Fixing skeletal muscle PO2 eliminates hyperinsulinemic microvascular blood flow response.

OBJECTIVE: To determine if insulin-mediated hyperemia is partially dependent on local muscle oxygen concentration. METHODS: Sprague-Dawley rats were anesthetized, and the extensor digitorum longus (EDL) was reflected onto an inverted microscope. Intravital video microscopy sequences were recorded during baseline and hyperinsulinemic euglycemia. The muscle was reflected over a glass stage insert (Experiment 1a and 1b), or over a gas exchange chamber (Experiment 2), and microvascular capillary blood flow was recorded during sequential changes (7%-12%-2%-7%) of oxygen (O2 ) concentration. Blood flow was measured by the red blood cell supply rate (SR) in number of cells per second. All animal protocols were approved by Memorial University's Institutional Animal Care Committee. RESULTS: In Experiment 1a, SR increased from 8.0 to 14.0 cells/s at baseline to euglycemia (p = .01), while no significant SR variation was detected after performing a sham hyperinsulinemic euglycemic clamp (Experiment 1b). In Experiment 2, SR decreased at 12% O2 and increased at 2% O2 , compared to 7% O2 , under both experimental conditions. Magnitude of SR responses to oxygen oscillations during euglycemia were not different to those at baseline at each O2 concentration (p > .9). CONCLUSIONS: Our results suggest that increased blood flow observed in response to insulin is eliminated if tissue oxygen microenvironment is fixed at a given oxygen concentration.

Optical method to determine in vivo capillary hematocrit, hemoglobin concentration, and 3-D network geometry in skeletal muscle.

OBJECTIVE: The aim of this study was to develop a tool to visualize and quantify hemodynamic information, such as hemoglobin concentration and hematocrit, within microvascular networks recorded in vivo using intravital video microscopy. Additionally, we aimed to facilitate the 3-D reconstruction of the microvascular networks. METHODS: Digital images taken from an intravital video microscopy preparation of the extensor digitorum longus muscle in rats for 25 capillary segments were used. The developed algorithm was used to delineate capillaries of interest, calculate the optical density for each pixel in the image, and reconstruct the 3-D capillary geometry using the calculated light path-lengths. Subsequently, the mean corpuscular hemoglobin concentration (MCHC), hemoglobin concentration, and hematocrit for these capillaries were calculated. We evaluated the hematocrit values determined by our methodology by comparing them to those obtained using a previously published method. RESULTS: The hematocrit values from the proposed optical method were strongly correlated with those calculated using published methods r2 (25) = .92, p < .001, and demonstrated excellent agreement with a mean difference of 1.3% and a coefficient of variation (CV) of 11%. The average MCHC, hemoglobin concentration, and light path-lengths were 23.83 g/dl, 8.06 g/dl, and 3.92 µm, respectively. CONCLUSION: The proposed methodology can quantify hemodynamic measurements and produce functional images for visualization of the microcirculation in vivo.

Zucker Diabetic-Sprague Dawley (ZDSD) rat: Type 2 diabetes translational research model.

