Journal of Veterinary Behavior (2012) 7, 225-232
RESEARCH
Harmonease Chewable Tablets reduces noise-induced fear
and anxiety in a laboratory canine thunderstorm
simulation: A blinded and placebo-controlled study
Theresa L. DePortera, Gary M. Landsbergb, Joseph A. Araujoc,d, Jennifer L. Ethierd,
David L. Bledsoee
a
Oakland Veterinary Referral Services, Bloomfield Hills, Michigan;
North Toronto Animal Clinic, Thornhill, ON, Canada;
c
Department of Pharmacology, University of Toronto, Toronto, ON, Canada;
d
CanCog Technologies Inc, Toronto, ON, Canada; and
e
Veterinary Products Laboratories, Phoenix, Arizona.
b
KEYWORDS:
anxiety;
fear;
thunderstorm;
Magnolia officinalis
and Phellodendron
amurense;
honokiol;
laboratory model
Abstract Thunderstorm simulation in the laboratory setting induces fearful and anxious behavior in
beagles, most notably manifested by increased inactivity (‘‘freezing’’), which, in a companion study,
was ameliorated by the anxiolytic diazepam (Araujo et al., 2009). Using this protocol, the present
study assessed the efficacy of Harmonease, a chewable oral anxiolytic botanical product containing
a proprietary blend of extracts of Magnolia officinalis and Phellodendron amurense. A balanced,
placebo-controlled, blinded, single crossover design including 20 healthy adult beagles was used for
this study. After a baseline thunderstorm test, subjects received Harmonease Chewable Tablets or placebo treatment daily and were reassessed on the treatment day 7. After a 7-day washout period, the
treatments were crossed over and a design identical to that used in the first phase was used. The
thunderstorm test was performed in an open-field arena (8 ft ! 9 ft) and consisted of three
3-minute phases: an anticipatory phase in which no stimulus was provided; the thunderstorm phase
in which a thunderstorm track was played over a speaker system; and a recovery phase in which no
stimulus was presented. Inactivity duration was considered the primary variable for assessing efficacy
which was measured by a trained observer. Difference in number of dogs improved versus worsened by
treatment group was significant at P , 0.05. Specifically, 12 of 20 (60%) dogs improved from baseline
when treated with Harmonease, whereas only 5 of 20 (25%) improved on placebo. Furthermore, 9 of
20 (45%) placebo dogs showed increased inactivity duration (worsened), whereas only 4 of 20 (20%)
treatment dogs worsened. Increases in distance travelled consistent with reduced inactivity were also
seen under Harmonease. Harmonease reduced fear-related inactivity or freezing in dogs in this thunderstorm simulation model. This supports past studies demonstrating that the combination of botanical
extracts in Harmonease is effective in dogs for the management of stress-related behaviors.
Ó 2012 Elsevier Inc. All rights reserved.
Harmonease is a registered trademark of Veterinary Products Laboratories, a division of Farnam Companies, Inc, Phoenix, AZ.
Address for reprint requests and correspondence: Theresa L. DePorter, BSc, DVM, Oakland Veterinary Referral Services, 1400 Telegraph Road, Bloomfield
Hills, Michigan 48302; Tel: 11-248-334-6877; Fax: 11-248-334-3693.
E-mail: [email protected]
1558-7878/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.jveb.2011.05.024
226
Introduction
Loud noises such as thunder and fireworks often cause
anxiety in dogs (Sheppard and Mills, 2003). Fear of stormrelated noises is a normal, adaptive, and self-preserving
mechanism in which a dog’s initial behavioral response is
generally to evade, avoid, and withdraw, and the continuum
of anxiety-based responses can range from fearful to phobic. Clinical signs associated with storm- or noise-related
fears or phobias include trembling, hiding, pacing, vocalizing, and destructiveness, which in the home environment
may be further influenced by the owner’s responses. Perception of danger, past experience, and association with
traumatic events and learning may contribute to the manifestation and severity of the response. In natural situations
and with repetitive exposure to the stimulus, dogs may begin to characterize the experience as being threatening,
which may result in continued avoidance and further sensitization, or as being nonthreatening, which may result in
habituation. This problem is frequently reported by dog
owners as a behavioral concern, either regarding their
dog’s general well-being with respect to anxiety or as
manifestations including nighttime waking, destruction, or
vocalization. In the clinical setting, dogs that are fearful
of loud noises, such as thunder, rarely habituate naturally,
making noise-induced anxiety one of the more vexing
conditions the dog owner and veterinarian may face
(Landsberg et al., 2003b).
