Novel protection strategy for pulmonary transplantation.

BACKGROUND: Ischemia-reperfusion injury continues to represent a significant challenge to successful lung transplantation. Traditional pulmonary ischemic protection is performed using hypothermic hyperkalemic depolarizing solutions to reduce the metabolic demands of the ischemic organ. Measures to further reduce the effects of ischemic injury have focused on the reperfusion period. We tested the hypothesis that novel physiologic hyperpolarizing solutions-using ATP-dependent potassium channel (K(ATP)) openers-given at the induction of ischemia, will reduce cellular injury and provide superior graft function even after prolonged periods of ischemia. METHODS: An isolated blood-perfused ventilated rabbit lung model was used to study lung injury. Airway, left atrial, and pulmonary artery pressures were measured continuously during the 2-h reperfusion period. Oxygenation, as a surrogate of graft function, was measured using intermittent blood gas analysis of paired left atrial and pulmonary artery blood samples. Graft function was measured by oxygen challenge technique (F(i)O(2) = 1.0). Wet-to-dry ratio was measured at the conclusion of the 2-h reperfusion period. Control (Group I) lungs were perfused with modified Euro-Collins solution (depolarizing) and reperfused immediately (no ischemia). Traditional protection lungs were perfused with modified Euro-Collins flush solution and stored for 4 h (Group II) or 18 h (Group III) at 4 degrees C before reperfusion. Novel protection (Group IV) lungs were protected with a hyperpolarizing solution containing 100 nM Aprikalim, a specific K(ATP) channel opener, added to the modified Euro-Collins flush solution and underwent 18 h of ischemic storage at 4 degrees C before reperfusion. RESULTS: Profound graft failure was measured after 18 h of ischemic storage with traditional protection strategies (Group III). Graft function was preserved by protection with hyperpolarizing solutions even for prolonged ischemic periods (Group IV). Wet-to-dry weight ratio, airway, left atrial, and pulmonary artery pressures were not significantly different between the groups. CONCLUSIONS: We have created a model of predictable lung injury. Membrane hyperpolarization with a K(ATP) channel opener (PCO) provides superior prolonged protection from ischemia-reperfusion injury in an in vitro model of pulmonary transplantation.

Myoelectric response of the small intestine to the orad presence of the tapeworm Hymenolepis diminuta.

During its 24-hr migratory cycle in the small intestine, Hymenolepis diminuta is located in the orad part of the small intestine during the early morning hours and then in the caudad part of the small intestine during the late afternoon and early evening. During the later period, tapeworm-induced alterations of interdigestive myoelectric activity, a correlate of smooth muscle contraction or intestinal motility, are most intense in the ileal region. The hypothesis tested was that the tapeworm-induced changes in intestinal motility are local responses of the intestine responding to the close proximity of the lumenally positioned tapeworm and to the nutritional state of the host. The small intestine was monitored before and for 20 days after infection using electrodes implanted on the serosa of the small intestine. Myoelectric recordings were analyzed for the frequency of the normal patterns of interdigestive myoelectric spiking patterns and the altered myoelectric spiking related to tapeworm infection. During the morning hours, when the tapeworms are situated in the orad small intestine, no changes were observed during the normal myoelectric pattern of the digestive phase in any region of the intestine. When examined after the conversion of the digestive to interdigestive phase of motility, only on day 10 postinfection was the interdigestive phase significantly altered. It was concluded that the presence of the tapeworm in the orad small intestine during the satiety stage of the rat causes no changes in the electric events of the small intestine, with the exception of day 10 postinfection. Because tapeworms in the orad small intestine do not induce the tapeworm-altered myoelectric activity observed in the afternoon and evening with caudally positioned tapeworms, tapeworm-altered motility is not simply a response of the small intestine to the local presence of the tapeworm.

Heat shock protects cardiomyocytes from hypoxia-mediated apoptosis by attenuation of nitric oxide.

BACKGROUND: Considerable data exists on the beneficial effect of Heat Shock (HS) on myocardial necrosis. However, the effect of HS on apoptosis or programmed cell death, the other mode of cell death in the heart, is unknown. Hypoxia leads to apoptosis and increased nitric oxide (NO) production in myocardium. NO has also been shown to cause apoptosis. We sought to investigate if HS could protect cardiomyocytes (CM) from hypoxia-induced apoptosis and if this protection was NO-mediated. MATERIALS AND METHODS: CM were isolated from 1-2 day old rats and incubated at 37 degrees C or exposed to HS at 42 degrees C for 90 minutes before overnight incubation. CM were then subjected to hypoxic incubation in an argon/CO2 chamber (O2 concentration between 0.8-1.3 parts per million) for 90 minutes, followed by reoxygenation for 24 hours. Apoptosis was measured at 48 hours by TUNEL assay for in-situ DNA fragmentation and by immunohistochemical detection of c-myc protein, a proto-oncogene upregulated with apoptosis (grade 0 = no stain; +++ = most dense stain). NO was detected using the Griess reaction (nM +/- SEM). N > 5 for each group, performed in triplicate. RESULTS: CM viability demonstrated by spontaneously beating cells in culture and trypan blue staining was > 85%. (*, # = p < 0.01, ANOVA).

