Cell Velocity Research Articles

Implementation of actin polymerization and depolymerization in a two-dimensional cell migration model and its implications on mammalian cell morphology and velocity

Cell migration, a pivotal process in wound healing, immune response, and even cancer metastasis, manifests through intricate interplay between morphology, speed, and cytoskeletal dynamics. Mathematical modeling emerges as a powerful tool to dissect these complex interactions. This work presents a two-dimensional immersed boundary model for mammalian cell migration, incorporating both filamentous actin (F-actin) and monomeric actin (G-actin) to explicitly capture polymerization and depolymerization. This model builds upon our previous one-dimensional efforts, now enabling us to explore the impact of G-actin on not just cell velocity but also morphology. We compare predictions from both models, revealing that while the one-dimensional model captures core dynamics along the cell’s axis, the two-dimensional model excels in portraying cell shape evolution and transverse variations in actin concentration and velocity. Our findings highlight the crucial role of including G-actin in shaping cell morphology. Actin velocity aligned with migration direction elongates the cell, while velocity normal to the membrane promotes spreading. Importantly, the model establishes a link between these microscopic aspects and macroscopic observables like cell shape, offering a deeper understanding of cell migration dynamics. This work not only provides a more comprehensive picture of cell migration but also paves the way for future studies exploring the interplay of actin dynamics, cell morphology, and biophysical parameters in diverse biological contexts.

Tire Rubber Based Microplastic Particles Cause Adverse on Quality Parameters of Rainbow Trout Sperm Cells.

In the present study, we aimed to determine the parameters of oxidative stress markers, motility and kinematics of rainbow trout (Oncorhynchus mykiss) sperm cells exposed to different doses (0.001, 0.01, 0.1, 1.0, and 10mg/L, in vitro 4h) of tire rubber based microplastic particles (TRMP-Ps) the leachates procedure of rubber pieces. First of all, TRMP-Ps were prepared by abrasion method in accordance with the literature. Structural and morphological features of TRMP-Ps were determined by Fourier transform infrared (FTIR) spectroscopy and scanning electron microscopy (SEM) methods, respectively. Energy dispersive X-ray (EDX) analysis technique was used to characterize the elemental composition of TRMP-Ps. Particle size of microplastic structures was measured hydrodynamically with dynamic light scattering analysis (DLS). After exposure, the effect of TRMP-Ps was defined by the observations of kinematics and antioxidant activities in sperm cells. Our findings showed that the straight line velocity, the curvilinear velocity, the angular path velocity, and the amplitude of lateral displacement of sperm cells decreased. Moreover, while the level of superoxide dismutase decreased dose-dependently against the toxicity of TRMP-Ps, no significant change was observed in the levels of malondialdehyde and total glutathione. The 4-h median effective concentrations (EC50) of TRMP-Ps based on mobility parameters of sperm ranged from 0.31mg/L for reduced straight line velocity of sperm cells to 0.51mg/L for reduced amplitude of lateral displacement of the spermatozoa head. Therefore, we concluded that TRMP-Ps can be a risk for the reproduction cycle of fish in aquatic environments.

In Vivo Visualization of Intravascular Patrolling Immune Cells in the Primate Eye.

Insight into the immune status of the living eye is essential as we seek to understand ocular disease and develop new treatments. The nonhuman primate (NHP) is the gold standard preclinical model for therapeutic development in ophthalmology, owing to the similar visual system and immune landscape in the NHP relative to the human. Here, we demonstrate the utility of phase-contrast adaptive optics scanning light ophthalmoscope (AOSLO) to visualize immune cell dynamics on the cellular scale, label-free in the NHP. Phase-contrast AOSLO was used to image preselected areas of retinal vasculature in five NHP eyes. Images were registered to correct for eye motion, temporally averaged, and analyzed for immune cell activity. Cell counts, dimensions, velocities, and frequency per vessel were determined manually and compared between retinal arterioles and venules. Based on cell appearance and circularity index, cells were divided into three morphologies: ovoid, semicircular, and flattened. Immune cells were observed migrating along vascular endothelium with and against blood flow. Cell velocity did not significantly differ between morphology or vessel type and was independent of blow flood. Venules had a significantly higher cell frequency than arterioles. A higher proportion of cells resembled "flattened" morphology in arterioles. Based on cell speeds, morphologies, and behaviors, we identified these cells as nonclassical patrolling monocytes (NCPMs). Phase-contrast AOSLO has the potential to reveal the once hidden behaviors of single immune cells in retinal circulation and can do so without the requirement of added contrast agents that may disrupt immune cell behavior.

