Effects of hypophyseal or thymic allograft on thymus development in partially decerebrate chicken embryos: expression of PCNA and CD3 markers.
ABSTRACT Changes in chicken embryo thymus after partial decerebration (including the hypophysis) and after hypophyseal or thymic allograft were investigated. Chicken embryos were partially decerebrated at 36-40 hr of incubation and on day 12 received a hypophysis or a thymus allograft from 18-day-old donor embryos. The thymuses of normal, sham-operated and partially decerebrate embryos were collected on day 12 and 18. The thymuses of the grafted embryos were collected on day 18. The samples were examined with histological method and tested for the anti-PCNA and anti-CD3 immune-reactions. After partial decerebration, the thymic cortical and medullary compartments diminished markedly in size. Anti-PCNA and anti-CD3 revealed a reduced immune-reaction, verified also by statistical analysis. In hypophyseal or grafted embryos, the thymic morphological compartments improved, the anti-PCNA and anti-CD3 immune-reactions recovered much better after the thymic graft, probably due to the thymic growth factors and also by an emigration of thymocytes from the same grafted thymus.
- SourceAvailable from: Sabrina Ceccariglia[Show abstract] [Hide abstract]
ABSTRACT: All jawed vertebrates share lymphocyte receptors that allow the recognition of pathogens and the discrimination between self and non-self antigens. The T cell transmembrane receptor (TcR) has a central role in the maturation and function of T lymphocytes in vertebrates via an important role in positive selection of the variable region of TcR αβ/γδ chains. In this study, the TcRβ transcript expression and TcRβ(+) cell distribution during the ontogeny of the immune system of sea bass (Dicentrarchus labrax, L.) were analysed. RT-PCR analysis of larvae during early development demonstrated that the β chain transcript is expressed by 19 days post-fertilisation (p.f.). RNA probes specific for the β chain were synthesised and used for in situ hybridisation experiments on 30 day p.f. to 180 day old juvenile larvae. A parallel immunohistochemical study was performed using the anti-T cell monoclonal antibody DLT15 developed in our laboratory [Scapigliati et al., Fish Shellfish Immunol 1996; 6:383-401]. The first thymus anlage was detectable at 32-33 days p.f. (Corresponding to about 27 days post-hatch). DLT15(+) cells were detected at day 35 p.f. in the thymus whereas TcRβ(+) cells were recognisable at day 38 p.f. in the thymus and at day 41 p.f. in the gut. TcRβ(+) cells were observed in capillaries from 41 to 80 days p.f. At day 46 p.f., TcRβ(+) cells were identified in the head kidney and were detected in the spleen 4 days later. The present results demonstrate that TcRβ(+) cells can be differentiated first in the thymus and then in other organs/tissues, suggesting potential TcRβ(+) cell colonisation from the thymus to the middle gut. Once the epithelial architecture of the thymus is completed with the formation of the cortical-medullary border (around 70-75 days p.f.), DLT15(+) cells or TcRβ(+) cells are confined mainly to the cortex and cortical-medullary border. In particular, a large influx of TcRβ(+) cells was observed at the cortical-medullary border from 72 to 90 days p.f., suggesting a role in positive selection for this thymic region during the ontogeny of the fish immune system. This study provides novel information about the primary differentiation and distribution of TcRβ(+) cells in sea bass larvae and juveniles.Fish & Shellfish Immunology 01/2011; 30(3):773-82. · 2.96 Impact Factor
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ABSTRACT: Histochemical journals represent a traditional forum where methodological and technological improvements can be presented and validated in view of their applications to investigate not only cytology and histology in normal and diseased conditions but to test as well hypotheses on more basic issues for life sciences, such as comparative and evolutionary biology. The earliest scientific journals on histochemistry began their publication in the first half of the '50s of the last century, and their readership did not probably change over the years; rather, the authors' interests may have progressively been changing as well as the main topics of their articles. This hypothesis is discussed, based on the subjects of the article published in the first and last ten years in the European Journal