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Antinociceptive effects of A1 and A2 type botulinum toxins on carrageenan-induced hyperalgesia in rat
Toxicon 64 (2013) 12–19
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Antinociceptive effects of A1 and A2 type botulinum toxins on carrageenan-induced hyperalgesia in rat Min-Chul Shin a,1, Takashi Yukihira a, b,1, Yushi Ito a, Norio Akaike a, * a b
Research Division for Life Sciences, Kumamoto Health Science University, Izumimachi 325, Kumamoto 861-5598, Japan Department of Physical Therapy, Teikyo University Faculty of Fukuoka Medical Technology, Fukuoka 836-8505, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 August 2012 Received in revised form 12 December 2012 Accepted 17 December 2012 Available online 24 December 2012
We performed a study on the antinociceptive effects of A1 and A2 type (A1LL and A2NTX, respectively) botulinum toxin on carrageenan-induced hyperalgesia in the rat. Both A1LL and A2NTX had antinociceptive effects in the carrageenan-induced inflammatory pain model, reducing the mechanical and thermal hyperalgesia. A2NTX also reduced the increase in c-fos immunoreactivity in L4–L5 spinal segments induced by carrageenan, suggesting that A2NTX inhibits the activation of spinal nociceptive afferent fibers that project to the CNS. Our results indicate that A2NTX may offer a new therapeutic tool to treat inflammatory pain. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: A2NTX A1LL Carrageenan c-Fos Pain
1. Introduction Botulinum toxins (BoNT/A-G, hereafter referred as BoNTs) are neurotoxins produced by the bacterium Clostridium botulinum, and are one of the most potent classes of naturally occurring poisons (Johnson, 1999; Schiavo et al., 2000). At present, however, BoNTs are widely used as tools to study the molecular events involved in the exocytosis of various neurotransmitters (see for example, Sudhof, 2004; Schiavo et al., 2000). Furthermore, BoNT/A, in small doses, is widely used clinically in a number of disorders, including neuromuscular and autonomic disorders arising from cholinergic hyper-reactivity (Dressler et al., 2005; Davletov et al., 2005; Truong and Jost, 2006; Montecucco and Molgo, 2005), facial wrinkles (see for example, Klein et al., 2008), and epilepsy (Costantin et al., 2005). In addition, long-lasting antinociception effects of peripherally applied BoNT/A have been reported in a variety of pain * Corresponding author. Tel.: þ81 96 275 2111; fax: þ81 96 245 3172. E-mail address: [email protected] (N. Akaike). 1 Min-Chul Shin & Takashi Yukihira equally contributed to the MS. 0041-0101/$ – see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.toxicon.2012.12.011
models including inflammatory pain induced by formalin (Cui et al., 2004), carrageenan and capsaicin (Bach-Rojecky et al., 2008; Bach-Rojecky and Lackovic, 2005), and in peripheral neuropathic pain (Bach-Rojecky and Lackovic, 2005; Luvisetto et al., 2007; Park et al., 2006; Kitamura et al., 2009; Kumada et al., 2012), post-surgical pain (Filipovic et al., 2010), experimental cystitis (Smith et al., 2004) and prostatitis (Chuang et al., 2008). Serotype BoNT/A1 (BOTOXÒ, Allergan Inc., hereafter referred as A1LL) has been mainly used in these clinical applications and in these studies on the antinociceptive effects of BoNT/A. As an alternative BoNT, we have isolated and developed the A2 type botulinum toxin (A2NTX) (Sakaguchi et al., 1981). We recently reported, using patch clamp recordings, that this A2NTX variant was extremely potent (0.01– 10 pM) in its ability to suppresses spontaneous and evoked neurotransmitter release from both inhibitory (glycinergic or GABAergic), and excitatory (glutamatergic) nerve terminals synapsing on to sacral dorsal commissural nucleus (SDCN) neurons in the rat spinal cord (Akaike et al., 2010). From a pharmacological point of view, paralysis of the wrong muscle group and allergic reaction are the two
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main adverse actions of BoNTs treatment, and A2NTX is much less immunogenic than A1LL, due to the removal of the hemagglutinin (HAs) component (Akaike et al., 2010; Goto et al., 2008). We have therefore examined whether A2NTX also has antinociceptive effects in the carrageenaninduced hyperalgesia model in rats and compared any such effect to that of the more commonly used A1LL. 2. Materials and methods 2.1. Experimental animals We used male Wistar rats (8 weeks of age, 240–250 g, n ¼ 40, Kyudo, Saga, Japan) that were maintained under controlled light/dark conditions and had free access to food and water. All experiments were performed in accordance with the Guiding Principles for Care and Use of Animals in The Field of Physiological Sciences of The Physiological Society of Japan and were approved by the Animal Ethics Committee of Kumamoto Health Science University.
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before the test, whereas indomethacin was injected (i.p.) 30 min before carrageenan administration. Each paw was measured 3 times at 10 min intervals, with the mean response calculated for each paw. 2.5. Behavioral tests 2.5.1. Mechanical allodynia Mechanical allodynia was assessed by applying a graded force to the plantar hind paw surface with a Von Frey filament, using a Dynamic Plantar Anesthesiometer (Model37450, Ugo Basile, Comerio, Italy). The cut-off force was set at 5 g. Rats were placed in a small transparent plastic cage for 10 min, to accommodate to the experimental environment, until they assumed their normal sniffing/no locomotion behavior. Mechanical nociceptive thresholds, expressed in grams, were measured by applying increasing pressure to the dorsal surface of the hind paw until paw withdrawal or overt struggling was observed. The measurements were performed bilaterally 3 times, alternating ipsilateral and contralateral paws in 10 min intervals.
