Neuroprotection of Retinal Ganglion Cells Suppresses Microglia Activation in a Mouse Model of Glaucoma

Sandeep Kumar; Abram Akopian; Stewart A. Bloomfield

doi:10.1167/iovs.64.7.24

Abstract

Purpose: Microglial activation has been implicated in many neurodegenerative eye diseases, but the interrelationship between cell loss and microglia activation remains unclear. In glaucoma, there is no consensus yet whether microglial activation precedes or is a consequence of retinal ganglion cell (RGC) degeneration. We therefore investigated the temporal and spatial appearance of activated microglia in retina and their correspondence to RGC degeneration in glaucoma.

Methods: We used an established microbead occlusion model of glaucoma in mouse whereby intraocular pressure (IOP) was elevated. Specific antibodies were used to immunolabel microglia in resting and activated states. To block retinal gap junction (GJ) communication, which has been shown previously to provide significant neuroprotection of RGCs, the GJ blocker meclofenamic acid was administered or connexin36 (Cx36) GJ subunits were ablated genetically. We then studied microglial activation at different time points after microbead injection in control and neuroprotected retinas.

Results: Histochemical analysis of flatmount retinas revealed major changes in microglia morphology, density, and immunoreactivity in microbead-injected eyes. An early stage of microglial activation followed IOP elevation, as indicated by changes in morphology and cell density, but preceded RGC death. In contrast, the later stage of microglia activation, associated with upregulation of major histocompatibility complex class II expression, corresponded temporally to the initial loss of RGCs. However, we found that protection of RGCs afforded by GJ blockade or genetic ablation largely suppressed microglial changes at all stages of activation in glaucomatous retinas.

Conclusions: Together, our data strongly suggest that microglia activation in glaucoma is a consequence, rather than a cause, of initial RGC degeneration and death.

Glaucoma, the second leading cause of blindness worldwide, is a neurodegenerative disease characterized by optic nerve atrophy and a progressive loss of retinal ganglion cells (RGCs) resulting in visual field deficits.¹^,² Elevated intraocular pressure (IOP) is considered a major risk factor in glaucoma, although loss of visual function may continue, despite therapeutic lowering of IOP.³ There is growing evidence that activation of microglia in multiple neurodegenerative diseases, including glaucoma, can drive pathogenic inflammation, thereby prompting investigation into new therapeutic strategies.⁴^,⁵

Microglia are morphologically and functionally dynamic resident cells that perform immune-like functions throughout the central nervous system (CNS), including retina.⁶^–⁸ In normal retina, microglia have a ramified shape, play a supportive role of neuron synaptic structure and function, and are associated with production of anti-inflammatory and neuroprotective factors.⁵ However, in response to a wide variety of insults, microglial cells transform to an activated phenotype. This is a complex multistage process that includes cell hypertrophy and proliferation; retraction of processes; expression of cell surface proteins such as CD68, CD86, and major histocompatibility complex class II (MHCII) glycoproteins; and production of pro-inflammatory cytokines. Activated microglia thereby play an essential role in cell loss associated with a number of neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases, amyotrophic lateral sclerosis and retinitis pigmentosa.⁹^–¹⁶ Activation of microglia has been suggested as a critical early event in cell degeneration associated with glaucoma.⁸^,¹⁰^,¹⁷^–²² In experimental glaucoma models, treatments that reduce activation of microglia have been shown to provide protection of RGCs and their axons in the optic nerve, consistent with the idea that microglia activation is detrimental to neuronal survivability.⁵^,¹⁹ In contrast, other studies have found that microglia activation is beneficial to or plays no role in retinal neuron degeneration.⁶^,¹²^,²³^,²⁴ Thus, the role of microglia activation in the cell loss associated with glaucoma remains unclear, including whether activation precedes or follows neurodegeneration.⁷

Here, we investigated the temporal and spatial appearance of activated microglia in a microbead occlusion mouse model of glaucoma and its relationship to RGC degeneration. We found that, although some early microglial changes, including altered morphology and increased cell number, followed IOP elevation and preceded RGC death, the later stage of activation, associated with upregulation of MHCII protein expression, corresponded temporally to initial RGC loss. However, we found that protection of RGCs in glaucomatous retinas afforded by gap junction (GJ) blockade or ablation²⁵ also largely prevented the early and late stages of microglia activation. These data strongly suggest that microglia activation in glaucoma is a consequence, rather than a cause, of RGC degeneration and death.

