Abstract
Purpose :
Retinal ganglion cell (RGC) replacement and optic nerve regeneration could restore vision for patients suffering from optic neuropathies. Yet, successful transplantation is hampered by the poor migration of donor cells into the retina due to their accumulation at the inner limiting membrane (ILM). We aim to significantly promote RGC transplantation by local disruption of the ILM using a light-based approach, i.e. photoporation.
Methods :
To perforate the ILM, 0.25 mg/ml of the photosensitizer indocyanine green was applied at the ILM side of bovine retinal explants followed by pulsed laser treatment (800 nm, 7 nanosecond pulses, 1.65 J/cm2) using a programmed laser pattern. Next, retinal explants were stained for laminin, enabling examination of ILM integrity using microscopy. To explore the impact of ILM photoporation on RGC transplantation, hiPSC-derived RGCs expressing TdTomato (20.000 per 5 mm explant) were co-cultured in an organotypic manner for 7 days on untreated (n=10) and photoporated explants (n=10). Fixation and subsequent staining of RGCs and human nuclei allowed us to quantify the number of explanted RGCs and assess their spatial distribution.
Results :
Analysis of high-resolution microscopy images with FIJI revealed that we are able of creating a distinct perforation pattern in the ILM with a mean pore size of 80 ± 22 µm resulting in the ablation of 34 ± 6% of the ILM surface. Applying this perforation pattern, donor RGC survival was significantly enhanced as compared to untreated explants (10.1±2.9% vs 5.4±3.3%; p<0.5), where treated explants also showed a higher donor cell density (106.5±32.6 cells/mm2 vs 41.8±26.7 cells/mm2; p<0.01). Interestingly, spatial metrics suggest that ILM photodisruption reduces donor cell clustering seeing that the nearest neighbor index increases from 0.38 (± 0.05) to 0.57 (± 0.07) in treated explants (p<0.001).
Conclusions :
Our results demonstrate that ILM photodisruption can ablate the ILM with an exceptionally high spatial control. Our preliminary data furthermore indicates that ILM photodisruption has a positive impact on donor RGC integration into the retina. Ongoing and future work involves exploration of the effect of different laser scanning patterns on RGC transplantation along with assessment of its impact on donor RGC connectivity within the host retinal neurocircuitry.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.