The effect of capsaicin on expression patterns of CGRP in trigeminal ganglion and trigeminal nucleus caudalis following experimental tooth movement in rats

ABSTRACT Objectives The aim of this study was to explore the effect of capsaicin on expression patterns of calcitonin gene-related peptide (CGRP) in the trigeminal ganglion (TG) and trigeminal subnucleus caudalis (Vc) following experimental tooth movement. Material and Methods Male Sprague-Dawley rats were used in this study and divided into small-dose capsaicin+force group, large-dose capsaicin+force group, saline+force group, and no force group. Closed coil springs were used to mimic orthodontic forces in all groups except for the no force group, in which springs were inactivated. Capsaicin and saline were injected into periodontal tissues. Rats were euthanized at 0 h, 12 h, 1 d, 3 d, 5 d, and 7 d following experimental tooth movement. Then, TG and Vc were obtained for immunohistochemical staining and western blotting against CGRP. Results Immunohistochemical results indicated that CGRP positive neurons were located in the TG, and CGRP immunoreactive fibers were distributed in the Vc. Immunohistochemical semiquantitative analysis and western blotting analysis demonstrated that CGRP expression levels both in TG and Vc were elevated at 12 h, 1 d, 3 d, 5 d, and 7 d in the saline + force group. However, both small-dose and large-dose capsaicin could decrease CGRP expression in TG and Vc at 1 d and 3 d following experimental tooth movement, as compared with the saline + force group. Conclusions These results suggest that capsaicin could regulate CGRP expression in TG and Vc following experimental tooth movement in rats.

The effect of capsaicin on expression patterns of CGRP in trigeminal ganglion and trigeminal nucleus caudalis following experimental tooth movement in rats.

Objectives: The aim of this study was to explore the effect of capsaicin on expression patterns of calcitonin gene-related peptide (CGRP) in the trigeminal ganglion (TG) and trigeminal subnucleus caudalis (Vc) following experimental tooth movement. Material and Methods: Male Sprague-Dawley rats were used in this study and divided into small-dose capsaicin+force group, large-dose capsaicin+force group, saline+force group, and no force group. Closed coil springs were used to mimic orthodontic forces in all groups except for the no force group, in which springs were inactivated. Capsaicin and saline were injected into periodontal tissues. Rats were euthanized at 0 h, 12 h, 1 d, 3 d, 5 d, and 7 d following experimental tooth movement. Then, TG and Vc were obtained for immunohistochemical staining and western blotting against CGRP. Results: Immunohistochemical results indicated that CGRP positive neurons were located in the TG, and CGRP immunoreactive fibers were distributed in the Vc. Immunohistochemical semiquantitative analysis and western blotting analysis demonstrated that CGRP expression levels both in TG and Vc were elevated at 12 h, 1 d, 3 d, 5 d, and 7 d in the saline + force group. However, both small-dose and large-dose capsaicin could decrease CGRP expression in TG and Vc at 1 d and 3 d following experimental tooth movement, as compared with the saline + force group. Conclusions: These results suggest that capsaicin could regulate CGRP expression in TG and Vc following experimental tooth movement in rats.

Analysis of pain in the rabbit temporomandibular joint after unilateral splint placement.

AIMS: To determine whether behavioral, anatomical, and physiologic endpoints widely used to infer the presence of pain in rodent models of temporomandibular disorders (TMD) were applicable to the rabbit model of TMD associated with altered joint loading. METHODS: Unilateral molar dental splints were used to alter temporomandibular joint (TMJ) loading. Changes in nociceptive threshold were assessed with a mechanical probing of the TMJ region on nine splinted and three control rabbits. Fos-like immunoreacitivty in the trigeminal subnucleus caudalis was assessed with standard immunohistochemical techniques from three splinted and six control animals. Retrogradely labeled TMJ afferents were studied with patch-clamp electrophysiologic techniques from three splinted and three control animals. Remodeling of TMJ condyles was assessed by histologic investigations of three splinted and three control animals. A Student t test or a Mann-Whitney U test was used with significance set at P < .05 to compare splinted to control samples. RESULTS: While variable, there was an increase in mechanical sensitivity in splinted rabbits relative to controls. The increase in Fos+ cells in splinted rabbits was also significant relative to naïve controls (86 ± 8 vs 64 ± 15 cells/section, P < .05). The rheobase (364 ± 80 pA) and action potential threshold (-31.2 ± 2.0 mV) were higher in TMJ afferents from splinted rabbits compared to controls (99 ± 22 pA and -38.0 ± 2.0 mV, P < .05). There was significant remodeling in the condylar fibrocartilage layers as manifested by a change in glycosaminoglycan distribution and a loss of defined cell layers. CONCLUSION: Behavioral and anatomical results were consistent with an increase in nociceptive signaling in concert with condylar remodeling driven by altered TMJ loading. Changes in excitability and action potential waveform were consistent with possible compensatory changes of TMJ afferents for an overall increase in afferent drive associated with joint degeneration. These compensatory changes may reflect pain-adaption processes that many patients with TMJ disorders experience.

