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. 2016 Jul;11(7):1108-14.
doi: 10.4103/1673-5374.187045.

Dynamic changes in neuronal autophagy and apoptosis in the ischemic penumbra following permanent ischemic stroke

Yi-Hao Deng  1 , Hong-Yun He  1 , Li-Qiang Yang  1 , Peng-Yue Zhang  1
Affiliations

Dynamic changes in neuronal autophagy and apoptosis in the ischemic penumbra following permanent ischemic stroke

Yi-Hao Deng et al. Neural Regen Res. 2016 Jul.

Abstract

The temporal dynamics of neuronal autophagy and apoptosis in the ischemic penumbra following stroke remains unclear. Therefore, in this study, we investigated the dynamic changes in autophagy and apoptosis in the penumbra to provide insight into potential therapeutic targets for stroke. An adult Sprague-Dawley rat model of permanent ischemic stroke was prepared by middle cerebral artery occlusion. Neuronal autophagy and apoptosis in the penumbra post-ischemia were evaluated by western blot assay and immunofluorescence staining with antibodies against LC3-II and cleaved caspase-3, respectively. Levels of both LC3-II and cleaved caspase-3 in the penumbra gradually increased within 5 hours post-ischemia. Thereafter, levels of both proteins declined, especially LC3-II. The cerebral infarct volume increased slowly 1-4 hours after ischemia, but subsequently increased rapidly until 5 hours after ischemia. The severity of the neurological deficit was positively correlated with infarct volume. LC3-II and cleaved caspase-3 levels were high in the penumbra within 5 hours after ischemia, and after that, levels of these proteins decreased at different rates. LC3-II levels were reduced to a very low level, but cleaved caspase-3 levels remained high 72 hours after ischemia. These results indicate that there are temporal differences in the activation status of the autophagic and apoptotic pathways. This suggests that therapeutic targeting of these pathways should take into consideration their unique temporal dynamics.

Keywords: LC3-II; autophagy; apoptosis; brain injuries; cleaved caspase-3; dynamic variations; immunofluorescence; infarct volume; nerve regeneration; neurological deficit score; permanent ischemic stroke; western blotting.

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Conflict of interest statement

Conflicts of Interest: None declared.

Figures

Figure 1
Figure 1
Effect of ischemic stroke on the neurological deficit score. Two hours following MCAO, the rats displayed a significant neurological deficit. The neurological deficit score was notably increased 5 hours after ischemia. **P < 0.01, vs. control, 2-h, 3-h and 4-h groups; ##P < 0.01, vs. 5-h and 6-h groups. Data are expressed as the mean ± SEM (n = 6; one-way analysis of variance and Dunnett’s test). MCAO: Middle cerebral artery occlusion; h: hour(s).
Figure 2
Figure 2
Effects of ischemic stroke on cerebral infarct volume. (A) The infarct volume was evaluated by TTC staining at 1, 2, 3, 4, 5, 6, and 12 h following permanent ischemic stroke. White and red regions indicate infarcted and normal tissue, respectively. (B) The infarcted area expands progressively over time. At 12 h, the infarct volume reaches a maximum and remains at this high level between 24 and 72 h after MCAO (the data for the last three time points is not shown). *P < 0.05, vs. control, 1-h, 2-h and 3-h groups; **P < 0.01, vs. control, 1-h, 2-h, 3-h and 4-h groups; ##P < 0.01, vs. 5-h and 12-h groups. Data are expressed as the mean ± SEM (n = 6; one-way analysis of variance and Dunnett’s test). MCAO: Middle cerebral artery occlusion; TTC: triphenyltetrazolium chloride; h: hour(s).
Figure 3
Figure 3
Effects of ischemic stroke on LC3-II protein levels in the rat ischemic penumbra (western blot assay). (A) Representative western blot assay. (B) Densitometric quantification showing that LC3-II protein was significantly expressed 1 h following stroke compared with the sham control group. LC3-II protein levels were stable from 2 to 5 h after permanent MCAO. Levels rapidly declined starting at 6 h, and were similar to those in the control group at 72 h. *P < 0.05, vs. 1-h and 12-h groups; **P < 0.01, vs. 12-h and 48-h groups; ##P < 0.01, vs. 24-h and 48-h groups. Data are expressed as the mean ± SEM (n = 6; one-way analysis of variance and Dunnett’s test). h: Hour(s).
Figure 4
Figure 4
Effects of ischemic stroke on cleaved caspase-3 levels in the ischemic penumbra (western blot assay). (A) Representative western blot. (B) Densitometric quantification showing that cleaved caspase-3 protein levels gradually increased between 1 and 5 h, reaching the highest level at 5 h after MCAO. The levels declined from 6 to 72 h after MCAO, but were persistently high compared with the sham control, indicating that the apoptotic rate remained high 72 h after stroke. *P < 0.05, vs. 1-h, 2-h, 3-h and 4-h groups; **P < 0.01, vs. 5 h and 6 h groups; ##P < 0.01, vs. 12 h and sham control groups. Data are expressed as the mean ± SEM (n = 6; one-way analysis of variance and Dunnett’s test). h: Hour(s).
Figure 5
Figure 5
Dynamic changes in autophagy and apoptosis after stroke. Within 5 h of permanent ischemic stroke, the LC3-II/cleaved caspase-3 ratio displayed a slight decreasing trend. From 12 to 72 h following ischemia, the ratio declined, showing that autophagy was reduced during this stage. At 72 h after stroke, more neurons underwent apoptosis, but not autophagy. *P < 0.05, vs. 1-h, 2-h and 12-h groups; **P < 0.01, vs. 24-h and 72-h groups. Data are expressed as the mean ± SEM (n = 6; one-way analysis of variance and Dunnett’s test). h: Hour(s).
Figure 6
Figure 6
Effects of ischemic stroke on the immunoreactivities for LC3-II and cleaved caspase-3 in the ischemic penumbra (immunofluorescence staining, × 200). (A, B) Representative fluorescence microscopic images of immunofluorescence staining for LC3-II and cleaved caspase-3. Abundant positive cells (including LC3-II and cleaved caspase-3-positive cells) were found in the penumbra 1–12 h following ischemic stroke; however, a few positive cells were detected in the corresponding area in the control group (C, D). Both the percentage of LC3-II-positive cells (C, **P < 0.01, vs. control, 3-h and 6-h groups; ##P < 0.01, vs. 6-h and 72-h groups) and cleaved caspase-3-positive cells (D, *P < 0.05, vs. 1-h group; **P < 0.01, vs. control group; ##P < 0.01, vs. 6-h and 72-h groups) reached the highest level 5 h after permanent ischemic stroke. Thereafter, the ratio of LC3-II-positive cells gradually declined, and remained at a very low level 72 h after MCAO. Comparatively, the percentage of cleaved caspase-3-positive cells only slightly decreased, and remained high between 12 and 72 h after MCAO. The arrow indicates LC3-II or cleaved caspase-3-positive cells (red-colored). Data are expressed as the mean ± SEM (n = 6; one-way analysis of variance and Dunnett’s test). MCAO: Middle cerebral artery occlusion; h: hour(s).
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