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Pathways of lymph flow through intestinal lymph nodes in the horse.

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THE ANATOMICAL RECORD 232:126-132 (1992)
Pathways of Lymph Flow Through Intestinal Lymph Nodes
in the Horse
STEPHEN A. NIKLES AND TREVOR J. HEATH
Department of Anatomy, University of Queensland, St. Lucia, 4067, Australia
ABSTRACT
In the horse, several thousand lymph nodes receive lymph from
the intestine, part of which is very large and contains microorganisms that enable
the animal to utilize refractory dietary constituents such as cellulose. The aim of
this study was t o describe the pathways by which lymph is delivered into,
traverses, and is drained from these lymph nodes. These pathways were studied
with either Microfill or methacrylate casting materials and with light and electron
microscopy.
The afferent lymphatic vessel delivering lymph into one of the nodes divides over
the capsular surface and within trabeculae into terminal branches, and these are
continuous with the subcapsular and trabecular sinuses through rounded holes up
to 30 pm across.
Lymph is conveyed from the subcapsular and trabecular sinuses through the
cortex by four types of sinuses: trabecular sinuses, cortical tubular sinuses, tubulelike sinuses with a network of stellate cell processes, and sinuses between cortical
cords. It is conveyed through the medulla by sinuses both within and between
medullary cords.
Lymph is drained from these sinuses by initial efferent lymphatics of three
types: those between medullary cords, those within the subcapsular sinus overlying medullary or cortical cords, and those within trabeculae. All three types are
continuous with surrounding sinuses through holes 5-30 pm across. These three
alternative routes for lymph drainage may ensure adequate lymph flow during
different intranodal conditions that may exist when the node is responding to
microorganisms or other foreign materials.
Major differences exist in the pathways by which
lymph traverses the lymph nodes of pigs, sheep, and
rodents (Heath and Brandon, 1983; Heath et al., 1986;
Heath and Spalding, 1987; Spalding and Heath, 1987).
Some information is available from the horse, but only
from the legs, on pathways by which lymph enters and
leaves lymph nodes (Heath and Perkins, 1989; Perkins
and Heath, 19901, but no information is available on
the pathways by which lymph flows through the node
parenchyma. This information could help provide a
better understanding of how antigens, such as those
derived from intestinal parasites and microorganisms
and carried in the intestinal lymph, interact with cells
of the immune system. This is of particular interest for
the horse intestine because of the vast population of
microorganisms in its huge cecum and colon, and because of the very large number, several thousand, of
lymph nodes that are associated with the intestine
(Schummer et al., 1981).
Our aim was to investigate the pathways by which
lymph is delivered into these lymph nodes by afferent
lymphatic vessels, traverses the node within lymph sinuses, and drains from these sinuses into efferent lymphatic vessels. This was undertaken using Microfil and
methacrylate casts and by light and electron microsCOPY.
0 1992 WILEY-LISS, INC
MATERIALS AND METHODS
Seven female and four male horses, 6-48-monthsold, were killed by shooting at a local knackery, and
their intestines were removed. These horses were also
used by Nikles and Heath (1990).
An intestinal lymphatic vessel was cannulated and
the lymph node on which it terminated was perfused
with either yellow or white Microfil Rubber Injection
Compound (Canton Biomedical Products, Boulder,
CO), or Batson’s #17 Anatomical Corrosion Compound
(Paul Valley Industrial Park, Warrington, PA) using
methods described by Spalding and Heath (1987).
Fixatives were perfused into the node through a cannula in a n afferent lymphatic. First, 0.5-1 ml of saline
was injected under manual pressure over 15-20 minutes, followed by 0.25-0.5 ml of fixative containing 2%
glutaraldehyde and 2% paraformaldehyde at pH 7.2,
and then 0.25-0.5 ml of a second fixative containing
4% glutaraldehyde, over a total of 50-60 minutes. This
Received September 5, 1990; accepted May 28, 1991.
Address reprint requests to Professor T.J. Heath, Department of
Anatomy, University of Queensland, St. Lucia, 4067, Australia.
LYMPH PATHWAYS IN HORSE LYMPH NODES
was followed by 0.01 ml of Evans Blue dye (0.05%) to
mark the perfused area of the node. The slow perfusion
rate was essential to avoid the distention of the lymph
sinuses. All measurements were made on nodes fixed
in this way.
