Rat Gingival Tissue Research Articles

Induction of RANTES and CCR5 through NF-κB Activation via MAPK Pathway in Aged Rat Gingival Tissues

Chemokine and chemokine receptor expression in gingival tissues plays a central role in periodontal disease during aging. In the present study, we explored the modulation of chemokines and chemokine receptors expression in aging rat gingival tissues. In the 24-month-old (Old) rat gingival tissues, RANTES and CCR5 mRNA and protein levels were 2-4 fold increased over those of the 6-month-old (Young) rats. The Old rats had considerable enhancement of all three of the studied MAPK activities: extracellular signal regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK. These results suggest that age-related increases in RANTES and CCR5 expression are associated with increased IkappaBalpha, nuclear NF-kappaB, and MAPK activity in gingival tissues.

Effect of Cyclosporin A on the Expression of Inducible Nitric Oxide Synthase in the Gingiva of Rats

The role of nitric oxide (NO) in the pathogenesis of cyclosporin A (CsA)-induced gingival overgrowth is still unknown. The purpose of this study was to evaluate the effect of CsA on the expression of nitric oxide synthases (NOS) in the gingival tissue of rats. Thirty male Sprague-Dawley rats were randomly assigned to a control and two test groups. Rats in each group received CsA (0, 10, or 30 mg/kg) daily by gastric feeding for 4 weeks. The plasma NO and the NOS enzyme activities were assayed at week 4 in the blood samples and in the gingiva and lung tissue specimens, respectively. The distribution of inducible nitric oxide synthase (iNOS) was further evaluated in tissues obtained from the gingiva and lung at the end of weeks 1 and 4 by immunohistochemistry. In the CsA-treated animals, increased levels of plasma nitrites/nitrates were measured in comparison to those in control rats. Significantly greater iNOS enzyme activities were detected in lung and gingival tissues obtained from CsA-treated animals than from control animals. In addition, cells positively staining for iNOS were clearly observed in both gingival and lung tissues obtained from the CsA-treated animals by immunohistochemistry, whereas a few stained cells were found in those from the control group. The quantity of cells positively stained for iNOS was greater in tissue from week 4 than week 1. The effect of CsA on gingival iNOS expression was evaluated in rats for 4 weeks. A greater iNOS expression in the gingiva was observed after CsA therapy by both enzyme activities and immunohistochemica staining. Therefore, we suggest that CsA can increase gingival iNOS expression, which may play an important role in cyclosporin-induced gingival overgrowth.

Activity of Nitric Oxide Synthase and Concentration of Nitric Oxide End Metabolites in the Gingiva under Experimental Pathological Conditions

Parameters of NO metabolism in the gingiva were studied during experimental periodontitis accompanied by alloxan diabetes and exogenous hypercholesterolemia. We measured activities of inducible and constitutive NO synthase and concentrations of stable NO end metabolites in rat gingival tissue (total contents of nitrite and nitrate). Under pathological conditions NO-metabolism significantly differed from the control. Treatment with mexidol for 14 days significantly decreased activity of inducible NO synthase in the gingiva of experimental animals.

Diltiazem did not induce gingival overgrowth in rats: a clinical, histological and histometric analysis

The administration of calcium channel blockers has been associated with gingival overgrowth. However, there are few studies in humans or animals that evaluated the effect of diltiazem on gingival tissues. The present study assessed the influence of diltiazem, at different dosages and treatment duration, on gingival tissues of rats, using clinical, histological and histometric analyses. Eighty young male rats were separated into eight groups according to the dosage and duration of treatment. Rats were treated for 20 or 40 days with a daily subcutaneous injection of 5, 20 or 50 mg/kg of body weight of diltiazem. The results confirmed that diltiazem did not induce gingival overgrowth in rats. For all animals, the evaluation did not show gingival alterations regardless of the dosages and periods of treatment. The histometric analysis showed no significant change in the area of epithelium and connective tissues, although after 40 days of treatment a decrease in the area of connective tissue was observed, without statistically significant difference from control groups. Within the limits of this study, we suggest that diltiazem did not induce gingival overgrowth.

