Entire Reperfusion Period Research Articles

Posthypoxic behavioral impairment and mortality of Drosophila melanogaster are associated with high temperatures, enhanced predeath activity and oxidative stress

Hypoxia is an underlying pathophysiological condition of a variety of devastating diseases, including acute ischemic stroke (AIS). We are faced with limited therapeutic options for AIS patients, and even after successful restoration of cerebral blood flow, the poststroke mortality is still high. More basic research is needed to explain mortality after reperfusion and to develop adjunct neuroprotective therapies. Drosophila melanogaster (D.m.) is a suitable model to analyze hypoxia; however, little is known about the impacts of hypoxia and especially of the subsequent reperfusion injury on the behavior and survival of D.m. To address this knowledge gap, we subjected two wild-type D.m. strains (Canton-S and Oregon-R) to severe hypoxia (<0.3% O2) under standardized environmental conditions in a well-constructed hypoxia chamber. During posthypoxic reperfusion (21% O2), we assessed fly activity (evoked and spontaneous) and analyzed molecular characteristics (oxidative stress marker abundance, reactive oxygen species (ROS) production, and metabolic activity) at various timepoints during reperfusion. First, we established standard conditions to induce hypoxia in D.m. to guarantee stable and reproducible experiments. Exposure to severe hypoxia under defined conditions impaired the climbing ability and reduced the overall activity of both D.m. strains. Furthermore, a majority of the flies died during the early reperfusion phase (up to 24 h). Interestingly, the flies that died early exhibited elevated activity before death compared to that of the flies that survived the entire reperfusion period. Additionally, we detected increases in ROS and stress marker (Catalase, Superoxide Dismutase and Heat Shock Protein 70) levels as well as reductions in metabolic activity in the reperfusion phase. Finally, we found that changes in environmental conditions impacted the mortality rate. In particular, decreasing the temperature during hypoxia or the reperfusion phase displayed a protective effect. In conclusion, our data suggest that reperfusion-dependent death might be associated with elevated temperatures, predeath activity, and oxidative stress.

Structural changes in skeletal muscles of the hind limbs of rats in acute ischemia-reperfusion and its correction by carbacetam, detected by polarization microscopy

Arterial tourniquets are used in clinical practice for angioplasty and arthroplasty, and in case of limb injuries, their use often occurs according to vital signs. After removing the tourniquet and blood supply restoration to the limb arises a multifactorial lesion of tissues both ischemic and distant from the site of ischemia. A number of publications have been devoted to the study of morphological disorders in muscle tissue in acute ischemia-reperfusion in the medical literature. However, the researches for effective means for drug correction of these disorders still continues. The aim of the study was to explore peculiarities of skeletal muscle remodeling of the hind limbs of rats, detected by polarization microscopy, in acute ischemia-reperfusion, caused by the application of an arterial tourniquet, and in the correction of reperfusion disorders by carbacetam. Microscopic examination of histological sections of skeletal muscles of the hind limbs of 60 rats below the site of application of the tourniquet under conditions of experimental acute ischemia-reperfusion was performed. Acute ischemia for all animals was caused by application of SWAT rubber bands on the hind limbs of animals, 5–6 mm in width, at the inguinal fold level within 2 hours under thiopental anesthesia. A reperfusion was modeled by removing the tourniquet. Half of the experimental animals in the reperfusion period for the purpose of correction intraperitoneally was administered the nootropic drug 1-oxo-3.3.6-trimethyl-1.2.3.4-tetrahydroindolo[2.3-c]quinoline (carbacetam) at a dose of 5 mg per kilogram of body weight once a day during the entire reperfusion period. The histological specimens of the skeletal muscles were stained with hematoxylin and eosin, and were examined with a light microscope with polarization nozzle. Studies with using the polarization microscopy have shown that in the early reperfusion period morphological criteria for skeletal muscle remodeling expressed by deformation and anisotropy of muscle fibers, disappearance of their transverse striation, cracks and ruptures of fibers, and in the most severe cases there were signs of necrosis of the fibers with their fragmentation into separate lumps. Subject to the correction of reperfusion disorders by carbacetam, there is a decrease in the degree of damage and consistent acceleration of restoration of the skeletal muscles structure, which was the most pronounced in groups of animals with reperfusion terms after 1 and 14 days. Complex of features indicates, that at the tissue level the administration of carbacetam as reduces the ischemic-reperfusion lesion of the muscular fibers, as also accelerates the mechanisms of reparative rhabdomyohistogenesis. Thus, structural changes in the skeletal muscles of the limb after two-hour ischemia and subsequent reperfusion increased in the early reperfusion period and reached its peak after 1 day of reperfusion, and in the late period of reperfusion their reverse development took place. With the correction of disorders by carbacetam, the degree of damage was reduced and the recovery of the skeletal muscle structure of the limb was accelerated.

