A quantitative method for determining polarization of neutrophil adhesion molecules associated with ischemia reperfusion

Ischemia-reperfusion-induced neutrophil adhesion to endothelium is CD18-dependent, but information regarding polarity of CD18 adhesion molecules remains speculative. This study evaluated neutrophil adhesion using an in vitro cell adhesion assay and introduces a quantitative method of measuring CD18 membrane distribution using confocal microscopy. Neutrophils from normal animals were isolated from whole blood and incubated with plasma from rat gracilis muscle flaps with no ischemia and reperfusion (nonischemic control, n = 10) or 4 hours of ischemia and 90 minutes of reperfusion (ischemia/reperfusion, n = 10), on coverslips pretreated with and without (phosphate-buffered saline) soluble intercellular adhesion molecules. Coverslips without intercellular adhesion molecules represented a negative control (intercellular adhesion molecules were required for adhesion). Percent adherence to intercellular adhesion molecules was expressed as a ratio of adherent cells/total cells. CD18 polarization was assessed by staining neutrophils with fluorescein isothiocyanate-labeled anti-CD11b, followed by confocal microscopy and Z-stack analysis. Membrane-associated CD18 was expressed as fluorescence intensity units in three equal areas of the cell membrane. Capping was defined as twice as much fluorescence in 33 percent of the cell membrane as in the remaining 67 percent. Neutrophils exposed to ischemia and reperfusion plasma showed a significant increase in adhesion (0.8 +/- 0.1 percent versus 16.7 +/- 2.2 percent, p < 0.001) and CD18 polarization (6.2 +/- 1.7 percent versus 43.9 +/- 12.2 percent, p = 0.0206) compared with controls. This article describes an in vitro assay that reliably reproduces the neutrophil adhesion phenomenon associated with ischemia-reperfusion injury. Results from confocal microscopy allowed for quantitative estimation of membrane-associated receptor polarization.     

Timing of microcirculatory injury from ischemia reperfusion

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

Prevention of ischemia-reperfusion injury in a rat skin flap model: the role of mast cells, cromolyn sodium, and histamine receptor blockade

The objective of this study was to examine the role of mast cells and their principal product, histamine, in ischemia/reperfusion injury. Cromolyn sodium, diphenhydramine, and cimetidine were administered to ischemic flaps just before reperfusion and evaluated for flap survival, mast cell count, neutrophil count, and myeloperoxidase levels. Epigastric island skin flaps were elevated in 49 rats; they were rendered ischemic by clamping the artery for 10 hours. Thirty minutes before reperfusion, the rats were treated with intraperitoneal saline (n = 11), cimetidine (n = 11), diphenhydramine (n = 11), or cromolyn sodium (n = 10). Flap survival was evaluated at 7 days. Neutrophil counts, mast cell counts, and myeloperoxidase levels were evaluated 12 hours after reperfusion. Flap necrosis in the sham group of animals (n = 6) was 0.0 percent, as expected, whereas the control group (saline-treated animals) had 47.3+/-33.4 percent necrosis. Animals treated with diphenhydramine and cimetidine demonstrated a significant decrease in flap necrosis to 17.7+/-8.8 percent and 19.4+/-14.7 percent, respectively. This protective effect was not seen with cromolyn sodium (44.3+/-35.6 percent). Both neutrophil and mast cell counts were significantly decreased in flaps from antihistamine-treated and sham animals versus both saline- and cromolyn sodium-treated groups. The administration of diphenhydramine and cimetidine before reperfusion can significantly reduce the extent of flap necrosis and the neutrophil and mast cell counts caused by ischemia/reperfusion. This protective effect is not seen with cromolyn sodium. The protective effect of antihistamines on flap necrosis might be related to the decrease in neutrophils and, possibly, mast cells within the flap.     

Reperfusion injury is reduced in skeletal muscle by inhibition of inducible nitric oxide synthase.

