Emergent behaviors in RBCs flows in micro-channels using digital particle image velocimetry.

The key points in the design of microfluidic Lab-On-a-Chips for blood tests are the simplicity of the microfluidic chip geometry, the portability of the monitoring system and the ease on-chip integration of the data analysis procedure. The majority of those, recently designed, have been used for blood separation, however their introduction, also, for pathological conditions diagnosis would be important in different biomedical contexts. To overcome this lack is necessary to establish the relation between the RBCs flow and blood viscosity changes in micro-vessels. For that, the development of methods to analyze the dynamics of the RBCs flows in networks of micro-channels becomes essential in the study of RBCs flows in micro-vascular networks. A simplification in the experimental set-up and in the approach for the data collection and analysis could contribute significantly to understand the relation between the blood non-Newtonian properties and the emergent behaviors in collective RBCs flows. In this paper, we have investigated the collective behaviors of RBCs in a micro-channel in unsteady conditions, using a simplified monitoring set-up and implementing a 2D image processing procedure based on the digital particle image velocimetry. Our experimental study consisted in the analysis of RBCs motions freely in the micro-channel and driven by an external pressure. Despite the equipment minimal complexity, the advanced signal processing method implemented has allowed a significant qualitative and quantitative classification of the RBCs behaviors and the dynamical characterization of the particles velocities along both the horizontal and vertical directions. The concurrent causes for the particles displacement as the base solution-particles interaction, particle-particle interaction, and the external force due to pressure gradient were accounted in the results interpretation. The method implemented and the results obtained represent a proof of concept toward the realization of a general-purpose microfluidic LOC device for in-vitro flow analysis of RBCs collective behaviors.

Control of overweight and obesity in childhood through education in meal time habits. The 'good manners for a healthy future' programme.

OBJECTIVE: Our aim is to determine the effect of paced eating, exposure to an educational programme that promotes healthy eating habits and allowing the satiety reflex to limit food intake in controlling weight gain in healthy adolescents. METHODS: Fifty-four healthy individuals consisting of 18 adolescent girls and 36 boys aged 12 ± 2 years were given recommendations for reducing eating rate without changing diet or meal size according to the educational programme 'good manners for a healthy future'. Each participant was provided with a 30-s portable hourglass to pace time between bites. Individuals using and not using the hourglass were placed either into an 'adhering' or a 'non-adhering' group, respectively. Control data were obtained from a similar population. RESULTS: Initially, the adhering group had higher weight compared with the non-adhering group (64.1 ± 13.2 vs. 56.2 ± 11.7 kg). Control group weight was no different from the study group at baseline (56.3 ± 10.3 kg). Weight in the adhering group decreased after the first semester of participation by 2.0 ± 5.7% and after a year by 3.4 ± 4.8%, while the non-adhering group gained weight by 5.8 ± 4.5% and 12.6 ± 8.3%. The control group increased weight after a year by 8.2 ± 6.5%. In total, 18 non-adhering and 14 adhering adolescents completed the study. CONCLUSIONS: This 1-year study shows a statistically significant association between rate of food intake and weight control in adherence to an educational programme directed at developing healthy eating habits. The proposed behavioural training may serve as an option for weight control in adolescents.

Red blood cells flows in rectilinear microfluidic chip.

The red blood cells flow in a controlled environment as a microfluidic chip with a rectilinear geometry was investigated. The optical monitoring performed by an automatic Particle Image Velocimetry procedure has allowed a quantitative analysis on flow features. Various parameters such as velocity, shear rate, strain rate, vorticity, divergence were extracted. The comparisons of the results obtained from the different experiments was used for the overall understanding of the RBC movements in different conditions and the establishment of the analysis procedure.

Loss of function in heparan sulfate elongation genes EXT1 and EXT 2 results in improved nitric oxide bioavailability and endothelial function.

