Publications by authors named "Nozomi Nishimura"

58 Publications

Causes and consequences of baseline cerebral blood flow reductions in Alzheimer's disease.

J Cereb Blood Flow Metab 2021 Jan 14:271678X20982383. Epub 2021 Jan 14.

Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, USA.

Reductions of baseline cerebral blood flow (CBF) of ∼10-20% are a common symptom of Alzheimer's disease (AD) that appear early in disease progression and correlate with the severity of cognitive impairment. These CBF deficits are replicated in mouse models of AD and recent work shows that increasing baseline CBF can rapidly improve the performance of AD mice on short term memory tasks. Despite the potential role these data suggest for CBF reductions in causing cognitive symptoms and contributing to brain pathology in AD, there remains a poor understanding of the molecular and cellular mechanisms causing them. This review compiles data on CBF reductions and on the correlation of AD-related CBF deficits with disease comorbidities (e.g. cardiovascular and genetic risk factors) and outcomes (e.g. cognitive performance and brain pathology) from studies in both patients and mouse models, and discusses several potential mechanisms proposed to contribute to CBF reductions, based primarily on work in AD mouse models. Future research aimed at improving our understanding of the importance of and interplay between different mechanisms for CBF reduction, as well as at determining the role these mechanisms play in AD patients could guide the development of future therapies that target CBF reductions in AD.
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http://dx.doi.org/10.1177/0271678X20982383DOI Listing
January 2021

High fat diet worsens Alzheimer's disease-related behavioral abnormalities and neuropathology in APP/PS1 mice, but not by synergistically decreasing cerebral blood flow.

Sci Rep 2020 06 18;10(1):9884. Epub 2020 Jun 18.

Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, USA.

Obesity is linked to increased risk for and severity of Alzheimer's disease (AD). Cerebral blood flow (CBF) reductions are an early feature of AD and are also linked to obesity. We recently showed that non-flowing capillaries, caused by adhered neutrophils, contribute to CBF reduction in mouse models of AD. Because obesity could exacerbate the vascular inflammation likely underlying this neutrophil adhesion, we tested links between obesity and AD by feeding APP/PS1 mice a high fat diet (Hfd) and evaluating behavioral, physiological, and pathological changes. We found trends toward poorer memory performance in APP/PS1 mice fed a Hfd, impaired social interactions with either APP/PS1 genotype or a Hfd, and synergistic impairment of sensory-motor function in APP/PS1 mice fed a Hfd. The Hfd led to increases in amyloid-beta monomers and plaques in APP/PS1 mice, as well as increased brain inflammation. These results agree with previous reports showing obesity exacerbates AD-related pathology and symptoms in mice. We used a crowd-sourced, citizen science approach to analyze imaging data to determine the impact of the APP/PS1 genotype and a Hfd on capillary stalling and CBF. Surprisingly, we did not see an increase in the number of non-flowing capillaries or a worsening of the CBF deficit in APP/PS1 mice fed a Hfd as compared to controls, suggesting that capillary stalling is not a mechanistic link between a Hfd and increased severity of AD in mice. Reducing capillary stalling by blocking neutrophil adhesion improved CBF and short-term memory function in APP/PS1 mice, even when fed a Hfd.
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http://dx.doi.org/10.1038/s41598-020-65908-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7303150PMC
June 2020

Impact of Chirality on Hydrogen-Bonded Supramolecular Assemblies and Photoconductivity of Diketopyrrolopyrrole Derivatives.

Chemistry 2020 Aug 17;26(44):9998-10004. Epub 2020 Jun 17.

CNRS, UPR22, University of Strasbourg, Institute Charles Sadron, 23 Rue du Loess, 67000, Strasbourg Cedex 2, France.

