Early recovery of microvascular perfusion induced by t-PA in combination with abciximab or eptifibatide during postischemic reperfusion

Background  

GPIIb/IIIa inhibitors abciximab and eptifibatide have been shown to inhibit platelet aggregation in ischemic heart disease. Our aim was to test the efficacy of abiciximab (Reo Pro) or eptifibatide (Integrilin) alone or in combination with plasminogen activator (t-PA) in an experimental model of ischemia reperfusion (I/R) in hamster cheek pouch microcirculation visualized by fluorescence microscopy. Hamsters were treated with saline, or abiciximab or eptifibatide or these drugs combined with t-PA infused intravenously 10 minutes before ischemia and through reperfusion. We measured the microvessel diameter changes, the arteriolar red blood cell (RBC) velocity, the increase in permeability, the perfused capillary length (PCL), and the platelet and leukocyte adhesion on microvessels.     

Vascular reactivity and thrombus formation during postischemic reperfusion

Reactivity, thrombogeneity, and thromboresistance of the rat mesentery microvessels were studied in postischemic reperfusion of the intestine, the brain, an extremity. Irrespective of ischemia localisation, an augmentation of the microvessels reactivity and reduction of their thromboresistance, were found. The microvessels thrombogeneity was depended on the ischemia localisation: an augmentation of the thrombogenity occurred in arterioles whereas it was reduced in venules following the brain and intestine reperfusion. A possible mechanism of the phenomenon may involve a deficiency of the nitric oxide synthesis.     

Human recombinant erythropoietin protects the striated muscle microcirculation of the dorsal skinfold from postischemic injury in mice

Erythropoietin (EPO) has been proposed as a novel cytoprotectant in ischemia-reperfusion (I/R) injury of the brain, heart, and kidney. However, whether EPO exerts its protection by prevention of postischemic microcirculatory deterioration is unknown. We have investigated the effect of EPO on I/R-induced microcirculatory dysfunctions. We used the mouse dorsal skinfold chamber preparation to study nutritive microcirculation and leukocyte-endothelial cell interaction in striated muscle of the dorsal skinfold by in vivo fluorescence microscopy before 3 h of ischemia and during 5 days of reperfusion. Animals were pretreated with EPO (5,000 U/kg body wt) 1 or 24 h before ischemia. Vehicle-treated I/R-injured animals served as controls. Additional animals underwent sham operation only or were pretreated with EPO but not subjected to I/R. I/R significantly (P < 0.05) reduced functional capillary density, increased microvascular permeability, and enhanced venular leukocyte-endothelial cell interaction during early reperfusion. These findings were associated with pronounced (P < 0.05) arteriolar constriction and diminution of blood flow during late reperfusion. Pretreatment with EPO induced EPO receptor and endothelial nitric oxide synthase expression at 6 h of reperfusion (P < 0.05). In parallel, EPO significantly (P < 0.05) reduced capillary perfusion failure and microvascular hyperpermeability during early reperfusion and arteriolar constriction and flow during late reperfusion. EPO pretreatment substantially (P < 0.05) diminished I/R-induced leukocytic inflammation by reducing the number of rolling and firmly adhering leukocytes in postcapillary venules. EPO applied 1 h before ischemia induced angiogenic budding and sprouting at 1 and 3 days of reperfusion and formation of new capillary networks at 5 days of reperfusion. Thus our study demonstrates for the first time that EPO effectively attenuates I/R injury by preserving nutritive perfusion, reducing leukocytic inflammation, and inducing new vessel formation.     

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.     

Capillary perfusion patterns in single alveolar walls.

We studied capillary perfusion patterns in single alveolar walls through a transparent thoracic window implanted in pentobarbital-anesthetized dogs. The capillaries were maximally opened by brief inflation of a balloon in the left atrium to raise pressure. After the balloon was deflated and pulmonary hemodynamics returned to zone 2 baseline conditions, the capillaries that remained perfused in the observed field were videotaped with the use of in vivo microscopy. The cycle of elevated pressure and baseline observation was repeated three times. Perfusion of different capillaries during each of the observations would imply that the capillaries had characteristics that permitted flow to switch between segments. Perfusion of a specific set of pathways through the network each time would demonstrate that flowing blood sought a unique and repeatable combination of segments, presumably with the least total pathway resistance. We found that the same capillary segments were perfused 79% of the time, a strong indication that a reproducible combination of individual segmental resistances determined the predominant pattern of pulmonary capillary perfusion.     

