Early and late effects of ischemic preconditioning on microcirculation of skeletal muscle flaps

The present study was designed to investigate the early and late effects of ischemic preconditioning on muscle flap perfusion and reperfusion-induced skeletal muscle damage. Thirty-six Sprague-Dawley rats were divided into six experimental groups of six animals each. The cremaster muscle flap model and the intravital microscopy system were used to observe microcirculatory changes associated with ischemia-reperfusion injury and ischemic preconditioning. In groups 1, 2, and 3, microcirculatory measurements were taken on the same day; however, in groups 4, 5, and 6, measurements were taken a day after surgery. Group 1 served as a control. The cremaster muscle was prepared as a tube flap, subjected to an hour of perfusion without ischemia. In group 2 (ischemic preconditioning + ischemia group), the cremaster muscle tube flap was subjected to 30 minutes of ischemia and 30 minutes of reperfusion, followed by 4 hours of total ischemia. In group 3 (ischemia alone), the flap was submitted to 4 hours of ischemia alone. In group 4 (control), the cremaster muscle flaps were dissected out, preserved in the subcutaneous tunnel, and submitted to 24 hours of perfusion only. In group 5 (ischemic preconditioning + 24 hours of perfusion + 4 hours of ischemia), the ischemic preconditioning protocol was followed by 24 hours of perfusion and 4 hours of ischemia. In group 6 (24 hours of perfusion + ischemia), the same protocol was used as in group 5 without ischemic preconditioning. Functional capillary perfusion, and the diameters of the arterioles of the first, second, and third order were significantly increased in the ischemic preconditioning group during the early period, but not after 24 hours of perfusion. No differences in the red blood cell velocities of arterioles of the first, second, or third order were found in either the early-effect or late-effect groups. The numbers of rolling, adhering, and transmigrating leukocytes, however, were significantly lower in the ischemic preconditioning group at both early and late follow-up. Ischemic preconditioning of the skeletal muscle flap has both an early and a late protective effect against reperfusion injury. Ischemic preconditioning at the early interval significantly improves muscle flow hemodynamics of the flap and attenuates leukocyte-mediated reperfusion injury. After 24 hours of reperfusion, however, ischemic preconditioning failed to improve the flow hemodynamics of the flap, yet it still protected the skeletal muscle flap from leukocyte-mediated reperfusion injury.     

The role of perfusion washout in limb revascularization procedures

Amputated rat hindlimbs were subjected to either normothermic (26 degrees C) or hypothermic (4 degrees C) ischemia. Experimental limbs had their microcirculation washed out (either before or after the ischemic insult) with a physiologic acellular plasma substitute previously reported to enhance flap survival following extended periods of warm ischemia. Control limbs were not washed out; i.e., stagnant blood remained in these limbs. Following the ischemic interval, amputated limbs were replanted. Monastral blue B, a colloidal pigment capable of labeling leaky blood vessels, was administered systemically to all rats just prior to vascular declamping. Limb biopsies of skin and muscle were harvested 30 minutes following revascularization in order to assess Monastral labeling and, therefore, the functional integrity of the microcirculation. Results confirm that stagnant blood under conditions of warm ischemia is detrimental to the functionality of the microcirculation in both skin (p less than 0.03) and muscle (p less than 0.007). Accordingly, perfusion washout, when performed prior to the ischemic period, enhances limb survival following 6 hours of warm ischemia (p less than 0.01). Hypothermia protects against the detrimental effects of stagnant blood; perfusion offers no benefit if hypothermic conditions prevail. Physiologic mechanisms responsible for these findings are discussed.     

