Neuropeptide release in the rat dorsal horn in models of persistent pain : effects of opioids

Abstract

Nerve injury and tissue inflammation may lead to exaggerated responses to both noxious stimuli (hyperalgesia) and innocuous stimuli (allodynia). Altered release pattems of neurotransmitters in the dorsal hom of the spinal cord have been suggested to contribute to hyperalgesia and allodynia. The cholecystokinin (CCK) gene- expression and CCK2-receptor gene-expression in dorsal root ganglion (DRG) cells is upregulated after sciatic nerve transection (axotomy). Since CCK acts as an anti-opioid peptide, an increased CCK release in the spinal cord following nerve lesions has been proposed to be of importance for the relatively low effectiveness of morphine in the treatment of persistent neuropathic pain. Peripheral inflammation induces increased levels of substance P (SP) in primary afferent neurons and there is also indirect evidence for increased release of SP in inflammation. The present study was undertaken to study the in vivo release of SP- and CCK-like immunoreactivity (-LI) in the dorsal hom of the spinal cord in models of persistent neuropathic (axotomy) and inflammatory pain.

All experiments were performed in the rat and the neuropeptide release was studied by microdialysis combined with radioimmunoassay. In control animals the release of CCK-LI was increased 5-fold by stimulation with potassium (100 mM in the perfusion fluid). In contrast, two to eight weeks after transection of the sciatic nerve, no significant CCK-LI release was detected. Three and seven days after axotomy, a tendency to an increased potassium-induced release of CCK-LI was observed. After subcutaneous administration of morphine (5mg/kg, s.c) to intact rats, an increased spinal release of CCKLI of the same order of magnitude as observed after potassium stimulation was detected. The morphineinduced CCK-LI release was TIX-sensitive and calcium-dependent and could be blocked by topical application of either the L-type calcium channel blocker verapamil or the N-type calcium channel blocker omega-conotoxin GVIA. The CCK-LI release induced by morphine was also completely inhibited by the deltaopioid antagonist naltrindole, but not by g-or kappa-opioid receptor antagonists. Furthermore, administration of the delta-opioid receptor agonist BW373U86 but not the µ-opioid receptor agonist DAMGO induced a significant increase of the spinal CCK-LI release. After peripheral axotomy, morphine, at doses that induced a release of CCK-LI in normal rats, failed to induce a significant release of CCK-LI. However, the ability of the delta-opioid receptor agonist BW373U86 to evoke a release of CCK-LI was not affected by the axotomy.

During induction of long term potentiation (LTP) in single wide dynamic range (WDR) neurons by a conditioning tetanic stimulation of the sciatic nerve, a short lasting significant four-fold increase of the SP-LI release was detected. However, the extracellular level of SP-LI was not altered upon single impulse nerve activation after LTP had been established. Perfusion of the microdialysis probe with capsaicin (50 or 100 µM), an agent known to activate vanilloid receptors on primary afferent C-fibers, induced a significant eight-fold increase of the extracellular SP-LI level. Spinal administration of the NMDA antagonist D-APV, but not the AMPA antagonist NBQX, completely prevented the SP-LI release induced by the low dose capsaicin (50 µM). Seven days after induction of peripheral inflammation by complete Freund's adjuvant (CFA), a dose of capsaicin (10 µM), previously insufficient to release SP-LI, induced a significant and D-APV reversible release of SP-LI.

In summary, spinal release of CCK-LI may be of importance for modulation of morphine analgesia and for the altered primary afferent processing during the first week after axotomy. However, the data do not support the concept of an increased spinal CCK release as a mechanism for morphine resistance of "phantom pain". The delta-opioid receptor is likely to mediate the opioid induced release of CCK-LI in intact animals and following sciatic nerve section. A contribution of SP in the induction, but not in the maintenance, of LTP in WDR neurons, is suggested. Moderate doses of capsaicin induce release of spinal SP-LI via an activation of the NMDA receptor. The ability of capsaicin to evoke a release of SP-LI in the dorsal hom is increased one week after CFA- induced inflammation.