Effects of hypophyseal or thymic allograft on thymus development in partially decerebrated chicken embryos: Expression of PCNA and CD3 markers

Article (PDF Available)inEuropean journal of histochemistry: EJH 54(3):e37 · September 2010with24 Reads
DOI: 10.4081/ejh.2010.e37 · Source: PubMed
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.
European Journal of Histochemistry 2010; volume 54:e37
Effects of hypophyseal or
thymic allograft on thymus
development in partially
decerebrated chicken embryos:
expression of PCNA and CD3
markers
M. Aita,
1
F. Benedetti,
1
E. Carafelli,
1
E. Caccia,
2
N. Romano
2
1
Department of Physiology and
Pharmacology “V. Erspamer”, Faculty
of Medicine, University “La Sapienza”,
Rome, Italy
2
Department of Environmental Science,
University of Tuscia, Viterbo, Italy
Abstract
Changes in chicken embryo thymus after
partial decerebration (including the hypoph-
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.
Introduction
The primary lymphatic organs in birds, thy-
mus and bursa of Fabricius, play a central role
in differentiating lymphocytes responsible for
specific immunological responses, such as cel-
lular (T lymphocytes, in thymus)
1,2
and humoral
(B lymphocytes, in bursa).
3
In 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-
tiated thymocytes or T-cells.
4-6
Chicken 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 cells
7,8
and cysts, of the intra- or inter-cellu-
lar type
8-11
are well visible, whereas macro -
phages, myoid cells and Hassall’s corpuscles are
rare. Lymphocytes are less numerous than in
the cortex.
The thymus, besides to have an immune
function, has also an endocrine function; in
fact endocrine-like cells were described in
embryonal chicken thymus.
9-12
The thymus
takes part in the mechanisms of reciprocal
effects between the immune and the neuro-
endocrine interactions in mammals and in
birds.
13-18
It has been characterized the influ-
ence of the hypothalamic-pituitary-gonadal
axis and that of thyroid hormone on the
immune system, in mammals
19-21
and in birds.
22
The production of several hormones has
been described in the thymus of mammals and
birds, as steroids,
23
glucocorticoids,
24-26
growth
hormone,
27
POMC-derived peptides,
28-30
and
neuropeptides.
31,32
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.
33-35
In birds, a specific hormonal factor,
the avian thymic hormone (ATH), a parvalbu-
min, has been extracted and localized only in
cortical reticular-epithelial cells.
36-38
The
thymic factor thymostimulin was extracted
from calf thymus
39
and detected, using an anti-
thymostimulin antibody, in mammals
40,41
and
humans.
42
In avian embryonal, post hatching
and aging thymuses
43-46
the 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.
49-51
Romano et
al.,
8
in 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 Romano
46
noted 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.,
52
making
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 cells
53
with a considerable
recovery of the cytology, even if a normal mor-
phology is not completely accomplished
8
and
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
embryos
8,17,21
and 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.
54,55
The 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 cycle
56-60
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.
62,63
Bravo et al.,
64
reported
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.
Tel. +39.06.49910734.
E-mail: mariangela.aita@uniroma1.it
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
doi:10.4081/ejh.2010.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
Experiments
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;
47
ii)
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.
71
We 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
embryos.
Thymic specimens
Two thymic lobes from right side and two
from left side of the neck from normal, sham-
operated, PD, PD+H and PD+Th, were fixed in
Bouin’s liquid for 8 h at room temperature,
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
The method used for the anti-PCNA/cyclin
antibody reaction has been reported in detail
in previous studies.
72,73
In 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
serum alone.
Anti-CD3 immune reaction
Thymic sections were treated with a PAP
method using an anti-CD3 antibody (a pan-T
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.
Statistical analysis
In each specimen, multiple sets of consecu-
tive sections were differentially immune-
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.
Original paper
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]
Results
Since no morphological or immune-histo-
chemical differences were found between thy-
muses from normal and sham-operated
embryos, we henceforward refer to both groups
as normal embryos.
Histological findings
In normal embryos of 12 d, the thymus was
set up by a cortex divided in lobes and a central
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
(Figure 3b).
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
Original paper
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
In normal thymus, an intense immune-reac-
tion was located in the nuclei of cortical thy-
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).
Anti-CD3 immune reaction
In normal thymus, the cortical thymocytes
revealed a strong membrane immune-staining,
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
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.
Original paper
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]
Discussion
This research provides new information on
the morpho-functional changes in the thymuses
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 hypophysis
8,46,50,53
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.
74
This 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
Original paper
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 researches
55,76
in the
thymus, in bursectomized embryos. These
results are in some way difficult to explain. In
fact, Kurki et al.
