H.-L. Amendta,*, N. Steffensena, F. J. Söbbelera, A. Schüttera, J. Tünsmeyera, K. Rohnb, S.B.R. Kästnera, A. Tipolda, V.M. Steina
a Department of Small Animal Medicine and Surgery, University of Veterinary Medicine Hannover
b Department of Biometry, Epidemiology and Information Processing, University of Veterinary Medicine Hannover, Hannover, Germany.
* Corresponding author: Hanna-Luise Amendt
Department of Small Animal Medicine and Surgery University of Veterinary Medicine Hannover Bünteweg 9
D-30559 Hannover Germany
Tel. 0049-511-953-6200
e-mail: hlamendt@tiho-hannover.de
Abstract
Ten healthy Beagle dogs without any neurological abnormalities underwent Transcranial Magnetic Stimulation (TMS) with a 50 mm circular coil and maximal intensity of 4.0 Tesla (=
100 %) to evoke magnetic motor evoked potentials (MMEPs). The influence of two sedation protocols on MMEPs measured at the M. extensor carpi radialis and the M. tibialis cranialis was evaluated. In a crossover design with a minimal wash out period of one week dogs received either acepromazine (AL) or dexmedetomidine (DL) both combined with levomethadone/fenpipramide. Parameters evaluated with TMS were onset latency and peak-to-peak amplitude.
Both sedation protocols allowed recordings of MMEPs in healthy Beagle dogs. However, the generation of reproducible measurements necessitated a stimulation intensity of at least 80
%. Onset latency decreased and peak-to-peak amplitude increased with enhancing the stimulation intensity from 70 % to 100 % maximal stimulating output. No significant differences in the MMEPs onset latencies and peak-to-peak amplitudes were detected comparing the two sedatives except of 100 % stimulation intensity.
In conclusion, MMEPs could be generated with both sedation protocols. As no clinical relevant difference in MMEPs could be detected, both drug combinations are suitable for use in clinical cases for TMS.
Keywords: Acepromazine; Dexmedetomidine; Magnetic Motor Evoked Potentials; Sedation;
Transcranial Magnetic Stimulation
Introduction
Transcranial Magnetic Stimulation (TMS) is a fast and non-invasive method to determine the functional integrity of the spinal cord (Van Soens et al., 2009). It is the only technique to evaluate the central motor pathways from motor cortex pyramidal cells to the muscle (Strain, 1990). The method of TMS has been described in numerous studies (Sylvestre et al., 1993;
Van Ham, 1994; Van Ham, 1995; da Costa et al., 2006). Michael Faraday´s principle of mutual induction where a magnetic field induces an electrical voltage in a circuit is the physical background of this technique (Barker, 1991). The magnetic field induces an electrical impulse and stimulates conductive regions such as nervous tissue (Van Soens et al., 2009). This electrical impulse leads to excitatory volleys inducing muscle twitches in the periphery recorded as magnetic motor evoked potentials (MMEPs) (De Decker et al., 2011).
Therefore, it is an ideal method to assess descending motor pathways of the spinal cord (Martin-Vaquero and da Costa, 2014).
Side effects of the TMS are rarely reported. Patients with epilepsy seem to have a higher risk to experience seizures as they might be triggered by TMS (Homberg and Netz, 1989;
Hufnagel et al., 1990), but such reports are rare (Classen et al., 1995; Nollet et al., 2003).
Although painless, TMS results in discomfort and necessitates the use of a sedation protocol that does not interfere with the measurements in dogs (Sylvestre et al., 1993). The phenothiazine acepromazine is commonly used in veterinary medicine for sedation and anxiolysis in dogs (Drynan et al., 2012) and enhances the effects of sedatives (Tobias et al., 2006). Levomethadone is a µ - receptor agonist and has the ability to improve the sedation caused by acepromazine (Tunsmeyer et al., 2012).