Comparison of cytosine arabinoside delivery to rat brain by intravenous, intrathecal, intraventricular and intraparenchymal routes of administration.

We evaluated the delivery of 14C-cytosine arabinoside (AraC) to rat brain by: 1) intravenous (IV) bolus, by 2) intrathecal (IT) and 3) intraventricular (IVT) infusion, and by 4) convection-enhanced delivery (CED) into the caudate nucleus. Plasma and brain AraC metabolites were measured with HPLC, and distribution and concentration of 14C-AraC in brain sections were measured by quantitative autoradiography. After IV administration, the alpha and beta plasma half-lives were 1.9 and 46.5 min, respectively. The blood-to-brain transfer constant of AraC was 2.5+/-1.4 microliter g(-1) min(-1), compatible with high water solubility. After IT and IVT administration, tissue levels were high at the brain and ventricular surfaces, but declined exponentially into brain. After CED, maximum brain levels were up to 10,000 times higher than the IV group, and the distribution pattern was one of high 14C-AraC concentration in the convective component, with exponentially declining concentrations outside this region. The rate loss constant from brain was 0.002+/-0.0004 min(-1), suggesting that AraC was accumulating in brain cells. AraC was metabolized into uracil arabinoside within the brain. 14C-AraC was infused into 1 dog and distributed widely in the ipsilateral hemisphere. These studies suggest that delivery of AraC to brain parenchyma by the IV, IT or IVT routes will be subtherapeutic. Delivery by CED can achieve, and maintain, therapeutic levels of AraC in the brain, and should be further evaluated as a potential method of drug delivery.

Hymenolepsis diminuta: mucosal mastocytosis and intestinal smooth muscle hypertrophy occur in tapeworm-infected rats.

The mechanisms mediating motility changes during noninvasive tapeworm infection have not been characterized. In contrast, host intestinal motility changes during invasive nematode infection are mediated by mucosal mast cells (MMC). The purpose of this study was to examine and the correlate onset of myoelectric alterations 8 days after initial tapeworm infection with changes in intestinal morphology, MMC numbers, and MMC secretory activity. Segments of the small intestine, the tapeworms normal habitat, along with stomach, colon, and bladder were taken from tapeworm-infected and control rats. Tissues were fixed and stained to identify MMC and for morphologic measurement. Tapeworm-infected and uninfected rats with chronically implanted intestinal electrodes were treated with ketotifen, a mast cell stabilizer, and in vivo myoelectric activity monitored. In tapeworm-infected rats, the muscularis externa, on day 20 postinfection, and crypts of Lieberkuhn, on day 26 postinfection, from the entire small intestine appeared thickened or deeper, respectively. Increased muscularis thickness was due to smooth muscle hypertrophy in both the circular and the longitudinal muscle layers. Mucosal mastocytosis was first observed on day 26 postinfection and occurred only in the ileum of tapeworm-infected rats. Pharmacologic stabilization of mast cells with ketotifen did not prevent onset of enteric myoelectric alterations during tapeworm infection. Stomach, colon, and bladder MMC numbers and tissue dimensions were not different between Hymenolepis diminuta-infected rats and uninfected controls. Initiation of myoelectric alterations 8 days after infection precedes and may be a contributing factor to the onset of both smooth muscle hypertrophy and mucosal mastocytosis. Taken together, our data indicate that mast cells are not an initiating factor nor chronic stimulus maintaining intestinal myoelectric alterations during H. diminuta infection.

Nitric oxide mediates fluid accumulation during cardiopulmonary bypass.

