影响脑缺血再灌注损伤细胞内钙离子超载的ATP酶

doi:10.3969/j.issn.1673-5374.2013.01.010

摘要/Abstract

摘要：

实验建立大脑中动脉栓塞脑梗死及再灌注大鼠模型，免疫组织化学染色和实时荧光定量聚合酶链反应结果显示，在大鼠大脑皮质和海马组织中，与假手术组相比，缺血期分泌途径衍生钙离子转运ATP酶2蛋白与基因的表达无显著变化，再灌注1，3，24 h其基因和蛋白表达均下降。说明脑组织中分泌途径衍生钙离子转运ATP酶2的蛋白与基因水平与脑缺血再灌注损伤存在明显的应答关系。再灌注期分泌途径衍生钙离子转运ATP酶2表达的下降将可使泵入高尔基体中的钙离子减少，继而胞质中钙离子超载，减轻脑缺血再灌注损伤。

关键词: 脑梗死, 脑损伤, 高尔基体, 钙离子流, 锰离子, 局灶性脑缺血, 免疫组化, 氧化应激, 基金资助文章, 图片文章

Abstract:

This study aimed to investigate changes in secretory pathway Ca²⁺ -ATPase 2 expression following cerebral ischemia/reperfusion injury, and to define the role of Ca²⁺ -ATPases in oxidative stress. A rat model of cerebral ischemia/reperfusion injury was established using the unilateral middle cerebral artery occlusion method. Immunohistochemistry and reverse transcription-PCR assay results showed that compared with the control group, the expression of secretory pathway Ca²⁺ -ATPase 2 protein and mRNA in the cerebral cortex and hippocampus of male rats did not significantly change during the ischemic period. However, secretory pathway Ca²⁺ -ATPase 2 protein and mRNA expression reduced gradually at 1, 3, and 24 hours during the reperfusion period. Our experimental findings indicate that levels of secretory pathway Ca²⁺ -ATPase 2 protein and mRNA expression in brain tissue change in response to cerebral ischemia/reperfusion injury.

Key words: neural regeneration, brain injury, cerebral infarction, secretory pathway Ca²⁺ -ATPase 2, Golgi apparatus, Ca²⁺ oscillations, manganese, focal cerebral ischemia, oxidative damage, Ca²⁺ -ATPase, grants-supported paper, photographs-containing paper, neuroregeneration

. 影响脑缺血再灌注损伤细胞内钙离子超载的ATP酶[J]. 中国神经再生研究(英文版), 2013, 8(1): 76-82.

Tonglin Lu, Zhiping Hu, Liuwang Zeng, Zheng Jiang. Changes in secretory pathway Ca²⁺ -ATPase 2 following focal cerebral ischemia/reperfusion injury[J]. Neural Regeneration Research, 2013, 8(1): 76-82.

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引言

材料方法

Design

A randomized, controlled animal experiment.

Time and setting

This experiment was performed in the Central Laboratory of the Second Xiangya Hospital in Central South University, China during March 2011 to December 2011.

Materials

Animals were provided by the Central Animal Care of Central South University, Changsha, China (license No. SCXK (Xiang) 2009-0004). Sixty male Sprague-Dawley rats weighing 200–250 g were given free access to food and water and kept in a 12-hour light/dark cycle at 22.0 ± 1.0°C. All rats received humane care in compliance with the guidelines of Central South University of China. The treatment of animals conformed to the Guidance Suggestions for the Care and Use of Laboratory Animals, published by the Ministry of Science and Technology of China^[38].

Methods

Establishment of MCAO models

The rat model of focal cerebral ischemia-reperfusion was used, and rats were treated according to a previously published method^[39]. Briefly, male Sprague-Dawley rats were anesthetized with 10% (v/v) chloral hydrate (3.5 mL/100 g, intraperitoneal injection). After the right common carotid artery was exposed through a midline neck incision, the common carotid artery, external carotid artery and internal carotid artery were carefully separated from the adjacent tissue and vagus nerve. Next, a 4-0 silk suture was tied loosely around the mobilized external carotid artery stump. A 4-cm length of fish wire was inserted through the incision of the common carotid artery into the internal carotid artery and then into the circle of Willis, effectively occluding the middle cerebral artery. The silk sutures around the external carotid artery were lifted until the suture reached the middle cerebral artery. The silk suture around the common carotid artery stump was then tightened around the intraluminal occluders, which were made of fish wire, to prevent bleeding. The diameter of the tip was 0.26 mm for rats below 250 g and 0.28 mm for rats above 250 g. After 90 minutes of MCAO, the fish wire was withdrawn from the internal carotid artery into the stump of the common carotid artery, stopping as soon as resistance was felt. Reperfusion then started in the ischemic brain area. The animals were then returned to their cages after recovering from anesthesia and allowed to recover, with free access to food and water. Sham control animals (n = 6) were prepared in the same way without carotid occlusion.

