The Effect And Underlying Mechanism Of (S)-4C3HPG Application Post Injury In Severe Traumatic Brain Injury Model Of Mice

Traumatic brain injury (traumatic brain injury TBI) is a prominent issue in modernsociety, and its high morbidity and mortality is a serious threat to human life and quality oflife. However secondary injury after traumatic brain injury has relative reversibility andplays a decisive role in prognosis. Therefore, the treatment of TBI is always people’sresearch focus to reduce secondary brain damage and neurological impairment.Currently, the toxic effects of excitatory amino acids is one of the importantmechanism of secondary injury, others including excessive inflammation, calcium overloadand oxygen free radical release. In the previous study, Glutamate excitotoxicity andinflammatory reaction, which are the important mechanisms of brain damage, can promoteeach other thus forming a vicious cycle aggravating the injury.(S)-4C3HPG is a hybridmetabotropic glutamate receptor modulators and has been demonstrated that (S)-4C3HPGhave good results in Alzheimer’s disease, Parkinson’s disease, schizophrenia, epilepsy andtreatment of extra pyramidal diseases, reducing cerebral ischemia after cerebral infarctionfoci of chronic diseases and neurological protection.Then we want to know whether(S)-4C3HPG can work in traumatic brain injury or not? Preliminary studies have confirmedthat (S)-4C3HPG can significantly alleviate injury secondary brain injury caused byneurological dysfunction. In more serious severe traumatic brain injury, the cerebralprotective effect of this drug is not clear. Clinical studies have shown that early diagnosisand treatment play a key role in trauma prognosis after traumatic brain injury. Therefore, onthe basis of preliminary studies, the use of gravity to impact injury law to build the mousesTBI model was combined with the dynamic changes in the cerebrospinal fluid glutamatelevels in mouse brain injury. In the acute phase after the injury, four time points (15min,1h,3h,6h) were selected to give (S)-4C3HPG to observe the effect of the acute phase injury ofsTBI and analysis of its relationship with the concentration of glutamate and inflammatory factors. The possibility of effective cerebral protective effect of regulating excitatory aminoacid toxic effects after sTBI will be hoped to confirm and thus (S)-4C3HPG applicationswill provide experimental basis for clinical treatment of traumatic severe traumatic braininjury.The main experimental methods, results and conclusions:1. The establishment of mice with traumatic severe brain injury model.Observing the effect after giving (S)-4C3HPG in mice in the acute phase after injury.Method：We selected the SPF level of C57BL/6male mice with reference to themethod established in this group. Pentobarbital sodium was used to anesthetize mice.4mm×4mm bone window was opened in mice between left parietal anterior fontanels and skull.Falling from50cm height,20g heavy objects impact on the left parietal to form the shape ofdiameter of3mm, depth of3mm. Then the injury model was established. Pieces of bonewere recovery and the skin was sutured. Experimental animals completely randomlydivided into six groups (n=10):①Sham group: only the skull and suture handling wereused to anesthetized mice;②The control group: only injury, without any drug intervention;③(S)-4C3HPG drug treatment group(4groups): after sTBI injury for15min,1h,3h,6h,(s)-4c3HPG was intraperitoneally injected5mg/kg respectively. With reference to themethod of our group, the neurological deficits assessment was proceed in each group24hafter injury. The mice were killed after the assessment of neurological function. Wet anddry weight method was used to detect the ipsilateral cortical tissue water content in eachanimals group.Result：After injury of24h, all mice were alive. But they have reduced activity, slowresponse, and with the extension of the delivery time, mouse brain tissue contusion wasbleeding and obviously injured. In light microscope, early neuron cell body was in narrowdeformation,nucleus condensation, deeply stained cytoplasm acute necrotizingchanges.Detection of (S)-4C3HPG drug treatment of15min and1h, neurological deficit(1.70±0.48)(1.90±0.56)] were significantly lower than control group (2.80±0.42)], P<0.01; Brain water content [(80.14±0.28)%(80.17±0.52)%] was significantly lower thancontrol group [(82.55±0.34)%], P <0.01. This shows that early giving (S)-4C3HPG canreduce the trauma of regional cerebral edema after impact injury in the brain and Obviousneuroprotective effect can be acquired if we use drug intervention as early as possible after injury.2. The relationship between glutamate concentration changes and expression ofinflammatory cytokines after using (s)-4c3HPG in the acute phase of traumatic braininjury.Method：Cerebrospinal fluid was extracted in each group of mice with traumaticsevere brain injury after24h. Gradient high performance liquid chromatography (HPLC)determined the concentration of glutamate in cerebrospinal fluid in animals with severeimpact injury model injury24hours.The detection of the injured cortex inflammation ofTNF-alpha, IL-1βmRNA content was performed by (RT-PCR) at24h after cortex injury.Result：compared with the sham group at24h after sTBI, glutamate concentration inthe cerebrospinal fluid of injured mice was significantly higher (P <0.01). But comparedwith the control group, the concentrations of glutamate in cerebrospinal fluid measured24hours after injury are significantly lower than the control group (P <0.01),which weretreated with (S)-4C3HPG drug in15min,1h. However, there is no significant difference(P>0.05) in (S)-4C3HPG drug treatment group and control group in3,6h. On the detectionof injured cortex inflammatory factor, the treatment with sTBI after24h and (S)-4C3HPGfor15min,1h group can significantly decrease the mRNA expression level of TNF-alpha,IL-1βcompared with the control group(P<0.01).But the IL-1β mRNA expression level invilification cortex after (S)-4C3HPG drug treatment of TNF-alpha for3,6h has nosignificantly difference in two groups (P>0.05). Experiments show that the degree ofneurological impairment and the extent of cerebral edema have closely relationship withglutamate concentration and inflammatory factors after injury.Conclusion：The early application of (S)-4C3HPG to regulate the release of glutamate cansignificantly reduce the toxic effects of excitatory amino acids, the inflammatory responseof induced factors and the damage of the nerve function. But inflammation may be thefactors focus in the acute phase after injury. So inhibiting glutamate toxicity can noteffectively alleviate the damage of the nervous system.In cerebrospinal fluid after sTBI, thelevel changes in Glutamate concentration and inflammatory cytokines has the sametropism.Glutamate will be rapidly released after severe traumatic brain injury. The effect of excitotoxicity can cause nerve ischemia, hypoxia, and induce the expression ofinflammatory factors.All those effects led to the series of complex trauma disorders ofcerebral blood flow, cerebral edema, neurological death and increase the formation ofneurological impairment after traumatic brain injury.