Red blood cells (RBCs) visualisation in bifurcations and bends

Bifurcating networks are commonly found in nature. One example is the microvascular system, composed of blood vessels consecutively branching into daughter vessels, driving the blood into the capillaries, where the red blood cells (RBCs) are responsible for delivering O 2 and up taking cell waste and CO 2 . In this preliminary study, we explore a microfluidic bifurcating geometry inspired by such biological models, for investigating RBC partitioning as well as RBC-plasma separation favored by the consecutive bifurcating channels. A biomimetic design rule [1] based on Murray’s law [2] was used to set the channels’ dimensions along the network, which consists of consecutive bifurcating channels of reducing diameter. The ability to apply differential flow resistances by controlling the flow rates at the end of the network allowed us to monitor the formation of a cell-free layer (CFL) for different flow conditions at haematocrits of 1% and 5%. We have also compared the values of CFL thickness determined directly by the measurement on the projection image created from a stack of images or indirectly by analyzing the intensity profile in the same projection. The results obtained from this study confirm the potential to study RBC partitioning along bifurcating networks, which could be of particular interest for the separation of RBCs from plasma in point-of-care devices.JF would like to thank Professor Graça Minas and her coworkers for providing the laboratory facilities and technical help during the experiments.info:eu-repo/semantics/publishedVersio

Visualization and measurement of the cell-free layer (CFL) in a microchannel network

In the past years, in vitro blood studies have revealed several significant hemodynamic phenomena that have played a key role in recent developments of biomedical microdevices for cells separation, sorting and analysis. However, the blood flow phenomena happening in complex geometries, such as microchannel networks, have not been fully understood. Thus, it is important to investigate in detail the blood flow behavior occurring at microchannel networks. In the present study, by using a high-speed video microscopy system, we have used two working fluids with different haematocrit (1% Hct and 15% Hct) and we have investigated the cell-free layer (CFL) in a microchannel network composed by asymmetric bifurcations. By using the Z Project method from the image analysis software ImageJ, it was possible to conclude that the successive bifurcations and confluences influence the formation of the CFL not only along the upper and lower wall of the microchannel but also at the region immediately downstream of the confluence apex.The authors acknowledge the financial support provided by the project POCI-01-0145 FEDER-016861 (with associated reference PTDC/QEQ-FTT/4287/2014), UID/EMS/00532/2013 and UID/CEC/00319/2013 funded by FCT (Foundation for Science and Technology), through national funds (PIDDAC), and FEDER through COMPETE2020 - Programa Operacional Competitividade e Internacionalização (POCI). D. Bento acknowledges the PhD scholarship SFRH/BD/91192/2012 granted by FCT. The authors also acknowledge the financial support provided by the project Nos. UID/EMS/00532/2013 and UID/EMS/04077/2013 and the project Nos. UID/EMS/00532/2013, UID/EMS/04077/2013, POCI-01-0145-FEDER-007043, UID/CEC/00319/2013info:eu-repo/semantics/publishedVersio

Two-photon NADH imaging exposes boundaries of oxygen diffusion in cortical vascular supply regions

Oxygen transport imposes a possible constraint on the brain's ability to sustain variable metabolic demands, but oxygen diffusion in the cerebral cortex has not yet been observed directly. We show that concurrent two-photon fluorescence imaging of endogenous nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation exposes well-defined boundaries of tissue oxygen diffusion in the mouse cortex. The NADH fluorescence increases rapidly over a narrow, very low pO2 range with a p50 of 3.4±0.6 mm Hg, thereby establishing a nearly binary reporter of significant, metabolically limiting hypoxia. The transient cortical tissue boundaries of NADH fluorescence exhibit remarkably delineated geometrical patterns, which define the limits of tissue oxygen diffusion from the cortical microcirculation and bear a striking resemblance to the ideal Krogh tissue cylinder. The visualization of microvessels and their regional contribution to oxygen delivery establishes penetrating arterioles as major oxygen sources in addition to the capillary network and confirms the existence of cortical oxygen fields with steep microregional oxygen gradients. Thus, two-photon NADH imaging can be applied to expose vascular supply regions and to localize functionally relevant microregional cortical hypoxia with micrometer spatial resolution

