Serial Two-photon Tomography Research Articles

Selective expansion of motor cortical projections in the evolution of vocal novelty.

Deciphering how cortical architecture evolves to drive behavioral innovations is a long-standing challenge in neuroscience and evolutionary biology. Here, we leverage a striking behavioral novelty in the Alston's singing mouse ( Scotinomys teguina ), compared to the laboratory mouse ( Mus musculus ), to quantitatively test models of motor cortical evolution. We used bulk tracing, serial two-photon tomography, and high-throughput DNA sequencing of over 76,000 barcoded neurons to discover a specific and substantial expansion ( ∼ 200%) of orofacial motor cortical (OMC) projections to the auditory cortical region (AudR) and the midbrain periaqueductal gray (PAG), both implicated in vocal behaviors. Moreover, analysis of individual OMC neurons' projection motifs revealed preferential expansion of exclusive projections to AudR. Our results imply that selective expansion of ancestral motor cortical projections can underlie behavioral divergence over short evolutionary timescales, suggesting potential mechanisms for the evolution of enhanced cortical control over vocalizations-a crucial preadaptation for human language.

Obesity and the cerebral cortex: Underlying neurobiology in mice and humans

Obesity is a major modifiable risk factor for Alzheimer’s disease (AD), characterized by progressive atrophy of the cerebral cortex. The neurobiology of obesity contributions to AD is poorly understood. Here we show with in vivo MRI that diet-induced obesity decreases cortical volume in mice, and that higher body adiposity associates with lower cortical volume in humans. Single-nuclei transcriptomics of the mouse cortex reveals that dietary obesity promotes an array of neuron-adverse transcriptional dysregulations, which are mediated by an interplay of excitatory neurons and glial cells, and which involve microglial activation and lowered neuronal capacity for neuritogenesis and maintenance of membrane potential. The transcriptional dysregulations of microglia, more than of other cell types, are like those in AD, as assessed with single-nuclei cortical transcriptomics in a mouse model of AD and two sets of human donors with the disease. Serial two-photon tomography of microglia demonstrates microgliosis throughout the mouse cortex. The spatial pattern of adiposity-cortical volume associations in human cohorts interrogated together with in silico bulk and single-nucleus transcriptomic data from the human cortex implicated microglia (along with other glial cells and subtypes of excitatory neurons), and it correlated positively with the spatial profile of cortical atrophy in patients with mild cognitive impairment and AD. Thus, multi-cell neuron-adverse dysregulations likely contribute to the loss of cortical tissue in obesity. The dysregulations of microglia may be pivotal to the obesity-related risk of AD.

High-Resolution Three-dimensional Whole-organ Tomography of Microbial Infections.

Most infections take place within three-dimensional host tissues with intricate anatomy and locally varying host physiology. The positioning of pathogen cells within this diverse environment significantly affects their stress levels, responses, fate, and contribution to the overall progression of the disease and treatment failure. However, due to the technical difficulties in locating µm-sized pathogen cells within cm-sized host organs, this area of research has been relatively unexplored. Here, we present a method for addressing this challenge. We employ serial two-photon tomography and AI-enhanced image analysis to locate individual Salmonella cells throughout the entire spleen, liver lobes, and whole lymph nodes of infected mice. Using fluorescent reporters and in vivo antibody administration, the replication rate of single Salmonella cells, their local interaction with specific immune cells, and bacterial responses to antibiotics can be determined. These methodologies open avenues for a comprehensive examination of infections, their prevention, and treatment within the three-dimensional tissue context.

Abstract TMP120: Contralesional Corticospinal Tract Reinnervates Cervical Spinal Anatomical Regions With Highest Pre-Lesion Synapse Density After Unilateral M1 Stroke in Mice

