Journal: Neural Regeneration Research
Article Title: Fibrotic scar formation after cerebral ischemic stroke: Targeting the Sonic hedgehog signaling pathway for scar reduction
doi: 10.4103/NRR.NRR-D-24-00999
Figure Lengend Snippet: Overexpression of Shh promotes fibrotic scar formation and improves outcome after MCAO/R injury. (A) Timeline of experiments in vivo . Created with BioRender.com. (B) Schematic representation of rat brain coronal section; tested areas are demarcated by squares. Created with BioRender.com. (C, D) Western blotting analysis of Shh protein expression and quantification data in the Con, rAd-Shh, and rAd-HK groups ( n = 3). (E, F) Laser speckle contrast imaging was used to assess cerebral blood flow in MCAO/R and MCAO/R + rAd-Shh groups. (G–I) Western blotting analysis of α-SMA and Fn protein expression and quantification data in the sham, MCAO/R, MCAO/R + rAd-Shh, and MCAO/R + rAd-HK groups ( n = 3). (J) Representative immunofluorescence images of α-SMA (red)/Shh (green)- and Fn (red)/Shh (green)-positive cells in the sham, MCAO/R, MCAO/R + rAd-Shh and MCAO/R + rAd-HK groups. Scale bars: 20 µm. (K, L) Representative images at 7 days following MCAO/R injury and quantitative analysis of infarct volumes in the sham, MCAO/R, MCAO/R + rAd-Shh, and MCAO/R + rAd-HK groups ( n = 3). The infarct volume was notably smaller in the MCAO/R + rAd-Shh group than the MCAO/R and MCAO/R + rAd-HK groups. Scale bar: 1 inch. (M) Neurological scores after stroke onset in the sham, MCAO/R, MCAO/R + rAd-Shh, and MCAO/R + rAd-HK groups. (N) Immunohistochemistry of synaptophysin (SY) protein in the sham, MCAO/R, MCAO/R + rAd-Shh and MCAO/R + rAd-HK groups. Scale bar: 2 µm. (O) TUNEL staining in the sham, MCAO/R, MCAO/R + rAd-Shh and MCAO/R + rAd-HK groups. Scale bar: 20 µm. Data are expressed as mean ± SD. * P < 0.05, vs . Sham group or the Con group; # P < 0.05, vs . MCAO/R group or the rAd-Shh group; & P < 0.05, vs . MCAO/R + rAd-Shh group (one-way analysis of variance followed by Tukey’s post hoc test). Con: Control; IF: immunofluorescence; IHC: immunohistochemistry; MCAO/R: middle cerebral artery occlusion/reperfusion; rAd-HK: adenoviral control vector; rAd-Shh: recombinant adenovirus-sonic hedgehog; Shh: sonic hedgehog; SY: synaptophysin; TTC: 2,3,5-triphenyltetrazolium chloride; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling; WB: western blot.
Article Snippet: The samples were incubated with rabbit anti-synaptophysin antibody (1:100, CST, Cat#30406T) at 4°C overnight followed by poly-horseradish peroxidase and polymer Helper goat anti-rabbit IgG (ready to use, Zhongshan Goldenbridge Biotechnology, Beijing, China, Cat# PV-9001, RRID: AB_2868452) at 37°C for 10 minutes.
Techniques: Over Expression, In Vivo, Western Blot, Expressing, Imaging, Immunofluorescence, Immunohistochemistry, TUNEL Assay, Staining, Control, Plasmid Preparation, Recombinant
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