Histological observations of age-related changes in the epiglottis associated with decreased deglutition function in older adults.

Although the epiglottis plays a vital role in deglutition, histological studies of the epiglottis and surrounding ligaments associated with swallowing dysfunction are limited. Therefore, we performed histological observations to clarify age-related changes in the morphological characteristics of the epiglottis and surrounding structures. Tissue samples comprising the epiglottis and surrounding structures were collected from corpses that were both orally fed and tube-fed during their lifetimes. Following hematoxylin and eosin, Elastica Van Gieson, and immunohistochemical staining procedures, the chondrocytes, connective tissue, and glandular tissue were observed under the epiglottis epithelium, and intervening adipose tissue was observed in the surrounding area. Fatty degeneration of acinar cells was also observed in the glandular tissue, possibly because of aging. Bundles of elastic fibers were present around the vascular wall in the peri-epiglottic ligament, but some were reduced. Furthermore, large amounts of collagen fibers ran toward and through the cartilage, whereas the mesh-like elastic fibers stopped in front of the cartilage. Microfibrils considered to be oxytalan fibers, which are thinner and shorter than elastic fibers, were observed around the vascular wall and in the fiber bundles. Age-related changes included connective tissue fibrosis shown by the large amount of collagen fibers, atrophy of salivary glands, and an accompanying increase in adipose tissue. Regarding stretchability and elasticity, the elastic fibers may have an auxiliary function for laryngeal elevation during deglutition. This suggests that disuse atrophy of the laryngeal organs with or without oral intake might reduce the amount of elastic fiber in older adults.

Expression Profiles of NOS Isoforms in Dental Pulp and Odontoblasts in nNOS Knockout Mice.

Active oxygen and free radicals are involved in metabolism in cells and tissues. Immunohistological studies of related enzymes are few, and the morphological dynamics of these enzymes in dental pulp and odontoblasts remain to be elucidated. Nitric oxide synthase (NOS) has 3 isoforms: nNOS, iNOS, and eNOS. The aim of this study was to investigate the profiles of NOS isoforms in the absence of nNOS in dental pulp and odontoblasts. Five-week-old male C57BL/6 and nNOS knockout (KO) mice were sacrificed and expression of nNOS, iNOS, and eNOS determined immunohistochemically. Expression of nNOS was positive, whereas that of iNOS was negative and eNOS weakly positive in the dental pulp and odontoblasts of the control mice. In nNOS KO mice, expression of iNOS was positive in dental pulp and strongly positive in odontoblasts, whereas that of eNOS was stronger in fibroblasts, endothelial cells in the vicinity of blood vessels in the dental pulp, and odontoblasts. Expression of nNOS was negative in the nNOS KO mice. This suggests that iNOS and eNOS compensate for nNOS deficiency in vascular endothelial cells and fibroblasts in the dental pulp and odontoblasts.

Immunohistochemical Analysis of Nerve Distribution in Mandible of Rats.

After review of the literature, there appears to be no report on the histology of the mandibular nerve fiber distribution. Therefore, using a Wistar rat model, immunohistochemical staining with protein gene product (PGP) and calcitonin gene-related peptide (CGRP) antibody for all nerves and only the pain-sensitive nerves, respectively, was performed. We also statistically compared the nerve distribution density by mandibular region. The section of the mandible from the alveolar crest to the mandibular canal was compartmentalized to several regions. Subsequently, nerve distribution density by region was measured microscopically in both the PGP- and CGRP-positive nerves. Furthermore, the ratio of CGRP- to PGP-positive nerves was measured in each region and statistically compared. In both the PGP- and CGRP-positive nerves, the nerve distribution density significantly increased vertically toward the mandibular canal from the alveolar crest and horizontally toward the periodontal ligament from the periosteum. From the CGRP- to PGP-positive nerve ratio, the pain-sensitive nerve accounted for approximately >70% in each region. Pain would therefore be more likely to develop when surgical invasiveness deepens toward the mandibular canal or periodontal ligament. Therefore, sufficient local anesthetic infiltration and/or combined use of conduction anesthesia or periodontal ligament injection may be required. These results may aid in the development of more effective surgical and anesthetic techniques for mandibular surgery.

Nitric Oxide Synthase (NOS) Isoform Expression after Peripheral Nerve Transection in Mice.

