Pharmacologic prophylactic treatment for perioperative protection of skeletal muscle from ischemia-reperfusion injury in reconstructive surgery.

BACKGROUND: In autogenous muscle transplantation, unpredictable complications can cause prolonged ischemia, resulting in ischemia-reperfusion injury. The authors investigated the efficacy and mechanism of nicorandil, a nitrovasodilator and adenosine triphosphate-sensitive potassium channel opener, in inducing perioperative protection of muscle flaps from ischemia-reperfusion injury. METHODS: Pigs (18.2 ± 2.4 kg) were assigned to one control and eight treatment groups. Bilateral latissimus dorsi muscle flaps were raised after saline administration (control) and 0, 4, 8, 12, 24, 48, 72, and 96 hours after nicorandil administration. Subsequently, flaps were subjected to 4 hours of ischemia and 48 hours of reperfusion. Viability was assessed, and biochemical probes were used to study nicorandil-induced infarct protection. RESULTS: Protection by nicorandil was biphasic. Infarction reduced from 40.2 ± 1.9 percent (control) to 27.3 ± 1.7 percent and 24.0 ± 2.3 percent (p < 0.05) 0 and 4 hours after nicorandil administration, respectively (early phase protection). No difference was seen between control and treatment groups between 8 and 12 hours after nicorandil administration compared with the control. Infarct protection increased again (p < 0.05) at 24 (22.4 ± 2.0 percent), 48 (25.1 ± 2.1 percent), and 72 hours (28.5 ± 2.1 percent) but not at 96 hours (43.9 ± 4.6 percent) compared with control (late phase protection). The sarcolemmal and mitochondrial channels played a central role in the trigger and mediator mechanisms, respectively. Late protection was associated with lower myeloperoxidase activity and mitochondrial calcium overload and higher adenosine triphosphate content (p < 0.05). CONCLUSIONS: Nicorandil induced 48-hour uninterrupted muscle infarct protection, starting 24 hours after intravenous administration. This category of clinical drug is a potential prophylactic treatment against skeletal muscle ischemia-reperfusion injury in reconstructive surgery.

Combination of hypoxic preconditioning and postconditioning does not induce additive protection of ex vivo human skeletal muscle from hypoxia/reoxygenation injury.

We previously demonstrated that hypoxic preconditioning (HPreC) or postconditioning (HPostC) protected ex vivo human skeletal muscle from hypoxia/reoxygenation injury. Here, we investigated if combined HPreC and HPostC could convey additive protection. Human rectus abdominis muscle strips were cultured in normoxic Krebs buffer for 5 hours (control) or in 3 hours hypoxic/2 hours normoxic buffer (treatment groups). HPreC and HPostC were induced by 1 cycle of 5 minutes hypoxia/5 minutes reoxygenation immediately before or after 3 hours hypoxia, respectively. Muscle injury, viability, and adenosine triphosphate (ATP) synthesis were assessed by measuring lactate dehydrogenase release, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide reduction, and ATP content, respectively. Hypoxia/reoxygenation caused lactate dehydrogenase to increase and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide reduction and ATP content to decrease (P < 0.05; n = 7). HPreC, HPostC, and combination of both were equally effective in protection of muscle from hypoxia/reoxygenation injury. Atractyloside (5 × 10 M), a mitochondrial permeability transition pore opener, abolished the protective effect of HPreC or HPostC. We conclude that HPreC and HPostC protect ex vivo human skeletal muscle against hypoxia/reoxygenation injury by closing the mitochondrial permeability transition pore. For that reason, they are equally effective and do not demonstrate an additive effect. Moreover, the potent effect of HPostC indicates ischemic postconditioning as an effective clinical intervention against reperfusion injury in autogenous skeletal muscle transplantation and replantation surgery.

Efficacy and mechanism of hypoxic postconditioning in salvage of ex vivo human rectus abdominis muscle from hypoxia/reoxygenation injury.

