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Modulation of formalin-induced pain-related behaviour by clonidine and
yohimbine in the Speke’s hinged tortoise (Kiniskys spekii)
C. M. MAKAU*
P. K. TOWETT
†
K. S. P. ABELSON
‡
&
T. I. KANUI
§
*School of Pure and Applied Sciences, Mount
Kenya University, Nakuru, Kenya;
†
Depart-
ment of Veterinary Anatomy and Physiol-
ogy, University of Nairobi, Nairobi, Kenya;
‡
Department of Experimental Medicine, Fac-
ulty of Health and Medical Sciences, Univer-
sity of Copenhagen, Copenhagen N,
Denmark;
§
School of Agriculture and Veteri-
nary Sciences, South Eastern Kenya Univer-
sity, Kitui, Kenya
Makau, C. M., Towett, P. K., Abelson, K. S. P., Kanui, T. I. Modulation of
formalin-induced pain-related behaviour by clonidine and yohimbine in the
Speke’s hinged tortoise (Kiniskys spekii). J. vet. Pharmacol. Therap. doi:
10.1111/jvp.12374.
The study was designed to investigate the involvement of noradrenergic and
serotonergic receptor systems in the modulation of formalin-induced pain-
related behaviour in the Speke’s hinged tortoise. Intradermal injection of
100 lL of formalin at a dilution of 12.5% caused pain-related behaviour
(hindlimb withdrawal) that lasted for a mean time of 19.28 min (monophasic
response). Intrathecal administration of clonidine (a
2
-adrenergic receptor ago-
nist) and yohimbine (a
2
-adrenergic receptor antagonist) at a dose of 40 lg/kg
and 37.5 lg/kg or 50 lg/kg, respectively, caused a highly significant reduc-
tion in the duration of the formalin-induced pain-related behaviour. The
effect of clonidine was reversed by intrathecal administration of yohimbine at
a dose of 26.7 lg/kg. The effect of yohimbine at a dose of 50 lg/kg was
reversed by intrathecal injection of 20 lg/kg of the serotonergic receptor
antagonist methysergide maleate. When performing antagonistic reactions,
the administration of the antagonist was followed immediately by that of the
agonist. The study indicates that for experimental purposes, intrathecal route
of drug administration through the atlanto-occipital joint is effective in tor-
toises. The data also suggest that testudines have noradrenergic and seroton-
ergic systems that appear to play a role in the modulation of pain in this
species.
(Paper received 18 March 2016; accepted for publication 19 September
2016)
Dr Christopher M. Makau, School of Pure and Applied Sciences, Mount Kenya
University, 17273 –20100, Nakuru, Kenya. E-mail: musembi06@yahoo.com
Chemical compounds studied in this study: Clonidine hydrochloride (PubChem
CID: 20179); yohimbine hydrochloride (PubChem CID: 6169); methysergide
maleate (PubChem CID: 5281073).
INTRODUCTION
The study of sensory systems in lower vertebrates is of great
interest in understanding the evolution of sensory function bet-
ter (Northcutt, 2002; Sneddon, 2004 and Haitao et al., 2008).
Reptiles display characteristic responses to painful stimulation
(Sneddon et al., 2014). The neuroanatomic components neces-
sary for nociception, the endogenous antinociceptive mecha-
nisms and modulation of pain with pharmacological agents
known to be analgesics in other species have also been demon-
strated in several reptiles (Craig, 2006). However, few nocicep-
tive tests have been applied to evaluate nociceptive behaviours
in the reptiles (Sneddon et al., 2014) and in particular the tes-
tudines. Studies are therefore needed to evaluate the efficacy
and adverse effects of analgesics and identify appropriate doses,
routes of administration and durations of action to optimize
testudine patient care.
vMost vertebrate animals have opioidergic, noradrenergic,
serotonergic and cholinergic pain-modulating systems in their
central nervous system. Pain modulation by opioidergic system
in the tortoise (Kinixyx spekii) has been reported (Wambugu
et al., 2010). Pain modulation by the noradrenergic and sero-
tonergic systems has, however, not been reported in this species.
