Protective effects of immunophilin ligands on testicular torsion/detorsion damage in rats

Article (PDF Available)inInternational Urology and Nephrology 41(1):93-99 · March 2009with38 Reads
DOI: 10.1007/s11255-008-9453-5
Abstract
Objectives: The purpose was to investigate the role of immunophilin ligands in ischemia/reperfusion (I/R)-induced germ cell apoptosis in the rat. Materials and methods: Sprague-Dawley rats were divided into five groups with ten animals in each. In animals undergoing torsion/detorsion, right testes were rotated 720 degrees for 1 h. A baseline group was for basal normal values. The sham-operated group served as a control group. The TD group underwent torsion/detorsion surgery alone; the cyclosporine-A group (TD-CsA) received intravenous cyclosporine injection (5 mg/kg) at the time of detorsion, and the FK-506 group (TD-FK) received intravenous FK-506 (3.5 mg/kg) at the time of detorsion. For measurement of lipid peroxidation and antioxidant enzyme activities, the right testes of five animals in each group were excised after 4-h reperfusion. Germ cell apoptosis indices were determined 24 h following detorsion in the right testes of the remaining five animals in each group. Results: Malondialdehyde (MDA) levels in the TD group were significantly higher compared to control and baseline groups. Moreover, testicular MDA values in TD-CsA and TD-FK groups were significantly lower than in TD. There were also significant decreases in catalase and superxide dismutase activities in the TD group compared to control and baseline groups. These values in TD-CsA and TD-FK groups were significantly higher than in TD. The mean germ cell apoptosis scores were significantly higher in TD animals compared to control and baseline groups; however, CsA and FK-506 treatment significantly reduced the apoptosis compared with the TD group. Conclusion: We have shown that administration of immunophilin ligands in testicular torsion decreases ischemia/reperfusion (I/R) cellular damage. The results of biochemical studies suggest that reduction of oxidative stress along with attenuated neutrophil accumulation by immunophilin ligands may have a major role in their cytoprotective effects.

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UROLOGY - ORIGINAL PAPER
Protective effects of immunophilin ligands on testicular
torsion/detorsion damage in rats
Behtash Ghazi Nezami Æ Sina Rahimpour Æ Taha Gholipour Æ
Seyedmehdi Payabvash Æ Reza Rahimian Æ Seyed Mohammad Tavangar Æ
Seyed Hassan Emami-Razavi Æ Ahmad Reza Dehpour
Received: 29 February 2008 / Accepted: 2 August 2008
Ó Springer Science+Business Media, B.V. 2008
Abstract
Objectives The purpose was to investigate the role
of immunophilin ligands in ischemia/reperfusion
(I/R)-induced germ cell apoptosis in the rat.
Materials and methods Sprague–Dawley rats were
divided into five groups with ten animals in each. In
animals undergoing torsion/detorsion, right testes
were rotated 7208 for 1 h. A baseline group was for
basal normal values. The sham-operated group served
as a control group. The TD group underwent torsion/
detorsion surgery alone; the cyclosporine-A group
(TD-CsA) received intravenous cyclosporine injec-
tion (5 mg/kg) at the time of detorsion, and the FK-
506 group (TD-FK) received intravenous FK-506
(3.5 mg/kg) at the time of detorsion. For measure-
ment of lipid peroxidation and antioxidant enzyme
activities, the right testes of five animals in each
group were excised after 4-h reperfusion. Germ cell
apoptosis indices were determined 24 h following
detorsion in the right testes of the remaining five
animals in each group.
Results Malondialdehyde (MDA) levels in the TD
group were significantly higher compared to control
and baseline groups. Moreover, testicular MDA
values in TD-CsA and TD-FK groups were signifi-
cantly lower than in TD. There were also significant
decreases in catalase and superxide dismutase activ-
ities in the TD group compared to control and
baseline groups. These values in TD-CsA and TD-FK
groups were significantly higher than in TD. The
mean germ cell apoptosis scores were significantly
higher in TD animals compared to control and
baseline groups; however, CsA and FK-506 treatment
significantly reduced the apoptosis compared with the
TD group.
