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Circumscribing Laser Cuts Attenuate Seizure Propagation in a Mouse Model of Focal Epilepsy

Seth Lieberman, Daniel A. Rivera, Ryan Morton, Amrit Hingorani, Teresa L. Southard, Lynn Johnson, Jennifer Reukauf, Ryan E. Radwanski, Mingrui Zhao, Nozomi Nishimura, Oliver Bracko, Theodore H. Schwartz, Chris B. Schaffer

Advanced Science (2024)

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In partial onset epilepsy, seizures arise focally in the brain and often propagate. Patients frequently become refractory to medical management, leaving neurosurgery, which can cause neurologic deficits, as a primary treatment. In the cortex, focal seizures spread through horizontal connections in layers II/III, suggesting that severing these connections can block seizures while preserving function. Focal neocortical epilepsy is induced in mice, sub-surface cuts are created surrounding the seizure focus using tightly-focused femtosecond laser pulses, and electrophysiological recordings are acquired at multiple locations for 3–12 months. Cuts reduced seizure frequency in most animals by 87%, and only 5% of remaining seizures propagated to the distant electrodes, compared to 80% in control animals. These cuts produced a modest decrease in cortical blood flow that recovered and left a ~20-µm wide scar with minimal collateral damage. When placed over the motor cortex, cuts do not cause notable deficits in a skilled reaching task, suggesting they hold promise as a novel neurosurgical approach for intractable focal cortical epilepsy.

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Extracellular vesicles incorporating retrovirus-like capsids for the enhanced packaging and systemic delivery of mRNA into neurons

Wenchao Gu, Sijin Luozhong, Simian Cai, Ketaki Londhe, Nadine Elkasri, Robert Hawkins, Zhefan Yuan, Kai Su-Greene, Yujie Yin, Margaret Cruz, Yu-Wei Chang, Patrick McMullen, Chunyan Wu, Changwoo Seo, Akash Guru, Wenting Gao, Tara Sarmiento, Chris Schaffer, Nozomi Nishimura, Richard Cerione, Qiuming Yu, Melissa Warden, Robert Langer, Shaoyi Jiang

Nature Biomedical Engineering (2024)

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The blood–brain barrier (BBB) restricts the systemic delivery of messenger RNAs (mRNAs) into diseased neurons. Although leucocyte-derived extracellular vesicles (EVs) can cross the BBB at inflammatory sites, it is difficult to efficiently load long mRNAs into the EVs and to enhance their neuronal uptake. Here we show that the packaging of mRNA into leucocyte-derived EVs and the endocytosis of the EVs by neurons can be enhanced by engineering leucocytes to produce EVs that incorporate retrovirus-like mRNA-packaging capsids. We transfected immortalized and primary bone-marrow-derived leucocytes with DNA or RNA encoding the capsid-forming activity-regulated cytoskeleton-associated (Arc) protein as well as capsid-stabilizing Arc 5’-untranslated-region RNA elements. These engineered EVs inherit endothelial adhesion molecules from donor leukocytes, recruit endogenous enveloping proteins to their surface, cross the BBB, and enter the neurons in neuro-inflammatory sites. Produced from self-derived donor leukocytes, the EVs are immunologically inert, and enhanced the neuronal uptake of the packaged mRNA in a mouse model of low-grade chronic neuro-inflammation.

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

Oliver Bracko , Jean C Cruz Hernandez, Laibaik Park, Nozomi Nishimura and Chris B Schaffer

Journal of Cerebral Blood flow and Metabolism (2021)

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

Oliver Bracko, Lindsay K. Vinarcsik, Jean C. Cruz Hernandez, Nancy E. Ruiz-Uribe, Mohammad Haft-Javaherian, Kaja Falkenhain, Egle M. Ramanauskaite, Muhammad Ali, Aditi Mohapatra, Madisen A. Swallow, Brendah N. Njiru, Victorine Muse, Pietro E. Michelucci, Nozomi Nishimura & Chris B. Schaffer

Scientific Reports (2020)

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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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Hyperspectral multiphoton microscopy for in vivo visualization of multiple, spectrally overlapped fluorescent labels

Amanda J. Bares, Menansili A. Mejooli, Mitchell A. Pender, Scott A. Leddon, Steven Tilley, Karen Lin, Jingyuan Dong, Minsoo Kim, Deborah J. Fowell, Nozomi Nishimura, and Chris B. Schaffer

Optica (2020)

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The insensitivity of multiphoton microscopy to optical scattering enables high-resolution, high-contrast imaging deep into tissue, including in live animals. Scattering does, however, severely limit the use of spectral dispersion techniques to improve spectral resolution. In practice, this limited spectral resolution together with the need for multiple excitation wavelengths to excite different fluorophores limits multiphoton microscopy to imaging a few, spectrally distinct fluorescent labels at a time, restricting the complexity of biological processes that can be studied. Here, we demonstrate a hyperspectral multiphoton microscope that utilizes three different wavelength excitation sources together with multiplexed fluorescence emission detection using angle-tuned bandpass filters. This microscope maintains scattering insensitivity, while providing high enough spectral resolution on the emitted fluorescence and capitalizing on the wavelength-dependent nonlinear excitation of fluorescent dyes to enable clean separation of multiple, spectrally overlapping labels, in vivo. We demonstrated the utility of this instrument for spectral separation of closely overlapped fluorophores in samples containing 10 different colors of fluorescent beads, live cells expressing up to seven different fluorescent protein fusion constructs, and in multiple in vivo preparations in mouse cortex and inflamed skin, with up to eight different cell types or tissue structures distinguished.

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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 Hernandez, 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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