| Objective:This study was designed to observe the situation of the delayed flap in the back of rat to simulate human reconstructive ear after hyperbaric oxygen therapy,about the scar adhesion between flap and basement,the ratio of collagen I to IIIL the number and diameter of blood vessel,the expression of VEGF and the change of blood flow,which is to certify the effect of hyperbaric oxygen therapy on the edge survival,the scar adhesion and the revascularization of delayed flap.We try to seek a safer and time-saving method for delayed flap,which offer a feasible method to cutting operation interval of forming delayed flap in ear reconstruction.Methods:1.skin flap modelIn this experiment,the reverse C skin flap model was reconstructed from the back of the SD rats.The pedicle was connected with the posterior superior iliac spine.The median line was extended from the middle to the left,and the width was 3 cm.The distal end of the flap was located on the head side and was 6cm long.The whole layer of skin was cut along the incision line,and the skin flap was separated by tissue cut between the membrane and the muscle membrane.The pedicle of the flap was used as the flap to provide blood transportat:ion.After careful hemostasis,the suture was in situ,and the line was disassembled 7 days after the operation.2.Grouping and processing50 healthy male SD rats were randomly divided into 2 groups:hyperbaric oxygen therapy group and control group.Each group was divided into 5 groups before and after operation,1,7,and 14,21 days.Each group contained 5 animals.The hyperbaric oxygen treatment group postoperative day with hyperbaric oxygen therapy for 2 times,the specific methods:animal animal experiments in the cabin,10 min up to 0.2MPa,voltage 36 min,oxygen concentration(86±2)%,stabilized during each of the 10 min 2min 15 min ventilation,decompression cabin,interval 2 among the above 4 h,for 5 consecutive days.In the control group,no other intervention was given after the operation.3.Collection treatment and observation index of specimen3.1 Measurement of blood flow of skin flapThe blood flow of flap was measured by laser Doppler in two groups,and blood flow of flap was recorded at first,seventh,fourteenth and 21 days before and after operation.Blood flow of flap was observed 21 days after operation,and the time of maximal blood flow after flap operation was recorded in two groups.3.2 Inflammatory response,scar tissue thickness and IⅠ/Ⅲ collagen ratio in basal flap of skin flapMaterials:The rats were anesthetized with intraperitoneal injection of 5%pentobarbital sodium(10 mg/kg weight)before and first,seventh,fourteenth,21 days after the operation.The skin flap and basal tissue samples were soaked in the volume fraction of 4%formaldehyde solution at the distal 1.0-2.0 cm from the distal 1.0-2.0 cm of the rat.Fine eosin staining was observed under microscope.Inflammatory reaction and morphological changes were observed at the base of flap.The thickness of the scar tissue between the sarcolemma and the sarcolemma at the base of the flap 21 days after operation was measured.The thickness of scar tissue was randomly measured under 40 times light microscope and the average thickness of scar tissue was taken as the thickness of scar tissue in the 5 groups.The embedded sections were stained with picric acid Sirius red and sliced under the polarizing microscope.Each tissue slice selected 5 horizons randomly under 200 times of polarized light.After slicing the photos,using the Image-Pro Plus 6 professional image analysis software,the ratio of I/III collagen was compared between the two groups.3.3 Observation of vascular number and vascular diameter of skin flapMaterials:all groups in preoperative and postoperative first,seventh,fourteenth,21 days,using intraperitoneal injection of 5%pentobarbital sodium(10 mg/kg BW)in anesthetized rats,1.0-2.0 cm at the distal cut 0.5 cm*0.5 cm flap and basal tissue samples were soaked in 4%formaldehyde solution volume fraction on the back flap in rats from the fixed,paraffin embedded,and sectioned to observe hematoxylin eosin staining under the microscope.Calculate the number of vessels in each slice 40 times under light microscope,the group average value as the number of blood vessels in the group;40 times under light microscope,randomly selected 3 vessels,each vessel diameter measurement of the long axis and short axis of the average value,average 3 values as the average diameter of the slice caliber of the vessel the average value of the diameter of the group.3.4 Detection of VEGF number of skin flapThe rats in the two groups were anesthetized with intraperitoneal injection of 5%pentobarbital(10 mg/kg body weight)before operation,first,seventh,fourteenth days and 21 days after operation,respectively.The whole skin and subcutaneous tissue of 0.5 cm cm 0.5 cm were collected from the distal 1.0-2.0 cm.With the volume fraction of 4%formaldehyde fixed,Rabbit anti rat VEGF monoclonal antibody was used for immunohistochemical staining.The staining method was performed according to the instructions of the kit.Finally,the cells with brown and yellow granules were stained with DAB.Each group of sections randomly selected 3 visual fields at 400 times,and counted the positive number,and the average number was the number of the positive cells.4.Statistical analysisThe data were statistically treated with SPSS 20 statistical software,and the results were expressed in X ±s.The variance analysis was compared between the two factors,and the 22 in the group was compared with the SNK test.The test level is a=0.05.Results:1.Measurement of blood flow of skin flapThe blood flow volume of the two groups decreased significantly on the 1 day after operation,and then the blood flow began to increase.The blood flow volume of the hyperbaric oxygen treatment group was higher than that of the control group at 1,7,14 and 21 days after operation,and the difference between the two groups was statistically significant(P<0.05).The maximum flow of blood flow in the hyperbaric oxygen therapy group was 10 ±1.20 days after operation.The maximum blood flow in the control group appeared at 13±1.40 days.The difference of the time of blood flow in the two groups of the largest flaps in the two groups was statistically different(P<0.05).2.Inflammatory response,scar tissue thickness and I/III collagen ratio in basal flap of skin flapThe edema of subcutaneous tissue in the control group was more obvious than that of the hyperbaric oxygen therapy group,and the inflammatory cells in the tissue were infiltrated more.On the 21 day after the operation,there were more fibroblasts in the basal part of the control group,the arrangement was disorderly,the density of the basal tissue was large,and the adhesion between the tissues was more obvious than that of the hyperbaric oxygen therapy group.The thickness of the basal tissue cicatricial adhesion in the control group was greater than that of the hyperbaric oxygen therapy group on the 21 day after the operation,and the difference between the two groups was statistically significant(P<0.05).The ratio of collagen I to III in the control group was greater than that of the hyperbaric oxygen therapy group.,and the difference between the two groups was statistically significant(P<0.05).3.Observation of vascular number and vascular diameter of skin flapThere was no significant difference in the number of flap vessels between the 1 days after operation(P>0.05).On the seventh day,14 days and 21 days after operation,the number of vessels in hyperbaric oxygen treatment group was higher than that in the normal group,the difference was statistically significant(P<0.05).The vessel diameters of the two groups were the smallest on the 1 day after operation,and the maximum diameter was 14 days after operation.The vessel diameter of the hyperbaric oxygen treatment group was larger than that of the control group on the 1 day after operation,and the difference between the two groups was statistically significant(P<0.05).4.Detection of VEGF number of skin flapThe VEGF numbers of the two groups began to increase on the 1 day after operation and reached the maximum at 14 days after operation.The VEGF values of the hyperbaric oxygen treatment group were higher than those of the control group at each observation time point,and the difference between the two groups was statistically significant(P<0.05).Conclusion1.Hyperbaric oxygen can reduce the scar adhesion between delayed skin flap and basal part in rats.2.Hyperbaric oxygen can increase the blood flow of delayed skin flap in rats,and its mechanism may be related to promoting angiogenesis. |
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