Distribution and organization of lymphatic vessels in the mouse gingiva: An immunohistochemical study with LYVE-1.

OBJECTIVE: This study introduced the usefulness of LYVE-1 immunoreactivity for identification of lymphatic vessels in decalcified tissues, and demonstrated the fine distribution and organization of these vessels in mouse gingiva. DESIGN: After confirming the specificity of anti-mouse LYVE-1, frozen sections of mouse decalcified gingiva were immunostained with the antibody. RESULTS: The LYVE-1-positive lymphatic vessels were clearly found in the connective tissue under the gingival epithelium; these vessels appeared to pass through the lamina propria of the gingiva toward the alveolar crest and run along the external surface of the alveolar bone. The lymphatic vessels were sparse and apart from the oral gingival and sulcular epithelia, while they were dense adjacent to the junctional epithelium. CONCLUSIONS: The dense network of the lymphatic vessels adjacent to the junctional epithelium, which is apparently exposed to foreign antigens, may act as an efficient drainage pathway of the excessive interstitial fluid and immune cells, and play an active role in the immune defense of the gingiva. The present study also revealed the absence of lymphatic connection between gingiva and periodontal ligament.

Growth of rat parotid glands is inhibited by liquid diet feeding.

This study investigated how liquid diet feeding affects the growth of parotid glands. We weaned 21-day-old rats and thereafter fed them a pellet diet (control group) or a liquid diet (experimental group) for 0, 1, 2, 4, or 8 weeks. Their parotid glands were excised, weighed, examined, and tested for 5-bromo-2'-deoxyuridine (BrdU) and cleaved caspase-3 (Casp-3) as markers of proliferation and apoptosis, respectively. Parotid gland weights were consistently smaller in experimental animals than in controls. Morphometrical analysis showed that control group acinar cells increased in area during the experiment, but experimental group acinar cells were almost unchanged. Labeling indices of BrdU in acinar cells in both groups declined during the experiment, but were consistently lower in the experimental group than in controls. Casp-3-positive acinar cells were rare in both groups, which consistently express significantly similar Casp-3 levels. Ultrastructurally, terminal portions of the experimental parotid glands consisted of a few acinar cells that were smaller than those in controls. Control acinar cells showed mitotic figures within short experimental periods, but not in experimental glands. These observations indicate that liquid diet feeding inhibits growth of parotid glands in growing rats through suppression of growth and proliferation of individual acinar cells, but not through apoptosis.

Involvement of apoptosis and proliferation of acinar cells in atrophy of rat parotid glands induced by liquid diet.

Parotid glands of experimental animals fed a liquid diet are reported to show atrophy (Hall and Schneyer 1964; Wilborn and Schneyer 1970; Hand and Ho 1981; Scott et al. 1990; Scott and Gunn 1991). To clarify whether apoptosis and proliferation of acinar cells participate in atrophy of rat parotid glands induced by liquid diet, rats were fed a liquid diet and compared to pellet-fed controls. Parotid glands were removed at 3, 7, 14 or 21 days, weighed, and examined using transmission electron microscopy (TEM), and studied immunohistochemically for cleaved-caspase-3 (Casp-3), a marker of apoptotic cells, and 5-bromo-2'-deoxyuridine (BrdU), a marker for proliferating cells. Body weights of experimental rats fed liquid diets were not significantly different from controls fed pellet diets; however weights of experimental parotid glands were smaller than those of controls. In the experimental parotid glands, structures like apoptotic bodies were histologically observed in acini at each time point; more Casp-3-positive acinar cells were identified in experimental parotid glands than in the controls on days 3, 7, and 14. Experimental glands showed fewer BrdU-positive acinar cells at each time point. TEM confirmed typical apoptotic acinar cells in the atrophic glands. These findings suggest that increased acinar cell apoptosis and reduced acinar cell proliferation occur in atrophic parotid glands of rats fed a liquid diet.

Morphological assessment of bone mineralization in tibial metaphyses of ascorbic acid-deficient ODS rats.

Osteogenic disorder shionogi (ODS) rats carry a hereditary defect in ascorbic acid synthesis, mimicking human scurvy when fed with an ascorbic acid-deficient (aa-def) diet. As aa-def ODS rats were shown to feature disordered bone formation, we have examined the bone mineralization in this rat model. A fibrous tissue layer surrounding the trabeculae of tibial metaphyses was found in aa-def ODS rats, and this layer showed intense alkaline phosphatase activity and proliferating cell nuclear antigen-immunopositivity. Many osteoblasts detached from the bone surfaces and were characterized by round-shaped rough endoplasmic reticulum (rER), suggesting accumulation of malformed collagen inside the rER. Accordingly, fine, fragile fibrillar collagenous structures without evident striation were found in aa-def bones, which may result from misassembling of the triple helices of collagenous α-chains. Despite a marked reduction in bone formation, ascorbic acid deprivation seemed to have no effect on mineralization: while reduced in number, normal matrix vesicles and mineralized nodules could be seen in aa-def bones. Fine needle-like mineral crystals extended from these mineralized nodules, and were apparently bound to collagenous fibrillar structures. In summary, collagen mineralization seems unaffected by ascorbic acid deficiency in spite of the fine, fragile collagenous fibrils identified in the bones of our animal model.