Introduction: Stroke leaves many patients with significant motor impairment. Following ischemic lesions of the rodent primary motor cortex, sprouting of the contralesional corticospinal tract (cCST) into the injured hemicord is associated with improved motor recovery. In adult mice, most corticospinal neurons project to spinal interneurons, and few synapse directly on alpha motor neurons. We examined the distribution of newly formed corticospinal synapses in the mouse spinal cord, using genetically encoded tracers, 3D microscopy, and a spinal atlas with machine learning-based automated classification and registration. Hypothesis: We tested the hypothesis that cCST neurons innervate homotopic regions in the injured hemicord after unilateral motor cortex stroke in adult mice. Method: We induced a photothrombotic motor cortex stroke or performed sham surgery (n=4 per group) in 8-11 week old male C57/B6 mice and administered a contralesional motor cortex injection of an anterograde adeno-associated virus expressing both membrane-targeted tdTomato and synaptically-targeted eGFP. Cervical spinal cords were subjected to volumetric imaging via serial two-photon tomography (TissueCyte 1000) to characterize synaptic and axonal density of cCST collaterals in whole cervical spinal cords in both stroke and sham mice. Unbiased, global quantification of axonal and synaptic density across the entire cervical cord was accomplished by the development of a custom automated image analysis pipeline, incorporating a novel 3D spinal cord reference volume, published spinal cord annotations comprising 47 distinct anatomical regions (SpinalJ), and machine learning based pixel classification. Results: We observed cCST synapses in the 6-week post-stroke injured hemicord innervating lamina IV & IX most densely at C7 & C8 (See Figure). Conclusions: Contralesional CST reinnervation of the denervated hemicord is targeted to both lamina and cervical level specific.

Abstract 12907: Aging Drives Cerebrovascular Network Remodeling and Functional Changes in the Mouse Brain

Introduction: Cerebrovascular dysfunction has been implicated in age-related cognitive decline and dementia, but the underlying vascular mechanisms are not well understood. An improved understanding of the nature of normal cerebrovascular aging is needed to help to establish the role that vascular dysfunction might play in cognitive decline and dementia. Methods: Here, we asked how normal aging differentially impacts the vascular structure and function in different brain areas in mice. We investigated structural changes in aged cerebrovascular networks and pericytes utilizing serial two-photon tomography (STPT). To further investigate potential remodeling of different vascular compartments and pericyte subtypes, we utilized tissue clearing, 3D immunolabeling, and light sheet fluorescence microscopy (LSFM) imaging. We also assessed how healthy aging impacts brain hemodynamics in response to voluntary locomotion and whisker stimulation in awake, head-fixed mice using wide field optical signal imaging and two-photon imaging. We tested mice of both sexes at 2-month, 18-month (early aging), and 24-month (late aging) of age. Results: Whole-brain vascular tracing using STPT showed an overall ~10% decrease in vascular length and branching density, and LSFM imaging with 3D immunolabeling further revealed increased arteriole tortuosity in aged brains. We also uncovered a selective vascular and pericyte loss in deep cortical layers, basal forebrain regions, and the hippocampal network. This may contribute to their regional vulnerabilities in neurodegenerative disorders. Moreover, our in vivo imaging in awake, head-fixed mice identified delayed neurovascular coupling response and inefficient oxygen delivery in aged brains. Conclusions: Our study reveals aging-related brain-wide changes in vascular and mural cell types that can explain vulnerability and resilience of different brain areas in normal aging. Moreover, we identified an age-related decrease in brain oxygenation and delayed neurovascular coupling responses which can be linked with cognitive impairment in aged brains. These aging-related changes will serve as a common factor to understand many neurodegenerative disorders and cognition decline in the elderly population.

Columnar and diffuse topographies in the marmoset prefrontal connectome

In this issue of Neuron, Watakabe etal.1 utilize serial two-photon tomography to reveal that the intra- and inter-regional prefrontal cortex projections in the marmoset brain terminate with two characteristic patterns, columnar and diffused, both of which display a topographically organized gradient.

Mouse brain MR super-resolution using a deep learning network trained with optical imaging data.

The resolution of magnetic resonance imaging is often limited at the millimeter level due to its inherent signal-to-noise disadvantage compared to other imaging modalities. Super-resolution (SR) of MRI data aims to enhance its resolution and diagnostic value. While deep learning-based SR has shown potential, its applications in MRI remain limited, especially for preclinical MRI, where large high-resolution MRI datasets for training are often lacking. In this study, we first used high-resolution mouse brain auto-fluorescence (AF) data acquired using serial two-photon tomography (STPT) to examine the performance of deep learning-based SR for mouse brain images. We found that the best SR performance was obtained when the resolutions of training and target data were matched. We then applied the network trained using AF data to MRI data of the mouse brain, and found that the performance of the SR network depended on the tissue contrast presented in the MRI data. Using transfer learning and a limited set of high-resolution mouse brain MRI data, we were able to fine-tune the initial network trained using AF to enhance the resolution of MRI data. Our results suggest that deep learning SR networks trained using high-resolution data of a different modality can be applied to MRI data after transfer learning.

Integrating multimodality magnetic resonance imaging to the Allen Mouse Brain Common Coordinate Framework.