Drug resistant pancreatic cancer cells exhibit altered biophysical interactions with stromal fibroblasts in imaging studies of 3D co-culture models

Interactions between tumor and stromal cells are well known to play prominent roles in progression of pancreatic ductal adenocarcinoma (PDAC). As knowledge of stromal crosstalk in PDAC has evolved, it has become clear that cancer associated fibroblasts can play both tumor promoting and tumor suppressive roles through a combination of paracrine crosstalk and juxtacrine interactions involving direct physical contact. Another major contributor to dismal survival statistics for PDAC is development of resistance to chemotherapy drugs, though less is known about how the acquisition of chemoresistance impacts upon tumor-stromal crosstalk. Here, we use time lapse imaging and image analysis to study how co-culture geometry impacts interactions between epithelial and stromal cells. We show that extracellular matrix (ECM) overlay cultures in which stromal cells (pancreatic stellate cells, or normal human fibroblasts) are placed adjacent to PDAC cells (PANC1) result in direct heterotypic cell adhesions accompanied by dramatic fibroblast contractility. We analyze these interactions in co-cultures using particle image velocimetry (PIV) analysis to quantify cell velocities over the course of time lapse movie sequences. We further contrast co-cultures of PANC1 with those containing a drug resistant subline (PANC1-OR) previously established in our lab and find that heterotypic cell–cell interactions are suppressed in the latter relative to the parental line. We use RNA-seq and bioinformatics analysis to identify differential gene expression in PANC1 and PANC1-OR, which shows that negative regulation of cell adhesion molecules, consistent with increased epithelial mesenchymal transition (EMT), is also correlated with reduction in the hetrotypic cell–cell contact necessary for the contractile behavior observed in drug naïve cultures. Overall these findings elucidate the role of drug-resistance in inhibiting an avenue of stromal crosstalk which is associated with tumor suppression and also help to establish cell culture conditions useful for further mechanistic investigation.

Nitroglycerin challenge identifies microcirculatory target for improved resuscitation in patients with circulatory shock

BackgroundCirculatory shock and multi-organ failure remain major contributors to morbidity and mortality in critically ill patients and are associated with insufficient oxygen availability in the tissue. Intrinsic mechanisms to improve tissue perfusion, such as up-regulation of functional capillary density (FCD) and red blood cell velocity (RBCv), have been identified as maneuvers to improve oxygen extraction by the tissues; however, their role in circulatory shock and potential use as resuscitation targets remains unknown. To fill this gap, we examined the baseline and maximum recruitable FCD and RBCv in response to a topical nitroglycerin stimulus (FCDNG, RBCvNG) in patients with and without circulatory shock to test whether this may be a method to identify the presence and magnitude of a microcirculatory reserve capacity important for identifying a resuscitation target.MethodsSublingual handheld vital microscopy was performed after initial resuscitation in mechanically ventilated patients consecutively admitted to a tertiary medical ICU. FCD and RBCv were quantified using an automated computer vision algorithm (MicroTools). Patients with circulatory shock were retrospectively identified via standardized hemodynamic and clinical criteria and compared to patients without circulatory shock.Results54 patients (57 ± 14y, BMI 26.3 ± 4.9 kg/m2, SAPS 56 ± 19, 65% male) were included, 13 of whom presented with circulatory shock. Both groups had similar cardiac index, mean arterial pressure, RBCv, and RBCvNG. Heart rate (p < 0.001), central venous pressure (p = 0.02), lactate (p < 0.001), capillary refill time (p < 0.01), and Mottling score (p < 0.001) were higher in circulatory shock after initial resuscitation, while FCD and FCDNG were 10% lower (16.9 ± 4.2 and 18.9 ± 3.2, p < 0.01; 19.3 ± 3.1 and 21.3 ± 2.9, p = 0.03). Nitroglycerin response was similar in both groups, and circulatory shock patients reached FCDNG similar to baseline FCD found in patients without shock.ConclusionCritically ill patients suffering from circulatory shock were found to present with a lower sublingual FCD. The preserved nitroglycerin response suggests a dysfunction of intrinsic regulation mechanisms to increase the microcirculatory oxygen extraction capacity associated with circulatory shock and identifies a potential resuscitation target. These differences in microcirculatory hemodynamic function between patients with and without circulatory shock were not reflected in blood pressure or cardiac index.