Inhibition of Growth of Human Gingival Fibroblasts by Chimeric DNA‐RNA Hammerhead Ribozyme Targeting Transforming Growth Factor‐β1

Transforming growth factor (TGF)-beta1 is involved in the pathogenesis of both drug-induced gingival overgrowth and hereditary gingival fibromatosis. Ribozymes enzymatically cleave target mRNAs and are expected to be utilized as the basis of novel nucleic acid-based therapies. We designed a chimeric DNA-RNA ribozyme targeting TGF-beta1 mRNA and examined its effect on growth of gingival fibroblasts in culture. Chimeric DNA-RNA hammerhead ribozyme with sequence complementary to the loop structure of human TGF-beta1 mRNA was used. We evaluated transfer of the chimeric ribozyme by hemagglutinating virus of Japan (HVJ)-envelope into cultured human gingival fibroblasts in vitro and rat gingival tissues in vivo. We then examined effects of the chimeric ribozyme to TGF-beta1 on proliferation and DNA synthesis in human gingival fibroblasts. We also examined effects of the chimeric ribozyme to TGF-beta1 on expression of TGF-beta1, type IV collagens, and fibronectin mRNAs and expression of TGF-beta1 protein in human gingival fibroblasts. Chimeric ribozyme was sufficiently distributed into human fibroblasts in vitro and rat gingivae in vivo. Chimeric ribozyme to TGF-beta1 significantly inhibited expression of TGF-beta1, type IV collagen, and fibronectin mRNAs and TGF-beta1 protein in human gingival fibroblasts. Mismatch ribozyme had no effect on expression of these molecules. Chimeric ribozyme to TGF-beta1 also significantly inhibited proliferation and DNA synthesis in gingival fibroblasts. Chimeric DNA-RNA ribozyme targeting TGF-beta1 may be a useful gene therapy agent for treatment of gingival hyperplasia.

Growth factors and proliferation of cultured rat gingival cells in response to cyclosporin A

The prominent side-effect of cyclosporin A, an immunosuppressive drug, in oral tissues is gingival outgrowth, although the exact mechanism underlying this side-effect is unclear. The main purposes of the present study were to determine whether cyclosporin A induced the gingival outgrowth by promoting proliferation of gingival cells and whether growth factors such as transforming growth factor-betas (TGF-betas), fibroblast growth factor-2 (FGF-2), platelet-derived growth factors (PDGFs), and insulin-like growth factors (IGFs) are involved in the possible changes in the proliferation of gingival cells induced by cyclosporin A. Cells isolated from rat gingival tissues were cultured with cyclosporin A or IGF-I for 3 days. The effects of cyclosporin A or IGF-I on the proliferation of cultured rat gingival cells were analyzed with a CellTiter 96 proliferation assay kit. The mRNA expression levels for TGF-betas, FGF-2, PDGFs, IGFs, insulin-like growth factor receptors (IGFRs), and insulin-like growth factor binding proteins (IGFBPs) in the rat gingival cells treated with cyclosporin A were measured using competitive reverse transcription-polymerase chain reaction (RT-PCR). Cyclosporin A induced 23-25% (p < 0.001) increases in the proliferation of rat gingival cells and approximately 130% (p < 0.05) and 60% (p < 0.05) elevations in the mRNA expression levels for TGF-beta1 and FGF-2, respectively. On the other hand, exogenous IGF-I induced 8-11% (p < 0.05) increases in the proliferation, but cyclosporin A induced 30-80% (p < 0.05-0.01) reductions in the mRNA expression levels for endogenous IGF-I, IGFR1, IGFBP2, IGFBP3, IGFBP5, and IGFBP6. Cyclosporin A stimulates the proliferation of rat gingival cells. TGF-beta1 and FGF-2 could be involved, but IGFs, IGFRs and IGFBPs could not be directly involved in this cyclosporin A induced-stimulation of the gingival cell proliferation.

