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1Department of Bioengineering, University of California-San Diego, La Jolla, California 92093; and 2Department of Mechanical Engineering, Universidad de los Andes, Bogotá, Colombia
Submitted 25 September 2003 ; accepted in final form 12 November 2003
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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11 mmHg; therefore, lymph fluid has the lowest PO2 of this tissue. The disparity between lymph and tissue PO2 is attributed to the microlymphatic vessel wall and lymphocyte oxygen consumption.
lymph oxygen; lymphatic wall; oxygen consumption
2 mmHg, whereas the tissue is regulated at a PO2 that is in the range of 20–22 mmHg. Oxygen exchange between blood and tissue, as well as the tissue oxygen level, is ultimately determined by the transference of oxygen from the tissue fluid to the cells, as discussed in detail by Farrell et al. (4). The presence of a significant perimicrovascular oxygen gradient (12) determines that tissue PO2 should always be significantly lower than capillary blood PO2 and therefore also venular and venous blood PO2. This configuration has been verified by measurements of tissue PO2 with the phosphorescence technique. It is noteworthy that this technique, which reveals significant oxygen gradients in the perimicrovascular tissue, tends to show a relatively uniform tissue PO2 environment.
These premises suggest that a better estimate of tissue PO2 could be obtained by sampling PO2 of tissue fluid or by measuring PO2 of the excess tissue fluid that returns to the circulation via the lymphatic channels as proposed by Bergofsky et al. (2). This concept was investigated using polarographic oxygen electrodes by Barankay et al. (1) in the lymphatics of the rabbit hindlimb and by Farrell et al. (4) in the mesenteric lymphatics of the dog. Microelectrode studies were carried out in relatively large lymphatic vessels yielding a minimum average lymph PO2 of 28 and 50 mmHg in the hind limb and mesentery, respectively. Both studies dealt with lymphatic vessels of 1 mm intraluminal diameter, and although precautions were taken to insure isolation from the environment, subsequent findings of PO2 of these tissues suggest that PO2 of the fluid in these vessels was not representative of tissue PO2 (1, 4).
Lymphatics are lined with endothelial and contractile cells (smooth muscle) that are energy consumers, because they power the lymphatic activity that, in a pump-like process, extracts fluid from the tissue (8, 9). Thus tissue fluid transiting from the tissue into the lymphatics should shed some of its oxygen in support of the metabolism of initial lymphatic endothelium and smooth muscle. Lymphatics were classified according to the terminology proposed by Schmid-Schönbein (7, 10) into initial and collecting lymphatics. Initial lymphatics are "microscopic lymph channels" without a smooth muscle coat, also denoted in the literature as a capillary lymphatic, a terminal lymphatic, or a prelymphatic. The initial lymphatics that we studied did not have valves. Collecting lymphatics, also referred to as collecting ducts, collectors, or conducting lymphatics, are "lymph channels with a continuous array of lymphangions and with smooth muscle intima." Valves were observed in these vessels. Therefore, initial lymphatic fluid should have the lowest PO2 in the tissue. Initial lymphatics are difficult to identify in most tissues with the exception of the bat wing (14) and human skin, as shown by Bollinger et al. (3); however, collecting lymphatic vessels of 100 µm diameter are readily observable in the mesentery. Although these vessels might not be fully representative of initial lymphatic fluid, they should give an indication of tissue PO2 and whether oxygen consumption at the tissue/lymph interface is a factor in determining lymph PO2.
To the investigate this problem, we measured PO2 in the tissue and collecting and initial lymphatics of the rat mesentery using the phosphorescence quenching technique.
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MATERIALS AND METHODS |
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Polyethylene catheters (PE-50, Becton-Dickinson; Parsippany, NJ) were placed into the right femoral vein and artery for blood sampling and for the injection of supplemental anesthesia and the Pd-phosphorescence probe, respectively. The animals were placed on the stage of an intravital microscope and kept warm with a heating pad (37°C). The mesentery was exteriorized from an epigastric midline incision, partially extended, and kept moist with dripped buffered Krebs-Henseleit solution, pH corrected by bubbling a gas mixture of 5% CO2-95% N2. Before experiments the mesenteric tissue, as well as the intestine, was placed between thin strips of transparent plastic film (Saran Wrap, S. C. Johnson & Son; Racine, WI) to prevent gas exchange (diffusion coefficient of 6.2 x 10–13 cm3 O2·cm–1· mmHg–1·s–1) and desiccation. The heated drip without gas was continued on the plastic to maintain the tissue at 37°C during the study.
