The relationship between measurements of cerebral blood oxygenation and neuronal activity is highly complex and depends on both neurovascular and neurometabolic biological coupling. While measurements of blood oxygenation changes via optical and MRI techniques have been developed to map functional brain activity, there is evidence that the specific characteristics of these signals are sensitive to the underlying vascular physiology and structure of the brain. Since baseline blood flow and oxygen saturation may vary between sessions and across subjects, functional blood oxygenation changes may be a less reliable indicator of brain activity in comparison to blood flow and metabolic changes. In this work, we use a biomechanical model to examine the relationships between neural, vascular, metabolic, and hemodynamic responses to parametric whisker stimulation under both normal and hypercapnic conditions in a rat model. We find that the relationship between neural activity and oxy- and deoxyhemoglobin changes is sensitive to hypercapnia-induced changes in baseline cerebral blood flow. In contrast, the underlying relationships between evoked neural activity, blood flow, and model-estimated oxygen metabolism changes are unchanged by the hypercapnic challenge. We conclude that evoked changes in blood flow and cerebral oxygen metabolism are more closely associated with underlying evoked neuronal responses.
Real-time investigation of cerebral blood flow (CBF), and oxy- and deoxyhemoglobin concentration (HbO, HbR) dynamics has been difficult until recently due to limited spatial and temporal resolution of techniques like laser Doppler flowmetry and magnetic resonance imaging (MRI). The combination of laser speckle flowmetry (LSF) and multispectral reflectance imaging (MSRI) yields high-resolution spatiotemporal maps of hemodynamic and metabolic changes in response to functional cortical activation. During acute focal cerebral ischemia, changes in HbO and HbR are much larger than in functional activation, resulting in the failure of the Beer-Lambert approximation to yield accurate results. We describe the use of simultaneous LSF and MSRI, using a nonlinear Monte Carlo fitting technique, to record rapid changes in CBF, HbO, HbR, and cerebral metabolic rate of oxygen (CMRO2) during acute focal cerebral ischemia induced by distal middle cerebral artery occlusion (dMCAO) and reperfusion. This technique captures CBF and CMRO2 changes during hemodynamic and metabolic events with high temporal and spatial resolution through the intact skull and demonstrates the utility of simultaneous LSF and MSRI in mouse models of cerebrovascular disease.
The analysis of fluorescence lifetime imaging microscopy (FLIM) data under complex biological conditions can be challenging. Particularly, the presence of short-lived autofluorescent aggregates can confound lifetime measurements in fluorescence energy transfer (FRET) experiments, where it can become confused with the signal from exogenous fluorophores. Here we report two techniques that can be used to discriminate the contribution of autofluorescence from exogenous fluorphores in FLIM. We apply the techniques to transgenic mice that natively express yellow fluorescence protein (YFP) in a subset of cortical neurons and to histological slices of aged human brain tissue, where we study the misfolding of intracellular tau protein in the form of neurofibrillary tangles.
Fluorescence lifetime measurement is widely used in the biological sciences due to its inherent sensitivity and concentration independence. Frequency domain high-throughput plate readers and time-resolved energy transfer (TRET) plate readers are in common use and have been successful in a variety of applications ranging from basic biochemistry to drug discovery. Time-domain systems would have advantages due to their ability to distinguish both FRETing and non-FRETing populations, but have been difficult to develop due to inherent difficulties with background autofluorescence and lifetime component separation. Using a modified commercial lifetime plate reader, we demonstrate a method for removal of the complex auto-fluorescent background decay, described using a stretched exponential function (StrEF). We develop a generalized multi-exponential fitting algorithm (GeMEF), which progressively accounts for confounding lifetime components in FRET-based assays using a series of control experiments. We demonstrate the separability of FRET strength and efficiency and apply the technique to protein–protein interactions and protein conformational assays in a cell-based format. Presenilin 1 (PS1) is known to be important in Amyloid Precursor Protein (APP) processing in Alzheimer's disease. Using transfected cells, we demonstrate APP-PS1 interactions by FRET in a cell-based, 96-well plate format.
Real time investigation of cerebral blood flow (CBF), and oxy/deoxy hemoglobin volume (HbO,HbR) dynamics has been difficult until recently due to limited spatial and temporal resolution of techniques like laser Doppler flowmetry and MRI. This is especially true for studies of disease models in small animals, owing to the fine structure of the cerebral vasculature. The combination of laser speckle flowmetry (LSF) and multi-spectral reflectance imaging (MSRI) yields high resolution spatio-temporal maps of hemodynamic changes in response to events such as sensory stimuli or arterial occlusion. Ischemia was induced by distal occlusion of the medial cerebral artery (dMCAO). Rapid changes in CBF, HbO, and HbR during the acute phase were captured with high temporal and spatial resolution through the intact skull. Hemodynamic changes that were correlated with vasoconstrictive events, peri-infarct spreading depressions (PISD), were observed. These experiments demonstrate the utility of LSF and Multi-spectral reflectance imaging (MSRI) in mouse disease models.
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