Final Term Paper kjk103

Author: Kayla Kindig

Introduction

Migraine is a type of headache that affects nearly ten percent of the population worldwide . Migraine is described as an intense, throbbing pain in the head that is often unilateral, with associated symptoms such as nausea, sensitivity to light (photophobia), and sensitivity to sound (phonophobia). About 1/3 of migraine patients experience a form of visual disturbance just before the headache, called an aura, in which a blind spot migrates across the patient’s field of vision. Known triggers of migraines include stress, lack of sleep, exercise, caffeine, and certain foods, though triggers vary between individuals . Migraines can be chronic or episodic, in which migraines occur >15 days per month or <14 days per month, respectively. Although there is some evidence to suggest that chronic and episodic migraines are distinct, it is common for patients who initially suffer episodic migraine to experience chronic migraines over time. A migraine headache can last for several hours or even days, with a fluctuating level of pain and associated symptoms.

Although the exact mechanism involved in migraine headache is largely unknown, there are key players and steps in the process that have been well-established. The trigeminal nerve is the fifth cranial nerve that relays sensory and motor information in the face and jaw, and also innervates blood vessels in the meninges. Most structures of the brain are not pain sensitive, but the meninges are an exception. The trigeminal nerve is thought to be capable of receiving or transmitting nociceptive information from the meninges. Perhaps the most prevalent and long-held theory as to the cause of migraine headaches is the vascular theory. Due to the throbbing nature of the pain and early observations of vasoconstriction in response to alleviation of headache, it was proposed that headache pain was due to vasodilation of meningeal blood vessels. It was thought that the stretching of the blood vessels was what caused the pain. Migraine can be invoked experimentally by the injection of a vasodilator, such as nitroglycerin. Another theory as to the cause of migraine headaches is more recent, and has been proposed as an explanation for the aura phase of headache in some individuals. Cortical spreading depression (CSD) is a phenomena in which a wave of depolarization spreads across the brain from anterior to posterior and causes a cascade of other effects. The speed of CSD propagation coincides approximately with the speed of aura progression across the visual field, and the ionic changes are thought to possibly explain the visual blind spot. More recent evidence suggests CSD might be linked to trigeminal activation and therefore may directly cause the headache pain, though this is contested. It should also be noted that vascular changes have been demonstrated to occur concomitantly with CSD, suggesting a possible overlap in the theories. Regardless of the theory, the trigeminal nerve is considered to be involved in migraine, and presumably some kind of sensitization occurs to cause nociception in the supposed absence of a noxious stimulus.

Many treatments for migraine were discovered to work retroactively, or developed under the assumptions of vascular theory. For example, ergotamine is a vasoconstrictor developed from the ergot fungus of the genus Claviceps. Derivatives of ergot were once used to assist with childbirth, but eventually became used to treat migraine headaches due to their vasoconstricting properties. Later, a class of medications called triptans were developed specifically to treat migraines. Triptans are serotonin agonists that target $\text{5-HT}_\text{1B}$ and $\text{5-HT}_\text{1D}$ receptors on cranial blood vessels and nerve endings to cause vasoconstriction. While triptans are effective at reducing migraine symptoms , there are unpleasant side effects with prolonged use, such as “rebound headaches” that occur when the medication wears off . Other side effects include feeling dizzy or lightheaded, nausea, and chest pain. Additionally, triptans do not work satisfactorily for all migraine patients, and they become less effective over time. Therefore, it is important to develop a migraine treatment that works well for most patients, retains its effectiveness over time, and has minimal side effects. Perhaps more importantly, there is a need to develop a preventative treatment for migraine; ergotamine and triptans are abortative treatments, and current preventative treatments are not migraine-specific. Creating the best possible treatment relies on understanding the mechanism responsible for generating migraine pain.

Calcitonin gene-related peptide (CGRP) is a neuropeptide created by alternative processing of the calcitonin gene. In neural tissue, it is found in highest concentration in the trigeminal nerve. The distribution led its initial discoverer to propose it was involved in nociception. CGRP was eventually observed to be a potent vasodilator, which potentially implicated it in the vascular theory. It is also shown to be released in the brain prior to CSD. CGRP activates a receptor complex that consists of a G-protein coupled receptor called calcitonin receptor-like receptor (CRLR or CLR), a receptor activity modifying protein (RAMP1), and a receptor component protein (RCP). Expression of mRNA of the receptor complex for CGRP has been observed in the brainstem of monkeys, which has been shown to activate during migraine headache. There is evidence to suggest that there is a significant increase in levels of CGRP in external jugular vein blood during migraine. More importantly, injection of CGRP consistently leads to a migraine headache in subjects who suffer from them . This evidence has been convincing enough to allow development and clinical trials CGRP antagonists for the treatment of migraine, which have all been demonstrated to be at least as effective as current migraine medications with fewer adverse effects .

