Elsevier

Neuroscience

Volume 94, Issue 4, November 1999, Pages 1219-1230
Neuroscience

Mechanisms of ischaemic damage to central white matter axons: a quantitative histological analysis using rat optic nerve

https://doi.org/10.1016/S0306-4522(99)00389-9Get rights and content

Abstract

The mechanism of ischaemic injury to white matter axons was studied by transiently depriving rat optic nerves in vitro of oxygen and glucose. Light and electron microscopic analysis showed that increasing periods of oxygen/glucose deprivation (up to 1 h) caused, after a 90-min recovery period, the appearance of increasing numbers of swollen axons whose ultrastructure indicated that they were irreversibly damaged. This conclusion was supported by experiments showing that the damage persisted after a longer recovery period (3 h). To quantify the axonal pathology, an automated morphometric method, based on measurement of the density of swollen axons, was developed. Omission of Ca2+ from the incubation solution during 1 h of oxygen/glucose deprivation (and for 15 min either side) completely prevented the axonopathy (assessed following 90 min recovery). Omission of Na+ was also effective, though less so (70% protection). The classical Na+ channel blocker, tetrodotoxin (1 μM), provided 92% protection. In view of this evidence implicating Na+ channels in the pathogenesis of the axonal damage, the effects of three different Na+ channel inhibitors, with known neuroprotective properties towards gray matter in in vivo models of cerebral ischaemia, were tested. The compounds used were lamotrigine and the structurally-related molecules, BW619C89 and BW1003C87. All three compounds protected the axons to varying degrees, the maximal efficacies (observed at 30 to 100 μM) being in the order: BW619C89 (>95% protection)>BW1003C87 (70%)>lamotrigine (50%). At a concentration affording near complete protection (100 μM), BW619C89 had no significant effect on the optic nerve compound action potential. Experiments in which BW619C89 was added at different times indicated that its effects were exerted during two distinct phases, one (accounting for about 50% protection) was during the early stage of oxygen/glucose deprivation itself and the other (also about 50%) during the first 15 min of recovery in normal incubation solution.

The results are consistent with a pathophysiological mechanism in which Na+ entry through tetrodotoxin-sensitive Na+ channels contributes to Na+ loading of the axoplasm which then results in a lethal Ca2+ overload through reversed Na+–Ca2+ exchange. The identification of BW619C89 as a compound able to prevent oxygen/glucose deprivation-induced injury to white matter axons without affecting normal nerve function opens the way to testing the importance of this pathway in white matter injury in vivo.

Section snippets

Optic nerves

Optic nerves were excised from adult Wistar rats (weighing 240 to 280 g) after decapitation. They comprised lengths of nerve (each about 9 mm long) running from immediately behind the eyeball to just in front of the optic chiasm. The nerves were incubated in Erlenmeyer flasks (50 ml capacity) containing 20 ml of an artificial cerebrospinal fluid (ACSF) solution composed of (mM): NaCl (120) KCl (2.0), CaCl2 (2.0), NaHCO3 (26), KH2PO4 (1.18), MgSO4 (1.19) and glucose (11), continuously gassed with

Light and electron microscopic observations

Rat optic nerves incubated under control conditions in vitro for at least 5 h showed very good preservation, in both the light (Fig. 1a) and electron microscopes (Fig. 2a). No major differences were discernible compared with nerves fixed in situ (see also Ref. 67).

Nerves were exposed to OGD for different periods of time (30, 45 and 60 min) followed, in each case, by 90 min of recovery in normal ACSF. This resulted in a graded axonopathy with the 60-min exposure causing the most severe effects, as

Methodological considerations

The primary method used to assess the integrity of optic nerve axons was histology combined with a quantitative morphometric analysis of the dimensions of axonal cross-sectional profiles. This is logical in view of abundant previous evidence that axonal swelling characterizes irreversible axonal pathology following metabolic inhibition.67 That the damage was irreversible in our experiments is indicated both by the severe disruption of the axonal cytoskeleton and mitochondria and by the finding

Conclusions

In the present study, OGD-induced axonopathy in isolated optic nerve was dependent on extracellular Ca2+ and Na+, and could be inhibited most effectively by TTX and BW619C89, and to a lesser degree, by lamotrigine and BW1003C87. The findings are consistent with a mechanism in which metabolic compromise results in a loading of the axoplasm with Na+ through non-inactivating, voltage-dependent Na+ channels and, subsequently, to reversed operation of the Na+–Ca2+ exchanger. Moreover, it is shown

Acknowledgements

This research was supported by The Wellcome Trust.

