Neuromuscular blocking effects of vecuronium in dogs with autosomal-recessive centronuclear myopathy OBJECTIVE To evaluate the potency of vecuronium and duration of vecuronium-induced neuromuscular blockade in dogs with centronuclear myopathy (CNM).
Manuel Martin-Flores DVM Monique D. Paré DVM Emily A.Tomak
ANIMALS 6 Labrador Retrievers with autosomal-recessive CNM and 5 age- and weightmatched control dogs.
Morgan L. Corn BSC Luis Campoy DVM Received April 16, 2014. Accepted October 20, 2014. From the Department of Clinical Sciences, College of Veterinary Medicine (Martin-Flores, Paré, Tomak, Campoy), and the Department of Animal Science, College of Agriculture and Life Sciences (Corn), Cornell University, Ithaca, NY 14853. Dr. Paré’s present address is Département de sciences cliniques, Faculté de médecine vétérinaire, Université de Montréal, SaintHyacinthe, QC J2S 2M2, Canada. Ms. Corn’s present address is Water4Dogs Rehabilitation Center, 77 Worth St, New York, NY 10013. Address correspondence to Dr. Martin-Flores (martinfl[email protected]).
PROCEDURES Dogs were anesthetized on 2 occasions (1-week interval) with propofol, dexmedetomidine, and isoflurane. Neuromuscular function was monitored with acceleromyography and train-of-four (TOF) stimulation. In an initial experiment, potency of vecuronium was evaluated by a cumulativedose method, where 2 submaximal doses of vecuronium (10 µg/kg each) were administered IV sequentially. For the TOF’s first twitch (T1), baseline twitch amplitude and maximal posttreatment depression of twitch amplitude were measured. In the second experiment, dogs received vecuronium (50 µg/kg, IV) and the time of spontaneous recovery to a TOF ratio (ie, amplitude of TOF’s fourth twitch divided by amplitude of T1) ≥ 0.9 and recovery index (interval between return of T1 amplitude to 25% and 75% of baseline) were measured. RESULTS Depression of T1 after each submaximal dose of vecuronium was not different between groups. Median time to a TOF ratio ≥ 0.9 was 76.7 minutes (interquartile range [IQR; 25th to 75th percentile], 66.7 to 99.4 minutes) for dogs with CNM and 75.0 minutes (IQR, 47.8 to 96.5 minutes) for controls. Median recovery index was 18.0 minutes (IQR, 9.7 to 23.5 minutes) for dogs with CNM and 20.2 minutes (IQR, 8 to 25.1 minutes) for controls. CONCLUSIONS AND CLINICAL RELEVANCE For the study dogs, neither potency nor duration of vecuronium-induced neuromuscular blockade was altered by CNM. Vecuronium can be used to induce neuromuscular blockade in dogs with autosomal-recessive CNM. (Am J Vet Res 2015;76:302–307)
C
entronuclear myopathy is a rare congenital condition in people that was first described in 1966 and is primarily associated with skeletal muscle weakness.1 The condition was originally named myotubular myopathy because the fibers resemble embryonic myotubes; their persistence may represent an arrest in normal myofiber development.1 Inherited forms of CNM can be X-linked (recessive) or autosomal (dominant and recessive).2 A form of the autosomal disease in dogs was first described in 1976 and originally named hereditary myopathy of the Labrador Retriever (the term CNM was proposed later).3 Since that original description, additional reports4–6 of affected dogs have become available. In dogs, CNM is transmitted as an autosomal recessive disease, and signs include hypotonia, generalized muscle weakness, abnormal postures, and ABBREVIATIONS CNM Centronuclear myopathy IQR Interquartile range (25th to 75th percentile) NMBA Neuromuscular blocking agent T1 First twitch of the TOF TOF Train-of-four 302
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exercise intolerance.7 Atrophy of skeletal muscles, in particular those of the head, can often be observed. A deficiency of type 2 fibers and a predominance of atrophic or hypertrophic type 1 fibers are also present.3,6,8 Histologically, skeletal muscle cells are characterized by centrally distributed myonuclei, which are frequently located in areas devoid of myofibrils with mitochondrial aggregation.7 Owing to the rarity of the disease, information on dogs with CNM requiring anesthesia is lacking. It is known that in humans, other myopathies can affect the duration of action of nondepolarizing NMBAs; although there is no difference in onset or potency, the duration of action of rocuronium or mivacurium is prolonged in people with Duchenne muscular dystrophy, compared with unaffected controls.9,10 Therefore, we decided to investigate the effects of neuromuscular blockade in dogs with CNM and our ability to monitor return of neuromuscular function as neuromuscular blockade diminished. The purpose of the study reported here was to evaluate the potency of vecuronium and duration of vecuronium-induced neuromuscular blockade in dogs with CNM by means of acceleromyography. The null hypothesis was that nei-
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ther the potency of vecuronium nor the duration of vecuronium-induced neuromuscular blockade would differ between affected and unaffected dogs. A secondary aim was to evaluate the performance of electromyography as an alternative monitoring technique for assessment of recovery from neuromuscular blockade in dogs with CNM.
Materials and Methods Animals
Six adult (1- to 5-year-old) Labrador Retrievers (4 males and 2 females) with CNM and 5 age-matched (1 to 5 years) and weight-matched dogs (4 Labrador Retrievers and 1 hound; 5 males) that were free of the disease (control group) were used in the study. For dogs with CNM, median body weight was 20.2 kg (IQR, 18.8 to 26.1 kg). For control dogs, median body weight was 27.4 kg (IQR, 25.3 to 32.5 kg). In the affected dogs, CNM was diagnosed through DNA testing by an independent laboratory.a Sample size was limited by availability of dogs with CNM and age- and weightmatched controls. All procedures were approved by an institutional animal care and use committee.
Experiments
Each dog was anesthetized on 2 occasions (1week interval). One experiment was performed to assess the potency of vecuronium in dogs with and without CNM. The other experiment was performed to determine the duration and recovery profile of vecuronium-induced neuromuscular blockade in dogs with and without CNM.
