Red Blood Cell Flow Research Articles

Curated Dataset for Red Blood Cell Tracking from Video Sequences of Flow in Microfluidic Devices

This work presents a dataset comprising images, annotations, and velocity fields for benchmarking cell detection and cell tracking algorithms. The dataset includes two video sequences captured during laboratory experiments, showcasing the flow of red blood cells (RBC) in microfluidic channels. From the first video 300 frames and from the second video 150 frames are annotated with bounding boxes around the cells, as well as tracks depicting the movement of individual cells throughout the video. The dataset encompasses approximately 20,000 bounding boxes and 350 tracks. Additionally, computational fluid dynamics simulations were utilized to generate 2D velocity fields representing the flow within the channels. These velocity fields are included in the dataset. The velocity field has been employed to improve cell tracking by predicting the positions of cells across frames. The paper also provides a comprehensive discussion on the utilization of the flow matrix in the tracking steps.

Acceleration of the Development of Microcirculation Embolism in the Brain due to Capillary Narrowing.

Cerebral microvascular obstruction is critically involved in recurrent stroke and decreased cerebral blood flow with age. The obstruction must occur in the capillary with a greater resistance to perfusion pressure through the microvascular networks. However, little is known about the relationship between capillary size and embolism formation. This study aimed to determine whether the capillary lumen space contributes to the development of microcirculation embolism. To spatiotemporally manipulate capillary diameters in vivo, transgenic mice expressing the light-gated cation channel protein ChR2 (channelrhodopsin-2) in mural cells were used. The spatiotemporal changes in the regional cerebral blood flow in response to the photoactivation of ChR2 mural cells were first characterized using laser speckle flowgraphy. Capillary responses to optimized photostimulation were then examined in vivo using 2-photon microscopy. Finally, microcirculation embolism due to intravenously injected fluorescent microbeads was compared under conditions with or without photoactivation of ChR2 mural cells. Following transcranial photostimulation, the stimulation intensity-dependent decrease in cerebral blood flow centered at the irradiation was observed (14%-49% decreases relative to the baseline). The cerebrovascular response to photostimulation showed significant constriction of the cerebral arteries and capillaries but not of the veins. As a result of vasoconstriction, a temporal stall of red blood cell flow occurred in the capillaries of the venous sides. The 2-photon excitation of a single ChR2 pericyte demonstrated the partial shrinkage of capillaries (7% relative to the baseline) around the stimulated cell. With the intravenous injection of microbeads, the occurrence of microcirculation embolism was significantly enhanced (11% increases compared to the control) with photostimulation. Capillary narrowing increases the risk of developing microcirculation embolism in the venous sides of the cerebral capillaries.

EFFECTS OF M101-AN EXTRACELLULAR HEMOGLOBIN-APPLIED DURING CARDIOPULMONARY RESUSCITATION: AN EXPERIMENTAL RODENT STUDY.

During and immediately after cardiac arrest, cerebral oxygen delivery is impaired mainly by microthrombi and cerebral vasoconstriction. This may narrow capillaries so much that it might impede the flow of red blood cells and thus oxygen transport. The aim of this proof-of-concept study was to evaluate the effect of M101, an extracellular hemoglobin-based oxygen carrier (Hemarina SA, Morlaix, France) derived from Arenicola marina , applied during cardiac arrest in a rodent model, on markers of brain inflammation, brain damage, and regional cerebral oxygen saturation. Twenty-seven Wistar rats subjected to 6 min of asystolic cardiac arrest were infused M101 (300 mg/kg) or placebo (NaCl 0.9%) concomitantly with start of cardiopulmonary resuscitation. Brain oxygenation and five biomarkers of inflammation and brain damage (from blood, cerebrospinal fluid, and homogenates from four brain regions) were measured 8 h after return of spontaneous circulation. In these 21 different measurements, M101-treated animals were not significantly different from controls except for phospho-tau only in single cerebellum regions ( P = 0.048; ANOVA of all brain regions: P = 0.004). Arterial blood pressure increased significantly only at 4 to 8 min after return of spontaneous circulation ( P < 0.001) and acidosis decreased ( P = 0.009). While M101 applied during cardiac arrest did not significantly change inflammation or brain oxygenation, the data suggest cerebral damage reduction due to hypoxic brain injury, measured by phospho-tau. Global burden of ischemia appeared reduced because acidosis was less severe. Whether postcardiac arrest infusion of M101 improves brain oxygenation is unknown and needs to be investigated.

