William A. Zamboni

Morphologic analysis of the microcirculation during reperfusion of ischemic skeletal muscle and the effect of hyperbaric oxygen.

The morphologic events in the microcirculation that lead to reperfusion injury of ischemic skeletal muscle remain incompletely understood. The purpose of this experiment was to evaluate leukocyte endothelial adherence characteristics and dynamic changes in microvessel caliber during reperfusion of an in vivo skeletal muscle ischemia preparation. In addition, the effect of hyperbaric oxygen treatment on these microcirculatory changes also was studied. An intravital microscopy preparation of a transilluminated gracilis muscle in 27 rats was used to observe a total of 101 arterioles and 63 venules (13 to 73 μm diameter). Baseline hemodynamics were videotaped for 30 minutes following muscle isolation. The animals were divided into six groups: (1) sham, no ischemia, (2) 4 hours of global ischemia only, (3) no ischemia plus hyperbaric oxygen (one 2.5 ATA/1 hour of treatment with 100% oxygen), (4) 4 hours of ischemia plus hyperbaric oxygen during ischemia, (5) 4 hours of ischemia plus hyperbaric oxygen immediately on reperfusion, and (6) 4 hours of ischemia plus hyperbaric oxygen 1 hour after reperfusion. Changes in arteriolar and venular diameters at specific times during 3 hours of reperfusion were recorded, and the number of adherent and slow-rolling leukocytes in 100-μm venular segments were counted and compared with baseline measurements. The proximity of arterioles to venules was classified as adjacent (<15 μm) or distant (>15 μm). No significant changes in leukocyte endothelial adherence or arteriolar diameter were noted in group 1 sham or group 3 nonischemic hyperbaric oxygen-treated rats when compared with baseline measurements. A significant increase in adherent leukocytes was observed in group 2 ischemic venules (+14.9 ± 2.5) within 5 minutes of reperfusion, which was maintained for 3 hours. Reperfusion measurements of arteriolar diameter in group 2 ischemic muscle preparations demonstrated an initial vasodilation that was followed at 1 hour by a progressive and severe vasoconstriction (−46.9 ±11.3 percent at 3 hours) in arterioles adjacent to venules that was not seen in distant arterioles. The increase in adherent leukocytes seen in group 2 ischemic venules was significantly reduced by hyperbaric oxygen treatment given during ischemia (group 4) or up to 1 hour during reperfusion (groups 5 and 6). In addition, the progressive ischemic arteriolar vasoconstriction was inhibited in all groups (4, 5, and 6) treated with hyperbaric oxygen. These results suggest that (1) leukocyte venular endothelial adherence and microarteriolar vasoconstriction are important morphologic events leading to reperfusion injury of skeletal muscle, (2) this vasoconstriction is seen primarily in arterioles that are in close proximity to venules, and (3) hyperbaric oxygen treatment does not exacerbate reperfusion injury, but rather appears to protect the microcirculation by reducing venular leukocyte adherence and inhibiting progressive adjacent arteriolar vasoconstriction.

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.

Hyperbaric oxygen and wound healing

Problem wounds, which fail to respond to traditional medical and surgical therapy, can be challenging to the plastic surgeon. Surgical, outpatient, and inpatient wound care costs can be exorbitant. Indirect costs, such as those related to patient productivity, disability, and premature death, can also be significant. The underlying problem in failure of a wound to heal is usually hypoxia and infection. HBO treatments in selected patients can facilitate healing by increasing tissue oxygen tension, thus providing the wound with a more favorable environment for repair. Therefore, HBO therapy can be an important component to any comprehensive wound care program.

The effect of hyperbaric oxygen on reperfusion of ischemic axial skin flaps: a laser Doppler analysis.

