Effect of periapical inflammation on calcium binding proteins and ERK in the trigeminal nucleus / Efecto de la inflamación periapical sobre las proteínas fijadoras de calcio y ERK en el núcleo trigeminal

Peripheral inflammation induces plastic changes in neurons and glia which are regulated by free calcium and calcium binding proteins (CaBP). One of the mechanisms associated with the regulation of intracellular calcium is linked to ERK (Extracellular Signal-Regulated Kinase) and its phosphorylated condition (pERK). ERK phosphorylation is important for intracellular signal transduction and participates in regulating neuroplasticity and inflammatory responses. The aim of this study is to analyse the expression of two CaBPs and pERK in astrocytes and neurons in rat trigeminal subnucleus caudalis (Vc) after experimental periapical inflammation on the left mandibular first molar. At seven days post-treatment, the periapical inflammatory stimulus induces an increase in pERK expression both in S100b positive astrocytes and Calbindin D28k positive neurons, in the ipsilateral Vc with respect to the contralateral side and control group. pERK was observed coexpressing with S100b in astrocytes and in fusiform Calbindin D28k neurons in lamina I. These results could indicate that neural plasticity and pain sensitization could be maintained by ERK activation in projection neurons at 7 days after the periapical inflammation.

La inflamación periférica induce cambios plásticos en las neuronas y en la glía, los cuales están regulados por el calcio libre y las proteínas fijadoras calcio (CaBP). Uno de los mecanismos asociados con la regulación del calcio intrace-lular está vinculado con la fosforilación de la pro teína quinasa ERK. Asimismo, ERK fosforilado es importante para la trans-ducción de señales intracelulares y participa en la regulación de la neuroplasticidad y las respuestas inflamatorias. El objetivo de este estudio es analizar la expresión de dos CaBPs y pERK en astrocitos y neuronas del subnúcleo caudal del trigémino (Vc) después de una inflamación periapical experimental en el primer molar inferior izquierdo en ratas. A los siete días posteriores al tratamiento, el estímulo inflamatorio periapical induce un aumento en la expresión de pERK, en el número de astrocitos positivos para la proteína marcadora astroglial S100b y en neuronas positivas para Calbindina D28k, en el Vc ipsilateral respecto del lado contralateral y el grupo de control. Además, se observó coexpresión de pERK tanto en astrocitos S100b positivos, como en neuronas fusiformes Calbindin D28k positivas, de la lámina I. Estas observaciones podrían indicar que la neuroplasticidad y la sensibilización al dolor podrían mantenerse mediante la activación de ERK en las neuronas de proyección a los 7 días de la inflamación periapical.

Orexin-A inhibits capsaicin-induced changes in cyclooxygenase-2 and brain-derived neurotrophic factor expression in trigeminal nucleus caudalis of rats

BACKGROUND: The trigeminal nucleus caudalis (Vc) is a primary central site for trigeminal transmitting. Noxious stimulation of the trigeminal nociceptors alters the central synaptic releases and neural expression of some inflammatory and trophic agents. Orexin-A and the orexin 1 receptor (OX1R) are expressed in pain pathways including trigeminal pain transmission. However, the the mechanism(s) underling orexin-A effects on trigeminal pain modulation have not been fully clarified. METHODS: Trigeminal pain was induced by subcutaneous injection of capsaicin in the upper lip in rats. The effect of trigeminal pain on cyclooxygenase-2 (COX-2) and brain-derived neurotrophic factor (BDNF) expression in the Vc of animals was determined by immunofluorescence. Subsequently, OX1R agonist (orexin-A) and antagonist (SB-334867-A) was administrated in the Vc to investigate the possible roles of the Vc OX1R on changes in COX-2 and BDNF levels following pain induction. RESULTS: The data indicated an increase in COX-2 and decrease in BDNF immuno-reactivity in the Vc of capsaicin, and capsaicin- pretreated with SB-334867-A (80 nM), groups of rat. However, the effect of capsaicin on COX-2 and BDNF expressions was reversed by a Vc microinjection of orexin-A (100 pM). CONCLUSIONS: Overall, the present data reveals that orexin-A can attenuate capsaicin-induced trigeminal pain through the modulation of pain effects on COX-2 and BDNF expressions in the Vc of rats.

Electrophysiological Evidence for Functional Astrocytic P2X₃ Receptors in the Mouse Trigeminal Caudal Nucleus

Recently, we reported that astrocytes in the trigeminal caudal nucleus (Vc) of the brain stem express a purinergic receptor P2X₃, which is involved in the craniofacial pathologic pain. Although we observed protein expression of P2X₃ receptors (P2X₃ Rs) in the astrocyte of the Vc, it is still unclear that astrocyte has functional P2X₃Rs in Vc. To address this issue, we recorded asrtocytic P2X₃Rs by using whole cell voltage-clamp recording in the Vc of the GFAP-GFP mice, which was used as a guide to astrocytes with green fluorescence. While measuring voltage ramp-induced astrocytic membrane current, we found the amplitude of the current was increased when we applied P2-purinoreceptor agonist, α,β-meATP. This increase was blocked by co-application of A317491, P2X₃R antagonist. These results demonstrate that astrocytes in the Vc express functional P2X₃Rs, which might be critical in craniofacial pathologic pain.

