animals

Article
Effect of Medetomidine, Dexmedetomidine,
and Their Reversal with Atipamezole on the
Nociceptive Withdrawal Reflex in Beagles
Joëlle Siegenthaler 1 , Tekla Pleyers 1 , Mathieu Raillard 1,2 , Claudia Spadavecchia 1
and Olivier Louis Levionnois 1, *
1 Section of Anaesthesiology and Pain Therapy, Department of Clinical Veterinary Sciences, Vetsuisse Faculty,
University of Berne, 3012 Bern, Switzerland; [email protected] (J.S.);
[email protected] (T.P.); [email protected] (M.R.);
[email protected] (C.S.)
2 University Veterinary Teaching Hospital, School of Veterinary Science, Faculty of Science,
The University of Sydney, Sydney 2006, Australia
* Correspondence: [email protected]




Received: 17 June 2020; Accepted: 17 July 2020; Published: 21 July 2020


Simple Summary: Medetomidine, an alpha-2 agonist routinely used to provide sedation and
pain relief in dogs, is a mixture of dexmedetomidine and levomedetomidine in equal proportions.
Dexmedetomidine, considered to be the only active component in the mixture, is also marketed
alone. Sedation caused by both formulations can be reversed using atipamezole, which shortens
recovery. Dexmedetomidine provides analgesic effects similar to medetomidine, but it remains
unclear at which dose and whether the analgesic effects of medetomidine or dexmedetomidine
disappear once atipamezole is injected. The present trial aimed at elucidating these uncertainties
using the nociceptive withdrawal reflex model. This model allows for quantification of analgesia by
measuring specific activity from muscles involved in limb withdrawal in response to mild electrical
stimulation. In eight beagles, the model was applied to compare the extent of pain relief provided by
medetomidine and dexmedetomidine and to investigate whether complete reversal occurs after the
administration of atipamezole. No difference in analgesic efficacy was identified between the two
formulations. Both sedation and pain relief terminated rapidly when atipamezole was administered.
These findings indicate that medetomidine and dexmedetomidine provide comparable levels of pain
relief and that additional analgesics may be necessary when atipamezole is administered to dogs
experiencing pain.

Abstract: The objectives were: (1) to compare the antinociceptive activity of dexmedetomidine and
medetomidine, and (2) to investigate its modulation by atipamezole. This prospective, randomized,
blinded experimental trial was carried out on eight beagles. During the first session, dogs received
either medetomidine (MED) (0.02 mg kg−1 intravenously (IV)] or dexmedetomidine (DEX) [0.01 mg
kg−1 IV), followed by either atipamezole (ATI) (0.1 mg kg−1 ) or an equivalent volume of saline (SAL)
administered intramuscularly 45 min later. The opposite treatments were administered in a second
session 10–14 days later. The nociceptive withdrawal reflex (NWR) threshold was determined using
a continuous tracking approach. Sedation was scored (0 to 21) every 10 min. Both drugs (MED
and DEX) increased the NWR thresholds significantly up to 5.0 (3.7–5.9) and 4.4 (3.9–4.8) times the
baseline (p = 0.547), at seven (3–11) and six (4–9) minutes (p = 0.938), respectively. Sedation scores
were not different between MED and DEX during the first 45 min (15 (12–17), p = 0.67). Atipamezole
antagonized sedation within 25 (15–25) minutes (p = 0.008) and antinociception within five (3–6)
minutes (p = 0.008). Following atipamezole, additional analgesics may be needed to maintain
pain relief.

Animals 2020, 10, 1240; doi:10.3390/ani10071240 www.mdpi.com/journal/animals

Animals 2020, 10, 1240 2 of 14

Keywords: antinociception; atipamezole; dexmedetomidine; dog; nociceptive withdrawal

