Mortality following Lower Limb Ischemia-Reperfusion: A Systemic Inflammatory Response?

n = 10); and (2) animals subjected to 3 hours of bilateral hind limb ischemia followed by reperfusion ( n = 10). Both groups were observed under standard conditions for 4 days. In a second experiment three groups of animals were studied: (I) control ( n = 12); (II) 3 hours of bilateral hind limb ischemia alone ( n = 12); and (III) 3 hours of bilateral hind limb ischemia followed by 2 hours of reperfusion ( n = 12). Animals subjected to bilateral hind limb ischemia followed by reperfusion had a significantly higher mortality rate (70%) than controls (0%) ( p < 0.005). Morphometric assessment of the small bowel showed a significant decrease in mean mucosal thickness in the ischemia-reperfusion group compared with that in the group of controls and the ischemia-alone group ( p < 0.05). Bilateral hind limb ischemia followed by reperfusion was associated with significantly increased plasma concentrations of endotoxin ( p < 0.05) and interleukin-6 ( p < 0.0001) compared with that of controls and ischemia alone. These results indicate that reperfusion of the acutely ischemic lower limb is accompanied by structural changes in the gut mucosa associated with increased systemic endotoxin concentrations and cytokine activation. Mortality following reperfusion of the acutely ischemic limb may be related to a systemic inflammatory response triggered by endotoxin of gut origin.     

Ischemic preconditioning before lower limb ischemia--reperfusion protects against acute lung injury

OBJECTIVE: Prolonged limb ischemia followed by reperfusion (I/R) is associated with a systemic inflammatory response syndrome and remote acute lung injury. Ischemic preconditioning (IPC), achieved with repeated brief periods of I/R before the prolonged ischemic period, has been shown to protect skeletal muscle against ischemic injury. The aim of this study was to ascertain whether IPC of the limb before I/R injury also attenuates systemic inflammation and acute lung injury in a fully resuscitated porcine model of hind limb I/R. METHODS: This prospective, randomized, controlled, experimental animal study was performed in a university-based animal research facility with 18 male Landrace pigs that weighed from 30 to 35 kg. Anesthetized ventilated swine were randomized (n = 6 per group) to three groups: sham-operated control group, I/R group (2 hours of bilateral hind limb ischemia and 2.5 hours of reperfusion), and IPC group (three cycles of 5 minutes of ischemia/5 minutes of reperfusion immediately preceding I/R). Plasma was separated and stored at -70 degrees C for later determination of plasma tumor necrosis factor-alpha and interleukin-6 with bioassay as markers of systemic inflammation. Circulating phagocytic cell priming was assessed with a whole blood chemiluminescence assay. Lung tissue wet-to-dry weight ratio and myeloperoxidase concentration were markers of edema and neutrophil sequestration, respectively. The alveolar-arterial oxygen gradient and pulmonary artery pressure were indices of lung function. RESULTS: In a porcine model, bilateral hind limb (I/R) injury significantly increased plasma interleukin-6 concentrations, circulating phagocytic cell priming, and pulmonary leukosequestration, edema, and impaired gas exchange. Conversely, pigs treated with IPC before the onset of the ischemic period had significantly reduced interleukin-6 levels, circulating phagocytic cell priming, and experienced significantly less pulmonary edema, leukosequestration, and respiratory failure. CONCLUSION: Lower limb IPC protects against systemic inflammation and acute lung injury in lower limb I/R injury.     

Thromboxane mediates the ischemia-induced neutrophil oxidative burst

Indirect evidence exists that the reperfusion of ischemic tissue activates white blood cells. Thus local and systemic reperfusion injuries are prevented by making animals leukopenic or by inhibiting white blood cell lung entrapment by blocking thromboxane A2 generation. This study tests directly whether ischemia and reperfusion activates neutrophils, as measured by their oxidative burst, and whether thromboxane mediates this event. Anesthetized rats underwent 4 hours of bilateral hind limb tourniquet ischemia followed by 60 minutes of reperfusion. Plasma thromboxane B2 levels increased to 2750 pg/ml at 5 minutes of reperfusion, higher than the sham control (n = 36) value of 370 pg/ml (p less than 0.01). In untreated ischemic animals (n = 30) the intracellular H2O2 production of circulating neutrophils, as assayed flow cytometrically by dichlorofluorescein oxidation, increased from a preischemic value of 133 to a peak of 251 femtomoles dichlorofluorescein/neutrophil at 5 minutes of reperfusion (p less than 0.01). Treatment of neutrophils with phorbol myristate acetate (PMA) 10(-7) mol/L led to a 91% increase in neutrophil H2O2 production before ischemia, and 5 minutes after reperfusion there was an enhanced response to PMA of 222% (p less than 0.01). Pretreatment of animals with the thromboxane-synthetase inhibitor OKY 046 (n = 36) prevented ischemia-induced thromboxane generation, neutrophil H2O2 production (p less than 0.05), as well as the enhanced response to PMA stimulation (p less than 0.05). Treatment with the thromboxane-receptor antagonist SQ 29,548 (n = 36) did not affect the increase in plasma thromboxane levels after ischemia but was as effective as OKY 046 in preventing the ischemia-induced increase in neutrophil H2O2 production and the enhanced response to PMA stimulation. These data indicate that lower-torso ischemia leads to neutrophil activation, manifest by H2O2 production, an event mediated by thromboxane.     

