Red Blood Cell Velocity Research Articles

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.

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.

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.

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.

Role of dietary interventions on microvascular health in South-Asian Surinamese people with type 2 diabetes in the Netherlands: A randomized controlled trial

Background/objectivesWe investigated whether dietary interventions, i.e. a fasting mimicking diet (FMD, Prolon®) or glycocalyx mimetic supplementation (EndocalyxTM) could stabilize microvascular function in Surinamese South-Asian patients with type 2 diabetes (SA-T2DM) in the Netherlands, a patient population more prone to develop vascular complications.Subjects/methodsA randomized, placebo controlled, 3-arm intervention study was conducted in 56 SA-T2DM patients between 18 and 75 years old, for 3 consecutive months, with one additional follow up measurement 3 months after the last intervention. Sublingual microcirculation was assessed with SDF-imaging coupled to the GlycoCheckTM software, detecting red blood cell velocity, capillary density, static and dynamic perfused boundary region (PBR), and the overall microvascular health score (MVHS). Linear mixed models and interaction analysis were used to investigate the effects the interventions had on microvascular function.ResultsDespite a temporal improvement in BMI and HbA1c after FMD the major treatment effect on microvascular health was worsening for RBC-velocity independent PBRdynamic, especially at follow-up. Glycocalyx supplementation, however, reduced urinary MCP-1 presence and improved both PBRdynamic and MVHSdynamic, which persisted at follow-up.ConclusionsWe showed that despite temporal beneficial changes in BMI and HbA1c after FMD, this intervention is not able to preserve microvascular endothelial health in Dutch South-Asian patients with T2DM. In contrast, glycocalyx mimetics preserves the microvascular endothelial health and reduces the inflammatory cytokine MCP-1.Clinical study registrationNCT03889236.

A homogenized two-phase computational framework for meso- and macroscale blood flow simulations

Background and ObjectiveOwing to the complexity of physics linked with blood flow and its associated phenomena, appropriate modeling of the multi-constituent rheology of blood is of primary importance. To this effect, various kinds of computational fluid dynamic models have been developed, each with merits and limitations. However, when additional physics like thrombosis and embolization is included within the framework of these models, computationally efficient scalable translation becomes very difficult. Therefore, this paper presents a homogenized two-phase blood flow framework with similar characteristics to a single fluid model but retains the flow resolution of a classical two-fluid model. The presented framework is validated against four different sets of experiments. MethodsThe two-phase model of blood presented here is based on the classical diffusion-flux framework. Diffusion flux models are known to be less computationally expensive than two-fluid multiphase models since the numerical implementation resembles single-phase flow models. Diffusion flux models typically use empirical slip velocity correlations to resolve the motion between phases. However, such correlations do not exist for blood. Therefore, a modified slip velocity equation is proposed, derived rigorously from the two-fluid governing equations. An additional drag law for red blood cells (RBCs) as a function of volume fraction is evaluated using a previously published cell-resolved solver. A new hematocrit-dependent expression for lift force on RBCs is proposed. The final governing equations are discretized and solved using the open-source software OpenFOAM. ResultsThe framework is validated against four sets of experiments: (i) flow through a rectangular microchannel to validate RBC velocity profiles against experimental measurements and compare computed hematocrit distributions against previously reported simulation results (ii) flow through a sudden expansion microchannel for comparing experimentally obtained contours of hematocrit distributions and normalized cell-free region length obtained at different flowrates and inlet hematocrits, (iii) flow through two hyperbolic channels to evaluate model predictions of cell-free layer thickness, and (iv) flow through a microchannel that mimics crevices of a left ventricular assist device to predict hematocrit distributions observed experimentally. The simulation results exhibit good agreement with the results of all four experiments. ConclusionThe computational framework presented in this paper has the advantage of resolving the multiscale physics of blood flow while still leveraging numerical techniques used for solving single-phase flows. Therefore, it becomes an excellent candidate for addressing more complicated problems related to blood flow, such as modeling mechanical entrapment of RBCs within blood clots, predicting thrombus composition, and visualizing clot embolization.

