Timing of microcirculatory injury from ischemia reperfusion

The low flow state that results from ischemia and reperfusion injury is a potentially reversible process that is important in numerous clinical situations. However, the point in time during the course of reperfusion where tissue injury becomes irreversible is unknown. This experiment evaluated the continuum of tissue damage in skeletal muscle after ischemic insult by quantifying the number of flowing capillaries and percentage muscle necrosis in a male Wistar rat skeletal muscle model. A gracilis muscle flap was raised on the vascular pedicle of 39 male Wistar rats and examined at 832x using intravital videomicroscopy. The numbers of flowing capillaries in five consecutive high-power fields were counted for baseline values. The flap was then subjected to 4 hours of global ischemia (except in sham animals, n = 7) by placing a microvascular clamp on the pedicle artery and vein. Upon reperfusion, flowing capillaries were counted in the same five high-power fields at intervals of 5, 15, 30, and 60 minutes, then at 2 to 8 (1-hour intervals), 24, and 48 hours. The gracilis muscle was then harvested at these intervals during reperfusion and assessed for viability. Compared with baseline, flowing capillaries from the ischemia and reperfusion group (mean +/- SEM) decreased significantly in the first 8 hours of reperfusion (7.7 +/- 0.2 to 3.2 +/- 0.3, p < 0.001) with minimal change noted from 8 to 48 hours. Percentage muscle necrosis increased progressively in ischemia and reperfusion preparations from 1 to 7 hours of reperfusion (16.5 +/- 2.6 percent to 38.9 +/- 1.2 percent, p < 0.001). No significant change in muscle necrosis in the ischemia and reperfusion group was noted between 7 and 48 hours. Sham preparations showed no change in the number of flowing capillaries through 3 hours of reperfusion, with a slight decrease at 24 hours. This rat gracilis microcirculation skeletal muscle model demonstrates a heterogeneous reperfusion injury. The decrease in flowing capillaries correlated with the increase in percentage necrosis and appeared to stabilize at the 7- to 8-hour interval. This finding may have important implications for the timing of interventions aimed at minimizing tissue damage from ischemia-reperfusion.     

The effect of low-molecular-weight heparin in the survival of a rabbit congested skin flap

Swelling and congestion of flaps are frequently seen postoperatively and can cause unexpected necrosis. According to previous reports, venous thrombosis seems to be a more frequent problem than arterial occlusion in both experimental and clinical surgery. Few satisfactory venous trauma models exist, and reports on experimental venous thrombosis are rare. The object of this study was to create a rabbit venous occlusion flap model and to evaluate the effect of low-molecular-weight heparin on this flap. Eight New Zealand rabbits were used in the pilot study, in which the ideal congested flap was investigated using a flap pedicle based on the central auricular artery with a skin pedicle 0, 1, 2, or 3 cm wide. The flap (3 x 6 cm) was designed on the central part of the left ear, and the central auricular vein and nerve, the former for venous return, were cut out at the base of the flap. The flaps with skin pedicles 0, 1, 2, or 3 cm wide showed mean necrosis length of 60.0, 9.3, 4.2, and 0.0 mm, respectively. The flaps with skin pedicles 0, 1, 2, or 3 cm wide showed mean necrosis of 100, 15.5, 7, and 0 percent, respectively. Therefore, the flap, based on a 1-cm-wide skin pedicle and the central auricular artery, was selected as an optimal congested flap model showing 15.5 percent necrosis. The congested flap was then elevated on the left ear of another 10 rabbits. Subcutaneous low-molecular-weight heparin (320 IU/kg) was administered immediately after surgery to five of the rabbits (the low-molecular-weight heparin group), and the remaining five were used as a control group. Fluorescein was injected 15 minutes after surgery to evaluate the circulatory territory of the flap, and the circulatory territory was measured 5 minutes after injection. The flaps were assessed 7 days after surgery by angiography, histology, and clinical findings. The circulatory territory was significantly greater in the low-molecular-weight heparin group (mean +/- SD, 39.2 +/- 3.0 mm) than the control group (mean +/- SD, 48.0 +/- 1.0 mm) (p < 0.001) assessed 7 days after surgery. The longest flap survival length in group A and group B ranged from 40 to 55 mm (mean +/- SD. 49.4 +/- 5.6 mm) and complete survival (mean +/- SD, 60.0 +/- 0.0 mm). The improvement in survival was statistically significant for group B compared with group A (p < 0.015). Histologic evaluation revealed moderate to severe venous congestion and inflammation in the control group, whereas there were minimal changes in the low-molecular-weight heparin group. Angiography of the flap revealed obvious venous occlusion in the periphery in the control group compared with the low-molecular-weight heparin group. The authors conclude that subcutaneous administration of low-molecular-weight heparin has a great potential to improve the survival length of a congested flap without major complications.     

