IOP induces upregulation of GFAP and MHC-II and microglia reactivity in mice retina contralateral to experimental glaucoma

Ocular hypertension is a major risk factor for glaucoma, a neurodegenerative disease characterized by an irreversible decrease of ganglion cells and their axons, the functional impact of which leads to a visual-field loss [1‐7].

Although the hypothesis is generally accepted that glaucomatous damage is a consequence of axonal degeneration that ends with the death of ganglion cells, recent studies have shown the important role played by glia in the pathogenic mechanism of the disease [8‐12].

Under normal conditions, astrocytes and Müller glia make contact with retinal neurons, providing stability to the neural tissue [13]. Physiological studies have demonstrated that both cell populations perform equivalent functions, including: storing glycogen, providing glucose to neurons, regulating the levels of extracellular potassium, playing a major role in the regulation and metabolism of neurotransmitters such as GABA, helping to remove CO₂ from the retina, and contributing to the maintenance of water homeostasis in the retina [12, 14‐18]. Furthermore, astrocytes as well as Müller cells can induce blood–brain barrier properties within the vascular endothelial cells [19].

Resident glia in the retina and optic-nerve head alter their gene-expression profile during activation, presumably exerting neuroprotective or damaging influences at different phases of disease progression [20]. In the glaucomatous optic neuropathy, glial cells from the retina and from the optic nerve show abnormal behavior. This results in the expression of glial fibrillary acid protein (GFAP) in Müller glia and the appearance of reactive astrocytes, which are characterized by a change in their form and their GFAP expression [21].

The neurofilaments from retinal ganglion cells (RGCs) undergo alterations in glaucoma. It has been reported that the excitatory neurotransmitter glutamate, which exhibits elevated extracellular levels in pathologies such as glaucoma, can enhance the phosphorylation of neurofilaments [22] and induce the accumulation of neurofilaments in the neuronal soma [23]. Additionally, the interference of axonal transport has been proposed as one possible mechanism of neurofilament-induced pathology and the disorganized neurofilaments can induce selective neuronal degeneration and death [24].

It has been suggested that reactive glial cells could help protect retinal ganglion cells, as they can be a source of neurotrophic factors [25]. On the contrary, reactive glial cells can exacerbate neuronal damage and may become one of the etiologies of glaucoma through the release of cytokines, reactive oxygen species, and functional disorders of the glutamate uptake in Müller cells [26, 27]. This could negatively influence ganglion cells, which could lose their normal functional support [28, 29]. In this regard, the colocalization of caspase 3 and GFAP in astrocytes and Müller glia in glaucomatous retina has indicated that these cells may be involved in the apoptosis process, in which the increase of nitric oxide (NO) and tumor-necrosis factor (TNF-α) produced by glial cells would lead to the death of retinal ganglion cells exposed to stressful conditions [30, 31].

Altered crosstalk between RGCs and microglia, astrocytes or oligodendrocytes has been proposed as an early factor in the pathophysiology of glaucoma [20]. The lack of agreement concerning the role played by the glia in ganglion cells has raised the need for research on both the location and the discrimination of responses which take place simultaneously in the RGCs and glia [8].

The present study analyzes a mouse model of ocular hypertension (OHT), both in the eye with laser-induced OHT (OHT-eyes) and in the contralateral eye. The aim was to determine i) concurrent responses of macroglial and retinal ganglion cells, using specific antibodies as markers against cytoskeletal proteins from both cells: GFAP (a major constituent of the astrocyte cytoskeleton [32] and NF-200kD (a major constituent of the neuronal cytoskeleton, which after axonal injury has an abnormal distribution in the soma and dendrites of the RGCs [33]; and ii) whether there is an inflammatory reaction to OHT by using anti-Iba 1 (a retinal microglial-specific calcium-binding adaptor protein) [34, 35] and an antibody against the class II major histocompatiblity complex (anti-MHC-II) (a marker for active antigen-presenting cells) [36] and then compare them with retinas from naïve eyes.

Experimental rodent models have been used to study glaucomatous neuropathy because they are inexpensive and easier to handle than other animal models (dog and rabbit). In comparison with the rat, the mouse has a major advantage, which is the possibility of being genetically manipulated [44]. In addition, although GFAP+ astrocytes were quantified in rats [45, 46], another advantage is that mouse astrocytes are farther apart from each other, allowing us to identify individual cells and count them one by one, more accurately than in rats [43].

Findings in mouse retinal glial cells reported here correspond to changes observed 15 days after lasering the treated eye. In the model of laser-induced OHT used in the present work, a substantial increase of the IOP was evident 24 hours after lasering which continued for four days and then gradually returned to the basal value after the fifth day, so that by one week after lasering, the IOP values in the treated animals were comparable for both eyes [40]. The experimental conditions of the present study constitute a model for human glaucomatous optic neuropathy and thus, can be used to investigate OHT-induced changes undergone by retinal macroglia, microglia, and ganglion cells.

