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Department of Bioengineering, The Whitaker Institute for Biomedical Engineering, University of California, San Diego, La Jolla, California 92093-0412, USA
1Correspondence: Department of Bioengineering, The Whitaker Institute for Biomedical Engineering, 9500 Gilman Dr., Engineering Bldg. I, Room 5606, University of California San Diego, La Jolla, CA 92093-0412, USA. E-mail: gwsc{at}bioeng.ucsd.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: initial lymphatics • lymphatic endothelium • lymphatic valves • rat • cremaster muscle • interstitium • permeability
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
.
Lymph pumping can be enhanced by cyclic compression and expansion of
the initial lymphatics, a process that depends on periodic motion of
tissue structures positioned in immediate proximity to the initial
lymphatics (3)
. Rhythmic tissue deformations such as
walking (4)
, arterial pulse and vasomotion
(5
6
7)
, intestinal peristalsis (8)
, muscle
movement (9)
, or skin massage (10
11
12)
serve
to enhance lymph flow.
An important unresolved issue in lymphology is the mechanism that
provides the unidirectional transport of fluid from the interstitium
into the initial lymphatics. Although reversal of motion of lymph fluid
inside the initial or collecting lymphatics is prevented by
a set of well-described intralymphatic valves (13
, 14)
,
these valves are insufficient to prevent fluid return into the
interstitium. They form a barrier only to reflow inside a lymphatic
lumen. We postulate here that a second valve system exists. We shall
refer to it as the primary valve system, since interstitial
fluid needs to first pass across these valves on its way into the
initial lymphatics. The traditional intralymphatic valves will be
referred to as the secondary valves, since fluid passes
through them after entry into the lymphatic lumen. The primary valve
system is required to prevent fluid escape from the initial lymphatics
back into the interstitial space.
We hypothesize that the primary valve system is located at the level of endothelial cells in the initial lymphatics. It permits easy entrance of fluid from the interstitial space into the lymphatics, but no significant escape back into the interstitial space. We provide here evidence for the existence of this primary lymph valve system in individual initial lymphatics of rat cremaster skeletal muscle by use of a set of microinjection and aspiration experiments with a fluorescent tracer.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Muscle preparation
The cremaster muscle was surgically exteriorized according to
the method by Baez (15)
and placed on a microscope stage
for transillumination and intravital microscopy. Throughout the
experiments, the muscle tissue was continuously superfused with
Krebs-Henseleit bicarbonate-buffered solution at 37°C (pH 7.4, 305
mOsm, saturated with a 95% N2 and 5%
CO2).
Microinjection of fluorescent microsphere
The cremaster muscle was visualized by an intravital microscope
(Ploempak, Leitz Wetzlar, Stuttgart, Germany) with a 3.5x objective
lens (Leitz). The transport of lymph fluid was monitored with
fluorescent microspheres (0.31 µm, FITC, estapor®, Bang
Laboratories) and injected into the cremaster muscle interstitium near
(within 
50 to 100 µm) larger arterioles (diameter 80 to 100
µm). This site was selected since the majority of arterioles in
skeletal muscle are accompanied by initial lymphatics (7)
.
The microspheres were injected via a micropipette mounted on a micromanipulator (Narishige Scientific Instrument Laboratory, Tokyo, Japan). Micropipettes with a tip diameter between 10 and 30 µm were prepared from capillary tubes (FHC, Brunswick, ME; 10 mm, OD 1 mm, ID 0.5 mm) with a pipette puller. Photobleaching of the fluorescent probe and oxidative damage to the tissue were minimized by limiting the observation period to four to six light exposures per hour, each less than 5 min. At each time point, bright-field and fluorescence images of the selected muscle areas were videotaped and viewed on a color monitor. Both bright-field and fluorescent images were recorded with a color-coupled charge device (CCD) camera (Model V1479, Optronics, Goleta, CA) and a videocassette recorder (Panasonic AG 1270).
