Retinal glial responses to optic nerve crush are attenuated in Bax-deficient mice and modulated by purinergic signaling pathways

The central hypothesis of secondary degeneration is that activation of the retinal neuroinflammatory response requires an initial wave of RGC somatic degeneration principally affected by axonal damage in the optic nerve. To test this hypothesis, we used Bax-deficient mice, which exhibit complete resistance to RGC soma death in an acute optic nerve crush paradigm. We first evaluated the Bax ^−/− mice for expression of the microglial activation marker, Aif1, and the microglial/macrophage marker Cd68 [23]. By 7 days post-injury, Aif1 expression in wild-type mice was dramatically elevated in the crushed eye over the contralateral eye, while Bax-deficient mice only showed a modest increase in Aif1 expression (Fig. 1a, P < 0.0001 for Bax ^−/− mice compared to wild types). By 14 days, Aif1 expression declined in wild-type mice but remained significantly higher than the mRNA levels in Bax-deficient retinas, and at 21 days, microglial activation trended towards baseline levels in both genotypes and was no longer statistically significant. The expression of Cd68 followed a similar trend as Aif1, with levels rising at 7 days post-crush to a significantly higher level in the wild-type mice relative to the Bax ^−/− mice (Fig. 1b, P < 0.005). By 14 days, expression began to decline in the crushed eye of both genotypes, although mRNA levels remained significantly higher in the injured retinas of wild-type mice. At 21 days, Cd68 expression in the knockout mice was slightly higher than the wild types, although the mRNA abundance was relatively low in both genotypes.

The QPCR data was supported by immunolabeling in retinas for AIF1 protein after optic nerve crush (Fig. 2). In wild-type mice (Fig. 2a–d), AIF1 positive cells were sparsely detected in the uncrushed contralateral eye, and the microglia present were small with branched processes, characteristic of resting “ramified” microglia [21, 29]. At 7 and 14 days, a population of AIF1-positive cells was abundant in the ganglion cell layer (GCL) and the associated nerve fiber layer (NFL), inner nuclear layer (INL), and outer nuclear layer (ONL). By 21 days after injury, AIF1 labeling decreased throughout the retina in wild-type mice, but still exhibited more labeling than the contralateral eye. The Bax ^−/− mice showed a similar baseline expression of AIF1 in the retina; however, after crush, the level of AIF1 labeling was markedly reduced at 7, 14, and 21 days relative to the wild-type mice (Fig. 2e‐h). The morphology of AIF1-expressing cells in the NFL was also examined in whole-mounted retina preparations (Fig. 3). In wild-type mice at 7 days after injury, the numbers of cells appeared to increase and then decrease by 14 and 21 days (Fig. 3b‐d). Crush injury did not appear to alter the density of the microglia in Bax ^−/− mice (Fig. 3e‐h). Quantification of cell density indicated that by 7 days post-crush, wild-type retinas showed a significant increase in the density of microglial cells in the NFL of crushed retinas (P < 0.0001 relative to contralateral eyes), which persisted through 14 days after injury (Fig. 4a). The density of AIF1-positive microglia peaked at 7–14 days post-crush, consistent with the peak in mRNA expression at 7 days (Fig. 1) and other studies [11]. By 21 days, the density of microglia was still significantly higher than the contralateral eye, but the numbers were declining. Unlike wild-type mice, Bax-deficient mice exhibited only a modest increase in microglial density, although the density of microglia gradually rose in both the injured and contralateral retinas of the knockout mice. Importantly, Bax ^−/− mice exhibited a significantly lower density of microglia, relative to injured retinas of wild-type mice, at both 7 and 14 days post-crush (P < 0.0001). Interestingly, control retinas of both Bax ^+/+ and Bax ^−/− mice showed a modest increase in microglial density over the time course examined, indicative of a contralateral eye effect from optic nerve injury, which has been described by others [82, 83].

A Sholl analysis was also conducted to quantify morphological changes in these cells after optic nerve damage. Quantification of retinas in each genotype, at different days, is shown in Additional file 1: Figure S1A–F. Figure 4b shows data for all genotypes at all days for processes crossing the 40-μm grid line. Cells in wild-type retinas show a significant increase in processes 7 days after optic nerve damage (compared to contralateral eyes, P < 0.001), while at 14 and 21 days, cells in both retinas had similar morphology, which was overall reduced in complexity relative to their shape at 7 days (P < 0.0005). Cells in Bax ^−/− retinas showed no change between eyes at 7 days after crush, but had significantly fewer processes in the damaged eyes by 14 days (P < 0.001). As with the wild-type retinas, there was an overall reduction in the numbers of processes in both eyes at 14 and 21 days, compared to 7 days (P < 0.005).