Hydrogen bonds can efficiently guide the self-assembly of organic materials, enabling to tune the properties of the aggregation processes. In the case of π-conjugated materials, several parameters such as temperature, concentration and solvent can be used to modify the aggregation state while tuning the optoelectronic properties. Chirality can be included within the impacting parameters due to the differences in molecular packing. Here, chiral and achiral thiophene-capped diketopyrrolopyrrole derivatives were designed and synthesized containing amide bonds, with the aim to study the interplay between chiral assemblies and their stabilization through hydrogen-bonding. Differences in aggregation properties were observed with spectroscopy and microscopy, and a contactless microwave-based technique was used to study their intrinsic charge carrier mobility. The positive role of hydrogen-bonding has been highlighted and the differences between chiral and achiral compounds have been elucidated.
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http://dx.doi.org/10.1002/chem.202001540DOI Listing
August 2020

Intravital Microscopy of the Beating Murine Heart to Understand Cardiac Leukocyte Dynamics.

Front Immunol 2020 4;11:92. Epub 2020 Feb 4.

Nancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, United States.

Cardiovascular disease is the leading cause of worldwide mortality. Intravital microscopy has provided unprecedented insight into leukocyte biology by enabling the visualization of dynamic responses within living organ systems at the cell-scale. The heart presents a uniquely dynamic microenvironment driven by periodic, synchronous electrical conduction leading to rhythmic contractions of cardiomyocytes, and phasic coronary blood flow. In addition to functions shared throughout the body, immune cells have specific functions in the heart including tissue-resident macrophage-facilitated electrical conduction and rapid monocyte infiltration upon injury. Leukocyte responses to cardiac pathologies, including myocardial infarction and heart failure, have been well-studied using standard techniques, however, certain questions related to spatiotemporal relationships remain unanswered. Intravital imaging techniques could greatly benefit our understanding of the complexities of leukocyte behavior within cardiac tissue, but these techniques have been challenging to apply. Different approaches have been developed including high frame rate imaging of the beating heart, explantation models, micro-endoscopy, and mechanical stabilization coupled with various acquisition schemes to overcome challenges specific to the heart. The field of cardiac science has only begun to benefit from intravital microscopy techniques. The current focused review presents an overview of leukocyte responses in the heart, recent developments in intravital microscopy for the murine heart, and a discussion of future developments and applications for cardiovascular immunology.
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http://dx.doi.org/10.3389/fimmu.2020.00092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7010807PMC
February 2020

An intravital window to image the colon in real time.

Nat Commun 2019 12 11;10(1):5647. Epub 2019 Dec 11.

Department of Biomedical Engineering, Duke University, Durham, NC, 27710, USA.

Intravital microscopy is a powerful technique to observe dynamic processes with single-cell resolution in live animals. No intravital window has been developed for imaging the colon due to its anatomic location and motility, although the colon is a key organ where the majority of microbiota reside and common diseases such as inflammatory bowel disease, functional gastrointestinal disorders, and colon cancer occur. Here we describe an intravital murine colonic window with a stabilizing ferromagnetic scaffold for chronic imaging, minimizing motion artifacts while maximizing long-term survival by preventing colonic obstruction. Using this setup, we image fluorescently-labeled stem cells, bacteria, and immune cells in live animal colons. Furthermore, we image nerve activity via calcium imaging in real time to demonstrate that electrical sacral nerve stimulation can activate colonic enteric neurons. The simple implantable apparatus enables visualization of live processes in the colon, which will open the window to a broad range of studies.
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http://dx.doi.org/10.1038/s41467-019-13699-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6906443PMC
December 2019

Computed optical coherence microscopy of mouse brain ex vivo.

J Biomed Opt 2019 11;24(11):1-18

Cornell University, Meinig School of Biomedical Engineering, Ithaca, New York, United States.

