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Aspirin treatment does not increase microhemorrhage size in young or aged mice

Sandy Chan, Morgan Brophy, Nozomi Nishimura, Chris B. Schaffer

PLoS ONE (2019)

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Microhemorrhages are common in the aging brain and are thought to contribute to cognitive decline and the development of neurodegenerative diseases, such as Alzheimer’s disease. Chronic aspirin therapy is widespread in older individuals and decreases the risk of coronary artery occlusions and stroke. There remains a concern that such aspirin usage may prolong bleeding after a vessel rupture in the brain, leading to larger bleeds that cause more damage to the surrounding tissue. Here, we aimed to understand the influence of aspirin usage on the size of cortical microhemorrhages and explored the impact of age. We used femtosecond laser ablation to rupture arterioles in the cortex of both young (2–5 months old) and aged (18–29 months old) mice dosed on aspirin in their drinking water and measured the extent of penetration of both red blood cells and blood plasma into the surrounding tissue. We found no difference in microhemorrhage size for both young and aged mice dosed on aspirin, as compared to controls (hematoma diameter = 104 +/- 39 (97 +/- 38) μm in controls and 109 +/- 25 (101 +/- 28) μm in aspirin-treated young (aged) mice; mean +/- SD). In contrast, young mice treated with intravenous heparin had an increased hematoma diameter of 136 +/- 44 μm. These data suggest that aspirin does not increase the size of microhemorrhages, supporting the safety of aspirin usage.

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Brain Capillary Networks Across Species: A few Simple Organizational Requirements Are Sufficient to Reproduce Both Structure and Function

Amy F. Smith, Vincent Doyeux, Maxime Berg, Myriam Peyrounette, Mohammad Haft-Javaherian, Anne-Edith Larue, John H. Slater, Frédéric Lauwer, Pablo Blinder, Philbert Tsai, David Kleinfeld, Chris B. Schaffer, Nozomi Nishimura, Yohan Davit and Sylvie Lorthois

Frontiers in physiology (2019)

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Despite the key role of the capillaries in neurovascular function, a thorough characterization of cerebral capillary network properties is currently lacking. Here, we define a range of metrics (geometrical, topological, flow, mass transfer, and robustness) for quantification of structural differences between brain areas, organs, species, or patient populations and, in parallel, digitally generate synthetic networks that replicate the key organizational features of anatomical networks (isotropy, connectedness, space-filling nature, convexity of tissue domains, characteristic size). To reach these objectives, we first construct a database of the defined metrics for healthy capillary networks obtained from imaging of mouse and human brains. Results show that anatomical networks are topologically equivalent between the two species and that geometrical metrics only differ in scaling. Based on these results, we then devise a method which employs constrained Voronoi diagrams to generate 3D model synthetic cerebral capillary networks that are locally randomized but homogeneous at the network-scale. With appropriate choice of scaling, these networks have equivalent properties to the anatomical data, demonstrated by comparison of the defined metrics. The ability to synthetically replicate cerebral capillary networks opens a broad range of applications, ranging from systematic computational studies of structure-function relationships in healthy capillary networks to detailed analysis of pathological structural degeneration, or even to the development of templates for fabrication of 3D biomimetic vascular networks embedded in tissue-engineered constructs.

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Deep convolutional neural networks for segmenting 3D in vivo multiphoton images of vasculature in Alzheimer disease mouse models

Mohammad Haft-Javaherian, Linjing Fang, Victorine Muse, Chris B. Schaffer, Nozomi Nishimura, Mert R. Sabuncu

PLoS ONE (2019)

