Experimental and theoretical studies of oxygen gradients in rat pial microvessels

doi:10.1038/jcbfm.2008.51

J Cereb Blood Flow Metab. Author manuscript; available in PMC 2012 December 23.

Published in final edited form as:

J Cereb Blood Flow Metab. 2008 September; 28(9): 1597–1604.

Published online 2008 May 28. doi: 10.1038/jcbfm.2008.51

PMCID: PMC3529159

NIHMSID: NIHMS419428

Experimental and theoretical studies of oxygen gradients in rat pial microvessels

Maithili Sharan,^1,⁵ Eugene P Vovenko,^2,⁵ Arjun Vadapalli,³ Aleksander S Popel,³ and Roland N Pittman⁴

¹Centre for Atmospheric Sciences, Indian Institute of Technology, Delhi, India

²I.Pavlov Institute of Physiology of the Russian Academy of Sciences, St Petersburg, Russia

³Department of Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland, USA

⁴Department of Physiology and Biophysics, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, Virginia, USA

Correspondence: Dr EP Vovenko, I.Pavlov Institute of Physiology, nab. Makarova, bld.6 199034, St Petersburg, Russia., Email: vovenko/at/infran.ru

⁵These authors contributed equally to this work.

The publisher's final edited version of this article is available at J Cereb Blood Flow Metab

See other articles in PMC that cite the published article.

Abstract

Using modified oxygen needle microelectrodes and intravital videomicroscopy, measurements were made of tissue oxygen tension (PO₂) profiles near cortical arterioles and transmural PO₂ gradients in the pial arterioles of the rat. Under control conditions, the transmural PO₂ gradient averaged 1.17 ± 0.06 mm Hg/μm (mean ± s.e., n = 40). Local arteriolar dilation resulted in a marked decrease in the transmural PO₂ gradient to 0.68 ± 0.04 mm Hg/μm (P < 0.001, n = 38). The major finding of this study is a dependence of the transmural PO₂ gradient on the vascular tone of the pial arterioles. Using a model of oxygen transport in an arteriole and experimental PO₂ profiles, values of radial perivascular and intravascular O₂ fluxes were estimated. Our theoretical estimates show that oxygen flux values at the outer surface of the arteriolar wall are approximately 10⁻⁵ mL O₂/cm² per sec, independent of the values of the arteriolar wall O₂ consumption within a wide range of consumption values. This also means that PO₂ transmural gradients for cerebral arterioles are within the limits of 1 to 2 mm Hg/μm. The data lead to the conclusion that O₂ consumption of the arteriolar wall is within the range for the surrounding tissue and O₂ consumption of the endothelial layer appears to have no substantial impact on the transmural PO₂ gradient.

Keywords: cortical microvessels, O₂ transport model, oxygen microelectrodes, tissue PO₂ profiles, transmural PO₂ gradient

Introduction

Oxygen molecules leaving microvessels diffuse along concentration gradients that are established by the spatial distribution of the vessels and the rate of oxygen consumption in the vascular wall and surrounding tissue. Capillaries are not the only source of oxygen to tissue (Duling and Berne, 1970). Arterioles and venules also supply oxygen to tissue and play a significant role in diffusional O₂ exchange between various segments of microvasculature. Extensive experimental studies have shown that arterioles could supply up to 50% (or even above) of all oxygen demand in resting skeletal muscle (Swain and Pittman, 1989; Ellsworth and Pittman, 1990). In cerebral tissue, arterioles supply up to 20% of all consumed oxygen (Vovenko, 1997, 1999).

