Effect of free radical scavenging on skeletal muscle blood flow during postischaemic reperfusion.

After 6-h tourniquet ischaemia of one hindlimb in male Sprague-Dawley rats, gastrocnemius muscle blood flow was measured following 10, 120 and 240 min of reperfusion using radiolabelled microspheres. A perfusion index was calculated (experimental limb: contralateral limb) for each of these times. Comparison of perfusion indices in ten control animals (6 h ischaemia, 4 h reperfusion) with similar measurements in ten normal rats with no ischaemia and in ten ischaemic animals with the tourniquet in situ demonstrated low median (interquartile range (i.q.r.)) reflow after 10 min (control 0.12 (0.02-0.43), ischaemia 0.04 (0.00-0.07), normal 1.05 (0.68-1.18); control versus ischaemia, P not significant; control versus normal, P < 0.01). Relative reperfusion occurred at 120 min (control 0.48 (0.11-0.70), ischaemia 0.02 (0.01-0.07), normal 0.97 (0.79-1.13); control versus ischaemia, P < 0.05; control versus normal, P < 0.05) and reperfusion injury after 240 min of revascularization, with muscle blood flow being little different from that in the ischaemic group (control 0.05 (0.01-0.38), ischaemia 0.03 (0.00-0.07), normal 1.01 (0.73-1.16); control versus ischaemia, P not significant; control versus normal, P < 0.01). Two groups of 12 rats were given either intravenous superoxide dismutase and catalase or dimethylthiourea 30 min before tourniquet release, continuing throughout the period of reperfusion. Superoxide dismutase and catalase reversed low reflow, producing a median (i.q.r.) perfusion index of 0.94 (0.54-1.12) (P < 0.01 versus control, P not significant versus normal), but had no effect on relative reperfusion (0.66 (0.42-1.01), P not significant versus control) or on reperfusion injury (0.27 (0.01-0.35), P not significant versus control). In contrast, dimethylthiourea had no effect on perfusion at either 10 min (0.10 (0.03-0.15), P not significant versus control) or 240 min (0.04 (0.00-0.11), P not significant versus control), but abolished the phase of relative reperfusion at 120 min (0.04 (0.02-0.21), P < 0.01 versus control). These results indicate that, although superoxide radicals are harmful during postischaemic reperfusion, hydroxyl radicals may be beneficial.     

Allopurinol--a free radical scavenger--reduces reperfusion injury in skeletal muscle

Reperfusion of ischaemic skeletal muscle may lead to increased vascular permeability, oedema and ultimately muscle necrosis. Oxygen-derived free radicals have been suggested as aetiological factors in reperfusion injury. Amputated rabbit hindlimbs were subjected to 4 h of ischaemia followed by 2 h or reperfusion with Krebs' buffer. One limb from each animal was reperfused with oxygen-saturated buffer (reoxygenated limb) while the other limb was reperfused with nitrogen-saturated buffer (non-reoxygenated limb). Six animals received allopurinol orally 2 days prior to the experiment and ten animals received no treatment. The energy charge dropped from 0.90 to 0.54 during ischaemia and increased to 0.82 after reperfusion with oxygenated perfusate. Oedema was determined by limb weight and water content in muscle biopsies and muscle injury was assessed by uptake of [Tc99]methylenediphosphonate ([Tc99]MDP). The results were expressed in ratios, between the reoxygenated and nonreoxygenated limb. Without allopurinol treatment, the increase in water content and limb weight in reoxygenated limbs exceeded (p less than 0.05) non-reoxygenated limbs (ratios = 1.73 and 1.89, respectively). Allopurinol treatment significantly reduced (p less than 0.05 and p less than 0.02, respectively) the increase in water content and limb weight (ratios = 0.54 and 1.01, respectively). Without treatment, [Tc99]MDP-uptake was greater (p less than 0.05) in reoxygenated limbs than in non-reoxygenated limbs (ratio = 1.60). Allopurinol treatment significantly reduced (p less than 0.002) [Tc99]MDP-uptake in reoxygenated limbs (ratio = 0.80). These results demonstrate that additional injury to ischaemic skeletal muscle occurs during reperfusion with oxygen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Do prostaglandins have a salutary role in skeletal muscle ischaemia-reperfusion injury?

