Characterisation of hypoglycaemic white matter injury : an electrophysiological, pharmacological and imaging study in the rodent brain

Abstract

Failure of white matter energy metabolism plays a major role in several neurological disorders. Over the years, several studies have focused on mechanisms of neuronal injury and death in the absence of sufficient substrate for oxidative metabolism. In so doing, a gap in the knowledge to central mechanisms of damage that are specific to white matter has been overlooked. In this study, we used the callosal brain slice from mice as a model of white matter so as to characterise the role of glucose deprivation (GD) and to uncover potential routes of injury for pharmacological intervention. Axonal loss or recovery of function was determined electrophysiologically by monitoring the evoked compound action potential (CAP). This technique was carefully combined with live twophoton microscopy to follow the sequential progression or recovery from injury during pharmacological block of key cellular events. Detailed histological assessment was determined through a combination of confocal, light and electron microscopy techniques. Results from this study show that 45 min of GD cause delayed structural disruption to YFP-expressing axons, that was preceded by the functional loss in electrical activity as observed by the loss in maintenance of the CAP. We therefore extended this time-window by stimulation of the glycogenolytic pathway through β-adrenergic afferents using clenbuterol. This protection was not mediated by enhanced lactate production as expected, but possibly mediated through the steroid’s anti-inflammatory and antioxidative properties. Inhibition of axonal lactate uptake by 4-CIN was associated with severe axonal injury, thus confirming the role of lactate as a central energy substrate for axons. In vivo microdialysis from mouse cerebellar white matter confirmed the continuous uptake of lactate by axons during normal brain physiology. Excitotoxicity has been shown to play a central role in hypoglycaemic white matter injury. Blocking AMPA/Kainate receptors with NBQX protects both axons and oligodendrocytes after GD as was seen through live-imaging and electron microscopy. Targeting AMPA/Kainate receptors might be a good strategy to preserve the integrity of oligodendrocytes and axons. Combined therapy involving the specific GluN2C/D-containing NMDA receptor antagonist (QNZ-46) and the non-competitive AMPAspecific receptor antagonist (CP-465,022) show excellent preservation of axon integrity and function following GD. Both drugs were able to cross the blood-brain-barrier, were non-toxic, and their presence confirmed through imaging as a result of their intrinsic fluorescence. This, together with the use-dependent features of QNZ-46 makes these drugs ideal candidates for therapeutic intervention in disorders that preferentially affect white matter. We further show that bath application of callosal axons exposed to micromolar levels of nitric oxide (NO) cause irreversible conduction block. Sequential live imaging of NO using fluorescent dyes during the course of GD shows that its increase preceded the loss in CAP. At the same time, blocking NO release during GD with L-NAME partially protects axons and suggests that this strategy can be a useful therapeutic target. It is to be determined whether QNZ-46 can also mitigate the block in NO by limiting the entry of Ca+ by way of the use-dependent activity of this drug. The findings from this study suggest a multimodal prophylactic therapy to protect patients that frequently suffer from hypoglycaemia and other forms of excitotoxic insults.