| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1Department of Bioengineering, University of California, San Diego, 2La Jolla Bioengineering Institute, La Jolla, California; and 3Division of General and Surgical Intensive Care Medicine, Department of Anesthesia and Critical Care Medicine, Leopold-Franzens-University of Innsbruck, Innsbruck, Austria
Submitted 13 September 2004 ; accepted in final form 22 November 2004
 |
ABSTRACT |
|---|
 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
vasoactivity; oxygen gradients; tissue oxygenation; microvessel metabolism
The diffusional oxygen exit from the arterioles is driven by the magnitude of the blood-tissue oxygen concentration gradient. The development of the phosphorescence decay technique (10) enabled a quantitative measurement of these gradients. Such measurements show that, in general, these gradients are relatively large and, therefore, commensurate with the high rates of oxygen exit measured in the arterioles of the hamster window model (7) and mesentery (19) and in skeletal muscle (13). These studies also showed that existence of such large gradients in conjunction with a large diffusional flux is possible if the tissue in which the gradient is present consumes oxygen.
The rate of oxygen consumption of the blood vessels has not been extensively studied in vivo. There is some evidence that it may be related to the tone that blood vessels maintain to regulate blood flow. According to the study of Ye et al. (23), when the vascular beds of the rat hindlimb, kidney, intestine, and mesentery constrict and develop tone from a relaxed state, their oxygen consumption virtually doubles if blood flow is maintained constant. This study indicated that perfused blood vessels in the mesentery consumed oxygen at a rate of 115 µmol·h–1·g blood vessel wt–1 at 25°C (4.3 x 10–2 ml O2·min–1·g tissue wt–1). On constriction, oxygen consumption increased by 75%. Vasoconstriction per se lowers tissue perfusion and functional capillary density (FCD) (16); therefore, it is not likely that the increased oxygen consumption was due to a better perfusion of the tissue, although the authors favored that interpretation in later studies (14, 15).
Arteriolar wall oxygen consumption was found to increase significantly during treatment with vasopressin, showing that vasoconstriction is a process that increases oxygen consumption of the vessel wall (5). The microcirculation in a tissue at rest supplies the tissue with its basal oxygen requirements; therefore, it is in a state of moderate vasoconstriction. Because further constriction from this condition increases oxygen consumption by the microvessels, we propose that inducing vasodilatation in a tissue at rest should lower arteriolar wall oxygen consumption. In the present study, we mapped the oxygen distribution in the microcirculation of the hamster chamber window preparation during administration of the Ca2+ channel blocker verapamil HCl (0.1 mg/kg bolus injection followed by 0.01 mg·kg–1·min–1 continuous infusion) to determine whether vasodilatation lowers the arteriolar vessel wall gradient and, presumably, vessel wall oxygen consumption.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
Investigations were performed in Syrian (golden) hamsters (Charles River Laboratories, Boston, MA). Animal handling and care were provided following the procedures outlined in the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996). The study was approved by the local Animal Subjects Committee. The hamster window chamber model is widely used for microvascular studies in the unanesthetized state, and the complete surgical technique is described in detail elsewhere (2, 4). Briefly, animals were anesthetized for chamber implantation with an injection of pentobarbital sodium (50 mg/kg ip; Nembutal, Abbott, IL). After hair removal, sutures were used to lift the dorsal skin away from the animal, and one frame of the chamber was positioned on the animal's back. A chamber consisted of two identical titanium frames with a 15-mm circular window. With the aid of backlighting and a stereomicroscope, one side of the skinfold was removed following the outline of the window until only a thin layer of retractor muscle and the intact subcutaneous skin of the opposing side remained. Saline and then a cover glass were placed on the exposed skin held in place by the other frame of the chamber. The intact skin of the other side was exposed to the ambient environment. The animal was allowed at least 2 days for recovery; then its chamber was assessed under the microscope for any signs of edema, bleeding, or unusual neovascularization. Barring these complications, the animal was anesthetized again with pentobarbital sodium. Arterial and venous catheters (PE-50) were implanted in the carotid artery and jugular vein. The catheters were filled with a heparinized saline solution (30 IU/ml) to ensure their patency at the time of the experiment. Catheters were tunneled under the skin and exteriorized at the dorsal side of the neck, where they were attached to the chamber frame with tape. The experiment was performed after at least 24 h but within 48 h of catheter implantation.
