The topology of the cerebral vasculature, which is the energy transport corridor of the brain, can be used to study cerebral circulatory pathways. Limited by the restrictions of the vascular markers and imaging methods, studies on cerebral vascular structure now mainly focus on either observation of the macro vessels in a whole brain or imaging of the micro vessels in a small region. Simultaneous vascular studies of arteries, veins and capillaries have not been achieved in the whole brain of mammals. Here, we have combined the improved gelatin-Indian ink vessel perfusion process with Micro-Optical Sectioning Tomography for imaging the vessel network of an entire mouse brain. With 17 days of work, an integral dataset for the entire cerebral vessels was acquired. The voxel resolution is 0.35×0.4×2.0 µm(3) for the whole brain. Besides the observations of fine and complex vascular networks in the reconstructed slices and entire brain views, a representative continuous vascular tracking has been demonstrated in the deep thalamus. This study provided an effective method for studying the entire macro and micro vascular networks of mouse brain simultaneously.

Cryo-imaging techniques have been widely used to measure the metabolic state of tissues by capturing reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) autofluorescence. However, NADH and FAD fluorescence is sensitive to changes in temperature, which may result in unreliable redox ratio calculations. Here, the relationship between the measured redox ratio and sample surface temperature was analyzed using a standard phantom solution and biological tissues. The results indicated that a temperature < - 100°C was a suitable cryo-imaging temperature window in which redox ratio measuring was immune to temperature fluctuations. These results may serve as a reference for designing and optimizing redox cryo-imaging experiments for quantitatively mapping the metabolic state of biological samples.

Rationale and Objectives Our long-term goals are to identify imaging biomarkers for hemorrhagic shock and to understand how the preservation of cerebral microcirculation works. We also seek to understand how the damage occurs to the cerebral hemodynamics and the mitochondrial metabolism during severe hemorrhagic shock. Materials and Methods We used a multimodal cerebral cortex optical imaging system to obtain 4-hour observations of cerebral hemodynamic and metabolic alterations in exposed rat cortexes during severe hemorrhagic shock. We monitored the mean arterial pressure, heart rate, cerebral blood flow (CBF), functional vascular density (FVD), vascular perfusion and diameter, blood oxygenation, and mitochondrial reduced nicotinamide adenine dinucleotide (NADH) signals. Results During the rapid bleeding and compensatory stage, cerebral parenchymal circulation was protected by inhibiting the perfusion of dural vessels. During the compensatory stage, although the brain parenchymal CBF and FVD decreased rapidly, the NADH signal did not show a significant increase. During the decompensatory stage, FVD and CBF maintained the same low level and the NADH signal remained unchanged. However, the NADH signal showed a significant increase after the rapid blood infusion. FVD and CBF rebounded to the baseline after the resuscitation and then declined again. Conclusions We present for the first time simultaneous imaging of cerebral hemodynamics and NADH signals in vivo during the process of hemorrhagic shock. This novel multimodal method demonstrated clearly that severe hemorrhagic shock imparts irreversible tissue damage that is not compensated by the autoregulatory mechanism. Hemodynamic and metabolic signatures including CBF, FVD, and NADH may be further developed to provide sensitive biomarkers for stage transitions in hemorrhagic shock.

Understanding the biological mechanism and identifying biomarkers of hemorrhagic shock is important for diagnosis and treatment. We aim to use optical imaging to study how the cerebral blood circulation and metabolism change during the progression of severe hemorrhagic shock, especially the decompensatory stage. We used a multi-parameter (blood pressure (BP), cerebral blood flow (CBF), functional vascular density (FVD), blood oxygenation and mitochondrial NADH signal) cerebral cortex optical imaging system to observe brain hemodynamic change and metabolic alteration of rats in vivo for 4 h. Cerebral circulation and mitochondrial metabolism could be well preserved in the compensatory stage but impaired during the decompensatory stage. The changes of brain hemodynamics and metabolism may provide sensitive indicators for various shock stages including the transition from compensatory stage to decompensatory stage. Our novel imaging observations of hemodynamic and metabolic signals in vivo indicated that the rat brains under hemorrhagic shock suffered irreversible damage which could not be compensated by the autoregulation mechanism, probably due to injured mitochondria.

