Fig 2: The effect of CR3 activation on oxidative stress after ICH. (A, C) Representative microphotographs and quantitative analysis of relative fluorescence intensity of DHE in perihematomal area and ipsilateral cerebral cortex. n = 4. **P <0.01, ***P <0.001 by one-way ANOVA. (B) Representative microphotographs of immunofluorescence double staining showing the localization of DHE with Iba-1 (microglia marker), NeuN (neuronal marker), and GFAP (astrocyte marker) in the perihematomal area after ICH. n = 4. (D) Representative microphotographs of immunofluorescence double staining showing the localization of DHE with Iba-1 (microglia marker), NeuN (neuronal marker), and GFAP (astrocyte marker) in the ipsilateral cerebral cortex after ICH7d. n = 4. Scale bar = 100 µm. Data are represented as mean ± SD.

Fig 3: CORM-3 ameliorates HSR-induced neuronal death in the hippocampus. (A) Representative photomicrographs of NeuN/PI/DAPI staining (NeuN, green; PI, red; DAPI, blue) demonstrating neuronal death in the hippocampal tissue induced by the indicated stimuli. Scale bar, 50 µm. (B) Percentages of neuronal death in the hippocampal tissue induced by the indicated stimuli. (C) LDH content in the hippocampal tissue induced by the indicated stimuli. Data are presented as the mean ± SD (n=6 per group). *P<0.05 vs. Sham; $P<0.05 vs. HSR vehicle and HSR iCORM-3. CORM, carbon monoxide-releasing molecule; HSR, hemorrhage shock and resuscitation; iCORM-3, inactive CORM-3; PI, propidium iodide; LDH, lactate dehydrogenase.

Fig 4: H&E, Nissl and FJC staining in brain tissue of mice subjected to tMCAO after administration of an S1PR3-specific antagonist. (A) H&E staining in brain tissue of mice subjected to tMCAO after administration of an S1PR3-specific antagonist. Vacuolated neuropil in brain tissue (*). The black arrow points to the glial cells and the white arrow points to the nucleus of neurons that were reduced and trachychromatic (?), n = 4. Scale bar = 50 µm. (B) Nissl staining in brain tissue of mice subjected to tMCAO after administration of an S1PR3-specific antagonist. The black arrow points to neurons with Nissl bodies. The white arrow points to neurons with absent Nissl bodies, n = 4. Scale bar = 50 µm. (C,D) DAPI (blue)/FJC (green)/NeuN (red) Representative immunofluorescence images and statistical results of mouse brain tissue slices after tMCAO (n = 4). Data are presented as the mean ± SEM. P-values were determined by ANOVA followed by the Tukey post hoc test, **P < 0.01; ***P < 0.001; and ns, not significant. Scale bar = 100 µm.

Fig 5: p-SFKs are expressed mainly in spinal microglia, very few in astrocytes but not in neurons of rats with NP. Double immunofluorescence staining (200×) in ipsilateral L4/5 spinal dorsal horn between p-SFKs (red: (a), (d), and (g)) and Iba-1 (a microglia marker, green: (b)); GFAP (an astrocyte marker, green: (e)); and NeuN (a neuronal marker, green: (h)) shows that p-SFKs are located mainly in microglia (c), very few in astrocytes (f) but not in neurons (i) of spinal dorsal horn five days after surgery. Scale bars (a)–(i) = 100 µm (n = 6/group). NeuN: neuronal specific nuclear protein; GFAP: glial fibrillary acidic protein; Iba-1: ionized calcium-binding adapter molecule 1; NP: nucleus pulposus; p-SFK: phosphorylated src-family kinase.