Capillary Red Blood Cell Velocity Research Articles

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<0.0001). RBC oxygen saturations (SO2) were significantly different beyond 3 s post-baseline in all [CO2] conditions ( p<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<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<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<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.

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 < 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 < 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.

Clamping Skeletal Muscle PO2 Eliminates Hyperinsulinemic Microvascular Blood Flow Response

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

Hyperinsulinemic Microvascular Blood Flow Response in Skeletal Muscle is Oxygen Mediated

ObjectiveTo determine if the blood flow response to insulin is partially mediated by local metabolic demand reflecting increased oxygen consumption required to fix glucose.MethodsTwo separate protocols were used to address the objective. 8 Sprague‐Dawley (171–204g) rats, 4 per protocol, were anesthetized with sodium pentobarbital via intraperitoneal injection, and mechanically ventilated. Arterial and venous catheters were introduced to maintain and monitor the animals’ cardiovascular state. The extensor digitorum longus (EDL) muscle was isolated and reflected onto the stage of an Olympus IX73 inverted microscope. Intravital video microscopy sequences were recorded during baseline and at hyperinsulinemic euglycemia in both protocols. Euglycemia was achieved through intravenous infusion of 2U insulin/kg/hr and variable rates of 50% D‐glucose were titrated against repeated blood glucose samples until stable normoglycemic levels were reached. In protocol 1, the EDL was reflected over a glass stage insert and isolated from room air with polyvinylidene film and a glass coverslip. In protocol 2, the muscle was instead reflected over a 100μm thick semi‐permeable membrane interfacing the EDL with a gas exchange chamber and similarly isolated from room air. An oxygen (O2) challenge was imposed in protocol 2 where O2 levels were oscillated at one minute intervals from 7%‐12%‐2%‐7%, with a stable 5% carbon dioxide concentration and the balance of gas composed of nitrogen. For both protocols, offline analysis of microvascular flow was conducted using custom MATLAB software yielding frame‐by‐frame measurements of capillary red blood cell (RBC) velocity, hematocrit, RBC supply rate (SR), and oxygen saturation (SO2). Animal protocols were approved by Memorial University’s Institutional Animal Care Committee.ResultsNo significant changes were found between glucose infusion rates (GIR) at euglycemia between both protocols, as well as blood glucose levels at both baseline and euglycemic conditions between experiments. In protocol 1, SR increased to 11.2 cells/s following euglycemic clamp, compared to 8.0 cells/s at baseline (p = 0.0255) consistent with flow increases reported in the literature. As expected, the imposed O2 challenges in protocol 2 caused significant changes in SO2 at 12% and 2% concentrations, which were found to be the same under both baseline and euglycemic conditions. In protocol 2, SR decreased at 12% O2 and increased at 2% O2 during the oscillation, compared to 7% O2, under both baseline and euglycemic conditions. SR was the same under euglycemia compared to baseline in the last 15 seconds of each step of the O2 oscillation from 7%‐12%‐2%‐7%, with p values >0.9999, >0.9999, 0.9984, and 0.9963 respectively.ConclusionsGas exchange chamber was effective at manipulating tissue oxygen levels, observed through profound SO2 changes during chamber oscillations at both baseline and euglycemic conditions. Our results suggest that the increase in muscle blood flow observed in response to insulin, as seen in experiment 1, is eliminated if the tissue oxygen environment is fixed at a given oxygen concentration as demonstrated in experiment 2.Support or Funding InformationProject funded through a CIHR project grant awarded to GM Fraser (PJT‐162119)Time series showing the change in capillary red blood cell oxygen saturation over the imposed oxygen oscillations under baseline and hyperinsulinemic euglycemic conditions (N=4 animals, 52 capillaries).Figure 1Time series of capillary red blood cell supply rate response to imposed oxygen oscillations under baseline and hyperinsulinemic euglycemic conditions (N=4 animals, 135 capillaries).Figure 2

Uncoupling of Microvascular Blood Flow and Capillary Density in Vascular Cognitive Impairment.