Microcirculatory effects of sildenafil in experimental testicular torsion in rats.

Investigate the short-term effect of sildenafil on microcirculation, especially the velocity, the pattern of the flow and the recruitment of the leukocyte in postcapillaries. In male Sprague-Dawley rats, the microcirculatory consequences of 60min experimental testicular torsion, followed by 240min of reperfusion, were examined. Using fluorescence intravital microscopy, changes in red blood cell velocity in post-capillary venules and rolling as well as adhesion of leukocytes in the postcapillary venules were examined before the torsion and every hour during the reperfusion period. Sildenafil was given 10min prior to reperfusion (iv 0.7mg/kg, n = 6), while control animals received saline vehicle (n = 5). The characteristic flow motion disappeared in the affected testicular during the torsion. Red blood cell velocity values were dramatically decreased (by > 50%) and both rolling and adhesion of leukocytes increased during the reperfusion phase. Sildenafil treatment resulted in significantly higher red blood cell velocity values during the entire reperfusion period, but exerted only a temporary positive effect on the plost-ischaemic leukocyte-endothelial interactions. Intraoperative administration of sildenafil during surgical detorsion may provide marked testicular microperfusion benefits, but failed to influence the overall leukocyte-driven microcirculatory inflammatory reactions.

Effects of Pre- and Postconditioning on Arrhythmogenesis in the In Vivo Rat Model

The antiarrhythmic potential of postconditioning in in vivo models remains poorly defined. We compared the effects of pre- and postconditioning on ventricular arrhythmogenesis against controls with and without reperfusion. Wistar rats (n = 40, 269 ± 3 g) subjected to ischemia (30 minutes)--reperfusion (24 hours) were assigned to the following groups: (1) preconditioning (2 cycles), (2) postconditioning (6 cycles), or (3) no intervention and were compared with (4) nonreperfused infarcts and (5) sham-operated animals. Infarct size was measured, and arrhythmogenesis was evaluated with continuous telemetric electrocardiographic recording, heart rate variability indices, and monophasic action potentials (MAPs). During a 24-hour observation period, no differences in mortality were observed. Reperfusion decreased infarct size and ameliorated sympathetic activation during the late reperfusion phase. Preconditioning decreased infarct size by a further 35% (P = .0017), but only a marginal decrease (by 18%, P = .075) was noted after postconditioning. Preconditioning decreased arrhythmias during ischemia and early reperfusion, whereas postconditioning almost abolished them during the entire reperfusion period. No differences were noted in MAPs or in the magnitude of sympathetic activation between the 2 interventions. Compared to postconditioning, preconditioning affords more powerful cytoprotection, but both interventions exert antiarrhythmic actions. In the latter, these are mainly evident during the ischemic phase and continue during early reperfusion. Postconditioning markedly decreases reperfusion arrhythmias during a prolonged observation period. The mechanisms underlying the antiarrhythmic effects of pre- and postconditioning are likely different but remain elusive.

The protective effect of rutin against ischemia/reperfusion-associated hemodynamic alteration through antioxidant activity

