Tumor necrosis factor-alpha mediates oligodendrocyte death and delayed retinal ganglion cell loss in a mouse model of glaucoma

Abstract

Glaucoma is a widespread ocular disease characterized by a progressive loss of retinal ganglion cells (RGCs). Previous studies suggest that the cytokine tumor necrosis factor-alpha (TNF-alpha) may contribute to the disease process, although its role in vivo and its mechanism of action are unclear. To investigate pathophysiological mechanisms in glaucoma, we induced ocular hypertension (OH) in mice by angle closure via laser irradiation. This treatment resulted in a rapid upregulation of TNF-alpha, followed sequentially by microglial activation, loss of optic nerve oligodendrocytes, and delayed loss of RGCs. Intravitreal TNF-alpha injections in normal mice mimicked these effects. Conversely, an anti-TNF-alpha-neutralizing antibody or deleting the genes encoding TNF-alpha or its receptor, TNFR2, blocked the deleterious effects of OH. Deleting the CD11b/CD18 gene prevented microglial activation and also blocked the pathophysiological effects of OH. Thus TNF-alpha provides an essential, although indirect, link between OH and RGC loss in vivo. Blocking TNF-alpha signaling or inflammation, therefore, may be helpful in treating glaucoma.

Figure 1.

Laser-induced angle closure leads to increased intraocular pressure and loss of RGCs and axons. A, Time course of IOP after laser-induced angle closure (n = 15 per time point). Pressure was measured at the indicated time points with an applanation tonometer. *p < 0.05 and **p < 0.01 compared with the contralateral control eye. B, DiI-labeled RGCs in flat-mounted retinas (top panels; scale bar, 100 μm) and axons (bottom panels; scale bar, 10 μm) of control or OH mice 4 weeks after increasing IOP. C, Quantitation of DiI-labeled RGCs in wild-type and TNF-α^−/− mice after elevation of IOP. **p < 0.01 and ***p < 0.001 compared with wild-type mice (n = 10 per time point).

Figure 2.

Loss of oligodendrocytes after OH. A, APC⁺ oligodendrocytes in retinas with or without OH at 8 weeks after surgery. Scale bar, 50 μm. B, Time course of oligodendrocyte survival after increasing IOP. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with normal controls (n = 8 per time point). Circle, Sham-operated controls (n = 6); triangle, OH; square, control.

Figure 3.

TNF-α levels increase after elevating IOP and result in a delayed loss of RGCs. A, Real-time PCR analysis of TNF-α mRNA in the retina at the indicated time points. Results represent fold-increase relative to normal controls. ***p < 0.001 compared with normal control (n = 6 per each time point). B, ELISA for TNF-α protein in the retina. **p < 0.01, ***p < 0.001 compared with normal controls. C, DiI-labeled RGCs in flat-mounted retinas with or without TNF-α injections at 2 or 8 weeks. Scale bar, 50 μm. D, Quantitation of RGC survival at 2 or 8 weeks after intravitreal injection of TNF-α. ***p < 0.001 compared with controls injected with PBS at 8 weeks (n = 8 per time point).

Figure 4.

TNF-α mediates the effect of increased IOP on oligodendrocytes. A–F, Representative merged photomicrographs showing APC⁺ oligodendrocytes (green) and DAPI nuclear staining (blue) in longitudinal sections through the optic nerve with or without TNF-α treatment. Shown are control (A) and 1 d (B), 4 d (C), and 14 d (D) after TNF-α injection. E, F, APC⁺ oligodendrocytes in the optic nerve after direct contact with a Spongel soaked in 1 ng/ml TNF-α solution (E) or PBS (F) after 14 d. Scale bar, 50 μm. G, Time course of oligodendrocyte degeneration after intravitreal administration of PBS (diamond) or TNF-α (square). Also shown are survival data 14 d after direct administration of PBS (triangle) or TNF-α (circle) to the optic nerve. **p < 0.01, ***p < 0.001 compared with PBS-treated controls (n = 8 per time point). H, APC⁺ oligodendrocytes in optic nerves treated with anti-TNF-α blocking antibody or normal goat serum (NGS) 14 d after inducing OH. Scale bar, 50 μm. I, Quantitation of OH-induced oligodendrocyte degeneration after 14 d. ***p < 0.001 compared with normal controls (n = 8). J, Quantitation of OH-induced oligodendrocyte degeneration 14 d after inducing OH in wild-type or TNF-α^−/− mice. ***p < 0.001 compared with normal control (n = 8).

Figure 5.

TNFR2 mediates the effect of increased IOP and of TNF-α on oligodendrocytes and RGCs. A, IOP elevation after angle closure by laser photo coagulation in wild-type, TNFR1^−/−, or TNFR2^−/− mice (n = 10 per group). Deletion of either TNFR gene does not alter IOP elevation. Solid line, laser-treated eye; dashed line, control eye. B, DiI-labeled RGCs (top panels; scale bar, 50 μm) or axons (bottom panels; scale bar, 20 μm) in wild-type or TNFR-deficient mice 4 weeks after inducing OH. C–E, Quantitation of DiI-labeled RGCs (C), optic nerve axons (D), or APC⁺ oligodendrocytes (E) 4 weeks after increasing IOP. **p < 0.01 and ***p < 0.001 compared with wild-type mice (n = 10).

Figure 6.

TNF-α -induced oligodendrocyte loss depends on TNFR2. A, APC⁺ oligodendrocytes in optic nerves of TNFR1^−/− or TNFR2^−/− mice treated with PBS or TNF-α. Scale bar, 100 μm. B, Quantitation of APC⁺ oligodendrocyte survival after intravitreal injection of TNF-α in TNFR1^−/− or TNFR2^−/− mice. ***p < 0.001 as compared with PBS-injected controls in TNFR1^−/− mice (n = 8).

Figure 7.

Microglia mediate the cytotoxic effects of TNF-α. A, CD11b immunostaining in optic nerve sections at the indicated times after intravitreal injections of TNF-α or PBS. Scale bar, 100 μm. B, Quantitation of CD11b⁺ microglia in optic nerves. **p < 0.01 as compared with PBS-treated controls at the same time points (n = 8 per time point). C–F, APC⁺ oligodendrocytes in optic nerves of Mac-1^−/− mice exposed to intravitreal injections of TNF-α (TNF-α, i.v.), direct application of TNF-α from a Spongel wrapped around the optic nerve (TNF-α on), or increased IOP (OH). Scale bar, 50 μm. G, Quantitation of APC⁺ oligodendrocytes 14 d after treatment. H, DiI-labeled RGCs in flat-mounted retinas of wild-type mice or Mac-1^−/− mice 4 weeks after increasing IOP. Scale bar, 100 μm.

Figure 8.

Schematic timeline of events. TNF-α is upregulated rapidly after increasing intraocular pressure. This upregulation is followed by a rapid increase in microglial activation in the nerve, and by week 2 oligodendrocyte loss can be seen. Significant RGC death appears by week 4.