Neuroimaging & Neurovascular Coupling
Using light, we are able to image the living brain in a number of different ways to be able to visualize both the neuronal and hemodynamic behavior of the brain at ensemble and cellular levels. Optical neuroimaging is a major focus of our lab, and in particular, we are seeking to use our imaging systems to better understand neurovascular coupling and cerebral metabolism.
6) Grand Unified Theory - Endothelium!
What is Neurovascular Coupling?
A local increase in cortical blood flow accompanies almost all neuronal responses to stimulus in the brain. This hemodynamic response is the basis of functional magnetic resonance imaging (fMRI). However, surprisingly little is understood about the interrelation between blood flow and the neuronal activity that underlies it. It would be of significant value to be able to interpret clinical fMRI data in terms of neuronal function. Yet more importantly, normal functioning of the brain seems to depend critically on the integrity of neurovascular coupling, so understanding the mechanistic basis of this coupling could yield therapeutic targets for a range of pathologies including Alzheimer's and age related neurodegeneration.
Studying neurovascular coupling is complex owing to the need for both neuronal interconnectivity and vascular networks to be intact. In-vivo imaging allows the complete neurovascular system to be observed, perturbed and directly linked to clinical measures such as fMRI. However in-vivo imaging presents significant challenges in terms of experimental paradigm and instrument design. Advanced imaging techniques are required to capture the behavior of the brain in real time, measuring both vascular and neuronal parameters in parallel, and probing beneath the surface of the cortex to resolve 3D and layer-specific interactions. We utilize all of our imaging tools described here to investigate neurovascular coupling in the living brain.
Characterizing the vascular compartments and timing of cortical hemodynamics
Our high-resolution, high-speed multispectral optical imaging techniques give us a unique view of the brain. While the fMRI response is typically observed at frame rates of less than 1Hz, and with voxel sizes exceeding 2-3mm, we can resolve the behaviors of individual vessels, informing the interpretation of the ensemble signal detected by fMRI. Multi-spectral optical intrinsic signal imaging (MS-OISI) can also distinguish between changes in oxy-, deoxy- and total hemoglobin (HbO, HbR and HbT). The fMRI blood oxygen level dependent (BOLD) signal predominantly shows only changes in HbR, which can make it difficult to disambiguate changes in total hemoglobin from changes in oxygenation.
Extending the above studies, we then sought to answer whether arteriolar dilation precedes or follows changes in the capillary bed, and whether we could resolve whether arteriolar dilation propagates and if so, in which direction (retrograde or anteriograde). Using statistical methods to determine the very earliest time of ‘response onset’ across the responding region imaged using MS-OISI we found that HbT changes in the parenchyma originating from the capillary bed precede dilation of pial arterioles by a few hundred milliseconds. We observed high speed (2-4 mm/s) retrograde propagation of vasodilation in the pial arteries feeding the responding region, and found that this dilation spread independent of the direction of blood flow in the vessels. Dilations over 1mm away were seen within 400 milliseconds of stimulus onset. We also evaluated the dynamics of the return to baseline, and found markedly different spatiotemporal characteristics, suggesting the interplay of two processes; one dilatory and one driving constriction over different spatiotemporal scales. Chen et al, 2011. These results provide benchmark characteristics of the hemodynamic response to give a better framework in which to evaluate different candidate cellular mechanisms for neurovascular control.
