The Metabolic Changes Of Intestine By Microdialysis During Trauma/Hemorrhagic Shock And Its Implication In Gut Barrier Dysfunction

Objective: Gut is the motor of MODS after hemorrhagic shock.The aim of this study was to determine the mechanism of gut barrier injury and the relationship between gut injury and metabolic products detected by intestinal microdialysis in a trauma/hemorrhagic shock rat model.Methods: Rats were randomized into two groups,trauma/hemorragic shock group(THS)and trauma/sham shock group(TSS).After intraperitoneal anesthesia,a femoral arterial and venous line was inserted.With arterial line for continous monitoring of mean blood pressure,we drew blood to keep mean arterial blood pressure at almost 30 mm Hg for 45 minutes.Then we resuscitated the rat with a mixture of 2 shed blood volume of normal saline and shed blood,which was infused back into the femoral vein in 2 hours with constant speed.We still made an observation of 6 hours after the end of resuscitation,also called recovery phase,not any fluids except for anesthetics in this period.To do laparotomy,we did a 5 cm incision in the midline of abdomen.In TSS group,the rats were undergoing the same procedure except for the shock part(blood withdraw and resuscitation).After laparotomy,two microdialysis catheters were placed on the mucosal and serosal side of ileum separately before shock,and were perfused with dextran-70 throughout the whole experiment period of almost 10 hours at a speed of 1ul per minute.Dialysate samples were collected in every 60 mins for future analysis of glycerol,glucose,lactate,pyruvate and glutamate.Blood routine test and blood gas analysis were analysized.Intestinal permeability was assessed using fluorescein isothiocyanate(FITC)-labeled dextran and serum was collected and fluorescence was quantified at an excitation wavelength of 485 nm and an emission wavelength of 535 nm.Intestinal tissues were sectioned and subjected to hematoxylin and eosin(H&E)staining and alcian blue(AB)staining.Histopathological analysis was performed in a blinded fashion for Chiu gut injury score,total score of crypt damage and inflammatory cell infiltration,height of villi.Mucus content was measured by calculating the intensity of the areas stained by AB and presented as mucus score.Immunohistochemistry was performed with antibody Muc-2 to determine the numbers of goblet cells per villi.Cytokine(IL-6,IL-1?,IL-4,IL-10,TNF-? and IFN-?)levels in the intestinal tissues were examined by enzyme-linked immunosorbent assay(ELISA)kit according to the manufacturer's instructions.Western blot analysis was performed to verify the protein of ZO-1,occludin?claudin-2 and glutaminase.Realtime PCR was performed to verify the gene expression of ZO-1,occludin,claudin-2 and Muc1,Muc2,Tff1,Tff3 and glutminase.High performance liquid chromatography was performed to analyze amino acids in rat plasma and mucosa tissue.Colorimetry was used for mucosal glutaminase activity test.Results: We performed a severe trauma /hemorrhagic shock model and tested by arterial blood gas analysis and blood routine test.We successfully performed the technique of microdialysis in this rat model and found that both mucosal microdialysis and serosal microdialysis could detect the increased change in concentration of glucose,lactate,glycerol and pyruvate but the elevation of glutamate could only be detected by mucoal microdialysis catheter.The altitude of glucose and lactate change by microdialysis was consistent with blood glucose and lactate change.The altitude of elevation of glucose,lactate,glycerol and pyruvate was similar in both mucosal microdialysis and serosal microdialysis.The concentration of all metabolites by microdialysis except for glutamate was elevated from shock period and peaking at the first hour of resuscitation period and decreased to baseline in early restoration period.But the elevation of glutamate was lagged after the other four metabolites as only could be detected by mucosal microdialysis for resuscitation period and last to late restoration period.The change of intestinal metabolites by microdialysis was accompanied by increased intestinal permeability,increased score of gut injury,decreased intestine villi height,destruction of tight junction,mucus and goblet cell number.Hemorrhagic shock induced intestinal inflammation tested by increased inflammatory cytokines of TNF–?,IL-6,IL-1?,IFN-?and decreased inflammatory cytokines of IL-4 and IL-10,together with infiltration of inflammatory cells to intestinal lamina propria.The injury to gut barrier function indicated by increased gut permeability,destruction of epithelial tight junction and decreased mucus content could possibly be regulated by decreased gene expression of tight junction protein ZO-1,occludin,claudin-2 and mucin gene Muc1,Muc2,Tff1,Tff3.An elevation of glutamate in the gut lumin was in consistent with elevation of glutamate in the gut mucosa,decresed plasma glutamine and increased glutaminase both in contentand in activity in the mucosa in gut restitution period.Conclusion: 1.Trauma/hemorrhagic shock could induce gut barrier dysfunction which was indicated with destruction of barrier structure and increased gut permeability.2.The change of gut permeability in hemorrhagic shock could be detected by microdialysis technique both in intestinal mucosal side and serosal side.3.The elevation of glutamate in intraluminal microdialysis catheter is inconsistent with an elevation of glutamate concentration in intestinal mucosa layer,together with an increased expression and activity of glutaminase in mucosa layer in intestinal restitution period which could transform glutamine to glutamate in the intestinal mucosa.