Conclusion We have shown that administration of
immunophilin ligands in testicular torsion decreases
ischemia/reperfusion (I/R) cellular damage. The
results of biochemical studies suggest that reduction
of oxidative stress along with attenuated neutrophil
accumulation by immunophilin ligands may have a
major role in their cytoprotective effects.
B. G. Nezami S. Rahimpour T. Gholipour
S. Payabvash A. R. Dehpour
Basic Medical Sciences Research Center, Imam Khomeini
Hospital, Tehran University of Medical Sciences, Tehran,
Iran
B. G. Nezami S. Rahimpour T. Gholipour
S. Payabvash R. Rahimian A. R. Dehpour (&)
Department of Pharmacology, School of Medicine,
Tehran University of Medical Sciences,
P.O. Box 13145-784, Tehran, Iran
e-mail: dehpour@yahoo.com
S. M. Tavangar
Department of Pathology, Shariati Hospital, Tehran
University of Medical Sciences, Tehran, Iran
S. H. Emami-Razavi
Iranian Tissue Bank Research Center, Imam Khomeini
Hospital, Tehran University of Medical Sciences,
Tehran, Iran
123
Int Urol Nephrol
DOI 10.1007/s11255-008-9453-5
Keywords Testicular torsion Ischemia
Apoptosis Antioxidant enzyme Cyclosporine
FK-506
Introduction
Testicular torsion is a pathologic condition that
renders the testis ischemic, and surgical intervention
is usually required to reestablish blood flow [1]. Even
with successful surgical repair, loss of spermatogen-
esis and a significant increase in germ cell apoptosis
may occur due to an increase in testicular oxidative
stress concomitant with reperfusion [2]. Identification
of pharmacological agents, administered as adjunc-
tive therapy to surgical repair for rescuing the testis
from ischemia/reperfusion (I/R) injury, is a clinically
important goal.
Cyclosporine and FK-506 (also known as tacrol-
imus) are classified as immunophilin ligand agents,
which have revolutionized transplantology due to
their ability to block the activation of lymphocytes
and immune system cells [3, 4]. Whereas they are
routinely used to impede graft rejection in transplan-
tation of organs and bone marrow [5], other
capabilities have been uncovered, most of which
concern a vigorous protecting effect against cell
damage in ischemic and stressed tissue [6]. The
classic known mechanism of action of these drugs in
cells is binding to specific intracellular proteins called
immunophilins and regulating the activity of calcine-
urine (a Ca
2?
/calmodulin-dependent phosphatase)
[7]. During the last decade, newer complexities have
emerged in the molecular aspects of action of these
drugs. Their potent biologic properties in inhibiting
programmed cell death and necrosis in stressed
tissues is thought to be through mitochondrial
enhancements via mitochondrial permeability transi-
tion pores (MPTP). This leads to an optimization in
cellular energy consumption in pathologic situations
[8, 9].
The aim of this study was to investigate the anti-
ischemic properties of CsA and FK-506 administered
after ischemia in a rat model of testicular torsion/
detorsion (T/D). It is also of clinical importance to
find a drug that can be used before testicular
detorsion to reduce testicular damage.
Materials and methods
Drugs
Drugs were purchased from following sources: CsA
(Zakaria Pharmaceutical, Tabriz, Iran), FR 900506
(placebo) and FK-506 (Fujisawa Pharmaceuticals,
Osaka, Japan), ketamine HCl (Gedoon Richter Ltd.,
Budapest, Hungary), and chlorpromazine (Bayer AG,
Leverkusen, Germany). CsA was dissolved in 0.1%
dimethylsulfoxide (DMSO), FK-506 and placebo, in
0.005% DMSO. All freshly prepared drug prepara-
tions were used the same day.
