Ischemia-reperfusion induced microvascular dysfunction in skeletal muscle: application of intravital video microscopy

Video microscopy of red cell flow in capillaries at the surface of skeletal muscle provided the opportunity to quantitate ischemia-reperfusion (I-R) induced microcirculatory changes, in vivo. Extensor Digitorum Longus (EDL) muscles of 22 male Wistar rats (300-400 g), anesthetized with sodium pentobarbital (Somnotol, 65 mg kg,-1 IP), were used to measure the number of perfused capillaries (CDper: mm-1) crossing lines drawn perpendicular to the muscle axis, and red blood cell velocity (VRBC: mm/s) within individual capillaries from controls (n = 6), and after 2 hr (n = 4), 3 hr (n = 4), and 4 hr (n = 5) of no-flow ischemia with the muscle temperature maintained at its normal value of 32 degrees C. Ischemia was induced by tightening a tourniquet placed around the limb above the EDL muscle. Measurements were made after 30, 60, and 90 min of reperfusion. To test the usefulness of this skeletal muscle model for evaluating proposed interventions in I-R, the effect of hypothermia (24 degrees C) on the microcirculation following 4 hr ischemia (n = 3) was measured. Edema formation was estimated from the wet/dry weight ratio of the ischemic and contralateral control EDL muscles. Capillary perfusion at the surface of the control muscles was remarkably stable over the 5 hr period studied, while significant changes occurred following the ischemic periods. Significantly lower CDper was measured 30 min following all periods of normothermic ischemia. However, unlike the 2 and 4 hr ischemic periods 3 hr normothermic ischemia resulted in a progressive decline in CDper throughout the reperfusion period. VRBC showed evidence of a hyperemic response following 2 hr normothermic ischemia (control: 0.12 mm/s +/- 0.19 compared to 0.26 mm/s +/- 0.03 following 90 min reperfusion; mean +/- sem). However, no such hyperemia was measured following either 3 or 4 hr normothermic ischemia (i.e., 3 hr control: 0.24 mm/s +/- 0.01 compared to 0.07 mm s +/- 0.003 following 90 min reperfusion). In fact, VRBC was essentially zero 90 min following 4 hr normothermic ischemia (0.01 mm/s +/- 0.01). However, when the muscle was allowed to cool to 24 degrees C during 4 hr ischemia no significant change in either VRBC or CDper was measured compared to pre-ischemic controls. Evidence of edema was found after 3 and 4 hr normothermic ischemia. This study establishes a skeletal muscle model of I-R, which may be useful in testing hypotheses regarding mechanisms of I-R injury, and effectiveness of proposed treatments of I-R.     

Ischemic preconditioning in surgery of extremities: experimental studies

Ischemic preconditioning (IP), using one or more brief periods of ischemia, each followed by a short reperfusion phase, improves tolerance of subsequent sustained ischemia in different organs. The aim of this experimental study was to evaluate the effects of IP on postischemic function in skeletal muscle. Right hindlimbs of anesthetized rats were pretreated with three cycles each of 10 min of ischemia and 10 min of reperfusion (n = 12). Non-preconditioned animals (n = 12) served as controls. These hindlimbs were then subjected to 3 h of ischemia and 2 h of reperfusion. IP resulted in a significant increase in postischemic skeletal muscle force (240 +/- 47 mN vs 409 +/- 63 mN), force-time integral (1081 +/- 242 mN*s vs 2546 +/- 481 mN*s) and endurance (29.6 +/- 3.4 s vs 48.0 +/- 5.0 s). These data support the potential of IP to reduce postischemic skeletal muscle damage in surgery of the extremities using tourniquet ischemia. The concept deserves clinical evaluation.     