OBJECTIVES: the effects of prostaglandins (PG) E1, E2, and the prostacyclin analogue iloprost with and without the addition of free-radical scavengers catalase and superoxide dismutase on gastrocnemius blood flow and oedema were studied in a rodent model of hindlimb ischaemia-reperfusion. METHODS: male Sprague-Dawley rats underwent 6-h hindlimb ischaemia with 4-h reperfusion. Prostaglandins were infused prior to reperfusion and their effects on limb blood flow and oedema examined. RESULTS: control animals exhibited a triphasic pattern of muscle blood flow during reperfusion compared to normal animals. PGE1 did not abolish low reflow at 10 min, relative reperfusion was preserved but reperfusion injury was abolished at 120 min. Muscle blood flow was increased at 240 min compared to controls. Increased limb swelling was also seen. Addition of free-radical scavengers caused the abolition of low reflow. Similar results were seen with iloprost. PGE2 abolished low reflow at 10 min and increased perfusion at 120 min but did not prevent reperfusion injury at 240 min. CONCLUSIONS: PGE1 and iloprost enhance muscle blood flow at 4-h reperfusion, though neither abolishes low reflow; PGE2 improved flow at 10 and 120 min but not after 240 min. This study demonstrates a potentially beneficial role for prostaglandins in improving muscle blood flow in skeletal muscle ischaemia-reperfusion injury.     

Skeletal Muscle Ischaemia-reperfusion Injury: Further Characterisation of a Rodent Model

BACKGROUND: Postischaemic damage in skeletal muscle may be reflected in changes to microvascular blood flow, vascular permeability, and subsequent tissue viability. Previous preclinical studies have not addressed all these parameters, and have not used periods of ischaemia and reperfusion relevant to the clinical setting. This study aimed to develop an animal model hindlimb ischaemia-reperfusion to simulate acute lower limb ischaemia. METHODS: A rodent model of hindlimb tourniquet-induced ischaemia-reperfusion was employed. Gastrocnemius muscle blood flow (GMBF; radio-labelled microspheres), oedema (GMO; using a wet:dry ratio method) and viability (GMV; histochemistry and computerised planimetry) were quantified. RESULTS: 6 h ischaemia per seresulted in neither muscle oedema nor loss of viability, but these changes were apparent following 4 h reperfusion. Early reperfusion at 10 min demonstrated low reflow, with GMBF improving at 120 min before declining sharply at 240 min. CONCLUSION: Prolonged hindlimb ischaemia followed by reperfusion in this rodent model caused significant reductions in gastrocnemius muscle blood flow, associated with muscle oedema and necrosis. These three parameters have not been previously reported together in the same model. This reproducible model could be used in the evaluation of potential therapeutic intervention strategies aimed at ameliorating skeletal muscle reperfusion injury.     

Microcirculatory effects of experimental acute limb ischaemia-reperfusion.

BACKGROUND: The object of this study was to develop an animal model in which changes in microvascular haemodynamics and leucocyte-vessel wall interactions due to acute limb ischaemia-reperfusion (I/R) can be measured in the skin. Furthermore, it was investigated whether these changes are related to local muscle injury. METHODS: Male Lewis rats were subjected to unilateral limb ischaemia for 1 h (n = 8) or 2 h (n = 8) by cuff inflation, or to a sham protocol (n = 6). Intravital video microscopic measurements of leucocyte-vessel wall interactions, venular diameter, red blood cell velocity and reduced velocity (which is proportional to wall shear rate) were performed in skin venules before ischaemia and at 0.5, 1, 2, 3 and 4 h after the start of reperfusion. Oedema and leucocyte infiltration of ischaemic/reperfused skeletal muscle were quantified histologically. RESULTS: In skin venules, both 1 and 2 h of ischaemia induced a significant increase in leucocyte rolling (six and five times baseline, respectively; P < 0.05) and adherence during reperfusion (eight and four times baseline; P < 0.05). No significant increase in muscular leucocyte infiltration was detected. After an initial hyperaemic response of 180 per cent of baseline values (P < 0.05), blood flow decreased to about 60 per cent after 4 h of reperfusion in skin venules of both experimental groups. I/R induced tibial muscle oedema, the severity of which depended on the ischaemic interval (wet to dry ratio: control, 4.0; 1 h, 4.5 (P not significant); 2 h, 5.8 (P < 0.05)). CONCLUSION: A non-invasive animal model was developed that enables investigation of the consequences of acute limb I/R.     

