Red Blood Cell Velocity Research Articles

Drag Reducing Polymers Improves Microvascular Perfusion After Extracorporeal Circulation

Drag reducing polymers (DRPs) have been used in various engineering applications, such as the transfer of crude oil or seawater in pipelines. The use of DRPs has crossed into the bioengineering sphere, with previous ex‐vivo and in‐vivo studies suggesting that the addition of DRPs decreases the erythrocyte cell free layer (CFL) or cell depleted layer near the vessel wall and increases microvascular blood flow. The objective of this study was to investigate whether the addition of DRPs would improve microvascular perfusion in a rodent model of extracorporeal circulation (ECC).Male Golden Syrian hamsters were instrumented with a dorsal skinfold window chamber, along with an arterial catheter into the right carotid artery to monitor blood pressure and heart rate. During ECC, animals were anesthetized with isoflurane, and a drainage cannula was inserted through the left jugular. The animal was connected to the circuit, primed with either lactate ringers (LR group) or 5 PPM 500 kDa polyethylene glycol in lactate ringers (DRP group). Blood was drained from the jugular cannula, run through a peristaltic pump and bubble trap, before entering the carotid catheter for reinfusion. The circuit was run for 90 minutes in total, with 60 minutes at a flowrate equivalent to 15% of the animal’s cardiac output. Measurements were taken before ECC (baseline, BL) and after 1, 2, and 24 hrs post‐ECC. At each timepoint, microvessel (arterioles and venules) diameter, red blood cell velocity and functional capillary density (FCD) were measured using intravital microscopy.Arteriole and venule hemodynamics are displayed in Figure 1. The arteriole results showed an interesting contrast between effects at 1 hr and 24 hrs post‐ECC. At 1 hr post‐ECC, the DRP group displayed a statistically lower arteriole diameter compared to the LR group, but no statistical difference in blood flow between the two groups. At 24 hrs post‐ECC, no difference in arteriole diameter was observed between groups, while arteriole blood flow was statistically higher in the DRP group compared to the LR group. In venules, no changes were observed between groups at 1 hr post‐ECC. However, at 2 and 24 hrs post‐ECC, the DRP group had a statistically significant higher diameter compared to the LR group. Venular blood flow was statistically elevated in the DRP group at 24 hrs post‐ECC compared to the LR group. Figure 2 shows the changes in FCD. FCD was lower for both DRP and LR groups at 1 and 2 hrs post‐ECC when compared to BL levels. The DRP group displayed a higher FCD at 2 hrs post‐ECC compared to the LR group. At 24 hrs however, while the DRP group was able to recover back to BL levels, the LR group was statistically lower than BL.From these results we conclude that the addition of DRPs to priming fluids for ECC procedures may be beneficial in preserving microvascular perfusion 24 hrs post‐ECC. This may help reduce the risk of ischemic injury, production of pro‐inflammatory markers, and development of multiorgan dysfunction. Further research is warranted to optimize the DRPs parameters, such as concentration and molecular weight.

Effects of pulmonary hypertension on microcirculatory hemodynamics in rat skeletal muscle.

Pulmonary hypertension (PH) has previously been characterized as a disease of the pulmonary vasculature that subsequently results in myocardial dysfunction. Heart failure compromises skeletal muscle microvascular function, which contributes to exercise intolerance. Therefore, we tested the hypothesis that such changes might be present in PH. Thus, we investigated skeletal muscle oxygen (O2) transport in the rat model of PH to determine if O2 delivery (Q̇O2) is impaired at the level of the microcirculation as evidenced via reduced red blood cell (RBC) flux, velocity, hematocrit, and percentage of capillaries flowing in quiescent muscle. Adult male Sprague-Dawley rats were randomized into healthy (n=9) and PH groups (n=9). Progressive PH was induced via a one-time intraperitoneal injection of monocrotaline (MCT; 50mg/kg) and rats were monitored weekly via echocardiography. Intravital microscopy in the spinotrapezius muscle was performed when echocardiograms confirmed moderate PH (preceding right ventricular (RV) failure). At 25±9days post-MCT, PH rats displayed RV hypertrophy (RV/(Left ventricle+Septum): 0.28±0.05 vs. 0.44±0.11), pulmonary congestion, and increased right ventricular systolic pressure (21±8 vs. 55±14mmHg) compared to healthy rats (all P<0.05). Reduced capillary RBC velocity (403±140 vs. 227±84μm/s; P=0.01), RBC flux (33±12 vs. 23±5 RBCs/s; P=0.04) and % of capillaries supporting continuous RBC flux at rest (79±8 vs. 56±13%; P=0.01) were evident in PH rats compared to healthy rats. When Q̇O2 within a given field of view was quantified (RBC flux x % of capillaries supporting continuous RBC flux), PH rats demonstrated lower overall Q̇O2 (↓ 50%; P=0.002). These data support that microcirculatory hemodynamic impairments (↓ Q̇O2 and therefore altered Q̇O2-to-V̇O2 matching) may compromise blood-myocyte O2 transport in PH. The mechanistic bases for decreased capillary RBC flux, velocity, and percentage of capillaries supporting RBC flow remains an important topic.