2.2. Drug injection A1LL (BOTOXÒ, Allergan Inc., Irvine, U.S.A) or A2NTX (The Chemo-Sero-Therapeutic Research Institute) were reconstituted in 0.9% NaCl solution. All the doses of A1LL and A2NTX were administered subcutaneously as a 50 ml bolus into the right hind paw while rats were under anesthesia with isoflurane (3–3.5%). The needle punctured the plantar skin and was placed in the subcutaneous space just proximal to the footpads. Four groups of animals were used to observe the dose–response effects for each of A1LL and A2NTX: doses of 1, 3 and 10 U/50 ml 0.9% saline, and, for control, 50 ml of 0.9% NaCl (saline). A1LL and A2NTX were administered 4 days prior to carrageenan injection. This long pretreatment time was based on the slow onset of action in producing muscle relaxation and based on a similar slow time course in our own preliminary studies in the rat carrageenan model. Indomethacin was also used for comparison, as a typical NSAID, and injected intraperitonealy 30 min before the application of carrageenan. 2.3. Carrageenan-induced hyperalgesia Following A1LL or A2NTX administration, each rat was weighed and placed inside a Plexiglas observation chamber for an acclimatization period of 30–60 min, following which time, a single 100 ml bolus of 1% l-carrageenan (w/v) was injected into the plantar surface of the right hind paw during anesthesia with isoflurane (3–3.5%). 2.4. Inflammatory edema Anti-inflammatory activity was assessed by quantifying the extent of carrageenan-induced edema of the rat paw. Injection of carrageenan elicited an increase in the paw volumes (ml) as measured using a water plethysmometer (model 7150; Ugo Basile, Italy). Paw volume measurements were made before treatment (t ¼ 0 as the baseline) and 1, 3 and 5 h after carrageenan administration. The subcutaneous injection of A1LL or A2NTX was performed 4 days
2.5.2. Thermal sensitivity test Rats were placed in a small transparent plastic cage for 10 min to accommodate to the experimental environment, and until they assumed their normal sniffing/no locomotion behavior. Thermal hyperalgesia was assessed as described previously (Clapper et al., 2010), measuring the latency to withdrawal of the hind paw from a focused beam of radiant heat (thermal intensity: infrared 3.0) applied to the plantar surface, using commercial apparatus (Model37370, Ugo Basile, Comerio, Italy). The radiant thermal stimulus was delivered through the bottom of the cage to the plantar side of each hind paw. A 60 s maximum cut-off time was used to prevent tissue damage. Each paw was measured 3 times at 10 min intervals, with the mean response calculated for each paw. 2.6. Immunohistochemistry 2.6.1. Tissue preparation Rats were sacrificed immediately after finishing the last behavioral test. Rats were fully anesthetized with Zoletil 50Ò (10 mg/kg, i.p.; Vibac, Carros, France), transcardially perfused with 50 mM phosphate-buffered saline (PBS), and then fixed with freshly prepared fixative: 100 mM phosphate buffer (PB, pH 7.4) containing 4% paraformaldehyde (PFA). Sections from the L4–L5 spinal cord segments were identified after following the nerve roots through the L4–L5 dorsal root ganglia. The L4–L5 segments of the spinal cord were excised, placed in 30% sucrose for 24 h, embedded in Tissue-Tek compound (Sakura Finetek, Torrance, CA) and flash-frozen. Frozen segments were sectioned at 40 mm transversely using a cryostat (Leica, Nussloch, Germany). 2.6.2. Immunohistochemistry for c-fos c-Fos positive cells in the L4–L5 spinal cord segments were detected by immunohistochemistry as previously described (Lee et al., 2003). Sections were incubated in PBS for 10 min, washed three times with PBS, and then incubated in 1% hydrogen peroxide (H2O2) for 30 min. After this,
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the sections were incubated overnight with rabbit anti-cfos antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a dilution of 1:1000. Sections were subsequently incubated for 1 h with anti-rabbit secondary antibody (1:200, Vector Laboratories, Burlingame, CA, USA) before incubation with an avidin–biotin–peroxidase complex (Vector Laboratories) for 1 h at room temperature. The immunoreactivity was visualized by incubating the sections in a solution consisting of 0.05% 3,30 -diaminobenzidine (DAB) and 0.01% H2O2 in 50 mM Tris-buffer (pH 7.6) for approximately 3 min. Sections were then mounted onto gelatin-coated glass slides, allowed to air dry overnight at room temperature, and then coverslips were mounted using PermountÒ. 2.7. Statistical analysis Results were presented as mean standard deviation. Statistical analysis was performed by the analysis of variance (ANOVA) followed by Tukey post-hoc test for between-group differences. p-values less than 0.05 were considered significant. 3. Results 3.1. Carrageenan response Subplantar injection of 100 ml carrageenan into the right hind paw of the rats produced unilateral thermal and
mechanical hyperalgesia that became evident 3–5 h after injection (Figs. 1Aa, Ba & 2Aa, Ba). 3.2. Effects of A1LL and A2NTX on thermal hyperalgesia Pretreatment of rats with saline (vehicle) injections into the ipsilateral or contralateral hind paw had no effect on the carrageenan induced responses. In contrast, rats with A1LL (>3 U/site), injected into the right (ipsilateral) hind paw, 4 days before the injection of carrageenan, showed a dose-dependent reduction in thermal hyperalgesia of the same ipsilateral hind paw. There was no effect of A1LL on the response to thermal stimulation of the contralateral hind paw, indicating no generalized effects of A1LL on thermal sensation or on muscle movement involved with the withdrawal response (Fig. 1A). Similarly, A2NTX (>3 U/ site) dose-dependently reduced the thermal hyperalgesia evoked in the ipsilateral paw, without affecting the response of the contralateral paw (Fig. 1B). 3.3. Effects of A1LL and A2NTX on mechanical hyperalgesia We similarly examined the effects of pretreatment of rats with A1LL and A2NTX on the mechanical hyperalgesia induced by carrageenan. Both BoTNs reduced the mechanical hyperalgesia of the ipsilateral hind paw, without affecting the contralateral hind paw response. A1LL, at doses at >3 U/site, had a significant effect at 3 and
Fig. 1. Analgesic effects of A1LL and A2NTX on thermal hyperalgesia. Effects of A1LL (A) and A2NTX (B) on thermal hyperalgesia in the carrageenan model of inflammation in rats. The latency of hind paw withdraw in response to thermal stimulation was measured prior to (D0), and 4 days (D4) after, A1LL or A2NTX injection and again at 1, 3 and 5 h after carrageenan injection on day 4. The relative latencies (n ¼ 6–7/group) were obtained and averaged. Each point represents the mean S.D. of the relative latency time for stimulation of the ipsilateral side (Aa and Ba) and the contralateral side (Ab and Bb). Red arrows indicate the time of injection of the carrageenan. *p < 0.05; **p < 0.01; ***p < 0.001 as compared with vehicle treatment. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 2. The effects of A1LL and A2NTX on mechanical hyperalgesia. Effects of A1LL (A) or A2NTX (B) on mechanical hyperalgesia in the carrageenan model of inflammation in rats. The mechanical nociceptive thresholds were measured prior to (D0), and 4 days (D4) after, A1LL or A2NTX injection and again at 1, 3 and 5 h after carrageenan injection on day 4. The relative paw withdrawal thresholds (n ¼ 6–7/group) were obtained and averaged. Each point represents the mean S.D. of the relative paw withdrawal thresholds for stimulation of the ipsilateral side (Aa and Ba) and the contralateral side (Ab and Bb). Red arrows indicate the time of injection of carrageenan. *p < 0.05; **p < 0.01; ***p < 0.001 as compared with vehicle treatment. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
5 h after the carrageenan injection (Fig. 2A). A2NTX was effective at lower doses (>1 U/site), and reduced the mechanical hyperalgesia at an earlier onset, being affective at 1, 3 and 5 h after the carrageenan injection (Fig. 2B). However, the direct comparison of the efficacy between A1LL and A2NTX is not technically feasible from pharmacological points of view, since at first the molecular sizes of A1LL and A2NTX are different and the units for A1LL or A2NTX is not exactly equivalent in the present study. 3.4. Effects of indomethacin on mechanical hyperalgesia Indomethacin (>0.5 mg/kg), injected 30 min before the carrageenan injection, dose-dependently reduced the mechanical hyperalgesia of the ipsilateral (right) hind paw, without affecting the mechanical response to the contralateral (left) hind paw (Fig. 3). Significant reductions in hyperalgesia were seen at 3 and 5 h after carrageenan injection. 3.5. Effects of A1LL, A2NTX and indomethacin on paw edema In addition to the thermal and mechanical hyperalgesia, carrageenan also caused a gradual increase in the volume of the injected paw, which reached the maximum value 5 h after injection, before slowly recovering to the normal, preinjection volume over the subsequent week. Indomethacin
(>0.5 mg/kg) dose-dependently reduced this paw edema, and at 2 mg/kg, the increase in paw volume was completely suppressed. In contrast, high doses (10 U/site) of either A1LL or A2NTX had no effect on the carrageenan-induced increases in paw volume (Fig. 4). 3.6. Effects of A1LL and A2NTX in c-fos immunoreactivity We next examined if A1LL and A2NTX affected activity of nociceptive afferents in the spinal cord by measuring expression of the immediate early gene, c-fos. Activation of the c-fos gene and the resultant expression of its protein product, fos, indicates neuronal activity in response to the carrageenan stimuli. Fig. 5 shows c-fos immunoreactivity in the dorsal horn of L4–L5 spinal cord segments 5 h after the applications of carrageenan, indicating enhanced activation of nociceptive afferents by carrageenan. A high dose of A1LL or A2NTX (at 10 U/site), or indomethacin (1 mg/kg), or vehicle, was administered to the plantar surface in each hind paw footpad prior to carrageenan injection, as for the hyperalgesia experiments. Five hours after carrageenan injection, mean total c-fos levels in the L4–L5 spinal cord segments increased to 450 35% (p < 0.001) of the control value seen in response to vehicle injection (normal saline). The increase in c-fos immunoreactivity induced by carrageenan injection was greatly suppressed by the A1LL or A2NTX (10 U/site each)
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Fig. 3. Effects of indomethacin on mechanical hyperalgesia. Effects of indomethacin on mechanical hyperalgesia in the carrageenan model of inflammation in rats. Indomethacin was injected intraperitonealy (i.p.) 30 min before the injection of carrageenan into the right hind paw. The mechanical nociceptive thresholds were measured before, and at 1, 3 and 5 h after carrageenan injection. Each point represents the mean S.D. of the relative latency of withdrawal in response to mechanical stimulation of the right (ipsilateral) hind paw (A) and the contralateral hind paw (B). Red arrows indicate the time of injection of carrageenan. *p < 0.05; **p < 0.01; ***p < 0.001 as compared with vehicle treatment. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
pretreatment (to 57 35% (p < 0.001) and 79 35% (p < 0.01) of the carrageenan only value, respectively). Similarly, pretreatment with indomethacin (1 mg/kg) also suppressed the c-fos response (to 60 35% of the carrageenan value (p < 0.001)). There were no significant effects of these drugs on the c-fos levels in contralateral dorsal horn at the same spinal level. These results indicate that A1LL or A2NTX inhibits the enhanced activity of the dorsal horn nociceptive nerve fibers seen in response to carrageenan.