The compromise between lateral resolution and usable imaging depth range is a bottleneck for optical coherence tomography (OCT). Existing solutions for optical coherence microscopy (OCM) suffer from either large data size and long acquisition time or a nonideal point spread function. We present volumetric OCM of mouse brain with a large depth coverage by leveraging computational adaptive optics (CAO) to significantly reduce the number of OCM volumes that need to be acquired with a Gaussian beam focused at different depths. We demonstrate volumetric reconstruction of mouse brain with lateral resolution of 2.2  μm, axial resolution of 4.7  μm, and depth range of ∼1.2  mm optical path length, using only 11 OCT data volumes acquired on a spectral-domain OCM system. Compared to focus scanning with step size equal to the Rayleigh length of the beam, this is a factor of 4 fewer datasets required for volumetric imaging. Coregistered two-photon microscopy confirmed that CAO-OCM reconstructions can visualize various tissue microstructures in the brain. Our results also highlight the limitations of CAO in highly scattering media, particularly when attempting to reconstruct far from the focal plane or when imaging deep within the sample.
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http://dx.doi.org/10.1117/1.JBO.24.11.116002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6880187PMC
November 2019

Microvessel occlusions alter amyloid-beta plaque morphology in a mouse model of Alzheimer's disease.

J Cereb Blood Flow Metab 2020 Oct 19;40(10):2115-2131. Epub 2019 Nov 19.

Nancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, USA.

Vascular dysfunction is correlated to the incidence and severity of Alzheimer's disease. In a mouse model of Alzheimer's disease (APP/PS1) using in vivo, time-lapse, multiphoton microscopy, we found that occlusions of the microvasculature alter amyloid-beta (Aβ) plaques. We used several models of vascular injury that varied in severity. Femtosecond laser-induced occlusions in single capillaries generated a transient increase in small, cell-sized, Aβ deposits visualized with methoxy-X04, a label of fibrillar Aβ. After occlusions of penetrating arterioles, some plaques changed morphology, while others disappeared, and some new plaques appeared within a week after the lesion. Antibody labeling of Aβ revealed a transient increase in non-fibrillar Aβ one day after the occlusion that coincided with the disappearance of methoxy-X04-labeled plaques. Four days after the lesion, anti-Aβ labeling decreased and only remained in patches unlabeled by methoxy-X04 near microglia. Histology in two additional models, sparse embolic occlusions from intracarotid injections of beads and infarction from photothrombosis, demonstrated increased labeling intensity in plaques after injury. These results suggest that microvascular lesions can alter the deposition and clearance of Aβ and confirm that Aβ plaques are dynamic structures, complicating the interpretation of plaque burden as a marker of Alzheimer's disease progression.
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http://dx.doi.org/10.1177/0271678X19889092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7786844PMC
October 2020

Mice lacking fucosyltransferase 2 show reduced innate allergic inflammation in the airways.

Allergy 2020 05 28;75(5):1253-1256. Epub 2019 Nov 28.

Department of Allergy and Clinical Immunology, Graduate School of Medicine, Chiba University, Chiba, Japan.

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http://dx.doi.org/10.1111/all.14101DOI Listing
May 2020

Rod-like transition first or chain aggregation first? ordered aggregation of rod-like poly(p-phenyleneethynylene) chains in solution.

Chem Commun (Camb) 2019 Nov 18;55(89):13342-13345. Epub 2019 Oct 18.

Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Nishikyo-ku, Kyoto 615-8510, Japan.

The rod-like configuration of conjugated polymer chains with its low energetic disorder is the key to utilizing the backbone as a highly electrically-conductive wire. An energetic disorder that is higher than 0.1 eV, coupled with vibronic modes of the chains, leads to the localization of charges. Herein, we have tracked precisely the rod-like transition of poly(p-phenyleneethynylene) (PPE) chains as a function of temperature in diluted solutions, and shown a steep increase in persistence length at 230 K. The resulting rod-like configuration of the PPE chains with its extended electronic conjugation exhibited an extremely small energetic disorder of ∼70 meV, and was stabilized by subsequent polymer aggregate formation.
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http://dx.doi.org/10.1039/c9cc06892aDOI Listing
November 2019

Advanced Circuit and Cellular Imaging Methods in Nonhuman Primates.

