William J. Gallagher

Isolated four-chamber working swine heart model

BACKGROUND Isolated heart models separate cardiac characteristics from systemic characteristics with subsequent findings used in cardiac research, including responses to pharmacologic, mechanical, and electrical components. The model objective was to develop the ability to represent in situ physiologic cardiac function ex vivo. METHODS Swine hearts were chosen over rat or guinea pig models due to their notably greater anatomical and physiologic similarities to humans. An in vitro apparatus was designed to work all four chambers under simulated in situ physiologic conditions. Using standard cardiac surgical techniques, 12 porcine hearts (mean weight 331 +/- 18 g) were explanted into the apparatus. Preload and afterload resistances simulated in situ input and output physiologic conditions. Hemodynamic characterizations, including cardiac output, max +/- dP/dt, and heart rate, were used to determine in situ function leading to explantation (prethoracic operation, postmedial sternotomy, and postperidectomy) and during in vitro function (t = 0, 60, 120, and 240 minutes). RESULTS In vitro performance decayed with time, with statistical differences from base line (t = 0) function at t = 240 minutes (p > 0.05). CONCLUSIONS An isolation and in vitro explantation protocol has been improved to aid in the study of isolated cardiac responses, and to determine cardiac hemodynamic function during open chest operation, transplantation, and in vitro reanimation with a crystalloid perfusate. The resulting model offers similar working physiologic function, with real-time imaging capabilities. The resulting model is advantageous in representing human cardiac function with regard to anatomic and physiologic functions, and can account for atrial and ventricular interactions.

Effects of sufentanil on cerebral circulation and metabolism in dogs

The cerebral and peripheral vascular effects of sufentanil (10–200 μg/kg) were examined in dogs. The cerebral blood flow (CBF) was measured continuously by an electromagnetic flow probe on the outflow of the posterior sagital sinus. Sufentanil at all doses significantly increased CBF that lasted for ∼20 min. The CBF then gradually decreased so that it was significantly below baseline levels by the end of the 60-min study period. The transient increase in CBF was accompanied by an equally transient statistically significant decrease in cerebrovascular resistance. Intracranial pressure did not change. Sufentanil produced an electroencephalographic pattern of deep anesthesia accompanied by a decrease in cerebral oxygen consumption significantly below baseline levels. At the end of the study tissue concentrations of metabolites taken from the cerebral hemispheres were within normal limits, indicative of a normal cerebral energy state. Sufentanil had little effect on systemic hemodynamics. The observation that sufentanil significantly increases CBF in the absence of seizure activity makes it unique among the narcotics. It is hypothesized that in the presence of decreased intracranial compliance, this sudden increase in CBF, although transient, may be detrimental if it is accompanied by an acute increase in intracranial pressure which could produce cerebral ischemia.

Opioid preconditioning: myocardial function and energy metabolism.

BACKGROUND Opioid receptor agonists are involved in ischemic preconditioning and natural hibernation. The aim of this study was to determine whether pretreatment with D-Ala2-Leu5-enkephalin or morphine confers cardioprotection in large mammalian hearts. We assessed myocardial functional recovery and global energy metabolism after ischemic cold storage. METHODS After pretreatment with D-Ala2-Leu5-enkephalin, morphine sulfate, or saline (n = 6 each), swine hearts were excised and stored for 75 minutes at 4 degrees C, then reperfused in a four-chamber isolated working heart apparatus. Serial myocardial biopsies were performed to assess cellular energy metabolism. RESULTS Improved systolic (cardiac output, contractility) and diastolic (tau) left ventricular functions were observed in hearts pretreated with D-Ala2-Leu5-enkephalin or morphine. These benefits were not correlated with changes in high-energy phosphate levels. Cardiac enzyme leakage (creatine kinase, troponin-I) was similar among treated and control groups. Lactate efflux increased significantly in controls, but not in opioid-pretreated hearts (p < 0.01) at 75 minutes of reperfusion. CONCLUSIONS D-Ala2-Leu5-enkephalin and morphine pretreatments improve postischemic function after cold storage of swine hearts. Postischemic lactate reduction, but not high-energy phosphate levels, may account for the observed cardioprotective effects.

