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Plasticity of innervation of the medulla of axillary lymph nodes in the rat after antigenic stimulation.

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THE ANATOMICAL RECORD 238:213-224 (1994)
Plasticity of Innervation of the Medulla of Axillary Lymph Nodes in
the Rat After Antigenic Stimulation
G.E.K. NOVOTNY, T. HEUER, A. SCHOTTELNDREIER, AND c. FLEISGARTEN
Department of Neuroanatomy, Dusseldorf University, Dusseldorf, Germany
ABSTRACT
The purpose of this investigation was to test the hypothesis
that activation of the immune system in rats will lead to changes in the
density of innervation in lymph nodes. In order to reduce the variability
between animals, the rats were reared under sterile conditions and immunostimulation was effected by subcutaneous application of bovine albumin
in a region draining to the axillary lymph nodes of both sides. Control
animals received an equivalent application of sterile physiological saline.
The animals were sacrificed 10 days and 27 days and 4 months after immunostimulation. The nerves in the axillary lymph nodes were quantified by
light microscopy in silver impregnated sections and at the ultrastructural
level on ultrathin sections. The survival times were chosen so that the first
group was in the ascending phase of antibody production, the second
group at the peak, and the third group in the declining phase. Both at the
light and ultrastructural levels, there were statistically significant differences in the density of innervation of medulla between the groups, with a
particularly pronounced increase in the group 4 months after immunostimulation. At the ultrastructural level, there was also an increase in the density of incompletely ensheathed axonal profiles in the parenchyma of the
medulla, while the nerves associated with blood vessels were not increased.
We conclude that immunostimulation leads to morphological changes in
the innervation of the medulla of axillary lymph nodes, that are consistent
with the concept of functional activation of the autonomic nervous system
through the immune system. o 1% Wiley-Liss, Inc.
Key words: Lymph node, Innervation, Immunostimulation, Silver impregnation, Ultrastructure
In recent years the question of interaction between
the immune and nervous systems has aroused interest.
The majority of workers in this field assume a humoral
axis from the central nervous system to the immune
competent organs, as first postulated by Besedovsky
(Besedovsky et al., 1988). However, several groups
have also investigated the possibility of a direct innervation of immune competent organs (see review by
Felten and Felten, 1991). Alterations in the density of
innervation of immune competent organs with age
have been reported (Ackerman et al., 1991a; Bellinger
et al., 1987, 1988; Novotny et al., 19931, and deficits in
the immune response have been shown after sympathectomy with 6-hydroxy dopamine (6-OHDA) (Ackerman et al., 1991b). Rearrangement of the innervation
within a given compartment with maintenance of the
total nerve content was reported for the spleen after
drug-induced immunosuppression (Carlson et al.,
1987). All these observations present suggestive evidence for a functional link between the peripheral innervation of lymphatic organs and the immune response. However, none of these investigations are
conclusive because: (1) little is known about age
changes in the peripheral innervation in general; (2)
0
1994 WILEY-LISS, INC
global sympathectomy with 6-OHDA may well affect
the immune response by unspecific mechanisms or via
the central nervous system; and (3) the alterations in
the spleen may be related to volume changes within
compartments after lymphocyte depletion. Thus, an effect on the immune response through humoral mechanisms via the hypothalamic-adrenal cortex axis, as postulated by Besedovsky et al. (19881, still remains the
most likely mechanism for immune regulation.
Functional plasticity is documented in the central
nervous system of invertebrates (Alkon et al., 1990)
and higher mammals (Keller et al., 19901, and alterations in the size of the fields of innervation have been
described in the peripheral nervous system after partial denervation (Terenghi et al., 1986; Navarro and
Kennedy, 1988; Taylor et al., 1988).
If there is a functional link between the peripheral
Received February 2, 1993; accepted September 10, 1993.
Address reprint requests to G.E.K. Novotny, Ph.D., Abteilung Neuroanatomie, Zentrum fur Anatomie und Hirnforschung, Med. Einrichtungen der Universitat Diisseldorf, Postfach 10 10 07, D-40001
Diisseldorf, Germany.
214
G.E.K. NOVOTNY E T AL.
Diagram 1: Experimental Design
GROUPS
DURATION IN DAYS
0 10 2027
120
10 day stim.
(N=10)
27 day stim
(N=10)
4 mo. stim.
(N=10)
Control
(N=10)
4 mo. cont.
(N=4)
Diagram 1. Diagrammatic presentation of treatment of the five animal groups. All animals were 3
months old at the start of the experiment (day 0 ) .
= subcutaneous injection, Ag = antigen, V =
Vehicle without antigen. The termination of the line indicates the time of sacrifice.
