Wei Z. Wang

Update on ischemia-reperfusion injury for the plastic surgeon: 2011.

Ischemia-reperfusion injury occurs when tissue is reperfused following a prolonged period of ischemia. It is a subject of interest to plastic surgeons involved in replantation, free tissue transfer, and composite tissue allotransplantation, as it can have a significant impact on the overall success of these procedures. The purpose of this article is to review the recent progress in the investigation of ischemia-reperfusion injury in skeletal muscle and skin and to highlight the potential clinical implications of therapeutic interventions aimed at reducing ischemia-reperfusion injury.

Melatonin reduces ischemia/reperfusion-induced superoxide generation in arterial wall and cell death in skeletal muscle

Abstract:  The purpose of this study was to determine the effect of melatonin on superoxide generation in arterial wall at an early phase of reperfusion and on endothelial dysfunction of microvasculature and cell viability of cremaster muscle at late phase of reperfusion (24 hr) after prolonged ischemia. Bilateral vascular pedicles which supply blood flow to the cremaster muscle were exposed. After surgical preparation, microvascular clamps were applied on the right iliac, femoral and spermatic arteries to create 4 hr of ischemia in both feeding vessels and the unexposed cremaster muscle. The vascular clamping was omitted on the left iliac, femoral and spermatic arteries and served as an internal control. Melatonin or Vehicle was via by intravenous injection at 10 min prior to reperfusion and 10 min after reperfusion. In the first experiment, the vascular pedicle was harvested after reperfusion to measure superoxide generation in real time by lucigenin‐derived chemiluminescence. In the second experiment, endothelial‐dependent and ‐independent vasodilatation was examined in the terminal arteriole of cremaster muscle which was then harvested to examine cell viability by WST‐1 assay on day 2. Superoxide generation in arterial wall peaked at first 5‐min of reperfusion and declined to near baseline after 60 min of reperfusion. Melatonin treatment significantly reduced superoxide generation in arterial walls and improved cell viability in cremaster muscles. Melatonin treatment also significantly reduced microvascular endothelial dysfunction which was still observable in the microcirculation of cremaster muscle after 24 hr of reperfusion. Melatonin reduces superoxide generation in the early phase of reperfusion resulting in attenuating endothelial dysfunction and muscle cell death in the late phase of reperfusion.

Melatonin attenuates I/R-induced mitochondrial dysfunction in skeletal muscle.

BACKGROUND Our recent studies have shown that ischemia/reperfusion (I/R) produces significant necrosis and apoptosis in the cells of skeletal muscle. Our previous studies also demonstrated that melatonin provides significant protection against superoxide generation, endothelial dysfunction, and cell death in the skeletal muscle after I/R. Mitochondria are essential for cell survival, because of their roles as ATP producers as well as regulators of cell death. However, the efficacy of melatonin on I/R-induced mitochondrial dysfunction in the skeletal muscle in vivo has not been demonstrated in the literature. MATERIALS AND METHODS Vascular pedicle isolated rat gracilis muscle model was used. After 4 h of ischemia followed by 24 h of reperfusion, gracilis muscle was harvested, and mitochondrial as well as cytosolic fractions were isolated. Mitochondrial dysfunction was determined by the alteration of mitochondrial membrane potential and the release of the proapoptotic protein, cytochrome c. Three groups were designed; sham I/R, I/R-V (I/R with vehicle), and I/R-Mel (I/R with melatonin). Melatonin or vehicle was given intravenously 10 min prior to reperfusion and 10 min after reperfusion. RESULTS We found that the capability of uptake of fluorescent JC-1 dye in skeletal muscle cells was substantially improved in I/R-Mel group compared with I/R-V group. Melatonin significantly inhibited the outflow of cytochrome c from mitochondria to cytoplasm, which was demonstrated in the I/R-V group. CONCLUSIONS Melatonin significantly attenuates I/R-induced mitochondrial dysfunction, such as the depolarization of mitochondrial membrane potential and the release of the proapoptotic protein, cytochrome c, from the mitochondria.

Microcirculatory effects of melatonin in rat skeletal muscle after prolonged ischemia.