Various forms of intervention may be used in the
treatment of anxiety, including environmental management
or stimulus avoidance, desensitization and counterconditioning, pharmaceuticals, and/or natural therapeutics such
as pheromones (Crowell-Davis and Murray, 2006; Levine
et al., 2007; Sheppard and Mills, 2003; Sherman and
Mills, 2008). For pet dogs with fears and phobias to thunder
or other loud noises, commercial or homemade recordings
that recreate the fear-evoking sound can be used for desensitization and counterconditioning (Levine et al., 2007).
Owners must also be counseled to be supportive of the
dog in an attempt to reduce anxiety because owner frustration and punishment is only likely to further contribute to
anxiety, and excessive reassurance does not intensify or
reinforce fearful responses (Dreschel and Granger, 2005).
Dogs, however, may still be frequently exposed to naturally
occurring thunderstorms or other unavoidable fear-evoking
noises (e.g., fireworks) during the desensitization period,
which may sabotage the desensitization program and
discourage owners from continuing. Drug therapy may be
applied in situations in which the anxiety is severe and/or
the stimulus cannot be effectively controlled or reduced
(Landsberg et al., 2003b). Commonly available and frequently prescribed veterinary phenothiazine tranquilizers,
such as acetylpromazine, are considered inappropriate
as they are not anxiolytic and may actually increase a
dog’s sensitivity to noises even though overt activity is
suppressed by sedation (Overall, 2001). Therefore,
Journal of Veterinary Behavior, Vol 7, No 4, July/August 2012
identifying more effective chemotherapeutic agents to reduce underlying anxiety is needed both to help the pet
cope and to improve the outcome of the behavior program
(Landsberg et al., 2003b).
This study was designed to scientifically assess the
anxiolytic benefits of a nutritional product, Harmonease
(Veterinary Products Laboratories, Phoenix, AZ), for effectiveness in reducing noise-related anxiety in dogs. Nutritional supplements or alternative medicine products are
often presented as beneficial despite the lack of scientific
documentation of safety and efficacy (Landsberg et al.,
2003a). People are eager to consider natural products for
themselves or their pets but the lack of placebo-controlled
trials should raise concern as to the efficacy of these
products (Scott et al., 2002; Overall and Dunham, 2009).
Harmonease contains a proprietary blend of extract from
the bark of Magnolia officinalis, which contains honokiol
and magnolol, and from the bark of Phellodendron
amurense, which contains berberine (Chen and Chen,
2004a, 2004b). It should be noted that P. amurense is a
species of tree in the family Rutaceae, commonly called
the Amur cork tree, and must not be confused with the
plants of the genus Philodendron in the Araceae family.
Philodendron includes several species of ornamental and indoor houseplants which are generally considered unhealthy
to consume due to the presence of oxalates. Phellodendron
shares a similar spelling but is a genus of deciduous trees in
the family Rutaceae (Chen and Chen, 2004a, 2004b). Harmonease has been developed for the treatment of anxiety
in dogs based on several studies performed in mice, rats,
chicks, and human beings, which have assessed the anxiolytic properties of honokiol, magnolol, and berberine individually and in combination (Kalman et al., 2008;
Maruyama and Kuribara, 2000; Sufka et al., 2001; Xu
et al., 2008). When tested in mice, honokiol demonstrated
anxiolytic activity similar to diazepam, with less sedation
(Kuribara et al, 1998, 1999). Although the antianxiety effects of honokiol may be similar to benzodiazepines, there
is a comparatively low risk of producing side effects such
as central depression, muscle relaxation, or amnesia, which
are frequently associated with benzodiazepines (Maruyama
and Kuribara, 2000). Using laboratory models for assessing
depression in rodents (i.e., the forced swim test and tail suspension test), the combination of honokiol and magnolol
was compared with vehicle control and was found to reduce
anxiety-related behaviors in both tests (Xu et al., 2008).
