Micro-optical Sectioning Tomography Research Articles

Mapping the Function of Whole-Brain Projection at the Single Neuron Level.

Axonal projection conveys neural information. The divergent and diverse projections of individual neurons implythe complexity of information flow. It is necessary to investigate the relationship between the projection and functional information at the single neuron level for understanding the rules of neural circuit assembly, but a gap remains due to a lack of methods to map the function to whole-brain projection. Here an approach is developed to bridge two-photon calcium imaging in vivo with high-resolution whole-brain imaging based on sparse labeling with the genetically encoded calcium indicator GCaMP6. Reliable whole-brain projections are captured by the high-definition fluorescent micro-optical sectioning tomography (HD-fMOST). A cross-modality cell matching is performed and the functional annotation of whole-brain projection at the single-neuron level (FAWPS) is obtained. Applying it to the layer 2/3 (L2/3)neurons in mouse visual cortex, the relationship is investigated between functional preferences and axonal projection features. The functional preference of projection motifs and the correlation between axonal length in MOs and neuronal orientation selectivity, suggest that projection motif-defined neurons form a functionally specific information flow, and the projection strength in specific targets relates to the information clarity. This pipeline provides a new way to understand the principle of neuronal information transmission.

A novel cholinergic projection from the lateral parabrachial nucleus and its role in methamphetamine-primed conditioned place preference.

Drug relapse is a big clinical challenge in the treatment of addiction, but its neural circuit mechanism is far from being fully understood. Here, we identified a novel cholinergic pathway from choline acetyltransferase-positive neurons in the external lateral parabrachial nucleus (eLPBChAT) to the GABAergic neurons in the central nucleus of the amygdala (CeAGABA) and explored its role in methamphetamine priming-induced reinstatement of conditioned place preference. The anatomical structure and functional innervation of the eLPBChAT–CeAGABA pathway were investigated by various methods such as fluorescent micro-optical sectioning tomography, virus-based neural tracing, fibre photometry, patch-clamp and designer receptor exclusively activated by a designer drug. The role of the eLPBChAT–CeAGABA pathway in methamphetamine relapse was assessed using methamphetamine priming-induced reinstatement of conditioned place preference behaviours in male mice. We found that the eLPBChAT neurons mainly projected to the central nucleus of the amygdala. A chemogenetic activation of the eLPBChAT neurons in vitro or in vivo triggered the excitabilities of the CeAGABA neurons, which is at least in part mediated via the cholinergic receptor system. Most importantly, the chemogenetic activation of either the eLPBChAT neurons or the eLPBChAT neurons that project onto the central nucleus of the amygdala decreased the methamphetamine priming-induced reinstatement of conditioned place preference in mice. Our findings revealed a previously undiscovered cholinergic pathway of the eLPBChAT–CeAGABA and showed that the activation of this pathway decreased the methamphetamine priming-induced reinstatement of conditioned place preference.

Multiscale Analysis of Cellular Composition and Morphology in Intact Cerebral Organoids.

Simple SummaryWe have established a pipeline to analyze the structures of intact millimeter-scale cerebral organoids. By using this pipeline, the morphological and spatial distribution of neurons and GFAP-positive cells in organoids, as well as the spatial distribution of cortical neuron subtypes, were obtained by using fMOST imaging. This study introduced a new approach to monitor cellular composition and morphology of cerebral organoids.Cerebral organoids recapitulate in vivo phenotypes and physiological functions of the brain and have great potential in studying brain development, modeling diseases, and conducting neural network research. It is essential to obtain whole-mount three-dimensional (3D) images of cerebral organoids at cellular levels to explore their characteristics and applications. Existing histological strategies sacrifice inherent spatial characteristics of organoids, and the strategy for volume imaging and 3D analysis of entire organoids is urgently needed. Here, we proposed a high-resolution imaging pipeline based on fluorescent labeling by viral transduction and 3D immunostaining with fluorescence micro-optical sectioning tomography (fMOST). We were able to image intact organoids using our pipeline, revealing cytoarchitecture information of organoids and the spatial localization of neurons and glial fibrillary acidic protein positive cells (GFAP+ cells). We performed single-cell reconstruction to analyze the morphology of neurons and GFAP+ cells. Localization and quantitative analysis of cortical layer markers revealed heterogeneity of organoids. This pipeline enabled acquisition of high-resolution spatial information of millimeter-scale organoids for analyzing their cell composition and morphology.