The role of the wall in microvessels has been the topic of several studies, as it participates in the exchange of gases between blood and tissue. Although some studies point to a very high consumption of O₂ by the endothelial and smooth muscle cells of the wall (Shibata et al, 2005; Tsai et al, 1998, 2003), other studies suggest a consumption rate closer to that of the surrounding parenchymal tissue (Golub et al, 2007; Pittman et al, 2005; Vadapalli et al, 2000). Various experimental methods have been developed that make it possible to conduct PO₂ measurements in the lumen (intravascularly), or on the outer wall of microvessels and in the surrounding tissue (Wilson et al, 1991; Ivanov et al, 1982; Duling et al, 1979). Using these methods, the transmural gradients of PO₂ have been measured on cat pial arterioles (Duling et al, 1979), rat mesenteric arterioles (Tsai et al, 1998), rat cremaster muscle (Shibata et al, 2001, 2005), and other tissues (Santilli et al, 2000; Crawford and Cole, 1985). These data vary significantly, from 1 to 10 mm Hg/μm and above. Such large values of transmural PO₂ gradients led to the hypothesis of a very high consumption rate of O₂ by endothelium and/or smooth muscle cells (Tsai et al, 1998, 2003). If this hypothesis is validated, it might result in significant revision of the current concept of oxygen transport to tissue and would require taking into consideration additional oxidative metabolism in the neighborhood of the blood–tissue interface.

The main goal of the present study was to test the hypothesis of a large transmural PO₂ gradient across the blood–tissue interface. We have studied transmural PO₂ gradients on the arterioles of rat brain cortex, a tissue with high oxidative metabolism and, consequently, high blood flow. Using these data, we performed theoretical estimates of O₂ consumption in the vascular (arteriolar) wall and estimated transmural PO₂ gradients have been assessed. In addition, using oxygen microelectrodes of a special design, we measured transmural PO₂ gradients in control and vasodilated pial arterioles. Our experimental results and theoretical estimates show that transmural PO₂ gradients for rat cortical arterioles are similar to those in surrounding tissue and are in the range of 1 to 2 mm Hg/μm under control conditions and below 1 mm Hg/μm in dilated vessels.

Materials and methods

Animals and Surgical Procedures

All the experiments were performed in male Wistar rats (Pavlov Institute of Physiology vivarium) weighing 250 to 320 g. The experiments were performed in accordance with ethics regulations of The Pavlov Institute, which are in agreement with the European Communities Council Directive (86/609/EEC) for the care and use of laboratory animals. Animals were initially anesthetized by an intra-peritoneal injection of sodium pentobarbital (60 mg/kg) and then by supplemental doses of 10 to 12 mg/kg per h. They were placed on a Plexiglas platform and the head was fixed using a special teeth rod mounted on a ball-and-socket joint. A tracheostomy was made to ensure proper spontaneous ventilation. A polyethylene catheter (PE-90) was inserted into the femoral artery and vein for collecting blood samples, for gas analysis (ABL-330, Radiometer, Denmark), for measurement of mean arterial pressure, and for blood and solute injections.

A 4 × 8 mm opening was made on the right parietal lobe of the animal’s scull. The dura mater was accurately removed and the exposed brain cortex was superfused with a solution of the following composition (in mmol/L): NaCl 118, KCl 4.5, CaCl₂ 2.5, KHPO₄ 1.0, MgSO₄ 1.0, NaHCO₃ 25, and glucose 6. The solution was equilibrated with a gas mixture of 5% CO₂/5% O₂/balance N₂ and its pH was adjusted to 7.38±0.05 at 37°C.

Experimental Setup

The design and layout of the setup has been described elsewhere (Vovenko, 1999). Briefly, the microscope and 3D-manipulated stage were mounted on a massive metal beam to minimize vibrations and displacements of the microelectrode during PO₂ measurements on microvessels. Any displacements of the animal relative to the microscope were performed using the stage under the microscope. A PO₂ microelectrode was manipulated by a 3D-micromanipulator that was mounted on the objective of the microscope (Figure 1). The microelectrode tip was positioned at the center of the frontal lens of the objective. A thermostabilizing system maintained a constant temperature (37°C±1°C) of the animal’s body, the objective of the microscope, and the calibration and superfusion solutions.

Figure 1

Local measurements of oxygen partial pressure using a fine-tip oxygen microelectrode. (A) Tip of the microelectrode (2), mounted on a 3D-micromanipulator (3), is positioned in the center of frontal lens of the contact epiobjective (1) of the microscope. (more ...)