The goal of biological samples' cryofixation is to trap a metabolic state as it exists in vivo by rapidly stopping internal reactions. However, obtaining perfect quality of cryofixation for large and high hypermetabolism organ/tissue (such as brain, heart) remains a challenge. The aim of this study was to develop and display a comprehensive and direct method to evaluate cryofixation's process and quality. Here, we adopt a delicate combination of homemade cryo-imaging system with a rat cardiac arrest model that can control cryofixation time optionally. We successfully evaluate the cryofixation time-related nicotinamide adenine dinucleotide (NADH) fluorescence pattern of several coronal sections in rat's brain that suffered from directional funnel cryofixation procedure. Through quantitative analysis of the distribution map of NADH fluorescence, we could obtain a relationship between cryofixation time and well cryofixation volume and then could deduce the cryofixation rates and quality at different time points. Our results also demonstrated that dissection of the temporal muscle of rat could significantly optimize the classical direct funnel cryofixation protocol.

Cortical spreading depression (CSD) is a self-propagating wave of cellular depolarization that plays an important role in the development of cerebral pathology following ischemia or trauma. Optical intrinsic signal (OIS) imaging has been widely used to investigate CSD. Sources of OIS are complex and related to the changes in brain tissue absorption and scattering. The absorbing chromophores may include oxy-hemoglobin, deoxy-hemoglobin, cytochromes, flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (NADH). Considering only one or part of these elements in studies involving OIS may cause inaccurate results. Thus, we simultaneously calculated changes in HbO, HbR, FAD, cytochrome c, cytochrome aa3 and light scattering during CSD by applying multi-spectral OIS imaging at 450, 470, 500, 530, 550, 570, 600, 630, and 650 nm in the rat brain. We also showed that the hemodynamic changes during CSD may not be correctly estimated if the scattering and other chromophores such as FAD, cytochrome c and cytochrome aa3, are not included in the fitting model of multi-wavelength data analysis. As shown in our results, if considering the changes in scattering and other chromophores in data fitting model, deoxy-hemoglobin (HbR) showed a triphasic change while only a monophasic decrease in HbR will be resolved without considering changes in scattering and other chromophores as reported in previous studies. Moreover, our results showed that changes in cytochrome c was tightly related to OIS at 550 nm, cytochrome aa3 was closely related to OIS at 450, 600 and 650 nm, and FAD was closely related to OIS at 450 and 470 nm during CSD. It indicates that if the contribution by these related chromophores is not considered, using OIS at these wavelengths to determine the hemoglobin changes during CSD may lead to inaccurate results.

Laser speckle spatial contrast analysis (LSSCA) is superior to laser speckle temporal contrast analysis (LSTCA) in monitoring the fast change in blood flow due to its advantage of high temporal resolution. However, the application of LSSCA which is based on spatial statistics may be limited when there is nonuniform intensity distribution such as fiber-transmitting laser speckle imaging. In this study, we present a normalized laser speckle spatial contrast analysis (nLSSCA) to correct the detrimental effects of nonuniform intensity distribution on the spatial statistics. Through numerical simulation and phantom experiments, it is found that just ten frames of dynamic laser speckle images are sufficient for nLSSCA to achieve effective correction. Furthermore, nLSSCA has higher temporal resolution than LSTCA to respond the change in velocity. LSSCA, LSTCA and nLSSCA are all applied in the fiber-transmitting laser speckle imaging system to analyze the change of cortical blood flow (CBF) during cortical spreading depression (CSD) in rat cortex respectively, and the results suggest that nLSSCA can examine the change of CBF more accurately. For these advantages, nLSSCA could be a potential tool for fiber-transmitting/endoscopic laser speckle imaging.