Ischemic and pressure-induced hyperemia: a comparison

OBJECTIVE: There is reason to question whether hyperemia after pressure occlusion is caused solely by local ischemia. This study quantitatively compared the response to the two forms of occlusion on the finger. DESIGN: Blood flow was measured by laser Doppler continuously before, during, and for 40 minutes after a 2-minute occlusion of flow at the finger dorsum and at the plantar surface of the finger tip (finger pulp), which has a much higher arteriolar density than the dorsum. Occlusion to the same low level was carried out either with a cuff at the base of the finger or by direct pressure of the laser Doppler probe head. Comparison experiments were performed with the probe head heated to 44 degrees C to elicit maximal local vasodilation. SETTING: Outpatient clinic. PARTICIPANTS: Eleven healthy volunteers. MAIN OUTCOME MEASURES: Magnitude and duration of skin blood flow after occlusion. RESULTS: Cuff occlusion at the base of the finger produced a typical, short-lived hyperemic response at both finger dorsum and finger pulp. The peak level at finger dorsum was 17.6 +/- 1.4mL/min/100g, approximately a twofold increase over the baseline flow level. The duration of the hyperemic response was 3.6 +/- 0.8 minutes. The baseline flow at the finger pulp was three times greater than at the finger dorsum, and peak flow after occlusion was also three times higher (44.3 +/- 2.6 mL/min/100g). The duration of hyperemia at finger pulp was 4.2 +/- 0.9 minutes. After pressure occlusion at the finger dorsum the hyperemic peak was higher (26.7 +/- 4.2 mL/min/100g; p < .05) and the duration of hyperemia was four times longer (16.9 +/- 2.3 minutes; p < .01) than after cuff occlusion. At the finger pulp, the pressure-induced hyperemic peak was also greater than the peak after cuff occlusion (56.3 +/- 1.7mL/min/100g; p < .05), with a longer duration than after cuff occlusion (11.1 +/- 1.1min; p < .01). Thermal stimulation significantly reduced the differences between cuff- and pressure-induced occlusion. There was a slow increase in flow over the 40-minute monitoring period. The maximal flow reached was approximately 100mL/min/100g at both finger dorsum and finger pulp. At both sites, however, the maximal flow level was attained more rapidly than the control condition without prior occlusion. CONCLUSIONS: These results confirmed that the pressure-induced hyperemic response is greater and of longer duration than that produced by flow ischemia alone. Thermal stimulation essentially abolishes the differences, suggesting that there is a common mechanism of vasodilatation. The mechanistic differences between pressure-induced and ischemic hyperemia remain to be determined.     

Cerebral protection by intermittent reperfusion during temporary focal ischemia in the rat

Temporary arterial occlusion has been routinely used as an adjunct in intracranial aneurysm surgery. This has commonly been performed using a protocol of multiple short periods of occlusion alternating with periods of restoration of normal circulation. Recently, the logical basis of this method has come under scrutiny. There is extensive experimental evidence to suggest that repetitive, brief periods of global ischemia may cause more severe cerebral injury than an equivalent single period of global ischemia. Only recently has this issue begun to be addressed with regard to focal ischemia. Hence, despite the common use of temporary clipping, little experimental data are available regarding the ischemic consequences of temporary arterial occlusion with periods of reperfusion versus uninterrupted temporary occlusion. To investigate this issue, a protocol of occlusion/reperfusion that simulates the temporal profile that occurs during surgery was performed in a rat model of focal ischemia. Sixteen anesthetized Sprague-Dawley rats were divided into two groups. The animals in Group I underwent 60 minutes of uninterrupted middle cerebral artery occlusion and the animals in Group II were subjected to six separate 10-minute occlusion periods with 5 minutes of reperfusion between occlusions. Histopathological analysis was performed 72 hours postischemia. Group I had significantly increased mean infarction volumes (50.0 +/- 12.1 mm3) compared to Group II (8.7 +/- 3.1 mm3) (p = 0.008). Injuries in Group I occurred in both the cortex and striatum, whereas Group II showed only striatal injuries. Furthermore, the extent of the injuries in Group II was less severe, characterized by ischemic neuronal injury rather than frank infarction. The results indicate that intermittent reperfusion is neuroprotective during temporary focal ischemia and support the hypothesis that intermittent reperfusion is beneficial if temporary clipping is required during aneurysm repair.     

