Sustained benefit of temporary limited reperfusion in skeletal muscle following ischemia

Limiting the rate of reperfusion blood flow following prolonged ischemia in skeletal muscle has been shown beneficial. However, the persistence of this benefit with reinstitution of normal blood flow remains undefined. We investigated the role of temporary limited reperfusion on ischemia-reperfusion injury in an isolated gracilis muscle model in six anesthetized dogs. Both gracilis muscles were subjected to 6 hr of ischemia followed by 2 hr of reperfusion. Reperfusion blood flow was limited for the first hour in one gracilis muscle to its preischemic rate followed by a second hour of normal reperfusion (LR/NR). The contralateral muscle underwent 2 hr of normal reperfusion (NR/NR). Muscle injury was quantified by technetium-99m pyrophosphate (TcPyp) uptake and by histochemical staining using triphenyltetrazolium chloride (TTC) with planimetry of the infarct size. Capillary permeability was evaluated by muscle weight gain. Results are reported as the mean +/- SEM: [table: see text] These data demonstrate a sustained benefit from temporary limited reperfusion. This methodology should be considered in the surgical management of the acutely ischemic limb.     

Rate of reperfusion blood flow modulates reperfusion injury in skeletal muscle

The mechanisms of ischemia-reperfusion (I-R) injury in skeletal muscle remain controversial. We investigated the effect of the rate of reperfusion blood flow on I-R injury in an isolated in vivo canine gracilis muscle model in six anesthetized dogs. In all animals, both gracilis muscles were subjected to 6 hr of ischemia followed by 1 hr of reperfusion. During reperfusion, one gracilis artery was partially occluded to limit the rate of reperfusion blood flow to its preischemic rate (limited reperfusion, LR), while the contralateral artery was allowed to perfuse freely at a normal rate (normal reperfusion, NR). Muscle injury was quantified by histochemical staining (triphenyltetrazolium chloride, TTC) with computerized planimetry of the infarct size, and by spectrophotometric determination of technetium-99m pyrophosphate uptake. Endothelial permeability was quantified by measurement of gracilis muscle weight gain and 125I-albumin radioactivity after intravenous injection. Results are presented as the means +/- SEM, and differences are considered to be statistically significant if P less than 0.05 by Student's t test for paired data. LR resulted in significantly less blood flow (9.7 +/- 1.7 cc/min/100 g) when compared to NR (55.7 +/- 11.6 cc/min/100 g). I-R injury was significantly reduced by LR as evidenced by a decrease in TTC infarct size from 41 +/- 7% to 11 +/- 5%, and a decrease in technetium-99m pyrophosphate uptake from 512 +/- 20 to 163 +/- 44 X 10(3) counts/min/g. LR also significantly decreased the postreperfusion edema formation as evidenced by a reduction in the muscle weight gain from 27 +/- 6 to 9 +/- 1 g, and a reduction in the 125I-albumin radioactivity from 45 +/- 14 to 32 +/- 8 counts/min/g. These data suggest that the hyperemic rate of reperfusion blood flow is a significant factor in the pathophysiology of postreperfusion edema and that clinical control of reperfusion injury in skeletal muscle may be achieved by limiting the rate of reperfusion blood flow.     

Interleukin-1 and thromboxane release after skeletal muscle ischemia and reperfusion

