TRPC expression in human periodontal ligament cells and the periodontal tissue of periodontitis mice: a preliminary study / 한국실험동물학회지

Background@#Transient receptor potential canonical (TRPC) channels are non-selective cationic channels with perme‑ ability to ­Ca2+ and ­Na+ . Despite their importance, there are currently few studies on TRPC in the periodontal ligament (PDL) and bone cells in the dental field. To provide biological information regarding TRPC in PDL cells and periodontal tissue, we evaluated TRPC channels expression in the osteoblast differentiation of PDL cells and periodontitis-induced tissue. Human PDL cells were cultured in osteogenic differentiation media for 28 days, and the expression of Runx2, osteocalcin (OCN), and TRPC1, 3, 4, and 6 was evaluated by real-time PCR. In ligature-induced periodontitis mice, the alveolar bone and osteoid areas, the osteoclast number, and the expression of Runx2, OCN, TRPC3, and TRPC6 was evaluated by H&E staining, TRAP staining, and immunohistochemistry, respectively. @*Results@#In the PDL cell differentiation group, TRPC6 expression peaked on day 7 and TRPC3 expression generally increased during differentiation. During the 28 days of periodontitis progression, alveolar bone loss and osteoclast numbers increased compared to the control group during the experimental period and the osteoid area increased from day 14. TRPC6 expression in the periodontitis group increased in the PDL area and in the osteoblasts compared to the control group, whereas TRPC3 expression increased only in the PDL area on days 7 and 28. @*Conclusions@#These results indicate changes of TRPC3 and TRPC6 expression in PDL cells that were differentiating into osteoblasts and in periodontitis-induced tissue, suggesting the need for research on the role of TRPC in osteo‑ blast differentiation or periodontitis progression.

Lipopolysaccharides of Fusobacterium nucleatum and Porphyromonas gingivalis increase RANKL-expressing neutrophils in air pouches of mice / 한국실험동물학회지

Increases of neutrophils and osteoclasts are pathological changes of periodontitis. RANKL is an osteoclast differentiation factor. The effect of periodontopathogen LPS on RANKL-expressing neutrophils has not been clarified yet. We evaluated numerical changes of RANKL-expressing neutrophils in air pouches of mice injected with LPSs of Fusobacterium nucleatum and Porphyromonas gingivalis. Mice with air pouches were assigned into saline (C)-, E. coli LPS- (Ec LPS)-, F. nucleatum LPS (Fn LPS)-, P. gingivalis LPS (Pg LPS)-, and Fn LPS and Pg LPS (Fn + Pg LPS)-injected groups. CD11b +Ly6G + neutrophils and CD11b +Ly6G+RANKL + neutrophils in blood and air pouch exudates were determined by flow cytometry. In blood, compared to the C group, the Fn LPS group showed increases of CD11b +Ly6G + neutrophils and CD11b +Ly6G +RANKL + neutrophils whereas the Pg LPS group showed no significant differences. These increases in the Fn LPS group were not different to those in the Ec LPS group. In exudates, Fn LPS and Pg LPS groups showed increases of CD11b +Ly6G + neutrophils and CD11b +Ly6G +RANKL + neutrophils compared to the C group. Increased levels in the Fn LPS group were not different to those in the Ec LPS group, but Pg LPS group was lower than those in the Ec LPS group. In blood and exudates, the Fn+ Pg LPS group showed no difference in levels of these neutrophils compared to the Ec LPS group. LPSs of F. nucleatum and P. gingivalis increased RANKL-expressing neutrophils although the degrees of increases were different. These suggest that periodontopathogen LPS can act as a stimulant to increase RANKL-expressing neutrophils.

