Michael J.M. Fischer

Cannabinoids mediate analgesia largely via peripheral type 1 cannabinoid receptors in nociceptors

Although endocannabinoids constitute one of the first lines of defense against pain, the anatomical locus and the precise receptor mechanisms underlying cannabinergic modulation of pain are uncertain. Clinical exploitation of the system is severely hindered by the cognitive deficits, memory impairment, motor disturbances and psychotropic effects resulting from the central actions of cannabinoids. We deleted the type 1 cannabinoid receptor (CB1) specifically in nociceptive neurons localized in the peripheral nervous system of mice, preserving its expression in the CNS, and analyzed these genetically modified mice in preclinical models of inflammatory and neuropathic pain. The nociceptor-specific loss of CB1 substantially reduced the analgesia produced by local and systemic, but not intrathecal, delivery of cannabinoids. We conclude that the contribution of CB1-type receptors expressed on the peripheral terminals of nociceptors to cannabinoid-induced analgesia is paramount, which should enable the development of peripherally acting CB1 analgesic agonists without any central side effects.

Calcitonin gene-related peptide receptor inhibition reduces neuronal activity induced by prolonged increase in nitric oxide in the rat spinal trigeminal nucleus.

Infusion of nitric oxide (NO) donors is known to induce delayed attacks of migraine and cluster headache or aggravate tension-type headaches in patients suffering from these primary headaches. Previously we have reported that infusion of NO donors in the rat causes delayed neuronal activity in the spinal trigeminal nucleus, which parallels the above clinical observations. Suggesting that endogenous NO production is involved in the generation of primary headaches, we used this animal model of meningeal nociception to determine whether a prolonged increase in NO levels causes an increase in neuronal activity. In anaesthetized rats spinal trigeminal neurons with afferent input from the exposed dura were recorded. Continuous intravenous infusion of the NO donors sodium nitroprusside (25 μg/kg/h) or glycerol trinitrate (250 μg/ kg/h) for 2h induced a persisting increase in neuronal activity but no change in systemic blood pressure. In this activated trigeminal system the calcitonin gene-related peptide (CGRP) receptor antagonist BIBN4096BS (900 μg/ kg) was infused. Spinal trigeminal activity was significantly reduced within minutes and to a similar extent as previously reported in animals not treated with NO. Slow continuous NO infusion may be a model of the active headache phase, and inhibition of CGRP receptors can reverse the induced neuronal activity.

Proton‐induced calcitonin gene‐related peptide release from rat sciatic nerve axons, in vitro, involving TRPV1

We have shown previously that rat sciatic nerve axons in vitro express sensitivity to capsaicin and heat and responded to these stimuli with a Ca2+‐dependent and graded immunoreactive calcitonin gene‐related peptide release. Morphological evidence for stimulated vesicular exocytosis and for the vanilloid receptor TRPV1 in the axolemma of the unmyelinated nerve fibres has also been presented. Here we used solutions of low pH, high K+ or 47 °C to stimulate isolated desheathed sciatic nerves measuring immunoreactive calcitonin gene‐related peptide release. pH 6.1 increased immunoreactive calcitonin gene‐related peptide release by 31% over baseline and pH 5.2 and 4.3 caused a log‐linear concentration‐dependent increase of 137 and 265%, respectively. The effect of pH 3.4 was out of the linear range and not reversible. Stimulation in Ca2+‐free solutions and under increased intracellular Ca2+ buffering capacity strongly reduced the proton responses. The TRPV1 antagonists capsazepine and ruthenium red substantially reduced the effects of pH 5.2 but not pH 6.1. Combining a stimulus of 60 mm K+ with the subliminal pH 6.3 reduced the axonal immunoreactive calcitonin gene‐related peptide response by 88%. The noxious heat response at pH 6.3, however, was only reduced by 39%, suggesting a hidden sensitization to heat by low pH. This was supported by an effect of capsazepine to reduce the combined response to half, indicative of an involvement of TRPV1 in the sensitization but not in the axonal heat response itself that was found to be resistant to capsazepine. Axonal calcitonin gene‐related peptide release is thought to play a physiological role in activity‐dependent autoregulation of endoneurial blood flow. Axonal sensitivity to and sensitization by protons may be a pathophysiological mechanism involved in certain peripheral neuropathies.

