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

Xenopusの細胞修復戦略を調査するための網膜変性の生成

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.

著者スポットライト:Xenopus Frogの網膜再生メカニズムの探索 

Xenopusオタマジャクシにおける網膜損傷モデルの生成

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  

細胞傷害性眼内CoCl2注射によるXenopusオタマジャクシの網膜細胞変性

浸漬網を使用して水槽からオタマジャクシを捕獲し、0.005%ベンゾカインの50ミリリットル溶液に移します。刺激に反応しなくなるまで、溶液の中にとどまるようにします。スプーンを使ってオタマジャクシをそっと拾い上げ、背側を上に向けて湿らせたティッシュを入れた小さなペトリ皿に入れます。次に、標本を実体顕微鏡の下に配置します。まず、鋭利なキャピラリーを取り、マイクロローダーチップを使用して10マイクロリットルの塩化コバルト溶液をロードします。その後、充填したキャピラリーをマイクロインジェクターハンドラーに入れてキャピラリーチップを切り離し、吐出量を約30ナノリットルに調整します。マイクロインジェクターと実体顕微鏡を使用して、塩化コバルトで満たされたキャピラリーをレンズの上から目に挿入します。毛細血管の先端で眼球の内側に到達したら、片眼あたり約30ナノリットルの滴を2滴吐出します。右目を注射した後、ペトリ皿を180度の角度に回して左目を注射します。注入したオタマジャクシを入れたシャーレを、飼育水1リットルの入った大きなタンクに移します。オタマジャクシが完全に目覚めるまで観察を続けます。10ミリモルの塩化コバルトを注射すると、錐体視細胞の特異的な細胞死が起こり、25ミリモルの注射では網膜の広域細胞死が起こりました。免疫染色により、10ミリモルの塩化コバルトを注入した網膜には錐体がなく、桿体はほとんど保存されていることが明らかになった。25ミリモルの塩化コバルトを注射した後、光受容体と双極細胞の両方が深刻な影響を受けました。

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 ...

発生生物学

Mechanical Retinal Puncture in Xenopus Tadpoles to Study Retinal Regeneration Mechanisms

Mechanical Retinal Puncture in Xenopus Tadpoles to Study Retinal Regeneration Mechanisms  

Xenopusオタマジャクシの網膜再生機構を研究するための機械的網膜穿刺

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

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

NTR-MTZシステムを用いたツ コツメガ ネオタマジャクシの条件付き桿体細胞アブレーション

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

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

CRISPR/Cas9を介したロドプシンノックアウトによるツコノプスオタマジャクシの桿体細胞変性