Comparison of the effects of isoflurane and thiopental on neurologic outcome and neuropathology after temporary focal cerebral ischemia in primates

In an attempt to determine whether one anesthetic might be clearly advantageous over another in clinical situations of temporary focal ischemia, isoflurane or thiopental (in concentrations producing equal suppression of cerebral function as measured by the electroencephalogram) were studied for their effects on neurologic outcome and cerebral infarct size in pigtailed monkeys exposed to temporary focal ischemia produced by 5 h of middle cerebral artery occlusion (MCAo). Burst suppression was produced for 15 min before MCAo and maintained throughout the ischemic period by 2.18 +/- 0.11% (mean +/- SE) end-expired isoflurane or 135 +/- 18 mg.kg-1 thiopental. Mean arterial pressure was supported with phenylephrine and maintained at approximately 90 mmHg in both groups throughout the ischemic period. At the end of the ischemic period, the isoflurane or thiopental was discontinued, allowing the animals to awaken. Intensive care was provided as needed. Neurologic function was scored for 8 days at the end of which surviving animals were killed and the brains were fixed in formalin and then examined for infarct size. There was no significant difference in final neurologic outcome between the animals receiving isoflurane and those receiving thiopental as determined by the Mann-Whitney rank sum test. Neurologic deficit scores ranged from normal (one of eight in the group receiving isoflurane and three of nine in the group receiving thiopental) to death resulting from brain injury (three in the isoflurane group and five in the thiopental-treated group). There also was no significant difference in infarct size between the two groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolism of Glucose, Glycogen, and High-energy Phosphates during Transient Forebrain Ischemia in Diabetic Rats: Effect of Insulin Treatment

Background Hyperglycemia associated with diabetes mellitus will exacerbate neurologic injury after global brain ischemia. Studies in a rat model of forebrain ischemia (bilateral carotid occlusion plus hypotension for 10 min) discovered that acute restoration of normoglycemia in diabetics, using an insulin infusion, resulted in a neurologic outcome that was similar to normoglycemic rats without diabetes. The current study evaluated cerebral glucose, glycogen, lactate, and high-energy phosphate concentrations to identify metabolic correlates that might account for an alteration in postischemic outcome. Methods Fifty-four pentobarbital-anesthetized Sprague-Dawley rats were assigned to three groups: chronically hyperglycemic diabetic rats (D; N = 18); insulin-treated, acutely normoglycemic diabetic rats (ID; N = 18); and nondiabetic rats (ND; N = 18). These groups were further divided into groups of six rats each that received either no ischemia, forebrain ischemia of 10 min duration without reperfusion, or ischemia plus 15 min of reperfusion. Brains were excised after in situ freezing, and metabolites were measured using enzymatic fluorometric techniques. Results Before ischemia, D rats had greater concentrations of brain glucose (12.18+/-2.67 micro mol/g) than did either ID (5.10 +/-1.33) or ND (3.20+/-0.27) rats (P < 0.05). Preischemic brain glycogen was similar in all groups. At the completion of ischemia, brain lactate concentrations in D were 86% greater than in ID and 61% greater than in ND (P < 0.05), reflecting a higher intraischemic consumption of glucose plus glycogen in D (P < 0.05). High-energy phosphate concentrations, as assessed by the energy charge of the adenylate pool, were better preserved in D (energy charge = 0.60 +/-0.28) than in either ID (0.29+/-0.09) or ND (0.36 +/-0.07; P < 0.05) rats. After 15 min of reperfusion, the energy charge returned to preischemic values (i.e., 0.91-0.92) in all groups. Conclusions These studies demonstrated greater intraischemic carbohydrate consumption and lactate production in D than in ID or ND rats. Under these conditions, intraischemic--but not postischemic-energy status was better in D rats. Acute insulin therapy in ID rats resulted in a metabolic profile that was similar to that of ND rats. These results suggest that, in this model, primary energy failure during ischemia is not the origin of greater injury in hyperglycemic diabetics, nor is energy enhancement the origin of improved outcome after acute insulin treatment.

The effect of chronic dexamethasone-induced hyperglycemia and its acute treatment with insulin on brain glucose and glycogen concentrations in rats.