Interleukin-1 and thromboxane are known to mediate the host response to sepsis, trauma, and myocardial ischemia. A well-established model of canine isolated gracilis muscle was used to evaluate whether cytokine (interleukin-1) played a role in skeletal muscle ischemia-reperfusion injury. Six adult mongrel dogs (25–30 kg) were subjected to six hours of muscle ischemia followed by reperfusion. Gracilis venous samples were collected pre-ischemia and at one hour of reperfusion. Systemic (arterial) blood samples were taken at one hour of reperfusion. Sera were analyzed for interleukin-1 by bioassay and thromboxane (B₂) by radio-immunoassay. The gracilis muscle of the operated limb was harvested in all the animals for assessment of the percentage of muscle necrosis. This was found to be 56.2±14.8% by serial transections, nitroblue tetrazolium staining, and computerized planimetry. Interleukin-1 levels in the gracilis venous effluent increased from 21.88±7.13 units/ml during pre-ischemic baseline to 50.42±9.12 units/ml after six hours of ischemia followed by one hour of reperfusion (p<0.04). Thromboxane B₂ levels were 2983±1083 pg/ml and 9483±2218 pg/ml at pre-ischemia and at one hour of reperfusion respectively (p<0.04). Systemic levels of both interleukin-1 and thromboxane B₂ at one hour of reperfusion were 0 units/ml and 1584±520 pg/ml respectively, which were significantly lower than the one hour reperfusion gracilis venous effluent levels (p<0.04). This is the first report in which cytokines have been implicated in skeletal muscle ischemia-reperfusion injury. Modulation of interleukin-1 may impact positively on muscle necrosis and systemic manifestations of reperfusion injury.Presented at the 16th Annual Meeting of the Peripheral Vascular Surgery Society, June 2, 1991, Boston, Massachusetts.     

The influence of arachidonic acid metabolites on leukocyte activation and skeletal muscle injury after ischemia and reperfusion.

Derivatives of arachidonic acid have been found to play a role in the reperfusion injury of various tissues. These compounds have a broad spectrum of activity, including modulation of white blood cell response to injured tissue. This study was designed to determine the effect of thromboxane and lipoxygenase derivatives on the local and systemic response to ischemia and reperfusion of skeletal muscle. Fifteen dogs were separated into three groups and subjected to gracilis muscle ischemia followed by 2 hours of reperfusion. One group served as controls, one group was treated with OKY-046 (a thromboxane synthetase inhibitor), and one group was treated with diethylcarbamazine (a lipoxygenase inhibitor). White blood cell activation as measured by superoxide anion production, and eicosanoid levels were measured both in the gracilis venous effluent and central venous circulation. These results were compared to infarct size in the gracilis muscle. OKY-046 significantly reduced thromboxane production in both the central venous (102 +/- 30 to 31 +/- 9 pg/ml, p less than 0.05) and gracilis samples (107 +/- 22 to 25 +/- 6 pg/ml, p less than 0.005). This was accompanied by a reduced white cell activation in the central venous blood (15 +/- 1 to 10 +/- 1 nmol O2-, p less than 0.05), but did not affect infarct size or white cell activation in the gracilis. Conversely, diethylcarbamazine significantly reduced both white cell activation (16 +/- 1 to 10 +/- 1 nmol O2-, p less than 0.005) and infarct size in the gracilis muscles (61.6% +/- 4.5% to 28.5% +/- 8.6%, p less than 0.01), as well as reduced systemic white blood cell activation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Technetium 99m pyrophosphate quantitation of skeletal muscle ischemia and reperfusion injury

The study of ischemia and reperfusion injury in the extremity has been hampered by lack of an accurate method of measuring skeletal muscle injury. We used a bilateral isolated in vivo canine gracilis muscle model in 15 anesthetized dogs. The experimental muscles had 4, 6, or 8 hours of ischemia and 1 hour of reperfusion. The contralateral gracilis muscle served as a control. Technetium 99m pyrophosphate (99mTc-PYP), an agent which localizes in injured muscle cells, was used to quantitate canine skeletal muscle damage. After 6 hours of ischemia and 1 hour of reperfusion, there was a significant increase of 215% of 99mTc-PYP uptake in the experimental vs the control muscle. Experimental muscle uptake was 8% greater than control after 4 hours and 405% more after 8 hours of ischemia and reperfusion. Segmental distribution of 99mTc-PYP uptake showed localization to be greatest in the middle of the muscle at the entry site of the gracilis artery. Electron microscopic evaluation also documented this area to have undergone the most severe injury. Distal portions of the muscle did not show increased damage. Our results show that 99mTc-PYP effectively quantitates skeletal muscle ischemia and reperfusion injury. The pattern of 99mTc-PYP uptake suggests that considerable injury is caused during reperfusion.     

