Capillary Blood Cell Velocity Research Articles

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.

Targeting Astrocyte Signaling Alleviates Cerebrovascular and Synaptic Function Deficits in a Diet-Based Mouse Model of Small Cerebral Vessel Disease.

Despite the indispensable role that astrocytes play in the neurovascular unit, few studies have investigated the functional impact of astrocyte signaling in cognitive decline and dementia related to vascular pathology. Diet-mediated induction of hyperhomocysteinemia (HHcy) recapitulates numerous features of vascular contributions to cognitive impairment and dementia (VCID). Here, we used astrocyte targeting approaches to evaluate astrocyte Ca2+ dysregulation and the impact of aberrant astrocyte signaling on cerebrovascular dysfunction and synapse impairment in male and female HHcy diet mice. Two-photon imaging conducted in fully awake mice revealed activity-dependent Ca2+ dysregulation in barrel cortex astrocytes under HHcy. Stimulation of contralateral whiskers elicited larger Ca2+ transients in individual astrocytes of HHcy diet mice compared with control diet mice. However, evoked Ca2+ signaling across astrocyte networks was impaired in HHcy mice. HHcy also was associated with increased activation of the Ca2+/calcineurin-dependent transcription factor NFAT4, which has been linked previously to the reactive astrocyte phenotype and synapse dysfunction in amyloid and brain injury models. Targeting the NFAT inhibitor VIVIT to astrocytes, using adeno-associated virus vectors, led to reduced GFAP promoter activity in HHcy diet mice and improved functional hyperemia in arterioles and capillaries. VIVIT expression in astrocytes also preserved CA1 synaptic function and improved spontaneous alternation performance on the Y maze. Together, the results demonstrate that aberrant astrocyte signaling can impair the major functional properties of the neurovascular unit (i.e., cerebral vessel regulation and synaptic regulation) and may therefore represent a promising drug target for treating VCID and possibly Alzheimer's disease and other related dementias.SIGNIFICANCE STATEMENT The impact of reactive astrocytes in Alzheimer's disease and related dementias is poorly understood. Here, we evaluated Ca2+ responses and signaling in barrel cortex astrocytes of mice fed with a B-vitamin deficient diet that induces hyperhomocysteinemia (HHcy), cerebral vessel disease, and cognitive decline. Multiphoton imaging in awake mice with HHcy revealed augmented Ca2+ responses in individual astrocytes, but impaired signaling across astrocyte networks. Stimulation-evoked arteriole dilation and elevated red blood cell velocity in capillaries were also impaired in cortex of awake HHcy mice. Astrocyte-specific inhibition of the Ca2+-dependent transcription factor, NFAT, normalized cerebrovascular function in HHcy mice, improved synaptic properties in brain slices, and stabilized cognition. Results suggest that astrocytes are a mechanism and possible therapeutic target for vascular-related dementia.

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

Spatiotemporal dynamics of red blood cells in capillaries in layer I of the cerebral cortex and changes in arterial diameter during cortical spreading depression and response to hypercapnia in anesthetized mice.

Control of red blood cell velocity in capillaries is essential to meet local neuronal metabolic requirements, although changes of capillary diameter are limited. To further understand the microcirculatory response during cortical spreading depression, we analyzed the spatiotemporal changes of red blood cell velocity in intraparenchymal capillaries. In urethane-anesthetized Tie2-green fluorescent protein transgenic mice, the velocity of fluorescence-labeled red blood cells flowing in capillaries in layer I of the cerebral cortex was automatically measured with our Matlab domain software (KEIO-IS2) in sequential images obtained with a high-speed camera laser-scanning confocal fluorescence microscope system. Cortical spreading depression repeatedly increased the red blood cell velocity prior to arterial constriction/dilation. During the first cortical spreading depression, red blood cell velocity significantly decreased, and sluggishly moving or retrograde-moving red blood cells were observed, concomitantly with marked arterial constriction. The velocity subsequently returned to around the basal level, whileoligemia aftercortical spreading depression with slight vasoconstriction remained. After several passages ofcortical spreading depression, hypercapnia-induced increase of red blood cell velocity, regional cerebral blood flow and arterial diameter were all significantly reduced, and the correlations among them became extremely weak. Taken together with our previous findings, these simultaneous measurements of red blood cell velocity in multiple capillaries, arterial diameter and regional cerebral blood flow support the idea that red blood cell flow might be altered independently, at least in part, from arterial regulation, that neuro-capillary coupling plays a role in rapidly meeting local neural demand.

