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Abstract

Background

Migraine is a disorder of the brain and is thought to involve activation of the trigeminovascular system, which includes the peripheral afferent projection to the nociceptive specific dura mater, as well as the central afferent projection to the trigeminal nucleus caudalis. Stimulation of the blood vessels of the dura mater produces pain in patients that is referred to the head similar to headache.

Headache mechanisms

The likely reason for the pain is because the vascular structures of the dura mater, including the superior sagittal sinus and middle meningeal artery, are richly innervated by a plexus of largely unmyelinated sensory nerve fibers from the ophthalmic division of the trigeminal ganglion.

Methodology

Stimulation of these nociceptive specific nerve fibers is painful and produces neuronal activation in the trigeminal nucleus caudalis. Preclinical models of headache have taken advantage of this primarily nociceptive pathway, and various animal models use dural trigeminovascular nociception to assay aspects of head pain. These assays measure responses at the level of the dural vasculature and the central trigeminal nucleus caudalis as a correlate of trigeminovascular activation thought to be involved in headache.

Summary

This review will summarize the history of the development of models of dural trigeminovascular nociception, including intravital microscopy and laser Doppler flowmetry at the level of the vasculature, and electrophysiology and Fos techniques used to observe neuronal activation at the trigeminal nucleus caudalis. It will also describe some of pitfalls of these assays and developments for the future.

Keywords

Migraine dura mater trigeminovascular nociception intravital microscopy electrophysiology Fos

Introduction

Migraine is a chronic and disabling disorder of the brain (1) that affects up to 15% of the population at any one time. It has a debilitating effect on the quality of a sufferer’s life and is estimated to cost the United States and European economies $19.6 billion and €27 billion a year in lost personnel hours, respectively (2 –4). This huge burden on the global economy is not reflected in the level of funding that is provided to research in understanding this condition (5,6), and yet over the last 25 years there have been huge steps in our understanding of the pathophysiology of this condition. While the exact pathophysiology of migraine is still not fully understood, it is thought to involve activation of the trigeminal afferents (1), which densely innervate dural structures and project to second order neurons in the trigeminal nucleus caudalis and C₁–C₂ region of the spinal cord (trigeminocervical complex, TCC). Studies in patients from the mid-20th century were able to demonstrate that while the brain is largely insensate, pain can be generated by stimulation of blood vessels of the dura mater in humans (7,8**) and this pain is referred to the head (9**). These discoveries led to the vascular hypothesis of migraine and an emphasis on cerebrovascular changes. There was also proliferation in preclinical research into the nerve fibers that innervate the dural vasculature, and the likely involvement of the trigeminovascular system (reviewed by Moskowitz in 1984 (10)), and the subsequent development of animal models of migraine. Ironically, with our greater understanding of the trigeminovascular system, the emphasis of migraine pathophysiology has moved to a more neural view, and how the brain may be involved in the modulation of the trigeminovascular system (11).

Trigeminovascular system – historical viewpoint

The trigeminovascular system (10) includes the pseudounipolar trigeminal ganglion that has central afferent projections to the trigeminal nucleus caudalis in the medullary spinal cord, and a peripheral projection, largely from the ophthalmic division of the trigeminal ganglion, which innervates the cranial blood vessels and other cranial structures, including the pain-sensitive dura mater. There is also a reflex connection from the trigeminal nucleus to the parasympathetic outflow to the craniovasculature (12 –14) (Figure 1). Several potent vasodilator peptides are present in the cell bodies of the trigeminal ganglion and project nerve fibers to cranial vessels, including calcitonin gene-related peptide (CGRP), substance P, neurokinin A and pituitary adenylate cyclase-activating peptide (PACAP) (15 –18).

Figure 1.

Overview of the trigeminovascular system. The trigeminovascular system relevant to headache includes the pseudounipolar trigeminal ganglion (TG) and its afferent projections to the trigeminal nucleus caudalis and C₁ and C₂ regions in the medullary and cervical spinal cord (TCC), and its peripheral afferent projections mainly from the ophthalmic division of the trigeminal nerve to the cranial blood vessels and other cranial structures, including the pain-sensitive dura mater. There is also a reflex connection from the trigeminal nucleus to the parasympathetic outflow to the craniovasculature (gray neuron) known as the trigeminal autonomic reflex arc, via the superior salivatory nucleus (SuS), which is the origin of cells of the parasympathetic vasodilator pathway. The parasympathetic projection to the craniovasculature is predominantly via the greater petrosal nerve branch (green) and the facial nerve (purple) and its synapse with the sphenopalatine (SPG, pterygopalatine – in human) ganglion. Electrical stimulation (ES) of the dural vasculature causes activation of the dural blood vessels, neuronal activation of the TCC and the SuS.

Sterile neurogenic inflammation

Given the hypothesis that migraine and other headache disorders are believed to involve activation of the trigeminovascular system, preclinical assays were developed to demonstrate this activation; it was believed that the changes that took place in animals were similar to those during primary headaches. Originally, studies that concentrated on understanding the anatomy and pharmacology of nociceptive activation of the trigeminovascular system used stimulation of the trigeminal ganglion. Trigeminal ganglion stimulation in animals and humans results in increase in cerebral blood flow (19,20), facial flushing and increase in skin temperature (21) and an increase in the concentration of CGRP and substance P levels in the external jugular vein (22). Additionally in rats, trigeminal ganglion stimulation results in dural neurogenic plasma protein extravasation (PPE) and vasodilation (23**). This led to the hypothesis that the initial trigger for migraine may be a sterile inflammatory response at the level of the dura mater, as a consequence of the release of substance P and neurokinin A, causing an increase in vascular permeability and activation of trigeminal afferents to stimulate the trigeminovascular system (24). This hypothesis was supported largely by the fact that anti-migraine treatments, ergot alkaloids and sumatriptan, were effective at inhibiting dural neurogenic PPE in rodents (25,26). However, the subsequent development and failure of specific extravasation antagonists (27,28) and neurokinin 1 receptor antagonists (29,30) based on this hypothesis has somewhat damaged this idea. The role of dural neurogenic PPE has recently been reviewed (31), and it is hard to sustain an argument that it plays an important role in migraine neurobiology.

While the use of trigeminal ganglion stimulation as a model of migraine is considered somewhat redundant today, the research that was conducted during the 1980s and early 1990s was crucial in improving our understanding of the anatomy and pharmacology of the trigeminovascular system, as well as proving an important screen for the development of the 5-HT_1B/1D, “triptan”, class of drugs. It was also the first assay to demonstrate neuronal activation at the level of the trigeminal nucleus caudalis (32**), using Fos immunoreactivity as a marker of activation and it is information that still guides us today.

Trigeminovascular dural nociception

As models of PPE and the use of trigeminal ganglion stimulation have slowly decreased in headache research, methods that activate trigeminovascular afferents via stimulation or manipulation of the dura mater have somewhat taken over. Many similarities between responses after dural manipulation techniques and migraine have been demonstrated. The dura mater is considered far more nociceptive specific, and in the cat stimulation of the superior sagittal sinus, whose stimulation causes pain in humans (33), produces greater increases in cerebral blood flow than trigeminal ganglion stimulation (34). Moreover, stimulation of the superior sagittal sinus also produces release of CGRP via trigeminal neurons and vasoactive intestinal peptide (VIP) (35**), presumably via activation of parasympathetic neurons (36). Interestingly, during severe migraine only increases of CGRP in the extracerebral vasculature have been demonstrated (37**), while VIP is additionally released during various trigeminal autonomic cephalalgias (TACs) (37**–40). There is one study that shows an exception to this (41**), where CGRP levels were not raised in patients with less developed migraine. It is noteworthy that during the studies exploring PPE, systemic application of substance P, neurokinin A and capsaicin were all able to induce PPE in the dura mater in rodents, whereas CGRP was the only molecule unable to demonstrate a PPE response (23**). These studies taken together illustrate the importance of CGRP in migraine and the dural trigeminovascular nociception assay and why this assay may offer greater similarities in exploring pathophysiological mechanisms of migraine than the PPE assay.

