The Contribution Of Autophagy-Lysosomal Pathway To Neuronal Death And Its Underlying Mechanisms In The Rat Models Of Permanent Focal Cerebral Ischemia

Aim: To investigate activation of autophagy by permanent focal cerebral ischemia, the effects of blocking autophagy-lysosomal pathway, and evaluate relationships between its neuroprotection and the expression of LC3 and cathepsin B and Bcl-2 in rat models of permanent focal cerebral ischemia.Methods: Permanent middle cerebral artery occlusion (pMCAO) model was induced by using intraluminal filament technique in rats. Activation of autophagy in neurons in ischemic cortex was observed using electron microscopy. 3-MA(150-600 nmol) or CBI (5-10μg) was injected intracerebroventricularly (icv.) immediately after the onset of ischemia. Twenty-four hours after the occlusion, the neuroprotections of 3-MA or CBI were analyzed by scoring neurological deficits, assessing brain infarction volume with 2,3,5-triphenyl tetrazolium chloride (TTC), determining the brain water content. Western blot was employed to determine alternations in LC3/Beclin 1/cathepsin B levels in ischemic cortex 0hr/1hr/3hr/6hr/12h/24hr after the occlusion, and alternations in LC3/Beclin 1/cathepsin B/Bcl-2 levels in ischemic 3hr cortex after given 3-MA or CBI. Results: Permanent focal cerebral ischemia induced the robust formation of autophagosome and enhanced presence of secondary lysosomes, fragmented endoplasmic reticulum, swollen mitochondria. Neurological deficits, infract areas and brain water content were significantly reduced in rats treated with 3-MA (300-600 nmol) or CBI (7.5-10μg); 3-MA (600 nmol) or CBI (10μg)could significantly reduce infract volume and brain water content. The expression of LC3/Beclin 1/cathepsin B was significantly increased and peak at 3h after pMACO, but the expression of Bcl-2 was significantly decreased 3h after the occlusion. 3-MA (600 nmol) or CBI (10μg) supressed the ischemia-induced increase in LC3/Beclin 1/cathepsin B, and recovered Bcl-2 levels in the ischemic cortex.Conclusions: 1. Permanent focal cerebral ischemia increased the formation of autophagosomes and actived lysosomes; 2. 3-MA (300 nmol and 600 nmol) and CBI (7.5 and 10μg) have protective effects on cerebral ischemia induced neuronal injury in rats; 3. 3-MA and CBI's protective effects in animal models of cerebral ischemia may due to abaissement in expression of LC3/Beclin 1/cathepsin B and rehabilitation in the expression of Bcl-2.