George J. Crystal

Elimination of recombinant hirudin by modified ultrafiltration during simulated cardiopulmonary bypass: Assessment of different filter systems

Recombinant hirudin (r-hirudin) is being used increasingly in patients with heparin-induced thrombocytopenia type II. Renal failure has been demonstrated to prolong the half-life of r-hirudin and to cause bleeding in patients who have undergone cardiopulmonary bypass (CPB). We assessed the ability of different filter systems for modified ultrafiltration to eliminate r-hirudin in vitro using simulated CPB. r-Hirudin concentration was measured (chromogenic laboratory standard plus ecarin clotting time) before and after filtration, and its elimination was calculated using both controlled system flow and arterial inflow (separate pump). Four hemofilters (Renoflow II, Baxter; Arylane H4, Cobe; Ultraflux AV 600, Fresenius; and BCS 110 Plus, Iostra) and two plasmapheresis filter systems (ASAHI Plasmaflow OP, Diamed; and PF 2000 N, Gambro) were assessed (5 filters of each brand = 30 filters) in a closed in vitro CPB system applying conditions usually occurring during CPB. Ten plasmapheresis filters showed a greater ability than 20 hemofilters to eliminate r-hirudin (60%–70% vs 15%–42%) within the shortest time (80 vs 180 s). Among the four hemofilter systems, the Arylane H4 filter provided the most effective (42%) r-hirudin elimination. Elimination of r-hirudin was markedly improved using plasmapheresis systems, compared with hemofilter systems. Our findings may be relevant to patients with impaired renal function, who have been administered r-hirudin during CPB. Implications Modified ultrafiltration may enhance the elimination of recombinant-hirudin, although plasmapheresis systems provide the most rapid and complete elimination of recombinant-hirudin during simulated cardiopulmonary bypass. The decision to use a specific system will ultimately depend on the prevailing clinical situation and overall health of the patient.

Chronic treatment with insulin-like growth factor I enhances myocyte contraction by upregulation of Akt-SERCA2a signaling pathway

Chronic treatment with insulin-like growth factor I (IGF-I) improves contractile function in congestive heart failure and ischemic cardiomyopathy. The present study investigated the effect of chronic treatment with IGF-I on intrinsic myocyte function and the role of the phosphatidylinositol (PI)3-kinase-Akt-sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)2a signaling cascade in these responses. Myocytes were isolated from 23 adult rats and cultured with and without IGF-I (10(-6) M). After 48 h of treatment, myocyte function was evaluated. IGF-I increased contractile function (percent contraction, 7.7 +/- 0.3% vs. 4.5 +/- 0.3%; P < 0.01) and accelerated relaxation time (time for 70% relengthening, 81 +/- 4 vs. 106 +/- 5 ms; P < 0.05) compared with untreated myocytes [control (Con)]. The enhanced function was associated with an increase in Ca(2+) transients assessed by fura-2 (340/380 nm; IGF-I, 0.42 +/- 0.02 vs. Con, 0.25 +/- 0.01; P < 0.01). The PI3-kinase inhibitor LY-249002 (10(-9) M) abolished the enhanced function caused by IGF-I. IGF-I increased both Akt and SERCA2a protein levels 2.5- and 4.8-fold, respectively, compared with those of Con (P < 0.01); neither phospholamban nor calsequestrin was affected. To evaluate whether the SERCA2a protein was directly mediated by Akt-SERCA2a signaling, IGF-I-induced changes in the SERCA2a protein were compared in myocytes transfected with adenovirus harboring either constitutively active Akt [multiplicity of infection (MOI), 15] or dominant negative Akt (dnAkt; MOI, 15). The ability of IGF-I to upregulate the SERCA2a protein in myocytes transfected with active Akt was absent in dnAkt myocytes. Taken together, our findings indicate that chronic treatment with IGF-I enhances intrinsic myocyte function and that this effect is due to an enhancement in intracellular Ca(2+) handling, secondary to the activation of the PI3-kinase-Akt-SERCA2a signaling cascade.