Localization of the nitric oxide (NO)-producing enzyme, nitric oxide synthase (NOS), and its functions are currently being investigated in several tissues and organs. It has been suggested that NO is involved in nerve cell death and the development of neurodegenerative disease. The purpose of this study was to immunohistochemically investigate expression of NOS to clarify its function in the degeneration and regeneration of transected mouse sciatic nerve. Scattered neuronal NOS (nNOS)-positive Schwann cells observed on the central side of the stump on day 1 after transection showed an increase in number on day 7. None were observed at the stump on day 14, however. Expression of nNOS was observed in axons extending from the stump. The number of nNOS-positive axons increased on day 21. Inducible NOS was expressed in inflammatory cells at the stump on day 1. This positive reaction subsequently weakened by day 7, however. Endothelial NOS was expressed in blood vessels at the stump on day 7, but decreased thereafter. The results of the present study suggest that NO is involved in the proliferation and migration of Schwann cells, as well as in axon regeneration at an early stage following nerve transection.

A Histological Study of Vasoconstriction by Local Anesthetics in Mandible.

To assess the effect of epinephrine-containing local anesthetics on vasoconstriction, we immunohistochemically measured the intravascular lumen area in different regions of the mandible. Twelve male Wistar rats were used. General anesthesia was induced and maintained with sevoflurane. Infiltration anesthesia was performed with 0.2 mL of epinephrine-free 2% lidocaine (E-) near the left mandibular first molar and with 0.2 mL of epinephrine-containing 2% lidocaine (E+) near the right mandibular first molar. After decalcification, the specimens were paraffinized, and thin sections were prepared and immunohistologically stained with an antismooth muscle actin antibody. The intravascular lumen area was measured in the mucosa, periodontal membrane, Haversian/Volkmann's canal, and bone marrow. A Mann-Whitney U test was used for statistical processing, and p < .05 was considered to indicate a statistically significant difference. In the oral mucosa and the periodontal membrane, E+ had a significantly smaller vascular lumen area than E-. In the Haversian/Volkmann's canal and the bone marrow, no significant intergroup difference was observed in the intravascular lumen area. We postulate that this is due to a low smooth muscle content of blood vessels in the mandible and suggest that the vasoconstrictive effect of epinephrine-containing local anesthetics within the mandible is ineffective.

Lymphangiogenesis and NOS Localization in Healing Process after Tooth Extraction in Akita Mouse.

Type I diabetes, an autoimmune disease, induces insulin deficiency, which then disrupts vascular endothelial cell function, affecting blood and lymphatic vessels. Nitric oxide (NO) is an immune-induced destructive mediator in type I diabetes, and inhibition of its production promotes arteriosclerosis. In this study, lymphangiogenesis and expression of NO synthase (NOS) during the healing process after tooth extraction were investigated immunohistochemically in control (C57BL) and Akita mice as a diabetes model. Between 1, 4, and 10 days after extraction, expression of NOS, vascular endothelial growth factor-C (VEGF-C), VEGF receptor-3 (VEGFR-3), and von Willebrand factor was strongest during the granulation tissue phase. This suggests that severe inflammation triggers regulation of NOS and these other angiogenic and lymphangiogenic factors. During the callus phase, a few days after extraction, induced osteoblasts were positive for VEGF-C and VEGFR-3 in both the control and Akita mice, suggesting that bone formation is active in this period. Bone formation in the Akita group exceeded that in the controls. Bone tissue formation was disrupted under hyperglycemic conditions, however, suggesting that such activity would be insufficient to produce new bone.

Fibroblast VEGF-receptor 1 expression as molecular target in periodontitis.

AIM: Degradation of extracellular matrices is an integral part in periodontitis. For antagonizing this pathophysiological mechanism, we aimed at identifying gene expression profiles in disease progression contributing periodontitis-associated fibroblasts (PAFs) versus normal gingival fibroblasts to determine their molecular repertoire, and exploit it for therapeutic intervention. MATERIALS AND METHODS: Applying an exploratory analysis using a small number of microarrays in combination with a three dimensional (3D) in vitro culture model that incorporates some aspects of periodontitis, PAFs were initially characterized by gene-expression analyses, followed by targeted gene down-regulation and pharmacological intervention in vitro. Further, immunohistochemistry was applied for phosphorylation analyses in tissue specimens. RESULTS: PAFs were characterized by 42 genes being commonly up-regulated >1.5-fold, and by five genes that were concordantly down-regulated (<0.7-fold). Expression of vascular endothelial growth factor (VEGF)-receptor 1 (Flt-1) was highly enhanced, and was thus further explored in in vitro culture models of periodontal fibroblasts without accounting for the microbiome. Phosphorylation of the VEGF-receptor 1 was enhanced in PAFs. Receptor inhibition by a specific VEGF-receptor inhibitor or intrinsic down-regulation by RNAi of the VEGF-receptor kinase in 3D gel cultures resulted in significant reduction in collagen degradation associated with increased tissue inhibitor of metalloproteinase expression, suggesting that Flt-1 may contribute to periodontitis. CONCLUSION: Based on the finding that VEGF-receptor kinase inhibition impaired collagen degradation pathways, Flt-1 may represent a candidate for therapeutic approaches in periodontitis.

