A morphological analysis of the motor neuron degeneration and microglial reaction in acute and chronic in vivo aluminum chloride neurotoxicity

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Abstract

The monthly intracisternal inoculation of aluminum chloride (AlCl3) to young adult New Zealand white rabbits induces motor neuron degeneration marked by intraneuronal neurofilamentous aggregates similar to that observed in amyotrophic lateral sclerosis (ALS). However, in contrast to ALS, this process occurs in the experimental paradigm in the absence of a glial response. In addition, whereas ALS is a fatal disorder, the cessation of aluminum exposure leads to both clinical and neuropathological recovery. Because microglia can influence neuronal regeneration, we have examined the effect of both acute and chronic aluminum exposure on microglial activation in vivo. We have studied microglial morphology in young adult New Zealand white rabbits receiving either single (1000 μg) or repeated sublethal (100 μg monthly) intracisternal inoculums of AlCl3. In addition, rabbits receiving 1000 μg AlCl3 inoculums were studied following an unilateral sciatic axotomy 48 h prior to the AlCl3 exposure. Our studies demonstrate that microglial activation in vivo is inhibited by AlCl3 exposure, and that a correlation exists between the extent of microglia suppression and the potential for recovery. This suggests that microglial activation is an important determinant of neuronal injury.

Introduction

In amyotrophic lateral sclerosis (ALS), the degeneration of upper and lower motoneurons results in a clinical syndrome of progressive weakness, culminating in respiratory failure and death. There are three variants of ALS, including a classical sporadic variant, a familial variant (accounting for 5–10% of ALS), and a western Pacific variant. The cause of ALS is unknown, although mutations in copper/zine superoxide dismutase (SOD-1) have been observed in approximately 15% of familial ALS cases (Rosen et al., 1993, Siddique and Deng, 1996). The unifying link amongst these three variants is the selective degeneration of motor neurons, often accompanied by the development of both neurofilamentous aggregates and ubiquitinated inclusions. In addition to this, microglial activation has been recognized in regions of neuronal degeneration, including the motor cortex, nuclei of motoneurons and ventral horn of the spinal cord (McGeer et al., 1993). The observation of significant microglial activation in transgenic mice harboring SOD-1 mutations lends support to the hypothesis that microglia may play a key role in this degenerative process (Hall et al., 1998). In this study, we have examined the role of microglia in an experimental model of motor neuron degeneration possessing many of the morphological and ultrastructural features of ALS (Wakayama et al., 1996).

Microglia, central nervous system resident macrophages, undergo profound functional changes in response to pathogenic stimuli, including activation from resting ramified microglia to amoebic, proliferating macrophages capable of participating in neuronal injury. It has been thought that microglial activation could be involved in mediating local immune responses in the CNS and in a number of neurodegenerative processes (Davis et al., 1994). However, these responses may be either beneficial or harmful (Kreutzberg, 1996). Morphologically, reactive microglia demonstrate enlarged cytoplasm with shortened and thickened processes (McGeer et al., 1993). At the same time, reactive microglia in ALS upregulate the immunoglobulin receptor Fc gamma R1, the complement receptor type 3 and 4, the class II major histocompatibility complex molecules HLA–DR, HLA–DP and HLA–DQ and common determinants of the class I HLA–A,B,C complex (Kawamata et al., 1992). In addition to this upregulation of cell surface markers, activated microglia release several secretary products, including proteinases, cytokines, reactive oxygen intermediates, and reactive nitrogen intermediates (Banati et al., 1993).

We have previously shown that the acute neurotoxicity of AlCl3 to spinal motoneurons in young adult New Zealand white rabbits is markedly enhanced by a sciatic axotomy 48 h preceding aluminum exposure (Strong and Gaytan-Garcia, 1996). Although the mechanism of the induction of this pathology is unclear, a prominent microglial proliferative response and activation occurs in response to axotomy (Streit et al., 1988). In a model of chronic AlCl3 neurotoxicity, induced by repeated monthly intracisternal inoculations of AlCl3, many of the clinical, histological and ultrastructural characteristics of ALS are reproduced (Strong and Garruto, 1991a, Wakayama et al., 1996). However, in contrast to ALS, these clinical and neuropathological features are reversible upon cessation of AlCl3 exposure, and there is a failure to induce a glial response (Strong et al., 1995). Because microglia can participate directly in neuronal injury and recovery (Thanos et al., 1993), we have now examined the relationship between the extent of neurofilamentous degeneration induced by acute AlCl3 exposure, the potential for recovery following chronic aluminum exposure, and the microglial response to the induction of neuronal pathology. Microglia were identified on the basis of lectin labeling (Sasaki et al., 1991) and morphological characteristics.

Section snippets

Animals and inoculation

All procedures were performed in accordance with the Canadian Council on Animal Care Guidelines. In total, 52 New Zealand white rabbits (Don Riemans Fur Ranch, Ste. Agathe, Ontario), at the age of 5–6 weeks, were used in the experiments.

Acute-axotomy model

Fig. 1 shows the number of microglia in the ventrolateral region of the lumbar cord section. The number of microglia increased significantly in the ventrolateral area ipsilateral to the axotomy at day 4 post-axotomy in both AlCl3- and NaCl-inoculated groups, compared with that of the contralateral side. However, the number of microglia present ipsilateral to the axotomy was significantly less following AlCl3 inoculation than following NaCl-inoculation (P<0.05). There was no statistical

Discussion

Motoneurons are uniquely sensitive to the toxicity of AlCl3 both in vivo and in vitro (Strong and Garruto, 1991b, Strong et al., 1995). In vivo, following the inoculation of either organic or inorganic aluminum compounds, affected neurons develop neurofilamentous aggregates in the neuronal soma, axons and dendrites. Acute high dose AlCl3 exposure induces widespread neurofibrillary tangle-like perikaryal inclusions throughout the CNS (Bugiani and Ghetti, 1982), and culminates in a fulminant

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