Additional file 1 of Rapid morphologic changes to microglial cells and upregulation of mixed microglial activation state markers induced by P2X7 receptor stimulation and increased intraocular pressure
Creators: name: Campagno, Keith E.
name: Lu, Wennan
name: Jassim, Assraa Hassan
name: Albalawi, Farraj
name: Cenaj, Aurora
name: Tso, Huen-Yee
name: Clark, Sophia P.
name: Sripinun, Puttipong
name: Gómez, Néstor Más
name: Mitchell, Claire H.
Publisher: name: figshare
Year: 2021
Subjects: subject: Biophysics
subject: Medicine
subject: Cell Biology
subject: Molecular Biology
subject: Neuroscience
subject: Physiology
subject: FOS: Biological sciences
subject: Pharmacology
subject: Immunology
subject: FOS: Clinical medicine
subject: Cancer

description: Additional file 1 : Supplementary Figure S1. Details of effect of intravitreal injection of BzATP on Sholl and other image analysis. a Representations of i- Iba1 staining; ii- image tracing, iii - concentric rings placed every 5 μm for analysis of crossing and length; and iv - conversion to a binary image. Bar = 25 μm. b, c Tracings were analyzed for distance from soma at which peak number of intersections with concentric rings (b) and farthest intersection with concentric ring (c) occurred (paired Student’s t-test; n = 6 retinae of 3 mice). d Total summed branch length averaged per mouse was reduced by approximately 10% (paired Student’s t-test; n = 6 retinae of 3 mice; Normalized to mean saline injected = 1.0000, mean 250 μM BzATP = 0.8914). e Sholl analysis performed with individual cells (2-way Repeated Measures ANOVA with Sidak’s Multiple Comparison’s Test; n = 41 Saline, 46 BzATP) cells from 6 retinae of 3 mice; significance represents Multiple Comparison’s, data measured every 5 μm). f, g Absolute (f) and normalized (g, to avg saline) of Sholl data from individual images confirms reduction in branch length of microglia exposed to BzATP (unpaired Student’s t-test; n = 41 Saline, 46 BzATP cells from 6 retinae of 3 mice; Normalized mean saline = 0.9941, mean BzATP = 0.8930). h Quantification of Iba1 intensity in 5 μm ring around soma of individual cells from 6 retinae from 3 mice (unpaired Student’s t-test; n = 60 saline, 55 BzATP cells from 6 retinae of 3 mice). i. Schematic diagram indicating approximate placement of images analyzed for microglia and RGCs. *p &lt; 0.05, **p &lt; 0.01, ****p &lt; 0.0001. Figure S2. Validation of observer scoring method of morphological microglial activation. a Representative images derived from Iba1-immunostained retinal whole mounts indicating a score of 1, 2, or 3. Bar = 50 μm. b A significant correlation between observer scoring and microglial Iba1-soma intensity was found. Observer scoring was normalized to the mean of 3 saline injected mice, while soma intensity was quantified in a 5 μm diameter ring as described in Figure 1. Each dot represents the mean value among masked, trained observers (Pearson’s correlation r = 0.6882 with p = 0.0006; n = 21 images, 6 retinae from 3 mice). c Scoring of microglial activation shows homogeneity between trained observers. d Observer scoring of images taken from saline- or BzATP-exposed retinae analyzed by image (unpaired Student’s t-test; n = 12 saline, 9 BzATP images, from 6 retinae of 3 mice). **p &lt; 0.01. Bar = 50 μm. Figure S3. Confirmation of cellular identity of isolated microglial cells. a Immunocytochemistry indicating absence of glial marker GFAP (top) or neuronal marker synaptophysin (Synapt, bottom) in primary cultures of retinal microglial cells. Bar = 20 μm. b Isolated brain microglial cells stained for Iba1 but not GFAP. Bar = 20 μm. c qPCR results from cultured retinal microglial cells exposed for 4 hrs to DMSO (Ctrl), 10 ng/ml LPS (LPS), or 10 ng/ml IL-4 (IL4); changes in relative expression of mRNA for Nos2, Tnfa, Arg1, and Chil1 were consistent with microglial cell polarization. (n = 6-9 samples from 2 to 3 biological replicates). Figure S4. Videos demonstrating microglial retraction with exposure to BzATP. 15 minute videos were recorded at 15 frames per second, with all solutions added at t = 3 min. a Addition of fresh control solution led to few morphological changes. b Addition of 250 μM BzATP resulted in rapid retraction of microglial extensions and rounding of cells. c In the presence of 10 μM A839977 (and following 30 min preexposure to 10 μM A839977), 250 μM BzATP induced little retraction. All solutions Mg2+-free to prevent block of the P2X7 receptor. Figure S5. a RGCs (left) and microglia (right) cell numbers were counted per image and averaged within superior inferior, nasal, or temporal regions. There was a significant difference in distribution of RGCs in peripheral retinal areas (RM 1-way ANOVA with Sidak’s MC test; n = 12 regions from 3 mice). b Quantification of morphological activation from individual images of microglia across central and middle regions suggests greater activation in C57Bl/6 J retinae than in P2X7−/− retinae (2-way ANOVA with Tukey’s MC test; n = 52 regions from 10 retinae derived from 5 mice per C57Bl/6 J, P2X7−/− strain; data normalized to unstimulated score for each strain). c RGC numbers counted in central and middle fields under normotensive (Ctrl) and increased IOP in mice from both genotypes. Statistical significance shown as ** p &lt; 0.01, ***p &lt; 0.001, ****p &lt; 0.0001, ns = not significant.