Am J Physiol Heart Circ Physiol 289: H1676 –H1682, 2005.
First published June 17, 2005; doi:10.1152/ajpheart.01084.2004.
Effective permeability of hydrophilic substances through walls of lymph
vessels: roles of endothelial barrier
Nobuyuki Ono,1 Risuke Mizuno,2 and Toshio Ohhashi2
1
Department of Electronics and Control Engineering, Nagano National College of Technology, Nagano;
and 2Department of Physiology, Shinshu University School of Medicine, Matsumoto, Japan
Submitted 25 October 2004; accepted in final form 7 June 2005
in part explain the higher concentration of protein observed in
the efferent lymph of the popliteal lymph node compared with
that in the afferent lymph (4, 22).
No report, however, exists to evaluate whether small-sized
lymph vessels can contribute to the mechanisms governing
concentration of protein in the lymph. Therefore, we have
attempted to study the permeability of hydrophilic substances
through the wall of small-sized lymph vessels isolated from
rats by using fluorescent dyes.
MATERIALS AND METHODS
of the lymphatic system is to return
plasma proteins from the tissue space to the bloodstream.
Chemical and electrophoretic analyses of blood plasma and
lymph have shown that all of the proteins are found in the
lymph, although in lower concentrations in most instances
(32). However, the precise mechanisms by which proteins are
transferred across the blood vessel wall to the tissue spaces and
ultimately to the lymph vessels are still unknown.
In previous studies, experimentally induced changes in the
concentration and output of lymphocytes in the efferent lymph
of sheep and dogs were shown to be accompanied by changes
in the concentration and output of proteins in the lymph (1, 13,
23, 24). These results suggest that a significant amount of
plasma protein may accompany the migration of lymphocytes
across the endothelium of postcapillary venules within the
lymph nodes and that this mechanism of protein transfer may
Seven- to eight-week-old male Wistar rats (⬃200 g body wt, n ⫽
29, SLC, Hamamatsu City, Japan) were used for the present study.
The rats were housed in an environmentally controlled vivarium and
fed a standard pellet diet and water ad libitum. All experimental
protocols were approved by the Animal Ethics Committee of Shinshu
University School of Medicine in accordance with the principles and
guidelines of the Japanese Physiological Society.
Isolation and cannulation of lymph vessels. The rats were anesthetized with pentobarbital sodium (50 mg/kg ip) and exsanguinated.
After an incision of the abdomen, the iliac lymph nodes and their
afferent lymph vessels were excised and placed on a Petri dish
containing cold Krebs bicarbonate solution (⬃4°C). The Krebs solution contained (in mM) 120.0 NaCl, 5.9 KCl, 2.5 CaCl2, 1.2 MgSO4,
1.2 NaH2PO4, 5.5 glucose, and 25.0 NaHCO3. With the use of
microsurgical instruments and an operating microscope, the lymph
vessels (n ⫽ 29, 173.5 ⫾ 6.7 ␮m in diameter, 3 mm in length) were
isolated and transferred to a vessel chamber (12 ml) containing two
glass micropipettes and the Krebs solution.
After each lymph vessel was mounted on a pipette (proximal) and
secured with a ⬃10-␮m nylon suture, the perfusion pressure was
raised to 3 cmH2O to flush out and clear the lumen from the vessel.
The other end of the vessel was then mounted to the outflow micropipette. The proximal (inflow) micropipette was connected with a
double-lumen Tygon tubing and a 50-ml syringe (inflow pressure
column). The distal (outflow) micropipette was connected to the
Tygon tubing and a 50-ml syringe (outflow pressure column). The
Krebs solution, bubbled with a gas mixture of 5% CO2-95% N2 to
give a pH 7.40 ⫾ 0.01 and a pO2 ⬃60 mmHg, was superfused over the
vessel. The flow rate of the superfusion solution was 18 ml/min
throughout the experiments. After cannulation of the lymph vessel,
the chamber was transferred to the stage of an inverted microscope
(Olympus IMT-2). The lymph vessels were then warmed slowly to
37°C and allowed to equilibrate for 30 min. During the equilibration
period, the inflow and outflow perfusion pressure of the vessels was
set at 6.5 and 5.5 cmH2O (mean intraluminal pressure of 6 cmH2O),
respectively.