Effect of nifedipine on the expression of bcl-2 protein in rat gingiva

Bcl-2 is a family of proteins involved in protecting the cell against death stimuli or in promoting cell death. The aim of the present study was to determine the effect of nifedipine treatment on the expression of bcl-2 protein in rat gingival tissues. Rats were given gastric intubation with various concentrations and durations of nifedipine. Nifedipine-untreated and dimethylsulfoxide (DMSO)-treated animals served as control groups. The gingival tissues were dissected and the expression of bcl-2 protein was determined immunohistochemically. The results showed that the numbers of bcl-2-positive cells in the gingiva of nifedipine-treated animals were significantly higher than in the control groups. These numbers increased parallel to increased concentration and duration of nifedipine treatment. The results suggest that nifedipine treatment may induce the expression of bcl-2 protein in rat gingival tissue in a dose- and duration-dependent fashion and that this proto-oncogenic protein may play a role in nifedipine-induced gingival hyperplasia.

Análise quantitativa dos tecidos gengivais de ratos tratados com fenitoína e ciclosporina

Os aumentos gengivais podem ser decorrentes de reações teciduais a estímulos idiopáticos, patológicos e farmacológicos. O objetivo desse trabalho foi avaliar morfometricamente e estereologicamente a ação da fenitoína (Fen) e ciclosporina (CsA) sobre os tecidos gengivais de ratos. Dez ratos receberam, por via intraperitonial, Fen na dose inicial de 2 mg/kg de peso corporal/dia, aumentando 2 mg a cada duas semanas, durante 60 dias. Em outros 10 ratos, administraram-se, por via subcutânea, 10mg/kg de peso corporal/dia de CsA, durante o mesmo período do grupo anterior. Os valores morfométricos e estereométricos dos tecidos gengivais dos ratos tratados com CsA foram significativamente maiores quando comparado com os valores dos tecidos gengivais do grupo tratado com Fen. Esses resultados sugerem que a CsA na dose utilizada é mais eficaz no desenvolvimento do aumento gengival em ratos, podendo estar atuando na proliferação de fibroblastos e no desequilíbrio fisiológico da síntese de fibras colágenas.

Effect of Nicotine on Rat Gingival Fibroblasts In Vitro

Tobacco smoking is considered a major risk factor for the development and progression of periodontal diseases (Haber, J. and Wattles, J. (1994). J. Periodontol. 64, 16-23). The purpose of this study was to determine the effects of nicotine on rat gingival fibroblasts (RGF) cultured in vitro. After ether anesthesia, rat gingival tissues were obtained from the attached gingiva of a Wistar rat. Small fragments of gingiva were maintained in culture in Petri dishes. Fibroblasts developing from these explants were collected to obtain monolayer cultures. After the fourth passage (T4), cells were supplemented with nicotine at various concentrations. Control and treated cells were examined under phase contrast or transmission electron microscopy. They were compared as regards their DNA content, mitochondrial activity, collagen and protein synthesis, and cell death by apoptosis or necrosis. Nicotine from 0.05 uM to 1 miVl did not affect the DNA content or protein and collagen synthesis. At concentrations between 3 and 5 niM. growth was significantly diminished and the survival rate reduced. Ultrastructural analysis revealed dilated mitochondria and vacuolization in treated cells, suggestive of necrosis, but increased apoptosis was also revealed by cytometry. On the basis of this in vitro study, it appears that tobacco, through its component nicotine, may directly affect various functions of RGF.

Graft of autologous fibroblasts in gingival tissue in vivo after culture in vitro. Preliminary study on rats.