Capillary red blood cell velocimetry by phase-resolved optical coherence tomography.

We present a phase-resolved optical coherence tomography (OCT) method to extend Doppler OCT for the accurate measurement of the red blood cell (RBC) velocity in cerebral capillaries. OCT data were acquired with an M-mode scanning strategy (repeated A-scans) to account for the single-file passage of RBCs in a capillary, which were then high-pass filtered to remove the stationary component of the signal to ensure an accurate measurement of phase shift of flowing RBCs. The angular frequency of the signal from flowing RBCs was then quantified from the dynamic component of the signal and used to calculate the axial speed of flowing RBCs in capillaries. We validated our measurement by RBC passage velocimetry using the signal magnitude of the same OCT time series data.

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.

In Vivo Two‐photon Fluorescence Microscopy Reveals Disturbed Cerebral Capillary Blood Flow and Increased Susceptibility to Ischemic Insults in Diabetic Mice

Diabetes mellitus increases the risk of stroke, but the mechanisms are unclear. The present study tested the hypothesis that diabetes mellitus disturbs the brain microcirculation and increases the susceptibility to cerebral damage in a middle cerebral artery occlusion (MCAO) model of ischemia. Diabetes was induced by streptozocin in mice expressing green fluorescent protein in endothelial cells (Tie2-GFP mice). Four weeks later, they were subjected to transient (20min) MCAO. In vivo blood flow was measured by two-photon laser-scanning microscopy (TPLSM) in cerebral arteries, veins, and capillaries. There was a significant decrease in red blood cell (RBC) velocity in capillaries in diabetic mice as assessed by TPLSM, yet the regional cerebral blood flow, as assessed by laser Doppler flowmetry, was maintained. Brain capillary flow developed turbulence after MCAO only in diabetic mice. These mice sustained increased neurological deficits after MCAO which were accompanied by an exaggerated degradation of tight junction proteins and blunted CaMKII phosphorylation in cerebral tissues indicating disruption of the blood-brain barrier and disturbed cognitive potential. Diabetic mice are more susceptible to disturbances of cerebral capillary blood flow which may predispose them to neurovascular defects following ischemia.

Abstract 93: In Vivo Two-photon Fluorescence Microscopy Reveals Disturbed Cerebral Capillary Blood Flow and Increased Susceptibility to Ischemic Insults in Diabetic Mice