Inclusion Criteria
Animals were suitable for the experiments if their systemic parameters were within normal range: 1) heart rate (HR) >320 beats/min, 2) mean systemic blood pressure (MAP) >80 mmHg, 3) systemic hematocrit (Hct) >45%, and 4) systemic arterial PO2 (PaO2) >50 mmHg. The microvasculature of the chamber tissue was examined under low (x150) and high (x650) magnification for edema and bleeding. Animals were excluded from the study when these signs of trauma were observed.
Systemic Parameters
MAP was tracked continuously over the entire experimental period, and HR was determined from the pressure trace (Spectramed Pressure Transducer, Biopac, Santa Barbara, CA). Systemic Hct was measured from centrifuged arterial blood samples taken in heparinized capillary tubes (Readacrit Centrifuge, Clay Adams, Division of Becton and Dickinson, Parsippany, NJ).
Blood Chemistry
Arterial blood was sampled from the carotid artery catheter into heparinized capillary tubes and immediately analyzed for PO2, PCO2, and pH at 37°C with a pH-blood gas analyzer (model 248, Bayer, Tarrytown, NY). Hemoglobin concentration was measured using a hand-held photometer (B-Hemoglobin, Hemocue).
Microhemodynamic Parameters
Microhemodynamics.
Arteriolar and venular blood flow velocities were measured online by using the photodiode cross-correlation method (8) (Photo Diode/Velocity Tracker, model 102B, Vista Electronics, San Diego, CA). The measured centerline velocity was corrected according to vessel size to obtain the mean RBC velocity (V) (12). The video image-shearing method was used to measure vessel diameter (D) (9). Blood flow (
) was calculated as follows: = V x 
(D/2)2. Changes in arteriolar and venular diameter from baseline were used as indicators of a change in vascular tone.
Arterioles and venules. Microvessels were classified according to their position in the microvascular network (11). Arterioles were grouped into small arteries (A0), large feeding arterioles (A1), small arcading arterioles (A2), transverse arterioles (A3), and terminal arterioles (A4). Veins and venules were classified as small veins (V0), large venules (Vl), and small collecting venules (Vc). When arterioles exhibited vasomotion, the diameter was measured for 1 min and averaged over this interval. Detailed mappings were made of the chamber vasculature, so that the same vessels were studied throughout the experiment.
FCD. Capillaries were considered functional if RBCs transit through the capillary segments during a 45-s period. FCD was tabulated from the capillary lengths with RBC transit in an area comprising 10 successive microscopic fields (420 x 320 µm2). FCD (cm–1) is the total length of RBC-perfused capillaries divided by the area of the microscopic field of view (18).
Microvascular PO2 Distribution
PO2 was measured using the Pd-phosphorescence quenching method developed by Wilson (22), which was implemented for microcirculatory studies according to the description of Kerger et al. (10). This noninvasive method is based on the oxygen-dependent quenching of phosphorescence emitted by albumin-bound metalloporphyrin complex after pulsed light excitation, where rate of decay of the light-excited phosphorescence is inversely proportional to PO2 according to the Stern-Volmer equation (21). The Pd-phosphorescence quenching method has been used in this preparation for intravascular and extravascular PO2 measurements, as albumin exchange between plasma and tissue allows for sufficient concentrations of albumin-bound dye within the interstitium to achieve an adequate signal-to-noise ratio (1, 19). Animals received a slow intravenous injection of Pd-meso-tetra(4-carboxyphenyl)porphyrin (Porphyrin Products, Logan, UT; 15 mg/kg body wt at 10.1 mg/ml). The dye was allowed to circulate for 10 min before oxygen measurements.