Perfusion of fluorescent dyes into intraluminal space of lymph
vessels. After the equilibration period, each dye was administered into
the intraluminal space of the lymph vessels. In the present study we
Address for reprint requests and other correspondence: T. Ohhashi, Dept. of
Physiology, Shinshu Univ. School of Medicine, 3-1-1 Asahi, Matsumoto,
390-8621 Japan (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.
endothelium; dextran; fluorescence
ONE OF THE MAJOR FUNCTIONS
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Ono, Nobuyuki, Risuke Mizuno, and Toshio Ohhashi. Effective permeability of hydrophilic substances through walls of lymph vessels: roles of
endothelial barrier. Am J Physiol Heart Circ Physiol 289: H1676–H1682,
2005. First published June 17, 2005; doi:10.1152/ajpheart.01084.2004.—
The wall effective permeability of hydrophilic substances labeled with
fluorescent dyes was evaluated in an isolated cannulated rat single
lymph vessel through a videomicroscope system. Sodium fluorescein
(NaFl; 332 mol wt) and FITC-dextrans (4,400, 12,000, and 71,200
mol wt) were administered into the intraluminal space of the lymph
vessels and then excited by a Xenon lamp. Changes in the fluorescence intensity of the dyes were continuously measured by a siliconintensified target camera through appropriate filters. The net flux of
each dye in the wall of the lymph vessels was calculated by the
relationship between the fluorescence intensity and the concentration
of the dyes. NaFl and FITC-dextran 4,400 in the intraluminal space of
isolated rat lymph vessels significantly penetrated the wall of the
lymph vessels. FITC-dextran 12,000 in the intraluminal space of
isolated rat lymph vessels slightly passed through the lymphatic wall,
whereas FITC-dextran 71,200 did not penetrate the wall. Intraluminal
pressures ranging from 4 to 8 cmH2O did not significantly affect the
net flux of dyes used in the present study. After administration of
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate into
the lymph vessels, the net flux of FITC-dextran 4,400 and 12,000 but
not 71,200 was augmented significantly. These results suggest that
small molecular hydrophilic substances (ⱕ4,400) are permeable from
the intraluminal to extraluminal space of isolated lymph vessels and
that the endothelial cell surface structure may play a barrier role in the
effective permeability of large molecular hydrophilic substances
(4,400 to 12,000) through the wall of the lymph vessels.
EFFECTIVE PERMEABILITY THROUGH LYMPHATIC WALL
AJP-Heart Circ Physiol • VOL
Effects of photobleaching on fluorescent dyes. To determine the
effects of photobleaching, the dyes (in ␮M: 0.5 NaFl, 10 4kD, 1 12kD,
and 0.5 70kD) were put into a glass pipette (250 ␮m in intraluminal
diameter) and then excited. After the maximal intensity of the dye in
the glass pipette was reached, the administration of the dye was
terminated. The changes in the intensity of the dyes in the glass
pipettes were evaluated with or without the shutter. Activation of
the shutter for 20 min allowed exposure of the dyes for 5 s in intervals
of 5 s.
Effects of intraluminal pressures on changes in intensity after
administration of dyes into lymph vessels. To determine the effects of
intraluminal pressures on changes in the intensity of each dye in the
intraluminal space of lymph vessels, NaFl (0.5 ␮M), 4kD (10 ␮M),
12kD (1 ␮M), and 70kD (0.5 ␮M) were put into the lymph vessels for
5 min and then excited for 20 min. After the maximal intensity of the
dye in the lymph vessels was reached, the outflow was stopped by
closure of the stopcock while the inflow pressure simultaneously
adjusted to each intraluminal pressure (4, 6, or 8 cmH2O) and
continued pressurizing the lymph vessels throughout the experiments.
The changes in the intensity of the images were evaluated for 20 min
with the shutter at 5 s exposure in intervals of 5 s.
Effects of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate on changes in intensity after administration of dyes into
lymph vessels. The changes in the intensity of the dyes in the lymph
vessels were also measured before and after treatment with 3-[(3cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS,
0.3%) at an intraluminal pressure of 6 cmH2O. CHAPS, which is a
detergent that can disrupt or eliminate the barrier of endothelial cells
without changing the responsiveness of smooth muscle cells (15, 16),
was perfused into the intraluminal space of the lymph vessels for 10
min at a mean intraluminal perfusion pressure of 6 cmH2O (6.5
cmH2O inflow pressure and 5.5 H2O outflow pressure). After CHAPS
was rinsed out for 5 min, the changes in the intensity of each dye were
also evaluated. At the end of each experiment, the intraluminal space
was rinsed with the Krebs bicarbonate solution without the dyes to
confirm a return to the image obtained before the administration of the
dyes.
