Dual orexin receptor antagonist 12 inhibits expression of proteins in neurons and glia implicated in peripheral and central sensitization.

Sensitization and activation of trigeminal nociceptors is implicated in prevalent and debilitating orofacial pain conditions including temporomandibular joint (TMJ) disorders. Orexins are excitatory neuropeptides that function to regulate many physiological processes and are reported to modulate nociception. To determine the role of orexins in an inflammatory model of trigeminal activation, the effects of a dual orexin receptor antagonist (DORA-12) on levels of proteins that promote peripheral and central sensitization and changes in nocifensive responses were investigated. In adult male Sprague-Dawley rats, mRNA for orexin receptor 1 (OX1R) and receptor 2 (OX2R) were detected in trigeminal ganglia and spinal trigeminal nucleus (STN). OX1R immunoreactivity was localized primarily in neuronal cell bodies in the V3 region of the ganglion and in laminas I-II of the STN. Animals injected bilaterally with complete Freund's adjuvant (CFA) in the TMJ capsule exhibited increased expression of P-p38, P-ERK, and lba1 in trigeminal ganglia and P-ERK and lba1 in the STN at 2 days post injection. However, levels of each of these proteins in rats receiving daily oral DORA-12 were inhibited to near basal levels. Similarly, administration of DORA-12 on days 3 and 4 post CFA injection in the TMJ effectively inhibited the prolonged stimulated expression of protein kinase A, NFkB, and Iba1 in the STN on day 5 post injection. While injection of CFA mediated a nocifensive response to mechanical stimulation of the orofacial region at 2h and 3 and 5 days post injection, treatment with DORA-12 suppressed the nocifensive response on day 5. Somewhat surprisingly, nocifensive responses were again observed on day 10 post CFA stimulation in the absence of daily DORA-12 administration. Our results provide evidence that DORA-12 can inhibit CFA-induced stimulation of trigeminal sensory neurons by inhibiting expression of proteins associated with sensitization of peripheral and central neurons and nociception.

CB1 and CB2 contribute to antinociceptive and anti-inflammatory effects of electroacupuncture on experimental arthritis of the rat temporomandibular joint.

Electroacupuncture (EA) and cannabinoids have been reported to have anti-inflammatory and antinociceptive effects in animal models of arthritis. Male Wistar rats were injected with saline or zymosan (2 mg) into the temporomandibular joint (TMJ). EA (10 Hz, 30 min) was performed 2 h after or 1 h before zymosan administration. AM251 or AM630 (3 mg/kg, i.p.)were administered before EA treatment. Mechanical hypernociception was accessed after zymosan administration. Rats were sacrificed 6 h after zymosan administration and the joint was removed for histopathological analysis. The gene expression of CB1 and CB2 receptors was assessed after sacrifice of the TMJ arthritic animals. EA inhibited zymosan-induced hypernociception (p < 0.05). AM251 reversed significantly the antinociceptive effect of EA, suggesting that the CB1 receptor is involved in this effect. AM630 reversed the anti-inflammatory effect of EA. CB1 and CB2 receptor gene expression was upregulated 6 h after zymosan-induced arthritis in the EA-treated group. We observed downregulation of CB2 receptor gene expression in the EA group at the 24th hour compared with the 6th hour. Higher CB1 receptor gene expression was also found compared with the 6th hour. EA produced antinociceptive and anti-inflammatory effects, and these effects appeared to be mediated through CB1 and CB2 receptor activation.

House-dust mite allergen and ozone exposure decreases histamine H3 receptors in the brainstem respiratory nuclei.

Allergic airway diseases in children are a common and a growing health problem. Changes in the central nervous system (CNS) have been implicated in contributing to some of the symptoms. We hypothesized that airway allergic diseases are associated with altered histamine H3 receptor expression in the nucleus tractus solitarius (NTS) and caudal spinal trigeminal nucleus, where lung/airway and nasal sensory afferents terminate, respectively. Immunohistochemistry for histamine H3 receptors was performed on brainstem sections containing the NTS and the caudal spinal trigeminal nucleus from 6- and 12-month-old rhesus monkeys who had been exposed for 5 months to house dust mite allergen (HDMA)+O3 or to filtered air (FA). While histamine H3 receptors were found exclusively in astrocytes in the caudal spinal trigeminal nucleus, they were localized to both neuronal terminals and processes in the NTS. HDMA+O3 exposure significantly decreased histamine H3 receptor immunoreactivity in the NTS at 6 months and in the caudal spinal trigeminal nucleus at 12 months of age. In conclusion, exposing young primates to HDMA+O3 changed histamine H3 receptor expression in CNS pathways involving lung and nasal afferent nerves in an age-related manner. Histamine H3 receptors may be a therapeutic target for allergic asthma and rhinitis in children.