of Histochemistry, as an example of old journal which started publication in 1954, being since then the official organ of the Italian Society of Histochemistry. This survey confirmed that histochemistry has provided and still offers unique opportunities for studying the structure, chemical composition and function of cells and tissues in a wide variety of living organisms, especially when the topological distribution of specific molecular components has diagnostic or predictive significance, as it occurs in human and veterinary biology and pathology. Some subjects (e.g. histochemistry applied to muscle cells or to mineralized tissues) have recently become rather popular, whereas a wider application of the histochemical approach may be envisaged for plant cells and tissues.European journal of histochemistry: EJH 01/2010; 54(4):e51. · 2.41 Impact Factor
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ABSTRACT: In the last three years, more than 70,000 scientific articles have been published in peer reviewed journals on the application of histochemistry in the biomedical field: most of them did not appear in strictly histochemical journals, but in others dealing with cell and molecular biology, medicine or biotechnology. This proves that histochemistry is still an active and innovative discipline with relevance in basic and applied biological research, but also demonstrates that especially the small histochemical Journals should likely reconsider their scopes and strategies to preserve their authorship. A review of the last three years volumes of the European Journal of Histochemistry, taken as an example of a long-time established small Journal, confirmed that the published articles were widely heterogeneous in their topics and experimental models, as in this Journal's tradition. This strongly suggests that a Journal of histochemistry should keep its role as a forum open to an audience as broad as possible, publishing papers on cell and tissue biology in a wide variety of models. This will improve knowledge of the basic mechanisms of development and differentiation, while helping to increase the number of potential authors since scientists who generally do not use histochemistry in their research will find hints for the applications of histochemical techniques to novel still unexplored subjects.European journal of histochemistry: EJH 01/2012; 56(4):e51. · 2.41 Impact Factor
European Journal of Histochemistry 2010; volume 54:e37
Effects of hypophyseal or
expression of PCNA and CD3
1Department of Physiology and
thymic allograft on thymus
Pharmacology “V. Erspamer”, Faculty
University of Tuscia, Viterbo, Italy
ysis) and after hypophyseal or thymic allograft
were investigated. Chicken embryos were par-
tially decerebrated at 36-40 h of incubation and
on day 12 received a hypophysis or a thymus
allograft from 18-day-old donor embryos. The
thymuses of normal, sham-operated and par-
tially decerebrate embryos were collected on
day 12 and 18. The thymuses of the grafted
embryos were collected on day 18. The samples
were examined with histological method and
tested for the anti-PCNA and anti-CD3
immune-reactions. After partial decerebration,
the thymic cortical and medullary compart-
ments diminished markedly in size. Anti-PCNA
and anti-CD3 revealed a reduced immune-
reaction, verified also by statistical analysis. In
hypophyseal or grafted embryos, the thymic
morphological compartments improved, the
anti-PCNA and anti-CD3 immune-reactions
recovered much better after the thymic graft,
probably due to the thymic growth factors and
also by an emigration of thymocytes from the
same grafted thymus.
development in partially
M. Aita,1F. Benedetti,1E. Carafelli,1
decerebrated chicken embryos:
E. Caccia,2N. Romano2
of Medicine, University “La Sapienza”,
2Department of Environmental Science,
Changes in chicken embryo thymus after
partial decerebration (including the hypoph-
in differentiating lymphocytes responsible for
specific immunological responses, such as cel-
lular (T lymphocytes, in thymus)1,2and humoral
(B lymphocytes, in bursa).3In thymus, the vari-
ous reticular-epithelial cells, the epithelial cysts
and humoral factors stabilise the correct milieu
involved in processing the T-cell precursors and
inducing them to become mature and differen-
The primary lymphatic organs in birds, thy-
mus and bursa of Fabricius, play a central role
tiated thymocytes or T-cells.4-6Chicken embry-
onal thymus is organized in cortex and medulla.
