Diagnostic value of plasma signal peptide-Cub-Egf domain-containing ...

American Journal of Emergency Medicine 33 (2015) 262–265

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American Journal of Emergency Medicine journal homepage: www.elsevier.com/locate/ajem

Original Contribution

Diagnostic value of plasma signal peptide-Cub-Egf domain-containing protein-1 (SCUBE-1) in an experimental model of acute ischemic stroke Suha Turkmen, MD a, Umut Eryigit, MD b,⁎, Yunus Karaca, MD a, Ahmet Mentese, Phd c, Uzun Aysegul Sumer, Phd c, Esin Yulug, MD d, Nurhak Aksut, MD e, Sibel Gazioglu, MD f, Abdulkadir Gunduz, MD a a

Karadeniz Technical University, Faculty of Medicine, Department of Emergency Medicine, Trabzon, Turkey Diyarbakır Selahaddin Eyyubi State Hospital, Department of Emergency Medicine, Diyarbakır, Turkey c Karadeniz Technical University, Faculty of Medicine, Department of Biochemistry, Trabzon, Turkey d Karadeniz Technical University, Faculty of Medicine, Department of Histology, Trabzon, Turkey e Manisa State Hospital, Department of Emergency Medicine, Manisa, Turkey f Karadeniz Technical University, Faculty of Medicine, Department of Neurology, Trabzon, Turkey b

a r t i c l e

i n f o

Article history: Received 10 August 2014 Received in revised form 17 November 2014 Accepted 26 November 2014

a b s t r a c t Objectives: This study was intended to examine possible diagnostic value of plasma Signal Peptide-Cub-Egf domain-containing protein-1 (SCUBE1) levels in an experimental model of acute ischemic stroke. Methods: Twenty-four female Sprague Dawley rats were divided into four groups. Blood and brain tissue specimens were collected immediately following artery ligation (control; Group 1), 1 h after ligation (Group 2), 2 h after ligation (Group 3) and 6 h after ligation (Group 4). SCUBE1 levels were investigated in the serum specimens. The brain samples were examined histopathologically. Correlation analysis was performed between the values. Results: Median SCUBE1 values were 1.75 ng/ml in the control group, 3.80 ng/ml, 3.71 ng/ml and 4.19 ng/ml in the groups 2, 3 and 4, respectively (n = 6 for each, P = 0.004, for each group compared to control values). Histopathological analysis revealed median atrophic neuron percentages of 16% (in group 1), 42%, 55% and 76% in group 2, 3 and 4 respectively (n = 6 for each, P = 0.004, for each group compared to control group). A higly significant correlation was determined between SCUBE-1 levels and percentage of atrophic neurons (r = 0.744 P = 0.000). Conclusions: In this experimental model of acute ischemic stroke plasma SCUBE1 levels rose from the 1st hour of induced stroke and remained high up to 6th hour tested. Results of this experimental study has a potential to become the basis for a clinical study to confirm whether SCUBE1 can be used as a biomarker in the early diagnosis of acute ischemic stroke patients. © 2014 Elsevier Inc. All rights reserved.

1. Introduction Considering the critical role of early action in management of stroke, there is still a need for a biomarker that would reliably assist in the early diagnosis and patient selection for tissue plasminogen activator therapy, which has provided a considerable success in terms of mortality and morbidity of patients with stroke. This is particularly important if this novel biochemical marker could give rapid results, specific for brain damage, and can be used in the emergency setting for early diagnosis and differentiation from other conditions that mimic some stroke findings [1,2]. Early diagnosis in patients presenting with stroke to the emergency department (ED) is essential in terms of mortality and morbidity. Considerable success has been achieved in early-stage treatment with the development of new therapeutic techniques, such as thrombolytic agents used ⁎ Corresponding author. Diyarbakır Selahaddin Eyyubi State Hospital, Department of Emergency Medicine, Diyarbakır, Turkey. Tel.:+90 5432162758. E-mail address: [email protected] (U. Eryigit). http://dx.doi.org/10.1016/j.ajem.2014.11.051 0735-6757/© 2014 Elsevier Inc. All rights reserved.