Fluid accumulation during cardiopulmonary bypass may be related to the production of endogenous vasoactive substances. We investigated the role of nitric oxide in mediating fluid accumulation during cardiopulmonary bypass. Normothermic cardiopulmonary bypass was carried out for 3 hours in male Sprague-Dawley rats with constant, nonpulsatile flow and hemodilution. Fluid accumulation (rate of change of external reservoir volume) was measured under three experimental conditions: saline solution control (n = 8), L-arginine infusion (n = 6), and N-nitro-L-arginine methyl ester infusion (n = 6). At the end of the experiments, body weight and organ wet/dry ratios were examined. Percentage weight gain was 77% greater in the N-nitro-L-arginine methyl ester group and 23% less in the L-arginine group compared with control values. Fluid accumulation was increased with N-nitro-L-arginine methyl ester after 30 minutes (p < 0.01) and reduced with L-arginine after 120 minutes (p < 0.01) compared with control animals. Water content was significantly decreased in the heart, lung, skin, muscle and peritoneum in rats receiving L-arginine. These data suggest that endogenous nitric oxide plays an important role in minimizing fluid accumulation during cardiopulmonary bypass.

A critical period of ventricular fibrillation more susceptible to defibrillation: real-time waveform analysis using a single ECG lead.

Previous studies have suggested that variations in the underlying ventricular fibrillation (VF) waveform may be one of the factors responsible for the probabilistic nature of defibrillation. The heart appeared to be more susceptible to defibrillation at higher absolute VF voltages (AVFV). This study investigated in an open-chest canine model (n = 8), a newly developed system that analyzed the VF waveform in real-time, instantaneously determined the time to shock, and immediately delivered a fixed low energy DC shock. A two parameter tracking technique using a running long-term and short-term AVFV average was devised to automatically identify a high voltage peak area of the VF waveform, which has been hypothesized to represent a critical period susceptible to defibrillation. Using a DC shock estimated at the 50% success level, the performance using this technique in 58 defibrillation trials was compared to the performance of the conventional method of shocking at a fixed time (random shock method) in 62 trials. Patch size, electrode location, and discharge voltage were kept constant while VF duration, transmyocardial resistance (TMR), energy delivered, and AVFV at the point of shock were measured. Shock energy and current, TMR, and VF duration were similar with both shock methods. A significantly higher AVFV was observed for trials performed with the peak shock method (0.66 +/- 0.02 mV) as compared to trials performed with the random shock method (0.25 +/- 0.09 mV) (P < 0.003). Using lead II as the only sensing lead, the success rate was increased in 6 of 8 dogs (75%) with the new method. One animal showed identical performance, and one animal a worse performance. The overall increase in success rate was 24% using a single ECG lead (range 0%-100%; P < 0.04). Our data document that using this algorithm a period of high VF voltage can be detected in real-time. The improved success in the majority of animals supports the hypothesis that a critical period susceptible to defibrillation exists during VF. However, the high AVFV detected using a single ECG lead did not translate to an improved success rate in all animals. This suggests that other factors in addition to the VF voltage measured on a single lead of the ECG are important in characterizing this critical period.

Integration of absolute ventricular fibrillation voltage correlates with successful defibrillation.

Previous work has suggested that at higher absolute ventricular fibrillation voltages (AVFV), the heart is more amenable to defibrillation. This study investigated in a canine model whether voltage integration of the AVFV is associated with the defibrillation success rate. The moving-average filter was used to process the ventricular fibrillation (VF) waveform recorded from Lead II of the electrocardiogram (ECG). In seven animals, defibrillation trials were analyzed using a dc shock (DCS) successful approximately 50% of the time when delivered randomly. For each of a total of 84 DCS (40% successes, 60% failures), the fibrillation waveform just prior to DCS was analyzed. The integration of the AVFV waveform was performed over various sample sizes including 1, 4, 8, 16, 64, and 128 ms, as well as the time equal to the mean VF cycle length. The results suggest that dc shocks delivered at instants of higher values of integrated AVFV over the various window sizes are associated with successful defibrillation. Window sizes less than 16 ms appeared to offer the best discrimination. The integration of AVFV over the entire VF cycle length was significantly higher for successful rather than unsuccessful DCS. This interesting observation is consistent with the clinical observation that "coarse" VF (high AVFV) is easier to defibrillate than "fine" VF (low AVFV). The use of voltage integration of AVFV may have potential implications in the improvement of defibrillation success in implantable devices.

Electrophysiologic consequences of hyperkalemic cardioplegia during surgical ischemia.