NEW FINDINGS: What is the topic of this review? The Zucker Diabetic-Sprague Dawley (ZDSD) rat is in the early adoption phase of use by researchers in the fields of diabetes, including prediabetes, obesity and metabolic syndrome. It is essential that physiology researchers choose preclinical models that model human type 2 diabetes appropriately and are aware of the limitations on experimental design. What advances does it highlight? Our review of the scientific literature finds that although sex, age and diets contribute to variability, the ZDSD phenotype and disease progression model the characteristics of humans who have prediabetes and diabetes, including co-morbidities. ABSTRACT: Type 2 diabetes (T2D) is a prevalent disease and a significant concern for global population health. For persons with T2D, clinical treatments target not only the characteristics of hyperglycaemia and insulin resistance, but also co-morbidities, such as obesity, cardiovascular and renal disease, neuropathies and skeletal bone conditions. The Zucker Diabetic-Sprague Dawley (ZDSD) rat is a rodent model developed for experimental studies of T2D. We reviewed the scientific literature to highlight the characteristics of T2D development and the associated phenotypes, such as metabolic syndrome, cardiovascular complications and bone and skeletal pathologies in ZDSD rats. We found that ZDSD phenotype characteristics are independent of leptin receptor signalling. The ZDSD rat develops prediabetes, then progresses to overt diabetes that is accelerated by introduction of a timed high-fat diet. In male ZDSD rats, glycated haemoglobin (HbA1c) increases at a constant rate from 7 to >30 weeks of age. Diabetic ZDSD rats are moderately hypertensive compared with other rat strains. Diabetes in ZDSD rats leads to endothelial dysfunction in specific vasculatures, impaired wound healing, decreased systolic and diastolic cardiac function, neuropathy and nephropathy. Changes to bone composition and the skeleton increase the risk of bone fractures. Zucker Diabetic-Sprague Dawley rats have not yet achieved widespread use by researchers. We highlight sex-related differences in the ZDSD phenotype and gaps in knowledge for future studies. Overall, scientific data support the premise that the phenotype and disease progression in ZDSD rats models the characteristics in humans. We conclude that ZDSD rats are an advantageous model to advance understanding and discovery of treatments for T2D through preclinical research.

Development and validation of a novel microfluidic device for the manipulation of skeletal muscle microvascular blood flow in vivo.

OBJECTIVE: To develop and validate a novel liquid microfluidic approach to deliver drugs to microscale regions of tissue while simultaneously allowing for visualization and quantification of microvascular blood flow. METHODS: Microfluidic devices were fabricated using soft lithographic techniques, molded in polydimethylsiloxane, and bound to a coverslip with a 600 × 300 µm micro-outlet. Sprague-Dawley rats, anesthetized with pentobarbital, were instrumented to monitor systemic parameters. The extensor digitorum longus muscle was dissected, externalized, and reflected across the device mounted on the stage of an inverted microscope. Doses (10-8 to 10-3  M) of adenosine triphosphate (ATP), acetylcholine, and phenylephrine (PE) were administered to the muscle via perfusion through the device. Microvascular blood flow directly overlying the micro-outlet was recorded at multiple focal depths. Red blood cell (RBC) velocity, supply rate, and hematocrit were measured from recordings. RESULTS: ATP significantly increased RBC velocity and supply rate. Increasing concentrations of PE caused a decrease in RBC velocity and supply rate. Perfusion changes were restricted to areas directly overlying the micro-outlet and within 500 µm. CONCLUSIONS: This novel microfluidic device allows for a controlled delivery of dissolved substances to constrained regions of microvasculature while simultaneously allowing for visualization and measurement of blood flow within discrete vessels and networks.

Evidence for role of capillaries in regulation of skeletal muscle oxygen supply.

How oxygen (O2 ) supply to capillaries is regulated to match the tissue's demand is unknown. Erythrocytes have been proposed as sensors in this regulatory mechanism since they release ATP, a vasodilator, in an oxygen saturation (SO2 )-dependent manner. ATP causes hyperpolarization of endothelial cells resulting in conducted vasodilation to arterioles. OBJECTIVE: We propose individual capillary units can regulate their own O2 supply by direct communication to upstream arterioles via electrically coupled endothelium. METHODS: To test this hypothesis, we developed a transparent micro-exchange device for localized O2 exchange with surface capillaries of intact tissue. The device was fabricated with an O2 permeable micro-outlet 0.2 × 1.0 mm. Experiments were performed on rat extensor digitorum longus (EDL) muscle using dual wavelength video microscopy to measure capillary hemodynamics and erythrocyte SO2 . Responses to local O2 perturbations were measured with only capillaries positioned over the micro-outlet. RESULTS: Step changes in the gas mixture %O2 caused physiological changes in erythrocyte SO2 , and appropriate changes in flow to offset the O2 challenge if at least 3-4 capillaries were stimulated. CONCLUSION: These results support our hypothesis that individual capillary units play a role in regulating their erythrocyte supply in response to a changing O2 environment.

