Intrathecally administered Sema3A protein attenuates neuropathic pain behavior in rats with chronic constriction injury of the sciatic nerve
Introduction
Peripheral nerve injury produces a number of neuroplastic changes in the spinal cord, including reorganization of neural circuits in the dorsal horn, that may contribute to the development of neuropathic pain (Ji and Strichartz, 2004, Obata et al., 2004a, Obata et al., 2004b, Yamanaka et al., 2004, Zimmermann, 2001). In the physiological condition, myelinated Aβ-afferent is activated by low threshold mechanical stimuli and it is thought to convey touch sensation and proprioception, not pain. On the other hand, unmyelinated C-afferents are activated by nociceptive polymodal stimuli (e.g., heat, acid, chemicals, local pressure and so on) and they are thought to convey the continuing, dull and burning pain. Unmyelinated C-afferents were included peptidergic fibers which contain substance P and calcitonin gene-related peptide (CGRP), and non-peptidergic fibers which are histochemically distinguished by isolectin B4 (IB4) binding.
On the other hand, in the pathophysiological condition like neuropathic pain, previous studies derived the idea that peripheral nerve injury causes Aβ afferents to sprout into the superficial layer of the dorsal horn where many neurons are nociception-specific (Doubell et al., 1997, Mannion et al., 1996, Woolf et al., 1992, Woolf et al., 1995). As a result, the neural inputs conducted through these Aβ afferents may provoke pain, although they would normally be perceived as non-nociceptive (e.g., tactile sensation).
Axon guidance factors play crucial roles in patterning neuronal projections during the development of the nervous system. Together with cell-contact inhibitory cues, semaphorin 3A (Sema3A), one of the secreted semaphorin families and repulsive axon guidance factors, prevents axons from innervating inappropriate territories in the developing nervous system (Goshima et al., 2000, Yu and Bargmann, 2001). Although the functions of axon guidance factors in the mature nervous system are still unclear, it has been reported recently that secretion of Sema3A and expression of its co-receptor Neuropilin (NPR) (Takahashi et al., 1999) were up-regulated around scar tissue at the site of the sectioned spinal cord and that Sema3A inhibited regeneration of injured nerve fibers and restoration of neural circuitry (Niclou et al., 2006, Shearer et al., 2003). Moreover, inhibition of Sema3A induced re-connection of transected axons of spinal cord neurons and restored motor function (Kaneko et al., 2006). With respect to pain, previous studies showed that Sema3A prevented the sprouting of unmyelinated sensory nerve endings and attenuated hyperalgesia in the injured cornea (Tanelian et al., 1997) and in the spinal cord of the nerve growth factor (NGF)-induced neuropathic pain model (Tang et al., 2004). However these studies pointed out the anti-allodynic effect of Sema3A, they focused only the sprouting of the peptidergic C-fiber nerve terminals, and the effects of Sema3A on the sprouting of myelinated nerve endings remain to be elucidated. Additionally, corneal hypersensitivity due to corneal injury did not involved in reorganization of sensory circuit in the central nervous system, and NGF-induced neuropathic pain model was not strictly neuropathic pain model because C-afferent nerve ending in the spinal dorsal horn was usually degenerated histochemically (Gardell et al., 2003) or at least did not sprouted into adjacent laminae (Nakamura and Myers, 1999) under the condition of peripheral nerve injury. So the mechanisms by which Sema3A modifies pain behavior and the sensory circuit of the spinal dorsal horn in neuropathic pain associated with peripheral nerve injury remain to be clarified. In this study, we used a chronic constriction injury (CCI) rat model to test the hypothesis that intrathecal administration of Sema3A protein would prevent development of neuropathic pain at least in part by reducing the sprouting of either myelinated or unmyelinated nerve fibers within the spinal cord dorsal horn. We found that intrathecal Sema3A protein administration attenuated neuropathic pain-like behaviors and the decrease of IB4-positive non-peptidergic unmyelinated sensory nerve terminals in lamina II of the dorsal horn. Contrary to our expectations, since Sema3A did not alter the extent of the sprouting of myelinated nerve terminals labeled with cholera toxin subunit b (CTb) and immunostained by anti-neurofilament-200 antibody in the lamina II 7 days after CCI, it is suggested that the inhibitory effect of Sema3A on the early development of neuropathic pain might be independent of prevention of the myelinated fiber sprouting.
Section snippets
Animals and surgery
Male Sprague-Dawley rats (220–300 g) were obtained from Japan SLC (Hamamatsu, Shizuoka, Japan). Animals were caged in small groups of 2–3 at a constant ambient temperature (23 ± 1 °C) under a 12-h light/dark cycle with free access to food and water.
All experimental procedures were approved by the Yokohama City University Animal Care and Use Committee of the Animal Research Center, Yokohama City University School of Medicine (approval number 04-56) and carried out in accordance with the guidelines
Expression of Neuropilin 1 and Plexin A, co-receptors of Sema3A, in dorsal horn of lumbar spinal cord
We first examined the expression of NPR1 and PlexA in the L4/5 dorsal part of the rat spinal cord and quantitatively compared those expression levels between naïve and CCI rats. We used CCI rats 7 days after operation. Both proteins were detected in the membrane fraction of either side (ipsilateral and contralateral) of the naïve and CCI rats’ dorsal spinal cords (Fig. 1A). Expression levels of NPR1 and PlexA in membrane fraction of pup forebrain were used for normalization of inter-membrane
Discussion
This is the first report that intrathecally administered Sema3A protein attenuated neuropathic pain-related behaviors induced by peripheral nerve injury, and partially prevented the decrease of IB-4-positive non-peptidergic nociceptive nerve terminals in the lamina II of the dorsal horn. However, contrary to our expectation, Sema3A had no effect on the magnitude of the myelinated nerve terminals sprouting labeled with CTb into the inner layer of lamina II and the superficial layer of lamina I
Acknowledgements
We thank Prof. H. Suzuki and Dr. A. Sakai (Department of Pharmacology, Nippon Medical School) for helpful advice and for teaching us the surgical procedures for the CCI model rat and tips for the behavioral testing. We also thank H. Ito, Y. Yuba and M. Kobayashi for excellent technical assistance in Western blotting analyses and immunohistochemical experiments, A. Yoshikawa for technical assistance and teaching in CTb microinjection to the sciatic nerves of experimental animals and Dr. K
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These authors contributed equally to this work.