Involvement of subtype 1 metabotropic glutamate receptors in apoptosis and caspase-7 over-expression in spinal cord of neuropathic rats

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Abstract

The effect of the non-selective, 1-aminoindan-1,5-dicarboxylic acid (AIDA), and selective (3,4-dihydro-2H-pyrano[2,3-b]quinolin-7-yl)-(cis-4-methoxycyclohexyl) methanone (JNJ16259685), metabotropic glutamate subtype 1 (mGlu1) receptor antagonists, on rat sciatic nerve chronic constrictive injury (CCI)-induced hyperalgesia, allodynia, spinal dorsal horn apoptosis, and gliosis was examined at 3 and 7 days post-injury. RT-PCR analysis showed increased expression of bax, apoptotic protease-activating factor-1 (apaf-1), nestin, GFAP, and caspase-7 mRNA in the dorsal horn spinal cord by 3 days post-CCI. At 7 days post-CCI, only over-expression of bcl-2, nestin and GFAP mRNA was observed. Administration of AIDA reduced thermal hyperalgesia and mechanical allodynia at 3 and 7 days post-CCI; administration of JNJ16259685 reduced thermal hyperalgesia at 3 and 7 days post-CCI, but not mechanical allodynia. AIDA decreased the mRNA levels of bax, apaf-1, GFAP and caspase-7 genes. JNJ16259685 increased the mRNA levels of bcl-2 and GFAP gene, and decreased APAF-1 and caspases-7 genes. Inhibiting mGlu1 receptors also reduced TUNEL-positive profiles and immunohistochemical reactivity for caspase-7. We report here that despite inhibiting CCI-induced over-expression of pro-apoptotic genes in the spinal cord dorsal horn, the selective mGlu1 receptor antagonist JNJ16259685 exerted only a slight and transient allodynic effect. Moreover, JNJ16259685, but not the non-selective AIDA, increased astrogliosis which may account for its decreased analgesic efficacy. This study provides evidence that the contemporary and partial blockade of group I and likely ionotropic glutamate receptors may be a more suitable therapy than selective blockade of mGlu1 subtype receptors condition to decrease neuropathic pain symptoms.

Introduction

Neuropathic pain is caused by an injury or dysfunction in the peripheral or central nervous system [1], [2], [3]. In neuropathic pain conditions, noxious stimuli are perceived as more painful (hyperalgesia), and normal, harmless stimuli may elicit pain (allodynia) [1]. The mechanisms that centrally control such spread of pain stem from neurochemical and functional changes, and therefore, neuropathic pain should be considered a neuropathological condition. Evidence for this classification is offered by several animal models of chronic pain showing that a sustained release of glutamate, cytokines and neurotrophic factors (i.e. neurokinins, BDNF, TNF-alpha, etc.) induces sensitization at both dorsal root ganglion neurons and second-order neurons in the dorsal horn [4], [5], [6]. Indeed, there is evidence that synaptic facilitation requires the release of neurokinins, glutamate, and tropomyosin-related kinase (TRK) family of neurotrophin receptors. Spinal N-methyl-d-aspartate (NMDA) glutamate receptor contributes to triggering intracellular signals that induce long lasting effects at the transcriptional level [3], [7], [8], [9], [10], [11], [12].

Moreover, not only central sensitization, but also a disinhibitory process associated either with excitotoxic-induced neuronal death [2], [10], [13], [14] or with a rearrangement in the pathophysiology of the endogenous antinociceptive pathway [15], have been suggested to be required for the induction of dorsal horn circuitry sensitization. Morphological studies suggested the occurrence of neuronal apoptosis in the spinal cord following peripheral nerve insult [16], [17]. Nevertheless, there is also evidence that astrocytes undergo apoptosis in the spinal cord of neuropathic rat [18]. We have shown that the occurrence of glutamate-dependent apoptosis may be an early and rapid event, as pro-apoptotic bax and bcl-xS mRNA levels increased in the spinal cord of rats within 2–3 days after sciatic nerve chronic constrictive injury (CCI) [19], [20].

