Quantitative estimates of stimulation-induced perfusion response using two-photon fluorescence microscopy of cortical microvascular networks
Imaging Research, Sunnybrook Research Institute, 2075 Bayview Avenue, Toronto, ON, Canada M4N 3M5.NeuroImage (Impact Factor: 6.36). 04/2012; 61(3):517-24. DOI: 10.1016/j.neuroimage.2012.04.009
Functional hyperemia, or the increase in focal perfusion elicited by neuronal activation, is one of the primary functions of the neurovascular unit and a hallmark of healthy brain functioning. While much is known about the hemodynamics on the millimeter to tenths of millimeter-scale accessible by MRI, there is a paucity of quantitative data on the micrometer-scale changes in perfusion in response to functional stimulation. We present a novel methodology for quantification of perfusion and intravascular flow across the 3D microvascular network in the rat somatosensory cortex using two-photon fluorescence microscopy (2PFM). For approximately 96% of responding microvessels in the forelimb representation of the primary somatosensory cortex, brief (~2s) forepaw stimulation resulted in an increase of perfusion 20±4% (mean±sem). The perfusion levels associated with the remaining 4% of the responding microvessels decreased 10±9% upon stimulation. Vessels irrigating regions of lower vascular density were found to exhibit higher flow (p<0.02), supporting the notion that local vascular morphology and hemodynamics reflect the metabolic needs of the surrounding parenchyma. High dispersion (~77%) in perfusion levels suggests high spatial variation in tissue susceptibility to hypoxia. The current methodology enables quantification of absolute perfusion associated with individual vessels of the cortical microvascular bed and its changes in response to functional stimulation and thereby provides an important tool for studying the cellular mechanisms of functional hyperemia, the spatial specificity of perfusion response to functional stimulation, and, broadly, the micrometer-scale relationship between vascular morphology and function in health and disease.
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ABSTRACT: The cortical microvessels are organized in an intricate, hierarchical, three-dimensional network. Superimposed on this anatomical complexity is the highly complicated signaling that drives the focal blood flow adjustments following a rise in the activity of surrounding neurons. The microvascular response to neuronal activation remains incompletely understood. We developed a custom two photon fluorescence microscopy acquisition and analysis to obtain 3D maps of neuronal activation-induced changes in the geometry of the microvascular network of the primary somatosensory cortex of anesthetized rats. An automated, model-based tracking algorithm was employed to reconstruct the 3D microvascular topology and represent it as a graph. The changes in the geometry of this network were then tracked, over time, in the course of electrical stimulation of the contralateral forepaw. Both dilatory and constrictory responses were observed across the network. Early dilatory and late constrictory responses propagated from deeper to more superficial cortical layers while the response of the vertices that showed initial constriction followed by later dilation spread from cortical surface toward increasing cortical depths. Overall, larger caliber adjustments were observed deeper inside the cortex. This work yields the first characterization of the spatiotemporal pattern of geometric changes on the level of the cortical microvascular network as a whole and provides the basis for bottom-up modeling of the hemodynamically-weighted neuroimaging signals.NeuroImage 01/2013; 71. DOI:10.1016/j.neuroimage.2013.01.011 · 6.36 Impact Factor
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ABSTRACT: Fluorescent imaging of acidic compartments in living cells was carried out successfully by a novel molecule (CAE) that contained a pyridine unit and a carbazole core. As the protonation of the pyridine N atom of CAE, pH-dependent absorption and fluorescence properties were shown with a pKa of 5.47 which matches the pH range of intracellular acidic compartments. Moreover, a large Stokes shift, a significant enhancement in ratios of I544nm/I460nm and a distinct two-photon turn-on character were exhibited in spectral analysis. Meanwhile, direct intracellular imaging and standard double-staining experiments of CAE and LTR (co-localization coefficient: 0.83) revealed that CAE is an effective one-photon ratiometric and two-photon acidic pH probe for imaging intracellular acidic compartments. The pH distribution pattern of intracellular acidic compartments can be obtained facilely by CAE. In especial, CAE possessed well membrane-permeability, brilliant selectivity among various bioanalyte and excellent counterstain compatibility with Hoechst 33342, MTR and LTR.Biosensors & Bioelectronics 06/2013; 50C:42-49. DOI:10.1016/j.bios.2013.05.060 · 6.41 Impact Factor
Conference Paper: Featured circular profile for vessel thresholding[Show abstract] [Hide abstract]
ABSTRACT: To analyze the characteristics of the blood vessels quantitatively is of great importance to the diagnosis of different diseases, e.g., stenosis, diabetic retinopathy, tumor and so on. However, the occurrence of imaging noise or illumination variation may include difficulty for image analysis, especially for accurate quantitative analysis. Therefore, more efficient algorithms for vessel image thresholding, segmentation or extraction have been studied. Nevertheless, these methods may still fail entirely in case of images with large noise or structural noise (such as spots that obscure and/or are brighter than vessels). To improve the algorithm performance on these issues, we propose a novel thresholding algorithm for vascular images by probing the polarity in the circular profiles (PCP) of image pixels. This can robustly distinguish tube-like objects from both cloud-like contaminations and structural noise. Extensive simulation studies based on multiple evaluation criteria suggest that the PCP algorithm typically has a superior performance over other representative approaches. Finally, we also demonstrate the satisfactory performance of the PCP method on real image data.2013 6th International Congress on Image and Signal Processing (CISP); 12/2013
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