Recombinant bactericidal/permeability-increasing protein attenuates the systemic inflammatory response syndrome in lower limb ischemia-reperfusion injury

OBJECTIVES: Hind limb ischemia-reperfusion (I/R) injury increases gut permeability, and resultant endotoxemia is associated with an amplified systemic inflammatory response syndrome leading to multiple organ dysfunction syndrome. We studied the potential role of recombinant bactericidal/permeability-increasing protein (rBPI(21) ), a novel antiendotoxin therapy, in modulating endotoxin-enhanced systemic inflammatory response syndrome in hind limb I/R injury. METHODS: In this prospective, randomized, controlled, experimental animal study, 48 male Wistar rats, weighing 300 to 350 g, were randomized to a control group (sham) and five groups undergoing 3 hours bilateral hind limb ischemia with 2 hours reperfusion (I/R) (n = 8 per group). The control and untreated I/R groups received thaumatin, a control-protein preparation, at 2 mg/kg. Treatment groups were administered rBPI(21) intravenously at 1, 2, or 4 mg/kg body weight at the beginning of reperfusion; an additional group was administered rBPI(21) intravenously at 2 mg/kg after 1 hour of reperfusion. Plasma interleukin-6 concentration was estimated by bioassay as a measure of systemic inflammation. Plasma endotoxin concentration was determined by use of an amebocyte lysate chromogenic assay. Crossreactive immunoglobulin G and M antibodies to the highly conserved inner core region of endotoxin were measured by use of an enzyme-linked immunosorbent assay. The lung tissue wet-to-dry weight ratio and myeloperoxidase concentration were used as markers of edema and neutrophil sequestration, respectively. RESULTS: I/R provoked highly significant elevation in plasma interleukin-6 concentrations (1351.20 pg/mL [860.16 - 1886.40 pg/mL]) compared with controls (125.32 pg/mL [87.76-157.52 pg/mL; P <.0001]), but treatment with rBPI(21) 2 mg/kg at onset of reperfusion (715.89 pg/mL [573.36-847.76 pg/mL]) significantly decreased interleukin-6 response compared with the nontreatment group ( P <.016). I/R increased plasma endotoxin concentrations significantly (21.52 pg/mL [6.20-48.23 pg/mL]), compared with control animals (0.90 pg/mL [0.00-2.30 pg/mL; P <.0001]), and treatment with rBPI(21) 4 mg/kg at reperfusion significantly decreased endotoxemia (1.30 pg/mL [1.20-2.20 pg/mL]), compared with the untreated group ( P <.001). The lung tissue myeloperoxidase level was significantly increased in the untreated I/R group (208.18% [128.79%-221.81%]), compared with in controls (62.00% [40.45%-80.92%; P <.0001]), and attenuated in those treated with rBPI(21) 2 mg/kg (129.54% [90.49%-145.78%; P <.05]). Data represent median and interquartile range, comparisons made with the nonparametric Mann-Whitney U test. CONCLUSIONS: These findings show that hind limb ischemia-reperfusion injury is associated with endotoxemia, elevations in plasma interleukin-6, and pulmonary leukosequestration. Treatment with rBPI(21) after ischemia reduces endotoxemia, the interleukin-6 response, and attenuates pulmonary leukosequestration in response to hind limb reperfusion injury.     

Dichloroacetate increases skeletal muscle pyruvate dehydrogenase activity during acute limb ischemia

The purpose of this study was to evaluate the effects of dichloroacetate sodium (DCA), a drug that inactivates pyruvate dehydrogenase kinase (PDH-K), on pyruvate dehydrogenase (PDH) activity, lactate level, and function of skeletal muscle in an experimental model of acute limb ischemia. Thirty-two male Sprague-Dawley rats underwent right iliac artery ligation to produce hindlimb ischemia. After 2 hours of ischemia, 16 animals received intravenous DCA (15 mg/100 g body weight) and 16 control animals received an equivalent volume of normal saline. After an additional 1 hour of ischemia (total 3 hours) tibialis anterior muscle from the ischemic limb and contralateral nonischemic limb was excised, rapidly freeze-clamped with Wallenberg tongs cooled in liquid nitrogen, and stored at -70 degrees C. Muscles specimens were subsequently assayed for PDH activity and lactate level by use of spectrophotometric techniques. An additional 16 animals (DCA-treated, n = 8; control, n = 8) underwent ex-vivo gastrocnemius muscle fatigue testing with a 10 g tension preload after 3 hours of limb ischemia. In ischemic hind limbs, DCA treatment significantly (p = 0.025) increased PDH activity (19.6 +/-1.6 micromol/min/g dry weight) compared to controls (13.1 +/-1.3 micromol/min/g dry weight). DCA treatment did not increase (p = 0.13) skeletal muscle PDH activity in the nonischemic limbs (9.6 +/-1.1 micromol/min/g dry weight, controls; 13.2 +/-1.3 micromol/min/g dry weight, DCA group). In DCA-treated animals, hind limb ischemia resulted in no significant increase in muscle lactate levels compared to the nonischemic limb, while control animals demonstrated a significant (p = 0.005) elevation in lactate level in ischemic limbs compared to contralateral nonischemic limb. Ischemia induced a significant decrease in time to muscle fatigue in both DCA-treated and control animals (p = 0.002 and 0.001, respectively). Time to muscle fatigue in DCA-treated animals was increased compared to controls (2.6 +/-0.3 versus 2 +/-0.6 minutes; p < 0.05)in ischemic limbs but was not significantly different in nonischemic limbs (DCA = 3.3 +/-0.5 minutes; control = 3.1 +/-0.6 minutes). Treatment with DCA during acute limb ischemia reduced the depression of PDH activity and lactate level of skeletal muscle. Ischemic muscle function was also improved by DCA treatment. Further investigation of the potential beneficial effects of DCA treatment on muscle injury during ischemia and reperfusion is warranted.     

