Mechanisms of Ageing and Development
96 (1997) 59 – 73

Premature thymic involution, observed at the
ultrastructural level, in two lineages of
human-SOD-1 transgenic mice
B. Nabarra a,*, M. Casanova b, D. Paris b, E. Paly b, K. Toyoma b,
I. Ceballos b, J. London b
a
b

U 345 INSERM, Institut Necker, 156 rue de Vaugirard, 75730 Paris Cedex 15, France
URA 1335 CNRS, Institut Necker, 156 rue de Vaugirard, 75730 Paris Cedex 15, France
Received 7 November 1996

Abstract
The human Cu/Zn superoxide dismutase (hSOD-1) gene, catalyses the dismutation of O2
to H2O2 and O2. It is located on chromosome 21 in q22.1 and is overexpressed in Down’s
syndrome (DS) patients. These patients present various abnormalities including mental
retardation, congenital heart disease, immunological deficits and premature aging. In order
to explore the potential role of SOD-1 overexpression in DS, we have generated two lineages
of transgenic mice for the hSOD-1 gene and studied, at the ultrastructural level, the effect of
hSOD-1 overexpression on the thymic microenvironment. Modification of the cellular
architecture and morphology associated with a lipidic invasion, signs of a premature
involution of the thymus, were observed in both lineages. A rupture of the filamentous
network in the extracellular and probably also in the intracellular matrix was first observed.
These results correlate the thymic alterations vizualized in light microscopy, on the thymus
from DS patients, and raise the question of the relationship between the SOD-1 overexpression and the different morphological alterations associated with the premature thymic
involution observed in SOD-1 transgenic mice. They suggest that thymic and immunological
impairments present in DS patients may be related to the SOD-1 gene dosage effect. © 1997
Elsevier Science Ireland Ltd.

* Corresponding author. Present address: Clinique Nephrologique, Hopital Necker, 161 rue de Sevres,
´
ˆ
`
75743 Paris Cedex 15, France.
0047-6374/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved.
PII S 0 0 4 7 - 6 3 7 4 ( 9 7 ) 0 1 8 9 2 - 7

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Keywords: hSOD-1 Transgenic mice; Ultrastructural study; Premature thymic involution;
Down’s syndrome

1. Introduction
Thymic damages (morphological, physiological, biochemical) associated with
aging, in human as well as in mice, are gathered under the denomination of
involution. This phenomenon corresponds to a suitable background involving
morphological irreversible alterations of the thymic microenvironment followed by
the general decline of immunocompetent functions. Indeed, thymic involution
occurs before the decline in the ability of the immune system and may thus be
responsible for this effect. Thus, the thymus has been considered to be implicated
as a ‘clock’ in the aging process of the immune system and of the general biological
functions of the organism [1 –9].
It is well known that oxygen free radicals (OFR) are involved in protein
modifications and membrane alterations [10,11]. Cu/Zn superoxide dismutase
’
(SOD-1), which catalyses the dismutation of superoxide radicals (O2−) into oxygen
and hydrogen peroxides (H2O2), is a key enzyme of ORF metabolism [12]. SOD-1
’
is also able to catalyse surrogate reactions such as the production of OH− [13] and
the nitrosylation of the tyrosine residues, present in proteins, by peroxynitrite [14].
Several reports have shown that an increase in Cu/Zn SOD activity may cause
oxidative damage [15,16].
Down’s syndrome (DS), the most frequent chromosomal abnormality in man,
results from the presence of three copies of chromosome 21. The SOD-1 gene
located in 21q22.1 is overexpressed by a factor of 1.5 in DS patients [17,18]. Apart
from symptoms including impairment in brain function and skeletal abnormalities,
DS patients show many signs of premature aging [19–23] including thymic premature involution observed in light microscopy [24] and various types of impairment
of T and B cell distribution and functions [25–28].
In an effort to explore the role of SOD-1 overexpression in DS patients,
transgenic mice for the human Cu/Zn SOD gene (hSOD-1) have been bred in
several laboratories [29 – 33] and analyzed for physiological consequences [34–37].
However, the general immunological status of these mice has not been investigated
except in two recent studies on some immunological aspects of the thymus and
bone marrow [38,39].
The primordial role of the thymus in the differentiation selection and maturation
of T cells and the necessity of an intact thymic microenvironment for the acquisition and the maintenance of immune functions [40–43] is well documented. Thus,
it appeared interesting to perform thymic morphological studies on these hSOD-1
transgenic mice, as a potential model of DS immunodeficiency.
Two different transgenic lines for the hSOD-1 gene have been bred in our
laboratory, one on the hybrid C57/B6-DBA/2 background (B6/D2) [30,31] and
another on the inbred FVB/N background [32,33].

