COX-2-Derived Prostaglandin E2 Produced by Pyramidal Neurons Contributes to Neurovascular Coupling in the Rodent Cerebral Cortex.

Vasodilatory prostaglandins play a key role in neurovascular coupling (NVC), the tight link between neuronal activity and local cerebral blood flow, but their precise identity, cellular origin and the receptors involved remain unclear. Here we show in rats that NMDA-induced vasodilation and hemodynamic responses evoked by whisker stimulation involve cyclooxygenase-2 (COX-2) activity and activation of the prostaglandin E2 (PgE2) receptors EP2 and EP4. Using liquid chromatography-electrospray ionization-tandem mass spectrometry, we demonstrate that PgE2 is released by NMDA in cortical slices. The characterization of PgE2 producing cells by immunohistochemistry and single-cell reverse transcriptase-PCR revealed that pyramidal cells and not astrocytes are the main cell type equipped for PgE2 synthesis, one third expressing COX-2 systematically associated with a PgE2 synthase. Consistent with their central role in NVC, in vivo optogenetic stimulation of pyramidal cells evoked COX-2-dependent hyperemic responses in mice. These observations identify PgE2 as the main prostaglandin mediating sensory-evoked NVC, pyramidal cells as their principal source and vasodilatory EP2 and EP4 receptors as their targets. SIGNIFICANCE STATEMENT: Brain function critically depends on a permanent spatiotemporal match between neuronal activity and blood supply, known as NVC. In the cerebral cortex, prostaglandins are major contributors to NVC. However, their biochemical identity remains elusive and their cellular origins are still under debate. Although astrocytes can induce vasodilations through the release of prostaglandins, the recruitment of this pathway during sensory stimulation is questioned. Using multidisciplinary approaches from single-cell reverse transcriptase-PCR, mass spectrometry, to ex vivo and in vivo pharmacology and optogenetics, we provide compelling evidence identifying PgE2 as the main prostaglandin in NVC, pyramidal neurons as their main cellular source and the vasodilatory EP2 and EP4 receptors as their main targets. These original findings will certainly change the current view of NVC.

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.

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.

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.

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.

Sub-millimeter resolution 3D optical imaging of living tissue using laminar optical tomography.

In-vivo imaging of optical contrast in living tissues can allow measurement of functional parameters such as blood oxygenation and detection of targeted and active fluorescent contrast agents. However, optical imaging must overcome the effects of light scattering, which limit the penetration depth and can affect quantitation and sensitivity. This article focuses on a technique for high-resolution, high-speed depth-resolved optical imaging of superficial living tissues called laminar optical tomography (LOT), which is capable of imaging absorbing and fluorescent contrast in living tissues to depths of 2-3 mm with 100-200 micron resolution. An overview of the advantages and challenges of in-vivo optical imaging is followed by a review of currently available techniques for high-resolution optical imaging of tissues. LOT is then described, including a description of the imaging system design and discussion of data analysis and image reconstruction approaches. Examples of recent applications of LOT are then provided and compared to other existing technologies.By measuring multiply-scattered light, Laminar Optical Tomography can probe beneath the surface of living tissues such as the skin and brain.

A system for high-resolution depth-resolved optical imaging of fluorescence and absorption contrast.

Laminar optical tomography (LOT) is a new three-dimensional in vivo functional optical imaging technique. Adopting a microscopy-based setup and diffuse optical tomography (DOT) imaging principles, LOT can perform both absorption- and fluorescence-contrast imaging with higher resolution (100-200 microm) than DOT and deeper penetration (2-3 mm) than laser scanning microscopy. These features, as well as a large field of view and acquisition speeds up to 100 frames per second, make LOT suitable for depth-resolved imaging of stratified tissues such as retina, skin, endothelial tissues and the cortex of the brain. In this paper, we provide a detailed description of a new LOT system design capable of imaging both absorption and fluorescence contrast, and present characterization of its performance using phantom studies.

Simultaneous multiwavelength laminar optical tomography.

Spatially resolved reflectance measurements can be used to characterize the depth-resolved optical properties of superficial tissues. However, until now, rapid acquisition of multiwavelength data has been hindered by multiplexing problems. We report on a novel multiwavelength laminar optical tomography system capable of acquiring data from multiple source-detector separations at three wavelengths simultaneously. Such data can allow in vivo depth-resolved spectroscopic imaging of absorbers, such as oxy- and deoxyhemoglobin, or of multiple fluorophores, that is unaffected by motion artifacts at frame rates exceeding 100 Hz. The system design and phantom validation studies are presented.

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.