Reperfusion of ischemic lower limbs increases pulmonary microvascular permeability

Lower torso ischemia and reperfusion has been shown to stimulate the generation of thromboxane (Tx)A2, leading to transient pulmonary hypertension and later to polymorphonuclear leukocyte accumulations in the lungs. This study investigated whether hind limb ischemia leads to increased pulmonary microvascular permeability. Anesthetized sheep (n = 6) previously prepared with a lung lymph fistula underwent 2 hr of tourniquet ischemia of both lower limbs. One minute following tourniquet release mean pulmonary arterial pressure (MPAP) rose from 14 +/- 1 to 36 +/- 4 mm Hg (p less than 0.05) and returned to baseline within 30 min. The pulmonary arterial wedge pressure of 4 +/- 1 mm Hg was unchanged. Plasma TxB2 levels rose from 211 +/- 21 to 304 +/- 52 pg/ml (p less than 0.05) 10 min after tourniquet release and were back to baseline at 30 min. Lymph flow (QL) rose from 4.3 +/- 0.6 ml/30 min to 8.3 +/- 1.8 ml/30 min (p less than 0.05); the lymph/plasma (L/P) protein ratio rose slightly but not significantly. In three sheep, inflation of a left atrial balloon increased left atrial pressure from 3 to 16 mm Hg. MPAP rose from 14 to 24 mm Hg. There was an increase in QL from 3.6 to 17 ml/30 min; the L/P protein ratio declined from 0.63 to 0.41. These results indicate that reperfusion following 2 hr of bilateral hind limb ischemia results in increased pulmonary microvascular permeability.     

Blockade of complement activation prevents local and pulmonary albumin leak after lower torso ischemia-reperfusion.

Lower torso ischemia and reperfusion leads to both local and remote tissue injuries. The purpose of this study was to assess the role of complement in mediating the local and remote microvascular permeability after bilateral hind limb tourniquet ischemia. Four hours of ischemia and 4 hours of reperfusion produced an increased skeletal muscle permeability index (muscle/blood 125I albumin ratio) of 2.90 +/- 0.35 compared with the index in nonischemic muscle of 0.25 +/- 0.02 (p < 0.01). Muscle wet-to-dry-weight ratio increased from 3.93 +/- 0.04 in sham to 5.55 +/- 0.09 in ischemic muscle (p < 0.0001). Lung permeability rose at 4 hours as indicated by the increased bronchoalveolar lavage (BAL)/blood 125I albumin ratio 4.36 +/- 0.41 x 10(-3) versus sham 2.64 +/- 0.28 x 10(-3) (p < 0.05) and neutrophil sequestration 0.28 +/- 0.02 U/g myeloperoxidase (MPO) versus sham 0.14 +/- 0.02 U/g (p < 0.001). Serum lytic activity of the classical but not the alternate complement pathway was reduced. The soluble complement receptor (sCR1) was used to inhibit complement activity and attenuated the increase in the permeability index after reperfusion in ischemic muscle 1.11 +/- 0.08 (p < 0.01) and reduced the lung BAL/blood 125I albumin ratio to sham levels 2.46 +/- 0.39 x 10(-3) (p < 0.05) at 6 mg/animal, without reducing the lung neutrophil sequestration, 0.24 +/- 0.02 U/g. The authors conclude that complement activation occurred during tourniquet ischemia and mediated permeability changes in the ischemic muscle and the lungs during reperfusion.     

Does iloprost mediate thromboxane activity and polymorphonuclear leukocyte sequestration in ischemic skeletal muscle?

Thromboxane is known to alter the endothelial cytoskeleton, thereby causing increased endothelial permeability and polymorphonuclear leukocyte (PMN) sequestration in the lungs. We investigated whether iloprost (a stable prostacyclin analog) can decrease thromboxane activity and consequently PMN sequestration because of its anti-platelet aggregation effect. This premise was investigated in a canine isolated gracilis muscle model using 18 animals. Six animals (group I) had the gracilis muscle subjected to 6 hours of complete ischemia followed by 48 hours of reperfusion. Group II (n = 6) received intravenous infusion of iloprost (0.45 micrograms/kg/hr) throughout the experiment (1 hour preischemia, 6 hours of ischemia and 1 hour of reperfusion) and boluses of 0.45 micrograms/kg 10 minutes before ischemia and reperfusion. Group III (n = 6) underwent a similar ischemic interval, but were given iloprost bolus of 0.45 micrograms/kg followed by intravenous infusion of 0.45 micrograms/kg/hr during 48 hours of reperfusion. Gracilis venous samples were obtained at preischemia (PI) and 1 hour of reperfusion (all 3 groups) and at 48 hours of reperfusion (groups I and III) to measure thromboxane (TXB2) levels. Muscle biopsies were taken at the same time to measure myeloperoxidase (MPO) activity, a marker of PMN infiltration. In group I, TXB2 level increased from a pre-ischemic value of 2983 +/- 1083 pg/ml to 9483 +/- 2218 pg/ml at 1 hour of reperfusion (p < 0.05) and then decreased to 2386 +/- 1533 pg/ml at 48 hours of reperfusion (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Ischemic preconditioning before lower limb ischemia—reperfusion protects against acute lung injury