Effect of L-arginine on leukocyte adhesion in ischemia-reperfusion injury

Nitric oxide has been reported to be beneficial in preserving muscle viability following ischemia-reperfusion injury. The purpose of this study was to evaluate the influence of nitric oxide via L-arginine on leukocyte adhesion following ischemia-reperfusion injury. Intravital videomicroscopy of rat gracilis muscle was used to quantify changes in leukocyte adherence. The gracilis muscle was raised on its vascular pedicle in 48 male Wistar rats. The animals were assigned to one of five groups: (1) nonischemic control; (2) ischemia-reperfusion; (3) ischemia-reperfusion and L-arginine; (4) ischemia-reperfusion and Nomega-nitro-L-arginine methyl ester (L-NAME); and (5) ischemia-reperfusion, L-NAME, and L-arginine. All groups that included ischemia-reperfusion were subjected to 4 hours of global ischemia followed by 2 hours of reperfusion. L-Arginine (10 mg/kg) and L-NAME (10 mg/kg) were infused into the contralateral femoral vein beginning 5 minutes before reperfusion, for a total of 30 minutes. The number of adherent leukocytes was counted at baseline and at 5, 15, 30, 60, and 120 minutes after reperfusion (reported as mean change from baseline, +/- SEM). Groups were compared by repeated-measures analysis of variance (five groups, five times). P < or =0.05 was accepted as significant. L-Arginine significantly reduced leukocyte adherence to venular endothelium during reperfusion when compared with the ischemia-reperfusion group (1.39 +/- 0.92 versus 12.78 +/- 1.43 at 2 hours, p < 0.05). Administration of L-NAME with L-arginine showed no significant difference in adherent leukocytes when compared with the ischemia-reperfusion group (10.28 +/- 2.03 at 2 hours). The nitric oxide substrate L-arginine appears to reduce the deleterious neutrophil-endothelial adhesion associated with ischemia-reperfusion injury. L-NAME (nitric oxide synthesis inhibitor) given concomitantly with L-arginine reversed the beneficial effect of L-arginine alone, indicating that L-arginine may be acting via a nitric oxide synthase pathway. These results suggest an important role for nitric oxide in decreasing the neutrophil-endothelial interaction associated with ischemia-reperfusion injury.     

Effects of arterial pressure on lung capillary pressure and edema after microembolism

The effect of increased arterial pressure (Pa) on microvessel pressure (Pc) and edema following microvascular obstruction (100-micron glass spheres) was examined in the isolated ventilated dog lung lobe pump perfused with blood. Lobar vascular resistance (PVR) increased 2- to 10-fold following emboli when either Pa or flow was held constant. Microbead obstruction increased the ratio of precapillary to total PVR from 0.60 +/- 0.05 to 0.84 +/- 0.02 (SE) or to 0.75 +/- 0.06 (n = 6), as determined by the venous occlusion and the isogravimetric capillary pressure techniques, respectively. Isogravimetric Pc (5.0 +/- 0.7) did not differ from Pc obtained by venous occlusion (3.8 +/- 0.2 Torr, n = 6). After embolism, Pc in constant Pa decreased from 6.2 +/- 0.3 to 4.4 +/- 0.3 Torr (n = 16). In the constant-flow group, embolism doubled Pa while Pc increased only 40% (6.7 +/- 0.6 to 9.2 +/- 1.4 Torr, n = 6) with no greater edema formation than in the constant Pa groups. These data indicate poor transmission of Pa to filtering capillaries. Microembolism, even when accompanied by elevated Pa and increased flow velocity of anticoagulated blood of low leukocyte and platelet counts, caused little edema. Our results suggest that mechanical effects alone of lung microvascular obstruction cause minimal pulmonary edema.     

Extended approach to the vascular pedicle of the gracilis muscle flap: anatomical and clinical study

Dissection of the proximal gracilis vascular pedicle proceeds in a dark tunnel-like space deep to the adductor longus. With the application of a previously described technique for an extended approach to the lateral arm free flap, the authors describe a novel technique that improves observation and thus facilitates dissection of the proximal gracilis vascular pedicle. A retrospective review of data for 18 consecutive patients who underwent gracilis muscle free flap harvesting with this modified technique between March of 1999 and October of 2001 was conducted, to assess flap viability and patient outcomes. A cadaveric dissection was also performed, to study the anatomical features of the region in depth and to test the proposed flap modification. After the standard incision has been made, the dominant pedicle is exposed on the medial aspect of the gracilis muscle, running in a fascial cleft between the adductor longus and the adductor magnus. Intramuscular branches to the adductor longus are divided. A space is bluntly created anterior and lateral to the adductor longus by separating the fibrous connections to the surrounding adductor and sartorius muscles on both sides of the vascular pedicle. The gracilis muscle is then divided and passed deep to the adductor longus, into this space. With this new position, the final dissection of the pedicle can easily be performed. The confluence of the venae comitantes is frequently encountered, providing a larger-caliber single vein for microvascular anastomosis. The ages of the patients ranged from 9 to 70 years. The majority (14 of 18 patients) had traumatic wounds. The free flap survival rate was 100 percent. One minor complication of a seroma at the donor site was observed. One major complication of venous thrombosis was detected on postoperative day 3, with complete flap salvage. No other complications were noted. This technique is safe and permits direct approach to and excellent observation of the proximal aspect of the gracilis pedicle, without the need for headlights or deep retractors. An additional benefit is the frequent finding of a single larger vein from the merging of the venae comitantes close to the deep femoral vessels.     

