Effect Of Nerve Growth Factor On Survival Of Cross-boundary Perforator Flap In Rats

Background and objective:Perforator flap as technology of the wound and tissue repairing,plays an important role in the clinical.Angiosome and Choke Vessels raised by Taylor are the anatomical basis for clinical application of perforator flaps,and it is the key of the survival of the distal end of the flap that the opening of the choke blood vessel between the adjacent angiosomes and the establishment of the effective circulation of the “Potential Territory”after transplantation.It was a difficult problem that cross-boundary perforator flaps would tend to necrosis in repairing larger wound intraoperatively and postoperatively.Surgical-Delay technique can improve the blood supply of the flap after transplantation by multi-stage operations with enlarged the repairing area of the skin flap effectively.The dilation of choke vessels and the establishment of collateral circulation or a combination of both mechanisms are currently considered to represent the major causes of flap survival in most of the studies.However,surgical delay can be augmented or even replaced by some drugs,cells and their secretions,as the result of it,a concept of chemical flap delay has emerged to improve flap healing.Nerve Growth Factor(NGF),a multifunctional polypeptide molecule,can trigger the proliferation and migration of endothelial cells through the endothelial progenitor cell tyrosine kinase receptor(Trk A)pathway,and maintain the endothelial function.Moreover,NGF is capable of regulating the expression of VEGF to promote angiogenesis directly or indirectly.In addition,EPCs are distributed on the vascular endothelial growth factor receptor-II(KDR),which combined with VEGF to play the role in angiogenesis.NGF is able to regulate the expression of KDR up,and has a synergistic effect with VEGF,which is another way of angiogenesis.However,it is not very clear whether NGF may influence the change of microvascular morphology in the choke flap area and the establishment of microcirculation in the potential territory through the mechanism mentioned above,so as to replace the surgical delay surgery to improve the large area of flap.Based on the above analysis,this study screened and established animal model,through the microscopic CT,to evaluate the nerve growth factor(NGF)effect on the change of microvascular morphology of dorsal cross-boundary perforator flap of SD rats,and to explore its mechanism,to provide theoretical support for improving clinical flap survival.Method:Part I: To observe the effect of nerve growth factor on vascular morphology in rat cross-boundary perforator flap by Micro-CT reconstruction technique.40 adult SD rats were divided into the experimental group and the control group equally.To screen the model of dorsal cross-boundary perforator flap of SD rats through constant anatomy.An area of about 3cm x10 cm cross-boundary perforator flap was harvested.The experimental group was injected subcutaneously with NGF solution(10nmol·ml-1·kg-1);the control group was injected with 0.1M PBS solution(1ml/kg).Day 3 and 7 after surgery,vascular perfusion with lead oxide-gelatin solution.Angiography was performed by the micro-CT to detect Three-dimensional morphology of microvasculature.Alteration of vascular volume and total length was analyzed Matlable software.At day 7 measuring survival area of the flap.Part II: Preliminary study on the effect of Nerve Growth Factor on survival of cross-boundary perforator flap in rats44 adult SD rats were divided into the experimental group and the control group equally,in the dorsum of which an area of about 3cm x10 cm cross-boundary perforator flap was produced.The experimental group was injected subcutaneously with NGF solution(10nmol·ml-1·kg-1);the control group was injected with 0.1M PBS solution(1ml/kg).Day 3 and 7 after surgery,the VEGF and CD34 of the local draw tissues of flap were tested via Blot Western;At day 7 the observation of expression of KDR and TrkA in vasculature by immunohistochemical stain.Result:1.The model of iliac lumbar artery as single perforator pedicle crossed the three vascular angiosomes of the designed flap could be provided with the constant necrosis area.Morphological analysis showed that there is an interaction between NGF and Time in the increase of vascular volume(F=33.304,P<0.05)and the total length of vessels(F=8.493,P=0.01);Compared with control group,the flap survival area of experimental group was significantly higher(P<0.05).2.It was found that NGF significantly promoted the expression of VEGF and CD34 protein in the flap.Day 3,There was no statistically significant differences in the expression of VEGF between two groups(P=0.088),the expression of CD34 in the experimental group was higher than that in the control group(P=0.004);Day 7,there were more highly expressed of VEGF and CD34 in the experimental group(P<0.05);NGF could induce expression of TrkA in microvessel of skin flap.Conclusion:The above results suggested that NGF can increase the density and length of microvessels in the choke region,and can promote the survival area of cross-boundary perforator flap.It could be the mechanism that NGF can regulate up the secretion of VEGF and CD34 and induce the expression of Trk A,thereby promoted choke angiogenesis,maintained their endothelial function,and ultimately improved the microcirculation for potential territory,via TrkA-VEGF pathway.