Neurovascular imaging

Laser speckle flowmetry (LSF) was initially developed to measure blood flow in the retina. More recently, its primary application has been to image baseline blood flow and activity-dependent changes in blood flow in the brain. We now describe experiments in the rat retina in which LSF was used in conjunction with confocal microscopy to monitor light-evoked changes in blood flow in retinal vessels. This dual imaging technique permitted us to stimulate retinal photoreceptors and measure vessel diameter with confocal microscopy while simultaneously monitoring blood flow with LSF. We found that a flickering light dilated retinal arterioles and evoked increases in retinal blood velocity with similar time courses. In addition, focal light stimulation evoked local increases in blood velocity. The spatial distribution of these increases depended on the location of the stimulus relative to retinal arterioles and venules. The results suggest that capillaries are largely unresponsive to local neuronal activity and that hemodynamic responses are mediated primarily by arterioles. The use of LSF to image retinal blood flow holds promise in elucidating the mechanisms mediating functional hyperemia in the retina and in characterizing changes in blood flow that occur during retinal pathology.

In the last few years two-photon microscopy has been used to perform in vivo high spatial resolution imaging of neurons, glial cells and vascular structures in the intact neocortex. Recently, in parallel to its applications in imaging, multi-photon absorption has been used as a tool for the selective disruption of neural processes and blood vessels in living animals. In this review we present some basic features of multi-photon nanosurgery and we illustrate the advantages offered by this novel methodology in neuroscience research. We show how the spatial localization of multi-photon excitation can be exploited to perform selective lesions on cortical neurons in living mice expressing fluorescent proteins. This methodology is applied to disrupt a single neuron without causing any visible collateral damage to the surrounding structures. The spatial precision of this method allows to dissect single processes as well as individual dendritic spines, preserving the structural integrity of the main neuronal arbor. The same approach can be used to breach the blood-brain barrier through a targeted photo-disruption of blood vessels walls. We show how the vascular system can be perturbed through laser ablation leading toward two different models of stroke: intravascular clot and extravasation. Following the temporal evolution of the injured system (either a neuron or a blood vessel) through time lapse in vivo imaging, the physiological response of the target structure and the rearrangement of the surrounding area can be characterized. Multi-photon nanosurgery in live brain represents a useful tool to produce different models of neurodegenerative disease.

Functional near infrared spectroscopy (fNIRS) is a portable monitor of cerebral hemodynamics with wide clinical potential. However, in fNIRS, the vascular signal from the brain is often obscured by vascular signals present in the scalp and skull. In this paper, we evaluate two methods for improving in vivo data from adult human subjects through the use of high-density diffuse optical tomography (DOT). First, we test whether we can extend superficial regression methods (which utilize the multiple source–detector pair separations) from sparse optode arrays to application with DOT imaging arrays. In order to accomplish this goal, we modify the method to remove physiological artifacts from deeper sampling channels using an average of shallow measurements. Second, DOT provides three-dimensional image reconstructions and should explicitly separate different tissue layers. We test whether DOT’s depth-sectioning can completely remove superficial physiological artifacts. Herein, we assess improvements in signal quality and reproducibility due to these methods using a well-characterized visual paradigm and our high-density DOT system. Both approaches remove noise from the data, resulting in cleaner imaging and more consistent hemodynamic responses. Additionally, the two methods act synergistically, with greater improvements when the approaches are used together.