+
innervation of immune competent organs and their immunological function, one could expect to find morphological changes of the innervation in response to extremes of immunological activity. One problem to be
anticipated in such a study is that each individual animal of a group is subject to an individual immunological history. If antigenic stimulation affects the innervation, then the individual history may well lead to
considerable differences in the innervation between
“normal” animals, which may be expected to obscure
small but consistent differences due to a single antigenic exposure with experimentally practicable group
sizes. To eliminate such a possible source of disturbance, this study was performed on animals reared under absolutely sterile conditions. The present publication reports on the light microscopical part of this study
and presents supporting evidence at the electron microscopical level from a study designed to investigate
ultrastructural changes under such conditons.
MATERIALS AND METHODS
Animals
Forty-four male HAN-Wistar rats were used. The animals were bred and reared under sterile conditions in
isolation units in the Tierversuchsanlage of Dusseldorf
University according to the regulations for housing of
experimental animals. Sterilized food and water were
available ad libitum. Because of the limited capacity of
the isolation units, the animals were reared and experimentally treated in eleven groups of four siblings
each. Ten of the sibling groups each contained one control animal and one animal of each of the three experimental groups. The eleventh group consisted of four
long-term controls that remained untreated. At the
start of the investigation all animals were 3 months
old.
prising 10 animals and three experimental groups also
of 10 animals each. The first experimental group received one application of antigen (see below) and was
sacrificed 10 days later (10 day stimulated). The second
group received two applications of antigen a t an interval of 20 days and was sacrificed 27 days after the first
application (27 day stimulated). The third group received identical treatment to the second group and was
sacrificed 4 months after the first antigen application
(4 month stimulated). The selected schedules and survival times ensure that the first group is in the ascending phase of antibody production, the second group is a t
the peak of antibody production, while the third group
is in the declining phase (Herbert, 1968). The control
group was matched with the second group a t the maximum of antibody production and received injections of
physiological saline at the same intervals. The fifth
group, added after another study in our laboratory had
revealed age effects on the innervation of lymph nodes
in the rat, consisted of only 4 animals as controls for
the long-term (4months) survival group. The animals
remained untreated and were sacrificed a t the same
age as the long-term survival group (4months control).
lmmunostimulation
Immunostimulation was achieved with the subcutaneous injection of purified bovine albumin (Serva) in
incomplete Freund’s adjuvans (Sigma Chemical Co.,
St. Louis, MO). One hundred microliters of this mixture, containing 100 pg albumin and 50 p1 incomplete
Freund’s adjuvant in sterile physiological saline, were
applied interscapularly. This ensured effective stimulation of an antigenic response with the appearance of
germinal centers and plasma cells in the axillary
lymph nodes (Figs. 1-4). Control injections consisted of
100 ~1 sterile physiological saline. Observation of the
animals after application of antigen did not give any
Experimental Design
indication of adverse effects, except in one animal
The animals were divided into five groups which which developed a slight granulomatous reaction at
were treated as summarized in Diagram 1. The pri- the site of injection. All animals survived the injections
mary experiment consisted of one control group com- for the predetermined period till sacrifice for perfusion.
LYMPH NODE IMMUNOSTIMULATION
Figs. 1-4. Micrographs of semithin sections of axillary lymph nodes
from control and experimental rats to illustrate the effect of immunostimulation. All calibrations 50 km.
Fig. I . Lymph node from a control animal, showing the homoge-
neous cortex (C) and the medulla (M) exhibiting many narrow vascular channels. x 70.
Fig. 2. Lymph node from a n animal 27 days after immunostimulation. Two germinal centers (arrows) are visible in the cortex (C). The
Light microscopy
Histological Procedures
All animals were sacrificed by transcardial perfusion
with a mixture of 1.25% glutaraldehyde and 1%paraformaldehyde in 0.1 M phosphate buffer of pH 7.4 at
0°C under deep anaesthesia (total areflexia) after intraperitoneal application of a lethal dose of 36% urethane. The initial perfusion pressure of 120 mm Hg
was reduced by 20 mm Hg a t 10-minute intervals, with
a total perfusion time of 30 minutes.
215
medulla (M)contains abundant wide vascular channels, many of
which are lymphatic. X 70.
Fig. 3. Lymph node from a n animal 4 months after immunostimulation. Plasma cells (arrows) are present in the medulla. x 600.
Fig. 4. Lymph node from a n animal 27 days after immunostimulation. Detail of a germinal center, showing mitotic cells (arrows).
x 500.
The axillary lymph nodes were removed immediately after the perfusion was completed and post-fixed
in 10% formaldehyde solution for 4 weeks. To avoid
damage to the lymph nodes, which were embedded as a
group in the adipose tissue of the axilla, they were only
separated from the fat sufficiently for identification
and isolation from each other. Thus, each tissue block
also contained some adipose tissue, and for this reason
it was not deemed practicable to determine the volumes or weights of the nodes. Subsequently, the lymph
216
G.E.K. NOVOTNY ET AL.
nodes were dehydrated over ethanol and embedded in
paraplast (Monoject Scientific Inc., Athy, Co. Kildare,
Ireland). One lymph node was chosen from each animal
and serial sections were cut at 10 pm, mounted on
slides, and silver impregnated as described (Novotny
and Gommert-Novotny, 1988). Out of each of these series, three slides (6 sections each) were taken from each
third of the series and stained with hematoxylin and
eosin or cresyl violet for the precise determination of
the presence of germinal centers or plasma cells, as a
control on the actual immune stimulation, germinal
centers being absent in non-stimulated animals, while
plasma cells are exceedingly rare.