Energy metabolism during microsurgical transfer of human skeletal muscle assessed by high-pressure liquid chromatography and by 31P-nuclear magnetic resonance.

The effect of ischaemia and reperfusion on human skeletal muscle was studied during free vascularised muscle transfer. Muscle biopsy specimens were taken from patients having microsurgical muscle transfer, 18 cases (17 patients; 12 men, 5 women). The biopsies were taken three times: before transfer of the muscle (control), at maximum ischaemic time, and one hour after revascularisation. The biopsy specimens were analysed for purine nucleotides, by high-pressure liquid chromatography (HPLC), and by nuclear magnetic resonance (NMR) at 500 MHz. Phosphocreatine (PCr) recovered only partially (79%) and adenosine triphosphate (ATP) did not differ significantly from normal control after revascularisation and a mean ischaemic time of 114 minutes. NMR measurements showed an accumulation of glucose-6-phosphate (G-6-P) during the ischaemic period, indicating anaerobic metabolism. After three hours of ischaemia and one hour of reperfusion the PCr recovery was less than 60% (r = 0.7). The results confirm those of previous animal studies, which set three hours normothermic ischaemia as a safe limit for tissue preservation when transferring skeletal muscle. Longer ischaemic times may cause serious postoperative healing problems and reduced muscle function.     

ENERGY METABOLISM DURING MICROSURGICAL TRANSFER OF HUMAN SKELETAL MUSCLE ASSESSED BY HIGH-PRESSURE LIQUID CHROMATOGRAPHY AND BY 31 P-NUCLEAR MAGNETIC RESONANCE

The effect of ischaemia and reperfusion on human skeletal muscle was studied during free vascularised muscle transfer. Muscle biopsy specimens were taken from patients having microsurgical muscle transfer, 18 cases (17 patients; 12 men, 5 women). The biopsies were taken three times: before transfer of the muscle (control), at maximum ischaemic time, and one hour after revascularisation. The biopsy specimens were analysed for purine nucleotides, by high-pressure liquid chromatography (HPLC), and by nuclear magnetic resonance (NMR) at 500 MHz. Phosphocreatine (PCr) recovered only partially (79%) and adenosine triphosphate (ATP) did not differ significantly from normal control after revascularisation and a mean ischaemic time of 114 minutes. NMR measurements showed an accumulation of glucose-6-phosphate (G-6-P) during the ischaemic period, indicating anaerobic metabolism. After three hours of ischaemia and one hour of reperfusion the PCr recovery was less than 60% ( r = 0.7). The results confirm those of previous animal studies, which set three hours normothermic ischaemia as a safe limit for tissue preservation when transferring skeletal muscle. Longer ischaemic times may cause serious postoperative healing problems and reduced muscle function.     

Acute embolic lower limb ischaemia is associated with decreased red cell deformability