Calculation for net flux of dyes through wall of lymph vessels. The
relationships between the concentrations of the dyes and the intensities of the digitized images enabled us to evaluate the concentrations
of the dyes in the intraluminal space of the lymph vessels. The
changes in the fluorescence intensities in the intraluminal space of the
lymph vessels reached a plateau after the perfusion of the dyes was
stopped at 5 min. Thus the net flux of the dyes from the intraluminal
to the extraluminal space of the lymph vessels was calculated with the
use of the equation
Net flux (for 5 min) ⫽ (C0 ⫺ C5)/␲d (␮M/␮m)
where C0 is the initial concentration of each dye in the intraluminal
space of the lymph vessels, C5 is the concentration of each dye in the
intraluminal space of the lymph vessels after the perfusion of dyes
was stopped at 5 min, and d is the diameter of the lymph vessels. We
also calculated the percent net flux as follows: percent net flux ⫽
100 ⫻ [(C0⫺C5)/C0].
A pressure gradient is maintained across the lymphatic wall in the
present study. Thus the net flux calculated in the isolated lymph
vessels reflects an effective permeability rather than permeability of
the solutes.
Drugs. All the salts for the Krebs bicarbonate solution (Wako),
NaFl (Alcon), FITC-dextrans (Sigma), and CHAPS (Dojindo) were
used in the present study. The dyes and CHAPS were directly diluted
into the Krebs bicarbonate solution. All salts and drugs were prepared
on the day of the experiment.
Statistics. The ejection fraction, a parameter of the spontaneous
beatings of the lymph vessels, was calculated as follows: [␲(Dmax/2)2 ⫺
␲(Dmin/2)2]/␲(Dmax/2)2 (21). The data are presented as means ⫾ SE,
and n indicates the number of vessels. Regression analysis was used
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used three sets of mean intraluminal perfusion pressure for the
administration of the dyes as follows: 4 cmH2O (4.5 cmH2O inflow
pressure and 3.5 H2O outflow pressure), 6 cmH2O (6.5 cmH2O inflow
pressure and 5.5 H2O outflow pressure), and 8 cmH2O (8.5 cmH2O
inflow pressure and 7.5 H2O outflow pressure). In these pressure
studies, we performed at a random order of the pressure. After the
intraluminal space was completely filled with the dyes for 5 min, the
outflow was stopped by closure of the stopcock while the inflow
pressure simultaneously adjusted to each intraluminal pressure and
continued pressurizing the lymph vessels throughout the experiments.
In the present study we could not measure an intraluminal pressure of
the lymph vessels directly. However, we measured indirectly an
intraluminal pressure via the inflow pressure column that was connected to a pressure transducer (Becton Dickinson) and an amplifier
(6M52, Sanei) through a MacLab data acquisition system (AD Instruments) and a personal computer (Power Macintosh 8500/1200, Apple). In the organ chamber used in the present study, the preparations
were exposed by an extraluminal hydrostatic pressure (0.5 cmH2O)
kept constant throughout the experiments. In addition, we used a large
inflow pressure column (50 ml) for preventing loss of pressure head.
Thus transmural pressure in the present study depends on the intraluminal pressure through the input column and is constant throughout
the experiments (4, 6, or 8 cmH2O). These ranges of the pressure are
suitable for producing stable spontaneous beatings of isolated rat
lymph vessels (19).
Measurements of intensity of dye and spontaneous beatings of
lymph vessels. The optical system contains a Xenon lamp (Olympus),
a 50% neutral density filter (Olympus), an electrically controlled
shutter (Sigma Kikai) and a dichroic mirror (420 nm). A fluorescence
image was obtained through a barrier filter (520 nm) with the use of
a silicon-intensified target camera (C-2400, Hamamatsu Photonics),
an objective ⫻10 lens (numerical aperture 0.30, Olympus), and a
photo ⫻3.3 eyepiece lens (NFK, Olympus). The images were displayed on both a video monitor (Hamamatsu Photonics) and an image
monitor (Nemco) through a video timer (For A, VTG-33). The images
were also recorded on a videocassette recorder (BR-S611, Victor).
The video monitor was used for measuring the maximum diameter
(Dmax, in micrometers), minimum diameter (Dmin, in micrometers),
and frequency (in minutes) of spontaneous beatings of the lymph
vessels by a video caliper (21). The changes in the intensity of the
fluorescence images were measured by an image analyzer (Epson
personal computer/AT compatible). The focus of the images was
adjusted onto the central plane of each lymph vessel. To measure the
changes in the intensity of fluorescence images, the image was
digitized (512 ⫻ 512 pixels and 8 bits density in each pixel) every 500
ms. A window displayed on the monitor with the fluorescence image
was adjusted to the center of each lymph vessel corresponding to the
longitudinal axis of the vessel and kept constant throughout each
experiment (Fig. 2). The width of the window was set at 30% to 60%
of the maximum diameter of the lymph vessels. The averaged intensity of the digitized images in the window was calculated by computer
and then recorded on a pen recorder.
