Abstract
To determine whether Porphyromonas gingivalis lipopolysaccharide (LPS) can directly activate trigeminal neurons, to identify which receptors are involved and to establish whether activation leads to secretion of the neuropeptide calcitonin gene-related peptide (CGRP) and/or the translocation of NF-κB. Mouse trigeminal ganglion (TG) cells were cultured in vitro for 2days. The effect of P.gingivalis LPS (20μgmL-1 ) on calcium signalling was assessed (by calcium imaging using Cal-520 AM) in comparison with the transient receptor potential channel A1 (TRPA1) agonist cinnamaldehyde (CA; 100μmolL-1 ), the TRP channel V1 (TRPV1) agonist capsaicin (CAP; 1μmolL-1 ) and high potassium (60mmolL-1 KCl). TG cultures were pre-treated with either 1μmolL-1 CLI-095 to block Toll-like receptor 4 (TLR4) signalling or with 3μmolL-1 HC-030031 to block TRPA1 signalling. CGRP release was determined using ELISA, and nuclear translocation of NF-κB was investigated using immunocytochemistry. Data were analysed by one-way analysis of variance, followed by Bonferroni's post hoc test as appropriate. Porphyromonas gingivalis LPS directly exerted a rapid excitatory response on sensory neurons and non-neuronal cells (P<0.001 to P<0.05). The effects on neurons appear to be mediated via TLR4- and TRPA1-dependent pathways. The responses were accompanied by an increased release of CGRP (P<0.001) and by NF-κB nuclear translocation (P<0.01). Porphyromonas gingivalis LPS directly activated trigeminal sensory neurons (via TLR4 and TRPA1 receptors) and non-neuronal cells, resulting in CGRP release and NF-κB nuclear translocation. This indicates that P.gingivalis can directly influence activity in trigeminal sensory neurons and this may contribute to acute and chronic inflammatory pain.
Highlights
Infection of the dental pulp initiates a variety of pathophysiological changes including the generation of pain
Our study demonstrated that approximately 13%, and 33% of mouse trigeminal ganglion (TG) neurons responded to CA and CAP, respectively
Neuronal cells show a very clear rapid rising and transient response to KCl resulting from depolarization and action potential-evoked calcium signals, the amplitude and duration of calcium transients being determined by the contribution of different ion channels contributing to the action potential (e.g. Mohammed et al 2017); in contrast, non-neuronal cells do not fire action potentials and where calcium signals are observed, these are notably slower, and are likely related to transporters
Summary
Infection of the dental pulp initiates a variety of pathophysiological changes including the generation of pain. While immune mechanisms are known to indirectly influence nociception, bacteria or their products have been reported to activate sensory neurons directly. A range of oral bacteria are known to infect dental pulps, many of which are Gramnegative, and amongst which is Porphyromonas gingivalis (Rôças et al 2002). A major component of Gram-negative bacterial surfaces is lipopolysaccharide (LPS) and previous investigators have shown that LPS might directly activate sensory neurons (Diogenes et al 2011, Tse et al 2014). P. gingivalis LPS differs from that of E. coli and it is not known whether this and other non-enterobacterial LPS that would be found in pulpal infection have the same effects and, if so, which neuronal receptors are involved
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