Fig 1: Induction of a fibrotic-like process after injury in TXA-treated zebrafish. a, b In vivo imaging and c, d morphological analysis of the laser area in uninjured untreated control (ctrl) and in untreated, vehicle ctrl and TXA-treated zebrafish at 14 dpli. a IR (left) and OCT (right) images of the laser burns. Arrowheads point to the central lesion on OCT depict the injury sites detected as hyper-reflective signal in both animal models. b Shown are zebrafish H&E-stained retinal sections of uninjured untreated ctrl and of untreated, vehicle ctrl and TXA-treated zebrafish at 14 dpli. The damaged area corresponds to photoreceptor layer discontinuity and cavity formation in the ONL and in the subretinal space (white frame). c Quantification of the laser damage width (mean ± SD). Significant differences (***p < 0.001) between uninjured untreated ctrl and untreated, vehicle ctrl and TXA-treated zebrafish at 14 dpli were determined by using two-tailed t test (n = 12). Representative scans were selected as indicated by the bold green line. d Quantification of cell nuclei in the ONL in uninjured and injured retinas at 14 dpli. Significant differences in structural changes after laser damage (***p < 0.001) between uninjured untreated ctrl and untreated, vehicle ctrl and TXA-treated zebrafish at 14 dpli were determined by using two-tailed t test (n = 12). e–i Analysis of Müller cell PAI1 expression in the TXA-treated zebrafish retinas at 14 dpli. e–h Shown are retinal sections at 14 dpli after laser damage induction stained for GS (red) and PAI1 (green). Cell nuclei were counterstained with DAPI (blue). i Histogram illustrating the mean ± SD of the number of PAI1+ cells normalized by the total number of GS+ cells expressed in percentage in the TXA-treated zebrafish. Significant differences (****p < 0.0001) between uninjured and injured animals were determined by using two-tailed t test (n = 12). j–n Analysis of Müller cells CTGF expression in the TXA-treated zebrafish retinas at 14 dpli. j–m Shown are retinal sections at 14 dpli after laser damage induction stained for GS (red) and CTGF (green). Cell nuclei were counterstained with DAPI (blue). n Histogram illustrating the mean ± SD of the number of CTGF+ cells normalized by the total number of GS+ cells expressed in percentage in the TXA-treated zebrafish. Significant differences (***p < 0.001) between uninjured and injured animals were determined by using two-tailed t test (n = 12). INL inner nuclear layer, ONL outer nuclear layer. Scale bar of H&E pictures equals 50 µm
Fig 2: Immunohistochemistry of PAI-1 and t-PA. A PAI-1 expression of immunohistochemistry in pleural-based masses. It showed strong stain in cytoplasm of histocytes. B PAI-1 expression of immunohistochemistry in patients without pleural-based masses. It was negative in all cells. C t-PA expression of immunohistochemistry in pleural-based masses. It only showed very weak stain in cytoplasm of parts of histocytes. D t-PA expression of immunohistochemistry in patients without pleural-based masses. Histocytes showed strong stain in cytoplasm
Fig 3: PAI1 upregulation in mouse is associated with retinal gliosis. a–j Analysis of PAI1+ Müller cells in zebrafish and murine retinas at1, 3, 7 and 14 dpli. Shown are retinal sections stained for GS (red) and PAI1 (green). Cell nuclei were counterstained with DAPI (blue). g–j Zoomed-in view of GS+/PAI1+ cells (right side) of the area defined by a white frame (left side). White arrowheads mark double-positive cells. k–l Histograms illustrating the mean ± SD of the number of PAI1+ cells normalized by the total number of GS+ cells expressed in percentage. Significant differences (**p < 0.01) between uninjured and injured animals were determined using a post hoc Bonferroni one-way ANOVA test (n = 12). m–n Heatmaps of pro-fibrotic genes differentially expressed genes in sorted cycling Müller cells. Data are expressed as fold-changes compared to negative controls (cycling Müller cells from uninjured retinas). o–x Detection of CTGF in Müller cells after laser induction and in uninjured retinas in both animal models. Shown are retinal sections stained for GS (red) and CTGF (green). u–x Zoomed-in view of GS+/CTGF+ cells (right side) of the area defined by a white frame (left side). White arrowheads mark double-positive cells. y–z Histograms illustrating the mean ± SD of the number of CTGF+ cells normalized by the total number of GS+ cells expressed in percentage. Significant differences (****p < 0.0001) between uninjured and injured animals were determined by using a post hoc Bonferroni one-way ANOVA test (n = 12). INL inner nuclear layer, ONL outer nuclear layer. Scale bar of the images equals 50 µm, while in the inserts corresponding to 150 µm
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