BackgroundIn the rat model of forebrain ischemia, long-term dexamethasone treatment is reported to cause hyperglycemia and worsen postischemic functional and histologic injury. This effect was assumed to result from glucose enhancement of intraischemic lactic acidosis within the brain. Short-term insulin therapy restored normoglycemia but did not return histologic injury completely to baseline values. Using a nonischemic rat model, the current study attempted to identify a metabolic basis for such outcome data. MethodsFifty-eight halothane-anesthetized (1.3% inspired) Sprague-Dawley rats were assigned randomly to be administered either no treatment (N = 18) or 2 mg/kg intraperitoneal dexamethasone (N = 40). The latter were administered dexamethasone 3 h before the study only (N = 8) or for 3 h before the study plus daily for 1 day (N = 8), 2 days (N = 8), or 4 days (N = 16). Of the rats treated with dexamethasone for 4 days, one half (N = 8) were administered an insulin-containing saline infusion subsequently to restore normoglycemia short-term. All other rats (N = 50) were administered an infusion of saline without insulin. Plasma glucose was quantified, and brains were excised after in situ freezing. Brain glucose and glycogen concentrations were measured using enzymatic fluorometric analyses. ResultsAfter 4 days of dexamethasone treatment, plasma glucose was 159% greater than in rats administered placebo (i.e., 22.01 ± 4.66 vs. 8.51 ± 1.65 &mgr;mol/ml; mean ± SD;P < 0.0001). Brain glucose concentrations increased parallel to plasma glucose. An insulin infusion for 27 ± 5 min restored normoglycemia but resulted in a brain-to-plasma glucose ratio that was 32% greater than baseline values (P < 0.01). Neither dexamethasone nor the combination of dexamethasone plus insulin affected brain glycogen concentrations. ConclusionsIn a nonischemic rat model, dexamethasone alone had no independent effect on the brain-to-plasma glucose ratio. However, short-term insulin therapy caused a dysequilibrium between plasma and brain glucose, resulting in an underestimation of brain glucose concentrations when normoglycemia was restored. The dysequilibrium likely was caused by the rapid rate of glucose reduction. The magnitude of the effect may account for the failure of insulin to reverse dexamethasone enhancement of neurologic injury completely in a previous report that used the rat model of forebrain ischemia.

4-chloro-m-cresol triggers malignant hyperthermia in susceptible swine at doses greatly exceeding those found in drug preparations.

BACKGROUND Chlorocresols are used as preservatives in numerous commercial drugs that have been shown to induce myoplasmic Ca2+ release; the most potent isoform is 4-chloro-m-cresol. The aims of this study were to (1) examine the in vivo effects of 4-chloro-m-cresol on swine susceptible to malignant hyperthermia and (2) contrast in vivo versus in vitro dose-response curves. METHODS Susceptible swine (weight: 38.5 kg+/-3.55 kg) were anesthetized and monitored for variations in physiological responses, including end-tidal CO2, heart rate, blood pressure, blood chemistry, and temperatures. In the first animals studied, 4-chloro-m-cresol, at equivalent cumulative doses of 0.14, 0.28, 0.57, 1.14, 2.27, 4.54, and 9.08 mg/kg (n = 3; 12.5, 25, 50, 100, 200, 400, and 800 micromol) were administered, and in a second group, larger doses were used: 1.14, 3.41, 7.95, 17.04 (n = 4), and/or 35.22 (n = 1) mg/kg (100, 300, 700, 1,500, and/or 3,100 micromol). For comparison, in vitro rectus abdominis muscle preparations obtained from normal and susceptible swine were exposed to 4-chloro-m-cresol, at cumulative concentrations of 6.25, 12.5, 25, 50, 100, 200, 400, 800, and 1,600 micromol; standard caffeine and halothane contracture testing was also performed. RESULTS Episodes of malignant hyperthermia were not triggered in response to administration of low doses of 4-chloro-m-cresol, but transient cardiovascular reactions (e.g., tachycardia, arrhythmias, and hypotension) were observed. Subsequently, episodes in these animals were triggered when halothane (0.87; 1 MAC) and succinylcholine (2 mg/kg) were given. Animals administered the higher doses of 4-chloro-m-cresol all had fulminant episodes of malignant hyperthermia that were fatal, when equivalent cumulative concentrations were greater than 1,500 micromol. The levels of 4-chloro-m-cresol in the plasma rapidly decreased: e.g., 5 min postadministration of the 1,500-micromol dose, the mean plasma level was only 52+/-18 micromol (n = 4). Hemolysis was detected following 4-chloro-m-cresol administration at concentrations > 200 micromol. In vitro, muscle from susceptible animals elicited contractures > 200 mg at 50-micromol bath concentrations of 4-chloro-m-cresol (n = 29), whereas normal muscle did not elicit such contractures until bath concentrations were > 800 micromol (n = 10). CONCLUSIONS 4-chloro-m-cresol is a trigger of malignant hyperthermia in susceptible swine, but only when serum concentrations are far above those likely to be encountered in humans. A relatively low concentration of 4-chloro-m-cresol, 50 micromol, is sufficient to activate sarcoplasmic [Ca+2] release in vitro (e.g., contractures); this same bolus dose administered in vivo (0.57 mg/kg) has minimal effects due to the rapid decrease in its plasma levels.