Effect of hyperbaric oxygen and medicinal leeching on survival of axial skin flaps subjected to total venous occlusion

This study evaluates the effect of hyperbaric oxygen and medicinal leeching on axial skin flaps subjected to total venous occlusion. Axial epigastric skin flaps (3 x 6 cm) were elevated on their vascular pedicles in 40 male Wistar rats. Total venous occlusion was achieved by division of all veins draining the skin flap. Arterial inflow was left intact. Animals were randomly assigned to one of five groups: sham (n = 8); control, total venous occlusion only (n = 8); occlusion with hyperbaric oxygen (n = 8); occlusion with leeching (n = 8); occlusion with leeching and hyperbaric oxygen (n = 8). The hyperbaric oxygen protocol consisted of 90-minute treatments, twice daily, with 100% O2 at 2.5 atmospheres absolute for 4 days. The leeching protocol consisted of placing medicinal leeches on the congested flaps for 15 minutes, once daily, for 4 days. Laser Doppler measurements of flap perfusion were recorded preoperatively, postoperatively, and on postoperative days 1 and 3. The percentage of flap necrosis was evaluated on postoperative day 3. Mean percentage necrosis and mean laser Doppler readings were compared between both groups. The flaps in the sham group demonstrated 99 percent survival, whereas the flaps in the occlusion-only group demonstrated 100 percent necrosis. The flaps in the occlusion with oxygen, the occlusion with leeching, and the occlusion with oxygen and leeching groups demonstrated 1, 25, and 67 percent survival, respectively. Sham laser Doppler readings remained within normal limits. Laser Doppler readings in the occlusion-only and the occlusion with oxygen groups decreased to negligible levels on postoperative day 1, and on postoperative day 3 no perfusion was demonstrated. In both the occlusion with leeching and the occlusion with leeching and oxygen groups, there was also a significant decrease in laser Doppler measurements after surgery, but perfusion remained stable throughout the remainder of the study. This study demonstrates that hyperbaric oxygen alone is not an effective treatment for skin flaps compromised by total venous occlusion. The combination of leeching and hyperbaric oxygen treatment of total venous occlusion results in a significant increase in flap survival above that found with leeching alone. It appears that hyperbaric oxygen is effective because of the venous outflow provided by leeching as demonstrated by laser Doppler flow readings.     

Tissue carbon dioxide tension: a putative specific indicator of ischemia in porcine latissimus dorsi flaps

Free flap surgical procedures are technically challenging, and anastomosis failure may lead to arterial or venous occlusion and flap necrosis. To improve myocutaneous flap survival rates, more reliable methods to detect ischemia are needed. On the basis of theoretical considerations, carbon dioxide tension, reflecting intracellular acidosis, may be suitable indicators of early ischemia. It was hypothesized that tissue carbon dioxide tension increased rapidly when metabolism became anaerobic and would be correlated with acute venoarterial differences in lactate levels, potassium levels, and acid-base parameters. Because metabolic disturbances have been observed to be less pronounced in flaps with venous occlusion, it was hypothesized that tissue carbon dioxide tension and venoarterial differences in lactate and potassium levels and acid-base parameters would increase less during venous occlusion than during arterial occlusion. In 14 pigs, latissimus dorsi myocutaneous flaps were surgically isolated, exposed to acute ischemia for 150 minutes with complete arterial occlusion (seven subjects) or venous occlusion (seven subjects), and reperfused for 30 minutes. After arterial occlusion, pedicle blood flow decreased immediately to less than 10 percent of baseline flow. Blood flow decreased more slowly after venous occlusion but within 3 minutes reached almost the same low levels as observed during arterial occlusion. Venous oxygen saturation decreased from approximately 70 percent to approximately 20 percent, whereas oxygen uptake was almost arrested. Tissue carbon dioxide tension increased to two times baseline values in both groups (p < 0.01). The venoarterial differences in carbon dioxide tension, pH, base excess, glucose levels, lactate levels, and potassium levels increased significantly (p < 0.01). Tissue carbon dioxide tension measured during the occlusion period were closely correlated with venoarterial differences in pH, base excess, glucose levels, lactate levels, and potassium levels (median r2, 0.67 to 0.92). After termination of arterial or venous occlusion, more pronounced hyperemia was observed in the arterial occlusion group than in the venous occlusion group (p < 0.05). Oxygen uptake (p < 0.05) and venoarterial differences in lactate and potassium levels (p < 0.05) were significantly more pronounced in the arterial occlusion group. In the venous occlusion group, with less pronounced hyperemia, venoarterial differences in acid-base parameters remained significantly different from baseline values before occlusion (p < 0.01). The data indicate that tissue carbon dioxide tension can be used to detect anaerobic metabolism, caused by arterial or venous occlusion, in myocutaneous flaps. The correlations between carbon dioxide tension and venoarterial differences in acid-base parameters were excellent. Because carbon dioxide tension can be measured continuously in real time, such measurements are more likely to represent a clinically useful parameter than are venoarterial differences.     