Visualization

The microvasculature of the brain cortex was visualized using a microscope LUMAM-K1 (LOMO, Russia) equipped with contact lens epiobjectives (×10/0.30, × 20/0.60). For visualization, the frontal lens of the objective was put in immediate contact with the brain tissue (no compression, the gap between lens and brain less than 10 μm). Focusing of the microscope was performed without displacement of the objective relative to tissue using a special lens, moved along the optical axis inside the microscope tube. The microscopic image was viewed on a TV monitor and, at the same time, on a PC monitor using a video camera (PIH-576, Taiwan) and capture card (Pinnacle Deluxe). The linear dimensions (microvascular luminal diameter, thickness of the vascular wall, and distance to the microelectrode tip) were determined during offline analysis using frame capture and a special screen caliper. The error of linear measurements was approximately ±2 μm.

Oxygen microelectrodes

Platinum oxygen microelectrodes with a tip diameter including glass insulation of 3 to 4 μm and a recess depth of 8 to 12 μm were used in the study. The polarographic current in physiologic solution saturated with room air at 37°C ranged from 5 × 10⁻¹¹ to 9 × 10⁻¹¹ A. Residual current was negligible. The sensitivity of the microelectrodes to fluid convection was tested by measuring the polarographic current in a stirred and unstirred solution. Convection was produced using a magnetic stirrer (linear velocity of the solution at the microelectrode tip was approximately ~100 mm/s). Oxygen microelectrodes with a recess of l/d = 8 to 10 showed an increase in current during the stirring of less than 2% to 3%, if any. The microelectrodes used in this study revealed a small ‘calibration error’ too, that is, they were not sensitive to variations in O₂ diffusion coefficient in the calibration and measurement media (Buerk, 2003; Craw-ford and Cole, 1985; Schneiderman and Goldstick, 1978). The microelectrode was calibrated several times during an experiment. To minimize measurement error before measurements, the electrode tip was inserted into the brain tissue or placed into the venular lumen for several minutes. Then, the electrode was placed into the calibration solution again. This procedure was repeated several times until a stable level of electrode sensitivity to oxygen was attained.

Experimental Protocol

The tissue was allowed to stabilize for a period of at least 10 to 15 mins before the acquisition of PO₂ data. In Series 1, measurements of radial tissue PO₂ profiles near the walls of rat precortical arterioles were performed. In Series 2, measurements of transmural PO₂ gradients were performed on rat pial arterioles. Group 1 of Series 2 consisted of transmural PO₂ measurements in control pial arterioles, whereas Group 2 of Series 2 consisted of transmural PO₂ measurements on dilated pial arterioles.

Measurements of radial PO₂ profiles in the vicinity of precortical arterioles (series 1)

To record a tissue PO₂ profile, representing oxygen diffusion across the wall of an arteriole, the PO₂ microelectrode was positioned so that the nearest large microvessel (on the course of the electrode track) was at least 70 to 80 μm away. The PO₂ microelectrode was positioned perpendicularly to the wall of the selected arteriole. PO₂ was then measured at the wall surface and at distances of 10, 20, 30 μm and so on from the vessel wall, sequentially (Figure 2). For reproducible measurements, the PO₂ microelectrode was again brought to the surface of the outer wall to recheck the values obtained.

Figure 2

Measurements of tissue PO₂ profiles near precortical arterioles of the rat. (A) The microelectrode tip (1) is 30 μm from the precortical arteriole (2) of luminal diameter 20 μm. Scale, 50 μm. (B) Example of recording of PO₂ radial (more ...)

Measurements of PO₂ transmural gradients on pial arterioles in control (series 2, group 1)

For assessing the transmural PO₂ gradient, it is necessary to know PO₂ values on the outer and inner wall of the arteriole and the wall thickness. The transmural PO₂ gradient was defined as the difference of intravascular and outer wall PO₂ relative to the displacement of the electrode (Δl) defined as the distance from the outer wall surface to the edge of the cell-free plasma layer and red blood cell core in the arteriolar lumen. Δl was measured offline during analysis of the recorded photomicrographs. Δl could be used also as an estimate of the arteriolar wall thickness ΔL (it exceeds wall thickness by the thickness of the cell-free plasma layer) used in the mathematical analysis. Cell-free plasma layer thickness has been measured in arterioles (Kim et al, 2007). To measure the transmural PO₂ gradient, the microelectrode tip was positioned on the outer surface of the wall in the midplane of an arteriole. An image of the field was captured and oxygen tension was recorded. The microvessel wall was punctured by quickly moving the microelectrode into the wall using a micromanipulator (Figure S1). Intravascular readings of PO₂ were relatively stable during the first 5 to 6 secs after penetration of the vessel. Then, thrombus formation began at the microelectrode tip and a progressive decline of PO₂ occurred. Therefore, all intravascular PO₂ measurements were obtained during the first 5 to 6 secs after wall puncture, when thrombus size was negligible and it did not disturb blood flow in the arteriole. Then, the microelectrode was withdrawn from the vessel to perform a calibration.

