Fig 2: Immunohistochemical staining of CARMIL3 in human infarct brains and stroke-free autopsied brains. (A–C) CARMIL3 expression in human infarct brain tissues. In the region most sever ely infarcted (right lower part of A), there were fewer neurons, with all the residual neurons strongly positive for CARMIL3. In the area surrounding the sever ely infarcted region, most neurons had strong CARMIL3 staining in their nuclei and cytoplasm. In the region with the least infarction severity (left upper part of A), most neurons had normal nuclear morphology with faint CARMIL3 staining mainly in the cytoplasm. (D) Relatively low CARMIL3 expression in the neurons of stroke-free autopsied brains. Scale bar = 20 μm.

Fig 3: Association of CARMIL3 with oxidative stress. (A) Representative images of double-labeling for CARMIL3 and 8-OHdG in the T2(+), mismatch, and control regions. CARMIL3(+) neurons (arrows) tended to have higher 8-OHdG intensity than CARMIL3(−) neurons (arrowheads). (B,C) 8-OHdG(+) neuron density (B) and 8-OHdG(+) neuron ratio [the number of 8-OHdG(+) neurons divided by total number of neurons] (C) in these three regions. The 8-OHdG(+) neuron ratios were significantly different among the three regions (p = 0.027). 8-OHdG(+) neuron ratios, but not densities, were higher in the T2(+) areas than in the contralateral areas (*p < 0.05). (D) Quantitative comparison of neuronal 8-OHdG intensity between CARMIL3(+) and CARMIL3(−) neurons. CARMIL3(+) neurons had higher 8-OHdG intensities than CARMIL3(−) neurons in all three regions (all p < 0.05). (E) Correlation between 8-OHdG and CARMIL3 intensities of all neurons in one image of each region in four rats. There were significant associations between neuronal 8-OHdG and CARMIL3 intensities in all three regions (all p < 0.001). There was a significant difference in the regression slopes between the mismatch and control regions (p = 0.003). Scale bar = 10 μm.

Fig 4: Expression pattern of MAP2 in the CARMIL3(+) profiles. (A) Double labeling of CARMIL3 and MAP2 in the T2(+), mismatch, and control regions after pMCAO. The CARMIL3(+) profiles were colocalized with MAP2, indicating that CARMIL3(+) profiles were located on neurites. CARMIL3(+) neurites (arrows) were thicker than CARMIL3(−) neurites (arrowheads) in the mismatch region. (B) CARMIL3(+) neurite ratio [the number of CARMIL3(+) neurites divided by that of all identifiable neurites] in the three regions. There were significantly higher ratios of CARMIL3(+) neurites in the mismatch regions compared with control areas (p = 0.035). (C) Comparison of the neurite diameters of CARMIL3(+) and CARMIL3(−) neurites. CARMIL3(+) neurites had significantly larger neurite diameters than CARMIL3(−) ones in mismatch regions (p = 0.036). *p < 0.05. N = 4 rats in each group. Scale bar = 10 μm.

Fig 5: Immunofluorescence staining of CARMIL3 in rat brains after pMCAO for 3 days. CARMIL3 expression at T2(+) and contralateral regions in the rat brains after pMCAO for 3 days. There were only minimal mismatch areas, thus they were not assessed. Many neurons in the T2(+) regions were swelling or deformed. CARMIL3 was expressed in more neurons at T2(+) regions compared with the contralateral regions, although the signal intensities of CARMIL3 in all neurons at this stage were very low compared with those in the T2(+) regions within 4.5 h after pMCAO [comparison of CARMIL3(+) count: p = 0.011]. Scale bar = 10 μm.