Several grafting techniques and guided tissue regeneration techniques (GTR) have been well-developed in periodontal surgery. However, these techniques could induce pain and side effects, such as a gingival recession during the healing period following the therapy. The graft of a small autologous connective tissue, using non-invasive surgical techniques could yield several benefits for the patients. Our preliminary study explores the feasibility of collecting healthy gingival tissues, culturing them in vitro to amplify rat gingival fibroblasts (RGF) and inoculating the obtained cells into autologous rat gingival tissues in vivo. Gingival tissues samples were cultured as explants as described by Freshney et al. and Adolphe. Confluent cells surrounding explants were detached after 7 d of culture from Petri dishes using 0.05% trypsin and designated "first transferred cells" (T1). At the third passage (T3), cells cultured as monolayer were either examined under microscopy--phase contrast, scanning, or transmission electron--or numerated after trypan blue exclusion test. Autologous RGF labelled with fluorochrome were inoculated at the vestibular and palatine site of gingival tissue close to the superior incisors. In this preliminary study, 12 Wistar rats were used; for each, 2 biopsies were dissected and fixed for phase contrast or fluorescence microscopy. On d 1, 3 and 7 after injection in rat gingival tissues, fluorochrome-labelled cells could be detected in all these.

Immunocytochemical localization of substance P neurokinin-1 receptors in rat gingival tissue.

The distributions of substance P (SP) and the neurokinin-1 receptor (NK1-R), the receptor preferentially activated by SP, were examined in rat gingiva by immunocytochemical methods with light and electron microscopy. SP-immunoreactive nerve fibers were located preferentially in the junctional epithelium (JE) but few in the other oral and oral sulcular epithelia. NK1-R immunoreactivity was found in the endothelial cells (capillaries and postcapillary venules underlying the JE). NK1-R-labeled and -unlabeled unmyelinated nerve fibers were located close to the blood vessels and partially or completely covered by a Schwann cell sheath. In the JE, labeled naked axons without Schwann cell sheaths were observed. Neutrophils and macrophages in the connective tissue underlying the JE and in the JE were also labeled with NK1-R. Furthermore, NK1-R was found in the JE cells. Basically, immunoreaction products for NK1-R were found throughout various cells (endothelial cells, neutrophils, and JE cells) at invaginations of the plasma membrane and in vesicular and granular structures that are probably endosomes and are found close to both the plasma membrane and the nucleus. This is a first report, demonstrating the presence of NK1-R in the gingival tissue in the normal nonstimulated condition. Furthermore, it is thought that SP may modulate the permeability of blood vessels beneath the JE, the production of antimicrobial agents in neutrophils, and the proliferation and endocytotic ability of JE cells through NK1-R.

Distribution of macrophage lineage cells in rat gingival tissue after topical application of lipopolysaccharide: an immunohistochemical study using monoclonal antibodies: OX6, ED1 and ED2.

To discuss the role of macrophage lineage cells on the periodontal tissue destruction, we immunohistochemically examined the phenotype and the dynamics of macrophage lineage cells 1 or 3 h or 1, 2, 3 or 7 d after topical application of LPS (5 mg/ml in physiological saline) from the rat gingival sulcus using 3 monoclonal antibodies: OX6 (antigen-presenting cells), ED1 (monocytes, macrophages and dendritic cells) and ED2 (resident macrophages). We could detect at least 3 different types of macrophage lineage cells, namely OX6+/ED1+/ED2- dendritic cells and exudate macrophages and ED2+ resident macrophages. After LPS application the majority of macrophage lineage cells accumulated in the subjunctional epithelial area were newly extravasated OX6+/ED1+/ED2- dendritic cells or macrophages. The number of these cells increased progressively with time and reached a maximum level at d 2. On the other hand, number and tissue distribution of ED2+ resident macrophages did not change. These results indicate that several types of macrophage lineage cells exist in rat gingival tissue and suggest that dendritic cells and exudate macrophages transiently accumulated after LPS application are responsible for various host immune response and tissue destruction caused by LPS.