The pain of migraine has been suggested to be due to the sensitization of meningeal afferent neurons, since migraine patients experience a heightened sensitivity to head movements and head/facial touch during the headache phase . It has been found that chemical stimulation of meningeal neurons with inflammatory mediators causes excitation and elevates their sensitivity so that they respond to stimuli previously below threshold . This could explain the theory that vasodilation is capable of causing migraines, in that the trigeminal projections around the meningeal vessels are sensitized to elicit a pain response to previously innocuous stimuli, such as the expansion of blood vessels. However, the question remains as to what causes the sensitization during migraine in the first place. The correlational data and potential functions of CGRP suggests it may play a causative role in the pain of migraine headache. The central hypothesis of this review is that CGRP sensitizes the trigeminal nerve, causing the reduced pain threshold that is likely responsible for migraines. Investigation of this subject would allow for a deeper understanding of the cause of migraine headache, and give researchers a specific target for treatments that would likely have greater effectiveness than current treatment options.

Arguments for the Hypothesis

Bennett et al. (2000) tested the role of CGRP8-37, a CGRP receptor antagonist, in reducing thermal and mechanical allodynia in a rat model of central neuropathic pain. In the experiment performed by Bennett and colleagues, the spinal cords of rats were hemisected and a catheter was inserted to allow for application of CGRP8-37 after surgical recovery. Spinal hemisection was chosen due to its presumed suitability as a model of chronic pain, and due to the localization of CGRP in portions of the spinal cord. Their control group received a similar operation without spinal hemisection, and received injections of a vehicle peptide in order to control for potential effects of injection stress. They tested the response of the rats to stimuli mimicking a light touch, a poke, and a pin prick to the fore and hind limbs, as well as the time taken to respond to an increase in temperature. In the test group, they found that the number of withdrawals increased from the baseline established before surgery to all stimuli except the pin prick. No significant changes were seen in the control group. They found that the highest dose of CGRP8-37 (50 nM) reduced the number of withdrawals in response to innocuous stimuli, but not the pin prick, for the experimental group. Again, there was no significant change for the control group. Responses to temperature were similar, with a decreased response time to an increase in temperature for the hemisected rats compared to baseline and controls, and the effect was alleviated by CGRP8-37. The authors established the potential pain causing action of CGRP by injecting it directly, which caused the rats to writhe, increase vocalization, and bite the site of the catheter. They determined that CGRP8-37 had no direct effect on motor function by testing motor scores at each drug injection, and these scores did not differ significantly from pre-drug values.

The results obtained by Bennett and colleagues suggest that CGRP plays a role in nociception that occurs with chronic pain. They saw no change in withdrawal from noxious stimuli or in controls, which provides no evidence for involvement in normal nociceptive pathways. The authors suggest this may be due to a chronic change in CGRP receptor activation following spinal injury. They propose that this increase in receptor activation could be due to an increase in neurotransmitter release. Interestingly, they explain that CGRP distribution in the spinal cord increases following spinal hemisection, thus suggesting an increase in the number of CGRP-containing synaptic terminals. It is not clear from this data what the mechanism of action is for CGRP, or even if it is pre- or postsynaptic. The authors propose an effect on other neurotransmitters, as CGRP has been shown to prolong the action of substance P , and stimulate the release of excitatory amino acids aspartate and glutamate . Glutamate acts on NMDA receptors, and NMDA receptors play a role in synaptic plasticity . Thus, CGRP could feasibly cause sensitization via the activation of NMDA, which could act to lower the nociceptive threshold of trigeminal neurons. Additionally, substance P is a peptide that has been linked to nociception. CGRP is thought to prolong the action of substance P by preventing its enzymatic breakdown, due to the two compounds sharing the same degradative enzymes. If there was an excess of CGRP, the enzymes would be more likely to degrade CGRP and substance P could remain in the synaptic cleft and act for a longer time. Alternatively, there is also evidence that CGRP can elicit pain transmission independently of substance P or glutamate, in that CGRP alone is capable of eliciting a depolarization in spinal cord dorsal horn neurons .

Although the results are consistent with CGRP being involved in sensitization, the behavior of CGRP in the central nervous system during migraine may differ from that in the spinal cord. Additionally, spinal injury was required for the relief of nociception by CGRP8-37, and while this is a general condition designed to evoke the sensitization of neurons to non-painful stimuli, it is unclear if the results are completely applicable to migraine, which presumably requires no physical injury to occur.

Sun et al. (2007) further investigated the role of CGRP in neuronal sensitization. In their study, rats were anesthetized, and a catheter was inserted into subarachnoid space. The rats were then paralyzed and the spinal cord exposed. For behavioral experiments, the experimenters injected the drugs, and for the electrophysiological experiments, the drugs were directly applied to the spinal cord. To assess behavioral changes to stimuli, they applied von Frey filaments of logarithmically increasing stiffnesses for 6-8 seconds and recorded if the paw of the animal was withdrawn. For the electrophysiological data, they recorded responses of wide dynamic range (WDR) neurons in the spinal cord that respond best to noxious stimuli. They performed extracellular recordings of WDR neurons in the L5 segment of the spinal cord, which all had receptive fields on the plantar surface of the hindpaw, determined by brush and pinch stimuli. The authors found that injection of CGRP reduced the threshold for paw withdrawal to von Frey filaments of about half the stiffness as before injection of CGRP or controls injected with DMSO. The effect was mitigated by addition of chelerythrine chloride (CC), which inhibits protein kinase C (PKC). The effect was also mitigated by application of chelerythrine (H89), which inhibits protein kinase A (PKA). They found that infusion of CGRP increased the firing rate of WDR neurons by about 50% of baseline in response to a press stimulus. They found no significant increase in firing rate from background or baseline levels when infused with CC or H89 30 minutes before infusion of CGRP.