Studies that use the dural vasculature as a means to activate trigeminovascular nociceptive neurons have allowed us to more fully characterize the anatomy of the trigeminovascular system and ascending projections that may be involved in migraine pathophysiology. This anatomy has been thoroughly described and reviewed in previous articles and therefore will not be covered here (11,42,43). In brief, though, stimulation of dural structures results in activation in the TCC and areas of the brainstem and diencephalon. This is important because as advanced technologies have allowed us to image patients’ brains during spontaneous and triggered migraine (see the review article in this special issue on “Pearls and pitfalls and imaging in headache”), there is a clear correlation with what is observed during migraine with what we know from animal models that use stimulation of the nociceptive-specific craniovasculature to activate the trigeminovascular system.

Dural electrical stimulation

Dural electrical stimulation takes advantage of the evidence that dural structures including the superior sagittal sinus and middle meningeal artery (MMA) are richly innervated by a plexus of largely unmyelinated nerve fibers from the ophthalmic division of the trigeminal ganglion (44). Therefore, electrical stimulation of the vascular structures they innervate has been used to produce antidromic stimulation of the peripheral projection of the trigeminal nerve, and orthodromic stimulation of the central projection to the trigeminal nucleus caudalis (Figure 1). There are various techniques that have been used to observe the responses of trigeminovascular nociception, which include intravital microscopy and blood flow monitoring at the level of the dural vasculature, and for neuronal responses electrophysiology and immunoreactivity of the early gene c-fos mRNA and its product Fos protein, which is a nuclear protein rapidly activated after neuronal activation.

Pearls of measuring changes in the cranial vasculature

Electrical stimulation of the dural vasculature and activation of the trigeminal nerve causes the release of vasoactive neuropeptides and subsequent changes in caliber of the vessels and blood flow. It is thought that during migraine activation of this peripheral afferent projection, the subsequent release of vasoactive peptides can cause vasodilation of cerebral and cranial blood vessels, which may contribute to further nociceptive transmission via activation of the trigeminal afferent projections to the trigeminal nucleus caudalis (45). This process can result in pain, or the perception of pain, as the sensory information is processed in higher-pain processing centers. Measuring the changes to the vasculature as an indication of activation of the trigeminal nerve provides considerable utility in identifying receptors that are capable of modulating nociceptive stimuli that are clinically relevant. The two methods used, intravital microscopy and laser Doppler flowmetry, vary slightly but carry many similarities.

Intravital microscopy

Intravital microscopy was developed by Williamson and colleagues and uses a thinned closed cranial window with video microscopy to visualize the dural and pial blood vessels, and allows direct measurement of changes to the diameter of the blood vessels (46**). Most commonly in rats, the MMA, or a branch of it, is measured. Placing bipolar stimulating electrodes onto the dural surface near to the MMA, but not touching it, and applying a nociceptive stimulus with electrical current (usually 10 Hz, 1–5 ms duration with 0.1–0.3 mA, equivalent to 30–50 V) causes a rapid and reproducible vasodilation of the dural meningeal blood vessels (Figure 2A). Maximum vasodilation can take up to 30 s, and slowly over several minutes the vessel caliber returns to baseline (Figure 2) (47**–49). It is thought that the vasodilation during neurogenic dural vasodilation is driven by local release of CGRP from activated nociceptive Aδ-fibers that innervate the dural vasculature, at the level of the vessel, by activating CGRP receptors on the smooth muscle of the vessel causing vasodilation (Figure 2B). Interestingly, while intravenous administration of vasodilator agents CGRP, substance P and neurokinin A are also able to evoke vasodilation of the meningeal vessels (46**), only systemic administration of CGRP antagonists, CGRP_8–37 or olcegepant, are able to significantly attenuate the neurogenic dural vasodilation response (Figure 2D), whereas NK₁ antagonists are ineffective (47**,49). This matches the clinical evidence that NK₁ antagonists demonstrate no clinical efficacy in either the acute or preventive treatment of migraine (29,30), while CGRP antagonists are extremely effective in the acute treatment of migraine (50,51**). The neurogenic dural vasodilation assay (or transcranial electrical stimulation assay) has also been able to predict the clinical efficacy of the 5-HT_1B/1D agonists, “triptan,” class of drugs, which must serve as the gold standard for any preclinical assay, as well as dihydroergotamine (47**,52,53), which are thought to inhibit the pre-synaptic release of CGRP from the peripheral afferent trigeminal nerve terminal (Figure 2B and C). There are also other compounds that inhibit neurogenic dural vasodilation and are clinically effective, including µ-opioid receptor antagonists (54), nitric oxide synthase inhibitors (48) as well as the migraine-preventive topiramate (55). This assay has been used to screen novel compounds, which may prove in the future to have clinical efficacy including cannabinoid CB₁ receptor agonists (56), orexinergic molecules (57) and calcium-activated potassium channel modulators (58). It has also helped dissect the varied pharmacology of the trigeminovascular system at the peripheral neuromuscular junction and therefore mechanisms that may underlie migraine, as well as identifying the actions of known and novel therapeutic compounds.

Figure 2.

Overview of craniovascular changes during dural trigeminovascular nociception. Measuring the vascular changes at the level of the dura mater after nociceptive electrical stimulation of the dural vasculature allows us to determine the extent of trigeminovascular activation. (a and b) Electrical stimulation (ES) of the dura mater causes the release of vasoactive neuropeptides, predominantly calcitonin gene-related peptide (CGRP), from peripheral trigeminal nerve endings, resulting in activation of post-junctional CGRP receptors on the vascular smooth muscle and vasodilation. Acute migraine treatments, 5-HT_1B/1D receptor agonists, “triptans” (b and c) (47**), are thought to act by preventing the release of CGRP from pre-junctional trigeminal nerve endings, and CGRP antagonists (49) (b and d) block the action of released CGRP at its receptor, inhibiting vasodilation and the subsequent further activation of the trigeminal afferent projection to the nucleus caudalis.

Laser Doppler flowmetry

Laser Doppler flowmetry is a very similar technique to intravital microscopy, but uses the measurement of changes in meningeal blood flow as an indication of activation of the trigeminovascular system, and it is essentially an indirect measure of vessel caliber. It was a precursor to intravital microscopy, using an open cranial window, with laser Doppler probes placed at the MMA to record changes in flow. Similar methods have used stimulation of the dural vasculature using electrical current (2–20 Hz, 0.5 ms, 10–20 V for 30 s) to cause a reproducible increase in meningeal blood flow (59 –61**). These increases are attenuated by the administration of 5-HT_1B/1D receptor agonists (59), CGRP receptor antagonists (60,61**) and nitric oxide synthase inhibitors (62), similar to neurogenic dural vasodilation. Additionally, these responses were not inhibited by an NK₁ antagonist (63), demonstrating that it is predictive of molecules that do and do not have clinical efficacy.