J Neurosci 2019 10;39(42):8267-8274

State University of New York Downstate Medical Center, Health Science Center at Brooklyn, New York 11203.

Novel genetically encoded tools and advanced microscopy methods have revolutionized neural circuit analyses in insects and rodents over the last two decades. Whereas numerous technical hurdles originally barred these methodologies from success in nonhuman primates (NHPs), current research has started to overcome those barriers. In some cases, methodological advances developed with NHPs have even surpassed their precursors. One such advance includes new ultra-large imaging windows on NHP cortex, which are larger than the entire rodent brain and allow analysis unprecedented ultra-large-scale circuits. NHP imaging chambers now remain patent for periods longer than a mouse's lifespan, allowing for long-term all-optical interrogation of identified circuits and neurons over timeframes that are relevant to human cognitive development. Here we present some recent imaging advances brought forth by research teams using macaques and marmosets. These include technical developments in optogenetics; voltage-, calcium- and glutamate-sensitive dye imaging; two-photon and wide-field optical imaging; viral delivery; and genetic expression of indicators and light-activated proteins that result in the visualization of tens of thousands of identified cortical neurons in NHPs. We describe a subset of the many recent advances in circuit and cellular imaging tools in NHPs focusing here primarily on the research presented during the corresponding mini-symposium at the 2019 Society for Neuroscience annual meeting.
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http://dx.doi.org/10.1523/JNEUROSCI.1168-19.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6794937PMC
October 2019

Author Correction: Intestinal crypts recover rapidly from focal damage with coordinated motion of stem cells that is impaired by aging.

Sci Rep 2019 Sep 30;9(1):13992. Epub 2019 Sep 30.

Nancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, 14853, USA.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
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http://dx.doi.org/10.1038/s41598-019-43805-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6768855PMC
September 2019

Special topic section: linkages among cerebrovascular, cardiovascular, and cognitive disorders: Preventing dementia by preventing stroke: The Berlin Manifesto.

Int J Stroke 2019 Sep 22:1747493019871915. Epub 2019 Sep 22.

Prevent Alzheimer Disease (PAD2020), Potamac, MD, USA.

The incidence of stroke and dementia are diverging across the world, rising for those in low-and middle-income countries and falling in those in high-income countries. This suggests that whatever factors cause these trends are potentially modifiable. At the population level, neurological disorders as a group account for the largest proportion of disability-adjusted life years globally (10%). Among neurological disorders, stroke (42%) and dementia (10%) dominate. Stroke and dementia confer risks for each other and share some of the same, largely modifiable, risk and protective factors. In principle, 90% of strokes and 35% of dementias have been estimated to be preventable. Because a stroke doubles the chance of developing dementia and stroke is more common than dementia, more than a third of dementias could be prevented by preventing stroke. Developments at the pathological, pathophysiological, and clinical level also point to new directions. Growing understanding of brain pathophysiology has unveiled the reciprocal interaction of cerebrovascular disease and neurodegeneration identifying new therapeutic targets to include protection of the endothelium, the blood-brain barrier, and other components of the neurovascular unit. In addition, targeting amyloid angiopathy aspects of inflammation and genetic manipulation hold new testable promise. In the meantime, accumulating evidence suggests that whole populations experiencing improved education, and lower vascular risk factor profiles (e.g., reduced prevalence of smoking) and vascular disease, including stroke, have better cognitive function and lower dementia rates. At the individual levels, trials have demonstrated that anticoagulation of atrial fibrillation can reduce the risk of dementia by 48% and that systolic blood pressure lower than 140 mmHg may be better for the brain. Based on these considerations, the World Stroke Organization has issued a proclamation, endorsed by all the major international organizations focused on global brain and cardiovascular health, calling for the joint prevention of stroke and dementia. This article summarizes the evidence for translation into action. © 2019 the Alzheimer's Association and the World Stroke Organisation. Published by Elsevier Inc. All rights reserved.
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http://dx.doi.org/10.1177/1747493019871915DOI Listing
September 2019

Preventing dementia by preventing stroke: The Berlin Manifesto.

