Abstract
Purpose :
Optic Nerve lamina region Neural progenitor cells (ONLR-NPC’s) (Bernstein et al, PNAS, 2020) may play an important role in maintaining normal optic nerve (ON) function. We are exploring approaches to selectively eliminate ONLR-NPC’s and determine the depleted ON’s robustness to stress response. We have developed a model to selectively express the diphtheria receptor (DTR) in NPC’s, which also express GFAP, using adeno-associated virus (AAV), followed by diphtheria toxin (DT) administration.
Methods :
We used homozygous Rosa26 flox (HBEGF) knockin mice with a floxed DTR. We intravitreally injected AAV pseudotype 2/5 with a GFAP promotor linked to active Cre recombinase allowing for the expression of DTR in NPC’s and other astrocyte types. One eye was used as experimental; the contralateral eye was used as an internal control. Mice were euthanized two weeks post injection by perfusion, and eyes and attached ON’s were post-fixed after isolation, cryoprotected in 30% sucrose and embedded in OCT. The retina and optic nerves were sectioned cross-sectionally. Sections were reacted with primary antibodies to Cre recombinase and to DTR followed by fluorescent labeled secondary antibodies. We examined immunolabeled sections using a confocal fluorescent microscope.
Results :
DTR was successfully restricted to both the GFAP(+) NPC’s in the optic nerve head and the GFAP(+) retinal Müller cells. DTR expression was robust in both cell groups. The more distal ON was not DTR(+).
Conclusions :
We can successfully induce strong DTR expression in ONLR-NPC’s. Using this paradigm, we can eliminate Müller cells and NPC’s using systemic DT or using intrathecal DT to selectively knock down the ONLR-NPC’s allowing evaluation of their role in suppression of retinal ganglion cell stress and other features.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.