Whole-Brain Connectome of GABAergic Neurons in the Mouse Zona Incerta

The zona incerta (ZI) is involved in various functions and may serve as an integrative node of the circuits for global behavioral modulation. However, the long-range connectivity of different sectors in the mouse ZI has not been comprehensively mapped. Here, we obtained whole-brain images of the input and output connections via fluorescence micro-optical sectioning tomography and viral tracing. The principal regions in the input-output circuits of ZI GABAergic neurons were topologically organized. The 3D distribution of cortical inputs showed rostro-caudal correspondence with different ZI sectors, while the projection fibers from ZI sectors were longitudinally organized in the superior colliculus. Clustering results show that the medial and lateral ZI are two different major functional compartments, and they can be further divided into more subdomains based on projection and input connectivity. This study provides a comprehensive anatomical foundation for understanding how the ZI is involved in integrating different information, conveying motivational states, and modulating global behaviors.

Brain-wide mapping of c-Fos expression with fluorescence micro-optical sectioning tomography in a chronic sleep deprivation mouse model

Chronic sleep deprivation (SD) is a common problem for humans and can lead to many deleterious effects, including depression, anxiety, stroke, permanent cognitive deficits, stress, and other physiological diseases. It is vital to acquire information about the relevant neural activities at the whole-brain level to systematically explore the mechanisms of brain dysfunction related to SD. Expression of the immediate-early gene (IEG) Fos in the mouse brain has been widely used as a functional marker of brain activity in the field of neuroscience. However, most previous studies only analyzed the change of c-Fos in several specific brain regions using traditional research methods or in short-term SD model. Here, we applied c-Fos mapping through the fluorescence micro-optical sectioning tomography (fMOST) technique and AAV-PHP.eB to comprehensive analysis the state of cumulative activation across the whole brain in a mouse model of chronic SD. The chronic rapid eyes movement (REM) SD model was induced by moving mice to a separate holding area filled with water. The experimental period lasted for 6 h per day. The results showed that after 14 days of SD, the mice displayed anxiety-like behaviors in open field test and elevated plus maze test, and displayed depression-like behaviors in tail suspension test and the sucrose preference test. The c-Fos + cells were detected in a maximum of 230 brain regions. SD-induced stress model evoked c-Fos expression in several brain regions compared to the control group. In particular, the isocortex-cerebral cortex plate area, including the retrosplenial, anterior cingulate, agranular insular, gustatory, and parasubiculum, appear to be the most sensitive regions after chronic REM SD.

Three-dimensional visualization of heart-wide myocardial architecture and vascular network simultaneously at single-cell resolution.

Obtaining various structures of the entire mature heart at single-cell resolution is highly desired in cardiac studies; however, effective methodologies are still lacking. Here, we propose a pipeline for labeling and imaging myocardial and vascular structures. In this pipeline, the myocardium is counterstained using fluorescent dyes and the cardiovasculature is labeled using transgenic markers. High-definition dual-color fluorescence micro-optical sectioning tomography is used to perform heart-wide tissue imaging, enabling the acquisition of whole-heart data at a voxel resolution of 0.32 × 0.32 × 1 μm3. Obtained structural data demonstrated the superiority of the pipeline. In particular, the three-dimensional morphology and spatial arrangement of reconstructed cardiomyocytes were revealed, and high-resolution vascular data helped determine differences in the features of endothelial cells and complex coiled capillaries. Our pipeline can be used in cardiac studies for examining the structures of the entire heart at the single-cell level.

Cryo-fluorescence micro-optical sectioning tomography for volumetric imaging of various whole organs with subcellular resolution

SummaryOptical visualization of complex microstructures in the entire organ is essential for biomedical research. However, the existing methods fail to accurately acquire the detailed microstructures of whole organs with good morphological and biochemical preservation. This study proposes a cryo-fluorescence micro-optical sectioning tomography (cryo-fMOST) to image whole organs in three dimensions (3D) with submicron resolution. The system comprises a line-illumination microscope module, cryo-microtome, three-stage refrigeration module, and heat insulation device. To demonstrate the imaging capacity and wide applicability of the system, we imaged and reconstructed various organs of mice in 3D, including the healthy tongue, kidney, and brain, as well as the infarcted heart. More importantly, imaged brain slices were performed sugar phosphates determination and fluorescence in situ hybridization imaging to verify the compatibility of multi-omics measurements. The results demonstrated that cryo-fMOST is capable of acquiring high-resolution morphological details of various whole organs and may be potentially useful for spatial multi-omics.

