大鼠模型中的视网膜病理生理学评估
Summary
糖尿病视网膜病变是失明的主要原因之一。组织学、血液-视网膜屏障破坏测定和荧光血管造影是了解视网膜病理生理学的宝贵技术，可以进一步提高针对糖尿病视网膜病变的药物筛查效率。
Abstract
像糖尿病视网膜病变这样的后段眼病会改变视网膜的生理学。糖尿病视网膜病变的特征是视网膜脱离、血液-视网膜屏障 （BRB） 破裂和视网膜血管生成。体内大鼠模型是检查视网膜结构和功能变化的宝贵实验工具。我们在大鼠模型中提出了三种不同的实验技术，以识别视网膜细胞，视网膜脉管系统和受损BRB的形态变化。视网膜组织学用于研究各种视网膜细胞的形态。此外，通过视网膜细胞计数和不同视网膜层的厚度测量进行定量测量。BRB分解测定用于确定由于BRB分解而导致的眼外蛋白从血浆泄漏到玻璃体组织。荧光血管造影用于通过使用FITC-葡聚糖染料可视化视网膜脉管系统来研究血管的血管生成和泄漏。
Introduction
糖尿病视网膜病变（DR）是糖尿病最复杂的继发性并发症之一。它也是全世界劳动年龄人口中可预防的失明的主要原因。在最近一项针对 3240 万盲人的荟萃分析中，有 830，000 人（2.6%）因 DR1 而失明。2015 年，糖尿病导致的视力丧失比例在全球排名第七，为 1.06%（0.15-2.38）2，3。
糖尿病视网膜病变由后眼组织血管异常诊断。临床上，它分为两个阶段 – 不增殖性DR（NPDR）和增殖性DR（PDR），基于视网膜中的血管形成。高血糖被认为是DR的有效调节剂，因为它涉及与视网膜中神经变性4，5，炎症6，7和微脉管8 有关的几种途径。由高血糖引起的多种代谢并发症包括晚期糖基化终产物 （AGEs）、多元醇途径、己糖胺途径和蛋白激酶-C 途径的积累。这些途径负责基于糖尿病视网膜病变不同阶段的细胞增殖（内皮细胞），迁移（周细胞）和凋亡（神经视网膜细胞，包细胞和内皮细胞）。这些代谢改变可导致生理变化，例如视网膜脱离、视网膜细胞丧失、血液-视网膜屏障 （BRB） 分解、动脉瘤和血管生成9。
链脲佐菌素（STZ）诱导的1型糖尿病是大鼠中评估糖尿病发病机制及其并发症的成熟和广泛接受的实践。STZ的致糖尿病作用是由于胰岛β细胞的选择性破坏10。结果，动物将经历胰岛素缺乏，高血糖，烦渴和多尿，所有这些都是人类1型糖尿病的特征11。对于重度糖尿病诱导，STZ在成年期静脉注射或腹膜内以40-65mg / kg体重给药。大约72小时后，这些动物的血糖水平大于250mg / dL10，12。
为了了解由于神经变性，炎症和血管生成引起的视网膜的生理变化，应在实验动物模型中优化不同的技术。视网膜细胞和视网膜血管的结构和功能变化可以通过各种技术进行研究，例如组织学，BRB分解测定和荧光血管造影。
组织学涉及在微观水平上研究细胞，组织和器官的解剖结构。它在细胞/组织的结构和功能之间建立相关性。执行几个步骤来可视化和识别组织结构的微观变化，从而比较健康和患病的对应物13。因此，必须一丝不苟地标准化组织学的每个步骤。视网膜组织学涉及的各种步骤包括标本固定，修剪标本，脱水，清除，用石蜡浸渍，石蜡包埋，切片和染色（苏木精和曙红染色）13，14。
在健康的视网膜中，分子在视网膜上的运输由BRB控制，BRB由内侧的内皮细胞和周围细胞以及外侧的视网膜色素上皮细胞组成。然而，在患病期间，BRB内皮细胞和周细胞开始退化，BRB也受到损害15。由于这种BRB分解，许多低分子量分子泄漏到玻璃体和视网膜组织中16。随着疾病的进展，许多其他蛋白质分子（低分子量和高分子量）也由于稳态紊乱而泄漏到玻璃体和视网膜组织中17。它导致各种其他并发症，最终导致黄斑水肿和失明。因此，量化玻璃体中的蛋白质水平并比较健康和糖尿病状态的测量会损害BRB。
荧光血管造影是一种用于使用荧光染料研究视网膜和脉络膜血液循环的技术。它用于 通过 静脉注射途径或心脏注射来可视化视网膜和脉络膜的脉管系统18。一旦染料被注射，它首先到达视网膜动脉，然后是视网膜静脉。染料的这种循环通常在注射染料19后5至10分钟内完成。它是诊断各种后段眼部疾病的重要技术，包括糖尿病视网膜病变和脉络膜新生血管形成20。它有助于检测正常和疾病条件下的主要和次要脉管系统变化。
Protocol
该协议遵循海得拉巴校区BITS-Pilani机构动物伦理委员会提供的所有动物护理指南。 1. 视网膜组织学 眼睛的去核和固定 使用通过腹膜内途径注射的高剂量戊巴比妥（150mg / kg）对2至3个月大的糖尿病Wistar雄性大鼠以及年龄匹配的对照组（14至15周龄）实施安乐死。没有可检测到的心跳在2-5分钟内确认死亡。 通过使用手术刀刀片在眼睛的鼻和颞叶?…
Representative Results
视网膜组织学在糖尿病视网膜中，视网膜细胞发生变性。此外，由于水肿22，视网膜层的厚度增加。苏木精和曙红染色后获得的图像可用于细胞计数和不同层厚度的测量，如图 2 所示，使用ImageJ。 血液-视网膜屏障分解测定由于糖尿病大鼠的BRB受损，泄漏变得突出，导致生物分子从血浆到视网膜和玻璃体?…
Discussion
组织学
进行视网膜组织学检查以可视化视网膜细胞和层的形态变化。需要优化各种步骤，包括固定溶液的选择、固定持续时间、脱水和石蜡浸渍。组织大小不应超过3毫米，因为固定剂渗透变得缓慢。常用的4%多聚甲醛即使在健康的眼睛中也会导致视网膜脱离，因为与房水和玻璃体房相比，溶液的渗透压相对较高，从而导致假阳性结果23。溶液的高渗透压导致体积?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
