Microvascular reperfusion injury: rapid expansion of anatomic no reflow during reperfusion in the rabbit

The aim was to define the degree and time course of reperfusion-related expansion of no reflow. In five groups of anesthetized, open-chest rabbits (30-min coronary occlusion and different durations of reperfusion), anatomic no reflow was determined by injection of thioflavin S at the end of reperfusion and compared with regional myocardial blood flow (RMBF; radioactive microspheres) and infarct size (triphenyltetrazolium). The area of no reflow progressively increased from 12.2 +/- 4.2% of the risk area after 2 min of reperfusion to 30.8 +/- 3.1% after 2 h and 34.9 +/- 3.3% after 8 h and significantly correlated with infarct size after 1 h of reperfusion (r = 0.88-0.97). This rapid expansion of no reflow predominantly occurred during the first 2 h, finally encompassing approximately 80% of the infarct size, and was accompanied by a decrease of RMBF within the risk area, being hyperemic after 2 min of reperfusion (3.78 +/- 0.75 ml x min(-1) x g(-1)) and plateauing at a level of approximately 0.9 ml x min(-1) x g(-1) by 2 and 8 h of reperfusion (preischemic RMBF: 2.06 +/- 0.01 ml x min(-1) x g(-1)). The development of macroscopic hemorrhage lagged behind no reflow, was closely correlated with it, and may be the consequence of microvascular damage.     

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.     

Inhibition of ischemia/reperfusion-induced damage by dexamethasone in isolated working rat hearts: the role of cytochrome c release

We investigated the contribution of dexamethasone treatment on the recovery of postischemic cardiac function and the development of reperfusion-induced arrhythmias in ischemic/reperfused isolated rat hearts. Rats were treated with 2 mg/kg of intraperitoneal injection of dexamethasone, and 24 hours later, hearts were isolated according to the 'working' mode, perfused, and subjected to 30 min global ischemia followed by 120 min reperfusion. Cardiac function including heart rate, coronary flow, aortic flow, and left ventricular developed pressure were recorded. After 60 min and 120 min reperfusion, 2 mg/kg of dexamethasone significantly improved the postischemic recovery of aortic flow and left ventricular developed pressure from their control values of 10.7 +/- 0.3 ml/min and 10.5 +/- 0.3 kPa to 22.2 +/- 0.3 ml/min (p < 0.05) and 14.3 +/- 0.5 kPa (p < 0.05), 19.3 +/- 0.3 ml/min (p < 0.05) and 12.3 +/- 0.5 kPa (p < 0.05), respectively. Heart rate and coronary flow did not show a significant change in postischemic recovery after 60 or 120 min reperfusion. In rats treated with 0.5 mg/kg of actinomycin D injected i.v., one hour before the dexamethasone injection, suppressed the dexamethasone-induced cardiac protection. Electrocardiograms were monitored to determine the incidence of reperfusion-induced ventricular fibrillation. Dexamethasone pretreatment significantly reduces the occurrence of ventricular fibrillation. Cytochrome c release was also observed in the cytoplasm. The results suggest that the inhibition of cytochrome c release is involved in the dexamethasone-induced cardiac protection.     

Ischemic postconditioning during reperfusion activates Akt and ERK without protecting against lethal myocardial ischemia-reperfusion injury in pigs

