Effects of physostigmine on the cardiopulmonary system of conscious pigs

FUNDAMENTAL

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TOXICOLOGY

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Effects of Physostigmine on the Cardiopulmonary System of Conscious Pigs’ FRED W. STEMLER,~ KEVIN D. CORCORAN, JOHN H. PARRISH, HOLCOMBE H. HURT, THERESA M. TEZAK-REID, ANDRIS KAMINSKIS, AND JAMES J. JAEGER United States Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, Maryland 21010-5425

Received February 7, 1989; accepted August 9, 1989 Effects of Physostigmine on the Cardiopulmonary CORCORAN,

K.

D.,

PARRISH.

J. H.,

HURT,

H.

System ofConscious Pigs. STEMLER,

H.,

TEZAK-REID,

T . M.,

KAMINSKIS.

F. W., A.,

AND

(1990). Fundam. Appl. Toxicol. 14, 96-103. Physostigmine, as a pretreatment candidate for nerve agent poisoning, was examined for cardiopulmonary side effects.Cardiovascular and pulmonary parameters were monitored in unanesthetized domestic pigs which received pulmonary arterial infusion of 5 rg/kg/min physostigmine salicylate for 2 hr. A level of 74% inhibition of red blood cell (RBC) acetylcholinesterase (AChE) activity was attained in 45 min. and this level of carbamylation increased only slightly during the remaining infusion period. In addition to this large change in AChE activity, minor changes were observed in hematocrit, heart rate, body temperature, mean aortic pressure, pulmonary arterial wedge pressure, and pulmonary artery pressure. Typically, these parameters showed a trend toward elevated levels. Blood gases, pH, respiratory rate, tidal and minute volume, cardiac output, nonelastic resistance, and dynamic compliance were not significantly different from baseline values. The unanesthetized pig responds to physostigmine in a manner similar to that reported for other species and appears to be a suitable model for evaluating cardiopulmonary effects of cholinesterase inhibitors. o 1990 society ofToxicology

JAEGER,

J. J.

Physostigmine, a centrally and peripherally acting carbamate, reversibly inhibits red blood cell (RBC) acetylcholinesterase (AChE) and plasma cholinesterase (ChE). Pretreatment with physostigmine or other cholinesterase inhibitors before exposure plus atropine therapy can provide protection from nerve agents in several species (Berry and Davis, 1970: Gordon et al., 1978; Dirnhuber

et al., 1979; Deyi et al., 198 1; Lennox et al., 1985; Leadbeater et al., 1985; Deshpande et al., 1986). The rationale for pretreatment

with physostigmine is to combine cholinesterase in blood and tissues as a carbamylated enzyme complex which temporarily protects it against irreversible cholinesterase inhibitors such as soman (Janowsky et al., 1986; Deyi et al., 198 1: Harris et al., 1980). Despite the extensive literature regarding cholinesterase inhibitors, selecting the best pretreatment compound for use in humans who have a potential for exposure to nerve agents is a difficult problem; obviously, severe poisoning by these agents cannot be prospectively studied in man. Physostigmine is known to have physiologic effects in man that are due to increased cholinergic activity and include bronchocon-

’ The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Army or the Department of Defense. In conducting the research described in this report, the investigators adhered to the Guide for the Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council. 2 To whom correspondence should be addressed. 0272-0590190 $3.00 Copyright 0 1990 by the Society ofToxlcology. All rights of reproduction in any form reserved.

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striction, bradycardia, tachycardia, hypertension, sahvation, nausea, emesis, bronchial secretion, and disturbed thermoregulation (Davis et al., 1976; Janowsky et al., 1985; Hartvig et al., 1986; Janowsky et al., 1986). The intravenous injection of 2-3 mg of physostigmine was reported to also produce a “physostigmine syndrome” consisting of decreased speech, slowed thoughts, mild sedation, expressionless faces, decreased spontaneous activity, and nausea in man (Davis et

