Role And Mechanism Of Autophagy Pathway In Early Brain Injury After Experimental Subarachnoid Hemorrhage

BackgroudSubarachnoid hemorrhage (SAH), mostly occurring secondary to aneurysms, accounts for5%～10%of all strokes. Considerable advances have been made in endovascular techniques, diagnostic methods and surgical and perioperative management paradigms, but outcome for patients with SAH remains poor. For decades, delayed vasospasm after subarachnoid hemorrhage has traditionally been considered the most important determinant of poor outcome. However, a multicenter clinical trial (CONSCIOUS) failed to show any effect on long-term outcome, despite significant reductions in angiographic vasospasm. Epidemiological studies demonstrate that two-thirds of the deaths occur within48hours, when delayed vasospasm occurs4days to2weeks after SAH induction. Thus, it is pointed out that early brain injury is the primary cause of mortality in SAH patients.The term "early brain injury" has been generated referring to the immediate injury within72h and includes secondary events to SAH before the development of cerebral vasospasm. Recent years, the possible mechanisms of early brain injury have been systematically studied, including elevation of intracranial pressure, reduction of cerebral blood flow, suppression of cerebral perfusion pressure, fall in brain oxygenation, blood-brain barrier breakdown, brain edema, and neuronal cell death. However, definitive mechanisms underlying early brain injury remain incompletely understood to date.Autophagy is a degradative mechanism mainly involved in the recycling and turnover of cytoplasmic constituents from eukaryotic cells. During autophagy, portions of the cytoplasm are sequestered by double membraned vesicles and degraded after fusion with lysosomes for subsequent recycling. The degradation of cytoplasmic material within the autolysosome can be reused by the cell to maintain energy production and protein synthesis. Autophagy has been demonstrated to play a significant role in diverse situations, including development, cancer, infections, and neurodegeneration. Although autophagy promotes cell survival under most circumstances, a growing number of studies have demonstrated that autophagy can trigger and mediate cell death. A detailed understanding of the potential contribution of autophagy to SAH-induced early brain injury is critical for future therapeutic strategies.In the current study we applied autophagy inducer and inhibitor to manuplate the autopahgic activity and subsequently assess early brain injury after SAH. Furthermore, we investigated the apoptosis related protein and explored the molecular mechanism of autophagy, which will provide new therapy strategies and targets.Methods1. SAH model was performed using endovascular perforation technique. Adult male Sprague Dawley rats were randomly assigned into sham group, SAH6H group,24h group and72h group. Neurological deficit scores and brain edema were evaluated. Three hallmarks of autophagy, microtubule-associated protein light chain3(LC3), autophagy-related gene5(ATG5) and Beclin lwere were measured by Western blot analysis. Immunofluorescent double labeling was used to identify the cell types expressing Beclin1. Transmission electron microscopy was performed to examine the ultrastructural changes in neural cells after SAH induction.2. Adult male Sprague Dawley rats were pretreated with intracerebral ventricle infusion of the autophagy inducer rapamycin or inhibitors3-methyladenine (3-MA) before the onset of SAH. The rats were randomly assigned into sham group, SAH+vehicle group, SAH+RAP group and SAH+3-MA group. Neurological deficit scores and brain edema were evaluated. Three hallmarks of autophagy, microtubule-associated protein light chain3(LC3), autophagy-related gene5(ATG5) and Beclin1were were measured by Western blot analysis.3. Adult male Sprague Dawley rats were randomly assigned into sham group, SAH+vehicle group, SAH+RAP group and SAH+3-MA group. Apoptosis was evalutaed by TUNEL and apoptosis-related protein (Bax, Bcl-2, Cytochrome C and Caspase3) were evaluated by Western blot analysis. Transmission electron microscopy was performed to examine the ultrastructural changes in-neural cells.Results:1. Western blot analysis showed that SAH resulted in conversion of LC3-Ⅰ to LC3-Ⅱ and increased Atg5and Beclin1levels (P<0.05). These changes peaked at24h after SAH. Belin1immunoreactivity is located in both neurons and astrocytes under laser scanning confocal microscopy. Autophagosomal vacuoles and double-membrane structures were manifested in contralateral brain cortex under transmission electron microscopy.2. Rapamycin treatment significantly increased the autophagic proteins Atg5and Beclin1expressions, the ratio of LC3-Ⅱ to LC3-Ⅰ, brain edema and neurological deficits after SAH. Conversely,3-MA treatment reversed these changes and increased neurological deficits compared with SAH group. 3. Rapamycin administration decreased Bax translocation to the mitochondria, the sequent cytochrome c release from the mitochondria to the cytosol, caspase-3activity and the number of TUNEL-positive cells. Conversely,3-MA treatment reversed these changes and increased neural cell apoptosis compared with SAH group.Conclusion:1. Autophagy pathway is activated in the acute phase after SAH and peaked at24h, which suggests that autophagy may play a key role in SAH induce early brain injury.2. Enhancing autophagy with rapamycin ameliorated early brain injury following SAH, whereas inhibiting autophagy with3-MA increased early brain injury.3. Activation of autophagic pathways reduces early brain injury after SAH. This neuroprotective effect is likely exerted by anti-apoptotic mechanisms via a mitochondrial pathway.