Enhanced neovascularization of rat tubed pedicle flaps with low perfusion of the wound margin

To study the role of ischemia due to low perfusion as the inciter of neovascularization, caudally based 3 X 9 cm skin flaps were created on the dorsum of 50 Sprague-Dawley rats. After injection of 0.2 ml 10% fluorescein, the animals were divided into two groups. In group I (n = 25), the distal margin of the flap tip was 1 cm proximal to the border of the fluorescence (good perfusion). In group II (n = 25), the flap was cut 1 cm distally in the nonfluorescent part (poor perfusion). The tips of the tubed flaps were transferred to a wound bed on the right flank. After 10 days, the pedicles were ligated, so that flap survival depended totally on the new vascular supply from the inset area of the flap. The flaps in group I showed a significantly higher rate of necrosis of 52.4 +/- 15.1 percent versus 1.7 +/- 1.4 percent in group II (p less than 0.0001), although the flap length in group I (5.85 +/- 1.16 cm) was less than in group II (7.15 +/- 0.95 cm; p = 0.0001). A nearly three times larger amount of tissue based on the new blood supply survived in group II compared to group I. Xerograms after injection of PbO2-gelatine on day 10 showed an increased ingrowth of blood vessels in group II. After excluding the delay phenomenon as the cause for the difference in necrosis rate, it is concluded that the only possible explanation is an enhancement of neovascularization by a perfusion gradient between the wound margins.     

Ischemia-reperfusion injury of adipofascial tissue: an experimental study evaluating early histologic and biochemical alterations in rats

Fat necrosis remains a serious complication in reconstructive flaps. In clinical setting, it is well known that fat tissue is more susceptible to ischemic events. We aimed to evaluate early histological and biochemical changes of adipofascial tissue in an experimental model. An epigastric flap model in rats was used to evaluate the effect of ischemia-reperfusion (I-R) injury on adipofascial tissue. Two groups of animals (one with ischemia alone and other ischemia-reperfusion group) were used to evaluate the degree of histological edema, congestion and extravascular bleeding, and early biochemical alterations within the adipofascial flaps. The biochemical parameters included glutathione (GSH) and malondialdehyde (MDA). In each group, contralateral groin subcutaneous adipose tissue served as control. These evaluations were compared to normal unmanipulated, contralateral abdominal subcutaneous adipose tissue. The ischemia-reperfused flap group showed histologically significantly much edema congestion and bleeding than the control groups (P<.0001). The control group showed less edema in fat tissue than the ischemia-alone group (P<.05). All of the flaps in the ischemia-only group showed significantly less bleeding and edema than I-R group (P<.001). The ratio of MDA/GSH was 33 in control, 37 in ischemia alone, and 82 in ischemia-reperfusion groups, respectively. This study confirms that significant histologic and biochemical alteration occurs after ischemia and ischemia-reperfusion events in adipose tissue. Marked drop in adipose tissue antioxidant levels after I-R suggested that preemptive measures to this decrease should be undertaken in clinical settings.     

Gene therapy by adenovirus-mediated vascular endothelial growth factor and angiopoietin-1 promotes perfusion of muscle flaps

