Capillary Red Blood Cell Flow Research Articles

Capillary oxygen regulates demand–supply coupling by triggering connexin40-mediated conduction: Rethinking the metabolic hypothesis

Coupling red blood cell (RBC) supply to O2 demand is an intricate process requiring O2 sensing, generation of a stimulus, and signal transduction that alters upstream arteriolar tone. Although actively debated, this process has been theorized to be induced by hypoxia and to involve activation of endothelial inwardly rectifying K+ channels (KIR) 2.1 by elevated extracellular K+ to trigger conducted hyperpolarization via connexin40 (Cx40) gap junctions to upstream resistors. This concept was tested in resting healthy skeletal muscle of Cx40-/- and endothelial KIR2.1-/- mice using state-of-the-art live animal imaging where the local tissue O2 environment was manipulated using a custom gas chamber. Second-by-second capillary RBC flow responses were recorded as O2 was altered. A stepwise drop in PO2 at the muscle surface increased RBC supply in capillaries of control animals while elevated O2 elicited the opposite response; capillaries were confirmed to express Cx40. The RBC flow responses were rapid and tightly coupled to O2; computer simulations did not support hypoxia as a driving factor. In contrast, RBC flow responses were significantly diminished in Cx40-/- mice. Endothelial KIR2.1-/- mice, on the other hand, reacted normally to O2 changes, even when the O2 challenge was targeted to a smaller area of tissue with fewer capillaries. Conclusively, microvascular O2 responses depend on coordinated electrical signaling via Cx40 gap junctions, and endothelial KIR2.1 channels do not initiate the event. These findings reconceptualize the paradigm of blood flow regulation in skeletal muscle and how O2 triggers this process in capillaries independent of extracellular K+.

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.

Spatiotemporal absorption fluctuation imaging based on U-Net.

.SignificanceFull-field optical angiography is critical for vascular disease research and clinical diagnosis. Existing methods struggle to improve the temporal and spatial resolutions simultaneously.AimSpatiotemporal absorption fluctuation imaging (ST-AFI) is proposed to achieve dynamic blood flow imaging with high spatial and temporal resolutions.ApproachST-AFI is a dynamic optical angiography based on a low-coherence imaging system and U-Net. The system was used to acquire a series of dynamic red blood cell (RBC) signals and static background tissue signals, and U-Net is used to predict optical absorption properties and spatiotemporal fluctuation information. U-Net was generally used in two-dimensional blood flow segmentation as an image processing algorithm for biomedical imaging. In the proposed approach, the network simultaneously analyzes the spatial absorption coefficient differences and the temporal dynamic absorption fluctuation.ResultsThe spatial resolution of ST-AFI is up to , and the temporal resolution is up to 0.032 s. In vivo experiments on 2.5-day-old chicken embryos were conducted. The results demonstrate that intermittent RBCs flow in capillaries can be resolved, and the blood vessels without blood flow can be suppressed.ConclusionsUsing ST-AFI to achieve convolutional neural network (CNN)-based dynamic angiography is a novel approach that may be useful for several clinical applications. Owing to their strong feature extraction ability, CNNs exhibit the potential to be expanded to other blood flow imaging methods for the prediction of the spatiotemporal optical properties with improved temporal and spatial resolutions.

Capillary Oxygen Triggers Conducted Vasodilation via Connexin 40 Independent of Extracellular K + and Endothelial K IR 2.1