Cerebral small vessel disease (cSVD) plays an important role in dementia and is a major cause for vascular cognitive impairment (VCI). Recent studies hypothesized that capillary dysfunction including reduction of capillary patency, rather than a flow-limiting pathology is crucial in cSVD. As cSVD is considered a systemic microvascular disease, we examined sublingual microvascular blood flow and capillary density in patients with VCI and controls. Fifteen patients with VCI due to cSVD and 15 controls underwent intravital microscopy of the sublingual microvessels. Microvascular blood flow and capillary density in high and low flow areas were determined for each participant. Flow-density coupling was examined by determining the ratio of density changes to flow changes, and the ratio of feed vessel red blood cell (RBC) velocity to capillary RBC velocity. These were compared between VCI and controls. In healthy controls, capillary density increased proportionally with feed vessel blood flow increase. In patients with VCI, no increase of capillary density was observed. Moreover, increase of feed vessel RBC velocity led to significant increase of capillary RBC velocity in VCI, whereas in controls, the capillary RBC increased only slightly. Flow-density coupling differed significantly between VCI and controls, also after correcting for age and hypertension. Our findings suggest uncoupling of microvascular blood flow and capillary density in patients with VCI. This uncoupling may impair oxygen and nutrients exchange when blood flow increases in response to increased metabolic demand, ultimately leading to tissue damage.

New alternative Mitrofanoff channel based on spiral intestinal lengthening and tailoring

The occasional lack of appendix and the increasing use of the Malone anterograde continence enema (MACE) procedure have expanded the need for alternative Mitrofanoff channels. The Monti procedure does not always provide adequate length, the anastomosis of the double Monti, and the potential kink of the Casale channel is not ideal for smooth catheterisation. We tested the concept of spiral intestinal lengthening and tailoring (SILT), we developed originally for short bowel syndrome, to create a long and straight alternative Mitrofanoff channel (Figure). After ethical approval five mini-pigs underwent spiral intestinal lengthening and tailoring (SILT) without any previous bowel dilatation procedure. (Mean bowel width was 20.5 ± 0.57 mm). The spiral line was marked on a 6-8-cm-long ileum approximately 15 mm apart with a 60° angle to the longitudinal axis of the bowel. When the incision was completed, the mesentery was incised perpendicularly where the spiral incision line met the mesentery. The maximum length segment hanging on a single 1.5-cm-wide well-vascularised mesentery was detached. The capillary red blood cell velocity (RBCV) and perfusion rate (PR) was measured at the edges of the opened bowel strip by in vivo microscopy using orthogonal polarising spectral imaging (Cytoscan A/R, Cytometrics, Philadelphia, PA, USA). The bowel strips have been reconstructed in spiral fashion over a 12F catheter and were implanted into the bladder. Viability, patency, and microcirculation were assessed 4 weeks later. Conventional microscopy with HE staining was performed. The mean length of the spiral channel (100 ± 26.4 mm) was longer than could have been achieved with the double Monti or Casale procedure (4 times the bowel width). A 17% and 8.3% reduction was measured in the median values of the RBCV and the PR at the edges of the bowel strip at the primary surgery. All implanted channels remained viable, straight, patent, and easily catheterisable after 4 weeks, with full recovery of the RBCV and PR. The histology showed no necrosis or fibrosis. The SILT concept is suitable for creating a long and straight alternative Mitrofanoff channel. However, the SILT technique has been reported to be successful in the clinical practice to tailor and lengthen dilated short bowel; in this study we first applied this technique on normal calibre intestine to create long alternative Mitrofanoff channel. The use of an animal model and the relative short-term observation are the limitations of this study.