Red cell deformability is an important determinant of peripheral blood flow. In this study the red cell filtration rate (RFR) was measured in 17 patients admitted for acute embolic lower limb ischaemia. The severity of the limb ischaemia on admission was scored from 0 to 6 depending on the degree of pain, and loss of motor and sensibility functions in the ischaemic extremity. Patients who presented with very severe ischaemia on admission underwent early embolectomy whereas those with less severe ischaemia (range from 0 to approximately 3 in ischaemic score) initially were treated conservatively with heparin. Red cell filtration rate was assessed with a standard microfiltration method and expressed in microliters/sec. On admission the RFR averages 30 +/- 5 microliters/sec (mean +/- S.D.), significantly lower than the corresponding value in a normal population of similar age (50 +/- 5 microliters/sec). There was an inverse relationship (r = 0.74, P less than 0.001 by use of linear regression analysis) between the ischaemic score and the RFR on admission, with low RFR values being related to high ischaemic scores. Forty-eight hours after revascularisation, the RFR had improved significantly in the patients having undergone successful revascularisation, whereas it was unchanged in conservatively treated patients. It is concluded that patients with acute embolic lower limb ischaemia have an impaired red cell filtration rate suggesting decreased red cell deformability, the impairment being related to the severity of the ischaemia. A poor red cell filtration rate on admission is related to increased risk of postoperative gangrene and/or cardiac death. Successful revascularisation, but not conservative heparin treatment is associated with increasing deformability.     

Postischaemic renal cortical microcirculation and tissue oxygenation in the pig

Porcine and human renal physiology are similar in important aspects. Renal cortical microcirculation and its relation to inulin clearance (CIn) was therefore studied before and after renal ischaemia in 28 pigs under continuous intravenous chlormethiazole-pancuronium anaesthesia. The anaesthesia used provided essential stability in central haemodynamics. The animals were studied for 90 min of reperfusion following 0, 30 or 60 min of renal ischaemia. Twelve of the animals (four were subjected to each duration of ischemia) were also studied 18 h after start of reperfusion. Regional blood flow in the superficial renal cortex was measured by laser Doppler flowmetry (LDF), and tissue oxygenation (PtO2) by surface microelectrode technique. These techniques allow continuous or repeated measurements. During the first 90 min of reperfusion, superficial renal cortical blood flow measured by LDF (Qsrc) underwent considerable temporal variation which followed a certain pattern. Thus, when the renal arterial blood flow was restored after ischaemia, we observed an instant peak in Qsrc followed by a decreasing flow until a minimum value (Qmin) was reached between 3 and 9 min after start of reperfusion (tQmin). Thereafter, Qsrc increased until a maximal value (Qmax) was reached between 11 and 64 min after start of reperfusion (tQmax). The parameters tQmin and tQmax were related to inulin clearance 18 h after start of reperfusion (P less than 0.05 and P less than 0.01, respectively). Thus, it might be possible soon after start of reperfusion--to evaluate the severity of ischaemic damage. This could be useful in the evaluation of different prophylactic strategies, since the full extent of the ischaemic damage, as assessed by clearance determinations, cannot be established until hours later.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of experimental lower-limb ischaemia-reperfusion injury on the mesenteric microcirculation

BACKGROUND: Ischaemia-reperfusion (I-R) of the leg is associated with functional and structural changes in the intestine. This study assessed whether acute hind-limb I-R in rats induced a reduction in perfusion and/or signs of an inflammatory response in the intestine. METHODS: Rats were subjected to 2 h of unilateral hind-limb ischaemia followed by 2 h of reperfusion (I-R group, n = 9) or to a sham procedure (control group, n = 9). Mesenteric microvascular diameters, red blood cell velocity, blood flow and leucocyte-vessel wall interactions during reperfusion were measured using intravital microscopy. RESULTS: Blood pressure and heart rate decreased from 30 min of reperfusion onwards in the I-R group compared with controls. From 15 min after the start of reperfusion, mesenteric arteriolar and venular red blood cell velocity and blood flow decreased by 40-50 per cent. Microvascular diameters and leucocyte-vessel wall interactions did not change. CONCLUSION: Restoration of blood flow to an acutely ischaemic hind limb led to a significant decline in the splanchnic microcirculatory blood flow. There were, however, no signs of an early inflammatory response in the gut.     

Skeletal muscle blood flow after prolonged tourniquet ischaemia and reperfusion with and without intervening reoxygenation: an experimental study in rats using laser Doppler perfusion imaging.