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The thymic aspect of these hSOD-1 transgenic mice has been studied by
electron microscopy, the best technique for a true identification of cellular modifications in most tissues. The morphological aspect of the thymus from transgenic mice
as well as controls have been compared at different ages. The controls are either
the murine strains used for transgenesis (C57Bl/6, DBA, B6/D2 and FVB/N)
which have been extensively studied for their thymic microenvironment morphology [44 – 48] or non transgenic mice from the littermates of transgenic hSOD-1
lineages.
In the first hSOD-1 transgenic lineage (PB line) which overexpresses SOD-1 in
the thymus by a factor of 1.5, we have previously described a premature thymic
involution (as early as 7 – 8 months-old) similar in morphological aspect to that
observed in the thymus of control aged mice (18–20 months) [39].
In this report, we described a similar morphology of premature thymic involution
in a second hSOD-1 transgenic lineage (KT line) obtained on a FVB/N background, and which overexpresses SOD-1 by a factor of 5 in blood [49] and by a
factor of 5.8 in the thymus [50].
The general involution phenomenon is similar in both transgenic strains, but
several differences in the onset of the involution process and differences in the
morphological aspects of the thymus in both lines is observed.

2. Materials and methods
The entire hSOD-1 gene under the control of its own promoter was microinjected
into the ovocytes of two murine strains B6/D2 and FVB/N for obtaining hSOD-1
transgenic animals [30 – 33].
The first lineage (named PB line in our nomenclature) has been obtained on a
B6/D2 background and has been described previously [30,31]. The animals were
shown to have integrated only one copy of the transgene and to have overexpressed
the SOD-1 enzyme by a factor of 1.5 in the thymus.
The second lineage (named KT line), was obtained by microinjecting zygotes of
FVB/N mice with a 11.5 kb EcoRI–BamH1 linear fragment of human genomic
DNA containing the entire hSOD-1 gene. The genome of the KT line carries 13/14
copies of the transgene integrated in tandem head to tail at a single autosomal locus
[32]. Overexpression of the SOD-1 was evaluated by enzymatic activity using the
classic test of the inhibition of nitroblue tetrazolium reduction by SOD-1 and
automatically assayed using a Ransod kit (Randox laboratories) and also at the
RNA level. The SOD-1 activity was increased by a factor of 5 in the blood and of
5 – 8 in the thymus as a function of age [49,50].
For the ultrastructural studies, two types of controls have been considered. First,
the B6/D2 and FVB/N strains used as transgenesis background at different ages (15
days, 2, 3, 5, 8, 10, 12, 14, 18, 20, 25, 30 months) and called ‘naive controls’.
Secondly, the thymus of non transgenic mice were obtained from crosses between
heterozygous SOD-1 transgenic mice and B6/D2 or FVB/N mice, these control
being called ‘transgenesis controls’.

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For electron microscopy preparations, a classic technique was used. Immediately
after sampling a first fixation of thymic fragments was performed for 1 h in 2%
glutaraldehyde and phosphate buffer pH 7.4, followed by a postfixation in 2%
osmium tetroxide in the same buffer for 1 h. Dehydration in alcohol series and
embedding in Epon follow the fixation. Ultrathin sections impregnated with uranyl
acetate and lead citrate were observed under a Philips EM300 electron microscope.
For each age, six mice of each strain were studied in the experimental and control
groups. Thymuses from 16 days to 12 months have been used for both transgenic
lines. For each group of mice, and for each age ten blocks of different thymic areas
(cortex and medulla) were analyzed.

3. Experimental results

3.1. Thymic microen6ironment morphology in control mice
The thymic morphology observed either in ‘naive’ control mice (C57/Bl6, DBA2,
B2/D6, FVB/N) or in ‘transgenesis control’ mice (issued from the same littermate
as transgenic animals) was similar according to the age considered.