Morphologically, the microglia in the uncrushed retinas appeared different between the two genotypes. Wild-type mice exhibited a range of morphologies for microglia varying from cells with thick cell bodies and large processes to cells with small somas and highly ramified fine processes. While a similar distribution was also evident in the Bax ^−/− mice, a greater proportion of cells appeared to have thick cell bodies and large processes. The average numbers of processes as assessed by the Sholl analysis (see Fig. 4b), and the average area of cells between the two genotypes, were not significantly different (P = 0.71 and 0.32, respectively). However, scoring of images for amoeboid-like or ramified-like morphologies showed that wild-type mice presented with ~58 % ramified-like microglia, while Bax ^−/− animals presented with ~60 % amoeboid-like cells, which was significantly different (P = 0.003).

The microglial response in the optic nerve head of the Bax ^+/+ and Bax ^−/− mice was also noticeably different. In control eyes, similar levels of AIF1-positive cells populated the ONH of both genotypes (Fig. 5a, d). In the wild-type mice, crush injury caused the number of microglia and intensity of AIF1 staining to increase at 7 and 14 days (Fig. 5b, c). In contrast, in the Bax ^−/− mice the amount of AIF1 labeling was quite attenuated despite an increase in the number of microglia in the injured relative to the control optic nerve head, compared to wild-type mice (Fig. 5e, f).

Mice deficient for Bax also exhibited a reduction in the expression of macroglial activation markers after optic nerve crush. As shown in Fig. 6a, at 7 days after crush, Gfap expression in the wild types was significantly elevated above the contralateral eye (P < 0.0001), while the injured retinas of Bax ^−/− mice showed only a modest increase that was greatly attenuated compared to wild-type mice (P < 0.0001). By 14 days, macroglial activation increased in both genotypes, however expression in the wild type mice still remained significantly elevated over Bax ^−/− mice (P < 0.0001). By 21 days, Gfap expression began to trend towards baseline levels in both genotypes, and no longer retained statistical significance (P = 0.28). A second macroglial activation marker, Nestin (Nes) [24, 26, 27, 84], was also analyzed by QPCR. Similar to Gfap, the expression of Nes at 7 and 14 days post-crush was attenuated in the crushed retinas of Bax ^−/− mice relative to the levels observed in the crushed wild-type retinas (Fig. 6b, P < 0.001). By 21 days, expression continued to decline in the wild-type mice, but rose slightly in the Bax ^−/− mice, although the expression levels were no longer statistically different between the two genotypes (P = 0.37).

Immunofluorescent labeling for GFAP performed on retinal sections showed that astrocytes expressed a baseline level of GFAP in the NFL of both Bax ^+/+ and Bax ^−/− mice (Fig. 7a, e). At 7 days after crush, the GFAP labeling intensified in the wild-type mice and fibers appeared through the retinal layers indicative of Müller cell expression. This expression pattern of GFAP persisted in the wild-type mice at 14 and 21 days, although at the latter time point, GFAP expression was markedly reduced. The Bax ^−/− retinas contrasted starkly with the wild-type mice at all time points analyzed (Fig. 7e–h). There was a complete absence of GFAP labeling in the Müller cells, resulting in a labeling pattern that did not appear to differ from the control eyes.

The attenuated glial response in Bax-deficient mice supports the hypothesis that RGC soma death is an important event in the signaling of retinal glial activation. To strengthen this interpretation, we next examined if retinal glia in Bax ^−/− mice could become activated using an alternative damage paradigm. While BAX has not been linked to glial activation, we wanted to confirm that Bax-deficient glia were capable of upregulating Aif1 and Gfap. In this experiment, we introduced the glutamate analog N-methyl-d-aspartate (NMDA) by an intravitreal injection, which has previously been shown to kill RGCs in both wild-type and Bax-deficient mice [50]. NMDA has also been reported to directly interact with NMDA receptors expressed by Müller cells [85]. Examination of both transcript levels and protein immunostaining showed a predictable microglial and macroglial response in both genotypes. By 7 days post-NMDA injection, Aif1 and Gfap mRNA expression were significantly elevated in both genotypes of mice (Fig. 8a, P < 0.0001 relative to contralateral eyes). Immunofluorescent labeling also revealed a clear increase in AIF1-positive cells in the GCL and plexiform layers of Bax ^−/− retinas (Fig. 8b, c), as well as an increase in GFAP labeling in the GCL and through the retinal layers, indicating Müller cell activation (Fig. 8d, e). Wild-type mice exhibited similar changes in protein expression following NMDA treatment (data not shown).