The total, safe, time available for operating during tourniquet ischaemia is thought to be prolonged by short, intervening episodes of reperfusion. However, animal experiments have suggested that this may cause a reduction of the postischaemic reperfusion injury. The purpose of the present study was to investigate the effect on final postischaemic reperfusion of intermittent, short periods of reperfusion compared with that of prolonged, continuous tourniquet ischaemia. A rat tourniquet model of total limb ischaemia and laser Doppler imaging to measure postischaemic microvascular perfusion in skeletal muscle was used in a total of 25 anaesthetised rats. Four were non-ischaemic controls. In 21 rats one hind leg was made totally ischaemic by a tourniquet. Fourteen were exposed to uninterrupted periods of either 1.5 hours or three hours, and seven to interrupted total ischaemia of three hours, with a 20 minutes reperfusion interval after 1.5 hours of ischaemia. The postischaemic blood flow was monitored 20 minutes after release of the tourniquet. Postischaemic skeletal muscle blood flow was significantly reduced after three hours of continuous ischaemia compared with the group that had an intervening short period of reoxygenation. The postischaemic reperfusion after three hours of ischaemia with a short reperfusion interval was not reduced after ischaemia compared with either that in controls or to that in the group exposed to only 1.5 hours of tourniquet ischaemia. These findings support the clinical practice of using intervening periods of reperfusion to prolong the total tourniquet time that can safely be used. There were no adverse effects on postischaemic reperfusion from intermittent reoxygenation.     

Microvascular transfer-related abrogation of capillary flow motion in critically reperfused composite flaps.

Capillary flow motion is defined as rhythmic fluctuations of blood flow in the capillaries. Although critical perfusion has been demonstrated to induce capillary flow motion, little is known about the role of capillary flow motion in microvascular free flaps. The aim of this study was to elucidate the tissue-confined incidence and consequence of capillary flow motion in microvascularly transferred composite flaps, using intravital fluorescence microscopy. In Wistar rats, transferred osteomyocutaneous flaps (n = 7), which were exposed to 1 h of ischaemia during the anastomotic procedure followed by 1 h of reperfusion, were subjected to critical perfusion by stepwise reduction of the femoral-artery blood flow to 0.15 ml min(-1), 0.10 ml min(-1) and 0.05 ml min(-1). Pedicled osteomyocutaneous flaps that were not subjected to ischaemia (n=8) served as controls. In pedicled flaps critical perfusion induced capillary flow motion in the muscle, but not in the skin, subcutis and periosteum. In these flaps, the functional capillary density was preserved in all tissues analysed, including the skeletal muscle. Additional sympathetic denervation of the pedicled flaps did not change the incidence or pattern of capillary flow motion. In contrast, after flap transfer capillary flow motion in muscle tissue did not occur during critical perfusion. As a consequence, a shutdown of perfusion of individual capillaries was observed, resulting in a significant reduction (P<0.05) in functional capillary density, not only in the subcutis, skin and periosteum but also in the muscle itself. Thus, our data suggest that the microcirculatory control of pedicled osteomyocutaneous flaps is preserved during critical perfusion by skeletal-muscle capillary flow motion, whereas this protective regulatory mechanism is lost during the initial reperfusion period after flap transfer, probably not because of denervation but because of surgery- and/or ischaemia-reperfusion-associated injury.     

Antibody-induced modulation of the leukocyte CD11b integrin prevents mild but not major renal ischaemic injury.

BACKGROUND: CD11/CD18 beta(2) integrins are involved in leukocyte adhesion to the activated endothelium, and therefore represent a possible therapeutic target in the prevention of ischaemic acute renal failure (ARF). METHODS: To assess the effect of an anti-CD11b monoclonal antibody (mAb) in ischaemic ARF, uninephrectomized Fischer rats were subjected to 45 or 60 min of warm renal ischaemia, then received 1 mg of anti-CD11b mAb 5 min before reperfusion. RESULTS: After 45 min of ischaemia, renal function tests at 24 and 48 h were less altered in mAb-treated than in control rats, but after 60 min of ischaemia the same level of renal insufficiency was observed in the two groups. In parallel, milder tubular necrosis and less leukocyte infiltration were observed in the treated group after 45 min of ischaemia, but no difference was seen after 60 min compared to the control group. The mAb was detected on blood neutrophils up to 48 h after infusion and a marked down-regulation of CD11b expression on neutrophil surfaces was documented by flow cytometry. CONCLUSION: These results indicate that anti-CD11b mAb administered prior to reperfusion decreases moderate ischaemic ARF but fails to prevent renal injury secondary to prolonged ischaemia in this model.     

