Neuroactive steroids protect retinal tissue through σ1 receptors
Neuroactive steroids protect retinal tissue through σ1 receptors
复制标题
DOI：
10.1111/j.1742-7843.2007.00044.x
复制
发表时间：
2007-03-01
影响因子：
3.1
通讯作者：
Drago, Filippo
中科院分区：
文献类型：
--
作者：
Bucolo, Claudio;Drago, Filippo
材料和方法。实验动物为雄性SD大鼠，体重250~300g。动物手术得到了卡塔尼亚大学(意大利卡塔尼亚)动物保护和利用委员会的批准。我们实验室在相同实验条件下的初步研究没有显示出任何性别差异。视网膜缺血/再灌流模型的制作如前所述[2]。简单地说，大鼠被麻醉，前房用一根30号的针连接到一个升高的盐水池上。升高眼压至120 mm Hg45min，造成视网膜缺血。用手持检眼镜对视网膜血管进行肉眼检查并验证缺血情况。45分钟后，降低生理盐水池，并允许眼内压和视网膜循环在10分钟内恢复正常。导管被从角膜上取出，动物们被允许康复。大鼠分别以0.1mgkg和1mgkg的剂量腹腔注射17β-雌二醇、脱氢表雄酮-S、σ-1受体激动剂Pre-084，30min。在短暂性脑缺血损伤前，用或不用BD1047{[2-(3，4-二氯苯基)乙基]-N-甲基-2-(二氨基)乙胺}(σ-1受体拮抗剂)预处理45min。在缺血损伤之前。本研究中使用的化合物的剂量是在先前工作的基础上选择的[2]。再灌注7d后处死动物，在0.1M磷酸盐缓冲液(pH 7.4；4℃过夜)中加入1%乙醛和4%福尔马林固定，包埋于副质粒中。5微米厚的切片穿过眼睛的视盘，进行苏木精/伊红染色。用连接到光学显微镜的数码相机将视神经头中心两侧约1.5 mm处的视网膜各层记录到硬盘上作为图像数据。测量两个不同视网膜层的厚度以评估缺血性视网膜损伤：(I)内视网膜层，从内界膜延伸到外丛状层和外核层的边界；(Ii)内丛状层。数据分析采用Newman-Keuls检验。在P<；0.05水平上接受统计学意义。形态计量学分析显示，缺血后7d，赋形剂治疗组缺血眼视网膜内层、内网状层均较正常眼薄(表1)。具有代表性的光学显微镜照片显示缺血诱导的视网膜
Materials and methods. Male Sprague-Dawley rats weighing 250–300 g were used. Animal procedures were approved by the Animal Care and Use Committee of the University of Catania (Catania, Italy). Preliminary studies in our laboratory under the same experimental conditions did not show any gender differences. Retinal ischaemia/reperfusion model has been performed as described previously [2]. Briefly, rats were anaesthetized and the anterior chamber was cannulated with a 30-gauge needle attached to a raised saline reservoir. Retinal ischaemia was induced by elevating the intraocular pressure to 120 mmHg for 45 min. A hand-held ophthalmoscope was used to visually inspect the retinal blood vessels and verify ischaemia. After 45 min., the saline reservoir was lowered and the intraocular pressure and retinal circulation was allowed to return to normal over a period of 10 min. The cannula was removed from the cornea and the animals were allowed to recover. Rats were injected (0.1 and 1 mg/kg intraperitoneally) with 17β-oestradiol, DHEA-S, PRE-084 (σ1 receptor agonist), 30 min. prior to the transient ischaemic insult, with or without a pre-treatment with BD1047 {[2-(3, 4-dichlorophenyl) ethyl]-N-methyl-2-(diamino) ethylamine}(σ1 receptor antagonist) 45 min. prior to the ischaemia damage. The dose of compounds used in this study has been chosen on the basis of previous work [2]. After ischaemia, the rats were left to recover for 7 days (reperfusion), then the animals were killed, and the globes were fixed in 1% gluteraldehyde and 4% formalin in 0.1 M phosphate buffer (pH 7.4; overnight at 4 C), and embedded in paraplast. Five-micrometer thick sections were cut through the optic disc of the eye and stained with haematoxylin/eosin. The retinal layers in each section at a distance of approximately 1.5 mm of either side of the centre of the optic nerve head were recorded onto hard-disk as image data using a digital camera connected to a light microscope. The thickness of two different retinal layers was measured to evaluate ischaemic retinal damage:(i) the inner retinal layer, which extends from the inner limiting membrane to the boundary of the outer plexiform layer and the outer nuclear layer and (ii) the inner plexiform layer. Data were analysed using Newman-Keuls test. Statistical significance was accepted at a level of P< 0.05.Results. As indicated by the morphometric analysis, the inner retinal layer and the inner plexiform layer in the ischaemic eye of vehicle-treated group were thinner than in the normal eye at 7 days after reperfusion (table 1). Representative photomicrographs demonstrating ischaemia-induced retinal