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Rat Strain Minimally Influences Anesthetic and Convulsant Requirements of Inhaled Compounds in Rats

Gong, Diane BS; Fang, Zexu MD; Ionescu, Pompiliu MD; Laster, Michael J. DVM; Terrell, Ross C. PhD; Eger, Edmond I II, MD

doi: 10.1213/00000539-199810000-00040
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We assessed the effect of rat strain on susceptibility to anesthesia and convulsions produced by inhaled compounds.We determined the minimum alveolar anesthetic concentration (MAC) of desflurane and nitrous oxide, and the convulsive 50% effective dose (ED (50)) of 1,2-dichlorohexafluorocyclobutane, flurothyl, and difluoromethyl-1-chlorotetrafluoroethyl ether in five strains (three inbred [Long Evans, Sprague-Dawley, and Wistar] and two outbred [Fischer and Brown Norway]). Strain had slight effects on anesthetic potency, the strains with the highest MAC values (Long Evans and Brown Norway) having values <or=to28% greater than the strains with the lowest values (Sprague Dawley and Wistar). MAC for nitrous oxide correlated directly with MAC for desflurane as a function of strain. MAC for either desflurane or nitrous oxide correlated inversely with the convulsive ED50 of 1,2-dichlorohexafluorocyclobutane, but correlated poorly (and directly) with the convulsive ED50 of the remaining compounds. Convulsivity varied little as a function of strain (greatest difference 21%) and did not vary consistently as a function of strain. No consistent difference was seen between inbred versus outbred strains. Implications: Rat strain has a minimal effect on the potency of inhaled anesthetics or the convulsant activity of inhaled compounds. It seems that the sites acted on by inhaled compounds to produce anesthesia and convulsions are conserved across common rat strains.

(Anesth Analg 1998;87:963-6)

Department of Anesthesia, University of California-San Francisco, San Francisco, California.

This work was supported in part by National Institutes of Health Grant 1P01GM47818-02 and by the Anesthesia Research Foundation.

Accepted for publication June 23, 1998.

Address correspondence to Dr. Eger, Department of Anesthesia, S-455, University of California, San Francisco, CA 94143-0464.

The determination of anesthetic potency provides information that has both clinical and theoretical implications. We may use minimum alveolar anesthetic concentration (MAC) to estimate anesthetic requirement and delivery [1]. MAC may be used to define the effect of physiological factors (e.g., age, temperature, pregnancy) on the anesthetic requirement. Difference or changes in the requirement must be explained by any relevant theory of narcosis.

Results across various studies suggest that species are one determinant of anesthetic potency (e.g., MAC), but the differences in MAC across species seem to be small [1]. Within a given species, variation from strain to strain seems to be still smaller but may be significant. For example, the nitrous oxide MAC for Long Evans rats (2.35 +/- 0.20 atm) exceeds that of Sprague-Dawley rats (2.21 +/- 0.19 atm) [2]. Sprague-Dawley rats also have a higher isoflurane MAC (1.57 +/- 0.08 atm) than spontaneously hypertensive (1.20 +/- 0.05 atm) or Wistar-Kyoto rats (1.26 +/- 0.07 atm) [3].

Other rodents, such as mice, can be selectively bred for resistance and susceptibility to the effects of nitrous oxide on the righting reflex [4]. Resistance to the anesthetic effect of inhaled compounds also may influence the vulnerability of an animal to the convulsant effects of inhaled compounds. Mice bred to have a greater susceptibility to nitrous oxide resist the convulsant effects of both inhaled (e.g., flurothyl) and injected (e.g., picrotoxin) convulsants [5]. Conversely, mice resistant to nitrous oxide are more susceptible to the action of convulsants.

General anesthetics depress the central nervous system's excitability through a mechanism that probably involves effects on synaptic ion channels, but the fundamental molecular nature of the site on which they act, and how they act on that site, is unknown. Similarly, little is known about the site and mechanism by which convulsant inhaled compounds exert their effects, or the possibility of a reciprocal relationship between anesthetic and convulsant effects of inhaled compounds. Although anesthetic and convulsant requirements across rat strains have been compared (see above), the numbers of such comparisons have been limited. Despite implications to mechanisms of anesthetic action, it is not known whether inhibitory or excitatory processes follow a consistent pattern in different strains of rats.

In the present study, we examined the effect of rat strain on the potency of inhaled anesthetics (as defined by MAC) and on the convulsivity of nonimmobilizers. Our hypotheses were that: (a) strain would affect MAC, but differences in MAC across strains would be small; (b) changes in MAC as a function of strain would be consistent across anesthetics (i.e., if it took a higher concentration of Anesthetic A to produce MAC for a certain strain, it would also take a higher concentration of Anesthetic B); and (c) MAC would correlate inversely with the concentration required to produce convulsions.