The results of this study further demonstrate that CGRP is involved in sensitization of neurons required to reduce the pain threshold. The authors demonstrate this sensitization both with behavioral and electrophysiological data. Additionally, this study provides a potential mechanism by which this occurs: activation of PKC and PKA. It has been demonstrated that the CGRP receptor activates cAMP as a second messenger, which leads to downstream activation of PKA . Due to the limitations of the study by Sun et al., it cannot be determined in what order PKC and PKA pathways are activated, but it is possible that they function together. PKA and PKC both have supported roles in neuron sensitization . Protein kinase C specifically has been demonstrated to enhance NMDA-evoked currents , possibly by regulation of receptor gating . There is evidence that NMDA EPSCs are dependent on 5-HT2A receptor activation, but only when PKC is active . This suggests a mechanism in which 5-HT2A receptor activation leads to activation of PKC, which causes the phosphorylation of NMDA receptor subunits and consequent upregulation of NMDA . Since NMDA affects synaptic plasticity, it is possible that this may lead to the enhanced excitation of nociceptive neurons. This mechanism could be able to explain why serotonin receptor agonists like Sumatriptan that are used to treat migraine headache can be ineffective and cause rebound headaches; although the primary function of triptan derivatives is to cause vasoconstriction, they may be inadvertently leading to neuronal excitation.

Gu and Yu (2007) investigated the co-localization of CGRP with AMPA receptors in the spinal cord. The authors used double immunofluorescence labeling to localize CGRP and AMPA receptors in the dorsal horn of the spinal cord in rats. The spinal cord was fixed, transected, and incubated with antibodies against a sequence at the C-terminal of the human CGRP receptor and an antibody against the GluR2 subunit of AMPA receptors. They found that there was significant overlap between the localization of CGRP receptors and AMPA receptors in the soma of dorsal horn neurons. They then performed a single-cell extracellular recording to confirm that the similar localization indicated a similar function. They exposed the L2-L4 dorsal surface of the spinal cord via laminectomy of anesthetized rats, and used varying grades of stimulation to determine the receptive field of the neuron. They recorded the average frequency per minute of discharge from a WDR neuron. They found that application of both CGRP and AMPA increased the average discharge frequency of the neuron by up to 75% from baseline.

The results of this study further provide evidence for the sensitization of neurons by CGRP, and also gives insight into a potential mechanism. AMPA receptors respond to glutamate, an excitatory neurotransmitter, and has been implicated in the transmission of sensory information. Both AMPA and CGRP increased the firing frequency of WDR neurons in the spinal dorsal horn, and both are localized to similar areas. However, this study does not provide any insight as to whether the site of action of CGRP is pre- or postsynaptic, since the drug is free to diffuse after application. Additionally, this study does not provide direct evidence for an interaction between AMPA and CGRP, but the functional and localization similarity is indirect evidence. It should be noted that since the authors did not apply a CGRP antagonist to see if that was able to stop the increased responsivity, it is unclear by looking at this study if CGRP is sufficient to cause the effects. However, taken with the results of other studies where CGRP antagonist did mitigate the effects, it can be presumed that CGRP is necessary. Hypothetically, if CGRP increases the quantity of AMPA receptors on nociceptive neurons, this could lead to the perception of pain by making the synapses of nociceptive neurons more excitable, as increased levels of AMPA receptors can alter protein synthesis over time and lead to synaptic remodeling . This could occur by a similar pathway as described for PKC and NMDA receptors, in that CGRP receptor activation could initiate a cascade involving the phosphorylation of AMPA receptors by PKC or PKA.

Adwaninkar et al. (2007) investigated the role of CGRP in the nociceptive signaling pathway of the amygdala. They induced arthritis in the knee of rats by injection of kaolin suspension and carrageenan . They performed single-cell extracellular recordings on amygdala neurons of the latero-capsular division of the central nucleus (CeLC). They stimulated the neurons by applying mechanical stimuli, delivered by forceps, to the receptive field, which included the knee and the ankle. They defined stimuli as innocuous (100 and 500 g/30mm^2) or noxious (1500 and 2000 g/30mm^2) based on whether it evoked the withdrawal reflex in conscious mice and if it was painful to experimenters. They chose to test neurons that respond more strongly to noxious stimuli than innocuous stimuli, and called them multireceptive (MR) neurons. They recorded neuron response to stimuli before and 6 hours after induction of arthritis in the knee. They applied the CGRP receptor antagonists CGRP8-37 and BIBN4096BS to control rats with no induced arthritis, and to arthritic rats 5-6 hours after injection. They applied mechanical stimulus to the hindpaw of rats and determined the threshold required for paw withdrawal, and recorded vocalizations made by the rats during stimulation. They found that firing rate was greater in arthritic mice for both innocuous and noxious stimuli, but was reduced by application of both CGRP8-37 and BIBN4096BS, and increased again after washout of the drugs. They found no effect of CGRP8-37 or BIBN4096BS on normal mice. Vocalization in both the audible and ultrasonic range was longer in duration for arthritic mice, but was decreased with application of both CGRP8-37 and BIBN4096BS. They found that the threshold for hindpaw withdrawal was lowered in arthritic mice, from about 1,000 g/30mm^2 in regular mice to ~600 g/30mm^2 in arthritic mice. This effect was reversed by CGRP8-37 and BIBN4096BS, restoring the threshold to approximately 1,000 g/30mm^2.

