Veterinary Anaesthesia and Analgesia, 2016, 43,

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1 Veterinary Anaesthesia and Analgesia, 2016, 43, doi: /vaa RESEARCH PAPER Isoflurane MAC determination in dogs using three intensities of constant-current electrical stimulation Marivaldo R Figueiro*, Joao HN Soares*,, Fabio O Ascoli*, Stephen Werre & Ignacio A Gomez de Segura *Veterinary Surgery and Clinics Graduation Program, Veterinary College, Fluminense Federal University, Niteroi, Brazil Department of Small Animal Clinical Sciences, Virginia Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA Laboratory for Study Design and Statistical Analysis, Virginia Maryland Regional College of Veterinary Medicine, Polytechnic Institute and State University, Blacksburg, VA, USA Department of Animal Medicine and Surgery, Faculty of Veterinary Medicine, University Complutense, Madrid, Spain Correspondence: Joao HN Soares, Department of Small Animal Clinical Sciences, Virginia Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA. jhsoares@vt.edu Abstract Objectives To compare isoflurane minimum alveolar concentrations (MACs) in dogs determined using three intensities of constant-current electrical stimulation applied at the tail, and thoracic and pelvic limbs, and to compare isoflurane MACs obtained with all combinations of electrical stimulation and anatomic site with those obtained using the tail clamp as the noxious stimulus. Study design Randomized trial. Animals Six mixed-breed, adult female dogs aged 1 2 years and weighing kg. Methods In each dog, MAC was determined by the bracketing method with the tail clamp (MAC TAILCLAMP ), and three electrical currents (10 ma, 30 ma, 50 ma) at three anatomic sites (thoracic limb, pelvic limb, tail). Each MAC achieved with electrical stimulation was compared with MAC TAILCLAMP using a mixed-model ANOVA and Dunnett s procedure for multiple comparisons. The effects of current intensity and anatomic site on isoflurane MAC were tested using a mixed-model ANOVA followed by Tukey s test for multiple comparisons (p < 0.05). Results Mean MAC TAILCLAMP was 1.69%. MACs achieved with currents of 30 ma and 50 ma did not differ independently of anatomic site. When currents of 10 ma were applied to the tail and thoracic limb, resulting MACs were lower than those obtained using currents of 30 ma and 50 ma. Currents of 30 ma and 50 ma provided MACs that did not differ from those of MAC TAILCLAMP, whereas a current of 10 ma achieved the same result only for the pelvic limb. Conclusions and clinical relevance Isoflurane MAC is affected by current intensity and anatomic site. Current intensities of 30 ma and 50 ma provided consistent results when applied to the tail, and thoracic and pelvic limbs that did not differ from those obtained using the tail clamp. Consequently, they can be used in place of the tail clamp in MAC studies in dogs. Keywords constant-current, dogs, isoflurane, minimum alveolar concentration, noxious stimulation. Introduction The minimum alveolar concentration (MAC) has been used as an index of inhalant anesthetic agent potency since 1963 (Merkel & Eger 1963), and is the 464

2 most widely employed index of the depth of anesthesia when such agents are administered. MAC has also facilitated investigations of the physiological effects of inhalation agents at equipotent concentrations and has helped to elucidate the mechanism of action of inhalant anesthetics (Saidman & Eger 1964; Quasha et al. 1980; Hornbein et al. 1982; Antognini & Schwartz 1993; Rampil et al. 1993; Antognini et al. 1994; Gomez de Segura et al. 1998; Jinks et al. 2002; Gerstin et al. 2003). In addition to tail clamping, electrical stimulation represents a noxious stimulus that is frequently used for MAC determination (Valverde et al. 1989, 2003; Antognini et al. 1994; Soares et al. 2004; Seddighi et al. 2011). Electrical stimulation has some advantages over tail clamping in that it facilitates easier quantification, standardization and reproducibility (Le Bars et al. 2001). Electrical stimulation may be an option when the tail clamping stimulus cannot be employed, such as when the animal is in a hyperbaric chamber (Antognini et al. 1994) or during investigations of segmental MAC-sparing effects (Valverde et al. 1989). Different modalities of noxious stimulation have been