Anesthesia
For each dog, food was withheld overnight (approx 12 hours) prior to anesthesia. Each dog was administered dexmedetomidine (1 µg/kg, IV). After approximately 3 minutes of O2 supplementation via face mask, anesthesia was induced with propofol administered IV to effect. Propofol was administered manually at a rate of approximately 2 mg/kg/min until the eyelash reflex could no longer be elicited and the dog’s mouth could be opened without resistance.The trachea was intubated with a cuffed tube when coughing or swallowing reflexes were not present, and anesthesia was maintained with isoflurane in O2 (end-tidal concentration, 1.2% to 1.5%). The lungs were ventilated to maintain normocapnia. Dexmedetomidine (1 µg/kg/h) and lactated Ringer’s solution (5 mL/kg/h) were infused throughout the experimental procedure. Electrocardiography and capnography were performed continuously, oxygen saturation as measured by pulse oximetry, end-tidal isoflurane concentration, oscillometric arterial blood pressure recordings, and esophageal temperature were monitored continuously. Esophageal temperature was maintained between 36° and 38°C by use of a forced warm air device.
Neuromuscular monitoring
Each dog was placed in left lateral recumbency. An acceleromyography monitorb was used on the
left thoracic limb. Subcutaneous needles were placed over the ulnar nerve and connected to the stimulating electrodes of the acceleromyograph. The acceleration transducer was taped to the ventral aspect of the paw to measure the evoked responses to nerve stimulation (flexion of the manus). To return the manus to a fixed position between muscular twitches as effectively as possible, a 150-g elastic preload was applied, similar to a procedure described previously.11,12 The acceleromyography monitor was calibrated with a calibration function that automatically detects supramaximal current and sets the amplitude of twitch acceleration to 100%. The acceleromyography monitor reports stimulating current and sensitivity (gain amplification), which are detected during calibration; these data were obtained in each experiment for analysis. Train-of-four stimulation (2 Hz) was begun at 15-second intervals. The amplitudes of T1 and fourth twitch of the TOF were measured, and the TOF ratio (ie, the amplitude of fourth twitch of the TOF divided by the amplitude of T1) was calculated.To allow for twitch potentiation and stabilization of twitch amplitude to occur, at least 15 minutes of TOF stimulation was delivered before data collection began.12 In each dog, stable twitch amplitudes were established for at least 3 to 5 minutes before vecuronium administration.13 All data regarding the potency and duration of action of vecuroniumc were evaluated with acceleromyography.
Evaluation of potency of vecuronium
To evaluate each dog’s susceptibility to vecuronium, a submaximal dose of 10 µg/kg was administered IV as a 5-second bolus. Depression of T1 amplitude was measured by acceleromyography until it reached its nadir. Once the T1 amplitude had no further reduction over 3 consecutive measurements, a second dose of 10 µg of vecuronium/kg was administered IV. If no depression of T1 amplitude was detected after the first dose, the second dose was administered 5 minutes later. The maximum decrease in T1 amplitude relative to baseline after each dose was recorded.
Duration of action and recovery profile of vecuronium
In a separate anesthetic period, vecuronium (50 µg/kg, IV) was administered as a single bolus to each dog.The time to onset of blockade (interval between vecuronium injection and establishment of complete neuromuscular blockade) was recorded, as were the times for T1 amplitude to spontaneously return to 25% and 75% of baseline following vecuronium injection, recovery index (interval between return of T1 amplitude to 25% and 75% of baseline), and the time to attain a TOF ratio ≥ 0.9 following vecuronium injection as measured by the acceleromyograph.
Electromyography
In each experiment to determine the duration of action and recovery profile of vecuronium, an electromyographd was also used to monitor neuromuscular
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function on the right pelvic limb of each dog. Subcutaneous stimulating needles were placed over the peroneal nerve of the nondependent right pelvic limb.The skin was clipped of hair and cleansed. An adhesive silver-silver chloride recording electrodee was placed over the belly of the right flexor cranial tibial muscle, and secured with flexible cohesive bandage.f The electromyograph was calibrated by means of the start-up function; similar to the acceleromyographic calibration process, the maximal response to nerve stimulation during calibration was set to 100%, and supramaximal current was detected automatically. Train-of-four stimulation every 15 seconds began after calibration.
Statistical analysis
The order of the 2 experimental procedures was randomized for all dogs. The dose of propofol administered to dogs with and without CNM in each experiment was compared with the Mann-Whitney U test.g Stimulating current and sensitivity (gain amplification) were obtained by means of the acceleromyography monitor for each dog in each experiment; pooled values of each were compared between groups by means of the Mann-Whitney U test. Baseline T1 amplitudes and TOF ratio were determined by averaging the 3 values recorded immediately prior to vecuronium administration. Depression of T1 amplitude (expressed as a percentage) during the potency experiment was calculated as follows:
pofol dose was 3.8 mg/kg (IQR, 3.2 to 4.3 mg/kg) for the dogs with CNM and 3.6 mg/kg (IQR, 2.8 to 4.7 mg/kg) for the control dogs (P = 0.9). Stimulating current and sensitivity obtained during calibration of the acceleromyography monitor in each experiment (to determine potency and duration of action of vecuronium) were pooled for each group of dogs. Stimulating current for the CNM (median, 39 mA; IQR, 33 to 54 mA) and control (median, 60 mA; IQR, 28 to 60 mA) groups did not differ significantly (P = 0.5). The pooled sensitivities (between 1 and 512, where 512 represents the highest sensitivity) were significantly (P = 0.001) higher for the CNM group (median, 351; IQR, 315 to 366) than for control dogs (median, 220; IQR, 174 to 235).
Evaluation of potency of vecuronium Depression of T1 amplitude after two 10 µg/kg doses of vecuronium in dogs with CNM and control dogs was evident (Figure 1). Depression of T1 amplitude did not differ between groups after each dose of vecuronium (all P > 0.7).
Duration of action and recovery profile of vecuronium
Administration of vecuronium (50 µg/kg, IV) resulted in complete ablation of responses to TOFs in all
(1 – [minimum T1 amplitude/baseline T1 amplitude]) X 100% where minimum T1 amplitude was the lowest T1 amplitude recorded after vecuronium administration. All acceleromyographic data were expressed relative to their baselines (ie, data were normalized to baseline). The significance of differences between groups for each neuromuscular variable was tested by means of the Mann-Whitney test. Values of P ≤ 0.05 were considered significant. Results are summarized as median and IQR. To assess the usefulness of electromyography as a neuromuscular monitor in clinical situations, we measured and compared the time to attain a TOF ratio ≥ 0.9 after administration of vecuronium (50 µg/kg) to dogs in both groups. The TOF ratios determined by means of electromyography were also normalized to their respective baselines.