This study evaluated the effects of the selective inducible nitric oxide synthase (iNOS) inhibitor N-[3-(aminomethyl)benzyl]acetamidine (1400W) on the microcirculation in reperfused skeletal muscle. The cremaster muscles from 32 rats underwent 5 h of ischemia followed by 90 min of reperfusion. Rats received either 3 mg/kg 1400W or PBS subcutaneously before reperfusion. We found that blood flow in reperfused muscles was <45% of baseline in controls but sharply recovered to near baseline levels in 1400W-treated animals. There was a significant (P < 0.01 to P < 0.001) difference between the two groups at each time point throughout the 90 min of reperfusion. Vessel diameters remained <80% of baseline in controls during reperfusion, but recovered to the baseline level in the 1400W group by 20 min, and reached a maximum of 121 +/- 14% (mean +/- SD) of baseline in 10- to 20-micro m arterioles, 121 +/- 6% in 21- to 40-micro m arterioles, and 115 +/- 8% in 41- to 70-micro m arteries (P < 0.01 to P < 0.001). The muscle weight ratio between ischemia-reperfused (left) and non-ischemia-reperfused (right) cremaster muscles was 193 +/- 42% of normal in controls and 124 +/- 12% in the 1400W group (P < 0.001). Histology showed that neutrophil extravasation and edema were markedly reduced in 1400W-treated muscles compared with controls. We conclude that ischemia-reperfusion leads to increased generation of NO from iNOS in skeletal muscle and that the selective iNOS inhibitor 1400W reduces the negative effects of ischemia-reperfusion on vessel diameter and muscle blood flow. Thus 1400W may have therapeutic potential in treatment of ischemia-reperfusion injury.     

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

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

Effects of L-NAME and L-arginine on ischemia-reperfusion injury in rat skeletal muscle

The involvement of nitric oxide in ischemia-reperfusion injury remains controversial and has been reported to be both beneficial and deleterious, depending on the tissue and model used. This study evaluated the effects of the nitric oxide synthase inhibitor N(G)-nitro-L-arginine-methyl ester (L-NAME) and the substrate for nitric oxide synthase, L-arginine on skeletal muscle necrosis in a rat model of ischemia-reperfusion injury. The rectus femoris muscle in male Wistar rats (250 to 500 g) was isolated on its vascular pedicle and subjected to 4 hours of complete arteriovenous occlusion. The animals were divided into five groups: (1) sham-raised control, no ischemia, no treatment (n = 6); (2) 4 hours of ischemia (n = 6); (3) vehicle control, 4 hours of ischemia + saline (n = 6); (4) 4 hours of ischemia + L-arginine infusion (n = 6); and (5) 4 hours of ischemia + L-NAME infusion (n = 6). The infusions (10 mg/kg) were administered into the contralateral femoral vein beginning 5 minutes before reperfusion and during the following 30 to 45 minutes. Upon reperfusion, the muscle was sutured in its anatomic position and all wounds were closed. The percentage of muscle necrosis was assessed after 24 hours of reperfusion by serial transections, nitroblue tetrazolium staining, digital photography, and computerized planimetry. Sham (group 1) animals sustained baseline necrosis of 11.9 +/- 3.0 (percentage necrosis +/- SEM). Four hours of ischemia (group 2) significantly increased necrosis to 79.2 +/- 1.4 (p < 0.01). Vehicle control (group 3) had no significant difference in necrosis (81.17 +/- 5.0) versus untreated animals subjected to 4 hours of ischemia (group 2). Animals treated with L-arginine (group 4) had significantly reduced necrosis to 34.6 +/- 7.5 versus untreated (group 2) animals (p < 0.01). Animals infused with L-NAME (group 5) had no significant difference in necrosis (68.2 +/- 6.7) versus untreated (group 2) animals. L-Arginine (nitric oxide donor) significantly decreased the severity of muscle necrosis in this rat model of ischemia-reperfusion injury. L-arginine is known to increase the amount of nitric oxide through the action of nitric oxide synthase, whereas L-NAME, known to inhibit nitric oxide synthase and decrease nitric oxide production, had comparable results to the untreated 4-hour ischemia group. These results suggest that L-arginine, presumably through nitric oxide mediation, appears beneficial to rat skeletal muscle subjected to ischemia-reperfusion injury.     