Influence of central command and ergoreceptors on the splanchnic circulation during isometric exercise

The splanchnic circulation can make a major contribution to blood flow changes. However, the role of the splanchnic circulation in the reflex adjustments to the blood pressure increased during isometric exercise is not well documented. The central command and the muscle chemoreflex are the two major mechanisms involved in the blood pressure response to isometric exercise. This study aimed to examine the behaviour of the superior mesenteric artery during isometric handgrip (IHG) at 30% maximal voluntary contraction (MVC). The pulsatility index (PI) of the blood velocity waveform of the superior mesenteric artery was taken as the study parameter. A total of ten healthy subjects [mean age, 21.1 (SEM 0.3) years] performed an IHG at 30% MVC for 90 s. At 5 s prior to the end of the exercise, muscle circulation was arrested for 90 s to study the effect of the muscle chemoreflex (post exercise arterial occlusion, PEAO). The IHG at 30% MVC caused a decrease in superior mesenteric artery PI, from 4.84 (SEM 1.57) at control level to 3.90 (SEM 1.07) (P = 0.015). The PI further decreased to 3.17 (SEM 0.70) (P = 0.01) during PEAO. Our results indicated that ergoreceptors may be involved in the superior mesenteric artery vasodilatation during isometric exercise.     

Local relation between oxidative metabolism and perfusion in leg muscles of patients with heart failure studied by magnetic resonance imaging and spectroscopy

BACKGROUND: We studied the local relation of muscle perfusion and metabolism in patients with severe chronic heart failure. Alterations of skeletal muscle blood flow and oxidative capacity contribute to exercise intolerance in these patients. The interdependence of both parameters has often been questioned. METHODS AND RESULTS: With the use of nuclear magnetic resonance, we quantified leg and muscle perfusion during reactive hyperemia in 7 patients with heart failure (New York Heart Association class III and IV) and 7 age-matched control subjects from the difference in longitudinal relaxation rate (1/T1). By using 31P nuclear magnetic resonance spectroscopy, we assessed oxidative metabolism from the creatine rephosphorylation time constant after a short ischemic exercise. Phosphocreatine recovery is slowed (74.6 +/- 11.3 vs 49.9 +/- 13.9 seconds, p = .002) and reactive hyperemic flow is reduced (48.5 +/- 24.9 vs 113 +/- 30.4 mL/100 mL per minute, p = .0005). CONCLUSIONS: By using a totally noninvasive protocol, we demonstrated that reactive hyperemic flow correlates with oxidative capacity in calf muscles from patients with heart failure, showing that exercise performance and local circulatory dysfunction are decreased in parallel in severe heart failure.     

Effects of intermittent reperfusion on brain pHi, rCBF, and NADH during rabbit focal cerebral ischemia

BACKGROUND AND PURPOSE: The use of intermittent reperfusion versus straight occlusion during neurovascular procedures is controversial. This experiment studied the effects of intermittent reperfusion and single occlusion on intracellular brain pH (pHi), regional cerebral or cortical blood flow, and nicotinamide adenine dinucleotide (NADH) fluorescence during temporary focal ischemia. METHODS: Twenty fasted rabbits under 1.0% halothane anesthesia were divided into four groups: (1) nonischemic controls, (2) 60 minutes of uninterrupted focal ischemia, (3) 2 x 30-minute periods of focal ischemia separated by a 5-minute reperfusion, and (4) 4 x 15-minute periods of focal ischemia separated by three 5-minute reperfusion periods. Focal ischemia was produced by occlusion of both the middle cerebral and ipsilateral anterior cerebral arteries. After the final occlusion, there was a 3-hour reperfusion period in all groups. Regional cerebral and cortical blood flow, brain pHi, and NADH fluorescence were measured with in vivo panoramic fluorescence imaging. RESULTS: During occlusion, regional cerebral and cortical blood flows and NADH fluorescence values were not different among the groups. Brain pHi was significantly lower in the 4 x 15-minute group compared with the 1 x 60-minute group (6.57 +/- 0.02 versus 6.73 +/- 0.06; P < .03) but not significant when compared with the 2 x 30-minute group. During the short reperfusion periods, all parameters returned to normal except for NADH fluorescence levels, which remained elevated. During the postischemic final reperfusion period, there was a mild brain alkalosis of approximately 7.1 in all groups. There were no significant differences in NADH fluorescence among groups during the final reperfusion. Regional cerebral and cortical blood flow returned to near normal values in all groups. CONCLUSIONS: This study demonstrates that intermittent reperfusion during temporary focal ischemia has different effects on the intracytoplasmic and the intramitochondrial compartments: worsening of brain cytoplasmic pHi but no significant differences in the oxidation/reduction level of mitochondrial NADH.     

