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Department of Bioengineering, University of California, San Diego, La Jolla, California
ABSTRACT I. INTRODUCTION II. METHODS FOR MEASUREMENT OF OXYGEN LEVELS IN THE MICROCIRCULATION A. Polarographic Methods 1. Microelectrode techniques 2. Surface electrodes B. Hemoglobin Spectrophotometric Methods C. Cryoscopic Hemoglobin and Myoglobin Measurements D. Porphyrin Phosphorescence Methods III. MICROCIRCULATORY PREPARATIONS A. Surgically Exposed Tissue Preparations B. Environment Isolated Preparations IV. LONGITUDINAL GRADIENTS IN THE MICROCIRCULATION A. Periarteriolar Oxygen Determinations With the Microelectrode Technique B. Intravascular Oxygen Determinations With the Spectroscopic Technique C. Cryoscopic Determination of Hemoglobin Saturation D. Intravascular Oxygen Determinations by Phosphorescence Quenching V. CAPILLARY LONGITUDINAL GRADIENTS VI. VENULAR LONGITUDINAL GRADIENTS VII. INTERACTION OF ARTERIOLAR AND VENULAR GRADIENTS VIII. LONGITUDINAL GRADIENTS: SUMMARY IX. RADIAL GRADIENTS A. Arterial Wall Radial Gradients B. Arteriolar Radial Gradients C. Capillary Radial Gradients D. Venular Radial Gradients E. Radial Gradients: Summary X. METHODOLOGICAL EFFECTS ON MEASUREMENTS OF GRADIENTS IN THE MICROCIRCULATION A. Measurement Techniques B. Experimental Conditions of Tissues Investigated XI. SIGNIFICANCE OF OXYGEN GRADIENTS A. Mass Balance Analysis of Oxygen Loss From the Microvessels B. Oxygen Distribution in the Vessel Wall C. Dependence of Results on Measurement Techniques Used D. Rate of Oxygen Loss E. Calculation of the Rate of Oxygen Consumption of the Arteriolar Vessel Wall XII. ESTIMATES OF OXYGEN CONSUMPTION OF THE VASCULATURE A. Organ Studies B. In Vitro Measurements of Oxidative Metabolism C. Possible Mechanisms of Elevated Oxygen Consumption in Arterioles D. Contribution of Capillary and Venular Endothelium to Vascular Oxygen Consumption XIII. CONCLUSIONS
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I. INTRODUCTION |
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As blood passes through the lung, oxygen diffuses down its partial pressure gradient from the alveoli into the bloodstream where it combines with hemoglobin in the red blood cells and is carried by convective transport through the heart and large and small arteries to the microcirculatory vessels where the partial pressure gradient favors diffusion from the red blood cell to the tissue. It has been assumed until recently that the unloading of oxygen from the blood to the tissue occurred to a significant degree only in the capillaries. The concept that capillaries are the sole suppliers of oxygen to the tissue is in fact a cornerstone of physiology that became crystallized with the work of Krogh and Erlangen in 1918, who developed the "Krogh cylinder model" (67). With the use of the capillary network of skeletal muscle as an example, this mathematical model describes how oxygen is delivered by a single capillary of a uniform array of capillaries to a surrounding tissue cylinder. At the time this model was developed there were no methods available to determine oxygen levels in the microcirculatory vessels and the tissue. The model therefore assumed that all oxygen exchange takes place at the capillary, with the PO2 at the entrance being that in the large artery and the PO2 at the exit being that in the large vein. This model described longitudinal and radial gradients at the capillary and surrounding tissue and has provided significant insight to the dynamics of oxygen delivery to the tissues. For example, it can be deduced from the model that under conditions of reduced blood flow or arterial oxygen level, the sites in the surrounding tissue cylinder at the greatest radial distance from the venous end of the capillary would be most vulnerable to oxygen lack (50, 68). At the same time, studies of this model have repeatedly noted that it may not fully predict tissue oxygenation (94), due to the underlying heterogeneity of capillary network and hemodynamics. An even more serious issue arises in respect to the assumption that oxygen exchange occurs only at the capillary level. This assumption was perhaps based on the notion that the thickness of the walls of the arterial and venous vessels and the high rate of blood flow would not permit significant loss of oxygen in those regions.
That the Krogh cylinder model has serious limitations as regards the assumed capillary entrance oxygen levels became evident with the advent of methods for localized measurement of microvascular and tissue oxygen levels. It was shown as early as the 1970s that the capillaries are not the only source of O2 in the microcirculation (25) since significant O2 loss was measured from the arterioles. This key observation did not immediately alter the prevailing concepts of oxygen exchange, perhaps in part because it was based on new methodology. However, subsequent studies by other investigators using polarographic microelectrodes and phosphorescence techniques to determine PO2 and spectrophotometric absorption techniques to determine intravascular hemoglobin saturation have consistently supported the finding that there is significant oxygen loss from the arteriolar network.
The decrease of oxygen content of the blood in the arterioles has been an unresolved problem until recently because the methodology did not reveal the oxygen gradients in the tissues needed to drive the outward flow of oxygen, nor the presence of oxygen sinks that could account for the measured oxygen loss from the arterioles. This situation led to the proposal that the diffusion in the arteriolar wall was an order of magnitude greater than that in tissue (or water) (51) and that the calculations of the oxygen loss had errors that caused a systematic overestimate of the oxygen deficit (135).