Heparin decreases ischemia-reperfusion injury in isolated canine gracilis model

The mechanisms of ischemia-reperfusion injury in skeletal muscle remain controversial. Some investigators have demonstrated that heparin can ameliorate ischemic injury to heart, brain, and renal tissue. We investigated the ability of heparin sodium to decrease ischemia-reperfusion injury in an isolated gracilis muscle model in ten anesthetized mongrel dogs. One gracilis muscle was perfused normally while the contralateral muscle was subjected to six hours of ischemia followed by one hour of reperfusion. Five dogs were given a preischemic bolus of heparin sodium (200 U/kg, intravenously followed by a continuous infusion (15 U/kg/h, intravenously), and five control dogs received no heparin. Quantitation of skeletal muscle ischemia-reperfusion injury was determined by histochemical staining with triphenyl tetrazolium-chloride and computerized planimetry of the infarct size. Results from the ischemic muscle demonstrate a significant beneficial effect of heparinization. The nonheparinized dogs had a 72% +/- 5% infarct size, which was significantly reduced to 24% +/- 8% in the heparinized dogs. The mechanism of this protective effect may be due to heparin's anticoagulant, antiplatelet, or anti-inflammatory action.     

The value and limitation of iloprost infusion in decreasing skeletal muscle necrosis.

Iloprost has been shown to minimize skeletal muscle necrosis when administered before the onset of ischemia in animal experiments, possibly by preventing neutrophil activation. Since patients with acute limb ischemia are seen after the process has begun, we investigated whether iloprost can be protective when given only during reperfusion. After anesthesia, 18 adult mongrel dogs underwent a standard isolated gracilis muscle preparation. In six control animals (group I) the gracilis muscle was subjected to 6 hours of ischemia followed by 48 hours of reperfusion. Group II animals (n = 6) received intravenous infusion of iloprost at a dose of 0.45 microgram/kg/hr beginning 1 hour before the onset of muscle ischemia and throughout the experiment (6 hours of ischemia and 1 hour of reperfusion). In addition to the continuous infusion, they received 0.45 microgram/kg intravenous boluses of iloprost 10 minutes before the induction of ischemia and 10 minutes before reperfusion. Group III animals (n = 6) had a similar ischemic interval, but were given a bolus of iloprost of 0.45 microgram/kg at end ischemia followed by continuous infusion of 0.45 microgram/kg/hr for 48 hours during reperfusion. Muscle biopsies were obtained at baseline and after 1 hour of reperfusion in all groups. Additional biopsies were obtained at 48 hours of reperfusion in groups I and III. Myeloperoxidase activity, a marker of neutrophil activation, was measured in all muscle biopsies. At the end of reperfusion, the gracilis muscle was harvested in all animals and weighed. Muscle necrosis was estimated by serial transection, nitroblue tetrazolium histochemical staining followed by computerized planimetry.(ABSTRACT TRUNCATED AT 250 WORDS)     

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)     

A clinically applicable method for long-term salvage of postischemic skeletal muscle.

The clinical significance and applicability of interventions aimed at reducing reperfusion injury in postischemic skeletal muscle remain unproven, since long-term muscle salvage has not been demonstrated by most treatment protocols that attenuate early reperfusion injury. We have shown that reperfusion of ischemic skeletal muscle results in an early and prolonged sequestration of white blood cells and activation of the alternative complement cascade. The purpose of this study was to determine if 40 minutes of reperfusion with blood depleted of white blood cells and complement proteins, followed by 2 days of normal perfusion, would reduce muscle necrosis after 5 hours of ischemia. The isolated paired canine gracilis muscle model was used. The treatment muscle was initially reperfused with arterial blood that had been spun, washed, passed through a leukocyte removal filter, and resuspended in hydroxyethyl starch (greater than 99.9% removal of white blood cells and the complement proteins factor B and C4). The contralateral control muscle was subjected to unaltered reperfusion. Blood flow (ml/min/100 gm) was measured by timed collection of gracilis venous blood. Myeloperoxidase activity (absorbance at 655 nm/min/mg tissue protein) in muscle biopsies was used to monitor white blood cell sequestration. After 48 hours of reperfusion in vivo, necrosis was quantified by nitroblue tetrazolium staining. Initial reperfusion with white blood cell and complement depleted blood significantly reduced muscle necrosis (53% +/- 3% vs 29% +/- 8%, p less than 0.0025, paired t test). Early blood flow was improved, (p = 0.0025, repeated measure-ANOVA), but subsequent white blood cell sequestration was not altered (p = 0.33, repeated measure-ANOVA). This suggests that a significant amount of white blood cell mediated injury occurs during the first 40 minutes of reperfusion. Preventing early complement activation and white blood cell mediated reperfusion injury is an intervention that is feasible during surgery and may result in clinically significant salvage of postischemic skeletal muscle.     