77
found 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.
78
tested 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 authors
78
indicated 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-
Bravo
59
in 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.
79
The kinetics
of T cell differentiation and maturation in the
thymus were studied in mammals and in
human
66,67,80
and 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.
References
1. Le Douarin NM, Dieterlen-Lievre F, Oliver
PD. Ontogeny of primary lymphoid organs
and lymphoid stem cells. Am J Anat 1984;
170:261-99.
2. Cooper MD, Chen CL, Bucy RP, Thompson
CB. Avian T cell ontogeny. Adv Immunol
1991;50:87-117.
3. Glick B. The Bursa of Fabricius: the evolu-
tion of a discovery. Poultry Sci 1994;73:
979-83.
4. Aita M. Regulation of cellular immunology:
general aspects of the function of the
thymic microenvironment. In: Wegmann
RJ and Wegmann MA eds. Recent Adv in
Cell Mol Biol, vol 1. Peeters Press, Leuven,
Belgium 1992, pp.1-10.
5. Boyd RL, Wilson TJ, Bean AG, Ward HA,
Gershwin ME. Phenotipic characterization
of chicken thymic stromal elements. Dev
Immunol 1992;2:51-66.
6. Wilson TJ, Davidson NJ, Boyd RL, Gershin
ME. Phenotypic analysis of the chicken
thymic microenvironment during onto-
genic development. Dev Immunol 1992;2:
19-27.
7. Kendall MD. Avian thymus gland: a
Review. Dev Comp Immunol 1980;4:191-
210.
8. Romano N, Casini P, Abelli L, Mastrolia L,
Aita M. Influence of partial decerebration
and hypophyseal allograft on differentia-
tion of thymic epithelial cells in chick
embryo and ultrastructural study. Anat
Embryol 1996;193:593-600.
9. Chan AS. Ultrastructural observations on
cytodifferentiation of thymic cystic cells of
chick embryo. Thymus 1991;17:115-22.
10. Chan AS. Ultrastructure of epithelial cells
of the chick embryo thymus. Acta
Anatomica 1994;150:96-103.
11. Aita M, Mazzone AM, Gabrielli F,
Evangelista A, Brenna S. Identification of
cells secreting a thymostimulin-like sub-
stance and examination of some histoen-
zymatic pathways in aging avian primary
lymphatic organs: I. Thymus. Eur J
Histochem 1995;39:289-300.
12. Crivellato E, Nico B, Ribatti D. Endocrine-
like cells in the Chick Embryo Thymus
express ultra structural features of piece-
meal degranulation. Anat Rec A Discov Mol
Cell Evol Biol 2005;282:106-9.
13. Comsa J, Leonhardt H, Wekerle H.
Hormonal coordination of the immune
response. Rev Physiol Biochem Pharmacol
1982;92:115-91.
14. Blalock JE. A molecular basis for bidirec-
tional communication between the
immune and neuroendocrine systems.
Physiol Rev 1989;69:1-32.
15. Marchetti B, Morale MC, Pelletier G. The
thymus gland as a major target for the cen-
tral nervous system and the neuroen-
docrine system: neuroendocrine modula-
tion of thymic β2-adrenergic receptor dis-
tribution as revealed by in vitro autoradi-
Original paper
[page 164] [European Journal of Histochemistry 2010; 54:e37]
ography. Mol Cell Neurosci 1990;1:10-9.
16. Marchetti B, Morale MC, Pelletier G.
Sympathetic nervous system control of rat
thymus gland maturation: autoradiograph-
ic localization of the β2-adrenergic recep-
tors in the thymus and presence of sexual
dimorphis during ontogeny. Progress
Neuroimmunol 1990;3:103-15.
17. Besedovsky HO, Del Rey A. Immune-
neuro-endocrine interactions: facts and
hypotheses. Endocr Rev 1996;17:64-102.
18. Savino W, Dardenne M. Neuroendocrine
control of thymus physiology. Endocr Rev
2000;21:412-43.
19. Marchetti B. Involvement of the thymus in
reproduction. Prog. Neuroendocri -
neimmunol 1989;2:64-9.
20. Marchetti B, Guarcello V, Morale MC,
Bartolini G, Farinella Z, Cordaro S, et al.
Luteinizing hormone-releasing-binding
sites in the rat thymus: characteristics and
biological function. Endocrinology 1989;
125:1025-36.