Lobar localization information in epilepsy patients: MEG—A useful tool in routine presurgical diagnosis

Epilepsy surgery is an established therapy for pharmacoresistant focal epilepsy. This study investigated the contribution of routinely used magnetoencepahlography (MEG) in addition to long term video-EEG-monitoring in presurgical evaluation. The distribution of localization results to anatomical lobes was compared with special focus to MEG spike localization results in cases without or with ambiguous EEG findings. A total of 105 consecutive patients with intractable focal epilepsy and epilepsy surgery after investigation by video-EEG-monitoring and MEG were included. The percentages of monolobar results were analysed and compared, especially with respect to the resection lobe. Postoperative outcome was used for further validation. No spikes were recorded on MEG in 30% (32 of 105). In cases with a diagnostic finding by the respective method, MEG localized in 82% (60 of 73 patients) within one anatomical lobe. Ictal EEG localized within one lobe in 72% (66 of 92 patients), interictal EEG in 60% (59 of 98 patients). In 25 of 105 patients (24%) no clear localization within one lobe was found either in interictal or in ictal EEG. In 11 of these cases MEG localized within the resection lobe. Six patients of these became seizure free, the other five had at least 50% reduction of their seizure rate 1 year after surgery. In summary MEG is a useful tool in the routine workup for epilepsy surgery contributing information to focus hypothesis in addition to video-EEG.

Calcitonin gene-related peptide release from intact isolated dorsal root and trigeminal ganglia.

Neuropeptides like calcitonin gene-related peptide (CGRP) and substance P are found in significant proportions of primary afferent neurons. Release of these neuropeptides as well as prostaglandin E(2) is an approved index for the activation of these primary afferents. Previous studies have used cultures of enzyme-treated and mechanically dissociated primary afferent neurons, fresh tissue slices or cubes. In the present study we demonstrate CGRP and prostaglandin E(2) release from intact isolated dorsal root and trigeminal ganglia. Stimulation with noxious heat, low pH, inflammatory mediators and high potassium concentration increased CGRP release. In conclusion, neuropeptide release from intact isolated ganglia is a reliable method to study the responsiveness of sensory neurons in situ in comparison with neuronal cell cultures.

Sensitization to heat through G‐protein‐coupled receptor pathways in the isolated sciatic mouse nerve

We have previously shown that isolated rat sciatic nerve axons express capsaicin, heat and proton sensitivity and respond to stimulation with a Ca++‐dependent and graded calcitonin gene related peptide (CGRP) release. There is morphological evidence for stimulated vesicular exocytosis and for capsaicin receptor, transient receptor potential vanilloid type‐1 (TRPV1, formerly VR1) translocation in the axolemma of unmyelinated nerve fibres. In sensory nerve terminals CGRP release in response to noxious heat can be sensitized by activation of G‐protein‐coupled receptors and related protein kinases. We present evidence that also in isolated mouse sciatic nerve axons the intracellular protein kinase A (PKA)‐ and C (PKC)‐dependent transduction pathways modulate heat‐induced (45 °C) CGRP release. This is demonstrated using the direct activators, forskolin and phorbol 12‐myristate 13‐acetate (PMA), as well as prostaglandin E2 (PGE2) and bradykinin acting through G‐protein‐coupled receptors. Inhibition at rest of protein kinases A or C left heat‐induced CGRP release unchanged. In TRPV1 knockout animals no sensitization to heat was observed using a combined stimulation by prostaglandin E2 and bradykinin. To a surprising degree, peripheral nerve axons resemble peripheral sensory terminals in their common properties of sensory and signal transduction.

Increase in CGRP- and nNOS-immunoreactive neurons in the rat trigeminal ganglion after infusion of an NO donor.

Background: Nitrovasodilators, such as glyceroltrinitrate (GTN), which produce nitric oxide (NO) in the organism, are known to cause delayed headaches in migraineurs, accompanied by increased plasma levels of calcitonin gene-related peptide (CGRP) in the cranial venous outflow. Increases in plasma CGRP and NO metabolites have also been found in spontaneous migraine attacks. In a rat model of meningeal nociception, infusion of NO donors induced activity of neurons in the spinal trigeminal nucleus. Methods: Isoflurane-anaesthetised rats were intravenously infused with GTN (250 µg/kg) or saline for two hours and fixed by perfusion after a further four hours. Cryosections of dissected trigeminal ganglia were immunostained for detection of CGRP and neuronal NO synthase (nNOS). The ganglion neurons showing immunofluorescence for either of these proteins were counted. Results: The proportions of CGRP- and nNOS- as well as double-immunopositive neurons were increased after GTN infusion compared to saline treatment in all parts of the trigeminal ganglion (CGRP) or restricted to the ophthalmic region (nNOS). The size of immunopositive neurons was not significantly different compared to controls. Conclusion: High levels of NO may induce the expression or availability of CGRP and nNOS. Similar changes may be involved in nitrovasodilator-induced and spontaneous headache attacks in migraineurs.