Animals
Fifty adult male Sprague–Dawley rats weighing 250–
300 g bred in the faculty animal reproduction labo-
ratory were used. All animals received care in
compliance with the recommendation of the animal
care committee of the university and in accordance
with the ‘Guide for the Care and Use of Laboratory
Animals’ (National Research Council, revised in
1996). Rats were housed in a temperature-controlled
room (24 ± 1°C) on a 12-h light/12-h dark cycle and
were given ad libitum access to food and water.
Groups
The baseline group was for basal normal values. The
sham operated group served as control group. The TD
group underwent 1 h testicular torsion. The cyclo-
sporine-A group (TD-CsA) received a single
intravenous cyclosporine injection (5 mg/kg), and
the FK-506 group (TD-FK) received intravenous
FK-506 (3.5 mg/kg), both at the time of detorsion.
Animal preparation and surgical procedure
The rats were divided into five groups of ten animals
each. Surgical procedures were done under general
anesthesia, induced by intraperitoneal injection of
ketamine HCl (50 mg/kg) and chlorpromazine
(25 mg/kg). The testis was rotated as described by
Lysiak et al. [2]. By use of sterile procedures, right
testes were exteriorized through a low midline
laparotomy. The gubernaculum was divided, with
the testicular and scrotal stumps remaining connected
by a loose, 4–0 silk suture. The membrane connecting
Int Urol Nephrol
123
the epididymis to the testis was divided up to the
testicular hilum, and the testis was rotated 720°. The
laparotomy was closed, and the testis was left in
torsion for 1 h. Then, the torsion was repaired by
opening the laparotomy, counter-rotating the testis to
the natural position, and joining the gubernacular
stumps with a 4–0 silk suture. At the time of repair, the
testes were examined to ensure that they had remained
in torsion, and the testes were scored qualitatively for
ischemic appearance. Additionally, testes were exam-
ined 5 min after counter-rotation to estimate the
quality of reperfusion. Testes were returned to the
scrotum until the time of tissue collection. Sham
operated animals received the same operation, but the
testicular rotation was relieved immediately. For
measurement of lipid peroxidation and antioxidant
enzyme activities, right testes of five animals in each
study group were excised 4 h after beginning of
reperfusion, and right testes of the remaining five
animals in each group was collected 24 h following
detorsion to determine germ cell apoptosis indices.
Malondialdehyde assay
Malondialdehyde (MDA) is the product of peroxida-
tive decomposition of polyenic fatty acids in the lipid
peroxidation process, and its accumulation in tissues is
indicative of the extent of lipid peroxidation. Tissue
MDA was measured using the thiobarbituric acid
(TBA)-reactive substance assay, as described by
Ohkawa et al. [10]. In brief, testis tissues were
homogenized in 1.15% KCl to make a 10% (w/v)
homogenate. To 0.1 ml of tissue homogenates were
added 0.9 ml of 1.8% sodium dodecyl sulfate (SDS),
1.5 ml of 20% acetic acid solution (pH 3.5), and 1.5 ml
of aqueous solution of TBA. The mixtures were heated
at 95°C for 60 min. After cooling with tap water, 5 ml
of the mixture of n-butanol and pyridine (15:1, v/v)
was added, shaken vigorously, and then centrifuged at
4,000 rpm for 10 min. The organic layer was taken,
and its absorbance at 532 nm was measured. 1,1,3,3-
Tetramethoxypropane (TMP) was used as an external
standard, and the level of lipid peroxides was
expressed as nmol of MDA per gram wet tissue [11].
Catalase activity
Tissue catalase (CAT) activity was measured accord-
ing to method of Aebi [12]. Tissue sections were
homogenized in triton X-100 (1%), and the homoge-
nates were diluted with potassium phosphate buffer.
