Journal: Scientific Reports
Article Title: Acute administration of catalase targeted to ICAM-1 attenuates neuropathology in experimental traumatic brain injury
Figure Lengend Snippet: CCI-TBI increases BBB permeability to fibrinogen and decreases tight junction protein detection with a rescue or preservation of barrier function with anti-ICAM-1/catalase. Immunohistochemical detection of plasma protein fibrinogen in brain parenchyma at 48 hr following moderate CCI-TBI. ( A,B ) Naive and sham controls show absent fibrinogen detection in the brain parenchyma, as the BBB is healthy and intact. Absence of staining maintained at 20X. ( C ) Following CCI-TBI, BBB hyperpermeability permits the extravasation of fibrinogen into the brain tissue. Black arrowheads indicate areas of dense fibrinogen staining in the perivascular space. ( D ) Anti-ICAM-1/catalase reduces parenchymal and perivascular fibrinogen detection in the brain by 48 hrs post-CCI-TBI. ( E , F ) Anti-ICAM-1 antibody and catalase alone do not appear to reduce fibrinogen extravasation as indicated by intense fibrinogen detection in the impact site with dense staining in perivascular space. Scale bar equals 50 microns. (Top panels 2X, bottom panels 20X). ( G ) Western blot analysis of tight junction protein expression for occludin and claudin-5 in the cortex ipsilateral to the site of CCI-TBI for sham, CCI-TBI and CCI-TBI+anti-ICAM-1/catalase groups. The whole membrane was cut at 75 kDa and between 37 and 25 kDa to minimize antibody use. Membranes were separately probed for occludin and claudin-5. The membrane probed for occludin was striped, reblocked, and then probed for GAPDH. Full length blots are presented in Supplementary Figure 1 . ( H , I ) Densitometry quantification for occludin and claudin-5 normalized to GAPDH presented as mean ± SD (Ordinary one-way ANOVA with multiple comparisons occludin F = 5.779, P = 0.0399. Claudin-5 F = 17.42, P = 0.0032).
Article Snippet: All sections were incubated in primary antibody prepared in Dako Antibody Diluent either for 1 hr at RT (NeuN, Iba1, Fibrinogen, 3-NT), or O/N at 4 °C (GFAP and ICAM-1) at the following dilutions: NeuN (1:500, Covance), Iba1 (1:400, Wako Chemicals), Fibrinogen (1:400, Dako), GFAP (1:2000, Cell Signaling), 3-NT (1:1000, Abcam) and ICAM-1 (1:250, Sino Biologicals).
Techniques: Permeability, Preserving, Immunohistochemistry, Staining, Western Blot, Expressing