Autophagy Elicits Neuroprotection at the Subacute Phase of Transient Cerebral Ischaemia but Has Few Effects on Neurological Outcomes After Permanent Ischaemic Stroke in Rats

Abstract

Objective

Autophagy was prominently activated by cerebral ischaemia. This study was to investigate the exact role of autophagy in ischaemic stroke.

Methods

Two rat models of transient middle cerebral artery occlusion (tMCAO) and permanent MCAO (pMCAO) were prepared. The brain tissues in the penumbra were obtained to observe the dynamic variations of autophagy activity with Beclin1 and LC3 antibodies by Western blotting. At the characteristic time points, when autophagy activity was markedly elevated or reduced, the autophagy activation signaling was intervened with rapamycin and 3-methyladenine, respectively. Thereafter, key proteins in the autopahgic/lysosomal pathway were detected with the antibodies of LC3, p62, ubiquitin, LAMP-1 and cathepsin B. Meanwhile, TTC staining, neurological score and immunofluorescence were performed to evaluate brain infarct volume, neurological deficit and neuron survival, respectively.

Results

Both Beclin1 and LC3 expression levels were remarkably altered at 6 h, 12 h, 2 days and 7 days after tMCAO. Interestingly, the dynamic changes of autophagy activity following pMCAO were identical to those after tMCAO. Neither autophagy induction nor autophagy inhibition was able to ameliorate the pMCAO-induced neurological injury due to lysosomal dysfunction, as indicated by low levels of LAMP-1 and cathepsin B, accompanied with the accumulation of LC3-II, ubiquitin and insoluble p62. Comparatively, autophagy induction elicited overt neuroprotection at 2 and 7 days after tMCAO, and this neuroprotection might be elicited by the enhancement of autophagy flux.

Conclusion

Our study suggests that autophagy confers neuroprotection at the subacute phase of tMCAO but has few effects on neurological outcomes after pMCAO.

Keywords

permanent cerebral stroke / transient cerebral ischaemia / autophagy induction / autophagy inhibition / neuroprotection

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