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

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

Effect of hyperbaric oxygen on neutrophil CD18 expression

Previous work has shown that treatment with hyperbaric oxygen significantly reduces neutrophil adhesion to postcapillary venules in a rat microcirculation model of ischemia-reperfusion injury. The mechanism of this process is unknown. The purpose of this study was to evaluate the effect of hyperbaric oxygen on neutrophil CD18 adhesion sites by flow cytometry in an animal model of ischemia-reperfusion injury. The gracilis muscle flap was raised in three groups of male Wistar rats: (1) a sham group (n = 25), (2) a group that underwent 4 hours of ischemia (n = 25), and (3) a group that underwent 4 hours of ischemia and received hyperbaric oxygen (100% 02, 2.5 atmospheres absolute, during the last 90 minutes of ischemia) (n = 25). Samples from one subgroup of each group (n = 5) were divided into two portions, and one portion was stimulated with phorbol-12 myristate 13-acetate (PMA). Samples from another subgroup of each group (n = 5) were treated in the same manner, and a flap flush was added at the end of reperfusion to determine the number of CD18 adhesion sites on adherent neutrophils remaining in the flap. Venous blood was drawn 10 minutes after the operation, at 5 minutes of reperfusion, and at 90 minutes of reperfusion. Hematocrit and white blood cell count were measured. Samples were analyzed by flow cytometry, and the antibody binding capacity was assessed using microbead standards and linear regression (antibody binding capacity was expressed as the mean number of sites per cell +/- SEM). Microbeads were used to align the flow cytometer and to provide external and internal standards. Ischemia-reperfusion injury increased the expression of CD18 by neutrophils (p < 0.05). Expression of CD18 was not decreased by hyperbaric oxygen treatment. Stimulation with PMA increased the expression of CD18 in all groups (p < 0.01). These results suggest that ischemia-reperfusion injury does increase the expression of CD18 by neutrophils. Hyperbaric oxygen, as administered in this experiment, did not prevent the increase in CD18 expression.     

Cardioprotective effect induced by brief exposure to nitric oxide before myocardial ischemia-reperfusion in vivo.

Administration of nitric oxide (NO) donors during ischemia and reperfusion protects from myocardial injury. However, whether administration of an NO donor during a brief period prior to ischemia protects the myocardium and the endothelium against ischemia-reperfusion injury in vivo is unknown. To study this possibility anesthetized pigs were subjected to 45-min ligation of the left anterior descending coronary artery (LAD) followed by 4h of reperfusion. In initial dose-finding experiments, vehicle or three different doses of the NO donor S-nitroso-N-acetyl-D,L-penicillamin (SNAP; 0.1; 0.5; 2.5 micromol) were infused into the LAD for 3 min starting 13 min during ischemia. Only the 0.5 micromol dose of SNAP reduced infarct size (from 85+/-3% of the area at risk in the vehicle group to 63+/-3% in the SNAP-treated group; p<0.01). There were no significant differences in hemodynamics in the vehicle and SNAP groups during ischemia-reperfusion. Endothelium-dependent dilatation of coronary microvasculature induced by substance P was larger in the SNAP group than in the vehicle group. Myeloperoxidase activity was lower in the ischemic/reperfused myocardial area of pigs given SNAP (4.97+/-0.61 U/g) than in vehicle-treated pigs (8.45+/-0.25 U/g; p<0.05). It is concluded that intracoronary administration of the NO donor SNAP for a brief period before ischemia reduces infarct size, attenuates neutrophil accumulation, and improves endothelial function. These results suggest that NO exerts a classic preconditioning-like protection against ischemia-reperfusion injury in vivo in a narrow concentration range.     

Neutrophil localization following reperfusion of ischemic skin flaps.