This study provides further support for the role of CGRP in sensitization of neurons. The fact that firing rate increased again in arthritic mice after washout of the CGRP receptor antagonist is compelling evidence, since it suggests the decrease in firing rate is not merely to the passage of time, but is a direct consequence of the CGRP antagonists. Again, the results of this study suggest that CGRP is not involved in normal perception of sensory information, but in chronic pain sensitization. This study also provides evidence of involvement of CGRP with a brain structure associated with responding to nociceptive information . The amygdala has been implicated in emotional responses that are associated with pain, such as fear and anxiety . It is perhaps worth noting that fear and anxiety are components of stress and/or causes of stress, and stress is a key trigger of migraine headaches for many patients . This could potentially explain why migraine patients are able to feel the headache pain without the presence of an external pain stimulus, in that feelings associated with pain could have the ability to initiate or mediate a response.

Storer et al (2004) investigated the effect of CGRP on trigeminocervical neurons. They performed a midline craniotomy and laminectomy of C1-C2 spinal segments. They identified trigeminal neurons by response to superior sagittal sinus stimulation and response to facial touch. The recording electrode was positioned at the dorsal root entry zone. They stimulated free-running neuronal activity with L-glutamate microiontophoresis to a frequency of 20 Hz, so that cell inhibition could be distinguished from random firing. For the control condition, they filled micropipette barrels with saline. For the experimental conditions, they filled micropipette barrels with 1 mM CGRP, 1mM CGRP8-37, or 20 mM BIBN4096BS (olcegepant). BIBN4096BS was also delivered intravenously via a catheter inserted in the femoral vein. The authors found that L-glutamate caused neuronal firing frequency to be sustained around 25 Hz at -60 nA, and co-application with CGRP caused no additional effect. The firing rate was reduced by application of BIBN4096BS and CGRP8-37 (to approximately 8 and 9 Hz, respectively), and could be increased again to near 25 Hz by application of CGRP.

This study provides direct evidence for the sensitization of trigeminal nerves by CGRP. L-glutamate is an excitatory neurotransmitter, which would allow for the activation of neurons. The ability of CGRP to have the same effect as glutamate but no additional effect when applied with glutamate suggests they act in the same pathway. Additionally, the ability of a CGRP receptor antagonist to reduce the trigeminal firing rate suggests that CGRP plays a direct role in enhancing the excitability of neurons by affecting glutamate in some way. This could potentially correspond with evidence examined earlier about the relationship of CGRP with AMPA and NMDA receptors.

Similarly to Storer et al., Fischer et al. (2005) investigated the effect of applying BIBN4096BS to nerves of the spinal trigeminal nucleus (STN) in the medulla. The authors anesthetized rats and cut a cranial window of ~6 x 4 mm in the parietal bone to expose the dura mater. They exposed the medullary brainstem by cutting a window in the atlanto-occipital ligament and the underlying dura mater, and inserted glass microelectrodes into the medulla. They delivered shock stimuli of 10-12 V at 0.2 Hz to the dura to identify trigeminal neurons, and also examined their response to mechanical stimulation of the dura. They recorded the spontaneous activity of neurons for 30 minutes before application of BIBN4096BS. They applied 1 mM BIBN4096BS to the exposed dura and observed effect for 15 minutes. They also diluted and intravenously injected 300 ug/kg BIBN4096BS, then 900 ug/kg BIBN4096BS 26 minutes later. They applied 2% lidocaine for comparison of effects. Additionally, the authors performed a thermal stimulation in which the temperature of the dura was progressively increased and decreased, to a maximum of 44C and a minimum of 20C. The authors found that direct application of BIBN4096BS to the dura had no effect on firing frequency. However, intravenous administration of BIBN4096BS led to a decrease in firing frequency of approximately 30% after 20 minutes at the 300 ug/kg dose, and by roughly 50% by 20 minutes after administration of the 900 ug/kg dose. They found that firing rate increased with temperature, seeing a 2.5 fold increase from spontaneous activity levels at 44C. BIBN4096BS was able to significantly reduce firing rate to near baseline at all temperatures.

