CLB-08811; No. of pages: 5; 4C: Clinical Biochemistry xxx (2014) xxx–xxx
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The association of inflammatory markers with cerebral vasoreactivity and carotid atherosclerosis in transient ischaemic attack Irena Martinic-Popovic a, Ana-Maria Simundic b, Lora Dukic b,⁎, Arijana Lovrencic-Huzjan a, Alek Popovic c, Vesna Seric a, Vanja Basic-Kes a, Vida Demarin a a b c
University Department of Neurology, Medical School University Hospital Sestre milosrdnice, Zagreb, Croatia University Department of Chemistry, Medical School University Hospital Sestre milosrdnice, Zagreb, Croatia University Department of Urology, Medical School University Hospital Sestre milosrdnice, Zagreb, Croatia
a r t i c l e
i n f o
Article history: Received 7 April 2014 Received in revised form 8 July 2014 Accepted 11 July 2014 Available online xxxx Keywords: Transient ischaemic attack Inflammation IL-6 CRP ICAM-1
a b s t r a c t Objectives: Inflammatory mediators have an important role in the pathogenesis of stroke. Increased activity of inflammatory mediators initiates the development of atherosclerosis independently of other risk factors, thus compromising brain microcirculation and causing transient ischaemic attack (TIA). The aim of our study was to evaluate the relationship between serum level of cellular adhesion molecules (ICAM-1), interleukin-6 (IL-6) and C-reactive protein (CRP) with carotid intima–media thickness (IMT) and breath-holding index (BHI) in subjects with transient ischaemic attack. We also aimed to assess the difference of those markers between TIA patients and disease-free control individuals. Design and methods: The study included 45 TIA patients and 36 disease-free controls matched for age, gender and vascular risk profile. The degree of carotid atherosclerosis was assessed by colour Doppler with measurements of carotid IMT. Transcranial Doppler (TCD) ultrasound was performed in order to assess BHI. IMT, TCD, BHI and serum concentrations of ICAM-1, IL-6, and CRP were measured for all study subjects. Results: Inflammatory markers IL-6, ICAM-1 and CRP were significantly higher in TIA patients than in disease-free controls (P b 0.001, P = 0.026, P b 0.001, respectively). TIA patients had significantly lower values of BHI and higher IMT relative to disease-free control individuals (P b 0.001). Conclusions: TIA is associated with higher ICAM-1, IL-6 and CRP, pointing to the marked inflammatory response to cerebral ischaemia. Inflammatory markers are associated with higher IMT and lower BHI, indicating the insufficient cerebral perfusion due to the underlying atherosclerotic disease. Our findings highlight the key significance of inflammation in the early response to ischaemia during the transitory ischaemic episode. © 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction Transient ischaemic attack (TIA) is traditionally defined as a brief episode of neurological dysfunction caused by focal ischaemia of the brain or the retina, with clinical symptoms lasting less than 1 h and without any objective evidence of acute infarction in the affected region of the brain or the retina [1,2]. The estimated prevalence of TIA in USA is approximately 5 million people [3]. According to recently proposed new concept termed acute cerebrovascular syndrome, acute TIA and acute ischaemic stroke can be considered as two entities within the same spectrum of acute ischaemic syndrome in the central nervous system [4]. TIA is a medical emergency and needs immediate evaluation and proper management. Although patients affected by TIA are well known to be at an increased risk of stroke and other severe adverse ⁎ Corresponding author at: University Department of Chemistry, Medical School University Hospital Sestre milosrdnice, Vinogradska c. 29, Zagreb 10000, Croatia. Tel./fax: + 385 1 3768280. E-mail address: [email protected] (L. Dukic).
vascular events, due to the underlying atherothrombotic disease, TIA is unfortunately still often managed without urgency [2,5]. This is probably due to the low level of the public awareness and knowledge of stroke definition, risk factors and symptoms [6,7]. Furthermore, one additional reason for the suboptimal quality of the evaluation and immediate management of patients with acute cerebrovascular ischaemic syndrome is probably due to the difficulty to make a confident and reliable diagnosis of acute ischaemic stroke and TIA [8,9]. Moreover, one of the limitations in the evaluation and management of patients presenting with symptoms indicative for acute cerebral ischaemia is the absence of a rapid and widely available laboratory based assay with high diagnostic sensitivity and specificity. Since diagnostic approach for the evaluation of acute cerebrovascular incident still mostly relies on clinical symptoms corroborated with patient history data and neuroimaging techniques, there is an ongoing search for an alternative strategy based on the evaluation of circulating serum biomarkers of cerebral tissue injury. Inflammatory mediators are nowadays acknowledged as key players in the development of cardiovascular and cerebrovascular
http://dx.doi.org/10.1016/j.clinbiochem.2014.07.010 0009-9120/© 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Please cite this article as: Martinic-Popovic I, et al, The association of inflammatory markers with cerebral vasoreactivity and carotid atherosclerosis in transient ischaemic attack, Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.07.010
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diseases [10,11]. Recent studies, mostly performed on patients with ischaemic stroke, have showed that molecular adhesion and cytokines production occur in early stages of brain ischaemia [12–17]. While the association of inflammatory markers with acute ischaemic stroke has been extensively studied for the last several years, the association between transient cerebral ischaemic events and inflammatory markers has not yet been confirmed. Since stroke and TIA, as already stated above, are proposed to share identical pathophysiology and symptoms of varying duration and intensity within the so-called acute cerebrovascular syndrome, one can expect that inflammatory response to TIA resembles the one in acute ischaemic stroke. We have therefore hypothesised that there is a measurable early biomarker response to cerebral ischaemia in patients with transient ischaemic attack. Furthermore, since inflammatory markers reflect the underlying atherosclerotic disease, we further hypothesised that a degree of vascular atherosclerotic changes would be associated with more pronounced inflammatory response. The aim of this case–control study was to assess the relationship between IL-6, CRP and ICAM-1 serum levels with carotid intima–media thickness (IMT) and cerebral vasoreactivity (evaluated by breathholding index technique) in subjects with transient ischaemic attack, within 12 h from the onset of symptoms. We also aimed to assess the differences of those markers between TIA patients and disease-free control individuals. Materials and methods Patient recruitment This study was done in the University Department of Neurology and University Institute of Chemistry at the Medical School University Hospital Sestre milosrdnice in Zagreb between September 2009 and March 2010. Enrolled were 50 consecutive patients diagnosed with first-ever TIA within 12 h from the occurrence of symptoms. Five patients were subsequently excluded (3 owing to the presence of malignancy and 2 due to the clinical signs of respiratory and urinary infection). A total of 45 TIA patients (median age 74; range 48–90 years) were finally included in the study. The control group consisted of 36 subjects matched for age, gender and vascular risk profile, hospitalized at the Department of Neurology for other neurological diagnoses with no clinical signs of respiratory, urinary or system infection. For both patients and controls, clinical history, detailed somatic and neurological status was noted and vascular risk factors were evaluated. Hypertension was defined as a systolic blood pressure of 140 mm Hg or higher and/or a diastolic blood pressure of 90 mm Hg or higher, or current treatment with antihypertensive drugs [18]. Diabetes mellitus was defined as a previous diagnosis of type I or type II diabetes, or at least two random glucose readings of N11.1 mmol/L or fasting blood glucose readings of N 7.0 mmol/L, or current use of standard oral blood sugarlowering drugs or insulin [19]. Hyperlipidemia was confirmed if total cholesterol concentration was 5.0 mmol/L or greater and a low density lipoprotein cholesterol concentration 3.0 mmol/L or higher [20]. Subjects were classified as having atrial fibrillation or being treated with anticoagulant therapy or statin on the basis of medical records. Participants were classified as smokers if they were current smokers or had quit cigarette smoking in the year before enrolment. All participants underwent brain computed tomography (CT; Sensation Multislice Computed Tomography scanner with 16-row detector layer, Siemens, Germany) which was interpreted by an expert neuroradiologist, blinded for other clinical and laboratory data. TIA was defined as a brief episode of neurological dysfunction caused by focal ischaemia of the brain or the retina, with clinical symptoms of less than 1 hour duration and without evidence of acute infarction on brain CT [1]. The study protocol was reviewed and approved by the Hospital Ethical Review Board (approval of Hospital Ethical Review Board, reference