Leukocyte activation in ischemia-reperfusion injury of skeletal muscle

Polymorphonuclear leukocyte (PMN) participation in the pathophysiology of the reperfusion injury following skeletal muscle ischemia has become recognized. We measured the activation of PMNs as evidenced by production of superoxide anion (O2-) in the isolated canine gracilis muscle preparation of ischemia-reperfusion injury. PMNs were isolated from the gracilis muscle venous effluent and central venous blood after 6 hr of bilateral gracilis ischemia and 1 hr of reperfusion in five dogs. Baseline samples were obtained prior to ischemia from the central venous circulation. Liberation of O2- from PMNs and from PMNs stimulated by opsonized zymosan was determined by measuring ferricytochrome reduction. Results are expressed as nanomoles of O2- produced/2 x 10(6) PMN +/- SEM. O2- production by unstimulated cells was increased from 0.33 +/- 0.15 nmole in the baseline samples to 0.96 +/- 0.08 nmole in the central venous sample (P less than 0.01). With stimulation by zymosan, production increased from 10.3 +/- 1.4 nmole in the baseline samples to 15.2 +/- 1.1 nmole in the central venous sample (P less than 0.05) and to 15.5 +/- 0.9 nmole in the gracilis venous sample (P less than 0.01). These increases in superoxide production were not seen in the three sham-operated animals. Mean infarct size determined by planimetry was 55%. O2- produced by PMNs from central venous blood correlated with infarct size (r = 0.934, P = 0.02). These data imply that PMNs are activated by muscular ischemia, and the degree of activation is directly related to the extent of the muscle infarction.     

Age-related differences of neutrophil activation in a skeletal muscle ischemia-reperfusion model

Free tissue transfers and replantation of amputated limbs are better tolerated by young adolescents than mature adults. The authors hypothesized that this observation may be, in part, because of an attenuated ischemia-reperfusion (IR) injury in younger patients. Because neutrophils have been identified as a critical cell line responsible for IR injury, the authors investigated the effects of animal age on the degree of neutrophil activation in a rat model. Activation was evaluated by monitoring expression of integrin surface markers (mean fluorescence intensity [MFI] of CD11b) and oxidative burst potential (MFI of dihydrorhodamine [DHR] oxidation) by flow cytometry in neutrophils analyzed after 4 hours of ischemia and 1, 4, and 16 hours of reperfusion in a gracilis muscle flap model in mature adult and young adolescent rats. Neutrophil activation was also evaluated in control sham-operated animals, which underwent elevation of gracilis muscle flaps without exposure to an ischemic insult. Muscle edema, determined by wet-to-dry muscle weight ratio, and muscle viability, determined by nitro blue tetrazolium (NBT) staining, were completed for gracilis muscles exposed to ischemia after 24 hours of reperfusion for each of the groups. Integrin expression, assessed by MFI of CD11b, was increased significantly in ischemic muscles of mature adult rats at 4 hours of reperfusion (71.10+/-3.53 MFI vs. 54.88+/-12.73 MFI, p=0.025). Neutrophil oxidative potential, assessed by MFI of DHR oxidation, was increased significantly in ischemic muscles of mature adult rats compared with young adolescent rats at 1 hour of reperfusion (78.10+/-9.53 MFI vs. 51.78+/-16.91 MFI, p=0.035) and 4 hours of reperfusion (83.69+/-15.29 MFI vs. 46.55+/-8.09 MFI, p=0.005). Increased edema formation was observed in the ischemic muscles of mature adult rats when compared with young adolescent rats (1.25+/-0.04 vs. 1.12+/-0.05, p=0.031) after 24 hours of reperfusion. A trend toward decreased muscle viability was observed in the mature adult rats when compared with young adolescent rats (23.7+/-3.1% NBT staining vs. 32.3+/-13.7% NBT staining, p=0.189) after 24 hours of reperfusion. The authors present evidence of an attenuated IR injury in young adolescent animals when compared with mature adult rats. These findings emphasize the importance that studies involving IR injury should be performed with consideration of animal age.     

