Preservation of Murine Whole Eyes With Supplemented UW Cold Storage Solution: Anatomical Considerations

Abstract

Purpose: Retinal ganglion cell (RGC) apoptosis and axon regeneration are the principal obstacles challenging the development of successful whole eye transplantation (WET). The purpose of this study was to create a neuroprotective cocktail that targets early events in the RGC intrinsic apoptotic program to stabilize RGCs in a potential donor eye.

Methods: University of Wisconsin (UW) solution was augmented with supplements known to protect RGCs. Supplements targeted tyrosine kinase signaling, histone deacetylase activity, K+ ion efflux, macroglial stasis, and provided energy support. Modified UW (mUW) solutions with individual supplements were injected into the vitreous of enucleated mouse eyes, which were then stored in cold UW solution for 24 hours. Histopathology, immunostaining of individual retinal cell types, and analysis of cell-specific messenger RNAs (mRNAs) were used to identify supplements that were combined to create optimal mUW solution.

Results: UW and mUW solutions reduced ocular edema and focal ischemia in globes stored in cold storage. Two major issues were noted after cold storage, including retinal detachment and reduction in glial fibrillary acidic protein staining in astrocytes. A combination of supplements resolved both these issues and performed better than the individual supplements alone. Cold storage resulted in a reduction in cell-specific mRNAs, even though it preserved the corresponding protein products.

Conclusions: Eyes treated with optimal mUW solution exhibited preservation of retinal and cellular architecture, but did display a decrease in mRNA levels, suggesting that cold storage induced cellular stasis.

Translational relevance: Application of optimal mUW solution lowers an important barrier to the development of a successful whole eye transplantation procedure.

Figure 1.

Images of whole eyes stored in mUW solutions for 24 hours at 4°C, stained with hematoxylin and eosin. These images are representative of the images that were used for evaluation in the scoring system listed in Table 6 (see Materials and Methods for a complete description of the scoring criteria). The optimal mUW solution had the best overall performance based on the subjective scoring protocol, and is the only solution, aside from the baseline, which did not demonstrate retinal detachment after cold storage. Scale bar, 1 mm.

Figure 2.

Corneal thickness was measured using ImageJ software and compared against the baseline. (A–L) Representative images of the corneas from all mUW solutions tested. Scale bar, 300 µm. (M) A graph showing the average corneal thickness from three samples each of all solutions. Data from eyes stored in PBS are also included (gray bar). All groups exhibited retinal swelling in the extracellular regions of the stroma (***P < 0.0001 by analysis of variance). Optimal mUW solution demonstrated the least swelling of all groups tested but was still significantly greater than baseline corneas (t test, ***P < 0.0001).

Figure 3.

Immunostaining of retinas for RBPMS (an RGC-specific marker) in freshly isolated eyes (A) or eyes kept in cold storage for 24 hours after injection of UW solution (B) or mUW solutions containing the supplement(s) indicated (see Tables 2 and 3) (C–L). Optimal mUW solution contained VPA, TEA, hydrocortisone, and L-lactate. The ganglion cell layer (GCL) and robust RBPMS staining appeared relatively normal in all conditions tested. Scale bar, 50 µm. 4′6-Diamidino-2-phenylindole (DAPI) counterstain; INL, inner nuclear layer; ONL, outer nuclear layer.

Figure 4.

Immunostaining of retinas for RHO (a rod photoreceptor cell specific marker) in freshly isolated eyes (A) or eyes kept in cold storage for 24 hours after injection of UW solution (B) or mUW solutions containing the supplement(s) indicated (see Tables 2 and 3) (C–L). Optimal mUW solution contained VPA, TEA, hydrocortisone, and L-lactate. RHO staining is most prominent in the outer segments (OS) of the rod photoreceptors, a pattern that was relatively preserved in each of the test conditions, although reduced intensity was noted in both hydrocortisone and L-lactate conditions. The intensity was robust, however, in optimal mUW solution, which contained both supplements. Scale bar, 50 µm. 4′6-Diamidino-2-phenylindole (DAPI) counterstain. INL, inner nuclear layer; IS, inner segments; ONL, outer nuclear layer.

Figure 5.

Immunostaining of retinas for PKCα (a rod bipolar cell specific marker) in freshly isolated eyes (A) or eyes kept in cold storage for 24 hours after injection of UW solution (B) or mUW solutions containing the supplement(s) indicated (see Tables 2 and 3) (C–L). Optimal mUW solution contained VPA, TEA, hydrocortisone, and L-lactate. Rod bipolar cell nuclei reside in the outermost region of the inner nuclear layer (INL) and send axons to the dendritic arbors of the RGCs in the ganglion cell layer (GCL). Compared with baseline retinas, these cells seemed to be disorganized and abnormal in mUW solutions containing sunitinib (C), SB203580 (D), and TEA (G). Retinas from eyes stored in optimal mUW solution, which contains TEA, exhibited cells virtually identical to baseline retinas (L). Scale bar, 25 µm. 4′6-Diamidino-2-phenylindole (DAPI) counterstain, which is shown as a separate channel on the left of each image so that bipolar cell morphology can be clearly shown. ONL, outer nuclear layer.

Figure 6.

Immunostaining of retinas for GFAP (a cell-specific marker for astrocytes and Müller cells, the latter being in a reactive state) in freshly isolated eyes (A) or eyes kept in cold storage for 24 hours after injection of UW solution (B) or mUW solutions containing the supplement(s) indicated (see Tables 2 and 3) (C–L). Optimal mUW solution contained VPA, TEA, hydrocortisone, and L-lactate. Astrocytes reside on the surface of the retina near the ganglion cell layer (GCL). Overall, cold storage in UW solution and most of the supplemented solutions resulted in a decrease or absence of GFAP staining except for (J) hydrocortisone, (K) L-lactate, and (L) optimal mUW solution. The combination of VPA and BaCl₂ (I) seemed to induce increased in expression in cells restricted to the astrocyte layer, and L-lactate also contained sporadic examples of Müller cell end feet processes containing GFAP (arrows in K). Scale bar, 50 µm. INL, inner nuclear layer; ONL, outer nuclear layer. 4′6-Diamidino-2-phenylindole (DAPI) counterstain.

Figure 7.

Assessment of cell-type specific mRNA abundance in retinas, 24 hours after cold storage. Heat map showing the relative change in transcript abundance compared to freshly isolated eyes. The heat scale bar on the right reflects the ratio of mean mRNA levels of experimental eyes divided by the same transcript level measured in the baseline eyes. Genes included in the mini-array are detailed in Table 4. qPCR results demonstrate a general decrease in mRNA abundance of all marker genes in all mUW groups, with the exception of VPA. The effect of VPA, when in combination with other supplements (including optimal mUW solution) was not retained, however. PRs, photoreceptors.