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Neuroprotection of the Inner Retina Also Prevents Secondary Outer Retinal Pathology in a Mouse Model of Glaucoma

Purpose We examined structural and functional changes in the outer retina of a mouse model of glaucoma. We examined whether these changes are a secondary consequence of damage in the inner retina and whether neuroprotection of the inner retina also prevents outer retinal changes. Methods We used an established microbead occlusion model of glaucoma whereby intraocular pressure (IOP) was elevated. Specific antibodies were used to label rod and cone bipolar cells (BCs), horizontal cells (HCs), and retinal ganglion cells (RGCs), as well as synaptic components in control and glaucomatous eyes, to assess structural damage and cell loss. ERG recordings were made to assess outer retina function. Results We found structural and functional damage of BCs, including significant cell loss and dendritic/axonal remodeling of HCs, following IOP elevation. The first significant loss of both BCs occurred at 4 to 5 weeks after microbead injection. However, early changes in the dendritic structure of RGCs were observed at 3 weeks, but significant changes in the rod BC axon terminal structure were not seen until 4 weeks. We found that protection of inner retinal neurons in glaucomatous eyes by pharmacological blockade of gap junctions or genetic ablation of connexin 36 largely prevented outer retinal damage. Conclusions Together, our results indicate that outer retinal impairments in glaucoma are a secondary sequalae of primary damage in the inner retina. The finding that neuroprotection of the inner retina can also prevent outer retinal damage has important implications with regard to the targets for effective neuroprotective therapy.

G laucoma is a neurodegenerative disease often associated with elevated intraocular pressure (IOP), which results in progressive degeneration of retinal ganglion cells (RGCs) and visual field deficits. 1,2 Although RGCs are the primary degenerative targets in glaucoma, the extent to which distal neurons are affected remains controversial. Some studies have found no evidence of outer retinal abnormalities, [3][4][5][6] but others have reported both structural and functional changes in cone photoreceptors, 7-11 bipolar cells (BCs), [12][13][14][15][16][17] and horizontal cells (HCs) 16,18 in both glaucoma patients and experimental animal models of glaucoma. Many parameters subserved by BCs and HCs are altered in glaucoma, consistent with functional deficits in outer retina. [19][20][21][22][23][24] Structural changes localized to the outer retina are temporally correlated with inner retinal pathology in glaucoma patients and therefore appear dependent on the state of disease progression. 8,25 However, it is unclear whether these abnormalities result directly from glaucomatous insult or are a secondary consequence of inner retina damage. Like other neurodegenerative central nervous system diseases, glaucoma often follows a course of initial cell death due to the primary insult followed by a progressive secondary loss. 26,27 Thus, understanding the extent and timing of outer retina dysfunction is necessary to construct a more complete picture of glaucoma pathophysiology necessary to define targets for neuroprotective therapies.
Here, we examine outer retinal changes in a mouse microbead occlusion model of glaucoma. We found that the structure and function of BCs, as well as the dendritic/axonal organization of HCs, show substantial damage following induction of elevated IOP. Importantly, we observed that early structural changes to RGC dendrites precede those to BC axon terminals in the inner plexiform layer (IPL). Finally, we found that protection of inner retinal neurons in glaucomatous eyes by the previously reported blockade of gap junctions (GJs) 28 significantly reduces abnormalities in the outer retina. These results indicate that outer retinal impairments in glaucoma are a secondary sequalae of primary damage in the inner retina, a finding that contributes

Immunohistochemistry
The immunohistochemical methods have been described previously. 28,31,32 Eyecups were fixed with 4% paraformaldehyde, cryoprotected, and embedded in tissue freezing medium (Electron Microscopy Sciences, Hatfield, PA, USA), and 10-to 15-μm-thick frozen sections were cut. The tissues were incubated with diluted primary antibodies overnight (sections) or for 48 hours (whole mounts), washed, and then incubated in secondary antibodies for 2 to 4 hours. Images of immunolabeled retinal sections or whole mounts from control and experimental groups were obtained with an Olympus FV1200 MPE confocal microscope (Olympus, Tokyo, Japan) using a 40× oil-immersion objective. Highresolution (1024 × 1024 pixels) z-stack images were taken using step sizes of 0.7 to 2.0 μm and compiled. The brightness and contrast of micrographs were adjusted using Photoshop CS6 (Adobe, San Jose, CA, USA). The primary antibodies were anti-PKCα (1:10,000; Sigma-Aldrich); anti-Chx10 (1:500; Santa Cruz Biotechnology, Dallas, TX, USA); anticalbindin (1:500; Santa Cruz Biotechnology); anti-SMI32