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With approval of our committee on animal research, we examined the effects of two anesthetics (nitrous oxide and desflurane) and three convulsants (two nonimmobilizers [1,2-dichlorohexafluorocyclobutane], also called 2N; and flurothyl, CF3 CH2 OCH2 CF3; and the transitional compound, CF3 CFCIOCHF2 [less potent than its lipid solubility would indicate]) on five rat strains: three outbred strains (Sprague-Dawley [Crl:CD[registered sign](SD)Br], Long Evans [Crl:(LE)Br], Wistar [Crl:(WI)Br]) and two inbred strains (Fischer [CDF[registered sign] (F-344)/CrlBR] and Brown Norway [BN/CrlBr]). Nitrous oxide was obtained from Puritan Bennet (Pleasanton, CA). Desflurane was donated by Baxter Pharmaceutical Products (Liberty Corner, NJ). 2N and flurothyl were obtained from PRC Corp (Gainsville, FL). CF3 CFCIOCHF (2) was synthesized by an investigator (RCT). All compounds were >99.5% pure.

The anesthetic ED50 (MAC) or convulsive ED50 values were measured in each rat strain for each drug. All rats were approximately 12-wk-old, male, specific pathogen-free, and obtained from Charles River Laboratories, Inc. (Wilmington, MA). Rats ate standard rat chow ad libitum and were housed in rooms maintained at 22-24[degree sign]C during a 12-h cycle of light and dark. Rats were studied one to three times with a minimum of a week interval between exposure to test compounds.

Pairs of rats were given nitrous oxide in a hyperbaric chamber constructed at our institution from a clear cast acrylic tube (Glaslex, Stirling, NJ) and metal end-plates forced by steel rods and bolts against rubber gaskets applied to the ends of the tube. Electrical pass-throughs and pressure fittings allowed 1) measurement of rectal temperatures and chamber pressures, 2) delivery and acquisition of gases, 3) delivery of electrical stimulation, and 4) delivery of power to a fan inside the chamber. A canister of soda lime next to the fan maintained CO2 levels below 0.008 atm (Capnomac Infrared Analysis; Datex Corporation, Helsinki, Finland). Rat rectal temperatures were maintained at 37.5 +/- 0.7[degree sign]C by applying ice or infrared light to the exterior of the chamber. MAC was determined using electrical stimulation of the tail after equilibration for 20 min at increasing step concentrations of nitrous oxide. The methods used for electrical stimulation were described previously [6]. The steps equaled 20%-30% of the preceding nitrous oxide partial pressure.

Studies of desflurane, 1,2-dichlorohexafluorocyclobutane, and CF3 CFCIOCHF2 were measured using a system described previously [7]. Rats were enclosed in individual cylinders that permitted control of respired gases (including resorption of carbon dioxide and replacement of oxygen), measurement of rectal temperature, and, for studies of anesthetic potency, access to the tail of the rat. MAC for a given rat was calculated as the average of the highest desflurane concentration permitting movement and the lowest concentration preventing movement. Tail clamping was used as the supramaximal stimulation in the determination of desflurane MAC.

Tests of convulsive activity were conducted in individually housed rats. For all studies, rectal temperature was measured and maintained at 36 to 39[degree sign]C by external heating or cooling. Each compound was introduced to provide an initial concentration at which no rat convulsed. The initial concentration was maintained for at least 30 min, after which the concentration was increased by steps of 20%-30% of the preceding concentration. Tonic-clonic seizures were identified by the sudden onset of arching of the back and neck, opening of the mouth and baring of the teeth, tight closure of the eyelids, piloerection, and repetitive rhythmic movement of the forelimbs. It was not difficult to distinguish convulsive activity from other muscle activity with either 2N or flurothyl. It was more difficult with CF3 CFCIOCHF (2) because the convulsive activity was less violent and often occurred only once in a given animal. We defined the ED50 for convulsions in an individual animal as the mean of the partial pressures in the chamber immediately below that causing and the succeeding higher concentration that caused convulsions.

The organic compounds were analyzed using a Gow Mac gas chromatograph (Gow-Mac Instrument Corporation, Bridgewater, NJ) equipped with a 15-foot long SF column at 100[degree sign]C. A nitrogen carrier stream of 10 mL/min was directed through the column to the detector. A flame ionization detector at 200[degree sign]C was supplied with hydrogen at 40 mL/min and with air at 0.3 mL/min. Samples were injected into a 0.2-mL gas sample loop. The chromatograph was calibrated with primary standards produced by injection of a liquid aliquot of the compound into a flask of known volume or by secondary (cylinder) standards that had been calibrated with primary standards.

