Immunohistochemical localisation of regulatory neuropeptides in human circumvallate papillae

The occurrence and distribution of neuropeptide-containing nerve fibres in the human circumvallate papillae were examined by the peroxidase–antiperoxidase immunolocalisation method using surgical specimens that had not been subjected to radiotherapy, and the abundance of neuropeptide-containing fibres was expressed as the percentage of total nerve fibres demonstrated by protein gene product (PGP) 9.5 immunoreactivity for a quantitative representation of these peptidergic fibres. Substance P (SP) and calcitonin gene-related peptide (CGRP) immunoreactive (IR) nerve fibres were densely distributed in the connective tissue core of the circumvallate papillae, and some SP and CGRP-IR fibres were associated with the taste buds. A moderate number of vasoactive intestinal polypeptide (VIP)-IR fibres and a few galanin (GAL)-IR fibres were also seen in the connective tissue core and subepithelial layer. There were, however, no VIP-IR or GAL-IR fibres associated with the taste buds. Neuropeptide Y (NPY)-IR fibres were few and were associated with the blood vessels. Within the epithelium of the circumvallate papillae, no peptidergic fibres were found, although a number of PGP 9.5-IR fibres were detected. The abundance of SP, CGRP, VIP, and GAL-IR fibres expressed as the percentage of total PGP 9.5 IR fibres was 25.35±3.45%, 22.18±3.26%, 10.23±1.18%, and 4.12±1.05%, respectively. The percentage of NPY-IR fibres was below 3%. In a deeper layer of the papillae, a few VIP, GAL, and NPY-IR ganglion cells were found, and VIP immunoreactivity was detected in a few cells of the taste buds. There was no somatostatin, leucine enkephalin, or methionine enkephalin immunoreactivity in the circumvallate papillae. These results suggest that the dense SP and CGRP-IR fibres within the connective tissue core of the human circumvallate papillae may be involved in the deep sensation of the tongue.     

Innervation of the triangular fibrocartilage complex of the human wrist: Quantitative immunohistochemical study

The distribution of neural elements in the triangular fibrocartilage complex (TFCC) of the human wrists was studied via immunohistochemical staining of protein gene product (PGP) 9.5 and calcitonin gene-related peptide (CGRP). Articular branches projecting to the TFCC arose from the dorsal branch of the ulnar nerve in all wrists examined. The TFCC is subdivided into the following six regions: the articular disc proper (ADP), meniscus homolog (MH), radio-ulnar ligament (RUL), loose part of ulnar collateral ligament (lUCL), dense part of ulnar collateral ligament (dUCL), and internal portion (IP). The IP consists of a mixture of dense and loose connective tissues enclosed by the ADP, MH, RUL, and UCL, and resides deep in the prestyloid recess, which is a pit in the MH. The densities of PGP 9.5-positive neural elements, including free nerve endings, single nerve fibers, nerve fascicles, and perivascular neural nets, were significantly higher in the IP than in other regions. Some of the neural elements except for the perivascular neural nets were positive for CGRP. The high density of neural elements in the IP suggests that sensory nerves projecting to the TFCC enter into the IP and from there distribute to adjacent regions such as the MH and RUL. Free nerve endings are responsible for pain transmission. The high density of free nerve endings in the IP suggests that the IP is a source of ulnar side wrist pain.     

The limits of tooth pulp evoked potentials for pain quantitation

Tooth pulp evoked potentials (TPEPs0 and subjective evaluation of painful dental stimuli have been recorded in healthy volunteers. The amplitude of TPEPs late components and the subjective rating have been studied in different psychological states, by changing the expectancy of pain with a placebo and by providing foreknowledge of stimulus timing with self-stimulation. The placebo induced a significant depression of TPEPs and pain sensation. The amplitude of TPEPs evoked by self-delivered stimuli was reduced but the subjective report remained unchanged. These results demonstrate that TPEPs are not a stable correlate of the pain perceived or of the painful input.     

