Botulinum neurotoxin (BoNT), a well-known lethal bacterial exotoxin produced by Clostridium botulinum, binds to the presynaptic cholinergic nerve terminals, and internalizes and blocks the calcium-dependent release of acetylcholine via cleavage of several parts of the exocytotic fusion complex (v-SNARE) of neurons.1 Immunologically distinct serotypes of BoNT (types A, B, C1, C2, D, E, F, and G) are homologous proteins consisting of a heavy chain and a light chain linked by an essential disulfide and noncovalent interactions. Among these serotypes, only type A (BoNT/A) and type B (BoNT/B) are clinically available.2,3
BoNT/A is commonly used for treating a variety of neuromuscular disorders such as strabismus, blepharospasm, hemifacial spasm, and torticollis.4,5 BoNT/A is also effective in relieving the symptoms of masticatory muscle hypertonicity.6 Several reports showed that BoNT/A can be used in the treatment of several chronic pain syndromes, including tension-type headache,7 migraine,8 chronic low back pain,9 and myofascial pain syndromes.10
Although the analgesic effect of BoNT/A is due to direct muscle relaxation, another analgesic mechanism of BoNT/A has been suggested. Several in vitro research studies suggested that BoNT/A can modulate the release of neurotransmitters implicated in migraines11 and involved in pain transmission peripherally.12 Furthermore, both peripheral injection into the hindpaw and intracisternal injection of BoNT/A resulted in the inhibition of antinociceptive pain behavior in mice.13 This suggests that the antinociceptive effect of BoNT/A may be associated with modulation in pain sensitization at both the peripheral and central levels.
In this study, we investigated the antinociceptive effect of intrathecally (i.t.) administered BoNT/A on formalin-induced inflammatory pain in mice. Our study shows for the first time that i.t. administered BoNT/A may exert an analgesic effect in the spinal cord on inflammatory pain.
These experiments were approved by Seoul National University Animal Care and Use Committee. All procedures were conducted in accordance with the guidelines specified in the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (NIH publication no. 86 to 23, revised 1985) as well as the Ethical Guidelines for Investigations of Experimental Pain in Conscious Animals.14 Male ICR mice (Orient Bio Inc., Sungnam, Republic of Korea) initially weighing 30 g were used for all the experiments. Animals were housed in a room maintained at 22°C ± 0.5°C with an alternating 12-hour light–dark cycle, and food and water were available ad libitum. The animals were allowed to adapt to the animal facility for at least 5 days before the experiments and were also habituated in the behavioral test room for at least 2 hours before testing. To reduce a bias of variation in circadian rhythm, we performed all experiments during the light phase of the cycle (10:00 AM to 17:00 PM).
Initially, BoNT/A (Botox®, Allergan Corporation, Irvine, California) was dissolved in 1 mL of cold sterile normal saline, aliquoted into 100-μL Eppendorf tubes and preserved in a 4°C refrigerator only for 3 days. For the 5 13,205 of final volume of BoNT/A, the stock was diluted again at a predetermined concentration (0.1, 0.01, and 0.001 U) with cold normal saline just before injection. The preliminary results showed that i.t.-administered 0.001-U BoNT/A was ineffective in reducing nociceptive behavior, whereas 0.1 U BoNT/A was fatal. After a preliminary study of BoNT/A dose adjusting, we decided to use 0.01 U BoNT/A for i.t. injection.
Intrathecal Injection of BoNT/A
The i.t. administration was performed in conscious mice following the previously established method15,16 using a 31-gauge needle directly connected to a 25-μL Hamilton syringe. Before the experiment, methylene blue dye tests were performed to check the consistency of the spread of injected drug. Intrathecal injection of methylene blue dye showed a uniform coloration near the tip. The i.t. injection volume was 5 μL, and the injection site was verified by injecting the same volume of 1% methylene blue solution and determining the distribution of the injected dye at L4 to 5. The dye injected i.t. was distributed both rostrally and caudally but within a short distance (about 0.5 to 1 cm) and injected dyes were not observed in the brain. The success rate for the injections was consistently >95% before the experiments were conducted. After confirming that a needle was placed i.t. with tail flick reflex, 0.01 U BoNT/A was slowly injected.
