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      Alterations of caveolin-1 expression in a mouse model of delayed cerebral vasospasm following subarachnoid hemorrhage

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          Abstract

          The aim of the present study was to evaluate the expression levels of caveolin-1 in the basilar artery following delayed cerebral vasospasm (DCVS) in a rat model of subarachnoid hemorrhage (SAH), in order to investigate the association between caveolin-1 and DCVS, and its potential as a treatment for DCVS of SAH. A total of 150 Sprague Dawley rats were randomly allocated into blank, saline and SAH groups. The SAH and saline groups were subdivided into days 3, 5, 7 and 14 following the establishment of the model. The murine model of SAH was established by double injection of autologous arterial blood into the cisterna magana and DCVS was detected using Bederson neurological severity scores. Hematoxylin and eosin (HE) staining was used to observe the inner perimeter of the basilar artery pipe and variations in the thickness of the basilar artery wall. Alterations in the levels of caveolin-1 protein in the basilar artery were measured using immunofluorescence and western blot analysis; whereas alterations in the mRNA expression levels of caveolin-1 were detected by reverse transcription-quantitative polymerase chain reaction. In the present study, 15 mice succumbed to SAH-induced DCVS in the day 3 (n=3), 5 (n=5) and 7 (n=2) groups. No mortality was observed in the blank control and saline groups during the process of observation in the SAH group, All mice in the SAH groups exhibited Bederson neurological severity scores ≥1; whereas no neurological impairment was detected in the blank and normal saline groups, demonstrating the success of the model. HE staining was used to assess vasospasm and the results demonstrated that the inner perimeter of the basal artery pipe decreased at day 3 in the SAH group; whereas values peaked in the day 7 group. The thickness of the basal artery wall significantly increased (P<0.05), as compared with the blank and saline groups, in which no significant alterations in the wall thickness and the inner perimeter of the basal artery pipe were detected. As detected by immunofluorescence and western blot analysis, the expression levels of caveolin-1 protein significantly decreased in the day 7 of SAH group, as compared with the blank and saline groups (P<0.01), in which no significant alterations were detected. Caveolin-1 mRNA expression levels significantly increased at the day 7 in the SAH group, as compared with the blank and the saline groups (P<0.01), as detected by RT-qPCR. Furthermore, significant differences were detected at day 14 in the SAH group, as compared with the blank and the saline groups (P>0.05), in which no significant alterations were detected. Therefore, the results of the present study demonstrated that caveolin-1 protein was downregulated in the basilar artery of a rat modeling SAH, which may be associated with DCVS. This suggested that caveolin-1 may be a potential target for the treatment of DCVS.

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          Most cited references41

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          Caveolin, a protein component of caveolae membrane coats.

          Caveolae have been implicated in the transcytosis of macromolecules across endothelial cells and in the receptor-mediated uptake of 5-methyltetrahydrofolate. Structural studies indicate that caveolae are decorated on their cytoplasmic surface by a unique array of filaments or strands that form striated coatings. To understand how these nonclathrin-coated pits function, we performed structural analysis of the striated coat and searched for the molecular component(s) of the coat material. The coat cannot be removed by washing with high salt; however, exposure of membranes to cholesterol-binding drugs caused invaginated caveolae to flatten and the striated coat to disassemble. Antibodies directed against a 22 kd substrate for v-src tyrosine kinase in virus-transformed chick embryo fibroblasts decorated the filaments, suggesting that this molecule is a component of the coat. We have named the molecule caveolin. Caveolae represent a third type of coated membrane specialization that is involved in molecular transport.
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            A new grading system evaluating bleeding scale in filament perforation subarachnoid hemorrhage rat model.