Transient episodes of ischemic preconditioning (PC) render myocardium protected against subsequent lethal injury after ischemia and reperfusion. Recent studies indicate that application of short, repetitive ischemia only during the onset of reperfusion after the lethal ischemic event may obtain equivalent protection. We assessed whether such ischemic postconditioning (Postcon) is cardioprotective in pigs by limiting lethal injury. Pentobarbital sodium-anesthetized, open-chest pigs underwent 30 min of complete occlusion of the left anterior descending coronary artery and 3-h reflow. PC was elicited by two cycles of 5-min occlusion plus 10-min reperfusion before the 30-min occlusion period. Postcon was elicited by three cycles of 30-s reperfusion, followed by 30-s reocclusion, after the 30-min occlusion period and before the 3-h reflow. Infarct size (%area-at-risk using triphenyltetrazolium chloride macrochemistry; means +/- SE) after 30 min of ischemia was 26.5 +/- 5.2% (n = 7 hearts/treatment group). PC markedly limited myocardial infarct size (2.8 +/- 1.2%, n = 7 hearts/treatment group, P < 0.05 vs. controls). However, Postcon had no effect on infarct size (37.8 +/- 5.1%, n = 7 hearts/treatment group). Within the subendocardium, Postcon increased phosphorylation of Akt (74 +/- 12%) and ERK1/2 (56 +/- 10%) compared with control hearts subjected only to 30-min occlusion and 15-min reperfusion (P < or = 0.05), and these changes were not different from the response triggered by PC (n = 5 hearts/treatment group). Phosphorylation of downstream p70S6K was also equivalent in PC and Postcon groups. These data do not support the hypothesis that application of 30-s cycles of repetitive ischemia during reperfusion exerts a protective effect on pig hearts subjected to lethal ischemia, but this is not due to a failure to phosphorylate ERK and Akt during early reperfusion.     

Cardioprotection by multiple preconditioning cycles does not require mitochondrial K(ATP) channels in pigs

To test whether cardioprotection induced by ischemic preconditioning depends on the opening of mitochondrial ATP-sensitive K(+) (K(ATP)) channels, the effect of channel blockade was studied in barbital-anesthetized open-chest pigs subjected to 30 min of complete occlusion of the left anterior descending coronary artery and 3 h of reflow. Preconditioning was elicited by two cycles of 5-min occlusion plus 10-min reperfusion before the 30-min occlusion period. 5-Hydroxydecanoate (5 mg/kg iv) was injected 15 min before preconditioning or pharmacological preconditioning induced by diazoxide (3.5 mg/kg, 1 ml/min iv). Infarct size (percentage of the area at risk) after 30 min of ischemia was 35.1 +/- 9.9% (n = 7). Preconditioning markedly limited myocardial infarct size (2.7 +/- 1.6%, n = 7), and 5-hydroxydecanoate did not abolish protection (2.4 +/- 0.9%, n = 8). Diazoxide infusion also significantly limited infarct size (14.6 +/- 7.4%, n = 7), and 5-hydroxydecanoate blocked this effect (30.8 +/- 8.0%, n = 7). Thus the opening of mitochondrial K(ATP) channels is cardioprotective in pigs, but these data do not support the hypothesis that opening of mitochondrial K(ATP) channels is required for the endogenous protection afforded by preconditioning.     

The role of thromboxane A2 in reperfusion injury

Thromboxane A2 (TXA2) receptor antagonists can limit infarct size in models of coronary occlusion and reperfusion, but it was unknown if these compounds can mitigate reperfusion injury. Anesthetized open chest dogs were subjected to left circumflex coronary (LCX) occlusion for 90 min. Two minutes before reperfusion, the dogs were given iv saline (0.9% NaCl) or the TXA2 antagonist SQ 29,548 (0.2 mg/kg + 0.2 mg/kg/hr). Reperfusion was instituted for 5 hr at which time infarct size was determined. Regional myocardial blood flow was determined before, during, and after occlusion. SQ 29,548 treatment resulted in a significant reduction in infarct size (57 +/- 7 and 34 +/- 8% of the left ventricular area at risk infarcted in the saline and SQ 29,548 groups, respectively). No differences in collateral flow during occlusion were observed between groups, but SQ 29,548 treatment resulted in a significantly higher subendocardial reperfusion flow (54 +/- 10 and 93 +/- 14 ml/min/100g for the saline and SQ 29,548 groups, respectively). Thus, TXA2 seems to play a role in exacerbating reperfusion injury and TXA2 receptor blockade may have potential as a mode of therapy for ischemia-reperfusion damage.     

Oxidative DNA damage precedes DNA fragmentation after experimental stroke in rat brain.

