Study And Application Of Decompression Sickness Bama Swine Model

With the change of China's strategic guiding ideology,especially the development of marine resources and the maintenance of maritime rights and interests,the strategy of navy has been shifting from "offshore defense" to "far-sea defense" with increasingly frequent maritime activities.Military submarine salvage,underwater special operations,and submarine escape are highly likely to occur decompression sickness(DCS).The number of recreational divers increases rapidly,and the underwater and high-pressure engineering operations increase sharply.All of them are faced with great risks of DCS.Compared with common clinical diseases,the number of DCS cases is limited.The exploration of the pathogenesis,pathophysiological processes and prophylactic-therapeutic measures more relies on animal models.Small experimental animals such as mice and rats have been widely used in the DCS study.However,the physiological structures of small animals are quite different from those of human beings,so it is necessary to make use of large animal models before in-depth observation of the prophylactic-therapeutic measures,exploration of special mechanisms or application of new technologies to human body.Among non-primate animals,swine are close to human beings in terms of gene level,anatomical structure and physiological characteristics,especially the cardiovascular system,respiratory system,skin tissue and spinal cord,which are closely related to the occurrence of DCS.However,compared with mice,rats and other animals,pigs are less used as DCS models at present,especially in this field in China.Bama swine is the only artificially bred miniature pig in China,with clear genetic background,stable physiological index and good reproducibility.It is an ideal animal for the establishment of DCS model.This project aims to systematically study the swine DCS model.Hyperbaric exposure schemes are optimized with stable morbidity and evaluation system was established.Aiming at the core pathophysiological changes of DCS,the rules of inflammatory response and endothelial injury were explored.And the clinical symptoms and mechanism of DCS skin lesion,the most characteristic manifestation of swine DCS model was studied.As the most important pathogenic factor of DCS,bubble has been throughout the study of the model,including the time course of bubble load and its correlation with the changes of indicators and the evolution of symptoms.The value of the model lies not only in the exploration of the mechanism of the disease itself,but also in the fundamental research on the prophylactic-therapeutic methods.In the second part of this study,hyperbaric exposure schemes with different DCS severity were selected for different interventions.According to the mechanism of intervention,appropriate evaluation indexes are selected from the evaluation system,and the study on the model itself is constantly improved.Hyperbaric oxygen(HBO)is a therapeutic measure for DCS,while the use of HBO in preventive measures is still limited to small animals.Escin,an endothelial protective agent,is widely used in chronic venous insufficiency,soft tissue edema and hemorrhoids.Our research group has proved that both of them have a prophylactic-therapeutic effect on DCS in rat models.Based on the bama swine DCS model,this project further explores the effects and mechanism of HBO preconditioning and escin on DCS,providing a theoretical basis for the future promotion to DCS treatment.Part ? Research on bama swine DCS model1.Establishment of bama swine DCS modelMethods : Bama swine underwent a simulated diving with several hyperbaric exposure schemes with different depth,exposure time and decompression speed.After decompression,limb motor function and spinal cord evoked potential were evaluated at fixed time points,and DCS spinal cord injury was determined by the combination.The amount of bubbles and cardio-pulmonary function were assessed by ultrasonography.The evolution of skin symptoms was observed and blood index was determined.A set of evalutating parameters was screened considering the overall behavior,histopathology,circulation,neurophysiology,bubble generation and inflammation response.Results: Each exposure produced the DCS model with different morbidity and mortality.After decompression,there were obvious air bubbles in the right cardiac system and decreases of blood flow and pressure across the valve in the pulmonary valve orifice were observed.Typical skin lesions appeared.White blood cells and platelets reduced,and the amount of D-dinner and fibrinogen degradation product increased.Prothrombin time was prolonged.Congestion and bleeding were noticed in the lung and spinal cord.The change of the above indicators could objectively reflect the degree of DCS injuries.Conclusions: The bama swine DCS model with stable morbidity and mortality was successfully established.The above indexes can objectively reflect the damage degree of the model.2.Time course of inflammatory factors and endothelial markers of bama swine DCSMethods:Ten bama swine were divided into two groups and exposed two schemes of hyperbaric exposure,“30 msw(meter sea water)-120 min,6 min decompression” and “40msw-35 min,11 min decompression”.Ultrasound was used to detect the bubble load,and blood samples were collected according to the established time points to detect the levels of various inflammatory factors including interleukin-1?