The neuropeptide calcitonin gene-related peptide (CGRP) prevents inflammatory liver injury in mice.

BACKGROUND/AIMS Calcitonin gene-related peptide (CGRP) is a potent vasodilator and supposed to be responsible for neurogenic inflammation involved in migraine. Its role in inflammatory diseases of other organs is controversial and poorly investigated regarding liver inflammation, although the organ is innervated by CGRP containing primary sensory nerve fibers. METHODS Male Balb/c and IL-10(-/-) mice were pretreated with either alphaCGRP or the CGRP receptor antagonists CGRP(8-37) or BIBN4096BS. Immune-mediated liver injury was induced by administration of lipopolysaccharide (LPS) or tumor necrosis factor-alpha (TNFalpha) to galactosamine (GalN)-sensitized mice and evaluated by serum transaminase activities and cytokine levels. Furthermore, intrahepatic CGRP receptor expression and hepatic CGRP concentrations were examined. RESULTS CGRP receptor 1 was expressed by immune cells and hepatocytes in human and murine liver. During liver injury CGRP receptor expression was increased whereas hepatic CGRP concentrations concomitantly decreased. While CGRP receptor antagonists failed to affect liver damage, pretreatment with alphaCGRP protected mice from GalN/LPS-induced liver injury by suppression of the pro-inflammatory cytokine response independently from IL-10 but related to the induction of the transcriptional repressor inducible cAMP early repressor (ICER). In contrast, alphaCGRP failed to protect against GalN/TNFalpha-induced liver failure. CONCLUSION In the liver, CGRP exerts anti-inflammatory properties, which are characterized by a reduced production of pro-inflammatory cytokines.

Spatial relationship of source localizations in patients with focal epilepsy: Comparison of MEG and EEG with a three spherical shells and a boundary element volume conductor model

Epilepsy surgery is an option for patients with pharmacoresistant focal epilepsies, but it requires a precise focus localization procedure. Magnetoencephalography (MEG) and electroencephalography (EEG) can be used for analysis of interictal activity. The aim of this prospective study was to compare clusters of source localization results with MEG and EEG using a three spherical shells (3SS) and a boundary element method (BEM) volume conductor model. The study was closed when 100 patients met the inclusion criteria. Simultaneous MEG and EEG were recorded during presurgical evaluation. Epileptiform signals were analyzed using an equivalent current dipole model. Centroids of source localizations from MEG, EEG, 3SS, and BEM in their respective combinations were compared. In a 3SS model, MEG source localizations were 5.6 mm inferior to those obtained by EEG, while in a BEM model MEG source localizations were 6.3 mm anterior and 4.8 mm superior. The mean scattering of source localizations between both volume conductor models was 19.5 mm for EEG and 9.6 mm for MEG. For MEG no systematic difference between BEM and 3SS source localizations was found. For EEG, source localizations with BEM were 5.9 mm posterior and 11.7 mm inferior to those determined using 3SS. No differences were found between the 46 temporal and the 54 extratemporal lobe epilepsy patients. The observed systematic differences of source localizations of epileptic spikes due to the applied source signal modality and volume conductor model should be considered in presurgical evaluation when only one source signal and volume conductor model is available. Hum Brain Mapp, 2007.

Release of CGRP from mouse brainstem slices indicates central inhibitory effect of triptans and kynurenate

BackgroundCGRP is contained in a substantial proportion of unmyelinated trigeminal neurons innervating intracranial tissues. Previously, we have described a hemisected rodent scull preparation and later the intact trigeminal ganglion to measure stimulated CGRP release from trigeminal afferents.MethodsHere, we establish a preparation for examining CGRP release from central trigeminal terminals using single fresh slices of the mouse medullary brainstem.ResultsBasal and stimulated amount of CGRP substantially exceeded the detection level. Experiments were designed as matched pairs of at least six brainstem slices per animal. Stimulation with high potassium induced calcium-dependent and reversible CGRP release. Capsaicin stimulation of TRPV1 provoked concentration-dependent CGRP release. The anti-migraine drug naratriptan did not inhibit capsaicin-induced CGRP release from peripheral terminals but inhibited the release from brainstem slices. The glutamate antagonist kynurenate showed a similar pattern of site-specific inhibition of CGRP release.ConclusionsAs observed earlier for other drugs used in the treatment of migraine this indicates that the central terminals in the spinal trigeminal nucleus may be the main site of action. The preparation allows evaluating the trigeminal brainstem as a pharmacological site of action.