Bioenergetics of contracting skeletal muscle after partial reduction of blood flow

The purpose of this study was to examine the bioenergetics and regulation of O2 uptake (VO2) and force production in contracting muscle when blood flow was moderately reduced during a steady-state contractile period. Canine gastrocnemius muscle (n = 5) was isolated, and 3-min stimulation periods of isometric, tetanic contractions were elicited sequentially at rates of 0.25, 0.33, and 0.5 contractions/s (Hz) immediately followed by a reduction of blood flow [ischemic (I) condition] to 46 +/- 3% of the value obtained at 0.5 Hz with normal blood flow. The VO2 of the contracting muscle was significantly (P < 0.05) reduced during the I condition [6.5 +/- 0.8 (SE) ml . 100 g-1 . min-1] compared with the same stimulation frequency with normal flow (11.2 +/- 1.5 ml . 100 g-1 . min-1), as was the tension-time index (79 +/- 12 vs. 123 +/- 22 N . g-1 . min-1, respectively). The ratio of VO2 to tension-time index remained constant throughout all contraction periods. Muscle phosphocreatine concentration, ATP concentration, and lactate efflux were not significantly different during the I condition compared with the 0. 5-Hz condition with normal blood flow. However, at comparable rates of VO2 and tension-time index, muscle phosphocreatine concentration and ATP concentration were significantly less during the I condition compared with normal-flow conditions. These results demonstrate that, in this highly oxidative muscle, the normal balance of O2 supply to force output was maintained during moderate ischemia by downregulation of force production. In addition, 1) the minimal disruption in intracellular homeostasis after the initiation of ischemia was likely a result of steady-state metabolic conditions having already been activated, and 2) the difference in intracellular conditions at comparable rates of VO2 and tension-time index between the normal flow and I condition may have been due to altered intracellular O2 tension.     

Near-infrared spectroscopy for monitoring of tissue oxygenation of exercising skeletal muscle in a chronic compartment syndrome model

Variations in the levels of muscle hemoglobin and of myoglobin oxygen saturation can be detected non-invasively with near-infrared spectroscopy. This technique could be applied to the diagnosis of chronic compartment syndrome, in which invasive testing has shown increased intramuscular pressure associated with ischemia and pain during exercise. We simulated chronic compartment syndrome in ten healthy subjects (seven men and three women) by applying external compression, through a wide inflatable cuff, to increase the intramuscular pressure in the anterior compartment of the leg. The tissue oxygenation of the tibialis anterior muscle was measured with near-infrared spectroscopy during gradual inflation of the cuff to a pressure of forty millimeters of mercury (5.33 kilopascals) during fourteen minutes of cyclic isokinetic dorsiflexion and plantar flexion of the ankle. The subjects exercised with and without external compression. The data on tissue oxygenation for each subject then were normalized to a scale of 100 per cent (the baseline value, or the value at rest) to 0 per cent (the physiological minimum, or the level of oxygenation achieved by exercise to exhaustion during arterial occlusion of the lower extremity). With external compression, tissue oxygenation declined at a rate of 1.4 +/- 0.3 per cent per minute (mean and standard error) during exercise. After an initial decrease at the onset, tissue oxygenation did not decline during exercise without compression. The recovery of tissue oxygenation after exercise was twice as slow with compression (2.5 +/- 0.6 minutes) than it was without the use of compression (1.3 +/- 0.2 minutes).     