identified as sources of variability in the determination of MAC in humans (Zbinden et al. 1994), dogs (Eger et al. 1965; Valverde et al. 2003) and cats (Ide et al. 1998). Electrical stimulation for the determination of isoflurane MAC in dogs resulted in higher values ( %) (Hellyer et al. 2001; Soares et al. 2004) than when tail clamping was used ( %) (Steffey & Howland 1977; Valverde et al. 2003; Pypendop et al. 2007). The level of electrical stimulation can be an additional source of variation because lower levels can provide lower MAC values as demonstrated in dogs (Eger et al. 1965). The most common type of electrical stimulation used in MAC studies is a constant-voltage stimulus, which may produce variations in stimulation intensity depending on the electrical resistance between the electrodes, as demonstrated in ponies (Levionnois et al. 2009). In contrast, constant-current stimulation should provide a more stable intensity of stimulation, independent of inter-electrode resistance, and thus facilitate a more repeatable pattern of stimulation (Levionnois et al. 2009). Different anatomic sites have been used for electrical stimulation in determinations of MAC in animals. These include the pelvic limb (Valverde et al. 1989, 2003; Soares et al. 2004), thoracic limb (Valverde et al. 1989; Seddighi et al. 2011), tail (Laster et al. 1993; Antognini et al. 1994; Valverde et al. 2003) and oral mucosa (Valverde et al. 2003; Levionnois et al. 2009). Isoflurane and halothane MAC in the dog did not differ when 50 V was applied to the pelvic or thoracic limbs, or the oral mucosa (Valverde et al. 1989, 2003). The same results for MAC were measured in ponies using 90 V applied to the gingiva or pelvic limb (Levionnois et al. 2009). However, different intensities of electrical stimulation at different anatomic locations have not been reported in determinations of MAC in the dog and deserve further investigation. The aims of the present study were: 1) to investigate the influence of anatomic site (tail, thoracic limb and pelvic limb) and electrical current intensity (10, 30 and 50 ma) of a constant-current electrical stimulation on the isoflurane MAC; and 2) to compare MAC values derived from all combinations of current intensity and anatomic site with MAC values established using the tail clamp (MAC TAILCLAMP ). The hypothesis of this study was that the isoflurane MAC values determined at different anatomic sites and with different intensities of current would differ. Materials and methods Animals The study was approved by the Institutional Animal Care and Use Committee of Fluminense Federal University. Six adult, female, mixed-breed dogs aged 1 2 years and weighing a mean standard deviation (SD) of kg were used in this study. Physical examinations, complete blood counts and biochemical and electrolyte analyses were performed before the experiments to assess the health status of each dog. Anesthesia and monitoring General anesthesia was induced with isoflurane (Isoforine; Cristalia Produtos Quımico e Farmac^euticos Ltda, Brazil) administered using a face mask, After orotracheal intubation, the endotracheal tube was connected to a circle system. Anesthesia was maintained during instrumentation with the isoflurane vaporizer set at 2%. The fresh gas flow was maintained at 2 L minute 1 during the whole experiment. A 20 gauge catheter (Safelet; Nipro Medical Ltda, Brazil) was inserted in a cephalic vein and 5 ml kg 1 hour 1 of lactated Ringer s solution 2016 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesia and Analgesia, 43,

3 was infused during the experiments. The dorsal pedal artery was catheterized using a 22 gauge catheter (Safelet; Nipro Medical Ltda) for blood pressure measurement (DX-10; Dixtal Biomedica Ind. & Com. Ltda, Brazil) and blood gas analysis. Arterial blood samples were anerobically collected every hour during the experiments and blood gases were measured immediately (ABL5; Radiometer Medical ApS, Denmark). Arterial pressure was measured using low-compliance plastic tubing filled with heparinized saline connected to a pressure transducer (DX-10; Dixtal Biomedica Ind. & Com. Ltda), which was zeroed and positioned at the level of the heart and its calibration verified with a mercury column (Tycos; Welch Allyn, Inc., NY, USA). A lead II electrocardiogram (ECG) was used