reflex; sedation

1. Introduction
Medetomidine and dexmedetomidine are alpha-2 adrenoreceptor agonists routinely used to
produce sedation and analgesia in dogs [1–4]. Medetomidine is a racemic mixture of two optical
isomers, levomedetomidine and dexmedetomidine [5]. As dexmedetomidine is considered the only
active isomer, it is commonly administered at half of the administered dose of medetomidine to obtain
similar levels of sedation [6,7] and antinociception [4,6]. However, contrasting evidence indicates that
dexmedetomidine induces weaker sedation [8] and stronger antinociception [3] than medetomidine,
thus raising some questions about the common assumption of equipotency.
So far, the antinociceptive effects of medetomidine and dexmedetomidine have been mainly
evaluated using behavioral models based on thermal, electrical or mechanical stimulation. These
models typically rely on the direct observation of nocifensive reactions as end-points [3,4,6,9–15].
When used to evaluate antinociception induced by alpha-2 adrenoreceptor agonists, the concomitant
sedation and muscle relaxation elicited by these drugs can introduce significant bias [2]. In addition,
the process of nociceptive threshold determination can generally be performed only at relatively wide
time intervals (every 10–30 min) [3,4,9–11,13,15]. A more accurate comparison of the antinociceptive
properties of these drugs and their temporal characteristics might be obtained with the nociceptive
withdrawal reflex (NWR) model [16]. Based on non-invasive transcutaneous electrical stimulation of
peripheral nerves and surface electromyographic (EMG) recordings, it allows a reliable determination
of the nociceptive threshold through the neurophysiological characterization of the evoked response.
The NWR model, validated in several animal species [16–18], has been applied to quantify the
antinociceptive effects of analgesic and anesthetic drugs [19–21], including alpha-2 agonists [22–24].
The recent introduction of an automated threshold tracking methodology, allowing an almost continuous
NWR threshold determination, further improved the model [25], making it ideal for highlighting
subtle temporal differences between drugs, as it would be required to reliably compare similar drugs
such as medetomidine and dexmedetomidine.
Atipamezole, an alpha-2 adrenoreceptor antagonist, is commonly administered to reverse
medetomidine- or dexmedetomidine-induced sedation and to shorten recovery. Previous work
suggests that atipamezole might contemporaneously reverse antinociception [4,26], which can be
considered a rather undesired property in clinical settings. To date, specific investigations on potential
differential effects of atipamezole on sedation versus antinociception are scarce and do not provide
conclusive evidence. Again, the NWR model could provide interesting quantitative data to fill this
clinically relevant knowledge gap.
The aims of this study were: (1) to compare the effects of intravenous (IV) dexmedetomidine and
medetomidine on sedation and antinociception in dogs, and (2) to investigate their differential
modulation by intramuscular (IM) atipamezole. We intended to test the hypotheses that (1)
dexmedetomidine at half the dose of medetomidine would evoke higher NWR thresholds than
medetomidine using the continuous NWR threshold tracking method, and (2) following atipamezole
administration, the NWR thresholds would return to baseline later than sedation scores.

2. Materials and Methods

This experiment was approved by the Cantonal Committee for Animal Experimentation (approval
number 30356). During the study, the investigators were unaware of the treatments administered
(blinded design).
Animals 2020, 10, 1240 3 of 14

2.1. Sample Size, Design

The first objective of the study was to detect a difference in peak NWR thresholds after
administration of medetomidine or dexmedetomidine. In previous studies it was shown that the
median NWR threshold of non-medicated dogs is approximately 2.5 (2–3) mA [19]. Medetomidine
was expected to increase it by 5 (4–6) times (personal experience). A 20% difference between the NWR
thresholds induced by dexmedetomidine compared to medetomidine was arbitrarily considered a
relevant endpoint. With a crossover design, eight dogs at least were considered necessary (Wilcoxon
signed-rank test, paired, two-tailed, effect size = 1.4, α = 0.05, 1-β = 0.9, GPower 3.1, Germany).
The second objective of the study was to detect a difference in duration of effect between
antinociception and sedation after administration of atipamezole. Reversal of sedation is expected to
occur within 10 (8–12) minutes [4]. A 10 min delay for reversal of antinociception was considered a
relevant endpoint. With a crossover design, three dogs at least were considered necessary (Wilcoxon
signed-rank test, paired, two-tailed, effect size = 5, α = 0.05, 1-β = 0.9, GPower 3.1, Germany).
A parallel study evaluating selected respiratory effects of medetomidine and dexmedetomidine
using electrical impedance tomography (EIT) was also carried out at the same time (data reported
elsewhere [27]). Absence of interference between EIT and NWR recordings was confirmed by
manufacturers of both devices and during the experiment.

2.2. Animals
A total of eight intact experimental beagles (two females and six males) were enrolled in this
study. Dogs were housed with other kennel-mates in groups of three, had access to an enriched indoor
and outdoor course. They were not involved in any other experiments, and did not receive any drug
other than anthelminthic in the two months preceding the trial. They were considered healthy based
on physical examination and selected hematology and blood chemistry analysis. Total body weight
(BW) was measured for each dog before each session. All the dogs had a body condition score of 5 or 6
on a scale of 9 (body condition system, Nestlé Purina, Vevey, Switzerland). Mandibular lymph nodes
were moderately increased in size in five animals; this was attributed to mild dental disease without
any sign of pain.

2.3. Randomization, Blinding and Treatment

Dogs were sedated twice (crossover design) with 10 to 14 days wash-out between the two
sessions. Each session included two consecutive phases: (1) sedation at time zero (T0 ), and (2)
reversal, 45 min later (T45 ). The sedation (Phase 1, T0 –T45 ) consisted of either medetomidine (“MED”;
Domitor, 1 mg mL−1 ; Orion Pharma, Switzerland; 0.02 mg kg−1 BW IV) or dexmedetomidine (“DEX”;
Dexdomitor, 0.5 mg mL−1 ; Orion Pharma, Switzerland; 0.01 mg kg−1 BW IV). The reversal (Phase 2,
T45 –T210 ) consisted of either atipamezole (“ATI”; Antisedan, 5 mg mL−1 ; Orion Pharma, Switzerland;
0.1 mg kg−1 BW IM) or saline (“SAL”; NaCl 0.9%, B Braun Melsungen AG, Melsungen, Germany;
volume equivalent to ATI, IM).
Before the experiment, dogs were randomly assigned (http://www.randomization.com) to one
of four blocks (MED/ATI, MED/SAL, DEX/ATI, DEX/SAL, n = 2 per block) for the first session.
The opposite treatments were administered during the second session. For example, a dog receiving
MED/ATI at the first session would receive DEX/SAL at the second. Treatments were prepared each
day by a person not involved in the data collection but familiar with the study design; similar volumes
of colorless solutions were administered so investigators were unaware of the treatments administered.
Experiments took place over a 4-week period in November and December, 2018. Each day, only
one dog was sedated. A quiet, dedicated room within the same facility where dogs were housed was
used. The dogs were acclimatized to the room and to the investigators the day before the experiment.
A standard meal was provided in the night before; water remained available overnight. Experiments
started around 09:00 a.m. for all dogs.
Animals 2020, 10, 1240 4 of 14