High-resolution magnetic resonance imaging (MRI) affords unique image contrasts to nondestructively probe the tissue microstructure; validation of MRI findings with conventional histology is essential to better understand the MRI contrasts. However, the dramatic difference in the spatial resolution and image contrast of these two techniques impedes accurate comparison between MRI metrics and traditional histology. To better validate various MRI metrics, we acquired whole mouse brain multigradient recalled-echo and multishell diffusion MRI datasets at 25-μm isotropic resolution. The recently developed Allen Mouse Brain Common Coordinate Framework (CCFv3) provides opportunities to integrate multimodal and multiscale datasets of the whole mouse brain in a common three-dimensional (3D) space. The T2*, quantitative susceptibility mapping, diffusion tensor imaging, and neurite orientation dispersion and density imaging parameters were compared with both serial two-photon tomography images and 3D Nissl staining images in the CCFv3 at the same spatial resolution. The correlation between MRI and Nissl staining strongly depends on different metrics and different regions of the brain. Integrating different imaging modalities to the same space may substantially improve our understanding of the complexity of the brain at different scales.

Mapping brain-wide excitatory projectome of primate prefrontal cortex at submicron resolution and comparison with diffusion tractography.

Resolving trajectories of axonal pathways in the primate prefrontal cortex remains crucial to gain insights into higher-order processes of cognition and emotion, which requires a comprehensive map of axonal projections linking demarcated subdivisions of prefrontal cortex and the rest of brain. Here, we report a mesoscale excitatory projectome issued from the ventrolateral prefrontal cortex (vlPFC) to the entire macaque brain by using viral-based genetic axonal tracing in tandem with high-throughput serial two-photon tomography, which demonstrated prominent monosynaptic projections to other prefrontal areas, temporal, limbic, and subcortical areas, relatively weak projections to parietal and insular regions but no projections directly to the occipital lobe. In a common 3D space, we quantitatively validated an atlas of diffusion tractography-derived vlPFC connections with correlative green fluorescent protein-labeled axonal tracing, and observed generally good agreement except a major difference in the posterior projections of inferior fronto-occipital fasciculus. These findings raise an intriguing question as to how neural information passes along long-range association fiber bundles in macaque brains, and call for the caution of using diffusion tractography to map the wiring diagram of brain circuits.

Small Extracellular Vesicles in Milk Cross the Blood-Brain Barrier in Murine Cerebral Cortex Endothelial Cells and Promote Dendritic Complexity in the Hippocampus and Brain Function in C57BL/6J Mice.

Human milk contains large amounts of small extracellular vesicles (sEVs) and their microRNA cargos, whereas infant formulas contain only trace amounts of sEVs and microRNAs. We assessed the transport of sEVs across the blood-brain barrier (BBB) and sEV accumulation in distinct regions of the brain in brain endothelial cells and suckling mice. We further assessed sEV-dependent gene expression profiles and effects on the dendritic complexity of hippocampal granule cells and phenotypes of EV depletion in neonate, juvenile and adult mice. The transfer of sEVs across the BBB was assessed by using fluorophore-labeled bovine sEVs in brain endothelial bEnd.3 monolayers and dual chamber systems, and in wild-type newborn pups fostered to sEV and cargo tracking (ECT) dams that express sEVs labeled with a CD63-eGFP fusion protein for subsequent analysis by serial two-photon tomography and staining with anti-eGFP antibodies. Effects of EVs on gene expression and dendritic architecture of granule cells was analyzed in hippocampi from juvenile mice fed sEV and RNA-depleted (ERD) and sEV and RNA-sufficient (ERS) diets by using RNA-sequencing analysis and Golgi-Cox staining followed by integrated neuronal tracing and morphological analysis of neuronal dendrites, respectively. Spatial learning and severity of kainic acid-induced seizures were assessed in mice fed ERD and ERS diets. bEnd.3 cells internalized sEVs by using a saturable transport mechanism and secreted miR-34a across the basal membrane. sEVs penetrated the entire brain in fostering experiments; major regions of accumulation included the hippocampus, cortex and cerebellum. Two hundred ninety-five genes were differentially expressed in hippocampi from mice fed ERD and ERS diets; high-confidence gene networks included pathways implicated in axon guidance and calcium signaling. Juvenile pups fed the ERD diet had reduced dendritic complexity of dentate granule cells in the hippocampus, scored nine-fold lower in the Barnes maze test of spatial learning and memory, and the severity of seizures was 5-fold higher following kainic acid administration in adult mice fed the ERD diet compared to mice fed the ERS diet. We conclude that sEVs cross the BBB and contribute toward optimal neuronal development, spatial learning and memory, and resistance to kainic acid-induced seizures in mice.

