Purpose.: Elevated intraocular pressure (IOP) is an important risk factor for glaucoma. Animal models often involve techniques for IOP elevation that are surgically invasive. Here the authors describe a novel and relatively simple method for inducing a highly consistent, modest, and repeatable elevation in IOP for rats and mice. Methods.: IOP was elevated unilaterally by injection of polystyrene microbeads into the anterior chamber to occlude aqueous outflow in rats (2.5–7 μL) and mice (1 μL). The fellow eye received an equivalent saline injection as internal control. The authors used tonometry to measure microbead-induced IOP elevations. Optic nerves were processed histologically to determine axon loss. Results.: For rats, a single injection of microbeads raised IOP by 21% to 34%, depending on volume, for approximately 2 weeks, though they were not tracked to full recovery. IOP in the saline-injected eye was constant. An additional injection (5 μL) extended the elevation to 8 weeks. Cumulative pressure exposure for both injections increased linearly. For mice, a single 1-μL injection of microbeads elicited a highly regular 30% elevation in IOP that persisted for more than 3 weeks, with a linear rise in cumulative pressure exposure. For both rats and mice, interanimal variability on a given day was modest, approximately 5% of the mean IOP measurement. Extended elevations (4–5 weeks) induced approximately a 20% loss of axons in both rats and mice. Conclusions.: These data support a novel and flexible model of modest ocular hypertension with axon loss. The maximal duration of IOP elevation will be further characterized in future studies.

An early hallmark of neuronal degeneration is distal transport loss and axon pathology. Glaucoma involves the degeneration of retinal ganglion cell (RGC) neurons and their axons in the optic nerve. Here we show that, like other neurodegenerations, distal axon injury appears early in mouse glaucoma. Where RGC axons terminate in the superior colliculus, reduction of active transport follows a retinotopic pattern resembling glaucomatous vision loss. Like glaucoma, susceptibility to transport deficits increases with age and is not necessarily associated with elevated ocular pressure. Transport deficits progress distal-to-proximal, appearing in the colliculus first followed by more proximal secondary targets and then the optic tract. Transport persists through the optic nerve head before finally failing in the retina. Although axon degeneration also progresses distal-to-proximal, myelinated RGC axons and their presynaptic terminals persist in the colliculus well after transport fails. Thus, distal transport loss is predegenerative and may represent a therapeutic target.

Glaucomatous optic neuropathy causes blindness through the degeneration of retinal ganglion cells (RGCs) and their axons, which comprise the optic nerve. Glaucoma traditionally is associated with elevated intraocular pressure, but often occurs or may progress with intraocular pressure in the normal range. Like other diseases of the CNS, a subset of glaucoma has been proposed to involve an autoimmune component to help explain the loss of RGCs in the absence of elevated intraocular pressure. One hypothesis involves heat shock proteins (HSPs), because increased serum levels of HSP autoantibodies are prominent in some glaucoma patients with normal pressures. In the first direct support of this hypothesis, we found that HSP27 and HSP60 immunization in the Lewis rat induced RGC degeneration and axon loss 1–4 months laterin vivoin a pattern with similarities to human glaucoma, including topographic specificity of cell loss. Infiltration of increased numbers of T-cells in the retina occurred much earlier, 14–21 d after HSP immunization, and appeared to be transient.In vitrostudies found that T-cells activated by HSP immunization induced RGC apoptosis via the release of the inflammatory cytokine FasL, whereas HSP immunization induced activation of microglia cells and upregulation of the FasL receptor in RGCs. In summary, our results suggest that RGC degeneration in glaucoma for selected individuals likely involves failed immunoregulation of the T-cell-RGC axis and is thus a disturbance of both proapoptotic and protective pathways.

