| Objective:Transplantation of extended skin flap in clinical practices often results in flap distal necrosis.This study aims to design a new model of three-vascular territory perforator flap supercharged in distal potential territory,explore the survival mechanism of the potential territory and compare with venous super-drainage to provide the basis for the design of safe flaps for patient benefit and reduce flap distal necrosis.Methods:1.6 rats were dissected and measured to compare the diameters of the thoracodorsal vein(TDV)and deep iliac circumflex vein(DCIV)with the inner core of clinical venipuncture needle as a reference.Statistical analysis was carried out to determine the diameters of TDV and DCIV.12 rats were divided randomly into two groups:Group A,the thoracodorsal artery(TDA)-flap with a territory based on the TDA and then extended to the intercostal artery(ICA)and deep circumflex iliac artery(DCIA)sequentially and Group B,the DCIA-flap with a territory based on the DCIA and then extended to the ICA and TDA sequentially.On postoperative day(POD)7,we compared the survival of flap between the two groups.Then imaged laser doppler blood flow to determine flap blood flow and gelatin-lead oxide arteriography showed vascular morphology.Finally,we identified which skin flap model was more beneficial to supercharge.2.Based on the model of rat dorsal three-vascular territory perforator flap,we designed a flap pedicled with the thoracodorsal vessel(TDA/TDV).This flap extended to the intercostal vessel(ICA/ICV)and deep iliac circumflex vessel(DCIA/DCIV)sequentially.We isolated and preserved DCIV and separated rat caudal artery.12 rats were randomly divided into two groups:Group A as the control group,without anastomosis of caudal artery,and the tail was severed and sutured at the point of rotation of the caudal artery;Group A:the distal non-physiological supercharge group,separated rat caudal artery and then anastomosed it with the deep iliac circumflex vein(DCIV)through the subcutaneous tunnel.On POD7,we observed and detected flap viability,and observed flap blood flow by laser doppler blood flow imaging and vascular morphology by gelatin-lead oxide angiography.3.96 rats were randomly divided into two groups.The flap design was the same as the second part.6 rats from each group were placed under anesthesia and scanned using a laser doppler instrument at each time point of postoperative hour(POH)1 3,6,12 and POD1,3,5 and 7 to observe whether there was blockage of blood flow signal in choke vessel,and its duration and release time.After laser doppler blood flow imaging,all rats underwent gelatin-lead oxide arteriography to observe the vascular morphological changes and the time of vascular morphological changes in the choke vessel.The differences between the two groups were compared.4.The grouping follows the third part.The images of laser doppler blood flow obtained in the third part were analysed by the Moor LDI Review software.Blood flow was quantified using perfusion units,and were calculated at POH1,3,6,12 and POD 1,3,5 and 7.The changes of choke vessel ?/?,DCIA and ICA blood flow at adjacent time points in each group were compared.The blood flow in the potential territory and dynamic territory was divided by the blood flow in the anatomical territory,and the corresponding ratios were obtained to compare the blood flow at different time points between the two groups.At the same time,blood flow of choke vessel ? to choke vessel ? was compared between the two groups at each time point.Statistical analysis was used to determine the effect of non-physiological supercharge on the blood flow in each area of the flap,especially in the early stage of the flap.5.The flap model was the same as the second part.24 rats were randomly divided into two groups:Group A:the TDA-flap;Group B:the non-physiological supercharge flap.The skin flaps were stained with hematoxlin-eosin(HE)to observe the micro-structure of potential territory and count the number of blood vessels in choke vessel ?.And we compared the effects of supercharge on the micro-structure andangiogenesis in the choke vessel.To evaluate the effect of distal non-physiological supercharge on angiogenesis at molecular level,we analysed vascular endothelial growth factor(VEGF)expressions detected by iminunohistochemical(IHC)staining and Western blotting in the choke vessel II of two groups.6.18 rats were used as Group C.On the basis of the second part,the deep circumflex iliac vein(DCIV),was retained as the distal