Evaluation of Neurotoxicity
A rotarod test was used to evaluate the potential neurotoxicity of i.t. BoNT/A. Before the test, the mice were trained for 2 consecutive days. The rotarod (Panlab, Spain), consisting of a nonslippery plastic rod (30 mm diameter) and 4 lines (50 mm wide), was set to a run mode (16 rotations per minute [rpm]) for >1 minute. The trained mice were allowed to run on the rod for at least 60 seconds. The rotarod test was performed 3 times at 1, 7, and 14 days after i.t. administration of 0.01 U BoNT/A and vehicle (normal saline), respectively. The time of maintaining equilibrium on the rod was used as an indicator of neurotoxicity.
Formalin-Induced Nociceptive Behavioral Test
This test was performed in mice with a previously published method.17 Ten microliters 1.0% formalin solution, prepared in physiologic saline (0.9% NaCl), was injected subcutaneously into the plantar of the left hindpaw. For the behavioral study, mice were habituated for 1 hour a day for 3 days in an acrylic observation chamber (20 cm in height, 20 cm in diameter) before the formalin test. A set of mice was grouped in accordance with the period after i.t. BoNT/A injection (1, 4, 7, 10, 14, 21, and 28 days). At 1, 4, 7, 10, 14, 21, and 28 days after the single injection of intrathecal BoNT/A, the formalin test was performed in each group.
After the intraplantar injection of formalin, the mice were immediately placed in an observation chamber. Using a camcorder, we observed total time of licking, shaking, and biting considered to be indicative of nociception at the injected paw. The observation of the nociceptive responses during the first phase was performed at 0 to 5 minutes and the second phase performed at 20 to 40 minutes after formalin injection.18 Nociceptive behaviors were observed by 2 observers who were blinded to this study. At 10 days after injection, experimental mice were used for further immunohistochemical and immunoblot analyses, because significant antinociceptive effects of i.t. BoNT/A were shown both in 1st and 2nd phases of the formalin test at that time.
All mice were deeply anesthetized with isoflurane and intracardially perfused with physiological saline followed with ice-cold phosphate-buffered 4% paraformaldehyde (pH 7.4). The lumbar 4th and 5th spinal cord was dissected and postfixed in the same fixative for 4 hours at 4°C. Samples were then cryoprotected in 30% sucrose for 24 hours at 4°C. The sections were cut with a sliding microtome at a thickness of 40 μm. Immunohistochemical staining for calcitonin gene-related peptide (CGRP), phosphorylated extracellullar signal-regulated kinases (p-ERK), and phosphorylated Ca2+/calmodulin-dependent protein kinase type 2 (p-CaMK-II) were performed with an Elite ABC Kit (Vector Laboratories, Burlingame, California). Sections were first rinsed with 0.1 M phosphate-buffered saline (PBS) 3 times for 10 minutes each, then preincubated in 0.1 M PBS containing 1% bovine serum albumin (BSA) and 0.2% Triton X-100 for 30 minutes. After rinsing twice with 0.1 M PBS containing 0.5% BSA for 10 to 15 minutes each, sections were incubated with antibodies against CGRP, p-ERK, and p-CaMK-II diluted with 0.1 M PBS containing 0.5% BSA and 0.05% sodium azide at 4°C. After overnight incubation, the sections were rinsed and incubated with biotinylated secondary antibody diluted 1:200 with 0.1 M PBS containing 0.5% BSA for 1 hour at room temperature. After rinsing, the sections were incubated with ABC reagent diluted 1:50 with PBS for 1 hour at room temperature and then rinsed with PBS followed with 0.1 M phosphate buffer. Finally, the sections were incubated in SIGMA FAST DAB kit (Sigma Chemical Co., St. Louis, Missour) until the desired stain intensity developed. The sections were rinsed with 0.1 M PB and then mounted on gelatin-coated slides, and dehydrated with alcohol and xylene. Antibody expression pattern was calculated and analyzed by the observer, who was blinded to the study using Image Pro 6.2 software (Media Cybernetics, Inc., Bethesda, Maryland).
