Regulation of Trigeminal Sensory Neuron Activity by the Inflammatory Mediators Interleukin-1β and Tumour Necrosis Factor-α

Sensory neurons originating in the trigeminal ganglia (TG) mediate sensations from the face and mouth. Dental pain often follows damage to the mineralised layers of the teeth and subsequent inflammation of the pulp in response to contact with material from the oral cavity. Sensitisation of nociceptive TG sensory afferents in the teeth has been proposed as an important causative factor in pulpal pain (1). However, the influence of the inflammatory microenvironment within the tooth pulp on TG neuron activity is not yet well understood. In this study we aimed to establish a system that allows the testing of inflammatory mediators relevant to dental pain for their ability to sensitise cultured TG neurons. The effects of the classical inflammatory cytokinesinterleukin-1β (IL-1β) and tumour necrosis factor-α (TNF-α)on the activity of the Transient Receptor Potential (TRP) ion channels TRPV1 and TRPA1, and the neuronal response to ATP, were examined.

TG neurons from adult male and female C57BL/6mice were isolated and cultured for 18-24 hours, and changes in intracellular Ca2+ concentration ([Ca2+]i) were detected using the Ca2+-sensitive dye Fura-2-AMand a microscope-based imaging system. Cells were pre-treated with TNF-α (30 ng/ml), IL-1β (10 ng/ml), or vehicle for 1 hour before exposure to concentrations of the TRPA1 agonist allyl isothiocyanate (AITC),TRPV1 agonist capsaicin (CAPS), or ATP that produced a sub-maximal increase in [Ca2+]i (2 µM, 20 nM, and 5 µM, respectively). Neurons that responded to the treatments with an increase in [Ca2+]i of at least 20% of the response to depolarisation with 50mM KCl were counted as responders. Sensitisation of TG neurons was detected as an increase in either the proportion of TG neurons responding to the agonists or the relative size of their response.

Pre-treatment of TG neurons with TNF-α caused a significant increase in the percentage of neurons responding to 20 nM CAPS (17.9% of 268 neurons in the vehicle-treated group vs. 28.5% of 298 neurons in the TNF-α group (p = 0.003, Fisher’s Exact test; n = 6-7). Moreover, the magnitude of mean TG neuron responses to 2 µM AITC increased from37.8 ± 2.4% to 67.2 ± 6.1% of KCl response (p = 0.001, unpaired t-test; n = 7 for each treatment group, at least 230 neurons in each group). However, pre-exposure to TNF-α caused a reduction from 33.9% of 257 neurons in the vehicle-treated group to 24.8% of 258 neurons in the TNF-α group in the proportion of neurons responding to 5 µM ATP(p = 0.026, Fisher’s Exact test; n = 5-6). In contrast to TNF-α, 1-hour pre-treatment with IL-1βfailed to significantly affect TG neuron responses to the same agonists, although a trend towards an increase(from 4.1% to 8.1%)in the proportion of TG neurons responsive to 2 µMAITC was observed (p = 0.051, Fisher’s Exact test; n = 5 (271-296 neurons in total)).

In conclusion, a single-cell calcium imaging-based system allowed the identification of agents capable of sensitising TG neurons. TNF-α sensitised both TRPV1 and TRPA1 ion channels in TG neurons, but the intracellular signalling pathways underlying this sensitisation remain to be determined. In contrast, the proportion of cultured TG neurons responding to ATP was reduced following exposure to TNF-α. The potentiating effect of IL-1β on TRPA1 activity in TG neurons still needs to be confirmed.

(1) Matsuura S et al. (2013). PLoS One. 8 (1): e52840.