Module-based multiscale simulation of angiogenesis in skeletal muscle

<p>Abstract</p> <p>Background</p> <p>Mathematical modeling of angiogenesis has been gaining momentum as a means to shed new light on the biological complexity underlying blood vessel growth. A variety of computational models have been developed, each focusing on different aspects of the angiogenesis process and occurring at different biological scales, ranging from the molecular to the tissue levels. Integration of models at different scales is a challenging and currently unsolved problem.</p> <p>Results</p> <p>We present an object-oriented module-based computational integration strategy to build a multiscale model of angiogenesis that links currently available models. As an example case, we use this approach to integrate modules representing microvascular blood flow, oxygen transport, vascular endothelial growth factor transport and endothelial cell behavior (sensing, migration and proliferation). Modeling methodologies in these modules include algebraic equations, partial differential equations and agent-based models with complex logical rules. We apply this integrated model to simulate exercise-induced angiogenesis in skeletal muscle. The simulation results compare capillary growth patterns between different exercise conditions for a single bout of exercise. Results demonstrate how the computational infrastructure can effectively integrate multiple modules by coordinating their connectivity and data exchange. Model parameterization offers simulation flexibility and a platform for performing sensitivity analysis.</p> <p>Conclusions</p> <p>This systems biology strategy can be applied to larger scale integration of computational models of angiogenesis in skeletal muscle, or other complex processes in other tissues under physiological and pathological conditions.</p

Maternal hypoxia decreases capillary supply and increases metabolic inefficiency leading to divergence in myocardial oxygen supply and demand

Maternal hypoxia is associated with a decrease in left ventricular capillary density while cardiac performance is preserved, implying a mismatch between metabolism and diffusive exchange. We hypothesised this requires a switch in substrate metabolism to maximise efficiency of ATP production from limited oxygen availability. Rat pups from pregnant females exposed to hypoxia (FIO2=0.12) at days 10-20 of pregnancy were grown to adulthood and working hearts perfused ex vivo. 14 C-labelled glucose and 3 H-palmitate were provided as substrates and metabolism quantified from recovery of 14CO2 and 3 H2O, respectively. Hearts of male offspring subjected to Maternal Hypoxia showed a 20% decrease in cardiac output (P<0.05), despite recording a 2-fold increase in glucose oxidation (P<0.01) and 2.5-fold increase (P<0.01) in palmitate oxidation. Addition of insulin to Maternal Hypoxic hearts, further increased glucose oxidation (P<0.01) and suppressed palmitate oxidation (P<0.05), suggesting preservation in insulin signalling in the heart. In vitro enzyme activity measurements showed that Maternal Hypoxia increased both total and the active component of cardiac pyruvate dehydrogenase (both P<0.01), although pyruvate dehydrogenase sensitivity to insulin was lost (NS), while citrate synthase activity declined by 30% (P<0.001) and acetyl-CoA carboxylase activity was unchanged by Maternal Hypoxia, indicating realignment of the metabolic machinery to optimise oxygen utilisation. Capillary density was quantified and oxygen diffusion characteristics examined, with calculated capillary domain area increased by 30% (P<0.001). Calculated metabolic efficiency decreased 4-fold (P<0.01) for Maternal Hypoxia hearts. Paradoxically, the decline in citrate synthase activity and increased metabolism suggest that the scope of individual mitochondria had declined, rendering the myocardium potentially more sensitive to metabolic stress. However, decreasing citrate synthase may be essential to preserve local PO2, minimising regions of hypoxia and hence maximising the area of myocardium able to preserve cardiac output following maternal hypoxia