Nitric oxide mediates protein kinase C isoform translocation in rat heart during postischemic reperfusion

It is controversial whether nitric oxide (NO) is protective or deleterious against ischemia-reperfusion injury. We examined the effect of NO on PKC isoform translocation and protection against ischemia-reperfusion injury in perfused heart. An NO synthase inhibitor L-NAME (NG-nitro-L-arginine methyl ester, 3.0 microM), administered only during reperfusion but not during ischemia, inhibited the translocation of PKC-alpha, -delta and -epsilon isoforms to the nucleus-myofibril fraction and the translocation of PKC-alpha to the membrane fraction after ischemia (20 min) and reperfusion (10 min) in the perfused rat heart. NO donors, 3-morpholinosydnonimine (SIN-1) or S-nitroso-N-acetylpenicillamine (SNAP) activated purified PKC in vitro. SIN-1 also induced PKC isoform translocation in perfused heart. On the other hand, PKC selective inhibitor, calphostin C (0.2 microM) or chelerythrine (1.0 microM), aggravated the contractile dysfunction of ischemic heart during reperfusion, when they were perfused during reperfusion. These data suggest that NO generated during reperfusion following ischemia activates PKC isoforms and may protect the heart against contractile dysfunction in the perfused rat heart.     

Regulatory proteins of lobster striated muscle.

The regulatory proteins of lobster muscles consist of tropomyosin and of troponin. Troponin contains a 17,000 chain weight component, two closely related components of about 30,000 and a 52,000 chain weight component. In addition to troponin, tropomyosin is required for the inhibition of the magnesium activated actomyosin ATPase activity in the absence of calcium and for the reversal of this inhibition by calcium. Lobster tropomyosin interacts with rabbit actin and lobster troponin interacts with rabbit tropomyosin. The 30,000 doublet component corresponds to the troponin-I of rabbit and inhibits the ATPase activity of actomyosin both in the presence and in the absence of calcium. The 17,000 component corresponds to the troponin-C of rabbit; it binds calcium and reverses the inhibition of the ATPase activity by troponin-I in the presence of calcium. No more than 1 mol of calcium is bound by a mole of troponin-C or by troponin. The 52,000 component interacts with tropomyosin and has been tentatively identified as troponin-T; however, it has not been demonstrated as yet that this component had a role in the regulation of lobster actomyosin.     

Interfilament forces in striated muscle

Evaluation of the Van der Waals energy per filament suggests that molecular dispersion forces should not be very important in determining the stability of the myofilament lattice in resting muscle. In order to explain the lattice stability and other important properties of the striated muscle, it is suggested that a balance between electrostatic forces and forces developed by some interfibrillar structures is mainly responsible.     

MCT1 confirmed in rat striated muscle mitochondria.

We sought to test the hypothesis that monocarboxylate transporter isoform 1 (MCT1) is the inner mitochondrial membrane lactate/pyruvate transporter, and, as such, contributes to functioning of the intracellular lactate shuttle. However, presence of a mammalian mitochondrially localized MCT1 (mMCT1) has been contested. We sought to confirm by Western blotting the mitochondrial localization of MCT1 in rat cardiac, soleus, and extensor digitorum longus muscles utilizing three different cell fractionation methods and three different antibodies. We performed Western blotting using antibodies to cell membrane glucose transporter isoform GLUT1, inner mitochondrial constituent cytochrome oxidase, the monocarboxylate transporter protein chaperone CD147, as well as custom and commercially available MCT1 antibodies. Western blots demonstrated similar results with each MCT1 antibody and two of three methods of fractionation. MCT1 was found in the mitochondria, as well as in the sarcolemmal membrane and whole muscle homogenates. Probing with GLUT1 and CD147 demonstrated that mitochondrial fractions were not contaminated with sarcolemmal remnants. Probing with cytochrome oxidase showed mitochondrial localization of MCT1. Comparison of these results to the findings of others indicates that the most likely source of discrepancy is the cell fractionation procedure utilized.     

The transformational potential of striated muscle in hydromedusae.

Isolated striated muscle tissue of the Anthomedusa Podocoryne carnea participates in the regeneration of a functional manubrium (the feeding organ of medusae) when it is combined homoclonally with endodermal cells of the medusa umbrella. The morphogenetic potential of striated muscle cells in this regeneration process was evaluated by combining nuclear labeled striated muscle cells with some unlabeled endoderm cells. Histological and autoradiographical results demonstrate that transformation of striated muscle cells into smooth muscle cells of the ectoderm and also into endoderm cells must have occurred in the regenerate. The potential for cell transformation of isolated striated muscle cells of Podocoryne carnea is discussed and it is postulated that under appropriate conditions all cell types necessary for the regeneration of a manubrium can be formed from striated muscle cells.     

Dinitrosyl iron complexes with glutathione in rat myocardial tissue during regional ischemia and postischemic reperfusion

Injection of dinitrosyl iron complexes with glutathione at the onset of 40-min regional myocardial ischemia in rat was shown to exert a clear cardioprotective action by decreasing the infarct size and suppressing the cardiac rhythm disturbance. After introducing the preparation, its effective accumulation with protein thiol-containing ligands in the myocardial tissue was registered be the EPR method. It was also found that in postischemic reperfusion, the rate of decrease in the content of these complexes in the ischemic area increases, which reflects effective scavenging of short-lived reactive oxygen species by the dinitrosyl iron complexes.     