High-resolution 3D demonstration of regional heterogeneity in the glymphatic system

Accumulating evidence indicates that the glymphatic system has a critical role in maintaining brain homeostasis. However, the detailed anatomy of the glymphatic pathway is not well understood, mostly due to a lack of high spatial resolution 3D visualization. In this study, a fluorescence micro-optical sectioning tomography (fMOST) was used to characterize the glymphatic architecture in the mouse brain. At 30 and 120 min after intracisternal infusion with fluorescent dextran (Dex-3), lectin was injected to stain the cerebral vasculature. Using fMOST, a high-resolution 3D dataset of the brain-wide distribution of Dex-3 was acquired. Combined with fluorescence microscopy and microplate array, the heterogeneous glymphatic flow and the preferential irrigated regions were identified. These cerebral regions containing large-caliber penetrating arteries and/or adjacent to the subarachnoid space had more robust CSF flow compared to other regions. Moreover, the major glymphatic vessels for CSF influx and fluid efflux in the entire brain were shown in 3D. This study demonstrates the regional heterogeneity in the glymphatic system and provides an anatomical resource for further investigation of the glymphatic function.

AIE-based fluorescent micro-optical sectioning tomography for automatic 3D mapping of β-amyloid plaques in Tg mouse whole brain

Rapid verification and three-dimensional (3D) mapping of β-amyloid (Aβ) plaques in mouse whole brain is of great significance in early diagnosis of Alzheimer’s disease (AD) due to the intricate relationship of Aβ plaques with their surrounding microenvironment. Previously reported fluorescent labelling to locate Aβ plaques usually require lengthy permeation/staining/washing procedures to eliminate non-specific binding and improve fluorescence signal-to-noise ratio, which is inaccessible to automatic brain wide 3D imaging. Here we report a kind of water-soluble fluorescent probes with aggregation-induced emission (AIE) activities, named AIEgens, which can rapidly permeate and stain Aβ plaques of transgenic (Tg) mouse brain sections into 8 μm of depth within 1 min. The excellent water-solubility, specificity and sensitivity of AIEgens for Aβ plaques enable rapid staining and washing-free imaging of brain sections. The automatic 3D mapping of Aβ plaques in Tg mouse’s whole brain is implemented through the cycling process of in situ real-time mechanical sectioning, staining and imaging with AIEgens in fluorescent micro-optical sectioning tomography (fMOST) system. The AIE-based fMOST, which integrates the in-situ staining/imaging/sectioning steps of Tg mouse’s whole brain, provides an automatic 3D mapping solution to access the size, morphology and 3D distribution of Aβ plaques in mouse whole brain, which would help to explore the correlation between Aβ structure and neurodegenerative activity.

Hippocampal Mitochondrial Abnormalities Induced the Dendritic Complexity Reduction and Cognitive Decline in a Rat Model of Spinal Cord Injury.

Spinal cord injury (SCI) is a progressive neurodegenerative disease in addition to a traumatic event. Cognitive dysfunction following SCI has been widely reported in patients and animal models. However, the neuroanatomical changes affecting cognitive function after SCI, as well as the mechanisms behind these changes, have so far remained elusive. Herein, we found that SCI accelerates oxidative stress damage of hippocampal neuronal mitochondria. Then, for the first time, we presented a three-dimensional morphological atlas of rat hippocampal neurons generated using a fluorescence Micro-Optical Sectioning Tomography system, a method that accurately identifies the spatial localization of neurons and trace neurites. We showed that the number of dendritic branches and dendritic length was decreased in late stage of SCI. Western blot and transmission electron microscopy analyses also showed a decrease in synaptic communication. In addition, a battery of behavioral tests in these animals revealed hippocampal based cognitive dysfunction, which could be attributed to changes in the dendritic complexity of hippocampal neurons. Taken together, these results suggested that mitochondrial abnormalities in hippocampal neurons induced the dendritic complexity reduction and cognitive decline following SCI. Our study highlights the neuroanatomical basis and importance of mitochondria in brain degeneration following SCI, which might contribute to propose new therapeutic strategies.

High-Resolution Digital Panorama of Multiple Structures in Whole Brain of Alzheimer's Disease Mice.