Background: Diabetes imposes an increased risk of microvascular complications. The present study aims to evaluate if diabetes and its disturbances in brain blood microcirculation increases the susceptibility to brain ischemic insults. Methods and Results: Diabetes was induced by streptozocin in mice expressing green fluorescent protein in endothelial cells (Tie2-GFP mice). Four weeks later the diabetic mice were subjected to transient (20 min) middle cerebral artery occlusion (MCAO). In vivo two-photon laser scanning microscopy (TPLSM) was applied to the brains of the mice, and blood flow was estimated in arteries, capillaries and veins in diabetic and control mice after the MCAO. We found a significant decrease in red blood cell (RBC) velocity in capillaries in diabetic mice (0.93 ± 0.11 mm/s, P &lt;0.001 versus control mice). Despite this no significant changes in regional blood flow was observed by simultaneously performed laser-Doppler flowmetry. Notably, we observed that a further decline in RBC velocity (0.62 ± 0.07 mm/s, P &lt;0.001 versus diabetic or ischemic mice) and capillary volume flux [(2.29 ± 0.27) × 10 -5 μl/s, P &lt;0.001 versus diabetic or ischemic mice] in diabetic-MCAO mice, indicating diabetes-aggravated brain ischemia-induced disturbance in cerebral capillary blood flow. Furthermore, our intravital TPLSM data demonstrated that brain capillary flow was more turbulent in diabetic-MCAO mice than in the other groups. Diabetic-MCAO mice also exhibited increased neurological deficit (3.36 ± 0.23, P &lt;0.01 versus MCAO mice), which was accompanied by exaggerated degradation of tight junction proteins and blunted CaMKII phosphorylation. Conclusions: The present data indicate that diabetic mice are more susceptible to disturbances of cerebral capillary blood flow and neurovascular defects during ischemic conditions.

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.

Modulation of Pre‐Capillary Arteriolar Pressure with Drag‐Reducing Polymers: A Novel Method for Enhancing Microvascular Perfusion

We have shown that drag-reducing polymers (DRP) enhance capillary perfusion during severe coronary stenosis and increase red blood cell velocity in capillaries, through uncertain mechanisms. We hypothesize that DRP decreases pressure loss from the aorta to the arteriolar compartment. Intravital microscopy of the rat cremaster muscle and measurement of pressure in arterioles (diameters 20-132 μm) was performed in 24 rats. DRP (polyethylene oxide, 1 ppm) was infused i.v. and measurements were made at baseline and 20 minutes after completion of DRP infusion. In a 10-rat subset, additional measurements were made three minutes after the start, and one to five and 10 minutes after completion of DRP. Twenty minutes after the completion of DRP, mean arteriolar pressure was 22% higher than baseline (from 42 ± 3 to 49 ± 3 mmHg, p < 0.005, n = 24). DRP decreased the pressure loss from the aorta to the arterioles by 24% (from 35 ± 6 to 27 ± 5 mmHg, p = 0.001, n = 10). In addition, there was a strong trend toward an increase in pressure at 10 minutes after the completion of DRP (n = 10). Drag-reducing polymers diminish pressure loss between the aorta and the arterioles. This results in a higher pre-capillary pressure and probably explains the observed DRP enhancement in capillary perfusion.

Deterioration of cutaneous microcirculatory status of Kawasaki disease

Kawasaki disease (KD) is associated with generalized vasculitis with a predilection for coronary artery leading to ectasia and aneurysm in some cases. The aim of this study was to noninvasively assess the cutaneous microcirculation and correlate it with the coronary artery diameter in these patients. Laser Doppler flowmetry and dynamic capillaroscopy were performed at the nailbeds to assess total cutaneous blood flow and microcirculation in children with KD, both in the afebrile phase (after the resolution of fever) and convalescent phases, in comparison to controls. The 100 subjects analyzed in this study included 64 patients with KD (33 in afebrile phase and 31 in convalescent phase) and 36 normal controls. In KD, the capillary morphology was abnormal when compared to controls, with a larger diameter of the arterial and venous limbs, a higher intercapillary distance and a decrease in the loop numbers. Significantly decreased capillary blood cell velocity was noted in afebrile phase but not in convalescent phase. In the afebrile phase, a decreased capillary blood cell velocity significantly correlated with an increased coronary artery diameter. In conclusion, KD patients, both in the afebrile and convalescent phases, exhibited morphologic alterations in the microcirculation when compared to the controls. The results indicate the potential role of dynamic capillaroscopy for the noninvasive survey of microcirculation abnormalities in patients with KD.

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.