Conduction velocities of single pulp nerve fibre units in the cat

Most experiments on dental nerves have been made using dentine recording technique and it seems that only the fast conducting myelinated pulpal nerve fibres can be recorded with this technique. However, histological studies have revealed that even 80% of intradental fibres in the cat are unmyelinated. In the present work we studied activation of intradental nerve fibres with monopolar electrical stimulation of the intact mandibular canine tooth crown of the cat. The purpose was to determine the proportion of responding intradental A-and C-fibres dissected from the inferior alveolar nerve. In 17 anesthetized cats 350 pulp nerve fibre units were identified and recorded. Conduction time (based on the shortest latency), distance between the stimulation and the recording sites and threshold current for each fibre unit were determined. The mean conduction velocity for all pulp nerve fibres was 9.2 m/s (SD=9.8). The number of C-fibres (conduction velocity ≤2.0 m/s) was 122 (34.9%) and the number of A-fibres was 228 (65.1 %). The mean conduction velocities of C- and A-fibres were 1.0 m/s (SD=0.4) and 13.4 m/s (SD=9.4), repectively. The mean threshold current was 22.8 μA (SD=20.1) for the whole group, 40.4 μA (SD=20.7) for C-fibres and 13.5 μA (SD=11.8) for A-fibres. Although the function of pulpal C-fibres is poorly known, it has been suggested that they have a distinct role in mediation of sensations from tooth, particularly in inflammatory processes. Using nerve dissection technique the activity of a considerable part of intradental C-fibres can be recorded and it seems to be the only available method to study the function of this fibre group.     

Theophylline attenuates hippocampal blood flow responses induced by tooth pulp stimulation in rats

In this study, we performed tests to determine whether tooth pulp stimulation (TPS) increases hippocampal blood flow (HBF), and if so, to investigate whether the increase in HBF is mediated via the activation of adenosine receptors. We measured HBF in urethane-anesthetized rats using laser Doppler flowmetry (LDF) and examined the effect of theophylline, a nonselective adenosine receptor antagonist, on TPS-induced HBF responses. TPS increased HBF, and its response was significantly attenuated by the intraperitoneal administration of theophylline (20 mg/kg). These results suggest that the HBF response induced by TPS may be, at least in part, produced through adenosine receptors.     

Immunohistochemical localization of endothelin-like immunoreactivity in human tooth germ and mature dental pulp

Summary The distribution in oral tissues of endothelin, a multifunctional peptide originally identified within endothelial cells, and subsequently in some epithelial cells, neurons and neuroendocrine cells, has not been investigated yet. We have studied the localization of endothelin-like immunoreactivity in human tooth germ and mature dental pulp by immunohistochemical techniques. Such immunoreactivity was detected only within endothelial cells in both mature dental pulp and developing tooth. Arteries and veins of various sizes as well as small thin vessels displayed endothelin-like immunoreactivity. In the tooth germ, the cells of the enamel organ or the precursors of the odontoblasts were found unreactive. In the mature pulp, no cells of the stroma or nerves displayed endothelin-like immunoreactivity. These findings suggest that vascular endothelium may be the only source of endothelin in human dental tissues. It is tentatively proposed that endothelin released in mature tooth pulp may participate in the regulation of the pulpal blood flow. Although the possible role of endothelin in developing tissues is far from being clear, the mitogenic effects and the proto-oncogenes expression induced by endothelin in some cells raise the possibility that this peptide might also play a role during tooth development.     

Expression survey of genes critical for tooth development in the human embryonic tooth germ.

In the developing murine tooth, the expression patterns of numerous regulatory genes have been examined and their roles have begun to be revealed. To unveil the molecular mechanisms that regulate human tooth morphogenesis, we examined the expression patterns of several regulatory genes, including BMP4, FGF8, MSX1, PAX9, PITX2, and SHOX2, and compared them with that found in mice. All of these genes are known to play critical roles in murine tooth development. Our results show that these genes exhibit basically similar expression patterns in the human tooth germ compared with that in the mouse. However, slightly different expression patterns were also observed for some of the genes at certain stages. For example, MSX1 expression was detected in the inner enamel epithelium in addition to the dental mesenchyme at the bell stage of the human tooth. Moreover, FGF8 expression remained in the dental epithelium at the cap stage, while PAX9 and SHOX2 expression was detected in both dental epithelium and mesenchyme of the human tooth germ. Our results indicate that, although slight differences exist in the gene expression patterns, the human and mouse teeth not only share considerable homology in odontogenesis but also use similar underlying molecular networks.     