al., 1976). In view of the side effects observed in these clinical studies, there is a military requirement to explore the extent of potential effects of cholinesterase inhibitors on cardiopulmonary parameters. If physostigmine is to be administered in anticipation of nerve agent poisoning, the pretreatment compound itself must not adversely affect the soldier or his performance. The pig was selected as an appropriate animal model for man because of reported similarities for many human functions, including cardiovascular and pulmonary parameters (Phillips and Tumbleson. 1986). Thus, an instrumented pig model was developed to monitor physiologic events in conscious pigs. In a study of the relationship between reversible AChE inhibition and efficacy against soman lethality, the best computed protective ratio in guinea pigs was obtained at 70% inhibition of AChE (the greatest level of inhibition studied by Lennox et al., 1985). On the other hand, in a 28-day subchronic study of continuous infusion of physostigmine, undesirable muscarinic (diarrhea and salivation) and nicotinic (tremors and circling) effects occurred at 70% inhibition of RBC AChE (Frost et al., 1988). Because of the significance of the two aforementioned studies to the medical chemical defense mission, this study was conducted to determine the effect of a physostigmine-induced 70% inhibition of RBC AChE on selected indices of cardiovascular and pulmonary function. METHODS Animals. Eight Chester White-Yorkshire Cross castrated male pigs (Sus scrofa) weighing between I5 and 20

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kg were used in these experiments. The pigs were obtained from a commercial breeder and, upon arrival, were maintained in quarantine for a period of 7 days. They were then maintained on a 12-hr light and dark cycle housed individually in metal cages. Water was provided ad libitum, and Purina porcine lab chow was hand fed twice a day. The pigs were touched and stroked while feeding to minimize stress during handling. Daily training sessions were conducted to familiarize pigs with attendants and also to accustom the pigs to restraint for several hours in a Panepinto sling (Panepinto el a/., 1983). While restrained in the sling, the pigs were also conditioned to accept an anesthesia cone which covered the nose and mouth and was attached to a pneumotachograph for measuring respiratory air flow during infusion of physostigmine. Since the pig sound levels in the laboratory exceeded 110 dB, all attendants were required to wear ear plugs. The daily training sessions continued for 7- IO days pre- and l-2 days postsurgery. Surgery. After an overnight fast, each pig received preanesthetic intramuscular injections of 0.08 mg/kg atropine sulfate, 2.2 mg/kg ketamine hydrochloride, and 2.2 mg/kg xylazine. An endotracheal tube (5F) was inserted, and anesthesia was maintained with a mixture of halothane, oxygen, and nitrous oxide. Through a ventral paramidline cervical incision, a polyvinyl catheter (0.28 cm o.d./O.l7 cm i.d.) was inserted into the left carotid artery and advanced into the aorta. A Gould heparincoated flow-directed thermodilution catheter (size 5F) was inserted into the left jugular vein and advanced into the pulmonary artery. Both catheters were tunneled subcutaneously to the dorsal midline and exited the skin through a common opening. The ends of the catheters were placed between two Velcro patches, one of which was sutured to the skin. After the catheters were placed. each was filled with heparinized saline (2.0 ml saline mixed with 1.0 ml of l:lO.OOO heparin sodium). The catheters were cleansed daily by withdrawal of fluid (blood plus heparinized saline) and filled with fresh heparinized saline. Either ampicillin sodium (22 mg/kg) or cephalothin sodium (25 mg/kg) was administered daily to prevent postoperative sepsis. Experimental prmednres. The pig, after an overnight fast, was placed in the Panepinto sling. One nostril was sprayed with xylocaine, and a polyethylene sleeve (P.E. 320 coated with xylocaine gel) containing a stylus was advanced through the nasopharynx to a position in the esophagus approximately midway between the cardiac sphincter and the glottis. The stylus was removed and replaced by a Millar Mikrotip catheter pressure transducer (PC-470, 7F), which extended beyond the tip of the sleeve for measurement of intraesophageal pressures. The nose cone was positioned and mounted securely over the snout of the pig. The pig was then allowed a 45to 60-min acclimatization period before beginning the experiment. During this interval all instrumentation was calibrated and all catheters were cleansed and refilled with heparinized saline. Baseline measurements were