Measurements of transmural PO₂ on pial arterioles during local vasodilation (series 2, group 2)

The vasodilator agent (~2 × 10⁻⁷ mmol/L sodium nitroprus-side in physiologic solution) was administered into the perivascular zone of the arteriole using a glass micropipette of diameter 25 to 30 μm (Figures 1C–1E). Transmural PO₂ measurements on the dilated arteriole were conducted according to the same protocol as above for the control series.

Data Analysis

All data are presented as mean ± s.e. All calculations and graphics were made using the program OriginPro ver. 7.5 (OriginLab Corp.).

Results

Tissue PO₂ Profiles (Series 1)

Sixteen male Wistar rats (250 to 280 g body weight) were used in the first series of experiments to measure radial PO₂ profiles in the vicinity of precortical arterioles.

Blood gas parameters of systemic blood were as follows: pH = 7.351 ± 0.002; PCO₂ = 37.6 ± 1.2 mm Hg; PO₂ = 78.7 ± 2.2 mm Hg (n = 19). Mean arterial pressure in the experiments of Series 1 averaged 126 ± 5 mm Hg (n = 57).

Tissue radial PO₂ profiles around precortical arterioles of the rat measured using oxygen micro-electrodes are shown in Figures 2D–2F. During measurements, the microelectrode tip was positioned within the brain tissue. The data showed that a marked decrease in tissue PO₂ was observed in the region near the arteriolar wall (within a tissue zone of 20 to 30 μm). The PO₂ profiles were more shallow farther from the arteriole.

Transmural PO₂ Gradients (Series 2)

A total of 31 rats (230 to 250 g body weight) were used in the second series of experiments to explore transmural PO₂ gradients in rat pial arterioles under control (Group 1, 17 animals) and vasodilated conditions (Group 2, 14 animals). Blood gas parameters of systemic arterial blood of both groups of animals were as follows: pH = 7.334 ± 0.006, PCO₂ = 43.7 ± 0.5 mm Hg, PO₂ = 87.3 ± 1.3 mm Hg (n = 34). Mean arterial pressure in Series 2 averaged 136 ± 2 mm Hg (n = 86).

To measure the transmural PO₂ gradient, the microelectrode tip was brought close to the outer arteriolar wall and the control level of PO₂ was recorded. Then, the microelectrode was moved into the arteriolar lumen and intra-vascular PO₂ was recorded. The relationship between Δl and arteriolar luminal diameter is presented in Figure 3A. The relationship between ΔPO₂ and the corresponding Δl is presented in Figure 3B. The slope of the regression line in Figure 3B is numerically equal to the transmural PO₂ gradient in rat pial arterioles (1.17 ± 0.06 mm Hg/μm, n = 40). Figure 3C shows the relationship of the transmural PO₂ gradient and measured arteriolar luminal diameter.

Figure 3

Results of transmural PO₂ measurements in rat pial arterioles during control (A to C) (Series 2, Group 1) and dilation (D to F) conditions (Series 2, Group 2). (A, D) Relationship between Δl and arteriolar luminal diameter. Δl is the distance (more ...)

The same measurements were performed on dilated arterioles to assess whether vascular tone has an impact on the transmural PO₂ gradient in rat pial arterioles. The results of Series 2, Group 2 are presented in Figures 3D–3F. The transmural PO₂ gradient in dilated arterioles was 0.68 ± 0.04 mm Hg/μm (P < 0.001, n = 38).