Mechanisms that fine-tune red blood cell (RBC) and O2 delivery to capillaries are critical to tissue function. Coupling RBC supply to demand is an intricate process requiring O2 sensing, generation of a capillary stimulus and triggering of a transduction process that alters the diameter of upstream arterioles. The sensor's identity, the stimulus it generates and the mechanisms that enable it are actively debated. Studies have implied the endothelial KIR2.1 channels drive O2 sensing whereas gap junctions, comprised of connexin 40, enable electrical signals to conduct upstream. This idea was tested in KIR2.1-/- and connexin 40-/- mice where the local O2 environment of skeletal muscle was precisely controlled. The extensor digitorum longus muscle was prepared for intravital microscopy; a custom stage insert with a gas chamber allowed for O2 control at the tissue surface. Second-by-second capillary RBC flow responses were recorded as O2 was: 1) reduced from 53 mmHg to 15 mmHg or 0 mmHg for 3 min; or 2) oscillated between 91 mmHg and 15 mmHg (1 cycle/min). Chamber O2 at 15 mmHg induces microvascular responses without diminishing mitochondrial function while 0 mmHg initiates additional metabolic responses. Dropping PO2 on the muscle surface (53 mmHg to 15 mmHg or 0 mmHg) significantly increased RBC supply rate in capillaries of control animals while elevated chamber O2 decreased capillary RBC supply rate in a graded manner. The RBC flow responses in control mice were rapid and tightly coupled to O2 levels as the chamber O2 was oscillated in a sinusoidal fashion. In stark contrast, this blood flow response failed to occur in connexin 40-/- mice. As this response was absent, capillary RBC O2 saturation was lower under resting conditions in the connexin 40-/- animals. Endothelial KIR2.1-/- mice, on the other hand, had normal resting RBC O2 saturation. Furthermore, KIR2.1-/- mice reacted normally to O2 changes, albeit an oscillation or a sustained O2 decrease (to 15 mmHg), with a rise in RBC supply rate. Likewise, the microcirculatory response to 0 mmHg O2 was similar between floxed control and endothelial KIR2.1-/- mice. We show that microvascular O2 responses depend on coordinated electrical signaling via gap junctions comprised of connexin 40 and that endothelial KIR2.1channels do not drive the initiating electrical event. These findings reform our understanding of blood flow regulation and how O2 initiates this process independent of metabolite production.

Channelrhodopsin Excitation Contracts Brain Pericytes and Reduces Blood Flow in the Aging Mouse Brain in vivo.

Brains depend on blood flow for the delivery of oxygen and nutrients essential for proper neuronal and synaptic functioning. French physiologist Rouget was the first to describe pericytes in 1873 as regularly arranged longitudinal amoeboid cells on capillaries that have a muscular coat, implying that these are contractile cells that regulate blood flow. Although there have been >30 publications from different groups, including our group, demonstrating that pericytes are contractile cells that can regulate hemodynamic responses in the brain, the role of pericytes in controlling cerebral blood flow (CBF) has not been confirmed by all studies. Moreover, recent studies using different optogenetic models to express light-sensitive channelrhodopsin-2 (ChR2) cation channels in pericytes were not conclusive; one, suggesting that pericytes expressing ChR2 do not contract after light stimulus, and the other, demonstrating contraction of pericytes expressing ChR2 after light stimulus. Since two-photon optogenetics provides a powerful tool to study mechanisms of blood flow regulation at the level of brain capillaries, we re-examined the contractility of brain pericytes in vivo using a new optogenetic model developed by crossing our new inducible pericyte-specific CreER mouse line with ChR2 mice. We induced expression of ChR2 in pericytes with tamoxifen, excited ChR2 by 488 nm light, and monitored pericyte contractility, brain capillary diameter changes, and red blood cell (RBC) velocity in aged mice by in vivo two-photon microscopy. Excitation of ChR2 resulted in pericyte contraction followed by constriction of the underlying capillary leading to approximately an 8% decrease (p = 0.006) in capillary diameter. ChR2 excitation in pericytes substantially reduced capillary RBC flow by 42% (p = 0.03) during the stimulation period compared to the velocity before stimulation. Our data suggests that pericytes contract in vivo and regulate capillary blood flow in the aging mouse brain. By extension, this might have implications for neurological disorders of the aging human brain associated with neurovascular dysfunction and pericyte loss such as stroke and Alzheimer’s disease.

