䡵 LABORATORY REPORT
Anesthesiology 2004; 101:796 – 8
© 2004 American Society of Anesthesiologists, Inc. Lippincott Williams & Wilkins, Inc.
Reversal of Minimum Alveolar Concentrations of Volatile
Anesthetics by Chromosomal Substitution
Thomas A. Stekiel, M.D.,* Stephen J. Contney, M.S.,† Zeljko J. Bosnjak, Ph.D.,‡ John P. Kampine, M.D., Ph.D.,§
Richard J. Roman, Ph.D,㛳 William J. Stekiel, Ph.D.㛳
GENETIC susceptibility to pharmacological agents,
termed “pharmacogenomics,” is an emerging field focused on genetic differences underlying alterations of
physiologic responses to pharmacological agents.1 The
objective of this study was to systematically compare
differences in the minimum alveolar concentration
(MAC) (defined as a movement response to a noxious
sensory stimulus by volatile anesthetics)2 in two genotypically distinct parental strains of rats (Dahl Salt Sensitive [SS] and control Brown Norway [BN]) and in two
consomic (chromosomal transfer) strains (SS.BN.13 and
SS.BN.16). The latter were two strains available from a
larger panel of consomics in which single chromosomes
from the BN strain have been introgressed into an otherwise unchanged SS genetic background.3 The underlying hypothesis of this study is that SS animals have a
greater overall sensitivity to the action of volatile anesthetics that is attributable to specific pharmacogenomic
mechanisms related to one or several chromosomes.
rane with an inspired oxygen concentration of 30% (in
an O2-N2 mixture). Isoflurane (Abbott Laboratories,
North Chicago, IL) was delivered via an Ohio Medical
Products vaporizer (Airco Inc., Madison, WI). Halothane
(Halocarbon Laboratories, River Edge, NJ) and sevoflurane (Abbott Laboratories) were delivered using a Drager
Vapor Model halothane vaporizer and a Drager Vapor
Model 19.1 sevoflurane vaporizer, respectively (Drägerwork Company, Lübeck, Germany). Surgical preparation
consisted of a tracheotomy and placement of femoral
arterial and venous cannulae.4 After preparation, endtidal volatile anesthetic concentration was decreased to
a level just sufficient to eliminate spontaneous movement. The MAC (defined as the level necessary to prevent movement response to a noxious stimulus)2 was
determined according to this level for isoflurane, halothane, or sevoflurane. “Standardized” stimuli were produced by a 15 cm rubbershod hemostat clamped to its
first ratchet point on the middle third of the tail for a
period of 1 min in a fashion previously described.5 Each
MAC value was calculated as the mean between end-tidal
volatile anesthetic concentration before and after elimination of movement in response to the standardized tail
clamp stimulus. Successive tail clamp stimuli were
placed more proximal to each previous site to avoid a
loss of response from potential local injury or accommodation from the previous clamp. MAC values were determined in separate animals under conditions of either
spontaneous or controlled ventilation using a Model 680
rodent respirator (Harvard Apparatus Co., South Natick,
MA). For the latter, end-tidal CO2 (measured with a
POET 2 infrared pantograph and an end-tidal agent monitor, Criticare Systems, Inc. Waukesha, WI) was maintained between 35 and 40 mm Hg. Initial results indicated that the MAC was not a function of the method of
ventilation. Therefore, data obtained during these two
modes of ventilation were pooled for each animal type.
To address the possibility of a difference in baseline
pain tolerance among the four strains studied, “awake”
tail flick responses6 were measured in each strain (n ⫽ 8,
10, 13, and 17 for SS, BN, SS.BN.16, and SS.BN.13,
respectively) using a Model TF6 tail flick device (EMDIE
Instrument Co., Maidens VA).
For each anesthetic the mean ⫾ SD MAC was calculated from measurements in individual animal preparations (n ⫽ 20 –22 for each strain). The significance of
differences between these means for the four strains
were determined with an analysis of variance using the
Materials and Methods
The parental and consomic rat strains used in this
study were developed and maintained under the direction of the Human and Molecular Genetics Center at
Medical College of Wisconsin (PhysGen program). Animals used in this study were 9 –12 week old males
(250 –350 g) maintained on a low (0.4% NaCl) rat chow.
