Elsevier

Neuroscience

Volume 103, Issue 1, 28 February 2001, Pages 203-218
Neuroscience

Oligodendroglial apoptosis occurs along degenerating axons and is associated with FAS and p75 expression following spinal cord injury in the rat

https://doi.org/10.1016/S0306-4522(00)00538-8Get rights and content

Abstract

Apoptosis or programmed cell death has been reported after CNS trauma. However, the significance of this mechanism in the pathophysiology of spinal cord injury, in particular at the cervical level, requires further investigation. In the present study, we used the extradural clip compression model in the rat to examine the cellular distribution of apoptosis following cervical spinal cord injury, the relationship between glial apoptosis and post-traumatic axonal degeneration and the possible role of apo[apoptosis]-1, CD95 (FAS) and p75 in initiating post-traumatic glial apoptosis. In situ terminal-deoxy-transferase mediated dUTP nick end labeling revealed apoptotic cells, largely oligodendrocytes as identified by cell specific markers, in grey and white matter following spinal cord injury. Apoptotic cell death was confirmed using electron microscopy and by the demonstration of DNA laddering on agarose gel electrophoresis. β-Amyloid precursor protein was used as a molecular marker of axonal degeneration on western blots and immunohistochemistry. Degeneration of axons was temporally and spatially co-localized with glial apoptosis. FAS and p75 protein expression was seen in astrocytes, oligodendrocytes and microglia, and was also seen in some apoptotic glia after cord injury. Both FAS and p75 increased in expression in a temporal course, which mirrored the development of cellular apoptosis. The downstream caspases 3 and 8, which are linked to FAS and p75, demonstrated activation at times of maximal apoptosis, while FLIP-L an inhibitor of caspase 8, decreased at times of maximal apoptosis.

We conclude that axonal degeneration after traumatic spinal cord injury is associated with glial, in particular oligodendroglial, apoptosis. Activation of the FAS and p75 death receptor pathways may be involved in initiating this process.

Section snippets

Spinal cord injury

All experimental protocols of this study were approved by the animal care committee of the Toronto Western Hospital Research Institute in accordance with the policies established in the guide to the care and use of experimental animals prepared by the Canadian Council of Animal Care. All efforts were made to minimize the number of animals used and their suffering.

SCI at C7–T1 was performed using the clip compression model in adult female Wistar rats (220–260 g; Charles River Laboratories,

DNA electrophoresis

To demonstrate the occurrence of internucleosomal DNA fragmentation characteristic of apoptotic cell degeneration, we electrophoresed 35S-labeled DNA isolated from normal and injured spinal cord in 1.5% agarose (Fig. 1). DNA isolated from injured tissue exhibited prominent banding of degraded DNA at lower molecular weights, (a characteristic of apoptosis resulting from internucleosomal DNA fragmentation), with a concomitant non-banding smear of DNA typical of necrotic degeneration. DNA from

Discussion

In this study we show using agarose electrophoresis, TUNEL and electron microscopy that apoptosis occurs following cervical SCI in the rat clip compression model. To date, this work is the first to demonstrate apoptosis in an animal model of cervical SCI, the most commonly injured level in humans.70 Ultrastructural data and double labeling immunomicroscopy revealed that most of the apoptotic cells are oligodendrocytes, and that these cells are spatially associated with degenerating axons. This

Acknowledgements

This work was supported by a Premier's Research Excellence Award Grant, Medical Research Council/Canadian Neurotrauma Research Program Grant and a Cervical Spine Research Society Grant. S. Casha is supported by an Ontario Neurotrauma Foundation Fellowship Award. M. G. Fehlings is the recipient of an Ontario Ministry of Health Career Scientist Award.

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