1 Department of Internal
Medicine, The free water content of lung tissue was
investigated by dielectric spectroscopy in normal lungs and in
pulmonary edema induced by oleic acid in rats. The dielectric
relaxation in a frequency range of
107 to
1010 Hz was measured
with the time domain reflectometry method at 25°C.
Three dielectric relaxation processes were analyzed for the lung
tissue. A high-frequency process around 10 GHz was attributed to the
orientation of free water molecules based on the relaxation time
[log
lung tissue water; oleic acid-induced lung injury
THE CHANGE IN LUNG TISSUE WATER in pulmonary
edema, which is defined as an accumulation of water in the
extravascular space, has been evaluated by gravimetric water content,
distribution of tracers (e.g., radionucleides), and morphometry. These
methods have been useful in evaluating the static state of lung tissue water. On the other hand, changes in the physical properties of water
molecules, i.e., alterations in the mobility of water molecules, in
pulmonary edema have rarely been investigated. Previously, Shioya and
colleagues (19-22) found changes in the nuclear
magnetic resonance (NMR) relaxation times T1 and T2 for various
experimental lung injuries, indicating changes in the physical state of
water in lung tissue. Increases in the NMR T2 relaxation time without accompanying increases in gravimetric water content suggested an
alteration in the free and bound water fractions of lung tissue water.
There should, therefore, be changes in the content of free water in
diseased lungs, especially in pulmonary edema.
The microwave dielectric method has been one of the most reliable
techniques for investigating the dynamic structure of water in
macromolecules such as deoxyribonucleic acids (26), globular proteins
(15, 16), and polysaccharides (24). Dielectric relaxation measurements
have been applied to various biological tissues (6). In the past,
Surowiec et al. (23) and Schwan and Kay (17) measured the dielectric
properties of lung tissue; however, they did not apply a high enough
frequency to observe the dielectric dispersion and absorption of free
water. Recent developments in the time domain reflectometry (TDR)
method have now made the measurement easy and fast, giving the
absorption and dispersion data continuously in a frequency range of 100 kHz to 10 GHz (2, 4, 14). In this study, we used the TDR method to
measure the free water content in normal lungs and in pulmonary edema
in rats, investigating the changes in lung tissue water. Our results
may provide new insights concerning water in the lung based on its
dynamic behavior.
Preparation of animals. Nine 250-g
Wistar-strain male rats were used. All the experiments were performed
in adherence with the guidelines for animal experiments in the School
of Medicine, Tokai University (Kanagawa, Japan). Rats were anesthetized
with 60 mg/kg of pentobarbital sodium intraperitoneally. Pulmonary edema was induced in four rats by injection of oleic acid (0.12 mg/kg)
into the tail vein, and the rats were used 4-5 h after injection.
Five rats served as normal controls. The left lung was removed for
experiments immediately after exsanguination via the abdominal aorta.
Dielectric relaxation measurements.
TDR (2, 4, 14) dielectric measurements in the frequency range of
107 to
1010 Hz were performed at
25°C. An incident pulse with a rise time of 35 ps was generated by
a pulse generator and passed through a semirigid coaxial cable, the top
of which was cut flat and plated with gold to prevent oxidation. The
top of the cable was in direct contact with the surface of the lung in
a sample bottle that was tightly sealed and placed in a water bath
(25°C) with a temperature control system. TDR measurement of the
lung samples was started within 5 min after exsanguination and
completed in 20 min. The reflected pulse from the sample was recorded
in the time domain at the sampling head (HP54121A, Hewlett-Packard) and
digitized by a digitizing oscilloscope mainframe (HP54121B,
Hewlett-Packard).
The reflected pulses from the unknown and reference samples were
measured, and the complex permittivity for each sample was obtained
after Fourier transformation of those pulse forms with a personal
computer (PC9800FA, NEC). If a sample with known permittivity (
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
h (in
s) =
11.03]. The dielectric
strength (
) of this high-frequency peak
(
h) should reflect the
amount of free water in the tissue. Because the measured
h depended on the air
content of the lung samples, the value of
h was corrected for the air
content of each sample as determined by the point-counting method in
the area where the time domain reflectometry data were obtained. The
lungs of rats that received an injection of oleic acid had a
significantly increased free water content
[(
h of lung/
of
pure water) × density of pure water] compared with that in
the normal lung (0.76 vs. 0.59 g/cm3). These results indicate
that free water occupies ~60% of the total volume of normal lung
tissue and that there is an increase in free water in pulmonary edema.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
) is used as a reference
sample, the permittivity of an unknown sample
(
) is given as
where
(1)
and
(2)
(3)
where
d is the geometric length;
(4)
d is the effective electric length
(0.18-0.22 mm);
rs(
) and
rx(
)
are the Fourier transforms of the reflected pulse from the reference
[Rs(t)]
and unknown [Rx(t)]
samples, respectively; j is the
imaginary unit;
is the angular frequency; and
c is the speed of propagation in
vacuo. The
d was
determined by measuring two known samples, water and acetone. The
measured values for water and acetone were adjusted to the known values
by selecting an appropriate value of
d.