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.

Three-Dimensional Iron Oxide Nanoparticle-Based Contrast-Enhanced Magnetic Resonance Imaging for Characterization of Cerebral Arteriogenesis in the Mouse Neocortex.

Purpose: Subsurface blood vessels in the cerebral cortex have been identified as a bottleneck in cerebral perfusion with the potential for collateral remodeling. However, valid techniques for non-invasive, longitudinal characterization of neocortical microvessels are still lacking. In this study, we validated contrast-enhanced magnetic resonance imaging (CE-MRI) for in vivo characterization of vascular changes in a model of spontaneous collateral outgrowth following chronic cerebral hypoperfusion.Methods: C57BL/6J mice were randomly assigned to unilateral internal carotid artery occlusion or sham surgery and after 21 days, CE-MRI based on T2*-weighted imaging was performed using ultra-small superparamagnetic iron oxide nanoparticles to obtain subtraction angiographies and steady-state cerebral blood volume (ss-CBV) maps. First pass dynamic susceptibility contrast MRI (DSC-MRI) was performed for internal validation of ss-CBV. Further validation at the histological level was provided by ex vivo serial two-photon tomography (STP).Results: Qualitatively, an increase in vessel density was observed on CE-MRI subtraction angiographies following occlusion; however, a quantitative vessel tracing analysis was prone to errors in our model. Measurements of ss-CBV reliably identified an increase in cortical vasculature, validated by DSC-MRI and STP.Conclusion: Iron oxide nanoparticle-based ss-CBV serves as a robust, non-invasive imaging surrogate marker for neocortical vessels, with the potential to reduce and refine preclinical models targeting the development and outgrowth of cerebral collateralization.

Combined whole-organ imaging at single-cell resolution and immunohistochemical analysis of prostate cancer and its liver and brain metastases.

SUMMARYEarly steps of cancer initiation and metastasis, while critical for understanding disease mechanisms, are difficult to visualize and study. Here, we describe an approach to study the processes of initiation, progression, and metastasis of prostate cancer (PC) in a genetically engineered RapidCaP mouse model, which combines whole-organ imaging by serial two-photon tomography (STPT) and post hoc thick-section immunofluorescent (IF) analysis. STPT enables the detection of single tumor-initiating cells within the entire prostate, and consequent IF analysis reveals a transition from normal to transformed epithelial tissue and cell escape from the tumor focus. STPT imaging of the liver and brain reveal the distribution of multiple metastatic foci in the liver and an early-stage metastatic cell invasion in the brain. This imaging and data analysis pipeline can be readily applied to other mouse models of cancer, offering a highly versatile whole-organ platform to study in situ mechanisms of cancer initiation and progression.

Brain microvascular damage linked to a moderate level of strain induced by controlled cortical impact

Cerebral blood vessels play an important role in brain metabolic activity in general and following traumatic brain injury (TBI) in particular. However, the extent to which TBI alters microvessel structure is not well understood. Specifically, how intracranial mechanical responses produced during impacts relate to vascular damage needs to be better studied. Therefore, the objective of this study was to investigate the biomechanical mechanisms and thresholds of brain microvascular injury. Detailed microvascular damage of mouse brain was quantified using Arterial Spin Labeling (ASL) magnetic resonance imaging (MRI) and ex vivo Serial Two-Photon Tomography (STPT) in seven mice that had undergone controlled cortical impact. Mechanical strains were investigated through finite element (FE) modeling of the mouse brain. We then compared the post-injury vessel density map with FE-predicted strain and found a moderate correlation between the vessel length density and the predicted peak maximum principal strains (MPS) (R2 = 0.52). High MPS was observed at the impact regions with low vessel length density, supporting the mechanism of strain-triggered microvascular damage. Using logistic regression, the MPS corresponding to a 50% probability of injury was found to be 0.17. Given the literature reporting MPS of over 0.2 in the human brain for mild TBI/concussion cases, it is highly recommended to consider microvascular damage when investigating mild TBI/concussion in the future.

3D Whole-Brain Imaging Approaches to Study Brain Tumors.