Lower limb ischaemia and reperfusion alters gut permeability.

The gut may be important in the aetiology of multiple organ failure (MOF) by amplifying of the inflammatory response to trauma. We investigated the effects of the reperfusion of ischaemic lower limbs on gut permeability. Male Wistar rats (n = 30) were randomised to (group 1) controls; (group 2) 3 h bilateral hind-limb ischaemia alone; or 3 h ischaemia followed by (group 3) 15 min reperfusion or (group 4) 2 h reperfusion. Gut permeability and plasma endotoxin were measured prior to tourniquet application, immediately before tourniquet release, and following reperfusion. To evaluate the effect of the hypotension that follows tourniquet release, (group 5) sodium nitroprusside was infused in further controls to maintain mean arterial pressure (MAP) at 75 mmHg for 2 h. Horseradish peroxidase was instilled into the isolated ileo-caecal loop 15 min before the animals were killed to measure permeability of horseradish peroxidase through mucosal intercellular tight junctions by electron microscopy. Mean arterial pressure increased from 105 +/- 5 mmHg to 136 +/- 4 mmHg on tourniquet application and fell to 79 +/- 7 mmHg following reperfusion (p less than 0.05). In group 1 (controls), group 2 (ischaemia alone animals) and group 5 (ischaemia and nitroprusside) one animal out of six demonstrated permeability to horseradish peroxidase. Following reperfusion, horseradish peroxidase permeability had not developed by 15 min (group 3) but was present in all animals by 2h (group 4) (p = 0.015 Fisher's exact test). Plasma endotoxin increased from 21.8 +/- 2.0 pg ml-1 to 30.7 +/- 2.6 pg ml-1 following 2 h reperfusion (p less than 0.05 Scheffe F-test).(ABSTRACT TRUNCATED AT 250 WORDS)     

Viability of hypothermic preserved muscle determined by gene expression levels.

We report a muscle viability index (MVI) that reflects mRNA degradation. The viability of hypothermically preserved (48C) rat skeletal muscle was evaluated using this MVI. To evaluate the hypothermic ischaemic insult of the muscle, 21 hind limbs of Fischer rats (three subgroups of seven limbs each) were hypothermically preserved for 1h, 3h and 6h, before harvesting the tibialis anterior muscle. To investigate reperfusion injury after hypothermic preservation, an additional 28 limbs were transplanted to recipient Fischer rats after hypothermic ischaemia for either 3h or 6h. The transplanted muscles were harvested on either day 3 or day 7 after transplantation. Seven fresh muscles were also harvested, and used as controls. In the 3h ischaemia group, the MVIs of both the hypothermic-ischaemia and the ischaemia-reperfusion subgroups were comparable to the controls. Likewise, there were no significant differences between the controls and the 6h hypothermic ischaemia and ischaemia-reperfusion subgroups. These results show that muscle viability is maintained with hypothermic preservation of up to 6h, and after reperfusion. Therefore, in clinical replantations the amputated extremity should be preserved under hypothermic conditions from the time of injury to the time of surgery.     

31P Nuclear magnetic resonance spectroscopy of acutely ischaemic limbs: the extent of changes and progress after reconstructive surgery

³¹P Nuclear magnetic resonance (NMR) spectroscopy of the tibialis anterior muscle was carried out on nine acutely ischaemic limbs in eight patients before and after revascularization, in 15 limbs of 13 claudicants at rest, in 17 patients with normal lower limb circulation suffering chronic renal failure, and in six healthy subjects. Claudicants, renal failure and healthy limbs showed similar inorganic phosphate/phosphocreatine ratios (Pi/PCr). Healthy volunteers after 30-min tourniquet ischaemia and patients with acutely ischaemic limbs showed significantly raised Pi/PCr ratios (P< 0.05). There was an association between Pi/PCr ratios and the systolic ankle:brachial pressure index in acutely ischaemic limbs. In the acute patients, the Pi/PCr ratios returned to normal after successful revascularization, the time course varying between 3 days and 3 months. Intracellular acidosis was observed in one patient who was also the only individual to develop reperfusion injury following reconstructive surgery. Acidosis may be a sign of muscle changes which lead to reperfusion injury.     

