TRPA1 receptor is upregulated in human oral lichen planus.

OBJECTIVE: Oral lichen planus (OLP) is a chronic inflammatory disease of unknown etiology with antigen-specific and non-specific mechanisms. Transient receptor potential ankyrin 1 (TRPA1) is a non-selective cation channel activated by noxious stimuli such as oxidative stress products evoking pain and release of proinflammatory mediators from sensory nerve endings culminating in neurogenic inflammation. Extraneuronal TRPA1s, for example, on immune cells possess yet unknown functions. SUBJECTS AND METHODS: We studied the buccal mRNA expression (qPCR) and protein localization (immunohistochemistry) of TRPA1 receptors and key OLP mediator transcripts in oral mucosa samples of healthy volunteers (n = 9), OLP patients (n = 43), and OLP-like hyperkeratotic patients (n = 12). RESULTS: We measured 27.7- and 25.5-fold TRPA1 mRNA increase in OLP and OLP-like hyperkeratotic patients compared to healthy controls. TRPA1 transcripts elevated 2.4-fold in hypertensive OLP but not in hyperkeratotic patients compared to counterparts, reduced by 1.6-fold by angiotensin-convertase inhibitor intake. TRPA1 messenger RNA was more coexpressed with transcripts of tumor necrosis factor α than with interferon γ. Keratinocytes, macrophages but not T cells expressed TRPA1. CONCLUSIONS: We provided evidence for the extraneuronal presence and upregulation of the proinflammatory TRPA1 receptor in buccal samples of patients with OLP. This may implicate the ion channel in the pathomechanism of OLP.

Estrogen-induced activation of the mitogen-activated protein kinase cascade in the cerebral cortex of estrogen receptor-alpha knock-out mice.

We have shown previously in the developing cerebral cortex that estrogen elicits the rapid and sustained activation of multiple signaling proteins within the mitogen-activated protein (MAP) kinase cascade, including B-Raf and extracellular signal-regulated kinase (ERK). Using estrogen receptor (ER)-alpha gene-disrupted (ERKO) mice, we addressed the role of ER-alpha in mediating this action of estrogen in the brain. 17beta-Estradiol increased B-Raf activity and MEK (MAP kinase/ERK kinase)-dependent ERK phosphorylation in cerebral cortical explants derived from both ERKO and their wild-type littermates. The ERK response was stronger in ERKO-derived cultures but, unlike that of wild-type cultures, was not blocked by the estrogen receptor antagonist ICI 182,780. Surprisingly, both the ER-alpha selective ligand 16alpha-iodo-17beta-estradiol and the ER-beta selective ligand genistein failed to elicit ERK phosphorylation, suggesting that a different mechanism or receptor may mediate estrogen-induced ERK phosphorylation in the cerebral cortex. Interestingly, the transcriptionally inactive stereoisomer 17alpha-estradiol did elicit a strong induction of ERK phosphorylation, which, together with the inability of the ER-alpha- and ER-beta-selective ligands to elicit ERK phosphorylation, and of ICI 182,780 to block the actions of estradiol in ERKO cultures, supports the hypothesis that a novel, estradiol-sensitive and ICI-insensitive estrogen receptor may mediate 17beta-estradiol-induced activation of ERK in the brain.

Novel mechanisms of estrogen action in the brain: new players in an old story.

