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Neuropathy-induced spinal GAP-43 expression is not a main player in the onset of mechanical pain hypersensitivity - PubMed

Comparative Study

. 2011 Dec;28(12):2463-73.

doi: 10.1089/neu.2011.1833. Epub 2011 Oct 20.

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Comparative Study

Neuropathy-induced spinal GAP-43 expression is not a main player in the onset of mechanical pain hypersensitivity

Robby J Jaken et al. J Neurotrauma. 2011 Dec.

Abstract

Structural plasticity within the spinal nociceptive network may be fundamental to the chronic nature of neuropathic pain. In the present study, the spatiotemporal expression of growth-associated protein-43 (GAP-43), a protein which has been traditionally implicated in nerve fiber growth and sprouting, was investigated in relation to mechanical pain hypersensitivity. An L5 spinal nerve transection model was validated by the presence of mechanical pain hypersensitivity and an increase in the early neuronal activation marker cFos within the superficial spinal dorsal horn upon innocuous hindpaw stimulation. Spinal GAP-43 was found to be upregulated in the superficial L5 dorsal horn from 5 up to 10 days after injury. GAP-43 was co-localized with calcitonin-gene related peptide (CGRP), but not vesicular glutamate transporter-1 (VGLUT-1), IB4, or protein kinase-γ (PKC-γ), suggesting the regulation of GAP-43 in peptidergic nociceptive afferents. These GAP-43/CGRP fibers may be indicative of sprouting peptidergic fibers. Fiber sprouting largely depends on growth factors, which are typically associated with neuro-inflammatory processes. The putative role of neuropathy-induced GAP-43 expression in the development of mechanical pain hypersensitivity was investigated using the immune modulator propentofylline. Propentofylline treatment strongly attenuated the development of mechanical pain hypersensitivity and glial responses to nerve injury as measured by microglial and astroglial markers, but did not affect neuropathy-induced levels of spinal GAP-43 or GAP-43 regulation in CGRP fibers. We conclude that nerve injury induces structural plasticity in fibers expressing CGRP, which is regarded as a main player in central sensitization. Our data do not, however, support a major role of these structural changes in the onset of mechanical pain hypersensitivity.

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Figures

FIG. 1.
FIG. 1.

Validation of the spinal nerve transection (SNT) model for neuropathic pain hypersensitivity. (A) Paw withdrawal thresholds (PWT) of ipsilateral hindpaws are reduced by about 60–70% by transection of the L5 spinal nerve (n=5); the contralateral PWT remained unaffected. (B–E) Expression of the immediate early marker cFos in neurons of the ipsilateral (B and D) and contralateral (C and E) L5 superficial dorsal horn at 5 days after SNT. (B and C) An animal without 10 min of innocuous hindpaw stimulation. (D and E) An animal with 10 min of innocuous hindpaw stimulation. (F) Graph showing that 10 min of innocuous hindpaw stimulation strongly increased the number of cFos-positive neurons in the superficial spinal dorsal horn ipsilateral, but not contralateral, to SNT (n=6 animals in both the non-stimulated and stimulated groups; PWT, paw withdrawal threshold in grams; time in days after SNT; t=0 days corresponds to presurgical baseline values; *p<0.05; **p<0.01).

FIG. 2.
FIG. 2.

Temporal upregulation of growth-associated protein-43 (GAP-43) ipsilateral to spinal nerve transection (SNT). (A) GAP-43 immunostaining of the L5 spinal cord at 5 days after SNT (SNT is on the left side). (B–D) GAP-43 expression in the superficial ipsilateral L5 spinal cord of a sham-operated rat (B), at 5 days after SNT (C), and at 21 days after SNT (D). Protein kinase-γ (PKC-γ) immunoreactivity demarcates the superficial dorsal horn; insets show spinal GAP-43 expression at higher resolution. (E) GAP-43 expression in the superficial dorsal horn of the L3, L4, and L5 spinal cord (expression is shown in relation to values of sham-operated animals; sham averages±standard error of the mean is indicated by dotted lines and grey shading; n=4 for sham animals; n=5 for 21 days after SNT; n=6 each for 3, 5, and 7 days after SNT; n=10 for 10 days after SNT; *p<0.05).

FIG. 3.
FIG. 3.

Identification of neuronal growth-associated protein-43 (GAP-43) immunoreactivity In order to investigate the cellular source of GAP-43 immunoreactivity following spinal nerve transection (SNT), several double immunostainings were performed. (A and B) Protein kinase-γ (PKC-γ)-immunostained sections were used to delineate the superficial dorsal horn. Insets in boxes A and B show the location in which double-stained sections were investigated. Combined immunohistochemical staining for GAP-43 with PKC-γ (C) and the primary afferent markers vesicular glutamate transporter-1 (VGLUT-1; D), isolectin B4 (IB4; E), and calcitonin-gene related peptide (CGRP; F) (all in red) at 5 days following SNT show a clear co-localization for CGRP/GAP-43 (yellow; F). High magnification of GAP-43 and CGRP shows an overlap in staining for fiber-like structures (G and H; indicated by white arrows). (I) Confocal analysis revealed an unambiguous co-localization of both GAP-43 and CGRP. (J and K) Double-staining of glial fibrillary acidic protein (GFAP) and GAP-43 shows no overlap in staining.

FIG. 4.
FIG. 4.

Propentofylline (PPF) attenuates mechanical hypersensitivity and microglial ionized calcium binding adaptor 1 protein (Iba-1) expression following spinal nerve transection (SNT). (A) SNT induced a nearly 60% reduction of mechanical hypersensitivity of the ipsilateral hindpaw compared to the contralateral hindpaw in saline-treated animals, while PPF-treated animals only showed a reduction of about 30%, which is similar to previous observations (Raghavendra, , #1268; n=10 for saline-treated SNT animals, and n=12 for PPF-treated SNT animals). (B, D–G): At 5 days after SNT, expression of Iba-1 was strongly upregulated in the ipsilateral spinal cord of SNT animals receiving daily intrathecal injections with saline (D and E). This was evidenced by a more than 30% increase of Iba-1 immunoreactivity compared to control animals (B). Daily intrathecal delivery of PPF for 5 days following SNT attenuated the expression of Iba-1 significantly (p<0.05; B, F and G). In contrast to Iba-1 immunoreactivity, glial fibrillary acidic protein (GFAP) immunoreactivity was not affected by SNT (C, H, and I). As a consequence, PPF did not affect GFAP expression in the dorsal horn of the lumbar spinal cord (C, J, and K; n=5 for saline-treated SNT animals, and n=5 for PPF-treated SNT animals; **p<0.01; *p<0.05; Ipsi, ipsilateral; contra, contralateral).

FIG. 5.
FIG. 5.

Propentofylline (PPF) does not affect spinal growth-associated protein-43 (GAP-43) expression following spinal nerve transection (SNT). (A) Spinal GAP-43 expression in sham-operated animals sacrificed at 5 days after sham surgery. (B and C) Spinal GAP-43 expression at 5 days after SNT in a vehicle-treated (B) and a PPF-treated (C) animal. (D) PPF does not affect spinal GAP-43 expression in the superficial dorsal horn of the L5 spinal cord after SNT (GAP-43 expression is shown in relation to values of sham-operated animals; n=4 for sham-operated animals, n=6 each for SNT animals with saline or PPF treatment). (E) Representative example of GAP-43/CGRP double staining in the superficial dorsal horn of a PPF-treated animal at 5 days after SNT (*p<0.05; CGRP, calcitonin gene-related peptide).

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