The expression of proinflammatory cytokines in the rat muscle flap with ischemia-reperfusion injury.

Ischemic-reperfusion injury mediated by free radicals and neutrophils is the principal pathway for tissue injury and death. Cytokines influence activity of various cell types during the inflammatory process. In this study, expression of selected proinflammatory cytokines was examined in primary and secondary ischemia in the rat gracilis flap model. Sixty Sprague-Dawley rats were used in the study. Primary ischemia of each gracilis flap was induced by clamping its vascular pedicle for 1 hour. The flap was then replaced and allowed to reperfuse. Twenty-four hours later, a secondary ischemia was induced via vascular clamping for 4 hours. All muscle flaps were biopsied at 4 hours and 18 hours after primary ischemia. After secondary ischemia, each flap was biopsied immediately postevent, at 4 hours, and at 18 hours. Expression of tumor necrosis factor (TNF-alpha), interleukin (IL-1beta), and platelet-derived growth factor (PDGF) mRNA was determined by RT-PCR in each case. An equal sample size of gracilis muscle flaps, elevated in an identical fashion but not subjected to vascular clamping, was examined for baseline gene expression. Results showed that TNF-alpha gene expression was significantly up-regulated at 18 hours after secondary ischemia. IL-1 gene expression was up-regulated at 4 hours after primary ischemia, and was greatest at 4 hours after secondary ischemia. PDGF expression was up-regulated immediately after secondary ischemia, then at 4 hours after secondary ischemia (P < 0.05), and down-regulated during reperfusion. This study delineated changes in the expression of TNF-alpha, IL-1beta, and PDGF mRNA, in both primary and secondary ischemia and reperfusion episodes at several critical time points.

Muscle flap mass preservation with end-to-side neurorrhaphy: an experimental study.

The authors examined the preservation of rat gracilis muscle flap mass after motor and sensory end-to-side neurorrhaphy. The rat gracilis muscle flap model was designed based on a previous study. Twenty-four Sprague-Dawley rats were divided into three groups. In Group 1 (n = 8), the flap was denervated by transecting the obturator nerve. In Group 2 (n = 8), the flap was reinnervated by coapting the proximal saphenous nerve to the distal obturator nerve. In Group 3 (n = 8), the flap was reinnervated by coapting the motor branch of the femoral nerve to the distal stump of the obturator nerve. At 6 months postoperatively, the gracilis muscle flaps were examined, harvested, and weighed individually. Results showed that the flaps with motor nerve reinnervation retained good bulk, with a weight of 634.0 +/- 65.1 gm, which was statistically significantly higher than the denervated group (457.5 +/- 125.3 gm, p < 0.01). However, muscle mass preservation in the sensory reinnervated group (606.9 +/- 209.1 gm) was not significantly different, compared to the denervated group. Histology revealed atrophic changes in the denervated group, compared to the sensory and motor-reinnervated groups. The authors concluded that muscle mass can be preserved by end-to-side nerve repair. Motor nerve reinnervation is able to better arrest atrophic changes of the muscle flaps.

Vascular endothelium growth factor, surgical delay, and skin flap survival.

OBJECTIVE: Cytokines may be a mechanism by which surgical delay can increase flap survival. We previously found that preoperative vascular endothelium growth factor (VEGF) administration in the rat transverse rectus abdominis myocutaneous (TRAM) flap could improve skin paddle survival. In this study, we used partial elevation of the rat TRAM flap as a surgical delay to assess endogenous cytokine expression and tissue survival comparable to undelayed TRAM flaps. METHODS: In Part I, TRAM flaps underwent surgical delay procedures; 7 days later, the flaps were completely elevated and reinset. At the same time, other flaps were raised and reinset without delay. Skin paddle survival in both groups was evaluated at 7 days. In Part II, skin biopsies from TRAM zones I to IV were taken at the time of delay and at intervals of 12, 24, 48, and 72 hours. Specimens were assessed for selected cytokine gene expression by reverse transcription-polymerase chain reaction analysis (TR-PCR). RESULTS: Surgical delay significantly (P < 0.001) increased skin paddle survival in the delayed TRAM flaps (16.14 +/- 1.53 cm, 81.9%) compared with undelayed flaps (7.68 +/- 3.16 cm, 40.9%). TGF-beta and PDGF expressions were not changed by surgical delay, but basic fibroblast growth factor (bFGF) and VEGF expressions increased significantly (P < 0.05 and P < 0.01) after delay. CONCLUSIONS: In the rat TRAM model, surgical delay resulted in increased VEGF expression and increased skin paddle survival. These results correlate with previous studies showing the preoperative injection of VEGF increases skin paddle survival. VEGF may be an important element in the delay phenomenon and may be an agent for pharmacological delay.

