Quantifying the effects of cerebral capillary flow disruptions with two photon microscopy

Abstract

The cerebral capillary network performs the critical role of delivering oxygen to tissue with a rich supply of oxygen bound red blood cells. Disruptions in this steady flow have been observed in healthy brains, but also at elevated levels in a number of disease models such as Alzheimer’s disease and stroke, both of which lack good treatment options. Reductions in these “stalls” in blood flow have been shown to improve cortical blood flow as well as disease outcomes in preclinical models. But the assessment of capillary stalling, and its potential as a therapeutic target, have been limited, in part due to limitations in technology. To address this, we develop and apply tools using two-photon microscopic imaging for improved estimation of stalling and its impact. We first utilize a custom built Bessel beam two photon microscope for extended depth of field imaging. We show that its high volumetric imaging rate allows for improved detection of capillary stalling events, and develop an algorithm for semi-automated analysis of stalls to increase analysis throughout and reliability. Next, we develop and validate a novel scanning strategy for estimation of red blood cell flow speed, for simultaneous estimation of capillary flow speeds across many capillaries. Our approach is easy to implement and readily adaptable to any researchers interested in capillary flow. Finally, we utilize phosphorescent lifetime imaging (PLIM) to measure changes in local oxygen around individual stalling events and demonstrate a rapid and consistent drop in oxygen as a result of stalled flow. Our measurements show that this drop likely extends to the local tissue, and in some cases reaches critically hypoxic levels