Seizure-induced neutrophil adhesion in brain capillaries leads to a decrease in postictal cerebral blood flow

Cerebral hypoperfusion has been proposed as a potential cause of postictal neurological dysfunction in epilepsy, but its underlying mechanism is still unclear. We show that a 30% reduction in postictal cerebral blood flow (CBF) has two contributing factors: the early hypoperfusion up to ∼30min post-seizure was mainly induced by arteriolar constriction, while the hypoperfusion that persisted for over an hour was due to increased capillary stalling induced by neutrophil adhesion to brain capillaries, decreased red blood cell (RBC) flow accompanied by constriction of capillaries and venules, and elevated intercellular adhesion molecule-1 (ICAM-1) expression. Administration of antibodies against the neutrophil marker Ly6G and against LFA-1, which mediates adhesive interactions with ICAM-1, prevented neutrophil adhesion and recovered the prolonged CBF reductions to control levels. Our findings provide evidence that seizure-induced neutrophil adhesion to cerebral microvessels via ICAM-1 leads to prolonged postictal hypoperfusion, which may underlie neurological dysfunction in epilepsy.

Mathematical Analysis for the Flow of Sickle Red Blood Cells in Microvessels for Bio Medical Application.

Sickle cell disease (SCD) is an inherited monogenic disease which is characterized by distorted red blood cells (RBCs) that cause vaso-occlusion and vasculopathy. In the pathogenesis of SCD, polymerized hemoglobin turn RBCs into fragile, less deformable cells, and are subsequently more susceptible to endothelial adhesion after deoxygenation. Presently, electrophoresis and genotyping are used as routine tests for diagnosis of SCD. These techniques are expensive and require specialized laboratories. Lab-on-a-chip technology is a low-cost microfluidics-based diagnostic tool which holds significant promise for rapid screening of RBC deformability. To explore the sickle RBC mechanics for screening purposes, we present a mathematical model for the flow of single RBC with altered rheological properties and slip effect on capillary wall in microcirculation. We consider single-file flow of cells through the axis symmetrical cylindrical duct, applying lubrication theory as plasma trapped between successive red blood cells. The rheological parameters used from published literature for normal RBC and corresponding variation has been taken for the purpose of this simulation to present the condition of the disease. An analytical solution has been found for realistic boundary conditions and results are simulated using MATLAB. We found that the height of plasma film in the capillary increases with increase in cell deformability and compliance which affects the forward flow velocity in the capillary. Rigid RBCs with increased adhesion between cell and capillary wall shows reduction in velocity and occurrence of vaso-occlusion events in extreme conditions. These rheological properties of the cells coupled with microfluidics mechanics can mimic the physiological condition and provides unique insights with novel possibilities for the design of microfluidics base diagnostic kit towards effective therapeutic intervention of SCD.

Microfluidic study of retention and elimination of abnormal red blood cells by human spleen with implications for sickle cell disease

The spleen clears altered red blood cells (RBCs) from circulation, contributing to the balance between RBC formation (erythropoiesis) and removal. The splenic RBC retention and elimination occur predominantly in open circulation where RBCs flow through macrophages and inter-endothelial slits (IESs). The mechanisms underlying and interconnecting these processes significantly impact clinical outcomes. In sickle cell disease (SCD), blockage of intrasplenic sickled RBCs is observed in infants splenectomized due to acute splenic sequestration crisis (ASSC). This life-threatening RBC pooling and organ swelling event is plausibly triggered or enhanced by intra-tissular hypoxia. We present an oxygen-mediated spleen-on-a-chip platform for in vitro investigations of the homeostatic balance in the spleen. To demonstrate and validate the benefits of this general microfluidic platform, we focus on SCD and study the effects of hypoxia on splenic RBC retention and elimination. We observe that RBC retention by IESs and RBC-macrophage adhesion are faster in blood samples from SCD patients than those from healthy subjects. This difference is markedly exacerbated under hypoxia. Moreover, the sickled RBCs under hypoxia show distinctly different phagocytosis processes from those non-sickled RBCs under hypoxia or normoxia. We find that reoxygenation significantly alleviates RBC retention at IESs, and leads to rapid unsickling and fragmentation of the ingested sickled RBCs inside macrophages. These results provide unique mechanistic insights into how the spleen maintains its homeostatic balance between splenic RBC retention and elimination, and shed light on how disruptions in this balance could lead to anemia, splenomegaly, and ASSC in SCD and possible clinical manifestations in other hematologic diseases.

