Increased production of tumor necrosis factor-alpha by glial cells exposed to simulated ischemia or elevated hydrostatic pressure induces apoptosis in cocultured retinal ganglion cells

Abstract

Although glial cells in the optic nerve head undergo a reactivation process in glaucoma, the role of glial cells during glaucomatous neurodegeneration of retinal ganglion cells is unknown. Using a coculture system in which retinal ganglion cells and glial cells are grown on different layers but share the same culture medium, we studied the influences of glial cells on survival of retinal ganglion cells after exposure to different stress conditions typified by simulated ischemia and elevated hydrostatic pressure. After the exposure to these stressors, we observed that glial cells secreted tumor necrosis factor-alpha (TNF-alpha) as well as other noxious agents such as nitric oxide into the coculture media and facilitated the apoptotic death of retinal ganglion cells as assessed by morphology, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, and caspase activity. The glial origin of these noxious effects was confirmed by passive transfer experiments. Furthermore, retinal ganglion cell apoptosis was attenuated approximately 66% by a neutralizing antibody against TNF-alpha and 50% by a selective inhibitor of inducible nitric oxide synthase (1400W). Because elevated intraocular pressure and ischemia are two prominent stress factors identified in the eyes of patients with glaucoma, these findings reveal a novel glia-initiated pathogenic mechanism for retinal ganglion cell death in glaucoma. In addition, these findings suggest that the inhibition of TNF-alpha that is released by reactivated glial cells may provide a novel therapeutic target for neuroprotection in the treatment of glaucomatous optic neuropathy.

Fig. 1.

Cultured retinal cells. After retrograde labeling by Fluoro-Gold and the selection of retinal ganglion cells by the use of an immunomagnetic separation method, the selected cells were immunolabeled with antibodies against Fluoro-Gold and Thy-1.1; they were examined with flow cytometry. A, Immunolabeling with Fluoro-Gold (FL1-H) and Thy-1.1 (FL3-H) antibodies was colocalized in >90% of these cells; >95% of these cells were positive for Thy-1.1.B, Unselected cells were negative for both Fluoro-Gold (FL1-H) and Thy-1.1 (FL3-H). Cultured retinal ganglion cells had round or oval cell bodies with a diameter of 10–20 μm, phase-bright appearance, and branched neuritis of uniform caliber and varying length. C, A retinal ganglion cell derived from newborn rat retina. D, Fluorescence microscope image of the retinal ganglion cell shown in C after labeling for neurofilament protein. E, Fluorescence microscope image of the retinal ganglion cell shown in C after labeling for Thy-1.1. F, Glial cells derived from newborn rat retina. G, Fluorescence microscope image of the retinal glial cells shown in F after labeling for glial fibrillary acidic protein. H, Fluorescence microscope image of the retinal glial cells shown in F after labeling for S-100. Scale bars: C–E, 20 μm;F–H, 60 μm.

Fig. 2.

Morphological analysis of apoptotic cell death in cocultures of retinal ganglion cells and glial cells.A–C, Phase-contrast microscope images of retinal ganglion cells that were incubated under different conditions for 72 hr. A, Normal conditions. B, Simulated ischemia. C, Elevated hydrostatic pressure. Fluorescence microscope images of TUNEL in D–F correspond to retinal ganglion cells seen in A–C, respectively.G–I, Phase-contrast microscope images of glial cells that were incubated under different conditions for 72 hr.G, Normal conditions. H, Simulated ischemia. I, Elevated hydrostatic pressure. Fluorescence microscope images of TUNEL in J–L correspond to glial cells seen in G–I, respectively. After the incubation of cocultures under stress conditions, apoptosis was induced in retinal ganglion cells, although there was no evidence of apoptosis in glial cells.

Fig. 3.

A, Quantitative analysis of positive TUNEL in retinal ganglion cells that were incubated under simulated ischemia or elevated hydrostatic pressure. After incubation in the presence of simulated ischemia or elevated hydrostatic pressure for 72 hr, the rate of positive TUNEL was higher in retinal ganglion cells in cocultures compared with that in retinal ganglion cells that were cultured alone (RGCs; p = 0.006 and p = 0.04, respectively). In addition, the rate of positive TUNEL was higher in retinal ganglion cells in cocultures exposed to simulated ischemia or elevated hydrostatic pressure for 72 hr, respectively, compared with that in retinal ganglion cells in cocultures incubated under normal conditions (Mann–WhitneyU test; p = 0.017 andp = 0.023, respectively). B, Quantitative analysis of positive TUNEL in retinal ganglion cells after passive transfer experiments. Conditioned medium of glial cells that were cultured alone was collected after their incubation in the presence or absence of simulated ischemia or elevated hydrostatic pressure for 72 hr. Then retinal ganglion cells that were cultured alone were incubated with the glial-conditioned medium for 24 hr. The rate of positive TUNEL was higher in retinal ganglion cells that were incubated with the conditioned medium of stressed glial cells as compared with that in retinal ganglion cells that were incubated with the conditioned medium of control glial cells (p = 0.04 and p = 0.02 for simulated ischemia and elevated hydrostatic pressure, respectively).