In reconstructive surgery, skeletal muscle may endure protracted ischemia before reperfusion, which can lead to significant ischemia/reperfusion injury. Ischemic postconditioning induced by brief cycles of reperfusion/reocclusion at the end of ischemia has been shown to salvage skeletal muscle from ischemia/reperfusion injury in several animal models. However, ischemic postconditioning has not been confirmed in human skeletal muscle. Using an established in vitro human skeletal muscle hypoxic conditioning model, we tested our hypothesis that hypoxic postconditioning salvages ex vivo human skeletal muscle from hypoxia/reoxygenation injury and the mechanism involves inhibition of opening of the mitochondrial permeability transition pore (mPTP) and preservation of ATP synthesis. Muscle strips (~0.5×0.5×15mm) from human rectus abdominis muscle biopsies were cultured in Krebs-Henseleit-HEPES buffer, bubbled with 95%N(2)/5%CO(2) (hypoxia) or 95%O(2)/5%CO(2) (reoxygenation). Samples were subjected to 3h hypoxia/2h reoxygenation. Hypoxic postconditioning was induced by one or two cycles of 5min reoxygenation/5min hypoxia after 3h hypoxia. Muscle injury, viability and ATP synthesis after 2h of reoxygenation were assessed by measuring lactate dehydrogenase (LDH) release, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reduction and ATP content, respectively. Hypoxic postconditioning or treatment with the mPTP-opening inhibitors Cyclosporine A (CsA, 5×10(-6)M) or N-Methyl-4-isoleucine Cyclosporine (NIM811, 5×10(-6)M) 10min before reoxygenation decreased LDH release, increased MTT reduction and increased muscle ATP content (n=7 patients; P<0.05). Conversely, treatment with the mPTP opener Atractyloside (5×10(-6)M) 10min before hypoxic postconditioning abolished its protective effect (n=7 patients; P<0.05). We conclude that hypoxic postconditioning effectively salvages human skeletal muscle from hypoxia/reoxygenation injury by inhibition of mPTP opening and preservation of ATP synthesis during reoxygenation.

Local hemodynamic effects of radiation on the rabbit orbitozygomatic complex with and without cytoprotection.

BACKGROUND: The authors have previously demonstrated that radiation-induced craniofacial bone growth inhibition may be ameliorated using the known cytoprotectant amifostine in the infant rabbit orbitozygomatic complex. The authors' hypothesis is that reduction in blood supply plays an important role in inhibiting craniofacial bone growth following radiotherapy and that cytoprotective pretreatment exerts its protective effect by maintaining blood supply. METHODS: Seven-week-old New Zealand male infant rabbits underwent single-dose orthovoltage irradiation to the right orbitozygomatic complex using established protocols: 0 Gy (sham), 35 Gy, and 35 Gy following pretreatment with amifostine (300 mg/kg administered intravenously). Blood flow to the orbitozygomatic complex, orbitozygomatic complex periosteum, masseter, hemimandible, and overlying skin was measured 1, 14, and 63 days after irradiation, using the modified 15-µm radioactive microsphere technique (n = 18 per group, n = 6 per time point). Orbitozygomatic complex bone specimens were harvested for blood vessel morphometry using safranin O stains at days 1 and 100 after irradiation (n = 20 per group, n = 10 per time point). RESULTS: Blood flow to the irradiated orbitozygomatic complex was significantly (p < 0.05) greater 1 day after single-dose orthovoltage irradiation compared with nonirradiated controls. This increase was not observed in the amifostine-pretreated animals and was also not seen 14 and 63 days after irradiation. No histomorphometric vessel changes were detected at any time point after irradiation in this study. CONCLUSIONS: Single-dose orthovoltage irradiation results in a temporary elevation in regional blood flow to the orbitozygomatic complex, returning to control levels within 14 days. Although pretreatment with amifostine attenuates this response, radiation-induced craniofacial bone growth inhibition in this model does not appear to be secondary to hemodynamic alterations.

Radiation-induced craniofacial bone growth inhibition: acute and long-term effects on bone histopathology with and without cytoprotection.

BACKGROUND: The authors previously established an animal model of radiation-induced craniofacial bone growth inhibition and demonstrated the effectiveness of cytoprotection in preserving growth using amifostine, but the mechanism is unclear. The objective of this study was to investigate the acute and long-term histopathologic effects of single-dose orthovoltage irradiation on craniofacial bone with and without cytoprotection. METHODS: Sixty infant New Zealand White rabbits (7-week-old) were randomized into three groups (n = 20 per group): group 1, 0-Gy, sham irradiation; group 2, 35-Gy single-dose orthovoltage irradiation; and group 3, cytoprotection with amifostine before irradiation. Orbitozygomatic complex bone was harvested from animals 12 hours after irradiation and at skeletal maturity (21 weeks of age). Histologic parameters measured included native bone cell (osteoblast, osteoclast, and osteocyte) populations, periosteal proliferation indices (MIB-1 stains), bone turnover rates [triple fluorochromes: tetracycline administered at 7 weeks of age (before irradiation), alizarin complexone at 12 weeks, and calcein at 16 weeks of age], and endosteal space fibrosis levels. RESULTS: Orthovoltage irradiation significantly (p < 0.05) reduced osteoblast and osteoclast counts 12 hours after irradiation (age, 7 weeks) with or without pretreatment with amifostine but had no effect on osteocyte populations. Long-term analysis at age 21 weeks demonstrated significantly (p < 0.05) increased osteoblast counts, reduced endosteal space fibrosis, reduced periosteal proliferation indices, and improved bone turnover (fluorochrome stains) in amifostine-treated animals. CONCLUSION: This study suggests that amifostine cytoprotection is mediated through a combination of reduced cellular injury with enhanced promotion of cellular bone rebuilding potential.