Assessment of Morphological Changes
Cell counts were made per lateral unit length of 300 μm in vertical sections, 10 to 15 μm thick. Measurements were made from the mid-periphery, 1.5 to 2.0 mm from the optic nerve head, within all four retinal quadrants (15-20 sections) and averaged, using at least three retinas for each condition. For measures of SMI32 labeling in flatmount retinas, three or four square areas (300 × 300 μm) in each quadrant in the mid-periphery were analyzed and averaged across four retinas each for control and experimental protocols. ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used to quantify the number of pixels with immunolabels above background. The number of pixels positive for a marker was then divided by the total pixel number and presented as a percentage of covered area. This parameter was independent of differences in the intensity of label within or across retinas, as the analysis was performed with grayscale thresholding. All data were imported into SigmaPlot software (Systat Software, San Jose, CA, USA), and histograms were constructed.

Electroretinogram Recording
The scotopic ERG responses were recorded from both eyes simultaneously. Visual stimuli consisted of brief (<5 ms) white Ganzfeld flashes ranging between -6.7 and 2.0 log scot cd·s/m 2 , produced by an array of light-emitting diodes (Diagnosys, Lowell, MA). Responses were averaged over 40 to 50 trials for weak stimuli and fewer trials for stronger stimuli. Signals were amplified, filtered (1-300 Hz), and digitized at 1 kHz with a resolution of 0.1 μV.

Statistical Analysis
Data are presented as mean ± SEM. Sample sizes (retinas, eyes, mice) were determined on the basis of our previous studies. 28,32 Sample sizes were determined using values of groups and common standard deviation. Calculations were performed using two-sided tests with alpha = 0.05 and power = 0.8. Samples were allocated to experimental groups according to genotype; therefore, there was no randomization. To compare two experimental groups, we used a twotailed Student's t-test. Comparisons between larger groups were analyzed using one-way analysis of variance followed by Tukey's multiple comparison test. Values of P < 0.05 were considered statistically significant.

