Objective To explore the effects of natural hirudin on the survival of dorsal random flap after early pedicle division in rats. Methods Thirty-eight adult Wistar rats, male or female, weighing 220-280 g, were selected and randomly divided into natural hirudin group (experimental group, n=19) and normal saline group (control group, n=19). The dorsal random flap of 9 cm × 3 cm in size was prepared on the back of the rats. Six symmetrical injection points were selected at 2, 4, and 8 cm from the pedicle, 0.5 mL (3 ATU) natural hirudin and equivalent normal saline were injected in experimental group and control group respectively every day to the end of the experiment. After 4 days, the pedicles were cut. The flap was observed, and the ratio of flap survival area was measured at 7 days after pedicle division. At immediately after flap preparation, at 1 day before pedicle division, and at 3 and 7 days after pedicle division, the specimens were harvested for histological staining, immunohistochemical staining, microvessel density (MVD) measurement, and detection of vascular endothelial growth factor (VEGF) mRNA expression by fluorescent quantitative RT-PCR. Results All rats of 2 groups survived to the completion of the experiment. After pedicle division, the flap necrosis area of experimental group was smaller than that of control group; at 7 days, the ratio of flap survival area in experimental group was 85.366% ± 2.872%, and was significantly higher than that in control group (75.252% ± 3.455%) (t=7.117, P=0.000). Histological staining showed that no significant difference was found in the number of capillaries between 2 groups at immediately after flap preparation; at 1 day before pedicle division and at 3 and 7 days after pedicle division, the capillary number of experimental group was significantly more than that of control group. Immunohistochemical staining showed that CD34 positive cells were observed in 2 groups; MVD of experimental group was significantly higher than that of control group at 1 day before pedicle division, and at 3 and 7 days after pedicle division (P lt; 0.05). At 7 days after pedicle division, the VEGF mRNA expression was 7.122 ± 0.503 in experimental group, and was 5.655 ± 1.174 in control group, showing significant difference (t=3.633, P=0.003). Conclusion Natural hirudin can promote the formation of new blood vessels, improve the blood supply of the flap, and increase the survival of random flap after early pedicle division by increasing VEGF expression.
Objective To investigate the effect of natural hirudin which is appl ied locally on vein congestion of random pattern skin flap in porcine models. Methods Three Guangxi Bama miniature pigs, including male and female aged 6-8 months and weighing 10-15 kg, were employed to establ ish animal model of vein congestive. Six dorsal random pattern skin flaps (three on each side) were prepared on each animal, 14 cm × 4 cm in size. According to the pharmacologic manipulations which were administered immediately and at 1, 2, and 3 days after operation respectively, the eighteen flaps were divided randomly into 3 groups (six in each group). In group A, isotonic Na chloride was locally appl ied as control group. In group B, 3 mL of 20 ATU natural hirudin was locally appl ied at each flap. In group C, 3 mL of 40 ATU natural hirudin was locally appl ied at each flap. Macroscopic observation (at 1 and 10 days postoperatively) and histological observation (at 1 and 7 days postoperatively) were made, the ratio of wet weight to dry weight of the congestive tissue (at 3 and 7 days postoperatively), the temperature of the surface of congestive flap (at 5 days postoperatively) and local blood flow of the flap (by Color Doppler Ultrasound at 7 days postoperatively) were measured. The survival rate of skin flaps was determined at 12 days postoperatively. Results Macroscopic observation showed that congestion of the flaps had no significance among three groups immediately after operation (P gt; 0.05); at 1 day postoperatively, the length of the congestion of the flap in group A (9.68 ± 0.43) cm was significantly longer than that in group B (6.81 ± 0.53) cm and group C (8.51 ± 0.64) cm (P lt; 0.05), while there was no significant difference between group B and group C (P gt; 0.05); at 10 days postoperatively, the necrosis at the distal end of flap in group A and group C were significantly longer than that in group B (P lt; 0.05), while there was no significant difference between group A and group C (P gt; 0.05). The histological observation revealed that the degree of erythrocyte agglutination in dermis capillary and veinule in group A was more serious than that of group B at 1 day postoperatively, and there was l ittle collagen and granulation tissue in group A when compared with group B at 7 days postoperatively. The ratio of wet weight to dry weight: at 3 days postoperatively, the value in group A (3.94 ± 0.14) was significantly higher than that of group B (3.43 ± 0.14) and group C (3.60 ± 0.19) (P lt; 0.05), and there was no significant difference between group B and group C (P gt; 0.05); at 7 days postoperatively, the value in group A (3.61 ± 0.11) was significantly higher than that of group B (3.08 ± 0.13) and group C (3.34 ± 0.21) (P lt; 0.05), and there was no significant difference between group B and group C (P gt; 0.05). The surface temperature of the congestive flap was (36.64 ± 0.70)℃ in group A, (38.61 ± 0.42)℃ in group B and (37.50 ± 0.46)℃ in group C at 5 days postoperatively; showing significant difference between group A and groups B, C (P lt; 0.05), and no significant difference between group B and group C (P gt; 0.05). The Color Doppler Ultrasound showed that the image of blood flow was very l ittle in group A, the image of venous return and perforator artery could be seen in group B and the image of arterial blood flow could be detected in group C. The survival rate of skin flaps was 45% ± 7% in group A, 67% ± 4% in group B and 52% ± 4% in group C at 12 days postoperatively; showing statistically significant difference between groups B, C and group A (P lt; 0.05), but no statistically significant difference between group B and group C (P gt; 0.05). Conclusion Local appl ication of natural hirudin can significantly improve the congestion of random pattern skin flap in a porcine model.
