Address all correspondence to Dr. Paul L. Durham, Department of Biology, Southwest Missouri State University, 225 Temple Hall, Springfield, MO 65804.
Objective.—To determine the effect of botulinum toxin type A on calcitonin gene-related peptide secretion from cultured trigeminal ganglia neurons.
Background.—The ability of botulinum toxins to cause muscle paralysis by blocking acetylcholine release at the neuromuscular junction is well known. Previous studies and clinical observations have failed to demonstrate sensory changes related to botulinum toxins or the disease of botulism. Recent studies, however, have suggested that botulinum toxin type A injected into pericranial muscles may have a prophylactic benefit in migraine. This observation has renewed the debate of a mechanism of sensory inhibition mediated by botulinum toxin type A.
Methods.—Primary cultures of rat trigeminal ganglia were utilized to determine whether botulinum toxin type A could directly decrease the release of calcitonin gene-related peptide, a neuropeptide involved in the underlying pathophysiology of migraine. Untreated cultures or cultures stimulated with a depolarizing stimulus (potassium chloride) or capsaicin, an agent known to activate sensory C fibers, were treated for 3, 6, or 24 hours with clinically effective doses of botulinum toxin type A or a control vehicle. The amount of calcitonin gene-related peptide secreted into the culture media following the various treatments was determined using a specific radioimmunoassay.
Results.—A high percentage (greater than 90%) of the trigeminal ganglia neurons present in 1- to 3-day-old cultures was shown to express calcitonin gene-related peptide. Treatment with depolarizing stimuli (potassium chloride), a mixture of inflammatory agents, or capsaicin caused a marked increase (4- to 5-fold) in calcitonin gene-related peptide released from the trigeminal neurons. Interestingly, overnight treatment of trigeminal ganglia cultures with therapeutic concentrations of botulinum toxin type A (1.6 or 3.1 units) did not affect the amount of calcitonin gene-related peptide released from these neurons. The stimulated release of calcitonin gene-related peptide following chemical depolarization with potassium chloride or activation with capsaicin, however, was greatly repressed by the botulinum toxin, but not by the control vehicle. A similar inhibitory effect of overnight treatment with botulinum toxin type A was observed with 1.6 and 3.1 units. These concentrations of botulinum toxin type A are well within or below the range of tissue concentration easily achieved with a local injection. Incubation of the cultures with toxin for 24, 6, or even 3 hours was very effective at repressing stimulated calcitonin gene-related peptide secretion when compared to control values.
Conclusions.—These data provide the first evidence that botulinum toxin type A can directly decrease the amount of calcitonin gene-related peptide released from trigeminal neurons. The results suggest that the effectiveness of botulinum toxin type A in the treatment of migraine may be due, in part, to its ability to repress calcitonin gene-related peptide release from activated sensory neurons.
Current pathophysiological models of migraine focus on the trigeminovascular system as an important generator of the sensory input leading to migraine. According to this model, trigeminal afferents innervating meningeal vessels are activated during migraine possibly by a wave of neuronal depression that spreads across the cerebral cortex.1 Consequently, afferents in the ophthalmic branch (V1) of the trigeminal nerve are stimulated to release various neuropeptides, including calcitonin gene-related peptide (CGRP). This results in vasodilation, focal areas of neurogenic inflammation, and a lowered threshold for sensory activation of the involved trigeminal afferent.2,3 The release of neuropeptides, and particularly CGRP, is considered an integral component in the pathophysiology of migraine.4
Calcitonin gene-related peptide is a multifunctional regulatory neuropeptide.5 Serum levels of CGRP are elevated during acute episodes of migraine and cluster headaches.6–9 Specific serotonin agonists, such as sumatriptan, lower CGRP levels in the jugular outflow, coincident with relief of head pain.10 Further, the release of CGRP from trigeminal afferents is inhibited by triptans through activation of the 5-HT1 receptors.11
While the use of triptan drugs has provided an effective means for treating most episodes of acute migraine, there remains a clinical need for more effective and better-tolerated prophylactic migraine drugs. Recently, several clinical reports as well as one large placebo-controlled, double-blinded clinical trial have suggested that botulinum toxin type A (BTXA) may provide prophylactic benefit in migraine.12–14 The mechanisms by which BTXA might function to prevent migraine, however, are unknown, and a subject of active investigation.