Results All dogs recovered uneventfully from both anesthetic episodes and experimental procedures. The dose of propofol administered during either phase of the study did not differ between groups. During the experiments to assess the potency of vecuronium, the median propofol dose was 3.4 mg/kg (IQR, 3.2 to 3.8 mg/kg) for the dogs with CNM and 3.3 mg/kg (IQR, 3.1 to 4.1 mg/kg) for the control dogs (P = 1.0). During the experiments to assess the duration of action and recovery profile of vecuronium, the median pro304
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Figure 1—Percentage depression of the T1 amplitude (relative to pretreatment baseline) after a first and second dose of vecuronium (10 µg/kg each, IV) in 6 dogs with CNM (black circles) and in 5 ageand weight-matched control dogs without CNM (white circles). Dogs were anesthetized with propofol, dexmedetomidine, and isoflurane. Neuromuscular function was monitored with acceleromyography and TOF stimulation. Train-of-four stimulation (2 Hz) was begun at 15-second intervals.To allow for twitch potentiation and stabilization of twitch amplitude to occur, at least 15 minutes of TOF stimulation was delivered before data collection began. In each dog, stable T1 amplitude was established for at least 3 to 5 minutes before vecuronium administration.The second dose of vecuronium was administered after the effects of the first dose had peaked. If no depression of T1 amplitude was detected after the first dose, the second dose was administered 5 minutes later. The lowest T1 amplitude detected after each dose was recorded. Depression of T1 amplitude (expressed as percentage) was calculated as follows: (1 – [minimum T1 amplitude/baseline T1 amplitude]) X 100%. For each grouping, the horizontal line indicates the median. Between groups, there was no difference in T1 amplitude depression after the first or second dose.
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dogs. Median time to onset of neuromuscular blockade was 157 seconds (IQR, 139 to 184 seconds) for dogs with CNM and 150 seconds (IQR, 120 to 165 seconds) for control dogs (P = 0.64).There were no differences between groups for any of the recovery variables.The median time for T1 amplitude to spontaneously return to 25% of baseline was 34.6 minutes (IQR, 22.1 to 41.2 minutes) for dogs with CNM and 31.5 minutes (IQR, 21.5 to 36.2 minutes) for control dogs (P = 0.64). The median time for T1 amplitude to spontaneously return to 75% of baseline was 60.2 minutes (IQR, 37.7 to 62.2 minutes) for dogs with CNM and 55.2 minutes (IQR, 29.5 to 59.6 minutes) for control dogs (P = 0.53). The median recovery index (interval between return of T1 amplitude to 25% and 75% of baseline) was 18.0 minutes (IQR, 9.7 to 23.5 minutes) for dogs with CNM and 20.2 minutes (IQR, 8.0 to 25.1 minutes) for control dogs (P = 0.83). The median time to attain a TOF ratio ≥ 0.9 was 76.7 minutes (IQR, 66.7 to 99.4 minutes) for dogs with CNM and 75.0 minutes (IQR, 47.8 to 96.5 minutes) for control dogs (P = 0.64).
Electromyography
For all dogs, electromyographic signals were obtained and the monitor was successfully calibrated at first attempt. By visual inspection, biphasic electromyographic curves obtained from dogs with CNM and control dogs were indistinguishable. The median time to attain a TOF ratio ≥ 0.9 during electromyography was 58 minutes (IQR, 43 to 68.5 minutes) for dogs with CNM and 61 minutes (IQR, 44 to 88.5 minutes) for control dogs (P = 0.53).
Discussion In the present study, there were no differences in the effects of vecuronium in dogs with autosomal recessive CNM and unaffected control dogs. The study was performed in 2 phases with the intent of evaluating both the susceptibility of these dogs to vecuronium and the duration of action and recovery profile of a clinically useful dose of the drug. Potency of an NMBA in a given species is typically established by measuring T1 amplitude depression following administration of submaximal doses of the drug. The results obtained by that approach can be managed in different ways. When only 1 submaximal dose is administered to each study animal, a minimal number of individuals must be evaluated before potency estimations are accurate.14 An alternative approach is to recruit fewer individuals and use a cumulative technique, in which doses of the NMBA are repeatedly administered to each study animal.15 The major limitation of the cumulative method is that a fraction of each dose might be redistributed or metabolized before the effects of the subsequent dose are measured (ie, a fraction of drug could be lost). Therefore, the cumulative method is usually reserved for long-lasting NMBAs, for which it is presumed that a short interval between dose administrations will have a lesser loss effect than it would for intermediate or short-acting drugs.
Despite this drawback, we chose to use a cumulative method in the present study because we had access to only 6 animals with CNM and because our objective was not to estimate the effective dose of vecuronium for a larger canine population, but rather to compare the response to submaximal doses between dogs with CNM and unaffected controls. The magnitude of T1 amplitude depression after each submaximal dose was the same between groups, suggesting that dogs with CNM are no more susceptible to vecuronium than dogs without CNM. Notwithstanding the limitations of the cumulativedose method, T1 amplitude depression after the second dose was approximately 95% in each group. This finding is not substantially different from previous findings for healthy Beagles, in which a vecuronium dose of 25 µg/kg (as a single bolus) produced T1 amplitude depression > 80%.16 Bom et al17 previously reported that, for vecuronium, the dose required to produce a 90% effect is 90 µg/kg. However, Kariman and Clutton18 showed that complete neuromuscular blockade in anesthetized dogs is achieved with 50 µg of vecuronium/kg. Moreover, when we studied recovery from vecuronium-induced neuromuscular blockade in the present study, a dose of 50 µg of vecuronium/kg produced complete neuromuscular blockade in both groups of dogs. Our results and those of Kariman and Clutton18 suggest that a 50 µg/kg dose is sufficient for producing complete neuromuscular blockade in this species. As part of the present investigation, the study dogs were administered a clinically useful dose of vecuronium that produced complete paralysis and descriptors of spontaneous recovery were measured. No differences were found in any indicator of recovery: following vecuronium injection, times for T1 amplitude to spontaneously return to 25% and 75% of baseline, recovery index, and the time to attain a TOF ratio ≥ 0.9 were each similar in the 2 groups.Taken together, these data suggested that dogs with autosomalrecessive CNM are not more sensitive to vecuronium than healthy dogs, and the duration of neuromuscular blockade or the recovery characteristics do not differ between dogs with and without CNM. In the present study, the acceleromyography monitor was used with an elastic preload, as described previously.12 During calibration, the monitor automatically detects supramaximal current and adjusts the gain of the acceleration signal so that the response is set to 100%; a value of sensitivity (gain amplification) is reported by the device. Hence, lower sensitivity indicates higher acceleration, and vice-versa. Sensitivity was consistently higher in dogs with CNM in the present study, indicating that the magnitude of the signal they produced was lower and that gain amplification was greater than findings in dogs without CNM. Although we did not measure force of contraction in the dogs with CNM, the responses to neuromuscular stimulation obtained appeared to be of a smaller magnitude than usual, especially in the most affected dogs.