Alternative ileal flap for bladder augmentation if mesentery is short

To date the clam ileocystoplasty is the preferred method of bladder augmentation in children when the urodynamic problem is non-compliance and/or detrusor overactivity. The key to this technique is the incision of the bladder wall deep into the pelvis down to the trigone in order to avoid a diverticulum like neobladder and to provide adequate margin for augmentation. The detubularised ileum flap therefore has to reach to the bottom of the divided bladder on a reliable vascular pedicle without significant tension. A short ileal mesentery caused by previous surgery, peritonitis, peritoneal dialysis or ventriculo-peritoneal shunt may complicate surgery and compromise outcome. We hypothesized we can rely on the communication of the intramural vessels within the intestine and can detubularise the ileum adjacent to the mesentery rather than along the antimesenteric line and this could be combined with ligation of some vasa recta (VR) in order to create alternative ileum flaps, which reach further into the pelvis. Our aim was to assess the viability of the alternative flaps detubularised along the paramesenteric line and measure how many VR could be sacrificed beyond the tertiary arcades. After ethical approval adjacent ileal segments were detubulirased along the antimesenteric line (Group 1) and along the paramesenteric line (Group 2) in 5 minipigs in general anaesthesia. Ligation of 0,1,2,3 and 4 VR has been performed starting from the free end of the segments. The length of the ileal flaps was recorded. The microcirculation of flap edges was detected by in vivo microscopy using orthogonal polarising spectral imaging (Cytoscan A/R Cytometrics, PA, USA). Clam ileocystoplasty was performed with the ileum detubularised along the paramesenteric line without ligation of VR. Specimens of the augmented bladder were obtained after 4 weeks and stained with Hematoxilin + Eosin. No alteration in capillary red blood cell velocity (RBCV) and perfusion rate (PR) was observed after paramesenteric detubularisation. The flaps in Group 2 reached 20.25 ± 0.5 mm longer vs. This is 98% of the mean bowel width (20.5 ± 0.57 mm) measured in the animals. Ligation of each VR further increased the length of both flaps (mean: 10.59 ± 3.18 mm) however ligation of more than 2 VR gradually decreased the microcirculation in both groups. All animals augmented with alternative flap survived, there was no urine leak or suture break down. Histology confirmed viable bowel flaps. Paramesenteric detubularisation of the ileum is fully tolerated and results in longer reaching ileal flap vs. Only limited ligation of VR is tolerated. This study showed the first time that clam ileocystoplasty is feasible with ileal flap detubularised along the paramesenteric line. The use of the animal model and the relative short postoperative observation are the main limitations of this study.

Systemic microvascular shunting through hyperdynamic capillaries after acute physiological disturbances following cardiopulmonary bypass.

Previously we showed that cardiopulmonary bypass (CPB) during cardiac surgery is associated with reduced sublingual microcirculatory perfusion and oxygenation. It has been suggested that impaired microcirculatory perfusion may be paralleled by increased heterogeneity of flow in the microvascular bed, possibly leading to arteriovenous shunting. Here we investigated our hypothesis that acute hemodynamic disturbances during extracorporeal circulation indeed lead to microcirculatory heterogeneity with hyperdynamic capillary perfusion and reduced systemic oxygen extraction. In this single-center prospective observational study, patients undergoing cardiac surgery with (n = 18) or without (n = 13) CPB were included. Perioperative microcirculatory perfusion was assessed sublingually with sidestream darkfield imaging, and recordings were quantified for microcirculatory heterogeneity and hyperdynamic capillary perfusion. The relationship with hemodynamic and oxygenation parameters was analyzed. Microcirculatory heterogeneity index increased substantially after onset of CPB [0.5 (0.0-0.9) to 1.0 (0.3-1.3); P = 0.031] but not during off-pump surgery. Median capillary red blood cell (RBC) velocity increased intraoperatively in the CPB group only [1,600 (913-2,500 μm/s) vs. 380 (190-480 μm/s); P < 0.001], with 31% of capillaries supporting high RBC velocities (>2,000 μm/s). Hyperdynamic microcirculatory perfusion was associated with reduced arteriovenous oxygen difference and systemic oxygen consumption during and after CPB. The current study provides the first direct human evidence for a microvascular shunting phenomenon through hyperdynamic capillaries following acute physiological disturbances after onset of CPB. The hypothesis of impaired systemic oxygen offloading caused by hyperdynamic capillaries was supported by reduced blood arteriovenous oxygen difference and low systemic oxygen extraction associated with CPB.

Multimodal Imaging in Rats Reveals Impaired Neurovascular Coupling in Sustained Hypertension

Arterial hypertension is an important risk factor for cerebrovascular diseases, such as transient ischemic attacks or stroke, and represents a major global health issue. The effects of hypertension on cerebral blood flow, particularly at the microvascular level, remain unknown. Using the spontaneously hypertensive rat (SHR) model, we examined cortical hemodynamic responses on whisker stimulation applying a multimodal imaging approach (multiwavelength spectroscopy, laser speckle imaging, and 2-photon microscopy). We assessed the effects of hypertension in 10-, 20-, and 40-week-old male SHRs and age-matched male Wistar Kyoto rats (CTRL) on hemodynamic responses, histology, and biochemical parameters. In 40-week-old animals, losartan or verapamil was administered for 10 weeks to test the reversibility of hypertension-induced impairments. Increased arterial blood pressure was associated with a progressive impairment in functional hyperemia in 20- and 40-week-old SHRs; baseline capillary red blood cell velocity was increased in 40-week-old SHRs compared with age-matched CTRLs. Antihypertensive treatment reduced baseline capillary cerebral blood flow almost to CTRL values, whereas functional hyperemic signals did not improve after 10 weeks of drug therapy. Structural analyses of the microvascular network revealed no differences between normo- and hypertensive animals, whereas expression analyses of cerebral lysates showed signs of increased oxidative stress and signs of impaired endothelial homeostasis upon early hypertension. Impaired neurovascular coupling in the SHR evolves upon sustained hypertension. Antihypertensive monotherapy using verapamil or losartan is not sufficient to abolish this functional impairment. These deficits in neurovascular coupling in response to sustained hypertension might contribute to accelerate progression of neurodegenerative diseases in chronic hypertension.

