Gyulladásos és neuropátiás fájdalom farmakológiai befolyásolása primér szenzoros neuronokon ható szerekkel = Pharmacological modulation of inflammatory and neuropathic pain by agents acting on primary sensory neurones

A kapszaicin-érzékeny, VR1/TRPV1 receptort expresszáló primér szenzoros neuronok afferens működésükön kívül lokális és szisztémás efferens funkciókkal is rendelkeznek. A belőlük felszabaduló calcitonin gén-rokon peptid (CGRP) és tachykininek (pl. P-anyag) neurogén gyulladást váltanak ki az innervációs területen, míg a szomatosztatin gyulladásgátló és antinociceptív hatásokkal rendelkezik az sst4 receptorokon keresztül. Ezen neuronok jelentős szerepet játszanak gyulladásos (ízületi, légúti) és neuropátiás állapotok patomechanizmusában. A neurogén gyulladás és a neuropátiás fájdalom kezelésére jelenleg nincs megfelelő terápiás lehetőség. Kísérleteinkben a TRPV1 kapszaicin receptor, az sst4 szomatosztatin receptor, a hipofízis adenilát cikláz aktiváló polipeptid (PACAP-38), valamint az endomorphin-1 szerepét és ezeken ható vegyületek hatásait vizsgáltuk különféle akut és krónikus gyulladás, valamint neuropátia modellekben. Bár bizonyos gyulladásos folyamatokban a TRPV1, a neurokinin és CGRP receptorok blokkolása, valamint a PACAP és az endomorphin-1 anti-inflammációs és anti-nociceptív hatásokat fejtett ki, az sst4 receptor bizonyult minden modellünkben a legígéretesebb célpontnak. Eredményeink alapján ezen az érzőideg végződéseken és gyulladásos sejteken egyaránt expresszálódó receptoron ható stabil, per os is hatékony szelektív agonisták egy teljesen új hatásmechanizmusú gyulladáscsökkentő és fájdalomcsillapító gyógyszercsoport kifejlesztésére adhatnak lehetőséget. | Capsaicin-sensitive, VR1/TRPV1 receptor-expressing primary sensory neurons have local and systemic efferent functions besides their afferent role. Calcitonin gene-related peptide and tachykinins (e.g. substance P) induce neurogenic inflammation in the innervated area, somatostatin exerts anti-inflammatory and anti-nociceptive actions via sst4 receptor activation. Therefore, these neurons play an important role in the pathological mechanisms of several inflammatory (arthritis, asthma) and neuropathic conditions. At present there is no appropriate pharmacological treatment for the neurogenic component of inflammatory reactions and neuropathic pain. In the present series of experiments the roles of TRPV1 capsaicin receptors, sst4 somatostatin receptors, the effects of pituitary adenylate-cyclase activating polypeptide (PACAP-38) and endomorphin-1 as well as agents acting at these targets were investigated in different acute and chronic inflammation and neuropathy models. Although in certain inflammatory processes the blockade of TRPV1, neurokinin and CGRP receptors, and also PACAP and endomorphin-1 exerted anti-inflammatory and anti-nociceptive actions, the sst4 receptor proved to be the most promising target. On the basis of our results stable, orally active, selective sst4 agonist acting on both sensory nerve terminals and several inflammatory and immune cells could provide perspectives for the development of a completely novel type of anti-inflammatory and analgesic drugs

The fluorescent dye 3,3′-diethylthiatricarbocyanine iodide is unsuitable for in vivo imaging of myelination in the mouse

There is a growing interest to use non-invasive optical imaging methods to study central nervous system diseases. The application of a myelin-binding fluorescent dye, 3,3-diethylthiatricarbocyanine iodide (DBT) was recently described for in vivo optical imaging of demyelination in the mouse. In the present study we aimed at adapting the method to our optical imaging systems, the IVIS Lumina II to measure epifluorescence and the fluorescent molecular tomograph (FMT) for 3-dimensional quantification of the fluorophore. Epifluorescent imaging was performed 5−30 min after DBT injection which was followed by FMT imaging at 40 min. Two mice also underwent micro-CT imaging in the FMT cassette for the purpose of FMT-CT co-registration. Ex vivo imaging of the brain and other tissues of the head and neck was carried out 1 h after injection. Both the FMT-CT co-registration and the ex vivo imaging of organs proved that DBT poorly crossed the blood-brain barrier. The dye did not accumulate in the myelin sheath of the sciatic nerve. In contrast, there was an intense accumulation in the pituitary and salivary glands. The FMT-CT co-registration unequivocally demonstrated that the signal localized to the head did not originate from beyond the blood-brain barrier. No myelin binding was demonstrated by the ex vivo imaging either. In conclusion, DBT is unsuitable for in vivo imaging of myelination due to its poor BBB penetration, accumulation in other structures of the head and neck region and lack of selective binding towards myelin in vivo