The technology now exists for obtaining quantitative information on the different components of the Krogh model, and therefore resolving some of the outstanding questions about how oxygen is managed in the tissue. The method consists of obtaining data that allows us to apply the principle of mass balance to the transport of oxygen in a blood vessel segment. This principle states that the changes in composition or concentration of a substance in a defined region is the sum of the balance between inflow and outflow of the given material from the region plus the amount of material that is either generated or consumed in the same region. Applying this concept to the oxygen transported in a blood vessel segment allows us to write the following relationships
Convective transport = diffusion flux out of the vessel = oxygen consumed
In this relationship we assume that 1) the blood vessel is cylindrical, with length L and internal radius R0; 2) there are no interactions with other vessels; and 3) all the oxygen diffusing out is consumed in a tissue region defined by the radius Rt. The convective transport is the total amount of oxygen entering the segment, minus that exiting the segment. The rate at which oxygen is delivered by blood in this segment is given by the product of blood flow Q times the oxygen concentration in blood (neglecting dissolved oxygen) times the change in fractional oxyhemoglobin saturation 
S, according to the expression
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, and the radial gradient in the partial pressure of oxygen at the blood/tissue interface. The third term is the oxygen consumed in the perivascular region defined by Rt and the average consumption rate of the cellular components in that region Mavg, which includes the endothelium, the vessel wall, and the parenchymal tissue.
This equation summarizes the different components of mass balance that will be discussed in this review, particularly the longitudinal gradients S/L, which have been measured with several methods that uniformly show that in a number of organs there is a significant exit from the arteriolar segments. New optical methods have produced data that allowed us to characterize radial gradient dPO2/dr, information that also showed the extend of regional differences of oxygen consumption in the tissue.
Because the interpretation of findings depends somewhat on an understanding of the methods involved and their limitations, we will next address the techniques used in these studies.
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II. METHODS FOR MEASUREMENT OF OXYGEN LEVELS IN THE MICROCIRCULATION |
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The polarographic electrode consists of a noble metal membrane-covered cathode where oxygen is reduced and a reference electrode submersed in the medium in which the measurements are to be made. A polarizing voltage causes current to flow in the electrode/medium circuit, where the current-voltage relationship is virtually independent of polarizing voltage in a plateau region of the polarogram. The measuring system is operated at this plateau voltage, where the cathode, an electrode donor, transfers electrons to the oxygen, generating a current proportional to the concentration of oxygen surrounding the electrode and the exposed reactive surface of the electrode. The metal surface is covered with a membrane to prevent the reduction of other molecules in the medium and to reduce the sensitivity to motion of fluid in the immediate vicinity of the electrode. An electrode of this type is usually referred to as a "Clark electrode," which was first described in a patent application (20).
The Clark electrode consumes oxygen generating a current that is the measure of the oxygen concentration in the medium. This current is determined by the oxygen concentration gradient between the electrode metal surface and the medium. This oxygen gradient is located in the boundary layer near the electrode surface, i.e., the catchment volume; thus any motion of the electrode or medium, or change of medium oxygen concentration, requires that a new stable boundary/diffusion layer be formed before an interpretable oxygen concentration measurement can be made. It is particularly difficult to establish a stable boundary layer at the tip of a microelectrode, which led Whalen et al. (141) to recess the metal surface from the glass micropipette tip, and to fill the tip with collodion, in such a fashion that a motion free layer is always in contact with the metal. Electrodes with tip diameters as small as 1 µm can be made by this process. These electrodes have low drift and oxygen consumption (on the order of 10-6 µl/min) and their time constant is of the order of 1 s. They are fragile to use, their introduction into the tissue introduces perturbations, and they become very noisy when used in flowing blood; however, they permit direct visualization of the location of measurement.
It is important to realize that the location of the microelectrode tip defines the center of the catchment volume whose surface PO2 is averaged by the electrode current; therefore, as an example, if the catchment volume includes a portion of an arteriole the current generated and therefore the oxygen measurement will reflect in part the presence of this high oxygen concentration region, even though the oxygen tension at the tip of the electrode may be significantly lower. Oxygen values could be affected significantly by the catchment volume in the case of the oxygen microelectrode method.
Recessed-type electrodes have superior characteristics when compared with exposed cathode electrodes and have advantages that are also reflected in smaller catchment volumes. This parameter is not specifically modeled in most analysis of electrode characteristics; however, it can be in part deduced when the oxygen concentration field around the electrode is known. Schneiderman and Goldstick (106) report the configuration of the oxygen field around the electrode tips with different ratios (l/d) of electrode recession (l, distance between the cathode surface and the tip of the glass micropipette) to electrode diameter (d), showing that shape of field is strongly dependent on this ratio for shallow depths of recession (smaller than the electrode diameter). In the case of exposed electrodes or membrane-covered electrodes, the catchment volume of the electrode is estimated to have a diameter that is about twice the diameter of the exposed area of the electrode (41). When recessed tip electrodes (Whalen electrodes) are used, the catchment volume would appear to be the same for l/d ratios of 0.5 and essentially insignificant for ratios of 5. Therefore, a precise interpretation of the data on tissue oxygen gradient measurement with microelectrodes cannot be made unless the l/d ratio is known.
The electrical properties of microelectrodes have been improved considerably with the development of surface Clark electrodes where both cathode and anode are sealed by a lipophilic membrane, thus providing a greater protection from impurities and, in principle, eliminating motion artifacts. However, they are implemented with electrodes of 
10–20 µm diameter, increasing the catchment volume to a sphere of 50 µm diameter and the time needed for establishing a stable boundary layer. These sensors are often configured in an array of independently connected sensors, whose signals are used to form a histogram of oxygen tensions. The arrays are calibrated by exposing them to a saline solution equilibrated with N2 and N2 + 10% oxygen. The response for each electrode in the array is computer memorized. Calibration is repeated every 3 h and applied to each reading for each electrode assuming linear drift between calibration points (63). These electrodes are placed over the field in which the measurements will be made, without the possibility of intentionally positioning them over specific microvascular structures. Therefore, the information is random and cannot be correlated with the underlying vessel types in the tissue under study. The histogram is obtained by moving the electrode array to multiple locations and is used to define a mean tissue PO2 and the dispersion of tissue PO2. However, shifting of the electrodes to different locations will not necessarily result in the formation of an identical, stable boundary/diffusion layer in the proximity of the electrode after each relocation, which changes the array calibration. This factor, in addition to the uncertainty of location of the electrodes and the large catchment volume, limits the usefulness of the data.