Both systems play important roles in the modulation of nocicep-
tive information in primary afferent neurons in the spinal cord
dorsal horn (Reddy & Yaksh, 1980; Proudfit, 1988). In other
animal species, activation of the endogenous noradrenergic sys-
tem results in analgesic effects (Jones, 1991; Read, 2004; Makau
©2016 John Wiley & Sons Ltd 1
J. vet. Pharmacol. Therap. doi: 10.1111/jvp.12374
et al., 2014). Several findings have highlighted the antinocicep-
tive effects of intrathecal administration of clonidine (a
2
-adrener-
gic agonist) in animal models of pain (Roh et al., 2008; Makau
et al., 2014). In addition, perineural and systemic administration
of clonidine showed anti-inflammatory effect related to immune
modulation (Lavand’homme & Eisenach, 2003; Romero-
Sandoval & Eisenach, 2006). Yohimbine, a relatively selective
a
2
-adrenergic receptor antagonist (Goldberg & Robertson,
1983), is frequently used to assess the involvement of a
2
-adre-
nergic receptors in the mechanism of action of drugs. Yohimbine
has also been shown to act as a partial 5-HT
1A
agonist in the for-
malin test (Shannon & Lutz, 2000).
The aim of this study was to investigate the involvement of
the noradrenergic and serotonergic systems in pain modulation
in the Speke’s hinged tortoise. Clonidine and yohimbine were
used to test the hypothesis that noradrenergic system is
involved in antinociception in the Speke’s hinged tortoise.
Yohimbine (which is also a serotonergic agonist) and Methy-
sergide maleate (serotonergic receptor antagonist) were used to
test the hypothesis that serotonergic system is involved in
antinociception in the Speke’s hinged tortoise. It was hypothe-
sized that intrathecal injection of clonidine would reduce for-
malin-induced pain-related behaviour and that the reduction
would be reversed by administration of yohimbine. Further-
more, the study tested the hypothesis that yohimbine in higher
concentrations would reduce formalin-induced pain-related
behaviour and that the reduction would be reversed by treat-
ment with methysergide maleate. The role of these drugs on
the motor activity was also investigated.
MATERIALS AND METHODS
Animals
A total of 58 Speke’s hinged tortoise sourced from Machakos
and Kangundo districts of Eastern Kenya were used. One ani-
mal was used for validation, 99 for nociceptive/antinociceptive
experiments and eighteen for assessment of the sensorimotor
performance/development of intrathecal method. The animals
used were adult males and females with a mean body weight
of 450 10 g (Mean SEM). They were housed in a well-
ventilated room, with translucent windows. The animals were
kept in open metallic tanks measuring 1.25 91.0 90.6 m.
The tanks were half-filled with sand, and stones were placed
on the sand to mimic their natural environment. Clean water
was provided ad libitum. The animals were fed four times in a
week on ProRep Tortoise food and vegetables, that is cabbages,
kales, carrots and tomatoes. Animals were housed under stan-
dard laboratory conditions with a 12/12-h light/dark cycle
and at a temperature of 26–30 °C. The animals were habitu-
ated to the laboratory conditions for 1 month before the start
of the experiments. During this period, they were handled daily
and adapted to a previously described restraining procedure
(Wambugu et al., 2010; Dahlin et al., 2012). The experimental
animals were acquired and cared for in accordance with the
guidelines published in the NIH Guide and use of laboratory
Animals (National institute of Health Publication No. 85-23
revised 1995). All animal protocols were approved by the
Committee of Veterinary Medicine Biosafety, Animal Care and
Use of the University of Nairobi.
Drugs
The drugs used were clonidine hydrochloride (a
2
-receptor ago-
nist), yohimbine hydrochloride (a
2
-receptor antagonist/5-HT
receptor agonist) and methysergide maleate (5-HT receptor
antagonist) (all from Sigma-Aldrich, Dorset, UK). The aim of
using methysergide maleate was to determine any interaction of
5-HT system with a
2
-antagonist (yohimbine). Clonidine
hydrochloride and methysergide maleate were dissolved in 0.9%
saline. Yohimbine was dissolved in 100% dimethylsulphoxide
(DMSO). Drugs or vehicles were administered intrathecally in a
volume of 100 lL using a 30-gauge needle. The dosages used
were based on preliminary investigations from our laboratory.
Fresh preparations of drugs were always used.
