Name: retinal pathophysiological evaluation in a rat model
Uploaded: 2022-05-06T15:00:00
Description: Watch this Scientific Journal Video about Retinal Pathophysiological Evaluation in a Rat Model at JoVE.com

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。

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
<ol><li>眼睛的去核和固定<ol>
<li>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。</li>
		<li>通过使用手术刀刀片在眼睛的鼻和颞叶?...

<ol>
	<li>Jonas, J. B., Sabanayagam, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+and+risk+factors+for+diabetic+retinopathy.">Epidemiology and risk factors for diabetic retinopathy.</a> Diabetic Retinopathy and Cardiovascular Disease. 27, 20-37 (2019).</li><li>Pandova, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Visual Impairment and Blindness. , (2019).</li><li>Mokdad, A. 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A., Dralands, L., Missotten, L., Al-Jadaan, I. A., Geboes, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+apoptosis+markers+in+the+retinas+of+human+subjects+with+diabetes.">Expression of apoptosis markers in the retinas of human subjects with diabetes.</a> Investigative Ophthalmology &amp; Visual Science. 45 (8), 2760-2766 (2004).</li><li>Schr&#246;der, S., Palinski, W., Schmid-Sch&#246;nbein, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activated+monocytes+and+granulocytes,+capillary+nonperfusion,+and+neovascularization+in+diabetic+retinopathy.">Activated monocytes and granulocytes, capillary nonperfusion, and neovascularization in diabetic retinopathy.</a> The American Journal of Pathology. 139 (1), 81 (1991).</li><li>Miyamoto, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+of+leukostasis+and+vascular+leakage+in+streptozotocin-induced+diabetic+retinopathy+via+intercellular+adhesion+molecule-1+inhibition.">Prevention of leukostasis and vascular leakage in streptozotocin-induced diabetic retinopathy via intercellular adhesion molecule-1 inhibition.</a> Proceedings of the National Academy of Sciences. 96 (19), 10836-10841 (1999).</li><li>Bhanushali, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linking+retinal+microvasculature+features+with+severity+of+diabetic+retinopathy+using+optical+coherence+tomography+angiography.">Linking retinal microvasculature features with severity of diabetic retinopathy using optical coherence tomography angiography.</a> Investigative Ophthalmology &amp; Visual Science. 57 (9), 519-525 (2016).</li><li>Wang, W., Lo, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diabetic+retinopathy:+pathophysiology+and+treatments.">Diabetic retinopathy: pathophysiology and treatments.</a> International Journal of Molecular Sciences. 19 (6), 1816 (2018).</li><li>Akbarzadeh, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+diabetes+by+streptozotocin+in+rats.">Induction of diabetes by streptozotocin in rats.</a> Indian Journal of Clinical Biochemistry. 22 (2), 60-64 (2007).</li><li>Weiss, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Streptozocin:+a+review+of+its+pharmacology,+efficacy,+and+toxicity.">Streptozocin: a review of its pharmacology, efficacy, and toxicity.</a> Cancer Treatment Reports. 66 (3), 427-438 (1982).</li><li>Karunanayake, E. H., Hearse, D. J., Mellows, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+metabolic+fate+and+elimination+of+streptozotocin.">The metabolic fate and elimination of streptozotocin.</a> Biochemical Society Transactions. 3 (3), 410-414 (1975).</li><li>Luna, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology. , (1968).</li><li>Okunlola, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histological+studies+on+the+retina+and+cerebellum+of+Wistar+rats+treated+with+Arteether.">Histological studies on the retina and cerebellum of Wistar rats treated with Arteether.</a> Journal of Morphological Sciences. 31 (01), 028-032 (2014).</li><li>Wallow, I., Engerman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permeability+and+patency+of+retinal+blood+vessels+in+experimental+diabetes.">Permeability and patency of retinal blood vessels in experimental diabetes.