Potassium‐induced cortical spreading depression bilaterally suppresses the electroencephalogram but only ipsilaterally affects red blood cell velocity in intraparenchymal capillaries

Cortical spreading depression (CSD) is a repetitive, propagating profile of mass depolarization of neuronal and glial cells, followed by sustained suppression of spontaneous neuronal activity. We have reported a long-lasting suppressive effect on red blood cell (RBC) velocities in intraparenchymal capillaries. Here, to test the hypothesis that the prolonged decrease of RBC velocity in capillaries is due to suppression of neuronal activity, we measured CSD-elicited changes in the electroencephalogram (EEG) as an index of neuronal activity. In isoflurane-anesthetized rats, DC potential, EEG, partial pressure of oxygen (PO₂), and cerebral blood flow (CBF) were simultaneously recorded in the temporo-parietal region. The velocities of fluorescently labeled RBCs were evaluated by high-speed camera laser scanning confocal fluorescence microscopy with our original software, KEIO-IS2. Transient deflection of DC potential and PO₂ and increase of CBF were repeatedly detected only in the ipsilateral hemisphere following topical KCl application. On the other hand, the relative spectral power of EEG was reduced bilaterally, showing the lowest value at 5 min after KCl application, when the other parameters had already returned to the baseline after the passage of CSD. Mean RBC velocity in capillaries was slightly but significantly reduced during and after passage of CSD in the ipsilateral hemisphere but did not change in the contralateral hemisphere in the same rats. We suggest that mass depolarization of neuronal and glial cells might transiently decelerate RBCs in nearby capillaries, but the sustained reduction of ipsilateral RBC velocity might be a result of the prolonged effect of CSD, not of neuronal suppression alone.

OCT methods for capillary velocimetry

To date, two main categories of OCT techniques have been described for imaging hemodynamics: Doppler OCT and OCT angiography. Doppler OCT can measure axial velocity profiles and flow in arteries and veins, while OCT angiography can determine vascular morphology, tone, and presence or absence of red blood cell (RBC) perfusion. However, neither method can quantify RBC velocity in capillaries, where RBC flow is typically transverse to the probe beam and single-file. Here, we describe new methods that potentially address these limitations. Firstly, we describe a complex-valued OCT signal in terms of a static scattering component, dynamic scattering component, and noise. Secondly, we propose that the time scale of random fluctuations in the dynamic scattering component are related to red blood cell velocity. Analysis was performed along the slow axis of repeated B-scans to parallelize measurements. We correlate our purported velocity measurements against two-photon microscopy measurements of RBC velocity, and investigate changes during hypercapnia. Finally, we image the ischemic stroke penumbra during distal middle cerebral artery occlusion (dMCAO), where OCT velocimetry methods provide additional insight that is not afforded by either Doppler OCT or OCT angiography.

Sustained Decrease and Remarkable Increase in Red Blood Cell Velocity in Intraparenchymal Capillaries Associated With Potassium‐Induced Cortical Spreading Depression

To examine changes in red blood cell (RBC) velocity in intraparenchymal capillaries of rat cerebral cortex in response to KCl-induced cortical spreading depression (CSD). In isoflurane-anesthetized rats, the velocity of fluorescently labeled RBCs flowing in capillaries in layer I was measured with a high-speed camera laser-scanning confocal fluorescence microscope, with simultaneous monitoring of DC potential, the electroencephalogram (EEG), partial pressure of oxygen (PO(2) ), and cerebral blood flow (CBF). After KCl application, a transient deflection of DC potential (i.e., CSD) repeatedly appeared concomitantly with depression of EEG, and was propagated in the distal direction. PO(2) transiently decreased and CBF was slowly elevated. The frequency distribution of RBC velocity was shifted downward during CSD and was still low after the passage of CSD. When we observed RBC velocity in 38 individual capillaries, 10 capillaries exhibited slowed-down RBC during CSD and RBC velocity remained low in 2 even after the passage of CSD. On the other hand, RBCs with moderately (<3 mm/sec) or remarkably (>3 mm/sec) increased velocities were seen in 10 and 5 capillaries, respectively. CSD-induced excitation of neurons may sustainably decrease or greatly increase RBC velocity in capillaries.