Pitfalls of measuring changes in the craniovasculature

Detailed methods for each assay and equipment used are outlined in the original articles and should be referred to; however, there are further considerations and cautions that experimenters should be aware of during these procedures. Any in vivo anesthetized procedure requires adequate anesthetic and reliable methods to assess the anesthesia depth and animal physiology; we refer the reader to the literature for detailed accounts (64). Measuring changes in the craniovasculature as a response to trigeminovascular activation can be very much affected by the cardiovasculature of each animal. Changes in blood pressure, either up or down, can alter both blood flow and vessel caliber, with increases in blood pressure often resulting in compensatory vasoconstriction and reduced blood flow, while decreases in blood pressure result in vasodilation and increase in blood flow. Likewise if the temperature of the animal is not regulated properly, the vessel size can change. It is therefore necessary to provide adequate and stable anesthesia throughout to maintain stable physiological parameters over the course of the study and have methods to continuously assess vital physiology, such as blood pressure and ventilation. Further, there has not been a systematic approach to study the effect of different kinds of anesthetics on the cranial vasculature per se, and in general different labs use different anesthetic approaches. In the published literature barbiturates, pentobarbital and thiopental, the hypnotic propofol, and urethane have all been used and demonstrate reliable vasodilation in response to chemical vasodilators and stimulation of peripheral trigeminal afferents, although it may be difficult to compare results with different anesthetics as the independent effects of each is unknown. Surgically the techniques differ in that intravital microscopy uses a closed cranial window whereas laser Doppler measurement has used an open window. A closed window is certainly more physiological; it is less likely to activate and irritate trigeminal afferents as a consequence of removal of the bone and less likely to create bleeding. Indeed anecdotal reports from Dr. Williamson during the development of the intravital assay state that measurement of vessel caliber with an open window was difficult because the blood vessels would remain dilated throughout the study. This might imply the peripheral afferent trigeminal nerve endings are irritated and there is release of neuropeptides beyond what might be induced by the assay itself, and therefore the vessel caliber cannot be adequately controlled. Therefore it is crucial that the skull not be broken during surgery as this is likely to induce excessive vasodilation. Recent improvements to the methods have used a combination of both intravital microscopy and laser Doppler flowmetry measurement, with a closed cranial window. Results show increases in blood flow with transcranial stimulation, but they do not achieve the same level of increase as in the open window (49), although lower current intensity was used. The open-window approach does offer one major advantage over the closed-window method, where substances can be applied locally to the dura mater to determine their effects on blood flow or blood vessel caliber. During trigeminal nerve stimulation, release of vasodilator peptides is locally at the level of the dura mater, and this does not affect the systemic cardiovasculature, therefore studies to understand only the local effects of these vasodilator peptides are helpful and use local dural application. However, the closed-window approach more often needs to use systemic administration of vasodilator peptides, as these molecules have little or no effect when administered locally to the cranium. Systemic application will inevitably affect the whole body, something that is unlikely to occur during their release in migraine. This method can produce profound cardiovascular affects that significantly alter dural blood flow and caliber and confound the observations made on the dural vasculature. More recently, though, intra-carotid injections have been demonstrated to significantly reduce these cardiovascular affects. On the other hand, with local open-window application these molecules have been demonstrated to be potent vasodilators, have no impact of the cardiovasculature, and perhaps more appropriately reflect their local release during trigeminovascular nerve activation and their possible release during migraine.

It is important to conduct the surgery carefully, and to minimize the amount of drilling used. Drilling will inevitably activate the peripheral trigeminal afferents and cause vasodilation in itself, so a rest period of an hour is recommended before experimentation begins. Also, care needs to be taken to prevent bleeding on the skull and over the dura mater. These bleeds can irritate and sensitize nerve endings, resulting in excessive vasodilation and a blood flow increase that does not recover. Bleeding from the skull during the preparation and drying out of the tissue during the course of the experiment can be reduced by application of bone wax to the skull, and hydration of the closed cranial window can be maintained under warm mineral oil. Finally, care needs to be taken with the placement of the bipolar stimulating electrodes. It is very important that once baselines are established that the same level of current be applied to the skull or dura mater each time. Placing the electrodes in an area free from blood and interstitial fluid will reduce changes in electrical current level applied and maintain consistency of response. The electrode should be placed so it is neither over the artery nor depressing the skull, which could impair dural blood flow. To check that a good contact between the skull and the electrode has been made, the cranial window should be stimulated with single pulses (10 V, 5 Hz 1–5 ms) and the delivered current can be calculated using an oscilloscope that measures the voltage drop across a 1 kΩ resistor in parallel, so that a 20 mV deflection represents current delivered to the skull of about 20 mA. If a poor connection between the electrode and skull is observed by high impedance, the electrode should be repositioned on the skull, or further drilling performed if it was considered the skull thickness is the cause of the poor contact.

The future

These assays provide a very good method for understanding the pharmacology of the peripheral junction of the trigeminal nerve and have proved to be excellent predictors of clinical efficacy, particularly for predicting the clinical efficacy of acute therapeutics, with an action thought to be in the trigeminovascular system. Migraine is thought of as a disorder of the brain, and therapeutic molecules that get into the brain are likely to be more clinically effective. This assay may therefore appear to be less relevant than neuronal methods described below. However, the importance of the cranial vasculature and the role of vasodilation and activation of meningeal nociceptors from the periphery in activating the trigeminovascular system are still hotly debated. Improved methods that have combined measurements of the craniovasculature alongside neuronal techniques provide interesting and conflicting results about the importance of the vessels and CGRP (65**,66**). There is also one example of neurogenic dural blood vessel changes in wild-type mice (67**), which opens up the possibility of studies in transgenic mouse migraine models in the future. These techniques are complicated in the smaller animals, and the dural blood vessels much less responsive, but mouse models can still provide a gateway to a better understanding of migraine mechanisms that we do not have with rats. It is certain that these methods will continue in the future and they serve as an excellent assay to screen novel targets of the trigeminovascular system, and particularly molecules that do not to cross the blood-brain barrier (BBB). They will continue to help us explore the importance of the vasculature and brain in the pathophysiology of migraine.

Pearls of measuring changes in the trigeminal nucleus caudalis and brain

It seems clear now that migraine is primarily a disorder of the brain. This includes not only the maintenance of migraine pain but also, more importantly, the initiation of a migraine attack. Experimental evidence from imaging studies in humans and experimental animals, as well as clinical symptoms such as the premonitory symptoms, which may appear 24–48 hours prior to migraine pain and therefore long before a migraine aura starts – in the case of migraine with aura – seem to confirm the importance of central processes in migraine. The TCC in the brain is a key relay center of the trigeminovascular system involved in the transmission of nociceptive information from the head and cranial vasculature to the brainstem and higher pain processing structures. Therefore measuring changes of neuronal activity in this structure in response to dural nociceptive stimulation is an attractive approach to study pathophysiological mechanisms and potential therapeutic agents in migraine. Neuronal activity in the TCC can be measured with indirect measurements, such as the analysis of Fos immunoreactivity, or with direct electrophysiological recordings placing an electrode directly in the relevant nucleus in the brainstem and measuring neuronal activity.

Fos immunoreactivity

Fos protein is the product of the c-fos gene, a nuclear protein that regulates the transcription of other target genes. Fos was first isolated directly from human tumors and therefore is categorized as an oncogene (68**,69). c-Fos is classified as an immediate-early gene as it is rapidly activated by a variety of stimuli including neuronal depolarization and neurotransmitters and transiently expressed in a variety of neurons (69,70). Its activation occurs within five mins and continues for 15–20 mins, and the synthesis of Fos protein follows mRNA expression and is detectable within 30 mins and is continuously expressed as long as the stimulus remains. Depolarization activates membrane receptors that initiate a second messenger cascade that induces long-term neuronal changes. Activation of the secondary messenger system results in the up-regulation of the immediate-early genes, including c-fos, which produces transcription factors including the c-fos protein, Fos. Fos is then transported to the nucleus, where it forms a heterodimeric complex with c-jun transcription factor, which in turn binds to the AP-1 DNA site and modulates the function of the late response genes (Figure 3). Fos protein has a half-life of two hours when the stimulus ceases (71), but the time of expression of Fos depends on the stimulus and species investigated (72,73). Therefore it is important prior to the study to understand the process and timing of when it will be optimal to detect a Fos signal in the study. The Fos protein is normally visualized using immunocytochemical techniques. Fos protein is a normal protein that can be observed at low levels in many cell types, including neurons and glia, and many studies show that Fos immunoreactivity can be used as a marker of nociception in the nociceptive-specific laminae of the spinal cord. Intuitively higher counts of Fos immunoreactive cells is indicative of higher neuronal activation, although a lack of Fos does not necessarily mean lack of neuronal activation, as in some cases other secondary messenger pathways are relevant. For example, staining for cyclic adenosine monophosphate (AMP)-responsive element binding protein (CREB) is often considered a more reliable marker in the trigeminal ganglion than Fos. Electrical, mechanical and chemical stimulation of dural structures result in Fos immunoreactivity in the TCC nociceptive-specific laminae (72,74**,75**,76**, 77**–81). This immunoreactivity is significantly reduced by migraine treatments such as triptans and dihydroergotamine (77**,78,82,83), and the lack of an inhibitory effect with neurokinin 1 receptor antagonists and extravasation inhibitors (84,85) illustrates the clinically predictive nature of the assay. A unique property of Fos is its ability to respond to polysynaptic activation, which allows mapping of functional pathways. Using this property, it has been possible to map the neuronal activation from dural stimulation to higher structures involved in the ascending and descending control of migraine pain, including the superior salivatory nucleus (86**), periaqueductal gray (86**,87**) and hypothalamus (88**,89**).

