Experimental Research On The Role Of Nature Hirudin In Rats’ Island Skin Flap With Ischemia-reperfusion Injury

Objective: Observing the flap survival area and the histological changesin tissue by making the ischemia-reperfusion （IR） injury model on the ratswhich interfered by nature hirudin of different concentrations to confirm thecontribution of nature hirudin to protect the flap from IR injury. Detecting thecontent of endothelin（ET） and malonaldehyde（MDA）, the activity ofsuperoxide dismutase（SOD）, the expression of nuclear factor（NF）-κB andtumor necrosis factor（TNF）-α in tissue by biochemistry andimmunohistochemistry to discuss the mechanism. Analysizing the conclusionmentioned above to provide a new method and experimental basis forresolving the problem that IR injury caused flap necrosis after transplantation.Method：1Animals and Group：The research comprised116healthy male Wistar ratsweighing（300±20）g.（provided by Hebei Medical University LaboratoryAnimal Center）.After weighted an ordered the rats were randomized to fourgroups of29rats: Non-ischemia-reperfusion group（Non-IR）, ischemia-reperfusion group （IR）,0.5mg/kg of nature Hirudin group(H_0.5)and1.0mg/kgof nature Hirudin group(H_1.0). Eight rats in each group were used to detect thesurvival rates of skin flap, one to observe the ultrastructure of basal cell andthe rest were divided into5subgroups equally（n=4） according to0h,8h,10h,16h,32h after operating.2Model preparation：The abdomen and groin were shaved, and the rats wereplaced supine on an operating board. The epigastric skin flap （6.0cm×3.0cmin size） based on the right superficial epigastric artery and vein was detachedwith its panniculus carnosus. The other branches of femoral artery weresutured to permit the flap circulation can be maintained only by the epigastricpedicle. In IR group and treatment group:8hours of ischemia was induced by clamping the proximal part of femoral artery beside the pedicle with amicro-vascular clamp. After the end of ischemia, the clamp was completelyreleased for full reperfusion. The elevated flap was sutured back to the originalsite; In Non-IR group：the epigastric skin flap was repositioned without anperiod of ischemia.3Drug administration： One day before operation and for the next7postoperative days, the flaps in H_0.5and H_1.0group were treated with0.5mg/kg or1.0mg/kg of nature hirudin by8-hourly subcutaneous injection,while IR group flaps were treated with normal saline at same volume. Alldrugs were applied to whole flap area. No treatment was given to Non-IRgroup.4To collect specimens：Two1.0cm×0.5cm×0.2cm sized tissue samples wereharvested at the middle of flaps of each subgroup at the appointed time. Onesample was divided into two pieces, one for biochemical analysis, one formaking paraffin section. One1.0mm×2.0mm×2.0mm sized tissue sampleswere harvested from rats prepared for TEM observation at16thhour. All ratswere sacrificed after taken samples.5Examination target:（1）Flap survival area：On the7thpostoperative day, eightflaps of each group were taken digital images after general observation. Thenecrotic area was adjusted by its color, texture and hair growth. The imageswere calculated by Image Analysis System on computer. The survival rate wasexpressed as a percent of the total flap area.（2）Histology: The paraffinsections were stained with hematoxylin and eosin for the histologicalevaluation under the light microscope.（3）TEM observation：The paraffinsections were stained withosmic acid to observe the ultrastructure changes inbasal cell.（4）Biochemical examination: Detecting the content of ET and MDA,the activity of SOD in the10%homogenized tissue.（5） Immunohistochemistryexamination：After immunohistochemistry stained,the expression and locationof NF-κB, TNF–α was observed under the light microscope. Averageoptical density（OD）of five400times fields which were selected randomlyfrom the vision were evaluated by Micro-Image Analysis Software2000。 6Statistical analysis：Data were presented as mean±standard deviation （SD）,and analyzed using SPSS13.0software package. Normal distribution wastested by the Kolmogorov-Smirnov and Shapiro-Wilk tests, and homogeneityof variance tested by the Levene statistic. One-way analysis of variance（One-Way ANOVA） was used to compare the variables among groups, andSNK-q test were used when the significant differences existed. P≤0.05wasaccepted as statistically significant.Results:1Flap condition：On the7thday, flaps of Non-IR group almost survived withpink color, soft texture and hair growth recovered. Conversely, black scab,hard texture, infection and flap contracture could be found on flaps of IRgroup which were almost necrosis. When necrosis was incised, no bleedingbut more inflammatory