Many studies have highlighted the involvement of metabotropic glutamate (mGlu) receptors in nociception control [21], [22], [23], [24], [25], [26]. Group I metabotropic glutamate receptors (mGluRs) (mGlu1 and 5) have been implicated in the processes of central sensitization and persistent nociception [27]. Treatment with selective antagonists for mGlu1 and mGlu5 receptors attenuated the development of mechanical allodynia and decreased extracellular glutamate in the spinal cord [28], [29]. We have previously shown that mGlu5 receptor blockade was transiently anti-allodynic and reduced spinal cord apoptosis in neuropathic rats [20], although an analysis of terminal caspase activation was not carried out in that study.

Caspases, a family of cysteine proteases, play a critical role in the execution phase of apoptosis and are responsible for many of the biochemical and morphological changes associated with apoptosis [30]. Procaspase-9 has been proposed as an initiator caspase that activates the effector caspases-3, -6, and -7 [31]. This apoptotic step is implicated in motorneuron degeneration produced by mutant superoxide dismutase-1 [32], in models of peripheral neuropathy [33], Alzheimer disease [34], [35], [36], Huntington disease [37], and neuropathic pain, suggesting a critical role for these proteases in numerous neurological conditions. To our knowledge, however, there is no study aimed at identifying the possible relationship between mGlu1 receptors, the activation of specific spinal caspases and the development of hyperalgesia/allodynia in neuropathic pain models.

In this study, we have addressed the issue of whether the blockade of mGlu1 receptors might modify early over-expression of pro-apoptotic and gliosis genes in the spinal cord dorsal horn, together with allodynia and hyperalgesia development by CCI of the sciatic nerve.

Section snippets

Animals

Male Wistar rats (Harlan, Udine, Italy) (250–300 g) were housed three per cage under controlled illumination (12:12 h light:dark cycle; light on 06.00 h) and environmental conditions (room temperature 20–22 °C, humidity 55–60%) for at least 1 week before the commencement of experiments. Rat chow and tap water were available ad libitum. Behavioural testing was performed before surgery to establish a baseline for comparison with post-surgical values. CCI and sham rats were assessed for thermal and

AIDA prevents thermal hyperalgesia and mechanical allodynia by 3 and 7 days CCI, JNJ16259685 prevents thermal hyperalgesia, but not mechanical allodynia

Post-surgery mean difference scores for thermal hyperalgesia were significantly lower for the CCI sciatic nerve groups at 3 and 7 days (Fig. 1A and B) than sham rats. The increased sensitivity on the CCI sides was not present on the contralateral sides (data not shown). Similarly, sciatic nerve CCI rats showed mechanical allodynia only on the ipsilateral sides of the nerve ligature at 3 and 7 days post-surgery (Fig. 1C and D). Administration of AIDA, a preferential mGlu1 receptor antagonist,

Discussion

It has been shown that increased expression of group I mGlu receptors parallels the development of thermal hyperalgesia and mechanical allodynia, and this change has been suggested to contribute to the development and maintenance of chronic central pain syndrome [22], [24], [29], [43]. This study provides molecular evidence that, in the model of sciatic nerve ligature in the rat [40], neuropathic pain is associated with marked activation of caspase-7 together with its transient increase in

Acknowledgements

This study was supported by grant from MIUR Italy (PRIN 2005). Dr. Kevin A. Roth was supported by NIH Grant (R01 NS35107).

References (76)

  • V. de Novellis et al.

    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

    Neuropharmacology

    (2004)
  • K. Fisher et al.

    Intrathecal administration of the mGluR compound, (S)-4CPG, attenuates hyperalgesia and allodynia associated with sciatic nerve constriction injury in rats

    Pain

    (1998)
  • S. Maione et al.

    Periaqueductal gray matter metabotropic glutamate receptors modulate formalin-induced nociception

    Pain

    (2000)
  • V. de Novellis et al.