21. Klecka AJ, Genaro AM, Gorelik G, Barreiro
Arcos ML, Silberman DM, Schuman M, et
al. Integrative study of hypothalamus-pitu-
itary-thyroid-immune system interaction:
thyroid hormone-mediated modulation of
lymphocyte activity through the protein
kinase C signaling pathway. J Endocrinol
2006;189:45-55.
22. Lutton B, Callard I. Evolution of reproduc-
tive-immune interactions. Integrative
Comp Biol 2006;46:1060-71.
23. Vacchio MS, Papadopoulus V, Ashwell JD.
Steroid production in the thymus: implica-
tions for thymocyte selection. J exp Med
1996;179:1835-46.
24. Ottaviani E, Franchini A, Franceschi C.
Presence of immunoreactive cortico -
tropin-releasing hormone and cortisol
molecole in invertebrate haematocytes
and lower and higher vertebrate thymus.
Histochem J 1998;30:61-7.
25. Lechner O, Dietrich H, Wiegers GJ,
Vacchio M, Wick G. Glucocorticoid produc-
tion in the chicken bursa and thymus. Int
Immunol 2001;13:769-76.
26. Gomez-Sanches CE. Glucocorticoid pro-
duction and regulation in thymus of mice
and birds. Endocrinology 2009;150:3977-9.
27. Hull KL, Thiagarajah A, Harvey S. Cellular
localization of growth hormone recep-
tors/binding proteins in immune tissues.
Cell Tissue Res 1996;286:69-80.
28. Ottaviani E, Franchini A, Franceschi C.
Evolution of neuroendocrine thymus: stud-
ies on POMC-derived peptides, cytokines
and apoptosis in lower and higher verte-
brates. J Neuroimmunol 1997;72:67-74.
29. Franchini A, Ottaviani E. Immunoreactive
POMC-derived peptides and cytokines in
the chicken thymus and bursa of Fabricius
microenvironments: age-related changes.
J Neuroendocrinol 1999;11:685-92.
30. De Luca A, Squillacioti C, Pero ME, Paino
S, Langella E, Mirabella. Urocortin-like
immuno reactivity in the primary lym-
phoid organs of the duck (Anas platyrhyn-
chos). Eur J Histochem 2009;53:167-76.
31. Atoji Y, Yamamoto Y, Suzuki Y. Neuro -
tensin-containing endocrine cells and
neurotensin receptor mRNA-expressing
epithelial cells in the chicken thymus.
Arch Histol Cytol 1996;59:197-203.
32. Atoji Y, Yamamoto Y, Komatsou T, Suzuki
Y. Localization of neuropeptides in
endocrine cells of the chicken thymus. J
Vet Med Sci 1997;59:601-3.
33. Goya RG, Brown OA, Bolognani F. The thy-
mus-pituitary axis and its changes during
aging. Neuroimmunomodulation 1999;6:
137-142.
34. Greeps RO (ed) Recent progress in hor-
mone research., vol. 37, 1981. Acad Press
London, UK.
35. Fabien N, Auger C, Monier J-C. Immuno -
localization of thymosin α1, thymopoietin
and thymulin in mouse thymic epithelial
cells of different stages of culture: a light
and electron microscopic study. Immu -
nology 1988;63:721-7.
36. Monier JC, Auger C, Fabien N. Les hor-
mones thymiques. Arch Int Physiol
Biochim 1988;96:A2-26.
37. Brewer JM, Wunderlich JK, Ragland WL.
The amino acid sequence of avian thymic
hormone, a parvalbumin. Biochimie 1990;
72:653-60.
38. Kiràly E, Celio MR. Parvalbumin and calre-
tinin in the avian thymus. Anat Embryol
(Berl) 1993;188:339-44.
39. Falchetti R, Bergesi G, Eshkol A, Cafiero C,
Adorini L, Caprino L. Pharmacological and
Biological properties of a calf thymus
extract (TP1). Drugs Exp Clin Res 1977;3:
39-47.
40. Aita M, Cocchia D, Minella AB, Amantea A.
Identification of thymostimulin secreting
cells in calf thymus by immunoperoxidase
method. Histochemistry 1984;80:207-11.
41. Aita M, Minella AB, Palermo D.
Localization of thymostimulin in mouse
and rat thymuses. Basic Appl Histochem
1986;30:53-9.
42. Aita M, Minella AB, Palermo D, Gabrielli F,
Franzé A. Localization of thymostimulin in
mammalian thymuses: comparative evalu-
ation. Cell Mol Biol 1989;35:137-45.
43. Aita M, Amantea A. Distribution of anti-
keratins and anti-thymostimulin antibod-
ies in normal and in Down’s syndrome
human thymuses. Thymus 1991;17:155-65.