Methods

Experimental Animals

Experiments were performed on adult C57BL/6 (WT) and connexin36 knockout (Cx36^−/−) mice, 3 to 4 months old, of both sexes. Animals were kept under a 12:12-hour ambient light cycle and fed ad libitum. For all experiments, animals were selected randomly within each group. All animal procedures were in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Institutional Animal Care and Use Committee at the SUNY College of Optometry.

Induction of Elevated IOP

The IOP was elevated by unilateral injection of 10-µm-diameter polystyrene microbeads (Invitrogen, Carlsbad, CA, USA) into the anterior chamber.²⁵ The intracameral injections of 2 µL of microbead suspension (containing 7.2 × 10⁶ beads) were performed using a glass micropipette connected to a microsyringe. An equivalent volume of PBS was injected into the contralateral eyes to provide control (sham) measurements. A second microbead injection was performed during the fourth week, which maintained elevated IOP for at least 8 weeks. All injections were performed on animals anesthetized with an intraperitoneal injection of a ketamine/xylazine mixture and topical application of proparacaine. The IOP measurements were performed weekly between 10 AM and 12 PM to minimize effects of diurnal IOP variation. Six measurements were obtained per eye and averaged. We found that microbead-injected eyes from all mouse strains under different experimental conditions showed a similar elevation in IOP over the 8-week experimental period (Fig. 1).

Figure 1.

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Microbead-induced elevation of IOP was similar for different mouse strains and experimental conditions. Histograms show the elevation of IOP for mouse strains under the different experimental conditions. A statistically significant increase in IOP was first seen at 1 week after the initial microbead injection, which was maintained over the entire 8-week experimental period. ***P < 0.001.

Blockade of Gap Junctions

The GJ blocker meclofenamic acid (MFA; 20 mg/kg/d; Sigma-Aldrich, St. Louis, MO, USA) was delivered by osmotic minipumps (model 2004; ALZET, Cupertino, CA, USA) as described previously.²⁵ Mice were anesthetized under isoflurane anesthesia, and pumps were implanted 1 day prior to microbead injection and replaced at 4 weeks.

Immunohistochemistry

The immunohistochemical methods have been described previously.²⁵ Eyecups were fixed with 4% paraformaldehyde; some were used for whole-mount preparation, and others were cryoprotected, embedded in tissue freezing medium (72592; Electron Microscopy Science, Hatfield, PA, USA), and cut into 10-µm-thick frozen sections. The tissues were then incubated with diluted primary antibodies overnight (sections) or 48 hours (whole mounts) at 4°C, washed, and then incubated in secondary antibodies for 2 to 4 hours at room temperature. The primary antibodies were anti-Iba1 (1:500; Abcam, Waltham, MA, USA), anti-MHCII (1:100; Invitrogen), and anti-Brn3a (1:500; Santa Cruz Biotechnology, Dallas, TX, USA). Secondary antibodies were donkey anti-rabbit, -mouse, and -goat conjugated with Alexa Fluor 488, 594, and 633, respectively (1:200; A-21206, R-37115, and A-21082; Life Technologies, Carlsbad, CA, USA).

Iba1 is expressed by cells of monocytic lineage, so Iba1 antibody cannot distinguish microglia in retina from infiltrating macrophages. However, retinal microglia in the resting state have a distinct appearance and ramified morphology distinguishable from the amoeboid structure of macrophages.²⁶^,²⁷ Further, a recent study of ocular hypertension mouse model reported that the large majority of Iba1+ cells were independently identified as microglia in both control of hypertensive eyes.²⁶ Although these data suggest that most Iba1+ cells in our study are microglia, their identification based solely on Iba1 immunolabeling is presumptive.

