Dynamics of PO2 and VO2 in resting and contracting rat spinotrapezius muscle.

This study examined changes in interstitial PO2, which allowed calculation of VO2 during periods of rest, muscle contraction and recovery using an in situ rat spinotrapezius muscle preparation. The PO2 was measured using phosphorescence quenching microscopy and the muscle VO2 was calculated as the rate of O2 disappearance during brief periods of muscle compression to stop blood flow with a supra-systolic pressure. The PO2 and VO2 measurements were made during "5 s compression and 15 s recovery" (CR) cycles. With all three stimulation frequencies, 1, 2 and 4 Hz, the fall in interstitial PO2 and rise in VO2 from resting values occurred within the first 20 s of contraction. The PO2 during contraction became lower as stimulation frequency increased from 1 to 4 Hz. VO2 was higher at 2 Hz than at 1 Hz contraction. With cessation of stimulation, PO2 began increasing exponentially towards baseline values. After 1 and 2 Hz contraction, the fall in muscle VO2 was delayed by one CR cycle and then exponentially decreased towards resting values. After 4 Hz stimulation, VO2 increased for 2 cycles and then decreased. The post-contraction transients of PO2 and VO2 were not synchronous and had different time constants. With further analysis two distinct functional responses were identified across all stimulation frequencies having PO2 during contraction above or below 30 mmHg. The corresponding VO2 responses were different - for "high" PO2, muscle VO2 reached high levels, while for the "low" PO2 data set muscle VO2 remained low. Recovery patterns were similar to those described above. In summary, local microscopic PO2 and VO2 were measured in resting and contracting muscle in situ and the post-contraction transients of PO2 and VO2 were all much slower than the onset transients.

Spike of interstitial PO2 produced by a twitch in rhythmically contracted muscle.

Oxygen (O2 ) exchange between capillaries and muscle cells in exercising muscles is of great interest for physiology and kinesiology. However, methodical limitations prevent O2 measurements on the millisecond scale. To bypass the constraints of quasi-continuous recording, progressive measurements of O2 partial pressure (PO2 ) in rhythmically contracting skeletal muscle were compiled to describe the O2 kinetics surrounding and including a single muscle contraction. Phosphorescence quenching microscopy measured PO2 in the interstitium of the rat spinotrapezius muscle. Measurements were triggered by contraction-inducing electrical pulses. For the first 60 seconds, measurement preceeded stimulation. After 60, measurement followed with a progressive 20 ms increment. Thus, the first 60 measurements describe the overall PO2 response to electrical stimulation initiated after a 10 second rest period, while 61-100 (stroboscopic mode) were compiled into a single 800 ms profile of the PO2 transient surrounding muscle contraction. Thirty seconds of stimulated contractions decreased interstitial PO2 from a baseline of 71 ± 1.4 mmHg to an "active" steady-state of 43 ± 1.5 mmHg. The stroboscopic mode compilation revealed an unexpected post-contractile rise in PO2 as a 205 ms spike with a maximum amplitude of 58 ± 3.8 mmHg at 68 ms, which restored 58% of the PO2 drop from baseline. Interpretation of this phenomenon is based on classical experiments by G.V. Anrep (1935), who discovered the rapid thrust of blood flow associated with muscle contraction. In addition to the metabolic implications during exercise, the physiological impact of these PO2 spikes may grow with an increased rate of rhythmical contractions in muscle or heart. NEW&NOTEWORTHY: The principal finding is a spike of interstitial PO2 , produced by a twitch in a rhythmically contracting muscle. A possible mechanism is flushing capillaries with arterial blood by mechanical forces. A technical novelty is the PO2 measurement with a "stroboscopic mode" and progressively increasing delay between stimulator pulse and PO2 measuring. That permitted a 20 ms time resolution for a 205 ms spike duration, using an excitation flash rate one per second.

Oxygen dependence of respiration in rat spinotrapezius muscle contracting at 0.5-8 twitches per second.

