RGC axonal retrograde transport and connectivity to the superior colliculi declines with age and IOP and serves as an early indicator of RGC pathology in the DBA/2J retina well before the onset of RGC death (Buckingham BP, et al.
IOVS 2006 47:ARVO E-Abstract 1237).
58 Thus, after having established that minocycline decreased microglia activation while leaving gliosis and anterior segment pathology unaltered, we determined whether minocycline treatment resulted in improved RGC axonal integrity. RGC somata were retrogradely labeled by bilateral injection of neuronal retrograde tracer in the superior colliculi. One week later, at dosing week 25, retinas from these mice were immunostained for NeuN, which strongly labeled all RGCs while it only weakly labeled displaced amacrine cells, which were excluded from this analysis. Healthy RGCs with normal axonal transport and functional projections to their target strata in the superior colliculi were double-labeled with neuronal retrograde tracer and NeuN, whereas those RGCs lacking normal connectivity or transport stained only for NeuN
(Figs. 4A 4B) . Stereology counts of both populations of RGCs provide a measure of relative RGC axonal connectivity (Buckingham BP, et al.
IOVS 2006 47:ARVO E-Abstract 1237).
36 37 38 Total NeuN-positive RGC numbers did not vary between vehicle (49,656 ± 421 cells) and minocycline (48,914 ± 2838 cells) groups, but minocycline-treated mice had significantly (
P < 0.05) more neuronal retrograde tracer-positive RGCs than vehicle-treated mice (47,990 ± 2924 vs. 39,552 ± 5461;
Fig. 4C ). This difference indicated that minocycline selectively protected the retrograde transport of neuronal retrograde tracer to RGC somata, suggesting improved axonal connectivity. Further supporting the beneficial effect of reducing microglia activation on RGC axon transport,
Figure 4Eillustrates the enhanced correlation between numbers of neuronal retrograde tracer-positive RGCs and microglia ramification detectable in the retinal midperiphery (1000 μm from ONH) after minocycline treatment. In this scatter plot, microglia ramification is highly correlated to neuronal retrograde tracer-positive RGC number for the minocycline treatment group (
r 2 = 0.73), suggesting that reduced microglial activation is associated with healthier RGCs. Although ramification is less correlated to neuronal retrograde tracer-positive RGC number for the vehicle treatment (
r 2 = 0.39), ramification is a good predictor of neuronal retrograde tracer-positive RGCs overall.