Figure 3.

The induction of c-fos. The immediate-early gene c-fos is rapidly and transiently expressed in neurons in response to a variety of stimuli. Depolarization activates membrane receptors that initiate a second messenger cascade that induces long-term neuronal changes. Activation of the second messenger system results in the up-regulation of the immediate-early genes, including c-fos, which produces transcription factors including the Fos protein. Fos is then transported to the nucleus, where it forms a heterodimeric complex with Jun, which in turn binds to the AP-1 DNA site and modulates the function of the late response genes (LRG).

Electrophysiological recordings within the TCC

Electrophysiological techniques provide a direct measure of neuronal activation and are a real-time assessment of these changes. These studies use lower stimulation thresholds to observe the responses of a depolarized nociceptive nerve fiber compared to observing Fos immunoreactivity, and therefore perhaps provide a more physiological approach. The surgery necessary for electrophysiological recording of neuronal activity requires exposure of brainstem areas, including the TCC, with careful dissection. After removal of the dura mater, a thin tungsten or stainless steel electrode with an impedance of 0.5–1.0 MΩ is placed in the TCC. One of the advantages of electrophysiology is that during the experiment one can assess the general placement of the electrode in the anatomical area of the trigeminal nucleus by assessing the cutaneous and dural receptive field response to brushing and pinching, due to the well-described somatotopical organization of the trigeminal nerve (Figure 4). In general the rostral-most portion, nearest the pons, of the trigeminal nucleus is more responsive to innocuous inputs than noxious, while the caudal portion is more nociceptive specific (see Figure 4A). Electrical stimulation of the dura mater provides a nociceptive-specific input to the TCC, and this explains why electrophysiological recording in the TCC take places predominantly in caudal regions of the trigeminal nucleus, as well as the C₁ and C₂ regions of the spinal cord. While the trigeminal nucleus does follow the curvature of the outer surface of the medulla, as indicated in Figure 4A, in general the dorsal-most portion of the trigeminal nucleus reflects mainly innervations from the V3 (mandibular) region of the trigeminal nerve. Moving ventrally through the nucleus is the V2 (maxillary) innervation of the trigeminal nerve, which reflects mainly the middle portion of the face including the nose and just below the eye. Finally, the V1 (ophthalmic) division of the trigeminal nerve, which predominantly innervates the area above the eye, including the intracranial dura mater, is found in the most ventral region of the dorsal horn (Figure 4A and B). It is important to note that these boundaries are not clearly separated, as there is overlap between the specific divisions of the trigeminal nerve and the regions of the face, head and intracranial structures that they innervate. The recorded signal is a result of neuronal activity of a cluster of neurons. The size of the clusters of neurons very much depends on the size of the tip and the impedance of the recording electrode. A smaller tip very often results in higher impedance of the electrode and translates into a smaller cluster of cells involved in the recorded signal. Most frequently wide-dynamic range (WDR) neurons are analyzed that respond to noxious pinch and innocuous brush in the receptive fields of facial branches of the trigeminal nerve, as well as a nociceptive signal from the dura mater. The experimental setup allows recording of neuronal background activity and thereby also offers the possibility to analyze neuronal sensitization or the expansion of a receptive field throughout the course of an experiment. This subject is covered in more detail in “Pearls and pitfalls of experimental in vivo studies in headache: Peripheral and central sensitization.” However, for the analysis of trigeminal activation with respect to headache research, the recording of stimulus-evoked activity of Aδ- and C-fibers is more specific as it more closely resembles the biochemical changes that occur during migraine, and the pharmacology of therapeutics have been shown to reliably affect these responses. Therefore, dural electrical stimulation offers some advantages over assays that record background activity and observe changes in response to chemical manipulation of the dura mater and the consequent sensitization of the trigeminovascular system, although these methods still have an important place in migraine research. In this model a cranial window is drilled and a bipolar stimulating electrode is placed proximate to the MMA to activate the trigeminal nerve fibers that innervate this vascular body during electrical stimulation. Usually the electrical stimulus consists of a series of square wave pulses (for example 20 pulses with a frequency of 0.5 Hz) each with up to 0.5 ms duration and a voltage that usually ranges between 5 and 15 V. Post-stimulus histograms (PSTH) are then established from the recorded signal in the TCC. Depending on the delay between the electrical stimulation of the dura mater and the response elicited in the TCC, cells are differentiated into neurons receiving either Aδ- or C-fiber inputs (Figure 4C) (90**). The advantage of this method lies in the fact that it directly analyzes central neuronal activity with a relatively low variability of the recorded signal, resulting in a lower number of experimental animals necessary. Furthermore, recordings allow an objective and observer-independent analysis of the data. This highly reliable in vivo model has been used for testing a broad range of compounds potentially effective in migraine treatment, such as the “triptans” and dihydroergotamine (91**,92**,93**) and CGRP receptor antagonists (94). It has also predicted the failure of NK₁ receptor antagonists (84) and extravasation inhibitors (95**,96). Dural electrical stimulation and electrophysiological recording is now used as a common assay to screen anti-migraine efficacy of novel molecules and to help understand the pathophysiology of migraine and other primary headache disorders.

Figure 4.

Anatomical organization of the trigeminal nerve. The trigeminal nerve and its projections to the head and face and trigeminal nucleus have a well-characterized somatotopical organization. (a) The rostral-most portion of the trigeminal nucleus is more responsive to innocuous inputs and moving caudally responses become more nociceptive specific. (a and b) While accepting the curvature of the trigeminal nucleus in general, the dorsal-most portion of the trigeminal nucleus receives innervations from mainly the V3 (mandibular) region of the trigeminal nerve, reflecting mainly the lower portion of the face. The V2 (maxillary) region of the trigeminal nerve innervates mainly the middle portion of the face, including the nose and just below the eye and projects ventral to the V3 region of the trigeminal nucleus. Finally, the V1 (ophthalmic) division of the trigeminal nerve projects mainly to the ventral portion of the dorsal horn, and innervates the area above the eye, including the intracranial dura mater. Nociceptive inputs coming from the dura mater of the ophthalmic division of the trigeminal nerve will most likely be found in the caudal portion of the trigeminal nucleus, in the ventral region of the dorsal horn. However, these boundaries are not clearly separated, and there is overlap between the specific divisions of the trigeminal nerve and the regions of the face, head and intracranial structures that they innervate. (c) An example of dural electrical nociceptive neuronal response in the caudal trigeminal nucleus with both an Aδ-fiber and C-fiber response.

Pitfalls of measuring changes in the trigeminal nucleus caudalis and brain

Fos immunoreactivity

Measurement of Fos immunoreactivity is the simplest method of quantifying neuronal activity but has substantial shortcomings. First of all, compared to electrophysiological recordings, it is only an indirect measurement as Fos expression is a result of neuronal activity. Fos expression is considered to be highly unspecific since many factors can induce its expression and the minimal nociceptive signal necessary to produce a Fos immunoreactive signal is undefined (97), and responses are not in real time. Therefore meticulous control of the depth of anesthesia is required to avoid Fos expression as a result of surgery-induced pain. We further recommend the use of locally applied lidocaine before making skin incisions as these can already induce a substantial amount of Fos expression in the areas of interest. One of the major limitations of the Fos technique is the assessment of a Fos cell. There are established published criteria for assessing Fos cells; essentially cells are considered positive if they are stained as dark brown or black (when using the DAB/Nickel method), round or ovoid structures with variable degree of staining intensity (87 **,98). Furthermore the dark stained nuclei of Fos-positive cells have to be clearly distinguishable from non-specific background staining in a magnification range of ×4 to ×20, using a light microscope, to be considered as positive. However, this judgment is extremely subjective despite having these standard guidelines and therefore results vary significantly between animals, and counting suffers from a significant observer-dependent bias. To ensure a reliable interpretation, the counters should be blinded to the treatment groups and at least two independent counters should analyze the tissue sections. In addition, the high variation in cell counting frequently requires a high number of experimental animals to ensure reliable results and interpretations. Finally, due to these methodological weaknesses the results do not allow quantification in exact numbers that correlate in a linear pattern with increasing neuronal activity. They allow only an ordinal interpretation. Therefore only non-parametric statistics should be used for data analysis.