A swine model of island latissimus dorsi myocutaneous and buttock cutaneous flaps was used to examine neutrophil localization and flap survival after 6 hours of global ischemia followed by 24 hours of reperfusion. Radioactivity from autotransfused neutrophils labeled with indium-111 enabled their localization. Radioactivity in ischemic latissimus dorsi flaps was increased by 101 +/- 30 percent over contralateral control latissimus dorsi flaps (n = 6, p = 0.01). Radioactivity in ischemic buttock flaps was increased by 142 +/- 40 percent over contralateral control buttock flaps (n = 6, p = 0.008). Despite increased neutrophil localization to ischemic flaps, the magnitude of tissue radioactivity failed to provide sufficient information to predict ischemic injury as measured by flap survival and tissue water content.     

Salvia miltiorrhiza treatment during early reperfusion reduced postischemic myocardial injury in the rat

Oxidative stress may play a causative role in myocardial ischemia-reperfusion injury. However, it is a relatively understudied aspect regarding an optimal timing of antioxidant intervention during ischemia-reperfusion. The present study investigates the effect of different treatment regimens of Salvia miltiorrhiza (SM) herb extracts containing phenolic compounds that possess potent antioxidant properties on postischemic myocardial functional recovery in the setting of global myocardial ischemia and reperfusion. Langendorff-perfused rat hearts were subjected to 40 min of global ischemia at 37 degrees C followed by 60 min of reperfusion, and were randomly assigned into the untreated control and 2 SM-treated groups (n = 7 per group). In treatment 1 (SM1), 3 mg/mL of water soluble extract of SM was given for 10 min before ischemia and continued during ischemia through the aorta at a reduced flow rate of 60 microL/min, but not during reperfusion. In treatment 2 (SM2), SM (3 mg/mL) was given during the first 15 min of reperfusion. During ischemia, hearts in the control and SM2 groups were given physiological saline at 60 microL/min. The SM1 treatment reduced the production of 15-F2t-isoprostane, a specific index of oxidative stress-induced lipid peroxidation, during ischemia (94 +/- 20, 43 +/- 6, and 95 +/- 15 pg/mL in the coronary effluent in control, SM1, and SM2 groups, respectively; p < 0.05, SM1 vs. control or SM2) and postponed the onset of ischemic contracture. However, SM2, but not the SM1 regimen, significantly reduced 15-F2t-isoprostane production during early reperfusion and led to optimal postischemic myocardial functional recovery (left ventricular developed pressure 51 +/- 4, 46 +/- 4, and 60 +/- 6 mmHg in the control, SM1, and SM2 groups, respectively, at 60 min of reperfusion; p < 0.05, SM2 vs. control or SM1) and reduced myocardial infarct size as measured by triphenyltetrazolium chloride staining (26% +/- 2%, 22% +/- 2%, and 20% +/- 2% of the total area in the control, SM1, and SM2 groups, respectively, p < 0.05, SM2 vs. control). It is concluded that S. miltiorrhiza could be beneficial in the treatment of myocardial ischemic injury and the timing of administration seems important.     

The role of prostaglandins in the antiarrhythmic effect of ischemic preconditioning

The role of prostaglandins in the antiarrhythmic effect of ischemic preconditioning (IP) was investigated in pentobarbital-anesthetized rats. In 5 unpreconditioned control rats, 30 min of occlusion of the left coronary artery elicited ventricular tachycardia (VT) and fibrillation (VF), with an average duration of VT and VF of 51 +/- 6 and 43 +/- 4 s, respectively. Frequent ventricular premature beats (VPBs; average 1,249 +/- 145) were also documented in these animals. Thirty minutes of reperfusion after the prolonged coronary occlusion in these animals caused more severe arrhythmias, including irreversible VF. In animals pretreated with IP (n = 5), which was achieved by 3 cycles of 3 min of occlusion followed by 5 min of reperfusion, 30 min of coronary artery occlusion caused neither VT nor VF, but occasional VPBs (average 2 +/- 1, p < 0.001 vs. control). Only occasional VPBs were observed during 30 min of reperfusion in this group. In animals pretreated with indomethacin (1 mg/kg i.v., n = 5) followed by IP, prolonged ischemia and reperfusion led to frequent VPBs but no VT or VF. The average number of VPBs during ischemia and reperfusion in this indomethacin-treated group was less than that of the controls but greater than the IP-only group (p < 0.01). In conclusion, prostaglandins appear to play a role in the protective effect of IP against VPBs during acute ischemia and reperfusion.     