References (72)

  • K. Morimoto et al.

    BW1003C87, phenytoin and carbamazepine elevate seizure threshold in the rat amygdala-kindling model of epilepsy

    Eur. J. Pharmac.

    (1997)
  • S.E. Smith et al.

    Excitatory amino acid antagonists, lamotrigine and BW 1003C87 as anticonvulsants in the genetically epilepsy-prone rat

    Epilepsy Res.

    (1993)
  • S.E. Smith et al.

    Long-term beneficial effects of BW619C89 on neurological deficit, cognitive deficit and brain damage after middle cerebral artery occlusion in the rat

    Neuroscience

    (1997)
  • A. Stefani et al.

    Lamotrigine inhibits Ca2+ currents in cortical neurons: functional implications

    Eur. J. Pharmac.

    (1996)
  • I. Steffensen et al.

    Immunolocalization of the Na+–Ca2+ exchanger in mammalian myelinated axons

    Brain Res.

    (1997)
  • P.K. Stys et al.

    Elemental composition and water content of rat optic nerve myelinated axons during in vitro post-anoxia reoxygenation

    Neuroscience

    (1996)
  • P.K. Stys et al.

    Effects of polyvalent cations and dihydropyridine calcium channel blockers on recovery of CNS white matter from anoxia

    Neurosci. Lett.

    (1990)
  • P.K. Stys et al.

    Compound action potential of nerve recorded by suction electrode: a theoretical and experimental analysis

    Brain Res.

    (1991)
  • F.Y. Sun et al.

    Neuroprotective effects of 619C89, a use-dependent sodium channel blocker, in rat traumatic brain injury

    Brain Res.

    (1995)
  • C.P. Taylor et al.

    Na+ channels as targets for neuroprotective drugs

    Trends pharmac. Sci.

    (1995)
  • S.G. Waxman et al.

    Ultrastructural concomitants of anoxic injury and early post-anoxic recovery in rat optic nerve

    Brain Res.

    (1992)
  • X.M. Xie et al.

    State-dependent inhibition of Na+ currents by the neuroprotective agent 619C89 in rat hippocampal neurons and in a mammalian cell line expressing rat brain type IIA Na+ channels

    Neuroscience

    (1996)
  • S.K. Agrawal et al.

    Mechanisms of secondary injury to spinal cord axons in vitro: role of Na+, Na+–K+–ATPase, the Na+–H+ exchanger, and the Na+–Ca2+ exchanger

    J. Neurosci.

    (1996)
  • O. Almkvist et al.

    White-matter hyperintensity and neuropsychological functions in dementia and healthy aging

    Archs Neurol.

    (1992)
  • J. Boiten et al.

    Lacunar infarcts. Pathogenesis and validity of the clinical syndromes

    Stroke

    (1991)
  • D.W. Choi et al.

    The role of glutamate neurotoxicity in hypoxic-ischemic neuronal death

    A. Rev. Neurosci.

    (1990)
  • R.C. Crumrine et al.

    Lamotrigine protects hippocampal CA1 neurons from ischemic damage after cardiac arrest

    Stroke

    (1997)
  • J. De Juan et al.

    Number, diameter and distribution of the rat optic nerve fibers

    Acta anat. basel

    (1978)
  • A.S. Eriksson et al.

    Pharmacokinetic interactions between lamotrigine and other antiepileptic drugs in children with intractable epilepsy

    Epilepsia

    (1996)
  • R. Fern et al.

    Axon conduction and survival in CNS white matter during energy deprivation: a developmental study

    J. Neurophysiol.

    (1998)
  • R. Fern et al.

    Pharmacological protection of CNS white matter during anoxia: actions of phenytoin, carbamazepine and diazepam

    J. Pharmac. exp. Ther.

    (1993)
  • R. Fern et al.

    Voltage-gated calcium channels in CNS white matter: role in anoxic injury

    J. Neurophysiol.

    (1995)
  • C.M. Fisher

    Lacunar strokes and infarcts: a review

    Neurology

    (1982)
  • F. Follis et al.

    Selective vulnerability of white matter during spinal cord ischemia

    J. cerebr. Blood Flow Metab.

    (1993)
  • J. Forrester et al.

    Nerve fibres in optic nerve of rat

    Nature

    (1967)
  • Garthwaite G., Goodwin D. A. and Garthwaite J. (1999) Nitric oxide stimulates cGMP formation in rat optic nerve axons,...
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