MOUSE STRAINS THAT LACK SPINAL DYNORPHIN UPREGULATION AFTER PERIPHERAL NERVE INJURY DO NOT DEVELOP NEUROPATHIC PAIN L. R. GARDELL, a M. IBRAHIM, b R. WANG, a Z. WANG, a1 M. H. OSSIPOV, a T. P. MALAN JR, a,b F. PORRECA a,b AND J. LAI a * a Department of Pharmacology, College of Medicine, University of Arizona Health Sciences Center, Tucson, AZ 85724, USA b Department of Anesthesiology, University of Arizona Health Sciences Center, Tucson, AZ 85724, USA Abstract—Several experimental models of peripheral neurop- athy show that a significant upregulation of spinal dynorphin A and its precursor peptide, prodynorphin, is a common consequence of nerve injury. A genetically modified mouse strain lacking prodynorphin does not exhibit sustained neu- ropathic pain after nerve injury, supporting a pronociceptive role of elevated levels of spinal dynorphin. A null mutation of the  isoform of protein kinase C (PKC KO [knockout]), as well as an inbred mouse strain, 129S6, also does not manifest behavioral signs of neuropathic pain following peripheral nerve injury. The objective of this study was to extend our observations to these genetic models to test the hypothesis that elevated levels of spinal dynorphin are essential for the maintenance of abnormal pain. In PKC wild-type mice and the outbred mouse strain ICR, ligation of the L5 and L6 spinal nerves (SNL) elicited both tactile hypersensitivity and ther- mal hyperalgesia. Both strains showed a significant elevation in dynorphin in the lumbar spinal dorsal horn following SNL. Spinal administration of an anti-dynorphin A antiserum blocked the thermal and tactile hypersensitivity in both strains of mice. However, the PKC KO mice and the 129S6 mice (which express PKC) did not show abnormal pain after SNL; neither strain showed elevated levels of spinal dynor- phin. The multiple phenotypic deficits in PKC KO mice con- found the interpretation of the proposed role of PKC-ex- pressing spinal neurons in neuropathic pain states. Addition- ally, the data show that the regulation of spinal dynorphin expression is a common critical feature of expression of neuropathic pain. © 2003 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: dynorphin, PKC, spinal nerve injury, transgenic mice, neuropeptide, opioid. Experimental models of peripheral neuropathy elicit abnor- mal pain behavior that is characterized by a persistent hypersensitivity to normally innocuous tactile stimuli as well as to noxious stimuli. Such sensory hypersensitivity may be sustained, at least in part, by the spontaneous firing of the injured nerves (Kajander et al., 1990; Liu et al., 2000; for review, see Gold, 2000) and by a sensitization of the CNS to sensory input (for review, see Woolf and Salter, 2000). The latter appears to be mediated by both spinal and supraspinal mechanisms that collectively confer a lower threshold to neuronal excitation (for reviews, see Urban and Gebhart, 1999; Ossipov et al., 2000; Porreca et al., 2002). One potential mediator of neuropathic pain is the endogenous opioid dynorphin, which is consistently upregulated in the spinal cord upon chronic inflammation (Iadarola et al., 1988; Draisci et al., 1991; Pohl et al., 1997), nerve injury (Cho and Basbaum, 1988; Kajander et al., 1990; Malan et al., 2000), and chronic opioid treatment (which paradoxically induces abnormal pain; Vanderah et al., 2000). Importantly, spinal administration of an anti- dynorphin antiserum can effectively block nerve injury- induced pain (Bian et al., 1999; Malan et al., 2000), as well as opioid induced pain and antinociceptive tolerance (Van- derah et al., 2000). A transgenic model that consists of a deletion mutation of dynorphin’s precursor peptide, pro- dynorphin, does not exhibit persistent abnormal pain be- havior after nerve injury (Wang et al., 2001), further sup- porting a role of spinal dynorphin in the maintenance of neuropathic pain (for review, see Lai et al., 2001). Pharmacological application of non-opioid fragments of dynorphin produces an enhancement in the evoked release of calcitonin gene-related peptide (CGRP), an ex- citatory transmitter found in the primary afferent (Gardell et al., 2002b). It is well established that non-opioid actions of dynorphin are pronociceptive and possibly excitotoxic (Skilling et al., 1992; Vanderah et al., 1996), and that these effects of dynorphin are consistent with excitatory effects on neurons (Hauser et al., 1999; Tang et al., 2000). A recent study in our laboratory shows that elevated levels of spinal dynorphin, and presumably its enhanced release, in morphine “tolerant rats” enhanced the evoked release of CGRP from primary afferents (Gardell et al., 2002b). This enhancing effect on excitatory neurotransmission by spinal dynorphin may underlie some aspects of opioid-induced abnormal pain and the expression of antinociceptive toler- ance to morphine. While the mechanisms that mediate the action of dynorphin is at present unknown, many excitatory signal transduction processes that may contribute to the manifestation of abnormal pain have been proposed; among these, the activation of the N-methyl-D-aspartate (NMDA) receptors and protein kinase C (PKC) has been 1 Present address: Department of Biopharmaceutical Sciences, Col- lege of Pharmacy, University of Illinois at Chicago, 833 South Wood Street, M/C874, Chicago, IL 60612, USA. *Corresponding author. Tel: +1-520-626-2147; fax: +1-520-626- 4182. E-mail address: lai@u.arizona.edu (J. Lai). Abbreviations: CGRP, calcitonin gene-related peptide; DLF, dorsal lateral funiculus; i.th., intrathecal; PKC, protein kinase C; PKC, pro- tein kinase C  isoform; SDS, sodium dodecyl sulfate; SNL, spinal nerve ligation; WT, wild type. Neuroscience 123 (2004) 43–52 0306-4522/04$30.00+0.00 © 2003 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2003.08.021 43