Astroglial Hmgb1 regulates postnatal astrocyte morphogenesis and cerebrovascular maturation.

Astrocytes are intimately linked with brain blood vessels, an essential relationship for neuronal function. However, astroglial factors driving these physical and functional associations during postnatal brain development have yet to be identified. By characterizing structural and transcriptional changes in mouse cortical astrocytes during the first two postnatal weeks, we find that high-mobility group box 1 (Hmgb1), normally upregulated with injury and involved in adult cerebrovascular repair, is highly expressed in astrocytes at birth and then decreases rapidly. Astrocyte-selective ablation of Hmgb1 at birth affects astrocyte morphology and endfoot placement, alters distribution of endfoot proteins connexin43 and aquaporin-4, induces transcriptional changes in astrocytes related to cytoskeleton remodeling, and profoundly disrupts endothelial ultrastructure. While lack of astroglial Hmgb1 does not affect the blood-brain barrier or angiogenesis postnatally, it impairs neurovascular coupling and behavior in adult mice. These findings identify astroglial Hmgb1 as an important player in postnatal gliovascular maturation.

Neonatal hyperoxia in mice triggers long-term cognitive deficits via impairments in cerebrovascular function and neurogenesis.

Preterm birth is the leading cause of death in children under 5 years of age. Premature infants who receive life-saving oxygen therapy often develop bronchopulmonary dysplasia (BPD), a chronic lung disease. Infants with BPD are at a high risk of abnormal neurodevelopment, including motor and cognitive difficulties. While neural progenitor cells (NPCs) are crucial for proper brain development, it is unclear whether they play a role in BPD-associated neurodevelopmental deficits. Here, we show that hyperoxia-induced experimental BPD in newborn mice led to lifelong impairments in cerebrovascular structure and function as well as impairments in NPC self-renewal and neurogenesis. A neurosphere assay utilizing nonhuman primate preterm baboon NPCs confirmed impairment in NPC function. Moreover, gene expression profiling revealed that genes involved in cell proliferation, angiogenesis, vascular autoregulation, neuronal formation, and neurotransmission were dysregulated following neonatal hyperoxia. These impairments were associated with motor and cognitive decline in aging hyperoxia-exposed mice, reminiscent of deficits observed in patients with BPD. Together, our findings establish a relationship between BPD and abnormal neurodevelopmental outcomes and identify molecular and cellular players of neonatal brain injury that persist throughout adulthood that may be targeted for early intervention to aid this vulnerable patient population.

An analysis of the influence of transfer learning when measuring the tortuosity of blood vessels.

BACKGROUND AND OBJECTIVE: Convolutional Neural Networks (CNNs) can provide excellent results regarding the segmentation of blood vessels. One important aspect of CNNs is that they can be trained on large amounts of data and then be made available, for instance, in image processing software. The pre-trained CNNs can then be easily applied in downstream blood vessel characterization tasks, such as the calculation of the length, tortuosity, or caliber of the blood vessels. Yet, it is still unclear if pre-trained CNNs can provide robust, unbiased, results in downstream tasks involving the morphological analysis of blood vessels. Here, we focus on measuring the tortuosity of blood vessels and investigate to which extent CNNs may provide biased tortuosity values even after fine-tuning the network to a new dataset under study. METHODS: We develop a procedure for quantifying the influence of CNN pre-training in downstream analyses involving the measurement of morphological properties of blood vessels. Using the methodology, we compare the performance of CNNs that were trained on images containing blood vessels having high tortuosity with CNNs that were trained on blood vessels with low tortuosity and fine-tuned on blood vessels with high tortuosity. The opposite situation is also investigated. RESULTS: We show that the tortuosity values obtained by a CNN trained from scratch on a dataset may not agree with those obtained by a fine-tuned network that was pre-trained on a dataset having different tortuosity statistics. In addition, we show that improving the segmentation accuracy does not necessarily lead to better tortuosity estimation. To mitigate the aforementioned issues, we propose the application of data augmentation techniques even in situations where they do not improve segmentation performance. For instance, we found that the application of elastic transformations was enough to prevent an underestimation of 8% of blood vessel tortuosity when applying CNNs to different datasets. CONCLUSIONS: The results highlight the importance of developing new methodologies for training CNNs with the specific goal of reducing the error of morphological measurements, as opposed to the traditional approach of using segmentation accuracy as a proxy metric for performance evaluation.