Iloprost infusion decreases skeletal muscle ischemia-reperfusion injury.

Iloprost (a long-acting prostacyclin analog) has been demonstrated to decrease cardiac muscle infarct size after ischemia-reperfusion injury. We investigated the ability of iloprost to decrease skeletal muscle injury and platelet sequestration after ischemia-reperfusion injury in a canine bilateral isolated gracilis muscle model. Anesthesized animals (n = 13) were subjected to 6 hours of gracilis muscle ischemia and 1 hour of reperfusion. Fifteen minutes before muscle reperfusion, the animals were infused with radium 111-labeled autogenous platelets. Experimental animals (n = 7) received a continuous preischemic intravenous infusion of iloprost (0.45 microgram/kg/hr) and two 0.45 microgram/kg intravenous injections of iloprost (10 minutes before the ischemic interval and 10 minutes before reperfusion). Muscle injury was measured with triphenyltetrazolium chloride histochemical staining. Platelet sequestration within ischemic muscle specimens was determined by measuring indium 111 activity in a gamma counter. Iloprost infusion decreased muscle infarct size from 57.0% +/- 12.6% in control animals to 15.8% +/- 4.4% in experimental animals (p less than 0.05). Platelet uptake in experimental and control muscle was 1.2 +/- 0.21 x 10(7) and 2.17 +/- 0.48 x 10(7) platelets/gm ischemic muscle, respectively (p = 0.1). Although platelet sequestration was not altered significantly in this experiment, a reduction in skeletal muscle injury was confirmed. Further investigation on the mechanisms of action of iloprost in chronic and acute skeletal muscle ischemia is warranted.     

Superoxide anion production by leukocytes exposed to post-ischemic skeletal muscle.

Superoxide anion (O2-) and polymorphonuclear leukocytes (PMNs) have been implicated in the genesis of skeletal muscle ischemia-reperfusion (I-R) injury, but the source of (O2-) has not been established. We studied PMNs as a potential source of O2- using a ferricytochrome reduction assay in 5 anesthetized dogs. Using a gracilis muscle model of I-R, 6 hours of ischemia was followed by 2 hours of reperfusion. The contralateral muscle served as control. Prior to ischemia and after 0.5 and 2.0 hours of reperfusion, PMNs were separated from the gracilis venous effluent of ischemic (I) and control (C) muscles. Central venous samples were also obtained prior to surgical preparation and after reperfusion. Assays for O2- were performed with and without zymosan (Z) activation. Results are expressed as nmol O2-/2 x 10(6) PMNs +/- SEM. Baseline production of O2- was 0.49 +/- 0.54 in central venous samples; Z increased the values to 6.77 +/- 2.13. After 2 hrs of reperfusion, central O2- was 1.57 +/- 0.75, which increased to 7.1 +/- 1.04 with Z. Gracilis venous samples O2- values with and without Z are reported in Table I. One way measures of analysis of variance showed no significant (p > 0.05) differences between samples. Our results demonstrate that PMNs are not the sole source of O2- in the pathophysiology of skeletal muscle I-R injury. PMN associated injury may be mediated by mechanisms other than O2- production.     