Dynamic cellular responses to gravitational forces: Exploring the impact on white blood cell(s).

In recent years, the allure of space exploration and human spaceflight has surged, yet the effects of microgravity on the human body remain a significant concern. Immune and red blood cells rely on hematic or lymphatic streams as their primary means of transportation, posing notable challenges under microgravity conditions. This study sheds light on the intricate dynamics of cell behavior when suspended in bio-fluid under varying gravitational forces. Utilizing the dissipative particle dynamics approach, blood and white blood cells were modeled, with gravity applied as an external force along the vertical axis, ranging from 0 to 2 g in parameter sweeps. The results revealed discernible alterations in the cell shape and spatial alignment in response to gravity, quantified through metrics such as elongation and deformation indices, pitch angle, and normalized center of mass. Statistical analysis using the Mann-Whitney U test underscored clear distinctions between microgravity (<1 g) and hypergravity (>1 g) samples compared to normal gravity (1 g). Furthermore, the examination of forces exerted on the solid, including drag, shear stress, and solid forces, unveiled a reduction in the magnitude as the gravitational force increased. Additional analysis through dimensionless numbers unveiled the dominance of capillary and gravitational forces, which impacted cell velocity, leading to closer proximity to the wall and heightened viscous interaction with surrounding fluid particles. These interactions prompted shape alterations and reduced white blood cell area while increasing red blood cells. This study represents an effort in comprehending the effects of gravity on blood cells, offering insights into the intricate interplay between cellular dynamics and gravitational forces.

Relaxation dynamics in the self-propelled Voronoi model for epithelial monolayers

Cell monolayers and epithelial tissues display slow relaxation dynamics during the reversible transition between mesenchymal and epithelial cells, a phenomenon directly relevant to embryogenesis, tumor metastases, and wound healing. In active cells, persistent motion induces collective dynamics that significantly influence relaxation behavior. To better understand the role of persistence in cell relaxations, we perform extensive simulations and employ cage-relative measures to address the previously overlooked system size effects in model active cells. We identify the glass transition at various persistence values, demonstrating conventional near-equilibrium supercooled dynamics at low persistence. However, highly persistent cells exhibit distinctive intermittent dynamics associated with intermittent local T1 transitions, where cell velocity correlates over space with a characteristic length ξ. More significantly, we formulate a universal relationship predicting the global relaxation time based on the T1 transition and the spatial velocity correlation across a wide range of persistence values. Specifically, the relaxation time demonstrates a power-law dependence on the irreversible T1 transition rate, multiplied by exp(ξ). Here, ξ vanishes at small persistence in nearly equilibrated cells, and the irreversible T1 transition rate diminishes towards the mode-coupling glass transition point. Published by the American Physical Society 2024

Drug resistant pancreatic cancer cells exhibit altered biophysical interactions with stromal fibroblasts in imaging studies of 3D co-culture models.

Interactions between tumor and stromal cells are well known to play a prominent roles in progression of pancreatic ductal adenocarcinoma (PDAC). As knowledge of stromal crosstalk in PDAC has evolved, it has become clear that cancer associated fibroblasts can play both tumor promoting and tumor suppressive roles through a combination of paracrine crosstalk and juxtacrine interactions involving direct physical contact. Another major contributor to dismal survival statistics for PDAC is development of resistance to chemotherapy drugs. Though less is known about how the acquisition of chemoresistance impacts upon tumor-stromal crosstalk. Here, we use 3D co-culture geometries to recapitulate juxtacrine interactions between epithelial and stromal cells. In particular, extracellular matrix (ECM) overlay cultures in which stromal cells (pancreatic stellate cells, or normal human fibroblasts) are placed adjacent to PDAC cells (PANC1), result in direct heterotypic cell adhesions accompanied by dramatic fibroblast contractility which leads to highly condensed macroscopic multicellular aggregates as detected using particle image velocimetry (PIV) analysis to quantify cell velocities over the course of time lapse movie sequences. To investigate how drug resistance impacts these juxtacrine interactions we contrast cultures in which PANC1 are substituted with a drug resistant subline (PANC1-OR) previously established in our lab. We find that heterotypic cell-cell interactions are highly suppressed in drug-resistant cells relative to the parental PANC1 cells. To investigate further we conduct RNA-seq and bioinformatics analysis to identify differential gene expression in PANC1 and PANC1-OR, which shows that negative regulation of cell adhesion molecules, consistent with increased epithelial mesenchymal transition (EMT), is also consistent with loss of hetrotypic cell-cell contact necessary for the contractile behavior observed in drug naïve cultures. Overall these findings elucidate the role of drug-resistance in inhibiting an avenue of stromal crosstalk which is associated with tumor suppression and also help to establish cell culture conditions useful for further mechanistic investigation.

