J Appl Physiol
89: 499–504, 2000.
Hemodynamic effects of 15-␮m-diameter microspheres
on the rat pulmonary circulation
ROBB W. GLENNY,1,2 SUSAN L. BERNARD,1 AND WAYNE J. LAMM1
Departments of 1Medicine and 2Physiology and Biophysics, University of Washington,
Seattle, Washington 98195–0005
Received 16 February 2000; accepted in final form 23 March 2000
Glenny, Robb W., Susan L. Bernard, and Wayne J.
Lamm. Hemodynamic effects of 15-␮m-diameter microspheres on the rat pulmonary circulation. J Appl Physiol 89:
499–504, 2000.—The microsphere method has been used
extensively to measure regional blood flow in large laboratory
animals. A fundamental premise of the method is that microspheres do not alter regional flow or vascular tone.
Whereas this assumption is accepted in large animals, it may
not be valid in the pulmonary circulation of smaller animals.
Three studies were performed to determine the hemodynamic effects of microspheres on the rat pulmonary circulation. Increasing numbers of 15-␮m-diameter microspheres
were injected into a fully dilated, isolated-lung preparation.
Vascular resistance increased 0.8% for every 100,000 microspheres injected. Microspheres were also injected into an
isolated-lung preparation in which vascular tone was increased with hypoxia. Microspheres did not induce vasodilatation, as reported in other vascular beds. Fluorescent microspheres were injected via tail veins into awake rats, and the
spatial locations of the microspheres were determined. Regional distributions remained highly correlated when microspheres of one color were injected after microspheres of
another color. This indicates that the initial injection did not
alter regional perfusion. We conclude that, when used in
appropriate numbers, 15-␮m-diameter microspheres do not
alter regional flow or vascular tone in the rat pulmonary
circulation.
effects of microspheres have not been rigorously studied in the rat pulmonary circulation.
The purpose of this study is to determine answers to
the following questions regarding the rat pulmonary
circulation. How many microspheres can be injected
before vascular resistance changes significantly? Do
microspheres cause vasodilatation as they do in other
circulations (5, 17)? And, do microspheres cause local
changes in the distribution of blood flow?
METHODS
WITH AN INCREASING FOCUS on “integrative physiology,”
tools for measuring physiology in large animals are
being applied to small animal models. The microsphere
method for measuring regional organ perfusion is one
of the tools being translated to small animal physiology
(9, 10, 12, 15). One of the guiding principles for reliable
measurements is that the injected microspheres must
be inert (3), causing no alteration in local blood flow.
Because smaller organ pieces are used in smaller animals, relatively more microspheres per organ volume
must be used to accurately determine local perfusion.
Recent studies in rats have shown that microspheres
do have significant hemodynamic effects when injected
into the systemic circulation (8). The hemodynamic
Materials. Colorless and fluorescent polystyrene microspheres, 15.5 ␮m in diameter, were obtained from Molecular
Probes (Eugene, OR). The manufacturer reported that they
had a specific gravity of 1.05 with a narrow distribution of
diameters (coefficient of variation ⫽ 0.02). The microspheres
were suspended in 0.02% Tween 80, and the fluorescent
microsphere suspension also contained 0.02% Thimerisol.
The numbers of microspheres injected were estimated from
the percent solids stated by the manufacturer, and these
estimates were confirmed using a hemocytometer. Microspheres containing green, yellow, and red fluorescent dyes
were used.
The Animal Care Committee at the University of Washington approved these animal studies. All animals were cared for
and handled in accordance with the guidelines established by
the National Institutes of Health (14). Twenty-one SpragueDawley rats (B and K Universal, Kent, WA) weighing 220–383
g were studied. Three different experimental designs were used
to determine the effects of intravenous microspheres on pulmonary vascular resistance, vasodilatation, and local blood flow
distribution.
Isolated rat lung. Isolated, perfused rat lungs were used to
determine changes in pulmonary vascular resistance due to
blockage by polystyrene microspheres. Rats were anesthetized by intraperitoneal injection of pentobarbital sodium
(50–100 mg/kg body wt). After a surgical plane of anesthesia
was obtained, a tracheotomy was performed to allow mechanical ventilation. A Harvard rodent ventilator (Harvard Apparatus, Natick, MA) provided 60 breaths/min of 5% CO2 in air,
at a tidal volume of 1–2 ml with a positive end expiratory
pressure of 2.5 cmH2O. A median sternotomy was performed,
and cannulae were placed in the pulmonary artery and left
atrium. The entire animal preparation was placed in a
warmed and humidified chamber. Depending on the experimental protocol, the lungs were perfused with either a Tris-
Address for reprint requests and other correspondence: R. W.