Phosphorescence quenching microscopy. Pd-phosphorescence quenching microscopy, based on the relationship between the decay rate of excited palladium-mesotetra-(4-carboxyphenyl)porphyrin (Porphyrin Products; Logan, UT) bound to albumin and the partial pressure of oxygen according to the Stern-Volmer equation, was used to measure PO2 in the microcirculation. The method is based on the technique described by Vanderkooi et al. (13). In our system, a xenon strobe arc (EG&G Electro Optics; Salem, MA; decay constant of 10 µs, 10-Hz flashing frequency, peak wave length 420 nm) excites the phosphorescence by epi-illumination of the tissue for 3 s. Phosphorescence emission from the target tissue is passed through an adjustable rectangular optical slit and light filter (630-nm cutoff) and is captured by a photomultiplier (Hamamatsu). Signals are visualized on an oscilloscope (TDS 2002, Tektronix; Beaverton, OR) and transferred to an analog input (BNC-2110, National Instruments; Austin, TX) connected to a high-performance data-acquisition board (PCI-6070E, National Instruments). Decay curves are analyzed off-line using a standard single-exponential least-squares numerical fitting technique, and the resultant time constants are applied to the Stern-Volmer equation to calculate PO2, where the quenching constant and the phosphorescence lifetime in the absence of O2, measured at pH 7.4 and a temperature of 37°C, are 325 mmHg–1·s–1 and 600 µs, respectively. 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 (6).
Interstitial and lymphatic PO2 measurement. The albumin-bound probe passes into the interstitium according to the exchange of albumin from blood to tissue (8). The resulting accumulation of albumin-bound dye within the tissue, which may contain up to 10% of the total albumin in the organism, allows the measurement of tissue and intravascular PO2 at high resolution with the same technique. Excess tissue fluid collected by the lymph contains the albumin-bound dye, as evidenced by the phosphorescence emission from the lymphatics within 20 min after injection, allowing us to carry out measurements of PO2, which are possible anywhere the palladium-porphyrin albumin-bound complex is located, if the signal-to-noise ratio is adequate.
Phosphorescence generated by the light excitation of the porphyrin probe consumes O2. This could be a factor affecting tissue O2 measurements made in slow-moving or stationary fluid of the lymphatics. This concern has already been addressed in the work of Tsai et al. (11) in the same tissue and led to the present protocol, which minimizes light excitation. The accuracy of the tissue PO2 measurements with the phosphorescence method was also examined in vivo using simultaneous continuous measurements with 5-µm-diameter recessed-tip gold cathode microelectrodes (15), and a maximum divergence of 2% was found between the methods over a tissue PO2 range of 5–40 mmHg. Extended flashing over a period of up to 1 min did not produce a detectable change in the microelectrode measurement, showing that microelectrode and phosphorescence quenching microscopy measurements yield the same information and that excitation of the porphyrin probe in the tissue does not affect tissue PO2.
Experimental protocol. The animals were placed on their side on an intravital microscope (BX-51, Olympus; New Hyde Park, NY) using a x20 objective (UMPlan Fl, Olympus; numerical aperture = 0.50) and equipped for trans- and epi-illumination. The Pd-phosphorescence probe was injected through the venous line. Measurements were started 20 min after the injection. Transillumination (halogen lamp, 12 V, 100 W) was used to identify the location of the lymphatics and the surrounding vessels. Oxygen measurements were obtained in vessels with sharp focus using an optical window of 5 x 20 µm using phosphorescence quenching microscopy. Oxygen measurements were carried out in the lymphatic fluid (intralymphatic measurements), perilymphatic tissue, in the adipose tissue at some distance from the lymphatics (>50 µm), and in the arteries and veins that paralleled the lymphatic vessels.
