The mechanisms of rat astrocytes survival in the medium without glucose and in hypoxic conditions in vitro

Увод. Познато је да астроцити преживљавају дуже излагања депривацији кисеоника и глукозе (OGD) и да преживљавају данима без нутријената у поређењу са изразито осетљивим неуронима. Разлози овако смањене осетљивости астроцита и даље нису довољно разјашњени. Шта више, промене мембранског потенцијала митохондрија (ψm) астроцита, као индикатора ћелијског енергетског метаболизма и вијабилности, нису испитане током симулиране реперфузије након дужег излагања OGD (односно 1 % О2 без глукозе у присуству ограничене количине алтернативних нутријената). Смањена осетљивост астроцита може бити последица коришћења алтернативних ванћелијских извора енергије, као и унутарћелијских залиха енергије у циљу одржања мембранског потенцијала митохондрија. Стратегија преживљавања астроцита током таквих метаболичких изазова и даље није јасна. Методе. У овој студији, култура астроцита изложена је депривацији глукозе (GD), OGD и њиховој сукцесивној комбинацији различитих трајања. Промене ψm, праћене путем промена у флуоресценцији JC-1, испитане су у току једног сата током симулиране реперфузије у циљу моделовања услова in vivo. Флуоресцентни обележивач митохондрија JC-1 улази у матрикс митохондрије у зависности од потенцијала мембране митохондрије, померајући при томе максимум флуоресценције из зеленог у црвени део спектра. Такође, ефекти фармаколошке инхибиције два битна метаболичка пута: аутофагије хлорокином (CQ) и липолизе орлистатом, испитани су током депривације нутријената. Праћен је утицај поменутих инхибиција на промене вијабилности астроцита бојењима са акридин наранџастим (AO) и пропидијум јодидом (PI), као и промене мембранског потенцијала митохондрија (JC-1). Резултати. Показали смо да су астроцити отпорни на дуже периоде OGD (у трајању од 6 и 8 часова) која је имала слаб утицај на ψm током симулиране реперфузије, док је GD довела до хиперполаризације мембранског потенцијала митохондрија астроцита...Introduction. Astrocytes are known to tolerate long periods of oxygen-glucose deprivation (OGD) and they survive nutrient deprivation (ND) for days as compared to rather vulnerable neurons. The reasons for this reduced vulnerability of astrocytes are not well understood. In fact, changes in mitochondrial membrane potential (ψm), as the indicator of the cellular energy metabolism and viability, have not been investigated during simulated reperfusion after extended OGD exposure (i.e. 1 % of О2 without glucose in the presence of limited alternative nutrients). This reduced vulnerability could be due to utilization of the alternative extracellular sources of energy, as well as utilization of the internal energy stores, in maintenance of mitochondrial membrane potential. The pro-survival strategy of astrocytes under such metabolic challenge is still not clear. Methods. Here, we subjected astrocytes in culture to glucose deprivation (GD), OGD and combinations of both conditions varying in duration and sequence. Changes in ψm, visualized by the change in the fluorescence of JC-1, were investigated within one hour after reconstitution of oxygen and glucose supply, intended to model in vivo reperfusion. Fluorescent probe JC-1 enters the mitochondrial matrix in a potential-dependent manner, thus shifting its emission from green to red. Furthermore, the effects of inhibition of two potential steps in energy acquisition during ND: autophagy using chloroqione (CQ) and lipolysis using orlistat were investigated. Changes in astrocytes viability were followed with acridine orange (AO) and propidium iodide (PI) staining, and ψm was followed with JC-1. Results. We showed that astrocytes were resilient to extended periods of OGD (6 and 8 h), which had little effect on ψm during reperfusion, whereas GD contributed to a more negative ψm. Subsequent chemical oxygen deprivation induced by sodium azide caused depolarization, which, however, was significantly delayed as compared to the normoxic group. When GD preceded OGD for 12 h, mitochondrial membrane hyperpolarization was induced by both GD and subsequent OGD, but significant interaction between these conditions was not detected..

