| Objective: Through observing the influence of salvia miltiorrhiza injection to the survival area and histological changes of rat's flap with ischemia-reperfusion, in order to confirm the function of salvia miltiorrhiza to prevent the flap necrosis and raise its transplant survival rate, and preliminarily discuss the mechanism, through test the concentration of MDA, MPO, Nuclear factor (NF)-κB, Intercellular Adhesion Molecule 1 (ICAM-1) in flap, to provide reliable experimental basis for clinical application.Method:1 Laboratory animals: 48 healthy Wistar rats, male or female, weighing 250~300g, (provided by Hebei medical university laboratory animal center).2 The group of laboratory animals: The rats were divided into 3 groups randomly. 16 animals were used in each treatment.①The non-ischemia-reperfusion group (Non-IR): The flaps were sutured, without other treatment.②The ischemia-reperfusion group (IR): The rats had been intraperitoneally injected physiological saline 2ml/kg, between 3 days before operation to 5 days after. After the flaps were formed, the Femoral Arteries beside which inferior epigastric arteries sending were blocked by vascular clamps, and then the flaps were sutured. The vascular clamp should be removed after 8h to restore the blood to supply.③The ischemia-reperfusion group treated with salvia miltiorrhiza (SM-IR): The rats were intraperitoneally injected salvia miltiorrhiza 2ml/kg injection instead of physiological saline and the other operations were the same as the IR group.3 Flap formation: The animals were depilated by the sodium sulfide three days before the operation and anesthetized by intraperitoneal injection of 3% Sodium pentobarbital (40mg/kg) when operation.Based on the methods described by Petry JJ[6] and so on, formed an about 3cm×6cm island flap under the right abdomen taking the abdominal wall shallow blood vessel as the peduncle, deep to subcutaneous dartos level. Full dissociated the Femoral Artery beside which inferior epigastric arteries sending and blocked it by vascular clamp, and then sutured it.4 Draw the materials from the flaps: The IR group and the SM-IR group's 8 rats were taken 1.0cm×0.5cm skin tissue at the middle of the flaps after rising instantly, and 2h, 8h, 24h after reperfusion. The Non-IR group's 8 rats were taken skin tissue in the same way, 10h, 16h, 32h after reperfusion, and then took partial tissue immediately to treat fixedly with 4% paraformaldehyde to Paraffin-embedded histology and Immunohistochemical staining. the others were frozen to preserve to biochemistry examination. Observed the survival flaps in 3 groups of the other 8 big rats 7d after the operation, and calculated the survival rates.5 Examination method and observation target:5.1 Observe the flaps roughly: 7d after the operation observed the survival situation of flaps roughly.5.2 Flap's survival rates: The necrosis of flap was adjusted by its appearance, color and texture. On the 7th day after operation, take photo of flap and entered into the computer image, calculating the survival rate (the proportion of the flap surviving area in total area) by using Image-Pro Plus.v6.0 software system.5.3 Histology inspection: The paraffin sections were stained of HE, to observe the changes.5.4 Biochemical examination: The content of malondialdehyde (MDA) and the activity of myeloperoxidase (MPO) were measured according to the instruction booklet of the reagent box. The content of protein in the flap was tested by coomassie brilliant blue.5.5 Test the Nuclear factor (NF)-κB, Intercellular Adhesion Molecule 1 (ICAM-1): After immunohistochemical staining the location and expression of ICAM-1, NF-κB was observed with a light microscope. Representative compass was selected from each slice. Using image analysis software UTHSCSA Image Tool 3.0 version, nonoverlapping five 400 times fields of visions was selected randomly. Count positive cells and then make its average.6 Analysis of results: Dealt with different kinds of results by using SAS V8 software.Results:1 General observation of flap7days after operation the flaps of Non-IR group were all survived, and their color were pink, good elasticity, showing that a small amount of hair