Central origin of the antinociceptive action of botulinum toxin type A
Bilateral antinociceptive effect of BTX-A
after unilateral application
Repeated intramuscular
acidic saline injections (pH 4.0) produce a long-lasting mechanical
hyperalgesia in rat (Sluka et al., 2001). Mechanical hyperalgesia from the
muscles spreads to the adjacent tissue (paw) and to the contralateral side,
i.e. a secondary hyperalgesia develops. Bilateral hyperalgesia was not abolished by a lidocaine
injection into the same gastrocnemius muscle nor it was affected by a
unilateral dorsal rhizotomy. It was assumed that the peripheral nervous system
has negligible if any effect in the bilateral pain induced by acidic saline
injections (Sluka et al., 2001). Bilateral effects of a
unilateral injury have been reported in other pain models like bee venom, capsaicin and
carrageenan (Chen et al., 2000;
Sluka, 2002; Radhakrishnan et al., 2003). It is widely accepted that contralateral
spread of hyperalgesia (mirror pain) depends most likely on the plastic changes
in the central nervous system (central sensitization) and that it might be
maintained by spinal and supraspinal mechanism (Koltzenburg et al., 1999;
Graven-Nielsen and Arendt-Nielsen, 2002). An increase in the release of
glutamate in the spinal cord was demonstrated after the second acidic saline
injection (Skyba et al., 2005). Furthermore, spinal
neurons show increased excitability after the acidic saline injections
characterized by bilateral spread of the receptive field (Sluka et al., 2003)
and bilateral increase in phosphorylation of the transcription factor CREB (Hoeger-Bement and Sluka, 2003).
Recent experiments have shown that a descending facilitatory input from the
rostral ventromedial medulla (RVM) are involved in initiation and maintenance of cutaneous and muscle
hyperalgesia associated with chronic muscle pain (Tillu et al., 2008).
It is generally accepted that BTX-A
acts peripherally and consequently, it is difficult to imagine any possibility
of BTX-A action on the
hyperalgesia on the side contralateral to its peripheral injection. In spite of that, in the present study BTX-A
injected into the rat hindpaw pad on the same side as i.m. acidic saline in a
dose 5 U/kg not only reduced secondary mechanical hyperalgesia on that side but
surprisingly on the
contralateral side as well. The effect on both sides was evident on day 5 and
was of similar intensity (Fig. 1). At the same time, BTX-A did
not affect the normal pain treshold on either side. This is in line with
previous observation of us and other authors that BTX-A
effectivelly reduces only pain hypersensitivity but not the acute normal pain
threshold (Bach-Rojecky and Lacković, 2005; Cui et al., 2004).
Antinociceptive effect in present experiments couldn’t be due to the
possible locomotor deficits induced with BTX-A.
Peripheral BTX-A injection into
the hindpaw pad in a dose 5 U/kg did not affect the locomotion in our
experiments (data not shown), nor in experiment done by Cui et al. (2004).
Obviously the antinociceptive effect of BTX-A
in this model cannot be explained only by the common assumption about the peripheral origin of BTX-A action and a local inhibition of neuropeptide
release from the sensory nerve endings. Bilateral effect of the unilateral
peripheral BTX-A injection
suggests the central action of BTX-A
after it’s peripheral application.
When BTX-A was injected into the hindpaw pad contralateral
to the pain induction side, it reduced mechanical hypersensitivity on that side
only (Fig. 2). The observation that BTX-A
is effective independently whether injected in the side with repeated tissue
damage or in the contralateral side without any local damage deserves further
investigation. However, this
result is an exception because in all other presented experiments, the effect
of BTX-A was bilateral as well as
the biochemical and physiological changes associated with this model of mirror
pain seem to bilateral (Hoeger-Bement and Sluka, 2003). Obviously the mechanism
of the BTX-A antinociceptive
action injected on the side of pain induction and injected in the contralateral
side are not equal. For now there is no answer to this puzzle since the contralateral
spread of hyperalgesia in this model of “mirror pain” is not understood.
Is
antinociceptive effect of BTX-A
independent of peripheral nerve endings?