Electron microscopy
For ultrastructural investigation, the whole lymph
nodes, excised from the animals, as described for light
microscopy, were post-fixed in 1%osmium tetroxide for
3 hours, dehydrated over acetone, and embedded in
Spurr’s medium. One lymph node was chosen from
each animal and completely sectioned, whereby ten ultrathin serial sections were gathered on 100 mesh copper grids at intervals of 100 pm throughout the total
extent of each lymph node. Semithin sections were collected for each group of ultrathin sections, to enable
comparisons between light and electron microscopy,
and to control for effective immunostimulation by the
presence of germinal centers and plasma cells. The ultrathin sections, contrasted with uranyl acetate and
lead citrate, were viewed in a Zeiss EM9 or Hitachi
H-600 at 60 or 75 kV and a primary magnification of
15,000 or 20,000.
Morphometric Procedure
Light microscopy
In a previous investigation on the density of innervation of lymph nodes (Kliche and Novotny, 1987) every 6th section was evaluated. By random selection of
sections from this existing pool, we determined that it
is sufficient to evaluate every 24th section only. To
reduce the variability in the present study, the sections
were not chosen totally randomly, but were selected
randomly in equal numbers from each third of the
lymph node. If the number of sections of the total
lymph node was fewer than necessary t o arrive a t a
total of 6 sections (e.g., 100 sections = 100/24 = 4.167),
then 6 sections were selected (i.e., 2 from each third);
otherwise the next full number of the computed figure
was taken (i.e., 10 sections if the computed number was
9.3). Each section was completely scanned in a meander pattern under oil immersion optics ( x 800) and the
length of all nerves encountered within the lymph node
(excluding the capsule) was measured under direct microscopic vision using a Zeiss Morphomat 10 digitalization tableau system. Additionally, the composition of
each microscope field (i.e., cortex or medulla) was noted
and the location of the nerves within these compartments was recorded. The total number of fields gave
the precise measure of the area of each section and
thus, together with the known section thickness, enabled the computation of the sampled volume of each
section. Summation of all the sections analyzed in one
animal gave the total volume sampled. The occurrence
of branching of the axons was also registered.
Measures were taken to determine whether the in-
vestigated lymph node was immunostimulated using
additional stains. This was in fact superfluous, since
the silver impregnated sections clearly revealed the
immunological status of the lymph node, so that the
observer could not fail to know to which group of animals a given lymph node belonged. For this reason it
was not possible to perform a blind morphometric analysis. However, to reduce observer bias no computations
whatever were performed on the raw data until after
all measurements for all groups were completed.
Electron microscopy
One lymph node from each animal was selected for
electron microscopy and completely sectioned. Series of
10 ultrathin sections were collected a t intervals of 100
pm. Between ultrathin series, semithin sections were
collected and stained with toluidin blue. These semithin sections were intended as controls, to ascertain the
immune reaction of the lymph node. In actual practice
this was superfluous, since the immune reaction was
also obvious on the ultrathin sections. Only 1 section
from each of the 10 sections comprising an ultrathin
series was chosen randomly and completely searched
for axonal sections, grid square by grid square. Series
for sampling were selected in a semi-random fashion to
ensure that all portions of the lymph node were equally
represented. Sampling from series was continued until
a total count of at least 150 incompletely ensheathed
axonal sections (varicosities) was attained for each
lymph node. The purpose of this procedure was to obtain quantitative data on the percentage representation of various categories of varicosities and their relation to cellular elements. These results will be
presented in detail elsewhere. Together with the aforementioned data, information was gathered on all axonal sections, including their location in cortex or medulla, as well as the total area of lymph node searched.
From this evaluation it is possible to derive information on the density of innervation and these results are
presented in the present publication. Sampling of each
individual lymph node was performed by four observers independently, with each observer aiming to contribute approximately equal proportions of the total
count. Since termination of the count with the attainment of the required figure will cause considerable bias
in the number of axonal sections per unit area, each
observer completed the evaluation of the section containing the axonal section number aimed at. Thus, usually more than the requisite number of axonal sections
was counted, and the summation over four observers
lead to totals of more than 150 axonal sections in almost all instances.
Again, the raw data was not analyzed until all the
data had been gathered, in order to reduce possible
observer bias. As the counting of a representative number of axons at the ultrastructural level is exceedingly
laborious, only those groups were evaluated that had
proved of interest a t the light microscopical level, i.e.,
both control groups and the 27 day and 4 month stimulated groups. This decision was taken by one author
(G.E.K.N) and the remaining observers were not informed as to the result of the light microscopic investigation until the gathering of the ultrastructural data
was completed.
