Central origin of the antinociceptive action of botulinum toxin type A

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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, sWillis et al., 1996) pinal 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.