ERK phosphorylation in rat Vc neurons by experimental orthodontic force

Objectives: The molecular mechanism of pain associated with orthodontic treatment remains to be elucidated, although an increasing evidence suggests that the extracellular signal-regulated kinase (ERK) signaling pathway is activated in dorsal horn neurons by noxious stimuli, such as capsaicin injection to the hind paw. We asked if experimental orthodontic force allows ERK phosphorylation (pERK) in the trigeminal spinal subnucleus caudalis (Vc). Methods: Experimental orthodontic force was performed by the insertion of an elastic between the first and second upper molars in rats anesthetized with sodium pentobarbital (50mg/kg i.p.) (referred to as Waldo's method). The pERK was analyzed in Vc neurons at 2, 5, 20, 60 and 120 minutes after elastic insertion using immunohistochemistry. For subcellular analysis of pERK, we carried out one plane scan analysis using confocal laser microscope. The anti-NeuN, anti-synaptophysin antibody and TO-PRO-3 were used for labeling of neurons, presynapses and nuclei, respectively. Results: Experimental orthodontic force resulted in rapid phosphorylation of ERK in the Vc. All pERK-like immmunoreactive (-LI) cells were labeled with NeuN antibody, indicating that the pERK-LI cells were confirmed as neuron. The number of the pERK-LI cells reached a peak at 2 minutes after the treatment and started to decrease in 5 minutes and then returned to basal level within 120 minutes. As a result of subcellular analysis, the pERK signals were found in dendritic shafts as a puncta. The pERK-positive puncta sometimes partially overlapped with synaptophysin-positive puncta, suggesting that pERK was induced in the synapses. Also, pERK signals were observed in nuclei (confirmed by TO-PRO-3 staining) of some neurons. Conclusion: The present findings suggest that ERK signaling pathway in Vc neurons is involved in orthodontic pain during clinical treatment and also involved in the synaptic function and pain-related gene expression.

Supported by Japan-Canada Joint Health Research Program grant#07033211-000165.