成年小鼠视网膜的视网膜外植体作为研究视网膜神经血管疾病的离 体 模型
Summary
该协议介绍并描述了从成年小鼠获得的视网膜外植体的分离，解剖，培养和染色的步骤。该方法作为研究不同视网膜神经血管疾病（如糖尿病视网膜病变）的 离体 模型是有益的。
Abstract
视网膜研究的挑战之一是研究不同视网膜细胞（如视网膜神经元、神经胶质细胞和血管细胞）之间的串扰。 体外分离 、培养和维持视网膜神经元具有技术和生物学局限性。培养视网膜外植体可以克服这些限制，并提供独特的 离体 模型来研究具有良好控制生化参数且独立于血管系统的各种视网膜细胞之间的串扰。此外，视网膜外植体是研究各种视网膜血管和神经退行性疾病（如糖尿病视网膜病变）的新型药物干预的有效筛选工具。在这里，我们描述了视网膜外植体长期分离和培养的详细方案。手稿还介绍了在此过程中的一些技术问题，这些问题可能会影响视网膜外植体培养的预期结果和可重复性。视网膜外植体培养开始2周后，视网膜血管、神经胶质细胞和神经元的免疫染色显示视网膜毛细血管和神经胶质细胞完整。这使视网膜外植体成为在模拟糖尿病视网膜病变等视网膜疾病的条件下研究视网膜血管系统和神经胶质细胞变化的可靠工具。
Introduction
已经提出了不同的模型来研究视网膜疾病，包括体内和体外模型。动物在研究中的使用仍然是一个持续的伦理和转化辩论问题1。涉及小鼠或大鼠等啮齿动物的动物模型通常用于视网膜研究2，3，4。然而，由于啮齿动物视网膜与人类不同的生理功能，例如黄斑缺失或色觉差异5，因此出现了临床问题。使用人类死后眼睛进行视网膜研究也存在许多问题，包括但不限于原始样本的遗传背景、捐赠者的病史以及捐赠者以前的环境或生活方式的差异6.此外，在视网膜研究中使用体外模型也有一些缺点。用于研究视网膜疾病的细胞培养模型包括利用人源细胞系、原代细胞或干细胞7。所使用的细胞培养模型已被证明在被污染、错误识别或去分化方面存在问题8，9，10，11。最近，视网膜类器官技术取得了重大进展。然而，体外构建高度复杂的视网膜有几个局限性。例如，视网膜类器官不具有与成熟的体内视网膜相同的生理和生化特征。为了克服这一限制，视网膜类器官技术必须整合更多的生物和细胞特征，包括平滑肌细胞、脉管系统和免疫细胞，如小胶质细胞12，13，14，15。
器官型视网膜外植体已成为研究糖尿病视网膜病变和退行性视网膜疾病等视网膜疾病的可靠工具16，17，18，19。与其他现有技术相比，视网膜外植体的使用通过添加独特的功能来研究相同生化参数下各种视网膜细胞之间的串扰，并且独立于系统变量，从而支持体外视网膜细胞培养和当前的体内动物模型。外植体培养物允许不同的视网膜细胞在同一环境中保持在一起，从而可以保存视网膜细胞间的相互作用20，21，22。此外，先前的一项研究表明，视网膜外植体能够保留培养的视网膜细胞的形态结构和功能23。因此，视网膜外植体可以为研究各种视网膜疾病的可能治疗靶点提供一个体面的平台24，25，26。视网膜外植体培养提供了一种可控的技术，并且非常灵活地替代了允许多种药理学操作并可以揭示多种分子机制的现有蛾体27。
本文的总体目标是将视网膜外植体技术作为 体外 细胞培养和 体内 动物模型之间的合理中间模型系统。这种技术可以比解离细胞更好地模仿视网膜功能。由于各种视网膜层保持完整，视网膜细胞间相互作用可以在实验室中在控制良好的生化条件下进行评估，并且独立于血管系统功能28。
Protocol
所有动物实验均由美国密歇根州罗切斯特奥克兰大学的机构动物护理和使用委员会（IACUC）批准，并遵循视觉和眼科研究协会（ARVO）关于在眼科和视觉研究中使用动物的声明制定的指南。 1. 动物制备 将动物饲养在光照受控的环境中，温度恒定。动物宿舍的温度设置应遵循《实验动物护理和使用指南》的指引。小鼠的温度范围在18°C至26°C之间。 保持湿度?…
Representative Results
视网膜外植体的神经元和血管视网膜细胞在体外培养基中长时间存活 通过使用我们的方案培养视网膜外植体，我们成功地维持了不同的视网膜细胞，这些细胞存活长达 2 周。为了验证不同视网膜细胞的存在，使用神经元细胞标志物（NeuN），神经胶质细胞标志物（GFAP）和血管标志物（异凝集素-B4）对视网膜外植体进行了免疫荧光染色（<strong class="xf…
Discussion
我们的实验室一直在研究促进视网膜微血管功能障碍的病理生理变化31，32，33，34，35，36年。视网膜外植体是可以用作研究视网膜疾病（如糖尿病视网膜病变或退行性视网膜疾病）的模型的技术之一。具有来自与治疗组或组相同的单个视网膜或动物…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