Isoflurane Inhibits Neutrophil-Endothelium Interactions in the Coronary Circulation: Lack of a Role for Adenosine Triphosphate-Sensitive Potassium Channels

Isoflurane protects myocardium during ischemia-reperfusion via a mechanism involving the adenosine triphosphate-sensitive potassium channels. We tested the hypothesis that an inhibition of the neutrophil-endothelium interactions by isoflurane contributes to this effect. Polymorphonuclear neutrophils and coronary artery segments were obtained from 35 healthy dogs. Superoxide production by neutrophils, stimulated with platelet-activating factor (PAF; 1.0 &mgr;M), was measured spectrophotometrically. Adherence of PAF-activated neutrophils to the endothelium of coronary segments was assessed by direct counting of neutrophils labeled with fluorescent dye. Coronary artery rings were exposed to PAF-activated neutrophils, and, after washing and preconstriction with U46619, they were evaluated for vasorelaxation responses to acetylcholine (endothelium dependent) and sodium nitroprusside (endothelium independent). Measurements were performed in the absence and presence of isoflurane (1 and 2 minimum alveolar anesthetic concentration) both with and without glibenclamide (10 &mgr;M). Isoflurane inhibited superoxide production and adherence by neutrophils and abolished neutrophil-induced reductions in coronary vascular relaxation responses to acetylcholine. Glibenclamide did not alter the effects of isoflurane on neutrophils or coronary artery endothelium. In conclusion, isoflurane had an inhibitory action on neutrophil-endothelium interactions and neutrophil-mediated coronary endothelial dysfunction—effects that may be involved in its cardioprotective action in vivo. These inhibitory actions of isoflurane were not mediated by adenosine triphosphate-sensitive potassium channels.

Isoflurane and sevoflurane precondition against neutrophil-induced contractile dysfunction in isolated rat hearts.

BackgroundThe authors tested the hypothesis that pretreatment with isoflurane or sevoflurane can protect the heart against neutrophil-induced contractile dysfunction. MethodsStudies were conducted in buffer-perfused and paced isolated rat hearts. Left ventricular developed pressure served as an index of contractility. Pretreatment consisted of administration of 1.0 minimum alveolar concentration isoflurane or sevoflurane for 15 min followed by a 10-min washout and was performed in the absence and presence of the adenosine triphosphate-sensitive potassium channel inhibitor glibenclamide (10 &mgr;m). Polymorphonuclear neutrophils and platelet-activating factor were then added to the perfusate for 10 min, followed by 30 min of recovery. Neutrophil retention was assessed from the difference between those administered and collected in coronary effluent and measurements of myeloperoxidase in myocardial samples. Isolated hearts were also used to assess the effect of volatile anesthetic pretreatment on cardiac dysfunction caused by enzymatically generated superoxide. In additional studies, the authors evaluated the effect of volatile anesthetic pretreatment on the adherence of neutrophils to isolated rat aortic segments. ResultsPlatelet-activating factor-stimulated neutrophils caused marked and persistent reductions (> 50%) in left ventricular developed pressure. Pretreatment with either isoflurane or sevoflurane abolished these effects, as well as the associated increases in neutrophil retention. Glibenclamide did not alter these actions of the anesthetics. Pretreatment with either volatile anesthetic attenuated the reductions in left ventricular developed pressure caused by exogenous superoxide and abolished the increases in neutrophil adherence in the aortic segments. ConclusionIsoflurane and sevoflurane preconditioned the heart against neutrophil-induced contractile dysfunction. This action was associated with an inhibition to neutrophil adherence and likely involved an increased resistance of the myocardium to oxidant-induced injury; the adenosine triphosphate-sensitive potassium channels played no apparent role.