Lipopolysaccharides of Fusobacterium nucleatum and Porphyromonas gingivalis increase RANKL-expressing neutrophils in air pouches of mice / 한국실험동물학회지

Increases of neutrophils and osteoclasts are pathological changes of periodontitis. RANKL is an osteoclast differentiation factor. The effect of periodontopathogen LPS on RANKL-expressing neutrophils has not been clarified yet. We evaluated numerical changes of RANKL-expressing neutrophils in air pouches of mice injected with LPSs of Fusobacterium nucleatum and Porphyromonas gingivalis. Mice with air pouches were assigned into saline (C)-, E. coli LPS- (Ec LPS)-, F. nucleatum LPS (Fn LPS)-, P. gingivalis LPS (Pg LPS)-, and Fn LPS and Pg LPS (Fn + Pg LPS)-injected groups. CD11b +Ly6G + neutrophils and CD11b +Ly6G+RANKL + neutrophils in blood and air pouch exudates were determined by flow cytometry. In blood, compared to the C group, the Fn LPS group showed increases of CD11b +Ly6G + neutrophils and CD11b +Ly6G +RANKL + neutrophils whereas the Pg LPS group showed no significant differences. These increases in the Fn LPS group were not different to those in the Ec LPS group. In exudates, Fn LPS and Pg LPS groups showed increases of CD11b +Ly6G + neutrophils and CD11b +Ly6G +RANKL + neutrophils compared to the C group. Increased levels in the Fn LPS group were not different to those in the Ec LPS group, but Pg LPS group was lower than those in the Ec LPS group. In blood and exudates, the Fn+ Pg LPS group showed no difference in levels of these neutrophils compared to the Ec LPS group. LPSs of F. nucleatum and P. gingivalis increased RANKL-expressing neutrophils although the degrees of increases were different. These suggest that periodontopathogen LPS can act as a stimulant to increase RANKL-expressing neutrophils.

Phagocytic osteoclasts in the alveolar bone of diabetic rats with periodontitis

Periodontitis is a bacteria-induced inflammatory disease associated with alveolar bone loss. Osteoclast is a macrophage-lineage cell that exhibits phagocytic activity; however, osteoclast phagocytic activity has not been demonstrated under pathological conditions. Diabetes is a pathological condition that exacerbates alveolar bone loss via periodontitis; therefore, we examined phagocytic osteoclasts in diabetic rats that had periodontitis. The rats were divided into the control (C), periodontitis (P), and diabetes with periodontitis (DP) groups. Diabetes and periodontitis were induced by streptozotocin injection and ligature of the mandibular first molars, respectively. On days 3 and 20 after the ligature, the rats were sacrificed, and osteoclasts containing inclusions were quantified by tartrate-resistant acid phosphatase staining. On day 3, there were more osteoclasts containing inclusions in the DP group than in the C group. Among inclusions, osteocyte-like cells and dense bodies were more frequently observed in the DP group than in the C group. Cytoplasm-like structures were elevated more in the DP group than in the C and P groups. However, no differences were observed on day 20. Interestingly, some osteoclasts were in contact with the osteocytes within the exposed lacunae and contained several inclusions within a large vacuole. Thus, the elevation of phagocytic osteoclasts in rats with diabetes and periodontitis provides insight into the role of osteoclast phagocytic activity under pathological conditions.

Eosinophilic encephalomyelitis in horses caused by protostrongylid parasites

Four thoroughbred horses showing lameness, ataxia, circling, depression, recumbency, and seizures, were examined. The horses had gross, pale- to dark-red manifestations and foci in the central nervous system (CNS). Multifocal to coalescing eosinophilic necrotizing encephalomyelitis was observed histologically in the CNS along with intact or degenerated nematodes. Nematodes had polymyarian-coelomyarian musculature, a smooth thin cuticle, and intestines lined by multinucleated cells with microvilli. These traits suggested the nematodes belonged to the family Protostrongylidae, which includes Parelaphostrongylus tenuis. It was concluded that the horses were infected by nematodes, presumably Parelaphostrongylus tenuis, resulting in eosinophilic necrotizing encephalomyelitis.

TNF-α Inhibitor Reduces Odontoclast Formation in Diabetes Rats with Ligature-Induced Periodontitis