Estrogen elicits a selective enhancement of the growth and differentiation of axons and dendrites (neurites) in the developing brain. Widespread colocalization of estrogen and neurotrophin receptors (trk) within estrogen and neurotrophin targets, including neurons of the cerebral cortex, sensory ganglia, and PC12 cells, has been shown to result in differential and reciprocal transcriptional regulation of these receptors by their ligands. In addition, estrogen and neurotrophin receptor coexpression leads to convergence or cross-coupling of their signaling pathways, particularly at the level of the mitogen-activated protein (MAP) kinase cascade. 17beta-Estradiol elicits rapid (within 5-15 min) and sustained (at least 2 h) tyrosine phosphorylation and activation of the MAP kinases, extracellular-signal regulated kinase (ERK)1, and ERK2, which is successfully inhibited by the MAP kinase/ERK kinase 1 inhibitor PD98059, but not by the estrogen receptor (ER) antagonist ICI 182,780 and also does not appear to result from estradiol-induced activation of trk. Furthermore, the ability of estradiol to phosphorylate ERK persists even in ER-alpha knockout mice, implicating other estrogen receptors such as ER-beta in these actions of estradiol. The existence of an estrogen receptor-containing, multimeric complex consisting of hsp90, src, and B-Raf also suggests a direct link between the estrogen receptor and the MAP kinase signaling cascade. Collectively, these novel findings, coupled with our growing understanding of additional signaling substrates utilized by estrogen, provide alternative mechanisms for estrogen action in the developing brain which could explain not only some of the very rapid effects of estrogen, but also the ability of estrogen and neurotrophins to regulate the same broad array of cytoskeletal and growth-associated genes involved in neurite growth and differentiation. This review expands the usually restrictive view of estrogen action in the brain beyond the confines of sexual differentiation and reproductive neuroendocrine function. It considers the much broader question of estrogen as a neural growth factor with important influences on the development, survival, plasticity, regeneration, and aging of the mammalian brain and supports the view that the estrogen receptor is not only a ligand-induced transcriptional enhancer but also a mediator of rapid, nongenomic events.

Estrogen-induced activation of mitogen-activated protein kinase in cerebral cortical explants: convergence of estrogen and neurotrophin signaling pathways.

We have shown that estrogen elicits a selective enhancement of the growth and differentiation of axons and dendrites (neurites) in the developing CNS. We subsequently demonstrated widespread colocalization of estrogen and neurotrophin receptors (trk) within developing forebrain neurons and reciprocal transcriptional regulation of these receptors by their ligands. Using organotypic explants of the cerebral cortex, we tested the hypothesis that estrogen/neurotrophin receptor coexpression also may result in convergence or cross-coupling of their signaling pathways. Estradiol elicited rapid (within 5-15 min) tyrosine phosphorylation/activation of the mitogen-activated protein (MAP) kinases, ERK1 and ERK2, that persisted for at least 2 hr. This extracellular signal-regulated protein kinase (ERK) activation was inhibited successfully by the MEK1 inhibitor PD98059, but not by the estrogen receptor (ER) antagonist ICI 182,780, and did not appear to result from estradiol-induced activation of trk. Furthermore, we also found that estradiol elicited an increase in B-Raf kinase activity. The latter and subsequent downstream events leading to ERK activation may be a consequence of our documentation of a multimeric complex consisting of, at least, the ER, hsp90, and B-Raf. These novel findings provide an alternative mechanism for some of the estrogen actions in the developing CNS and could explain not only some of the very rapid effects of estrogen but also the ability of estrogen and neurotrophins to regulate the same broad array of cytoskeletal and growth-associated genes involved in neurite growth and differentiation.

Gonadotropin-releasing hormone neuroblasts from one olfactory placode can be present in both hemispheres in the clawed toad Xenopus laevis.

Ontogenetic differentiation of the GnRH-immunoreactive (GnRHir) neuron system was studied in the clawed toad Xenopus laevis by immunocytochemistry employing polyclonal antibodies against mammalian GnRH and chicken type II GnRH, and monoclonal antibodies against GnRH exhibiting wide cross-reactivity over animal classes. Toads at different stages of differentiation as well as postmetamorphic toads subjected to uni- or bilateral ablation of the olfactory placode (OPX) between developmental stages 25 and 30 were studied. GnRHir neurons and nerve fibers could not be detected before metamorphosis. Following metamorphosis, at stage 65-66, hemi-OPX toads did not exhibit any side differences in the number and overall distribution of the GnRHir neuronal structures; however, the total number of GnRHir neurons was approximately 50% of that counted in intact controls at the same developmental stages. These findings indicate that GnRHir neuroblasts differentiating on one side in the olfactory placode can appear on both sides of the brain in the course of their migration.

Distribution of Fluoro-Gold (FG) in the central nervous system of the rat following its injection into the cerebello-medullary cistern.