Quantitative Analysis of Activated Microglia

We determined the changes in cell morphology, cell density, and upregulation of expression of MHCII, which are considered reliable indicators of microglial activation in glaucoma.¹⁸^,²⁸ Images of immunolabeled tissues were taken using a Olympus FV1200MPE confocal microscope with 40× (oil immersion) objectives (Olympus, Tokyo, Japan). High-resolution (1024 × 1024 pixels) Z-stack images were taken using a step size of 0.7 to 2.0 µm, compiled to a single plane, and analyzed quantitatively. Three square areas of 300 × 300 µm in each quadrant within the mid-peripheral region were selected for analysis, and values were averaged across at least four control and four experimental retinas for each protocol. All cell numbers provided below are converted to per mm² area. The brightness and contrast of micrographs were adjusted using Photoshop CS6 (Adobe, San Jose, CA, USA). The following parameters were measured using ImageJ software (National Institutes of Health, Bethesda, MD, USA) to characterize microglial activation: (1) cell soma area; (2) length of the longest arbor segment; (3) number of arbor branches per cell; and (4) arbor area of microglial cell. As a descriptor of microglia activation, the length of the longest arbor segment extending directly from the cell body under control and experimental conditions was measured with the freehand selection tool. The arbor area was measured by connecting terminal endings of processes in each cell using the polygon tool. Measurements were made for 20 to 50 cells selected from the mid-periphery and averaged across at least three retinas each for control and experimental protocols. All data were imported into SigmaPlot software (Systat Software, San Jose, CA, USA) for histogram construction.

Statistical Analysis

Data are presented as mean ± SEM. Sample sizes (eyes) were determined based on our previous studies.²⁵^,²⁹^,³⁰ Calculations were performed using two-sided tests with α = 0.05 and power = 0.8. Samples were allocated to experimental groups according to genotype, and there was no randomization. To compare two experimental groups, we used a two-tailed Student's t-test. Comparisons among more than two groups were analyzed using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. Values of P < 0.05 were considered statistically significant.

Results

Time Course of IOP-Induced Changes in Microglia

Vertical retinal sections taken from microbead-injected eyes were double immunolabeled at different time points with anti-Iba1 to visualize presumptive steady-state microglia³¹ and with anti-MHCII antibody to identify activated microglia.⁵^,³² In control retinas, Iba1+/MHCII− cells were found predominantly in the inner plexiform layer (IPL) and outer plexiform layer (OPL), with smaller numbers visualized in the ganglion cell layer (GCL) (Fig. 2A). The density of Iba1+/MHCII− cells increased at 2 weeks after the initial microbead injection, but no MHCII+ cells were observed at this time point (Fig. 2B). However, by week 4 after microbead injection, we found a few Iba1+ cells that were activated as indicated by MHCII immunoreactivity (Fig. 2C). At week 8, the density and distribution of Iba1+ cells dramatically changed, with a large number appearing in the GCL/nerve fiber layer (NFL) (Fig. 2D). Most of these Iba1+ cells were found to express MHCII, indicating widespread activation of microglia.

Figure 2.

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Microglia cell changes following microbead injection. (A) Representative vertical sections from the mid-peripheral retinas of control mice and experimental mice at 2 weeks (B), 4 weeks (C), and 8 weeks (D) after the initial microbead injection. Sections were double immunolabeled with anti-Iba1 (red) to visualize presumed microglia and anti-MHCII (green) to identify activated microglia. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Iba1+ cells co-expressing MHCII first appeared at 4 weeks of microbead injection. Scale bars: 50 µm for all panels. OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.

A quantitative analysis of flat mount retinas revealed three major changes of microglial cells relevant to their morphology, density, and immunoreactivity in glaucomatous retinas. In the IPL of control retinas, microglia had small round or triangular somata and exhibited ramified morphology with smooth, continuous Iba1 labeling of processes extending tens of microns (Figs. 3A, 3B). All presumed microglia cells showed Iba1+/MHCII− expression under control conditions and formed a regular mosaic arrangement with non-overlapping neighboring cells. By 1 week after microbead injection, the number of Iba1+ cells increased by 23% from 111 ± 8 under control conditions to 144 ± 7 (P < 0.01, n = 4 eyes), and cells appeared with altered morphology exhibiting larger somata and retracted, thicker processes (Figs. 3C, 3D, 3M). Virtually no MHCII+ cells were observed at this time point (Fig. 3N). At 4 weeks after initial microbead injection, the soma shape was altered markedly and the cell density was increased further by 45% (177 ± 5 cells) relative to control values (P < 0.05, n = 4 eyes) (Figs. 3E, 3F, 3M). We observed 18% (P < 0.001, n = 4 eyes) of cells with Iba1+/MHCII+ immunoreactivity at this phase of disease progression (Fig. 3N). At 8 weeks after microbead injection, the morphology of presumed microglial cells was altered dramatically with fewer processes and elongated somata (Figs. 3G, 3H). The density of Iba1+ cells increased by 82% to 222 ± 9 cells (P < 0.001, n = 6 eyes) relative to controls (Fig. 3M), and most cells (79%; P < 0.001, n = 6 eyes) showed Iba1+/MHCII+ immunolabeling (Fig. 3N). It is important to note that we observed dense clustering of MHCII+ cells in the marginal regions of the retina at 4 and 8 weeks of microbead injection, which may have been a result of a direct mechanical trauma.³³^,³⁴ Therefore, we excluded retinal edges and used regions primarily from the mid-periphery in our quantitative analyzes. Interestingly, the timing of MHCII protein expression, indicating microglial activation, beginning at week 4 closely coincides with the earliest loss of RGCs (Figs. 3I–3L, 3O) reported previously for the microbead occlusion mouse model used here.³⁰