"There's plenty of room at the bottom": deep brain imaging with holographic endo-microscopy.

Over more than 300 years, microscopic imaging keeps providing fundamental insights into the mechanisms of living organisms. Seeing microscopic structures beyond the reach of free-space light-based microscopy, however, requires dissection of the tissue-an intervention seriously disturbing its physiological functions. The hunt for low-invasiveness tools has led a growing community of physicists and engineers into the realm of complex media photonics. One of its activities represents exploiting multimode optical fibers (MMFs) as ultra-thin endoscopic probes. Employing wavefront shaping, these tools only recently facilitated the first peeks at cells and their sub-cellular compartments at the bottom of the mouse brain with the impact of micro-scale tissue damage. Here, we aim to highlight advances in MMF-based holographic endo-microscopy facilitating microscopic imaging throughout the whole depth of the mouse brain. We summarize the important technical and methodological prerequisites for stabile high-resolution imaging in vivo. We showcase images of the microscopic building blocks of brain tissue, including neurons, neuronal processes, vessels, intracellular calcium signaling, and red blood cell velocity in individual vessels. This perspective article helps to understand the complexity behind the technology of holographic endo-microscopy, summarizes its recent advances and challenges, and stimulates the mind of the reader for further exploitation of this tool in the neuroscience research.

Network response of brain microvasculature to neuronal stimulation

Neurovascular coupling (NVC), or the adjustment of blood flow in response to local increases in neuronal activity is a hallmark of healthy brain function, and the physiological foundation for functional magnetic resonance imaging (fMRI). However, it remains only partly understood due to the high complexity of the structure and function of the cerebrovascular network. Here we set out to understand NVC at the network level, i.e. map cerebrovascular network reactivity to activation of neighbouring neurons within a 500×500×500 μm3 cortical volume (∼30 high-resolution 3-nL fMRI voxels). Using 3D two-photon fluorescence microscopy data, we quantified blood volume and flow changes in the brain vessels in response to spatially targeted optogenetic activation of cortical pyramidal neurons. We registered the vessels in a series of image stacks acquired before and after stimulations and applied a deep learning pipeline to segment the microvascular network from each time frame acquired. We then performed image analysis to extract the microvascular graphs, and graph analysis to identify the branch order of each vessel in the network, enabling the stratification of vessels by their branch order, designating branches 1–3 as precapillary arterioles and branches 4+ as capillaries. Forty-five percent of all vessels showed significant calibre changes; with 85 % of responses being dilations. The largest absolute CBV change was in the capillaries; the smallest, in the venules. Capillary CBV change was also the largest fraction of the total CBV change, but normalized to the baseline volume, arterioles and precapillary arterioles showed the biggest relative CBV change. From linescans along arteriole-venule microvascular paths, we measured red blood cell velocities and hematocrit, allowing for estimation of pressure and local resistance along these paths. While diameter changes following neuronal activation gradually declined along the paths; the pressure drops from arterioles to venules increased despite decreasing resistance: blood flow thus increased more than local resistance decreases would predict. By leveraging functional volumetric imaging and high throughput deep learning-based analysis, our study revealed distinct hemodynamic responses across the vessel types comprising the microvascular network. Our findings underscore the need for large, dense sampling of brain vessels for characterization of neurovascular coupling at the network level in health and disease.

Absolute retinal blood flow in healthy eyes and in eyes with retinal vein occlusion

