This research was supported by grants to M.P. from the Association Retina France, Fondation de France, FMR (Fondation Maladies Rares), BBS (Association du syndrome de Bardet-Biedl), and UNADEV (Union Nationale des Aveugles et D&#233;ficients Visuels) in partnership with ITMO NNP (Institut Th&#233;matique Multi-Organisme Neurosciences, sciences cognitives, neurologie, psychiatrie) / AVIESAN (Alliance Nationale pour les sciences de la vie et de la sant&#233;).

Generating Retinal Degeneration to Investigate Cellular Repair Strategies in &lt;em&gt;Xenopus&lt;/em&gt;

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The authors have no conflicts of interest to disclose.

Advantages and disadvantages of various retinal injury paradigms in Xenopus tadpoles
Mechanical retinal injury 
Various surgical injuries of the neural retina have been developed in Xenopus tadpoles. The neural retina may either be entirely removed15,16 or only partly excised16,17. The mechanical injury presented here doe...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1,2-Propanediol (propyl&#232;ne glycol)</td>
			<td>Sigma-Aldrich</td>
			<td>398039</td>
			<td></td>
		</tr>
		<tr>
			<td>Absolute ethanol &#8805;99.8%</td>
			<td>VWR chemicals</td>
			<td>20821-365</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Cleaved Caspase 3 antibody (rabbit)</td>
			<td>Cell signaling</td>
			<td>9661S</td>
			<td>Dilution 1/300</td>
		</tr>
		<tr>
			<td>Anti-GFP antibody (chicken)</td>
			<td>Aveslabs</td>
			<td>GFP-1020</td>
			<td>Dilution 1/500</td>
		</tr>
		<tr>
			<td>Anti-M-Opsin antibody (rabbit)</td>
			<td>Sigma-Aldrich</td>
			<td>AB5405</td>
			<td>Dilution 1/500</td>
		</tr>
		<tr>
			<td>Anti-mouse secondary antibody, Alexa Fluor 594 (goat)</td>
			<td>Invitrogen Thermo Scientific</td>
			<td>A11005</td>
			<td>Dilution 1/1,000</td>
		</tr>
		<tr>
			<td>Anti-Otx2 antibody (rabbit)</td>
			<td>Abcam</td>
			<td>Ab183951</td>
			<td>Dilution 1/100</td>
		</tr>
		<tr>
			<td>Anti-rabbit secondary antibody, Alexa Fluor 488 (goat)</td>
			<td>Invitrogen Thermo Scientific</td>
			<td>A11008</td>
			<td>Dilution 1/1,000</td>
		</tr>
		<tr>
			<td>Anti-rabbit secondary antibody, Alexa Fluor 594 (goat)</td>
			<td>Invitrogen Thermo Scientific</td>
			<td>A11012</td>
			<td>Dilution 1/1,000</td>
		</tr>
		<tr>
			<td>Anti-Recoverin antibody (rabbit)</td>
			<td>Sigma-Aldrich</td>
			<td>AB5585</td>
			<td>Dilution 1/500</td>
		</tr>
		<tr>
			<td>Anti-Rhodopsin antibody (mouse)</td>
			<td>Sigma-Aldrich</td>
			<td>MABN15</td>
			<td>Dilution 1/1,000</td>
		</tr>
		<tr>
			<td>Anti-S-Opsin antibody (rabbit)</td>
			<td>Sigma-Aldrich</td>
			<td>AB5407</td>
			<td>Dilution 1/500</td>
		</tr>
		<tr>
			<td>Apoptotis detection kit (Dead end fluorimetric TUNEL system)</td>
			<td>Promega</td>
			<td>G3250</td>
			<td></td>
		</tr>
		<tr>
			<td>Benzocaine&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>E1501</td>
			<td>Stock solution 10%</td>
		</tr>
		<tr>
			<td>bisBenzimide H 33258 (Hoechst)</td>
			<td>Sigma-Aldrich</td>
			<td>B2883</td>
			<td>Stock solution 10 mg/mL</td>
		</tr>
		<tr>
			<td>Butanol-1 &#8805;99.5%</td>
			<td>VWR chemicals</td>
			<td>20810.298</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate (CaCl2, 2H2O)</td>
			<td>Sigma-Aldrich (Supelco)</td>
			<td>1.02382</td>
			<td>Use at 0.1 M</td>
		</tr>
		<tr>
			<td>Cas9 (EnGen Spy Cas9 NLS)</td>
			<td>New England Biolabs</td>
			<td>M0646T</td>
			<td></td>
		</tr>
		<tr>
			<td>Clark Capillary Glass model GC100TF-10</td>
			<td>Warner Instruments (Harvard Apparatus)</td>
			<td>30-0038</td>
			<td></td>
		</tr>
		<tr>
			<td>Cobalt(II) chloride hexahydrate (CoCl2, 6H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>C8661</td>
			<td>Stock solution 100 mM</td>
		</tr>
		<tr>
			<td>Coverslip 24 x 60 mm</td>
			<td>VWR</td>
			<td>631-1575</td>
			<td></td>
		</tr>
		<tr>
			<td>Dako REAL ab diluent&#160;</td>
			<td>Agilent</td>
			<td>S202230-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr>
			<td>Electronic Rotary Microtome</td>
			<td>Thermo Scientific</td>
			<td>Microm HM 340E&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin 1% aqueous</td>
			<td>RAL Diagnostics</td>
			<td>312740</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein lysine dextran&#160;&#160;</td>
			<td>Invitrogen Thermo Scientific</td>
			<td>D1822</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent stereomicroscope</td>
			<td>Olympus</td>
			<td>SZX 200</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamycin</td>
			<td>Euromedex</td>
			<td>EU0410-B</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerin albumin acc. Mallory</td>
			<td>Diapath</td>
			<td>E0012</td>
			<td>Use at 3% in water</td>
		</tr>
		<tr>
			<td>Hematoxylin (Mayer&#39;s Hemalun)</td>
			<td>RAL Diagnostics</td>
			<td>320550</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES potassium salt</td>
			<td>Sigma-Aldrich</td>
			<td>H0527</td>
			<td></td>
		</tr>
		<tr>
			<td>Human chorionic gonadotropin hormone</td>
			<td>MSD Animal Health</td>
			<td>Chorulon 1500</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid fuming, 37% (HCl)</td>
			<td>Sigma-Aldrich (SAFC)</td>
			<td>1.00314</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Cysteine hydrochloride monohydrate</td>