The oxygen dependence of respiration was obtained in situ in microscopic regions of rat spinotrapezius muscle for different levels of metabolic activity produced by electrical stimulation at rates from 0.5 to 8 Hz. The rate of O2 consumption (V̇o2) was measured with phosphorescence quenching microscopy (PQM) as the rate of O2 disappearance in a muscle with rapid flow arrest. The phosphorescent oxygen probe was loaded into the interstitial space of the muscle to give O2 tension (Po2) in the interstitium. A set of sigmoid curves relating the Po2 dependence of V̇o2 was obtained with a Po2-dependent region below a characteristic Po2 (~30 mmHg) and a Po2-independent region above this Po2. The V̇o2(Po2) plots were fit by the Hill equation containing O2 demand (rest to 8 Hz: 216 ± 26 to 636 ± 77 nl O2/cm3 s) and the Po2 value corresponding to O2 demand/2 (rest to 8 Hz: 22 ± 4 to 11 ± 1 mmHg). The initial Po2 and V̇o2 pairs of values measured at the moment of flow arrest formed a straight line, determining the rate of oxygen supply. This line had a negative slope, equal to the oxygen conductance for the O2 supply chain. For each level of tissue blood flow the set of possible values of Po2 and V̇o2 consists of the intersection points between this O2 supply line and the set of V̇o2 curves. An electrical analogy for the intraorgan O2 supply and consumption is an inverting transistor amplifier, which allows the use of graphic analysis methods for prediction of the behavior of the oxygen processing system in organs. NEW & NOTEWORTHY The sigmoidal shape of curves describing oxygen dependence of muscle respiration varies from basal to maximal workload and characterizes the oxidative metabolism of muscle. The rate of O2 supply depends on extracellular O2 tension and is determined by the overall oxygen conductance in the muscle. The dynamics of oxygen consumption is determined by the supply line that intersects the oxygen demand curves. An electrical analogy for the oxygen supply/consumption system is an inverting transistor amplifier.

Barometric calibration of a luminescent oxygen probe.

The invention of the phosphorescence quenching method for the measurement of oxygen concentration in blood and tissue revolutionized physiological studies of oxygen transport in living organisms. Since the pioneering publication by Vanderkooi and Wilson in 1987, many researchers have contributed to the measurement of oxygen in the microcirculation, to oxygen imaging in tissues and microvessels, and to the development of new extracellular and intracellular phosphorescent probes. However, there is a problem of congruency in data from different laboratories, because of interlaboratory variability of the calibration coefficients in the Stern-Volmer equation. Published calibrations for a common oxygen probe, Pd-porphyrin + bovine serum albumin (BSA), vary because of differences in the techniques used. These methods are used for the formation of oxygen standards: chemical titration, calibrated gas mixtures, and an oxygen electrode. Each method in turn also needs calibration. We have designed a barometric method for the calibration of oxygen probes by using a regulated vacuum to set multiple PO2 standards. The method is fast and accurate and can be applied to biological fluids obtained during or after an experiment. Calibration over the full physiological PO2 range (1-120 mmHg) takes ∼15 min and requires 1-2 mg of probe.

Simultaneous sampling of tissue oxygenation and oxygen consumption in skeletal muscle.

Under physiologic conditions, microvascular oxygen delivery appears to be well matched to oxygen consumption in respiring tissues. We present a technique to measure interstitial oxygen tension (PISFO2) and oxygen consumption (VO2) under steady-state conditions, as well as during the transitions from rest to activity and back. Phosphorescence Quenching Microscopy (PQM) was employed with pneumatic compression cycling to achieve 1 to 10 Hz sampling rates of interstitial PO2 and simultaneous recurrent sampling of VO2 (3/min) in the exteriorized rat spinotrapezius muscle. The compression pressure was optimized to 120-130 mmHg without adverse effect on the tissue preparation. A cycle of 5s compression followed by 15s recovery yielded a resting VO2 of 0.98 ± 0.03 ml O2/100 cm(3)min while preserving microvascular oxygen delivery. The measurement system was then used to assess VO2 dependence on PISFO2 at rest and further tested under conditions of isometric muscle contraction to demonstrate a robust ability to monitor the on-kinetics of tissue respiration and the compensatory changes in PISFO2 during contraction and recovery. The temporal and spatial resolution of this approach is well suited to studies seeking to characterize microvascular oxygen supply and demand in thin tissues.