There is growing evidence that injured neurons release ATP as a distress signal into the extracellular space, so we next wanted to determine if ATP analogs triggered glial activation in the retina. An intraocular injection of BzATP, an agonist selective for P2X receptors was delivered to wild-type mice, and microglial and macroglial activation markers were analyzed 48 h later. RGC gene expression was also monitored as a sign of injury, as previous studies have shown that this agonist can trigger RGC degeneration [75, 76]. While BzATP had no effect on inducing Aif1 expression in microglia (Fig. 9a), this agonist triggered macroglial activation as indicated by a clear rise in Gfap mRNA expression (Fig. 9b), and an increase in GFAP protein labeling, particularly in the Müller glia (Fig. 9c‐e). BzATP was also delivered to Bax ^−/− mice to confirm the functionality of purinergic receptors in the absence of BAX. By 48 h, Gfap expression was significantly elevated in the injected eye of both wild-type and Bax ^−/− mice, relative to the contralateral eye (Fig. 9f). To determine if P2X receptor activation affected the RGCs, and cause indirect macroglial activation, we evaluated if it resulted in a decrease in RGC-specific gene expression, which is a sensitive indicator of damage to these cells [54, 86‐88]. BzATP had no effect on mRNA levels of Nrn1 or Sncg (Fig. 9e), suggesting that RGCs were not adversely affected by this agonist, within the 48-h time frame evaluated in this experiment. This observation, added to the lack of microglial activation, indicated that BzATP was acting principally on the macroglia.

While the previous results show that purinergic signaling can mediate activation of retinal macroglia, they do not verify if this mechanism of signaling is active in the optic nerve crush paradigm of damage. To test this, we examined if a P2X receptor antagonist (oxATP) was able to block macroglial activation after crush. We delivered an intraocular injection of the drug 24 h prior to, or 3 days post-crush, and analyzed for glial activation 7 days after surgery when peak activation is known to occur. The expression of Gfap was significantly elevated in the crushed eye of all treatment groups (Fig. 10, P < 0.0005, relative to the contralateral eye); however, both pre-treating and post-treating, mice with an intraocular injection of oxATP attenuated Gfap expression by about half (Fig. 10a, e). Immunostaining in retinal sections showed that neither pre- nor post-crush treatment with oxATP was able to completely block GFAP expression in Müller cells, while there was a marked decrease in apparent staining in the NFL (Fig. 10b–d and f–h), suggesting that the 50 % decrease in Gfap expression may be attributable to astrocytes. Consistent with no effect of BzATP on activating Aif1 expression in microglia, oxATP had no effect on suppressing Aif1 expression after optic nerve crush (Fig. 9a).

It appears that purinergic signaling may play a role in triggering Gfap upregulation after crush, although the source of extracellular ATP in response to optic nerve damage is not known. We hypothesized that ATP was released from RGCs undergoing apoptosis, and wanted next to explore the mechanism by which ATP release may occur. One possible mechanism of ATP release involves the PANX1 hemichannel, which is expressed across the retinal cell types and is highly enriched in RGCs [89]. This mechanism is consistent with the attenuated glial response observed in Bax ^−/− mice, since dying cells are known to release ATP through caspase 3/7-mediated activation of the PANX1 protein [60, 90]. Since BAX activation is required for activation of caspases, it is reasonable to predict that Bax-deficient RGCs would not be able to activate PANX1 and release ATP. To test for whether glial activation is dependent on Panx1 after injury, the Panx1 gene was selectively disabled in either RGCs or all cell types in Panx1-LoxP mice using cell type-specific expression of CRE recombinase (see the “Methods” section).

The retinas were first evaluated for Panx1 mRNA by QPCR, which showed a complete abrogation of Panx1 expression in Panx1 ^−/− mice, while RGC Panx ^−/− retinas showed about a 50 % reduction in mRNA expression (Fig. 11a). Conditional knockout and wild-type mice were then subjected to optic nerve crush and evaluated for changes in macroglial Gfap expression. Wild-type mice and Panx1 ^−/− mice followed similar trends in Gfap expression, with levels peaking at 7 days and then declining at 14 days and again at 21 days (Fig. 11b). The RGC Panx1 ^−/− mice, however, consistently exhibited higher and sustained Gfap expression after crush relative to wild-type and Panx1 ^−/− mice. While Gfap expression was not statistically different from wild-type mice at 7 days, the mRNA levels peaked at 14 days and remained elevated at 21 days relative to wild type and RGC Panx1 ^−/− mice (P < 0.0005). Retinal sections were further evaluated by immunolabeling, which showed an increase in GFAP expression in the nerve fiber layer and through the retinal layers, consistent with astrocyte and Müller cell activation (Fig. 12). The increase in GFAP was present in all genotypes, and at all post-crush time points examined, while the control eyes only exhibited baseline levels of GFAP by astrocytes. GFAP labeling appeared to be most prominent at 7 and 14 days, although Müller cell activation was still prevalent by 21 days.