Clamping Skeletal Muscle PO2 Eliminates Hyperinsulinemic Microvascular Blood Flow Response

ObjectiveTo determine if insulin mediated hyperemia is partially dependent on local muscle oxygen concentration.HypothesisThe blood flow responses to insulin are partially mediated by the local metabolic demand reflecting the increased oxygen consumption required to uptake and convert glucose into glycogen in skeletal muscle.Methods16 male Sprague Dawley rats (161g ‐ 204g) were anesthetized with sodium pentobarbital, the carotid artery and jugular vein were then cannulated for blood pressure monitoring and fluid resuscitation. Following cannulation, the extensor digitorum longus (EDL) was isolated and reflected onto an inverted microscope. Intravital video microscopy sequences of the EDL microcirculation were recorded during baseline and following hyperinsulinemic euglycemia or sham in all experiments. The muscle was reflected over a glass stage insert during Experiment 1a and 1b which allowed for direct observation of microvascular blood flow response to systemic insulin infusion in Experiment 1a. Experiment 1b consisted of a sham hyperinsulinemic euglycemic clamp which involved infusing saline at similar rates as insulin and glucose in standard clamp procedures. In Experiment 2, the EDL was reflected over a gas exchange chamber and microvascular capillary blood flow was recorded during sequential changes (7%‐12%‐2%‐7%) of oxygen concentration [O2] within the chamber during baseline and euglycemic conditions. The gas exchange chamber is fitted into the stage of an inverted microscope that interfaces directly with the EDL via a gas permeable polydimethylsiloxane membrane. By adjusting the concentration of gases flowing through the gas exchange chamber we have direct control of the [O2] within the overlying muscle. Hemodynamic measurements including capillary red blood cell (RBC) velocity, supply rate (SR), hematocrit, and oxygen saturation (SO2) were taken from in focus capillaries in the EDL during each experiment and analyzed offline using custom MATLAB software. All animal protocols were approved by Memorial University’s Institutional Animal Care Committee.ResultsIn Experiment 1a, the hyperinsulinemic euglycemic clamp caused a significant increase in SR from 8.9 ± 3.1 to 14.2 ± 4.9 cells/s at baseline to euglycemia (p = 0.01; N = 6, 677 capillaries at baseline and 552 at clamp), while no significant SR variation was detected after performing a sham clamp in Experiment 1b (11.0 ± 1.6 cells/sec at baseline and 11.4 ± 3.7 cells/sec at sham clamp; N = 6, 837 capillaries at baseline and 724 at sham clamp). In Experiment 2 using the gas exchange chamber, SR significantly decreased at 12% O2 (p = 0.01, p &lt; 0.03) and significantly increased at 2% O2 (p = 0.01, p &lt; 0.04), compared to baseline 7% O2, under both baseline and euglycemic conditions respectively (N = 6, 313 capillaries at baseline and 195 at clamp). SR responses to oxygen oscillations during euglycemia were not different to those at baseline for each O2 concentration (p &gt; 0.9). SO2 significantly increased at 12% O2 and significantly decreased at 2% O2 under both baseline and euglycemic conditions (p &lt; 0.05).ConclusionsOur saturation measurements demonstrate the effectiveness of using a gas exchange device to manipulate local oxygen concentrations in the muscle. Our SR results suggest the increase in blood flow observed in response to insulin is eliminated if the tissue oxygen microenvironment is fixed at a given [O2].