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)     

Regional hypothermia protects against ischemia-reperfusion injury in isolated canine gracilis muscle

Regional hypothermia is known to protect many tissues from ischemic injury. We investigated the relationship between regional hypothermia and skeletal muscle ischemia-reperfusion injury in a bilateral in vivo isolated canine gracilis muscle model. In five anesthetized dogs, one gracilis muscle was subjected to 6 hours of ischemia followed by 1 hour of reperfusion while the contralateral muscle served as a nonischemic control. Localization and quantitation of skeletal muscle injury was determined by histochemical staining with triphenyl tetrazolium chloride (TTC) followed by computerized planimetry of the infarct size. Muscle pH and temperature were monitored continuously in the proximal, middle, and distal segments by using pH electrodes and needle thermistors. Muscle pH was calculated by use of the Nernst equation with temperature correction, and hydrogen ion washout rates (H+) were derived from the observed change in muscle pH during reperfusion. A significant (p less than 0.05) regional hypothermia was observed in the distal third of the muscle. The preischemic temperature in the distal muscle was 27 +/- 2 degrees (SEM) C, compared to 34 +/- 1 degree and 32 +/- 2 degrees C in the proximal and middle segments of muscle, respectively. This temperature gradient was sustained throughout the experiment. The distal third of the ischemic muscle demonstrated significantly less (p less than 0.05) injury than the proximal and middle thirds as measured by TTC infarct size (31 +/- 10%, compared to 71 +/- 3% and 78 +/- 6%, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Heparinization reduces endothelial permeability and hydrogen ion accumulation in a canine skeletal muscle ischemia-reperfusion model

Skeletal muscle injury after revascularization (ischemia-reperfusion) continues to be a major clinical problem. Although heparinization has been recommended, its action in an experimental model of I-R has not been evaluated. We investigated the ability of heparinization to decrease I-R injury in 10 anesthetized dogs (nonheparinized, n = 5; heparinized, n = 5), subjecting one gracilis muscle to 6 hours of ischemia followed by 1 hour of reperfusion while the identically prepared contralateral muscle served as a nonischemic control. Skeletal muscle infarction was determined by Tc-PYP uptake. Endothelial permeability was quantified by measurement of skeletal muscle 125I-Alb activity after intravenous injection. Interstitial hydrogen ion (H+) accumulation was determined by a miniature pH electrode inserted into the gracilis muscle. Isotopic activities from the ischemic muscle were calculated as a percentage of the contralateral nonischemic muscle (mean +/- SEM). Nonheparinized ischemic muscles had an increase in the activities of Tc-PYP and 125I-Alb of 684% +/- 149% and 742% +/- 130%, which were reduced to 218% +/- 54% and 378% +/- 85% by heparinization, respectively (p less than 0.05). During ischemia, the nonheparinized muscles accumulated 1223 +/- 121 nmol of H+ compared with 785 +/- 95 nmol in the heparinized animals (p less than 0.01). This significant reduction in I-R injury may be causally related to diminished endothelial permeability and H+ accumulation.     

Permeability changes following ischemia-reperfusion injury in the rabbit hindlimb.

Changes in permeability following ischemia-reperfusion injury were assessed in the intact rabbit hindlimb by measuring the transvascular clearance of 125I-labeled rabbit serum albumin. Ischemia was induced for periods of 1 or 2 hours by use of a pneumatic tourniquet inflated to 300 mmHg. Following ischemia, the limb was reperfused for 1, 2, or 3 hours. The albumin clearance in the gastrocnemius muscle of control rabbits was 5.1 +/- 0.7 (mean +/- SEM) microliters/hr/g dry weight. Following 1 hour of ischemia and reperfusion, muscle albumin clearance rose to 71.4 +/- 26 microliters/hr/g dry weight which was not significantly different from those animals that underwent 2 hours of ischemia. Muscle albumin clearance continued to be elevated following 2 hours of reperfusion; however, it returned toward control levels after 3 hours of reperfusion. These data suggest there is a transient increase in albumin permeability following ischemia-reperfusion injury in skeletal muscle.     