2.4. Animal Preparation

The day before the experiment, hair was clipped over stimulation and recording sites on the left
hindlimb, at the site of insertion of IV cannulas and around the thorax for the EIT belt.
On the day of the experiment, dogs were weighed, the identification number (electronic chip)
was checked and a physical examination was performed. If the animal presented with abnormalities
at physical examination or developed unexpected complications during the study, it would be
excluded and replaced. A eutectic mixture of local anesthetics (Emla cream 5%; Aspen Pharma GmbH,
Switzerland) was applied over both right and left cephalic veins and covered with an occlusive dressing
approximately one hour before placement of intravenous catheters (22 gauge, 25mm, Optiva 2IV
Catheter; Smiths Medical International Ltd., Lower Pemberton, UK).
Electrode positioning was standardized to avoid differences among body region and biological
function [28–30]. The stimulation and recording sites were shaved and degreased and gentle abrasion
was performed with abrasive tape (Red Dot Trace Prep; 3M, Switzerland). Two self-adhesive stimulation
electrodes (Bluesensor N; Ambu, Germany) were placed 0.5 cm apart over the left lateral plantar digital
nerve with the anode in the distal position. Two self-adhesive recording electrodes (Bluesensor N;
Ambu, Germany) were placed 0.5 cm apart over the left tibialis cranialis muscle, 3 cm distal to the
knee joint. A self-adhesive ground electrode (Bluesensor VL; Ambu, Germany) was placed over the
latero-distal femoral epicondyle. The electrodes were fixed with bandages to avoid displacement.
Electrode impedance was measured continuously, and the electrodes were replaced if above 3 kΩ.
The dogs were positioned and gently maintained in right lateral recumbency on a soft pillow.
At the end of the experiment (latest T210 ), the electrodes were removed and a layer of multipurpose
antiseptic cream (Bepanthen Onguent; Bayer AG, Switzerland) was applied to avoid skin irritation
at the electrode sites. A heparin-based cream (Hirudoid cream; Medinova AG, Switzerland) was
applied at the sites of venous catheterization to prevent phlebitis. The dogs were monitored for a
further 4 h to prevent complications, discomfort or re-sedation, before being re-introduced to their
normal environment.

2.5. NWR Threshold Determination

After instrumentation, electrical stimulation and EMG recording were initiated using a dedicated
unit (Dolosys pain tracker; Dolosys GmbH, Germany). Each stimulation consisted of a train of five
rectangular pulses (1 millisecond, 200 Hz). The EMG was recorded at 1 kHz over 500 milliseconds,
starting 100 milliseconds before stimulation. The 100-millisecond time window preceding stimulation
was used to evaluate the EMG background noise (“noise range”). The time window between 30 and
100 milliseconds after stimulation was used to evaluate the NWR (“NWR range”). The response
was considered positive when the interval peak Z score was above 10, meaning that the peak EMG
amplitude within the NWR range had to exceed the mean EMG amplitude within the noise range by
at least 10 times its standard deviation. The first stimulation started at 1 mA. The NWR threshold was
then evaluated following an up-and-down bracketing design with maximal cut-off intensity set at 50
mA. It was possible to stop stimulation at any time in case of evident discomfort or pain (vocalization
or escape movement). Stimulations were repeated every 10 s (with 30% interval randomization)
changing intensity by steps of 0.3 mA. When the intensity increased or decreased three consecutive
times, the step became 0.5 mA until the intensity changed direction again. A measurement was
automatically discarded when the EMG amplitude exceeded 10 µV within the noise range. In this case,
stimulation was repeated at the same intensity. The NWR threshold was automatically estimated after
each stimulation through a logistic regression of the last 12 valid stimuli [25].
A stable baseline NWR threshold measurement for at least 5 min was obtained before the first
treatment (phase 1). Medetomidine or dexmedetomidine were injected over a minute in the right
cephalic IV catheter. The end of the injection was defined as T0 . The threshold was then continuously
determined until it returned to baseline, or until the dogs became intolerant to the experimental settings
(impatient to move, not staying quiet or lying), or until 210 min after T0 (T210 ). If the measurements
Animals 2020, 10, 1240 5 of 14

were discontinued before T210 , the threshold was arbitrarily given the value of its baseline in the
remaining time period to avoid missing data for group comparison.

2.6. Sedation
The depth of sedation was scored at baseline and every 10 min until the end of the experiments
using a previously described descriptive scale [31]. Seven items were evaluated. The scores attributed
to each individual item were summed to obtain a total sedation score ranging from 0 (no sedation) to
21 (deepest possible sedation). According to the scoring system, sedation was defined as mild with
a score of 4 to 6, moderate with a score of 6 to 12 and profound with a score of 13 to 21. The same
two investigators, both unaware of the administered treatment, always scored the depth of sedation
together (agreement for each item score).