Quantitative Hemodynamic Measurements in Cortical Vessels Using Functional Ultrasound Imaging.

Red blood cell velocity (RBCv), cerebral blood flow (CBF), and volume (CBV) are three key parameters when describing brain hemodynamics. Functional ultrasound imaging is a Doppler-based method allowing for real-time measurement of relative CBV at high spatiotemporal resolution (100 × 110 × 300 μm3, up to 10 Hz) and large scale. Nevertheless, the measure of RBCv and CBF in small cortical vessels with functional ultrasound imaging remains challenging because of their orientation and size, which impairs the ability to perform precise measurements. We designed a directional flow filter to overpass these limitations allowing us to measure RBCv in single vessels using a standard functional ultrasound imaging system without contrast agents (e.g., microbubbles). This method allows to quickly extract the number of vessels in the cortex that was estimated to be approximately 650/cm3 in adult rats, with a 55–45% ratio for penetrating arterioles versus ascending venules. Then, we analyzed the changes in RBCv in these vessels during forepaw stimulation. We observed that ∼40 vessels located in the primary somatosensory forelimb cortex display a significant increase of the RBCv (median ΔRBCv ∼15%, maximal ΔRBCv ∼60%). As expected, we show that RBCv was higher for penetrating arterioles located in the center than in the periphery of the activated area. The proposed approach extends the capabilities of functional ultrasound imaging, which may contribute to a better understanding of the neurovascular coupling at the brain-wide scale.

Measurement of Blood Velocity With Laser Scanning Microscopy: Modeling and Comparison of Line-Scan Image-Processing Algorithms.

Laser scanning microscopy is widely used to measure blood hemodynamics with line-scans in physiological and pathological vessels. With scans of broken lines, i.e., lines made of several segments with different orientations, it also allows simultaneous monitoring of vessel diameter dynamics or the activity of specific cells. Analysis of red blood cell (RBC) velocity from line-scans requires specific image-processing algorithms, as angle measurements, Line-Scanning Particle Image Velocimetry (LSPIV) or Fourier transformation of line-scan images. The conditions under which these image-processing algorithms give accurate measurements have not been fully characterized although the accuracy of measurements vary according to specific experimental parameters: the vessel type, the RBC velocity, the scanning parameters, and the image signal to noise ratio. Here, we developed mathematical models for the three previously mentioned line-scan image-processing algorithms. Our models predict the experimental conditions in which RBC velocity measurements are accurate. We illustrate the case of different vessel types and give the parameter space available for each of them. Last, we developed a software generating artificial line-scan images and used it to validate our models.

CapillaryNet: An automated system to quantify skin capillary density and red blood cell velocity from handheld vital microscopy

Capillaries are the smallest vessels in the body which are responsible for delivering oxygen and nutrients to surrounding cells. Various life-threatening diseases are known to alter the density of healthy capillaries and the flow velocity of erythrocytes within the capillaries. In previous studies, capillary density and flow velocity were manually assessed by trained specialists. However, manual analysis of a standard 20-s microvascular video requires 20 min on average and necessitates extensive training. Thus, manual analysis has been reported to hinder the application of microvascular microscopy in a clinical environment. To address this problem, this paper presents a fully automated state-of-the-art system to quantify skin nutritive capillary density and red blood cell velocity captured by handheld-based microscopy videos. The proposed method combines the speed of traditional computer vision algorithms with the accuracy of convolutional neural networks to enable clinical capillary analysis. The results show that the proposed system fully automates capillary detection with an accuracy exceeding that of trained analysts and measures several novel microvascular parameters that had eluded quantification thus far, namely, capillary hematocrit and intracapillary flow velocity heterogeneity. The proposed end-to-end system, named CapillaryNet, can detect capillaries at ~0.9 s per frame with ~93% accuracy. The system is currently used as a clinical research product in a larger e-health application to analyse capillary data captured from patients suffering from COVID-19, pancreatitis, and acute heart diseases. CapillaryNet narrows the gap between the analysis of microcirculation images in a clinical environment and state-of-the-art systems.

