| Objective: From the research of combined with Dexamethasone and bFGF on rat's random skin flap, observe that the influence of the survival area of random skin flap and histological varieties, and infirm that the effects on preventing and curing the putrescence of flap. In order to enhance the rate of the transplantation of skin flap and explore a new effective approach. Trough the quantificational measurement of the concentration of TNF-αin blood and the ICAM-1 on skin flap. Preliminarily discuss the mechanism of their role to protecting flap from necrosis, and providing reliable experimental basis for clinical application.Method1 Laboratory animals: 96 healthy Wistar rats (provide by Hebei medical university laboratory animal center, weighing 280±10g), male or female.2 The group of laboratory animals: The rats were divided into 4 groups randomly. 24 animals were used in each treatment .Group A was control group: Intraperitoneal injection of saline 0.2ml, and instillation of saline 0.25ml under the flap; Group B was dexamethasone group: Intraperitoneal injection of dexamethasone 0.2ml(5mg/kg), and instillation of saline 0.25ml under the flap; Group C was bFGF group: Intraperitoneal injection of saline 0.2ml, and instillation of the saline which contain 1750IU bFGF(155IU/cm2) 0.25ml under the flap; Group D was dexamethasone + bFGF group: Intraperitoneal injection of dexamethasone 0.2ml, and instillation of the saline which contain 1750IU bFGF 0.25ml under the flap.3 Skin flap formation: All the animals were anesthetized with 2% Sodium pentobarbital (35~40mg/kg) by intraperitoneal injection. When induce anesthesia successfully, depilate the back hair, Prone fixed, disinfected by iodine and alcohol. The line connected with the iliac crest on the center of rat's dorsal as the base line, the back of midline as the axis, made a random skin flap with a size of 7.5cm×1.5cm(including muscle lipid membrane, the length and breadth ratio was 5:1), the pedicel was on the rump. Close the incision at the original position by 3-0 Suture. Group B,D gave the one-off intraperitoneal injection of dexamethasone immediate after operation. Group C,D gave the instillation of the bFGF equably under the flap immediate after operation. The control group gave the saline instead, ditto time and method.4 The detection method and observation marker: 4.1 The general observation: Observe the survival situation of skin flap on the 7th day after operation.4.2 The observation of flap surviving rate: Flap necrosis adjust by its appearance,color and texture. The 7th day after operation, take photo of flap and enter into computer image, calculating the ratio of surviving flap( the proportion of the flap surviving area in total area) by using Image-Pro Plus.v6.0 software system.4.3 The observation through light microscope: Take 0.5cm×0.2cm skin tissue at the middle of the flap on the 3rd and the 7th day after operation in each group, 4% formalin-fixed, Paraffin-embedded histotomy, stain of HE.4.3.1 Observe the varies of tissue structure.4.3.2 Capillary calculate. Method: All the vascular were included in. Five histotomis in every group and five high multiples visual fields were selected randomly from central and peripheral area, calculating the number of capillary and obtaining the mean value.4.3.3 Fibroblast calculate. Five histotomis in every group and five high multiples visual fields were selected randomly from central and peripheral area, calculating the number of fibroblast and obtaining the mean value.4.4 Concentration of TNF-αin serum: Phlebotomize from cardiac blood in each group after 0h,1h,4h,8h,12h,24h operation, and get serum after centrifugation, measure the concentration of TNF-αby using double-antibody sandwich ELISA method.4.5 Immunohistochemical staining of Intercellular Adhesion Molecule 1(ICAM-1): Take 0.5cm×0.2cm skin tissue at the middle of the flap after 4h,8h,12h operation in each group, 4% formalin-fixed, Paraffin-embedded histotomy, stain of immunohistochemical. The location and expression of ICAM-1 were observed with a light microscope. Representative compass was selected from each slice. Nonoverlapping five 400 times fields of visions was selected randomly. Optical density value 200 as background, take picture by photomicroscope and the image analysis was performed by Image-Pro Plus.v6.0 software system. Measure integral optical density(IOD) value and then conduct statistical treatment the values by computers.5 Analysis of results: Dealt with different kinds of results by using SPSS14.0 software.Results1 The general observation: The 7th day after operation, treated groups: the near section of flap: Light red, good elasticity, and a little back hair grew up can be seen; middle section: The color of the center flap gradually darken. There were not scab formation on surface, no hair grew. The color was black in peripheral area and covered with a little scabs. The elasticity was diminished. Far section: The color was black, and covered with scabs, bad elasticity. There can be seen bleeding activity when lift up the