Muscle contraction increases interstitial nitric oxide as predicted by a new model of local blood flow regulation.

The prevailing metabolic theory of local blood flow regulation suggests the dilatation of arterioles in response to tissue hypoxia via the emission of multiple metabolic vasodilators by parenchymal cells. We have proposed a mechanism of regulation, built from well-known components, which assumes that arterioles are normally dilated in metabolically active tissues, due to the emission of NO by the endothelium of microvessels. Regulation of local blood flow aims at preventing an excessive supply of oxygen (O2) and glucose to the tissue and thus provides an adequate supply, in contrast to the metabolic regulation theory which requires permanent hypoxia to generate the metabolic vasodilators. The mediator of the restrictive signal is superoxide anion (O2(-)) released by membrane NAD(P)H oxidases into the interstitial space, where it neutralizes NO at a diffusion-limited rate. This model predicts that the onset of muscle contraction will lead to the cessation of O2(-) production, which will cause an elevation of interstitial NO concentration and an increase in fluorescence of the NO probe DAF-FM after its conversion to DAF-T. The time course of DAF-T fluorescence in contracting muscle is predicted by also considering the washout from the muscle of the interstitially loaded NO indicator. Experiments using pulse fluorimetry confirmed an increase in the interstitial concentration of NO available for reaction with DAF-FM during bouts of muscle contraction. The sharp increase in interstitial [NO] is consistent with the hypothesis that the termination of the neutralizing superoxide flow into the interstitium is associated with the activation of mitochondria and a reduction of the interstitial oxygen tension. The advantage of the new model is its ability to explain the interaction of metabolic activity and local blood flow through the adequate delivery of glucose and oxygen.

Bang-bang model for regulation of local blood flow.

The classical model of metabolic regulation of blood flow in muscle tissue implies the maintenance of basal tone in arterioles of resting muscle and their dilation in response to exercise and/or tissue hypoxia via the evoked production of vasodilator metabolites by myocytes. A century-long effort to identify specific metabolites responsible for explaining active and reactive hyperemia has not been successful. Furthermore, the metabolic theory is not compatible with new knowledge on the role of physiological radicals (e.g., nitric oxide, NO, and superoxide anion, O2 (-) ) in the regulation of microvascular tone. We propose a model of regulation in which muscle contraction and active hyperemia are considered the physiologically normal state. We employ the "bang-bang" or "on/off" regulatory model which makes use of a threshold and hysteresis; a float valve to control the water level in a tank is a common example of this type of regulation. Active bang-bang regulation comes into effect when the supply of oxygen and glucose exceeds the demand, leading to activation of membrane NADPH oxidase, release of O2 (-) into the interstitial space and subsequent neutralization of the interstitial NO. Switching arterioles on/off when local blood flow crosses the threshold is realized by a local cell circuit with the properties of a bang-bang controller, determined by its threshold, hysteresis, and dead-band. This model provides a clear and unambiguous interpretation of the mechanism to balance tissue demand with a sufficient supply of nutrients and oxygen.

Oxygen dependence of respiration in rat spinotrapezius muscle in situ.