The results of this study are mostly consistent with those observed by Storer et al. in 2004, but differs in one important way. Storer and colleagues used a glutamate to increase firing rate before seeing if this elevated firing rate could be reduced with BIBN4096BS, whereas Fisher et al. administered the antagonist at resting activity level and saw a response. This is in contradiction to studies examined earlier, which found that CGRP antagonists can reduce firing rate or increase threshold stimulus response for models of chronic pain but not controls . However, there are studies that support their result . Also, ut us worth considering that exposure of the dura itself could have some effect on neuronal activity. It is unclear as to why the authors observed an effect of BIBN4096BS when administered intravenously but not when applied directly to the dura. The answer may be that communication with other structures of the nervous system not in the dura is necessary for the maintenance of the response, as intravenous injection would allow BIBN4096BS to circulate as opposed to being confined to the dura.

Arguments Against the Hypothesis

In order to prove that CGRP plays a causative role in migraines, it is first necessary to establish correlation between CGRP and migraines. As mentioned previously, this was accomplished by demonstrating that heightened levels of CGRP are found in the external jugular vein (EJV) during a migraine attack, that administration of anti-migraine medication can lower CGRP levels in the EJV , and that injection of CGRP induces a migraine attack in migraine patients. This paper has provided evidence as to the mechanism of CGRP in migraine headache by establishing a correlation between the involvement of CGRP in sensitization of the spinal cord, and the presence of CGRP and receptors in high levels in the trigeminal nerve, suggesting that the mechanism of action in the spinal cord can be the mechanism of action in the trigeminal nerve and that this is what causes migraine. Then, evidence of sensitization specifically in the trigeminal nerve was provided, demonstrating its sufficiency in the response. However, there is evidence that suggests CGRP is not substantially correlated with migraine attacks, or that it is not necessary to evoke a migraine attack, and this kind of data would refute the assumptions of the hypothesis.

A study conducted by Tvedskov et al. in 2005 attempted to replicate the experiment in which CGRP was observed at elevated levels in the EJV during migraine. The authors recruited 39 migraine patients to participate in the study, all of whom met the International Headache Society guidelines for migraine headache without aura. Patients had to have experienced migraines 1 to 6 times a month for the past three months, be between 18-65 years of age, and be in general good health in order to be included in the study. Patients were excluded if they had more than 10 hours of migraine attacks per month and if they took headache medication more than 12 days per month. Patients contacted a physician at the onset of a migraine attack, and blood samples were taken within one hour, as long as the patient had not taken anti-migraine medications in the previous 72 hours. Blood samples were taken in the patient’s homes and after 15 minutes of rest in the supine position, to eliminate stress. Blood was drawn from the patient’s dominant side if the headache was bilateral or from the affected side for unilateral headaches. Control samples were taken within 7 days on the same side in the same manner, on a day when the patient had been headache free for 72 hours and had not taken a serotonin agonist for at least 48 hours. Samples were not used if patient contacted the physician within 24 hours indicating that they had developed a headache after sampling. The authors performed two different radioimmunoassays to detect CGRP in blood, one of which was used in the previous study that reported CGRP increase in EJV blood during migraine. The authors found that there was no significant increase in CGRP concentration in EJV blood or peripheral cubital blood during migraine for either assay. The mean difference between migraine and non-migraine levels for the first assay was 1.81 pmol/L (p=0.44) in the EJV and -0.79 pmol/L (p=0.69) in the cubital vein. For the second assay, the mean difference was 2.00 pmol/L (p=0.416) and 1.53 pmol/L (p=0.431) in the EJV and cubital vein, respectively.

The authors of the previous landmark study claiming an increase in CGRP concentration in the EJV blood during migraine did not measure the non-migraine CGRP blood level in the test subjects; they instead used sample blood from an unspecified healthy subject as a measure of resting CGRP level . They found blood levels of CGRP to be 86±4 pmol/L for migraine patients and <40 pmol/L for the control . Levels of CGRP have been demonstrated to vary between individuals and circumstances, as CGRP levels can be heightened during exercise , and there is evidence to suggest a difference between males and females and between resting levels of migraine patients and healthy subjects . The experimental design of Tvedskov and colleagues eliminated inter-patient differences in CGRP concentrations. Additionally, in the study by Goadsby et al., samples were taken in a clinical setting, which could cause considerable stress, and patients could have taken medication before arriving for their sample. Taking samples in the subjects’ homes eliminates some stress, which is important because stress could possibly result in elevated CGRP levels. The differences in methods between these two studies seem to explain the differences in results, with the study by Tvedskov most likely being more accurate. The authors suggest that the reduced CGRP levels in the EJV seen by administration of sumatriptan are not necessarily migraine specific, and that the reduction may be of a normal CGRP level or a stress-induced level.

Previous studies implied that CGRP concentration increases during migraine to such a degree that it is flushed out via the EJV, which is responsible for drainage of much of the head. Such high levels during the pain phase of a chronic condition would suggest an important role in the mechanism. However, the results of the study by Tvedskov and colleagues do not indicate that CGRP is not involved in migraine, just that CGRP does not appear in elevated levels in the EJV blood during migraine. Their results suggest that is unlikely that the level of CGRP in the meninges could diffuse in significant volumes through the blood brain barrier (BBB) to substantially increase the concentration in the EJV blood. Indeed, although BBB permeability has been demonstrated to increase in conjunction with the migraine-related phenomenon cortical spreading depression , the BBB only allows diffusion of lipophilic molecules between 400-600 Da in weight under normal conditions and CGRP weighs approximately 3.4 kDa . There are areas of the brainstem not protected by the BBB where CGRP receptor mRNA has been located, but it is understandable that there would not be a substantial increase of CGRP in these areas during migraine if the site of excitation is primarily trigeminal. A more direct implication of the study by Tvedskov et al. might be that CGRP does not increase as substantially as previously thought during migraine headache. It might be that the difference between migraine patients and healthy volunteers is a naturally higher level of CGRP and/or enhanced sensitivity to CGRP. One important conclusion to make from this study is that increased level of CGRP in the blood is likely not a reliable indication of an accurate animal model of migraine.

