Sustained morphine-mediated pain sensitization and antinociceptive tolerance are blocked by intrathecal treatment with Raf-1-selective siRNA.
OBJECTIVE
Long-term morphine treatment enhances pain neurotransmitter [such as calcitonin gene-related peptide (CGRP)] levels in the spinal cord. It has been suggested previously that increased spinal CGRP may contribute to sustained morphine-mediated paradoxical pain sensitization and antinociceptive tolerance. Previous in vitro studies from our group indicated that Raf-1 kinase-mediated adenylyl cyclase superactivation played a crucial role in sustained morphine-mediated augmentation of basal and evoked CGRP release from cultured primary sensory neurons. The present study was aimed to evaluate the physiological significance of this molecular mechanism in vivo, in rats.


METHODS
Rats were intrathecally (i.th) injected with a Raf-1-selective small interfering RNA (siRNA) mixture for 3 days and were subsequently infused with saline or morphine, s.c. for 7 days. Thermal and mechanical sensory thresholds of the animals were assessed by daily behavioural tests. After final behavioural testing (day 6), spinal cords were isolated from each animal group and spinal CGRP and Raf-1 protein levels were measured using elisa and immunohistochemistry.


RESULTS
Selective knockdown of spinal Raf-1 protein levels by i.th Raf-1-selective siRNA pretreatment significantly attenuated sustained morphine-mediated up-regulation of CGRP immunoreactivity in the spinal cord of rats and prevented the development of thermal hyperalgesia, mechanical allodynia and antinociceptive tolerance.


CONCLUSIONS
Raf-1 played a significant role in sustained morphine-mediated paradoxical pain sensitization and antinociceptive tolerance in vivo. These findings suggest novel pharmacological approaches to improve the long-term utility of opioids in the treatment of chronic pain.
Tumati
S
S
Department of Pharmacology, The University of Arizona, Tucson, AZ, USA.
Roeske
W R
WR
Largent-Milnes
T
T
Wang
R
R
Vanderah
T W
TW
Varga
E V
EV
D000818:Animals; D006930:Hyperalgesia / Q000139:chemically induced* / Q000517:prevention & control; D007278:Injections, Spinal; D020930:MAP Kinase Kinase Kinases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D008297:Male; D009020:Morphine / Q000494:pharmacology*; D019908:Proto-Oncogene Proteins c-raf; D034741:RNA, Small Interfering / Q000008:administration & dosage / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013116:Spinal Cord / Q000187:drug effects / Q000378:metabolism
D034741:RNA, Small Interfering; D009020:Morphine; D019908:Proto-Oncogene Proteins c-raf; C546782:Raf1 protein, rat; D020930:MAP Kinase Kinase Kinases