Mechanism Research Of Autonomic Nervous System Dysfunction In Acute Traumatic Coagulopathy

Background:Following severe traumatic injury and sustained tissue hypoperfusion, up to one third of trauma patients are subject to acute traumatic coagulopathy(ATC) early in the accident sites. As recently confirmed endogenous acute coagulopathy, ATC is mainly characterized by hypocoagulable state and hyperfibrinolysis and is independently associated with higher mortality and greater transfusion requirements. Conversely, timely management strategies against ATC, e.g. the use of tranexamic acid or damage control resuscitation, could promote the reduction of blood transfusion and improvement in overall survival. However, the exact pathogenesis of ATC still needs to be elucidated.In addition to activation of protein C serving for a major trigger in ATC, studies have also indicated that the disturbances of immune, endocrine and nervous systems are actively involved in development of ATC, contributing to worse outcome. In the instances of trauma and shock, the human body promptly initiates physiological responses to counteract stress state, which are primarily regulated by the autonomic nervous system(ANS) via the sympathetic and vagus nerve.On account of the generally opposing effects generated by the sympathetic and the parasympathetic nervous systems, the equilibrium between the sympathetic and vagus nerve activity, namely ANS function, plays the pivotal role in various pathologic conditions. Nevertheless, heightened sympathetic activation and inhibition of vagal tone often occurs early in many apparently disparate pro-inflammatory conditions, such as sepsis, trauma and hemorrhagic shock, resulting in autonomic system disfunction. Multiple researches have showed that reduced heart rate variability(HRV) could reflect the ANS failure in trauma patients, which could predict mortality and is closely associated with early inflammatory response, multiple organ failure and posttraumatic coagulopathy. Now it has been widely accepted that inflammatory response and coagulation system are closely related and interact with each other. However, the relationship between ANS function and coagulation disturbance in ATC has not been investigated yet.Studies have clarified that suppression of sympathetic hyperactivity and enhancement of vagal tone in pro-inflammatory state could alleviate autonomic dysfunction, attenuate systemic inflammation, and improve organ damage, contributing to better survival and clinical outcome. To our knowledge, there is still no study regarding whether attenuation of autonomic dysfunction could improve coagulation disturbance and exert protective effects in ATC.Purposes of our research: 1. evaluate the ANS function in ATC, as well as its relevance to coagulation disturbance; 2. analyze the impact of sympathetic suppression or vagus activation on coagulation dysfunction in rats with ATC, and its possible mechanism as well.Contents of research: Part I: Relation between autonomic function and coagulation disturbance in ATC ratsObjective: To investigate the autonomic function and its relevance to coagulation disturbance in ATC.Methods: Male Sprague-Dawley rats were randomized into control or ATC groups. ATC was induced by trauma and phlebotomy. Control group underwent the same procedure but without trauma or hemorrhage. HRV and serum catecholamine(ELISA) was recorded at baseline, and 0h, 1h and 2h after shock. At 2h after shock rats were euthanized, and then thrombelastography(TEG) and conventional coagulation test were performed. Serum pro-inflammatory cytokine TNF-α and IL-6 were also measured by ELISA. Independent T-test was applied to compare data of two groups, and Pearson correlation analysis was conducted to test the relation between HRV and coagulation parameters.Results: Compared with the sham group, the ATC group had significantly prolonged PT, APTT and sustained increased serum catecholamine concerntration after shock. TEG demonstrated a prolongation of R time and K time, accompanied by a reduction of α angle and MA in the ATC group. HRV showed a decrease of total power and high frequency(HF), along with an increase of low frequency(LF) and LF:HF ratio in the ATC group, as compared with those of the sham group. The ATC group had evidently higher levels of TNF-a and IL-6 than those of the sham rats. Analysis of correlation between HRV and TEG parameters showed that LF correlated negatively with MA(r=-0.89), and LF:HF ratio also correlated well with MA(r=-0.64).Conclusions: Catecholamine surge and hypocoagulable state both occurred early in ATC rats. Rats with ATC showed autonomic dysfunction including both sympathetic excitation and vagal suppression. Moreover, the parameters of HRV correlated well with coagulation indexes. Part II: Impact of sympathetic suppression on inflammation and coagulation disturbances in ATC ratsObjective: To investigate whether suppression of sympathetic activity in ATC exerts beneficial effects on inflammatory markers and coagulation function.Methods: Male Sprague-Dawley rats were randomised to control, ATC and ATC + sympathectomy groups. In ATC + sympathectomy group, rats were intraperitoneally injected with 6-hydroxydopamine(100 mg/kg) for twice in one week before the study. After trauma and shock, HRV was assessed in rats with or without chemical sympathectomy by using 6-hydroxydopamine. Following HRV examination, rats were euthanized at baseline, and 0h, 1h and 2h after shock. And then blood gas, coagulation mornitoring and parameters of coagulation, fibrinolysis, endothelial damage, inflammation responses and sympathoadrenal activation were all determined.Results: In comparison with the control group, the ATC group had obviously higher PT, APTT and augmented catecholamine concentrations after shock. HRV demonstrated a decrease in total power and high frequency(HF), and a increase in low frequency(LF) and LF:HF ratio in the ATC group, which both were suppressed by sympathectomy in the