Blockade of glutamate mGlu5 receptors in a rat model of neuropathic pain prevents early over-expression of pro-apoptotic genes and morphological changes in dorsal horn lamina II

Abstract

We used rats with a sciatic nerve chronic constrictive injury (CCI) and combined behavioural, molecular and morphological approaches to assess the involvement of mGlu5 receptors in neuropathic pain-associated hyperalgesia and spinal cord neuron apoptosis. Mechanical and thermal hyperalgesia developed 2–3 days after surgery. Morphological changes in the ipsilateral L4–L5 lamina II consisted of: (i) cell loss (38±5%), (ii) increased TUNEL-positive profiles, (iii) decreased SP-immunoreactive primary afferents, and (iv) reactive gliosis. Molecular expression data suggested a bi-phasic response of bcl-2 family genes in CCI. An early (2–3 days post-CCI) E2F1- and p53-independent apoptosis appeared in the spinal cord as the pro-apoptotic bax gene increased (320±19%), followed by an increased expression of the anti-apoptotic bcl-2 and bcl-xL genes (60±11% and 110±15%, respectively) 7 days from CCI. The selective mGlu5 receptor antagonist, MPEP (2 mg/kg i.p. twice daily), prevented the development of thermal hyperalgesia and transiently reduced mechanical hyperalgesia. Despite the MPEP treatment, which normalised bax/bcl-2 and bcl-xL/bcl-xS ratios at all times post-CCI, mechanical hyperalgesia reappeared by 7 days after CCI. Similarly, MPEP was cytoprotective at 3, but not 7 days post-CCI. This study shows that: (a) spinal cord neuron loss may be triggered by a p53- and E2F1-independent apoptosis in lamina II with the participation of glutamate mGlu5 receptors, (b) these receptors seem to be involved transiently, as their blockade was no longer protective by 7 days CCI, and (c) this delayed cell death occurred in the absence of Bax activation, suggesting the involvement of an alternative death pathway.

Introduction

Neuropathic pain is a devastating and difficult to manage consequence of peripheral nerve injury, the main aspect of which is enhanced transmission of nociceptive messages (Bonica, 1970). In this condition, noxious stimuli are perceived as more painful (hyperalgesia), and normal, harmless stimuli may elicit pain (allodynia). Neuropathic pain is a progressive nervous system disease that results from poorly defined neurophysiological and neurochemical changes. Support for this hypothesis is obtained from animal models of chronic pain in which a long-lasting increase (central sensitisation) in the excitability of spinal dorsal horn neurons is observed.

Glutamate is a likely mediator of central sensitisation in neuropathic pain (Willis, 2001). Spinal NMDA glutamate receptors clearly trigger intracellular signals that induce long-lasting effects on gene transcription (Sher and Mitchell, 1990, Cumberbatch et al., 1994, Dickenson et al., 1997, Martin et al., 2001). Pain hypersensitivity has also been suggested to be a consequence of glutamate-induced excitotoxicity and neuronal death resulting in change in dorsal horn circuitry and disinhibitory processes (Sugimoto et al., 1990). A dramatic NMDA-receptor-dependent decrease in the efficacy of spinal opioid analgesia was observed following sciatic nerve injury in rats (Mayer et al., 1999) and this may, at least in part, reflect the loss of interneurons and changes in lamina II cytoarchitecture (Sugimoto et al., 1990, Woolf et al., 1992, Moore et al., 2002). Morphological studies have suggested the occurrence of apoptosis in the spinal cord following peripheral nerve insult (Azkue et al., 1998, Whiteside and Munglani, 2001), and we have shown in the spinal superficial laminae of neuropathic rats, that altered bcl-2 family gene expression may modulate cell death in a model of neuropathic pain (Maione et al., 2002). Whether or not changes in the expression of apoptosis-associated genes occur in the spinal cord during the first days leading to the establishment of neuropathic pain syndrome is unknown. Similarly, although there is growing evidence in support of a role for metabotropic glutamate receptor 5 (mGlu5) in nociception and pain behaviours, its involvement in neuropathic pain pathogenesis is unknown. It has been shown that single administration of MPEP reverses inflammatory and thermal hyperalgesia, but has no effect on mechanical hyperalgesia in rats with established neuropathic pain (Dogrul et al., 2000, Walker et al., 2001, Hudson et al., 2002). Identification of a role for mGlu5 receptors in neuropathic pain pathogenesis would represent a critical step in developing pharmacological strategies to treat or prevent this syndrome. Therefore, we used a rat model of neuropathic pain to correlate the development of thermal and mechanical hyperalgesia with morphological changes in dorsal horn lamina II and changes in mRNA expression levels of several apoptosis-associated genes including: bax, bcl-2, bcl-xL, bcl-xS, p53, E2F1, and cathepsin D in lumbar L4–L5 spinal cords of rats 1, 3 and 7 days after sciatic nerve chronic constrictive injury (CCI). We report that a selective blockade of mGlu5 receptors by MPEP prevents thermal hyperalgesia, transiently delays mechanical hyperalgesia, and inhibits the early over-expression of pro-apoptotic genes and neuron cell loss following CCI.