Electrophysiological recording

Electrophysiological techniques have many benefits over using the Fos method as a marker of neuronal activation. It is a direct measurement of neuronal changes, and the exact dynamics of the stimulus are known that create a neuronal nociceptive signal. In general the signal necessary to evoke a nociceptive response is considerably less than required in Fos studies. It is also a real-time assessment of neuronal activity changes. Despite being one of the most reliable and predictive models for headache research, electrophysiological recordings have their pitfalls. Electrophysiological recordings require expensive equipment and a substantial amount of training until useful data acquisition can be achieved. One of the main practical issues that affect the outcome is noise from surrounding equipment. Therefore meticulous care should be exerted in establishing good insulation throughout the whole experimental setup as well as removal of unnecessary electrical equipment. Moreover, the minimum level of surgery to provide adequate exposure of targets sites should be performed to prevent extensive skin and muscle lesions as well as bleeding, which may sensitize neurons and as a consequence lead to an increase in background activity that might significantly affect stimulus-evoked recordings. The animal should be given time to rest after surgery before experimentation begins, usually an hour. This allows time for recovery of cells after surgery and for physiological parameters to stabilize. As always, maintaining stable anesthesia and having a measure of physiological output will aid the success of experiments, as one can respond to subtle changes quickly. Movement of the experimental setup or animal should be avoided since minuscule movements at the spinal cord can change the location of the recording electrode, leading to a different response or even complete loss of the neuronal activity of the recorded cluster of cells. Care should also be taken to avoid fluid accumulation in the cranial window where the stimulating electrode is placed. Fluid accumulation leads to a spread of the applied current leading to a loss of signal and/or stimulation of the surrounding muscles affecting the recorded signal or even leading to muscular movements that might alter the position of the recording electrode. To prevent fluid accumulation or drying out of the dura mater, a few drops of mineral oil should be applied on the cranial window. If muscular contraction cannot be avoided, the use of a neuromuscular blocker such as pancuronium bromide can be considered; this will also prevent movement artifacts at the level of the recording electrode. However, in this case even more precautions should be taken to ensure a sufficient and constant depth of anesthesia. As mentioned above, it is of crucial importance that the vital parameters (blood pressure, temperature, etc.) be kept at constant physiological levels as, for example, a relatively small reduction of blood pressure can induce a significant reduction of neuronal activity. Finally, it is recommended that several methods be used in parallel to ensure reliable and interpretable results. For example, if only receptive field analysis is performed the results are less reliable since even when using von Frey hairs as a standardized stimulus, it is very difficult to always apply the tactile stimulus at a comparable pressure and duration. Moreover, the simple approximation with the hand and the brush or von Frey tool to the recording electrode usually causes noise that might significantly affect the small effects that this stimulation induces per se. Therefore, measurement of (electrical) stimulus-evoked activity in conjunction with background activity in the TCC is probably the most accurate and reliable method of trigeminal activation for headache research.

Measuring neuropeptide changes

Neuropeptides, especially CGRP, are of crucial importance in the pathophysiology of migraine. The discovery of elevated plasma levels of only CGRP during severe migraine by Goadsby et al. (37**,99), which return to normal levels after sumatriptan administration, has greatly enhanced the understanding of migraine. Interestingly, experimental animal models that used dural electrical stimulation demonstrate clear correlation with this, with release of CGRP, alongside vasoactive intestinal peptide, important in TACs (35**), while trigeminal ganglion stimulation in humans and cats caused CGRP release but also the NK₁ agonist, substance P (22**). Following this, studies have demonstrated that infusion of human CGRP triggers migraine attacks in migraineurs in the same pattern and time frame as nitric oxide donors do, which is the induction of an immediate headache followed by a delayed migraine-like headache (100**). Despite its strong vasodilatory effects, probably central neuronal actions of CGRP are of crucial importance in the pathophysiology of migraine. In experimental in vivo models, Storer et al. demonstrated that microiontophoresed CGRP onto TCC neurons increase activity (94). The ongoing pursuit over the last 20 years to understand the exact role of CGRP in the pathophysiology of migraine and its effects on the trigeminovascular system culminated in the development of CGRP receptor antagonists, and the proof of concept of their clinical efficacy was proven in a clinical trial using the intravenously administered CGRP receptor antagonist BIBN4096BS (51**). Therefore, since CGRP is released into the extracerebral circulation upon trigeminal activation, measuring CGRP levels in plasma is an obvious approach for in vivo models in headache research.

Pitfalls of measuring neuropeptide changes

Despite the positive clinical and experimental studies, measurement of CGRP has been proven to be more difficult and less reliable for the prediction of potential migraine treatments than initially thought and is covered in greater detail in “Pearls and pitfalls of neuropeptide studies of headache.” The release of CGRP during spontaneous migraine attacks has been the subject of controversial discussions. However, discrepancies in the published data may be related to difficulties in probe handling and analysis (101**). CGRP has a very short half-life of a few minutes. Therefore, immediately after blood sampling the plasma needs to be cooled and ethylenediaminetetraacetic acid (EDTA), as well as protease and peptidase inhibitors (for example, aprotinin), have to be added to prevent degradation of the peptide. Blood samples then need to be stored at −80°C until further processing. Another difficulty lies in the fact that the increase of CGRP concentration in plasma is relatively small and suffers from significant inter-individual variations, requiring a high sample number and a sensitive assay for probe analysis. This problem is further aggravated if blood samples are taken from distal sites such as the radial or femoral artery as the small amounts of released CGRP get further diluted. Furthermore, it is not known at which point during a spontaneous migraine attack CGRP concentrations increase in the extracerebral circulation and how long the increase lasts, hampering an adequate sample collection. This may explain the negative results of Tvedskov et al. (41**), analyzing CGRP concentrations in spontaneous migraine attacks, since in this study blood samples were drawn many hours after the initiation of the painful phase of the migraine attack (41**).

In animal models, CGRP measurement has further difficulties. Electrical stimulation of the trigeminal ganglion does induce CGRP release but applying such a strong stimulus into the trigeminal ganglion probably induces mechanisms that are not necessarily important in spontaneous migraine attacks. This is demonstrated by the fact that substance P and neurokinin A are also released, but not during migraine (22**,37**). Hence the dural electrical stimulation method does seem optimal, as it demonstrates clear translational response and predicts the clinical failure of NK₁ antagonists and extravasation inhibitors (102,103). In general, stimulus-induced CGRP release is short lasting, peaking within a few minutes after stimulation, making sample collection even more difficult. Furthermore, in rodent plasma and cerebrospinal fluid (CSF) the CGRP concentration ranges are close to the sensitivity levels of commercially available enzyme-linked immunosorbent assay (ELISA) kits. Due to the required volume for analysis, CSF samples and plasma samples in mice need to be diluted to achieve the necessary amount. However, the dilution makes analytic results less exact, and if they fall below the detection limit of the kit, even impossible to assess. Commercially available radioimmunoassay (RIA) kits have a slightly better sensitivity but include all the pitfalls as well as regulatory and safety issues that come along with handling radioactive substances.

Finally, from a pathophysiological standpoint it has to be considered that evidence suggests that CGRP does not cross the BBB as its infusion induces a dilatation of the MMA but not of the MCA (104). Therefore, if the BBB remains intact, which might be questioned in spontaneous migraine attacks, can it be evaluated in the experimental setting? CGRP measured in the extracerebral circulation must originate from the peripheral parts of the trigeminal system. However, since CGRP does not activate peripheral meningeal afferents (65**) and local application of a CGRP receptor antagonist onto the dura mater does not affect spinal trigeminal activity (105), its peripheral site of action is probably not important for the generation of migraine. Therefore, interpretations on the basis of CGRP release in the extracerebral circulation have to be taken with caution.