Intrathecal administration
The intrathecal drug administration technique (through the
atlanto-occipital joint) was developed through a similar proce-
dure previously described by Makau et al. (2014). Six Speke’s
hinged tortoises were used for developing this technique. Lido-
caine hydrochloride (100 lL) was used to develop the tech-
nique. A metal rod was used to gently rub the animal at its
back to make it relax. The head was then held at the neck
region and an injection was made by gently inserting a 30-
gauge, one-cm-long needle at the termination of the occipital
process in line with the midline of the head. The needle was
inserted at an angle of about 45
°
until atlanto-occipital mem-
brane was hit. The needle was pushed almost halfway its
length to pass through the atlanto-occipital membrane into the
subarachnoid space where the drug was delivered. Prior to
drug injection, aspiration was performed in order to avoid
intravascular injection. Immediately the needle went through
the atlanto-occipital membrane, the animal normally reacted
by jerking its head. The general behaviour and muscle tension
were observed and recorded. Prior to drug injections, one ani-
mal was used to validate the intrathecal injection technique.
The animal was injected intrathecally with Evans blue dye and
then killed by injecting sodium pentobarbitone (200 mg/kg)
intravenously into the jugular vein. The animals were declared
dead when spontaneous blinking had ceased and the corneal
reflex was absent (Nevarez et al., 2014). Once the animal was
confirmed dead, it was placed on a dissecting dish and slit with
the use of a scalpel at the point where Evans blue was injected.
Pictures of the sections were taken immediately and the loca-
tion of the dye identified histologically.
Formalin test
The animal was restrained on a stand with a string tied
around its shell and then positioned facing away from the
©2016 John Wiley & Sons Ltd
2C. M. Makau et al.
investigator, that is facing the wall (Wambugu et al., 2010;
Dahlin et al., 2012). The procedure was performed in a sound-
attenuated room. The animal was then injected with 100 lL
of 12.5% formalin (
w
/
v
) into the inter-claw space of the hin-
dlimb using a micro-litre syringe and a 29-gauge needle. The
controls for formalin were given 100 lL of saline (0.9% NaCl)
or dimethylsulphoxide (DMSO) in a similar manner to that of
formalin. The total time spent lifting the injected limb (referred
to as hindlimb withdrawal) was recorded. Recording was per-
formed in 12 blocks of 5 min and the data were recorded as
total time spent in pain-related behaviour after the injection of
formalin or vehicle. Animal reuse was allowed only after at
least two weeks washout period, and in that case, injection
was performed on an alternative paw. The experiments were
always performed at a room temperature of 26–28 °C and
between 10.00 a.m. and 2.00 p.m.
Antinociceptive testing
The animals were randomly put in twelve groups each consist-
ing of six to eight animals. Each group was assigned a certain
dosage level of the drug being tested. The injection site was
aseptically prepared before the drugs were injected intrathe-
cally. The animals were injected with clonidine (10, 20 or
40 lg/kg), yohimbine (25, 37.5 or 50 lg/kg), a combination
of clonidine (40 lg/kg) and yohimbine (25 lg/kg) or a combi-
nation of methysergide maleate (20 lg/kg) and yohimbine
(50 lg/kg). Five minutes after drug injection, the animals were
injected with formalin 12.5% on the hind paw and recording
started immediately. The 5-min lapse between drug injection
and formalin injection and dose levels were chosen based on
published data (Kanui et al., 1993; Makau et al., 2014) and
preliminary studies. The controls were injected with saline or
DMSO intrathecally.
Assessment of the sensorimotor performance
Muscle tension and locomotion were used to assess the sensori-
motor performance. After the injection of a drug or vehicle,
the animal was placed in an observation cage and observed for
one hour. Locomotion of the animal was assessed by monitor-
ing its movement across a marked line drawn on the floor of
the cage. To test the muscle tension, a pair of forceps was used
to stretch the hind and front feet and the animal’s response
was noted. Assessment of the effects of the drugs on the ten-
sion of the muscle and locomotion was based on arbitrary
scale of 0–4 as follows:
0- No muscle tension/hypoactivity.
2- Normal tension/activity.
4- High muscle tension/hyperactivity.
Data analysis
Data collected following formalin test and antinociceptive test-
ing were tested for equal variance and normal distribution.
The data were analysed using one-way ANOVA with a two-sided
Dunnett’s post hoc test using IBM Corp. SPSS V21.0, Armonk,
NJ, USA. Sensorimotor performance data were ordinal scale
data, and the effects of the drugs on sensorimotor performance
were analysed using Kruskal–Wallis ANOVA by ranks. P-values
lower than or equal to 0.05 were considered statistically signif-
icant. For the antinociceptive experiments, results are pre-
sented as means standard error of the mean (SEM).