Unbiased analysis of mouse brain endothelial networks from two- or three-dimensional fluorescence images.

Significance: A growing body of research supports the significant role of cerebrovascular abnormalities in neurological disorders. As these insights develop, standardized tools for unbiased and high-throughput quantification of cerebrovascular structure are needed. Aim: We provide a detailed protocol for performing immunofluorescent labeling of mouse brain vessels, using thin ( 25 µ m ) or thick (50 to 150 µ m ) tissue sections, followed respectively by two- or three-dimensional (2D or 3D) unbiased quantification of vessel density, branching, and tortuosity using digital image processing algorithms. Approach: Mouse brain sections were immunofluorescently labeled using a highly selective antibody raised against mouse Cluster of Differentiation-31 (CD31), and 2D or 3D microscopy images of the mouse brain vasculature were obtained using optical sectioning. An open-source toolbox, called Pyvane, was developed for analyzing the imaged vascular networks. The toolbox can be used to identify the vasculature, generate the medial axes of blood vessels, represent the vascular network as a graph, and calculate relevant measurements regarding vascular morphology. Results: Using Pyvane, vascular parameters such as endothelial network density, number of branching points, and tortuosity are quantified from 2D and 3D immunofluorescence micrographs. Conclusions: The steps described in this protocol are simple to follow and allow for reproducible and unbiased analysis of mouse brain vascular structure. Such a procedure can be applied to the broader field of vascular biology.

Maternal high-fat diet in mice induces cerebrovascular, microglial and long-term behavioural alterations in offspring.

Various environmental exposures during pregnancy, like maternal diet, can compromise, at critical periods of development, the neurovascular maturation of the offspring. Foetal exposure to maternal high-fat diet (mHFD), common to Western societies, has been shown to disturb neurovascular development in neonates and long-term permeability of the neurovasculature. Nevertheless, the effects of mHFD on the offspring's cerebrovascular health remains largely elusive. Here, we sought to address this knowledge gap by using a translational mouse model of mHFD exposure. Three-dimensional and ultrastructure analysis of the neurovascular unit (vasculature and parenchymal cells) in mHFD-exposed offspring revealed major alterations of the neurovascular organization and metabolism. These alterations were accompanied by changes in the expression of genes involved in metabolism and immunity, indicating that neurovascular changes may result from abnormal brain metabolism and immune regulation. In addition, mHFD-exposed offspring showed persisting behavioural alterations reminiscent of neurodevelopmental disorders, specifically an increase in stereotyped and repetitive behaviours into adulthood.

Gland context networks: A novel approach for improving prostate cancer identification.

Prostate cancer (PCa) is a pervasive condition that is manifested in a wide range of histologic patterns in biopsy samples. Given the importance of identifying abnormal prostate tissue to improve prognosis, many computerized methodologies aimed at assisting pathologists in diagnosis have been developed. It is often argued that improved diagnosis of a tissue region can be obtained by considering measurements that can take into account several properties of its surroundings, therefore providing a more robust context for the analysis. Here we propose a novel methodology that can be used for systematically defining contextual features regarding prostate glands. This is done by defining a Gland Context Network (GCN), a representation of the prostate sample containing information about the spatial relationship between glands as well as the similarity between their appearance. We show that such a network can be used for establishing contextual features at any spatial scale, therefore providing information that is not easily obtained from traditional shape and textural features. Furthermore, it is shown that even basic features derived from a GCN can lead to state-of-the-art classification performance regarding PCa. All in all, GCNs can assist in defining more effective approaches for PCa grading.