In addition to conflicting correlational evidence, there is evidence to suggest that CGRP is not sufficient to cause migraine. Migraine is often induced experimentally by a general vasodilator, such as nitroglycerin, and nitroglycerin injected animals have been determined to be a decent migraine model by measurements of facial allodynia and trigeminal nucleus caudalis c-fos expression, which is an indicator of neuronal activation . This information suggests the mechanism of action for CGRP could be its action as a vasodilator and that it does not act to sensitize neurons, though there is the possibility that there are multiple players in the pathway, or that the pathway is not unidirectional. Nitroglycerin can be converted to nitric oxide, a known neurotransmitter , which could function along with CGRP. Both nitric oxide synthase inhibitors and CGRP-receptor inhibitors are effective at reducing migraine, though we do not know exactly how they act.

A paper by Capuano and colleagues in 2014 investigated the role of CGRP in a nitroglycerin-induced model of trigeminal sensitization. To test the effects of nitroglycerin and CGRP, the authors injected either 1 ug of CGRP or 50 ul of sterile water into the upper lip of rats . This was performed for both rats pretreated with 10 mg/kg nitroglycerin and untreated rats. The rats were placed into transparent observation cages and their behavior was video recorded for one hour. The researchers recorded the number of seconds the rats spent rubbing the site of injection. Additionally, the experimenters performed an experiment to measure the levels of CGRP in different areas of the nervous system following nitroglycerin injection. They decapitated rats at time points of 2, 4, and 24 hours after injection and collected the trigeminal ganglion, brainstem, hypothalamus and hippocampus. They assessed CGRP levels with a radioimmunoassay. The authors found that injection of CGRP alone into the whisker pad did not result in facial rubbing behavior that differed significantly from control. Injection of nitroglycerin alone also did not cause a change in facial rubbing behavior compared to control. However, CGRP and nitroglycerin injected together resulted in an increase in the time rats spent rubbing their faces. The increase was more prolonged when nitroglycerin was injected 24 hours before CGRP, compared to when it was injected 4 hours before CGRP. The authors also found that CGRP levels increased after injection of nitroglycerin in the trigeminal ganglia and brainstem, and the levels were sustained for 24 hours, but there was no effect on CGRP levels in the hypothalamus or hippocampus.

The results of this study suggest that CGRP is not sufficient for trigeminal sensitization, requiring nitric oxide to invoke a pain response. This implies that nitric oxide may be responsible for trigeminal sensitization, and CGRP merely mediates the response. This could explain why healthy volunteers do not experience migraines when injected with CGRP . However, inconsistent with the results of this experiment, injection of nitric oxide into healthy volunteers does cause them to experience a headache, though it does not meet the International Headache Society criteria for a migraine . The authors of this experiment used rats injected with nitroglycerin into the whisker pad due to the presence of trigeminal afferents in the face. Rubbing of the face was used as an indication of pain, but the extent to which this is analogous to migraine pain is unclear. Behavioral measures are limited in their application; the authors' results would be more convincing if they had looked at brain responses. Additionally, since nitric oxide functions as both a neurotransmitter and a vasodilator, and because nitric oxide is a gas that diffuses freely, it is unclear which action is responsible for the observed effects and where precisely it is occurring. It is entirely possible that CGRP sensitizes trigeminal neurons in this situation, but pain was not observed because there was no stimulus, until nitric oxide was injected and caused vasodilation of adjacent blood vessels. Since nitric oxide has been demonstrated to cause nociceptive activation on its own , it raises the question as to why injection of nitroglycerin alone caused no behavioral change in rats. The authors claim that nitroglycerin causes sensitization, but data from other authors have suggested that migraine patients are sensitized to nitric oxide, as seen by migraine patients developing migraines upon injection of nitroglycerin , implying that nitric oxide is a trigger and there is another substance that has a sensitizing effect.

