Abstract
Chronic relapsing experimental allergic encephalomyelitis (CREAE) is an
autoimmune model of multiple sclerosis1. Although both these
diseases are typified by relapsing-remitting paralytic episodes, after CREAE
induction by sensitization to myelin antigens1 Biozzi ABH mice
also develop spasticity and tremor. These symptoms also occur during multiple
sclerosis and are difficult to control. This has prompted some patients to
find alternative medicines, and to perceive benefit from cannabis use2. Although this benefit has been backed up by small clinical studies,
mainly with non-quantifiable outcomes3, 4, 5, 6, 7, the value
of cannabis use in multiple sclerosis remains anecdotal. Here we show that
cannabinoid (CB) receptor agonism using R(+)-WIN 55,212, 
9-tetrahydrocannabinol, methanandamide and JWH-133 (ref. 8) quantitatively ameliorated both tremor and spasticity
in diseased mice. The exacerbation of these signs after antagonism of the
CB1 and CB2 receptors, notably the CB1 receptor,
using SR141716A and SR144528 (ref. 8) indicate
that the endogenous cannabinoid system may be tonically active in the control
of tremor and spasticity. This provides a rationale for patients' indications
of the therapeutic potential of cannabis in the control of the symptoms of
multiple sclerosis2, and provides a means of evaluating more
selective cannabinoids in the future.
High doses of 9-tetrahydrocannabinol THC; (the major
psychoactive component of cannabis) can inhibit the development of CREAE in
rodents9, 10, but this has been attributed to immunosuppression
preventing the conditions that lead to the development of paralysis, rather
than to a direct effect on the paralysis itself9, 10. However,
the action of cannabinoids on experimental spasticity and tremor remains uncertain
because there have so far been no behavioural data on the effects of cannabinoids
in animal models relevant to these symptoms of multiple sclerosis.
It is well established that repeated neurological insults occur during
CREAE; these are associated with increasing primary demyelination and axonal
loss in the central nervous system (CNS)1. However, it was also
evident that CREAE animals can develop additional clinical signs, including
unilateral or bilateral fore- and hindlimb tremor (Fig. 1)
and hindlimb spasticity (Fig. 2). These accumulate with
disease duration and activity. Tremor was associated with voluntary limb movements,
but in more severe cases it was persistent at a frequency of 
40 Hz
(Fig. 1e). Although considerably faster than encountered
in humans (6 Hz), this frequency is consistent with tremor electromyography
in mutant spastic (GlrbSpa) mice11.
These animals develop episodes of rapid tremor and rigidity of the limb and
trunk muscles12. However, unlike the GlrbSpa
mouse, spasticity in CREAE mice need not be triggered by sudden
disturbance12. The effects of cannabis are mediated through
the CB1, CB2 and putative CB2-like receptors13, 14. CB1 is predominant in the CNS and is the main target
for psychoactivity, but it is also expressed at lower levels in many peripheral
tissues. The CB2 receptor is expressed at high levels on leucocytes,
but there is also evidence for limited CB2 receptor expression
in mouse brain13, 4. The administration of a full CB1
and CB2 agonist, R(+)-WIN 55,212 (ref.
8), to post-relapse remission mice resulted in a rapid (within 1–10 min)
amelioration of the frequency and amplitude of tremor in both the fore- and
hindlimbs of CREAE mice (Fig. 1). This was visually
evident at 5 mg kg-1 (Fig. 1a–d
; n = 10/10) and 1 mg kg
-1 intraperitoneal (i.p.) (n = 6/6). In addition,
9-THC (10 mg kg-1 intravenous (i.v.))
also ameliorated this response (n = 5/5). Tremor returned
within hours after treatment. As 9-THC was observed
to be relatively ineffective when injected intraperitoneally (i.p.), as seen
in other studies10, all subsequent compounds were injected intravenously.
Furthermore, as 9-THC is a partial CB1 agonist
but provides more limited CB2 agonist activity, these results suggest
that the effect on tremor is mainly mediated by the brain CB1 receptor8.
Figure 1: Cannabinoid receptor agonism inhibits tremor in autoimmune encephalomyelitis1.
Mice with hindlimb (a, b) or fore- and hindlimb (c, d) tremor both before (a, c) and after (b, d) treatment with 5 mg kg-1 i.p. with R(+)-WIN 55,212. e, Power spectra of hindlimb tremors recorded with the foot suspended above a strain gauge before (thick line) and after (thin line) 5 mg kg -1 i.p. R(+)-WIN 55,212 injection. Inset, snapshot of raw record over 0.5 s.