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The health and function of tissue rely on its vasculature network to provide reliable blood perfusion. Volumetric imaging approaches, such as multiphoton microscopy, are able to generate detailed 3D images of blood vessels that could contribute to our understanding of the role of vascular structure in normal physiology and in disease mechanisms. The segmentation of vessels, a core image analysis problem, is a bottleneck that has prevented the systematic comparison of 3D vascular architecture across experimental populations. We explored the use of convolutional neural networks to segment 3D vessels within volumetric in vivo images acquired by multiphoton microscopy. We evaluated different network architectures and machine learning techniques in the context of this segmentation problem. We show that our optimized convolutional neural network architecture with a customized loss function, which we call DeepVess, yielded a segmentation accuracy that was better than state-of-the-art methods, while also being orders of magnitude faster than the manual annotation. To explore the effects of aging and Alzheimer’s disease on capillaries, we applied DeepVess to 3D images of cortical blood vessels in young and old mouse models of Alzheimer’s disease and wild type littermates. We found little difference in the distribution of capillary diameter or tortuosity between these groups, but did note a decrease in the number of longer capillary segments (>75μm) in aged animals as compared to young, in both wild type and Alzheimer’s disease mouse models.

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Endothelial cells promote 3D invasion of GBM by IL-8-dependent induction of cancer stem cell properties

Michael G. McCoy, Dennis Nyanyo, Carol K. Hung, Julian Palacios Goerger, Warren R. Zipfel, Rebecca M. Williams, Nozomi Nishimura & Claudia Fischbach

Scientific Reports (2019)

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Rapid growth and perivascular invasion are hallmarks of glioblastoma (GBM) that have been attributed to the presence of cancer stem-like cells (CSCs) and their association with the perivascular niche. However, the mechanisms by which the perivascular niche regulates GBM invasion and CSCs remain poorly understood due in part to a lack of relevant model systems. To simulate perivascular niche conditions and analyze consequential changes of GBM growth and invasion, patient-derived GBM spheroids were co-cultured with brain endothelial cells (ECs) in microfabricated collagen gels. Integrating these systems with 3D imaging and biochemical assays revealed that ECs increase GBM invasiveness and growth through interleukin-8 (IL-8)-mediated enrichment of CSCs. Blockade of IL-8 inhibited these effects in GBM-EC co-cultures, while IL-8 supplementation increased CSC-mediated growth and invasion in GBM-monocultures. Experiments in mice confirmed that ECs and IL-8 stimulate intracranial tumor growth and invasion in vivo. Collectively, perivascular niche conditions promote GBM growth and invasion by increasing CSC frequency, and IL-8 may be explored clinically to inhibit these interactions.

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Neutrophil adhesion in brain capillaries reduces cortical blood flow and impairs memory function in Alzheimer’s disease mouse models

Jean C. Cruz Hernández, Oliver Bracko, Calvin J. Kersbergen , Victorine Muse , Mohammad Haft-Javaherian, Maxime Berg, Laibaik Park , Lindsay K. Vinarcsik, Iryna Ivasyk, Daniel A. Rivera, Yiming Kang, Marta Cortes-Canteli, Myriam Peyrounette, Vincent Doyeux, Amy Smith , Joan Zhou, Gabriel Otte, Jeffrey D. Beverly, Elizabeth Davenport, Yohan Davit, Charles P. Lin, Sidney Strickland, Costantino Iadecola , Sylvie Lorthois , Nozomi Nishimura , Chris B. Schaffer 

Nature Neuroscience (2019)

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Cerebral blood flow (CBF) reductions in Alzheimer’s disease patients and related mouse models have been recognized for decades, but the underlying mechanisms and resulting consequences for Alzheimer’s disease pathogenesis remain poorly understood. In APP/PS1 and 5xFAD mice we found that an increased number of cortical capillaries had stalled blood flow as compared to in wild-type animals, largely due to neutrophils that had adhered in capillary segments and blocked blood flow. Administration of antibodies against the neutrophil marker Ly6G reduced the number of stalled capillaries, leading to both an immediate increase in CBF and rapidly improved performance in spatial and working memory tasks. This study identified a previously uncharacterized cellular mechanism that explains the majority of the CBF reduction seen in two mouse models of Alzheimer’s disease and demonstrated that improving CBF rapidly enhanced short-term memory function. Restoring cerebral perfusion by preventing neutrophil adhesion may provide a strategy for improving cognition in Alzheimer’s disease patients

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Surgical preparations, labeling strategies, and optical techniques for cell-resolved, in vivo imaging in the mouse spinal cord