LYMPH NODE IMMUNOSTIMULATION
Statistical Procedures
The various parameters obtained were compared between the groups using the Kruskal-Wallace non-parametric analysis of variance (H-test) a t a significance
level of P < 0.05. Global comparisons attaining this
significance level were further analyzed using the comparison of pairs according to Schaich & Hamerle (Bortz
et al., 1990).
RESULTS
General Obsewations
Light microscopy
Lymph nodes from non-immunostimulated control
animals show a homogeneous cortex and a medulla
lacking prominent vascular channels or plasma cells
(Fig. 1). All axillary lymph nodes from animals surviving the application of antigen by 10 or 27 days before
sacrifice contained germinal centers (Figs. 2,4). Four
months after immunostimulation germinal centers
could only be found in few lymph nodes. However,
plasma cells could be located in the medulla in numbers not present in the control animals (Fig. 3).
Nerves were present in the medulla of all lymph
nodes examined. However, 4 months after immunostimulation their number seems to be distinctly increased and branching of the nerves is more frequent
(Figs. 5,6).
Electron microscopy
As already noted at the LM level, immunostimulated
lymph nodes contained germinal centers and large
numbers of plasma cells, which were also obvious a t
the ultrastructural level. Nerve bundles could be found
throughout the medulla and on the borderline between
medulla and cortex. The remaining cortex and all germinal centers were devoid of innervation.
The axons within the nerves were either completely
ensheathed by Schwann cell processes (axonal sections), or partially denuded (varicosities) (Figs. 7-9).
Some axons were myelinated. Both varicosities or axonal sections could contain vesicles, but vesicles were
never found in myelinated axons. As previously described for normal animals (Novotny, S988), varicosities were predominantly associated with reticular cells
(Figs. 7,9). Of the lymphocytic population, plasma cells
were most frequently related to varicosities (Fig. 81,
and associations of varicosities with lymphocytes
(which were not further characterized) were rare.
Quantitative Results
Volume changes
Since a fixed proportion of the sections of each lymph
node was analyzed at the light microscopical level, the
actually scanned volume of each lymph node is approximately proportional to its total volume. The result is
presented graphically in Figure 10 and demonstrates
an increase in the analyzed total volume of stimulated
lymph nodes 10 days after the first antigen application,
which is statistically not significant (P = 0.2700).
There is a subsequent statistically not significant decline to below control values in the 4 month survival
group. As may be further noted, the alteration in analyzed volumes is solely due to changes in the volume of
217
the cortex, the medullary volume remaining remarkably constant with only an insignificant increase in the
4 month immunostimulated group (P = 0.6560). The
volume changes in the cortex do not attain statistical
significance (P = 0.0953) mainly because of scatter,
indicated by the large standard deviations, reflecting
considerable differences in the size of the randomly selected lymph nodes from individual animals. The relation between volume of cortex and volume of the medulla is demonstrated in Figure 11. The ratio of cortex
to medulla increases up to 27 days after antigenic stimulation and declines after longer intervals. Here there
is a significant difference in global comparison (P =
0.0018) and there are significant pair differences between the 4 month stimulated group on the one hand
and the 10 day and 27 day immunostimulated groups
on the other (both with P < 0.05). This is due to the
relatively constant relationship between medulla and
cortex within each group, despite large differences in
total size of the lymph nodes. The time course of the
change in the ratio of the volumes roughly corresponds
to that of the production of antibodies by our immunization schedule, as based upon data in the literature
(Herbert, 1968; Harlow and Lane, 1988).
Innervation
As shown in Figure 12, the average total length of
nerves measured per lymph node in each group increases from the control to the 4 month survival group,
but is lowest in the 4 month control group. The total
length of nerves in the whole lymph node, as well as
the total values for cortex and medulla, all show significant differences in global comparison (total: P =
0.0053; medulla: P = 0.0022; cortex: P = 0.0114). In
post-test pair comparison, there is no significant pair
difference for the cortex, for the total lymph node there
is a significant pair difference between the 10 day and
4 month stimulated groups (P < 0.011, and in the medulla there is a significant pair difference between the
control and 4 month stimulated groups (P< 0.05) and
between the 10 day stimulated and 4 month stimulated
groups (P < 0.0s). When related to the total volume as
density of innervation, there is an increase in the
groups 27 days and 4 months after antigenic stimulation, with a slight decrease in the 10 day group (Fig.
13). The groups differ significantly in global comparison (P = 0.00009), with significant pair differences (P
< 0.05) between the control, 10 day stimulated, and 27
day stimulated groups on the one hand and the 4
month stimulated group on the other. There is no significant difference between the 4 month stimulated
group and the 4 month control group or between the
latter and the original control group, which may be
attributed to the few animals available for the 4 month
control group. For the innervation of the medulla the
global difference is equally pronounced (P = 0.00008),
with significant pair differences between the control,
SO day stimulated, and 4 month control groups vs. the
4 month stimulated group. There is no significant difference for the medullary innervation between the
short-term control group and the 4 month control
group.
