Experimental Research Of The Neuroprotection Of α-lipoic Acid-plus And The Relevant Neuroprotective Mechanism On Early Brain Injury Following Experimental Subarachnoid Hemorrhage In Rats

objective: As a natural antioxidant, α-lipoic acid(LA) has been shown to play neuroprotective effects on subarachnoid hemorrhage(SAH). α-lipoic acid-plus(LAP), an amine derivative of LA, could protect cells against oxidant challenges via interacting with intralysosomal iron. However, whether LAP could inhibit the early brain injury(EBI) after SAH and the underlying mechanisms were still not well known. This study was designed to evaluate the potential protective effects of LA and LAP on the EBI and the underlying mechanisms in a rat model of SAH.Methods: In experiment 1, 42 Sprague-Dawley(SD) rats were randomly divided into 7 groups of 6 rats each: sham group, SAH + vehicle group, SAH + LA(100 mg/kg)group, SAH + LAP(50 mg/kg) group, SAH + LAP(100 mg/kg) group, SAH + LAP(150mg/kg), and SAH + LAP(200 mg/kg). LAP was synthesized by LA, Either LA or LAP was administered through an orogastric tube at 4 h after the induction of SAH and continued for 72 h once a day. Here, LAP was chosen in different dosages as a result of almost no literature report concerning LAP application in animal model. At the 72 th hour of operation, behavioral activity examinations were performed in all groups. all the brain cortex of six rats in each group was sacrificed for double immunofluorescence analysis. In experiment 2, 90 male SD rats were randomly divided into 5 groups of 18 rats each: sham group,SAH + vehicle group, SAH + LA(100 mg/kg) group, SAH + LAP(100 mg/kg)group, and SAH + LAP(150 mg/kg) group. The dosages of LAP were in accordance with the result of experiment 1. After 72 hours, behavioral activity examinations were performed in all groups. The brain cortex of six rats was taken for terminal deoxynucleotidyl transferase-mediated d UTP nick end labeling(TUNEL) method,immunofluorescence analysis, and iron staining. Another six rats were exsanguinated and decollated. The partial brain sample was frozen in liquid nitrogen for Western blot analysis.The remaining part was collected, the fresh brain was homogenized mixed with0.9 % normal saline(4 °C) immediately, and used for assay of oxidative stress indicators.The left six rats were for BBB injury and brain edema evaluation.Results: In experiment 1, we found that lysosome amounts in neurons decreased significantly in SAH + vehicle group, and LAP(100 mg/kg) could stabilize lysosomal membrane markedly based on lysosomal-associated membrane protein-1(LAMP-1)expression in neurons by immunofluorescence. In experiment 2, At 72 h after SAH,western blot analysis showed that the protein levels of Cathepsin B/D, caspase-3, Bax,ferritin and HO-1(heme oxygenase-1) were markedly increased in SAH+vehicle group,which were further confirmed by double immunofluorescence staining and reversed by LA and LAP treatments. In addition, compared with SAH+ vehicle group, LA and LAP treatments also reduced oxidative stress and iron deposition in brain tissue. Furthermore,LA and LAP treatments significantly ameliorated brain edema, blood–brain barrier(BBB)injury, cortical apoptosis and neurological behaviour impairment induced by SAH. Finally,it is noteworthy that, LAP exerted more significant effects than LA on these parameters as described above.Conclusions: LAP probably exerted the neuroprotection in EBI post-SAH in rats and the underlying mechanisms involved LAP inhibiting signaling pathways mediated by cathepsins via interacting with intralysosomal iron.