The combination of honokiol and magnolol assessed by
Xu et al. (2008) also reduced corticosterone concentrations
in serum as well as the Chronic Mild Stress model–induced
elevation of serum cortisone concentrations in chronically
stressed rats. Honokiol and magnolol have each been
identified in vitro as modulators of gamma-aminobutyric
acid (GABAA) receptors (Patocka et al., 2006), which
may explain the diazepam-like effects of derivatives of
M. officinalis. The proprietary extracts of M. officinalis
and P. amurense contained in Harmonease were assessed
DePorter et al
A laboratory canine thunderstorm simulation
separately and each one reduced stress in a standardized
chick anxiety model (Sufka et al., 2001). Berberine has
been shown to have similar anxiolytic effects to diazepam
and buspirone in experimental anxiety models in mice
(Peng et al., 2004). A pilot human clinical trial concluded
that the combination of extracts of P. amurense and M. officinalis provided measurable relief in premenopausal
women exhibiting anxiety (Kalman et al., 2008). On the basis of these studies from other species, and favorable pilot
canine data (Central Life Sciences, 2008), the need for a
placebo-controlled assessment of Harmonease for anxiety
relief in dogs was identified.
Recently CanCog (CanCog Technologies Inc., Toronto,
ON, Canada) developed a laboratory model of noiseinduced anxiety that uses an open-field testing room and
a recording of thunderstorm sounds (Araujo et al., 2009).
The recording is a compilation of commercially available
tracks and effectively reproduces the sounds of a storm.
Similar recordings are used in integrated behavior modification programs for desensitization and counterconditioning. This thunderstorm soundtrack is played over a sound
system under standardized conditions (Araujo et al.,
2009). The model measures stress-related clinical signs
during the test such as distance travelled by the dog, inactivity duration, inactivity frequency, and time the dog
spends by the door; the first 3 of which are related to freezing responses and are the most robust measures of anxiety
in this test (Araujo et al., 2009). In the present study, the
effectiveness of Harmonease was evaluated in this model
of simulated thunderstorms, and the authors hypothesized
that treatment with Harmonease would reduce the noiseinduced anxiety of beagles in this model.
Materials and methods
Test article and treatment
Harmonease contains a proprietary blend of extracts of
M. officinalis and P. amurense. Each tablet contains 500 mg
of the proprietary blend, which is standardized to contain
9.5 mg honokiol. Each dog received the label dose of
half a tablet daily for dogs weighing up to 22.7 kg; all
dogs in this study weighed ,22.7 kg. Placebo tablets
were similar in appearance, including size, shape, weight,
and palatability, and were administered in exactly the
same manner as the test article (half a tablet daily).
227
complete blood counts. Using testing procedure (lowest
inactivity duration on baseline testing during thunder),
2 dogs were deemed to be the least sensitive to the thunderstorm were eliminated from the study before receiving
treatment, leaving 20 dogs for the study; 14 males and
6 females.
The test facility was licensed by the Province of Ontario
and operated in accordance with guidelines set forth by the
Canadian Council on Animal Care. Animal housing and
care met or exceeded all applicable provincial and Federal
regulations, and the study protocol was approved by the test
facility’s Animal Care Committee. Dogs were grouphoused either 3 or 4 per pen, with each pen measuring
5 ft ! 16 ft (80 ft2 in area). Each pen contained a single
2 ft ! 4 ft elevated perch. Pens were constructed from galvanized steel with open-sided mesh walls on epoxy-sealed
concrete floors. All pens were located in an animal containment room, which was approximately 40 ft ! 80 ft with a
10-ft ceiling. The room temperature was electronically controlled to maintain a temperature between 15 C and 28 C,
and a ventilation system provided approximately 12-15
filtered air changes per hour. Natural and incandescent
lighting was provided for the dogs in appropriate duration
to mimic the seasonal photoperiod. Various toys were provided for environmental enrichment. All animals were fed
their standard diet (Purina Pro Plan Chicken and Rice, Nestlé Purina PetCare, St. Louis, MO) to maintain body
weight. Feed was provided in stainless steel bowls once
daily, and water was provided ad libitum through an automatic nipple watering system and/or stainless steel bowls.
