| Background:Traumatic brain injury(TBI)refers to brain damage caused by external mechanical forces(such as fall,car crash,sports collision,firearm attack and explosion,etc.)acting on the brain.In some developed countries,TBI is accepted as the leading cause of mortality or disability among people under the age of 45.In the United States alone,as many as 1.7million people suffer from varying degrees of TBI each year,in which more than 50,000 die from trauma,and at least 35% of the 230,000 hospitalized survivors suffer from long-term disability.With the improvement of social and economic level and the development of transportation systems,the incidence of TBI and the mortality and disability rates caused by TBI are gradually increasing in China.TBI can lead to temporary or permanent physical movement disorders,cognitive dysfunction and psychological dysfunction,which are often encountered in clinical practice.Blast induced traumatic brain injury(b TBI)is an important and unique type of TBI,which often occurs in local wars,anti-terrorism and civilian environments.TBI induced by improvised explosive devices(IEDs)is the main cause of civilian and soldier casualties in the current international local wars(including anti-terrorism).TBI induced by IEDs can lead to varying degrees of cognitive and behavioral impairment,but the exact pathophysiological mechanism of brain damage remains not clear and the ideal animal model is still lacking.Different from other TBI animal models,both controlled cortical impact injury(CCI)model and fluid percussion injury(FPI)model have widely accepted commercial animal models.Because of the differences of explosion sources,principles and exposed parts of b TBI animal models,the b TBI models made by different laboratories are quite different,which brings great difficulties to the consistency and interpretation of the studies of b TBI.Therefore,there is an urgent need to establish a small animal model which is easy to replicate and better resembles human brain damage in the real world,in order to further clarify the pathogenesis of b TBI and find a new and effective treatment strategy.Cognitive dysfunction is an important sequela of TBI,especially b TBI,seriously affecting the life quality of patients and being a major issue in clinical practice,which has attracted more and more scholars' attention.Epidemiological and experimental data show that shock wave exposure can lead to long-term behavioral changes and memory impairments as characterized by rapid impairment of learning and memory,which has been considered to be a signature brain damage in recent military conflicts in Iraq and Afghanistan.Different from other neurodegenerative diseases such as Alzheimer's disease,cognitive impairment after b TBI or TBI is charactized by early appearance,rapid progress,duration with the latter half of life and impaired working memory.Therefore,there is an urgent need to clarify how to improve cognitive impairment after b TBI.Although more and more animal studies focus on the pathophysiological mechanism of b TBI,there are no effective and promising drugs for b TBI up until now.Therefore,it is of great significance and urgent need to deeply investigate the pathogenesis of b TBI and find effective intervention strategies or drugs to improve the neurological and cognitive impairment caused by b TBI,which has important application prospects in both military and civilian fields.Thymosin ?1(T?1)is a small molecular peptide composed of 28 amino acids isolated and purified from thymopoietin component V.T?1 is a pleiotropic drug,which can promote cellular immunity and possess anti-tumor and anti-inflammatory properties.In addition,it has been accepted as a promising drug in clinical practice in patients with infectious diseases,immune deficiency and malignancies.In addition,Ta1 has been shown to reduce pancreatic lesions and diabetes,possess anti-atherosclerotic effects,induce endothelial cell migration and wound healing and exhibit antioxidant stress effect in atherosclerotic rabbit models.Thymectomy not only leads to immune deficiency in mice,but also results in impairements of their learning and memory,suggesting that thymic peptides may be involved in learning and memory.Moreover,it has been demonstrated that T?1 can promote neural regeneration and improve cognitive function of newborn normal mice,suggesting that T?1 can regulate the cognitive function of normal mice.Tau protein is a member of microtubule-associated proteins(MAPs).The phosphorylation of tau proteins evokes aggregation and further induces the formation of neurofibrillary tangles(NFTs),which has been widely considered as a common pathological characterics of many neurodegenerative diseases and brain injuries.Previous studies and our unpublished data suggest that T?1 may regulate miniature excitatory postsynaptic potential(m EPSP),indicating that T?1 may affect nervous system function.TBI or b TBI is accompanied by severe sequelaes,such as cognitive impairment and neuroinflammatory response.However,it remains unclear whether T?1 can improve cognitive dysfunction and tau protein dysfunction after b TBI.The present study is designed to establish a new type of real world b TBI model based on compressed gas with only craniocerebral exposure,and to further investigate whether T?1treatment can improve neurological and cognitive dysfunction caused by b TBI,and explore its possible underlying mechanisms.This study is expected to provide a reliable animal model for the study of b TBI,and provide new academic insights