PurposeTo measure non-invasively retinal venous blood flow (RBF) in healthy subjects and patients with retinal venous occlusion (RVO). MethodsThe prototype named AO-LDV (Adaptive Optics Laser Doppler Velocimeter), which combines a new absolute laser Doppler velocimeter with an adaptive optics fundus camera (rtx1, Imagine Eyes®, Orsay, France), was studied for the measurement of absolute RBF as a function of retinal vessel diameters and simultaneous measurement of red blood cell velocity. RBF was measured in healthy subjects (n = 15) and patients with retinal venous occlusion (RVO, n = 6). We also evaluated two softwares for the measurement of retinal vessel diameters: software 1 (automatic vessel detection, profile analysis) and software 2 (based on the use of deep neural networks for semantic segmentation of vessels, using a M2u-Net architecture). ResultsSoftware 2 provided a higher rate of automatic retinal vessel measurement (99.5 % of 12,320 AO images) than software 1 (64.9 %) and wider measurements (75.5 ± 15.7 μm vs 70.9 ± 19.8 μm, p < 0.001). For healthy subjects (n = 15), all the retinal veins in one eye were measured to obtain the total RBF. In healthy subjects, the total RBF was 37.8 ± 6.8 μl/min. There was a significant linear correlation between retinal vessel diameter and maximal velocity (slope = 0.1016; p < 0.001; r2 = 0.8597) and a significant power curve correlation between retinal vessel diameter and blood flow (3.63 × 10−5 × D2.54; p < 0.001; r2 = 0.7287). No significant relationship was found between total RBF and systolic and diastolic blood pressure, ocular perfusion pressure, heart rate, or hematocrit. For RVO patients (n = 6), a significant decrease in RBF was noted in occluded veins (3.51 ± 2.25 μl/min) compared with the contralateral healthy eye (11.07 ± 4.53 μl/min). For occluded vessels, the slope between diameter and velocity was 0.0195 (p < 0.001; r2 = 0.6068) and the relation between diameter and flow was Q = 9.91 × 10−6 × D2.41 (p < 0.01; r2 = 0.2526). ConclusionThis AO-LDV prototype offers new opportunity to study RBF in humans and to evaluate treatment in retinal vein diseases.

Quantitative analyses of RBC movement in whole blood exposed to DC and ELF electric field.

For the study of biological effects of direct current (DC) and extremely low frequency (ELF) electric fields, we have quantitatively analyzed red blood cell (RBC) movement in whole blood. Considering the inhomogeneous distribution of electric fields in vivo, five different electric field distributions were generated under a microscope. For theoretical analyses, we assumed electrophoresis and dielectrophoresis as basic motive forces and obtained the spatial distribution of blood cell velocity. The RBC velocity was measured using video image analysis. The spatial dependence of the velocity showed good agreement with that predicted by theoretical analysis. This result suggests the validity of the theoretical model based on electrophoresis and dielectrophoresis for the study of ELF electric field exposure to inhomogeneous animal and human bodies. Next, using the same measurement system, we attempted to find the electric field strength at which these effects occur. The threshold values were found to be 0.40and 1.6 kV/m, respectively, for DC and AC electric field exposures. Furthermore, we investigated the reproducibility of the field effects in more realistic conditions of human exposure. The RBCs in microchannels were exposed to the electric field generated in capacitive coupling using electrodes separated by an air gap. Even in the new condition, similar effects were observed, which also verified the validity of the analysis described above. These results will provide useful information for the safety assessment of field exposure and for the future biomedical applications of electric fields to manipulate RBCs in vivo.

Astrocyte Ca2+ dysregulation and neurovascular coupling deficits in a diet‐based mouse model of small cerebral vessel disease

