Experimental Research Of The Pathogenesis And Treatment On The Early Onset Of Acute Lung Injury Induced By Seawater-immersion Following Open Chest Trauma

ObjectiveIn the modern sea conflict, soldiers with open chest trauma were very susceptible to fall into the sea, and immersed in seawater. Subsequently, acute lung injury (ALI) would happen following open chest trauma and seawater-immersion of the thoracic cavity. Acute lung injury induced by seawater-immersion following open chest trauma (SI-ALI), which further developed to acute respiratory distress syndrome (ARDS) and the multiple organ dysfunction syndrome (MODS), is associated with high mortality. Unfortunately, the pathogenesis and management of these complex and often lethal syndromes have been unclear. Though the lung would be injuried heavily and resulted in severe and intricate physiopathologic changes, there is rare report from overseas and a few from domestic about this special ALI. Li Hui and colleagues reported the clinical manifestations and drew up some measures to the trauma on the early onset of SI-ALI. Professor Duan and our team expended considerable effort to study the comprehensive mechanisms and treatment measures of acute lung injury following open chest trauma and seawater-immersion.Pathogenic factors, such as seawater, cytokines, may play crucial role in the pethogenesis of SI-ALI. Higher osmotic pressure, lower temperature and abundance of salts are the mainly characteristics of seawater. On the physiological action, the role of VEGF on endothelial cell permeability and proliferation, have been demonstrated in studies. But there are no simple answers to the pathological issue, such as ALI, which require an understanding on the roles not only VEGF lead to acute pulmonary edema, but also VEGF protects the epithelial cells from cell death and activate endothelial cell proliferation. Pre-B cell colony-enhancing factor (PBEF), highly conserved in evolution, is a 52-kDa protein found in living species from bacteria to humans and is an endogenous inflammatory mediator. Recently, PBEF was discussed as a novel biomarker for ALI because of its inhibitting apoptosis of neutrophils in ALI. Despite extensive research and increasing awareness of ALI, few studies have been done on the relation between PBEF and ALI induced by seawater-immersion after trauma. We knew a little about their roles in SI-ALI. Therefore, in this study, we would focus on discussing the role of these pathogenic factors in the pathogenesis of SI-ALI. Furthermore, we want to give evidence that dexamethasone attenuate the inflammatory response, at least partially. Today's aim is, firstly, to investigate the pathophysiological changes through comparing the differences of the injury respectively by seawater-immersion or freshwater-immersion following open chest trauma in dog models; secondly, to investigate the levels of VEGF and PBEF in SI-ALI and further explore whether the changes of VEGFs and PBEF are connected with the pathogenesis of SI-ALI. Thirdly, to evaluate whether the low-dose dexamethasone treatment on the early onset of SI-ALI could act as an inhibitor of systemic inflammation and prevent or attenuate the lung injury following open chest trauma and seawater-immersion in dog models.MethodsAll investigations involving experimental dogs were reviewed and approved by the Institutional Review Board (IRB) of Beijing Navy General Hospital Animal Care and Use Committee. We prefer large animals for their essential tolerance to fulfill the hard experimental process. Dogs were randomly divided into four groups:control group (CG), seawater group (SG), freshwater group (FG) and dexamethasone treatment group (DG). All dogs were anesthetized with intramuscular injection of ketamine (20mg/kg) and assisted ventilation with endotracheally intubated. A 0.5 cm diameter incision was made with a sharp instrument between the 4 th and 5 th ribs and into the right chest and a hollow plastic tube was placed into the incision for 5 min and then pull it out to form an opened pneumothorax. The dogs in CG only suffered from pneumothorax, whereas seawater or freshwater (35ml/kg) was slowly infused into the pleural cavity of animals in SG or FG, respectively. The incision skins of all experimental dogs were sutured 10 min after trauma. The right carotid artery and jugular vein were cannulated for drawing blood. Fluid or air in the chest made it difficult to breathe. Arterial blood gas values were monitored and the oxygen index (PaO2/FiO2)<300 mmHg is the standard to prove the success of the ALI model and all experimental dogs met the criteria for ALI.Blood samples were collected at 0 h before trauma and 2,4,6 and 8 h after trauma. At 8 h, lungs were lavaged thrice with 15 ml of saline through the endotracheal tube and gently aspirating back to