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Pathways of lymph flow through superficial inguinal lymph nodes in the pig.

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THE ANATOMICAL RECORD 217:188-195 (1987)
Pathways of Lymph Flow Through Superficial
lnguinal Lymph Nodes in the Pig
School of Veterinary Science, University of Queensland, St. Lucia, Queensland 4067,
The pig lymph node has a n unusual structure in that tissue containing lymph nodules generally occupies a central position. Our aim was to describe
the lymphatic pathways through this node. We studied the structure of these pathways with light and electron microscopy, made casts of lymphatic vessels and
sinuses with Microfil, and studied the distribution within the node of subcutaneously
injected carbon particles.
Most afferent lymphatics penetrate deeply within the node, where they give off
several branches to peritrabecular sinuses that ramify through centrally located
nodular tissue. However, where a n afferent lymphatic enters the node there is a
subcapsular sinus over a n area of nodular tissue that occupies a conventional
superficial position. Some lymph reaches this sinus from the central peritrabecular
sinuses, but there can also be direct communications between this sinus and the
afferent lymphatic.
After flowing through sinuses in nodular tissue, lymph enters tissue that is
analogous to medullary tissue in other species. This tissue is of two types, one
consisting mainly of a diffuse network of reticular cells around spaces up to 10-12
pm across, and one that more closely resembles conventional medullary tissue.
Lymph then flows to collecting ducts, which lack valves, and then to efferent
Our findings do not support suggestions that a purely physical obstruction of
lymphocytes in the lymph node accounts for the dearth of lymphocytes in efferent
lymph of pigs.
Major differences exist between lymph nodes of the
pig and those of most other species. In the pig, most of
the tissue that resembles the nodal cortex in other species is located centrally, whereas “medullary” tissue is
generally found a t the periphery (Bouwman, 1959;
McFarlin and Binns, 1973; Binns, 1980, 1982). The pig
lymph node also lacks a hilus (Bouwman, 1959; McFarlin and Binns, 1973).
Another unusual feature of the pig is that recirculation of lymphocytes occurs predominantly from afferent
lymph to blood, rather than blood to lymph as is the
case in other species (Binns and Hall, 1966; McFarlin
and Binns, 1973; Bennell and Husband, 1981a,b; Binns,
1980, 1982). It has been suggested that a lack of open
lymphocytes in the “medulla” may impede the traffic of
lymphocytes through the node (McFarlin and Binns,
1973; Binns, 1980, 1982). Unfortunately, published descriptions of lymph flow through the pig lymph node are
brief and conflict on a number of points (Trautmann and
Fiebiger, 1952; Bouwman, 1959; Preuss, 1977; Binns,
The aim of this study was to describe the pathways by
which lymph passes through lymph nodes in the pig. It
was undertaken using Microfil casts, light and electron
microscopy, and by studying the fate of carbon particles
reaching the node in the lymph.
0 1987 ALAN R. LISS, INC.
Eight Landracebarge White-cross pigs weighing between 20 and 40 kg-were obtained-from a minimal
disease piggery.
All pigs were anesthetised with intravenous sodium
thiopentone (Pentothal, Ceva Chemicals Australia R y
Ltd, Hornsby, N.S.W. Australia), and some also received
Acepromazine (Promex 10, Apex Laboratories Pty Ltd,
St. Mary’s, N.S.W.) intramuscularly. For procedures involving cannulation of lymph vessels, anesthesia was
maintained using halothane (Fluothane, ICI Australia
Operations Pty Ltd, Villawood, N.S.W.) and oxygen in a
closed circuit. The pigs did not regain consciousness but
were killed by exsanguination through a carotid cannula or by a n overdose of thiopentone.
Lymphatic Casts
Evans’ Blue dye (1 ml of 1%)was injected subcutaneously over the medial aspect of the stifle joint. An incision was made through the skin about 5 cm proximal to
this, and superficial lymphatics containing Evans’ Blue
were identified. One of these was cannulated with clear
vinyl tubing (Dural Plastics and Engineering Pty Ltd,
Received December 13, 1985; accepted July 25, 1986.
fewer lymphocytes and is penetrated by a n interlacing
network of sinuses. This diffuse tissue has a somewhat
similar appearance to medullary tissue seen in other
species, but is generally located superficial to the nodular tissue.
Afferent lymph vessels, one to four in number, enter
the node either together, or a t different sites over the
nodal surface. Most penetrate the node through a small
indentation in its surface (Figs. 1-3). Within the node
they are surrounded by a large trabeculum, and they
divide into several branches that connect with peritrabecular sinuses in the centrally located dense nodular
tissue (Fig. 3). However, in one specimen a n afferent
lymphatic divided before it reached the lymph node.