Sympathetic neurovascular transduction following acute hypoxia.

PURPOSE: Following an acute exposure to hypoxia, sympathetic nerve activity remains elevated. However, this elevated sympathetic nerve activity does not elicit a parallel increase in vascular resistance suggesting a blunted sympathetic signaling [i.e. blunted sympathetic neurovascular transduction (sNVT)]. Therefore, we sought to quantify spontaneous sympathetic bursts and related changes in total peripheral resistance following hypoxic exposure. We hypothesized that following hypoxia sNVT would be blunted. METHODS: Nine healthy participants (n = 6 men; mean age 25 ± 2 years) were recruited. We collected data on muscle sympathetic nerve activity (MSNA) using microneurography and beat-by-beat total peripheral resistance (TPR) via finger photoplethysmography at baseline, during acute hypoxia and during two periods of recovery (recovery period 1, 0-10 min post hypoxia; recovery period 2, 10-20 min post hypoxia). MSNA burst sequences (i.e. singlets, doublets, triplets and quads+) were identified and coupled to changes in TPR over 15 cardiac cycles as an index of sNVT for burst sequences. A sNVT slope for each participant was calculated from the slope of the relationship between TPR plotted against normalized burst amplitude. RESULTS: The sNVT slope was blunted during hypoxia [Δ 0.0044 ± 0.0014 (mmHg/L/min)/(a.u.)], but unchanged following termination of hypoxia [recovery 1, Δ 0.031 ± 0.0019 (mmHg/L/min)/(a.u.); recovery 2, Δ 0.0038 ± 0.0014 (mmHg/L/min)/(a.u.) compared to baseline (Δ 0.038 ± 0.0015 (L/min/mmHg)/(a.u.)] (main effect of group p = 0.012). CONCLUSIONS: Contrary to our hypothesis, we have demonstrated that systemic sNVT is unchanged following hypoxia in young healthy adults.

Blunted sympathetic neurovascular transduction is associated to the severity of obstructive sleep apnea.

PURPOSE: Obstructive sleep apnea (OSA) is a common disorder (~ 4%) that augments sympathetic nerve activity (SNA) and elevates blood pressure. The relationship between sympathetic vasomotor outflow and vascular responsiveness, termed sympathetic neurovascular transduction (sNVT), has been sparsely characterized in patients with OSA. Therefore, we sought to quantify spontaneous sympathetic bursts and related changes in diastolic pressure. METHODS: Twelve participants with variable severities of OSA were recruited. We collected muscle sympathetic nerve activity (MSNA) (microneurography) and beat-by-beat diastolic pressure (finger photoplethysmography) during normoxia (FiO2 = 0.21) and hyperoxia (FiO2 = 1.0) to decrease MSNA burst frequency. MSNA burst sequences (i.e. singlets, doublets, triplets and quadruplets) were identified and coupled to changes in diastolic pressure over 15 cardiac cycles as an index of sNVT. sNVT slope for each individual was calculated from the slope of the relationship between peak responses in outcome plotted against normalized burst amplitude. RESULTS: sNVT slope was unchanged during hyperoxia compared to normoxia (normoxia 0.0024 ± 0.0011 Δ mmHg total activity [a.u.]-1 vs. hyperoxia 0.0029 ± 0.00098 Δ mmHg total activity [a.u.]-1; p = 0.14). sNVT slope was inversely associated with burst frequency during hyperoxia (r = -0.58; p = 0.04), but not normoxia (r = -0.11; p = 0.71). sNVT slope was inversely associated with the apnea-hypopnea index (AHI) (r = -0.62; p = 0.030), but not after age was considered. CONCLUSIONS: We have demonstrated that the prevailing MSNA frequency is unmatched to the level of sNVT, and this can be altered by acute hyperoxia.

Sex differences in dynamic blood pressure regulation: beat-by-beat responses to muscle sympathetic nerve activity.

