Lithium Treatment Exhibits Neuroprotection After Neonatal Hypoxic-ischemic Brain Injury

Perinatal asphyxia-induced brain injury is one of the most common causes of morbidity and mortality in term and preterm neonates. The treatment has been purely supportive including stabilizing cardio-respiratory functions and treating convulsions. In recent years, although the clinical control studies demonstrated that hypothermia and erythropoietin therapy could provide an improvement in children with hypoxic-ischemic brain injury in the prognosis, however, it is still the top reason of child handicape. It is still a hot topic to investigate the mechanisms and potential therapy methods for neonatal hypoxic-ischemic brain injury.Lithium was discovered in 1817. It has been used as an effective treatment for bipolar disorder since 1970. Recent in vitro and in vivo studies have implicated lithium as a neuroprotective agent in preventing neuronal cell death. Growing evidences indicate that lithium is neuroprotective against a variety of insults in adult brain injury models, but no report yet in the immature brain injury model. The purpose of this study was to evaluate the effect of lithium on neonatal hypoxic-ischemic brain injury and possible mechanisms.Material and MethodNine-day-old male Wistar rats were anesthetized with isoflurane. The left common carotid artery was cut between double ligatures of prolene sutures. After the surgical procedure, the wounds were filled with lidocaine for local analgesia. The rats were returned to their cages for 1 h and then placed in a chamber perfused with a humidified gas mixture (7.8% oxygen in nitrogen) for 50 min at 36oC. Following hypoxic exposure, the rats were injected either 2 mmol/kg lithium chloride or equal volume saline i.p. immediately after the insult. Addentional injections,1 mmol/kg, were administered at 24 h intervals. The rat pups were sacrificed at 6h,24h or 72h after HI either by perfusion with 4% formaldehyde or by decapation. Paraffin embed tissue were cut with 5μm and stained with MAP2, caspase-3, AIF, cytochrome c, FBDP and LC3 antibodies. The brains were rapidly dissected out on a bed of ice. The parietal cortex and striatum were dissected out from each hemisphere and ice-cold isolation buffer was added. Homogenization was performed gently by hand. The homogenates were centrifuged to separate to nuclear pellet, cytosolic and mitochondrial fractions for enzyme assay or immunoblotting.Brain injury was evaluated by infarct volume, atrophy volume and neuropathological scoring according to MAP2 immunoblotting at 72h after HI. The infarct volume with lithium treatment (n=28) was 13.8±3.3 mm3, reduced by 44.1% compared with vehicle treatment (24.7±2.9 mm3) at 72h (P<0.05). The atrophy volume in lithium treatment (n=28) was 24.8±3.3 mm3, reduced by 41.5% compared with vehicle treatment (42.4±2.4 mm3) (P<0.05). The injury was reduced significantly in multiple brain regions including cortex (P<0.05), hippocampus (P<0.01), thalamus (P<0.001) and striatum (P<0.01). Phosphorylated glycogen synthase kinase-3β(GSK-3β) is the inactive form of GSK-3β, which enhances apoptosis. Phosphorylated glycogen synthase kinase-3βincreased significantly at 6h after HI in the ipsilateral with lithium treatment as indicated by immunoblotting (P<0.05). Extracellular signal-regulated kinases (ERK), plays an important role in cell survival. Phosphorylated ERK is the active form of ERK, which increased significantly at 6h after HI in the ipsilateral after lithium treatment (P<0.05).Fodrin is a cytoskeleton protein, which can be degraded into 150 kDa degradation products (FBDP) by calpain or 120kDa fragment by caspase3. FBDP positive cells were significantly decreased after lithium treatment, which indicated that calpain activation was inhibited by the lithium treatment.Caspase-3 positive cells and caspase-3 activity measurement as well as immunoblotting indicated Caspase-3 activity decreased at 6h after lithium treatment, but did not show significant difference between vehicle and lithium treatment. The more pronounced difference was seen at 24h after HI as indicated by activity measurement, caspase-3 positive cells counting and immunoblotting (P<0.001)Cyt c locates in the mitochondrial membrane space in the normal cells. Cyt c released from mitochondria to cytoplasm after insult which leading to caspases activation and neuronal cell death. The release of Cyt c increased with prolongation of reperfusion after HI and the release was reduced after lithium treatment as indicated by immnoblotting (P<0.05). The number of Cyt c positive cells decreased significantly in the ipsilateral hemisphere at 24h after lithium treatment compared with vehicle (P<0.05)AIF locates in the membrane space of mitochondria in the normal cells and translocated to nuclei in the injury cells leading to DNA fragmentation. This study demonstrated that lithium treatment inhibits AIF release from mitochondria and translocated to nuclei. The number of AIF positive cells decreased significantly even more pronounced in the hippocampus at 24h after lithium treatment compared with vehicle (P<0.05)The diffuse cytoplasmic staining of LC3 is a sign of autophagy, the number of LC3 positive cells increased after HI and decreased after lithium treatment and more pronounced at 72h after HI (P<0.001)Conclusion1. Lithium treatment exhibits neuroprotection after neonatal hypoxic-isc hemic brain injury2. Lithium inhibits glycogen synthase kinase-3βand activates ERK3. Lithium inhibits calpain and caspases-3 activation after HI4. Lithium inhibits the release of AIF and Cyt c from mitochondria...