B. Hemoglobin Spectrophotometric Methods
The partial pressure of oxygen in the blood in microvessels can be determined by a technique that evaluates oxygen saturation of hemoglobin through measurements of light absorption at different wavelengths of the hemoglobin absorption spectrum. This technique has been implemented utilizing two and three wavelengths, where the choice depends on the need to correct for light scattering. This indirect technique is attractive because it utilizes optical means that are easily implemented at the microscope (90). However, with respect to its use to determine PO2, it depends on a precise knowledge of the hemoglobin absorption spectrum and the relationship between the oxygen dissociation curve for hemoglobin and PO2, a relation that is strongly influenced by local carbon dioxide concentration and pH, parameters that are not easily determined in blood in the microcirculation. A further limitation of this technique is that it cannot be used to measure tissue PO2.
The PO2 values obtained with the spectrophotometric technique were found to agree with periarteriolar microelectrode measurements in vivo by Pittman and Duling (90) and Steenbergen et al. (119). The latter group compared periarteriolar PO2 measured with the microelectrode method and the value estimated from oxygen saturation of the red blood cells in the arteriolar lumen of the rat intestine and found a difference of <1 mmHg over a range of 0–100 mmHg. These investigators used microelectrodes of 5- to 8-µm tip diameter, and therefore, as a consequence of the extended catchment volume, their measurements are most likely representative of the PO2 in blood, and not the external wall of the vessel itself.
C. Cryoscopic Hemoglobin and Myoglobin Measurements
Estimates of the level of oxygen in the vascular lumen and parenchymal cells can be obtained by cryoscopic measurements of hemoglobin and myoglobin saturation from thin sections of rapidly frozen tissue as described by Gayeski and Honig (36). In their studies, a copper plate cooled with liquid nitrogen was rapidly applied to the surface of a skeletal muscle, cooling the tissue 500 µm below the surface to the freezing point in 50 ms. Three isosbestic wavelengths and a measuring wavelength in the region of 544–579 nm were used. This method has the advantage that one can obtain measurements at a variety of vascular and tissue sites at a fixed time point, as opposed to the single site measurements possible with in vivo methods. However, the rate of cooling is not sufficient to prevent water crystallization (92), which limits optical resolution and thus the accuracy of oxygen gradient determinations over short distances.
D. Porphyrin Phosphorescence Methods
High spatial resolution measurements of oxygen tension in the microvessels and the surrounding tissue by optical means are now possible through the development of the phosphorescence quenching technique. Because this technique has yielded extensive information on the oxygen gradients in the microcirculation, it will be described here in some detail. The phosphorescence method (114, 115, 129, 136, 142) is based on the relationship between the rate of decay rate of excited phosphorescence from palladium-mesotetra-(4-carboxyphenyl)porphyrin (Porphyrin Products, Logan, UT) bound to albumin and the partial pressure of oxygen according to the Stern-Volmer equation (136, 142). In this method, animals receive a slow intravenous injection of the porphyrin dye (15 mg/kg body wt) at a concentration of 10 mg/ml 10 min before PO2 measurements. The dye is made to phosphoresce by excitation with light flashes, and the oxygen concentration in an adjustable optical window that delineates the area where the measurement is to be made is deduced from the rate of decay of the phosphorescence, which depends on the amount of oxygen that surrounds the dye.
Phosphorescence is the emission of photons due to the electronic transition in molecules that are excited into a triplet state by absorbing light and then passing from this state to a singlet ground state (114, 136). Molecules such as Pd-porphyrin either release the absorbed energy as light or transfer this energy to oxygen, which prevents light emission, therefore "quenching" the phosphorescence. The intensity of light emission I(t) from many molecules is described by an exponential decay of the form
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, and the local partial pressure PO2 of oxygen) is give by the Stern-Volmer equation
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0 is the phosphorescence decay constant in absence of oxygen and KQ is the quenching constant. An advantage of this method is that mixing Pd-porphyrin with excess albumin leads to the formation of a probe whose sensitivity to oxygen quenching is independent of the probe concentration. In other words, the decay constant becomes independent of the concentration of the albumin/porphyrin complex. Another advantage of this method is that calibrations performed in vitro can be subsequently applied to measurements obtained in vivo, since oxygen is the only chemical species that significantly influences the rate of decay of the excited phosphorescence (136). This method has been used most frequently to determine intravascular microcirculatory blood oxygen levels, starting with the report of Wilson et al. (143), Shonat et al. (115), Torres Filho and Intaglietta (129), Shonat and coworkers (113, 114), Zheng et al. (147), Kerger et al. (62), Sinaasappel et al. (117), and Helmlinger et al. (45). Regarding extravascular measurement, the blood-borne albumin-bound probe passes into the interstitium at a rate that depends on the reflection coefficient of albumin in the vascular network under study (86). The resulting accumulation of albumin-bound dye within the tissue, which may contain up to 10% of the total albumin in the organism, allows us to measure tissue PO2 at high resolution with the same technique, if the signal-to-noise ratio is adequate.
Phosphorescence generated by the light excitation of the porphyrin probe consumes O2. The amount consumed depends on the concentration of the dye and the total energy delivered by the light source. With a very intense illumination, it is possible to make a determination of oxygen level with a single flash (147). In this implementation, the flash lamp employed had a 25-µs decay constant which precluded the acquisition of phosphorescence decay data before 80 µs after flash extinction, and therefore prevented reliable measurements of PO2 above 50 mmHg, which corresponds to a phosphorescence decay time of the similar duration. The emission obtained with this technique is intense, and the phosphorescence decay curve may be the summation of signals from adjoining areas that would not normally have sufficient intensity to affect the principal component present, particularly if the measurement is made in the neighborhood of a microvessel where the oxygen field is not uniform. In addition, oxygen is consumed as the phosphorescence decays, further distorting the decay signal. These problems have been reported to be amenable to solution by deconvolving the decay signal by mathematical techniques, an approach that may be useful if signal noise is very small (137, 147). Other laboratories have circumvented the problem of oxygen consumption due to the measurement technique by using repeated light excitation of low intensity over a period that allows diffusion to replenish the consumed oxygen, and averaging the signal to obtain the final PO2 measurement (114, 129).