This study evaluates the microvascular reperfusion of ischemic skin flaps with and without acute hyperbolic oxygen (HBO) treatment. Thirty-two axial pattern epigastric skin flaps (3 x 6 cm) in male Wistar rats were subjected to 8 hours of global ischemia by pedicle clamp occlusion. The rats were divided into the following control and two experimental groups: Control (n = 12) with ischemia, no HBO; Group 1 (n = 11) with HBO treatment (three 1.75-hour dives, 2.5 absolute atm, 100% O2) during ischemia; and Group 2(n = 9) with HBO treatment (two 1.75-hour dives) immediately after ischemia. Laser Doppler flows were recorded in two distal standardized flap locations at 0.5, 2, 4, and 18 hours after reperfusion in control rats and Group 1 rats and at 18 hours only in Group 2 rats, using a Med-Pacific 6000 laser Doppler unit. Mean distal flap laser Doppler flows (mV) were Control: 0.5 hours=23.2 ± 11.9, 2 hours=52.8 ± 27.3,4hours=53.6 ± 32.1,18hours=40.2 ± 36.2; Group 1: 0.5 hours=71.8 ± 30.9 (p<0.05 vs. control), 2 hours=74.3 ± 27.3, 4 hours=67.4 ± 20.6, 18 hours=79.1 ± 40.3 (p<0.05 vs. control); and Group 2:18 hours=90.3 ± 47.9 (p<0.05 vs. control). It is concluded that acute HBO treatment of ischemic rat skin flaps improves distal microvascular perfusion as measured by laser Doppler flowmetry. This effect is observed for HBO treatment given either during or immediately after prolonged global ischemia. Although the mechanism of this beneficial effect on the microcirculation remains unknown, these results correlate with earlier findings that HBO treatment improves survival of rat axial skin flaps subjected to prolonged global ischemia.

Ischemia-reperfusion injury in skeletal muscle: CD 18-dependent neutrophil-endothelial adhesion and arteriolar vasoconstriction.

&NA; The purpose of this study was to evaluate if the venular neutrophil‐endothelial adhesion associated with ischemia‐reperfusion of skeletal muscle is dependent on leukocyte adhesion glycoprotein CD18 function and to determine if this interaction influences the vasoactive response in nearby arterioles. An in vivo microscopy preparation of transilluminated gracilis muscle in 13 male Wistar rats was used for this experiment. Observations of nonischemic muscle (sham) demonstrated this preparation to be stable for 8 hours with negligible change in neutrophil adherence or arteriole diameter. Three groups were evaluated in this study: (1) sham, no ischemia, no treatment (n = 5, 20 arterioles, 20 venules), (2) 4 hours of global ischemia only (n = 4, 19 venules, 22 arterioles), and (3) 4 hours of ischemia plus monoclonal antibody against CD18 (n = 4, 12 venules, 9 arterioles). The murine monoclonal antibody (WT.3, Seikagaku America, Inc.), which binds the rat leukocyte function antigen 1 CD18 chain, was infused into the contralateral femoral vein 30 minutes prior to reperfusion. The number of leukocytes rolling and adherent to endothelium (15 seconds of observation) was counted in 100‐&mgr;m venular segments, and arteriole diameters were measured at various times during reperfusion. All counts and measurements were normalized to baseline preischemic readings for each animal. Mean changes from baseline were compared between groups. The increase in ischemia‐reperfusion—induced neutrophil‐endothelial adherence in venules was blocked by monoclonal antibody, but rolling behavior was not changed. The ischemia‐reperfusion—induced progressive vasoconstriction in arterioles was blocked by monoclonal antibody. These results suggest that (1) neutrophil‐endothelial adherence function associated with ischemia‐reperfusion in this model is CD18‐dependent, (2) neutrophil rolling function does not appear to be dependent on CD18, and (3) neutrophil CD18 function is a prerequisite for ischemia‐reperfusion‐induced arteriolar vasoconstriction. These findings provide important mechanistic information that may help explain the deleterious microcirculatory events associated with ischemia‐reperfusion injury of skeletal muscle. (Plast. Reconstr. Surg. 99: 2002, 1997.)

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.

Effects of L-NAME and L-arginine on ischemia-reperfusion injury in rat skeletal muscle.