Pericytes in capillaries are contractile in vivo, but arterioles mediate functional hyperemia in the mouse brain

Modern functional imaging techniques of the brain measure local hemodynamic responses evoked by neuronal activity. Capillary pericytes recently were suggested to mediate neurovascular coupling in brain slices, but their role in vivo remains unexplored. We used two-photon microscopy to study in real time pericytes and the dynamic changes of capillary diameter and blood flow in the cortex of anesthetized mice, as well as in brain slices. The thromboxane A(2) analog, 9,11-dideoxy-9α,11α-methanoepoxy Prostaglandin F2α (U46619), induced constrictions in the vicinity of pericytes in a fraction of capillaries, whereas others dilated. The changes in vessel diameter resulted in changes in capillary red blood cell (RBC) flow. In contrast, during brief epochs of seizure activity elicited by local administration of the GABA(A) receptor antagonist, bicuculline, capillary RBC flow increased without pericyte-induced capillary diameter changes. Precapillary arterioles were the smallest vessels to dilate, together with penetrating and pial arterioles. Our results provide in vivo evidence that pericytes can modulate capillary blood flow in the brain, which may be important under pathological conditions. However, our data suggest that precapillary and penetrating arterioles, rather than pericytes in capillaries, are responsible for the blood flow increase induced by neural activity.

Counterpoint: There is not capillary recruitment in active skeletal muscle during exercise

The notion that a substantial proportion of capillaries do not contain moving red blood cells (RBCs) in muscle at rest but are “recruited,” i.e., begin flowing with RBCs during contractions, is one basis for our present understanding of blood-muscle exchange during exercise ([20][1], [28][2]).

Microcirculation in rat soleus muscle after eccentric exercise: the effect of nifedipine

This paper explores the role of the calcium entry blocker nifedipine in the explanation of eccentric exercise-induced fibre damage by changes in skeletal muscle microcirculation. Eccentric exercise (EE) was induced by indirect stimulation of rat soleus muscle in its lengthening phase during cycling. Muscle damage was assessed by histology, electron microscopy and muscle tension 48 h later. Diameters of arterioles and venules, their response to dilator and constrictor stimuli and pattern of capillary flow were measured in epiiluminated muscles using intravital microscopy. Tetanic tension developed by EE muscles was lower (8.60 +/- 1.02, means +/- SEM, n = 8 N g(-1) wet weight compared to 12.25 +/- 0.56 in controls, P < 0.01). Electron microscopy showed changes similar to those in muscles exposed to EE by downhill running (Z line streaming, disruption of sarcolemma, swollen tubules). A total of 16% of muscle fibres were damaged, and fibre areas and interstitial space were enlarged. Capillary red blood cell flow showed tendency to a greater intermittency. Large venules were narrower, but arterioles and smaller venules had diameters similar to control muscles. Vessel dilatation to topically applied 10(-4) M adenosine was attenuated. Daily administration of calcium entry blocker nifedipine by gavage (2 mg/kg/day in two equal doses) removed the narrowing of venules, restored the dilator response of all vessels to adenosine and increased capillary:fibre ratio. The percentage of damaged fibres decreased to 4.7 and the size of the interstitial space and fibre areas was normalized. Thus muscle damage caused by eccentric exercise was attenuated by nifedipine due to its beneficial effect on muscle microcirculation, which was impaired by eccentric exercise.

A New Video Image Analysis System to Study Red Blood Cell Dynamics and Oxygenation in Capillary Networks