(IL-1?),IL-6,IL-8,monocyte chemo-attractant protein-1(MCP-1),and endothelial markers including endothelin-1(ET-1),vascular cell adhesion molecule-1(VCAM-)and intercellular cell adhesion molecule-1,(ICAM-1).The time course of the two types of index and their correlation with the bubble loading were observed.Results: After decompression,the bubbles peak at 30 min,the first detection time point,and there are still a few bubbles at 6 h.The bubble load of scheme 1 is higher than that of scheme 2.Decompression induces a significant increase in the levels of IL-1?,IL-6,IL-8,MCP-1,ET-1,VCAM-1 and ICAM-1.The time courses of indicators caused by different bubble loads are the same,and the time courses of each indicator are significantly correlated with the bubble loads except VCAM-1.Conclusions: After hyperbaric exposure,the amount of bubbles is highly correlated with inflammatory factors and endothelial damage markers,which proves that bubbles are the trigger factors for inflammation and endothelial damage.Inflammatory cytokines IL-1?,IL-6,IL-8,MCP-1,and endothelial injury markers ET-1,VCAM-1 and ICAM-1 can be used to assess the level of stress and the amount of bubbles in the body.3.Skin lesions in bama swine DCS: clinical appearance and pathogenesissMethods:Thirteen Bama swine underwent “40 msw-35 min,11 min decompression”hyperbaric exposure.After decompression,chronological changes in the appearance of skin lesions,skin ultrasound,temperature,tissue nitric oxide(NO)levels,and histopathology were studied in each stage.Meanwhile bubbles and central nervous system(CNS)function were monitored.Results: All animals developed skin lesions and two died abruptly possibly due to cardiopulmonary failure.A staging approach was developed to divide the appearance into six consecutive stages,which could help diagnosing the progress of skin lesions.Bubbles were only seen in the right but not left heart chambers.There were strong correlations between bubble load,lesion area,latency to lesion appearance and existence of cutaneous lesions.Even though local skin temperature did not change significantly,skin thicknessincreased,NO elevated and histological changes were observed.Increased vessel echo-reflectors in lesion areas were detected ultrasonically.No CNS dysfunction was detected by treadmill walking and evoked potential.Conclusions: The typical DCS skin lesions present a stage symptom evolution.The present results suggest skin lesions mainly result from local bubbles and not CNS injuries or arterial bubbles.Part ? Application of bama swine DCS model in the prophylactic-therapeutic research of DCS1.Benefits of hyperbaric oxygen pretreatment for DCS in Bama pigsMethods:Six swine were subjected to a session of “15 msw-1 h” HBO treatment.HSP32,60,70 and 90 were detected,before and at 6,12,18,24 and 30 h following exposure in lymphocytes.In the second part of our investigation,another 10 swine were randomly assigned into two groups(five per group).All swine were subjected to two “40msw-35 min,11 min decompression”hyperbaric exposures in a hyperbaric chamber with an interval of 7 days.Eighteen hours before each dive,the swine were pretreated with HBO or air: the first group received air pretreatment prior to the first hyperbaric exposure and HBO pretreatment prior to the second;the second group were pretreated with HBO first and then air.Bubble loads,skin lesions,inflammation(IL-8,MCP-1)and endothelial markers(ET-1,VCAM-1)were detected after each dive.Results: In lymphocytes,all HSPs increased significantly(P<0.05),with the greatest expression appearing at 18 h for HSP32 and 70.HBO pretreatment significantly reduced all the determined changes compared with air pretreatment.Conclusions: The results demonstrate that a single exposure to HBO 18 h prior to diving effectively protects against DCS in the swine model,possibly via induction of HSPs.2.Benefits of escin for DCS in Bama pigs by endothelia-targeting protectionMethods:Sixteen swine were subjected to a two-stage experiment with an interval of7 days.In each stage,seven days before a hyperbaric exposure,the swine were treated with0.4 mg/kg/d escin(0.16 g/L)or saline.The first group received a successive administration of escin for 7 d prior to the first hyperbaric exposure and saline for 7 d prior to the second;the second group was treated with saline and then escin.After decompression,signs ofDCS and circulating bubbles were monitored,and blood was sampled for platelet count and determination of inflammatory(IL-1?,IL-6)and endothelia related indices(ET-1,ICAM-1).Results: The death rate of DCS was markedly decreased in swine treated with escin compared with that in animals treated with saline,though not statistically significant due to the limited number of animals.Escin had no effect on bubble load but significantly ameliorated platelet reduction and endothelial dysfunction,as well as oxidative and inflammatory responses.Conclusions: The results suggest the beneficial effects of escin on DCS by its endothelia-protective properties.Endothelial injury is one of the important pathogenesis of DCS,which can be effectively countered by endothelial protection.