| ObjectiveTo investigate the effect of natural hirudin and recombinant hirudin which are applied locally to the vein congestion on random skin flap in rats models, and their effect on the flap of VEGF and CD34 are the same or not.Methods1. Establishment of animal model:Thirty Wistar rats, including male and female,were employed to establish animal model of vein congestive flap. All rats were anaesthetized by 10% chloral hydrate (300mg/kg). The hair of dorsal skin was removed with 8% sodium sulfide and the skin was sterilized with betadine. Caudally based dorsal flaps (10 cm×3 cm) in size were raised under sterile conditions. The palpable hip joints were used as anatomical landmarks to define the base of the flap. The flap was dissected and detached from its panniculus carnosus and reattached in the native position with separate sutures. The sites of injection were 1 ~ 3 cm distal to the flaps. The time of injection was immediately after surgery and re-injected at day 3 and day 5 post-operation. The injection spot was at the hypodermic level. The rats were divided randomly into 3 groups (ten in each group). In group A, 5 U of natural hirudin was locally applied to each flap. In groups B, 5 U of recombinant hirudin was locally applied to each flap. In group C, isotonic NaCl was locally applied to each flap as a control group.2. Make continuous clinical observation and record the back flap of the rats during 7 days after operation. Observe whether there is immunological rejection , such as diarrhea, Camponotus, Alice hair,depression and skin ulcers to the experimental animals. Observe the flap color,thickness, elasticity, exudation,hair growth, necrosis area and acupuncture bleeding situation,then record in detail.3. Histopathological evaluation: The samples were preserved in 10% formalin solution. Transverse sections were taken at the flap base 3 cm (viable area) or 7 cm (necrosis area) distal to the viable-necrotic area boundaries. The tissue samples were embedded in paraffin blocks; 0.5cm×0.5cm thick sections were cut and stained with haematoxylin and eosin and evaluated with a light microscope.4. VEGF and CD34 levels were measured on postoperative day 3,5 and 7. VEGF and CD34 were determined by immunohistochemistry. VEGF mRNA expression measured by fluorescence RT-PCR.5. To determine the skin flap survival rate, photographs of the skin flaps 7 days after surgery were analyzed using the Image-Pro 6.0 software. The flap survival rate was calculated as the percentage of the surviving flap area over the total flap area.Results1.General observation of the flap survival All rats survived until the end of the study without infection. Seven days after surgery, the necrotic area was stabilized with clear boundaries between the viable and necrotic areas. In the natural and recombinant hirudin treated groups, parts of the crust shells of the flaps were inconsistent in depth and were gray or light gray in color. These soft scar tissue can be easily peeled off revealing the wound and bleeding underneath. In contrast, the crust shells in the control group were hard with deep dark brown color.and contained local fester which can not be easily peeled off.2. Histopathological observation of the flapsIn the natural hirudin group, no atrophy was observed. Only mild hyperkeratosis and inflammatory cell infiltration were present in the flap tissue. There was an increase in subcutaneous vessel densities in the treated group. In contrast, subcutaneous flap edema and local ulceration were seen in the control group, with fewer subcutaneous vessels observed. The histopathological features of the recombinant hirudin group were intermediate among the three groups.3. The VEGF positive vessel density in flapImmunohistochemical coloring well. Good dyeing effect. Clean background pollution. Positive cells displayed clear. Postoperative day 3, VEGF staining the number of positive vessels: natural hirudin group (A): 41.71±3.08, recombinant hirudin group (B): 33.05±2.23, control group (C): 14.71±2.67. Both treatment groups have significantly higher numbers of VEGF positive vessels compared to control group (P<0.05). The largest number of VEGF positive vessels stained was group A . Group B followed it .Group C was the least. Postoperative day 5, VEGF staining the number of positive vessels: natural hirudin group (A): 55.24±3.96, recombinant hirudin group (B) : 41.63±3.97, control group (C): 8.63±2.59. Both treatment groups have significantly higher numbers of VEGF positive vessels compared to control group (P<0.05). The largest number of VEGF positive vessels stained was group A . Group B followed it. Group C was the least. Postoperative day 7, VEGF staining the number of positive vessels: natural hirudin group (A) : 40.76±3.69, recombinant hirudin group (B) : 32.93±2.54, control group (C): 3.58±2.97. Both treatment groups have significantly higher numbers of VEGF positive vessels compared to control group (P<0.05). The largest number of VEGF positive vessels stained was group A . Group B followed it .Group C was the least. In group A, the 5th day VEGF positive vessels stained compared with the 3th and 7th day ,P <0.05, peak at postoperative day 5; In group B, the 5th day VEGF positive vessels stained compared with the 3th and 7th day P <0.05, peak at postoperative day 5; In the C group, the 5th day VEGF positive vessels stained compared with the 3th and 7th day P <0.05, peak at postoperative day 3.4. The CD34 positive vessel density in flapPostoperative day 3, CD34 staining the number of positive vessels: natural hirudin group (A): 56.83±3.19, recombinant hirudin group (B): 