角膜缘血管丛全圆烧灼治疗啮齿动物手术诱发的青光眼
Summary
该协议的目的是表征一种基于角膜缘血管丛 360° 热烧灼的青光眼性神经退行性变性的新模型，诱导亚急性高眼压症。
Abstract
青光眼是全球第二大失明原因，是一组异质性眼部疾病，其特征是视神经结构损伤和视网膜神经节细胞 （RGC） 变性，通过中断视觉信息从眼睛到大脑的传递而导致视觉功能障碍。眼压升高是最重要的危险因素;因此，已经通过遗传或实验方法在啮齿动物中开发了几种高眼压模型来研究疾病的原因和影响。其中，已经报告了一些局限性，例如手术侵入性、功能评估不足、需要广泛培训以及视网膜损伤的高度可变扩展。本工作描述了一种简单、低成本和高效的诱发啮齿动物高眼压的方法，该方法基于角膜缘血管丛的低温、全圆烧灼，角膜缘血管丛是房水引流的主要成分。新模型提供了一种技术上简单、无创且可重复的亚急性高眼压症，与进行性 RGC 和视神经变性相关，以及独特的术后临床恢复率，允许通过电生理学和行为学方法 进行体内 功能研究。
Introduction
医学文献将青光眼理解为一组异质性视神经病，其特征是视网膜神经节细胞 （RGC）、树突、体细胞和轴突进行性变性，导致视盘结构拔罐（挖掘）和视神经功能退化，通过中断视觉信息从眼睛到大脑的传递，导致不受控制的病例黑朦1.青光眼是目前全球不可逆转性失明的最常见原因，预计到 2040 年将达到约 1.118 亿人2，从而严重影响患者的生活质量 （QoL），并导致重大的社会经济问题3。
眼内压 （IOP） 升高是青光眼发生和进展的最重要且唯一可改变的危险因素之一。在多种类型的青光眼中，除正常张力型青光眼 （NTG） 外，所有青光眼在临床病史中的某个时间都与眼压升高有关。尽管在靶向眼压和减缓或阻止疾病进展方面取得了显着的临床和手术进展，但患者仍然因青光眼而失明 4,5。因此，彻底了解这种疾病的复杂和多因素病理生理学对于开发更有效的治疗方法至关重要，特别是为RGC提供神经保护。
在了解疾病机制的各种实验方法中，基于高眼压症（OHT）的动物模型与人类青光眼最为相似。啮齿动物模型特别有用，因为它们成本低，易于操作，可以进行基因操作，寿命短，并且具有与人类相当的眼部解剖学和生理学特征，例如房水产生和引流6,7,8,9,10,11,12,13 .目前使用的模型包括将高渗盐水注射到巩膜外静脉后小梁网硬化14、前房内注射微珠 15 或粘弹性物质 16、涡静脉烧灼 17、用氩激光光凝小梁网 18、环缘缝合19，以及使用年龄相关 OHT 的转基因模型（DBA/2J 小鼠）8.然而，侵入性、术后角膜混浊、眼前节破坏、广泛的学习曲线、昂贵的设备和高度可变的术后眼压是与当前模型相关的少数报告陷阱之一，因此需要开发 OHT 的替代模型来克服这些问题20,21,22。
本方案正式确定了一种新的外科手术，以诱导 OHT 作为青光眼的代理，基于啮齿动物的角膜缘丛烧灼 （LPC）23。这是一种简单、可重复、可访问且非侵入性的模型，可提供高效率和低 IOP 升高的变异性，与独特的高临床完全恢复率相关，因此在减少每个实验中使用的动物数量的情况下提供 体内 功能评估。手术技术诱导亚急性 OHT，并在几天内逐渐恢复到基线水平，这模拟了急性闭角型青光眼中可见的高血压发作。此外，模型中的眼压恢复之后是持续的青光眼性神经变性，这对于未来 RGC 继发性变性的机制研究很有用，尽管 IOP 得到充分控制，但 RGC 的继发性变性发生在几例人类青光眼病例中。
Protocol
所有程序均按照视觉和眼科研究协会 （ARVO） 的《在眼科和视觉研究中使用动物的声明》进行，并得到里约热内卢联邦大学健康科学中心科学中心动物使用伦理委员会的批准（协议 083/17）。在本研究中，使用了两性李斯特帽大鼠，年龄为2-3个月，体重为180-320克。然而，该程序可以适用于不同年龄范围的不同大鼠品系。 1. 高眼压手术及临床随访 家畜在受?…
Representative Results
定量变量表示为平均值±均值标准误差 （SEM）。除了OHT组和对照组之间的IOP动态比较（图1F）外，使用双向方差分析进行统计分析，然后进行Sidak的多重比较检验。p 值< 0.05 被认为具有统计学意义。 图 1 说明了全圆角膜缘神经丛烧灼术 （LPC） 模型的手术步骤，具有重要的标志，例如 360° 热诱导的角膜缘血管消失，以及手术结束时?…
Discussion
角膜缘丛烧灼术 （LPC） 是一种新型的后小梁模型，其优点是它针对不需要结膜或肌腫夹层的易于接近的血管结构17,28。与涡旋静脉烧灼模型（一种基于脉络膜静脉引流手术损伤的著名 OHT 模型）不同，静脉淤血预计不会影响 LPC 模型中的眼压升高，因为角膜缘静脉位于房水流出的上游。此外，它在技术上易于学习且成本低廉，主要需要低温热烧灼器。此…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢我们的实验室技术人员 José;尼尔森·多斯桑托斯、戴安·曼达里诺·托雷斯、何塞·弗朗西斯科·蒂布尔西奥、吉尔多·布里托·德索萨和卢西亚诺·卡瓦尔坎特·费雷拉。这项研究由 FAPERJ、CNPq 和 CAPES 资助。
Materials
| Acetone | Isofar | 201 | Used for electron microscopy tissue preparation (step 5) |
| Active electrode for electroretinography | Hansol Medical Co | – | Stainless steel needle 0.25 mm × 15 mm |
| Anestalcon | Novartis Biociências S/A | MS-1.0068.1087 | Proxymetacaine hydrochloride 0.5% |
| Calcium chloride | Vetec | 560 | Used for electron microscopy tissue preparation (step 5) |
| Cautery Low Temp Fine Tip 10/bx | Bovie Medical Corporation | AA00 | Low-temperature ophthalmic cautery |
| Cetamin | Syntec do Brasil Ltda | 000200-3-000003 | Ketamine hydrochloride 10% |
| DAKO | Dako North America | S3023 | Antifade mounting medium |
| DAPI | Thermo Fisher Scientific | 28718-90-3 | diamidino-2-phenylindole; blue fluorescent nuclear counterstain; emission at 452±3 nm |
| Ecofilm | Cristália Produtos Químicos Farmacêuticos Ltda | MS-1.0298.0487 | Carmellose sodium 0.5% |