44. Aita M, Brenna S, Mazzone AM. Thymo -
stimulin-like immunoreactions in avian
thymus and Bursa of Fabricius. Basic Appl
Histochem 1989;3:suppl 9.
45. Aita M, Mazzone AM, Gabrielli F,
Evangelista A, Brenna S. Identification of
cells secreting a thymostimulin-like sub-
stance and examination of some hystoen-
zimatic pathways in aging avian primary
lymphatic organs: I. Thymus. Eur J
Histochem 1995;39:289-300.
46. Aita M, Romano N. Effects of partial decer-
ebration and hypophyseal allograft in the
thymus of chicken embryos: thymostim-
ulin localization and enzymatic activities.
Eur J Histochem 2006;50:69-78.
47. Fugo NW. Effects of hypophysectomy in the
chick embryo. J exp Zool 1940;85:271-91.
48. Betz TW. The effects of embryonic pars
distalis grafts on the development of
hypophysectomized chick embryos. Gen
comp Endocrinol 1967;9:172-186.
49. Jankovic BD, Isakovic K, Micic M,
Knezevic Z. The embryonic-lympho-neuro-
endocrine relationship. Clin Immunol
Immunopathol 1981;18:108-20.
50. Mastrolia L, Aita M, Romano N, Gallarello
F, Jamele M, Manelli H. Histological study
of the primary lymphatic organs in partial-
ly decerebrated chicken embryos. Acta
Embryol Morphol Exper 1986;7:112.
51. Herradòn PG, Razquin B, Zapata Ag.
Effects of early partial decapitation on the
ontogenic development of chicken lym-
phoid organs. I. Thymus. Am J Anat 1991;
191:57-66.
52. Moreno J, Vicente A, Varras A, Zapata A G.
T-cell development in early partially
decapited chicken embryos. Dev Immunol
1995;4:211-26.
53. Mastrolia L, Aita M, Romano N,
Evangelista A, Manelli F, Manelli H. Effects
of hypophyseal grafts on thymus and bursa
of Fabricius in partially decerebrated chick
embryos Acta Embryol Morphol Exper
1987;8:419-26.
54. Aita M, Carafelli E, Zamponi F, Alfei L.
Distribution of calcium-binding proteins
and CD3, PCNA/cyclin in thymuses of par-
tially decerebrated and thymic-grafted
chicken embryos. 1999, Congress of
Società Italiana di Fisiologia, Rome, Italy,
p 195 (abstr).
55. Aita M, Carafelli E, Alfei L, Caronti B.
Thymic development in surgically bursec-
tomized embryonic chicken: expression of
PCNA, CD3, CD4 and CD8 markers. Eur J
Histochem 2007;51:241-50.
56. Bravo R. Coordinated synthesis of the
nuclear protein cyclin and DNA in serum-
stimulated quiescent 3T3 cells. FEBS Lett
1984;169: 185-8.
57. Bravo R. Synthesis of the nuclear protein
cyclin (PCNA) and its relationship with
DNA replication. Exp Cell Res 1986;163:
287-93.
Original paper
[European Journal of Histochemistry 2010; 54:e37] [page 165]
58. Bravo R, Graf T. Synthesis of the nuclear
protein cyclin does not correlate directly
with transformation in quail embryo
fibroblasts. Exp Cell Res 1985;156:450-4.
59. Bravo R, Macdonald-Bravo H. Existence of
two populations of cyclin/proliferative cell
nuclear antigen during the cell cycle: asso-
ciation with DNA replication sites. J Cell
Biol 1987;105:1549-54.
60. Bravo R, Fey SJ, Bellatin J, Larsen PM,
Arevalo J, Celis JE. Identification of a
nuclear and of a cytoplasmic polipeptide
whose relative proportions are sensitive to
changes in the rate of cell proliferation.
Exp Cell Res 1981;136:311-9.
61. Mathews MB, Bernstein RM, Franza BR jr,
Garrels JI. Identity of the proliferating cell
nuclear antigen and cyclin. Nature 1984;
309:374-6.
62. Crute JJ, Wahl AF, Bambara RA.
Purification and characterization of two
new high molecular weight forms of DNA
polymerase δ. Biochemistry 1986;25:26-36.
63. Tan CK, Castillo C, So AG, Downey KM. An
auxiliary protein for DNA polymerase-
delta from fetal calf thymus. J Biol Chem
1986;261:12310-16.
64. Bravo R, Frank R, Blundell PA, Macdonald-
Bravo H. Cyclin/PCNA is the auxiliary pro-
tein of DNA polymerase-delta. Nature
1987;326:515-7.