Ramacharndran et al. performed a study in 2014 further testing the sufficiency of nitric oxide and CGRP in trigeminal activation using nitric oxide synthase inhibitor L-NAME and CGRP receptor inhibitor Olcegepant. They anesthetized rats and inserted cannula for infusion of drugs . Seven days after surgery, rats were permitted to move freely in cages, and drugs were administered two days later. They administered nitroglycerin at 4 mg/kg/min for 20 minutes at different time points. Sumatriptan was infused at 0.6 mg/kg for 3 minutes or L-NAME at 40 mg/kg for 20 minutes followed 5 minutes later by nitroglycerin infusion. Olcegepant at 1 mg/ kg was infused over three minutes, and was followed by nitroglycerin ten minutes later. Additionally, olcegepant was infused over 30 minutes at the start of nitroglycerin infusion to compare olcegepant pretreatment with postreatment. L-733060, a substance P receptor antagonist, was infused at 1 mg/kg for 3 minutes and followed by nitroglylcerin infusion 10 minutes later. As a control, rats were infused with saline or vehicle after two hours and four hours. They recorded baseline mean arterial blood pressure in 3 rats. After perfusion of drugs, they isolated the dura mater, trigeminal ganglion, and trigeminal nucleus caudalis and fixed them. The tissues were processed for immunofluorescence staining. They used antibodies against fos, nitric oxide synthase, and CGRP, and an observer blind to the treatments counted the cells that expressed the tagged substance. They found that fos expression in the tissues analyzed was substantially elevated for the nitroglycerin treatment compared to controls, and application of L-NAME reduced the fos levels to near baseline. Application of olcegepant after nitroglycerin infusion did not decrease the elevated fos expression caused by nitroglycerin, but application of olcegepant before nitroglycerine reduce fos expression to near control levels. Pretreatment with L-733060 significantly reduced c-fos expression. Levels of nitric oxide synthase expression increased in dura mater with nitrocglycerin infusion, and these levels were not significantly altered by pretreatment with sumatriptan, L-NAME, or L-733060. They saw an increase in CGRP immunoreactivity in nerve fibers of the dura four hours after nitroglycerin infusion, which was reduced with pretreatment of L-733060 but not with pretreatment of sumatriptan or L-NAME. Similarly, there was an increase in CGRP immunoreactivity in the trigeminal nucleus caudalis, but the levels were reduced by pretreatment with L-NAME, sumatriptan, and L-733060.

The results of this study suggest that both nitric oxide and CGRP play a role in sensitization, though the order and way in which they act is less clear. The fact that nitroglycerin alone induces neuron activation and that CGRP expression is increased after infusion of nitroglycerin suggests that nitric oxide acts sooner in the pathway than CGRP. The authors propose that nitric oxide may sensitize peripheral afferent nerves, which activates second order neurons. They also suggest that nitric oxide might amplify afferent nociceptive signals. The authors focus on nitric oxide and do not consider functions of CGRP at length, instead seeming to assume it acts solely as a vasodilator. Although it is uncertain if experimental animals could experience migraine, the measurement of trigeminal activation should be satisfactorily analogous to migraine. Additionally, the authors kept the rats completely awake during the process so as to control for effects of sedatives and procedural stress on neuronal activation, and most human patients infused with nitroglycerin during migraine studies are awake, implying that the authors' results are close to what would be observed in humans. Perhaps the most interesting result of this study is that a CGRP receptor antagonist prevents trigeminal activation when applied before nitroglycerin, but not when applied after. This result could indicate multiple things, but importantly it suggests that CGRP may still precede nitric oxide in the activation pathway and be responsible for neuronal sensitization. Assume for a moment that CGRP sensitizes trigminal neurons. The author's results still make sense, because blocking CGRP receptors before nitroglycerin infusion can block sensitization to nitric oxide, but blocking CGRP receptors after nitric oxide has already diffused has no effect because nitroglycerin has already increased CGRP expression substantially, and the expression of nitric oxide synthase has already been elevated. This proposed explanation suggests that nitric oxide is sufficient for generating trigemial activation, due to its regulatory impact on CGRP, but is not necessary because it cannot increase activation without CGRP. Considering a migraine patient in which endogenous levels of CGRP may be elevated, nitric oxide may be especially unnecessary. A mechanism that makes sense with the results of this paper would be that CGRP causes sensitization to ntiric oxide, and nitric oxide may be responsible for relaying or amplifying nociceptive information. There are likely to be a number of other steps in the pathway where nitric oxide could act to amplify nociceptive information, since what little we understand of the mechanism has already proven to be complex. Other compounds such as substance P are probably involved, as suggested by the fact that inhibition of substance P receptors results in a decline of neuronal activation. All of these compounds could potentially be involved in a positive feedback loop where each stimulates production of the other, which makes it especially difficult to parse out their place in the mechanism. However, the direct sensitization of trigeminal neurons by CGRP has not been ruled out.

Figure 1. Proposed mechanism of CGRP sensitization in migraine attacks, based on experimental data. Not previously mentioned was evidence suggesting CGRP has an autocrine effect, promoting its own production. Thick arrows represent upregulation, whereas thin arrows represent advancing steps in pathway. Uncertain mechanisms written on arrows with question marks, unknown mechanisms indicated by question marks alone. CGRP may act as a sensitizing peptide and a vasodilator. Nitric oxide may act as a neurotransmitter and/or a vasodilator. Substance P likely acts as a neurotransmitter. This pathway proposes a positive regulatory effect of CGRP and nitric oxide on one another, as well as each on itself. This regulation may be due to activation of receptors, inhibiting degradating enzymes, a cascade leading to increase in expression, etc.