The authors present a Measurement and Analysis System for Capillary Oxygen Transport (MASCOT) to study red blood cell (RBC) dynamics and oxygenation in capillary networks. The system enables analysis of capillaries to study geometry and morphology and provides values for capillary parameters such as diameter and segment length. It also serves as an analysis tool for capillary RBC flow characteristics, including RBC velocity, lineal density, and supply rate. Furthermore, the system provides a means of determining the oxygen saturation of hemoglobin contained within RBCs, by analysis of synchronized videotapes containing images at two wavelengths, enabling the quantification of the oxygen content of individual RBCs. Video recordings of RBC flow at two wavelengths, 420 nm (isosbestic) and 436 nm (oxygen sensitive), are made using a dual camera video microscopy system. The 420-nm recording is used to generate images based on the variance of light intensity fluctuations that help to identify capillaries in a given field of view that are in sharp focus and exhibit flow of individual RBCs separated by plasma gaps. A region of interest enclosing the desired capillary is defined and a fixed number of successive video frames at the two wavelengths are captured. Next a difference image is created, which delineates the RBC column, whose width is used to estimate the internal diameter of the capillary. The 420-nm images are also used to identify the location and centroid of each RBC within the capillary. A space-time image is generated to compute the average RBC velocity. Lineal density is calculated as the number of RBCs per unit length of a capillary segment. The mean optical density (OD) of each RBC is calculated at both wavelengths, and the average SO(2) for each cell is determined from OD(436)/OD(420). MASCOT is a robust and flexible system that requires simple hardware, including a SGI workstation fitted with an audio-visual module, a VCR, and an oscilloscope. Since the new system provides information on an individual cell basis from entire capillary segments, the authors believe that results obtained using MASCOT will be more accurate than those obtained from previous systems. Due to its flexibility and ease of extension to other applications, MASCOT has the potential to be applied widely as an analysis tool for capillary oxygen transport measurements.

Capillary red blood cell flow and activation of white blood cells in chronic muscle ischemia in the rat.

Increased activity of ischemic skeletal muscles in which functional hyperemia is impaired has been linked with capillary endothelial swelling postcapillary white blood cell (WBC) adherence. The perfusion pattern of capillaries under these conditions and time course of WBC activation is not known. Capillary microcirculation was studied by videomicroscopy at rest and after muscle contractions (1 Hz, 10 min) in extensor digi-torum longus muscles of pentobarbital sodium-anesthetized rat during the early stages of chronic ischemia (unilateral ligation of the common iliac artery for 3 days) and in ischemic muscles subjected to increased activity (7 days of ischemia or 3 days of ischemia plus indirect electrical stimulation via planted electrodes, 10 Hz, 7 x 10 min on-90 min off/day) to investigate how perfusion was affected. All ischemic muscles had more intermittently flowing capillaries than did unoperated control) muscles. Temporal heterogeneity of perfusion at rest, assessed by velocity, time spent stationary, and stop/start frequency of red blood cells, was similar to control values in ischemic muscles but greater in ischemic muscles subjected to additional activity. Hyperemic responses to contractions were severely blunted in all ischemic groups. The portion of morphologically nonspherical WBCs, taken to indicate activation, was 24 +/- 3% in venous blood after 3 days of ischemia vs. 14 +/- 1% in control muscles and increased further by 7 days (42 +/- 2%) when activated cells were also found in arterial blood. Thus increased muscular activity may exacerbate the adverse effects of ischemia on capillary perfusion, and WBC activation, evident before endothelial swelling is apparent, provides the potential as a circulating signal for capillary swelling in the ischemic and other muscles.

In vivo microvascular structural and functional consequences of muscle length changes.

As muscles are stretched, blood flow and oxygen delivery are compromised, and consequently muscle function is impaired. We tested the hypothesis that the structural microvascular sequellae associated with muscle extension in vivo would impair capillary red blood cell hemodynamics. We developed an intravital spinotrapezius preparation that facilitated direct on-line measurement and alteration of sarcomere length simultaneously with determination of capillary geometry and red blood cell flow dynamics. The range of spinotrapezius sarcomere lengths achievable in vivo was 2.17 +/- 0.05 to 3.13 +/- 0.11 microns. Capillary tortuosity decreased systematically with increases of sarcomere length up to 2.6 microns, at which point most capillaries appeared to be highly oriented along the fiber longitudinal axis. Further increases in sarcomere length above this value reduced mean capillary diameter from 5.61 +/- 0.03 microns at 2.4-2.6 microns sarcomere length to 4.12 +/- 0.05 microns at 3.2-3.4 microns sarcomere length. Over the range of physiological sarcomere lengths, bulk blood flow (radioactive microspheres) decreased approximately 40% from 24.3 +/- 7.5 to 14.5 +/- 4.6 ml.100 g-1.min-1. The proportion of continuously perfused capillaries, i.e., those with continuous flow throughout the 60-s observation period, decreased from 95.9 +/- 0.6% at the shortest sarcomere lengths to 56.5 +/- 0.7% at the longest sarcomere lengths and was correlated significantly with the reduced capillary diameter (r = 0.711, P < 0.01; n = 18). We conclude that alterations in capillary geometry and luminal diameter consequent to increased muscle sarcomere length are associated with a reduction in mean capillary red blood cell velocity and a greater proportion of capillaries in which red blood cell flow is stopped or intermittent. Thus not only does muscle stretching reduce bulk blood (and oxygen) delivery, it also alters capillary red blood cell flow dynamics, which may further impair blood-tissue oxygen exchange.