Production and physiological role of NO in the oral cavity.

Nitric oxide (NO) is a free radical which is produced from a wide variety of cells and tissues in the human body. NO is involved in the regulation of many physiological processes, such as vascular relaxation, neurotransmission, immune regulation, and cell death. NO is generated by nitric oxide synthase (NOS), which has three identified isoforms: neuronal type NOS (nNOS), endothelial type NOS (eNOS), and inducible type NOS (iNOS). Different isoforms are expressed depending on the organs, tissues, and cells, and investigation of the types and functions of enzymes expressed in various tissues is underway. The oral cavity is a space in which marked changes have been detected in NO levels, and each tissue is constantly influenced by NO. NO is a component of saliva and is produced by oral bacteria in the oral cavity and released by NOS expressed in oral mucosa. NOS isoforms expressed under normal conditions differ among the oral organs. In addition, the overexpression of NOS was involved in carcinogenesis and tumor growth progression. This review summarized the expression of NOS and functions of NO in oral cavity organs, and their roles in diseases and the influences of treatments.

Immunohistochemical localization of Nox in mouse circumvallate papillae.

Recently it has been reported that reactive oxygen species plays an important role in several physiological processes. Reactive oxygen species are generated by reactive oxygen-synthesizing enzymes (Nox). We immunohistochemically investigated expression and localization of the Nox family in a mouse circumvallate papillae. In the epithelium of the circumvallate papilla, Nox 1, 2, 3, and 4, Noxo1, and Noxa1 were expressed. In the circumvallate papilla, Nox2 was more weakly expressed in the lateral than in the upper part, and Nox3 was not expressed. In the taste buds, Nox 1, 2, 3, and 4, Noxo1, and Noxa1 were expressed; the Nox expression pattern varied with the cell type. In type II cells, Nox 1, 3, and 4, Noxo1, and Noxa1 were expressed. In type III cells, Nox2, besides Nox 1, 3, and 4, Noxo1, and Noxa1, were specifically expressed, unlike in other taste bud cells. In the mouse circumvallate papilla, Nox is always expressed, suggesting the generation of reactive oxygen species. Of note, all cells comprising taste buds expressed Nox, with each showing a specific Nox expression pattern.

Immunohistochemical localization of Nox1, Nox4 and Mn-SOD in mouse femur during endochondral ossification.

Enzymes synthesizing reactive oxygen (Nox family) have recently been identified. Elucidation of the production mechanism has been initiated, and the involvement of reactive oxygen in metabolism, intracellular transport, signal transmission and apoptosis has been reported. We immunohistochemically investigated expression and localization of the Nox family in endochondral ossification using a normal mouse femur. Weakly positive reactions with Nox1, Noxa1, and Noxo1 were observed in the zones of proliferative and prehypertrophic chondrocytes at 3 weeks of age. Nox4 was widely positive from the resting over the hypertrophic cell zone. At 18 weeks of age, none of the Nox types was expressed in chondrocytes as the zones disappeared. On the other hand, positive reactions with Nox1, Noxa1, Noxo1, and Nox4 were observed in osteoblasts in the zone of ossification at 3 weeks of age, and each Nox was also positive in osteoblasts arranged on the bone marrow side in the epiphyseal cartilage at 18 weeks of age. In addition, a reactive oxygen-eliminating enzyme, Mn-SOD, was observed only in prehypertrophic chondrocytes at 3 weeks of age, and not detected in osteoblasts. It was suggested that the Nox family is closely associated with endochondral ossification of the mouse femur, and Nox1 and Nox4 are closely involved in the chondrocyte maturation process and bone matrix formation.