In addition to histologic analysis, immunolabeling was used to enable direct quantification of RGC density. Approximately 40% to 60% of cells in the mouse GCL are estimated to be displaced amacrine cells.
18–21 Although we did not directly quantify the survival of amacrine cells, no reduction was found in the thickness of the INL, 40% of which is composed of amacrine cells.
19 This observation would argue that amacrine cells are not vulnerable in LIOH, consistent with previous reports in other rodent models of glaucoma.
22,23 Considering the existence of displaced amacrine cells, the total number of nuclei in GCL does not provide an accurate measure of RGC survival after laser treatment. To overcome this limitation, Brn-3b antibody was used to specifically label RGCs in retinal whole-mounts (
Fig. 3). Brn-3b is a transcription factor expressed in a subset of RGCs
15,24 distributed uniformly across the retina, allowing for direct visualization and convenient RGC counting in immunostained retinal whole-mounts. Because previous studies demonstrated that no specific class of RGCs is selectively impacted in mouse glaucoma,
22 we used the density of Brn-3b
+ nuclei as a surrogate measure of the overall density of surviving RGC somas. The results showed that Brn-3b
+ RGC density at 1 week after laser treatment was not significantly different from that of control (
Fig. 3F). However, there was a substantial (42%) loss of Brn-3b
+ RGCs compared with control retinas by 2 weeks after laser treatment. By 4 weeks, the average density of Brn-3b
+ RGCs in laser-treated eyes was reduced by 91% of controls. Thus the loss of Brn-3b expression appeared to commence, on average, between 1 to 2 weeks after laser treatment. Although it is possible that the loss of Brn-3b protein by itself may not mark the death of RGCs, histologic evidence of reduced cell numbers in the GCL and diminution of the NFL (
Fig. 2B) indicates that death does occur in at least a proportion of RGCs (see Discussion). In addition, DAPI staining of experimental retinal whole-mounts revealed substantial reductions in cell number (CTF, DS, unpublished observation, 2008), suggesting that changes in Brn-3b immunolabeling mirrored the overall pattern of RGC degeneration. Furthermore, the loss of Brn-3b
+ RGCs was nonuniform across the retina and often appeared sectorial in nature (
Fig. 3E). This pattern of RGC damage in laser-treated CD-1 mice is similar to what has been observed in DBA/2J animals using RGC-specific ROSA3 βGeo reporter or retrograde labeling
22,25,26 and is consistent with an insult to discrete axon bundles at the ONH.