2.7. Physiological Variables

Heart rate (HR, beats minute−1 ), respiratory rate (f R, breaths minute−1 ) and temperature (T, ◦ C)
were measured (thorax auscultation and rectal thermometer) and recorded every 10 min. Thoracic
impedance was continuously recorded by electrical impedance tomography (Swisstom BB2, Swisstom
AG, Switzerland) [27]. Blood samples (2 mL) were obtained from the left IV catheter into EDTA
collection tubes at 2, 4, 8, 16, 30, 60, 90, 120 and 180 min after T0 for future pharmacokinetic analysis
(not performed yet).

2.8. Data Analysis

To ease comparison between animals and treatments, the mean baseline NWR threshold was
calculated over the 5 min immediately preceding drug administration. The mean relative NWR threshold
(absolute threshold divided by its baseline) [32] was then calculated for every minute after T0 .
Data are presented as median (interquartile range). In accordance with the recommendations
of the American Statistical Association [33,34], differences in median are presented together with the
p-value for the respective statistical test (Sigmaplot v14.0; Systat software Inc., San Jose, CA, USA).
Since p-values below 0.001 could not be determined, they are reported as p < 0.001.
For phase 1 (T0 –T45 ), differences between treatments (paired data, MED versus DEX) for baseline
NWR thresholds, time to relative NWR threshold peak and peak intensity were tested using a Wilcoxon
signed-rank test. Differences between treatments for NWR thresholds and sedation scores were tested
using a two-way ANOVA for repeated measures, accounting for effect of time and treatment within
each subject. Post hoc pairwise analysis was performed with a Holm–Sidak test.
For phase 2 (T45 –T210 ), differences between treatments (paired data, ATI versus SAL) for relative
NWR thresholds at T44 , time to relative NWR threshold ≤ 1.5, time to sedation score ≤ 3 and comparison
of the two latter variables were tested using a Wilcoxon signed-rank test. Differences between treatments
for NWR thresholds and sedation scores were tested using a two-way ANOVA for repeated measures,
accounting for effect of time and treatment within each subject. Post hoc pairwise analysis was
performed with a Dunnett’s test.
Additionally, differences between MED/SAL (n = 4) and DEX/SAL (n = 4) subgroups and between
MED/ATI (n = 4) and DEX/ATI (n = 4) subgroups for time to relative NWR threshold ≤ 1.5 and time to
sedation score ≤ 3 were evaluated. Two-way ANOVA for repeated measures and Wilcoxon signed-rank
tests were used. Correlation between NWR thresholds and sedation scores was tested using Pearson
product moment and linear regression.

3. Results
The dogs were seven (5–8) years old and weighed 12.9 (11.7–15.8) kg. One female presented signs
of proestrus during the second session of the trial but was not excluded. One male dog vomited shortly
after administration of the alpha-2 adrenergic agonist on both sessions (once with medetomidine,
once with dexmedetomidine), without further complication. All dogs tolerated well the experimental
Animals 2020, 10, 1240 6 of 14
Animals 2020, 10, x 6 of 14

the experimental
setting setting in lateral
in lateral recumbency. recumbency.
Electrical Electrical
stimulations couldstimulations could
be continued bedogs
in all continued in all dogs
until return to a
until return
relative NWR tothreshold
a relative NWR
of onethreshold of one (0.9–1.1)
(0.9–1.1) within within
the duration of the duration of the
experiment, experiment,
except except
in one dog on
in one
one dog on(group
occasion one occasion
MED/SAL) (group
when MED/SAL) when were
measurements measurements
stopped atwere
T210 stopped
while theatrelative
T210 while
NWRthe
relative NWR
threshold was still > 1.5 and
threshold wassedation
still > 1.5score was > 5. score was > 5.
and sedation

3.1.
3.1. Phase
Phase 11 (T
(T00–T 44,, MED
–T44 MED Versus
Versus DEX)
DEX)

3.1.1.
3.1.1. Sedation
Sedation
Sedation scores>> 13
Sedationscores 13 (profound
(profound sedation)
sedation) were
were observed
observedin inall
all dogs
dogs within
withinaa minute
minuteafter
afterthe
the end
end
of
of the IV injection of both MED and DEX. Peak sedation scores of 17 (16–17) were reached 10after
the IV injection of both MED and DEX. Peak sedation scores of 17 (16–17) were reached 10 min min
Tafter
0 in all
T0 dogs. Sedation
in all dogs. scores scores
Sedation varied varied
significantly over time
significantly (p <time
over 0.001),
(p <but werebut
0.001), notwere
different
not between
different
treatments (MED versus DEX, p = 0.67, Figure 1a).
between treatments (MED versus DEX, p = 0.67, Figure 1a).