There is practically no innervation of the cortex, the
few nerves located in this compartment are close to the
border of the medulla and are of no consequence to the
218
G.E.K. NOVOTNY ET AL.
Figs. 5-6
LYMPH NODE IMMUNOSTIMULATION
Figs. 7-9. Electron micrographs illustrating axons and varicosities
with their typical relationships to cells. All calibrations 1 pm.
Fig. 7. A nerve within a group of plasma cells in a lymph node of an
animal 27 days after immunostimulation. There are two varicosities
(arrows),the upper of which is associated with a plasma cell, while the
lower one (possibly sensory) is approximated to the process of a reticular cell. The third profile (arrowhead) is completely ensheathed and
thus axonal. x 16,000.
Figs. 5-6. Silver impregnated sections from control and experimen.
tal animals to show nerves. All calibrations 50 pm.
Fig. 5. Lymph node from control animal. A representative field from
the medulla showing two nerves in focus (arrows). The arrowheads
indicate the position of nerves present in the visual field, but out of
the focal plane at this setting. Note that none of the nerves branch.
x 500.
Fig. 6. Lymph node from an animal 4 months after immunostimulation. An equivalent field of the medulla to that shown in Figure 5,
with nerves (small arows) and branching of nerves (large arrows).
Note the distinct increase in density of innervation after 4 months of
immunostimulation. x 500.
219
Fig. 8. Animal 27 days after immunostimulation. A fine nerve accompanying a blood vessel (bottom of illustration), with two varicosities containing vesicles (arrows). The upper varicosity is closely apposed to a plasma cell, whereas the lower one is not closely associated
with any cell. x 20,000.
Fig. 9. Animal 4 months after immunostimulation. A nerve containing two axons (arrowheads) and three varicosities farrows),the one on
the left with vesicles. All of the varicosities are associated with reticular cells. x 12,000.
cortex as a whole. The innervation of the cortex shows
a marginally significant difference in the global comparison between the groups (P = 0.050991,with no significant pair differences. It should be noted that the
density of innervation increases for the total lymph
node and the medulla, while it decreases for the cortex.
As shown in Figure 14, the frequency of axonal
branching per unit volume of medulla is distinctly increased in the 4 month stimulated group. The global
group comparison is very significant (P = 0.0024), the
220
G.E.K. NOVOTNY ET AL.
Average Volumes per Lymph Node in all Groups
(Actual volumes analyzed)
ri
Average Length of Nerves Measured
10000
[zlTotal
Cortex
Medulla
L
m'
E
8000
l
.r
[71 control
@$ 1Od.
stirn.
[IIII 27d.
stirn.
6000
A
.wZ
rn
f
4000
2000
0
Groups
Control
10d. stim. 27d. stim.
4mO.
stim. 4mO. cont.
Groups
Figs. 10-20. Bar diagrams showing quantitative changes in lymph
nodes after immunostimulation. The error bars show the standard
deviations. Significance of pair comparisons in post-test, after global
comparison attaining P < 0.05, is indicated by identical symbols
above related bars. One symbol indicates P < 0.05, two symbols indicate P < 0.01, and three symbols indicate P < 0.001.
Fig. 10. Diagrammatic representation of the average volume of
lymph node analyzed at the light microscopic level in each group of
animals, with a fixed proportion of sections selected for each lymph
node. Thus, the volumes shown give a n approximate estimate of the
relative volumes of the actual lymph nodes. In addition to the total
volumes, the volumes for cortex and medulla are also indicated. Note
the minimal change in the volume of medulla analyzed in all the
groups. There are no significant differences.
Fig. 12. Showing the average total length of nerves measured per
lymph node at the light microscopic level in all animal groups.
Nerve Length/Vohe for all Groups
400F
3001
Ratio CwCx/Medulla
:i- i i
8
0
.r
m
Iy
0
5
0Control
10d. stim.
Control
10d. stim. 27d. stim. 4mo. stim. 4mo. cont
Groups
m27d. stim.
memo.
stim.
m4rno. cont.
4
3
2
Fig. 13. Light microscopic density of nerves in the various compartments of axillary lymph nodes in all animal groups. Abbreviations:
NN, = nerve density per total lymph node volume; NN, = nerve
density related to volume of cortex; N/V, = density of innervation in
medulla. There is a distinct increase in the innervation for the total
lymph node and the medulla 4 months after immunostimulation,
whereas the density of innervation in the cortex is unchanged over all
groups.