RESULTS
Formalin test
Subcutaneous injection of 100 lL of formalin (12.5%) induced
a highly significant pain-related behaviour (hindlimb with-
drawal) compared to that for animals injected with saline or
DMSO (P≤0.001, F-test). The mean time spent in pain-related
behaviour following 12.5% formalin administration was
19.3 0.4 min (n=8). The injection of formalin caused
monophasic pain-related behaviour. Other behaviours observed
after formalin injection were defecation, salivation and urina-
tion. The mean time spent in hindlimb withdrawal following
injection with saline or DMSO (5 lg/kg) was 0.4 1.3 min
(n=6) and 0.2 0.1 min (n=6), respectively. The range of
the time spent in hindlimb withdrawal following injection with
saline or DMSO (5 lg/kg) is 0.5–1.2 min. Neither saline nor
DMSO caused any significant change in the position of the
injected limb (Fig. 1).
0
5
10
15
20
25
Formalin (12.5%) Saline (0.9%) DMSO
Mean hind limb withdrawal time (min)
***
Fig. 1. Effect of subcutaneous injection of formalin (12.5%), saline
(0.9%) or dimethylsulphoxide (DMSO) on the mean hindlimb with-
drawal time in the tortoise. Bars represent means SEM. and n=6–8
in each group. Treatment means were compared using Dunnett’s (two-
sided) test, subsequent to ANOVA.***denotes P<0.001 (saline or DMSO
group verses formalin group).
©2016 John Wiley & Sons Ltd
Descending pain modulation in the tortoise 3
The mean time spent in hindlimb withdrawal after intrathe-
cal administration of saline (100 lL) or clonidine at doses of
10 lg/kg, 20 lg/kg or 40 lg/kg was 19.3 1.3, 17.1 0.3,
15.1 0.3 and 9.1 0.7 min, respectively (Fig. 2). Intrathe-
cal administration of clonidine at a dose of 40 lg/kg induced a
highly significant [F
ANOVA
(3, 20) =11.344; P<0.001]
decrease in the mean hindlimb withdrawal time compared to
the saline control. Intrathecal administration of clonidine
10 lg/kg or 20 lg/kg did not induce a significant decrease in
hindlimb withdrawal time.
The mean time spent in hindlimb withdrawal after intrathe-
cal administration of DMSO (100 lL) or yohimbine at doses of
25 lg/kg, 37.5 lg/kg or 50 lg/kg was 19.3 0.7,
17.5 0.2, 13.1 0.2 and 8.1 0.3 min, respectively
(Fig. 3). Yohimbine at doses of 37.5 lg/kg or 50 lg/kg
induced a highly significant [F
ANOVA
(3, 21) =19.439;
P<0.05] decrease in the hindlimb withdrawal time compared
to DMSO control. Intrathecal administration of yohimbine at a
dose of 25 lg/kg did not induce a significant decrease in hin-
dlimb withdrawal time.
The mean time spent in hindlimb withdrawal after intrathe-
cal administration of yohimbine at a dose of 25 lg/kg followed
immediately by administration of clonidine at a dose of 40 lg/kg
was 18 0.3 min (Fig. 4). The mean time spent in hindlimb
withdrawal for the combined treatment (clonidine, 40 lg/kg +
yohimbine, 25 lg/kg) was significantly higher when compared
to that of clonidine alone [F
ANOVA
(2, 15) =19.935; P<0.001].
0
5
10
15
20
25
Saline 10 20 40
Mean hind limb withdrawal time (min)
Clonidine (µg/kg)
***
Fig. 2. Effect of intrathecal administration of saline or clonidine (10,
20 or 40 lg/kg) on the mean hindlimb withdrawal time in the forma-
lin test in the Speke’s hinged tortoise. Bars represent means S.E.M.
and n=6 in each group. Treatment means were compared using Dun-
nett’s (two-sided) test, subsequent to ANOVA.***denotes P<0.001
(treatment group verses saline group).
0.00
5.00
10.00
15.00
20.00
25.00
DMSO 25 37.5 50
Mean hind limb withdrawal time (min)
Yohimbine (µg/kg)
***
***
Fig. 3. Effect of intrathecal administration of dimethylsulphoxide
(DMSO) or yohimbine (25, 37.5 or 50 lg/kg) on the mean hindlimb
withdrawal time in the formalin test in the Speke’s hinged tortoise.
