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

Generering av näthinnedegeneration för att undersöka cellulära reparationsstrategier hos Xenopus

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.

Författare i fokus: Utforska mekanismer för näthinneregenerering hos Xenopus-grodan 

Generering av modeller för näthinneskador hos Xenopus-grodyngel

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  

Retinal celldegeneration i Xenopus grodyngel genom cytotoxiska intraokulära CoCl 2-injektioner

Använd en håv för att fånga grodyngel från deras akvarier och överför dem till en 50 ml-lösning av 0,005 % bensokain. Låt dem stanna kvar i lösningen tills de inte längre svarar på stimuli. Använd en sked för att försiktigt plocka upp ett grodyngel och placera det med ryggsidan uppåt i en liten petriskål som innehåller en fuktig näsduk.Placera sedan provet under stereomikroskopet. För att börja, ta en vässad kapillär, använd en mikroladdningsspets för att ladda 10 mikroliter koboltkloridlösning. Placera sedan den fyllda kapillären i mikroinjektorhanteraren för att bryta av kapillärspetsen för att justera utmatningsvolymen till cirka 30 nanoliter.Använd mikroinjektorn och ett stereomikroskop och för in kapillären fylld med koboltklorid över linsen i ögat. När du når insidan av ögat med kapillärspetsen, mata ut två droppar på cirka 30 nanoliter per öga. Efter att det högra ögat har injicerats, vrid petriskålen till en 180 graders vinkel för att injicera det vänstra ögat.Överför petriskålen med grodyngel till ett stort akvarium som innehåller en liter odlingsvatten. Fortsätt att observera grodynglen tills de har vaknat helt. Injektion av 10 millimolär koboltklorid ledde till specifik celldöd av tappfotoreceptorer, medan 25 millimolar ledde till bred celldöd i näthinnan.Immunfärgning avslöjade frånvaron av tappar i 10 millimolar koboltklorid injicerade näthinnor medan stavar i stort sett bevarades. Efter injektioner av 25 millimolär koboltklorid påverkades både fotoreceptorer och bipolära celler allvarligt.

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 ...

Mechanical Retinal Puncture in Xenopus Tadpoles to Study Retinal Regeneration Mechanisms

Mechanical Retinal Puncture in Xenopus Tadpoles to Study Retinal Regeneration Mechanisms  

Mekanisk retinal punktion hos Xenopus grodyngel för att studera näthinneregenereringsmekanismer

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

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

Villkorlig stavcellsablation i Xenopus-grodyngel med hjälp av NTR-MTZ-systemet

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

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

Rod Cell Degeneration i Xenopus grodyngel genom CRISPR/Cas9-medierad rodopsinknockout