The Neuroprotective Effect Of Sodeium Pyruvate On Hypoxic-Ischemic Injury To Neonatal Rat Brain

PartⅠNeuroprotective Effect of Sodium Pyruvate on Hypoxic-ischemic Brain InjuryObjective:In this study, the protective effect of sodium pyruvate against hypoxia-ischemic brain injury was investigated in neonatal rats.Methods:Both in vivo (Hypoxia ischemia, HI) and in vitro (oxygen and glucose deprivation, OGD) models were established in this study.In vivo HI model employed unilateral (left/ipsilateral) carotid ligation in postnatal day 7 rats with exposure to 8% hypoxia for 2.5h. And in vitro OGD model employed primary cortical neurons on 7 DIV from neonatal rats subjected to oxygen and glucose deprivation for 2.5h. In vitro, cell death and cell viability were assessed using LDH and MTT kits respectively in OGD model. In vivo, Fluoro-Jade B (FJB) staining,2,3,5-triphenyltetrazolium chloride monohydrate (TTC) staining, cresyl violet (CV) staining were used to detect neuronal apoptosis and brain damage at different time points after HI injury.Results:In vitro study suggested LDH release increased and MTT levels decreased at 24 hours after OGD while SP reversed the effect in a dose dependent manner.2mM and 4mM rescued neurons from OGD induced injury to almost normal levels. In vivo study, SP at concentrations ranging from 125 to 1000 mg/kg markedly reduced hypoxic-ischemic injury to the immature brain and the maximal protection was achieved at 500 mg/kg if SP was administered at 5 min post-HI injury. Compared to the vehicle-treated group, FJB-positive neurons in the left/ipsilateral cortex at 24 h post-HI were significantly reduced by SP treatment. On brain morphology shown by cresyl violet staining, SP treatment increased the surviving brain tissues from 45.3±11.9% to 80±5.5% (cortex) and 39.2±8.7% to 67.3±9%(hippocampus).Conclusion:Both in vivo and in vitro studies showed SP took a neuroprotective effect on hypoxic-ischemic brain damage. PartⅡEffect of Sodium Pyruvate on Long-term Behavior After Hypoxic-ischemic Brain InjuryObjective:To determine whether SP can improve long-term functional recovery after hypoxic-ischemic brain injury on neonatal rats.Methods:The foot-fault test was performed at 3 weeks post-HI to assess sensorimotor function. Numbers of foot-faults with right forelimb and right hind limb per 50 steps was counted within 5 min. Morris Water maze (MWM) test were performed at 8 weeks post-HI to assess memory function. The learning trials are conducted over 4 days on rats. At the end of learning trials, a probe trial and a cued trial are given on day 5. Latency, the time spent in the platform quadrant and velocity were recorded. After MWM, tissue survival in the cortex and the hippocampus was quantified using cresyl violet staining.Results:In comparison to the sham group, rats in the vehicle-treated group showed significantly increased foot fault in right side at 3 weeks post HI. SP treatment resulted in a profound improvement in both right forelimb and hind limb foot faults. The latency in the vehicle-treated group was significantly increased, whereas SP treatment significantly reduced the latency. The time spent in the platform quadrant was reduced by 2 times in the vehicle-treated group (p<0.05) and SP treatment significantly increased the time spent in the platform quadrant (p<0.05). There is no significant difference in velocity among groups in the water maze test (p>0.05). The long-term effect of SP treatment significantly increased the cortical survival from 51%±9.9% in vehicle-treated to 87.2%±3.9% in SP-treated and the hippocampus survival from 28.7%±7.4% in vehicle-treated to 77.9%±3.6% in SP-treatedConclusion:SP improved sensorimotor and memory functions on rats subject to HI insult. PartⅢThe Mechanism of Neuroprotective Effects of Sodium Pyruvate on Hypoxic-ischemic Brain InjuryObjective:To study the relevant mechanism of neuroprotective effects of sodium pyruvate on Hypoxic-ischemic Brain InjuryMethods:ATP and ROS were measured to evaluate cerebral metabolism and oxidative stress both in vivo and in vitro. Bax, a pro-apoptotic signal in the outer mitochondrial membrane for immature neurons and cleaved Caspase-3, an apoptosis signal were detected both in vivo and in vitro. The expression of cleaved Caspase-3, total Caspase-3, phosphoralated Akt, total Akt, Bax were detected by western blot. A key survival signaling kinase, phosphoralated Akt was also tested.Results:Both in vivo and in vitro studies demonstrated that hypoxia-ischemic induced brain injury result in ROS accumulation and ATP depletion, and also increased Bax and cleaved caspase-3 expression, and decreased phosphoralated Akt. Sodium pyruvate attenuated hypoxia-ischemic induced brain injury by preventing an increase in intracellular reactive oxygen species (ROS) levels and maintaining ATP levels. Meanwhile, sodium pyruvate decreased Bax and cleaved caspase-3 expression, while activating P-Akt expression.Conclusion:Both in vivo and in vitro studies demonstrated SP protected neonatal brain from hypoxic-ischemic injury through maintaining cerebral metabolism and mitochondrial function.