Fig. 4. Effects of A1LL, A2NTX and indomethacin on carrageenan-induced paw edema. Effects of A1LL, A2NTX and indomethacin on carrageenaninduced edema. A1LL and A2NTX were injected s.c. into the right hind paw 4 days prior to injection of carrageenan (1%, 100 ml) into the same hind paw. Injection of carrageenan resulted in a significant increase in paw volume that increased over the 1st 4 h and subsided by about 1 week later. 10 U/site A1LL or A2NTX did not affect this carrageenan-induced edema (n ¼ 6–7/group). In contrast, indomethacin dose-dependently reduced the carrageenan-induced edema (n ¼ 6–7/group). Insets show sample photographs of carrageenan-induced paw edema. Data are expressed as mean S.D.
4. Discussion Our present study has initially confirmed previous observations that A1LL (BOTOXÒ) has antinociceptive effects in various animal models of inflammatory pain, but does not have direct anti-inflammatory actions (BachRojecky et al., 2008; Bach-Rojecky and Lackovic, 2005; Filipovic et al., 2012; Luvisetto et al., 2007; Aoki, 2005; Cui et al., 2004). The novel observation is that we show that A2NTX also has an antinociceptive effect in the carrageenan-induced inflammatory pain model (Figs. 1 and 2). As with A1LL, A2NTX did not show any antiinflammatory effects, assessed by the measurement of hind paw volume and hence is similarly not mediating the reduced hyperalgesia by reducing inflammation directly. As a control, the anti-inflammatory NSAID indomethacin (1 mg/kg) completely suppressed the local inflammation (Fig. 3) and thereby likely reduced the hyperalgesia. Initially, it was considered that A1LL reduces pain primarily in conditions with concomitant muscle contraction, such as painful dystonia (Tsui, 1986). Specifically, A1LL significantly reduces pain associated with craniocervical dystonia, with the analgesic effect considered secondary to A1LL induced muscle relaxation (Brin and Benabou, 1999; Gobel et al., 2001). However, dissociation between muscle relaxation and pain relief has been observed in the clinical use of A1LL. For example, pain alleviation may begin before significant muscle relaxation, and endures for a longer period. Furthermore, pain relief by A1LL has been observed in conditions not associated with muscle hypercontraction; including migraine (Brin and Benabou, 1999; Binder et al., 2000; Silberstein et al., 2000; Gobel, 2004), trigeminal neuralgia (Allam et al., 2005), neuropathic pain (Ranoux et al., 2008), refractory joint pain (Mahowald et al., 2006) and low-back pain (Jabbari, 2008). Inflammatory pain is another example. Cui et al. (2004) were the first to demonstrate that subcutaneous injection of A1LL into the
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Fig. 5. Effects of A1LL and A2NTX on c-fos expression in L4–L5 spinal cord segments. Upper panels show sample sections immunostained for c-fos from the ipsilateral and contralateral L4–L5 spinal cord. Sample sections are shown from rats receiving saline injection or carrageenan injection into the ipsilateral hind paw alone, or 4 days following 10 U/site A1LL or 10 U/site A2NTX injection into the same hind paw, or carrageenan following i.p. 1 mg/kg indomethacin injection. The lower graph displays the averaged number of c-fos positive cells in the indicated L4–L5 spinal cord segments. Note the significant decrease in c-fos expression in the L4–L5 segments of A1LL and A2NTX treated rats, as compared to carrageenan. **p < 0.01; ***p < 0.001 for comparisons as indicated by the dark bars. n.s., no significance.
rat hind paw decreases formalin-induced inflammatory pain. The A1LL effect in inflammatory pain model is associated with reduced formalin-induced glutamate release in the dialysate of the hind paw, reduced number of fos immunoreactive cells in the dorsal horn of the spinal cord, and reduced excitation of wide dynamic range dorsal horn neurons (Aoki, 2005; Cui et al., 2004). It was subsequently reported that A1LL (5 U/kg) also reduces inflammatory hyperalgesia, but not local edema or protein extravasation, induced by carrageenan and capsaicin injections into the rat hind paw (Bach-Rojecky et al., 2008; Bach-Rojecky and Lackovic, 2005). Results of several in vitro experiments have indicated that the mechanism of A1LL-induced antinociception might be inhibition of the release of neuropeptide transmitters from the primary sensory neurons, such as substance P and calcitonin gene-related peptide (CGRP) (Durham et al., 2004; Welch et al., 2000). The release of substances from the peripheral branch of these neurons also occurs, such as glutamate release in hind paw dialysate (Cui et al., 2004). Kitamura et al. (2009) have recently demonstrated that intradermal A1LL injection in the area of infraorbital branch of the trigeminal nerve reduces CGRP release from trigeminal ganglion neurons in vitro, thereby proposed to relieve neuropathic pain behavior in response to infraorbital nerve constriction in rats. Taken together, there is strong evidence that A1LL inhibits neurotransmitter release from the sensory neuron nerve endings. Interestingly, a recent study indicates that BoNT/A
undergoes retrograde axonal transport, like tetanus toxin (TeNT) (Lalli et al., 2003), and is trans-cytosed to afferent neurons from the periphery. For example, injections of BoNT/A into the optic tectum led to the appearance of BoNT/A-truncated SNAP-25 in synaptic terminals within the retina, and cleaved SNAP-25 also appeared in the facial nucleus within three days after injection of toxin into rat whisker muscle (Antonucci et al., 2008). Furthermore, when the neurotoxin was injected into the rat left foreleg muscle, compound muscle action potentials and grip strength on both the ipsilateral or contralateral side were reduced remarkably 2–3 days after injection. The inhibition was completely prevented by either denervation or colchicine treatment immediately after the neurotoxin injection, both preventing retrograde transport (Torii et al., 2011). Hence it is likely that alterations in central spinal neurotransmission also occur following hind paw injection. Further support for a central action of BoTNs, might come from studies on “mirror pain”, where a unilateral intramuscular injections of acidic saline produce a bilateral, long-lasting hyperalgesia, which is proposed to be centrally mediated (Sluka et al., 2001). Unilateral subcutaneous A1LL application diminished pain on both the ipsilateral and contralateral sides. In conclusion, although A1LL also reduces peripheral release of transmitters, it seems likely that central actions of A1LL are involved in the antinociceptive actions. Regardless of the extent to which the BoTNs are affecting peripheral or central transmitter release, the outcome is a clear reduction
