A critical role for the vascular endothelium in functional neurovascular coupling in the brain.

BACKGROUND: The functional modulation of blood flow in the brain is critical for brain health and is the basis of contrast in functional magnetic resonance imaging. There is evident coupling between increases in neuronal activity and increases in local blood flow; however, many aspects of this neurovascular coupling remain unexplained by current models. Based on the rapid dilation of distant pial arteries during cortical functional hyperemia, we hypothesized that endothelial signaling may play a key role in the long-range propagation of vasodilation during functional hyperemia in the brain. Although well characterized in the peripheral vasculature, endothelial involvement in functional neurovascular coupling has not been demonstrated. METHODS AND RESULTS: We combined in vivo exposed-cortex multispectral optical intrinsic signal imaging (MS-OISI) with a novel in vivo implementation of the light-dye technique to record the cortical hemodynamic response to somatosensory stimulus in rats before and after spatially selective endothelial disruption. We demonstrate that discrete interruption of endothelial signaling halts propagation of stimulus-evoked vasodilation in pial arteries, and that wide-field endothelial disruption in pial arteries significantly attenuates the hemodynamic response to stimulus, particularly the early, rapid increase and peak in hyperemia. CONCLUSIONS: Involvement of endothelial pathways in functional neurovascular coupling provides new explanations for the spatial and temporal features of the hemodynamic response to stimulus and could explain previous results that were interpreted as evidence for astrocyte-mediated control of functional hyperemia. Our results unify many aspects of blood flow regulation in the brain and body and prompt new investigation of direct links between systemic cardiovascular disease and neural deficits.

Calcium imaging of infrared-stimulated activity in rodent brain.

Infrared neural stimulation (INS) is a promising neurostimulation technique that can activate neural tissue with high spatial precision and without the need for exogenous agents. However, little is understood about how infrared light interacts with neural tissue on a cellular level, particularly within the living brain. In this study, we use calcium sensitive dye imaging on macroscopic and microscopic scales to explore the spatiotemporal effects of INS on cortical calcium dynamics. The INS-evoked calcium signal that was observed exhibited a fast and slow component suggesting activation of multiple cellular mechanisms. The slow component of the evoked signal exhibited wave-like properties suggesting network activation, and was verified to originate from astrocytes through pharmacology and 2-photon imaging. We also provide evidence that the fast calcium signal may have been evoked through modulation of glutamate transients. This study demonstrates that pulsed infrared light can induce intracellular calcium modulations in both astrocytes and neurons, providing new insights into the mechanisms of action of INS in the brain.

Resolving the transition from negative to positive blood oxygen level-dependent responses in the developing brain.

The adult brain exhibits a local increase in cortical blood flow in response to external stimulus. However, broadly varying hemodynamic responses in the brains of newborn and young infants have been reported. Particular controversy exists over whether the "true" neonatal response to stimulation consists of a decrease or an increase in local deoxyhemoglobin, corresponding to a positive (adult-like) or negative blood oxygen level-dependent (BOLD) signal in functional magnetic resonance imaging (fMRI), respectively. A major difficulty with previous studies has been the variability in human subjects and measurement paradigms. Here, we present a systematic study in neonatal rats that charts the evolution of the cortical blood flow response during postnatal development using exposed-cortex multispectral optical imaging. We demonstrate that postnatal-day-12-13 rats (equivalent to human newborns) exhibit an "inverted" hemodynamic response (increasing deoxyhemoglobin, negative BOLD) with early signs of oxygen consumption followed by delayed, active constriction of pial arteries. We observed that the hemodynamic response then matures via development of an initial hyperemic (positive BOLD) phase that eventually masks oxygen consumption and balances vasoconstriction toward adulthood. We also observed that neonatal responses are particularly susceptible to stimulus-evoked systemic blood pressure increases, leading to cortical hyperemia that resembles adult positive BOLD responses. We propose that this confound may account for much of the variability in prior studies of neonatal cortical hemodynamics. Our results suggest that functional magnetic resonance imaging studies of infant and child development may be profoundly influenced by the maturing neurovascular and autoregulatory systems of the neonatal brain.

Analysis of skin lesions using laminar optical tomography.

Evaluation of suspicious skin lesions by dermatologists is usually accomplished using white light examination and direct punch or surgical biopsy. However, these techniques can be imprecise for estimating a lesion's margin or level of dermal invasion when planning surgical resection. Laminar optical tomography (LOT) is an imaging technique capable of acquiring depth-sensitive information within scattering tissues. Here, we explore whether LOT data can be used to predict the depth and thickness of pigmented lesions using a range of simulations and phantom models. We then compare these results to LOT data acquired on normal and malignant skin lesions in vivo.

Simultaneous multiplane in vivo nonlinear microscopy using spectral encoding.

Conventional point-by-point imaging schemes for laser scanning microscopy limit acquisition speeds, particularly when imaging three-dimensional volumes. We report a novel approach that achieves parallelization of multiple fields of view through the use of spectral encoding. By focusing two or more beams of different wavelengths at different positions within a suitable tissue, fluorescence or second/third harmonic generation emissions from these regions can be uniquely separated. We demonstrate that this approach can allow simultaneous in vivo imaging of fluorescence in two planes within the living rodent cortex, and of second harmonic generation in fresh tissue.