Distribution of macrophage lineage cells in rat gingival tissue after topical application of lipopolysaccharide: an immunohistochemical study using monoclonal antibodies: 0X6, ED1 and ED2

To discuss the role of macrophage lineage cells on the periodontal tissue destruction, we immunohistochemically examined the phenotype and the dynamics of macrophage lineage cells 1 or 3 h or 1, 2, 3 or 7 d after topical application of LPS (5 mg/ml in physiological saline) from the rat gingival sulcus using 3 monoclonal antibodies: OX6 (antigen‐presenting cells), EDI (monocytes, macrophages and dendritic cells) and ED2 (resident macrophages). We could detect at least 3 different types of macrophage lineage cells, namely 0X6+ /ED1+ /ED2− dendritic cells and exudate macrophages and ED2+ resident macrophages. After LPS application the majority of macrophage lineage cells accumulated in the subjunctional epithelial area were newly extravasated 0X6+/EDI+/ED2− dendritic cells or macrophages. The number of these cells increased progressively with time and reached a maximum level at d 2. On the other hand, number and tissue distribution of ED2+ resident macrophages did not change. These results indicate that several types of macrophage lineage cells exist in rat gingival tissue and suggest that dendritic cells and exudate macrophages transiently accumulated after LPS application are responsible for various host immune response and tissue destruction caused by LPS.

Phospholipase A2 in rat gingival tissue

Phospholipase A2 (PLA2) is a proinflammatory enzyme in the synovial fluids of all--and sera of some--patients with rheumatoid arthritis. Due to the similarities in pathogenesis between rheumatoid arthritis and periodontitis, we sought to study the enzymatic properties of PLA2 in periodontal tissue. In this study, we demonstrated PLA2 activity in rat gingival tissue, about 80% of which was present in the cytosolic fraction. We characterized the cytosolic PLA2 enzyme with respect to substrate specificity, sensitivity to detergent, Ca2+ ion dependency and optimum pH. We found that phosphatidylethanolamine, rather than phosphatidylcholine, was the preferred substrate, the Ca2+ ion was essential for the expression of PLA2 activity, the enzyme was active over a broad pH range, with the optimum at pH 9.0, and sodium-deoxycholate inhibited the enzyme activity strongly in a concentration-dependent manner. These results are consistent with those which have been obtained with synovial fluid PLA2 and suggest that gingival PLA2 may be involved in the pathogenic processes of gingivitis and periodontitis.

Effect of toxic doses of cholecalciferol (vitamin D3) on alkaline phosphatase levels in serum and gingival tissues of rats.

Toxic doses of cholecalciferol (vitamin D3) were injected subcutaneously (25 micrograms/day) into rats for 5 weeks, causing a significant increase of serum alkaline phosphatase (ALP) levels (p less than 0.01). Gingival tissue alkaline phosphatase levels were also elevated (p less than 0.01). On the other hand, a significant difference was detected between serum calcium levels in experimental and control groups (p less than 0.02).

Gingival tissue-produced inhibition of platelet aggregation and the loss of inhibition in streptozotocin-induced diabetic rats.

Addition of medium incubated with normal rat gingival tissue to platelet‐rich plasma inhibited ADP‐induced platelet aggregation. The ability of rat gingiva to produce activity inhibiting platelet aggregation was enhanced by the addition of arachidonic acid. Diabetic rat gingiva failed to inhibit platelet aggregation but did produce the anti‐platelet aggregating activity in the presence of arachidonic acid. Indomethacin blocked the production of anti‐platelet aggregating activity. There was no difference in conversion of [1‐14C]arachidonic acid to prostaglandins by normal and diabetic rat gingiva. These results suggest that an arachidonic acid metabolite released from gingiva during incubation inhibits platelet aggregation, and the synthesis of the metabolite is impaired in diabetic rat gingiva. A decrease in availability of arachidonic acid may be a causal factor of the defect in diabetic rat gingiva.