An experimental study was conducted to investigate the potential use of intravascular gene therapy with adenovirus-mediated (Ad) vascular endothelial growth factor (VEGF) or angiopoietin-1 (Ang-1) for the enhancement of muscle flap perfusion and to evaluate the effect of therapy on microcirculatory hemodynamics and microvascular permeability in vivo by using a cremaster muscle flap model in the rat. The cremaster tube flap was left intact after isolation of the pudo-epigastric pedicle. A total of 90 male Sprague-Dawley rats were divided into five groups of 18 each, according to the type of intraarterial treatment. Control flaps received phosphate-buffered saline. Group 2 (the control gene encoding green fluorescent protein, Ad-GFP) served as the adenovirus control. In Groups 3, 4, and 5, flaps were pretreated with Ad-VEGF, Ad-Ang-1, and Ad-Ang-1 + Ad-VEGF, respectively. Flaps were preserved in a subcutaneous pocket in the hindlimb for evaluation of functional capillary density and microvascular permeability indices at 3, 7, and 14 days by intravital microscopy system. At day 7 and 14, Ad-VEGF, Ad-Ang-1, and combined treatment groups showed significantly higher numbers of capillary densities when compared with control and Ad-GFP groups (p < 0.05). At day 14, Ad-VEGF was the superior treatment group compared with Ad-Ang-1 and Ad-VEGF + Ad-Ang-1 (p < 0.05). Overall, there was a linear increase in the number of functional capillaries in all treatment groups (p < 0.05). At day 3 after Ad-Ang-1 therapy, a significantly lower permeability index was found when compared with Ad-VEGF + Ad-Ang-1 and Ad-VEGF alone treatment (p < 0.05). At day 7, the Ad-VEGF group had the highest score of permeability index compared with control, combined, and Ad-Ang-1 groups (p < 0.05). Histologic evaluation of muscle flaps demonstrated mild focal inflammation. There was evidence of mild vasculitis in all flaps except control muscles. Intravascular angiogenic therapy with Ad-VEGF or Ad-Ang-1 was technically feasible, as demonstrated by expression of the control gene, GFP, along the vascular tree. All treatment groups increased perfusion of the muscle flap over a period of 14 days, indicating a long-lasting effect of gene therapy. Ang-1 alone or in combination with VEGF was as effective as VEGF alone in augmenting muscle perfusion with more stable vessels 1 week after gene therapy.     

Increase in skin-flap survival by the vasoactive drug buflomedil

The effect of buflomedil to protect skin tissue from ischemia and necrosis was studied in random cutaneous flaps. Measurements were performed by intravital microscopy on the microcirculatory level of capillary perfusion in a flap model in the hairless mouse. In 30 hairless mice, single-pedicle flaps measuring 6 x 16 mm were raised perpendicular to the spine of the animal. This flap develops a reliable amount of necrosis at its distal edge over a period of 7 days. A group of 10 mice received intravenous injections of buflomedil in doses of 3 mg/kg per day diluted in 0.1 ml normal saline beginning 4 hours before flap elevation and for 6 consecutive days postoperatively. In addition, 10 further animals received the same treatment except that it was started 5 minutes after flap elevation. In 10 mice serving as controls, normal saline in equal volumes as in the experimental groups was applied. By means of intravital microscopy, functional vessel density (FVD) was determined in 2.5-mm increments from the flap's base to its distal edge at 1, 6, and 24 hours after elevation. Skin-flap survival was quantified by measuring the necrotic area on day 7 by means of digital planimetry. Functional vessel density was preserved in the distal flap of animals pretreated with buflomedil, revealing a higher functional vessel density at 10.0 mm (p less than 0.01), 12.5 mm (p less than 0.05), and 15.0 mm (p less than 0.001) from the flap's base as compared with controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lidocaine-containing Euro-Collins solution prevents renal injury in the isolated perfused canine kidney exposed to prolonged cold ischemia

Previous studies have reported a decreased incidence of delayed graft function after cadaveric transplantation with the use of lidocaine pretreatment of the donor. We evaluated the effects of lidocaine on prolonged cold ischemia and reperfusion injury in a canine model of isolated kidney perfusion (IPK). The purpose of this study was to evaluate the renal function of isolated perfused canine kidneys after 48 h of cold storage with Euro-Collins (EC) solution or EC solution plus lidocaine. Isolated perfused canine kidneys were randomized into four groups which contained six kidneys: I) cold flush with EC solution and immediately reperfused, II) cold flush with EC solution plus lidocaine and immediately reperfused, III) 48 h of cold storage with EC and reperfusion, IV) 48 h of cold storage with EC solution plus lidocaine and reperfusion. The measured renal functions were glomerular filtration rate, urine production, perfusate flow, urinary lactic dehydrogenase (ULDH), Na reabsorptive capacity, and tissue MDA levels. Histological examination was performed after reperfusion. The tubular functions of kidneys preserved with EC solution containing lidocaine were better when compared with the kidneys preserved with EC alone. Tubular injury marker levels (ULDH) in group IV were significantly lower than in group III and lidocaine also reduced lipid peroxidation during reperfusion. This is in agreement with the histological results. The results of the present study can be taken as evidence of the cytoprotective effect of lidocaine, which may therefore be accepted as a useful agent for kidney preservation.     