The oxygen dependence of respiration in striated muscle in situ was studied by measuring the rate of decrease of interstitial Po(2) [oxygen disappearance curve (ODC)] following rapid arrest of blood flow by pneumatic tissue compression, which ejected red blood cells from the muscle vessels and made the ODC independent from oxygen bound to hemoglobin. After the contribution of photo-consumption of oxygen by the method was evaluated and accounted for, the corrected ODCs were converted into the Po(2) dependence of oxygen consumption, Vo(2), proportional to the rate of Po(2) decrease. Fitting equations obtained from a model of heterogeneous intracellular Po(2) were applied to recover the parameters describing respiration in muscle fibers, with a predicted sigmoidal shape for the dependence of Vo(2) on Po(2). This curve consists of two regions connected by the point for critical Po(2) of the cell (i.e., Po(2) at the sarcolemma when the center of the cell becomes anoxic). The critical Po(2) was below the Po(2) for half-maximal respiratory rate (P(50)) for the cells. In six muscles at rest, the rate of oxygen consumption was 139 ± 6 nl O(2)/cm(3)·s and mitochondrial P(50) was k = 10.5 ± 0.8 mmHg. The range of Po(2) values inside the muscle fibers was found to be 4-5 mmHg at the critical Po(2). The oxygen dependence of respiration can be studied in thin muscles under different experimental conditions. In resting muscle, the critical Po(2) was substantially lower than the interstitial Po(2) of 53 ± 2 mmHg, a finding that indicates that Vo(2) under this circumstance is independent of oxygen supply and is discordant with the conventional hypothesis of metabolic regulation of the oxygen supply to tissue.

The rate of O2 loss from mesenteric arterioles is not unusually high.

The O(2) disappearance curve (ODC) recorded in an arteriole after the rapid arrest of blood flow reflects the complex interaction among the dissociation of O(2) from hemoglobin, O(2) diffusivity, and rate of respiration in the vascular wall and surrounding tissue. In this study, the analysis of experimental ODCs allowed the estimation of parameters of O(2) transport and O(2) consumption in the microcirculation of the mesentery. We collected ODCs from rapidly arrested blood inside rat mesenteric arterioles using scanning phosphorescence quenching microscopy (PQM). The technique was used to prevent the artifact of accumulated O(2) photoconsumption in stationary media. The observed ODC signatures were close to linear, in contrast to the reported exponential decline of intra-arteriolar Po(2). The rate of Po(2) decrease was 0.43 mmHg/s in 20-µm-diameter arterioles. The duration of the ODC was 290 s, much longer than the 12.8 s reported by other investigators. The arterioles associated with lymphatic microvessels had a higher O(2) disappearance rate of 0.73 mmHg/s. The O(2) flux from arterioles, calculated from the average O(2) disappearance rate, was 0.21 nl O(2)·cm(-2)·s(-1), two orders of magnitude lower than reported in the literature. The physical upper limit of the O(2) consumption rate by the arteriolar wall, calculated from the condition that all O(2) is consumed by the wall, was 452 nl O(2)·cm(-3)·s(-1). From consideration of the microvascular tissue volume fraction in the rat mesentery of 6%, the estimated respiration rate of the vessel wall was ∼30 nl O(2)·cm(-3)·s(-1). This result was three orders of magnitude lower than the respiration rate in rat mesenteric arterioles reported by other investigators. Our results demonstrate that O(2) loss from mesenteric arterioles is small and that the O(2) consumption by the arteriolar wall is not unusually large.

Phosphorescence quenching microrespirometry of skeletal muscle in situ.

We have developed an optical method for the evaluation of the oxygen consumption (Vo(2)) in microscopic volumes of spinotrapezius muscle. Using phosphorescence quenching microscopy (PQM) for the measurement of interstitial Po(2), together with rapid pneumatic compression of the organ, we recorded the oxygen disappearance curve (ODC) in the muscle of the anesthetized rats. A 0.6-mm diameter area in the tissue, preloaded with the phosphorescent oxygen probe, was excited once a second by a 532-nm Q-switched laser with pulse duration of 15 ns. Each of the evoked phosphorescence decays was analyzed to obtain a sequence of Po(2) values that constituted the ODC. Following flow arrest and tissue compression, the interstitial Po(2) decreased rapidly and the initial slope of the ODC was used to calculate the Vo(2). Special analysis of instrumental factors affecting the ODC was performed, and the resulting model was used for evaluation of Vo(2). The calculation was based on the observation of only a small amount of residual blood in the tissue after compression. The contribution of oxygen photoconsumption by PQM and oxygen inflow from external sources was evaluated in specially designed tests. The average oxygen consumption of the rat spinotrapezius muscle was Vo(2) = 123.4 ± 13.4 (SE) nl O(2)/cm(3) · s (N = 38, within 6 muscles) at a baseline interstitial Po(2) of 50.8 ± 2.9 mmHg. This technique has opened the opportunity for monitoring respiration rates in microscopic volumes of functioning skeletal muscle.