3.1.1. Young mice
The ultrastructural aspect of the normal murine thymic microenvironment has
been largely described previously [44–48]. The basic structure of the thymus
appeared as a network organization of different stroma cells distributed in two
zones and forming a cellular microenvironment enclosing numerous lymphocytes.
The cortical area contained one type of epithelial cells (Type I) whereas the
medullary area contained three types of epithelial cells morphologically different
(type I, II and III) (Fig. 1aFig. 2a and b). These cells associated with macrophages
(in both zones) and interdigitated cells (in the cortico–medullary junction and in
the medulla) form the thymic microenvironment (Fig. 1bFig. 2c). This organization,
surrounded by fibrous capsula, was maintained by fibrous trabeculae carrying
vessels and innervations.
Some differences in the medullary zone were observed between the two murine
strains used in this work for transgenesis. Thus, FVB/N mice seemed to have less
thymocytes in the medullary zone than other murine strains. This allows a better
observation of the stroma and the identification of an undifferentiated cell which
may be a precursor of the epithelial lineage [48].
3.1.2. In6olution and aged thymuses
A detailed description has been previously reported [8]. Briefly, we observed
progressive lesions beginning at about 10–12 months characterized by a vacuolisation of a few epithelial cells, scattered in normal zones, which progressively formed
large altered zones infiltrated by lipofuscin pigment and small lipidic globules. Then
the number of thymocytes decreased and the architecture of the organ was altered
with the loss of separation between the cortical and medullary zones. The whole

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microenvironment appeared disorganized. Many cells lost their morphological
characteristics and appeared as undifferentiated cells, with rupture of plasma
membranes. They appeared to form a large clear cytoplasmic syncytium, containing

Fig. 1. Control: thymic cortical microenvironment in 3 month old normal mouse. (a) Type I epithelial
cell showing its morphological characteristics (arrow heads: few ‘clear intracytoplasmic vacuoles’
containing dense granulations) surrounded by numerous lymphocytes. (b) Macrophage with numerous
dense, homogeneous primary and secondary lysosomes. E: type I epithelial cell, L: lymphocyte. (a)
× 8000; (b) × 15 000.

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Fig. 2. Control: thymic medullary microenvironment in 5 month-old normal mouse. (a) Type II
epithelial cell with its morphological characteristic: an intracytoplasmic ‘alveolar’ network (arrow heads).
(b) Type III epithelial cell showing a large intracytoplasmic, ciliated cavity (arrows heads). (c)
Interdigitated cell with clear cytoplasm and numerous compressed digitations on the peripheral area
(arrow heads). The section does not involve the nucleus. (a) ×16 500; (b) × 18 000; (c) ×7500.

few cellular organelles and lipidic globules and to engulf the few remaining
thymocytes (Fig. 3). In the terminal stage of involution, at around 18–20 months,
the thymus was quite reduced in cell number and in weight and was practically

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transformed into a mass of adipose tissue containing scattered islands of more or
less normal parenchyma in small cellular nests and/or bordering cystic cavities and
very few lymphocytes.

Fig. 3. Control: involuted thymic microenvironment in 16 month-old mice. (a) Epithelial cell (arrow
heads: tonofilaments) showing disrupted part of limiting membrane ( “ ) and a clarified cytoplasm. Few
thymocytes, plasma cell ( [ ) and lipofuscin pigment (Lip.) are present. (b) Disorganized cytoplasmic
clear layer with a few scattered cellular organites and few thymocytes engolfed in thymus of 20
month-old mice. Large lipidic globules (arrow heads). (a, b) × 7500.