Simultaneously visualizing Amyloid-β (Aβ) plaque with its surrounding brain structures at the subcellular level in the intact brain is essential for understanding the complex pathology of Alzheimer's disease, but is still rarely achieved due to the technical limitations. Combining the micro-optical sectioning tomography (MOST) system, whole-brain Nissl staining, and customized image processing workflow, we generated a whole-brain panorama of Alzheimer's disease mice without specific labeling. The workflow employed the steps that include virtual channel splitting, feature enhancement, iso-surface rendering, direct volume rendering, and feature fusion to extract and reconstruct the different signals with distinct gray values and morphologies. Taking advantage of this workflow, we found that the denser-distribution areas of Aβ plaques appeared with relatively more somata and smaller vessels, but show a dissimilar distributing pattern with nerve tracts. In addition, the entorhinal cortex and adjacent subiculum regions present the highest density and biggest diameter of plaques. The neuronal processes in the vicinity of these Aβ plaques showed significant structural alternation such as bending or abrupt branch ending. The capillaries inside or adjacent to the plaques were observed with abundant distorted micro-vessels and abrupt ending. Depicting Aβ plaques, somata, nerve processes and tracts, and blood vessels simultaneously, this panorama enables us for the first time, to analyze how the Aβ plaques interact with capillaries, somata, and processes at a submicron resolution of 3D whole-brain scale, which reveals potential pathological effects of Aβ plaques from a new cross-scale view. Our approach opens a door to routine systematic studies of complex interactions among brain components in mouse models of Alzheimer's disease.

Whole-Brain Direct Inputs to and Axonal Projections from Excitatory and Inhibitory Neurons in the Mouse Primary Auditory Area.

Neurons in the primary auditory area (AUDp) innervate multiple brain regions with long-range projections while receiving informative inputs for diverse functions. However, the brain-wide connections of these neurons have not been comprehensively investigated. Here, we simultaneously applied virus-based anterograde and retrograde tracing, labeled the connections of excitatory and inhibitory neurons in the mouse AUDp, and acquired whole-brain information using a dual-channel fluorescence micro-optical sectioning tomography system. Quantified results showed that the two types of neurons received inputs with similar patterns but sent heterogeneous projections to downstream regions. In the isocortex, functionally different areas consistently sent feedback-dominated projections to these neurons, with concomitant laterally-dominated projections from the sensory and limbic cortices to inhibitory neurons. In subcortical regions, the dorsal and medial parts of the non-lemniscal auditory thalamus (AT) were reciprocally connected to the AUDp, while the ventral part contained the most fibers of passage from the excitatory neurons and barely sent projections back, indicating the regional heterogeneity of the AUDp-AT circuit. Our results reveal details of the whole-brain network and provide new insights for further physiological and functional studies of the AUDp.

Extension of Endocardium-Derived Vessels Generate Coronary Arteries in Neonates.

Unraveling how new coronary arteries develop may provide critical information for establishing novel therapeutic approaches to treating ischemic cardiac diseases. There are 2 distinct coronary vascular populations derived from different origins in the developing heart. Understanding the formation of coronary arteries may provide insights into new ways of promoting coronary artery formation after myocardial infarction. To understand how intramyocardial coronary arteries are generated to connect these 2 coronary vascular populations, we combined genetic lineage tracing, light sheet microscopy, fluorescence micro-optical sectioning tomography, and tissue-specific gene knockout approaches to understand their cellular and molecular mechanisms. We show that a subset of intramyocardial coronary arteries form by angiogenic extension of endocardium-derived vascular tunnels in the neonatal heart. Three-dimensional whole-mount fluorescence imaging showed that these endocardium-derived vascular tunnels or tubes adopt an arterial fate in neonates. Mechanistically, we implicate Mettl3 (methyltransferase-like protein 3) and Notch signaling in regulating endocardium-derived intramyocardial coronary artery formation. Functionally, these intramyocardial arteries persist into adulthood and play a protective role after myocardial infarction. A subset of intramyocardial coronary arteries form by extension of endocardium-derived vascular tunnels in the neonatal heart.