Some of its branches joined a subcapsular sinus, while
others penetrated into the node (Fig. 4).Valves are present in virtually all afferent lymphatics, both outside the
nodes and within them.
Microfil casts of peritrabecular sinuses have a tubular
and often broad, flattened shape, which corresponds to
the histological appearance of the trabeculae. Often they
partly encircle rounded areas of tissue, which can be
shown by histology to include nodules, and which contain only a sparse network of Microfil-containing sinuses. The central trabeculae divide into many small
branches as they approach the junction between the
dense nodular and diffuse zones, and they do not usually
penetrate deeply into the diffuse tissue.
Casts of the sinuses in the diffuse tissue form a dense,
sponge-like pattern and are larger towards the periphery of the node. The diffuse tissue generally surrounds
the dense nodular tissue except where a n afferent lymphatic enters the node. Here, dense nodular tissue is
present in a subcapsular position. This is continuous
with the central nodular tissue along the line of the
afferent lymphatic (Figs. 1,2). However, the superficial
layer of nodular tissue becomes thinner with increasing
distance from the afferent lymphatic, and here it overlays diffuse tissue (Figs. 1,2).
The dense nodular tissue that is located superficially
is covered by a subcapsular sinus, and this is continuous
with the central peritrabecular sinuses. Casts of these
subcapsular sinuses have a n undulating surface (Fig. 5).
This was shown by histology to be due to lymph nodules
beneath the sinus and to small trabeculae projecting
inwards from the capsule. These trabeculae are much
smaller than the trabeculae that ramify through the
centrally placed diffuse nodular tissue, and they usually
just penetrate the peripheral layer of dense nodular
tissue. Accordingly, casts of their related sinuses extend
only a short distance into the node (Fig. 5).
In areas where the diffuse tissue is located at the
periphery of the node, sinuses immediately beneath the
capsule range in appearance from that of a conventional
subcapsular sinus to that of sinuses in the diffuse tissue
deeper within the node.
There appear to be two types of diffuse tissue, with a
gradual transition between them (Figs. 6-8). The tissue
adjacent to the dense nodular tissue consists of a loose
network of reticular cells, among which are many sinuses up to 10-12 pm or more in diameter (Figs. 6,7).
These sinuses are much more obvious in scanning electron micrographs of tissue fixed either by immersion or
perfusion than in light micrographs. The diffuse tissue
situated further from the nodular tissue contains larger
Dural, N.S.W.), initially 0.6 mm outer diameter (OD),
which had been drawn out over a gentle flame. Through
this cannula 0.5-1.0 ml of Microfil was injected by hand
over a period of 5-10 minutes. The pig was then killed.
The Microfil was allowed to set, then the superficial
inguinal lymph nodes were dissected from the carcass.
They were dehydrated in a graded series of ethanol
solutions and cleared in methyl salicylate. Dissection of
the lymph nodes and photography were done using a
stereomicroscope. Tissue was taken for histology from
some nodes to confirm the identity of the cleared tissue.
Studies With Carbon Particles
In three pigs, 1.5-2.0 ml of a mixture of equal parts of
China Ink (Rotring) and 1% Evans’ Blue dye was injected into the skin overlying the medial aspect of each
stifle joint. The superficial inguinal lymph nodes were
removed either 1,2, or 24 hours later.
A piece of tissue (2 x 5 x 5 mm) from nodes removed
at 1and 2 hours was cut immediately into 2-3-mm cubes
and immersed in a mixture of 2.5% glutaraldehyde and
4% paraformaldehyde at pH 7.3. This tissue was postfixed in 1%osmium tetroxide for 2 hours and stained in
5%aqueous uranyl acetate, then dehydrated in a graded
series of ethanol solutions, infiltrated with propylene
oxide, and embedded in Araldite. Thick sections were
stained with toluidine blue for light microscopy. Ultrathin sections were cut using a diamond knife and were
mounted on uncoated copper grids, stained with lead
citrate, and then examined using a Jeol 100s transmission electron microscope.
The remaining lymph nodes were fixed in Bouin’s
fluid, then 10-pm sections were cut at intervals of 2-3
mm and stained with hematoxylin and eosin.