in ischemic stroke in particular [1]. There is therefore a need for easily available novel biochemical markers that give rapid results, that reveal brain damage in a reliable manner, and that can be used in the ED for early diagnosis of patients with stroke and to rule out stroke mimics [2]. The inflammatory process is an important step in atherosclerosis in the development of stroke and one in which levels of several serum markers are known to increase. Experimental studies of cerebral ischemia have reported increased release of numerous molecules, such as vascular cell adhesion molecules. Measurement of levels of adhesion molecules in plasma provides important information concerning atherogenesis developing as a result of endothelial dysfunction or inflammation [3]. Some studies have suggested that signal peptide-CubEgf domain-containing protein-1 (SCUBE1), levels of which increase following thrombocyte activation and which is released by thrombocytes, can be used in cerebrovascular and cardiovascular events [4]. The purpose of this study was to evaluate changes in SCUBE1 levels in an experimentally induced stroke model in the early diagnosis of ischemic stroke and to compare these with histopathological scores.

S. Turkmen et al. / American Journal of Emergency Medicine 33 (2015) 262–265

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Table 1 Groups’ median SCUBE1 and atrophic neuron percentages Groups

Group 1

Group 2

Group 3

Group 4

SCUBE1 (ng/mL) ANP (%)

1.75 (1.64-1.87)a,b,c 16 (12–19)a,b,c

3.80 (3.54-4.29)a 42 (40–44)a

3.71 (3.21-5.08)b 55 (52–60)b

4.19 (2.18-5.80)c 76 (73–79)c

Values are reported as median (25%-75%). ANP: atrophic neuron percentage. For SCUBE1: aP = .004, bP = .004, cP = .004. For ANP: aP = .004, bP = .004, cP = .004.

2. Methods

3.2. Histopathological examination

2.1. Study design

Brains of all rats were removed at the end of the study. Tissue specimens containing all layers of the brain were collected from the same anatomical region in the central part of the brain in all groups. These were then fixed in 10% neutral formalin, rendered transparent in xylene, and embedded in paraffin blocks. Sections 5 μm in thickness were made using a fully automatic microtome (Leica RM 2255, Tokyo, Japan). Sections were stained with hematoxylin-eosin and Cresyl violet for detailed assessment of general histological structure. Preparates were evaluated by an experienced histologist blinded to the different groups using a light microscope (Olympus BX-51; Olympus Optical Co, Tokyo, Japan). Percentage of degenerative pyramidal neurons was calculated by counting 100 pyramidal cells under a light microscope at ×200 magnification on the Analysis 5 Research program (Olympus Soft Imaging Solutions, Münster, Germany). Cells with body shrinkage, loss of Nissl substance in cytoplasm, and eosinophilia and those with a darkcolored, contracted nucleus were regarded as degenerative neurons. The degenerative neurons identified were expressed as proportion of atrophic neurons to all neurons [5].

This experimental study was performed following approval from the Animal Experiments Local Ethical Committee.

2.2. Study setting and population Twenty-four female Sprague-Dawley rats weighing 250 to 300 g and bred in the Surgical Research Center were used. The rats were kept in steel cages until the day of the study at room temperature (22 °C) and were given water and standard rat chow. For the last 12 hours before the study, they were given only water.

2.3. Study protocol The rats were randomly divided into 4 experimental groups of 6 rats each. Following the establishment of the experimental group, all rats were given 10 mg/kg xylazine and 90 mg/kg ketamine hydrochloride intraperitoneally for general anesthesia. Rats’ body temperatures were measured using a rectal thermometer and were kept at approximately 37 °C. Rats were fixed on the operating table in the supine position, and the neck was shaved along the midline. Once the operation site had been disinfected, a midline incision was made. Following superficial microdissection, we progressed toward the right main carotid artery with deep microdissection. The trachea was visualized, and the right main carotid artery was accessed by dissecting the paratracheal muscles. Ligation was performed with 2/0 silk suture 1 cm proximal to the carotid bifurcation, and the right main carotid artery remained ligated throughout the procedure. Blood and brain tissue specimens in group 1 were collected immediately following artery ligation. Blood and brain tissue specimens were collected 1 hour after ligation in group 2, 2 hours after ligation in group 3, and 6 hours after ligation in group 4.

