Fig. 3

3D imaging shows reduced perfused vasculature network in the spinal cord of EAE mice. A Scheme represents the method followed in three steps: 1. Staining with the vascular marker lectin and PFA fixation; 2. Spinal column decalcification and iDISCO+ clearing for 3D imaging of the lumbar cord using LSFM; 3. Segmentation of the spinal cord (excluding the vertebrae, indicated with an asterisk, and surrounding tissue) and deep learning-based segmentation of the blood vessels of the spinal cord followed by 3D reconstruction, feature extraction and analysis. B 3D rendering of the lumbar spinal cord segmentation from a control mouse. C 3D rendering of the lumbar spinal cord segmentation from a EAE mouse. D Left: single slice of vessel segmentation from a representative area from a EAE mouse and a control; right: graphic representation of the features extracted and analyzed from the vessel segmentation. E–G Quantification of the vasculature length, average vessel radius and number of bifurcations from EAE and control mice (unpaired t test, n = 9–13 per group, mean ± SEM). *p < 0.05, **p < 0.01, ***p < 0.001. Scale bar in B and C, 500 μm. Abbreviations: iDISCO, Immunolabeling-enabled three-dimensional imaging of solvent-cleared organs; LSFM, light sheet fluorescence microscopy; VesSAP, Vessel Segmentation & Analysis Pipeline