Substance P, tyrosine hydroxylase and serotonin terminals in the rat caudal nucleus ambiguus.

Substance P (SP), tyrosine hydroxylase (TH) and serotonin inputs onto laryngeal motoneurons (LMNs) are known to exist, but the distribution of their terminals in the caudal nucleus ambiguus (NA), remains unclear. Using immunofluorescence and confocal microscopy, we assessed simultaneously the distribution of SP, TH, serotonin and synaptophysin immunoreactive (ir) terminals in the caudal NA. SP, TH and serotonin-ir varicosities were considered to represent immunoreactive synapses if, using confocal microscopy, they were co-localized with the presynaptic protein, synaptophysin. Relative to the total number of synapses, we found only a modest number of SP, TH or serotonin-ir synaptic terminals in the caudal NA. The density of SP-ir synaptic terminals was higher than that of TH-ir and serotonin-ir synaptic terminals. Our results suggest that SP, TH, and serotonin-ir inputs may play only a modest role in regulating the activity of LMN. We conclude that SP, TH and serotonin are not always co-localized in terminals forming inputs with LMN and that they arise from separate subpopulations of neurons.

Astroglia in medullary dorsal horn (trigeminal spinal subnucleus caudalis) are involved in trigeminal neuropathic pain mechanisms.

The aim of this study was to investigate whether astroglia in the medullary dorsal horn (trigeminal spinal subnucleus caudalis; Vc) may be involved in orofacial neuropathic pain following trigeminal nerve injury. The effects of intrathecal administration of the astroglial aconitase inhibitor sodium fluoroacetate (FA) were tested on Vc astroglial hyperactivity [as revealed by glial fibrillary acid protein (GFAP) labeling], nocifensive behavior, Vc extracellular signal-regulated kinase phosphorylation (pERK), and Vc neuronal activity in inferior alveolar nerve-transected (IANX) rats. Compared with sham-control rats, a significant increase occurred in GFAP-positive cells in ipsilateral Vc at postoperative day 7 in IANX rats, which was prevented following FA administration. FA significantly increased the reduced head withdrawal latency to high-intensity heat stimulation of the maxillary whisker pad skin in IANX rats, although it did not significantly affect the reduced escape threshold to low-intensity mechanical stimulation of the whisker skin in IANX rats. FA also significantly reduced the increased number of pERK-like immunoreactive cells in Vc and the enhanced Vc nociceptive neuronal responses following high-intensity skin stimulation that were documented in IANX rats, and glutamine administration restored the enhanced responses. These various findings provide the first documentation that astroglia is involved in the enhanced nociceptive responses of functionally identified Vc nociceptive neurons and in the associated orofacial hyperalgesia following trigeminal nerve injury.

Vagus nerve stimulation in awake rats reduces formalin-induced nociceptive behaviour and fos-immunoreactivity in trigeminal nucleus caudalis.