super-drainage vein.On POD7,we observed and detected flap viability,and the necrotic area was further identified by laser doppler blood flow imaging and gelatin-lead oxide angiography.At the same time,we performed HE staining to count mean vascular density and IHC staining and Western blotting to determine the expression of VEGF.Finally,we compared the effects of distal super-drainage and distal non-physiological supercharge on the survival of three-vascular territory perforator flap combined with the data of the fifth part.Results:1.The mean outer diameter of the two perforator veins under the microscope were:TDV:0.427±0.10,DCIV:0.632±0.06.Difference was found between these two perforator veins.DCIA-flaps showed significantly higher relative viable area than TDA-flaps(88.8±5.3%vs.81.2±6.3%,p=0.047).Both laser doppler blood flow imaging and gelatin-lead oxide arteriography demonstrated flap distal ischemic changes in two groups.2.Group A flaps showed significantly lower relative viable area than Group B flaps(81.9±5.4%vs.100%,p<0.001).Both laser doppler blood flow imaging and gelatin-lead oxide arteriography demonstrated flap distal ischemic changes in Group A,while Group B flaps showed normal blood flow signal and good vascular morphology in the distal.3.Laser doppler blood flow imaging showed there was a blood flow blockage in choke vessel I in Group A and the obstruction disappeared after 6-12 hours;The blood flow in choke vessel II did not pass until 3 days.In Group B,a blood flow blockage in choke vessel I was observed and disappeared after 6-12 hours.However,after non-physiological supercharge in choke vessel ?,the blood flow signal in the potential territory was weak within 1 day after surgery.After 1 day,the blood flow passed through the choke vessel ?,and the blood flow signal was significantly enhanced as the time went by.Gelatin-lead oxide angiography showed that the vessels in choke vessel ? were mainly connected by choke vessel at 3 hours after surgery in the Group A with a small amount of fine true anastomosis,and with true anastomosis within 1 day after surgery;During this period,a large number of slender vascular structures appeared.The large true anastomosis appeared within 3-7 days,while the slender blood vessels disappeared gradually.The gelatin-lead oxide angiography of Group B in the choke vessel ? was basically the same as that of Group A.In Group A,the choke vessel ? were mainly choke vessel after surgery,a little fine true anastomosis appeared at 12 hours,and much fine true anastomosis appeared on POD1,and lasted until POD7.In Group B,the choke vessel? were mainly choke vessel within 3hours after surgery;The fine true anastomosis appeared at 6 hours and gradually increased to a large number at 12hours.On POD1,3,5 and 7,a small number of large true anastomosis connected DCIA and ICA territory.4.Compared blood flow in choke vessel ?/? at each time point in the two groups,we found that both groups showed an increasing trend,but with differences between these two growth trends.In Group A,blood flow in choke vessel ?/?increased mildly within 12 hours and showed significantly differences between J 2h and 1d(p<0.05),and gradually stabilized from POD3-7.In Group B,blood flow in choke vessel ?/? showed significantly differences between 1h and 3h,1d and 3d(p<0.01),but not other adjacent time points;After 3 days,blood flow in choke vessel?/? increased mildly and gradually stabilized.Observed the blood flow curves at different time points in Group A and B,it was found that the blood flow in each vascular territory of Group A and B showed an upward trend and gradually stabilized from POD3-7.Specifically,DCIA blood flow in Group A increased slowly,and ICA blood flow increases showed significantly differences between 3h and 6h,12h and 1d,1d and 3d in Group A(p<0.01),but no significantly difference between adjacent time points of other vascular territories blood flow increases;TDA blood flow increases in Group B showed no significantly difference between the adjacent time points;There were significantly differences in ICA blood flow increases between 3h and 6h,12h and 1d,1d and 3d(p<0.01),but not other adjacent time points;And DCIA blood flow increased significantly in Group B and showed differences between 1 h and 3 h,6 h and 12 h,12 h and 1 d,1 d and 3 d(p<0.01).Compared blood flow in choke vessel ?