Alzheimers Dement 2019 07;15(7):961-984

Prevent Alzheimer Disease (PAD2020), Potamac, MD, USA.

The incidence of stroke and dementia are diverging across the world, rising for those in low- and middle-income countries and falling in those in high-income countries. This suggests that whatever factors cause these trends are potentially modifiable. At the population level, neurological disorders as a group account for the largest proportion of disability-adjusted life years globally (10%). Among neurological disorders, stroke (42%) and dementia (10%) dominate. Stroke and dementia confer risks for each other and share some of the same, largely modifiable, risk and protective factors. In principle, 90% of strokes and 35% of dementias have been estimated to be preventable. Because a stroke doubles the chance of developing dementia and stroke is more common than dementia, more than a third of dementias could be prevented by preventing stroke. Developments at the pathological, pathophysiological, and clinical level also point to new directions. Growing understanding of brain pathophysiology has unveiled the reciprocal interaction of cerebrovascular disease and neurodegeneration identifying new therapeutic targets to include protection of the endothelium, the blood-brain barrier, and other components of the neurovascular unit. In addition, targeting amyloid angiopathy aspects of inflammation and genetic manipulation hold new testable promise. In the meantime, accumulating evidence suggests that whole populations experiencing improved education, and lower vascular risk factor profiles (e.g., reduced prevalence of smoking) and vascular disease, including stroke, have better cognitive function and lower dementia rates. At the individual levels, trials have demonstrated that anticoagulation of atrial fibrillation can reduce the risk of dementia by 48% and that systolic blood pressure lower than 140 mmHg may be better for the brain. Based on these considerations, the World Stroke Organization has issued a proclamation, endorsed by all the major international organizations focused on global brain and cardiovascular health, calling for the joint prevention of stroke and dementia. This article summarizes the evidence for translation into action.
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http://dx.doi.org/10.1016/j.jalz.2019.06.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7001744PMC
July 2019

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

Sci Rep 2019 06 21;9(1):9069. Epub 2019 Jun 21.

Nancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, 14853, United States.

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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http://dx.doi.org/10.1038/s41598-019-45535-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6588602PMC
June 2019

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

Front Physiol 2019 26;10:233. Epub 2019 Mar 26.

Institut de Mécanique des Fluides de Toulouse (IMFT), Université de Toulouse, CNRS, Toulouse, France.

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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http://dx.doi.org/10.3389/fphys.2019.00233DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6444172PMC
March 2019

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

PLoS One 2019 13;14(3):e0213539. Epub 2019 Mar 13.

Nancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, United States of America.

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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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0213539PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6415838PMC
December 2019

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

Nat Neurosci 2019 03 11;22(3):413-420. Epub 2019 Feb 11.

Nancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, USA.

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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http://dx.doi.org/10.1038/s41593-018-0329-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6508667PMC
March 2019

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

PLoS One 2019 4;14(1):e0204295. Epub 2019 Jan 4.

Meinig School of Biomedical Engineering, Cornell University, Ithaca, New York, United States of America.

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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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0204295PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6319729PMC
September 2019

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

Cereb Cortex 2019 07;29(8):3415-3426

Department of Neurological Surgery, Weill Cornell Medicine of Cornell University, 525 East 68th Street, Box 99, New York, NY, USA.

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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http://dx.doi.org/10.1093/cercor/bhy210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6644864PMC
July 2019

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

Front Physiol 2018 31;9:969. Epub 2018 Jul 31.

Nancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, United States.

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 , cell-resolved measurements of calcium dynamics and vascular function possible in motionless organs such as the brain. In heart, however, studies of 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. 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. 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 (rise-time and tau) at varying positions within the ventricle wall showed no significant depth dependence. 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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http://dx.doi.org/10.3389/fphys.2018.00969DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6079295PMC
July 2018

Intestinal crypts recover rapidly from focal damage with coordinated motion of stem cells that is impaired by aging.