number 40-1/2003) and written informed consent was obtained from all participants. Blood sampling and laboratory methods Venous blood samples were collected at admission for all patients included in the study. Blood was collected in serum tubes (Becton Dickinson, Franklin Lakes, NJ) and in the citrate tubes for the determination of erythrocyte sedimentation rate (ESR). Serum samples were centrifuged, on a bench top centrifuge, for 10 min at 1800 g. Venous blood glucose, total cholesterol, HDL-cholesterol, LDLcholesterol, triglycerides and CRP were determined for all participants in the study. Serum aliquots for determination of IL-6 and ICAM-1 were stored at − 20 °C until analysis. ESR was determined by the Wintergreen method. Biochemical analyses were performed on Beckman Coulter AU 2700 biochemical analyser (Beckman Coulter Inc., Brea, CA, USA), using the original Beckman Coulter reagents (Beckman Coulter Biomedical Limited, Co Clare, Ireland). LDL-cholesterol level was determined using the Friedewald formula for the triglyceride levels b3.0 mmol/L. For the patient samples with triglyceride levels N3.0 mmol/L, LDL-cholesterol test was determined by a direct cholesterol oxidase and cholesterol esterase assay, using the original Beckman Coulter reagents. Concentration of CRP was determined with high-sensitive application for CRP Latex Beckman Coulter immuno-turbidimetric assay. Linearity of the test was 0.08–80 mg/L and intra- and inter-assay precisions were 5.73% and 5.76% respectively for concentration of 0.23 mg/L. IL-6 concentration was determined by Roche electrochemiluminescence immunoassay (Roche Diagnostics GmbH, Mannheim, Germany) on Cobas e 411 analyser (Roche Diagnostics GmbH, Mannheim, Germany). Intra-assay and inter-assay precisions declared by the manufacturer were 2.5% and 3.2%, respectively. Serum concentration of ICAM-1 was measured using the enzymelinked immunosorbent Human sICAM-1 Platinum ELISA assay (Bender MedSystems GmbH, Vienna, Austria). According to the manufacturers' declaration, intra-assay and inter-assay coefficients of variation were 4.1% and 7.7%, respectively. QC results were within the acceptance limits for both IL-6 and ICAM-1. Extracranial colour Doppler ultrasound and IMT assessment In all study subjects colour Doppler flow imaging (CDFI) of both carotid and vertebral arteries (high resolution B-mode, colour Doppler, and pulse Doppler ultrasonography) was performed with a ProSound SSD Alpha 10 system (Aloka Co., Ltd., Tokyo, Japan) with linear 10 MHz transducer according to well defined procedure and diagnostic protocol [21]. Colour Doppler findings of carotid arteries were classified according to the degree of atherosclerosis: 0 — normal findings–no atherosclerotic changes, 1 — mild stenosis (30–40%), 2 — moderate stenosis (50–60%), 3 — severe stenosis (70–99%) and 4 — carotid occlusion. Colour Doppler findings of vertebral arteries were classified as positive if vertebral stenosis or occlusion was present. After the carotid arteries were examined, the probe was rotated 90° to obtain a longitudinal image of the anterior and posterior vessel walls. The maximum IMT was measured according to the protocol at the near and far walls of the common carotid artery, the bifurcation, and the internal carotid arteries and was expressed as a mean aggregate value (mean-IMT) [22]. Transcranial Doppler sonography and BHI All subjects underwent TCD assessment of intracranial vessels using TCD DWL Multidrop X4 instrument (ScanMed Medical, Gloucestershire, United Kingdom) with 2 MHz hand-held pulsed-wave probe. TCD examination of vessels of the circle of Willis and vertebrobasilar system was performed bilaterally, in supine position, in a quiet room, after 5 min of bed rest. Cerebrovascular reactivity to hypercapnia was
Please cite this article as: Martinic-Popovic I, et al, The association of inflammatory markers with cerebral vasoreactivity and carotid atherosclerosis in transient ischaemic attack, Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.07.010
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evaluated by means of BHI, with mean blood flow velocity (BFV) of middle cerebral artery (MCA) being continuously monitored during the test [23,24]. After an initial 5 minute resting period, a continuous mean BFV during the next 30 s were noted as baseline BFV value (vmean). After normal inspiratory breath, subjects were asked to hold their breath for 30 s and mean BFV of the last 3 s of breath holding period were recorded (vmax). The same procedure was repeated after 2 min. BHI was calculated as the percentage increase in mean BFV occurring during breath holding divided by the time (seconds) for which the subject hold his/her breath [23]. Statistical analysis Qualitative data were presented as counts and proportions. Quantitative variables were assessed for normality using the Kolmogorov– Smirnov test. Normally distributed variables were described with mean and standard deviation. Data sets which were not distributed normally, were described with median and interquartile range (IQR). Age was presented as median and range (min–max). Relative differences in frequencies between groups were assessed by Chi-square test [25]. If the data were normally distributed and if sample size was ≥30, t-test was used for testing the statistical significance between two groups. Otherwise (small sample (N b 30) or if data are not normally distributed), Mann–Whitney test was used. The difference among 3 and more groups was tested with Kruskal–Wallis test [26]. Spearman correlation was used to test the strength of association between variables. Logistic regression analysis was done to identify most significant predictors of acute transitory ischaemia. All variables which were statistically significant in univariate regression, were tested in multivariate model, by stepwise method. Level of significance was set at b0.05. Statistical analysis was done using the MedCalc Statistical software version 12.1.4.0 (MedCalc Software, Mariakerke, Belgium). Results Demographic profile, vascular risk factors, CDFI findings of carotid and vertebral arteries, neurosonological parameters, blood glucose and lipid profile in patients and controls are presented in Table 1. Patients with TIA and disease-free controls did not differ significantly relative to the different demographic characteristics and traditional vascular risk factors. Colour Doppler findings of vertebral arteries have demonstrated that steno-occlusive changes were more common in TIA patients than in disease-free controls. This was particularly observed in right carotid artery (P = 0.030). In both, right and left carotid artery, there were 4 patients with complete carotid occlusion, whereas such findings were not observed in disease-free controls. The advanced atherosclerotic changes in TIA patients were further confirmed using carotid intima–media thickness (IMT) and cerebral vasoreactivity (evaluated by breathholding index technique) measurements. In TIA patients, IMT and BHI findings suggested advanced atherosclerosis, as opposed to diseasefree controls. IMT was significantly higher in TIA patients than in disease-free controls (P b 0.001). Due to the increased thickness of the temporal bones, TCD and subsequent BHI calculations were performed only in 24/45 (0.53) patients and in 22/36 (0.61) disease-free controls. BHI values were significantly lower in patients with TIA (P b 0.001). Inflammatory markers, in TIA patients and disease-free controls are presented in Table 2. Three (CRP, ICAM-1 and IL-6) out of four inflammatory markers were significantly higher in TIA patients compared to disease-free controls. IL-6 was the only inflammatory marker associated with both BHI and IMT values in TIA patients (Table 3). All inflammatory and endothelial markers and BHI, IMT were identified as significant predictors of TIA by univariate logistic regression analysis. When all variables which were statistically significant in
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Table 1 Demographic data, neurosonological parameters and CDFI findings of carotid and vertebral arteries in TIA patients and disease-free controls. P
Patients (N = 45)
Disease-free controls (N = 36)
74 (48–90)
69 (55–85)
0.203
22 (0.49) 40 (0.89) 17 (0.38) 8 (0.18) 8 (0.18) 3 (0.06)
15 (0.42) 29 (0.81) 6 (0.16) 18 (0.5) 5 (0.14) 3 (0.08)
0.686 0.486 0.053 0.152 0.951 0.929
19 (0.42) 5.6 (5.05–6.73) 6.6 (5.7–8.3) 1.1 (1.1–1.4) 3.8 ± 1.1 1.8 (1.8–2.4) 1.22 ± 0.17 (N = 45) 0.63 (0.54–0.89) (N = 24)
21 (0.58) 0.227 5.6 ± 1.3 0.485 6.05 (5.05–6.8) 0.028 1.2 (1.1–1.6) 0.993 3.4 ± 0.96 0.101 1.8 (1.2–2.3) 0.845 0.86 ± 0.19 b0.001 (N = 36) 1.2 (0.80–1.48) b0.001 (N = 22)
Left ICA 0 (N/proportion) 1 (N/proportion) 2 (N/proportion) 3 (N/proportion) 4 (N/proportion)
33 (0.73) 5 (0.11) 3 (0.06) 0 (0) 4 (0.08)
27 (0.75) 7 (0.19) 2 (0.05) 0 (0) 0 (0)
0.259
Right ICA 0 (N/proportion) 1 (N/proportion) 2 (N/proportion) 3 (N/proportion) 4 (N/proportion) VA occlusion (N/proportion)
34 (0.75) 2 (0.04) 3 (0.06) 2 (0.04) 4 (0.08) 7 (0.15)
30 (0.83) 6 (0.16) 0 (0) 0 (0) 0 (0) 3 (0.08)
0.030
Age/years [median (range)] Females (N/proportion) Arterial hypertension (N/proportion) Diabetes mellitus (N/proportion) Statin therapy (N/proportion) Atrial fibrillation (N/proportion) Anticoagulant therapy (N/proportion) Smoking (N/proportion) Total cholesterol (mmol/L) Blood glucose (mmol/L) HDL cholesterol (mmol/L) LDL cholesterol (mmol/L) Triglycerides (mmol/L) IMT/mm (mean ± SD) BHI/%/s (median (Q1–Q3))
0.534
0 — no stenosis; 1 — mild stenosis (30–49%); 2 — moderate stenosis (50–69%); 3 — severe stenosis (70–99%) and 4 — carotid occlusion.