Yu-Ting Cheng, Kawasi M.Lett, Chris B.Schaffer

Experimental Neurobiology (2019)

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n vivo optical imaging has enabled detailed studies of cellular dynamics in the brain of rodents in both healthy and diseased states. Such studies were made possible by three advances: surgical preparations that give optical access to the brain; strategies for in vivo labeling of cells with structural and functional fluorescent indicators; and optical imaging techniques that are relatively insensitive to light scattering by tissue. In vivo imaging in the rodent spinal cord has lagged behind than that in the brain, largely due to the anatomy around the spinal cord that complicates the surgical preparation, and to the strong optical scattering of the dorsal white matter that limits the ability to image deep into the spinal cord. Here, we review recent advances in surgical methods, labeling strategies, and optical tools that have enabled in vivo, high-resolution imaging of the dynamic behaviors of cells in the spinal cord in mice. Surgical preparations that enable long-term optical access and robust stabilization of the spinal cord are now available. Labeling strategies that have been used in the spinal cord tend to follow those that have been used in the brain, and some recent advances in genetically-encoded labeling strategies remain to be capitalized on. The optical imaging methods used to date, including two photon excited fluorescence microscopy, are largely limited to imaging the superficial layers of the spinal cord by the optical scattering of the white matter. Finally, we show preliminary data that points to the use of higher-order nonlinear optical processes, such as three photon excited fluorescence, as a means to image deeper into the mouse spinal cord.

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Apoε4 disrupts neurovascular regulation and undermines white matter integrity and cognitive function

Kenzo Koizumi, Yorito Hattori, Sung Ji Ahn, Izaskun Buendia, Antonio Ciacciarelli, Ken Uekawa, Gang Wang, Abigail Hiller, Lingzhi Zhao, Henning U. Voss, Steven M. Paul, Chris Schaffer, Laibaik Park & Costantino Iadecola

Nature Communications (2018)

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The ApoE4 allele is associated with increased risk of small vessel disease, which is a cause of vascular cognitive impairment. Here, we report that mice with targeted replacement (TR) of the ApoE gene with human ApoE4 have reduced neocortical cerebral blood flow compared to ApoE3-TR mice, an effect due to reduced vascular density rather than slowing of microvascular red blood cell flow. Furthermore, homeostatic mechanisms matching local delivery of blood flow to brain activity are impaired in ApoE4-TR mice. In a model of cerebral hypoperfusion, these cerebrovascular alterations exacerbate damage to the white matter of the corpus callosum and worsen cognitive dysfunction. Using 3-photon microscopy we found that the increased white matter damage is linked to an enhanced reduction of microvascular flow resulting in local hypoxia. Such alterations may be responsible for the increased susceptibility to hypoxic-ischemic lesions in the subcortical white matter of individuals carrying the ApoE4 allele.

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Diverse Inflammatory Response After Cerebral Microbleeds Includes Coordinated Microglial Migration and Proliferation

Sung Ji Ahn, Josef Anrather, Nozomi Nishimura, Chris B. Schaffer

Stroke (2018)

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Cerebral microbleeds are linked to cognitive decline, but it remains unclear how they impair neuronal function. Infarction is not typically observed near microbleeds, suggesting more subtle mechanisms, such as inflammation, may play a role. Because of their small size and largely asymptomatic nature, real-time detection and study of spontaneous cerebral microbleeds in humans and animal models are difficult. We used in vivo 2-photon microscopy through a chronic cranial window in adult mice to follow the inflammatory response after a cortical microhemorrhage of ≈100 μm diameter, induced by rupturing a targeted cortical arteriole with a laser. The inflammatory response included the invasion of blood-borne leukocytes, the migration and proliferation of brain-resident microglia, and the activation of astrocytes. Nearly all inflammatory cells responding to the microhemorrhage were brain-resident microglia, but a small number of CX3CR1+ and CCR2+ macrophages, ultimately originating from the invasion of blood-borne monocytes, were also found near the lesion. We found a coordinated pattern of microglia migration and proliferation, where microglia within 200 μm of the microhemorrhage migrated toward the lesion over hours to days. In contrast, microglia proliferation was not observed until ≈40 hours after the lesion and occurred primarily in a shell-shaped region where the migration of microglia decreased their local density. These data suggest that local microglia density changes may trigger proliferation. Astrocytes activated in a similar region as microglia but delayed by a few days. By 2 weeks, this inflammatory response had largely resolved.Although microhemorrhages are small in size, the brain responds to a single bleed with an inflammatory response that involves brain-resident and blood-derived cells, persists for weeks, and may impact the adjacent brain microenvironment.