All implementations use a probe injection of 20–30 mg porphyrin/kg body wt, leading to a blood concentration of 0.3 mg/ml, and tissue fluid concentration smaller than 0.1 mg/ml. The multi-flash system of Torres Filho and Intaglietta (129) with a flash decay constant of 10 µs requires 100 flashes to obtain an interpretable signal in blood, where each flash consumes oxygen, causing the concentration of oxygen in stationary plasma to decrease by 0.01 mmHg/flash at steady state. Thus a single flash system that gives an interpretable signal will introduce an error <1 mmHg when used in stationary tissue fluid. This error may be lower in tissue because the amount of probe present is about one-third that of plasma, but this causes a proportional decrease in phosphorescence signal, requiring a significant increase of flash intensity and a corresponding increase in the consumption of oxygen, which may explain why not all systems are able to obtain tissue measurements. Therefore, systems built according to the method of Golub et al. (40) that use few flashes of high intensity and long flash duration are adequate for measurement of intravascular PO2 where the moving blood replenishes the consumed oxygen, if the blood PO2 is low; however, they cannot be used to measure PO2 values greater than about 50 mmHg, since light from the flash illumination is superposed to the phosphorescent emission. Furthermore, this method cannot measure tissue PO2 due to the high oxygen consumption of the flash, a process that affects the signal as the phosphorescence is emitted, introducing a highly variable, dye concentration-dependent perturbation into the measurement.
Comparison of in vivo PO2 measurements at the same site with the multi-flash phosphorescence method and the microelectrode technique were obtained by two groups of investigators using avascular tissue areas of the hamster skinfold preparation (17) and rat skeletal muscle (112). In the hamster preparation superfused with Krebs solution bubbled with 100% N2, there was a maximum divergence of 2% between the methods over the tissue PO2 range of 5–40 mmHg. Similar results were obtained in rat skeletal muscle over a range of 7–90 mmHg. In addition, multiple excitation of phosphorescence by repeated flashes at a frequency of 30/s over a period of up to 1 min did not produce a detectable change in value obtained with the microelectrode measurement (17).
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III. MICROCIRCULATORY PREPARATIONS |
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Most of the studies reviewed herein were performed on acutely exposed tissues in anesthetized animals. Pentobarbital sodium is the most commonly used anesthetic, and anesthesia is typically maintained in the surgical plane. However, the precise level of anesthesia may vary among laboratories as well as in a single preparation during the course of an experiment. A commonly used tissue is the cremaster muscle of the rat or the hamster. Surgical preparation involves removal of the skin and exposure of the muscle under a physiological salt solution (PSS) followed by removal of the testicle or moving it up into the body cavity. The animal and muscle are then mounted on a platform which provides an elevated area on which the muscle is spread out for viewing the microcirculatory vessels. PSS equilibrated either with 100% N2 or with 95% N2-5% O2 is usually suffused over the muscle. Such suffusate oxygen levels are found to have minimal effect on the tissue oxygen level as discussed in section XB. Some investigators also add 5% CO2 to the gas mixture if HCO3-buffered PSS is used. Measurements with the oxygen microelectrode require continuous bathing of the tissue surface with the suffusing solution. While the suffusate solution may affect the degree of hydration of the tissue, it also enables the investigator to alter the oxygen level at the tissue surface by changing the oxygen concentration of the suffusate. Elevating the oxygen level in the suffusate will increase the degree of arteriolar tone (24, 121) and decrease functional capillary density (96) as described in sections IIIB, X, and XIC. Thus, under the conditions of such studies, the suffusing solution may influence blood flow and oxygen gradients in the tissue. If the oxygen measurements are optical in nature, then the muscle may be covered with polyvinyl film or the tissue enclosed in a chamber and the suffusing solution discontinued.
B. Environment Isolated Preparations
Introduction of the window chamber technique provided an additional refinement since the tissue is allowed to recover from the acute effects of surgery for 2–4 days before study. The animal is studied in the unanesthetized state, and it develops its own "milieu interieur," independently of most conditions in the environment. This type of preparation was originally developed for studies using the rabbit ear (19, 103) and was implemented in rodents by Algire (1), but initially depended on the formation of scar tissue to fill the chamber. The model was modified by Reinhold (97), who implemented a skinfold window chamber in the rat where the window chamber supported and protected a thin layer of preexisting tissue in the dorsal region. This was one of the first models that allowed the long-term study of a preestablished microvascular system in subcutaneous connective tissue and skeletal muscle. The model was further elaborated by Papenfuss et al. (85) and was adapted by Endrich et al. (31) to the hamster, by Smith et al. (118) to the rat, and by Leunig et al. (73) to the mouse. A cranial window developed by Levasseur et al. (75) has similar features. These preparations allow the study of the tissue after the initial surgical trauma has subsided and in the conscious animal. The tissue is isolated from the environment, forms its own milieu interieur, and can be studied for periods as long as a week in the hamster according to our own experience and 1 mo in the rat as indicated by Smith et al. (118), before the tissue begins to exhibit features of scar tissue, including edema, increased tissue optical density (lower light transmission), and venules become tortuous.
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IV. LONGITUDINAL GRADIENTS IN THE MICROCIRCULATION |
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As described below, measurements of local PO2 levels in the microcirculation have provided evidence that a longitudinal gradient is present to a greater or lesser degree in the arteriolar network of almost all vascular beds studied. It must be borne in mind that a variety of measurement techniques were employed and the actual values that were obtained may have reflected the vagaries of surgical preparation, level of anesthesia, and oxygen level of the suffusate, if used.