Glenny, Karolinska Hospital and Institute, Dept. of Anesthesia and
Intensive Care, Bldg. F2, 00, SE-171 76, Stockholm, Sweden (E-mail:
[email protected]).
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.
blood flow; fluorescent cryomicrotome; perfusion
http://www.jap.org
8750-7587/00 $5.00 Copyright © 2000 the American Physiological Society
499
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MICROSPHERES IN RAT PULMONARY CIRCULATION
buffered Tyrode solution or autologous blood with Tris-buffered Tyrode. A Harvard mini-peristaltic pump (Harvard
Apparatus) provided a constant flow rate. The perfusate
temperature was maintained at 38°C. The pump rate in each
preparation was set so that the baseline pulmonary artery
pressure was ⬃20 cmH2O and ranged from 13.0 to 14.2
ml/min. The venous outflow pressure was set at ⫹5 cmH2O
by raising the venous reservoir to the appropriate height.
Because the lungs were perfused at a constant rate, changes
in pulmonary artery pressure reflected changes in pulmonary vascular resistance. Pulmonary arterial and airway
pressures were continuously measured and recorded on a
PowerPC Macintosh using MacLab/8s (ADInstruments, Castle Hill, Australia) at 40 samples/s. The isolated lung preparation was allowed to stabilize for at least 15 min before
measurements and interventions were initiated. Three control rats were prepared as described above and observed for
70 min to determine the stability of the preparation. Control
injections consisting of 1 ml of Tyrode’s solution were injected
at the same frequency and rate as in the interventions
outlined below.
Vascular resistance protocol. To determine the effects of
microspheres on vascular resistance, four rats were studied
using the isolated lung preparation. Papaverine (3 mg/kg
body wt; American Regent Labs, Shirel, NY) was given intravenously before exsanguination to fully dilate the vascular bed so that changes in vascular resistance would be due
only to physical blockage of capillaries by microspheres. The
lungs were perfused from a reservoir containing 100 ml of
Tyrode’s solution, 5.5 ml of autologous blood, and 5 mg of
papaverine. Adenosine (7.5 mM, Sigma-Aldrich, MO) was
infused at a rate of 0.125 ml/min to demonstrate that the
vascular bed was not able to further vasodilate.
Aliquots of colorless microspheres were injected via a side
port of the circulation system distal to the roller pump. The
numbers of microspheres per injection were as follows:
100,000 ⫻ 4; 200,000 ⫻ 3, and 500,000 ⫻ 8. At least 1 min
elapsed between injections, and pulmonary arterial pressures were observed to be stable before each injection. Microspheres were vortexed, sonicated, and suspended in 1 ml of
perfusate.
Vasodilatation protocol. To determine whether microspheres cause vasodilatation, four rats were studied using
the isolated lung preparation. The lungs were perfused with
8–10 ml of autologous blood mixed with 6 ml of Tyrode
solution. The hematocrit of the perfusate ranged from 25.3 to
26.5%. The perfusate was recirculated to the pulmonary
artery through a bubble trap and was maintained at 38°C.
Blood gases were determined with a radiometer (ABL4,
Copenhagen, Denmark). Perfusate pH was determined with
a Corning pH meter. Papaverine was not used in this preparation.
Because the pulmonary vascular bed of the rat has little
vasoconstrictor tone under resting conditions, pulmonary
arterial pressure was actively increased so that a vasodilator
response could be observed. The vascular tone was increased
by ventilating the lungs with a hypoxic gas mixture with O2,
CO2, and N2 fractions of 0.035, 0.050, and 0.915, respectively.
The lungs were preconditioned with three episodes of hypoxia before the studies began. To demonstrate that the isolated lung was able to vasodilate, 7.5 mM adenosine was
infused for 1–2 min at a rate of 0.125 ml/min at the beginning
and end of each experiment.
Aliquots of 50,000 colorless microspheres were injected via
a side port of the circulation system distal to the roller pump.
Microspheres were vortexed, sonicated, and suspended in 1
ml of perfusate. One milliliter of perfusate was injected as a
control. Two microsphere aliquots, separated by 2 min, were
injected into each lung preparation. Changes in the perfusion
pressure were observed after each injection.
Changes in local flow. To determine whether injected microspheres affected the local distribution of blood flow, 10
rats were studied using a new high-resolution method that
determines the spatial distribution of fluorescent microspheres. Because microspheres lodge in organ regions in
proportion to the amount of blood flow to that region (1),
microspheres of two different colors that are simultaneously
injected should be distributed similarly. If injected microspheres are inert, they should not affect local perfusion, and
serially injected microspheres should also have similar distributions.