to monitor heart rate and rhythm (DX-10; Dixtal Biomedica Ind. & Com. Ltda). Body temperature was measured using an esophageal temperature probe (DX-10; Dixtal Biomedica Ind. & Com. Ltda) and maintained at C by an electric thermal pad (Brasmed Veterinaria, Brazil). A thin catheter was placed within the endotracheal tube so that its distal opening was at the end of the tube. End-tidal isoflurane concentration (FE 0 Iso) and carbon dioxide partial pressure (PE 0 CO 2 ) were measured continuously during the experiments by a sidestream monitor (Multigas monitor 9100; BCI International, Inc., WI, USA) using piezoelectric and infrared techniques, respectively. The gas analyzer provided the FE 0 Iso measurement to two decimal places. The monitor was internally calibrated (onepoint calibration) before and hourly during each experiment according to a reference sample (10% CO 2, 50% N 2 O, 1.5% isoflurane, balanced with N 2 ) and a protocol provided by the manufacturer (BCI International, Inc.). The experiments were performed at sea level. The FE 0 Iso used in each step of MAC determination was measured from a 15 ml end-tidal gas sample hand-collected with a glass syringe from an endotracheal tube catheter encompassing at least five expirations. This sample was collected before each stimulus trial, after disconnecting the sidestream sampling port of the monitor, and introduced immediately in the gas analyzer. Isoflurane MAC determination Isoflurane MAC values were determined using the bracketing method and duplicated in each animal for each stimulus. The mechanical noxious stimulus was the tail clamp performed with a Carmalt forceps (17 cm long with 5 cm blade tips covered with plastic tubing, reaching the first ratchet on the forceps but not closing it) applied to the unclipped proximal third of the tail. The tail was moved continuously during the clamping process. The 10 ma, 30 ma and 50 ma electrical stimuli (square wave and 50 Hz) were delivered by a constantcurrent electrical stimulator (NS242; Fisher & Paykel Healthcare Ltd, New Zealand) connected to a pair of stainless steel needles, located 3 cm apart and inserted subcutaneously on the ventral base of the tail, as well as on the cranial aspects of the carpus and tarsus. Each stimulus was applied for a maximum of 60 seconds or stopped as soon as a positive response was elicited. Three observers assessed the response by direct visual observation. Movements of the stimulated anatomic site and swallowing, blinking or increased respiratory effort were not considered positive responses. Any obvious movement of another part of the body (mainly the head or limbs) occurring during the noxious stimulation was considered to indicate a positive response. In the event of any disagreement among the three observers, the response was discarded and the stimulation repeated. The output of the electrical stimulator was verified using a digital multimeter (Fluke 28 II; Fluke Corp., WA, USA). If the output differed by >5% from the targeted current intensity, the response was discarded, the stimulation needles readjusted and the stimulation repeated. Ten isoflurane MACs were determined in each animal on each experiment day: MAC TAILCLAMP ; tail MAC for 10 ma (MAC TAIL10 ); tail MAC for 30 ma (MAC TAIL30 ); tail MAC for 50 ma (MAC TAIL50 ); thoracic limb (TL) MAC for 10 ma (MAC TL10 ); TL MAC for 30 ma (MAC TL30 ); TL MAC for 50 ma (MAC TL50 ); pelvic limb (PL) MAC for 10 ma (MAC PL10 ); PL MAC for 30 ma (MAC PL30 ), and PL MAC for 50 ma (MAC PL50 ). Each MAC was determined as the mean between two pairs of consecutive FE 0 Iso values (rounded to the second decimal place) eliciting a negative and a positive response independently of the order. The duplicated MAC for each combination of current intensity and anatomic site was obtained by duplicating the whole sequence of stimulation, which is described below. The first sequence of noxious stimulation was performed with FE 0 Iso of %. For each sequence of stimulation, the order of stimulation modalities was randomized by picking a piece of paper from an opaque envelope containing all combinations. Within each sequence, successive Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesia and Analgesia, 43,