For intravascular measurements, an optical rectangular window (
5 x 40 µm) was placed longitudinally within the vessel of interest, with the longest side of the rectangle positioned parallel to the vessel wall. Tissue PO2 was measured within intercapillary spaces in regions without large vessels with an optical window size of 6 x 6 µm. The decay curves were analyzed offline by means of a standard single-exponential least-squares numerical fitting technique, and the resultant time constants were applied to the Stern-Volmer equation (21) to calculate PO2 with use of parameters corrected for this animal model. The phosphorescence decay due to quenching at a specific PO2 yields a single decay constant, and in vitro calibration has been demonstrated to be valid for in vivo measurements.
Oxygen Saturation Curve for Hamster Blood
Oxygen saturation of hemoglobin was investigated by deoxygenation of oxygen-equilibrated oxyhemoglobin in a Hemox buffer (pH 7.4) at 37.6°C using a Hemox analyzer (TCS Scientific, New Hope, PA), which measures PO2 with a Clark-type O2 electrode (Yellow Springs Instrument, Yellow Springs, OH) and simultaneously calculates the hemoglobin saturation via a dual-wavelength spectrophotometer. PO2 at which hemoglobin is half-saturated (P50) was obtained directly from the oxygen equilibrium curves. Oxygen equilibrium curves for hamster RBCs were determined from freshly collected blood.
Experimental Design
The unanesthetized animal was placed in a restraining tube, which was attached to the stage of an inverted microscope (model IMT-2, Olympus, New Hyde Park, NY) equipped with a x20 objective (Olympus SPlan, NA = 0.46). The tissue image was projected onto a charge-coupled device camera (model 4815-2000, COHU, San Diego, CA) connected to a video cassette recorder (Panasonic AG-7355, Matsushita Electric Industries, Osaka, Japan) and viewed on a monitor (model PVM-1271Q, Sony, Tokyo, Japan). Sites of investigation were chosen on the basis of their visual acuity and vessel type within the network. The same sites of measurement were followed throughout the experiments, so that comparisons could be made directly with baseline levels.
Baseline systemic parameters, blood gas analysis, vascular diameter, RBC velocity, and FCD measurements commenced 30 min after the animal had become accustomed to the restraining tube. The Pd-porphyrin dye was injected intravenously over a 1-min period and allowed 10 min to distribute. Intravascular PO2 in the vessels chosen for study was measured along with vessel diameter and RBC velocity. Microvascular characterization and intravascular PO2 measurements were followed by interstitial tissue PO2 measurements. Blood gas analysis was repeated to ensure that stable oxygen levels were maintained during the observation period.
From a pilot study of the vascular diameter and blood pressure response, it was determined that the chosen dosage was able to sustain a moderate level of arteriolar dilatation (15%) without a substantial decrease in blood pressure for 
2–3 h. After 1 h of sustained dilatation, FCD was assessed, and capillary fields were recorded for 1 min for later offline analysis of RBC velocity. Next, the phosphorescence dye was infused into the venous catheter and allowed to circulate, and then intravascular and perivascular PO2 of arterioles and venules was measured. Flow and diameter were also measured online in these larger vessels. MAP and HR were monitored throughout the experiment.
Control data on microvascular PO2 distribution were obtained from 5 animals.
Data Analysis
The value presented for each parameter of microvascular hemodynamics and oxygenation (vessel diameter, flow velocity, flow, blood PO2, and FCD) is the average for all the animals in the study (n = 9). In the case of systemic parameters, each animal in the study is characterized by a value that is then averaged for the population of this study (n = 9). Results are presented as means ± SD unless otherwise noted. Data are presented as absolute values and ratios relative to baseline values: a ratio of 1.0 would signify no change from baseline, whereas lower and higher numbers are indicative of changes from baseline (i.e., 1.5 would mean a 50% increase from baseline levels and 0.5 would mean a 50% decrease in baseline levels). All measurements were compared with their levels at baseline. Changes are considered statistically significant if P 
0.05.