flap at the original position, and the quantity is large; There were no hematocele under the flap, little effusion, and vessels were abundance. The flap color and elasticity of combination group was better than the group that use the drug individually.The control group: the near section of flap: The color was l light red and gradually darken from near to far. A little hair grew at the pedicel of flap; Middle and far section: The color was black and covered with scabs, bad elasticity. There can be seen a little bleeding when lift up the flap at the original position, much inflammatory exudates under the flap, vessels relatively sparse.According to the results of observation, the survival situation of group B,C,D was quiet better than group A; and group D is a little better than group B and C.2 The ratio of surviving skin flap: dexamethasone + bFGF group (58.2±5.2)%﹥bFGF group (50.5±3.9)%﹥dexamethasone group (48.7±4.2)%﹥control group (39.5±3.7)%, The four groups had very significant difference (F=32.08, P﹤0.01). Post hoc multiple comparisons, dexamethasone group had no significant difference compared with bFGF group (P﹥0.05), and other groups all had significant differences (P﹤0.01). 3 The observation through light microscope3.1 The 3rd day after operation, subcutaneous tissue edema, vasodilator, congestive, lightly inflammatory response, no signs of necrosis. The 7th day after operation, every treated groups: Cuticle atrophy was not obvious or partly damaged, cutis'structure had not obvious changing, neutrophils infiltration. There can be seen freshmen capillary in cutis, subcutaneous fibrous tissue hyperplasia, fiber array rules. The control group: Cuticle and cutis were completely necrosis, the structure was disordered, a mount of neutrophils infiltration, muscles fiber obviously necrotizing.3.2 Capillary calculate: The 3rd day after operation, the mean value of the number of capillary on every high multiples visual fields: dexamethasone + bFGF group(7.4±0.55)﹥bFGF group(6.8±1.09)﹥dexamethasone group(4.6±0.55)﹥control group(3.2±0.84), The four groups had very significant difference(F=32.40, P﹤0.01). Post hoc multiple comparisons, dexamethasone + bFGF group had no significant difference compared with bFGF group(P﹥0.05), and other groups all had significant differences(P﹤0.01). The 7th day after operation, dexamethasone + bFGF group(7.0±0.71)﹥bFGF group(3.8±1.09)﹥dexamethasone group (2.6±0.55)﹥control group(0.4±0.55), The four groups had very significant difference(F=65.94, P﹤0.01). Post hoc multiple comparisons, there were significant differences between every two groups (P﹤0.05).3.3 Fibroblast calculate: The 3rd day after operation, the mean value of the number of fibroblast on every high multiples visual fields: dexamethasone + bFGF group(27.8±3.56)﹥bFGF group(26.4±3.21)﹥dexamethasone group(11.8±2.86)﹥control group(11.2±2.39), The four groups had very significant difference(F=44.18, P﹤0.01). Post hoc multiple comparisons, the amount of the number of fibroblast in dexamethasone + bFGF group was larger than bFGF group, the difference was obvious (P﹤0.01); dexamethasone + bFGF group compared with bFGF group, dexamethasone compared with the control group, all had no significant difference(P﹥0.05). The 7th day after operation, dexamethasone + bFGF group(48.2±7.33)﹥bFGF group(23.6±4.45)﹥dexamethasone group(15.0±1.87)﹥control group(0.6±0.89), The four groups had very significant difference(F=102.47, P﹤0.01). Post hoc multiple comparisons, there were significant differences between every two groups (P﹤0.01).4 Concentration of TNF-αin serum: The concentration of TNF-αof the control group and bFGF group reach the peak value at the time 4 hours after operation, then gradually declined, and decreased at the time 24 hours after operation. The concentration of TNF-αof dexamethasone group and dexamethasone + bFGF group reach the peak value at the time 8 hours after operation, however, the peak value was much less than the control group and bFGF group(P﹤0.01). And also decreased at the time 24 hours after operation.5 The results of immunohistochemical staining of ICAM-1:4 hours after operation, in the cytoplasm of the epithelial cells, endothelial cells and fibroblasts of flap in each group all showed ICAM-1 mRNA expression. The peak value of ICAM-1 protein positive expression appeared at 8 hours after operation. The peak value of four groups had very obvious differences(F=224.30, P﹤0.01). Post hoc multiple comparisons, the peak value of the control group and bFGF group were much higher than dexamethasone group and dexamethasone + bFGF group (P﹤0.01). Then gradually declined, but still maintaining a relatively high level at 12 hours after operation.Conclusions: Dexamethasone and bFGF both capable of improving circulation, reducing the inflammatory response, stimulating the granulation tissue to grow and promoting surviving of skin flap; and combined with dexamethasone and bFGF to promote the survival of skin flap was better than use dexamethasone or bFGF individually.
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