| EPON Resin | Polysciences, Inc. | – | Epoxy resin used for electron microscopy, composed of a mixture of four reagents: Poly/Bed 812 Resin (CAT#08791); DDSA – Dodecenylsuccinic Anhydride (CAT#00563); NMA – Nadic Methyl Anhydride (CAT#00886); DMP-30 – 2,4,6-tris(dimethylaminomethyl)phenol (CAT#00553) |
| Glutaraldehyde | Electron Microscopy Sciences | 16110 | Used for electron microscopy tissue preparation (step 5) |
| Hyabak | União Química Farmacêutica Nacional S/A | MS-8042140002 | Sodium hyaluronate 0.15% |
| Icare Tonolab | Icare Finland Oy | TV02 (model number) | Rebound handheld tonometer |
| IgG donkey anti-mouse antibody + Alexa Fluor 555 | Thermo Fisher Scientific | A31570 | Secondary antibody solution |
| LCD monitor 23 inches | Samsung Electronics Co. Ltd. | S23B550 | Model LS23B550, for electroretinogram recording |
| LSM 510 Meta | Carl Zeiss | – | Confocal epifluorescence microscope |
| Maxiflox | Cristália Produtos Químicos Farmacêuticos Ltda | MS-1.0298.0489 | Ciprofloxacin 3.5 mg/g |
| MEB-9400K | Nihon Kohden Corporation | – | System for electroretinogram recording |
| monoclonal IgG1 mouse anti-Brn3a | MilliporeSigma | MAB-1585 | Brn3a primary antibody solution |
| Neuropack Manager v08.33 | Nihon Kohden Corporation | – | Software for electroretinogram signal processing |
| Optomotry | CerebralMechanics | – | System for optomotor response analysis |
| Osmium tetroxide | Electron Microscopy Sciences | 19100 | Used for electron microscopy tissue preparation (step 5) |
| Potassium ferrocyanide | Electron Microscopy Sciences | 20150 | Used for electron microscopy tissue preparation (step 5) |
| Reference and ground electrodes for electroretinography | Chalgren Enterprises | 110-63 | Stainless steel needles 0.4 mm × 37 mm |
| Sodium cacodylate buffer | Electron Microscopy Sciences | 12300 | Used for electron microscopy tissue preparation (step 5) |
| Ster MD | União Química Farmacêutica Nacional S/A | MS-1.0497.1287 | Prednisolone acetate 0.12% |
| Terolac | Cristália Produtos Químicos Farmacêuticos Ltda | MS-1.0497.1286 | Ketorolac trometamol 0.5% |
| Terramicina | Laboratórios Pfizer Ltda | MS-1.0216.0024 | Oxytetracycline hydrochloride 30 mg/g + polymyxin B 10,000 U/g |
| Tono-Pen XL | Reichert Technologies | 230635 | Digital applanation handheld tonometer |
| TO-PRO-3 | Thermo Fisher Scientific | T3605 | Far red-fluorescent nuclear counterstain; emission at 661 nm |
| Triton X-100 | Sigma-Aldrich | 9036-19-5 | Non-ionic surfactant |
| Uranyl acetate | Electron Microscopy Sciences | 22400 | Used for electron microscopy tissue preparation (step 5) |
| Xilazin | Syntec do Brasil Ltda | 7899 | Xylazine hydrochloride 2% |
| Carl Zeiss | – | Stereo microscope for surgery and retinal dissection |
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Cite This Article
Lani-Louzada, R., Abreu, C. A., Araújo, V. G., Dias, M. S., Petrs-Silva, H., Linden, R. Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents. J. Vis. Exp. (180), e63442, doi:10.3791/63442 (2022).