Direct, Live Imaging of Cortical Spreading Depression and Anoxic Depolarisation Using a Fluorescent, Voltage-Sensitive Dye

Perilesion depolarisations, whether transient anoxic depolarisation (AD) or spreading depression (SD), occur in stroke models and in patients with acute brain ischaemia, but their contribution to lesion progression remains unclear. As these phenomena correspond to waves of cellular depolarisation, we have developed a technique for their live imaging with a fluorescent voltage-sensitive (VS) dye (RH-1838). Method development and validation were performed in two different preparations: chicken retina, to avoid any vascular interference; and cranial window exposing the cortical surface of anaesthetised rats. Spreading depression was produced by high-K medium, and AD by complete terminal ischaemia in rats. After dye loading, the preparation was illuminated at its excitation wavelength and fluorescence changes were recorded sequentially with a charge-coupled device camera. No light was recorded when the VS dye was omitted, ruling out the contribution of any endogenous fluorophore. With both preparations, the changes in VS dye fluorescence with SD were analogous to those of the DC (direct current) potential recorded with glass electrodes. Although some blood quenching of the emitted light was identified, the VS dye signatures of SD had a good signal-to-noise ratio and were reproducible. The changes in VS dye fluorescence associated with AD were more complex because of additional interferents, especially transient brain swelling with subsequent shrinkage. However, the kinetics of the AD-associated changes in VS dye fluorescence was also analogous to that of the DC potential. In conclusion, this method provides the imaging equivalent of electrical extracellular DC potential recording, with the SD and AD negative shifts translating directly to fluorescence increase.