for the investigation of b TBI mechanisms and new drugs or targets.Objective:1.The present study is designed to establish a new b TBI model with highly biomechanical and biomedical stability based on compressed gas with only craniocerebral exposure,and to explore the underlying mechanism of cognitive impairment and the pathophysiological mechanism of b TBI.2.To elucidate whether T?1 can improve cognitive impairment after b TBI and clarify its possible underlying mechanism,so as to provide new academic insights for the investigation of b TBI mechanisms and new drugs or targets.Methods:1.The design of compressed gas driven shock wave generator.1.1 To perform an innovative design based on the working basis of our research group and the reported animal model of b TBI in the literature.1.2 To develop a rat b TBI animal model that better resembles battlefield environment.The prelimitary experiment of identifying the TNT mediated b TBI model was carried out in a large and open outdoor environment in Hebei province.The whole process of explosion was shooted with high speed camera.PCB sensors were placed at the same height and on the circumference to measure shock wave characteristics by using DEWESoft shock wave data acquisition system.1.3 To design a compressed gas driven b TBI model and complete the verification of biomechanical indexes and corresponding operation instructions.To pay more attention to solve the stability of rupture disc.Explore the stability and consistency of the rupture disc made of aluminum sheet with different groove thicknesses.The shock wave overpressure characteristics were detected by PCB sensors via DEWESoft shock wave data acquisition system.The peak overpressure,rise time,duration and positive impulse were also recorded.The feasibility analysis of the b TBI model was also performed.According to different groove thicknesses,the experiments for recording the shock wave overpressure characteristics of the shock wave generator were divided into 0.5mm,0.6mm,0.7mm and 0.8mm groups.According to different distances from the blast sources,the experiments for recording the shock wave overpressure characteristics of the shock wave generator were divided into 20 cm,35cm,50 cm,65cm,80 cm and 95 cm groups.2.To establish a new animal model of b TBI in rats.2.1 Dose effect of s single blast: 0.6mm thickness grooved aluminum sheets were used as rupture disc.The part of experiment was divided into four groups: Sham: sham explosion group(except not receiving explosion shock,the rest were the same as other groups),L: low intensity explosion group(distance to explosion source was 80cm),M: medium intensity explosion group(distance to explosion source was 50cm),H: high intensity explosion group(distance to explosion source was 20cm).2.2 Time effect of s single blast: 0.6mm thickness grooved aluminum sheets were used as rupture disc.According to the sampleing time,the experiments were divided into 6 groups:Sham: sham explosion group(except not receiving explosion shock,the rest were the same as other groups),2h: 2 hours after explosion group,12h: 12 hours after explosion group,24h: 24 hours after explosion group,3d: 3 days after explosion group,7d: 7 days after explosion injury group.2.3.The Morris Water Maze test(MWM test)was performed to study the effects of a single blast on the acquisition and retention of spatial memory of rats after blast injury.According to the distances from explosion source,the experiments were divided into four groups: Sham: sham explosion group(except not receiving explosion shock,the rest were the same as other groups),L: low intensity explosion group(distance to explosion source was80cm),M: medium intensity explosion group(distance to explosion source was 50cm),H:high intensity explosion group(distance to explosion source was 20cm).2.4.Western Blotting was performed to investigate the effect of a single blast on tau protein expression and phosphorylation in the hippocampus.2.5 HE staining was used to observe the effect of a single blast on the microscopic pathology of rat brain tissue,and the effect was observed according to the dose effect and time effect.2.6 Flow cytometry was performed to detect the effects of a single blast on the percentages of CD4+T cells,CD8+T cells and Treg T cells in the blood of rats.2.7 Blood routine automatic analyzer was used to detect the effects of a single blast on the percentage of lymphocytes and neutrophils in the blood of rats.2.8.ELISA was performed to clarify the effect of a single blast on inflammatory factor IL-6 in the blood of rats.2.9 Weighing method was used to detect the effect of a single blast on brain water content in rats.3.The effect and mechanism of T?1 on cognitive dysfunction after craniocerebral explosive injury.3.1 To determine the effect of T?1 on 24-hour mortality of rats subjected to b TBI.Rats were subjected to T?1 therapy at a dose of 200 ?g/kg in 1 ml saline(twice a day)or NS therapy with the same frequency and volume.According to the experimental design,the rats were divided into four groups: sham explosion group(except not receiving explosion shock,the rest were the same as other groups),explosion group(distance from explosion source was20cm),explosion+normal saline(NS)treatment group(distance from explosion source was20cm),explosion + T?1 treatment group(distance from explosion source was 20cm).The24-hour mortality of each group was calculated to determine whether T?1 exhibits protective effect on rats after b TBI.3.2 MWM test was performed to study the effect of T?1 on the cognitive