47.12±2.13, control group (C):21.82±2.56. Both treatment groups have significantly higher numbers of CD34 positive vessels compared to control group (P<0.05). The largest number of CD34 positive vessels stained was group A. Group B followed it. Group C was the least. Postoperative day 5, CD34 staining the number of positive vessels: natural hirudin group (A):70.13±3.85, recombinant hirudin group (B): 52.74±3.08, control group (C):16.52±2.48. Both treatment groups have significantly higher numbers of CD34 positive vessels compared to control group (P<0.05). The largest number of CD34 positive vessels stained was group A .Group B followed it. Group C was the least. Postoperative day 7, CD34 staining the number of positive vessels: natural hirudin group (A):55.87±3.75, recombinant hirudin group (B):45.93±3.65, control group (C):11.47±2.87.Both treatment groups have significantly higher numbers of CD34 positive vessels compared to control group (P<0.05). The largest number of CD34 positive vessels stained was group A . Group B followed it. Group C was the least. In group A, the 5th day CD34 positive vessels stained compared with the 3th and 7th day P <0.05, peak at postoperative day 5; In the B group, the 5th day CD34 positive vessels stained compared with the 3th and 7th day P <0.05, peak at postoperative day 5; In the C group, the 5th day CD34 positive vessels stained compared with the 3th and 7th day P <0.05, peak at postoperative day 3.5. Fluorescent RT-PCR to detect the expression of flap tissue VEGFmRNAPostoperative day 3, VEGFmRNA expresses:natural hirudin group (A):28.69±2.07, recombinant hirudin group (B) : 23.94±3.12, control group (C):19.64±1.92. Both treatment groups have significantly higher numbers of VEGFmRNA compared to control group (P<0.05). The largest number of VEGFmRNA expresses was group A, Group B followed it. Group C was the least. Postoperative day 5, VEGFmRNA expresses:natural hirudin group (A):33.13±4.07, recombinant hirudin group (B):29.52±2.89, control group (C):15.52±3.48. Both treatment groups have significantly higher numbers of VEGFmRNA compared to control group (P<0.05). The largest number of VEGFmRNA expresses was group A. Group B followed it. Group C was the least. Postoperative day 7, VEGFmRNA expresses:natural hirudin group (A):27.87±2.58, recombinant hirudin group (B):22.93±3.43, control group (C):10.47±2.86. Both treatment groups have significantly higher numbers of VEGFmRNA compared to control (P<0.05). The largest number of VEGFmRNA expresses was group A. Group B followed it. Group C was the least. In group A, the 5th day VEGFmRNA expresses compared with the 3th and 7th day P <0.05, peak at postoperative day 5; In group B, the 5th day VEGFmRNA expresses compared with the 3th and 7th day P <0.05, peak at postoperative day 5; In group C, the 5th day VEGFmRNA expresses compared with the 3th and 7th day P <0.05, peak at postoperative day 3.6. Flap survivalSurface area of flap survival: natural hirudin group (A): 26.64±2.24 (cm~2), recombinant hirudin group (B): 23.64±2.02 (cm~2), control group (C): 20.71±1.41 (cm~2); flap survival rate (%): natural hirudin group (A): 88.87±2.24, recombinant hirudin group (B):79.97±2.02, control group (C):69.07±1.42. Pairwise comparison between three groups, P <0.05.ConclusionVEGF and CD34 expression in random skin flap can be increased by natural hirudin and recombinant hirudin which are conducive to angiogenesis and can improve the survival rate of random skin flap. Natural hirudin is superior than recombinant hirudin. OBJECTIVETo investigate the effect of natural hirudin and recombinant hirudin which are applied locally to the vein congestion on random skin flap in rats models. And the flap of SOD, MDA and ET expression are the same or not.METHODS1,Animal model: It is the same with the first.2. Make continuous clinical observation and record the back flap of the rats during 7 days after operation. It is the same with the first.3. Histopathological evaluation: It is the same with the first.4. SOD, ET and MDA levels were measured after surgery. In the light microscope, the flap cell morphological changes were observed.Flap survival 7 days after surgery was calculated. MDA was detected through thiobarbituric acid method. SOD was detected using hydroxylamine method. ET was detected by enzyme linked immunosorbent assay method. Results1.General observation of the flap survival It is the same with the first.2.Histopathological observation of the flaps It is the same with the first.3. MDA, SOD, and ET levels in the flaps Postoperative day 7, MDA content of the flap: Natural hirudin group (A): 10.20±3.41 (nmol / mgprot), Recombinant hirudin group (B): 22.12±3.84 (nmol / mgprot), Control group (C) : 32.65±3.91 (nmol / mgprot); MDA is significant different from each other (P <0.05). Postoperative day 7, SOD levels of the flap: natural hirudin group (A): 72.59±17.48 (U / mgprot), recombinant hirudin group (B): 36.68±8.32 (U / mgprot), the control group (C) : 27.66±11.34 (U / mgprot); SOD is significant different from each other (P <0.05). ET levels of the flap: Natural hirudin group (A): 85.67±37.90, Recombinant hirudin group (B): 163.55±43.45, Control group (C): 247.56±38.04; ET is significant different from each other (P <0.05).4. Flap survivalIt is the same with the first.CONCLUSIONSHirudin can improve the flap survival rate and SOD content; reduce the rate of flap necrosis, MDA, and ET levels. The experimental group were significantly different from the control group. Natural hirudin demonstrated more pronounced effects than recombinant hirudin.
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