There is theoretical
possibility that BTX-A produces
antinociceptive effect acting primarily on SNAP-25 in the peripheral nerve
endings, while indirectly triggering some long lasting changes in the CNS. Several
papers have indeed described changes at the level of the CNS in man and animals
treated intramuscularly with BTX-A
(Garner et al., 1993; Giladi, 1997; A bbruzzese and Berardelli, 2006). These
changes were usually ascribed to plastic rearrangements subsequent to
denervation or alterations in the sensory input after the toxin local
application. To
elaborate participation of periphery, BTX-A
was injected directly into the n. ishiadicus which was cut distally
to the site of injection. In this experiment attention was paid that no BTX-A leaks outside the nerve. Even after such an
injection, BTX-A produced a
significant antinociceptive effect on the contralateral side (Fig. 3). In line with the common knowledge,
transection of the n. ishiadicusu produced flaccid paralysis on the ipsilateral
side. Because nerve transection prevents
BTX-A to reach the peripheral
nerve endings on that side, this experiment demonstrates that the
antinociceptive effect of BTX-A could not be associated with the ipsilateral SNAP-25 cleavage in the
peripheral cholinergic or any other peripheral nerve endings. Seems that the
only explanation for the observed phenomeneon might be that the anitnociceptive
effect on the contralateral side results from the central action of BTX-A after its retrograde axonal transport from the nerve trunk.
Evidence of an
axonal transport of BTX-A
Antonucci et al. (2008) have recently detected a
time-dependent bilateral SNAP-25 cleavage and blockade of neuronal activity
after a unilateral toxin injection (0.2 ml of 10 nM toxin solution which corresponds to ~0.3 ng,
i.e. 6 U of the toxin per animal) into the rat hippocampus.
Additionally, retrograde appearance of
the BTX-A truncated SNAP-25 in the retina after the
toxin injection into the optic tectum was prevented by the microtubule
depolymerizing agent colchicine. Furthermore, a cleaved SNAP-25 appeared
in the facial nucleus after the injection of the toxin (135 pg ~ 2.8 U) into the rat whisker
muscles. Although using an indirect approach, Antonucci et al. (2008) were the
first to offer novel pathways of BTX-A
trafficking in neurons. From a clinical point of view, these findings
raise the question whether BTX-A
injected into muscles or cutis might induce unexpected central actions, and
whether these actions might have clinical relevance (Currà and Berardelli,
2009). Nowadays there is evidence that after i.m. injection BTX-A might exert CNS effects, partially ascribed
to plastic rearrangements subsequent to the peripheral blockade and partially
due to retrograde axonal transport and direct BTX-A
central effects (Caleo et al, 2009). At present, the functional consenquence of
BTX-A axonal transport through
motor and central neurons is not understood. Up to now, to our knowledge, there
has been neither molecular nor behavioural evidence for the axonal transport of
BTX-A within the sensory nerves.
Results of our experiments cannot be explained without the
assumption that BTX-A is
transported from the site of injection to the CNS. In our experiments, when
colchicine, an axonal transport blocker, was injected into the n. ishiadicus
before the BTX-A application into
the hindpaw pad, it completely prevented the effect of BTX-A
on both sides (Tab. 1.). To exclude the theoretical possibility of anterograde
axonal transport of BTX-A from the
CNS to the contralateral peripheral nerve endings, in one group of animals
colchicine was injected into the n. ishiadicus on the side contralateral to the
s.c. BTX-A injection. In that experiment colchicine did
not affect the toxin's antinociceptive activity on either side (Tab.1.), thus eliminating
possible contribution of the contralateral peripheral nerve endings to the BTX-A effect.
Antinociceptive effect of BTX-A
after intrathecal application
A BTX-A
intrathecal injection in a dose 1 U/kg
abolished the acidic saline induced mechanical hypersensitivity on both
sides (Fig. 4). Luvisetto et
al. (2006) were first to demonstrate antinociceptive effect after central
(intracerebroventricular) injection of small doses of BTX-A
(1.8-3.5 pg/mice, which corresponds to ~1-2 U/kg). They suggested that BTX-A might act not only at peripheral but also at
the central level. Although this is only circumstantial argument, our results
after intrathecal injection in the model of “mirror pain” are in line with
suggestion of Luvisetto et al. (2006) and support the central site of the BTX-A antinociceptive action.
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