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Two photon microscopy and optical coherence tomography (OCT) are two standard methods for measuring flow speeds of red blood cells in microvessels, particularly in animal models. However, traditional two photon microscopy lacks the depth of field to adequately capture the full volumetric complexity of the cerebral microvasculature and OCT lacks the specificity offered by fluorescent labeling. In addition, the traditional raster scanning technique utilized in both modalities requires a balance of image frame rate and field of view, which severely limits the study of RBC velocities in the microvascular network. Here, we overcome this by using a custom two photon system with an axicon based Bessel beam to obtain volumetric images of the microvascular network with fluorescent specificity. We combine this with a novel scan pattern that generates pairs of frames with short time delay sufficient for tracking red blood cell flow in capillaries. We track RBC flow speeds in 10 or more capillaries simultaneously at 1 Hz in a 237 µm × 237 µm × 120 µm volume and quantified both their spatial and temporal variability in speed. We also demonstrate the ability to track flow speed changes around stalls in capillary flow and measure to 300 µm in depth. [ABSTRACT FROM AUTHOR]

Ever since the introduction of thrombolysis and the subsequent expansion of endovascular treatments for acute ischemic stroke, it remains to be identified why the actual outcomes are less favorable despite recanalization. Here, by high spatio-temporal resolution imaging of capillary circulation in mice, we introduce the pathological phenomenon of dynamic flow stalls in cerebral capillaries, occurring persistently in salvageable penumbra after reperfusion. These stalls, which are different from permanent cellular plugs of no-reflow, were temporarily and repetitively occurring in the capillary network, impairing the overall circulation like small focal traffic jams. In vivo microscopy in the ischemic penumbra revealed leukocytes traveling slowly through capillary lumen or getting stuck, while red blood cell flow was being disturbed in the neighboring segments under reperfused conditions. Stall dynamics could be modulated, by injection of an anti-Ly6G antibody specifically targeting neutrophils. Decreased number and duration of stalls were associated with improvement in penumbral blood flow within 2–24 h after reperfusion along with increased capillary oxygenation, decreased cellular damage and improved functional outcome. Thereby, dynamic microcirculatory stall phenomenon can be a contributing factor to ongoing penumbral injury and is a potential hyperacute mechanism adding on previous observations of detrimental effects of activated neutrophils in ischemic stroke. [ABSTRACT FROM AUTHOR]

Optical coherence tomography (OCT) allows label-free imaging of red blood cell (RBC) flux within capillaries with high spatio-temporal resolution. In this study, we utilized time-series OCT-angiography to demonstrate interruptions in capillary RBC flux in mouse brain in vivo. We noticed ∼7.5% of ∼200 capillaries had at least one stall in awake mice with chronic windows during a 9-min recording. At any instant, ∼0.45% of capillaries were stalled. Average stall duration was ∼15 s but could last over 1 min. Stalls were more frequent and longer lasting in acute window preparations. Further, isoflurane anesthesia in chronic preparations caused an increase in the number of stalls. In repeated imaging, the same segments had a tendency to stall again over a period of one month. In awake animals, functional stimulation decreased the observance of stalling events. Stalling segments were located distally, away from the first couple of arteriolar-side capillary branches and their average RBC and plasma velocities were lower than nonstalling capillaries within the same region. This first systematic analysis of capillary RBC stalls in the brain, enabled by rapid and continuous volumetric imaging of capillaries with OCT-angiography, will lead to future investigations of the potential role of stalling events in cerebral pathologies. [ABSTRACT FROM AUTHOR]