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This was expected because CNM results in muscle atrophy. In accordance with our observations, reduced force of contraction during TOF stimulation has been reported in a human subject with CNM requiring general anesthesia.19 To our knowledge, this is the first prospective investigation of an NMBA in dogs with CNM. There are only isolated reports of the use of NMBAs in humans with CNM; in most of those patients, administration of an NMBA was in fact avoided during anesthesia. The use of atracurium in a patient with X-linked CNM has been reported.20 A cumulative dose of 0.5 mg of atracurium/kg was administered before TOF response was abolished; however, a clinically relevant increased sensitivity was not reported.20 In the present study, we evaluated the performance of an electromyography monitor because during our pilot investigations, we experienced substantial difficulties with the acceleromyography monitor; we were unable to calibrate the acceleromyography monitor when placed on the pelvic limb in several of the affected dogs that were subsequently used in the study. Such difficulties had not been encountered during our previous experiences with acceleromyography in healthy dogs. Judging by the degree of muscle atrophy and the poor ability to support body weight, the pelvic limbs appeared to be substantially more affected than the thoracic limbs in the dogs with CNM. Difficulties calibrating the acceleromyography monitor in human neonates and infants have been reported; for those individuals, the magnitude of evoked twitches are presumably small.21 It is likely that low signal intensity was the reason that calibration was difficult to obtain in the dogs with CNM in the present study. Unlike acceleromyography monitoring, the electromyography signal does not depend on movement but on muscle cell activation (compound motor action potential); therefore, we speculated that some of the difficulties encountered during acceleromyography would not occur during electromyography. The electromyography monitor was successfully used and calibrated in all dogs. This monitor displays the biphasic curves that are detected during neuromuscular stimulation and reports the amplitude of T1 and the TOF ratio. By inspection, the electromyographic curves of all dogs with CNM were identical to those obtained from dogs without CNM in the present study. Furthermore, no differences were found when the duration of neuromuscular blockade as measured by electromyography was compared between dogs with CNM and control dogs. It appears that electromyography may offer some advantages when measurement of neuromuscular function of muscles that produce low signals (small contraction) is required. In the present study, we elected to assess the return of the TOF ratio to ≥ 0.9 with electromyography to simplify the amount of data presented. The objective for evaluating electromyography was 306
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to assess its usefulness as a means to clinically monitor recovery from neuromuscular blockade when muscle contraction is decreased due to myopathy, but not to evaluate the bias or agreement with acceleromyography. Results of the present study indicated that there was no difference in the potency or duration of action of vecuronium in dogs with autosomal-recessive CNM and unaffected control dogs. Acceleromyography was useful for quantifying neuromuscular transmission in dogs with myopathy, although calibration of the acceleromyography monitor in the pelvic limbs of some dogs was difficult. Electromyography was successfully calibrated in the pelvic limbs of all study dogs, even in limbs for which acceleromyography calibration had failed.
Footnotes a. b. c. d. e. f. g.
DDC Veterinary, Fairfield, Ohio. TOF-Watch SX, Organon, Swords, County Dublin, Ireland. Vecuronium bromide, SUN Pharmaceutical Industries, Gujarat, India. Datex Ohmeda AS/3, GE Healthcare, Helsinki, Finland. Cleartrace, Conmed Corp, New York, NY. PetFlex, Andover Healthcare, Salisbury, Mass. Minitab, version 16.2.4, Minitab Inc, State College, Pa
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Spiro AJ, Shy GM, Gonatas NK. Myotubular myopathy. Persistence of fetal muscle in an adolescent boy. Arch Neurol 1966;14:1–14. Jungbluth H, Wallgren-Pettersson C, Laporte J. Centronuclear (myotubular) myopathy. Orphanet J Rare Dis 2008;3:26– 1172–3-26. Kramer JW, Hegreberg GA, Bryan GM, et al. A muscle disorder of Labrador Retrievers characterized by deficiency of type II muscle fibers. J Am Vet Med Assoc 1976;169:817–820. McKerrell RE, Braund KG. Hereditary myopathy in Labrador Retrievers: clinical variations. J Small Anim Pract 1987;28:479– 489. Watson AD, Farrow BR, Middleton DJ, et al. Myopathy in a Labrador Retriever. Aust Vet J 1988;65:226–227. Gortel K, Houston DM, Kuiken T, et al. Inherited myopathy in a litter of Labrador Retrievers. Can Vet J 1996;37:108–110. Pelé M, Tiret L, Kessler JL, et al. SINE exonic insertion in the PTPLA gene leads to multiple splicing defects and segregates with the autosomal recessive centronuclear myopathy in dogs. Hum Mol Genet 2005;14:1417–1427. Bley T, Gaillard C, Bilzer T, et al. Genetic aspects of Labrador Retriever myopathy. Res Vet Sci 2002;73:231–236. Muenster T, Schmidt J, Wick S, et al. Rocuronium 0.3 mg X kg–1 (ED95) induces a normal peak effect but an altered time course of neuromuscular block in patients with Duchenne’s muscular dystrophy. Paediatr Anaesth 2006;16:840–845. Schmidt J, Muenster T,Wick S, et al. Onset and duration of mivacurium-induced neuromuscular block in patients with Duchenne muscular dystrophy. Br J Anaesth 2005;95:769–772. Kopman AF, Kumar S, Klewicka MM, et al. The staircase phenomenon: implications for monitoring of neuromuscular transmission. Anesthesiology 2001;95:403–407. Martin-Flores M, Lau EJ, Campoy L, et al. Twitch potentiation: a potential source of error during neuromuscular monitoring with acceleromyography in anesthetized dogs. Vet Anaesth Analg 2011;38:328–335. Fuchs-Buder T, Claudius C, Skovgaard LT, et al. Good clinical research practice in pharmacodynamics studies of neuromuscular blocking agents II: the Stockholm revision. Acta Anaesthesiol Scand 2007;51:789–808. Kopman AF, Lien CA, Naguib M. Neuromuscular dose-response
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studies: determining sample size. Br J Anaesth 2011;106:194– 198. 15. Donlon JV Jr, Savarese JJ, Ali HH, et al. Human dose-response curves for neuromuscular blocking drugs: a comparison of two methods of construction and analysis. Anesthesiology 1980;53:161–166. 16. Martin-Flores M, Sakai DM, Campoy L, et al. Recovery from neuromuscular block in dogs: restoration of spontaneous ventilation does not exclude residual blockade. Vet Anaesth Analg 2014;41:269–277. 17. Bom A, Hope F, Rutherford S, et al. Preclinical pharmacology of sugammadex. J Crit Care 2009;24:29–35.