Scanning Electron Microscopy
Three milliliters of glutaraldehyde/paraformaldehyde
fixative was perfused over a period of 20 minutes into
a n afferent lymphatic on the medial aspect of the hind
leg. This was followed by 0.5 ml of Evans’ Blue solution
to mark the part of the node that was perfused. A superficial inguinal node that contained areas with and without Evans’ Blue was removed and immersed in fixative
for 24 hours. It was then dehydrated with ethanol, infiltrated with xylol, and mounted in wax. A cut face was
prepared with a microtome, wax was removed with xy101, and the node was dried by the critical-point method.
It was coated with gold and examined with a Phillips
505 scanning electron microscope.
The superficial inguinal lymph nodes are adjacent to
the external pudendal vessels, and there are up to 12 on
each side. They vary in diameter from 2 to 30 mm, and
many of the larger ones have a lobular structure. When
Microfil was injected through a n afferent lymphatic, it
was uneven in its distribution within individual nodes,
being present in some lobules but not others.
Each lymph node is surrounded by a capsule from
which trabeculae penetrate into the parenchyma. This
parenchyma can be divided broadly into two types (Figs.
1,2): dense lymphoid tissue that contains nodules and is
similar in appearance to the cortical tissue of lymph
nodes in other species, but which is located mainly in
the center of the node, and diffuse tissue that contains
Fig. 1. Schematic diagram showing a n afferent lymphatic entering a the superficial part of the dense nodular tissue. The diffuse lymphoid
lymph node and penetrating within a trabeculum into dense nodular (“medullary”) tissue extends to the periphery over most of the node,
(“cortical”) tissue. Some dense nodular tissue extends over the periph- and collecting ducts emerge from the sinuses of this tissue and join
ery of the node from the point of entry of the afferent lymphatic. This efferent lymphatics. The portion of dense nodular tissue shown at left
tissue is overlain by a subcapsular sinus, and this is continuous with is thought to be derived from a different node anlage, and at the lower
the peritrabecular sinus. To avoid cluttering the diagram, we did not left collecting ducts emerge from deep within the node along what is
show the small trabeculae, each surrounded by a peritrabecular sinus, believed to be the line of fusion of the two node anlages. Major pathwhich penetrate dense nodular tissue in two locations: as branches of ways for the flow of lymph are represented by arrows.
the central trabeculum, and extending inward from the capsule over
lymph sinuses that resemble typical medullary sinuses
in other species (Figs. 6,8).
Efferent pathways from the node are of a variety of
types (Fig. 9). Some sinuses of the diffuse tissue connect
more or less directly with efferent lymphatics. More
commonly they join collecting ducts, which, unlike the
efferent lymphatics, do not possess valves (Fig. 9). Collecting ducts are extensively distributed at the surface
of the node where diffuse tissue is peripherally located.
They are also seen more deeply within the node, often
near what is apparently the plane of fusion between
adjacent node anlages (Fig. 1).At the surface, collecting
ducts are sometimes confluent with subcapsular sinuses
overlying diffuse tissue, but they do not connect with
the subcapsular sinus where it overlies dense nodular
The collecting ducts join efferent lymphatics either
after a very short course or after passing over the node
surface and joining with other collecting ducts. Efferent
Afferent lymphatic
Efferent lymphatic
Subcapsular sinus
Diffuse (“medullary”) lymphoid tissue
Dense nodular (“cortical”) lymphoid tissue
Collecting duct
Peritrabecular sinus
lymphatics from different nodes converge and interconnect, forming up to five or sometimes more lymphatic
trunks that accompany the external pudendal vessels.
In pigs these vessels pass through the inguinal canal.
Studies With Carbon Particles
Carbon particles were usually not present within all
lobules of a node. One or 2 hours after injection, most
carbon particles were in peritrabecular sinuses or in
accumulations at the junction between dense nodular
and diffuse tissue (Fig. 10). The latter were always adjacent to heavy deposits in peritrabecular sinuses. At 24
hours there were far fewer carbon particles in these
sinuses, but the diffuse accumulations at the junction
between the two types of tissue had persisted (Fig. 11).
Most of the carbon in the heavy deposits in the peritrabecular sinuses was extracellular, but at 24 hours when
less carbon was present in the sinus, more was within
macrophages and reticular cells. No other significant
variations were noted in the distribution of carbon between lymph nodes removed at different times.
A small amount of carbon was in the dense nodular
tissue. Most of this was intracellular, and some particles
were seen amongst collagen fibres (Fig. 12). The lymph
nodules contained very few carbon particles in their
central parts, but more were distributed in a mesh-like
pattern around their periphery.