Abstract:  The purpose of this study was to determine microcirculatory effects and response of nitric oxide synthase (NOS) to melatonin in skeletal muscle after prolonged ischemia. A vascular pedicle isolated rat cremaster muscle model was used. Each muscle underwent 4 hr of zero‐flow warm ischemia followed by 2 hr of reperfusion. Melatonin (10 mg/kg) or saline as a vehicle was given by intraperitoneal injection at 30 min prior to reperfusion and the same dose was given immediately after reperfusion. After reperfusion, microcirculation measurements including arteriole diameter, capillary perfusion and endothelial‐dependent and ‐independent vasodilatation were performed. The cremaster muscle was then harvested to measure endothelial NOS (eNOS) and inducible NOS (iNOS) gene expression and enzyme activity. Three groups of rats were used: sham‐ischemia/reperfusion (I/R), vehicle + I/R and melatonin + I/R. As compared with vehicle + I/R group, administration of melatonin significantly enhanced arteriole diameter, improved capillary perfusion, and attenuated endothelial dysfunction in the microcirculation of skeletal muscle after 4 hr warm ischemia. Prolonged warm ischemia followed by reperfusion significantly depressed eNOS gene expression and constitutive NOS activity and enhanced iNOS gene expression. Administration of melatonin did not significantly alter NOS gene expression or activity in skeletal muscle after prolonged ischemia and reperfusion. Melatonin provided a significant microvascular protection from reperfusion injury in skeletal muscle. This protection is probably attributable to the free radical scavenging effect of melatonin, but not to its anti‐inflammatory effect.

Hyperbaric Oxygen Inhibits Ischemia-reperfusion–induced Neutrophil Cd18 Polarization by a Nitric Oxide Mechanism

Background: Hyperbaric oxygen decreases ischemia-reperfusion–induced neutrophil/intercellular adhesion molecule-1 adhesion by blocking CD18 polarization. The purpose of this study was to evaluate whether this hyperbaric oxygen effect is nitric oxide dependent and to determine whether nitric oxide synthase is required. Methods: A gracilis muscle flap was raised in nine groups of male Wistar rats. Global ischemic injury was induced by clamping the gracilis muscle pedicle artery and vein for 4 hours. The hyperbaric oxygen treatment consisted of 100% oxygen at 2.5 atm absolute during the last 90 minutes of ischemia. Groups were repeated with and without various nitric oxide synthase inhibitors and carboxy-2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide (C-PTIO), a nitric oxide scavenger. Normal neutrophils were exposed to activated plasma on intercellular adhesion molecule-1–coated coverslips (percentage adherent) and labeled with fluorescein isothiocyanate/antirat-CD11b for confocal microscopy (percentage polarized). The percentage of adherent and polarized cells was reported as mean ± SEM. Statistical analysis was by analysis of variance. A value of p ≤ 0.05 was considered significant. Results: C-PTIO–treated ischemia-reperfusion/hyperbaric oxygen plasma showed a significant increase in the percentage polarization of CD18 compared with ischemia-reperfusion/hyperbaric oxygen–untreated plasma from 4.1 ± 2.5 percent to 33.7 ± 7.7 percent (p ≤ 0.05). The nitric oxide scavenger C-PTIO also increased the percentage of adherent cells from 1.6 ± 0.4 percent to 20.3 ± 5.9 percent (p ≤ 0.05). Administration of NG-nitro-L-arginine methyl ester and other nitric oxide synthase inhibitors before hyperbaric oxygen treatment restored neutrophil adhesion and CD18 polarization to ischemia-reperfusion control values, significantly greater than ischemia-reperfusion/hyperbaric oxygen alone. Conclusion: These results suggest that the hyperbaric oxygen reduction of ischemia-reperfusion–induced neutrophil polarization of CD18 and adherence to intercellular adhesion molecule-1 is mediated through a nitric oxide mechanism that requires nitric oxide synthase.