Label-free imaging of red blood cells and oxygenation with color third-order sum-frequency generation microscopy

Mapping red blood cells (RBCs) flow and oxygenation is of key importance for analyzing brain and tissue physiology. Current microscopy methods are limited either in sensitivity or in spatio-temporal resolution. In this work, we introduce a novel approach based on label-free third-order sum-frequency generation (TSFG) and third-harmonic generation (THG) contrasts. First, we propose a novel experimental scheme for color TSFG microscopy, which provides simultaneous measurements at several wavelengths encompassing the Soret absorption band of hemoglobin. We show that there is a strong three-photon (3P) resonance related to the Soret band of hemoglobin in THG and TSFG signals from zebrafish and human RBCs, and that this resonance is sensitive to RBC oxygenation state. We demonstrate that our color TSFG implementation enables specific detection of flowing RBCs in zebrafish embryos and is sensitive to RBC oxygenation dynamics with single-cell resolution and microsecond pixel times. Moreover, it can be implemented on a 3P microscope and provides label-free RBC-specific contrast at depths exceeding 600 µm in live adult zebrafish brain. Our results establish a new multiphoton contrast extending the palette of deep-tissue microscopy.

Measuring capillary flow dynamics using interlaced two-photon volumetric scanning

Two photon microscopy and optical coherence tomography (OCT) are two standard methods for measuring flow speeds of red blood cells in microvessels, particularly in animal models. However, traditional two photon microscopy lacks the depth of field to adequately capture the full volumetric complexity of the cerebral microvasculature and OCT lacks the specificity offered by fluorescent labeling. In addition, the traditional raster scanning technique utilized in both modalities requires a balance of image frame rate and field of view, which severely limits the study of RBC velocities in the microvascular network. Here, we overcome this by using a custom two photon system with an axicon based Bessel beam to obtain volumetric images of the microvascular network with fluorescent specificity. We combine this with a novel scan pattern that generates pairs of frames with short time delay sufficient for tracking red blood cell flow in capillaries. We track RBC flow speeds in 10 or more capillaries simultaneously at 1 Hz in a 237 µm × 237 µm × 120 µm volume and quantified both their spatial and temporal variability in speed. We also demonstrate the ability to track flow speed changes around stalls in capillary flow and measure to 300 µm in depth.

Modeling of Red Blood Cells in Capillary Flow Using Fluid–Structure Interaction and Gas Diffusion

Red blood cell (RBC) distribution, RBC shape, and flow rate have all been shown to have an effect on the pulmonary diffusing capacity. Through this study, a gas diffusion model and the immersed finite element method were used to simulate the gas diffusion into deformable RBCs running in capillaries. It has been discovered that when RBCs are deformed, the CO flux across the membrane becomes nonuniform, resulting in a reduced capacity for diffusion. Additionally, when compared to RBCs that were dispersed evenly, our simulation showed that clustered RBCs had a significantly lower diffusion capability.

Aβ1‐42 oligomers induce pericyte dysfunction via p75 Neurotrophin Receptor

AbstractBackgroundAmyloid beta (Aβ) aggregates and tau tangles are hallmark pathologies of Alzheimer’s disease (AD). There is growing evidence that vascular dysfunction may contribute to the pathophysiology of AD. Pericyte injury and/or deterioration leading to blood‐brain barrier (BBB) breakdown occur in normal aging and are associated with cognitive impairment in AD patients. Furthermore, there is reduced cerebral blood flow in AD. Because vascular resistance occurs in capillaries, we hypothesize that Aβ1‐42 causes aberrant pericyte contraction and cell death via p75 neurotrophin receptor (p75NTR).Methodp75NTR levels were quantified via Western blotting in mouse vascular brain pericytes treated with either artificial cerebrospinal fluid (aCSF) as a control or 72 nM Aβ1‐42 oligomers for either 20 minutes or 24 hours. Furthermore, LIVE/DEAD assays were performed to quantify pericyte deterioration after exposure to 72 nM Aβ1‐42 for various time points. In mouse models, changes in red blood cell (RBC) flow due to pericyte contraction around capillaries were measured using intravital microscopy in the somatosensory cortex of anesthetized C57 mice and applying either topical aCSF or 72 nM Aβ1‐42.ResultAfter 20 minutes of aCSF or Aβ1‐42 treatment, there is a statistically significant increase in p75NTR levels (n = 3, p = 0.016, two‐tailed t‐test), while there is a statistically significant decrease by 43% in p75NTR levels after 24 hours (n = 6, p = 0.0028, two‐tailed t‐test). After 48 hours of aCSF or Aβ1‐42 treatment, 75.6% of the control pericytes were viable, whereas only 65.3% of the Aβ1‐42 treated pericytes were viable. Preliminary in vivo studies revealed that 72 nM Aβ1‐42 treatment stalled RBC flow.ConclusionPericytes were confirmed to express p75NTR. Aβ1‐42 treatment upregulates p75NTR expression after 20 minutes while downregulating its expression after 24 hours. This suggests that acute increases in p75NTR levels may mediate increased pericyte contraction and play a role in reduced cerebral blood flow during the progression of AD. Because the details of this mechanism are unknown, future in vivo studies will involve pericyte‐specific p75NTR knock‐out mice.

