The interphase of the formalin test

The formalin test still surprises with its biphasic pain-related behavior resulting from a quiescent interphase that does not occur with other algogenic compounds and remains unexplained. The first phase has been attributed to TRPA1-mediated excitation of nociceptors, the second phase to their inflammatory and/or spinal sensitization. We show that the second and interphase require higher formaldehyde concentrations to emerge, and that from 12 mM on calcium influx is induced in TRPA1-deficient sensory neurons as well as in native HEK293T cells. After a short depolarizing and excitatory period in a subset, all wild-type neurons showed a concentration-dependent hyperpolarization, a reduction of voltage-activated sodium currents, and a progressive increase of the input resistance, which, after about 10 min restored the transiently lost excitability, enabling smaller and wider action potentials to be evoked than before formaldehyde (30 mM). The hyperpolarizing effect was absent if extracellular sodium was replaced, and largely prevented by a high but not low concentration of tetrodotoxin. In rat skin in vivo, the spatiotemporal redistribution of injected formalin and the plasma extravasation were studied using Evans blue. The parameters gained were entered into a computational model to predict the activation pattern of primary afferents. The model supports a peripherally generated biphasic response, the time course matching the behavioral results. In conclusion, the interphase is a result of hyperpolarization and transient inactivation by formaldehyde of the surviving neurons; their recovery and the centrifugal spread of formalin in the skin induce a second phase of nociceptive activity before the formalin concentration falls below threshold.