Fig. 3

TSP1 inhibits RD-induced leukocyte infiltration and prevents cone loss. a Representative cytometry plots of mouse retina 7 days after RD without (upper panels) or with subretinal injection of TSP1 (lower panels). CD45⁺CD3⁺ cells define T cells and CD45⁺CD11b⁺ cells define microglia and myeloid cells with CD45^lowCD11b⁺ representing microglia and CD45^highCD11b⁺ representing infiltrating myeloid cells. CD45^highCD11b⁺ were further defined with Ly6C and Ly6G with Ly6C^highLy6C^- cells representing monocytes, Ly6C^lowLy6C^- representing macrophages and Ly6G⁺ representing neutrophils. b Flow cytometry quantification of T cells, MCs, Mos, and Mφs in mouse retina without RD and 7 days after RD without or with subretinal injection of TSP1 (n = 8–24/group; one-way Anova/Bonferroni test *p = 0.0018, **p = 0.0003, and ***p < 0.0001 versus the control group, ^$p = 0.0032 and †p < 0.0005 versus the PBS group). c Quantitative RT-PCR of Il-6, Ifn-γ, Ccl2, Ccl3, and Ccl4 mRNAs normalized to S26 mRNA in mouse retina without RD and 4 days after RD with or without subretinal injection of TSP1 (n = 6–8/group; one-way Anova/Bonferroni test *p < 0.003, **p < 0.001, and ***p < 0.0001 versus the control group, ^$p < 0.01, ^‡p < 0.007, and †p < 0.0005 versus the PBS group). d Representative images of peanut agglutinine- (PNA, red), cone arrestin- (CAR, green), and ionized calcium-binding adapter molecule 1- (IBA-1, white) stained retinal flatmounts of C57BL6/J mice 7 days after RD without or with subretinal injection of TSP1. Quantification of e subretinal IBA-1+ cells, f PNA+ cones, and g CAR+ cones in retinal flatmounts of C57BL6/J mice without RD and 7 days after RD without or with subretinal injection of TSP1 (n = 6–12/group; e one-way Anova/Bonferroni test *p < 0.0001 versus the control group, ^$p = 0.0002 versus the PBS group; f one-way Anova/Bonferroni test *p < 0.0001 versus the control group, ^$p = 0.0470 versus the PBS group; g one-way Anova/Bonferroni test *p < 0.0001 versus the control group, ^$p = 0.0448 versus the PBS group). h Live cone counts per well of cone-enriched cultures from chicken embryos treated with PBS or the indicated TSP1 concentration for 7 days (n = 15 for PBS and n = 4 for each TSP1 concentration one-way Anova/Bonferroni test *p = 0.0219, †p < 0.0001 versus PBS). RD, retinal detachment; PBS, phosphate-buffered saline; TSP1, thrombospondin-1; Myeloid, myeloid cells; Mos, monocytes; MCs, microglial cells; Mφs, macrophages; Neutros, neutrophils; PNA, peanut agglutinine; CAR, cone arrestin; IBA-1, ionized calcium-binding adapter molecule 1. Scale bar (d) = 50 μm. All values are reported as mean ± SEM