A Quantitative Method for Determining Polarization of Neutrophil Adhesion Molecules Associated with Ischemia Reperfusion

Ischemia-reperfusion–induced neutrophil adhesion to endothelium is CD18-dependent, but information regarding polarity of CD18 adhesion molecules remains speculative. This study evaluated neutrophil adhesion using an in vitro cell adhesion assay and introduces a quantitative method of measuring CD18 membrane distribution using confocal microscopy. Neutrophils from normal animals were isolated from whole blood and incubated with plasma from rat gracilis muscle flaps with no ischemia and reperfusion (nonischemic control, n = 10) or 4 hours of ischemia and 90 minutes of reperfusion (ischemia/reperfusion, n = 10), on coverslips pretreated with and without (phosphate-buffered saline) soluble intercellular adhesion molecules. Coverslips without intercellular adhesion molecules represented a negative control (intercellular adhesion molecules were required for adhesion). Percent adherence to intercellular adhesion molecules was expressed as a ratio of adherent cells/total cells. CD18 polarization was assessed by staining neutrophils with fluorescein isothiocyanate–labeled anti-CD11b, followed by confocal microscopy and Z-stack analysis. Membrane-associated CD18 was expressed as fluorescence intensity units in three equal areas of the cell membrane. Capping was defined as twice as much fluorescence in 33 percent of the cell membrane as in the remaining 67 percent. Neutrophils exposed to ischemia and reperfusion plasma showed a significant increase in adhesion (0.8 ± 0.1 percent versus 16.7 ± 2.2 percent, p < 0.001) and CD18 polarization (6.2 ± 1.7 percent versus 43.9 ± 12.2 percent, p = 0.0206) compared with controls. This article describes an in vitro assay that reliably reproduces the neutrophil adhesion phenomenon associated with ischemia-reperfusion injury. Results from confocal microscopy allowed for quantitative estimation of membrane-associated receptor polarization.

Reperfusion-induced neutrophil CD18 polarization: effect of hyperbaric oxygen.

BACKGROUND Hyperbaric oxygen (HBO) inhibits ischemia reperfusion (IR) -induced neutrophil adhesion to endothelium through an unknown mechanism. This study evaluates the effect of HBO on IR-stimulated neutrophil adhesion and polarization of expressed CD18 adhesion molecules using a novel in vitro adhesion assay and confocal microscopy. MATERIALS AND METHODS Neutrophils from normal animals were isolated from whole blood and incubated with plasma from rat gracilis muscle flaps on coverslips pretreated with ICAM. Percent adherence to ICAM and CD18 polarization was evaluated in the following five groups: (1) Nonischemic control, n = 15; (2) 4 h ischemia (IR, n = 15); (3) 4 h ischemia with HBO treatment (100% oxygen at 2.5 atmospheres absolute (IR + HBO, n = 15)); (4) 4 h ischemia with 100% oxygen at room temperature and pressure (RTP) (IR + normobaric hyperoxia, n = 5); and (5) 4 h ischemia with 8% oxygen at 2.5 atmospheres absolute (IR + hyperbaric normoxia, n = 5). Direct HBO treatment of neutrophils was also evaluated. RESULTS Neutrophils exposed to IR plasma showed a significant increase in percent adherent (0.8 +/- 0.1% versus 16.7 +/- 2.2%, P < 0.05) and polarized cells (6.2 +/- 1.7% versus 43.9 +/- 12.2%, P < 0.05) compared to controls. Hyperbaric oxygen significantly reduced the adhesion and polarization to 1.6 +/- 0.3 and 4.1 +/- 2.5%, respectively (P = < 0.05). Normobaric hyperoxia and hyperbaric normoxia did not affect neutrophil adherence or CD18 polarization following IR. Direct HBO treatment of neutrophils did not change the percent of polarized cells in IR. CONCLUSIONS Hyperbaric oxygen inhibits IR-induced neutrophil adhesion by blocking CD18 surface polarization and requires plasma exposure to HBO. Treatment with oxygen or pressure alone is not effective.

Analysis for apoptosis and necrosis on adipocytes, stromal vascular fraction, and adipose-derived stem cells in human lipoaspirates after liposuction.