Quantification of retinal blood leakage in fundus fluorescein angiography in a retinal angiogenesis model.

Blood leakage from the vessels in the eye is the hallmark of many vascular eye diseases. One of the preclinical mouse models of retinal blood leakage, the very-low-density-lipoprotein receptor deficient mouse (Vldlr-/-), is used for drug screening and mechanistic studies. Vessel leakage is usually examined using Fundus fluorescein angiography (FFA). However, interpreting FFA images of the Vldlr-/- model is challenging as no automated and objective techniques exist for this model. A pipeline has been developed for quantifying leakage intensity and area including three tasks: (i) blood leakage identification, (ii) blood vessel segmentation, and (iii) image registration. Morphological operations followed by log-Gabor quadrature filters were used to identify leakage regions. In addition, a novel optic disk detection algorithm based on graph analysis was developed for registering the images at different timepoints. Blood leakage intensity and area measured by the methodology were compared to ground truth quantifications produced by two annotators. The relative difference between the quantifications from the method and those obtained from ground truth images was around 10% ± 6% for leakage intensity and 17% ± 8% for leakage region. The Pearson correlation coefficient between the method results and the ground truth was around 0.98 for leakage intensity and 0.94 for leakage region. Therefore, we presented a computational method for quantifying retinal vascular leakage and vessels using FFA in a preclinical angiogenesis model, the Vldlr-/- model.

An Exercise Mimetic Approach to Reduce Poststroke Deconditioning and Enhance Stroke Recovery.

Evidence supports early rehabilitation after stroke to limit disability. However, stroke survivors are typically sedentary and experience significant cardiovascular and muscular deconditioning. Despite growing consensus that preclinical and clinical stroke recovery research should be aligned, there have been few attempts to incorporate cardiovascular and skeletal muscle deconditioning into animal models of stroke. Here, we demonstrate in rats that a hindlimb sensorimotor cortex stroke results in both cardiovascular and skeletal muscle deconditioning and impairments in gait akin to those observed in humans. To reduce poststroke behavioral, cardiovascular, and skeletal muscle perturbations, we then used a combinatorial intervention consisting of aerobic and resistance exercise in conjunction with administration of resveratrol (RESV), a drug with exercise mimetic properties. A combination of aerobic and resistance exercise mitigated decreases in cardiovascular fitness and attenuated skeletal muscle abnormalities. RESV, beginning 24 hours poststroke, reduced acute hindlimb impairments, improved recovery in hindlimb function, increased vascular density in the perilesional cortex, and attenuated skeletal muscle fiber changes. Early RESV treatment and aerobic and resistance exercise independently provided poststroke benefits, at a time when individuals are rapidly becoming deconditioned as a result of inactivity. Although no additive effects were observed in these experiments, this approach represents a promising strategy to reduce poststroke behavioral impairments and minimize deconditioning. As such, this treatment regime has potential for enabling patients to engage in more intensive rehabilitation at an earlier time following stroke when mechanisms of neuroplasticity are most prevalent.

Vascular contributions to 16p11.2 deletion autism syndrome modeled in mice.

While the neuronal underpinnings of autism spectrum disorder (ASD) are being unraveled, vascular contributions to ASD remain elusive. Here, we investigated postnatal cerebrovascular development in the 16p11.2df/+ mouse model of 16p11.2 deletion ASD syndrome. We discover that 16p11.2 hemizygosity leads to male-specific, endothelium-dependent structural and functional neurovascular abnormalities. In 16p11.2df/+ mice, endothelial dysfunction results in impaired cerebral angiogenesis at postnatal day 14, and in altered neurovascular coupling and cerebrovascular reactivity at postnatal day 50. Moreover, we show that there is defective angiogenesis in primary 16p11.2df/+ mouse brain endothelial cells and in induced-pluripotent-stem-cell-derived endothelial cells from human carriers of the 16p11.2 deletion. Finally, we find that mice with an endothelium-specific 16p11.2 deletion (16p11.2ΔEC) partially recapitulate some of the behavioral changes seen in 16p11.2 syndrome, specifically hyperactivity and impaired motor learning. By showing that developmental 16p11.2 haploinsufficiency from endothelial cells results in neurovascular and behavioral changes in adults, our results point to a potential role for endothelial impairment in ASD.

