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

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

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

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, which we call DeepVess, yielded a segmentation accuracy that was better than both the current state-of-the-art and a trained human annotator, while also being orders of magnitude faster. 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 ($>75\mu m$) in aged animals as compared to young, in both wild type and Alzheimer's disease mouse models.Comment: 34 pages, 9 figure

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

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

Two-photon microscopy-guided femtosecond-laser photoablation of avian cardiogenesis: Noninvasive creation of localized heart defects

Yalçın, Hüseyin Çağatay (Dogus Author)Embryonic heart formation is driven by complex feedback between genetic and hemodynamic stimuli. Clinical congenital heart defects (CHD), however, often manifest as localized microtissue malformations with no underlying genetic mutation, suggesting that altered hemodynamics during embryonic development may play a role. An investigation of this relationship has been impaired by a lack of experimental tools that can create locally targeted cardiac perturbations. Here we have developed noninvasive optical techniques that can modulate avian cardiogenesis to dissect relationships between alterations in mechanical signaling and CHD. We used two-photon excited fluorescence microscopy to monitor cushion and ventricular dynamics and femtosecond pulsed laser photoablation to target micrometer-sized volumes inside the beating chick hearts. We selectively photoablated a small (∼100 μm radius) region of the superior atrioventricular (AV) cushion in Hamburger-Hamilton 24 chick embryos. We quantified via ultrasound that the disruption causes AV regurgitation, which resulted in a venous pooling of blood and severe arterial constriction. At 48 h postablation, quantitative X-ray microcomputed tomography imaging demonstrated stunted ventricular growth and pronounced left atrial dilation. A histological analysis demonstrated that the laser ablation produced defects localized to the superior AV cushion: a small quasispherical region of cushion tissue was completely obliterated, and the area adjacent to the myocardial wall was less cellularized. Both cushions and myocardium were significantly smaller than sham-operated controls. Our results highlight that two-photon excited fluorescence coupled with femtosecond pulsed laser photoablation should be considered a powerful tool for studying hemodynamic signaling in cardiac morphogenesis through the creation of localized microscale defects that may mimic clinical CHD

Interdisciplinary Class in Okayama University on the Climate Environment around Japan in Association with the Variety of Seasonal Feeling (with Attention to the Seasonal Cycle of Rainfall in the Warm Season)

The present study is a part of the activity to develop an interdisciplinary class on the climate environment around the Japan Islands in association with the “seasonal feeling”, with attention to the seasonal cycle of the weather systems and the rainfall characteristics during the warm season. Firstly, the school song “Wa-ka-ba” (which means the deep green leaves) was analyzed as a typical song expressing the season just between the spring and the Baiu, for developing study materials for the joint activity of meteorology to music. Next, seasonal difference of the rainfall characteristics around the Japan Islands was reviewed together with the new preliminary analyses, with attention to the contribution of the “heavy rainfall days” to the total precipitation amount. Finally, the joint activity of the art with meteorology for the class at the Faculty of Education, Okayama University was reported, together with the analyses of the students’ works expressing the rainfall event in a specified season by themselves

Cortical Microhemorrhages Cause Local Inflammation but Do Not Trigger Widespread Dendrite Degeneration

Microhemorrhages are common in the aging brain, and their incidence is correlated with increased risk of neurodegenerative disease. Past work has shown that occlusion of individual cortical microvessels as well as large-scale hemorrhages can lead to degeneration of neurons and increased inflammation. Using two-photon excited fluorescence microscopy in anesthetized mice, we characterized the acute and chronic dynamics of vessel bleeding, tissue compression, blood flow change, neural degeneration, and inflammation following a microhemorrhage caused by rupturing a single penetrating arteriole with tightly-focused femtosecond laser pulses. We quantified the extravasation of red blood cells (RBCs) and blood plasma into the brain and determined that the bleeding was limited by clotting. The vascular bleeding formed a RBC-filled core that compressed the surrounding parenchymal tissue, but this compression was not sufficient to crush nearby brain capillaries, although blood flow speeds in these vessels was reduced by 20%. Imaging of cortical dendrites revealed no degeneration of the large-scale structure of the dendritic arbor up to 14 days after the microhemorrhage. Dendrites close to the RBC core were displaced by extravasating RBCs but began to relax back one day after the lesion. Finally, we observed a rapid inflammatory response characterized by morphology changes in microglia/macrophages up to 200 µm from the microhemorrhage as well as extension of cellular processes into the RBC core. This inflammation persisted over seven days. Taken together, our data suggest that a cortical microhemorrhage does not directly cause significant neural pathology but does trigger a sustained, local inflammatory response