In the cortex, lymphocytes overspread the retic-
ular-epithelial cells, and in the medulla, at the
centre of the lobe, isolated or clustered epithe-
lial cells7,8and cysts, of the intra- or inter-cellu-
lar type8-11are well visible, whereas macro -
phages, myoid cells and Hassall’s corpuscles are
rare. Lymphocytes are less numerous than in
The thymus, besides to have an immune
function, has also an endocrine function; in
fact endocrine-like cells were described in
embryonal chicken thymus.9-12The thymus
takes part in the mechanisms of reciprocal
effects between the immune and the neuro-
endocrine interactions in mammals and in
birds.13-18It has been characterized the influ-
ence of the hypothalamic-pituitary-gonadal
axis and that of thyroid hormone on the
immune system, in mammals19-21and in birds.22
The production of several hormones has
been described in the thymus of mammals and
birds, as steroids,23glucocorticoids,24-26growth
In accordance with the reciprocity of the
actions between immune and neuro endocrine
system, several thymic factors were found to be
active on nervous and endocrine system,
(reviewed in 33) and localized in mammalian
thymus.33-35In birds, a specific hormonal factor,
the avian thymic hormone (ATH), a parvalbu-
min, has been extracted and localized only in
cortical reticular-epithelial cells.36-38The
thymic factor thymostimulin was extracted
from calf thymus39and detected, using an anti-
thymostimulin antibody, in mammals40,41and
humans.42In avian embryonal, post hatching
and aging thymuses43-46the thymostimulin-like
immune reactivity was observed in vacuolar
and cystic epithelial cells, around an epithelial
cluster or arranged in small groups of epithe-
lial cells. The importance of the lack of hypoph-
ysis for the endocrine glands development in
the chicken embryo was described by Fugo,47
without noting any histological changes in
thymic structure. The effects of embryonic
pars distalis grafts on different endocrine
glands after a chick embryos hypophysectomy
was evidenced by Betz.48
On the contrary, in our experience, early
partial decerebration including the ablation of
hypophyseal anlage, caused thymus underde-
velopment with a reduction in the number and
differentiation of reticular-epithelial cells as
compared with normal embryos.49-51Romano et
al.,8in an ultrastructural study, found evident
variations in the cortical zone and in the small
medullary zone; there were few clusters and
few cysts of the intracellular type with some
modifications compared with normal thymus.
Aita and Romano46noted also the lack of the
synthesis of the thymic factor “thymostimulin”
and the reduction of some enzymatic pathways
as ATP ase and SDH. Regarding the differenti-
ation of T-cell subsets, Moreno et al.,52making
a partial decapitation at 33-38 hours of incuba-
tion, found that T-cell subsets were affected
with a decline of CD4+and CD8+ cells and
TCRαβ-expressing cells. A hypophyseal graft
in partially decerebrate chick embryo deter-
mined an increase of total thymus size and of
medullary epithelial cells53with a considerable
recovery of the cytology, even if a normal mor-
phology is not completely accomplished8and
with the recovery of the synthesis of thy-
mostimulin and of the enzymatic pathways.46
The aim of this research is: i) to verify if an
allograft of thymus in partially decerebrate
embryos may improve the morphological varia-
tions described in partially decerebrate
embryos8,17,21and compare these results with
the normal thymus, ii) to ascertain if a partial
decerebration, followed by a hypophyseal or
thymic allograft may influence the expression
of the nuclear PCNA/cyclin marker or the
expression of the membrane CD3 marker in
thymocytes and to compare these results with
the normal thymus and examine thoroughly
previous research conducted in our laborato-
ry.54,55The cyclin, an acidic polypeptide of MW
36 kDa, is present in several cells nucleus. Its
synthesis is followed by DNA synthesis and its
level increase in S phase of the cell cycle56-60
PCNA (the proliferating cell nuclear antigen)
was demonstrated to be identical with cyclin.61
Thereafter, an auxiliary protein specific for
DNA polymerase δ, was extracted from foetal
or adult calf thymus.62,63Bravo et al.,64reported
that cyclin/PCNA is the auxiliary protein of
DNA polymerase δ. The marker CD3 is
expressed in the cortical and medullary zones
of the thymus during the ontogenic period, by
[page 158][European Journal of Histochemistry 2010; 54:e37]
Correspondence: Mariangela Aita, Department of
Physiology and Pharmacology “Vittorio Erspamer”,
Faculty of Medicine University La Sapienza, P.le
A. Moro 5, 00185 Roma, Italy.
Key words: hypophysectomy, hypophyseal and
thymic allograft, chicken embryonal thymus,
PCNA, CD3 markers.
Received for publication: 3 February 2010.