It has been suggested that sex differences in acute blood pressure fluctuations occur during the periods of time between bursts of muscle sympathetic nerve activity. Therefore, we tested the hypothesis that men experience more dynamic changes in mean arterial pressure (Finometer MIDI) than women during acute sympathoinhibition (i.e., slow breathing) in which bursts of sympathetic activity occur more infrequently than at rest. We tested healthy women (n = 9) and men (n = 9) of similar age (22 ± 2 vs. 23 ± 3 yr, P = 0.6). Custom software was used to calculate beat-by-beat changes in blood pressure following sympathetic burst and nonburst sequences (recorded using microneurography) during 10 min of supine rest and a 15-min bout of slow breathing. During slow breathing following nonburst sequences, women demonstrated smaller overall reductions in mean arterial pressure compared with men over the subsequent 15 cardiac cycles (P < 0.01). In addition, following a burst of sympathetic activity, women experienced greater overall increases in mean arterial pressure compared with men over the following 15 cardiac cycles (P < 0.01). Despite these differences, the peak and nadir changes in arterial pressure following burst and nonburst sequences were not different between the sexes (P = 0.45 and P = 0.48, burst and nonburst sequences, respectively). As such, these data suggest that women respond to a burst of sympathetic activity with more sustained increases in blood pressure than men, coupled with improved maintenance of blood pressure during acute periods of sympathetic quiescence. In other words, these findings suggest that men rely more on frequent bursts of sympathetic activity to acutely regulate arterial pressure than women.NEW & NOTEWORTHY We demonstrate that during acute sympathoinhibition, women demonstrate more sustained increases in blood pressure following sympathetic bursts of activity than men. Likewise, during prolonged sympathetic quiescence, blood pressure is less labile in women than men. This suggests that lower overall blood pressure in young women may not be mediated by smaller beat-by-beat changes in blood pressure in response to sympathetic outflow but may instead be mediated by a lower frequency of sympathetic bursts.

Highs and lows of sympathetic neurocardiovascular transduction: influence of altitude acclimatization and adaptation.

High-altitude (>2,500 m) exposure results in increased muscle sympathetic nervous activity (MSNA) in acclimatizing lowlanders. However, little is known about how altitude affects MSNA in indigenous high-altitude populations. Additionally, the relationship between MSNA and blood pressure regulation (i.e., neurovascular transduction) at high-altitude is unclear. We sought to determine 1) how high-altitude effects neurocardiovascular transduction and 2) whether differences exist in neurocardiovascular transduction between low- and high-altitude populations. Measurements of MSNA (microneurography), mean arterial blood pressure (MAP; finger photoplethysmography), and heart rate (electrocardiogram) were collected in 1) lowlanders (n = 14) at low (344 m) and high altitude (5,050 m), 2) Sherpa highlanders (n = 8; 5,050 m), and 3) Andean (with and without excessive erythrocytosis) highlanders (n = 15; 4,300 m). Cardiovascular responses to MSNA burst sequences (i.e., singlet, couplet, triplet, and quadruplet) were quantified using custom software (coded in MATLAB, v.2015b). Slopes were generated for each individual based on peak responses and normalized total MSNA. High altitude reduced neurocardiovascular transduction in lowlanders (MAP slope: high altitude, 0.0075 ± 0.0060 vs. low altitude, 0.0134 ± 0.080; P = 0.03). Transduction was elevated in Sherpa (MAP slope, 0.012 ± 0.007) compared with Andeans (0.003 ± 0.002, P = 0.001). MAP transduction was not statistically different between acclimatizing lowlanders and Sherpa (MAP slope, P = 0.08) or Andeans (MAP slope, P = 0.07). When resting MSNA is accounted for (ANCOVA), transduction was inversely related to basal MSNA (bursts/minute) independent of population (RRI, r = 0.578 P < 0.001; MAP, r = -0.627, P < 0.0001). Our results demonstrate that transduction is blunted in individuals with higher basal MSNA, suggesting that blunted neurocardiovascular transduction is a physiological adaptation to elevated MSNA rather than an effect or adaptation specific to chronic hypoxic exposure.NEW & NOTEWORTHY This study has identified that sympathetically mediated blood pressure regulation is reduced following ascent to high-altitude. Additionally, we show that high altitude Andean natives have reduced blood pressure responsiveness to sympathetic nervous activity (SNA) compared with Nepalese Sherpa. However, basal sympathetic activity is inversely related to the magnitude of SNA-mediated fluctuations in blood pressure regardless of population or condition. These data set a foundation to explore more precise mechanisms of blood pressure control under conditions of persistent sympathetic activation and hypoxia.