Reactive oxygen species exert toxic effects during ischemia-reperfusion (I/R) injury of various organs. This study was designed to evaluate the preventive effects of various isoflavonoids such as biochanin A, daidzein, genistein, rutin and quercetin. These compounds are wellknown naturally occurring compounds with beneficial health effects and antioxidant activity. Free radical scavenging activity was measured by 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay and superoxide dismutase (SOD) assay. Among the isoflavonoids tested, biochanine A, quercetin and rutin showed significant DPPH free radical scavenging activity. Similarly, treatment of biochanine A, genistein and rutin significantly increased SOD activity in neonant rat heart myocyte primary cells as well as in H9C2 cells. For ex vivo study, hearts from Sprague-Dawley rats were perfused in Langendorff apparatus with Krebs-Henseleit solution with a gas mixture of 95% O(2) and 5% CO(2). Hearts were subjected to 20 min of pre-ischemia followed by 20 min of global ischemia, and then 50 min of reperfusion at 37°C. The test compounds were perfused 10 min before ischemia and during the entire reperfusion period. Among the isoflavonoids tested, only rutin significantly increased left ventricular developed pressure (LVDP) and increased maximum positive and negative dP/dt (+/- dP/dtmax). In left ventricular end diastolic pressure (LVEDP) analysis, rutin, daidzein and biochanin A were effective. Among the isoflavonoids, rutin had consistent protective effects in I/R injury by affecting cardiac dynamic factors as well as by enhancing SOD and DPPH activity.

Acute treatment with polyphenol quercetin improves postischemic recovery of isolated perfused rat hearts after global ischemia

Quercetin is a plant-derived bioflavonoid with potentially beneficial effects on the cardiovascular system. Studies focused on the efficiency of flavonoids against ischemia-reperfusion (I/R) injury have demonstrated that quercetin exerts robust protective effects in renal, cerebral, and hepatic I/R models. However, there is only limited evidence about the effect of quercetin on myocardial I/R injury. Therefore, the aim of the current study was to examine the effect of quercetin on isolated rat heart during ischemia and reperfusion. Rat hearts perfused according to Langendorff at 37 degrees C were examined during 25 min global ischemia followed by 120 min reperfusion. Quercetin (15 micromol/L) was administered either 15 min before ischemia (group Q1), or during the entire reperfusion period (group Q2). Changes in functional parameters of the hearts were measured during the initial 40 min of reperfusion. At the end of the experiment, the hearts were stained with tetrazolium to estimate the size of infarction (IS). Our study showed that quercetin improved postischemic recovery of functional parameters of isolated hearts in both treated groups. This improvement was manifested by significantly higher values of left ventricular developed pressure (LVDP) and the maximal rates of pressure development and fall (+(dP/dt)max and -(dP/dt)max) and by significantly lower increase of end-diastolic pressure. Coronary flow was not significantly changed during reperfusion in the group treated before ischemia, but was significantly increased in the group treated during reperfusion. Quercetin also significantly reduced IS in both groups, more markedly in postischemically treated group. We conclude that acute quercetin treatment exerts significant positive effects on isolated hearts during I/R injury. These results are consistent with the beneficial effects of quercetin and other flavonoids on the cardiovascular system.

The Rocky Road from Bench to Bedside

The Rocky Road from Bench to Bedside

In vivo Visualization of Early Microcirculatory Changes following Ischemia/Reperfusion Injury in Human Kidney Transplantation

To determine whether microcirculatory changes following ischemia/reperfusion (I/R) may serve as predictors for subsequent graft dysfunction, we used noninvasive orthogonal polarization spectral (OPS) imaging to directly visualize and quantify cortical kidney microcirculation. In a total of 13 combined kidney/pancreas recipients, following reperfusion (5/30 min) microcirculatory parameters such as capillary diameter, functional capillary density (FCD) and red-blood-cell velocity (V_RBC) of the renal graft were analyzed. From these parameters, a heterogeneity index (HI) and volumetric capillary blood flow (vCBF) were calculated. In addition, the extent of graft injury was determined by daily analysis of serum creatinine, blood urea nitrogen, C-reactive protein and systemic leukocyte count for 7 days post-transplant. At early reperfusion, a heterogeneous perfusion pattern with oscillating flow and scattered microvascular thrombosis of peritubular capillaries, resembling a ‘no reflow’, was observed. FCD was constant throughout the entire reperfusion period, whereas HI, capillary diameters, V_RBC and vCBF increased. The latter showed a significant positive correlation with creatinine changes between days 1 and 3. So far our finding of a positive correlation of early microvascular changes (vCBF) and clinical parameters (creatinine) indicate a possible therapeutic implication of OPS imaging to predict early I/R-induced renal graft dysfunction.