Immediately after the perfusion of fixative, the tissue was cut into slices 1-2 mm thick, then immersed in
the second fixative for a further 24 hours.
Tissues for electron microscopy were prepared a s described by Heath et al. (1986) and Spalding and Heath
(1987) and examined in a Phillips 505 scanning electron microscope. Sections for light microscopy included
some 7 pm thick stained with hematoxylin and eosin,
and some 1 pm stained with toluidine blue.
A total of 282 lymph nodes were examined. These
came from the jejunum and ileum (26 nodes), cecum
(64), right ventral colon (5), left ventral colon (381,pelvic flexure (4),left dorsal colon (12), right dorsal colon
(16), descending colon (9), and near the root of the mesentery in the region of the cranial mesenteric artery
(108). The latter region is referred to by Schummer et
al. (1981) as the cranial mesenteric root. This term will
be used here.
RESULTS
Studies with light and scanning electron microscopy
and with Microfil casts revealed that each lymph node
was composed of parenchyma surrounded by a capsule
from which trabeculae extended inwards at about a
right angle, and then branched extensively. The cortical parenchyma consisted of a relatively continuous peripheral layer which was continuous with a series of
cortical cords. These cords, which contained fewer nodules, varied considerably in width. For example, they
were 10-350 pm (87 16; n = 34) wide in one node
from the cranial mesenteric root. It was not possible to
identify clearly a separate paracortical region.
The medullary parenchyma was arranged in cords
which were generally smaller than the cortical cords;
most were 20-50 pm across.
Both cortical and medullary parenchyma was penetrated by a labyrinth of lymph sinuses. Lymph was
delivered into these sinuses by afferent lymphatics and
drained from them by efferent lymphatics (Fig. 1).
*
Afferent Lymph Pathways
Afferent lymphatic vessels generally divided into 24 primary branches and less commonly into as many as
seven (Fig. 1).Valves were present in the afferent vessels and in their branches that were more than 40 pm
in diameter. Most branches remained on the node surface dividing progressively into terminal vessels, and
these penetrated obliquely through the capsule. Sometimes the afferent vessels on the node surface were
closely associated with efferent vessels.
Other afferent lymphatics, up to 600 pm in diameter,
penetrated deeply into the node within trabeculae, and
then divided to yield terminal vessels some of which
were 10 p m or less across (Figs. 1,2). Terminal afferent
lymphatics were continuous with the surrounding sinuses through many rounded holes, and these varied
from about 10-30 pm across (Fig. 2).
127
Cortical Lymph Sinuses
The subcapsular sinus extended over the whole of the
cortical parenchyma (Fig. 1)but varied in width from
less than 10 pm to more than 200 pm. It was traversed
by a complex network of stellate cell processes, which
were generally 0.5-8 p.m across but were occasionally
as wide as 13 pm.
Sinuses of four other types conveyed lymph through
the cortex. These were:
Trabecular sinuses
These surrounded trabeculae and penetrated into the
cortical parenchyma, branching extensively and becoming progressively narrower. In one node, for example, they ranged in width from 275 pm at their origin
from the subcapsular sinus to 15 pm in terminal regions. A network of processes, similar to that within
the subcapsular sinus, pervaded the lumen (Fig. 2).
Cortical tubular sinuses
These sinuses were generally circular in cross section and about 10-30 pm in diameter. In one node from
the cranial mesenteric root, 20 measurements revealed
a mean width of 17 i 1.3 pm. The lumen contained
only occasional stellate cell processes (Fig. 3), and the
wall was punctuated by rounded holes, generally 6-10
pm across their major axis (Figs. 3, 4).Processes 1-4
pm across radiated outward from the sinus wall (Fig.
4).
Cortical tubular sinuses were mainly distributed
through the more-continuous parenchyma and were
only occasionally present within the cortical cords (Fig.
1). They were often the predominant sinus in the vicinity of nodules.