4 stimulations were separated by intervals of 1 10 minutes. After each completed sequence of stimulation, the FE 0 Iso was modified by approximately 10%, but never by more than 20%, and maintained for 15 minutes. After the first sequence of stimulation, FE 0 Iso was increased or decreased according to the responses observed. If the majority of responses were positive, it was increased. If the majority of responses were negative, it was decreased. In the event of equal numbers of positive and negative responses, the increase or decrease was randomly decided. Subsequently, FE 0 Iso was changed progressively in the same direction so that it was increased or decreased until MAC was obtained for the stimulations that had the majority of responses. The FE 0 Iso was finally modified in order to obtain the pairs of consecutive FE 0 Iso values eliciting, respectively, a negative and a positive response (or vice versa) to determine MAC for the stimulations associated with the minority of responses in the initial sequence. However, responses to stimulation intensities of 30 ma and 50 ma were assumed to be positive and the stimulation modality was not applied when a positive response had been previously recorded at a lower intensity of stimulation for the same FE 0 Iso and the same stimulation site. Therefore, some positive responses used to calculate some of the reported MAC values (at 30 and 50 ma) were assumed and not actually observed. The duration of the determination of all 10 MAC values was recorded as the time from the end of instrumentation until the last response observed. Recovery At the end of the experiment, ketoprofen (2.0 mg kg 1 ; Ketofen; Merial Saude Animal Ltda, Brazil) was intravenously administered and isoflurane delivery was stopped. The dogs were kept under observation for 1 week after the experiments during which they were checked for behavior changes each day. Statistical analysis The primary outcome was isoflurane MAC determined for all 10 patterns of noxious stimulation. Normal probability plots showed that all MAC values followed a normal distribution. Data were then expressed as the mean SD. Subsequently, the effects of current intensity and anatomic site on MAC were tested using mixed-model analyses of variance (ANOVAs) followed by Tukey s procedure for multiple comparisons. The linear model specified current intensity, anatomic site, and the interaction between current intensity and anatomic site as fixed effects. To compare the three current intensities within each of the anatomic sites and to compare anatomic sites within each current intensity, the slicediff option in the SAS procedure PROC GLIMMIX was applied to the interaction between current intensity and anatomic site. To assess average bias from MAC TAILCLAMP, values for MAC TAILCLAMP were compared with those for each combination of current intensity and anatomic site (a total of nine treatments) using mixed-model ANOVAs followed by Dunnett s procedure for multiple comparisons. The linear model specified treatment as a fixed effect. Options common to all ANOVA models included dog identification as a random effect (adjusts for clusters of measurements within each dog) and Kenward Roger approximation as the denominator degrees of freedom. For each model, residual plots were inspected to verify that the errors followed an approximately normal distribution with a constant variance. A Pearson product moment correlation coefficient between MAC TAILCLAMP and the number of clamp stimulations required to achieve it was calculated. The ANOVA models were fitted using SAS Version 9.4 (SAS Institute, Inc., NC, USA). A p-value of <0.05 was used to reject the null hypothesis. Results All dogs had normal serum sodium concentrations before the experiments. No macroscopic lesions were observed on the tails of the dogs at the end of the experiments. All isoflurane MAC values were normally distributed. Mean SD MAC TAILCLAMP was %, which was significantly different from values for MAC TAIL10 and MAC TL10. There was a significant interaction between current intensity and anatomic site of electrical stimulation. MAC values did not differ significantly among current intensities when stimulating at the pelvic limb (Table 1). At the tail and at the thoracic limb, MAC values obtained with stimulation at 10 ma were significantly lower than those obtained with stimulation at 30 and 50 ma. No significant differences in MAC values obtained with 30 and 50 ma, respectively, emerged among anatomic sites. The mean SD duration of anesthesia for the determination of the 10 MAC values was minutes. Tail clamping was applied four 2016 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesia and Analgesia, 43,