Accepted for publication: 12 July 2010.
This work is licensed under a Creative Commons
Attribution 3.0 License (by-nc 3.0).
©Copyright M. Aita et al., 2010
Licensee PAGEPress, Italy
European Journal of Histochemistry 2010; 54:e37
[European Journal of Histochemistry 2010; 54:e37][page 159]
T lymphocytes, during their differentiation
until becoming mature thymocytes.1,5,55,65-70
Materials and Methods
White Leghorn chicken embryos (Gallus gal-
lus domesticus) were used for three series of
experiments: i) the prosencephalon, part of
mesencephalon including the hypophyseal
anlage and the presumptive anlage of the
Rathke pouch were removed from 36-40 h
embryos (PD) using Fugo’s technique;47ii)
some PD embryos, at d 12 of incubation,
received a hypophyseal allograft onto the
chorio-allantoic membrane from an 18 day-old
donor embryo, (PD+H); iii) some PD embryos,
at d 12 of incubation, received a thymic allo-
graft onto the chorio-allantoic membrane from
an 18 day-old donor embryo (PD+Th).
The experimental thymuses were compared
with normal and sham-operated embryos. For
the sham-operation a small window was
opened in the embryonic shell, at 36 h and
covered with a sterilized tape at 40 h. On d 12,
the sham-operated embryos were opened for
the second time and then covered and sealed
again. Thymuses of normal, sham operated
and PD embryos were collected at the d 12 and
18 of incubation. Every experimental chick
was examined to ascertain hypophyseal
removal. Thymuses of PD+H, PD+Th embryos
were collected at d 18. In PD+H the grafted
hypophysis as well as in PD+Th, the grafted
thymus were histologically examined. The
embryos’ stage was evaluated by the days of
incubation and by Lillie’s tables of develop-
ment.71We repeated experiments for 4 times.
We utilized 50 eggs for normal and sham oper-
ated embryos, removed at d 12 and 18, in total
n=36 surviving embryos. For the experiments,
a total amount of 1200 eggs was used. After
the partial decerebration at 36-40 h, the mor-
tality was high. The surviving embryos at d 12
were 96. Ten of them were removed as (PD)
embryos. From the remaining 86, 30 embryos
were used for grafts, 15 grafted with hypoph-
ysis, surviving at day 18 n=10 (PD+H)
embryos, 15 grafted with thymus, surviving at
day 18, n=9 (PD+Th) embryos; among them, 5
embryos had the thymic graft with a remnant
of histological structure. The remaining 56 PD
embryos were left till d 18, surviving 9
operated, PD, PD+H and PD+Th, were fixed in
Bouin’s liquid for 8 h at room temperature,
Two thymic lobes from right side and two
from left side of the neck from normal, sham-
dehydrated and embedded in paraffin wax.
Transversal serial sections, from a whole
thymic lobe, of 5 μm thick were stained by
haematoxylin-eosin for histological examina-
tion. Other sections were processed with
immune-histological methods (anti PCNA/
cyclin and anti-CD3, antibodies).
Anti-PCNA/cyclin immune reaction
in previous studies.72,73In brief, the thymic sec-
tions were incubated with the primary anti-
body (anti-PCNA, PC 10 mouse monoclonal
antibody (moAb) IgG Sigma, n. P-8825) at the
dilution of 1:1000 in 1% normal horse serum in
phosphate buffer, pH 7.2, overnight at 4°C. The
immune reaction was revealed by 0.05% 3-3’
diaminobenzidine (DAB) tetrahydrochloride
with 1% nickel-sulfate and 0.01% hydrogen
peroxide in 0.05% M Tris-HCl buffer (Sigma-
Aldrich). No counterstain was used. Slides
were then dehydrated and coverslipped using
Entellan (Merck, Germany). The specificity of
the immune staining was tested by replacing
the primary antiserum with immune horse
The method used for the anti-PCNA/cyclin
antibody reaction has been reported in detail
Anti-CD3 immune reaction
marker, clone UCHT1 by DAKO). The immune
reaction was revealed by 3-amino-9-ethyl-car-
bazole (AEC-immune staining DAKO PAP kit).
