Root canal contamination or exposure to lipopolysaccharide differentially modulate prostaglandin E 2 and leukotriene B 4 signaling in apical periodontitis.

Purpose To evaluate the kinetics of apical periodontitis development in vivo , induced either by contamination of the root canals by microorganisms from the oral cavity or by inoculation of bacterial lipopolysaccharide (LPS) and the regulation of major enzymes and receptors involved in the arachidonic acid metabolism. Methodology Apical periodontitis was induced in C57BL6 mice (n=96), by root canal exposure to oral cavity (n=48 teeth) or inoculation of LPS (10 µL of a suspension of 0.1 µg/µL) from E. coli into the root canals (n= 48 teeth). Healthy teeth were used as control (n=48 teeth). After 7, 14, 21 and 28 days the animals were euthanized and tissues removed for histopathological and qRT-PCR analyses. Histological analysis data were analyzed using two-way ANOVA followed by Sidak's test, and qRT-PCR data using two-way ANOVA followed by Tukey's test (α=0.05). Results Contamination by microorganisms led to the development of apical periodontitis, characterized by the recruitment of inflammatory cells and bone tissue resorption, whereas inoculation of LPS induced inflammatory cells recruitment without bone resorption. Both stimuli induced mRNA expression for cyclooxygenase-2 and 5-lipoxygenase enzymes. Expression of prostaglandin E 2 and leukotriene B 4 cell surface receptors were more stimulated by LPS. Regarding nuclear peroxisome proliferator-activated receptors (PPAR), oral contamination induced the synthesis of mRNA for PPARδ, differently from inoculation of LPS, that induced PPARα and PPARγ expression. Conclusions Contamination of the root canals by microorganisms from oral cavity induced the development of apical periodontitis differently than by inoculation with LPS, characterized by less bone loss than the first model. Regardless of the model used, it was found a local increase in the synthesis of mRNA for the enzymes 5-lipoxygenase and cyclooxygenase-2 of the arachidonic acid metabolism, as well as in the surface and nuclear receptors for the lipid mediators prostaglandin E2 and leukotriene B4.


Introduction
The importance of microorganisms for the genesis of apical periodontitis was demonstrated in a classic study in which the dental pulp, when exposed to the oral environment in a germ-free mice, did not lead to bone loss; whereas in conventional laboratory animals lesions were detected after 15 days of exposure. 1 Until the 1970s, the isolation of microorganisms from root canals demonstrated a predominantly aerobic and anaerobic facultative composition. 2 Subsequently, it was observed that most of the microorganisms present in the infections of the root canal system of teeth with chronic periapical lesions were anaerobes, 3,4 particularly Gram-negative. 2 Gram-negative bacteria present bacterial lipopolysaccharide (LPS) or endotoxin as a component of the cellular wall, and contain both lipid components and polysaccharide moieties, with lipid A being considered the toxic portion of the molecule. 5 LPS is released during the occurrence of cellular stress, multiplication or bacterial death, stimulating a tissue immune-inflammatory reaction [5][6][7] and bone resorption. 6,[8][9][10] Studies that perform the apical periodontitis induction procedure in their methodology show variations, either by the inoculation of a mixture of pathogens, 11 a single species such as Fusobacterium nucleatum, 12 LPS 6 or by the contamination of oral root canals by microorganisms from the oral cavity. 13

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Rossi, et al. 11 (2008)  In another study, carried out to analyze the effect of calcium hydroxide on bacterial endotoxins, the process of inoculating LPS into the root canal promoted extensive bone resorption. 6 Notwithstanding, another way of inducing periapical lesion is to perform a coronary opening, remove the pulp tissue and then leave the cavity open for contamination by microorganisms from the oral cavity. 13,14 During this response, biochemical mediators are released locally with the aim of stimulating cellular and humoral immune responses. Among these inflammatory mediators are eicosanoids, synthesized from the metabolism of arachidonic acid, produced by the action of phospholipase enzymes on the phospholipids present in cellular membrane. Cyclooxygenase (COX) and lipoxygenase (LO) enzymes cause structural modifications in arachidonic acid chain, leading to the synthesis of prostaglandins and thromboxanes or leukotrienes and lipoxins, respectively. [15][16][17][18] Prostaglandins are produced via COX-1 and COX-2 enzymes. 19  In the presence of FLAP, a membrane-associated nuclear protein, the 5-LO enzyme is activated resulting in the oxidation of arachidonic acid to generate leukotriene B 4 (LTB 4 ). 21  Complimentary DNA (cDNA) was synthesized from 1300 ng of total RNA using random primers (High Quality cDNA Reverse Transcriptase Kits, Applied Biosystems, Foster City, CA). Aliquots of 2 µl of the total cDNA were amplified by qRT-PCR using primers An increase in periapical space due to bone resorption was found in root canals exposed to oral contamination at 21 and 28 days after exposure, differently from root canals submitted to LPS inoculation or healthy teeth with a normal periodontal ligament and alveolar bone structure (p<0.05, Figure 3).
Signalling signature of arachidonic acid metabolism enzymes and receptors modulated by contamination or inoculation of LPS into the root canals  Ptger4, which encodes EP4 receptor, was not modulated from 7 to 21 days (p>0.05) but was inhibited at 28 days (p<0.05). In contrast, inoculation of LPS in root canals inhibited expression of Ptger1 at 7 days and stimulated expression at 21 and 28 days (p<0.05).
Ptger2 and Ptger4 were stimulated in all experimental periods (p<0.05) and Ptger3 was inhibited at 7 days and stimulated at 21 and 28 days (p<0.05) (Figure 4).      Thus, we reject the initial hypothesis that induction of apical periodontitis is similar when using inoculation with LPS or contamination of the root canal by oral cavity microorganisms.
Our study was carried out to induce apical periodontitis by coronary access, insertion of a file into the root canals and pulp extirpation, according to previous studies. 13,14,27 However, to achieve the induction of the apical periodontitis, other studies use either coronary opening and disorganization of the coronary pulp, 28,29 a pool of several microorganisms into the root canals, 13 a combination of a pool of bacteria and oral contamination, 11 a single specific bacteria such as F. nucleatum 12 or bacterial products such as LPS. 6,8 In this study, contamination or inoculation of