Tubule-like sinuses with a luminal network
These sinuses differed from cortical tubular sinuses
in having a complex network of luminal stellate cell
processes, and in being larger. For example, in one
node from the cranial mesenteric root they were 25-85
pm (43 i 3.6 pm n = 25) in diameter (Fig. 5).They were
the predominant sinus within the inner regions of the
cortex and appeared to be extensions of the sinuses
between cortical cords.
Sinuses between cortical cords
Although occurring mainly between cortical cords
some of these sinuses penetrated deeply into the cortical parenchyma (Fig. 1).They contained a complex network of luminal stellate cell process, and occasional
trabeculae, up to 30 pm across. These sinuses varied
considerably in size; in one node from the left ventral
colon they were found to be 25-225 pm across. The
lining of these sinuses was variable: in some places it
appeared continuous, while in other there were many
rounded holes, 1-9 pm or more across.
Medullary Lymph Sinuses
Lymph was conveyed through the medullary parenchyma by sinuses both within and between medullary
cords.
128
S.A. NIKLES AND T.J. HEATH
1
CORTICAL TUBULAR SINUS
I
TRABECULAR SINUS
PENETRATING AFFERENT
SINUS WI
MEDULLARY
EFFERENT
LYMPHATICS
2. FROM MEDULLARY SINUS
3. WITHIN TRABECULUM
Fig. 1. Diagrammatic representation of the lymph pathways
through an intestinal lymph node of the horse. This is based on light
and electron micrographs, and Microfil casts. An afferent lymphatic
vessel approaches the dorsal surface of the node and branches over a
large portion of the surface. Some of these branches remain on the
node surface, dividing into terminal vessels which deliver lymph into
the subcapsular sinus. Others penetrate into the node within trabeculae and deliver lymph into trabecular sinuses. Lymph flows from the
subcapsular and trabecular sinuses to the sinuses between cortical
cords and medullary cords. It is conveyed there either directly or
through cortical tubular sinuses or tubule-like sinuses with networks
of stellate cell processes. Initial efferent lymphatics arise from sinuses
between medullary cords, within trabeculae in sinuses between cortical or medullary cords, and from the subcapsular sinus overlying
cortical and medullary cords. Only one of each type of initial efferent
lymphatic is shown.
Sinuses within medullary cords
These were rounded in cross section and less than 10
pm across (8 f 2.2 pm, n = 11 in a node from the
cranial mesenteric root) (Fig. 6). Occasional processes
0.5-3 pm across, extended across the lumen, and the
sinus lining appeared continuous (Fig. 6B).
great as 12 pm in diameter, extended across their lumen. The processes of surrounding sinuses were attached to the outer wall of these initial efferent lymphatics. Rounded holes, 5-25 pm across (16 2.0 pm
n = lo), were present in the walls of these vessels.
These holes were generally smaller than those in terminal afferent lymphatics, and they were present in
greater numbers and distributed more extensively
along the length of the vessel. As the efferent vessels
increased in size, the numbers of holes in the wall and
of luminal processes decreased, but valves appeared.
The smallest vessel observed with valves was 90 pm in
diameter.
Sinuses between medullary cords
These sinuses contained a network of luminal stellate cell processes, similar to that within the sinuses
between cortical cords, with which these sinuses were
continuous (Fig. 6A). The sinus width varied considerably: it was 70-200 pm in one node from the cranial
mesenteric root. Initial and larger efferent lymphatics,
and trabeculae, passed through some of these sinuses.
Efferent Lymph Pathways
Lymph was drained from lymph sinuses by initial
efferent lymphatic vessels of three types. These were:
Vessels between medullary cords
These vessels were similar to those described by
Heath and Perkins (1989). They were 40-100 pm
across with processes, mostly 0.5-5 pm, but some a s
*
Vessels within trabeculae
Some of the initial efferent lymphatics within trabeculae were as narrow a s 9 km across. They were
continuous with the surrounding sinuses through
rounded holes in the surface of the trabeculae; these
were generally 5-30 pm (11 1.2 pm, n = 22) but
occasionally as large as 85 km across their major axis.
Luminal processes were present, but their frequency
varied considerably between vessels.
The initial efferents often formed complex labyrinths
*
LYMPH PATHWAYS IN HORSE LYMPH NODES
129
medullary and cortical cords. They were continuous
with the sinus through holes in their wall, and these
were similar to those in the initial efferent vessels described above (Fig. 8). After penetrating the capsule,
the vessels joined with other efferent vessels and
formed an interlacing network on the surface.
