NGF message and protein distribution in the injured rat spinal cord

https://doi.org/10.1016/j.expneurol.2004.03.017Get rights and content

Abstract

Nerve growth factor (NGF) content of the spinal cord is increased after cord injury. NGF can cause central sprouting of sensory fibers after spinal cord injury (SCI), leading to autonomic dysfunction and pain. NGF also can promote the death of oligodendroglia after SCI. Knowing the source of intraspinal NGF would benefit strategies for minimizing abnormal plasticity and cell death after SCI. We identified these sources, using RNA in situ hybridization to detect NGF mRNA and double-labeling immunocytochemistry for NGF and cell-marking antigens. In uninjured and sham-injured rats, we identified NGF mRNA in leptomeningeal cells and in neurons in the intermediate grey matter, whereas NGF protein was observed only in leptomeningeal cells. At 3–7 days after transection or clip-compression SCI, NGF mRNA and protein were expressed in the lesion and throughout the intermediate grey matter and white matter rostral and caudal to the injury site. Transection-SCI was used to permit comparisons to previous studies; clip-compression injury was used as a more clinically relevant model. mRNA and protein in adjacent sections were expressed in ramified microglia, astrocytes, intermediate grey neurons, pial cells, and leptomeningeal and Schwann cells in the lateral white matter and the lesion site. Rounded macrophages in the lesion were immunoreactive (Ir) for NGF, but the cells expressing NGF mRNA were not in the same areas of the lesion and were not stained by a macrophage marker. Our data demonstrate that glia, neurons, meningeal cells and Schwann cells but not macrophages contribute to the increased intraspinal NGF after SCI.

Introduction

Nerve growth factor (NGF) concentrations are very low in the normal spinal cord and increase at least 4-fold within a week after spinal cord injury (SCI) Bakhit et al., 1991, Beattie et al., 2002, Marsh et al., 2002, Murakami et al., 2002, Widenfalk et al., 2001. Although exogenous NGF can have trophic actions on sensory neurons after SCI Grill et al., 1997, Menei et al., 1998, Ramer et al., 2000, Romero et al., 2001, Tuszynski et al., 1996, recent findings suggest that NGF contributes to the pathology of spinal cord injury. NGF has been implicated in changes leading to autonomic dysfunction and pain and to apoptotic cell death. Expansion of the primary afferent arbours of sensory neurons expressing calcitonin gene-related peptide is associated with autonomic dysreflexia Jacob et al., 2001, Krenz et al., 1999 and pain (Romero et al., 2000). Strategies to block the actions of intraspinal NGF reduce these disabling secondary consequences of SCI Krenz et al., 1999, Marsh et al., 2002. In the absence of the high-affinity trkA neurotrophin receptor, effects of NGF mediated by the low-affinity p75NTR (neurotrophin receptor) induce apoptosis of oligodendrocytes, contributing to demyelination Casaccia-Bonnefil et al., 1996, Yoon et al., 1998. Furthermore, pro-NGF binds with high affinity to p75NTR, leading to apoptosis even in the presence of trkA receptors Beattie et al., 2002, Lee et al., 2001. To date, anti-NGF strategies using blocking antibodies Christensen and Hulsebosch, 1997, Gwak et al., 2003, Krenz et al., 1999 or TrkA-IgG fusion proteins (Marsh et al., 2002) have reduced autonomic dysfunction and pain after SCI. Complementing these studies, delivery of cellular and viral vector transplants engineered to express NGF Cameron et al., 2003, Romero et al., 2000, Tuszynski et al., 1996 has lead to sprouting of nociceptive sensory fibers, induction of hyperalgesia and augmented autonomic dysreflexia. On the other hand, direct NGF infusion can improve sensory neuron regeneration (Sayer et al., 2002). More precise control over NGF expression in the injured cord, by controlling its transcription and/or translation in particular cell types, should allow us to minimize its deleterious effects and perhaps to maximize its pro-regenerative effects. To accomplish this goal, the cellular sources of this neurotrophin after SCI must be ascertained.

In a previous study, we demonstrated immunoreactivity for NGF in cells within the injured rat spinal cord at 7 and 14 days after injury by complete transection (Krenz and Weaver, 2000). However, that study employed limited double-labeling methods, identifying with certainty only the presence of NGF in ramified microglia and its absence in oligodendrocytes. Since NGF could either be produced locally or imported, we sought to determine the source of NGF by carrying out RNA in situ hybridization studies in the injured spinal cord of rats at 1–7 days after SCI. Using double-labeling immunofluorescence methods, we also characterized the cells that expressed NGF protein. A transection model of SCI was used in the initial experiments as we have demonstrated robust immunoreactivity for NGF the spinal cord after transection injury (Krenz and Weaver, 2000). We also examined expression of NGF after clip-compression SCI as this model has neurological and histopathological characteristics that closely resemble the human injury Bruce et al., 2002, Weaver et al., 2001.

Section snippets

Surgical preparation of the rat spinal cord injury models

All protocols for these experiments were approved by the University of Western Ontario Animal Care Committee in accordance with the policies established by the Canadian Council on Animal Care. In total, 44 male and female adult Wistar rats (210–300 g) were used in this study. Control groups consisted of nine rats that did not undergo any surgery (unoperated) and six rats that had sham injury as described below. In 19 rats, the spinal cord was fully transected (transection-SCI) at the level of

NGF mRNA expression in uninjured and sham-injured rats

Little or no NGF mRNA was observed in the white matter of sham-injured (n = 6) or unoperated (n = 6) control rats (Figs. 2A, B). In contrast, NGF mRNA was clearly expressed in leptomeningeal cells and in cells within the intermediate grey matter (n = 10, Figs. 2A, B). Although these cells were present throughout the grey matter, they were mostly in lamina VII, and were characteristically round with diameters of at least 25 μm, suggestive of neurons. To confirm that they were neurons, serial

Discussion

To determine the cellular sources of NGF in the injured spinal cord, we compared NGF mRNA and protein expression patterns after SCI in the rat, using transection and clip-compression models of injury. Robust expression of NGF mRNA was present in the injured rat spinal cord within 3 days of injury and was maintained for at least 1 week. The intensity of NGF mRNA and protein expression was greatest within the lesion site and leptomeninges. In the white matter close to, or far from, the injury

Acknowledgements

This research was supported by grants from the Heart and Stroke Foundation of Ontario (T4053) and Canadian Institutes of Health Research. Dr. A. Brown is supported by a New Investigator Award from the Heart and Stroke Foundation of Canada. The anti-NGF antibody used in this study was kindly provided by Dr. James Conner, University of California, San Diego. We thank Dr. Canio Polosa for his critical evaluation of this work.

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