3.3. Data analysis Statistical analysis was performed on SPSS (Statistical Package for Social Sciences for Windows v.13.0; SPSS, Chicago, IL) software. Categorical variables were calculated as median and percentage quartiles. Nonparametric tests were used for analyzing data. The Mann-Whitney U test with Bonferroni correction was used for intergroup comparisons. Spearman correlation analysis was used to analyze correlation between variables. Results were assessed with a 95% confidence interval with significance set at P b .05.

3. Measurements 3.1. SCUBE-1 measurement Plasma SCUBE1 levels were measured using the enzyme-linked immunosorbent assay technique with a commercial kit following the manufacturer’s instructions (Cusabio Biotech Co, catalog no. CSBE16229R, Wuhan, PR China).

Table 2 Correlation analysis of groups’ SCUBE1 and atrophic neuron percentages ANP SCUBE1 ⁎ P b .01.

r value P value

0.744(⁎) .000 Fig. 1. Box chart of groups’ median, minimum, and maximum values.

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Fig. 2. Photomicrograph of brain tissue cortex (hematoxylin-eosin, × 400) (A, control group; B, 1-hour ischemia group; C, 2-hour ischemia group; D, 6-hour ischemia group). Pyramidal neurons with normal morphology (↑), degenerative pyramidal neurons (Δ).

Fig. 3. Photomicrograph of brain tissue cortex (Cresyl violet, ×400) (A, control group; B, 1-hour ischemia group; C, 2-hour ischemia group; D, 6-hour ischemia group). Pyramidal neurons with normal morphology (↑), degenerative pyramidal neurons (Δ).


4. Results Median SCUBE1 and atrophic neuron percentages were calculated for all groups. The results are given as median (25%-75%) values in Table 1. (See Table 2.) Median, minimum, and maximum SCUBE1 values are shown in a box plot for comparative purposes (Fig. 1). Comparison of SCUBE1 levels among the groups revealed a significant difference between group 1 and groups 2, 3, and 4 (P = .004, P = .004, and P = .004). No significant difference was determined in terms of median SCUBE1 values between groups 2, 3, and 4 (P N .05). At analysis of brain tissue, a normal brain cortex and pyramidal neuron histological structure were observed in group 1. Nissl granules were observed in cortex cytoplasm in group 2, whereas, in addition to normal neurons with a euchromatic nucleus, pyramidal neurons with occasional shrinkage in cytoplasm and loss of Nissl granules were also observed. In group 3, there was a predominance of degenerative pyramidal neurons with eosinophilic cytoplasm and pyknotic nuclei. Neurons with a normal histological structure were also observed in places. A widespread degenerative pyramidal neuron structure was observed in all layers of the cortex in group 4. Degenerative pyramidal neurons increased with length of ischemia, and degenerative pyramidal neurons were also seen in areas of the cortex close to the white matter margin (Figs. 2 and 3). Brain tissues were examined histopathologically, and atrophic neuron percentages were calculated. Median atrophic neuron percentages were 16% in group 1, 42% in group 2, 55% in group 3, and 76% in group 4. Analysis revealed a significant difference in terms of atrophic neuron percentages between group 1 and groups 2, 3, and 4 (P = .004, P = .004, and P = .004). Spearman correlation analysis was performed to determine correlations between SCUBE1 levels and atrophic neuron percentage. There was a significant, strong positive correlation between SCUBE1 levels and atrophic neuron percentage (r = 0.744, P = .000). 5. Discussion The first result from this study shows that SCUBE1 levels rose significantly compared to the control group from the first hours of ischemic stroke. The SCUBE1 levels rose significantly compared to the control group at the first, second, and sixth hours of ischemia. This elevation beginning in the first hour continued in a stable manner. This may be associated with SCUBE1 levels increasing after thrombosis, which initiates the thrombotic ischemic event. Dai et al [4] determined that increase in plasma SCUBE1 levels was detectable after 6 hours at the earliest and sustained until the 84th hour in patients with acute ischemic stroke. These results imply that SCUBE1 levels rise in chronic stable coronary artery disease and when platelet activation takes place. In agreement but with shifted time course, in our experimental study, the rise in SCUBE1 levels was detectable from as early as 1 hour. Although the mechanism of atherogenesis was not the primary focus of our study, our results implicate that SCUBE1 may be a novel molecule playing a role in cardiovascular and cerebrovascular events through platelet endothelial adhesion [6].