Experimental stroke using a focal cerebral ischemia and reperfusion (FCIR) model was induced in male Long-Evans rats by a bilateral occlusion of both common carotid arteries and the right middle cerebral artery for 30-90 min, followed by various periods of reperfusion. Oxidative DNA lesions in the ipsilateral cortex were demonstrated using Escherichia coli formamidopyrimidine DNA N-glycosylase (Fpg protein)-sensitive sites (FPGSS), as labeled in situ using digoxigenin-dUTP and detected using antibodies against digoxigenin. Because Fpg protein removes 8-hydroxy-2'-deoxyguanine (oh8dG) and other lesions in DNA, FPGSS measure oxidative DNA damage. The number of FPGSS-positive cells in the cortex from the sham-operated control group was 3 +/- 3 (mean +/- SD per mm(2)). In animals that received 90 min occlusion and 15 min of reperfusion (FCIR 90/15), FPGSS-positive cells were significantly increased by 200-fold. Oxidative DNA damage was confirmed by using monoclonal antibodies against 8-hydroxy-guanosine (oh8G) and oh8dG. A pretreatment of RNase A (100 microg/ml) to the tissue reduced, but did not abolish, the oh8dG signal. The number of animals with positive FPGSS or oh8dG was significantly (P<0.01) higher in the FCIR group than in the sham-operated control group. We detected few FPGSS of oh8dG-positive cells in the animals treated with FCIR of 90/60. No terminal UTP nicked-end labeling (TUNEL)-positive cells, as a detection of cell death, were detected at this early reperfusion time. Our data suggest that early oxidative DNA lesions elicited by experimental stroke could be repaired. Therefore, the oxidative DNA lesions observed in the nuclear and mitochondrial DNA of the brain are different from the DNA fragmentation detected using TUNEL.     

Suppressive effect of coenzyme Q10 on phospholipase A2 activation in cardiac cells after prolonged swimming.

Phospholipase A2 (PLA2) activity is elevated in cardiac microsomal fractions and phospholipids (PL) are much reduced in both the cardiac mitochondria and microsomal fractions from rats subjected to prolonged swimming. Preadministration of coenzyme Q10 (CoQ10 i.v. 30 mg/kg) significantly suppressed these changes. Two groups of 8-week-old male Wistar rats were trained to swim, receiving 30 min of training for 4 days. On the fifth day they were given an intravenous injection of either 30 mg/kg CoQ10 in saline or 1 ml saline. Thirty minutes later they began to swim for 3 hours carrying a weight representing 3% of body weight. On completion of the swim they were sacrified by instantaneous decapitation, and cardiac mitochondria were isolated. Mitochondria were also prepared from saline injected, unexercised control rats. Phosphatidylethanolamine (PE) and phosphatidylcholine (PC) concentrations were measured with HPLC and PLA2 activity was assayed fluorometrically. The mitochondrial concentrations (means +/- SEM, n = 6) of PE and PC were respectively 126 +/- 22 and 140 +/- 22 nmol/mg protein in the exercise-CoQ10 group against 66 +/- 4 and 50 +/- 10 nmol/mg protein in the exercise-saline group. The specific PLA2 activities (expressed as nmol degraded dipyrene phosphorylethanolamine substrate/hr/mg protein) in the microsomes was 0.20 +/- 0.02 in the exercise-CoQ10 group against 0.30 +/- 0.02 in the exercise-saline group. These results suggest CoQ10 has a protective effect against an excessive reduction in mitochondrial membrane phospholipids during prolonged exercise.     