Cerebral blood flow (CBF) is critical for the maintenance of cerebral function by guaranteed constant oxygen and glucose supply to brain. Collateral channels (CCs) are recruited to provide alternatives to CBF to ischemic regions once the primary vessel is occluded during ischemic stroke. However, the knowledge of the relationship between dynamic evolution of collateral flow and the distribution of regional blood flow remains limited. In this study, laser speckle imaging was used to assess dynamic changes of CCs and regional blood flow in a rat cortex with permanent middle cerebral artery occlusion (MCAo). We found that CCs immediately provided blood flow to ischemic territories after MCAo. More importantly, there were three kinds of dynamic changes of CCs during acute stroke: persistent CC, impermanent CC, and transient CC, respectively, related to different distributions of regional blood flow. Although there was the possible occurrence of peri-infarct depolarization (PID) during ischemia, there was no obvious significance about the onset time and duration of CCs between rats with and without PID. These results suggest that the initial arising of CCs does not ensure their persistence, and that collateral flow could be varied with distribution of regional blood flow in acute ischemic stroke, which may facilitate the understanding of collateral recruitment and promote the development of collateral therapeutics in the future.

Cortical spreading depression (CSD) plays an important role in trauma, migraine and ischemia. CSD could induce pronounced hemodynamic changes and the disturbance of pH homeostasis which has been postulated to contribute to cell death following ischemia. In this study, we described a fluorescence-corrected multimodal optical imaging system to simultaneously monitor CSD associated intracellular pH (pH(i)) changes and hemodynamic response including hemoglobin concentrations and cerebral blood flow (CBF). CSD was elicited by application of KCl on rat cortex and direct current (DC) potential was recorded as a typical characteristic of CSD. The pH(i) shift was mapped by neutral red (NR) fluorescence which was excited at 516-556 nm and emitted at 625 nm. The changes in hemoglobin concentrations were determined by dual-wavelength optical intrinsic signal imaging (OISI) at 550 nm and 625 nm. Integration of fluorescence imaging and dual-wavelength OISI was achieved by a time-sharing camera equipped with a liquid crystal tunable filter (LCTF). CBF was visualized by laser speckle contrast imaging (LSCI) through a separate camera. Besides, based on the dual-wavelength optical intrinsic signals (OISs) obtained from our system, NR fluorescence was corrected according to our method of fluorescence correction. We found that a transient intracellular acidification followed by a small alkalization occurred during CSD. After CSD, there was a prolonged intracellular acidification and the recovery of pH(i) from CSD took much longer time than those of hemodynamic response. Our results suggested that the new multimodal optical imaging system had the potential to advance our knowledge of CSD and might work as a useful tool to exploit neurovascular coupling under physiological and pathological conditions.

Automatic separation of arteries and veins in optical cerebral cortex images is important in clinical practice and preclinical study. In this paper, a simple but effective automatic artery-vein separation method which utilizes single-wavelength coherent illumination is presented. This method is based on the relative temporal minimum reflectance analysis of laser speckle images. The validation is demonstrated with both theoretic simulations and experimental results applied to the rat cortex. Moreover, this method can be combined with laser speckle contrast analysis so that the artery-vein separation and blood flow imaging can be simultaneously obtained using the same raw laser speckle images data to enable more accurate analysis of changes of cerebral blood flow within different tissue compartments during functional activation, disease dynamic, and neurosurgery, which may broaden the applications of laser speckle imaging in biology and medicine.

Cortical spreading depression (CSD) is a wave of neuronal and glial depolarization that propagates across the cortex at a rate of 2–5 mm/min accompanied by reversible electroencephalogram (EEG) suppression, a negative shift of direct current (DC) potential, and change of optical intrinsic signals (OIS). Propagation velocity of CSD is an important parameter used to study this phenomenon. It is commonly determined in an electrophysiological way that measures the time required for a CSD wave to pass along two electrodes. Since the electrophysiology technique fails to reveal the spreading pattern of CSD, velocity calculated in this manner might be inaccurate. In this study, we combined the electrophysiological recording and OIS imaging (OISI) for detecting changes in DC potential and OIS during CSD simultaneously. An optical method based on OISI to determine the CSD velocity, which is measured by generating a series of regions of interest (ROI) perpendicular to the advancing wavefront along propagation direction of CSD at different time points and then dividing by the distance between ROIs over time, is presented. Comparison of the accuracy of the two approaches in determining the CSD velocity is made as well. The average rate of 33 CSDs is 3.52 ± 0.87 mm/min by use of the optical method and 4.36 ± 1.65 mm/min by use of the electrophysiological method. Because of the information about spreading pattern of CSD provided optically, the velocity determined by OISI is of smaller deviation and higher accuracy.