RBM10 loss induces aberrant splicing of cytoskeletal and extracellular matrix mRNAs and promotes metastatic fitness.

RBM10 modulates transcriptome-wide cassette exon splicing. Loss-of-function RBM10 mutations are enriched in thyroid cancers with distant metastases. Analysis of transcriptomes and genes mis-spliced by RBM10 loss showed pro-migratory and RHO/RAC signaling signatures. RBM10 loss increases cell velocity. Cytoskeletal and ECM transcripts subject to exon-inclusion events included vinculin (VCL), tenascin C (TNC) and CD44. Knockdown of the VCL exon inclusion transcript in RBM10-null cells reduced cell velocity, whereas knockdown of TNC and CD44 exon-inclusion isoforms reduced invasiveness. RAC1-GTP levels were increased in RBM10-null cells. Mouse Hras G12V /Rbm1O KO thyrocytes develop metastases that are reversed by RBM10 or by combined knockdown of VCL, CD44 and TNC inclusion isoforms. Thus, RBM10 loss generates exon inclusions in transcripts regulating ECM-cytoskeletal interactions, leading to RAC1 activation and metastatic competency. Moreover, a CRISPR-Cas9 screen for synthetic lethality with RBM10 loss identified NFkB effectors as central to viability, providing a therapeutic target for these lethal thyroid cancers.

Hemodynamics regulate spatiotemporal artery muscularization in the developing circle of Willis.

Vascular smooth muscle cells (VSMCs) envelop vertebrate brain arteries and play a crucial role in regulating cerebral blood flow and neurovascular coupling. The dedifferentiation of VSMCs is implicated in cerebrovascular disease and neurodegeneration. Despite its importance, the process of VSMC differentiation on brain arteries during development remains inadequately characterized. Understanding this process could aid in reprogramming and regenerating dedifferentiated VSMCs in cerebrovascular diseases. In this study, we investigated VSMC differentiation on zebrafish circle of Willis (CoW), comprising major arteries that supply blood to the vertebrate brain. We observed that arterial specification of CoW endothelial cells (ECs) occurs after their migration from cranial venous plexus to form CoW arteries. Subsequently, acta2+ VSMCs differentiate from pdgfrb+ mural cell progenitors after they were recruited to CoW arteries. The progression of VSMC differentiation exhibits a spatiotemporal pattern, advancing from anterior to posterior CoW arteries. Analysis of blood flow suggests that earlier VSMC differentiation in anterior CoW arteries correlates with higher red blood cell velocity and wall shear stress. Furthermore, pulsatile flow induces differentiation of human brain PDGFRB+ mural cells into VSMCs, and blood flow is required for VSMC differentiation on zebrafish CoW arteries. Consistently, flow-responsive transcription factor klf2a is activated in ECs of CoW arteries prior to VSMC differentiation, and klf2a knockdown delays VSMC differentiation on anterior CoW arteries. In summary, our findings highlight blood flow activation of endothelial klf2a as a mechanism regulating initial VSMC differentiation on vertebrate brain arteries.

Hemodynamics regulate spatiotemporal artery muscularization in the developing circle of Willis