Mast cell degranulation activates a pain pathway underlying migraine headache.

Intracranial headaches such as that of migraine are generally accepted to be mediated by prolonged activation of meningeal nociceptors but the mechanisms responsible for such nociceptor activation are poorly understood. In this study, we examined the hypothesis that meningeal nociceptors can be activated locally through a neuroimmune interaction with resident mast cells, granulated immune cells that densely populate the dura mater. Using in vivo electrophysiological single unit recording of meningeal nociceptors in the rat we observed that degranulation of dural mast cells using intraperitoneal administration of the basic secretagogue agent compound 48/80 (2 mg/kg) induced a prolonged state of excitation in meningeal nociceptors. Such activation was accompanied by increased expression of the phosphorylated form of the extracellular signal-regulated kinase (pERK), an anatomical marker for nociceptor activation. Mast cell-induced nociceptor interaction was also associated with downstream activation of the spinal trigeminal nucleus as indicated by an increase in c-fos expression. Our findings provide evidence linking dural mast cell degranulation to prolonged activation of the trigeminal pain pathway believed to underlie intracranial headaches such as that of migraine.

Substance P receptor (NK1)-immunoreactive neurons projecting to the periaqueductal gray: distribution in the spinal trigeminal nucleus and the spinal cord of the rat.

Substance P receptor (SPR)-immunoreactive neurons projecting to the periaqueductal gray (PAG) were examined in the rat spinal trigeminal nucleus and spinal cord by a retrograde tracing method combined with immunofluorescence histochemistry. After injection of Fluoro-gold (FG) into the PAG, SPR-immunoreactive neurons labeled with FG were observed mainly in the lateral spinal nucleus and lamina I of the medullary and spinal dorsal horns and additionally in laminae V and X of the spinal cord.

Substance P receptor-expressing neurons in the medullary and spinal dorsal horns projecting to the nucleus of the solitary tract in the rat.

By using substance P receptor (SPR) immunofluorescence histochemistry combined with fluorescent retrograde labeling, we examined the distribution of the trigeminal and spinal neurons with SPR-like immunoreactivity (-LI) projecting to the nucleus of the solitary tract in the rat. After injection of Fluoro-Gold (FG) into the nucleus of the solitary tract, FG-labeled neurons showing SPR-LI were mainly seen in lamina I of the medullary and spinal dorsal horns, lamina V and the lateral spinal nucleus of the spinal cord. The present results suggest that the trigeminal and spinal neurons with SPR-LI, especially those in lamina I may be involved in the transmission of somatic and/or visceral nociceptive information from the medullary and spinal dorsal horns to the nucleus of the solitary tract.

Adrenalectomy enhances Fos-like immunoreactivity within the spinal trigeminal nucleus induced by noxious thermal stimulation of the cornea.

