Effects Of Planned Peritoneal Lavage On Septic Rats With Cecal Ligation And Puncture

Damage Control Surgery(DCS)has been used in the clinical treatment of abdominal sepsis more and more.However,the feasibility and effectiveness of planned damage control operations in abdominal septic animal models have not been reported.In this study,the abdominal septic rat model was set up with cecal ligation and puncture,which was then treated with multiple planned peritoneal lavage,and the effects on rats were explored from the aspects of general performance,survival,inflammatory immunity and organ function,providing basis for the research and clinical practice.PART 1: To establish a septic rat model with cecal ligation and puncture,and to explore its characteristics and test its stabilityObjective:(1)A cecal ligation and perforation(CLP)method was chosen to establish a rat model of abdominal sepsis.(2)The survival performance and general condition of the rats were recorded and analyzed.(3)The hepatic function changes and pathological states were recorded and analyzed.(4)ELISA(enzyme-linked immunosorbent assay)was used to detect changes in serum endotoxin and cytokines TNF-α(tumor necrosis factor-α)and IL-10(interleukin-10)at different time after surgery.Methods:(1)20 SPF grade,8-10 weeks old,weight 300±20g,male SD rats were randomly divided into two groups(n=10),namely SHAM group and CLP group.SHAM group only underwent laparotomy,and CLP group were treated with the cecal ligation and puncture method to establish an abdominal septic model.To observe the survival,general conditions of the postoperative animal models,and calculate MSS(murine sepsis score).(2)Seventy rats were randomly divided into SHAM group(n=20)and CLP group(n=50).Rats in each group were sacrificed at different time points after operation at 3h,6h,12 h,24h,48 h,blood was taken from the inferior vena cava,and liver function was detected,including ALT(alanine aminotransferase)and AST(aspartate aminotransferase).Hepatic specimens were taken for HE staining of paraffin sections to observe pathological changes.(3)As above,rats in each group were sacrificed at different time after surgery,blood was taken,and endotoxin,cytokines TNF-α and IL-10 were tested by ELISA.Results:(1)Within 7 days after CLP,SHAM group all survived.The mortality of CLP group was significantly increased at 24h(70%),and all died before 7d.CLP group showed signs of pilling,blinking,lack of energy,and decreased activity,and it worsened with time.The MSS in the CLP group was significantly higher than that in SHAM group(P<0.05).(2)The hepatic function indexes,ALT and AST,showed no significant changes in SHAM group,while increasing significantly with time in CLP group.HE staining showed that the normal hepatic lobule structure was damaged,the central venule was dilated,the liver cells underwent edema,vacuolar degeneration and necrosis,and the inflammatory cells infiltrated in a large amount,and the tendency was aggravated with time.(3)Compared with SHAM group,the endotoxin level in the CLP group increased significantly at after 6h,,and reached the top at 48h(P<0.05).The TNF-α and IL-10 levels in SHAM group kept stable,while TNF-α increased significantly at 6h and peaked at 24 h in CLP group(P<0.05).The early increase of anti-inflammatory factor IL-10 was slightly slower,increased significantly at 12 h,and peaked at 48h(P<0.05).Conclusion: The lower survival level and typical infectious symptoms in CLP group showed that the acute severe abdominal septic model was successfully established.The biochemical and pathological results after modeling showed that the liver function continued to deteriorate,reflecting the occurrence of organ failure in sepsis.The release of inflammatory factors after modeling suggested the early promotion of inflammatory response induced by TNF-α,and the enhanced anti-inflammatory response mediated by IL-10 in the later period.The results above provided relevant evidence for subsequent reoperation and immune mechanism studies.PART 2: Planned peritoneal lavage in the rat model of abdominal sepsis was performed to explore its effects in different aspectsObjective: To investigate the effects of DCS-directed planned relaparotomy on abdominal septic rat model in survival,organ function,inflammation and immunity.Methods:(1)55 SD rats were randomly divided into 4 groups,namely SHAM(n=10),CLP(n=15),SPL(n=15)and MLP(n=15).SHAM and CLP groups were treated as before.SPL group underwent peritoneal lavage at 6h after CLP only for once.MPL group underwent planned reoperations of peritoneal lavage at 6h,18 h,and 36 h after CLP.(2)The survival performance and general condition of each group were observed and recorded.(3)Rats were sacrificed at 48 h after CLP.Blood was taken from the inferior vena cava and ALT,AST,endotoxin,cytokines TNF-α and IL-10 were detected.(4)At 48 h after CLP,liver samples were taken for HE staining,to observe pathological changes.(5)Rats in each group were sacrificed at 48 h after CLP,spleen was taken to prepare mononuclear cell suspension,and Tregs level was detected with flow cytometry.Results:(1)At 48 h after CLP,all the SHAM group survived,and the CLP srrvival rate was 26.7%.The survival rates of the SPL and MPL groups were better than that of the CLP group at 48 h,which were 33.3% and 46.7% respectively.(2)The hepatic function indexes of ALT and AST were the lowest in the SHAM group,the highest in the CLP group,and the MPL group was lower significantly compared to SPL(P<0.05).Endotoxin and serum cytokine TNF-α and IL-10 were the lowest in SHAM group,highest in CLP group.Only endotoxin and IL-10 in MPL group are much lower than in SPL group(P<0.05),while the TNF-αlevel in MPL group was a little higher than SPL group,but with no significance(P>0.05).(3)Hepatic HE staining results showed that SHAM group was normal.In CLP group there were obvious cell necrosis,hepatic lobular structure damage and inflammatory cell infiltration.These changes in SPL and MPL groups were improved with MPL group improved more.(4)Tregs level at 48 h after CLP was significantly higher in CLP than in other 3 groups(P<0.05),the lowest in SHAM,and significantly lower in MPL group than in SPL group(P<0.05).Conclusion: Planned multiple peritoneal lavage based on DCS can effectively improve the survival of severe abdominal septic rat model,reduce the bacteria and endotoxin release in infectious location and blood by debridement and unobstructed drainage,and moderate the systemic inflammatory response caused by infection,so as to protect organ function,improve immune disorder,and provide reliable reference for the study of pathophysiological mechanism and clinical practice for severe abdominal sepsis.