1
0
Groups
Fig. 11. Diagram showing the changes in the average ratio between
cortex and medulla for each group at the light microscopic level. Note
the increase up to the 27 day immunostimulated group and the distinct decline to the 4 month immunostimulated group. The time
course of these volume changes parallels the time course of changes in
antibody production. However, it should be noted that the antibody
production 4 months after immunostimulation is still considerably
elevated over control levels.
pair difference between the 4 month stimulated and 4
month control group attains P < 0.01 and the difference between the 4 month stimulated and 10 days stimulated groups is also significant (P < 0.05). Even when
related to the length of nerves in the medulla, the increase in branching remains significant (Fig. 15) with
P = 0.032, but no single pair difference attains significance. A slightly higher significance is obtained for
axonal branching in the total lymph node (branches
per unit length nerve: P = 0.0252), since the values for
the cortex serve to amplify the effects (not shown).
Electron microscopy
The areas of the lymph nodes analyzed at the electron microscopical level are shown in Figure 16 for all
groups. Since the aim was to register a certain minimum number of varicosities per animal, the area of
lymph node analyzed roughly reflects the density of
varicosities. The relationship is not quite precise, since
the independent activity of four observers, each completing the evaluation of the section containing the
varicosity number to be attained for the individual
quota, resulted in variably higher total counts. The
analyzed total areas differ significantly in global com-
221
LYMPH NODE IMMUNOSTIMULATION
Nerve Branches per Unit Volume Medulla
EM Areas Analyzed in Groups
/Total
Control
“E
E
T
l.5i
L
I
“ I
ln
1.0
T
area
Medulla
N
E
u 4 m o stim
€
3
.-C
m
E 2
a
1
0
Groups
Control
Fig. 14. Showing the frequency of nerve branching per unit volume
medulla at the light microscopic level for all groups. There is a
marked increase in branching 4 months after immunostimulation.
5r
Nerve Branching in all Groups
-r
1
CJ control
1Od. stim.
27d. stim. 4mo. stim.
4mo. cont.
Groups
Fig. 16. Graphic presentation of the average total area of ultrathin
sections analyzed for each group, together with the distribution of the
area into cortex and medulla.
Nerves in Medulla
27d. stim.
L 3
01
a
111
2
0
2
C
e
m i
0
Groups
Fig. 15. Nerve branching related to unit length of nerve in medulla
at the light microscopic level for all groups. Even when related to unit
length of nerve the global comparison of the groups attains the significance level, but none of the pair comparisons is significant.
parison (P = 0.03181, with no significant pair differences. The areas of medulla analyzed differ more distinctly (P = 0.00047) with a significant pair difference
only between the control group and the 4 month immunostimulated group, whereas the cortical areas do
not differ significantly. At the electron microscopical
level, nerves are found almost exclusively in the medulla. The few instances of axonal profiles located in
the cortex are always close to the border of the medulla,
in grid squares containing both cortex and medulla. In
the following, the innervation is thus only related to
the area of medulla analyzed.
The number of nerve sections ( = bundles of axons)
per unit area medulla is shown in Figure 17. Global
comparison reveals a highly significant difference (P =
0.00172), with a significant increase between the control and 4 month immunostimulated groups. Significance is not attained between the 4 month control
group and the 4 month immunostimulated group,
which may be attributed to the small size of the 4
month control group. The number of axonal sections
per unit area is shown in Figure 18. The results closely
Groups
Fig. 17. Showing the average density of nerves in the medulla of the
lymph nodes of all animal groups at the electron microscopic level.
The relations between the groups should be compared with those presented in Figure 13, derived at the light microscopic level, which are
almost identical. Note that a “nerve” contains a variable number of
axons, and that the number of nerves to be found a t the light microscopic level is considerably fewer than those identifiable at the electron microscopic level.
resemble those for nerve sections, the global difference
between groups giving a value of P = 0.0134. Again
only the 4 month immunostimulated and control
groups differ significantly. This is reflected in the almost constant number of axons per nerve over all
groups (maximum = 4.5; minimum = 3.21).
Nerves closely applied to blood vessels, i.e., with no
intervening cell processes, were classified as vessel
nerves. Figure 19 shows the incidence of these nerves
in the medulla for all groups. Global comparison reveals a significant difference between groups (P =
0.016561, the difference being caused by the drastic reduction of such nerves in the 4 month control group
with significant differences between this group and the
4 month and 27 day stimulated groups (P < 0.05).
222
G.E.K. NOVOTNY ET AI,.
Control
d
m27d. stim.
a
m4mo. stim.
+++
T
>
400
0
$
300
9)
0
a 2 7 d . stim.
CzJ4mo. stim.
9)
.-0L
vl
C
E
0
X
200
100
0
Groups
Groups
Fig. 18. Graphic demonstration of the changes in the density of
axons at the electron microscopic level in the medulla of the various
groups. The increase in the number of axons after immunostimulation
is not quite as pronounced as that of nerves, indicating that the average number of axons per nerve is slightly reduced. For the longterm control group, there is a slight increase in the number of axons
per nerve, when compared with the younger control group.