18. Kariman A, Clutton RE. The effects of medetomidine on the action of vecuronium in dogs anaesthetized with halothane and nitrous oxide. Vet Anaesth Analg 2008;35:400–408. 19. Breslin D, Reid J, Hayes A, et al.Anaesthesia in myotubular (centronuclear) myopathy. Anaesthesia 2000;55:471–474. 20. Costi D, van der Walt JH. General anesthesia in an infant with X-linked myotubular myopathy. Paediatr Anaesth 2004;14:964– 968. 21. Driessen JJ, Robertson EN, Booij LH. Acceleromyography in neonates and small infants: baseline calibration and recovery of the responses after neuromuscular blockade with rocuronium. Eur J Anaesthesiol 2005;22:11–15.
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Manuel Martin-Flores DVM Monique D. Paré DVM Emily A.Tomak
ANIMALS 6 Labrador Retrievers with autosomal-recessive CNM and 5 age- and weightmatched control dogs.
Morgan L. Corn BSC Luis Campoy DVM Received April 16, 2014. Accepted October 20, 2014. From the Department of Clinical Sciences, College of Veterinary Medicine (Martin-Flores, Paré, Tomak, Campoy), and the Department of Animal Science, College of Agriculture and Life Sciences (Corn), Cornell University, Ithaca, NY 14853. Dr. Paré’s present address is Département de sciences cliniques, Faculté de médecine vétérinaire, Université de Montréal, SaintHyacinthe, QC J2S 2M2, Canada. Ms. Corn’s present address is Water4Dogs Rehabilitation Center, 77 Worth St, New York, NY 10013. Address correspondence to Dr. Martin-Flores (martinfl[email protected]).
PROCEDURES Dogs were anesthetized on 2 occasions (1-week interval) with propofol, dexmedetomidine, and isoflurane. Neuromuscular function was monitored with acceleromyography and train-of-four (TOF) stimulation. In an initial experiment, potency of vecuronium was evaluated by a cumulativedose method, where 2 submaximal doses of vecuronium (10 µg/kg each) were administered IV sequentially. For the TOF’s first twitch (T1), baseline twitch amplitude and maximal posttreatment depression of twitch amplitude were measured. In the second experiment, dogs received vecuronium (50 µg/kg, IV) and the time of spontaneous recovery to a TOF ratio (ie, amplitude of TOF’s fourth twitch divided by amplitude of T1) ≥ 0.9 and recovery index (interval between return of T1 amplitude to 25% and 75% of baseline) were measured. RESULTS Depression of T1 after each submaximal dose of vecuronium was not different between groups. Median time to a TOF ratio ≥ 0.9 was 76.7 minutes (interquartile range [IQR; 25th to 75th percentile], 66.7 to 99.4 minutes) for dogs with CNM and 75.0 minutes (IQR, 47.8 to 96.5 minutes) for controls. Median recovery index was 18.0 minutes (IQR, 9.7 to 23.5 minutes) for dogs with CNM and 20.2 minutes (IQR, 8 to 25.1 minutes) for controls. CONCLUSIONS AND CLINICAL RELEVANCE For the study dogs, neither potency nor duration of vecuronium-induced neuromuscular blockade was altered by CNM. Vecuronium can be used to induce neuromuscular blockade in dogs with autosomal-recessive CNM. (Am J Vet Res 2015;76:302–307)
C
entronuclear myopathy is a rare congenital condition in people that was first described in 1966 and is primarily associated with skeletal muscle weakness.1 The condition was originally named myotubular myopathy because the fibers resemble embryonic myotubes; their persistence may represent an arrest in normal myofiber development.1 Inherited forms of CNM can be X-linked (recessive) or autosomal (dominant and recessive).2 A form of the autosomal disease in dogs was first described in 1976 and originally named hereditary myopathy of the Labrador Retriever (the term CNM was proposed later).3 Since that original description, additional reports4–6 of affected dogs have become available. In dogs, CNM is transmitted as an autosomal recessive disease, and signs include hypotonia, generalized muscle weakness, abnormal postures, and ABBREVIATIONS CNM Centronuclear myopathy IQR Interquartile range (25th to 75th percentile) NMBA Neuromuscular blocking agent T1 First twitch of the TOF TOF Train-of-four 302
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exercise intolerance.7 Atrophy of skeletal muscles, in particular those of the head, can often be observed. A deficiency of type 2 fibers and a predominance of atrophic or hypertrophic type 1 fibers are also present.3,6,8 Histologically, skeletal muscle cells are characterized by centrally distributed myonuclei, which are frequently located in areas devoid of myofibrils with mitochondrial aggregation.7 Owing to the rarity of the disease, information on dogs with CNM requiring anesthesia is lacking. It is known that in humans, other myopathies can affect the duration of action of nondepolarizing NMBAs; although there is no difference in onset or potency, the duration of action of rocuronium or mivacurium is prolonged in people with Duchenne muscular dystrophy, compared with unaffected controls.9,10 Therefore, we decided to investigate the effects of neuromuscular blockade in dogs with CNM and our ability to monitor return of neuromuscular function as neuromuscular blockade diminished. The purpose of the study reported here was to evaluate the potency of vecuronium and duration of vecuronium-induced neuromuscular blockade in dogs with CNM by means of acceleromyography. The null hypothesis was that nei-
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ther the potency of vecuronium nor the duration of vecuronium-induced neuromuscular blockade would differ between affected and unaffected dogs. A secondary aim was to evaluate the performance of electromyography as an alternative monitoring technique for assessment of recovery from neuromuscular blockade in dogs with CNM.