DISCUSSION
Afferent Lymph Pathways
The pathways by which lymph from the intestine of
the horse is delivered into lymph nodes by afferent
lymphatics, then conveyed through the parenchyma in
lymph sinuses and drained through efferent lymphatics, have some similarities to those in the pig and the
sheep, but they are much more complex than the traditional concepts derived from work on rodents.
In the horse, each intestinal lymph node, like each
superficial node (Perkins and Heath, 1990), is generally supplied with lymph from a single afferent vessel.
By contrast, the superficial nodes of the sheep and pig,
the only ones that have been described in any detail,
receive lymph from a number of different vessels: 6-12
in the sheep, and 1-4 in the pig (Heath and Brandon,
1983; Spalding and Heath, 1987).
The terminal afferent lymphatics in the horse are
continuous with the subcapsular and trabecular sinuses through rounded holes up to 30 pm across. In
this respect the situation in the horse is similar to that
in sheep (Heath and Brandon, 1983). However in the
horse nodes, both more and larger vessels penetrate
into the substance of the nodes. Furthermore, they penetrate deeply into the node in a manner reminiscent of
that in the pig (Spalding and Heath, 1987).These penetrating afferent lymphatics in the horse have the potential to deliver a large proportion of the lymph deep
within the node, and in the general vicinity of some
initial efferent lymphatics. The effect of this on the
phagocytic and immune capacity of the node can only
be speculated upon.
Lymph Sinuses
Fig. 2. Scanning electron micrograph of a lymph node from the
cranial mesenteric root showing a n afferent lymphatic (a) within a
trabeculum (asterisks). A shows a terminal branch of the afferent
lymphatic that has a hole about 10 pm across in its wall. Detail of this
hole is shown in B. This hole allows communication between the
vessel and the surrounding trabecular sinus (s). cp, cortical parenchyma. A, X 400; B, x 2,400.
within the trabeculae before coalescing to form larger
vessels (Fig. 7). Direct connections between the lumen
of these larger vessels, up to 130 pm across, and the
sinuses were observed. Valves were present in vessels
as small as 40 pm in diameter. Luminal processes became less common as the vessels size increased up to
100 pm and were generally absent in still larger vessels (Fig. 7). These vessels emerged from the node over
a large area of its surface, and occasionally from a deep
groove in the node.
Vessels within subcapsular sinus
Initial efferent lymphatics, 12-25 pm across,
drained lymph from the subcapsular sinus overlying
The lymph flows through a labyrinth of sinuses of far
greater complexity than in nodes that have been described from pigs, sheep, and rodents.
Lymph from afferent lymphatics that has entered
either the subcapsular sinus or trabecular sinus flows
through the cortex via an extensive network of trabecular sinuses. It may then flow directly into the sinuses
between cords of parenchymal tissue, or be further distributed throughout the cortical parenchyma within either the cortical tubular sinuses, or the tubule-like sinuses. Some lymph may also flow around the periphery
of the node within the subcapsular sinus, and from
there into sinuses between either cortical or medullary
cords.
The possible alternative pathways for lymph flow in
rodents are more restricted (Sainte-Marie et al., 1982).
In the rat, for example, Sainte-Marie and Sin (1968)
reported that the subcapsular sinus occasionally yields
narrow extensions that run across the medulla and
transform into the wider and irregular medullary sinuses. In the superficial nodes of sheep, the subcapsular and trabecular sinuses, and the sinuses between
medullary cords, are comparable to those described in
S.A. NIKLES AND T.J. HEATH
130
Fig. 6. Scanning electron micrographs showing a region of medullary parenchyma in a lymph node from the left ventral colon. A shows
sinuses (s) between medullary cords (m), which are traversed by a
complex network of luminal stellate cell processes. A sinus within the
medullary cord is indicated by the arrow, and B shows luminal detail
of one of these sinuses. The sinus contains few luminal processes (p)
and the sinus lining appears continuous. A, x 400; B, x 2,400.
Figs. 3,4. Scanning electron micrographs of cortical tubular sinuses
within the cortical parenchyma (cp) from a lymph node from the left
ventral colon. Figure 3 shows a longitudinal section and Figure 4 a
transverse section, with processes (p) radiating outward from the sinus wall. Arrows indicate holes up to 30 km across in the sinus wall.
Figure 3, x 800; Figure 4, x 1,200.
Figs. 3-5.
Fig. 5. Scanning electron micrograph of a lymph node from the left
ventral colon showing two tubule-like sinuses which are traversed by
a network of stellate cell processes (asterisks), and a cortical tubular
sinus which is smaller (arrow). This sinus does not contain luminal
stellate cell processes. cp, cortical parenchyma. x 320.
LYMPH PATHWAYS I N HORSE LYMPH NODES
131
Kurokawa and Ogata, 1980; Heath and Spalding,
1987). The tubular cortical sinuses of horses, though
smaller, resemble those of sheep (Heath et al., 1987).
They are also similar to the sinuses located within the
medullary cords in the horse-sinuses which do not
appear to have a counterpart in the other species that
have been studied.
The complex labyrinth of sinuses, coupled with the
lack of structural compartmentalisation, should allow
lymph that enters through the single afferent lymphatic to be distributed extensively throughout the parenchyma of the horse intestinal lymph node. This
would have important implications for the ability of
the node to remove and respond to foreign material
carried from the intestine in the lymph. The real significance of this, and the relationship of this feature to
the vast microbial population of the horse intestine, is
not known.
It appears that these features are not common to all
species. In some, for example, the cortical parenchyma
may be divided into functionally separate units. These
include the “deep cortical units” or “physiological
units” of the rat, each of which is related to the opening
of the afferent lymphatic, the “lobular units” of the
rabbit, and, on a somewhat broader scale, the node anlagen of the pig (Kelly, 1975; Sainte-Marie et al., 1982;
Spalding and Heath, 1987). Functional studies using
the pig and the rat have shown that lymph from each
afferent lymphatic terminating on a node is regionally
distributed within the parenchyma (Sainte-Marie et
al., 1982; Spalding and Heath, 1987). It would appear
that in these animals, in contrast to the horse, the
lymph does not normally interact with all the parenchyma of the node, but only that in the region to which
it is delivered by afferent lymphatics.
Efferent Lymph Pathways
Lymph is drained from lymph sinuses in the horse by
initial efferent lymphatic vessels of three types: those
Fig. 7. Scanning electron micrograph of an efferent lymphatic (e) within sinuses between medullary cords, those within
from a lymph node from the right ventral colon, showing points of the subcapsular sinus overlying cortical or medullary
entry of smaller efferent lymphatics (asterisks). c, capsule; s, sinuses
cords, and those within trabeculae. These lymphatics
between medullary cords; v, valve. x 200.
are continuous with the surrounding sinuses through
Fig. 8. Scanning electron micrograph of a lymph node from the rounded holes in their walls. Most of these holes are
5-30 pm across, and are smaller than those described
cranial mesenteric root showing the initial efferent lymphatic vessel
(e) within the subcapsular sinus (s).The vessel is continuous with the
in the sheep (20-50 pm) and pig (80 pm or more)
sinus through holes in its wall. c, capsule. x 760.
(Heath and Spalding, 1987; Spalding and Heath, 1989).
The initial efferent lymphatics within sinuses have
thin walls but are supported by processes extending
this paper (Heath et al., 1986; Heath and Spalding, from their outer walls into the network of the surrounding sinuses (Heath and Perkins, 1989). However,
1987).
However, in contrast to the sheep, the cortex of the the initial lymphatics within trabeculae appear to be
horse intestinal nodes has two types of tubular sinuses: well buttressed by the connective tissue of the trabeccortical tubular sinuses, which have a circular cross- ulum. These mechanisms should help keep the lumen
section and few luminal processes, and tubule-like si- of the vessel open if the intranodal pressure rises as
nuses, which have a complex network of stellate pro- may occur when the node responds t o foreign material
carried t o it in the lymph.
cesses in their lumen. These latter sinuses are 25-85
Initial efferent lymphatics arising within the node
pm across, and so are larger than the cortical tubular
sinuses; these vary between 10-30 pm across, and they have been reported from sheep (Heath and Spalding,
can be readily distinguished with the scanning electron 19871,but not rats. In this species, as Sainte-Marie and
Sin (1968) reported, the medullary sinuses join tomicroscope.