Botulinum is a potent neurotoxin that causes muscle paralysis. The principal mechanism by which all serotypes of BTX inhibits muscle fiber activity is to prevent the motor neurons from releasing acetylcholine into the neuromuscular junction. This inhibitory action is due to the ability of BTXA to block the docking of synaptic vesicles in motor neuron terminals. The cellular mechanism involves cleavage of SNAP-25, a protein essential for binding the vesicle to the nerve terminal.15
Historically, BTXA has treated medical conditions such as cervical dystonia and blepharospasm, which are characterized by excessive muscle contraction.16 While muscle pain is a common complaint during acute episodes of migraine and tension-type headache, studies have failed to convincingly demonstrate abnormalities of muscle physiology as being central to the pathophysiology of either disorder.17,18 Thus, the muscle paralysis produced by BTXA does not easily translate into a mechanism likely to explain the toxin's clinically observed prophylactic benefit.
Recently, antinociceptive effects of BTXA have been postulated. Interestingly, experimental evidence did not demonstrate direct antinociceptive effects of BTXA in terms of pain threshold to temperature or electrical stimulation.19 This observation does not rule out the possibility that the clinically observed benefit of BTXA in treating pain syndromes results from suppression of inflammatory mechanisms. Rather, this hypothesis is particularly appealing for disorders such as migraine where neuroinflammatory mechanisms are thought to play a central role in the pathophysiology of the disease.
Based on previous in vitro studies, it is likely that BTXA affects the release of neuropeptides such as CGRP from sensory neurons in the trigeminal afferents.20,21 To test this hypothesis, primary cultures of rat trigeminal ganglia previously shown to be responsive to sumatriptan (and other 5-HT1 agonists) and mediate inhibition of CGRP secretion were incubated with BTXA,22 and the release of CGRP was measured under basal and potassium chloride (KCl) or capsaicin-stimulated conditions. Results from these studies demonstrate that BTXA can directly repress the stimulated, but not unstimulated, basal release of CGRP from cultured trigeminal neurons in a dose- and time-dependent manner. To our knowledge, this is the first evidence that therapeutic concentrations of BTXA can directly decrease the secretion of CGRP from sensory trigeminal neurons. The clinical implications of our findings for the use of BTXA in the prophylactic treatment of migraine and other types of headache will be discussed.
MATERIALS AND METHODS
Cell Culture.— Trigeminal ganglia cultures were established based on a previous utilized protocol.23,24 Ganglia isolated from approximately 20 to 24 3- or 4-day-old Sprague-Dawley rats were washed in 10 mL cold plating medium (L-15, pH 7.2 to 7.4, Leibovitz, Sigma, St. Louis, Mo) and collected at 100g for 2 to 3 minutes. The ganglia were resuspended in 10 mL plating medium containing 10 mg/mL dispase II (Invitrogen Life Technologies, Gaithersburg, Md), then split into two 15-mL tubes (5 mL each) for 30 minutes at 37°C. The cell suspension was collected by centrifugation at 100g for 3 minutes. The pellets were resuspended and further dissociated in 5 mL plating medium by vigorous trituration (approximately 15 times) using a 5-mL pipette. After allowing larger fragments to settle (about 1 minute), the suspensions from each tube were combined into a new 15-mL tube. The remaining large fragments were pooled and pipeted up and down approximately 15 times in 5 mL plating medium. After the trituration step, any remaining fragments were mechanically removed by gently swirling a Pasteur pipette in the suspension. The suspension was combined with the cells obtained following the first trituration step (15 mL total) and centrifuged at 100g for 3 minutes. The cell pellet was resuspended in 7.2 mL L-15 medium containing 10% fetal bovine serum, 50 μM glucose, 250 μM ascorbic acid, 8 μM glutathione, 2 μM glutamine, and 10 ng/mL mouse 2.5 S nerve growth factor (Alomone Labs, Israel) at 37°C at ambient CO2 levels. For secretion studies, the cells were plated on 24-well tissue culture plates (Becton Dickinson, Franklin Lakes, NJ) coated with poly-D-lysine (MW 30,000-70,000; Sigma, St. Louis, Mo). Penicillin (100 units/mL), streptomycin (100 μg/mL), and amphotericin B (2.5 μg/mL, Sigma) were added to the L-15 medium unless otherwise noted. The culture medium was changed after 24 hours and every other day thereafter. Botox was purchased from Allergan, Inc (Irvine, Calif) while capsaicin, histamine, bradykinin, prostaglandin E2, and serotonin were obtained from Sigma. In all studies, cells were treated with equivalent amounts of the vehicle.