</a> Investigative Ophthalmology &amp; Visual Science. 16 (5), 447-461 (1977).</li><li>do Cartmo, A., Ramos, P., Reis, A., Proen&#231;a, R., Cunha-Vaz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breakdown+of+the+inner+and+outer+blood+retinal+barrier+in+streptozotocin-induced+diabetes.">Breakdown of the inner and outer blood retinal barrier in streptozotocin-induced diabetes.</a> Experimental Eye Research. 67 (5), 569-575 (1998).</li><li>Shires, T., Faeth, J., Pulido, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+levels+in+the+vitreous+of+rats+with+streptozotocin-induced+diabetes+mellitus.">Protein levels in the vitreous of rats with streptozotocin-induced diabetes mellitus.</a> Brain Research Bulletin. 30 (1-2), 85-90 (1993).</li><li>D'amato, R., Wesolowski, E., Smith, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscopic+visualization+of+the+retina+by+angiography+with+high-molecular-weight+fluorescein-labeled+dextrans+in+the+mouse.">Microscopic visualization of the retina by angiography with high-molecular-weight fluorescein-labeled dextrans in the mouse.</a> Microvascular Research. 46 (2), 135-142 (1993).</li><li>Gupta, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescein+angiography+refresher+course:+Here's+how+to+interpret+the+findings+of+this+useful+diagnostic+tool.">Fluorescein angiography refresher course: Here's how to interpret the findings of this useful diagnostic tool.</a> Review of Optometry. 138 (11), 60-65 (2001).</li><li>Edelman, J. L., Castro, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+image+analysis+of+laser-induced+choroidal+neovascularization+in+rat.">Quantitative image analysis of laser-induced choroidal neovascularization in rat.</a> Experimental Eye Research. 71 (5), 523-533 (2000).</li><li>Szab&#243;, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histological+evaluation+of+diabetic+neurodegeneration+in+the+retina+of+Zucker+diabetic+fatty+(ZDF)+rats.">Histological evaluation of diabetic neurodegeneration in the retina of Zucker diabetic fatty (ZDF) rats.</a> Scientific Reports. 7 (1), 1-17 (2017).</li><li>Margo, C. E., Lee, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fixation+of+whole+eyes:+the+role+of+fixative+osmolarity+in+the+production+of+tissue+artifact.">Fixation of whole eyes: the role of fixative osmolarity in the production of tissue artifact.</a> Graefe's Archive for Clinical and Experimental Ophthalmology. 233 (6), 366-370 (1995).</li><li>Tokuda, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+fixative+solution+for+retinal+morphology:+a+comparison+with+Davidson's+fixative+and+other+fixation+solutions.">Optimization of fixative solution for retinal morphology: a comparison with Davidson's fixative and other fixation solutions.</a> Japanese Journal of Ophthalmology. 62 (4), 481-490 (2018).</li><li>Luna, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology. Third edition. , (1968).</li><li>Skeie, J. M., Tsang, S. H., Mahajan, V. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evisceration+of+mouse+vitreous+and+retina+for+proteomic+analyses.">Evisceration of mouse vitreous and retina for proteomic analyses.</a> Journal of Visualized Experiments. (50), e2795 (2011).</li><li>D'Amato, R., Wesolowski, E., Smith, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscopic+visualization+of+the+retina+by+angiography+with+high-molecular-weight+fluorescein-labeled+dextrans+in+the+mouse.">Microscopic visualization of the retina by angiography with high-molecular-weight fluorescein-labeled dextrans in the mouse.</a> Microvascular Research. 46 (2), 135-142 (1993).</li><li>Atkinson, E. G., Jones, S., Ellis, B. A., Dumonde, D. C., Graham, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+size+of+retinal+vascular+leakage+determined+by+FITC-dextran+angiography+in+patients+with+posterior+uveitis.">Molecular size of retinal vascular leakage determined by FITC-dextran angiography in patients with posterior uveitis.</a> Eye (Lond). 5, 440-446 (1991).</li></ol>