The rats were physically restrained while fluorescent microspheres were injected via a tail vein. In five control rats,
⬃75,000 microspheres of two different colors (150,000 microspheres total) were simultaneously injected. Before injection,
the microspheres were sonicated, vortexed, and mixed in the
same syringe. In another five rats, ⬃150,000 microspheres of
two different colors were serially injected (one color after the
other). A catheter was constructed to allow serial injections
without having to remove the tail-vein needle. Before injection, the microspheres were sonicated, vortexed, and retained in separate syringes. The microspheres were suspended in 0.24 ml of saline, injected over ⬃15 s, and flushed
with 0.2 ml of saline. The second color of microspheres was
injected 2–3 s after the initial color and flushed again with
0.2 ml of saline.
After the final microsphere injection, the rats were deeply
anesthetized with an intraperitoneal injection of sodium pentobarbital, their chests were widely opened, and the rats
were exsanguinated. The lungs were removed from the chest,
filled via the trachea with OCT media, and frozen. The
spatial locations of all microspheres were determined using
an imaging cryomicrotome (Barlow Scientific, Olympia, WA).
Details of the instrument configuration have been previously
reported (6). The instrument consisted of a Kodak Megaplus
4.2 CCD video camera (Eastman Kodak, San Diego, CA), a
computer (Dell Computer, Round Rock, TX), a metal halide
lamp (HTI 403W/24, Osram, Sylvania), an excitation filterchanger wheel, an emission filter-changer wheel, and a cryostatic microtome. Fluorescence images were acquired with
the Kodak digital camera (2,000 ⫻ 2,000 pixel array) with a
200-mm Nikkor lens (Nikon, Tokyo, Japan) using a macrofocusing baffle. A computer controled two motorized filter
wheels containing excitation and emission filters. A customdesigned microtome was outfitted with an asynchronous
stepper motor to serially section frozen organs. A virtual
instrument written in LabVIEW 5.0.1 (National Instruments) controlled the microtome motor, filter wheels, image
capture, and display.
The cryomicrotome serially sectioned through the frozen
lung with a slice thickness of 30 ␮m. Digital images of the
tissue surface (en face) were acquired with appropriate excitation and emission filters to isolate each fluorescent color.
Images at each fluorescence excitation/emission wavelength
pair were collected and processed so that X, Y, and Z locations of each colored microsphere in each slice were determined and saved in a text file. The spatial resolution of the
system depended on the size of the lung being processed, but
was typically 17 ␮m in the X and Y directions and 30 ␮m in
the Z direction. Images of lung cross sections produced a
three-dimensional binary map defining the spatial location of
parenchyma.
The organ can be mathematically dissected into multiple
spherical regions with diameters of 1,000 ␮m. The general
MICROSPHERES IN RAT PULMONARY CIRCULATION
approach is to choose x, y, and z coordinates from a pseudorandom number generator (Unix, Berkeley, CA). This random spatial point is then located in the binary map of the
organ. If ⬎95% of the sampling sphere lies within the organ,
the spherical region is considered adequate to sample. Sampled volumes cannot overlap any portion of a previously
sampled region. This sampling process continues until no
other spherical regions can be found within the organ. The
numbers of microspheres of each different color are determined for each sampled volume.
Statistics. Data are presented as means ⫾ SD, except
where noted otherwise. Pearson’s correlation coefficient (r)
was used to quantify the similarities in the spatial distribution of microspheres. The observed Pearson correlation (robs)
was calculated from the observed data and pairs of values (Xi,
Yi,) for each piece i and for all n pieces, as
n
兺 (Y ⫺ Y ) 䡠 (X ⫺ X )
i
r obs ⫽
冑兺
n
i
i⫽1
(1)
n
n
(Y i ⫺ Y ) 2 n
i⫽1
䡠
兺 (X ⫺ X )
i
2
n
i⫽1
Because microspheres are discrete particles, Poisson noise is
inherent in their distribution (1). The smaller the number of
microspheres counted in each lung region, the greater the
amount of noise. The value of robs is a biased estimate of the
true correlation (rtrue), due to the inflation of the denominator of robs by Poisson noise. The Poisson noise can also be
adjusted “out” of the correlation using the equation
r true ⫽
r obs 䡠 S X 䡠 S Y
冑[ ( ) ] [ ( ) ]
S 2X ⫺
n⫺1
n⫺1
䡠 X 䡠 S Y2 ⫺
䡠Y
n
n
(2)
where SX and SY are the population standard deviations for
the X and Y observations, respectively (18). If this formula
501
leads to a number ⬎1, the correlation estimate should be set
to 1. Mathematical dissection and determination of regional
microsphere counts was repeated five times in each lung to
provide an average correlation coefficient.
