Name: Retinal Cell Degeneration in Xenopus Tadpoles by Cytotoxic Intraocular CoCl2 Injections
Uploaded: 2023-10-13T12:00:00
Description: Watch this Scientific Journal Video about Retinal Cell Degeneration in Xenopus Tadpoles by Cytotoxic Intraocular CoCl2 Injections at JoVE.com

retinal cell degeneration in xenopus tadpoles by cytotoxic intraocular cocl2 injections

产生视网膜变性以研究非洲爪蟾的细胞修复策略

Millions of people worldwide are afflicted with various retinal degenerative diseases leading to blindness, such as retinitis pigmentosa, diabetic retinopathy, or age-related macular degeneration (AMD). To date, these conditions remain largely untreatable. Current therapeutic approaches under evaluation include gene therapy, cell or tissue transplantations, neuroprotective treatments, optogenetics, and prosthetic devices. Another emerging strategy is based on self-regeneration through the activation of endogenous cells with stem cell potential. M&#252;ller glial cells, the major glial cell type of the retina, are among cellular sources of interest in this context. Upon injury, they can dedifferentiate, proliferate, and generate neurons1,2,3. Although this process is very effective in zebrafish or Xenopus, it is largely inefficient in mammals.
Nonetheless, it has been shown that appropriate treatments with mitogenic proteins or overexpression of various factors can induce mammalian M&#252;ller glia cell-cycle re-entry and, in some cases, trigger their subsequent&#160;neurogenesis commitment1,2,3,4,5. This remains, however, largely insufficient for treatments. Hence, increasing our knowledge of the molecular mechanisms underlying regeneration is necessary to identify molecules able to efficiently turn M&#252;ller stem-like cell properties into new cellular therapeutic strategies.
With this aim, we developed several injury paradigms in Xenopus that trigger retinal cell degeneration. Here, we present (1) a mechanical retinal injury that is not cell type-specific, (2) a conditional and reversible cell ablation model using the NTR-MTZ system that targets rod cells, (3) a CRISPR/Cas9-mediated rhodopsin knockout, a model of retinitis pigmentosa that triggers progressive rod cell degeneration, and (4) a CoCl2-induced cytotoxic model that according to the dose can specifically target cones or lead to broader retinal cell degeneration. We highlight the particularities, advantages, and disadvantages of each paradigm.

作者聚焦：探索非洲爪蟾的视网膜再生机制 

在 非洲爪蟾 蝌蚪中生成视网膜损伤模型

My lab works on retinal regeneration. We are using the Xenopus frog as a model system. This amphibian is indeed quite fascinating because unlike mammals, like us, it can regenerate its retina very efficiently in case of injury.And we are studying the underlying mechanism because, in the future, it could be useful to trigger retinal regeneration in human patients afflicted with retinal neurodegenerative diseases. We have recently discovered that in addition to stem cells in the periphery of the retina and the retinal pigmented epithelium, neuroglial cells can also be recruited for retinal regeneration in case of injury. And so we are now studying the links between neuroinflammation and the regenerative capacity of these cells.Indeed, it seems that the neuroinflammatory niche is a key player in the modulation of the regeneration of the retina. To study the cellular and molecular mechanisms involved in retinal regeneration, we developed several retinal injury paradigms in Xenopus. The first is a mechanical retinal injury.The second is a transgenic line allowing for nitroreductase-mediated photoreceptor conditional ablation. The third is a retinitis pigmentosa model based on CRISPR/Cas9-mediated rhodopsin knockout, and, to finish, a cytotoxic model driven by intraocular injection of cobalt chloride or CoCl2. My host laboratory has showed that although Xenopus can regenerate its retina, the efficiency is highly variable and depends on the stages of the tadpole or on species, Xenopus laevis or tropicalis.This makes Xenopus a fantastic model for illustrating the molecular mechanisms that trigger or limit retinal regeneration.

Watch this Scientific Journal Video about Retinal Cell Degeneration in Xenopus Tadpoles by Cytotoxic Intraocular CoCl2 Injections at JoVE.com

Retinal Cell Degeneration in Xenopus Tadpoles by Cytotoxic Intraocular CoCl2 Injections  

非洲爪蟾蝌蚪通过细胞毒性眼内 CoCl2 注射的视网膜细胞变性

使用浸网从水箱中捕获蝌蚪，并将它们转移到 50 毫升 0.005% 苯佐卡因溶液中。让它们留在溶液中，直到它们不再对刺激做出反应。用勺子轻轻地拿起一只蝌蚪，将其背面朝上放入装有湿纸巾的小培养皿中。然后将标本置于体视显微镜下。首先，取一个锋利的毛细管，使用微型装载器尖端装载 10 微升氯化钴溶液。之后，将填充的毛细管放入微型注射器处理器中以折断毛细管尖端，以将射出体积调节在约 30 纳升。使用显微注射器和体视显微镜，将晶状体上充满氯化钴的毛细管插入眼睛。毛细血管尖端到达眼睛内部后，每只眼睛喷出两滴约 30 纳升。右眼注射后，将培养皿转动 180 度角注射左眼。将含有注射蝌蚪的培养皿转移到装有一升饲养水的大水箱中。继续观察蝌蚪，直到它们完全苏醒。注射 10 毫摩尔氯化钴导致视锥光感受器的特异性细胞死亡，而 25 毫摩尔导致广泛的视网膜细胞死亡。免疫染色显示注射 10 毫摩尔氯化钴的视网膜中没有视锥细胞，而视杆细胞基本保留。注射25毫摩尔氯化钴后，光感受器和双极细胞均受到严重影响。

Research

JoVE Journal

Developmental Biology

Retinal Cell Degeneration in Xenopus Tadpoles by Cytotoxic Intraocular CoCl2 Injections

Generating Retinal Injury Models in Xenopus Tadpoles

Retinal neurodegenerative diseases are the leading causes of blindness. Among numerous therapeutic strategies being explored, stimulating self-repair ...

科研

发育生物学

Mechanical Retinal Puncture in Xenopus Tadpoles to Study Retinal Regeneration Mechanisms

Mechanical Retinal Puncture in Xenopus Tadpoles to Study Retinal Regeneration Mechanisms  

非洲爪蟾蝌蚪的机械性视网膜穿刺研究视网膜再生机制

Conditional Rod Cell Ablation in Xenopus Tadpoles Using the NTR-MTZ System

Conditional Rod Cell Ablation in Xenopus Tadpoles Using the NTR-MTZ System  

使用NTR-MTZ系统对 非洲爪蟾 蝌蚪进行条件性杆细胞消融

Rod Cell Degeneration in Xenopus Tadpoles by CRISPR/Cas9-Mediated Rhodopsin Knockout

Rod Cell Degeneration in Xenopus Tadpoles by CRISPR/Cas9-Mediated Rhodopsin Knockout  

非洲 爪蟾 蝌蚪通过CRISPR/Cas9介导的 视紫红质 敲除变性