Besides its well-established efficacy in epilepsy, vagus nerve stimulation (VNS) may be of potential interest in pain treatment. It has, however, not yet been assessed in animal pain models with the devices and stimulation protocols used in humans. We have therefore studied in awake rats the effects of left cervical VNS on trigeminal nociception using an implantable electrode and stimulator (NCP-Cyberonics). VNS was applied for 24h at 2 mA intensity, 20 Hz frequency, 0.5 ms pulse width and a duty cycle of 20s ON/18s OFF. As a nociceptive stimulus, we injected formalin into the left mystacial vibrissae, assessed behaviour for 45 min and sacrificed the animals 45 min later. Fos-immunoreactive (Fos-Ir) neurons were counted in laminae I-II of trigeminal nucleus caudalis (TNC) on both sides. We used three groups of control animals: VNS without formalin, formalin without VNS and sham VNS (implanted without stimulation or formalin). Whereas sham VNS had no significant effect, VNS alone increased Fos expression in ipsilateral TNC in addition to the expected increase in nucleus tractus solitarius. It also significantly attenuated the increase of Fos-Ir neurons observed in ipsilateral TNC laminae I-II after formalin injection. If the proper VNS effect on Fos-expression was subtracted, the reduction of formalin-induced nociceptor activation was 55%. VNS also reduced nociceptive behaviour on average by 96.1% during the early phase (0-6 min) and by 60.7% during the late phase (6-45 min) after the formalin injection. These results suggest that VNS applied with a device used in human therapy may have in awake rats a significant antinociceptive effect in a model of trigeminal pain.

Preproenkephalin-like immunoreactive and calcium-binding proteins-like immunoreactive double-labelled neurons in the spinal trigeminal nucleus caudalis of the rat.

Immunofluorescence histochemical double-staining for preproenkephalin (PPE) and calbindin-D28k (CB), calretinin (CR) or parvalbumin (PV) were performed in the spinal trigeminal nucleus caudalis (Vc) of the rat. Neuronal cell bodies exhibiting PPE-like immunoreactivity were present in all laminae of the Vc, with a higher concentration in lamina II. Most of the CB-, CR- and PV-like immunoreactive neurons were located in lamina II, and some of them were also found in laminae I and III of the Vc. Some PPE-like immunoreactive neurons also showed CB-, CR-, or PV-like immunoreactivities. CB/PPE, CR/PPE and PV/PPE double-labelled neurons were mainly observed in lamina II. The percentages of CB/PPE double-labelled neurons in the total numbers of the CB- and PPE-like immunoreactive neurons were 3.5-1.5% and 3.3-15.7%, respectively. Of all CR- and PPE-like immunoreactive neurons, 4.7-13.5% and 3.7-14.2% showed both CR- and PPE-like immunoreactivities. The ratios of PV/PPE double-labelled neurons in all PV- and PPE-like immunoreactive neurons were 9.7-28.1% and 2.1-8.7%, respectively. The present results indicate that some enkephalinergic neurons in the Vc of the rat also contain calcium-binding proteins.

Role of neuronal nicotinic-acetylcholine receptors in the activation of neurons in trigeminal subnucleus caudalis by nicotine delivered to the oral mucosa.

To characterize the role of neuronal nicotinic acetylcholine receptors (nAChRs) in oral irritation and pain, we employed the method of c-fos immunohistochemistry to map the locations and numbers of brainstem neurons that express the immediate-early gene, c-fos, after application of nicotine to the tongue, either alone or after pretreatment with cholinergic antagonists. Groups of anesthetized rats received the following chemicals delivered bilaterally to the dorsal tongue: (1) 0.9% NaCl followed by nicotine (1%, 61 mM), (2) the nAChR antagonist, mecamylamine 0.1% (= 4.9 mM) followed by nicotine, (3) the muscarinic antagonist atropine (0.1% 1.46 mM) followed by nicotine, (4) atropine (1%, 14.6 mM) followed by nicotine, (5) 0.9% NaCl as a control, and (6) unstimulated controls. Two hours later, animals were perfused with phosphate-buffered saline followed by 4% paraformaldehyde through the aorta. Post-fixed brainstems were cut in 50-micron frozen sections and immunohistochemically processed for fos-like immunoreactivity (FLI). Following application of nicotine, there were significant increases in FLI compared with saline-treated controls in dorsomedial and ventrolateral aspects of the trigeminal caudalis. Pretreatment with either mecamylamine or the high (1%) concentration of atropine significantly reduced nicotine-evoked FLI in these areas, while pretreatment with the low (0.1%) atropine concentration did not significantly affect FLI. These results are consistent with the idea that nicotine activates nAChRs residing on lingual nociceptive fibers, which, in turn, excite neurons in trigeminal caudalis.