Immediate Effects of Monochromatic Infrared Energy on Microcirculation in Healthy Subjects

The purpose of this study was to evaluate the influence of monochromatic infrared energy (MIRE) on the microcirculation of the skin surface of the feet in healthy subjects. Near-infrared energy was shown to increase microcirculation in an animal study. In humans, only one case study demonstrated that MIRE increases microcirculation in the skin of the lower limbs. Thirty healthy volunteers were recruited and randomly allocated into three groups to receive either: (1) active MIRE; (2) sham MIRE (placebo group); or (3) warm packs (control group) on the feet. The MIRE device comprised an array of 60 x 890 nm LEDs attached to flexible pads (3×7.5 cm). Each diode spot size was 0.2 cm(2), and each LED power was 12 mW with a power density of 60 mW/cm(2). The arrays were placed in direct contact with the skin for 30 min delivering a total fluence of 108 J/cm(2) over an area of 22.5 cm(2). Capillary blood cell velocity (CBV) and superficial skin blood flow (flux) were recorded before and after intervention. Significant differences among the three groups were recorded in both CBV and flux (both p<0.05). Post-hoc comparisons indicated that a significantly greater increase in both CBV and flux occurred in the active MIRE group than in the placebo group and control group (all p<0.05). A 30-min MIRE produced a significantly greater increase in the CBV and flux of the feet in the active MIRE group than in the placebo and control groups.

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.

Lack of association between flow-mediated endothelium-dependent vasodilatation and biomarkers of endothelial dysfunction in patients with severe rheumatoid arthritis

Accelerated atherogenesis is one of the main reasons for the increased mortality observed in patients with rheumatoid arthritis (RA) [1]. Progressive impairment of macrovascular and microvascular endothelium-dependent vasodilatation is associated with increased risk of cardiovascular complications. Endothelial function can be noninvasively assessed by postocclusive reactive hyperemia, either as increase in the brachial artery blood flow, as flowmediated vasodilatation of the afferent artery (macrovascular), or as increase in skin microcirculatory flow detected by laser Doppler. Both macrovascular and microvascular studies have confirmed the presence of endothelial dysfunction in patients with long-standing RA [2, 3]. Elevated circulating levels of soluble adhesion molecules are also associated with cardiovascular risk factors and predict atherosclerosis and CV events [4, 5]. Measurement of these biomarkers of endothelial activation and dysfunction has confirmed the presence of endothelial dysfunction in patients with RA [6]. Since administration of anti-TNF-a resulted in a rapid improvement in endothelial function in patients with this chronic inflammatory disease [7], and reduction in some biomarkers of endothelial dysfunction was also observed in these patients following anti-TNF-a drugs [8], an issue of potential interest may be to establish whether a correlation between the levels of soluble biomarkers of endothelial dysfunction and the presence of impairment of endothelium-dependent vasodilatation exists in patients with RA. In this regard, Meyer et al. [9] assessed whether microvascular changes in capillary blood cell velocity had correlation with levels of soluble adhesion molecules in 30 patients with RA. However, no correlation between capillary blood cell velocity and serum levels of soluble intercellular adhesion molecule-1 (sICAM-1), soluble vascular cell adhesion molecule-1 (sVCAM-1), soluble E-selectin (sE-selectin), and soluble P-selectin (sP-selectin) was found. To further investigate into this issue, we aimed to determine whether levels of serum biomarkers of endothelial dysfunction might correlate with values of macrovascular flowmediated endothelium-dependent vasodilatation in 14 patients with severe RA refractory to conventional diseasemodifying anti-rheumatic drugs. Ethical approval and informed consent were obtained. All patients had severe disease (DAS28 in all cases greater than 3.2), refractory to conventional disease-modifying anti-rheumatic drugs including methotrexate, and were receiving periodical treatment with infliximab therapy. Evaluation of brachial M. A. Gonzalez-Gay (&) Department of Rheumatology, Hospital Universitario Marques de Valdecilla, IFIMAV, Avenida de Valdecilla s/n, 39008 Santander, Spain e-mail: miguelaggay@hotmail.com

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.