function of rats after b TBI,and they were divided into four groups: sham explosion group(except not receiving explosion shock,the rest was the same as other groups),explosion group(distance from explosion source was 20cm),explosion+NS treatment group(distance from explosion source was 20cm),explosion+T?1 treatment group(distance from explosion source was20cm).3.3 Western blotting technique was used to detect the effect of T?1 on the expression and phosphorylation of tau protein in the hippocampus after b TBI in rats.3.4 HE staining was performed to observe the effect of T?1 on the microscopic pathology of brain tissue after b TBI in rats.3.5 Flow cytometry was performed to detect the effect of T?1 on Treg cells in the blood of rats after b TBI.3.6.ELISA was performed to determine the effect of T?1 on inflammatory factor IL-6 in blood after b TBI in rats.3.7 Weighing method was performed to clarify the effect of T?1 on brain water content in rats with craniocerebral explosive injury.Results1.Based on the aluminum disc with grooves as the rupture disc,the newly designed compressed gas driven shock wave generating device can produce highly biomechanical stable blast.2.In this study,a new rat b TBI model based on compressed gas explosion was successfully established.2.1 Rats were exposed at different distances from the explosion origin to receive a single blast(0.6mm thickness V-grooved aluminum sheets).As the distance increases from 20 cm to95 cm,the overpressure decreased from 136.44 k Pa to 26.77 k Pa and the 24-hour mortality decreased from 19.6% to 0.A dose-response linear regression model with the prediction model being P=1/1+e(5.50-0.026X)(P: 24-hour mortality;X: overpressure)was established to predict 24-hour mortality via shock wave overpressure.Receiver operating characteristic(ROC)curves for this model exhibited good predictive power as demonstrated by the fact that areas under ROC curves are 0.833(P < 0.001).2.2 A high level blast exposure decreased the learning and memory ability of rats,which was characterized by the prolongation of escape latency during the acquisition phase and the decrease of crossing times over the platform during the probe phase.2.3 There were no obvious signs of contusion,necrosis,hematoma,hemrrhage or other types of microscopic brain tisue damages from 2h to 7d after exposure to a single blast injury.More over,different degrees of blast injury from low intensity to high intensity did not cause significant neuropathology change,ischemic neurons or tissue damages.2.4 A single blast exposure significantly increased tau protein phosphorylation of Thr205 epitope in a dose-and time-dependent manner.However,tau protein phosphorylation at Ser404 and Ser262 epitopes and total tau expression did not change when the rats were exposed to different degrees of blast(from low intensity to high intensity)or the same high intensity blast exposure at 2h,12 h,24h,3d and 7d post injury.2.5 The percentages of CD4+T cells,CD8+T cells,Treg T cells and lymphocytes were significantly decreased on 7d,2h,3d and 12 h post a single blast injury,while the percentage of neutrophils was significantly increased at 12 h after a single blast injury.2.6 The change of inflammatory cytokine IL-6 level in plasma of rat subjected to a single blast injury exhibited a time dependent manner.The plasma IL-6 levels were significanly increased at 12 h,24 h and 3 d after blast injury.2.7 A single blast exposure can lead to increased brain water content at 2h,12 h,24h and3 d after craniocerebral explosive injury in rats.As the intensity of blast increased,the brain water content was significantly increased.3.The effect of T?1 on cognitive dysfunction after craniocerebral explosive injury and its possible mechanism.3.1 T?1 treatment could significantly improve the cognitive impairment after craniocerebral explosive injury,which resulted in a decrease in the escape latency during the acquisition phase and an increase in the numbers of crossing the platform during the probe trial.However,T?1 did not significantly affect the swimming speed of rats in each day of the whole process.3.2 T?1 had a tendency to reduce the 24-hour mortality of rats after explosive injury,but there was no statistical significance.3.3 T?1 did not significantly change the histopathology of the brain after craniocerebral explosive injury.3.4 T?1 significantly inhibited the phosphorylation of Thr205 epitope of tau protein,but had no significant effect on the phosphorylation of Ser404 and Ser262 epitopes of tau protein.3.5 T?1 increased the percentage of Treg cells in 3 days after explosive injury,3.6 T?1 inhibited the production of plasma IL-6 in rats subjected to b TBI.3.7 T?1alleviated brain edema in rats exposed to b TBI,as demonstrated by decreasing the water content of brain tissues.Conclusions:1.In the present study,a compressed gas explosion driven and new b TBI model with only craniocerebral exposure was established,which was characterized by persistent cognitive impairment,phosphorylation of Thr205 site of tau protein,decreased percentages of Treg T cells,increased inflammatory response and brain edema.2.T?1 improved the learning and memory ability of rats after b TBI,inhibited the phosphorylation of tau protein Thr205 epitopes,increased Treg cells,and inhibited inflammation and brain edema without affecting the microscopic pathology of rat brain tissue.T?1 is expected to be a new and promising drug for the treatment of craniocerebral explosive injury. |
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