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