A previous study showed that acepromazine in combination with methadone is useful to measure MMEPs with TMS (Van Ham, 1994). However, various contraindications for acepromazine exist. The most important side effect of acepromazine is the peripheral alpha-adrenergic blockade and depression of the hypothalamic vasomotor center. This is leading to reduced blood pressure due to the provoked vasodilation (Thurmon et al., 2007). Other rarely seen side effects are reduction in packed cell volume by sequestration in the spleen at the time of effect, transient bradycardia due to sympathicolysis, long duration of action in dogs with liver dysfunction based on liver metabolism of the drug, renal hypertension, and depression of thermoregulation (Clarke, 2014). Large breeds such as St Bernard, Newfoundland, Swiss
Mountain dog, and especially Boxer show a specific sensitivity and develop bradycardia (Hall, 2001). As the measurement of MMEPs in dogs with a higher risk to experience side effects might still be useful, a sedation protocol using another drug with a differing mode of action was evaluated in healthy Beagle dogs to explore feasibility and to receive reference values for MMEPs.
Dexmedetomidine is an alpha-2-adrenergic agonist and an enantiomer of medetomidine which has sedative effects (Bloor et al., 1992). It is associated with cardiovascular effects classically described as an increase in systemic vascular resistance (Bell et al., 2011), peripheral vasoconstriction, bradycardia, decrease in CO2 (Rocchi et al., 2013), increased central venous pressure, hypertension followed by hypotension, and decreased oxygen delivery (Congdon et al., 2013). Despite these side effects it is a potent sedative, analgesic, anesthetic sparing drug and can be antagonized by an alpha-2- adrenergic antagonist like atipamezole (Bell et al., 2011).
Different sedatives might have a different impact on TMS findings (Van Soens et al. 2009).
Therefore, the objectives of the study were (1) to generate physiological TMS values for onset latency and peak-to-peak amplitude in healthy Beagle dogs, (2) to compare two different sedation protocols in respect to their effects on MMEPs parameters, and (3) to identify which drug combination is more useful for conducting TMS under clinical conditions.
Material and Methods Dogs
Ten healthy Beagle dogs, nine male and one female, from the colony of the Department of Small Animal Medicine and Surgery, University of Veterinary Medicine Hannover, Germany were recruited for TMS with two different sedation protocols. The procedures and examinations were approved and performed according to ethical approval and national regulations for animal welfare (animal experiment number 33.14-42502-04-13/1277). The dogs mean body weight was 16.7 kg (range: 10.2 - 20.6 kg) and the mean age was 32 months (range: 27 - 73 months). All dogs underwent regular general physical and blood examinations with unremarkable results and did not have a history of neurological disease. Before the TMS procedure all dogs were subjected to a routine neurological examination and did not show any
neurological deficits. Dogs were deprived of food but not water on the day of the TMS examination.
Sedation protocols
An 18 gauge intravenous catheter (B. Braun Melsungen AG, Melsungen, Germany) was percutaneously placed in one V. saphena lateralis of the right or left pelvic limb. Two different sedation protocols were tested in each dog in a crossover design with a washout phase of at least one week between the two protocols. The protocols consisted of either acepromazine (AL) (0.05 mg/kg, Vetranquil® 1%, CEVA Tiergesundheit GmbH, Düsseldorf, Germany) given slowly intravenously (IV) or dexmedetomidine (initial bolus 5 µg/kg intramuscularly (IM); Dexdomitor®, Orion Pharma, Bad Homburg, Germany; DL) followed by 1 µg/kg/h dexmedetomidine IV continuous rate infusion into the lateral saphenous vein each in combination with levomethadone/fenpipramide (0.2 mg/kg for AL IV, 0.2 mg/kg for DL IM; L-Polamivet®, Intervet Deutschland GmbH, Unterschleißheim, Germany). A crystalline continuous rate infusion (B. Braun Melsungen AG) was given IV in both sedation protocols. The dogs were positioned in lateral recumbency as soon as permitted by their sedative state. In both sedation protocols TMS could be started a few minutes after drug administration.
Transcranial magnetic stimulation of the motor cortex (TMS)
The TMS was performed as described by Van Ham et al. 1994 with minor modifications.
The MMEPs were generated by a Magstim 200² (Magstim Company Limited, Carmarthenshire, UK) with a 50 mm circular coil and a peak magnetic field capacity of 4.0 Tesla (= 100 % intensity). The Magstim 200² is a single pulse monophasic stimulator for the use of cortical and peripheral stimulation with a maximum frequency at maximum power of 0.25 Hz. The upper frequency and the maximum frequency burst were 0.5 Hz. The minimal pulse interval between each measurement was two seconds and the total recording time was maximal one hour.
