Elsevier

Brain Research

Volume 1075, Issue 1, 23 February 2006, Pages 1-12
Brain Research

Research Report
Microarray analysis of gene expression patterns in adult spinal motoneurons after different types of axonal injuries

https://doi.org/10.1016/j.brainres.2005.12.060Get rights and content

Abstract

Three experimental models of axonal injuries in adult rat spinal motoneurons were established to investigate changes of gene expression in response to such injuries. We took advantage of cDNA microarray analysis to determine the differential expression of genes in injured motoneurons following distal axotomy or root avulsion in the absence or presence of BDNF. The major finding was that, in response to proximal axonal injury (avulsion), expression of genes that are known to facilitate neuronal survival and axonal regeneration (e.g., IGFRII, PI3K, IGFBP-6, GSTs, GalR2) were down-regulated; but following treatment with BDNF they were up-regulated. In addition, the expression of genes known to be involved in apoptosis and DNA damage (e.g., ANX5, TS, ALR) were down-regulated in BDNF-treated animals with avulsion. Furthermore, many functional families of genes previously shown to play roles in the pathophysiology of axonal injury, including SNAP-25A, SV2B, Ras-related ras3a/4b, ERK1/2, 14-3-3 proteins, proteasome proteins, oncogenes, GAP-43, and NMDAR1, were altered after either distal axotomy or avulsion injury. Some of the changes in gene expression, including Lim-2, FRAG1, GlaR2, GSTs, ALR, TS, ANX3/5, and nhe1/2, are first reported here in injured motoneurons. The differential expression of genes identified by the expression arrays was confirmed by gene-specific RT-PCR for eight genes (GAP-43, IGFR II, Lim-2, MIF, NDAP1, TS, PCC3, and FRAG1) and by in situ hybridization for Lim-2. These results suggest that abnormal regulation of particular biochemical pathways may induce motoneuron death after ventral root avulsion in adult animals. This study presents an approach for selecting specific genes and their products that may be involved in motoneuron degeneration following axonal injuries.

Introduction

Neurons may die or survive following axonal injury. Mechanisms underlying neuronal death are not clear. It has been suggested that injured neurons must express a program, presumably involving the sequential switching of genes, that either controls axonal regeneration or induces cell death (Fawcett, 1992). During the last decade, increasing evidence has indicated that specific gene expression is induced in neurons after axonal injury (Hu et al., 2002, Islamov et al., 2003, Jacobsson et al., 1996, Nakamura and Bregman, 2001, Tachibana et al., 2002, Wu, 1996, Zhao et al., 1998). These changes in gene expression can lead to the activation of different signaling pathways, which results in neuronal death or regeneration (Dolcet et al., 1999, Hu et al., 2002, Martin and Liu, 2002, Morrison et al., 2000). Previously, in a spinal root avulsion injury model, we found that up-regulation of neuronal nitric oxide synthase (nNOS) coincides with the death of spinal motoneurons (Wu et al., 1994a, Wu et al., 2003), while up-regulation of c-jun coincides with their regeneration (Wu, 1996, Wu et al., 1994a). Moreover, treatment with neurotrophic factors such as brain-derived neurotrophic factor (BDNF) alters gene expression and enhances survival and regeneration of motoneurons following root avulsion (Chai et al., 1999, Dolcet et al., 1999, Novikova et al., 1997). These studies suggest that regulation of gene expression plays an important role in the neuronal response to injury. Furthermore, these investigations have shown that the expression of genes can be manipulated, which is very important for the treatment of neuronal injury as well as neural degenerative diseases. However, a more comprehensive understanding of gene expression changes involved in neuronal degeneration and regeneration is required before viable therapies can be developed. The aim of the present study was to investigate how spinal motoneurons respond to axonal injury in terms of gene expression changes, and how these gene expression changes relate to neuronal death.

In the present study, three experimental models were established to investigate the gene expression changes in spinal motoneurons following axonal injury: distal axotomy, root avulsion and root avulsion followed by treatment with BDNF. It has been shown that distal axotomy does not cause motoneuron death in adult animals, but the lesion does cause intracellular molecular changes(Gu et al., 1997, Wu, 1996, Wu et al., 2003). Ventral root avulsion, in contrast, causes motoneuron death in adult animals (Li et al., 1995, Wu et al., 2003), while treatment with BDNF can rescue motoneurons in this model (Chai et al., 1999, Nakamura and Bregman, 2001, Wu et al., 1994b). Therefore, by taking advantage of cDNA microarray technology, the present study focused on global gene expression changes in adult spinal motoneurons following the three experimental manipulations.

Section snippets

Gene expression profiles

Understanding of the molecular mechanisms in response to motoneuron death and regeneration has long been a goal of neuroscience. One of these major efforts is identification of required proteins or specific molecular pathways on which motoneuron death and regeneration dependent in. High-density oligonucleotide microarray Atlas™ 1.2 Rat cDNA Expression Array provide the ability to measure gene expression of 1176 known genes simultaneously. Taking advantage of the microarray, Atlas™ 1.2 Rat cDNA

Discussion

In order to understand the molecular mechanisms of motoneuron death, cDNA expression arrays were employed to identify gene expression patterns in the ventral horn of spinal cord after three different types of axonal injury in adult rats. We found that 147 were differentially expressed at least 2-fold in ventral horns after injury in comparison to normal controls. These genes related to growth factors/cytokines, receptors/signal pathways, neurotransmitter receptors, transporters, release

Animal models

All procedures were approved by the committee on the use of live animals in teaching and research of the University of Hong Kong. Adult male Sprague–Dawley rats (250–300 g) were anesthetized with ketamine (80 mg/kg, i.m.) and xylazine (8 mg/kg, i.m.) and underwent one of the following experimental procedures (6 rats per group).

Acknowledgments

We express our gratitude to Prof. Nora Perrone-Bizzozero and Ann Githinji for their helpful comments and critical reading of the manuscript. This study was supported by grants from the University of Hong Kong, the Research Grant Council of Hong Kong and the National Key Basic Research Programme grant Of China (2003CB515303).

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