Hyperinsulinemia does not cause de novo capillary recruitment in rat skeletal muscle.

OBJECTIVE: The effect of insulin on blood flow distribution within muscle microvasculature has been suggested to be important for glucose metabolism. However, the "capillary recruitment" hypothesis is still controversial and relies on studies using indirect contrast-enhanced ultrasound (CEU) methods. METHODS: We studied how hyperinsulinemia effects capillary blood flow in rat extensor digitorum longus (EDL) muscle during euglycemic hyperinsulinemic clamp using intravital video microscopy (IVVM). Additionally, we modeled blood flow and microbubble distribution within the vascular tree under conditions observed during euglycemic hyperinsulinemic clamp experiments. RESULTS: Euglycemic hyperinsulinemia caused an increase in erythrocyte (80 ± 25%, P < .01) and plasma (53 ± 12%, P < .01) flow in rat EDL microvasculature. We found no evidence of de novo capillary recruitment within, or among, capillary networks supplied by different terminal arterioles; however, erythrocyte flow became slightly more homogenous. Our computational model predicts that a decrease in asymmetry at arteriolar bifurcations causes redistribution of microbubble flow among capillaries already perfused with erythrocytes and plasma, resulting in 25% more microbubbles flowing through capillaries. CONCLUSIONS: Our model suggests increase in CEU signal during hyperinsulinemia reflects a redistribution of arteriolar flow and not de novo capillary recruitment. IVVM experiments support this prediction showing increases in erythrocyte and plasma flow and not capillary recruitment.

Blunted sympathetic neurovascular transduction during normotensive pregnancy.

KEY POINTS: Normotensive pregnancy is associated with elevated sympathetic nervous system activity yet normal or reduced blood pressure. It represents a unique period of apparent healthy sympathetic hyperactivity. The present study models the blood pressure and heart rate (ECG R-R interval) responses to fluctuations in sympathetic nervous system activity aiming to understand neurocardiovascular transduction. The reported data clearly demonstrate that transduction of sympathetic nervous system signalling to systemic cardiovascular outcomes is reduced in normotensive pregnancy. These data are important for understanding how blood pressure regulation adapts during normotensive pregnancy and set the foundation for exploring similar mechanisms in hypertensive pregnancies. ABSTRACT: Previously, we described sympathetic nervous system hyperactivity yet decreased blood pressure responses to stress in normotensive pregnancy. To address the hypothesis that pregnant women have blunted neurocardiovascular transduction we assessed the relationship between spontaneous bursts of sympathetic nerve activity (SNA) and fluctuations in mean arterial blood pressure and R-R interval. Resting SNA, blood pressure and ECG were obtained in pregnant (third trimester, n = 18) and non-pregnant (n = 18) women matched for age and pre-/non-pregnant body mass index. Custom software modelled beat-by-beat pressure (photoplethysmography) and R-R interval in relation to sequences of SNA bursts and non-bursts (peroneal microneurography). Sequences were grouped by the number of bursts and non-bursts [singlets, doublets, triplets and quadruplet (four or more)] and mean blood pressure and R-R interval were tracked for 15 subsequent cardiac cycles. Similar sequences were overlaid and averaged. Peak mean pressure in relation to sequences of SNA was reduced in pregnant vs. non-pregnant women (doublets: 1.6 ± 1.1 mmHg vs. 3.6 ± 3.1 mmHg, P < 0.05; triplets: 2.4 ± 1.2 mmHg vs. 3.4 ± 2.1 mmHg, P < 0.05; quadruplets: 3.0 ± 1.0 mmHg vs. 5.5 ± 3.7 mmHg, P < 0.05). The nadir R-R interval following burst sequences was also smaller in pregnant vs. non-pregnant women (singlets: -0.01 ± 0.01 s vs. -0.04 ± 0.04 s, P < 0.05; doublets: -0.02 ± 0.03 s vs. -0.05 ± 0.04 s, P < 0.05; triplets: -0.02 ± 0.01 s vs. -0.07 ± 0.04 s, P < 0.05; quadruplets: -0.01 ± 0.01 s vs. -0.09 ± 0.09 s, P < 0.05). There were no differences between groups in the mean arterial pressure and R-R interval responses to non-burst sequences. Our data clearly indicate blunted systemic neurocardiovascular transduction during normotensive pregnancy. We propose that blunted transduction is a positive adaptation protecting pregnant women from the cardiovascular consequences of sympathetic hyperactivity.

