Early Optic Nerve Head Glial Proliferation and Jak-Stat Pathway Activation in Chronic Experimental Glaucoma

Abstract

Purpose We previously reported increased expression of cell proliferation and Jak-Stat pathway–related genes in chronic experimental glaucoma model optic nerve heads (ONH) with early, mild injury. Here, we confirm these observations by localizing, identifying, and quantifying ONH cellular proliferation and Jak-Stat pathway activation in this model. Methods Chronic intraocular pressure (IOP) elevation was achieved via outflow pathway sclerosis. After 5 weeks, ONH longitudinal sections were immunolabeled with proliferation and cell-type markers to determine nuclear densities in the anterior (unmyelinated) and transition (partially myelinated) ONH. Nuclear pStat3 labeling was used to detect Jak-Stat pathway activation. Nuclear density differences between control ONH (uninjected) and ONH with either early or advanced injury (determined by optic nerve injury grading) were identified by ANOVA. Results Advanced injury ONH had twice the nuclear density ( P < 0.0001) of controls and significantly greater astrocyte density in anterior ( P = 0.0001) and transition ( P = 0.006) ONH regions. An increased optic nerve injury grade positively correlated with increased microglia/macrophage density in anterior and transition ONH ( P < 0.0001, both). Oligodendroglial density was unaffected. In glaucoma model ONH, 80% of anterior and 66% of transition region proliferating cells were astrocytes. Nuclear pStat3 labeling significantly increased in early injury anterior ONH, and 95% colocalized with astrocytes. Conclusions Astrocytes account for the majority of proliferating cells, contributing to a doubled nuclear density in advanced injury ONH. Jak-Stat pathway activation is apparent in the early injury glaucoma model ONH. These data confirm dramatic astrocyte cell proliferation and early Jak-Stat pathway activation in ONH injured by elevated IOP.

The Role of Aquaporin-4 in Optic Nerve Head Astrocytes in Experimental Glaucoma

“…In this study, we confirmed the proliferation of both microglia and astrocytes in the UON of mouse after 3 days of IOP elevation. While some investigators have stated that most proliferating cells in the glaucoma ON are Iba1 positive microglia, 25 our findings agree with those of Lozano and co-workers that, while microglia and astrocytes both proliferate 56 , astrocytes remain the majority of glia in the glaucomatous ON. In chronic experimental glaucoma in monkey, the total number of glia appeared constant, though the proportionate area of glia increased with axonal loss.…”

“…51 Astrocytes are the most numerous glia cell in mammalian ONH 52 and the reaction of astrocytes to injurious stimuli is now recognized to be either beneficial or detrimental. 53,54,55 Designated as A2 astrocytes, the beneficial phenotype activates the JAK-STAT pathway 56 and STAT knockout in mouse glaucoma leads to greater ganglion cell loss 25 . The detrimental A1 astrocytes arise from stimulation from microglia with interleukin 1α, tumor necrosis factor α (TNFα) , and complement component subunit 1q (C1q) .…”

Pressure-Induced Changes in Astrocyte GFAP, Actin, and Nuclear Morphology in Mouse Optic Nerve

“… 62 However, significant astrocyte proliferation has been reported by Ki67 labeling in mice 1 week after IOP elevation 46 and in rat glaucoma models. 63 Thus, some of the increase in nuclear clusters may be caused by a proliferation of astrocytes or microglia. Because external stresses are transmitted from the plasma membrane via the cytoskeleton to the nucleus, a change in nuclear shape or conformation is expected, as we observed and has been previously reported.…”

“…A recent study of rat ONH showed that the ON consists of 78% astrocytes. 63 The GFAP antibodies had limited depth penetration in sections, necessitating our image capture from the more superficial layer of each section. The axonal compartments were inferred from the negative space of binarized actin and nuclear images, which could include extracellular fluids or voids.…”

Astrocyte responses to experimental glaucoma in mouse optic nerve head

“…A general retraction of astrocytes from PPS followed by complete astrocyte loss was reported in one rat glaucoma model [ 66 ]. This finding was contradicted by abundant astrocyte division found in another rat glaucoma model [ 67 ]. In mouse glaucoma and in a monkey glaucoma model [ 68 ], there was minimal astrocyte death, as indicated by only occasional apoptotic ultrastructure and TUNEL labeling.…”