5 Table 1 Mean SD isoflurane minimum alveolar concentration in six dogs obtained with three electrical current intensities (10 ma, 30 ma and 50 ma) applied to the thoracic limb, pelvic limb or tail Anatomic site to seven times in order to calculate MAC TAILCLAMP and the correlation between the number of clamping applications and MAC TAILCLAMP was very weak (r = ). During all determinations, all physiological variables measured remained within ranges that are not expected to affect MAC (Quasha et al. 1980). During MAC determination, the averaged mean SD arterial pressure was 66 6 mmhg, arterial ph was , arterial partial pressure of oxygen (PaO 2 ) was mmhg ( kpa), arterial partial pressure of carbon dioxide (PaCO 2 )was mmhg ( kpa) and body temperature was C. Anesthesia recovery was without complications in all dogs and no behavior abnormalities were detected in the week after the experiments. Discussion Current intensity 10 ma 30 ma 50 ma Thoracic Ba Aa Aa limb Pelvic limb Aa Aa Aa Tail Bb Aa Aa Values are expressed as mean SD in percentage of 1 atmosphere. Different upper case letters indicate a significant difference between current intensities for a given anatomic site (p < 0.05). Different lower case letters indicate a significant difference between anatomic sites for a given current intensity (p < 0.05). The present study shows that isoflurane MAC can differ depending on the stimulation applied according to its modality, site or intensity. The main findings of the study were: 1) applying electrical stimulation of 10 ma current at the tail elicited a lower isoflurane MAC compared with stimulation of the thoracic and pelvic limbs, and no difference was observed for higher intensities (30 ma and 50 ma); 2) when electrical stimulation was applied to the tail and thoracic limb, isoflurane MAC was lower with currents of 10 ma than with currents of 30 ma or 50 ma; and 3) the tail clamp elicited MACs similar to those obtained by electrical stimulation of 30 ma and 50 ma at all sites, whereas lower MACs were obtained with stimulations of 10 ma at the tail and the thoracic limb. In view of the importance of MAC and its widespread use as an index of inhalant anesthetic potency, standardization of the noxious stimulus used during its determination is highly desirable in order to facilitate repeatable results and valid comparisons among independent studies. Different types and levels of noxious stimulation have been reported to provide different MAC results (Eger et al. 1965; Zbinden et al. 1994; Ide et al. 1998; Valverde et al. 2003). The present study indicates that constantcurrent electrical stimuli can be applied at different sites and evoke comparable responses if the intensity of the current is 30 ma. The values derived from these stimuli were also similar to those obtained using the tail clamp. This information should be considered when designing a MAC study in which the objective of the investigation includes stimulation in different anatomic sites (Valverde et al. 1989; Antognini et al. 1994). The concept of supramaximal stimulation for MAC studies was developed by Eger et al. (1965), who demonstrated that MAC values are directly proportional to the intensity of stimulation up to a certain level, after which increasing the intensity does not result in further increases in MAC. Thus, the current intensities of 30 ma and 50 ma could be considered supramaximal because they did not produce different MACs for any of the anatomic sites investigated. However, the current study was not designed to verify similarity among MAC values derived from electrical stimulation and hence the lack of significance in the differences between MAC values obtained using currents of 30 ma and 50 ma should not be immediately interpreted as indicating similarity. In fact, it is questionable whether a supramaximal stimulus is necessary to guarantee stimulation repeatability (Sonner et al. 2003; Eger et al. 2008), and lower stimulation intensity may decrease the risk for injury (e.g. burning), as well as stimulus habituation (Laster et al. 1993). No macroscopic skin injury was observed in any dogs during the present study. Electrical stimulations of 15 V in rats and 30 V or 50 V applied to the tail in dogs have been reported to be equivalent to tail clamping and are considered to represent noxious stimuli adequate for MAC studies in, respectively, rats and dogs (Eger et al. 1965; Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesia and Analgesia, 43,

