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- MATERIALS AND METHODS
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.
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- MATERIALS AND METHODS
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.