ATC + sympathectomy group. Sympathectomy also evidently inhibited the augmentation of pro-inflammatory cytokines TNF-a and the fibrinolysis markers tPA and PAP in ATC rats. Moreover, increased serum catecholamine, syndecan-1 and soluble thrombomodulin in rats with ATC were all clearly inhibited by sympathectomy.Conclusions: Autonomic dysfunction developed early in ATC and had apparent impact on systemic inflammation, glycocalyx shedding and coagulation disturbance. Moreover, sympathectomy could attenuate systemic inflammation, suppress hyperfibrinolysis and alleviate endothelial damage. Part III: Effects of β-adrenoceptor blockade on systemic inflammation and coagulation disturbance in ATC ratsObjective: To test the hypothesis that β-adrenoceptor blockade has evident impact on inflammation, endothelial and hemostatic systems in ATC.Methods: Male Sprague-Dawley rats were randomised to control, ATC and ATC + β-blocker groups. In ATC + β-blocker group, rats were intraperitoneally injected with propranolol(7.5 mg/kg) before modeling. SDNN, a time-domain parameter of HRV, and markers of inflammation, coagulation and endothelial function were all measured at baseline, and 0h, 1h and 2h after shock. Western blotting analysis of nuclear factor(NF)-κB was performed at 2h after shock. Meanwhile, small bowel mesentery was infused with lanthanum-based fixative solution and was detected by electron microscopy to reveal the structure of endothelial glycocalyx. Additional ATC and ATC + β-blocker groups were observed to compare survival time after shock.Results: Beta-adrenoceptor blockade significantly reduced heart rate of ATC rats without alteration of their mean blood pressure. HRV test showed evidently decreased SDNN, reflecting sympathetic hyperactivity in the ATC group, which was suppressed by the β-blocker. Beta-adrenoceptor blockade obviously inhibited the enhancement of pro-inflammatory cytokines TNF-α, fibrinolysis markers tPA and PAP and liver NF-κB activity. Moreover, increased soluble thrombomodulin and syndecan-1 were reduced by β-adrenoceptor blockade. Electron microscopy revealed apparent shedding of the endothelial glycocalyx after shock in the ATC group, whereas β-blocker exerted obvious protective effect on endothelial glycocalyx. There was no significant difference in survival time after shock between ATC and ATC + β-blocker groups.Conclusions: Beta-adrenoceptor blockade could suppress sympathetic hyperactivity, alleviate acute inflammatory reaction, attenuate endothelial damage and inhibit hyperfibrinolysis, confirming the pivotal role of β-adrenoceptor activation in the mechanism of ATC. Part IV: Effects of electrical vagus nerve stimulation on circulating HMGB1 and coagulation disturbance in ATC rats and its mechanismObjective: Both severe trauma and hemorrhagic shock could induce early release of high mobility group box1 protein(HMGB1) into circulation, which triggers sterile inflammation via the pattern recognition receptors and correlates well with posttraumatic coagulopathy. In this study, we aimed to investigate the impact of vagus nerve stimulation on serum HMGB1 and coagulation dysfunction in ATC.Methods: Male Sprague-Dawley rats were randomly assigned into control, ATC and ATC + VNS(vagus nerve stimulation) groups. ATC rats received sham operation or electrical vagus nerve stimulation(5V, 2ms) for 10 min after modeling. Animals were euthanized at 2h after shock for blood withdrawing and organism collection. Then, HRV and TEG were applied, and western blotting analysis was performed to determine the expression of liver NF-κB and HMGB1. Serum TNF-α, IL-6 and HMGB1 was assessed by ELISA. Lung tissues were collected for evaluation of histological damage, cell surface syndecan-1 expression, HMGB1 and syncecan-1 mRNA, and the immunohistochemistry expression of HMGB1 and receptor for advanced glycation end products(RAGE).Results: VNS did not alter the HR and MAP in ATC rats. VNS effectively improved the decreased vagal tone and attenuated the increased K time and decreased MA the ATC group. Serum levels of HMGB1 and pro-inflammatory cytokines were obviously reduced by VNS. Lung tissue immunostaining showed that cell surface syndecan-1 expression was significantly reduced in the ATC group but enhanced by VNS. Pulmonary syndecan-1 mRNA expression was lower and HMGB1 mRNA expression was higher in the ATC group as compared to the control group, while VNS only evidently enhanced syndecan-1 mRNA expression. Compared to the control group, liver NF-κB and HMGB1 protein levels were both higher in ATC group, whereas VNS just significantly reduced liver NF-κB expression. But VNS has no impact on pulmonary HMGB1 mRNA expression and liver HMGB1 protein level. Trauma and shock also resulted in significant lung injury in the ATC group, which was mitigated by VNS. Immunohistochemistry showed that HMGB1 and RAGE expression in the lungs were both upregulated in the ATC group, which were both suppressed by VNS.Conclusions: VNS could attenuate systemic inflammation, suppress serum HMGB1 and alleviate endothelial damage and hypocoagulable state in ATC rats. The mechanism seemed to be mediated partly through the NF-κB and RAGE pathway.Summary 1. ATC rats are subject to ANS disfunction, manifested by enhancement of sympathetic nerve activity and inhibition of vagal tone. Moreover, autonomic dysfunction correlates well with coagulation disturbance in ATC. 2. Inhibition of sympathetic nerve activity could attenuate systemic inflammation, suppress hyperfibrinolysis and alleviate endothelial damage in ATC rats. 3. Beta-adrenoceptor blockade could suppress sympathetic hyperactivity and protect ATC rats against inflammation, hyperfibrinolysis and endothelial damage, demonstrating its key role in the mechanism of ATC. 4. Electrical vagus nerve stimulation may attenuate excessive inflammatory reaction and coagulation dysfunction through the inhibition of serum HMGB1 and the downregulation of the signal transductions of the NF-κB and RAGE pathway.