The direct effects of enflurane on coronary blood flow, myocardial oxygen consumption, and myocardial segmental shortening in in situ canine hearts

Previous in vivo studies of the coronary vascular effects of halothane (HAL) were complicated by varying hemodynamic conditions and global cardiac work demands.Accordingly, the current study evaluated changes in coronary blood flow (CBF) and associated variables during selective intracoronary administrations of HAL in in situ canine hearts using an extracorporeal-controlled pressure perfusion system. Findings during HAL were compared to those during isoflurane (ISO). The left anterior descending coronary artery (LAD) of 8 open-chest dogs anesthetized with fentanyl and midazolam was perfused at constant pressure (109 +/- 2 mm Hg) with HAL-free arterial blood or with blood equilibrated in an extracorporeal oxygenator with HAL (0.5%, 1.0%, 2.0% in 95% O2-5.0% CO2). In the LAD bed, measurements of CBF were obtained with an electromagnetic flowmeter and used to calculate myocardial oxygen consumption (MVO2). Percent segmental shortening (%SS) was measured with ultrasonic crystals. Changes in CBF by HAL were compared to those during maximal vasodilation with adenosine. Separate studies (n = 5) were performed using 1.4% [1 minimum alveolar anesthetic concentration (MAC)] ISO and the findings compared to those during an equianesthetic (1.0%) concentration of HAL. HAL caused concentration-dependent increases in CBF, and decreases in MVO2 and %SS. With 2.0% HAL, the level of CBF was 50% of the maximal adenosine-induced response. At equianesthetic concentrations, HAL caused increases in CBF that were one-third of those caused by ISO, while the decreases in MVO2 and %SS caused by the drugs were not significantly different. We conclude that HAL has a direct concentration-dependent relaxing action on vascular smooth muscle in the coronary circulation of the in situ canine heart. The ability of HAL to increase CBF significantly while it was reducing local myocardial O2 requirements by a direct negative inotropic effect attests to the potency of this vasodilator action. HAL was a less potent direct coronary vasodilator than ISO, whereas it had a comparable direct negative inotropic effect. (Anesth Analg 1995;80:256-62)

Regional hemodynamics and oxygen supply during isovolemic hemodilution in the absence and presence of high-grade β-adrenergic blockade

Studies were performed in 16 pentobarbital-anesthetized dogs to evaluate regional circulatory effects of isovolemic hemodilution in the absence (group 1) and presence (group 2) of high-grade beta-adrenergic blockade with propranolol. Regional blood flow measured with 15 microm radioactive microspheres was used to calculate regional oxygen supply. In group 1, hemodilution with 5% dextran (40,000 molecular weight) reduced arterial hematocrit and oxygen content by approximately one half and had heterogeneous effects on regional blood flows. Blood flow was unchanged in the renal cortex, liver, and spleen, and it increased in the pancreas, duodenum, brain, and myocardium; however, only in the brain and myocardium were increases in blood flow sufficient to maintain oxygen supply at baseline (pre-hemodilution) levels. In group 2, intravenous administration of propranolol (1 mg/kg) itself decreased blood flow in the spleen and myocardium and had no other regional effects. Hemodilution after propranolol caused regional circulatory changes that were essentially similar to those in the absence of propranolol. It is concluded that (1) during isovolemic hemodilution, oxygen supply to the brain and myocardium is maintained at the expense of oxygen supply to less critical organs, and (2) this pattern of regional circulatory response during hemodilution remains intact in the presence of high-grade beta-adrenergic blockade with propranolol.

Nitric oxide does not modulate the increases in blood flow, O2 consumption, or contractility during CaCl2 administration in canine hearts

OBJECTIVEnEndothelium-derived nitric oxide (EDNO) has been shown to have vascular, metabolic, and contractile effects in the heart. We evaluated these effects during intracoronary (i.c.) administration of CaCl2 in dogs.nnnMETHODSnThe left anterior descending coronary artery of nine anesthetized, open-chest dogs was perfused at controlled pressure (80 mm Hg) with arterial blood. Coronary blood flow (CBF) was measured with a Doppler transducer and segmental shortening (SS) with ultrasonic crystals. Myocardial oxygen consumption (MVO2) and oxygen extraction (EO2) were calculated. Responses were assessed during i.c. infusions of CaCl2 (5, 10, 15 mg min-1) before and after administration of the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 300 micrograms min-1 for 15 min, i.c.).nnnRESULTSnBefore L-NAME, CaCl2 caused dose-dependent, proportional increases in SS and MVO2. Although CBF also increased, these responses were less than proportional to those in MVO2, and thus EO2 increased. L-NAME did not alter the cardiac effects of CaCl2.nnnCONCLUSIONSn(1) CaCl2 had direct inotropic and coronary vasoconstricting effects. (2) The vasoconstricting effect impaired coupling of CBF to the augmented metabolic demands by local vasodilating mechanisms. (3) EDNO did not modulate the increases in CBF, MVO2, or SS during administration of CaCl2.