Apoptosis of odontoclasts under physiological root resorption of human deciduous teeth.

This study was designed to establish the apoptosis of odontoclasts during physiological root resorption of human deciduous teeth. Deciduous teeth were fixed, decalcified, and embedded in paraffin for immunohistochemical (IHC) observations and in Epon for transmission electron microscopy (TEM). Apoptotic cells were identified by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick-end labeling (TUNEL), and then tartrate-resistant acid phosphatase (TRAP) activity was determined on the same sections. Epon-embedded specimens were sectioned serially into 0.5-microm semithin sections; some of these sections were re-embedded in Epon, sectioned into 0.1-microm ultrathin sections, and observed by TEM. IHC revealed that the nuclei of TRAP-positive odontoclasts on the dentine were generally TUNEL-negative. Around these odontoclasts, a few TRAP-positive structures were present together with TUNEL-positive structures, e.g., a TRAP-positive structure with one TUNEL-positive nucleus, a TRAP-positive structure with one TUNEL-positive nucleus plus one or two TUNEL-negative nuclei, or a TRAP-positive structure with no nucleus. By TEM, some odontoclasts showed nuclear fragments including compacted chromatin. The results suggest that, during apoptosis, odontoclasts fragment into variously sized cellular parts including three or fewer nuclei.

Effects of eight antibacterial agents on cell survival and expression of epithelial-cell- or cell-adhesion-related genes in human gingival epithelial cells.

OBJECTIVE AND BACKGROUND: Our previous studies suggest that little adverse effect on the growth of the periodontal ligament would be expected, if tetracycline, minocycline, ofloxacin, roxithromycin, clarithromycin, and azithromycin were topically administered to the periodontal pocket at their MIC90 doses required to inhibit the growth of 90% of periodontopathic bacteria, including Porphyromonas gingivalis, Prevotella intermedia, and Actinobacillus actinomycetemcomitans. In the present study, we investigated the cytocidal effects of eight antibacterial agents on the human gingival epithelial cell line NDUSD-1. We also used NDUSD-1 cells to examine the effects of these agents on the mRNA and protein expressions of genes associated with the proliferation, differentiation, or cellular adhesion important to the epithelial regeneration of the periodontal attachment. METHODS: The cytocidal effect of the test agents was measured as a decrease in cell survival. To obtain a quantitative measure of the cytocidal effect, the LD50, i.e. the concentration which results in a 50% decrease in cell survival relative to the controls, was extrapolated from the concentration-response curves. The effects of the agents on the mRNA and protein expressions in NDUSD-1 cells were studied by reverse transcription-polymerase chain reaction (RT-PCR) and western blot analyses, respectively. RESULTS: The cytocidal effect increased in a concentration-dependent manner as the concentration of each of the eight test agents increased. The order of the agents according to their cytocidal effects (LD50) was minocycline > tetracycline > enoxacin > clarithromycin > roxythromycin approximately ofloxacin > azithromycin > erythromycin. The cytocidal effects of minocycline, tetracycline, enoxacin, clarithromycin, roxythromycin, ofloxacin, and azithromycin ranged from 1.2 to 23.2 times greater than that of erythromycin. The maximum non-cytocidal concentrations (MNCCs) of these agents for NDUSD-1 cells were: 0.3 microm for minocycline, 1 microm for tetracycline, 3 microm for ofloxacin and erythromycin, 10 microm for enoxacin, clarithromycin, and azithromycin, and 100 microm for roxythromycin. The MNCCs of ofloxacin, azithromycin, clarithromycin, and roxythromycin were greater than their MIC90 concentrations for periodontopathic bacteria described above. The effects on the mRNA and protein expressions of epithelial-cell- or cell-adhesion-related genes were examined in NDUSD-1 cells exposed to clarithromycin, roxythromycin, ofloxacin, and azithromycin at their MNCCs. None of the agents affected the mRNA expressions of five genes: keratinocyte growth factor receptor, keratin 18, integrin beta1, integrin beta4, and laminin 5gamma2. Clarithromycin and ofloxacin slightly decreased the protein expression of integrin beta4. Roxythromycin markedly decreased the protein expressions of integrin beta4 and laminin 5gamma2. Azithromycin had little inhibitory effects on the protein expressions of any of the five genes. CONCLUSIONS: These results suggest that little, if any, adverse effects on growth, differentiation, and adhesion of basal epithelial cells would be expected with topical administration of clarithromycin, ofloxacin or azithromycin to the periodontal pocket at a dose equivalent to the MIC90. It is important to note, however, that the extrapolation of these findings to in vivo conditions has yet to be undertaken.