青光眼诱导在程序<em>在体内</em>大鼠模型和全安装视网膜准备
Summary
Glaucoma is characterized by damage to retinal ganglion cells. Inducing glaucoma in animal models can provide insight into the study of this disease. Here, we outline a procedure that induces loss of RGCs in an in vivo rat model and demonstrates the preparation of whole-mount retinas for analysis.
Abstract
青光眼是一种影响视网膜神经节细胞（RGC）的中枢神经系统的疾病。研资局轴突组成视神经进行视觉输入到大脑的视觉感受。损害视网膜神经节细胞和它们的轴突导致视力丧失和/或失明。尽管青光眼的具体原因不明，为疾病的主要危险因素之一是升高的眼内压。在动物模型青光眼诱导过程是一个有价值的工具，研究人员在研究RGC死亡的机制。这样的信息可导致可能在预防视力丧失的有助于有效的神经保护治疗的发展。本文中的协议描述诱导的青光眼的方法-像，其中50微升2M的高渗盐水注入巩膜静脉丛在体内大鼠模型的条件。血管的热烫表示成功注入。此过程会导致视网膜神经节细胞的损失来模拟青光眼。一个月以下注射后，将动物处死，眼睛被除去。接着，角膜，晶状体和玻璃体被除去以使一个眼罩。视网膜然后从眼睛的后部剥离和钉扎到使用仙人掌针SYLGARD菜肴。在这一点上，在视网膜的神经元可染色进行分析。从这个实验结果表明，相对于内部控制时，视网膜神经节细胞的约25％是在过程的一个月内丢失。这个过程允许在体内大鼠青光眼模型的视网膜神经节细胞死亡的定量分析。
Introduction
青光眼是一组影响的神经元中的视网膜，具体而言，视网膜神经节细胞1-2眼疾。这些细胞的轴突收敛到成为视神经承载可视信息，其中视觉感知的大脑。因此损害视网膜神经节细胞和其轴突导致视觉缺陷。
与青光眼疾病相关的主要特征是RGC变性和死亡，增加的眼内压（IOP），并视盘拔罐和萎缩。这些特点导致视野丧失或完全，不可逆转的失明。目前，青光眼全球7000万人造成3失明。因此，它是世界上第三大致盲4的原因。
RGC死亡的青光眼的确切机制尚不清楚。许多研究已经完成，以解开这个谜。然而，已经知道的是，青光眼的主要危险因素之一是增加我Ñ眼压由于房水（AH）在眼睛的前房的不规则循环。 AH充当眼睛的缺血性前房无色透明的替代血液。它滋养周围的细胞，分泌删除废物从代谢过程，运输的神经递质，并允许在病理状态1的药物和眼内炎症细胞的循环。
房水流通的维护包括睫状体和小梁网。房水由睫状体产生的。然后，它流入前房维持眼组织的总体健康状况。 75 – 房水外流的80％时，流体通过在睫状肌海绵组织的三层过滤正在积极通过非颜料睫状体上皮分泌的。穿过小梁网并通过施累姆氏管其制止所述流体排出独立实体进入血液系统5。其余20 -流出的25％绕过小梁网和通过超滤和扩散通过uveo巩膜途径被动分泌。这一途径似乎是相对独立的眼压1。
当房水的生产和流出都失去平衡，压力增大眼内。如上所述，这种增加的眼内压是在青光眼的发展的主要危险因素。这种压力导致在眼睛的回视网膜神经元的复杂的层的损伤。损伤视神经的视网膜神经节细胞轴突导致大脑不再接收准确的视觉信息。其结果是，视觉的知觉丧失，并且可以发生完全失明。
到目前为止，还没有治愈青光眼。不同的治疗方法存在的主要目的是降低眼压。这些措施包括外用药物类，如β1的肾上腺素能受体阻断剂，或局部前列腺素类似物。 β阻滞剂通过减少房水的产生7的降低眼内压。前列腺素的作用，通过增加房水8-14外流以减少眼压。 α-肾上腺素能激动剂和碳酸酐酶抑制剂也可作为治疗的辅助方法。阿尔法肾上腺素受体 激动剂通过葡萄膜巩膜途径15-17加大流出。碳酸酐酶抑制剂，通过抑制酶的18降低生产AH的。更侵入性程序也被用于治疗青光眼。激光小梁成形术是用于增加房水19的流出。另一个手术疗法，称为小梁切除术，创建了一个另类的排水现场时，传统的小梁途径被阻断20-21过滤啊。
这些治疗方案已经知道EFFectively眼压降低。然而，青光眼患者的高达40％的正常显示眼压水平表明需要更完整的治疗方法。22,23此外，在青光眼看到视网膜神经节细胞的死亡是不可逆的，一旦开始，目前的治疗不停止疾病的进展24-28。这强调了靶向神经元本身的生存有效的神经保护治疗的需要。青光眼模型的发展是这一发展的关键。
在这项研究中，我们证明使用最初由莫里森29中概述的改性过程诱导成年龙Evans大鼠青光眼样作用的方法。在此过程中，2M的高渗盐水注射到巩膜静脉丛被瘢痕组织引起青光眼样的条件，以减少在小梁网，导致增加的眼内压和视网膜神经节细胞的w的显著损失水状液流出ithin程序30-31的一个月。青光眼诱导程序，如这里描述的，可能是关键解锁在青光眼治疗的新发展。
Protocol
使用受试动物的所有程序已经按照动物护理和使用委员会（IACUC）在西密歇根大学研究所的标准。 1.动物使用雄性和雌性大鼠3个月大了这项研究。 保持12小时光/暗周期动物自由获取食物和水。 2. KAX鸡尾酒动物麻醉的制备在5ml氯胺酮（100毫克/毫升）与1ml乙酰丙嗪（10毫克/毫升）和3毫升蒸馏水溶解50mg的甲苯噻嗪（20毫克/毫升）?…
Representative Results
本节说明用于诱导在体内大鼠青光眼模型青光眼样条件的装置组件和过程。我们表明用于执行高渗盐水注射导致眼内压增加的个体的工具和设备。我们展示了注入以其特有的漂白效果和前房导致的浑浊外观巩膜静脉丛。我们还描述了视网膜去除和平面安装丢失RGC的分析过程。最后，我们显示在视网膜神经节细胞存活的注射的效果。作为视网膜神经节细胞的分布是在?…
Discussion
这个协议描述诱导在体内大鼠模型青光眼样病症的方法。此过程使用高渗盐水的注射诱导疤痕在小梁网29，32。发展疤痕组织闭塞房水的流出这增加前房压力。随着流出和压力降低建立，通过弹性悬挂韧带镜头推回到玻璃体腔。玻璃体然后施加压力到视网膜损害脆弱的视网膜神经细胞。使用此过程我们的结果表明，视网膜神经节细胞数开始以2周1个月后的过程神经节细胞损失显著损?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
C. Linn is supported by an NIH grant (NIH NEI EY022795).
