Antioxidant Treatment Limits Neuroinflammation in Experimental Glaucoma

Abstract

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.

Figure 1

Assessment of the experimentally induced IOP elevation in mice. Shown are the IOP curves during an experimental period of 6 weeks. Control groups of C57BL/6J WT or SOD1^−/− mice that received physiologic saline injection into the anterior chamber had a steady level of IOP that was maintained at an average value of 10.8 ± 1.2 mm Hg through the experimental period. However, the microbead/viscoelastic-injected mice, including both WT and SOD1^−/− animals, exhibited a transient course of ocular hypertension. Also shown are the treatment groups including the WT ocular hypertensive mice that received either antioxidant Tempol or the saline vehicle alone (n = 12/group). Data are presented as mean ± SD.

Figure 2

Assessment of treatment effects on oxidative stress. The mouse retina (A) and optic nerve (B) protein samples were analyzed for antioxidant capacity, protein carbonyls, and HNE adducts by specific assays. Presented is the fold-change (mean ± SD) in Tempol-treated versus vehicle-treated groups, or SOD1^−/− versus WT controls (C57BL/6J). The antioxidant capacity was increased and the oxidative stress end products were decreased after Tempol treatment of ocular hypertensive mice relative to ocular hypertensive controls that received only the vehicle (Mann-Whitney U test; P < 0.01). In contrast, ocular hypertensive SOD1^−/− mice exhibited decreased antioxidant capacity and increased protein oxidation compared with ocular hypertensive WT controls (P < 0.01). Data represent at least six mice for each group.

Figure 3

Retina and optic nerve inflammation in ocular hypertensive mice. To determine the inflammatory status of the retina and optic nerve, cytokine titers were analyzed by ELISA. The bar graphs show the fold-change (mean ± SD) in ocular hypertension–induced cytokine production. (A) Fold decrease in retina cytokine titers in Tempol-treated ocular hypertensive mice compared with ocular hypertensive controls that received only the saline vehicle. (B) Fold increase in retina cytokine titers in SOD1^−/− mice with ocular hypertension compared with WT (C57BL/6J) ocular hypertensive mice. (C) Fold decrease in optic nerve cytokine titers in Tempol-treated versus vehicle groups of ocular hypertensive mice. (D) Fold increase in optic nerve cytokine titers in SOD1^−/− versus WT ocular hypertensive mice. Presented data represent at least six mice per group. *P < 0.05; **P < 0.01 (Mann-Whitney U test).

Figure 4

Activity of NF-KB, a transcriptional regulator of inflammation, in experimental mouse glaucoma. (A) For Western blot analysis of protein expression, retina and optic nerve protein samples were probed with a phosphorylation site-specific antibody to NF-κB subunit, p65 (p-p65). This analysis indicated that Tempol treatment of the ocular hypertensive mice (relative to ocular hypertensive controls that received the saline vehicle alone) resulted in a prominent decrease in p-p65 expression. However, SOD1^−/− mice (relative to C57BL/6J WT controls) exhibited a prominent increase in ocular hypertension–induced p-p65 expression. (B) Nuclear factor–κB DNA binding activity assay similarly indicated over 3-fold decreased activity of NF-κB in Tempol-treated ocular hypertensive samples relative to ocular hypertensive controls that received only the vehicle (mean ± SD). However, NF-κB activity was significantly increased in ocular hypertensive SOD1^−/− mice relative to ocular hypertensive WT controls. Presented data represent at least six mice per group.

Figure 5

Immunohistochemical analysis of glial cytokine production in the ocular hypertensive mouse retina. Specific antibodies to astroglia (GFAP) or microglia (Iba1) markers were used to determine the retinal localization of TNF-α, an important proinflammatory cytokine. Presented include retina images from C57BL/6J WT mice with or without induced ocular hypertension, WT ocular hypertensive mice treated with Tempol, and SOD1^−/− mice with induced ocular hypertension. Compared with the normotensive retina (thin arrows), hypertrophic astrocytes in the ocular hypertensive retina exhibited increased immunolabeling for GFAP (red). As seen in the image from SOD1^−/− mice, the ocular hypertension–induced glial response also included GFAP immunolabeling of the Müller glia located in the inner nuclear layer (arrowheads). Besides astroglia, the glial activation response to ocular hypertension included the microglia. The weakly labeled microglia for Iba1 in the normotensive control retina (thin arrows) were mainly localized around the blood vessels in the ganglion cell (GCL) or inner nuclear (INL) layers. However, ocular hypertensive retinas exhibited increased number and Iba1 immunolabeling (red) of the microglia that were distributed throughout the inner retina. Tumor necrosis factor-α immunolabeling was not detectable in the normotensive retina; however, ocular hypertensive retinas were prominently immunolabeled for this proinflammatory cytokine (green), which was remarkably decreased after Tempol treatment. The glia in the ocular hypertensive SOD1^−/− mice presented the most prominent immunolabeling for TNF-α (thick arrows). The increased TNF-α immunolabeling in the glaucomatous retina was localized to GFAP+ astroglia and Iba1+ microglia. Blue indicates nuclear DAPI staining. Data represent three different samples for each group (scale bar: 100 μm).

Figure 6

Assessment of treatment effects on neuron injury. (A) Analysis of optic nerve cross-sections and whole-mounted retinas indicated prominent injury to RGC somas and axons with experimentally induced glaucoma in mice. Compared with C57BL/6J WT mice, ocular hypertension induced greater injury to neurons in SOD1^−/− mice. The red arrow indicates degenerating axons and myelin debris; blue asterisk indicates the areas of prominent axon loss. Optic nerve axons were counted in cross-sections, and RGCs were counted in whole-mounted retinas after β-III-tubulin immunolabeling. To estimate the neuron loss in each mouse, axon and RGC counts in the ocular hypertensive eye were adjusted to the normotensive fellow eye. Bar graphs in (B) and (C) indicate the percentage of axon or RGC loss in (1) WT ocular hypertensive mice (relative to WT normotensive controls); (2) Tempol-treated WT ocular hypertensive mice (relative to WT ocular hypertensive controls that received only the saline vehicle); and (3) ocular hypertensive SOD1^−/− mice (relative to ocular hypertensive WT controls). P values are based on Mann-Whitney U test. Presented data (mean ± SD) represent 12 mice per group for axon counts and at least 3 mice per group for RGC counts.