Before initiation of experiments, all techniques and protocols were reviewed and approved by the Animal Care
and Use Committee at the Medical College of Wisconsin.
General anesthesia was induced and maintained by
inhalation of 2% halothane, 2% isoflurane, or 3% sevoflu㛳 Professor, Department of Physiology, The Medical College of Wisconsin,
Milwaukee, Wisconsin. * Associate Professor of Anesthesiology, Medical College of Wisconsin and The Zablocki Veterans Affairs Medical Center, Milwaukee,
Wisconsin. † Research Scientist, the Medical College of Wisconsin, Milwaukee,
Wisconsin. ‡ Professor of Anesthesiology and Physiology, the Medical College
of Wisconsin, Milwaukee, Wisconsin. § Professor and Chairman, Department of
Anesthesiology, Professor, Department of Physiology, The Medical College of Wisconsin and The Zablocki Veterans Affairs Medical Center, Milwaukee, Wisconsin.
Received from the Departments of Anesthesiology and Physiology, the Medical
College of Wisconsin and The Zablocki Veterans Affairs Medical Center, Milwaukee, Wisconsin. Submitted for publication November 26, 2002. Accepted for
publication May 5, 2004. Supported by Grant No. HL56290 from the National
Institutes of Health, Bethesda, Maryland. Presented in part at the Annual Meeting
of the American Society of Anesthesiologists in New Orleans, Louisiana, October
16, 2001 and in Orlando, Florida, October 15, 2002.
Address correspondence to Dr. Stekiel: Anesthesia Research, M4280, Medical
College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, Wisconsin
53226. Address electronic mail to: [email protected]. Individual article reprints
may be purchased through the Journal Web site, www.anesthesiology.org.
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Fig. 1. Strain differences in MAC levels for isoflurane. Significantly smaller mean (ⴞ SD) end-tidal isoflurane concentrations
were required to eliminate movement response to tail clamp in
Dahl Salt Sensitive rats (SS) and SS.BN.16 consomics compared
with Brown Norway (BN) and SS.BN.13 animals. No differences
exist between SS versus SS.BN.16 or between BN versus
SS.BN.13. *P < 0.05 versus BN and SS.BN.13, pooled (spontaneous and controlled ventilation); n ⴝ 20 –22 for each group
Bonferroni-Dunn post hoc test in the Stat-View program
for Macintosh computers (SAS Institute, Cary, NC). A
similar analysis was made for baseline tail flick values. All
differences were considered significant at P ⱕ 0.05.
Results
Figures 1–3 illustrate that the respective MAC values
for isoflurane, halothane, and sevoflurane were significantly less in SS and SS.BN.16 compared with BN and
SS.BN.13. No difference was observed in the baseline
(awake) tail flick mean latency times (2.3 ⫾ 0.2, 2.2 ⫾
0.1, 2.5 ⫾ 0.3, and 2.2 ⫾ 0.7 s for BN, SS, SS.BN.13, and
SS.BN.16 respectively).
Discussion
The observed MAC differences verify that the effect of
volatile anesthetics on movement sensitivity elicited by a
Fig. 2. Strain differences in MAC levels for sevoflurane. Significantly smaller mean (ⴞ SD) end-tidal sevoflurane were required
to eliminate movement response to tail clamp in Dahl Salt
Sensitive rats (SS) and SS.BN.16 consomics compared with
Brown Norway (BN) and SS.BN.13 animals. There were no differences between SS versus SS.BN.16 or between BN versus
SS.BN.13. *P < 0.05 versus BN and SS.BN.13, pooled (spontaneous and controlled ventilation;) n ⴝ 20 –22 for each group.