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in activity of nociceptive neurons in the spinal cord that project centrally. Our current results demonstrating reduced dorsal horn c-fos immunoreactivity, indicate that both A1LL and A2NTX have this same effect. In conclusion, our results indicate that A2NTX, in addition to A1LL, elicits antinociceptive effects independent of any effects on muscle relaxation or on local inflammation. Our results suggest that A2NTX may offer a new therapeutic tool to treat pain. Ethical statement Rats were prepared for experiments under the ethical treatments according to the Guiding Principles for Care and Use of Animals in The Field of Physiological Sciences of The Physiological Society of Japan and were approved by the Animal Ethics Committee of Kumamoto Health Science University. Acknowledgments This work was supported by grant to M.C. Shin, Y. Ito and N. Akaike from Kumamoto Health Science University. Conflict of interest There is no conflict of interest. References Akaike, N., Ito, Y., Shin, M.C., Nonaka, K., Torii, Y., Harakawa, T., Ginnaga, A., Kozaki, S., Kaji, R., 2010. Effects of A2 type botulinum toxin on spontaneous miniature and evoked transmitter release from the rat spinal excitatory and inhibitory synapses. Toxicon 56, 1315–1326. Allam, N., Brasil-Neto, J.P., Brown, G., Tomaz, C., 2005. Injections of botulinum toxin type a produce pain alleviation in intractable trigeminal neuralgia. Clin. J. Pain 21, 182–184. Antonucci, F., Rossi, C., Gianfranceschi, L., Rossetto, O., Caleo, M., 2008. Long-distance retrograde effects of botulinum neurotoxin A. J. Neurosci. 28, 3689–3696. Aoki, K.R., 2005. Review of a proposed mechanism for the antinociceptive action of botulinum toxin type A. Neurotoxicology 26, 785–793. Bach-Rojecky, L., Lackovic, Z., 2005. Antinociceptive effect of botulinum toxin type a in rat model of carrageenan and capsaicin induced pain. Croat. Med. J. 46, 201–208. Bach-Rojecky, L., Dominis, M., Lackovic, Z., 2008. Lack of antiinflammatory effect of botulinum toxin type A in experimental models of inflammation. Fundam. Clin. Pharmacol. 22, 503–509. Binder, W.J., Brin, M.F., Blitzer, A., Schoenrock, L.D., Pogoda, J.M., 2000. Botulinum toxin type A (BOTOX) for treatment of migraine headaches: an open-label study. Otolaryngol. Head Neck Surg. 123, 669–676. Brin, M.F., Benabou, R., 1999. Cervical dystonia (Torticollis). Curr. Treat. Options Neurol. 1, 33–43. Chuang, Y.C., Yoshimura, N., Huang, C.C., Wu, M., Chiang, P.H., Chancellor, M.B., 2008. Intraprostatic botulinum toxin a injection inhibits cyclooxygenase-2 expression and suppresses prostatic pain on capsaicin induced prostatitis model in rat. J. Urol. 180, 742–748. Clapper, J.R., Moreno-Sanz, G., Russo, R., Guijarro, A., Vacondio, F., Duranti, A., Tontini, A., Sanchini, S., Sciolino, N.R., Spradley, J.M., Hohmann, A.G., Calignano, A., Mor, M., Tarzia, G., Piomelli, D., 2010. Anandamide suppresses pain initiation through a peripheral endocannabinoid mechanism. Nat. Neurosci. 13, 1265–1270. Costantin, L., Bozzi, Y., Richichi, C., Viegi, A., Antonucci, F., Funicello, M., Gobbi, M., Mennini, T., Rossetto, O., Montecucco, C., Maffei, L., Vezzani, A., Caleo, M., 2005. Antiepileptic effects of botulinum neurotoxin E. J. Neurosci. 25, 1943–1951. Cui, M., Khanijou, S., Rubino, J., Aoki, K.R., 2004. Subcutaneous administration of botulinum toxin A reduces formalin-induced pain. Pain 107, 125–133.
Davletov, B., Bajohrs, M., Binz, T., 2005. Beyond BOTOX: advantages and limitations of individual botulinum neurotoxins. Trends Neurosci. 28, 446–452. Dressler, D., Saberi, F.A., Barbosa, E.R., 2005. Botulinum toxin: mechanisms of action. Arq. Neuropsiquiatr. 63, 180–185. Durham, P.L., Cady, R., Cady, R., 2004. Regulation of calcitonin generelated peptide secretion from trigeminal nerve cells by botulinum toxin type A: implications for migraine therapy. Headache 44, 35–42. Filipovic, B., Bach-Rojecky, L., Lackovic, Z., 2010. Lasting reduction of postsurgical hyperalgesia after single injection of botulinum toxin type A in rat. Fundam. Clin. Pharmacol. 24, 43–45. Filipovic, B., Matak, I., Bach-Rojecky, L., Lackovic, Z., 2012. Central action of peripherally applied botulinum toxin type A on pain and dural protein extravasation in rat model of trigeminal neuropathy. PLoS One 7, e29803. Gobel, H., Heinze, A., Heinze-Kuhn, K., Austermann, K., 2001. Botulinum toxin A for the treatment of headache disorders and pericranial pain syndromes. Nervenarzt 72, 261–274. Gobel, H., 2004. Botulinum toxin in migraine prophylaxis. J. Neurol. 251 (Suppl. 1), I8–I11. Goto, Y., Torii, Y., Nakahira, S., Harakawa, T., Ginnaga, A., Takahashi, M., Sakamoto, T., Kozaki, S., Kaji, R., 2008. The therapeutic window in rats of botulinum neurotoxin subtype A2 is wider than that of subtype A1, regardless of the presence of nontoxic proteins. Int. Conf. 2008, Suppl. 1 (51), 12. Jabbari, B., 2008. Botulinum neurotoxins in the treatment of refractory pain. Nat. Clin. Pract. Neurol. 4, 676–685. Johnson, E.A., 1999. Clostridial toxins as therapeutic agents: benefits of nature’s most toxic proteins. Annu. Rev. Microbiol. 53, 551–575. Kitamura, Y., Matsuka, Y., Spigelman, I., Ishihara, Y., Yamamoto, Y., Sonoyama, W., Kamioka, H., Yamashiro, T., Kuboki, T., Oguma, K., 2009. Botulinum toxin type a (150 kDa) decreases exaggerated neurotransmitter release from trigeminal ganglion neurons and relieves neuropathy behaviors induced by infraorbital nerve constriction. Neuroscience 159, 1422–1429. Klein, A.W., Carruthers, A., Fagien, S., Lowe, N.J., 2008. Comparisons among botulinum toxins: an evidence-based review. Plast. Reconstr. Surg. 121, 413e–422e. Kumada, A., Matsuka, Y., Spigelman, I., Maruhama, K., Yamamoto, Y., Neubert, J.K., Nolan, T.A., Watanabe, K., Maekawa, K., Kamioka, H., Yamashiro, T., Kuboki, T., Oguma, K., 2012. Intradermal injection of Botulinum toxin type A alleviates infraorbital nerve constrictioninduced thermal hyperalgesia in an operant assay. J. Oral Rehabil. 39, 63–72. Lalli, G., Bohnert, S., Deinhardt, K., Verastegui, C., Schiavo, G., 2003. The journey of tetanus and botulinum neurotoxins in neurons. Trends Microbiol. 11, 431–437. Lee, T.H., Jang, M.H., Shin, M.C., Lim, B.V., Kim, Y.P., Kim, H., Choi, H.H., Lee, K.S., Kim, E.H., Kim, C.J., 2003. Dependence of rat hippocampal c-fos expression on intensity and duration of exercise. Life Sci. 72, 1421–1436. Luvisetto, S., Marinelli, S., Cobianchi, S., Pavone, F., 2007. Anti-allodynic efficacy of botulinum neurotoxin A in a model of neuropathic pain. Neuroscience 145, 1–4. Mahowald, M.L., Singh, J.A., Dykstra, D., 2006. Long term effects of intraarticular botulinum toxin A for refractory joint pain. Neurotox. Res. 9, 179–188. Montecucco, C., Molgo, J., 2005. Botulinal neurotoxins: revival of an old killer. Curr. Opin. Pharmacol. 