Vasorelaxation effect and mechanism of action of vascular endothelial growth factor-165 in isolated perfused human skin flaps.

BACKGROUND: Experimental evidence is accumulating to indicate that local acute vascular endothelial growth factor-165 (VEGF(165)) therapy is effective in attenuation of skin ischemia and increase in skin viability in rat skin flap surgery and the mechanism involves vasodilation induced by VEGF(165). So far, the vasodilator effect and mechanism of action of VEGF(165) have not been studied in human skin. The objective of this project is to test the hypothesis that VEGF(165) is also a potent vasodilator in human skin vasculature. MATERIALS AND METHODS: We used an established isolated perfused human skin flap model and pharmacologic probes to demonstrate that VEGF(165) is a potent vasodilator in human skin vasculature and the mechanism involves activation of receptors and postreceptor signaling pathway, which in turn stimulates local synthesis/release of endothelial vasodilators. RESULTS: We observed that VEGF(165) induced a concentration-dependent vasorelaxation in human skin flaps preconstricted with norephinephrine (8 × 10(-7)M; n = 7) or endothelin-1 (3 × 10(-9)M; n = 6). The vasorelaxation potency of VEGF(165) (pD(2) = 12.02 ± 0.25; n = 7) was higher (P < 0.05) than that of acetylcholine (pD(2) = 6.76 ± 0.06; n = 5) in human skin flaps preconstricted with 8 x 10(-7)M of norepinephrine. Using pharmacologic probes, we also detected that the vasorelaxation effect of VEGF(165) in the isolated perfused human skin flaps (n = 4) was triggered by activation of VEGF receptor-2. Furthermore, the postreceptor signaling pathway involved activation of Src family tyrosine kinase, phospholipase C, protein kinase C, an increase in inositol 1,4,5-triphosphate activity, a release of the intracellular Ca(2+) store, and finally synthesis/release of the endothelial nitric oxide (eNO) and prostacyclin and eNO predominantly mediated the vasodilator effect of VEGF(165) in the effector mechanism. CONCLUSION: These findings support our hypothesis that VEGF(165) is a potent vasodilator in human skin vasculature and also provide important insights into the clinical study of local acute VEGF(165) therapy for prevention/treatment of skin ischemia in skin flap surgery.

Pathogenesis of radiation-induced capsular contracture in tissue expander and implant breast reconstruction.

BACKGROUND: Capsular contracture is the main complication in postmastectomy tissue expander and implant breast reconstruction in patients requiring radiotherapy. There is evidence that the wingless signaling pathway plays a central role in the pathogenesis of fibroproliferation in fibromatosis and hyperplastic skin wounds, involving multiple linked events leading to up-regulation of target genes and fibroproliferation. Here, the authors tested their hypothesis that the wingless signaling pathway may also regulate radiotherapy-induced fibroproliferation in capsular tissue around expanders/implants in breast reconstruction. METHODS: Biopsies of the periprosthetic capsule were obtained from patients undergoing bilateral expander breast reconstruction in which one side was radiated and the other side was not radiated. Capsular biopsies were snap-frozen and stored at -80 degrees C for Western blot assays to determine protein content of phospho-glycogen-synthase-kinase-3beta (phospho-GSK-3beta), total GSK-3beta, beta-catenin, cyclooxygenase-2 (COX-2), and collagen types I and III (n = three to five patients), normalized to beta-actin. Immunostaining for beta-catenin in radiated and nonradiated capsular tissue was also performed. Slides were scanned and analyzed using Zeiss Mirax Scan. RESULTS: The following protein content levels were significantly (p < 0.01) increased in radiated capsule compared with nonradiated capsule: phospho-GSK-3beta (6.7-fold), total GSK-3beta (3.0-fold), beta-catenin (2.3-fold), COX-2 (2.8-fold), and collagen type I (1.6-fold) and type III (1.8-fold). Immunohistochemical staining demonstrated increased fibroblast cytosolic beta-catenin staining and evidence of beta-catenin nuclear translocation in radiated compared with nonradiated capsular tissue. CONCLUSION: Results from this study highlight the importance of the wingless signaling pathway in the pathogenesis of radiation-induced fibroproliferation associated with capsular contracture in expander/implant breast reconstruction.