Pentoxifylline Decreases Up-Regulated Nuclear Factor kappa B Activation and Cytokine Production in the Rat Retina following Transient Ischemia

Aim: To investigate whether pentoxifylline (PTX) could influence the increased cytokine gene expression in the retina flowing transient ischemia, and if so, whether it acts through the modulation of nuclear factor kappa B (NF-ĸB) activation. Methods: Sprague-Dawley rats were randomly divided into three equal groups: control group, saline-treated group, and PTX-treated group. Increased intraocular pressure was applied for 90 min to induce retinal ischemia, and reperfusion was established by lowering the bottle to eye level. The reperfusion period lasted for 48 h. In the PTX-treated group, an initial dose of 20 mg PTX was injected via tail vein at the beginning of reperfusion. Then the rat received infusion of PTX at a rate of 6 mg/kg/h throughout the entire reperfusion period. The retinal tissues were collected at the end of 1, 6, 12, 24, and 48 h of reperfusion, respectively, for biochemical analysis. Histological examination was done on the tissues collected at the end of 48 h after reperfusion. Results: Histological examination revealed reduction of overall retinal thickness and thinning of the inner retinal layer in saline-treated rats after 48-hour reperfusion. However, PTX treatment significantly reduced the loss of overall retinal thickness and thinning of inner retinal layers. Dramatic increase in NF-ĸB activation, tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) production and mRNA expression were observed in the saline-treated group after reperfusion, with the peak reached around 12 h. In the PTX-treated group, NF-ĸB activation, TNF-α and IL-1β production and mRNA expression were significantly reduced at each corresponding time point compared to the saline-treated group. Conclusion: PTX decreased the up-regulated activation of NF-ĸB and the expression of proinflammatory cytokines, TNF-α and IL-1β in rat retinas following ischemia/reperfusion. This may contribute to significantly reduce the loss of overall retinal thickness and thinning of inner retinal layers.

Gender differences in ischemia-reperfusion-induced microcirculatory and epithelial dysfunctions in the small intestine

Female sex hormones have been reported to preserve endothelial integrity and to reduce inflammation. However, gender-related differences in the intestinal mucosal barrier function during compromised perfusion after ischemia and transplantation have not been defined. Herein, we applied intravital microscopy to determine the mucosal epithelial and intestinal microcirculatory responses in ileal villus and longitudinal muscle layers in a murine model of 30-min intestinal ischemia and 90-min reperfusion. In male animals, the entire reperfusion period was characterized by a significantly increased epithelial permeability. This was associated with an early leukocytic inflammatory response and late alterations in functional capillary density, capillary red blood cell velocity and mitochondrial redox state. In contrast, the female intestine exhibited a delayed increase in epithelial permeability during postischemic reperfusion. This was associated with a late leukocytic inflammatory response which did not affect the microcirculatory function. Nonetheless, at the end of the 90-min reperfusion period, the neutrophilic infiltration and structural mucosal disintegration in the female intestine were found to be pronounced to a similar extent as in the male intestine. These results suggest that in small intestinal ischemia–reperfusion the leukocytic inflammatory response and microcirculatory dysfunction develop more rapidly and are initially more pronounced in males, but the hormonal status in females is not capable of preventing the final manifestations of reperfusion injury.

Acute Microvascular Action of Vascular Endothelial Growth Factor in Skeletal Muscle Ischemia/Reperfusion Injury