Figure 1. Median (interquartile range) sedation scores (from 0 to 21) recorded in 8 beagles after
Figure 1. Median (interquartile range) sedation scores (from 0 to 21) recorded in 8 beagles after
administration in a crossover design of (a) intravenous medetomidine (MED, 0.02 mg kg−1−1, n = 8) or
administration in a crossover design of (a) intravenous medetomidine (MED, 0.02 mg kg , n = 8) or
dexmedetomidine (DEX, 0.01 mg kg−1 , n = 8) at T , and (b) intramuscular atipamezole (ATI, 0.1 mg
dexmedetomidine (DEX, 0.01 mg kg−1, n = 8) at T00, and (b) intramuscular atipamezole (ATI, 0.1 mg
kg−1 ) or an equivalent volume of saline (SAL) at T . Data are presented slightly apart from the actual
kg−1) or an equivalent volume of saline (SAL) at T45 45. Data are presented slightly apart from the actual
measurement time points to ease readability.
measurement time points to ease readability.
3.1.2. Nociceptive Withdrawal Reflexes
3.1.2. Nociceptive Withdrawal Reflexes
Baseline NWR thresholds were similar between groups: 1.1 (0.9–1.3) mA for MED and 1.2 (1.1–1.5)
mA forBaseline
DEX (pNWR thresholds
= 0.313). wereNWR
Relative similar between varied
thresholds groups:over
1.1 (0.9–1.3)
time (p < mA for MED
0.001) andbetween
but not 1.2 (1.1–
1.5) mA for DEX (p = 0.313). Relative NWR thresholds varied over time (p < 0.001)
treatments (MED versus DEX, p = 0.317). Peak relative NWR thresholds were reached at seven (3–11) but not between
treatments
minutes for(MED versus
MED and sixDEX,
(4–9) pminutes
= 0.317).for
Peak
DEXrelative NWR Peaks
(p = 0.938). thresholds
of 4.4were reached
(3.9–4.8) timesat the
seven (3–11)
baseline
minutes for MED and six (4–9) minutes for DEX (p = 0.938). Peaks of 4.4 (3.9–4.8) times
and 5.0 (3.7–5.9) times the baseline were reached for MED and DEX, respectively (p = 0.547, Figure 2a). the baseline
and corresponded
This 5.0 (3.7–5.9) times the baseline
to a difference were which
of 14%, reached forbelow
was MEDthe andpredefined
DEX, respectively (p = 0.547,ofFigure
relevant endpoint 20%.
2a). This corresponded to a difference of 14%, which was below the predefined relevant endpoint of
20%.
Animals 2020, 10, 1240 7 of 14
Animals 2020, 10, x 7 of 14

Figure 2. Median (interquartile range) relative nociceptive withdrawal reflex (NWR) thresholds
Figure 2. Median (interquartile range) relative nociceptive withdrawal reflex (NWR) thresholds
recorded in 8 beagles after administration in a crossover design of (a) intravenous medetomidine (MED,
recorded
0.02 mg kgin
−1 8
, nbeagles
= 8) orafter administration(DEX,
dexmedetomidine in a crossover
0.01 mg kgdesign of8)(a)
−1 , n = at intravenous medetomidine
T0 , and (b) intramuscular
(MED, 0.02 mg kg −1, n = 8) or dexmedetomidine (DEX, 0.01 mg kg−1, n = 8) at T0, and (b) intramuscular
−1
atipamezole (ATI, 0.1 mg kg ) or an equivalent volume of saline (SAL) at T45 . Data are presented only
atipamezole
for (ATI,time
representative 0.1 mg kg−1(while
points ) or an actually
equivalent volumeevery
recorded of saline (SAL)
minute) toat T45.readability.
ease Data are presented only
for representative time points (while actually recorded every minute) to ease readability.
3.1.3. Physiological Variables
3.1.3. Physiological Variables
After treatment administration, the HR decreased from 106 (100–118) beats minute−1 at T0 to 45
After
(34–60) beats minute−1
treatment administration,
at T44 for MEDthe < 0.001),
(p HR decreased from100
and from 106(96–104)
(100–118)beats
beatsminute
minute−1−1at
atTT00to
to44
45
(34–60)beats
(34–57) beatsminute −1
minute−1 atat TT44 MED(p(p<< 0.001).
44 for DEX 0.001), There
and from
was 100 (96–104)difference
no relevant beats minute −1 at Ttreatments
between 0 to 44 (34–