6 Laster et al. 1993; Valverde et al. 2003). In the present study, MAC TAILCLAMP did not differ from MACs induced by stimulation with currents of 30 ma or 50 ma at any of the anatomic sites investigated, which suggests that MAC values achieved with these current intensities can be compared with those induced by the tail clamp method. Moreover, these results suggest that currents of 30 ma or 50 ma could be used in place of the tail clamp in MAC studies. Isoflurane MAC values obtained with the tail clamp and currents of 30 ma and 50 ma applied to all anatomic sites studied were in the upper range of previously reported data in dogs ( %) (Eger et al. 1965; Steffey & Howland 1977; Hellyer et al. 2001; Valverde et al. 2003; Soares et al. 2004; Pypendop et al. 2007). This range of isoflurane MAC values reported in the dog is higher than the expected intraspecies variation in MACs (Quasha et al. 1980) and may be associated with methodological differences. In studies in which only tail clamping was applied, isoflurane MACs varied from 1.27% to 1.44% (Steffey & Howland 1977; Valverde et al. 2003; Pypendop et al. 2007), and were thus lower than the mean MAC TAILCLAMP of 1.69% found in the present study. There are two main hypotheses to account for the higher MAC TAILCLAMP found in the present study: 1) differences in the positive response assessment; and 2) populational variability in MAC. The original definition of a positive response in MAC studies is a gross purposeful movement in response to noxious stimulation, excluding swallowing, blinking or increased respiratory effort (Eger et al. 1965). This definition can add some subjectivity to the interpretation of a positive response and consequently introduce some bias in the identification of a positive response during MAC determination. More recent reports (Sonner et al. 2003; Eger et al. 2008) do not include gross and purposeful movements in their definitions of responses, but only movement in response to noxious stimulation, which was the definition used in the present study. Although this definition may potentially produce higher MAC, the MAC TAILCLAMP achieved in the present study (1.69%) was higher than the isoflurane MAC determined considering any movement (MAC NM 1.42%) as the end point for a positive response (Seddighi et al. 2011), which suggests that other factors played a role in the present higher MAC TAILCLAMP. Regardless, the same definition of positive response was applied to all MAC determinations and was very unlikely to affect the main results and conclusions of the present study. In the present authors opinion, the most likely cause of the higher MAC TAILCLAMP found in this study is a population-based variation in inhalant anesthetic response, which has been previously demonstrated in mice and cats (Koblin et al. 1980; Ilkiw et al. 1997; Escobar et al. 2012). Isoflurane MACs in cats have been reported at % when using the tail clamp as the noxious stimulus, even when determined by the same research groups (Ilkiw et al. 1997; Escobar et al. 2012). Other potential causes of increased MAC TAILCLAMP, such as a true increase in MAC or an overestimation of arterial partial pressure of isoflurane from the FE 0 Iso measurements, were excluded in the present study. Hyperthermia may increase MAC, but did not occur during the study reported here. The presence of peripheral or central sensitization leading to a higher MAC, which has been demonstrated in rabbits (Sobair et al. 1993), was unlikely to have occurred in this study because the correlation between MAC TAILCLAMP and the number of clamp stimulations was very weak in each dog. Finally, overestimation of arterial partial pressure of isoflurane because of significant venous admixture was excluded because the ratio between PaO 2 and its inspired fraction (PaO 2 /FIO 2 ) was always higher than 450 mmhg (60 kpa) during the experiments. Although some authors (Sobair