The magnetic coil was placed tangentially to the skull with the center of the coil placed over the vertex and with close contact to the skin for stimulating the motorcortex (Fig. 1). The induced electric field ran in a clockwise direction across the nerve roots and generated a
muscle contraction which was measurable by an electromyograph Nicolet NicVue 2.9.1 (Natus Medical Incorporated).
Figure 1: The magnetic coil was placed tangentially to the skull with the centre of the coil placed over the vertex and with close contact with the skin to stimulate the motorcortex.
Magnetic motor evoked potential recordings (MMEPs)
Four individual measurements each with different field capacity intensities (70 %, 80 %, 90
%, and 100 %) were performed in the extensor carpi radialis and the tibialis cranialis muscles, respectively, on each side after stimulating the contralateral cortex. Monopolar needle electrodes (Natus Medical Incorporated, Planegg, Germany) with a length of in total 1 m and a non-insulated needle of 23 mm were inserted into the muscle belly with the tip of the recording electrode positioned in the middle of the muscle. For the thoracic limbs the position of the muscle needle was cranial to the lateral humeral epicondyle directly in the middle of the M. extensor carpi radialis. In case of the pelvic limbs the position was laterally to the distal end of the tibial crest in the middle of the M. tibialis cranialis. The recording electrode was connected to an electromyograph. The reference electrode (CareFusion, Hoechberg, Germany) was a subdermal needle and positioned subcutaneously at the level of the carpal and the tarsal joints approximately 1 cm distally to the muscle needle, respectively. The ground electrode (CareFusion) was placed subcutaneously axially at the level of Th1-Th3 for the thoracic limbs and L4-L6 for the pelvic limbs. Recordings were obtained using the electromyograph (Natus Medical Incorporated) and the VikingSelect-Software Version 11.0 (Viasys healthcare, CareFusion, Höchberg, Germany).
Parameters measured during TMS were onset latency and peak-to-peak amplitude of MMEPs that were assessed by using the cursors on the oscilloscope. Onset latency was measured as distance between stimulus artefact and deflection from the baseline in a positive or negative direction. The peak-to-peak amplitude was defined between the two largest peaks of adverse polarity and expressed in millivolt (mV). The neuronal path length of each dog was assessed by determination of the distance between the vertex to the recording electrode in M. extensor carpi radialis and M. tibialis cranialis contralateral to the stimulated site, respectively.
Monitoring of the dogs
Dogs were monitored at the AL protocol for heart rate by auscultation while the drug was given IV until deep sedation was reached. Body temperature was documented every ten minutes in both sedation protocols. Due to more expected side effects in case of the DL protocol the monitoring was more complex with an anesthesia device (Dräger, Drägerwerk AG, Lübeck, Germany) including an additional permanent electrocardiogram (GE healthcare Finland Oy, Helsinki, Finland) to detect bradycardia, a pulse oximeter placed on the tongue or flew (GE healthcare Finland Oy) to control oxygen saturation and surveillance of the respiratory rate for monitoring respiratory depression.
Response to TMS
Dogs were observed if TMS resulted in discomfort like raising the head, moving the limbs or being awake after stimulation.
Statistical analysis
All MMEPs for each stimulation intensity of the left and right side were summed to obtain a mean value for the M. extensor carpi radialis and the M. tibialis cranialis, respectively.
The objective of the study was to identify possible differences in mean onset latency and mean peak-to-peak amplitude depending on stimulation intensity (70 %, 80 %,90 %, and 100%), side (left and right), sedation (AL and DL) and limb position (thoracic and pelvic limbs).
All data were included into a descriptive analysis. Normal distribution of the model residuals of onset latency and peak-to-peak amplitude were confirmed by Kolmogorov-Smirnov-Test
and visual assessment of q-q-plots. Both endpoints were right skewed distributed therefore logarithmic transformation was performed prior to analysis; hence the results were tabulated on the original scale after exponential retransformation.
For analysing the four described effects to the endpoints, a four way ANOVA was calculated for repeated measurements, considering interactions between effects. Post-hoc tukey test was calculated for multiple pair wise comparisons, regarding familywise error rate.
Analyses were carried out with the statistical software SAS, version 9.3 (SAS Institute, Cary, NC, USA). For the analysis of the linear model, the MIXED procedure was used. Differences were considered to be significant if P < 0.05.
