Microvascular PO2 Research Articles

Transcapillary PO2 Gradients in Contracting Muscles of Type I Diabetic Rats.

This study aimed to clarify the effect of Type I diabetes (DIA) on transcapillary PO2 gradients, which are oxygen-driving factors between the blood and the interstitium, in the contracting muscle of rats. Wistar male rats were divided into the diabetic (streptozocin i.p.) and sham groups. Microvascular and interstitial PO2 were measured in the extensor digitorum longus muscle during electrical stimulation-induced muscle contraction, using the phosphorescence quenching method. Transcapillary PO2 gradient, ΔPO2, was calculated as microvascular minus interstitial PO2. Resting microvascular PO2 was higher in the diabetic group than in the sham group (6.3 ± 1.7 vs. 4.7 ± 0.9 mmHg, p < 0.05) and remained for 180 s. Interstitial PO2 from rest to muscle contraction did not differ between the groups. The ΔPO2 was higher in the diabetic group than in the sham group at rest and during muscle contraction (4.03 ± 1.42 vs. 2.46 ± 0.90 mmHg at rest; 3.67 ± 1.51 vs. 2.22 ± 0.65 mmHg during muscle contraction, p < 0.05). Marked muscle atrophy was observed in the diabetic group. DIA increased microvascular and transcapillary PO2 gradients in the skeletal muscle. The enhanced PO2 gradients were maintained from rest to muscle contraction in diabetic muscle.

Treatment of Hypotension with Phenylephrine Increases Cerebral Microvascular Perfusion in a Rat Model of Acute Hemodilution

Intraoperative blood loss is associated with reduced mean arterial blood pressure (MAP), which may endanger vital organ perfusion, including the brain. Phenylephrine (PE), an α1-receptor agonist, is clinically utilized to treat intraoperative hypotension through vasoconstriction of resistance arterioles. Clinical studies suggest that raising MAP with PE may decrease perfusion to vital organs such as the brain. Experimental studies suggest that lower α-receptor density in the cerebral resistance arteries may lead to preserved brain perfusion after PE administration. We hypothesize that treatment of hypotension with PE will result in preserved brain perfusion due to different microvascular blood flow and tissue oxygen tension (pO2) responses in the brain and skeletal muscle in an experimental rat model. With Animal Care and Use Committee approval in accordance with ARRIVE-2 guidelines, spontaneously breathing anesthetized rats (1.5% isoflurane) underwent hemodilution by exchanging 40-50% estimated blood volume 1:1 with saline over 10 minutes to achieve a 10-20 mmHg drop in MAP. MAP (tail artery) was measured continuously. Hypotension was treated with PE (10-20 μg/kg/min iv) to increase MAP to a target above baseline MAP. Shed blood was re-infused after stopping the PE infusion. Microvascular blood flow (Oxyflo; Oxford Optronix, Oxford, UK, n=7) or microvascular pO2 (OxyLED, Oxyphor G4, Oxygen Enterprises Ltd., Philadelphia, PA, n=6) were measured continuously in the cerebral cortex (burr hole) and biceps femoris muscle simultaneously in each rat. Results were analyzed via one or two-way ANOVA, with significance achieved at p&lt;0.05. Baseline and post-hemodilution MAP values were comparable in both groups. PE infusion increased MAP to a mean level near 100 mmHg in both groups (p&lt;0.05). Microvascular blood flow and tissue pO2 were greater in the brain as compared to the muscle at all time points (p&lt;0.01 for both). PE infusion resulted in an increase in cerebral microvascular blood flow from baseline (1465±689 vs. 687±453 blood perfusion units (BPU); p&lt;0.001) that was sustained after discontinuing PE and during blood reinfusion. No changes in muscle microvascular flow were observed after PE infusion (294±129 vs. 168±113 BPU). A sustained increase in cerebral microvascular pO2 was measured following PE infusion (50.2±8.6 vs. 40.6±4.6 mmHg; p=0.02), whereas muscle tissue pO2 did not change (22.2±5.6 vs. 25.0±2.5 mmHg). A differential blood flow and microvascular pO2 response was observed following PE infusion in brain and skeletal muscle. Both cerebral microvascular blood flow and tissue pO2 demonstrated sustained increases after PE infusion. By contrast, no changes were observed in muscle microvascular blood flow and pO2. These results suggest that treatment of hypotension with PE administration preferentially increased cerebral vs. muscle perfusion in an experimental model of acute blood loss and fluid resuscitation. Canadian Anesthesiologists’ Society and University of Toronto Merit Award Support. 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.

Bilateral nephrectomy impairs cardiovascular function and cerebral perfusion in a rat model of acute hemodilutional anemia.

Anemia and renal failure are independent risk factors for perioperative stroke, prompting us to assess the combined impact of acute hemodilutional anemia and bilateral nephrectomy (2Nx) on microvascular brain Po2 (PBro2) in a rat model. Changes in PBro2 (phosphorescence quenching) and cardiac output (CO, echocardiography) were measured in different groups of anesthetized Sprague-Dawley rats (1.5% isoflurane, n = 5-8/group) randomized to Sham 2Nx or 2Nx and subsequently exposed to acute hemodilutional anemia (50% estimated blood volume exchange with 6% hydroxyethyl starch) or time-based controls (no hemodilution). Outcomes were assessed by ANOVA with significance assigned at P < 0.05. At baseline, 2Nx rats demonstrated reduced CO (49.9 ± 9.4 vs. 66.3 ± 19.3 mL/min; P = 0.014) and PBro2 (21.1 ± 2.9 vs. 32.4 ± 3.1 mmHg; P < 0.001) relative to Sham 2Nx rats. Following hemodilution, 2Nx rats demonstrated a further decrease in PBro2 (15.0 ± 6.3 mmHg, P = 0.022). Hemodiluted 2Nx rats did not demonstrate a comparable increase in CO after hemodilution compared with Sham 2Nx (74.8 ± 22.4 vs. 108.9 ± 18.8 mL/min, P = 0.003) that likely contributed to the observed reduction in PBro2. This impaired CO response was associated with reduced fractional shortening (33 ± 9 vs. 51 ± 5%) and increased left ventricular end-systolic volume (156 ± 51 vs. 72 ± 15 µL, P < 0.001) suggestive of systolic dysfunction. By contrast, hemodiluted Sham 2Nx animals demonstrated a robust increase in CO and preserved PBro2. These data support the hypothesis that the kidney plays a central role in maintaining cerebral perfusion and initiating the adaptive increase in CO required to optimize PBro2 during acute anemia.NEW & NOTEWORTHY This study has demonstrated that bilateral nephrectomy acutely impaired cardiac output (CO) and microvascular brain Po2 (PBro2), at baseline. Following acute hemodilution, nephrectomy prevented the adaptive increase in CO associated with acute hemodilution leading to a further reduction in PBro2, accentuating the degree of cerebral tissue hypoxia. These data support a role for the kidney in maintaining PBro2 and initiating the increase in CO that optimized brain perfusion during acute anemia.

Plasma resuscitation improves and restores intestinal microcirculatory physiology following haemorrhagic shock.

Intestinal ischaemia-reperfusion injury following resuscitated haemorrhagic shock (HS) leads to endothelial and microcirculatory dysfunction and intestinal barrier breakdown. Although vascular smooth muscle machinery remains intact, microvascular vasoconstriction occurs secondary to endothelial cell dysfunction, resulting in further ischaemia and organ injury. Resuscitation with fresh frozen plasma (FFP) improves blood flow, stabilizes the endothelial glycocalyx and alleviates organ injury. We postulate these improvements correlate with decreased tissue CO2 concentrations, improved microvascular oxygenation and attenuation of intestinal microvascular endothelial dysfunction. Male Sprague-Dawley rats were randomly assigned to groups (n = 8/group): (1) sham, (2) HS (40% mean arterial blood pressure [MAP], 60 min) + crystalloid resuscitation (CR) (shed blood saline) and (3) HS + FFP (shed blood + FFP). MAP, heart rate (HR), ileal perfusion, pO2 and pCO2 were measured at intervals until 4 h post-resuscitation (post-RES). At 4 h post-RES, the ileum was rinsed in situ with Krebs solution. Topical acetylcholine and then nitroprusside were applied for 10 min each. Serum was obtained, and after euthanasia, tissues were harvested and snap-frozen in liquid N2 and stored at -80°C. FFP resuscitation resulted in sustained ileal perfusion as well as rapid sustained return to baseline microvascular pO2 and pCO2 values when compared to CR (p < 0.05). Endothelial function was preserved relative to sham in the FFP group but not in the CR group (p < 0.05). FFP-based resuscitation improves intestinal perfusion immediately following resuscitation, which correlates with improved tissue oxygenation and decreased tissue CO2 levels. CR resulted in significant damage to endothelial vasodilation response to acetylcholine, while FFP preserved this function.

Combination of hyperoxia and a right-shifted HbO2 dissociation curve delays V̇o2 kinetics during maximal contractions in isolated muscle.

Acute enhancement of peripheral O2 diffusion may accelerate skeletal muscle O2 uptake (V̇o2) kinetics and lessen fatigue during transitions from rest to maximal contractions. Surgically isolated canine gastrocnemius muscles in situ (n = 6) were studied during transitions from rest to 4 min of electrically stimulated isometric tetanic contractions at V̇o2peak, in two conditions: normoxia (CTRL) and hyperoxia ([Formula: see text] = 1.00) + administration of a drug (RSR-13), which right shifts the Hb-O2 dissociation curve (Hyperoxia + RSR-13). Before and during contractions, muscles were pump-perfused with blood at constant elevated flow ([Formula: see text]) and infused with the vasodilator adenosine. Arterial ([Formula: see text]) and muscle venous ([Formula: see text]) O2 concentrations were determined at rest and at 5- to 7-s intervals during contractions; V̇o2 was calculated as [Formula: see text]·([Formula: see text] - [Formula: see text]). Po2 at 50% of Hb saturation (standard P50) and mean microvascular Po2 ([Formula: see text]) were calculated by the Hill equation and a numerical integration technique. P50 [42 ± 7 (means ± SD) mmHg vs. 33 ± 2 mmHg, P = 0.02] and [Formula: see text] (218 ± 73 mmHg vs. 49 ± 4 mmHg, P = 0.003) were higher in Hyperoxia + RSR-13. Muscle force and fatigue were not different in the two conditions. V̇o2 kinetics (monoexponential fitting) were unexpectedly slower in Hyperoxia + RSR-13, due to a longer time delay (TD) [9.9 ± 1.7 s vs. 4.4 ± 2.2 s (P = 0.001)], whereas the time constant (τ) was not different [13.7 ± 4.3 s vs. 12.3 ± 1.9 s (P = 0.37)]; the mean response time (TD + τ) was longer in Hyperoxia + RSR-13 [23.6 ± 3.5 s vs. 16.7 ± 3.2 s (P = 0.003)]. Increased O2 availability deriving, in Hyperoxia + RSR-13, from higher [Formula: see text] and from presumably greater intramuscular O2 stores did not accelerate the primary component of the V̇o2 kinetics, and delayed the metabolic activation of oxidative phosphorylation.NEW & NOTEWORTHY In isolated perfused skeletal muscle, during transitions from rest to V̇o2peak, hyperoxia and a right-shifted oxyhemoglobin dissociation curve increased O2 availability by increasing microvascular Po2 and by presumably increasing intramuscular O2 stores. The interventions did not accelerate the primary component of the V̇o2 kinetics (as calculated from blood O2 unloading) and delayed the metabolic activation of oxidative phosphorylation. V̇o2 kinetics appear to be mainly controlled by intramuscular factors related to the use of high-energy "buffers."

Impact of cell‐free hemoglobin on oxygen pressure dynamics in muscles comprised primarily of fast‐twitch fibers

Cell free hemoglobin (Hb), released due to hemolysis, reduces the bioavailability of nitric oxide (NO) and the microvascular PO2 (PO2mv, the pressure head that facilitates blood‐myocyte O2 flux) during contractions in mixed fiber‐type skeletal muscle. Despite the known impacts of cell‐free Hb on mixed fiber type muscles, its effect within muscles composed primarily of fast‐twitch fibers remains unknown. Thus, the purpose of this investigation was to test the hypothesis that cell free Hb would lower the PO2mv in the white (fast‐twitch) portion of the gastrocnemius muscle during muscle contractions with no effect at rest or during the rest‐contraction transition. Six young‐adult male Sprague‐Dawley rats had the white portion of the gastrocnemius muscle surgically exposed (100% type IIb + dx). PO2mv was measured at rest and during 180 s of electrically induced contractions (1 Hz, 6‐8 v) under control and following 50 mg of Hb infusion (tail‐artery catheter). Hb resulted in a 48% increase in mean arterial pressure at the onset of muscle contractions (control: 97.16 ± 6.5, Hb 143.40 ± 9.0 mmHg, p&lt;0.01), significantly shortened time‐delay (control 14.3 ± 2.0, Hb 4.8 ± 1.6 s, p&lt;0.02), and mean response time (control 24.9 ± 1.2, Hb 16.4 ± 2.0 s, p&lt;0.02). There were no between‐condition differences in steady‐state (control 10.4 ± 1.4, Hb 14.1 ± 1.4 mmHg, p&gt;0.05) or baseline PO2mv (control 16.4 ± 0.2, Hb 19.1 ± 0.01). Acute exposure to cell‐free Hb resulted in a faster fall in the PO2mv during the rest‐contraction transition. However, despite a significant increase in mean arterial pressure, there were no differences in the PO2mv observed during the contracting steady state. These results suggest that cell free Hb significantly increases vascular resistance within fast‐twitch muscle, which causes O2 delivery‐to‐utilization mismatch at the onset of muscle contractions. These findings provide a potential mechanism by which hemolytic diseases like sickle cell disease impair exercise tolerance. Given that slow‐twitch muscles rely more heavily on NO to support O2 delivery at rest and during contractions, it is likely that the effects observed in the present investigation will be exacerbated in highly oxidative muscles.

Bilateral Nephrectomy Impairs Cerebral Oxygen Delivery After Acute Hemodilution Anemia in Rats

HypothesisWe hypothesize that renal sensing of hypoxia is essential for activation of cardiovascular responses needed to maintain cerebral oxygen delivery during acute anemia and that bilateral nephrectomy will negatively affect brain oxygen delivery during acute hemodilutional anemia.MethodsAll experiments were approved by the Animal Care and Use Committee at St. Michael’s Hospital and conducted in accordance with ARRIVE‐2 guidelines. Sham or bilateral nephrectomy (n=5) were performed on isoflurane anesthetized Sprague‐Dawley rats prior to performing acute hemodilution (50% estimated blood volume) 1:1 with 6% hydroxyethyl starch. Heart rate (HR), mean arterial pressure (MAP) and rectal temperature were monitored continuously. Arterial blood gases and cooximetry samples were measured before and after hemodilution. Continuous measurements of brain microvascular oxygen tension (PbrO2) were measured via a phosphorescence quenching method using an intravascular oxygen probe, Oxyphor PdG4, and an OxyLED phosphorometer. Data were tested for normality and analysis performed by two‐way ANOVA with p&lt;0.05 taken to be significant.ResultsBilateral nephrectomy resulted in a reduction in baseline HR (p&lt;0.004). Hemodilution resulted in a comparable reduction in hemoglobin concentration in both groups (Sham: 5.1±0.3 g/dL; Nephrectomy: 4.2±0.4 g/dL) with no differences in arterial PaO2 or PaCO2. After hemodilution, HR increased significantly in both groups (p&lt;0.05). MAP tended to be lower in the bilateral nephrectomy group and decreased transiently in both groups following hemodilution (p&lt;0.01). Brain microvascular pO2 (PbrO2)was not significantly different between nephrectomy and sham at baseline. Post‐hemodilution the PbrO2 was significantly lower in nephrectomised rats (11.5±5.1 mmHg) compared to sham operated rats (25.4±11.3 mmHg) (p=0.031).ConclusionsWe observed that bilateral nephrectomy resulted in changes in baseline physiology, including a lower HR. Hemodilution resulted in a comparable reduction in hemoglobin concentration, without difference in PaO2 or PaCO2. MAP decreased more profoundly immediately after hemodilution in nephrectomised animals, and recovered with time. Nephrectomy resulted in a significant drop in brain microvascular pO2 (PbrO2) immediately after hemodilution. This was not observed in sham controls. These data suggest that the kidney serves an important sensing role during acute anemia which supports cardiovascular responses and preserves the adequacy of brain tissue oxygen delivery. Hypoxia sensing by the kidney, and other integrative systemic mechanisms, may be central to these findings.

Increased Hepatic Microvascular Density, Oxygenation, and VEGF in the Hypertrophic Lobe following Portal Vein Embolization in Rabbits

Introduction: The microvascular events following portal vein embolization (PVE) are poorly understood despite the pivotal role of the microcirculation in liver regeneration and tumor progression. We aimed to assess the changes in hepatic microvascular perfusion and neo-angiogenesis after experimental PVE. Methods: PVE of the cranial liver lobes was performed in 12 New Zealand White rabbits divided into 2 groups of permanent (P-PVE) and reversible PVE (R-PVE), respectively. Hepatobiliary scintigraphy and CT were used to evaluate hepatic function and volume. Hepatic microcirculation was assessed using a handheld vital microscope (Cytocam) to measure microvascular density (total vessel density; TVD) before PVE, right after PVE, and 20 min after PVE, as well as at 14 days (D14 post-PVE) and 35 days (D35 post-PVE). Additionally, on D35, microvascular PO₂ and liver parenchymal VEGF were assessed. Results: Eleven rabbits were included after PVE (R-PVE, n = 5; P-PVE, n = 6). TVD in the nonembo­lized (hypertrophic) lobes was higher than in the embolized (atrophic) lobes of the P-PVE group at D35 post-PVE (36.7 ± 7.2 vs. 23.4 ± 4.9 mm/mm²; p < 0.05). In the R-PVE group, TVD in the nonembolized lobes was not increased at D35. Function and volume were increased in the nonembolized lobes of the P-PVE group compared to the embolized lobes, but not in the R-PVE group. Likewise, the mmicrovascular PO₂ and VEGF staining rate were higher in the nonembolized lobes of the P-PVE group at D35 post-PVE. Discussion/Conclusion: Successful volumetric and functional hypertrophy of the nonembolized lobe was accompanied by microvascular alterations featuring increased neo-angiogenesis, microvascular density, and microvascular oxygen pressure following P-PVE.

Renal Oxygen Sensing Mechanisms May Contribute to Maintaining Cerebral Perfusion During Acute Anemia

Introduction The integrative physiological response to anemia is complex and incompletely understood. We utilized data from studies of acute anemia in rodents to determine the relationship between changes in blood oxygen content (CaO2) and the heterogenous response of the kidney, heart and brain. We hypothesize that renal hypoxia sensing mechanisms contribute to adaptive physiological responses to maintain cerebral oxygen delivery (DO2) during acute anemia. Methods With animal care committee approval, we synthesized novel and published data from 5 previously published studies. Outcomes included: assessment of the relationship between CaO2 and microvascular renal and brain PO2 (phosphorescence quenching of oxyphor G4); cardiac output (CO) and renal and cerebral blood flow (ultrasound Doppler); hypoxia induced cellular responses (brain and kidney erythropoietin (EPO) mRNA and serum protein levels (ELISA)). Statistical analysis (SigmaPlot 14) was performed by ANOVA, Holm-Sidak and Mann-Whitney rank sum test when appropriate. Significance was assigned at p<0.05. Results In two models of anemia (hemodilution and RBC antibody mediated), acute reductions in blood CaO2 were associated with larger decreases in renal microvascular PO2, relative to brain microvascular PO2 (p<0.05). After acute hemodilution, there was a strong relationship between CaO2 and renal microvascular PO2 (r2=0.75). The magnitude of reduction in renal microvascular PO2 correlated with the degree of renal EPO mRNA expression and serum EPO protein levels. The magnitude of the increase in EPO mRNA was much larger in the kidney than in the brain (p<0.03). While no change in renal blood flow was observed in either model, a significant increase in common carotid and internal carotid blood flow was observed in both models (p<0.012). When DO2 was assessed, the kidney DO2 was reduced at all levels of anemia (p<0.01) whereas brain tissue DO2 was maintained in mild and moderate (Hb 90 and 70 g/L) (p=0.44) anemia but reduced in severe anemia (Hb 50 g/L) (p<0.02). The role of active cardiovascular increases in brain blood flow and maintained DO2 during anemia was impaired by systemic beta blockade, suggesting that active cardiovascular mechanisms are required to maintain optimal brain DO2 during anemia. Discussion Our analysis demonstrated: evidence of quantitative renal PO2 sensing of changes in CaO2; the clamping of renal blood flow (reduced DO2) during anemia may be a central mechanism allowing for sensing of changes in CaO2; reduced arterial CaO2 resulted in a local renal hypoxia response (increased serum EPO) and may have initiated the cardiovascular response to increase cerebral blood flow and maintain cerebral DO2. Inhibition of the adrenergic system impaired these responses and resulted in reduced brain DO2. Understanding the heterogeneous adaptive responses to acute anemia may inform clinical practice and optimize management of acutely anemia patients.

Impact Of Cell-free Hemoglobin On Exercising Muscle Vascular Control In Rats

Hemolysis associated with Sickle Cell Disease (SCD) compromises nitric oxide (NO) bioavailability and results in a plethora of cardiopulmonary and skeletal muscle complications causing severe exercise intolerance. Recent evidence suggests that cell-free Hb reduces NO bioavailability and lowers the skeletal muscle microvascular PO2 during contractions, likely due to a reduction in blood flow. Despite these observations, the effects of Hb on skeletal muscle vascular control during locomotory exercise remain unknown. PURPOSE: We tested the hypothesis that acute exposure to Hb would increase mean arterial pressure (MAP) and decrease hindlimb muscle blood flow in the exercising rat. METHODS: MAP and hindlimb skeletal muscle blood flow (fluorescent microspheres) were measured in male Sprague-Dawley rats (3-6 months, n=8) during submaximal treadmill running (20 ml/min, 5% grade) following a vehicle (0.2 ml of saline) and Hb (50 mg/kg) infusion. RESULTS: Relative to control, Hb resulted in a significantly greater exercising MAP (control: 137 ± 3, Hb: 150 ± 3 mmHg, p<0.05) and blood [lactate] (control: 2.51 ± 0.25, Hb: 3.13 ± 0.42 mM, p<0.05). Total exercising hindlimb skeletal muscle blood flow (control: 179 ± 14, Hb: 111 ± 7, ml/min/100 g, p<0.05) and vascular conductance (control: 1.34 ± 0.13, Hb: 0.75 ± 0.05 ml/min/100 g/mmHg, p<0.05) were lower following Hb infusion when compared to control. CONCLUSION: These data support the hypothesis that free Hb impairs vascular control and lowers skeletal muscle O2 delivery during exercise and provides a potential mechanism by which hemolytic diseases like SCD impair exercise tolerance in humans. Support: NIH-P30DK048520 (SKF), NIH-1R01HL125642-01A1 (DCI)

Skeletal muscle interstitial Po2 kinetics during recovery from contractions.

The oxygen partial pressure in the interstitial space (Po2 is) drives O2 into the myocyte via diffusion, thus supporting oxidative phosphorylation. Although crucial for metabolic recovery and the capacity to perform repetitive tasks, the time course of skeletal muscle Po2 is during recovery from contractions remains unknown. We tested the hypothesis that Po2 is would recover to resting values and display considerable on-off asymmetry (fast on-, slow off-kinetics), reflective of asymmetric capillary hemodynamics. Microvascular Po2 (Po2 mv) was also evaluated to test the hypothesis that a significant transcapillary gradient (ΔPo2 = Po2 mv - Po2 is) would be sustained during recovery. Po2 mv and Po2 is (expressed in mmHg) were determined via phosphorescence quenching in the exposed rat spinotrapezius muscle during and after submaximal twitch contractions (n = 12). Po2 is rose exponentially (P < 0.05) from end-contraction (11.1 ± 5.1), such that the end-recovery value (17.9 ± 7.9) was not different from resting Po2 is (18.5 ± 8.1; P > 0.05). Po2 is off-kinetics were slower than on-kinetics (mean response time: 53.1 ± 38.3 versus 18.5 ± 7.3 s; P < 0.05). A significant transcapillary ΔPo2 observed at end-contraction (16.6 ± 7.4) was maintained throughout recovery (end-recovery: 18.8 ± 9.6; P > 0.05). Consistent with our hypotheses, muscle Po2 is recovered to resting values with slower off-kinetics compared with the on-transient in line with the on-off asymmetry for capillary hemodynamics. Maintenance of a substantial transcapillary ΔPo2 during recovery supports that the microvascular-interstitium interface provides considerable resistance to O2 transport. As dictated by Fick's law (V̇o2 = Do2 × ΔPo2), modulation of O2 flux (V̇o2) during recovery must be achieved via corresponding changes in effective diffusing capacity (Do2; mainly capillary red blood cell hemodynamics and distribution) in the face of unaltered ΔPo2.NEW & NOTEWORTHY Capillary blood-myocyte O2 flux (V̇o2) is determined by effective diffusing capacity (Do2; mainly erythrocyte hemodynamics and distribution) and microvascular-interstitial Po2 gradients (ΔPo2 = Po2 mv - Po2 is). We show that Po2 is demonstrates on-off asymmetry consistent with Po2 mv and erythrocyte kinetics during metabolic transitions. A substantial transcapillary ΔPo2 was preserved during recovery from contractions, indicative of considerable resistance to O2 diffusion at the microvascular-interstitium interface. This reveals that effective Do2 declines in step with V̇o2 during recovery, as per Fick's law.

Managing the power grid: how myoglobin can regulate PO2 and energy distribution in skeletal muscle.

Managing the power grid: how myoglobin can regulate PO2 and energy distribution in skeletal muscle.

Skeletal muscle interstitial O2 pressures: bridging the gap between the capillary and myocyte.

The oxygen transport pathway from air to mitochondria involves a series of transfer steps within closely integrated systems (pulmonary, cardiovascular, and tissue metabolic). Small and finite O2 stores in most mammalian species require exquisitely controlled changes in O2 flux rates to support elevated ATP turnover. This is especially true for the contracting skeletal muscle where O2 requirements may increase two orders of magnitude above rest. This brief review focuses on the mechanistic bases for increased microvascular blood-myocyte O2 flux (V̇O2 ) from rest to contractions. Fick's law dictates that V̇O2 elevations driven by muscle contractions are produced by commensurate changes in driving force (ie, O2 pressure gradients; ΔPO2 ) and/or effective diffusing capacity (DO2 ). While previous evidence indicates that increased DO2 helps modulate contracting muscle O2 flux, up until recently the role of the dynamic ΔPO2 across the capillary wall was unknown. Recent phosphorescence quenching investigations of both microvascular and novel interstitial PO2 kinetics in health have resolved an important step in the O2 cascade between the capillary and myocyte. Specifically, the significant transmural ΔPO2 at rest was sustained (but not increased) during submaximal contractions. This supports the contention that the blood-myocyte interface provides a substantial effective resistance to O2 diffusion and underscores that modulations in erythrocyte hemodynamics and distribution (DO2 ) are crucial to preserve the driving force for O2 flux across the capillary wall (ΔPO2 ) during contractions. Investigation of the O2 transport pathway close to muscle mitochondria is key to identifying disease mechanisms and develop therapeutic approaches to ameliorate dysfunction and exercise intolerance.

Impact of cell‐free hemoglobin on contracting skeletal muscle oxygen pressure dynamics: Potential therapeutic role of haptoglobin

Free hemoglobin (Hb) associated with hemolytic diseases (i.e., sickle cell anemia) extravasates into vascular tissue and depletes nitric oxide (NO), which leads to impaired vascular function and could disrupt skeletal muscle metabolic control during exercise. We tested the hypothesis that: 1) free Hb would extravasate into skeletal muscle tissue impairing the contracting skeletal muscle O2 delivery/O2 utilization ratio (microvascular PO2, PO2mv) to a similar extent as that observed following NO synthase (NOS) blockade, and 2) that the Hb scavenging protein haptoglobin (Hp) would prevent Hb extravasation and inhibit these skeletal muscle tissue effects. PO2mv was measured in eight healthy rats (phosphorescence quenching) at rest and during 180s of electrically induced (1‐Hz) twitch spinotrapezius muscle contractions (experiment 1). A second subset of seven rats was also used to investigate the effects of Hb+Hp (experiment 2). For both experiments, measurements were made: 1) during control conditions, 2) following a bolus infusion of either Hb (50 mg/kg) or Hb+Hp (50 mg/kg, bound Hb), and 3) following local superfusion of NG‐nitro‐l‐arginine methyl ester (L‐NAME; 10 mg/kg). Additional experiments were completed to visualize Hb extravasation into the muscular tissue using Click chemistry techniques. There were no significant differences in the PO2mv observed at rest for any condition in either experiment (p&gt;0.05 for all). In experiment 1, both Hb and L‐NAME reduced the PO2mv significantly during the steady‐state of muscle contractions when compared to control conditions with no differences between Hb and L‐NAME conditions (control: 24 ± 1, Hb: 21 ± 1, L‐NAME: 20 ± 1 mmHg, p&lt;0.05). In experiment 2, only L‐NAME resulted in a significantly lower PO2mv during the steady‐state of muscle contractions (control: 25 ± 1, Hb+Hp: 22 ± 2, L‐NAME: 18 ± 1 mmHg, p&lt;0.05). Free Hb lowered the blood‐myocyte O2 driving force to a level not significantly different from L‐NAME. However, infusing Hb bound to Hp resulted in no significant differences in steady‐state PO2mv during muscle contractions when compared to control. Surprisingly, we did not observe Hb accumulation in skeletal muscle tissue. Taken together these data suggest that free Hb impairs O2 delivery/utilization via a NO scavenging effect which would presumably disrupt oxidative metabolism and shorten the onset of fatigue during exercise. Furthermore, the unchanged PO2mv steady‐state observed following Hb+Hp further suggests that therapeutic administration of the Hb binding protein haptoglobin may restore skeletal muscle metabolic control and potentially exercise tolerance in those afflicted with hemolytic diseases.Support or Funding InformationFunded by NIH (R01HL125642) to DCI and Colorado Nutrition and Obesity Research Center (P30DK048520) award to SKF.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Impact of chronic free hemoglobin infusion on skeletal muscle microvascular PO2 dynamics in rats

Patients with hemolytic diseases (i.e. sickle cell anemia) exhibit severely compromised exercise capacity and quality of life. This is likely due, at least in part, to chronic exposure to free hemoglobin (Hb) which results in a robust reduction in nitric oxide (NO) bioavailability and, particularly when combined with systemic hypoxia, enacts a proinflammatory cascade that causes vascular dysfunction and remodeling within the pulmonary circulation. Similar downstream effects may occur which could disrupt the QO2/VO2 relationship within skeletal muscle and thus impair tolerance to exercise. Therefore, the purpose of this investigation was to test the hypothesis that chronic exposure to free Hb would lower the microvascular PO2 (PO2mv, the pressure head that facilitates blood‐myocyte O2 flux) at rest and during muscle contractions with a greater deficit observed in rats exposed to hypoxia+Hb. Using programmable micropump technology, 8 young‐adult male Sprague‐Dawley rats were housed in normoxia (Hb+Nx, n=4) or hypoxia (Hb+Hx, n=4) while exposed to 60 mg/day Hb for 18 days while 4 rats were outfitted with dummy infusion pumps (SHAM, n=4) and were housed in normoxia. Following infusion, all rats performed a graded treadmill exercise test to fatigue for the determination of endurance capacity. PO2mv was then measured during 180s of electrically induced (1‐Hz) twitch spinotrapezius muscle contractions. There were no between group differences in exercise tolerance (average run time 16–18 min, p&gt;0.05). While there were no significant differences in any PO2mv kinetic parameter, exposure to hypoxia and Hb resulted in a significantly greater resting baseline (SHAM: 28.3 ± 4, Hb+Nx: 31.5 ± 2, Hb+Hx: 42.8 ± 3 mmHg) and contracting steady‐state PO2 (SHAM: 20.70 ± 4, Hb+Nx: 26 ± 2, Hb:Hx: 36.3 ± 2 mmHg) when compared to SHAM and Hb+Nx conditions, while the contracting steady‐state PO2 was also significantly higher in Hb+Nx vs. SHAM (p&lt;0.05). Blood hematocrit was significantly higher in Hb+Hx when compared to SHAM and Hb+Nx with no significant differences between SHAM and Hb+Nx (SHAM: 48.5 ± 3, Hb+Nx: 43.1 ± 3, Hb+Hx: 54.7 ± 2%, p&lt;0.05). Contrary to our original hypothesis, combined exposure to free Hb and hypoxia resulted in a greater baseline and contracting steady‐state PO2mv while Hb alone raised the steady‐state PO2mv relative to SHAM operated rats. Given that free Hb results in oxidative stress and cellular damage, the elevated PO2mv in both Hb groups likely reflects an impaired ability to utilize oxygen (↓VO2) within the skeletal muscle. This is supported by the lack of differences in exercise tolerance in Hb vs. SHAM despite an increased Hct (i.e. oxygen carrying capacity) observed in the Hb+Hx rats and significantly greater steady‐state PO2mv in both Hb groups. These results carry robust implications for those suffering from hemolytic diseases and provides a potential mechanism by which chronic exposure to free Hb impacts exercise tolerance and quality of life.Support or Funding InformationFunded by NIH (R01HL125642) to DCI and Colorado Nutrition and Obesity Research Center (P30DK048520) award to SKF.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Exercise (In)Tolerance in Sickle Cell Disease: Potential Disruptive Role of Free Hemoglobin on Skeletal Muscle Oxygen Delivery/Utilization Matching and Functional Capacity

Sickle cell disease (SCD) reduces exercise tolerance likely due to vascular and skeletal muscle abnormalities stemming from increased free hemoglobin (Hb, a known scavenger of nitric oxide). However, there has been little advancement in our understanding of the precise mechanisms responsible for the reduction in physical functionality. Therefore, the purpose of this investigation was 1) determine the degree by which exercise tolerance is impaired in a mouse model of SCD (BERK) and 2) examine the impact of free Hb on the skeletal muscle microvascular PO2 (PO2mv, the principal driving force that facilitates blood-muscle O2 flux) at rest and during muscle contractions in rats exposed to acutely elevated free [Hb]. We hypothesized that exercise capacity, as measured by the running speed/exercise duration relationship (critical speed, CS), would be lower in BERK relative to wild-type mice (WT) with a lower PO2mv observed during muscle contractions following Hb infusion. Twenty female mice (WT, n=10 and BERK, n=10) performed 4 constant-speed treadmill tests that resulted in fatigue within 1.5 to 20 min. Time to fatigue vs. treadmill speed were fit to a hyperbolic model to determine CS. PO2mv was measured (phosphorescence quenching) at rest and during 180 s of electrically induced (1-Hz) spinotrapezius muscle contractions during control, following free Hb infusion (Hb, 50 mg), and L-nitro arginine methyl ester superfusion (L-NAME, 1.5 mM) conditions in 9 rats. Speed and time to exhaustion for WT and BERK conformed to a hyperbolic relationship (WT: r2 = 0.93 ± 0.02, BERK: r2 = 0.97 ± 0.01, p>0.05). CS was significantly lower in BERK when compared to WT (WT: 33.1 ± 1.5, BERK: 25.2 ± 0.7 m/min, p<0.05). Following the onset of muscle contractions, Hb and L-NAME significantly increased the amplitude of the fall in PO2mv when compared to control, with no significant differences between Hb and L-NAME conditions (Δ1PO2mv: control: 9.5±0.7, Hb: 11.7±1, L-NAME 10.4±0.8 mmHg, p<0.05). The increased Δ1PO2mv resulted in a significantly lower PO2mv during the steady-state of muscle contractions in both Hb and L-NAME conditions, with no differences between Hb and L-NAME (PO2mv(steady-state): control: 24.1±0.9, Hb: 21.3±0.7, L-NAME: 19.6±1 mmHg, p<0.05). To summarize, exercise tolerance, as measured via CS, was significantly lower in BERK mice relative to WT. Furthermore, the lower PO2mv(steady-state) in Hb and L-NAME represents a compromised blood-myocyte O2 driving force during muscle contractions. Collectively, these data suggest that SCD impacts physical capacity via a disruption in the tight matching between oxygen delivery and utilization within the skeletal muscle and that this dysfunction could be mediated by a reduced nitric oxide bioavailability consequent of elevated free [Hb]. Support or Funding Information Funded by NIH (R01HL125642) to DCI and Colorado Nutrition and Obesity Research Center (P30DK048520) award to SKF. Mean percent PO2mv for the spinotrapezius muscle for control, Hb, and L-NAME conditions. Time “zero” represents the onset of electrically stimulated 1-hz twitch muscle contractions. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Impact of cell-free hemoglobin on contracting skeletal muscle microvascular oxygen pressure dynamics

Free hemoglobin (Hb) associated with hemolysis extravasates into vascular tissue and depletes nitric oxide (NO), which leads to impaired vascular function and could impair skeletal muscle metabolic control during exercise. We tested the hypothesis that: 1) free Hb would extravasate into skeletal muscle tissue, reducing the contracting skeletal muscle O2 delivery/O2 utilization ratio (microvascular PO2, PO2mv) to a similar extent as that observed following NO synthase (NOS) blockade, and 2) that the Hb scavenging protein haptoglobin (Hp) would prevent Hb extravasation and inhibit these skeletal muscle tissue effects. PO2mv was measured in eight rats (phosphorescence quenching) at rest and during 180 s of electrically induced (1-Hz) twitch spinotrapezius muscle contractions (experiment 1). A second group of seven rats was also used to investigate the effects of Hb + Hp (experiment 2). For both experiments, measurements were made: 1) during control conditions, 2) following a bolus infusion of either Hb (50 mg/kg) or Hb + Hp (50 mg/kg), and 3) following local superfusion of NG-nitro-l-arginine methyl ester (L-NAME; 10 mg/kg). Additional experiments were completed to visualize Hb extravasation into the muscular tissue using Click chemistry techniques. There were no significant differences in the PO2mv observed at rest for any condition in either experiment (p > 0.05 for all). In experiment 1, both Hb and L-NAME reduced the PO2mv significantly during the steady-state of muscle contractions when compared to control conditions with no differences between Hb and L-NAME (control: 24 ± 1, Hb: 21 ± 1, L-NAME: 20 ± 1 mmHg, p < 0.05). In experiment 2, only L-NAME resulted in a significantly lower PO2mv during the steady-state of muscle contractions (control: 25 ± 1, Hb + Hp: 22 ± 2, L-NAME: 18 ± 1 mmHg, p < 0.05). Free Hb lowered the blood-myocyte O2 driving force to a level not significantly different from L-NAME. However, infusing Hb bound to Hp resulted in no significant differences in steady-state PO2mv during muscle contractions when compared to control. Surprisingly, we did not observe Hb accumulation in skeletal muscle tissue. Taken together these data suggests that free Hb impairs O2 delivery/utilization via a NO scavenging effect. Furthermore, the unchanged PO2mv steady-state observed following Hb + Hp further indicates that vascular compartmentalization of Hb by the scavenger protein haptoglobin may improve skeletal muscle metabolic control and potentially exercise tolerance in those afflicted with hemolytic diseases.

Dynamics of Skeletal Muscle Microvascular and Interstitial PO2 from Rest to Contractions

Oxygen pressure (PO2) gradients within the microcirculation are required for diffusive O2 transport and, thus, to support oxidative metabolism. The greatest resistance to O2 flux into the skeletal muscle is considered to reside in the short distance between the red blood cell and the adjacent sarcolemma. Given the high flux density and absence of an O2 carrier within this diffusion path, we tested the hypothesis that PO2 gradients between muscle microvascular and interstitial spaces (PO2mv and PO2is; respectively) would be present at rest and maintained or increased during contractions. Oxyphor probes G2 (Pd‐meso‐tetra‐(4‐carboxyphenyl)‐porphyrin; i.a.) and G4 (Pd‐meso‐tetra‐(3,5‐dicarboxyphenyl)‐tetrabenzoporphyrin; tissue microinjection) were used to determine PO2mv and PO2is, respectively, via phosphorescence quenching in the exposed rat spinotrapezius muscle at rest and during submaximal twitch contractions (1 Hz, 6 V, 3 min). Due to overlapping spectral features of G2 and G4, separate measurements of PO2mv (right spinotrapezius; second protocol) and PO2is (left spinotrapezius; first protocol) were performed in the same animals (Sprague‐Dawley; n=7). There were no differences in mean arterial pressure or heart rate during the rest‐contraction transient between protocols (P&gt;0.05 for all). PO2mv was higher than PO2is in all instances from rest to contractions (baseline: 34.3±2.3 vs. 13.8±1.4; steady‐state: 27.4±1.8 vs. 9.3±1.6 mmHg, respectively; P&lt;0.05 for both) such that the mean PO2 gradient throughout the entire protocol was 18.3±2.2 mmHg (z‐test P&lt;0.05). No differences in the magnitude of the PO2 fall induced by contractions were observed between the microvascular and interstitial spaces (amplitude: 9.3±1.4 vs. 11.1±0.9 mmHg, respectively; P&gt;0.05). The speed of the PO2is fall during contractions was slower than that of PO2mv (time constant: 13.0±1.9 vs. 9.6±2.0 s, respectively; P&lt;0.05). Consistent with our hypothesis, a significant gradient between PO2mv and PO2is was sustained (but not increased) from rest to contractions in skeletal muscle. These data support that the carrier‐free region is the site of a substantial PO2 gradient driving blood‐myocyte O2 flux. Therefore, Oxyphor G2 and G4 probes allow discrete investigation of O2 exchange in the microvascular and interstitial spaces. Differences in PO2mv and PO2is kinetics illustrate the potential for muscle microcirculatory O2 exchange dynamics at sites closer to the mitochondria to provide novel insights into the control of muscle oxidative function in health and dysfunction in disease.Support or Funding InformationNIH HL‐2‐108328

Microvascular oxygen pressures in muscles comprised of different fiber types: Impact of dietary nitrate supplementation

Nitrate (NO3−) supplementation via beetroot juice (BR) preferentially improves vascular conductance and O2 delivery to contracting skeletal muscles comprised predominantly of type IIb + d/x (i.e. highly glycolytic) fibers following its reduction to nitrite and nitric oxide (NO). To address the mechanistic basis for NO3− to improve metabolic control we tested the hypothesis that BR supplementation would elevate microvascular PO2 (PO2mv) in fast twitch but not slow twitch muscle. Twelve young adult male Sprague–Dawley rats were administered BR ([NO3−] 1 mmol/kg/day, n = 6) or water (control, n = 6) for 5 days. PO2mv (phosphorescence quenching) was measured at rest and during 180 s of electrically-induced 1-Hz twitch contractions (6–8 V) of the soleus (9% type IIb +d/x) and mixed portion of the gastrocnemius (MG, 91% type IIb + d/x) muscles. In the MG, but not the soleus, BR elevated contracting steady state PO2mv by ~43% (control: 14 ± 1, BR: 19 ± 2 mmHg (P < 0.05)). This higher PO2mv represents a greater blood–myocyte O2 driving force during muscle contractions thus providing a potential mechanism by which NO3− supplementation via BR improves metabolic control in fast twitch muscle. Recruitment of higher order type II muscle fibers is thought to play a role in the development of the V˙O2 slow component which is inextricably linked to the fatigue process. These data therefore provide a putative mechanism for the BR-induced improvements in high-intensity exercise performance seen in humans.

Oxygenation measurement by multi-wavelength oxygen-dependent phosphorescence and delayed fluorescence: catchment depth and application in intact heart.

Oxygen delivery and metabolism represent key factors for organ function in health and disease. We describe the optical key characteristics of a technique to comprehensively measure oxygen tension (PO(2)) in myocardium, using oxygen-dependent quenching of phosphorescence and delayed fluorescence of porphyrins, by means of Monte Carlo simulations and ex vivo experiments. Oxyphor G2 (microvascular PO(2)) was excited at 442 nm and 632 nm and protoporphyrin IX (mitochondrial PO(2)) at 510 nm. This resulted in catchment depths of 161 (86) µm, 350 (307) µm and 262 (255) µm respectively, as estimated by Monte Carlo simulations and ex vivo experiments (brackets). The feasibility to detect changes in oxygenation within separate anatomical compartments is demonstrated in rat heart in vivo. Schematic of ex vivo measurements.