RIPC provides neuroprotection against ischemic stroke by suppressing apoptosis via the mitochondrial pathway

Abstract

Ischemic stroke is a common disease with high morbidity and mortality. Remote ischemic preconditioning (RIPC) can stimulate endogenous protection mechanisms by inducing ischemic tolerance to reduce subsequent damage caused by severe or fatal ischemia to non-ischemic organs. This study was designed to assess the therapeutic properties of RIPC in ischemic stroke and to elucidate their underlying mechanisms. Neurobehavioral function was evaluated with the modified neurological severity score (mNSS) test and gait analysis. PET/CT was used to detect the ischemic volume and level of glucose metabolism. The protein levels of cytochrome c oxidase-IV (COX-IV) and heat shock protein 60 (HSP60) were tested by Western blotting. TUNEL and immunofluorescence staining were used to analyze apoptosis and to observe the nuclear translocation and colocalization of apoptosis-inducing factor (AIF) and endonuclease G (EndoG) in apoptotic cells. Transmission electron microscopy (TEM) was used to detect mitochondrial-derived vesicle (MDV) production and to assess mitochondrial ultrastructure. The experimental results showed that RIPC exerted significant neuroprotective effects, as indicated by improvements in neurological dysfunction, reductions in ischemic volume, increases in glucose metabolism, inhibition of apoptosis, decreased nuclear translocation of AIF and EndoG from mitochondria and improved MDV formation. In conclusion, RIPC alleviates ischemia/reperfusion injury after ischemic stroke by inhibiting apoptosis via the endogenous mitochondrial pathway.

Figure 1

Histopathological structure of the brain tissue in the different groups. The brain tissue around the ischemic area in the RIPC + I/R group was more intact and compact than that in the I/R group. Bar = 100 μm.

Figure 2

Behavioral evaluation by the mNSS test. Compared with that of the I/R group, the score of the RIPC + I/R group was significantly reduced. ****P < 0.0001.

Figure 3

Changes in gait parameters in the different groups. (a) Representative footprints of the rats in the different groups obtained by the analysis software. Green, FR (front right foot); dark blue, FL (front left foot); red, RR (rear right foot); sky blue, RL (rear left foot). (b–p) The data show significant differences in stance time, swing time, stride length, running speed, foot pressure, print area and stride number among the 4 groups. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 4

Results of ¹⁸F-FDG micro-PET/CT scans in the different groups. (a) Representative coregistered PET/CT (left) and PET (right) scan images, including axial, coronal and sagittal (R) images, of the mouse brains. (b) Quantitative analysis of glucose metabolism in ischemic foci. (c) Infarct volumes in the I/R and RIPC + I/R groups. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 5

Apoptotic cells were analyzed by TUNEL staining. (a) Apoptotic cells were labeled with fluorescein isothiocyanate (red), and all nuclei were stained with DAPI (blue). (b) Percentage of apoptotic cells relative to total cells. **P < 0.01, ****P < 0.0001. Bar = 50 μm.

Figure 6

The protein expression levels of COX IV and HSP60 were examined by Western blotting. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 7

The colocalization of AIF and EndoG proteins in apoptotic cells was determined by TUNEL and immunofluorescence staining. (a) Immunofluorescence staining for AIF (green), EndoG (green) and DAPI (blue) and staining of apoptotic cells (red). (b,c) Proportions of TUNEL-positive cells exhibiting colocalization of AIF and EndoG that translocated from mitochondria to nuclei relative to the total TUNEL-positive cells in the different groups. ****P < 0.0001. Bar = 50 μm.

Figure 8

Formation of MDVs. The entire process of MDV formation (arrows) was observed from germination to exfoliation (a–d). Bar = 100 nm.

Figure 9

Identification of mitochondrial budding events by TEM. Mitochondrial buds are marked with arrows, and mitochondrial vacuoles are marked with asterisks (a–d represent the sham, RIPC, I/R and RIPC + I/R groups, respectively). Bar = 500 nm.