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?...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Histology</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Abbott</td>
			<td></td>
			<td>Anesthesia agent</td>
		</tr>
		<tr>
			<td>Ketamine hydrochloride</td>
			<td>Troikaa Pharmaceuticals</td>
			<td></td>
			<td>Anesthesia agent</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Indian Immunologicals Limited</td>
			<td></td>
			<td>Anesthesia agent</td>
		</tr>
		<tr>
			<td>Pentobarbital sodium</td>
			<td>Zora Pharma</td>
			<td></td>
			<td>Euthanesia agent</td>
		</tr>
		<tr>
			<td>Fixative solution (1 % formaldehyde, 1.25 % Glutaraldehyde</td>
			<td>HiMedia, Avra</td>
			<td>MB059, ASG2529</td>
			<td>Prepared in-house</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Hayman</td>
			<td>F204325</td>
			<td>Dehydration</td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>HiMedia</td>
			<td>MB-180</td>
			<td>Clearing of ethanol or paraffin</td>
		</tr>
		<tr>
			<td>Paraffin wax</td>
			<td>HiMedia</td>
			<td>GRM10702</td>
			<td>used for embedding tissue</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>HiMedia</td>
			<td>TC503</td>
			<td>To prepare albumin coated slides. Glycerol and egg albumin is mixed in 1:1 ratio to coat on slides</td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Sisco Research laboratories Pvt. Ltd.</td>
			<td>65955</td>
			<td>For preparation of 1 % acid alcohol</td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>HiMedia</td>
			<td>AS119</td>
			<td>For preparation of eosin</td>
		</tr>
		<tr>
			<td>Scotts water</td>
			<td>Leica</td>
			<td>3802900</td>
			<td>Bluing reagent</td>
		</tr>
		<tr>
			<td>Papanicolaou&#39;s solution 1b Hematoxylin solution</td>
			<td>Sigma</td>
			<td>1.09254.0500</td>
			<td>Staining of nuclei</td>
		</tr>
		<tr>
			<td>Eosin</td>
			<td>HiMedia</td>
			<td>GRM115</td>
			<td>Staining of cytoplasm, 0.25 % solution was prepared in-house</td>
		</tr>
		<tr>
			<td>DPX Mountant media</td>
			<td>Sigma</td>
			<td>6522</td>
			<td>Visualization and protection of retinal sections</td>
		</tr>
		<tr>
			<td>Equipments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glassware</td>
			<td>Borosil</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Corneal forcep</td>
			<td>Stephens Instruments</td>
			<td>S5-1200</td>
			<td>Dissection</td>
		</tr>
		<tr>
			<td>Colibri forcep</td>
			<td>Stephens Instruments</td>
			<td>S5-1135</td>
			<td>Dissection</td>
		</tr>
		<tr>
			<td>Curved micro scissor</td>
			<td>Stephens Instruments</td>
			<td>S7-1311</td>
			<td>Dissection</td>
		</tr>
		<tr>
			<td>Vannas scissor</td>
			<td>Stephens Instruments</td>
			<td>S7-1387</td>
			<td>Dissection</td>
		</tr>
		<tr>
			<td>Iris scissor</td>
			<td>Stephens Instruments</td>
			<td>S7-1015</td>
			<td>Dissection</td>
		</tr>
		<tr>
			<td>Cassettes</td>
			<td>HiMedia</td>
			<td>PW1292</td>
			<td>To hold tissue during histology processing</td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>GT Sonic</td>
			<td>GT Sonic-D9</td>
			<td>Temperature maintenance</td>
		</tr>
		<tr>
			<td>Paraffin embedding station</td>
			<td>Myr</td>
			<td>EC 350</td>
			<td>Preparation of paraffin blocks</td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Zhengzhou Nanbei Instrument Equipment Co., Ltd.</td>
			<td>YD-335A</td>
			<td>Sectioning</td>
		</tr>
		<tr>
			<td>Blades</td>
			<td>Leica</td>
			<td>Leica 818</td>
			<td>Sectioning</td>
		</tr>
		<tr>
			<td>Slides</td>
			<td>HiMedia</td>
			<td>BG005</td>
			<td>Holding paraffin-tissue sections</td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>HiMedia</td>
			<td>BG014C</td>
			<td>To cover tissue after adding mounting media</td>
		</tr>
		<tr>
			<td>Blood Retinal Barrier breakdown</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Abbott</td>