Mechanisms of increased right and left ventricular oxygen uptake during inotropic stimulation

AIMSnFew studies have investigated oxygen supply/demand relations in right ventricle (RV). The current study compared changes in myocardial oxygen uptake (MVO2) and its determinants, myocardial blood flow (MBF) and oxygen extraction (EO2), in RV and left ventricle (LV) during stimulation with inotropic drugs.nnnMAIN METHODSnTwenty-one anesthetized dogs were studied. MBF was measured with radioactive microspheres. A-VO2 difference was determined separately for RV and LV, and used to calculate MVO2 (Fick equation) and EO2. Responses to iv. infusions of isoproterenol (0.1 μg/kg/min), dobutamine (5 μg/kg/min), and amrinone (1mg/kg+10 μg/kg/min) were assessed.nnnKEY FINDINGSnBaseline MVO2 in RV was 50-60% of that in LV. The inotropes increased MVO2 in both RV and LV (range 32-63%; P<0.05). With isoproterenol, the increased MVO2 in the RV was due to increases in both MBF and EO2, whereas in LV it was due only to increases in MBF. With dobutamine and amrinone, the increased MVO2 in RV was due to increased MBF while EO2 was constant (dobutamine) or decreased (amrinone), and that in the LV was due to increased MBF while EO2 decreased.nnnSIGNIFICANCEnRV possesses EO2 reserve that contributed to increased MVO2 during isoproterenol infusion, whereas LV depended on increased MBF alone. Because dobutamine and amrinone caused direct coronary vasodilation, i.e., luxuriant perfusion, it was not necessary to recruit the EO2 reserve. Thus, it remained available to meet further increases in MVO2 or to maintain MVO2 in the face of reductions in MBF.

In vivo gene delivery of XIAP protects against myocardial apoptosis and infarction following ischemia/reperfusion in conscious rabbits

AIMSnWe tested the hypothesis that an in vivo gene delivery of the pro-survival protein XIAP (X-chromosome linked inhibitor of apoptosis protein) protects against myocardial apoptosis and infarction following ischemia/reperfusion.nnnMAIN METHODSnNineteen rabbits were chronically instrumented with a hydraulic occluder placed around the circumflex coronary artery. Adenovirus harboring XIAP (Ad.XIAP; 1×10(10)pfu/ml) or β-galactosidase (5×10(9)pfu/ml), as a control, was constructed and transfected into the heart using a catheter placed into the left ventricle accompanied by cross-clamping. 1-2weeks after gene delivery, myocardial ischemia was induced by a 30-min occlusion followed by reperfusion for four days. Protein expression was determined by Western blot and apoptosis (% of myocytes) was quantified by TUNEL staining.nnnKEY FINDINGSnMyocardial infarct size, expressed as a fraction of the area at risk, was reduced in Ad.XIAP (n=5) compared to control (n=7) rabbits (21±3% vs. 30±2%, p<0.05). Apoptosis was reduced in Ad.XIAP rabbits compared to control rabbits (2.96±0.68% vs. 8.98±1.84%, p<0.01). This was associated with an approximate 60% decrease in the cleaved caspase-3 level in Ad.XIAP rabbits compared to control rabbits.nnnSIGNIFICANCEnThe current findings demonstrate that overexpression of XIAP via in vivo delivery in an adenovirus can reduce both myocardial apoptosis and infarction following ischemia/reperfusion, at least in part, due to the ability of XIAP to inhibit caspase-3. These findings confirm previous work suggesting a link between myocardial apoptosis and infarction i.e., anti-apoptotic therapy was effective in reducing myocardial infarct size.