Animals were observed twice daily by personnel masked
to the treatment condition throughout the study to ensure
that they remained in good general health.
Study schedule and general design
The study was divided into 4 periods: a baseline period
(days 23 through 21) in which physical examinations,
clinical pathology testing, and baseline (pretreatment)
thunderstorm tests were conducted (day 23) and baseline
(pretreatment) thunderstorm tests were repeated on day 21;
the first 7-day treatment period (day 0 through 6) on the
conclusion of which the first treatment assessment thunderstorm test was conducted; a 7-day washout period (day 7
through 13); and the second 7-day treatment period (day 14
through 20), on the conclusion of which the second
treatment assessment thunderstorm test was conducted.
See Table 1 for overview of the schedule.
Test animals and their management
Thunderstorm testing procedure
Twenty-two beagle dogs of either sex were selected from
the test facility’s colony for potential enrollment into this
study. All dogs were between 4 and 8 years of age and were
confirmed to be in good general health through baseline
physical examinations, serum chemistry profiles, and
Thunderstorm testing for each dog occurred on days 23,
21 (baseline), 6 (on the completion of the first treatment
period), 13, and 20 (the beginning and completion, respectively, of the second treatment period). Subjects were
228
Table 1
Journal of Veterinary Behavior, Vol 7, No 4, July/August 2012
Overview of the study design including baseline assessments and a single crossover study design
Study day
Procedure
Day23
Day21
Baseline thunder test period 1
Blocking and group assignment
Treatment period 1
Washout period
Baseline thunder test period 2
Treatment period 2
Thunderstorm test
(assessment of response to treatment)
X
X
X
placed in an open-field arena––a room approximately
8 ft ! 9 ft––for 9 minutes. Before each subject entered
the room, it was cleaned with a multipurpose concentrated
industrial cleaner (Dynamite Big Job Cleaner, CP Industries, Fergus, ON). Subjects were recorded through a video
device for a total of 9 minutes; a 3-minute anticipatory
phase, a 3-minute thunderstorm phase, and a 3-minute
recovery phase, respectively. Dogs entered the room by
passing through a dog door. During the anticipatory phase,
all dogs entered and explored freely over the first 3 minutes
with no external stimuli. During the thunderstorm phase,
which occurred over the subsequent 3 minutes, subjects
were exposed to a thunder track played over a stereo
speaker. The sounds of thunderstorm were reproduced
using a commercially available sound desensitization
compact disc (Sounds Scary!, Sound Therapy 4 Pets Ltd,
Upton Chester, England, www.soundtherapy4pets.com),
which has been shown to be effective for behavior modification programs for noise-related anxieties (Levine et al.,
2007; Levine and Mills, 2008). For this trial, the thunderstorm sounds were edited to remove the rain portions and
produce a 3-minute thunderstorm segment. Volume, treble,
and bass were standardized. During the recovery phase, subjects were kept in the room for the final 3 minutes with no
external stimuli. The behavioral parameters measured for
each dog included time spent inactive, distance travelled, inactivity frequency, and time spent within 0.5 m of the door,
and each served as a dependent variable. Parameters were
measured during all 3 phases of the thunderstorm test (anticipatory phase, thunderstorm phase, and recovery phase) of
each test session and were analyzed by phase.
The Ethovision 3.1 (Noldus Information Technology,
Leesburg, VA) behavioral analysis software was used to
measure the dependent variables. This software was calibrated to the measurements of the room and measured
distance travelled automatically as well as time spent near
the door. The inactivity variable was recorded in real time by a
trained and blinded observer through key presses. Inactivity
was defined as sitting, lying down, or standing in the absence
of any other overt behavior such as sniffing, grooming, or
urinating. Timing of inactivity was done manually by pressing
Days 0-6
Day 6
Days 7-13
Day 13
Days 14-20
Day 20
X
X
X
X
X
X
a computer key at the beginning and again at the end of the
period of inactivity. All other parameters were recorded
automatically by the software and analyzed at a later time.