In vivo 3D morphology of astrocyte-vasculature interactions in the somatosensory cortex: implications for neurovascular coupling.

Astrocytes are increasingly believed to play an important role in neurovascular coupling. Recent in vivo studies have shown that intracellular calcium levels in astrocytes correlate with reactivity in adjacent diving arterioles. However, the hemodynamic response to stimulation involves a complex orchestration of vessel dilations and constrictions that spread rapidly over wide distances. In this work, we study the three-dimensional cytoarchitecture of astrocytes and their interrelations with blood vessels down through layer IV of the mouse somatosensory cortex using in vivo two-photon microscopy. Vessels and astrocytes were visualized through intravenous dextran-conjugated fluorescein and cortically applied sulforhodamine 101 (SR101), respectively. In addition to exploring astrocyte density, vascular proximity, and microvascular density, we found that sheathing of subpial vessels by astrocyte processes was continuous along all capillaries, arterioles, and veins, comprising a highly interconnected pathway through which signals could feasibly be relayed over long distances via gap junctions. An inner SR101-positive sheath noted along pial and diving arterioles was determined to be nonastrocytic, and appears to represent selective SR101 staining of arterial endothelial cells. Our findings underscore the intimate relationship between astrocytes and all cortical blood vessels, and suggest that astrocytes could influence neurovascular regulation at a range of sites, including the capillary bed and pial arterioles.

High-speed vascular dynamics of the hemodynamic response.

While a range of cellular mechanisms have been proposed to underlie control of neurovascular coupling, a comprehensive, reconciliatory model has yet to be determined. To fit with such a model, it is essential that candidate mechanisms exhibit reaction times, spatial ranges, and speeds of propagation that are consistent with the vascular manifestations of the 'hemodynamic response'. Understanding these vascular dynamics is therefore a critical step towards developing a robust model of neurovascular coupling. In this study, we utilize high-speed optical imaging of exposed rodent somatosensory cortex to explore and characterize the spatiotemporal dynamics of surface vessels during functional hyperemia. Our high-speed, high-resolution optical imaging approach allows us to study the hemodynamic response independently in individual vessels, and in discrete regions of the parenchyma with enough resolution to precisely characterize subtle spatial and temporal features of the response. Specifically, we explore when and where the first hemodynamic changes occur in response to stimuli, the direction and speed at which these changes propagate in arterioles and regions of the parenchyma, and the relative timing at which each of these compartments returns to its original baseline state. From these results, we are able to conclude that the hemodynamic response appears to initiate in the parenchyma and then spreads rapidly to surface arterioles. Following the initial onset we find evidence that the response spreads spatially outwards via the dilation of targeted arterioles. This propagation of vasodilation is independent of the direction of blood flow within each arteriole. We also find evidence of a decay phase that acts with a more uniform spatial dependence, rather than along targeted vessels, causing the periphery of the responding region to return to baseline first. We hypothesize that different underlying cellular mechanisms/signaling pathways are responsible for the response initiation and the response decay. Our results advance the fundamental understanding of the hemodynamic response, as well as our ability to evaluate potential cellular mechanisms for their involvement in neurovascular coupling.

Fiber-optic and articulating arm implementations of laminar optical tomography for clinical applications.

Laminar optical tomography (LOT) is a recently developed technique for depth-resolved in vivo imaging of absorption and fluorescence contrast. Until now, LOT has been implemented in a benchtop configuration, limiting accessibility to tissues and restricting imaging applications. Here we report on LOT implemented through an articulating arm and a fiber optic image bundle allowing flexible imaging for a range of clinical applications. We quantify the performance of these two implementations by imaging a tissue mimicking phantom.

Ultra-fast multispectral optical imaging of cortical oxygenation, blood flow, and intracellular calcium dynamics.

Camera-based optical imaging of the exposed brain allows cortical hemodynamic responses to stimulation to be examined. Typical multispectral imaging systems utilize a camera and illumination at several wavelengths, allowing discrimination between changes in oxy- and deoxyhemoglobin concentration. However, most multispectral imaging systems utilize white light sources and mechanical filter wheels to multiplex illumination wavelengths, which are slow and difficult to synchronize at high frame rates. We present a new LED-based system capable of high-resolution multispectral imaging at frame rates exceeding 220 Hz. This improved performance enables simultaneous visualization of hemoglobin oxygenation dynamics within single vessels, changes in vessel diameters, blood flow dynamics from the motion of erythrocytes, and dynamically changing fluorescence.

Hyperspectral in vivo two-photon microscopy of intrinsic contrast.

In vivo two-photon imaging of intrinsic contrast can provide valuable information about structural tissue elements such as collagen and elastin and fluorescent metabolites such as nicotinamide adenine dinucleotide. Yet low signal and overlapping emission spectra can make it difficult to identify and delineate these species in vivo. We present a novel approach that combines excitation scanning with spectrally resolved emission two-photon microscopy, allowing distinct structures to be delineated based on their characteristic spectral fingerprints. The amounts of intrinsic fluorophores present in each voxel can also be evaluated. We demonstrate our method using in vivo imaging of nude mouse skin.