Unilateral testis torsiyonu yapılan ratlarda karşı testiste prednizolon tedavisinin etkileri ve oluşan histopatolojik değişiklikler

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Introduction
The testicular torsion is an
acute disease, which appears especially in male adolescents and
observed in 1 of 158 males under
25 years old (1). Although the
diagnosis of the testicular torsion
is easy, the period that passes
between the beginning of torsion
and the treatment of the disease is
very important (2,3) The effects
of unilateral testicular torsion on
the contralateral testis and fertility
have been searched by several scientists but there is no consensus
on the level of these effects, the
treatment period and approaches
(1,2,4).
Some authors suggest that differences in pathological findings
* 3rd Urology Clinic, Ankara Dýþkapý Education
and Research Hospital, Ankara
**Department of Pathology, Ankara University
Faculty of Veterinary Medicine, Ankara
Reprint request: Dr. A.Levent Saðnak, Angoraevleri, Altýnbel Villalarý, Hitit Bulvarý No: 38/A,
Beysukent, Ankara
E-mail: leventsagnak@yahoo.com
Date submitted: November 15, 2006
Accepted: March 6, 2007
ARAÞTIRMA/ORIGINAL ARTICLE Gülhane Týp Dergisi 2007; 49: 67-71
© Gülhane Askeri Týp Akademisi 2007
Summary
We aimed to determine the effects of
unilateral testicular torsion on the contralateral testis and to evaluate different
treatment approaches. Forty pubertal
Sprague Downey rats were randomized
into 8 groups (Five in each group). In the
control group (Group 1), the sham operation was performed. In Group 2, the left
testis underwent torsion and was
returned to the scrotum. In the other
groups, the left testis underwent torsion
at 720o
counterclockwise and was fixed
to the scrotum wall. Then the surgical
procedures like detorsion (Group 3, 24
hours later) and orchiectomy (Group 4,
24 hours later and Group 5, 48 hours
later); intraperitoneal prednisolone injection in addition to orchiectomy (Group
6, 24 hours later and group 8, 48 hours
later), and intraperitoneal prednisolone
injection in addition to detorsion (Group
7, 24 hours later) were performed. All
the contralateral testes were removed 4
weeks after the torsion and evaluated
histopathologically. We observed degeneration at varying intensities in contralateral testes of all groups except for
the control group. We found a significant
difference in the diameters of seminiferous tubules of the contralateral testes in
Groups 2, 3, 4, 5 and 8 compared with
the control group. In Groups 6 and 7, no
significant changes were observed in the
diameters of seminiferous tubules of the
contralateral testes, and hyperemia,
edema and degeneration were less
prominent. We demonstrated that the
histopathological damage can be prevented by prednisolone even in the
detorsioned group by suppressing the
immunologic response.
Key words: Contralateral testis, histopathology, rat, testicular torsion
Özet
Unilateral testis torsiyonu yapýlan ratlarda karþý testiste prednizolon tedavisinin etkileri ve oluþan histopatolojik
deðiþiklikler
Bu çalýþmada unilateral testis torsiyonunun kontralateral testise olan etkilerinin araþtýrýlmasý ve deðiþik tedavi yaklaþýmlarýnýn deðerlendirilmesi amaçlanmýþtýr. Bu amaçla pubertal (yaklaþýk 2.5-
3 aylýk) 40 adet Sprague-Downey sýçan
rastlantýsal olarak 5'erli 8 gruba ayrýldý.
Kontrol grubunda (Grup 1) testislere
yalancý operasyon uygulandý. Grup 2'de
sol testis torsiyone edilip býrakýldý. Diðer
gruplarda sol testisler 720o
saat yelkovaný
tersine torsiyone edilip tespit edildi.
