Abstract
Purpose :
High-power laser models of murine injury have been used to create clinically visible lesions, the study of which has illuminated angiogenesis targets for wet age-related macular degeneration (wet-AMD). We developed a laser injury model featuring subclinical lesions in one quadrant of the retina to elicit retinal gliosis. We also exploited a genetic model of retinal degeneration (JR5558 mouse line), investigating gliosis and the peptidyl arginine deiminase-4 (PAD4)-citrullination axis in both these models.
Methods :
A cluster low-power laser protocol (CLPLP) was established where C57Bl6 mice received 9 consecutive pulses of 50 mW for 5 seconds each applying 5 such burns in a cluster to one quadrant of the retina. The conventional laser protocol (CLP; 250 mW 100 msec) was also employed. Non-injured mice and laser injured cohorts were sacrificed along a 7 to 30 day post-injury time course. JR5558 mice were sacrificed at 30 and 60 days of age. Eyes were cryosectioned, antibody stained for glial fibrillary acidic protein (GFAP), citrullination (F95), citrullinated-GFAP and PAD4, then analyzed by epifluorescence microscopy.
Results :
In the CLP, fundus imaging reveals bright autofluorescence of the lesion sites at time of injury, remaining visible for extended time periods. In the CLPLP, cluster injury is barely noticeable and rapidly resolved, suggesting the CLPLP models subclinical levels of injury. When retinas from CLPLP were co-stained for GFAP and F95, Muller cell-specific expression was observed at the lesion. PAD4 expression was also observed, however with reduced expression compared to CLP. In the JR5558 mouse line spontaneous lesions revealed striking upregulation of GFAP in Muller cell processes spanning the retina. Remarkably, GFAP citrullination along Muller cell processes starting at the endfeet was observed along with PAD4 expression.
Conclusions :
We previously reported that retinal gliosis and hypercitrullination initiates in the endfeet following the CLP, and citrullinated GFAP is also seen in human wet-AMD maculae. We extend this study showing sub-clinical features of retinal pathology created with CLPLP also feature these biomarkers. Furthermore, as the PAD4-hypercitrullination axis is also engaged in the endfeet of JR5558 mice, we propose that Muller glial endfeet are a ‘citrullination bunker’ that initiates and sustains citrullination in retinal degeneration.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.