Immunohistochemistry.— One-day-old cultures were briefly rinsed in phosphate-buffered saline (PBS) and fixed in 100% methanol for 5 minutes at −20°C. The fixed cells were incubated for 30 minutes in PBS with 5% donkey serum, rinsed with PBS, and incubated for 1 hour with rabbit anti-rat CGRP polyclonal antibodies (1:1000 dilution in PBS, Sigma). Calcitonin gene-related peptide immunoreactivity was detected using FITC-conjugated goat anti-rabbit IgG antibodies (1:100, Jackson ImmunoResearch Laboratories, Inc, West Grove, Pa).
Calcitonin Gene-Related Peptide Assays.— For the unstimulated CGRP secretion studies, cells were incubated in HBS (22.5 mM HEPES, 135 mM NaCl, 3.5 mM KCl, 1 mM MgCl, 2.5 mM CaCl, 3.3 mM glucose, and 0.1% BSA, pH 7.4) (Vasko et al, 1994), and the amount of CGRP released from trigeminal neurons into the culture media was determined using a specific CGRP radioimmunoassay (Bachem, San Carlos, Calif). As a control, the basal (unstimulated) rate of CGRP secreted into the media in 1 hour was determined for each well, and these values were used to normalize for differences between individual wells and dishes.
For stimulated release studies, 1-day-old cultures were treated for 1 hour with either HEPES-buffered saline (HBS) (control), 60 mM KCl, 2 μM capsaicin, or a cocktail of known inflammatory agents. The inflammatory cocktail contained 10 μM each of bradykinin, prostaglandin, and serotonin, and 50 μM histamine in HBS at pH 5.5. This combination and concentration of agents was based on previous studies that elicited physiological responses.25,26 Although it is difficult to know what the local concentrations of these agents would be during neurogenic inflammation, high hydrogen ion concentrations have been found in inflammation, pH 5.4, and the relatively high, perhaps unphysiological, concentrations of the chemical agents have been reported to be necessary for in vitro responses.25
For the BTXA studies, 1-day-old cultures were pretreated with either 1.6 or 3.1 units of BTXA or the vehicle (the solution used to resuspend the toxin) for 3, 6, or 24 hours before addition of HBS (control), KCl, capsaicin, or a cocktail of known inflammatory agents. Each experimental condition was performed in duplicate and repeated in at least 3 independent experiments. Statistical analyses were performed using the Student t test (2-tailed, unpaired samples).
Calcitonin Gene-Related Peptide Secretion From Primary Trigeminal Ganglia Neurons.— Activation of the trigeminovascular system is thought to be a common pathway involved in many primary headache disorders. Therapeutic efforts that prevent or reverse activation of the trigeminovascular mechanisms involved in migraine may be therapeutically beneficial.
The major purpose of this study was to determine whether BTXA could directly decrease CGRP secretion from trigeminal nerves. Primary cultures of rat trigeminal ganglia neurons have been used as an in vitro model to elucidate cellular mechanisms by which sumatriptan controls the synthesis and release of CGRP.22 The culturing conditions utilized in this study allowed for the enrichment of CGRP-positive neuronal cells (Figure 1). It was estimated that more than 90% of the neuronal cells, as identified by morphological characteristics, expressed immunoreactive CGRP.
To further validate the use of this model for studying the effect of BTXA, the level of CGRP released from untreated trigeminal cultures or cultures treated with known stimulatory agents was determined by radioimmunoassay. The mean (SD) basal rate of CGRP released from unstimulated control cultures was 26 pg/h per well (2) (Figure 2). The secretion rate for each condition was normalized to the basal rate for each well, which did not vary appreciably within or between experiments.