Effect of total venous occlusion on capillary flow and necrosis in skeletal muscle

Limb replantation and microvascular transfer of flaps are sometimes complicated by postoperative venous thrombosis. Total venous occlusion can lead to complete shutdown of microvascular perfusion, resulting in failure of the transfer or replantation. Once venous return stops, it must be restored within a critical period of time for tissue survival. The purpose of this experiment was to delineate this critical period of time at which no reflow and irreversible muscle necrosis occurs by the use of a rat gracilis flap microcirculation model. The gracilis muscle of 40 male Wistar rats (135.3 +/- 37.2 g) was elevated on its vascular pedicle and mounted on a raised platform for videomicroscopic analysis. Animals were randomly assigned to one of four groups: (1) sham (no total venous occlusion), (2) 10 minutes of total venous occlusion, (3) 30 minutes of total venous occlusion, and (4) 60 minutes of total venous occlusion. Total venous occlusion was established by placing a microvascular clamp across the femoral vein at the junction of the gracilis pedicle. The number of flowing capillaries in five consecutive high-power fields (832x) were counted at baseline and at 5, 15, 30, 60, 120, 180 minutes, and 24 hours after reperfusion. At 24 hours after reperfusion, the gracilis muscles were harvested and stained with nitroblue tetrazolium. Percentage of muscle necrosis was measured by using computer planimetry. The data were reported as mean +/- standard error of mean and were compared between groups by analysis of variance and appropriate post hoc comparisons. Total venous occlusion for 10, 30, and 60 minutes showed a significant decrease in the number of flowing capillaries through 24-hour postreversal. There was a significant drop (p < 0.01) in the number of flowing capillaries from 30 minutes of total venous occlusion to 60 minutes of total venous occlusion at all times. Muscle necrosis was significantly increased in all three groups of total venous occlusion compared with the sham group (36.1 +/- 1.7 percent, 45.5 +/- 3.4 percent, 74.1 +/- 4.7 percent versus 14.3 +/- 1.7 percent, and p < 0.01). These results indicate that irreversible tissue damage occurs in a very short time interval (60 minutes) in this model, making the early detection of venous occlusion critical to the successful correction of this complication.     

Dichloroacetate improves cardiac efficiency after ischemia independent of changes in mitochondrial proton leak

Dichloroacetate (DCA) is a pyruvate dehydrogenase activator that increases cardiac efficiency during reperfusion of ischemic hearts. We determined whether DCA increases efficiency of mitochondrial ATP production by measuring proton leak in mitochondria from isolated working rat hearts subjected to 30 min of ischemia and 60 min of reperfusion. In untreated hearts, cardiac work and efficiency decreased during reperfusion to 26% and 40% of preischemic values, respectively. Membrane potential was significantly lower in mitochondria from reperfused (175.6 +/- 2.2 mV) versus aerobic (185.8 +/- 3.1 mV) hearts. DCA (1 mM added at reperfusion) improved recovery of cardiac work (1.9-fold) and efficiency (1.5-fold) but had no effect on mitochondrial membrane potential (170.6 +/- 2.9 mV). At the maximal attainable membrane potential, O(2) consumption (nmol O(2) x mg(-1) x min(-1)) did not differ between untreated or DCA-treated hearts (128.3 +/- 7.5 and 120.6 +/- 7.6, respectively) but was significantly greater than aerobic hearts (76.6 +/- 7.6). During reperfusion, DCA increased glucose oxidation 2.5-fold and decreased H(+) production from glucose metabolism to 53% of untreated hearts. Because H(+) production decreases cardiac efficiency, we suggest that DCA increases cardiac efficiency during reperfusion of ischemic hearts by increasing the efficiency of ATP use and not by increasing the efficiency of ATP production.     

Hyperglycemic Damage to Mitochondrial Membranes During Cerebral Ischemia: Amelioration by Platelet-Activating Factor Antagonist BN 50739

Abstract: The Pulsinelli-Brierley four-vessel occlusion model was used to study the consequences of hyperglycemic ischemia and reperfusion. Rats were subjected to either 30 min of normo- or hyperglycemic ischemia or 30 min of normo- or hyperglycemic ischemia followed by 60 min of reperfusion. In some animals, 2 mg/kg BN 50739, a platelet-activating factor receptor antagonist, was administered intraarterially either before or after the ischemic insult. The changes in mitochondrial membrane free fatty acid levels, phosphatidylcholine fatty acyl composition, and thiobarbituric acid-reactive material (TBAR) content plus the mitochondrial respiratory control ratio (RCR) were monitored. When the platelet-activating factor antagonist was present during normoglycemia, (a) the mitochondrial free fatty acid release both during and after ischemia was slowed, (b) reacylation of phosphatidylcholine following ischemia was promoted, and (c) TBAR accumulation during and following ischemia was decreased. The detrimental effects of hyperglycemia were muted when BN 50739 was present during ischemia. The RCR was preserved and phosphatidylcholine hydrolysis during ischemia was decreased. TBAR levels were consistently higher in hyperglycemic brain mitochondria both during and after ischemia. The RCR correlated directly with mitochondrial phosphatidylcholine polyunsaturated fatty acid content during ischemia and reperfusion. BN 50739 protection of mitochondrial membranes in brain may be influenced by tissue pH.     

