| Objective: The non-lethal mechanical trauma, such as soft tissue injuries, traffic accident injuries, and confessions obtained by torture, often lead to MODS, and even death. Because this kind of injury alone is not enough to cause the death, the cause of death are often not identified, which result in the delay judgment and seriously affect the national judicial credibility. Therefore, investigating the mechanism of death caused by non lethal lethal mechanical injury becames a science and technology problem demanding prompt solution in forensic medicine.In addition to inflammation, distant organ damage caused by local tissue injury, trauma, as a stressor, could lead to systemic stress response. Especially before and during trauma, the injured organism would have a bad mood, such as anxiety, helplessness,terror or depression, which can cause an extraordinary psychological stress response. As a defense compensatory response,stress play an important role in maintaining the homeostasis of body. But overgenerous or persistent stressor will cause the body dysfunction, metabolic disorders and tissue damage [1]. Physical stress and Psychological stress result from the non lethal mechanical damage may induce excessive stress response. To clarify the damage effect of stress on organism has an important significance for revealing the mechanisms of death caused by non lethal mechanical damage.Tyrosine hydroxylase(TH) is the rate limiting enzyme for catecholamine synthesis process [2]. The increased expression level of TH followed by the secretion of catecholamines had become the main material basis by which locus coeruleus- the sympathetic adrenal medulla system exert central and peripheral effects in stress. The locus coeruleus has a close relationship between respiratory and cardiovascular center. Extensive destruction of locus coeruleus neurons can lead to respiratory failure, cardiac arrest, and even sudden death [3,4]. Therefore, to study the influence of excessive stress on the locus coeruleus morphology and function has very important significance for revealing the relationship between stress and death. Catecholamine released from the presynaptic membrane was reuptaked, which produced lots of aldehydes resulted from oxidation by monoamine oxidase. The aldehydes can cause neuronal injury and apoptosis. Both whether excessive stress can cause the locus coeruleus injury and whether locus coeruleus injury is related to catecholamine remain unknowned.The present study aimed to explore the role of catecholamine in the damage effects of stress on rat locus coeruleus on the basis of establishing the compound model of restraint stress and crush injury, which will provide experimental basis for revealing the underlying mechanisms of death caused by non-lethal mechanical trauma.Methods:1 After were adaptively fed for 7 days, 162 male Sprague-Dawley rats(weight: 240-260g), were randomly divided into six groups(27 rats in each group).①control group: neither food nor water was given for daily 8h or 10 h alternatly.②restraint stress group: The rats were removed from their cage and restrained in the same standardized restraining boxes for daily 8h or 10 h, alternately.③crush injury group: the rats were subjected to the mechanical pressure(24kg per kilogram of body weight) to induce a crush injury for 6 hours.④compound model group : after crush for 6 hours, the rats were subjected to restraint stress for 8h on the same day, and followed by daily restraint for 8h or 10 h alternately. ⑤ compound model group+AMPT(alpha-methyl-p-tyrosine, AMPT) group: the rats were injected AMPT 2 hours before daily restraint stress.⑥AMPT group: the rats were injected AMPT daily at the same time. Except compound model group+AMPT and AMPT administration group execution rats at 7d later, rats in the other groups were killedat 1d、3d、5d and 7d respectively.Blood samples were saved at-80 degrees to assay E、NE levels, six fixed brain tissue each group were subjected to thionin staining and simultaneously immunohistochemistry to detect the c-Fos, Caspase-3 and phosphorylated TH expression levels. In addition, the remaining 3 rats of each group were subjected to observe ultrastructural change.2 The data were presented as the mean(SD) and analyzed with analysis of variance and least significant difference using SPSS version 16.0. A level of P<0.05 wasconsidered statistically significant.Results:1 The changes of body weight in ratsBody weight of rats increased gradually in control group.The weight gain in RS groups were slowed down compared with control groups.The results show that the successful establishment of restraint stress model.2 Changes of E、NE concentration in rat plasma1d, 3d after establishing model, compared with the control group, the concentration of E、NE in crush injury group was significantly increased. Compared with crush injury group, the concentration of E、NE in compound group further increase. But 5d,7d after establishing model, no statistical significance was shown between control group and crush injury group. Howerer, a significant increase of concentration E,NE still was shown in compound group compared with the control group.3 The results of phosphorylated TH immunohistochemistry staining of locus ceruleus tissueThe change tendency in expression of phosphorylated TH protein was in accord with changes in concentration of E,NE which suggested that stress response intensity caused by double stressors was much higher than intensity caused by simple crush.4 The locus coeruleus of thionin stainedThe most serious pathologic changes were observed 7 days later. Control group showed normal neuron morphology. Compared with control group, crush injury group showed different degrees of edema and deformation in neuron.In addition to visible edema, deformation, restraint stress group also showed disorganized neurons, necrosis and other pathological changes. Compared with the crush injury group, pathologic histological changes in the compound model group became more obvious, showing neurons necrosis, karyopyknosis, more severous arranged disorder. Pre application of AMPT significantly reduced damage caused by combined action of the crush and restraint, which suggested that restraint stress aggravated the locus coeruleus injury in the crush injury rat and the possible mechanism was related to excessive activation of TH.5 The locus ceruleus tissue c-Fos immunohistochemistry staining results1d, 3d, 5d after establishing model, no positive expression were observed. c-Fos immunohistochemical staining results 7d later are as follows: between control group and crush injury group, no positive expression was discovered. Restraint stress group showed weak positive expression; But compound model group displayed the positive expression of c-Fos.But pre-application of AMPT maked the positive expression of c-Fos into negative expression.6 The locus ceruleus tissue caspase-3 immunohistochemistry staining results and the results of electron microscope1d, 3d, 5d after establishing model, no positive expression were observed. Positive expression was only displayed in compound model group 7d later. But pre-application of AMPT maked the positive expression of caspase-3 into negative expression.Electron microscopy results are as follows: the control group and the crush injury group showed mild edema. Restraint stress group displayed moderately severe edema. But early apoptosis appear in compound model group. Compared with compound model group, compound model +AMPT group ultrastructural change reduced,which was consistent with the caspase-3 results, suggesting that combined action of restraint stress and crush injury could induce apoptosis of neurons in the locus coeruleus, and possible mechanism may be related to excessive activation of TH.Conclusions:A rat stressful injury model was successful established for the first time in the present study. Pathologic and ultrastructure changes in locus coeruleus were observed; TH,c-Fos and caspase-3 protein expression were tested; plasma concentration of E, NE change was assayed,The conclusion can be reached as follows:restraint stress aggravated locus coeruleus injury in the crush injury rat, which was be related to excessive activation of synthesis rate limiting enzyme TH followed by increased CA production. |
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