An understanding of the sequence of events being executed in damaged RGCs is relevant to understanding how retinal glia respond. We have monitored the glial response using the markers Aif1 and Gfap. We previously determined that mRNA levels for these markers become elevated between 3 and 5 days after crush [55]. Importantly, both glial populations appeared to be synchronized with RGC pathology that followed the interval of BAX protein activation, which is estimated to occur by 3 days after acute optic nerve damage [91, 92] (M. Maes and R. Nickells, unpublished data), suggesting that apoptotic events downstream of the BAX-dependent step were linked to the signaling mechanism leading to the glial response. This, in fact, appears to be the case, since both microglial and macroglial responses are significantly suppressed in the absence of RGC death in Bax-deficient mice. A caveat in our study is that microglial and macroglial activation rose modestly in the injured and contralateral retinas of both genotypes. Additional activation signals, while minimally influential, may be released prior to the Bax-dependent step of the apoptotic program, or originate from a source other than dying RGCs.

Given the prerequisite for RGC death and the timing of the glial response, we reasoned that RGCs could be communicating with the glia via purinergic signaling pathways. We show that an injection of BzATP into the mouse eye stimulates Gfap expression in retinal macroglia, including Müller cells. This is particularly intriguing, since BzATP is historically considered a specific agonist for the P2X7 receptor [93], and there is conflicting evidence about the expression of this receptor by Müller cells [94‐96]. How then, do the Müller glia respond to BzATP? It is possible that different purinergic receptors on Müller cells are responding to the agonist, especially at the relatively high concentrations that were used for intravitreal injection. BzATP is a similarly potent agonist for the P2X1 receptor, and has partial activity for multiple P2X and P2Y1 receptors [97‐99]. While there is evidence that P2X7R is not expressed by mammalian Müller cells, there are reports that Müller cells in the lower vertebrate species do express this receptor [96]. Additionally, we cannot rule out that this receptor is not upregulated in Müller cells under stress conditions [100]. Lastly, it may be possible that the Müller cell response was secondary to the action of BzATP on either RGCs or astrocytes, which do contain P2X7 receptors [94, 101]. Activation of P2X7R on other cell types may lead to ATP release that then affect the Müller cells via a different purinergic receptor [102]. Experiments using P2X antagonists shed little light on this mechanism. While we show that oxATP, also considered a relatively selective antagonist for the P2X receptors [103], can partially suppress the macroglial response, this does not rule out a secondary activating mechanism from the astrocytes or RGCs to the Müller cells. Additionally, suppression of glial activation using oxATP appears to selectively affect the response in astrocytes when assessed by immunostaining. Further studies are necessary to help elucidate the purinergic signaling network in the retina after optic nerve damage.

While purinergic signaling appears to play a role in macroglial activation, our data do not show an effect on microglial activation, at least in the context of upregulating Aif1 expression. Microglia reportedly express the P2X7 receptor [104, 105], and activation of this receptor has been directly linked to microglial inflammatory responses in rat primary hippocampal cultures [71]. Nevertheless, experiments using both BzATP and oxATP appear to exclude purinergic signaling to activate these cells after optic nerve damage, and it is not possible to rule out an effect on the microglia based on other metrics typical of their activation response. It is clear, however, that Bax-deficient mice exhibit a suppression of microglial activation in this damage paradigm, which is consistent with a recent study using an acute ethanol toxicity model, in which microglial activation was also suppressed in Bax ^−/− mice [106]. The signaling pathway linking RGCs and microglia was not a focus of this study, but we hypothesize that it likely occurs via Toll-like receptors (TLRs). This is supported by three lines of experimental evidence. Retinal microglia upregulate TLR expression as an early response to glaucomatous damage [107], polymorphisms in both TLR2 and TLR4 are risk alleles for glaucoma [108, 109], and deletion of Tlr4 in mice reduces RGC death after optic nerve damage [110] and ischemia-reperfusion [111]. TLRs respond to endogenously derived “damage-associated molecular pattern” (DAMP) molecules released from damaged or dying cells [3]. For example, TLR9 additionally recognizes mitochondrial DNA [112], which would only be released from cells undergoing mitochondrial breakdown, a feature expected only in cells with a functional BAX protein. Perhaps, not coincidentally, this receptor has also been linked to glial activation in the retina [113].