Temporal Profiles of Nerve Growth Factor β-Subunit Level in Rat Brain Regions After Transient Ischemia

To determine the role of nerve growth factor (NGF) in ischemic brain damage, we measured the temporal and regional changes in the level of NGF in the hippocampal subfields, the cerebral cortex, the striatum, and the septum at 1, 2, 7, and 30 days after transient forebrain ischemia using a highly sensitive sandwich-type enzyme immunoassay system for the beta-subunit of mouse 7S NGF (beta-NGF). We also analyzed glial fibrillary acidic protein immunoreactivity in the hippocampus to ascertain the contribution of reactive astrocytes to NGF production after an ischemic insult. In the CA1 subfield of the hippocampus, the level of beta-NGF decreased slightly 2 days after ischemia (not significant), at which time CA1 pyramidal cell loss began to occur, and increased by 40% 30 days after ischemia (p less than 0.05). A marked increase in glial fibrillary acidic protein-positive astrocytes in the CA1 subfield 2-30 days after ischemia suggests that the reactive astrocytes participated in a gradual increase in the level of beta-NGF after recirculation. The level of beta-NGF in the dentate gyrus decreased transiently 2 days (p less than 0.05) and 7 days (p less than 0.01) after ischemia, followed by recovery to the level of control animals 30 days after ischemia. The level of beta-NGF in the septum gradually decreased 7 days (-27%, p less than 0.05) and 30 days (-43%, p less than 0.01) after ischemia. The levels of beta-NGF in the cerebral cortex and striatum remained unaltered throughout the observation period.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ductal injection does not increase the islet yield or function after cold storage in a vascular perfusion model

Several studies have reported that pancreatic ductal preservation greatly improved the islet yield and function after cold storage. However, these studies were devoid of appropriate controls, such as vascular perfusion, which is routinely performed to preserve organs in the clinical setting. In this study, we created a vascular perfusion model using inbred rats, and investigated the effect of ductal injection on the islet yield and function after cold storage. Rat pancreases after 10 h cold ischemia were classified as follows: without ductal/vascular perfusion; with ductal injection; with vascular perfusion; and with ductal/vascular perfusion. The islet yield, function, viability, release of inflammatory mediators, and pathological changes in the exocrine tissues were assessed in the Hanks' Balanced Salt Solution (HBSS) model. The islet yield was also assesed by introducing University of Wisconsin Solution (UWS) and Histidine-Tryptophan-Ketoglutarate solution (HTK), which are the standard clinical preservation solutions. In the HBSS model, ductal injection and vascular perfusion significantly improved the islet yield compared with the control group. However, ductal injection showed no additional effects on the islet yield, function, viability and suppressing the release of inflammatory mediators when vascular perfusion was performed. Although ductal injection significantly decreased the apoptosis of exocrine cells, no beneficial effect on vacuolation was observed. In contrast, vascular perfusion significantly suppressed vacuolation in the exocrine tissues. Likewise, in the UWS and HTK model, ductal injection and vascular perfusion improved the islet yield compared with the control group. Nevertheless, the combination group showed no additional effects. These data suggest that ductal injection has no additional effect on islet yield and function after cold storage in a vascular perfusion model. We propose that ductal injection can be an effective and simple alternative for vascular perfusion prior to pancreas harvest, but is not necessary in most cases, since vascular perfusion is routinely performed.     

