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PO2 measurements in the rat intestinal microcirculation

M. Sinaasappel, C. Donkersloot, J. van Bommel, and C. Ince

Department of Anesthesiology, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Microvascular partial oxygen pressure (PO2) data measured with the quenched phosphorescence of palladium-porphyrin (Pd-porphyrin) with the use of optical fibers have provided new insight into the behavior of the microvascular oxygenation in models of shock. However, the actual microcirculatory compartment measured by this fiber technique has not yet been demonstrated. The purpose of this study was to investigate whether the PO2 of the intestines, as measured using a fiber phosphorometer, reflects the microvascular compartment. To this end, a new intravital phosphorometer with an improved sensitivity to high-PO2 levels (up to 180 mmHg) was developed and validated. With this setup, PO2 values were measured at different inspired oxygen fractions (15, 25, and 50% oxygen) in first-order arterioles, capillaries, and venules of the ileum of rats. Simultaneously, the PO2 was measured with an optical fiber attached to another phosphorometer. PO2 measurements with the fiber phosphorometer show excellent correlation with the PO2 in the capillaries and first-order venule vessels (r2 = 0.94, slope 0.99). We therefore conclude that values measured with the fiber phosphorometer correlate with the capillary and venular PO2.

intravital microscopy; palladium-porphyrin; phosphorescence quenching


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE DEVELOPMENT OF THE oxygen-dependent quenching of palladium-porphyrin (Pd-porphyrin) phosphorescence technique by Wilson and co-workers (27, 31) has led to its application in the investigation of the oxygenation of the microvascular bed. Initially, this was accomplished with optical fibers attached to phosphorometers for delivery of the excitation and emission light (7-9, 13-17, 20-22, 28). Later, phosphorometers were attached to intravital microscopes, and it was possible to measure oxygen pressures in single capillaries venules and arterioles (6, 9, 12, 18, 24-26, 32). The advantage of using fiber optics for providing partial oxygen pressures (PO2) in vivo is that measurements can be made under conditions and locations not easily accessible to intravital microscopes. Such conditions include measurements in large animal models, with which systemic parameters of oxygen transport can be better monitored than in small animal models, and measurements of moving organs such as the heart (20) or diaphragm (17). Furthermore, fiber phosphorometers allow the possibility of measuring microvascular PO2 simultaneously in different organ beds, such as in the heart and gut (20). We have used the fiber-optic phosphorometer to investigate the efficacy of hemoglobin solutions for improving microvascular oxygenation following hemorrhagic shock (22, 28) and the disassociation of microvascular and venous PO2 in the progress of experimental sepsis (9). However, the true microcirculatory compartment measured with the fiber phosphorometer, although considered to be at the end-capillary level (17), has yet to be established.

This study aimed to determine which microvascular compartment best corresponds to the microvascular PO2, as measured by fiber phosphorometry. To this end, both techniques were combined to investigate which microvascular compartment was sensed with the fiber phosphorometer. The PO2 values measured with the fiber phosphorometers ranged from the low end of the scale, around 0-10 mmHg found in shock, to around 60 mmHg in mechanically ventilated animals using inspired oxygen fractions of 30% oxygen (22, 28). To allow a comparison between the fiber phosphorometer and the microscope phosphorometer, both devices have to be able to measure over the same range of values. This imposes a problem for the intravital phosphorometer at the high end of the PO2 scale. Phosphorometers attached to microscopes to measure the PO2 of single capillaries have shown maximal PO2 values of ~55 mmHg (18, 24, 32). Therefore, a new intravital phosphorometer was introduced in this study that has a very short excitation pulse (1 µs) to measure high-PO2 levels, which works in conjunction with an integrating amplifier to enhance the signal-to-noise ratio. We chose to compare the two techniques in the gut because of our group's interest surrounding the oxygenation of this organ under conditions of shock (9, 20, 22, 28).