Carbon particles were sometimes present in the subcapsular sinus where this overlays dense nodular tissue
(Fig. 10). They also occurred in the sinuses of small
peripheral trabeculae and in diffuse tissue where these
sinuses terminated. The deposits of carbon in peripheral
dense nodular tissue and its related sinuses were much
less dense than those in similar tissue located deep
within the node.
In the diffuse tissue most of the carbon was near the
junction with the dense nodular tissue, and deposits did
not extend to the efferent collecting ducts. Most of the
carbon was within macrophages (Fig. 13), and free particles were much less common than in the dense nodular
tissue. However, some extracellular particles were found,
either apparently adhering to the surface of macrophages and sometimes trapped between their cytoplasmic projections or lying free in the sinuses.
Fig. 2.Photomicrograph showing an afferent lymphatic in a depression on the surface of a lymph node and a trabeculum surrounded by
dense nodular tissue extending from this depression into the node. The
dense nodular tissue also extends from the depression around part of
the periphery of the node, and in this region is overlain by a subcapsular sinus. Diffuse lymphoid tissue lies between the superficial and
deep (trabecular) portions of the dense nodular tissue, and extends to
the periphery of the node beyond the limits of the superficial portion
of dense nodular tissue (indicated by arrowhead). ~ 2 5 .
The flow of lymph through the lymph node in the pig
has been described as being similar to that in other
species (Bouwman, 1959; Calhoun and Brown, 1975).
That is, lymph flows first through “cortical” tissue, albeit in a central position, and then proceeds through
“medullary” tissue (Bouwman, 1959; Binns, 1982). Although this general pattern was evident in our experiments, the flow pattern appears to be rather more
Before discussing the lymph flow, mention should be
made of terminology. The lymphoid tissue that has been
referred to as “cortex” and “medulla” by other authors
resembles tissue in the cortex and medulla in conventional nodes (Bouwman, 1959; Binns, 19821, but these
Fig.4.Microfil cast of a n afferent lymphatic that divides into several
Fig. 3.Photograph of a section through a lymph node that contains
Microfil that was injected into the afferent lymphatic. Near the center branches before reaching the lymph node. Some of its branches (large
of the node the afferent lymphatic divides into several branches (ar- arrows) penetrate into the node, but others (small arrows) merge with
rows), and these connect with peritrabecular sinuses in the dense a subcapsular sinus. X2O.
nodular tissue. The sinuses that penetrate through this tissue merge
with a dense network of sinuses in the diffuse tissue. x 10.
Fig. 5. Photograph of a portion of subcapsular sinus of the type found
over peripheral dense nodular tissue. This sinus is filled with Microfil,
and this has flowed along small peritrabecular sinuses (arrows) into
the node. ~ 2 0 .
Fig. 6. Scanning electron micrograph of that part of a lymph node
that was fixed by a perfusion through a n afferent lymphatic. Peritrabecular sinuses (small arrows) can be seen, as well as large sinuses in
the diffuse tissue adjacent to the capsule (large arrow). The diffuse
tissue adjacent to the dense nodular tissue contains many smaller
sinuses. ~ 8 5 .
Fig. 7. Scanning electron micrograph of diffuse tissue that lay adja-
terms do not accurately reflect the position of these
tissues in the pig node. Although some “cortical” tissue
is located at the periphery of the node, most is located
centrally. As this tissue contains densely packed cells
and is the site of nodules, we have referred to it as
“dense nodular.” The “medullary” tissue, which generally lies superficially, contains fewer cells and many
sinuses, and we have referred t o it as “diffuse.”
Where afferent lymphatics penetrate into the node,
Some lymph passes through the adjacent peritrabecular
Sinus to a subcapsular sinus overlying peripheral dense
nodular tissue. This lymph then flows through small
peritrabecular sinuses, which penetrate the peripheral
nodular tissue and drain into underlying diffuse tissue.
cent to dense nodular tissue. This tissue was fixed by immersion. The
diffuse tissue contains many sinuses of up to 10-12 pm across. x 1,000,
Fig. 8. Light micrograph of lymph node tissue that was fixed by
immersion. The right side comprises diffuse tissue in which many
sinuses can be seen. However, sinuses are not obvious in the diffuse
tissue that lies adjacent to those parts of the dense tissue that lack
nodules (A). x200.
Fig. 9. Photographs of Microfil casts of efferent pathways from lymph nodes. A,B: Sinuses in diffuse
tissue join with collecting ducts that lack valves, and these in turn join with efferent lymphatics that do
contain valves (arrows). C: A collecting duct joins with a subcapsular sinus overlying diffuse tissue. D: An
efferent lymphatic joins directly with sinuses in diffuse tissue. A) ~ 2 5B)
, X 11, C) X 3 9 , D) x25.