Vascular smooth muscle cells (VSMCs) envelop vertebrate brain arteries and play a crucial role in regulating cerebral blood flow and neurovascular coupling. The dedifferentiation of VSMCs is implicated in cerebrovascular disease and neurodegeneration. Despite its importance, the process of VSMC differentiation on brain arteries during development remains inadequately characterized. Understanding this process could aid in reprogramming and regenerating dedifferentiated VSMCs in cerebrovascular diseases. In this study, we investigated VSMC differentiation on zebrafish circle of Willis (CoW), comprising major arteries that supply blood to the vertebrate brain. We observed that arterial specification of CoW endothelial cells (ECs) occurs after their migration from cranial venous plexus to form CoW arteries. Subsequently, acta2+ VSMCs differentiate from pdgfrb+ mural cell progenitors after they were recruited to CoW arteries. The progression of VSMC differentiation exhibits a spatiotemporal pattern, advancing from anterior to posterior CoW arteries. Analysis of blood flow suggests that earlier VSMC differentiation in anterior CoW arteries correlates with higher red blood cell velocity and wall shear stress. Furthermore, pulsatile flow induces differentiation of human brain PDGFRB+ mural cells into VSMCs, and blood flow is required for VSMC differentiation on zebrafish CoW arteries. Consistently, flow-responsive transcription factor klf2a is activated in ECs of CoW arteries prior to VSMC differentiation, and klf2a knockdown delays VSMC differentiation on anterior CoW arteries. In summary, our findings highlight blood flow activation of endothelial klf2a as a mechanism regulating initial VSMC differentiation on vertebrate brain arteries.

Integrated Single Camera µPTV And Florescence Imaging For Cell Tracking And Flow Investigation In Centrifugal Microfluidic Devices

A micro-hydrocyclone is investigated as a high throughput particle/cell sorting microfluidic device. A complex flow structure has been reported by limited numerical works that increases the importance of undertaking a comprehensive experimental investigation. In addition to the flow, utilizing damageable bio cells in a micro-hydrocyclone, requires a deep understanding of the interaction between the cells entering the device, the flow structure and its instabilities forming in different operational phases. In such cases the implementation of multiple measurement techniques can be challenging due to the small scale of devices, here &lt; 5 mm diameter. Therefore, for this complex flow an experimental approach is introduced to capture the flow structure and the motion of cells. In this method, tracer particles and fluorescent stained cells are captured simultaneous by a single camera in the same flow field. A custom image processing scheme is used for partitioning cells and particles from the raw data. To determine velocity vectors, µPTV is employed on the segmented data sets to study the effect of flow on trajectory and velocity of each individual cell in the system. By a combined investigation in a vast range of Reynolds number (50&lt;Re&lt;800) the dynamics of cells are investigated in response to different flow structures and flow regimes.

Computational modeling of the physical features that influence breast cancer invasion into adipose tissue.

Breast cancer invasion into adipose tissue strongly influences disease progression and metastasis. The degree of cancer cell invasion into adipose tissue depends on both biochemical signaling and the mechanical properties of cancer cells, adipocytes, and other key components of adipose tissue. We model breast cancer invasion into adipose tissue using discrete element method simulations of active, cohesive spherical particles (cancer cells) invading into confluent packings of deformable polyhedra (adipocytes). We quantify the degree of invasion by calculating the interfacial area At between cancer cells and adipocytes. We determine the long-time value of At vs the activity and strength of the cohesion between cancer cells, as well as the mechanical properties of the adipocytes and extracellular matrix in which adipocytes are embedded. We show that the degree of invasion collapses onto a master curve as a function of the dimensionless energy scale Ec , which grows linearly with the cancer cell velocity persistence time and fluctuations, is inversely proportional to the system pressure, and is offset by the cancer cell cohesive energy. When , cancer cells will invade the adipose tissue, whereas for , cancer cells and adipocytes remain de-mixed. We also show that At decreases when the adipocytes are constrained by the ECM by an amount that depends on the spatial heterogeneity of the adipose tissue.

Cardiac stereotactic body radiotherapy to treat malignant ventricular arrhythmias directly affects the cardiomyocyte electrophysiology