The reaction was initiated by the addition of hydrogen
peroxide (H
2
O
2
) to reaction mixture, and the level of
enzyme activity was quantitated according to ability of
tissue CAT to decompensate H
2
O
2
by monitoring of
the decrease in absorbance at 240 nm. The value of log
A1/A2 for a measured time interval was used for unit
definition due to the first-order reaction of enzyme.
Glutathione peroxidase activity
Tissue glutathione peroxidase (GPx) activity was
measured using the modified method of Paglia and
Valentine [13]. Using glutathione (GSH) as a reducing
reagent, the GPx enzymes catalyze the reduction of
hydrogen peroxide (H
2
O
2
) to water. In the assay,
oxidized glutathione is reduced to glutathione by the
enzyme glutathione reductase, which oxidizes
NADPH (nicotinamide adenine dinucleotide phos-
phate, reduced form) to NADP in the catalytic cycle.
The change in absorbance at 340 nm resulting from
the oxidation of NADPH is the basis for quantitating
tissue GPx activity.
Superoxide dismutase activity
Tissue superoxide dismutase (SOD) activity was
measured according to the method of Paoletti and
Mocali [14]. In brief, the superoxide anions were
generated from manganese (II) chloride and mercap-
thoethanol in the presence of acid-EDTA. The level
of SOD was measured based on its ability to inhibit
NADH oxidation in reaction mixture after addition of
tissue homogenate. NADH oxidation was measured
by monitoring of decrease in absorbance at 340 nm
during the reaction.
Detection of apoptosis
Cleavage of genomic DNA during apoptosis may yield
single-strand breaks (nicks) in high molecular weight
DNA. Apoptotic nuclei in tissue sections were iden-
tified using the in situ terminal deoxynucleotidyl
transferase (TdT)-mediated deoxyuridine triphosphate
(dUTP) biotin nick-end labeling (TUNEL) technique
that identified DNA strand breaks by labeling their free
3
0
-OH termini. We used the In Situ Cell Death
Detection Kit (Roche Diagnostic, Mannheim,
Int Urol Nephrol
123
Germany). The method distinguishes apoptotic cells
from those undergoing necrosis, because damaged
DNA in the former presents a different distribution of
staining and nuclear morphology. Paraffin-embedded
tissue sections were treated with proteinase K, and the
endogenous peroxidase activity was blocked by
hydrogen peroxide. The sections were incubated at
37°C with terminal TdT and nucleotides mixture in
reaction mixture for 1 h. Then, the reaction was
stopped, and the slides were rinsed with PBS. Nuclear
labeling was developed with horseradish peroxidase
and diaminobenzidine. Hematoxylin dye was used for
counterstaining. Quantitative evaluation of the apop-
totic index was performed by manual counting of
positively stained nuclei at 9100 magnification
(Fig. 1). One hundred seminiferous tubule cross
sections were evaluated for the appearance of apop-
totic nuclei, and the mean number of apoptotic nuclei
per tubule cross section was used for statistical
analysis. Only circular tubular cross sections cut in
bold face were used in these studies [11].
Statistical analysis
All data are expressed as the mean ± SD. The one-
way analysis of variance (ANOVA) was used for
statistical analysis of data among all groups. Multiple
comparisons were made using Tukey’s procedure
with P \ 0.05 considered statistically significant.
Results
The values of MDA, CAT, and SOD of the control
group did not show significant changes compared
with the baseline group (Table 1). The MDA level
was significantly increased in the TD group versus
control group. There were significant decreases in
CAT and SOD activities in the TD group compared
to baseline and control groups.
Cyclosporine reduces the oxidative stress
and apoptosis indices in ischemia/
reperfusion injury of testis
The MDA level in the cyclosporine group was
significantly lower than in the TD group, while it
was not significantly different from the control group
(Table 1). CAT and SOD activities in the cyclospor-
ine group were significantly higher than those in the
TD group, while the activities of GPx were not
significantly different among all groups (Table 1).
