Research reportThe behavioral and neuroanatomical effects of IB4-saporin treatment in rat models of nociceptive and neuropathic pain
Introduction
Sensory information is transmitted from the periphery to the spinal cord by distinct subsets of primary afferent neurons, including small diameter unmyelinated C-fibers [48]. Although many C-fibers express TRPV1 (VR1 receptor) and are therefore considered nociceptors [43], a rich heterogeneity exists within this population. Two major classes of C-fibers are distinguished by their ability to bind the lectin IB4, by their termination pattern in the superficial laminae of the spinal cord dorsal horn and by their sensitivity to neurotrophins [32], [38], [40]. In general, sensory neurons that contain substance P (SP) and calcitonin gene-related peptide (CGRP) terminate in lamina I and the outer portion of lamina II (IIo) in the spinal cord and do not bind IB4 (IB4−), whereas IB4-positive fibers (IB4+) express fluoride-resistant acid phosphatase (FRAP), P2X3 receptors and terminate in the inner portion of lamina II (IIi) [15], [32], [38], [39]. IB4− and IB4+ nociceptors also express distinct neurotrophin receptors, TrkA and the GDNF receptor complex, which confer sensitivity to NGF and GDNF, respectively [6], [30], [31]. In addition to unique histochemical and anatomical signatures, IB4− and IB4+ neurons have distinct electrophysiological properties with respect to action potential threshold and duration as well as the proportion of TTX-resistant sodium currents and sensitivity to heat stimuli [41].
While many of the differences between IB4− and IB4+ neurons are now appreciated, the functional consequences of these differences for nociceptive signaling are less well-understood. The involvement of IB4+ sensory neurons derives largely from studies focused on interneurons in lamina IIi, onto which IB4-positive sensory neurons terminate. Previous work suggested that neurons in the inner part of lamina II (IIi) facilitate nociceptive transmission in the setting of injury. For example, PKCγ-expressing interneurons in lamina IIi have been implicated in both inflammatory and neuropathic pain [18], [26], [27], [34] and PKCγ, itself, is required for prolonged injury-induced hyperexcitability of lamina V neurons in the spinal cord [28]. Thus, enhanced activity in lamina IIi interneurons is essential for central sensitization and the full expression of persistent pain.
Selective elimination of IB4+ sensory neurons with intrasciatic injection of a cytotoxin, saporin, conjugated to IB4 (IB4-SAP) increases acute nociceptive thresholds to noxious thermal and mechanical stimuli in uninjured rats [44], but the contribution of these neurons to the development and maintenance neuropathic pain behavior has not been examined. In this study, we used the same targeted neuronal lesioning approach [47] to selectively eliminate IB4+ sensory neurons [44] and assessed directly the contribution of IB4+ sensory neurons in neighboring L4 primary afferents to the behavioral and anatomical consequences of L5 spinal nerve injury [23].
Section snippets
Methods
All experiments were approved by the MRL-Rahway Institutional Animal Care and Use Committee and adhere to the guidelines established by the International Association for the Study of Pain [49]. Adult male Sprague–Dawley rats (150–250 g at start of study; Charles River Laboratories, Raleigh, NC) were housed in pairs on a 12-h light/dark cycle and were given free access to food and water.
IB4-SAP treatment in uninjured rats: anatomical effects
Two to four weeks after intrasciatic injection of IB4-SAP, IB4 staining in the superficial dorsal horn of L4 and L5 spinal segments was significantly reduced. The loss of IB4 staining was restricted to, and essentially complete in, the zone of innervation of the sciatic nerve, i.e. the medial portion of the dorsal horn (Fig. 2). Compared to non-injected animals, IB4-labeling was reduced by 55% 3 weeks following IB4-SAP treatment. By contrast, IB4-labeling was only modestly reduced after
Discussion
In this study, we attempted to define the functional significance of IB4+ sensory fibers in nociceptive and neuropathic pain using a conjugated toxin technique. We showed that intrasciatic injection of IB4-SAP only reduced IB4 binding and P2X3 immunoreactivity within the sciatic nerve territory of the spinal cord. The deletion of IB4+ sensory fibers increased modestly thermal and mechanical nociceptive thresholds and significantly reduced NGF-induced hyperalgesia. Furthermore, administration of
Conclusion
The neurochemical heterogeneity across C-fibers has made it difficult to define a functional role for either of the IB4− or IB4+ subclasses beyond that which can be gleaned from their termination pattern in the spinal cord or by studying the individual receptor elements of the subclasses. Our results support the functional distinction between IB4− or IB4+ subclasses and illustrate that selective deletion of a GDNF-dependent, IB4+, subset of sensory neurons mitigates injury-induced behavioral
Acknowledgments
We thank Drs. M. Leitner and C. Abbadie for helpful comments on this manuscript.
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