SPECIAL COMMUNICATION
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 |
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 |
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 |
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.
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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
|
(1)
|
Here,
0 (in µs) is the decay time
at zero oxygen and
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
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
|
(2)
|
Equation 2 gives the relation between
the oxygen concentration and the
PO2 (30).
In this equation,
is the Bunsen coefficient and
Vm is the molar volume at 0°C
(22.4 mol/l). For in vivo calculation, the Bunsen coefficient (
) 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/
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 |
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
0/
(see
Eq. 1) to the oxygen pressure as
measured with an oxygen electrode. Figure 3
shows that
0/
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 ( ) 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
( 0)/ 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 0
at these temperatures were taken from previous calibration experiments
(21).
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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).
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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 ( = 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.
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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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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.
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 |
DISCUSSION |
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.
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