Nerve growth factor enhances cholinergic innervation and contractile response to electric field stimulation in a murine in vitro model of chronic asthma

Background Asthma is a chronic inflammatory disease characterized by airway hyper‐responsiveness. Alterations in the neurogenic control are believed to contribute to the pathogenesis. Yet, the long‐term interaction between nerves and inflammatory mediators, such as the neurotrophin nerve growth factor (NGF), are not fully understood much due to the absence of appropriate experimental assays. Objective To develop an ex vivo mouse organ culture assay and to investigate the effects of NGF on nerve‐mediated airway contractions. Method Mouse tracheal segments were cultured in periods of up to 16 days. Their contractile responses to electric field stimulation (EFS) were investigated. In addition, the effect of 4 days of NGF treatment was analysed using EFS and immunohistochemistry. Results EFS (0.2–25.6 Hz) induced reproducible and frequency‐dependent cholinergic contractions of both fresh and cultured tracheal segments. The main part of the EFS response was blocked by tetrodotoxin or atropine. After 4 days in culture, regional differences appeared, with stronger EFS responses in distal than in proximal segments. More nerve fibres were seen in distal segments than in proximal segments. Treatment with NGF during 4 days of culture increased the innervation of the proximal segments, at the same time as the cholinergic contractile responses to EFS were enhanced dose‐dependently. Conclusion Culture of tracheal segments appears to be a suitable assay for the examination of long‐term effects induced by inflammatory mediators on neurally mediated airway contractions. NGF treatment enhanced the cholinergic, nerve‐dependent contractions and increased the amount of nerve fibres seen in the murine tracheal segments, suggesting a role for NGF in the development of airway hyper‐responsiveness.     

Morphological Analysis for Neuron‐Like Cells in the Vomeronasal Organ of Human Fetuses at the Middle of Gestation

The vomeronasal organ (VNO) of 5‐month‐old fetuses was examined immunohistochemically by the use of an antiserum to protein gene product 9.5 (PGP). The purpose was to identify if the human fetal VNO is lined by neuroepithelium. The PGP antiserum labeled abundant cells within the vomeronasal epithelium (VE), nerve fiber bundles in its lamina propria, and cells associated with these bundles. PGP‐immunoreactive (ir) vomeronasal epithelial cells were classified into three subtypes. Type I cells, about 44% of the total cells observed, did not have any processes and tended to be located in the basal layer of the VE. Type II cells, about 37% had a single apical process that projected toward the lumen, ending at the epithelial surface. Type III cells sent a prominent process mainly toward the basement membrane, and occupied about 19% of the total cells observed. In the lamina propria, a considerable number of PGP‐ir cells was observed. Some of them were present in nerve fiber bundles and contained processes parallel to the bundles. In addition, PGP‐ir nerve fiber bundles and cells associated with them were even present in the portion of the nasal septal mucosa that was very close to the brain. The present results strongly suggested that the VE in human fetuses at mid‐gestation is a neuroepithelium and that the VE may produce migrating cells toward the brain. Anat Rec, 299:88–97, 2016. © 2015 Wiley Periodicals, Inc.     