5, 274–279. Park, H.J., Lee, Y., Lee, J., Park, C., Moon, D.E., 2006. The effects of botulinum toxin A on mechanical and cold allodynia in a rat model of neuropathic pain. Can. J. Anaesth. 53, 470–477. Ranoux, D., Attal, N., Morain, F., Bouhassira, D., 2008. Botulinum toxin type A induces direct analgesic effects in chronic neuropathic pain. Ann. Neurol. 64, 274–283. Sakaguchi, G., Ohishi, I., Kozaki, S., 1981. Purification and oral toxicities of Clostridium botulinum progenitor toxins. In: Lewis, G.E. (Ed.), Biomedical Aspects of Botulism. Academic Press, New York, pp. 21–34. Schiavo, G., Matteoli, M., Montecucco, C., 2000. Neurotoxins affecting neuroexocytosis. Physiol. Rev. 80, 717–766. Silberstein, S., Mathew, N., Saper, J., Jenkins, S., 2000. Botulinum toxin type A as a migraine preventive treatment. For the BOTOX Migraine Clinical Research Group. Headache 40, 445–450. Sluka, K.A., Kalra, A., Moore, S.A., 2001. Unilateral intramuscular injections of acidic saline produce a bilateral, long-lasting hyperalgesia. Muscle Nerve 24, 37–46. Smith, C.P., Radziszewski, P., Borkowski, A., Somogyi, G.T., Boone, T.B., Chancellor, M.B., 2004. Botulinum toxin a has antinociceptive effects in treating interstitial cystitis. Urology 64, 871–875.
M.-C. Shin et al. / Toxicon 64 (2013) 12–19 Sudhof, T.C., 2004. The synaptic vesicle cycle. Annu. Rev. Neurosci. 27, 509–547. Torii, Y., Akaike, N., Harakawa, T., Kato, K., Sugimoto, N., Goto, Y., Nakahira, S., Kohda, T., Kozaki, S., Kaji, R., Ginnaga, A., 2011. Type A1 but not type A2 botulinum toxin decreases the grip strength of the contralateral foreleg through axonal transport from the toxin-treated foreleg of rats. J. Pharmacol. Sci. 117, 275–285.
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Truong, D.D., Jost, W.H., 2006. Botulinum toxin: clinical use. Parkinsonism Relat. Disord. 12, 331–355. Tsui, J.K., Eisen, A., Stoessl, A.J., Calne, S., Calne, D.B., 1986. Double-blind study of botulinum toxin in spasmodic torticollis. Lancet 2, 245–247. Welch, M.J., Purkiss, J.R., Foster, K.A., 2000. Sensitivity of embryonic rat dorsal root ganglia neurons to Clostridium botulinum neurotoxins. Toxicon 38, 245–258.
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Toxicon journal homepage: www.elsevier.com/locate/toxicon
Antinociceptive effects of A1 and A2 type botulinum toxins on carrageenan-induced hyperalgesia in rat Min-Chul Shin a,1, Takashi Yukihira a, b,1, Yushi Ito a, Norio Akaike a, * a b
Research Division for Life Sciences, Kumamoto Health Science University, Izumimachi 325, Kumamoto 861-5598, Japan Department of Physical Therapy, Teikyo University Faculty of Fukuoka Medical Technology, Fukuoka 836-8505, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 August 2012 Received in revised form 12 December 2012 Accepted 17 December 2012 Available online 24 December 2012
We performed a study on the antinociceptive effects of A1 and A2 type (A1LL and A2NTX, respectively) botulinum toxin on carrageenan-induced hyperalgesia in the rat. Both A1LL and A2NTX had antinociceptive effects in the carrageenan-induced inflammatory pain model, reducing the mechanical and thermal hyperalgesia. A2NTX also reduced the increase in c-fos immunoreactivity in L4–L5 spinal segments induced by carrageenan, suggesting that A2NTX inhibits the activation of spinal nociceptive afferent fibers that project to the CNS. Our results indicate that A2NTX may offer a new therapeutic tool to treat inflammatory pain. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: A2NTX A1LL Carrageenan c-Fos Pain
1. Introduction Botulinum toxins (BoNT/A-G, hereafter referred as BoNTs) are neurotoxins produced by the bacterium Clostridium botulinum, and are one of the most potent classes of naturally occurring poisons (Johnson, 1999; Schiavo et al., 2000). At present, however, BoNTs are widely used as tools to study the molecular events involved in the exocytosis of various neurotransmitters (see for example, Sudhof, 2004; Schiavo et al., 2000). Furthermore, BoNT/A, in small doses, is widely used clinically in a number of disorders, including neuromuscular and autonomic disorders arising from cholinergic hyper-reactivity (Dressler et al., 2005; Davletov et al., 2005; Truong and Jost, 2006; Montecucco and Molgo, 2005), facial wrinkles (see for example, Klein et al., 2008), and epilepsy (Costantin et al., 2005). In addition, long-lasting antinociception effects of peripherally applied BoNT/A have been reported in a variety of pain * Corresponding author. Tel.: þ81 96 275 2111; fax: þ81 96 245 3172. E-mail address: [email protected] (N. Akaike). 1 Min-Chul Shin & Takashi Yukihira equally contributed to the MS. 0041-0101/$ – see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.toxicon.2012.12.011
models including inflammatory pain induced by formalin (Cui et al., 2004), carrageenan and capsaicin (Bach-Rojecky et al., 2008; Bach-Rojecky and Lackovic, 2005), and in peripheral neuropathic pain (Bach-Rojecky and Lackovic, 2005; Luvisetto et al., 2007; Park et al., 2006; Kitamura et al., 2009; Kumada et al., 2012), post-surgical pain (Filipovic et al., 2010), experimental cystitis (Smith et al., 2004) and prostatitis (Chuang et al., 2008). Serotype BoNT/A1 (BOTOXÒ, Allergan Inc., hereafter referred as A1LL) has been mainly used in these clinical applications and in these studies on the antinociceptive effects of BoNT/A. As an alternative BoNT, we have isolated and developed the A2 type botulinum toxin (A2NTX) (Sakaguchi et al., 1981). We recently reported, using patch clamp recordings, that this A2NTX variant was extremely potent (0.01– 10 pM) in its ability to suppresses spontaneous and evoked neurotransmitter release from both inhibitory (glycinergic or GABAergic), and excitatory (glutamatergic) nerve terminals synapsing on to sacral dorsal commissural nucleus (SDCN) neurons in the rat spinal cord (Akaike et al., 2010). From a pharmacological point of view, paralysis of the wrong muscle group and allergic reaction are the two
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main adverse actions of BoNTs treatment, and A2NTX is much less immunogenic than A1LL, due to the removal of the hemagglutinin (HAs) component (Akaike et al., 2010; Goto et al., 2008). We have therefore examined whether A2NTX also has antinociceptive effects in the carrageenaninduced hyperalgesia model in rats and compared any such effect to that of the more commonly used A1LL. 2. Materials and methods 2.1. Experimental animals We used male Wistar rats (8 weeks of age, 240–250 g, n ¼ 40, Kyudo, Saga, Japan) that were maintained under controlled light/dark conditions and had free access to food and water. All experiments were performed in accordance with the Guiding Principles for Care and Use of Animals in The Field of Physiological Sciences of The Physiological Society of Japan and were approved by the Animal Ethics Committee of Kumamoto Health Science University.
