A methoxyflurane delivery system for stereotaxic surgery

Brain Research Bulletin. Vol. 13, pp. 457a,

1984. 0 A&ho hmmtional

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BRIEF COMMUNICATION

A Methoxyflurane Delivery System for Stereotaxic Surgery’ PHILLIP S. LASITER* AND JOHN GARCIA Department

of Psychology

and Brain Research Institute, University of California, Los Angeles Los Angeles, CA 90024 Received 13 July 1W

P. S. AND J. GARCIA. A mefhoxyfluranc delivery system .for stereotaxic surgery. BRAIN RES BULL 13(3) 457-460,1!%4.-Methoxyflurane (2,2dichlorol,l-difluro-ethyl methyl ether; Metofane”) is a potent general inlmktkn anesthetic that is well-suited for small animal surgery. Methoxytlurane is particularly attractive as an anest&& agent in neurological stereotaxic surgery, because methoxyflurauc does not marhedly attenuate the rate of auterograde or retro grade trausport of horseradish peroxidase, or reduce the consistency at&u extent of excitatory neurotoxin damage. Methoxyfhunne also is non-fkmmahk when mixed with Oz or air at anesthetic concentrations. The use of methoxyfIurane anesthesia iu stereotaxic surgery has heen limited hecause methoxyfiurane must he delivered via a vapor&r system that will easily interface with standard stereotaxic headholders. The present report describes a simpk, rcliabk and inexpensive methoxyfhunue delivery system for stereotaxic surgery. LASITER,

Animal anesthesia Tract-tracing

Horseradish peroxidase

SODIUM pentobarbital (NembutaP, ployed as a parcnteral anestbctic

Methoxyflurane

Neurotoxiu

Second,

SagataI) is widely emagent in small animal

several

Stereotaxic surgery

species,

patticularly

theguineapig . . ..

(Cavia cobaya) and rabbit (Oryctolagus cuntcurl), are ex-

surgery. However, barbiturate anesthesia is known to produce a number of profound physiological complications during surgical procedures. First, barbiturates produce anesthesia via hypnotic effects, that is, responses normally elicited by pain are suppressed by an alteration of consciousness (e.g., [5,6]). Barbiturates provide little or no analgesia, and therefore relatively large doses of barbiturates must be used in many species to achieve surgical planes of anesthesia [a]. Large doses of barbiturates markedly depress medullary autonomic centers, lowering body core temperature, reducing respiratory tidal volume, and increasing pulmonary dead space and tracheobronchial secretions. Attenuations of respiratory function, and hence alveolar gas exchange, are particularly dangerous during barbiturate anesthesia, insofar as blood Op partial pressure decreases (hypoxia) and CO2 partial pressure increases (hypercarbia). Hypoxia and hypercarbia each depress the myocardium, producing abnormal deviations in cardiovascular function. Hypoxia is exacerbated in the presence of abnormal tracheobronchial secretions that result from chronic respiratory disease in rats and mice, and antisialagogues such as atropine sulfate are often employed as adjuncts to barbiturate anesthesia. Despite such therapeutic regimens barbiturate anesthesia is usually associated with high mortality.

quisitely sensitive to parenteral it+ctions of barbiturates. In those species the overaII margin of safety afforded by barbi-

turate anesthesia is questionable [a. Dissociative agents (e.g., Ketamine HCl) or tratquillixers (e.g., chlorpromazine, acepromaxine), used alone or in conjunction with reduced doses of barbiturates, may be employed in those species to attain surgical planes of anesthesia. However, dissociative agents or ttanquillixers may not provide &qua!e ana&sia during surgery, and preanesthetic regimens have the inherent disadvantage of extending anesthesia Muction time. Third, the rate of uptake and axonal transport of horseradish peroxidase (HRP) is attenuated by barbiturate anesthesia (see [14] for a review). For instance, Rogers, Butcher and Novin [ll] reported that pentobarbital anesthesia (50 mg&g, IP) in rats reduces the rate of anterogntde and retrograde tmnsport of HKP, as compared to anesthesia induced by ethyl carbamate (methane; 1.5 g/kg, IP). Although results of Kogers et al. [ll] indicate that urethane anesthesia is acceptable for tract-tracing experiments, the mutagenic and carcinogenic properties of that agent present a hazard to the experimental subject and laboratory personnel. Finally, the consistency of results obtamed from manipulations involving excitatory neurotoxins (e.g., kainic acid,

‘Supported by NIHNCDS Grant No. ROl-11618_ to ~. J.G. ... - _ *Requests for reprints should be addressed to Dr. Phillip S. Lasiter, Mental Retardation Research Center, Room 58-228, University of California, Los Angeles, 760 Westwood Plaza, Los Augeles, CA 90024.