Na+/H+ exchange inhibitor cariporide attenuates skeletal muscle infarction when administered before ischemia or reperfusion.

Administration of Na(+)/H(+) exchange isoform-1 (NHE-1) inhibitors before ischemia has been shown to attenuate myocardial infarction in several animal models of ischemia-reperfusion injury. However, controversy still exists as to the efficacy of NHE-1 inhibitors in protection of myocardial infarction when administered at the onset of reperfusion. Furthermore, the efficacy of NHE-1 inhibition in protection of skeletal muscle from infarction (necrosis) has not been studied. This information has potential clinical applications in prevention or salvage of skeletal muscle from ischemia-reperfusion injury in elective and trauma reconstructive surgery. The objective of this research project is to test our hypothesis that the NHE-1 inhibitor cariporide is effective in protection of skeletal muscle from infarction when administered at the onset of sustained ischemia or reperfusion and to study the mechanism of action of cariporide. In our studies, we observed that intravenous administration of cariporide 10 min before ischemia (1 or 3 mg/kg) or reperfusion (3 mg/kg) significantly reduced infarction in pig latissimus dorsi muscle flaps compared with the control, when these muscle flaps were subjected to 4 h of ischemia and 48 h of reperfusion (P < 0.05; n = 5 pigs/group). Both preischemic and postischemic cariporide treatment (3 mg/kg) induced a significant decrease in muscle myeloperoxidase activity and mitochondrial-free Ca(2+) content and a significant increase in muscle ATP content within 2 h of reperfusion (P < 0.05; n = 4 pigs/group). Preischemic and postischemic cariporide treatment (3 mg/kg) also significantly inhibited muscle NHE-1 protein expression within 2 h of reperfusion after 4 h of ischemia, compared with the control (P < 0.05; n = 3 pigs/group). These observations support our hypothesis that cariporide attenuates skeletal muscle infarction when administered at the onset of ischemia or reperfusion, and the mechanism involves attenuation of neutrophil accumulation and mitochondrial-free Ca(2+) overload and preservation of ATP synthesis in the early stage of reperfusion.

Radiation effects and radioprotection in MC3T3-E1 mouse calvarial osteoblastic cells.

BACKGROUND: Little is known about the mechanisms and treatment of radiation-induced inhibition of craniofacial bone growth. In an earlier study, the radioprotector amifostine (WR-2721) administered to rabbits before irradiation radioprotected cultured orbitozygomatic complex periosteal osteoblast-like cells. This study assessed the effects of amifostine and its active metabolite on the radiation survival, function, and phenotype of mouse calvarial osteoblast-like cells in a cell culture model. METHODS: MC3T3-E1 newborn mouse calvarial osteoblast-like cells underwent gamma-radiation (0 to 10 Gy) in the presence or absence of either WR-2721 or WR-1065, its active metabolite (10 to 10 M). The effects of radiation with and without drugs were assessed using endpoints of colony-forming ability, cell viability, alkaline phosphatase activity, and expression of osteoblastic phenotype genes (alkaline phosphatase, collagen type I, osteocalcin, and osteopontin). All experiments were replicated at least in triplicate. RESULTS: Irradiation resulted in a dose-dependent inhibition of clonogenic cell survival. Pretreatment with WR-1065, but not WR-2721, resulted in a significant improvement of osteoblast-like cell survival. Specifically, maximum radioprotection was observed with 10 M WR-1065 at a clinically relevant 2-Gy dose of irradiation. No significant radioprotection was observed at the lower (5 x 10 M) concentration of WR-1065. Furthermore, radiation seemed to suppress the expression of osteoblastic phenotype-related genes in a dose-dependent manner. CONCLUSIONS: This study reveals improved survival after irradiation in osteoblast-like cells treated with WR-1065 in vitro and corroborates previous findings from animal models. Further studies using this agent and similar drugs are important for devising strategies to prevent radiation-induced inhibition of craniofacial bone growth.

Postconditioning for salvage of ischemic skeletal muscle from reperfusion injury: efficacy and mechanism.