Influence of acetylsalicylic acid (Aspirin) on cutaneous microcirculation

The protective effect of acetylsalicylic acid (aspirin) in primary and secondary prophylaxis of cardiovascular events is attributed to the inhibition of platelet cyclooxygenase (COX). However, a recent animal study found a vasodilating and blood pressure lowering effect of aspirin independent of COX, but mediated by inhibition of the RhoA/Rho kinase signaling pathway. Prospective, randomized, double-blind, placebo-controlled cross-over study. In each instance 5 healthy volunteers received either aspirin 500 mg/d or placebo for 7 days. Capillary red blood cell velocity (vRBC) at rest and after postischemic hyperemia was determined on day 1 and 7 by means of nailfold capillary microscopy. In the aspirin group after 7 days a significant increase of vRBC was found at rest and during hyperemia. In the placebo group vRBC did not change. The finding was confirmed by the cross-over design of the study. Aspirin at a dosage of 500 mg/d has an impact on vasoregulation in the microcirculation. At present, the underlying mode of action in humans is unknown.

Effects of Hot and Cold Foods on Signals of Heart Rate Variability and Nail Fold Microcirculation of Healthy Young Humans: A Pilot Study

In traditional Chinese medicine, hot- and cold-attribute of food ingredients are a major part of dietary therapy. The aim of this study was to establish a suitable scientific methodology to define the attributes of food ingredients by investigating the relationship between food attributes and the physiological signals produced in healthy young subjects with different constitutions. Thirty subjects were grouped into hot and cold constitutions by Chinese medical doctors. Every subject took water, aged ginger tea and coconut water, which are well recognized as having neutral-, hot- and cold-attribute, respectively, on different visits. The different physiological signals induced by the samples were observed using skin and axillary temperature sensors, a heart rate variability analyzer and a laser Doppler anemometer. We found that the capillary red blood cell (RBC) velocity in nail fold microcirculation (NFM) of the subjects with hot constitution accelerated significantly after taking the hot-attribute aged ginger tea, which might be the result of elevated vagal activity leading to arteriole dilation in these subjects. In contrast, in subjects with cold constitution, capillary RBC velocity decelerated significantly and skin temperature decreased markedly after taking the cold-attribute coconut water, which might have been induced by sympathetic nerve activation causing the arteriole to be constricted. Accordingly, the use of capillary RBC velocity of NFM measured by laser Doppler anemometer may be a promising way to classify attributes of food ingredients commonly used in Chinese medicine dietary therapy in accordance with different personal constitutions.

Rat pial microvascular responses to melatonin during bilateral common carotid artery occlusion and reperfusion

The present study assessed the in vivo rat pial microvascular responses induced by melatonin during brain hypoperfusion and reperfusion (RE) injury. Pial microcirculation of male Wistar rats was visualized by fluorescence microscopy through a closed cranial window. Hypoperfusion was induced by bilateral common carotid artery occlusion (BCCAO, 30 min); thereafter, pial microcirculation was observed for 60 min. Arteriolar diameter, permeability increase, leukocyte adhesion to venular walls, perfused capillary length (PCL), and capillary red blood cell velocity (V(RBC) ) were investigated by computerized methods. Melatonin (0.5, 1, 2 mg/kg b.w.) was intravenously administered 10 min before BCCAO and at the beginning of RE. Pial arterioles were classified in five orders according to diameter, length, and branchings. In control group, BCCAO caused decrease in order 2 arteriole diameter (by 17.5 ± 3.0% of baseline) that was reduced by 11.8 ± 1.2% of baseline at the end of RE, accompanied by marked leakage and leukocyte adhesion. PCL and capillary V(RBC) decreased. At the end of BCCAO, melatonin highest dosage caused order 2 arteriole diameter reduction by 4.6 ± 2.0% of baseline. At RE, melatonin at the lower dosages caused different arteriolar responses. The highest dosage caused dilation in order 2 arteriole by 8.0 ± 1.5% of baseline, preventing leakage and leukocyte adhesion, while PCL and V(RBC) increased. Luzindole (4 mg/kg b.w.) prior to melatonin caused order 2 arteriole constriction by 12.0 ± 1.5% of baseline at RE, while leakage, leukocyte adhesion, PCL and V(RBC) were not affected. Prazosin (1 mg/kg b.w.) prior to melatonin did not significantly change melatonin's effects. In conclusion, melatonin caused different responses during hypoperfusion and RE, modulating pial arteriolar tone likely by MT1 and MT2 melatonin receptors while preventing blood-brain barrier changes through its free radical scavenging action.