A new model for molecule exchange in the brain microvascular system: consequences of capillary occlusions in Alzheimer's disease

The brain microvascular system is a key actor in Alzheimer’s disease (AD) development. Indeed, a significant decrease of cerebral blood flow is the earliest biomarker of AD. In vivo TPLSM of cortical vasculature in APP/PS1 mice suggests the mechanism underlying the blood flow reduction is capillary occlusions. Leucocytes adhere to inflamed vessel walls and limit the flow. The impact of capillary occlusions on blood flow has been quantified numerically in large (>10000 vessels) anatomical networks in humans and mice. The regional blood flow has been found to depend linearly with no threshold effect on the fraction of capillary occlusions, so that a small fraction of stalls (2-4%) yields a significant decrease in blood flow (5-12%). Such flow decrease has a strong impact on nutrient delivery and waste clearance. That is why we devised a new model to study the effect of capillary stalling on molecule transport. The geometry of anatomical networks is too complex to use classic numerical approaches like finite elements. Instead, our model, inspired by pore-network approaches, reduces computational costs while capturing most of the underlying physics. To derive this model, we apply upscaling methods to the 3D transport equations within each vessel to obtain 1D average equations along the axis. Contrary to previous models, this new formulation describes accurately radial concentration gradients, capturing effects like longitudinal dispersion. We further use a Green’s function formulation to calculate the concentration fields inside the tissue where diffusion and reaction occur. The coupling between vessels and tissues is modelled using a membrane condition representing the blood brain barrier. This new molecule transport model is coupled with our previously validated blood flow model to examine the effects of capillary stalling on molecular exchange in transient and stationary regimes in anatomical networks. In particular, in stationnary regimes, we demonstrate an increase of the extraction coefficient with the proportion of stalled capillaries, which does not compensate for the associated blood flow reduction

Pressure Effect on Transport Properties of EuNi(Si1-xGex)3 Compounds

AbstractThe compounds of EuNi(Si1−xGex)3 order antiferromagnetically. At the temperature TC below the Ńeel temperature TN, EuNiSi3 (x = 0) shows an additional magnetic transition into ferro-magnetic state. TN decreases monotonously with increasing the Ge composition x. The Curie temperature TC decreases rapidly with increasing x and vanishes at the critical composition x ≈ 0.3. We have measured the electrical resistivity and thermopower of EuNi(Si0.8Ge0.2)3, which is a compound near to the boundary between the ferromagnetic and antiferromagnetic ground states in the phase diagram for EuNi(Si1−xGex)3 system, under pressures up to 1.8GPa at temperatures from 2 to 300K. The anomalies in ρ(T) and S(T) curves of EuNi(Si0.8Ge0.2)3 are observed at TC = 16K and TN = 34K at ambient pressure. Both TC and TN increase linearly with increasing pressure. The temperature variations of ρ and S of EuNi(Si0.8Ge0.2)3 at P = 1.8GPa are almost the same as those of EuNi(Si0.9Ge0.1)3 (x=0.1) at ambient pressure, revealing that the effect of pressure on TN and TC is the same as that of the increase of Si concentration. The pressure and atomic composition dependences of the magnetic transition temperatures TN and TC can be expressed by using the Grüneisen parameters. These results indicate that the changes of TN and TC are attributed to the change of atomic volume induced by the applying pressure or the atomic substitution

Comparison of brain vasculature network characteristics between wild type and Alzheimer’s disease mice using topological metrics

There is a strong clinical correlation between Alzheimer’s disease (AD) and microvascular disorders. In mouse models of AD, our lab has found blood flow dysfunction in brain capillaries, suggesting the need to study the function of vascular networks at the capillary level. However, the ability to deliver blood flow continuously to all neurons also depends on connections between vessels, requiring that we also characterize the topology of brain vascular networks. Here, we use graph theory and topological metrics to characterize the connectivity of brain capillary networks in AD and control mice