Migraines are a type of headache that specifically act on only one side of the head, although about 30% of patients with migraine may experience a bilateral headache. Migraine is a brain disorders that typically involve issues of the typical sensory processing that takes place in the brainstem. Possible causation has been linked to issues in blood vessels, blood flow, and oxygen levels in the brain. Migraine can be described in three phases, and common throughout the three phases is the importance of the neuropeptide CGRP and its role in migraine pathogenesis. CGRP increases in plasma have been linked to migraine headaches, and specific treatment plans have been tailored to account for this. CGRP is a vasodilator that causes dilation of cranial blood vessels and can lead to possible neurogenic inflammation in the periphery of its release while activating the pain pathway in the brainstem. The primary treatment for migraines is currently drugs from the triptan family and NSAIDs, as well as prophylactic drugs including antiepileptic drugs, beta-blockers, and Ca2+ channel blockers. The experiment conducted for this project aimed to determine the effects of a specific CGRP polyclonal antibody and CGRP receptor antagonist when it is with capsaicin, which stimulates sensory nerves. In an ex-vivo experiment using cell culture medium, the dura mater of mice is given either rabbit polyclonal antibody or a CGRP receptor antagonist or both, and then is challenged with capsaicin. CGRP positive (expressing) fibers and nerve terminals are examined under a fluorescent microscope in the dura mater of the mice.

Indiana University-Purdue University Indianapolis (IUPUI) / Nerve growth factor (NGF) is a neurotrophin upregulated with injury and inflammation. Peripheral administration of NGF causes hyperalgesia and allodynia in animals. Blocking NGF signaling reverses these effects. At the cellular level, chronic exposure of sensory neurons to NGF enhances expression the neurotransmitter, calcitonin gene-related peptide (CGRP). Acute exposure to NGF increases capsaicin-evoked CGRP release from sensory neurons in culture. Thus, NGF increases peptide release from neurons by: (1) increasing expression of peptides, and/or (2) altering their sensitivity. The increase in peptide outflow by either mechanism could contribute to development of hyperalgesia and allodynia. The signaling cascades mediating the actions of NGF in sensory neurons are unclear. Therefore, experiments were designed to determine which pathways regulate changes in iCGRP content and evoked release from primary sensory neurons in culture. The Ras/MEK/ERK cascade was identified as a possible regulator of iCGRP expression in response to NGF. To test this pathway, it was manipulated in neurons by (1) expression of dominant negative or constitutively active isoforms of Ras, (2) farnesyltransferase inhibition, (3) manipulation of the RasGAP, synGAP, and (4) blocking MEK activity. When the pathway was blocked, the NGF-induced increase in iCGRP expression was attenuated. When the Ras pathway was activated, iCGRP expression increased. These data indicate that Ras, and downstream signaling kinases, MEK and ERK, regulate the NGF-induced increases in CGRP in sensory neurons. To determine which pathway(s) regulate the increase in capsaicin-evoked iCGRP release upon brief exposure to NGF, the Ras/MEK/ERK pathway was manipulated as described above, and pharmacological inhibitors of the PI3 kinase, PLC, and Src kinase pathways were used. There were no differences observed in NGF-sensitization when the Ras and PI3 kinase pathways were inhibited, suggesting these two pathways were not involved. However, when the Src kinase inhibitor PP2 was used, the NGF-induced increase in release was completely blocked. Furthermore, the PKC inhibitor, BIM, also inhibited the sensitization by NGF. This data indicate Src and PKC regulate of sensitivity of sensory neurons in response to brief exposure to NGF. Thus, there is differential regulation of iCGRP content and evoked release from sensory neurons in response to NGF.

Die bakterielle Meningitis (BM) ist trotz antibiotischer Therapie eine Erkrankung mit einer hohen Mortalität und Morbidität. Kopfschmerzen und Meningismus sind Hauptsymtome und ein klinischer Hinweis für die Aktivierung trigeminaler Fasern. Ziel dieser Arbeit war es zu prüfen ob die freigesetzten Neuropeptide einen proinflammatorischen Effekt auf zerebrale Endothelzellen, einen wesentlichem Bestandteil der Blut-Hirn-Schranke haben. Wir verwendeten primär kultivierte zerebrale Kapillarendothelzellen (BMEC) der Ratte und als Stimulus Neuropeptide und/oder Pneumokokkenzellwände (PCW). Beide Neuropeptide, CGRP mehr als SP, verstärken den Effekt von PCW auf die mRNA Expression und Freisetzung von TNF-alpha, IL-1beta, IL-6, IL-10 und MIP-2 aus den BMEC. CGRP und SP haben nur eine geringe Wirkung. PCW regulieren die Dichte der CRLR (CGRP1-R) bzw. NK-1 Rezeptoren und erklären damit die kostimulatorische Wirkung. Zudem untersuchten wir den Effekt von PCW und/oder CGRP auf die Adrenomedullin (AM)- Synthese. AM ist ein vasodilatorisch wirkendes Peptid, dass vorwiegend in Endothelzellen konstitutiv gebildet wird und am CRLR Rezeptor wirkt. PCW und CGRP verstärken die Synthese von AM. Mit dieser Arbeit konnte gezeigt werden, dass PCW zur Hochregulation von Neuropeptidrezeptoren führt und CGRP und SP über diese Rezeptoren einen modulatorischen Effekt auf die Zytokinproduktion in BMEC haben. Ein genaues Verständnis dieser Interaktionen könnte die Entwicklung immunmodulatorischer Interventionen und damit eine Verbesserung der Prognose der bakteriellen Meningitis bewirken. / Despite antibiotic treatment bacterial meningitis is still associated with a high mortality and morbidity. Headache and meningismus as key symptoms, provide clear evidence for the activation of trigeminal nerve fibers. Aim of the study was to test whether the released neuropeptides have a proinflammatory effect in cerebral endothelial cells the major compartment of the blood brain barrier. We used primary brain microvascular endothelial cells of the rat (BMEC) which were stimulated with CGRP, SP and/or pneumococcal cell walls (PCW). Both neuropeptides CGRP more than SP enhanced PCW-induced mRNA expression and the release of TNF-alpha, IL-1-beta, IL-6, IL-10 and MIP-2. Neuropeptides alone were not able to induce these cytokines. PCW upregulate the density of CRLR receptor and regulate the NK-1 receptor and therefore may explain the costimulatory effect. Furthermore the effect of PCW and/or CGRP on adrenomedullin synthesis in BMEC was investigated. Adrenomedullin is a vasodilatatory peptide, which is constitutivly produced by endothelial cells and act on the CRLR receptor. PCW as well as CGRP enhance the synthesis of AM. Our data suggest that PCW upregulate neuropeptide receptors and modulate via these specific receptors the cytokine production. A detailed understanding of these interactions may open new immunmodulatory interventions and therefore may contribute to a better prognosis of bacterial meningitis.