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Histological comparison of the thymus of germfree (axenic) and conventional CFW mice.

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Histological Comparison of the Thymus of Germfree
(Axenic) and Conventional CFW Mice
'r2
M. BEALMEAR3 AND R. WILSON
Lobund Laboratory, Biology Department, University of Notre Dame,
Notre Dame, Indiana
ABSTRACT
The histology of the thymus of both germfree and conventional mice
at days 0, 14, 50, and 168 supports the interpretation of the growth pattern of the
thymus as a reflection of the lymphocyte population of the cortex and medulla. In
general the conventional thymus has more smaU lymphocytes in the cortex and more
lymphocytes migrating into the medulla than in its germfree counterpart. Hence it
would seem that antigenic challenge stimulates lymphocyte production in the thymus
cortex and initiates a chain of events that may eventually result in thymus lymphocytes populating the other lymphoid tissues of the body, or that may result in lymphocyte degeneration in the thymus and the subsequent releas of a humoral factor
that may stimulate the other lymphoid tissue to lymphocytopoiesis.
The association between the thymus and
the immunological mechanisms of the
mammal has become the subject of much
investigation in recent years (Good and
Gabrielsen, '64; Defendi and Metcalf, '64).
On the evidence produced by this new wave
of research regarding the function of the
thymus, it has been proposed that the
thymus is the lymphoid organ primarily
responsible for the ultimate development
of immunological competence in most
vertebrates (Miller et al., '62; Finstad et al.,
'64). Two hypotheses of thymus function
have been proposed: ( 1 ) cellular, and ( 2 )
humoral. In most animals this function is
purportedly complete before the end of
gestation or shortly thereafter (Miller, '63;
Parrott and East, '64).
Although there has been some inquiry
into the ability of the cellular elements of
the thymus itself to produce antibodies
against antigens inoculated into it (Marshall and White, ' S l ) , there has been
very little investigation of the correlation
between thymus activity and antigenic
stimuli originating from the natural environment of the animal. Recently we
demonstrated that the thymus of germfree
mice grows at a slower rate during the
two-week period following birth than those
of conventional (non-germfree ) mice and
that the thymus of germfree mice never
gets as large as that of conventional mice
(Wilson et al., '65). It was postulated that
the increased size of the conventional
ANAT, REC., 1 5 4 : 261-274.
thymus is a function of the stimulation of
the production of lymphocytes in the
thymus by antigens arising from microorganisms in contact with the host. The
germfree status is responsible for a lower
degree of development of lymphoid tissue
and lower beta and gamma globulin levels
in germfree animals, but they are capable
of immunological response (Wostmann,
'59). The difference between germfree
and conventional animals is probably
quantitative rather than qualitative (Thorbecke, '59).
Since the response of the thymus is
basically a cellular one, a histological comparison of the thymus of the germfree and
the conventional animal would be more
meaningful than gross comparisons. Consequently in the study reported here we
chose to examine and compare the histology of germfree and conventional thymus
tissues at significant points on the growth
curve and to correlate the microscopic
anatomy with the gross appearance and
growth of the thymus.
MATERIALS AND METHODS
The thymuses prepared for this study
were those of mice in the conventional and
1Sup orted by funds from the Office of Naval
Researcx Nonr-1623( 15).
SThis work is a part of the disseFtation submitted
to the Graduate School of the Unlverslty of Notre
Dame in partial fulfillment of the requirements for
the degree of Doctor of Philosophy by one of the
authors (M.B.).
3 Supported, by predoctoral fellowship from the
National Institutes of Health, 1-F1-GM-23.
261
262
M . BEALMEAR AND R. WILSON
germfree colonies of CFW mice maintained at the Lobund Laboratory. Both
groups were caged in plastic cages, fed the
same sterilized diet, and subjected to the
same routine of animal care. The cages
of germfree mice were contained within
Trexler flexible film isolators (Trexler and
Reynolds, '57). Conventional mice were
derived from germfree mice which had
been adapted to the microflora of the
animal quarters.
The thymuses were removed from both
germfree and conventional mice 0, 14, 50,
and 168 days of age. These ages were
selected to give a base line (0 days - day
of birth) and to present the picture at the
times of fastest growth (14 days), largest
size (50 days), and regression (168 days).
For the study at 0 days, three female and
five male germfree mice and four female
and four male conventional mice were
used; at 1 4 days, five female and three
male germfree and three female and five
male conventionals; at 50 days, four female and four male germfree mice were
used and four female and four male conventional; and at 168 days, three female
and four male germfree and five female
and two male conventional mice were used.
There seemed to be no obvious histological
differences between the thymuses of males
and females at any of the ages studied.
Representative sections from tissues at
these age periods are shown in plates 1-4;
these illustrate some of the characteristics
enumerated in table 1. At the time of
removal the thymuses were fixed in Bouin's
solution and transferred to 70% ethanol
for storage. The tissues were dehydrated
in dioxane, embedded in paraffin, sectioned
at 6 p on a rotary microtome, and stained
with hematoxylin and eosin.
RESULTS
Grossly the thymuses of both germfree
and conventional mice are similar; the two
lobes of the thymus are covered with
fibrous connective tissue from which septa
project into the organ producing lobulation without subdividing it into distinct
lobules. Microscopically both thymuses
have an outer deeply staining cortex and
inner less dense medulla. The four typical
thymus cell types described by Sainte-Marie
and Leblond ('64) can be observed in the
thymuses of all ages. These cell types include reticular cells, large lymphocytes,
medium lymphocytes, and small lymph@
cytes. The Hassall's corpuscles described
by Fekete ('41) can be found in the
thymuses of both conventional and germfree mice at all the ages studied (plate 2
C,D) including the day of birth.
At 0 days (plate 1 ) there are many
mitotic figures in evidence with 7-8 mitotic
figures per field in the conventional thymus
(plate 1,C) and 5-6 per field in the germfree thymus (plate1,D). The mitotic index
was not determined. The medulla is larger
in the germfree than in the conventional
and contains proportionately fewer small
lymphocytes and more reticular cells.
There are many small lymphocytes in the
small but somewhat diffuse medulla of the
conventional.
A smaller medulla and larger densely
staining cortex are found in the germfree
thymus than in conventional at 14 days
(plate 2). The cortex of the germfree is
densely populated with small lymphocytes,
but few can be found among the reticular
cells of the medulla. By contrast, in the
conventional thymus at 14 days it is difficult to see a sharp division between cortex
and medulla with the medulla occupying a
large area and containing many small
lymphocytes. Reticular cells are also perceptible in the cortex.
When the thymus actually reaches its
maximum size at 50 days of age (plate 3),
more small lymphocytes are still found in
the cortex of the germfree than in the conventional thymus. The medulla is well defined in germfree, but more diffuse and
larger in conventional. The number of
mitotic figures decreases with age in both
germfree and conventional thymuses.
At 168 days of age (plate 4) when involution is in progress, there are few
mitotic figures, more reticular cells, and
an increase in the number of macrophages
in the medulla of both germfree and conventional thymuses; but the cortex is still
more sharply delineated from the medulla
in the germfree. Some large cells that contain one or more usually eccentric nuclei
seem to fit the description of the chromolipoid cells or lipid-laden foamy cells described by Loewenthal ('52) in aging
thymuses. There is no adipose tissue de-
Many small lymphocytes; medulla large, but
not sharply defined.
Few small lymphocytes; medulla sharply
defined.
Medulla diffuse.
Medulla well defined.
Increase i n reticular cells, macrophages, and
chromolipoid cells.
Increase i n reticular cells, macrophages, and
chromolipoid cells.
Many small lymphocytes; reticular cells
Many small lymphocytes; no reticular
cells evident
Fewer small lymphocytes
More small lymphocytes
Not sharply delineated from medulla;
many lymphocytes
Sharply delineated from medulla;
many lymphocytes
less than at 0 days
less than at 0 days
less than at 14 days
less than at 14 days
less than at 50 days
less than at 50 days
Germfree
50 Days
Convention a1
Germfree
168 Days
Conventional
Germfree
Fewer small lymphocytes; medulla large.
Fewer small lymphocytes
5-6 per field
Germfree
14 Days
Conventional
Many small lymphocytes; medulla small,
but diffuse.
Medulla
Many small lymphocytes
Cortex
7-8 per field
Mitotic figures
0 Days
Conventional
Age
Histological comparison of the thymuses of germfree and conventional CFW mice
TABLE 1
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264
M. BEALMEAR AND R. WILSON
posited in the involuted mouse thymus. A
comparison of germfree and conventional
thymuses at the days studied is summarized in table 1, noting in particular the
relative numbers of mitotic figures and of
lymphocytes in the cortex and medulla.