Immunocytochemical expression of protein gene product (PGP) 9.5 in the cat bronchopulmonary neuroendocrine cells and nerves

Variously fixed, wax-embedded lung and gastrointestinal serial tissue sections from newborn to adult cats were stained with hematoxylin-eosin (H&E), Grimelius' silver, and immunohistochemical techniques using antisera to protein gene product (PGP) 9.5, a neuron-specific protein under strong evolutionary constraints. PGP 9.5 is revealed as a pan-neuroendocrine marker useful for tracing the pulmonary diffuse neuroendocrine system (PDNES) and studying the relationships between neuronal and neuroendocrine elements at various stages of life. Its occurrence is also compared in the pulmonary and the gastrointestinal tract. In spite of a close resemblance to already described neuroepithelial bodies (NEB) of other mammals, cat NEB feature typical constitutional and distributional difference, illustrating interspecies differences. The number of PGP 9.5 immunopositive pulmonary neuroendocrine cells declines gradually after 3 weeks and throughout adult life. Immunoreactivity in neuronal elements is lost after 1 week of age. In gastrointestinal tissues, only neuronal lelements immunostain, suggesting functional variations or a separate embryological origin for enteroendocrine cells. © 1993 Wiley-Liss, Inc.     

Protein gene product (PGP) 9.5 as a reliable marker in primitive neuroectodermal tumours—an immunohistochemical study of 21 childhood cases

A number of antibodies to neural proteins have been used to demonstrate neuronal differentiation in primitive neuroectodermal tumours. One of them is protein gene product (PGP) 9.5, a neuronal protein isolated from brain, whose function is unknown at present. We have studied differentiation in 21 cases of primitive neuroectodermal tumours of the CNS in children. Immunocytochemical staining was performed for such neuronal markers as: PGP 9.5, neuron specific enolase and synaptophysin, a glycosylated protein associated with synaptic vesicles. Positive staining for PGP 9.5 was present in 16 cases (strong staining in 12), for neuron-specific enolase in 16 cases (strong staining in 10) and for synaptophysin in 10 cases (strong staining in six). Both PGP 9.5 and synaptophysin showed a clear staining pattern with less non-specific background than with neuron-specific enolase. Our findings demonstrate the value of using more than one antibody marker in assessing neuronal differentiation in tumours. The high incidence of positive staining with antibody to PGP 9.5 suggests that this is an essential marker in the panel of antibodies used for the identification of primitive neuroectodermal tumours.     

泛素羧基末端水解酶-1的研究进展

泛素羧基末端水解酶-1（UCH-L1），又名蛋白基因产物9.5，是泛素-蛋白酶体系统中的重要成员，它除了去泛素化作用外，还具有泛素连接酶和稳定细胞内泛素单体的功能。UCH-L1通过泛素相关途径调节细胞的增生、分化和凋亡，并可能参与与之相关的神经退行性疾病、肿瘤等的发生发展过程。最近UCH-L1的特异性底物的发现为探讨其作用的具体机制开辟了新的途径。     

Immunoreactivities to protein gene product 9.5, neurofilament protein and neuron specific enolase in the ovary of the sexually immature ostrich (Struthio camelus)

The innervation of the ovary has been studied in various species of birds and mammals. Despite the fact that the innervation of any organ is an essential factor in controlling its growth and function, no information is available on the distribution of nerve fibers in the ovary of the sexually immature ostrich. Thus, the present study was undertaken to investigate the distribution of nerve fibers in the ovary of the sexually immature ostrich, using antibodies against neurofilament protein type M of 160 kD (NP), protein gene product 9.5 (PGP 9.5) and neuron specific enolase (NSE). A total of 26 sexually immature female ostriches, aged between 12 and 14 months were used in the present study. Immunostaining was performed using a LSAB plus kit (Dakocytomation, Denmark). Antibodies against NP and PGP 9.5 were used at dilutions of 1:25 and 1:50, respectively. A ready-to-use solution containing antibodies against NSE was also used. Strong immunostaining for NP, PGP 9.5 and NSE was observed in nerve bundles, which coursed through the ovarian stalk and extended into the medulla and cortex. In addition, NSE immunoreactive nerve cell bodies were observed in the cortex and medulla. NP, PGP 9.5 and NSE immunoreactive nerve fibers were present in the thecal layer of the follicular wall. The current study has highlighted the distribution of NP, PGP 9.5 and NSE-immunoreactive nerve fibers in the ovary of the sexually immature ostrich. The findings of the present study suggest that the distribution of nerve fibers in the immature ostrich is similar to that of the domestic fowl.     