Graphical Approach to Model Reduction for Nonlinear Biochemical Networks

Model reduction is a central challenge to the development and analysis of multiscale physiology models. Advances in model reduction are needed not only for computational feasibility but also for obtaining conceptual insights from complex systems. Here, we introduce an intuitive graphical approach to model reduction based on phase plane analysis. Timescale separation is identified by the degree of hysteresis observed in phase-loops, which guides a “concentration-clamp” procedure for estimating explicit algebraic relationships between species equilibrating on fast timescales. The primary advantages of this approach over Jacobian-based timescale decomposition are that: 1) it incorporates nonlinear system dynamics, and 2) it can be easily visualized, even directly from experimental data. We tested this graphical model reduction approach using a 25-variable model of cardiac β1-adrenergic signaling, obtaining 6- and 4-variable reduced models that retain good predictive capabilities even in response to new perturbations. These 6 signaling species appear to be optimal “kinetic biomarkers” of the overall β1-adrenergic pathway. The 6-variable reduced model is well suited for integration into multiscale models of heart function, and more generally, this graphical model reduction approach is readily applicable to a variety of other complex biological systems

Modeling intracranial aneurysm stability and growth: An integrative mechanobiological framework for clinical cases

We present a novel patient-specific fluid-solid-growth framework to model the mechanobiological state of clinically detected intracranial aneurysms (IAs) and their evolution. The artery and IA sac are modeled as thick-walled, non-linear elastic fiber-reinforced composites. We represent the undulation distribution of collagen fibers: the adventitia of the healthy artery is modeled as a protective sheath whereas the aneurysm sac is modeled to bear load within physiological range of pressures. Initially, we assume the detected IA is stable and then consider two flow-related mechanisms to drive enlargement: (1) low wall shear stress; (2) dysfunctional endothelium which is associated with regions of high oscillatory flow. Localized collagen degradation and remodelling gives rise to formation of secondary blebs on the aneurysm dome. Restabilization of blebs is achieved by remodelling of the homeostatic collagen fiber stretch distribution. This integrative mechanobiological modelling workflow provides a step towards a personalized risk-assessment and treatment of clinically detected IAs

3D Multi-Cell Simulation of Tumor Growth and Angiogenesis

We present a 3D multi-cell simulation of a generic simplification of vascular tumor growth which can be easily extended and adapted to describe more specific vascular tumor types and host tissues. Initially, tumor cells proliferate as they take up the oxygen which the pre-existing vasculature supplies. The tumor grows exponentially. When the oxygen level drops below a threshold, the tumor cells become hypoxic and start secreting pro-angiogenic factors. At this stage, the tumor reaches a maximum diameter characteristic of an avascular tumor spheroid. The endothelial cells in the pre-existing vasculature respond to the pro-angiogenic factors both by chemotaxing towards higher concentrations of pro-angiogenic factors and by forming new blood vessels via angiogenesis. The tumor-induced vasculature increases the growth rate of the resulting vascularized solid tumor compared to an avascular tumor, allowing the tumor to grow beyond the spheroid in these linear-growth phases. First, in the linear-spherical phase of growth, the tumor remains spherical while its volume increases. Second, in the linear-cylindrical phase of growth the tumor elongates into a cylinder. Finally, in the linear-sheet phase of growth, tumor growth accelerates as the tumor changes from cylindrical to paddle-shaped. Substantial periods during which the tumor grows slowly or not at all separate the exponential from the linear-spherical and the linear-spherical from the linear-cylindrical growth phases. In contrast to other simulations in which avascular tumors remain spherical, our simulated avascular tumors form cylinders following the blood vessels, leading to a different distribution of hypoxic cells within the tumor. Our simulations cover time periods which are long enough to produce a range of biologically reasonable complex morphologies, allowing us to study how tumor-induced angiogenesis affects the growth rate, size and morphology of simulated tumors

