Hongyang Lu

Laser speckle contrast imaging of cerebral blood flow in freely moving animals

We designed a miniature laser speckle imager that weighs ∼20 g and is 3.1-cm high for full-field high-resolution imaging of cerebral blood flow (CBF) in freely moving animals. Coherent laser light illuminates the cortex through a multimode optical fiber bundle fixed onto the supporting frame of the imager. The reflected lights are then collected by a miniature macrolens system and imaged by a high-resolution CMOS camera at a high frame rate (50 fps). Using this miniature imager, we achieve high spatiotemporal resolution laser speckle contrast imaging of CBF in freely moving animals in real time.

Predicting the ischemic infarct volume at the first minute after occlusion in rodent stroke model by laser speckle imaging of cerebral blood flow

Abstract. Stroke is a worldwide medical emergency and an important issue in stroke research is looking for the early pathophysiological markers which can predict the severity of brain injury. Decreased cerebral blood flow (CBF) has been serving as the most important indicator of ischemic stroke. Particular attention is paid to study the spatio-temporal CBF changes immediately after the onset of stroke in a rat intraluminal filament middle cerebral artery occlusion (MCAO) model and investigation of its correlation with brain infarct volume after 24 h. We implement an on-line laser speckle imaging (LSI) system, which could provide real time high spatio-temporal resolution CBF information before, during, and immediately after the rat MCAO surgery. We found a significant correlation between the affected area with 50% CBF reduction (CBF50) at the first minute after occlusion with the infarct volume. To the best of our knowledge, this is the earliest CBF marker for infarct volume prediction. Based on such a CBF–infarct volume correlation, LSI may be used as a real time guidance for improving the consistency of intraluminal filament MCAO model since the depth of filament insertion could be adjusted promptly and those unsuccessful models could be excluded in the earliest stage.

Induction and imaging of photothrombotic stroke in conscious and freely moving rats.

Abstract. In experimental stroke research, anesthesia is common and serves as a major reason for translational failure. Real-time cerebral blood flow (CBF) monitoring during stroke onset can provide important information for the prediction of brain injury; however, this is difficult to achieve in clinical practice due to various technical problems. We created a photothrombotic focal ischemic stroke model utilizing our self-developed miniature headstage in conscious and freely moving rats. In this model, a high spatiotemporal resolution imager using laser speckle contrast imaging technology was integrated to acquire real-time two-dimensional CBF information during thrombosis. The feasibility, stability, and reliability of the system were tested in terms of CBF, behavior, and T2-weighted magnetic resonance imaging (MRI) findings. After completion of occlusion, the CBF in the targeted cortex of the stroke group was reduced to 16±9% of the baseline value. The mean infarct volume measured by MRI 24 h postmodeling was 77±11  mm3 and correlated well with CBF (R2=0.74). This rodent model of focal cerebral ischemia and real-time blood flow imaging opens the possibility of performing various fundamental and translational studies on stroke without the influence of anesthetics.

Directly measuring absolute flow speed by frequency-domain laser speckle imaging.

Laser speckle contrast imaging (LSCI) is a simple yet powerful tool to image blood flow. However, traditional LSCI has limited quantitative analysis capabilities due to various factors affecting flow speed evaluation, including illumination intensity, scattering from static tissues, and mathematical complexity of blood flow estimation. Here, we present a frequency-domain laser speckle imaging (FDLSI) method that can directly measure absolute flow speed. In phantom experiments, the measured flow speed agreed well with the preset actual values (10% deviation). Furthermore, in vivo experiments demonstrated that FDLSI was minimally affected by illumination condition changes.

Real-time high resolution laser speckle imaging of cerebral vascular changes in a rodent photothrombosis model

The study of hemodynamic and vascular changes following ischemic stroke is of great importance in the understanding of physiological and pathological processes during the thrombus formation. The photothrombosis model is preferred by researchers in stroke study for its minimal invasiveness, controllable infarct volume and lesion location. Nevertheless, there is a lack in high spatiotemporal resolution techniques for real time monitoring of cerebral blood flow (CBF) changes in 2D-profile. In this study, we implemented a microscopic laser speckle imaging (LSI) system to detect CBF and other vascular changes in the rodent model of photothrombotic stroke. Using a high resolution and high speed CCD (640 × 480 pixels, 60 fps), online image registration technique, and automatic parabolic curve fitting, we obtained real time CBF and blood velocity profile (BVP) changes in cortical vessels. Real time CBF and BVP monitoring has been shown to reveal details of vascular disturbances and the stages of blood coagulation in photothrombotic stroke. Moreover, LSI also provides information on additional parameters including vessel morphologic size, blood flow centerline velocity and CBF spatiotemporal fluctuations, which are very important for understanding the physiology and neurovascular pathology in the photothrombosis model.