			<td>B506</td>
			<td>Anesthesia</td>
		</tr>
		<tr>
			<td>Dry ice</td>
			<td>Not applicable</td>
			<td>Not applicable</td>
			<td>Dissection</td>
		</tr>
		<tr>
			<td>Bradford reagent</td>
			<td>Sigma</td>
			<td>B6916</td>
			<td>Protein quantification</td>
		</tr>
		<tr>
			<td>Equipments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Corneal forcep</td>
			<td>Stephens Instruments</td>
			<td>S5-1200</td>
			<td>Dissection</td>
		</tr>
		<tr>
			<td>Colibri forcep</td>
			<td>Stephens Instruments</td>
			<td>S5-1135</td>
			<td>Dissection</td>
		</tr>
		<tr>
			<td>Curved micro scissor</td>
			<td>Stephens Instruments</td>
			<td>S7-1311</td>
			<td>Dissection</td>
		</tr>
		<tr>
			<td>Vannas scissor</td>
			<td>Stephens Instruments</td>
			<td>S7-1387</td>
			<td>Dissection</td>
		</tr>
		<tr>
			<td>Iris scissor</td>
			<td>Stephens Instruments</td>
			<td>S7-1015</td>
			<td>Dissection</td>
		</tr>
		<tr>
			<td>Glassware</td>
			<td>Borosil</td>
			<td>Not applicable</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA coated tubes</td>
			<td>J.K Diagnostics</td>
			<td>Not applicable</td>
			<td>Separate plasma from whole blood</td>
		</tr>
		<tr>
			<td>Homogenization tubes</td>
			<td>MP Biomedicals</td>
			<td>SKU: 115076200-CF</td>
			<td>Homogenization of vitreous</td>
		</tr>
		<tr>
			<td>Homogenization caps</td>
			<td>MP Biomedicals</td>
			<td>SKU: 115063002-CF</td>
			<td>Homogenization of vitreous</td>
		</tr>
		<tr>
			<td>Glass beads</td>
			<td>MP Biomedicals</td>
			<td>SKU: 116914801</td>
			<td>Homogenization of vitreous</td>
		</tr>
		<tr>
			<td>Homogeniser</td>
			<td>Bertin Instruments</td>
			<td>P000673-MLYS0-A</td>
			<td>Homogenization of vitreous</td>
		</tr>
		<tr>
			<td>96-well plate - Transparent</td>
			<td>Grenier</td>
			<td>GN655101</td>
			<td>Protein quantification</td>
		</tr>
		<tr>
			<td>Plate reader</td>
			<td>Molecular devices</td>
			<td>SpectrMax M4</td>
			<td>Absorbance measurement</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>REMI</td>
			<td>CPR240 Plus</td>
			<td>Centrifugation</td>
		</tr>
		<tr>
			<td>Fluorescence Angiography</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Abbott</td>
			<td>B506</td>
			<td>Anesthesia</td>
		</tr>
		<tr>
			<td>FITC-dextran 70 kD (FITC, Dextran, Dibutylin dilaurate, DMSO</td>
			<td>FITC, Dextran and Dibutylin dilaurate from Sigma; DMSO from HiMedia</td>
			<td>FITC-F3651,Dextran-31390,Dibutylin dilaurate -29123, DMSO-TC185</td>
			<td>Prepared in-house</td>
		</tr>
		<tr>
			<td>Fluoroshied</td>
			<td>Sigma</td>
			<td>F6182</td>
			<td>Anti-fading mounting medium</td>
		</tr>
		<tr>
			<td>Equipments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Corneal forcep</td>
			<td>Stephens Instruments</td>
			<td>S5-1200</td>
			<td>Dissection</td>
		</tr>
		<tr>
			<td>Colibri forcep</td>
			<td>Stephens Instruments</td>
			<td>S5-1135</td>
			<td>Dissection</td>
		</tr>
		<tr>
			<td>Curved micro scissor</td>
			<td>Stephens Instruments</td>
			<td>S7-1311</td>
			<td>Dissection</td>
		</tr>
		<tr>
			<td>Vannas scissor</td>
			<td>Stephens Instruments</td>
			<td>S7-1387</td>
			<td>Dissection</td>
		</tr>
		<tr>
			<td>Iris scissor</td>
			<td>Stephens Instruments</td>
			<td>S7-1015</td>
			<td>Dissection</td>
		</tr>
		<tr>
			<td>Glassware</td>
			<td>Borosil</td>
			<td>Not applicable</td>
			<td></td>
		</tr>
		<tr>
			<td>Slides</td>
			<td>HiMedia</td>
			<td>BG005</td>
			<td>Flatmount preparation</td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>HiMedia</td>
			<td>BG014C</td>
			<td>To cover tissue after adding mounting media</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Leica</td>
			<td>DMi8</td>
			<td>Visualization of flatmount</td>
		</tr>
	</tbody>
</table>