We tested our hypothesis that postischemic conditioning (PostC) is effective in salvage of ischemic skeletal muscle from reperfusion injury and the mechanism involves inhibition of opening of the mitochondrial permeability transition pore (mPTP). In bilateral 8x13 cm pig latissimus dorsi muscle flaps subjected to 4 h ischemia, muscle infarction increased from 22+/-4 to 41+/-1% between 2 and 24 h reperfusion and remained unchanged at 48 (38+/-6%) and 72 (40+/-1%) h reperfusion (P<0.05; n=4 pigs). PostC induced by four cycles of 30-s reperfusion/reocclusion at the onset of reperfusion after 4 h ischemia reduced muscle infarction from 44+/-2 to 22+/-2% at 48 h reperfusion. This infarct protective effect of PostC was mimicked by intravenous injection of the mPTP opening inhibitor cyclosporin A or NIM-811 (10 mg/kg) at 5 min before the end of 4 h ischemia and was abolished by intravenous injection of the mPTP opener atractyloside (10 mg/kg) at 5 min before PostC (P<0.05; n=4-5 pigs). PostC or intravenous cyclosporin A injection at 5 min before reperfusion caused a decrease in muscle myeloperoxidase activity and mitochondrial free Ca2+ concentration and an increase in muscle ATP content after 4 h ischemia and 2 h reperfusion compared with the time-matched controls. These effects of PostC were abolished by intravenous injection of atractyloside at 5 min before PostC (P<0.05; n=6 pigs). These observations support our hypothesis that PostC is effective in salvage of ischemic skeletal muscle from reperfusion injury and the mechanism involves inhibition of opening of the mPTP.

Radiation-induced craniofacial bone growth inhibition: in vitro cytoprotection in the rabbit orbitozygomatic complex periosteum-derived cell culture.

BACKGROUND: Radiotherapy for the management of head and neck cancer in pediatric patients results in severe inhibition of craniofacial bone growth. Previously, the infant rabbit orbitozygomatic complex was established as an experimental model. Amifostine, a cytoprotective agent, was found effective in preventing radiation-induced bone growth inhibition. This study was designed to investigate the effects radiation on osteogenic cells from infant rabbit orbitozygomatic complex periostea and to assess the effects of cytoprotection in vitro. METHODS: Infant New Zealand White rabbits (n = 18) were randomized into three groups and received radiation (0, 10, or 15 Gy) to both orbitozygomatic complexes. Cell cultures were developed from orbitozygomatic complex periostea, and cell numbers, proliferation, alkaline phosphatase, and collagen type I expression and mineralization were assessed. Subsequently, rabbits (n = 18) were randomized into three groups to receive either radiation at the effective dose, pretreatment with amifostine (300 mg/kg, intravenously, 20 minutes before irradiation) with the effective radiation dose, or no treatment. Cell cultures were developed and tested for proliferation and alkaline phosphatase expression. RESULTS: Irradiation resulted in a significant inhibition of cell numbers (p < 0.001) and proliferation (p < 0.01) at the 15-Gy dose and no statistically significant changes in alkaline phosphatase activity. Collagen type I expression and mineralization were also significantly reduced at the 15-Gy dose. Pretreatment with amifostine significantly (p < 0.05) enhanced the number of surviving cells. CONCLUSIONS: Amifostine is capable of protecting orbitozygomatic complex periosteum-derived osteogenic cells from the deleterious effects of radiation. This study provides the basis for understanding the cellular mechanisms of radiation-induced craniofacial bone growth inhibition and cytoprotection by amifostine.

Radioprotection of craniofacial bone growth.

In this review, the potential of pharmacologic therapy for prevention of radiation-induced bone growth inhibition is discussed. Significant radioprotection using the radioprotector Amifostine has been achieved in animal models of radiation-induced retardation of long and craniofacial bone growth. Moreover, radioprotection in vitro has been achieved in a number of cell lines, including osteoblast-like, endothelial, and fibroblastic. This evidence may support future clinical investigations of radioprotector Amifostine or similar substances for radioprotection of the growing craniofacial skeleton.

Radiation-induced craniofacial bone growth disturbances.

Multimodality treatment, including radiotherapy, chemotherapy, and surgery, is required for the management of head and neck cancer in pediatric patients. Despite the modern advances in radiation dosing and targeting techniques, the radiation damage to the growing craniofacial skeleton in children remains a significant clinical problem. The first part of this review summarizes the clinical effects of radiotherapy on craniofacial bone growth in children. Experimental evidence on therapeutic radiation effects on bone growth in in vivo and in vitro models is reviewed. The second part of this review focuses on prevention of radiation-induced craniofacial bone growth inhibition using radioprotective agents.

An in vitro model of radiation-induced craniofacial bone growth inhibition.