BackgroundPromising as a treatment option for life-threatening ventricular arrhythmias, cardiac stereotactic body radiotherapy (cSBRT) has demonstrated early antiarrhythmic effects within days of treatment. The mechanisms underlying the immediate and short-term antiarrhythmic effects are poorly understood. ObjectiveWe hypothesize that cSBRT has a direct antiarrhythmic effect on cellular electrophysiology through reprogramming of ion channel and gap junction protein expression. MethodsAfter exposure to 20 Gy of x-rays in a single fraction, neonatal rat ventricular cardiomyocytes were analyzed 24 and 96 hours postradiation to determine changes in conduction velocity, beating frequency, calcium transients, and action potential duration in both monolayers and single cells. In addition, the expression of gap junction proteins, ion channels, and calcium handling proteins was evaluated at protein and messenger RNA levels. ResultsAfter irradiation with 20 Gy, neonatal rat ventricular cardiomyocytes exhibited increased beat rate and conduction velocity 24 and 96 hours after treatment. Messenger RNA and protein levels of ion channels were altered, with the most significant changes observed at the 96-hour mark. Upregulation of Cacna1c (Cav1.2), Kcnd3 (Kv4.3), Kcnh2 (Kv11.1), Kcnq1 (Kv7.1), Kcnk2 (K2P2.1), Kcnj2 (Kir2.1), and Gja1 (Cx43) was noted, along with improved gap junctional coupling. Calcium handling was affected, with increased Ryr2 ryanodin-rezeptor 2 and Slc8a1 Na+/Ca2+ exchanger expression and altered properties 96 hours posttreatment. Fibroblast and myofibroblast levels remained unchanged. ConclusioncSBRT modulates the expression of various ion channels, calcium handling proteins, and gap junction proteins. The described alterations in cellular electrophysiology may be the underlying cause of the immediate antiarrhythmic effects observed after cSBRT.

Checkpoint inhibition enhances cell contacts between CD4+ T cells and Hodgkin-Reed-Sternberg cells of classic Hodgkin lymphoma.

Although checkpoint molecules like CTLA-4 and PD1 have been described several years ago, checkpoint inhibitors such as nivolumab (an anti-PD-1 antibody) have only recently been used to treat classic Hodgkin lymphoma (cHL). Several studies have shown convincing therapeutic effects of nivolumab in cHL. However, the mechanism of action of nivolumab in cHL is not fully understood. The aim of this study was to monitor changes in cell motility and cell contacts after administration of nivolumab to an in vitro model of cHL as well as to native hyperplastic lymphoid tissue and native human tissue from cHL. In both tissue and in vitro, CD4+, CD8+, CD30+ and CD20+ cell velocities were unchanged after nivolumab incubation. In contrast, in primary cHL tissue, the duration of cell contacts between CD4+ T cells and Hodgkin-Reed-Sternberg cells was significantly increased after 5 hours of nivolumab treatment, and the number of contacts with HRS cells was also slightly increased for CD4+ T cells (not significant), suggesting that CD4+ T cells in particular contribute to the cytotoxicity observed as a result of nivolumab therapy. There was no change in the duration of cell contacts in the hyperplastic lymphoid tissue after nivolumab incubation. In conclusion, we show here for the first time by imaging of native lymphoma tissue an enhanced interaction of CD4+ T cells and Hodgkin-Reed-Sternberg cells in cHL after nivolumab administration.

An-Gong-Niu-Huang-Wan (AGNHW) regulates cerebral blood flow by improving hypoperfusion, cerebrovascular reactivity and microcirculation disturbances after stroke

BackgroundThe restoration of cerebrovascular regulation and improvement of cerebral blood flow in ischaemic regions are crucial for improving the clinical prognosis after stroke. An-Gong-Niu-Huang-Wan (AGNHW) is a famous traditional compound Chinese medicine that has been used for over 220 years to treat acute ischaemic stroke; however, its role in the regulation of cerebral blood flow is still unclear. The aim of the present study was to investigate the regulatory effect of AGNHW on cerebral blood flow and microcirculation after ischaemic stroke and to elucidate the underlying mechanisms involved.MethodsMale C57BL/6 mice were subjected to distal middle cerebral artery occlusion (dMCAO) and randomly assigned to the sham, MCAO, or AGNHW groups. AGNHW was administered intragastrically 1 h after dMCAO. The rotarod test was utilized to evaluate behavioural function; TTC was used to determine the infarct volume; and ischaemic injury was assessed by detecting brain levels of SOD, MDA and NO. Then, cortical perfusion and acetazolamide-induced cerebrovascular reactivity were assessed using laser speckle contrast imaging, and the velocity and flux of red blood cells in cortical capillaries were detected using two-photon laser scanning microscopy. In addition, we employed RNA-Seq to identify variations in gene expression profiles and assessed endothelium-dependent changes in microcirculatory dysfunction by measuring vasoactive mediator levels.ResultsAGNHW significantly increased cerebral blood flow, reduced the infarct volume, and promoted functional recovery after cerebral ischaemia. AGNHW increased the velocity and flux of red blood cells in capillaries and improved cerebrovascular reactivity in the ischaemic cortex. Furthermore, AGNHW regulated endothelium-dependent microcirculation, as evidenced by decreases in the expression of endothelins (Edn1, Edn3 and Ednrb) and the ratios of brain and serum TXB2/6-keto-PGF1α and ET-1/CGRP.ConclusionsAGNHW improved cerebral hypoperfusion, regulated cerebrovascular reactivity and attenuated microcirculatory dysfunction within the ischaemic cortex after stroke. This outstanding effect was achieved by modulating the expression of genes related to vascular endothelial cell function and regulating endothelium-dependent vasoactive mediators.