The future

The measurement of functional changes in the TCC will continue to be an outstanding tool for further understanding of the pathophysiology of migraine and for the screening of potential anti-migraine drugs. In that sense direct electrophysiological recordings are clearly superior to the measurement of Fos immunoreactivity. Electrophysiological studies are and will continue to gain importance as migraine is more and more seen as a disorder of central origin rather than being the result of peripheral or vascular events. The dissection of peripheral versus central effects of tested compounds recordings within the TCC will complement intravital microscopy, which clearly addresses peripheral parts of the trigeminovascular system. Analysis of neuropeptides will probably focus mainly on CGRP, as the other neuropeptides are of only secondary importance.

Footnotes

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Conflict of interest

None declared.

References

Lipton RB, Stewart WF, Diamond S, et al. Prevalence and burden of migraine in the United States: data from the American Migraine Study II. Headache 2001; 41: 646–657.

Stewart

Ricci

Chee

. Lost productive time and cost due to common pain conditions in the US workforce. JAMA 2003; 290: 2443–2454.

Lipton

Stewart

Diamond

. Prevalence and burden of migraine in the United States: Data from the American Migraine Study II. Headache 2001; 41: 646–657.

Andlin-Sobocki

Jonsson

Wittchen

. Cost of disorders of the brain in Europe. Eur J Neurol 2005; 12(Suppl 1): 1–27.

Shapiro

Goadsby

. The long drought: The dearth of public funding for headache research. Cephalalgia 2007; 27: 991–994.

Olesen

. Response to Dr Balak and Dr Elmaci. Eur J Neurol 2007; 14: e16–e16.

McNaughton

Feindel

Innervation of intracranial structures: A reappraisal. In: Rose

(ed.) Physiological aspects of clinical neurology, Oxford: Blackwell Scientific, 1977, pp. 279–293.

Penfield

McNaughton

. Dural headache and innervation of the dura mater. Arch Neurol Psychiatry 1940; 44: 43–75.**.

Ray

Wolff

. Experimental studies on headache. Pain-sensitive structures of the head and their significance in headache. Arch Surg 1940; 41: 813–856.**. **Refs 8 and 9 are seminal papers that determined the stimulation of dural structures in humans is painful and results in pain referred to the head..

10.

Moskowitz

. The neurobiology of vascular head pain. Ann Neurol 1984; 16: 157–168.

11.

Akerman

Holland

Goadsby

. Diencephalic and brainstem mechanisms in migraine. Nat Rev Neurosci 2011; 12: 570–584.

12.

Goadsby

Lambert

Lance

. Stimulation of the trigeminal ganglion increases flow in the extracerebral but not the cerebral circulation of the monkey. Brain Res 1986; 381: 63–67.

13.

Goadsby

Lambert

Lance

. The peripheral pathway for extracranial vasodilatation in the cat. J Auton Nerv Syst 1984; 10: 145–155.

14.

Goadsby

. Effect of stimulation of facial nerve on regional cerebral blood flow and glucose utilization in cats. Am J Physiol 1989; 257(Pt 2): R517–R521.

15.

Edvinsson

Brodin

Jansen

. Neurokinin A in cerebral vessels: Characterization, localization and effects in vitro. Regul Pept 1988; 20: 181–197.

16.

Uddman

Edvinsson

Ekman

. Innervation of the feline cerebral vasculature by nerve fibers containing calcitonin gene-related peptide: Trigeminal origin and co-existence with substance P. Neurosci Lett 1985; 62: 131–136.

17.

Uddman

Goadsby

Jansen

. PACAP, a VIP-like peptide: Immunohistochemical localization and effect upon cat pial arteries and cerebral blood flow. J Cereb Blood Flow Metab 1993; 13: 291–297.

18.

Uddman

Edvinsson

. Neuropeptides in the cerebral circulation. Cerebrovasc Brain Metab Rev 1989; 1: 230–252.

19.

Goadsby

Duckworth

. Effect of stimulation of trigeminal ganglion on regional cerebral blood flow in cats. Am J Physiol 1987; 253(Pt 2): R270–R274.

20.

Tran-Dinh

Thurel

Cunin

. Cerebral vasodilation after the thermocoagulation of the trigeminal ganglion in humans. Neurosurgery 1992; 31: 658–662.

21.

Drummond

Gonski

Lance

. Facial flushing after thermocoagulation of the Gasserian ganglion. J Neurol Neurosurg Psychiatry 1983; 46: 611–616.

22.

Goadsby

Edvinsson

Ekman

. Release of vasoactive peptides in the extracerebral circulation of humans and the cat during activation of the trigeminovascular system. Ann Neurol 1988; 23: 193–196. **First demonstration of CGRP and substance P release in human and cats during trigeminovascular activation. .

23.

Markowitz

Saito

Moskowitz

. Neurogenically mediated leakage of plasma protein occurs from blood vessels in dura mater but not brain. J Neurosci 1987; 7: 4129–4136.**.

24.

Moskowitz

. Basic mechanisms in vascular headache. Neurol Clin 1990; 8: 801–815.

25.

Buzzi

Moskowitz

. The antimigraine drug, sumatriptan (GR43175), selectively blocks neurogenic plasma extravasation from blood vessels in dura mater. Br J Pharmacol 1990; 99: 202–206.

26.

Markowitz

Saito

Moskowitz

. Neurogenically mediated plasma extravasation in dura mater: Effect of ergot alkaloids. A possible mechanism of action in vascular headache. Cephalalgia 1988; 8: 83–91.

27.

Roon

Olesen

Diener

. No acute antimigraine efficacy of CP-122,288, a highly potent inhibitor of neurogenic inflammation: Results of two randomized, double-blind, placebo-controlled clinical trials. Ann Neurol 2000; 47: 238–241.

28.

Earl

McDonald

Lowry

. Efficacy and tolerability of the neurogenic inflammation inhibitor, 4991W93, in the acute treatment of migraine. Cephalalgia 1999; 19: 357–357.

29.

Goldstein

Offen

Klein

. Lanepitant, an NK-1 antagonist, in migraine prevention. Cephalalgia 2001; 21: 102–106.

30.

Goldstein

Wang

Saper

. Ineffectiveness of neurokinin-1 antagonist in acute migraine: A crossover study. Cephalalgia 1997; 17: 785–790.

31.

Peroutka

. Neurogenic inflammation and migraine: Implications for the therapeutics. Mol Interv 2005; 5: 304–311.

32.

Uhl

Walther

Nishimori

. Jun B, c-jun, jun D and c-fos mRNAs in nucleus caudalis neurons: Rapid selective enhancement by afferent stimulation. Brain Res Mol Brain Res 1991; 11: 133–141. **Demonstration of neuronal activation in the trigeminal nucleus caudalis that helped describe both peripheral and central projections from the trigeminal ganglion..

33.

Feindel

Penfield

McNaughton

. The tentorial nerves and localization of intracranial pain in man. Neurology 1960; 10: 555–563.

34.

Goadsby

Knight

Hoskin

. Stimulation of an intracranial trigeminally-innervated structure selectively increases cerebral blood flow. Brain Res 1997; 751: 247–252.

35.

Zagami

Goadsby

Edvinsson

. Stimulation of the superior sagittal sinus in the cat causes release of vasoactive peptides. Neuropeptides 1990; 16: 69–75.**.

36.

Goadsby

MacDonald

. Extracranial vasodilation mediated by vasoactive intestinal polypeptide (VIP). Brain Res 1985; 329: 285–288.

37.

Goadsby

Edvinsson

Ekman

. Vasoactive peptide release in the extracerebral circulation of humans during migraine headache. Ann Neurol 1990; 28: 183–187.**.

38.

Goadsby

Edvinsson

. Human in vivo evidence for trigeminovascular activation in cluster headache. Neuropeptide changes and effects of acute attacks therapies. Brain 1994; 117(Pt 3): 427–434.**.

39.

Goadsby

Edvinsson

. Neuropeptide changes in a case of chronic paroxysmal hemicrania – evidence for trigemino-parasympathetic activation. Cephalalgia 1996; 16: 448–450.**. **Refs 37–39 demonstrated that CGRP levels are increased in the extracerebral circulation during migraine, cluster headache and paroxysmal hemicrania, and VIP in TACs, implying they are important components of primary headache pathophysiology..