|
RESULTS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
Systemic Parameters
Systemic parameters at baseline and after verapamil injection are shown in Table 1. Baseline MAP of 121 ± 7 mmHg was reduced to 116 ± 9 mmHg at 1 h after verapamil treatment (P < 0.05). HR was 413 ± 39 beats/min at baseline and 393 ± 36 beats/min 1 h after verapamil treatment, a change that was not statistically significant.
|
Microvascular Hemodynamics
Arteriolar diameter increased to 1.20 ± 0.17 (n = 100, P < 0.001) and venular diameter was 1.11 ± 0.19 (n = 93, P < 0.001) after verapamil treatment (Fig. 1A). Arterioles dilated more than venules, but the difference was not significant; however, all changes were significantly greater than baseline. A3 vessels showed the largest dilatation.
|
Calculated flows increased to 2.00 ± 1.38 (P < 0.005) of baseline in arterioles and 1.56 ± 0.65 (P < 0.005) in venules (Fig. 1C). Flow changes from baseline at each vessel level were 2.04 ± 0.70 in A0 (P < 0.001), 1.71 ± 1.09 in A1 (P < 0.001), 1.85 ± 0.73 in A2 (P < 0.01), 2.48 ± 1.00 in A3 (P < 0.001), 1.74 ± 0.54 in A4 (P < 0.001), 1.61 ± 0.77 in Vc (P < 0.001), 1.44 ± 0.47 in Vl (P < 0.001), and 1.71 ± 0.35 in V0 (P < 0.01).
FCD
FCD in the verapamil-treated group was increased 1.05 ± 0.02 (P < 0.005), a change that was significant compared with baseline.
Oxygen Distribution in the Microvascular Network
Oxygen distribution in the different vessel orders in the control group is shown in Fig. 2. Intravascular and perivascular PO2 were measured in arterioles and venules and in the surrounding tissue. Intravascular PO2 was 56.8 ± 3.2 mmHg in A0 (n = 17), 51.8 ± 5.8 mmHg in A1 (n = 12), 44.3 ± 3.8 mmHg in A2 (n = 11), 39.9 ± 4.7 mmHg in A3 (n = 11), 37.2 ± 3.2 mmHg in A4 (n = 17), 31.0 ± 3.6 mmHg in Vc (n = 28), 35.1 ± 2.7 mmHg in Vl (n = 16), 41.1 ± 3.3 mmHg in V0 (n = 12), and 25.5 ± 4.1 mmHg in the tissue (n = 46). Perivascular PO2 was 36.9 ± 6.4 mmHg in A0, 36.7 ± 7.5 mmHg in A1, 31.9 ± 5.5 mmHg in A2, 29.8 ± 4.1 mmHg in A3, 29.3 ± 3.9 mmHg in A4, 26.4 ± 2.8 mmHg in Vc, 26.9 ± 2.4 mmHg in Vl, and 28.7 ± 4.0 mmHg in V0.
|
The oxygen gradient at the vascular wall was estimated from the difference between PO2 measured in blood at a position adjacent to the blood vessel wall interface and PO2 measured in the tissue in the immediate vicinity of the vessel wall. This difference in PO2 was not normalized to a length scale; therefore, the quantity reported is not actually a gradient and is referred to as the vessel wall PO2 difference (Fig. 2). The wall PO2 difference was 19.9 ± 5.3 mmHg in A0, 15.1 ± 3.6 mmHg in A1, 12.4 ± 4.1 mmHg in A2, 10.1 ± 4.2 mmHg in A3, 7.9 ± 3.5 mmHg in A4, 4.7 ± 2.7 mmHg in Vc, 8.3 ± 2.5 mmHg in Vl, and 12.4 ± 3.5 mmHg in V0 in the control group and 14.9 ± 6.7 mmHg in A0, 9.5 ± 3.5 mmHg in A1, 7.4 ± 4.1 mmHg in A2, 6.6 ± 2.8 mmHg in A3, 6.3 ± 2.5 mmHg in A4, 4.2 ± 2.8 mmHg in Vc, 5.2 ± 3.5 mmHg in Vl, and 7.4 ± 2.3 mmHg in V0 in the verapamil-treated group. The vessel wall PO2 difference in the verapamil-treated group was significantly decreased compared with control in A0 (P < 0.05), A1 (P < 0.001), A2 (P < 0.01), A3 (P < 0.05), V1 (P < 0.01), and V0 (P < 0.005). The vessel wall PO2 difference also decreased in the A4 and Vc vessels, but the change was not significant. The vessel wall PO2 difference during verapamil treatment is compared with control and vasopressin treatment according to data from Friesenecker et al. (5) in Table 2.