Inability of dimethylthiourea to limit tissue necrosis during acute myocardial infarction in rabbits.

This study examined the effect of treatment with dimethylthiourea (DMTU), a highly cell-permeable scavenger of hydroxyl radicals, on tissue necrosis in rabbit hearts during myocardial ischemia and reperfusion. Sixty-two rabbits underwent 45 minutes of coronary occlusion with, or without, coronary reperfusion for 3 hours. A saline vehicle, or DMTU (500 mg/kg intravenously [iv]) was administered over 45 minutes starting either 10 minutes before or 10 minutes after coronary occlusion, or 10 minutes before coronary reperfusion. Anatomic risk zone size was assessed using microsphere autoradiography, and the area of necrosis was determined using tetrazolium staining. Cardiac hemodynamics and risk zone size were similar for all treatment groups. No differences were observed in the extent of tissue necrosis (normalized to risk zone size) for saline- and DMTU-treated rabbits subjected to 45 minutes (61.2 +/- 23.1% vs. 70.6 +/- 16.5%) or 225 minutes (82.8 +/- 5.4% vs. 78.3 +/- 5.9%) of permanent coronary occlusion without reperfusion. Similarly, tissue necrosis in rabbits with 45 minutes coronary occlusion followed by 3 hours reperfusion was not significantly reduced when DMTU was administered either 10 minutes before coronary occlusion, 10 minutes after coronary occlusion, or 10 minutes before coronary reperfusion (67.0 +/- 9.9%; 57.6 +/- 10.6%; 68.3 +/- 13.3%) compared to saline-treated controls (76.6 +/- 10.5%). These results demonstrate that the hydroxyl radical scavenger DMTU does not appear to influence the progression of myocyte injury in this experimental model of acute myocardial infarction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on hepatic injury and antioxidant enzyme activities in rat subcellular organelles following in vivo ischemia and reperfusion

The activities of rat hepatic subcellular antioxidant enzymes were studied during hepatic ischemia/reperfusion. Ischemia was induced for 30 min (reversible ischemia) or 60 min (irreversible ischemia). Ischemia was followed by 2 or 24 h of reperfusion. Hepatocyte peroxisomal catalase enzyme activity decreased during 60 min of ischemia and declined further during reperfusion. Peroxisomes of normal density (d = 1.225 gram/ml) were observed in control tissues. However, 60 min of ischemia also produced a second peak of catalase specific activity in subcellular fractions corresponding to newly formed low density immature peroxisomes (d = 1.12 gram/ml). The second peak was also detectable after 30 min of ischemia followed by reperfusion for 2 or 24 h. Mitochondrial and microsomal fractions responded differently. MnSOD activity in mitochondria and microsomal fractions increased significantly (p < 0.05) after 30 min of ischemia, but decreased below control values following 60 min of ischemia and remained lower during reperfusion at 2 and 24 h in both organelle fractions. Conversely, mitochondrial and microsomal glutathione peroxidase (GPx) activity increased significantly (p < 0.001) after 60 min of ischemia and was sustained during 24 h of reperfusion. In the cytosolic fraction, a significant increase in CuZnSOD activity was noted following reperfusion in animals subjected to 30 min of ischemia, but 60 min of ischemia and 24 h of reperfusion resulted in decreased CuZnSOD activity. These studies suggest that the antioxidant enzymes of various subcellular compartments respond to ischemia/reperfusion in an organelle or compartment specific manner and that the regulation of antioxidant enzyme activity in peroxisomes may differ from that in mitochondria and microsomes. The compartmentalized changes in hepatic antioxidant enzyme activity may be crucial determinant of cell survival and function during ischemia/reperfusion. Finally, a progressive decline in the level of hepatic reduced glutathione (GSH) and concomitant increase in serum glutamate pyruvate transaminase (SGPT) activity also suggest that greater tissue damage and impairment of intracellular antioxidant activity occur with longer ischemia periods, and during reperfusion.     

