Capsaicin-szenzitív neurogén mechanizmusok a meningeális nocicepcióban és a dura mater vaszkuláris reakcióinak modulációjában = Role of capsaicin-sensitive neurogenic mechanisms in meningeal nociception and in the modulation of vascular reactions of the dura mater

Kísérleteink során morfológiai és funkcionális módszerekkel igazoltuk a trigemino-vaszkuláris capsaicin szenzitív CGRP-immunreaktív nociceptív afferens idegek jelenlétét patkány kemény agyhártyájában. Igazoltuk, hogy kísérletes diabetes mellitusban drámaian károsodik a capsaicin-szenzitív idegek nociceptív és szenzoros vasomotor funkciója és csökken a TRPV1-immunreaktív meningeális idegrostok száma. Kimutattuk, hogy a capsaicin-szenzitív idegek szerepet játszanak a PAR-2 aktiválódását kísérő CGRP- és NO-függő vazodilatáció kialakulásában. A neurogén szenzoros vazodilatáció károsodásának patofiziológiai következménye a gyulladásos metabolitok eltávolítását szolgáló protektív mechanizmusok károsodása, ami magyarázhatja a diabéteszes betegek fejfájással kapcsolatos fokozott panaszait. Megfigyeléseink a kemoszenzitív érző neuronok szerepét bizonyítják a kemény agyhártya nociceptív és szenzoros vazomotor működésében és a fejfájások patofiziológiájában. Patkány elsődleges érző ganglionsejt-kultúrákon végzett vizsgálatainkban bizonyítottuk a gangliozidok nociceptív folyamatokban betöltött szerepét. Kimutattuk, hogy a gangliozid szintézis kulcsenzime, a glucosylceramid szintáz gátlását követően - az NGF szignalizáció és/vagy a TRPV1 receptor membrán lokalizációjának változásai miatt - jelentősen és szignifikánsan csökken az érző neuronok capsaicinnel történő aktiválhatósága és a TRPV1 receptor expressziója. Eredményeink a fájdalomcsillapítás új farmakológiai lehetőségeire utalnak. | Morphological studies demonstrated a rich innervation of the rat dura mater by chemosensitive afferent nerves which express the TRPV1 receptor. Retrograde labelling revealed that trigeminal afferent nerves innervating the meningeal vessels exhibit strong immunoreactivity for the potent vasodilator peptide CGRP. Studies on streptozotocin diabetic rats revealed a significant impairment in nociceptive and vasomotor functions and a reduction in the number of TRPV1-positive afferent nerves. The role of chemosensitive afferent nerves in the mediation of the vasodilator effects of PAR-2 activation was also established. The effects of PAR-2 are dependent on the release of CGRP and NO. Impairment of sensory neurogenic vasodilatation may result in a decreased protective effect due to the insufficient removal of inflammatory mediators from the meningeal tissues, which might explain the increased incidence of headache complaints in diabetics. Studies on cultured adult rat primary sensory neurons furnished evidence for the role of membrane gangliosides in the transmission of nociceptive stimuli. Inhibition of glucosylceramide synthase, the key enzyme of ganglioside metabolism resulted in a substantial reduction in neuronal capsaicin sensitivity and in the number of neurons which expressed the TRPV1 receptors due to impaired NGF-signaling and changes in the expression and/or membrane localization of TRPV1. The findings point to new pharmacological possibilities in the treatment of pain

Perineural Capsaicin Treatment Inhibits Collateral Sprouting of Intact Cutaneous Nociceptive Afferents

Perineural treatment of peripheral nerves with capsaicin produces a long-lasting selective regional thermo- and chemo-analgesia and elimination of the neurogenic inflammatory response involving degeneration of nociceptive afferent fibers. In this study, we examined longitudinal changes in mustard oil-induced sensory neurogenic vasodilatation and plasma extravasation following perineural capsaicin treatment of the rat saphenous nerve utilizing scanning laser Doppler imaging and vascular labeling with colloidal silver. Capsaicin treatment resulted in a marked decrease in mustard oil-induced vasodilatation in the skin area served by the saphenous nerve. Repeated imaging of the vasodilatatory response showed no recovery for at least 7 weeks. However, following transection and ligation of the capsaicin-treated saphenous nerve, a substantial recovery of the vasodilatatory response was observed, suggesting a reinnervation of the chemodenervated skin area by collateral sprouting of neighboring intact sciatic nerve afferents. Elimination of the recovered vascular reaction by capsaicin treatment of the sciatic nerve supported this conclusion. Similar results have been obtained by using the vascular labeling technique. These findings indicate an inhibitory effect of persisting cutaneous nerve fibers on the collateral sprouting of intact nerve fibers into the chemodenervated skin area. These observations may bear implications for the development of sensory disturbances following peripheral nerve injuries

