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THE minimum alveolar concentration (MAC) has been extensively used to study and compare the effects of volatile anesthetics. The concept of MAC has also become widely accepted in clinical practice. Age has an important effect on the MAC of inhalational anesthetics, particularly in the pediatric range. As age decreases, MAC increases, reaching a maximum value in infants 1–6 months of age, and decreases thereafter with decreasing age. Although the neonatal rat has become widely used as an experimental laboratory animal, especially for cardiovascular and respiratory physiology and pharmacology research, there is no precise information available on the relative potency and MAC values in rats during postnatal maturation of the main volatile anesthetics that are used in pediatric practice (halothane, isoflurane, and sevoflurane). To validly compare the effects of these anesthetic agents at equipotent anesthetic concentrations in experimental studies during postnatal maturation, one needs to know the MAC values of these anesthetics at different steps during postnatal maturation. Therefore, this study was undertaken to determine the MAC values of halothane, isoflurane, and sevoflurane during postnatal maturation in the rat.
Minimum alveolar concentration was determined using the tail-clamp technique as previously described. Animals were tested at the same time of day (2:00–8:00 pm), to minimize variations in anesthetic requirements induced by circadian rhythm. MAC determination was performed in one brood of rat (12–15 rat pups by brood) or in adult rats (15–20 rats by group) in each experiment. A minimal interval of 4 days was required between two experiments involving the same animals.
Each group of rat was studied with only one volatile anesthetic on any given day of their postnatal maturation ( e.g., the same neonatal rats were used to determine MAC values for sevoflurane on days 2, 9, and 30 of their postnatal maturation). Spontaneously breathing rats were exposed to halothane, isoflurane, or sevoflurane in individual chambers (20 × 10 × 12 cm) closed by a thin plastic sheet (Polyethylen film, size 712M29; Manutan, Paris, France). The volatile agent was vaporized with a calibrated vaporizer (Model Fluotec 4, Isotec 3, or Sevotec 5; Ohmeda, Steeton, United Kingdom) in 100% O 2as the carrier gas, with fresh gas flow of 12 l/min. Concentrations of the volatile anesthetic in each chamber were measured with an infrared calibrated analyzer (Artema Model MM 206SD; Taema, Antony, France).
The infrared analyzer was calibrated daily according to the manufacturer guidelines using anesthetic mixtures of known concentration. The anesthetic mixture (volatile anesthetic in oxygen) was rewarmed to 30.0°C before entering the chamber, and temperature of the chamber was continuously monitored. Body temperature of one adult rat in each experiment was continuously monitored with a rectal probe (Harvard Apparatus, Inc., South Natick, MA). When the rectal temperature of this monitored rat dropped by 1.0°C, the chamber was rewarmed using a heating lamp and a warming blanket until its temperature was restored to 37.0°C.
The rectal temperature of newborn rats in the nest varies between 32 and 39°C, depending on environmental temperature and the presence of the dam. Because changes in body temperature influence MAC values in a linear manner, we have also measured the rectal temperature (Compact JKT thermometer; Fisher Bio-block Scientific, Tanneries, France) of 8–10 rats of each age group (days 2, 9, and 30 and adult) at baseline (before putting the rats in the individual chambers) and after a 2-h exposure to halothane at an inspired concentration closed to the MAC values (1.0–1.4%) of the age group under study, in otherwise the same experimental conditions as those used to determine MAC values. The values of rectal temperature measured in these conditions were used to correct the MAC values at 37°C, if needed. We have applied a correction of ± 5% to the MAC value for each increase or decrease of ± 1°C in body temperature, as previously reported.
A hemostatic clamp (De Bakey clamp; Harvard Apparatus, Inc.) was applied for 45 s to the first ratchet position on the mid portion of the tail without wiggling the clamp. The hemostatic clamp was applied through a small hole so as not to modify the anesthetic concentration in the experiment chamber. An animal was considered to have moved if it made a “gross purposeful muscular movement,” usually of the hind limb or the head, or both. The anesthetic concentration was increased in steps of 0.1% (halothane and isoflurane) to 0.2% (sevoflurane), and the testing sequence was repeated after 30 min of each concentration exposure, meaning that steady-state FI/FA ratios close to 1.0 could be reasonably achieved.
No experiment required exposure to more than seven consecutive increased anesthetic concentrations; therefore, the total anesthetic exposure was kept to less than 8 h, although MAC determination is not affected by the duration of anesthetic exposure. At the end of the procedure, anesthetic administration was stopped, and rats awoke while breathing 100% O 2. During MAC determination no rats exhibited respiratory distress, and all animals recovered without obvious untoward effect. The original MAC concept of Eger et al.