Group assignment
On day 21, the second baseline thunderstorm assessment
was performed on the twenty two dogs initially selected and
the dogs were subsequently ranked on the basis of their
inactivity duration during this thunderstorm test. Inactivity
duration response was chosen for ranking as it is believed to
be the most robust parameter in this model (Araujo et al.,
2012). The 2 dogs with the least inactivity on day
21 were removed from the study, and the remaining dogs
were assigned to ranks 1 through 20 in which the dog demonstrating the least inactivity received the rank of 1. Dogs
that were assigned ranks 1, 2, 3, and 4 were placed into treatment groups 1, 2, 2, 1, respectively, and those assigned ranks
5, 6, 7, and 8 were placed into treatment groups 1, 2, 2, 1,
respectively. This pattern continued until all subjects were
assigned to a treatment group.
One group received the placebo during the first treatment
period and the other received Harmonease during the first
treatment period. On days 0 through 6 (first treatment period),
dogs received their assigned treatment. Each dog received its
daily dose at the same time each day (630 minutes);
however, dosing times were spread out over a 6-hour period
to accommodate the test schedule on day 6. On day 6, all dogs
were subjected to the thunderstorm testing, each test occurring 75 minutes (615 minutes) after treatment administration.
On days 7 through 13 (washout period), dogs did not receive
treatment. On day 13, dogs were again subjected to the
thunderstorm testing and the treatment groups were crossed
over so that dogs receiving Harmonease during the first
treatment period received the placebo during the second
treatment period and vice versa. Dogs were treated with their
appropriate treatment on days 14 through 20, per the dosing
schedule described earlier in the text. On day 20, a final
thunderstorm test was conducted and the in-life portion of the
study concluded. All animals were returned to the test facility
colony upon conclusion of the study.
DePorter et al
A laboratory canine thunderstorm simulation
229
Calculations and statistical analyses
Both parametric and nonparametric analyses were used
to evaluate effects of treatment during the thunderstorm test.
To evaluate magnitude of effect, an analysis of variance was
conducted in which treatment (placebo vs. Harmonease),
test phase (anticipatory vs. thunderstorm vs. recovery), and
time-point (baseline vs. treatment) served as within-subject
variables, and test order (placebo first vs. Harmonease first)
served as a between-subject variable. Because the data were
skewed toward high levels of inactivity, a nonparametric
statistical approach, in which the number of subjects that
showed increases and decreases from baseline, was also
used. These data were then subjected to a chi-square
analysis. Subjects that showed no change were excluded
from these analyses. For all analyses, the Statistical Software Package 6.0 (StatSoft, Intl., Tulsa, OK) was used
and P , 0.05 was considered significant.
As past research indicated that inactivity duration was
the most robust variable in this model (Araujo et al., 2012),
it was the parameter used in the ranking procedure before
group assignment and was considered the primary variable
for assessing treatment efficacy. Decreases in this variable
under treatment were considered a positive result. A
treatment-related decrease in near door duration and a
treatment-related increase in distance travelled were also
considered positive results.
Results
At baseline, mean inactivity duration of the groups
during the thunderstorm phase did not differ statistically,
confirming groups were balanced. For the group receiving
placebo first, the day 21 baseline inactivity duration was
M 6 SEM 5 114 seconds 6 6.4 seconds and for group
receiving Harmonease first the baseline inactivity duration
was M 6 SEM 5 130 seconds 6 12.9 seconds. Regardless
of treatment group, there was a significant phase effect
(P , 0.001) as dogs were significantly more inactive during the thunderstorm and recovery phases than during the
anticipatory phase (Figure 1), confirming that inactivity
duration parameter is highly affected by the thunderstorm
sound and may serve as a sensitive indicator of therapeutic
response.