Life span and ovarian dynamics of the pine beauty moth, Panolis flammea (D&S): the effect of low temperatures after adult emergence on reproductive success

Abstract Adult female Panolis flammea were kept at 2^oC either immediately after emergence from the pupae or immediately after mating, for periods ranging from 5 to 20 days and then placed at 15^oC together with a male moth, food supply and suitable oviposition site ( Pinus contorta foliage). Weight loss over the storage period was directly proportional to the time spent at 2^oC irrespective of whether the moths were virgin or mated. Pre-oviposition period decreased significantly from the control after more than 5 days at 2^oC, but there was no significant difference between late mated and early mated moths. The post-storage life span of late mated and early mated moths decreased in proportion to the time spent at 2^oC but late mated moths had significantly longer life spans than early mated moths. Moths mated prior to storage were significantly less fecund than moths mated after storage except those moths kept at 2^oC for 20 days where the fecundities of both treatments were identical (c. 45 eggs/female). These results are discussed in relation to the conditions likely to be experienced by P.flammea in the field and their bearing on the population dynamics of this insect.     

Hypothermic renal perfusion during aortic surgery reduces the presence of lipocalin-2 and preserves renal extraction of dimethylarginines in rats

Cold perfusion through the renal arteries during renal ischemia has been suggested to diminish postoperative renal damage after juxtarenal aortic aneurysm repair. As the kidneys play a key role in dimethylarginine metabolism, which in turn is associated with renal hemodynamics, we hypothesized that the protective effect of cold perfusion is associated with a preserved renal extraction of dimethylarginines. Renal ischemia was induced in three groups of anesthetized Wistar rats (n = 7/group), which underwent suprarenal aortic clamping (45 min) with no perfusion (group 1), renal perfusion with 37°C saline (group 2), or renal perfusion with 4°C saline (group 3), respectively, followed by 90 min of renal reperfusion in all groups. The sham group had no clamping. In group 3 (renal ischemia with cold perfusion), postoperative serum creatinine levels as well as the presence of luminal lipocalin-2 and its associated brush-border damage were lower compared with groups 1 and 2 (P < 0.05). Also, renal extraction of asymmetrical (ADMA) and symmetrical (SDMA) dimethylarginine as well as the arginine/ADMA ratio, which defines the bioavailability of nitric oxide, remained intact in group 3 only (P < 0.04). The arginine/ADMA ratio correlated with cortical flow, lipocalin-2, and creatinine rises. Warm and cold renal perfusion (groups 2 and 3) during ischemia were similarly effective in lowering protein nitrosylation levels, renal leukocyte accumulation, neutrophil gelatinase-associated lipocalin (NGAL) expression in distal tubules, and urine NGAL (P < 0.05). These data support the use of cold renal perfusion during renal ischemia in situations where renal ischemia is inevitable, as it reduces tubular damage and preserves renal extraction of dimethylarginines. Renal perfusion with saline per se during renal ischemia is effective in diminishing renal leukocyte accumulation and oxidative stress.     

The hypothetical role of potassium conductance on the genesis of R-wave amplitude increase during ischemia in the isolated rat heart.

The effect of increased potassium conductance on the genesis of R-wave amplitude increase during acute myocardial ischemia has been studied in the isolated perfused rat heart by simultaneously recording the R-wave amplitude of epicardial electrograms (VEE), heart rate (HR), coronary flow rate (CFR), left ventricular diastolic pressure (LVDP), and left ventricular systolic pressure (LVSP). The experiments were performed during basal and partial or total ischemic conditions at spontaneous or fixed HR. In some experiments, potassium conductance was increased by means of high-calcium (8 mM) or acetylcholine chloride (10(-6) M) perfusion. In the control experiments, partial ischemic perfusion produced an increase in VEE and LVDP and a decrease in HR, CFR, and LVSP; total ischemic perfusion exaggerated these variations. High-calcium perfusion provoked an increase in VEE and LVDP and a decrease in HR, CFR, and LVSP during basal conditions (p less than 0.01 vs. control experiment); these modifications increased progressively during partial ischemic perfusion (p less than 0.01 vs. control experiment) and during total ischemic perfusion (p less than 0.01 vs. control experiment). Perfusion with acetylcholine chloride produced variations similar to those observed in high-calcium solution except that LVDP under basal conditions remained unchanged from control. When the HR was maintained at a constant value by means of atrial pacing the results were similar to those observed in the unpaced hearts. In conclusion, in the isolated perfused rat heart, increasing potassium conductance may influence the genesis of R-wave amplitude increasing during acute myocardial ischemia.     

