GENETIC susceptibility to pharmacological agents, termed “pharmacogenomics,” is an emerging field focused on genetic differences underlying alterations of physiologic responses to pharmacological agents.1The 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)2in 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.3The 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.
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% sevoflurane with an inspired oxygen concentration of 30% (in an O2-N2mixture). 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.4After preparation, end-tidal 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)2was 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.5Each 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 responses6were 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 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 ≤ 0.05.
Figures 1–3illustrate 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).
The observed MAC differences verify that the effect of volatile anesthetics on movement sensitivity elicited by a 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 .7Subsequently, Dietrich et al. observed differential sensitivity to volatile anesthetics in rats selectively bred for sensitivity or resistance to ethanol8and more recently Sonner et al. described naturally occurring significant differences in MAC values of halothane, isoflurane, and desflurane between different strains of mice.9These 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 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.10What 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 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).