Hans Suchy

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.

Experimental pretransfer expansion of free-flap donor sites. I: Flap viability and expansion characteristics

Expansion of cutaneous and myocutaneous free-flap donor sites prior to elevation is possible in the pig model. There is no significant difference in survival between control and expanded cutaneous buttock and myocutaneous latissimus dorsi flaps after elevation solely on their axial pedicles. Axial-pattern flap expansion appears to augment capillary blood flow. The maximum amount of expansion occurs directly over the center of the expander and decreases toward the periphery. There is virtually no expansion of skin adjacent to the expander.

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.)

Local hypothermia during early reperfusion protects skeletal muscle from ischemia-reperfusion injury.

Amputated tissue maintained in a hypothermic environment can endure prolonged ischemia and improve replantation success. The authors hypothesized that local tissue hypothermia during the early reperfusion period may provide a protective effect against ischemia-reperfusion injury similar to that seen when hypothermia is provided during the ischemic period. A rat gracilis muscle flap model was used to assess the protective effects of exposing skeletal muscle to local hypothermia during ischemia only (p = 18), reperfusion only (p = 18), and both ischemia and reperfusion (p = 18). Gracilis muscles were isolated and exposed to hypothermia of 10 degrees C during 4 hours of ischemia, the initial 3 hours of reperfusion, or both periods. Ischemia-reperfusion outcome measures used to evaluate muscle flap injury included muscle viability (percent nitroblue tetrazolium staining), local edema (wet-to-dry weight ratio), neutrophil infiltration (intramuscular neutrophil density per high-power field), neutrophil integrin expression (CD11b mean fluorescence intensity), and neutrophil oxidative potential (dihydro-rhodamine oxidation mean fluorescence intensity) after 24 hours of reperfusion. Nitroblue tetrazolium staining demonstrated improved muscle viability in the experimental groups (ischemia-only: 78.8 +/- 3.5 percent, p < 0.001; reperfusion-only: 80.2 +/- 5.2 percent, p < 0.001; and ischemia-reperfusion: 79.6 +/- 7.6 percent, p < 0.001) when compared with the nonhypothermic control group (50.7 +/- 9.3 percent). The experimental groups demonstrated decreased local muscle edema (4.09 +/- 0.30, 4.10 +/- 0.19, and 4.04 +/- 0.31 wet-to-dry weight ratios, respectively) when compared with the nonhypothermic control group (5.24 +/- 0.31 wet-to-dry weight ratio; p < 0.001, p < 0.001, and p < 0.001, respectively). CD11b expression was significantly decreased in the reperfusion-only (32.65 +/- 8.75 mean fluorescence intensity, p < 0.001) and ischemia-reperfusion groups (25.26 +/- 5.32, p < 0.001) compared with the nonhypothermic control group (62.69 +/- 16.93). There was not a significant decrease in neutrophil CD11b expression in the ischemia-only group (50.72 +/- 11.7 mean fluorescence intensity, p = 0.281). Neutrophil infiltration was significantly decreased in the reperfusion-only (20 +/- 11 counts per high-power field, p = 0.025) and ischemia-reperfusion groups (23 +/- 3 counts, p = 0.041) compared with the nonhypothermic control group (51 +/- 28 counts). No decrease in neutrophil density was observed in the ischemia-only group (40 +/- 15 counts per high-power field, p = 0.672) when compared with the nonhypothermic control group (51 +/- 28 counts). Finally, dihydrorhodamine oxidation was significantly decreased in the reperfusion-only group (45.83 +/- 11.89 mean fluorescence intensity, p = 0.021) and ischemia-reperfusion group (44.30 +/- 11.80, p = 0.018) when compared with the nonhypothermic control group (71.74 +/- 20.83), whereas no decrease in dihydrorhodamine oxidation was observed in the ischemia-only group (65.93 +/- 10.3, p = 0.982). The findings suggest a protective effect of local hypothermia during early reperfusion to skeletal muscle after an ischemic insult. Inhibition of CD11b expression and subsequent neutrophil infiltration and depression of neutrophil oxidative potential may represent independent protective mechanisms isolated to local tissue hypothermia during the early reperfusion period (reperfusion-only and ischemia-reperfusion groups). This study provides evidence for the potential clinical utility of administering local hypothermia to ischemic muscle tissue during the early reperfusion period.