作者希望承认印度医学研究理事会（ICMR;ITR-2020-2882）为Nirmal J博士提供资金支持。我们还要感谢大学委员会赠款为Manisha Malani和海得拉巴校区BITS-Pilani中央分析实验室设施提供初级研究奖学金，以提供基础设施。
Materials
| Histology | |||
| Reagents | |||
| Isoflurane | Abbott | Anesthesia agent | |
| Ketamine hydrochloride | Troikaa Pharmaceuticals | Anesthesia agent | |
| Xylazine | Indian Immunologicals Limited | Anesthesia agent | |
| Pentobarbital sodium | Zora Pharma | Euthanesia agent | |
| Fixative solution (1 % formaldehyde, 1.25 % Glutaraldehyde | HiMedia, Avra | MB059, ASG2529 | Prepared in-house |
| Ethanol | Hayman | F204325 | Dehydration |
| Xylene | HiMedia | MB-180 | Clearing of ethanol or paraffin |
| Paraffin wax | HiMedia | GRM10702 | used for embedding tissue |
| Glycerol | HiMedia | TC503 | To prepare albumin coated slides. Glycerol and egg albumin is mixed in 1:1 ratio to coat on slides |
| Hydrochloric acid | Sisco Research laboratories Pvt. Ltd. | 65955 | For preparation of 1 % acid alcohol |
| Acetic acid | HiMedia | AS119 | For preparation of eosin |
| Scotts water | Leica | 3802900 | Bluing reagent |
| Papanicolaou's solution 1b Hematoxylin solution | Sigma | 1.09254.0500 | Staining of nuclei |
| Eosin | HiMedia | GRM115 | Staining of cytoplasm, 0.25 % solution was prepared in-house |
| DPX Mountant media | Sigma | 6522 | Visualization and protection of retinal sections |
| Equipments | |||
| Glassware | Borosil | ||
| Corneal forcep | Stephens Instruments | S5-1200 | Dissection |
| Colibri forcep | Stephens Instruments | S5-1135 | Dissection |
| Curved micro scissor | Stephens Instruments | S7-1311 | Dissection |
| Vannas scissor | Stephens Instruments | S7-1387 | Dissection |
| Iris scissor | Stephens Instruments | S7-1015 | Dissection |
| Cassettes | HiMedia | PW1292 | To hold tissue during histology processing |
| Water bath | GT Sonic | GT Sonic-D9 | Temperature maintenance |
| Paraffin embedding station | Myr | EC 350 | Preparation of paraffin blocks |
| Microtome | Zhengzhou Nanbei Instrument Equipment Co., Ltd. | YD-335A | Sectioning |
| Blades | Leica | Leica 818 | Sectioning |
| Slides | HiMedia | BG005 | Holding paraffin-tissue sections |
| Coverslips | HiMedia | BG014C | To cover tissue after adding mounting media |
| Blood Retinal Barrier breakdown | |||
| Reagents | |||
| Isoflurane | Abbott | B506 | Anesthesia |