The microcirculation of myocutaneous island flaps in pigs studied with radioactive blood volume tracers and microspheres of different sizes.

In order to further improve the understanding of hemodynamic changes in the immediate postoperative phase after elevation of myocutaneous flaps, regional blood flow and arteriovenous (A-V) shunting were measured in rectus abdominis island flaps in 8 pigs. Radioactive microspheres of two sizes (15 and 50 micron) were used. Approximately half (53.4 +/- 6 percent) of the 15-micron microspheres and one-fourth (24.1 +/- 6 percent) of the 50-micron microspheres entering the flap appeared in the venous outflow. Compared with the control area, A-V shunting was significantly increased in muscle and substantially more pronounced in skin. Nutritional blood flow, total blood flow, and vascular volume were increased in muscle and unchanged in skin and subcutis. The lowest tissue hematocrit of 7 +/- 1 percent was found in skin as compared with a central hematocrit of 35 +/- 2 percent. Tissue hematocrit in flap muscle was decreased to 17 +/- 2 percent when compared with control muscle (22 +/- 3 percent), and the mean transit time for blood was correspondingly decreased. Thus vasodilation provided increased perfusion through muscular capillaries and through A-V shunts. Shunting of 15-micron microspheres appeared to take place not only in skin, but also in subcutis and muscle, which challenges the widespread belief that A-V shunting does not occur in muscle.     

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.     

Ascorbate prevents microvascular dysfunction in the skeletal muscle of the septic rat.

Septic patients have low plasma ascorbate concentrations and compromised microvascular perfusion. The purpose of the present experiments was to determine whether ascorbate improves capillary function in volume-resuscitated sepsis. Cecal ligation and perforation (CLP) was performed on male Sprague-Dawley rats. The concentration of ascorbate in plasma and urine, mean arterial blood pressure, and density of continuously perfused capillaries in the extensor digitorum longus muscle were measured 24 h after surgery. CLP caused a 50% decrease (from 56 +/- 4 to 29 +/- 2 microM) in plasma ascorbate concentration, 1,000% increase (from 46 +/- 13 to 450 +/- 93 microM) in urine ascorbate concentration, 20% decrease (from 115 +/- 2 to 91 +/- 2 mmHg) in mean arterial pressure, and 30% decrease (from 24 +/- 1 to 17 +/- 1 capillaries/mm) in the density of perfused capillaries, compared with time-matched controls. A bolus of intravenous ascorbate (7.6 mg/100 g body wt) administered immediately after the CLP procedure increased plasma ascorbate concentration and restored both blood pressure and density of perfused capillaries to control levels. In vitro experiments showed that ascorbate (100 microM) inhibited replication of bacteria and prevented hydrogen peroxide injury to cultured microvascular endothelial cells. These results indicate that ascorbate is lost in the urine during sepsis and that a bolus of ascorbate can prevent microvascular dysfunction in the skeletal muscle of septic animals. Our study supports the view that ascorbate may be beneficial for patients with septic syndrome.     

Minimally invasive harvest of the gracilis muscle.

Acceptance of minimally invasive plastic surgery has been predicated on decreasing morbidity while maintaining the quality and costs of outcomes. The major patient complaint about the gracilis muscle donor site has almost solely been related to the length of the thigh scar, and thus would appear to be an ideal indication for outcome improvement using minimally invasive techniques. A method of endoscopically assisted gracilis muscle harvest, therefore, was developed, starting with a transverse incision just proximal to the knee to identify the gracilis tendon. This endoscopic port allows retrograde subfascial dissection of the muscle and precise identification of its anatomic course, whereupon a small proximal medial thigh incision can be made secondarily for direct access to the vascular pedicle. This variation has now been used successfully in 10 patients. The mean proximal thigh scar length was 8.30 +/- 0.74 (SD) cm, and total surgical scars measured 11.84 +/- 0.95 cm, compared with 27.73 +/- 9.55 cm for 16 patients for whom an open method had been used. This diminished scar length was a statistically significant improvement (p < 0.05), verifying the value of the surgical endoscope as an adjunct for harvest of the gracilis muscle as a free flap.     

