Rat Model Of Apical Periodontitis Research Articles

Effect of the Progression of Fusobacterium nucleatum–induced Apical Periodontitis on the Gut Microbiota

IntroductionFusobacterium nucleatum, which is involved in the development of periodontal disease and apical lesions, can be transmitted to the colon and metastasize to colorectal cancer, suggesting a link between oral and systemic diseases. We analyzed the effects of F. nucleatum on bacterial flora in the gut and surrounding organs in a rat model of apical periodontitis and analyzed the infection route to the gut and distant organs. MethodsWe induced apical periodontitis in rat molars by infecting the dental pulp with F. nucleatum and then took X-ray images and performed histopathologic analyses. Next, we removed the maxilla, gut, heart, liver, and kidney from the rats at 0, 2, 4, and 8 weeks postsurgery and then extracted DNA samples and performed polymerase chain reaction and microbiome analyses using the Illumina MiSeq (Illumina Co, Tokyo, Japan). ResultsThe presence of inflammatory cell infiltration confirmed apical periodontitis from 2–8 weeks. Polymerase chain reaction and microbiome analyses revealed F. nucleatum in the rat gut from 2 weeks and in the kidney from 8 weeks. The rat gut, heart, liver, and kidney exhibited altered bacterial flora, including a marked decrease in Verrucomicrobia and an increase in Proteobacteria after 2 weeks and increases in Bacteroidetes and Firmicutes after 4 weeks. ConclusionsThe onset of F. nucleatum–induced apical periodontitis changed the bacterial flora in the rat gut, heart, liver, and kidney, with a confirmed progressing infection in the large intestines.

Blockade of fatty acid signalling inhibits lipopolysaccharide-induced macrophage recruitment and progression of apical periodontitis.

To examine the role of palmitic acid in lipopolysaccharide (LPS)-stimulated chemotaxis of macrophages and the potential contribution of saturated fatty acid in signalling during the pathogenesis of apical periodontitis. J774, a mouse macrophage cell line, was used in the experiments. After treatment with LPS, proteolytic maturation of sterol regulatory element-binding protein-1c (SREBP-1c) and expression of fatty acid synthase (FASN) were examined by Western analysis. Levels of palmitic acid were measured by reverse phase-high performance liquid chromatography-mass spectrometry. Knockdown of SREBP-1c and FASN was accomplished by small interfering RNA technology. Secretion of CC-chemokine ligand 2 (CCL2) and cellular chemotaxis were assessed by enzyme-linked immunosorbent assay and transwell migration assay, respectively. Sulfo-N-succinimidyl oleate (SSO) treatment was used to inhibit fatty acid signalling in vitro and also in a rat model of apical periodontitis. All data were first subjected to Levene's test. In vitro data were then analysed using ANOVA followed by Tukey's multiple comparison test. Data from animal experiments were analysed by independent t-tests. The significant level was set at 0.05. LPS stimulated proteolytic maturation of SREBP-1c and FASN expression in macrophages and significantly enhanced palmitic acid synthesis (P<0.05). Knockdown of SREBP-1c attenuated LPS-enhanced FASN expression. Knockdown of FASN significantly suppressed LPS-enhanced palmitic acid synthesis (P<0.05). LPS and exogenous palmitic acid significantly enhanced CCL2 secretion and macrophage chemotaxis (all P<0.05). Inhibition of FASN expression significantly alleviated LPS-augmented CCL2 secretion (P<0.05). SSO significantly suppressed CCL2 secretion and macrophage chemotaxis augmented by LPS and palmitic acid (all P<0.05). In a rat model of induced apical periodontitis, SSO treatment significantly attenuated progression of apical periodontitis and macrophage recruitment (all P<0.05). LPS/SREBP-1c/FASN/palmitic acid signalling contributed to tissue destruction caused by bacterial infection. Modulation of lipid metabolism and signalling may be helpful for the management of apical periodontitis.

A Pilot Study of Chronological Microbiota Changes in a Rat Apical Periodontitis Model

Apical periodontitis caused by microbial infection in the dental pulp is characterized by inflammation, destruction of the pulpal and periradicular tissues, and alveolar bone resorption. We analyzed the chronological changes in microbiota using a pyrosequencing-based approach combined with radiologic and histopathologic changes in a rat apical periodontitis model. During the three-week observation, the pulp and periapical area showed a typical progress of apical periodontitis. A total of 27 phyla, 645 genera, and 1276 species were identified. The root apex had a lower bacterial species diversity than the pulp chamber. Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria were dominant phyla in both the pulp chamber and root apex. Remarkably, bacterial communities showed a tendency to change in the root apex based on the disease progression. At the genus level, Escherichia, Streptococcus, Lactobacillus, Rodentibacter, and Bacteroidetes were dominant genera in the pulp chamber. The most abundant genera in the root apex were Bradyrhizobium, Halomonas, and Escherichia. The species Azospirillum oryzae increased in the pulp chamber, whereas the species Bradyrhizobium japonicum and Halomonas stevensii were highly observed in the root apex as the disease progressed. The experimental rat model of apical periodontitis demonstrated a relationship between the microbiota and the apical periodontitis progression.