Background: Adipose-derived stem cells have become the most studied adult stem cells. The authors examined the apoptosis and necrosis rates for adipocyte, stromal vascular fraction, and adipose-derived stem cells in fresh human lipoaspirates. Methods: Human lipoaspirate (n = 8) was harvested using a standard liposuction technique. Stromal vascular fraction cells were separated from adipocytes and cultured to obtain purified adipose-derived stem cells. A panel of stem cell markers was used to identify the surface phenotypes of cultured adipose-derived stem cells. Three distinct stem cell subpopulations (CD90+/CD45−, CD105+/CD45−, and CD34+/CD31−) were selected from the stromal vascular fraction. Apoptosis and necrosis were determined by annexin V/propidium iodide assay and analyzed by flow cytometry. Results: The cultured adipose-derived stem cells demonstrated long-term proliferation and differentiation evidenced by cell doubling time and positive staining with oil red O and alkaline phosphatase. Isolated from lipoaspirates, adipocytes exhibited 19.7 ± 3.7 percent apoptosis and 1.1 ± 0.3 percent necrosis; stromal vascular fraction cells revealed 22.0 ± 6.3 percent of apoptosis and 11.2 ± 1.9 percent of necrosis; stromal vascular fraction cells had a higher rate of necrosis than adipocytes (p < 0.05). Among the stromal vascular fraction cells, 51.1 ± 3.7 percent expressed CD90+/CD45−, 7.5 ± 1.0 percent expressed CD105+/CD45−, and 26.4 ± 3.8 percent expressed CD34+/CD31−. CD34+/CD31− adipose-derived stem cells had lower rates of apoptosis and necrosis compared with CD105+/CD45− adipose-derived stem cells (p < 0.05). Conclusions: Adipose-derived stem cells had a higher rate of apoptosis and necrosis than adipocytes. However, the extent of apoptosis and necrosis was significantly different among adipose-derived stem cell subpopulations.

Nitrite attenuates ischemia-reperfusion-induced microcirculatory alterations and mitochondrial dysfunction in the microvasculature of skeletal muscle.

Background: Recently, nitrite has been rediscovered as a physiologically relevant storage reservoir of nitric oxide in blood and it can readily be converted to nitric oxide under hypoxic and acidic conditions. In this study, the authors evaluated the therapeutic efficacy of nitrite on reperfusion-induced microcirculatory alterations and mitochondrial dysfunction in the microvasculature of skeletal muscle. Methods: The authors used a vascular pedicle isolated rat cremaster model that underwent 4 hours of warm ischemia followed by 2 hours or 17 hours of reperfusion. At 5 minutes before reperfusion, normal saline, sodium nitrite (0.20 &mgr;M/minute/kg), or nitrite mixed with 2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl (potassium salt) (0.2 mg/minute/kg) was infused into the microcirculation of ischemic cremaster by means of intraarterial infusion. Ischemia-reperfusion–induced microcirculatory alterations were measured after 2 hours of reperfusion. Microvasculature of the cremaster muscle including the vascular pedicle was harvested to determine the mitochondrial dysfunction. The blood concentration of methemoglobin was also measured to determine the toxicity of nitrite. Results: The authors found that nitrite significantly attenuated ischemia-reperfusion–induced vasoconstriction, arteriole stagnation, and capillary no-reflow in the early phase of reperfusion and the depolarization of mitochondrial membrane potential and cytochrome c release in the late phase of reperfusion. Nitrite-induced protection was significantly blocked by a nitric oxide scavenger (potassium salt). The methemoglobin results showed that the doses of nitrite we used in the present study were safe. Conclusion: The supplementation of a low dose of nitrite, directly into the microcirculation of ischemic muscle through local intraarterial infusion, significantly attenuated ischemia-reperfusion–induced microcirculatory alterations in vivo and mitochondrial dysfunction in vitro in the microvasculature of skeletal muscle.

Objective evaluation of skill acquisition in novice microsurgeons.

INTRODUCTION Medical training is increasingly focused on patient safety, limiting the ability to practice technical skills in the operative arena. Alternative methods of training residents must be designed and implemented. METHODS Three expert microsurgeons were solicited to develop two drills to help residents acquire the basic subset of skills in microsurgery. The first drill was performance of five consecutive simple interrupted sutures on a rubber glove. Expert proficiency was considered a drill time of two standard deviations from expert mean. The drill was performed up to 10 times until completion of the task at expert proficiency. The second drill was performance of an anastomosis on silastic tubing. Residents performed the drill sequentially until performing two consecutive drills at expert proficiency. RESULTS Eight residents with no microsurgical experience volunteered. Six of the eight residents were able to perform the rubber glove drill at expert proficiency within 10 attempts, with an average of 5.3. All of the residents were able to perform two consecutive silastic tubing drills at expert proficiency within nine attempts, with an average of 5.4. CONCLUSION Residents were able to acquire a basic subset of microsurgical skills within a reasonable time period using these drills.