Weihua Luo

Spatiotemporal laser speckle contrast analysis for blood flow imaging with maximized speckle contrast

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.

Time-varying spreading depression waves in rat cortex revealed by optical intrinsic signal imaging.

This study aimed to investigate the variation of propagation patterns of successive spreading depression (SD) waves induced by K+ in rat cortex. SD was elicited by 1 M KCl solution in the frontal cortex of 18 Sprague-Dawley rats under alpha-chloralose/urethane anesthesia. We applied optical intrinsic signal imaging (OISI) at an isosbestic point of hemoglobin (550 nm) to examine regional cerebral blood volume (CBV) changes in the parieto-occipital cortex. In 6 of the 18 rats, OISI was performed in conjunction with DC potential recording of the cortex. CBV changes appeared as repetitive propagation of wave-like hyperemia at a speed of 3.7+/-0.4 mm/min, which was characterized by a significant negative peak (-14.3+/-3.2%) in the reflectance signal. Among the observed 186 SDs, the first wave always propagated through the entire imaged cortex in every rat, whereas following waves were likely to bypass the medial area of the imaged cortex (partially propagated waves, n=65, 35%). Correspondingly, DC potential shifts showed non-uniform in the medial area, and they seemed closely related to the changes in reflectance. For partially propagated SD waves, the mean time interval to the previous SD wave (217.0+/-24.3 s) was significantly shorter than for fully propagated SD waves (251.2+/-29.0 s). The results suggest that the propagation patterns of a series of SD waves are time-varying in different regions of rat cortex, and the variation is related to the interval between SD waves.

Simultaneous monitoring of intracellular pH changes and hemodynamic response during cortical spreading depression by fluorescence-corrected multimodal optical imaging.

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.

Hyperosmotic chemical agent’s effect on in vivo cerebral blood flow revealed by laser speckle

We investigated the influence of a hyperosmotic agent (glycerol) on the normal physiological function of tissue by applying the glycerol in vitro and in vivo to rabbit dura mater to assess the changes in the tissues optical properties. We used a laser speckle imaging technique to study the effect of epidurally applied glycerol on resting cerebral blood flow (CBF). Our results showed that resting CBF decreased as the transparency of the dura mater increased. The challenges for the design of an optical clearing technique were not only the clearing effects and the duration of the action of the chemical agents but also the influence of the glycerol on the tissues normal physiological function.

Spatiotemporal characteristics of cerebral blood volume changes in rat somatosensory cortex evoked by sciatic nerve stimulation and obtained by optical imaging

The spatiotemporal characteristics of changes in cerebral blood volume associated with neuronal activity were investigated in the hindlimb somatosensory cortex of alpha-chloralose-urethane anesthetized rats (n=10) with optical imaging at 570 nm through a thinned skull. Activation of the cortex was carried out by electrical stimulation of the contralateral sciatic nerve with 5-Hz, 0.3-V pulses (0.5 ms) for 2 s. The stimulation evoked a monophasic decrease in optical reflectance at the cortical parenchyma and arterial sites soon after the onset of stimulation, whereas no similar response was observed at vein compartments. The optical signal changes reached 10% of the peak response 0.70 +/- 0.32 s after the start of stimulation, and no significant time lag in this 10% start latency time was observed between the response at the cortical parenchyma and artery compartments. The decrease in optical reflectance reached a peak (0.25 +/- 0.047%) 2.66 +/- 0.61 s after stimulus onset at parenchymal sites, which is 0.40 +/- 0.20 s earlier (P<0.05) than that at arterial sites (0.50 +/- 0.068% 3.06 +/- 0.70 s). Varying the locations within the cortical parenchyma and arterial compartments did not significantly affect the temporal characteristics of the evoked signal. These results suggest that stimulation of the sciatic nerve evokes an increase in local blood volume in both capillaries (cortical parenchyma) and arterioles soon after the onset of a stimulus, but the blood volume increase evoked in capillaries could not be entirely accounted for by the dilation of arterioles.

Simultaneous detection of hemodynamics, mitochondrial metabolism and light scattering changes during cortical spreading depression in rats based on multi-spectral optical imaging

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.

Acute hyperglycemia compromises cerebral blood flow following cortical spreading depression in rats monitored by laser speckle imaging

Hyperglycemia and cortical spreading depression (CSD) are possible factors that worsen the outcome of ischemic stroke, and it is probable that there is a longterm cooperative effect of hyperglycemia and CSD on cerebral blood flow (CBF). Long-lasting and full-field observation of changes in CBF following CSD in vivo during acute hyperglycemia in rats might show whether this is the case. Here, we utilized laser speckle imaging to study influences of acute hyperglycemia on CBF at the level of individual vascular compartments for 3 h in normal rats and those with CSD. It is shown that there are extensive increases of CBF at the arteriole and parenchyma over the normal rat cortex during acute hyperglycemia, whereas there is no significant change in CBF at the venule. We also find that, at all vascular compartments, after the glucose administration there is a stepwise reduction of CBF following CSD, but after saline injection CBF following CSD is close to the baseline. Our results indicate that acute hyperglycemia could aggravate the severity of decrease in CBF following CSD, suggesting possible mechanisms by which hyperglycemia exacerbates cerebral damage after ischemic stroke.

Dynamic change of collateral flow varying with distribution of regional blood flow in acute ischemic rat cortex

Abstract. 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.

A modified mini-stroke model with region-directed reperfusion in rat cortex.

Mini-ischemia localized into a specific brain area has promoted understanding of the mechanisms underlying brain recovery in stroke. However, the conventional mini-stroke model adopted permanent arterial ligations but lacked controllable reperfusion, which is crucial for the outcome of delayed functional recovery. In this study, we devised a new rat mini-stroke model in which the vascular ligations can be easily reversed to induce targeted reperfusion. Specifically, a flexible ring was incorporated into the conventional small arterial ligations to tighten the ligating loops and facilitate cutting the ligatures for sufficient reperfusion afterwards. The distribution of cerebral blood flow was explored directly through a cranial window using laser speckle contrast imaging. A distinct ischemic core, which well fits the profile of the ligated ring, was bordered by a penumbral zone and then together surrounded by nonischemic tissue immediately after the arterial ligations involving the ring. After cutting the ligatures, post-recanalization hyperperfusion occurred in the previous ischemic core and to a greater extent at 24 h after reperfusion. In contrast, recirculation of common carotid artery in the conventional mini-stroke model hardly altered hypoperfusion status within the ischemic core. Evidence from two kinds of control groups indicated that the ring might produce a compression effect on the underlying cortex and then contribute to the more highly localized infarct that was identified by triphenyltetrazolium chloride staining. Our data suggest that this model provides opportunities for investigating the neurovascular dynamics in acute stroke and rehabilitation, especially with emerging optical imaging techniques.

Correcting the detrimental effects of nonuniform intensity distribution on fiber-transmitting laser speckle imaging of blood flow

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.