A. Periarteriolar Oxygen Determinations With the Microelectrode Technique
Duling and Berne (25) used Whalen-type polarographic microelectrodes (2- to 6-µm tip diameter, Ref. 141) and estimated blood PO2 by placing the microelectrode on the external wall of the arterioles of the hamster cheek pouch preparation and the rat and hamster cremaster muscle. The PO2 of arterioles of 60–100 µm diameter in the hamster cheek pouch was 35 ± 4 mmHg (67 ± 8% estimated HbO2 saturation assuming intraluminal and periarteriolar PO2 are the same) and fell to 24 ± 3 mmHg (44 ± 6% saturation) in terminal arterioles of 10–20 µm diameter, 20 ± 3 mmHg (33 ± 6% saturation) in arterial capillaries, and 8 ± 2 mmHg in the tissues, using a suffusion solution equilibrated at a PO2 of 39 mmHg and when animals breathed room air. When the hamster was respired with 95% oxygen, the PO2 values increased to a greater degree in the proximal vessels, being 152 ± 13 mmHg (100% saturation) in the 60- to 100-µm arterioles and 37 ± 9 mmHg (73 ± 18% saturation) in arterial capillaries. In the rat breathing room air, the longitudinal PO2 gradient in cremaster muscle arterioles was also present, although absolute values were significantly higher in all vessels compared with the hamster cheek pouch, probably reflecting the higher P50 value for rat red blood cells compared with those of the hamster. In a subsequent study on the hamster cheek pouch, Duling (24) reported periarteriolar PO2 values of 48 ± 2 mmHg in 30- to 50-µm arterioles, 39 ± 2 mmHg in 10- to 20-µm arterioles, 30 ± 3 mmHg in 7- to 12-µm arterioles, and 18 ± 2 mmHg in 4- to 6-µm arterioles.
Lash and Bohlen (72) observed a periarteriolar PO2 longitudinal gradient in the resting rat spinotrapezius muscle where oxygen tension fell from 50 ± 12 mmHg (72 ± 17% estimated saturation) in first-order (A1) arterioles (41 µm mean diameter) to 46 ± 3 mmHg (61 ± 4% saturation) in second-order (A2) arterioles (28 µm mean diameter) and 31 ± 3 mmHg (40 ± 4% saturation) in third-order (A3) and fourth-order (A4) arterioles (11 to 7 µm mean diameter). They also examined the effect of muscle contraction on the PO2 at these sites. Somewhat surprisingly, during a 5-min period of 2- to 12-Hz electrical stimulation of the muscle, periarteriolar PO2 initially fell but returned to control levels at all stimulation frequencies. Flow rose by as much as 150% above control during muscle contraction. Thus it appears that the increased flow counterbalanced the effect of increased tissue oxygen consumption on periarteriolar and tissue oxygen tension, and the longitudinal gradient was maintained.
Boegehold and Johnson (9) studied the exteriorized sartorius muscle of the cat suffused with a solution equilibrated with 95% N2-5% CO2 and obtained periarteriolar PO2 values of 52 ± 3 mmHg adjacent to second-order arterioles and 40 ± 2 mmHg for fifth-order arterioles. During reduced blood flow with sympathetic nerve stimulation, the periarteriolar PO2 values were 25 ± 4 mmHg in second-order arterioles and 20 ± 4 mmHg in fifth-order arterioles. Thus reducing blood flow dropped both the absolute values and the longitudinal gradient of periarteriolar PO2 in the arteriolar network. Adding 10% oxygen to the suffusate solution did not change the periarteriolar PO2 significantly due to arteriolar constriction and reduced blood oxygen delivery.
Duling et al. (26) measured the periarteriolar PO2 distribution in the pial microvessels of the cat and found a systematic longitudinal decrease from 99 ± 11 mmHg (98 ± 1% saturation) in vessels of 230 µm diameter to 73 ± 4 mmHg (94 ± 2% saturation) in vessels of 22 µm diameter. In the pial vessels of the rat, Ivanov et al. (55) reported periarteriolar PO2 values of 75 ± 3 mmHg (87 ± 2% saturation) in 35 µm arterioles and 43 ± 3 mmHg (78 ± 2% saturation) in 8–12 µm arterioles. Blood samples from the femoral artery averaged 95 mmHg (99% saturation). They estimated that 21% of the oxygen present in arterial blood is lost before the capillary network. In a later study on rat pial vessels, the periarteriolar PO2 of first-order arterioles (45 ± 7 µm) was 81 ± 6 mmHg or 94 ± 2% HbO2 saturation and fell to 76 ± 11 mmHg (93 ± 6% saturation) in third-order arterioles (26 ± 7 µm) and 62 ± 12 mmHg (84 ± 11% saturation) in fifth-order arterioles (8 ± 2 µm) (138). The systemic HbO2 saturation averaged 95%. It is notable that a significant longitudinal gradient was found in arterioles of such a tissue, which is normally subjected to high flow conditions. When rats were respired with 100% oxygen the periarteriolar PO2 values rose to 241 ± 27 (SE) mmHg in 30 µm arterioles and 154 ± 11 mmHg in 10 µm arterioles (56). Blood samples from the femoral artery averaged 345 ± 6 mmHg. All PO2 values indicate 100% saturation. A significant longitudinal gradient of arteriolar PO2 was also observed in retinal vessels of the cat where perivascular PO2 of a 50 µm arteriole fell an estimated 2.4–3.8 mmHg over a distance of 500 µm (16).
It should be noted that in these measurements, no allowance or correction was made for the effect of the catchment volume of the microelectrode, which varies with tip size. As a result, the periarteriolar PO2 measured with different size microelectrodes provides varying estimates of intravascular PO2 depending on the relative position of the microelectrode tip and the blood tissue interface. This problem was in part resolved by the introduction of microvessel blood spectrophotometry.