| Dry ice | Not applicable | Not applicable | Dissection |
| Bradford reagent | Sigma | B6916 | Protein quantification |
| Equipments | |||
| Corneal forcep | Stephens Instruments | S5-1200 | Dissection |
| Colibri forcep | Stephens Instruments | S5-1135 | Dissection |
| Curved micro scissor | Stephens Instruments | S7-1311 | Dissection |
| Vannas scissor | Stephens Instruments | S7-1387 | Dissection |
| Iris scissor | Stephens Instruments | S7-1015 | Dissection |
| Glassware | Borosil | Not applicable | |
| EDTA coated tubes | J.K Diagnostics | Not applicable | Separate plasma from whole blood |
| Homogenization tubes | MP Biomedicals | SKU: 115076200-CF | Homogenization of vitreous |
| Homogenization caps | MP Biomedicals | SKU: 115063002-CF | Homogenization of vitreous |
| Glass beads | MP Biomedicals | SKU: 116914801 | Homogenization of vitreous |
| Homogeniser | Bertin Instruments | P000673-MLYS0-A | Homogenization of vitreous |
| 96-well plate – Transparent | Grenier | GN655101 | Protein quantification |
| Plate reader | Molecular devices | SpectrMax M4 | Absorbance measurement |
| Centrifuge | REMI | CPR240 Plus | Centrifugation |
| Fluorescence Angiography | |||
| Reagents | |||
| Isoflurane | Abbott | B506 | Anesthesia |
| FITC-dextran 70 kD (FITC, Dextran, Dibutylin dilaurate, DMSO | FITC, Dextran and Dibutylin dilaurate from Sigma; DMSO from HiMedia | FITC-F3651,Dextran-31390,Dibutylin dilaurate -29123, DMSO-TC185 | Prepared in-house |
| Fluoroshied | Sigma | F6182 | Anti-fading mounting medium |
| Equipments | |||
| Corneal forcep | Stephens Instruments | S5-1200 | Dissection |
| Colibri forcep | Stephens Instruments | S5-1135 | Dissection |
| Curved micro scissor | Stephens Instruments | S7-1311 | Dissection |
| Vannas scissor | Stephens Instruments | S7-1387 | Dissection |
| Iris scissor | Stephens Instruments | S7-1015 | Dissection |
| Glassware | Borosil | Not applicable | |
| Slides | HiMedia | BG005 | Flatmount preparation |
| Coverslips | HiMedia | BG014C | To cover tissue after adding mounting media |
| Confocal microscope | Leica | DMi8 | Visualization of flatmount |
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Citer Cet Article
Malani, M., Nirmal, J. Retinal Pathophysiological Evaluation in a Rat Model. J. Vis. Exp. (183), e63111, doi:10.3791/63111 (2022).