Fig. 20. Diagram showing the incidence of only partially ensheathed
axonal sections (varicosities) for all groups of animals. The group of
animals 4 months after immunostimulation exhibits a particularly
pronounced increase of such axonal sections, when compared with the
short-term control group.
DISCUSSION
d Nerves of Medulh
V
Control
T
m27d. stim.
+
a 4 m o . stim.
Groups
Fig. 19. Showing the average density of nerves accompanying blood
vessels, and not separated from these by intervening cells processes,
in the medulla of the lymph nodes of the various animal groups. Note
that there is not such a n increase after immunostimulation, as was
found for nerves not associated with blood vessels. However, the longterm control animals possess very few vessel nerves, so that the maintenance of this category of nerve in the 4 month immunostimulated
group may reflect some activation of this class of nerves.
The functionally relevant aspect of lymph node innervation may be presumed to be reflected by the nonensheathed axonal profiles, which we have defined as
“varicosities,” irrespective of their content of synaptic
vesicles. Figure 20 illustrates the incidence of varicosities in the medulla for all groups. Global comparison
shows significant differences tP = 0.00064), with a
highly significant difference between the 4 month immunostimulated and control groups (P < 0.001). The
remaining differences do not attain the significance
lev 4.
The main finding in this study is an increase in the
density of innervation in the medulla of axillary lymph
nodes 4 months after immunostimulation, at both the
light and the electron microscopical level. However,
the total volumes analyzed at the light microscopical
level, and hence also the volumes of the lymph nodes,
are not identical; they are less in the 4 month group,
although this difference is not statistically significant.
The question therefore arises, whether the increased
density of innervation is genuine, or a spurious effect,
due to the reduction in volume, with the innervation
simply maintained at its absolute value. Figure 10
shows the volumes of cortex and medulla scanned in all
groups. It may be seen that the actually scanned volumes of the medulla, where the changes in density of
innervation are most pronounced, are almost identical
between the groups, with a slight tendency to the
greatest volume in the 4 month immunostimulated
group. At the ultrastructural level the areas analyzed
differ significantly. However, this difference is simply
a reflection of the increase in the number of varicosities, since this was the parameter determining the termination of the registration. It is thus evident that
alterations in volume cannot be responsible for the increase in the density of innervation that is found in the
medulla.
The density of innervation of axillary lymph nodes in
the rat is increased in animals aged over 2 years (Novotny et al., 1993). Our original control group was
matched to the 27 day survival group, because the
greatest effect on the innervation was anticipated at
the height of antibody production. The 4 month survival animals are thus 3 months older than the controls. For this reason, a further control group, comprising only four animals, was introduced to match the
long-term survival group in age. It is quite evident that
this control group shows not the slightest tendency to
an increase in the density of innervation. This finding
LYMPH NODE IMMUNOSTIMULATION
agrees well with the results of an unpublished pilot
study (Krucken), where the young adult group of animals, aged approximately 4 months, also exhibited a
slightly lower density of innervation than a juvenile
group, although the aged animals (> 2 years) had the
highest density of innervation. Together with the
present results, this suggests that the density of innervation does not change appreciably during adolescence
and young adulthood and only increases distinctly in
old age. Interestingly, the values for innervation density, obtained by identical methods, are higher in the
animals from a sterile environment than from normally reared animals (Novotny et al., 1993). However,
comparison is difficult, because the ages are not identical, and we are currently engaged in determining the
precise time course of age changes.
A further problem of this study is the inability to
conceal from the observers, from which group of animals each lymph node derived. At the light microscopic
level, it is impossible to reliably identify nerves without actually focusing through a suspected structure in
the microscope. Identification of all nerves on photographs is impossible. It would have been conceivable to
let an observer evaluate a microscopic field chosen by
another person, so that the whole section cannot be
viewed, but this would have caused a drastic prolongment of an already dreary procedure. Also, the question of selection of a field of view would have arisen.
Such a course is not practicable. At the electron microscopical level it would have been theoretically possible
to take random photographs to be evaluated by another
observer. However, the density of innervation is so low
that many thousands of photographs would have to
have been made, the vast majority of them without
nerves. The time consumption and costs would have
been prohibitive. In our view, there was no alternative
to the procedure we have adopted, even if this entails
the danger of introducing bias into the evaluation (for
precautions see the Materials and Methods section).
Additionally, in the ultrastructural investigation four
observers obtained their results independently, with no
knowledge of possible trends. Finally, the actual results obtained do not conform to those anticipated. As
evidenced by the design of this experiment, the greatest effect was expected to be found a t the peak of antibody production, i.e., in the 27 day immunostimulated
group. Maybe naively, anticipated changes were expected to revert to control values in the 4 month immunostimulated group. These expectations are clearly
negated by the results.