Development of an Extracorporeal Perfusion Device for Small Animal Free Flaps

Background

Extracorporeal perfusion (ECP) might prolong the vital storage capabilities of composite free flaps, potentially opening a wide range of clinical applications. Aim of the study was the development a validated low-cost extracorporeal perfusion model for further research in small animal free flaps.

Methods

After establishing optimal perfusion settings, a specially designed extracorporeal perfusion system was evaluated during 8-hour perfusion of rat epigastric flaps followed by microvascular free flap transfer. Controls comprised sham-operation, ischemia and in vivo perfusion. Flaps and perfusate (diluted blood) were closely monitored by blood gas analysis, combined laser Doppler flowmetry and remission spectroscopy and Indocyanine-Green angiography. Evaluations were complemented by assessment of necrotic area and light microscopy at day 7.

Results

ECP was established and maintained for 8 hours with constant potassium and pH levels. Subsequent flap transfer was successful. Notably, the rate of necrosis of extracorporeally perfused flaps (27%) was even lower than after in vivo perfusion (49%), although not statistically significant (P = 0,083). After sham-operation, only 6% of the total flap area became necrotic, while 8-hour ischemia led to total flap loss (98%). Angiographic and histological findings confirmed these observations.

Conclusions

Vital storage capabilities of microvascular flaps can be prolonged by temporary ECP. Our study provides important insights on the pathophysiological processes during extracorporeal tissue perfusion and provides a validated small animal perfusion model for further studies.     

Oxygen radical scavengers improve vascular patency and bone-muscle cell survival in an ischemic extremity replant model

We examined the use of oxygen radical scavengers in preventing the no-reflow phenomenon and improving bone-muscle cell survival in an ischemic extremity replant model. A total of 70 Lewis rat modified hindlimb replants were performed after specific periods of cold ischemia and intraarterial perfusion with either superoxide dismutase and catalase, specific oxygen free-radical scavengers, or a control solution. Ischemic hindlimbs treated with superoxide dismutase and catalase showed a statistically significant (p less than 0.05) improvement in vascular patency after prolonged cold ischemia when compared to controls. Histologically, experimental extremities demonstrated greater osteoblast, osteocyte, and muscle cell survival in replanted hindlimbs with patent vascular anastomoses. The perfusion of severed limbs and digits and free vascularized tissue transfers with superoxide dismutase and catalase after a period of ischemia has already occurred may prolong the ischemic "time window" tolerated for successful tissue survival.     

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.     

Bile analysis as a tool for assessing integrity of biliary epithelial cells after cold ischemia--reperfusion of rat livers

Previous morphological studies failed to show appreciable injury of biliary epithelial cells (BEC) after cold ischemia of rat liver, although recent evidence indicated that BEC integrity and function were impaired in this model. We tested the hypothesis that analysis of bile for enzymes, such as lactate dehydrogenase (LDH), alanine transaminase (ALT), and aspartate transaminase (AST), can be used for assessing cold ischemic injury of BEC. Furthermore, we examined whether biliary gamma-glutamyltransferase (GGT) reflects warm ischemic injury of BEC and whether normothermic reperfusion aggravates the negative effect of cold ischemia on BEC integrity and function. Rat livers were reperfused after different periods of cold or warm ischemia using a blood-free perfusion model. Compared with controls, perfusate LDH, ALT, and AST levels and parameters of hepatocyte function, including hepatocyte tight junction permeability, were not significantly altered by 18-h cold ischemia. On the other hand, 9-h cold ischemia markedly increased biliary LDH, ALT, and AST levels. However, only LDH release into the bile was strongly dependent on the time of cold storage. Biliary GGT, LDH, and glucose levels decreased during the reperfusion period following 18-h cold ischemia. The results suggest that biliary LDH can be used for assessing injury of BEC in cold-preserved livers and that normothermic reperfusion does not aggravate preservation-induced injury of BEC after cold ischemic storage.     