Results Sedatives
Dogs tolerated TMS with both sedation protocols and no manual restraint of the dogs was necessary due to a deep sedation in 10/10 dogs. The body temperature decreased during TMS after sedation with acepromazine and levomethadone (Table 1). However, thermoregulation was impaired in the dogs when sedated with AL also some hours after TMS. Bradycardia was observed with the DL sedation protocol in 10/10 dogs as well as sticky and pale mucosae after the DL bolus was given IM but not to a degree that intervention was necessary (Table 1).
Furthermore, respiratory depression was documented in the DL protocol in 10/10 dogs.
However, no machine assisted ventilation was necessary in any of the dogs. Slightly decreased oxygen saturation was detected when performing TMS under DL sedation which was most probably due to vasoconstriction (Table 1). The dogs showed a more constant deep sedation during the TMS with the DL sedation compared to the AL protocol where the dogs were easily aroused and disturbed by the auditory stimuli generated by the magnetic stimulator. No side effects during or subsequent to the TMS were noted in any of the patients.
sedation-protocol body temperature saturation measured during Transcranial Magnetic Stimulation (TMS) in ten healthy Beagle dogs under sedation with acepromazine combined with levomethadone (AL) or dexmedetomidine plus levomethadone (DL). No data for heart rate, respiratory rate, and oxygen saturation was documented for the AL protocol.
Assessment of MMEPs
MMEPs could be evoked by TMS in each Beagle dog with both sedation protocols. However, a stimulation intensity of at least 80 % was necessary to achieve reproducible MMEPs in 10 of 10 Beagle dogs whereas a stimulation intensity of 70 % only resulted in a generation of MMEPs in all 4 limbs in 5 dogs. Therefore, a stimulation intensity of 80-100 % was used to determine mean and standard deviation of physiological values. Increasing the stimulation intensity of the TMS (70 %, 80 %, 90 %, and 100 %) induced a reciprocal effect on onset latency and peak-to-peak amplitude. Onset latency decreased and peak-to-peak amplitude increased significantly when increasing the maximal stimulation output from 70 % to 100 % (Fig. 2).
MTC
Figure 2: Four measurements with different intensities (70 %, 80 %, 90 %, and 100 %) were performed in the right and left M. extensor carpi radialis (MECR) (not shown in Fig. 2) and the M. tibialis cranialis (MTC) and averaged to obtain single values under sedation with acepromazine and levomethadone (AL). Only one MMEP was measurable at stimulation intensity of 70 % in dog number 4. In dog 8, 9, and 10 no MMEPs could be evaluated at a stimulation intensity of 70 %. Onset latency decreased and peak-to-peak amplitude increased significantly (P < 0.001) when increasing the maximal stimulation output from 70 % to 100 % maximal stimulating output. Moreover, peak-to-peak amplitude illustrated more individual variability.
The waveform of the MMEPs was in most cases biphasic with a large positive deflection and a subsequent negative deflection from the baseline, or vice versa (Fig. 3). Incidentally polyphasic waves occurred and occasionally there was variation of the dimension in peak-to-peak amplitude of the individuals.
Figure 3: The Magnetic Motor Evoked Potentials` (MMEPs) waveform at a stimulation intensity of 80 % from the maximal output of 4.0 Tesla recorded from a Beagle sedated with dexmedetomidine and levomethadone is mostly biphasic with a large positive deflection and a subsequent negative deflection from the baseline. Vertical bars indicate onset latency measured by using the cursor on the oscilloscope and describes the time from stimulus artefact to deflection. Horizontal bars indicate peak-to-peak amplitude measured between the two largest peaks of adverse polarity.
As expected, no statistical difference was found between right and left side MMEPs for thoracic and pelvic limbs, respectively and onset latency and peak-to-peak amplitude of the MMEPs in the thoracic limbs were significantly lower than in the pelvic limbs (Table 2). The mean of neuronal path length of the thoracic limbs was 50.3 cm with a range of 34.0 – 60.0 cm and for the pelvic limbs 85.9 cm (85.0–96.0 cm).
Two different sedation protocols were evaluated in this study. With the AL protocol slightly shorter onset latencies of the MMEPs could be observed in all stimulation intensities. For the stimulation intensity of 100 % this difference reached the level of significance (thoracic limbs P < 0.0267; pelvic limbs P < 0.0452) although the difference was only marginal.