retinal pathophysiological evaluation in a rat model

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。

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?...

Watch this Scientific Journal Video about Retinal Pathophysiological Evaluation in a Rat Model at JoVE.com

Retinal Pathophysiological Evaluation in a Rat Model

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。

大鼠模型中的视网膜病理生理学评估

A posterior segment eye disease like diabetic retinopathy alters the physiology of the retina. Diabetic retinopathy is characterized by a retinal ...

这些技术的总体目标是评估链脲佐菌素诱导的糖尿病视网膜病变大鼠模型中的视网膜病理生理学。糖尿病视网膜病变是失明的主要原因之一。持续的搜索正在进行中，以发现具有更好疗效和更长作用的药物来治疗糖尿病视网膜病变。在临床前动物模型中发现药理剂的过程起着至关重要的作用。这些动物模型有助于了解眼睛后节段组织的结构和功能改变。因此，我们将展示三种不同的技术来研究后段问题的这些变化。我们正在展示组织学以研究层中视网膜细胞的形态变化，BRB分解测定通过量化血浆中玻璃体中泄漏的蛋白质来研究受损的BRB，以及荧光血管造影术以使用FITC-葡聚糖染料可视化视网膜脉管系统。使用高剂量的戊巴比妥对大鼠实施安乐死，并确认其死亡，没有可检测到的心跳。通过使用手术刀刀片在眼睛的鼻和颞叶区域做切口来修饰眼睛。然后沿着其边缘切割，将眼睛从眼眶窝中取出。去除眼睛周围的多余脂肪，并将其置于5毫升固定溶液中。将眼睛在固定溶液中孵育24至48小时。固定后，将眼睛从固定溶液中取出，转移到装有10 mL PBS的培养皿中。使用微型镊子，用视神经握住眼睛，并在微型剪刀的帮助下在pars plana上划痕。切开角膜的整个边缘，将眼的前杯和后杯分开。取下晶状体并切断视神经。将后杯切成两半，穿过视神经。将每半部分转移到单独的盒式磁带中。通过将乙醇的浓度从50%逐渐增加到100%乙醇来使组织脱水。最后，用二甲苯清除乙醇。用60摄氏度的预热石蜡浸渍组织以取代组织中的二甲苯。使用钢模准备石蜡块，以将组织和盒作为底座。在准备砌块时，眼睛的视盘部分应面向钢模的底部。用石蜡填充盒，然后将其转移到阴凉的表面上。将刀片式服务器和胶盒安装到切片机上的相应支架上。使用切片机修剪多余的石蜡，并切割五到六个厚度为5微米的丝带。将丝带转移到预温水浴的表面上以展开丝带。在涂有白蛋白的载玻片上收集切片。让载玻片在37摄氏度下干燥过夜。要进行苏木精和曙红染色，请将载玻片在60摄氏度下预热1小时以融化石蜡。按照手稿中给出的染色程序进行。从组织补液开始，然后用苏木精染色，然后用曙红染色。现在，使组织脱水并在切片上添加安装介质。将盖玻片放在切片上，并在光学显微镜下可视化。为了进行血液 - 视网膜屏障分解测定，使用氯胺酮和木拉嗪麻醉大鼠，并通过脚趾捏确认麻醉状态。定位心脏并用24号针头插入1 mL注射器，通过心脏穿刺抽血。一旦收集了足够的血液，将其转移到EDTA涂层的管中。轻轻混合以防止溶血。对眼睛进行去核，并立即将其转移到干冰上。在冷冻条件下，通过去除角膜和晶状体开始解剖眼睛。使用镊子从眼睛后段拉出玻璃体，而不会受到视网膜的任何污染。将其转移到带有三到四个珠子的均质管中。使用珠状均质机以中速均质10秒对样品进行均质。将血液和玻璃体液样品转移到微量离心管中以进一步处理。将两个样品在4摄氏度下以5， 200 g离心10分钟。使用多通道移液器，将250微升布拉德福德试剂加入孔中。使用PBS稀释玻璃体样品10次，血浆样品稀释20次。将5微升稀释的样品加入布拉德福德试剂中并充分混合。将样品孵育20至30分钟，然后使用96孔板读数仪测量590纳米处的吸光度。样品的强度与蛋白质的浓度成正比。为了进行荧光血管造影，使用3%异氟醚麻醉大鼠，并通过脚趾冲头确认麻醉状态。在注入染料之前，将尾巴浸入温水中。将1 mL每mL FITC-葡聚糖溶液50mg注入侧尾静脉。让染料循环5至10分钟。使用戊巴比妥对大鼠实施安乐死，并通过无法检测到的心跳确认其死亡。对眼睛进行去核，然后进行平贴准备。要准备视网膜扁平支架，请将眼睛放在无纤维的Kimwipe上，并切除眼睛的前部。慢慢地将晶状体和玻璃体液从眼睛的后段拉出。将后段转移到充满PBS的培养皿中并切断视神经。现在，将尖镊子的尖端放在巩膜和视网膜之间。沿着杯子的边缘轻轻移动它，以确保视网膜在任何时候都不会附着在巩膜上。在视盘附近切开，使视网膜完全脱离巩膜。一旦确认视网膜未附着在任何一侧的巩膜上，将其缓慢推入PBS溶液中。在镊子的帮助下，将视网膜转移到干净的载玻片上，而不会对其造成任何损害。使用微型剪刀或手术刀刀片，在视网膜上做四个切口，如图所示，将其分成四个象限。从视网膜周围去除多余的PBS，并在视网膜平坦支架上添加防褪色安装介质。用盖玻片盖住它，并在共聚焦显微镜下可视化平面支架。这里需要注意的是，荧光血管造影的整个过程都是在最小的光线下进行的，以避免来自FITC-葡聚糖染料的荧光猝灭。在糖尿病大鼠中，视网膜细胞发生变性，导致进一步的并发症。这些结构变化可以通过组织学技术可视化，以确定视网膜的细胞数量和厚度。在糖尿病大鼠的情况下，视网膜层的厚度由于水肿而增加。因此，该技术有助于筛选各种潜在的药理学药物来治疗糖尿病视网膜病变。糖尿病引起的视网膜代谢变化导致的周细胞丢失和血液 - 视网膜屏障的破坏。由于蛋白质和其他生物分子泄漏到视网膜和玻璃体中，糖尿病大鼠玻璃体中总蛋白质的水平增加。预防血液 - 视网膜屏障的破坏可能会显着阻碍糖尿病视网膜病变的进展。在糖尿病视网膜病变中，血管内皮生长因子的表达增加，导致新血管的形成。这些血管会扰乱正常的视网膜结构，因此其神经支配是治疗糖尿病视网膜病变的首要问题。荧光血管造影技术用于可视化新血管和血管泄漏，如本文重点介绍的那样。然而，血管生成仅在糖尿病诱导 6 至 12 个月后才显现出来。该技术有助于我们量化药物治疗对血管生成和血管渗漏神经支配的影响。糖尿病视网膜病变的未来治疗需要更有效的药物，但未来的治疗只能通过更好地了解疾病的发病机制来实现。掌握这些技术可以帮助研究人员更好地了解糖尿病视网膜病变的发病机制，这可能导致发现更好的药物干预。