Simple SummaryBrain tumors integrate into the brain and consist of tumor cells with different molecular alterations. During brain tumor pathogenesis, a variety of cell types surround the tumors to either inhibit or promote tumor growth. These cells are collectively referred to as the tumor microenvironment. Three-dimensional and/or longitudinal visualization approaches are needed to understand the growth of these tumors in time and space. In this review, we present three imaging modalities that are suitable or that can be adapted to study the volumetric distribution of malignant or tumor-associated cells in the brain. In addition, we highlight the potential clinical utility of some of the microscopy approaches for brain tumors using exemplars from solid tumors.Although our understanding of the two-dimensional state of brain tumors has greatly expanded, relatively little is known about their spatial structures. The interactions between tumor cells and the tumor microenvironment (TME) occur in a three-dimensional (3D) space. This volumetric distribution is important for elucidating tumor biology and predicting and monitoring response to therapy. While static 2D imaging modalities have been critical to our understanding of these tumors, studies using 3D imaging modalities are needed to understand how malignant cells co-opt the host brain. Here we summarize the preclinical utility of in vivo imaging using two-photon microscopy in brain tumors and present ex vivo approaches (light-sheet fluorescence microscopy and serial two-photon tomography) and highlight their current and potential utility in neuro-oncology using data from solid tumors or pathological brain as examples.

Abstract P367: Brain-Wide Quantification of Amyloid in Transgenic Mice Using Volumetric Imaging and Automated Image Analysis

Localizing and quantifying amyloid in three dimensions furthers understanding of spatial patterns of amyloid deposition and disease progression in experimental models of cerebral amyloid angiopathy (CAA). Serial two-photon tomography (STPT) is ideal for this purpose because it produces 3D datasets of the whole mouse brain in a matter of hours. We present an optimized pipeline for fluorescent whole-brain labeling of amyloid and vasculature, STPT imaging of labeled brains, machine learning-aided classification of vascular and parenchymal amyloid, registration of images to a standard reference atlas, and automated quantification of these signals across brain regions. We used traditional histological sectioning and slide mounted imaging to compare and optimize published methods of fluorescent amyloid and vascular labeling. An intraperitoneal injection of methoxy-X04 delivered the day before euthanasia was chosen to label amyloid. Cerebral vessels are visualized by transcardial perfusion of rhodamine-B isothiocyanate in gelatin. Labeled brains are imaged by STPT and rendered in 3D to appreciate regional amyloid distribution. Volumetric image datasets are quantified using a modification of our custom image analysis pipeline. Pixels are classified as vascular or parenchymal amyloid with supervised machine learning methods. Classified signals are registered into the Allen Institute for Brain Science Common Coordinate Framework version 3.0 for quantification. This strategy produces high resolution volumetric images amenable to automatic quantification of parenchymal and vascular amyloid deposits throughout the brain, appropriate for studies involving large numbers of experimental brains in rodent models of CAA. Figure 1: A 3D rendering of prediction probability maps of a whole labelled mouse brain depicting fluorescent signals that were automatically classified as parenchymal amyloid (cyan), vascular amyloid (yellow) and brain parenchyma.

Characterizing region-specific lymphocyte migration within the post-stroke brain

Abstract Background Studies in our lab show that while B cell-depleted mice exhibit motor deficits, impaired neurogenesis, increased anxiety, and spatial memory deficits after stroke, CD8 T cell depletion reduces post-stroke motor deficits. Therefore, we hypothesized that B cells migrate into brain regions outside the infarct to promote post-stroke motor and cognitive recovery, unlike pro-inflammatory CD8 T cells that migrate to the area of injury. Methods Naïve donor B or CD8 T cells were labeled with e450 dye and injected i.v. into recipient mice at 7, 24, 48 and 72h after receiving a 60-minute transient middle cerebral artery occlusion (tMCAo). Peripheral and brain lymphocyte migration was assessed using flow cytometry and serial two-photon tomography (STPT) 96h after tMCAo. Results e450+ lymphocytes were detected in the periphery 96h after stroke. STPT co-registered with the Common Coordinate Framework (Allen Institute) created a 3D brain reconstruction that identified lymphocyte diapedesis. B cells were found bilaterally in areas outside the ischemic penumbra, such as the olfactory areas (p<0.05) and dentate gyrus (p<0.05), whereas CD8 T cells migrated into ipsilesional areas including the somatosensory cortex (p<0.01) and brainstem (p<0.01). Conclusions B cell diapedesis differs distinctly from that of CD8 T cells throughout the ischemic brain. While B cells migrate bilaterally into brain regions mediating cognitive deficits and neurogenesis, CD8 T cells exhibit greater unilateral migration into the injured hemisphere, including motor and sensory-related areas. Future directions will determine if the spatial location of CD8 T cells or B cells mediates post-stroke pathology or supports functional recovery, respectively.