(p = beats
57) 0.114).minute −1 at T44rates
Respiratory for DEX (p < 0.001). There
and temperatures was noinrelevant
remained differencerange.
the physiological between treatments (p
= 0.114). Respiratory rates and temperatures remained in the physiological range.
3.2. Phase 2 (T45 –T210 , ATI Versus SAL)
3.2. Phase 2 (T45–T210, ATI Versus SAL)
3.2.1. Sedation
Sedationscores were lower in the dogs treated with ATI than SAL (p < 0.001). Time to sedation
3.2.1.Sedation
≤ 3 was 25
score Sedation (15–25)
scores minutes
were lower for ATIdogs
in the and treated
90 (68–118)
withminutes SAL(p(p<=0.001).
forSAL
ATI than 0.008,Time
Figure
to1b).
sedation
score ≤ 3 was 25 (15–25) minutes for ATI and 90 (68–118) minutes for SAL (p = 0.008, Figure 1b).
3.2.2. Nociceptive Withdrawal Reflexes
3.2.2.The relative NWR
Nociceptive thresholds
Withdrawal recorded during the first phase (T0 –T44 ) were not different for
Reflexes
groups ATI and SAL (p = 0.293). The relative NWR thresholds were also similar at T44 : 2.5 (1.9–3.3) for
The relative NWR thresholds recorded during the first phase (T0–T44) were not different for
ATI and 2.3 (1.9–3.1) for SAL (p = 0.461, Figure 2a). Between T45 and T210 , the relative NWR thresholds
groups ATI and SAL (p = 0.293). The relative NWR thresholds were also similar at T44: 2.5 (1.9–3.3)
differed between groups (p < 0.001). Time to a relative NWR threshold ≤ 1.5 was five (3–6) minutes for
for ATI and 2.3 (1.9–3.1) for SAL (p = 0.461, Figure 2a). Between T45 and T210, the relative NWR
ATI and 41 (28–97) minutes for SAL (p = 0.008). In dogs receiving ATI, a relative NWR threshold ≤ 1.5
thresholds differed between groups (p < 0.001). Time to a relative NWR threshold ≤ 1.5 was five (3–
was reached 18 (11–22) minutes before sedation scores returned to values ≤ 3 (p = 0.008, Figure 2b).
6) minutes for ATI and 41 (28–97) minutes for SAL (p = 0.008). In dogs receiving ATI, a relative NWR
threshold
3.2.3. ≤ 1.5 wasVariables
Physiological reached 18 (11–22) minutes before sedation scores returned to values ≤ 3 (p =
0.008, Figure 2b).
After atipamezole administration, HR increased to 88 (80–107) beats minute−1 within 5 min.
Respiratory rates andVariables
3.2.3. Physiological temperatures did not change.

After atipamezole administration, HR increased to 88 (80–107) beats minute−1 within 5 min.

Respiratory rates and temperatures did not change.

3.3. Additional Observations on the Effect Offset

 and 115 (88–135) minutes for and DEX/SAL (p = 0.057).
Relative NWR thresholds did not differ between MED/SAL and DEX/SAL subgroups (p = 0.241,
Figure 4); similarly, time to relative NWR threshold ≤ 1.5 did not differ between the groups, at 116
(75–158) minutes for MED/SAL and 80 (73–118) minutes for DEX/SAL (p = 0.686). This difference of
30% was above the relevant endpoint of 20%, but at least 12 animals per group would have been
Animals 2020, 10, 1240 8 of 14
required to confirm this result (Mann–Whitney test, unpaired, two-tailed, effect size = 1.3, α = 0.05, 1-
β = 0.9, GPower 3.1, Germany).
In the SAL
3.3. Additional groups, the
Observations ontime to sedation
the Effect Offset scores ≤ 3 was 135 (113–163) minutes, whereas the time
to relative NWR threshold ≤ 1.5 was 86 (73–142) minutes (p = 0.008), suggesting that sedation lasts
In the
longer thantime interval T45 –T210
antinociception. , sedation scores
A significant were
positive higher forbetween
correlation MED/SAL than
these forvariables
two (p =found
DEX/SALwas 0.02,
Figure Furthermore, time to sedation scores ≤
(p = 0.005, p = 0.87) and intercept-free linear regression had a slope of 0.81 (r2 = 0.74, Figure 5). 115
3). 3 were 155 (133–200) minutes for MED/SAL and
(88–135)
Nominutes
relevantfor and DEX/SAL
difference (p = 0.057).
was observed between MED/ATI and DEX/ATI subgroups.

Figure3.3.Median
Figure Median(interquartile
(interquartile range)
range) sedation
sedation scores
scores (from
(from 00 to
to 21)
21) recorded
recorded from
from TT45 to T210 in
45 to T210
in 8
8 beagles
beagles sedated
sedated at
at T with either intravenous medetomidine (MED/SAL, 0.02 mg kg −1 , n = 4) or
0
T0 with either intravenous medetomidine (MED/SAL, 0.02 mg kg , n = 4) or −1

mgkgkg−1, ,nn==4).
dexmedetomidine −1
dexmedetomidine(DEX/SAL,
(DEX/SAL,0.010.01mg 4). Data
Dataare
arepresented
presentedslightly
slightlyapart
apartfrom
fromthe theactual
actual
measurement time points to ease readability.
measurement time points to ease readability.

Relative NWR thresholds did not differ between MED/SAL and DEX/SAL subgroups (p = 0.241,
Figure 4); similarly, time to relative NWR threshold ≤ 1.5 did not differ between the groups, at 116
(75–158) minutes for MED/SAL and 80 (73–118) minutes for DEX/SAL (p = 0.686). This difference
of 30% was above the relevant endpoint of 20%, but at least 12 animals per group would have been
required to confirm this result (Mann–Whitney test, unpaired, two-tailed, effect size = 1.3, α = 0.05,
1-β = 0.9, GPower 3.1, Germany).
In the SAL groups, the time to sedation scores ≤ 3 was 135 (113–163) minutes, whereas the time
to relative NWR threshold ≤ 1.5 was 86 (73–142) minutes (p = 0.008), suggesting that sedation lasts
longer than antinociception. A significant positive correlation between these two variables was found
(p = 0.005, p = 0.87) and intercept-free linear regression had a slope of 0.81 (r2 = 0.74, Figure 5).
 Animals 2020, 10, x 9 of 14
Animals 2020, 10, 1240 9 of 14
Animals 2020, 10, x 9 of 14