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before the test, whereas indomethacin was injected (i.p.) 30 min before carrageenan administration. Each paw was measured 3 times at 10 min intervals, with the mean response calculated for each paw. 2.5. Behavioral tests 2.5.1. Mechanical allodynia Mechanical allodynia was assessed by applying a graded force to the plantar hind paw surface with a Von Frey filament, using a Dynamic Plantar Anesthesiometer (Model37450, Ugo Basile, Comerio, Italy). The cut-off force was set at 5 g. Rats were placed in a small transparent plastic cage for 10 min, to accommodate to the experimental environment, until they assumed their normal sniffing/no locomotion behavior. Mechanical nociceptive thresholds, expressed in grams, were measured by applying increasing pressure to the dorsal surface of the hind paw until paw withdrawal or overt struggling was observed. The measurements were performed bilaterally 3 times, alternating ipsilateral and contralateral paws in 10 min intervals.
2.2. Drug injection A1LL (BOTOXÒ, Allergan Inc., Irvine, U.S.A) or A2NTX (The Chemo-Sero-Therapeutic Research Institute) were reconstituted in 0.9% NaCl solution. All the doses of A1LL and A2NTX were administered subcutaneously as a 50 ml bolus into the right hind paw while rats were under anesthesia with isoflurane (3–3.5%). The needle punctured the plantar skin and was placed in the subcutaneous space just proximal to the footpads. Four groups of animals were used to observe the dose–response effects for each of A1LL and A2NTX: doses of 1, 3 and 10 U/50 ml 0.9% saline, and, for control, 50 ml of 0.9% NaCl (saline). A1LL and A2NTX were administered 4 days prior to carrageenan injection. This long pretreatment time was based on the slow onset of action in producing muscle relaxation and based on a similar slow time course in our own preliminary studies in the rat carrageenan model. Indomethacin was also used for comparison, as a typical NSAID, and injected intraperitonealy 30 min before the application of carrageenan. 2.3. Carrageenan-induced hyperalgesia Following A1LL or A2NTX administration, each rat was weighed and placed inside a Plexiglas observation chamber for an acclimatization period of 30–60 min, following which time, a single 100 ml bolus of 1% l-carrageenan (w/v) was injected into the plantar surface of the right hind paw during anesthesia with isoflurane (3–3.5%). 2.4. Inflammatory edema Anti-inflammatory activity was assessed by quantifying the extent of carrageenan-induced edema of the rat paw. Injection of carrageenan elicited an increase in the paw volumes (ml) as measured using a water plethysmometer (model 7150; Ugo Basile, Italy). Paw volume measurements were made before treatment (t ¼ 0 as the baseline) and 1, 3 and 5 h after carrageenan administration. The subcutaneous injection of A1LL or A2NTX was performed 4 days
2.5.2. Thermal sensitivity test Rats were placed in a small transparent plastic cage for 10 min to accommodate to the experimental environment, and until they assumed their normal sniffing/no locomotion behavior. Thermal hyperalgesia was assessed as described previously (Clapper et al., 2010), measuring the latency to withdrawal of the hind paw from a focused beam of radiant heat (thermal intensity: infrared 3.0) applied to the plantar surface, using commercial apparatus (Model37370, Ugo Basile, Comerio, Italy). The radiant thermal stimulus was delivered through the bottom of the cage to the plantar side of each hind paw. A 60 s maximum cut-off time was used to prevent tissue damage. Each paw was measured 3 times at 10 min intervals, with the mean response calculated for each paw. 2.6. Immunohistochemistry 2.6.1. Tissue preparation Rats were sacrificed immediately after finishing the last behavioral test. Rats were fully anesthetized with Zoletil 50Ò (10 mg/kg, i.p.; Vibac, Carros, France), transcardially perfused with 50 mM phosphate-buffered saline (PBS), and then fixed with freshly prepared fixative: 100 mM phosphate buffer (PB, pH 7.4) containing 4% paraformaldehyde (PFA). Sections from the L4–L5 spinal cord segments were identified after following the nerve roots through the L4–L5 dorsal root ganglia. The L4–L5 segments of the spinal cord were excised, placed in 30% sucrose for 24 h, embedded in Tissue-Tek compound (Sakura Finetek, Torrance, CA) and flash-frozen. Frozen segments were sectioned at 40 mm transversely using a cryostat (Leica, Nussloch, Germany). 2.6.2. Immunohistochemistry for c-fos c-Fos positive cells in the L4–L5 spinal cord segments were detected by immunohistochemistry as previously described (Lee et al., 2003). Sections were incubated in PBS for 10 min, washed three times with PBS, and then incubated in 1% hydrogen peroxide (H2O2) for 30 min. After this,
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the sections were incubated overnight with rabbit anti-cfos antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a dilution of 1:1000. Sections were subsequently incubated for 1 h with anti-rabbit secondary antibody (1:200, Vector Laboratories, Burlingame, CA, USA) before incubation with an avidin–biotin–peroxidase complex (Vector Laboratories) for 1 h at room temperature. The immunoreactivity was visualized by incubating the sections in a solution consisting of 0.05% 3,30 -diaminobenzidine (DAB) and 0.01% H2O2 in 50 mM Tris-buffer (pH 7.6) for approximately 3 min. Sections were then mounted onto gelatin-coated glass slides, allowed to air dry overnight at room temperature, and then coverslips were mounted using PermountÒ. 2.7. Statistical analysis Results were presented as mean standard deviation. Statistical analysis was performed by the analysis of variance (ANOVA) followed by Tukey post-hoc test for between-group differences. p-values less than 0.05 were considered significant. 3. Results 3.1. Carrageenan response Subplantar injection of 100 ml carrageenan into the right hind paw of the rats produced unilateral thermal and
mechanical hyperalgesia that became evident 3–5 h after injection (Figs. 1Aa, Ba & 2Aa, Ba). 3.2. Effects of A1LL and A2NTX on thermal hyperalgesia Pretreatment of rats with saline (vehicle) injections into the ipsilateral or contralateral hind paw had no effect on the carrageenan induced responses. In contrast, rats with A1LL (>3 U/site), injected into the right (ipsilateral) hind paw, 4 days before the injection of carrageenan, showed a dose-dependent reduction in thermal hyperalgesia of the same ipsilateral hind paw. There was no effect of A1LL on the response to thermal stimulation of the contralateral hind paw, indicating no generalized effects of A1LL on thermal sensation or on muscle movement involved with the withdrawal response (Fig. 1A). Similarly, A2NTX (>3 U/ site) dose-dependently reduced the thermal hyperalgesia evoked in the ipsilateral paw, without affecting the response of the contralateral paw (Fig. 1B). 3.3. Effects of A1LL and A2NTX on mechanical hyperalgesia We similarly examined the effects of pretreatment of rats with A1LL and A2NTX on the mechanical hyperalgesia induced by carrageenan. Both BoTNs reduced the mechanical hyperalgesia of the ipsilateral hind paw, without affecting the contralateral hind paw response. A1LL, at doses at >3 U/site, had a significant effect at 3 and
Fig. 1. Analgesic effects of A1LL and A2NTX on thermal hyperalgesia. Effects of A1LL (A) and A2NTX (B) on thermal hyperalgesia in the carrageenan model of inflammation in rats. The latency of hind paw withdraw in response to thermal stimulation was measured prior to (D0), and 4 days (D4) after, A1LL or A2NTX injection and again at 1, 3 and 5 h after carrageenan injection on day 4. The relative latencies (n ¼ 6–7/group) were obtained and averaged. Each point represents the mean S.D. of the relative latency time for stimulation of the ipsilateral side (Aa and Ba) and the contralateral side (Ab and Bb). Red arrows indicate the time of injection of the carrageenan. *p < 0.05; **p < 0.01; ***p < 0.001 as compared with vehicle treatment. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 2. The effects of A1LL and A2NTX on mechanical hyperalgesia. Effects of A1LL (A) or A2NTX (B) on mechanical hyperalgesia in the carrageenan model of inflammation in rats. The mechanical nociceptive thresholds were measured prior to (D0), and 4 days (D4) after, A1LL or A2NTX injection and again at 1, 3 and 5 h after carrageenan injection on day 4. The relative paw withdrawal thresholds (n ¼ 6–7/group) were obtained and averaged. Each point represents the mean S.D. of the relative paw withdrawal thresholds for stimulation of the ipsilateral side (Aa and Ba) and the contralateral side (Ab and Bb). Red arrows indicate the time of injection of carrageenan. *p < 0.05; **p < 0.01; ***p < 0.001 as compared with vehicle treatment. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