Early microsurgical reconstruction of complex trauma of the extremities

Five hundred and thirty-two patients underwent microsurgical reconstruction following trauma to their extremities. They were divided into three groups for the purpose of review. Group 1 underwent free-flap transfer within 72 hours of the injury, group 2 between 72 hours and 3 months of the injury, and group 3 between 3 months and 12.6 years, with a mean of 3.4 years. The results were analyzed with respect to flap failure, infection, bone-healing time, length of hospital stay, and number of operative procedures. The flap failure rate was 0.75 percent in group 1, 12 percent in group 2, and 9.5 percent in group 3 (p less than 0.0005 early versus delayed; p less than 0.0025 early versus late). Postoperative infection occurred in 1.5 percent of group 1, 17.5 percent of group 2, and 6 percent of group 3. Bone-healing time was 6.8 months in group 1, 12.3 months in group 2, and 29 months in group 3. The average length of total hospital stay was 27 days for group 1, 130 days for group 2, and 256 days for group 3. The number of operations averaged 1.3 for group 1, 4.1 for group 2, and 7.8 for group 3.     

Efficacy of intravenous infusion of prostacyclin (PGI2) or prostaglandin E1 (PGE1) in augmentation of skin flap blood flow and viability in the pig

The dose-response effects of 6-h intravenous infusion of PGI2 (0, 5, 10, 25 or 75 ng/kg/min) or PGE1 (0, 25, 50, 100 or 300 ng/kg/min) on skin hemodynamics and viability were studied in 4 x 10 cm random pattern skin flaps (n = 24) raised on both flanks of the pig. Infusion of PGI2 or PGE1 was started immediately after intravenous injection of a loading dose 30 min before skin flap surgery. PGI2 infusion significantly (P less than 0.05) increased the total skin flap capillary blood flow at the dose of 10 ng/kg/min, compared with the control. However, the distance of blood flow along the skin flap from the pedicle to the distal end, i.e. perfusion distance, was not increased. Consequently, the length and area of skin flap viability was also not significantly increased. The effect of PGI2 infusion on skin blood flow was biphasic. Specifically, higher doses (greater than or equal to 25 ng/kg/min) of intravenous PGI2 infusion produced no beneficial effect on the skin flap capillary blood flow. PGI2 infusion at the dose of 10 or 75 ng/kg/min did not significantly increase plasma renin activities or plasma levels of norepinephrine compared with the control, therefore the biphasic effect of PGI2 on skin flap blood flow was not related to circulating levels of norepinephrine or angiotensin. Intravenous infusion of PGE1 did not produce any therapeutic effect on the skin capillary blood flow in the random pattern skin flaps at all doses tested. At the dose of 300 ng/kg/min, the mean arterial blood pressure was 17% lower (P less than 0.05) than the control, but the skin capillary flow still remained similar to the control. It was concluded that intravenous infusion of PGI2 or PGE1 was not effective in augmentation of distal perfusion or length of skin viability in the porcine random pattern skin flaps. Drug treatment modalities for prevention or treatment of skin flap ischemia is discussed.     

