37.1)	29(41.43)
MR scale-severe	44(62.9)	41(58.57)

 

Table 3. Type of ischemia and infarcts size in patients with ischemic stroke.

 

	N(%)	Pentraxin-3 (ng/ml)
Type of ischemia
Cardioembolic	14(20%)	17.43(8.90-21.63)
Atherosclerotic	24(34.3%)	11.88(4.27-20.96)
Lacunar	28(40%)	5.56(3.31-19.80)
Undetermined etiology	4(5.7%)	12.03(5.52-18.43)
Infarct size
Small (less than 1.5 cm)	26(37.1%)	5.17(3.31-9.37)
Large (more than 1.5 cm)	44 (62.9%)	17.28(5.33-21.63)

 

 

 

Figure 1. Pentraxin-3 (PTX-3) in different stroke severity assessment scales in patients with ischemic stroke.

 

 

 

 

Figure 2. Pentraxin-3 (PTX-3) in different stroke severity assessment scales in patients with hemorrhagic stroke.

 

 

 

Figure 3. Correlation between plasma PTX3 and NIHSS in ischemic stroke patients.

 

 

 

Figure 4. Correlation between plasma PTX3 and NIHSS in hemorrhagic stroke patients.

 

 

 

Figure 5. Receiver operating characteristic (ROC) curve of PTX3.

 

 

DISCUSSION

 

Stroke is a world health problem with great direct and indirect cost on countries' economy. Over the last two decades, stroke has begun to be both preventable and treatable disease rather than a direct aging consequence that end with disability or death. This was achieved by better identification of people at risk of stroke and mechanisms that makes them susceptible for stroke or the likelihood of severe disability thereafter¹⁰.

Pentraxin-3 is one of the pentraxin family that is well-identified as acute inflammatory cardiovascular biomarker and was incriminated in the pathogenesis of acute coronary syndrome , congestive heart failure, sleep apnea syndrome, heart valvular disease, and atherosclerosis¹¹. Atherosclerosis exerts a state of chronic inflammation of the arterial wall and stroke itself leads to perpetuation of this inflammatory response that produce PTX-3 locally to prevent more platelet adhesion and stimulate antiapoptotic mechanism and help regeneration by mediating neurogenesis and angiogenesis¹². The aim of this study was to explore the role of PTX-3 in patients with cerebral stroke. We found significantly higher PTX-3 in all stroke patients whether hemorrhagic or ischemic type. Zanier et al has detected elevated PTX-3 in all subarachnoid hemorrhage in plasma and CSF with acute peak in the first 48 hours and another peak with the development of vasospasm¹³. Sezer et al. reported also significantly higher PTX-3 in patients with acute ischemic stroke when compared to control subjects¹⁴.

Our results showed that patients with hypertension, diabetes mellitus and hypercholestrolemia have higher PTX-3 level. This is in agreement with Parlak et al who beautifully investigated the relationship between systolic and diastolic blood pressure and PTX-3 level. They found higher PTX-3 level in both systolic and diastolic hypertension. Moreover, on regression analysis PTX-3 levels strongly affected blood pressure causing increase in both of them¹⁵. Also Karakas et al found a correlation between PTX-3 and metabolic syndrome and its level increase with increasing the number of metabolic syndrome criteria and severity¹⁶. Cigarette smoking increases pulmonary PTX-3 expression in an IL-1 dependant manner¹⁷. The later has an established role in the atherosclerosis state of chronic inflammation. Our results showed that cigarette smokers with ischemic stroke but not hemorrhagic one has higher PTX-3 when compared to ischemic stroke non-smokers.

A higher level of PTX-3 was found with severe stroke as measured clinically by NIHSS and by the infarct volume and type. This is in agreement with sezer et al (14) that showed similar trends and Ryu et al.¹⁸, who showed that higher levels of PTX-3 are independently associated with increased mortality.

This is the only study, to our knowledge, that comprised PTX-3 assessment in both hemorrhagic and ischemic stroke and shedding light on its role as a cause of severe forms of stroke; clinically and radiologically and its relationship with the common stroke risk factors. The limitations of our study were the small number of patients to explore different severity forms of stroke and also the fact that we didn't follow up the mortality and the long-term disability of patients with high PTX3 level.

More studies in this field are essential as PTX3 is emerging biomarker and a possible target for stroke prevention and treatment.

 

[Disclosure: Authors report no conflict of interest]

 

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