Materials and Methods Animals
Six adult (1- to 5-year-old) Labrador Retrievers (4 males and 2 females) with CNM and 5 age-matched (1 to 5 years) and weight-matched dogs (4 Labrador Retrievers and 1 hound; 5 males) that were free of the disease (control group) were used in the study. For dogs with CNM, median body weight was 20.2 kg (IQR, 18.8 to 26.1 kg). For control dogs, median body weight was 27.4 kg (IQR, 25.3 to 32.5 kg). In the affected dogs, CNM was diagnosed through DNA testing by an independent laboratory.a Sample size was limited by availability of dogs with CNM and age- and weightmatched controls. All procedures were approved by an institutional animal care and use committee.
Experiments
Each dog was anesthetized on 2 occasions (1week interval). One experiment was performed to assess the potency of vecuronium in dogs with and without CNM. The other experiment was performed to determine the duration and recovery profile of vecuronium-induced neuromuscular blockade in dogs with and without CNM.
Anesthesia
For each dog, food was withheld overnight (approx 12 hours) prior to anesthesia. Each dog was administered dexmedetomidine (1 µg/kg, IV). After approximately 3 minutes of O2 supplementation via face mask, anesthesia was induced with propofol administered IV to effect. Propofol was administered manually at a rate of approximately 2 mg/kg/min until the eyelash reflex could no longer be elicited and the dog’s mouth could be opened without resistance.The trachea was intubated with a cuffed tube when coughing or swallowing reflexes were not present, and anesthesia was maintained with isoflurane in O2 (end-tidal concentration, 1.2% to 1.5%). The lungs were ventilated to maintain normocapnia. Dexmedetomidine (1 µg/kg/h) and lactated Ringer’s solution (5 mL/kg/h) were infused throughout the experimental procedure. Electrocardiography and capnography were performed continuously, oxygen saturation as measured by pulse oximetry, end-tidal isoflurane concentration, oscillometric arterial blood pressure recordings, and esophageal temperature were monitored continuously. Esophageal temperature was maintained between 36° and 38°C by use of a forced warm air device.
Neuromuscular monitoring
Each dog was placed in left lateral recumbency. An acceleromyography monitorb was used on the
left thoracic limb. Subcutaneous needles were placed over the ulnar nerve and connected to the stimulating electrodes of the acceleromyograph. The acceleration transducer was taped to the ventral aspect of the paw to measure the evoked responses to nerve stimulation (flexion of the manus). To return the manus to a fixed position between muscular twitches as effectively as possible, a 150-g elastic preload was applied, similar to a procedure described previously.11,12 The acceleromyography monitor was calibrated with a calibration function that automatically detects supramaximal current and sets the amplitude of twitch acceleration to 100%. The acceleromyography monitor reports stimulating current and sensitivity (gain amplification), which are detected during calibration; these data were obtained in each experiment for analysis. Train-of-four stimulation (2 Hz) was begun at 15-second intervals. The amplitudes of T1 and fourth twitch of the TOF were measured, and the TOF ratio (ie, the amplitude of fourth twitch of the TOF divided by the amplitude of T1) was calculated.To allow for twitch potentiation and stabilization of twitch amplitude to occur, at least 15 minutes of TOF stimulation was delivered before data collection began.12 In each dog, stable twitch amplitudes were established for at least 3 to 5 minutes before vecuronium administration.13 All data regarding the potency and duration of action of vecuroniumc were evaluated with acceleromyography.
Evaluation of potency of vecuronium
To evaluate each dog’s susceptibility to vecuronium, a submaximal dose of 10 µg/kg was administered IV as a 5-second bolus. Depression of T1 amplitude was measured by acceleromyography until it reached its nadir. Once the T1 amplitude had no further reduction over 3 consecutive measurements, a second dose of 10 µg of vecuronium/kg was administered IV. If no depression of T1 amplitude was detected after the first dose, the second dose was administered 5 minutes later. The maximum decrease in T1 amplitude relative to baseline after each dose was recorded.
Duration of action and recovery profile of vecuronium
In a separate anesthetic period, vecuronium (50 µg/kg, IV) was administered as a single bolus to each dog.The time to onset of blockade (interval between vecuronium injection and establishment of complete neuromuscular blockade) was recorded, as were the times for T1 amplitude to spontaneously return to 25% and 75% of baseline following vecuronium injection, recovery index (interval between return of T1 amplitude to 25% and 75% of baseline), and the time to attain a TOF ratio ≥ 0.9 following vecuronium injection as measured by the acceleromyograph.
Electromyography
In each experiment to determine the duration of action and recovery profile of vecuronium, an electromyographd was also used to monitor neuromuscular
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function on the right pelvic limb of each dog. Subcutaneous stimulating needles were placed over the peroneal nerve of the nondependent right pelvic limb.The skin was clipped of hair and cleansed. An adhesive silver-silver chloride recording electrodee was placed over the belly of the right flexor cranial tibial muscle, and secured with flexible cohesive bandage.f The electromyograph was calibrated by means of the start-up function; similar to the acceleromyographic calibration process, the maximal response to nerve stimulation during calibration was set to 100%, and supramaximal current was detected automatically. Train-of-four stimulation every 15 seconds began after calibration.