FUNCTIONAL MICROVASCULAR EFFECTS OF STREPTOZOTOCIN (STZ)-INDUCED DIABETES IN RAT SKELETAL MUSCLE 853

The efficacy of blood-tissue oxygen and substrate exchange is determined in large part by capillary geometry and flow dynamics. As detailed in the companion paper (Kindig et al. “InVivo Structural Microvascular Consequences of Diabetes in Skeletal Muscle”), STZ-induced diabetes produces muscle atrophy and microvascular remodeling (i.e. straighter, narrower capillaries). Using intravital microscopy, we evaluated the effect of this condition on capillary red blood cell (RBC) dynamics within the spinotrapezius muscle. Compared with control (C), diabetic (D) muscles evidenced a lower proportion of continuously flowing capillaries(D=55±4% vs C=85±5%, P<0.01). In D muscles, there was a significant positive correlation between mean capillary diameter and percent flowing capillaries (r=0.775, P<0.05). In D muscles, there was an average of 0.83±0.16 flowing capillaries per fiber compared with 1.41±0.11 in C (P<0.05). However, because of the severe fiber atrophy, the lineal density of flowing capillaries was similar (D=26; C=30 cap/mm). Systemic hematocrit was unchanged in D, however, within the flowing capillaries, RBC velocities and fluxes were markedly reduced. These impaired flow dynamics were not the result of altered blood viscosity in D, at least over the range of shear rates from 11.25-450 s-1. We conclude that STZ-induced diabetes impairs spinotrapezius capillary RBC flow dynamics in proportion to the reduction in mean capillary diameter. While the lineal density of flowing capillaries remains unaffected, the uniformity of the distribution of RBC flow within and among capillaries of D muscles is compromised. This scenario is likely to reduce the capacity for blood-tissue O2 and substrate exchange.

Regulation of oxygen supply in the cerebral circulation.

The dynamics and regulation of red blood cell flow in the cerebral microcirculation was studied by intravital fluorescence video-microscopy in a closed cranial window preparation in the rat. The studies revealed that capillary perfusion in the brain is essentially continuous but a stationary difference from capillary to capillary within the same microvascular network exists. The main mechanism of an increase in flow in cerebral capillaries is an increase in linear velocity with no or minor role for classical capillary recruitment. While cyclic opening and closing of capillaries is not evident, low frequency oscillations in capillary flow velocity are present when perfusion or oxygen supply to tissue is challenged. In hypoxic hypoxia and moderate hypercapnia, RBC velocity increases in all capillaries while in severe hypercapnia, redistribution of RBC velocity in the capillary network occurs. Both systemic hypotension and severe hypercapnia are accompanied by an increase in the homogeneity of capillary flow; this change involves the redistribution of RBC flow between thoroughfare channels and exchange capillaries. Thoroughfare channels may thus provide a recruitable flow reserve in the cerebral microcirculation. The capillary flow response to hypoxic and anemic hypoxia depends on the activity neuronal nitric oxide synthase. These findings suggest the presence of a physiological regulatory mechanism of cerebral capillary red blood cell flow and oxygen supply which may involve neuronal nitric oxide as a mediator.