| Objective: In this study,a model of rat＇s epigastric axial flaps withischemia reperfusion injury was performed, and Sodium Ferulate was injectedinto intraperitoneal of rat,to preliminarily discuss the mechanism of protectiveeffects of Sodium Ferulate on rat＇s flaps with ischemia reperfusion injury andto provide a reliable experimental basis for clinical application throughobserve its influence on the surviral areas and the change of histolgicalstructure of rat＇s epigastric axial flaps with ischemia reperfusion injury, andobserve its concentration change of Malondialdehyde (MDA), superoxidedismutase(SOD), nitric oxide(NO),endothelin(ET) in the flap tissue and itsinfluence on the expression of nuclear factor κB (NF-κB),tumor necrosisfactor(TNF) in the flaps＇endothelial cells before and after reperfusion.Method：Selected96healthy Wistar rats, male or female, weighing300~350g,(provided by laboratory animal center Hebei medicaluniversity).The rats were divided into4groups randomly with each group24rats. A group,which was not involved ischemia reperfusion injury(None-IRcontrol group)；B group,which was control group involved ischemiareperfusion injury with normal saline treatment(NS-IR control group)；Cgroup,which was experiment group involved ischemia reperfusion injury with20mg/Kg Sodium Ferulate treatment(Small-SF-IR experiment group)；Dgroup,which was experiment group involved ischemia reperfusion injury with40mg/Kg Sodium Ferulate treatment(Large-SF-IR experiment group)。The rats were depilated by the8%Sodium sulfide two days before theoperation and anesthetized by intraperitoneal injection of3%chloral hydrate(30mg/kg) when operation.Based on the methods described by Petry JJ et.al, an3cm×6cm axial flapwas performed under the right abdomen taking the abdominal wall shallow blood vessel as the pedicle. Full dissociated the Femoral Artery beside whichinferior epigastric arteries sending and blocked it by micro vascular clamp,and the flap was then repositioned to its orginal location with silk sutures.10hours after flap ischemia,the rats were reanesthetized and the micro vascularclamp was removed.The flaps were examined under the operative microscopeto confirm reperfusion of the tissue.0.5ml Nomal Saline was injected intointraperitoneal of rat10minutes before operation and reperfusion, and8h afterreperfusion. A1.0cm×0.5cm skin tissue at the margin middle of the flaps weretaken from8rats of this group after rising flap instantly and2h afterreperfusion. And then took partial tissue immediately to treat fixedly with4%paraformaldehyde to be Paraffin-embedded to HE staining to examine thehistological structure change of the flaps tissue and Immunohistochemicalstaining to examine the expression of NF-κB,TNF-a in the flaps＇endothelial cells, the others were preserved by frozen until tissue homogenatewas operated,and then SOD,MDA,NO,ET,TP were examined.Simultaneously,2h after reperfusion two rats were selected randomly whichflaps were taken0.1cm×0.3cm skin tissue at the middle of the flaps above afroze plate. The specimen were fixed with3%glutaraldehyde to be madeultrathin sections to examine the ultrastructure change of the flaps tissue underTEM. The others8rats the specimen were harvested at the same way,and thesame assays were examined. The others8rats were not harvested specimenand the survival flaps were observed7d after the operation, and survival rateswere calculated.Small-SF-IR experiment group, Large-SF-IR experiment group were givenintraperitoneal injection of20mg/Kg and40mg/Kg Sodium Ferulate, The restof the operations were same with NS-IR control group.None-IR control group the flaps were repositioned to its orginal locationwhen the flap was performed, no others ways was treated.8rats thespecimen were harvested at the same way at the moment the flap wasperformed,12h after operation,The others8rats the specimen wereharvested at the same way18h,34h after operation. The rest of the operations were same with NS-IR group.Survival situation of flaps were observed roughly7d after operation.The necrosis of flap was adjusted by its appearance, color and texture. Onthe7th day after operation, the necrosis situation was draw in transparentpaper.The flap surviral was caculated by weight transparent paper method。The all Biochemical examination test values of SOD,MDA,NO,ETwere corrected by TP proteinAfter immunohistochemical staining the location and expression ofNF-κB,TNF-a was observed with a light microscope. Using HMIAS-2000high-definition color medical graphic analysis system software,5nonoverlapping400times magnification fields of visions was selectedrandomly in each slice,and to measured the average brightness value ofpositive cells, and calculate the average.Different kinds of results were dealt with by using SPSS V13.0software.One-way ANOVA was used to compare the difference between different groupmean. P<0.05was with a significant difference.Results:1General observation of flap7days after operation the flaps of Non-IR group were all survived, andtheir color were pink, good elasticity,not atrophy,and the temperture of flapswere normal. In the NS-IR control group, most of middle and distal flaps werenecrosis,the appearances were pale black to dark brown, some crusting, poorflexibility, cold skin temperture.And the flaps were atrophy and weresignificantly smaller.Rising off the distal flap, observed a little inflammatorysecretions under the scab. The flaps necrosis of Small-SF-IR experimentgroup,Large-SF-IR experiment group was similar. Partial distal flap wasnecrosis, the necrotic area was smaller than Non-IR group.The appearancesof proximal Flap were pink, texture were soft,.the distal were dark brown,texture were harden, flexibility were poor. It could be seen that None-IRcontrol group flaps survival significantly better than the