Gabrb3 endothelial cell-specific knockout mice display abnormal blood flow, hypertension, and behavioral dysfunction

Our recent studies uncovered a novel GABA signaling pathway in embryonic forebrain endothelial cells that works independently from neuronal GABA signaling and revealed that disruptions in endothelial GABAA receptor-GABA signaling from early embryonic stages can directly contribute to the origin of psychiatric disorders. In the GABAA receptor β3 subunit endothelial cell conditional knockout (Gabrb3ECKO) mice, the β3 subunit is deleted selectively from endothelial cells, therefore endothelial GABAA receptors become inactivated and dysfunctional. There is a reduction in vessel densities and increased vessel morphology in the Gabrb3ECKO telencephalon that persists in the adult neocortex. Gabrb3ECKO mice show behavioral deficits such as impaired reciprocal social interactions, communication deficits, heightened anxiety, and depression. Here, we characterize the functional changes in Gabrb3ECKO mice by evaluating cortical blood flow, examine the consequences of loss of endothelial Gabrb3 on cardiac tissue, and define more in-depth altered behaviors. Red blood cell velocity and blood flow were increased in the cortical microcirculation of the Gabrb3ECKO mice. The Gabrb3ECKO mice had a reduction in vessel densities in the heart, similar to the brain; exhibited wavy, myocardial fibers, with elongated ‘worm-like’ nuclei in their cardiac histology, and developed hypertension. Additional alterations in behavioral function were observed in the Gabrb3ECKO mice such as increased spontaneous exploratory activity and rearing in an open field, reduced short term memory, decreased ambulatory activity in CLAMS testing, and altered prepulse inhibition to startle, an important biomarker of psychiatric diseases such as schizophrenia. Our results imply that vascular Gabrb3 is a key player in the brain as well as the heart, and its loss in both organs can lead to concurrent development of psychiatric and cardiac dysfunction.

Analysis of Red Blood Cell Movement in Whole Blood Exposed to DC and ELF Electric Fields.

To evaluate hematological effects of direct current (DC) and alternating current (AC) extremely low frequency (ELF) electric field exposure, this study investigated red blood cell (RBC) movement in whole blood. Video images of RBCs were recorded under a microscope using specially designed electrode systems. Video analysis software was then used to measure the RBC velocity. The noise level and measurement system stability were confirmed based on results of a no‐field exposure experiment. Using the electrode system to produce a non‐homogeneous electric field, different movements were found to occur in DC and AC field exposure. The RBCs moved in the directions of the electric field and the gradient of field distribution, respectively, in the DC and AC fields. Dependences of the RBC velocity on the field strength were, respectively, linear and quadratic in the DC and AC fields. These results suggest that electrophoretic and dielectrophoretic movements were, respectively, dominant in the DC and AC fields. The magnitude of the electric field necessary to cause these effects was found to be 103–105 times greater than the internationally publicized guideline for human safety. © 2022 Bioelectromagnetics Society.

Post-occlusive reactive hyperemia and skeletal muscle capillary hemodynamics.

In female Sprague-Dawley rats (n=6), the spinotrapezius muscle was exteriorized for evaluation of capillary hemodynamics pre-occlusion, 5min of feed artery occlusion (Occ), and 5min of reperfusion (Post-Occ). There were no differences in mean arterial pressure (MAP) or capillary diameter (Dc) between pre-occlusion and post-occlusion (P>0.05). During 30s of PORH, capillary fRBC was increased (pre: 59±4 vs. 30s-post: 77±2 cells/s; P<0.05) and VRBC was not changed (pre: 300±24 vs. 30s post: 322±25μm/s; P>0.05). Capillary hematocrit (Hctcap) was unchanged across the pre- to post-occlusion conditions (P>0.05). Following occlusion, there was a 20-30% decrease in the number of capillaries supporting RBC flow at 30s and 300s-post occlusion (pre: 92±2%; 30s-post: 66±3%; 300s-post: 72±6%; both P<0.05). Short-term feed artery occlusion (i.e. 5min) resulted in a more heterogeneous capillary flow profile with the presence of capillary no-reflow, decreasing the percentage of capillaries supporting RBC flow. A complex interaction between myogenic and metabolic mechanisms at the arteriolar level may play a role in the capillary no-reflow with PORH. Measurements at the level of the conduit artery mask significant alterations in blood flow distribution in the microcirculation.