Preparation and characterization of human gingival cells.

A procedure was developed for the preparation of single cell suspensions from gingival and periodontal tissues using collagenase digestion followed by gentle mechanical disruption. The procedure was evaluated on rat gingival tissues. One‐hour incubation was found to produce the greatest number of cells with the highest viability, and the largest recovery of mononuclear cells. Collagenase did not appear to effect recovery, viability or B cell surface markers on human peripheral blood cells. Human gingival and periodontal tissues were obtained from 35 patients: 20 with adult periodontitis; 8 with juvenile periodontitis; 7 with normal gingivae or chronic gingivitis. Scaling, followed by probing measurements and then surgery of the affected sites was routinely performed. In single cell suspensions from tissue obtained from surgery, significantly fewer mononuclear cells were recovered from the tissues of normal/chronic gingivitis patients than cells/mg of tissue recovered from adult periodontitis or juvenile periodontitis groups. The maximum contribution of blood mononuclear cells to the gingival cells averaged 0.5+0.2%. This method is useful for recovering gingival (periodontal) cells for phenotypic and functional studies.

コンカナバリン A 接種による歯肉組織内のリンパ球と T 細胞 subset の経日変動に関する研究

コンカナバリン A 接種による歯肉組織内のリンパ球と T 細胞 subset の経日変動に関する研究

Fibrinolytic activities in saliva and gingival tissue of rats with experimental gingivitis.

Periodontal disorders are accompanied by a series of clinical symptoms such as formation of dental plaque, deposition of dental calculus, changes in the colour of gingiva, morbid abnormal formation of the gingival pocket, purulent discharge and mobility of teeth. Among these, bleeding from the perigingival area is a rather marked symptom. There are many outpatients who visit the department of dentistry with perigingival bleeding as the chief complaint. Thus, the problem of bleeding and hemostasis is, along with the inflammatory reactions, an important issue that is frequently encountered.Fibrinolysis is a reaction involving a series of enzymes including plasmin. Plasmin exists in vivo as an inactive plasminogen and is activated by tissue activators, bacteriokinase, mental stress, and antigen-antibody reaction. Fibrinolytic activities are reported mainly in association with fibrinolysis in saliva, mandibular cyst walls and gingival tissues.The present study, examined the relationship between the inflammation process and plasminogen activator activity, using gingival tissue, saliva and blood samples collected from the rats with experimentally induced gingivitis.

Effect of the nature and amount of dietary energy on lipid composition of rat gingival tissue.

1. The effect of the nature and amount of dietary energy on the lipid composition of rat gingival tissue was studied. Male weanling rats were given one of three iso-energetic diets: high-carbohydrate, high-protein and extremely high-protein, or a fourth high-fat diet, for 49 d. 2. The high-carbohydrate, extremely high-protein and high-fat diets caused significant increases in the gingival levels of total lipids compared with the normal-protein diet. These increases in total lipids were due primarily to increases in the levels of triglycerides and cholesterol esters. There were no significant differences in the fatty acid composition of either non-polar or polar lipids among rats given the high-carbohydrate diet and those given the high-protein diet. 3. A composition of the fatty acid composition of lipids of rats given the extremely high-protein diet and the other two iso-energetic diets revealed that the proportion of palmitic acid was higher and the proportion of oleic acid was lower to animals given the extremely high-protein diet than in animals given the other two diets. Compared with the three iso-energetic low-fat diets, the high-fat diet caused decreases in the proportion of palmitic and palmitoleic acids and increases in the proportion of linoleic, arachidonic and docosapentaenoic acids in total fatty acids of both no-polar and polar lipids. It should be noted that the high-fat diet contained a high proportion of linoleic acid and it is expected that this diet would raise the 18:2 fatty acid content of the lipids and also would raise the 20:4 and 22:5 levels as 18:2 is an essential fatty acid and will, with its metabolites, be directly incorporated into tissue lipids.