    Periaqueductal grey cb1 cannabinoid and metabotropic glutamate subtype 5 receptors modulate changes in rostral ventromedial medulla neuronal activities induced by subcutaneous formalin in the rat

    Neuroscience

    (2005)
  • S.M. Srinivasula et al.

    Autoactivation of procaspase-9 by Apaf-1 mediated oligomerization

    Mol Cell

    (1998)
  • C. Stadelmann et al.

    Activation of caspase-3 in single neurons and autophagic granules of granulovacuolar degeneration in Alzheimer's disease. Evidence for apoptotic cell death

    Am J Pathol

    (1999)
  • H. Lavreysen et al.

    JNJ16259685, a highly potent, selective and systemically active mGlu1 receptor antagonist

    Neuropharmacology

    (2004)
  • D. Siniscalco et al.

    Role of reactive oxygen species and spinal cord apoptotic genes in the development of neuropathic pain

    Pharmacol Res

    (2007)
  • T.E. Salt et al.

    Evaluation of agonists and antagonists acting at group I metabotropic glutamate receptors in the thalamus in vivo

    Neuropharmacology

    (1999)
  • M.W. Jones et al.

    Interactions between metabotropic and ionotropic glutamate receptor agonists in the rat spinal cord in vitro

    Neuropharmacology

    (1995)
  • A. Ugolini et al.

    Potentiation of NMDA and AMPA responses by group I mGluR in spinal cord motoneurons

    Neuropharmacology

    (1997)
  • G. Miglio et al.

    Stimulation of group I metabotropic glutamate receptors evokes calcium signals and c-jun and c-fos gene expression in human T cells

    Biochem Pharmacol

    (2005)
  • X. Liu et al.

    Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c

    Cell

    (1996)
  • D.M. Finucane et al.

    Bax-induced caspase activation and apoptosis via cytochrome c release from mitochondria is inhibitable by Bcl-xL

    J Biol Chem

    (1999)
  • H. Zou et al.

    An APAF-1 cytochrome c multimeric complex is a functional apoptosome that activates procaspase-9

    J Biol Chem

    (1999)
  • C. Young et al.

    Role of caspase-3 in ethanol-induced developmental neurodegeneration

    Neurobiol Dis

    (2005)
  • M.Y. Stepanichev et al.

    Central administration of a caspase inhibitor impairs shuttle-box performance in rats

    Neuroscience

    (2005)
  • L.H. Barbeito et al.

    A role for astrocytes in motor neuron loss in amyotrophic lateral sclerosis

    Brain Res Rev

    (2004)
  • L. Watkins et al.

    Glial activation: a driving force for pathological pain

    Trends Neurosci

    (2001)
  • K. Inoue et al.

    ATP- and adenosine-mediated signaling in the central nervous system: chronic pain and microglia: involvement of the ATP receptor P2X4

    J Pharmacol Sci

    (2004)
  • M. Tsuda et al.

    Neuropathic pain and spinal microglia: a big problem from molecules in “small” glia

    Trends Neurosci

    (2005)
  • J.J. Bonica

    Advances in pain research and therapy

    (1970)
  • D. Siniscalco et al.

    Neuropathic pain: is the end of suffering starting in the gene therapy?

    Curr Drug Targets

    (2005)
  • Willis W.D. Role of neurotransmitters in sensitization of pain responses. In: Sorg B.A., I.R. Bell (Eds.). Spinal cord...
  • D.J. Mayer et al.

    Cellular mechanisms of neuropathic pain, morphine tolerance, and their interactions

    Proc Natl Acad Sci USA

    (1999)
  • W.J. Martin et al.

    PKCgamma contributes to a subset of the NMDA-dependent spinal circuits that underlie injury-induced persistent pain

    J Neurosci

    (2001)
  • L. Fang et al.

    Calcium-calmodulin-dependent protein kinase II contributes to spinal cord central sensitisation

    J Neurosci

    (2002)
  • G.T. Whiteside et al.

    Cell death in the superficial dorsal horn in a model of neuropathic pain

    J Neurosci Res

    (2001)
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