Critical role of somatostatin receptor 2 in the vulnerability of the central noradrenergic system: new aspects on Alzheimer's disease

Alzheimer's disease and other age-related neurodegenerative disorders are associated with deterioration of the noradrenergic locus coeruleus (LC), a probable trigger for mood and memory dysfunction. LC noradrenergic neurons exhibit particularly high levels of somatostatin binding sites. This is noteworthy since cortical and hypothalamic somatostatin content is reduced in neurodegenerative pathologies. Yet a possible role of a somatostatin signal deficit in the maintenance of noradrenergic projections remains unknown. Here, we deployed tissue microarrays, immunohistochemistry, quantitative morphometry and mRNA profiling in a cohort of Alzheimer's and age-matched control brains in combination with genetic models of somatostatin receptor deficiency to establish causality between defunct somatostatin signalling and noradrenergic neurodegeneration. In Alzheimer's disease, we found significantly reduced somatostatin protein expression in the temporal cortex, with aberrant clustering and bulging of tyrosine hydroxylase-immunoreactive afferents. As such, somatostatin receptor 2 (SSTR2) mRNA was highly expressed in the human LC, with its levels significantly decreasing from Braak stages III/IV and onwards, i.e., a process preceding advanced Alzheimer's pathology. The loss of SSTR2 transcripts in the LC neurons appeared selective, since tyrosine hydroxylase, dopamine beta-hydroxylase, galanin or galanin receptor 3 mRNAs remained unchanged. We modeled these pathogenic changes in Sstr2 -/- mice and, unlike in Sstr1 -/- or Sstr4 -/- genotypes, they showed selective, global and progressive degeneration of their central noradrenergic projections. However, neuronal perikarya in the LC were found intact until late adulthood (<8 months) in Sstr2 -/- mice. In contrast, the noradrenergic neurons in the superior cervical ganglion lacked SSTR2 and, as expected, the sympathetic innervation of the head region did not show any signs of degeneration. Our results indicate that SSTR2-mediated signaling is integral to the maintenance of central noradrenergic projections at the system level, and that early loss of somatostatin receptor 2 function may be associated with the selective vulnerability of the noradrenergic system in Alzheimer's disease

Nitrogén-monoxid, neuropeptidek és más nem-adrenerg, nem-cholinerg átvivőanyagok szerepe zsigeri funkciókban, gyógyszeres befolyásolásuk ép és gyulladásos körülmények közt = Role of nitric oxide, neuropeptides and other non-adrenergic, non-cholinergic mediators in visceral functions; their modulation by drugs under normal and inflammatory conditions