One noteworthy observation in our study involving the microglia response in wild-type mice after optic nerve damage is the increase in ramifications of these cells at 7 days after injury. The classic activation response of microglia is to convert to a more amoeboid state by the retraction of processes, after which they enter a transitional/motile phenotype by extending new processes [21]. It is possible that our observations at 7 days correspond to cells already in this transitional phase.

Based on the suppressed glial response in Bax-deficient mice, and the pharmacologic evidence suggesting a role for purinergic signaling in macroglial activation, we hypothesized that ATP was released from dying RGCs via activated PANX1 channels. This hypothesis was particularly attractive given that mechanical stress applied to RGCs elicits ATP release via PANX1 hemichannels [65]. Additionally, activated caspases proteolytically activate PANX1 [60]; therefore, PANX1 activation would not occur in Bax-deficient cells, which fail to stimulate caspases. However, Panx1 ablation in RGCs failed to reduce the macroglial response invalidating our hypothesis. Several lines of evidence implicate PANX1 as playing a role in RGC pathology, although none of these studies evaluated the effects after optic nerve damage. In ischemia damage models, deletion of Panx1 in RGCs provides a protective response [78], including attenuation of neurotoxic Ca²⁺ uptake. Panx1 ^−/− RGCs also exhibited impaired activation of the inflammasome. Tied with this is evidence that prolonged exposure to ATP analogs, including BzATP, stimulates RGC loss [75, 76], presumably through the interaction of P2X7 receptors and PANX1 [114] on RGCs. In preliminary experiments, however, we did not detect a similar protective effect for RGCs after optic nerve damage when Panx1 was selectively deleted in these neurons (data not shown). Conversely, optic nerve crush in the eyes with Panx1 ^−/− RGCs resulted in what appeared to be an elevated and sustained macroglial response, measured as a function of Gfap expression. This argues for a role of PANX1 channels in paracrine signaling between RGCs and activated macroglia to modulate or shutdown the activation response, although the nature of this paracrine response is yet to be determined.

A major caveat to our observations is that the sustained activation of macroglia after crush was not observed in the complete Panx1 ^−/− mouse as well. Currently, we have not been able to reconcile these conflicting results. One possible explanation, however, is the developmental timing of the Panx1 deletion. In complete (zygotic) knockout mice generated by crossing animals with a floxed Panx1 allele to the CMV-Cre transgenic line, the disabling of Panx1 alleles occurs as early as the 1-2 cell stage of development [115]. Under these conditions, it is conceivable that Panx1 ^−/− mice develop compensatory mechanisms to overcome the loss of PANX1 hemichannels. Consistent with this, multiple functions attributed to Panx1 have only been observed in mice with both Panx1 and Panx2 deleted [116]. In contrast, RGC-specific deletion of Panx1 by AAV2-Cre injection is achieved well into adulthood, thereby excluding the development of a compensatory mechanism in these cells. We posit that these experiments more accurately reflect a role for Panx1 in these cells.

We used Bax ^−/− mice to assess the relationship between RGC death and retinal glial activation, principally because RGCs lacking this proapoptotic member of the Bcl2 gene family exhibit complete and sustained prevention of RGC loss in optic nerve damage paradigms [50‐54]. It is relevant, however, to consider how the loss of Bax affects the glial population in our interpretation of the results of this study. This is especially important, given the well-documented phenomenon that Bax-deficiency leads to supernumerary numbers of some classes of neurons in the mouse [117‐119]. Surprisingly, there is very little information regarding glial populations in the CNS of Bax-deficient mice. White and colleagues [118] found no evidence of reduced cell death of glial populations in these mice, presumably because they also express a functional proapoptotic BAK protein, unlike neurons, which alternatively splice the Bak transcript [120]. Conversely, oligodendrocyte cell density in the optic nerves of Bax-deficient mice is greater, retaining a normal ratio with the increased axon number [121]. Our comparison of microglial density in the peripheral retinal nerve fiber layers of wild-type and Bax-deficient mice suggests that there is no increase in microglial cells commensurate with the reported increase in RGCs [50, 122, 123]. The microglial population in the retinas of Bax-deficient mice is different from wild-type littermates, however, and appears to be enriched in cells with more amoeboid-like characteristics. The reason for this enrichment is not known, but perhaps Bax deficiency provides a bias towards a subclass of microglia [124] by preventing programmed cell death during development, similar to the phenomenon exhibited by neuronal populations. Importantly, however, our experiments using the excitotoxic agent NMDA to induce RGC death indicate that both microglia and macroglia in Bax ^−/− mice were able to upregulate markers consistent with a normal activation response.