Experimental protocol. All experimental protocols were conducted
in a dark room.
Relationship between concentrations of dyes and intensities of
digitized images. To determine an optimal concentration of the dyes
used in the present study, the relationships between the concentrations
of the dyes and the intensities of the digitized images were evaluated.
The dyes used were sodium fluorescein (NaFl; from 0 to 0.5 ␮M, 332
mol wt), FITC-dextran 4,400 (hereinafter referred to as 4kD; 4,400
mol wt, from 0 to 10 ␮M), FITC-dextran 12,000 (hereinafter referred
to as 12kD; 12,000 mol wt, from 0 to 1 ␮M), and 71,200 (hereinafter
referred to as 70kD; 71,200 mol wt, from 0 to 0.5 ␮M). Each dye was
continuously administered dose dependently through a glass pipette
(250 ␮m in intraluminal diameter) and then excited. The intensities in
the pipette were measured to evaluate the correlation.
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EFFECTIVE PERMEABILITY THROUGH LYMPHATIC WALL
to determine the relationship between the concentration of the dyes
and the intensity of the digitized images. Significant differences (P ⬍
0.05) were determined by ANOVA, followed by Scheffé’s post hoc
test or paired Student’s t-test, as appropriate.
RESULTS
Dye
n
Excitation
NaFl
NaFl
4kD
4kD
12kD
12kD
70kD
70kD
4
4
4
4
5
5
4
4
Before
After
Before
After
Before
After
Before
After
Ejection Fraction
21.3⫾1.5
20.8⫾1.5*
19.4⫾1.1
19.8⫾0.8*
19.5⫾0.5
19.4⫾0.8*
17.5⫾0.8
18.0⫾0.9*
0.72⫾0.03
0.71⫾0.04*
0.69⫾0.04
0.69⫾0.02*
0.66⫾0.05
0.66⫾0.04*
0.72⫾0.02
0.73⫾0.03*
Values are means ⫾ SE; n, number of vessels; NaFl, sodium fluorescein;
4kD, FITC-dextran 4,400; 12kD, FITC-dextran 12,000; 70kD, FITC-dextran
71,200; *No significant difference from before excitation.
Changes in fluorescence intensities of lymph vessels after
administration of dyes. The frequency and ejection fraction of
the spontaneous beatings in isolated lymph were not significantly different before and after the excitation of each dye.
Table 1 shows the results of the frequency and ejection fraction
of spontaneous beatings in isolated lymph at an intraluminal
pressure of 6 cmH2O.
Figure 2 shows representative transilluminated and fluorescence images of the intraluminal space of lymph vessels before
and after perfusion with 0.5 ␮M NaFl (Fig. 2, A–C) and 0.5
␮M 70kD (D–F). The fluorescence intensities of the lymph
vessels increased after the administration of NaFl (Fig. 2B) and
70kD (Fig. 2E). After the administration of NaFl was stopped,
the fluorescence intensity decreased (Fig. 2C). There was,
however, no reduction in the fluorescence intensity of the lymph
vessel after the administration of 70kD (Fig. 2F) was stopped.
These results indicate that NaFl permeated the wall of the lymph
vessels, whereas 70kD stayed in the intraluminal space.
Effects of intraluminal pressure on net flux of dyes. With the
elevation of intraluminal pressure ranging from 4 to 6 to 8
cmH2O, the frequency and ejection fraction of the beatings
significantly increased and decreased, respectively. The frequency values at intraluminal pressures of 4 and 8 cmH2O
were 16.2 ⫾ 0.7 min⫺1 (n ⫽ 17) and 21.6 ⫾ 0.6 min⫺1 (n ⫽
Fig. 1. A: relationship between concentrations of dyes and fluorescence intensities of
digitized images in glass pipette. Sodium
fluorescein (NaFl; F, 0 to 1 ␮M), FITCdextran 4,400 (4kD; }, 0 to 10 ␮M), FITCdextran 12,000 (12kD Œ, 0 to 1 ␮M), and
FITC-dextran 71,200 (70kD; , 0 to 1 ␮M)
were administered into glass pipette in dosedependent manner. B: photobleaching effects on NaFl (E and F), 4kD ({ and }),
12kD (‚ and Œ), and 70kD (ƒ and ) in glass
pipette with shutter (closed symbols) and
without shutter (open symbols).