Purpose. Elevated hydrostatic pressure induces retinal ganglion cell (RGC) apoptosis in culture. The authors investigated whether the transient receptor potential vanilloid 1 (TRPV1) channel, which contributes to pressure sensing and Ca²⁺-dependent cell death in other systems, also contributes to pressure-induced RGC death and whether this contribution involves Ca²⁺. methods. trpv1 mRNA expression in RGCs was probed with the use of PCR and TRPV1 protein localization through immunocytochemistry. Subunit-specific antagonism (iodo-resiniferatoxin) and agonism (capsaicin) were used to probe how TRPV1 activation affects the survival of isolated RGCs at ambient and elevated hydrostatic pressure (+70 mm Hg). Finally, for RGCs under pressure, the authors tested whether EGTA chelation of Ca²⁺ improves survival and whether, with the Ca²⁺ dye Fluo-4 AM, TRPV1 contributes to increased intracellular Ca²⁺. results. RGCs express trpv1 mRNA, with robust TRPV1 protein localization to the cell body and axon. For isolated RGCs under pressure, TRPV1 antagonism increased cell density and reduced apoptosis to ambient levels (P ≤ 0.05), whereas for RGCs at ambient pressure, TRPV1 agonism reduced density and increased apoptosis to levels for elevated pressure (P ≤ 0.01). Chelation of extracellular Ca²⁺ reduced RGC apoptosis at elevated pressure by nearly twofold (P ≤ 0.01). Exposure to elevated hydrostatic pressure induced a fourfold increase in RGC intracellular Ca²⁺ that was reduced by half with TRPV1 antagonism. Finally, in the DBA/2 mouse model of glaucoma, levels of TRPV1 in RGCs increased with elevated IOP. conclusions. RGC apoptosis induced by elevated hydrostatic pressure arises substantially through TRPV1, likely through the influx of extracellular Ca²⁺.

Purpose.: To characterize a glaucoma model of mice, the authors adopted and modified a method of inducing the chronic elevation of intraocular pressure (IOP) by anterior chamber injection of polystyrene microbeads. Methods.: Chronic elevation of IOP was induced unilaterally in adult C57BL/6J mice by injecting polystyrene microbeads to the anterior chamber. Effectiveness of microbeads of different sizes (small, 10 μm; large, 15 μm) on inducing IOP elevation was compared, and IOP was measured every other day using a tonometer. After maintaining elevated IOP for 2, 4, or 8 weeks, the degree of RGC and axon degeneration was assessed quantitatively using electron microscopy, fluorogold, retrograde labeling, and immunohistochemistry. Results.: Eighty-one of 87 mice that received anterior chamber injection of microbeads exhibited consistent IOP elevation above that of control eyes. Injection of small microbeads induced longer and higher peak value of IOP elevation compared with that of the large microbeads. A single injection of small microbeads resulted in a 4-week elevation of IOP that was maintained to an 8-week period after a second injection of microbeads in week 4. As the duration of IOP elevation increased, RGC bodies and their axons degenerated progressively and reached an approximately 50% loss after an 8-week elevation of IOP. Conclusions.: Anterior chamber injection of microbeads effectively induced IOP elevation and glaucomatous optic neuropathy in mice. Development of an inducible mouse model of elevated IOP will allow applications of mouse genetic technology to the investigation of the mechanisms and the evaluation of treatment strategies of glaucoma.

Purpose. The response of retinal ganglion cells (RGCs) to ocular pressure in glaucoma likely involves signals from astrocytes and microglia. How glia-derived factors influence RGC survival at ambient and elevated pressure and whether the inflammatory cytokine interleukin-6 (IL-6) is a contributing factor were investigated. methods. Primary cultures of retinal astrocytes, microglia, and RGCs were prepared using immunomagnetic separation. Comparisons were made of RGC survival at ambient and elevated pressure (+70 mm Hg) and with pressure-conditioned medium from glia with, and depleted of, IL-6. results. Pressure elevated for 24 to 48 hours reduced RGC density, increased TUNEL labeling, and upregulated several apoptotic genes, including the early immediate genes c-jun and jun-B. Pressure-conditioned medium from astrocytes reduced RGC survival another 38%, while microglia medium returned RGC survival to ambient levels. These effects were unrelated to IL-6 in microglia medium. Neither astrocyte- nor microglia-conditioned medium affected ambient RGC survival unless depleted of IL-6, which induced a 63% and a 18% decrease in RGCs, respectively. Recombinant IL-6 equivalent to levels in glia-conditioned medium doubled RGC survival at elevated pressure. conclusions. For RGCs at ambient pressure, IL-6 secreted from astrocytes and microglia under pressure is adequate to abate other proapoptotic signals from these glia. For RGCs challenged by elevated pressure, decreased IL-6 in astrocyte medium is insufficient to counteract these signals. Increased IL-6 in microglia medium counters not only proapoptotic signals from these cells but also the pressure-induced apoptotic cascade intrinsic to RGCs.