Characterizing the in-vivo morphology of perivascular cells
Astrocytes possess many properties that make them attractive candidates for involvement in neurovascular coupling. They can sense the presence of local glutamate release via their metabotropic glutamate receptors, and can release a range of vasoactive products including prostaglandins and potassium ions. They are also well documented to be present around vessels in the brain. Astrocytes have been shown to exhibit transient increases in intracellular calcium during stimulation, and photolysis-induced changes in intracellular calcium have been shown to correlate with dilation of local diving arterioles (See Takano et al, 2005 and Wang et al, 2006). However, the morphology of astrocyte connectivity with the cortical vasculature can be difficult to evaluate using ex-vivo histology, where blood vessels are not perfused and the pial surface is rarely intact or considered. We therefore undertook to survey the relative interactions between astrocytes and their endfeet and the different vascular compartments of the in-vivo cortical vasculature. Astrocytes were labeled with sulforhodamine 101 (SR101) and the vasculature was loaded with FITC dextran. Extensive 3D analysis revealed that astrocytes enstheath all sub-pial vessels; diving arterioles, capillaries and ascending veins. There is little difference between this sheathing except that more astrocyte cell bodies, on average, are present on diving arterioles. The extensiveness and continuity of this sheathing suggests that it could act as a conduit for signal transduction. Sheathing of diving veins was seen to extend from the glia limitans superficialis with no SR101 staining seen on pial veins. On diving arterioles, SR101 staining was observed, but was seen as an inner sheath, with a second external sheath of astrocyte processes joining arterioles as they dive through the glia limitans.
Immunohistochemistry confirmed that the inner staining of the pial and diving arterioles corresponds to uptake of SR101 into arterial endothelial cells, and does not indicate the presence of astrocytes. This endothelial uptake was not seen in veins. Figure 4 below shows further results from this study where the depth-dependent density of astrocyte cell bodies and microvasculature were compared and found to exhibit little correlation. However, despite the substantial variations in relative density, astrocytes were found to be located at relatively consistent distances from one another as a function of depth (averaging ~ 6 microns). See McCaslin et al, 2011.
In-vivo imaging of intracellular calcium dynamicsIn ongoing studies, we are using dynamic in-vivo two-photon microscopy of calcium sensitive dyes to map the neuronal and astrocytic responses to somatosensory stimulation. By establishing accurate spatiotemporal assessment of their responsivities, we hope to clarify the role of astrocytes in neurovascular coupling. An example of this approach is shown below in Figure 5 which shows wide-field MS-OISI imaging of the exposed cortex after multiple intracortical injections of Oregon Green 499 BAPTA-1 AM calcium sensitive dye. After observing the locations of both the hemodynamic and neuronal responses, we can use two-photon microscopy in the same animal to zoom in an evaluate the responses of individual cells at different depths.
In collaboration with Aniruddha Das and Yevgeniy Sirotin, we completed a study using MS-OISI data acquired on the exposed visual cortex of awake behaving primates to demonstrate an important confound in conventional optical intrinsic signal imaging (OISI). In conventional OISI, many investigators use only a single wavelength of light to image the cortex, typically between 610-630nm. It is expected that this wavelength is predominantly sensitive to changes in deoxy-hemoglobin (HbR) since only oxy-hemoglobin (HbO) only contributes a small amount to absorption at these wavelengths (see Figure 6 below). However, data acquired at these ‘oximetric’ wavelengths typically has a biphasic shape, initially showing a decrease in detected light, followed by an increase. Simple interpretation of this pattern would suggest that HbR levels are initially increasing (termed the ‘initial dip’) and then subsequently decrease – and this pattern has been widely interpreted as measuring an initial, local increase in oxygen consumption (converting HbO to HbR) followed by a wash-in effect due to active local increases in blood flow and therefore HbT.
Grand Unified Theory - Endothelium!
We recently demonstrated that the vascular endothelium plays an important role in neurovascular coupling in the brain.
We recently demonstrated that the vascular endothelium plays an important role in neurovascular coupling in the brain (Chen et al, 2014, Hillman, 2014). We propose this model based on our many observations of high-speed propagated dilation of brain arterioles.