High resolution image and legend (42K)Figure 2: Spasticity develops in autoimmune encephalomyelitis1.
a, Spastic hindlimb showing full extension, including the digits. These were pressed against a strain gauge to measure the force required to bend the leg to full flexion. b, Increased resistance to flexion in post-relapse remission animals with spasticity (n = 12 mice) compared with age-matched mice without evidence of spasticity ( n = 5 mice; asterisk, = P < 0.001), or during active paralytic relapse episodes (n = 6; two asterisks, = P < 0.001).
High resolution image and legend (43K)Pretreatment (10 min) of animals with 5 mg kg
-1 i.v. of both selective CB1 (SR141716A) (ref. 15) and CB2 (SR144528) (ref.
16) receptor antagonists eliminated the capacity of 5 mg kg
-1 i.p. R(+)-WIN 55,212 to inhibit tremor (n
= 5/5). However animals with residual paresis and mild spasticity became
significantly more spastic after such CB receptor antagonism (
Fig. 3). This was associated with uncontrolled leg crossing (Fig. 3c and d) and severe tail spasms.
These showed gross curling which is atypical of post-remission animals, in
which the tail generally hangs limply (Fig. 3e). Animals
also show hindlimb extension (Fig. 3c), including a
significant (P < 0.0001) increase in resistance
to flexion (Fig. 3a, f).
This was not observed in vehicle-treated controls (Fig. 3a
). These signs were also not evident in similarly injected normal mice
(n = 0/5) or normal-appearing pre-acute EAE animals
(hindlimb resistance to flexion 0.159 
0.013N compared with
0.206 0.022N in treated mice (n = 12
limbs, P > 0.05) and in animals with paresis/paralysis
without evidence of spasticity (n = 0/5 treated with
SR141716A and SR144528, n = 0/4 treated with SR141716A
or SR144528 alone). When mildly spastic animals without tremor were injected
with 5 mg kg-1 i.v. CB1 antagonist,
not only did significant hindlimb (P < 0.001; Fig. 3a) and tail spasticity (n = 18/18
, P < 0.001) develop compared with vehicle
treated controls (n = 0/6), but forelimb tremor also
became evident in 3 out of 10 mice. This suggests a role for CB1
in the control of tremor. After injection of 5 mg kg
-1 i.v. CB2 antagonist, some animals (n
= 10/14) seemed to show a mild increase in tail spasticity (
P < 0.02) and showed a small but significant (
P < 0.05) increase in resistance to hindlimb flexion (Fig. 3a). However, when the CB2 antagonist was
injected into animals previously made more spastic (P
< 0.01) by CB1 antagonism, spasticity increased significantly
(P < 0.001) compared with animals treated with
SR141716A alone, whereas this was resolved in animals treated with vehicle.
This suggests that both CB receptors may control spasticity (
Fig. 3f). However, it is possible that the effects of SR144528 could
be mediated by CB2-like (rather than CB2) receptors
as previously proposed17, or that at the dose used, SR144528
may have produced additional CB1 antagonism because it has some
limited capacity to bind to CB1 (ref. 8).
These observations may indicate the continual release of endogenous cannabinoid
receptor agonists such as anandamide and 2-arachidonylglycerol which are present
within the brain and exhibit neurotransmitter function18. Alternatively,
or in addition, they may reflect the presence of precoupled, constitutively
active cannabinoid receptors, as there is evidence that SR141716A and SR144528
are both inverse agonists that are capable of producing inverse cannabimimetic
effects by reducing the proportion of cannabinoid recetors that exist in a
precoupled state8, 15, 16. In comparison to some studies in
which the antagonists affected the exogenous agonists17, the
actions of the antagonists seen here were relatively short-lived (
Fig. 3f). This may reflect the fact that the animals were attempting
to compensate for the antagonist effect, and would be consistent with tonic
control of the endogenous cannabinoid system. These data provide compelling
evidence that CB receptors are involved in the control of spasticity in an
environment of existing neurological damage, and that exogenous agonism may
be beneficial.
