| Objective: This study was undertaken to investigate the effects of tanshinone?A on survival of multi-territory perforator flaps in ratsand further explore the mechanism,provide theoretical basis for improving perforators flap survival rate in the clinical.Method: 1.Group Dividing and Flap Model Designing:Select 84 healthy male SD rats,weighting about 200-250 g,divided into experiment group and control grouprandomly,with 42 rats in each group.Choosing the midline of the dorsum as the medial border,2.5 cm lateral to the midline of the back as the lateral border,a line joining the medial and lateral borders at the subscapular angle as the cranial border,a line joining the lateral and medial border at the posterior superior iliac spine as the cranial border,to design a 9.5cm x 2.5cm rectangle type axial perforator flap.In the experimental group,the intraperitoneal injection of tanshinone?A injection was 3.2ml/kg for 7 days after the surgery.In the control group,the same dose of physiological saline was administered at the same time for intraperitoneal injection.2.Measurement Indicators 2.1Flap appearance and Percentage of Survival Area All flaps were observed for 7 days postoperatively,including color,texture,swelling,blood flow and hair conditions.Anesthetizing rats and using a digital camera to takehigh quality photos on dorsal skin of rat in the 7th day.The survival area of the flap was measured as a percentage of the total flap area as calculated by Image Pro Plus6.0.2.2Microangiography At 7 days after surgery,choose6 rats respectively from the experimental group and control group.All rats were anesthetized and underwent whole-body angiography according to a modified lead oxide-gelatin injection technique to observe the vascular condition of the flap.2.3Histology in Choke Zone II At 7 days after surgery,choose 6 rats respectively from the experimental group and control group.Take transverse sections of tissue specimenswhichobtained from the second choke zone of the flaps and fixed in 4% paraformaldehyde to measure vascular density.2.4Flap perfusion laser Doppler imaging Using a Laser doppler scanning to evaluate flap perfusion at 1day,3 days,7days after surgery.The rats(6 rats in each group)were anaesthetized and maintained in an environment of 25?.2.5 qRT-PCR Using real time PCR to detect VEGF?HIF-1??e NOS?SOD-1?Caspase3?Bcl-2?Bax mRNA of tissue specimens obtained from the second choke zone of the flaps at1 day,3 days,7days after surgery(6 rats in each group).2.6Statistical analyses SPSS software version 24.0(SPSS,Chicago,IL,USA)was used for the statistical analyses.All data in the normal distribution are expressed as mean and standard deviation.The data of two groups were compared using the independent Student t-test and two-way repeated measures analysis of variance.Statistical significance was accepted at p<0.05.Results: 1.General Observation and Percentage of Survival Area After one day,the distal area of flap of two groups were dark purple and swelling.At the third day,flaps of two groups showed sepia and patchy necrosis.At the seventh day,the boundary between survival and necrosis was clear,the percentages of survival area were 85.53 % ±5.68 % and 78.62%±5.36% in the treated group and the contrast group,respectively.The difference was statistically significant(P < 0.05).2.Microangiography At 7 days post-surgery,vascular structure in the second choke zones of the experimental group was more clear and showed more neovascularization than the contrast group.3.Histology in Choke Zone II The flaps in the experimental group(27.90±2.86)/mm2 had higher microvascular density per square millimeter in the second choke zone than those in the control group(19.77±3.17/mm2;P<0.05)at 7 days after surgery.4.Blood flow The perfusion of flaps show significant difference between the experimental group and the contrast group(60.28±8.62 vs.36.20±8.15 perfusion units at one day after surgery;82.86±9.02 vs.65.88±4.41 perfusion units at three days after surgery;128.39±10.61 vs.101.11±5.39 perfusion units at seven days after surgery,respectively,all p<0.05)5.qRT-PCR The expression of VEGF?HIF-1??eNOS?SOD-1?Caspase3?Bcl-2?Bax show no significant difference betweenthe two groups at one day after surgery.But at three and seven days after surgery,the experimental group show high expression of VEGF?HIF-1??ENOS?SOD-1?Bcl-2 while low expression of Caspase3 and Bax compared to the contrast group.Conclusions: The above results suggested that tanshinone?A can increase the blood flow and density of microvessels in the second choke zones,and promote the survival area of perforator flap.It could be the mechanism that tanshinone?A can promote angiogenesis,reduce ischemia-reperfusion injury and reduce apoptosis. |
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