Extended Data Fig. 9: Intra-cerebrospinal fluid injection of CHIT1 induces accelerated MN senescence in the spinal cords in nude mice.
From: CHIT1-positive microglia drive motor neuron ageing in the primate spinal cord
a, Schematic diagram showing the time course of intra-cerebrospinal fluid injection of the vehicle or CHIT1 in 2-month-old nude mice. Every other day injection per mouse. The dose of CHIT1 was the same as that used for the C57BL/6 mice. b, Immunofluorescence staining of p-SAMD2 and ChAT in spinal cords from vehicle- or CHIT1-treated nude mice. Representative images (left) and the mean intensity of p-SMAD2 fluorescence per (ChAT+) MN (right) are shown. The arrows indicate the MNs. Scale bars, 50 μm. c, Immunofluorescence staining of ChAT in spinal cords from vehicle- or CHIT1-treated nude mice. Representative images (left) and the number of ChAT+ MNs in the spinal vGM (right) are shown. The dashed lines show the boundary of the vGM region. Scale bars, 100 μm. d, SA-β-Gal staining of the spinal vGM sections from vehicle- or CHIT1-treated nude mice. Representative images (left) and the percentage of SA-β-Gal-positive areas in total spinal vGM area (right) are shown. Scale bars, 100 μm. e-f, Co-staining for SA-β-Gal and ChAT in the spinal cords of vehicle- or CHIT1-treated nude mice. Representative images are shown in (e). The percentage of SA-β-Gal+ MNs in total MNs in vehicle- or CHIT1-treated nude mice and the percentage of SA-β-Gal+ MNs or SA-β-Gal+ non-MN cells in total SA-β-Gal+ cells in CHIT1-treated spinal vGM are shown in (f). Scale bars, 100 μm. g, Immunofluorescence staining of lamin B2 and ChAT in spinal cords from vehicle- or CHIT1-treated nude mice. Representative images (left) and the mean lamin B2 intensity per (ChAT+) MN (right) are shown. The arrows indicate the MNs. Scale bars, 20 μm. h, Grip strength test of the four limbs of nude mice treated with vehicle or CHIT1. Data (b-h) are shown as the mean ± s.e.m. P values were calculated using two-tailed unpaired t-tests. Three discontinuous cross-sectional slices per mouse (b-g) and n = 9 nude mice per group (a-h).