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458

ibotenic acid or quinolinic acid) may be affected by barbiturate anesthesia [8]. Presumably, barbiturate anesthesia reduces the consistency and extent of neurotoxin damage by competing for receptor sites in glutamate and/or GABA projection systems [S]. In combination, the foregoing observations indicate that barbiturate anesthesia may be contraindicated in various experimental surgical procedures. Methoxyflurane (2,2-dichloro-l , 1-difluro-ethyl methyl ether; Metofane@) (Pitman-Moore Co., Washington Crossing NJ 08560) is a potent general inhalation anesthetic that can be employed when barbiturate anesthesia is contraindicated. Several chemical properties of methoxyflurane render it attractive as an anesthetic agent in small animal surgery. For instance, unlike diethyl ether, methoxyflurane is nonflammable when mixed with O2 or air at anesthetic concentrations. Methoxyflurane is highly volatile and reaches saturated concentration in air or O2 at standard temperature and pressure (ca. 3.5% v/v). Methoxyflurane also produces desirable physiological effects during anesthesia. The high solubility of methoxyflurane in blood (blood/gas partition coefficient; BGPC) affords relatively rapid absorption as compared to parenteral anesthetic agents such as sodium pentobarbital. Methoxyflurane is a potent analgesic, as measured by minimum alveolar concentration (MAC) required for analgesia (0.2% v/v: rat [6]), and analgesia induced by methoxyflurane persists for some time during postoperative recovery. Methoxyflurane is non-irritating to the tracheobronchial lining, and surgical levels of anesthesia can be attained without marked depressions of brainstem autonomic centers or the myocardium proper. The high BGPC and MAC of methoxyflurane also make it difficult to administer an overdose during surgery. Finally, methoxyflurane is neither mutagenic nor carcinogenic [ 11. Although methoxyflurane is an anesthetic agent of choice for small animal surgery 161, the use of methoxyflurane in stereotaxic surgery has been limited by methodological factors. For example, while it is possible to induce anesthesia for brief periods of time (ca. 15 min) using vaporized methoxyflurane in a closed container (“bell-jar” technique), stereotaxic surgery usually necessitates the maintenance of anesthesia for 30 min to 2 hr. Commercial methoxyfhtrane delivery systems are expensive, require tracheal intubation, and are not readily adapted to most stereotaxic headholders. Spencer [ 121described a methoxyflurane delivery system for stereotaxic surgery that provides precise control of anesthetic concentration during neurophysiological experiments lasting up to 15 hours. However, the limitations of that system in most stereotaxic procedures are that (a) a mixture of halothane and methoxyflurane is used to induce anesthesia, (b) a halothane vaporizer system (“wick” type) is required, and (c) several flowmeters and mixing valves are required. The inherent cost of halothane, vaporizer equipment, flowmeters and mixing valves precludes the use of Spencer’s [ 121 system in most laboratories. Moreover, precise control of methoxyflurane concentration may not be required in many experimental surgical procedures. The present report describes a reliable, simple and inexpensive methoxyllurane delivery system for routine stereotaxic surgery (i.e., lesion production, tract-tracing injections, etc.). This system can be used in surgical applications that do not require precise control of blood and brain methoxyflurane concentration. A schematic diagram of the methoxyflurane delivery system is shown in Figs. 1 and 2. The distinguishing feature of the present system is that methoxyflurane gas is delivered

LASITER

AND GARClA

AIR INLEl

NOSECOK OUTLET

FL FUSE

IISVAC

FIG. 1. Schematic diagram of regulated vaporizer system and forced-air purging pump.

intermittently to the animal via a non-recirculating nosecone system. The rationale for intermittent delivery of methoxyflurane is as follows. Methoxyflurane is very suluble in blood and brain, and the solubility of an inhalation anesthetic agent in body tissue largely determines the rate of absorption and elimination. For instance, highly soluble anesthetic agents, such as methoxyflurane and diethyl ether, are rapidly taken up by pulmonary blood, and the alveolar partial pressure of the agent falls rapidly. In that situation arterial blood and brain partial pressures equilibrate with alveolar partial pressure, increasing anesthesia induction time. Conversely, alveolar tension rises quickly with insoluble agents such as N20, giving rise to relatively rapid induction of anesthesia. Anesthetic solubility similarly influences elimination, whereby brain concentrations of an anesthetic agent decrease in proportion to alveolar partial pressure. Soluble anesthetic agents are eliminated relatively slowly and insoluble anesthetic agents are eliminated rapidly. Because brain concentrations of methoxyflurane increase relatively slowly, high concentrations (ca. 3.5% v/v) are typically used for induction of anesthesia, whereas low concentrations (ca. 0.2% v/v) are used for maintenance of anesthesia (e.g., [ 121). High methoxyflurane concentrations increase the alveolar partial pressure gradient during induction, reducing induction time. However, because the minimum alveolar concentration (MAC) necessary for analgesia is low with methoxyflurane, anesthetic concentration must be reduced immediately following induction. Thus, in constant-flow systems (e.g., [ 12]) methoxyfhtrane concentration must be reduced following induction of anesthesia. Such procedures require calibrated flowmeters and/or airgas mixing valves. The unusual attribute of methoxyflurane is that intermittent anesthetic deliveries can maintain surgica.l planes of anesthesia. For example, a brief delivery of concentrated methoxyflurane gas rapidly increases blood and brain