The main reason to use whole-mounted preparations of the retina was to quantify the population of astrocytes and NF-200+RGCs in the whole mouse retina and also to analyze the behavior of glial cells and NF-200+RGCs in different retinal zones. In addition, whole-mounted preparations allow the differentiation of astrocytes from Müller glial cell end-feet which otherwise are not readily distinguishable in a sectional profile [47, 48], allowing astrocyte quantification.

The intermediate filament protein GFAP of astrocytes is considered an early marker for retinal injury and is commonly used as an index of gliosis-hypertrophy [21, 49, 50]. Two relevant morphological alterations in gliotic Müller cells are hypertrophy and the expression of the filament protein GFAP [51].

It has been reported that experimental diabetes in rats induces a differential GFAP expression pattern in the macroglial cells of the retina, reduces GFAP-IR in astrocytes, and increases GFAP-IR in Müller cells [47]. Such opposite reactions in astrocytes and Müller cells in terms of GFAP-IR has been reported in OHT-eyes of two models of experimental glaucoma in rats [43, 45]. In the present study, a similar behavior took place in the macroglial of OHT-eyes in mice.

Numerous in vitro and in vivo studies have shown GFAP to be essential for several astrocyte functions such as proliferation, differentiation, extension of processes, vesicle trafficking, astrocyte-neuron interaction [52], astrogliosis [53] and protection from cerebral ischemia [52].

In comparison to the naïve group, in some astrocytes of OHT-eyes the secondary processes could not be identified at 10x magnification. This difference could have contributed to the decreased GFAP-RA found in OHT-retinas. In addition, the intensity of the GFAP-IR in OHT-eyes was significantly higher than in naïve eyes. Whether or not astrocyte differences in OHT-eyes reflect a functional change, that is, preparation for migration or a switch from neuroprotective functions to immunogenic functions or astrocyte damage, is unknown. Astrocyte changes in OHT-eyes could impair the neurosupportive role of astrocytes [54] and participate in the death of RGCs reported in a recent parallel study using a comparable methodology to induce OHT [40].

It bears noting that the astrocyte number was similar in the three groups studied (OHT-eyes, contralateral, and naïve eyes). This leads us to postulate that in the retina of OHT-eyes a reactive, non-proliferative gliotic response takes place, similar to that reported in mouse [34, 55] and rat [43, 55]. It has been suggested that a gliotic non-proliferative response is the consequence of slow degeneration, while rapid degeneration leads to a proliferative gliosis [34, 55, 56]. However, it should be stressed that, although the number of astrocytes in OHT eyes did not differ significantly from naïve, 37.8% of the astrocytes exhibited morphological changes that could account for the decrease of GFAP-RA in eyes with OHT.

NF-200 is a component of the neuronal cytoskeleton. Under normal conditions, anti-NF-200 labels axons of the RGCs but rarely labels RGC somas [57]. Both staining patterns were observed in our naïve group. It is known that an elevated IOP has been associated with the disruption of the axonal transport [58] in different animal models [2, 59‐62]. A factor deeply involved in axonal transport is phosphorylation of the heavy neurofilament subunit (NF-H) [63‐65]. In a monkey model of chronic ocular hypertension [66], most NF-Hs in RGC axons in the glaucomatous eyes were significantly dephosphorylated by high IOP, which may be a sign of damaged axonal transport. In the present study, the number of NF-200+RGCs was significantly increased in OHT-eyes in comparison to naïve and contralateral eyes. These RGCs that had accumulated NF-200 in their cell bodies, proximal axon, and primary dendrites most probably represented functionally impaired RGCs as a consequence of the disruption of the axonal transport. The possibility that NF-200+RGCs represent damaged cells is supported by a recent study using comparable methodology to induce OHT, in which eyes with OHT had RT97+RGCs showing typical signs of axotomy-induced neuronal degeneration [40]. Notably, the NF-200+RGC somas of the OHT-eyes in our study tended to be more abundant in areas of the retina having less GFAP-RA. It is possible that an impairment of axonal support exerted by astrocytes could increase the vulnerability of the axon to IOP-induced stress [67]. A possible explanation could be the observations reported by Dibas et al. [68] according to which retinas with OHT showed a downregulation of AQP4 protein and an accumulation of ubiquitin in astrocytes which might not be appropriately transferred to adjacent RGCs. The attenuation of ubiquitination in axons may result in the accumulation of several proapoptotic proteins (that is, caspases, Bax and Bad) [69] and thus contributes to axonal degeneration in glaucoma.

It is known that severe axon insult can result in a rapid Wallerian degeneration of the distal axon [70]. On the contrary, milder insults may result in degeneration via the slower process of axonal dying-back and greater functional connectivity between the soma, proximal axon, and the distal axon segments [71‐76]. This situation involving the NF-200+RGCs of the present work, as reported by Soto et al. [56, 77] could represent RGCs that have suffered an insult but retain their fundamental homeostatic mechanisms, which might provide an opportunity for therapeutic rescue in the human disease.