The video images were analyzed off line and digitized in black and
white with image analysis software (Optimas 6.5, Media Cybernetics,
Silver Spring, MD). Fluorescent intensities were measured in terms of
optical density (0–255 gray levels) and expressed in terms of the
light intensity
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Lymphatic cross sections
For histological examination, 15 tissue samples from 8 cremaster
muscles were fixed by superfusion of 10% formalin for 15 min at the
same time that an oscillatory muscle compression was applied. Three
tissue samples from three different rats were fixed under the same
conditions but without application of an oscillatory compression. The
tissues were postfixed in 1% OsO4, dehydrated,
and embedded in araldite resin (Polysciences, Warrington, PA); 1
µm-thick sections were stained with toluidine blue and examined by
light microscopy (Diavert, Leitz) with and without oil immersion
objectives (10x, 40x, and 100x).
Cyclic cremaster muscle compression
The cremaster muscle is a thin, flat muscle of almost uniform
thickness (800 µm). During the intravital observations, it rests
on a rigid glass slide. To enhance lymph pumping, the muscle tissue was
compressed with a glass coverslip placed on its top surface. An
inflatable latex tubing (OD 4 mm, ID 2 mm) was mounted on the top
coverslip to serve as the compression device. The latex tubing was
inflated in a sinusoidal pattern by a pump such that the amplitude and
frequency of the coverslip displacement could be adjusted to
preselected values. The compression device was mounted on a
micromanipulator (Narishige) to permit precise positioning over the
muscle. The use of the coverslip permits observation of the tissue
during periods of compression. The frequency of compression was set at
1 Hz, a typical physiological order-of-magnitude frequency.
After injection of microspheres into the muscle interstitium, the
microvasculature was observed with and without oscillatory compression
for selected periods. The position of the glass coverslip was adjusted
with the micromanipulator to achieve a surface displacement between
100 and 150 µm. The microvasculature in the cremaster muscle was
observed to assure that the tissue compression did not result in a
compromise of the circulation through the arterioles and venules in the
center of the muscle.
Lymphatic outflow obstruction
In selected experiments, the lymphatic outflow channels draining
the cremaster muscle were obstructed with a clamp at the insertion
point of the muscle. These occlusion sites were in close proximity to
the major arteriolar/venular pair feeding the muscle microcirculation
and typically at a distance of 3 cm from the microsphere injection
sites.
Statistics
The measurements are presented as mean ± SD.
Group comparisons were carried out by a two-tailed Student’s test for
unpaired data. A probability P < 0.05 was considered
statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 9)
. The initial lymphatics exhibit no inherent smooth muscle
activity. Collecting lymphatics with smooth muscle media are located
outside the muscle tissue.
A small volume (1 µl) of fluorescent microspheres was deposited
into the interstitium 250 µm from the arterioles and venules
(Fig. 1A
, B
, C
). In the absence of muscle compression, no entry of
microspheres into initial lymphatics was observed over a period of
1.5 h (Fig. 1D
, E
). Application of oscillatory surface
compressions for periods of several minutes led to filling of adjacent
initial lymphatics with microspheres (Fig. 1F
). This local
entry of the microspheres occurred irrespective of the particular
interstitial position along the length of an initial lymphatic where
the microspheres were deposited. Thus, the initial lymphatics absorb
material anywhere along their length. The filling of initial lymphatics
could be further enhanced by microinjection of a larger volume of
microsphere suspension (300 µl), a situation that causes
significant local swelling of the muscle interstitium. The initial
lymphatics are positioned along an arteriolar/venular pair
(Fig. 2A
).
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Microsphere retention without and with lymphatic pressure elevation
No microspheres could be detected to escape the lumen along the
length of the initial lymphatic duct back into the interstitium whether
the microspheres had entered the lymphatics by periodic muscle
compression or interstitial volume expansion.