This was demonstrated by the accumulation of carbon
particles at the ends of the peripheral trabeculae.
Usually lymph enters the subcapsular sinus from centrally located peritrabecular sinuses. However, direct
communications can occur between branches of afferent
lymphatics and the subcapsular sinus over dense nodular tissue. In this case, the pathway of lymph into the
node would be similar to that in conventional lymph
nodes (Heath and Brandon, 1983).
In most nodes, however, efferent lymphatics penetrate
deeply within the node, at one or more sites, either
singly or in groups. In the central dense nodular tissue,
these afferent vessels branch extensively before connecting with the peritrabecular sinuses. These findings concur with those of Bouwman (1959). They conflict,
however, with the statement of Trautmann and Fiebiger
(1952)that afferent lymphatics rarely penetrate the node
at more than one site. Furthermore, no evidence was
found to support the claim by Preuss (1977) that the
afferent lymphatics converge into a common cistern
within the lymph node.
The larger lymph nodes appear to be formed by the
fusion of several node anlages (Bouwman, 1959; Kampmeier, 1969). This observation, together with the distribution of Microfil and carbon particles within lymph
nodes, supports the view of Binns (1982) that the node
comprises several systems, each of which receives a separate lymph supply. However, we found that more than
one afferent lymphatic may contribute to each system,
and that small nodes may form a single lymph system.
It has been suggested that “medullary” (diffuse)tissue
in pigs is relatively impermeable, and that this may
account for the dearth of lymphocytes in the efferent
lymph (McFarlin and Binns, 1973; Binns, 1982). However, Microfil flowed freely through this tissue, and the
size of the casts and the appearance of the tissue on
scanning electron microscopy indicated that physical
obstruction of lymphocytes is unlikely. This is also supported by the fact that erythrocytes pass freely through
the node in lymph (McFarlin and Binns, 1973).
The lymph sinuses associated with the diffuse tissue
include segments of subcapsular sinuses of the type
found in conventional lymph nodes, but these are not
present over all areas of diffuse tissue (Bouwman, 1959).
The sinuses of the diffuse tissue occasionally connect
directly with efferent lymphatics, but they usually drain
first into collecting ducts. These ducts, which lack valves,
course extensively over the surface of the node and also
deep within it, where they drain adjacent lymph systems. We did not find collecting ducts in areas at the
surface overlying superficial dense nodular tissue. Nor
was there any evidence from the studies with carbon
particles that lymph flowed from a subcapsular sinus
over superficial dense nodular tissue to adjacent efferent
lymph vessels, as was suggested by Bouwman (1959).
The carbon particles were mainly carried to the node
in the free form, as occurs in sheep (Heath et al., 19861,
and they appeared to enter the node at the peritrabecular and subcapsular sinuses of the dense nodular tissue.
Some particles then crossed the sinus wall into the pa-
Fig. 10. Light micrograph of a lymph node 2 hours after it received
China ink from a subcutaneous injection site. Carbon particles can be
seen in the peritrabecular sinuses and at the termination of the trabeculae (large arrow) near the junction of the centrally placed dense
nodular and diffuse tissues. Carbon particles are also present in a
subcapsular sinus that overlies a thin layer of peripheral dense nodular tissue (small arrow). ~ 4 0 .
Fig. 11. Light micrograph of a lymph node 24 hours after the injection of China ink. Deposits of carbon (arrows) near the junction of the
dense nodular tissue and diffuse tissue are present, but carbon cannot
be seen in sinuses peripheral to the trabeculae. x50.
Fig. 12. Transmission electron micrograph of a reticular cell in the
dense nodular region of a lymph node removed 2 hours after the
subcutaneous injection of China ink. Carbon particles (arrows) can be
seen within the reticular cell, and amongst collagen fibres (CF).
x 12,000.
Fig. 13. Transmission electron micrograph of a macrophage in a
sinus within the diffuse tissue of a lymph node removed 2 hours after
the subcutaneous injection of China ink. Many vesicles (arrows) containing carbon particles are present within this cell. ~ 6 , 5 0 0 .
renchvma. and some of these were distributed around
nodulks. However, most of the carbon within the trabecular sinuses was carried in the lymph to the junction of
the dense nodular and diffuse tissue. This is the site of
concentration of macrophages (Bouwman, 19591, and by
24 hours most of the carbon particles were within these
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Paul Addison with histology, Lynn Tolley and Dr. John
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