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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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Label-free imaging of atherosclerotic plaques using third-harmonic generation microscopy

David M. Small, Jason S. Jones, Irwin I. Tendler, Paul E. Miller, Andre Ghetti, and Nozomi Nishimura

Biomedical Optics Express (2018)

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Multiphoton microscopy using laser sources in the mid-infrared range (MIR, 1,300 nm and 1,700 nm) was used to image atherosclerotic plaques from murine and human samples. Third harmonic generation (THG) from atherosclerotic plaques revealed morphological details of cellular and extracellular lipid deposits. Simultaneous nonlinear optical signals from the same laser source, including second harmonic generation and endogenous fluorescence, resulted in label-free images of various layers within the diseased vessel wall. The THG signal adds an endogenous contrast mechanism with a practical degree of specificity for atherosclerotic plaques that complements current nonlinear optical methods for the investigation of cardiovascular disease. Our use of whole-mount tissue and backward scattered epi-detection suggests THG could potentially be used in the future as a clinical tool.

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Experimentally constrained circuit model of cortical arteriole networks for understanding flow redistribution due to occlusion and neural activation

Tejapratap Bollu, Nathan R Cornelius, John Sunwoo, Nozomi Nishimura, Chris B Schaffer and Peter C Doerschuk

Journal of Cerebral Blood Flow & Metabolism (2017)

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Computations are described which estimate flows in all branches of the cortical surface arteriole network from two-photon excited fluorescence (2PEF) microscopy images which provide the network topology and, in selected branches red blood cell (RBC) speeds and lumen diameters. Validation is done by comparing the flow predicted by the model with experimentally measured flows and by comparing the predicted flow redistribution in the network due to single-vessel strokes with experimental observations. The model predicts that tissue is protected from RBC flow decreases caused by multiple occlusions of surface arterioles but not penetrating arterioles. The model can also be used to study flow rerouting due to vessel dilations and constrictions.

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In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain

Ouzounov DG, Wang T, Wang M, Feng D, Horton NG, Cruz Hernández JC, Cheng Y, Reimer J, Tolias A, Nishimura N, Xu C

Nature Methods (2017)

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High-resolution optical imaging is critical to understanding brain function. We demonstrate that three-photon microscopy at 1,300-nm excitation enables functional imaging of GCaMP6s-labeled neurons beyond the depth limit of two-photon microscopy. We record spontaneous activity from up to 150 neurons in the hippocampal stratum pyramidale at ~1-mm depth within an intact mouse brain. Our method creates opportunities for noninvasive recording of neuronal activity with high spatial and temporal resolution deep within scattering brain tissues.

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 Supplement 1

Spatio-temporal dynamics of cerebral capillary segments with stalling red blood cells

Sefik Evren Erdener, Jianbo Tang, Amir Sajjadi, Kıvılcım Kılıc, Sreekanth Kura, Chris B Schaffer and David A Boas

Journal of Cerebral Blood Flow & Metabolism (2017)

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Optical coherence tomography (OCT) allows label-free imaging of red blood cell (RBC) flux within capillaries with high spatio-temporal resolution. In this study, we utilized time-series OCT-angiography to demonstrate interruptions in capillary. RBC flux in mouse brain in vivo. We noticed 7.5% of 200 capillaries had at least one stall in awake mice with chronic windows during a 9-min recording. At any instant, 0.45% of capillaries were stalled. Average stall duration was 15 s but could last over 1 min. Stalls were more frequent and longer lasting in acute window preparations. Further, isoflurane anesthesia in chronic preparations caused an increase in the number of stalls. In repeated imaging, the same segments had a tendency to stall again over a period of one month. In awake animals, functional stimulation decreased the observance of stalling events. Stalling segments were located distally, away from the first couple of arteriolar-side capillary branches and their average RBC and plasma velocities were lower than nonstalling capillaries within the same region. This first systematic analysis of capillary RBC stalls in the brain, enabled by rapid and continuous volumetric imaging of capillaries with OCTangiography, will lead to future investigations of the potential role of stalling events in cerebral pathologies. Ke

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A circuit motif in the zebrafish hindbrain for a two alternative behavioral choice to turn left or right

M. Koyama, F. Minale, J. Shum, N. Nishimura, C. B. Schaffer, and J. R. Fetcho

eLife (2016)

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Animals collect sensory information from the world and make adaptive choices about how to respond to it. Here, we reveal a network motif in the brain for one of the most fundamental behavioral choices made by bilaterally symmetric animals: whether to respond to a sensory stimulus by moving to the left or to the right. We define network connectivity in the hindbrain important for the lateralized escape behavior of zebrafish and then test the role of neurons by using laser ablations and behavioral studies. Key inhibitory neurons in the circuit lie in a column of morphologically similar cells that is one of a series of such columns that form a developmental and functional ground plan for building hindbrain networks. Repetition within the columns of the network motif we defined may therefore lie at the foundation of other lateralized behavioral choices.