The retention of microspheres in the lymphatic lumen was observed even
after occlusion of the proximal outflow of the initial lymphatics at
the root of the cremaster muscle in combination with
oscillatory muscle compression over the microinjection site (Fig. 2B
). No direct micropressure measurements inside the initial
lymphatics were carried out. However, previous studies have shown that
such a procedure serves to raise the intralymphatic fluid pressures to
values of 30 to 40 cmH2O and even higher,
depending on the force applied during the periodic tissue compression
(16)
. Microsphere dispersion in the tissue was measured in
terms of the linear dimensions of the microsphere pool over the
interstitial injection site, and the linear dimensions within the
lymphatic lumen measured normal to the lymphatic long axis (Fig. 2A
). Whereas dispersion of microspheres inside the
interstitial space was observed during lymph outflow obstruction and
muscle compression (for 30 min), the width of the microsphere column
inside the lymphatic did not change significantly (Fig. 3
).
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Microsphere retention in lymphatics during interstitial fluid
aspiration
We used two kinds of micromanipulation studies to examine whether
the initial lymphatic endothelium may serve as an one-way barrier.
First, microspheres were injected via a micropipette (Fig. 4A
) and aspirated shortly afterward (within 5 s) at
the same site into the same micropipette by application of a
negative fluid pressure (-100 mmHg) (Fig. 4B
). A fraction
of microspheres remained in the tissue at the injection site, but a
significant amount of microsphere suspension could be reaspirated from
the interstitium back into the micropipette (large arrows). In
contrast, microspheres that had entered the lumen of the initial
lymphatics remained inside these lymph vessels (Fig. 4C
,
small arrows). This pattern was confirmed by direct fluorescent light
intensity measurements over the lymphatic channel and over the adjacent
interstitial injection site. There was no change in fluorescence light
intensity over the lymphatic vessel during fluid aspiration whereas the
light intensity in the interstitium decreased on average 50%
(Fig. 5
). This local valve action could be observed whatever the location along
the initial lymphatics.
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Second, initial lymphatics were filled by injection of 300 µl of
microsphere suspension as described above (Fig. 2
, Fig. 6A
). Next, a fresh micropipette was placed with its tip
outside the wall of an initial lymphatic and an aspiration pressure of
10 cm H2O was applied for 5 min (Fig. 6B
). No significant number of microspheres could be
collected into the micropipette under these circumstances. We saw the
same phenomenon even when the aspiration pressure was lowered to -100
mmHg. But when the pipette tip was advanced to where it punctured the
lymphatic vessel, fluorescent microspheres could be removed from a
local segment of the initial lymphatic within a fraction of a second
(Fig. 6C
). Measurements of fluorescent light intensity
(Fig. 7
) confirm this observation. This evidence indicates that the
microspheres maintain their mobility in the lymphatic lumen, an
observation in line with the unrestricted transport of microspheres
along lymphatic channels.
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Lymphatic endothelial flap junctions
Histological sections through the initial lymphatics exhibited no
openings when the muscle was fixed in a noncontracting resting state.
In contrast, when the muscles were fixed during muscle contraction, we
encountered many open junctions (Fig. 8
). The endothelial openings were frequently larger than 1 µm and serve
as open channels for fluid or particulate transport. The density of
these openings is significantly increased if the muscles are fixed
during active contraction (Table 1
). No openings could be seen in any of the adjacent microvascular
endothelial cells of arterioles, capillaries, or venules.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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did not remove a significant number
of microspheres out of the lymphatic lumen. No microspheres inside the
lumen could be reaspirated even within seconds after they had entered
the initial lymphatics or at any subsequent time, indicating the
capacity for rapid opening and closing action by the primary valve
mechanism. Neither could microspheres be removed anywhere else along
the length of an initial lymph channel by aspiration with a
micropipette located adjacent but outside its endothelium (Fig. 6)
. All
parts of the initial lymphatics possess the valve-like features. In
contrast, puncture of the initial lymphatic endothelium rapidly led to
aspiration of tracer microspheres from the lymph lumen. Thus, the
initial lymphatic endothelium may serve as a unidirectional transport
barrier.