Perfluorocarbon in microcirculation during ischemia reperfusion

BACKGROUND: The effects of perfluorocarbon (PFC) emulsions administered at a nonhemodiluting dose were studied in the hamster window chamber model to determine the difference in ischemia-reperfusion injury associated with PFC delivery before and after an ischemic episode. STUDY DESIGN: Ischemia was induced by compressing the periphery of the window chamber for 1 hour. Vessel diameter, red blood cell velocity, rolling and adherent leukocytes, and functional capillary density (FCD) were assessed by intravital microscopy. The animals received an infusion (10% blood volume) of PFC emulsion or equivalent volumes of saline, before or after ischemia. Two groups were studied in each experimental protocol: A, infusion after ischemia; and B, infusion before ischemia, where a fraction of the infused material stagnated in the ischemic zone during the occlusion time. Measurements were made before induced ischemia and at 0.5, 2, and 24 hours of reperfusion. RESULTS: Animals treated with PFC after ischemia had substantially decreased leukocytes rolling and sticking in postcapillary venules and recovered functional capillary density and blood flow when compared with saline-treated controls. Conversely, administration of PFC before ischemia considerably reduced functional capillary density and increased leukocyte activation after reperfusion. CONCLUSIONS: Results indicate that PFC without stagnation within an ischemic zone attenuates postischemic reperfusion injury of striated skin muscle, presumably through the reduction of leukocyte-endothelial cell interactions. Accordingly, PFC effects on ischemia-reperfusion injury are determined mainly by the time of administration relative to the ischemic episodes.     

The expression of proinflammatory cytokines in the rat muscle flap with ischemia-reperfusion injury

Ischemic-reperfusion injury mediated by free radicals and neutrophils is the principal pathway for tissue injury and death. Cytokines influence activity of various cell types during the inflammatory process. In this study, expression of selected proinflammatory cytokines was examined in primary and secondary ischemia in the rat gracilis flap model. Sixty Sprague-Dawley rats were used in the study. Primary ischemia of each gracilis flap was induced by clamping its vascular pedicle for 1 hour. The flap was then replaced and allowed to reperfuse. Twenty-four hours later, a secondary ischemia was induced via vascular clamping for 4 hours. All muscle flaps were biopsied at 4 hours and 18 hours after primary ischemia. After secondary ischemia, each flap was biopsied immediately postevent, at 4 hours, and at 18 hours. Expression of tumor necrosis factor (TNF-alpha), interleukin (IL-1beta), and platelet-derived growth factor (PDGF) mRNA was determined by RT-PCR in each case. An equal sample size of gracilis muscle flaps, elevated in an identical fashion but not subjected to vascular clamping, was examined for baseline gene expression. Results showed that TNF-alpha gene expression was significantly up-regulated at 18 hours after secondary ischemia. IL-1 gene expression was up-regulated at 4 hours after primary ischemia, and was greatest at 4 hours after secondary ischemia. PDGF expression was up-regulated immediately after secondary ischemia, then at 4 hours after secondary ischemia (P < 0.05), and down-regulated during reperfusion. This study delineated changes in the expression of TNF-alpha, IL-1beta, and PDGF mRNA, in both primary and secondary ischemia and reperfusion episodes at several critical time points.     

肢体缺血再灌注损伤的新型动物模型制作

[目的]用充气止血带制作肢体缺血再灌注损伤的新型动物模型,研究其对周围神经和骨骼肌损伤的影响.[方法]选择健康新西兰大白兔6个月龄,30只,体重(3.5 ±0.3) kg,雌雄不限,在家兔左侧后肢环扎充气止血带,于不同时间点松开,造成左侧后肢缺血再灌注损伤的模型.随机分为3组,每组10只.A组:对照组,B组:缺血2h,C组:缺血4h.对照组不扎充气止血带,第1、2、3、4、5、6h检测肢体的神经电生理学指标,B组、C组于再灌注(松开止血带,血供恢复后)的1、2、3、4、5、6h检测肢体的神经电生理学指标,A组于第6h观察骨骼肌的形态,B、C组于再灌注(松开止血带,血供恢复后)的第6h观察骨骼肌的形态,每组于术后第5d评估左侧后肢的行走功能.[结果]随着缺血后再灌注时间的延长,B、C和A组相比较,周围神经的潜伏期延长、波幅降低,传导速度降低,三组之间的潜伏期、波幅、传导速度差异均有统计学意义(P＜0.05),光镜观察骨骼肌可见(B、C组):横纹紊乱、肌纤维断裂、间质血管扩张充血、大量中性粒细胞浸润.[结论]经过缺血期和再灌注损伤的交互作用后,肢体的功能性损伤进一步加重,出现了不可逆的病损.该模型制作对动物的损伤较小,更贴近临床.