Materials
| Xylazine hydrochloride, Minimum 99% | Sigma, Life Science | X1251-1G | |
| Ketamine hydrochloride injection, USP, 100mg/mL | Putney, Inc | NDC 26637-411-01 | 10 mL bottle |
| Acepromazine Maleate, 10mg/mL | Phoenix Pharmaceutical, Inc | NDC 57319-447-04, 670008L-03-0408 | 50 mL bottle |
| Serum bottle, 10 mL | VWR | 16171319 | Borosilicate glass |
| 1 mL insulin syringe | VWR | BD329410 | 28 gauge needle |
| Sodium chloride | Sigma | S7653 | 2 M Solution |
| Microelectrode Puller | Narishige Group | PP-830 | |
| Heavy Polished Standard and Thin Walled Borosilicate Tubing | Sutter Instruments | B150-86-10HP | without filament, 0.86 mm |
| Microfil syringe needle for filling micropipettes | World Precision Instruments, Inc | MF28G | |
| 18 gauge Luer-Lock needle | Fisher Scientific | 1130421 | Syringe needle |
| Flexible Polyethylene Tubing | Fisher Scientific | 22046941 | 0.034 inch diameter, approximately 10 inches |
| Proparacaine Hydrochloride Opthalmic Solution, USP, 0.5% | Akorn, Inc | NDC 17478-263-12 | 15 mL sterile bottle |
| Curved Scissors | Fine Science Tools | 14061-11 | |
| Microscope | Leica | StereoZoom 4 | |
| Hemostat Clamp | Fine Science Tools | 1310912 | curved edge |
| Triple Antibiotic Ointment | Fisher Scientific | NC0664481 | |
| Scalpel handle | Fine Science Tools | 10004-13 | |
| Scalpel blade # 11 | Fine Science Tools | 10011-00 | |
| 60 mm x 15 mm Disposable Petri Dish | VWR | 351007 | |
| Phosphate Buffered Saline 10x Concentrate | Sigma, Life Science | P7059-1L | 1x dilution |
| Spring Scissors | Fine Science Tools | 15009-08 | |
| Forceps (2), Dumont # 5 | Fine Science Tools | 11251-30 | |
| 3 mL Transfer Pipets, polyethylene, non sterile | BD Biosciences | 357524 or 52947-948 | 1 and 2 mL graduations |
| 35 mm x 10 mm Easy Grip Petri Dish | BD Biosciences | 351008 | |
| Sylgard 184 | VWR | 102092-312 | |
| Cactus Needles | N/A | N/A | |
| Paraformaldehyde | EMD Millipore | PX0055-3 or 818715.0100 | Made into a 4% solution |
| Triton X-100 | Sigma | T9284-100 mL | Made into both a 1% and 0.1% solution |
| Fetal Bovine Serum | Atlanta Biological | S11150 | 500 ml |
| Purified Mouse Anti-Rat CD90/mouse CD90.1 | BD Pharmingen | Cat 554892 | 1:300 dilution |
| Alexa Fluor 594 goat anti-mouse | Life Technologies | A11005 | 1:300 dilution |
| Microscope Slides | Corning | 2948-75×25 | |
| Glycerol | Sigma | G5516-100 mL | 50% glycerol to 50% PBS, by weight |
| Coverglass | Corning | 2975-225 | Thickness 1 22 x 50 mm |
| Confocal Microscope | Nikon | C2 Eclipse Ti |
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Citazione di questo articolo
Gossman, C. A., Linn, D. M., Linn, C. Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation. J. Vis. Exp. (109), e53831, doi:10.3791/53831 (2016).