METHOXYFLURANE

DELIVERY

SYSTEM

FlG. 2. Coastruction details for nose cone used with a Kopf series 900 stereotaxic headholder and a mature rat or guinea pig subject. Lower drawing shows placement of nose cone with regard to bite bar.

methoxyflurane concentrations. When gas delivery is terminated, methoxyflurane concentration decreases relatively Slowly, maintaining the MAC. If intermittent high concentration “pulses” ofmethoxyflurane are delivered as required to establish the MAC, surgical levels of anesthesia will be maintained. Intermittent delivery of methoxyflurane eliminates the need for anesthetic concentration adjustments after induction, and therefore flowmeters and/or calibrated air-gas metering systems are not required. Construction and operation of the delivery system is quite simple. Compressed air is delivered to a low pressure regulator (Norgren RO4-270-RNAA), that limits pressures in subsequent vaporizer lines to approximately 0.07-0.14 kg/cm’. It is essential to incorporate the regulator into the system for safety. Unregulated pressure surges in the compressed air feeder line may produce an explosion of glass vaporizer parts, causing injury to the surgeon or attending personnel.

The output line from the regulator is fed into an electric solenoid gas valve (MINAC No. ill-11lB) (low-pressure regulator and solenoid valve can be obtained from H & R Co., 401 E. Erie Ave., Philadelphia, PA 19134) which contains a 0.78 mm orlice. The gas valve provides air to the vaporizer when actuated. The solenoid valve is normally closed, preventing air delivery to the vaporizer. Actuation of a foot switch opens the regulated air supply line, forcing air to the vaporizer. The vaporizer is constructed with a 50 ml glass graduated cylinder (Corning 3029, and a silicon “rubber” stopper. A fritted glass gas bubbler (Corning 39533; 40-60 micron pore size) is positioned at the bottom of the cylinder. The outlet of the vaporizer consists of a glass,elbow tube that extends 3-4 mm into the cylinder. During operation the level of methoxytlurane is adjusted to cover the bubbler by approximately l-l.5 cm. The outlet line of the vaporizer is fed to a Y connector which also receives air from an aquarium pump. The aquarium pump provides a forced-air purge of the nose cone line during the “off” cycle of the vaporizer. A final line