			<td>Sigma-Aldrich</td>
			<td>C7880</td>
			<td>Use at 2% in 0.1x MBS (pH 7.8 - 8.0)</td>
		</tr>
		<tr>
			<td>Magnesium Sulfate Heptahydrate (MgSO4, 7H2O)</td>
			<td>Sigma-Aldrich (Supelco)</td>
			<td>1.05886</td>
			<td></td>
		</tr>
		<tr>
			<td>Metronidazole&#160;</td>
			<td>Sigma-Aldrich (Supelco)</td>
			<td>M3761</td>
			<td>Use at 10 mM</td>
		</tr>
		<tr>
			<td>Microloader tips</td>
			<td>Eppendorf</td>
			<td>5242956003</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette puller (P-97 Flaming/Brown)</td>
			<td>Sutter Instrument Co.</td>
			<td>Model P-97</td>
			<td>Program : Heat 700 / Pull 100 / Vel 75 / Time 90 / Unlocked p = 500</td>
		</tr>
		<tr>
			<td>Mounting medium to preserve fluorescence, FluorSave Reagent</td>
			<td>Millipore</td>
			<td>345789</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting medium, Eukitt</td>
			<td>Chem-Lab</td>
			<td>CL04.0503.0500</td>
			<td></td>
		</tr>
		<tr>
			<td>MX35 Ultra Microtome blade</td>
			<td>Epredia</td>
			<td>3053835</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle Agani 25 G x 5/8&#39;&#39;</td>
			<td>Terumo</td>
			<td>AN*2516R1</td>
			<td></td>
		</tr>
		<tr>
			<td>Nickel Plated Pin Holder</td>
			<td>Fine Science Tools</td>
			<td>26016-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon filtration tissue (sifting fabric) NITEX, mesh opening 1,000 &#181;m</td>
			<td>Sefar</td>
			<td>06-1000/44</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin histowax without DMSO</td>
			<td>Histolab</td>
			<td>00403</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde solution (32%)</td>
			<td>Electron Microscopy Sciences</td>
			<td>EM-15714-S</td>
			<td>Use at 4% in 1x PBS pH 7.4</td>
		</tr>
		<tr>
			<td>Peel-A-Way Disposable Embedding Molds</td>
			<td>Epredia</td>
			<td>2219</td>
			<td></td>
		</tr>
		<tr>
			<td>Pestle</td>
			<td>VWR</td>
			<td>431-0094</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dish 100 mm</td>
			<td>Corning Gosselin</td>
			<td>SB93-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dish 55 mm</td>
			<td>Corning Gosselin</td>
			<td>BP53-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffer Saline Solution (PBS) 10x</td>
			<td>Euromedex</td>
			<td>ET330-A</td>
			<td></td>
		</tr>
		<tr>
			<td>PicoSpritzer Microinjection system</td>
			<td>Parker Instrumentation Products</td>
			<td>PicoSpritzer III</td>
			<td></td>
		</tr>
		<tr>
			<td>Pins&#160;</td>
			<td>Fine Science Tools</td>
			<td>26002-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Polysucrose (Ficoll PM 400 )</td>
			<td>Sigma-Aldrich</td>
			<td>F4375</td>
			<td>Use at 3% in 0.1x MBS</td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>Sigma-Aldrich</td>
			<td>P3911</td>
			<td></td>
		</tr>
		<tr>
			<td>Powdered fry food : sera Micron Nature</td>
			<td>sera</td>
			<td>45475 (00720)</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors dissection</td>
			<td>Fine Science Tools</td>
			<td>14090-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide Superfrost&#160;</td>
			<td>&#160;KNITTEL Glass</td>
			<td>VS11171076FKA&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide warmer</td>
			<td>Kunz instruments</td>
			<td>HP-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium citrate trisodium salt dihydrate (C6H5Na3O7, 2H2O)</td>
			<td>VWR chemicals</td>
			<td>27833.294</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydrogen carbonate (NaHCO3)</td>
			<td>Sigma-Aldrich (Supelco)</td>
			<td>1.06329</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide 30% aqueous solution (NaOH)</td>
			<td>VWR chemicals</td>
			<td>28217-292</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Zeiss</td>
			<td>Stemi 2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringes Omnifix-F Solo Single-use Syringes 1 mL</td>
			<td>B-BRAUN</td>
			<td>9161406V</td>
			<td></td>
		</tr>
		<tr>
			<td>trans-activating crRNA (tracrRNA)</td>
			<td>Integrated DNA Technologies</td>
			<td>1072533</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>X-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
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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 recently emerged as particularly appealing. A cellular source of interest for retinal repair is the M&#252;ller glial cell, which harbors stem cell potential and an extraordinary regenerative capacity in anamniotes. This potential is, however, very limited in mammals. Studying the molecular mechanisms underlying retinal regeneration in animal models with regenerative capabilities should provide insights into how to unlock the latent ability of mammalian M&#252;ller cells to regenerate the retina. This is a key step for the development of therapeutic strategies in regenerative medicine. To this aim, we developed several retinal injury paradigms in Xenopus: a mechanical retinal injury, a transgenic line allowing for nitroreductase-mediated photoreceptor conditional ablation, a retinitis pigmentosa model based on CRISPR/Cas9-mediated rhodopsin knockout, and a cytotoxic model driven by intraocular CoCl2 injections. Highlighting their advantages and disadvantages, we describe here this series of protocols that generate various degenerative conditions and allow the study of retinal regeneration in Xenopus.

