Mechanisms of Ageing and Development
94 (1997) 165 – 175

Alterations in sympathetic innervation of thymus
and spleen in aged mice
Kelley S. Madden *, Denise L. Bellinger, Suzanne Y. Felten,
Eric Snyder, Mary E. Maida, David L. Felten
Department of Neurobiology and Anatomy and the Center for Psychoneuroimmunology Research,
Uni6ersity of Rochester School of Medicine and Dentistry, 601 Elmwood A6enue, Rochester,
NY 14642, USA
Received 7 November 1996; received in revised form 14 December 1996; accepted 14 December 1996

Abstract
Aging is associated with reduced immune reactivity, contributing to increased rates of
infectious disease and cancer in old age. We have begun to assess the potential for
sympathetic nervous system involvement in age-related immune dysfunction by characterizing sympathetic noradrenergic (NA) innervation in lymphoid organs in old animals. In the
present study noradrenergic innervation of spleen and thymus was examined histologically
and neurochemically in 2-, 12- and 24-month old BALB/c mice. In the thymus of 2-month
old animals, NA nerve fibers were found in the subcapsular, cortical, and cortico – medullary
regions associated with blood vessels and septa; occasional branches from these nerve fibers
entered the parenchyma. With increasing age and thymic involution, NA nerve fibers
increased in density; by 24 months of age, dense plexuses were compacted among septa and
blood vessels, and numerous linear, varicose nerve fibers were observed branching into the
parenchyma. Thymic norepinephrine (NE) concentration (per mg wet weight) increased
approximately 4-fold in 12-month old animals and 15-fold in 24-month old animals. Taking
the reduced thymus weight into account, total thymic NE at 12- and 24-month of age was
equivalent to total thymic NE at 2-month of age, suggesting that NA innervation is
maintained as the thymus involutes. In the spleen from 2-month old animals, NA innervation entered the white pulp with the central artery to innervate the periarteriolar lymphatic
Abbre6iations: NA, noradrenergic; NE, norepinephrine; SNS, sympathetic nervous system; TH,
tyrosine hydroxylase.
* Corresponding author.
0047-6374/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved.
PII S 0 0 4 7 - 6 3 7 4 ( 9 6 ) 0 1 8 5 8 - 1

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sheath and the marginal zone. At 12-month of age, histologically and neurochemically there
was no change in splenic NA innervation. By 24-month of age, NE was increased significantly, independent of changes in spleen weight. Histologically, increased catecholamine-containing fibers were apparent at 24-month of age, particularly in the parenchyma surrounding
the central artery. The alterations in sympathetic NA innervation of lymphoid organs with
age suggest that the sympathetic nervous system and NE may play a role in age-associated
immune dysregulation. Alternatively, the changes in NA innervation may be secondary to
functional changes within the immune system. © 1997 Elsevier Science Ireland Ltd.
Keywords: Noradrenergic innervation; Spleen; Sympathetic nervous system; Thymus

1. Introduction
We have demonstrated sympathetic noradrenergic (NA) innervation of bone
marrow, thymus, spleen, lymph nodes, and gut-associated lymphoid tissues in
numerous species [1 – 3]. A component of the sympathetic nervous system (SNS),
NA nerve fibers convey signals originating in the brain via the spinal cord to the
periphery. We have previously reported dramatic age-related changes in sympathetic innervation of thymus and spleen in Fischer 344 (F344) rats (reviewed in [4]).
In the rat thymus, sympathetic NA nerve fibers and norepinephrine (NE) levels
increased progressively with age [5]. In contrast, NA innervation in the spleen was
diminished and NE levels were reduced in old animals [6,7]. We were interested in
exploring the functional significance of these age-related changes, particularly in the
thymus. We chose to assess the potential functional significance of NA innervation
in the mouse because of the large body of knowledge concerning thymic T cell
development in this species and the availability of a wide range of cell-specific
antibodies for immunocytochemical staining and flow cytometry. First, we needed
to extend our previous work in young mice and characterize NA innervation of
lymphoid organs in the aged mouse [8–10]. In the present report, we demonstrate
age-related changes in thymic and splenic NA innervation in BALB/c mice histologically and neurochemically.