Fluoro-Gold (FG) injected into the cerebello-medullary cistern (CMC) is accumulated in several nuclei and neurons of the brain. These structures include: taenia tecta, some fusiform perikarya in the diagonal band of Broca, pyramidal cells in the fifth layer of the cerebral cortex, paraventricular nuclei, zona incerta, medial habenular nuclei, red nuclei, dorsal tegmental nuclei, median raphe nucleus, nuclei pontis, cochlear and vestibular nuclei and some Purkinje cells in the most lateral segment of the cerebellar cortex. The supraoptic nucleus and the median eminence are free of FG. Many nuclei in the myelencephalon and large groups of neurons in the dorsal, intermediate and anterior horns of the spinal cord are also labeled with FG, but exact identification of these cell groups needs further experimentation. Our findings indicate that FG can be picked up from the subarachnoid space into many cell groups of the brain and spinal cord. It is concluded that possible leaking of FG into the subarachnoid space following its injection into the central nervous system has to be taken into account whenever FG is used in tract tracing experiments.

The gonadotropin-releasing hormone (GnRH) neuron system of the clawed toad Xenopus laevis.

The GnRH immunoreactive (GnRH-ir) neuronal system of the Clawed toad Xenopus laevis was studied and compared with the GnRH-ir system of the frog Rana esculenta. Polyclonal antibodies against mammalian (mGnRH) and chicken type-II GnRH (cGnRH-II), and monoclonal antibodies against mGnRH were used in the study. In the Xenopus laevis, most of the immunopositive neuronal cell bodies were located in telencephalic (35-50 per cent) and diencephalic areas (50-65 per cent). About 15-20 per cent of the GnRH perikarya appeared in mesencephalic tegmental regions. Besides the larger GnRH fiber tracts present also in mammals, the toad has rich mGnRH immunopositive axon population in the mesencephalon and in the upper part of the medulla. A similar distribution of the GnRH-ir neuronal elements exists in Rana esculenta, but the number of stained cells and fibers was less. Specificity of the staining of cGnRH-IIir structures located in the lower brainstem could not be proved and therefore the study is only restricted to the findings with mGnRH-antibodies.

Ontogenesis of the GnRH neuron system in the rat. A quantitative immunohistochemical study with special reference to the extra cerebral GnRH-positive cells and the occupation of intracerebral termination fields.

The distribution of GnRH immunopositive cells in the rat was studied from day 13 of fetal life to day 10 of postnatal life with the aid of a GnRH antibody (H-16) generated against the decapeptide p-Glu1-LHRH in the rabbit and following a PAP immunohistochemical staining protocol on 70 um serial Vibratome sections. The first appearance of GnRH cells was observed simultaneously in both intra- and extra-cerebral regions on day 16 of fetal life. At all stages of development, at least 10 times more GnRH cells were found in the brain than in extracerebral areas. The population of GnRH cells was maximum in the nasal mucosa on day 20, and in intracranial, extracerebral regions (along the anterior cerebral artery and the terminal nerve) on day 22 of fetal life: it declined thereafter. The population of GnRH cells in the brain continued to increase until the day of birth and remained unchanged thereafter. Our findings are in accordance with the theory of nasal placodal origin of the GnRH neuronal system, but also suggest that in the rat, shifting of the cells from the nasal placode to the brain is somewhat preceding the onset of GnRH synthesis. Our other field of endeavor was to examine the GnRH axon terminal fields in the course of development. On day 18 of fetal life, the vascular organ of the lamina terminalis, the median eminence and the medial habenular nuclei received their GnRH axon terminals. On day 19 the subfornical organ, mammillary nuclei and the central nucleus of the amygdala, and on day 21 the periaqueductal gray and the interpeduncular nucleus became invaded by GnRH axons. These data serve the better understanding of the onset of the different functional activities of the GnRH system in the rat.

Translingual approach of the basal surface of the diencephalon of the rat and retrograde labeling from the median eminence.