40.

Gallai

Sarchielli

Floridi

. Vasoactive peptide levels in the plasma of young migraine patients with and without aura assessed both interictally and ictally. Cephalalgia 1995; 15: 384–390.

41.

Tvedskov

Lipka

Ashina

. No increase of calcitonin gene-related peptide in jugular blood during migraine. Ann Neurol 2005; 58: 561–568. **This paper encouraged debate on the role of CGRP in migraine as no increase in CGRP levels was demonstrated..

42.

Goadsby

Charbit

Andreou

. Neurobiology of migraine. Neuroscience 2009; 161: 327–341.

43.

Bartsch

Goadsby

. Anatomy and physiology of pain referral in primary and cervicogenic headache disorders. Headache Currents 2005; 2: 42–48.

44.

McNaughton

The innervation of the intracranial blood vessels and dural sinuses. In: Cobbs

Frantz

Penfield

(eds). The circulation of the brain and spinal cord, New York: Hafner Publishing Co. Inc., 1966, pp. 178–200.

45.

Goadsby

Lipton

Ferrari

. Migraine— current understanding and treatment. N Engl J Med 2002; 346: 257–270.

46.

Williamson

Hargreaves

Hill

. Intravital microscope studies on the effects of neurokinin agonists and calcitonin gene-related peptide on dural vessel diameter in the anaesthetized rat. Cephalalgia 1997; 17: 518–524.**.

47.

Williamson

Hargreaves

Hill

. Sumatriptan inhibits neurogenic vasodilation of dural blood vessels in the anaesthetized rat – intravital microscope studies. Cephalalgia 1997; 17: 525–531.** **Refs 46 and 47 demonstrated the first use of the intravital microscopy assay, and that neurogenic dural vasodilation is predominantly via CGRP release, and triptans block the release of CGRP from prejunctional nerve endings...

48.

Akerman

Williamson

Kaube

. Nitric oxide synthase inhibitors can antagonize neurogenic and calcitonin gene-related peptide induced dilation of dural meningeal vessels. Br J Pharmacol 2002; 137: 62–68.

49.

Petersen

Birk

Doods

. Inhibitory effect of BIBN4096BS on cephalic vasodilatation induced by CGRP or transcranial electrical stimulation in the rat. Br J Pharmacol 2004; 143: 697–704.

50.

Mannix

Fan

. Randomized controlled trial of an oral CGRP receptor antagonist, MK-0974, in acute treatment of migraine. Neurology 2008; 70: 1304–1312.

51.

Olesen

Diener

Husstedt

. Calcitonin gene-related peptide receptor antagonist BIBN 4096 BS for the acute treatment of migraine. N Engl J Med 2004; 350: 1104–1110. **This proof of concept clinical study demonstrated that CGRP receptor antagonists were efficacious in the acute treatment of migraine without cardiovascular implications, and may represent a new class of compound for migraineurs..

52.

Williamson

Hill

Shepheard

. The anti-migraine 5-HT(1B/1D) agonist rizatriptan inhibits neurogenic dural vasodilation in anaesthetized guinea-pigs. Br J Pharmacol 2001; 133: 1029–1034.

53.

Williamson

Hargreaves

. Neurogenic inflammation in the context of migraine. Microsc Res Tech 2001; 53: 167–178.

54.

Williamson

Shepheard

Cook

. Role of opioid receptors in neurogenic dural vasodilation and sensitisation of trigeminal neurones in anaesthetised rats. Br J Pharmacol 2001; 133: 807–814.

55.

Akerman

Goadsby

. Topiramate inhibits trigeminovascular activation: An intravital microscopy study. Br J Pharmacol 2005; 146: 7–14.

56.

Akerman

Kaube

Goadsby

. Anandamide is able to inhibit trigeminal neurons using an in vivo model of trigeminovascular-mediated nociception. J Pharmacol Exp Ther 2004; 309: 56–63.

57.

Holland

Akerman

Goadsby

. Orexin 1 receptor activation attenuates neurogenic dural vasodilation in an animal model of trigeminovascular nociception. J Pharmacol Exp Ther 2005; 315: 1380–1385.

58.

Akerman

Holland

Lasalandra

. Inhibition of trigeminovascular dural nociceptive afferents by Ca(2+)-activated K(+) (MaxiK/BK(Ca)) channel opening. Pain 2010; 151: 128–136.

59.

Messlinger

Hotta

Pawlak

. Effects of the 5-HT1 receptor agonists, sumatriptan and CP 93,129, on dural arterial flow in the rat. Eur J Pharmacol 1997; 332: 173–181.

60.

Troltzsch

Denekas

Messlinger

. The calcitonin gene-related peptide (CGRP) receptor antagonist BIBN4096BS reduces neurogenic increases in dural blood flow. Eur J Pharmacol 2007; 562: 103–110.

61.

Kurosawa

Messlinger

Pawlak

. Increase of meningeal blood flow after electrical stimulation of rat dura mater encephali: Mediation by calcitonin gene-related peptide. Br J Pharmacol 1995; 114: 1397–1402. **Early demonstration of meningeal blood flow is increased with trigeminovascular activation..

62.

Messlinger

Suzuki

Pawlak

. Involvement of nitric oxide in the modulation of dural arterial blood flow in the rat. Br J Pharmacol 2000; 129: 1397–1404.

63.

Carmody

Pawlak

Messlinger

. Lack of a role for substance P in the control of dural arterial flow. Exp Brain Res 1996; 111: 424–428.

64.

Flecknell

. Laboratory animal anaesthesia: A practical introduction for research workers and technicians, 2nd edn. London, UK; San Diego, CA: Academic Press, 1996.

65.

Levy

Burstein

Strassman

. Calcitonin gene-related peptide does not excite or sensitize meningeal nociceptors: Implications for the pathophysiology of migraine. Ann Neurol 2005; 58: 698–705.**.

66.

Cumberbatch

Williamson

Mason

. Dural vasodilation causes a sensitization of rat caudal trigeminal neurones in vivo that is blocked by a 5-HT1B/1D agonist. Br J Pharmacol 1999; 126: 1478–1486.** **Refs 65 and 66 demonstrate the debate surrounding the possible role of CGRP in sensitising trigeminovascular neurons..

67.

Gupta

Akerman

van den Maagdenberg

. Intravital microscopy on a closed cranial window in mice: A model to study trigeminovascular mechanisms involved in migraine. Cephalalgia 2006; 26: 1294–1303.**.

68.

Morgan

Curran

. Stimulus-transcription coupling in the nervous system: Involvement of the inducible proto-oncogenes fos and jun. Annu Rev Neurosci 1991; 14: 421–451.** **Refs 67 and 68 provide an early description of the use of the immediate-early gene Fos as a marker on neuronal activation via secondary messenger activation..

69.

Morgan

Curran

. Stimulus-transcription coupling in neurons: Role of cellular immediate-early genes. Trends Neurosci 1989; 12: 459–462.

70.

Zimmermann

. Immediate-early genes in the nervous system – molecular steps in hyperalgesia and chronic pain?, Amsterdam: Elsevier Science Publishers, 1993.

71.

Svendsen

Lykkegaard

. Neuronal c-Fos immunoreactivity as a quantitative measure of stress or pain. Acta Agric Scand, Sect A Animal Sci 2001(Suppl 30): 131–134.

72.

Hoskin

Goadsby

. Exposure and isolation of the superior sagittal sinus elicits Fos in the trigeminal nucleus caudalis and dorsal horn of the cervical spinal cord: How long should you wait? Brain Res 1999; 824: 133–135.

73.

Le Doare

Akerman

Holland

. Occipital afferent activation of second order neurons in the trigeminocervical complex in rat. Neurosci Lett 2006; 403: 73–77.

74.

Goadsby

Zagami

. Stimulation of the superior sagittal sinus increases metabolic activity and blood flow in certain regions of the brainstem and upper cervical spinal cord of the cat. Brain 1991; 114: 1001–1011.**.

75.

Kaube

Keay

Hoskin

. Expression of c-Fos-like immunoreactivity in the caudal medulla and upper cervical spinal cord following stimulation of the superior sagittal sinus in the cat. Brain Res 1993; 629: 95–102.**.