et al. 1993; Antognini & Carstens 1998) have suggested methods to provide a uniform intensity of mechanical noxious stimulation in MAC studies, the majority of studies using the tail clamp provide a poor description of its standardization. Consequently, the relatively high variability found between isoflurane MAC studies using the tail clamp ( %) (Steffey & Howland 1977; Valverde et al. 2003; Pypendop et al. 2007) can be partially explained by this lack of standardization. Although the pressure applied to the tail was not measured in the present study and may be a confounding factor when using the tail clamp, it was standardized by engaging the first ratchet on the forceps but not closing it, as reported in the original description of MAC in dogs (Eger et al. 1965) and by other authors (Steffey & Howland 1977). Alternatively, electrical noxious stimulation can allow easier quantification and standardization (Le Bars et al. 2001) and can thus potentially generate MAC results with better reproducibility and repeatability. These advantages are reinforced by using constant-current stimulation (Levionnois et al. 2009) as the variable resistance between the stimulation electrodes leads 2016 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesia and Analgesia, 43,

7 to a variable stimulation output when a constantvoltage modality is used. However, most of the MAC studies using electrical noxious stimulation applied a constant voltage but did not report the actual current intensity (Eger et al. 1965; Hornbein et al. 1982; Valverde et al. 1989, 2003; Laster et al. 1993; Antognini et al. 1994; Soares et al. 2004). The small sample size (n = 6) used in the present study may have impaired its ability to detect further significant differences between current intensities and anatomic sites. Nevertheless, significant differences of at least 0.24% (14.2% of MAC TAILCLAMP ) were detected in the present study design (MAC TL10 compared with MAC TAILCLAMP ). The application of the results of this study in other species and with other inhalant anesthetics should be undertaken with caution and the issue warrants further evaluation. In conclusion, different estimations of isoflurane MAC were obtained by changes in the intensity of electrical currents used for stimulation of different anatomic sites. The results suggest that electrical current intensities of 30 ma and 50 ma applied to the tail, or the thoracic or pelvic limbs can be used as alternative stimuli to the tail clamp method to determine isoflurane MAC in dogs. Acknowledgements This study was supported by grants from the Brazilian Council for Scientific and Technology Development (CNPq) / The authors thank Dr Firmino Marsico Filho, formerly of the Veterinary Surgery and Clinics Graduation Program, Veterinary College, Fluminense Federal University, since deceased, for his dedication to his research group and contributions to the accomplishment of this study; his presence and inspiration are greatly missed. The authors also thank Dr Tatiana Henriques Ferreira currently at the Department of Surgical Sciences, University of Wisconsin, Madison, WI, USA and Dr Fabio de Oliveira Monteiro from the Veterinary Surgery and Clinics Graduation Program, Veterinary College, Fluminense Federal University for their technical assistance during the experiments. Authors contributions MRF, JHNS, FOA: study design, data acquisition and analysis, manuscript preparation. SW: data analysis, manuscript preparation. IAGdS: study design, reviewed data analysis, manuscript preparation. All authors approved the final manuscript. JHNS archived the study files. References Antognini JF, Carstens E (1998) A simple, quantifiable, and accurate method for applying a noxious mechanical stimulus. Anesth Analg 87, Antognini JF, Schwartz K (1993) Exaggerated anesthetic requirements in the preferentially anesthetized brain. Anesthesiology 79, Antognini JF, Lewis BK, Reitan JA (1994) Hypothermia minimally decreases nitrous oxide anesthetic requirements. Anesth Analg 79, Eger EI II, Saidman LJ, Brandstater B (1965) Minimum alveolar anesthetic concentration: a standard of anesthetic potency. Anesthesiology 26, Eger EI II, Raines DE, Shafer SL et al. (2008) Is a new paradigm needed to explain how inhaled anesthetics produce immobility? Anesth Analg 107, Escobar A, Pypendop BH, Siao