In distribution, both types of sinus resemble “para- gether with the end-portion of the subcapsular sinus at
cortical” and “tubular cortical” sinuses in rabbits, the hilus to form the efferent lymphatic vessels. The
“mudstreams” of rodents, and “tubular” sinuses of pig, which differs from most other animals in the censheep (Sonderstrom and Stenstrom, 1969; Kelly, 1975; tral location of its nodular tissue, has some initial lym-
S.A. NIKLES AND T.J. HEATH
132
phatics which are similar to those within trabeculae in
the horse (Spalding and Heath, 1987). It is thought
that in the pig these vessels were once on the node
surface, but after fusion of node anlagen come to lie
within remnants of the capsule in the enlarged, adult
node (Spalding and Heath, 1989). There is no evidence
for a similar origin for the initial efferent lymphatics
within trabeculae in the horse. Furthermore, those
within trabeculae in the horse are present in far
greater numbers and form more complex networks
than those in the pig.
In the horse, the efferent lymphatics generally
emerge over a large area of the node surface. This arrangement is comparable to that seen in the pig and
the sheep, but it contrasts to that in the rat, where a
single efferent lymphatic leaves the node at a clearly
defined hilus (Sainte-Marie and Sin, 1968; Heath and
Brandon, 1983; Spalding and Heath, 1987).
ACKNOWLEDGMENTS
We are grateful to Gary Godbold for help with the
horses and the Director and staff of the UQ Electron
Microscope Centre for their assistance. We thank the
Queensland Equine Research Foundation for financial
support.
LITERATURE CITED
Heath, T., and R. Brandon 1983 Lymphatic and blood vessels of the
popliteal node in sheep. Anat. Rec., 207;461-472.
Heath, T.J., R.L. Kerlin, and H.J. Spalding 1986 Afferent pathways of
lymph flow within the popliteal node in sheep. J. Anat., 149:
65-75.
Heath, T.J., and H.J. Spalding 1987 Pathways of lymph flow to and
from the medulla of lymph nodes in sheep. J . Anat., 155:177-188.
Heath, T.J., and N.R. Perkins (1989) Pathways between lymph vessels and sinuses in lymph nodes: A study in horses. Anat. Rec.,
223,420-424,
Kelly, R.H. 1975 Functional anatomy of lymph nodes. I. The paracortical cords. Inter. Arch. Allergy Appl. Immunol., 48:836-849.
Kurokawa, T., and T. Ogata 1980 A scanning electron microscope
study on the lymphatic microcirculation of the rabbit mesenteric
lymph node: A corrosion cast study. Acta Anat., 107:439-466.
Nikles, S.A., and T.J. Heath (1990) Pathways of lymph flow from the
intestine of the horse. Anat. Rec., 229521-524.
Perkins, N.R., and T.J. Heath 1990 Pathways of lymph flow from the
superficial tissues in the legs of horses. Res. Vet. Sci., 48:119123.
Sainte-Marie, G., F.S. Peng, and C. Belisle 1982 Overall architecture
and patterns of lymph flow in the rat lymph node. Am. J. Anat.,
164:275-309.
Sainte-Marie, G., and Y.M. Sin 1968 Structures ofthe lymph node and
their possible function during the immune response. Rev. Can.
Biol., 27:191-207.
Schummer, A., H. Wilkens, B. Vollmerhaus, and K.-H. Habermehl
1981 The Circulatory System, the Skin, and the Cutaneous Organs of the Domestic Animals. Verlag Paul Parey, Berlin, pp.
328-330,432-434.
Sonderstrom, N., and A. Stenstrom 1969 Outflow paths of cells from
the lymph node parenchyma to the efferent lymphatics. Scand. J.
Haematol., 6t186-196.
Spalding, H.J., and T. Heath 1987 Pathways of lymph flow through
superficial inguinal lymph nodes in the pig. Anat. Rec., 217:188195.
Spalding, H.J., and T.J. Heath 1989 The fine structure of lymph pathways in nodes from the superficial inguinal lymph centre in the
pig. J . Anat., 166:43-54.
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