gamma-aminobutyric acid- and glycine-immunoreactive neurons postsynaptic to substance P-immunoreactive axon terminals in the superficial layers of the rat medullary dorsal horn.

gamma-Aminobutyric acid (GABA)ergic and glycinergic neurons were examined light- and electron-microscopically in laminae I and II of the medullary dorsal horn (MDH, i.e. spinal trigeminal nucleus caudalis in the rat). The majority of GABA- and glycine (Gly)-immunoreactive (-ir) neurons showed both GABA- and Gly-immunoreactivities (-IRs). Noxious stimulation (subcutaneous injection of formalin into perioral regions) induced Fos-IR in some of GABA- and Gly-ir neurons. GABA- and Gly-ir neuronal profiles were postsynaptic to substance P-ir axon terminals. These results suggest that nociceptive information being carried by primary afferent SP-fibers may be relayed directly to GABAergic and glycinergic neurons in laminae I and II of the MDH.

Calcium-binding protein-immunoreactive projection neurons in the caudal subnucleus of the spinal trigeminal nucleus of the rat.

It has been reported that calcium-binding proteins are good markers for different sets of neurons in various brain regions. We examined expression of the main calcium-binding proteins in projection neurons in the rat medullary dorsal horn (MDH) by combining immunofluorescence histochemistry for calbindin D28k (CB), calretinin (CR) and parvalbumin (PV) with the retrograde tract-tracing method. A fluorescence tracer, tetramethylrhodamine-dextran amine (TMR-DA), was injected into the parabrachial, thalamic or hypothalamic region. After such injections, a number of PV-, CR-, and/or CB-immunoreactive MDH neurons were labeled retrogradely with TMR-DA. Triple-immunofluorescence histochemistry further revealed that a number of CB-, CR-, or PV-immunoreactive TMR-DA-labeled MDH neurons showed immunoreactivity for substance P receptor (NK1), and that they expressed immunoreactivity for c-fos protein in the rats which were injected with formalin into the lips. Thus, it was indicated that some of CB-, CR-, or PV-containing projection neurons in the MDH might be involved in the transmission of nociceptive stimuli.

Distribution of the low-affinity neurotrophin receptor (p75) in the developing trigeminal brainstem complex in the rat.

The low-affinity neurotrophin receptor (p75) binds all members of the neurotrophin family. In the rat, during the first week postpartum, dense p75-immunoreactivity (IR) is present throughout all components of the trigeminal brainstem complex (TBC), largely associated with primary sensory afferents. Within subnucleus caudalis (SpC) of the TBC, intense p75-IR is present in all laminae at birth. During the second and third postnatal weeks, p75-IR in SpC gradually fades within the deeper laminae, becoming generally restricted in the adult to laminae I and II. Similar declines in p75-IR intensity occur in the subnucleus oralis (SpO); in the SpO in the adult, p75-IR is confined to the dorsalmost portion of SpO. In subnucleus interpolaris, an emerging, vibrissa-related pattern of p75-IR is detectable on PD0 (first 24 hr postpartum), which becomes fully differentiated during PD4-PD7. However, this pattern gradually disappears during the third postnatal week. Ventrally in the nucleus principalis (PrV), a pattern of p75-IR that mirrors the topographical arrangement of the vibrissae is detectable by PD0-PD1, is fully differentiated by the end of the first postnatal week, and persists into adulthood. Perinatal unilateral sectioning of the infraorbital nerve on PD0-PD1, but not as late as PD4, disrupts p75-IR patterning in the adult PrV. Although p75 appears to be associated with primary afferent pattern formation, to determine whether it is essential, we examined mutant mice unable to form functional p75. In the TBC of these knockout mice, examined as adults, patterns of cytochrome oxidase staining (which parallel those of p75-IR) appeared to be normal. In summary, during early development, p75 is widely expressed in the TBC during periods of active synaptogenesis and pattern formation, whereas in the adult, its expression is restricted to association with populations of primary sensory afferents. However, the absence of functional p75 in genetically altered mice does not appear to prevent primary afferent pattern formation.

Differential expression of Fos protein after transection of the rat infraorbital nerve in the trigeminal nucleus caudalis.