Long‐Term Effects of Neonatal Capsaicin Treatment on Intraepidermal Nerve Fibers and Keratinocyte Proliferation in Rat Glabrous Skin

Innervation is required to preserve several aspects of skin homeostasis. Previous studies in rodents have shown that sciatic nerve transection leads to epidermal thinning and reduced keratinocyte proliferation. As the sciatic nerve is composed of sensory and motor axons, it is not clear whether skin alterations reflect motor or sensory disturbances. In this study, we used neonatal capsaicin treatment to evaluate whether sensory chemical denervation affects keratinocyte proliferation at 1, 3, and 6 months of age. Using design‐based stereological methods, we estimated the total length of intraepidermal nerve fibers (IENF) that were of peptidergic type and the number of bromodeoxyuridine‐labeled (BrdU⁺) nuclei in the hind paw glabrous epidermis of control and capsaicin‐treated rats. We found that the treatment decreased the total fiber length of IENF immunoreactive for both protein gene product 9.5 (PGP⁺) and of IENF immunoreactive for calcitonin gene‐related peptide (CGRP⁺). The length of PGP⁺ fibers decreased by 83%, 81%, and 77% and that of CGRP⁺ fibers decreased by 48%, 58%, and 58% at 1, 3, and 6 months, respectively. Double‐immunofluorescence staining for neural beta III tubulin and CGRP revealed that the majority of the remaining fibers in the epidermis after capsaicin treatment were of peptidergic type. The number of BrdU⁺ nuclei was similar in both groups. Our findings suggest that IENF present after capsaicin treatment are sufficient to maintain epidermal replacement. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.     

JNK MAPK is involved in BMP-2-induced odontoblastic differentiation of human dental pulp cells

Bone morphogenetic protein-2 (BMP-2) is a multi-functional growth factor belonging to the transforming growth factor β superfamily that has a broad range of activities that affect many different cell types. BMP-2 induces odontoblastic differentiation of human dental pulp cells (DPCs), but the underlying mechanism remains unclear. In this study, we investigated the potential role of the JNK mitogen-activated protein kinases (MAPK) pathway in BMP-2-induced odontoblastic differentiation of DPCs. The levels of phosphorylated and unphosphorylated JNK MAPK were quantified by Western blot analysis following treatment with BMP-2 and the JNK inhibitor SP600125. The role of JNK MAPK in the BMP-2-induced odontoblastic differentiation of DPCs was determined by measuring alkaline phosphatase (ALP) activity and by examining the expression of odontoblastic markers using quantitative real-time polymerase chain reaction analysis. The effect of JNK MAPK silencing on odontoblastic differentiation was also investigated. BMP-2 upregulated the phosphorylation of JNK in DPCs in a dose- and time-dependent manner. Early markers of odontoblastic differentiation, including ALP activity, osteopontin and dentin matrix protein-1, were not inhibited by the JNK inhibitor. However, the JNK inhibitor, SP600125, significantly inhibited late-stage differentiation of odontoblasts, including the gene expression of osteocalcin, dentin sialophosphoprotein and bone sialoprotein, and also reduced the formation of mineralized nodules in BMP-2-treated DPCs. Consistent with this observation, silencing of JNK MAPK also decreased late-stage odontoblastic differentiation. Taken together, these findings suggest that JNK activity is required for late-stage odontoblastic differentiation induced by BMP-2.     

Effect of pentobarbital on the strength duration curve of the jaw opening reflex to tooth pulp stimulation in cats

Brain stem field potentials and digastric electromyographic responses to tooth pulp stimulation were recorded in six chronically prepared adult cats. Strength duration curves were derived for both responses under conditions of wakefulness and anesthetic levels of sodium pentobarbital (35 mg/kg). For the muscle response, the slope of the strength duration curve for short duration pulses became more negative, and the chronaxies were significantly increased under anesthesia as compared to the awake state. For the field potential response, the same parameters were not significantly altered. The results suggest that the alteration in strength duration curve seen in the muscle response may be determined beyond the level of the first synapse.