Numerical investigation on red blood cell flow based on unstructured grid.

Prediction of blood cell flow is known as the difficult research by reason of the complexity of blood vessel. In this study, considering the complex structure of blood vessels, a mechanical model for red blood cell (RBC) based on unstructured grid has been established to study the flow characteristics of RBCs in complex blood vessels. In the model, the strain-energy function by Skalak is employed to model the shear elasticity and surface-area conservation of the membrane, and the hinge spring is used to describe the forces originating from local bending of the membrane. The immersed boundary method is utilized to couple the interphase force. Using the model, the stretching test of RBC is compared with the experiment data, and the good agreement verified the validation of the present model. The morphology of red blood cell and the blood viscosity in micro-vessel are studied. RBCs move with a symmetric shape (parachute shape) in small blood vessels, and the buckling instability is observed when the RBC flow slowly through a micro-vessel or a converging-diverging capillary. When the vessel diameter is around 10μm, the reverse Fahraeus-Lindqvist effect is presented. The blood apparent viscosity shows linear increase with the blood hematocrit. In addition, Malaria infection can make the RBC deformability decreased and the blood apparent viscosity increased.

Spatiotemporal analysis of blood plasma and blood cell flow fluctuations of cerebral microcirculation in anesthetized rats

Cerebral hemodynamics fluctuates spontaneously over broad frequency ranges. However, its spatiotemporal coherence of flow oscillations in cerebral microcirculation remains incompletely understood. The objective of this study was to characterize the spatiotemporal fluctuations of red blood cells (RBCs) and plasma flow in the rat cerebral microcirculation by simultaneously imaging their dynamic behaviors. Comparisons of changes in cross-section diameters between RBC and plasma flow showed dissociations in penetrating arterioles. The results indicate that vasomotion has the least effect on the lateral movement of circulating RBCs, resulting in variable changes in plasma layer thickness. Parenchymal capillaries exhibited slow fluctuations in RBC velocity (0.1 to 0.3 Hz), regardless of capillary diameter fluctuations (<0.1 Hz). Temporal fluctuations and the velocity of RBCs decreased significantly at divergent capillary bifurcations. The results indicate that a transit of RBCs generates flow resistance in the capillaries and that slow velocity fluctuations of the RBCs are subject to a number of bifurcations. In conclusion, the high-frequency oscillation of the blood flow is filtered at the bifurcation through the capillary networks. Therefore, a number of bifurcations in the cerebral microcirculation may contribute to the power of low-frequency oscillations.

Flows of healthy and hardened RBC suspensions through a micropillar array.

Red blood cell (RBC) deformability is an important haemorheological factor; it is impaired in many pathologies leading to microvascular complications. Several microfluidic platforms have been utilized to examine the role of deformability in RBC flows but their geometries tend to be simplified. In the present study, we extend our previous work on healthy RBC flows in micropillar arrays [1] to probe the effect of impaired RBC deformability on the velocity and haematocrit distributions in microscale RBC flows. Healthy and artificially hardened RBC suspensions at 25% haematocrit were perfused through the micropillar array at various flow rates and imaged. RBC velocities were determined by Particle Image Velocimetry (PIV) and haematocrit distributions were inferred from the image intensity distributions. The pillars divide the flow into two distinct RBC streams separated by a cell-depleted region along the centreline and in the rear/front stagnation points. RBC deformability was not found to significantly affect the velocity distributions; the shape of the velocity profiles in the interstitial space remained the same for healthy and hardened RBCs. Time-averaged and spatiotemporal intensity distributions, however, reveal differences in the dynamics and local distributions of healthy and hardened cells; hardened cells appear to enter the cell-depleted regions more frequently and their interstitial distributions are more uniform. The study highlights the importance of local RBC distributions and the impact of RBC deformability on cell transport in complex microscale flows.

