The Study On The Protective Effects Of Exogenous Zine In Rat Flaps With Ischemia-reperfusion Injury

Objective: To observe the expressive location and quantityof metallothionein(MT) in the rat flap with ischemia-reperfusioninjury through the method of immunohistochemistry. To observethe changes of the ultrastructure in skin flap with transmissionelectron microscope. To study the protective effects that theexogenous zinc has on the ischemical reperfusion injury flapand its mechanism, through which a new method that can lessenischemical reperfusion injury clinically was explored.Method: 1. laboratory animal: 48 male and healthy Wisterrats were weighed and ordered. 2. The creation method of flap:The rats were anesthetized with 2% sodium pentobarbital(40mg/Kg)by intraperitoneal injection, their limbs were fixedand their hairs on abdmon were sheared, the abdmen wassterilized by 75% alcohol after the hairs were cut down. Aright low abdominal island flap, which size was 3cm×6cm, wascreated according to the method of Petry JJ's. 3. The rats weredivided into three groups randomly. The first group was controlgroup,which included 16 rats. There were no other treatmentsafter creating the flap in this group. The second group wasischemia-reperfusion group(IR group), which included 16 rats.The operation was done 24 hours after 0.3ml saline was injectedinto the rats'abdominal cavity. The distally of the point that thesuperficial epigastric artery arised from the femoral artery wasligated with 5-0 suture silk. The proximal end of the point wasoccluded by a vascular clamp .The flap was sewed up with 3-0suture silk. The vascular clamp was taken out in anotheroperation after 8 hours. The third group was Zn-IR group, whichincluded 16 rats. The operation was done 24 hours after 4.4%ZnSO₄·7H₂O(1ml/Kg)was injected into the rats'abdominalcavity. The following treatments were same with the IR group. 4.To draw the materials from the flaps: Full-thickness of the flapwhich size was about 1cm×0.5cm was taken respectively at themoment when the flap was created, 1 hour and 24 hour afterischemia-reperfusion in the IR group and the Zn-IR group. Inthe control group, the sample of flap was taken at the momentwhen the flap was created, 9 hour and 33 after the operation.Part of the obtained flap was conserved in 4% paraformaldehydein order to commit paraffin section and dyeing ofimmunohistochemistry. Part of the obtained flap was conservedin a freezer in order to detected the content ofmalondialdehyde(MDA) and the activity ofmyeloperoxidase(MPO). Full-thickness of the flap that wasabout 0.3cm×0.3cm was taken respectively 24 hour afterischemia-reperfusion from the IR group and the Zn-IR group.The obtained flap was conserved in 4% glutaral in order toobserve the changes of the ultrastructure. The flap viability inthese three groups was observed seven days after the operation.5. The observation marker (1) According to the specification ofthe test kit, the content of MDA and the activity of MPO weremeasured . The quantity of the protein in the obtained flap wasmeasured with the test kit of coomassie light blue. (2) Sevendays after the operation, the flap viability was observed. Theareas of the total skin flap and the survival skin flap weredepicted on a sulfuric acid paper. The papers on behalf of theflap area were sheared and were weighted with the electronicscale. The flap area was reprensented by the paper's weight.Then the flap viability was calculated. (3) The method of imageanalysis: The location and the pigmenting of MT were observedwith a light microscope. The image analysis was performed byusing Motic Med 6.0 digital medical image analysis system.Four sections were selectd from every group. The integratialoptical density of MT was measured. (4) The ultrastructure ofskin flap was observed with transmission electron microscope. 6.Analysis of results: Differinet kinds of results were dealed withby using SAS6.12 software.Results: 1. At the beginning of flap creation, the contentsof MDA and MPO in these three groups have nodifferents(P>0.05). At reperfusion 1h and 24h, the level of MDAin rats IR group increased by 62.2%, 136.4%(P<0.01), theactivity of MPO increased by 238.4%, 503.4%(P<0.01),compared with the control group, respectively. Compared withthe IR group, at reperfusion 1h and 24h, the level of MDA inZn-IR group decreased by 11.3%,33.2%(P<0.05) , the activity...