Cultures treated for 1 hour with 60 mM KCl, a stimulus that causes depolarization of neurons, caused an almost 5-fold increase in the amount of CGRP released from trigeminal neurons (Figure 2). The cultured neurons were equally responsive to a cocktail of inflammatory agents and capsaicin. Treatment of trigeminal cultures with an inflammatory mixture that contained bradykinin, histamine, serotonin, and prostaglandin E2 at pH 5.5 resulted in a 5-fold increase in relative CGRP levels when compared to unstimulated control values. Calcitonin gene-related peptide levels were also greatly stimulated (approximately 5-fold) by capsaicin, an agent known to activate sensory C fibers. Taken together, these data support the use of trigeminal cultures as an in vitro model to study the direct effect of BTXA on CGRP secretion.
Botulinum Toxin Type A Does Not Inhibit Unstimulated Calcitonin Gene-Related Peptide Release.— To determine whether BTXA could inhibit the basal (unstimulated) release of CGRP from trigeminal neurons, cultures were incubated for 24 hours in the presence of 1.6 or 3.1 units of BTXA. Interestingly, BTXA treatment did not decrease the relative levels of CGRP when compared to unstimulated control levels (Figure 3). As a control, cultures were also treated with a solution that contained all the ingredients found in the Botox preparation, with the exception of the toxin itself. Similarly, CGRP levels in the vehicle-treated samples were not appreciably different than those for the control sample. These data demonstrate that BTXA does not inhibit the basal release of CGRP from trigeminal neurons.
Stimulated Calcitonin Gene-Related Peptide Secretion Is Repressed by Botulinum Toxin Type A.— Sensitization and activation of trigeminal nerves contributes to migraine pathology by increasing the release of neuropeptides that mediate inflammation. Recently, BTXA treatment has been shown to be efficacious as a migraine prophylactic. The mechanism may involve an inhibition of neuropeptide secretion from sensory neurons. To determine whether BTXA could inhibit CGRP release from stimulated trigeminal nerves, cultures were co-treated with BTXA and 60 mM KCl, which chemically depolarizes the nerve cells, and secreted CGRP levels were measured. Overnight treatment of cultures with either 1.6 or 3.1 units of BTXA greatly repressed KCl-stimulated CGRP secretion levels when compared to vehicle-treated cultures (Figure 4). These results are in contrast to our finding that overnight BTXA treatment did not inhibit basal CGRP secretion (Figure 3).
Time-Dependent Inhibition of Calcitonin Gene-Related Peptide Secretion by Botulinum Toxin Type A.— Having shown that an overnight (24 hours) incubation could markedly repress stimulated CGRP release from trigeminal neurons, the acute effect of BTXA was investigated. Treatment of trigeminal cultures with 3.1 units of BTXA for 6 hours, or as short as 3 hours caused a similar magnitude of repression as seen with the overnight incubation (Figure 5). At each of the time points, CGRP levels were reduced to near basal levels by the toxin. Similar to our previous data, incubation of the cultures with the control vehicle solution did not repress the stimulated release of CGRP caused by KCl-mediated chemical depolarization.
Botulinum Toxin Type A Repression of Capsaicin-Stimulated Calcitonin Gene-Related Peptide Release.— Capsaicin is known to selectively activate sensory C fibers via the vanilloid receptor type 1 (VR1). The majority of small-diameter sensory neurons, such as thinly myelinated A and unmyelinated C fibers, function as nociceptors. These neurons are activated in response to noxious thermal, mechanical, and chemical stimuli. Our primary cultures of trigeminal ganglia were shown to contain small-diameter, capsaicin-responsive neurons (Figures 1 and 2). As shown in Figure 6, overnight treatment of trigeminal cultures with 3.1 units of BTXA repressed stimulated CGRP release in response to 2 μM capsaicin. The level of repression was similar to that seen in response to KCl stimulation. Thus, BTXA can repress the level of CGRP secretion from trigeminal nerves activated by chemical depolarization, and presumably, from C fibers of the trigeminal ganglia that are involved in nociception.