Radiation-induced craniofacial bone growth inhibition is a consequence of therapeutic radiation in the survivors of pediatric head and neck cancer. Previously, the infant rabbit orbitozygomatic complex (OZC) was established as a reliable animal model. The purpose of this study was to develop a cell culture model from the rabbit OZC to study the effects of radiation in the craniofacial skeleton. Infant (7-week-old) New Zealand white rabbits were used in this study. Periostea from both OZC were harvested in sterile conditions, introduced into cell culture by way of sequential digestion, and subcultured at confluence. Cultures were analyzed for cellular proliferation (methylthiazoletetrazolium assay), alkaline phosphatase activity, collagen type I expression, and mineralization. Electron microscopy was performed to reveal the in vitro ultrastructure. Subsequently, rabbits were irradiated with sham or 15 Gy radiation, and cell cultures were developed and analyzed for cell numbers. Cell cultures, grown from OZC periostea, expressed osteoblast-like phenotype, with high alkaline phosphatase activity, collagen type 1 expression, and mineralization in an osteogenic environment. Electron microscopy confirmed the characteristic ultrastructural features of osteogenesis in vitro. Finally, significantly (P < 0.01) fewer cells were obtained from animals treated with 15 Gy radiation compared with those from control animals.A primary cell culture with osteoblast-like cellular phenotype was developed from infant rabbit OZC periosteum. This cell culture system responded to in vivo administered radiation by a significant decrease in cell numbers. This in vitro model will be subsequently used to study the cellular mechanisms of radiation and radioprotection in craniofacial osteoblast-like cells.

Development of an in vitro model for study of the efficacy of ischemic preconditioning in human skeletal muscle against ischemia-reperfusion injury.

Ischemia-reperfusion (I/R) injury causes skeletal muscle infarction and ischemic preconditioning (IPC) augments ischemic tolerance in animal models. To date, this has not been demonstrated in human skeletal muscle. This study aimed to develop an in vitro model to investigate the efficacy of simulated IPC in human skeletal muscle. Human skeletal muscle strips were equilibrated in oxygenated Krebs-Henseleit-HEPES buffer (37 degrees C). Aerobic and reperfusion phases were simulated by normoxic incubation and reoxygenation, respectively. Ischemia was simulated by hypoxic incubation. Energy store, cell viability, and cellular injury were assessed using ATP, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), and lactate dehydrogenase (LDH) assays, respectively. Morphological integrity was assessed using electron microscopy. Studies were designed to test stability of the preparation (n = 5-11) under normoxic incubation over 24 h; the effect of 1, 2, 3, 4, or 6 h hypoxia followed by 2 h of reoxygenation; and the protective effect of hypoxic preconditioning (HPC; 5 min of hypoxia/5 min of reoxygenation) before 3 h of hypoxia/2 h of reoxygenation. Over 24 h of normoxic incubation, muscle strips remained physiologically intact as assessed by MTT, ATP, and LDH assays. After 3 h of hypoxia/2 h of reoxygenation, MTT reduction levels declined to 50.1 +/- 5.5% (P < 0.05). MTT reduction levels in HPC (82.3 +/- 10.8%) and normoxic control (81.3 +/- 10.2%) groups were similar and higher (P < 0.05) than the 3 h of hypoxia/2 h of reoxygenation group (45.2 +/- 5.8%). Ultrastructural morphology was preserved in normoxic and HPC groups but not in the hypoxia/reoxygenation group. This is the first study to characterize a stable in vitro model of human skeletal muscle and to demonstrate a protective effect of HPC in human skeletal muscle against hypoxia/reoxygenation-induced injury.

Efficacy and mechanism of adenovirus-mediated VEGF-165 gene therapy for augmentation of skin flap viability.