AbstractBackgroundMice exposed to a diet deficient in B6/B12 vitamins and enriched in methionine exhibit hyperhomocysteinemia (HHcy) concomitant with many pathologic features of vascular cognitive impairment and dementia: a very common Alzheimer’s disease related dementia. Here, we evaluated the impact of astrocyte Ca2+ dysregulation and astrocytic Ca2+ signaling pathways in the context of functional hyperemia in fully awake mice exposed to HHcy diet.MethodAdult C57/BL6 mice were fed for 15 weeks with control chow, or chow deficient in vitamins B6/B12 and enriched in methionine (HHcy diet). Spontaneous and evoked (via air puff whisker stimulation) Ca2+ transients in astrocytes of fully awake mice were investigated in barrel cortex using the Ca2+ sensor GCaMP6f (expressed specifically in astrocytes using AAV) and two‐photon imaging. In other experiments, mice were treated with AAV‐Gfa2‐VIVIT to inhibit the Ca2+‐sensitive calcineurin/NFAT pathway in astrocytes, which regulates reactive astrocyte phenotypes. Two‐photon imaging was then used to assess diet and VIVIT effects on functional hyperemia in barrel cortex of fully awake mice.ResultCa2+ transients in individual barrel cortex astrocytes were significantly augmented (greater in amplitude, with faster rise/decay kinetics) in mice treated with HHcy diet. However, the functional connectivity in astrocyte networks of HHcy mice was significantly impaired, particularly during engagement of brain activity with whisker stimulation. Compared to control diet mice, HHcy mice also showed impaired functional hyperemia during whisker stimulation characterized by impaired arteriole dilations and reduced red blood cell velocity in nearby capillaries. Inhibition of reactive astrocyte signaling in HHcy diet mice with VIVIT significantly ameliorated these neurovascular coupling deficits.ConclusionThe results show that astrocyte Ca2+ dysregulation is a major feature of HHcy and contributes to cerebrovascular dysfunction through hyperactivation of the astrocytic CN/NFAT pathway.

The Role of P-Selectin in Microvascular Hemodynamics and Cerebral Brain Volumes in Aging Sickle Cell Mice

Cerebral vasculopathy has been linked to the development of cognitive impairment in sickle cell disease (SCD). We have previously demonstrated that 13-month-old sickle cell mice recapitulate features of cerebral vasculopathy such as tortuous vessels, microthrombi, and hypoperfusion seen in adults with SCD. Histologically, we also showed these mice have increased cerebral microvascular deposition of P-selectin and more frequent and larger microinfarcts compared to matched controls. We further showed that these mice developed evidence of cognitive deficit compared to matched controls. Based on these observations, we hypothesized P-selectin may play a critical role in development of cerebral micro vasculopathy and downstream pathologic sequalae such as microinfarcts by enhancing leukocyte-endothelial interactions. Furthermore, abundant evidence from non-sickle mice shows that microinfarcts can exacerbate small vessel changes including gray matter volume loss. Building on our prior work, in this study, we examined the effect of P-selectin deficiency on cerebral microvascular hemodynamics and leukocyte-endothelial interaction and well as changes in cortical and subcortical gray matter volume in aged sickle mice. Male, P-selectin knockout (Psel KO) or intact (Psel WT) chimeric mice were generated by transplanting control (AA) or sickle (SS) bone marrow into P-selectin knockout (Psel -/-) or C57BL/6 (B6) recipients. To assess the impact of vasculopathy and brain gray matter volume changes longitudinally, male mice were assessed at 12-, 14- or 16-month post-transplant. Hemodynamics (mean velocity, red blood cell (RBC) flux, white blood cell stalls in capillaries as well as venules) were obtained from line-scan images acquired using in-vivo two-photon microscopy, using custom and validated MATLAB scripts. Gray matter volumes in 26 cortical and subcortical brain regions with relevance to cognitive function, were derived from parcellation of T2-weighted magnetic resonance imaging after non-linear registration to Allen brain atlas T1 anatomical space. Parcellation was accomplished by registering the Paxinos atlas to the mouse T2 now in Allen space. Brain volume analyses was accomplished using Analyze 12 software. After the completion of in-vivo studies, brains were extracted for immunohistochemistry analysis. Statistical analysis was performed using two-way ANOVA. The breakdown of the mice included in this study are; 14 B6 AA, 12 B6 SS, 12 Psel KO AA and 13 Psel KO SS. Given the high attrition rate in older mice, data from 12-, 14- and 16-month-old cohorts were combined. Mean age of B6 AA cohort was 13.1 months, 12.8 months for B6 SS, 13. 9 months for Psel KO AA and 13.6 months for Psel KO SS mice and were not statistically different. Psel KO sickle mice had a significantly lower mean capillary RBC velocity (1.733 mm/s vs 4.717 mm/s, p &amp;lt; 0.01) compared to Psel WT SS mice. In addition, Psel KO SS mice had relatively smaller (more normal) capillary diameter compared to Psel WT SS mice (6.248 um vs 8.613 um, p &amp;lt; 0.001). Interestingly, there was no statistically significant difference in RBC flux between Psel KO SS and Psel WT SS mice, however Psel KO SS mice show a lower red blood cell flux similar to that seen in Psel WT AA mice ( Figure 1). Of the 26 brain regions that were assessed, Psel KO SS mice had larger measured gray matter volume in the frontal association cortex (3.01 ± 0.77 vs 3.66± 0.33 mm 3, p= 0.0083), prelimbic cortex (1.72 ± 0.27 vs 1.95 ± 0.17 mm 3, p=0.0079), hippocampus (17.67 ± 1.56 vs 16.21± 1.97 mm 3, p=0.01)( Figure 1) including other regions such as lateral (2.12 ± 0.19 vs 1.88 ± 0.31 vs mm 3 p=0.0096) medial (1.84± 0.16 vs 1.64 ± 0.26 mm 3, p=0.01), and ventral orbital cortex( 1.95± 0.17 vs1.72 ± 0.27 mm 3, p=0.01) and dorsolateral olfactory tract (1.46 6± 0.13 vs 1.31 ± 0.20 mm 3, p=0.023 compared to the Psel WT SS mice (data not shown). In conclusion, non-hematopoetic P-selectin deficiency improves cerebral vasculopathy as demonstrated by normalization of hemodynamics in sickle mice. We also show that Psel KO SS mice had larger brain gray matter volume in regions involved in cognitive function. Additional studies are needed to identify the underlying mechanism of improvement in brain gray matter volume as well as whether P-selectin could be potential target for reducing SCD associated neurological complications.