Daha sonra bu testise detorsiyon (Grup
3'de 24 saat sonra), orþiyektomi (Grup
4'de 24 saat sonra ve Grup 5'de 48 saat
sonra) gibi cerrahi müdahaleler ile bazý
gruplarda orþiyektomi (Grup 6'da 24 saat
sonra ve Grup 8'de 48 saat sonra) ve
detorsiyona (Grup 7'de 24 saat sonra)
ilaveten intraperitoneal kortizon uygulandý. Tüm kontralateral testisler torsiyondan 4 hafta sonra orþiyektomi ile alý-
narak histopatolojik deðerlendirmeleri
yapýldý. Histopatojik olarak kontrol grubu
dýþýnda tüm kontralateral testislerde
deðiþen þiddetlerde dejenerasyon mevcuttu. Seminiferöz tübül çaplarý arasýndaki
fark, Grup 6 ve Grup 7 dýþýnda önemli bulundu. Bu iki grupta kontralateral hiperemi,
ödem ve dejenerasyon da belirgin olarak
azdý. Bu bulgularýn eþliðinde, prednizolon
ile histopatolojik hasarýn detorsiyone grup
da dahil olmak üzere immunolojik cevabýn
baskýlanmasý yoluyla önlenebileceðini
söyleyebiliriz.
Anahtar kelimeler: Kontralateral testis,
histopatoloji, sýçan, testis torsiyonu68 · Haziran 2007 · Gülhane TD Saðnak et al.
after the torsion in the contralateral testis are due to decreased
blood flow and oxidative stress as
a result of sympathetic activation
(5-7), which is explained by
immunological mechanisms (8).
In this study, we examined the
effects of unilateral testicular torsion on the contralateral testis and
evaluated different treatment
approaches in an experimental rat
model.
Material and Methods
For this study, 40 pubertal
(2.5-3 months old) SpragueDowney rats which are known to
reach puberty 50±10 days after
the birth were used. The experiment was performed on the rats
by the permission of Ankara
University Veterinary Faculty
Ethic Commission. The rats were
randomized into 8 groups (Five in
each group) as follows: Group 1
(control): Sham operation was
performed to the left testis. Group
2 (torsion): Left testis was subjected to 720o
counterclockwise torsion and fixed to the scrotum subcutaneously with non-absorbable
sutures. Then, the scrotum was
closed with absorbable sutures.
Group 3 (torsion/detorsion 24
hours later): The left testis was
subjected to torsion with the same
surgical procedures performed in
Group 2. Twenty four hours later,
left testis was returned to its normal position (detorsion) and the
scrotum was closed with
absorbable sutures. Group 4 (torsion/ orchiectomy 24 hours later):
The left testis was subjected to
torsion with the same surgical
procedures performed in Group
2. Left orchiectomy was performed 24 hours after the torsion
and the scrotum was closed.
Group 5 (torsion/orchiectomy 48
hours later): The left testis was
subjected to torsion with the same
surgical procedures performed in
Group 2. Left orchiectomy was
performed 48 hours after the torsion and the scrotum was closed.
Group 6 (torsion/orchiectomy 24
hours later/prednisolone): 2 mg/
kg/day methyl prednisolone was
administered intraperitoneally to
the rats for 4 weeks after the same
procedures performed in Group
4. Group 7 (torsion/detorsion 24
hours later/prednisolone): 2 mg/
kg/day methyl prednisolone was
administered intraperitoneally to
the rats for 4 weeks after the same
procedures performed in Group
3. Group 8 (torsion/orchiectomy
48 hours later/prednisolone): 2
mg/kg/day methyl prednisolone
was administered intraperitoneally to the rats for 4 weeks after the
same procedures performed in
Group 5. Rats were anesthetized
with intraperitoneal penthobarbital sodium (30 mg/kg body
weight) and placed in the supine
position. After local cleansing
with povidone iodine, the left
testes of the rats were explored
following a scrotal incision. In the
control group, after the scrotal
incision and release of the left
testis, the scrotum was closed
with absor-bable sutures (Sham
operation). After the surgical procedures, all rats were put into separate cages for 24 hours and then
they were united with their
groups.