Persistency and pathway of isoflurane-induced inhibition of superoxide production by neutrophils

BackgroundOur previous work has demonstrated that treatment with isoflurane has a preconditioning-like inhibitory effect on superoxide production (SOP) by polymorphonuclear neutrophils. The current objectives were to determine persistency of this effect and to clarify where in the signalling pathway this inhibition of SOP occurred. The latter was accomplished using two receptor-dependent neutrophil agonists, platelet activating factor (PAF) and formyl-methionyl-leucyl-phenylalanine (fMLP), and two receptor-independent neutrophil stimuli, the protein-kinase C stimulator, phorbol myristate acetate (PMA), and the calcium ionophore, A23187.MethodsArterial blood samples were obtained from eight dogs under baseline condition (conscious state), during isoflurane (1 MAC) administration, and 24 and 48xa0hr post-isoflurane (also in conscious state). Neutrophils were isolated and stimulated with 1xa0μM concentrations of PAF, fMLP, PMA, and A23187. SOP was measured spectrophotometrically.ResultsIsoflurane administration caused (1) an approximate 50% decrease in SOP during PAF or fMLP (Pxa0<xa00.01 vs baseline), which remained evident from 24 to 48xa0hr following isoflurane; (2) an initial 29% decrease in SOP during PMA (Pxa0<xa00.05 vs baseline), which returned to baseline by 24xa0hr following isoflurane; and (3) no change in SOP during A23187 (Pxa0>xa00.05 vs baseline).ConclusionsIsoflurane administration caused prolonged (from 24 to 48xa0hr) decreases in agonist-induced SOP by neutrophils. This effect involved inhibition at site(s) in the signalling pathway upstream from protein kinase C. The current findings suggest that the intraoperative use of isoflurane may result in an extended impairment to the antibacterial host defense mechanism and that neutrophil inhibition may play a role in the delayed tissue protection afforded by treatment with volatile anesthetics.RésuméContexteNos travaux précédents ont démontré qu’un traitement à base d’isoflurane a un effet de type pré-conditionnement sur la production de superoxyde (SOP) par les neutrophiles polynucléaires. Les objectifs de cette étude étaient de déterminer la persistance de cet effet et de clarifier l’endroit où cette inhibition avait lieu sur la voie de signalisation. Nous avons effectué cette clarification à l’aide de deux agonistes des neutrophiles dépendants du récepteur, le facteur d’activation plaquettaire (PAF) et le formyl-méthionyl-leucyl-phénylalanine (fMLP), et de deux stimuli des neutrophiles indépendants du récepteur, le stimulateur de la protéine kinase C, le phorbol myristate acétate (PMA), et l’ionophore de calcium A23187.MéthodeDes échantillons de sang artériel ont été obtenus chez huit chiens avant (état conscient), pendant (1 MAC), et 24 et 48xa0h après l’administration d’isoflurane (également en état conscient). Les neutrophiles ont été isolés et stimulés avec des concentrations d’1xa0μM de PAF, fMLP, PMA et A23187. La SOP a été mesurée par spectrophotométrie.RésultatsL’administration d’isoflurane a causé 1) une réduction d’environ 50% de la SOP pendant la stimulation par PAF ou fMLP (Pxa0<xa00,01 vs valeur de base), ce qui est demeuré évident de 24 à 48xa0h après l’administration d’isoflurane; 2) une réduction initiale de 29% de la SOP pendant la stimulation par PMA (Pxa0<xa00,05 vs valeur de base), laquelle est revenue à la valeur de base au plus tard 24xa0h après l’administration d’isoflurane; et 3) aucune modification de la SOP avec la stimulation par A23187 (Pxa0>xa00,05 vs valeur de base).ConclusionL’administration d’isoflurane a provoqué des réductions prolongées (de 24 à 48xa0h) de la SOP induite par un agoniste par les neutrophiles. Cet effet a impliqué l’inhibition sur un/des site(s) de la voie de signalisation en amont de la protéine kinase C. Ces résultats suggèrent que l’utilisation peropératoire d’isoflurane pourrait avoir pour résultat une détérioration prolongée du mécanisme de défense hôte antibactérien, et que l’inhibition des neutrophiles pourrait jouer un rôle dans la protection tissulaire retardée que procure un traitement à l’aide d’agents anesthésiques volatils.