Nitrous oxide was analyzed by oxygen difference with an oxygen analyzer (Model E2; Beckman Instruments, Fullerton, CA; calibrated with 100% oxygen and 100% nitrous oxide). The chamber always was flushed with enough oxygen to ensure >98% elimination of nitrogen from the system. Knowledge of the oxygen concentration and the pressure within the system (measured by using a Bourdon gage) and the concentration of any additional compound permitted the calculation of the nitrous oxide partial pressure.

Differences within strains were tested for significance using analysis of variance with Student-Newman-Kuels multiple comparison tests, or using unpaired t-tests with Bonferroni's adjustment for multiple comparisons. We accepted P < 0.05 as significant for the former and P < 0.005 for the latter.

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Small but significant differences were found for anesthetic potency (Table 1). The highest versus lowest MAC values for desflurane and nitrous oxide differed by only 28%. Despite these small differences, significance was found because of the small variability of the MAC values for a given strain: the coefficient of variation for the 10 measurements made was 9.3% +/- 4.6%. Potency for the two anesthetics correlated directly as a function of strain with r2 = 0.80 (Figure 1). Inbred versus outbred rats did not seem to have different MAC values.

Table 1

Table 1

Figure 1

Figure 1

Small differences were found for the convulsivity of the three test compounds, with the difference between extremes being less than 21% (Table 1) and the coefficient of variation across the 15 measurements equalling 10.9% +/- 6.1%. For flurothyl and CF3 CFCIOCHF2, the differences were not significant. Although the differences were not greater than for flurothyl and CF3 CFCIOCHF2, the extremes did differ significantly for 2N. The convulsive ED50 for 2N correlated inversely with anesthetic potency for desflurane (r2 = 0.90) (Figure 2) and nitrous oxide (r2 = 0.50). For flurothyl and CF3 CFCIOCHF2, the correlations with desflurane and nitrous oxide tended to be less (r2 = 0.32-0.52) and were constantly direct rather than inverse. No consistent correlations were found among the convulsant compounds, and the correlation coefficients were small (r2 = 0.03-0.39). As for MAC, inbred versus outbred rats did not seem to differ in susceptibility to convulsivity of the test compounds.

Figure 2

Figure 2

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Results from the present study suggest that anesthetic and convulsive potencies can vary as a function of the test strain for a given species. Thus, in any comparison of results from different studies, the species used must be considered as a variable. However, the absolute changes found in the present study, despite their sometimes statistical significance (and more statistically significant differences may have been found if we had studied more animals), were small (a maximum of 28% for the desflurane or nitrous oxide MAC data and a maximum of 21% for data for the convulsive ED50 for each of the three test compound). Thus, even considering differences in strain, comparisons across studies may be made, particularly if such comparisons take into account specific strain differences.

Our data for desflurane and nitrous oxide versus 2N are consistent with a hypothesis that predicts a reciprocal, strain-related receptivity to the excitatory and anesthetic potencies of the test compounds. For compounds with anesthetic but no obvious excitatory properties (i.e., nitrous oxide and desflurane) we found directly correlated MAC differences across strains (Figure 1). These differences correlated inversely with the convulsant effect of 2N (Figure 2). However, for compounds that were convulsant without any anesthetic effect (i.e., 2N, flurothyl), differences in the convulsant ED50 values across strains correlated poorly. Similarly, for the single transitional compound studied (CF3 CFCIOCHF2, a compound with both anesthetic and convulsant properties), we found no differences across strains.

As indicated above, there seems to be a strain-related reciprocal relationship of MAC and the convulsive potencies of 2N (Figure 2). This larger anesthetic requirement and lower ED50 for convulsions associated with a given animal strain may result from a generalized increase in central nervous system excitability and/or from specific structural differences immediately related to the mechanism of anesthetic action. An animal's response to a specific inhaled compound may depend, at least in part, on a balance between the depressant and the excitatory effects of that compound. The balance that seems to exist between anesthetics and 2N suggests the possibility of common sites of action. If the anesthetic properties of desflurane and nitrous oxide and the convulsant effects of 2N have a common site of action, it may be possible to determine the structure of that site that is crucial to the effect of the compounds by an examination of the differences in structure among different strains. One might correlate the changes in convulsant and anesthetic potencies with differences in structure. However, such an examination is not likely to easily produce convincing results because the differences in MAC and convulsive potency among strains are so small. Furthermore, given the poor correlation found for strain versus convulsant potency, the generalizability of any correlation found may be limited.

It seems that the sites acted on by inhaled compounds to produce anesthesia and convulsions are conserved across inbred and outbred rat strains.

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