Multi-modal imaging of anoxic depolarization and hemodynamic changes induced by cardiac arrest in the rat cerebral cortex

We have reported previously that, in otherwise physiological conditions, spreading depression (SD) can be visualized directly by using a fluorescent, voltage-sensitive (VS) dye. However, in stroke models, where depolarizations occur spontaneously near the ischemic core, marked hemodynamic changes interfere significantly with VS dye imaging. This study provides the scientific basis necessary for accurate interpretation of VS dye images captured from ischemic brains. Using two cameras and carefully selected illuminations, multiple image sequences of the cortex were captured through a cranial window during cardiac arrest and subsequent anoxic depolarization (AD). This multi-modal strategy, used in anesthetized rats, allowed the study of synchronous changes in the following variables: (i) membrane potential (VS dye method); (ii) cerebral blood volume (CBV) with green (540-550 nm) illumination; (iii) hemoglobin (Hb) deoxygenation with red (620-640 nm) illumination, and cerebral blood flow (CBF) by laser speckle contrast imaging. Careful analysis of the data and their relationship revealed two important points: (i) as long as hemoglobin deoxygenation is not too pronounced, vascular changes interfere little with VS dye signals; (ii) in contrast, when the local, blood oxygen carrying capacity is close to exhaustion, higher absorption of both red light excitation and VS dye emission by deoxy-Hb, results in marked decreases of VS dye signals. Multiple, synchronous imaging of cellular depolarization, CBF, CBV and Hb deoxygenation is required for reliable data interpretation - but this combination is a powerful tool to examine the coupling between membrane potential and hemodynamic changes, with high spatial and temporal resolution.