B. Intravascular Oxygen Determinations With the Spectroscopic Technique
The spectroscopic technique for measuring blood PO2 in the microvessels was developed and used by Pittman and Duling (90) to map oxygen distribution in the arteriolar network of the hamster cheek pouch and corroborated the findings based on microelectrode measurements. Similar studies were carried out in the hamster retractor muscle by Swain and Pittman (123), showing, in essence, that in connective tissue and muscle a significant amount of oxygen has left the circulation before blood arrives to the capillaries. These authors reported that while femoral artery saturation was 87%, first-order arterioles (60 µm) had a saturation of 71 ± 7% (43 mmHg estimated PO2) while fourth-order arterioles had a saturation of 60% (33 mmHg). These authors reported that oxygen saturation fell by 2%/mm vessel length in the first-order arterioles and 18%/mm vessel length in fourth-order vessels. Kobayashi and Takizawa (66) reported an oxygen saturation of 98.6 ± 5.4% (90–160 mmHg) in 1A arterioles of the rat cremaster muscle(110 ± 22 µm) and a gradual reduction along the network to 64.2 ± 4.5% (44.2 mmHg) in the 4A arterioles (20 ± 4 µm) when the animal respired 30% oxygen in nitrogen. They also measured intravascular pH using a pH-sensitive dye, 1-hydroxypyrene-1,3,6,8-trisulfonic acid, which does not penetrate the cell membrane and found a significant reduction from 7.39 ± 0.02 in 1A arterioles to 7.26 ± 0.05 in 4A vessels. When breathing 12% O2 in N2, oxygen saturation fell from 47.0 ± 5.5% (36.6 mmHg) in 1A vessels to 25.1 ± 3.8% (23.0 mmHg) in 4A arterioles. The change in pH was considerably greater, falling from 7.36 ± 0.01 in 1A arterioles to 7.01 ± 0.06 in 4A vessels. This finding suggests that the Bohr effect may aid in unloading oxygen from red blood cells in the arteriolar network either due to CO2 with normoxia as suggested by Pittman and Duling (91) or to lactic acid production during hypoxia.
Seiyama et al. (109) found a drop in O2 saturation from 73 ± 8% upstream to 66 ± 8% downstream at two points averaging 73 ± 13 µm apart in 7–11 µm arterioles of the rat exocrine pancreas. The difference in O2 saturation more than doubled with secretin administration, and red cell velocity also rose, indicating an increase in oxygen consumption of the acinar cells, assuming that vessel diameter did not decrease greatly at the same time causing a reduction of blood flow.
Bohlen and Lash (11) found that arteriolar saturation between 2A arterioles and the villus tip in the small intestine decreased by 10% in rats and 15% in rabbits. The oxygen loss in the inflow vessels was 11.5% saturation/mm vessel length in rats and 7%/mm vessel length in rabbits. The fall in saturation occurred mainly in the terminal arterioles, but the authors noted that flow rates in the larger arterioles were very high. It would be expected that for arterioles of the same diameter, the fall in saturation would be less in arterioles with greater flow.
C. Cryoscopic Determination of Hemoglobin Saturation
Gayeski and Honig (36) used this method to determine the myoglobin and hemoglobin saturation in skeletal and cardiac muscle at various levels of activity. In dog gastrocnemius muscles that were electrically stimulated at 4 Hz, they found that oxyhemoglobin saturation was 88% in arterioles of 100 µm diameter and 86% in 40 µm diameter arterioles (48). Similarly, there was not a significant HbO2 saturation gradient in the arteriolar network with reduced flow and with free flow during maximal exercise. In working dog heart, this group found that the oxyhemoglobin saturation was not significantly different for arterioles of 180 µm diameter (95%) and arterioles of 20 µm diameter (93%) (47).
D. Intravascular Oxygen Determinations by Phosphorescence Quenching
Studies in the awake hamster model (62) showed that the mean arteriolar PO2 for first order (A1) arterioles was 52 ± 10 mmHg, a reduction of 19 mmHg from systemic arterial PO2 (71 ± 11 mmHg). When vessels were grouped in orders according to the position in the network, a strong positive and significant correlation was found between internal arteriolar diameter and intravascular PO2. Information on the magnitude of the oxygen loss in individual arterioles of the hamster cheek pouch was reported by Torres Filho et al. (130), who found that the average decrease in PO2 from first- to fourth-order arterioles was 18 mmHg (or 39% of saturation). While a significant PO2 decrease was found in moving downstream between vessels of different orders, the longitudinal PO2 gradient was small for a given arteriole, suggesting that the diffusion pattern at the bifurcations may be significantly altered by the geometry of the bifurcation and the increase in surface area. In 15 arterioles, intravascular PO2
measurements were made sequentially at 70- to 600-µm intervals in the direction of blood flow. In most cases there was a decrease in PO2. Dividing each PO2 difference by the distance between the measurement locations yielded an average PO2 gradient for each arteriole, which was converted to percent of saturation according to the oxygen dissociation curve for hamster blood (134). The mean longitudinal saturation gradient (%/mm) obtained for the arteriolar network was 3.4 ± 0.4%/mm, or 3.0 ± 0.5 mmHg/mm. Fourth-order arteriolar PO2 was 34 ± 2 mmHg.
Data on longitudinal gradients in the arteriolar network for a number of these studies are shown in Figure 1, left. As is evident from the preceding descriptions and Figure 1, there are substantial differences in the longitudinal gradients in different vascular beds; the gradient is much less in the brain compared with the hamster window preparation, for example. There also appear to be differences dependent on the measurement technique employed; the gradient was reported to be much less in resting skeletal muscle with the cryoscopic method than that reported with the microelectrode or phosphorescence techniques.