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made during the 30 min prior to the start of the physostigmine infusion. Each pig received a slow pulmonary artery infusion of 12.0 ml of a physostigmine salicylate solution (5 rg/kg/min) over a 2-hr period by means of a motor-driven infusion pump (Harvard Apparatus Co.). The physostigmine salicylate was obtained from Walter Reed Army Institute of Research (WRAIR Lot No. BL 25591). Solutions were prepared shortly before the start of infusion. Data were collected during the infusion period and for 2 hr after the termination of the infusion. Cardiopulmonary measurements. Aortic and pulmonary arterial pressures were recorded continuously by attachment of vascular catheters to pressure transducers (Statham Model P50). Pulmonary arterial wedge pressures were obtained at half-hour intervals by inflation of the balloon on the tip of the Gould catheter. Pulmonary artery temperature was also recorded. Pulmonary artery and pulmonary wedge pressure measurements were taken from the recording at end expiration. Electrocardiograms were obtained from patch electrodes attached to the skin. Thermal dilution cardiac output using 3 ml of iced 5% dextrose solution was measured in triplicate every 30 min. Cardiac outputs were calculated by a Sorenson Model No. 4 1234-O 1 cardiac output computer. Tidal airflow was measured by a heated No. 2 Fleish pneumotachograph attached to the animal’s nose cone. Inspired and expired flow was integrated electronically to obtain tidal volumes. Transpulmonary pressure was obtained by subtraction of esophageal pressure from oral pressure. Pulmonary nonelastic resistance to airflow and pulmonary dynamic compliance were measured by techniques described by Rodarte and Rehder (1986). Arterial blood samples for the measurement of pO2, KO, , and pH were drawn at 15-min intervals during the control period and the first hour of infusion and at 30-min intervals for the rest of the experiment. Additionally, a 2-ml arterial blood sample was collected in a syringe containing 2% EDTA for determination of microhematocrit and cholinesterase activities. RBC and plasma specimens were used to determine AChE and ChE activities, respectively. Total cholinesterase activity was obtained from whole blood analyses. These analyses were performed immediately on a Technicon Auto Analyzer II system by the method of Groff et al. (1976). Statistical analysis. Data are presented as means f SEM with a significance level at p < 0.05. The data were analyzed by one-way ANOVA with repeated measures using BMDP program P2V (Jennrich et al., 1983) followed by Dunnett’s test (Winer, 197 1).

RESULTS Clinical signs. All pigs were restless during the first hour of infusion but appeared to be sedated during the second hour of infusion through the end of the experiment. In some

ET AL

pigs, the restlessness was marked and accompanied by defecation. Salivation was common in all pigs but emesis was never observed. Muscle fasciculations in the head and neck region, which occurred between 30 and 60 min after the start of infusion, eventually extended to the whole body. The fasciculations continued for almost an hour after infusion was stopped. Enzyme activity. The time course of significant changes of plasma ChE, RBC AChE, and blood total cholinesterase activities in pigs in response to infusion of physostigmine salicylate (5 pg/kg/min) is shown in Fig. 1. Activities of AChE in RBC, total cholinesterase in blood, and ChE in plasma during the control period were 3.46 ? 0.13, 1.5 1 +- 0.06, and 0.35 f 0.02 pM/ml/min, respectively. Inhibition of AChE activity (approximately 74%) was reached in about 45 min, and inhibition in whole blood total cholinesterase tended to parallel RBC AChE with time. In contrast, inhibition of ChE in plasma increased throughout the period of infusion of physostigmine but never exceeded 45% inhibition. Following infusion of physostigmine, regression analysis of the recovery of RBC AChE and plasma ChE activity in a semilog plot (not shown) of percentage inhibition vs time indicated a first-order reaction. After infusion was terminated, the half-time for enzyme recovery (decarbamylation) in RBC and plasma was determined to be 77.8 and 88.2 min, respectively. Cardiovascular measurements. Figures 2, 3, and 4 show statistically significant increases in heart rate, hematocrit, mean aortic, pulmonary artery, and pulmonary artery wedge pressures. The ECG showed an occasional premature ventricular depolarization and inverted T-wave as well as a few instances of A-V blocks. Sinus bradycardia was never observed. Pulmonary measurements. Lung mechanics were altered to some degree, but no consistent pattern of response was found in the four pigs in which data were successfuliy obtained. The lung mechanics were difficult to obtain in unanesthetized pigs due to vocalization.

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TOME(MINUTES) FIG. 1. Mean activities in blood total ChE, RBC AChE, and plasma ChE in pigs in response to infusion of 5 pg/kg/min physostigmine. IV = 8. Although not indicated by asterisks, all values are significantly different from baseline. p < 0.05.