Figure 3.

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Time course of microglia activation in the IPL and RGC degeneration following microbead injection. (A, B) Flatmount view of control retina showing that presumed microglia were Iba1+/MHCII− and displayed ramified morphology. Arrowhead in panel A points to microglia cell shown at higher magnification in panel B. (C, D) One week after microbead injection, the number of Iba1+ cells markedly increased, and cells appeared with altered morphology. No MHCII+ cells were observed by this time point. Arrowhead in panel C points to microglia cell shown at higher magnification in panel D. (E, F) At 4 weeks after microbead injection, the number of Iba1+ cells increased further relative to control, and now ∼18% of cells were Iba1+/MHCII+. Arrowhead in panel E points to microglia cell shown at higher magnification in panel F. (G, H) At 8 weeks after microbead injection, the number of Iba1+ cells increased even further relative to control, with ∼79% of cells being Iba1+/MHCII+. Arrowhead in panel A points to cell shown at higher magnification in panel B. Scale bars: 100 µm for panels A, C, E, and G; 30 µm for panels B, D, F, and H. (I–L) Time course of Brn3a⁺ RGC loss following initial microbead injection. Scale bar: 50 µm for all panels. (M) Histogram showing the number of Iba1+ cells in the IPL of control retinas and at 1, 4, and 8 weeks after initial microbead injection. (N) Histogram showing the number of Iba1+/MHCII+ cells in the IPL of control retinas and at 1, 4, and 8 weeks of microbead injection. (O) Scatterplot comparing the timing of the appearance of activated microglia expressing MHCII protein and the loss of RGCs in microbead-injected retinas. Data are presented as mean ± SEM. *P < 0.05, ***P < 0.001, two-tailed Student's t-test; n = 6 eyes for control data, n = 4 eyes for weeks 1 and 4 data, n = 6 eyes for week 8 data.

Microglial Changes in the OPL of Glaucomatous Retinas

In the OPL of control retinas, Iba1+ cells adopted a regular mosaic-like arrangement but exhibited a bushy morphology, which differed from the predominantly, primary long processes expressed by Iba1+ cells in the IPL (Figs. 4A, 4B). We observed a significant increase in the density of Iba1+ cells from 100 ± 6 in control retinas to 165 ± 7 (65%; P < 0.001, n = 4 eyes) as early as 1 week after the initial microbead injection (Figs. 4C, 4D, 4I). Although we found no significant change in the density of Iba1+ cells between weeks 1 and 8 (Figs. 4C, 4D; see also Fig. 3I), there was a dramatic appearance of MHCII+ expression in nearly half of the Iba1+ cells at week 8 (42%; P < 0.001, n = 4 eyes) (Figs. 4G, 4H, 4J). Similar to the data presented above for the IPL (Figs. 3G, 3H), cell morphology in the OPL was characterized by elongated somata and fewer processes at week 8 (Figs. 4G, 4H).

Figure 4.