Laser speckle contrast imaging is a technique used for imaging blood flow without scanning. Though several studies have attempted to combine spatial and temporal statistics of laser speckle images for reducing image noise as well as preserving acceptable spatiotemporal resolution, the statistical accuracy of these spatiotemporal methods has not been thoroughly compared. Through numerical simulation and animal experiments, this study investigates the changes in the mean speckle contrast values and the relative noise of the speckle contrast images computed by these methods with various numbers of frames and spatial windows. The simulation results show that the maximum relative error of the mean speckle contrast computed by the spatiotemporal laser speckle contrast analysis (STLASCA) method, in which the speckle contrast images are computed by analyzing the 3-D spatiotemporal speckle image cube, is approximately 5%, while it is higher than 13% for other methods. Changes in the mean speckle contrast values and the relative noise computed by these methods for animal experiment data are consistent with the simulation results. Our results demonstrate that STLASCA achieves more accurate speckle contrast, and suggest that STLASCA most effectively utilizes the number of pixels, thus achieving maximized speckle contrast, and thereby maximizing the variation of the laser speckle contrast image.

Cortical spreading depression (CSD) is an important pathological model to study cerebral ischemia and migraine. In pharmacological studies of CSD, dimethylsulfoxide (DMSO) is an efficient solvent for water‐insoluble drugs. Previous studies indicated that DMSO could prevent pial arteriolar dilation induced by oxidants. Therefore, it was very important to study the effect of DMSO on hemodynamics during CSD so that optimization of dose of DMSO as solvent can be made.

Cortical spreading depression (CSD) is a pathophysiological phenomenon. There are sufficient evidences to prove that CSD plays an important role in some neurological disorders. However, exact mechanisms of its initiation and propagation are still unclear. Previous studies showed that glutamate receptors could be concerned with CSD, but those studies were mostly performed oriented to ionotropic glutamate receptors (iGluRs). There is relatively little report about effects of metabotropic glutamate receptors (mGluRs) on CSD. Here, we applied optical intrinsic signal imaging (OISI) combined with direct current (DC) potential recording to examine influences of some mGluRs antagonist (or agonist) on CSD propagation in rat's brain, to indirectly validate actions of some mGluRs on CSD. We found that N-acetyl-L-aspartyl-L-glutamate (NAAG, an agonist at mGluR3) inhibited the propagation of CSD, and the inhibition was gradually developed with time. However, 6-methyl-2-phenylethynyl-pyridine (MPEP, an antagonist of mGluR5) did not produce any significant alterations with the CSD propagation. Our findings suggest that mGluR3 could play an important role in the CSD propagation, but the activity of mGluR5 was comparatively weak. These findings can help to understand the propagation mechanism of CSD, and consider the therapy of some neurological diseases involved with CSD.

Cortical spreading depression (CSD), which is a significant pathological phenomenon that correlates with migraines and cerebral ischemia, has been characterized by a wave of depolarization among neuronal cells and propagates across the cortex at a rate of 2–5 mm/min. Although the propagation pattern of CSD was well-investigated using high-resolution optical imaging technique, the variation of propagation speed of CSD across different regions of cortex was not well-concerned, partially because of the lack of ideal approach to visualize two-dimensional distribution of propagation speed of CSD over the whole imaged cortex. Here, we have presented a method to compute automatically the propagation speed of CSD throughout every spots in the imaged cortex. In this method, temporal clustering analysis (TCA) and least square estimation (LSE) were first used to detect origin site where CSD was induced. Taking the origin site of CSD as the origin of coordinates, the data matrix of each image was transformed into the corresponding points based on the polar-coordinate representation. Then, two fixed-distance regions of interest (ROIs) are sliding along with the radial coordinate at each polar angle within the image for calculating the time lag with correlating algorithm. Finally, we could draw a two-dimensional image, in which the value of each pixel represented the velocity of CSD when it spread through the corresponding area of the imaged cortex. The results demonstrated that the method can reveal the heterogeneity of propagation speed of CSD in the imaged cortex with high fidelity and intuition.