Changes in Bipolar Cell Structure and Function in Glaucomatous Eyes
Unilateral injections of microbeads made at weeks 0 and 4 resulted in a sustained elevation of IOP from ∼12 to 20 mm Hg for the entire 8-week period (P < 0.001 for all time points; n = 10 eyes/group) (Fig. 1A). We detected no sex-specific differences in mean IOP elevation. To study the effect of elevated IOP on outer retina function, we recorded and at 8 weeks after the initial microbead injection (red). There was a significant decrease of the peak amplitude (C) and an elevation of the time-to-peak latency (D) of the b-wave in glaucomatous eyes as compared with controls. Data are presented as mean ± SEM. P < 0.05 for all intensities; n = 5 eyes/group. (E-H) Confocal images of vertical sections from control and glaucomatous retinas at 8 weeks after the initial microbead injection immunostained with anti-PKCα for rod BCs and anti-Chx10 for cone BCs (asterisks). Arrowheads in G and H show rod BC dendrites. Projection of eight images, z = 0.7 μm. (I, J) Histograms quantify the reduction of rod and cone BCs (per 300-μm lateral distance) in microbead-injected eyes, 8 weeks after initial injection, compared with controls. Data are presented as mean ± SEM. ** P < 0.01; n = 5 eyes/group. (K) Histogram showing the density of rod BC axon terminals in IPL of glaucomatous retinas as compared with control eyes. Data are presented as mean ± SEM. ** P < 0.01; n = 5 eyes/group. Scale bar: 10 μm in all panels. OPL, outer plexiform layer; INL, inner nuclear layer; IP, inner plexiform layer; GCL, ganglion cell layer.
scotopic ERGs weekly for 8 weeks following the initial microbead injection. Comparisons were made to control recordings from contralateral eyes with sham injections, as well as eyes from completely untreated animals. We found no significant differences in amplitude and shape of ERG waveforms obtained from the two control cohorts (P > 0.1 for both parameters). We analyzed the a-wave and bwave profiles of the ERGs to evaluate changes in photoreceptor and BC function, respectively. 33,34 At 8 weeks after microbead injection, we found no significant change in the amplitude of the a-wave (P > 0.1; data not shown). Consistent with this finding, photoreceptor nuclei stained with 4 ,6diamidino-2-phenylindole showed no apparent loss in glaucomatous eyes (P > 0.1; data not shown). In contrast, elevation of IOP in WT mice resulted in a significant reduction in amplitude (Figs. 1B, 1C) and implicit time to peak (Fig. 1D) of the b-wave (P < 0.05; n = 5 eyes/group), indicative of changes in ON BC activity. 35 Next, we assessed structural changes in BC structure in glaucomatous eyes. Vertical retinal sections of control and microbead-injected eyes from WT mice were immunostained with anti-PKCα, a selective marker for rod BCs (RBCs). [36][37][38] We then double-labeled retinas with anti-Chx10 to visualize BC nuclei. 36,39 Cone BCs (CBCs) were differentiable as Chx10-positive/PKCα-negative profiles (Figs. 1E, 1F). Under the control condition, PKCα-positive RBC somata formed two or three cell layers at the outer inner nuclear layer (INL) border with long, bushy dendrites (Figs. 1E, 1G). The RBC axons ended deep in layer 5 of the IPL as dense terminals with lateral varicosities (Fig. 1E). In glaucomatous retinas, we found a significant reduction in the number of RBC somata that, in contrast to controls, were mainly confined to a single layer adjacent to the outer plexiform layer (OPL) (Figs. 1F, 1I). We observed a 32% loss (P < 0.001; n = 8 eyes/group) at 8 weeks after microbead injection. The overall density and size of RBC axon terminals were reduced in glaucomatous eyes and showed a substantial reduction in the number of lateral varicosities (P < 0.001; n = 8 eyes/group) (Fig. 1K). There was also a marked loss of CBCs in glaucomatous eyes. We observed a 12% loss of cone BCs (P < 0.05; n = 3-5 eyes/group) by 5 weeks, which increased to a 22% loss (P < 0.001; n = 8 eyes/group) by 8 weeks.

Timing of Bipolar Cell Structural Changes in Glaucoma
We have reported that the first significant loss of RGCs and amacrine cells (ACs) occurs at 4 weeks after initial microbead injection. 32 In the next series of experiments, we examined BC structure at weekly intervals after microbead injection to compare the time course with changes in the inner retina. The first significant loss of RBCs, a 13% reduction from control values (P < 0.05; n = 3-5 eyes/group), was at 4 weeks (Figs. 2A, 2C, 2E, 2G, 2M), whereas a significant loss of CBCs, a 12% reduction (P < 0.01; n = 3-5 eyes/group), first occurred at 5 weeks after microbead injection (Fig. 2N).
Prior to complete cell loss, neurons show structural changes to dendrites and axon terminals. 40,41 We have reported that αRGCs in glaucomatous mouse eyes that survive at 8 weeks show a significant loss of dendrites. 28 We extended this finding by calculating the dendritic lengths of anti-SMI32-labeled αRGCs at weekly intervals after microbead injection. We found the first significant change in dendritic length at 3 weeks, a 40% reduction from control values (P < 0.01; n = 3-5 eyes/group) (Figs. 2I-2L, 2P). In contrast, the first significant loss of RBC axon terminals was a 20% reduction (P < 0.01; n = 3-5 eyes/group) at 4 weeks (Figs. 2B, 2D, 2F, 2H, 2O). Clearly, early structural changes of RGC dendrites preceded those of BC axon terminals.

Neuroprotection of the Outer Retina in Glaucoma
To test whether the BC changes were a consequence of prior degenerative changes in the inner retina, we leveraged the neuroprotective actions of GJ blockade or ablation reported in a number of retinopathies, including glaucoma. 28,31,32,[42][43][44] We showed previously that selective genetic deletion of connexin 36 (Cx36) protein largely prevented loss of RGCs and ACs, preserved optic nerve axons, and reduced reactive gliosis in a mouse model of glaucoma. 28 Importantly, this neuroprotective action was not associated with a lowering of IOP.