The influence of adrenergic blockade in rats with apical periodontitis under chronic stress conditions

ObjectiveTo investigate the influence of chronic stress and adrenergic blockade in a rat model of apical periodontitis. MethodsThirty-two Wistar rats were submitted to an animal model of periapical lesion and randomly divided into 4 groups (n = 8): no stress (NS); stress + saline solution (SS); stress + β-adrenergic blocker (Sβ); stress + α-adrenergic blocker (Sα). The SS, Sβ and Sα groups were submitted to an animal model of chronic stress for 28 days and received daily injections of saline solution, propranolol (β adrenergic blocker) and phentolamine (α adrenergic blocker), respectively. After 28 days the animals were euthanized and the following analyses were carried out: a) serum corticosterone levels through Radioimmunoassay; b) measurement of serum levels of IL-1B, IL-6, IL-10 and IL-17 by enzyme-linked immunosorbent assay (ELISA); c) volume of periapical bone resorption by micro-computed tomography; d) histomorphometric analysis by staining with hematoxylin and eosin; e) expression of β-AR, α-AR, receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin (OPG) by immunohistochemistry; f) tartrate-resistant acid phosphatase (TRAP) staining; g) ex-vivo cytokine release followed by the stimulation with LPS in superfusion system, by ELISA. ResultsSS group displayed significantly higher corticosterone levels than NS group (non-stressed). Higher IL-1β serum level was observed in the NS group (p < .05); compared to all stressed groups. Other cytokines were present in similar amounts in the serum of all groups. All groups presented similar periapical lesions. All groups presented moderate inflammatory infiltrate, without statistically significant differences between them. No differences were observed regarding β-AR, α-AR, Rank-L and OPG expression. The number of TRAP-positive cells was significantly decreased in the groups that received daily injections of adrenergic blockers. The IL-1β release followed LPS stimulation was significantly suppressed when the superfusion media contained propranolol (p < .05). Perfusion containing phentolamine induced a greater release of IL-10. TGF-β was significantly suppressed by phentolamine perfusion in the NS group (p < .05). ConclusionsChronic stress can significantly change the inflammatory cytokines release. Rank-L/OPG system and periapical lesion volume were not affected following the current method applied. The administration of adrenergic blockers was not able to modulate the inflammatory response but presented effectivity in reducing the number of osteoclasts in the periapical region.

The expression of MCP-1 and CCR2 in induced rats periapical lesions

ObjectiveMonocyte chemotatic protein-1 (MCP-1) has been found to promote chemotaxis, differentiation, and activation of osteoclasts. The aim of this study is to detect and localize the expressions of MCP-1 and its receptor CCR2 in a rat model of apical periodontitis. DesignAfter exposing the pulp of the first mandibular molars, 25 Wistar rats were killed on days 0, 7, 14, 21, 28, and 35. Rat jaws containing the first molar were obtained and routinely prepared for histological analysis, immunohistochemistry, enzyme histochemistry and immunofluorescence staining. ResultsLesions expanded from day 0 to day 28, and stabilized thereafter. Most of the MCP-1-positive cells were inflammatory cells, and their amount increased from day 7 to day 28. Aside from inflammatory cells, CCR2 was also detected in osteoclasts in the lesions. From day 7 to day 14, the number of osteoclasts increased. From day 21 to day 35, fewer osteoclasts could be observed, and the number of osteoclast was negatively correlated with positive MCP-1 expression. ConclusionThese findings showed that MCP-1/CCR2 could be observed and might possibly be involved in the development of periapical lesions.

Vascular Endothelial Growth Factors and Receptors Are Up-regulated during Development of Apical Periodontitis

IntroductionApical periodontitis is a common inflammatory disease caused by persistent root canal infection and is characterized by bone resorption. Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) have been described in many pathologic and inflammatory conditions, but their involvement in the development of apical periodontitis has not been thoroughly investigated. The aim of this study was to quantify gene expression and localize VEGF-A, VEGF-C, and VEGF-D and VEGFR-2 and VEGFR-3 in a rat model of apical periodontitis. MethodsMolar pulps were unilaterally exposed to the oral cavity for 10 or 21 days. Jaw sections were used for localization of VEGFs and VEGFRs with immunohistochemistry and identification of cells with double immunofluorescence. Gene expression analysis for VEGF-A, VEGF-C, and VEGFR-3 of periapical tissues was performed with quantitative real-time polymerase chain reaction. ResultsAll investigated factors and receptors were expressed immunohistochemically in blood vessels at the periodontal ligament of control teeth and were up-regulated during lesion development. In apical lesions, macrophages and neutrophils expressed all studied factors and receptors, with macrophages being an important source of VEGF-C and VEGF-D. Osteoclasts expressed VEGFR-2 and VEGFR-3, and the latter was also identified in fibroblast-like cells in the lesions. VEGF-A and VEGFR-3 gene expression was up-regulated at days 10 and 21 (P < .05). ConclusionsThe current findings indicate that the VEGF family and receptors are involved in vascular remodeling and immune functions during disease development. The presence of VEGFR-2 and VEGFR-3 on osteoclasts indicates that bone resorbing activity is influenced by VEGFs.