Western Blot Analysis
After dissecting the L4 to 5 spinal cord, the tissues were washed twice in cold PBS and homogenized with a sonicator in lysis buffer (50 mM Tris-HCl, pH 7.4, 1% NP-40, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA) supplemented with protease inhibitors. An aliquot (30 or 40 μg of protein per lane) was resolved by 10% or 12% SDS-PAGE and blotted to a polyvinyl difluoride transfer membrane. Western immunoblotting was performed with primary antibodies diluted in 5% nonfat dry milk in Tris-buffered saline and then with the secondary antibodies of hydroxyperoxide-linked antirabbit or antimouse IgG by using ECL systems (Amersham Biosciences, Piscataway, New Jersey). Monoclonal antibodies to p-ERK and p-CaMKII were purchased from Cell Signaling Technology (Beverly, Massachusetts) and polyclonal antibodies to β-actin were obtained from Santa Cruz Biochemical (Santa Cruz, California).
Behavioral analysis data were presented as the mean ± SEM. The statistical significance of differences between groups was assessed with a 1-way analysis of variance (ANOVA) with Bonferroni's post hoc test. P < 0.05 was considered to be statistically significant.
Intrathecally Administered BoNT/A Attenuated the Nociceptive Behaviors Induced by Formalin in Mice
In the preliminary experiment, 0.01 U BoNT/A was applied to mice because the antinociceptive effect was observed in the formalin test without any obvious side effects such as sensory and motor deficits. To evaluate the antinociceptive effect of i.t. BoNT/A on inflammatory pain, we performed the formalin test. Before conducting the formalin test, we first i.t. pretreated the mice with vehicle (saline) or BoNT/A (0.01 U), respectively. As is shown in Figure 1, the total time of the nociceptive responses— such as shaking, licking, and biting, elicited by subcutaneous formalin injection—was significantly decreased by BoNT/A pretreatment in the first phase (0 to 5 minutes) at 10 and 14 days (Fig. 1). However, there was no significant difference in total time of nociceptive behavior during the first phase before 10 days after injection between groups (Fig. 1). Comparably, the significant difference in total time of nociceptive behavior during the second phase (20 to 40 minutes) between groups was present throughout the observational period except during 4 weeks (Fig. 1). These differences peaked at 7 days and declined at 10 days after formalin injection (Fig. 1).
Intrathecally Administered BoNT/A Inhibited the Expression of CGRP Induced by Subcutaneous Formalin in the Spinal Cord
The release of CGRP in the spinal cord increases from the noxious stimuli.19 The CGRP have been recognized as an important mediator in facilitating synaptic plasticity from noxious stimulation in the spinal dorsal horn.20 Accumulating evidence supports the role of CGRP in central sensitization in the spinal cord.21,22 To investigate the inhibitory effects of BoNT/A for central sensitization, we measured expression of CGRP. Immunohistochemical analysis was performed to observe the change in the expression of CGRP in L4 to 5 dorsal horn. To quantify the expression of CGRP in the superficial layers (lamina I and II) of L4 to 5, we measured the expression of CGRP at 1 hour after intrathecal BoNT injections and we averaged it per area. Immunoreactivity for CGRP in the ipsilateral dorsal horn in the BoNT/A-treated group was higher than was that in the control group (Fig. 2). The averaged per-area expression of CGRP was 0.040 ± 0.005 in the control group and 0.018 ± 0.004 in the BoNT/A-treated group (P < 0.05). These data suggest that BoNT/A inhibits inflammatory pain-induced up-regulation of CGRP in the spinal cord, contributing to inhibited central sensitization in the spinal cord.