Disturbed fluid flow reinforces endothelial tractions and intercellular stresses

Disturbed fluid flow is well understood to have significant ramifications on endothelial function, but the impact disturbed flow has on endothelial biomechanics is not well understood. In this study, we measured tractions, intercellular stresses, and cell velocity of endothelial cells exposed to disturbed flow using a custom-fabricated flow chamber. Our flow chamber exposed cells to disturbed fluid flow within the following spatial zones: zone 1 (inlet; length 0.676–2.027 cm): 0.0037 ± 0.0001 Pa; zone 2 (middle; length 2.027–3.716 cm): 0.0059 ± 0.0005 Pa; and zone 3 (outlet; length 3.716–5.405 cm): 0.0051 ± 0.0025 Pa. Tractions and intercellular stresses were observed to be highest in the middle of the chamber (zone 2) and lowest at the chamber outlet (zone 3), while cell velocity was highest near the chamber inlet (zone 1), and lowest near the middle of the chamber (zone 2). Our findings suggest endothelial biomechanical response to disturbed fluid flow to be dependent on not only shear stress magnitude, but the spatial shear stress gradient as well. We believe our results will be useful to a host of fields including endothelial cell biology, the cardiovascular field, and cellular biomechanics in general.

Carbon Dioxide Concentration Alters the Dynamics of Oxygen-Mediated Capillary Blood Flow Response s in Skeletal Muscle

Objective: We sought to quantify how different muscle carbon dioxide concentrations ([CO2]) alter the dynamics of oxygen-mediated blood flow responses in skeletal muscle capillaries. Methods: 6 male Sprague Dawley rats (164-215g) were anaesthetized, instrumented for systemic monitoring, and mechanically ventilated. The extensor digitorum longus muscle was isolated via blunt dissection, externalized, and reflected over a microfluidic gas exchange chamber mounted in the stage of an inverted microscope. Intravital video of capillary blood flow was recorded during 4-minute O2 challenges consisting of a 1-minute baseline at 7% oxygen concentration ([O2]), followed by 3 minutes at 2% [O2]. Before recordings, the muscle was equilibrated to constant background [CO2] of either 2%, 5%, or 8% for 5 minutes. Recordings were analyzed offline using custom MATLAB software. The time transients () of capillary hemodynamic responses were determined using a least-squared regression fit (LSRF) to single- and double-exponential models. All animal protocols were approved by Memorial University’s Animal Care Committee. Results: Baseline red blood cell (RBC) velocity was significantly different at 5% [CO2] (213.2±179.4 μm/s) compared to 2% [CO2] (177.7±162.6 μm/s, p=0.0016), and 8% [CO2] (250.3±193.6 μm/s, p&lt;0.0001). RBC oxygen saturations (SO2) were significantly different beyond 3 s post-baseline in all [CO2] conditions ( p&lt;0.02, =3.2-4.3s). Minimum RBC SO2 was 52%, 46%, and 38% below baseline at 12s, 14s, and 12s for 2%, 5%, and 8% [CO2] conditions, respectively. RBC velocity was significantly increased during 2% [CO2] by 3 s post-baseline (177.7±162.6 vs 188.7±168.6 μm/s, p&lt;0.0001), during 5% [CO2] by 6 s post-baseline (213.2±179.4 vs 224.2±189.9 μm/s, p=0.0190), and during 8% [CO2] by 12 s post-baseline (250.3±193.6 vs 267.0±199.9 μm/s, p=0.0062). Time transients of the fast component of the oxygen-mediated velocity response, as determined by LSRF, were 1.9s for 2% [CO2], 4.8s for 5% [CO2], and 9.8s for 8% [CO2]. Velocity was increased at the end of the O2 challenge by 39%, 33%, and 36% during 2%, 5%, and 8% [CO2] conditions, respectively. Baseline RBC supply rate (SR) was significantly different at 5% [CO2] (13.5±18.9 cells/s) compared to 2% [CO2] (9.5±15.3 cells/s, p=0.0090), and 8% [CO2] (19.0±23.6 cells/s, p&lt;0.0001). At 2% [CO2], SR increased significantly by 9 s post-baseline (9.5±15.2 vs 10.9±16.9 cells/s, p=0.0001), by 10 s post-baseline for 5% [CO2] (13.5±18.9 vs 14.9±20.6 cells/s, p=0.0099), and by 13 s post-baseline during 8% [CO2] (19.0±23.6 vs 21.1±25.2 cells/s, p=0.0204). The fast component of the oxygen-mediated SR response had a of 2.7s for 2% [CO2], 4.5s for 5% [CO2], and 6.9s for 8% [CO2] as determined by LSRF. Overall SR was increased by 74%, 51%, and 39% at the end of the O2 challenge during 2%, 5%, and 8% [CO2], respectively. As well, capillary hematocrits were significantly different during baseline at 5% [CO2] (21.4±16.1%) compared to 2% [CO2] (18.2±22.2%, p=0.0113), and 8% [CO2] (27.2±21.4%, p&lt;0.0001). Conclusion: We have quantified the fast component of oxygen-mediated capillary hemodynamic responses to an acute decrease in skeletal muscle [O2]. The time transients of O2-mediated responses vary with tissue [CO2] such that increasing CO2 results in slower response dynamics. As well, increasing background [CO2] provokes higher capillary RBC velocity, SR, and hematocrit both at baseline and during low O2 challenges as expected. This project was supported by a Natural Sciences and Engineering Research Council of Canada Discovery Grant awarded to GMF. Student support was provided by Memorial University's School of Graduate Studies and Faculty of Medicine. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Numerical simulation of cellular blood flow in curved micro-vessels with saccular aneurysms: Effect of curvature degree and hematocrit level.