univariate regression were tested in multivariate model, only IMT and BHI remained statistically significant predictors of TIA (P = 0.018 and 0.254). However, after adjusting the model for inflammatory and endothelial markers none of the investigated variables have remained significant (P = 0.078 and 0.263). Discussion With this study we have demonstrated that transient ischaemic attack is associated with substantially higher concentrations of soluble adhesion molecule ICAM-1, pro-inflammatory cytokine IL-6 and CRP, compared to disease-free controls, suggesting a marked inflammatory response to cerebral ischaemia. The presence of inflammatory markers was associated with higher IMT and lower BHI, indicating the insufficient cerebral perfusion in those individuals mostly due to the underlying atherosclerotic disease. Our findings highlight the key significance of inflammation in the early response to ischaemia during the transitory ischaemic episode.
Table 2 Inflammatory markers in TIA patients and disease-free controls.
CRP (mg/L) ESR (mm/h) IL-6 (pg/mL) ICAM-1 (ng/mL)
Patients (N = 45)
Disease-free controls (N = 36)
P
8.5 (6.7–14.9) 14 (7.75–27.5) 6.76 (2.72–18.3) 371.6 ± 161.8
4.5 (2.8–5.8) 8.5 (5.0–13.1) 2.46 (1.5–4.61) 301.9 ± 91.6
b0.001 0.054 b0.001 0.026
Please cite this article as: Martinic-Popovic I, et al, The association of inflammatory markers with cerebral vasoreactivity and carotid atherosclerosis in transient ischaemic attack, Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.07.010
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Table 3 Correlations of inflammatory and neurosonological parameters in TIA patients.
BHI r (p) IMT r (p) Total cholesterol r (p) Blood glucose r (p) HDL cholesterol r (p) LDL cholesterol r (p) Triglycerides r (p) CRP r (p) IL-6 r (p) ICAM-1 r (p)
IL-6
ICAM-1
CRP
ESR
−0.419 (0.041) 0.334 (0.025) −0.418 (0.004) ns ns −0.405 (0.005) ns 0.394 (0.007) NA NA
ns ns ns ns ns ns ns −0.049 (0.668) 0.030 (0.790) NA
ns 0.317 (0.034) ns ns ns ns ns NA −0.005 (0.966) NA
ns 0.474 (0.001) ns ns ns ns ns 0.532 (b0.001) 0.239 (0.036) 0.290 (0.011)
ns—not significant. NA—not applicable.
Whereas CRP increase has already been demonstrated, our study is, to the best of our knowledge, the first one to show the evidence for the presence of elevated levels of ICAM-1 and IL-6 in TIA patients. There are only a few laboratory tests which are of some use in diagnosis of patients with TIA. Laboratory workup is usually done to exclude some common TIA mimics. Most common laboratory assays employed in TIA laboratory workup are glucose, electrolytes, complete blood count and coagulation tests for detecting hypoglycaemia, metabolic disorders and some prothrombotic hypercoagulable states, respectively [27]. Currently, laboratory assays are used neither for diagnosis nor for prognosis of TIA. Nevertheless, the evidence that support the increase of neutrophil and monocyte count, lipoprotein-associated phospholipase A(2), prothrombotic markers, NT-proBNP and some other molecules in the early hours in TIA patients exist [28–31]. However, as no single blood marker has so far been shown to improve the diagnostic performance of commonly used clinical stroke scales, it seems possible that a panel of biomarkers could eventually provide aid in diagnosis of TIA. Several markers have been demonstrated as having potentially predictive or prognostic significance in TIA patients. Serum copeptin has also proven to be predictive of the stroke subsequent to first-ever TIA [32]. Elevated levels of serum hsCRP have also been shown to predict further ischaemic events following TIA [33]. On the other hand, Selvarajah and colleagues have not been able to show the predictive role of CRP and IL-6 for the recurrence of stroke or other vascular events in patients suffering from transient ischaemic attack [34]. Hence, it remains to be assessed whether routine copeptin or CRP measurement could be a useful tool for identifying TIA patients at increased risk of developing subsequent cerebrovascular ischaemic events, either repeated TIAs or stroke. In our study IL-6 was associated with CRP and ESR. Moreover, ESR was associated with CRP, IL-6 and ICAM-1. This shows that additionally to the increased basal levels of inflammatory markers, there probably occurs an immediate increase of IL-6 triggered by the acute ischaemia and subsequently of all other investigated downstream inflammatory markers. Serum biomarkers able to detect cerebral ischaemia are not yet available and the search is ongoing. Several recent lines of evidence clearly indicate that biomarkers might be identified somewhere upstream or downstream the pathophysiological events prior to or subsequent to cerebral ischaemia [35]. Zhan and colleagues have explored gene expression profiles in the circulation of TIA patients using the Affymetrix microarray technology [36]. They have found 449 genes differentially expressed between TIA patients (N = 26) and controls (N = 26), out of which 34 genes have been able to discriminate TIA patients from controls with 100% sensitivity and 100% specificity. Identified genes were associated with systemic inflammation, platelet activation, and prothrombin activation. This is in line with our observations of the higher levels of IL-6 and CRP in TIA patients, compared to controls.
So far the increased levels of ICAM-1 have not yet been reported in TIA. ICAM-1 was found to be increased in acute stroke and to be associated with stroke severity and infarct volume [13,37], whereas Frijns et al. have found that P- and E-selectin, but not ICAM-1 and VCAM-1, are increased in patients with ischaemic stroke and carotid stenosis [38]. Increased concentration of ICAM-1 in TIA patients indicates endothelial changes and adhesion molecule activation early in the course of ischaemic cerebral event. In our study, IMT and BHI findings in TIA patients were suggesting advanced atherosclerosis. TIA patients had significantly higher IMT and lower BHI compared to disease-free controls. Moreover, IL-6 proven to be associated with both BHI and IMT values. High carotid IMT and low BHI are commonly described as vascular risk markers defining the individual susceptibility to unfavourable cerebrovascular events [39,40]. With our study we provide additional evidence for clustering of inflammatory mediators and impaired microcirculation in patients suffering from TIA. The question remains whether the level of inflammatory markers could possibly be used in assessing the short-term stroke risk following TIA. Some future larger studies should be performed to evaluate the possible association of the level of inflammatory markers with the severity of TIA and subsequent short-term stroke risk. Nevertheless, if proven to be useful, inflammatory markers could be of great assistance in evaluation of patients suspected for TIA who should undergo immediate etiologic workup and treatment. Limitations Some limitations of our study should be acknowledged. First, the size of the studied group is limited, and the results of such analyses should be interpreted with caution. Brain CT was performed only in TIA patients on inclusion, and clinical follow-up of patients with TIA for possible stroke was not done.
Conclusions Transient ischaemic attack is associated with higher sICAM-1, IL-6 and CRP, pointing to the marked inflammatory response to cerebral ischaemia. Inflammatory markers are associated with higher IMT and lower BHI, indicating the insufficient cerebral perfusion due to the underlying atherosclerotic disease. Our findings highlight the key significance of inflammation in the early response to cerebral ischaemia during the transitory ischaemic episode. Acknowledgments This study was supported by Ministry of Science, Education and Sports, Republic of Croatia, projects #134-1340227-0200, #1341340036-0034, #134-1340036-0035 and # 022-1340036-2083.