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Relationship between concentrations of dyes and intensities
of digitized images. Figure 1A shows the relationships between
concentrations of the dyes and the intensities of the digitized
images of the glass pipette. After the administration of the dyes
into the glass pipette, the fluorescence intensities increased,
ranging in gray scale value from 50 to 240. The regression
analyses demonstrate that the relationships were dose dependent and linear to the concentrations of NaFl (y ⫽ 188x ⫹ 66,
P ⬍ 0.05, r2 ⫽ 0.99), 4kD (y ⫽ 12x ⫹ 62, P ⬍ 0.05, r2 ⫽
0.99), 12kD (y ⫽ 111x ⫹ 53, P ⬍ 0.05, r2 ⫽ 0.98), and 70kD
(y ⫽ 269x ⫹ 88, P ⬍ 0.05, r2 ⫽ 0.99). Thus we determined
optimal concentrations of the dyes for evaluating changes in
the fluorescence intensities of the intraluminal space of lymph
vessels as NaFl (0.5 ␮M), 4kD (10 ␮M), 12kD (1 ␮M), and
70kD (0.5 ␮M) in the present study. The digitized fluorescence
intensity of the glass pipette after the administration of each
dye was kept in a range from 150 to 240 on the gray scale
because this was a submaximal and suitable value for analysis
in the optical system. According to the regression analyses,
we could determine the concentration of dyes in the intraluminal space of the lymph vessels and could calculate net flux
of the dyes.
Effects of photobleaching on dyes. Figure 1B shows the
effects of photobleaching on percent changes in the fluorescence intensity of the glass pipette with (closed symbols) or
without (open symbols) use of the shutter. Without use of the
shutter, the percent changes in the fluorescence intensities of
NaFl (0.5 ␮M), 12kD (1 ␮M), and 70kD (0.5 ␮M) were ⬍5%
during the 20 min. Without the shutter, the administration of
4kD (10 ␮M) results in a ⬎5% reduction of percent changes in
the fluorescence intensity after administration of the dye is
stopped. There was no significant photobleaching in the percent changes of the fluorescence intensity with use of the
shutter (closed symbols).
Table 1. Effects of excitation of dye on frequency
and ejection fraction of spontaneous beatings of lymph
vessels at 6 cmH2O intraluminal pressure
EFFECTIVE PERMEABILITY THROUGH LYMPHATIC WALL
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17, P ⬍ 0.05 vs. 4 cmH2O), respectively. The ejection fraction
values at intraluminal pressures of 4 cmH2O and 8 cmH2O
were 0.78 ⫾ 0.02 (n ⫽ 17) and 0.59 ⫾ 0.02 (P ⬍ 0.05 vs. 4
cmH2O; n ⫽ 17), respectively.
Figure 3 shows the effects of intraluminal pressure (4, 6, and
8 cmH2O) on the net flux of the dyes (Fig. 3A, NaFl, 0.5 ␮M,
n ⫽ 4; Fig. 3B, 4kD, 10 ␮M, n ⫽ 4; Fig. 3C, 12kD, 1 ␮M, n ⫽
5; Fig. 3D, 70kD, 0.5 ␮M, n ⫽ 4). The net flux of NaFl, 4kD,
12kD, and 70kD at an intraluminal pressure of 6 cmH2O was
0.56 ⫾ 0.11 ⫻ 10⫺3, 3.43 ⫾ 0.33 ⫻ 10⫺3, 0.09 ⫾ 0.03 ⫻
Fig. 3. Net flux (10⫺3 ␮M/␮m) of dyes (A, 0.5 ␮M NaFl, n ⫽
4; B, 10 ␮M 4kD, n ⫽ 4; C, 1 ␮M 12kD, n ⫽ 5; D, 0.5 ␮M
70kD, n ⫽ 4) in lymph vessels at 4, 6, or 8 cmH2O intraluminal
pressure.
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Fig. 2. Transilluminated images of lymph vessels before administration of 0.5 ␮M NaFl (A) and 0.5 ␮M 70kD (D). Fluorescence images of lymph vessels just
before administration of 0.5 ␮M NaFl (B) and 0.5 ␮M 70kD (E) is stopped. After administration of 0.5 ␮M NaFl (C) and 0.5 ␮M 70kD (F) is stopped at 20
min, fluorescence intensity of lymph vessel administered with NaFl (C) decreased. A window (white boxes, B, C, E, and F) displayed on the monitor with the
fluorescence image was adjusted to center of each lymph vessel corresponding to longitudinal axis of the vessel and kept constant throughout each experiment.