Statistical analysis
The order of the 2 experimental procedures was randomized for all dogs. The dose of propofol administered to dogs with and without CNM in each experiment was compared with the Mann-Whitney U test.g Stimulating current and sensitivity (gain amplification) were obtained by means of the acceleromyography monitor for each dog in each experiment; pooled values of each were compared between groups by means of the Mann-Whitney U test. Baseline T1 amplitudes and TOF ratio were determined by averaging the 3 values recorded immediately prior to vecuronium administration. Depression of T1 amplitude (expressed as a percentage) during the potency experiment was calculated as follows:
pofol dose was 3.8 mg/kg (IQR, 3.2 to 4.3 mg/kg) for the dogs with CNM and 3.6 mg/kg (IQR, 2.8 to 4.7 mg/kg) for the control dogs (P = 0.9). Stimulating current and sensitivity obtained during calibration of the acceleromyography monitor in each experiment (to determine potency and duration of action of vecuronium) were pooled for each group of dogs. Stimulating current for the CNM (median, 39 mA; IQR, 33 to 54 mA) and control (median, 60 mA; IQR, 28 to 60 mA) groups did not differ significantly (P = 0.5). The pooled sensitivities (between 1 and 512, where 512 represents the highest sensitivity) were significantly (P = 0.001) higher for the CNM group (median, 351; IQR, 315 to 366) than for control dogs (median, 220; IQR, 174 to 235).
Evaluation of potency of vecuronium Depression of T1 amplitude after two 10 µg/kg doses of vecuronium in dogs with CNM and control dogs was evident (Figure 1). Depression of T1 amplitude did not differ between groups after each dose of vecuronium (all P > 0.7).
Duration of action and recovery profile of vecuronium
Administration of vecuronium (50 µg/kg, IV) resulted in complete ablation of responses to TOFs in all
(1 – [minimum T1 amplitude/baseline T1 amplitude]) X 100% where minimum T1 amplitude was the lowest T1 amplitude recorded after vecuronium administration. All acceleromyographic data were expressed relative to their baselines (ie, data were normalized to baseline). The significance of differences between groups for each neuromuscular variable was tested by means of the Mann-Whitney test. Values of P ≤ 0.05 were considered significant. Results are summarized as median and IQR. To assess the usefulness of electromyography as a neuromuscular monitor in clinical situations, we measured and compared the time to attain a TOF ratio ≥ 0.9 after administration of vecuronium (50 µg/kg) to dogs in both groups. The TOF ratios determined by means of electromyography were also normalized to their respective baselines.
Results All dogs recovered uneventfully from both anesthetic episodes and experimental procedures. The dose of propofol administered during either phase of the study did not differ between groups. During the experiments to assess the potency of vecuronium, the median propofol dose was 3.4 mg/kg (IQR, 3.2 to 3.8 mg/kg) for the dogs with CNM and 3.3 mg/kg (IQR, 3.1 to 4.1 mg/kg) for the control dogs (P = 1.0). During the experiments to assess the duration of action and recovery profile of vecuronium, the median pro304
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Figure 1—Percentage depression of the T1 amplitude (relative to pretreatment baseline) after a first and second dose of vecuronium (10 µg/kg each, IV) in 6 dogs with CNM (black circles) and in 5 ageand weight-matched control dogs without CNM (white circles). Dogs were anesthetized with propofol, dexmedetomidine, and isoflurane. Neuromuscular function was monitored with acceleromyography and TOF stimulation. Train-of-four stimulation (2 Hz) was begun at 15-second intervals.To allow for twitch potentiation and stabilization of twitch amplitude to occur, at least 15 minutes of TOF stimulation was delivered before data collection began. In each dog, stable T1 amplitude was established for at least 3 to 5 minutes before vecuronium administration.The second dose of vecuronium was administered after the effects of the first dose had peaked. If no depression of T1 amplitude was detected after the first dose, the second dose was administered 5 minutes later. The lowest T1 amplitude detected after each dose was recorded. Depression of T1 amplitude (expressed as percentage) was calculated as follows: (1 – [minimum T1 amplitude/baseline T1 amplitude]) X 100%. For each grouping, the horizontal line indicates the median. Between groups, there was no difference in T1 amplitude depression after the first or second dose.
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dogs. Median time to onset of neuromuscular blockade was 157 seconds (IQR, 139 to 184 seconds) for dogs with CNM and 150 seconds (IQR, 120 to 165 seconds) for control dogs (P = 0.64).There were no differences between groups for any of the recovery variables.The median time for T1 amplitude to spontaneously return to 25% of baseline was 34.6 minutes (IQR, 22.1 to 41.2 minutes) for dogs with CNM and 31.5 minutes (IQR, 21.5 to 36.2 minutes) for control dogs (P = 0.64). The median time for T1 amplitude to spontaneously return to 75% of baseline was 60.2 minutes (IQR, 37.7 to 62.2 minutes) for dogs with CNM and 55.2 minutes (IQR, 29.5 to 59.6 minutes) for control dogs (P = 0.53). The median recovery index (interval between return of T1 amplitude to 25% and 75% of baseline) was 18.0 minutes (IQR, 9.7 to 23.5 minutes) for dogs with CNM and 20.2 minutes (IQR, 8.0 to 25.1 minutes) for control dogs (P = 0.83). The median time to attain a TOF ratio ≥ 0.9 was 76.7 minutes (IQR, 66.7 to 99.4 minutes) for dogs with CNM and 75.0 minutes (IQR, 47.8 to 96.5 minutes) for control dogs (P = 0.64).
Electromyography
For all dogs, electromyographic signals were obtained and the monitor was successfully calibrated at first attempt. By visual inspection, biphasic electromyographic curves obtained from dogs with CNM and control dogs were indistinguishable. The median time to attain a TOF ratio ≥ 0.9 during electromyography was 58 minutes (IQR, 43 to 68.5 minutes) for dogs with CNM and 61 minutes (IQR, 44 to 88.5 minutes) for control dogs (P = 0.53).
Discussion In the present study, there were no differences in the effects of vecuronium in dogs with autosomal recessive CNM and unaffected control dogs. The study was performed in 2 phases with the intent of evaluating both the susceptibility of these dogs to vecuronium and the duration of action and recovery profile of a clinically useful dose of the drug. Potency of an NMBA in a given species is typically established by measuring T1 amplitude depression following administration of submaximal doses of the drug. The results obtained by that approach can be managed in different ways. When only 1 submaximal dose is administered to each study animal, a minimal number of individuals must be evaluated before potency estimations are accurate.14 An alternative approach is to recruit fewer individuals and use a cumulative technique, in which doses of the NMBA are repeatedly administered to each study animal.15 The major limitation of the cumulative method is that a fraction of each dose might be redistributed or metabolized before the effects of the subsequent dose are measured (ie, a fraction of drug could be lost). Therefore, the cumulative method is usually reserved for long-lasting NMBAs, for which it is presumed that a short interval between dose administrations will have a lesser loss effect than it would for intermediate or short-acting drugs.