No sections were counterstained. Slides were
then dehydrated and coverslipped with Tissue
Adhesive PC-380 k (Ortho). A non-immune
serum (DAKO) was used as a negative control.
Thymic sections were treated with a PAP
method using an anti-CD3 antibody (a pan-T
stained with the anti-PCNA and anti-CD3 anti-
bodies. Counts of immune reactive cells
(nucleated only, cell area ranging from 4 to 100
mm²) in 1 mm² area of thymuses were per-
formed with a computer-assisted image analy-
sis system (Axoscope-KS300-Zeiss) by an
external observer, unaware of treatments. The
number of immune reactive cells was then cal-
culated by averaging the cell numbers from 4
specimens per treatment, and expressed as the
mean±standard deviation (SD). Numerical
results were analysed by means of analysis of
two-tail-T student test.
In each specimen, multiple sets of consecu-
tive sections were differentially immune-
Figure 1. Haematoxylin-Eosin. Thymuses from 12 day-old embryos. (a) Normal thymus.
Note the division in cortex (C) and medulla (M). (b) Normal thymus. Small epithelial
clusters with few cells (arrow), in the medulla. (c) PD thymus. Note the reduction of the
thymic size. (d) PD thymus, in the medulla no epithelial cells are present. Scale bar: a,c:
200 µm; b: 40 µm; d: 16 µm.
[page 160][European Journal of Histochemistry 2010; 54:e37]
muses from normal and sham-operated
embryos, we henceforward refer to both groups
as normal embryos.
Since no morphological or immune-histo-
chemical differences were found between thy-
medulla with small epithelial clusters made up
of few cells (Figure 1a, b). In PD embryos of 12
d, the thymus was underdeveloped and the cor-
tical lobes width was reduced. At the centre of
the organ the medulla was very small, no
epithelial cells were present (Figure 1c, d).
In normal embryos, the thymus of 18 d
(Figure 2a, b) revealed a capsular line of con-
nective tissue, which gives rise to the septa
dividing the cortex in lobes rich in thymocytes,
veiling the presence of the reticular-epithelial
cells. The medulla, mainly at the centre of the
thymus, presented a less number of thymo-
cytes, making so more distinct the epithelial
cells; few of them were isolated, whereas the
majority was assembled forming large clusters,
where light and dark cells were visible, or
epithelial cells with inside vacuoles, or cysts of
the intra or intercellular type. Hassall’s corpus-
cles and myoïd cells were rare.
In PD embryos of 18 d the thymus was
always underdeveloped, no epithelial cluster
was present in the medulla (Figure 2c, d).
In every grafted embryos, either with
hypophysis or with thymus, no host immune
reaction was detected.
In PD+H embryos, the cortex was enriched
in thymocytes. The medulla showed an
improvement of epithelial clusters, without
anyway reaching the number of the normal
thymus (Figure 2e, f).
The histological control of the grafted
hypophysis stained with haematoxilin-eosin
showed a well-preserved organization as cellu-
lar cord. The graft contained mainly adenohy-
pophyseal tissue, with scarce neuro-hypophy-
seal fibres (data not shown).
In PD+Th experiments we examined the
respective thymic grafts. Two types of residual
grafts were observed: i) a thymic fragment
with a remnant of the histological structure
(Figure 3a); ii) a thymic fragment without the
typical histological structure, but only with few
thymocytes, connective and adipose tissue
In PD+Th thymus, collected from the
embryo where the graft still had a histological
structure, we found a good recovery of the
thymic size, even if the cortical lobes were not
well separated, the medulla at the centre was
In normal embryos of 12 d, the thymus was
set up by a cortex divided in lobes and a central
Figure 2. Haematoxylin and eosin. Thymuses from 18 day-old embryos. (a) Normal thy-
mus. (b) The medullary clusters are made up of epithelial cells, some have cystic appear-
ance of the intra-or intercellular type. (c) PD thymus. The size is clearly reduced. (d) PD
thymus. In the medulla no clusters of epithelial cells are present. (e) PD+H thymus. The
cortex (C) is similar to that of normal thymus and there is an improvement of the
medullary zone (M). (f) In the medulla the epithelial clusters are present (arrow) without
reaching the number of the normal thymus. (g) PD+Th thymus. Note the good recovery
of the total size. (h) PD+Th thymus. Good improvement of the epithelial cluster in the
medulla (arrow). Scale bar: a,c,e,g: 100 µm; b,d,h: 40 µm; f: 25 µm.