The Allen Mouse Brain Common Coordinate Framework: A 3D Reference Atlas

Recent large-scale collaborations are generating major surveys of cell types and connections in the mouse brain, collecting large amounts of data across modalities, spatial scales, and brain areas. Successful integration of these data requires a standard 3D reference atlas. Here, we present the Allen Mouse Brain Common Coordinate Framework (CCFv3) as such a resource. We constructed an average template brain at 10μm voxel resolution by interpolating high resolution in-plane serial two-photon tomography images with 100μm z-sampling from 1,675 young adult C57BL/6J mice. Then, using multimodal reference data, we parcellated the entire brain directly in 3D, labeling every voxel with a brain structure spanning 43 isocortical areas and their layers, 329 subcortical gray matter structures, 81 fiber tracts, and 8 ventricular structures. CCFv3 can be used to analyze, visualize, and integrate multimodal and multiscale datasets in 3D and is openly accessible (https://atlas.brain-map.org/).

B cells migrate into remote brain areas and support neurogenesis and functional recovery after focal stroke in mice

Lymphocytes infiltrate the stroke core and penumbra and often exacerbate cellular injury. B cells, however, are lymphocytes that do not contribute to acute pathology but can support recovery. B cell adoptive transfer to mice reduced infarct volumes 3 and 7 d after transient middle cerebral artery occlusion (tMCAo), independent of changing immune populations in recipient mice. Testing a direct neurotrophic effect, B cells cocultured with mixed cortical cells protected neurons and maintained dendritic arborization after oxygen-glucose deprivation. Whole-brain volumetric serial two-photon tomography (STPT) and a custom-developed image analysis pipeline visualized and quantified poststroke B cell diapedesis throughout the brain, including remote areas supporting functional recovery. Stroke induced significant bilateral B cell diapedesis into remote brain regions regulating motor and cognitive functions and neurogenesis (e.g., dentate gyrus, hypothalamus, olfactory areas, cerebellum) in the whole-brain datasets. To confirm a mechanistic role for B cells in functional recovery, rituximab was given to human CD20+ (hCD20+) transgenic mice to continuously deplete hCD20+-expressing B cells following tMCAo. These mice experienced delayed motor recovery, impaired spatial memory, and increased anxiety through 8 wk poststroke compared to wild type (WT) littermates also receiving rituximab. B cell depletion reduced stroke-induced hippocampal neurogenesis and cell survival. Thus, B cell diapedesis occurred in areas remote to the infarct that mediated motor and cognitive recovery. Understanding the role of B cells in neuronal health and disease-based plasticity is critical for developing effective immune-based therapies for protection against diseases that involve recruitment of peripheral immune cells into the injured brain.

Abstract 121: Visualization and Quantification of Post-Stroke Neural Connectivity in Mice Using Serial Two-Photon Tomography

Background: It is a challenge to characterize post-stroke changes in neural connectivity at microscopic scale across the entire rodent brain. Serial two-photon tomography (STPT) is an advanced laser-scanning microscopy technique which collects serial fluorescence images across the brain and reconstructs 3D datasets. We examined changes in motor connectivity after cortical infarcts in mice, using retrograde viral tract tracing, STPT imaging, automated registration workflow, and machine learning algorithms. Methods: Young male C57/B6 mice received a photothrombotic motor cortex (M1) stroke (n=3) or sham surgery (n=3). 15 days later, a retrograde pseudorabies trans-synaptic virus encoding fluorescent protein was injected into the left forelimb flexor muscles to label motor system projections. Mice were sacrificed 3 weeks post-stroke. STPT images were analyzed using supervised machine learning (pixel-wise random forest via the “ilastik” software package) and datasets were mapped to the Allen Mouse Brain Atlas for region-specific visualization and quantification of fluorescent signals. Results: Machine learning algorithms successfully identified neuronal cell bodies, neuronal processes, and ischemic tissue throughout the brain. The fluorescent signal of cells and neuronal processes was higher in the right M1 and SS of uninjured mice than the left M1 and SS. After stroke, this signal was diminished in the right M1 and SS. Labeled neurons were also reduced in the left M1 suggesting the presence of secondary transcortical connections. Conclusions: STPT generates whole brain datasets that when analyzed with ML algorithms show early alterations in post-stroke neural connectivity in the corticospinal tract. Further studies utilizing monosynaptic and conditional viral tracers will better assess the full spectrum of connectivity changes during post-stroke functional recovery.