DISCUSSION
The difference in thymus growth patterns in germfree and conventional mice
probably results from the fact that the
germfree animal is freed from appreciable
antigenic stimulation from its environment
after birth. The germfree animal is the
object of some antigenic stimulation from
killed antigens in the food and bedding and
possibly undetected or latent viruses, but
this is minimal when compared with the
conventional.
The data for growth and regression of
the germfree and conventional thymus correlate well with the histological picture.
At 0 days in the germfree thymus the
medulla is large with few lymphocytes, and
the cortex is not as densely packed with
small lymphocytes as it is in the conventional. This observation is consistent with
the cellular hypothesis proposed by SainteMarie and Leblond ('64) and others according to which the germfree thymus,
free from appreciable antigenic stimulation, would not produce as many small
lymphocytes in the cortex to migrate eventually to the medulla and finally to the
other lymphoid organs via the blood stream.
By the same reasoning, the conventional
thymus would be stimulated to produce
more small lymphocytes in the cortex,
which would in turn be forced into the
medulla and finally into the blood stream
as the population of small lymphocytes increases in the medulla. The presence of
more mitotic figures in the conventional
thymus than in germfree at 0 days parallels the tripling of the ratio of thymus
weight to body weight in conventional in
the first 24 hours after birth and the doubling of the ratio in germfree. The lack of a
sharp demarcation between the cortex and
the medulla in the conventional mice may
be explained on the basis of cell migration.
If many small lymphocytes are constantly
migrating from the cortex to the medulla
in the conventional, then it would obvi-
ously be difficult to observe a sharp line of
demarcation between cortex and medulla.
The histological scene at 14 days corresponds well with the growth curve. At
this time the ratio is almost at its peak
in both germfree and conventional, the
conventional being slightly higher. Many
small lymphocytes are found in the cortex
in both germfree and conventional, but the
conventional small lymphocyte population
must be greater because many small
lymphocytes have been crowded out of the
cortex into the medulla, and again the
medulla is not as well defined in conventional as in germfree.
When the thymus reaches its period of
maximum growth at 50 days, histological
examination shows a reduced number of
mitotic figures in both germfree and conventional. The medulla of the conventional
is still diffuse with lymphocytes migrating
into it from the cortex.
Finally at 168 days when involution is
in progress, few mitotic figures can be
found in the histological sections. Antigenic stimulation is apparently still influencing small lymphocyte production in
the conventional and subsequent migration
to the medulla; however, this activity is
now somewhat reduced and both thymuses
show an increase in the number of macrophages, which remove cellular debris, and
an increase in the number of lipid-laden
cells.
This picture of small lymphocyte production in the cortex and migration to the
medulla is not completely incompatible
with the humoral hypothesis of thymus
function postulated by Levey and associates (Levey, Trainin and Law, ' 6 3 ) and
Osoba and Miller ('63). Parrott and East
('64) suggest that the humoral factor could
be a breakdown product of the lymphocytes
themselves and not a product of the
reticuloendothelial cells because thymic
lymphocytes are known to disintegrate
and disappear during their sojourn in
grafted diffusion chambers. If this is so,
in the normal animal the lymphocytes
whether residing in the thymus or migrating to other lymphoid organs could induce
lymphocytosis; some degenerating lymphocytes are in evidence in thymus sections
from mice at all the ages studied.
HISTOLOGY OF THE GERMFREE THYMUS
The thymus growth patterns and hist&
logica1 picture in germfree and 'Onventional mice tend to give Some credence
to the cellular hypothesis of thymic function, but do not necessarily rule out the
humoral hypothesis; the two need not be
mutually exclusive.
LITERATURE CITED
Defendi, V., and D. Metcalf 1964 The Thymus.
The Wistar Institute press, Philadelphia, pa.
145 pp.
Fekete, E. 1941 Histology. In: Biology of the
Laboratory Mouse, George D. Snell, (ed.),
Dover Publications, Inc., New York, pp. 89-167.
Finstad, J., B. W. Papermaster and R. A. Good
1964 Evolution of the immune response. 11.
Morphologic studies on the origin of the thymus
and organized lymphoid tissue. Lab. Invest.,
23: 490-512.
Good, R. A., and A. E. Gabrielsen 1964 The
Thymus in Immunobiology. Hoeber Medical
Division, Harper and Row, New York, 778 pp.