[European Journal of Histochemistry 2010; 54:e37] [page 161]
large and the improved clusters were formed
by few cells not reaching the form and the
number of the normal thymus (Figure 2g, h).
In PD+Th thymus, collected from the
embryo where the graft was without the histo-
logical structure, the thymic size was
increased in a considerable way, the medulla
was large and located not only in the centre,
but also inside the cortical lobes, the epithelial
clusters are improved without anyway reach-
ing the form and the number of the normal thy-
mus (data not shown).
Anti-PCNA immune reaction
mocytes, indicating an efficient proliferation
activity. The medullary thymocytes were not
immune-reactive (Figure 4a).
In PD thymus, the nuclei of few thymocytes
of the external cortical zone were immune-
reactive; the nuclei of the other thymocytes
were weakly immune-stained. In the medulla
no thymocyte was reactive (Figure 4b).
In PD+H thymus, there was a recovery of
the total size of the cortex and the nuclei of
many thymocytes, spread in the total cortex,
were positive. No thymocyte was reactive in
the medulla (Figure 4c).
In PD+Th thymus, there was a notable
recovery of the number of the total cortical thy-
mocytes, more evident in the situation of the
graft without the histological structure. The
nuclei of thymocytes were positive to this
immune reaction. No positive thymocyte in the
medulla (Figure 4d).
Image analyses and calculation of PCNA
positive thymocytes reported in Figure 5
revealed that there was a significant decre-
ment in PD and PD+H (P<0.05) compared
with normal thymus and a significant incre-
ment in the group of PD+Th compared with
the other groups (P<0.001).
In normal thymus, an intense immune-reac-
tion was located in the nuclei of cortical thy-
Anti-CD3 immune reaction
making up the characteristic ring. The
medullary thymocytes were also immune-reac-
tive (Figure 6a). In PD thymus, there was a
reduction of the number of the reactive thymo-
cytes. Few medullary thymocytes revealed the
membrane immune-reactivity (Figure 6b). In
PD+H thymus, there was a recovery of the total
number of reactive thymocytes. Also in the
medulla, the thymocytes were reactive (Figure
6c). In PD+Th thymus, also in both experimen-
tal samples, there was a good recovery of the
number of the immune-reactive thymocytes as
in the cortex and as in the medulla (Figure 6d).
Image analyses and calculation of CD3 positive
thymocytes reported in Figure 7 revealed that in
In normal thymus, the cortical thymocytes
revealed a strong membrane immune-staining,
PD and in PD+H there was no significant differ-
ence in cellular density compared with normal
thymus. On the contrary, in the PD+Th there
was a significant increment (P<0.001) of the
number of CD3 positive cells respect to other
experimental groups, together with an enhance-
ment of the number of thymocytes. In the medul-
la the groups of PD+H and PD+Th revealed both
a significant increment (P<0.001) of positive
cells as compared with normal and PD groups.
Figure 3. Two types of thymic residual grafts. (a) It is possible to note some remnant of
the histological structure. (b) Note the presence of some thymocytes (arrows), connective
(C) and adipose (A) tissue. Scale bar: a: 80 µm; b: 120 µm.
Figure 4. Anti-PCNA reaction. (a) Intense immune reaction in the normal cortical (C)
thymocytes, M=Medulla. Inset: magnification of the cortical positive thymocytes. (b) PD
thymus. In cortex, (C) few thymocytes of the external zone are strongly positive, the other
thymocytes are weakly stained, M=Medulla. (c) PD+H thymus. Good immune reaction in
the total cortical (C) thymocytes, M=Medulla. (d) PD+Th thymus. Considerable recovery
of the immune staining in the total cortical (C) thymocytes, M=Medulla. Scale bar: a,d:
37 µm; b: 220 µm; c: 250 µm.
[page 162][European Journal of Histochemistry 2010; 54:e37]
taken from partial decerebrate (including
hypophysis) chicken embryos and from
embryos grafted with a hypophysis or a thymus.