other threegroups,Small-SF-IR experiment group, Large-SF-IR experiment group were better than the NS-IR control group. Large-SF-IR experiment group wasslightly better than the Small-SF-IR experiment group.2Flap's survival rate:The Non-IR group (100±0.0)%>Large-SF-IR experiment group (81.2士4.3)%> Small-SF-IR experiment (67.4士5.2)%>NS-IR control group (36.3士4.5)%,the difference between different group mean significant weredifference(P<0.05)3,Laboratory examination results3.1The observation under light microscope:None-IR control group in different time piont the layers structure of flaptissue were clear, nucleus were deeply stained and not inflammatory edemaapparently. NS-IR control group:2h after reperfusion, a small amount ofneutrophils were infiltration;8h after reperfusion, subcutaneous tissue edemawas aggressive,the tissue structure was disorganized, and a large number ofneutrophil adhered to the vessel wall, the integrity of the capillary vessel wallwas damaged;24h after reperfusion, the situation was further aggressive, alarge number of neutrophil infiltrated to the tissue space, blurred layersstructure of flap tissue, part of the muscle fiber necrosis. Small-SF-IRexperiment group and the Large-the SF-IR experiment group were slightlycompared with the NS-IR control group at different time piont.24h afterreperfusion the situation was better than before, but still a large number ofneutrophil cell infiltrated3.2ultrathin sections observed by transmission electron microscopy:None-IR group endothelial cell structure in the flap tissue were stillintact, showing that the mitochondrial cristae, cristae and membrane structurewere still clear, the endoplasmic reticulum to see a small amount ofdegranulation. Small-SF-IR experimental group and the Large-SF-IRexperimental group observed part of the mitochondrial cristae disappearedand fused with membrane, desmosomes between the basal cell,endoplasmicreticulum degranulation. The NS-IR control group: observed endothelial cellsseverely damaged, showing that the mitochondria fused with membrane,the structure was obscure, part of the cristae was cleft and missing.4Organization of biochemical examination4.1,MDA Determination:In the four groups,after raising flap instantly MDA concentration wasabout3.2nmol/mgp, no significant difference (P>0.05). Reperfusion after2h,8h,24h, the MDA value increased as reperfusion time procceed, the meanshowing: the NS-IR control group> Small-SF-IR experiment group>Large-SF-IR experiment group> None-IR group. Comparisons between eachgroup were statistically significant (P <0.05)4.2,SOD Determination:In the four groups,after raising flap instantly SOD concentration was about80.5U/mgp, no significant difference (P>0.05). Reperfusion after2h,8h,24h,the mean showing: None-IR group> Large-SF-IR experiment group>Small-SF-IR experiment group> the NS-IR control group. Comparisonsbetween each group were statistically significant (P <0.05).4.3,ET Determination:In the four groups,after raising flap instantly ET concentration was about18.2pgl/mgp, no significant difference (P>0.05). Reperfusion after2h,8h,24h the mean showing: the NS-IR control group> Small-SF-IR experimentgroup> Large-SF-IR experiment group> None-IR group. Comparisonsbetween each group were statistically significant (P <0.05).4.4,NO Determination:In the four groups,after raising flap instantly NO concentration was about6.65nmol/mgp, no significant difference (P>0.05). Reperfusion after2h,8h,24h the mean showing: None-IR group> Large-SF-IR experiment group>Small-SF-IR experiment group> the NS-IR control group. Comparisonsbetween each group were statistically significant (P <0.05)5Immunohistochemical staining results:5.1Nuclear factor NF-κB determination:NF-κB expression in vascular endothelial cells and neutrophils. In the fourgroups after raising flap instantly there were no NF-κB positive cells. the positive expression of the None-IR control group were weak at each timepoint. Small-SF-IR experiment group, Large-SF-IR experiment group andNS-IR control group after reperfusion2h,8h,24h a large number ofNF-κB-positive cells could be seen. NS-IR control group expressed strongestafter2h reperfusion, Small-SF-IR experiment group, Large-SF-IRexperimental group expressed the strongest after8h reperfusion. the meanshowing: the NS-IR control group> Small-SF-IR experiment group>Large-SF-IR experiment group> None-IR group. Comparisons between eachgroup were statistically significant (P <0.05).5.2Tumor necrosis factor (TNF-a) determination:TNF-a expression in vascular endothelial cells. In the four groups afterraising flap instantly there were few TNF-a positive cells. the positiveexpression of the None-IR group were weak at each time point. Small-SF-IRexperiment group, Large-SF-IR experiment group and NS-IR control groupafter reperfusion2h,8h,24h a large number of NF-κB-positive cells could beseen. NS-IR control group expressed strongest after2h reperfusion,Small-SF-IR experiment group, Large-SF-IR experimental group expressedthe strongest after8h reperfusion. the mean showing: the NS-IR controlgroup> Small-SF-IR experiment group> Large-SF-IR experiment group>None-IR group. Comparisons between each group were statisticallysignificant (P <0.05).Conclusions:1.Sodium Ferulate maybe through increase concentration of NO byinhibiting the action of endotheline and then decrease activation of NF-κBresult in inhibiting cascade of inflammatory response, alleviate flapischemia-reperfusion injury and result in improving flap tissue survival2.Sodium Ferulate can scvenge free radical directly and then alleviateinflammatory response and decrease activation of NF-κB result in inhibitingcascade of inflammatory response,decrease flap ischemia-reperfusion injuryand result in improving flap tissue survival... |
PDF Full Text Request