Dependence of red blood cell dynamics in microvessel bifurcations on the endothelial surface layer's resistance to flow and compression.

Red blood cells (RBCs) make up 40-45% of blood and play an important role in oxygen transport. That transport depends on the RBC distribution throughout the body, which is highly heterogeneous. That distribution, in turn, depends on how RBCs are distributed or partitioned at diverging vessel bifurcations where blood flows from one vessel into two. Several studies have used mathematical modeling to consider RBC partitioning at such bifurcations in order to produce useful insights. These studies, however, assume that the vessel wall is a flat impenetrable homogeneous surface. While this is a good first approximation, especially for larger vessels, the vessel wall is typically coated by a flexible, porous endothelial glycocalyx or endothelial surface layer (ESL) that is on the order of 0.5-1 µm thick. To better understand the possible effects of this layer on RBC partitioning, a diverging capillary bifurcation is analyzed using a flexible, two-dimensional model. In addition, the model is also used to investigate RBC deformation and RBC penetration of the ESL region when ESL properties are varied. The RBC is represented using interconnected viscoelastic elements. Stokes flow equations (viscous flow) model the surrounding fluid. The flow in the ESL is modeled using the Brinkman approximation for porous media with a corresponding hydraulic resistivity. The ESL's resistance to compression is modeled using an osmotic pressuredifference. One cell passes through the bifurcation at a time, so there are no cell-cell interactions. A range of physiologically relevant hydraulic resistivities and osmotic pressure differences are explored. Decreasing hydraulic resistivity and/or decreasing osmotic pressure differences (ESL resistance to compression) produced four behaviors: (1) RBC partitioning nonuniformity increased slightly; (2) RBC deformation decreased; (3) RBC velocity decreased relative to blood flow velocity; and (4) RBCs penetrated more deeply into the ESL. Decreasing the ESL's resistance to flow and/or compression to pathological levels could lead to more frequent cell adhesion and clotting as well as impaired vascular regulation due to weaker ATP and nitric oxide release. Potential mechanisms that can contribute to these behaviors are also discussed.

Discrete phase model of blood flow in a roughness microchannel simulating the formation of pseudointima

Purpose: The goal of the present study was the development of discrete phase model to simulate the phenomenon of backfilling a morphologically complex surface by red blood cells (RBCs) in a flow microchannel and to anticipate the conditions of forming a pseudointima. The objective of the experimental studies that inspired the development of the simulation was to create a surface that stimulates the formation of the pseudointima layer. Methods: The finite volume method (FVM) and discrete particle method (DPM) were applied to develop the target model. In addition, a mixture model and a roughness model of bottom layer were tested in the present study to show their influence on simulation the phenomenon of backfilling a morphologically complex surface by RBCs in a flow microchannel. Results: Numerical models were developed including: a) FVM models to compare the effect of applying boundary conditions with/without roughness and cubes, as well as the analysis of their influence on blood velocity and shear stress; b) mixture models to compare the effect of applying different boundary conditions and cubes on computed results; c) DPM models to compare the effect of applying and not applying roughness as a boundary condition; d) DPM models with a morphologically complex surface and RBCs collisions to present RBCs concentration, velocity and time distributions during flow in a channel. Conclusions: The analysis carried out for the developed numerical models indicates that DPM model with cubes computes the best results. It also shows the backfilling of a morphologically complex surface of the bottom microchannel with RBCs.

Peripheral microvascular dysfunction is also present in patients with ischemia and no obstructive coronary artery disease (INOCA).