Regulation of inducible nitric oxide synthase in ischemic preconditioning of muscle flap in a rat model.

PURPOSE: Ischemic preconditioning has been shown to influence flap tolerance to prolonged ischemia. Nitric oxide (NO) synthesis is one of the proposed mechanisms involved in ischemic preconditioning. In this study, the molecular marker of NO is examined in correlation with ischemic preconditioning on improving muscle flap survival. METHODS: Fifty male Sprague-Dawley rats were randomized into experimental and control groups. The gracilis muscle flap with femoral vascular pedicle was used as a flap model. Ischemic preconditioning consisted of 3 sequences of clamping the pedicle for 10 minutes followed by 10 minutes of reperfusion for a total of 1 hour. In part I, the experimental group (n = 10) underwent ischemic preconditioning for 1 hour. In the control group (n = 10), the flaps were dissected without clamping of the pedicle. Both groups were then subjected to 4 hours of global ischemia by continuous pedicle clamping, after which the flaps were sutured to their beds. On postoperative day 3, flap survival was determined by gross and histologic examinations. The evaluators were blinded to the treatment. In part II, the experimental group (n = 12) underwent ischemic preconditioning, while the control group (n = 12) did not. The flaps from each group were harvested for inducible nitric oxide synthase (iNOS) gene expression using reverse transcriptase-polymerase chain reaction at the end of 1 hour after reperfusion and at 4 hours of global ischemia. RESULTS: The results indicated a significantly higher survival rate in the experimental group than in the control group (90 versus 50%, P < 0.05). iNOS gene expression was significantly higher in the experimental group than in the control group at 1 hour after ischemic preconditioning (0.73+/-0.18 versus 0.26+/-0.11, P < 0.01). However, after 4 hours of global ischemia, iNOS expression in the control group was statistically higher than in the experimental group (0.83+/-0.16 versus 0.26+/-0.07, P < 0.01). CONCLUSIONS: We conclude that ischemic preconditioning can enhance flap tolerance to ischemia-reperfusion injury and improve flap viability rate. This study provides evidence that the regulation of NOS may play a role in ischemic preconditioning phenomenon and warrants further investigation.

Porcine small intestinal submucosa as a carrier for skin flap prefabrication.

The feasibility of porcine small intestinal submucosa (SIS) as a carrier in skin flap prefabrication was examined in this study. Thirty-eight rats were randomly divided into five groups. The saphenous vascular bundle was used as the vascular carrier. In group 1 (n = 8), an arteriovenous fistula was made by anastomosis of distal saphenous artery and vein. A SIS patch (1.5 x 2 cm2) was placed underneath the vascular bundle. In group 2 (n = 8), the vascular bundle was isolated and laid over the SIS patch. The distal saphenous vessels were ligated when the flap was raised. In group 3 (n = 8), an arteriovenous fistula was made without SIS implant. In group 4 (n = 8), the flap was raised with only the vascular bundle with the distal end ligated. After 2 weeks of maturation, the flap was raised with only the vascular bundle. In group 5 (n = 6), SIS was implanted and the flap including the SIS patch was raised and replaced without the vascular pedicles. The survival of the flaps and histology were evaluated at 5 days after flap replacement. The results showed that the average survival area in group 1 was 99% +/- 3% and the survival area in group 2 was 86% +/- 16%. The mean survival areas in group 3 and 4 were 60% +/- 9% and 25% +/- 10%, respectively. No flap survival was observed in the group 5. These were significantly lower than in groups 1 and 2 (p < 0.05, p < 0.01). Histology showed that SIS patch was incorporated into the adjacent connective tissue and increased amounts of neovascularization were seen between the collagenous sheets and dermis. In conclusion, this study demonstrated that porcine SIS can incorporate into the adjacent tissue and induce angiogenesis in flap prefabrication. This biomaterial can provide a scaffold for supporting and enhancing the survival of vascular prefabricated skin flap.