|
Oxygen release to the microcirculation was calculated as follows
 | (1) |
where HbRBC is the amount of hemoglobin, 
a-v% is the difference in O2 saturation between arterioles and venules given by O2 dissociation curve, and is the average flow through the paired arteriolar and venular vessels. Results are given in Table 2, where flow is normalized relative to control.
|
DISCUSSION |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
The intravascular distribution pattern of PO2 in the control group (Fig. 2) is the same as that found in previous studies of this tissue (6). The pattern of oxygen distribution was not significantly different after verapamil treatment, although there was a tendency for intravascular PO2 to be somewhat lower. Tissue PO2 was 32.0 ± 3.7 mmHg, which was significantly elevated relative to control (25.5 ± 4.1 mmHg). The same elevated level was found in a previous study on the effects of hyperoxia due to 100% inspired oxygen, where tissue PO2 was 31.9 ± 0.9 mmHg (17). In the case of hyperoxia, oxygen delivery remained constant compared with normoxia, but intravascular arteriolar and venular PO2 values were higher, and tissue equilibrated with the higher values of venular PO2.
In the present study, oxygen release to the tissue was approximately the same for control and verapamil treatment when computed between A1 and V1 vessels and lower vessel order pairings (Table 2). Oxygen release between A0 and V0 vessels after verapamil treatment was about double that in the control condition; however, these vessels are rather large (87.2–165.3 µm diameter for A0 and 165.8–383.6 µm diameter for V0), and their oxygen content may not reflect events of the tissue in the window chamber, where microvascular parameters were measured.
The PO2 difference across the microvascular wall was significantly decreased relative to control and arginine vasopressin. In the studies of Tsai et al. (19), Shibata et al. (13), and Friesenecker et al.(5), this difference in PO2 is 18 mmHg in A1 arterioles in control conditions, which is similar to the value found in the present study (16 mmHg). As a consequence of the cylindrical geometry, it can be shown that the minimum PO2 difference in A1 arterioles is 12 mmHg, assuming that the vessel wall tissue consumes oxygen at the same rate as most tissues or 10–2 ml O2·min–1·g tissue–1 (20). This theoretical lower limit appears to be reached in the present study, because in A1 vessels the PO2 difference is 10 mmHg.
Subtraction of the vessel wall PO2 difference from the intraluminally measured PO2 shows the PO2 of the outer microvessel wall, which is the oxygen source "seen" by the tissue, which governs the exchange of oxygen between the microvessels and the tissue. In control baseline conditions, the PO2 of the outer surface of the vessel wall of arterioles and venules is always greater than tissue PO2 (Fig. 2), indicating that all microvessels release oxygen to the tissue. Conversely, verapamil treatment causes the PO2 of the outer vessel wall of arterioles from A2 to V0 venules to not be significantly different from tissue PO2. Therefore, these vessels are in oxygen equilibrium with the tissue, indicating that tissue is primarily oxygenated by the oxygen released from the A0 and A1 vessels, whereas the oxygen released from the remainder of the microvessels is used by the microvessels per se. The effects found using the same protocol while the tissue was treated with the vasoconstrictor arginine vasopressin (5) are also shown in Fig. 2.