Please cite this article as: Martinic-Popovic I, et al, The association of inflammatory markers with cerebral vasoreactivity and carotid atherosclerosis in transient ischaemic attack, Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.07.010
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References [1] Easton JD, Saver JL, Albers GW, Alberts MJ, Chaturvedi S, Feldmann E, et al. The American Academy of Neurology affirms the value of this statement as an educational tool for neurologists. Stroke 2009;40:2276–93. [2] Gállego J, Muñoz R, Martínez-Vila E. Emergent cerebrovascular disease risk factor weighting: is transient ischemic attack an imminent threat? Cerebrovasc Dis 2009; 27(Suppl. 1):88–96. [3] Lloyd-Jones D, Adams RJ, Brown TM, et al. Heart disease and stroke statistics—2010 update: a report from the American Heart Association. Circulation 2010;121:e46-215. [4] Uchiyama S. The concept of acute cerebrovascular syndrome. Front Neurol Neurosci 2014;33:11–8. [5] Lovrenčić-Huzjan A, Strineka M, Šodec-Šimičević D, et al. Management of patients with transient ischemic attack (TIA) at Sestre milosrdnice University Hospital Center. Acta Clin Croat 2011;50:367–73. [6] Hong KS, Bang OY, Kim JS, Heo JH, Yu KH, Bae HJ, et al. Stroke statistics in Korea: part II stroke awareness and acute stroke care, a report from the Korean stroke society and clinical research center for stroke. J Stroke 2013;15:67–77. [7] Soulleihet V, Nicoli F, Trouve J, Girard N, Jacquin L. Optimized acute stroke pathway using medical advanced regulation for stroke and repeated public awareness campaigns. Am J Emerg Med 2014;32:225–32. [8] Barber PA, Zhang J, Demchuk AM, Hill MD, Buchan AM. Why are stroke patients excluded from tPA therapy? An analysis of patient eligibility. Neurology 2001; 56:1015–20. [9] Castle J, Mlynash M, Lee K, Caulfield AF, Wolford C, Kemp S, et al. Agreement regarding diagnosis of transient ischemic attack fairly low among stroke-trained neurologists. Stroke 2010;41:1367–70. [10] Ross R. Atherosclerosis: an inflammatory disease. N Engl J Med 1999;340:115–26. [11] Faxon DP, Fuster V, Libby P, Beckman JA, Hiatt WR, Thompson WR, et al. Atherosclerotic vascular disease conference: writing group III: pathophysiology. Circulation 2004;109:2617–25. [12] Xia W, Han J, Huang G, Ying W. Inflammation in ischemic brain injury: current advances and future perspectives. Clin Exp Pharmacol Physiol 2010;37:253–8. [13] Simundic AM, Basic V, Topic E, Demarin V, Vrkic N, Kunovic B, et al. Soluble adhesion molecules in acute ischemic stroke. Clin Invest Med 2004;27:86–92. [14] Vila N, Castillo J, Davalos A, Esteve A, Planas AM, Chamorro A, et al. Levels of antiinflammatory cytokines and neurological worsening in acute ischemic stroke. Stroke 2003;34:671–5. [15] Domac FM, Somay G, Misirli H, Erenoglu NY. Tumor necrosis factor alpha serum levels and inflammatory response in acute ischemic stroke. Neurosciences 2007; 12:25–30. [16] Zhu XD, Chen JS, Zhou F, Liu QC, Chen G, Zhang JM. Detection of copeptin in peripheral blood of patients with aneurysmal subarachnoid hemorrhage. Crit Care 2011; 15:R288. [17] Dobsa L, Cullen Edozien K. Copeptin and its potential role in diagnosis and prognosis of various diseases. Biochem Med 2013;23:172–92. [18] Chobanian AV, Bakris GL, Black HR, Cushman WC, Green LA, Izzo Jr JL, et al. Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure. National Heart, Lung, and Blood Institute; National High Blood Pressure Education Program Coordinating Committee. Seventh report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure. Hypertension 2003;42:1206–52. [19] Expert committee on the diagnosis, classification of diabetes mellitus. Report of the expert committee on the diagnosis and classification of diabetes mellitus. Diabetes Care 1997;20:1183–97.
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[20] Grundy SM, Cleeman JI, Merz CN, Brewer HB, Clark LT, Hunninghake DB, et al. Implications of recent clinical trials for the National Cholesterol Education Program Adult Treatment Panel III guidelines. Circulation 2004;110:227–39. [21] Lovrenčić-Huzjan A, Vuković V, Demarin V. Neurosonology in stroke. Acta Clin Croat 2006;45:385–401. [22] Silvestrini M, Cagnetti C, Pasqualetti P, Albanesi A, Altamura C, Lanciotti C, et al. Carotid wall thickness and stroke risk in patients with asymptomatic internal carotid stenosis. Atherosclerosis 2010;210:452–7. [23] Markus HS, Harrison MJG. Estimation of cerebrovascular reactivity using transcranial Doppler, including the use of breath-holding as the vasodilatory stimulus. Stroke 1992;23:668–73. [24] Tang SC, Huang YW, Shieh JS, Huang SJ, Yip PK, Jeng JS. Dynamic cerebral autoregulation in carotid stenosis before and after carotid stenting. J Vasc Surg 2008;48:88–92. [25] McHugh ML. The Chi-square test of independence. Biochem Med 2013;23:125–31. [26] Simundic AM. Practical recommendations for statistical analysis and data presentation in Biochemia Medica journal. Biochem Med 2012;22:15–23. [27] Siket MS, Edlow J. Transient ischemic attack: an evidence-based update. Emerg Med Pract 2013;15:1–28. [28] Ross AM, Hurn P, Perrin N, Wood L, Carlini W, Potempa K. Evidence of the peripheral inflammatory response in patients with transient ischemic attack. J Stroke Cerebrovasc Dis 2007;16:203–7. [29] Cucchiara BL, Messe SR, Sansing L, MacKenzie L, Taylor RA, Pacelli J, et al. Lipoproteinassociated phospholipase A2 and C-reactive protein for risk-stratification of patients with TIA. Stroke 2009;40:2332–6. [30] Prodan CI, Vincent AS, Dale GL. Coated-platelet levels are elevated in patients with transient ischemic attack. Transl Res 2011;158:71–5. [31] Whiteley W, Wardlaw J, Dennis M, Lowe G, Rumley A, Sattar N, et al. Blood biomarkers for the diagnosis of acute cerebrovascular diseases: a prospective cohort study. Cerebrovasc Dis 2011;32:141–7. [32] Katan M, Nigro N, Fluri F, Schuetz P, Morgenthaler NG, Jax F, et al. Stress hormones predict cerebrovascular re-events after transient ischemic attacks. Neurology 2011; 76:563–6. [33] Purroy F, Montaner J, Molina CA, Delgado P, Arenillas JF, Chacon P, et al. C-reactive protein predicts further ischemic events in transient ischemic attack patients. Acta Neurol Scand 2007;115:60–6. [34] Selvarajah JR, Smith CJ, Hulme S, Georgiou R, Sherrington C, Staniland J, et al. Does inflammation predispose to recurrent vascular events after recent transient ischaemic attack and minor stroke? The North West of England transient ischaemic attack and minor stroke (NORTHSTAR) study. Int J Stroke 2011;6:187–94. [35] Cucchiara B, Nyquist P. Blood markers in TIA: array of hope? Neurology 2011; 77:1716–7. [36] Zhan X, Jickling GC, Tian Y, Stamova B, Xu H, Ander BP, et al. Transient ischemic attacks characterized by RNA profiles in blood. Neurology 2011;77:1718–24. [37] Clark WM, Coull BM, Briley DP, Mainolfi E, Rothlein R. Circulating intercellular adhesion molecule-1 levels and neutrophil adhesion in stroke. J Neuroimmunol 1993;44:123–5. [38] Frijns CJ, Kappelle LJ, van Gijn J, Nieuwenhuis HK, Sixma JJ, Fijnheer R. Soluble adhesion molecules reflect endothelial cell activation in ischemic stroke and in carotid atherosclerosis. Stroke 1997;28:2214–8. [39] Buratti L, Viticchi G, Falsetti L, Cagnetti C, Luzzi S, Bartolini M, et al. Vascular impairment in Alzheimer's disease: the role of obstructive sleep apnea. J Alzheimers Dis 2014;38:445–53. http://dx.doi.org/10.3233/JAD-131046. [40] Rodríguez-Flores M, García-García E, Cano-Nigenda CV, Cantú-Brito C. Relationship of obesity and insulin resistance with the cerebrovascular reactivity: a case control study. Cardiovasc Diabetol 2014;13:2. http://dx.doi.org/10.1186/1475-2840-13-2.