To attain better precision in the gigahertz region, a bilinear analysis was employed (1, 14). This analysis provides the unknown permittivity as
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(5) |
Histological examination. After the TDR measurements, lung tissue samples were fixed with 10% Formalin-saline and prepared for light microscopy examination with hematoxylin and eosin staining.
Normalization of the dielectric strength for the
actual tissue volume. The complex permittivity (*)
for biological tissues is represented as the sum of several relaxation
processes of the Havriliak-Negami (8) equation
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(6) |
In the TDR measurement of lung tissue, the measured is a value
for a unit volume of lung tissue including the air, and the air, for
which the permittivity is almost equal to 1, decreases the apparent
dielectric constant of the lung. To compare the values of
obtained from lung samples of various air content, we normalized the
measured
with the actual lung tissue volume without alveolar air. To make the TDR data and the measured actual tissue volume consistent, several microscopy sections were prepared in the area where the TDR electrode was attached (Fig.
1A).
The actual tissue volume for each sample was determined by the
point-counting method with light microscopy as [points on
tissue/total points (sum of points on tissue and air)] (27). With
a micrometer (U-OCM SQ 10/10, Olympus), a total of 242 points (500 µm
in width and 250 µm in depth) were counted under high power
(×400) in the area where the TDR measurement was performed. For
each sample, measurements were made at least three times, and the
averaged values are used as the actual tissue volumes.
|
To verify this normalization, we compared the normalized for
passively collapsed lungs with some air and a degassed lung without
air. When the chest is opened under ambient pressure, the lung
passively collapses because the chest cavity is normally maintained
under negative pressure. In the degassed lung, the
should be
affected less by air than by the passively collapsed lung. The degassed
lung was prepared by inserting a tracheal tube into an anesthetized rat
and allowing it to breath 100% O2
for 20 min. The tracheal tube was clamped shut for 5 min to allow the
complete absorption of intra-alveolar
O2. In contrast to air, which
consists of 21% O2 and 79%
inactive nitrogen, 100% O2 is completely removed from the alveoli by the circulating blood. The heart
continues to beat actively for ~5 min even after ventilation ceases.
After exsanguination from the abdominal aorta, the degassed lung was
excised for TDR measurement.
Gravimetric measurement of water
content. A lung tissue sample for the measurement of
gravimetric water content was excised from the same lung used for the
TDR measurement, placed in a glass tube, and sealed tightly until
measured. The samples were placed in a vacuum desiccator to dehydrate
for 24 h. Water content (in percent) was calculated from the difference
in sample weight before and after desiccation as [(wet weight dry weight)/wet weight] × 100.
Statistical analysis. Data are expressed as means ± SD. Comparisons for the data between groups were performed with a Mann-Whitney U-test. A correlation between the water content and dielectric strength was analyzed by linear regression. A value of P < 0.05 was considered significant.
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RESULTS |
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Figure 1 shows microscopic views of a sample of normal passively collapsed lung with a tissue volume of 0.64 (A), a sample of degassed lung with a tissue volume of 0.97 (B), and a lung 4 h after injection of oleic acid with a tissue volume of 0.92 (C). The degassed lung shows almost complete absorption of alveolar air by the inhalation of 100% O2. The oleic acid-treated lung shows massive pulmonary edema with alveoli filled with edema fluid. The alveolar walls are necrotic.
Dielectric dispersion (', real part) and absorption
(
'', imaginary part) curves observed for a normal rat
lung at 25°C are shown in Fig.
2A. Three
relaxation processes, low (l), intermediate (m), and high (h)
frequency, were analyzed for the lung tissue in a frequency range from
1 MHz to 10 GHz. The permittivity is thus described by
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(7) |
|
Figure 2B shows a magnified view of
the high-frequency region. The high-frequency process around 10 GHz,
which is well separated from the other two lower-frequency processes,
can be attributed to the orientation of free water based on its
relaxation time of log h (in s) =
11.03. Although our measurements did not cover the frequency
at this peak,
h and
h were determined with a high level of accuracy by assuming a Cole-Cole-type relaxation process,
with the value
determined
by least squares fitting. The value
did not vary in normal
lungs and pulmonary edema, and the theoretical curves agreed well with
the measured data as shown in Fig. 2. The parameters analyzed for this
relaxation process are shown in Table 1.
The values for a degassed lung were close to the mean values for the
normal lung, except for the value of
h and the actual tissue
volume. The similar normalized
h
(
n) values (means ± SD) for the passively collapsed lung and a degassed lung indicate that
normalization of
h with the actual tissue volume is effective.
|
In oleic acid-induced injury, the
n was significantly greater
compared with that in the normal lung. The gravimetric water content
was markedly increased for the oleic acid-treated lung. The increase in
n
(Y) was significantly correlated
with the increase in water content
(X):
Y =
82.53 + 1.61X
(r = 0.925; P < 0.001;
n = 8 lungs).
