Figure 3shows micrographs of retinas that were obtained 14 days after exposure to damaging light and underwent ISH for AChE-R mRNA
(Figs. 3a 3c) , or histochemical staining for total AChE activity
(Figs. 3b 3d) . Sections were derived from experimental rats treated with Monarsen
(Figs. 3c 3d) , and from control rats treated with saline
(Figs. 3a 3b) . It should be noted that in preliminary experiments, Monarsen treatment, either by intravitreal injection or by intraperitoneal injection, exerted no effects on the retinas of rats, which were raised under a 12-hour light–dark cycle and were not exposed to bright, damaging light (data not shown), similar to its null effects in the control brain.
37 In contrast, averaged densitometry measurements from retinas of four rats of each group in this experiment (right of each micrograph) demonstrated that Monarsen, injected intraperitoneally, predictably crossed the blood–retinal barrier, reached the retina, and suppressed the light-induced expression of AChE-R mRNA in all retinal cells, having the strongest effect in the photoreceptor inner segments (
Fig. 3c , arrowheads). Correspondingly, AChE activity in the photoreceptor inner segments was considerably weaker in the retina from rats treated with Monarsen (
Fig. 3d , arrowheads) compared with those injected with saline (
Fig. 3b , arrowheads). An important finding is that Monarsen did not alter AChE activity in the proximal retina (IPL and GC), suggesting that these layers express AChE-S, which is considerably less sensitive to this agent. This supports previous observations in rat muscle,
27 mouse brain,
36 and primate motor neurons,
35 indicating that nascent mRNA transcripts of AChE are more susceptible to the antisense treatment, probably because they are not yet covered with the protective bulky structure of the ribosomes and translational protein complexes which engulf functioning mRNA chains. Reproducible AChE-R suppression occurred in four rats treated with Monarsen compared with four control rats. Densitometry measurements were further used to calculate the effects of saline and Monarsen treatment on AChE-R mRNA expression in the different retinal layers and on AChE activity in the photoreceptor inner segments
(Table 1) . The suppressive effects of Monarsen on light-induced expression of AChE-R mRNA were statistically significant in photoreceptor inner segments (IS) and in neurons of the INL, but not in ganglion cells. Monarsen’s effect was also statistically significant for AChE activity in photoreceptor inner segments (IS).