Fig. 5
Transient elevation of IOP releases ATP and activates microglia through P2X7 receptor involvement. a Representative image of staining for Iba1 in a retinal whole mount from an unpressurized C57Bl/6J mouse eye (Ctrl, left) and from an analogous region 20 h after elevation of IOP to 57 mmHg for 4 h (right). Retinal microglia subject to IOP elevation showed increased soma size, increased staining for Iba1, and shorter, thicker projections. Bar = 20 μm. b Increase in ATP concentration of posterior vitreous humor after elevation of IOP (paired Student’s t test; retinal pairs from 3 mice). c Iba1 staining from a P2X7−/− mouse indicates reduced morphological activation after elevation of IOP (right) when compared to unpressurized retina (left). d Quantification of morphological activation of microglia across central and middle regions suggests IOP elevation triggered greater morphological activation in C57Bl/6J than P2X7−/− retinae (2-way ANOVA with Tukey’s MC test; dots are n = 10 retinae from 5 mice per C57Bl/6J, P2X7−/− strain; data normalized to unpressurized score for each strain). e qPCR showing increased expression of Nos2, Tnfa, Arg1, and Chil3 in the retina after elevation of IOP in C57Bl/6J (e) mice. Dots represent change in expression from a single mouse, with expression normalized to the average ΔΔCT value of unpressurized contralateral eyes (paired Student’s t tests; Nos2: n = 5 mice, Tnfa, Arg1, Chil3: n = 6 mice). f A small but significant increase was observed in retinal gene expression of Arg1, and Tnfa, but not Nos2 or Chil3 after IOP elevation in P2X7−/− mice (Paired Student’s t tests; Nos2 n = 6 retinae from 3 mice, Tnfa, Arg1, Chil3, n = 12 retinae from 6 mice). g Relative change in retinal expression of key genes after elevation of IOP in C57Bl/6J mice compared to P2X7−/− mice. Values represent mean ΔΔCT levels for each gene compared to unpressurized control retinae (unpaired Student’s t test; n = 5 genes). Statistical significance shown as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant