Red Blood Cell Flux Research Articles

Women with a history of preeclampsia have preserved sensory nerve-mediated dilatation in the cutaneous microvasculature.

What is the central question of this study? Are sensory nerve-mediated vasodilatation and the NO-dependent contribution to that response attenuated in the cutaneous microvasculature of women who have had preeclampsia? What is the main finding and its importance? Women who have had preeclampsia demonstrate attenuated microvascular endothelium-dependent dilatation compared to women with a history of uncomplicated pregnancy. However, there are no differences in sensory nerve-mediated vasodilatation between groups. This suggests that the neurogenic response is not altered following preeclampsia, and that the NO-dependent vasodilatation of the neurogenic response is not related to endothelium-dependent NO-mediated dilatation in these women. Women who have had preeclampsia (PE) demonstrate microvascular endothelial dysfunction, mediated in part by reduced nitric oxide (NO)-dependent mechanisms. Localized heating of the skin induces a biphasic vasodilatation response: a sensory nerve-mediated initial peak, followed by a sustained endothelium-dependent plateau. We have previously shown that the endothelium-dependent plateau is attenuated in PE. However, it is unknown if the sensory nerve-mediated initial peak is similarly attenuated. Therefore, the purpose of this study was to examine the effect of PE history on sensory nerve-mediated vasodilatation and the NO-dependent contribution to that response. We hypothesized that PE would have an attenuated initial peak and a reduced NO-dependent contribution to that response compared to women with a history of normotensive pregnancy (healthy controls, HC). Nine HC (31±4years) and nine PE (28±6years) underwent a standard local heating protocol (42°C; 0.1°Cs-1 ). Two intradermal microdialysis fibres were placed in the skin of the ventral forearm for the continuous local delivery of lactated Ringer solution alone (control) or 15-mM NG -nitro-l-arginine methyl ester for nitric oxide synthase (NOS) inhibition. Red blood cell flux was measured at each site by laser Doppler flowmetry (LDF). Cutaneous vascular conductance was calculated (CVC=LDF/mean arterial pressure) and normalized to maximum (%CVCmax ; 28-mM SNP + local heat 43°C). There were no differences in the initial peak between groups (HC: 79±8 vs. PE: 80±10%CVCmax ; P=0.936). NOS inhibition attenuated the initial peak in both HC (57±18% CVCmax ; P=0.003) and PE (54±10%CVCmax ; P=0.002). However, there were no differences in the NO-dependent portion of the initial peak (HC: 23±16 vs. PE: 24±9%; P=0.777). The local heating plateau (HC: 99±4 vs. PE: 88±7%CVCmax ; P=0.001) and NO contribution to the plateau (HC: 31±9 vs. PE: 17±14%; P=0.02) were attenuated in PE. There was no relation between NO-dependent dilatation in the initial peak and NO-dependent dilatation in the plateau across groups (R2 =0.005; P=0.943). Women who have had PE demonstrate attenuated microvascular endothelium-dependent dilatation. However, there are no differences in sensory nerve-mediated vasodilatation following PE, suggesting that the NO-dependent vasodilatation of the neurogenic response is not related to endothelium-dependent NO-mediated dilatation in these women.

Menthol-induced activation of TRPM8 receptors increases cutaneous blood flow across the dermatome.

Topical menthol-based analgesics increase skin blood flow (SkBF) through transient receptor potential melastatin 8 (TRPM8) receptor-dependent activation of sensory nerves and endothelium-derived hyperpolarization factors. It is unclear if menthol-induced TRPM8 activation mediates a reflex change in SkBF across the dermatome in an area not directly treated with menthol. The purpose of this study was to determine the effects of localized topical menthol application on SkBF across a common dermatome. We hypothesized that SkBF would be increased with menthol at the site of application and across the dermatome (contralateral limb) through a spinal reflex mechanism. In a double blind, placebo controlled, cross-over design, 15 healthy participants (7 men; age=22±1yrs) were treated with direct application (3ml over 8×13cm) of 5% menthol gel (Biofreeze™) or placebo gel on the L4 dermatome, separated by 48h. Red blood cell flux was measured using laser Doppler flowmetry over the area of application, on the contralateral leg of the same dermatome, and in a separate dermatome (L5/S1) to serve as control. Cutaneous vascular conductance was calculated for each measurement site (CVC=flux/MAP). At baseline there were no differences in CVC between menthol and placebo gels, or among sites (all p>0.05). After 30±6min, CVC increased at the treated site with menthol (0.12±0.02 vs. 1.36±0.19flux/mmHg, p<0.01) but not the placebo (0.10±0.01 vs. 0.18±0.04flux/mmHg, p=0.91). There was a modest increase in CVC at the contralateral L4 dermatome with menthol gel (0.16±0.04 vs. 0.29±0.06flux/mmHg, p<0.01), but not placebo (0.11±0.02 vs. 0.15±0.03flux/mmHg, p=0.41). There was no effect on SkBF from either treatments at the L5/S1 control dermatome (both, p>0.05), suggesting the lack of a systemic response. In conclusion, menthol containing topical analgesic gels increased SkBF at the treated site, and modestly throughout the dermatome. These data suggest menthol-induced activation of the TRPM8 receptors induces an increase in SkBF across the area of common innervation through a localized spinal reflex mechanism.

Contrast Ultrasound Assessment of Skeletal Muscle Recruitable Perfusion after Permanent Left Ventricular Assist Device Implantation: Implications for Functional Recovery

In heart failure with reduced ejection fraction (HFrEF), abnormal regulation of skeletal muscle perfusion contributes to reduced exercise tolerance. The aim of this study was to test the hypothesis that improvement in functional status after permanent left ventricular assist device (LVAD) implantation in patients with HFrEF is related to improvement in muscle perfusion during work, which was measured using contrast-enhanced ultrasound (CEUS). CEUS perfusion imaging of calf muscle at rest and during low-intensity plantar flexion exercise (20W, 0.2Hz) was performed in patients with HFrEF (n=22) at baseline and 3months after placement of permanent LVADs. Parametric analysis of CEUS data was used to quantify muscle microvascular blood flow (MBF), blood volume index, and red blood cell flux rate. For subjects alive at 3months, comparisons were made between those with New York Heart Association functional class I or II (n=13) versus III or IV (n=7) status after LVAD. Subjects were followed for a median of 5.7years for mortality. Echocardiographic data before and after LVAD placement and LVAD parameters were similar in subjects classified with New York Heart Association functional class I-II versus functional class III-IV after LVAD. Skeletal muscle MBF at rest and during exercise before LVAD implantation was also similar between groups. After LVAD placement, resting MBF remained similar between groups, but during exercise those with New York Heart Association functional class I or II had greater exercise MBF (111±60 vs 52±38 intensity units/sec, P=.03), MBF reserve (median, 4.45 [3.95 to 6.80] vs 2.22 [0.98 to 3.80]; P=.02), and percentage change in exercise MBF (median, 73% [-28% to 83%] vs -45% [-80% to 26%]; P=.03). During exercise, increases in MBF were attributable to faster microvascular flux rate, with little change in blood volume index, indicating impaired exercise-mediated microvascular recruitment. The only clinical or echocardiographic feature that correlated with post-LVAD exercise MBF was a history of diabetes mellitus. There was a trend toward better survival in patients who demonstrated improvement in muscle exercise MBF after LVAD placement (P=.05). CEUS perfusion imaging can quantify peripheral vascular responses to advanced therapies for HFrEF. After LVAD implantation, improvement in functional class is seen in patients with improvements in skeletal muscle exercise perfusion and flux rate, implicating a change in vasoactive substances that control resistance arteriolar tone.

Differential Hemodynamic Response of Pial Arterioles Contributes to a Quadriphasic Cerebral Autoregulation Physiology.

BackgroundCerebrovascular autoregulation (CA) regulates cerebral vascular tone to maintain near‐constant cerebral blood flow during fluctuations in cerebral perfusion pressure (CPP). Preclinical and clinical research has challenged the classic triphasic pressure‐flow relationship, leaving the normal pressure‐flow relationship unclear.Methods and ResultsWe used in vivo imaging of the hemodynamic response in pial arterioles to study CA in a porcine closed cranial window model during nonpharmacological blood pressure manipulation. Red blood cell flux was determined in 52 pial arterioles during 10 hypotension and 10 hypertension experiments to describe the pressure‐flow relationship. We found a quadriphasic pressure‐flow relationship with 4 distinct physiological phases. Smaller arterioles demonstrated greater vasodilation during low CPP when compared with large arterioles (P<0.01), whereas vasoconstrictive capacity during high CPP was not significantly different between arterioles (P>0.9). The upper limit of CA was defined by 2 breakpoints. Increases in CPP lead to a point of maximal vasoconstriction of the smallest pial arterioles (upper limit of autoregulation [ULA] 1). Beyond ULA1, only larger arterioles maintain a limited additional vasoconstrictive capacity, extending the buffer for high CPP. Beyond ULA2, vasoconstrictive capacity is exhausted, and all pial arterioles passively dilate. There was substantial intersubject variability, with ranges of 29.2, 47.3, and 50.9 mm Hg for the lower limit, ULA1, and ULA2, respectively.ConclusionsWe provide new insights into the quadriphasic physiology of CA, differentiating between truly active CA and an extended capacity to buffer increased CPP with progressive failure of CA. In this experimental model, the limits of CA widely varied between subjects.

Capillary hemodynamics and contracting skeletal muscle oxygen pressures in male rats with heart failure: Impact of soluble guanylyl cyclase activator

In heart failure with reduced ejection fraction (HFrEF), nitric oxide-soluble guanylyl cyclase (sGC) pathway dysfunction impairs skeletal muscle arteriolar vasodilation and thus capillary hemodynamics, contributing to impaired oxygen uptake (V̇O2) kinetics. Targeting this pathway with sGC activators offers a new treatment approach to HFrEF. We tested the hypotheses that sGC activator administration would increase the O2 delivery (Q̇O2)-to-V̇O2 ratio in the skeletal muscle interstitial space (PO2is) of HFrEF rats during twitch contractions due, in part, to increases in red blood cell (RBC) flux (fRBC), velocity (VRBC), and capillary hematocrit (Hctcap). HFrEF was induced in male Sprague-Dawley rats via myocardial infarction. After 3 weeks, rats were treated with 0.3 mg/kg of the sGC activator BAY 60–2770 (HFrEF + BAY; n = 11) or solvent (HFrEF; n = 9) via gavage b.i.d for 5 days prior to phosphorescence quenching (PO2is, in contracting muscle) and intravital microscopy (resting) measurements in the spinotrapezius muscle. Intravital microscopy revealed higher fRBC (70 ± 9 vs 25 ± 8 RBC/s), VRBC (490 ± 43 vs 226 ± 35 μm/s), Hctcap (16 ± 1 vs 10 ± 1%) and a greater number of capillaries supporting flow (91 ± 3 vs 82 ± 3%) in HFrEF + BAY vs HFrEF (all P < 0.05). Additionally, PO2is was especially higher during 12–34s of contractions in HFrEF + BAY vs HFrEF (P < 0.05). Our findings suggest that sGC activators improved resting Q̇O2 via increased fRBC, VRBC, and Hctcap allowing for better Q̇O2-to-V̇O2 matching during the rest-contraction transient, supporting sGC activators as a potential therapeutic to target skeletal muscle vasomotor dysfunction in HFrEF.

Piezo1 is a mechanosensor channel in CNS capillaries

Cerebral blood flow (CBF) is exquisitely controlled to meet the ever-changing demands of active neurons in the brain. Brain capillaries are equipped with sensors of neurovascular coupling agents released from neurons/astrocytes onto the outer wall of a capillary. While capillaries can translate external signals into electrical and Ca2+ changes, control mechanisms from the lumen are less clear. The continuous flux of red blood cells and plasma through narrow-diameter capillaries imposes mechanical forces on the luminal (inner) capillary wall. Whether-and, if so, how-the ever-changing CBF could be mechanically sensed in capillaries is not known. Here, we propose and provide evidence that the mechanosensitive Piezo1 channels operate as mechanosensors in CNS capillaries to ultimately regulate CBF. Patch clamp electrophysiology confirmed the expression and function of Piezo1 channels in brain cortical and retinal capillary endothelial cells. Mechanical or pharmacological activation of Piezo1 channels evoked currents that were sensitive to Piezo1 channel blockers. Using genetically encoded Ca2+ indicator (Cdh5-GCaMP8) mice, we observed that Piezo1 channel activation triggered Ca2+ signals in endothelial cells. An ex vivo pressurized retina preparation was employed to further explore the mechanosensitivity of capillary Piezo1-mediated Ca2+ signals. Genetic and pharmacologic manipulation of Piezo1 in endothelial cells had significant impacts on CBF, reemphasizing the crucial role of mechanosensation in blood flow control. In conclusion, this study shows that Piezo1 channels act as mechanosensors in capillaries, and that these channels initiate crucial Ca2+ signals. We further show that Piezo1 modulates CBF, an observation of profound significance for the control of brain blood flow in health and in disorders where hemodynamic forces are disrupted, such as hypertension.

Hydrogen sulfide-dependent microvascular vasodilation is improved following chronic sulfhydryl-donating antihypertensive pharmacotherapy in adults with hypertension.

Hypertension is characterized by systemic microvascular endothelial dysfunction, in part due to a functional absence of hydrogen sulfide (H2S)-mediated endothelium-dependent dilation. Treatment with a sulfhydryl-donating ACE inhibitor (SH-ACE inhibitor) improves endothelial function in preclinical models of hypertension. To date, no studies have directly assessed the effects of SH-ACE-inhibitor treatment on H2S-dependent vasodilation in humans with hypertension. We hypothesized that SH-ACE-inhibitor treatment would improve H2S-mediated endothelium-dependent vasodilation. Ten adults with hypertension [1 woman and 9 men; 56 ± 9 yr; systolic blood pressure (SBP): 141 ± 8.5 mmHg; diastolic blood pressure (DBP): 90.3 ± 6 mmHg] were treated (16 wk) with the SH-ACE-inhibitor captopril. Red blood cell flux (laser-Doppler flowmetry) was measured continuously during graded intradermal microdialysis perfusion of the endothelium-dependent agonist acetylcholine (ACh; 10-10 to 10-1 M) alone (control) and in combination with an inhibitor of enzymatic H2S production [10-3 M aminooxyacetate (AOAA)] preintervention and postintervention. Cutaneous vascular conductance (CVC; flux/mmHg) was calculated and normalized to the site-specific maximal CVC (0.028 M sodium nitroprusside and local heat to 43°C). Area under the curve was calculated using the trapezoid method. The 16-wk SH-ACE-inhibitor treatment resulted in a reduction of blood pressure (systolic BP: 129 ± 10 mmHg; diastolic BP: 81 ± 9 mmHg, both P < 0.05). Preintervention, inhibition of H2S production had no effect on ACh-induced vasodilation (316 ± 40 control vs. 322 ± 35 AU AOAA; P = 0.82). Captopril treatment improved ACh-induced vasodilation (316 ± 40 pre vs. 399 ± 55 AU post; P = 0.04) and increased the H2S-dependent component of ACh-induced vasodilation (pre: -6.6 ± 65.1 vs. post: 90.2 ± 148.3 AU, P = 0.04). These data suggest that SH-ACE-inhibitor antihypertensive treatment improves cutaneous microvascular endothelium-dependent vasodilation in adults with hypertension, in part via H2S-dependent mechanisms.NEW & NOTEWORTHY This is the first study to prospectively assess the effects of sulfhydryl antihypertensive treatment on microvascular endothelial function in adults with hypertension. Our data suggest that 16 wk of SH-ACE-inhibitor antihypertensive treatment improves cutaneous microvascular endothelium-dependent vasodilation in middle-aged adults with hypertension, in part via H2S-dependent mechanisms.

ACUTE DIETARY NITRATE MAY IMPAIR CUTANEOUS VASCULAR CONDUCTANCE DURING LOCAL SKIN HEATING IN POSTMENOPAUSAL WOMEN

Declining estradiol production during menopause is linked to increased oxidative stress and reduced endothelial-dependent vasodilation. It is unknown whether supplementation with dietary nitrate may improve nitric oxide (NO) bioactivity and microvascular function in postmenopausal women. PURPOSE: To determine the effects of acute dietary nitrate ingestion on NO-mediated cutaneous vascular conductance (CVC) in a cohort of postmenopausal women. METHODS: In eight women (mean ± SD: age = 62 ± 4 y, BMI = 31 ± 2 kg/m2), two laser-Doppler flowmetry probes were affixed to the dorsal aspect of the forearm for measurement of red blood cell (RBC) flux during local skin heating to 39 °C and 44 °C. Assessments were made before and 3 h after ingesting 140 mL of beetroot juice containing 12.9 mmoL of inorganic nitrate. Fractional exhaled nitric oxide (FeNO) concentrations were measured at the same time points. Cutaneous vascular conductance (CVC) at 39 °C was calculated as RBC flux/ mean arterial pressure then normalized as a proportion of maximal CVC at 44°C. During the plateau phase at 39 °C, median spectral power of the endothelial NO-dependent frequency interval was analyzed using a Morlet wavelet transform. Pre-post comparisons were performed using within-sample t-tests. RESULTS: Following beetroot juice ingestion, FeNO concentrations (mean ± SD: 18 ± 8 ppb vs. 35 ± 16 ppb; p = 0.006, dz = 1.4) and spectral power of the NO-dependent frequency interval (0.01 - 0.02 Hz; p = 0.018, dz = 1.1) increased. Despite evidence for greater NO bioavailability, CVC at 39 °C was reduced (76 ± 12% vs. 50 ± 18%; p < 0.001, dz = 2.3). There was no change in maximal CVC at 44 °C (p = 0.287). CONCLUSION: A functional consequence of increased NO-bioavailability with acute dietary nitrate ingestion appears to be a diminished NO-mediated CVC response to local skin heating in our cohort of postmenopausal women with overweight and obesity. Future research should consider the effects of dietary nitrate coupled with an antioxidant supplement as means to further support NO-mediated endothelial function in such populations.

Impact of cutaneous blood flow on NIR-DCS measures of skeletal muscle blood flow index.

Near-infrared diffuse correlation spectroscopy (NIR-DCS) is an optical technique for estimating relative changes in skeletal muscle perfusion during exercise but may be affected by changes in cutaneous blood flow, as photons emitted by the laser must first pass through the skin. Accordingly, the purpose of this investigation was to examine how increased cutaneous blood flow affects NIR-DCS blood flow index (BFI) at rest and during exercise using a passive whole body heating protocol that increases cutaneous, but not skeletal muscle, perfusion in the uncovered limb. BFI and cutaneous perfusion (laser-Doppler flowmetry) were assessed in 15 healthy young subjects before (e.g., rest) and during 5 min of moderate-intensity handgrip exercise in normothermic conditions and after cutaneous blood flow was elevated via whole body heating. Hyperthermia significantly increased both cutaneous perfusion (∼7.3-fold; P ≤ 0.001) and NIR-DCS BFI (∼4.5-fold; P ≤ 0.001). Although relative BFI (i.e., fold-change above baseline) exhibited a typical exponential increase in muscle perfusion during normothermic exercise (2.81 ± 0.95), there was almost no change in BFI during hyperthermic exercise (1.43 ± 0.44). A subset of eight subjects were subsequently treated with intradermal injection of botulinum toxin-A (Botox) to block heating-induced elevations in cutaneous blood flow, which 1) nearly abolished the hyperthermia-induced increase in BFI and 2) restored BFI kinetics during hyperthermic exercise to values that were not different from normothermic exercise (P = 0.091). Collectively, our results demonstrate that cutaneous blood flow can have a substantial, detrimental impact on NIR-DCS estimates of skeletal muscle perfusion and highlight the need for technical and/or pharmacological advancements to overcome this issue moving forward.NEW & NOTEWORTHY We used passive whole body heat stress, in combination with local intradermal botulinum toxin type A treatment, to experimentally manipulate cutaneous blood flow and investigate its impact on NIR-DCS measures of skeletal muscle BFI at rest and during exercise. Collectively, the results show that cutaneous blood flow, which was augmented in response to passive whole body heat stress, markedly affects NIR-DCS-derived BFI, such that the BFI signal becomes dominated by changes in cutaneous red blood cell flux.

Quantifying Intestinal Capillary Oxygenation Using Two‐photon Phosphorescence Lifetime Microscopy

Impaired oxygen delivery reduces the diversity of the gut microbiota1,2. Quantifying the microvascular oxygenation in the small intestines may help us better understand progression of the intestinal diseases such as inflammatory bowel disease (IBD), their relation with the changes in the gut microbiota, as well as implications to the microbiota–gut–brain axis. To start addressing these questions, we applied two-photon phosphorescence lifetime microscopy (2PLM) to quantify partial pressure of oxygen (pO2) and Red Blood Cell (RBC) flux in the capillary plexus through entire cecum wall in healthy, 2-month-old, female C57BL6 mice. Mice were anesthetized with isoflurane mixed with air (3% during surgery and 2% during imaging). The animals were breathing spontaneously through a loose mask. A small incision was made into the abdomen and the cecum was gently pulled out and positioned into a custom-made chamber with a cover glass on top. The exposed intestine was kept moist with a warmed 1x phosphate buffered saline (PBS) solution. Both core body temperature and temperature over the cecum were maintained at 37 °C. Experiments were terminated when the peristaltic movement prevented microscopic measurements. We measured pO2 and RBC flux during normoxia (n=4 mice, mean pO2 = 60 ± 6 mmHg, mean capillary RBC flux = 23 ± 8 RBCs/s) and hyperoxia (100% O2 inhalation, n=3, mean pO2= 120 ± 15 mmHg, mean capillary RBC flux = 31 ± 17 RBCs/s), as a function of depth in the cecum wall from 0 to 120 µm. Our results show expected significant increase in pO2 in response to hyperoxia, small capillary mean RBC flux values, and approximately uniform capillary pO2 across the thickness of the cecum wall during normoxia. Future steps include investigation of the capillary oxygenation and RBC flux in pathological conditions and in relation with the diversity of the gut microbiome.

Cutaneous Microvascular Impairment in 5‐Fluorouracil Treated Cancer Patients

INTRODUCTION Despite frequent use and high efficacy in treatment of several cancers, 5-Fluorouracil (5-FU) chemotherapy is associated with acute and late occurring cardiotoxicity, ranging from vasospastic angina to increased risk of overt cardiovascular disease mortality. To date, the mechanisms by which these toxicities occur are not well understood, with leading theories implicating coronary vascular dysfunction by way of endothelial-dependent vasodilator impairment. PURPOSE & HYPOTHESIS Therefore, the purpose of the present research was to evaluate multiple mechanisms of endothelial function within the cutaneous microcirculation of 5-FU treated cancer patients (5-FU) and matched controls (CON) using the noninvasive measure of laser Doppler flowmetry (LDF). We hypothesized that 5-FU would attenuate both acetylcholine (ACh)-induced and local heating-induced cutaneous vasodilation. METHODS 17 5-FU (13 M, 4 F; age = 63.5 ± 12 yr) and 16 CON (8 M, 8 F; age = 58.1 ± 16 yr) volunteered for this study. Red blood cell flux was acquired via LDF in the forearm at baseline and following 1) iontophoresis of 200 µL of 2% ACh and 2) ~ 35 min of localized heating at 42°C. CVC was calculated from red blood cell flux divided by mean arterial pressure and presented as the percent increase from baseline to peak (%CVC). RESULTS ACh resulted in a significantly increased CVC in both groups, with no differences in %CVC between groups (CON = 920 ± 107%, 5-FU = 842 ± 113%; p = .62). Local heating, however, elicited a significantly lower %CVC in 5-FU treated patients (CON = 567.5 ± 77.7%, 5-FU = 256.7 ± 71.8%; p = .012). CONCLUSION Given the primary role of endothelial nitric oxide in local heating-induced cutaneous vasodilation, but not during ACh, our findings suggest that 5-FU chemotherapy treatment may result in endothelial dysfunction through nitric oxide mediated mechanisms. Experiments to corroborate these findings through iontophoresis of N-ω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) and in human coronary endothelial cell culture are ongoing by our group and will provide additional mechanistic insight into 5-FU induced cardiotoxicity.

Women with a History of Preeclampsia Have Preserved Neurogenically‐Mediated Dilation in the Cutaneous Microvasculature

Women who have had preeclampsia (PrEC) demonstrate microvascular endothelial dysfunction, mediated in part by reduced nitric oxide (NO)-dependent mechanisms. Localized heating of the skin induces a biphasic vasodilation response: a neurogenically-mediated initial peak, followed by a sustained endothelium-dependent plateau. We have previously shown that the endothelium-dependent plateau is attenuated in PrEC, due to attenuated NO-dependent dilation. However, it is unknown if the same is true concerning the neurogenically-mediated initial peak. The purpose of this study was to (1) examine the effect of preeclampsia history on neurogenically-mediated vasodilation and the NO-dependent contribution to that response, and (2) examine the relation between NO-dependent neurogenic dilation and the NO-dependent portion of the endothelium-dependent plateau. We hypothesized that PrEC would have an attenuated initial peak response and a reduced NO-dependent contribution to that response compared to women with a history of normotensive pregnancy (HC). Six HC (31±2yrs, 7±2 months post-partum) and 7 PrEC (29±2yrs, 7±3months post-partum) underwent a standard local heating protocol (42°C; 0.1°C·s-1). Two intradermal microdialysis fibers were placed in the skin of the ventral forearm for the continuous local delivery of Lactated Ringer's alone (control) or 15mM NG-nitro-L-arginine methyl ester (L-NAME) for nitric oxide synthase (NOS)-inhibition. Red blood cell flux was measured at each site via laser-Doppler flowmetry (LDF). Cutaneous vascular conductance was calculated (CVC=LDF/mean arterial pressure) and normalized to maximum (%CVCmax; 28mM SNP + local heat 43°C). NO-dependent dilation was calculated within-subject as the difference between control and NOS-inhibited sites. There were no differences in the initial peak between groups (HC: 80±5 vs PrEC: 79±5 %CVCmax; p=.88). NOS-inhibition attenuated the initial peak in both HC (Control: 80±5 vs L-NAME: 57±9 %CVCmax; p=0.02) and PrEC (Control: 79±5 vs L-NAME: 53±6 %CVCmax; p=0.004). However, there were no group differences in the NO-dependent portion of initial peak (HC: 23±8 vs PrEC: 24±5 %CVCmax; p=0.97). The local heating plateau (HC: 99±2 vs PrEC: 85±1 %CVCmax; p<0.001) and NO-contribution to the plateau (HC: 47±5 vs PrEC: 14±5 %CVCmax; p<0.001) were attenuated in PrEC compared to HC. There was no relation between NO-dependent dilation in the initial peak and NO-dependent dilation in the plateau across groups (R2 = 0.05; p = 0.51). Women who have had preeclampsia demonstrate attenuated microvascular endothelium-dependent dilation compared to women with a history of uncomplicated pregnancy. However, there are no differences in neurogenically-mediated vasodilation between the groups, suggesting that the NO-dependent vasodilation of the neurogenic response is not related to endothelium-dependent NO-mediated dilation in these women.

Effects of Pulmonary Hypertension on Microcirculatory Hemodynamics in Rat Skeletal Muscle

Introduction Pulmonary hypertension (PH) has previously been characterized as a disease of the pulmonary vasculature resulting subsequently in myocardial dysfunction. However, because heart failure compromises skeletal muscle microvascular function, contributing to exercise intolerance, we tested the hypothesis that such changes might be present in PH. Thus, we investigated skeletal muscle oxygen (O2) transport in the rat model of PH to determine if O2 delivery (Q̇O2) is impaired at the level of the microcirculation as evidenced via reduced red blood cell (RBC) flux, velocity, hematocrit, and percentage of capillaries flowing at rest. Methods Adult male Sprague-Dawley rats were randomized into healthy (n=9) and PH groups (n=4). Progressive PH was induced via a one-time intraperitoneal injection of monocrotaline (MCT; 50 mg/kg) and disease progression was monitored via echocardiography. Intravital microscopy in the resting spinotrapezius muscle was performed when moderate PH (preceding right ventricular (RV) failure) became evident. Results At 23 ± 1 days post-MCT, PH rats displayed RV hypertrophy (RV/Left ventricle+Septum: 0.28 ± 0.01 vs. 0.44 ± 0.04), pulmonary congestion, increased right ventricular systolic pressure (20 ± 3 vs. 48 ± 6 mmHg), and arterial hypoxemia (PaO2: 84 ± 3 vs. 64 ± 4 mmHg) compared to healthy rats (all P ≤ 0.05). Reduced capillary RBC velocity (403 ± 140 vs. 216 ± 88 µm/s; P = 0.03) and% of capillaries supporting RBC flux at rest (87 ± 5 vs. 75 ± 11%; P = 0.017) were evident in PH rats compared to healthy rats. Interestingly, no differences were observed in capillary RBC flux (P > 0.05) which was due, in part, to increased capillary hematocrit (10 ± 3 vs. 16 ± 4%; P < 0.05) in PH. However, when we quantified total Q̇O2 within a given field of view (RBC flux x % of capillaries supporting continuous RBC flux), PH rats demonstrated lower overall Q̇O2 (↓ 43%; P = 0.04). Conclusions These data support that microcirculatory hemodynamic impairments (↓ Q̇O2 and altered Q̇O2-to-V̇O2 matching) may compromise blood-myocyte O2 flux in PH. The mechanistic bases for the decreased capillary RBC velocity, elevated hematocrit, and reduced percentage of capillaries supporting RBC flux remains an important topic for future investigations.

Investigation of red blood cell partitioning in an in vitro microvascular bifurcation.

There is a long history of research examining red blood cell (RBC) partitioning in microvasculature bifurcations. These studies commonly report results describing partitioning that exists as either regular partitioning, which occurs when the RBC flux ratio is greater than the bulk fluid flowrate ratio, or reverse partitioning when the RBC flux ratio is less than or equal to that of the bulk fluid flowrate. This paper presents a study of RBC partitioning in a single bifurcating microchannel with dimensions of 6 to 16μm, investigating the effects of hematocrit, channel width, daughter channel flowrate ratio, and bifurcation angle. The erythrocyte flux ratio, N*, manifests itself as either regular or reverse partitioning, and time-dependent partitioning is much more dynamic, occurring as both regular and reverse partitioning. We report a significant reduction in the well-known sigmoidal variation of the erythrocyte flux ratio (N*) versus the volumetric flowrate ratio (Q*), partitioning behavior with increasing hematocrit in microchannels when the channel dimensions are comparable with cell size. RBCs "lingering" or jamming at the bifurcation were also observed and quantified in vitro. Results from trajectory analyses suggest that the RBC position in the feeder channel strongly affects both partitioning and lingering frequency of RBCs, with both being significantly reduced when RBCs flow on streamlines near the edge of the channel as opposed to the center of the channel. Furthermore, our experiments suggest that even at low Reynolds number, partitioning is affected by the bifurcation angle by increasing cell-cell interactions. The presented results provide further insight into RBC partitioning as well as perfusion throughout the microvasculature.

Regulation of capillary hemodynamics by KATP channels in resting skeletal muscle.

ATP‐sensitive K+ channels (KATP) have been implicated in the regulation of resting vascular smooth muscle membrane potential and tone. However, whether KATP channels modulate skeletal muscle microvascular hemodynamics at the capillary level (the primary site for blood‐myocyte O2 exchange) remains unknown. We tested the hypothesis that KATP channel inhibition would reduce the proportion of capillaries supporting continuous red blood cell (RBC) flow and impair RBC hemodynamics and distribution in perfused capillaries within resting skeletal muscle. RBC flux (f RBC), velocity (V RBC), and capillary tube hematocrit (Hctcap) were assessed via intravital microscopy of the rat spinotrapezius muscle (n = 6) under control (CON) and glibenclamide (GLI; KATP channel antagonist; 10 µM) superfusion conditions. There were no differences in mean arterial pressure (CON:120 ± 5, GLI:124 ± 5 mmHg; p > 0.05) or heart rate (CON:322 ± 32, GLI:337 ± 33 beats/min; p > 0.05) between conditions. The %RBC‐flowing capillaries were not altered between conditions (CON:87 ± 2, GLI:85 ± 1%; p > 0.05). In RBC‐perfused capillaries, GLI reduced f RBC (CON:20.1 ± 1.8, GLI:14.6 ± 1.3 cells/s; p < 0.05) and V RBC (CON:240 ± 17, GLI:182 ± 17 µm/s; p < 0.05) but not Hctcap (CON:0.26 ± 0.01, GLI:0.26 ± 0.01; p > 0.05). The absence of GLI effects on the %RBC‐flowing capillaries and Hctcap indicates preserved muscle O2 diffusing capacity (DO2m). In contrast, GLI lowered both f RBC and V RBC thus impairing perfusive microvascular O2 transport (Q̇m) and lengthening RBC capillary transit times, respectively. Given the interdependence between diffusive and perfusive O2 conductances (i.e., %O2 extraction∝DO2m/Q̇m), such GLI alterations are expected to elevate muscle %O2 extraction to sustain a given metabolic rate. These results support that KATP channels regulate capillary hemodynamics and, therefore, microvascular gas exchange in resting skeletal muscle.

Cyclic Hypoxia: An Update on Its Characteristics, Methods to Measure It and Biological Implications in Cancer.

Simple SummaryThe term hypoxia is used to describe biological situations where insufficient levels of oxygen exist. Hypoxia and hypoxic states can occur in a range of diseases including in cancer. Researchers have understood for numerous years that the hypoxia found in tumors leads to more aggressive and harder to treat disease and ultimately, poor patient outcome. While much research is, and has been, carried out to investigate the effects of hypoxia, this is usually done using experimental models which employ stable levels of hypoxia. However, we know that tumor hypoxia is not static but instead is rapidly changing through complex processes involving both rapid and slower fluctuations in oxygen concentrations. These dynamic changes in oxygen, known as cyclic hypoxia, are challenging to model experimentally and therefore our understanding of these processes has been limited. This review seeks to outline the known causes of cyclic hypoxia and the best ways to measure it both experimentally and clinically. How cancer cells respond to cyclic hypoxic compared to stable levels will also be discussed.Regions of hypoxia occur in most if not all solid cancers. Although the presence of tumor hypoxia is a common occurrence, the levels of hypoxia and proportion of the tumor that are hypoxic vary significantly. Importantly, even within tumors, oxygen levels fluctuate due to changes in red blood cell flux, vascular remodeling and thermoregulation. Together, this leads to cyclic or intermittent hypoxia. Tumor hypoxia predicts for poor patient outcome, in part due to increased resistance to all standard therapies. However, it is less clear how cyclic hypoxia impacts therapy response. Here, we discuss the causes of cyclic hypoxia and, importantly, which imaging modalities are best suited to detecting cyclic vs. chronic hypoxia. In addition, we provide a comparison of the biological response to chronic and cyclic hypoxia, including how the levels of reactive oxygen species and HIF-1 are likely impacted. Together, we highlight the importance of remembering that tumor hypoxia is not a static condition and that the fluctuations in oxygen levels have significant biological consequences.

Sural Nerve Perfusion in Mice.

Peripheral nerve function is metabolically demanding and nerve energy failure has been implicated in the onset and development of diabetic peripheral neuropathy and neuropathic pain conditions. Distal peripheral nerve oxygen supply relies on the distribution of red blood cells (RBCs) in just a few, nearby capillary-sized vessels and is therefore technically challenging to characterize. We developed an approach to characterize distal sural nerve hemodynamics in anesthetized, adult male mice using in vivo two-photon laser scanning microscopy. Our results show that RBC velocities in mouse sural nerve vessels are higher than those previously measured in mouse brain, and are sensitive to hindlimb temperatures. Nerve blood flow, measured as RBC flux, however, was similar to that of mouse brain and unaffected by local temperature. Power spectral density analysis of fluctuations in RBC velocities over short time intervals suggest that the technique is sufficiently sensitive and robust to detect subtle flow oscillations over time scales from 0.1 to tens of seconds. We conclude that in vivo two-photon laser scanning microscopy provides a suitable approach to study peripheral nerve hemodynamics in mice, and that local temperature control is important during such measurements.

Validation of red blood cell flux and velocity estimations based on optical coherence tomography intensity fluctuations

We present a validation of red blood cell flux and speed measurements based on the passage of erythrocytes through the OCT’s focal volume. We compare the performance of the so-called RBC-passage OCT technique to co-localized and simultaneously acquired two-photon excitation fluorescence microscopy (TPEF) measurements. Using concurrent multi-modal imaging, we show that fluctuations in the OCT signal display highly similar features to TPEF time traces. Furthermore, we demonstrate an overall difference in RBC flux and speed of 2.5 ± 3.27 RBC/s and 0.12 ± 0.67 mm/s (mean ± S.D.), compared to TPEF. The analysis also revealed that the OCT RBC flux estimation is most accurate between 20 RBC/s to 60 RBC/s, and is severely underestimated at fluxes beyond 80 RBC/s. Lastly, our analysis shows that the RBC speed estimations increase in accuracy as the speed decreases, reaching a difference of 0.16 ± 0.25 mm/s within the 0–0.5 mm/s speed range.

August Krogh's theory of muscle microvascular control and oxygen delivery: a paradigm shift based on new data.

August Krogh twice won the prestigious international Steegen Prize, for nitrogen metabolism (1906) and overturning the concept of active transport of gases across the pulmonary epithelium (1910). Despite this, at the beginning of 1920, the consummate experimentalist was relatively unknown worldwide and even among his own University of Copenhagen faculty. But, in early 1919, he had submitted three papers to Dr Langley, then editor of The Journal of Physiology in England. These papers coalesced anatomical observations of skeletal muscle capillary numbers with O2 diffusion theory to propose a novel active role for capillaries that explained the prodigious increase in blood-muscle O2 flux from rest to exercise. Despite his own appraisal of the first two papers as "rather dull" to his friend, the eminent Cambridge respiratory physiologist, Joseph Barcroft, Krogh believed that the third one, dealing with O2 supply and capillary regulation, was"interesting". These papers, which won Krogh an unopposed Nobel Prize for Physiology or Medicine in 1920, form the foundation for this review. They single-handedly transformed the role of capillaries from passive conduit and exchange vessels, functioning at the mercy of their upstream arterioles, into independent contractile units that were predominantly closed at rest and opened actively during muscle contractions in a process he termed 'capillary recruitment'. Herein we examine Krogh's findings and some of the experimental difficulties he faced. In particular, the boundary conditions selected for his model (e.g. heavily anaesthetized animals, negligible intramyocyte O2 partial pressure, binary open-closed capillary function) have not withstood the test of time. Subsequently, we update the reader with intervening discoveries that underpin our current understanding of muscle microcirculatory control and place a retrospectroscope on Krogh's discoveries. The perspective is presented that the imprimatur of the Nobel Prize, in this instance, may have led scientists to discount compelling evidence. Much as he and Marie Krogh demonstrated that active transport of gases across the blood-gas barrier was unnecessary in the lung, capillaries in skeletal muscle do not open and close spontaneously or actively, nor is this necessary to account for the increase in blood-muscle O2 flux during exercise. Thus, a contemporary model of capillary function features most muscle capillaries supporting blood flow at rest, and, rather than capillaries actively vasodilating from rest to exercise, increased blood-myocyte O2 flux occurs predominantly via elevating red blood cell and plasma flux in already flowing capillaries. Krogh is lauded for his brilliance as an experimentalist and for raising scientific questions that led to fertile avenues of investigation, including the study of microvascular function.

Healthy active older adults have enhanced K+ channel-dependent endothelial vasodilatory mechanisms.

Microvascular endothelial dysfunction, a precursor to atherosclerotic cardiovascular disease, increases with aging. Endothelium-derived hyperpolarizing factors (EDHFs), which act through K+ channels, regulate blood flow and are important to vascular health. It is unclear how EDHFs change with healthy aging. To evaluate microvascular endothelial reliance on K+ channel-mediated dilation as a function of age in healthy humans. Microvascular function was assessed using intradermal microdialysis in healthy younger (Y; n = 7; 3 M/4 W; 26 ± 1 yr) and older adults (O; n = 12; 5 M/7 W; 64 ± 2 yr) matched for V̇o2peak (Y: 39.0 ± 3.8, O: 37.6 ± 3.1 mL·kg-1·min-1). Participants underwent graded local infusions of: the K+ channel activator Na2S (10-6 to 10-1 M), acetylcholine (ACh, 10-10 to 10-1 M), ACh + the K+ channel inhibitor tetraethylammonium (TEA; 25 or 50 mM), and ACh + the nitric oxide synthase-inhibitor l-NAME (15 mM). Red blood cell flux was measured with laser-Doppler flowmetry and used to calculate cutaneous vascular conductance (CVC; flux/mean arterial pressure) as a percentage of each site-specific maximum (%CVCmax, 43°C+28 mM sodium nitroprusside). The %CVCmax response to Na2S was higher in older adults (mean, O: 51.7 ± 3.9% vs. Y: 36.1 ± 5.3%; P = 0.03). %CVCmax was lower in the ACh+TEA vs. the ACh site starting at 10-5 M (ACh: 34.0 ± 5.7% vs. ACh+TEA: 19.4 ± 4.5%; P = 0.002) in older and at 10-4 M (ACh: 54.5 ± 9.4% vs. ACh+TEA: 31.2 ± 6.7%; P = 0.0002) in younger adults. %CVCmax was lower in the ACh+l-NAME vs. the ACh site in both groups starting at 10-4 M ACh (Y: P < 0.001; O: P = 0.02). Healthy active older adults have enhanced K+ channel-dependent endothelial vasodilatory mechanisms, suggesting increased responsiveness to EDHFs with age.