Assessment of group means did not reveal significant
treatment or order effects when analyzed using an analysis
of variance. However, inspection of the data revealed
a large degree of variability. Furthermore, the data were
not distributed normally, with 4 of 20 dogs having
maximum inactivity during the thunderstorm phase while
on the Harmonease treatment (i.e., inactive during the
entire 3-minute phase) and 5 of 20 dogs inactive for the
entire 3-minute thunderstorm test while on the placebo.
Consequently, a chi-square analysis was conducted on
the number of subjects that either improved or worsened
Figure 1 Inactivity duration by phase during the thunderstorm
test demonstrating the significant phase effect (P , 0.001) in
inactivity duration regardless of treatment group. The inactivity
duration was the most robust measurement for this laboratory
canine thunderstorm simulation.
(as defined a priori) in each test parameter. The data are
presented in Table 2.
When changes in inactivity duration as compared with
baseline was examined, a significant treatment effect was
found during the thunderstorm phase (P 5 0.03), but not
during the anticipatory or recovery phases of the thunderstorm test. The effect was because of an overall improvement under treatment and worsening under placebo
(Table 2). Specifically, 12 of 20 subjects (60%) showed
reduced inactivity duration during the thunderstorm phase
(as compared with baseline, i.e., improvement) while receiving Harmonease, as compared with 5/20 subjects (25%)
while receiving placebo (Figure 2). An increase in inactivity from baseline (i.e., worsening) was seen in 9/20 subjects
(45%) while receiving placebo as compared with 4/20
subjects (25%) while receiving Harmonease. The remaining subjects, 6 during placebo treatment and 4 during
Harmonease treatment, showed no change from baseline.
No other significant effects were found. However, the
distance travelled measured during thunderstorm phase
closely approached significance (P 5 0.0565), which reflected increased distance travelled in 14 of 20 dogs during
Harmonease treatment as compared with 8 of 20 dogs during placebo. The improvements observed on the distance
travelled measure are consistent with those found on inactivity duration and also indicative of a reduction in anxiety
for dogs treated with Harmonease. To ensure that order
effects did not confound these findings, an identical analysis was conducted in which number of subjects that showed
changes over both the first 2 and last 2 tests was analyzed.
No significant order effects were found on either during
thunder inactivity duration or on distance travelled. In
general, the chi-square test results indicate a consistent
treatment-related improvement on inactivity duration during thunder that is not related to the effect size.
230
Journal of Veterinary Behavior, Vol 7, No 4, July/August 2012
Table 2 Number of subjects that showed changes from baseline measurements of inactivity duration and distance travelled in each of
the 3 phases of the thunderstorm test (anticipatory, thunderstorm, and recovery phase)
Test measure
Anticipatory phase
Inactivity duration
Distance travelled
Thunderstorm phase
Inactivity duration
Distance travelled
Recovery phase
Inactivity duration
Distance travelled
Response
Placebo
Treatment
P-value
Increased
Decreased
No change
Increased
Decreased
7
13
0
10
10
8
12
0
12
S
0.744
Increased
Decreased
No change
Increased
Decreased
9
5
6
8
12
4
12
4
14
6
0.03
Increased
Decreased
No change
Increased
Decreased
6
7
7
7
13
7
5
8
7
13
0.5425
0.525
0.0565
1.000
Improvement in anxiety is reflected by a decrease in inactivity duration. Similarly, reduction of anxiety is reflected by an increase in distance travelled.
Numbers in bold are significant.
Discussion
In the present study, laboratory beagle dogs were used to
evaluate the anxiolytic effects of Harmonease Chewable
Tablets in a blinded and placebo-controlled single crossover
study using an artificial thunderstorm model. In natural
situations, dogs can become sensitized to repeat exposures
to noises that may result in a variable pattern of responses
(Scott et al., 2002; Overall, 1997). To ensure the population
comprised dogs that all showed increased inactivity to thunder, the 2 subjects that demonstrated the least inactivity duration when exposed to the thunder were excluded. The
remaining subjects were then ranked on this variable and
assigned to balanced groups, which did not differ at baseline. Harmonease significantly reduced inactivity duration
Figure 2 Response to thunderstorm model inactivity duration
during the thunderstorm phase illustrating not only the improvement in dogs treated with Harmonease but also the increase in
anxiety displayed by dogs receiving the placebo treatment.
in more dogs as compared with the placebo group. Similarly, distance travelled was increased by Harmonease,
with results approaching significance. Collectively, the
data suggest a consistent, anxiolytic effect of Harmonease
in this model.
