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 sam- ples 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.

(C) 2014

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 dam- age, and can be used in the emergency setting for early diagnosis and dif- ferentiation 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. Consid- erable success has been achieved in early-stage treatment with the devel- opment of new therapeutic techniques, such as thrombolytic agents used

* Corresponding author. Diyarbakir Selahaddin Eyyubi State Hospital, Department of Emergency Medicine, Diyarbakir, Turkey. Tel.:+90 5432162758.

E-mail address: [email protected] (U. Eryigit).

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 ische- mia 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 in- flammation [3]. Some studies have suggested that signal peptide-Cub- Egf domain-containing protein-1 (SCUBE1), levels of which increase following thrombocyte activation and which is released by thrombo- cytes, 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 ische-

mic stroke and to compare these with histopathological scores.

http://dx.doi.org/10.1016/j.ajem.2014.11.051

0735-6757/(C) 2014

S. Turkmen et al. / American Journal of Emergency Medicine 33 (2015) 262–265 263

Table 1

Groups’ median SCUBE1 and atrophic neuron percentages

Groups	Group 1	Group 2	Group 3	Group 4
SCUBE1 (ng/mL)	1.75 (1.64-1.87)a,b,c	3.80 (3.54-4.29)a	3.71 (3.21-5.08)b	4.19 (2.18-5.80)c
ANP (%)	16 (12-19)a,b,c	42 (40-44)a	55 (52-60)b	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.

Methods

Study design

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

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.

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 mus- cles. 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.

Measurements

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. CSB- E16229R, Wuhan, PR China).

Histopathological examination

Brains of all rats were removed at the end of the study. Tissue spec- imens 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 um in thickness were made using a fully automatic microtome (Leica RM 2255, Tokyo, Japan). Sec- tions 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 x200 mag- nification on the Analysis 5 Research program (Olympus Soft Imaging Solutions, Munster, Germany). Cells with body shrinkage, loss of Nissl substance in cytoplasm, and eosinophilia and those with a dark- colored, contracted nucleus were regarded as degenerative neurons.

The degenerative neurons identified were expressed as proportion of atrophic neurons to all neurons [5].

Data analysis

Statistical analysis was performed on SPSS (Statistical Package for Social Sciences for Windows v.13.0; SPSS, Chicago, IL) software. Cate- gorical 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.

Table 2

Correlation analysis of groups’ SCUBE1 and atrophic neuron percentages

ANP

SCUBE1 r value 0.744(?)

P value .000

* P b .01. Fig. 1. Box chart of groups’ median, minimum, and maximum values.

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

Fig. 2. Photomicrograph of brain tissue cortex (hematoxylin-eosin, x 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 (?).

Fig. 3. Photomicrograph of brain tissue cortex (Cresyl violet, x400) (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 (?).

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

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 neu- ron 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 correla- tions 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).

Discussion

The first result from this study shows that SCUBE1 levels rose signifi- cantly 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 be- ginning in the first hour continued in a stable manner. This may be asso- ciated with SCUBE1 levels increasing after thrombosis, which initiates the thrombotic ischemic event. Dai et al [4] determined that increase in plas- ma 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 ar- tery 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 impli- cate that SCUBE1 may be a novel molecule playing a role in cardiovascular and Cerebrovascular events through platelet endothelial adhesion [6].

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 cerebro- vascular 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 stud- ies 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 lim- ited, we believe that SCUBE1 levels may be a guide in the diagnosis of is- chemic stroke.

Limitations

Signal peptide-Cub-Egf domain-containing protein-1 is a novel bio- marker whose levels are significantly affected by a range of physiologi- cal 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.

Conclusion

The results from this experimental study model show that SCUBE1 levels that rise as of the early period of ischemic stroke have the poten- tial to be used as a biomarker in the early diagnosis of ischemic stroke. Higher SCUBE1 levels in patients with high histopathological scores in- dicate a powerful correlation between SCUBE1 levels and neuronal inju- ry. Further studies involving larger case series as well as clinical studies are needed to corroborate these findings.

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