The analysis is based on the concept that only the vessels with a greater PO2 on the outer surface than in the tissue can supply oxygen to the tissue. This is a statistical consideration based on averages and, not necessarily, restricted to A0 and A1 vessels. Local variations will cause some of the vessels of most vessel orders to participate in this process. An important caveat is that oxygen release to the tissue cannot be estimated solely from the analysis of the changes of blood oxygen saturation, because blood oxygenates the tissue through the vessel wall, which extracts a portion of oxygen in relation to its tone. It is not possible to establish the exact proportion of vessel wall oxygen consumption vs. tissue oxygen consumption of the different conditions of vasoconstriction and vasorelaxation; however, this work and related studies show that vasoconstriction and vasorelaxation have an influence on tissue oxygenation, independently of the associated changes in local blood flow. An unknown in these calculations is the exact oxygen saturation at the venous exit from the microcirculation, a parameter determined from the oxygen saturation curve measured in the Hemox analyzer that is significantly changed by the Bohr effect. This effect, however, should be the same for normal baseline conditions and verapamil treatment, because there were no changes in pH.
Lowering of the vessel wall gradient could also be the consequence of the increase in the diffusion constant for oxygen across the vessel wall caused by verapamil; however, there is no mechanism to account for this phenomenon.
In conclusion, verapamil lowers the PO2 difference across the vessel wall (also referred to as the vessel wall gradient) in the microcirculation. This phenomenon leads to the increase in tissue PO2 to the level obtained with 100% inspired oxygen as more oxygen is available to the tissue and less oxygen appears to be consumed in the vessel wall. Calculation of the oxygen released to the tissue and the microcirculation in control and during verapamil treatment shows that this parameter remains unchanged. In control conditions, all vessels in the microcirculation deliver oxygen to the microvessels and the tissue; during verapamil treatment, the outside of the vessel wall surface of microvessels from A2 to V0 is in equilibrium with the tissue. The source of oxygen to the tissue is the blood-blood vessel combination taken as a unit. Therefore, most of the oxygen is delivered to the tissue during verapamil treatment by A0 and A1 vessels, and this oxygen delivery is sufficient to elevate tissue PO2. The present findings support the hypothesis that the vessel wall consumes a significant amount of oxygen and that this process is modulated by vasodilatation as well as vasoconstriction, as shown previously.
|
GRANTS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
|
FOOTNOTES |
|---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
|
REFERENCES |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  C.-L. Leong, W. P. Anderson, P. M. O'Connor, and R. G. Evans Evidence that renal arterial-venous oxygen shunting contributes to dynamic regulation of renal oxygenation Am J Physiol Renal Physiol, June 1, 2007; 292(6): F1726 - F1733. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Shibata, K. Qin, S. Ichioka, and A. Kamiya Vascular wall energetics in arterioles during nitric oxide-dependent and -independent vasodilation J Appl Physiol, June 1, 2006; 100(6): 1793 - 1798. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Cabrales, A. G. Tsai, P. C. Johnson, and M. Intaglietta Oxygen release from arterioles with normal flow and no-flow conditions J Appl Physiol, May 1, 2006; 100(5): 1569 - 1576. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Cabrales, A. G. Tsai, and M. Intaglietta Nitric oxide regulation of microvascular oxygen exchange during hypoxia and hyperoxia J Appl Physiol, April 1, 2006; 100(4): 1181 - 1187. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Shibata, S. Ichioka, and A. Kamiya Nitric oxide modulates oxygen consumption by arteriolar walls in rat skeletal muscle Am J Physiol Heart Circ Physiol, December 1, 2005; 289(6): H2673 - H2679. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Shibata, S. Ichioka, and A. Kamiya Estimating oxygen consumption rates of arteriolar walls under physiological conditions in rat skeletal muscle Am J Physiol Heart Circ Physiol, July 1, 2005; 289(1): H295 - H300. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