Four-Dimensional Microvascular Analysis Reveals That Regenerative Angiogenesis in Ischemic Muscle Produces a Flawed Microcirculation.

RATIONALE: Angiogenesis occurs after ischemic injury to skeletal muscle, and enhancing this response has been a therapeutic goal. However, to appropriately deliver oxygen, a precisely organized and exquisitely responsive microcirculation must form. Whether these network attributes exist in a regenerated microcirculation is unknown, and methodologies for answering this have been lacking. OBJECTIVE: To develop 4-dimensional methodologies for elucidating microarchitecture and function of the reconstructed microcirculation in skeletal muscle. METHODS AND RESULTS: We established a model of complete microcirculatory regeneration after ischemia-induced obliteration in the mouse extensor digitorum longus muscle. Dynamic imaging of red blood cells revealed the regeneration of an extensive network of flowing neo-microvessels, which after 14 days structurally resembled that of uninjured muscle. However, the skeletal muscle remained hypoxic. Red blood cell transit analysis revealed slow and stalled flow in the regenerated capillaries and extensive arteriolar-venular shunting. Furthermore, spatial heterogeneity in capillary red cell transit was highly constrained, and red blood cell oxygen saturation was low and inappropriately variable. These abnormalities persisted to 120 days after injury. To determine whether the regenerated microcirculation could regulate flow, the muscle was subjected to local hypoxia using an oxygen-permeable membrane. Hypoxia promptly increased red cell velocity and flux in control capillaries, but in neocapillaries, the response was blunted. Three-dimensional confocal imaging revealed that neoarterioles were aberrantly covered by smooth muscle cells, with increased interprocess spacing and haphazard actin microfilament bundles. CONCLUSIONS: Despite robust neovascularization, the microcirculation formed by regenerative angiogenesis in skeletal muscle is profoundly flawed in both structure and function, with no evidence for normalizing over time. This network-level dysfunction must be recognized and overcome to advance regenerative approaches for ischemic disease.

A computational model of the effect of capillary density variability on oxygen transport, glucose uptake, and insulin sensitivity in prediabetes.

OBJECTIVES: The purpose of this study was to model how CD variability affects tissue oxygenation under resting and exercise conditions. Additionally, we examine how CD impacts glucose and insulin transport in skeletal muscle. METHODS: We applied an established 3D finite difference model of oxygen transport to predict tissue oxygenation using FCD, hemodynamics, and SO2 measurements from a previous study. A 2D finite element model of glucose transport was applied to predict glucose and insulin uptake in PP and fasting conditions using the same range of CD. RESULTS: Control simulations used CD ranging from 562.5 to 781.3 capillaries/mm2 , whereas prediabetic densities ranged from 375.0 to 593.8 capillaries/mm2 . Mean tissue PO2 was 30.6±4.6 to 40.5±3.6 mm Hg for rest and 19.6±6.7 to 33.27±4.7 mm Hg for control and prediabetic simulations, respectively. Mean PP glucose concentrations were 5.85±1.13 mmol/L in the control group and 5.11±1.28 in the prediabetic simulations. Glucose uptake rates were 35% lower in the lowest capillary CD case compared to the high CD simulation. CONCLUSIONS: Our simulations predict that CD decreases can have a substantial effect on oxygen delivery and glucose disposal across the observed physiological ranges of capillarization.