Effects of a muscle exercise program on exercise capacity in subjects with osteoarthritis

Maximal aerobic power and muscle function have been shown to decrease with age and to be even lower in patients with osteoarthritis (OA). This study was designed to determine if subjects with OA who underwent only a muscle exercise program had improved exercise capacity and cardiovascular fitness. A maximal graded exercise test was given before and after 3 months of exercise (isometric, isotonic, and isometric force generated as a function of time contractions, three times a week). Maximal strength and the tension-time index improved significantly. Peak aerobic power increased from 15.99 +/- 3.96 mL.kg-1.min-1 to 20.34 +/- 3.29 mL.kg-1.min-1. On average, maximal walking speed increased from 2.0 +/- 0.6 mph to 2.4 +/- 0.7mph. Exercise time increased 22%, from 9.2 +/- 2.3 minutes to 11.2 +/- 2.7 minutes. There were significant reductions in submaximal heart rate (15b.min-1) and systolic blood pressure (15mmHg) after training. It would appear that the reduction in aerobic fitness of subjects with OA is secondary to their reduced muscle function. By improving muscle function, increases in exercise capacity and aerobic fitness occurred.     

Ischemic preconditioning at a distance: reduction of myocardial infarct size by partial reduction of blood supply combined with rapid stimulation of the gastrocnemius muscle in the rabbit

BACKGROUND: Limitation of myocardial infarct size by an earlier brief complete occlusion of a coronary artery is defined as ischemic preconditioning. However, myocardial protection also can be achieved by partial reduction of coronary flow, rapid cardiac pacing, or brief ischemia-reperfusion of a remote region of the heart. Our study assesses the effect on myocardial infarct size of preconditioning at a distance induced by partial reduction of blood flow to a hind limb with or without increase of demand by electrical stimulation of a skeletal muscle. METHODS AND RESULTS: Anesthetized rabbits were randomized to 30 minutes of waiting period (controls), 55% to 65% reduction of femoral artery blood flow (stenosis), electrical stimulation of the gastrocnemius muscle at a rate of one per second (stimulation), or stenosis+stimulation. Thereafter, rabbits underwent 30 minutes of coronary artery occlusion and 4 hours of reperfusion. Each group included 8 rabbits. Risk zones were comparable among groups. However, the ratio of infarct size to risk zone was smaller in the stenosis+stimulation group (0.09+/-0.02) compared with the control (0.26+/-0.03), stenosis (0.36+/-0.05), and stimulation (0.30+/-0.05) groups (P=.0006). ANCOVA performed on the fraction of infarction (infarct size/left ventricular weight) and the fraction of risk zone revealed a significant group effect (P=.0004). CONCLUSIONS: Remote ischemia of a skeletal muscle induced by muscle stimulation combined with restriction of blood flow preconditioned the myocardium. The combination of muscle stimulation with reduction of femoral arterial blood flow but not muscle stimulation without blood flow restriction or of flow restriction without muscle stimulation reduced myocardial infarct size considerably.     

Ischemic-reperfused rat skeletal muscle: the effect of vasoactive intestinal peptide (VIP) on contractile force, oxygenation and antioxidant enzyme systems

The effect of vasoactive intestinal peptide (VIP) on the nerve-stimulated contraction, tissue oxygenation, lipid peroxidation and antioxidant enzymes activities-superoxide dismutase and catalase was investigated in the rat gastrocnemius muscle exposed to 4 h ischemia-4hr reperfusion. Ischemia caused significant decrease in muscle contractile force, oxygenation and superoxide dismutase enzyme activity. Reperfusion of ischemic muscle increased the muscle contractile force and restored the tissue oxygenation to the baseline level. Superoxide dismutase and catalase activities of reperfused muscle increased significantly. However neither ischemia nor reperfusion affected gastrocnemius muscle malondialdehide (MDA) levels. VIP administration at the onset of reperfusion significantly increased skeletal muscle contractile force and tissue oxygenation even higher than baseline and reperfusion values. VIP also normalized the increased superoxide dismutase and catalase activities of reperfused skeletal muscle. In conclusion, VIP, acting as a powerful antioxidant and preserving contractile machinery seems to be a promising endogenous peptide that can salvage the skeletal muscle from severe ischemia-reperfusion injury.     