In patients with ischemia and no obstructive coronary artery disease (INOCA), coronary microvascular dysfunction is associated with higher rate of major adverse cardiovascular events. To demonstrate if microvascular dysfunction present in coronary microcirculation of patients with INOCA may be detected noninvasively in their peripheral circulation. 25 patients with INOCA and 25 apparently healthy individuals (controls) were subjected to nailfold videocapillaroscopy (NVC) and venous occlusion plethysmography (VOP) to evaluate peripheral microvascular function and blood collection for biomarkers analysis, including soluble vascular cell adhesion molecule-1 (sVCAM-1), endothelin-1 (ET-1) and C-reactive protein (CRP). Red blood cell velocity (RBCV) before and after ischemia (RBCVmax) were significantly lower in patients with INOCA (p = 0.0001). Time to reach maximal red blood cell velocity (TRBCVmax) was significantly longer in INOCA group (p = 0.0004). Concerning VOP, maximal blood flow (p = 0.004) and its relative increment were significantly lower in patients with INOCA (p = 0.0004). RBCVmax showed significant correlations with sVCAM-1 (r = -0.38, p < 0.05), ET-1 (r = -0.73, p < 0.05) and CRP (r = -0.33, p < 0.05). Relative increment of maximal post-ischemic blood flow was significantly correlated with sVCAM-1 (r = -0.42, p < 0.05) and ET-1 (r = -0.48, p < 0.05). The impairment of microvascular function present in coronary microcirculation of patients with INOCA can be also detected in peripheral microcirculation.

Microfluidic Characterization of Red Blood Cells Microcirculation under Oxidative Stress.

Microcirculation is one of the basic functional processes where the main gas exchange between red blood cells (RBCs) and surrounding tissues occurs. It is greatly influenced by the shape and deformability of RBCs, which can be affected by oxidative stress induced by different drugs and diseases leading to anemia. Here we investigated how in vitro microfluidic characterization of RBCs transit velocity in microcapillaries can indicate cells damage and its correlation with clinical hematological analysis. For this purpose, we compared an SU-8 mold with an Si-etched mold for fabrication of PDMS microfluidic devices and quantitatively figured out that oxidative stress induced by tert-Butyl hydroperoxide splits all RBCs into two subpopulations of normal and slow cells according to their transit velocity. Obtained results agree with the hematological analysis showing that such changes in RBCs velocities are due to violations of shape, volume, and increased heterogeneity of the cells. These data show that characterization of RBCs transport in microfluidic devices can directly reveal violations of microcirculation caused by oxidative stress. Therefore, it can be used for characterization of the ability of RBCs to move in microcapillaries, estimating possible side effects of cancer chemotherapy, and predicting the risk of anemia.

Continuum microhaemodynamics modelling using inverse rheology

Modelling blood flow in microvascular networks is challenging due to the complex nature of haemorheology. Zero- and one-dimensional approaches cannot reproduce local haemodynamics, and models that consider individual red blood cells (RBCs) are prohibitively computationally expensive. Continuum approaches could provide an efficient solution, but dependence on a large parameter space and scarcity of experimental data for validation has limited their application. We describe a method to assimilate experimental RBC velocity and concentration data into a continuum numerical modelling framework. Imaging data of RBCs were acquired in a sequentially bifurcating microchannel for various flow conditions. RBC concentration distributions were evaluated and mapped into computational fluid dynamics simulations with rheology prescribed by the Quemada model. Predicted velocities were compared to particle image velocimetry data. A subset of cases was used for parameter optimisation, and the resulting model was applied to a wider data set to evaluate model efficacy. The pre-optimised model reduced errors in predicted velocity by 60% compared to assuming a Newtonian fluid, and optimisation further reduced errors by 40%. Asymmetry of RBC velocity and concentration profiles was demonstrated to play a critical role. Excluding asymmetry in the RBC concentration doubled the error, but excluding spatial distributions of shear rate had little effect. This study demonstrates that a continuum model with optimised rheological parameters can reproduce measured velocity if RBC concentration distributions are known a priori. Developing this approach for RBC transport with more network configurations has the potential to provide an efficient approach for modelling network-scale haemodynamics.