BACKGROUND: Heparanase is the major enzyme involved in degradation of endothelial heparan sulfates, which is associated with impaired endothelial nitric oxide synthesis. However, the effect of heparan sulfate chain length in relation to endothelial function and nitric oxide availability has never been investigated. We studied the effect of heterozygous mutations in heparan sulfate elongation genes EXT1 and EXT2 on endothelial function in vitro as well as in vivo. METHODS AND RESULT: Flow-mediated dilation, a marker of nitric oxide bioavailability, was studied in Ext1(+/-) and Ext2(+/-) mice versus controls (n=7 per group), as well as in human subjects with heterozygous loss of function mutations in EXT1 and EXT2 (n=13 hereditary multiple exostoses and n=13 controls). Endothelial function was measured in microvascular endothelial cells under laminar flow with or without siRNA targeting EXT1 or EXT2. Endothelial glycocalyx and maximal arteriolar dilatation were significantly altered in Ext1(+/-) and Ext2(+/-) mice compared to wild-type littermates (glycocalyx: wild-type 0.67±0.1 µm, Ext1(+/-) 0.28±0.1 µm and Ext2(+/-) 0.25±0.1 µm, P<0.01, maximal arteriolar dilation during reperfusion: wild-type 11.3±1.0%), Ext1(+/-) 15.2±1.4% and Ext2(+/-) 13.8±1.6% P<0.05). In humans, brachial artery flow-mediated dilation was significantly increased in hereditary multiple exostoses patients (hereditary multiple exostoses 8.1±0.8% versus control 5.6±0.7%, P<0.05). In line, silencing of microvascular endothelial cell EXT1 and EXT2 under flow led to significant upregulation of endothelial nitric oxide synthesis and phospho-endothelial nitric oxide synthesis protein expression. CONCLUSIONS: Our data implicate that heparan sulfate elongation genes EXT1 and EXT2 are involved in maintaining endothelial homeostasis, presumably via increased nitric oxide bioavailability.

Hemodynamics and tissue oxygenation after hemodilution with ultrahigh molecular weight polymerized albumin.

BACKGROUND: Compared to blood transfusion, plasma expanders (PEs) are more cost effective, have a longer shelf-life, and elicit a milder immune response. High molecular weight (MW) dextrans preserve microvascular function during extreme hemodilution. Dextrans, however, evokes negative hemostatic effects, including red blood cell (RBC) aggregation and reduce platelet adhesion, that limit their clinical use. Therefore, polymerization of human serum albumin (HSA) presents a simple strategy to increase HSA's molecular size. METHODS: This study was designed to test the hypothesis that polymerized HSA (PolyHSA) biophysical properties improves systemic and microvascular hemodynamics when used as a PE under anemic conditions. The study was implemented using the hamster window chamber model. Animals were first hemodiluted to 18% hematocrit (Hct) using 6% dextran 70 kDa and then to 11% Hct using either 10% PolyHSA, 10% unpolymerized HSA, or 6% dextran 70 kDa. Systemic and microvascular hemodynamics, including cardiac output (CO), mean arterial blood pressure (MAP), functional capillary density (FCD), microvascular perfusion, and oxygen tension were measured. RESULTS: Posthemodilution, PolyHSA improved MAP, CO, and oxygen delivery compared to HSA and dextran. Additionally, PolyHSA improved microvascular function in terms of blood flow and FCD. Although oxygen carrying capacity is limited at 11% Hct, tissue pO2 and oxygen delivery were higher for PolyHSA compared to HSA and dextran. CONCLUSION: PolyHSA during extreme anemia supported systemic and microvascular hemodynamics by increasing plasma viscosity without increasing vascular resistance. These findings can aid to design of studies to understand the role of the PE biophysical properties in clinical scenarios.

Impact of enzymatic degradation of the endothelial glycocalyx on vascular permeability in an awake hamster model.