Objective: Prolonged limb ischemia followed by reperfusion (I/R) is associated with a systemic inflammatory response syndrome and remote acute lung injury. Ischemic preconditioning (IPC), achieved with repeated brief periods of I/R before the prolonged ischemic period, has been shown to protect skeletal muscle against ischemic injury. The aim of this study was to ascertain whether IPC of the limb before I/R injury also attenuates systemic inflammation and acute lung injury in a fully resuscitated porcine model of hind limb I/R. Methods: This prospective, randomized, controlled, experimental animal study was performed in a university-based animal research facility with 18 male Landrace pigs that weighed from 30 to 35 kg. Anesthetized ventilated swine were randomized (n = 6 per group) to three groups: sham-operated control group, I/R group (2 hours of bilateral hind limb ischemia and 2.5 hours of reperfusion), and IPC group (three cycles of 5 minutes of ischemia/5 minutes of reperfusion immediately preceding I/R). Plasma was separated and stored at −70° C for later determination of plasma tumor necrosis factor-α and interleukin-6 with bioassay as markers of systemic inflammation. Circulating phagocytic cell priming was assessed with a whole blood chemiluminescence assay. Lung tissue wet-to-dry weight ratio and myeloperoxidase concentration were markers of edema and neutrophil sequestration, respectively. The alveolar-arterial oxygen gradient and pulmonary artery pressure were indices of lung function. Results: In a porcine model, bilateral hind limb (I/R) injury significantly increased plasma interleukin-6 concentrations, circulating phagocytic cell priming, and pulmonary leukosequestration, edema, and impaired gas exchange. Conversely, pigs treated with IPC before the onset of the ischemic period had significantly reduced interleukin-6 levels, circulating phagocytic cell priming, and experienced significantly less pulmonary edema, leukosequestration, and respiratory failure. Conclusion: Lower limb IPC protects against systemic inflammation and acute lung injury in lower limb I/R injury. (J Vasc Surg 2002;35:1264-73.)     

Adenosine analogue reduces spinal cord reperfusion injury in a time-dependent fashion

BACKGROUND: We hypothesized that inflammation during spinal cord reperfusion worsens ischemic injury. ATL-146e, an adenosine A(2A) agonist with known anti-inflammatory properties, was used to test this hypothesis at varied intervals to determine the time course of reperfusion injury. METHODS: Forty rabbits underwent cross-clamping of the infrarenal aorta for 45 minutes. One group (n = 14 animals) received 0.06 microg/kg/min systemic ATL-146e over 3 hours, beginning after 30 minutes of ischemic time. A second group (n = 6 animals) received ATL-146e over 1.5 hours. A third group (n = 3 animals) received ATL-146e over 1 hour, and a fourth group (n = 17 animals) received saline solution. All animals were assessed at 48 hours for hind limb motor function (Tarlov scale, 0-5). RESULTS: Animals that received ATL-146e for 3 hours (Tarlov score, 4.3 +/- 0.22; P <.001) or 1.5 hours (Tarlov score, 2.7 +/- 0.6; P <.05) had improved neurologic outcomes compared with rabbits that received saline solution (Tarlov score, 0.6 +/- 0.29). Animals that received ATL-146e for 1 hour (Tarlov score, 0.7 +/- 0.8) were not significantly different from those animals that received saline solution. CONCLUSIONS: Systemic ATL-146e, given during reperfusion, results in time-dependent improvement in spinal cord function after ischemia. This implies that the mechanism of spinal reperfusion injury includes leukocyte-mediated inflammation at a critical post-ischemic time interval.     

Thromboxane A2 moderates permeability after limb ischemia.

Reperfusion after limb ischemia results in muscle edema as well as excess secretion of thromboxane A2 (TxA2), an agent associated with permeability increase in other settings. This study tests whether TxA2 moderates the permeability following limb ischemia. A tourniquet inflated to 300 mmHg was applied for 2 hours around the hind limb of four groups of dogs. In untreated animals (N = 25), 2 hours following tourniquet release, plasma TxB2 values rose from 320 pg/ml to 2416 pg/ml (p less than 0.001), and popliteal lymph values rose from 378 pg/ml to 1046 pg/ml (p less than 0.001). Platelet TxB2 was unaltered and plasma 6-keto-PGF1 alpha levels did not vary. Following ischemia, lymph flow (QL) increased from 0.07 to 0.37 ml/h (p less than 0.05), while the lymph/plasma (L/P) protein ratio was unchanged at 0.41. These measurements indicate increased permeability since increase in hydrostatic pressure in a second group by tourniquet inflation to 50 mmHg (N = 7) led to a rise in QL from 0.07 to 0.22 ml/h, but a fall in the L/P ratio to 0.32, a value lower than the ischemic group (p less than 0.05). Pretreatment with the imidazole derivative ketoconazole (N = 11) reduced platelet Tx synthesis from 42 ng to 2 ng/10(9) platelets, but lymph TxB2 levels rose to 1703 pg/ml after ischemia, indicating an extravascular or vessel wall site of synthesis not inhibited by ketoconazole. Pretreatment with a lower molecular weight imidazole derivative OKY 046 (N = 9) inhibited all Tx synthesis after ischemia. Prior to tourniquet inflation, both OKY 046 and ketoconazole lowered plasma TxB2 levels as well as the L/P ratio (p less than 0.05). After ischemia, OKY 046, but not ketoconazole, maintained the L/P ratio at 0.33, a value below that of untreated animals (p less than 0.05). These results indicate that nonplatelet-derived TxA2 modulates both baseline and ischemia-induced increases in microvascular permeability in the dog hind limb.     