Periodic compression and expansion of the initial lymphatics serves as
a key mechanism for lymph pumping (7
, 9)
. Pulse pressure
oscillations and/or vasomotion, for example, provide such periodic
motion. Lymph formation can be enhanced by application of oscillatory
pressure on the muscle surface, low enough not to interrupt the muscle
microcirculation. Such periodic muscle compression may occur under
physiological conditions of tissue movement, as during walking, skin
massage, intestinal peristalsis, or respiration.
Two phases make up a single pump cycle in the initial lymphatics. During expansion, an initial lymphatic channel is filled with interstitial fluid. This is a period when the primary lymphatic valves need to be open and the secondary valves inside the lymphatics are closed to prevent reflow of fluid inside the lymphatic. During lymphatic channel compression, fluid is transported along the lumen of the initial lymphatics in a proximal direction toward the contractile lymphatics and nodes. The primary valves need to be closed during this phase of the cycle to prevent escape of fluid back into the interstitium while the secondary valves along the lymphatic lumen are open. In this respect, the lymphatic system may work not unlike a manual pair of bellows at a fireplace. Bellows also require cyclic expansion and compression and two valves for unidirectional airflow.
What mechanism may constitute a primary lymphatic valve system? The
paucity of tight cell junctions and adhesion molecules such as
VE-catherins (17
18
19)
between lymphatic endothelial cells
suggests that the junctions may have the form of cellular flaps that
could act as valves. Neighboring lymphatic endothelial cells have at
their junctions overlapping cytoplasmic extensions
(20
21
22
23
24)
that may be separated from each other due to the
lack of interendothelial adhesion molecules. We hypothesize that such
overlapping junctions should be opened during expansion of an initial
lymphatic when the intralymphatic pressure falls below the ambient
tissue fluid pressure. During compression, however, when the
intralymphatic pressure rises, the overlapping endothelial junctions in
initial lymphatics are compressed and thereby become sealed. Thus,
lymphatic endothelium is leaky when fluid enters from the interstitial
space but is tight for fluid transport in the reverse direction.
Cross sections through the initial lymphatics show open lymphatic
endothelial flaps in muscle that has been periodically compressed
during tissue fixation. No open junctions can be observed on cross
sections of any initial lymphatic fixed in a resting state without
oscillatory muscle compression (Table 1)
.
We have observed endothelial openings in histological sections with
dimensions of up to 2 µm. Thus, colloidal material can readily
enter into the initial lymphatics across the primary valves. Transport
studies with larger tracers show that even cells the size of
lymphocytes are carried in large numbers during periodic tissue
compression along the lymphatics (25)
. This may suggest
that the junctions between endothelial cells in initial lymphatics may
open to even larger dimensions than could be demonstrated with the
tissue fixation technique described here.
The interendothelial junctions can readily be separated if tension is
applied to the lymphatic wall, e.g., by overinflation of the lymphatic
lumen (21)
. There are local regions along the
interendothelial junctions where neighboring endothelial cells can be
separated when tension is applied to them. When these junctions are
stretched open, one can identify endothelial attachment to the
underlying basement membrane and to anchoring filaments, and detect
large openings between endothelial cells that permit free fluid entry
in and out of the initial lymphatics (21)
. The primary
lymphatic valves are expected to cease functioning under such edematous
conditions.
In the presence of a primary and secondary valve systems, the initial lymphatics serve as an efficient unidirectional transport system that depends on periodic compression and expansion provided by surrounding tissue structures. The primary lymphatic valves appear to be provided by the specialized junctions in the endothelium of initial lymphatics that are depleted of interendothelial adhesion molecules. Further work is under way to analyze the ultrastructure of these junctions and their biomechanical properties.
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ACKNOWLEDGMENTS |
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Received for publication February 9, 2001.
Revision received April 5, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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