5 h after the carrageenan injection (Fig. 2A). A2NTX was effective at lower doses (>1 U/site), and reduced the mechanical hyperalgesia at an earlier onset, being affective at 1, 3 and 5 h after the carrageenan injection (Fig. 2B). However, the direct comparison of the efficacy between A1LL and A2NTX is not technically feasible from pharmacological points of view, since at first the molecular sizes of A1LL and A2NTX are different and the units for A1LL or A2NTX is not exactly equivalent in the present study. 3.4. Effects of indomethacin on mechanical hyperalgesia Indomethacin (>0.5 mg/kg), injected 30 min before the carrageenan injection, dose-dependently reduced the mechanical hyperalgesia of the ipsilateral (right) hind paw, without affecting the mechanical response to the contralateral (left) hind paw (Fig. 3). Significant reductions in hyperalgesia were seen at 3 and 5 h after carrageenan injection. 3.5. Effects of A1LL, A2NTX and indomethacin on paw edema In addition to the thermal and mechanical hyperalgesia, carrageenan also caused a gradual increase in the volume of the injected paw, which reached the maximum value 5 h after injection, before slowly recovering to the normal, preinjection volume over the subsequent week. Indomethacin
(>0.5 mg/kg) dose-dependently reduced this paw edema, and at 2 mg/kg, the increase in paw volume was completely suppressed. In contrast, high doses (10 U/site) of either A1LL or A2NTX had no effect on the carrageenan-induced increases in paw volume (Fig. 4). 3.6. Effects of A1LL and A2NTX in c-fos immunoreactivity We next examined if A1LL and A2NTX affected activity of nociceptive afferents in the spinal cord by measuring expression of the immediate early gene, c-fos. Activation of the c-fos gene and the resultant expression of its protein product, fos, indicates neuronal activity in response to the carrageenan stimuli. Fig. 5 shows c-fos immunoreactivity in the dorsal horn of L4–L5 spinal cord segments 5 h after the applications of carrageenan, indicating enhanced activation of nociceptive afferents by carrageenan. A high dose of A1LL or A2NTX (at 10 U/site), or indomethacin (1 mg/kg), or vehicle, was administered to the plantar surface in each hind paw footpad prior to carrageenan injection, as for the hyperalgesia experiments. Five hours after carrageenan injection, mean total c-fos levels in the L4–L5 spinal cord segments increased to 450 35% (p < 0.001) of the control value seen in response to vehicle injection (normal saline). The increase in c-fos immunoreactivity induced by carrageenan injection was greatly suppressed by the A1LL or A2NTX (10 U/site each)
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Fig. 3. Effects of indomethacin on mechanical hyperalgesia. Effects of indomethacin on mechanical hyperalgesia in the carrageenan model of inflammation in rats. Indomethacin was injected intraperitonealy (i.p.) 30 min before the injection of carrageenan into the right hind paw. The mechanical nociceptive thresholds were measured before, and at 1, 3 and 5 h after carrageenan injection. Each point represents the mean S.D. of the relative latency of withdrawal in response to mechanical stimulation of the right (ipsilateral) hind paw (A) and the contralateral hind paw (B). Red arrows indicate the time of injection of carrageenan. *p < 0.05; **p < 0.01; ***p < 0.001 as compared with vehicle treatment. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
pretreatment (to 57 35% (p < 0.001) and 79 35% (p < 0.01) of the carrageenan only value, respectively). Similarly, pretreatment with indomethacin (1 mg/kg) also suppressed the c-fos response (to 60 35% of the carrageenan value (p < 0.001)). There were no significant effects of these drugs on the c-fos levels in contralateral dorsal horn at the same spinal level. These results indicate that A1LL or A2NTX inhibits the enhanced activity of the dorsal horn nociceptive nerve fibers seen in response to carrageenan.
Fig. 4. Effects of A1LL, A2NTX and indomethacin on carrageenan-induced paw edema. Effects of A1LL, A2NTX and indomethacin on carrageenaninduced edema. A1LL and A2NTX were injected s.c. into the right hind paw 4 days prior to injection of carrageenan (1%, 100 ml) into the same hind paw. Injection of carrageenan resulted in a significant increase in paw volume that increased over the 1st 4 h and subsided by about 1 week later. 10 U/site A1LL or A2NTX did not affect this carrageenan-induced edema (n ¼ 6–7/group). In contrast, indomethacin dose-dependently reduced the carrageenan-induced edema (n ¼ 6–7/group). Insets show sample photographs of carrageenan-induced paw edema. Data are expressed as mean S.D.
4. Discussion Our present study has initially confirmed previous observations that A1LL (BOTOXÒ) has antinociceptive effects in various animal models of inflammatory pain, but does not have direct anti-inflammatory actions (BachRojecky et al., 2008; Bach-Rojecky and Lackovic, 2005; Filipovic et al., 2012; Luvisetto et al., 2007; Aoki, 2005; Cui et al., 2004). The novel observation is that we show that A2NTX also has an antinociceptive effect in the carrageenan-induced inflammatory pain model (Figs. 1 and 2). As with A1LL, A2NTX did not show any antiinflammatory effects, assessed by the measurement of hind paw volume and hence is similarly not mediating the reduced hyperalgesia by reducing inflammation directly. As a control, the anti-inflammatory NSAID indomethacin (1 mg/kg) completely suppressed the local inflammation (Fig. 3) and thereby likely reduced the hyperalgesia. Initially, it was considered that A1LL reduces pain primarily in conditions with concomitant muscle contraction, such as painful dystonia (Tsui, 1986). Specifically, A1LL significantly reduces pain associated with craniocervical dystonia, with the analgesic effect considered secondary to A1LL induced muscle relaxation (Brin and Benabou, 1999; Gobel et al., 2001). However, dissociation between muscle relaxation and pain relief has been observed in the clinical use of A1LL. For example, pain alleviation may begin before significant muscle relaxation, and endures for a longer period. Furthermore, pain relief by A1LL has been observed in conditions not associated with muscle hypercontraction; including migraine (Brin and Benabou, 1999; Binder et al., 2000; Silberstein et al., 2000; Gobel, 2004), trigeminal neuralgia (Allam et al., 2005), neuropathic pain (Ranoux et al., 2008), refractory joint pain (Mahowald et al., 2006) and low-back pain (Jabbari, 2008). Inflammatory pain is another example. Cui et al. (2004) were the first to demonstrate that subcutaneous injection of A1LL into the
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Fig. 5. Effects of A1LL and A2NTX on c-fos expression in L4–L5 spinal cord segments. Upper panels show sample sections immunostained for c-fos from the ipsilateral and contralateral L4–L5 spinal cord. Sample sections are shown from rats receiving saline injection or carrageenan injection into the ipsilateral hind paw alone, or 4 days following 10 U/site A1LL or 10 U/site A2NTX injection into the same hind paw, or carrageenan following i.p. 1 mg/kg indomethacin injection. The lower graph displays the averaged number of c-fos positive cells in the indicated L4–L5 spinal cord segments. Note the significant decrease in c-fos expression in the L4–L5 segments of A1LL and A2NTX treated rats, as compared to carrageenan. **p < 0.01; ***p < 0.001 for comparisons as indicated by the dark bars. n.s., no significance.
