| Weapons exploded under water can cause serious damage to the combatants,which is known as underwater explosive injury.Underwater explosive injury is one of the major types of injury in the naval war and also the focus of military medical research around the world.Nowadays,there are numerous reports about the chest and abdomen injuries caused by underwater explosion.However,few studies have been conducted regarding the characteristics and mechanisms of traumatic brain injury(TBI)caused by underwater explosion.As the headquarters of human body,brain has complex structures and functions,injuring of which would lead to headache,dizziness,decreased reaction speed,temporary loss of consciousness,or even coma,paralysis,and death.The mechanism of TBI is relatively complex and underwater explosion test is difficult to implement.Moreover,there is no experimental method and experience for us to follow.Therefore,it is urgent to explore the characteristics and mechanism of TBI caused by underwater explosion.As an effective component of Cordyceps sinensis,cordycepin(3'-deoxyadenosine)has a wide range of pharmacological functions such as anti-inflammation,anti-tumor,inhibition of apoptosis,scavenging of free oxygen radicals and so on.Moreover,it is demonstrated that cordycepin has neuroprotective effect.It can inhibit the release of inflammatory factors,alleviate the infiltration of inflammatory cells and macrophages,and maintain the integrity of blood-brain barrier after injury.However,the mechanism by which cordycepin exerts its neuroprotective effect is unclear and needs further studies.In the current study,we built a stable underwater explosion-induced TBI model of rats at first,and the features of this type of TBI were studied.Then the protective effect of cordycepin on TBI induced by underwater explosion was confirmed.Lastly,we explored the protective mechanism of cordycepin involving TLR4/My D88 pathway on TBI.We hoped the works we did would provide some valuable information for the treatment of such brain injury.Section I.Establishment of TBI model of rats induced by underwater explosion and study of pathological features.Objective: To establish a stable and repetitive model of TBI in rats induced by underwater explosion,and to study the pathological features of TBI.Methods: A total of 120 Sprague Dawley(SD)rats were equally divided into four groups according to the explosion distances: 0.5m group,1.0m group,1.5m group,and Control group.Before the explosion,anesthetized rats were fixed on the self-made underwater explosion instrument,and the thorax and abdomen were covered with air bubble film.All rats except for those in Control group were injured by the explosion of 10 g TNT with different distances.The general condition,pathological slices and water content of brain tissue at 6h,12 h,24h,48 h,and 72 h after injury were examined.Then,a total of 60 SD rats were equally divided into two groups: Model group and Control group.Rats in the Model group were injured in the same way as 1.0m group and the Control group was not injured.Pathological slices of brain tissue were examined.Moreover,Western blot(WB)was used to measure the expression of inflammatory factors TNF-a and IL-1? at 6h,12 h,24h,48 h,and 72 h after injury,and cell apoptosis was detected by TUNEl method.Results: All rats in 0.5m group were dead in 48 h,while rats in 1.0m group and 1.5m group were alive through the whole experiment.According to the pathological slices,there were significant injuries for brain tissue among 1.0m group,while the injuries were slight among 1.5m group.The brain water content in 1.0m group began to increase at 12 h after injury and reached the peak at 48 h.While the brain water content in 1.5m group was significantly lower than that in 1.0m group.HE staining examination confirmed that rats were suffered from diffuse brain injury which mainly manifested as brain tissue structure disorder,edema,local hemorrhage,and congestion.WB results showed that the expression of TNF-a and IL-1? increased 12 h after injury and reached the peak at 48 h.At the same time,apoptotic cells appeared 12 h after TBI in the Model group,and the number of apoptotic cells reached the peak at 48 h.Conclusion: When the TBI was caused by 10 g TNT with the explosion distance being 1.0m,a stable model of rats was successfully created.Underwater explosion can cause diffuse brain injury to rats.Section II.The protective effects of 3'-deoxyadenosine on TBI induced by underwater explosion.Objective: To explore the protective effects of 3'-deoxyadenosine on TBI induced by underwater explosion and the underlying mechanisms.Methods: A total of 90 rats were equally divided into three groups: Model group,Cordycepin group,and Control group.Rats in Model and Cordycepin group were injured in the same way as the Section I,and those in Cordycepin group were administrated with 3'-deoxyadenosine(20mg/kg)by intravenous injection 30 minutes after the injury.Rats in the Control group were not injured.The brain water content,expression of TNF-a?IL-1b?TLR4 and My D88,as well as cell apoptosis,were examined at 6h,12 h,24h,48 h and 72h after the injury.Results: The brain water content,expression of TNF-a,IL-1b,TLR4,MyD88,as well as apoptotic cells in Model group increased evidently compared to the Control group at 12 h,24h,48 h,and 72 h.In contrast with Model group,the brain water content,expression of TNF-a,IL-1b,TLR4,My D88,and number of apoptotic cells in Cordycepin group decreased significantly at the same time points.Conclusion: 3'-deoxyadenosine has protective and therapeutic effects on TBI caused by underwater explosion,TLR4/My D88 signaling pathway might be involved in this process.Section III.In vivo research of the protective mechanism of 3'-deoxyadenosine on TBI induced by underwater explosion.Objective: To explore the protective mechanism of cordycepin on TBI induced by underwater explosion.Methods: A total of 30 rats were equally divided into five groups: Control group,Model group,Cordycepin group,TLR4 blocking group,and My D88 blocking group.Rats in the Model group and Cordycepin group were injured in the same way as Section III.Rats in TLR4 blocking group and My D88 blocking group were administrated with Resatorvid and MIP before injury,and then these rats were managed in the same way as the Cordycepin group.The brain water content,expression of TNF-a and IL-1b,as well as cell apoptosis,were examined 48 h after the injury.Results: Compared to the Cordycepin group,rats in TLR4 blocking group and My D88 blocking group had increased brain water content,expression of TNF-a and IL-1b,as well as number of apoptotic cells at 48 h after the injury.Conclusion: The protective effect of 3'-deoxyadenosine on TBI induced by underwater explosion might be mediated by TLR4/My D88 signaling pathway.Section IV.In vitro study of the protective mechanism of 3'-deoxyadenosine on TBI induced by underwater explosion.Objective: To explore whether 3'-deoxyadenosine alleviates inflammation by regulating TLR4/My D88 signaling pathway in microglia.Methods: Microglia were randomly divided into three groups: Control group,Lipopolysaccharides(LPS)group and Cordycepin group.Microglia in LPS group were treated with LPS,and 3'-deoxyadenosine was administrated after LPS stimulation in Cordycepin group.The secretion of TNF-a,IL-1b was measured by Elisa 48 h after the cultivation.Moreover,the protein and m RNA level of TLR4 and My D88 were examined in all groups.Results: Compared to the Control group,the secretion of TNF-a and IL-1b,as well as the protein and m RNA level of TLR4 and My D88 elevated significantly in the LPS group.These indicators decreased evidently in Cordycepin group compared to those in the LPS group.Conclusion: 3'-deoxyadenosine could inhibit the up-regulation of TNF-a and IL-1b secretion in microglia stimulated by lipopolysaccharide.TLR4/My D88 signaling pathway was involved in this process. |
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