Microscopic Studies of Neurovascular Coupling During Epilepsy in the Mouse Brain

Neurovascular coupling (NVC) is the mechanism that links a transient neural activity to the corresponding increase of cerebral blood flow (CBF). It underlies the local increase in blood flow during neural activity, forms the basis of functional brain imaging and is altered in epilepsy. For the last decades, functional imaging using BOLD fMRI, IOS and fNIRS and others have been applied to epilepsy, and yielded good correlation between epileptic activity and the measured signal. However, most previous work on epilepsy focused on the measurement of hemoglobin changes which sometimes leads to non-linear phenomena and does not quantify oxygen delivery in tissue. The aim of this thesis is to study oxygen delivery using microscopy with new oxygen sensitive molecular probes during epileptic events in the mouse somatosensory cortex. First, a confocal phosphorescence lifetime microscopy system for measuring brain oxygen partial pressure (PO2) was developed. This system enabled minimally invasive measurements of oxygen partial pressure in cerebral tissue with high spatial and temporal resolution using a dendritic phosphorescent probe, Oxyphor G4. Significant changes of PO2 in tissue were found at the epileptic focus and in remote areas during 4-aminopyridine (4-AP) induced epilepsy. The spatio-temporal distribution of the “initial dip” in PO2 near the injection site and along nearby arterioles was characterized by investigating epileptic events. A positive correlation between the percent change in the PO2 signal during the “initial dip” and the duration of seizure-like activity was revealed in this work, which may help localize the epileptic focus and predict the length of seizures. Because astrocytic calcium signalling is involved in neurovascular coupling, the second study investigated the role of this pathway in epilepsy. The free calcium concentration in astrocytic endfeet and diameter of adjacent arterioles were simultaneously monitored with the calcium-sensitive indicator OGB-1 by two-photon fluorescence lifetime measurements following 4-AP injection. Our results revealed that, increases in calcium concentration induced vasodilation for each ictal event in the focus. In the remote area, increases in calcium concentration correlated with vasoconstriction at the onset of seizure and vasodilation during the later part of the seizures. Furthermore, a slow increase in absolute calcium concentration following multiple seizures was observed, which in turn, caused a trend of arteriolar constriction both at the epileptic focus and remote areas. These observations confirmed the role of astrocytes in the control of local microcirculation and suggest a modulating role for baseline absolute calcium concentration in astrocytic endfeet. Since the confocal phosphorescence microscopy system was not able to measure PO2 deep in the cortex or resolve capillaries, two-photon phosphorescence microscopy was then used in the last project to study the PO2 delivery during epilepsy in deep tissue and vessels. Significant changes of PO2 in tissue and vasculature were observed during epileptic events. The spatial landscape of “initial dip” in PO2 signals around arterioles, veins and tissue near the injection site was characterized. These results may have profound implications for evaluating microvascular oxygen delivery capacity to support cerebral tissue in disease. The results of this thesis confirmed the potential of using microscopy to study neurovascular coupling during epilepsy.