Study On The Changes Of MCP-1and MIP-1α In Wound Healing Of Sepsis Rats

PART I Establishment of the sepsis annimal modelObjective Establishing the method for the building of sepsis rat model.Methods40Sprague-Dawley rats of200-250g were divided into two groups randomly,Shamgroup and CLP group.The CLP group rats were made by being ligatured the distal1/3of cecum,and then being punctured single with the12th needle at the midpoint of the ligation segment,andits direction was perpendicular mesenteric. A little of stool was squeezed into abdominalcavity.After operation,we subcutaneously injected saline(5ml/100g) to resuscit the rats.Whilethe Sham group was the same of the CLP group,except ligature and puncture the cecum of therats.At6,12,24,and48hour post-operation,we recorded the symptoms,mortality,organpathological changes,and the levels of LPS of rats.And blood cultures was assessed at12hpost-operation.Homogeneity test of variance used Levene test, statistical significance werecompared with two independent samples T-test.Results The CLP group rats appeared the symptoms of irritability,chills, hair shaft,eating anddringking reduction at12h post-operation.But the Sham group rats were normal.The mortality ofthe CLP group was25%.The sham group rats were not dead.The organs of CLP group rats wereextravasated blood,some of which were adhesion with napes.The enterocoelia of CLP group hadmuch purulent exudate,with malodorous.Stomach and small intestine of CLP group werepneumatosis and expansion.The distal of cecum of CLP group swollen significantly,andavascular necrosis like purple.But the Sham group rats were normal.The texture of pulmonary ofCLP group rats were visible,the wall of pulmonary alveoli was thick obviously and some ofwhich was collapse.There were many inflammatory cells in pulmonary interstitial.The liver cellsof the CLP group rats were necrosis obviously.The cord of liver cells were dissociation.Therewere many inflammation cells in the hepatic sinusoidal and all of the cells showed obviousinflammatory changes at12h and24h points after operation.At each of time point,the level ofLPS in the CLP group rats was higher than that of Sham group rats(p<0.01).LPS peakingappeared at12h post-oparative(4.839±0.050Eu/ml),and then declined gradually to the normallevel.Many bacteria, such as Enterococcus faecalis, E. coli and other Enterobacter, were detectedin blood cultures of CLP group, but were not in Sham group at12h postoperative.Conclusions The rats which are ligatured and punctured appeared obviously thepathophysiologic symptoms of sepsis. This method was a good method for the building of sepsisannimal model. PARTⅡ The influence of sepsis on wound healing in ratsObjective To investigate the influence of sepsis on wound healing by observing the woundhealing processes of sepsis rats.Methods All rats were received full thickness skin wound (1.5cm×1.5cm) at the middle ofback.After two days which was equivalent to clinical burn shock stage,one group was receivedcecal ligation and punture(CLP group, n=25),the other was received sham(Sham group,n=25),the rest of rats was not treated(Sham group, n=25).At different time point post-injure,werecorded the time of wound healing and the size of wound by digitally photographe by randomlyselecting5rats in each group, scored the wound with the standard of wound healing score, anddetected histological structre of wound, collagen formation and angiogenesis.Homogeneity testof variance used SNK test, if variance was homogeneity, statistical significance were comparedby completely randomized design analysis of variance; If variance was heterogeneity,statisticalsignificance were compared by multiple independent samples to compare Kruskal-Wallis H test.Results The time of wound healing in CLP group rats was13±1.87d, was delayed significantlycomparing with the Sham group and the N group.At3d and5d of post-injure,the rate of woundhealing in CLP group rats was lower than the N group(p<0.05),but there was no difference withthe Sham group.But at the7d and10d post-injure,the CLP group (52±8%，76±10%) was lowerthan that of the Sham group（71±10%，94±2%）(p<0.05).while no different at14d.At3dpost-injure,the score of wound in CLP group was no different with the Sham group and the Ngroup (P>0.05),but at the other time point,the CLP group was lower than other twogroups.Except the3d post-injure,the formation of collagen and angiogenesis in CLP group werelower than that of Sham and N groups in other time points.(p<0.05).Conclusions Sepsis delayed would healing significantly in sepsis rats.PARTⅢ The changes of chemokine of MCP-1and MIP-1α in wound healing of sepsis rats.Objective Elucidate the influence of MCP-1and MIP-1α on wound healing of sepsis rats bychecking the expressions of them at protein and mRNA levels in wound healing process.Methods Annimals and groups were the same as the part Ⅱ.The expression of MCP-1andMIP-1α protein in wound were measured by ELISA,and their mRNA were measured byRT-PCR.Homogeneity test of variance used SNK test, if variance was homogeneity, statisticalsignificance were compared by completely randomized design analysis of variance,If variancewas heterogeneity,statistical significance were compared by multiple independent samples tocompare Kruskal-Wallis H test. Results The peak expression of MCP-1and MIP-1α protein were at3day post-injure in allgroups,and declined gradully to the normal level at10d post-injure.At3d and5d post-injure,theexpression of MCP-1in CLP group were higher than that of N group(p<0.05),but there was nodifferent with the Sham group(P>0.05).While at other time points,the CLP group were higherthan that of Sham and N group(p<0.05). At3d post-injure,the expression of MIP-1α was higherthan N group signcially,but no different with Sham group.At5d post-injure,the expression ofMIP-1α in CLP group was higher than Sham group and N group.And at7d post-injure,there wasno different among these three groups(P>0.05).But at10d post-injure,the expression ofMIP-1α(208.94±65.33pg/ml) in CLP group increased suddenly, then declined rapidly.And at14dpost-injure,the CLP group was lower than Sham group and N group.While the changes ofmRNA of MCP-1and MIP-1α were consistency with the expression of their proteins.Conclusions The expression of MCP-1was high in early stage and low in later stage in woundhealing of sepsis rat and the expression of MCP-1was the reason that delayed wound healing ofsepsis rats. But MIP-1α was not the key factor which influences would healing of sepsis rat.