RESULTS
On average, the vascular resistances in the isolated
perfused rat lungs used as controls increased by 1.3 ⫾
0.9% over 70 min of observation. The perfusate pH was
⬎7.3 in all preparations without adding a buffer. Airway pressures did not change in any of the experiments.
Vascular resistance protocol. Baseline perfusion
pressure in the isolated lung was 14.8 ⫾ 1.3 cmH2O
and did not change with adenosine infusion. Perfusion
pressure and vascular resistance increased as microspheres were injected. Figure 1 shows the change in
vascular resistance with each aliquot of injected microspheres. Vascular resistance increased by ⬍10% after
the injection of 1 ⫻ 106 microspheres. As expected from
a fully dilated vascular system, the increase in resistance was linear with respect to the number of microspheres injected. Vascular resistance increased 0.81%
for every 100,000 microspheres injected.
Vasodilatation protocol. Baseline perfusion pressure
in the isolated lungs was 20.4 ⫾ 1.9 cmH2O, and
vascular resistance increased by an average of 72.9%
when ventilated with the hypoxic gas (Fig. 2). Adenosine infusion decreased the vascular resistance in each
isolated lung by an average of 29.3% (Fig. 2). Perfusion
pressure increased immediately after control injection
of 1 ml of perfusate but returned to baseline within
30 s. This change in pressure was due to a momentary
increase in the perfusion rate caused by injecting the
1-ml volume. A similar pressure increase was noted
Fig. 1. In a fully dilated, pump-perfused rat lung, vascular resistance increases as microspheres are injected into
the circulation. A: change in vascular resistance with each aliquot of microspheres injected. B: relationship
between change in vascular resistance and number of microspheres injected is linear. n, No. of animals; R2,
correlation coefficient.
502
MICROSPHERES IN RAT PULMONARY CIRCULATION
provide good estimates of the correlation coefficients
corrected for Poisson noise (18).
DISCUSSION
Fig. 2. Perfusion pressures and changes in vascular resistance after
adenosine infusion and microsphere injections. Open bars, perfusion
pressure just before each intervention; solid bars, change in vascular
resistance after infusion of adenosine or microspheres. Values are
means ⫾ SD, n ⫽ 4.
with each microsphere injection. There were no significant changes in vascular resistance with microsphere
injections (Fig. 2).
Changes in local flow. The correlation coefficients
between simultaneously and serially injected microspheres were the same. Table 1 presents the number of
microspheres counted in each lung, the average number of samples obtained from each lung, the average
number of microspheres counted in the sampled regions, and the average correlation coefficients before
and after correction for Poisson noise. These data demonstrate that microspheres lodging in capillary beds
did not alter the regional distribution of a second microsphere injection. Adequate numbers of regions were
sampled, and enough microspheres were counted to
This study demonstrates that, when used in appropriate numbers, 15-␮m-diameter microspheres do not
significantly alter regional vascular resistance or local
blood flow distribution in rat lungs. Microspheres can,
therefore, be used in small laboratory animals to measure regional pulmonary blood flow.
The microsphere method has been used extensively
for more than 20 years (19) to measure regional distribution of organ blood flow in large laboratory animals.
The microsphere method is based on the fundamental
principle that labeled microspheres lodge within capillary beds, where they can later be quantified, in proportion to local blood flow. Because 15-␮m-diameter
microspheres lodge in small vessels, they must alter
vascular resistance. However, it is argued that microspheres occlude only a small fraction of capillaries and
do not, therefore, significantly alter local vascular resistance. This assumption may not be acceptable in
small laboratory animals where relatively larger numbers of microspheres must be used to obtain adequate
counting statistics in smaller regions of interest.
We commonly use between 50,000 and 100,000 microspheres per injection to study blood flow distributions in rat lungs. The results of this study suggest that
injecting these numbers of microspheres increases vascular resistance by ⬍1%. The change in vascular resistance is likely to be even less because capillary recruitment may offset some of the increase in vascular
resistance due to microsphere occlusion.
Studies performed by Hales and Cliff (2) demonstrated that microspheres injected into arteries lodge
in capillaries that have diameters considerably less
than those of the microspheres. They also showed that
red blood cells continue to flow past lodged microspheres. Recent histology studies with fluorescent microspheres (11) clearly show that 15-␮m-diameter mi-
Table 1. Data from simultaneous and serial injections of 15-␮m microspheres
Rat No.