To determine the effect of the tumor necrosis factor-α (TNF-α) in odontoclast formation, we administrated a TNF-α inhibitor in rats with diabetes rats with periodontitis. The rats included in the study were divided into three groups: control rats without diabetes or periodontitis (the C group), rats with periodontitis and diabetes (the PD group), and rats with periodontitis and diabetes treated by infliximab, the TNF inhibitor (the PD+infliximab group). The PD and PD+ infliximab groups received intravenous administrations of streptozotocin (STZ, 50 mg/kg) to induce diabetes. After 7 days of STZ injections, the mandibular first molars were ligatured to induce periodontitis. The PD+infliximab group was intrapenitoneally administrated by infliximab (5 mg/kg). On days 3 and 20 after the ligature administration, odontoclast formation along root surfaces was evaluated by tartrate resistant acid phosphatase (TRAP) staining and cathepsin K immunohistochemistry. On day 3, the number of TRAP- and cathepsin K-positive cells increased more so in the PD group than in the C group. The PD+infliximab group showed a lower number of positive cells than the PD group. There was no difference in all the groups on day 20. On day 3, the cathepsin-K positive multinucleated and mononucleated cells were higher in the PD group than in the C group. The number of cathepsin-K positive multinucleated cells was lower in the PD+infliximab group than in the PD group. The PD group showed more cathepsin K-positive cells in the furcation and distal surfaces than the c group. The Cathepsin K-positive cells of the PD+infliximab group were lower than that of the PD group in furcation. These results suggest that TNF-α stimulates odontoclast formation in diabetes with periodontitis.

Mongolian Gerbil as a Novel Animal Model for Ligature-induced Periodontitis

Inflammation from chronic and acute infections of distal organs and tissues such as periodontitis is a risk factor for atherosclerotic vascular processes. Recently, a new model of atherosclerosis with vascular pathologies was developed in the Mongolian gerbil. In this study, we attempted to develop a model of ligature-induced periodontitis in gerbils and compared the characteristics of that periodontitis model with that in rats and mice. Each gerbil, rat, and mouse was randomly assigned to groups of control and periodontitis. A thread was placed around the cervix of the right and left first molars in the mandible with knots placed on the mesial side of each molar. At day 14 after the ligation, the animals were sacrificed and their mandibles were dissected. To measure alveolar bone loss along with inflammation, histopathological and micro-CT analyses were carried out. Gerbils showed tooth characteristics of deeper gingival crevice, longer cusp, longer root trunk and shorter root than those of rats and mice. The increased CEJ-ABC distance in distal and PDL area in furcation was also observed in ligated gerbils. An inflammatory response in the connective tissue under the junctional epithelium was also shown in all the animals. As a result, we confirmed the induction of periodontitis by ligature in the gerbils. We therefore consider the gerbil to be a useful model for investigating relationship between periodontitis and vascular disease in the same animal.

Pox viral infection in a rufous turtle dove

A dead dove was found on the road and submitted for diagnosis. The bird was severely emaciated, with deformation in its facial area. Grossly, white coalescing nodules were seen on the cut surface of the nasal cavity. Histopathologically, epithelial cells of the upper respiratory tract were markedly proliferated, with ballooning degeneration, down growth of the rete ridge, and large eosinophilic intracytoplasmic inclusion bodies. Parakeratotic hyperkeratosis and focal necrotic focus was present in the proliferative area. The facial bones showed partial bone resorption. Transmission electron microscopy revealed numerous viral particles in epithelial cells with dumbbell-shaped bodies, consistent with poxvirus.

Root Resorption in Streptozotocin-induced Diabetic Rats with Ligature-induced Periodontitis

To determine the effect of diabetes on root resorption in periodontitis, we investigated odontoclast formation and root resorption in diabetic rats with periodontitis. Odontoclast formation was observed in three groups of F344 rats: Controls (C) were normal rats without diabetes or periodontitis; the periodontitis (P) group had mandibular first molars to be ligatured; the periodontitis with diabetes (PD) group was intravenously administered streptozotocin (50 mg/kg) to induce diabetes and had mandibular first molars to be ligatured. On days 3, 10, and 20 after ligature, tumor necrosis factor (TNF)-alpha and receptor activator of nuclear factor-kappaB ligand (RANKL) expression, odontoclast formation, and root resorption areas were evaluated by immunohistochemistry, tartrate-resistant acid phosphatase staining, and hematoxylin and eosin staining, respectively. The PD group showed frequent urination, weight loss, and hyperglycemia. Numbers of TNF-alpha- and RANKL-positive cells were higher in the P and PD groups than in the C group. It was more prevalent in PD group on day 3. Odontoclast formation was greater in the P and PD groups than in the C group on days 3 and 10, then decreased to same level as the C group by day 20. Root resorption in the PD and P groups showed increases on days 3 and 10, respectively, compared to the C group. These results suggest that diabetes may transiently increase root resorption on day 3 with high expression of TNF-alpha and RANKL after periodontitis induction. This study could aid the understanding of root resorption in diabetic patients with periodontitis.