A spatial and cellular distribution of rabies virus infection in the mouse brain revealed by fMOST and single‐cell RNA sequencing

BackgroundNeurotropic virus infection can cause serious damage to the central nervous system (CNS) in both humans and animals. The complexity of the CNS poses unique challenges to investigate the infection of these viruses in the brain using traditional techniques.MethodsIn this study, we explore the use of fluorescence micro‐optical sectioning tomography (fMOST) and single‐cell RNA sequencing (scRNA‐seq) to map the spatial and cellular distribution of a representative neurotropic virus, rabies virus (RABV), in the whole brain. Mice were inoculated with a lethal dose of a recombinant RABV encoding enhanced green fluorescent protein (EGFP) under different infection routes, and a three‐dimensional (3D) view of RABV distribution in the whole mouse brain was obtained using fMOST. Meanwhile, we pinpointed the cellular distribution of RABV by utilizing scRNA‐seq.ResultsOur fMOST data provided the 3D view of a neurotropic virus in the whole mouse brain, which indicated that the spatial distribution of RABV in the brain was influenced by the infection route. Interestingly, we provided evidence that RABV could infect multiple nuclei related to fear independent of different infection routes. More surprisingly, our scRNA‐seq data revealed that besides neurons RABV could infect macrophages and the infiltrating macrophages played at least three different antiviral roles during RABV infection.ConclusionThis study draws a comprehensively spatial and cellular map of typical neurotropic virus infection in the mouse brain, providing a novel and insightful strategy to investigate the pathogenesis of RABV and other neurotropic viruses.

Brain-Wide Mapping of c-Fos Expression with Fluorescence Micro-Optical Sectioning Tomography in a Chronic Sleep Deprivation Mouse Model

Brain-Wide Mapping of c-Fos Expression with Fluorescence Micro-Optical Sectioning Tomography in a Chronic Sleep Deprivation Mouse Model

Whole-Brain Reconstruction of Neurons in the Ventral Pallidum Reveals Diverse Projection Patterns.

The ventral pallidum (VP) integrates reward signals to regulate cognitive, emotional, and motor processes associated with motivational salience. Previous studies have revealed that the VP projects axons to many cortical and subcortical structures. However, descriptions of the neuronal morphologies and projection patterns of the VP neurons at the single neuron level are lacking, thus hindering the understanding of the wiring diagram of the VP. In this study, we used recently developed progress in robust sparse labeling and fluorescence micro-optical sectioning tomography imaging system (fMOST) to label mediodorsal thalamus-projecting neurons in the VP and obtain high-resolution whole-brain imaging data. Based on these data, we reconstructed VP neurons and classified them into three types according to their fiber projection patterns. We systematically compared the axonal density in various downstream centers and analyzed the soma distribution and dendritic morphologies of the various subtypes at the single neuron level. Our study thus provides a detailed characterization of the morphological features of VP neurons, laying a foundation for exploring the neural circuit organization underlying the important behavioral functions of VP.

Cross-modal coherent registration of whole mouse brains.

Recent whole-brain mapping projects are collecting large-scale three-dimensional images using modalities such as serial two-photon tomography, fluorescence micro-optical sectioning tomography, light-sheet fluorescence microscopy, volumetric imaging with synchronous on-the-fly scan and readout or magnetic resonance imaging. Registration of these multi-dimensional whole-brain images onto a standard atlas is essential for characterizing neuron types and constructing brain wiring diagrams. However, cross-modal image registration is challenging due to intrinsic variations of brain anatomy and artifacts resulting from different sample preparation methods and imaging modalities. We introduce a cross-modal registration method, mBrainAligner, which uses coherent landmark mapping and deep neural networks to align whole mouse brain images to the standard Allen Common Coordinate Framework atlas. We build a brain atlas for the fluorescence micro-optical sectioning tomography modality to facilitate single-cell mapping, and used our method to generate a whole-brain map of three-dimensional single-neuron morphology and neuron cell types.

Whole-brain connectivity atlas of glutamatergic and GABAergic neurons in the mouse dorsal and median raphe nuclei.

The dorsal raphe nucleus (DR) and median raphe nucleus (MR) contain populations of glutamatergic and GABAergic neurons that regulate diverse behavioral functions. However, their whole-brain input-output circuits remain incompletely elucidated. We used viral tracing combined with fluorescence micro-optical sectioning tomography to generate a comprehensive whole-brain atlas of inputs and outputs of glutamatergic and GABAergic neurons in the DR and MR. We found that these neurons received inputs from similar upstream brain regions. The glutamatergic and GABAergic neurons in the same raphe nucleus had divergent projection patterns with differences in critical brain regions. Specifically, MR glutamatergic neurons projected to the lateral habenula through multiple pathways. Correlation and cluster analysis revealed that glutamatergic and GABAergic neurons in the same raphe nucleus received heterogeneous inputs and sent different collateral projections. This connectivity atlas further elucidates the anatomical architecture of the raphe nuclei, which could facilitate better understanding of their behavioral functions.