The purpose of this study was to investigate the acute action of vascular endothelial growth factor (VEGF) in the microcirculation of skeletal muscle subject to ischemia/reperfusion in vivo and to determine the role of nitric oxide synthase in VEGF-induced microvascular protection. A vascular pedicle isolated rat cremaster muscle model coupled with local intraarterial infusion technique was used. Each muscle underwent 4 hours of zero-flow warm ischemia followed by 2 hours of reperfusion. Femoral artery cannulation was performed before reperfusion. The infusate was administered by continuous infusion into the arterial tree of the muscle beginning at 1 minute before reperfusion and at the rate of 0.1 ml/hour throughout the entire reperfusion period. Three groups were designed: (1) the ischemia/reperfusion group, with infusion normal saline; (2) the VEGF plus ische-mia/reperfusion group, with infusion of recombinant human VEGF165 protein; and (3) the L-NA plus VEGF plus ischemia/reperfusion group, with infusion of N-nitro-L-arginine (L-NA; a nonselective nitric oxide synthase antagonist) mixed with VEGF165 protein. After 2 hours of reperfusion, microcirculation measurements including arteriole diameter, capillary perfusion, and endothelium-dependent and endothelium-independent vasodilatation were performed. The muscle was harvested and processed for reverse-transcriptase polymerase chain reaction for measuring eNOS and endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) gene expression. Reperfusion caused significant microvascular alterations including vasoconstriction, poor capillary perfusion, and endothelial dysfunction in the skeletal muscle. These alterations were significantly attenuated by intraarterial infusion of VEGF during reperfusion, but the beneficial effect of VEGF was reduced significantly by coadministration of L-NA. Reverse-transcriptase polymerase chain reaction study revealed that ischemia/reperfusion depressed eNOS mRNA expression but enhanced iNOS mRNA expression. Intraarterial infusion of VEGF during reperfusion amplified mRNA expression of eNOS but not of iNOS. Local intraarterial infusion of VEGF produced significant microvascular protection from skeletal muscle ischemia/reperfusion injury. The VEGF-induced enhancement of eNOS may play an important mechanistic role.

Pharmacologic pre-conditioning and controlled reperfusion prevent ischemia–reperfusion injury after 30 minutes of hypoxia/ischemia in porcine hearts

Background Hearts from non–heart-beating organ donors are not transplanted because of risk of ischemia-reperfusion injury. We tested whether pharmacologic pre-conditioning with adenosine and the Na +/H + exchanger inhibitor, cariporide, combined with controlled reperfusion, would prevent injury in porcine hearts that had sustained 30 minutes of hypoxia/ischemia in closed-chest animals. Methods Hearts from Yorkshire pigs (100 kg) were studied in 3 groups. Group 1 (control) hearts were surgically removed while beating. Group 2 hearts were harvested from animals made hypoxic by discontinuing mechanical ventilation for 30 minutes. Group 3 hearts were hypoxic as in Group 2, but these animals received adenosine (40 mg) and cariporide (400 mg) 10 minutes before stopping ventilation. Cardiac function in all groups was assessed ex vivo in a working heart apparatus in which pressure and flow measurements were made over 3 hours. Controlled reperfusion in Group 3 hearts used leukocyte-depleted blood perfusate containing free radical scavengers. Myocardial injury was assessed on the basis of perfusate creatine phosphokinase activity and histopathologically determined injury score. Results Groups 1 and 3 hearts could be resuscitated to perform work equivalently during the entire reperfusion period and showed positive responses to increases in pre-load and norepinephrine. Group 2 hearts could not perform work. After 3 hours, Group 2 hearts showed significantly higher creatine phosphokinase and histopathologic injury scores compared to with Groups 1 and 3, which were not significantly different from each other. Conclusions Pharmacologic pre-conditioning and controlled reperfusion effectively protect non-beating porcine hearts from injury after 30 minutes of hypoxia/ischemia in situ.

Effect of estrogen on global myocardial ischemia-reperfusion injury in female rats.

We investigated the effects of estrogen on global myocardial ischemia-reperfusion injury in rats that were ovariectomized (Ovx), sham-operated, or ovariectomized and then given 17beta-estradiol (E(2)beta) supplementation (Ovx+E(2)beta). Hearts were excised, cannulated, perfused with and then immersed in chilled (4 degrees C) cardioplegia solution for 30 min, and then retrogradely perfused with warm (37 degrees C), oxygenated Krebs-Henseleit bicarbonate buffer for 120 min. The coronary flow rate, first derivative of left ventricular pressure, and nitrite production were all significantly lower in Ovx than in sham-operated or Ovx+E(2)beta hearts. However, coronary flow rates or nitrate production were not consistently different throughout the entire reperfusion period. Ca(2+) accumulated more in Ovx rat hearts than in sham-operated or Ovx+E(2)beta hearts, and mitochondrial respiratory function was lower in Ovx hearts than in hearts from the other two groups. Marked interstitial edema and contraction bands were seen in hematoxylin-eosin-stained sections of Ovx rat hearts but not in hearts from either of the other groups. Hematoxylin-basic fuchsin-picric acid-stained sections revealed fewer viable myocytes in hearts from the Ovx group than from the sham or Ovx+E(2)beta group. Transmission electron microscopy demonstrated more severely damaged mitochondria and ultrastructural damage to myocytes in Ovx rat hearts. Our results indicate that estrogen plays a cardioprotective role in global myocardial ischemia-reperfusion injury in female rats.