Background. The inside of the endothelium is covered by a glycocalyx layer, and enzymatic degradation of this layer induces vascular leakage ex vivo. We hypothesized that enzymatic degrading of the glycocalyx in an in vivo, whole body model, would induce plasma leakage and affect the microcirculation. Methods. Golden Syrian hamsters were divided into an enzyme (hyaluronidase) and a control group. Mean arterial pressure (MAP), heart rate (HR), hematocrit (Hct), base excess (BE), and plasma volume were obtained before, 45 and 120 min after enzyme/saline treatment. Plasma volume was evaluated by the distribution volume of indocyanine green and the microcirculation by functional capillary density (FCD). The enzymatic effect was determined by measuring plasma levels of hyaluronan (HA). Results. There were no differences in MAP, HR, Hct, and BE between the two groups. Enzyme treatment did not induce changes in plasma volume but reduced FCD. There was a 50-100-fold increase in plasma HA, but no relationship was found between HA levels and plasma volume or FCD. Conclusion. Vascular leakage was not confirmed in an in vivo, whole body model after degradation of the endothelial glycocalyx. The microcirculation was affected, but no relationship between plasma levels of HA and FCD was seen.

Effects on cardiac function of a novel low viscosity plasma expander based on polyethylene glycol conjugated albumin.

BACKGROUND: Plasma expanders have become increasingly advantageous when compared to blood transfusion, due to their long shelf-life and cost-effectiveness. A new generation of plasma expander based on polyethylene glycol (PEG) conjugated to human serum albumin (PEG-HSA) has shown positive microvascular effects during extreme hemodilution and fluid resuscitation from severe hemorrhagic shock. PEG conjugation increases uniformly albumin molecular weight (MW) and colloidal osmotic pressure, with minor effects on viscosity. METHODS: This study was designed to test the hypothesis that PEG-HSA improves and maintains cardiac function during anemic condition, independently of its lower viscosity, compared to plasma expanders with higher viscosity. To accomplish this objective, we compared PEG-HSA to colloids of different MWs and viscosities, dextran 70 kDa (moderate viscosity plasma expander, MVPE) and dextran 2000 kDa (high viscosity plasma expander, HVPE). Cardiac function was analyzed using indices derived from left ventricular pressure volume, and were assessed using a miniaturized conductance catheter, in two experimental models: 1) hemodilution and 2) resuscitation from hemorrhagic shock. RESULTS: After hemodilution, PEG-HSA increased cardiac output compared to MVPE through the entire observation period, and HVPE increased stroke work compared to MVPE. After resuscitation, PEG-HSA increased stroke work compared to HVPE through the entire observation period. In both experimental protocols, cardiac functional changes induced by PEG-HSA were sustained over the observation time. CONCLUSION: PEG-HSA, a low viscosity plasma expander, had beneficial effects on cardiac function when compared to conventional colloidal plasma expanders with higher viscosities. Maintenance of homeostasis during hemodilution and resuscitation from hemorrhagic shock using PEG-HSA will lead to a significant decrease of the use of blood, thus alleviating in part, forecasted blood shortages, and significantly reducing morbidity and mortality associated with the use of blood in transfusion medicine.

Cardiovascular benefits in moderate increases of blood and plasma viscosity surpass those associated with lowering viscosity: Experimental and clinical evidence.

Decreasing blood viscosity has been proposed since the advent of hemodilution as a means for increasing perfusion in many pathological conditions, and increased plasma viscosity is associated with the presence of pathological conditions. However, experimental studies show that microvascular functions as represented by functional capillary density in conditions of significantly decreased viscosity is impaired, a problem corrected by increasing plasma and blood viscosity. Blood viscosity, primarily dependent on hematocrit (Hct) is a determinant of peripheral vascular resistance, and therefore blood pressure. In the healthy population Hct presents a variability, which is not reflected by the variability of blood pressure. This is due to a regulatory process at the level of the endothelium, whereby the increase of Hct (and therefore blood viscosity) leads to increased shear stress and the production of the vasodilator nitric oxide (NO), a finding supported by experimental studies showing that the acute increase of Hct lowers blood pressure. Studies that in the healthy population show that blood pressure and Hct have a weak positive correlation. However, when the effect of blood viscosity is factored out, blood pressure and Hct are negatively and significantly correlated, indicating that as blood viscosity increases, the circulation dilates. Conversely, lower Hct and blood viscosity conditions lead to a constricted circulation, associated with a condition of decreased NO bioavailability, and therefore a pro-inflammatory condition.