Oxygen free radicals are required for ischemia-induced leukotriene B4 synthesis and diapedesis.

Hind limb ischemia and reperfusion have been shown to result in high plasma levels of leukotriene B4 (LTB4) and polymorphonuclear neutrophil (PMN) sequestration in the pulmonary microvasculature. This study tests whether LTB4 is derived from PMNs and its role in mediating ischemic plasma-induced diapedesis. Plasma derived from rabbit hind limbs after 3 hours of tourniquet ischemia and 10 minutes of reperfusion (n = 6) showed an increased LTB4 level of 560 pg/ml, higher than sham plasma values of 106 pg/ml (p less than 0.05). Introduction of ischemic plasma in abraded skin chambers placed on the dorsum of normal rabbits (n = 6) led after 3 hours to PMN diapedesis of 1175 PMN/mm3, associated with a further increase in LTB4 levels to 820 pg/ml (both p less than 0.05). In contrast, ischemic plasma derived from neutropenic animals (n = 4; nitrogen mustard, 2 mg/kg; PMNs less than 30/mm3) contained lower levels of LTB4, 160 pg/ml (p less than 0.05). When introduced in skin chambers in normal rabbits (n = 4), this plasma induced accumulations of only 163 PMN/mm3, accompanied by a smaller increase in LTB4 levels in the blister fluid after 3 hours, 397 pg/ml (both p less than 0.05). A correlation was found between LTB4 levels in ischemic plasma and PMN accumulations in blister fluid (r = 0.92; p less than 0.05). Intravenous pretreatment of rabbits (n = 4) used in the blister chamber bioassay with the LT receptor antagonist FPL-55712, 40 micrograms/kg/hr, attenuated diapedesis induced by ischemic and ischemic-neutropenic plasma, 103 and 35 PMN/mm3, respectively (both p less than 0.05). Pretreatment with superoxide dismutase, 1500 units/kg, and catalase, 5000 units/kg, both conjugated to polyethylene glycol (n = 4), prevented ischemic plasma-induced LTB4 synthesis, as well as ischemic plasma-induced diapedesis, 12 PMN/mm3 (p less than 0.05). Finally, pretreatment with allopurinol, 25 mg/kg, was similarly effective in preventing LTB4 synthesis and PMN migration. These data suggest that oxygen free radicals are essential for ischemia-induced PMN synthesis of LTB4 that in turn mediates their diapedesis.     

Thromboxane release after reperfusion of chronically ischemic limbs in patients

We hypothesized that chronic ischemia of peripheral vascular disease would lead to increased thromboxane A2 (TxA2) and decreased prostacyclin (PGI2) production and surgical correction of the ischemia would stabilize TxA2 and PGI2 at normal levels. TxA2 and PGI2 concentrations were determined in 22 patients before, during, and after arterial reconstruction for limb salvage and in 10 control subjects. Control samples and preoperative patient samples had no detectable TxA2 or PGI2 (less than 26 pg/ml). Five minutes after reperfusion TxA2 increased (TxA2 = 76.27 +/- 48.9 pg/ml, mean +/- SEM) and persisted at 1 day (TxA2 = 190.1 +/- 80.1 pg/ml), 2 days (TxA2 = 224.7 +/- 131.7 pg/ml), 5 days (TxA2 = 334.8 +/- 272.8 pg/ml), and 7 days postoperatively (TxA2 = 256.6 +/- 149.0 pg/ml). Elevated TxA2 production was not associated with chronic ischemia of peripheral vascular disease. Reperfusion of the severely ischemic limb caused significant TxA2 release.     

Do cytokines play a role in skeletal muscle ischemia and reperfusion?