Intraoperative laser speckle contrast imaging improves the stability of rodent middle cerebral artery occlusion model

Abstract. Rodent middle cerebral artery occlusion (MCAO) model is commonly used in stroke research. Creating a stable infarct volume has always been challenging for technicians due to the variances of animal anatomy and surgical operations. The depth of filament suture advancement strongly influences the infarct volume as well. We investigated the cerebral blood flow (CBF) changes in the affected cortex using laser speckle contrast imaging when advancing suture during MCAO surgery. The relative CBF drop area (CBF50, i.e., the percentage area with CBF less than 50% of the baseline) showed an increase from 20.9% to 69.1% when the insertion depth increased from 1.6 to 1.8 cm. Using the real-time CBF50 marker to guide suture insertion during the surgery, our animal experiments showed that intraoperative CBF-guided surgery could significantly improve the stability of MCAO with a more consistent infarct volume and less mortality.

Single-trial estimation of the cerebral metabolic rate of oxygen with imaging photoplethysmography and laser speckle contrast imaging.

Cortical cerebral metabolic rate of oxygen (CMRO(2)) could conventionally be measured by combining laser Doppler flowmetry and multispectral reflectance imaging across multiple trials of stimulation, which compromises the real-time capacity. Monitoring transient change of CMRO(2) has been challenging. In this Letter, imaging photoplethysmography (iPPG) and laser speckle contrast imaging were combined into a multi-modal optical imaging system for single-trial estimation of CMRO(2). In a physiologically stable experiment, the iPPG-based method showed a less than 4% variance in comparison with the conventional method over 20 trials, and its temporal stability could be comparable to that by conventional method over 6 trials. While the oxygen supply was decreased deliberately, the new method was able to detect the transient changes of CMRO(2) in real time, which could not be revealed by the conventional method.

Intraoperative cerebral blood flow imaging of rodents

Intraoperative monitoring of cerebral blood flow (CBF) is of interest to neuroscience researchers, which offers the assessment of hemodynamic responses throughout the process of neurosurgery and provides an early biomarker for surgical guidance. However, intraoperative CBF imaging has been challenging due to animals motion and position change during the surgery. In this paper, we presented a design of an operation bench integrated with laser speckle contrast imager which enables monitoring of the CBF intraoperatively. With a specially designed stereotaxic frame and imager, we were able to monitor the CBF changes in both hemispheres during the rodent surgery. The rotatable design of the operation plate and implementation of online image registration allow the technician to move the animal without disturbing the CBF imaging during surgery. The performance of the system was tested by middle cerebral artery occlusion model of rats.

Separation of cortical arteries and veins in optical neurovascular imaging

Separation of arteries and veins in the cerebral cortex is of significant importance in the studies of cortical hemodynamics, such as the changes of cerebral blood flow, perfusion or oxygen concentration in arteries and veins under different pathological and physiological conditions. Yet the cerebral vessel segmentation and vessel-type separation are challenging due to the complexity of cortical vessel characteristics and low spatial signal-to-noise ratio. In this work, we presented an effective full-field method to differentiate arteries and veins in cerebral cortex using dual-modal optical imaging technology including laser speckle imaging (LSI) and optical intrinsic signals (OIS) imaging. The raw contrast images were acquired by LSI and processed with enhanced laser speckle contrast analysis (eLASCA) algorithm. The vascular pattern was extracted and segmented using region growing algorithm from the eLASCA-based LSI. Meanwhile, OIS images were acquired alternatively with 630 and 870 nm to obtain an oxyhemoglobin concentration map over cerebral cortex. Then the separation of arteries and veins was accomplished by Otsu threshold segmentation algorithm based on the OIS information and segmentation of LSI. Finally, the segmentation and separation performances were assessed using area overlap measure (AOM). The segmentation and separation of cerebral vessels in cortical optical imaging have great potential applications in full-field cerebral hemodynamics monitoring and pathological study of cerebral vascular diseases, as well as in clinical intraoperative monitoring.

Imaging the early cerebral blood flow changes in rat middle cerebral artery occlusion stroke model

Intraluminal middle cerebral artery occlusion (MCAO) model in rats has been widely used to mimic human ischemic stroke and serves as an indispensable tool in the stroke research field. One limitation of this model is its high variability in infarct volume. The cerebral blood flow (CBF) information after cerebrovascular occlusion may reflect the availability of collateral circulation, which serves as a key factor for brain infarct volume. Laser speckle contrast imaging (LSCI) is a valuable tool for full-field imaging of CBF with high spatial and temporal resolution. In this paper, we investigated the spatio-temporal changes of CBF in rat MCAO stroke model using our self-developed real-time LSCI system. CBF images of adult male Sprague Dawley rats (n=13) were recorded before surgery, during first 1.5 hours after surgery, and 24 hours after stroke. We compared the CBF changes of different functional vessels during this period. In the ipsilateral hemisphere, CBF of veins and arteries both decreased as expected, while CBF of veins increased after occlusion in the contralateral hemisphere. Moreover, we found a linear correlation between early-stage CBF after occlusion and brain infarct volume, which can be utilized for surgery guidance to improve the uniformity of rat MCAO stroke models.