Abstract
Purpose :
Organotypic tissue cultures are valuable tools to investigate various diseases, however, traditional ocular tissue culture systems fail to preserve optic nerve head(ONH) which is vulnerable to pathological insult and has several unique characteristics. We established a new retinal explant culture system to maintain retinal ganglion cells(RGCs) integrity and reserve ONH for In vitro studies.
Methods :
Tissue from newborn mice were cultured in vitro for 7 days by two preparation methods: first, only the neuroretina was saved; second, the neuroretina attached with optic nerve(ON) were saved. The number of living cell on RGCs layer were checked at several time points during the culture, and viability of RGCs was assessed by multielectrode array technology for both two methods. After then, only explants with preserved ON were used for the followed experiment for ONH observation. Morphology of ONH, glial activation of retina and ONH were checked.
Results :
Similar patterns of cell apoptosis and morphological changes were observed in both new and traditional culture system. Yet, RGCs loss in retinal explants with preserved ON was significantly lower than in retinas that did not preserve the ON. After 8 h in culture, the local field potentials (LFPs) of explants could be recorded in the retinas that had the ON attached. There were no LFPs detected in the retinas without ON. In the new culture method, glial lamina structure can be well observed under confocal microscope with glial fibrillary acidic protein (GFAP) staining, and with the culture went on the glia lamina became structural disordered. Astrocyte hypertrophy was observed in retinal ganglion cell layer. GFAP was up regulated in both retina and ONH.
Conclusions :
The optic nerve-preserving culture helps preserve the integrity of RGCs and provide an opportunity to study the unique structure of ONH and the extended ganglion axons in vitro.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.