Utility of UW-based solutions for reducing apoptotic signaling and retinal ganglionic cell death in a whole eye transplantation rodent model

Abstract

Purpose : Overcoming retinal ganglionic cell (RGC) death induced by ischemia reperfusion injury and optic nerve transection is necessary for successful whole eye transplantation (WET). Current organ transplants use tissue preservatives like University of Wisconsin (UW) solution. We tested UW and a modified version including BaCl₂ and valproic acid (mUW) as RGC preservation solutions in an orthotopic rodent WET.

Methods : Untreated naïve eyes from 14-to-16-week-old male Brown Norway rats (n=32) were harvested and retinas were stained by immunohistochemistry (IHC) for Brn3a to quantify RGC preservation and phospho-c-Jun (PNJ) for apoptotic signaling. Donor eyes were then intravitreally injected with 10 μL of heparinized saline (n=3), UW (n=5), mUW (n=5), or not injected at all (n=3) and hemifacial flaps with donor eyes were harvested for WET. At POD2, transplanted and native (recipient, untreated) eyes were enucleated and retinas were prepared for IHC analysis. Continuous variables are presented as mean (±standard error) and compared using one-way ANOVA and two sample Z tests.

Results : Naïve retinas expressed 102.4±3.2k Brn3a+ RGCs with a 12% background expression of PNJ+ signal. At POD2, native recipient retinas show 107.8±4.0k Brn3a+ RGCs with 9% co-staining for PNJ+. In the untreated WET condition, retinas show 114.6k±5.6k Brn3a+ RGCs with 39% co-staining for PNJ+, a significant increase from the naïve condition (P<0.05). In the saline condition, retinas show 90.4±8.8k Brn3a+ RGCs with 21% co-staining for PNJ+, a significant increase from the naïve condition (P<0.05). UW treated retinas show 78.7±4.5k Brn3a+ RGCs with 1% co-staining for PNJ+. mUW treated retinas show 88.9±4.9k Brn3a+ RGCs with 2% co-staining for PNJ+. UW and mUW are not statistically different from one another (P>0.05) and both provided a significant reduction in RGC apoptosis in the setting of WET at POD2 (P<0.05).

Conclusions : We have established a novel system to assess RGC preservation and apoptosis post-WET. Injecting UW-based preservation solutions represents a promising strategy for RGC survival post WET. UW and mUW reduced apoptotic signaling compared to no treatment and saline in donor eyes at POD2 after WET. Future research will include monitoring Brn3a+ and PNJ+ expression at other timepoints and analyzing other apoptotic markers.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.