Please cite this article as: Martinic-Popovic I, et al, The association of inflammatory markers with cerebral vasoreactivity and carotid atherosclerosis in transient ischaemic attack, Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.07.010
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The association of inflammatory markers with cerebral vasoreactivity and carotid atherosclerosis in transient ischaemic attack Irena Martinic-Popovic a, Ana-Maria Simundic b, Lora Dukic b,⁎, Arijana Lovrencic-Huzjan a, Alek Popovic c, Vesna Seric a, Vanja Basic-Kes a, Vida Demarin a a b c
University Department of Neurology, Medical School University Hospital Sestre milosrdnice, Zagreb, Croatia University Department of Chemistry, Medical School University Hospital Sestre milosrdnice, Zagreb, Croatia University Department of Urology, Medical School University Hospital Sestre milosrdnice, Zagreb, Croatia
a r t i c l e
i n f o
Article history: Received 7 April 2014 Received in revised form 8 July 2014 Accepted 11 July 2014 Available online xxxx Keywords: Transient ischaemic attack Inflammation IL-6 CRP ICAM-1
a b s t r a c t Objectives: Inflammatory mediators have an important role in the pathogenesis of stroke. Increased activity of inflammatory mediators initiates the development of atherosclerosis independently of other risk factors, thus compromising brain microcirculation and causing transient ischaemic attack (TIA). The aim of our study was to evaluate the relationship between serum level of cellular adhesion molecules (ICAM-1), interleukin-6 (IL-6) and C-reactive protein (CRP) with carotid intima–media thickness (IMT) and breath-holding index (BHI) in subjects with transient ischaemic attack. We also aimed to assess the difference of those markers between TIA patients and disease-free control individuals. Design and methods: The study included 45 TIA patients and 36 disease-free controls matched for age, gender and vascular risk profile. The degree of carotid atherosclerosis was assessed by colour Doppler with measurements of carotid IMT. Transcranial Doppler (TCD) ultrasound was performed in order to assess BHI. IMT, TCD, BHI and serum concentrations of ICAM-1, IL-6, and CRP were measured for all study subjects. Results: Inflammatory markers IL-6, ICAM-1 and CRP were significantly higher in TIA patients than in disease-free controls (P b 0.001, P = 0.026, P b 0.001, respectively). TIA patients had significantly lower values of BHI and higher IMT relative to disease-free control individuals (P b 0.001). Conclusions: TIA is associated with higher ICAM-1, IL-6 and CRP, pointing to the marked inflammatory response to cerebral ischaemia. Inflammatory markers are associated with higher IMT and lower BHI, indicating the insufficient cerebral perfusion due to the underlying atherosclerotic disease. Our findings highlight the key significance of inflammation in the early response to ischaemia during the transitory ischaemic episode. © 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction Transient ischaemic attack (TIA) is traditionally defined as a brief episode of neurological dysfunction caused by focal ischaemia of the brain or the retina, with clinical symptoms lasting less than 1 h and without any objective evidence of acute infarction in the affected region of the brain or the retina [1,2]. The estimated prevalence of TIA in USA is approximately 5 million people [3]. According to recently proposed new concept termed acute cerebrovascular syndrome, acute TIA and acute ischaemic stroke can be considered as two entities within the same spectrum of acute ischaemic syndrome in the central nervous system [4]. TIA is a medical emergency and needs immediate evaluation and proper management. Although patients affected by TIA are well known to be at an increased risk of stroke and other severe adverse ⁎ Corresponding author at: University Department of Chemistry, Medical School University Hospital Sestre milosrdnice, Vinogradska c. 29, Zagreb 10000, Croatia. Tel./fax: + 385 1 3768280. E-mail address: [email protected] (L. Dukic).
vascular events, due to the underlying atherothrombotic disease, TIA is unfortunately still often managed without urgency [2,5]. This is probably due to the low level of the public awareness and knowledge of stroke definition, risk factors and symptoms [6,7]. Furthermore, one additional reason for the suboptimal quality of the evaluation and immediate management of patients with acute cerebrovascular ischaemic syndrome is probably due to the difficulty to make a confident and reliable diagnosis of acute ischaemic stroke and TIA [8,9]. Moreover, one of the limitations in the evaluation and management of patients presenting with symptoms indicative for acute cerebral ischaemia is the absence of a rapid and widely available laboratory based assay with high diagnostic sensitivity and specificity. Since diagnostic approach for the evaluation of acute cerebrovascular incident still mostly relies on clinical symptoms corroborated with patient history data and neuroimaging techniques, there is an ongoing search for an alternative strategy based on the evaluation of circulating serum biomarkers of cerebral tissue injury. Inflammatory mediators are nowadays acknowledged as key players in the development of cardiovascular and cerebrovascular
http://dx.doi.org/10.1016/j.clinbiochem.2014.07.010 0009-9120/© 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Please cite this article as: Martinic-Popovic I, et al, The association of inflammatory markers with cerebral vasoreactivity and carotid atherosclerosis in transient ischaemic attack, Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.07.010
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diseases [10,11]. Recent studies, mostly performed on patients with ischaemic stroke, have showed that molecular adhesion and cytokines production occur in early stages of brain ischaemia [12–17]. While the association of inflammatory markers with acute ischaemic stroke has been extensively studied for the last several years, the association between transient cerebral ischaemic events and inflammatory markers has not yet been confirmed. Since stroke and TIA, as already stated above, are proposed to share identical pathophysiology and symptoms of varying duration and intensity within the so-called acute cerebrovascular syndrome, one can expect that inflammatory response to TIA resembles the one in acute ischaemic stroke. We have therefore hypothesised that there is a measurable early biomarker response to cerebral ischaemia in patients with transient ischaemic attack. Furthermore, since inflammatory markers reflect the underlying atherosclerotic disease, we further hypothesised that a degree of vascular atherosclerotic changes would be associated with more pronounced inflammatory response. The aim of this case–control study was to assess the relationship between IL-6, CRP and ICAM-1 serum levels with carotid intima–media thickness (IMT) and cerebral vasoreactivity (evaluated by breathholding index technique) in subjects with transient ischaemic attack, within 12 h from the onset of symptoms. We also aimed to assess the differences of those markers between TIA patients and disease-free control individuals. Materials and methods Patient recruitment This study was done in the University Department of Neurology and University Institute of Chemistry at the Medical School University Hospital Sestre milosrdnice in Zagreb between September 2009 and March 2010. Enrolled were 50 consecutive patients diagnosed with first-ever TIA within 12 h from the occurrence of symptoms. Five patients were subsequently excluded (3 owing to the presence of malignancy and 2 due to the clinical signs of respiratory and urinary infection). A total of 45 TIA patients (median age 74; range 48–90 years) were finally included in the study. The control group consisted of 36 subjects matched for age, gender and vascular risk profile, hospitalized at the Department of Neurology for other neurological diagnoses with no clinical signs of respiratory, urinary or system infection. For both patients and controls, clinical history, detailed somatic and neurological status was noted and vascular risk factors were evaluated. Hypertension was defined as a systolic blood pressure of 140 mm Hg or higher and/or a diastolic blood pressure of 90 mm Hg or higher, or current treatment with antihypertensive drugs [18]. Diabetes mellitus was defined as a previous diagnosis of type I or type II diabetes, or at least two random glucose readings of N11.1 mmol/L or fasting blood glucose readings of N 7.0 mmol/L, or current use of standard oral blood sugarlowering drugs or insulin [19]. Hyperlipidemia was confirmed if total cholesterol concentration was 5.0 mmol/L or greater and a low density lipoprotein cholesterol concentration 3.0 mmol/L or higher [20]. Subjects were classified as having atrial fibrillation or being treated with anticoagulant therapy or statin on the basis of medical records. Participants were classified as smokers if they were current smokers or had quit cigarette smoking in the year before enrolment. All participants underwent brain computed tomography (CT; Sensation Multislice Computed Tomography scanner with 16-row detector layer, Siemens, Germany) which was interpreted by an expert neuroradiologist, blinded for other clinical and laboratory data. TIA was defined as a brief episode of neurological dysfunction caused by focal ischaemia of the brain or the retina, with clinical symptoms of less than 1 hour duration and without evidence of acute infarction on brain CT [1]. The study protocol was reviewed and approved by the Hospital Ethical Review Board (approval of Hospital Ethical Review Board, reference