Sci Rep 2018 Jul 20;8(1):10989. Epub 2018 Jul 20.

Nancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, 14853, USA.

Despite the continuous renewal and turnover of the small intestinal epithelium, the intestinal crypt maintains a 'soccer ball-like', alternating pattern of stem and Paneth cells at the base of the crypt. To study the robustness of the alternating pattern, we used intravital two-photon microscopy in mice with fluorescently-labeled Lgr5+ intestinal stem cells and precisely perturbed the mosaic pattern with femtosecond laser ablation. Ablation of one to three cells initiated rapid motion of crypt cells that restored the alternation in the pattern within about two hours with only the rearrangement of pre-existing cells, without any cell division. Crypt cells then performed a coordinated dilation of the crypt lumen, which resulted in peristalsis-like motion that forced damaged cells out of the crypt. Crypt cell motion was reduced with inhibition of the ROCK pathway and attenuated with old age, and both resulted in incomplete pattern recovery. This suggests that in addition to proliferation and self-renewal, motility of stem cells is critical for maintaining homeostasis. Reduction of this newly-identified behavior of stem cells could contribute to disease and age-related changes.
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http://dx.doi.org/10.1038/s41598-018-29230-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6054609PMC
July 2018

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

Stroke 2018 07 29;49(7):1719-1726. Epub 2018 May 29.

From the Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY (S.J.A., N.N., C.B.S.)

Background And Purpose: 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.

Methods: 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.

Results: 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.

Conclusions: 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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http://dx.doi.org/10.1161/STROKEAHA.117.020461DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6019563PMC
July 2018

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

Biomed Opt Express 2018 01 13;9(1):214-229. Epub 2017 Dec 13.

Nancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, 526 N. Campus Rd., Ithaca, NY 14853, USA.

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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http://dx.doi.org/10.1364/BOE.9.000214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5772576PMC
January 2018

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

J Cereb Blood Flow Metab 2018 01 13;38(1):38-44. Epub 2017 Nov 13.

1 Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, USA.

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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http://dx.doi.org/10.1177/0271678X17741086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5757444PMC
January 2018

Impaired prosaposin lysosomal trafficking in frontotemporal lobar degeneration due to progranulin mutations.

Nat Commun 2017 05 25;8:15277. Epub 2017 May 25.

Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, New York 14853, USA.

Haploinsufficiency of progranulin (PGRN) due to mutations in the granulin (GRN) gene causes frontotemporal lobar degeneration (FTLD), and complete loss of PGRN leads to a lysosomal storage disorder, neuronal ceroid lipofuscinosis (NCL). Accumulating evidence suggests that PGRN is essential for proper lysosomal function, but the precise mechanisms involved are not known. Here, we show that PGRN facilitates neuronal uptake and lysosomal delivery of prosaposin (PSAP), the precursor of saposin peptides that are essential for lysosomal glycosphingolipid degradation. We found reduced levels of PSAP in neurons both in mice deficient in PGRN and in human samples from FTLD patients due to GRN mutations. Furthermore, mice with reduced PSAP expression demonstrated FTLD-like pathology and behavioural changes. Thus, our data demonstrate a role of PGRN in PSAP lysosomal trafficking and suggest that impaired lysosomal trafficking of PSAP is an underlying disease mechanism for NCL and FTLD due to GRN mutations.
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http://dx.doi.org/10.1038/ncomms15277DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5477518PMC
May 2017

A Notch positive feedback in the intestinal stem cell niche is essential for stem cell self-renewal.

Mol Syst Biol 2017 04 28;13(4):927. Epub 2017 Apr 28.