Numerical Model Validation of the Blood Flow through a Microchannel Hyperbolic Contraction.

A computational fluid dynamics (CFD) model of blood flow through hyperbolic contraction with a discrete phase model (DPM) was experimentally validated. For this purpose, the positions and velocities of red blood cells (RBCs) flowing in a microchannel with hyperbolic contraction were experimentally assessed using image analysis techniques, and were subsequently compared with the numerical results. The numerically and experimentally obtained velocity fields were in good agreement, with errors smaller than 10%. Additionally, a nearly constant strain rate was observed in the contraction region, which can be attributed to the quasilinear increase in the velocity along the hyperbolic contraction. Therefore, the numerical technique used was validated due to the close similarity between the numerically and experimentally obtained results. The tested CFD model can be used to optimize the microchannel design by minimizing the need to fabricate prototypes and evaluate them experimentally.

Cortical microvascular blood flow velocity mapping by combining dynamic light scattering optical coherence tomography and two-photon microscopy.

The accurate large-scale mapping of cerebral microvascular blood flow velocity is crucial for a better understanding of cerebral blood flow (CBF) regulation. Although optical imaging techniques enable both high-resolution microvascular angiography and fast absolute CBF velocity measurements in the mouse cortex, they usually require different imaging techniques with independent system configurations to maximize their performances. Consequently, it is still a challenge to accurately combine functional and morphological measurements to co-register CBF speed distribution from hundreds of microvessels with high-resolution microvascular angiograms. We propose a data acquisition and processing framework to co-register a large set of microvascular blood flow velocity measurements from dynamic light scattering optical coherence tomography (DLS-OCT) with the corresponding microvascular angiogram obtained using two-photon microscopy (2PM). We used DLS-OCT to first rapidly acquire a large set of microvascular velocities through a sealed cranial window in mice and then to acquire high-resolution microvascular angiograms using 2PM. The acquired data were processed in three steps: (i)2PM angiogram coregistration with the DLS-OCT angiogram, (ii)2PM angiogram segmentation and graphing, and (iii)mapping of the CBF velocities to the graph representation of the 2PM angiogram. We implemented the developed framework on the three datasets acquired from the mice cortices to facilitate the coregistration of the large sets of DLS-OCT flow velocity measurements with 2PM angiograms. We retrieved the distributions of red blood cell velocities in arterioles, venules, and capillaries as a function of the branching order from precapillary arterioles and postcapillary venules from more than 1000 microvascular segments. The proposed framework may serve as a useful tool for quantitative analysis of large microvascular datasets obtained by OCT and 2PM in studies involving normal brain functioning, progression of various diseases, and numerical modeling of the oxygen advection and diffusion in the realistic microvascular networks.