Discussion

We have presented experimental measurements of tissue PO₂ profiles around rat cortical arterioles and transmural PO₂ measurements in pial arterioles using specially designed fine-tip O₂ microcathodes. Our results showed that under control conditions radial and transmural PO₂ gradients did not exceed 2 mm Hg/μm. In dilated arterioles, transmural PO₂ gradients are ~0.7 mm Hg/μm. Our theoretical estimates showed that oxygen flux values at the outer surface of the arteriolar wall are around 10⁻⁵ mL O₂/cm² per sec, independent of the values of the arteriolar wall O₂ consumption within a wide range of consumption values. This also means that transmural PO₂ gradients for cerebral arterioles are within the limits of 1 to 2 mm Hg/μm.

In this study, attention was focused on O₂ diffusion through the arteriolar wall—an interface between blood and tissue. Arterioles do play a significant role in oxygen exchange between these two compartments. Arterioles are powerful sources of O₂ to tissue (high PO₂, blood flow). Nerve cells located nearby arterioles might favor under the conditions of restricted O₂ supply. In addition, arterioles take part in diffusional oxygen exchange of respiratory gases between venules and capillaries. Direct PO₂ measurements showed that anatomical capillaries deliver ~60% of all O₂ consumed by brain tissue, whereas cortical arterioles of diameter 7 to 20 μm deliver approximately 20% and arterioles of diameter 30 μm and larger deliver less than 10%. The data imply that cerebral microvasculature of caliber ≤20 μm is a primary site of gas transfer between the blood and the tissue (Vovenko, 1997, 1999).

There are a few data in the literature concerning measurements of transmural PO₂ gradients in brain arterioles. Duling et al (1979), using Whalen-type oxygen microcathodes in cat pial arterioles, found transmural PO₂ gradients of ~0.91 mm Hg/μm. Our measurements of transmural PO₂ gradients are smaller than those reported in other tissues using the optical phosphorescence quenching method (Tsai et al, 1998, 2003). This discrepancy might be because of the differences in the tissues studied and differences in the methodology used. Several studies point out that extravascular PO₂ measurements using the phosphorescence quenching method might contain a measurement artifact—underestimation of the actual tissue PO₂ because of an oxygen consumption artifact (O₂ consumption caused by the excitation flash impulse) (Golub and Pittman, 2005).

Shibata et al (2001, 2005), using the phosphorescence quenching method, measured the decrease in PO₂ in the arterioles of rat cremaster muscle and found that the PO₂ difference was approximately 15 to 20 mm Hg over a distance of 10 to 12 μm, so that the measured transmural PO₂ gradient was within 1 to 2 mm Hg/μm. A pronounced PO₂ difference (40 to 45 mm Hg) over a distance of 2 μm was found in the rabbit infra-renal aorta using a microelectrode technique (Santilli et al, 2000). This steep decrease in PO₂ over such a small distance, however, could be because of tissue compression/distortion during microelectrode penetration of the dense tissue (Crawford and Cole, 1985).

It is worth noting that the oxygen microelectrode technique is probably the only method enabling local PO₂ measurements in three different compartments in the brain cortex: tissue (extravascular), vascular wall (perivascular), and vessel lumen (intravascular). The method of phosphorescence quenching is applicable for intravascular (intravascular infusion of phosphor) and extravascular (topical application of phosphor) measurements only, because of the practical impermeability of the blood–brain barrier for extravasation of the albumin-bound phosphorescence probe.

Despite the advantages indicated above, the use of oxygen microelectrodes has a number of limitations. The first limitation is because of the invasive nature of the measurements. The scull is open (no closed cranial window), making the cortical tissue susceptible to the development of edema. However, the duration of the experiments was within 60 to 90 mins, after opening of the skull, and no significant edema was observed during this time.

It is extremely difficult to penetrate the wall of an arteriole of diameter ≤20 to 25 μm, even using thin and specially sharpened microelectrodes (with tip diameter including glass insulation ~3 μm). This is the reason why all our data on transmural gradients were obtained on microvessels of larger caliber (the data in Series 1 on smaller vessels did not involve puncturing the arteriolar wall).