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Time course of microglia activation in the OPL following microbead injection. (A, B) In control retinas, Iba1+ cells displayed bushy morphology and adopted a regular mosaic-like arrangement. Arrowhead in panel A points to the cell shown at higher magnification in panel B. (C, D) The number of Iba1+ cells was markedly increased as early as 1 week after microbead injection. Arrowhead in panel C points to cell shown at higher magnification in panel D. (E, F) There was no statistically significant increase in the number of Iba1+ cells between weeks 1 and 4 and no detectable MHCII+ cells. (G, H) There was no statistically significant increase in the number of Iba1+ cells between weeks 4 and 8; however, at 8 weeks after microbead injection, nearly 42% of cells were Iba1+/MHCII+. Arrowhead in panel G points to cell shown at higher magnification in panel H. Scale bars: 100 µm for panels A, C, E, and G; 30 µm for panels B, D, F, and H. (I) Histogram showing the number of Iba1+ cells in the OPL of control retinas and at different timepoints after initial microbead injection. (J) Histogram showing the number of Iba1+/MHCII+ cells in the OPL of control retinas and at different time points after microbead injection. Data are presented as mean ± SEM. ***P < 0.001, two-tailed Student's t-test; n = 6 eyes for control data, n = 4 eyes for weeks 1 and 4 data, n = 6 eyes for week 8 data.

Effect of Gap Junction Blockade/Deletion on Microglia Activation

As mentioned, we reported previously that loss of RGCs in the mouse model of glaucoma occurs first at 4 weeks after microbead injection and increases further by 8 weeks.²⁵ The temporal correspondence between the expression of MHCII by activated microglia in the IPL of glaucomatous retinas and the onset of RGC loss (Fig. 3O) prompted us to investigate whether microglia activation at this stage is the cause or the consequence of RGC death.

One group of mice was unilaterally injected with microbeads, whereas a second group was treated with the GJ blocker MFA³⁵ just prior to microbead injection. Application of MFA has been shown to provide neuroprotection in glaucomatous eyes, largely preventing RGC and more proximal neuron degeneration associated with microbead-induced IOP elevation.²⁵^,³⁰ A comparative histological analysis showed that retinas from microbead-injected eyes that were also treated with MFA failed to show the phenotypic changes in microglia in both plexiform layers associated with induction of glaucoma (Fig. 5). At 8 weeks after initial microbead injection and MFA application, we observed no significant change in the density of Iba1+ cells in the IPL (Fig. 5I) or OPL (Fig. 5K), and no significant expression of MHCII, indicating no microglia activation in the IPL (Fig. 5J) or OPL (Fig. 5L).

Figure 5.

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Suppression of microglia activation by GJ blockade or genetic deletion of the GJ subunit Cx36. (A, B) Confocal images of Iba1+ cells in the IPL and OPL of control retinas. (C, D) Images of Iba1+ and MHCII+ cells in the IPL and OPL at 8 weeks after the initial microbead injection. (E, F) Images of Iba1+ cells in the IPL and OPL of wild-type mice at 8 weeks after initial microbead injection and application of the GJ blocker MFA. (G, H) Images of Iba1+ microglia in the IPL and OPL of retinas of Cx36^−/− mice at 8 weeks after the initial microbead injection. Scale bars: 100 µm for all panels. (I, K) Histogram showing the number of Iba1+ cells in the IPL and OPL of retinas from control (n = 6 eyes), MFA-treated (n = 4 eyes), and Cx36^−/− (n = 4 eyes) mice at 8 weeks after initial microbead injection. (J, L) Histograms showing the percentage of Iba1+/MHCII+ cells from the IPL and OPL of retinas from control (n = 6 eyes), MFA-treated (n = 4 eyes), and Cx36^−/− (n = 4 eyes) mice at 8 weeks after initial microbead injection. Data are presented as mean ± SEM. *P < 0.05, ***P < 0.001, 1-way ANOVA followed by Tukey's multiple comparisons test.

These results suggest that RGC degeneration in glaucomatous eyes leads to activation of microglia in both plexiform layers and that protection of RGCs by GJ blockade by MFA prevents such activation. However, MFA also manifests nonsteroidal anti-inflammatory properties,³⁶^,³⁷ which could suppress microglia activation independent of its GJ-mediated neuroprotective action. To test this possibility, we performed a similar immunohistochemical analysis using microbead-injected eyes of Cx36^–/– mice, where selective GJ ablation also provides neuroprotection to RGCs and more proximal neurons.²⁵^,³⁰ Retinas in which Cx36 was ablated showed the same suppression of microglia activation seen in MFA-treated eyes (Figs. 5G–5L).

