Effects Of Nitric Oxide On The Second Choke Zone In Extended Perforator Flap Of A Rat Model

Background Since the late nineteen eighties,the appearance of perforator flap has explored a new field for the development of microsurgery and plastic surgery.Taylor's research demonstrated that the adjacent perforator were linked by choke vessels,and anastomosis generally take one of three forms,including true anastomosis,choke anastomosis and potential anastomosis.Extended perforator flap is generally elevated to repair the large skin defects which cannot be covered by flaps only containing one perforator.In a perforator flap,encompassing three vascular territories,distal circulation is supplied by blood stream flowed through the first and second choke zone.Flow through a single choke zone is often sufficient to supply the neighboring perforasome,but flow through a second choke zone may be inadequate.The behavior of the second choke zone seems to be associated with necrosis in the third territory,the second choke zone appears to operate as a watershed against blood flow.NO appears to be an important factor for arteriole vasodilation and skin flap perfusion in the process of skin flap healing.Constitutive production of NO is important for blood flow in the skin and flap tissue for the survival of skin flaps,and the activity of the produced NO could be isolated to the skin flap.In this study,a rat dorsal extended perforator flap model was made to explore the effect of NO on the second choke zone after elevating skin flap.Objective This study was performed to explore the mechanism of NO's promotion on vascular growth and dilatation in second choke zone and effect on flap's survival after flap elevation.Methods Overall 108 male Sprague Dawley rats were divided into three groups(n=36),L-Arg [400 mg /(kg·d)]was injected intraperitoneally in L-Arg group,L-NAME [40 mg /(kg·d)] was injected intraperitoneally in L-NAME group,the same volume of saline was injected intraperitoneally in Control group at immediately and one to seven days after operation.A three-territory flap was made.(1)The survival flap area was calculated as a percentage of total flap dimensions and blood vessel shape and distribution was observed on the 7th postoperative day.(2)Tissue samples were harvested from choke zones to measure NO concentration quantitatively using Nitrate reductase method on the1 st,3th,5th and 7th postoperative day.(3)Angiograph and imaging were performed to observe microvascular growth and anastomosis of the choke zones on the 7th postoperative day.(4)Tissue samples from choke zones were sliced to performed histological examinations,microvascular density and diameter were recorded on the 7th postoperative day.(5)Tissue samples from the second choke zone were sliced to examine proteins expression including VEGF,ICAM-1,MMP-2 and CD11 b by immunohistochemistry on the 1st,3th,5th and 7th postoperative day.(6)Tissue samples from the second choke zone were homogenized to examine proteins expression including VEGF,ICAM-1 and MMP-2 by Western blot.Results(1)On the 7th postoperative day,true anastomosis was visualized in all three groups' first choke zone,while choke vessels dilated extensively in L-Arg group.Axial vessels extending to the distal end of the flap could be observed in rats receiving L-Arg.No visible structure of choke vessels in the second choke zone could be found in the rat treated with L-NAME.Mean flap survival area in L-Arg and Control group were statistically larger than that in L-NAME group [(t=8.045,P<0.001)and(t=3.289,P=0.009)].(2)NO concentration of Choke zone in L-Arg group and control group was basically the same trend,reaching the peak on the 3rd postoperative day,the difference between the two groups at same time points were statistically significant(P<0.05).The NO concentration of the first Choke zone in L-NAME group significantly decreased,follow-up was fluctuating in a small range.In the second Choke zone,postoperative NO concentration significantly decreased within 3 days after operation,the difference with L-Arg group and control group at the same time points were statistically significant(P<0.05).(3)On the 7th postoperative day,vascular structure of Choke zones in L-Arg group were clear,vascular reaching true anastomosis extended to the end of flap.Vascular structure of the first Choke zone in L-NAME group and control group was clear.Vascular structure of the second Choke zone in L-NAME group and control group was out of shape.(4)On the 7th postoperative day,difference of microvascular density and diameter in first choke zone among all three groups were not statistically significant(P>0.05).Microvascular density and diameter in second choke zone in L-Arg group were statistically higher than those in L-NANE group[(t=4.038,P<0.001)and(t=7.496,P<0.001)].Microvascular density and diameter in second choke zone in Control group were statistically higher than those in L-NANE group [(t=4.038,P<0.001)and (t=5.064,P<0.001)].(5)On the 3th and 7th postoperative days,VEGF and MMP-2 were significantly expressed in L-Arg group and the control group,the expression of VEGF was weak in L-NAME group;3 days after operation,CD11 b were significantly expressed in all three groups,7 days after operation,CD11 b were significantly expressed in L-NAME and control groups,expression in L-Arg group of CD11 b was weak;3 days after operation,ICAM-1in all three groups was obvious expression,7 days after operation,ICAM-1 expression in L-NAME and control groups was significant,expression of ICAM-1 in L-Arg group was not significant.(6)Tissue samples from the second choke zone were homogenized to examine proteins expression by Western blot.On the 3th postoperative day,expression of VEGF in L-Arg and Control group were statistically higher than that in L-NAME group [(t=3.329,P=0.014)and(t=4.76,P=0.005)].Expression of ICAM-1 in L-Arg and Control group were statistically higher than that in L-NAME group [(t=26.465,P<0.001)and(t=28.566,P<0.001)].Expression of MMP-2 in Control group was not statistically higher than that in L-NAME group(t=0.283,P=0.789),expression of MMP-2 in L-Arg group was statistically higher than that in L-NAME group(t=4.053,P=0.010).On the 7th postoperative day,expression of VEGF in L-Arg and Control group were statistically higher than that in L-NAME group [(t=2.61,P=0.048)and(t=3.836,P=0.012)].Expression of ICAM-1 in Control group was statistically higher than that in L-NAME group(t=-2.766,P=0.04),expression of ICAM-1in L-Arg group was not statistically higher than that in L-NAME group(t=4.053,P=0.010).Expression of MMP-2 in L-Arg and Control group were not statistically higher than that in L-NAME group [(t=1.602,P=0.17)and(t=1.148,P=0.303)].Conclusions Receiving exogenous L-arginie intraperitoneally could increase NO concentration in choke zones of a rat dorsal extended perforator flap model.NO could promote microvascular growth and dilatation in second choke zone,which was beneficial to survival of skin flap,arteriogenesis and inflammation may play a key role in the microvascular changes.