To determine the effects of nerve injury on Fos expression, temporal and spatial distributions of Fos-positive neurons in the trigeminal nucleus caudalis were examined after tissue injury for isolation of the infraorbital nerve as controls and transection of this nerve as well as noxious chemical stimulation by formalin injection in adult rats. Fos immunoreactivity was markedly elevated in laminae I and II of the only ipsilateral nucleus caudalis 2 h after these surgical procedures and noxious chemical stimulation. The distributions of Fos-positive neurons were restricted rostro-caudally following formalin injection and tissue injury compared to transection of the infraorbital nerve. One day after tissue injury and nerve transection, however, Fos-positive neurons were distributed bilaterally in laminae III and IV extending rostro-caudally and medio-laterally in this nucleus, and this persisted over the 2-week study period. The number of Fos-positive neurons in the side ipsilateral to nerve transection was markedly less than that in the contralateral side whereas positive neurons in the tissue injured rats were distributed symmetrically along the rostro-caudal axis. There was no difference in the contralateral sides between nerve transection and tissue injury groups. The rostro-caudal level showing reduction in Fos expression corresponded roughly to the sites of central termination of the injured nerve in this nucleus, suggesting a role for the primary afferents in the reduction of Fos expression in laminae III and IV neurons of the ipsilateral nucleus caudalis.

Central origins of substance P-like immunoreactive fibers and terminals in the spinal trigeminal caudal subnucleus in the rat.

After trigeminal rhizotomy, some substance P-like immunoreactive (SP-LI) fibers and terminals in the spinal trigeminal caudal subnucleus (Vc), specially in its superficial laminae (laminae I and II), still remained in the rat. Employing a combination of Fluoro-Gold retrograde tracing and immunofluorescence histochemical staining for SP, we found that the main central origins of these SP-LI fibers and terminals were midbrain periaqueductal gray (PAG), nucleus raphe magnus (NRM) and other raphe nuclei, and nucleus reticularis gigantocellularis pars alpha; all of them are important structures of the endogenous pain control system. The present results provided morphological evidence for PAG or NRM stimulation could inhibit neuronal activities in the Vc evoked by orofacial nociceptive stimulation and also suggested that SP might be an important neurotransmitter or neuromodulator for endogenous pain control system.

trk-like immunoreactivity in the human trigeminal ganglion and subnucleus caudalis.

The immunohistochemical occurrence of trkA, trkB and trkC receptors was examined in the human trigeminal ganglion and spinal nucleus of subjects at all ages and compared with that of substance P (SP) and calcitonin gene-related peptide (CGRP), trk-like immunoreactive (LI) material was detectable in discrete subpopulations of primary sensory neurones from 25 weeks of gestation to adult life. Each subpopulation overlapped partially with those immunoreactive to SP and CGRP, trkA- and trkC-positive filamentous and punctate elements occurred in the trigeminal subnucleus caudalis. While immunostaining for trkC was restricted to rare isolated elements, that for trkA outlined the superficial laminae of the nucleus and was more intense early in life than in adults.

Trigeminoparabrachial projection neurons showing substance P receptor-like immunoreactivity in the rat.

By means of substance P receptor (SPR) immunofluorescence histochemistry combined with Fluoro-Gold fluorescent retrograde labeling, SPR-like immunoreactive neurons in the caudal subnucleus of the spinal trigeminal nucleus of the rat were observed to send their axons to the nucleus of Kölliker-Fuse and ventrolateral part of the lateral parabrachial nucleus bilaterally with a clear ipsilateral dominance. These neurons were distributed mainly in lamina I, and additionally in lamina III.

Morphological characterization of substance P receptor-immunoreactive neurons in the rat spinal cord and trigeminal nucleus caudalis.