Capillary hemodynamics and contracting skeletal muscle oxygen pressures in male rats with heart failure: Impact of soluble guanylyl cyclase activator

In heart failure with reduced ejection fraction (HFrEF), nitric oxide-soluble guanylyl cyclase (sGC) pathway dysfunction impairs skeletal muscle arteriolar vasodilation and thus capillary hemodynamics, contributing to impaired oxygen uptake (V̇O2) kinetics. Targeting this pathway with sGC activators offers a new treatment approach to HFrEF. We tested the hypotheses that sGC activator administration would increase the O2 delivery (Q̇O2)-to-V̇O2 ratio in the skeletal muscle interstitial space (PO2is) of HFrEF rats during twitch contractions due, in part, to increases in red blood cell (RBC) flux (fRBC), velocity (VRBC), and capillary hematocrit (Hctcap). HFrEF was induced in male Sprague-Dawley rats via myocardial infarction. After 3 weeks, rats were treated with 0.3 mg/kg of the sGC activator BAY 60–2770 (HFrEF + BAY; n = 11) or solvent (HFrEF; n = 9) via gavage b.i.d for 5 days prior to phosphorescence quenching (PO2is, in contracting muscle) and intravital microscopy (resting) measurements in the spinotrapezius muscle. Intravital microscopy revealed higher fRBC (70 ± 9 vs 25 ± 8 RBC/s), VRBC (490 ± 43 vs 226 ± 35 μm/s), Hctcap (16 ± 1 vs 10 ± 1%) and a greater number of capillaries supporting flow (91 ± 3 vs 82 ± 3%) in HFrEF + BAY vs HFrEF (all P < 0.05). Additionally, PO2is was especially higher during 12–34s of contractions in HFrEF + BAY vs HFrEF (P < 0.05). Our findings suggest that sGC activators improved resting Q̇O2 via increased fRBC, VRBC, and Hctcap allowing for better Q̇O2-to-V̇O2 matching during the rest-contraction transient, supporting sGC activators as a potential therapeutic to target skeletal muscle vasomotor dysfunction in HFrEF.

Measuring Temporal and Spatial Variability of Red Blood Cell Velocity in Human Retinal Vessels.

PurposeThe retinal circulation regulates blood flow through various internal and external factors; however, it is unclear how locally these factors act within the retinal microcirculation. We measured the temporal and spatial variability of blood velocity in small retinal vessels using a dual-beam adaptive optics scanning laser ophthalmoscope.MethodsIn young healthy subjects (n = 3), temporal blood velocity variability was measured in a local vascular region consisting of an arteriole, capillary, and venule repeatedly over 2 days. Data consisted of 10 imaging periods separated into two sessions: (1) five 6-minute image acquisition periods with 30-minute breaks, and (2) five 6-minute image acquisition periods with 10-minute breaks. In another group of young healthy subjects (n = 5), spatial distribution of velocity variability was measured by imaging three capillary segments during three 2-minute conditions: (1) baseline imaging condition (no flicker), (2) full-field flicker, and (3) no flicker condition again.ResultsBlood velocities were measurable in all subjects with a reliability of about 2%. The coefficient of variation (CV) was used as an estimate of the physiological variability of each vessel. Over 2 days, the average CV in arterioles was 7% (±2%); in capillaries, it was 19% (±6%); and, in venules, it was 8% (±2%). During flicker stimulation, the average capillary CV was 16% during baseline, 15% during flicker stimulation, and 18% after flicker stimulation.ConclusionsCapillaries in the human retina exhibit spatial and temporal variations in blood velocity. This inherent variation in blood velocity places limits on studying the vascular regulation of individual capillaries, and the study presented here serves as a foundation for future endeavors.

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice &lt;em&gt;In Vivo&lt;/em&gt;

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to correlate this with local hemodynamics. This protocol uses transgenic mice that have been prepared with a chronic cranial window and express the genetically encoded calcium indicator, RCaMP1.07, under the α-smooth muscle actin promoter to specifically label mural cells, such as vascular smooth muscle cells and ensheathing pericytes. Steps are outlined on how to prepare a tail vein catheter for intravenous injection of fluorescent dyes to trace blood flow, as well as how to measure brain pericyte calcium and local blood vessel hemodynamics (diameter, red blood cell velocity, etc.) by two photon microscopy in vivo through the cranial window in ketamine/xylazine anesthetized mice. Finally, details are provided for the analysis of calcium fluctuations and blood flow movies via the image processing algorithms developed by Barrett et al. 2018, with an emphasis on how these processes can be adapted to other cellular imaging data.

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo.

