NCAM-mediated locomotor recovery from spinal cord contusion injury involves neuroprotection, axon regeneration, and synaptogenesis

https://doi.org/10.1016/j.neuint.2010.03.023Get rights and content

Abstract

The expression level of neural cell adhesion molecule (NCAM), which plays a critical role in pathways involving development and plasticity of the nervous system, changes markedly after spinal cord injury (SCI). However, the significance of NCAM-involved mechanisms in SCI remains elusive. The present study demonstrates that NCAM-deficient (ND) mice exhibited significantly poorer locomotor activity than wildtype (WT) littermates with the same injury intensity by the contusion model. To determine detailed contribution of NCAM, quantitative immunohistochemistry examination was performed on the injured spinal cord of 6 mm along the rostrocaudal axis in the animals for up to 5 weeks after SCI. Overall level of NCAM decreased initially in the lesion site but increased around the center of the injury thereafter. At acute stage, more apoptotic cells were found in the gray and white matter in ND mice. Between the two animal groups, no obvious difference in expression levels of GFAP (astrocytosis marker) and MBP (remyelination marker) was detected. However, diverse expression trends of NF200 (axon marker), GAP-43 (synaptogenesis indicator) and phosphorylated ERK (active signal molecule) were observed in the area encompassing the lesion site, and remarkable differences were illustrated between WT mice and ND littermates. Detailed analysis indicates that NCAM-mediated pathways may be involved in the activation of ERK at acute stages and bi-phasic upregulation of GAP-43 expression at acute and sub-acute stages after SCI to promote cell survival, outgrowth of regenerated axons, synaptogenesis, and function recovery.

Introduction

Spinal cord injury (SCI) causes serious loss of neural cells, tissue destruction and attenuation of neuronal function (reviewed by Kwon et al., 2004). A large variety of molecules are involved in the pathological developments. Some of them showed direct beneficial or detrimental effects on functional recovery (Profyris et al., 2004) but the mechanisms are poorly understood. From accumulated evidences it is known that spontaneous axonal growth occurs in experimental SCI animals (Okano et al., 2007, Hill et al., 2001). The axon outgrowth is believed to establish new circuits and contribute to the limited functional recovery (Bareyre et al., 2004, Bregman et al., 2002). Thus, elucidating influences of the activated neurotrophic factors on axon outgrow after SCI may provide clues to treatment.

Neural cell adhesion molecule (NCAM), which belongs to the immunoglobulin super-family, plays important roles in mediating cell migration, survival, neurite growth and synaptic plasticity (Rønn et al., 1998). There are three major isoforms of NCAM identified in most tissues with distinct molecular weight of 180, 140 and 120 kDa (Chuong and Edelman, 1984). All NCAM isoforms are transcribed from a single gene via mRNA alternative splicing mechanism (Santoni et al., 1987). Through homo- and heter-philic binding, NCAM associates with cell structural molecules and evokes several signaling cascades (Ditlevsen and Kolkova, 2008; Büttner et al., 2003). CAMP, Ca+, PKC and MAPK pathways play important roles in NCAM-stimulated neurite outgrowth (Maness and Schachner, 2007). Recent studies have revealed that NCAM actively participates in the regeneration of muscle (Dubois et al., 1994) and nervous system (Walsh and Doherty, 1996, Zhang et al., 2008). In completely transacted spinal cords, the expression level of NCAM was markedly changed (Tzeng et al., 2001), indicating its involvement in the pathological development after SCI. Moreover, polysialylated NCAM promotes selective targeting of regenerated motor neurons (Franz et al., 2005). These findings have aroused our interest to investigate the change in NCAM expression after SCI and its possible correlation with functional recovery of the spinal cord. NCAM-deficient (ND) mice and their wildtype (WT) littermates were employed in the present study of NYU weight-drop induced contusion spinal cord injury model. The locomotor ability of their hind limb was evaluated for up to 5 weeks after SCI. Apoptosis, astrocytosis, axon regeneration and synaptogenesis in the spinal cord were examined.

Section snippets

Surgical procedure and animal care

A total of 24 female B6.129P2-NCAM1tm1Cgn/J mice (ND) and 95 female wildtype (WT) littermates (Jackson Laboratory, Bar Harbor, ME, USA, 15–20 g) were used in this study. Animals were randomly selected for sham or injury groups. The New York University (NYU) model is favored in the present study because the lesions created seem to accurately mimic those seen in humans (Anderson and Stokes, 1992). Mice were anesthetized with pentobarbital (60 mg/kg) and their body temperatures were maintained by a

Expression and distribution of NCAM change dynamically following SCI

In the spinal cord, expression of NCAM was mainly demonstrated in the superficial dorsal horn of the sham animals (Fig. 1A), which is consistent with previous report (Seki and Arai, 1993). However, at 2 weeks after SCI, NCAM was highly expressed in gray and white matters of the injured spinal cord and hardly detectable in the scar area (Fig. 1A). To determine the overall NCAM expression in the damaged spinal cords, Western blot has been performed with spinal cord segment containing the injury

Discussion

Spinal cord injury was characterized by the drastic tissue destruction, cell death, scar formation and neural function loss. It has been widely accepted that the functional outcomes following SCI are determined by severity of the exogenous damage and capacity of the endogenerous recovery, which were attributed to the effects of tremendous number of factors (Profyris et al., 2004). Recent studies demonstrated that the endogenous function recovery is probably benefited from the limited axonal

Acknowledgments

We are grateful to Dr Yu Wei Ping in the National Neuroscience Institute of Singapore for his kind help with cryostat and thank Dr. Chew Sing Yian in the School of Chemical and Biomedical Engineering, Nanyang Technological University for critical reading the manuscript. This work was funded by Biomedical Research Council Grant (BMRC 06/1/33/19/479) of Singapore.

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