Experimental Evaluation of Oxygen-free Radical Scavengers in the Prevention of Reperfusion Injury to Skeletal Muscle

A prolonged preoperative ischemic interval decreases the chances for successful replantation of an amputated limb. Skeletal muscle is especially sensitive to periods of prolonged ischemia. It is now hypothesized that significant tissue injury occurs during reperfusion, when oxygen-rich blood contacts anaerobic metabolites forming toxic oxygen free radicals. A replantation model, using the rabbit hind limb tibialis anterior muscle, was developed to assess muscle function and histological appearance following ischemic intervals of five and eight hours. Muscle strength five weeks after injury was used as a functional measurement of tissue damage. The effects of the superoxide free radical scavenger superoxide dismutase (SOD) and the hydroxyl radical scavenger dimethylsulfoxide (DMSO), administered systemically just before reperfusion, were studied. Muscle treated with SOD following five hours of ischemia had essentially normal strength and histological appearance; however, there was no protective effect after eight hours. DMSO treatment had no beneficial effects after five hours of ischemia, but after eight hours DMSO-treated muscle had significantly better function than untreated muscle. Histological examination confirmed the functional results. Clinical treatment of ischemic limbs with free radical scavengers before revascularization may aid in avoiding reperfusion injury and may improve survival and later muscle function.

The effects of chronic ketorolac tromethamine (toradol) on wound healing.

Intramuscular ketorolac is a commonly used nonsteroidal anti-inflammatory (NSAI) agent for analgesia in surgical patients. Increasing numbers of surgical patients are chronically taking some form of an NSAI drug. We examined the effects of “chronic” intramuscular ketorolac on the healing of a closed linear surgical wound in the rat. Wistar rats were pretreated with 4 mg per kilogram per day ketorolac intramuscularly prior to receiving dorsal incisional wounds. The ketorolac treatment was continued and after 2 weeks the wounds were excised and separated with a tensiometer to measure mechanical properties. Breaking strength was directly measured, tensile strength was calculated, and collagen concentrations at the wound site were determined. A significant decrease in the mean breaking strength was seen in the ketorolac-treated animals when compared to controls. The ketorolac-treated animals had a mean tensile strength less than the controls, although this difference did not reach statistical significance. The mean collagen concentration of the ketorolac-treated wounds was significantly less than the untreated wounds. Use of ketorolac for just 1 week prior to surgery in rats produced a significant decrease in the breaking strength of their wounds. With the increasing use of ketorolac in surgical patients as well as the increasing use of oral NSAI drugs, more study of this effect is warranted.

Vitamin A-soaked gelfoam sponges and wound healing in steroid-treated animals.

Previous work has shown improved wound healing after the administration of systemic vitamin A in patients on chronic steroids. In contrast there have been mixed reports on the effect of topical vitamin A on wound healing in steroid-treated patients. Previous laboratory work has suggested that the topical application of vitamin A may be beneficial to wound healing in a sutured wound in a steroid-treated rat. Due to some inconsistencies in previous studies and steroid animal models, we sought to develop a better wound-healing model in a steroid-treated rat and to assess the effect of topical vitamin A as part of the wound closure. With preliminary studies, we developed a consistent and reliable wound-healing model in a steroid-treated rat using dexamethasone in contrast to cortisone acetate, which had been used in previous studies. Next, rats were randomized into 8 groups, some of which received steroids. Wounds were treated with saline or vitamin A topically or via a soaked gelfoam sponge. Rats were wounded 1 week after the commencement of steroid administration. Wounds were repaired and allowed to heal for 2 weeks. Strips of the wounds were then harvested and tested for tensile strength and breaking strength using a tensiometer. Wound edges were then fixed and wound surface area was measured using digital planimetry. Steroid treatment resulted in consistent weight loss and failure to gain weight as well as decreased breaking strength. Tensile strength was not decreased. Vitamin A applied for 10 minutes before wound closure and a gelfoam sponge alone placed before wound closure both resulted in an increased breaking strength and tensile strength. Vitamin A applied via a gelfoam sponge did not statistically increase breaking or tensile strength.

Early growth response factor-1: expression in a rabbit flexor tendon scar model.

Background: Adhesion formation limits functional recovery after flexor tendon repair. Various growth factors have been implicated in the adhesion scar process. Early growth response factor-1 (EGR-1), a transcription factor associated with synthesis of a variety of key fibrotic growth factors and expression of extracellular matrix genes, has never been identified in a tendon repair model. Methods: Thirty New Zealand White rabbit forepaws underwent laceration and repair of the middle digit flexor digitorum profundus equivalent in zone II. Sodium morrhuate, a topical sclerosing agent, or phosphate-buffered saline, a standard control, was applied to the repair during closure of the tendon sheath. Tendons were harvested from operated and unoperated forepaws at increasing time intervals (1, 3, 7, 14, and 28 days). Tissues were analyzed by immunohistochemistry and Masson trichrome staining. Results: Immunohistochemistry demonstrated that EGR-1 is expressed at the site of tendon repair, along the epitenon of the tendon, and in the infiltrate of inflammatory cells in the surrounding sheath-scar matrix. Control, unoperated tendons demonstrated baseline EGR-1 expression within epitenon cells. EGR-1 was maximally expressed on postoperative day 7. Sodium morrhuate and phosphate-buffered saline demonstrated no difference in their ability to augment tendon adhesion scar formation. Conclusions: Findings demonstrate the following: (1) EGR-1 expression is increased in the tendon wound environment after flexor tendon laceration repair; (2) normal epitenon cells have low, baseline levels of EGR-1 expression; and (3) sodium morrhuate does not augment scar matrix production more than phosphate-buffered saline. The ideal tendon scar model was not generated.