growth.The flaps of SM-IR group were pink, from near to far began to emerge bad blood supply and necrosis, expressed gradual dark, even black; Texture gradually harden and distal necrotic area formed on the surface crust shell, elastic poor; In-situ off flaps ,they showed a slight bleeding under the existence of a small amount of dartos inflammatory exudates.The average necrotic area of IR group was appreciably larger than the SM-IR group showing that the majority necrosis and forming large black scab shell, poor elasticity. Only small amount of necrotic tissue was not, but the color was a little dark and its blood circulation was bad. In-situ off flap, it could be seen bleeding less and much more inflammatory secretions under the dartos.It can be seen that the Non-IR group flap was obviously better than the other two groups, and SM-IR group was better than IR group.2 Flap's survival rate: The Non-IR group (100%±0.0) > SM-IR group (60.2±3.8)% > IR group (38.3±2.6)%, SM-IR group and the IR group, significant difference (P<0.01).3 Histology3.1 In Non-IR group at various time the subcutaneous tissue of flaps showed edema and light inflammation, no signs of necrosis. In SM-IR group after 2h, the epidermis began to appear partially damaged and some of neutrophil infiltration; after 8h severe subcutaneous edema, skin layers of loose, disordered and a large number of neutrophils adhesion and aggregation in the blood vessel wall. Part of the capillaries walls were damaged, showing the integrity damaged, and a large number of neutrophils infiltrating into the tissue space, a small amount of muscle fiber necrosis; after 24h, although slight improvement, there was still a large number of neutrophil infiltration.IR group: At all time higher than those of SM-IR group. after 24h it can not seen any significant improvement, sustained increase. Partial necrosis of skin layer, the structure is unclear. A large number of capillaries walls were damaged in the integrity of destruction. A large number of neutrophils infiltrated and some muscle fiber mortified.4 Organization of biochemical examination4.1 MDA Determination: In the three groups after raising flap instantly MDA was no significant difference (P>0.1), after reperfusion 2h, 8h, 24h, in the various points MDA values were presented: IR group>SM-IR group>Non-IR group (P all<0.01), and the difference was statistically significant.4.2 MPO Determination: In the three groups after raising flap instantly MPO was no significant difference (P>0.1), after reperfusion 2h, 8h, 24h, in the various points MDA values were presented: IR group>SM-IR group>Non-IR group (P all<0.01), and the difference was statistically significant.5 Immunohistochemical staining results:5.1 Nuclear factor (NF)-κB determination: In the three groups after raising flap instantly there were no NF-κB positive cells. The Non-IR group, IR group and SM-IR group in 2h, 8h, 24h, mainly in PMN (Polymorph nuclear neutrophil) and vascular endothelial cells showed positive expression. The Non-IR group number of positive cells (2.00±1.20, 3.88±0.99, 5.00±0.76), IR group: the numbers of positive cells (18.29±1.38, 16.86±0.69, 19.43±0.98), SM-IR group of positive cells (10.13±1.25, 12.13±1.25, 12.88±1.13), IR group>SM-IR group>Non-IR group (P all<0.01), the difference was statistically significant.5.2 Intercellular Adhesion Molecule 1 (ICAM-1) determination: In the three groups after raising flap instantly there all were few ICAM-1 positive cells in the vascular endothelial cells, it was too small, not quantitative. The Non-IR group number of positive cells (1.13±0.83, 2.89±0.78, 4.00±0.70), IR group number of positive cells (19.13±1.13, 20.25±1.28, 20.29±0.76), SM-IR group of positive cells (10.83±1.30, 11.14±1.07, 10.25±1.04), IR group>SM-IR group>Non-IR group (P all<0.01), the difference was statistically significant.Conclusions:Salvia miltiorrhiza maybe by inhibiting NF-κB to activate the inflammatory response pathway ring and its own that has the ability of anti-oxidation and to improve the body's overall antioxidant capacity, can reduce flap ischemia-reperfusion injury and promote of flap survival.
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