Additional file 1 of Pathological hemodynamic changes and leukocyte transmigration disrupt the blood–spinal cord barrier after spinal cord injury
Creators: name: Zhou, Rubing
name: Li, Junzhao
name: Chen, Zhengyang
name: Wang, Ruideng
name: Shen, Yin
name: Zhang, Rong
name: Zhou, Fang
name: Zhang, Yong
Publisher: name: figshare
Year: 2024
Subjects: subject: Biochemistry
subject: Physical Sciences not elsewhere classified
subject: Medicine
subject: Cell Biology
subject: Molecular Biology
subject: Neuroscience
subject: Physiology
subject: FOS: Biological sciences
subject: Biotechnology
subject: Chemical Sciences not elsewhere classified
subject: Information Systems not elsewhere classified

description: Additional file 1: Fig. S1. a A homemade imaging installation. The chamber was cemented by dental cement after using the clamps to hold the vertebral column of the mouse. This installation was used for rapid imaging of the injury site before and after SCI. b A schematic timeline of in vivo imaging. c Temporal explanation of the steps of BSCB disruption after SCI. d Representative maximum projection about the extravasation of 40kDa TRITC-dextran 30 min post-SCI. The frames were manually drawn to quantify the extravascular fluorescence density leaked from dSVor dAVin ImageJ. e Quantification of fluorescence intensity extravasated from dSV in the epicenter from Fig. 1c. Data are the mean ± SEM, n.s. no significance; nested, general estimated equation. f Representative immunohisto-chemistry images of serum immunoglobulinextravasation near the epicenter. A cystappeared 4 hours post-SCI. Scale bar = 1 mm. g The membrane component obtained by the Mem-PER kitwas detected by ATPase and β-actin antibodies. ATPase is a pump of hydrogen and potassium ions located on the membrane and was used as a membrane protein loading control; the β-actin was used as a plasma protein loading control. The loading lysis was diluted to 10-3 μg/μl by the optimized detection concentration for ATPase. Fig. S2. Representing graph view about chemiluminescence intensity of junctional proteins measured by automated capillary western assay 4 hours post-SCI. a claudin-5; b occludin; c ZO-1; d tricellulin; e CD31; f ATPase; g vinculin. Vinculin was used as a loading control for ZO-1, and ATPase was used as a loading control for the other proteins. The expression of proteins was calculated automatically by the peak-area methodin Compass software. Fig. S3. Gap formation in TJs at non-capillaries small vessels post-SCI. a–c Representative images of claudin-5, occludinand ZO-1 at non-capillary small vessels post-SCI. The same images were merged with CD31. Scale bar = 25 μm. The gaps in TJs were detected on these vessels. TJs on small vessels at different time-point post-SCI with NT or TTM.; cldn-5 on spinal cord 15, 30, 60 minutes post-SCI, total 58, 63, 68 vessels respectively). TJs at small vessels manipulated with NONOate 4 hours post-SCI..TJs at the small vessels 24 hours after LPS injection or manipulated with CXCR2 antagonist SB225002 4 hours post-SCI. The gap was measured by the line intensity methodin LasX software. The discontinuity characterized as a more than 70% decrease in fluorescence intensity&lt; 0.05, nested, one-way ANOVA; the Mann–Whitney test. Fig. S5. a Representative images about the PE-conjugated GR-1 labeled leukocytes were activated at the vascular junction between the dSV and spinal radial vein post-SCI under a CCD cameraand two-photon microscope. Scale bar: left = 250 μm, right = 100 μm. Many leukocytes quickly got deformed or connected to other leukocytes. b Density of leukocytes rolling on the lumen of dSV in epicenter and penumbra from. c Density of leukocytes adhered on the lumen of dSV in epicenter and penumbra from Fig. 6c. d, e Images about the most extreme case of YFP-positive cells adhered to the lumen. The ROI under CCD cameraand under two photon microscope. The extravascular LysM positive cells decreased over time, and the outline of dAVs gradually became invisible. Scale bar: left = 150, right = 200 μm. f YFP labeled leukocytes and/or microglia in epicenter and penumbra from Fig. 6e. g, h Density of leukocytes rolling or adhering on the lumen of dSV. i Density of YFP labeled leukocytes and/or microglia in the extravascular area. The vasoactive agent NONOate does not have an apparent effect on leukocyte transmigration. Data are the mean ± SEM; n.s. no significance, #P &lt; 0.05, ##P &lt; 0.01, ####P &lt; 0.0001. nested, Kruskal–Wallis test and two-way ANOVA. Fig. S6. a Representative images of GR-1 positive leukocyteson cross-sections 6 hours post-SCI. Scale bar = 500 μm. b Quantification of GR-1 positive leukocytes in multiple spinal segments 6 hours post-SCI. The value was adjusted by the number of DAPI positive nuclei. c A representative image about GR-1 labeled neutrophilsreleased nuclear DNA/histone complexand cytoplasmic neutrophils elastaseinto nearby tissue 4 hours post-SCI. The extracellular neutrophils elastase formed a spiky-like structure, and the cell-free DNA/histone formed a cloudy-like structure. Scale bar = 25 μm. d Representative images about AlexaFluor 700 conjugated GR-1 labeled leukocytesactivated after LPS injection. The plasma was labeled by 40 kDa FITC-dextran. The leakage related to leukocytes transmigration was less at tubular dSVthan at vascular junction between the dSV and spinal radial vein in. Scale bar: left = 250 μm, right = 100 μm. Data are the mean ± SEM; *P &lt; 0.05, *P &lt; 0.0001. nested, one-way ANOVA. Table S1. Antibodies or fluorescence materials used in this study.