Effect of vascular delay on viability, vasculature, and perfusion of muscle flaps in the rabbit

The delay procedure is known to augment pedicled skin or muscle flap survival. In this study, we set out to investigate the effectiveness of vascular delay in two rabbit muscle flap models. In each of the muscle flap models, a delay procedure was carried out on one side of each rabbit (n = 20), and the contralateral muscle was the control. In the latissimus dorsi flap model, two perforators of the posterior intercostal vessels were ligated. In the biceps femoris flap model, a dominant vascular pedicle from the popliteal artery was ligated. After the 7-day delay period, the bilateral latissimus dorsi flaps (based on the thoracodorsal vessels) and the bilateral biceps femoris flaps (based on the sciatic vessels) were elevated. Animals were divided into three groups: part A, assessment of muscle flap viability at 7 days using the tetrazolium dye staining technique (n = 7); part B, assessment of vascular anatomy using lead oxide injection technique (n = 7); and part C, assessment of total and regional capillary blood flow using the radioactive microsphere technique (n = 6). The results in part A show that the average viable area of the latissimus dorsi flap was 96 +/- 0.4 percent (mean +/- SEM) in the delayed group and 84 +/- 0.7 percent (mean +/- SEM) in the control group (p < 0.05, n = 7), and the mean viable area of the biceps femoris flap was 95 +/- 2 percent in the delayed group and 78 +/- 5 percent in the control group (p < 0.05, n = 7). In part B, it was found that the line of necrosis in the latissimus dorsi flap usually appeared at the junction between the second and third vascular territory in the flap. Necrosis of the biceps femoris flap usually occurred in the third territory, and occasionally in both the second and the third territories. In Part C, total capillary blood flow in delayed flaps (both the latissimus dorsi and biceps femoris) was significantly higher than that in the control flaps (p < 0.05). Increased regional capillary blood flow was found in the middle and distal regions, compared with the control (p < 0.05, n = 6). In conclusion, ligation of either the dominant vascular pedicle in the biceps femoris muscle flap or the nondominant pedicle in the latissimus dorsi muscle flap in a delay procedure 1 week before flap elevation improves capillary blood flow and muscle viability. Vascular delay prevents distal flap necrosis in two rabbit muscle flap models.     

Phalloidin attenuates postischemic neutrophil infiltration and increased microvascular permeability.

The aim of this study was to determine whether phalloidin (1 microM) or antamanide (1 microM), cyclic peptides that stabilize dense peripheral band and stress fiber F-actin in endothelium, would attenuate the increase in microvascular permeability induced by 4 h of ischemia and 30 min of reperfusion (I/R) in the isolated canine gracilis muscle. Changes in microvascular permeability (1 - sigma) were assessed by determining the solvent drag reflection coefficient for total plasma proteins (sigma) in muscles subjected to 4.5 h of continuous perfusion (nonischemic controls), I/R alone, I/R + phalloidin, or I/R + antamanide. Muscle neutrophil content was assessed by determination of myeloperoxidase (MPO) activity in tissue samples obtained at the end of the experiments. Fluorescent detection of nitrobenzoxadiazole-phallicidin in endothelial cell monolayers confirmed that phalloidin enters these cells. I/R was associated with marked increases in microvascular permeability and muscle neutrophil content (1 - sigma = 0.45 +/- 0.07; MPO = 8.9 +/- 0.5 units/g) relative to control (4.5 h continuous perfusion) preparations (1 - sigma = 0.12 +/- 0.03; MPO = 0.5 +/- 0.8 unit/g). These I/R-induced changes were largely prevented by administration of phalloidin (1 - sigma = 0.19 +/- 0.02; MPO = 0.8 +/- 0.4 U/g) or antamanide (1 - sigma = 0.07 +/- 0.11; MPO = 0.9 +/- 0.3 unit/g) at reperfusion. Similar results were obtained when phalloidin was administered before ischemia (1 - sigma = 0.24 +/- 0.04; MPO = 1.2 +/- 1.0 units/g). Although antamanide decreased superoxide production (by approximately 60%) and adherence to plastic (by approximately 75%) by activated neutrophils in vitro, phalloidin failed to alter these aspects of granulocyte function.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inability of dimethylthiourea to limit tissue necrosis during acute myocardial infarction in rabbits.