Diminution of "reperfusion injury" in reperfused ischemic myocardium by phenothiazines. A quantitative morphological study

The effect of pretreatment by phenothiazines--Chlorpromazine (CPR) /Spofa/ and Trifluoperazine (TFP) /Smith Kline and French/ on reperfusion injury of ischemic myocardium were studied. Reperfusion of ischemic myocardium following an ischemic period exceeding 40 min resulted in morphological, physiological and biochemical changes identical with those induced by enhanced cytosolic Ca2+ concentration. Left descending coronary ligation was performed on 70 dogs divided into four group. Group I: permanent occlusion (5 dogs--60 min, 5 dogs--120 min, 5 dogs--180 min); group II: 15 dogs (60 min occlusion + 120 min reperfusion); group III: 20 dogs (60 min occlusion, 15 mg CPR, reperfusion 120 min); group IV: 20 dogs (60 min occlusion, 2 mg TFP + 120 min reperfusion). CPR or TFP were administered 30 min after the ligation. The effect of drugs was quantified on tetrazolium stained gross sections and studied from physiological, biochemical and ultrastructural points of view. Treatment of animals with phenothiazines, known as calmodulin inhibitors, considerably improved the ultrastructure of myocytes in area at risk, and allowed for the recovery of at least 60 per cent of injured myocytes after reflow restoration. Ultrastructural findings tightly correlate with physiological and biochemical results.     

Naloxone effect on the neurological deficit induced by forebrain ischemia in rats

The effect of naloxone upon neurologic deficit was evaluated in a model of transient forebrain ischemia in rats. Awake male Wistar rats were subjected to a 30 minute ischemia by occluding both common carotid arteries 8 days after cauterizing vertebral arteries. Administration of naloxone 1 or 5 mg/kg iv 10 minutes after carotid occlusion or 1 mg/kg iv one hour after clamp removal failed to reduce immediate and tardive neurologic postischemic deficits. On the other hand, in rats treated by a dose of 1 mg/kg naloxone 10 minutes after carotid occlusion and perfused with an additional dose of 2 mg/kg/h for 80 minutes, neurologic score was improved one hour after ischemia. However mortality was not decreased whatever was the modality of naloxone administration. This result confirms previous data showing that naloxone exerts a protective effect when given at sufficiently high dosage.     

Tissue and plasma levels of endothelin in free flaps

The goal of the study was to assess whether endothelin-1 levels are increased in tissue and plasma in free flaps. To assess this hypothesis, blood samples were taken from the general circulation before and after reperfusion and from the flap after reperfusion in 20 patients undergoing breast reconstruction with free transverse rectus abdominis musculocutaneous or deep inferior epigastric perforator flaps. Tissue samples were also taken from the flap before and after the period of ischemia. Peripheral blood samples of 10 ml each were taken before the vessels were clamped and at 10 minutes and 1 hour after the flap was recharged. The flap vein was catheterized with a smooth catheter to avoid endothelial trauma, and ischemic blood from the flap was obtained immediately after the artery was unclamped and 10 minutes later. Two skin samples of 2 cm each were taken: one after dissection of the flap before division of the vessels and one after reanastomosis of the veins (one or two veins). Statistical analyses were performed with the (nonparametric) Wilcoxon signed rank test. Flap ischemia time, from vessel division to the completion of the arterial anastomosis, ranged from 35 to 120 minutes (mean, 48 minutes). The plasma endothelin-1 level extracted from the flap was 4.34 +/- 0.85 pg/ml, significantly higher than baseline, 3.87 +/- 0.81 pg/ml (p < 0.0001). There was a small increase, 4.5 +/- 1.03 pg/ml (p = NS), 10 minutes after reperfusion. The peripheral level after venous anastomosis was 3.78 +/- 0.79 pg/ml, not significantly different from the peripheral plasma level, before the flap was raised. The peripheral plasma level 1 hour after reperfusion was 3.83 +/- 0.8 pg/ml, with no difference from baseline. The tissue level of endothelin-1 before clamping was 3.8 +/- 0.8 pg/mg and in postischemic tissue, 5.2 +/- 0.6 pg/mg, a statistically significant increase. The authors concluded that endothelin-1 levels are elevated in free flaps. This could be an explanation for vasospasm and may lead to therapy directed against the no-reflow phenomenon.     