A novel approach for comparative study of periosteum, muscle, subcutis, and skin microcirculation by intravital fluorescence microscopy

Herein, we report a new model, which allows comparative study of the microcirculation of different peripheral tissues, i.e., periosteum, skeletal muscle, subcutis, and skin. Using dextran-insensitive Wistar rats gracilis and semitendinosus muscles of the left hindlimb were prepared in association with their appertaining tibial fragments, subcutis, and skin. Blood supply was guaranteed by the femoral artery via the saphenous vessels. High-resolution intravital epi-illumination microscopy of the two muscles displayed the typical microvascular architecture with the capillaries running in parallel to each other (capillary density (CD) 128.4 +/- 4.5 cm-1). In subcutis and skin, capillaries were found arranged as interconnecting mesh-like networks with a density, which was significantly higher (P < 0.05) in subcutis (191.0 +/- 5.5 cm-1) compared with skin (108.9 +/- 3.3 cm-1). Analysis of periosteal tissue revealed two distinct types of arrangements of microvascular architecture. Adjacent to the major feeding and draining vessels of the periosteum, capillaries were organized in densely meshed shunt-like networks, revealing the highest capillary density (242.7 +/- 13.2 cm-1; P < 0.05) of all tissues studied. Periosteal capillaries distant from the major feeding and draining vessels were arranged in parallel to the longitudinal axis of the tibial bone and presented with a density similar to that of the skeletal muscle (128. 6 +/- 9.4 cm-1). Topical application of acetylcholine for analysis of physiological reactivity of the microvasculature showed dose-dependent arteriolar dilation. Moreover, a 3-min upstream femoral artery occlusion demonstrated an appropriate hyperemic response in all tissues studied, indicating intact myogenic control. A prolonged period of ischemia (120 min) followed by reperfusion (60 min) caused massive (P < 0.05) leukocyte-endothelial cell interaction in postcapillary venules, similarly as reported in other microvascular tissue preparations. We propose that the model presented provides a good approach to all peripheral tissues for both the analysis of the physiology of tissue-confined microvascular control and the development of novel therapeutic strategies to counteract manifestation of nutritional dysfunction and inflammatory response in disease.     

Estimation of active force-length characteristics of human vastus lateralis muscle

The length and angles of fascicles were determined for the vastus lateralis muscle (VL) using ultrasonography in 6 subjects performing ramp isometric knee extension. The subject increased torque from zero (relax) to maximum (MVC) with the knee positioned every 15 degrees, from 10 degrees to 100 degrees flexion (0 degrees = full extension). As the knee was positioned closer to extension, fascicle length was shorter [116 +/- 4.7 (mean +/- SEM) mm at 100 degrees vs. 88 +/- 4.1 mm at 10 degrees (relax)]. The fascicle length of the VL decreased with increasing torque at each knee position [116 +/- 4.7 (relax) to 92 +/- 4.3 mm (MVC) at 100 degrees]. On the other hand, fascicle angles increased with an increase in torque. These changes reflected the compliance of the muscle-tendon complex which increased as the knee reached a straight position. The estimated muscle force of the VL was maximal (2,052 +/- 125 N) for a fascicle length of 78 +/- 2.7 mm (i.e. optimum length) with the knee positioned at 70 degrees of flexion. The relationship between muscle force and fascicle length indicated that the VL uses the ascending (knee < 70 degrees), plateau (70 degrees), and descending regions (> 70 degrees) of the force-length curve.     