Symmetry breaking and functional incompleteness in biological systems

Symmetry-based explanations using symmetry breaking (SB) as the key explanatory tool have complemented and replaced traditional causal explanations in various domains of physics. The process of spontaneous SB is now a mainstay of contemporary explanatory accounts of large chunks of condensed-matter physics, quantum field theory, nonlinear dynamics, cosmology, and other disciplines. A wide range of empirical research into various phenomena related to symmetries and SB across biological scales has accumulated as well. Led by these results, we identify and explain some common features of the emergence, propagation, and cascading of SB-induced layers across the biosphere. These features are predicated on the thermodynamic openness and intrinsic functional incompleteness of the systems at stake and have not been systematically analyzed from a general philosophical and methodological perspective. We also consider possible continuity of SB across the physical and biological world and discuss the connection between Darwinism and SB-based analysis of the biosphere and its history

Symmetry breaking and functional incompleteness in biological systems

Symmetry-based explanations using symmetry breaking (SB) as the key explanatory tool have complemented and replaced traditional causal explanations in various domains of physics. The process of spontaneous SB is now a mainstay of contemporary explanatory accounts of large chunks of condensed-matter physics, quantum field theory, nonlinear dynamics, cosmology, and other disciplines. A wide range of empirical research into various phenomena related to symmetries and SB across biological scales has accumulated as well. Led by these results, we identify and explain some common features of the emergence, propagation, and cascading of SB-induced layers across the biosphere. These features are predicated on the thermodynamic openness and intrinsic functional incompleteness of the systems at stake and have not been systematically analyzed from a general philosophical and methodological perspective. We also consider possible continuity of SB across the physical and biological world and discuss the connection between Darwinism and SB-based analysis of the biosphere and its history

Combined segmentation and classificationbased approach to automated analysis of biomedical signals obtained from calcium imaging

Automated screening systems in conjunction with machine learning-based methods are becoming an essential part of the healthcare systems for assisting in disease diagnosis. Moreover, manually annotating data and hand-crafting features for training purposes are impractical and time-consuming. We propose a segmentation and classification-based approach for assembling an automated screening system for the analysis of calcium imaging. The method was developed and verified using the effects of disease IgGs (from Amyotrophic Lateral Sclerosis patients) on calcium (Ca2+) homeostasis. From 33 imaging videos we analyzed, 21 belonged to the disease and 12 to the control experimental groups. The method consists of three main steps: projection, segmentation, and classification. The entire Ca2+ time-lapse image recordings (videos) were projected into a single image using different projection methods. Segmentation was performed by using a multi-level thresholding (MLT) step and the Regions of Interest (ROIs) that encompassed cell somas were detected. A mean value of the pixels within these boundaries was collected at each time point to obtain the Ca2+ traces (time-series). Finally, a new matrix called feature image was generated from those traces and used for assessing the classification accuracy of various classifiers (control vs. disease). The mean value of the segmentation F-score for all the data was above 0.80 throughout the tested threshold levels for all projection methods, namely maximum intensity, standard deviation, and standard deviation with linear scaling projection. Although the classification accuracy reached up to 90.14%, interestingly, we observed that achieving better scores in segmentation results did not necessarily correspond to an increase in classification performance. Our method takes the advantage of the multi-level thresholding and of a classification procedure based on the feature images, thus it does not have to rely on hand- crafted training parameters of each event. It thus provides a semi-autonomous tool for assessing segmentation parameters which allows for the best classification accuracy

The mechanisms of rat astrocytes survival in the medium without glucose and in hypoxic conditions in vitro