Tempol reduces infarct size in rodent models of regional myocardial ischemia and reperfusion.

Reactive oxygen species (ROS) contribute to ischemia-reperfusion injury of the heart. This study investigates the effects of tempol, a membrane-permeable radical scavenger on (i) the infarct size caused by regional myocardial ischemia and reperfusion of the heart in vivo (rat, rabbit) and in vitro (rat), and (ii) the cell injury caused by hydrogen peroxide (H2O2) in rat cardiac myoblasts (H9c2 cells). In the anesthetized rat, tempol reduced the infarct size caused by regional myocardial ischemia (25 min) and reperfusion (2 h) from 60 +/- 3% (control, n = 8) to 24 +/- 5% (n = 6, p < .05). In the anesthetized rabbit, tempol also attenuated the infarct size caused by myocardial ischemia (45 min) and reperfusion (2 h) from 59 +/- 3% (control, n = 6) to 39 +/- 5% (n = 5, p < .05). Regional ischemia (35 min) and reperfusion (2 h) of the isolated, buffer-perfused heart of the rat resulted in an infarct size of 54 +/- 4% (control n = 7). Reperfusion of hearts with buffer containing tempol (n = 6) caused a 37% reduction in infarct size (n = 6, p < .05). Pretreatment of rat cardiac myoblasts with tempol attenuated the impairment in mitochondrial respiration caused by H2O2 (1 mM for 4 h). Thus, the membrane-permeable radical scavenger tempol reduces myocardial infarct size in rodents.     

Myocardial ischemia-reperfusion injury is enhanced in a model of systemic allergy and asthma

Despite epidemiological evidence of cardiovascular complications in asthmatics, the direct contribution of asthmatic pathophysiology to cardiovascular effects is unknown. Considering parallels in underlying pathophysiology, we tested the hypothesis that presence of systemic allergy and asthma worsens the outcome of myocardial ischemia-reperfusion injury. Systemic allergy and asthma were created in rabbits by repeated intraperitoneal injections of allergen with adjuvant, followed by an airway challenge in two groups. Nonsensitized animals served as controls. In situ myocardial ischemia-reperfusion was induced in anesthetized animals by a 30-min ligation of a coronary artery, followed by 3 h of reperfusion. Ischemia-reperfusion was done at 24 h after intraperitoneal boost (1 DB) and 7 days (7 DB) after the last intraperitoneal injection and at 24 h (1DAWCH) and 7 days (7DAWCH) after airway challenge. The infarct size (determined by 2,3,5-triphenyltetrazolium chloride staining, normalized to area at risk) was significantly higher in all sensitized groups compared with control (1DB, 31 +/- 4; 7DB, 28.9 +/- 2.6; 1DAWCH, 66.1 +/- 4.1; 7DAWCH, 28.9 +/- 9.2; control, 16.7 +/- 3.2; means +/- SE; P < 0.01 by ANOVA; n = 6). The 1DAWCH group showed significantly greater infarct than all other groups (P < 0.05). Myocardial neutrophil infiltration was significantly higher in the sensitized groups compared with control (P < 0.01). Tissue neutrophil counts showed a strong positive correlation to infarct sizes (r2 = 0.9). These observations indicate that the presence of systemic allergy and asthma is associated with increased myocardial neutrophil infiltration during acute ischemia-reperfusion and increased size of the resulting infarct.     

Reperfusion injury in skeletal muscle is reduced in inducible nitric oxide synthase knockout mice.