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Fig. 3. Strain differences in MAC levels for halothane. Significantly smaller mean (ⴞ SD) end-tidal halothane were required
to eliminate movement response to tail clamp in Dahl Salt
Sensitive rats (SS) and SS.BN.16 consomics compared with
Brown Norway (BN) and SS.BN.13 animals. There were no differences between SS versus SS.BN.16 or between BN versus
SS.BN.13. *P < 0.05 versus BN and SS.BN.13, pooled (spontaneous and controlled ventilation); n ⴝ 20 –22 for each group.
noxious stimulus is greater in SS compared with BN
animals. The observation that this effect is reversed by
introgression of BN chromosome 13 (but not 16) into an
otherwise unchanged SS genetic background strongly
suggests that factors associated with chromosome 13 are
involved in producing this difference. The lack of a
difference in “awake” tail flick responses further suggests that the greater effect of volatile anesthetics on
movement sensitivity in SS and SS.BN.16 animals is not
merely the result of a higher baseline pain threshold in
the BN or the SS.BN.13.
The first evidence of a genetic basis for altered physiologic responses to volatile anesthetics was the identification of an ether-resistant strain of Drosophila.7 Subsequently, Dietrich et al. observed differential sensitivity to
volatile anesthetics in rats selectively bred for sensitivity
or resistance to ethanol8 and more recently Sonner et al.
described naturally occurring significant differences in
MAC values of halothane, isoflurane, and desflurane between different strains of mice.9 These results are very
similar to the differences in MAC that have been observed in the present study between parental (SS and
BN) and between consomic (SS.BN.13 and SS.BN.16)
strains of rats.
Two related questions arise from these similar results.
First, are differences in anesthetic sensitivity among
these strains really the result of genetic differences in
homeostatic control mechanisms that are affected by
anesthetics? Second, which genes are involved in the
action of anesthetic agents on these physiologic mechanisms? Regarding the first question, in previous studies
using the SS rat strain over the past decade, we (and our
colleagues) have anecdotally observed that the dose required for surgical anesthesia in the SS animals (without
overanesthetization) is substantially lower than that required for other strains (e.g., Sprague-Dawley and BN).
The observed elevated analgesic effect of anesthetics in
normotensive SS animals maintained on a low salt diet
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798
strongly suggest that this increased sensitivity is not
related to salt-induced hypertension but rather to a heritable difference in the effects of anesthetics on central
and/or peripheral neural regulation of pain and movement in SS versus BN animals.
With regard to the question of specific genes, the
present study represents only an initial step in this approach to clarify the genetic basis for mechanisms of
anesthetic action. The elimination of tail clamp response
does not constitute the definition of anesthesia or even
the “correct” definition of MAC. The concept of anesthesia can have multiple meanings based on the phenotype (or physiologic mechanism) of interest. Thus, MAC
is (at best) an imprecise term dependent upon the chosen end point and the test protocol employed.10 What is
important to note in the present study is that significant
differences exist for anesthetic modulation of movement
responses to quantifiable, standardized, and reproducible pain and/or pressure stimuli in inbred parental
strains of rats and as a function of a specific chromosomal substitution between them.
Currently, the consomic strains developed by our Animal Resource Facility under the direction of the Human
and Molecular Genetics Center at Medical College of
Wisconsin are considered 100% complete. However, the
development of the consomic panel has been ongoing
and at the time the present studies were conducted, the
SS.BN.13 and SS.BN.16 strains were almost but not completely (approximately 95%) consomic, meaning that
they could have contained some BN genome markers
from segments other than the chromosomal substitution
of interest (i.e., 13 or 16). Thus, it is possible that mechanisms related to genetic factors other than those residing on chromosome 13 could be involved in the production of the MAC differences we observed in this study.
Regardless, it is reasonable to assume that the complex
mechanisms underlying both neural and cardiovascular
responses to anesthetics are almost certainly multifactorial and that genetic factors modulating their control do
exist on other chromosomes. Thus, further clarification
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of the physiologic effects of anesthetics will require
further studies using other consomic strains. Even
though any particular consomic strain enables study of
the effect of nothing more specific than a single chromosomal substitution, the entire consomic panel (as a
group), greatly facilitates the study of multifactorial traits
by studying other consomics to examine the impact of
more than one locus.
Thus, the major significance of these results is not the
identification of a particular MAC in SS or BN but rather
the demonstration of a significant and reproducible difference in anesthetic sensitivity between these two
strains that can be changed by a single chromosomal
substitution. This knowledge provides a basis for selectively studying genetic factors associated with mechanisms of anesthetic sensitivity related to this chromosomal substitution (and others as they are identified).
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