Fig. 2

RIPC upregulates SIRT3 expression and the neuroprotective effect of RIPC is reversed in KO mice. A, B Western blot was used to detect the expression of SIRT3 at different time points after spinal cord I/R. Relative expression of SIRT3 was normalized to the level of control. N = 5/group, *p < 0.05, **p < 0.01, compared to the former timepoint. C, D Western blot was used to detect the expression of SIRT3 in different groups. Relative expression of SIRT3 was normalized to the level of the sham group (n = 5/group). E, F Immunofluorescence was used to detect the expression of SIRT3 (green) in neurons (red) in different groups. Nuclear was stained with DAPI (in blue). Scale bar = 100 μm. Relative fluorescence intensity was normalized to the level of the sham group (n = 5/group). G The KO mice were functionally scored up to 3d postinjury using BMS (n = 5/group). H, I Representative footprint images of KO mice on the third-day post-injury and quantification of the stride length and width. Blue: frontpaw print; red: hindpaw print (n = 5/group). J, K MEP analysis was used for electrophysiological assessment at day3 postinjury and quantification of the peak-to-peak MEP amplitudes in KO mice (n = 5/group). L, M Representative images of Nissl staining of neurons in the anterior horn of the spinal cord and quantification of the number of integrated Nissl bodies per section from KO mice (n = 5/group). Scale bar = 100 μm. N, O Representative images of TUNEL-positive apoptotic cells (in red) in spinal cord sections and quantification of the number of apoptotic cells in each group from KO mice at day 3 postinjury (n = 5/group). Neuron was stained with NeuN (in green) and Nuclear stained with DAPI (in blue). Scale bar = 100 μm. P MnSOD, GSH, CAT, and MDA were measured to reflect the level of oxidative stress in each group of KO mice (n = 5/group). Statistical analysis: mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001