            The endovascular perforation rodent model for experimental subarachnoid hemorrhage (SAH) studies is criticized for lack of control over bleeding. Presently, there is no practical grading system to categorize the severity of SAH depending on the amount of blood. We outline a simple and objective novel SAH grading system by examining the subarachnoid blood clots in the basal cisterns, and evaluate for correlation with neurological status and cerebral vasospasm. Effects of simvastatin, known to reduce vasospasm, were examined using this grading system. Seventy-seven adult male Sprague-Dawley rats were divided randomly into three groups: sham-operated (n=24), SAH (n=32), and SAH+simvastatin (n=25). High-resolution brain pictures were used to grade the severity of SAH and categorize animals into mild, moderate and severe groups. The SAH grades were compared with neurological scores and internal carotid artery parameters such as diameter, perimeter and wall thickness at 24h. Two investigators verified the grading system independently. The SAH grade showed linear correlation functionally with neurological status (r=0.42, p 0.7, p<0.01), and also between two independent investigators (r=0.937, p<0.001). Simvastatin improved neurological score in moderate and severe (p<0.05) but not mild SAH groups (p=0.28). This grading system has the potential to be adopted for SAH experimental rodent models.
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              Cellular and subcellular localization of endogenous nitric oxide in young and senescent pea plants.

              The cellular and subcellular localization of endogenous nitric oxide (NO.) in leaves from young and senescent pea (Pisum sativum) plants was studied. Confocal laser scanning microscopy analysis of pea leaf sections with the fluorescent probe 4,5-diaminofluorescein diacetate revealed that endogenous NO. was mainly present in vascular tissues (xylem and phloem). Green fluorescence spots were also detected in the epidermal cells, palisade and spongy mesophyll cells, and guard cells. In senescent leaves, NO. generation was clearly reduced in the vascular tissues. At the subcellular level, by electron paramagnetic resonance spectroscopy with the spin trap Fe(MGD)(2) and fluorometric analysis with 4,5-diaminofluorescein diacetate, NO. was found to be an endogenous metabolite of peroxisomes. The characteristic three-line electron paramagnetic resonance spectrum of NO., with g = 2.05 and a(N) = 12.8 G, was detected in peroxisomes. By fluorometry, NO. was also found in these organelles, and the level measured of NO. was linearly dependent on the amount of peroxisomal protein. The enzymatic production of NO. from l-Arg (nitric oxide synthase [NOS]-like activity) was measured by ozone chemiluminiscence. The specific activity of peroxisomal NOS was 4.9 nmol NO. mg(-1) protein min(-1); was strictly dependent on NADPH, calmodulin, and BH(4); and required calcium. In senescent pea leaves, the NOS-like activity of peroxisomes was down-regulated by 72%. It is proposed that peroxisomal NO. could be involved in the process of senescence of pea leaves.
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                Author and article information

                Journal
                Exp Ther Med
                Exp Ther Med
                ETM
                Experimental and Therapeutic Medicine
                D.A. Spandidos
                1792-0981
                1792-1015
                October 2016
                04 August 2016
                04 August 2016
                : 12
                : 4
                : 1993-2002
                Affiliations
                Department of Neurosurgery, First Affiliated Hospital of Wenzhou Medical College, Wenzhou, Zhejiang 325000, P.R. China
                Author notes
                Correspondence to: Dr Xian-Xi Tan, Department of Neurosurgery, First Affiliated Hospital of Wenzhou Medical College, 2 Fuxue Lane, Wenzhou, Zhejiang 325000, P.R. China, E-mail: tanxiangxi_txx@ 123456163.com
                Article
                ETM-0-0-3568
                10.3892/etm.2016.3568
                5038886
                27703494
                8eab863c-2f13-4fd5-902a-4a40edbbaa1b
                Copyright: © Xiong et al.

                This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

                History
                : 15 November 2014
                : 13 January 2016
                Categories
                Articles

                Medicine
                subarachnoid hemorrhage,delayed cerebral vasospasm,caveoion-1
                Medicine
                subarachnoid hemorrhage, delayed cerebral vasospasm, caveoion-1

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