Quantitative ultrastructural criteria of myocardial damage in postischemic reperfusion

Swollen clarified mitochondria were found during postischemic reperfusion in cardiomyocytes with and without signs of myofibril relaxation. The values of morphometric parameters of mitochondria were similar in both groups of cells studied. Mean indexes characterizing the condition of cardiomyocyte mitochondria may serve as quantitative criteria of the severity of myocardial damage: surface to volume, ratio, visual index of mitochondrial condition and relative volume density, i.e. volume density with respect to control cell volume.     

Oxygen reperfusion is limited in the postischemic hypertrophic myocardium

Studies have shown that hypertrophied hearts are unusually vulnerable to ischemia. Compromised O2 supply has been postulated as a possible explanation for this phenomenon on the basis of elongated O2 diffusion distance and altered coronary vasculature found in hypertrophied myocardium. To examine the postulate, perfused heart experiments followed the metabolic and functional responses of hypertrophic myocardium to ischemia. 1H/31P NMR was used to measure cellular oxygenation and energy level during ischemia-reperfusion. The left ventricles from spontaneously hypertensive rats (SHR) were enlarged by 48%. With this moderate degree of hypertrophy, cellular O2 and energy levels were normal during baseline perfusion. After an ischemic episode, however, cellular O2 was severely deprived in the SHR hearts compared with the normal hearts. Depressed postischemic O2 reperfusion correlated well with depressed energetic and functional recovery. The results from the current study thus demonstrate a critical relationship between reperfused O2 level and functional recovery in hypertrophic myocardium. The role of reperfused O2, however, is time dependent. During early reperfusion, factor(s) other than O2 appear to limit functional recovery. It is when the mechanical function of the heart approaches a new steady state that O2 becomes a dominant factor. Meanwhile, the finding of a normal O2 level in preischemic SHR hearts defies the notion of preexisting hypoxia as a primer of ischemic damage.     

Reduced 4-hydroxynonenal degradation in hearts of spontaneously hypertensive rats during normoxia and postischemic reperfusion

4-Hydroxynonenal (HNE) degradation was investigated in isolated perfused rat hearts of the WKY and SHR strains before and after ischemia. HNE (10 μmoles l^?1) were infused and the concentration of HNE in the effluent was determined. The rate of initial consumption was about 50 nmoles min^?1 g^?1 wet weight in hearts of both the WKY and SHR rats. In the WKY rat hearts, this rate of HNE degradation did not change during several minutes of HNE infusion and also remained constant during postischemic reperfusion. In the hearts of the SHR rats the HNE degradation rate declined within 5 min to 25 nmoles min^?1 g^?1 wet weight. Also during postischemic reperfusion, there was a lower HNE degradation rate in the SHR rat hearts than in the WKY rat hearts. The influence of hypertrophy on the rate of HNE degradation is discussed. It is suggested that the low degradation of the cytotoxic lipid peroxidation product, HNE, in hypertrophic hearts may contribute to reduced antioxidant defence in those hearts.     

Microvascular oxygenation,oxidative stress,NO suppression and superoxide dismutase during postischemic reperfusion

Increased formation of reactive oxygen species (ROS) on reperfusion after ischemia underlies ischemia-reperfusion (I/R) damage. We measured, in real time, oxygen tension in both microvessels and tissue and oxidant stress during postischemic reperfusion in the hamster cheek pouch microcirculation. We measured Po2 by using phosphorescence quenching microscopy and ROS production in the systemic blood. We evaluated the effects of a nitric oxide synthase inhibitor (NG-monomethyl-L-arginine, L-NMMA) and SOD on the oxidative stress during reperfusion. Microvascular injury was assessed by measuring diameter change, the perfused capillary length (PCL), and leukocyte adhesion. During early reperfusion, arteriolar Po2 was significantly lower than baseline, whereas capillary Po2 varied between 7 and 0 mmHg. Arterial blood flow did not regain baseline values, whereas Po2 returned to baseline in arterioles and tissue after 30 min of reperfusion. During 5 and 15 min of reperfusion, ROS increased by 72 and 89% versus baseline, respectively, and declined to baseline after 30 min of reperfusion. Pretreatment with SOD maintained ROS at normal levels, increased arteriolar diameter, blood flow, and PCL, and decreased leukocyte adhesion (P < 0.05). L-NMMA decreased ROS only within 5 min of reperfusion, which increased significantly by 72% later during reperfusion. L-NMMA worsened leukocyte adhesion (P < 0.05). In conclusion, our results show that the early reperfusion is characterized by low Po2 linked to increased production of ROS. At early reperfusion both SOD and L-NMMA decreased ROS production, whereas only SOD reduced it during later reperfusion. We suggest that low-flow hypoxia profoundly affects vascular endothelial damage during reperfusion through changes in ROS and nitric oxide production.