Abstract
Purpose :
While axolotls (Ambystoma mexicanum) are studied for their remarkable ability to regenerate, there is a lack in understanding on their ability to regenerate their retina and the source of the progenitors responsible. It is also not known if the regeneration process is damage specific and if all retinal cell classes and types can regenerate. Previous work has shown that the axolotl retina regenerates following mechanical injury and that there was an increase in cell proliferation observed in the Retina Pigment Epithelium (RPE), Ciliary Marginal Zone (CMZ), and Müller glia. This study serves to investigate the regenerative potential of the axolotl retina following chemical injury. We hypothesize that the Müller glia, RPE, and CMZ are involved in regeneration following NMDA injury.
Methods :
The hypothesis was tested through two major avenues, single cell RNA sequencing (scRNAseq) and histology: samples were collected 27 days following 1µL intravitreal injections of 100µM NMDA into the left eye of seven 12-15cm animals using a 29.5-gauge needle both eyes were harvested and retina ganglion cell (RGC) death was assessed by staining for Brn3b on whole mounts and scRNAseq was performed on the total unsorted dissociated retina cell population. Cell fate and pseudotime analysis was conducted using scFates and Velocity approaches. For the second experiment we injected 1/2µL of 100µM, 300µM or 500µM NMDA or vehicle into one eye of 3-5cm animals with 6 replicates in each group. Next an EdU pulse-chase experiment with Immunohistochemistry (IHC) and hybridization chain reaction fluorescent in situ hybridization (HCR-FISH) was performed to identify proliferating cell types following injury. This was performed on 3-5cm animals at 7, 15, 30, and 60, and 90 dpi. The EdU was pulsed once with a 3-hour long chase.
Results :
Our initial analysis showed that 100 µM NMDA injections lead to 30% reduction in RGC number. Through the methods described above it was discovered that axolotls contain all the major cell types of the vertebrate retina, have an active CMZ zone as adults, they can regenerate their retina following NMDA injury, and there was an increase in cell proliferation in the CMZ, Müller glia, and RPE.
Conclusions :
Our finding suggests that all three cell niches – RPE, CMZ, and Müller glia – are involved in retinal regeneration following NMDA injury, which opens new avenues to explore cell reprograming for retinal ganglion cell loss.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.