Although there is considerable evidence that primary afferent-derived substance P contributes to the transmission of nociceptive messages at the spinal cord level, the population of neurons that expresses the substance P receptor, and thus are likely to respond to substance P, has not been completely characterized. To address this question, we used an antibody directed against the C-terminal portion of the rat substance P receptor to examine the cellular distribution of the receptor in spinal cord neurons. In a previous study, we reported that the substance P receptor decorates almost the entire dendritic and somatic surface of a subpopulation of spinal cord neurons. In the present study we have taken advantage of this labeling pattern to identify morphologically distinct subpopulations of substance P receptor-immunoreactive neurons throughout the rostral-caudal extent of the spinal cord. We observed a dense population of fusiform substance P receptor-immunoreactive neurons in lamina I at all segmental levels. Despite having the highest concentration of substance P terminals, the substantia gelatinosa (lamina II) contained almost no substance P receptor-immunoreactive neurons. Several distinct populations of substance P receptor-immunoreactive neurons were located in laminae III-V; many of these had a large, dorsally directed dendritic arbor that traversed the substantia gelatinosa to reach the marginal layer. Extensive labeling was also found in neurons of the intermediolateral cell column. In the ventral horn, we found that labeling was associated with clusters of motoneurons, notably those in Onuf's nucleus in the sacral spinal cord. Finally, we found no evidence that primary afferent fibers express the substance P receptor. These results indicate that relatively few, but morphologically distinct, subclasses of spinal cord neurons express the substance P receptor. The majority, but not all, of these neurons are located in regions that contain neurons that respond to noxious stimulation.

[The role of zinc ions in the pathogenesis of trigeminal neuralgia (experimental and clinical research)]. / Rol' ionov tsinka v patogeneze trigeminal'noi nevralgii (éksperimental'no-klinicheskoe issledovanie).

In order to make clear the role of zinc (Zn) ions in the pathogenesis of trigeminal neuralgia (TN) the investigations of rats with the compression model of TN as well as on false operated animals were carried out. In 1.5 months after the intraorbital nerve compression the increase of Zn concentration in caudal trigeminal nucleus and (to a less degree) in hippocampus as well as behavioural and pathophysiological pain correlates were revealed. The exogenous Zn introduction led to an increase of its level in these structures simultaneously with the exacerbation of all sings of the TN syndrome. The addition of chelating agents Xydiphone which bound bivalent cations in soft tissues, into drinking water, normalized the Zn content in rats brain and reduced the pathological symptoms. The use of Xydiphone in the complex therapy of 25 patients with the TN tolerant to carbamazepine reduced the pain syndrome in 15 cases. Thus the local changes of Zn level in the CNS may play a certain role in the manifestation of the TN pain syndrome.

Caudal portions of the spinal trigeminal complex are necessary for autonomic responses and display Fos-like immunoreactivity after corneal stimulation in the cat.

Corneal input to the spinal trigeminal nucleus (Vsp) was assessed by examining Fos-like immunoreactivity (Fos-LI) after chemical irritant stimulation by mustard oil in chloralose-anesthetized cats. The distribution of Fos-LI within the ipsilateral Vsp was bimodal: a dominant group of cells within the superficial laminae at caudal levels of subnucleus caudalis and a second group of cells within the ventrolateral pole of Vsp at obex levels and within the adjacent interstitial islands. Few Fos-positive cells were seen within the Vsp rostral to the mid-portion of subnucleus interpolaris or within the contralateral Vsp. To assess the involvement of caudal portions of the Vsp in mediating the adrenal and autonomic responses to corneal stimulation, mustard oil was applied before and after lidocaine blockade of the Vsp at obex levels in a second group of cats. Corneal stimulation alone increased significantly (P < 0.001) the adrenal secretion of catecholamines, adrenal blood flow, mean arterial pressure and heart rate. With the exception of heart rate, the adrenal and autonomic responses to mustard oil were greatly attenuated or abolished by lidocaine blockade of the ipsilateral Vsp at the level of the obex, a region that displayed a high number of Fos-positive cells after corneal stimulation. These results indicate that neurons within the Vsp at or more caudal than the level of the obex process chemical irritant input from the cornea and are necessary for corneal-evoked changes in adrenal and autonomic function.

Biochemical and anatomical consequences of adult infraorbital nerve transection for serotonergic afferents within rat trigeminal subnuclei interpolaris and caudalis.

Immunocytochemistry and high-performance liquid chromatography with electrochemical detection (HPLC-ED) were used, more than 76 days after infraorbital nerve (ION) transection, to examine the distribution and density of serotonin-immunoreactive (5-HTIR) axons, as well as serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) content, within the infraorbital (IO) regions of subnuclei caudalis (SpVc) and interpolaris (SpVi). In SpVi, increases in 5-HT concentration and in density of 5-HTIR axonal varicosities were observed on the lesioned side. No changes were seen in SpVc.