secretion could be seen under the flap. The proximalpedicle area was a little better than the distal. The flap condition of H_0.5andH_1.0group was between IR and Non-IR group’s. The color became dark fromproximal to distal. Some scab distributed on necrosis area and sloughed offeasily. When necrosis were incised, slight bleeding and some secretion couldbe seen under the flap.Flap survival area: The Non-IR group（98.00±1.08）%＞the H_0.5group（65.32±3.33）%and the H_1.0group （70.52±4.93）%＞the IR group（38.47±5.42）%.The difference between any two groups was statisticallysignificant （F=82.20，P＜0.05）,except the combination like H_0.5and H_1.0group（P＞0.05）.2Histology observation：In Non-IR group, the structure of tissue was intact,slight edema, vasodilator, minimal leukocyte infiltration, connective tissuearranged regularly and no sign of necrosis. In IR group after8h’s ischemia, theepidermis appeared thicken, severe edema and vasodilator and part of vessel’swall lose continuity. After8h’s reperfusion, the damage was aggravated andirreversible. The epidermis appeared atrophy, partially discontinuity and vaguestructure. In dermis, neutrophil infiltrated widely and part of vessels wasobliteration. In H_0.5 and H_1.0 group after8h’s ischemia, tissue appeared edema, some neutrophil infiltrated in dermis. After8h’s reperfusion, part ofepidermis thickened, the number of capillary increased, thrombosis could befound in few of vessel and connective tissue arranged loosely.3TEM observation：At the16thhour，in Non-IR group, the ultrastructure ofbasal cells were almost complete with lots of mitochondria. In IR group, basalcells were highly edema, mitochondria swelling, cristae vague or disappeared,rough endoplasmic reticulum expansion and degranulation. In H_0.5 and H_1.0group, the changes in basal cells were lighter than IR group, expressed as:mitochondria light swelling, few cristae vague, rough endoplasmic lightexpansion and rare degranulation, much more free ribosomes and desmosomesbetween cells.4Biochemical examinations4.1Flap ET determination：Immediately after surgery, the mean ET of eachgroup was no significant difference（F=0.50，P＞0.05）and the value was about35.06pg/ml. After the8th,10th,16th,32thhour，the values of ET increasedgradually. The comparison expressed as: The IR group＞the H_0.5group andthe H_1.0group＞the Non-IR group. The difference between any two groupswas statistically significant （P＜0.01）,except the combination of H_0.5and H_1.0group （P＞0.05）.4.2Flap SOD activity：Immediately after surgery, the SOD values expressedas: The H_0.5group＞the H_1.0group＞the Non-IR group and the IR group. Nosignificant difference between Non-IR and IR group（P＞0.05）. After the8th,10th,16th,32thhour， the activity of SOD decreased gradually. Thecomparison expressed as: The H_0.5group＞the H_1.0group and＞the Non-IRgroup＞the IR group. The difference between any two groups wasstatistically significant （P＜0.01）.4.3Flap MDA determination：Immediately after surgery, the mean MDA ofeach group was no significant difference（F=0.80，P＞0.05）and the value wasabout2.5-3.0nmol/mgprot. After the8th,10th,16th,32thhour，the values of MDAincreased gradually. The comparison expressed as: The Non-IR group＞theH_0.5group and the H_1.0group＞the IR group. The difference between any two groups was statistically significant （P＜0.01）, except the combination of H_0.5and H_1.0group （P＞0.05）.5Immunohistochemistry examination: Immediately after surgery, theexpression of TNF-α and NF-κB were few and inaccuracy were big. So it wasnot involved in statistical analysis. During the period of ischemia andreperfusion, the various expression of TNF-α and NF-κB could be found inbasal cells, sebaceous glands, follicles, microvascular endothelial cells andleukocytes. The positive cells of TNF-α were centered in microvascularendothelial cells, while that of NF-κB were centered in basal cells,microvascular endothelial cells and leukocytes. The average OD of TNF-α andNF-κB both expressed as：The IR group＞the H_0.5group and the H_1.0group＞the Non-IR group. The difference between any two groups was statisticallysignificant （P＜0.01）,except the combination of H_0.5and H_1.0group （P＞0.05）.Conclusions:1Nature hirudin may improve the ischemia anoxic condition of islandskin flap before reperfusion, and prevent the release of proinflammatoryfactor by inhibiting vasospasm.2Nature hirudin may reduce inflammatory cascade and inflammationspread which caused by the expression of TNF-α and NF—κB duringischemia reperfusion.3Hirudin may increase the SOD activity before injury to contribute toactivate the anti-oxidation capacity.4That the protective effect of nature hirudin on ischemia reperfusioninjury is dose-dependent is needed to be further evaluated.