School of Electrical and Computer Engineering, Cornell University, Ithaca, NY, USA

The intestinal epithelium is the fastest regenerative tissue in the body, fueled by fast-cycling stem cells. The number and identity of these dividing and migrating stem cells are maintained by a mosaic pattern at the base of the crypt. How the underlying regulatory scheme manages this dynamic stem cell niche is not entirely clear. We stimulated intestinal organoids with Notch ligands and inhibitors and discovered that intestinal stem cells employ a positive feedback mechanism via direct Notch binding to the second intron of the Notch1 gene. Inactivation of the positive feedback by CRISPR/Cas9 mutation of the binding sequence alters the mosaic stem cell niche pattern and hinders regeneration in organoids. Dynamical system analysis and agent-based multiscale stochastic modeling suggest that the positive feedback enhances the robustness of Notch-mediated niche patterning. This study highlights the importance of feedback mechanisms in spatiotemporal control of the stem cell niche.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5408779PMC
http://dx.doi.org/10.15252/msb.20167324DOI Listing
April 2017

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

Nat Methods 2017 Apr 20;14(4):388-390. Epub 2017 Feb 20.

School of Applied and Engineering Physics, Cornell University, Ithaca, New York, USA.

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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http://dx.doi.org/10.1038/nmeth.4183DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6441362PMC
April 2017

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

Elife 2016 08 9;5. Epub 2016 Aug 9.

Department of Neurobiology and Behavior, Cornell University, Ithaca, United States.

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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http://dx.doi.org/10.7554/eLife.16808DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4978520PMC
August 2016

Simultaneous optical and electrical in vivo analysis of the enteric nervous system.

Nat Commun 2016 06 7;7:11800. Epub 2016 Jun 7.

School of Electrical and Computer Engineering, Cornell University, Ithaca, New York 14853, USA.

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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http://dx.doi.org/10.1038/ncomms11800DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4899629PMC
June 2016

Use of Tethered Enzymes as a Platform Technology for Rapid Analyte Detection.

PLoS One 2015 25;10(11):e0142326. Epub 2015 Nov 25.

Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Hungerford Hill Rd., Ithaca, NY 14853, United States of America.

Background: Rapid diagnosis for time-sensitive illnesses such as stroke, cardiac arrest, and septic shock is essential for successful treatment. Much attention has therefore focused on new strategies for rapid and objective diagnosis, such as Point-of-Care Tests (PoCT) for blood biomarkers. Here we use a biomimicry-based approach to demonstrate a new diagnostic platform, based on enzymes tethered to nanoparticles (NPs). As proof of principle, we use oriented immobilization of pyruvate kinase (PK) and luciferase (Luc) on silica NPs to achieve rapid and sensitive detection of neuron-specific enolase (NSE), a clinically relevant biomarker for multiple diseases ranging from acute brain injuries to lung cancer. We hypothesize that an approach capitalizing on the speed and catalytic nature of enzymatic reactions would enable fast and sensitive biomarker detection, suitable for PoCT devices.

Methods And Findings: We performed in-vitro, animal model, and human subject studies. First, the efficiency of coupled enzyme activities when tethered to NPs versus when in solution was tested, demonstrating a highly sensitive and rapid detection of physiological and pathological concentrations of NSE. Next, in rat stroke models the enzyme-based assay was able in minutes to show a statistically significant increase in NSE levels in samples taken 1 hour before and 0, 1, 3 and 6 hours after occlusion of the distal middle cerebral artery. Finally, using the tethered enzyme assay for detection of NSE in samples from 20 geriatric human patients, we show that our data match well (r = 0.815) with the current gold standard for biomarker detection, ELISA-with a major difference being that we achieve detection in 10 minutes as opposed to the several hours required for traditional ELISA.

Conclusions: Oriented enzyme immobilization conferred more efficient coupled activity, and thus higher assay sensitivity, than non-tethered enzymes. Together, our findings provide proof of concept for using oriented immobilization of active enzymes on NPs as the basis for a highly rapid and sensitive biomarker detection platform. This addresses a key challenge in developing a PoCT platform for time sensitive and difficult to diagnose pathologies.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0142326PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4659663PMC
June 2016