Plasma fibronectin during myocardial ischemia-reperfusion: effects of magnesium, diltiazem, and a novel Mac-1 inhibitor.

The important role of fibronectin (Fn) has been recognized in patients with ischemic heart disease. However, serial changes of Fn during both brief and prolonged ischemia-reperfusion are poorly known. Plasma Fn was measured during acute myocardial infarction (AMI) and myocardial stunning (MS), and in the absence of myocardial injury. The effects of magnesium (Mg), diltiazem, and a Mac-1 inhibitor on the level of Fn were elucidated. Forty-nine swine underwent prolonged (50 min) or brief (8 min) coronary artery occlusion followed by reperfusion, while six control animals were free of ischemia. During the AMI experiments, plasma Fn underwent a significant progressive increase. Mg or diltiazem similarly affects the plasma Fn, reducing its release during the entire reperfusion period, and did not influence the plasma Fn in the absence of myocardial injury. Contrarily, Mac-1 inhibition resulted in the Fn elevation in controls, and during the occlusion phase, with no significant effect during reperfusion. There were no changes in the plasma Fn during MS, while inhibition of Mac-1 was associated with the significant increase of Fn during ischemia-reperfusion. Ability of Mg, diltiazem, and leumedins to modulate plasma Fn level may have direct clinical implications for the use of these agents in patients with coronary artery disease.

The response of heat shock proteins 25 and 72 to ischaemia in different kidney zones.

Induction of heat shock proteins (HSPs) following cell injury contributes to the protection of vital cell functions. It was, therefore, of interest to study the effects of transient renal ischaemia on the abundance and distribution of two HSPs, HSP25 and HSP72, in renal tissue using Western-blot techniques. Analyses were performed on the supernatant (HSP25, HSP72) and pellet (HSP25) of homogenates obtained from cortex (CX) and outer (OM) and inner (IM) medulla of the rat kidney immediately after 60 min of ischaemia followed by varying periods of reperfusion. Ischaemia of the left kidney caused HSP25 contents to decrease in CX, OM and IM by 73, 89 and 54% respectively, compared with the corresponding zones of the contralateral control kidney. This initial decrease in supernatant HSP25 was accompanied by an increased abundance of HSP25 in the pellet. Following reperfusion, HSP25 contents in the supernatant gradually increased in CX and OM, reaching, after 24 h, values that were 5.4- and 2.5-fold higher, respectively, than those in the control kidneys. After 7 or 14 days of reperfusion, HSP25 contents had not completely normalised in CX, but had reached control levels in OM. In IM, the HSP25 content remained below control throughout the entire reperfusion period. HSP72 (supernatant) was below the detection limit in the CX of the control kidney. Similar to the level of HSP25, that of HSP72 was also markedly lower in OM and IM immediately after ischaemia. The intrarenal distribution of HSP72 and the sequence of zonal changes in HSP72 contents were similar to those observed for HSP25. These results are compatible with the view that, during ischaemia and the initial reperfusion period, HSP25 migrates from the cytoplasmic compartment (supernatant) into the nucleus and/or associates with cytoskeletal structures. The observation that both HSP25 and HSP72 are transiently induced in CX and OM, but not in IM, may be explained by the fact that, while all kidney cells are exposed to ischaemic stress, only inner medullary cells experience a major postischaemic attenuation of osmotic stress.

Serial changes of natural antithrombotics during myocardial ischemia-reperfusion in swine. Effects of magnesium, diltiazem, and a novel Mac-1 inhibitor.