The rats were housed and
maintained in specific pathogenfree conditions. At the end of 4
weeks, orchiectomy was performed on the right testes of all
rats. The rats were necropsied,
and body weights and testicular
weights were determined.
The removed right testes were
fixed in Bouin's solution for 24
hours, sectioned, processed and
embedded in paraffin. Sections
(4- to 6-µm thick) of paraffinembedded tissues were stained
with Hematoxylin and Eosin
(H&E) and evaluated under the
light microscopy. For histopathological evaluation, we used a
method similar to three-grade
system of Mikuz (9). In this
method, the histopathologic findings such as edema, hyperemia,
degeneration and spermatogenesis
are evaluated in most simple
method as mild (+), moderate
(++) and severe (+++) in all
samples. According to this, in
mild (+) grade, the testicular
parenchyma shows edema of
interstice, slight blood extravasation and desquamation of the
germ cells. The moderate (++)
grade shows, diffuse hemorrragic
saturation of the interstice and
partial necrosis of germ cells. The
severe (+++) grade shows fully
developed hemorrhagic infarction
of the testis. The diameters of 10
seminiferous tubules were measured in each animal by the ocular
micrometer under the light
microscopy. The Kruskal Wallis
Anova with the Mann Whitney
U-test were then used for comparison.
Results
Histopathological changes appearing in contralateral testes in
all groups are shown in Table I.
The average testicular weights,
body weights and the diameters of
seminiferous tubules are summarized in Table II. We observed a
decrease in the diameters of seminiferous tubules in all groups
except for the control group
(Figure 1).
In Group 1, seminiferous
tubules in all contralateral testes
had regular structure demonstrating normal spermatogenesis
(Figure 1a).Cilt 49 · Sayý 2 · Gülhane TD Effects of torsion on contralateral testis · 69
In Group 2, mild hyperemia
and interstitial edema were
observed in all testes except for
one animal. There were vacuolar
and hydropic degenerations at
varying degrees in germinal
epithelium of the seminiferous
tubules. In some tubules, the germinal epithelium was completely
detached and the basal membrane
was in wavy structure. Spermatogenesis was observed in two
animals in 1-2 tubules (Figure
1b).
In Group 3, mild hyperemia
and interstitial edema were
observed in the testes of four animals. In one animal hyperemia
and edema were moderate. There
were vacuolar and hydropic
degenerations at varying degrees
in germinal epithelium. Spermatogenesis was observed in all animals.
In Group 4, mild to moderate
hyperemia and interstitial edema
were observed in the testes of four
animals. In one animal the edema
was severe. The basal membrane
was in wavy structure and the germinal epithelium of seminiferous
tubules was completely detached.
There were vacuolar and hydropic degenerations at varying
degrees in germinal epithelium
(Figure 1c). In one animal, the
degenerative changes were severe.
Spermatogenesis was observed in
one animal in 1-2 tubules.
In Group 5, mild to moderate
hyperemia and interstitial edema
were observed. Degenerative
changes at varying degrees were
observed in the germinal epithelium (Figure 2a). In some portions,
the tubular diameter was quite
diminished and the basal membrane was in wavy appearance.
Spermatogenesis was observed in
two animals in 1-2 tubules.
In Group 6, mild hyperemia
and interstitial edema were
observed in two animals. In all
seminiferus tubules, mild degenerative changes were observed
(Figure 2b). No spermatogenesis
was observed in two animals.
1a 1b
1c 1d
Figure 1a. Seminiferous tubules demonstrating normal spermatogenesis in the contralateral testis of a rat from the control group (Group 1). H&E x 90. 1b. Disordered
appearance of seminiferus tubules in the contralateral testis of a rat from the torsion
group (Group 2). The germinal epithelium that covers some tubules are completely
detached. H&E x 90. 1c. Moderate to severe degenerative changes in the germinal
epithelium and interstitial edema in the contralateral testis of a rat from the
torsion/orchiectomy 24 hours later group (Group 4). H&E x 90. 1d. Large nuclear cells
in the degenerated germinal epithelium with interstital edema in the contralateral testis
of a rat from the torsion/detorsion 24 hours later/prednisolone group (Group 5). H&E
x 90.