At the best of our knowledge, this is the first
time that a thymic graft was made on hypophy-
sectomized chicken embryos. As well as con-
firming previous histological observations for
the hypophysectomized chicken embryos and
for the embryos grafted with a hypophysis8,46,50,53
this study provides new data, showing that also
the graft of a thymus on the hypophysectomized
embryos brings to an improvement of the size of
cortex, medulla and of the epithelial clusters.
Anyway, both grafts do not recover completely
the morphological structure, probably because
after the hypophysectomy, the thymus is seri-
ously reduced and damaged already at d 12 and
so the functional contribution of grafts do not
allow the complete thymic maturation and dif-
ferentiation in 6 days.
Concerning PCNA immune-reaction, few
papers deal with thymocytes and thymus, in
contrast with the immune localization in dif-
ferent tissues.74This study confirms the data
on the PCNA immune-reactivity in normal
embryonic thymus, previously described.55,75
The strong PCNA thymocytes labelling we
detected throughout the control cortex is in
contrast with the hypophysectomized thymus,
where the strong PCNA labelling was present
only in the external cortex, whereas in the
other thymocytes of the remaining cortex the
PCNA reaction was faint, as confirmed by
This research provides new information on
the morpho-functional changes in the thymuses
Figure 5. Anti-PCNA-Density (number of
cells/mm²) of PCNA+thymocytes (detect-
ed by immune histochemistry) in thymus
of normal (N), partially decerebrated
(PD), partially decerebrated + hypophy-
seal graft (PD+H) and partially decerebrat-
ed + thymus graft (PD+Th). Data refer to
quantitative analysis on tissue section and
are expressed as mean ± SD. The two-tail
Student t-test for unpaired data shows a
significantly increase number of positive
cells in cortex (a: PD+Th vs. N, P<0.001;
b: PD+Th vs. PD, P<0.001; c: PD+Th vs.
PD+H, P<0.001). Note the decrement of
PD and PD+H vs. N (P<0.05).
Figure 6. Anti-CD3 immune reaction. (a) Normal thymus. Strong membrane immune
staining in the cortical (C) and medullary (M) thymocytes. Inset: magnification of posi-
tive cortical thymocytes. (b) PD thymus. Reduction of the number of cortical (C) and
medullary (M) reactive thymocytes. Inset: magnification of positive cortical thymocytes.
(c) PD+H thymus Recovery of the total number of cortical (C) and medullary (M) reac-
tive thymocytes. Inset: magnification of positive cortical thymocytes. (d) PD+Th thymus.
Consistent recovery of the number of immune reactive thymocytes in the cortex and in
the medulla. Scale bar: a: 28 µm; b,d: 40 µm; c: 50 µm.
Figure 7. Anti-CD3- Density (number of cells/mm²) of CD3+ thymocytes (detected by
immune histochemistry) in thymic cortex and medulla of normal (N), partially decere-
brated (PD), partially decerebrated+ hypophyseal graft (PD+H) and partially decerebrat-
ed + thymus graft (PD+Th). Data refer to quantitative analysis on tissue section and are
expressed as mean ± SD. The two-tail Student t-test for unpaired data shows a significant-
ly increase number of positive cells in cortex (a: PD+Th vs. N, P<0.001; b: PD+Th vs. PD,
P<0.001; c: PD+Th vs. PD+H, P<0.001). In medulla, PD+H and PD+Th evidence a sig-
nificant increment vs. N (a, P<0.001) and PD (b, P<0.001).
[European Journal of Histochemistry 2010; 54:e37] [page 163]
image analysis. These results are similar to the
ones found in previous researches55,76in the
thymus, in bursectomized embryos. These
results are in some way difficult to explain. In
fact, Kurki et al.77found that unstimulated
human peripheral blood T-lymphocytes were
PCNA negative and their expression was evi-
dent only after stimulation. The PCNA immune
reaction was thus indicated as a marker for T-
lymphocytes committed to DNA synthesis and
occuring later in G1 phase of the cell cycle;
whereas Turka et al.78tested peripheral blood T
lymphocytes and cell suspension of thymic tis-
sue from children under 3 years of age. They
found that PCNA protein higher level was pres-
ent in immature double positive thymocytes,
than both in single positive thymocytes and in
peripheral blood T lymphocytes. The PCNA dou-
ble positive thymocytes displayed low RNA con-
tent, characteristic of the resting cells in G0
phase. The authors78indicated that the high
levels of PCNA in these resting cells might
mean a differential regulation during lymphoid
development and contribute to the process of
thymic selection and found that thymocytes,
more or less expressed PCNA reaction.