Reduced uterine perfusion pressure decreases functional capillary density in skeletal muscle.

The purpose of this study was to examine the functional and structural capillary density in the reduced uterine perfusion pressure (RUPP) model, which when performed during pregnancy is an established animal model of preeclampsia. We hypothesized that the RUPP model would be associated with capillary rarefaction and impaired capillary perfusion, which would be more pronounced in the pregnant state. Female Sprague-Dawley rats (n = 32) were randomized to nonpregnancy (Nonpregnant) or breeding (Pregnant) at 12 wk of age and again to RUPP or SHAM surgeries on gestational day (GD) 14 (or equivalent age in nonpregnant rats). On GD 20 (or equivalent), capillary structure and perfusion of the extensor digitorum longus were imaged using digital intravital video microscopy. Functional videos were analyzed by a blinded observer to measure capillary density, expressed as capillaries per millimeter intersecting three staggered reference lines (200 µm). Flow was scored as the percentage of capillaries having 1) continuous, 2) intermittent, or 3) stopped flow. Total capillary density was not different between groups. There was a main effect of RUPP surgery resulting in decreased continuous flow vessels (P < 0.01) and increased stopped flow (P < 0.01), which was driven by differences between pregnant animals (Continuous flow: pregnant SHAM 80.1 ± 7.8% vs. pregnant RUPP 67.8 ± 11.2%, P < 0.05) (Stopped flow: pregnant SHAM 8.7 ± 3.2% vs. pregnant RUPP 17.9 ± 5.7%, P < 0.01). Our results demonstrate that the RUPP surgery is associated with a decrease in functional capillary density in skeletal muscle that is more pronounced in the pregnant state, which may contribute to the vascular pathophysiology observed in preeclampsia.

Impact of Incremental Perfusion Loss on Oxygen Transport in a Capillary Network Mathematical Model.

OBJECTIVES: To quantify how incremental capillary PL, such as that seen in experimental models of sepsis, affects tissue oxygenation using a computation model of oxygen transport. METHODS: A computational model was applied to capillary networks with dimensions 84 × 168 × 342 (NI) and 70 × 157 × 268 (NII) µm, reconstructed in vivo from rat skeletal muscle. FCD loss was applied incrementally up to ~40% and combined with high tissue oxygen consumption to simulate severe sepsis. RESULTS: A loss of ~40% FCD loss decreased median tissue PO2 to 22.9 and 20.1 mmHg in NI and NII compared to 28.1 and 27.5 mmHg under resting conditions. Increasing RBC SR to baseline levels returned tissue PO2 to within 5% of baseline. HC combined with a 40% FCD loss, resulted in tissue anoxia in both network volumes and median tissue PO2 of 11.5 and 8.9 mmHg in NI and NII respectively; median tissue PO2 was recovered to baseline levels by increasing total SR 3-4 fold. CONCLUSIONS: These results suggest a substantial increase in total SR is required in order to compensate for impaired oxygen delivery as a result of loss of capillary perfusion and increased oxygen consumption during sepsis.

Comparison of generated parallel capillary arrays to three-dimensional reconstructed capillary networks in modeling oxygen transport in discrete microvascular volumes.