/? at each time point between the two groups,we found that the ratio of blood flow in Group B was significantly higher than that in Group A from 1 hour to 3 days(p<0.01);It was still significantly higher in Group B than that in Group A on POD5 and 7(p<0.05).Compared the ratio of ICA blood flow/TDA blood flow,we found that the ratio in Group B was significantly higher than that in Group A at 1 h,3 h,6 h and 12 h(p<0.05),but no significant difference between the two groups after 1day.Compared the ratio of DCIA blood,flow/TDA blood flow,we found all ratio in Group at each time point was significantly higher than that in Group A(p<0.05).5.HE staining showed significantly more microvessels in the Group B,with higher mean vessel density than the Group A(219.54±18.26/mm2 vs.103.33±13.35/mm2,p<0.001).In the Group B,both IHC staining and western blotting showed VEGF expression was significantly higher than in the Group A(p<0.001).6.Group B flaps showed significantly higher relative viable area than Group A and Group C flaps(100%vs.81.9±5.4%and 92.8±4.1%,p<0.001 and p<0.05).Meanwhile,we found significantly higher relative viable area in the Group C than in the Group A(p<0.05).Both laser doppler blood flow imaging and gelatin-lead oxide arteriography demonstrated flap distal ischemic changes in Group A and Group C,while Group B flaps showed normal blood flow signal and good vascular morphology in the distal.HE staining showed the mean vessel density in the Group C was 148.547±29.94/mm2;There were significantly differences compare with Group A(103.33 ± 13.35/mm2)and Group B(219.54 ± 18.26/mm2)respectively(p<0.05).In addition,both IHC staining and western blotting showed VEGF expression in the Group C was significantly higher than in the Group A,while significantly lower than in the Group B.Conclusions:When TDA was used as the pedicle,DCIV was thicker and more suitable for vascular anastomosis.In addition,the distal necrosis area of the TDA-flap was larger,which was easier to observe flap distal necrosis.Therefore,the TDA-flap model was more suitable for non-physiological supercharge at the distal of extended dorsal three-vascular territory perforator flap models in rats.This supercharged skin flap model was confirmed that non-physiological supercharge at the flap distal can:improve flap distal blood flow and promote the survival of the extended perforator 'flap.This study confirmed that the morphological changes of choke vessel I did not occur obviously after arterialization of veins in potential territory,while the large true anastomosis of choke vessel ? appeared earlier than that of TDA-flap.In non-physiological supercharge model,choke vessel ? opened slowly until 12 hours postoperatively and built blood signal connection between dynamic area and potential territory;the blood flow in the potential territory tended to balance with the anatomic area until 3 days postoperatively.In the early stage after surgery,the potential territory was still in the low perfusion state.Especially within 1 day,laser doppler blood flow imaging and gelatin-lead oxide arteriography both confirmed that the blood perfusion in choke vessel ? was not good.Laser doppler blood flow imaging revealed that the potential territory blood flow was lower than that in the proximal two areas,but it could significantly improve the distal blood flow of the flaps.At the same time,the blood flow in choke vessel ? increased significantly at 3 h.It can be seen that the blood flow could be refluxed after supercharging in the potential territory by opening some microvessels,which solved the early swelling and alleviated the blood stasis to promote the early survival of the skin flaps.In addition,this kind of non-physiological supercharge model effectively improved the overall blood supply of the flaps via angiogenesis,vascular remodeling and dilatation in choke vessel ?.In this case,even if the supercharged blood vessel was occluded,it could still effectively maintain the distal blood flow of the flaps,which may be an important mechanism for the long-term survival of skin flaps.Finally,super-drainage could partially improve the blood perfusion in a certain area of the single vascular territory in the potential territory of the flaps and promote the survival of the flap in a certain range,but not the whole vascular territory.In contrast,we confirmed that the survival of the whole perforator territory could be achieved by non-physiological supercharge of venae comitantes of the perforator vessels in the flap potential territory,which could be used as an optimal method to promote flap distal blood perfusion in clinical practices. |
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