Effect of vascular endothelial growth factor on rat Achilles tendon healing.

This study evaluated the effect of exogenous vascular endothelial growth factor (VEGF) on tendon healing and regulation of other growth factors in a rat Achilles tendon model. Fifty Sprague-Dawley rats were used. In the experimental group, the left Achilles tendon was transected and repaired with the modified Kessler suture technique, and the right Achilles tendon was transected and repaired with resection of plantaris tendon. VEGF, 100 mul (50 mug/ml), was injected into each tendon at the repair site. The same surgical procedures were performed in the control group, with the same volume of saline injected into the repair sites. At intervals of 1, 2, and 4 weeks, the animals were killed and the tendons were harvested and evaluated for tensile strength (1, 2, and 4 weeks) and gene expression (postoperative day 4). At 1 week postoperatively, when plantaris tendon was preserved, the tensile strength of the repaired tendons with VEGF treatment (3.63 +/- 0.62 MPa) was significantly higher than the tensile strength of the repaired tendons with saline treatment (2.20 +/- 0.36 MPa). There was no difference in tensile strength between the two groups without the plantaris tendon support. At 2 weeks postoperatively, the tensile strength was 11.34 +/- 3.89 MPa in the group with VEGF treatment and plantaris tendon preservation, which was significantly higher than the tensile strength in the other groups. There was no significant difference in tensile strength among the groups at 4 weeks postoperatively. The gene expression showed that transforming growth factor-beta in the VEGF-treated tendon was up-regulated in the early stage of tendon healing, whereas expression of platelet-derived growth factor, basic fibroblast growth factor, and insulin-like growth factor-1 was not significantly different among the groups. In conclusion, administration of exogenous VEGF can significantly improve tensile strength early in the course of the rat Achilles tendon healing and was associated with increased expression of transforming growth factor-beta.

Evaluation of the mechanism of vascular endothelial growth factor improvement of ischemic flap survival in rats.

This study evaluated the effects of exogenous vascular endothelial growth factor (VEGF) on the regulation of cytokines in a rat dorsal ischemic skin flap model. Exogenous VEGF (1 microg/ml) was injected subdermally into the flaps of 12 rats before the flaps were sutured back in place. Another 12 rats with flaps received saline injections, as a control group. Biopsy specimens were obtained from the flaps treated with VEGF or saline solution, at positions 2.5, 5.5, and 8.5 cm from the distal edge of the flaps, at 12 hours (n = 6 for each group) and 24 hours (n = 6 for each group) after suturing of the flaps. Expression of cytokine, growth factor, and inducible nitric oxide synthase was measured. The results demonstrated that expression of tumor necrosis factor-alpha and nitric oxide synthase in the distal part of the VEGF-treated flaps was significantly decreased, compared with the control values, at 12 and 24 hours postoperatively. It was concluded that administration of exogenous VEGF could protect flaps from ischemia-reperfusion injury through the regulation of proinflammatory cytokines and the inhibition of cytotoxic nitric oxide production.

Clinical case experience using microsurgical venous flaps for soft-tissue coverage of the lower extremity.

Numerous venous flap models have been experimentally introduced and clinically attempted in reconstruction of soft-tissue defects. The authors report experience in soft-tissue coverage with microsurgical saphenous venous flap transfers in five patients with lower-extremity wounds. Four of these flaps failed postoperatively. Unhealthy recipient beds and inflamed veins were potential causes for these flap failures. Easy dissection without sacrifice of the artery would seem to provide an ideal situation for venous flap utilization, but this rate of failure prohibits recommendation of this flap in large, traumatic wounds.