Migraine is a painful and frequently debilitating neurological disorder that affects 10% of the adult population in the United States.27,28 Although the specific cause remains unknown, current theories suggest that the initiation of migraine involves a primary central nervous system dysfunction with subsequent activation of the trigeminovascular system.1,3,28 Activation of trigeminal neurons is known to elevate CGRP levels during migraine.3,8 The ability of acute antimigraine drugs, such as sumatriptan, to return serum CGRP levels to normal coincident with alleviation of pain suggests that CGRP is involved in the underlying pathology of migraine.10 More recently, evidence in support of a causative role for CGRP in migraine was demonstrated by an in vivo study in which administration of CGRP was shown to cause headache and migraine in migraineurs.29
In this study, we have shown that CGRP secretion from stimulated trigeminal neurons is directly inhibited by BTXA. It follows that with CGRP release inhibited, the vascular and inflammatory changes mediated by CGRP would likewise be limited. This could suggest potential mechanisms by which BTXA could prevent the initiation and propagation of migraine. It is of interest that basal secretion of CGRP from trigeminal neurons is unaffected by BTXA, whereas the KCl-stimulated secretion is significantly reduced. This implies that BTXA may inhibit trigeminal release of CGRP only under physiological conditions where the trigeminal system is activated. Again, this condition is presumed necessary in the genesis and propagation of migraine.
These results need to be interpreted carefully. The neuronal cells were harvested from rats, and although these cells have been shown to be responsive to sumatriptan-mediated inhibition of CGRP, it cannot automatically be assumed that human trigeminal cells would behave similarly. Further, the clinical trials using BTXA for migraine prophylaxis were conducted with the neurotoxin injected into discreet muscles, which are anatomically distant from the trigeminally innervated meningeal vessels believed to be involved in migraine. Evidence of peripheral allodynia occurring in the skin innervated by the first division of the trigeminal nerve has been demonstrated, however, to occur early in the migraine episode at least for some migraineurs.30 Whether early cutaneous allodynia could act as a marker for responsiveness to BTXA has not yet been defined, but is likely worthy of future study. In addition, if these results are confirmed, it would suggest that cultures of trigeminal cells could be used as an in vitro model to elucidate cellular mechanisms of neurogenic inflammation and to study novel acute and preventive effects of pharmacological agents.
ACKNOWLEDGMENTS: This work was supported by grants from the National Institutes of Health 1 R15 NS44033-01 (P. L. D.) and the National Headache Foundation (P. L. D.). Appreciation is expressed to Ms. Tonya Fielder for technical support.
1Bolay H, Reuter U, Dunn AK, et al.Intrinsic brain activity triggers trigeminal meningeal afferents in a migraine model. Nat Med. 2002;8: 136-142.
5Van Rossum D, Hanisch UK, Quirion R. Neuroanatomical localization, pharmacological characterization and functions of CGRP, related peptides and their receptors. Neurosci Biobehav Rev. 199x;21: 649-678.
9Fanciullacci M, Alessandri M, Figini M, et al.Increase in plasma calcitonin gene-related peptide from the extracerebral circulation during nitroglycerine-induced cluster headache attack. Pain. 1995;60: 119-123.
20Duggan MJ, Quinn CP, Chaddock JA, et al.Inhibition of release of neurotransmitters from rat dorsal root ganglia by a novel conjugate of a Clostridium botulinum toxin A endopeptidase fragment and erythrina cristagalli lectin. J Biol Chem. 2002;277: 34846-34852.
24Durham PL, Russo AF. Stimulation of the calcitonin gene-related peptide enhancer by mitogen-activated protein kinase and repression by an antimigraine drug in trigeminal ganglia neurons. J Neurosci. In press.
30Burstein R, Cutrer MF, Yarnitsky D. The development of cutaneous allodynia during a migraine attack: clinical evidence for the sequential recruitment of spinal and supraspinal nociceptive neurons in migraine. Brain. 2000;123: 1703-1709.