Data analysis. Statistical analysis was performed using Excel (Microsoft; Redmond, WA) and Prism (GraphPad; San Diego, CA) software. Differences between two means were assessed using the t-test. Comparison between three or more means was performed using one-way ANOVA and Tukey's multiple-comparison test. Changes were considered statistically significant if P < 0.05. All data are presented as means ± SD.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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200 µm apart along their length, leading to a total of 49 lymphatic PO2 measurements. There was no consistent pattern of longitudinal variability, and each measurement was taken to represent a vessel segment adding up to a total of measurements for each lymphatic vessel type. Additionally, tissue PO2 was measured in a total of 143 locations of the mesenteric tissue. Lymphatics in the mesenteric tissue could be divided into four categories depending on their position relative to the adipose tissue and connective tissue (Fig. 1). Accordingly, PO2 measurements were made in the centerline of the following vessel types: 1) lymphatic vessels in adipose tissue (Ladipose; nL = 14, n = 20; Fig. 2); 2) lymphatic vessels at the edge of the adipose tissue, between the adipose tissue and mesenteric connective tissue free of adipose tissue (Ledge; nL = 9, n = 11; Fig. 3); 3) lymphatic vessels in the mesenteric connective tissue (Lconnective; nL = 5, n = 13; Fig. 4); and 4) lymphatic vessels in the mesenteric connective tissue without surrounding blood vessels (Linitial; nL = 2, n = 5; Fig. 5). The wall of most lymphatic vessels (except Linitial) was usually in close proximity to arterioles, capillaries, and venules.
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PO2 was measured in four different regions of the mesenteric adipose tissue, namely: 1) the adipose tissue next to a lymphatic vessel (Tlymph; n = 47; Figs. 2 and 3); 2) next to an arteriole at location Tartery (n = 16); 3) next to a venule at location Tvein (n = 22; Fig. 2); and 4) in a region void of vessels (Tadipose; n = 39; Fig. 2). PO2 was also measured in connective tissue, namely: the connective tissue per se (Tconnective; n = 14; Figs. 4 and 5) and in the connective tissue in close proximity to adipose tissue (Tconn/adi; n = 5; Fig. 3). Perilymphatic (Tlymph) oxygen measurements in the adipose tissue were made as paired measurements in lymphatic vessels Ladipose or Ledge.
Measurement of PO2. The PO2 in the different lymphatic vessels according to our classification was 24.9 ± 6.9 mmHg in Ladipose, 22.4 ± 6.8 mmHg in Ledge, 20.2 ± 4.3 mmHg in Lconnective, and 0.8 ± 0.2 mmHg in Linitial. The PO2 in Linitial was significantly lower than the PO2 in the other groups (P < 0.001). There was no significant difference in lymph PO2 among vessels Ladipose, Ledge, and Lconnective, as shown in Fig. 6A.
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The results of PO2 measurements in the mesenteric adipose tissue are shown in Fig. 6B. The tissue PO2 was 32.8 ± 6.9 mmHg in Tlymph, 40.6 ± 7.9 mmHg in Tartery, 33.0 ± 7.8 mmHg in Tvein, and 33.7 ± 7.9 mmHg in Tadipose. The tissue PO2 was significantly higher in Tartery, and there were no statistical differences among the other groups (P < 0.05). The tissue PO2 in Tconnective was 3.0 ± 3.2 mmHg, which was significantly lower than the other groups (P < 0.001).
The mean lymph PO2 value of all the groups was 20.6 ± 9.1 mmHg, which was significantly lower than the mean tissue PO2 in all groups, which was 30.5 ± 12.3 mmHg (P < 0.001), as shown in Fig. 7A.
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Because PO2 in the adipose tissue next to blood vessels could be affected by the vessel PO2, we compared the mean value of tissue PO2 in Tlymph and the mean PO2 of the lymphatic fluid of Ladipose and Ledge. The mean PO2 of Ladipose and Ledge was 24.0 ± 6.8 mmHg, which was significantly lower than the tissue PO2 in Tlymph and Tadipose, which was 32.8 ± 6.9 mmHg (P < 0.001; Fig. 7B).
PO2 in the arterioles that surrounded lymphatic vessels was 44.8 ± 6.3 mmHg, which was significantly higher than the average lymph PO2 of Ladipose, Ledge, and Lconnective, which was 22.9 ± 6.4 mmHg (P < 0.001).