Plasma antithrombin-III (AT-III), protein S, and protein C were measured during myocardial stunning (MS) and acute myocardial infarction (AMI). The effects of magnesium (Mg), diltiazem, and a Mac-1 inhibitor on their plasma levels were elucidated. Forty-nine open-chest swine underwent brief (8 min) or prolonged (50 min) coronary artery occlusion followed by reperfusion. During MS an increase in the plasma AT-III (from 98.5 +/- 3.38% to 138.1 +/- 3.6%) during the early occlusion phase, without any further changes was observed. The profile of total protein S was not changed during MS. Protein C increased at the end of occlusion (from 45.3 +/- 1.8% to 55.7 +/- 1.4%) reaching a peak (64.5 +/- 1.4%) at the beginning of reperfusion. When compared with controls, no significant differences were found in the antithrombotics profile during MS after pretreatment with Mac-1 inhibitor. For the AMI, the AT-III decreased during occlusion (from 98.5 +/- 3.4% to 61.0 +/- 3.6%). The protein S decreased during occlusion with the lowest level at 1 h of reperfusion (from 71.8 +/- 2.2% to 46.7 +/- 1.0%), followed by an increase during late reperfusion (59.2 +/- 1.5%). Contrarily, protein C increased during occlusion and early reperfusion (from 44.7 +/- 2.6% to 79.4 +/- 2.4%), but declined to 49.6 +/- 2.5% thereafter. In both Mg and diltiazem-treated swine, protein C was higher at the end of occlusion and during the entire reperfusion period compared with controls. Mg and diltiazem therapy was associated with the slight elevation of plasma AT-III. The patterns for protein S level during ischemia-reperfusion were similar with the controls. Protein S was higher at the end of occlusion and through the entire reperfusion in the NPC 15669-treated animals when compared with the controls. Mac-1 inhibition was associated with the elevated protein C during late reperfusion. Ability of Mg, diltiazem, and Mac-1 inhibitor to favorably modulate the plasma level of antithrombotics have direct clinical implications for the use of these agents in patients with acute coronary artery syndromes.

Protection Against Reperfusion-Induced Arrhythmias by Human Thioredoxin

Adult T-cell leukemia-derived factor (ADF), identified in the supernatant of adult T-cell leukemia (ATL) cell culture, is a human homologue of thioredoxin and consists of 104 amino acids; it has two redox-active half-cysteine residues in an exposed active center. Human thioredoxin has many biological activities, including growth promotion, cell activation, and a catalase-like radical scavenging activity. We examined the protective effect of human thioredoxin (h-thioredoxin) against reperfusion-induced arrhythmias in an isolated rat heart model with 10-min regional ischemia followed by 30-min reperfusion. Male Wistar rats were assigned to six groups: a control, a superoxide dismutase (SOD 8 x 10(4) IU/L), and a catalase group (1 x 10(6) IU/L), and three groups treated with h-thioredoxin [approximately .01 microM (TRX-I group), approximately 0.1 microM (TRX-II group), and approximately 1 microM (TRX-III group)]. In the early reperfusion period, h-thioredoxin reduced the incidence of ventricular fibrillation (VF) to 8% in the TRX-II group (p < 0.01) from the control value of 75%. SOD and catalase reduced the incidence of VF to 43 and 33%, respectively (NS). During the entire reperfusion period, the incidence of VF in the SOD group was 79%, as compared to 83% in the control group. In the catalase and TRX-II groups, the incidence of VF was significantly reduced to 42 and 25%, respectively. These findings indicate that SOD failed to protect against the reperfusion-induced arrhythmias. h-Thioredoxin exerted a protective effect against these arrhythmias; a concentration of approximately 0.1 micro was the most effective.

Platelet-Activating Factor Mediates Pulmonary Macromolecular Leak Following Intestinal Ischemia–Reperfusion

Platelet-activating factor (PAF) causes hypotension, cardiac dysfunction, increased vascular permeability, intestinal necrosis, and pulmonary microvascular injury when administered experimentally. Receptor antagonism attenuates or abolishes many of these effects in animal models of bacteremia, endotoxemia, and intestinal ischemia/reperfusion (I/R). The purpose of this study was to further examine the role of PAF in intestinal I/R-induced pulmonary injury using the PAF receptor antagonist WEB 2086. Sprague–Dawley rats were anesthetized and cannulated for measurement of mean arterial pressure, heart rate, and cardiac output. Laparotomy and thoracotomy were performed and the superior mesenteric artery was occluded for 45 min and reperfused for 120 min. Sham animals were treated similarly but without I/R. In the treatment groups, iv WEB 2086 (20 mg/kg/1 cc NS) was administered as a bolus 15 min prior to reperfusion. Hemodynamic and videomicroscopic data were obtained before and during ischemia, and after reperfusion at 30-min intervals. Alveolar leak index was calculated off-line via computer analysis of videomicroscopic images. Intestinal I/R caused pulmonary macromolecular leakage and hemodynamic instability. Treatment with WEB 2086 attenuated the pulmonary leak during the entire reperfusion period but improved cardiac output only during the first 30 min of reperfusion and had no effect on other hemodynamic variables. These data suggest that PAF is an important, but not the exclusive, mediator of pulmonary injury after intestinal I/R. PAF appears to play a minor role in the hemodynamic derangements observed after rat intestinal I/R.