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EFFECTIVE PERMEABILITY THROUGH LYMPHATIC WALL
10⫺3, and 0.00 ⫾ 0.01 ⫻ 10⫺3 ␮M/␮m, respectively. The
percent net flux of NaFl, 4kD, 12kD, and 70kD at an intraluminal pressure of 6 cmH2O was 52.3 ⫾ 8.1%, 21.2 ⫾ 1.3%,
5.1 ⫾ 1.5%, and ⫺0.4 ⫾ 0.9%, respectively. These results
indicate that NaFl and 4kD significantly (⬎5% of the initial
concentration) penetrated the wall of the lymph vessels and
that 12kD slightly (⬃5% of the initial concentration) penetrated the wall. On the other hand, 70kD did not pass through
the lymphatic walls (⬃0% of the initial concentration). In
addition, there were no significant differences in the net flux of
NaFl, 4kD, or 12kD among the intraluminal pressures (Fig. 3,
A–C).
Effect of CHAPS on net flux of dyes in lymph vessels. There
were no significant differences in the frequency and ejection
fraction of spontaneous beatings in the isolated lymph vessels
at an intraluminal pressure of 6 cmH2O before and after an
administration of 0.3% CHAPS. Thus the frequency and ejection fraction after the treatment with CHAPS were 23.0 ⫾ 1.7
min⫺1 (n ⫽ 12, not significant vs. before 23.6 ⫾ 1.6 min⫺1)
and 0.68 ⫾ 0.03 (n ⫽ 12, not significant from before 0.67 ⫾
0.03), respectively.
Figure 4 shows the net flux of the dyes in the lymph vessels
at an intraluminal pressure of 6 cmH2O after stopping the
administration (Fig. 4A, 10 ␮M 4kD; Fig. 4B, 1 ␮M 12kD; Fig.
4C, 0.5 ␮M 70kD) obtained before (open columns) and after
(shaded columns) the treatment with 0.3% CHAPS. After the
treatment with CHAPS, the net flux of 4kD (Fig. 4A) or 12kD
(Fig. 4B) was significantly augmented. Thus the percent net
flux of 4kD and 12kD after the treatment with 0.3% CHAPS
was 29.9 ⫾ 2.2% (P ⬍ 0.05 vs. before treatment; 19.2 ⫾ 1.9%)
and 18.6 ⫾ 1.4% (P ⬍ 0.05 vs. before treatment; 4.2 ⫾ 1.8%),
respectively. On the other hand, the treatment with 0.3%
CHAPS did not increase net flux of 70kD in the wall of the
lymph vessels (Fig. 4C).
DISCUSSION
The major findings of the present study are summarized as
follows: 1) NaFl and 4kD in the intraluminal space of isolated
rat lymph vessels significantly penetrated the wall of lymph
vessels; 2) 12kD in the intraluminal space of isolated rat lymph
vessels slightly permeated the lymphatic wall, whereas 70kD
did not penetrate the wall; 3) intraluminal pressure ranging
from 4 to 8 cmH2O did not significantly affect the net flux of
the dyes used in the present study; and 4) after the adminisAJP-Heart Circ Physiol • VOL
tration of CHAPS into the lymph vessels, the net flux of 4kD
and 12kD but not 70kD was augmented significantly. These
results suggest that hydrophilic substances with smaller molecular weight (ⱕ4,400) are able to permeate from the intraluminal to the extraluminal space through the walls of lymph
vessels and that the endothelial cell layer may be a barrier for
hydrophilic substances with larger molecular weight (4,400 to
12,000) in small-sized lymph vessels.
Development of technique for evaluating permeability of
hydrophilic substances through walls of isolated lymph vessels.
Several experimental approaches have been made to analyze
macromolecular transport and permeability through the walls
of blood (5, 8, 9) and lymph vessels (6, 20, 26, 27) in vivo with
the use of techniques with fluorescent dyes. Although these in
vivo studies seem to demonstrate functions under physiological
conditions, it is difficult to identify the roles of neural, humoral, and local metabolic factors. The normal environment
and pressures of afferent lymph vessels in vivo changed rapidly
due to endogenous nitric oxide by lymphatic flow or chemical
mediators by lymphocytes in the lymph. In the in vivo condition, it is possible to apply fluorescent dyes into the intraluminal space of lymph vessels via the interstitial space. However,
it is quite difficult for us to directly infuse the dyes into the
intraluminal space of rat lymph vessels in vivo. On the other
hand, isolated preparations of blood and lymph vessels enabled
us to directly administer the dyes into the intraluminal space of
the vessels and to quantitatively evaluate the mechanisms for
wall permeability in detail under controlled conditions (15, 16,
33). Therefore, we first attempted to construct an isolated
small-sized lymph vessel preparation suitable for evaluating
effective permeability of hydrophilic substances through the
wall of lymph vessels.