Botulinum Toxin Type A: A Neuromodulatory Mechanism of Action in Migraine.— Recent studies have shown that treatment with botulinum toxin type A (BTXA) provides substantial relief of pain in patients with migraine and other headache types,1–3 as well as a variety of other painful neuromuscular disorders.4 The exact mechanisms by which BTXA alleviates pain, however, are unclear. Indeed, there is ongoing debate regarding the extent to which muscle relaxation alone and other nonmuscle-related sensory pathways are involved in its observed analgesic effects. Pain reduction produced by BTXA therapy for cervical dystonia and other spasmodic dystonias would be expected simply based on toxin-induced reversal of the underlying abnormal muscular contractions. Temporal dissociation between the postinjection onset of muscle relaxation and pain relief suggests, however, that the effects of BTXA extend beyond those related to muscle relaxation: the pain relief precedes and outlasts the relaxation of the muscle.5
Botulinum toxin type A is thought to exert its therapeutic effects by modulating specific antinociceptive signaling pathways, although these mechanisms are not fully understood.6–11 In this issue of Headache, Durham et al report the results of a study designed to evaluate the effect of BTXA on the secretion of calcitonin gene-related peptide (CGRP) from cultured trigeminal ganglia neurons.12 Using primary cultures of trigeminal neurons under conditions that simulate migraine pathology and therapy, Durham and coworkers have demonstrated a role of CGRP and its controlling pathways in migraine and other headaches.13,14 Specifically, their results provide direct evidence that BTXA attenuates the release of CGRP from trigeminal neurons stimulated by chemical depolarization with potassium chloride or activation with capsaicin. The data suggest that, by inhibiting CGRP secretion, BTXA achieves its clinical efficacy in migraine by suppressing CGRP-mediated vascular and inflammatory changes.
Previous studies have shown BTXA inhibits the release of substance P (mediated by cleavage of the intracellular effector SNAP-25)15 and glutamate, another neurotransmitter involved in nociceptive processing.16,17 Thus, the putative antinociceptive effects of BTXA may, in part, be explained by a common mechanism—namely, suppression of neurotransmitter release.9 Furthermore, the finding that BTXA did not affect basal CGRP secretion but rather appears to inhibit trigeminal CGRP release only under conditions in which the trigeminal system is activated, suggests a neuromodulatory role of BTXA in the cascade of events involved in the onset and propagation of migraine. Taken together with other recent advances in our understanding of the antinociceptive properties of BTXA,6 the results of Durham et al elucidate the likely effects of BTXA on sensory nerves and support the concept that, beyond chemodenervation of skeletal muscle, BTXA inhibits neurotransmission of pain signals from the periphery to the cortex. Additional research is needed to clarify further the impact of BTXA on the neuronal signaling pathways activated during migraine.
Andrew J. Blumenfeld, MD San Diego, California
1Silberstein S, Mathew N, Saper J, Jenkins S. Botulinum toxin type A as a migraine preventive treatment. For the BOTOX® Migraine Clinical Research Group. Headache. 2000;40: 445-450.
10Giladi N. The mechanism of action of botulinum toxin type A in focal dystonia is most probably through its dual effect on efferent (motor) and afferent pathways at the injected site. J Neurol Sci. 1997;152: 132-135.
14Durham PL, Russo AF. Stimulation of the calcitonin gene-related peptide enhancer by mitogen-activated protein kinases and repression by an antimigraine drug in trigeminal ganglia neurons. J Neurosci. 2003;23: 807-815.
15Purkiss J, Welch M, Doward S, Foster K. Capsaicin-stimulated release of substance P from cultured dorsal root ganglion neurons: involvement of two distinct mechanisms. Biochem Pharmacol. 2000;59: 1403-1406.
16Cui M, Li S, Khanijou S, Aoki K. Subcutaneous BOTOX inhibits formalin-induced local neurotransmitter release and spinal nociceptive processing. Presented at: the 10th World Congress on Pain; August 17-22, 2002; San Diego , CA , USA .
17Ishikawa T, Nakanishi O, Funatsu N, Kameyama H. Nerve growth factor inducer, 4-methyl catechol, potentiates central sensitization associated with acceleration of spinal glutamate release after mustard oil paw injection in rats. Cell Mol Neurobiol. 1999;19: 587-596.