Role of the thromboxane A2 receptor in the vasoactive response to ischemia-reperfusion injury.

Neutrophil-endothelial adhesion in venules and progressive vasoconstriction in arterioles seem to be important microcirculatory events contributing to the low flow state associated with ischemia-reperfusion injury of skeletal muscle. Although the neutrophil CD-18 adherence function has been shown to be a prerequisite to the vasoconstrictive response, the vasoactive substances involved remain unknown. The purpose of this study was to evaluate the role of thromboxane A2 receptor in the arteriole vasoactive response to ischemia-reperfusion injury. An in vivo microscopy preparation of transilluminated gracilis muscle in male Wistar rats (175 +/- 9 g) (n = 12) was used for this experiment. Three experimental groups were evaluated in this study: (1) sham, flap raised, no ischemia (20 venules, 20 arterioles), (2) 4 hours of global ischemia only (19 venules, 22 arterioles), and (3) 4 hours of global ischemia + thromboxane A2 receptor antagonist (ONO-3708) (17 venules, 20 arterioles). ONO-3708 (5 mg/kg), a specific competitive antagonist of thromboxane A2 receptor, was infused at a rate of 0.04 ml/minute into the contralateral femoral vein 30 minutes before reperfusion. Mean arterial blood pressure was not changed at this dose of ONO-3708 (88 +/- 6 mmHg before infusion, 81 +/- 4 mmHg after infusion, n = 3). The number of leukocytes rolling and adherent to endothelium (15-sec observation) were counted in 100-microm venular segments, and arteriole diameters were measured at 5, 15, 30, 60, and 120 minutes of reperfusion. Leukocyte counts and arteriole diameters were analyzed with two-way factorial analysis of variance for repeated measures and Duncans post hoc mean comparison. Statistical significance was indicated by a p < or = 0.05. The ischemia-reperfusion-induced vasoconstriction was significantly reduced by the thromboxane A2 receptor antagonist (ONO-3708). The mean arteriole diameters at 30, 60, and 120 minutes reperfusion were significantly greater in the treated animals than in the ischemia-reperfusion controls. Despite a significant increase in treated mean arteriole diameters, 30 percent of arterioles still demonstrated vasoconstriction. Neutrophil-endothelial adherence was not reduced by ONO-3708. Thromboxane A2 receptor blockade significantly reduces but does not eliminate ischemia-reperfusion-induced vasoconstriction in this model. This finding suggests that additional and perhaps more important vasoactive mediators contribute to vasoconstriction. Furthermore, thromboxane A2 receptor blockade has no effect on polymorphonuclear endothelial adherence.

Transgene expression in a model of composite tissue allotransplantation.

Background: Composite tissue allografting may be an ideal solution to many problems requiring reconstructive surgery. Unfortunately, complications associated with chronic immunocompromise are major impediments to widespread use of composite tissue allografting. Current immunosuppressive and immunomodulatory paradigms focus on modification of the recipient through global immunosuppression or donor/recipient chimerism. Alternatively, modifying the allograft to block rejection or promote tolerance could confine deleterious immunosuppressive effects to the graft or eliminate graft rejection. However, a technique introducing genetic information into the transplant is needed. The authors demonstrate the first model for expressing a gene of interest locally in a hind-limb transplant. Methods: Using a rat hind-limb transplant model, the authors tested the ability of naked DNA infusion, cationic polymer/DNA complex transfection, and adenoviral vector transduction to introduce genetic material into the composite tissue allograft. The marker genes luciferase and green fluorescent protein were used to follow gene expression. Results: Recombinant adenovirus showed rapid, high-level expression of marker genes in the graft, with no detectable expression in recipient animals. Expression was detectable at 18 hours and peaked at 7 days. Levels of expression were lower but above baseline at 4 weeks. Conclusions: Using an adenoviral vector system, the authors have selectively introduced a marker gene (luciferase) into a transplanted hind-limb rat model. Expression was rapid and seen in a variety of cell types. Adenovirus infection had no impact on limb rejection. This method may be a powerful tool for genetically modifying composite tissue allografts and may contribute to immune tolerance and more widespread use of composite tissue allograft surgery.