A unique surgical procedure is described by which the basal surface of the diencephalon can be exposed without cannulation of the trachea. The basal surface of the diencephalon is exposed through a midsagittal incision by splitting the oral diaphragm, tongue, and soft palate respectively. Then a small hole is drilled in the base of the skull. After manipulation on the base of the brain is completed, the hole in the skull is plugged with Histoacryl and the soft palate and tongue are sutured. Rats having such interventions survive in excellent condition for days without the need of intensive medical care.

Fetal pituitary graft is capable of initiating hormone synthesis in median eminence removed adult rat.

Rathke's pouches of 12- and 13-day-old rat embryos were implanted beneath the kidney capsule of adult male rats subjected to the removal of median eminence or to sham-operation. Host animals were sacrificed 28 days after grafting and the implanted pituitaries were processed for immunohistological examination. ACTH, LH-beta, FSH-beta, TSH-beta, GH and PRL immunopositive cells could be observed in fetal grafts of all experimental groups. However, the number and staining intensity of different hormone containing cells largely varied and presumably depended on the hormonal state of host animals. The results indicate that undifferentiated fetal pituitary does not require hypothalamic hypophysiotrophic neurohormones for proliferation and cytodifferentiation and that its development might be modulated by circulating trophic hormones of host animals.

Combined retrograde tracing and immunocytochemical identification of luteinizing hormone-releasing hormone- and somatostatin-containing neurons projecting to the median eminence of the rat.

LHRH and somatostatin or somatotropin-release inhibiting factor (SRIF) are produced by neurons whose cell bodies are located in telencephalic and diencephalic regions in the rat. Many, but not all, of these neurons project to the external zone of the median eminence (ME), where the peptides are released from the nerve terminals into hypophysial portal vessels. In the present study, we identified these neurons by in vivo injection of a retrograde tracer, the lectin wheat germ agglutinin (WGA), into the external zone of the ME. Subsequently, colchicine was given into the lateral ventricle 10-24 h after the WGA injection. The animals were killed 24-48 h after the WGA injection. Vibratome sections of the brains were stained for both WGA and LHRH or SRIF with a dual immunocytochemical technique. Approximately 70% of the LHRH neurons in the septum and the anterior hypothalamus and about 70% of the SRIF neurons in the medial preoptic area, the anterior periventricular area, and the paraventricular nucleus were double labeled, indicating that they projected to the ME. None of the SRIF neurons in the ventromedial and arcuate nuclei were labeled with WGA. Double labeled LHRH cells were either smooth and fusiform or spiny. WGA-accumulating LHRH or SRIF perikarya were intermixed with single labeled LHRH or SRIF cells, which apparently did not project to the ME. The results indicate that there are at least two populations of LHRH neurons in the preoptic-septal region and two populations of SRIF neurons in the medial preoptic and anterior periventricular areas and the paraventricular nucleus of the rat brain: one with access to the portal capillaries of the ME and, therefore, functionally related to the regulation of the pituitary, and another without access to portal capillaries, perhaps functionally related to intracerebral neurotransmission or modulation. Moreover, some hypophysiotropic LHRH and SRIF neurons may have axon collaterals reaching multiple targets within the central nervous system.

Identification of hypophysiotropic luteinizing hormone-releasing hormone (LHRH) neurons by combined retrograde labeling and immunocytochemistry.

The decapeptide luteinizing hormone-releasing hormone (LHRH) is produced by telencephalic and diencephalic neurons and transported to the median eminence (ME). After having been released from nerve terminals, it is carried by the hypophysial portal vessels to the anterior pituitary, where it stimulates the production and release of luteinizing hormone (LH) and follicle stimulating hormone (FSH). Those LHRH neurons which project to the ME represent the final common pathway for the regulation of the pituitary/gonadal axis. We identified these neurons by injecting a retrograde tracer, the lectin wheat germ agglutinin (WGA), into the external zone of the ME. Eight to 24 hours later colchicine was given into the lateral ventricle and 24-48 hours after the WGA injection the animals were sacrificed. Vibratome sections of the brains were stained simultaneously for WGA and LHRH with a dual immunocytochemical technique. Approximately 70% of the LHRH neurons in the septum and the anterior hypothalamus were double-labeled, indicating that they projected to the ME. Double labeled LHRH cells were either smooth, fusiform or "spiny". WGA-accumulating LHRH perikarya were intermixed with single-labeled LHRH cells. The remaining 30% of the LHRH neurons which were not labeled with WGA appeared to project to different hypothalamic and extrahypothalamic areas of the brain. Our results suggest that there are at least two populations of LHRH neurons, one with access to the portal capillaries of the ME and functionally related to the regulation of the pituitary, and one without access to capillaries of the ME, functionally probably related to intracerebral neurotransmission or modulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Ontogenesis of the three parts of the fetal rat adenohypophysis. A detailed immunohistochemical analysis.

