Elsevier

Neuroscience

Volume 82, Issue 1, 25 September 1997, Pages 21-32
Neuroscience

Mechanisms of calcium and sodium fluxes in anoxic myelinated central nervous system axons

https://doi.org/10.1016/S0306-4522(97)00230-3Get rights and content

Abstract

Electron probe X-ray microanalysis was used to measure water content and concentrations of elements (i.e. Na, K, Cl and Ca) in selected morphological compartments of rat optic nerve myelinated axons. Transaxolemmal movements of Na+ and Ca2+ were modified experimentally and corresponding effects on axon element and water compositions were determined under control conditions and following in vitro anoxic challenge. Also characterized were effects of modified ion transport on axon responses to postanoxia reoxygenation. Blockade of Na+ entry by tetrodotoxin (1 μM) or zero Na+/Li+-substituted perfusion reduced anoxic increases in axonal Na and Ca concentrations. Incubation with zero-Ca2+/EGTA perfusate prevented axoplasmic and mitochondrial Ca accumulation during anoxia but did not affect Na increases or K losses in these compartments. Inhibition of Na+–Ca2+ exchange with bepridil (30 μM) selectively prevented increases in intra-axonal Ca, whereas neither nifedipine (5 μM) nor nimodipine (5 μM) influenced the effects of anoxia on axonal Na, K or Ca. X-ray microanalysis also showed that prevention of Na and Ca influx during anoxia obtunded severe elemental deregulation normally associated with reoxygenation.

Results of the present study suggest that during anoxia, Na+ enters axons mainly through voltage-gated Na+ channels and that subsequent increases in axoplasmic Na+ are functionally coupled to extra-axonal Ca2+ import. Na+i-dependent, Ca2+o entry is consistent with reverse operation of the axolemmal Na+–Ca2+ exchanger and we suggest this route represents a primary mechanism of Ca2+ influx. Our findings also implicate a minor route of Ca2+ entry directly through Na+ channels.

Section snippets

In vitro incubation of rat optic nerve in modified artificial cerebrospinal fluid solutions

All aspects of this study were in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the local animal care committee. In the present studies, experimental procedures that modify transaxolemmal movements of Na+ and Ca2+ (e.g., perfusion with zero Na+ solutions or bepridil) were used to evaluate routes of Ca2+ entry during in vitro anoxia and subsequent reoxygenation. Long–Evans rats (Charles River, Montreal) aged 50–70 days were anaesthetized with 80% CO

Zero Na+/Li+-substituted perfusion

If influx of extraaxonal Na+ along its inwardly directed electrochemical gradient is functionally linked to entry of extra-axonal Ca2+ during anoxia, then removing external Na+ should prevent axonal accumulation of both Na and Ca. Therefore, isolated optic nerves were perfused with zero Na+/Li+-substituted medium during an anoxic challenge. Elemental composition was determined in axons exposed to zero Na+ solutions at the end of a 60 min anoxic period, and in reoxygenated fibres incubated for an

Discussion

In a previous study using electron probe X-ray microanalysis, we demonstrated that in vitro anoxic challenge of rat optic nerve caused an early derangement of elemental composition characterized by significant accumulation of Ca in axoplasm and mitochondria of myelinated axons.[29]Although substantial evidence strongly implicates a pivotal role for Ca2+ in the pathophysiology of anoxia-induced axon injury, the corresponding route of entry has not been conclusively identified. Pharmacological

Conclusions

The present study provides direct support for the following scenario of anoxia-induced CNS white matter injury:29, 43, 46during periods of oxygen deprivation and reduced aerobic energy production, myelinated axons lose K+ while intra-axonal Na+ concentrations increase following Na+ entry primarily through voltage-gated, TTX-sensitive channels. Elevated axoplasmic Na+ and axolemmal depolarization promote Ca2+ entry mediated primarily by reverse operation of the Na+–Ca2+ exchanger. Ca2+ influx

Acknowledgements

This research was made possible by an NIEHS grant RO1-ES03830 (R.M.L.) and by a grant from the Heart and Stroke Foundation of Ontario No. NA3201 (P.K.S.). The authors thank Dr Ellen Lehning for her insightful comments during the preparation of this manuscript.

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