2. Methods

2.1. Mice
BALB/cJNIA male mice (National Institute on Aging) aged 2 months, 12
months, and 24 months were housed 2 per cage with food and water available at all
times. Animals who showed overt signs of illness, including tumors or
splenomegaly, were not included in the study. This strain of mouse was chosen
because BALB/c male mice have been characterized in our studies of neural–immune interactions. BALB/c mice are known for the predominance of TH2 activity
in certain immune responses. Maximal life span in this strain is 30 months of age.

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2.2. Histochemical techniques
2.2.1. Fluorescence histochemistry for catecholamines
Animals were sacrificed by cervical dislocation. The thymus was rapidly removed and frozen on dry ice. Tissue was stored at − 80°C until sectioning.
Fresh frozen tissue was cut at a thickness of 16 vm in a − 20°C cryostat
and thaw mounted onto glass slides. This technique employs a modification of
the glyoxylic acid condensation method of de la Torre [11]. The slides were
dipped in a phosphate buffer containing 1% glyoxylic acid and 7.5% sucrose (pH
7.4), and dried with blow dryers in a cool stream of air for 15 min. Tissue
sections were covered with mineral oil, placed in an oven at 37°C for 2.5 min,
and cover slipped with fresh oil. Fluorescence was examined and photographed
using a Nikon fluorescence microscope equipped with epi-illumination accessories.
2.2.2. Immunocytochemistry (ICC) for tyrosine hydroxylase (TH)
Mice were anesthetized with Chloropent and perfused transcardially with
4% paraformaldehyde and 0.2% picric acid in phosphate buffered saline
(PBS). The thymus was dissected, postfixed in the perfusion fixative for 24 h at
4°C, and transferred into 0.15 M phosphate buffer (pH 7.4) with 10, 20 and
30% sucrose for an additional 24 h at 4°C for each sucrose concentration.
Samples were frozen on dry ice, and stored at − 80°C. Tissue was mounted onto
the freezing chuck of a sliding microtome, and 40 vm sections were mounted
on gelatin-coated slides. All steps were carried out in 0.15 M phosphate buffer
at 25°C using gentle agitation, unless otherwise indicated. Sections were rinsed in
buffer and incubated in 10% normal goat serum (NGS). Rabbit anti-TH
antibody (Chemicon) was diluted 1:100 in 0.15 M phosphate buffer containing 0.4% Triton X-100 and 0.25% bovine serum albumin. Sections were incubated in the primary antibody for 24 h at 4°C. On day 2, sections were rinsed
6 ×10 min in buffer, incubated for 30 min in 10% NGS, and then incubated
in the secondary antibody (goat-anti-rabbit IgG, 1:8000; Vector) for 90 min.
Sections were rinsed 4× 10 min in buffer and incubated in 2.5% methanol
with 5% hydrogen peroxide for 30 min to remove endogenous peroxidase activity. Following 6 ×10 min rinses, sections were incubated in avidin–biotin–
peroxidase complex (ABC, 1:8000; Vector Elite kit) for 90 min. Sections were
rinsed 4 × 10 min in buffer, followed by 2 × 10 min in 0.05 M sodium acetate and 0.03 M imidazole buffer containing 0.25 g/100 ml nickel (II) sulfate,
0.04 g/100 ml 3,3%-diaminobenzidine (DAB), and 0.005% hydrogen peroxide for
15 – 20 min. Nickel intensification changes the normal brown DAB reaction
product to a blue – black color. All sections then were rinsed 2 × 10 min in
acetate – imidazole buffer, followed by 4 × 10 min rinses in phosphate buffer.
Sections were counter-stained with 0.6% thionin, rinsed, dried, dehydrated
through a series of graded ethanols, cleared in xylene, and coverslipped in Permount.