number 40-1/2003) and written informed consent was obtained from all participants. Blood sampling and laboratory methods Venous blood samples were collected at admission for all patients included in the study. Blood was collected in serum tubes (Becton Dickinson, Franklin Lakes, NJ) and in the citrate tubes for the determination of erythrocyte sedimentation rate (ESR). Serum samples were centrifuged, on a bench top centrifuge, for 10 min at 1800 g. Venous blood glucose, total cholesterol, HDL-cholesterol, LDLcholesterol, triglycerides and CRP were determined for all participants in the study. Serum aliquots for determination of IL-6 and ICAM-1 were stored at − 20 °C until analysis. ESR was determined by the Wintergreen method. Biochemical analyses were performed on Beckman Coulter AU 2700 biochemical analyser (Beckman Coulter Inc., Brea, CA, USA), using the original Beckman Coulter reagents (Beckman Coulter Biomedical Limited, Co Clare, Ireland). LDL-cholesterol level was determined using the Friedewald formula for the triglyceride levels b3.0 mmol/L. For the patient samples with triglyceride levels N3.0 mmol/L, LDL-cholesterol test was determined by a direct cholesterol oxidase and cholesterol esterase assay, using the original Beckman Coulter reagents. Concentration of CRP was determined with high-sensitive application for CRP Latex Beckman Coulter immuno-turbidimetric assay. Linearity of the test was 0.08–80 mg/L and intra- and inter-assay precisions were 5.73% and 5.76% respectively for concentration of 0.23 mg/L. IL-6 concentration was determined by Roche electrochemiluminescence immunoassay (Roche Diagnostics GmbH, Mannheim, Germany) on Cobas e 411 analyser (Roche Diagnostics GmbH, Mannheim, Germany). Intra-assay and inter-assay precisions declared by the manufacturer were 2.5% and 3.2%, respectively. Serum concentration of ICAM-1 was measured using the enzymelinked immunosorbent Human sICAM-1 Platinum ELISA assay (Bender MedSystems GmbH, Vienna, Austria). According to the manufacturers' declaration, intra-assay and inter-assay coefficients of variation were 4.1% and 7.7%, respectively. QC results were within the acceptance limits for both IL-6 and ICAM-1. Extracranial colour Doppler ultrasound and IMT assessment In all study subjects colour Doppler flow imaging (CDFI) of both carotid and vertebral arteries (high resolution B-mode, colour Doppler, and pulse Doppler ultrasonography) was performed with a ProSound SSD Alpha 10 system (Aloka Co., Ltd., Tokyo, Japan) with linear 10 MHz transducer according to well defined procedure and diagnostic protocol [21]. Colour Doppler findings of carotid arteries were classified according to the degree of atherosclerosis: 0 — normal findings–no atherosclerotic changes, 1 — mild stenosis (30–40%), 2 — moderate stenosis (50–60%), 3 — severe stenosis (70–99%) and 4 — carotid occlusion. Colour Doppler findings of vertebral arteries were classified as positive if vertebral stenosis or occlusion was present. After the carotid arteries were examined, the probe was rotated 90° to obtain a longitudinal image of the anterior and posterior vessel walls. The maximum IMT was measured according to the protocol at the near and far walls of the common carotid artery, the bifurcation, and the internal carotid arteries and was expressed as a mean aggregate value (mean-IMT) [22]. Transcranial Doppler sonography and BHI All subjects underwent TCD assessment of intracranial vessels using TCD DWL Multidrop X4 instrument (ScanMed Medical, Gloucestershire, United Kingdom) with 2 MHz hand-held pulsed-wave probe. TCD examination of vessels of the circle of Willis and vertebrobasilar system was performed bilaterally, in supine position, in a quiet room, after 5 min of bed rest. Cerebrovascular reactivity to hypercapnia was
Please cite this article as: Martinic-Popovic I, et al, The association of inflammatory markers with cerebral vasoreactivity and carotid atherosclerosis in transient ischaemic attack, Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.07.010
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evaluated by means of BHI, with mean blood flow velocity (BFV) of middle cerebral artery (MCA) being continuously monitored during the test [23,24]. After an initial 5 minute resting period, a continuous mean BFV during the next 30 s were noted as baseline BFV value (vmean). After normal inspiratory breath, subjects were asked to hold their breath for 30 s and mean BFV of the last 3 s of breath holding period were recorded (vmax). The same procedure was repeated after 2 min. BHI was calculated as the percentage increase in mean BFV occurring during breath holding divided by the time (seconds) for which the subject hold his/her breath [23]. Statistical analysis Qualitative data were presented as counts and proportions. Quantitative variables were assessed for normality using the Kolmogorov– Smirnov test. Normally distributed variables were described with mean and standard deviation. Data sets which were not distributed normally, were described with median and interquartile range (IQR). Age was presented as median and range (min–max). Relative differences in frequencies between groups were assessed by Chi-square test [25]. If the data were normally distributed and if sample size was ≥30, t-test was used for testing the statistical significance between two groups. Otherwise (small sample (N b 30) or if data are not normally distributed), Mann–Whitney test was used. The difference among 3 and more groups was tested with Kruskal–Wallis test [26]. Spearman correlation was used to test the strength of association between variables. Logistic regression analysis was done to identify most significant predictors of acute transitory ischaemia. All variables which were statistically significant in univariate regression, were tested in multivariate model, by stepwise method. Level of significance was set at b0.05. Statistical analysis was done using the MedCalc Statistical software version 12.1.4.0 (MedCalc Software, Mariakerke, Belgium). Results Demographic profile, vascular risk factors, CDFI findings of carotid and vertebral arteries, neurosonological parameters, blood glucose and lipid profile in patients and controls are presented in Table 1. Patients with TIA and disease-free controls did not differ significantly relative to the different demographic characteristics and traditional vascular risk factors. Colour Doppler findings of vertebral arteries have demonstrated that steno-occlusive changes were more common in TIA patients than in disease-free controls. This was particularly observed in right carotid artery (P = 0.030). In both, right and left carotid artery, there were 4 patients with complete carotid occlusion, whereas such findings were not observed in disease-free controls. The advanced atherosclerotic changes in TIA patients were further confirmed using carotid intima–media thickness (IMT) and cerebral vasoreactivity (evaluated by breathholding index technique) measurements. In TIA patients, IMT and BHI findings suggested advanced atherosclerosis, as opposed to diseasefree controls. IMT was significantly higher in TIA patients than in disease-free controls (P b 0.001). Due to the increased thickness of the temporal bones, TCD and subsequent BHI calculations were performed only in 24/45 (0.53) patients and in 22/36 (0.61) disease-free controls. BHI values were significantly lower in patients with TIA (P b 0.001). Inflammatory markers, in TIA patients and disease-free controls are presented in Table 2. Three (CRP, ICAM-1 and IL-6) out of four inflammatory markers were significantly higher in TIA patients compared to disease-free controls. IL-6 was the only inflammatory marker associated with both BHI and IMT values in TIA patients (Table 3). All inflammatory and endothelial markers and BHI, IMT were identified as significant predictors of TIA by univariate logistic regression analysis. When all variables which were statistically significant in
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Table 1 Demographic data, neurosonological parameters and CDFI findings of carotid and vertebral arteries in TIA patients and disease-free controls. P
Patients (N = 45)
Disease-free controls (N = 36)
74 (48–90)
69 (55–85)
0.203
22 (0.49) 40 (0.89) 17 (0.38) 8 (0.18) 8 (0.18) 3 (0.06)
15 (0.42) 29 (0.81) 6 (0.16) 18 (0.5) 5 (0.14) 3 (0.08)
0.686 0.486 0.053 0.152 0.951 0.929
19 (0.42) 5.6 (5.05–6.73) 6.6 (5.7–8.3) 1.1 (1.1–1.4) 3.8 ± 1.1 1.8 (1.8–2.4) 1.22 ± 0.17 (N = 45) 0.63 (0.54–0.89) (N = 24)
21 (0.58) 0.227 5.6 ± 1.3 0.485 6.05 (5.05–6.8) 0.028 1.2 (1.1–1.6) 0.993 3.4 ± 0.96 0.101 1.8 (1.2–2.3) 0.845 0.86 ± 0.19 b0.001 (N = 36) 1.2 (0.80–1.48) b0.001 (N = 22)
Left ICA 0 (N/proportion) 1 (N/proportion) 2 (N/proportion) 3 (N/proportion) 4 (N/proportion)
33 (0.73) 5 (0.11) 3 (0.06) 0 (0) 4 (0.08)
27 (0.75) 7 (0.19) 2 (0.05) 0 (0) 0 (0)
0.259
Right ICA 0 (N/proportion) 1 (N/proportion) 2 (N/proportion) 3 (N/proportion) 4 (N/proportion) VA occlusion (N/proportion)
34 (0.75) 2 (0.04) 3 (0.06) 2 (0.04) 4 (0.08) 7 (0.15)
30 (0.83) 6 (0.16) 0 (0) 0 (0) 0 (0) 3 (0.08)
0.030
Age/years [median (range)] Females (N/proportion) Arterial hypertension (N/proportion) Diabetes mellitus (N/proportion) Statin therapy (N/proportion) Atrial fibrillation (N/proportion) Anticoagulant therapy (N/proportion) Smoking (N/proportion) Total cholesterol (mmol/L) Blood glucose (mmol/L) HDL cholesterol (mmol/L) LDL cholesterol (mmol/L) Triglycerides (mmol/L) IMT/mm (mean ± SD) BHI/%/s (median (Q1–Q3))
0.534
0 — no stenosis; 1 — mild stenosis (30–49%); 2 — moderate stenosis (50–69%); 3 — severe stenosis (70–99%) and 4 — carotid occlusion.
