My lab works on retina 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, Muller glia 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 similar 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 rhodopsin knockout. And to finish, cytotoxic model driven by intraocular injection of cobalt chloride, or CoCl2.
My host laboratory has showed that although Xenopus regenerate its retina, the efficiency is highly variable and depends on the stages of the tadpole or species, Xenopus laevis or tropicalis. This make Xenopus a fantastic model for illustrating the molecular mechanisms that trigger or limit retinal regeneration. 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 stereomicroscope.
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. To begin, use a transgenic line, expressing GFP and nitro reductase or NTR under a rod photoreceptor-specific promoter.
NTR converts the pro drug 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 stereomicroscope 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 stereomicroscope 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 immunostaining. To begin, take a sharpened capillary, use a microloader tip to load two to 10 microliters of the CRISPR Cas9 ribonucleoprotein complex or control solution. Afterward, place the filled capillary into the microinjector handler.
Break off the capillary tip with fine forceps to adjust the ejection volume at 10 nanoliters. Position 1-cell stage embryos on a grid glued in a 100 millimeter Petri dish with 3%polysucrose solution. Using the microinjector and under a stereomicroscope, 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 stereomicroscope 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 immunostaining analysis with photoreceptor markers showed the degeneration of rod outer segments. 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 stereomicroscope. To begin, take a sharpened capillary, use a microloader tip to load 10 microliters of cobalt chloride solution.
Afterward, place the filled capillary into the microinjector handler to break off the capillary tip to adjust the ejection volume at about 30 nanoliters. Using the microinjector and a stereomicroscope, 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. Immunostaining 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.