Germ cell apoptosis indices were significantly higher
in the TD and cyclosporine groups versus the control
Fig. 1 TUNEL staining of
nuclei in the seminiferous
tubules of rat testes in the
control group (a) and TD
group (b)
Table 1 MDA, CAT, SOD, and GPx levels in the ipsilateral testes
Groups MDA (nm/g wet tissue) Catalase (IU/g wet tissue) SOD (IU/g wet tissue) GPx (IU/g wet tissue)
Normal 117.666 ± 19.633
a
366.006 ± 35.595
c
1931.002 ± 129.588
e
712.000 ± 80.729
g
Sham 123.550 ± 20.615
a
349.056 ± 34.705
c
1892.382 ± 126.996
e
709.177 ± 48.975
g
T/D 187.666 ± 26.089
b
237.602 ± 38.902
d
1547.447 ± 83.299
f
582.545 ± 75.729
g
Cyclosporine 142.666 ± 15.161
a
319.480 ± 41.527
c
1851.646 ± 147.753
e
683.232 ± 88.082
g
Tacrolimus 136.333 ± 13.170
a
342.480 ± 43.575
c
1773.899 ± 116.809
e
625.697 ± 83.272
g
Means sharing the same superscript alphabet (a to g) in each column are not significantly different (P [ 0.05)
Int Urol Nephrol
123
and baseline groups; however, cyclosporine treatment
significantly reduced the apoptosis in the cyclospor-
ine group compared to the TD group (Table 2).
FK-506 reduces the oxidative stress and apoptosis
indices in ischemia/reperfusion injury of testis
CAT and SOD activities were significantly increased
in the FK-506 group compared to the TD group
(Table 1). The activities of GPx were not signifi-
cantly different among all groups (Table 1). Germ
cell apoptosis indices were significantly higher in FK-
506 groups versus control and baseline groups;
however, FK-506 treatment significantly reduced
the apoptosis in the FK-506 group compared with
the TD group (Table 2).
Discussion
Testicular torsion is a medical emergency occurring
primarily in adolescent males and young men with an
incidence estimated to be as high as 1 in 158 males by
the age of 24 years. Surgical detorsion should be done
promptly to avoid loss of function of the ipsilateral
testis. Even with successful surgical repair, however,
testicular atrophy is a common clinical outcome and is
a significant urological issue [15]. To date, a number
of chemicals and drugs have been successfully used to
reduce the I/R injury in animal models of testicular
torsion, but few of them are currently in clinical use
[11, 16, 17]. Here, for the first time, we report that
cyclosporine A and FK-506 (tacrolimus) could induce
a protective effect against I/R injury in the rat testis.
It is well established that 1-h 720° testicular torsion
leads to a decrease in antioxidant enzyme levels as
well as an increase in MDA level compared to the
sham operated group when measured 4 h after reper-
fusion. In addition, germ cell-specific apoptosis
significantly increases when assessed by in situ
TUNEL technique 24 h after detorsion. The primary
pathophysiological event in testicular torsion is ische-
mia of the testis followed by reperfusion upon repair.
I/R injury in many aspects resembles an inflammatory
response characterized by recruitment of activated
neutrophils to the testis and an increase in oxidative
stress [2]. There is an increase in tumor necrosis factor-
alpha (TNF-a) and interleukin-1-beta (IL-1b) expres-
sion after reperfusion of the testis. IL-1 b may be
responsible for neutrophil recruitment from the testic-
ular vasculature, and activated reperfusing neutrophils
may be potent generators of ROS [15]. Calcium influx
into neutrophils during ischemia causes increased
NADPH oxidase activity in the cell, which leads to the
release of free radicals during reperfusion [18].
Intravenous administration of cyclosporine and
FK-506 at the time of detorsion could normalize the
antioxidant enzymes and MDA to levels that were
significantly improved compared to the T/D group.
