Neuropeptides, sensory neurons and pain modulation

Tesis

Resumen *

In the present thesis work we have analyzed the expression, regulation and function of two neuropeptides: galanin and the neuropeptide tyrosine (NPY). Their associated receptors, galanin receptor- 1 and -2 (GalR1, -R2) and NPY Y1- and Y2R, were also studied. The final goal of this thesis was to establish a relationship between the above mentioned molecules and receptors, and neuropathic pain modulation. To this end, several nerve lesion models were employed to induce pain in rodents, including the single ligature nerve constriction (SLNC), a model developed in our laboratories (Paper IV). Immunohistochemistry to study protein expression; in situ hybridization for the analysis of messenger ribonucleic acid (mRNA) and pharmacological manipulation of the galanin receptors through the intrathecal application of different drugs for correlation analysis with pain behavior were used. Different tests for pain behavior were employed to assess the consequences of peripheral nerve injury and for correlation with the morphological and pharmacological findings. In Paper I, we demonstrate a receptor subtype-specific action of pharmacological doses of galanin on pain behavior, and that this depends on the condition of the rat, e.g. unlesioned vs. neurophatic. Thus, GalR2 would account for a pronociceptive effect in unlesioned animals, whereas GalR1 appears to be related to the analgesic effect of galanin in neuropathic rats. In Papers II-III, we analyze the role of galanin in pain modulation in GalOE mice under the control of the dopamine beta-hydroxylase promoter. We show that these animals are less reactive to pain when compared to WT mice, both in normal conditions or during the course of neuropathic pain. In addition, we found that GalOE mice exhibit higher galanin expression in DRG neurons than normally observed, and interestingly also in large neurons, which usually do not express the peptide. These findings suggest that the antinociceptive phenotype observed in the transgenic animals could be due to the overexpression of galanin in primary afferent neurons. In Paper IV, we present a model of peripheral nerve lesion with the aim of illustrating the effect of different degrees of constriction of the sciatic nerve (strong, medium, light) on the extent of immunohistochemical (NPY and Y1R expression) and behavioral changes in rats. We conclude that the stronger the degree of lesion, the more dramatic changes in expression of NPY (up) and the Y1R (down). In addition, a medium constriction is the most effective in inducing pain-like behavior and is accompanied by changes in NPY and Y1R expression in DRGs, suggesting a role for the NPYergic system in chronic pain mechanisms. In Paper V, we demonstrate the axonal transport of the Y1R towards the peripheral (sciatic nerve) and central projections (dorsal roots) of rodent DRG neurons, providing evidence for a presynaptic action on pain mechanisms for this receptor. Indeed, Y1R+ fibers were shown to penetrate the epidermal layers of the glabrous skin, and some primary afferents in the dorsal horn of the spinal cord exhibited Y1R immunoreactivity. In Paper VI we show, for the first time, the pattern of expression of the Y2R protein in mouse sensory neurons and their central and peripheral projections. The antibody is specific, and it was tested with immunoadsorption and application onto tissue from KO mice. Our results indicate that the receptor is present in peptidergic and non-peptidergic lumbar DRG neurons. Interestingly, Y2R+ DRG neurons seem to specifically project to hairy skin. Therefore, the Y2R, as well as the Y1R, may have a role in presynaptic neurotransmission and pain mechanisms. In Paper VII we analyze the expression of the Y1R in the different layers of the dorsal horn of rat. We found that the receptor is expressed by neurons in basically all laminae of the dorsal horn and, importantly, also in projection neurons found in deep layers of the dorsal horn. Finally, in Paper VIII we have analyzed the expression of tyrosine hydroxylase (TH) in mouse DRG neurons. We have found that a subpopulation of DRG neurons express TH, most are small in size, and are non-peptidergic, but still do not bind IB4 either. Chronic pain generated by peripheral nerve lesion is a major clinical problem and many patients wait for an adequate treatment. The present studies, focusing on mechanisms underlying and maintaining neuropathic pain, but also dealing with the anatomical and cellular distribution of different molecules important for pain modulation, will hopefully lead to the development of novel treatment strategies for this type of pain. Información suministrada por el agente en SIGEVA