Changes in vessel ultrastructure during ischemia and reperfusion of rabbit hindlimb: implications for therapeutic intervention

Peripheral ischemia was induced in the rabbit by occlusion of the left iliac artery for 6 hr, followed by 24 hr of reperfusion. Biochemical and morphological investigations were performed to evaluate the extent of vascular and tissue injury. Blood samples for plasma enzyme determinations (creatine kinase (CK) and lactate dehydrogenase (LDH) activities) were obtained at times t = 0, t = 6, t = 30 hr. Plasma CK and LDH activities in ischemic animals were approximately twice as high as those in sham-operated animals at the end of reperfusion, although no difference was observed at the end of the period of ischemia. Morphological and morphometric analysis of extensor digitorum longus muscle from ischemic animals showed a reduction in the number of patent capillary vessels per muscle fiber (1.54 +/- 0.1 and 1.04 +/- 0.09, P < 0.05, in sham and ischemic groups, respectively; mean +/- SEM). In addition, the number of microvilli on endothelial surfaces were considerably increased in the ischemic group (0.14 +/- 0.02 and 0.41 +/- 0.01 microns -2, P < 0.001, in sham and ischemic groups, respectively). A great number of adhered leucocytes were found on the vessel surface with some leucocytes having migrated through the vessel wall. Microcirculatory damage was accompanied by the formation of microthrombi which sometimes occluded the entire vessel lumen. The infusion of 1 mg/kg/hr of cloricromene for 6 hr prevented ischemic injury in microvessels and also prevented swelling of muscle mitochondria. In the treated group the number of patent capillaries per muscle fiber was very similar to that found in sham-operated animals (1.49 +/- 0.08; P < 0.01 vs. ischemic control). In conclusion, several different cell types are involved in the pathophysiological changes which occur in microvessels during ischemia/reperfusion injury. Pharmacological interventions, which inhibit the interactions of blood cells with endothelium, may be of value in the treatment of peripheral ischemia/reperfusion injury.     

Superoxide anion production during reperfusion is reduced by an antineutrophil antibody after prolonged cerebral ischemia

Neutrophils may be involved in the pathophysiology of reperfusion injury following cerebral ischemia. One potential mechanism of reperfusion injury by neutrophils is through production of the superoxide anion. We hypothesized that, due to progressive endothelial damage during ischemia, neutrophil activation would be more prominent after longer periods of ischemia prior to reperfusion. Thus, neutrophils would contribute more to pathological processes such as superoxide anion formation after longer than after shorter periods of ischemia. A reversible middle cerebral artery occlusion model in rats was employed and superoxide anion concentration was measured with a cytochrome c coated electrode placed on the cortical penumbral region. Occlusion times were varied from 60 min to 2 h, and neutrophils were inhibited with an antiCD18 antibody administered prior to occlusion. Neutrophil accumulation and reduction with antibody treatment was confirmed immunohistochemically. Superoxide anion (O2*-) concentration was detected during the hours following 60 min of occlusion, and increased further with 2 h of occlusion. Treatment with the antiCD18 antibody had no effect on O2*- concentration during reperfusion in the 60-90 min occlusion groups, but O2*- concentration was significantly lower in the antiCD18 antibody treated group than in the control group during reperfusion after 120 min of ischemia. The antibody also reduced cortical neutrophil accumulation in the 120 min ischemia group. These results indicate for the first time that superoxide production by neutrophils becomes more important with longer periods of ischemia, and other quantitatively less important sources of superoxide predominate with shorter periods of ischemia. This phenomenon may explain some of the variation seen between different models of ischemia with different durations of ischemia when targeting reactive oxygen species, and supports an approach to combination therapy to extend the therapeutic window and reduce the deleterious effects of reperfusion.     

A dynamic model for simulating movements of the elbow, forearm, an wrist

We developed a dynamic model of the upper extremity to simulate forearm and wrist movements. The model is based on the skeletal structure of the arm and is capable of elbow flexion/extension, forearm pronosupination, and wrist flexion/extension and radial/ulnar deviation movements. Movements are produced by activation of a Hill-type model of muscle, and limits on joint motion are imposed by passive moments modeled after experimental results. We investigated the muscle output force sensitivity, as well as wrist flexion/extension motion sensitivity to parameter variations. The tendon slack length and muscle fiber length were found to have the greatest influence on muscle output and flexion/extension wrist motion. The model captured the direction of the moment vectors at the wrist well, but predicted much higher moments than were measured by stimulating the paralyzed muscles of one tetraplegic subject.     