Measurement of oxygen in the microcirculation using phosphorescence quenching microscopy.

According to the classical concept of Krogh, O(2) is delivered to the tissues solely by capillaries and intra-capillary resistance to O(2) diffusion is negligible. Over the past three decades longitudinal PO(2) and SO(2) gradients in arterioles have been observed with a transmural PO(2) gradient in small arterioles of only 1-2 mmHg. Application of phosphorescence quenching microscopy to measurements of PO(2) in arterioles of the rat mesentery by Tsai et al. (1998) found a large transmural PO(2) in these arterioles. That led to the provocative conclusion that the arteriolar wall is the major sink for O(2) in the microcirculation. Our studies indicate that many of these results can be explained by photo-activated O(2) consumption following phosphor excitation, combined with a large excitation area and high frequency of flash excitation. We have developed the basic principles for phosphorescence quenching microscopy including the need to use a small excitation area, a low excitation frequency and a scanning excitation for stationary samples.

PO2 measurements in the microcirculation using phosphorescence quenching microscopy at high magnification.

In phosphorescence quenching microscopy (PQM), the multiple excitation of a reference volume produces the integration of oxygen consumption artifacts caused by individual flashes. We analyzed the performance of two types of PQM instruments to explain reported data on Po2 in the microcirculation. The combination of a large excitation area (LEA) and high flash rate produces a large oxygen photoconsumption artifact manifested differently in stationary and flowing fluids. A LEA instrument strongly depresses Po2 in a motionless tissue, but less in flowing blood, creating an apparent transmural Po2 drop in arterioles. The proposed model explains the mechanisms responsible for producing apparent transmural and longitudinal Po2 gradients in arterioles, a Po2 rise in venules, a hypothetical high respiration rate in the arteriolar wall and mesenteric tissue, a low Po2 in lymphatic microvessels, and both low and uniform tissue Po2. This alternative explanation for reported paradoxical results of Po2 distribution in the microcirculation obviates the need to revise the dominant role of capillaries in oxygen transport to tissue. Finding a way to eliminate the photoconsumption artifact is crucial for accurate microscopic oxygen measurements in microvascular networks and tissue. The PQM technique that employs a small excitation area (SEA) together with a low flash rate was specially designed to avoid accumulated oxygen photoconsumption in flowing blood and lymph. The related scanning SEA instrument provides artifact-free Po2 measurements in stationary tissue and motionless fluids. Thus the SEA technique significantly improves the accuracy of microscopic Po2 measurements in the microcirculation using the PQM.

Microvascular oxygen tension in the rat mesentery.

Longitudinal Po(2) profiles in the microvasculature of the rat mesentery were studied using a novel phosphorescence quenching microscopy technique that minimizes the accumulated photoconsumption of oxygen by the method. Intravascular oxygen tension (Po(2), in mmHg) and vessel diameter (d, in microm) were measured in mesenteric microvessels (n = 204) of seven anesthetized rats (275 g). The excitation parameters were as follows: 7 x 7-microm spot size; 410 nm laser; and 100 curves at 11 pulses/s, with pulse parameters of 2-micros duration and 80-pJ/microm(2) energy density. The mean Po(2) (+/- SE) was 65.0 +/- 1.4 mmHg (n = 78) for arterioles (d = 18.8 +/- 0.7 microm), 62.1 +/- 2.0 mmHg (n = 38) at the arteriolar end of capillaries (d = 7.8 +/- 0.3 microm), and 52.0 +/- 1.0 mmHg (n = 88) for venules (d = 22.5 +/- 1.0 microm). There was no apparent dependence of Po(2) on d in arterioles and venules. There were also no significant deviations in Po(2) based on d (bin width, 5 microm) from the general mean for both of these types of vessels. Results indicate that the primary site of oxygen delivery to tissue is located between the smallest arterioles and venules (change of 16.3 mmHg, P = 0.001). In conclusion, oxygen losses from mesenteric arterioles and venules are negligible, indicating low metabolic rates for both the vascular wall and the mesenteric tissue. Capillaries appear to be the primary site of oxygen delivery to the tissue in the mesenteric microcirculation. In light of the present results, previously reported data concerning oxygen consumption in the mesenteric microcirculation can be explained as artifacts of accumulated oxygen consumption due to the application of instrumentation having a large excitation area for Po(2) measurements in slow moving and stationary media.