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2.3. Neurochemical measures
Thymuses were dissected free of fat, weighed, frozen on dry ice, and stored at
−80°C. A sonic dismembrator was used to homogenize the tissue in 0.1 M
perchloric acid containing the internal standard 3,4-dihydroxybenzylamine (DHBA;
0.25 vM), 1 mM EDTA, and 50 mM metabisulfite. After centrifugation at
13 000×g for 10 min, supernatants were collected for aluminum oxide extraction.
Samples from all ages were extracted at the same time. The alumina-extracted
samples were placed randomly in an autosampler, and neurochemical analysis was
performed using high performance liquid chromatography with electrochemical
detection (LCEC) as described previously [5].

2.4. Statistics
Group means were compared by one-way analysis of variance. Significant main
effects (P B .05) were analyzed post-hoc by Fisher’s least significant difference
(LSD) test.

3. Results

3.1. Age-related changes in sympathetic NA inner6ation of the mouse thymus
Fluorescence histochemistry to detect catecholamine-containing nerve fibers provided initial evidence for increased NA innervation in the aged murine thymus.
Scattered NA nerve fibers in the thymus of 2-month old BALB/c male mice were
observed in the capsular region with very few fibers penetrating into the cortex (Fig.
1A). By 12 and 24 months of age, fluorescent NA nerve fibers increased in
frequency in the capsule and extended into the center of the thymus as dense,
tangled nerve fibers (Fig. 1B and C). In the 12-month and 24-month old animals,
NA nerve fibers were often associated with yellow autofluorescent cells, most likely
macrophages, which increase in number and intensity as the thymus involutes. In
some sections, the density of NA innervation in 12-month old animals appeared
equivalent to that of 24-month old animals (compare 1B–C). However, high
density NA innervation was not maintained in all sections obtained from 12-month
old thymuses, compared to the high density of catecholamine-containing fibers
observed in the majority of sections from 24-month old thymuses. Thus, fluorescence histochemistry for catecholamines indicated that thymic innervation in 12month old animals was greater than that of 2-month old animals, but thymic
innervation in 12-month old animals was less than that of 24-month old animals.
Immunocytochemistry for tyrosine hydroxylase (TH), the rate-limiting enzyme in
NE synthesis, was used with thionin counter-staining to differentiate the densely
packed cortical region from the more loosely packed medullary region of the
thymus. At 2 months of age, TH + nerve fibers were associated with septa and
blood vessels coursing through the cortex, the cortico-medullary junction, and the

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Fig. 1. Fluorescence histochemistry for catecholamine-containing fibers in BALB/c mouse thymus. At 2
months (A), a few fluorescent profiles are present in the capsule (arrows) with a paucity of innervation
present in the cortex (× 100). By 12 months (B), and 24 months (C) of age, fluorescent nerve fibers
become more apparent extending from the capsule toward the center of the thymus ( ×100). CA denotes
capsule. Note increase in yellow autofluorescent cells with age.

medulla (Fig. 2A and B). At this age, TH+ nerve fibers surrounded most blood
vessels and septa, but only sparse innervation was observed in the parenchyma of
the cortex. By 12 months of age, increased thymic NA innervation was observed
associated with septa and blood vessels as well as increased numbers of TH+ nerve
fibers entering the parenchyma (Fig. 2C and 2D). By 24 months of age, the thymus
consisted primarily of the medullary region with shrunken cortical regions (Fig.
2E). At this age, compacted networks of TH+ nerve fibers were present in the
capsule and surrounding septa and blood vessels (Fig. 2E and F). Numerous TH+
linear profiles were readily apparent coursing through fields of thymocytes.
Neurochemical analysis by LCEC confirmed the increased density of innervation
apparent histologically. Thymic NE concentration (per mg wet weight) increased
approximately 4-fold in 12-month old animals and 15-fold in 24-month old animals
compared to 2-month old animals (Fig. 3A). When the age-associated decrease in
thymic weight was taken into account, total NE per thymus did not differ
significantly between the three age groups (Fig. 3B and C), suggesting that NA
innervation is maintained as the thymus involutes.