This study examined the effect of treatment with dimethylthiourea (DMTU), a highly cell-permeable scavenger of hydroxyl radicals, on tissue necrosis in rabbit hearts during myocardial ischemia and reperfusion. Sixty-two rabbits underwent 45 minutes of coronary occlusion with, or without, coronary reperfusion for 3 hours. A saline vehicle, or DMTU (500 mg/kg intravenously [iv]) was administered over 45 minutes starting either 10 minutes before or 10 minutes after coronary occlusion, or 10 minutes before coronary reperfusion. Anatomic risk zone size was assessed using microsphere autoradiography, and the area of necrosis was determined using tetrazolium staining. Cardiac hemodynamics and risk zone size were similar for all treatment groups. No differences were observed in the extent of tissue necrosis (normalized to risk zone size) for saline- and DMTU-treated rabbits subjected to 45 minutes (61.2 +/- 23.1% vs. 70.6 +/- 16.5%) or 225 minutes (82.8 +/- 5.4% vs. 78.3 +/- 5.9%) of permanent coronary occlusion without reperfusion. Similarly, tissue necrosis in rabbits with 45 minutes coronary occlusion followed by 3 hours reperfusion was not significantly reduced when DMTU was administered either 10 minutes before coronary occlusion, 10 minutes after coronary occlusion, or 10 minutes before coronary reperfusion (67.0 +/- 9.9%; 57.6 +/- 10.6%; 68.3 +/- 13.3%) compared to saline-treated controls (76.6 +/- 10.5%). These results demonstrate that the hydroxyl radical scavenger DMTU does not appear to influence the progression of myocyte injury in this experimental model of acute myocardial infarction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolic characteristics of experimental free vascularized canine gracilis muscle transfers

A canine gracilis model was used to study muscle energy metabolism and enzyme activities after free vascularized muscle transfer. Fifteen male mongrel dogs underwent orthotopic, free transfer of the left gracilis with microneurovascular anastomosis. After a minimum of 10 months' recovery, muscle biopsy specimens were obtained from the transfers and the contralateral controls and analyzed for relative fiber type areas and maximum activities of phosphorylase, hexokinase, phosphofructokinase, glycerol-3-phosphate dehydrogenase (GPDH), pyruvate kinase, lactate dehydrogenase, citrate synthase, succinate dehydrogenase, 3-hydroxyacyl coenzyme A dehydrogenase (HAD), and creatine phosphokinase. Biopsy specimens obtained before and after a 10 minute, 20-Hz contraction were analyzed for glucose, glycogen, glycolytic intermediates, phosphocreatine, total creatine, and adenine nucleotides (adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, inosine monophosphate, and inosine). There was no significant transfer versus control difference in type I relative fiber area (45 +/- 4 percent versus 44 +/- 3 percent). Total creatine was significantly reduced in the transferred muscles relative to control (83.1 +/- 3.0 mmol/kg versus 100.6 +/- 5.1 mmol/kg dry weight). Maximal activities of phosphorylase, pyruvate kinase, lactate dehydrogenase, citrate synthase, succinate dehydrogenase, HAD, and creatine phosphokinase were diminished in transfers relative to controls, although hexokinase activity was significantly higher in the freely transferred gracilis muscles. During the 20-Hz contraction, muscle transfers produced less force initially, although the force/time integral over the 10-minute stimulation was similar in transfers (277 +/- 25 N/g/second) and controls (272 +/- 24 N/g/second). The contraction was associated with significant glvcogen use and lactate accumulation in both transfers and controls, although this was less pronounced for the transfers. Glycolytic flux appeared muted in the transfers relative to controls. Significant, similar high-energy phosphagen reductions and inosine monophosphate accumulation were noted during the contraction in both groups. Contractile activity is associated with the expected pattern of muscle metabolite changes following free vascularized transfer, indicating the components of cellular energy metabolism are not qualitatively altered after microneurovascular muscle transfer. In contrast, quantitative differences suggest that free vascularized muscle transfer can be associated with a muscle enzyme profile consistent with deconditioning and the presence of denervated muscles fibers in the absence of fiber type profile changes.     

Can angiogenesis induced by chronic electrical stimulation enhance latissimus dorsi muscle flap survival for application in cardiomyoplasty?

In cardiomyoplasty, the latissimus dorsi muscle is lifted on its primary neurovascular pedicle and wrapped around a failing heart. After 2 weeks, it is trained for 6 weeks using chronic electrical stimulation, which transforms the latissimus dorsi muscle into a fatigue-resistant muscle that can contract in synchrony with the beating heart without tiring. In over 600 cardiomyoplasty procedures performed clinically to date, the outcomes have varied. Given the data obtained in animal experiments, the authors believe these variable outcomes are attributable to distal latissimus dorsi muscle flap necrosis. The aim of the present study was to investigate whether the chronic electrical stimulation training used to transform the latissimus dorsi muscle into fatigue-resistant muscle could also be used to induce angiogenesis, increase perfusion, and thus protect the latissimus dorsi muscle flap from distal necrosis. After 14 days of chronic electrical stimulation (10 Hz, 330 microsec, 4 to 6 V continuous, 8 hours/day) of the right or left latissimus dorsi muscle (randomly selected) in 11 rats, both latissimus dorsi muscles were lifted on their thoracodorsal pedicles and returned to their anatomical beds. Four days later, the resulting amount of distal flap necrosis was measured. Also, at predetermined time intervals throughout the experiment, muscle surface blood perfusion was measured using scanning laser Doppler flowmetry. Finally, latissimus dorsi muscles were excised in four additional stimulated rats, to measure angiogenesis (capillary-to-fiber ratio), fiber type (oxidative or glycolytic), and fiber size using histologic specimens. The authors found that chronic electrical stimulation (1) significantly (p < 0.05) increased angiogenesis (mean capillary-to-fiber ratio) by 82 percent and blood perfusion by 36 percent; (2) did not reduce the amount of distal flap necrosis compared with nonchronic electrical stimulation controls (29 +/- 5.3 percent versus 26.6 +/- 5.1 percent); (3) completely transformed the normally mixed (oxidative and glycolytic) fiber type distribution into all oxidative fibers; and (4) reduced fiber size in the proximal and middle but not in the distal segments of the flap. Despite the significant increase in angiogenesis and blood perfusion, distal latissimus dorsi muscle flap necrosis did not decrease. This might be because of three reasons: first, the change in muscle metabolism from anaerobic to aerobic may have rendered the muscle fibers more susceptible to ischemia. Second, because of the larger diameter of the distal fibers in normal and stimulated latissimus dorsi muscle, the diffusion distance for oxygen to the center of the distal fibers is increased, making fiber survival more difficult. Third, even though angiogenesis was significantly increased in the flap, cutting all but the single vascular pedicle resulted in the newly formed capillaries not receiving enough blood to provide nourishment to the distal latissimus dorsi muscle. The authors' findings indicate that chronic electrical stimulation as tested in these experiments could not be used to prevent distal latissimus dorsi muscle flap ischemia and necrosis in cardiomyoplasty.     