Hemodynamic analysis of capillary in finger nail-fold using computational fluid dynamics and image estimation

Red blood cell (RBC) dynamics in capillaries is a useful diagnostic tool for many diseases. Previous study showed that optical flow estimation (OFE) is capable of accurately calculating RBC velocities using image registration technique. The computational fluid dynamics (CFD) method is explored in this study to calculate the RBC velocity in capillaries of finger nail-fold for six cases. The two-dimensional capillary images were reconstructed to three-dimensional, assuming circular cross sections. The no-slip boundary conditions were applied on the vessel walls. The initial velocity of the RBC going into each capillary was calculated by OFE. The velocities of multiple points along each capillary calculated by CFD, V CFD, were compared with OFE calculations, V OFE. The calculated RBC velocity was in the range of 56–685 μm/s. The average difference ( V CFD − V OFE) with one standard deviation is −2.66 ± 18.61 μm/s for all the 48 calculation points, and 0.03 ± 0.12 μm/s for all except one points (47 points), indicating that CFD can provide a reasonable accuracy in RBC velocity calculation in finger nail-fold capillaries.

Association between shear stress, angiogenesis, and VEGF in skeletal muscles in vivo.

To investigate the hypothesis that capillary proliferation in skeletal muscles, induced by a long-term increase in blood flow which elevates capillary shear stress, is associated with capillary expression of vascular endothelial growth factor (VEGF). Adult rats received prazosin in drinking water ( approximately 2 mg per day) or had extensor digitorum longus (EDL) muscles stimulated by implanted electrodes for up to 14 days. At intervals, serial frozen sections of EDL were stained for alkaline phosphatase to identify capillaries, proliferating cell nuclear antigen (PCNA), and VEGF-A protein. Shear stress was estimated from capillary red blood cell velocities and diameters, measured by direct observation of epi-illuminated EDL. Chronic stimulation and prazosin treatment both increased capillary: fiber ratio by approximately 40% after 14 days. In stimulated muscles, the percentage of capillaries positively stained for VEGF increased within 3 to 4 days, while the density of PCNA-positive capillaries had increased 20-fold after 2 days. With prazosin, VEGF-positive capillaries increased after 2 and 4 days, accompanied by a threefold increase in PCNA. By 14 days, PCNA labeling and VEGF were still high in stimulated muscles, but no longer different from controls with prazosin. After 3 to 4 days of treatment, capillary shear stress in resting muscle was 57% higher than in controls as a result of stimulation, but 4 times higher with prazosin. Higher capillary shear stress with prazosin than with stimulation may upregulate VEGF expression in the early stages of treatment. Greater proliferation of capillaries preceding a higher proportion of VEGF-positive capillaries in stimulated muscles, in the presence of a modest increase in shear stress, suggests that angiogenesis was initiated by other factors in addition to shear stress.

Validation of near-infrared laser speckle imaging for assessing microvascular (re)perfusion