Microvascular benefits of increasing plasma viscosity and maintaining blood viscosity: counterintuitive experimental findings.

The circulation is adapted to specific levels of blood viscosity resulting in a balance that simultaneously sets peripheral vascular resistance, blood pressure and cardiac output, factors in part mediated by the production of nitric oxide by the endothelium. Although it is generally perceived that decreasing blood viscosity is beneficial for cardiovascular function, small increases of blood viscosity in normal healthy experimental subjects significantly improve cardiovascular function. These changes are within the normal variations of viscosity due to the variations of hematocrit in the healthy population. Hemodilution reduces blood viscosity, which is proposed to be physiologically beneficial. However, in extreme hemodilution, increased plasma viscosity via the use of viscogenic plasma expanders sustains microvascular and tissue function at significantly reduced levels of oxygen delivery. Studies in hemorrhagic shock resuscitation using oxygen carrying and non-carrying red blood cells show that restoration of blood viscosity is as important as restoration of oxygen carrying capacity. It is concluded that although hemodilution is indicated for reducing abnormally high blood viscosities, it is beneficial to increase plasma viscosity when hematocrit is reduced. Furthermore small increases in hematocrit may be beneficial due to the related increase in blood viscosity, independently of the increase of oxygen delivery capacity.

Beneficial effects due to increasing blood and plasma viscosity.

Increased plasma and blood viscosity are usually associated with pathological conditions; however there are several situations in which the elevation of both parameters results in increased perfusion and the lowering of peripheral vascular resistance. In extreme hemodilution blood viscosity is too low and insufficient to maintain functional capillary density, a problem that in experimental studies is shown to be corrected by increasing plasma viscosity up to 2.2 cP. This effect is mediated by Nitric oxide (NO) production via restoration of shear stress at the endothelium as shown by microelectrode perivascular measurements of NO concentration. Moderate elevations of blood viscosity by increasing hematocrit (approximately 10% of baseline) result in reductions of blood pressure by 10 mmHg of baseline. This effect is also NO mediated since it is absent after N-nitro-L-arginine methyl ester (L-NAME) treatment and in endothelial NO synthase deficient mice. These results show that the rheological properties of plasma affect vessel diameter in the microcirculation leading to counterintuitive responses to the increase in viscosity.

Mechanotransduction and the homeostatic significance of maintaining blood viscosity in hypotension, hypertension and haemorrhage.

The increase of plasma and blood viscosity is usually associated with pathological conditions; however, elevation of both parameters often results in increased perfusion and the lowering of peripheral vascular resistance. In extreme haemodilution, blood viscosity is too low and insufficient to maintain functional capillary density, a problem that in experimental studies is shown to be corrected by increasing plasma viscosity up to 2.2 cP. This effect is mediated by mechanotransduction-induced nitric oxide (NO) production via shear stress in the endothelium as shown by microelectrode perivascular measurements of NO concentration. Moderate elevations of blood viscosity by increasing haematocrit ( approximately 10%) result in comparable reductions of blood pressure and peripheral vascular resistance, an effect also NO-mediated as it is absent after Nomega-nitro-L-arginine methyl ester treatment and in endothelial nitric oxide synthase-deficient mice. These findings show that the rheological properties of plasma affect vessel diameter in the microcirculation leading to counterintuitive responses to the changes in blood and plasma viscosity. Application of these findings to haemorrhagic shock resuscitation leads to the concept of hyperosmotic-hyperviscous resuscitation as a modality for maintaining the recovery of microvascular function.