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 Influence of Glutaraldehyde Concentration on Electron Microscopic Multiple Immunostaining

The present study was aimed to evaluate the influence of glutaraldehyde (GA) concentration on multiple electron microscopic (EM) immunostaining using pre-embedding peroxidase and post-embedding immunogold method. Influence of various concentrations of GA included in the fixative on immuoreactivity was assessed in the multiple immunostaining using antisera against anti-transient receptor potential vanilloid 1 (TRPV1) for peroxidase staining and anti-GABA for immunogold labeling in the rat trigeminal caudal nucleus. Anti-TRPV1 antiserum had specificity in pre-embedding peroxidase staining when tissues were fixed with fixative containing paraformaldehyde (PFA) alone. Immunoreactivity for TRPV1 was specific in tissues fixed with fixative containing 0.5% GA at both perfusion and postfixation steps, though the immunoreactivity was weaker than in tissues fixed with fixative containing PFA alone. Tissues fixed with fixative containing 0.5% GA at the perfusion and postfixation steps showed specific immunogold staining for GABA. The results of the present study indicate that GA concentration is critical for immunoreactivity to antigens such as TRPV1 and GABA. This study also suggests that the appropriate GA concentration is 0.5% for multiple immunostaining with peroxidase labeling for TRPV1 and immunogold labeling for GABA.

Effect of orofacial inflammatory pain on p38 mitogen-activated protein kinase activation in trigeminal caudal nucleus of rats / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To evaluate the potential role of p38 mitogen-activated protein kinase (MAPK) in the orofacial inflammatory pain.</p><p><b>METHODS</b>SD rats received subcutaneous injection of 2.5% formalin 50 µl in the left vibrissa pad to establish the inflammatory pain model. The rats were grouped into the control group, the formalin group (FOR group), the formalin + saline group (FOR + NS group) and the formalin + SB203580 group (FOR + SB group). SB203580 or saline was inserted into the rat's cisterna magna 20 minutes prior to the formalin injection, then the behavioral changes were tested. The immunofluorescence staining and Western blotting analysis were performed to examine c-fos, p38MAPK and phosphorylated p38 (p-p38) activity in Vc at 20, 60, 120, 180 minutes after formalin injection.</p><p><b>RESULTS</b>p38MAPK was constitutively expressed in Vc (P > 0.05) and p38MAPK was activated following formalin injection.Compared with the control group at 20 min (0.12 ± 0.01), the level of p-p38 in FOR group (0.66 ± 0.04) and FOR + NS group (0.64 ± 0.04) increased significantly (P < 0.001). The expression of p-p38 peaked at 20 minutes, and then declined in each group. Intracisterna magna pretreatment of p38MAPK inhibitor SB203580 resulted in potent attenuation of phase II of pain behavior (P < 0.05), while the expression of c-fos was also inhibited, especially at the point of 120 min (P < 0.01).</p><p><b>CONCLUSIONS</b>Activation of p38 mitogen-activated protein kinase played a major role in the development of orofacial inflammatory pain and it was verified by the experimental result that p38MAPK inhibitor SB203580 inhibited the formalin-induced orofacial pain.</p>

Ultrastructural analysis of low-threshold mechanoreceptive vibrissa afferent boutons in the cat trigeminal caudal nucleus / 대한해부학회지

Ultrastructural parameters related to synaptic release and their correlation with synaptic connectivity were analyzed in the low-threshold mechanoreceptive vibrissa afferent boutons in laminae III and IV of the trigeminal caudal nucleus (Vc). Rapidly adapting vibrissa afferents were intra-axonally labeled, and quantitative ultrastructural analyses with serial sections were performed on the labeled boutons and their presynaptic endings (p-endings). The volume of the labeled boutons was widely distributed from small to large ones (0.8~12.3 microm3), whereas the p-endings were small and uniform in size. The volume of the labeled boutons was positively correlated with the ultrastructural parameters such as mitochondrial volume (correlation coefficient, r=0.96), active zone area (r=0.82) and apposed surface area (r=0.79). Vesicle density (r=-0.18) showed little correlation to the volume of labeled boutons, suggesting that the total vesicle number of a bouton is proportional to its volume. In addition, the bouton volume was positively correlated with the number of p-endings (r=0.52) and with the number of dendrites postsynaptic to the labeled bouton (r=0.83). These findings suggest that low-threshold mechanoreception conveyed through vibrissa afferents is processed in a bouton size-dependent manner in the Vc, which may contribute to the sensory-motor function of laminae III/IV in Vc.

