So, this animal will underwent cardiorest and then treated with GBC and all neural stem cells and evaluated was throughout 72 hours after resuscitation. And from the left side, we can see more inactive morphology in microglia were founded in the human neural stem cell treated animals. Human neural stem cell treated animal restored the maximum resting microglia, which is type 1 here and minimal the activated, but non-phagocytic microglia, which is type 3 compared other two groups, which is GBC group and all control group. Additionally, quantitative analysis of immunofluorescence and the western blots demonstrated the express of TLR4, NLRP3, inflammatory pathway-related protein, which includes TLR4, NF-GAP-beta, NLRP3, CAPASE-1, and IL-1-beta were significantly suppressed by both treatments and were more significantly suppressed by human stem cell therapies. Inflammation due to recruitment of dimensional microglia cells exacerbates brain injury and worsens neurological functional outcome, which also leads to our potential therapeutic target neural inflammation. S1P receptors have been linked to microglia activation and contributes to ischemic brain injury. JTE013 as S1P2 antagonist showed the neuroproductive function in a stroke model. However, its effect in global ischemic for cardiac arrest is unknown. Well, since there's a minimal therapeutic strategy available after cardiac arrest that targeting inflammatory pathway. So, in this study, we want to evaluate the effect of JTE013 targeting neural inflammation pathway and evaluate its effect. First, we evaluated JTE's effect in-vitro using an OGD model for three hours. We treated with normal cell or normal media or JTE, and then we evaluated the axon loss, myelin loss, and the synaptic loss by immunocytochemistry assessment. And then, in an in vivo model, we're using asphyxial cardiac arrest model. So, the injection will be three hours after return or response as circulation. And this one, we can see here, we started monitoring EEG throughout the end of treatment, which is the end point is 72 hours after resuscitation. And NDS will be evaluated every day. We start rest divided by two group treated with JTE013 or PBS three hours after resuscitation via ICV injection in an eight minutes experimental asphyxial cardiac arrest model. Three days after cardiac arrest resuscitation, EEG, quantitative EEG, will be monitored. And the neurological functional outcome by NDS or immunofluorescent staining will be quantified to evaluate the therapeutic effects of JTE013. Immunocytochemistry was performed to analyze axon loss using NF200, myelin loss using MBP, and the synaptic loss using Synapsin-1. In vitro study shows the quantified cell positive area and intensity of antibody fluorescence showed more cell positive areas and higher intensity of NF200, MBP, and synapsin in neurons cultured with JTE013 compared to the control medium. Neuro-deficit score NDS reviewed improved neurological outcome of JTE013 treated rats compared to the control condition rats with an increased survival rate among the JTE013 treated animal. In conclusion, our in vitro model shows JTE013 protects neuron, myelination, and synapses in in vitro ODD model. In vivo study shows rats that underwent cardiac arrest had a better neurological functional outcome when treated with JTE013. Thus, JTE013 proposes a potential neuroprotective intervention for ischemic brain injury induced by cardiac arrest. I appreciate the hard work from our research team in my lab and this R1 support from NIH. Thank you for your attention.

neuroinflammation

ischemic brain injury

JTE013

neuroprotective treatment

cardiac arrest