From a methodological point of view, the intra-vascular PO₂ measurements represented a serious and difficult task. First of all, the puncture should induce only minimal injury of the arteriolar wall. The microelectrode tip diameter should be small (2 to 4 μm) and the shaft taper of the tip should be ≤3° and be sharpened similarly to a syringe needle (sharpening angle ~15° to 18°). The angle of beveling of the tip plays a critical role in minimizing trauma to the wall (even for microelectrodes with a tip diameter of 3 to 4 μm). In addition, the polaro-graphic current of the microelectrode should not depend on fluid convection (for correct measurements in flowing blood). The microelectrode readings should not be dependent on the O₂ permeability coefficient of the measurement and calibration media. Finally, the calibration characteristics of the O₂ cathodes should be relatively stable and reproducible. The oxygen microelectrodes used in this study complied with the above requirements.

Dilation of the arteriolar wall resulted in a decrease of the transmural PO₂ gradient to 0.68 ± 0.04 mm Hg/μm. This means that the vascular tone and, accordingly, the level of oxidative metabolism of the smooth muscle cells could have an impact on the transmural PO₂ gradient. It is possible that endothelial cells also contribute to the observed decrease in oxygen tension in the arteriolar wall. Nevertheless, our data do not provide support for the hypothesis of an important role for the endothelium in the transmural PO₂ gradients in arterioles.

Experimental measurements (Vovenko, 1999) yield an intravascular O₂ flux J_i of the order of 10⁻⁵ mL O₂/cm² per sec (Figure S2). Our theoretical estimates of J_i are consistent with those based on measurements when the PO₂ gradients are within 1 to 2 mm Hg/μm. Calculation of J_i in individual vessel segments also supports a value of J_i~10⁻⁵ mL O₂/cm² per sec. In addition, a majority of the computed values of J_i in microvessels, compiled by Vadapalli et al (2000), and based on in vivo measurements of longitudinal hemoglobin oxygen saturation or PO₂ gradients in arteriolar segments, are ~10⁻⁵ mL O₂/cm² per sec, consistent with those based on the observations.

The estimate of O₂ flux from the rat cremaster muscle (Shibata et al, 2005) results in J_i values of an order of magnitude higher than that in the present measurements. This estimation is based on morphologic assumptions about vessel length; however, they have reported oxygen gradients between 1 and 2 mm Hg/μm. In their analysis, it was assumed that the entire O₂ flux was consumed by the wall and, accordingly, they calculated an O₂ consumption in the wall that was 100 times higher than in the tissue.

For the pial arteriolar network in rats, the global estimate also yields a value of J_i = 1.2 × 10⁻⁵ mL O₂/cm² per sec (Figure S3). Using morphologic data for the rat mesentery network (Pries et al, 1990), the computed value of J_i is 1.03 × 10⁻⁵ mL O₂/cm² per sec. Global estimates of intravascular flux for the pial and mesenteric arteriolar networks in rats support a value of J_i ~ 10⁻⁵ mL O₂/cm² per sec. However, global estimates of J_i for the rat spinotrapezius muscle with two sets of morphologic data for that arteriolar network (Engelson et al, 1985) are 3.026 × 10⁻⁴ and 9.85 × 10⁻⁵ mL O₂/cm² per sec. The foregoing discussion supports theoretical estimates of J_i ~ 10⁻⁵ mL O₂/cm² per sec, consistent with the observations when oxygen gradients are within 1 to 2 mm Hg/μm.

In summary, theoretical estimates based on experimental data of tissue PO₂ profiles around rat cortical arterioles and direct measurements of transmural PO₂ gradients on pial arterioles showed that the decrease in PO₂ across the arteriolar wall is within 1 to 2 mm Hg/μm under control conditions and approximately ~0.7 mm Hg/μm under vasodilated conditions. The data presented lead to the conclusion that the O₂ consumption of the arteriolar wall is within the range for the surrounding tissue and that the O₂ consumption of the endothelial layer, apparently, does not have a substantial impact on the transmural PO₂ gradient.

Supplementary Material

Sharan et al_Supplemental Data

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Acknowledgments

This study was supported in part by Grants 03-04-49470 and 06-04-49274 from the Russian Basic Research Foundation and by Grant HL18292 from the National Institutes of Health.

Footnotes

Supplementary Information accompanies the paper on the Journal of Cerebral Blood Flow & Metabolism website (http://www.nature.com/jcbfm)

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