Levey, R. H., N. Trainin and L. W. Law 1963
Evidence for function of thymic tissues in diffusion chambers implanted i n neonatally thymectomized mice. J. Nat. Cancer Inst., 31: 199217.
Loewenthal, L. A., and C. Smith 1952 Studies
on the thymus of the mammal. IV. Lipid-
265
laden foamy cells in the involuting thymus of
the mouse Anat. Rec., 112: 1-15.
Marshall, A. 'H. E., and R. G. White 1961 Experimental thymic lesions resembling those of
myasthenia gravis. Lancet, I: 1030-1031.
Miller, J. F. A. P. 1963 Role of the thymus in
immunity. Brit. Med. J., 2: 459464.
Osoba, D., and J. F.A. P. Miller 1963 Evidence
for a humoral thymus factor responsible for the
maturation of immunological faculty. Nature,
199: 653-654.
Parrott, D. M. v., and J. East
The thymus
immunity. Proc. ROY. SOC. Med., 57: 147-
"rd
131.
Sainte-Marie, G., and C. P. Leblond 1964
Thymus-cell population dynamics. In : The
Thymus i n Immunobiology, A. Good and Ann
E. Gabrielsen, (ed.), Hoeber Medical Division,
Harper and Row, New York, pp. 207-235.
Thorbecke, G. J. 1959 Some histological and
functional aspects of lymphoid tissue in germfree animals. I. Morphological studies. Ann.
N. Y. Acad. Sci., 78: 237-246.
Trexler, P. C., and L. I. Reynolds 1957 Flexible
film apparatus for the rearing and use of
germfree animals. Appl. Microbiol., 5: 406412.
Wilson, R., M. Bealmear and R. Sobonya 1965
Growth and regression of the germfree (axenic)
thymus. Proc. SOC.Exp. Biol. Med., 118: 97-99.
Wostmann, B. S. 1959 Serum proteins in germfree: vertebrates. Ann. N. Y. Acad. Sci., 78:
254-260.
PLATE 1
EXPLANATION OF FIGURES
Thymus
266
of
CFW Mouse at 0 Days
A
Conventional ( X 51) with many small lymphocytes in the cortex
and a small but diffuse medulla.
B
Germfree ( X 5 1 ) with fewer small lymphocytes in the cortex and a
large distinct medulla.
C
Conventional ( X 197) taken from a dense area of the cortex to illustrate the large number of mitotic figures in evidence.
D
Germfree ( X 197) also taken from a dense area o i the cortex to illustrate the number of mitotic figures i n evidence.
HISTOLOGY OF T H E GERMFREE THYMUS
M. Bealmear and R. Wilson
PLATE 1
267
PLATE 2
EXPLANATION OF FIGURES
Thymus of CFW Mouse at 14 Days
A
Conventional ( X 40) with many small lymphocytes and reticular cells
in the cortex and a large but not sharply defined medulla.
B
Germfree ( X 40) with many small lymphocytes in the cortex and a
small sharply defined medulla.
C
Conventional ( X 197) showing Hassall's corpuscle ( H ) .
D
Germfree ( X 197) showing Hassall's corpuscle ( H ) .
HISTOLOGY OF THE GERMFREE THYMUS
M. Bealmear and R. Wilson
PLATE 2
269
PLATE 3
EXPLANATION OF FIGURES
Thymus
270
of C F W
Mouse at 50 Days
A
Conventional ( X 4 0 ) with fewer small lymphocytes in the cortex
and a very diffuse medulla.
B
Germfree ( X 40) with more small lymphocytes in the cortex.
C
Conventional ( X 197) with Hassall's corpuscles and an increase in
the number of reticular cells.
D
Germfree ( x 197) with lymphocytes obscuring the reticular cells.
HISTOLOGY OF THE GERMFREE THYMUS
M. Bealmear and R. Wilson
PLATE 3
PLATE 4
EXPLANATION OF FIGURES
Thymus of CFW Mouse at 168 Days
272
A
Conventional ( x 40) with many lymphocytes in the cortex and a
diffuse medulla.
B
Germfree ( X 40) with many lymphocytes and well defined medulla.
C
Conventional ( X 197) with an increase in reticular cells and macrophages.
D
Germfree ( X 197) with a slight increase in reticular cells and macrophages.
HISTOLOGY OF THE GERMFREE THYMUS
M. Bealmear and R. Wilson
PLATE 4
273
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