Figure 3: Control of spasticity by the cannabinoid system.
a, Forces required to flex individual spastic hindlimbs against
a strain gauge before and after injection with vehicle (0.1 ml i.v.,
n = 14), SR141716A (5 mg kg-1 i.v.,
n = 32), SR144528 (5 kg kg-1 i.v.,
n = 32), SR141716A and SR144528 (n = 21
limbs), R(+)-WIN 55,212 (5 mg kg-1 i.p.,
n = 16) or S(-)-WIN 55 55,212 (5 mg kg
-1 i.p., n = 19). b–e,
Cannabinoid receptor antagonism increased spasticity. Before (b) and
after (c, e) SR141716A and SR144528 or after SR141716A (
d) administration. c, d, Extension and crossing of limbs;
e, spastic tail. f, Resistance to flexion forces 5 min after
SR141716A or SR144528 administration. 10 min later, mice were re-injected
(5 mg kg-1 i.v.) with either SR144528 (
n = 10), vehicle (n = 15) or SR141716A
(n = 18 limbs) and the resistance to flexion assessed
after 5 min. g, Cannabinoid receptor agonism in spastic mice
after either R(+)-WIN 55,212 (n = 16 limbs),
9-THC (n = 18), methanandamide (
n = 23) or cannabidiol (n = 22). Asterisk,
= P < 0.001 compared with baseline. h, Spasticity
was ameliorated (i) by treatment with R(+)-WIN 55,212.
Indeed, in mice with significant spasticity, 5 mg kg
-1 i.p. R(+)-WIN 55,212 reduced severity both visually (
n = 7/7; Fig. 3g, h and i) and after assessment of resistance to hindlimb flexion
(P < 0.001) (Fig. 3a and i). This was also evident with 2.5 mg kg
-1 i.p. R(+)-WIN 55,212 (Resistance of flexion of both limbs
being reduced (P < 0.05) from 0.384
0.096N to 0.276 0.063N, n = 7,
P < 0.05). Similar treatment with 5 mg kg
-1 i.p. of the inactive enantiomer S(-)-WIN 55,212 failed to
significantly affect the spastic resonse (Fig. 3a).
In contrast, 10 mg kg-1 i.v. 9
-THC and 5 mg kg-1 i.v. methanandamide
(CB1-selective; Ki for CB1 
20 nM and Ki for CB2 815 nM
)8 induced a significant (P < 0.001
) amelioration in spasticity (Fig. 3g). Coupled
with the observations using SR141716A, this may suggest further that CB
1 is a main target for control of spasticity. Currently there are no
compounds which are totally CB1 or CB2 receptor specific,
but the lack of effect after 10 mg kg-1 i.v.
cannabidiol (main non-psychoactive component of cannabis. Ki
for CB1 = 4350 nM)8 suggested a subthreshold
dose for CB1 stimulation for treatment of spasticity. Using the
CB2-selective agonist JWH-133 (1.5 mg kg-1
i.v. Ki for CB1 680 nM
and Ki for CB2 3 nM)8, 19 spasticity was reduced both 10 min (P
< 0.05) and 30 min (P < 0.001) after
injection at a time when 0.05 mg kg-1 i.v. (dose
selected to exhibit similar CB1 activity to JWH-133) methanandamide
was not active (Fig. 4). It is possible that sedative
effects may have contributed (though CB1 receptors) to cannabinoid-mediated
effects in these assays, but there was no hypothermia, indicative of 'sedation'
after JWH-133 administration (37.1 0. °C (baseline),
37.2 0.4 °C (10 min) 37.1 0.2 °C
(30 min)). That non-CB1 receptors may also control
spasticity is further indicated by the transient inhibition of spasticity
with the endocannabinoid palmitoylethanolamide (Fig. 4).
This compound has no significant affinity for CB1 but may have
activity for CB2-like receptors8. The involvement
of non-CB1 receptors may be definitively resolved through the use
of CB receptor subtype-specific compounds or CB-receptor-deficient mice.
Figure 4: Treatment of spasticity in autoimmune encephalomyelitis1 with non-CB1 receptor agonists.
Forces (mean s.e.m.) required to flex individual spastic
hindlimbs against a strain gauge after i.v. injection with either low-dose
methanandamide (n = 9 limbs), JWH-133 (
n = 9) or palmitoylethanolamide (n = 14).
Asterisk, P < 0.05; two asterisks,
P < 0.001 compared with baseline.
