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Vascular contributions to cognitive impairment and dementia including Alzheimer’s disease

H. M. Snyder, R. A. Corriveau, S. Craft, J. E. Faber, S. Greenberg, D. Knopman, B. T. Lamb, T. J. Montine, M. Nedergaard, C. B. Schaffer, J. A. Schneider, C. Wellington, D. M. Wilcock, G. J. Zipfel, B. Zlokovic, L. J. Bain, F. Bosetti, Z. S. Galis, W. Koroshetz, M. C. Carrillo

Alzheimer’s and Dementia (2015)

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Scientific evidence continues to demonstrate the linkage of vascular contributions to cognitive impairment and dementia such as Alzheimer's disease. In December, 2013, the Alzheimer's Association, with scientific input from the National Institute of Neurological Disorders and Stroke and the National Heart, Lung and Blood Institute from the National Institutes of Health, convened scientific experts to discuss the research gaps in our understanding of how vascular factors contribute to Alzheimer's disease and related dementia. This manuscript summarizes the meeting and the resultant discussion, including an outline of next steps needed to move this area of research forward.

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Constitutively active Notch4 receptor elicits brain arteriovenous malformations through enlargement of capillary-like vessels

Patrick A. Murphya, Tyson N. Kima, Lawrence Huanga, Corinne M. Nielsena , Michael T. Lawtonb , Ralf H. Adamsc , Chris B. Schafferd , and Rong A. Wanga,

Proceedings of the National Academy of Sciences (2014)

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Arteriovenous (AV) malformation (AVM) is a devastating condition characterized by focal lesions of enlarged, tangled vessels that shunt blood from arteries directly to veins. AVMs can form anywhere in the body and can cause debilitating ischemia and life-threatening hemorrhagic stroke. The mechanisms that underlie AVM formation remain poorly understood. Here, we examined the cellular and hemodynamic changes at the earliest stages of brain AVM formation by time-lapse two-photon imaging through cranial windows of mice expressing constitutively active Notch4 (Notch4*). AVMs arose from enlargement of preexisting microvessels with capillary diameter and blood flow and no smooth muscle cell coverage. AV shunting began promptly after Notch4* expression in endothelial cells (ECs), accompanied by increased individual EC areas, rather than increased EC number or proliferation. Alterations in Notch signaling in ECs of all vessels, but not arteries alone, affected AVM formation, suggesting that Notch functions in the microvasculature and/or veins to induce AVM. Increased Notch signaling interfered with the normal biological control of hemodynamics, permitting a positive feedback loop of increasing blood flow and vessel diameter and driving focal AVM growth from AV connections with higher blood velocity at the expense of adjacent AV connections with lower velocity. Endothelial expression of constitutively active Notch1 also led to brain AVMs in mice. Our data shed light on cellular and hemodynamic mechanisms underlying AVM pathogenesis elicited by increased Notch signaling in the endothelium.

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TRAIL-coated leukocytes that kill cancer cells in the circulation

Michael J. Mitchell, Elizabeth Wayne, Kuldeepsinh Rana, Chris B. Schaffer, and Michael R. King

Proceedings of the National Academy of Sciences USA (2014)

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Metastasis through the bloodstream contributes to poor prognosis in many types of cancer. Mounting evidence implicates selectin- based adhesive interactions between cancer cells and the blood vessel wall as facilitating this process, in a manner similar to leukocyte trafficking during inflammation. Here, we describe a unique approach to target and kill colon and prostate cancer cells in the blood that causes circulating leukocytes to present the cancer-specific TNF-related apoptosis inducing ligand (TRAIL) on their surface along with E-selectin adhesion receptor. This approach, demonstrated in vitro with human blood and also in mice, mimics the cytotoxic activity of natural killer cells and increases the sur- face area available for delivery of the receptor-mediated signal. The resulting “unnatural killer cells” hold promise as an effective means to neutralize circulating tumor cells that enter blood with the potential to form new metastases.

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In vivo three-photon microscopy of subcortical structures within an intact mouse brain

Nicholas G. Horton, Ke Wang, Demirhan Kobat, Catharine G. Clark, Frank W. Wise, Chris B. Schaffer and Chris Xu

Nature Photonics 7, 205 (2013)

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Two-photon fluorescence microscopy enables scientists in various fields including neuroscience, embryology and oncology to visualize in vivo and ex vivo tissue morphology and physiology at a cellular level deep within scattering tissue. However, tissue scattering limits the maximum imaging depth of two-photon fluorescence microscopy to the cortical layer within mouse brain, and imaging subcortical structures currently requires the removal of overlying brain tissue or the insertion of optical probes. Here, we demon- strate non-invasive, high-resolution, in vivo imaging of subcor- tical structures within an intact mouse brain using three-photon fluorescence microscopy at a spectral excitation window of 1,700nm. Vascular structures as well as red fluorescent protein-labelled neurons within the mouse hippocampus are imaged. The combination of the long excitation wavelength and the higher-order nonlinear excitation overcomes the limit- ations of two-photon fluorescence microscopy, enabling biological investigations to take place at a greater depth within tissue.