Author Spotlight: Exploring Retinal Regeneration Mechanisms in the Xenopus Frog 

Generating Retinal Injury Models in Xenopus Tadpoles

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.

Mechanical retinal injury 
Retinal sections of tadpoles subjected to the mechanical injury described in protocol section 1 show that the retinal lesion encompasses all layers of the tissue while remaining limited to the puncture site (Figure 2A,B).
Conditional rod cell ablation using the NTR-MTZ system 
The eyes of anesthetized Tg(rho:GFP-NTR) transgenic tadpoles treated with MTZ treatment, as d...

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We have developed several protocols to induce retinal damage or retinal degeneration in Xenopus laevis tadpoles. These models offer the possibility of studying retinal regeneration mechanisms.

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

generating-retinal-injury-models-in-xenopus-tadpoles

Research

JoVE Journal

Developmental Biology

Mechanical Retinal Puncture in Xenopus Tadpoles to Study Retinal Regeneration Mechanisms

Mechanical Retinal Puncture in Xenopus Tadpoles to Study Retinal Regeneration Mechanisms  

Begin by using a dip net to capture the tadpoles from their tanks and transfer them to a fresh tank filled with 0.005%benzocaine solution for anesthesia. Then wait for a few minutes to allow the tadpoles to stop reacting to stimuli. Now, capture the anesthetized tadpole with a spoon and carefully place it with its dorsal side up in the center of a small Petri dish lined with moist tissue.Then position the specimen under the stereo microscope. Using a 0.2 millimeter diameter pin attached to a pin holder, carefully insert the tool on the dorsal side of the right eye, piercing the retina, and passing through completely. Then rotate the Petri dish 180 degrees and puncture the left eye.Carefully immerse the Petri dish containing the lesioned tadpole into a large tank with one liter of rearing water. Monitor the tadpoles until they are fully awakened. Retinal sections of tadpoles subjected to mechanical injury showed the retinal lesion in all layers of the tissue while remaining limited to the puncture site.