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to correlate this with local hemodynamics. This protocol uses transgenic mice that have been prepared with a chronic cranial window and express the genetically encoded calcium indicator, RCaMP1.07, under the α-smooth muscle actin promoter to specifically label mural cells, such as vascular smooth muscle cells and ensheathing pericytes. Steps are outlined on how to prepare a tail vein catheter for intravenous injection of fluorescent dyes to trace blood flow, as well as how to measure brain pericyte calcium and local blood vessel hemodynamics (diameter, red blood cell velocity, etc.) by two photon microscopy in vivo through the cranial window in ketamine/xylazine anesthetized mice. Finally, details are provided for the analysis of calcium fluctuations and blood flow movies via the image processing algorithms developed by Barrett et al. 2018, with an emphasis on how these processes can be adapted to other cellular imaging data.

Oral L-Arginine (5 g/day) for 14 Days Improves Microcirculatory Function in Healthy Young Women and Healthy and Type 2 Diabetes Mellitus Elderly Women

Objective: The aim of this study was to investigate the effect of oral supplementation with L-arginine on serum biochemical profile, blood pressure, microcirculation, and vasoreactivity/endothelial function in young controls, and elderly women with and without type 2 diabetes mellitus (T2DM). Methods: Healthy young (n = 25), healthy elderly (n = 25), and elderly women with type 2 diabetes mellitus (T2DME, n = 23, glycated Hb ≥6.4% and mean of 7.7 years for duration of the disease), aged 18–30 and older than 65 years, respectively, were included in the study. All patients underwent biochemical analysis (fasting glycemia and lipidogram), arterial blood pressure, nailfold videocapillaroscopy (capillary diameters, functional capillary density [FCD], peak red blood cell velocity [RBCV_max] after 1 min ischemia, time to reach peak RBCV [TRBCV_max]), and venous occlusion plethysmography (vasoreactivity), before and after 14 days of oral supplementation with L-arginine (5 g/day). Results: L-Arginine did not change fasting glycemia and lipidogram, but it decreased systolic, diastolic, and mean arterial pressure in elderly women, increased RBCV_max in all groups, and did not decrease TRBCV_max in T2DME. Capillary diameters and FCD remained unchanged in all groups. L-Arginine improved vasoreactivity during reactive hyperemia and after sublingual nitroglycerin (0.4 mg) in all groups. Conclusion: L-Arginine supplementation (5g/day during 14 days) was able to improve vascular/microvascular health in the elderly women with or without T2DM.

Abstract 10086: Cardiac Intravital Microscopy Enables Quantitative Mapping of Capillary-Level Blood Flow in the Murine Heart

Introduction: Coronary microvascular disease (CMD) is increasingly appreciated as a contributor to adverse cardiovascular outcomes, but underlying mechanisms of CMD remain poorly understood. Investigation of CMD in animal models is limited by a lack of techniques to quantify flow in the heart. We assessed the hypothesis that intravital microscopy (IVM) can enable in vivo imaging of red blood cell (RBC) flow profiles in the coronary microcirculation at cellular resolution. Methods: C57BL/6 mice (n=20) were used to develop cardiac IVM protocols. Cardiomyocyte membrane staining was achieved using intravenous injection of a voltage dye (di-2-anepeq). RBC labeling was done by ex-vivo staining of cells with Ter119 followed by transfusion. IVM was performed through a thoracotomy with animals anesthetized and ventilated. Confocal and two-photon microscopy was done using a custom tissue stabilizer and an Olympus FV1000MPE microscope synchronized to physiologic signals using custom hardware and software and cardiac pacing protocols. Images were analyzed in MATLAB to extract myocyte contractile properties, capillary diameter, and capillary flow properties. Results: Using cardiac and respiratory gating, images with cellular resolution could be measured in the beating heart at all phases of the cardiac cycle. This enabled quantification of cardiomyocyte contractile function and capillary diameter and widefield imaging by stitching of several images (Fig. 1A). Tracking of RBCs over serial images allowed quantification of RBC velocity across multiple capillaries simultaneously (Fig. 1B). Conclusion: Cardiac IVM can provide quantification of cardiac function and blood flow at the single cell scale and will provide an exciting tool for investigating CMD in models of heart disease.