The dynamics of cellular blood flow in curved vessels considerably differ from those in straight vessels. It is reported that clotting development is significantly affected by vessel shape irregularities. Thus, the current study aims to investigate the effect of curvature degree and hematocrit level on cellular blood flow in a curved micro-vessel with a saccular aneurysm. Accordingly, a three-dimensional numerical simulation is performed using a validated code developed for cellular blood flow problems. The obtained results show that the cell-free layer thickness is highly dependent on the curvature degree and hematocrit level, which may have a remarkable impact on the apparent viscosity of blood as well as the dynamics of other particles such as drug particulates. The near-wall region exhibits the highest degree of cell deformation, whereas the red blood cells within the aneurysm zone remain nearly undeformed. Meanwhile, the velocity of the red blood cells decreases with the increase in curvature degree, which can affect the quality of the oxygenation process. Because of the saccular aneurysm, a considerable decrease in plasma velocity is predicted. Moreover, no secondary flows are detected in the curved vessel except in the aneurysm zone. An increase in the curvature degree is expected to reduce the blood flow rate by about 10%. Furthermore, low wall shear stress values are predicted in the straight case compared to the values at the apex of the curved vessel, which may affect the structure and function of the endothelial cells of the vessel wall and, hence, increase the aneurysm rupture possibility.

SMORES: a simple microfluidic operating room for the examination and surgery of Stentor coeruleus

Ciliates are powerful unicellular model organisms that have been used to elucidate fundamental biological processes. However, the high motility of ciliates presents a major challenge in studies using live-cell microscopy and microsurgery. While various immobilization methods have been developed, they are physiologically disruptive to the cell and incompatible with microscopy and/or microsurgery. Here, we describe a Simple Microfluidic Operating Room for the Examination and Surgery of Stentor coeruleus (SMORES). SMORES uses Quake valve-based microfluidics to trap, compress, and perform surgery on Stentor as our model ciliate. Compared with previous methods, immobilization by physical compression in SMORES is more effective and uniform. The mean velocity of compressed cells is 24 times less than that of uncompressed cells. The compression is minimally disruptive to the cell and is easily applied or removed using a 3D-printed pressure rig. We demonstrate cell immobilization for up to 2 h without sacrificing cell viability. SMORES is compatible with confocal microscopy and is capable of media exchange for pharmacokinetic studies. Finally, the modular design of SMORES allows laser ablation or mechanical dissection of a cell into many cell fragments at once. These capabilities are expected to enable biological studies previously impossible in ciliates and other motile species.