Myocardial protection strategies use cardioplegic solutions to reduce the injury induced by surgical ischemia and reperfusion. However, there is a high incidence of electrophysiologic abnormalities after cardioplegic arrest. A computerized epicardial mapping system in a porcine cardiopulmonary bypass model was used to measure the electrophysiologic consequences of different myocardial protection techniques. Both warm and cold, crystalloid and blood cardioplegic solutions were compared. The effects of hypothermia and prolonged cardiopulmonary bypass were examined in a control group that underwent a 2-hour period of hypothermia without cardioplegia or aortic cross-clamping, followed by 2 hours of normothermic reperfusion. Isochronous activation maps, unipolar electrograms, ventricular refractory periods, and pacing thresholds were measured before cardioplegic arrest and during reperfusion. Compared with the control group, crystalloid cardioplegia, but not blood cardioplegia, was accompanied by large changes in the pattern of ventricular activation and by persistent (> 2 hours) and significant slowing of the time required for complete ventricular activation. This was not the result of hypoxia. Moreover, the effective refractory period and the pacing threshold were unchanged by any cardioplegia. Our data suggest that crystalloid cardioplegia increases myocardial resistance to current flow leading to a derangement of electrical impulse propagation that may underlie arrhythmogenesis.

The effect of left ventricular intracavitary volume on the unipolar electrogram.

In order to examine the effects of ventricular distention on the unipolar electrogram (UEG), an isolated rabbit heart modified Langendorff preparation was utilized. Left ventricular (LV) volume was adjusted using ionically permeable (PB = 9 hearts) or ionically impermeable balloons (IB = 4 hearts). LV UEGs, LV end-diastolic pressure (EDP), and LV minor axis dimension (MAD), as measured by ultrasonic transducers, were recorded. Three hundred twenty-five electrograms were digitized and analyzed with custom-designed software. In the PB group, a significant inverse linear relationship was found between UEG amplitude and changes in MAD (P < 0.0001). For each animal, this relationship had an R value > 0.8 and a P value < 0.0001. There was also a significant inverse linear relationship between UEG slope and changes in MAD (P < 0.01). UEG amplitude and slope also exhibited a significant inverse relationship to changes in LV EDP, which were best described by a third order polynomial function. In the IB group, no significant relationship was found between either UEG amplitude or slope and MAD or EDP. In this study, intracavitary volume exerted a profound and significant influence on UEG amplitude and slope. This effect was due to increases in conductive intraventricular volume and not to myocardial stretch.

Elective cardiac arrest with a hyperpolarizing adenosine triphosphate-sensitive potassium channel opener. A novel form of myocardial protection?

BACKGROUND: Hyperkalemic depolarized cardiac arrest has been the cornerstone of myocardial protection during cardiac surgery for more than 30 years. Many of the advances in myocardial protection seek to minimize the cellular damage and to reduce the ongoing metabolic processes occurring as a direct consequence of the depolarized state. Ideally, cardiac arrest at hyperpolarized cellular membrane potentials--the natural resting state of the heart--will meet all the requirements of modern cardioplegia, namely, electromechanical asystole and cardiac relaxation, while preserving the vital integrity of the heart itself. METHODS AND RESULTS: To determine whether activation of adenosine triphosphate-sensitive potassium channels by pharmacologic agents could produce hyperpolarized cardiac arrest, we tested the ability of aprikalim, a known adenosine triphosphate-sensitive potassium channel opener, to arrest the intact beating heart. In a normothermic (37 degrees C) isolated rabbit heart preparation, aprikalim was found to rapidly shorten the action potential duration and produce cardiac asystole that was maintained during 20 minutes of "no-flow" global ischemia without a rise in end-diastolic pressure. Cardiac rhythm and function were fully restored by reperfusion alone (developed pressure was 100.6% +/- 7.9% of prearrest value after 30 minutes of reperfusion). In contrast, 20 minutes of unprotected normothermic global ischemia resulted in a 2.7 +/- 0.55 mmHg rise in end-diastolic pressure and only 58.2% +/- 3.8% recovery of developed pressure after 30 minutes of reperfusion. By way of comparison, 20 minutes of standard hyperkalemic depolarized normothermic rest was accompanied by a 1.2 +/- 0.6 mmHg rise in end-diastolic pressure and only 80.8% +/- 2.6% recovery of developed pressure after 30 minutes of reperfusion. To directly compare hyperkalemic depolarized cardiac arrest to hyperpolarized cardiac arrest induced by potassium channel openers and to better define the characteristics of such hyperpolarized arrest, we studied a fixed (4 mmHg rise in end-diastolic pressure--contracture) ischemic injury model. The time to development of the contracture was prolonged by hyperkalemic arrest (35.8 +/- 1.7 minutes) and significantly more so by hyperpolarized arrest (47.0 +/- 3.3 minutes) when compared with that of unprotected hearts (24.0 +/- 1.2 minutes). Moreover, aprikalim resulted in significantly better postischemic recovery of function (developed pressure was 69.0% +/- 6.7% of prearrest value after 30 minutes of reperfusion) than after no cardioplegia (45.4% +/- 7.5%) or standard hyperkalemic cardioplegia (44.3% +/- 5.7%). CONCLUSIONS: Pharmacologic activation of adenosine triphosphate-sensitive potassium channels can result in predictable and sustainable hyperpolarized cardiac arrest that is reversible by reperfusion. This method of myocardial protection was found to fully preserve cardiac electromechanical function after a 20-minute period of global normothermic ischemia. Furthermore, hyperpolarized arrest induced by potassium channel openers significantly prolonged the period to the development of contracture and afforded a significantly better postischemic recovery of function than obtained in either hearts protected with hyperkalemic depolarized arrest or those not protected by any form of cardioplegia.