PO2 in the lymphatic vessels not surrounded by blood vessels and located in connective tissue at least 100 µm from the nearest mass of adipose tissue was 0.8 ± 0.2 mmHg, whereas the perilymphatic tissue had a PO2 of 0.7 ± 0.1 mmHg. Connective tissue at different locations of the mesentery at a distance of at least 100 µm from the adipose tissue had a PO2 of 3.7 ± 3.3 mmHg. The Tconn/adi group had a PO2 of 12.1 ± 0.9 mmHg.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The finding that lymph fluid PO2 is consistently lower than the surrounding tissue is indicative of the presence of an oxygen-consuming compartment between tissue and the lymphatic fluid. This may be constituted by the lymphatic endothelium, smooth muscle, and lymphocytes, otherwise the lymph fluid should be in equilibrium with tissue fluid, as hypothesized in the attempt to establish a baseline value for tissue PO2 discussed in the introduction. Analysis of the disposition of lymphatic and microvascular vessels shows that the lymphatics in the rat mesentery are in part juxtaposed to arterioles whose average blood PO2 is of the order of 45 mmHg and that give rise to a capillary network that is mostly adjacent to the lymphatic wall. The oxygen contribution of these vessels is probably small considering that arterioles have a vessel wall gradient of the order of 12 mmHg in the rat mesentery (11). However, the presence of this paralymphatic microvascular network, a sort of "vasa lymphorum," suggests that the cellular components of the lymphatic wall may have oxygen requirements that are not provided by diffusion from the surrounding tissue.
Initial lymphatics were rarely observed in the connective tissue of the mesentery, in regions void of microvessels and adipose tissue, as shown by our finding only 2 of this type of vessels (Fig. 5) of 25 independent observations of all categories of microlymphatics. These microlymphatics had virtually no cellular components in the vessel wall, and their lymph PO2 was in equilibrium with the surrounding tissue, which was significantly lower than the overall tissue PO2 measured in the connective tissue of the mesentery (0.8 ± 0.2 vs. 3.7 ± 3.3 mmHg). One may speculate that the very low PO2 in this region limited the development of most cellular species.
The noted distribution of tissue PO2 may be in part a consequence of how the tissue is exposed for analysis. In situ, the thin mesenteric membrane void of adipose tissue and blood vessels is probably folded and in close proximity to the remainder of the tissue, in such a fashion that the connective tissue avascular areas are in direct contact with the surface of highly vascularized adipose tissue, leading to more uniform tissue PO2. The disparity between lymph PO2 and tissue PO2, however, should be maintained because the lymph fluid analyzed in this study in the mesenteric adipose tissue and in the borderline adipose/connective tissue areas originates from either the gut or the adipose tissue per se.
Although oxygen consumption may be the cause of why the lymph fluid PO2 is 10–11 mmHg lower than the surrounding tissue, the actual rate of oxygen consumption by the lymphatic microvessel may not be significant for the overall oxygen management for the organism. This may be established approximately by considering that lymph flow in an adult person is of the order of one blood volume per day, or 5 l/24 h. The oxygen solubility in lymph can be assumed to be the same in water and not affected by potential differences in the concentration of lipids (2.14 x 10–5 ml O2·cm–3·mmHg–1). Therefore, if the whole lymphatic system undergoes the same oxygen consumption process, we can estimate the order of magnitude of the oxygen consumption of the lymphatic system, which is 1 ml O2/day.
The very low rate of oxygen consumption by the lymphatic system, however, suggests that there may be conditions in which the lymphatic fluid PO2 may be reduced to close to anaerobic conditions. This situation may occur if a highly metabolic cellular species were to be introduced into the lymphatic system, such as lymphatic metastasis or a bacterial infection were to develop in this system. Conversely, systemic measurements of lymphatic fluid PO2 greater than those reported here suggest that lymph from other organs may equilibrate at higher PO2 values or that oxygen shunts may be present in the larger lymphatic vessels.
In conclusion, the present study shows that the phosphorescence oxygen quenching method can be successfully used to map PO2 in microlymphatic vessels. Measurements show that lymph fluid in the rat mesentery has a systematically lower PO2 than the surrounding tissue, the difference being 10–11 mmHg, a phenomenon that can be attributed to the oxygen consumption by the cellular component of the lymphatic wall and lymphocytes. The overall oxygen supply and consumption of the lymphatic system appears to be very low, suggesting that lymph fluid PO2 may be manipulated to reach anaerobic conditions. Finally, the diversity of oxygen sinks and sources that affect the final PO2 outcome of lymph fluid appears to prevent using this parameter as an indicator of tissue PO2.
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ACKNOWLEDGMENTS |
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This study was supported by National Heart, Lung, and Blood Institute Bioengineering Research Partnership Grant R24-HL-64395 and Grants R01-HL-62354 and R01-HL-62318 (to M. Intaglietta).
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FOOTNOTES |
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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.
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REFERENCES |
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P < 0.001). PO2 in the connective tissue close to the adipose tissue was significantly lower than that in any part of the adipose tissue (
P < 0.001).