Ischemic preconditioning by brief extremity ischemia before flap ischemia in a rat model

Ischemic preconditioning is a protective endogenous mechanism to reduce ischemia/reperfusion injury and is defined as a brief period of ischemia the authors term "preclamping." This is followed by tissue reperfusion and is believed to increase the ischemic tolerance. The objective of this study was to determine whether acute remote ischemic preconditioning, which has been reported to be successful for other organs, such as the heart, kidney, intestine, and liver, will also result in an enhancement of survival in flaps, and whether remote ischemic preconditioning is as effective as preclamping. Forty male Wistar rats were divided into four experimental groups. An extended epigastric adipocutaneous flap (6 x 10 cm) was raised, based on the left superficial epigastric artery and vein. In the control group, a 3-hour flap ischemia was induced. In the preclamping group, a brief ischemia of 10 minutes was induced by clamping the flap pedicle, followed by 30 minutes of reperfusion. Ischemia of the right hind limb was induced in the femoral ischemia group by clamping the femoral artery and vein for 10 minutes after flap elevation. The limb was then reperfused for 30 minutes. Thereafter, flap ischemia was induced as in the control group. A similar protocol was used in the tourniquet group. A tourniquet was used to induce hind-limb ischemia. The experiment was then performed as in the femoral ischemia group. Mean flap necrosis area was assessed for all groups on the fifth postoperative day using planimetry software. Average flap necrosis area was 68.2 +/- 18.1 percent in the control group, 11 +/- 8.38 percent in the preclamping group, 12.5 +/- 5.83 percent in the femoral ischemia group, and 24 +/- 11.75 percent in the tourniquet group. All preconditioned animals demonstrated a significantly lower area of flap necrosis than the control group (p < 0.001, one-way analysis of variance, post hoc Tukey's test). The data show that ischemic preconditioning and enhancement of flap survival can be achieved not only by preclamping of the flap pedicle but also by induction of an ischemia/reperfusion event in a body area distant from the flap before harvest. These findings indicate that remote ischemic preconditioning is a systemic phenomenon, leading to an enhancement of flap survival. The exact mechanism is not yet completely understood. The data suggest that remote ischemic preconditioning could be performed simultaneously with flap harvest in the clinical setting, resulting in an improved flap survival without prolongation of the operation. This may decrease the rate of partial flap loss or fat necrosis, especially in high-risk groups such as smokers, those with irradiated tissues, and obese patients.     

Microvascular remodeling and accelerated hyperemia blood flow restoration in arterially occluded skeletal muscle exposed to ultrasonic microbubble destruction

We showed previously that microbubble destruction with pulsed 1-MHz ultrasound creates a bioeffect that stimulates arteriogenesis and a chronic increase in hyperemia blood flow in normal rat muscle. Here we tested whether ultrasonic microbubble destruction can be used to create a microvascular remodeling response that restores hyperemia blood flow to rat skeletal muscle affected by arterial occlusion. Pulsed ultrasound (1 MHz) was applied to gracilis muscles in which the lateral feed artery was occluded but the medial feed artery was left intact. Control muscles were similarly occluded but did not receive ultrasound, microbubbles, or both. Hyperemia blood flow and number of smooth muscle (SM) alpha-actin-positive vessels, >30-mum arterioles, and capillaries per fiber were determined 7, 14, and 28 days after treatment. In ultrasound-microbubble-treated muscles, lateral region hyperemia blood flow was increased at all time points and restored to normal at day 28. The number of SM alpha-actin vessels per fiber was increased over control in this region at days 7 and 14 but decreased by day 28, when larger-diameter arterioles became more prevalent in the medial region. The number of capillaries per fiber was increased over control only at day 7 in the lateral region and only at days 7 and 14 in the medial region, indicating that the angiogenesis response was transient and likely did not contribute significantly to flow restoration at day 28. We conclude that ultrasonic microbubble destruction can be tailored to stimulate an arteriogenesis response that restores hyperemia blood flow to skeletal muscle in a rat model of arterial occlusion.     