Local Oxidative and Nitrosative Stress Increases in the Microcirculation during Leukocytes-Endothelial Cell Interactions

Leukocyte-endothelial cell interactions and leukocyte activation are important factors for vascular diseases including nephropathy, retinopathy and angiopathy. In addition, endothelial cell dysfunction is reported in vascular disease condition. Endothelial dysfunction is characterized by increased superoxide (O2•−) production from endothelium and reduction in NO bioavailability. Experimental studies have suggested a possible role for leukocyte-endothelial cell interaction in the vessel NO and peroxynitrite levels and their role in vascular disorders in the arterial side of microcirculation. However, anti-adhesion therapies for preventing leukocyte-endothelial cell interaction related vascular disorders showed limited success. The endothelial dysfunction related changes in vessel NO and peroxynitrite levels, leukocyte-endothelial cell interaction and leukocyte activation are not completely understood in vascular disorders. The objective of this study was to investigate the role of endothelial dysfunction extent, leukocyte-endothelial interaction, leukocyte activation and superoxide dismutase therapy on the transport and interactions of NO, O2•− and peroxynitrite in the microcirculation. We developed a biotransport model of NO, O2•− and peroxynitrite in the arteriolar microcirculation and incorporated leukocytes-endothelial cell interactions. The concentration profiles of NO, O2•− and peroxynitrite within blood vessel and leukocytes are presented at multiple levels of endothelial oxidative stress with leukocyte activation and increased superoxide dismutase accounted for in certain cases. The results showed that the maximum concentrations of NO decreased ∼0.6 fold, O2•− increased ∼27 fold and peroxynitrite increased ∼30 fold in the endothelial and smooth muscle region in severe oxidative stress condition as compared to that of normal physiologic conditions. The results show that the onset of endothelial oxidative stress can cause an increase in O2•− and peroxynitrite concentration in the lumen. The increased O2•− and peroxynitrite can cause leukocytes priming through peroxynitrite and leukocytes activation through secondary stimuli of O2•− in bloodstream without endothelial interaction. This finding supports that leukocyte rolling/adhesion and activation are independent events

Elongation, proliferation & migration differentiate endothelial cell phenotypes and determine capillary sprouting

<p>Abstract</p> <p>Background</p> <p>Angiogenesis, the growth of capillaries from preexisting blood vessels, has been extensively studied experimentally over the past thirty years. Molecular insights from these studies have lead to therapies for cancer, macular degeneration and ischemia. In parallel, mathematical models of angiogenesis have helped characterize a broader view of capillary network formation and have suggested new directions for experimental pursuit. We developed a computational model that bridges the gap between these two perspectives, and addresses a remaining question in angiogenic sprouting: how do the processes of endothelial cell elongation, migration and proliferation contribute to vessel formation?</p> <p>Results</p> <p>We present a multiscale systems model that closely simulates the mechanisms underlying sprouting at the onset of angiogenesis. Designed by agent-based programming, the model uses logical rules to guide the behavior of individual endothelial cells and segments of cells. The activation, proliferation, and movement of these cells lead to capillary growth in three dimensions. By this means, a novel capillary network emerges out of combinatorially complex interactions of single cells. Rules and parameter ranges are based on literature data on endothelial cell behavior in vitro. The model is designed generally, and will subsequently be applied to represent species-specific, tissue-specific in vitro and in vivo conditions.</p> <p>Initial results predict tip cell activation, stalk cell development and sprout formation as a function of local vascular endothelial growth factor concentrations and the Delta-like 4 Notch ligand, as it might occur in a three-dimensional in vitro setting. Results demonstrate the differential effects of ligand concentrations, cell movement and proliferation on sprouting and directional persistence.</p> <p>Conclusion</p> <p>This systems biology model offers a paradigm closely related to biological phenomena and highlights previously unexplored interactions of cell elongation, migration and proliferation as a function of ligand concentration, giving insight into key cellular mechanisms driving angiogenesis.</p