Neuropeptides, sensory neurons and pain modulation

Sammanfattning: In the present thesis work we have analyzed the expression, regulation and function of two neuropeptides: galanin and neuropeptide tyrosine (NPY). Their associated receptors, galanin receptor-1 and -2 (GaIR1, -R2) and NPY Y1- and Y2R, were also studied. The goal of this thesis was to histochemically define sensory systems and to create a basis for studies on the 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, that is unlesioned vs. neurophatic. Thus, GaIR2 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 a galanin overexpressig (GalOE) mouse. These animals are less reactive to pain when compared to WT mice, both in normal and in neuropathic pain conditions. In addition, GalOE mice exhibit a higher galanin expression in dorsal root ganglion (DRG) neurons than WT mice. 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 based on different degrees of a single constriction of the sciatic nerve (strong, medium, light) in rat. NPY and Y1R expression and behavioral changes are recorded. We conclude that the stronger the degree of lesion, the more dramatic are the changes in expression of NPY (up) and the Y1R (down) in DRG neurons, whereby the medium constriction is the most effective in inducing pain-like behavior. These findings suggest a role for the NPY ergic system in chronic pain mechanisms. In Paper V, we demonstrate axonal transport of the Y1R into the peripheral branches (sciatic nerve and skin) and into the central projections (dorsal roots) of rat DRG neurons, providing evidence for a presynaptic action of NPY on pain mechanisms. In Paper VI we show the pattern of expression of the Y2R protein in mouse sensory neurons and their central and peripheral projections. The antibody is specific, as shown in immunoadsorption tests and by using tissue from KO mice. Our results indicate that the receptor is present in peptidergic and non-peptidergic lumbar DRG neurons, specifically projecting to hairy skin. Therefore, also the Y2R 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 in deep layers of the dorsal horn. Finally, in Paper VIII we found that a subpopulation of DRG neurons express tyrosine hydroxylase (TH), the rate-limiting enzyme in the cathecolamine synthesis. These are mostly small in size, and are nonpeptidergic, and still do not bind isolectin B4. 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 contribute to the development of novel treatment strategies for this type of pain.