Research

JoVE Journal

Medicine

A Model of Chronic Nutrient Infusion in the Rat

在大鼠模型慢性营养输液

Chronic exposure to excessive levels of nutrients is postulated to  affect the function of several organs and tissues and to contribute to the  ...

科研

医学

Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model

在皮下肿瘤模型的评估肿瘤浸润白细胞亚群

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or ...

Quantitative Fundus Autofluorescence for the Evaluation of Retinal Diseases

定量自体荧光眼底视网膜疾病的评价

The retinal pigment epithelium (RPE) is juxtaposed to the overlying sensory retina, and supports the function of the visual system. Among the tasks ...

Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation

Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation

青光眼诱导在程序<em&gt;在体内</em&gt;大鼠模型和全安装视网膜准备

Glaucoma is a disease of the central nervous system affecting retinal ganglion cells (RGCs). RGC axons making up the optic nerve carry visual input to the ...

Evaluation of Cerebral Blood Flow Autoregulation in the Rat Using Laser Doppler Flowmetry

使用激光多普勒流测量测量大鼠脑血流自动调节的评估

When investigating the body&#39;s mechanisms for regulating cerebral blood flow, a relative measurement of microcirculatory blood flow can be obtained ...

Standardized Histomorphometric Evaluation of Osteoarthritis in a Surgical Mouse Model

手术小鼠模型中骨关节炎的标准化形态学评价

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, ...

Oxygen-Induced Retinopathy Model for Ischemic Retinal Diseases in Rodents

罗登斯缺血性视网膜疾病的氧诱导视网膜病变模型

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR ...

Acupuncture in a Rat Model of Asthma

哮喘鼠模型中的针灸

A high platform can fix rats without restriction and completely expose the acupoints on the back during acupuncture manipulation. This article describes ...

Retinal Pigment Epithelium Transplantation in a Non-human Primate Model for Degenerative Retinal Diseases

非人灵长类动物模型中视网膜色素上皮移植治疗退行性视网膜疾病

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These ...

Evaluation of Hepatic Glucose Production in a Polycystic Ovary Syndrome Mouse Model

多囊卵巢综合征小鼠模型中肝葡萄糖产生的评价

Polycystic ovary syndrome (PCOS) is a common disease that results in disorders of glucose metabolism, such as insulin resistance and glucose intolerance. ...