The involvement of nitric oxide in ischemia-reperfusion injury remains controversial and has been reported to be both beneficial and deleterious, depending on the tissue and model used. This study evaluated the effects of the nitric oxide synthase inhibitor N(G)-nitro-L-arginine-methyl ester (L-NAME) and the substrate for nitric oxide synthase, L-arginine on skeletal muscle necrosis in a rat model of ischemia-reperfusion injury. The rectus femoris muscle in male Wistar rats (250 to 500 g) was isolated on its vascular pedicle and subjected to 4 hours of complete arteriovenous occlusion. The animals were divided into five groups: (1) sham-raised control, no ischemia, no treatment (n = 6); (2) 4 hours of ischemia (n = 6); (3) vehicle control, 4 hours of ischemia + saline (n = 6); (4) 4 hours of ischemia + L-arginine infusion (n = 6); and (5) 4 hours of ischemia + L-NAME infusion (n = 6). The infusions (10 mg/kg) were administered into the contralateral femoral vein beginning 5 minutes before reperfusion and during the following 30 to 45 minutes. Upon reperfusion, the muscle was sutured in its anatomic position and all wounds were closed. The percentage of muscle necrosis was assessed after 24 hours of reperfusion by serial transections, nitroblue tetrazolium staining, digital photography, and computerized planimetry. Sham (group 1) animals sustained baseline necrosis of 11.9 +/- 3.0 (percentage necrosis +/- SEM). Four hours of ischemia (group 2) significantly increased necrosis to 79.2 +/- 1.4 (p < 0.01). Vehicle control (group 3) had no significant difference in necrosis (81.17 +/- 5.0) versus untreated animals subjected to 4 hours of ischemia (group 2). Animals treated with L-arginine (group 4) had significantly reduced necrosis to 34.6 +/- 7.5 versus untreated (group 2) animals (p < 0.01). Animals infused with L-NAME (group 5) had no significant difference in necrosis (68.2 +/- 6.7) versus untreated (group 2) animals. L-Arginine (nitric oxide donor) significantly decreased the severity of muscle necrosis in this rat model of ischemia-reperfusion injury. L-arginine is known to increase the amount of nitric oxide through the action of nitric oxide synthase, whereas L-NAME, known to inhibit nitric oxide synthase and decrease nitric oxide production, had comparable results to the untreated 4-hour ischemia group. These results suggest that L-arginine, presumably through nitric oxide mediation, appears beneficial to rat skeletal muscle subjected to ischemia-reperfusion injury.

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.

Contributing Variables to Post Mastectomy Tissue Expander Infection

Abstract:  Tissue expander breast reconstruction is a common post mastectomy breast procedure performed by plastic surgeons. The purpose of this study was to define the incidence of breast reconstruction prosthetic infection, relate patient characteristics with increased rate of infection, and analyze the influence of postoperative complications to expander/implant infection. A retrospective, single‐institution chart review of 195 women with post mastectomy tissue expander/implant reconstructions performed from 2006 through 2008 was conducted. Total periprosthetic infection rate was calculated. Patient factors, operative technique, and noninfectious complications were analyzed and related to increased periprosthetic infection rate. A binary logistic regression model was fitted using periprosthetic infection as the dependent variable and 12 patient characteristics as independent variables, followed by a step‐wise model for best fit with a limited number of independent variables. The overall periprosthetic infection rate per patient over the 2 year period was 5.1%. The incidence of periprosthetic infection per reconstructed breast was 3.2%. Odds ratio estimates indicated that the presence of cellulitis increased the odds of periprosthetic infection more than 200 times (p = <0.0001), and inpatient procedures increased the odds 16 times (p = 0.02). Other variables (i.e., age > 65, DM, flap necrosis, smoking, dehiscence, AlloDerm, etc) failed to reach statistical significance (p > 0.05). Postoperative breast cellulitis and inpatient status appear to be significant risk factors for increased periprosthetic infection. No significant increase in periprosthetic infection rate was noted with other variables in this model.

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.