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Mixing injector enables time-resolved crystallography with high hit rate at X-ray free electron lasers

G. D. Calvey, A. M. Katz, C. B. Schaffer, and L. Pollack,

Structural dynamics (2016)

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Knowledge of protein structure provides essential insight into function, enhancing our understanding of diseases and enabling new treatment development. X-ray crystallography has been used to solve the structures of more than 100 000 proteins; however, the vast majority represent long-lived states that do not capture the functional motions of these molecular machines. Reactions triggered by the addition of a ligand can be the most challenging to detect with crystallography because of the difficulty of synchronizing reactions to create detectable quantities of transient states. The development of X-ray free electron lasers (XFELs) and serial femtosecond crystallography (SFX) enables new approaches for solving protein structures following the rapid diffusion of ligands into micron sized protein crystals. Conformational changes occurring on millisecond timescales can be detected and time-resolved. Here, we describe a new XFEL injector which incorporates a microfluidic mixer to rapidly combine reactant and sample milliseconds before the sample reaches the X-ray beam. The mixing injector consists of bonded, concentric glass capillaries. The fabrication process, employing custom laser cut centering spacers and UV curable epoxy, ensures precise alignment of capillaries for repeatable, centered sample flow and dependable mixing. Crystal delivery capillaries are 50 or 75 μm in diameter and can contain an integrated filter depending on the demands of the experiment. Reaction times can be varied from submillisecond to several hundred milliseconds. The injector features rapid and uniform mixing, low sample dilution, and high hit rates. It is fully compatible with existing SFX beamlines.

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Simultaneous Optical and Electrical In Vivo Analysis of the Enteric Nervous System

Rakhilin N, Barth B, Choi J, Munoz N, Kulkarni S, LaVinka C, Dong X, Spencer M, Pasricha P, Nishimura N, Jones J, Small D, Cheng YT, Cao Y, Kan E, Shen X

Nat Commun (2016)

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The enteric nervous system (ENS) is a major division of the nervous system and vital to the gastrointestinal (GI) tract and its communication with the rest of the body. Unlike the brain and spinal cord, relatively little is known about the ENS in part because of the inability to directly monitor its activity in live animals. Here, we integrate a transparent graphene sensor with a customized abdominal window for simultaneous optical and electrical recording of the ENS in vivo. The implanted device captures ENS responses to neurotransmitters, drugs and optogenetic manipulation in real time.

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The origin and implementation of the Broadening Experiences in Scientific Training programs: an NIH common fund initiative

F. J. Meyers, A. Mathur, C. N. Fuhrmann, T. C. O’Brien, I. Wefes, P. A. Labosky, D. S. Duncan, A. August, A. Feig, K. L. Gould, M. J. Friedlaner, C. B. Schaffer, A. Van Wart, R. Chalkley

FASEB Journal (2016)

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Recent national reports and commentaries on the current status and needs of the U.S. biomedical research workforce have highlighted the limited career development opportunities for predoctoral and postdoctoral trainees in academia, yet little attention is paid to preparation for career pathways outside of the traditional faculty path. Recognizing this issue, in 2013, the U.S. National Institutes of Health (NIH) Common Fund issued a request for application titled "NIH Director's Biomedical Research Workforce Innovation Award: Broadening Experiences in Scientific Training (BEST)." These 5-yr 1-time grants, awarded to 17 single or partnering institutions, were designed to develop sustainable approaches to broaden graduate and postgraduate training, aimed at creating training programs that reflect the range of career options that trainees may ultimately pursue. These institutions have formed a consortium in order to work together to develop, evaluate, share, and disseminate best practices and challenges. This is a first report on the early experiences of the consortium and the scope of participating BEST programs. In this report, we describe the state of the U.S. biomedical workforce and development of the BEST award, variations of programmatic approaches to assist with program design without BEST funding, and novel approaches to engage faculty in career development programs. To test the effectiveness of these BEST programs, external evaluators will assess their outcomes not only over the 5 yr grant period but also for an additional 10 yr beyond award completion.

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TRAIL-coated leukocytes that prevent the bloodborne metastasis of prostate cancer,”

W.C. Wayne, S. Chandrasekaran, M. J. Mitchell, M. F. Chan, R. E. Lee, C. B. Schaffer, M. R. King

Journal of Controlled Release (2016)

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Prostate cancer, once it has progressed from its local to metastatic form, is a disease with poor prognosis and limited treatment options. Here we demonstrate an approach using nanoscale liposomes conjugated with E-selectin adhesion protein and Apo2L/TRAIL (TNF-related apoptosis-inducing ligand) apoptosis ligand that attach to the surface of leukocytes and rapidly clear viable cancer cells from circulating blood in the living mouse. For the first time, it is shown that such an approach can be used to prevent the spontaneous formation and growth of metastatic tumors in an orthotopic xenograft model of prostate cancer, by greatly reducing the number of circulating tumor cells. We conclude that the use of circulating leukocytes as a carrier for the anti-cancer protein TRAIL could be an effective tool to directly target circulating tumor cells for the prevention of prostate cancer metastasis, and potentially other cancers that spread through the bloodstream.

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