Effect of μ and κ opioids on injury-induced microglial accumulation in leech CNS:: Involvement of the nitric oxide pathway

Damage to the leech or mammalian CNS increases nitric oxide (NO) production and causes accumulation of phagocytic microglial cells at the injury site. Opioids have been postulated to modulate various parameters of the immune response. Morphine and leech morphine-like substance are shown to release NO and suppress microglial activation. Regarding the known immuno-modulatory effects of selective mu and kappa ligands, we have assessed the effect of these agents on accumulation of microglia at the site of injury in leech CNS. Leech nerve cords were dissected, crushed with fine forceps and maintained in different concentrations of opiates in culture medium for 3 h and then fixed and double stained with Hoechst 33258 and monoclonal antibody to endothelial nitric oxide synthase (NOS). Morphine and naloxone (>= 10(-3) M) but not selective mu agonist, DAMGO [d-Ala2, N-Me-Phe-Gly5(ol)-enkephalin] and antagonist, CTAP [D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2] inhibited the microglial accumulation. The effect of morphine was abrogated by pre-treatment with naloxone and also non-selective NOS inhibitor, L-NAME [N-omega-nitro-L-arginine-methyl-ester; 10(-3) M] implying an NO-dependent and mu-mediated mechanism. These results are similar to properties of recently found mu-3 receptor in leech, which is sensitive to alkaloids but not peptides. Both selective kappa agonist, U50,488 [3,4-dichloro-N-methyl-N-(2-(1- -pyrrolidinyl)cyclohexyl)-benzeneacetamide; >= 10(-3) M], and antagonist, nor-binaltorphimine (nor-BNI; 10-3 M), inhibited the accumulation. The effect of nor-BNI was reversed by L-NAME. Immunohistochemistry showed decreased endothelial NOS expression in naloxone and U50,488-treated cords. Since, NO production at the injury site is hypothesized to act as a stop signal for microglias, opiold agents may exert their effect via changing of NO gradient along the cord resulting in disruption of accumulation. These results suggest an immuno-modulatory role for mu and kappa Opioid receptors on injury-induced microglial accumulation which may be mediated via NO. (c) 2006 IBRO. Published by Elsevier Ltd. All rights reserved.