Conclusion

The cause of migraine headaches remains to be determined, though important structures and substances have been identified and investigated. One potential mechanism by which migraine headache might be generated is the sensitization of the trigeminal nerve , which would lower the threshold of meningeal nociceptors and cause a pain response without the presence of noxious stimuli. CGRP has been found in correlation with migraine, and thus has been a subject of extensive testing. The central hypothesis of this review is that CGRP causes migraine headache pain by sensitizing meningeal trigeminal afferents.

In favor of this hypothesis is evidence such as the application of CGRP receptor antagonists reducing thermal and chemical allodynia in hemisected rat models of neuropathic pain, behavior indicative of pain following injection of CGRP, reduced paw withdrawal threshold in CGRP injected rats, increased neuronal firing of WDR neurons after application of CGRP, and increased withdrawal threshold in arthritic mice upon application of a CGRP receptor antagonist. Thus, there is sufficient evidence to suggest that CGRP is capable of neuron sensitization. Further evidence in support of the hypothesis comes from studies directly analyzing the effect of CCGRP on the trigeminal nerve or the meninges, which include reduced firing of spinal trigeminal nucleus neurons in response to thermal stimulus after application of a CGRP receptor antagonist to the dura mater, and increased firing of cat trigeminal neurons after application of CGRP that can be reduced by application of CGRP receptor antagonists.

Evidence against the hypothesis includes a study that found no elevation of CGRP levels in EJV blood during migraine headache, which negates the correlation of CGRP with migraine. Additional evidence opposing the hypothesis is the finding that CGRP alone was not sufficient to cause facial rubbing behavior that indicates a lowered trigeminal pain threshold, and that application of nitroglycerin causes elevated neuronal activity that cannot be reduced if a CGRP antagonist is applied afterwards. These studies call into question the necessity and sufficiency of CGRP in causing neuronal sensitization.

The evidence in favor of the hypothesis is somewhat more compelling than that against it. The original study claiming that CGRP levels were elevated during migraine has been used as a rationale for further investigation of CGRP in migraine pathogenesis in that it supports correlation of CGRP with migraine. However, the result that CGRP levels were not elevated in the EJV blood during migraine, which was obtained using a more suitable control than the previous study, does not allow us to conclude that CGRP does not play a role in migraine, merely that it is not elevated in the blood. The study by Capuano et al. in 2014 used nitroglycerin to evoke trigeminal sensitization, though they used behavioral assessments to determine sensitization and injected compounds into the facial whisker pad, which would contain trigeminal projections but would also contain blood vessels upon which nitric oxide could also act. Ramacharndran et al. (2014) measured trigeminal activation via cfos expression, and determined that nitroglycerin increases neuron activation, which cannot be reduced by application of a CGRP receptor antagonist after nitroglycerin application, but can be reduced if the antagonist is applied before nitroglycerin. This does not negate the sufficiency of CGRP as much as it suggests that nitric oxide also functions in the pathway and that there could be positive feedback in which CGRP increases production or receptor binding of nitric oxide. Studies supporting the hypothesis demonstrate both behavioral evidence of neuron sensitization and direct evidence via electrophysiological recording of neurons, both in spinal cord nociceptors and in parts of the trigeminal nerve. These studies have shown that application of CGRP increases neuron sensitization, and that application of a CGRP receptor antagonist decreases sensitization.

Future studies should be done to further support or refute the hypothesis, specifically with regards to the sufficiency of CGRP. Since CGRP, nitric oxide, and substance P have all been suggested to have a role, it would be advantageous to test their function in trigeminal sensitization separately to determine if CGRP is sufficient and if the others occur further down the same pathway. For these experiments, it would be necessary to block CGRP with a receptor antagonist such as olcegepant or CGRP8-37 to see if nitric oxide and substance P can elicit the same effects on their own, which is similar to what Ramacharndran et al. did in their 2014 study in which they found that nitroglycerin-induced neuronal activation was blocked with pretreatment of olcegepant. Then, nitric oxide synthase should be blocked with L-NAME to see if CGRP is still able to elicit sensitization, and then substance P should be blocked, such as with substance P receptor inhibitor L-733060 to see if CGRP can elicit the same effect.

To determine if these substances are in the same pathway, one should be blocked while the remaining two are applied to see if their effects are linearly additive, indicating that they function in separate pathways, or if they saturate at a certain level, indicating that they act in the same pathway. It would be advantageous to perform these experiments on projections of the trigeminal nerve as opposed to other neurons in order to assess their immediate relevance, and direct measures of neuron sensitization should be used, such as electrophysiological measurements or cfos expression. Recording from neurons does have its drawbacks in that animals usually must be anesthetized, which can potentially confound the data, so it may be useful to supplement direct measurements with behavioral evidence in freely moving awake animals. Most importantly, experimenters should use human subjects where possible, since even the best animal model is still just a model, and humans are the subject for which treatments will ultimately be necessary. While it may be impractical or unethical to record directly from neurons in the human brain, perhaps magnetic resonance imaging could be of use to determine activation in particular areas of the brain. Additionally, while it is unlikely to be safe for researchers to block substance P or CGRP in the human brain due to possible side effects, experiments have already been conducted in which CGRP or nitric oxide are injected into awake human patients, so it should be plausible to try administering both at the same time to observe the effect.

References