Bars represent means SEM. and n=6 in each group. Treatment
means were compared using Dunnett’s (two-sided) test, subsequent to
ANOVA.***denotes P<0.001(DMSO group verses treated groups).
0
2
4
6
8
10
12
14
16
18
20
Yoh (25) Cl (40) Yoh and Cl (25 and 40)
Mean hind limb withdrawal time (min)
Treatment (µg/kg)
***
Fig. 4. Effects of intrathecal yohimbine (Yoh, 25 lg/kg), clonidine (Cl,
40 lg/kg) or a combination of yohimbine and clonidine (Yoh, 25 lg/
kg +Cl, 40 lg/kg) on the mean hindlimb withdrawal time in the for-
malin test in the Speke’s hinged tortoise. Bars represent means SEM.
and n=6 in each group. Treatment means were compared using Dun-
nett’s (two-sided) test, subsequent to ANOVA.***denotes P<0.001
(clonidine 40 lg/kg group verses the combined treatment group).
©2016 John Wiley & Sons Ltd
4C. M. Makau et al.
The mean time spent in hindlimb withdrawal after the
administration of methysergide maleate at a dose of 20 lg/kg
followed immediately by administration of yohimbine at a dose
of 50 lg/kg was 19.4 0.2 min (Fig. 5). The mean time
spent in hindlimb withdrawal for the combined treatment
(methysergide maleate, 20 lg/kg +yohimbine, 50 lg/kg) was
significantly higher when compared to that of yohimbine alone
[F (2, 15) =26.788; P<0.001]. Administration of methy-
sergide maleate (20 lg/kg) alone caused a mean hindlimb
withdrawal time of 19.8 min which was insignificant as com-
pared to the combined treatment (Fig. 6). Administration of
methysergide maleate at a dose of 20 lg/kg followed 5 min
later by injection of formalin had no significant effect on hin-
dlimb withdrawal time (Fig. 6). None of the doses of the drugs
used in the experiments had any significant effect on locomo-
tion and muscle tension.
DISCUSSION
In most animal species studied, formalin test is characterized
by a biphasic pain response, with distinct first and second
phases (Reddy & Yaksh, 1980). In this study, the tortoises
showed a monophasic pain-related behaviour response (hin-
dlimb withdrawal) following injection with formalin. The pain-
related behaviour lasted for approximately 19 min (Fig. 1).
This confirms previous findings of formalin test in the tortoise
(Wambugu et al., 2010). The results indicate that similar to
crocodiles (Kanui et al., 1990), testudines do not show the sec-
ond phase of pain-related behaviour in the formalin test.
Recently, a similar finding was reported in the marsh terrapin
(Makau et al., 2014).
The first phase of the formalin test is commonly attributed to
acute nociception caused by direct activation of nociceptive
fibres (Puig & Sorkin, 1996), whereas the second phase is
attributed to tonic nociception resulting from tissue inflamma-
tion (Haley et al., 1989; Miyata et al., 2003). The lack of sec-
ond phase in the tortoise suggests that formalin perhaps does
not cause significant tissue inflammation necessary for tonic
nociception attributed to development of the phase. These ani-
mals may be lacking or having different functions of neuro-
transmitters such as substance P, calcitonin gene-related
peptide, NMDA and NK-1 which are known to play a role in
the development of the second phase of the formalin test (Allen
et al., 2002). Previous studies have demonstrated that PLCb4
is crucial for the formalin-induced inflammatory pain but not
acute pain (Miyata et al., 2003). Therefore, there is a
0.00
5.00
10.00
15.00
20.00
25.00
DMSO Yoh (50) Met and Yoh
(20 and 50)
Mean hind limb withdrawal time (min)
Treatment (µg/kg)
***
Fig. 5. Effect of administration of DMSO, yohimbine (Yoh, 50 lg/kg) or
a combination of methysergide maleate (Met - 20 lg/kg) and yohim-
bine (Yoh, 50 lg/kg) on the mean hindlimb withdrawal time in
the formalin test in the Speke’s hinged tortoise. Yohimbine was
administered 5 min prior to the injection of formalin. Yohimbine was
intrathecally administered immediately after methysergide maleate
administration. Bars represent means SEM. and n=6. Treatment
means were compared using Dunnett’s (two-sided) test, subsequent to
ANOVA.***denotes P<0.01 (yohimbine 50 lg/kg group verses the
combined treatment group).