Table I. Histopathological findings in contralateral testis
_____________________________________________________________________
Groups Hyperemia Edema Degeneration Spermatogenesis
_____________________________________________________________________
1 -/- -/- -/- ++/+++
2 -/+ -/+ ++/+++ -/+
3 +/++ +/++ +/++ -/+
4 +/++ +/+++ +/+++ -/+
5 +/++ +/++ +/+++ -/+
6 -/+ -/+ +/+ -/+
7 -/+ -/+ +/++ -/++
8 -/++ -/+++ +/+++ -/++
_____________________________________________________________________
+: Mild, ++: Moderate, +++: Severe
Table II. The body weights, average contralateral testis weights, and the diameters
of seminiferous tubules
_____________________________________________________________________
Groups Body weight (gr) Testis weight (gr) Seminiferous tubule
(Mean±SD) (Mean±SD) diameter (micron) (Mean±SD)
_____________________________________________________________________
1 229.1±18.92 2.58±0.115 197.0±6.67
2 220.0±16.78 2.49±0.117 *178.8±10.77
3 255.6±8.78 2.88±0.219 *177.4±6.52
4 275.6±16.0 2.99±0.111 *160.8±7.64
5 263.8±8.53 3.12±0.103 *160.2±2.82
6 237.4±7.83 2.62±0.084 192.2±4.97
7 268.2±19.0 2.89±0.235 193.4±9.10
8 268.8±14.5 2.42±0.216 *173.8±9.34
_____________________________________________________________________
*: p<0.05 compared with the control group (Group 1)70 · Haziran 2007 · Gülhane TD Saðnak et al.
In Group 7, mild hyperemia
and interstitial edema were observed in three animals. Degenerative changes in the germinal
epithelium were observed at varying degrees. In one animal, we
observed large nuclear cells in the
degenerated epithelial cells
(Figure 1d). There was no spermatogenesis in one animal, and
the others had spermatogenesis at
varying degrees.
In Group 8, hyperemia and
interstitial edema were observed
in three animals at varying
degrees. Degenerative changes
were severe in two animals. In
these animals, the germinal
epithelium was completely detached and in some tubules numerous large nuclear cells were
observed. There was no spermatogenesis in these two animals
and the others had spermatogenesis at varying degrees.
The differences in the body
weights and testicular weights
between the groups were not significant (p>0.05). The difference
in the diameters of seminiferous
tubules between the groups was
evaluated with Mann-Whitney U
test. Significant decreases were
found in Groups 2, 3, 4, 5, and 8
compared with the control group
(p<0.05).
Discussion
Unilateral testicular torsion
has been shown to affect the contralateral testis histopathologically
in varying intensities depending
on the treatment method (9,10).
However, there is no consensus
on when the effects of the torsion
starts on the contralateral testis
and how the treatment approach
affects the outcome. A number of
studies showed that 4-6 hours of
torsion is adequate to affect the
contralateral testis (11). In addition, the degree of rotation is also
important. Sessions et al. reported
varying degrees of torsion ranging
from 180
o
to 1080
o
in patients who
underwent surgical exploration
for testicular torsion (12). It has
been reported that medium level
pathological changes start with
360 degree torsion, and full
infarction is seen after 4 hours of
720 degree torsion (13).
Nagler et al. demonstrated that
detorsion did not protect the
contralateral testis from atrophy
while orchiectomy could prevent
the contralateral testis damage (7).
Testicular torsion leads to an
immunologic response that damages the contralateral testis.