In the present adopted technique, the relationship between
the concentrations of the dyes and the intensities of the images
in the glass pipettes were dose dependent and linear for all dyes
used. Thus the changes in the fluorescence intensities indicate
changes in the concentrations of the dyes. It is also known that
the dye has a photobleaching effect that results in a decrease in
fluorescence intensity. Figure 1B show there is no significant
reduction in the intensity through the photobleaching effect
with use of the shutter. The photoactivation of the dyes causes
an oxygen free radical-mediated inhibition of spontaneous
beatings of rat mesenteric lymph vessels in vivo (34). In the
present study, however, there were no significant effects of
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Fig. 4. Net flux (10⫺3 ␮M/␮m) of dyes (A, 10 ␮M 4kD, n ⫽ 4; B, 1 ␮M 12kD, n ⫽ 4; C, 0.5 ␮M 70kD, n ⫽ 4) in lymph vessels before and after treatment
with 0.3% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) at 6 cmH2O intraluminal pressure. *Significant difference from before
treatment with 0.3% CHAPS.
EFFECTIVE PERMEABILITY THROUGH LYMPHATIC WALL
AJP-Heart Circ Physiol • VOL
strongly support the present findings that NaFl and 4kD but not
12kD and 70kD could significantly permeate from the intraluminal to the extraluminal space of the lymph vessels. The
present study is the first demonstration of the transport of small
molecular hydrophilic substances through the walls of a single
isolated lymph vessel. In addition, the nonpermeability of
70kD in the walls of the lymph vessels may reflect the nature
of albumin (69,000 mol wt) collection in the lymphatic system.
In conclusion, the walls of small-sized lymph vessels may play
a crucial role in condensing the concentration of lymph protein,
which seems to affect the release of lymphocytes from regional
lymph nodes.
Intraluminal pressure ranging from 4 to 8 cmH2O did not
significantly affect the net flux of the dyes used in the present
study. However, the net flux values of 12kD at an intraluminal
pressure of 8 cmH2O were slightly higher than those obtained
with pressures of 4 or 6 cmH2O (Fig. 3C). These results
suggest that higher intraluminal pressures (⬎8 cmH2O) may
facilitate the increase in the net flux of the dyes (12,000 –
70,000 mol wt) in the lymph vessels. A pressure gradient is
maintained across the lymphatic wall in the present study. Thus
we suggest that the net flux calculated in isolated lymph vessels
reflects an effective permeability rather than permeability of
solutes. It is also still unclear that the main transport mechanism of the dyes in the walls of the lymph vessels is diffusion,
which is the concentration gradient driving transport in the
present study. We used one concentration in each dye being
available for evaluating changes in the intensity because our
experimental condition used in the present study has a limitation for intensity range. Thus the driving forces for the net flux
across the lymphatic wall, be it a concentration or a pressure
gradient, do not control the effective permeability but do
control the net flux. The present study, however, at least shows
there is a size-limited effective permeability of the dyes in the
lymph vessels.
Role of endothelium in transport of hydrophilic substances
through walls of isolated lymph vessels. It is well known that
the permeability of macromolecule substances depends on the
structure of the capillary endothelium in the organs and tissues
(14, 25). No information exists regarding the permeability of
hydrophilic substances through the walls of lymph vessels and
the crucial role of the lymphatic endothelium in the permeability of the substances. CHAPS is known to be a detergent that
can disrupt or eliminate the endothelial barrier without changing the responsiveness of smooth muscle cells (15, 16). In fact,
treatment with CHAPS did not affect the spontaneous beatings
of the lymph vessels in the present study. The treatment with
CHAPS significantly increased the net flux of 4kD. Interestingly, although the net flux of 12kD was not identified and was
slight in the control, the treatment with CHAPS significantly
increased the net flux of 12kD. On the other hand, 70kD did not
permeate the wall of lymph vessels before or after the treatment with CHAPS. These results suggest that an endothelial
barrier in the lymph vessels may be disrupted by the treatment
with CHAPS, resulting in the enhancement of effective permeability for hydrophilic substances in the lymphatic wall. The
treatment with CHAPS (the concentration used was the same
as in the present study) influenced the intimal permeability of
the dyes in the isolated arterioles (15).