Electrophysiological consequences of hypothermic hyperkalemic elective cardiac arrest.

While the development of pharmacological cardioplegic solutions for myocardial protection during cardiopulmonary bypass (CPB) have significantly lengthened the safe operating time for cardiac surgical procedures, the introduction of hypothermic hyperkalemic cardioplegia (CPG) has markedly increased the incidence of postoperative arrhythmias and conduction abnormalities. Using a customized modification of a computerized mapping system, we have developed a large animal porcine model of CPB that is exquisitely sensitive to the electrophysiological (EP) derangements imposed by ischemia and cardiac arrest. This model is able to measure spatial and temporal parameters of ventricular activation with high resolution, using an array of up to 84 epicardial electrodes that can be reproducibly placed on the surface of the heart utilizing known epicardial anatomical markers (e.g., coronary arteries). With this system we have measured the spectrum of clinically observed EP disturbances caused by CPG, from slowed intraventricular conduction to complete heart block. Compared to the control group of hypothermia alone, 2 hours of crystalloid CPG arrest had a significant slowing effect on ventricular activation (p < 0.05). CPG was accompanied, in each animal, by profound changes in the spatial distribution of ventricular activation and persistent slowing of ventricular activation. Traditional EP parameters of effective refractory period and pacing threshold were unchanged by CPG. Smaller temporal and spatial changes were observed in the control group, but were always reversed by 90 minutes of warm reperfusion. We conclude that CPG induces injury of the specialized conducting system and, to a lesser degree, the myocardium. This model will afford us the opportunity to test new methods of CPG to further improve myocardial preservation during CPB.

Preparation and properties of highly enriched cardiac sarcolemma from isolated adult myocytes.

Isolated myocytes were prepared from adult canine hearts using a combined technique of myocardial perfusion followed by incubation with collagenase. More than 60% of the cells routinely excluded trypan blue dye. Disruption of the myocytes was accomplished using high pressure nitrogen cavitation. After differential and sucrose gradient centrifugation, the peak sarcolemmal fraction averaged 100-fold enrichment in ouabain-inhibited K+-stimulated p-nitrophenyl phosphatase and 82-fold in ouabain-inhibited (Na+,K+)-ATPase. These sarcolemmal membranes are enriched in phospholipid phosphorus (1.98 mumol/mg of protein) and more than 4-fold in sphingomyelin and cholesterol. Polyacrylamide gels revealed three major protein peaks at 50,000, 91,000, and 140,000 apparent molecular weights. This work demonstrates the feasibility of preparing highly pure cardiac sarcolemma from isolated adult myocytes. The problem of cellular cross-contamination due to heterogeneity of cell types in whole myocardial tissue has been circumvented. The level of enrichment exceeds all reported preparations of cardiac sarcolemma from whole myocardium and cultured myocytes. This preparation should prove to be useful as an in vitro model for studies of physiological, pharmacological, and pathological perturbations of sarcolemmal structure and function.

The effect of lithium and potassium on the transient state kinetics of the (Ca + Mg)-ATPase of cardiac sarcoplasmic reticulum.

KCl or LiCl, when added in 100 mM concentrations to cardiac sarcoplasmic reticulum incubated at 17 degrees C with 5 micron [gamma-32P]ATP, 1 mM MgCl2, and 9.1 micron M Ca2+, increased the apparent phosphorylation rate constant from 14.5 s-1 to 23.8 s-1 (100 mM LiCl) or to 44.1 s-1 (100 mM KCl). These same monovalent cations also increased the apparent rate constant for the hydrolysis of the phosphorylated sarcoplasmic reticulum from 0.51 s-1 to 1.12 s-1 (100 mM LiCl) or to 1.71 s-1 (100 mM KCl). Although there was a small burst in Pi production, rate constant of 0.97 s-1, when 100 mM KCl was added, the burst when LiCl or no monovalent cation was added was either nonexistent or so small as to make its detection unreliable. KCl thus appears to induce an intermediate which is either nonexistent when omitted or in such low concentration as not to be readily detected.