univariate regression were tested in multivariate model, only IMT and BHI remained statistically significant predictors of TIA (P = 0.018 and 0.254). However, after adjusting the model for inflammatory and endothelial markers none of the investigated variables have remained significant (P = 0.078 and 0.263). Discussion With this study we have demonstrated that transient ischaemic attack is associated with substantially higher concentrations of soluble adhesion molecule ICAM-1, pro-inflammatory cytokine IL-6 and CRP, compared to disease-free controls, suggesting a marked inflammatory response to cerebral ischaemia. The presence of inflammatory markers was associated with higher IMT and lower BHI, indicating the insufficient cerebral perfusion in those individuals mostly due to the underlying atherosclerotic disease. Our findings highlight the key significance of inflammation in the early response to ischaemia during the transitory ischaemic episode.
Table 2 Inflammatory markers in TIA patients and disease-free controls.
CRP (mg/L) ESR (mm/h) IL-6 (pg/mL) ICAM-1 (ng/mL)
Patients (N = 45)
Disease-free controls (N = 36)
P
8.5 (6.7–14.9) 14 (7.75–27.5) 6.76 (2.72–18.3) 371.6 ± 161.8
4.5 (2.8–5.8) 8.5 (5.0–13.1) 2.46 (1.5–4.61) 301.9 ± 91.6
b0.001 0.054 b0.001 0.026
Please cite this article as: Martinic-Popovic I, et al, The association of inflammatory markers with cerebral vasoreactivity and carotid atherosclerosis in transient ischaemic attack, Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.07.010
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Table 3 Correlations of inflammatory and neurosonological parameters in TIA patients.
BHI r (p) IMT r (p) Total cholesterol r (p) Blood glucose r (p) HDL cholesterol r (p) LDL cholesterol r (p) Triglycerides r (p) CRP r (p) IL-6 r (p) ICAM-1 r (p)
IL-6
ICAM-1
CRP
ESR
−0.419 (0.041) 0.334 (0.025) −0.418 (0.004) ns ns −0.405 (0.005) ns 0.394 (0.007) NA NA
ns ns ns ns ns ns ns −0.049 (0.668) 0.030 (0.790) NA
ns 0.317 (0.034) ns ns ns ns ns NA −0.005 (0.966) NA
ns 0.474 (0.001) ns ns ns ns ns 0.532 (b0.001) 0.239 (0.036) 0.290 (0.011)
ns—not significant. NA—not applicable.
Whereas CRP increase has already been demonstrated, our study is, to the best of our knowledge, the first one to show the evidence for the presence of elevated levels of ICAM-1 and IL-6 in TIA patients. There are only a few laboratory tests which are of some use in diagnosis of patients with TIA. Laboratory workup is usually done to exclude some common TIA mimics. Most common laboratory assays employed in TIA laboratory workup are glucose, electrolytes, complete blood count and coagulation tests for detecting hypoglycaemia, metabolic disorders and some prothrombotic hypercoagulable states, respectively [27]. Currently, laboratory assays are used neither for diagnosis nor for prognosis of TIA. Nevertheless, the evidence that support the increase of neutrophil and monocyte count, lipoprotein-associated phospholipase A(2), prothrombotic markers, NT-proBNP and some other molecules in the early hours in TIA patients exist [28–31]. However, as no single blood marker has so far been shown to improve the diagnostic performance of commonly used clinical stroke scales, it seems possible that a panel of biomarkers could eventually provide aid in diagnosis of TIA. Several markers have been demonstrated as having potentially predictive or prognostic significance in TIA patients. Serum copeptin has also proven to be predictive of the stroke subsequent to first-ever TIA [32]. Elevated levels of serum hsCRP have also been shown to predict further ischaemic events following TIA [33]. On the other hand, Selvarajah and colleagues have not been able to show the predictive role of CRP and IL-6 for the recurrence of stroke or other vascular events in patients suffering from transient ischaemic attack [34]. Hence, it remains to be assessed whether routine copeptin or CRP measurement could be a useful tool for identifying TIA patients at increased risk of developing subsequent cerebrovascular ischaemic events, either repeated TIAs or stroke. In our study IL-6 was associated with CRP and ESR. Moreover, ESR was associated with CRP, IL-6 and ICAM-1. This shows that additionally to the increased basal levels of inflammatory markers, there probably occurs an immediate increase of IL-6 triggered by the acute ischaemia and subsequently of all other investigated downstream inflammatory markers. Serum biomarkers able to detect cerebral ischaemia are not yet available and the search is ongoing. Several recent lines of evidence clearly indicate that biomarkers might be identified somewhere upstream or downstream the pathophysiological events prior to or subsequent to cerebral ischaemia [35]. Zhan and colleagues have explored gene expression profiles in the circulation of TIA patients using the Affymetrix microarray technology [36]. They have found 449 genes differentially expressed between TIA patients (N = 26) and controls (N = 26), out of which 34 genes have been able to discriminate TIA patients from controls with 100% sensitivity and 100% specificity. Identified genes were associated with systemic inflammation, platelet activation, and prothrombin activation. This is in line with our observations of the higher levels of IL-6 and CRP in TIA patients, compared to controls.
So far the increased levels of ICAM-1 have not yet been reported in TIA. ICAM-1 was found to be increased in acute stroke and to be associated with stroke severity and infarct volume [13,37], whereas Frijns et al. have found that P- and E-selectin, but not ICAM-1 and VCAM-1, are increased in patients with ischaemic stroke and carotid stenosis [38]. Increased concentration of ICAM-1 in TIA patients indicates endothelial changes and adhesion molecule activation early in the course of ischaemic cerebral event. In our study, IMT and BHI findings in TIA patients were suggesting advanced atherosclerosis. TIA patients had significantly higher IMT and lower BHI compared to disease-free controls. Moreover, IL-6 proven to be associated with both BHI and IMT values. High carotid IMT and low BHI are commonly described as vascular risk markers defining the individual susceptibility to unfavourable cerebrovascular events [39,40]. With our study we provide additional evidence for clustering of inflammatory mediators and impaired microcirculation in patients suffering from TIA. The question remains whether the level of inflammatory markers could possibly be used in assessing the short-term stroke risk following TIA. Some future larger studies should be performed to evaluate the possible association of the level of inflammatory markers with the severity of TIA and subsequent short-term stroke risk. Nevertheless, if proven to be useful, inflammatory markers could be of great assistance in evaluation of patients suspected for TIA who should undergo immediate etiologic workup and treatment. Limitations Some limitations of our study should be acknowledged. First, the size of the studied group is limited, and the results of such analyses should be interpreted with caution. Brain CT was performed only in TIA patients on inclusion, and clinical follow-up of patients with TIA for possible stroke was not done.
Conclusions Transient ischaemic attack is associated with higher sICAM-1, IL-6 and CRP, pointing to the marked inflammatory response to cerebral ischaemia. Inflammatory markers are associated with higher IMT and lower BHI, indicating the insufficient cerebral perfusion due to the underlying atherosclerotic disease. Our findings highlight the key significance of inflammation in the early response to cerebral ischaemia during the transitory ischaemic episode. Acknowledgments This study was supported by Ministry of Science, Education and Sports, Republic of Croatia, projects #134-1340227-0200, #1341340036-0034, #134-1340036-0035 and # 022-1340036-2083.