0
5
10
15
20
25
Met (20) Yoh (50) Met and Yoh
(20 and 50)
Mean hind limb withdrawal time (min)
Treatment (µg/kg)
***
Fig. 6. Effect of administration of methysergide maleate (Met, 20 lg/kg),
yohimbine (Yoh, 50 lg/kg) or a combination of methysergide maleate
(Met, 20 lg/kg) and yohimbine (Yoh, 50 lg/kg) on the mean hindlimb
withdrawal time in the formalin test in the Speke’s hinged tortoise.
Methysergide maleate (20 lg/kg) and yohimbine (50 lg/kg) were
administered 5 min prior to the injection of formalin. For antagonistic
reaction, Yohimbine was intrathecally administered immediately after
methysergide maleate administration. Bars represent means SEM.
and n=6. Treatment means were compared using Dunnett’s (two-
sided) test, subsequent to ANOVA.*** denotes P<0.01 (yohimbine
50 lg/kg group verses the combined treatment group).
©2016 John Wiley & Sons Ltd
Descending pain modulation in the tortoise 5
possibility that the animals could be lacking or having limited
proteins in their nociceptive pathways critical for the formalin-
induced inflammatory pain. This is an area that requires inves-
tigation to determine the mechanism behind the lack of the
second phase.
In this study, intrathecal method of drug delivery was used
effectively for administration of the study drugs during the
antinociceptive testing experiments. Formalin injection in the
hind leg activates primary afferent fibres of the lower segments.
In this study, doses of clonidine and yohimbine were delivered
intrathecally and are thought to have acted spinally or
supraspinally by blocking the transmission of nociceptive signal
through the ascending tracts. There is evidence that intrathe-
cally administered clonidine and yohimbine act spinally and
supraspinally (Buerkle & Yaksh, 1998). Intrathecal injection
involves drug administration into the subarachnoid space,
directly into the area occupied by cerebrospinal fluid (CSF).
Previous studies have indicated that testudines spinal cord is
covered by three meninges (Carvalho et al., 2011) just like the
mammalian and bird species. A well-developed cerebrospinal
fluid-filled intrathecal (subdural) space directly surrounds the
spinal cord and allows for intrathecal administration of various
anaesthetic and analgesic drugs (Rivera et al., 2011). Owing to
the presence of the carapace and the fusion of the vertebral
column to the carapace, access to the intrathecal space is lim-
ited in chelonians to the cervical and coccygeal vertebrae. The
site of injection (termination of the occipital process in line
with the midline of the head) allows for proper restraint in
conscious tortoises. Careful insertion of the needle should carry
minimal risk of damage to the spinal cord. During the study,
there was no spinal cord injury as evidenced by normal senso-
rimotor performance by all the study animals.
In this study, clonidine, administered intrathecally, produced
a dose-dependent decrease in the mean duration of hindlimb
withdrawal (Fig. 2), suggesting antinociceptive effect. This find-
ing is in agreement with previous reports observed in the for-
malin test in rats (Kanui et al., 1993), in Haffner test in mice
(Ossipov et al., 1988), in tail-flick test in rats and mice (Ossipov
et al., 1988), in paw pressure test in normal and arthritic rats
(Kayser et al., 1992), in carrageenan-induced inflammation/
hyperalgesia test in rats (Hylden et al., 1991), in writhing test
in amphibians (Brenner et al., 1994), in radiant heat-evoked
hind paw withdrawal in rats (Naguib & Yaksh, 1994) and
recently in the formalin test in marsh terrapins (Makau et al.,
2014). Clonidine, a a
2
-adrenergic receptor agonist, has been
used in various clinical settings and has been shown to exert
excellent analgesic effects, especially when administered by
epidural or intrathecal route (Steriade & McCarley, 1990).
Although intrathecal clonidine produced antinociceptive effect
in testudines in the current study, Fujimoto and Arts (1990)
reported no antinociception in the tail-flick test in mice when
the drug was administered by intracerebroventricular route.
Similarly, Eisenach et al. (1998) reported a lack of effect of
clonidine, injected intravenously, in an experimentally thermal
or capsaicin-induced pain and hyperalgesia in normal volun-
teers. The differences might be explained on the basis of the
route of administration, type of algesiometric test used, the spe-
cies studied and perhaps the experimental protocol.
