Title: LPS-induced Neuronal Activation and TRPA1 Sensitization is TLR4 Dependent
Benoit Michot, New York University
Sharon Casey, New York University College of Dentistry
Jennifer Gibbs (Presenter)
New York University
Objectives: Painful pulpitis is caused by bacterial proliferation near or in the pulp subsequent to enamel/dentin fracture or caries. Previous studies have shown that the bacterial toxin lipopolysaccharide (LPS) directly activates and sensitizes 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 critically involved in pain perception and 3) the involvement of TLR4 receptor in LPS effects.
Methods: Trigeminal ganglion neurons from mice were cultured for 3h, then washed and incubated with the calcium indicator fura-2 AM for the real-time imaging of intracellular calcium concentration. Then neurons were pre-treated with LPS (1, 10, 100µg/ml), then stimulated with the TRPA1 agonist acyl-isothiocyanate (AITC, 250µM) for 1 min. In antagonist studies, a TLR4 antgonist (LPS-RS, TAK242; multiple doses) or a TRPA1 antagonist (HC-030031; multiple doses) was administered prior to and during LPS application. The primary endpoint was proportion of neurons activated by LPS.
Results: LPS 1, 10 and 100µg/ml, increased intracellular calcium concentration in 10%, 16% and 40% of trigeminal ganglion neurons respectively. LPS 10µg/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). Blockade of TLR4 receptor with the antagonists LPS-RS and TAK242 prevented both the LPS-induced increase in intracellular calcium concentration and the LPS-induced TRPA1 sensitization in trigeminal sensory neurons. The TRPA1 antogonist HC-030031 completely blocked LPS activation of sensory neurons.
Conclusions: LPS induced both activation and sensitization of trigeminal neurons, and these effects are mediated by the TLR4 receptor. Further, TRPA1 is essential for LPS activation of trigeminal neurons. In summary, TLR4 and TRPA1 are important therapeutic targets for interferance of neuronal sensitization during painful pulpitis.