Fig. 1

Retinal P2X7 receptor stimulation leads to activated microglia morphology and gene expression. Representative images from retinae fixed 24 h after intravitreal injection with saline (a) or 250 μM BzATP (b) indicated BzATP exposure led to greater Iba1 expression and altered morphology. c, d Z-projections of retinal whole mounts demonstrate increased Iba1 staining in the OPL, IPL and RGC layers of the retina with BzATP exposure. e Representation of tracing and conversion of Iba1 staining to a binary image for Sholl analysis. f Sholl analysis indicated reduced branching complexity of microglia exposed to BzATP (2-way RM ANOVA with Sidak’s MC test; n = 3 mice; significance represents interaction value of ANOVA). g Summed branch length is reduced in microglia exposed to BzATP as compared to saline (paired Student’s t test; n = 3 mice). Cell soma size and Iba1 intensity are determined in circled area (yellow ring, 5 μm diameter) surrounding microglia cell body from retinae exposed to h saline vs. i BzATP. j Quantification of Iba1 intensity in circled area (paired Student’s t test; n = 3 mice). k Observer scoring of images taken from saline- or BzATP-exposed retinae showing Iba1-positive microglia are activated with BzATP exposure (paired Student’s t test; n = 3 mice; normalized to average saline value). l Expression of both classical M1- activation genes Nos2, Tnfa, and alternative M2 activation genes Arg1, Chil3 are elevated in retinae exposed to BzATP (paired Student’s t test; n = 7 mice). Statistical significance shown at *p < 0.05, **p < 0.01, ****p < 0.0001. Scale bars represent 40 μm (a), 15 μm (d), and 25 μm (e, h)