Skin ischemic necrosis due to vasospasm and/or insufficient vascularity is the most common complication in the distal portion of the skin flap in reconstructive surgery. This project was designed to test our hypothesis that preoperative subdermal injection of adenoviral vectors encoding genes for vascular endothelial growth factor-165 (Ad.VEGF-165) or endothelial nitric oxide (NO) synthase (Ad.eNOS) effectively augments skin viability in skin flap surgery and that the mechanism of Ad.VEGF-165 gene therapy involves an increase in synthesis/release of the angiogenic and vasodilator factor NO. PBS (0.5 ml) or PBS containing Ad.VEGF-165, Ad.eNOS, or adenovirus (Ad.Null) was injected subdermally into the distal half of a mapped rat dorsal skin flap (4 x 10 cm) 7 days preoperatively, and skin flap viability was assessed 7 days postoperatively. Local subdermal gene therapy with 2 x 10(7)-2 x 10(10) plaque-forming units of VEGF-165 increased skin flap viability compared with PBS- or Ad.Null-injected control (P < 0.05). Subdermal Ad.VEGF-165 and Ad.eNOS gene therapies were equally effective in increasing skin flap viability at 5 x 10(8) plaque-forming units. Subdermal Ad.VEGF-165 therapy was associated with upregulation of eNOS protein expression, Ca2+ -dependent NOS activity, synthesis/release of NO, and increase in capillary density and blood flow in the distal portion of the skin flap. Injection of the NOS inhibitor Nomega-nitro-L-arginine (15 mg/kg im), but not the cyclooxygenase inhibitor indomethacin (5 mg/kg im), 45 min preoperatively completely abolished the increase in skin flap blood flow and viability induced by Ad.VEGF-165 injected subdermally into the mapped skin flap 7 days preoperatively. We have demonstrated for the first time that 1) Ad.VEGF-165 and Ad.eNOS mapped skin flap injected subdermally into the mapped skin flap 7 days preoperatively are equally effective in augmenting viability in the rat dorsal skin flap compared with control, 2) the mechanism of subdermal Ad.VEGF-165 gene therapy in augmenting skin flap viability involves an increase in NO synthesis/release downstream of upregulation of eNOS protein expression and Ca2+ -dependent NOS activity, and 3) the vasodilating effect of NO may predominantly mediate subdermal Ad.VEGF gene therapy in augmenting skin flap blood flow and viability.

Inducing late phase of infarct protection in skeletal muscle by remote preconditioning: efficacy and mechanism.

We have previously demonstrated that remote ischemic preconditioning (IPC) by instigation of three cycles of 10-min occlusion/reperfusion in a hindlimb of the pig elicits an early phase of infarct protection in local and distant skeletal muscles subjected to 4 h of ischemia immediately after remote IPC. The aim of this project was to test our hypothesis that hindlimb remote IPC also induces a late phase of infarct protection in skeletal muscle and that K(ATP) channels play a pivotal role in the trigger and mediator mechanisms. We observed that pig bilateral latissimus dorsi (LD) muscle flaps sustained 46 +/- 2% infarction when subjected to 4 h of ischemia/48 h of reperfusion. The late phase of infarct protection appeared at 24 h and lasted up to 72 h after hindlimb remote IPC. The LD muscle infarction was reduced to 28 +/- 3, 26 +/- 1, 23 +/- 2, 24 +/- 2 and 24 +/- 4% at 24, 28, 36, 48 and 72 h after remote IPC, respectively (P < 0.05; n = 8). In subsequent studies, hindlimb remote IPC or intravenous injection of the sarcolemmal K(ATP) (sK(ATP)) channel opener P-1075 (2 microg/kg) at 24 h before 4 h of sustained ischemia (i.e., late preconditioning) reduced muscle infarction from 43 +/- 4% (ischemic control) to 24 +/- 2 and 19 +/- 3%, respectively (P < 0.05, n = 8). Intravenous injection of the sK(ATP) channel inhibitor HMR 1098 (6 mg/kg) or the nonspecific K(ATP) channel inhibitor glibenclamide (Glib; 1 mg/kg) at 10 min before remote IPC completely blocked the infarct- protective effect of remote IPC in LD muscle flaps subjected to 4 h of sustained ischemia at 24 h after remote IPC. Intravenous bolus injection of the mitochondrial K(ATP) (mK(ATP)) channel inhibitor 5-hydroxydecanoate (5-HD; 5 mg/kg) immediately before remote IPC and 30-min intravenous infusion of 5-HD (5 mg/kg) during remote IPC did not affect the infarct-protective effect of remote IPC in LD muscle flaps. However, intravenous Glib or 5-HD, but not HMR 1098, given 24 h after remote IPC completely blocked the late infarct-protective effect of remote IPC in LD muscle flaps. None of these drug treatments affected the infarct size of control LD muscle flaps. The late phase of infarct protection was associated with a higher (P < 0.05) muscle content of ATP at the end of 4 h of ischemia and 1.5 h of reperfusion and a lower (P < 0.05) neutrophilic activity at the end of 1.5 h of reperfusion compared with the time-matched control. In conclusion, these findings support our hypothesis that hindlimb remote IPC induces an uninterrupted long (48 h) late phase of infarct protection, and sK(ATP) and mK(ATP) channels play a central role in the trigger and mediator mechanism, respectively.

Radiation-induced craniofacial bone growth inhibition: efficacy of cytoprotection following a fractionated dose regimen.