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Intracerebral haemorrhage associated with antithrombotic treatment: translational insights from experimental studies

Arne Lauer, Waltraud Pfeilschifter, Chris B Schaffer, Eng H Lo, Christian Foerch

Lancet Neurology 12, 394 (2013)

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Little is known about the pathophysiology of intracerebral haemorrhage that occurs during anticoagulant treatment. In observational studies, investigators have reported larger haematoma volumes and worse functional outcome in these patients than in those with intracerebral haemorrhage and a normal coagulation status. The need to prevent extensive haematoma enlargement by rapid reversal of the anticoagulation seems intuitive, although no evidence is available from randomised clinical trials. New oral anticoagulants, such as the direct thrombin inhibitor dabigatran and the factor Xa inhibitor rivaroxaban, have been approved recently; however, intracerebral haemorrhage during dabigatran or rivaroxaban anticoagulation has not been characterised, and whether anticoagulation reversal can be beneficial in this scenario is unknown. In a translational approach, new experimental models have been developed to study anticoagulation-associated intracerebral haemorrhage in more detail and to test treatment strategies. Vitamin k antagonists enlarge haematoma volumes and worsen functional outcome in animal models. Rapid reversal of anticoagulation in the experimental setting prevents prolonged haematoma expansion and improves outcome. The new oral anticoagulants increase intracerbral haemorrhage volumes less than does warfarin. Haemostatic approaches that have been used for vitamin k-associated intracerebral haemorrhage also seem to be effective in intracerebral haemorrhage associated with the new anticoagulants. These experimental studies are valuable for filling gaps in knowledge, but the results need careful translation into routine clinical practice.

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Intracerebral haemorrhage associated with antithrombotic treatment: translational insights from experimental studies.

A. Lauer, W. Pfeilschifter, C. B. Schaffer, E. H. Lo, and C. Foerch

Lancet Neurology (2013)

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Little is known about the pathophysiology of intracerebral haemorrhage that occurs during anticoagulant treatment. In observational studies, investigators have reported larger haematoma volumes and worse functional outcome in these patients than in those with intracerebral haemorrhage and a normal coagulation status. The need to prevent extensive haematoma enlargement by rapid reversal of the anticoagulation seems intuitive, although no evidence is available from randomised clinical trials. New oral anticoagulants, such as the direct thrombin inhibitor dabigatran and the factor Xa inhibitor rivaroxaban, have been approved recently; however, intracerebral haemorrhage during dabigatran or rivaroxaban anticoagulation has not been characterised, and whether anticoagulation reversal can be beneficial in this scenario is unknown. In a translational approach, new experimental models have been developed to study anticoagulation-associated intracerebral haemorrhage in more detail and to test treatment strategies. Vitamin k antagonists enlarge haematoma volumes and worsen functional outcome in animal models. Rapid reversal of anticoagulation in the experimental setting prevents prolonged haematoma expansion and improves outcome. The new oral anticoagulants increase intracerbral haemorrhage volumes less than does warfarin. Haemostatic approaches that have been used for vitamin k-associated intracerebral haemorrhage also seem to be effective in intracerebral haemorrhage associated with the new anticoagulants. These experimental studies are valuable for filling gaps in knowledge, but the results need careful translation into routine clinical practice.

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Intravenous tPA Therapy Does Not Worsen Acute Intracerebral Hemorrhage in Mice

Christian Foerch, Nathanael L. Rosidi, Frieder Schlunk, Arne Lauer, Flor A. Cianchetti, Emiri Mandeville, Ken Arai, Kazim Yigitkanli, Xiang Fan, Xiaoying Wang, Klaus van Leyen, Helmuth Steinmetz, Chris B. Schaffer, Eng H. Lo

Public Library of Science ONE 8, e54203 (2013)

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Tissue plasminogen activator (tPA) is the only FDA-approved treatment for reperfusing ischemic strokes. But widespread use of tPA is still limited by fears of inadvertently administering tPA in patients with intracerebral hemorrhage (ICH). Surprisingly, however, the assumption that tPA will worsen ICH has never been biologically tested. Here, we assessed the effects of tPA in two models of ICH. In a mouse model of collagenase-induced ICH, hemorrhage volumes and neurological deficits after 24 hrs were similar in saline controls and tPA-treated mice, whereas heparin-treated mice had 3-fold larger hematomas. In a model of laser-induced vessel rupture, tPA also did not worsen hemorrhage volumes, while heparin did. tPA is known to worsen neurovascular injury by amplifying matrix metalloproteinases during cerebral ischemia. In contrast, tPA did not upregulate matrix metalloproteinases in our mouse ICH models. In summary, our experimental data do not support the assumption that intravenous tPA has a deleterious effect in acute ICH. However, due to potential species differences and the inability of models to fully capture the dynamics of human ICH, caution is warranted when considering the implications of these findings for human therapy.