Inducible nitric oxide synthase (iNOS) participates in many pathological events, and selective inhibition of iNOS has been shown to reduce ischemia-reperfusion (I/R) injury in different tissues. To further confirm its role in this injury process, I/R injury was observed in denervated cremaster muscles of iNOS-deficient (iNOS-/-) and wild-type mice. After 3-h ischemia and 90-min reperfusion, blood flow in reperfused muscle was 80 +/- 8.5% (mean +/- SE) of baseline at 10-min reperfusion and completely returned to the preischemia baseline after 20 min in iNOS-/- mice. In contrast, blood flow was 32 +/- 7.4% at 10 min and increased to 60 +/- 20% of the baseline level at 90 min in wild-type mice (P < 0.001 vs. iNOS-/- mice at all time points). The increased muscle blood flow in iNOS-/- mice was associated with significantly less vasospasm in all three sizes of arterial vessel size categories. The weight ratio to the contralateral muscle not subjected to I/R was greater in wild-type mice (173 +/- 11%) than in iNOS-/- mice (117 +/- 3%; P < 0.01). Inflammation and neutrophil extravasation were also more severe in wild-type mice. Western blot analysis demonstrated an absence of iNOS protein band in iNOS-/- mice and upregulation of iNOS protein expression in wild-type mice. Our results confirm the importance of iNOS in I/R injury. Upregulated iNOS exacerbates I/R injury and appears to be a therapeutic target in protection of tissues against this type of injury.     

Role of the thromboxane A2 receptor in the vasoactive response to ischemia-reperfusion injury.

Neutrophil-endothelial adhesion in venules and progressive vasoconstriction in arterioles seem to be important microcirculatory events contributing to the low flow state associated with ischemia-reperfusion injury of skeletal muscle. Although the neutrophil CD-18 adherence function has been shown to be a prerequisite to the vasoconstrictive response, the vasoactive substances involved remain unknown. The purpose of this study was to evaluate the role of thromboxane A2 receptor in the arteriole vasoactive response to ischemia-reperfusion injury. An in vivo microscopy preparation of transilluminated gracilis muscle in male Wistar rats (175 +/- 9 g) (n = 12) was used for this experiment. Three experimental groups were evaluated in this study: (1) sham, flap raised, no ischemia (20 venules, 20 arterioles), (2) 4 hours of global ischemia only (19 venules, 22 arterioles), and (3) 4 hours of global ischemia + thromboxane A2 receptor antagonist (ONO-3708) (17 venules, 20 arterioles). ONO-3708 (5 mg/kg), a specific competitive antagonist of thromboxane A2 receptor, was infused at a rate of 0.04 ml/minute into the contralateral femoral vein 30 minutes before reperfusion. Mean arterial blood pressure was not changed at this dose of ONO-3708 (88 +/- 6 mmHg before infusion, 81 +/- 4 mmHg after infusion, n = 3). The number of leukocytes rolling and adherent to endothelium (15-sec observation) were counted in 100-microm venular segments, and arteriole diameters were measured at 5, 15, 30, 60, and 120 minutes of reperfusion. Leukocyte counts and arteriole diameters were analyzed with two-way factorial analysis of variance for repeated measures and Duncan's post hoc mean comparison. Statistical significance was indicated by a p < or = 0.05. The ischemia-reperfusion-induced vasoconstriction was significantly reduced by the thromboxane A2 receptor antagonist (ONO-3708). The mean arteriole diameters at 30, 60, and 120 minutes reperfusion were significantly greater in the treated animals than in the ischemia-reperfusion controls. Despite a significant increase in treated mean arteriole diameters, 30 percent of arterioles still demonstrated vasoconstriction. Neutrophil-endothelial adherence was not reduced by ONO-3708. Thromboxane A2 receptor blockade significantly reduces but does not eliminate ischemia-reperfusion-induced vasoconstriction in this model. This finding suggests that additional and perhaps more important vasoactive mediators contribute to vasoconstriction. Furthermore, thromboxane A2 receptor blockade has no effect on polymorphonuclear endothelial adherence.     