In this study, we determined the microvascular bases of the PO2 values measured by the fiber phosphorometer by simultaneously measuring arteriolar, capillary, and venular PO2, which were measured with the intravital phosphorometer in the rat gut under different inspired oxygen fractions.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Intravital phosphorometer. The optical system was built on an intravital microscope configured for epifluorescence (Fig. 1) similar to that described previously (29). Long distance objectives with ×50 and ×20 magnification were used to observe the microvasculature. Images were recorded by an image-intensified charge-coupled device camera (MXRI 5051, HCS Vision Technology, Eindhoven, The Netherlands). For Pd-porphyrin measurements, a xenon flashlamp with a 100-nF capacitor (EG&G, type FX249, Salem, MA) was used; this flashlamp provided a flash with a duration of 1 µs. This short pulse duration was necessary to prevent disturbances on the phosphorescence decay by the flashlamp decay. The excitation wavelength was selected by a 400 ± 20-nm interference filter (Omega Optical, Salem, MA). The excitation light was coupled in the light path of the illumination system via a dichroic mirror with a cutoff wavelength at 430 nm (Omega Optical), which illuminated the complete field of view. A rectangular diaphragm of adjustable size was placed in the tissue-image plane in front of the photomultiplier tube (PMT; R928, Hamamatsu) to select the area of interest. The light passing through the diaphragm was filtered by a RG630 long-pass glass filter (Omega Optical) and projected onto the PMT. The area of interest can be identified by illumination of this area with an additional light source via a mirror between the diaphragm and the PMT (see Fig. 1). This way, the diaphragm can be projected onto the image plane to determine the precise location and size. The phosphorescence was measured by a PMT and recorded with a PC equipped with a data-acquisition board (AT-MIO16E-1, National Instruments).


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Fig. 1.   A schematic diagram of the intravital phosphorometer. Setup consists of an intravital microscope, illumination to define the region of interest, and a xenon flashlamp for excitation of the palladium (Pd)-porphyrin. Measurement of the phosphorescence is accomplished by a phosphorometer consisting of a photomultiplier tube (PMT), a preamplifier, and 2 integrating amplifiers. A computer performed phosphorescence decay and conversion to partial oxygen pressure (PO2) levels. BP, band pass; ICCD, image-intensified charge-coupled device camera; LP, long pass.

In addition to the intravital measurements, the PO2 was measured with a fiber phosphorometer as described in Ref. 21. This phosphorometer consisted of a flashlamp (L4634, Hamamatsu, pulse duration of 1 µs), a photomultiplier (R928, Hamamatsu), and an optical fiber. The decay traces were collected using the same 1-MHz data-acquisition board (AT-MIO16E-1, National Instruments). In front of the flashlamp, a 520 ± 20-nm interference filter (Omega Optical) selected the excitation wavelength and a 700 ± 40-nm interference filter selected the emission wavelength.

Phosphorescence measurements. The quenching of the phosphorescence of Pd-porphyrin by oxygen is based on the principle that a Pd-porphyrin molecule excited by light can either release the absorbed energy as light (phosphorescence) or transfer the absorbed energy to oxygen molecules. This process causes the phosphorescence intensity and decay time to decrease in an oxygen-dependent manner as described by the Stern-Volmer relation
<FR><NU>&tgr;<SUB>0</SUB></NU><DE>&tgr;</DE></FR> = 1 + &tgr;<SUB>0</SUB><IT>k</IT><SUB>q</SUB>[O<SUB>2</SUB>] (1)
Here, tau 0 (in µs) is the decay time at zero oxygen and tau  is the decay time in the presence of a given concentration of oxygen [O2] (in µM). The second-order rate constant, kq (1/µM per µs), relates the oxygen concentration to the collision frequency between Pd-porphyrin and oxygen. The calibration constants tau 0 and kq are temperature dependent and were determined elsewhere (21). To convert the oxygen concentration from Eq. 1 to PO2 (mmHg) the following relation was used
P<SC>o</SC><SUB>2</SUB> = <FR><NU>V<SUB>m</SUB> 760</NU><DE>&agr;</DE></FR> [O<SUB>2</SUB>] (2)
Equation 2 gives the relation between the oxygen concentration and the PO2 (30). In this equation, alpha  is the Bunsen coefficient and Vm is the molar volume at 0°C (22.4 mol/l). For in vivo calculation, the Bunsen coefficient (alpha ) for serum divided by Vm amounts to 1.4 µM/mmHg at 37°C (4).

From the Stern-Volmer relation (Eq. 1), it can be seen that the phosphorescence intensity and decay time are inversely proportional to the oxygen concentration. Therefore, high-PO2 values that occur in the arterioles are expected to give short decay times and low phosphorescence intensities. To measure such short decay times from these small phosphorescence signals, an integrating amplifier was added to the phosphorometer.

At very low light levels, the PMT will detect single photons if the used PMT and preamplifier have a sufficiently wide bandwidth. The PMT and preamplifier used in this study can record a single photon as a pulse with a full width at a half maximum of ~20 ns. Conventionally, an analog-to-digital (A/D) converter would collect the signal in 65 ns and uses the rest of the sampling interval (935 ns) for conversion and transfer of the data to the memory of the computer. During this 935 ns, the A/D converter is not recording any photons. Therefore, a conventional A/D converter records only 10% of the photons detected by the PMT. In the present integrating preamplifier configuration, the signal is integrated during 1 µs before the A/D converter samples it, thus making more efficient use of the available phosphorescence. This results therefore in an increase of about a factor 10 in the signal-to-noise ratio.

The integrating amplifier consists of two integrators with a time constant of 0.2 µs. The signal is switched between the integrators in such a way that while one is integrating during 1 µs the other is being read by the A/D converter and subsequently reset to zero. This procedure therefore increases the signal-to-noise ratio and thus allows measurement of higher PO2 levels. A decay time constant is calculated from a decay trace following an excitation flash (21) only when the phosphorometer detects a change in phosphorescence from the baseline. In this way, arbitrary fits of noisy signals are rejected.

In vitro experiments. For testing the linearity between 1/tau and the PO2, a sample containing 20 µM Pd-porphyrin-albumin complex was bubbled with various gas mixtures of oxygen and nitrogen. The Pd-porphyrin-albumin complex was prepared in such a manner that its phosphorescence properties became pH independent (21). The PO2 was measured by a solid-state needle electrode (GMS, Kiel-Mielkendorf), which was calibrated in water saturated with 0%, 21%, and 100% oxygen at 39 and 20°C.

Animal preparation. Five male Wistar rats weighing 280-300 g were anesthetized with an intraperitoneal injection of pentobarbital sodium (60 mg/kg). After a tracheotomy, they were mechanically ventilated with a mixture of oxygen and nitrogen. After cannulation of the jugular vein, the animals were anesthetized by a continuous infusion of 6 mg · kg-1 · h-1 pentobarbital sodium. The carotid artery was cannulated to measure the mean arterial pressure and for sampling arterial blood gases. The cannula in the jugular vein was used for sampling the mixed venous PO2 and for administration of Ringer-lactate (30 ml · kg-1 · h-1) as a maintenance fluid. The blood gases were analyzed for PO2 using an ABL505 blood gas analyzer (Radiometer, Copenhagen, Denmark). A length of ileum was extracted from the peritoneal cavity via a midline laparotomy and placed in a closed saline-filled chamber kept at 38°C (11). An observation window was placed in contact with the tissue, preventing oxygen from the surrounding air from influencing the measurements. Finally, the animals were injected with 12 mg/kg Pd-meso-tetra(4-carboxyphenyl) porphine (Porphyrin Products, Logan, UT) bound to BSA (4%).

The PO2 was measured in the first-order arterioles, first-order venules, and capillaries of the ileum, which were denoted as 1A, 1V, and capillary vessels (1) at three different values of inspired oxygen fractions (FIO2 of 0.15, 0.25, and 0.5, respectively). At each FIO2, the PO2 was measured in two first-order arterioles and first-order venules and four capillaries. Values obtained from the different vessels were compared with the readings obtained by the fiber phosphorometer, which measures the phosphorescence over an area of ~1 cm2. This was done by placing the fiber of the phosphorometer (21) on the same ileal segment and measuring the PO2 for each FIO2. These measurements were compared with the central arterial and venous PO2 measured by blood gas analysis.

All procedures described in this study were approved by the Animal Ethical Committee of the Academic Medical Center of the University of Amsterdam.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Validation of the intravital phosphorometer. The accuracy of the estimation of the decay time is dependent on the noise of the measured phosphorescence decay. Improvement of the signal-to-noise ratio can be achieved by averaging multiple decay traces. A disadvantage of this procedure is that it limits the temporal resolution of the measurement and increases the exposure of the preparation to excitation light. The integrating amplifier introduced in this study decreased the noise per decay trace, thereby requiring fewer flashes to make a reliable measurement. This property directly translates itself to more reproducible measurements for a fixed number of averaged flashes. To validate this, a sample containing 20 µM Pd-porphyrin-albumin complex was placed under the microscope at 20°C in contact with air. The decay traces of a number of flashes were averaged for estimation of a decay time. This procedure was repeated five times to calculate the mean ± SD of the decay time as a function of the number of flashes (Fig. 2). As Fig. 2 shows, the integrator does not change the average decay time. The integrator (Fig. 2B) improves the standard deviation of the estimated decay time, which reflects the reproducibility of the measurement. Analysis of the standard deviations shows that, with five times less flashes averaged, the same standard deviation was obtained using the integrator. Next, the intravital phosphorometer was calibrated by comparison of tau 0/tau (see Eq. 1) to the oxygen pressure as measured with an oxygen electrode. Figure 3 shows that tau 0/tau is linear with respect to the PO2 up to 260 mmHg at 20°C and up to 180 mmHg at 39°C.


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Fig. 2.   Average decay time (tau ) and standard deviation as a function of the number of flashes (N) without integrator (A) and with integrator (B). For each number of flashes, the experiment was repeated 5 times; error bars represent SD. Decrease in SD shows that the integration increases the reproducibility of the measurement.



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Fig. 3.   Decay time at zero oxygen (tau 0)/tau measured with the present intravital phosphorometer as function of the PO2, as measured by a solid-state oxygen electrode at 20°C () and 39°C (). PO2 was altered by bubbling different mixtures of oxygen and nitrogen through a solution containing 20 µM Pd-porphyrin. Temperature was kept constant at 20 or 39°C. Values for tau 0 at these temperatures were taken from previous calibration experiments (21).

To test the in vitro correlation between the microscope setup and the fiber phosphorometer, the decay time was estimated using both systems at different PO2 levels, obtained by leading different gas mixtures through a medium containing 20 µM Pd-porphyrin bound to albumin (Fig. 4). The slope of the fitted line through the measured points was 0.99 ± 0.004 (r2 = 1.00).


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Fig. 4.   Correlation between decay times measured by intravital phosphorometer and fiber phosphorometer. PO2 was altered by bubbling different mixtures of oxygen and nitrogen through a solution containing 20 µM Pd-porphyrin. Line fitted through the measured points shows that the PO2 levels as measured by both setups are linearly correlated (r2 = 1.00, slope 0.99 ± 0.004).

Intravital PO2 measurements. For intravital measurement of intravascular PO2 of different order vessels in the serosa of the rat, animals were instrumented as described in MATERIALS AND METHODS. Subsequently, they were infused with the Pd-porphyrin-albumin complex. The experiments were performed with a slit dimension of 5 × 10 µm. The decay time was estimated from a decay curve composed of the average of 50 decays. A typical trace obtained from a first-order arteriole is shown in Fig. 5. In Fig. 5, bottom, the difference between the fit and the measured decay trace (the residue) is shown. Figure 5 shows that the residue obtained with the present setup is well estimated with a single exponential fit and does not require a more complex fit as proposed by others (32).


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Fig. 5.   An example of a decay trace from a first-order arteriole. Dimension of the slit was 5 × 10 µm; decay curve represents the average of 50 flashes. Top: decay curve and exponential function obtained by the fit procedure (tau  = 50 µs, correlation coefficient = 0.97). Bottom: residue of the fit, which is obtained by subtracting the measured decay from the calculated single exponential function. Phos, phosphorescence; A.U., arbitrary unit.

The PO2 was measured in first-order arterioles, first-order venules, and capillaries on the serosal side of the ileum. Arterial and venous blood samples were taken for conventional blood gas analysis. These values were compared with the PO2 that was measured simultaneously with a fiber-optic phosphorometer on the same gut segment to establish the microvascular basis of the fiber measurement.

The results are summarized in Table 1 and Fig. 6. Table 1 shows that the arteriole PO2 is higher than the venous and capillary PO2 (P < 0.05, Student's t-test) for every FIO2 but lower than the systemic arterial PO2 (P < 0.05). No significant difference was found between the venular (1V) and capillary PO2 and total microcirculatory PO2, as measured by the fiber phosphorometer (P > 0.1). To test the correlation between fiber measurements of first-order arteriole, first-order venule, and capillary PO2, the data from all animals and all FIO2 values were plotted in a scatter plot (Fig. 6). The regression lines showed slopes near unity for capillaries and first-order venule vessels against the fiber measurements (1.00 ± 0.04 and 0.95 ± 0.04, respectively), whereas the slope for the first-order arteries against the fiber measurements was significantly larger (1.34 ± 0.11).

                              
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Table 1.   PO2 at different inspired oxygen fractions in the ileum of the rat



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Fig. 6.   A: correlation between the measurements obtained by fiber phosphorometer and PO2 in first-order arteriole vessels (1A). PO2 was altered by changing the inspired oxygen fractions. Line fitted through the measured points has a slope of 1.34 ± 0.11 (r2 = 0.82). B: as in A, correlation of the fiber phosphorometer measurements and first-order venule (1V; ) and capillary () vessel PO2. Regression line for 1V: slope of 0.95 ± 0.04, r2 = 0.94; for capillaries: slope of 1.00 ± 0.04, r2 = 0.94.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