Fig. 4.

Examination of caspase activity in cocultures incubated under simulated ischemia or elevated hydrostatic pressure. A, Western blot analysis of caspase-8 expression in cocultures. B, Western blot analysis of caspase-3 expression cocultures. Column 1, Control retinal ganglion cells; column 2, retinal ganglion cells incubated under simulated ischemia for 72 hr; column 3, retinal ganglion cells incubated under elevated hydrostatic pressure for 72 hr; column 4, control glial cells; column 5, glial cells incubated under simulated ischemia for 72 hr;column 6, glial cells incubated under elevated hydrostatic pressure for 72 hr. Western blots revealed that the 55 kDa immunoreactive band corresponding to caspase-8 cleaved to 30 and 20 kDa products in retinal ganglion cells that were incubated under stress conditions. In addition, 32 kDa pro-enzyme caspase-3 cleaved to a 17 kDa active subunit in retinal ganglion cells. No cleavage of caspase-8 or caspase-3 was detected with the use of the extracts of glial cells. Caspase activation also was examined, in situ, using Phiphilux-G₆D₂ in retinal ganglion cells that were incubated under different conditions for 72 hr. C, Normal conditions. D, Simulated ischemia.E, Elevated hydrostatic pressure. Fluorescence microscope images seen in F–H correspond to phase-contrast images of the retinal ganglion cells seen inC–E, respectively. Rhodamine fluorescence (red) indicates caspase-3-like activity in retinal ganglion cells that were incubated under stress conditions.I, The amount of DEVD-AMC cleaving activity with the use of fluorometric analysis was higher in retinal ganglion cells in cocultures that were incubated under simulated ischemia or elevated hydrostatic pressure for 72 hr as compared with cocultures that were incubated under normal conditions (Mann–Whitney U test;p = 0.006 and p = 0.04, respectively).

Fig. 5.

Examination of TNF-α and iNOS expression in cocultures that were incubated under simulated ischemia or elevated hydrostatic pressure. Both Western blot analysis (A, B) and immunocytochemistry (C–H) revealed increased expression of TNF-α and iNOS in glial cells, but not in retinal ganglion cells, in cocultures that were incubated under stress conditions. A, Western blot analysis of TNF-α expression. B, Western blot analysis of iNOS expression.Column 1, Control retinal ganglion cells; column 2, retinal ganglion cells that were incubated under simulated ischemia for 72 hr; column 3, retinal ganglion cells that were incubated under elevated hydrostatic pressure for 72 hr;column 4, control glial cells; column 5, glial cells that were incubated under simulated ischemia for 72 hr;column 6, glial cells that were incubated under elevated hydrostatic pressure for 72 hr. C–E, TNF-α expression in glial cells that were incubated under different conditions for 72 hr. C, Normal conditions. D, Simulated ischemia. E, Elevated hydrostatic pressure.F–H, iNOS expression in glial cells that were incubated under different conditions for 72 hr. F, Normal conditions. G, Simulated ischemia. H, Elevated hydrostatic pressure.

Fig. 6.

Measurement of TNF-α and end products of NO in conditioned medium of cocultures that were incubated under stress conditions. A, Titers of TNF-α in conditioned medium as measured by ELISA. B, Titers of end products of NO in conditioned medium as measured by a colorimetric assay. Levels of both TNF-α and NO end products were higher in the conditioned medium of cocultures that were exposed to simulated ischemia or elevated hydrostatic pressure as compared with cocultures that were incubated under normal conditions (Mann–Whitney U test;p = 0.003 and p = 0.003, respectively).

Fig. 7.

Inhibition of apoptosis in retinal ganglion cells in cocultures that were incubated under stress conditions in the presence of specific inhibitors of TNF-α or iNOS. Treatment of cocultures with a specific antibody neutralizing the activity of TNF-α (10 μg/ml) or with a selective inhibitor of iNOS, 1400W (2.5 μm), decreased the rate of positive TUNEL after incubation under stress conditions (Mann–Whitney Utest; p = 0.0002 and p = 0.003, respectively). Inhibition of apoptosis by a neutralizing antibody against TNF-α was more prominent than that by 1400W (p = 0.008).