Today we know the important role of neutrophils in
the development of tissue ischemia/reperfusion
injury. Triggered neutrophils release substances such
as products of arachidonic acid metabolism, granular
elements, and oxygen radicals that cause tissue
damage [19, 20]. Accumulation of neutrophils is
mediated by the local generation of chemotactic
agents, which regulate neutrophil migration from the
vascular compartment to the tissues. The tissue-
derived neutrophil-activating peptide, which is iden-
tical to interleukin-8 (IL-8), has been cloned [21].
The effects of IL-8 on human neutrophil function
include enhanced chemotaxis, enzyme release,
expression of surface adhesion molecules, and induc-
tion of the respiratory burst [22, 23].
Although no substance homologous to IL-8 has
been identified in the rat, potent neutrophil chemo-
tactic activity has been detected in rat inflammatory
exudate induced by lipo-polysaccharide (LPS), tumor
necrosis factor-alpha, or interleukin-1 [
24]. Cytokine-
induced neutrophil chemo-attractant (CINC) is an
8-kDa polypeptide originally identified in the condi-
tioned media of interleukin-1 beta-stimulated rat
glomerular epithelial cells and subsequently purified
from NRK52E rat epithelial cells [25].
Calcium-dependent signal transduction is essential
to the induction of cytokine expression by stimuli.
Table 2 Apoptotic germ cell indices in the ipsilateral testes
Mean apoptotic nuclei/
tubule
Apoptotic tubules
(%)
Normal 0.56 ± 0.28
a
15.05 ± 3.89
d
Sham 0.71 ± 0.29
a
16.71 ± 2.61
d
T/D 10.1 ± 0.91
b
54.72 ± 5.91
e
Cyclosporine 3.29 ± 1.02
c
34.92 ± 4.91
f
Tacrolimus 4.03 ± 1.77
c
41.26 ± 5.15
f
Means sharing the same superscript alphabet (a, b, and c) are
not significantly different (P [ 0.05)
Int Urol Nephrol
123
CsA inhibits calcineurin, a Ca
2?
-activated serine/
threonine phosphatase necessary for the nuclear
import of NF-AT transcription factors [26]. In fact,
downstream signals, such as induction of interferon-
gamma and interleukin-2 mRNA by the calcium
ionophore A23187, are inhibited by CsA. These
observations suggest that CINC production may be
affected by Ca
2?
-dependent signal transduction. In
their study, Matsuda et al. demonstrated that pre-
treatment with CsA and FK506 significantly
decreased serum CINC concentrations after ische-
mia/reperfusion, resulting in reduced neutrophil
accumulation and attenuated tissue injury [27, 28].
Lymphocyte chemotaxis, but not neutrophil or
monocyte chemotaxis, is reported to be impaired by
immunosuppressive agents [29]. CsA does not influ-
ence several important manifestations of the
neutrophil’s antimicrobial defense system, including
chemotaxis, adherence to plastic, superoxide produc-
tion, and degranulation [30]. Furthermore, CsA does
not cause neutropenia in vivo [31]. Thus, it is unclear
by what mechanisms CsA might diminish neutrophil
infiltration in the reperfused tissue.
Inhibition of cytokine production and neutrophil
adhesion to rat liver sinusoidal cells, with diminished
generation of reactive free radicals, may be one of the
most plausible mechanisms by which CsA protects
against injury. Matsuda et al. demonstrated that CsA
significantly decreases CINC production by Kupffer
cells and attenuates neutrophil accumulation in the
liver after reperfusion. CsA inhibits the LPS-induced
activation of the NF-kB and decreases the nuclear
translocation of NF-kB. Pyrrolidine dithiocarbamate,
a potent inhibitor of NF-kB, has been found to
completely inhibit in vitro CINC production by
macrophages stimulated by LPS. Therefore, CINC
expression is dependent on NF-kB activation [32, 33].