QS16. The Effects of Variations in the Skin Surface Temperature on Capillary Blood Flow Using Video-capillaroscopy

Purpose: Video-capillaroscopy (VC) can be used to evaluate real-time flow of red blood cells in capillaries at a depth of 1-mm from the skin surface. Therefore, this device could be used in microsurgery to assess the perfusion of flaps, replanted fingers, skin grafts, and keloids. The details of capillary changes during skin cooling under VC observations are not known. By investigating the adaptations of capillaries after cooling using VC, we determined the effects of temperature on skin capillaries with those at room temperature, establishing temperature criteria for future VC applications. Therefore, we observed and compared VC findings on skin areas often used for flap harvest at a normal and lower body temperature. Methods: Twenty healthy Japanese adults were included. Skin capillaries were observed at the lateral thigh, forearm, mid-axillary line, abdomen, and fingertip using VC (GOKO Bscan-Z). Ice packs were used to lower the skin temperature to less than 35°C. The VC findings were recorded for three minutes before and after cooling. By means of the ImageJ software, we measured the total blood vessel area (pixels) as a percentage of the total area of the visual field (pixels). Also, we measured the blood flow velocity (μm/s) using GOKO-VIP software and the results for both temperatures (normal and after cooling) were then compared. Results: The average age and BMI were 37.5±9.72 years and 22.4±2.45 kg/m2 respectively. The mean arterial pressure was 89.4±3.1 mmHg. In accordance with the Fitzpatrick phenotype, eleven patients were type II, five were type III, and four were type IV. The average skin temperature before and after cooling were 36.4±0.2°C and 34.7±0.8°, respectively. The blood vessel areas were significantly different before and after cooling in all anatomical areas (p <.001). The average reduction rate of the vessel area after cooling was 63% for the thigh, 30% for the forearm, 43.3% for the mid-axillary line, 35% for the abdomen, and 65% for fingertips. The mean reduction rate of the vessel area was significantly different among anatomic regions (p <.001). The average blood flow velocities were significantly different before and after cooling in all anatomical areas (p <.001). The average blood flow velocity reduction rate after cooling was 75.7% for the thigh, 55.3% for the forearm, 68.9% for the mid-axillary line, 61.6% for the abdomen, and 79.2% for fingertips. The average blood flow velocity reduction rate after cooling was significantly different among anatomic regions (p <.001). Conclusion: Decrease in skin surface temperature resulted in capillary vasoconstriction and a decrease of capillary blood flow velocity in all areas. When VC is used for flap monitoring, it is important to keep the observation area warm since decreasing the temperature in the monitored area might result in the false diagnosis of arterial occlusion.

High-Throughput Imaging of Blood Flow Reveals Developmental Changes in Distribution Patterns of Hemodynamic Quantities in Developing Zebrafish.

Mechanical forces from blood flow and pressure (hemodynamic forces) contribute to the formation and shaping of the blood vascular network during embryonic development. Previous studies have demonstrated that hemodynamic forces regulate signaling and gene expression in endothelial cells that line the inner surface of vascular tubes, thereby modifying their cellular state and behavior. Given its important role in vascular development, we still know very little about the quantitative aspects of hemodynamics that endothelial cells experience due to the difficulty in measuring forces in vivo. In this study, we sought to determine the magnitude of wall shear stress (WSS) exerted on ECs by blood flow in different vessel types and how it evolves during development. Utilizing the zebrafish as a vertebrate model system, we have established a semi-automated high-throughput fluorescent imaging system to capture the flow of red blood cells in an entire zebrafish between 2- and 6-day post-fertilization (dpf). This system is capable of imaging up to 50 zebrafish at a time. A semi-automated analysis method was developed to calculate WSS in zebrafish trunk vessels. This was achieved by measuring red blood cell flow using particle tracking velocimetry analysis, generating a custom-made script to measure lumen diameter, and measuring local tube hematocrit levels to calculate the effective blood viscosity at each developmental stage. With this methodology, we were able to determine WSS magnitude in different vessels at different stages of embryonic and larvae growth and identified developmental changes in WSS, with absolute levels of peak WSS in all vessel types falling to levels below 0.3 Pa at 6 dpf. Additionally, we discovered that zebrafish display an anterior-to-posterior trend in WSS at each developmental stage.