leads from the Y connector to the nose cone that is fitted tightly around the animal’s nares. Construction details for thenosecoae(Fig.2)applytoaKopfBCri68~stCrtOtLUtic h~andamatureratorguineapigsul$ect,buta similar nose cone may be developed for other stereotaxic headholders and/or species. All lines are constructed with 5 mm i.d. Tygon tubing, and wherever possibk glass or teflon connectors are used. Do not use natural rubber tubing, inasmuch as methoxyflurane is soluble in rubber. The outlet line of the nose cone is fed to a 2 liter canister (not illustrated) that contains activated charcoal (Norit). It is essential to vent the nose cone outlet line to a charcoal filter to prevent accumulation of methoxyflurane gas in the surgical theatre. Do not vent methoxyfIurane into room air conditioner returns or outside the laboratory. After approximately 12-15 hr of operation the activated charcoal is discarded and the canister is refilled. To induce anesthesia the animal is placed in a desiccating chamber or similar air-tight container that contains a small gauze pad saturated with approximately 1 ml of methoxyflurane. In practice, we place the gauze pad in the chamber approximately 5-10 mitt before induction to allow saturation of enclosed air. When the animal has been placed in the chamber we added 50-100 ml of 0, to the chamber air (1.b 3.0% v/v), since blood 0, tension may drop considerably with prolonged exposure to high concentrations of methoxyflurane (ca. 3.5% v/v for 10-15 min). However, it should be mentioned that we have induced anesthesia without 0, delivery with no fatalities. Induction is complete after 8-10 min in the chamber. Next, the animal is removed from the induction chamber, positioned in the stereotaxic headholder and fitted with the nose cone. Once positioned, intermittent gas delivery (5001000 ml/mitt) is begun “to effect” (level 3 anesthesia). With experience in operation respiration rate provides the best index of anesthesia; respiration rate decreases slightly, yet tidal volume is not markedly depressed. Responses to peripheral stimulation, such as an ear pinch, should rarely occur with experience in operation. During the first 10 minutes of surgery methoxyflurane is delivered at an approximate rate of 5-15 set every 4-5 minutes. The frequency of gas delivery decreases as surgical time increases, because methoxyflurane accumulates gradually in blood and brain. At the conclusion of surgery the animal is removed from the stereotaxic unit. It is not advantageous to administer 0, during recovery, since recovery times are generally unaffected. In our experience mature rats are ambulatory approximately 15-20 minutes following the last gas delivery. Infant rats and guinea pigs are ambulatory approximately 8-10 minutes following the last gas delivery. With the materials described herein construction cost is approximately $50.00. With careful attention to gas delivery rate a single 125 ml bottle of methoxyfhuane should be sufficient for approximately 5&75 45 min surgeries. The cost of anesthesia for each animal is approximately $0.40. The most wasteful portion of the procedure is induction, and thus, we save saturated “induction” pads in a small specimen jar at 4°C when not used. We have used the present system in over 300 surgeries involving HRP injections, neurotoxin injections (ibotenic acid) and lesion placements, with only one fatality resulting from preoperative respiratory complications (rat). Peroxidase transport rate compares favorably to that observed with urethane, and the consistency and extent of neurotoxininduced damage (neostriatum, agranular insular neocortex)

460

LASITER

is increased, as compared to sodium pentobarbital anesthesia. Several concluding comments are warranted concerning suspected nephrotoxicity resulting from exposure to methoxyflurane [6]. The microsomal biotransformation of fluorinated hydrocarbons produces free fluoride as at least one blood-borne metabolite, and it has been suggested that hepatic fluoride turnover may produce renotoxic effects (e.g., [7]). However, observations in human [4, 9, 131 and infrahuman subjects [3] indicate that low-dose anesthetic concentrations of methoxyflurane are not sufficient to

AND GARCIA

produce renal dysfunction. Nephrotoxic effects can OCCUI when fluorinated hydrocarbons are presented during conditions of elevated hepatic microsomal activity. For instance, barbiturates and ethanol increase hepatic microsomal activity, and methoxyflurane presentations to animals receiving barbiturate or ethanol pretreatments produce nephrotoxic effects [2,10]. Therefore, the extant of clinical and experimental evidence indicates that anesthetic doses of methoxyflurane do not produce nephrotoxic effects when hepatic microsomal activity is normal.

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ether

2. Baden, J. M., S. A. Rice and R. I. Maze. Deuterated methoxyflurane anesthesia and renal function in Fischer 344 rats. Anesthesiology 56: 203-206,

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3. Cook. T. L.. W. J. Benou. B. A. Hitt. J. C. Kosek and R. I. Mazze. A cdmparison 0; renal effects and metabolism of sevoflurane and methoxyflurane in enzyme-induced rats. Anesth Analg (C/eve) 54: 829-835, 1975. 4. Cousins. M. J.. L. R. Greenstein. B. A. Hitt and R. I. Mazze. Metabolism anh renal effects of e&uane in man. Anesthesiology 44: 44-53,

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Goodman, L. S. and A. Gilman. The Pharmacological Basis oj Therapeutics. New York: Macmillan Co., 1970. Green, C. J. Animal Anesthesia. London: Laboratory Animals Ltd., 1979. Kluwe, W. M. and J. B. Hook. Metabolic activation of nephrotoxic haloalkanes. Fed Proc 39: 3129-3133. 1980. Moore, R. Y. Methods for selective, restricied lesion placement in the central nervous system. In: Neuroanatomical TracrTracing Methods, edited by L. Heimer and M. J. Robards. New York: Plenum Press, 1981, pp. 55-90.

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Penthrane (Methoxy-Fluorane)-air anesthesia system for small mammals. Physiol Behav 16: 501504, 1976. 13. Stoelting, R. K. and C. Peterson. Methoxyflurane anesthesia in pediatric patients: Evaluation of anesthetic metabolism and renal function. Anesthesiology 42: 26-29, 1975. 14. Warr, B. W., J. de Olmos and L. Heimer. Horseradish peroxidase: The basic procedure. In: Neuroanatomical Tract-Tracing Methods, edited by L. Heimer and M. J. Robards. New York: Plenum Press, 1981, pp. 207-262.