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30 mmHg. Also, it was found in this preparation that fourth-order arteriolar PO2 was 34 ± 2 mmHg and tissue PO2 between the capillaries was 25 ± 6 mmHg, showing that tissue and capillary PO2 is relatively uniform in this preparation. In the study of Torres Filho et al. (130), the change in O2 saturation from A4 arterioles to venules was only 8%. These data show that in this unanesthetized hamster model the whole microvascular network participates in tissue oxygenation and that the capillaries do not play a major role in the process. In the studies of Pittman and Duling (90) on the hamster cheek pouch preparation, it was found that the mean change in blood saturation as blood traverses the capillaries was 9 ± 2%, a value which reduced to 5% when the contribution of each vessel was weighted according to flow rate. This correction was introduced to reduce the bias caused by the large change in oxygen saturation of slow flowing capillaries, which otherwise do not contribute significantly to the tissue oxygen delivery. In the intestinal muscle of rats, Bohlen (10) found that tissue PO2 measured with a microelectrode at a point 15 µm from an adjacent capillary fell from 25 ± 1 (SE) mmHg near arterial capillaries to 22 ± 1 mmHg near venous capillaries. Lash and Bohlen (72), in the resting rat spinotrapezius muscle, reported levels of 31 ± 3 mmHg in third- and fourth-order arterioles (11 to 7 µm) and 28 ± 14 mmHg in tissue sites between capillaries at the midlength of these vessels. During a 5-min period of electrical stimulation of the muscle at 2–12 Hz, capillary and periarteriolar PO2 initially fell and then fully recovered during the stimulation period at all stimulation frequencies except at 12 Hz. Somewhat larger reductions in PO2 in the capillary region were found by other investigators. Boegehold and Johnson (9), using the microelectrode technique, found a drop from 40 ± 2 mmHg at periarteriolar sites for fifth-order arterioles to 23 ± 3 mmHg at tissue sites between venous capillaries in cat sartorius muscle. During reduced blood flow with sympathetic nerve stimulation, the PO2 values were 20 ± 4 mmHg in fifth-order arterioles and 9 ± 2 mmHg at the tissue sites. Adding 10% oxygen to the suffusate solution did not change periarteriolar PO2 but increased the tissue PO2 to 34 ± 3 mmHg. Ellsworth and co-workers (29, 20), using a two-wavelength spectrophotometric method to determine hemoglobin oxygen saturation, found that HbO2 saturation in capillaries decreased from 61% at the arterial end to 40% at the venous end in hamster retractor muscle. Average capillary length was 412 µm. The rate of oxygen loss per unit capillary length calculated from these estimates (0.051% SO2/µm) is about one-half that measured in short segments of arterial and venous capillaries, possibly due to oxygen transfer between adjacent capillaries. In a separate study, Ellsworth and Pittman (28) found that an arteriole crossing the capillary bed would significantly reduce or even reverse this gradient locally while a venule crossing the bed would cause it to increase.
The largest fall in HbO2 saturation across the capillary network was reported by Honig et al. (48), who used the cryoscopic technique. In that study, saturation fell from 86% to 14% between 20 µm arterioles and 20 µm venules of the dog gracilis muscle that was electrically stimulated at 4 Hz. Somewhat surprisingly, the findings were similar in this muscle at rest when blood flow was reduced to 25% of that for free flow in resting muscle. Saturation fell from 93% in 20 µm arterioles to 38% in 20 µm venules of the dog heart, and from 86% to 45% in 20 µm arterioles to 20 µm venules of the rat heart (47). These findings are consistent with the very small drop in saturation in the arteriolar network found with this technique as described in section IVC.
In the brain the longitudinal capillary gradient appears to be substantial. In pial microvessels of the cat, Duling et al. (26) found that perivascular PO2 was 73 ± 4 mmHg in 22 µm arterioles while tissue PO2 was only 11 mmHg. Rubin and Bohlen (101) found that tissue PO2 in the capillary bed of the cerebral cortex of the rat averaged 13 ± 1 mmHg (SE), but over 20% of the values fell below 5 mmHg while a few (5%) were 30 mmHg or higher. Ivanov et al. (55) reported that PO2 dropped from 43 ± 3 mmHg in the terminal arterioles of the rat pia to 26 ± 3 mmHg in small venules, representing a fall of 38% in blood oxygen content. Vovenko (138) found that perivascular PO2 in the pial microcirculation of the rat was 58 ± 11 mmHg (82 ± 9% saturation) at the arterial end of capillaries and fell to 41 ± 11 mmHg (59 ± 18% saturation) at a site 260 µm downstream in the capillaries and dropped further to 38 ± 12 mmHg (54 ± 18% saturation) in fifth-order venules (13 ± 6 µm diameter). In this case the rate of oxygen loss was 0.16% SO2/µm, or about three times greater than seen in hamster retractor muscle at rest across the full capillary network. This difference is not unexpected, considering the higher metabolic rate of the brain (3.5 x 10-2 ml O2 · min-1 · g-1, Ref. 3) compared with resting skeletal muscle (4.4 x 10-3 ml O2 · min-1 · g-1, Ref. 87).
Bohlen and Lash (11) found that HbO2 saturation in the intestinal mucosa decreased by 5% along the small arterioles and villus capillaries in the intestine of the rat and by 15% in rabbits. In both species, 70–90% of the total oxygen loss in the intestinal mucosa occurred in the small arteriole and capillary region.
Seiyama et al. (109) found in hepatic sinusoids of the rat (which are supplied from both the hepatic artery and portal vein) the HbO2 saturation decreased from 39 ± 9 to 26 ± 8% over a 70 µm distance with normal systemic hematocrit or 0.19% SO2/µm, a rate of oxygen loss similar to that seen in the brain. The difference in saturation between upstream and downstream sites almost doubled with moderate anemia, although volume flow did not change, perhaps reflecting in part the reduced oxygen content of the blood.
It has been pointed out by several investigators that the oxygen transport process from blood to tissue at the capillary level may be significantly impeded by the presence of plasma gaps between red blood cells (33, 43). In this regard, it is of interest that Zheng et al. (147) utilized the phosphorescence signal from the plasma in the hamster retractor muscle capillary to estimate a PO2 gradient of 4 mmHg/µm in the plasma gap between red blood cells. This comparatively large gradient suggests that one of the limitations to complete extraction of oxygen from the red blood cell in exercising muscle is the diffusional resistance in the capillaries (122, 139), a limitation that may be further augmented by the PO2 gradient between the red blood cell and the plasma.