The immunosuppressants can be classified as
inhibitors of transcription (CsA and FK506), of
nucleotide synthesis (azathiopurine), of growth factor
signal transduction (rapamycin), and of differentia-
tion (15-deoxyspergualin) [34]. CsA and FK506 bind
to their intracellular receptors, immunophilins, creat-
ing composite surfaces that block the activity of
specific targets. The cyclophilin family of immuno-
philins binds CsA, whereas the FK506-binding
protein (FKBP) family binds FK506 and rapamycin
[35]. The target for these complexes is calcineurin.
Calcineurin, a Ca
2?
/calmodulin-dependent protein
phosphatase, stimulates the NF-kB element by
enhancing inactivation of I kappa B/MAD3, an
inhibitor of NF-kB.
The immunosuppressants CsA and FK506 modu-
late intracellular calcium by disrupting the interaction
between immunophilins and Ca
2?
channel receptors.
Calcium plays an important role as a crucial intracel-
lular messenger in signal transduction. An increase
in the intracellular Ca
2?
levels induces NF-kB
activation. The endoplasmic reticulum-resident
Ca
2?
-ATPase inhibitor thapsigargin, which triggers a
rapid efflux of Ca
2?
from the endoplasmic reticulum,
can potently activate NF-kB. Shatrov et al. also demon-
strated that intracellular calcium mobilization induced
by sphingosine-1-phosphate activates transcription
factor NF-kB [36]. Contrarily, the Ca
2?
channel
blocker or chelator inhibits NF-kB activation [37].
These observations along with our results may indicate
the possible mechanisms by which CsA and FK506
protect the testis from ischemia/reperfusion injury.
Conclusion
In conclusion, we demonstrated that both CsA and
FK-506 (tacrolimus) have anti-ischemic properties,
even if administered in single and low doses;
however, further studies are required. The immuno-
suppressants CsA and FK506 significantly reduced
testicular reperfusion injury possibly due to attenu-
ated neutrophil accumulation.
Acknowledgement This work was supported by a research
grant provided by the Tehran University of Medical Sciences.
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    • "Therefore, testicular torsion followed by repair produces a reperfusion injury involving complex and multifaceted molecular signalling pathways. Experimental pharmacological approaches for treatments of testicular ischemia–reperfusion injury, including the use of several antioxidants (Wei et al., 2007; Payabvash et al., 2007; Pekcetin et al., 2007; Beheshtian et al., 2008; Nezami et al., 2008 ), dehydroepiandrosterone (Aksoy et al., 2007) and erythropoietin (Yazihan et al., 2007; Akcora et al., 2007) have proved to be effective. The early events which results in reperfusion injury, however, are still not fully understood. "
    [Show abstract] [Hide abstract] ABSTRACT: Nuclear factor kappa-B (NF-kappaB), mitogen-activated protein kinase3/MAPK1 and MAPK8 are involved in testicular ischemia reperfusion injury (testicular-I/R). NF-kappaB knock-out mice (KO) subjected to testicular-I/R have a reduced testicular damage, blunted MAPK8 activation and enhanced MAPK3/MAPK1 activity. To better understand the role of MAPK3/MAPK1 up-regulation during testicular-I/R, we investigated the effects of PD98059, an inhibitor of MAPK3/MAPK1, in KO mice during testicular-I/R. KO and wild-type (WT) animals underwent 1 h testicular ischemia followed by 24 h reperfusion or a sham testicular-I/R. Animals received either PD98059 (5 mg/kg/ip) or its vehicle. MAPK3/MAPK1, BAX, caspase-3 and -9 and TNF-alpha expression were assessed along with histological examination and an immunostaining for protein of apoptosis. Testicular-I/R caused a greater increase in MAPK3/MAPK1 in KO than in WT animals in both testes. KO mice had a lower expression of the apoptotic proteins and TNF-alpha as well as reduced histological damage compared to WT. Immunostaining confirmed the lower expression of BAX in the Leydig cells of KO mice. Administration of PD98059, abrogated MAPK3/MAPK1 expression and slightly reduced TNF-alpha but did not improve or reverse the histological damage in KO. PD98059 significantly reduced the histological damage in WT mice and markedly reduced the apoptotic proteins in KO and WT mice. These results suggest that testicular-I/R triggers also a pathway of organ damage involving MAPK3/MAPK1, TNF-alpha, BAX, caspase-3 and -9 that activates an apoptotic machinery in an NF-kappaB independent manner. These findings should contribute to better understand testicular torsion-induced damage.