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Differences in the beginning of the involution exist between the FVB/N mice and
the other murine strains considered here. Indeed, in C57/Bl6, DBA2 and B6/D2
mice, the morphological involution of the thymus starts at about 10–12 months
while the more complete involuted thymus can be visualized at about 18–20
months. In FVB/N mice, the involuted process appeared at about 7–8 months and
was complete by 12 months. In spite of this difference, the evolutive steps of the
microenvironmental morphology modifications and the final picture were similar in
FVB/N and other murine strains, and correspond to the previous description of the
aged thymus [8].
3.2. Thymic microen6ironment morphology in transgenic mice o6erexpressing
hSOD-1
Thymuses from both lineages of hSOD-1 transgenic mice exhibit a premature
involution in comparison with controls. The main difference between the two types
of transgenic mice was the onset of the premature involuting process. In the PB
transgenic line (bred on the hybrid B6/D2 background and overexpressing SOD-1
by a factor of 1.5 in the thymus), the microenvironment involution started at about
7 – 8 months [44] whereas in the KT transgenic line (bred on the inbred FVB/N
background and overexpressing SOD-1 by a factor of 5–8 in the thymus), these
morphological modifications appeared earlier, at 4–5 months. The architectural
modifications as well as the cellular alterations were the same at the final stage of
the involution process in both transgenic lineages and corresponded to the pictures
observed in control aged thymus.
The primary event was a large vacuolisation of epithelial cells (Fig. 4a) followed
by an invasion of the epithelial cells by a few lipofuscin and lipidic globules.
Thymuses from 2 – 3 month-old transgenic mice exhibited a peculiar aspect in
their cellular network. The architectural organization of the thymus in two zones
was still conserved, but in some areas, the microenvironment network appeared to
be completely retracted and often broken. The lymphocytes were completely
separated from the epithelial cells as if they were ‘floating’ in large, clear, empty
‘holes’. Numerous cytoplasmic finger-like processes which seemed to originate from
the epithelial cells were observed (Fig. 4b). This aspect of epithelial network
retraction appeared less extended in the PB transgenic line than in the KT line.
Around 3 – 4 months, hSOD-1 transgenic mice presented thymic areas modified in
different ways: some of these areas displayed ‘holes’ in the stroma, some vacuolized
epithelial cells and others areas presented a cellular involuted aspect with a clarified
cytoplasm and few clusters of dense lipofuscin material or small lipidic globules
(Fig. 5a). Finally few areas showed normal cells. Few adipocytes invaded the
capsule and fibrous septae.
At 5 months, in the KT transgenic mice, and at 7 months in the PB mice, the
architecture was already modified and the separation in cortical and medulla zones
was mostly blurred. The epithelial cells had lost part of their limiting membranes
and formed a continuous layer of clear poorly differentiated cytoplasm with few
organelles and with inclusions of lipids and lipofuscin. A small number of
lymphocytes were present in this cytoplasmic layer (Fig. 5).

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Fig. 4. Premature involution in the thymus of 3 month-old hSOD-1 transgenic mice. (a) Type I epithelial
cell showing cytoplasm with numerous vacuoles and dilated perinuclear endoplasmic reticulum. (b)
Aspect of ‘hole’in the thymic stroma with disrupted epithelial cells (arrow heads), numerous finger-like
processes (“ ) and lymphocytes. (a) ×9500; (b) ×8000.

Alterations similar to those usually present in the involuted thymus of ‘naive’
mice have been observed in the PB transgenic mice from the age of 7–8 months and
from 5 – 6 months in the KT line. However, the lipidic invasion, was the same in the
KT mice and in aged control mice, and was less extant in the PB mice.

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4. Discussion
Thymic involution is a normal process which occurs during aging. Electron

Fig. 5. Premature involution in the thymus of hSOD-1 transgenic mice. The reticulum appears as a large,
poorly differentiated, unlimited cytoplasmic layer containing lipofuscin material and small lipidic
globules (arrow heads). Few resting lymphocytes are engulfed in this cytoplasmic syncitium. (a) 8
month-old transgenic mouse (PB lineage) × 7500; (b) 6 month-old transgenic mouse (KT lineage)
× 7500.