In addition, there is very good agreement between
the light microscopic and the electron microscopic analysis (compare Figs. 13 and 17).The ratio of innervation
density for the medulla between the controls and the 4
month stimulated group is 1:l.g for the light microscopic analysis and 1:2.1 at the electron microscopic
level. It is hard to conceive the independent bias of four
observers, without knowledge of the trends of the evaluation, producing such consistent results.
We therefore conclude that the immunostimulation
has produced a genuine increase in the density of innervation of the medulla of axillary lymph nodes 4
months after immunostimulation. The electron microscopical observations demonstrate that the increase in
the density of innervation is accompanied by an
223
equally pronounced increase in the number of varicosities, which argues that the observed changes are functionally relevant.
The time-course of the changes found is an important
factor. Our immunization schedule was designed to
produce a response maximum a t 27 days, with a gradual decline in antibody production to the 4 month immunostimulated group. If the innervation of the axillary lymph nodes serves to stimulate antibody
production (which was our original assumption), then
one would anticipate a pronounced increase in the innervation before the peak of antibody production is attained (i.e., during the phase of greatest acceleration of
antibody production), or at least in synchrony with antibody production. However, the greatest change was
actually observed after the peak of antibody production
was passed. This means that the morphologically visible changes in the innervation lag behind the immunological events. We thus consider it to be unlikely
that activation of the direct innervation of axillary
lymph nodes is concerned with the mounting of an antigenic response in these nodes, unless the morphological changes take a long time to become visible. The
first reports of neuronal plasticity suggest such a long
time course (3-6 months) before morphological alterations can be registered (Raisman, 1969), but more recent studies have demonstrated recognizable changes
after intervals of only 4-8 days (Taylor et al. 1988;
Alkon et al. 1990). We are therefore inclined to believe
that the lack of a pronounced effect 27 days after immunostimulation reflects the late response of the peripheral nervous system. Nevertheless, groups between 27 days and 4 months, as well as periods longer
than 4 months, would have been of great interest.
If the innervation of lymph nodes is not concerned
with the stimulation of antigen production, the question arises as to what function may be attributed to it.
The data in the literature are seemingly in conflict,
which may be illustrated with just two examples. Stimulation of the sympathetic outflow leads to increased
outflow of lymphocytes from popliteal lymph nodes
(McHale and Thornbury, 1990), which may be classified as activation of the immune system. On the other
hand, sympathectomy has been reported to enhance
immune responses (Besedovsky et al., 19881, which
may be interpreted as indicating an inhibitory action of
the nervous system on the immune system. However,
such conflicting reports may be reconciled. The first
instance is an example of cellular mobilization,
whereas the second example is of an inhibitory effect
on antibody producing cells, i.e., the humoral immune
response mediated by plasma cells. Seen in this light,
the increase in innervation, which we have found
within the plasma cell regions of the axillary lymph
nodes, may reflect the activation of a negative feedback circuit concerned with the down-regulation of the
humoral immune response. This hypothesis agrees
well with two of our previous observations. Firstly, we
have consistently observed nerves to be concentrated in
plasma cell areas of lymph nodes, and ultrastructural
axonal varicosities to be associated with reticular cells
and plasma cells (Novotny and Niche, 1986; Novotny,
1988; Novotny et al., 1993). Secondly, there is a
marked increase in the density of innervation of axillary lymph nodes in aged rats (Novotny et al., 19931,
224
G.E.K. NOVOTNY ET AL.
and the reduced reactivity of the immune system in old
age is well known. It thus seems most likely that for
the lymphocytic cell population the innervation of axillary lymph nodes in the rat is primarily concerned
with plasma cells and not with T lymphocytes. In this
connection it is of interest that B cells have been shown
to possess a higher density of nerve growth factor receptors (NGF-R) than T cells, and to be stimulated to
IgM production via these receptors (Otten et al., 1989).
Inflammatory stimuli significantly increase NGF production, and denervation has been shown to be accompanied by an increase in NGF levels, which are decreased by renewed innervation (Weskamp and Otten,
1987). It could thus be conceived that the innervation
of lymph nodes is concerned in such a NGF mediated
regulation of antibody production. However, the time
course of events reported by Weskamp and Otten is in
the range of hours, as compared to weeks for the events
described here.
A very high proportion of axonal varicosities is associated with fibroblastic reticular cells of the medulla
(Novotny, 1988). In the medulla of lymph nodes these
cells also possess NGF receptors (Thompson et al.,
1989). It is thus possible that the innervation may be
concerned with regulative mechanisms via these cells.
It is thus clear that much work remains to be done to
elucidate the precise mechanisms by which the increase in innervation is attained, and to determine the
functional significance of such changes.
ACKNOWLEDGMENTS
The authors express their debt to Dr. A. Treiber for
helpful discussions of immunization schedules, to Ms.
A. Spitz for most exceptionally competent breeding,
maintenance, and handling of the sterile animals, and
to Ms. I. Mertens for expert histological and technical
assistance. This work was supported by the Deutsche
Forschungsgemeinschaft.
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