Chemically induced vasospasm: the effect of ischemia, vessel occlusion, and adrenergic blockade

Vasospasm in revascularized tissue can compromise tissue perfusion even though the microsurgical anastomoses remain patent. Circulating catecholamine stimulates peripheral vasoconstriction. Chemical vasospasm was induced by the intraarterial administration of norepinephrine to denervated rat hind limbs. Heel pad blood flow was assessed by laser-Doppler velocimetry. Mean blood flow was 463 +/- 106 in the denervated leg and 337 +/- 50 in the opposite (intact) leg (p less than 0.01). Flow in the denervated leg decreased 78 percent (463 +/- 106 to 100 +/- 23) within 5 minutes of norepinephrine administration and did not return to normal for 30 minutes. Norepinephrine administration in the presence of 1 and 3 hours of ischemia decreased flow at 5 minutes to 6.6 and 6.0 percent of normal, respectively (31 +/- 14, 28 +/- 14, control 100 +/- 23; p less than 0.001). Administration of intraarterial norepinephrine distal to a femoral artery occluded for 1 and 5 minutes reduced flow following clamp release to 11.2 and 7.1 percent of normal 5 minutes after clamp release (52 +/- 9, 33 +/- 7, control 100 +/- 23; p less than 0.001). Comparison of the 1-minute and 5-minute groups to each other showed a significant flow decrease in the 5-minute group (p less than 0.007). This indicates that the observed decrease in flow was related both to the presence of a vessel occlusion and to the length of the occlusion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Blockade of platelet endothelial cell adhesion molecule-1 (PECAM-1) protects against ischemia-reperfusion injury in muscle flaps at microcirculatory level

Several lines of evidence show that platelet endothelial cell adhesion molecule-1 (PECAM-1), a component of endothelial cell junctions, is required for leukocyte transmigration through endothelial cell monolayers. Polymorphonuclear leukocytes play an important role in ischemia-reperfusion injury. We sought to determine whether administering an anti-PECAM-1 antibody would prevent or attenuate ischemia-reperfusion injury in a rat cremaster muscle flap injury model. Eighteen male Sprague-Dawley rats were divided into three groups. Group I (control): Cremaster muscle island flaps were dissected for baseline measurements of eight indicators: numbers of rolling, sticking, and transmigrating neutrophils, numbers of rolling and sticking lymphocytes, number of perfused capillaries, endothelial edema, and vessel permeability. Group II: The prepared cremaster flap was subjected to 4 hours of ischemia and 24 hours of reperfusion. Group III: The muscle flap was subjected to ischemia and reperfusion as in group II, and anti-PECAM-1 antibodies (1 mg/kg) were injected subcutaneously 15 minutes before reperfusion. Blood vessels were observed in vivo under an intravital microscopy system. Microvascular permeability was made visible with injected fluorescein isothiocyanate-labeled albumin and evaluated with Kontron Elektronik computer software. The ischemia-reperfusion-alone group (group II) presented a 225-percent increase in the activation of sticking leukocytes (2.4 +/- 0.4 to 7.8 +/- 0.8, p < 0.05) (p < 0.01). This leukocyte activation was reduced by 83 percent following anti-PECAM-1 monoclonal antibody treatment (1.3 +/- 0.5 per 100 microm) (p < 0.01). At 24 hours, endothelial injury in group II was confirmed by a 4-fold increase in the number of transmigrating leukocytes into the interstitial space (7.6 +/- 1.2 per field versus 1.9 +/- 0.4 per field in controls). This phenomenon was reduced by 85 percent following anti-PECAM-1 monoclonal antibody treatment (1.1 +/- 0.2 per field) (p < 0.01). Analysis showed that the number of flowing capillaries was 67 percent lower in group II (6.8 +/- 0.3 to 2.2 +/- 0.7, p < 0.01). Anti-PECAM-1 antibody treatment caused a 2.5-fold increase in this number (5.6 +/- 0.5, p < 0.01). Microcirculatory permeability index showed a 180-percent increase in group II (p < 0.05) when compared with baseline values. This increased albumin leakage was effectively attenuated by antibody treatment (p < 0.05). Blocking the action of PECAM-1 in vivo by administering monoclonal antibodies significantly attenuated ischemia-reperfusion injury, presumably by inhibiting transendothelial migration of neutrophils and by increasing capillary perfusion at a muscle flap microcirculatory level.