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Purpose Besides primary neurotoxicity, oxidative stress may compromise the glial immune regulation and shift the immune homeostasis toward neurodegenerative inflammation in glaucoma. We tested this hypothesis through the analysis of neuroinflammatory and neurodegenerative outcomes in mouse glaucoma using two experimental paradigms of decreased or increased oxidative stress. Methods The first experimental paradigm tested the effects of Tempol, a multifunctional antioxidant, given through osmotic mini-pumps for drug delivery by constant infusion. Following a 6-week treatment period after microbead/viscoelastic injection-induced ocular hypertension, retina and optic nerve samples were analyzed for markers of oxidative stress and cytokine profiles using specific bioassays. We also analyzed a redox-sensitive transcriptional regulator of neuroinflammation, namely NF-κB. The second paradigm included a similar analysis of the effects of overloaded oxidative stress on retina and optic nerve inflammation in mice knockout for a major antioxidant enzyme (SOD1(-/-)). Results Increased antioxidant capacity and decreased protein carbonyls and HNE adducts with Tempol treatment verified the drug delivery and biological function. Among a range of cytokines measured, proinflammatory cytokines, including IL-1, IL-2, IFN-γ, and TNF-α, exhibited more than 2-fold decreased titers in Tempol-treated ocular hypertensive eyes. Antioxidant treatment also resulted in a prominent decrease in NF-κB activation in the ocular hypertensive retina and optic nerve. Although pharmacological treatment limiting the oxidative stress resulted in decreased neuroinflammation, ocular hypertension-induced neuroinflammatory responses were increased in SOD1(-/-) mice with defective antioxidant response. Conclusions These findings support the oxidative stress-related mechanisms of neuroinflammation and the potential of antioxidant treatment as an immunomodulation strategy for neuroprotection in glaucoma.

To delineate astrocyte-mediated inflammatory processes in glaucoma, we analyzed proteomic responses of retinal astrocytes in an experimental rat model using a cell-specific approach.IOP elevation was induced in rats by hypertonic saline injections into episcleral veins. Enriched samples of astrocytes were isolated through the immunomagnetic cell selection process established originally for retinal ganglion cell (RGC) sampling. Ocular hypertensive and control samples were collected by pooling from rat eyes matched for the cumulative IOP exposure. Protein expression was analyzed complementarily by quantitative two-dimensional capillary liquid chromatography and linear ion trap mass spectrometry (LC-MS/MS) followed by quantitative Western blot analysis and retinal tissue immunolabeling using specific antibodies to selected proteins.Following validation of enriched astrocyte samples, LC-MS/MS analysis resulted in the identification of over 2000 proteins with high confidence. Bioinformatic comparison analysis of the high-throughput MS/MS data along with the findings of immunoblotting and immunohistochemistry supported distinct responses of ocular hypertensive astrocytes during the experimental paradigm, which exhibited predominantly cellular activation and immune/inflammatory responses as opposed to activation of cell death signaling in ocular hypertensive RGCs. Inflammatory responses of astrocytes in experimental glaucoma included up-regulation of a number of immune mediators/regulators linked to TNF-α/TNFR signaling, nuclear factor kappa-B (NF-κB) activation, autophagy regulation, and inflammasome assembly.These findings validate an astrocyte-specific approach to quantitatively identify proteomic alterations in experimental glaucoma, and highlight many immune mediators/regulators characteristic of the inflammatory responses of ocular hypertensive astrocytes. By dissecting the complexity of prior data obtained from whole tissue, this pioneering approach should enable astrocyte responses to be defined and new treatments targeting astrocytes to be developed.