Clustering algorithms: A comparative approach.

Many real-world systems can be studied in terms of pattern recognition tasks, so that proper use (and understanding) of machine learning methods in practical applications becomes essential. While many classification methods have been proposed, there is no consensus on which methods are more suitable for a given dataset. As a consequence, it is important to comprehensively compare methods in many possible scenarios. In this context, we performed a systematic comparison of 9 well-known clustering methods available in the R language assuming normally distributed data. In order to account for the many possible variations of data, we considered artificial datasets with several tunable properties (number of classes, separation between classes, etc). In addition, we also evaluated the sensitivity of the clustering methods with regard to their parameters configuration. The results revealed that, when considering the default configurations of the adopted methods, the spectral approach tended to present particularly good performance. We also found that the default configuration of the adopted implementations was not always accurate. In these cases, a simple approach based on random selection of parameters values proved to be a good alternative to improve the performance. All in all, the reported approach provides subsidies guiding the choice of clustering algorithms.

The dynamics of knowledge acquisition via self-learning in complex networks.

Studies regarding knowledge organization and acquisition are of great importance to understand areas related to science and technology. A common way to model the relationship between different concepts is through complex networks. In such representations, networks' nodes store knowledge and edges represent their relationships. Several studies that considered this type of structure and knowledge acquisition dynamics employed one or more agents to discover node concepts by walking on the network. In this study, we investigate a different type of dynamics adopting a single node as the "network brain." Such a brain represents a range of real systems such as the information about the environment that is acquired by a person and is stored in the brain. To store the discovered information in a specific node, the agents walk on the network and return to the brain. We propose three different dynamics and test them on several network models and on a real system, which is formed by journal articles and their respective citations. The results revealed that, according to the adopted walking models, the efficiency of self-knowledge acquisition has only a weak dependency on topology and search strategy.

Hyperfiltration in ubiquitin C-terminal hydrolase L1-deleted mice.

Neuronal ubiquitin C-terminal hydrolase L1 (UCHL1) is a deubiquitinating enzyme that maintains intracellular ubiquitin pools and promotes axonal transport. Uchl1 deletion in mice leads to progressive axonal degeneration, affecting the dorsal root ganglion that harbors axons emanating to the kidney. Innervation is a crucial regulator of renal hemodynamics, though the contribution of neuronal UCHL1 to this is unclear. Immunofluorescence revealed significant neuronal UCHL1 expression in mouse kidney, including periglomerular axons. Glomerular filtration rate trended higher in 6-week-old Uchl1-/- mice, and by 12 weeks of age, these displayed significant glomerular hyperfiltration, coincident with the onset of neurodegeneration. Angiotensin converting enzyme inhibition had no effect on glomerular filtration rate of Uchl1-/- mice indicating that the renin-angiotensin system does not contribute to the observed hyperfiltration. DCE-MRI revealed increased cortical renal blood flow in Uchl1-/- mice, suggesting that hyperfiltration results from afferent arteriole dilation. Nonetheless, hyperglycemia, cyclooxygenase-2, and nitric oxide synthases were ruled out as sources of hyperfiltration in Uchl1-/- mice as glomerular filtration rate remained unchanged following insulin treatment, and cyclooxygenase-2 and nitric oxide synthase inhibition. Finally, renal nerve dysfunction in Uchl1-/- mice is suggested given increased renal nerve arborization, decreased urinary norepinephrine, and impaired vascular reactivity. Uchl1-deleted mice demonstrate glomerular hyperfiltration associated with renal neuronal dysfunction, suggesting that neuronal UCHL1 plays a crucial role in regulating renal hemodynamics.