There is evidence that systematically administered clonidine
induces antinociception by spinal action (Steriade & McCarley,
1990). Clonidine influences nociceptive transmission by exert-
ing inhibitory effects on C-fibre terminals in the spinal cord
(Calvillo & Ghignone, 1986), by decreasing the release of glu-
tamate or substance P from primary afferent nerve terminals
(Pang & Vasko, 1986), and by mimicking the action of spinally
released norepinephrine from descending noradrenergic inhibi-
tory pathways (Sladky et al., 2007). In addition, clonidine can
act postsynaptically to hyperpolarize dorsal horn wide dynamic
range neurons (Fleetwood-Walker et al., 1985) and increase
acetylcholine in the dorsal horn of the spinal cord (Klimscha
et al., 1995; Abelson & H€
oglund, 2004). The mechanisms reg-
ulating a
2
-adrenergic-mediated antinociception in testudines
are not clear at the moment but it is assumed to be similar to
those of other vertebrates.
Yohimbine, a a
2
-adrenergic receptor antagonist (Goldberg &
Robertson, 1983) is frequently used to assess the involvement
of a
2
-adrenergic receptors in the mechanism of action of ago-
nistic cholinergic drugs. Yohimbine at a dose of (25 lg/kg) sig-
nificantly antagonized the antinociceptive effects of clonidine
(40 lg/kg) (Fig. 4). The dose (25 lg/kg) of yohimbine chosen
for antagonistic reaction had no effect when administered
alone on the mean hindlimb withdrawal time. The data there-
fore suggest that clonidine induced antinociception in the
Speke’s hinged tortoise by interacting with the a
2
-noradrener-
gic system. This conclusion is concordant with previous find-
ings reported in other nociceptive tests in different animal
models (Howe et al., 1983; Ossipov et al., 1988; Kanui et al.,
1993; Naguib & Yaksh, 1994; Dharmananda, 2005; Makau
et al., 2014). Several studies have indicated that yohimbine
does not completely antagonize the effects of clonidine in the
formalin test (Rosland et al., 1990; Dharmananda, 2005), but
instead it may act as an analgesic agent (Rosland et al., 1990).
This agrees well with the current study, where the higher
doses (37.5 or 50 lg/kg) of yohimbine showed antinociceptive
effects (Fig. 3). This is also in agreement with reports where
intrathecal (i.t) yohimbine inhibited nociception in the hot
plate and the formalin tests in rats and mice (Dennis et al.,
1980; Kanui et al., 1993). This effect can be partly explained
by the fact that yohimbine also has an affinity for serotonin
(5-HT
1A
) receptors (Millan et al., 2000). This is supported by
the present data showing that the antinociceptive effects of
yohimbine in the formalin test were reversed by the nonselec-
tive 5-HT receptor antagonist methysergide maleate (Fig. 5).
We therefore suggest that the yohimbine-induced antinocicep-
tion is mediated through the agonistic activity at 5-HT recep-
tors. Further experiments are needed to explore the
involvement of 5-HT receptors in antinociception in the
tortoise.
Some researchers have suggested that clonidine may not
have analgesic properties, but merely impair the ability of the
animal to respond to the nociceptive stimulation (Izenwasser &
Kornetsky, 1990). Clonidine induced hypnosis (Mizobe et al.,
©2016 John Wiley & Sons Ltd
6C. M. Makau et al.
1996), hypotension and motor blockade (Klimscha et al.,
1995) and altered thermoregulation (LoPachin & Rudy, 1981).
Although some of these parameters were not measured, the
treated animals appeared normal as the control ones. The sen-
sorimotor studies were carried out to assess the motor function
and consequently sedation in these animals.
In conclusion, this study shows that the intrathecal method
of drug delivery is a suitable and effective method of drug
administration in the Speke’s hinged tortoise in a research set-
ting. The study also supports previous finding that the formalin
test is a good test for studying nociception and antinociception
in the Speke’s hinge-back tortoise. It is evident from the pre-
sent data that the noradrenergic and serotonergic systems are
involved in antinociception in this species. Further research is
required to address the mechanisms of the unique monophasic
pain in the formalin test in reptiles.
ACKNOWLEDGMENTS
We express our gratitude to James Bisiker, Brenda Bisiker and
Lindsay Bisiker for funding this work. We also gratefully thank
Margaret Kagina, Kavoi, Stanley Wambugu and Manyi who
gave us technical support during the course of the study.
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