Dysregulation of blood flow with subsequent tissue hypoxia, secondary to or independent from elevated intraocular pressure (IOP) in glaucoma, has been implicated as a component of the pathogenic mechanisms of optic nerve degeneration and retinal ganglion cell (RGC) loss. Many patients with glaucoma exhibit vascular abnormalities such as vasospasm, systemic hypotension, angiographic vascular perfusion defects, and alterations in blood flow parameters that may result in reduced vascular perfusion in the optic nerve head and retina.1–5 Besides these clinical findings, hypoxic tissue stress is evident in the glaucomatous optic nerve head and retina by increased expression of a hypoxia-induced transcription factor, hypoxia-inducible factor (HIF)-1α.6 HIF-1α is known to activate transcription of a wide variety of genes with products that increase oxygen delivery and represent an adaptive response to hypoxia.7 Of interest, the retinal regions exhibiting increased HIF-1α immunolabeling in some of the donor eyes with glaucoma have been found to exhibit a close concordance with the location of visual field defects recorded in these eyes.6 It has long been known that the vertebrate retina consumes large amounts of oxygen and that the main oxygen consumption in the inner retina takes place in RGCs.8 However, although the maintenance of an adequate oxygen supply is critical for neuronal viability and function, current understanding of RGC oxygenation is incomplete. The oxygen demand of RGCs is supplied mainly by retinal arteries in the retina and short posterior ciliary arteries in the optic nerve head. Despite a heterogeneity in vascular response to increased IOP, oxygen tension in the inner retina is relatively unaffected by IOP changes, owing to effective autoregulation of retinal circulation.8–10 Similarly, optic nerve head perfusion pressure is adequately compensated by vascular autoregulation under normal conditions.11 However, in glaucomatous eyes, several risk factors compromising the autoregulatory control have been proposed to reduce blood flow, particularly in intermittent episodes affected by circadian variations in IOP, systemic blood pressure, and ocular perfusion pressure.1–5 In addition to these vascular factors, another important aspect of tissue oxygen delivery that is not well understood is extravascular oxygen transport. Although tissue perfusion is known to involve gas diffusion depending on the oxygen tension gradient and diffusion distance, it is unclear whether there is a facilitated mechanism for oxygen transport to RGCs from retinal capillaries, which are known to be surrounded by glial cells. This mechanism may be particularly important in glaucomatous conditions in which glial cells are in an activated state and may require increased oxygen consumption.12 Regarding the optic nerve head, the load-bearing connective tissue encasing the lamina cribrosa vasculature possibly makes RGC axons even more dependable for a facilitated oxygen transport. Besides a limited understanding of tissue oxygen transport within the retina and optic nerve head tissues, mechanisms controlling intracellular oxygen transport to mitochondria for oxidative phosphorylation also remain unclear. Hemoglobin (Hb) is a more than 600 million-year-old respiratory protein that reversibly binds oxygen and mediates oxygen transport function in blood erythrocytes.13 Repeated detection of Hb in our proteomic studies and recent discovery of Hb expression in the retinal pigment epithelium14 stimulated us to determine whether this hemeprotein may also be present in the inner retina and optic nerve head and whether the oxygen transport function of Hb may be involved in RGC oxygenation in normal or glaucomatous eyes. To answer these questions, we performed a series of experiments determining expression and cellular localization of Hb in animal tissues as well as in glaucomatous and nonglaucomatous human donor eyes, with particular attention to RGCs and macroglia. To determine whether this protein is regulated by ambient oxygen concentrations, we also performed in vitro experiments with primary cultures of rat RGCs and macroglia incubated in the absence and presence of hypoxia. Herein, we present new evidence that Hb is expressed by these cell types as repeatedly detected in the retinal proteome and exhibits an upregulation in ocular hypertensive rat eyes and glaucomatous human donor eyes. Our in vitro findings revealed that hypoxia boosts glial Hb expression through glial erythropoietin (EPO), a well-known target of hypoxic HIF-1α signaling. In addition to this autocrine loop, hypoxia also upregulates Hb expression in RGCs in a paracrine manner. These findings support that in addition to tissue gas diffusion, Hb is likely to be involved in tissue oxygen transport in the inner retina and optic nerve head and that, besides vascular autoregulation, this oxygen-binding protein may provide an intrinsic mechanism for regulation of cellular oxygenation. Based on other predicted functions of Hb in free radical scavenging and nitric oxide detoxification, such an intrinsic protective mechanism against hypoxic/oxidative injury may have important implications in glaucomatous neurodegeneration.