Cytokines, interleukin-1 (IL-1) and tumor necrosis factor (TNF) are known to mediate host cell response to sepsis, trauma, and myocardial ischemia. We have previously found increased levels of IL-1 in the venous effluent during the reperfusion phase of skeletal muscle ischemia in a canine model. This study was done to evaluate whether TNF also played a role in skeletal muscle ischemia-reperfusion injury since IL-1 and TNF have inter-related functions. In twelve adult mongrel dogs (28-32 kg) one gracilis muscle was subjected to six hours of normothermic ischemia followed by normothermic reperfusion. The contralateral side served as a control and remained normally perfused throughout the experiment. Gracilis venous samples were collected at pre-ischemia and one hour of reperfusion. Systemic (arterial) blood samples were taken simultaneously with the venous samples at one hour of reperfusion. At the end of the experiment the muscles were harvested and amount of necrosis quantitated by serial transections, nitroblue tetrazolium staining and computerized planimetry. Muscle necrosis on the experimental side was found to be 48.86 +/- 5.37%. Sera were analyzed for TNF activity using a bioassay. TNF levels in the gracilis venous effluent at one hour of reperfusion were not significantly different from the simultaneous systemic (arterial) levels (27.15 +/- 5.05 pg/ml vs 18.23 +/- 4.27 pg/ml). Pre-ischemic levels of TNF were 96.50 +/- 20.12 pg/ml, which was significantly higher than either venous or arterial levels obtained after one hour of reperfusion (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Ischemia activates neutrophils but inhibits their local and remote diapedesis.

Hindlimb ischemia and reperfusion results in local limb and distant lung injury. This study tests whether the mechanism of injury is by ischemia mediated polymorphonuclear leukocyte (PMN) activation and diapedesis. Anesthetized rabbits were subjected to three hours of hindlimb ischemia (n = 8) or sham ischemia (n = 4). PMN derived solely from the reperfused ischemic limb and assayed flow cytometrically displayed an oxidative burst of 135 /- 8 fentamoles dichlorofluorescein (fmDCF)/cell compared to preischemc levels of 74 +/- 14 fmDCF/cell (p less than 0.05). Additional aliquots of isolated neutrophils were treated with phorbol myristate acetate (PMA) 10(-7) M. In contrast to a 162% increase in oxidative burst before ischemia, neutrophils at ten minutes of reperfusion had an enhanced response to PMA of 336% (p less than 0.05). Plasma collected from the ischemic hindlimb at ten minutes of reperfusion when introduced into an abraded skin chamber or intratracheally induced diapedesis in nonischemic animals. PMN accumulations in the skin chamber were 1636 +/- 258 PMN/mm3 after three hours (n = 8) compared to 63 +/- 18 PMN/mm3 induced by sham plasma (n = 4, p less than 0.05). Introduction of ischemic plasma intratracheally into a lobar bronchus (n = 4) induced PMN accumulations after three hours, measured by bronchoalveolar lavage fluid of 19 +/- 2 X 10(4) PMN/mm3 compared to 5 +/- 1 X 10(4) PMN/mm3 with sham plasma (n = 4, p less than 0.05). Diapedesis was completely prevented (0-3 PMN/mm3, p less than 0.05) by introducing ischemic plasma into skin chambers in animals whose hindlimbs had been made ischemic (n = 6) or into chambers located on skin regions that had been previously made ischemic (n = 6). Similarly after hindlimb ischemia, lavage of the lung with ischemic plasma yielded few PMN 0-3/mm3 (p less than 0.05). These data indicate that ischemia and reperfusion lead to generation of a circulating component in plasma that causes an oxidative burst in PMN and inhibits their diapedesis but promotes diapedesis when applied extravascularly to a naive animal.     

Cytokine responses in patients undergoing coronary artery bypass surgery after ischemic preconditioning

OBJECTIVE: The release of proinflammatory cytokines has been shown to be associated with the development of complications after coronary artery bypass grafting with cardiopulmonary bypass. The purpose of the present study was to establish whether ischemic preconditioning (IP) could limit inflammatory cytokines release in patients undergoing elective coronary artery bypass surgery. METHODS: Twenty-two patients with multiple-vessel coronary artery disease and stable angina admitted for first-time elective coronary artery bypass surgery were randomized into control or ischemic preconditioning groups. Patients in the IP group were exposed to two cycles of two-minute myocardial ischemia, followed by three minutes of reperfusion, at the beginning of the revascularization operation, before the cross-clamping and ischemic period used for coronary artery bypass graft anastomosis. Peripheral plasma levels of TNF-alpha, IL-6, IL-8 and IL-10 were measured perioperatively. RESULTS: Significant elevation of IL-6, IL-8 and IL-10 were observed in both groups after reperfusion. Ischemic preconditioning has no effect on cytokine release in the early stage after reperfusion. Arterial blood IL-6 levels in the preconditioning group were significantly lower than in controls at 20 h after declamping (52.93 +/- 9.79 vs 96.04 +/- 17.56 pg/ml, p < 0.05). CONCLUSIONS: The results indicate that ischemic preconditioning results in no effect on systemic inflammatory cytokine release in the early stage but a delayed reduction in IL-6 levels at 20 h after reperfusion.     