Disturbances in glycaemia can significantly alter brain functions. Several brain-mapping techniques have been developed to characterize complex functional topography of the brain. This chapter introduces basic principles and instrumentations of two optical imaging techniques used for investigating cerebral hemodynamics: intrinsic optical signal imaging and laser speckle imaging. Especially, these two imaging techniques are used to study influences of acute hyperglycaemia on cerebral blood flow and spreading depression in rat cerebral cortex.

Bursts are electrical spikes firing with a high frequency, which are the most important property in synaptic plasticity and information processing in the central nervous system. However, bursts are difficult to identify because bursting activities or patterns vary with physiological conditions or external stimuli. In this paper, a simple method automatically to detect bursts in spike trains is described. This method auto-adaptively sets a parameter (mean inter-spike interval) according to intrinsic properties of the detected burst spike trains, without any arbitrary choices or any operator judgment. When the mean value of several successive inter-spike intervals is not larger than the parameter, a burst is identified. By this method, bursts can be automatically extracted from different bursting patterns of cultured neurons on multi-electrode arrays, as accurately as by visual inspection. Furthermore, significant changes of burst variables caused by electrical stimulus have been found in spontaneous activity of neuronal network. These suggest that the mean inter-spike interval method is robust for detecting changes in burst patterns and characteristics induced by environmental alterations.

In this paper, neurons were cultured on a substrate above a multielectrode array, so the changes of electrophysiological activity patterns during development of the neuronal network or in response to environmental perturbations were monitored. But the complexity of these spontaneous activity patterns is not well understood. In order to solve the problem, a comprehensive method (approximate entropy (ApEn) in combination with a "sliding window" over the data) is introduced to quantify the complexity of four spontaneous activity patterns (sporadic spikes, tonic spikes, pseudobursts, and typical bursts) in cultured hippocampal neuronal networks. The results show that the dynamic curves of ApEn illustrate vivid differences between the four patterns and the values of ApEn fall into different ranges. Among these patterns, the complexity of tonic spikes is the highest while that of pseudobursts is the lowest. This suggests that the proposed method is a valid procedure for tracking the dynamic variation in neuronal signals and can distinguish the different firing patterns of neuronal networks in terms of their complexity.

以激光散斑衬比分析为基础的激光散斑成像技术,是一种无需扫描的全场光学成像方法,在监测生理及病理状态下组织血流动态变化中的应用日益广泛.在实际应用中,像面散斑平均尺寸等多种因素影响散斑衬比值,使得该技术在反映血流变化的准确性方面受到影响.采用一种成像散斑计算机模拟方法研究了像面散斑平均尺寸对成像散斑统计特性的影响,分析了成像参数与像面散斑尺寸的定量关系,并通过物理模型实验对模拟结果进行了验证.研究结果确认了合理的像面散斑平均尺寸计算公式,证实了散斑衬比值随像面散斑平均尺寸增大而减小的现象,并为确定合理的成像参数提供了依据.

The collateral circulation is crucial for the pathophysiology and outcome of acute cortical ischemia. Current understanding of collateral circulation still remains sparse, largely due to prior limitations of spatial or/and temporal resolution in methods to evaluate these diminutive redistributive routes of cerebral blood flow (CBF) especially in leptomeningeal anastomoses that connected cortical arteries. In the study, based on a mini-stroke model, laser speckle imaging with high spatiotemporal resolution was used to assess the dynamic evolution of the collateral circulation around a mini-ischemia in the rat cortex. We found that the blood flow and diameter in the intra-arterial anastomoses were enhanced immediately after the ligation of one branch of middle cerebral artery and recovered to baseline level as arterial recirculation was performed. Whereas the communicative flow-through of the posterior cerebral artery and the middle cerebral artery anastomoses was not significant enough to be determined. This is the evidence that intra-arterial anastomoses were the primary routes to restore blood flow into the ischemic territory during the acute phase of ischemia, and laser speckle imaging method was proven as a powerful tool to be potential for subserving further investigation of the collateral circulation.