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

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

To begin, use a transgenic line expressing GFP and nitro reductase, or NTR, under a rod photoreceptor specific promoter. NTR converts the prodrug metronidazole, or MTZ, into a cytotoxic agent for selective ablation of NTR expressing rods. For GFP monitoring, use a spoon to gently catch one anesthetized tadpole and cautiously place it onto a moist tissue within a small Petri dish.Observe the GFP fluorescence under a fluorescent stereo microscope and capture photographic images of the eyes. Carefully immerse the Petri dish with the tadpole into a large tank with rearing water until fully awakened. For individual monitoring of retinal degeneration, place each tadpole in a small container containing 30 milliliters of 10 millimolar MTZ, or transgenic siblings in the control solution.For batch treatment, place the transgenic tadpoles in a larger container with one liter of MTZ, or control solution. Raise the tadpoles for one week at 20 degrees Celsius under darkness to avoid degradation of MTZ. After seven days, observe the GFP under the fluorescent stereo microscope and capture photographic images of the eye.A decrease in fluorescence intensity, or even a virtual absence of fluorescence indicates the degradation of rods. The eyes of transgenic tadpoles treated with MTZ showed a decrease in GFP fluorescence compared to the controls, which is further confirmed on retinal sections by GFP immuno staining.

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

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

To begin, take a sharpened capillary, use a micro loader tip to load 2 to 10 microliters of the CRISPR Cas9 ribonucleoprotein complex or control solution. Afterward, place the filled capillary into the micro injector handler. Break off the capillary tip with fine forceps to adjust the ejection volume at 10 nanoliters.Position one cell stage embryos on a grid glued in a 100 millimeter Petri dish with 3%polysucrose solution. Using the micro injector, and under a stereo microscope, inject 10 nanoliter of the solution into the cortical region, specifically at the animal pole level under the cytoplasmic membrane. After 24 hours, visualize fluorescein, lysine, dextrin under a fluorescent stereo microscope, and select well-injected rho crispant embryos.Caspase 3 labeling of rho crispant tadpole retinas showed that some rods undergo apoptosis. Histological staining revealed global preservation of nuclear layers, but a severe shortening of photoreceptor outer segments. Further immuno staining analysis with photoreceptor markers showed the degeneration of rod outer segments.

Retinal Cell Degeneration in Xenopus Tadpoles by Cytotoxic Intraocular CoCl2 Injections

Retinal Cell Degeneration in Xenopus Tadpoles by Cytotoxic Intraocular CoCl2 Injections  

Use a dip net to capture tadpoles from their tanks and transfer them into a 50 milliliter solution of 0.005%benzocaine. Allow them to remain in the solution until they no longer respond to stimuli. Use a spoon to gently pick up a tadpole and place it with its dorsal side facing up into a small Petri dish containing a damp tissue.Then position the specimen under the stereo microscope. To begin, take a sharpened capillary, use a micro loader tip to load 10 microliters of cobalt chloride solution. Afterward, place the filled capillary into the micro injector handler to break off the capillary tip to adjust the ejection volume at about 30 nanoliters.Using the micro injector and a stereo microscope, insert the capillary filled with cobalt chloride over the lens into the eye. Upon reaching the inside of the eye with the capillary tip, eject two drops of about 30 nanoliters per eye. After the right eye has been injected, turn the Petri dish to a 180 degree angle to inject the left eye.Transfer the Petri dish containing the injected tadpole to a large tank containing one liter of rearing water. Keep observing the tadpoles until they have fully awakened. Injection of 10 millimolar cobalt chloride led to specific cell death of cone photoreceptors, while 25 millimolar led to broad retinal cell death.Immuno staining revealed the absence of cones in 10 millimolar cobalt chloride injected retinas while rods were largely preserved. After injections of 25 millimolar cobalt chloride, both photoreceptors and bipolar cells were severely affected.

849 Views.  Université Paris-Saclay.  We have developed several protocols to induce retinal damage or retinal degeneration in Xenopus laevis tadpoles. These models offer the possibility of studying retinal regeneration mechanisms.

Generating Retinal Degeneration to Investigate Cellular Repair Strategies in Xenopus

概要