LPS-induced neuronal activation and TRPA1 sensitization in trigeminal sensory neurons is dependent to TLR4 receptor

Benoit Michot, Sharon M Casey, Caroline Lee, Jennifer L Gibbs

Abstract: Painful pulpitis is an inflammation of the dental pulp caused by bacterial proliferation near the pulp after tooth damage or caries. Previous studies have shown that the bacterial toxin lipopolysaccharide (LPS) can directly activate and sensitize sensory neurons but the underlying mechanisms are not fully understood. In this study, we evaluated the effects of LPS on 1) neuronal activity, 2) neuronal sensitization through the activity of the ion channel TRPA1 which is involved in pain perception and 3) the involvement of TLR4 receptor in LPS effects. Trigeminal ganglion neurons from mice were cultured for 3h, then washed and incubated with the fluorescent calcium indicator fura-2 AM for the real-time imaging of intracellular calcium concentration. Then neurons were pre-treated for 3 min with LPS (1, 10, 100 µg/ml), washed for 3 min and then stimulated with the TRPA1 agonist acyl-isothiocyanate (AITC, 250 µM) for 1 min. LPS 1, 10 and 100 µg/ml increased intracellular calcium concentration in 10%, 16% and 40% of trigeminal ganglion neurons respectively. LPS 10 µg/ml induced neuronal sensitization, increasing the proportion trigeminal ganglion neurons sensitive to AITC compared to vehicle treated neurons (63% and 40% of neurons responding to AITC respectively). On the contrary, LPS 100 µg/ml did not significantly affected the proportion of AITC responding neurons. Blockade of TLR4 receptor with the antagonists LPSRS and TAK242 prevented both the LPS-induced increase in intracellular calcium concentration and the LPS-induced TRPA1 sensitization in trigeminal sensory neurons. In conclusion, LPS induced both activation and sensitization of trigeminal neurons that are mediated by TLR4 receptor. These data suggest that TLR4 would be important for neuronal sensitization and the development painful pulpitis.