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DISCUSSION |
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In the dielectric relaxation measurement of lung tissue, three relaxation processes were analyzed in a frequency range from 1 MHz to 10 GHz, the low, intermediate, and high processes. The low-frequency process around 1 MHz could be an electrode polarization (6). The intermediate-frequency relaxation process, with a peak between 10 and 100 MHz, may be explained by the reorientation of water molecules restricted by the macromolecules in the tissue (15, 16, 26). The Maxwell-Wagner effect occurring on the cell interface and the micro-Brownian motion of the other biomolecules might also contribute to this peak (12). These two processes would overlap with each other in these measurements, and their origins have not yet been determined for biological tissues. The high-frequency relaxation process around 10 GHz was well separated from the other two lower-frequency processes and was attributed to the orientation of free water. A recent microwave dielectric study (13) has revealed that the elementary structure of water is cyclic, consisting of six molecules linked together by hydrogen bonding. In view of the dielectric properties, the structure of free water in the tissues was not different from that of pure water, judging from the relaxation time.
In the dielectric relaxation measurements for lung tissue containing
air in the alveoli, as shown in Fig.
1A, the measured should be
underestimated. Although a significantly greater
was measured in
pulmonary edema compared with the normal lung, the increase could be
affected by the increase in tissue volume due to the obliteration of
air spaces by the accumulation of edema fluid in the alveoli. To
normalize the effects of varied tissue volume on
h, we corrected the measured
with the actual tissue volume for each sample. The actual tissue
volume was determined in the area where the TDR measurements were
performed so that these data are consistent. The
n for the oleic acid-treated lung was significantly greater than that for the normal lung. These
results indicate an actual increase in free water in the edematous lung.
An important parameter in dielectric spectroscopy is . For the
relaxation process due to the orientation of free water, the value of
depends on the number of water molecules measured by the TDR
method, if it is assumed that the local structure of free water is the
same as that of pure water. This behavior was observed in the polymer
solution. In this case, the content of the free water per unit volume
[Cf (in
g/cm3)] can be estimated
from the
h as
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(8) |
With the use of n, the
Cf can be calculated using
w = 73.16 at
25°C (9) and Cw = 0.997 g/cm3. The calculated values are
shown in Table 1. The Cf value of 0.59 g/cm3 for normal lung tissue
is close to the reported value of 0.54 g/cm3 for the gray matter of brain
tissue (10). These results suggest that water with the same physical
properties as pure water occupies ~60% of the tissue volume in the
normal lung. With theoretical approaches other than
Eq. 8, the calculated results may
differ from our data. However, the trends in water content for
edematous tissue would be the same regardless of the approach. It has
been supposed that the water in biological tissues has a characteristic structure and physicochemical properties different from those of pure
water (11, 25). However, recent dielectric studies (5, 6, 11) with the
TDR method have also suggested the existence of water with the features
of pure water in biological materials.
Oleic acid-induced lung injury is one of the most popular animal models
of acute permeability pulmonary edema and fatty embolism (3). The early
stage of oleic acid-induced lung injury is characterized by massive
pulmonary edema with cellular necrosis and alveolar flooding. The
histology observed in this study was consistent with that reported by
other investigators (3). In measurements of the oleic acid-treated
lung, the of the free water process as well as the
n was significantly greater
than those of the normal lung. The calculated value of
Cf was markedly increased. These
results quantitatively indicate that an increase in free water is an
essential feature of pulmonary edema.
Although TDR measurements are not destructive and not invasive, it may be difficult to apply this method to lungs in vivo unless the air content for a sample is known. In addition, TDR data can be obtained only for a limited area just under the electrode. The advantage of this method is that fractional changes in free water can be evaluated for the tissue. In contrast, gravimetry only measures net changes in the weight of water, and this method cannot be used to measure water content in vivo. With NMR measurements, dynamic changes in tissue water caused by macromolecular derangement can be detected as changes in relaxation times, and this method can also be used for measurements of living tissues by magnetic resonance imaging (18). However, the data obtained by NMR do not differentiate free water fractions from other more restricted water fractions in the tissue. NMR and TDR gave different results that can be explained by differences in the experimental time scale (7). TDR has a time resolution on the order of a few picoseconds, in contrast to NMR with its time resolution of a few milliseconds, making it possible to observe the relaxation process due to the orientation of free water in aqueous solutions with a high solute concentration.
In conclusion, we have, for the first time, quantified the amount of free water in lung tissue with the TDR method, showing that normal lung tissue has a free water content of 0.59 g/cm3. In the pulmonary edema induced by injection of oleic acid, there was a significant increase in the free water content of the lung.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Chizuko Tsuji for technical assistance.
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FOOTNOTES |
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This study was supported by a Research Promotion Grant from the Tokai University (Kanagawa, Japan) General Research.
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: S. Shioya, Dept. of Internal Medicine, Tokai Univ. School of Medicine, Isehara, Kanagawa 259-1193, Japan.
Received 2 February 1998; accepted in final form 11 September 1998.
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