The increase in inactivity duration during thunderstorm
phase in this model is mainly attributed to an increase in
freezing behavior that occurs in response to a loud noise
(Araujo et al., 2009). In the baseline thunderstorm test before placebo treatment, there were 5 of 20 dogs that were
inactive for the entire thunderstorm phase. None of these
dogs improved with the placebo, and 1 additional dog
worsened to this 100% inactivity level while on the placebo. This maximum response to the stimulus suggests a
relevant and realistic thunderstorm anxiety to this
simulated model. Similarly, before treatment with Harmonease, 7 of 20 dogs were inactive for the entire 3 minutes
during baseline thunderstorm phase, which may represent
phobic behavior. However, unlike the dogs given the
placebo, 3 of these 7 Harmonease-treated dogs improved.
The positive response in the 3 Harmonease-treated dogs
and the lack of any response in placebo-treated dogs suggests potential efficacy on maximal manifestations of
anxiety in this test.
In an earlier study, reduced inactivity frequency was
considered a significant indicator of reduced anxiety
(Araujo et al., 2009). However, reduced inactivity was
problematic to interpret in this investigation, as some
dogs displayed a low inactivity frequency (e.g., as low as
1) in combination with a prolonged duration of inactivity
(e.g., the entire 3-minute thunderstorm phase). In the
DePorter et al
A laboratory canine thunderstorm simulation
present study, those dogs inactive for the entire thunderstorm phase showed a ceiling effect, confounding interpretation of the inactivity frequency variable. Ideally, the use
of this variable is best suited for dogs that do not show
the extremely high levels of inactivity seen in the present
study, which may be partially because of the selection criterion used. It is noteworthy that this anxious or fearful behavior was only evident during the thunderstorm test; no
observable differences were noted outside of the open field.
Furthermore, the dogs were observed to behave typically
before and after the test and did not display notable hesitancy to accompany the tester or enter the test room.
In this investigation, baseline thunder testing was
provided on days 23 and 21 with the rationale of
sensitizing the dogs to the thunderstorm test and thereby
reducing the potential confound of dogs showing increased inactivity, or sensitization, from the baseline test
to the second treatment test, which was reported previously and could confound interpretation of treatment
effects (Araujo et al., 2012). Instead, the dogs in the present study were highly inactive, which allowed us to
largely avoid the potential confound of sensitization but
also resulted in a skewing of the data toward maximal
levels of inactivity, therefore, potentially reducing the
ability of the model to detect treatment differences using
parametric statistical approaches. Consistent with the
greater percentage of animals showing decreased inactivity duration under treatment, there was a trend for more
dogs showing an increase in distance traveled under treatment. It would be expected that a decrease in inactivity
would correlate with an increase in distance traveled.
However, stress may induce a hyperactive response in
which distance travelled may increase, which can occur
in conjunction with increases in inactivity duration
(Araujo et al., 2012). Although this effect was not observed in this study, it should be recognized as a potentially significant limitation to the model. Moreover,
results of the test should be interpreted cautiously when
evaluating response to compounds that may potentially
stimulate motor function. Similarly, drugs with sedative
effects would be expected to produce increases in inactivity and reduce distance travelled, which would confound
the ability of this test to accurately assess anxiolytic effects. In past studies on mice using combinations of honokiol and magnolol, doses which supported antianxiety
effects did not affect ambulatory or rearing behavior in
the mouse open-field test; thus, the behavioral actions of
Harmonease are unlikely to be related to stimulation of locomotor activity (Xu et al., 2008). Changes in activity
were found only on the thunder phase of the test, which
is consistent with an absence of motor stimulating effects
of treatment; motor stimulating effects would have likely
increased activity across all phases of the test (Araujo
et al., 2012). Thus, we conclude that the distance travelled, although of marginal statistical significance, is supportive of the anxiolytic response revealed by the primary
231
outcome measure, reduced inactivity duration during
thunder.
