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D.A. Reichstein, L. Ren, T. Mittag, J. Danias; Visualization of Retinal Ganglion Cell Apoptosis in Aging Glaucomatous DBA/2J Mice . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1259.
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Purpose: To detect retinal ganglion cells (RGC) undergoing apoptosis in the retina of glaucomatous DBA/2J mice in vivo, and to determine whether RGC apoptosis occurs in a focal or diffuse pattern. Methods: Groups of female DBA/2J mice of ages 3, 9–10, and 12 months (n=6, 7, and 8 retinas respectively) were used. Animals received intravitreal injections of Annexin V bound to Alexa–488 (1 ul injection) one hour before euthanasia. Retinas were fixed and flatmounted. Annexin V–positive RGCs in the hemiretina opposite the site of injection were counted, and their positions relative to the optic nerve head were recorded. Numbers of Annexin V–binding cells per hemiretina in the various age groups were compared using ANOVA. Control experiments to validate the method were performed on adult female rats that had previously undergone optic nerve ligation and on C57 mice that had previously undergone either optic nerve ligation or intravitreal injection of 30 uM NMDA (1 ul injection). Results:Annexin V reliably labeled apoptotic RGCs in eyes after either optic nerve ligation or intravitreal NMDA injection. Retinas from DBA/2J mice aged 12 months showed significantly higher numbers of apoptotic cells than those from DBA/2J mice aged 3 or 9–10 months (ANOVA, p<0.001, post–hoc Bonferoni). In addition, visual inspection of the position of apoptotic cells within each retina indicated that apoptotic RGCs tended to form clusters. Cluster analysis using medoid partitioning revealed the presence of between 3 and 14 clusters of at least 3 cells each in retinas of 12 month old glaucomatous mice. Conclusions: Annexin V can be used reliably to visualize RGC apoptosis in vivo in a murine retina. Apoptotic RGC death in DBA/2J mice appears to increase with age to 12 months and occurs in clusters.
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