Extremity
Table 2: Mean onset latencies and peak-to-peak amplitudes for Magnetic Motor Evoked Potentials (MMEPs) with Transcranial Magnetic Stimulation (TMS) recorded from the thoracic and pelvic limbs with stimulation intensities of 80 %, 90 %, and 100 % (= 4.0 Tesla) in ten healthy Beagle dogs sedated with acepromazine (AL) or dexmedetomidine (DL) in combination with levomethadone.
95 % confidence interval.
arecorded from the right and left extensor carpi radialis muscle
brecorded from the right and left tibialis cranialis muscle
*statistical significance P < 0.05.
The means of onset latency for the thoracic limbs were for AL protocol 12.02 ms and for DL 12.65 ms. In case of the pelvic limbs the mean for the AL protocol was 17.41 and 18.18 ms for the DL protocol (Fig. 4; Table 2). Also, similarly, the peak-to-peak amplitude was slightly higher in the AL protocol compared to the DL sedation protocol. However, no significant
(A)
(B)
Figure 4: (A) Onset latencies and (B) peak-to-peak amplitude results of both sedation protocols from 80 % to 100 % (4.0 Tesla) in the extensor carpi radialis muscle (MECR) are illustrated. With a stimulation intensity of at least 80 % of 4.0 Tesla, reproducible MMEPs were measurable in all 10 Beagle dogs. The acepromazine sedation protocol (AL) combined with levomethadone generated a slightly higher peak-to-peak amplitude at each stimulus intensity (80 %, 90 %, and 100 %) and slightly shorter onset latency compared to the dexmedetomidine sedation protocol (DL) combined with levomethadone. However, the differences are only marginal.
Discussion
Two sedation protocols were compared in the present study, acepromazine and dexmedetomidine, both in combination with levomethadone. Previous studies evaluated the effects of the sedatives acepromazine and medetomidine combined with methadone in dogs.
Van Ham (1994) detected a significant difference for peak-to-peak amplitude, but not for onset latency. On the other hand, Van Soens et al. (2009) identified no significant difference in MMEP parameters between acepromazine plus methadone and medetomidine. In this study TMS generated reproducible MMEPs in each dog with the two sedatives acepromazine and dexmedetomidine which is the dextro-rotary isomer of medetomidine with potent sedative and analgesic effects. Though, the sedation with acepromazine in combination with levomethadone (AL) results in a slightly larger peak-to-peak amplitude and shorter onset latency compared to dexmedetomidine combined with levomethadone (DL), this did not reach the level of statistical significance (Table 2). A possible explanation for the slightly decreased onset latency and increased peak-to-peak amplitude is dexmedetomidine’s action on alpha-2-adrenergic receptors that leads to muscle relaxation (Nagore et al., 2013). Furthermore, differences were assessed for stimulation intensity of 100 % (thoracic limbs P < 0.0267;
pelvic limbs P < 0.0452) between the AL and DL sedation protocols (Table 2). However, the differences in numerical values are very small and reached the level of significance only due to a very low standard deviation. Moreover, the reciprocal effect of decreasing onset latency and increasing peak-to-peak amplitude with increasing the TMS stimulation intensity was seen in both sedatives. Therefore, these differences are rather mathematically and physiologically irrelevant and do not represent an effect of the sedative drugs on the MMEPs generated.
Both, the AL and DL protocols provided a safe and sufficient sedation in the dogs to perform the TMS. The dogs treated with DL were constantly deep sedated during the whole process of TMS without any signs of stress or unpleasant moments. Although the monitoring of the DL sedation protocol was more complex due to the potential side effects, it offers the possibility to antagonize the effects of dexmedetomidine with the alpha-2-adrenergic antagonist atipamezol (Nguyen et al., 1992). However, in this study, the noted side effects
Both, the AL and DL protocols provided a safe and sufficient sedation in the dogs to perform the TMS. The dogs treated with DL were constantly deep sedated during the whole process of TMS without any signs of stress or unpleasant moments. Although the monitoring of the DL sedation protocol was more complex due to the potential side effects, it offers the possibility to antagonize the effects of dexmedetomidine with the alpha-2-adrenergic antagonist atipamezol (Nguyen et al., 1992). However, in this study, the noted side effects