Figure 4. Median (interquartile range) relative nociceptive withdrawal reflex (NWR) thresholds
recorded
Figure from T45(interquartile
4. Median to T210 in 8 beagles
range)after administration
relative nociceptiveatwithdrawal
T0 of either reflex
intravenous
(NWR)medetomidine
thresholds
Figure 4. Median
(MED/SAL, 0.02 (interquartile
mg kgin−1, n = 4)range) relative nociceptive
orafter
dexmedetomidine withdrawal reflex
mg kg(NWR) thresholds
recorded from T 45 to T210 8 beagles administration (DEX
at T0 of/ SAL,
either0.01
intravenous −1, n = 4). Data are
medetomidine
recorded
(MED/SAL, from
presented0.02 T 45 to T−1
onlymgfor 210 in 8 beagles after administration at T0 of either intravenous
, n = 4) or dexmedetomidine
kgrepresentative time points (while(DEX / SAL, recorded
actually −1
0.01 mg kgevery ,n= medetomidine
minute)
4). Datatoare
ease
(MED/SAL, only0.02
readability.
presented mg kg−1, n = 4)
for representative or points
time dexmedetomidine (DEX
(while actually / SAL,
recorded 0.01minute)
every mg kg−1 to, ease
n = 4). Data are
readability.
presented only for representative time points (while actually recorded every minute) to ease
readability.

Figure 5. Intercept-free linear regression between the time to recover from sedation (sedation score ≤ 3)
andFigure 5. Intercept-free
the duration linear regression
of antinociception (relative between thewithdrawal
nociceptive time to recover
reflexfrom sedation
(NWR) (sedation
threshold ≤ 1.5)score
in 8 ≤
3) andafter
beagles the administration
duration of antinociception
at T0 of either(relative nociceptive
intravenous withdrawal
medetomidine reflex (NWR)
(MED/SAL, 0.02 mgthreshold
kg−1 , n =≤4)1.5)
Figure
in 8 5. Intercept-free
beagles after linear regression
administration at T betweenintravenous
0 of either
the time to recover from sedation
medetomidine (sedation
(MED/SAL, 0.02 score
mg kg≤−1,
or dexmedetomidine (DEX/SAL, 0.01 mg kg , n = 4). −1
3)nand the duration of antinociception (relative nociceptive
= 4) or dexmedetomidine (DEX/SAL, 0.01 mg kg , n = 4). −1 withdrawal reflex (NWR) threshold ≤ 1.5)
in 8 beagles after administration at T0 of either intravenous medetomidine (MED/SAL, 0.02 mg kg−1,
No relevant difference was observed between MED/ATI and DEX/ATI subgroups.
n = 4) or dexmedetomidine (DEX/SAL, 0.01 mg kg−1, n = 4).
Animals 2020, 10, 1240 10 of 14

4. Discussion

4.1. Interpretation
The administration of a single IV dose of medetomidine (0.02 mg kg−1 ) or dexmedetomidine (0.01
mg kg−1 ) rapidly induced comparable sedation and antinociception in dogs. Dexmedetomidine did
not induce significantly higher NWR thresholds than medetomidine. Both effects rapidly terminated
after IM administration of atipamezole and the NWR thresholds decreased earlier than sedation scores.
In dogs, the sedative effects of dexmedetomidine administered alone [35] or as part of the racemic
mixture [9,36] have already been reported. In our study, profound sedation (sedation scores ≥ 13 out
of 21) and peak sedation scores were rapidly achieved after treatment administration. There was no
difference in onset and quality of sedation between treatments. This is in contrast with Raszplewicz et
al. [8], who reported that more dogs achieved profound sedation after medetomidine (0.01 mg kg−1 )
than after dexmedetomidine (0.005 mg kg−1 ) administered IM in combination with butorphanol. On
the other side, in our study, the duration of drugs’ action was different: sedation lasted longer after
medetomidine compared to dexmedetomidine. Similar results were reported in cats receiving high IM
doses of alpha-2 agonists [37]. Other studies could not demonstrate any difference in sedation quality
nor in duration of action between medetomidine and dexmedetomidine [3,4,15], but the sedation scores
applied were poorly sensitive (numerical subjective scale from zero to three) compared to the scoring
system used here [38]. Differences in duration of action probably depend on the doses administered,
the route of administration and the co-administration of other drugs.
Differences in effect between dexmedetomidine administered alone and medetomidine as a
racemic mixture are most probably due to the addition of levomedetomidine. When administered alone
to dogs, levomedetomidine did not cause any behavioral effect [15] or decrease halothane minimum
alveolar concentration (MAC) [39]. Only at high dosages, it moderately impaired the quality of the
sedation induced by dexmedetomidine [15]. Therefore, the longer duration of sedation observed in
our study after medetomidine is unlikely to be the result of a sedative effect from levomedetomidine.
However, a pharmacokinetic interaction between the two enantiomers could occur, for instance,
a slower elimination of dexmedetomidine in presence of levomedetomidine. While this has not been
investigated yet, differences between dex- and levomedetomidine concentrations were observed after
administration of the racemic mixture [13], suggesting an enantioselective pharmacokinetic.
Antinociceptive properties did not significantly differ between treatments in the present study.
Previous results comparing analgesia induced by either medetomidine or dexmedetomidine were
inconclusive. Longer lasting analgesia was reported with dexmedetomidine (0.02 mg kg−1 IV)
compared to medetomidine (0.04 mg kg−1 IV) in response to toe pinch (assessed by a subjective score
of the nocifensive response) [3]. Dexmedetomidine was not different from medetomidine at decreasing
halothane MAC in dogs [39]. Likewise, no difference in nocifensive reaction to toe pinch was observed
between dexmedomidine (0.04 mg kg−1 IM) and medetomidine (0.08 mg kg−1 IM) in both dogs [4] and
cats [40]. These results would also benefit from being interpreted in perspective with pharmacokinetic
studies. If sedation is hypothesized to be shorter when dexmedetomidine is administered alone due to
lower plasma concentrations, as mentioned above, similar levels of antinociception could potentially
support a stronger intrinsic analgesic efficacy for dexmedetomidine compared to medetomidine.
Anxiety may be difficult to observe in trained beagles. It could have potentially occurred during
baseline measurements when the dogs were unmedicated, which could affect our results. Anxiety has
been reported to increase central hyperexcitability and to potentially lower the NWR threshold [41,42],
though this is controversial [43,44]. Efforts were made to acclimatize the dogs to the experimental
room, staff and methodology. No sign of anxiety was noticed during the experiment. Baseline values
for NWR threshold were comparable to previous studies [20] and to final values towards the end of
the measurements. It is unlikely that anxiety affected the results presented here.
One objective of the present study was to apply a methodology that could potentially highlight
differences in duration and efficacy between treatments. Using the automated continuous tracking
Animals 2020, 10, 1240 11 of 14