Grunting, especially during the first hour of infusion of physostigmine, interfered with data collection in about half of the pigs. Arterial blood gases and pH showed no significant changes during infusion of physostigmine. Tidal volume, respiratory rate, and minute volume were also not significantly altered by physostigmine.

Temperature measurements. The temperature measured with the Gould catheter increased during the physostigmine infusion as shown in Fig. 5. DISCUSSION The results of this study indicate that pigs can tolerate 5 pg/kg/min of physostigmine

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FIG.2. Mean heart rate in pigs in response to infusion of 5 &kg/min lines indicate tSEM. p < 0.05.

physostigmine. N = 8. Barred

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FIG. 3. Mean hematocrit in pigs in response to infusion of 5 pg/kg/min physostigmine. N = 8. Barred lines indicate SEM. p < 0.05.

without serious cardiopulmonary side effects. Each pig received 1 mg every 10 min. This is comparable with rates of administration of physostigmine used in clinical studies in man. However, the total dose administered to pigs over time was approximately fourfold higher than that administered to human subjects by slow infusion (Risch et al., 198 1; Janowsky et al., 1973, 1986; Davis et al., 1976). All of the above human subjects also received scopolamine or atropine to curb nausea and vomiting, whereas the pigs received no additional medication. Therefore, in the pig study, the cardiovascular effects of physostigmine are not masked by other medication. The primary purpose of this study was to evaluate potential cardiovascular side effects of physostigmine in an unanesthetized animal. In pigs, behavioral responses to physical and social environment are prominent according to Dantzer and Mormede ( 1986) and Hannon (1986). These authors indicated that the relative state of anxiety or calm of the pig could influence cardiovascular parameters. Hannon (1986) showed that minor physical activity in a pig could result in increases in plasma epinephrine concentration accompanied by minor increases in arterial pressure, heart rate, and hemoglobin concentration.

Similar effects may have occurred in our study and may have caused some of the elevations in pressure and heart rate. An effort was made, through training and gentle handling, to avoid producing anxiety or discomfort in the pigs, but the possible effects of these sensations cannot be excluded in unanesthetized pigs. The cardiovascular and temperature changes appear to correlate temporally with the infusion of physostigmine. However, the sensation of muscle fasciculations, nausea, and perhaps abdominal cramps, rather than the direct systemic or central nervous effect of physostigmine itself, may have resulted in these cardiovascular changes. A parallel control study utilizing pigs not receiving physostigmine might have indicated whether the cardiovascular changes observed were a result of psychological stress associated with the procedure. A striking behavioral difference was observed in the pigs between the first and second hours of infusion. During the first period, spontaneous movement and periods of restlessness were evident. This changed to inactivity during the remainder of the experiment. Somewhat similar behavioral changes in cats given injections of physostigmine into the right cerebral ventricle have been re-

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TIME(MINUTES) FIG. 4. Mean aortic, pulmonary artery, and pulmonary artery wedge pressures in pigs in response to infusion of 5 pg/kg/min physostigmine. N = 8. Barred lines indicate +SEM. p -e 0.05.

ported by Feldberg and Sherwood (1954a). The injection of physostigmine into the cerebral ventricles of sheep by Palmer ( 1959) produced excitability without symptoms of stupor. The effects of intraventricular injection of acetylcholine have also been studied by several authors. Physostigmine is known to cross the blood-brain barrier in the rat (Somani and Khalique 1987) and dog (Giacobini et al., 1987). It would be expected, through ChE inhibition, to elevate intracerebra1 levels of acetylcholine after intravenous administration. Thus physostigmine as used in the present study may provide an elevated level of intracerebral acetylcholine similar to direct intraventricular injection of acetylcholine. The response of awake cats (Feldberg and Sherwood, 1954b) and dogs (Lang and Rush, 1973) to acetylcholine injected into the cerebral ventricles has been reported. They found that the procedure produced initial hyperactivity, which was followed by drowsiness and lethargy, as was found in our study. These considerations suggest that systemic administration of these amounts of physostigmine in the pig results in initial excitement, followed by drowsiness, and that these effects may be due to the accumulation of excess acetylcholine in the brain.