OBJECTIVE: We compare RMN to PCA under several simulated physiological conditions to determine how the use of different vascular geometry affects oxygen transport solutions. METHODS: Three discrete networks were reconstructed from intravital video microscopy of rat skeletal muscle (84 × 168 × 342 µm, 70 × 157 × 268 µm, and 65 × 240 × 571 µm), and hemodynamic measurements were made in individual capillaries. PCAs were created based on statistical measurements from RMNs. Blood flow and O2 transport models were applied, and the resulting solutions for RMN and PCA models were compared under four conditions (rest, exercise, ischemia, and hypoxia). RESULTS: Predicted tissue PO2 was consistently lower in all RMN simulations compared to the paired PCA. PO2 for 3D reconstructions at rest were 28.2 ± 4.8, 28.1 ± 3.5, and 33.0 ± 4.5 mmHg for networks I, II, and III compared to the PCA mean values of 31.2 ± 4.5, 30.6 ± 3.4, and 33.8 ± 4.6 mmHg. Simulated exercise yielded mean tissue PO2 in the RMN of 10.1 ± 5.4, 12.6 ± 5.7, and 19.7 ± 5.7 mmHg compared to 15.3 ± 7.3, 18.8 ± 5.3, and 21.7 ± 6.0 in PCA. CONCLUSIONS: These findings suggest that volume matched PCA yield different results compared to reconstructed microvascular geometries when applied to O2 transport modeling; the predominant characteristic of this difference being an over estimate of mean tissue PO2. Despite this limitation, PCA models remain important for theoretical studies as they produce PO2 distributions with similar shape and parameter dependence as RMN.

Mapping 3-D functional capillary geometry in rat skeletal muscle in vivo.

We have developed a novel mapping software package to reconstruct microvascular networks in three dimensions (3-D) from in vivo video images for use in blood flow and O2 transport modeling. An intravital optical imaging system was used to collect video sequences of blood flow in microvessels at different depths in the tissue. Functional images of vessels were produced from the video sequences and were processed using automated edge tracking software to yield location and geometry data for construction of the 3-D network. The same video sequences were analyzed for hemodynamic and O2 saturation data from individual capillaries in the network. Simple user-driven commands allowed the connection of vessel segments at bifurcations, and semiautomated registration enabled the tracking of vessels across multiple focal planes and fields of view. The reconstructed networks can be rotated and manipulated in 3-D to verify vessel connections and continuity. Hemodynamic and O2 saturation measurements made in vivo can be indexed to corresponding vessels and visualized using colorized maps of the vascular geometry. Vessels in each reconstruction are saved as text-based files that can be easily imported into flow or O2 transport models with complete geometry, hemodynamic, and O2 transport conditions. The results of digital morphometric analysis of seven microvascular networks showed mean capillary diameters and overall capillary density consistent with previous findings using histology and corrosion cast techniques. The described mapping software is a valuable tool for the quantification of in vivo microvascular geometry, hemodynamics, and oxygenation, thus providing rich data sets for experiment-based computational models.

Microvascular flow modeling using in vivo hemodynamic measurements in reconstructed 3D capillary networks.

OBJECTIVE: We describe a systematic approach to modeling blood flow using reconstructed capillary networks and in vivo hemodynamic measurements. Our goal was to produce flow solutions that represent convective O(2) delivery in vivo. METHODS: Two capillary networks, I and II (84 × 168 × 342 and 70 × 157 × 268 µm(3)), were mapped using custom software. Total network red blood cell supply rate (SR) was calculated from in vivo data and used as a target metric for the flow model. To obtain inlet hematocrits, mass balances were applied recursively from downstream vessels. Pressure differences across the networks were adjusted to achieve target SR. Baseline flow solutions were used as inputs to existing O(2) transport models. To test the impact of flow redistribution, asymmetric flow solutions (Asym) were generated by applying a ± 20% pressure change to network outlets. RESULTS: Asym solutions produced a mean absolute difference in SR per capillary of 27.6 ± 33.3% in network I and 33.2 ± 40.1% in network II vs. baseline. The O(2) transport model calculated mean tissue PO(2) of 28.2 ± 4.8 and 28.1 ± 3.5 mmHg for baseline and 27.6 ± 5.2 and 27.7 ± 3.7 mmHg for Asym. CONCLUSIONS: This outcome illustrates that moderate changes in flow distribution within a capillary network have little impact on tissue PO(2) provided that total SR remains unchanged.