The sequential changes in the histological pattern of anterior pituitary cytodifferentiation of the rat are described. The first labeled cells were ACTH positive and were detected in the pars tuberalis on postconceptual day 13. On day 14 ACTH cells also appeared in the ventral periphery of the pars distalis. On fetal day 15 the pars tuberalis anlage was characterized by numerous well-stained ACTH cells and by some weakly labeled FSH-beta, LH-beta, TSH-beta, GH and PRL cells while the pars distalis showed only ACTH positivity. On day 16 of gestation the ACTH cells were equally distributed throughout the whole pars distalis, while LH-beta, FSH-beta, TSH-beta, PRL and GH immunoreactive cells were localized either in the ventral region of the pars distalis only or were evenly distributed throughout the pars distalis. The present immunocytochemical data suggest that in the pars distalis the hypophyseal cell differentiation follows a clear rostrocaudal, ventrodorsal direction and that the time sequence of the functional differentiation of the adenohypophysis is pars tuberalis, pars distalis and pars intermedia.

Axovascular relationships in developing median eminence of perinatal rats with special reference to luteinizing hormone-releasing hormone projections.

Topographical relationships of neurosecretory axons with the capillaries of the primary portal plexus were studied in the median eminence of rats from the 14th fetal till the 9th postnatal day by means of electron microscopy combined with morphometric analysis. Special attention was given to the light and electron microscopic immunocytochemical examination of luteinizing hormone-releasing hormone projections to the median eminence. Neurosecretory axons possessing secretory granules and clear microvesicles were first observed in the median eminence at the 14th fetal day. However, all of them were situated at a distance from the primary portal plexus. By the 20th fetal day, neurosecretory axons reached the external basal lamina of the primary portal plexus giving rise to so-called axovascular contacts. Some axons even penetrated into the perivascular space, apparently facilitating the neurohormone delivery into the hypophysial portal circulation. From that time on, both the number of the axons abutting on the external basal lamina and the entire area of axovascular contacts increased gradually. As to luteinizing hormone-releasing hormone axons, they grew into the median eminence from the 18th fetal day concentrating in older fetuses and neonates either over the primary portal plexus, or around the infundibular recess of the 3rd ventricle. After birth, the concentration and distribution of luteinizing hormone-releasing hormone axons within the median eminence became similar to those of adults. Luteinizing hormone-releasing hormone axons were found to arise from the neurons of septopreoptic area including the diagonal band of Broca. These data suggest the onset of neurohormone release in the median eminence from the 14th fetal day followed by the establishment of the hypothalamic control over the pituitary functions during the perinatal period in rats.

Evidence for local corticotropin releasing factor (CRF)-immunoreactive neuronal circuits in the paraventricular nucleus of the rat hypothalamus. An electron microscopic immunohistochemical analysis.