Geometry effect in multi-step crossflow microfluidic devices for red blood cells separation and deformability assessment

The efficient separation of blood components using microfluidic systems can help to improve the detection and diagnosis of several diseases, such as malaria and diabetes. Therefore, a novel multi-step microfluidic device, based on passive crossflow filters was developed. Three different designs were proposed, fabricated and tested in order to evaluate the most suitable geometry to perform, simultaneously, blood cells separation and cell deformability measurements. All the proposed geometries include a main channel and three sequential separation steps, all comprised of symmetrical crossflow filters, with multiple rows of pillars, to reduce the amount of red blood cells (RBCs) flowing to the outlets of the microfluidic device (MD). Sets of hyperbolic constrictions located at the outlets allow the assessment of cells deformability. Based on the proposed geometries, the three correspondent MD were evaluated and compared, by measuring the RBCs velocities, the cell-free layer (CFL) effect through the microchannels and by quantifying the amount of RBCs at the outlets. The results suggest that the proposed MD 3 configuration was the most effective one for the desired application, due to the formation of a wider CFL. As a result, a minor amount of RBCs flow through the hyperbolic contraction at the third separation level of the device. Nevertheless, for all the proposed geometries, the existence of three separation levels shows that it is possible to achieve a highly efficient cell separation. If needed, such microdevices have the potential for further improvements by increasing the number of separation levels, aiming the total separation of blood cells from plasma.

A potent luminogen with NIR-IIb excitable AIE features for ultradeep brain vascular and hemodynamic three-photon imaging

Three-photon excited fluorescence microscopy (3PEFM) has emerged as a promising protocol for visualizing deep-brain vasculature and hemodynamics. However, the current situation is still far from satisfactory, due to small excitation action cross-section and short excitation wavelength of those previously reported 3PEFM luminogens. Herein, we manipulated molecular engineering by subtly regulating structural planarization/twisting to achieve ingenious integration of large three-photon absorption cross-section, high fluorescence quantum yield, ultralong near-infrared IIb excitation, and aggregation-induced emission features. The resulting molecule, namely DPCZ-BT, exhibited as high as 50.6% of fluorescence quantum yield and as large as 2.0 × 10−81 cm6s2/photon2 of three-photon absorption cross-section, which can be excited by 1665 nm fs laser and presents a recorded penetration depth of 1860 μm for deep-brain vascular structural imaging with high spatiotemporal resolution and signal-to-background ratio. Moreover, DPCZ-BT having good photostability and excellent biocompatibility is capable of impressively approaching 1600 μm depth in monitoring red blood cells flow velocity with extraordinary clarity for hemodynamics.

Conducted Capillary Signaling Enables Oxygen Responses in Skeletal Muscle Independent of Metabolite Production

Red blood cell (RBC) flow through the microvasculature is closely matched to tissue O2 requirements. At a fundamental level, O2 demand‐supply coupling entails the sensing of PO2, and the generation of a stimulus that alters upstream arteriolar tone. However, where and how O2 needs are sensed in the microcirculation is presently contested. One idea centers on hypoxia triggering the release of K+, activating endothelial KIR2.1 channels and initiating a hyperpolarization that conducts upstream via gap junctions, comprised of connexins (Cx). We tested this idea in skeletal muscle where we controlled the local tissue O2 environment using live animal imaging in Cx40‐/‐and endothelial KIR2.1‐/‐ mice. The extensor digitorum longus muscle positioned overtop a gas control chamber allowed second‐by‐second monitoring of capillary RBC flow responses as O2 was altered around its physiological set point of 53 mmHg. A stepwise drop in PO2 at the muscle surface (53 to 15 or 0 mmHg) increased RBC supply rate in control capillaries while elevated chamber O2 elicited the opposite response; these capillaries robustly expressed Cx40. The RBC flow responses were rapid and tightly coupled to O2 levels as readily observed when chamber O2 was oscillated in a sinusoidal manner. In contrast, this blood flow response was significantly diminished in Cx40‐/‐ mice and translated into lower capillary RBC O2 saturation. KIR2.1‐/‐mice, on the other hand, had normal resting RBC O2 saturation and reacted normally to O2 changes, albeit an oscillation or a sustained stepwise decrease. Furthermore, we confirmed that RBC flow responses are conserved in endothelial KIR2.1‐/‐mice even when the low O2 challenge is applied to a restricted number of surface capillaries in the muscle; interestingly, these responses were dominated by capillary hematocrit changes in both control and endothelial KIR2.1‐/‐mice. Modelling this phenomenon supported the idea that the number of capillaries stimulated is indeed tied to conduction distance and tissue RBC distribution. In conclusion, we demonstrate that microvascular O2 responses depend on coordinated electrical signaling via gap junctions comprised of Cx40 and that endothelial KIR2.1channels do not drive the initiating electrical event. These findings reconceptualize our understanding of blood flow regulation and how O2 initiates this process at the capillary level independent of metabolite production.