Induction of IL-6 and IL-8 Expression by Leptin Treatment in Periodontal Ligament Cells and Gingival Fibroblasts

Leptin is one of the adipocytokines produced from adipose tissue but its functions in periodontal tissue have not previously been investigated. In our current study, we examined the effects of leptin on the expression of interleukin (IL)-6 and IL-8 in periodontal ligament (PDL) cells and gingival fibroblasts. Leptin receptor expression was evaluated by RT-PCR and the production of cytokines was measured by ELISA. The phosphorylation of Akt and Erk1/2 was assessed by western blotting. mRNA of long and short form leptin receptors were detected in both PDL cells and gingival fibroblasts. Leptin was found to increase the production of IL-6 and IL-8 in both of these cell types, an effect which was not blocked by polymyxin B, an inhibitor of lipopolysaccharide (LPS). Leptin did not alter the production of IL-6 and IL-8 induced by LPS in PDL cells but increased Akt and Erk1/2 phosphorylation in these cells. These results suggest that leptin acts as an inducer of IL-6 and IL-8 in PDL cells and gingival fibroblasts.

Sequential hepatic ultrastructural changes and apoptosis in rabbits experimentally infected with Korean strain of rabbit hemorrhagic disease virus (RHDVa) / 대한수의학회지

In this study, to understand the pathogenesis of new rabbit hemorrhagic disease virus (RHDVa) serotype, we carried out to administrate RHDVa to rabbits, and to examine sequential electron microscopic changes and relationship between pathogenesis and apoptosis. TUNEL-positive cells began to be observed from 24 hours after inoculation (HAI) and the number of positive cells was slightly increased with the course of time. Whereas marked increase of positive cells was seen in the liver from the rabbits died acutely. Typical viral particles with cup-like projections and a diameter of 30~40 nm were detected in homogenized liver samples and tissues at 36 and 48, and 48 HAI, respectively. Ultrastructurally, glycogen deposition was observed from the first stage of hepatocellular degeneration by RHDVa infection and then, swelling and disruption of cristae of mitochondria by viral particles, swelling of smooth endoplasmic reticulum, vacuoles and vesicles were detected. Condensation, margination and fragmentation of chromatin were observed in degenerative hepatocytes at 36 and 48 HAI, indicating apoptotic bodies. These data offer that hepatocytic apoptosis by RHDV infection could be closely related with mitochondrial impairment in the hepatocytes.

Sequential pathologic changes and viral distribution in rabbits experimentally infected with new Korean strain of rabbit hemorrhagic disease virus (RHDVa) / 대한수의학회지

Rabbit hemorrhagic disease is a highly acute and fatal viral disease caused by rabbit hemorrhagic disease virus (RHDV). Since first outbreak in Korea 1987, RHDV has been continually affected in the country, but the pattern of outbreak seem to be changed. In this study, to understand the pathogenesis of the new RHDVa serotype, we therefore carried out to inoculate RHDVa to rabbits, and to examine the sequential histopathologic changes and viral distribution. Macroscopically, various sized dark red or white spots or appearance were observed in the liver, lung, kidney uterus and ureter. In euhanized rabbits, significant pathologic findings such as infiltration of heterophils and mononuclear cells were observed at 24 hours after inoculation (HAI), and these were sequentially extended periportal to centrilobular area. However, in dead rabbits, severe hepatic degeneration and/or necrosis with relatively weak inflammatory responses were observed. RHDV antigens began to detect in liver, spleen, and lung from 12 HAI by PCR. Immunohistochemically, RHDV positive cells were seen in only liver from 24 HAI, and the degree of immunogen reactivity was stronger in dead rabbits than in euthanized ones. In conclusion, RHDVa caused the subacute or chronic infection accompanying low mortality and moderate to severe inflammatory reaction in rabbits, suggesting the possibility that RHD could become endemic.