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microscopy is a powerful tool to identify the numerous cellular modifications occurring from the onset through the various stages of the involution
process.
The most striking ultrastructural morphological changes observed in the aging
thymus ([8] and references in this publication) are related to the distribution,
architectural arrangement, and cytologic appearance of the different cellular components of the thymic microenvironment. After a stage of vacuolisation, epithelial
cells lose their limiting cellular membranes and a large, clear, poorly differentiated
cellular syncytium is formed, followed by a progressive replacement of this thymic
tissue by adipose tissue. At the terminal stage, lipidic invasion by fat cells with
enormous globules appeared surrounding few areas of epithelial cell islands organized in small nests and bordering cystic cavities [8].
In this thymic morphological study from two different transgenic lines expressing
the hSOD-1 gene, ultrastructural modifications in comparison with various control
thymus is observed. They showed an aspect which appeared, for a great part,
similar to that observed in aged mice. These modifications are specific of the
hSOD-1 transgenic mice since they are not present in different controls of the same
age and are observed in the two transgenic lines obtained with the same construct
but on different murine backgrounds with different levels of both transgene copy
numbers and SOD-1 overexpression activity.
Three important differences are visualized in the hSOD-1 transgenic mice compared to the aged control mice: first, the beginning of the thymic involution
occurred around 7 – 8 months in the PB transgenic line and around 4–5 months in
the KT one.
A second difference has to do with the rupture of the cellular and extracellular
filamentous network and the formation of ‘holes’ by retraction of the cellular
network which formed the stroma. The cellular network appeared retracted with
broken epithelium leaving large ‘holes’ containing fragments of cytoplasm in
finger-like cellular protrusions scattered in empty zones. These alterations, not
observed in aged controls, appeared early in the thymic involution process of the
transgenic mice and are then followed by more ‘classic’ involution modifications.
This aspect is rarely described and perhaps corresponds to the description, in light
microscopy, of the empty areas in diabetic rodents strains [51,52]. We suggest that
such morphological alterations are perhaps due to the rupture of the cytoskeleton
network of epithelial cells and extracellular matrix as a consequence of a SOD-1
overexpression which is more drastic in the KT transgenic line than in the PB
transgenic line. It is interesting to correlate these observations on thymic involution
in hSOD-1 transgenic mice with the modifications in the extracellular matrix and
cellular microfilament network in DS patients thymus. Indeed, in observation of DS
thymus morphology by immunofluorescence [53], a rupture and retraction of the
cellular skeleton are also reported.
The third difference concerns the lipidic invasion always observed in the involuted thymic microenvironment. Contrary to the very large lipidic invasion
throughout the thymic tissue in aged mice, we noticed in the thymus of PB
transgenic mice only a moderate amount of lipofuscin pigment and lipidic globules

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[39]. However in the thymus of the KT lineage the lipidic invasion is
more extent and similar to the thymus of aged control mice. The lipidic
invasion of the thymus and the organ transformations are poorly understood. All aging phenomena are associated with increase of lipidic components in the tissues and it is suggested that in the thymus lipidic material
may reflect storage of different components after drastic thymocyte lysis occurs in the aged thymus [54,55].
Our observations raise the question of a possible relationship between the
overexpression of the hSOD-1 transgene and the premature involution observed in the thymus of these transgenic mice. As our results have been
obtained in two different lineages of transgenic mice for the hSOD-1 gene
and on two different backgrounds, the thymic involution described here can
be correlated with the transgene expression and not with a possible modification of the murine genome at the transgene insertion site.
The present results are more or less in agreement with the recent results
published by Peled-Karman et al. [38] showing that hSOD-1 transgenic
mice injected with lipopolysaccharide exhibit a loss of thymic architecture
and an increase in thymocyte apoptosis. Unfortunately, this study has been
performed only with light microscopy which does not allow precise identification of the cellular and chromatin alterations. Nevertheless, it is possible
that these observations are related to an involution process.
In conclusion, the overexpression of SOD-1 gene appears involved in the
process of premature thymic involution observed in the two different transgenic lines, but we cannot overlook the fact that the enzyme effect may
be either direct or indirect, as it is known that the SOD-1 gene may have
alternatively protective or deleterious functions in different tissues and physiological conditions [56 – 58].
Furthermore, the morphological and cellular modifications observed in the
thymus during aging both in control mice and in hSOD-1 transgenic mice,
suggest the possible implication of the superoxide dismutase overexpression
in the etiology of premature thymus involution and related immunological
impairments observed in DS patients [50].
As the thymus is considered as a putative ‘clock’ for numerous physiological phenomena we suggest, in thymus involution and in various more
general aging processes, the involvement of the level of SOD-1 or the implication of reactive oxygen species (ROS). Furthermore the balance between the levels of the main enzymes involved in ROS elimination (Cu/Zn
SOD, Mn/SOD, catalase and glutathione peroxydase) might also be very
important. Further studies on the expression of these enzymes in the different areas of thymus should give more information for a better understanding of SOD-1 transgenic mice as a model for DS and suggest the
necessity of similar approaches to understand the general mechanism of aging in DS patients.

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Acknowledgements
We wish to thank I. Andrianarison and A. Nicol for their technical work, T.
Sacksick and P. Lemoine for their photographic work, and Sonia Hamon for help
in preparing the manuscript.

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