Увод. Познато је да астроцити преживљавају дуже излагања депривацији кисеоника и глукозе (OGD) и да преживљавају данима без нутријената у поређењу са изразито осетљивим неуронима. Разлози овако смањене осетљивости астроцита и даље нису довољно разјашњени. Шта више, промене мембранског потенцијала митохондрија (ψm) астроцита, као индикатора ћелијског енергетског метаболизма и вијабилности, нису испитане током симулиране реперфузије након дужег излагања OGD (односно 1 % О2 без глукозе у присуству ограничене количине алтернативних нутријената). Смањена осетљивост астроцита може бити последица коришћења алтернативних ванћелијских извора енергије, као и унутарћелијских залиха енергије у циљу одржања мембранског потенцијала митохондрија. Стратегија преживљавања астроцита током таквих метаболичких изазова и даље није јасна. Методе. У овој студији, култура астроцита изложена је депривацији глукозе (GD), OGD и њиховој сукцесивној комбинацији различитих трајања. Промене ψm, праћене путем промена у флуоресценцији JC-1, испитане су у току једног сата током симулиране реперфузије у циљу моделовања услова in vivo. Флуоресцентни обележивач митохондрија JC-1 улази у матрикс митохондрије у зависности од потенцијала мембране митохондрије, померајући при томе максимум флуоресценције из зеленог у црвени део спектра. Такође, ефекти фармаколошке инхибиције два битна метаболичка пута: аутофагије хлорокином (CQ) и липолизе орлистатом, испитани су током депривације нутријената. Праћен је утицај поменутих инхибиција на промене вијабилности астроцита бојењима са акридин наранџастим (AO) и пропидијум јодидом (PI), као и промене мембранског потенцијала митохондрија (JC-1). Резултати. Показали смо да су астроцити отпорни на дуже периоде OGD (у трајању од 6 и 8 часова) која је имала слаб утицај на ψm током симулиране реперфузије, док је GD довела до хиперполаризације мембранског потенцијала митохондрија астроцита...Introduction. Astrocytes are known to tolerate long periods of oxygen-glucose deprivation (OGD) and they survive nutrient deprivation (ND) for days as compared to rather vulnerable neurons. The reasons for this reduced vulnerability of astrocytes are not well understood. In fact, changes in mitochondrial membrane potential (ψm), as the indicator of the cellular energy metabolism and viability, have not been investigated during simulated reperfusion after extended OGD exposure (i.e. 1 % of О2 without glucose in the presence of limited alternative nutrients). This reduced vulnerability could be due to utilization of the alternative extracellular sources of energy, as well as utilization of the internal energy stores, in maintenance of mitochondrial membrane potential. The pro-survival strategy of astrocytes under such metabolic challenge is still not clear. Methods. Here, we subjected astrocytes in culture to glucose deprivation (GD), OGD and combinations of both conditions varying in duration and sequence. Changes in ψm, visualized by the change in the fluorescence of JC-1, were investigated within one hour after reconstitution of oxygen and glucose supply, intended to model in vivo reperfusion. Fluorescent probe JC-1 enters the mitochondrial matrix in a potential-dependent manner, thus shifting its emission from green to red. Furthermore, the effects of inhibition of two potential steps in energy acquisition during ND: autophagy using chloroqione (CQ) and lipolysis using orlistat were investigated. Changes in astrocytes viability were followed with acridine orange (AO) and propidium iodide (PI) staining, and ψm was followed with JC-1. Results. We showed that astrocytes were resilient to extended periods of OGD (6 and 8 h), which had little effect on ψm during reperfusion, whereas GD contributed to a more negative ψm. Subsequent chemical oxygen deprivation induced by sodium azide caused depolarization, which, however, was significantly delayed as compared to the normoxic group. When GD preceded OGD for 12 h, mitochondrial membrane hyperpolarization was induced by both GD and subsequent OGD, but significant interaction between these conditions was not detected..

Structural and Functional Modulation of Perineuronal Nets: In Search of Important Players with Highlight on Tenascins

The extracellular matrix (ECM) of the brain plays a crucial role in providing optimal conditions for neuronal function. Interactions between neurons and a specialized form of ECM, perineuronal nets (PNN), are considered a key mechanism for the regulation of brain plasticity. Such an assembly of interconnected structural and regulatory molecules has a prominent role in the control of synaptic plasticity. In this review, we discuss novel ways of studying the interplay between PNN and its regulatory components, particularly tenascins, in the processes of synaptic plasticity, mechanotransduction, and neurogenesis. Since enhanced neuronal activity promotes PNN degradation, it is possible to study PNN remodeling as a dynamical change in the expression and organization of its constituents that is reflected in its ultrastructure. The discovery of these subtle modifications is enabled by the development of super-resolution microscopy and advanced methods of image analysis

Astrocytic mitochondrial membrane hyperpolarization following extended oxygen and glucose deprivation

Astrocytes can tolerate longer periods of oxygen and glucose deprivation (OGD) as compared to neurons. The reasons for this reduced vulnerability are not well understood. Particularly, changes in mitochondrial membrane potential (Δψm) in astrocytes, an indicator of the cellular redox state, have not been investigated during reperfusion after extended OGD exposure. Here, we subjected primary mouse astrocytes to glucose deprivation (GD), OGD and combinations of both conditions varying in duration and sequence. Changes in Δψm, visualized by change in the fluorescence of JC-1, were investigated within one hour after reconstitution of oxygen and glucose supply, intended to model in vivo reperfusion. In all experiments, astrocytes showed resilience to extended periods of OGD, which had little effect on Δψm during reperfusion, whereas GD caused a robust Δψm negativation. In case no Δψm negativation was observed after OGD, subsequent chemical oxygen deprivation (OD) induced by sodium azide caused depolarization, which, however, was significantly delayed as compared to normoxic group. When GD preceded OD for 12 h, Δψm hyperpolarization was induced by both GD and subsequent OD, but significant interaction between these conditions was not detected. However, when GD was extended to 48 h preceding OGD, hyperpolarization enhanced during reperfusion. This implicates synergistic effects of both conditions in that sequence. These findings provide novel information regarding the role of the two main substrates of electron transport chain (glucose and oxygen) and their hyperpolarizing effect on Δψm during substrate deprivation, thus shedding new light on mechanisms of astrocyte resilience to prolonged ischemic injury

Schematic representation of the experimental design and most important results.