Biological network border detection.

Complex networks have been widely used to model biological systems. The concept of accessibility has been proposed recently as a means to organize the nodes of complex networks as belonging to its border or center. Such an approach paves the way to investigating how the functional and structural properties of nodes vary with their respective position in the networks. In this work, we approach such a problem in a biological context applying border detection to Protein-Protein Interaction networks from four organisms of the Mycoplasma genus. We found evidence that the borderness of proteins bears a relation with their spatial organization and molecular function specificity.

The aPKC-CBP Pathway Regulates Post-stroke Neurovascular Remodeling and Functional Recovery.

Epigenetic modifications have emerged as attractive molecular substrates that integrate extrinsic changes into the determination of cell identity. Since stroke-related brain damage releases micro-environmental cues, we examined the role of a signaling-induced epigenetic pathway, an atypical protein kinase C (aPKC)-mediated phosphorylation of CREB-binding protein (CBP), in post-stroke neurovascular remodeling. Using a knockin mouse strain (CbpS436A) where the aPKC-CBP pathway was defective, we show that disruption of the aPKC-CBP pathway in a murine focal ischemic stroke model increases the reprogramming efficiency of ischemia-activated pericytes (i-pericytes) to neural precursors. As a consequence of enhanced cellular reprogramming, CbpS436A mice show an increased transient population of locally derived neural precursors after stroke, while displaying a reduced number of i-pericytes, impaired vascular remodeling, and perturbed motor recovery during the chronic phase of stroke. Together, this study elucidates the role of the aPKC-CBP pathway in modulating neurovascular remodeling and functional recovery following focal ischemic stroke.

Analysis of Scanning Electron Microscopy Images To Investigate Adsorption Processes Responsible for Detection of Cancer Biomarkers.

Adsorption processes are responsible for detection of cancer biomarkers in biosensors (and immunosensors), which can be captured with various principles of detection. In this study, we used a biosensor made with nanostructured films of polypyrrole and p53 antibodies, and image analysis of scanning electron microscopy data made it possible to correlate morphological changes of the biosensor with the concentration of cells containing the cancer biomarker p53. The selectivity of the biosensor was proven by distinguishing images obtained with exposure of the biosensor to cells containing the biomarker from those acquired with cells that did not contain it. Detection was confirmed with cyclic voltammetry measurements, while the adsorption of the p53 biomarker was probed with polarization-modulated infrared reflection absorption (PM-IRRAS) and a quartz crystal microbalance (QCM). Adsorption is described using the Langmuir-Freundlich model, with saturation taking place at a concentration of 100 Ucells/mL. Taken together, our results point to novel ways to detect biomarkers or any type of analyte for which detection is based on adsorption as is the case of the majority of biosensors.

Temporal modulation of collective cell behavior controls vascular network topology.

Vascular network density determines the amount of oxygen and nutrients delivered to host tissues, but how the vast diversity of densities is generated is unknown. Reiterations of endothelial-tip-cell selection, sprout extension and anastomosis are the basis for vascular network generation, a process governed by the VEGF/Notch feedback loop. Here, we find that temporal regulation of this feedback loop, a previously unexplored dimension, is the key mechanism to determine vascular density. Iterating between computational modeling and in vivo live imaging, we demonstrate that the rate of tip-cell selection determines the length of linear sprout extension at the expense of branching, dictating network density. We provide the first example of a host tissue-derived signal (Semaphorin3E-Plexin-D1) that accelerates tip cell selection rate, yielding a dense network. We propose that temporal regulation of this critical, iterative aspect of network formation could be a general mechanism, and additional temporal regulators may exist to sculpt vascular topology.

Seeking maximum linearity of transfer functions.