Immunocytochemistry was used to assess the distribution of neurons within the spinal trigeminal nucleus that expressed the protein product of the proto-oncogene c-fos after thermal stimulation of the cornea in barbiturate-anesthetized rats. The influence of adrenal steroids on Fos-like immunoreactivity induced by corneal stimulation also was examined by comparison of the results obtained in adrenal intact rats to those in adrenalectomized rats and to those in adrenalectomized rats given corticosterone replacement therapy. Stimuli (42 or 52 degrees C, 20 s per min, 15 min) were applied unilaterally to the cornea by a contact thermode. At 2 h after stimulation animals were perfused with 4% paraformaldehyde and tissue sections were incubated with primary antiserum against the Fos protein and processed with the avidin-biotin method. The pattern of Fos-like immunoreactivity after 52 degrees C stimulation revealed a dominant group of cells ipsilaterally within the superficial laminae of the caudalmost portion of trigeminal subnucleus caudalis that was greatly enhanced in adrenalectomized rats. Low-intensity stimulation did not induce Fos-like immunoreactivity among cells in this caudal region. A second significant group of cells was seen more rostrally at periobex levels within the ventrolateral pole of the nucleus. The number of cells in the periobex grouping was increased after 52 or 42 degrees C corneal stimulation when compared to unstimulated controls and was not affected by levels of corticosterone. The results indicated a discontinuous pattern of Fos-like immunoreactivity within the spinal trigeminal nucleus after thermal stimulation of the cornea and a differential effect of adrenal steroids. The appearance of Fos-like immunoreactivity within caudal portions of the nucleus was increased only by noxious intensities of stimulation and was further enhanced in animals with low levels of corticosterone. In contrast, the Fos-like immunoreactivity at periobex levels was increased after noxious and innocuous thermal stimuli and was independent of the level of corticosterone. The results were consistent with the hypothesis that glucocorticoids modify the expression of immediate early genes among a select group of central trigeminal neurons. Such steroid modulation may contribute to the mechanisms that underlie long-term adaptation to noxious sensory input.

Loss of substance P immunoreactivity in the nucleus of the spinal trigeminal tract after intradural tumour compression of the trigeminal nerve.

The peptide, substance P (SP), is suspected of being a mediator of nociception. Immunoreactive SP is conspicuously present in areas of the mammalian central nervous system which receive nociceptive input. We have observed that compression of the trigeminal nerve by an intradural tumour deposit results in the ipsilateral depletion of SP immunoreactivity in human spinal trigeminal nucleus. These results expand previous experimental animal studies which associate this peptide with a subpopulation of primary sensory neurones and, in addition, demonstrate a correlation between the levels of SP immunoreactivity and premortem clinical sensory alterations.

Localization of Met-enkephalin-like immunoreactivity within pain-related nuclei of cervical spinal cord, brainstem and midbrain in the cat.

Met-enkephalin immunoreactivity was investigated with an indirect immunoperoxidase technique in the cervical spinal cord, brainstem and midbrain of the cat, paying special attention to pain-related nuclei. Different technical conditions were used to reveal preferentially met-enkephalin-containing fibres and terminals or perikarya. Immunoreactive fibres and terminals were revealed optimally in sections from control animals incubated with detergent (Triton X-100). Immunoreactive perikarya were revealed in colchicine treated animals. Comparison between different routes of administration showed that local injections of colchicine are needed to reveal optimally immunoreactive perikarya in nuclei located far from the ventricles. Met-enkephalin-containing fibres and terminals are widely distributed in the posterior brain and spinal cord. The densest network of immunoreactive fibers are observed in the superficial layers of the cervical spinal cord and the caudal trigeminal nucleus, in the nucleus of the solitary tract, the nucleus of the facial nerve, the nucleus of the prepositus hypoglossi, the nucleus raphe pallidus, the medial vestibular nucleus, the interpedoncular nucleus and the substantia nigra. A moderate staining of fibres is observed in various nuclei including the ventral horn of the spinal cord and caudal trigeminal nucleus, the brainstem and midbrain reticular formation, the inferior olivary complex, the nucleus of the descending trigeminal tract and the periaqueductal grey. Met-enkephalin-containing perikarya are present in all the nuclei cited before, except in the inferior olivary complex. The densest aggregation of enkephalin-like perikarya is observed in the nucleus raphe magnus, nucleus raphe obscurus, nucleus raphe pallidus, nucleus reticularis gigantocellularis pars alpha and nucleus reticularis lateralis. The general distribution of enkephalin-containing structures in the cervical spinal cord, brainstem and midbrain of the cat appears very similar to that of the rat except in the substantia nigra where met-enkephalin cell bodies are found in the cat but not in the rat. In particular the pain-related nuclei present a similar distribution of the peptide in the two species; however, met-enkephalin-containing cell bodies are much more numerous in the cat than in the rat (notably in the reticular formation). Similar types of met-enkephalin innervation occur in the dorsal and intermediate grey of the spinal cord and of the caudal trigeminal nucleus supporting further that the functional organizations of these regions are closely related.