Microspheres
in Lung*
No. of Sampled
Volumes†
Microspheres
in Sampled
Volumes‡
Observed
Correlation
Correlation
Corrected for
Poisson Noise
19,647
24,271
20,538
14,848
20,947
20,050
0.67
0.62
0.62
0.68
0.69
0.66
0.98
1.00
1.00
1.00
0.99
0.99
19,849
20,511
17,694
17,393
20,264
19,142
0.76
0.66
0.78
0.76
0.67
0.73
1.00
1.00
1.00
1.00
1.00
1.00
Simultaneous injections
1
2
3
4
5
Mean
59,693, 72,734
65,964, 75,275
59,673, 74,681
47,032, 57,085
74,697, 76,317
61,412, 71,218
413
607
467
496
517
500
1
2
3
4
5
Mean
59,448, 75,056
59,096, 74,570
68,511, 71,796
60,714, 66,907
63,660, 75,561
62,285, 72,778
589
640
400
412
461
500
Serial injections
* Two injected colors; † average of 5 repetitions; ‡ average of both colors over 5 repetitions.
MICROSPHERES IN RAT PULMONARY CIRCULATION
crospheres lodge in alveolar capillaries that have
diameters ⬍7–8 ␮m. The linear relationship between
vascular resistance and numbers of microspheres injected in this study suggests that ⬃6 ⫻ 106 microspheres must be injected before half of the vascular bed
is occluded. Because there are ⬃20 ⫻ 106 alveoli in a
rat lung (13), with many capillaries per alveoli, some of
the 15-␮m diameter microspheres must lodge in vessels upstream from the alveolar capillaries.
Microspheres have been shown to induce vasodilatation in systemic vascular beds. Hori et al. (4) demonstrated that embolization with 15-␮m diameter
spheres caused a hyperemic response in canine myocardium and postulated an adenosine-related mechanism. Using sheep, Pearse and co-workers (17) documented a dose-dependent vasodilatation of the
bronchial circulation after injection of 15-␮m-diameter
microspheres. Bronchial artery resistance decreased
by 15 and 36% after injection of 100,000 and 1 ⫻ 106
microspheres, respectively. The effect lasted for up to
30 min. They were very careful to exclude diluents,
detergents, and dyes as possible causes. A second study
to identify the mechanism of the microsphere-induced
vasodilatation did not support a role for adenosine and
suggested that prostacyclin release may cause vasodilatation (16). Kobayahsi and co-workers (8) found a
significant decrease in blood pressure when 2 ⫻ 106
microspheres were injected into the left atrium of rats.
They later determined that some of the observed hypotension may have been due to the suspension used
with the microspheres (7). Our current study found no
significant change in vascular resistance after injection
of 15-␮m-diameter microspheres into the pulmonary
circulation. We can only speculate that the pulmonary
circulation does not produce a vasodilating substance
or does not have the receptors for a vasodilating product. It is also possible that the hypoxic conditions used
in this study may prevent some other O2-dependent
vasodilation that is stimulated by microspheres. Alternatively, hypoxia might affect a different size of
pulmonary vessel upstream from the site of microsphere-induced vasodilation, thereby preventing the
measurement of pulmonary vasodilation.
The excellent correlation between serially injected
microspheres demonstrates that an initial injection of
75,000 microspheres does not alter the regional distribution of a second microsphere injection. If microspheres faithfully represent regional perfusion, then
this is clear evidence that microspheres do not alter
regional blood flow. We intentionally kept the times
between the serial injections short to minimize the
local fluctuations in perfusion that naturally occur over
time. Kuwahira and colleagues (10) used nonradioactive microspheres and X-ray fluorescence spectroscopy
to study the regional distribution of pulmonary blood
flow in conscious rats. They injected 600,000 microspheres of one label and another 600,000 microspheres
of a second label 30 min apart. The lungs were physically dissected into 28 samples, and flow to each piece
was determined at the two separate timepoints. They
found a correlation coefficient of 0.92 for flow within
503
each piece at the different timepoints. The lower correlation coefficient compared with our present study
may be due to the error of the X-ray fluorescent
method, the large numbers of injected microspheres, or
the temporal variation in pulmonary blood flow over
time.
In summary, when 15-␮m-diameter polystyrene microspheres are injected into an isolated rat lung preparation that has a fully dilated vascular bed, vascular
resistance increases linearly as a function of the number of microspheres injected. Vascular resistance increases ⬍1% after the injection of 100,000 microspheres. In contrast to other systemic vascular beds,
microsphere injections do not cause vasodilatation in
the rat pulmonary circulation. Injection of 75,000 microspheres does not change the local distribution of
pulmonary blood flow in awake rats.
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