Inhibitory Effect of SPA0355, a Thiourea Analogue, on Inflammation and Alveolar Bone Loss in Rats with Ligature-Induced Periodontitis

It has been documented that SPA0355 exerts anti-inflammatory effects via the inhibition of nuclear factor-kappaB activation. In present study, we investigated the inhibitory effects of SPA0355 on periodontitis in an animal model. Periodontitis was induced by ligation of the cervix of the 1st molar in the left mandible in rats. After ligature, the rats were randomly divided into four groups and topically applied with SPA0355 (0.5, 1, and 2%) or the vehicle alone once daily for 10 days. Body weight and food intake were measured daily throughout the experimental period. At day 10 post-ligature, the infiltration of inflammatory cells and distance of the cementoenamel junction (CEJ) to the alveolar bone crest (ABC) in the distal area of ligatured tooth were estimated histopathologically. No changes in body weight or food intake were found between the control and SPA0355 groups. The degree of inflammation was decreased in all three SPA0355 application groups. A decrease CEJ-ABC distance was observed in the 0.5% and 1% SPA0355 groups. These results indicate that SPA0355 inhibits the infiltration of inflammatory cells and alveolar bone resorption and suggests its potential as a therapeutic agent for periodontitis.

Effect of globular adiponectin on interleukin-6 and interleukin-8 expression in periodontal ligament and gingival fibroblasts

PURPOSE: Globular adiponectin (gAd) is a type of adipocytokine, which is mainly produced by adipose tissue. It has been reported that gAd acts as a pro- as well as an anti-inflammatory factor. Interleukin (IL)-6 and IL-8 are pro-inflammatory cytokines. To investigate the role of gAd on periodontal tissues, the expression of adiponectin receptor 1 (AdipoR1) and the effect of gAd on the expression of IL-6 and IL-8 were investigated in periodontal ligament (PDL) and gingival fibroblasts. METHODS: PDL and gingival fibroblasts were cultured from human periodontal tissues. gAd derived from Escherichia coli and murine myeloma cells were used. The expression of AdipoR1 was estimated by reverse transcription-polymerase chain reaction and western blot. The expression of cytokines was measured by enzyme-linked immunosorbent assay. RESULTS: PDL and gingival fibroblasts expressed both mRNA and protein of AdipoR1. gAd derived from E. coli increased the production of IL-6 and IL-8, but polymyxin B, an inhibitor of lipopolysaccharide (LPS), inhibited IL-6 and IL-8 production induced by gAd in both types of cells. gAd derived from murine myeloma cells did not induce IL-6 and IL-8 production in those cells. gAd derived from E. coli contained higher levels of LPS than gAd derived from murine myeloma cells. LPS increased production of IL-6 and IL-8 in PDL and gingival fibroblasts, but pretreatment of cells with gAd derived from murine myeloma cells did not inhibit LPS-induced IL-6 and IL-8 expression. CONCLUSIONS: Our results suggest that PDL and gingival fibroblasts express AdipoR1 and that gAd does not act as a modulator of IL-6 and IL-8 expression in PDL and gingival fibroblasts.

The influence of diabetes mellitus on periodontal tissues: a pilot study

PURPOSE: The purpose of this study was to preliminarily evaluate the influence of diabetes mellitus (DM) on periodontal tissue without establishment of periodontitis. METHODS: Seven-week-old db/db mice were used for the diabetic experimental group and systematically healthy mice of the same age were used as controls. After 1 week of acclimatization, the animals were sacrificed for hard and soft tissue evaluation. The pattern of bone destruction was evaluated by stereomicroscope evaluation with alizarin red staining and radiographic evaluation by microscopic computerized tomography images. Histological evaluation was performed with hematoxylin and eosin stain for evaluation of soft tissue changes. RESULTS: In both stereomicroscope evaluation and radiograph image analysis, aggressive form of bone destruction was observed in diabetic animals when compared to the systematically healthy controls. In histological evaluation, apical migration of junctional epithelium with slight inflammatory cell infiltration was observed with disarrangement of connective tissue fibers. CONCLUSIONS: Within the limits of this study, diabetic animals presented distortion in periodontal attachment and an aggressive bone loss pattern when compared to the healthy controls, suggesting that DM has an independent effect on periodontal tissue destruction irrespective of the presence or absence of periodontal disease.