Postconditioning fails to improve no reflow or alter infarct size in an open-chest rabbit model of myocardial ischemia-reperfusion

Postconditioning (PoC) with brief intermittent ischemia after myocardial reperfusion has been shown to lessen some elements of postischemic injury including arrhythmias and, in some studies, the size of myocardial infarction. We hypothesized that PoC could improve reflow to the risk zone after reperfusion. Anesthetized, open-chest rabbits were subjected to 30 min of coronary artery occlusion followed by 3 h of reperfusion. In protocol 1, rabbits were randomly assigned to the control group (n = 10, no further intervention after reperfusion) or to the PoC group, which consisted of four cycles of 30-s reocclusions with 30 s of reperfusion in between starting at 30 s after the initial reperfusion (4 x 30/30, n = 10). In protocol 2, rabbits were assigned to the control group (n = 7) or the PoC group, which received PoC consisting of four cycles of 60-s intervals of ischemia and reperfusion starting at 30 s after the initial reperfusion (4 x 60/60, n = 7). No reflow was determined by injecting thioflavine S (a fluorescent marker of capillary perfusion), risk zone by blue dye, and infarct size by triphenyltetrazolium chloride. In protocol 1, there were no statistical differences in hemodynamics, ischemic risk zone, or infarct size (35 +/- 6% of the risk zone in the PoC group vs. 29 +/- 4% in the control group, P = 0.38) between the groups. Similarly, in protocol 2, PoC failed to reduce infarct size compared with the control group (45 +/- 4% of the risk zone in the PoC group vs. 42 +/- 6% in the control group, P = 0.75). There was a strong correlation in both protocols between the size of the necrotic zone and the portion of the necrotic zone that contained an area of no reflow. However, PoC did not affect this relationship. PoC did not reduce infarct size in this model, nor did it reduce the extent of the anatomic zone of no reflow, suggesting that this intervention may not impact postreperfusion microvascular damage due to ischemia.     

Flow heterogeneity following global no-flow ischemia in isolated rabbit heart

The purpose of this study was to evaluate flow heterogeneity and impaired reflow during reperfusion after 60-min global no-flow ischemia in the isolated rabbit heart. Radiolabeled microspheres were used to measure relative flow in small left ventricular (LV) segments in five ischemia + reperfused hearts and in five nonischemic controls. Relative flow heterogeneity was expressed as relative dispersion (RD) and computed as standard deviation/mean. In postischemic vs. preischemic hearts, RD was increased for the whole LV (0.92 +/- 0.41 vs. 0.37 +/- 0.07, P < 0.05) as well as the subendocardium (Endo) and subepicardium considered separately (1.28 +/- 0.74 vs. 0.30 +/- 0.09 and 0.69 +/- 0.22 vs. 0.38 +/- 0.08; P < 0.05 for both comparisons, respectively) during early reperfusion. During late reperfusion, the increased RD for the whole LV and Endo remained significant (0.70 +/- 0.22 vs. 0.37 +/- 0.07 and 1.06 +/- 0.55 vs. 0.30 +/- 0.09; P < 0.05 for both comparisons, respectively). In addition to the increase in postischemic flow heterogeneity, there were some regions demonstrating severely impaired reflow, indicating that regional ischemia can persist despite restoration of normal global flow. Also, the relationship between regional and global flow was altered by the increased postischemic flow heterogeneity, substantially reducing the significance of measured global LV reflow. These observations emphasize the need to quantify regional flow during reperfusion after sustained no-flow ischemia in the isolated rabbit heart.     