我们要感谢美国国立卫生研究院（NIH）向国家眼科研究所（R01 EY030054）提供的资助，感谢Mohamed Al-Shabrawey博士。我们要感谢Kathy Wolosiewicz帮助我们进行视频解说。我们要感谢奥克兰大学眼科研究所儿科视网膜研究实验室的Ken Mitton博士在使用手术显微镜和记录期间提供的帮助。该视频由Khaled Elmasry博士编辑和导演。
Materials
| Adult C57Bl/6J mice | The Jackson Laboratory, Bar Harbor, ME, 04609, USA | 664 | |
| All-in-One Fluorescence Microscope | KEYENCE CORPORATION OF AMERICA, IL, 60143, U.S.A. | BZ-X800 | |
| B27 supplements | Thermo scientific. Waltham, MA, 02451, USA | Gibco #17504-04 | |
| Blockade blocking solution | Thermo scientific. Waltham, MA, 02451, USA | B10710 | |
| DMEM F12 | Thermo scientific. Waltham, MA, 02451, USA | Gibco #11320033 | |
| Goat anti-Rabbit IgG. | Thermo scientific. Waltham, MA, 02451, USA | F-2765 | |
| GSL I, BSL I (Isolectin) | Vector Laboratories. Burlingame, CA 94010,USA | B-1105-2 | |
| Hanks Ballanced Salt Solution (HBSS) | Thermo scientific. Waltham, MA, 02451, USA | Gibco #14175095 | |
| Micro Scissors, 12 cm, Diamond Coated Blades | World Precision Instruments,FL 34240, USA | Straight (503365) | |
| N2 supplements | Thermo scientific. Waltham, MA, 02451, USA | Gibco #17502-048 | |
| Nunc Polycarbonate Cell Culture Inserts in Multi-Well Plates | Thermo scientific. Waltham, MA, 02451, USA | 140652 | |
| Paraformaldehyde 4% in PBS | BBP, Ashland, MA, 01721 USA | C25N107 | |
| Penicillin-Streptomycin (10,000 U/mL) | Thermo scientific. Waltham, MA, 02451, USA | 15140148 | |
| PROLONG DIAMOND ANTIFADE 4′,6-diamidino-2-phenylindole (DAPI). | Thermo scientific. Waltham, MA, 02451, USA | P36962 | |
| Rabbit Anti-NeuN Antibody | Abcam.,Cambridge, UK | ab177487 | |
| Rabbit Glial Fibrillary Acidic Protein (GFAP) Antibody | Dako,Carpinteria, CA 93013, USA. | Z0334 | |
| Texas Red | Vector Laboratories. Burlingame, CA 94010,USA | SA-5006-1 | |
| TritonX | BioRad Hercules, CA, 94547,USA | 1610407 |
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Citazione di questo articolo
Elmasry, K., Moustafa, M., Al-Shabrawey, M. Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases. J. Vis. Exp. (190), e63966, doi:10.3791/63966 (2022).