Differential responses in human subcutaneous and skeletal muscle vascular beds to critical limb ischaemia.

OBJECTIVES: To investigate the effects of chronic ischaemia on the subcutaneous and the skeletal muscle resistance vasculature. To understand the redistribution of available blood in the ischaemic limb. METHODS: Human subcutaneous and skeletal muscle resistance arteries were obtained from limbs amputated for critical limb ischaemia and studied under isobaric conditions using pressure myography. Morphological measurements of wall and lumen were analysed using light microscopy and image analysis. Vasoconstrictor responses to potassium and adrenoceptor agonists were used to measure functional status. Noradrenaline re-uptake mechanisms and alpha(1)-selectivity were investigated. RESULTS: Both human skeletal muscle and subcutaneous resistance arteries undergo a severe atrophy of the arterial wall in ischaemic conditions. However, whereas subcutaneous resistance arteries become less able to vasoconstrict to adrenoceptor stimulation, the response of skeletal muscle resistance arteries becomes exaggerated and significantly augmented. This is true in response to both the endogenous vasoconstrictor noradrenaline and the alpha(1)-selective adrenoceptor agonist phenylephrine. CONCLUSIONS: Hypersensitivity to circulating catecholamines in the skeletal muscle vascular resistance bed may contribute to the progression of ischaemic disease by differentially diverting available blood to the subcutaneous tissue to the detriment of skeletal muscle perfusion.     

Remote ischaemic preconditioning of the hind limb reduces experimental liver warm ischaemia-reperfusion injury

BACKGROUND: Direct ischaemic preconditioning of the liver reduces ischaemia-reperfusion injury (IRI). Remote ischaemic preconditioning (RIPC) of a limb has been shown to reduce IRI to the heart. This study determined the effect of brief remote ischaemia to the limb in reducing early liver warm IRI. METHODS: Twenty-eight male rabbits were allocated to four groups: sham operated, RIPC alone, IRI alone, and RIPC plus IRI. RIPC was induced in the leg with a tourniquet, before liver IRI, by three alternate cycles of 10 min ischaemia followed by 10 min reperfusion. Liver IRI was produced by total inflow occlusion for 25 min. Markers of liver injury and systemic and hepatic haemodynamics were measured for 2 h after reperfusion. RESULTS: At 2 h, IRI alone was associated with increased serum levels of aminotransferases, and reduced mean arterial blood pressure, hepatic blood flow and peripheral oxygen saturation. There was significant improvement in these variables in animals that had RIPC before liver IRI, and hepatic venous nitrate/nitrite levels were also significantly higher. CONCLUSION: In this experimental model RIPC appeared to reduce liver IRI.     

L-Arginine given after ischaemic preconditioning can enhance cardioprotection in isolated rat hearts.