Effects of oxidative stress on the expression of antioxidative defense enzymes in spontaneously hypertensive rat hearts

Little is known concerning the effect of oxidative stress on the expression of antioxidative enzymes in the decompensated cardiac hypertrophy of spontaneously hypertensive rats (SHR), considered as a model of dilative cardiomyopathy in man. Superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx) were characterized in isolated perfused hearts of 18 month old SHR and the age-matched normotensive control Wistar-Kyoto (WKY) rats, before and after 30 min infusion of 25 microM H(2)O(2). After infusion of H(2)O(2), aortic flow decreased in WKY from 26.2 +/- 2.2 to 16.0 +/- 0.8 ml/min (p <.05) but not in SHR (18.2 +/- 1.9 vs. 20.7 +/- 2.2 ml/min). This protection was related to the higher myocardial activities of GPx, MnSOD and CuZnSOD in SHR, compared with those of the WKY group. Although total SOD activity in the SHR fell after H(2)O(2) exposure (to 1.81 +/- 0.13 from 3.56 +/- 0.49 U/mg of protein), catalase activity increased (to 2.46 +/- 0.34 from 1.56 +/- 0.29 k min(-1)mg(-1)protein), compared with the pre-infusion period (p <.05 in each case). In additional studies, hearts were subjected to 30 min of global ischemia followed by 30 min of reperfusion. The results obtained in ischemic/reperfused hearts show the same changes in enzyme activities measured as it was observed in H(2)O(2) perfused hearts, indicating that oxidative stress is independent of the way it was induced. The higher catalase activity derived from elevated mRNA synthesis. The antioxidative system in dilative cardiomyopathic hearts of SHR is induced, probably due to episodes of oxidative stress, during the process of decompensation. This conditioning of the antioxidative potential may help overcome acute stress situations caused by reactive oxygen species in the failing myocardium.     

Chronically administered acetaminophen and the ischemia/reperfused myocardium

Male and female Hartley strain guinea pigs weighing 280 +/- 10 g were given acetaminophen-treated water ad libitum for 10 days. Sham-treated control animals were given similar quantities of untreated tap water (vehicle-treated control group). On Day 10, hearts were extracted, instrumented, and exposed to an ischemia (low-flow, 20 min)/reperfusion protocol. Our objective was to compare and contrast ventricular function, coronary circulation, and selected biochemical and histological indices in the two treatment groups. Left ventricular developed pressure in the early minutes of reperfusion was significantly greater in the presence of acetaminophen, e.g., at 1 min, 40 +/- 4 vs 21 +/- 3 mmHg (P < 0.05). Coronary perfusion pressure was significantly less from 3 to 40 min of reperfusion in the presence of acetaminophen. Creatine kinase release in vehicle-treated hearts rose from 42 +/- 14 (baseline) to 78 +/- 25 units/liter by the end of ischemia. Corresponding values in acetaminophen-treated hearts were 36 +/- 8 and 44 +/- 14 units/liter. Acetaminophen significantly (P < 0.05) attenuated release of creatine kinase. Chemiluminescence, an indicator of the in vitro production of peroxynitrite via the in vivo release of superoxide and nitric oxide, was also significantly attenuated by acetaminophen. Electron microscopy indicated a well-preserved myofibrillar ultrastructure in the postischemic myocardium of acetaminophen-treated hearts relative to vehicle-treated hearts (e.g., few signs of contraction bands, little or no evidence of swollen mitochondria, and well-defined light and dark bands in sarcomeres with acetaminophen; opposite with vehicle). We conclude that chronic administration of acetaminophen provides cardioprotection to the postischemic, reperfused rodent myocardium.