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Fig. 2. Immunocytochemistry for TH in aging BALB/c mouse thymus. (A,B) In 2-month old animals,
NA nerve plexuses surround blood vessels and intralobular septa in cortex (arrowheads) and medulla
(small arrows in B) with occasional branching into parenchymal regions (arrow in B). (C,D) At 12
months of age, septal/vessel innervation becomes more prominent (arrowheads). Note branching into
parenchymal regions (arrow in D). (E,F) By 24 months of age, the involuted thymus contains numerous
tangled TH+ plexuses surrounding septa and vessels (arrowheads) with long branches extending into
the parenchyma (arrows). (A,C,E × 100; B,D,F × 200)

3.2. Age-related changes in sympathetic NA inner6ation of the spleen
At 2-month of age, fluorescent histochemistry of the spleen revealed NA innervation associated with the central artery and its branches as fluorescent profiles
scattered throughout the white pulp surrounding the artery (Fig. 4A). At 12 months
of age, no change in catecholamine-containing fibers in the parenchyma was
observed but an increase in NA nerve fibers surrounding the central artery was

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171

apparent (Fig. 4B). This finding was present in several animals examined, but
remains to be quantified by morphometric analysis. By 24 months of age, NA nerve
fibers in the white pulp of the spleen appeared to increase (Fig. 4C). In Fig. 4C, NA
innervation immediately surrounding the central artery appeared reduced compared
to 2- and 12-month old animals, although this was not found in all 24-month old
animals. Both NE concentration (NE/mg wet weight) and total NE were significantly increased by 24 months of age, with no significant change in spleen weight
(Fig. 5).

Fig. 3. Age-associated changes in NE concentration in BALB/c thymus. (A) Thymic NE concentration
increases with age (ANOVA, P50.0001). (B) Total NE per thymus does not significantly differ between
2-, 12- and 24-month old mice (ANOVA, P= 0.1). (C) Thymic weight decreases with age (ANOVA,
P5 0.0001). 2 months, n= 14; 12 months, n =15; 24 months, n =10. * Indicates significantly different
from 2-month old by post-hoc analysis with Fisher’s LSD test (P B0.05).

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Fig. 4. Fluorescence histochemistry for catecholamines in aging BALB/c mouse spleen. Fluorescent NA
nerve fibers are associated with the central artery (arrowheads) and are scattered throughout the
surrounding parenchymal regions (arrows). No obvious difference in NA innervation is observed
between 2 and 12 months of age (A,B). By 24 months of age, a marked increase in NA innervation of
the parenchyma surrounding the central artery is observed (C). (A – C, ×200)

4. Discussion
Age-related alterations in sympathetic NA innervation and NE concentration in
thymus and spleen of BALB/c mice may contribute to age-related immune dysfunction. The dramatic increase in NA innervation in the involuting thymus has been
previously reported by our laboratory in F344 rats [5]. This increase manifests itself
as an increase in the density of NA nerves associated with septa and blood vessels
and greater penetration of parenchymal regions as early as 12 month of age, and is
quite extensive in the 24-month old thymus. The observation that NE concentration
increased with age, but total thymic NE was not altered suggests that thymic NA
innervation is maintained as the thymic involutes.
The precise anatomical definition of the target cell(s) for thymic NA innervation
are currently under investigation using double-label immunocytochemistry and
electron microscopy. Sympathetic NA nerve fibers communicate with target cells

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Fig. 5. Age-associated changes in NE levels in BALB/c mouse spleen. (A,B) Splenic NE concentration
(ANOVA, P = 0.008) and total splenic NE (ANOVA, P= 0.02) increases at 24 months of age. (C)
Splenic weight is unchanged with age (ANOVA, P =0.3472). 2 months, n =7; 12 months, n =7; 24
months, n= 6. Asterisk indicates significantly different from 2-month old by post-hoc analysis with
Fisher’s LSD test (P B0.05).