BACKGROUND: Severe craniofacial growth disturbances are noted in 66 to 100 percent of children with head and neck cancers who received radiotherapy during their growing years. The authors have previously demonstrated the prevention of radiation-induced craniofacial bone growth inhibition following single-dose orthovoltage radiation to the orbitozygomatic complex in an infant rabbit model through the administration of the cytoprotective agent amifostine (WR-2721) before radiation treatment. The purpose of this study was to investigate the efficacy of cytoprotection using a fractionated dose regimen that better approximates the clinical application of radiation therapy. METHODS: Thirty 7-week-old male New Zealand rabbits were randomized into three groups (n = 10), each receiving six fractions of orthovoltage radiation to the right orbitozygomatic complex: group C, sham irradiation control; group F35, total dose of 35 Gy; and group F35A, total dose of 35 Gy with administration of amifostine 200 mg/kg intravenously 20 minutes before each fraction. Bone growth was evaluated up to skeletal maturity (age 21 weeks) with serial radiographs and computed tomography scans for cephalometric analysis, bone volume, and bone density measurements. RESULTS: Fractionated radiation resulted in significant (p < 0.05) bone growth inhibition compared with sham radiation in 16 of 21 cephalometric parameters measured and significantly (p < 0.05) reduced bone volume of the rabbit orbitozygomatic complex. Pretreatment with amifostine before each radiation fraction prevented growth deformities in four cephalometric parameters and significantly (p < 0.05) attenuated these effects in another seven parameters compared with radiated animals. Bone volumes were also significantly (p < 0.05) improved in F35A animals compared with F35 animals. CONCLUSIONS: This study establishes that fractionation of orthovoltage radiation does not prevent the development of growth disturbances of the rabbit craniofacial skeleton and also demonstrates that preirradiation administration of amifostine is highly effective in the prevention and attenuation of radiation-induced craniofacial bone growth inhibition.

Mitochondrial KATP channels in hindlimb remote ischemic preconditioning of skeletal muscle against infarction.

We previously demonstrated in the pig that instigation of three cycles of 10 min of occlusion and reperfusion in a hindlimb by tourniquet application (approximately 300 mmHg) elicited protection against ischemia-reperfusion injury (infarction) in multiple distant skeletal muscles subsequently subjected to 4 h of ischemia and 48 h of reperfusion, but the mechanism was not studied. The aim of this project was to test our hypothesis that mitochondrial ATP-sensitive potassium (KATP) (mKATP) channels play a central role in the trigger and mediator mechanisms of hindlimb remote ischemic preconditioning (IPC) of skeletal muscle against infarction in the pig. We observed in the pig that hindlimb remote IPC reduced the infarct size of latissimus dorsi (LD) muscle flaps (8 x 13 cm) from 45 +/- 2% to 22 +/- 3% (n = 10; P < 0.05). The nonselective KATP channel inhibitor glibenclamide (0.3 mg/kg) or the selective mKATP channel inhibitor 5-hydroxydecanoate (5-HD, 5 mg/kg), but not the selective sarcolemmal KATP (sKATP) channel inhibitor HMR-1098 (3 mg/kg), abolished the infarct-protective effect of hindlimb remote IPC in LD muscle flaps (n = 10, P < 0.05) when these drugs were injected intravenously at 10 min before remote IPC. In addition, intravenous bolus injection of glibenclamide (1 mg/kg) or 5-HD (10 mg/kg) at the end of hindlimb remote IPC also abolished the infarct protection in LD muscle flaps (n = 10; P < 0.05). Furthermore, intravenous injection of the specific mKATPchannel opener BMS-191095 (2 mg/kg) at 10 min before 4 h of ischemia protected the LD muscle flap against infarction to a similar extent as hindlimb remote IPC, and this infarct-protective effect of BMS-191095 was abolished by intravenous bolus injection of 5-HD (5 mg/kg) at 10 min before or after intravenous injection of BMS-191095 (n = 10; P < 0.05). The infarct protective effect of BMS-191095 was associated with a higher muscle content of ATP at the end of 4 h of ischemia and a decrease in muscle neutrophilic myeloperoxidase activity at the end of 1.5 h of reperfusion compared with the time-matched control (n = 10, P < 0.05). These observations led us to conclude that mKATP channels play a central role in the trigger and mediator mechanisms of hindlimb remote IPC of skeletal muscle against infarction in the pig, and the opening of mKATP channels in ischemic skeletal muscle is associated with an ATP-sparing effect during sustained ischemia and attenuation of neutrophil accumulation during reperfusion.