ObjectiveTo investigate the effect of natural hirudin combined with hyperbaric oxygen therapy on the survival of transplanted random-pattern skin flap in rats.MethodsA random-pattern skin flap in size of 10.0 cm×2.5 cm was elevated on the dorsum of 72 Sprague Dawley rats. Then the 72 rats were randomly divided into 4 groups (n=18) according to the therapy method. At immediate and within 4 days after operation, the rats were treated with normal saline injection in control group, normal saline injection combined with hyperbaric oxygen treatment in hyperbaric oxygen group, the natural hirudin injection in natural hirudin group, and the natural hirudin injection combined with hyperbaric oxygen treatment in combined group. The flap survival was observed after operation, and survival rate was evaluated at 6 days after operation. The skin samples were collected for histological analysis, microvessel density (MVD) measurement, and evaluation of tumor necrosis factor α (TNF-α) expression level by the immunohistochemical staining at 2 and 4 days after operation.ResultsPartial necrosis occurred in each group after operation, and the flap in combined group had the best survival. The survival rate of flap was significantly higher in hyperbaric oxygen group, natural hirudin group, and combined group than that in control group, and in combined group than in hyperbaric oxygen group and natural hirudin group (P<0.05). There was no significant difference between hyperbaric oxygen group and natural hirudin group (P>0.05). At 2 days, more microvascular structure was observed in hyperbaric oxygen group, natural hirudin group, and combined group in comparison with control group; while plenty of inflammatory cells infiltration in all groups. At 4 days, the hyperbaric oxygen group, natural hirudin group, and the combined group still showed more angiogenesis. Meanwhile, there was still infiltration of inflammatory cells in control group, inflammatory cells in the other groups were significantly reduced when compared with at 2 days. At 2 days, the MVD was significantly higher in hyperbaric oxygen group, natural hirudin group, and combined group than that in control group (P<0.05); the expression of TNF-α was significantly lower in hyperbaric oxygen group, natural hirudin group, and combined group than that in control group (P<0.05). There was no significant difference in above indexes between hyperbaric oxygen group, natural hirudin group, and combined group (P>0.05). At 4 days, the MVD was significantly higher in hyperbaric oxygen group, natural hirudin group, and combined group than that in control group, in natural hirudin group and combined group than in hyperbaric oxygen group (P<0.05). The expression of TNF-α was significantly lower in hyperbaric oxygen group, natural hirudin group, and combined group than that in control group, in combined group than in natural hirudin group and hyperbaric oxygen group (P<0.05).ConclusionHyperbaric oxygen and natural hirudin therapy after random-pattern skin flap transplantation can improve the survival of flaps. Moreover, combined therapy is seen to exhibit significant synergistic effect. This effect maybe related to promotion of angiogenesis and the reduction of inflammation response.
ObjectiveTo investigate the effect of natural hirudin on revascularization of ischemic skin flap in rats using Micro-CT and three-dimensional (3D) reconstruction.MethodsThirty-two Sprague Dawley rats were prepared a ischemic skin flap (8.0 cm×1.8 cm) model on the back and randomly divided into hirudin group and control group (16 rats in each group). At immediate and within 3 days after operation, the rats were treated with hypodermic injection of natural hirudin 0.3 mL (including natural hirudin 6 ATU) every day in hirudin group and the equal amount of normal saline in control group. At 6 days after operation, the survival rate of skin flap was evaluated, histological changes were observed by HE staining, and the volemia, length of blood vessels, and number of blood vessels were analyzed with Micro-CT 3D reconstruction.ResultsBoth groups of rats survived to the end of the experiment without infection. Different degrees of necrosis occurred in the distal part of the skin flaps in both groups at 6 days after operation, but the flap survival rate of the hirudin group (72.11%±8.97%) was significantly higher than that of control group (58.94%±4.02%) (t=3.280, P=0.008). Histological observation showed that the histological hierarchy of the hirudin group was clearer than that of the control group, with more microangiogenesis and less inflammatory response and inflammatory cell infiltration. Micro-CT 3D reconstruction showed that the flap vessels in the hirudin group were more and denser, and the volemia, length of blood vessels, and number of blood vessels were significantly higher than those in the control group (P<0.05).ConclusionNatural hirudin can reduce the inflammation of tissue, promote the regeneration and recanalization of blood vessels in ischemic skin flap, so as to improve the survival rate of the flap.