Microcirculatory window for early detection of allograft rejection

The purpose of this study is to introduce a technical detail on a transplantation model for in vivo evaluation of microcirculatory changes during the acute phase of allograft rejection. The cremaster muscle is incorporated and transplanted along with the hind limb to detect and study ischemia/reperfusion injury and the acute phase of allograft rejection in rats. Thirty-six animals were studied in three experimental groups of 12 animals each. Each group was divided into subgroups and microcirculatory measurements were taken at two different time periods: 24 and 72 hours. In the ischemic control group (N = 12), cremaster muscles were denervated, prepared as a tube flap, and submitted to the same interval of ischemia as the other groups but without transplantation. In the isograft group (N = 12), rat hind limb-cremaster grafts were transplanted between genetically identical Lewis rats (RT11). In the allograft group (N = 12), 12 transplantations were performed across a major histocompatibility barrier between Lewis Brown-Norway (RT-11+/-n) and Lewis (RT 11) rats. The diameters of first-, second-, and third-order arterioles and venules; red blood cell velocities; and functional capillary density were recorded at 24 and 72 hours after transplantation. Daily follow-up observations were continued until 3 days after the first clinical signs of graft rejection. The mean number of perfused capillaries in the two transplantation groups was significantly lower than in the control group at both 24 hours and 72 hours (p < 0.05). Those results were as follows: 8.2 +/- 2.1 at 24 hours, 7.7 +/- 0.85 at 72 hours in the ischemic control group; 5.4 +/- 0.9 at 24 hours, 6 +/- 0.6 at 72 hours in the isograft group; and 5 +/- 0.9 at 24 hours, 5.5 +/- 0.3 at 72 hours in the allograft group. Red blood cell velocities and vessel diameters in the main arteries were also decreased in transplant groups at 24 hours (p < 0.05) but returned to normal 72 hours after the operation (p > 0.05). The composite rat hind limb-cremaster model presented in this study introduces a reproducible in vivo approach to monitor the differences in microcirculatory hemodynamics of ischemia/reperfusion injury and acute graft rejection. The model allows the study of the timing, sequence, and correlation between clinical and hemodynamic signs during the acute phase of allograft rejection.     

Influence of groin incision, duration of ischemia, and prostaglandin E1 on ischemia-reperfusion injury of the lower limb

OBJECTIVE: The influences of groin incision, duration of ischemia, and the effects of prostaglandin E1 (PGE1) on ischemia-reperfusion (I/R) injury of the hind limb in rabbits were evaluated. DESIGN: A prospective study. SETTING: Laboratory. PARTICIPANTS: In 64 rabbits, bilateral hind limb ischemia was induced by occlusion of the abdominal aorta. Volume changes, neuromuscular function of the hind limb, and creatine kinase (CK) release were measured as variables of tissue injury. INTERVENTIONS: Eight rabbits served as untreated controls (CON). In 2 groups (each n = 14), 3 hours of ischemia were followed by 3 hours of reperfusion (I/R). In 2 different groups (each n = 14), 45 minutes of ischemia were followed by 2 hours of reperfusion. To determine effects of PGE1, 1 I/R group of each ischemia duration was treated intravenously with 80 ng/kg/min of PGE1 starting 30 minutes after the onset of ischemia (I/R-PGE1). To determine effects of groin incision on edema formation, volume changes were determined in the "operated" right (CON-R, 3h-R, 3h-PGE1-R and 45 min-R, 45 min-PGE1-R) or in the "nonoperated" left hind limb (CON-L, 3h-L, 3h-PGE1-L and 45 min-L, 45 min-PGE1-L), representing a subgroup analysis. MEASUREMENTS AND MAIN RESULTS: Volume changes after I/R occurred only in operated legs after ischemia (3h-R: 2.3 +/- 0.3 mL, p < 0.0001 v CON-R and 3h-L; 45 min-R: 0.8 +/- 0.2 mL, p < 0.01 v 45 min-L). PGE1 reduced edema formation in the operated legs (3h-PGE1-R: 1.0 +/- 0.4 mL, p < 0.0001 v 3h-R; 45 min-PGE1-R: 0.5 +/- 0.3 mL, p = 1.0 v 45 min-R). Groin incision without I/R had no effect on edema formation (CON-R: -0.13 +/- 0.17 mL of baseline). The increase of CK release from 616 +/- 584 U/L in controls to 5,921 +/- 2,156 U/L after 3 hours of ischemia (p < 0.001) was attenuated by treatment with PGE1 (3,732 +/- 2,653, p < 0.05 v I/R). Forty-five minutes of ischemia did not lead to cellular damage as measured by CK release (I/R: 606 +/- 364 U/L). Recovery of neuromuscular function was not affected by PGE1. CONCLUSION: Development of edema during I/R depends on groin incision of the hind limb and on the duration of ischemia. The I/R injury is attenuated by PGE1 treatment, in terms of reduced edema formation and CK release, but not in terms of neuromuscular function.     