collect the broncho-alveolar lavage fluids(BALF).Whereafter, the dogs received an intracardiac injection of 15 ml of 15% KCl and were then sacrificed. The lungs were removed for histological evaluation. The levels of total protein both in plasma and BALF were measured to calculate the pulmonary vascular permeability index (PVPI). Lung injury was assessed by histology. The levels of VEGF, PBEF and inflammatory cytokines in plasma were measured by ELISA Kit. The levels of PBEF and VEGF protein in lung tissue were evaluated with immunohistochemistry and Western blotting. Quantification both of VEGF mRNA and PBEF mRNA were evaluated in lung tissue by real-time reverse transcriptase-PCR. Data were compared between groups using ANOVA analysis.Results1. The SI-ALI dog model copied successfully. All animals in "SG" represented increasing breath frequency and breath distress following trauma. Arterial oxygen partial pressure (PaO2) decreased markedly. Arterial oxygen partial pressure/suction gas oxygen concentration (PaO2/FiO2) which oxygenation index dramatically reduced in 2 h and continuely reduced in 6-8 h after trauma, and maintained less than 300 mmHg, which met the criteria of ALI. The pathological changes in the "SG" demonstrated a marked lung injury, represented by hemorrhage, edema, thickened alveolar septum, formation of hyaline membranes and the infiltration of inflammatory cells in alveolar spaces.2. Though arterial oxygen partial pressure (PaO2) and arterial oxygen partial pressure/suction gas oxygen concentration (PaO2/FiO2) decreased in "FG", they didn't meet the criteria of ALI. Compared with the samples from "FG", plasma osmotic pressure (POP, mmol/L) in "SG "was significantly increased (SG vs. FG: 326.66±3.45 vs.268.61±4.40, P<0.05); the pulmonary vascular permeability index (PVPI) significantly increased in "SG "(SG vs. FG:0.055±0.002 vs.0.034±0.007, P <0.05); the cytokines levels (pg/ml) were markedly increased in "SG" (IL-1β: 123.82±46.39 vs.66.53±13.88; IL-8:79.91±8.24 vs.57.69±8.37; vWf:1.03±0.09 vs. 0.64±0.08, P<0.05); the levels of PVPI, IL-1βand IL-8 were significantly positively correlated with the plasma osmotic pressure (rho:0.743,0.616 and 0.638, P<0.05).3. Compared with animals in "CG", the levels of both IL-1βand IL-8, POP, PVPI significantly increased in "SG", P<0.05; The plasma levels of both VEGF and sVEGFR-1 (pg/ml) significantly increased in "SG" (SG vs. CG, VEGF:72.20±25.41 vs.27.28±2.27, P<0.05; sVEGFR-1:1.92±0.32 vs.0.61±0.13, P<0.05); The level of VEGF protein in lung tissue was increased in "SG", (SG vs. CG,0.2375±0.036 vs.0.1649±0.031, P<0.05); The level of VEGF mRNA synthesis in lung tissue was increased in "SG", (SG vs. CG, VEGF mRNA:5.04±0.29 vs.0.25±0.04, VEGFR-2 mRNA:5.08±0.20 vs.0.64±0.02, P<0.05). The levels of VEGF was significant correlatiom with POP and PVPI.4. Compared with animals in "CG", PBEF levels (pg/ml) significantly increased in "SG" (plasma:3014.16±883.47 vs.1060.94±251.08, P<0.05; BALF:1373.35±102.53 vs.997.77±70.31, P<0.05; lung tissue:1455.22±71.74 vs.921.43±118.13, P<0.05); PBEF level were positively correlated with IL-1βand IL-8 (IL-1β: rho is 0.820, P<0.05; IL-8:rho is 0.842,P<0.05).5. Compared with the samples in "SG", the levels of IL-1βand IL-8 from plasma (pg/ ml)were markedly decreased in "DG". (IL-1β:90.97±41.99 vs.144.11±29.72; IL-8: 45.21±16.39 vs.88.26±6.66, P<0.05); In lung tissue, the levels of IL-1β(40.18±10.59 vs.60.72±10.87, P<0.05) and IL-8 (34.63±5.55 vs.49.39±8.61,P <0.05) were decreased significantly in 8 h after treatment. The PVPI was markedly decreased too (0.039±0.006 vs.0.055±0.002, P<0.05); But there was no significant difference of electrolyte concentration and POP between these two groups (P>0.05).Conclusions1. The injury of immersion in seawater following open chest trauma is severer than that of in freshwater. It is attributed to the seawater's characteristic of higher osmotic pressure and abundance of salts, which increasing the PVPI and aggravating the inflammatory response.2. Higher levels of VEGF and it's receptor in plasma and lung tissue are distinguishing characteristics during the early onset of SI-ALI. Furthermore, the levels of VEGF in the early onset of SI-ALI were significantly positive correlated with POP and PVPI. Early release of VEGFs increase pulmonary vascular permeability and partially lead to the development of SI-ALI. VEGFs may have a crucial role in the early onset of SI-ALI.3. Higher levels of PBEF in the early onset of SI-ALI were significantly positive correlated with inflammatory factors. As a proinflammmatory and destructive mediator, PBEF have important roles in the development of SI-ALI.4. low-dose dexamethasone have an protection on the early onset of SI-ALI through decreasing the inflammatory response and improving the pulmonary permeability.