    Full-text · Article · Feb 2009
  • [Show abstract] [Hide abstract] ABSTRACT: To investigate the effects of transplanted endothelial progenitor cells (EPCs) on the spermatogenic functions in testicular detorsion. Bone-marrow-derived EPCs were obtained from rats and transfected by enhanced green fluorescent protein adenovirus (Ad-eGFP). The rats were divided into 3 groups (n = 6 each). In the sham group, left testis was not twisted. In the ischemia reperfusion injury (IRI) group, 1 ml saline was injected into the femoral vein of each rat after testicular detorsion. In the EPCs group, 1 ml EPCs suspension (1.0 × 10(6) EPCs) was injected into each rat after testicular detorsion. The Ad-eGFP transfected EPCs were injected into the 3 additional rats of testicular torsion-detorsion. At Day 5 post-transplantation, the characteristics of transplanted EPCs homing were detected. And the pathological changes and apoptotic cells/seminiferous tubules in left testis were examined. When the value of multiplication of infection (MOI) was at 50, the transfection rate of EPCs by Ad-eGFP exceeded 73.7%. At Day 5 post-treatment, the cells exhibiting green fluorescence were detected in left testis. The germ cells in rats of the sham group were normal. And the ratio of apoptotic cells to seminiferous tubules was 0.09 ± 0.02. The germ cells in rats of the IRI group were much fewer. And the ratio of apoptotic cells to seminiferous tubules was 2.82 ± 0.81. As compared with the IRI group, seminiferous epithelium was thicker in the EPCs group. And the ratio of apoptotic cells to seminiferous tubules was 0.32 ± 0.09 in the EPCs group. It was much smaller than that in the IRI group. There was significant difference (P < 0.01). The transplantation of EPCs is effective for treating the spermatogenic dysfunctions caused by testicular torsion so as to greatly enhance the spermatogenic functions.
    Article · Aug 2011
  • [Show abstract] [Hide abstract] ABSTRACT: To evaluate biochemically and histopathologically the effects of Nigella sativa (NS) in experimental ischemia and ischemia/reperfusion (I/R) injury in rat ovaries. Thirty-six female rats were divided into 6 groups: group I = sham operation; group II = 500 mg/kg NS + sham operation; group III = bilateral ovarian ischemia; group IV = 500 mg/kg NS + ischemia; group V = 3-hour period of ischemia + 3-hour reperfusion, and group VI: 3-hour period of ischemia + 500 mg/kg NS 2.5 h after the induction of ischemia + 3-hour reperfusion. At the end of ischemia, the bilateral vascular clips were removed, and 3-hour reperfusion was continued. IL-1β, IL-6, and TNF-α cytokine levels in serum, and superoxide dismutase (SOD), myeloperoxidase (MPO), glutathione (GSH), and malondialdehyde (MDA) levels were determined. I/R increased the MDA level and MPO activity while significantly decreasing the SOD activity and GSH level when compared to the sham. The 500-mg/kg dose of NS before I/R reversed the trend in MDA levels, MPO activity, SOD activity, and GSH levels. Ischemia and I/R increased the serum levels of IL-1β, IL-6, and TNF-α, while the administration of NS decreased the serum levels of these cytokines. The administration of NS is effective in reversing tissue damage induced by ischemia and/or I/R in ovaries.
    Full-text · Article · Mar 2012
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