Medium-sized FITC-dextran (50.7kD) slowly penetrated the
endothelial cell layer of the mesenteric artery but did not
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excitation of the dyes on the frequency and ejection fraction of
the lymphatic beatings. Thus these results indicate that the
changes in the fluorescence intensity reflect changes in the
concentrations of the dyes in the intraluminal space of lymph
vessels and that the present experimental conditions have no
significant photobleaching effect and no significant effect on
the spontaneous beatings of the lymph vessels.
In the present study, the values of frequency and ejection
fraction of the lymphatic beatings in the control condition and
the response of the lymphatic pump activity to increasing
intraluminal pressure were quite similar to those obtained with
previous studies (19). We believe that the mechanical properties of isolated rat lymph vessels of iliac lymph nodes have
been maintained during the preparation. Minor damage, however, to the vessel may be involved in the permeability of the
dyes in the wall of isolated lymph vessels. Thus further
methodological improvement including more intact in vivo
study will be needed to evaluate the permeability of solutes in
the walls of lymph vessels as well as initial lymphatics.
The spontaneous changes in the diameter of lymph vessels
may affect the intensity of the dyes. In fact, the intensity of the
fluorescence dyes in the intraluminal space of lymph vessels
was reported to be higher during the dilated phase than during
the constricted state (20). Thus, in the present study, we
evaluated the fluorescence intensity of the lymph vessels obtained in the maximal dilated phase only. The shutter use
condition, in which the vessels are exposed for 5 s in intervals
of 5 s, enabled us to measure the fluorescence intensity obtained only in the maximum dilated phase of spontaneous
beatings.
Size-limited effective permeability of dyes through walls of
isolated lymph vessels. It is well known that lymph protein
increases during the transport of the lymph from peripheral
lymph vessels to thoracic ducts (7, 10, 12). Heterogeneity in
the concentration of lymph protein between the afferent and
efferent lymph vessels of the regional lymph nodes has also
been reported (1–3, 24). These results suggest that the concentration of lymph protein is actively and/or passively modulated
while lymph returns to the systemic circulation. These results
of the changes in the concentration of lymph protein are based
on in vivo studies and the collection of lymph fluids and could
not demonstrate the possibility of a single lymph vessel condensing the concentration of lymph albumin. According to the
regression analyses of the relationship between concentrations
of the dyes and the intensities of the digitized images in the
glass pipettes, we could determine the concentration of the
dyes of the intraluminal space of the lymph vessels and could
calculate the net flux of the dyes.
The present study indicates that NaFl and 4kD significantly
(⬎5% of the initial concentration) passed through the wall of
lymph vessels, and 12kD slightly (⬃5% of the initial concentration) penetrated the wall. On the other hand, 70kD did not
permeate the lymphatic walls (⬃0% of the initial concentration). Macromolecules with a molecular weight as high as or
higher than 6,000 remained in the lymph vessels and are
returned to systemic circulation via the regional lymph nodes
and thoracic ducts. On the other hand, smaller molecules
including water, sodium, and urea leave from the intraluminal
space to extracellular matrix tissues. Thus the limit of permeability through the walls of the lymph vessels is considered to
be between 2,300 and 6,000 mol wt (17, 18). These results
H1681
H1682
EFFECTIVE PERMEABILITY THROUGH LYMPHATIC WALL
GRANTS
This study was supported by Grants-in-Aid for Scientific Research
(15700325 and 14580842) from the Japanese Ministry of Education, Science,
Sports, and Culture.
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penetrate the arterial wall, whereas small-sized FITC-dextran
(4.4kD) quickly passed through the endothelial cell layer and
accumulated in the arterial wall (29). The size- and endothelialdependent transport mechanisms of hydrophilic substances in
the arterial wall were disrupted by phototoxic stress (29). A
surface layer of membrane-associated glycocalyx in arterioles
and capillaries significantly contributes to the permeability of
physiological substances (11, 31). In an in vivo study of rat
cremaster muscles (28), microspheres (0.31 ␮m) pass from the
interstitium across the endothelium into the lumen of the initial
lymph vessels. Once inside the lymphatic lumen, the microspheres cannot be forced out of the lumen. Thus endothelial
cells act as a primary valve system for particles such as
microspheres. It is also known that in endothelial cells, F-actin,
a cytoskeleton protein, strongly contributes to the permeability,
and inflammatory mediators facilitate the increase in the permeability due to the reorganization of F-actin (30). In conclusion, the present study may suggest that the endothelial surface
structure of the lymph vessels plays a key role as a barrier for
the movement of hydrophilic substances through the walls of
lymph vessels. However, further investigation will be needed
to study the role of F-actin in contributing to the permeability
of the lymphatic endothelial cells.