bearing cell surface adrenergic receptors (adrenoceptors) via the neurotransmitter
NE. Mature T and B lymphocytes, macrophages, and other cells of the immune
system possess h- and i-adrenergic adrenoceptors [12–17]. Unfractionated thymocytes express very low levels of i-adrenoceptors, and i-adrenoceptor expression
may be up-regulated with thymocyte maturation ([15,17], Madden, unpublished
data). For example, Fuchs et al. reported that enriched populations of mature
thymocytes (cortisone-resistant or peanut non-agglutinating thymocytes) expressed
equivalent numbers of i-adrenoceptors as mature T cells in the periphery [15].
Using in vitro autoradiography, Marchetti et al. demonstrated i-adrenergic receptors localized primarily to the medulla of the rat thymus [16]. Stromal elements in
the thymus, including epithelial cells, macrophages, and dendritic cells may also
express h- or i-adrenoceptors and thus may serve as targets of NA innervation.
The finding that splenic NA innervation is enhanced at 24 months of age in the
mouse is directionally opposite to the marked diminution in splenic NA innervation

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in the old rat. C57BL/6 mice also showed no reduction in splenic NE levels through
24 months of age (data not shown), suggesting that this finding is not limited to
BALB/c mice. It is possible that mice older than the ones used in this study (\ 24
months of age) may show reduced NA innervation. In this study, a reduction in
NA innervation surrounding the central artery in 24-month old animals suggests
that neuronal loss may be occurring at this age. Alternatively, the differences in
age-related changes in NA innervation may reflect microenvironmental differences
between mouse and rat spleen, which may lead to ‘protection’ of NA nerve fibers
in the aged mouse. More detailed morphometric analysis and double-labeling of
TH + NA nerve fibers and specific lymphocyte populations in the aged mouse
spleen by ICC may help clarify any differences between rat and mouse splenic NA
innervation.
The age-related increase in sympathetic NA innervation in mouse thymus and
spleen suggests that NE may play a progressively greater role in signaling potential
targets in spleen and in thymus as the animal ages. This hypothesis predicts that
pharmacological manipulation of the SNS may have a more profound effect on
immune reactivity in old animals compared to young animals. We found that
sympathectomy-induced alterations in immune reactivity were more apparent in old
rats [18]. In preliminary studies, chronic i- or h-blockade had no effect on
thymocyte differentiation in 2-month or 12-month old mice, but had significant
effects on thymocyte CD4/CD8 co-expression and proliferation in 24-month old
animals (data not shown). Age-related changes in adrenoceptor expression and
signaling in selected cell populations need to be investigated in both spleen and
thymus to understand the potential for immunomodulation in aged animals.
Together, these results suggest that pharmacological manipulation of the SNS may
enhance immune reactivity to pathogens in the aging host and possibly under
conditions of immunosuppression in younger hosts.

Acknowledgements
This work was supported by grants from the Rochester Area Pepper Center on
Aging, the Markey Charitable Trust, and NIH Grant MH42076. We thank Thanh
Nguyen, Don Henderson and Charles Richardson for excellent technical assistance.

References
[1] D.L. Felten, S.Y. Felten, D.L. Bellinger et al., Noradrenergic sympathetic neural interactions with
the immune system: structure and function. Imm. Re6., 100 (1987) 225 – 260.
[2] S.Y. Felten, D.L. Felten, D.L. Bellinger and J.A. Olschowka, Noradrenergic and peptidergic
innervation of lymphoid organs. In J.E. Blalock (ed.), Chemical Immunology: Neuroimmunoendocrinology, S. Karger, Basel, 1992, pp. 25 – 48.
[3] S.Y. Felten and D.L. Felten, Innervation of lymphoid tissue. In R. Ader, D.L. Felten and N. Cohen
(eds.), Psychoneuroimmunology-II, Academic Press, San Diego, 1991, pp. 27 – 68.