A vállalt munka fő céljai: Izolált szervi kísérletekben megismerni a zsigerek mozgásválaszainak mechanizmusait, az állatokon kapott eredményeket humán preparátumokkal összevetve. Szenzoros és más eredetű nem-adrenerg, nem-kolinerg (NANC) transzmitterek azonosítása (funkcionális vizsgálatok és a transzmitter-felszabadulás mérése). Kóros zsigeri működések modellezése. (Egyes irányokban komplett, másokban tájékozódó kísérletek). Közölt eredmények: Új humán adatok közlése mellett összefoglaltuk a kapszaicin zsigeri hatásaival, transzmittereivel kapcsolatos jelentősebb eredményeket. Alappal fölvetettük annak lehetőségét, hogy emberben?és bizonyos állatfajokban is?az NO szenzoros transzmitter (Barthó et al. 2004?Eur J Pharmac; Benkó et al. 2005?Life Sci). Mind állati, mind (világelsőként) humán GI preparátumokban bizonyítottuk az ATP közvetítő szerepét NANC válaszokban (Undi et al. 2005?Bas Clin Pharmac; 2006?Brain Res Bull; Benkó et al 2006?NS Arch Pharmac, 2007?Neurosci). Nem találtunk bizonyítékot VIP szerepére emberi bél kapszaicinnel kiváltott gátló válaszában, a CO szerepére perisztaltikus reflexben (kongresszusi közlés), ill a CO szerepére a NANC gátló válaszban állati és humán GI preparátumokon (ld. fenti közlemények). Közlésre vár: P-anyag és CGRP-IR felszabadulás bélből; szenzoros izgató mustárolaj és H2S hatásmechanizmusa; a passzív szenzibilizáció/antigén-expozíció hatásainak elemzése állati és emberi GI és légúti simaizomzaton stb. | Aims of the project Experiments on isolated tissues for elucidating the mechanisms behind some evoked movements of viscera of animals and man. Identifying sensory and other non-adrenergic, non-cholinergic (NANC) transmitters. Measuring neurotransmitter release. Modelling pathophysiological processes of viscera. (Planned were complete series of experiments in some and pilot experiments in other directions.) Published results Review, containing original results, on visceral effects of capsaicin and the transmitters thereof. Providing indirect evidence that NO is a sensory neurotransmitter (Bartho et al. 2004?Eur J Pharmac; Benko et al. 2005?Life Sci). Proving the presence of purinergic innervation of human (Undi et al. 2006?Brain Res Bull; Benko et al. 2007?Neurosci) and rat intestine (Benko et al. 2006?NS Arch Pharmac). No evidence for a mediating role of VIP in the inhibitory effect of capsaicin in the human gut, a role of CO in the peristaltic reflex (congress presentations) or in the evoked NANC relaxation in animal or human GI preparations (papers as above). To be published Release of substance P- or CGRP-like IR from the gut; mechanisms of action of the sensory stimulants mustard oil and H2S; effects of passive sensitization/antigen exposure on GI and respiratory smooth muscles of animals and man, etc

Capsaicin-Sensitive Peptidergic Sensory Nerves Are Anti-Inflammatory Gatekeepers in the Hyperacute Phase of a Mouse Rheumatoid Arthritis Model

Capsaicin-sensitive peptidergic sensory nerves play complex, mainly protective regulatory roles in the inflammatory cascade of the joints via neuropeptide mediators, but the mechanisms of the hyperacute arthritis phase has not been investigated. Therefore, we studied the involvement of these afferents in the early, "black box" period of a rheumatoid arthritis (RA) mouse model. Capsaicin-sensitive fibres were defunctionalized by pretreatment with the ultrapotent capsaicin analog resiniferatoxin and arthritis was induced by K/BxN arthritogenic serum. Disease severity was assessed by clinical scoring, reactive oxygen species (ROS) burst by chemiluminescent, vascular permeability by fluorescent in vivo imaging. Contrast-enhanced magnetic resonance imaging was used to correlate the functional and morphological changes. After sensory desensitization, both early phase ROS-burst and vascular leakage were significantly enhanced, which was later followed by the increased clinical severity scores. Furthermore, the early vascular leakage and ROS-burst were found to be good predictors of later arthritis severity. We conclude that the anti-inflammatory role of peptidergic afferents depends on their activity in the hyperacute phase, characterized by decreased cellular and vascular inflammatory components presumably via anti-inflammatory neuropeptide release. Therefore, these fibres might serve as important gatekeepers in RA

Transcriptional Alterations in the Trigeminal Ganglia, Nucleus and Peripheral Blood Mononuclear Cells in a Rat Orofacial Pain Model

Orofacial pain and headache disorders are among the most debilitating pain conditions. While the pathophysiological basis of these disorders may be diverse, it is generally accepted that a common mechanism behind the arising pain is the sensitization of extra- and intracranial trigeminal primary afferents. In the present study we investigated gene expression changes in the trigeminal ganglia (TRG), trigeminal nucleus caudalis (TNC) and peripheral blood mononuclear cells (PBMC) evoked by Complete Freund's Adjuvant (CFA)-induced orofacial inflammation in rats, as a model of trigeminal sensitization. Microarray analysis revealed 512 differentially expressed genes between the ipsi- and contralateral TRG samples 7 days after CFA injection. Time-dependent expression changes of G-protein coupled receptor 39 (Gpr39), kisspeptin-1 receptor (Kiss1r), kisspeptin (Kiss1), as well as synaptic plasticity-associated Lkaaear1 (Lkr) and Neurod2 mRNA were described on the basis of qPCR results. The greatest alterations were observed on day 3 ipsilaterally, when orofacial mechanical allodynia reached its maximum. This corresponded well with patterns of neuronal (Fosb), microglia (Iba1), and astrocyte (Gfap) activation markers in both TRG and TNC, and interestingly also in PBMCs. This is the first description of up- and downregulated genes both in primary and secondary sensory neurones of the trigeminovascular system that might play important roles in neuroinflammatory activation mechanisms. We are the first to show transcriptomic alterations in the PBMCs that are similar to the neuronal changes. These results open new perspectives and initiate further investigations in the research of trigeminal pain disorders

