Benchmark III kjk103

Author: Kayla Kindig

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. So far, 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. 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 showed 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 4pmol/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 since 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 3.4 kD . 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. Additionally, 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 nitroglycerine 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 linear. Nitroglycerine 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. 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.

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. Perhaps nitric oxide acts to directly or indirectly promote substance P in a similar manner to how CGRP may prolong the effect of substance P by preventing its enzymatic degradation . 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.

To further understand the exact steps involved in generating a migraine, more rigorous experiments that sequentially block potential players in the pathway will need to be conducted, and efforts should be taken to use human models where possible or to use an animal model that mimics human migraine by as many measures as possible.

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