Synaptic Changes Associated With Rod Bipolar Cells in Glaucomatous Retinas
We next examined changes in synaptic structures associated with RBC dendrites and axon terminals in microbead-injected eyes. Retinas of WT mice were double immunolabeled for PKCα and anti-CtBP2, an established marker for photoreceptor synaptic ribbons. 45 For the first 3 weeks following microbead injection, the dendritic architecture of RBCs appeared similar to that in controls (Figs. 4A-4C). This included processes projecting to CtBP2-positive, horseshoe-shaped presynaptic ribbons (Fig. 4B, inset).
However, structural changes in the OPL were first observed at 4 weeks and progressed over the next 4 weeks (Figs.  4D-4F). These changes included a retraction of dendrites and a 27% (P < 0.01, n = 3 eye/group) reduction in CtBP2positive synaptic ribbons (Fig. 4J), with the spared ribbons  remodeled to a punctate shape (Fig. 4E, inset), which were no longer clearly associated with dendritic tips of RBCs. Despite these changes to the photoreceptor synaptic complex, there was no measurable loss of photoreceptors over the 8-week experimental period. We occasionally observed RBC dendrites sprouting (Figs. 4D, 4F), a process of remodeling, 46,47 presumably to make ectopic contact with photoreceptor terminals. In contrast, we observed no significant changes in RBC dendritic structure, nor in the number and shape of the CtBP2-positive synaptic ribbons, in the OPL of microbead-injected Cx36 −/− mice (Figs. 4G-4J).
We next investigated changes in the distribution of vesicular glutamate transporter 1 (VGluT1) in glaucomatous eyes, which is responsible for the uploading of glutamate into synaptic vesicles. 48 Consistent with earlier studies, 49,50 immunostaining of control retinas (Fig. 5A) showed discrete punctate labeling for VGluT1 throughout the IPL (Fig. 5B). Double-labeling experiments identified large PKCα-positive RBC axon terminals in distal layer 5 of the IPL with VGluT1 expression (Fig. 5A). In glaucomatous retinas, the density of VGluT1-positive structures was reduced in the IPL by 34% (P < 0.05; n = 4 eyes/group) (Figs. 5C, 5D, 5I). This reduction was evident for VGluT1-positive patches that were colocalized with the PKCα-positive terminals deep in layer 5 of the IPL (Fig. 5E). The reduced VGluT1 expression throughout both sublamina in the IPL strongly suggests synaptic changes to CBC axon terminals, as well.

Altered Morphology of Horizontal Cells in Glaucomatous Retinas
A single type of axon-bearing HCs in mouse makes synaptic contacts with cone and rod photoreceptors via dendritic and axonal endings, respectively. 51,52 The HCs form an electrically coupled network important to contrast sensitivity, 53 a feature often impaired in glaucoma patients. 54,55 We therefore looked for morphological changes in HC structure in glaucomatous retinas by labeling vertical sections with anticalbindin (CB) 37 (Fig. 6A). We found no significant difference between the number of HC somata in control retinas and those in microbead-injected eyes (P > 0.5; n = 3 eyes/group), but we did see structural changes in dendritic and axonal processes. In addition to the thick proximal dendritic processes, distal punctate terminal endings could be visualized at the level of photoreceptor terminals (arrowheads). However, we found that to see the terminal puncta it was necessary to increase the gain of the photomultiplier of the confocal microscope, which resulted in a saturated image. Although these synaptic terminals have been described previously as dendritic tips, 16 it is possible that some puncta may be axon terminals. Nevertheless, at 8 weeks after microbead injection, we found that CB-positive terminal puncta showed a clear change in structure (Fig. 6B). Although background CB label could be visualized, there was a clear loss of the punctate structure seen in control retinas. In contrast, retinas from microbead-injected eyes of Cx36 −/− mice or WT mice treated with MFA maintained the punctate structure of terminal endings seen in control retinas (Figs. 6C, 6D).

DISCUSSION
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 PKCαpositive 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][63][64][65][66][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][78][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.