The rats underwent 90 minutes of ischemia (n = 6), followed by 1, 3, and 24 hours of reperfusion (n = 6 each). Neurological examinations were performed 90 minutes after ischemia. The neurological findings were scored on a five-point scale: a score of 0 indicated no neurological deficit, a score of 1 (failure to extend left forepaw fully) indicated a mild focal neurological deficit, a score of 2 (circling to the left) revealed moderate focal neurological deficit, and a score of 3 (falling to the left) indicated a severe focal deficit; rats with a score of 4 did not walk spontaneously and had a depressed level of consciousness. Rats scoring 2–3 were chosen for the study.

Identification of models

At 24 hours after ischemia, histological staining was performed using 0.2% (w/v) 2,3,5-triphenyltetrazolium chloride. Normal tissues were red, while ischemic tissue was pale. Brains were cut into approximately 2-mm thick coronal slices starting 1 mm from the frontal pole. The infarct region was visualized by staining with 2,3,5-triphenyltetrazolium chloride solution for 15– 30 minutes at 37°C under dark conditions. The stained sections were then photographed.

Real-time fluorescence quantitative PCR assay for SPCA2 gene changes

Real-time PCR and data analysis were performed in a total volume of 20 μL using 48-well micro well plates and a StepOne™ Real-Time PCR System (Applied BioSystems, Foster City, CA, USA). RNA was extracted from the cortex of the experimental animals under RNAse-free conditions. RNA was extracted using Trizol (Beijing CoWin Biotech Co., Ltd., Beijing, China) and phenol-chloroform with subsequent reverse transcription. Reverse transcription was performed using the PrimeScript RT reagent Kit system (TaKaRa Biotechnology (Dalian) Co., Ltd., Dalian, Liaoning Province, China) with 1 µg of RNA. Multiplex PCR for SPCA2 and β-actin was carried out with 500 ng of each cDNA in a final volume of 10 μL containing 2 μL of 5 × PrimeScript Buffer (for Real Time), 0.5 μL PrimeScript RT Enzyme Mix 1, 0.5 μL Oligo dT Primer (50 µM), and 0.5 μL Random 6 mers (100 µM). The forward and reverse primers used to amplify SPCA2 and β-actin are shown in Table 1.

The primers were all purchased from Invitrogen, China. The PCR reaction conditions were: 30 seconds of denaturation at 95°C, followed by 45 cycles of 5 seconds of denaturation at 95°C, and 31 seconds of annealing at 62°C. The 2^–ΔΔCt method was used to determine the different expression levels.

Immunohistochemistry for SPCA2 protein changes

For immunohistochemical analysis, animals were anesthetized with 10% (v/v) chloral hydrate (3.5 mL/100 g body weight), followed by 4% (v/v) polyoxymethylene solution in 0.1 M PBS (pH 7.2). Brains were removed and fixed in 4% (v/v) polyoxymethylene solution for 8–12 hours at 4°C. Tissue samples were dehydrated and embedded in paraffin following routine procedures^[40]. After standard histological processing and embedding in paraffin, 4-mm-thick sections were prepared. All the following steps were carried out in a moist chamber. Deparaffinized and rehydrated sections were processed as follows: 2 × 10 minutes with xylene; 3 × 2 minutes with 100% (v/v) ethanol; 2 minutes with 80% (v/v) ethanol; 2 minutes with 70% (v/v) ethanol; and 3 × 3 minutes with PBS. Sections were then blocked for 10 minutes with 3% (v/v) peroxide at room temperature for endogenous peroxidase ablation. The method of antigen retrieval is shown below: sections were fixed in citric acid-citrate buffer solution (pH 6.0) for 15–20 minutes at 92–98°C. The sections were incubated with a rabbit anti-SPCA2 polyclonal antibody (1:50, sc-134761; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 6–8 hours at 4°C. The sections were then incubated for 30 minutes at 37°C with goat anti-rabbit IgG (PV-6001; Beijing Zhongshan Golden Bridge Company, China). Diaminobenzidine staining was enhanced by treating the sections for 10 seconds with DAB Enhancing Solution (Beijing Zhongshan Golden Bridge Company). Sections were washed, counterstained with Gill’s hematoxylin, cleared, mounted with Aquamount (Polysciences), and then examined by light microscopy with a Motic 5000 microscope (Berlin, Germany).

Protein determination

Five sections of each brain tissue sample were selected for image analysis. Under the optical microscope (magnification × 200), two areas of the frontal cortex and hippocampus were selected for imaging. The locations of the chosen field of vision of each slice were consistent. Image Pro-Plus 6.0 (Media Cybernetics, Silver Spring, USA) software was used for analysis after the photos had been inputted into the computer. The software measured the integrated absorbance of positive cells (part of the yellow) in each picture.

Statistical analysis

Data are expressed as mean ± SEM. All statistical analyses were performed using SPSS Statistics 17.0 (SPSS, Chicago, IL, USA). One-way analysis of variance followed by the Fisher’s least significant difference test was used for statistical analysis to determine significant differences between the control, ischemic and reperfusion groups. A P-value less than 0.05 was considered significant.

影响脑缺血再灌注损伤细胞内钙离子超载的ATP酶

Changes in secretory pathway Ca²⁺ -ATPase 2 following focal cerebral ischemia/reperfusion injury

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