Retinal ganglion cell death in a DBA/2J mouse model of glaucoma Microglial activation and intraocular pressure

BACKGROUND: Retinal microglia has been shown to reactivate in a murine model of pigmentary glaucoma. However, the relationship between microglial activation and intraocular pressure (IOP) elevation and retinal ganglion cell (RGC) death is still unclear. OBJECTIVE: To verify that microglial activation and tumor necrosis factor alpha (TNF-alpha) expression is involved in RGC death with elevated IOP and prolonged time of glaucomatous optic nerve lesion in a DBA/2J mouse model of glaucoma. DESIGN, TIME AND SETTING: This randomized, controlled, animal experiment was performed at the Peking University Third Hospital, Peking University Eye Center, China between December 2006 and May 2008. MATERIALS: DBA/2J mice and C57BL/6J mice (Jackson Laboratory, USA), rat anti-mouse CD11b monoclonal antibody (Serotec, UK), and goat anti-TNF-alpha polyclonal antibody (Sigma, USA) were used in this study. METHODS: A total of 100 female, DBA/2J mice at 3, 6, 9, 12, and 14 months of age (20 mice per age group) were used for the glaucoma model, and 18 C57BL/6J mice at 3, 9, 14 months of age (6 mice per age group) were used as normal controls. The anterior segment of the eye was observed using a slit-lamp biomicroscope. IOP was measured using a microneedle system. Morphology and number of retinal microglia were observed using immunohistochemistry. RGCs were quantified using Nissl staining. Co-localization of TNF-alpha and microglia was observed using double-labeling immunofluorescence. Excavation of the optic nerve head was observed utilizing hematoxylin-eosin staining. MAIN OUTCOME MEASURES: The following parameters were measured: IOP levels, numbers of RGCs and activated microglia, and TNF-alpha expression. RESULTS: In 6-month-old DBA/2J mice, dispersed pigment was observed, and some mice developed increased IOP. At 9 months of age, IOP levels reached a peak. In 3-month-old DBA/2J mice, microglia were activated. In 6-month-old DBA/2J mice, the number of activated microglia was significantly increased and migrated to the outer retinal layer. In 9-month-old mice, TNF-alpha expression was co-localized with microglia. Significant RGC loss occurred in mice aged 9 to 14 months, with the presence of optic nerve fiber loss and optical nerve head excavation. IOP returned to normal levels at 12 months of age, but microglia remained activated, which was consistent with RGC loss. CONCLUSION: Retinal microglial activation was partially attributed to increased IOP. Activated microglia might be mainly responsible for RGC loss. TNF-alpha expression was evident in the inner retinal layer. However, the relationship between TNF-alpha and RGC loss remains poorly understood.