PO2 profiles near arterioles and tissue oxygen consumption in rat mesentery.

A scanning phosphorescence quenching microscopy technique, designed to prevent accumulated O(2) consumption by the method, was applied to Po(2) measurements in mesenteric tissue. In an attempt to further increase the accuracy of the measurements, albumin-bound probe was topically applied to the tissue and an objective-mounted pressurized bag was used to reduce the oxygen transport bypass through the thin layer of fluid over the mesentery. Po(2) was measured at multiple sites perpendicular to the blood/wall interface in the vicinity of 84 mesenteric arterioles (7-39 microm in diameter) at distances of 5, 15, 30, 60, 120, and 180 microm in seven anesthetized Sprague-Dawley rats, thereby creating Po(2) profiles. Interstitial Po(2) above and immediately beside arterioles was found to agree with known intravascular values. No significant difference in Po(2) profiles was found between small and large arterioles, indicating a small longitudinal Po(2) gradient in the precapillary mesenteric microvasculature. In addition, the Po(2) profiles were used to calculate oxygen consumption in the mesenteric tissue (56-65 nl O(2) x cm(-3) x s(-1)). Correction of these values for contamination with ambient oxygen yielded an oxygen consumption rate of 60-68 nl O(2) x cm(-3) x s(-1), the maximal limit for consumption in the mesentery. The results were compared with measurements made by other workers in regard to the employed techniques.

Erythrocyte-associated transients in capillary PO2: an isovolemic hemodilution study in the rat spinotrapezius muscle.

Mathematical simulations of oxygen delivery to tissue from capillaries that take into account the particulate nature of blood flow predict the existence of oxygen tension (Po(2)) gradients between erythrocytes (RBCs). As RBCs and plasma alternately pass an observation point, these gradients are manifested as rapid fluctuations in Po(2), also known as erythrocyte-associated transients (EATs). The impact of hemodilution on EATs and oxygen delivery at the capillary level of the microcirculation has yet to be elucidated. Therefore, in the present study, phosphorescence quenching microscopy was used to measure EATs and Po(2) in capillaries of the rat spinotrapezius muscle at the following systemic hematocrits (Hct(sys)): normal (39%) and after moderate (HES1; 27%) or severe (HES2; 15%) isovolemic hemodilution using a 6% hetastarch solution. A 532-nm laser, generating 10-micros pulses concentrated onto a 0.9-microm spot, was used to obtain plasma Po(2) values 100 times/s at points along surface capillaries of the muscle. Mean capillary Po(2) (Pc(O(2)); means +/- SE) significantly decreased between conditions (normal: 56 +/- 2 mmHg, n = 45; HES1: 47 +/- 2 mmHg, n = 62; HES2: 27 +/- 2 mmHg, n = 52, where n = capillary number). In addition, the magnitude of Po(2) transients (DeltaPo(2)) significantly decreased with hemodilution (normal: 19 +/- 1 mmHg, n = 45; HES1: 11 +/- 1 mmHg, n = 62; HES2: 6 +/- 1 mmHg, n = 52). Results suggest that the decrease in Pc(O(2)) and DeltaPo(2) with hemodilution is primarily dependent on Hct(sys) and subsequent microvascular compensations.

Effect of oxygen consumption by measuring method on PO2 transients associated with the passage of erythrocytes in capillaries of rat mesentery.