Intrathecally Administered BoNT/A Attenuated Increase of Expression of p-ERK and p-CaMK Induced by Formalin Injection in the Spinal Cord
The upregulation of p-ERK in the spinal cord occurs in response to noxious stimuli.23 Normally expressed in the spinal cord,24 p-CaMK-II plays a key role in central sensitization in the spinal cord.25 Choi et al.26 reported that p-ERK and p-CaMK-II were induced by subcutaneous injection of formalin in ICR mice.26 To investigate the effect of BoNT/A-induced protein expression at the spinal cord after formalin injection, we first examined the expression of p-ERK and p-CaMK-II in the lumbar spinal cord. In agreement with previous results,26 immunoreactivity for both p-ERK and p-CaMK-II in the ipsilateral dorsal horn was significantly increased after formalin injection (Figs. 3 and 4). Meanwhile, i.t. BoNT/A treatment reduced immunoreactivity for both p-ERK and p-CaMK-II significantly (Figs. 3 and 4). The averaged per-area expression of p-ERK and p-CaMK-II was 0.017 ± 0.005, 0.037 ± 0.004, in the control group, and 0.005 ± 0.001, 0.013 ± 0.002 in the BoNT/A-treated group, respectively (P < 0.05). These data suggest that the antinociceptive activity of BoNT/A injection induces a central inhibitory effect on the superficial layers in the spinal cord, which is important to process pain signals. To confirm whether decreased levels of p-ERK and p-CaMK-II after BoNT/A injection arise from immunohisochemistry, we measured the expression of p-ERK and p-CaMK-II by Western blot analyses. The expression of p-ERK and p-CaMK-II significantly increased at 30 minutes and at 2 hours after formalin injection. (Fig. 5) Meanwhile, i.t. injection of BoNT/A significantly decreased the expression of p-ERK and p-CaMK-II in the spinal cord. Both the expression of p-ERK and of p-CaMK-II declined significantly at 30 minutes and at 2 hours after BoNT/A i.t. injection (Fig. 5). These data were compatible with immunohistochemical results.
Intrathecally Administered BoNT/A Did Not Attenuate the Balance Time on the Rotarod
To investigate the potential neurotoxic effects of i.t.-administered BoNT/A at the selected dose (0.01 U), we performed the rotarod test. As is shown in Figure 6, the time on the rod was not different between the i.t. BoNT/A (0.01 U)–treated mice and the controls, thus indicating that the reduction of behavioral responses after BoNT/A administration may not be due to a BoNT/A effect on motor coordination (Fig. 6). To investigate whether the lack of motor disturbance in the BoNT/A group was due to a failure of the rotarod test to detect the alterations of motor functions, we performed an additional experiment using MK-801, which evokes motor dysfunction when i.t. administered. As was expected, MK-801 reduced the balancing time significantly (Supplementary Fig. 1). This indicates that BoNT/A at the tested dose (0.01 U) had no effect on motor functions.
A single i.t. injection of BoNT/A attenuated formalin-induced inflammatory pain for 14 days after the injection. Interestingly, the antinociceptive effect of BoNT/A was accompanied by decreased expression levels of CGRP, p-ERK, and p-CaMK-II, which are normally induced in the spinal cord during the formalin test in mice. To our knowledge, this is the first study to show the antinociceptive effect of BoNT/A on formalin-induced inflammatory pain at the spinal level.
The formalin test is a useful animal pain model to evaluate and assess the grade of moderate and continuous pain generated by injured tissue induced by formalin injection.27,28 Two distinct phases of response are followed by injection of formalin into the paw. Formalin injection causes localized inflammation and pain. After the injection, the mice show typical pain behaviors such as licking, shaking, and biting at the injected area. Two distinct periods of pain behaviors can be identified, the first phase lasting 5 minutes and the second phase lasting 20 to 40 minutes after the injection of formalin. The first phase seems to be caused by C-fiber activation due to peripheral stimuli, whereas the second phase may depend on the combination of an inflammatory reaction in the peripheral tissue and functional changes in the dorsal horn of the spinal cord leading to central sensitization.29
Consistent with previous studies in which BoNT/A was injected subcutaneously and intracisternally,30,31 our study showed that i.t. injected BoNT/A significantly inhibited the second phase more than did the first phase of the formalin test, as is shown in Figure 1. Because BoNT/A does not affect excitability of sensory neurons, BoNT/A itself does not completely block noxious chemical–induced pain.29 This could be one explanation of the difference of pain response between the 1st phase and the 2nd phase of the formalin test. Indeed, even a single i.t. injection of BoNT/A caused a long-lasting effect in the 2nd phase (up to 21 days) in accordance with previous results reported by Cui et al.29 BoNT/A i.t. injection may result in modulation of the pain sensitization process in the spinal cord.