When viewed in flatmount, we found that blockade of GJs with MFA or genetic ablation of Cx36-expressing GJs also prevented the structural changes in Iba1+ cells associated with microbead injection and induction of glaucoma (Fig. 6). Quantitative measures confirmed this observation, showing no significant retraction of processes or structural elongation of Iba1+ cells following GJ blockade/ablation as indicated by no reduction in the arbor area (Fig. 6I); no reduction in the longest arbor segment (Fig. 6J); no increase in the soma area (Fig. 6K); and no reduction in the arbor branch number (Fig. 6L). In addition, we found that application of MFA or genetic ablation of Cx36 prevented the structural change to a rod-like or amoeboid appearance in the GCL/NFL layers, described above for glaucomatous eyes of untreated wild-type mice.

Figure 6.

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Blockade of GJs with MFA or genetic ablation of the GJ subunit Cx36 prevents morphological changes associated with activation of microglia induced by microbead injection. (A, B) Confocal images of Iba1+ cells in the IPL and the OPL of control retinas. (C, D) Images of Iba1+ and MHCII+ cells in the IPL and OPL of retinas in wild-type mice at 8 weeks after microbead injection. (E, F) Images of Iba1+ and MHCII+ cells in the IPL and OPL of retinas in wild-type mice at 8 weeks after microbead injection and MFA application. (G, H) Images of Iba1+ and MHCII+ cells in the IPL and OPL of retinas in Cx36^−/− mice at 8 weeks after microbead injection. Scale bars: 40 µm in all panels. (I) Histogram showing that arbor area reduction of microglia in the IPL and OPL seen after microbead injection was prevented by GJ blockade with MFA or genetic ablation of Cx36. (J) Histogram showing that largest arbor segment reduction of microglia in the IPL and OPL seen after microbead injection was prevented by GJ blockade with MFA or genetic ablation of Cx36. (K) Histogram showing that the increased soma area of microglia in the IPL and OPL seen after microbead injection was prevented by GJ blockade with MFA or genetic ablation of Cx36. (L) Histogram showing that arbor area reduction of microglia in the IPL and OPL seen after microbead injection was prevented by GJ blockade with MFA or genetic ablation of Cx36 of retinas from control eyes and 8 weeks after microbead injection under indicated conditions. Data are presented as mean ± SEM. *P < 0.05; ***P < 0.001, 1-way ANOVA followed by Tukey's multiple comparisons test; n = 45 cells for control retinas (5 eyes), n = 35 cells for MFA-treated retinas (3 eyes), n = 20 cells for Cx36^−/− mouse retinas (3 eyes).

Discussion

Despite the widely recognized association of microglia activation and RGC degeneration, the exact relation between microgliosis and RGC death remains obscure.⁷^,¹¹^,³⁸^–⁴⁰ A major finding of this study, using a mouse microbead occlusion model of glaucoma, is the demonstration that late-stage of microglial activation associated with upregulation of MHCII, appears to be a consequence rather than an initial determinant of RGC death.

Activation of microglia is a complex multistage process that includes changes in density, soma size and shape, degree of branching, length/thickness of processes, and expression of activation markers such as MHCII.¹⁵^,⁴¹ The spatiotemporal relationship between neurodegeneration and the staged microglial changes has been unclear. We observed changes in cell morphology and the density within 1 week after the initial microbead injection, the time point when early degenerative changes in RGCs occur, such as synaptic and dendritic remodeling, but prior to detectable cell death.²⁵^,⁴² This finding is consistent with studies using other glaucoma models, showing that early microglia transformation precedes RGC death.¹⁸^,²⁰ Results of experimental glaucoma indicate that late expression of the surface protein MHCII in activated microglia is also associated with RGC degeneration, but, again, whether the expression is a cause or consequence of cell loss was uncertain.⁷^,⁴⁰^,⁴³^,⁴⁴ We first observed MHCII expression by microglia in the IPL at 4 weeks after microbead injection, which increased significantly by week 8. Interestingly, the timing of MHCII expression coincided with the onset of RGC loss, which as we previously reported occurs at week 4 after microbead injection.²⁵ In the same study, we showed that blockade of GJs or ablation of Cx36 prevented both dendritic remodeling and RGC death in this mouse model of glaucoma. Thus, it is reasonable to suggest that, whereas early microglial changes are associated with dendritic degeneration of RGCs, the appearance of MHCII expression corresponds temporally to RGC death. This is in agreement with previous reports that microglial MHCII is immunohistochemically undetectable in healthy tissue but upregulated following neurodegeneration.¹⁸^,⁴⁰ In the GCL/NFL of glaucomatous retinas, activated microglia adopted several morphological phenotypes, including amoeboid and rod-like, at week 8. Rod-like microglia appear restricted to eyes with neuronal damage caused by ocular hypertension or elevated IOP.¹⁶^,⁴⁵^,⁴⁶

