da Silva de Jesus Maria Clara, Cruz Douglas Penaforte, León Greysa Saraí Barrios, Crisóstomo Millena Costa, Magalhães Camila Feitosa, Ribeiro de Mattos Aline Estacio, Fragel-Madeira Lucianne
Neural Development and Regeneration Laboratory, Department of Neurobiology, Science and Biotechnology Graduate Program, Institute of Biology, Fluminense Federal University.
Neural Development and Regeneration Laboratory, Department of Neurobiology, Neuroscience Graduate Program, Institute of Biology, Fluminense Federal University.
J Vis Exp. 2025 Jul 22(221). doi: 10.3791/68324.
Retinopathies affect hundreds of millions of people worldwide and are significant causes of visual impairment and blindness. Among these diseases, retinitis pigmentosa (RP) is a hereditary ocular disorder characterized by progressive retinal degeneration, affecting approximately 1.5 to 2 million people worldwide. Proof of concept studies in animal models of hereditary retinal dystrophies form an important basis of evidence for studies in pathophysiological mechanisms of the disease, treatment effectiveness and safety. Intravitreal injection in mice and cryostat sectioning are widely utilized techniques for investigating drug effects in RP models, as they enable a precise delivery of substances into vitreous humor and preparation of high-quality histological samples for detailed analysis of retinal alterations. Also, cell counting through immunofluorescence using antibodies against recoverin, a calcium-dependent protein expressed in retinal photoreceptors, has been recently employed to study cellular changes associated with retinal degeneration in RP models, because this approach facilitates evaluation of photoreceptor loss and potential cellular preservation following therapeutic interventions. The intravitreal injection has been optimized to ensure ocular integrity and reliable results, utilizing a fine needle to administer the solution into the vitreous chamber. After a predefined period to assess drug effects, histological processing is performed, with samples sectioned at 10 μm thickness using a cryostat, followed by immunofluorescence labeling with recoverin antibodies. Finally, cell counting is performed to assess whether the treatment with drugs exerted a protective effect, as evidenced by preservation of photoreceptors. Although these techniques have been extensively applied to generate consistent data in retinopathy research, challenges persist in achieving precise tissue handling and processing quality. This protocol provides a standardized approach to optimize each step, from intravitreal injection to the preparation of histological sections, ensuring reproducible and high-quality results.
视网膜病变影响着全球数亿人，是导致视力损害和失明的重要原因。在这些疾病中，视网膜色素变性（RP）是一种遗传性眼部疾病，其特征是视网膜进行性退化，全球约有150万至200万人受其影响。遗传性视网膜营养不良动物模型的概念验证研究是该疾病病理生理机制、治疗效果和安全性研究的重要证据基础。在RP模型中，向小鼠玻璃体内注射和低温恒冷切片是广泛用于研究药物作用的技术，因为它们能够将物质精确地递送至玻璃体液中，并制备高质量的组织学样本以详细分析视网膜改变。此外，最近还采用了通过使用针对恢复蛋白（一种在视网膜光感受器中表达的钙依赖性蛋白）的抗体进行免疫荧光细胞计数，来研究RP模型中与视网膜变性相关的细胞变化，因为这种方法有助于评估治疗干预后光感受器的损失和潜在的细胞保存情况。玻璃体内注射已得到优化，以确保眼部完整性和可靠结果，使用细针将溶液注入玻璃体腔。在经过预定时间以评估药物效果后，进行组织学处理，使用低温恒冷切片机将样本切成10μm厚，然后用恢复蛋白抗体进行免疫荧光标记。最后，进行细胞计数以评估药物治疗是否发挥了保护作用，光感受器的保存可证明这一点。尽管这些技术已广泛应用于视网膜病变研究以生成一致的数据，但在实现精确的组织处理和加工质量方面仍然存在挑战。本方案提供了一种标准化方法，可优化从玻璃体内注射到组织学切片制备的每个步骤，确保可重复的高质量结果。