Orchiectomy results in removal
of the antigenic stimulus, which
in turn protects against the development of contralateral testicular
damage (14). However, detorsion
of the damaged testis might further increase the damage to the
contralateral testis due to the
remaining immune response. In
the present study, we have
demonstrated that unilateral testicular torsion caused a significant
decrease in the diameters of seminiferous tubules of the contralateral testis with degenerative
changes. Detorsion or orchiectomy performed 24 or 48 hours
after the torsion did not protect
the contralateral testis.
In experimental models of testicular torsion, it has been shown
that antispermatozooal humoral
immunity is activated following
testicular torsion in rats (15).
Immunocytochemical studies have demonstrated that IgM antibodies cross the blood-testis barrier of the contralateral testis 7
days after the infarction, which
are then replaced by IgG antibodies after 28 days, affecting the
spermatogenic and the supporting
cells of the seminiferous tubules
(16). In another study, anti-rat
IgG antibodies against spermatozoa antigens were identified in the
contralateral testes of animals subjected to unilateral testicular torsion for 12 and 24 hours, which
were then subjected to detorsion
(8). However, no antibody formation was detected in animals that
underwent orchiectomy 24 hours
following unilateral torsion. Immunosuppressive treatment with
anti-lymphocyte globulin (ALG)
has been shown to decrease the
contralateral testicular damage
induced by spermatic cord torsion, whereas ALG treatment plus
splenectomy prevents the contralateral testicular damage completely (7). In addition, Gulmez et
al. demonstrated that adjuvant
2a 2b
Figure 2a. Intensive degeneration in the germinal epithelium in the contralateral testis
of a rat from the torsion/orchiectomy 48 hours later group (Group 6). H&E x 380. 2b.
Normal spermatogenesis together with moderate degenerative changes in the contralateral testis of a rat from the torsion/orchiectomy 24 hours later/prednisolone group
(Group 7). H&E x 380.Cilt 49 · Sayý 2 · Gülhane TD Effects of torsion on contralateral testis · 71
immunotherapy with prednisolone following detorsion protected the contralateral testis from
ipsilateral testicular antigenic
stimulation (14). In our study, we
also noted that in groups that had
undergone orchiectomy or detorsion (Groups 6 and 7) 24 hours
following torsion, addition of
prednisolone treatment protected
the contralateral testes from
immunologic damage. We clearly
observed less hyperemia and
interstitial edema in these two
groups compared to the other
groups. The histopathological
findings correlated with minimum changes in the average seminiferous tubule diameters,
which were close to the control
group. Nevertheless, in Group 8,
orchiectomy performed 48 hours
following torsion plus prednisolone treatment did not prevent the contralateral testicular
damage confirming that the duration of torsion is the most important factor that determines the
degree of contralateral damage.
On the other hand, in some
experimental animal studies, no
damage to the contralateral testis
was reported following unilateral
testicular torsion with no changes
in the semen parameters (1,15). In
some publications, the immunological damage in the contralateral
testis after the unilateral torsion
were not also supported (1,17,18).
Nagler et al. studied the effects of
testicular torsion in prepubertal
rats (19). They found no damage
in the contralateral testis and concluded that prepubertal testicular
torsion may not have the same
implications as postpubertal torsion since the antisperm antibodies which cause immunologic
damage to the contralateral testis
do not appear before puberty.
Cosentino et al. have also worked
with prepubertal rats, and found
that the decrease in spermatogenesis following torsion is more
prominent in the contralateral
testis of pubertal rats compared to
the younger ones (20).
We conclude that the extent of
histological damage to the contralateral testis is dependent on the
duration of the torsion and the
treatment approach. These changes are observed less intensively in
animals that undergo orchiectomy, showing that the presence of
torsioned testis might be responsible for the contralateral damage
mediated by immunologic mechanisms. However, we have demonstrated that the histopathological damage can be prevented
by prednisolone even in the
detorsioned group by suppressing
the immunologic response.