The present study was conducted to compare laser speckle imaging (LSI) with sidestream dark field (SDF) imaging (i.e., capillary microscopy) so as to validate the use of LSI for assessing microvascular (re)perfusion. For this purpose, LSI and SDF measurements were performed on the human nail fold during gradual occlusion of the upperarm circulation to modify nail fold perfusion under controlled circumstances. Additionally, a vascular occlusion test was performed to test the ability of LSI to detect rapid changes in tissue perfusion during reactive hyperemia and a hyperthermic challenge was performed to measure LSI perfusion at maximum functional capillary density. Normalized LSI measurements (i.e., normalized to baseline is 100%) were shown to correlate positively with normalized SDF measurements (Pearson's r = 0.92). This was supported by linear regression analysis (slope of 1.01, R 2 = 0.85, p < 0.001). During the vascular occlusion test, LSI perfusion decreased from 307 ± 90 AU (baseline) to 42 ± 8 AU (ischemia). Peak perfusion during reperfusion was 651 ± 93 AU (212% of baseline), which had returned to baseline after 2 min. Hyperthermia increased LSI perfusion from 332 ± 90 AU to 1067 ± 256 AU (321% of baseline). The main finding was that changes in perfusion as measured by LSI correlated well with changes in capillary red blood cell velocities as measured by SDF imaging during controlled reduction of the (micro)vascular perfusion. It was further shown that LSI is capable of measuring tissue perfusion at high temporal and spatial resolution. In conclusion, LSI can be employed to accurately quantitate microvascular reactivity following ischemic and hyperthermic challenges.

Effects of propionyl-L-carnitine on ischemia–reperfusion injury in hamster cheek pouch microcirculation

Background and purpose Propionyl-l-carnitine (pLc) exerts protective effects in different experimental models of ischemia–reperfusion (I/R). The aim of the present study was to assess the effects of intravenously and topically applied pLc on microvascular permeability increase induced by I/R in the hamster cheek pouch preparation. Methods The hamster cheek pouch microcirculation was visualized by fluorescence microscopy. Microvascular permeability, leukocyte adhesion to venular walls, perfused capillary length, and capillary red blood cell velocity (VRBC) were evaluated by computer-assisted methods. E-selectin expression was assessed by in vitro analysis. Lipid peroxidation and reactive oxygen species (ROS) formation were determined by thiobarbituric acid-reactive substances (TBARS) and 2′-7′-dichlorofluorescein (DCF), respectively. Results In control animals, I/R caused a significant increase in permeability and in the leukocyte adhesion in venules. Capillary perfusion and VRBC decreased. TBARS levels and DCF fluorescence significantly increased compared with baseline. Intravenously infused pLc dose-dependently prevented leakage and leukocyte adhesion, preserved capillary perfusion, and induced vasodilation at the end of reperfusion, while ROS concentration decreased. Inhibition of nitric oxide synthase prior to pLc caused vasoconstriction and partially blunted the pLc-induced protective effects; inhibition of the endothelium-derived hyperpolarizing factor (EDHF) abolished pLc effects. Topical application of pLc on cheek pouch membrane produced the same effects as observed with intravenous administration. pLc decreased the E-selectin expression. Conclusions pLc prevents microvascular changes induced by I/R injury. The reduction of permeability increase could be mainly due to EDHF release induce vasodilatation together with NO. The reduction of E-selectin expression prevents leukocyte adhesion and permeability increase.

Mechanical Circulatory Support Devices Improve Tissue Perfusion in Patients With End-Stage Heart Failure or Cardiogenic Shock

This study evaluated the effects of mechanical circulatory support (MCS) on sub-lingual microcirculation as a surrogate for splanchnic microvascular perfusion. Between May 2008 and April 2009, 10 consecutive patients received an MCS device or extracorporeal membrane oxygenation for end-stage chronic heart failure (n = 6) or cardiogenic shock (n = 4). Microcirculation was investigated using a hand-held Sidestream Dark Field imaging device. Perfused capillary density (PCD) and capillary red blood cell velocity (cRBCv) were assessed before device implantation (T0), immediately after implantation (T1), and 1 day after implantation (T2). Median patient age was 45 years (interquartile range [IQR] 38-52 years) and 70% were men. MCS significantly decreased pulmonary capillary wedge pressure (p = 0.04). Median cardiac power index increased (0.29 [IQR, 0.21-0.34] W/m(2) at T0 vs 0.48 [IQR, 0.39-0.54] W/m(2) at T1, p = 0.005) as well as median central venous oxygen saturation (54% [IQR, 46%-61%] at T0 vs 78% [IQR, 67%-85%] at T1, p = 0.007). There was a 3-fold increase in tissue perfusion index (sub-lingual PCD x cRBCv) during mechanical circulatory support (573 [IQR, 407-693] at T0 vs 1909 [IQR, 1771-2835] at T1, p = 0.005). Microcirculatory parameters remained improved at T2. Mechanical circulatory support for severe heart failure is associated with a consistent, significant, and sustained improvement in tissue perfusion, as measured at the bedside by a 2-dimensional microcirculation imaging technique.