Hemokinin-1 Gene Expression Is Upregulated in Trigeminal Ganglia in an Inflammatory Orofacial Pain Model: Potential Role in Peripheral Sensitization

A large percentage of primary sensory neurons in the trigeminal ganglia (TG) contain neuropeptides such as tachykinins or calcitonin gene-related peptide. Neuropeptides released from the central terminals of primary afferents sensitize the secondary nociceptive neurons in the trigeminal nucleus caudalis (TNC), but also activate glial cells contributing to neuroinflammation and consequent sensitization in chronic orofacial pain and migraine. In the present study, we investigated the newest member of the tachykinin family, hemokinin-1 (HK-1) encoded by the Tac4 gene in the trigeminal system. HK-1 had been shown to participate in inflammation and hyperalgesia in various models, but its role has not been investigated in orofacial pain or headache. In the complete Freund's adjuvant (CFA)-induced inflammatory orofacial pain model, we showed that Tac4 expression increased in the TG in response to inflammation. Duration-dependent Tac4 upregulation was associated with the extent of the facial allodynia. Tac4 was detected in both TG neurons and satellite glial cells (SGC) by the ultrasensitive RNAscope in situ hybridization. We also compared gene expression changes of selected neuronal and glial sensitization and neuroinflammation markers between wild-type and Tac4-deficient (Tac4-/-) mice. Expression of the SGC/astrocyte marker in the TG and TNC was significantly lower in intact and saline/CFA-treated Tac4-/- mice. The procedural stress-related increase of the SGC/astrocyte marker was also strongly attenuated in Tac4-/- mice. Analysis of TG samples with a mouse neuroinflammation panel of 770 genes revealed that regulation of microglia and cytotoxic cell-related genes were significantly different in saline-treated Tac4-/- mice compared to their wild-types. It is concluded that HK-1 may participate in neuron-glia interactions both under physiological and inflammatory conditions and mediate pain in the trigeminal system

Differential Regulatory Role of Pituitary Adenylate Cyclase–Activating Polypeptide in the Serum-Transfer Arthritis Model

OBJECTIVE: Pituitary adenylate cyclase-activating polypeptide (PACAP) expressed in capsaicin-sensitive sensory neurons and immune cells has divergent functions in inflammatory and pain processes. This study was undertaken to investigate the involvement of PACAP in a mouse model of rheumatoid arthritis. METHODS: Arthritis was induced in PACAP(-/-) and wild-type (PACAP(+/+) ) mice by K/BxN serum transfer. General features of the disease were investigated by semiquantitative scoring, plethysmometry, and histopathologic analysis. Mechano- and thermonociceptive thresholds and motor functions were also evaluated. Metabolic activity was assessed by positron emission tomography. Bone morphology was measured by in vivo micro-computed tomography, myeloperoxidase activity and superoxide production by bioluminescence imaging with luminol and lucigenin, respectively, and vascular permeability by fluorescent indocyanine green dye study. RESULTS: PACAP(+/+) mice developed notable joint swelling, reduced grasping ability, and mechanical (but not thermal) hyperalgesia after K/BxN serum transfer. In PACAP(-/-) mice clinical scores and edema were significantly reduced, and mechanical hyperalgesia and motor impairment were absent, throughout the 2-week period of observation. Metabolic activity and superoxide production increased in the tibiotarsal joints of wild-type mice but were significantly lower in PACAP(-/-) animals. Myeloperoxidase activity in the ankle joints of PACAP(-/-) mice was significantly reduced in the early phase of arthritis, but increased in the late phase. Synovial hyperplasia was also significantly increased, and progressive bone spur formation was observed in PACAP-deficient mice only. CONCLUSION: In PACAP-deficient mice with serum-transfer arthritis, joint swelling, vascular leakage, hyperalgesia, and early inflammatory cell accumulation are reduced; in the later phase of the disease, immune cell function and bone neoformation are increased. Elucidation of the underlying pathways of PACAP activity may open promising new avenues for development of therapy in inflammatory arthritis. © 2014 The Authors. Arthritis & Rheumatology is published by Wiley Periodicals, Inc. on behalf of the American College of Rheumatology