OBJECTIVE: Ischaemic or pharmacological preconditioning with L-arginine has been reported to be insufficient for optimal cardioprotection. The ability of nitric oxide (NO) to enhance ischaemic preconditioning was assessed, and the role of L-arginine-induced ischaemic preconditioning in myocardial protection was determined. METHODS: Isolated rat hearts were prepared and divided into six groups: control hearts (control, n=6) were perfused without global ischaemia at 37 degrees C for 160 min; global ischaemia hearts (GI, n=6) were subjected to ischaemia for 20 min and reperfusion for 120 min; ischaemic preconditioned hearts (IP, n=6) received 2 min of zero-flow global ischaemia followed by 5 min reperfusion, before 20 min of global ischaemia; L-arginine hearts (ARG, n=6) received 1 mmol/l L-arginine for 5 min, before 20 min of global ischaemia; ischaemic preconditioning plus nitro-L-arginine methyl ester hearts (IP+L-NAME, n=6) received 2 min of ischaemic preconditioning and 5 min reperfusion with 3 mmol/l L-NAME in Krebs-Henseleit buffer, before 20 min of global ischaemia; and ischaemic preconditioning plus L-arginine hearts (IP+ARG, n=6) received 2 min of ischaemic preconditioning and 5 min reperfusion with 1 mmol/l L-arginine in Krebs-Henseleit buffer. Haemodynamic parameters and coronary flow were recorded continuously. Nitrites and nitrates (NOx) were measured 5 and 60 min after reperfusion, and infarct size was also determined. RESULTS: In the IP+ARG group, significant amelioration and preservation of left ventricular peak developed pressure and coronary flow was observed compared with the GI, IP, ARG and IP+L-NAME groups. Infarct size in the IP+ARG group was reduced significantly compared with that in the GI, IP, ARG and IP+L-NAME groups. Significant preservation of NOx was observed during reperfusion in the IP+ARG group compared with the GI group. CONCLUSIONS: Inhibition of NO synthase with L-NAME had little impact on ischaemic preconditioning, suggesting that endogenous NO is not a major mediator of ischaemic preconditioning. Nevertheless, enhancement of the effects of ischaemic preconditioning can be achieved with L-arginine, a precursor of NO, improving post-ischaemic functional recovery and infarct size in the isolated rat heart.     

Studies of reperfusion injury in skeletal muscle: preserved cellular viability after extended periods of warm ischemia

Four hours of complete normothermic ischemia in the rat hindlimb has been thought to produce extensive and irreversible damage and no possibility of salvage by reperfusion. This study tests the hypothesis that, in contrast to conventional wisdom, the cellular integrity is preserved after 4 hours of complete warm ischemia and control of the initial reperfusion can restore immediate contractility in these limbs. Ninety-two rat hindlimbs were isolated and 26 of the 92 did not undergo ischemia or reperfusion and served as controls. Sixty-six limbs were subjected to 4 hours of complete warm ischemia; of those 34 were assessed after the ischemic period without reperfusion and 32 were reperfused after the ischemic period. Nineteen hindlimbs were reperfused with Krebs-Henseleit buffer at a pressure of 100 mmHg to simulate embolectomy (uncontrolled reperfusion). In 13 legs a modified reperfusate at a pressure of 60 mmHg was used during the initial 30 minutes followed by an additional 30 minutes of reperfusion with 100 mmHg using Krebs-Henseleit buffer (controlled reperfusion). At the end of each experimental protocol, limbs were assessed by the following methods: muscle contraction, water content, volume, high energy phosphate content, muscle pH, effluent pH, mitochondrial function, ultrastructure, flow, and creatinkinase activity in the effluent. Data are expressed as mean +/- SEM. Significant differences were defined as probabilities for each test of p less than 0.05. Four hours of complete warm ischemia resulted in a severe reduction of adenosine triphosphate (4.0 +/- 0.8 vs 27.1 +/- 6.7 mumol/gm protein, p less than 0.001) and no contractions could be stimulated (0.0 +/- 0.0% CC). Muscle pH fell to 6.3 +/- 0.1 (p less than 0.001), and ultrastructural damage occurred (score 3.3 +/- 0.4 vs 0.8 +/- 0.1, p less than 0.002). However, there was only a slight increase in water content of the soleus muscle (78.7 +/- 0.2% vs 74.8 +/- 1.1%, p less than 0.05) without increase in limb volume (103.6 +/- 0.6% CV). In addition mitochondrial function was preserved well: mitochondrial oxidative phosphorylation capacity remained at 94% of control levels, ST3 at 93%, and ADP/O at 100% of control. Most importantly, controlled reperfusion restored immediate contractility in all limbs and was superior in all parameters investigated compared to uncontrolled reperfusion. These data support our inference that necrosis of skeletal muscle does not invariably occur after four hours of complete warm ischemia and suggest that muscle salvage by controlled reperfusion is possible after at least 4 hours of warm ischemia.