Quantifying Longitudinal Network Geometry Alterations in Rat Skeletal Microvasculature Subject to Ischemia

Study objectiveIntra‐vital video microscopy (IVVM) data of rat subcutaneous skeletal muscle microvasculature under ischemic conditions at two time points (0 hours, 3 hours) was obtained from our collaborator. The objectives of this study are to elicit network geometries from the IVVM data and quantify the alterations in the network statistics of the geometries due to ischemia.HypothesisWe hypothesize that ischemia causes blood vessel de‐recruitment.MethodsIVVM data from six experiments was obtained, where each experiment provided a video at the start (0 hours) and end (3 hours) of the experiment. Using consecutive still images from the videos, vessel centerlines were manually drawn using Procreate and the NIH software, ImageJ. The included vessels were chosen based on two features: (1) visible red blood cell flow and (2) clearly defined edges. The centerlines were used to guide thresholding by identifying appropriate vessels. This was followed by the generation of binarized representations of the microvascular networks. The network statistics were quantified by measuring vessel segment lengths and diameters, counting the number of bifurcations, and calculating vessel density in ImageJ.ResultsAt 3 hours, the average vessel length changed from 283.91 + 252.14 pixels to 316.75 + 292.37 pixels, the average diameter changed from 16.02 + 7.99 pixels to 15.42 + 7.50 pixels, the average number of bifurcations changed from 32.42 to 18.92 and the average vessel density changed from 0.0085 + 0.0017 to 0.0061 + 0.0024.ConclusionThere is a clear decrease in average diameter, vessel density and the number of bifurcations characterizing the microvascular networks. However, an increase in the average vessel length accompanies these findings. The data indicates that ischemia may cause de‐recruitment of blood vessels.

Erysense, a Lab-on-a-Chip-Based Point-of-Care Device to Evaluate Red Blood Cell Flow Properties With Multiple Clinical Applications.

In many medical disciplines, red blood cells are discovered to be biomarkers since they “experience” various conditions in basically all organs of the body. Classical examples are diabetes and hypercholesterolemia. However, recently the red blood cell distribution width (RDW), is often referred to, as an unspecific parameter/marker (e.g., for cardiac events or in oncological studies). The measurement of RDW requires venous blood samples to perform the complete blood cell count (CBC). Here, we introduce Erysense, a lab-on-a-chip-based point-of-care device, to evaluate red blood cell flow properties. The capillary chip technology in combination with algorithms based on artificial neural networks allows the detection of very subtle changes in the red blood cell morphology. This flow-based method closely resembles in vivo conditions and blood sample volumes in the sub-microliter range are sufficient. We provide clinical examples for potential applications of Erysense as a diagnostic tool [here: neuroacanthocytosis syndromes (NAS)] and as cellular quality control for red blood cells [here: hemodiafiltration (HDF) and erythrocyte concentrate (EC) storage]. Due to the wide range of the applicable flow velocities (0.1–10 mm/s) different mechanical properties of the red blood cells can be addressed with Erysense providing the opportunity for differential diagnosis/judgments. Due to these versatile properties, we anticipate the value of Erysense for further diagnostic, prognostic, and theragnostic applications including but not limited to diabetes, iron deficiency, COVID-19, rheumatism, various red blood cell disorders and anemia, as well as inflammation-based diseases including sepsis.