Used a “bracketing approach” in humans and animals. In animal studies it is possible to apply the tail clamp stimuli on multiple occasions. Thus, an appropriate mathematical technique to quantify the relationship between MAC and response versus no response data is the logistic regression analysis. Such analyses show the probability of a binary outcome ( i.e., yes or no response) as a linear function of the exponential part of logit of the logistic function.
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This model may be applied to MAC determination. This produces values for MAC comparable to those produced with the bracketing technique and enables an extrapolation of the probability of response to any given anesthetic concentration within the curve. For each age group and for each volatile anesthetic, median MAC values were calculated using logistic regression (NCSS 6.0 software; Statistical Solutions, Cork, Ireland), and the 95% confidence interval limits were calculated. All P values were two-tailed, and a P value less than 0.05 was considered significant.
The inspired median MAC values (95% confidence intervals) of volatile anesthetics in rats during postnatal maturation are presented in. Baseline rectal temperatures, measured at an ambient temperature between 22 and 25°C, were as follows: 33.1 ± 1.4, 35.0 ± 0.4, 37.7 ± 0.4, and 37.8 ± 0.3°C, respectively in days 2, 9, and 30 and adult rats. Rectal temperatures, measured after a 2-h exposure to halothane at an inspired concentration between 1.0 and 1.4% were as follows: 32.8 ± 1.3, 33.7 ± 0.9, 39 ± 1.0, and 39.5 ± 0.6°C, respectively in days 2, 9, and 30 and adult rats. The inspired median MAC values (95% confidence intervals) corrected at 37°C are also shown in. As age decreased, MAC increased, reaching the greatest value in 9-day-old rats, and decreased thereafter with decreasing age, remaining still above adult MAC values. Thus, inspired MAC values of halothane at 37°C were increased by 75% ( P. Percentage of animals with no movement for halothane ( A ), isoflurane ( B ), and sevoflurane ( C ) in each age group.
The numbers of rats studied were 14 neonates and 16 adults, 15 neonates and 20 adults, and 12 neonates and 15 adults, respectively, for halothane, isoflurane, and sevoflurane. The curves were estimated by logistic regression of probability of no movement fitted for halothane, isoflurane, and sevoflurane concentrations, in each age group. The minimum alveolar concentration and its 95% confidence interval (horizontal line) are shown on each graph. Percentage of animals with no movement for halothane ( A ), isoflurane ( B ), and sevoflurane ( C ) in each age group.
The numbers of rats studied were 14 neonates and 16 adults, 15 neonates and 20 adults, and 12 neonates and 15 adults, respectively, for halothane, isoflurane, and sevoflurane. The curves were estimated by logistic regression of probability of no movement fitted for halothane, isoflurane, and sevoflurane concentrations, in each age group. The minimum alveolar concentration and its 95% confidence interval (horizontal line) are shown on each graph.
Neonatal rats have become widely used as experimental laboratory animals, especially for cardiovascular and respiratory physiology and pharmacology research. However, the MAC values of volatile anesthetics in rats during postnatal maturation are poorly understood or are not known. Recently, Prakash et al., comparing the effects of volatile anesthetics on actin–myosin cross-bridge cycling in neonatal versus adult cardiac muscle, used adult rat MAC values in the neonates, because of the lack of MAC values for neonatal rats. However, because MAC values are increased in neonatal rats, the magnitude of the effects of volatile anesthetics on actin–myosin cross-bridge cycling could have been underestimated in neonatal rats. To determine MAC values in rats, we used the tail-clamp technique initially described in the dog by Eger et al. And then applied to rodents.
Because various test stimuli have been used for rodents in previous studies, we decided to use the same stimuli as Mazze et al. In mice and rats, and the 6-inch hemostat clamp was applied for 45 s across the mid portion of the rat tail. Quasha et al. Have shown that MAC determination is more precise when using 10% rather than 20% step change in anesthetic concentration.
Therefore, in the current study, we increased the anesthetic concentration by 10% steps. The total anesthetic exposure time was kept to 8 h or less, although MAC determination is not affected by the duration of anesthetic exposure. Have suggested that, when only inspired anesthetic concentrations are measured, it is preferable to go from high to low concentrations because this technique results in lower inspired-alveolar concentration difference, but they averaged the results of increased and decreased concentrations. In our study, we used only increased anesthetic concentrations. However, the 30-min exposure time to each anesthetic concentration meant that steady-state FI/FA ratios close to 1.0 could be reasonably achieved. Our MAC values in adult rats were consistent with MAC values previously determined in rats.