TRP Channels in the Focus of Trigeminal Nociceptor Sensitization Contributing to Primary Headaches

Pain in trigeminal areas is driven by nociceptive trigeminal afferents. Transduction molecules, among them the nonspecific cation channels transient receptor potential vanilloid 1 (TRPV1) and ankyrin 1 (TRPA1), which are activated by endogenous and exogenous ligands, are expressed by a significant population of trigeminal nociceptors innervating meningeal tissues. Many of these nociceptors also contain vasoactive neuropeptides such as calcitonin gene-related peptide (CGRP) and substance P. Release of neuropeptides and other functional properties are frequently examined using the cell bodies of trigeminal neurons as models of their sensory endings. Pathophysiological conditions cause phosphorylation, increased expression and trafficking of transient receptor potential (TRP) channels, neuropeptides and other mediators, which accelerate activation of nociceptive pathways. Since nociceptor activation may be a significant pathophysiological mechanism involved in both peripheral and central sensitization of the trigeminal nociceptive pathway, its contribution to the pathophysiology of primary headaches is more than likely. Metabolic disorders and medication-induced painful states are frequently associated with TRP receptor activation and may increase the risk for primary headaches

CGRP outflow into jugular blood and cerebrospinal fluid and permeance for CGRP of rat dura mater

Background Calcitonin gene-related peptide (CGRP) is released from activated meningeal afferent fibres in the cranial dura mater, which likely accompanies severe headache attacks. Increased CGRP levels have been observed in different extracellular fluid compartments during primary headaches such as migraine but it is not entirely clear how CGRP is drained from the meninges. Methods We have used an in vivo preparation of the rat to examine after which time and at which concentration CGRP applied onto the exposed parietal dura mater appears in the jugular venous blood and the cerebrospinal fluid (CSF) collected from the cisterna magna. Recordings of meningeal (dural) and cortical (pial) blood flow were used to monitor the vasodilatory effect of CGRP. In a new ex vivo preparation we examined how much of a defined CGRP concentration applied to the arachnoidal side penetrates the dura. CGRP concentrations were determined with an approved enzyme immunoassay. Results CGRP levels in the jugular plasma in vivo were slightly elevated compared to baseline values 5-20 min after dural application of CGRP (10 μM), in the CSF a significant three-fold increase was seen after 35 min. Meningeal but not cortical blood flow showed significant increases. The spontaneous CGRP release from the dura mater ex vivo was above the applied low concentration of 1 pM. CGRP at 1 nM did only partly penetrate the dura. Conclusions We conclude that only a small fraction of CGRP applied onto the dura mater reaches the jugular blood and, in a delayed manner, also the CSF. The dura mater may constitute a barrier for CGRP and limits diffusion into the CSF of the subarachnoidal space, where the CGRP concentration is too low to cause vasodilatation

Semi-Automated Recording of Facial Sensitivity in Rat Demonstrates Antinociceptive Effects of the Anti-CGRP Antibody Fremanezumab

Migraine pain is frequently accompanied by cranial hyperalgesia and allodynia. Calcitonin gene-related peptide (CGRP) is implicated in migraine pathophysiology but its role in facial hypersensitivity is not entirely clear. In this study, we investigated if the anti-CGRP monoclonal antibody fremanezumab, which is therapeutically used in chronic and episodic migraines, can modify facial sensitivity recorded by a semi-automatic system. Rats of both sexes primed to drink from a sweet source had to pass a noxious mechanical or heat barrier to reach the source. Under these experimental conditions, animals of all groups tended to drink longer and more when they had received a subcutaneous injection of 30 mg/kg fremanezumab compared to control animals injected with an isotype control antibody 12–13 days prior to testing, but this was significant only for females. In conclusion, anti-CGRP antibody, fremanezumab, reduces facial sensitivity to noxious mechanical and thermal stimulation for more than one week, especially in female rats. Anti-CGRP antibodies may reduce not only headache but also cranial sensitivity in migraineurs