Thoracic Vagal Ganglion and Referred Craniofacial Pain: a Case Report and Review / Ganglio Torácico Vagal y Dolor Craneofacial Referido: Reporte de Caso y Revisión

The presence of a ganglion-like tumefaction is reported in the mediastinal course of the right vagus nerve at Tl level in a cadaver in the Universidad Industrial de Santander's morphology laboratory. The vagal ganglion was located next to hyperplasic lymphoid nodes in para-tracheal and tracheal-bronchial levels, agglomerating in a large lymphoid mass in the carina and the pulmonary hilum. Anatomical-pathological study revealed a marked, diffusely distributed, predominantly histo-lymphocyte mixed inflammation, separating the epineurium, producing lysis of the vagus nerve fibers. This finding showed the degeneration of this cranial par by mediastinal pathology. This provided a possible explanation for the physiopathology of pain referring to the head and neck in inflammatory or neoplastic pathology involving compression and degeneration by inflammatory infiltration of the vagus nerve. Pons-medullar trigeminal afferent tracts and connectivity, supra-spinal pathways for processing somatic-visceral pain, possible somatic-vegetative responses and the integration of the trigeminal system in the physiology of pain concerning the vagus nerve are all discussed.

Se reporta la presencia de una tumefacción a manera de "ganglio" en el trayecto mediastínico del nervio vago derecho, a nivel de TI, en un cadáver en el Laboratorio de Morfología de la Universidad Industrial de Santander, Colombia. El "ganglio" vagal se encuentra adyacente a nodos linfoides hiperplásicos, en niveles para-traqueales y tráqueo-bronquiales que se aglomeran en una gran masa linfoide a la altura de la carina e hilio pulmonar. En el estudio anatomopatológico, se encontró marcado proceso inflamatorio mixto de predominio histo-linfocitario el cual se distribuye de manera difusa separando el epineuro y produciendo lisis de las fibras del nervio vago. Este hallazgo muestra la degeneración de este par craneal por patologías a nivel mediastínico. Esto brinda una posible explicación de la fisiopatología del dolor referido a cabeza y cuello, en patologías inflamatorias o neoplásicas que involucran la compresión y degeneración por infiltración inflamatoria del nervio vago. Se discute los tractos y la conectividad de las aferencias vagales a nivel ponto-medular, las vías supraespinales para el procesamiento del dolor sómato-visceral, las posibles respuestas sómato-vegetativas y la integración del sistema trigeminal en la fisiología del dolor en el nervio vago.

Nissl study of lower motor neurons of mylohyoid muscle

To determine the somatotopic organization of lower motor neurons of the mylohyoid muscle in the motor trigeminal nucleus. Design: Histological study of the rat hind-brain by fluorescence microscopy and Nissl staining. Setting: Neuro-histological laboratory, College of Medicine, King Saud University, Riyadh, Kingdom of Saudi Arabia. Materials: The mylohyoid muscle in twenty albino rats were injected with retrogradely transported fluorescent material DAPI-Pr. Fluorescence microscopy was performed, followed by subsequent staining of these sections with the Nissl stain, thionine. The fluorescent cells were found to occupy the rostro-medial part of the ipsilateral motor trigeminal nucleus. Subsequent staining of the sections with the Nissl stain thionine confirmed the rostro-medial location of the labeled cells. The entire lot of cells in this 'rostro-medial subgroup' was found labeled, representing the domain area of the lower motor neurons of the mylohyoid muscle. The rostro-medial subgroup of the trigeminal motor nucleus is the only one to supply the mylohyoid muscle

Peptidos opioides endogeneos an la medula espinal y el núcleo caudal del trigemino / Endogenous opioid peptides in the spinal cord and the trigeminal caudal nucleus

El presente trabajo es un estudio de la distribución de tres peptidos opioides endogenos, la leu-encefalina, la met-encefalina-arg-gli-leu y la clinorfina B, en neuromas, fibras y terminaciones nerviosas de diversos segmentos de la médula espinal y el núcleo caudal del Trigémino del gato. Los resultados de esta investigación permitieron identificar semejanzas y diferencias en la distribución de la inmunorreactividad para dinorfina B, leucina-encefalina y met-encefalina-arg-gli-leu en la médula espinal y el núcleo caudal del Trigémino del gato. Los materiales usados fueron: catorce gatos adultos de ambos sexos jovenes de 2 a 3 kg. de peso. Reactivos, anticuerpos de cabra contra globulina del conejo, soluciones y un fotomicroscopio zeiss provisto de camara lácida. El método empleado fue la administración de colchicina. La compremsión de los resultados de este trabajo requiere la realización de estudios adicionales