Inhibition of Autophagy Contributes to Ischemic Postconditioning-Induced Neuroprotection against Focal Cerebral Ischemia in Rats

<div><h3>Background</h3><p>Ischemic postconditioning (IPOC), or relief of ischemia in a stuttered manner, has emerged as an innovative treatment strategy to reduce programmed cell death, attenuate ischemic injuries, and improve neurological outcomes. However, the mechanisms involved have not been completely elucidated. Recent studies indicate that autophagy is a type of programmed cell death that plays elusive roles in controlling neuronal damage and metabolic homeostasis. This study aims to determine the role of autophagy in IPOC-induced neuroprotection against focal cerebral ischemia in rats.</p> <h3>Methodology/Principal Findings</h3><p>A focal cerebral ischemic model with permanent middle cerebral artery (MCA) occlusion plus transient common carotid artery (CCA) occlusion was established. The autophagosomes and the expressions of LC3/Beclin 1/p62 were evaluated for their contribution to the activation of autophagy. We found that autophagy was markedly induced with the upregulation of LC3/Beclin 1 and downregulation of p62 in the penumbra at various time intervals following ischemia. IPOC, performed at the onset of reperfusion, reduced infarct size, mitigated brain edema, inhibited the induction of LC3/Beclin 1 and reversed the reduction of p62 simultaneously. Rapamycin, an inducer of autophagy, partially reversed all the aforementioned effects induced by IPOC. Conversely, autophagy inhibitor 3-methyladenine (3-MA) attenuated the ischemic insults, inhibited the activation of autophagy, and elevated the expression of anti-apoptotic protein Bcl-2, to an extent comparable to IPOC.</p> <h3>Conclusions/Significance</h3><p>The present study suggests that inhibition of the autophagic pathway plays a key role in IPOC-induced neuroprotection against focal cerebral ischemia. Thus, pharmacological inhibition of autophagy may provide a novel therapeutic strategy for the treatment of stroke.</p> </div

Effects of IPOC on autophagy induction in the penumbra at 6 h and 24 h postischemia.

<p>Extracts from the sham-operated and ischemic cerebral cortex in I/R and IPOC groups were separated for immunoblotting at 6 h and 24 h postischemia. (A–C) The expression changes of LC3, Beclin 1 and p62 at 6 h and 24 h after postconditioning treatment. Autophagy is activated in both I/R and IPOC groups, and IPOC eliminated the induction of LC3/Beclin 1 and reversed the reduction of p62 at 24 h but not 6 h postischemia. Levels of β-actin protein were used as the loading control. n = 5 for each group in each time point; *<i>p</i><0.05 vs. the Sham group; ^#<i>p</i><0.05 vs. I/R-24 h group.</p

TTC staining and brain edema measurement from rat brains in different groups.

<p>Rats were treated with an i.c.v. injection of 35 pmol rapamycin 15 min before postconditioning, and then followed by 24 h reperfusion. (A) Representative infarcts stained with TTC in the Sham, I/R-24 h, IPOC-24 h, IPOC+rapa and IPOC+Veh groups 24 h after stroke. (B) Quantification of infarct size from each group at 24 h after ischemia. (C) Quantification of water content from each group at 24 h after ischemia. IPOC reduced infarct size and mitigated brain edema after stroke, whereas rapamycin partially eliminated the neuroprotection of IPOC. n = 8 for each group. *<i>p</i><0.05 vs. the Sham group; ^#<i>p</i><0.05 vs. I/R-24 h group; ^$<i>p</i><0.05 vs. IPOC+rapa group.</p

Time-dependent changes of LC3, Beclin 1 and p62 expression in the penumbra following ischemia.

<p>Rats were subjected to 30 min ischemia followed by 1, 6, 12, 24, and 48 h reperfusion. Extracts from the sham-operated and ischemic cerebral cortex were separated for immunoblotting. (A–B) Changes of LC3 and Beclin 1 expressions at different times of reperfusion. The ratio of LC3-II/LC3-I and the expression of Beclin 1 increased from 1 h to 48 h with a significant increase starting from 6 h and peaking at 24 h postischemia. (C) Changes of p62 expression at different times of reperfusion. The expression of p62 decreased significantly at 6 h and lasted until 48 h, with a maximum reduction at 24 h postischemia. Levels of β-actin protein were used as the loading control. Bar represents mean ± SEM from 5 rats in each time point. *<i>p</i><0.05 vs. the Sham group; ^#<i>p</i><0.05 vs. I/R-24 h group.</p

Rapamycin partially attenuated the autophagic inhibition effects induced by IPOC.

<p>(A) The formation of autophagosomes in cerebral cortex in the Sham, I/R-24 h, IPOC-24 h, IPOC+rapa and IPOC+Veh groups at 24 h after ischemia. Rats were perfused with 4% PFA and 1% glutaradehyde and processed for electron microscopic examination. The photomicrographs showed represent samples taken from 3 rats in each group. Arrows indicate autophagosomes. Arrowheads indicate lysosomes. Insets in the pictures were enlarged autophagosomes taken from the areas indicated by arrows. Scale bars: 1 µm. (B–D) Western blot analysis of the expressions of LC3, Beclin 1 and p62 in the penumbra in the Sham, I/R-24 h, IPOC-24 h, IPOC+rapa and IPOC+Veh groups at 24 h after ischemia. Rapamycin upregulated the expressions of LC3/Beclin 1 and downregulated p62 when compared with that in IPOC group. Levels of β-actin protein were used as the loading control. n = 5 for each group. *<i>p</i><0.05 vs. the Sham group; ^#<i>p</i><0.05 vs. I/R-24 h group; ^$<i>p</i><0.05 vs. IPOC+rapa group.</p

Cellular localization of LC3 and Beclin 1 after postconditioning.

<p>(A) Cellular localization of LC3 after postconditioning. (B) Cellular localization of Beclin 1 after postconditioning. Rats were fixed with 4% PFA, dehydrated in alcohol, and the brain sections of the ipsilateral hemisphere were double-labeled with the anti-LC3 antibody (green) and DAPI (blue) or anti-Beclin 1 antibody (red) and DAPI (blue). The LC3 immunoreactivity was weaker in IPOC-24 h and IPOC+Veh groups than that in I/R-24 h group. When rapamycin was administrated before postconditioning, the LC3 immunoreactivity was relatively enhanced, but weaker than that in I/R-24 h group. Similarly, rapamycin plus postconditioning treatment increased Beclin 1 immunoreactivity, but not as strong as that in I/R-24 group. n = 3 in each group. Brain sections were viewed under a fluorescence microscopy. Insets in the pictures were amplified immunofluorecence staining taken from the areas indicated by arrowheads. Scale bar: 400 µm.</p