device, even subtle variation in the NWR thresholds can be quantified over time. Moreover,
the EMG-based NWR model is less likely to be influenced by sedation and muscle relaxation than visual
scoring of a gross movement in response to stimulation. Finally, the evaluation can be repeated at high
frequency (every 10 s) without risking habituation, sensitization or skin damage. Our results confirm
that there is no difference in antinociceptive efficacy between medetomidine and dexmedetomidine in
dogs at the doses used.
Another objective of this study was to compare the time courses of sedation and analgesia, in
particular after atipamezole. A relevant observation was that the NWR thresholds returned to baseline
before sedation, indicating that analgesic coverage is no longer guaranteed once sedation subsides.
Intercept-free linear regression (r2 = 0.74, Figure 5) suggest that the duration of antinociception (relative
NWR thresholds >1.5) approximates 80% of the duration of sedation (sedation score ≤ 3).
Antinociception was rapidly reversed by atipamezole at the dosage recommended by the
manufacturer. The continuous NWR threshold determination tracked precisely the onset of
antinociception reversal, which was approximately 6 min after atipamezole administration for both
medetomidine and dexmedetomidine. This implies that appropriate analgesia needs to be considered
whenever this antagonist is administered to animals experiencing pain.

4.2. Limitations
The comparison between MED and DEX followed a crossover design with eight subjects (paired)
for the first 45 min only. Thereafter, the output variables (i.e., NWR threshold and sedation) in absence
of reversal were compared between two groups of four dogs each (subgroups MED/SAL and DEX/SAL,
no cross-over). The observations resulting from this comparison seem to indicate that DEX and MED
may differ in duration of action more than in their potency, but this finding requires further validation.
Two female and six male intact dogs were used in the study. An equal sex distribution would
have been preferable, as sex is known to potentially affect sensitivity to pain and thus nociceptive
thresholds [45]. One female dog presented signs of proestrus during the second session of the trial.
For this individual no difference was observed between the baseline values recorded during the two
sessions but a certain influence on the nociceptive threshold course after drug administration cannot
be excluded.
In the present study, antinociception was investigated using the non-invasive NWR model. A
drug-related increase in the NWR threshold cannot be directly interpreted as clinical analgesia, as the
complexity of clinical pain goes clearly beyond a spinal nociceptive reflex. Nevertheless, in humans the
NWR threshold is known to correlate well to the threshold for pain perception [46], justifying the use of
this model to assess pharmacologically induced analgesia in pain-free subjects in experimental settings.

4.3. Harm
No adverse events other than one female dog vomiting after both alpha-2-agonist injections was
observed. Vomiting is common after administration of alpha-2 agonists as a result of their action at the
chemoreceptor trigger zone [9,47,48].

5. Conclusions
Although sedation appeared to be slightly longer lasting after medetomidine, sedation quality and
antinociceptive efficacy were similar for dexmedetomidine and medetomidine. Antinociception did
not outlast sedation, and atipamezole was able to rapidly reverse both sedation and antinociception.

Author Contributions: Conceptualization, J.S., T.P., M.R., C.S. and O.L.L.; methodology, J.S., T.P., M.R., C.S.
and O.L.L.; formal analysis, J.S., T.P., M.R., C.S. and O.L.L.; writing—original draft preparation, J.S. and O.L.L.;
writing—review and editing, J.S., T.P., M.R., C.S. and O.L.L. All authors have read and agreed to the published
version of the manuscript.
Funding: This research was partially funded by the Specialisation Committee of the Vetsuisse Faculty at the
University of Berne.
Animals 2020, 10, 1240 12 of 14

Acknowledgments: The authors would like to warm-heartedly acknowledge the support of the staff from the
experimental station for provision and care of the dogs.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.

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