Microcirculatory Monitoring in Children with Congenital Heart Disease Before and After Cardiac Surgery

In this prospective observational study, we investigated whether congenital heart disease (CHD) affects the microcirculation and whether the microcirculation is altered following cardiac surgery with cardiopulmonary bypass (CPB). Thirty-eight children with CHD undergoing cardiac surgery with CPB and 35 children undergoing elective, non-cardiac surgery were included. Repeated non-invasive sublingual microcirculatory measurements were performed with handheld vital microscopy. Before surgery, children with CHD showed similar perfused vessel densities and red blood cell velocities (RBCv) but less perfused vessels (p < 0.001), lower perfusion quality (p < 0.001), and higher small vessel densities (p = 0.039) than children without CHD. After cardiac surgery, perfused vessel densities and perfusion quality of small vessels declined (p = 0.025 and p = 0.032), while RBCv increased (p = 0.032). We demonstrated that CHD was associated with decreased microcirculatory perfusion and increased capillary recruitment. The microcirculation was further impaired after cardiac surgery. Decreased microcirculatory perfusion could be a warning sign for altered tissue oxygenation and requires further exploration.Graphical abstract

PRMT7 can prevent neurovascular uncoupling, blood-brain barrier permeability, and mitochondrial dysfunction in repetitive and mild traumatic brain injury

Mild traumatic brain injury (TBI) comprises the largest percentage of TBI-related injuries, with pathophysiological and functional deficits that persist in a subset of TBI patients. In our three-hit paradigm of repetitive and mild traumatic brain injury (rmTBI), we observed neurovascular uncoupling via decreased red blood cell velocity, microvessel diameter, and leukocyte rolling velocity 3 days post-rmTBI via intra-vital two-photon laser scanning microscopy. Furthermore, our data suggest increased blood-brain barrier (BBB) permeability (leakage), with corresponding decrease in junctional protein expression post-rmTBI. Mitochondrial oxygen consumption rates (measured via Seahorse XFe24) were also altered 3 days post-rmTBI, along with disrupted mitochondrial dynamics of fission and fusion. Overall, these pathophysiological findings correlated with decreased protein arginine methyltransferase 7 (PRMT7) protein levels and activity post-rmTBI. Here, we increased PRMT7 levels in vivo to assess the role of the neurovasculature and mitochondria post-rmTBI. In vivo overexpression of PRMT7 using a neuronal specific AAV vector led to restoration of neurovascular coupling, prevented BBB leakage, and promoted mitochondrial respiration, altogether to suggest a protective and functional role of PRMT7 in rmTBI.

Ten-kilohertz two-photon microscopy imaging of single-cell dendritic activity and hemodynamics in vivo.

The studying of rapid neuronal signaling across large spatial scales in intact, living brains requires both high temporal resolution and versatility of the measurement device. We introduce a high-speed two-photon microscope based on a custom-built acousto-optic deflector (AOD). This microscope has a maximum line scan frequency of 400kHz and a maximum frame rate of 10,000 frames per second (fps) at . For stepwise magnification from population view to subcellular view with high spatial and temporal resolution, we combined the AOD with resonance-galvo (RS) scanning. With this combinatorial device that supports both large-view navigation and small-view high-speed imaging, we measured dendritic calcium propagation velocity and the velocity of single red blood cells (RBCs). We measured dendritic calcium propagation velocity ( ) in OGB-1-labeled single cortical neurons in mice in vivo. To benchmark the spatial precision and detection sensitivity of measurement in vivo, we also visualized the trajectories of single RBCs and found that their movement speed follows Poiseuille's law of laminar flow. This proof-of-concept methodological development shows that the combination of AOD and RS scanning two-photon microscopy provides both versatility and precision for quantitative analysis of single neuronal activities and hemodynamics in vivo.