KT et al. (2012) Effect of dexmedetomidine on the minimum alveolar concentration of isoflurane in cats. J Vet Pharmacol Ther 35, Gerstin KM, Gong DH, Abdallah M et al. (2003) Mutation of KCNK5 or Kir3.2 potassium channels in mice does not change minimum alveolar anesthetic concentration. Anesth Analg 96, Gomez de Segura IA, Criado AB, Santos M et al. (1998) Aspirin synergistically potentiates isoflurane minimum alveolar concentration reduction produced by morphine in the rat. Anesthesiology 89, Hellyer PW, Mama KR, Shafford HL et al. (2001) Effects of diazepam and flumazenil on minimum alveolar concentrations for dogs anesthetized with isoflurane or a combination of isoflurane and fentanyl. Am J Vet Res 62, Hornbein TF, Eger EI II, Winter PM et al. (1982) The minimum alveolar concentration of nitrous oxide in man. Anesth Analg 61, Ide T, Sakurai Y, Aono M et al. (1998) Minimum alveolar anesthetic concentrations for airway occlusion in cats: a new concept of minimum alveolar anesthetic concentration airway occlusion response. Anesth Analg 86, Ilkiw JE, Pascoe PJ, Fisher LD (1997) Effect of alfentanil on the minimum alveolar concentration of isoflurane in cats. Am J Vet Res 58, Jinks SL, Antognini JF, Martin JT et al. (2002) Isoflurane, but not halothane, depresses c-fos expression in rat spinal cord at concentrations that suppress reflex movement after supramaximal noxious stimulation. Anesth Analg 95, Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesia and Analgesia, 43,

8 Koblin DD, Dong DE, Deady JE et al. (1980) Selective breeding alters murine resistance to nitrous oxide without alteration in synaptic membrane lipid composition. Anesthesiology 52, Laster MJ, Liu J, Eger EI II et al. (1993) Electrical stimulation as a substitute for the tail clamp in the determination of minimum alveolar concentration. Anesth Analg 76, Le Bars D, Gozariu M, Cadden SW (2001) Animal models of nociception. Pharmacol Rev 53, Levionnois OL, Spadavecchia C, Kronen PW et al. (2009) Determination of the minimum alveolar concentration of isoflurane in Shetland ponies using constant current or constant voltage electrical stimulation. Vet Anaesth Analg 36, Merkel G, Eger EI II (1963) A comparative study of halothane and halopropane anesthesia including method for determining equipotency. Anesthesiology 24, Pypendop BH, Solano A, Boscan P et al. (2007) Characteristics of the relationship between plasma ketamine concentration and its effect on the minimum alveolar concentration of isoflurane in dogs. Vet Anaesth Analg 34, Quasha AL, Eger EI II, Tinker JH (1980) Determination and applications of MAC. Anesthesiology 53, Rampil IJ, Mason P, Singh H (1993) Anesthetic potency (MAC) is independent of forebrain structures in the rat. Anesthesiology 78, Saidman LJ, Eger EI II (1964) Effect of nitrous oxide and of narcotic premedication on the alveolar concentration of halothane required for anesthesia. Anesthesiology 25, Seddighi R, Egger CM, Rohrbach BW et al. (2011) The effect of midazolam on the end-tidal concentration of isoflurane necessary to prevent movement in dogs. Vet Anaesth Analg 38, Soares JHN, Ascoli FO, Gremiao IDF et al. (2004) Isoflurane sparing action of epidurally administered xylazine hydrochloride in anesthetized dogs. Am J Vet Res 65, Sobair AT, Cottrell DF, Camburn MA (1993) A mechanical stimulator for the determination of the minimum alveolar concentration (MAC) of halothane in the rabbit. Vet Res Commun 17, Sonner JM, Antognini JF, Dutton RC et al. (2003) Inhaled anesthetics and immobility: mechanisms, mysteries, and minimum alveolar anesthetic concentration. Anesth Analg 97, Steffey EP, Howland D (1977) Isoflurane potency in the dog and cat. Am J Vet Res 38, Valverde A, Dyson DH, McDonell WN (1989) Epidural morphine reduces halothane MAC in the dog. Can J Anaesth 36, Valverde A, Morey TE, Hernandez J et al. (2003) Validation of several types of noxious stimuli for use in determining the minimum alveolar concentration for inhalation anesthetics in dogs and rabbits. Am J Vet Res 64, Zbinden AM, Maggiorini M, Petersen-Felix S et al. (1994) Anesthetic depth defined using multiple noxious stimuli during isoflurane/oxygen anesthesia. I. Motor reactions. Anesthesiology 80, Received 4 May 2015; accepted 13 November Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesia and Analgesia, 43,