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Chronic in vivo imaging in the mouse spinal cord using an implanted chamber

Matthew J Farrar, Ida M Bernstein, Donald H Schlafer, Thomas A Cleland, Joseph R Fetcho, and Chris B Schaffer

Nature Methods (2012)

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Understanding and treatment of spinal cord pathology is limited in part by a lack of time-lapse in vivo imaging strategies at the cellular level. We developed a chronically implanted spinal chamber and surgical procedure suitable for time-lapse in vivo multiphoton microscopy of mouse spinal cord without the need for repeat surgical procedures. We routinely imaged mice repeatedly for more than 5 weeks postoperatively with up to ten separate imaging sessions and observed neither motor-function deficit nor neuropathology in the spinal cord as a result of chamber implantation. using this chamber we quantified microglia and afferent axon dynamics after a laser-induced spinal cord lesion and observed massive microglia infiltration within  d along with a heterogeneous dieback of axon stumps. By enabling chronic imaging studies over timescales ranging from minutes to months, our method offers an ideal platform for understanding cellular dynamics in response to injury and therapeutic interventions.

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Cyclic strain anisotropy regulates valvular interstitial cell phenotype and tissue remodeling in three-dimensional culture

Russell A. Gould, Karen Chin, Thom P. Santisakultarm, Amanda Dropkin, Jennifer M. Richards, Chris B. Schaffer, Jonathan T. Butcher

Acta Biomaterialia (2012)

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Many planar connective tissues exhibit complex anisotropic matrix fiber arrangements that are critical to their biomechanical function. This organized structure is created and modified by resident fibroblasts in response to mechanical forces in their environment. The directionality of applied strain fields changes dramatically during development, aging, and disease, but the specific effect of strain direction on matrix remodeling is less clear. Current mechanobiological inquiry of planar tissues is limited to equibiaxial or uniaxial stretch, which inadequately simulates many in vivo environments. In this study, we implement a novel bioreactor system to demonstrate the unique effect of controlled anisotropic strain on fibroblast behavior in three-dimensional (3-D) engineered tissue environments, using aortic valve interstitial fibro- blast cells as a model system. Cell seeded 3-D collagen hydrogels were subjected to cyclic anisotropic strain profiles maintained at constant areal strain magnitude for up to 96 h at 1 Hz. Increasing anisotropy of biaxial strain resulted in increased cellular orientation and collagen fiber alignment along the principal directions of strain and cell orientation was found to precede fiber reorganization. Cellular proliferation and apoptosis were both significantly enhanced under increasing biaxial strain anisotropy (P < 0.05). While cyclic strain reduced both vimentin and alpha-smooth muscle actin compared to unstrained con- trols, vimentin and alpha-smooth muscle actin expression increased with strain anisotropy and corre- lated with direction (P < 0.05). Collectively, these results suggest that strain field anisotropy is an independent regulator of fibroblast cell phenotype, turnover, and matrix reorganization, which may inform normal and pathological remodeling in soft tissues.

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Line-Scanning Particle Image Velocimetry: An Optical Approach for Quantifying a Wide Range of Blood Flow Speeds in Live Animals

Tyson N. Kim, Patrick W. Goodwill, Yeni Chen, Steven M. Conolly, Chris B. Schaffer, Dorian Liepmann, Rong A. Wang

Public Library of Science ONE 7, e38590 (2012)

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Background: The ability to measure blood velocities is critical for studying vascular development, physiology, and pathology. A key challenge is to quantify a wide range of blood velocities in vessels deep within living specimens with concurrent diffraction-limited resolution imaging of vascular cells. Two-photon laser scanning microscopy (TPLSM) has shown tremendous promise in analyzing blood velocities hundreds of micrometers deep in animals with cellular resolution. However, current analysis of TPLSM-based data is limited to the lower range of blood velocities and is not adequate to study faster velocities in many normal or disease conditions. Methodology/Principal Findings: We developed line-scanning particle image velocimetry (LS-PIV), which used TPLSM data to quantify peak blood velocities up to 84 mm/s in live mice harboring brain arteriovenous malformation, a disease characterized by high flow. With this method, we were able to accurately detect the elevated blood velocities and exaggerated pulsatility along the abnormal vascular network in these animals. LS-PIV robustly analyzed noisy data from vessels as deep as 850 mm below the brain surface. In addition to analyzing in vivo data, we validated the accuracy of LS-PIV up to 800 mm/s using simulations with known velocity and noise parameters. Conclusions/Significance: To our knowledge, these blood velocity measurements are the fastest recorded with TPLSM. Partnered with transgenic mice carrying cell-specific fluorescent reporters, LS-PIV will also enable the direct in vivo correlation of cellular, biochemical, and hemodynamic parameters in high flow vascular development and diseases such as atherogenesis, arteriogenesis, and vascular anomalies.

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