Tissue Oxygenation Around Capillaries: Effects of Hematocrit and Arteriole Oxygen Condition.

Oxygen transfer in the microvasculature is a complex phenomenon that involves multiple physical and chemical processes and multiple media. Hematocrit, the volume fraction of red blood cells (RBCs) in blood, has direct influences on the blood flow as well as the oxygen supply in the microcirculation. On the one hand, a higher hematocrit means that more RBCs present in capillaries, and thus, more oxygen is available at the source end. On the other hand, the flow resistance increases with hematocrit, and therefore, the RBC motion becomes slower, which in turn reduces the influx of oxygen-rich RBCs entering capillaries. Such double roles of hematocrit have not been investigated adequately. Moreover, the oxygen-hemoglobin dissociation rate depends on the oxygen tension and hemoglobin saturation of the cytoplasm inside RBCs, and the dissociation kinetics exhibits a nonlinear fashion at different oxygen tensions. To understand how these factors and mechanisms interplay in the oxygen transport process, computational modeling and simulations are favorite since we have a good control of the system parameters and also we can access to the detailed information during the transport process. In this study, we conduct numerical simulations for the blood flow and RBC deformation along a capillary and the oxygen transfer from RBCs to the surrounding tissue. Different values for the hematocrit, arteriole oxygen tension, tissue metabolism rate and hemoglobin concentration and affinity are considered, and the simulated spatial and temporal variations of oxygen concentration are analyzed in conjunction with the nonlinear oxygen-hemoglobin reaction kinetics. Our results show that there are two competing mechanisms for the tissue oxygenation response to a hematocrit increases: the favorite effect of the higher RBC density and the negative effect of the slower RBC motion. Moreover, in the low oxygen situations with RBC oxygen tension less than 50mmHg at capillary inlet, the reduced RBC velocity effect dominates, resulting in a decrease in tissue oxygenation at higher hematocrit. On the opposite, for RBC oxygen tension higher than 50mmHg when entering the capillary, a higher hematocrit is beneficial to the tissue oxygenation. More interestingly, the pivoting arteriole oxygen tension at which the two competing mechanisms switch dominance on tissue oxygenation becomes lower for higher oxygen-hemoglobin affinity and lower hemoglobin concentration. This observation has also been analyzed based on the oxygen supply from RBCs and the oxygen-hemoglobin reaction kinetics. The results and discussions presented in this article could be helpful for a better understanding of oxygen transport in microcirculation.

Novel tools for neuronal activity, vessel diameter and red blood cell velocity imaging

Healthy brain function requires a delicate balance between neural activity and local vascular dynamics. Perturbations to this balance, such as vessel diameter changes or fluctuations in red blood cell velocity, are thought to underlie a number of neurodegenerative and neurovascular diseases, including migraine, stroke, and epilepsy. Here, for the first time, we demonstrate high fidelity simultaneous acquisition of vascular and neural circuit dynamics in freely-moving animals using our newly developed nVue dual color miniscope. Our system enables simultaneous imaging of blood flow and neural activity in both superficial and deep-brain structures in awake, behaving rodents. By multiplexing two LEDs at up to 100hz, the nVue enables direct, real-time visualization of both hemodynamics and calcium activity in two channels. Additionally, we have augmented the Inscopix Data Processing Software package to capture time-varying estimates of vessel diameter and red blood cell (RBC) velocity in specific vessels. Vessel diameter is estimated as the full-width half maximum of a Lorentzian function fit to vessel cross-section, while RBC velocity is calculated based on time-lagged correlations between seed-pixels within individual regions of interest and all immediately neighboring pixels. Distances between correlation peaks are then plotted against time. Manual annotation of blood flow data was performed to establish a ground truth against which to validate the algorithm. The nVue blood flow imaging application offers a powerful, turn-key, multisystem workflow for simultaneously interrogating hemodynamics and neural circuit function in freely behaving animals which will profoundly expand the neurovascular coupling toolkit not only for basic and translational research but also for the development of novel preclinical therapeutics. Commercial This is the full abstract presented at the American Physiology Summit 2023 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.