Despite findings of abnormalities in the outer retina of human patients and animal models with glaucoma,
9,14 it has remained unclear whether these changes result directly from glaucomatous insult or are secondary consequences of inner retinal pathology.
25 Our findings support and extend the evidence for outer retinal damage in experimental glaucoma but also provide strong evidence that outer retinal changes occur subsequent to and result as a consequence of inner retinal damage.
Consistent with earlier glaucoma studies,
10,14,57–61 we found that sustained elevation of IOP negatively affected the ERG b-wave in WT mice, suggesting a reduction of ON BC activity.
35 We found a corresponding loss of both RBCs and CBCs in glaucomatous eyes. A loss of RBC immunoreactivity for PKCα has been reported earlier in a vein cauterization model of glaucoma in rats, but it was not clear whether these cells died or had stopped expressing the molecular marker.
13 The decreased number of PKCα-positive RBCs, as well as Chx10-positive CBCs, in our study is most likely attributable to cell death rather than a change in protein expression, as surviving BCs showed intensity of immunolabeling comparable to that in control retinas. Structural changes to dendrites and axon terminals have been reported in a number of ocular pathologies, including glaucoma.
14,46,62–67 Consistent with these studies, we found that dendrites of surviving RBCs were less profuse than in controls, with some cells almost devoid of processes. In the IPL, RBC axon terminals showed decreased branching with smaller varicosities, similar to changes described in the DBA/2J mouse model of congenital glaucoma.
66
We observed a significant alteration in CtBP2 immunostaining in the OPL in glaucomatous retinas, indicating damage to the photoreceptor synaptic complex. Such changes corresponded to dendritic changes of second-order BCs and HCs, which included retraction of tip processes and occasional abnormal growth. The morphological alterations of structures in the OPL are consistent with the functional impairment indicated by changes in the ERG b-wave. In the IPL, surviving RBC axon terminal endings in glaucomatous retinas retained PKCα expression but lost the VGluT1 immunoreactivity found in control retinas. This suggests that synaptic release of glutamate from BCs to postsynaptic targets is diminished, as well.
The first significant loss of RBCs was first observed 4 weeks after microbead injection, which coincides with the earliest reported loss of RGCs and ACs.
28,32 In contrast, significant loss of CBCs was first seen at 5 weeks after microbead injection. Changes in dendritic architecture preceding neuronal loss have been described in animal models of glaucoma.
40,41 We found significant loss of dendritic processes in RGCs as early as 3 weeks after the initial microbead injection. However, damage to RBC axon terminals was first observed at 4 weeks, a significant difference in timing relative to our 8-week model.
Overall, these findings support the idea that structural changes to outer retinal neurons occur subsequent to those in the inner retina but do not address whether they result as a consequence. To examine this question, we determined whether neuroprotection of inner retinal neurons could also prevent damage in the outer retina. Indeed, we found that genetic ablation of Cx36 or blockade of GJs with MFA prevented structural damage to BCs and HCs, preserved the ERG b-wave at control levels, and sustained synaptic structures in the OPL and IPL of glaucomatous retinas. Combined with our previous finding that ablation of Cx36-expressing GJs protects RGCs and ACs,
28 these results support the idea that outer retina damage is a secondary consequence of inner retinal pathology in glaucoma. Yet, the question may still be raised whether GJ blockade or, in particular, Cx36 ablation could directly protect the outer retina independent of actions in the inner retina. A number of points argue against this possibility. First, HC GJs in the mouse retina express Cx57 but not Cx36.
68 Second, although photoreceptors do express Cx36,
69 our finding of no significant loss in glaucoma indicates that bystander cell death did not occur at the ONL level. Third, RBCs do not express GJs. Fourth, although some OFF CBCs may express Cx36,
70 ON CBCs mainly express Cx45
71,72 and are coupled to glycinergic AII ACs,
73,74 which are relatively unaffected in the glaucoma mouse model.
32 These findings thus strongly suggest that bystander-mediated neuronal loss occurred exclusively in the inner retina. Our conclusion that outer retinal damage follows that in the inner retina is supported by the findings for glaucoma patients and DBA/2J mice that outer retina damage was found prominently in regions overlying patches of inner retina with prominent RGC loss.
8,16,25
The mechanism by which damage to the inner retina in glaucoma can lead to changes in outer retina is unclear. However, as BCs are presynaptic to vulnerable RGCs and ACs, a likely mechanism is trans-neuronal or trans-synaptic degeneration.
27,75,76 In this scheme, the degeneration of BCs does not result from the primary insult but rather from the loss of their postsynaptic target cells, consistent with attenuated VGluT1 expression in BC axon terminals. This mechanism has been linked to cell death in numerous CNS neurological diseases, including recently in glaucoma.
27,77–79 Although CBCs synapse directly with degenerating RGCs, RBCs do not; however, RBCs make synaptic contacts with ACs, which have been shown to degenerate in glaucomatous eyes with a time course similar to that for the loss of RGCs.
28 In turn, the structural changes in BC dendrites could then lead to the observed alterations in photoreceptor terminal synaptic complexes that, in turn, initiate the postsynaptic degeneration of HC dendritic and axonal processes.
Another potential mechanism for the spread of retinal damage is reactive gliosis, which is often manifested as an increase in glial fibrillary acidic protein expression in astrocytes lying adjacent to the nerve fiber layer and in Müller cell processes that extend vertically through retinal layers.
28,80,81 Reactive gliosis can alter environmental homeostasis and release toxins that can lead to progressive neuronal degeneration.
82,83 Interestingly, ablation of Cx36 or blockade of GJs, which we show here can prevent outer retinal changes in glaucoma, has been reported to prevent gliosis.
28
Although IOP-lowering drugs remain the mainstay treatment for glaucoma, recent studies have turned to neuroprotective strategies to prevent cell death and subsequent vision loss.
84 Importantly, therapies providing protection to the inner retina but leaving outer retinal damage unaffected would be pointless, as dysfunctional BC signaling to RGCs would still lead to vision defects. It is therefore fortuitous that our findings show that protection of the inner retina by GJ blockade can also prevent damage to outer retinal neurons, including their synaptic connections. Our findings thus reveal inner retinal neurons as potential targets for effective neuroprotective therapies to sustain vision in glaucoma patients.