The cytotoxicity of zinc oxide nanoparticles to 3D brain organoids results from excessive intracellular zinc ions and defective autophagy.

Although the neurotoxicity of ZnO nanoparticles (NPs) has been evaluated in animal and nerve cell culture models, these models cannot accurately mimic human brains. Three-dimensional (3D) brain organoids based on human-induced pluripotent stem cells have been developed to study the human brains, but this model has rarely been used to evaluate NP neurotoxicity. We used 3D brain organoids that express cortical layer proteins to investigate the mechanisms of ZnO NP-induced neurotoxicity. Cytotoxicity caused by high levels of ZnO NPs (64μg/mL) correlated with high intracellular Zn ion levels but not superoxide levels. Exposure to a non-cytotoxic concentration of ZnO NPs (16μg/mL) increased the autophagy-marker proteins LC3B-II/I but decreased p62 accumulation, whereas a cytotoxic concentration of ZnO NPs (64μg/mL) decreased LC3B-II/I proteins but did not affect p62 accumulation. Fluorescence micro-optical sectioning tomography revealed that 64μg/mL ZnO NPs led to decreases in LC3B proteins that were more obvious at the outer layers of the organoids, which were directly exposed to the ZnO NPs. In addition to reducing LC3B proteins in the outer layers, ZnO NPs increased the number of micronuclei in the outer layers but not the inner layers (where LC3B proteins were still expressed). Adding the autophagy flux inhibitor bafilomycin A1 to ZnO NPs increased cytotoxicity and intracellular Zn ion levels, but adding the autophagy inducer rapamycin only slightly decreased cellular Zn ion levels. We conclude that high concentrations of ZnO NPs are cytotoxic to 3D brain organoids via defective autophagy and intracellular accumulation of Zn ions.

Dl-3-N-Butylphthalide Promotes Angiogenesis in an Optimized Model of Transient Ischemic Attack in C57BL/6 Mice.

Transient ischemic attack (TIA) has been widely regarded as a clinical entity. Even though magnetic resonance imaging (MRI) results of TIA patients are negative, potential neurovascular damage might be present, and may account for long-term cognitive impairment. Animal models that simulate human diseases are essential tools for in-depth study of TIA. Previous studies have clarified that Dl-3-N-butylphthalide (NBP) promotes angiogenesis after stroke. However, the effects of NBP on TIA remain unknown. This study aims to develop an optimized TIA model in C57BL/6 mice to explore the microscopic evidence of ischemic injury after TIA, and investigate the therapeutic effects of NBP on TIA. C57BL/6 mice underwent varying durations (7, 8, 9 or 10 min) of middle cerebral artery occlusion (MCAO). Cerebral artery occlusion and reperfusion were assessed by laser speckle contrast imaging. TIA and ischemic stroke were distinguished by neurological testing and MRI examination at 24 h post-operation. Neuronal apoptosis was examined by TUNEL staining. Images of submicron cerebrovascular networks were obtained via micro-optical sectioning tomography. Subsequently, the mice were randomly assigned to a sham-operated group, a vehicle-treated TIA group or an NBP-treated TIA group. Vascular density was determined by immunofluorescent staining and fluorescein isothiocyanate method, and the expression of angiogenic growth factors were detected by western blot analysis. We found that an 8-min or shorter period of ischemia induced neither permanent neurological deficits nor MRI detectable brain lesions in C57BL/6 mice, but histologically caused neuronal apoptosis and cerebral vasculature abnormalities. NBP treatment increased the number of CD31+ microvessels and perfused microvessels after TIA. NBP also up-regulated the expression of VEGF, Ang-1 and Ang-2 and improved the cerebrovascular network. In conclusion, 8 min or shorter cerebral ischemia induced by the suture MCAO method is an appropriate TIA model in C57BL/6 mice, which conforms to the definition of human TIA, but causes microscopic neurovascular impairment. NBP treatment increased the expression of angiogenic growth factors, promoted angiogenesis and improved cerebral microvessels after TIA. Our study provides new insights on the pathogenesis and potential treatments of TIA.