Our study also showed that the first and second phases of the formalin test diminished at 10 and 14 days after BoNT/A injection. Our observation indicates that BoNT/A injected i.t. may affect the processing of nociceptive inputs from the sensory afferents in the formalin-injected paw. This finding raises a question: why does BoNT/A injected i.t. affect the first phase of the formalin test in the relatively chronic phase (10 to 14 days), but not in the acute phase? In agreement with our observation, the antinociceptive behaviors remained unaltered in the 1st phase of the formalin test 3 days (a relatively acute period) after subplantar Botox injection.13 A recent report has shown that the first phase of the formalin test has a tendency to decrease 10 to 12 days after Botox injection, although the difference is not statistically significant.30 From the findings of our study and the previous reports, we speculate that BoNT/A takes a longer time than expected to modulate the nociceptive inputs. Interestingly, it has been reported that Botox can be retrogradely transported via central neurons and motorneurons.31 This finding may suggest that BoNT/A may be retrogradely transported to the peripheral sensory afferents to have a central action on modulating the nociceptive inputs, although this is quite speculative. A further immnunohistochemical and immunoblot study is warranted to investigate our hypothesis.
Several studies have demonstrated that p-ERK and p-CaMK-II are associated with the regulation of nociception induced by formalin in rats or mice.32–34 In this study, phosphorylation of ERK could have led to enhanced hypersensitivity of inflammatory pain, which is consistent with a previous report.35 Nociceptive behavior was decreased by i.t. injection of PD98059, an inhibitor of ERK, in mice. The activation of MAPK/ERK kinase, upstream of the ERK pathway, is also related to neuronal synaptic plasticity.36 Thus, we suggest that the ERK pathway, one of the intracellular pain signaling pathways, may be associated with the antinociceptive activity of BoNT/A in the spinal cord. A regulator for the calcium signaling pathway, p-CaMK-II, modulates synaptic transmission by phosphorylating the intracellular transcription protein. This p-CaMK-II is closely associated with neuronal plasticity and long-term enhancement. One study revealed that protein and mRNA expression of p-CaMK-II was increased by formalin injection into the rat hindpaw.37 Increased expression and phosphorylation of CaMK-II induced by intradermal capsaicin injection contribute to development of central sensitization in spinal dorsal neurons.38 Also, intrathecal injection of KN-93, an inhibitor of p-CaMK-II, also inhibited nociceptive behaviors in formalin-induced inflammatory pain.26 We showed that the expression level of p-CaMK-II in the spinal cord was increased by formalin injection, whereas i.t.-injected BoNT/A attenuated the increase of p-CAMK-II expression. This suggests that the p-CaMK-II pathway may be critical to the antinociceptive mechanism of intrathecally administered BoNT/A. Interestingly, p-CaMK-II is closely associated with intracellular calcium regulation through which ERK activation is stimulated. Activated CaMK-II modulates ERK activation to function on various kinds of pain signal–related cells in the spinal cord. Taken together, our results suggest that i.t. injection of BoNT/A attenuates ERK and CAMK-II pathways simultaneously, in turn attenuating the process of central sensitization induced by formalin injection at the spinal dorsal horn.
CGRP is involved with the pain mechanism at the peripheral and spinal levels.39–41 Noxious stimuli releases CGRP in the spinal dorsal horn in agreement with our findings.42,43 CGRP increases the excitability of neuronal cells and facilitates synaptic plasticity with postsynaptic action in the spinal dorsal horn.20 In this study, BoNT/A interfered with the expression of CGRP in the dorsal horn. The antinociceptive effect of BoNT/A could possibly be mediated through inhibition of synaptic plasticity in which CGRP plays a pivotal role. There are several studies reporting a connection of the ERK44 and CaMK-II45 pathways to CGRP release to control various cellular functions. Further investigations are needed to understand the mechanism in antinociception of i.t.-administered BoNT/A.
In conclusion, i.t.-administered BoNT/A may exert a central analgesic effect on central and other inflammatory pain. Further studies on another antinociceptive mechanism of BoNT/A and the potential clinical use of i.t. BoNT/A in pain management are needed.
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