<p>Figure illustrates crucial steps in investigation of Δψ_m changes during simulated reperfusion within one hour after extended exposure of astrocytes to GD or OGD. The culturing media are designated as: high glucose (hG), low glucose (lG) or no glucose (nG). During the experiments cells were cultured either in normoxic (normO₂) or hypoxic (hypoO₂) conditions. Ellipsoid shapes show conditions the cells were subjected to. Text on connector lines shows whether we found significant effect of given experimental condition(s). Octagons indicate conclusions we made.</p

OGD in lG partly preserves Δψ_m (i.e. delays depolarization) caused by subsequent NaN₃ treatment.

<p>(<b>A</b>) Sodium azide (NaN₃) causes concentration-dependent decline of Δψ_m in both experimental conditions. However, OGD in lG medium significantly delayed the decrease in JC-1 fluorescence ratio caused by treatment with 5 mM NaN₃ during simulated reperfusion. (<b>B</b>) Expectedly, both NaN₃ and H₂O₂ show concentration-dependent effect by lowering the JC-1 red/green fluorescence ratio. Astrocytes were stained with JC-1 either before or during the treatment with NaN₃ (marked as pre-dye loading and dye loading under ETC inhibition, respectively). NaN₃ has not affected the cell membrane organization allowing at the same time JC-1 to enter the cytoplasm and mitochondria within. (<b>C</b>) Representative fluorescent micrographs of astrocytes labeled with JC-1 Original micrographs were converted to tritanope color palette (ImageJ 1.48a). Depolarization is visible in the normoxic group (b, c) (seen as concentration-dependent decrease in magenta and increase in blue color), but it is more pronounced than in OGD group after treatment with NaN₃ (e, f). Some mitochondria remained partly depolarized. Data are expressed as a percentage normalized to the red/green fluorescence ratio values of untreated control (the first bar from left). Significant differences are indicated by **p<0.01 with respect to untreated control, ##p<0.01 between treatment and its respective control, ΔΔp<0.01 between different inhibitor concentrations.</p

Glucose deprivation leads to hyperpolarization of Δψ_m after both normoxic and OGD conditions.

<p>(<b>A</b>) Glucose deprivation for 8 h increases the ratio of JC-1 fluorescence. This effect was not detected when combining GD and OD, but level of significance was noticable low (p = 0.059). (<b>B</b>) Preconditioning for 12 h with media containing reduced or no glucose promotes hyperpolarization after OGD during simulated reperfusion. After preconditioning, astrocytes were subjected to hypoxic conditions for additional 6 h (grey bars) or they were maintained in normoxic conditions (white bars). (<b>C</b>) Lowering glucose in the incubation medium leads to an increased ratio of JC-1 fluorescence during simulated reperfusion after normoxic and OGD conditions. There was significant interaction between effect of glucose in the culturing media and the effect of OGD. Astrocytes were incubated for two days either in hG or lG medium. Subsequently, OGD was conducted for 6 h (grey bars). (<b>D</b>) Schematic representation of the experimental design of C. The duration of each step is shown in brackets. (<b>E</b>) Representative red signal from fluorescent micrographs of astrocytes labeled with JC-1. Original micrographs were converted to rainbow pseudocolor pallete using LUTs (ImageJ 1.48a). Increase in red fluorescence is observed when astrocytes are incubated in OGD in nG medium as compared to normoxic conditions in lG medium (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090697#pone-0090697-g002" target="_blank">Fig. 2B</a>). The scale bar represents 100 µm. Data are expressed as a percentage normalized to the JC-1 red/green fluorescence ratio values of untreated control astrocytes (first bar on the left). Significant differences are indicated by **p<0.01 with respect to control (normoxia in hG for Fig. 3A and 3C, normoxia in lG for Fig. 3B), ##p<0.01 between normoxia and OGD (in lG or nG), ΔΔp<0.01 between two OGD treatments.</p