Obviously we cannot deduce if the strong
reaction in every cortical thymocyte of the nor-
mal thymus may represent PCNA content only
in resting cells or in S phase cells, or in both.
On the contrary, in the thymocytes of the PD
embryos the faint reaction may indicate that
the thymocytes are in a different cell-division
cycle, as indicated by Bravo and MacDonald-
Bravo59in an immune fluorescence study in
3T3 cells. The authors described the presence
of two populations of PCNA/cyclin expressed in
resting cells and during the S phase from G1 to
G2, evident when formaldehyde fixation was
used, as we did in our research. In the medul-
la, thymocytes of both normal and PD embryos
do not express PCNA immune-reaction. This
finding, in our opinion, reveals that thymo-
cytes ceased to proliferate, or that the reaction
is very weak and no detectable.
Hypophyseal or thymic grafts stress differ-
ent aspects. In the case of the graft of hypoph-
ysis, the recovery of the localization of PCNA
thymocytes are similarly distributed as in the
normal thymus; however, the number of posi-
tive cells is dramatically inferior to the normal,
as revealed by statistical analysis. On the con-
trary, very interesting data are observed in
PD+Th (particularly in those with the graft
without histological structure), where an
enhancement of the number of PCNA positive
thymocytes, throughout the cortex, is surpris-
ingly higher than that found in normal thymus,
confirmed also by statistical analysis.
Like PCNA, the expression of the CD3 mark-
er also differs, to some extent, in normal and
in the experimental thymuses. In all the thy-
mocytes of normal embryos, throughout the
cortical zone, and the medulla, CD3 was well
expressed. This finding fits in with evidence
that at the time when prothymocytes prolifer-
ate and differentiate in the thymus, the cellu-
lar expression of both the T cell receptor (TCR)
and the non-polymorphic parts of the TCR
complex, are referred to as CD3 antigens.67
The TCR, associated with the molecular com-
plex CD3, is necessary for the membrane
expression of the αβ heterodimer and serves
to transmit the signal generated at the cell sur-
face to the interior and thereby to induce the
appropriate effectual function.79The kinetics
of T cell differentiation and maturation in the
thymus were studied in mammals and in
human66,67,80and it was shown that the
immune system of the chicken functions in a
similar way to that of mammals and human.65,81
In our experiments, in the cortex of PD and
PD+H, there is not a foreseeable decrease of
CD3 expression, as shown also by statistical
analysis. This finding could be explained by
the fact that the hypophysis does not interfere
in the expression of this marker on thymo-
cytes. On the contrary, in the cortex of PD+Th
there is a statistical significant increment of
positive cells, underlining the difference
between the poor role of the hypophysis and
the greater contribution of the thymus.
However, in the medulla, the graft of hypoph-
ysis or thymus, both provoked a strong statisti-
cal increment of positive cells vs. normal and
PD. The detected increment of positive cells in
cortex and in medulla of PD+Th, compared
with control, may be due to thymic growth fac-
tors and probably also to thymocytes coming
from the graft, permitting a better thymic dif-
ferentiation. It is more difficult to explain why
in the medulla of PD+H the increment
observed is statistical greater than that
observed in PD and mostly in the control. We
may suppose that the density per area of CD3
cells is greater because thymocytes do not dif-
ferentiate further on. In our opinion, these
data confirm that the lack of hypophysis caus-
es the thymic morphological under-develop-
ment and decreases the possibility of cortical
thymocytes to proliferate and differentiate.
The hypophyseal graft allows a partial recovery
of the morphological but not of the functional
aspect, while the thymic graft, mostly that
without histological structure, may substan-
tially influence the recovery of the thymic
functions, due to thymic growth factors and
probably also to an emigration of thymocytes.
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