In Vitro Contracture Testing for Determination of Susceptibility to Malignant Hyperthermia: A Methodologic Update

The in vitro contracture test determines the sensitivity of freshly obtained skeletal muscle specimens to caffeine or halothane applied to a bathing solution. Muscles from persons susceptible to malignant hyperthermia have lower contracture thresholds for these agents than do normal muscle. Thus, known concentrations of these agents must be accurately administered. We describe a system in which the bath concentrations of halothane (0 to 3%) are reproducible within 0.2% halothane from month to month. With use of this system, which includes an on-line gas analyzer, the measured halothane concentration within the bathing solution was independent of the performance of the vaporizer. The system is inexpensive and stable. The methods used to determine the bath concentrations of halothane and caffeine are reviewed. Finally, experimental equipment and a new recording system are described.

Fructose-1,6-bisphosphate and fructose-2,6-bisphosphate do not influence brain carbohydrate or high-energy phosphate metabolism in a rat model of forebrain ischemia

Phosphorylated fructose compounds have been reported to lessen neuronal injury in in vitro models of hypoxia and in vivo models of ischemia. Although a variety of mechanisms have been proposed to account for this finding, it is unknown if intracellular uptake and incorporation of these compounds into the glycolytic pathway contribute to the benefit. We evaluated phosphorylated fructose administration in an adult rat model of transient, near-complete cerebral ischemia to determine its impact on brain metabolism before, during, and after ischemia. Fifty-four pentobarbital anesthetized rats were randomly assigned to receive IV infusions of either fructose-1,6-bisphosphate, fructose-2,6-bisphosphate, or 0.9% saline. After 2 hours of infusion, 18 rats (6/treatment group) were subjected to brain harvesting before any ischemia, 18 additional rats had brain harvesting at the completion of 10 minutes of forebrain ischemia (2-vessel occlusion plus induced hypotension), and 18 rats had harvesting after ischemia and 15 minutes of reperfusion. Cortical brain samples were analyzed for ATP, ADP, AMP, phosphocreatine, glucose, and glycogen. When compared with placebo, neither phosphorylated fructose compound altered preischemic, intraischemic, or postischemic concentrations of brain high-energy phosphates, glucose, glycogen, or lactate, nor did they influence the intraischemic metabolism of endogenous brain glucose or glycogen. On the basis of these results, we conclude that mechanisms other than augmented carbohydrate metabolism are responsible for previous reports of neuronal protection by the bisphosphonates.

Measurement of Impact Loads Applied to an Implanted Drug Pump Connector in a Porcine Cadaver Specimen

Anecdotal and documented reports from both patients and doctors have described unanticipated breaks in connections between implanted catheters and drug pumps. In extreme cases, such disconnections in patient-required therapies could result in either withdrawal symptoms or possible deaths. Patients typically attribute such device failures to falls or impacts associated with vigorous physical activity; subsequent failure analyses most often have indicated pump connector uncouplings. We fabricated a facsimile of the Medtronic® SynchroMed® II pump that included both an accelerometer and a force sensor. The force sensor measured forces imparted on the pump connector via the attached catheter and surrounding tissues. The test pump was implanted in the lower left abdominal areas of porcine cadavers in various orientations. Wire-reinforced catheters were tunneled for 20-25 cm under the abdominal epidermis, anteriorly toward the head, and the non-connector pump ends were secured by sutures. Following each simulated implant, the cadaver specimens were loaded into a harness and hoisted to a height where either their buttocks or backs were 80-86 cm above the floor, simulating a worst-case scenario in which a patient might have fallen down a flight of stairs or off a step stool. The cadavers were then quick released from the hoist attachment, while forces (X, Y, and Z) and accelerations (X, Y, and Z) versus time were simultaneously recorded. Six porcine cadaver specimens were utilized for a total of 72 trials. Subsequent Monte Carlo analyses allowed us to model the variation in stress imparted onto the pump connectors and the estimated variation of the pump connector strength, as a means of predicting required connector retention impact specification for future designs. The recorded forces applied onto the connectors, including data from all three connector axes (X, Y, and Z), were typically within the range of 4.5-9 N. However, in several trials, applied forces ranged as high as 30-49 N. Monte Carlo modeling provided a maximum resultant load specification of 100.4 N for a 0.033 msec duration. Based on this value, due to predicted impact events, subsequent failures of future designs would be estimated at 7 ppm. Based on our data, a new design requirement has been generated to ensure that implantable drug pump connector assemblies will, in high probability, perform their intended functions.