Acetazolamide reduces peripheral afferent transmission in humans

Carbonic anhydrase has been localized in skeletal muscle and nerve, thus, inhibition with acetazolamide (ACZ) may alter nerve and/or muscle function in healthy humans. ACZ (3 oral doses 14, 8, and 2 h prior to testing) reduced isometric force (37%) and peak to peak electromyographic (EMG) amplitude (1.38 mV to 0.83 mV), while increasing EMG latency associated with a unilateral Achilles tendon-tap. Reflex recovery profiles, following a contralateral conditioning tap, were similar in both placebo and ACZ experiments. ACZ led to significant changes in Hmax/Mmax ratio (52.19/14.42 to 45.73/15.65) and H-reflex latency (34.18 +/- 2.54 ms to 35.24 +/- 2.74 ms). Motor nerve conduction velocity and maximal voluntary isometric torque (knee extensors) were unaltered by ACZ. These data suggest that inhibition of the tendon-tap reflex and associated isometric force, following ACZ, is related to impairment of synaptic integrity between la fibers of the muscle spindle and the alpha motor neuron and not impairment of the muscle spindle or force-generating capacity.     

Differential nitric oxide and TNF-alpha production of murine Kupffer cell subfractions upon priming with IFN-gamma and TNF-alpha

The effects of a nitric oxide (NO) donor on microcirculation and contractile function of reperfused skeletal muscle were studied. Rat cremaster muscles underwent 5 hours of ischemia and 90 minutes of reperfusion and were divided into two groups systemically infused with S-nitroso-N-acetylcysteine (SNAC, 100 nmol/min) and phosphate-buffered saline (PBS), respectively. The results showed that the vessels in the SNAC group had more rapid and complete recovery than that in controls. A significant difference was found from 10 to 40 minutes and at 90 minutes in 10-20-microm arterioles, from 10 to 90 minutes in 20-40-microm arterioles, and at 10 and 90 minutes in 40-70-microm arteries. When compared to controls, SNAC-treated muscles showed larger fluorescein filling areas at 15, 30, 60, and 90 minutes and greater isometric tetanic contractile forces in response to stimulation frequencies of 40, 70, 100, and 120 Hz. The data indicate that supplementation of exogenous NO could effectively improve microcirculation and contractile function of skeletal muscle during early reperfusion.     

Inhibition of neutrophil adhesion does not prevent ischemic spinal cord injury

Paraplegia may occur after transient aortic occlusion as a consequence of primary ischemia to the spinal cord or injury during the reperfusion period. In animal models of ischemia/reperfusion there is evidence that reperfusion injury may be modulated partially by neutrophils. The efficacy of the neutrophil adherence blocking murine monoclonal antibody (MAb 60.3) was assessed in spinal cord ischemia/reperfusion in rabbits. Spinal cord ischemia was accomplished by balloon catheter occlusion of the infrarenal aorta. Neurologic assessment was graded as normal, partial neurologic deficit, or complete paralysis. Electrophysiologic monitoring with somatosensory evoked potentials was used to determine the optimal length of time of occlusion. Animals were treated randomly with 2 mg/kg of intravenous Mab 60.3 (n = 8) or saline solution (n = 9) with the investigator unaware of treatment. Mean occlusion times were no different between groups (control, 32.7 +/- 3.6 minutes versus MAb, 32.4 +/- 6.0 minutes). Five (55%) saline-treated and four (50%) MAb 60.3-treated animals became paraplegic. Animals with initial paraparesis all progressed to flaccid paraplegia within 24 hours. We conclude that spinal cord injury after transient aortic occlusion is independent of the CD11/CD18 glycoprotein complex of the neutrophil. Injury in this setting may occur during ischemia and thus may not be dependent on neutrophils or reperfusion.