rat hind paw decreases formalin-induced inflammatory pain. The A1LL effect in inflammatory pain model is associated with reduced formalin-induced glutamate release in the dialysate of the hind paw, reduced number of fos immunoreactive cells in the dorsal horn of the spinal cord, and reduced excitation of wide dynamic range dorsal horn neurons (Aoki, 2005; Cui et al., 2004). It was subsequently reported that A1LL (5 U/kg) also reduces inflammatory hyperalgesia, but not local edema or protein extravasation, induced by carrageenan and capsaicin injections into the rat hind paw (Bach-Rojecky et al., 2008; Bach-Rojecky and Lackovic, 2005). Results of several in vitro experiments have indicated that the mechanism of A1LL-induced antinociception might be inhibition of the release of neuropeptide transmitters from the primary sensory neurons, such as substance P and calcitonin gene-related peptide (CGRP) (Durham et al., 2004; Welch et al., 2000). The release of substances from the peripheral branch of these neurons also occurs, such as glutamate release in hind paw dialysate (Cui et al., 2004). Kitamura et al. (2009) have recently demonstrated that intradermal A1LL injection in the area of infraorbital branch of the trigeminal nerve reduces CGRP release from trigeminal ganglion neurons in vitro, thereby proposed to relieve neuropathic pain behavior in response to infraorbital nerve constriction in rats. Taken together, there is strong evidence that A1LL inhibits neurotransmitter release from the sensory neuron nerve endings. Interestingly, a recent study indicates that BoNT/A
undergoes retrograde axonal transport, like tetanus toxin (TeNT) (Lalli et al., 2003), and is trans-cytosed to afferent neurons from the periphery. For example, injections of BoNT/A into the optic tectum led to the appearance of BoNT/A-truncated SNAP-25 in synaptic terminals within the retina, and cleaved SNAP-25 also appeared in the facial nucleus within three days after injection of toxin into rat whisker muscle (Antonucci et al., 2008). Furthermore, when the neurotoxin was injected into the rat left foreleg muscle, compound muscle action potentials and grip strength on both the ipsilateral or contralateral side were reduced remarkably 2–3 days after injection. The inhibition was completely prevented by either denervation or colchicine treatment immediately after the neurotoxin injection, both preventing retrograde transport (Torii et al., 2011). Hence it is likely that alterations in central spinal neurotransmission also occur following hind paw injection. Further support for a central action of BoTNs, might come from studies on “mirror pain”, where a unilateral intramuscular injections of acidic saline produce a bilateral, long-lasting hyperalgesia, which is proposed to be centrally mediated (Sluka et al., 2001). Unilateral subcutaneous A1LL application diminished pain on both the ipsilateral and contralateral sides. In conclusion, although A1LL also reduces peripheral release of transmitters, it seems likely that central actions of A1LL are involved in the antinociceptive actions. Regardless of the extent to which the BoTNs are affecting peripheral or central transmitter release, the outcome is a clear reduction
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in activity of nociceptive neurons in the spinal cord that project centrally. Our current results demonstrating reduced dorsal horn c-fos immunoreactivity, indicate that both A1LL and A2NTX have this same effect. In conclusion, our results indicate that A2NTX, in addition to A1LL, elicits antinociceptive effects independent of any effects on muscle relaxation or on local inflammation. Our results suggest that A2NTX may offer a new therapeutic tool to treat pain. Ethical statement Rats were prepared for experiments under the ethical treatments according to the Guiding Principles for Care and Use of Animals in The Field of Physiological Sciences of The Physiological Society of Japan and were approved by the Animal Ethics Committee of Kumamoto Health Science University. Acknowledgments This work was supported by grant to M.C. Shin, Y. Ito and N. Akaike from Kumamoto Health Science University. Conflict of interest There is no conflict of interest. References Akaike, N., Ito, Y., Shin, M.C., Nonaka, K., Torii, Y., Harakawa, T., Ginnaga, A., Kozaki, S., Kaji, R., 2010. Effects of A2 type botulinum toxin on spontaneous miniature and evoked transmitter release from the rat spinal excitatory and inhibitory synapses. Toxicon 56, 1315–1326. Allam, N., Brasil-Neto, J.P., Brown, G., Tomaz, C., 2005. Injections of botulinum toxin type a produce pain alleviation in intractable trigeminal neuralgia. Clin. J. Pain 21, 182–184. Antonucci, F., Rossi, C., Gianfranceschi, L., Rossetto, O., Caleo, M., 2008. Long-distance retrograde effects of botulinum neurotoxin A. J. Neurosci. 28, 3689–3696. Aoki, K.R., 2005. Review of a proposed mechanism for the antinociceptive action of botulinum toxin type A. Neurotoxicology 26, 785–793. Bach-Rojecky, L., Lackovic, Z., 2005. Antinociceptive effect of botulinum toxin type a in rat model of carrageenan and capsaicin induced pain. Croat. Med. J. 46, 201–208. Bach-Rojecky, L., Dominis, M., Lackovic, Z., 2008. Lack of antiinflammatory effect of botulinum toxin type A in experimental models of inflammation. Fundam. Clin. Pharmacol. 22, 503–509. Binder, W.J., Brin, M.F., Blitzer, A., Schoenrock, L.D., Pogoda, J.M., 2000. Botulinum toxin type A (BOTOX) for treatment of migraine headaches: an open-label study. Otolaryngol. Head Neck Surg. 123, 669–676. Brin, M.F., Benabou, R., 1999. Cervical dystonia (Torticollis). Curr. Treat. Options Neurol. 1, 33–43. Chuang, Y.C., Yoshimura, N., Huang, C.C., Wu, M., Chiang, P.H., Chancellor, M.B., 2008. Intraprostatic botulinum toxin a injection inhibits cyclooxygenase-2 expression and suppresses prostatic pain on capsaicin induced prostatitis model in rat. J. Urol. 180, 742–748. Clapper, J.R., Moreno-Sanz, G., Russo, R., Guijarro, A., Vacondio, F., Duranti, A., Tontini, A., Sanchini, S., Sciolino, N.R., Spradley, J.M., Hohmann, A.G., Calignano, A., Mor, M., Tarzia, G., Piomelli, D., 2010. Anandamide suppresses pain initiation through a peripheral endocannabinoid mechanism. Nat. Neurosci. 13, 1265–1270. Costantin, L., Bozzi, Y., Richichi, C., Viegi, A., Antonucci, F., Funicello, M., Gobbi, M., Mennini, T., Rossetto, O., Montecucco, C., Maffei, L., Vezzani, A., Caleo, M., 2005. Antiepileptic effects of botulinum neurotoxin E. J. Neurosci. 25, 1943–1951. Cui, M., Khanijou, S., Rubino, J., Aoki, K.R., 2004. Subcutaneous administration of botulinum toxin A reduces formalin-induced pain. Pain 107, 125–133.
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