Mathematical models have predicted the existence of Po(2) gradients between erythrocytes in capillaries in the usual case where plasma contributes substantial resistance to oxygen diffusion. According to theoretical predictions, these gradients could be detected as rapid Po(2) fluctuations (erythrocyte-associated transients, EATs) along the capillary. However, verification of a model and correct choice of its parameters can be made only on the basis of direct experimental measurements. We used phosphorescence quenching microscopy to measure Po(2) in 52 capillaries of rat mesentery to obtain plasma Po(2) values 100 times/s at a given point along a capillary. A 532-nm laser generated 10-mus pulses of light, concentrated by a x100 objective, onto a spot 0.9 mum in diameter. The presence of erythrocytes in the excitation region was detected on the basis of phosphorescence amplitude (PA), proportional to the amount of plasma encountered by the laser beam, and on the basis of the intensity of transmitted laser light (LT), detected by a photodiode placed under the capillary. The data revealed correlated waveforms in PA, LT, and Po(2) in capillaries. The magnitude of the Po(2) gradients between erythrocytes and plasma was correlated with average capillary Po(2). EATs in Po(2) were more readily detected in capillaries with relatively low oxygenation. The correlation coefficients between PA and Po(2) for the half of the capillaries (n = 26) below the median Po(2) (mean Po(2) = 17 mmHg; R = -0.72) was higher than that for the other half (mean Po(2) = 39 mmHg; R = -0.38). These results support the theoretical predictions of EATs and plasma Po(2) gradients in capillaries.

Erythrocyte-associated transients in PO2 revealed in capillaries of rat mesentery.

Mathematical models have predicted the existence of Po(2) gradients between erythrocytes in capillaries in the usual case where plasma contributes substantial resistance to oxygen diffusion. According to theoretical predictions, these gradients could be detected as rapid Po(2) fluctuations (erythrocyte-associated transients, EATs) along the capillary. However, verification of a model and correct choice of its parameters can be made only on the basis of direct experimental measurements. We used phosphorescence quenching microscopy to measure Po(2) in 52 capillaries of rat mesentery to obtain plasma Po(2) values 100 times/s at a given point along a capillary. A 532-nm laser generated 10-micros pulses of light, concentrated by a x100 objective, onto a spot 0.9 microm in diameter. The presence of erythrocytes in the excitation region was detected on the basis of phosphorescence amplitude (PA), proportional to the amount of plasma encountered by the laser beam, and on the basis of the intensity of transmitted laser light (LT), detected by a photodiode placed under the capillary. The data revealed correlated waveforms in PA, LT, and Po(2) in capillaries. The magnitude of the Po(2) gradients between erythrocytes and plasma was correlated with average capillary Po(2). EATs in Po(2) were more readily detected in capillaries with relatively low oxygenation. The correlation coefficients between PA and Po(2) for the half of the capillaries (n = 26) below the median Po(2) (mean Po(2) = 17 mmHg; R = -0.72) was higher than that for the other half (mean Po(2) = 39 mmHg; R = -0.38). These results support the theoretical predictions of EATs and plasma Po(2) gradients in capillaries.

Rate of decrease of PO2 from an arteriole with arrested flow.

When flow to a region is arrested, the amount of oxygen contained within the stationary blood decreases at a rate dependent on the oxygen utilization of the surrounding tissue. We used phosphorescence quenching microscopy to measure arteriolar PO2 in the mesentery of male Sprague-Dawley rats. Flow was quickly stopped (< 1 s) by occluding the microvessels using an inflatable Saran bag attached to the microscope objective. The rate of decline in PO2 following occlusion yielded a calculated initial flux of oxygen out of the vessel lumen of 8.0 x 10(-7) ml O2 cm(-2) sec(-1). An upper limit on the oxygen consumption of the arteriolar wall was calculated by assuming that all of the oxygen in the lumen was consumed by the wall at the initial rate. This value was 2.5 x 10(-3) ml O2 cm(-3) sec(-1) and is an overestimate since the oxygen consumption of the nearby parenchymal cells was neglected. The calculated maximum oxygen consumption of the wall is more than an order of magnitude smaller than that reported previously for arterioles in the rat mesentery (6.5 x 10(-2) ml O2 cm(-3) sec(-1)). We conclude that oxygen consumption of the arteriolar wall is similar to previous values for other vascular tissues.