Several mechanisms or a combination of mechanisms could explain the cardiovascular findings of the current study but no definitive explanations can be ascribed. Lang and Rush ( 1973) in their study of dogs given physostigmine into cerebral ventricles found some increases in heart rate and blood pressure. They reported similar findings with acetylcholine injections. The authors concluded that both muscarinic and nicotinic cholinergic mechanisms led to these cardiovascular effects, but that the sites of action and peripheral mechanisms were not elucidated. Brezenoff ( 1973) studied pressor responses resulting from the intravenous or cerebral intraventricular administration of physostigmine in anesthetized rats. Amelioration of the response by intravenous atropine sulfate (considered to cross the blood-brain barrier) but not by intravenous atropine methyl bromide (which does not readily cross the blood-brain barrier) suggested that the response was due to central muscarinic action. Adrenalectomy abolished the pressor response to intraventricular, but not intravenous injection of physostigmine. This was interpreted to mean that the release of adrenal catecholamines explained the response to intraventricular, but not intravenous physostigmine. The response

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FIG. 5. Mean body temperatures in pigs in response to infusion of 5 &kg/min Barred lines indicate ?SEM. p < 0.05.

to intravenous physostigmine was attributed to a general sympathetic discharge exclusive of the adrenal medulla. In view of these considerations, the pressor responses observed following intravascular administration of physostigmine in pigs may likewise result from general sympathetic discharge resulting from central cholinergic stimulation due to accumulating acetylcholine in the brain. General sympathetic discharge may have been responsible for the tachycardia also observed. Several explanations of the temperature elevations may be considered. The continuous muscle fasciculations due to nicotinic effects of acetylcholine accumulation may have produced some temperature rise. Initial excitement and hyperactivity may have contributed. Temperature elevations have been reported in animals exposed to physostigmine by injection into the CNS. Sheep given intraventricular physostigmine demonstrated temperature elevations (Palmer, 1959; Darling et al., 1974). Direct injection of acetylcholine or a mixture of acetylcholine and physostigmine into the hypothalamus of monkeys produced dose-related hyperthermia (Myers and Yaksh, 1969).

240 physostigmine. N = 8.

In summary, the administration of amounts of physostigmine sufficient to cause greater than 70% inhibition of RBC AChE in pigs was accompanied by only moderate cardiopulmonary side effects. Moderate temperature elevations and changes in state of alertness also occurred. The unanesthetized pig appears to be a suitable model for evaluating cardiopulmonary effects of cholinesterase inhibitors. REFERENCES BERRY, W. K., AND DAVIES, D. R. (1970). The use of carbamates and atropine in the protection of animals against poisoning by 1,2,2-trimethylpropyl-methyl phosphonoflouridate. Biochem. Pharmacol. 19,927934.

BREZENOFF,H. E. (1973). Centrally induced pressor responses to intravenous and intraventricular physostigmine evoked via different pathways. Eur. J. Pharmacol. 23,290-292.

DANTZER, R., AND MORMEDE, P. (1986). The behavior of swine and its relevance to cardiovascular research. In Swine in Cardiovascular Research (H. C. Stanton and H. J. Mersmann, Eds.), Vol. I, pp. 61-73. CRC Press, Boca Raton, FL. DARLING, K. F., FINDLAY, J. D., AND THOMPSON, G. E. (1974). Effect of intraventricular acetylcholine and eserine on the metabolism of sheep. Pfrugers Arch. 349, 235-245.

PHYSOSTIGMINE DAVIS, K. L., HOLLISTER, L. E., OVERALL, J., JOHNSON, A., AND TRAIN, K. (1976). Physostigmine: Effects on cognition and affect in normal subjects. Psychopharmacology 51,23-27. DESHPANDE, S. S.. VIANA, G. B., KAUFFMAN, F. C., RICKETT, D. L., AND ALBUQUERQUE. E. X. (1986). Effectiveness of physostigmine as a pretreatment drug for protection of rats from organophosphate poisoning. Fundam. Appl. Toxicol. 6,566-577. DEYI, X., LINXIU, W., AND SHUQIU, P. (198 1). The inhibition and protection of cholinesterase by physostigmine and pyridostigmine against soman poisoning in vivo. Fundam. Appt. Toxicol. I,2 17-22 1. DIRNHUBER, P.. FRENCH, M. C.. GREEN, D. M., LEADBEATER, L., AND STRATTON, J. A. (1979). The protection of primates against soman poisoning by pretreatment with pyridostigmine. J. Pharm. Pharmacol. 31, 295-299.

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