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Another finding from this experimental study is the powerful correlation between atrophic neuron percentages and SCUBE1 levels at histopathological examination of the brain in ischemia of thrombotic origin. This correlation shows that SCUBE1 levels increase with severity of organ damage. In clinical practice, elevated SCUBE1 levels in patients with suspected ischemic stroke show the scale of cerebrovascular damage. A study involving an experimentally induced model of mesenteric ischemia showed significantly higher SCUBE1 levels at the sixth hour of mesenteric ischemia compared to the control group [7]. Existing studies therefore suggest that SCUBE1 levels rise in thrombotic-ischemic events in particular. Therefore, in situations in which definitive diagnosis cannot be made by physical examination in the ED and when imaging facilities are limited, we believe that SCUBE1 levels may be a guide in the diagnosis of ischemic stroke. 5.1. Limitations Signal peptide-Cub-Egf domain-containing protein-1 is a novel biomarker whose levels are significantly affected by a range of physiological variables. We were unable to control all of the variables that might potentially affect SCUBE-1 levels. Our study involved some limitations in terms of the model used. The study was controlled but may not mimic typical acute ischemic stroke cases seen in practice. 6. Conclusion The results from this experimental study model show that SCUBE1 levels that rise as of the early period of ischemic stroke have the potential to be used as a biomarker in the early diagnosis of ischemic stroke. Higher SCUBE1 levels in patients with high histopathological scores indicate a powerful correlation between SCUBE1 levels and neuronal injury. Further studies involving larger case series as well as clinical studies are needed to corroborate these findings. References [1] Bivard A, Lin L, Parsonsb MW. Review of stroke thrombolytics. J Stroke 2013;15:90–8. [2] Prugger C, Luc G, Haas B, Morange PE, Ferrieres J, Amouyel P, et al. Multiple biomarkers for the prediction of ischemic stroke: the PRIME study. Arterioscler Thromb Vasc Biol 2013;33:659–66. [3] Lindemann S, Kramer B, Seizer P, Gawaz M. Platelets, inflammation and atherosclerosis. J Thromb Haemost 2007;5(Suppl. 1):203–11. [4] Dai DF, Thajeb P, Tu CF, Chiang FT, Chen CH, Yang RB, et al. Plasma concentration of SCUBE1, a novel platelet protein, is elevated in patients with acute coronary syndrome and ischemic stroke. J Am Coll Cardiol 2008;51:2173–80. [5] Garman RH. Histology of the central nervous system. Toxicol Pathol 2011;39:22–35. [6] Tu CF, Su YH, Huang YN, Tsai MT, Li LT, Chen YL, et al. Localization and characterization of a novel secreted protein SCUBE1 in human platelets. Cardiovasc Res 2006;71: 486–95. [7] Turkmen S, Mentese S, Mentese A, Sumer AU, Saglam K, Yulug E, et al. The value of signal peptide-CUB-EGF domain-containing protein 1 and oxidative stress parameters in the diagnosis of acute mesenteric ischemia. Acad Emerg Med 2013; 20:257–64.