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The slight differences observed in MAC values in adult rats between our study and some other studies may be related to the variation usually observed for different determinations within the same animals (less than 10%) and to difference in rat strain.2,20 In the study by Gong D et al., assessing the effect of rat strain on MAC, adult Sprague-Dawley rats had MAC values for desflurane that were 18% higher than in adult Wistar rats. Moreover, our MAC ratio values in adult rats (MAC ratios of halothane to isoflurane, 0.78; sevoflurane to isoflurane, 1.75; and sevoflurane to halothane, 2.25) were also in agreement with those reported in humans, rats, and other rodents. The percentage of increase in MAC values with increasing age observed in the current study is in the range of those previously reported for desflurane in rats and for sevoflurane in children. We observed that the increase in MAC values with decreasing age reached the greatest value in 9-day-old rats and decreased thereafter in 2-day-old rats. These results are in agreement with the hypothesis of Gregory, who speculated that MAC in preterm neonates may be significantly less than in full-term neonates and older infants, and with the results of the study by LeDez and Lerman, showing that the MAC of isoflurane in preterm neonates of less than 32 weeks’ gestation was significantly less than in preterm neonates of 32–37 weeks’ gestation. Taking into consideration the demonstration of similarities between rat and human somatosensory development, as well as a good correspondence between infant rats behavioral measurements and analogous behavioral measurements in human infants, 2-day-old rats may be considered as preterm human neonates (approximately 24-week-old premature humans), 9-day-old rats as full-term neonates, and 30-day-old rats as human teenagers.
It has been suggested that there is a fairly consistent effect of aging on anesthetic requirement for conventional inhaled anesthetics. A meta-analysis of studies from different institutions on 12 clinical inhalation anesthetics found no significant difference in the slope of the regression of the log 10of MAC on age in humans among the drugs, for age greater than 1 yr. These data are consistent with hypothesis that the age-dependence of MAC for clinical inhaled anesthetics has a common basis. On the other hand, the factors responsible for the increase in MAC from preterm neonates to full-term infants remain speculative. Progesterone, endorphins, and structural changes in the central nervous system have all been implicated to explain these changes in MAC, but all remain unproven. Other authors have found that the generalized decrease in anesthetic requirement with age paralleled several physiologic variables that also decreased with age, including cerebral blood flow, cerebral oxygen consumption, and neuronal density.
Other authors, observing that the concentration of halothane in the brain at anesthesia was lower in 15-day-old rats than in 30- or 60-day-old rats, have suggested that this difference in brain concentration was, most likely, attributable to the difference in water content in the younger rats. Indeed, a higher partial pressure of anesthetics ( i.e., MAC) may be necessary in the younger animals to compensate for the high water content of the developing brain. In addition, MAC depends on a spinally mediated reflex withdrawal in response to a noxious stimulus. If general anesthesia, as defined by MAC, is attributable to anesthetic actions on a limited number of receptors and ion channels, age-dependence per se implies that the representation of the anesthetically critical ion channels is different in adult and neonatal spinal cord.
Glutamate and γ-aminobutyric acid A (GABAA) receptors have been proposed as probable target sites for inhaled anesthetics actions, and functional evidence suggests the importance of glutamate and GABAA receptors to spinal cords function. Ontogenetically, GABA receptor subtypes and functional properties, as well as concentration of N -methyl-d-aspartate receptors change from embryo to adult. Therefore, changes in receptor and ion channel subtypes with postnatal maturation can provide suggestive evidence for possible bases for age-dependent changes in MAC values. As in most previous determinations of MAC in rodents, inspired rather than alveolar anesthetic concentrations were measured; thus, in theory, a correction factor should be applied.
However, we did not use correction factors to calculate the exact MAC values. Indeed, these correction factors are unknown for sevoflurane, as well as for neonatal rats. Moreover, these correction factors might have been different in rat pups because they are only 3–20% of the weight of the adult rat, according to the postnatal age. Because the equilibration time was long, we assumed that FI/FA ratios were close to 1.0. Percentage of animals with no movement for halothane ( A ), isoflurane ( B ), and sevoflurane ( C ) in each age group. The numbers of rats studied were 14 neonates and 16 adults, 15 neonates and 20 adults, and 12 neonates and 15 adults, respectively, for halothane, isoflurane, and sevoflurane.
The curves were estimated by logistic regression of probability of no movement fitted for halothane, isoflurane, and sevoflurane concentrations, in each age group. The minimum alveolar concentration and its 95% confidence interval (horizontal line) are shown on each graph. Percentage of animals with no movement for halothane ( A ), isoflurane ( B ), and sevoflurane ( C ) in each age group. The numbers of rats studied were 14 neonates and 16 adults, 15 neonates and 20 adults, and 12 neonates and 15 adults, respectively, for halothane, isoflurane, and sevoflurane. The curves were estimated by logistic regression of probability of no movement fitted for halothane, isoflurane, and sevoflurane concentrations, in each age group. The minimum alveolar concentration and its 95% confidence interval (horizontal line) are shown on each graph.