Figure 7 Adding the vascular endothelium to our picture of neurovascular coupling. We note that endothelial cells can feasibly use the same pathways to alter vascular smooth muscle tone as astrocytes. Endothelial cells can also propagated vasodilation long distances along a vessel, utilizing at least two different processes: high-speed endothelial hyperpolarization and a slower process accompanied by a calclium wave that elicits dilation via prostanoids, NO and / or EETs. This intravascular pathway for propagating vasodilation makes it feasible that neurovascular coupling is initiated at the level of capillaries, and propagates rapidly to upstream penetrating and pial arterioles. Brain endothelial cells express a wide range of receptors that have yet to be explored as possible components in neurovascular coupling. See Chen et al 2014 and for full legend.
To demonstrate the role of the endothelium, we developed an in-vivo light-dye technque to cause spatially selective damage to the endothelium of pial arteries, leaving the rest of the vessel intact.
Figure 8. FITC within the vessel releases oxygen free radicals upon blue light illumination. These directly (and selectively) damage the vessel's inner endothelial layer, while leaving the rest of the vessel intact. The use of light means that different patterns of endothelial disruption can be achieved in the intact, in-vivo brain.
In Chen et al 2014, we showed that pial arteries that previously dilated in response to somatosensory stimulation did not dilate beyond the point of light-dye damage (shown below). Wide-field light-dye prevented any dilation of pial arteries, which led to a significant reduction in functional hyperemia (as measured by HbT).
Figure 9. A fine line of laser light disrupts a small section of arterial endothelium in the somatosensory cortex. Before disruption, the targeted pial arteriole dilated rapidly following somatosensory stimulation. After light-line damage, dilation abruptly halts at the point of endothelial damage. See Chen et al 2014 and for full legend.
Control experiments confirmed that the damaged part of the vessel still dilated in response to sodium nitroprusside, confirming that the vessel's smooth muscle was still intact. Acetylcholine evoked dilation of all parts of the pial arterioles except the regions directly illuminated during light-dye (including the 'distal' portion of the arteriole in light-line experiments that had failed to dilate in response to somatosensory stimulation).
We conclude that the vascular endothelium forms an essential conduit for conduction of vasodilatory signals along the arteries and arterioles feeding active neurons. This finding could resolve many of the discrepancies in earlier models of neurovascular coupling.
Resting state v/s stimulus-evoked hemodynamics
Functional connectivity mapping, or resting state analysis is a popular new technique for fMRI analysis in humans (fc-MRI). Instead of invoking blood flow responses in the brain using specific stimuli, subjects simply rest for a period of around 6 minutes while fMRI data is acquired throughout the brain. Baseline fluctuations in blood flow are observed throughout the brain in this resting state. While these fluctuations typically appear random on a local level, it was noted that correlations in these fluctuations could be observed within spatially separated brain regions. Regions found to be strongly correlated (or anti-correlated) are inferred to have some form of functional connection, either directly between each other, or as a result of a common connection to another brain region. Not only do these results suggest the presence of innate networks within the brain that had not previously been detectable; intriguing results have suggested that these networks, while fairly repeatable in the healthy brain, are affected in a range of pathologies from Alzheimer’s to autism.
In a recent collaboration with Drs Guy McKhann, Jeffrey Bruce and Peter Canoll at Columbia, we have developed an MS-OISI system capable of acquiring high-resolution, high-speed data on the exposed cortex of humans during neurosurgery. This data, combined with intrasurgical electrocorticography is giving us unique insights into resting state and stimulus-evoked fluctuations in the anesthetized and awake human brain.
Optogenetics, Neonatal Development, and Glioma
Additional projects in progress in the lab include using optogenetics to understand hemodynamic control. Our neonatal development project aims to understand the way in which cortical blood flow responses to stimulus manifest and evolve during the first weeks of life. This project is based on a range of studies in human neonates that have reported both positive and inverted fMRI BOLD and near infrared spectroscopy responses. Our glioma project combines findings in human studies as described above, with studies of animal models of glioma developed by Dr Peter Canoll. Figure 9 below shows an in-vivo two-photon microscopy image of a green fluorescent protein (GFP)-expressing tumor.
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