Nitric oxide donors improve gut function after prolonged hypothermic ischemia.

The objective of this study was to determine whether the nitric oxide (NO) donors, spermine NO and 3-morpholinosydonimine-N-ethyl-carbamide (SIN1), alter the mucosal and microvascular responses of the feline small intestine to 6 hr of hypothermic ischemia and 2 hr of normothermic reperfusion. Intestinal mucosal permeability was monitored using the blood-to-lumen clearance of 51Cr-EDTA. Lymph flow and lymphatic protein clearance estimates were used to assess intestinal microvascular fluid filtration and vascular protein leakage, respectively. Spermine NO (0.1 mmol/L) or SIN1 (0.5 mmol/L) was added to the luminal perfusate during the entire reperfusion period. Both NO donors were effective in attenuating the increased mucosal permeability to 51Cr-EDTA and the depressed net water absorption, relative to untreated intestinal preparations exposed to the same protocol. Intestinal lymph flow, lymphatic protein clearance, and capillary hydrostatic pressure were increased by a greater extent in preparations treated with spermine NO. These findings suggest that NO donors may improve mucosal function in intestinal allografts subjected to prolonged hypothermic ischemia. This protective effect on mucosal epithelium appears to be unrelated to an action of the NO donors on the microvasculature.

Intracellular compartmentation of troponin T: Release kinetics after global ischemia and calcium paradox in the isolated perfused rat heart

A. Remppis, T. Scheffold, J. Greten, M. Haas, T. Greten, W. Kübler and H. A. Katus. Intracellular Compartmentation of Troponin T: Release Kinetics After Global Ischemia and Calcium Paradox in the Perfused Rat Heart. Journal of Molecular and Cellular Cardiology (1995) 27, 793–803. The marked differences in troponin T serum concentrations observed in patients with reperfused and non-reperfused myocardial infarction may be due to a perfusion dependent wash-out of an unbound fraction of cardiac troponin T. To test the release kinetics of troponin T experimentally, the isolated rat heart (Langendorff preparation) was damaged either by the calcium paradox or by no-flow ischemia. Following membrane damage by the calcium paradox troponin T (TNT) showed the same release kinetics in the coronary effluent as the cytosolic markers creatine kinase (CK) or lactate dehydrogenase (LDH). Peak levels of troponin T (282 ± 58 μg/l), CK (6754 ± 1642 U/l), and LDH (5817 ± 1730 U/l) occurred 5 min after onset of reperfusion with calcium containing buffers and returned to 9.9%, 1.3%, and 1% of their respective peak levels within 55 min of reperfusion. During reperfusion after no-flow ischemia different release kinetics were found for cytosolic enzymes and troponin T. After 60 min of ischemia, troponin T levels in the coronary effluent increased over the entire reperfusion period of 55 min, almost doubling the 5 min value (191%). In contrast, cardiac enzymes rapidly declined to 18% (CK) and 23% (LDH) of their respective 5 min values at the end of reperfusion. Light microscopy after reperfusion with carbon black revealed a complete and homogenous reperfusion of Langendorff hearts after no-flow ischemia. Immunoblot analysis confirmed the release of an undegraded 39 kDa troponin T molecule, both after global ischemia and the calcium paradox. These data indicate that prolonged ischemia induces a continuous liberation of cardiac troponin T, most probably from disintegrating myofibres, whereas membrane damage leads almost exclusively to leakage of a functionally unbound troponin T pool. These findings may explain the biphasic serum concentration changes of cardiac troponin T in patients with reperfused myocardial infarction.