Technical variations of the bipedicled TRAM flap in unilateral breast reconstruction: effects of conventional versus microsurgical techniques of pedicle transfer on complications rates

In cases of unilateral breast reconstruction with a transverse rectus abdominis musculocutaneous (TRAM) flap, poorly perfused tissue, which is normally excised to avoid subsequent fat necrosis, must sometimes be used to achieve adequate breast size and projection. In such cases, incorporation of a second vascular pedicle into the flap design improves perfusion. The authors retrospectively examined their experience with bipedicled TRAM flap-based unilateral breast reconstruction to determine whether the use of microsurgical rather than conventional (nonmicrosurgical) techniques for flap transfer resulted in lower incidences of flap-site fat necrosis and donor-site hernia/bulge. The authors retrospectively reviewed the medical records of all patients who underwent unilateral breast reconstruction with a bipedicled TRAM or deep inferior epigastric perforator flap between January of 1991 and March of 2001. Group 1 consisted of patients who had undergone flap transfer using a conventional technique for both pedicles; group 2, patients who had flap transfer using a conventional technique for one pedicle and a microsurgical technique for the other; and group 3, patients who had flap transfer using a microsurgical technique for both pedicles. Of the 863 patients identified, 72 (8.3 percent) had undergone reconstruction using a bipedicled flap. There were 43 patients in group 1, 24 patients in group 2, and five patients in group 3. Only one case of total flap loss had occurred (group 1). Partial flap loss occurred in two patients in group 1 (5 percent) and three patients in group 2 (13 percent). Fat necrosis occurred more frequently in groups 1 (23 percent) and 2 (29 percent) than in group 3 (0 percent) (p = 0.5, Fisher's exact test). Similarly, bulge or hernia was more common in groups 1 (12 percent) and 2 (4 percent) than in group 3 (0 percent) (p = 0.6, Fisher's exact test). In this study, patients who received a bipedicled TRAM flap using microsurgical techniques alone (group 3) appeared to have better flap perfusion and less frequent hernia/bulge than did patients who underwent flap transfer using conventional (group 1) or combined techniques (group 2). However, these differences were not statistically significant, and this trend must be verified in a larger study.     

Vascular endothelial growth factor expression in pig latissimus dorsi myocutaneous flaps after ischemia reperfusion injury

Exogenous administration of vascular endothelial growth factor (VEGF) improves long-term viability of myocutaneous flaps. However, endogenous expression of this substance in flaps following ischemia-reperfusion injury has not been reported previously. Endogenous production of VEGF was measured in myocutaneous pig latissimus dorsi flaps after ischemia-reperfusion injury. Latissimus dorsi myocutaneous flaps (15 x 10 cm) were simultaneously elevated bilaterally in six Yorkshire-type male pigs (25 kg). Before elevation, three flap zones (5 x 10 cm) were marked according to their distance from the vascular pedicle. After isolation of the vascular pedicle, ischemia-reperfusion injury was induced in one flap by occlusion of the thoracodorsal artery and vein for 4 hours, followed by 2 hours of reperfusion. The contralateral flap served as a control. Perfusion in each zone was monitored by laser Doppler flowmetry at baseline, during ischemia, and during reperfusion. At the end of the protocol, skin and muscle biopsies of each flap zone and adjacent tissues were obtained for later determination of VEGF protein levels. VEGF concentrations were quantified using the Quantikine human VEGF immunoassay. Skin perfusion was similar among all flap zones before surgery. Flow fell in all flaps immediately after flap elevation. After 4 hours of ischemia, blood flow in the ischemic flaps was significantly decreased (p < 0.05) compared with nonischemic control flaps. After 2 hours of reperfusion, flow in ischemic flap skin recovered to levels similar to those in control flaps. VEGF protein concentrations in muscle tissue exceeded concentrations in skin and decreased from zones 2 to 3 in control and ischemic flaps. No significant differences in VEGF concentrations between ischemic and control muscle zones were observed. However, the concentration of VEGF in all muscle zones was significantly higher (p < 0.05) than muscle adjacent to the flap. Concentrations in skin zones 1 and 2 were significantly higher (p < 0.05) in ischemic flaps than in control flaps, but levels in zone 3 (most ischemic flaps) showed no significant difference.