迷走神经电刺激后处理联合肢体远隔缺血后处理对大鼠心肌缺血再灌注损伤的影响

目的 评价迷走神经电刺激后处理联合肢体远隔缺血后处理对大鼠心肌缺血再灌注损伤的影响.方法 雄性SD大鼠100只,8周龄,体重250～350 g,采用随机数字表法,将其随机分为5组(n=20):假手术组(S组)、缺血再灌注组(I/R组)、迷走神经电刺激后处理组(POES组)、肢体远隔缺血后处理组(Rp组)、迷走神经电刺激后处理联合肢体远隔缺血后处理组(POES-RP组).I/R组、POES组、RP组和POES-RP组采用结扎冠状动脉左前降支30 min和再灌注120 min的方法制备心肌缺血再灌注损伤模型.POES组和POES-RP组于心肌缺血15 min时对右侧迷走神经干实施电刺激30min,电刺激参数:波宽2ms,频率1O Hz,电流强度随HR进行调整,以保持HR较刺激前降低10％.RP组和POES-RP组于心肌缺血20 min时采用止血带结扎双后肢10 min后恢复血流灌注.各组随机取10只大鼠,于再灌注120 min时采集颈动脉血样,采用ELISA法检测血清cTnI、CK-MB、TNF-α、高迁移率组蛋白1( HMGB1)、细胞间粘附分子1(ICAM-1)、IL-1、IL-6和IL-10的浓度;颈动脉采血后,采用伊文氏蓝和TTC双重染色法测定心肌梗死体积.再灌注120 min时,各组随机处死10只大鼠,取缺血区和非缺血区心肌组织,采用ELISA法检测TNF-α、HMGB-1、ICAM-1、IL-1、IL-6和IL-10的含量.结果 与S组比较,I/R组心肌梗死体积、血清cTnI、CK-MB、TNF-α、HMGB1、ICAM-1、IL-1和IL-6的浓度升高,缺血区和非缺血区心肌组织TNF-α、HMGB1、ICAM-1、IL-1、IL-6和IL-10的含量升高(P＜0.05).与I/R组比较,POES组、RP组POES-RP组心肌梗死体积、血清cTnI、CK-MB、TNF-α、HMGB1、ICAM-1、IL-1和IL-6的浓度降低,缺血区和非缺血区心肌组织TNF-α、HMGB1、ICAM-1、IL-1和IL-6的含量降低,POES组和POES-RP组缺血区和非缺血区心肌组织IL-10含量升高(P＜0.05).与POES组比较,POES-RP组心肌梗死体积、血清cTnI、CK-MB、TNF-α和ICAM-1的浓度、缺血区心肌组织ICAM-1和IL-1含量降低,非缺血区心肌组织IL-10含量升高(P＜0.05).与RP组比较,POES-RP组心肌梗死体积、血清cTnI、CK-MB、TNF-α、HMGB1、ICAM-1、IL-1和IL-6的浓度、缺血区心肌组织TNF-α、ICAM-1、IL-1和IL-6的含量降低,IL-10含量升高,非缺血区心肌组织HMGB1、ICAM-1、IL-1和IL-6的含量降低,IL-10含量升高(P＜0.05).结论 迷走神经电刺激后处理联合肢体远隔缺血后处理可减轻大鼠心肌缺血再灌注损伤,且联合应用的效果强于单独应用,其机制可能与抑制局部和全身炎性反应有关.     

Ischemia-induced neutrophil activation and diapedesis is lipoxygenase dependent

Ischemia and reperfusion lead to eicosanoid- and neutrophil (PMN)-dependent injury. This study tests the role of ischemia-induced lipoxygenase activity in mediating PMN activation and diapedesis. Anesthetized rabbits (n = 8) underwent 3 hours of bilateral hindlimb ischemia. At 10 minutes of reperfusion, leukotriene B4 (LTB4) levels in femoral venous effluent were 0.49 +/- 0.05 ng/ml compared with 0.04 +/- 0.07 ng/ml in sham-treated animals (n = 10) (p less than 0.05). Intracellular H2O2 production of circulating PMNs assayed flow cytometrically by dichlorofluorescein (DCF) oxidation, increased from a preischemic value of 74 +/- 14 femtomoles DCF/cell to 135 +/- 8 fmol DCF/cell (p less than 0.05). PMNs were treated with phorbol myristate acetate (PMA), 10(-7) mol/L. In contrast to a 162% increase in H2O2 production before ischemia, PMNs at 10 minutes of reperfusion had an enhanced response to PMA of 336% (p less than 0.05). Addition of authentic LTB4 (0.5 ng/ml) to PMN from sham-treated animals led to their activation, manifest by an oxidative burst, 127 +/- 12 fmol DCF/cell, and an enhanced response of 337% to PMA stimulation. To study diapedesis, plasma collected at 10 minutes of reperfusion was introduced into plastic chambers taped atop skin abrasions in rabbits (n = 8). After 3 hours, 1610 +/- 246 PMN/mm3 accumulated and LTB4 levels in blister fluid were 0.83 +/- 0.03 ng/ml, higher than values of 44 +/- 23 PMN/mm3 (p less than 0.05) and 0.04 +/- 0.03 ng LTB4/ml (p less than 0.05) with saline solution and 68 +/- 16 PMN/mm3 (p less than 0.05) and 0.19 +/- 0.02 ng/ml (p less than 0.05) with nonischemic plasma. The introduction of LTB4, 3.3 ng/ml, into the chambers resulted in an accumulation of 536 +/- 352 PMN/mm3 (p less than 0.05). Pretreatment of animals before hindlimb ischemia (n = 5) with the lipoxygenase inhibitor diethylcarbamazine abolished PMN activation (51 +/- 12 fmol DCF/cell) and ischemic plasma-induced diapedesis into the plastic chamber (38 +/- 18 PMN/mm3). Pretreatment of nonischemic animals (n = 13) used for the dermabrasion bioassay with diethylcarbamazine abolished diapedesis into the plastic chambers induced by ischemic plasma (n = 5) (32 +/- 24 PMN/mm3) or LTB4 (n = 3) (36 +/- 28 PMN/mm3). These data indicate that PMN activation after reperfusion of ischemic tissue is mediated by a lipoxygenase product, perhaps LTB4, and that both reperfusion plasma and authentic LTB4 induce diapedesis by stimulating de novo lipoxygenase activity.