| BackgroundA skin flap is a transferable tissue unit with its own blood supply,composed of skin,muscle,bone,fat and fascia.These days,skin flaps are widely used to cover skin and subcutaneous tissue defects.The pedicled island perforator flap is a flap formed by surgery.With the widespread use of this flap,partial necrosis of the flap has become a serious problem.However,apart from the method of surgical delay,there is currently not effective treatment that can protect flaps from necrosis.Although surgical delay is still the most effective option,it is not easily accepted by patients as an invasive treatment method.Therefore,in order to find more effective,safe,and easy-to-operate treatment methods,scholars have conducted more extensive researches,including physical factor pretreatment,growth factors treatment and drugs treatments.Heat shock protein 32(HSP 32)is also called Heme oxygenase-1(HO-1),which is an enzyme that exists in microsomes.HO-1 can be induced by a variety of stimuli,including heavy metals,thermal pretreatment,inflammatory stimuli,heme and its derivatives,stress,hypoxia,and biological hormones.HO-1 is the rate-limiting enzyme of heme catabolism,which breaks down heme into biliverdin,carbon monoxide(CO)and iron.This study selects a non-invasive physical factor therapy which is called transcutaneous electrical nerve stimulation pretreatment.We will observe whether TENS pretreatment can go through the HO-1 to convert the choke anastomosis of the pedicled island perforator flap into a true anastomosis which thereby improves the survival rate of the flap.Furthermore,we will observe how HO-1 changes the endothelial cells once it starts to work on human endothelial cells.Objects1.This study explored whether transcutaneous electrical nerve stimulation(TENS)pretreatment of the pedicled island perforator flap can prevent partial necrosis of the flap by converting the obstructed anastomosis into a real anastomosis.2.This study is also to explore whether transcutaneous electrical nerve stimulation(TENS)pretreatment can induce HO-1 to prevent partial necrosis of the pedicled island perforator flap before flap surgery.Methods1.(1)Use infrared thermal imaging and skin flap necrosis rate to select a best TENS pretreatment plan.(2)Use infrared thermal imaging,postmortem arteriography,CD31 immunohistochemical staining,and flap necrosis rate as evaluating methods to compare the effect of the treatment of TENS group(biphasic pulse,25 m A,80 Hz,200 μs,1 h)and the treatment of surgical delay group(PIC artery perforator ligation on the first day).(3)Use western blot to compare the molecular mechanisms after TENS pretreatment and surgical delay treatment.2.(1)Seventy-six SD rats are randomly divided into control group,percutaneous neuroelectric pretreatment group,percutaneous neuroelectric preconditioning + heme oxygenase-1 inhibitor tin protoporphyrin(Sn PP,50 μmol/kg)group and Cutaneous nerve electrical preconditioning + saline group.The flap donor is the unilateral dorsal.The skin flap necrosis was observed on the six rats in each group,and the blood vessel changes were observed by arteriography on five of them after death.The rest one rat in each group,we used HE staining,immunohistochemistry process as well as western blot to detect blood vessel and molecular changes.(2)After the human endothelial cells grow to 90% in a six-well culture dish,a 10 μl pipette tip is used to form a uniform scratch on the bottom of each well of the culture dish.The 6 wells are randomly divided into two groups,the control group and the experimental group(HO-1 was added in the operation with a concentration of 5 ng/μl).Then 0 h,24 h,and 48 h after,we select a 1 cm×0.4 cm rectangular area(scratch in the center)to take pictures,then we use Image J software to calculate the relative migration area,the result equals to this following formula: the relative migration area =(0 h area-24 h(or 48 h)area)/0 h area.The above experiments use SPSS 25 for statistical software,and there is a statistical difference in P<0.05.Results1.(1)Stimulation frequency 80 Hz,current intensity 25 m A,pulse duration200μs,treatment last for 1 hour.The bidirectional low-frequency pulse electrical pretreatment can effectively convert choked anastomosis into real anastomosis on the1/3/4 days.(2)Infrared thermal imaging,arterial angiography and CD31 immunohistochemical staining showed that the relative temperature,number of transverse arterioles and microvessel density significantly increase after pretreatment.Compared to the control group,the area of necrosis in the TNES pretreatment group and the surgical delay treatment group is significantly reduced(P<0.01).The area of necrosis in the control group is 22.95±1.35%,the TENS group is 4.05±0.91%,and the surgical delay group is 8.79±0.90%.(3)HO-1 in the TENS pretreatment group increases significantly on the 1/3/4 days,and the vascular endothelial growth factor(VEGF)in the surgical delay group increases significantly.2.(1)Compared to the control group,TENS pretreatment significantly reduces the area of flap necrosis(P<0.01);The tin protoporphyrin(Sn PP)completely deduces this effect(TENS+ Sn PP vs.TENS,P<0.01).Infrared thermal imaging,postmortem arteriography and HE staining and CD31 immunohistochemical staining showed that TENS pretreatment can significantly increase the relative temperature,the number of transverse arterioles,the number of blood vessels larger than 0.1 mm,and the density of microvessels after pretreatment.But the tin protoporphyrin(Sn PP)efficiently eliminates this phenomenon.The results of immunohistochemistry and western blot shows that compared to the control group,the level of heme oxygenase-1 in TENS pretreatment,inhibitor group and the saline group is significantly increased.Western blot results of SCG and PKG shows that compared to the control group,only the TENS pretreatment group and the saline group has significantly increased protein expression.However,the expression of the above two proteins is significantly reduced after application of HO-1 inhibitor.(2)Add HO-1 to human endothelial cells,it shows as mean±standard deviation,24 hours after(HO-1 group: 0.89±0.03 VS,control group: 0.05±0.05,P<0.001),48 hours after compared with the control group(HO-1 group: 0.98±0.1 VS control group: 0.21±0.07,P<0.001).Conclusions1.TENS pretreatment transforms choke anastomosis into a true anastomosis and protects the necrosis of the pedicled island perforator flap.2.In the rat model,TENS pretreatment can prevent partial necrosis of the pedicled island perforator flap through the HO-1. |
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