We found that the morphological and activation dynamics of microglia in the IPL and OPL of glaucomatous retinas differed during the disease progression. These data are consistent with reports of microglial structural heterogeneity dependent on their location in the plexiform layers, suggesting distinct functions both under normal condition and during ocular disease.¹³^,⁴⁷ In the OPL, where bipolar and horizontal cells extend dendritic processes, microglia expressing MHCII were not detected until 8 weeks after the initial microbead injection. In contrast, approximately 18% of Iba1+ cells in the IPL showed MHCII expression at week 4. The late expression of MHCII in the outer retina is consistent with the correspondingly late degeneration of outer retinal neurons in glaucomatous eyes, as it occurs as a secondary sequalae of primary damage in the inner retina.³⁰ Overall, the earliest MHCII expression by activated microglia in the IPL temporally correlates with the loss of RGCs, whereas later expression by microglia in the OPL coincides with the degeneration of bipolar and horizontal cells in the outer retina.²⁵^,³⁰

We demonstrated that GJ blockade by MFA application, which we showed previously provides significant neuroprotection in the mouse model of glaucoma,²⁵ largely prevented structural changes and activation of microglia associated with glaucoma. These data suggest that microglial activation may be secondary to RGC degeneration; however, MFA also has anti-inflammatory action, which could also suppress microglia activation.³⁶^,³⁷ To differentiate the two effects, we determined whether genetic ablation of GJ subunits in the Cx36^−/− mouse, which largely prevent RGC degeneration but have no clear anti-inflammatory action,²⁵ can also reduce microglia activation. Indeed, we did not detect any significant changes in the morphology of Iba1+ cells, their number, or their expression of MHCII in microbead-injected Cx36^–/– mice. Although we did not study the time course of microglia activation in Cx36^−/− mice, the finding that late stages of activation seen in wild-type mice at 8 weeks were prevented suggests that the changes seen at earlier time points following microinjection were prevented, as well.

Overall, our results strongly indicate that neuroprotection of RGCs precludes late stage activation of microglia associated with MHCII expression. Although our examination of the effects of GJ blockade/deletion on microglia was limited to 8 weeks after microbead injection, the early stages of activation, indicated by changes in the cell morphology and density that precede RGC death, were likely largely suppressed by GJs blockade/ablation, as well. We therefore posit that early microglia changes may be in response to the early degenerative RGC dendritic and somatic atrophy that precedes their ultimate loss.

Alternatively, it has been proposed that microglial activation is supported by the transfer of inflammatory messengers through GJ channels; therefore, their blockade/ablation may suppress microglial activation and prevent neuronal damage and death during neuroinflammation.⁴⁸^,⁴⁹ It has been further suggested that microglia express Cx36 or Cx43 that are upregulated in various pathologies when microglia shift from resting to activated state.⁴⁹^–⁵² However, whether microglia express functional GJs that can support intercellular communication is unclear.⁵⁰^,⁵³^–⁵⁵ We found that Iba1+ cells in retina, whether in the resting or activated state, never showed the punctate immunoreactivity signature of Cx36-expressing GJs (data not shown), nor did Iba1+ neighbors show overlapping processes necessary for functional GJ communication. These findings argue that MFA or Cx36 ablation treatments did not directly affect microglia coupling but rather support our position that microglia activation was a consequence rather than a determinant of neuronal degeneration and death in glaucomatous eyes. This conclusion is consistent with reports of microglial activation secondary to neurodegeneration in various CNS pathologies such as amyotrophic lateral sclerosis⁵⁶ and Parkinson's diseases,⁵⁷ as well as eye pathologies related to retinal axotomy, optic nerve crush, and acute ocular hypertension.³⁸^,⁵⁶^,⁵⁸^–⁶⁰

Acknowledgments

Supported by grants from the National Institutes of Health (EY026024 and EY007360 to SAB).

Disclosure: S. Kumar, None; A. Akopian, None; S.A. Bloomfield, Connexin Therapeutics (I, P)

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Figure 1.