Induction of endotoxin tolerance improves lung function after warm ischemia in dogs

In shock models, induction of endotoxin tolerance (ET) is known to have a protective effect. The present study was designed to explore if ET is effective in protecting lungs from reperfusion injury. Twelve foxhounds were used as experimental animals. After a left thoracotomy, the left hilum was clamped for 3 h, followed by 8 h of reperfusion. In the treatment group (ET, n = 6), dogs were pretreated with incremental daily endotoxin doses of up to 60 microg/kg on day 6. The ischemia and reperfusion experiment was carried out on day 9. Control group animals (n = 6) were not subjected to endotoxin. After 8 h of observation, functional parameters of the reperfused lung of the ET and the control group were statistically different (P < 0.05) with respect to Po(2) [ET vs. control: 172.7 +/- 12.9 vs. 66.1 +/- 7.2 (SE) mmHg], compliance (16.0 +/- 1.2 vs. 8.3 +/- 1.0 ml/0.1 kPa), and the wet-to-dry ratio (9.4 +/- 0.8 vs. 16.7 +/- 1.2). After 3 h of warm ischemia and 8 h of reperfusion, pulmonary function and lung water content improved in the endotoxin-tolerant group.     

亚低温联合镁剂对脑缺血再灌注大鼠保护作用的研究

目的:rt-PA溶栓为缺血性卒中最有效的治疗方法,脑血流再通后挽救濒临死亡的神经细胞同时,也可能发生更为严重而持久的脑缺血再灌注损伤。本研究探讨联合应用局部亚低温(32-35℃)及硫酸镁对局灶性脑缺血再灌注大鼠的保护作用及其可能机制。方法:通过线栓法建立大鼠大脑中动脉阻塞(MCAO)及再通模型,将50只雄性Wistar大鼠随机分为假手术组、常温组、亚低温组、硫酸镁组、亚低温+硫酸镁组,每组10例,采用Longa神经功能评分、TTC染色、干湿重法、TUNEL技术,检测和比较各组脑缺血再灌注后大鼠的神经功能、脑梗死体积、脑组织含水量及凋亡细胞数。结果:与常温组相比,亚低温组与亚低温+硫酸镁组的梗死体积、神经功能评分、脑组织含水量、凋亡细胞数均明显降低,差异有显著意义(P0.05);而与亚低温组相比,亚低温+硫酸镁组局灶脑缺血大鼠的脑梗死体积、神经功能评分、脑组织含水量、凋亡细胞数均显著减少,差异有显著意义(P0.05)。结论:与单独应用亚低温相比,局部亚低温与硫酸镁联合应用,对局灶性脑缺血再灌注大鼠可发挥更有效的脑保护作用。其机制可能与抑制脑缺血再灌注后凋亡及减轻脑水肿有关。二者联用可能为缺血性卒中患者提供一种减轻溶栓后再灌注损伤的有效脑保护方法。     

Effects of hypothermia on neuronal-vascular function after cerebral ischemia in piglets

We determined whether cerebral arteriolar dilation to N-methyl-d-aspartate (NMDA), a response dependent on stimulation of cortical neurons and inhibited by anoxic stress, would be preserved by hypothermia during and following ischemia. Pial arteriolar diameters in anesthetized piglets were determined via intravital microscopy. Arteriolar responses to NMDA (10, 50, and 100 micromol/l) were measured before and 1 h after 10 min of global ischemia. Piglets were exposed to either total body or selective brain cooling (33-34 degrees C). Arteriolar dilation to lower doses or to 100 micromol/l NMDA was not affected by hypothermia alone (51 +/- 3 vs. 46 +/- 7%, normothermia vs. hypothermia; n = 7) in nonischemic animals. However, arteriolar responses to 100 micromol/l NMDA were clearly attenuated after ischemia despite body cooling during ischemia (53 +/- 3 vs. 32 +/- 6%; n = 8), hypothermia during ischemia and early reperfusion (49 +/- 10 vs. 20 +/- 3%; n = 8), or selective brain cooling (48 +/- 5 vs. 20 +/- 5%; n = 10). In contrast, pretreatment with indomethacin resulted in complete preservation of NMDA-induced vasodilation after ischemia. Thus, hypothermia fails to protect against neuronal dysfunction during ischemia.