The Fåhraeus effect may be another factor that significantly influences the longitudinal gradient in the arterioles, although this possibility has not been emphasized in the literature, which focuses on how this phenomenon influences the fluid dynamics of the microcirculation (39). The reduction of hematocrit in small-diameter blood vessels and the formation of a plasma layer lowers the intrinsic oxygen-carrying capacity of blood in the arterioles, which increases the fall in oxygen content in these vessels as oxygen diffuses out. Conversely, as discussed by Hellums (43) and Hellums et al. (44), the formation of a plasma layer constitutes an additional barrier to the outward diffusion of oxygen, in part offsetting the effect of lowered hematocrit on the longitudinal gradient.
Data on longitudinal gradients in the capillary network for a number of these studies are shown in Figure 1, middle. As noted for the longitudinal gradients in the arteriolar network, there are differences that appear to be related to the specific vascular bed (brain vs. hamster window preparation) and to the technique employed (cryoscopic method vs. microelectrode and phosphorescence techniques).
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Shonat and Johnson (113) studied the oxygen tension in the venous microcirculation of the rat spinotrapezius muscle utilizing the phosphorescence decay technique. They found that the mean intravascular PO2 levels in postcapillary venules (diameter: 11 ± 4 µm), venular vessels (diameter: 31 ± 9 µm), and arcading venules (diameter: 60 ± 22 µm) rose sequentially from 22 ± 9 to 26 ± 10 to 33 ± 8 mmHg, respectively, thus corroborating the presence of an increasing mean oxygen level in progressing downstream in this venular microcirculation.
Swain and Pittman (123) also found higher mean blood oxygen saturations levels in the 147 µm I.D. first-order vessels in the venular network of the hamster retractor muscle compared with 28 µm fourth-order vessels. These investigators found that blood oxygen saturation was distributed in such a fashion that vessels with high saturation had high flow rates and vice versa. To obtain the average values for the oxygen saturation in each branching order flow they weighted each saturation measurement, i.e., multiplied each saturation measurement by the corresponding flow rate and divided by the sum of all flow rates measured, with the effect that a vessels with low saturation and flow had little effect on the calculated average saturation for a given vessel order. Thus mean PO2 values at upstream and downstream sites alone may not provide sufficient information to draw conclusions regarding the gain or loss of oxygen in the venular network. Regarding the mechanism behind the increase of blood PO2 in the large venules seen in some tissues, Stein et al. found that mean PO2 and oxygen saturation were both significantly higher in large (first-order) venules compared with end-capillary values in the hamster retractor muscle when breathing 30 or 21% O2 but not when breathing 10% O2. They suggested that a diffusional shunt from arterioles to venules at the level of first- and/or second-order arterioles when breathing 30 or 21% O2 was responsible for the elevated oxygen levels in the large venules. In special circumstances, direct evidence of countercurrent exchange between arterioles and venules has been obtained as in the retinal circulation where the venous blood oxygen level rises as the two vessels travel side by side (16). This situation is somewhat unique since the vitreous humor adjacent to the venule of the eye does not consume oxygen and thus may allow countercurrent exchange to be manifest. There is no significant oxygen exchange between parallel arterioles and venules in the intestinal villus of the rat and rabbit perhaps because of the 300- to 500-µm separation and the oxygen consumption of the intervening tissue (11). In the submucosa of the rat and rabbit there is a fall (although modest) in oxygen saturation as the blood flows through the venular network. In the rat, this finding could reflect mixing of blood with higher saturation from the intestinal muscle with blood of lower saturation from the villi, but this explanation would not apply to the rabbit where the saturation in second-order venules is less than that from the villi and third-order venules of the intestinal muscle.
Gayeski and Honig (37) found a slight increase in mean HbO2 saturation from 38 to 44% between venules of 25 and 180 µm in the dog heart and no apparent change in saturation between 20 and 160 µm venules of the rat heart using the cryoscopic technique. Also, in the working dog gracilis muscle (4 Hz electrical stimulation), there was a slight rise from 14 to 20% saturation between venules of 20 and 140 µm (48). Saturations varied widely among the small venules in these studies.
Vovenko (138) found in the venular network of the brain that PO2 levels were very heterogeneous at each branching order, varying by as much as 60 mmHg. In the large venules intraluminal PO2 measurements showed distinct flow streams from different tributaries with different oxygen levels, suggesting that the oxygen extraction from the deeper regions of the brain may be greater than those near the surface. Average values of PO2 rose slightly from 38 ± 12 mmHg (54 ± 18% saturation) in 13 ± 6 µm fifth-order venules to 40 ± 9 mmHg (57 ± 18% saturation) in 71 ± 24 µm third-order venules, and 41 ± 10 mmHg (59 ± 14% saturation) in 258 ± 31 µm first-order venules. Despite the slight rise in average PO2 apparent in the data presented above, venules of 10–40 µm diameter appear to supply oxygen to surrounding tissue as discussed in the section VIII. It may be speculated that the higher levels of oxygen in the larger venules and veins, whatever the mechanism, may serve a functional purpose. In contrast to the small collecting venules, the latter contain significant quantities of vascular smooth muscle (98) and may require a greater supply of oxygen for the oxidative metabolism of this tissue.
Data on longitudinal gradients in the venular network for a number of these studies are shown in Figure 1, right. Notable in this graph is the gradual rise in PO2 for most vascular beds except for the venular networks of the hamster skinfold and rat spinotrapezius muscle where there is a substantial rise and the rabbit muscosa where there is a slight fall.
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VII. INTERACTION OF ARTERIOLAR AND VENULAR GRADIENTS |
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VIII. LONGITUDINAL GRADIENTS: SUMMARY |
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IX. RADIAL GRADIENTS |
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