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In Vivo Calcium Imaging of Cardiomyocytes in the Beating Mouse Heart With Multiphoton Microscopy

Jason S. Jones, David M. Small and Nozomi Nishimura

Frontiers in physiology (2018)

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Background: Understanding the microscopic dynamics of the beating heart has been challenging due to the technical nature of imaging with micrometer resolution while the heart moves. The development of multiphoton microscopy has made in vivo, cell-resolved measurements of calcium dynamics and vascular function possible in motionless organs such as the brain. In heart, however, studies of in vivo interactions between cells and the native microenvironment are behind other organ systems. Our goal was to develop methods for intravital imaging of cardiac structural and calcium dynamics with microscopic resolution. Methods: Ventilated mice expressing GCaMP6f, a genetically encoded calcium indicator, received a thoracotomy to provide optical access to the heart. Vasculature was labeled with an injection of dextran-labeled dye. The heart was partially stabilized by a titanium probe with a glass window. Images were acquired at 30 frames per second with spontaneous heartbeat and continuously running, ventilated breathing. The data were reconstructed into three-dimensional volumes showing tissue structure, vasculature, and GCaMP6f signal in cardiomyocytes as a function of both the cardiac and respiratory cycle. Results: We demonstrated the capability to simultaneously measure calcium transients, vessel size, and tissue displacement in three dimensions with micrometer resolution. Reconstruction at various combinations of cardiac and respiratory phase enabled measurement of regional and single-cell cardiomyocyte calcium transients (GCaMP6f fluorescence). GCaMP6f fluorescence transients in individual, aberrantly firing cardiomyocytes were also quantified. Comparisons of calcium dynamics (risetime and tau) at varying positions within the ventricle wall showed no significant depth dependence. Conclusion: This method enables studies of coupling between contraction and excitation during physiological blood perfusion and breathing at high spatiotemporal resolution. These capabilities could lead to a new understanding of normal and disease function of cardiac cells.

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In Vivo Femtosecond Laser Subsurface Cortical Microtransections Attenuate Acute Rat Focal Seizures

Shivathmihai Nagappan, Lena Liu, Robert Fetcho, John Nguyen, Nozomi Nishimura, Ryan E. Radwanski, Seth Lieberman, Eliza Baird-Daniel, Hongtao Ma2,3, Mingrui Zhao, Chris B. Schaffer and Theodore H. Schwartz

Cerebral Cortex (2018)

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Recent evidence shows that seizures propagate primarily through supragranular cortical layers. To selectively modify these circuits, we developed a new technique using tightly focused, femtosecond infrared laser pulses to make as small as ~100 µm-wide subsurface cortical incisions surrounding an epileptic focus. We use this “laser scalpel” to produce subsurface cortical incisions selectively to supragranular layers surrounding an epileptic focus in an acute rodent seizure model. Compared with sham animals, these microtransections completely blocked seizure initiation and propagation in 1/3 of all animals. In the remaining animals, seizure frequency was reduced by 2/3 and seizure propagation reduced by 1/3. In those seizures that still propagated, it was delayed and reduced in amplitude. When the recording electrode was inside the partially isolated cube and the seizure focus was on the outside, the results were even more striking. In spite of these microtransections, somatosensory responses to tail stimulation were maintained but with reduced amplitude. Our data show that just a single enclosing wall of laser cuts limited to supragranular layers led to a significant reduction in seizure initiation and propagation with preserved cortical function. Modification of this concept may be a useful treatment for human epilepsy.

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