| Objective:In judicial practice, some litigants suffered from a variety of nonfatal mechanical damage, such as traffic accidents, kidnapping, torture, etc, often turned out multiple organ dysfunction syndrome and even death. Owing to this kind of injury itself is not enough serious to cause death, it was used to cause the death becoming intangible and controversial, which delayed the case. Thereby, it is meaningful to investigate the impact of morphology and function on vital organs by this kind of damage and further to reveal the exact mechanism of the death.The stress response often occurred after damage to avoid injury. Neuroendocrine responses to stress are mainly for the activation of locus ceruleus-norepinephrine system and hypothalamus-pituitary-adrenal cortex system. Subsequent increase of stress hormones in blood caused a series of functional metabolic changes, which has an important significance compensatory defense. However, excessive or persistent stress may cause dysfunction, metabolic disorders and tissue damage [1]. As physical stressors, damage can cause stress response; In addition to, damage will produce anxiety, tension and depression and other negative emotion are defined as psychological stress, these spiritual and psychological factors play a more important role in stress response. The restraint stress model is an accepted animal model to reproduce psychological stress. Our previous studies indicated that restraint stress could aggravate heart, liver and kidney damage in rats with crush injury [2] and further lead to MODS, which suggest that heavy psychological stress may play critical role in the process of death caused by non-lethal mechanical injury.Nucleus tractus solitaries(NTS), a medulla oblongata dorsal nucleus, located in the vagus nerve dorsolateral. Because NTS contains many catecholamine neurons and fiber terminals, NTS is one of the central regulating center of cardiovascular reflex, but also is the important center of the stress response[3~5]. Previous studies confirmed that damaged solitary nucleus neurons of rats NTS could decrease the baroreceptor reflex, cause arrhythmias and even sudden death. Therefore, NTS plays an important role in maintaining the homeostasis of the cardiovascular system [6]. Cardiovascular system dysfunction is one of common deaths mechanisms in the judicial practice. However, it is still unclear that damaged NTS suffered from stress is is involved in the mechanism of death result from non-fatal injury.The crush injury model is an accepted animal model to copy soft tissue injury. Thus, the present study will establish the complex model consisting of restraint stress and crush injury to explore combined action of the psychological stress and physical stress on rats NTS, which can provide experimental basis for revealing the underlying mechanism for death caused by nonfatal mechanical damage.Methods:1 Healthy male Sprague Dawley(SD) rats(220 ~ 240g), adaptively fed for a week, were randomly divided into four groups(n=24). ① control group(Con): rats were fasted water at a fixed time every day(8h,10 h and 12 h alternately) activating and drinking freely the rest time. ②restraint stress group(Rs): rats were put at a fixed time every day(8h,10 h and 12 h alternately) into the restraint stress mold, fasting water. ③crush injury group(CI): rats anesthesiaed with anhydrous diethyl ether, were subjected to crush with 24 kg weight on their double hind limbs for six hours, activating and drinking freely the rest time. ④ composite model group(Com): rats were continuously subjected to crushed for six hours, and suffered to restraint stress at a fixed time every day(8h,10 h and 12 h alternately). The weight before the experiment each day were recorded to evaluate whether to establish a model of restraint stress. The partial performance and station were observed to assay whether to successfully establish the crush injury model. Rat were measured blood pressure by non-invasive blood pressure monitor BP-98 A at a fixed time. After restraint stress continuous for 21 days, intraperitoneal injection of rats were anesthesiaed with 10% chloral hydrate ip. Blood was collected from abdominal aorta, and the supernatant was used to detect catecholamine concentrations. Brain tissues were removed rapidly and put into 10% paraformaldehyde solution to fix. Brains paraffin sections were prepared, and carried out thionine staining for histopathological observation, immunohistochemical staining(IHC) for observation expression changes of tyrosine hydroxylase(TH), glial fibrillary acidic protein(GFAP) and apoptosis executor Caspase-3 protein in brains.2 The data were presented as Mean±SD and analyzed with one way ANOVA and least significant difference test(LSD) or Tamhane’s by SPSS 21.0 statistical program. A level of P<0.05 was supposed to be statistically significant.Results:1 The changes of rat body weightsBody weight of rats increased gradually in Con group. The weight gain in Rs group showed a significant slow increase compared with Con group(P<0.001), which confirmed that the present study successfully established the rat restraint stress model.2 NE, E levels changes in rat plasma samplesNo significant difference was noted in plasma concentration NE and E between Con group and Crush injury group. Compared with crush injury group, NE and E concentration increased significantly in the composite model group, P<0.001.3 Immunohistochemical staining results of THCon group showed a small amount of expression in TH protein. Compared with Con group, a significant increase was observed in the other groups. Compared with crush injury group, TH positive cells increased significantly in composite model group, P<0.001. Combined with blood catecholamine levels, the present study confirmed that compared with the simple crush injury, double stressors can cause higher strength stress response.4 Thionin staining results in nucleus tractus solitariesCon group was showed an uniformly distributed in Nissl bodies. Nissl bodies disappearing or shifting, and different degrees of edema, deformation were noted in crush injury group. Individual neurons appeared shrinkage, necrosis or simolar pathological changes in restraint stress group. Compared with crush injury group, Nissl body unevenly distributed, disappeared, more neurons deformation, pyknosis, hyperchromatic were observed in composite model group.5 Immunohistochemical staining results of GFAPGlial fibrillary acid protein(GFAP) is the astrocyte-specific marker. When damage in the central nervous system occurs, astrocytes activate followed by increase of GFAP expression. Con group showed little expression of GFAP protein. Compared with Con group, a significant increase was observed in the other groups. Compared with crush injury group, GFAP positive cells increased significantly and projections grow longer in composite model rats, P<0.001.6 Immunohistochemical staining results of executor Caspase3Thionine staining showed neurons shrinkage, necrosis, so the present study further examined apoptosis-executor Caspase3 expression level. Con group showed little expression of Caspase3 protein. Compared with Con group, a significant increase was observed in the other groups. Compared with crush injury group, Caspase3 positive cells increased significantly, P<0.001.7 The results of blood pressure measurementIncrease blood pressure fluctuations regarded as one of symbols solitary for NTS damage. Blood pressure of Con group showed less degree of fluctuation. Compared with Con group, blood pressure appeared a significant drop after extrusion on the second day, then gradually increased in Crush injury group. No significant difference was noted in blood pressure fluctuation 14 d later between Con group and Crush injury group. Blood pressure fluctuations turn out a increase degree in restraint stress group, compared with Con group, P<0.001. Compared with crush injury group, a more significant increase in blood pressure variability degree-was observed in composite model group, p<0.05.Conclusions:The present study successfully established the composite model consisting of restraint stress and crush injury. By observing histopathological changes in NTS; assaying the blood pressure variation degree and the CA concentration in plasma; as well as detecting the expression levels of TH, GFAP and Casepase3 protein in solitary nucleus, the present study drawed a conclusion: restraint stress aggravates the damage of nucleus tractus solitarius in rat with crush injury. |
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