Spasticity in patients with multiple sclerosis can be very difficult to
control despite the use of oral baclofen, dantrolene, diazepam and tizanidine,
continuous intrathecal baclofen infusion, and selective injection of botulinum
toxin20. There is a need for more effective oral or systemic
antispasticity agents. The hydrophobic nature of cannabinoids allows their
rapid access to the CNS. Although the effects of chronic administration and
dose dependency of CB receptor agonists on experimental spasticity remain
to be investigated further, the data presented here provide evidence for the
rational assessment of cannabinoid derivatives in the control of spasticity
and tremor in multiple sclerosis, in placebo-controlled trials. The observation
that CB1 appears to be the main therapeutic target suggests that
it may be difficult to dissociate the full benefit from undesirable psychoactive
elements using 9-THC or cannabis. It is also consistent
with the unpleasant side effects experienced by some patients at the doses
required for potential therapy by existing cannabinoids3. The
use of selective CB2 agonists may provide some symptomatic benefit
without significant psychoactive effects. Furthermore, it may be possible
to upregulate endogenous produced cannabinoids18 to mediate
therapeutic benefit. This CREAE model provides a means of evaluating and controlling
the pathophysiology of spasticity in a chronic inflammatory environment relevant
to the control of multiple sclerosis.
Methods
Induction of CREAE
Biozzi ABH mice, bred at the Institute of Ophthalmology, were injected with 1 mg of mouse spinal cord homogenate emulsified in Freund's complete adjuvant on days 0 and 7 (ref. 1). Animals injected for CREAE, before the onset of acute phase CREAE1 (usually occurring 15–20 days post inoculation (p.i.)) were used as normal CREAE controls. Paralysed CREAE animals were selected during the acute phase or first relapse (typically occurring 34–45 days p.i.), and remission animals used for the assessment of tremor and spasticity were used after the second or third relapse 40–80 days p.i.).
Chemicals
R(+)-WIN 55,212, S(-)-WIN
55,212, 9-THC, methanandamide and cannabidiol were purchased
from RBI/Sigma (Poole, UK). Palmitoylethanolamide was purchased from Tocris
Cookson Ltd (Bristol, UK). SR141716A (ref. 15)
and SR144528 (ref. 16) were supplied by M. Mossé
and F. Barth (Sanofi Research, Montpellier, France). JWH-133 (3-(1'1'dimethylbutyl)-1-deoxy-
8-THC) was synthesised as described19. All compounds
were dissolved at 0.5 mg ml-1 in ethanol containing
1 mg ml-1 Tween 80 (Sigma). The ethanol was removed
by vacuum drying, and samples were reconstituted with phosphate buffered saline
to a concentration of 2 mg ml-1. Similar preparations
without active drugs were used as vehicle controls. Suspensions (0.1 ml)
were injected either i.v. or i.p. after CREAE induction.
Assessment of Clinical Signs
Spasticity and tremor
were initially assessed by blinded analysis of video recordings. Digital images
were sampled from video at 0.04 s. Signs of tail spasticity (flicking
and curling) were assessed visually as being either present or absent. Spasticity
was confirmed by assessing limb spasticity against a small purpose-build strain
gauge. Limbs of animals without clinical evidence of spasticity (propensity
to full extend the limb after tension on the leg) or the propensity to cross
were not examined in drug studies. The analogue signal was amplified and digitally
converted using an Amplicon card (Brighton, UK). This was captured using dacquire
V10 software (D. Buckwell, MRC HMBU, Institute of Neurology) and analysed
using Spike 2 software (Cambridge Electronic Design, UK). The hindlimbs were
fully extended twice then moved to full flexion against the strain gauge.
Each hindlimb was individually assessed by a blinded operator. The mean of
4–8 individual readings per limb was taken. Tremor frequency and severity
were also recorded by holding the limb 5 mm above the strain gauge.
Tremor lead to the foot knocking the strain gauge. The strain gauge output
was notch filtered at 50 Hz. The device had a resonance frequency of
95 Hz. The frequency of limb tremor was also confirmed using a lightweight
unidirectional accelerometer (EGA XT-50, Entrain, UK) mounted over the foot.
Statistical Analysis
Results are expressed as means
of individual feet or animals s.e.m. per group. The data were
assessed using either a t-test, paired t-test for flexion data
or nonparametric Mann–Whitney U-test using SigmaStat 2.0 software
(Jandel Corp, San Rafael, California, USA).