Despite this drawback, we chose to use a cumulative method in the present study because we had access to only 6 animals with CNM and because our objective was not to estimate the effective dose of vecuronium for a larger canine population, but rather to compare the response to submaximal doses between dogs with CNM and unaffected controls. The magnitude of T1 amplitude depression after each submaximal dose was the same between groups, suggesting that dogs with CNM are no more susceptible to vecuronium than dogs without CNM. Notwithstanding the limitations of the cumulativedose method, T1 amplitude depression after the second dose was approximately 95% in each group. This finding is not substantially different from previous findings for healthy Beagles, in which a vecuronium dose of 25 µg/kg (as a single bolus) produced T1 amplitude depression > 80%.16 Bom et al17 previously reported that, for vecuronium, the dose required to produce a 90% effect is 90 µg/kg. However, Kariman and Clutton18 showed that complete neuromuscular blockade in anesthetized dogs is achieved with 50 µg of vecuronium/kg. Moreover, when we studied recovery from vecuronium-induced neuromuscular blockade in the present study, a dose of 50 µg of vecuronium/kg produced complete neuromuscular blockade in both groups of dogs. Our results and those of Kariman and Clutton18 suggest that a 50 µg/kg dose is sufficient for producing complete neuromuscular blockade in this species. As part of the present investigation, the study dogs were administered a clinically useful dose of vecuronium that produced complete paralysis and descriptors of spontaneous recovery were measured. No differences were found in any indicator of recovery: following vecuronium injection, times for T1 amplitude to spontaneously return to 25% and 75% of baseline, recovery index, and the time to attain a TOF ratio ≥ 0.9 were each similar in the 2 groups.Taken together, these data suggested that dogs with autosomalrecessive CNM are not more sensitive to vecuronium than healthy dogs, and the duration of neuromuscular blockade or the recovery characteristics do not differ between dogs with and without CNM. In the present study, the acceleromyography monitor was used with an elastic preload, as described previously.12 During calibration, the monitor automatically detects supramaximal current and adjusts the gain of the acceleration signal so that the response is set to 100%; a value of sensitivity (gain amplification) is reported by the device. Hence, lower sensitivity indicates higher acceleration, and vice-versa. Sensitivity was consistently higher in dogs with CNM in the present study, indicating that the magnitude of the signal they produced was lower and that gain amplification was greater than findings in dogs without CNM. Although we did not measure force of contraction in the dogs with CNM, the responses to neuromuscular stimulation obtained appeared to be of a smaller magnitude than usual, especially in the most affected dogs.
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This was expected because CNM results in muscle atrophy. In accordance with our observations, reduced force of contraction during TOF stimulation has been reported in a human subject with CNM requiring general anesthesia.19 To our knowledge, this is the first prospective investigation of an NMBA in dogs with CNM. There are only isolated reports of the use of NMBAs in humans with CNM; in most of those patients, administration of an NMBA was in fact avoided during anesthesia. The use of atracurium in a patient with X-linked CNM has been reported.20 A cumulative dose of 0.5 mg of atracurium/kg was administered before TOF response was abolished; however, a clinically relevant increased sensitivity was not reported.20 In the present study, we evaluated the performance of an electromyography monitor because during our pilot investigations, we experienced substantial difficulties with the acceleromyography monitor; we were unable to calibrate the acceleromyography monitor when placed on the pelvic limb in several of the affected dogs that were subsequently used in the study. Such difficulties had not been encountered during our previous experiences with acceleromyography in healthy dogs. Judging by the degree of muscle atrophy and the poor ability to support body weight, the pelvic limbs appeared to be substantially more affected than the thoracic limbs in the dogs with CNM. Difficulties calibrating the acceleromyography monitor in human neonates and infants have been reported; for those individuals, the magnitude of evoked twitches are presumably small.21 It is likely that low signal intensity was the reason that calibration was difficult to obtain in the dogs with CNM in the present study. Unlike acceleromyography monitoring, the electromyography signal does not depend on movement but on muscle cell activation (compound motor action potential); therefore, we speculated that some of the difficulties encountered during acceleromyography would not occur during electromyography. The electromyography monitor was successfully used and calibrated in all dogs. This monitor displays the biphasic curves that are detected during neuromuscular stimulation and reports the amplitude of T1 and the TOF ratio. By inspection, the electromyographic curves of all dogs with CNM were identical to those obtained from dogs without CNM in the present study. Furthermore, no differences were found when the duration of neuromuscular blockade as measured by electromyography was compared between dogs with CNM and control dogs. It appears that electromyography may offer some advantages when measurement of neuromuscular function of muscles that produce low signals (small contraction) is required. In the present study, we elected to assess the return of the TOF ratio to ≥ 0.9 with electromyography to simplify the amount of data presented. The objective for evaluating electromyography was 306
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to assess its usefulness as a means to clinically monitor recovery from neuromuscular blockade when muscle contraction is decreased due to myopathy, but not to evaluate the bias or agreement with acceleromyography. Results of the present study indicated that there was no difference in the potency or duration of action of vecuronium in dogs with autosomal-recessive CNM and unaffected control dogs. Acceleromyography was useful for quantifying neuromuscular transmission in dogs with myopathy, although calibration of the acceleromyography monitor in the pelvic limbs of some dogs was difficult. Electromyography was successfully calibrated in the pelvic limbs of all study dogs, even in limbs for which acceleromyography calibration had failed.
Footnotes a. b. c. d. e. f. g.
DDC Veterinary, Fairfield, Ohio. TOF-Watch SX, Organon, Swords, County Dublin, Ireland. Vecuronium bromide, SUN Pharmaceutical Industries, Gujarat, India. Datex Ohmeda AS/3, GE Healthcare, Helsinki, Finland. Cleartrace, Conmed Corp, New York, NY. PetFlex, Andover Healthcare, Salisbury, Mass. Minitab, version 16.2.4, Minitab Inc, State College, Pa
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