A nociceptív érző neuronok plaszticitása a szomato-szenzoros rendszert érintő fiziológiás, gyulladásos és degeneratív/neuropathiás folyamatok kapcsán = Plasticity of nociceptive sensory neurons associated with physiological, inflammatory and degenerative/neuropathic changes affecting the somatosensory system

A lezárult OTKA támogatással végzett kutatási periódus főbb eredményeinek a következőket tartom: 1) Elsőként állapítottuk meg és további adatokat szolgáltattunk arra vonatkozóan, hogy a perifériás idegek sérülését követő ún. gerincvelői sprouting jelenség morfológiai szubsztrátumát nem a vastag myelinhüvelyes spinális afferens rostok ill. azok burjánzása, hanem a velőtlen, nociceptív capsaicin-érzékeny C-típusú afferensek képezik. Ezzel jelentősen hozzájárultunk a fájdalomkutatásban mintegy egy évtizedig uralkodó dogma megdöntéséhez; 2) Kvantitatív in situ hibridizációs és immunhisztokémiai vizsgálatainkban kimutattuk, hogy a capsaicin receptor fehérje expressziójának tartós, és a TRPV1 mRNS részleges és tranziens down-regulációja szerepet játszhat a capsaicin és más vanilloidok, mint a resiniferatoxin antinociceptív hatásában; 3) Elektrofiziológiai és immunhisztokémiai módszerekkel karakterizáltuk az inzulin receptor-pozitív ganglionsejtek új populációit; 4) Felismertük, hogy a glukozilceramide-szintáz gátlása jelentősen befolyásolja a nociceptív érző ganglionsejtek capsaicin-érzékenységét. Megfigyelésünk utat nyithat egy új kutatási területhez, a glykolipideknek fájdalomérzés modulációjában betöltött szerepének tanulmányozásához. | The main findings of the past research period may be summarized as follows: 1) We provided evidence for the first time for unmyelinated nociceptive C-fibre afferents being the anatomical substrate of nerve lesion induced increased transganglionic transport of choleratoxin B subunit, a highly specific marker and receptor of GM1 ganglioside and membrane lipid rafts. This observation disproved the decade old dogma of pain research which suggested that myelinated primary afferent sprouting may be responsible for this phenomenon which, in turn, may play a role in the development of chronic pain states; 2) Using quantitative immunohistochemistry and in situ hybridization techniques we demonstrated that permanent elimination of the TRPV1 (capsaicin) receptor protein and transient and partial down regulation of TRPV1 mRNA may significantly contribute to the antinociceptive, pain relieving effect of perineurally administered capsaicin and similar vanilloids such as resiniferatoxin; 3) By using electrophysiological and immunohistochemical techniques we characterized new populations of primary sensory neurons which express the insulin receptor; 4) We demonstrated that inhibition of neuronal glucosylceramide synthase markedly reduced the sensitivity of cultured dorsal root ganglion cells to capsaicin. This finding may lead to new observations concerning pain modulation and pain relief

The Anti-Calcitonin Gene-Related Peptide (Anti-CGRP) Antibody Fremanezumab Reduces Trigeminal Neurons Immunoreactive to CGRP and CGRP Receptor Components in Rats

Treatment with the anti-CGRP antibody fremanezumab is successful in the prevention of chronic and frequent episodic migraine. In preclinical rat experiments, fremanezumab has been shown to reduce calcitonin gene-related peptide (CGRP) release from trigeminal tissues and aversive behaviour to noxious facial stimuli, which are characteristic pathophysiological changes accompanying severe primary headaches. To further decipher the effects of fremanezumab that underlie these antinociceptive effects in rats, immunohistochemistry and ELISA techniques were used to analyse the content and concentration of CGRP in the trigeminal ganglion, as well as the ratio of trigeminal ganglion neurons which are immunoreactive to CGRP and CGRP receptor components, 1–10 days after subcutaneous injection of fremanezumab (30 mg/kg) compared to an isotype control antibody. After fremanezumab treatment, the fraction of trigeminal ganglion neurons which were immunoreactive to CGRP and the CGRP receptor components calcitonin receptor-like receptor (CLR) and receptor activity modifying protein 1 (RAMP1) was significantly lowered compared to the control. The content and concentration of CGRP in trigeminal ganglia were not significantly changed. A long-lasting reduction in CGRP receptors expressed in trigeminal afferents may contribute to the attenuation of CGRP signalling and antinociceptive effects of monoclonal anti-CGRP antibodies in rats.</jats:p