K.S. Madden et al. / Mechanisms of Ageing and De6elopment 94 (1997) 165–175

175

[4] K.D. Ackerman, D.L. Bellinger, S.Y. Felten and D.L. Felten, Ontogeny and senescence of
noradrenergic innervation of the rodent thymus and spleen. In R. Ader, D.L. Felten and N. Cohen
(eds.), Psychoneuroimmunology-II, Academic Press, San Diego, 1991, pp. 71 – 125.
[5] D.L. Bellinger, S.Y. Felten and D.L. Felten, Maintenance of noradrenergic sympathetic innervation
in the involuted thymus of the aged Fischer 344 rat. Brain Beha6. Immun., 2 (1988) 133 – 150.
[6] D.L. Bellinger, S.Y. Felten, T.J. Collier and D.L. Felten, Noradrenergic sympathetic innervation of
the spleen: IV. Morphometric analysis in adult and aged F344 rats. J. Neurosci. Res., 18 (1987)
55–63.
[7] D.L. Bellinger, K.D. Ackerman, S.Y. Felten and D.L. Felten, A longitudinal study of age-related
loss of noradrenergic nerves and lymphoid cells in the rat spleen. Exp. Neurol., 116 (1992) 295 – 311.
[8] J.M. Williams and D.L. Felten, Sympathetic innervation of murine thymus and spleen: a comparative histofluorescence study. Anat. Rec., 199 (1981) 531 – 542.
[9] J.M. Williams, R.G. Peterson, P.A. Shea, J.F. Schmedtje, D.C. Bauer and D.L. Felten, Sympathetic
innervation of murine thymus and spleen: evidence for a functional link between the nervous and
immune systems. Brain Res. Bull., 6 (1981) 83 – 94.
[10] D.L. Felten, S.Y. Felten, S.L. Carlson, J.A. Olschowka and S. Livnat, Noradrenergic and
peptidergic innervation of lymphoid tissue. J. Immunol., 135 (1985) 755s – 765s.
[11] J.C. de la Torre, Standardization of the sucrose-potassium phosphate-glyoxylic acid histofluorescence method for tissue monoamines. Neurosci. Lett., 17 (1980) 339 – 340.
[12] R.N. Spengler, R.M. Allen, D.G. Remick, R.M. Strieter and S.L. Kunkel, Stimulation of alphaadrenergic receptor augments the production of macrophage-derived tumor necrosis factor. J.
Immunol., 145 (1990) 1430–1434.
[13] C.K. Abrass, S.W. O’Connor, P.J. Scarpace and I.B. Abrass, Characterization of the beta-adrenergic receptor of the rat peritoneal macrophage. J. Immunol., 135 (1985) 1338 – 1341.
[14] A.M. Genaro and E. Borda, Alloimmunization-induced changes in b-adrenoceptor expression and
cAMP on B lymphocytes. Immunopharmacology, 18 (1989) 63 – 70.
[15] B.A. Fuchs, J.W. Albright and J.F. Albright, b-adrenergic receptors on murine lymphocytes:
density varies with cell maturity and lymphocyte subtype and is decreased after antigen administration. Cell. Immunol., 114 (1988) 231 – 245.
[16] B. Marchetti, M.C. Morale and G. Pelletier, Sympathetic nervous system control of rat thymus
gland maturation: autoradiographic localization of the i2-adrenergic receptor in the thymus and
presence of sexual dimorphism during ontogeny. Prog. Neuro. Endocrin. Immunol., 3 (1990)
103–115.
[17] T. Radojcic, S. Baird, D. Darko, D. Smith and K. Bulloch, Changes in b-adrenergic receptor
distribution on immunocytes during differentiation: an analysis of T cells and macrophages. J.
Neurosci. Res., 30 (1991) 328–335.
[18] K.S. Madden, S.Y. Felten, D.L. Felten and D.L. Bellinger, Sympathetic nervous system-immune
system interactions in young and old Fischer 344 rats. Ann. N.Y. Acad. Sci., 771 (1995) 523 – 534.