Trigeminal pain transmission requires reactive oxygen species production

Abstract

Three experiments were conducted in order to investigate the possible involvement of the reactive oxygen species in the nociception within the subnucleus caudalis of the spinal trigeminal nucleus (Vc). In the first experiment the extracellular level of hydrogen peroxide was evaluated by microdialysis in the Vc of two groups of six rats before and after a formalin (group 1) or saline solution (group 2) injection into the upper lip. In the second experiment the formalin test was conducted in three groups of 6 rats after a microinjection of 2-methoxyestradiol (2-ME, a superoxide-dismutase inhibitor; group 1) or N-acetylcysteine (NAC, an oxygen intermediate scavenger; group 2) or saline solution (group 3) into the Vc. In the third experiment an histochemical assay for superoxide dismutase activity was performed on two groups of 4 rats each 2 h after a formalin (group1) or saline solution (group 2) injection into the upper lip. The results showed that (1) the level of hydrogen peroxide increases into the Vc during facial pain (134% of baseline); (2) the inhibition of superoxide dismutase or the removal of oxygen intermediate within the Vc decreases the sensibility to facial pain stimuli; and (3) persistent facial pain stimuli decrease the superoxide activity into the Vc (90% of counter-lateral). These data indicate that reactive oxygen species are produced in the Vc during persistent facial pain and are necessary for the transmission of pain.

Introduction

The subnucleus caudalis of the spinal trigeminal nucleus (Vc) is the major relay for the transmission of orofacial pain information to other brain nuclei. This nucleus operates a complex and still not completely understood elaboration of the painful stimuli before the retransmission of the “filtered” information to superior neural centers [2], [8], [27], [45], involving a lot of different neurotransmitters [7], [14], [40], [44]. Differently from other subnuclei, the Vc is characterized by a marginal layer and a substantia gelatinosa (laminae I and II) resembling the upper cervical dorsal horn with which it is continuous. For these reasons, the Vc has been named medullary dorsal horn [13]. Disconnection of the Vc in humans results in loss of pain and thermic sensation with a moderate tactile deficit [11]. These clinical features and a wealth of experimental data [17] have pointed out a specialization of the Vc in nociceptive processing, although the trigeminal nociceptive pathways have been demonstrated to be more complex than originally thought [10].

The formalin test is one of the most widely used models of tonic pain, due to tissue injury and inflammation [33], [34]. Formalin solution, when injected subcutaneously, induces an initial strong activation of small primary afferent fibers that subside over the next 10 min at a low but continued level of activity. In man, an immediate burning pain is experienced, followed by a troubling pain over a 30- to 60-min period. In rats, a formalin injection produces a characteristic biphasic response of face rubbing. The first phase is a result of direct activation of peripheral nociceptors, while the second phase is believed to be mediated by a combination of low ongoing activity in primary afferent fibers and an increased sensitivity of spinal cord neurons.

Both hydrogen peroxide (H₂O₂) and superoxide anion (O₂⁻), that are reactive oxygen species (ROS), are often considered as tissue-damaging agents related with oxidative stress, aging, and neurodegenerative diseases. ROS could also be considered as an additional class of cellular messenger that are physiologically synthesized by the cell metabolism. Pellmar et al. [32] reported that long incubations of hippocampal slices with H₂O₂ prevented the full expression of LTP [19]. Similarly, Auerbach and Segal [1] found that long incubations of hippocampal slices with a lower concentration of H₂O₂ can attenuate the full expression of LTP. On the other hand, a brief incubation of hippocampal slices with H₂O₂ results in improved synaptic transmission. Slices incubated with catalase (an H₂O₂ scavenger) show attenuated LTP [3], [20]. Also O₂⁻ is implicated in the expression of hippocampal LTP. Bindokas reported that NMDA activation in area CA1 of hippocampal slices results in the production of O₂⁻[3]. Subsequently it was found that LTP is blocked by the incubation of hippocampal slices with cell-permeable scavenger of O₂⁻, and that hippocampal slices exhibited deficient LTP in transgenic mice over-expressing the O₂⁻ scavenger CuZn-superoxide dismutase (SOD) [15].

Since there are similarities in the neuronal plasticity phenomena between LTP and tonic inflammatory pain [47], the aim of the present experiments was to evaluate the following: (1) the production of H₂O₂ into the Vc through microdialysis during the formalin test; (2) the effect, on the formalin test, of the administration of 2-methoxyestradiol (2-ME), an inhibitor of SOD [18], or N-acetylcysteine (NAC), a non-enzymatic antioxidant [37] into the Vc; and (3) the enzymatic activity of SOD after the formalin test by mean of a histochemical methods.