A striking feature of the contralateral eyes was that macroglia exhibited morphological signs of reactivity that differed from naïve and OHT-eyes: astrocytes were more robust, formed a honeycomb-like network, and had an increase in GFAP-RA. In addition, the percentage of the retinal area occupied by GFAP+ cells with intensities above 3,000 was higher than in naïve eyes but did not differ from that of OHT-eyes. By contrast, the contralateral retinas of two experimental models of glaucoma in rats exhibited a decrease in both the retinal area occupied by astrocytes [43] and the GFAP-IR in astrocytes [43, 45]. The different behavior of the retinal macroglia of the contralateral eyes between mice and rats could be species related or could depend on the experimental model such as differences in time to increase pressure or time to return to normal values, among others [67].

It has been reported that the glial-activation response in glaucomatous eyes involves the activation of a glial immunoregulatory function and antigen-presenting ability [10, 78]. The expression of MHC-II in glial cells, required for antigen presentation to T cells, is upregulated in the glaucomatous human retina and optic-nerve head [10, 79, 80]. In addition, microglial activation has been demonstrated in human eyes with glaucoma [81], in experimental models of OHT [8, 80, 82, 83] and in a genetic mouse model of glaucoma [34]. Our study confirms the microglial activation in eyes with OHT, as evidenced by their morphological changes and stronger expression of MHC-II in most microglial cells compared to naïve eyes. Also, MHC-II immunoreaction in Müller cells supports the idea of immune activation in eyes with OHT.

A lack of relation between immune responses in DBA/2J mice retina and greater IOP has been reported [84] and recently corroborated by Bosco et al. [20], who saw no strict correlation between higher IOP and early microglia activation. They concluded that a rise in IOP may not be a contributory factor in these initial changes [20]. It bears mentioning that most microglial cells of the contralateral eyes (normal values of IOP) showed morphological changes and MHC-II upregulation in comparison with naïve eyes. In an experimental model of laser-induced OHT in rats, the contralateral eyes reportedly had a marginal increase in OX42 and OX6 (a MHC-II marker) two hours post-operation before returning to almost normal or normal, respectively, at three days [82]. The microglia of the contralateral retinas of the present study exhibited an upregulation of MHC-II immunoreaction that was widespread and more persistent than in the study of Wang et al. [82], given that in our retinas, an intense MHC-II immunoreaction of the microglia was detected throughout the retina after 15 days of lasering the treated eye. In addition, MHC-II upregulation was observed in both macroglial cell types, preferentially in astrocytes, a fact not reported by Wang et al. [82]. The MHC-II upregulation that we detected in the three retinal glial types of our study, which was not temporarily related to surgical eyeball manipulations, led us to postulate that an immunologically mediated process was taking place in contralateral retinas. This glial response may reflect an attempt to maintain tissue homeostasis, perhaps in an effort to protect optic axons from a compromised blood–brain barrier [20, 81]. A more sustained insult or prolonged neuronal stress may lead to glial changes that could potentially contribute to neuronal decline.

In the present experiments, we did not estimate RGCs survival, but in a recent parallel study using a comparable methodology to induce OHT, we documented RGC loss using a retrograde tracer applied to both superior colliculi one week prior to animal processing. In that study, the contralateral retinas showed the typical distribution of RGCs throughout the retina [40]. Based on this data, macroglial activation as well as MHC-II expression on astrocytes and Müller cells of our contralateral eyes could have exerted a neuroprotective effect. It has been suggested that the expression of modest levels of MHC-II may inhibit the activation of invading T cell, whereas overexpression of these molecules may promote the activation of autoimmune T cells, thereby augmenting the inflammatory cascade leading to tissue damage [10, 85]. This could be the case for our OHT-eyes in which GFAP-IR and MHC-II expression of Müller cells notably increased in comparison to naïve and contralateral eyes.

Another finding supporting the possible contribution of retinal macroglia to neuronal homeostasis in contralateral eyes and the harmful response of this population in OHT-eyes could be the difference between the contralateral and OHT-eyes in the NF-200+RGC count.

No previous studies are available on the expression of MHC-II on macroglia and microglia in the contralateral eye of adult Swiss mice after 15 days of laser-induced OHT. MHC-II upregulation in contralateral eyes could be secondary to IOP-induced changes in OHT-eyes. Furthermore, the absence of NF-200+RGCs (sign of RGC degeneration) leads us to postulate that the expression of modest levels of MHC-II in macroglial cells in contralateral eyes could offer a protective role.

In summary, after 15 days of unilateral laser-induced OHT, widespread and persistent changes in GFAP and MHC-II expression took place both in the contralateral and OHT-eyes of adult Swiss mice. MHC-II upregulation in Iba-1+ retinal cells was similar in both eyes; however, in the retinal macroglia, MHC-II expression was preferentially located in astrocytes of the contralateral eye and appeared to be restricted to Müller cells in the OHT-eye. The increased antigen-presenting activity in macroglial and microglial cells may be key in the role of the immune system in glaucoma. Knowledge of this role could lead to the development of more effective neuroprotective treatments to modulate the immune response to achieve, on the one hand, tissue repair and neuronal survival, and on the other, a decrease in the immune-mediated neurodegenerative damage.

On the basis of the alterations in the contralateral eye, we conclude that it should not be used as a control eye in this experimental model of laser-induced OHT. Further research is needed to understand the behavior of contralateral eyes in other models of experimental OHT.