Please cite this article as: Martinic-Popovic I, et al, The association of inflammatory markers with cerebral vasoreactivity and carotid atherosclerosis in transient ischaemic attack, Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.07.010
I. Martinic-Popovic et al. / Clinical Biochemistry xxx (2014) xxx–xxx
References [1] Easton JD, Saver JL, Albers GW, Alberts MJ, Chaturvedi S, Feldmann E, et al. The American Academy of Neurology affirms the value of this statement as an educational tool for neurologists. Stroke 2009;40:2276–93. [2] Gállego J, Muñoz R, Martínez-Vila E. Emergent cerebrovascular disease risk factor weighting: is transient ischemic attack an imminent threat? Cerebrovasc Dis 2009; 27(Suppl. 1):88–96. [3] Lloyd-Jones D, Adams RJ, Brown TM, et al. Heart disease and stroke statistics—2010 update: a report from the American Heart Association. Circulation 2010;121:e46-215. [4] Uchiyama S. The concept of acute cerebrovascular syndrome. Front Neurol Neurosci 2014;33:11–8. [5] Lovrenčić-Huzjan A, Strineka M, Šodec-Šimičević D, et al. Management of patients with transient ischemic attack (TIA) at Sestre milosrdnice University Hospital Center. Acta Clin Croat 2011;50:367–73. [6] Hong KS, Bang OY, Kim JS, Heo JH, Yu KH, Bae HJ, et al. Stroke statistics in Korea: part II stroke awareness and acute stroke care, a report from the Korean stroke society and clinical research center for stroke. J Stroke 2013;15:67–77. [7] Soulleihet V, Nicoli F, Trouve J, Girard N, Jacquin L. Optimized acute stroke pathway using medical advanced regulation for stroke and repeated public awareness campaigns. Am J Emerg Med 2014;32:225–32. [8] Barber PA, Zhang J, Demchuk AM, Hill MD, Buchan AM. Why are stroke patients excluded from tPA therapy? An analysis of patient eligibility. Neurology 2001; 56:1015–20. [9] Castle J, Mlynash M, Lee K, Caulfield AF, Wolford C, Kemp S, et al. Agreement regarding diagnosis of transient ischemic attack fairly low among stroke-trained neurologists. Stroke 2010;41:1367–70. [10] Ross R. Atherosclerosis: an inflammatory disease. N Engl J Med 1999;340:115–26. [11] Faxon DP, Fuster V, Libby P, Beckman JA, Hiatt WR, Thompson WR, et al. Atherosclerotic vascular disease conference: writing group III: pathophysiology. Circulation 2004;109:2617–25. [12] Xia W, Han J, Huang G, Ying W. Inflammation in ischemic brain injury: current advances and future perspectives. Clin Exp Pharmacol Physiol 2010;37:253–8. [13] Simundic AM, Basic V, Topic E, Demarin V, Vrkic N, Kunovic B, et al. Soluble adhesion molecules in acute ischemic stroke. Clin Invest Med 2004;27:86–92. [14] Vila N, Castillo J, Davalos A, Esteve A, Planas AM, Chamorro A, et al. Levels of antiinflammatory cytokines and neurological worsening in acute ischemic stroke. Stroke 2003;34:671–5. [15] Domac FM, Somay G, Misirli H, Erenoglu NY. Tumor necrosis factor alpha serum levels and inflammatory response in acute ischemic stroke. Neurosciences 2007; 12:25–30. [16] Zhu XD, Chen JS, Zhou F, Liu QC, Chen G, Zhang JM. Detection of copeptin in peripheral blood of patients with aneurysmal subarachnoid hemorrhage. Crit Care 2011; 15:R288. [17] Dobsa L, Cullen Edozien K. Copeptin and its potential role in diagnosis and prognosis of various diseases. Biochem Med 2013;23:172–92. [18] Chobanian AV, Bakris GL, Black HR, Cushman WC, Green LA, Izzo Jr JL, et al. Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure. National Heart, Lung, and Blood Institute; National High Blood Pressure Education Program Coordinating Committee. Seventh report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure. Hypertension 2003;42:1206–52. [19] Expert committee on the diagnosis, classification of diabetes mellitus. Report of the expert committee on the diagnosis and classification of diabetes mellitus. Diabetes Care 1997;20:1183–97.
5
[20] Grundy SM, Cleeman JI, Merz CN, Brewer HB, Clark LT, Hunninghake DB, et al. Implications of recent clinical trials for the National Cholesterol Education Program Adult Treatment Panel III guidelines. Circulation 2004;110:227–39. [21] Lovrenčić-Huzjan A, Vuković V, Demarin V. Neurosonology in stroke. Acta Clin Croat 2006;45:385–401. [22] Silvestrini M, Cagnetti C, Pasqualetti P, Albanesi A, Altamura C, Lanciotti C, et al. Carotid wall thickness and stroke risk in patients with asymptomatic internal carotid stenosis. Atherosclerosis 2010;210:452–7. [23] Markus HS, Harrison MJG. Estimation of cerebrovascular reactivity using transcranial Doppler, including the use of breath-holding as the vasodilatory stimulus. Stroke 1992;23:668–73. [24] Tang SC, Huang YW, Shieh JS, Huang SJ, Yip PK, Jeng JS. Dynamic cerebral autoregulation in carotid stenosis before and after carotid stenting. J Vasc Surg 2008;48:88–92. [25] McHugh ML. The Chi-square test of independence. Biochem Med 2013;23:125–31. [26] Simundic AM. Practical recommendations for statistical analysis and data presentation in Biochemia Medica journal. Biochem Med 2012;22:15–23. [27] Siket MS, Edlow J. Transient ischemic attack: an evidence-based update. Emerg Med Pract 2013;15:1–28. [28] Ross AM, Hurn P, Perrin N, Wood L, Carlini W, Potempa K. Evidence of the peripheral inflammatory response in patients with transient ischemic attack. J Stroke Cerebrovasc Dis 2007;16:203–7. [29] Cucchiara BL, Messe SR, Sansing L, MacKenzie L, Taylor RA, Pacelli J, et al. Lipoproteinassociated phospholipase A2 and C-reactive protein for risk-stratification of patients with TIA. Stroke 2009;40:2332–6. [30] Prodan CI, Vincent AS, Dale GL. Coated-platelet levels are elevated in patients with transient ischemic attack. Transl Res 2011;158:71–5. [31] Whiteley W, Wardlaw J, Dennis M, Lowe G, Rumley A, Sattar N, et al. Blood biomarkers for the diagnosis of acute cerebrovascular diseases: a prospective cohort study. Cerebrovasc Dis 2011;32:141–7. [32] Katan M, Nigro N, Fluri F, Schuetz P, Morgenthaler NG, Jax F, et al. Stress hormones predict cerebrovascular re-events after transient ischemic attacks. Neurology 2011; 76:563–6. [33] Purroy F, Montaner J, Molina CA, Delgado P, Arenillas JF, Chacon P, et al. C-reactive protein predicts further ischemic events in transient ischemic attack patients. Acta Neurol Scand 2007;115:60–6. [34] Selvarajah JR, Smith CJ, Hulme S, Georgiou R, Sherrington C, Staniland J, et al. Does inflammation predispose to recurrent vascular events after recent transient ischaemic attack and minor stroke? The North West of England transient ischaemic attack and minor stroke (NORTHSTAR) study. Int J Stroke 2011;6:187–94. [35] Cucchiara B, Nyquist P. Blood markers in TIA: array of hope? Neurology 2011; 77:1716–7. [36] Zhan X, Jickling GC, Tian Y, Stamova B, Xu H, Ander BP, et al. Transient ischemic attacks characterized by RNA profiles in blood. Neurology 2011;77:1718–24. [37] Clark WM, Coull BM, Briley DP, Mainolfi E, Rothlein R. Circulating intercellular adhesion molecule-1 levels and neutrophil adhesion in stroke. J Neuroimmunol 1993;44:123–5. [38] Frijns CJ, Kappelle LJ, van Gijn J, Nieuwenhuis HK, Sixma JJ, Fijnheer R. Soluble adhesion molecules reflect endothelial cell activation in ischemic stroke and in carotid atherosclerosis. Stroke 1997;28:2214–8. [39] Buratti L, Viticchi G, Falsetti L, Cagnetti C, Luzzi S, Bartolini M, et al. Vascular impairment in Alzheimer's disease: the role of obstructive sleep apnea. J Alzheimers Dis 2014;38:445–53. http://dx.doi.org/10.3233/JAD-131046. [40] Rodríguez-Flores M, García-García E, Cano-Nigenda CV, Cantú-Brito C. Relationship of obesity and insulin resistance with the cerebrovascular reactivity: a case control study. Cardiovasc Diabetol 2014;13:2. http://dx.doi.org/10.1186/1475-2840-13-2.
Please cite this article as: Martinic-Popovic I, et al, The association of inflammatory markers with cerebral vasoreactivity and carotid atherosclerosis in transient ischaemic attack, Clin Biochem (2014), http://dx.doi.org/10.1016/j.clinbiochem.2014.07.010