When intravital measurements in rat intestines are compared with PO2 measurements using the fiber phosphorometer, it is apparent that the fiber phosphorometer measures predominantly the PO2 of the capillary and venular compartment. The intravital phosphorometer developed for this study is able to measure PO2 values up to 180 mmHg at 39°C. This was achieved by the introduction of an integrating amplifier and the use of a flashlamp with short pulse duration. This intravital phosphorometer allows the measurement of arteriolar PO2 in intestinal microcirculation of the rat. To date, four intravital systems have been described (6, 19, 24, 32) for measuring the quenching of phosphorescence by oxygen. The maximally attainable PO2 values that could be measured with these systems were, respectively, 38 (6), 55 (19), 30 (24), and 55 mmHg (32). Two studies included detailed information about the setup, which revealed two possible reasons why these devices were constricted to relatively low maximum oxygen pressures: 1) use was made of lamps with relatively long flash durations (i.e., 25 µs) (32), and 2) there was a deliberate application of low-pass filtering of the phosphorescence signal (low pass at 50 kHz) (19). Low-pass filtering was introduced to improve signal-to-noise ratio by removing high-frequency components from the phosphorescence signal. This resulted, however, in a system with decreased response time, thus unable to measure the short decay times occurring at high-PO2 levels. In the present study, enhanced signal-to-noise ratio was achieved without the use of low-pass filtering via integration of the phosphorescence signal sequential every 1 µs by switching between two integrating circuits. In this way, the sensitivity of the phosphorometer was increased (Fig. 2) without compromising the response time, which occurs when low-pass filtering is applied. In combination with the short flash duration of the flashlamp, this new approach allowed us to map the PO2 values in the arteriolar compartment of the ileal microcirculation in which values in excess of 60 mmHg were found (Table 1, Fig. 6).

Quenching of Pd-porphyrin phosphorescence causes the excitation of oxygen to its singlet state, which can oxidize tryptophan groups within the albumin (27). If the rate at which oxygen is removed by this oxidative reaction is comparable to the diffusion of oxygen in the medium, the oxygen concentration at the measured site can decrease. Whether illumination with the flashlamp decreases the PO2 at the measured site depends on both the energy per flash and the repetition rate. Thus the oxygen concentration decreases as the amount of flashes increases. Figure 2 shows that the oxygen concentration is not affected by measurement with the repetition rate and flash energy used by the intravital setup described in this study.

The PO2 levels in the first-order arterioles are approximately a factor two lower than the central arterial PO2 for all imposed FIO2 levels (Table 1). This loss has been attributed to loss of oxygen along the arteriolar vessels (2, 5, 10, 23). The difference in the PO2 levels for the venules compared with the capillaries found in the rat gut was small for all FIO2 levels (Table 1, Fig. 6B). This result is in agreement with earlier findings by Shonat and Johnson (18) on rat muscle preparation. This small capillary venous oxygen gradient was attributed to oxygen diffusion from nearby postcapillaries to the venules (11, 23).

The phosphorescence measured with the fiber phosphorometer is a combination of arteriolar, capillary, and venular PO2. Because the Pd-porphyrin is dissolved in serum, the contribution of the different order vessels will be proportional to the volumes of these vessels. In a histological study, Burton (3) presented data concerning this volume distribution over the different order vessels. The relative volumes of the arteriolar, capillary, and venular compartments were taken from the data of Burton (3) as 13%, 31%, and 56%, respectively. Using these factors to calculate an average PO2 from the data presented in Table 1 gives a weighted average PO2 of 25, 51, and 104 mmHg at a FIO2 of 0.15, 0.25, and 0.5, respectively. These values correspond well with the fiber measurements (Table 1). Because ~85% of the blood volume is in the capillaries and venules and only 15% of the blood volume is present in the arterioles, the fiber phosphorometer will predominately measure the venular and capillary compartment.

When the PO2 is measured in moving organs like the heart or at multiple positions simultaneously, it is not possible to use microscopic techniques. The finding that the PO2 values measured with the fiber phosphorometer can be interpreted as the capillary and venular PO2 is therefore important because it allows the use of fiber phosphorometers as an instrument to measure the venular and capillary PO2 values in vivo.


    ACKNOWLEDGEMENTS

We thank O. Eerbeek for assistance.


    FOOTNOTES

This study was supported in part by a grant from the Netherlands Science Foundation (Grant 900-519-110).

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: M. Sinaasappel, Dept. of Anesthesiology, Academic Medical Center, Univ. of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands (E-mail: m.sinaasappel{at}amc.uva.nl).

Received 25 August 1998; accepted in final form 25 February 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Gastroint Liver Physiol 276(6):G1515-G1520
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