Near door duration may represent escape behaviors, as
the dog recognizes the way out of the stressful environment.
This outcome was unaffected in the present study, and
additional research with this model is needed to determine
whether this variable is sufficiently sensitive to detect a
treatment effect with most compounds. An additional consideration is that inactivity near the door may limit the utility
of this variable because it is confounded by the dogs which
were both inactive and near the door simultaneously.
Prospective, blinded, and placebo-controlled clinical
trials are rightly judged to be the ‘‘gold standard’’ in
evidence-based efficacy testing and are generally agreed to
yield the strongest evidence (Green and Byar, 1984). A prospective, blinded, and placebo-controlled laboratory model
allows tight control of confounding variables and avoids
the difficulty in case enrollment sometimes seen in clinical
trials; clients may be reluctant to enroll their pet in a negatively controlled behavioral study because of the severe
nature of the animal’s anxiety. Furthermore, clinical trials
having nonquantifiable (observational and subjective)
outcomes may be plagued by a large placebo effect (Mills
and Cracknell, 2008), making interpretation of results difficult. In the open-field laboratory model of thunderstorm
simulation the dog does not display a variety of behaviors
as would be seen in a home setting, such as seeking a safe
refuge or soliciting attention from family members. The
use of a laboratory model thus reduces the possibility of
behavioral changes in response to owner cues. This thunderstorm simulation was intended to use a standardized model,
which results in a predictable anxiety-based response, to
assess the effectiveness of Harmonease as an anxiolytic.
The thunderstorm simulation was not intended to recreate
all aspects of a thunderstorm, nor can we propose that this
simulation would induce anxiety in pet dogs with an established diagnosis of thunderstorm anxiety. Applications to
real life phobias and anxieties in dogs are suggested by
this study, but specific indications, such as car ride anxiety,
veterinary visits, separation anxiety, or even noise-induced
anxieties and phobias to natural thunderstorms, cannot be
concluded without clinical trials for these indications.
All research studies contain inherent limitations and one
limitation in the current study was the inclusion of only
20 dogs, which may have limited our ability to detect
significant group effects. Furthermore, the use of 7-day
treatment and washout periods was empirical but supported
by a study in mice, which demonstrated anxiolytic properties of honokiol when mice were treated orally for 7 days
and evaluated in an elevated plus-maze test (Maruyama
et al., 1998). The fact that at least one outcome measure
demonstrated statistical significance suggests that this treatment duration was reasonable. Thunderstorm testing was
conducted approximately 75 minutes after treatment. This
was in part based on the study performed by Sufka et al.
(2001), which suggested that the active ingredients in
232
Harmonease have the potential to rapidly exert an anxiolytic effect. Future research testing alterations in one or
more of these parameters is warranted. Another concern
is that our ability to interpret canine behavior based solely
on measurements of activity is limited. Therefore, future
studies should include randomized blinded clinical trials
to determine the clinical efficacy of the product for patients
with anxieties and phobias and to further support the use of
models of this type.
The results of this study indicate that Harmonease was
effective in reducing inactivity in this model; the model was
robust and realistic; Harmonease may yield anxiolytic
effects; and Harmonease may be a beneficial addition to
behavior modification programs for treating anxiety-related
behavior in dogs.
Conclusion
In the present study, Harmonease Chewable Tablets reduced anxiety in laboratory beagles exposed to a simulated
thunderstorm. These results support the clinical and anecdotal evidence for the use of Harmonease in treating
anxiety-related behavior in dogs. Further investigation and
clinical trials are indicated to validate the clinical effectiveness of Harmonease for naturally occurring anxietyrelated behaviors in pet dogs.
Acknowledgments
This project was sponsored by Veterinary Products
Laboratories, which provided both funding and the nutraceutical supplement. The study was conducted under
contract by CanCog Technologies Inc. It was approved by
the Local Animal Care and Use Committee and conducted
in accordance with the Guidelines of the Canadian Council
on Animal Care.
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