The interrelationships of corticotropin-releasing factor (CRF) immunoreactive neuronal cell bodies and processes have been examined in the paraventricular nucleus (PVN) of adrenalectomized-dexamethasone treated rats. Antisera generated against ovine CRF (oCRF) were used in the peroxidase-anti-peroxidase-complex (PAP)-immunocytochemical method at both the light and electron microscopic levels. In this experimental model, a great number of CRF-immunoreactive neurons were detected in the parvocellular subdivisions of the PVN and a few scattered labelled parvocellular neurons were also observed within the magnocellular subunits. Characteristic features of immunolabeled perikarya included hypertrophied rough endoplasmic reticulum with dilated endoplasmic cisternae, well developed Golgi complexes and increased numbers of neurosecretory granules. These features are interpreted to indicate accelerated hormone synthesis as a result of adrenalectomy. Afferent fibers communicated with dendrites and somata of CRF-immunoreactive neurons via both symmetrical and asymmetrical synapses. Some neurons exhibited somatic appendages and these structures were also observed to receive synaptic terminals. Within both the PVN and its adjacent neuropil, CRF-immunoreactive axons demonstrated varicosites which contained accumulations of densecore vesicles. CRF-containing axons were observed to branch into axon collaterals. These axons or axon collaterals established axo-somatic synapses on CRF-producing neurons in the parvocellular regions of the PVN, while in the magnocellular area of the nucleus they were found in juxtaposition with unlabeled magnocellular neuronal cell bodies or in synaptic contact with their dendrites. The presence of CRF-immunoreactive material in presynaptic structures suggests that the neurohormone may participate in mechanisms of synaptic transfer. These ultrastructural data indicate that the function of the paraventricular CRF-synthesizing neurons is adrenal steroid hormone dependent. They also provide morphological evidence for the existence of a neuronal ultrashort feed-back mechanism within the PVN for the regulation of CRF production and possibly that of other peptide hormones contained within this complex.

Application of the silver-gold intensified 3,3'-diaminobenzidine chromogen to the light and electron microscopic detection of the luteinizing hormone-releasing hormone system of the rat brain.

A highly sensitive, recently developed immunohistological method is introduced in the present study with special emphasis on the luteinizing hormone-releasing hormone system of the rat brain. The method utilizes the specific capability of the diaminobenzidine endproduct, a frequently used chromogen in immunocytochemistry, to produce and bind silver grains from a special physical developer, following suppression of the argyrophilia of the nervous tissue by thioglycolic acid. Metal deposition into the immunolabelled structures results in a real Golgi-like appearance of immunoreactive profiles. Specificity of this silver method was confirmed by ultrastructural analysis, which showed that unlabelled elements did not bind silver. Using this method, more immunoreactive neurons and fibres were visualized than compared with the results of the traditional peroxidase-antiperoxidase method. The luteinizing hormone-releasing hormone-synthesizing neurons proved to be fusiform, exhibiting either smooth or rough surfaced contour. Unlabelled terminals established axo-somatic synapses on labelled perikarya. The juxtaposition of immunoreactive luteinizing hormone-releasing hormone profiles suggest the possibility of self-regulation within the luteinizing hormone-releasing hormone system. The main advantages of the method are the increased sensitivity with preserved selectivity and wide applicability in different fields of neuroscience (peptide and transmitter immunocytochemistry, double labelling, horseradish peroxidase tract tracing, X-ray analysis).

Gonadotropin-releasing hormone (GnRH) neurons and pathways in the rat brain.

Gonadotropin-releasing hormone (GnRH) neurons and their pathways in the rat brain were localized by immunocytochemistry in 6- to 18-day-old female animals, by use of thick frozen or vibratome sections, and silver-gold intensification of the diaminobenzidine reaction product. GnRH-immunoreactive perikarya were observed in the following regions: olfactory bulb and tubercle, vertical and horizontal limbs of the diagonal band of Broca, medial septum, medial preoptic and suprachiasmatic areas, anterior and lateral hypothalamus, and different regions of the hippocampus (indusium griseum, Ammon's horn). In addition to the known GnRH-pathways (preoptico-terminal, preoptico-infundibular, periventricular), we also observed GnRH-immunopositive processes in several major tracts and areas of the brain, including the medial and cortical amygdaloid complex, stria terminalis, stria medullaris thalami, fasciculus retroflexus, medial forebrain bundle, indusium griseum, stria longitudinalis medialis and lateralis, hippocampus, periaqueductal gray of the mesencephalon, and extracerebral regions, such as the lamina cribrosa, nervus terminalis and its associated ganglia. By use of the silver-gold intensification method we present Golgi-like images of GnRH perikarya and their pathways. The possible distribution of efferents from each GnRH cell group is discussed.