65. Chan MM, Chen CH, Ager LL, Cooper MD.
Identification of the avian homologues of
mammalian CD4 and CD8 antigens. J
Immunol 1988;140:2133-8.
66. Strominger JL. Developmental Biology of T
cell receptors. Science 1989;244:943-50.
67. Janossy G, Campana D, Akbar A. Kinetics
of T lymphocyte development. Curr Top
Pathol 1989;79:59-99.
68. Dunon D, Allioli N, Vainio O, Ody C, Imhof
BA. Renewal of thymocyte progenitors and
emigration of thymocytes during avian
development. Dev Comp Immunol 1998;22:
279- 87.
69. Dunon D, Imhof BA. T cell migration dur-
ing ontogeny and T cell repertoire genera-
tion. Curr Top Microbiol Immunol 1996;
212:79-93.
70. Dunon D, Imhof BA. The role of cell traffic
in the emergence of the T lymphoid sys-
tem. Semin Immunol 2000;12:429-33.
71. Hamilton H. Lillie’s development of the
chick: an introduction to embryology. Halt
H (ed.) NY, USA, 1952, pp. 78-91.
72. Waseem NH, Lane DP. Monoclonal anti-
body analysis of the proliferating cell
nuclear antigen (PCNA). Structural con-
servation and the detection of a nucleolar
form. J Cell Sci 1990;196:121-9.
73. Alunni A, Pierucci F, Aita M, Margotta V, De
Vita R, Alfei L. Proliferative activity and
motoneurone recruitment persist at the
spinal cord central canal during larval and
some postlarval stages in the rainbow
trout (Oncorhynchus mykiss). Eur J
Histochem 2001;45:191-202.
74. Yu CC-W, Woods AL, Levison DA. The
assessment of cellular proliferation by
Immunohistochemistry: a review of cur-
rently available methods and their applica-
tions. Histochem J 1991;24:121-31.
75. Aita M, Caccia E, Romano N. Thymus
development is influenced by hypophyseal
and thymic allograft after hypophysecto-
my: expression of PCNA and CD3 markers.
Invertebr Surv J 2008;5:38.
76. Aita M, Romano N. Thymic morpho-func-
tional changes after hypophysectomy and
bursectomy in chicken embryos. Invertebr
Surv J 2007;4:29-30.
77. Kurki P, Lotz M, Ogata K, Tan EM.
Proliferating cell nuclear antigen (PCNA)/
cyclin in activated human T lymphocytes. J
Immunol 1987;138:4114-20.
78. Turka LA, Gratiot-Deans J, Keim D,
Bandukwala R, Green J, Strahler J, et al.
Elevated proliferating cell nuclear antigen
levels in immature thymocytes. J Immunol
1993;150:2746-52.
79. Blackman M, Kappler J, Marrack P. The
role of the T cell receptor in positive and
negative selection of developing T cells.
Science 1990;248:1335-41.
80. Campana D, Janossy G, Coustan-Smith E,
Amlot PL, Tian WT, Ip S, et al. The expres-
sion of T cell receptor-associated proteins
during T cell ontogeny in man. J Immunol
1989;142:57-66.
81. Vainio O, Imhof BA. The immunology and
developmental biology of the chicken.
Immunol Today 1995;16:365-70.
Original paper
    • "Although the intrathymic selection process is only partially understood in fish, interactions between developing thymocytes and stromal elements have been evidenced during ontogeny [5,11,16,25]. Essentially, to support the maturation process, the epithelial milieu must be efficiently differentiated, as shown in mammals [28,29], birds [30] and fish [11,31]. In fish, including the sea bass, different compartments of the thymus (cortex, medulla and corticalemedullary border, BCM) are recognisable by histology and cytology of epithelial/lymphoid elements [6,10; personal observation]. "
    [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.
    Full-text · Article · Mar 2011
    • "The potential of histochemical and microscopical techniques for investigating stem cell biology and embryonic development was underlined during a symposium held in Pavia (Italy) in 2008,118 and several articles have been devoted to differential gene expression during the embryogenesis of organs and systems from different animal species,6,119–128 with special attention to the lymphoid organs in birds,129,130 and mammals,35,43 during their development under unperturbed or experimental conditions. Interestingly, the functional role of haemocytes has also been studied in some Invertebrates131,132 providing important clues on the origin and evolution of the immune system in vertebrates. "
    [Show abstract] [Hide abstract] 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.
    Full-text · Article · Dec 2010
  • [Show abstract] [Hide abstract] 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.
    Full-text · Article · Oct 2012

  • undefined · undefined
  • undefined · undefined
  • undefined · undefined