Linearity is an important and frequently sought property in electronics and instrumentation. Here, we report a method capable of, given a transfer function (theoretical or derived from some real system), identifying the respective most linear region of operation with a fixed width. This methodology, which is based on least squares regression and systematic consideration of all possible regions, has been illustrated with respect to both an analytical (sigmoid transfer function) and a simple situation involving experimental data of a low-power, one-stage class A transistor current amplifier. Such an approach, which has been addressed in terms of transfer functions derived from experimentally obtained characteristic surface, also yielded contributions such as the estimation of local constants of the device, as opposed to typically considered average values. The reported method and results pave the way to several further applications in other types of devices and systems, intelligent control operation, and other areas such as identifying regions of power law behavior.

Thermodynamic characterization of networks using graph polynomials.

In this paper, we present a method for characterizing the evolution of time-varying complex networks by adopting a thermodynamic representation of network structure computed from a polynomial (or algebraic) characterization of graph structure. Commencing from a representation of graph structure based on a characteristic polynomial computed from the normalized Laplacian matrix, we show how the polynomial is linked to the Boltzmann partition function of a network. This allows us to compute a number of thermodynamic quantities for the network, including the average energy and entropy. Assuming that the system does not change volume, we can also compute the temperature, defined as the rate of change of entropy with energy. All three thermodynamic variables can be approximated using low-order Taylor series that can be computed using the traces of powers of the Laplacian matrix, avoiding explicit computation of the normalized Laplacian spectrum. These polynomial approximations allow a smoothed representation of the evolution of networks to be constructed in the thermodynamic space spanned by entropy, energy, and temperature. We show how these thermodynamic variables can be computed in terms of simple network characteristics, e.g., the total number of nodes and node degree statistics for nodes connected by edges. We apply the resulting thermodynamic characterization to real-world time-varying networks representing complex systems in the financial and biological domains. The study demonstrates that the method provides an efficient tool for detecting abrupt changes and characterizing different stages in network evolution.

Automated high-content morphological analysis of muscle fiber histology.

In the search for a cure for many muscular disorders it is often necessary to analyze muscle fibers under a microscope. For this morphological analysis, we developed an image processing approach to automatically analyze and quantify muscle fiber images so as to replace today's less accurate and time-consuming manual method. Muscular disorders, that include cardiomyopathy, muscular dystrophies, and diseases of nerves that affect muscles such as neuropathy and myasthenia gravis, affect a large percentage of the population and, therefore, are an area of active research for new treatments. In research, the morphological features of muscle fibers play an important role as they are often used as biomarkers to evaluate the progress of underlying diseases and the effects of potential treatments. Such analysis involves assessing histopathological changes of muscle fibers as indicators for disease severity and also as a criterion in evaluating whether or not potential treatments work. However, quantifying morphological features is time-consuming, as it is usually performed manually, and error-prone. To replace this standard method, we developed an image processing approach to automatically detect and measure the cross-sections of muscle fibers observed under microscopy that produces faster and more objective results. As such, it is well-suited to processing the large number of muscle fiber images acquired in typical experiments, such as those from studies with pre-clinical models that often create many images. Tests on real images showed that the approach can segment and detect muscle fiber membranes and extract morphological features from highly complex images to generate quantitative results that are readily available for statistical analysis.

Approximate von Neumann entropy for directed graphs.

In this paper, we develop an entropy measure for assessing the structural complexity of directed graphs. Although there are many existing alternative measures for quantifying the structural properties of undirected graphs, there are relatively few corresponding measures for directed graphs. To fill this gap in the literature, we explore an alternative technique that is applicable to directed graphs. We commence by using Chung's generalization of the Laplacian of a directed graph to extend the computation of von Neumann entropy from undirected to directed graphs. We provide a simplified form of the entropy which can be expressed in terms of simple node in-degree and out-degree statistics. Moreover, we find approximate forms of the von Neumann entropy that apply to both weakly and strongly directed graphs, and that can be used to characterize network structure. We illustrate the usefulness of these simplified entropy forms defined in this paper on both artificial and real-world data sets, including structures from protein databases and high energy physics theory citation networks.