76.

Kaube

Hoskin

Goadsby

. Activation of the trigeminovascular system by mechanical distension of the superior sagittal sinus in the cat [see comments]. Cephalalgia 1992; 12: 133–136.** **Refs 74–76 were the first demonstration that stimulation of dural structures results in metabolic changes and neuronal activation in the trigeminal nucleus caudalis..

77.

Hoskin

Kaube

Goadsby

. Sumatriptan can inhibit trigeminal afferents by an exclusively neural mechanism. Brain 1996; 119(Pt 5): 1419–1428. **The first clear demonstration that triptans act on neuronal pathways, not just at the level of the peripheral vasculature..

78.

Hoskin

Kaube

Goadsby

. Central activation of the trigeminovascular pathway in the cat is inhibited by dihydroergotamine. A c-Fos and electrophysiological study. Brain 1996; 119(Pt 1): 249–256.

79.

Hoskin

Zagami

Goadsby

. Stimulation of the middle meningeal artery leads to Fos expression in the trigeminocervical nucleus: A comparative study of monkey and cat. J Anat 1999; 194(Pt 4): 579–588.

80.

Strassman

Vos

Mineta

. Fos-like immunoreactivity in the superficial medullary dorsal horn induced by noxious and innocuous thermal stimulation of facial skin in the rat. J Neurophysiol 1993; 70: 1811–1821.

81.

Goadsby

Hoskin

. The distribution of trigeminovascular afferents in the nonhuman primate brain Macaca nemestrina: A c-fos immunocytochemical study. J Anat 1997; 190(Pt 3): 367–375.

82.

Shepheard

Williamson

Williams

. Comparison of the effects of sumatriptan and the NK1 antagonist CP-99,994 on plasma extravasation in dura mater and c-fos mRNA expression in trigeminal nucleus caudalis of rats. Neuropharmacology 1995; 34: 255–261.

83.

Hoskin

Goadsby

. Comparison of more and less lipophilic serotonin (5HT1B/1D) agonists in a model of trigeminovascular nociception in cat. Exp Neurol 1998; 150: 45–51.

84.

Goadsby

Hoskin

Knight

. Substance P blockade with the potent and centrally acting antagonist GR205171 does not effect central trigeminal activity with superior sagittal sinus stimulation. Neuroscience 1998; 86: 337–343.

85.

Goadsby

Hoskin

. Differential effects of low dose CP122,288 and eletriptan on Fos expression due to stimulation of the superior sagittal sinus in cat. Pain 1999; 82: 15–22.

86.

Knight

Classey

Lasalandra

. Patterns of fos expression in the rostral medulla and caudal pons evoked by noxious craniovascular stimulation and periaqueductal gray stimulation in the cat. Brain Res 2005; 1045: 1–11.**.

87.

Hoskin

Bulmer

DCE

Lasalandra

. Fos expression in the midbrain periaqueductal grey after trigeminovascular stimulation. J Anat 2001; 198: 29–35.**.

88.

Benjamin

Levy

Lasalandra

. Hypothalamic activation after stimulation of the superior sagittal sinus in the cat: A Fos study. Neurobiol Dis 2004; 16: 500–505.**.

89.

Malick

Jakubowski

Elmquist

. A neurohistochemical blueprint for pain-induced loss of appetite. Proc Natl Acad Sci U S A 2001; 98: 9930–9935.**.

90.

Burstein

Yamamura

Malick

. Chemical stimulation of the intracranial dura induces enhanced responses to facial stimulation in brain stem trigeminal neurons. J Neurophysiol 1998; 79: 964–982. **Refs 86–90 demonstrate that stimulation of the dura mater results in neuronal activation higher brain structures, beyond the TCC, other than the thalamic nuclei..

91.

Goadsby

Hoskin

. Serotonin inhibits trigeminal nucleus activity evoked by craniovascular stimulation through a 5HT1B/1D receptor: A central action in migraine? Ann Neurol 1998; 43: 711–718.**.

92.

Goadsby

Hoskin

. Inhibition of trigeminal neurons by intravenous administration of the serotonin (5HT)1B/D receptor agonist zolmitriptan (311C90): Are brain stem sites therapeutic target in migraine? Pain 1996; 67: 355–359.**.

93.

Kaube

Hoskin

Goadsby

. Inhibition by sumatriptan of central trigeminal neurones only after blood-brain barrier disruption. Br J Pharmacol 1993; 109: 788–792.** **Refs 91–93 and 95 demonstrate clearly that triptans have central sites of action in the trigeminal nucleus caudalis, using electrophysiological techniques..

94.

Storer

Akerman

Goadsby

. Calcitonin gene-related peptide (CGRP) modulates nociceptive trigeminovascular transmission in the cat. Br J Pharmacol 2004; 142: 1171–1181.

95.

Goadsby

Akerman

Storer

. Evidence for postjunctional serotonin (5-HT1) receptors in the trigeminocervical complex. Ann Neurol 2001; 50: 804–807.**.

96.

Storer

Akerman

Connor

. 4991W93, a potent blocker of neurogenic plasma protein extravasation, inhibits trigeminal neurons at 5-hydroxytryptamine (5-HT1B/1D) agonist doses. Neuropharmacology 2001; 40: 911–917.

97.

Levy

Moskowitz

Nuseda

. Activation of the migraine pain pathway by cortical spreading depression: Do we need more evidence? Cephalalgia 2012; 32: 581–582.

98.

Hammond

Presley

Gogas

. Morphine or U-50,488 suppresses Fos protein-like immunoreactivity in the spinal cord and nucleus tractus solitarii evoked by a noxious visceral stimulus in the rat. J Comp Neurol 1992; 315: 244–253.

99.

Goadsby

Edvinsson

. The trigeminovascular system and migraine: Studies characterizing cerebrovascular and neuropeptide changes seen in humans and cats. Ann Neurol 1993; 33: 48–56.

100.

Lassen

Haderslev

Jacobsen

. CGRP may play a causative role in migraine. Cephalalgia 2002; 22: 54–61. **Seminal work that demonstrates that CGRP induces delayed migraine in patients..

101.

Edvinsson

Ekman

Goadsby

. Measurement of vasoactive neuropeptides in biological materials: Problems and pitfalls from 30 years of experience and novel future approaches. Cephalalgia 2010; 30: 761–766.**.

102.

Knight

Edvinsson

Goadsby

. Blockade of calcitonin gene-related peptide release after superior sagittal sinus stimulation in cat: A comparison of avitriptan and CP122,288. Neuropeptides 1999; 33: 41–46.

103.

Knight

Edvinsson

Goadsby

. 4991W93 inhibits release of calcitonin gene-related peptide in the cat but only at doses with 5HT(1B/1D) receptor agonist activity? Neuropharmacology 2001; 40: 520–525.

104.

Asghar

Hansen

Kapijimpanga

. Dilation by CGRP of middle meningeal artery and reversal by sumatriptan in normal volunteers. Neurology 2010; 75: 1520–1526.

105.

Fischer

Koulchitsky

Messlinger

. The nonpeptide calcitonin gene-related peptide receptor antagonist BIBN4096BS lowers the activity of neurons with meningeal input in the rat spinal trigeminal nucleus. J Neurosci 2005; 25: 5877–5883.

Pearls and pitfalls in experimental in vivo models of migraine: Dural trigeminovascular nociception

Abstract

Background

Headache mechanisms

Methodology

Summary

Keywords

Introduction

Trigeminovascular system – historical viewpoint

Sterile neurogenic inflammation

Trigeminovascular dural nociception

Dural electrical stimulation

Pearls of measuring changes in the cranial vasculature

Intravital microscopy

Laser Doppler flowmetry

Pitfalls of measuring changes in the craniovasculature

The future

Pearls of measuring changes in the trigeminal nucleus caudalis and brain

Fos immunoreactivity

Electrophysiological recordings within the TCC

Pitfalls of measuring changes in the trigeminal nucleus caudalis and brain

Fos immunoreactivity

Electrophysiological recording

Measuring neuropeptide changes

Pitfalls of measuring neuropeptide changes

The future

Footnotes

Funding

Conflict of interest

References