2. Volume 43, Number 2/April 2021 53
Effects of Nebivolol on Ischemia-Reperfusion
shown that free oxygen radicals are responsible
for ischemia-reperfusion injury in tissue.1 Free
oxygen radicals can disrupt the structural ele-
ments of tissue in the organism and cause harm-
ful effects. It has been reported that the effects of
torsion-detorsion injury in many organs and tis-
sues can be reduced by antioxidant therapy.2 The
accumulation of reactive oxygen species (ROS) is
the underlying pathologic mechanism of testicu-
lar torsion followed by ischemia, and it has been
ob
served to lead to infertility in several cases. ROS
is known to react with proteins, lipids, carbohy-
drates, and nucleic acids, causing impaired cell
function, DNA damage, and apoptosis.3 Germ cell
apoptosis occurs through two major pathways,
involving either mitochondria (intrinsic) or cell
surface death receptors (extrinsic). The mitochon-
drial pathway of apoptosis involves the Bcl-2
group of proteins, and different members of this
group are involved in diverse situations.4 Apop-
totic cell death has been reported to play an impor
tant role in the limitation of the testicular germ
cell population following the testicular ischemia-
reperfusion (IR) injury and is associated with male
infertility. Microbe cell homeostasis after testicular
IR has been reported to depend on the balance
between cell production, germ cell differentiation,
and programmed cell death from a constantly
renewed testicular stem cell population.5 The cell
death receptor pathway involves members of the
TNF receptor superfamily.6 TNF-α is a multifunc-
tional cytokine with effects only in the proinflam-
matory response7 and apoptosis.
Nebivolol is a relatively new highly cardio se-
lective β‐adrenergic receptor antagonist that pos-
sesses endothelium‐dependent vasodilator prop-
erties and antioxidant capacities.8 In addition to
its vasodilator effects, nebivolol has been found to
counteract oxidative stress. De Groot et al in 20049
showed that nebivolol protects against injury from
hydroxyl radical (·OH) in the right ventricular rab-
bit heart trabeculae and explanted human hearts
from the demembranized muscles.
The aim of this study is to investigate the anti-
oxidant effects of nebivolol in spermatogenic cells
against apoptotic and testicular inflammation in
torsion-detorsion injury in rats by using the TUNEL
assay and immunohistochemical methods.
Materials and Methods
All experimental protocols were conducted accord-
ing to the National Institutes of Health Guidelines
for the Care and Use of Laboratory Animals. The
study was approved by the local ethics commit-
tee. Forty male Wistar albino rats with a mean
weight of 200–250 g were used. They were housed
in an air-conditioned room with 12-hour/12-hour
light and dark cycles, where the temperature
(23±2°C) and relative humidity (65–70%) were
kept constant.
In this study, all surgical procedures were per-
formed after intramuscular injection of 50 mg/kg
ketamine hydroxide (Ketalar, Pfizer, Turkey) and
10 mg/kg xylazine (Rompun, Bayer, Germany)
for general anesthesia. All operations were per-
formed under sterile conditions. The animals were
randomly divided into 4 groups. Forty adult male
rats were used in each group.
In the control group the animals did not under-
go any surgical operation and were sacrificed at
the end of the experiment. In the torsion group,
ischemia injury was induced by torsion of the
left testis, with a 720° twisting of the spermatic
cord so as to produce a total occlusion of the tes-
tis for 2.5 hours. The left testicles were reperfused
for 3 hours after 2.5 hours of ischemia. In the
torsion/detorsion group, the left testicles were
reperfused for 4 hours after 3 hours of ischemia.
In the torsion/detorsion+nebivolol group, the left
testicles were twisted clockwise 720°, and 10 mg/
kg nebivolol was administered orally 60 min be-
fore ischemia was induced in the animals. The
dose of nebivolol was chosen on the basis of a pre
vious study.9 The scrotal sutures were opened and
the left testicles returned to the normal position.
The scrotums were closed again, and the testicles
were reperfused for 4 hours. All animals were sac-
rificed at the end of the reperfusion period.
Surgical Procedure
The rats were anesthetized under aseptic con-
ditions by intramuscular injection of 50 mg/kg
ketamine hydroxide and 10 mg/kg xylazine. The
tunica was removed with the help of a forceps to
make the vaginal testicular tissue visible. A scrotal
pocket was created to place the testicles back into
the scrotum after twisting. The left testicle was
rotated 720° degrees clockwise for 3 hours around
the longitudinal axis of the spermatic cord to cre
ate torsion. To prevent deterioration, the testis
dartos and testicular tunica albuginea were fixed
in the scrotal pocket by passing a 4/0 nontraumatic
absorbable suture. After the torsion procedure, a
return was made for 4 hours for detorsion.
3. 54 Analytical and Quantitative Cytopathology and Histopathology®
Taş et al
Biochemical Analysis
MDA, SOD, CAT, and GSH levels were exam-
ined in testicular tissue. Tissue samples were
homogenized with ice-cold 150 mMKC. MDA lev-
els were assayed for products of lipid peroxida-
tion, and the results were expressed as nmol
MDA/g tissue.10 The SOD activity in the tissue
was measured using the RANSOD kit (Randox
Laboratories, Crumlin, UK). GSH was determined
by the spectrophotometric method based on the
use of Ellman’s reagent, and the results were
expressed as μmol glutathione/g tissue).11 CAT
activity was determined by the spectrophotome-
tric method based on the ability of hydrogen per
oxide to form a stable stained complex with mo-
lybdenum salts.12
TUNEL Assay Analysis
Testicular apoptosis was analyzed using the termi-
nal deoxynucleotidyl transferase enzyme-mediated
dUTP nick-end labeling (TUNEL) method. Sec-
tions 4–6 µm thick were cut from the paraffin
blocks of the samples. TUNEL staining of the sec-
tions was done using ApopTag Plus Peroxidase in
Situ Apoptosis Kit (catalog no. #S7101, Millipore,
Burlington, Massachusetts, USA) in accordance
with the manufacturer’s instructions. Sections
were dewaxed in xylene, rehydrated, and incu-
bated with proteinase K for 10 minutes and rinsed
in distilled water. Endogenous peroxidase activ-
ity was inhibited by 3% hydrogen peroxide. Sec-
tions were then incubated for 10–15 seconds with
equilibration buffer and TdT enzyme in a moist
atmosphere at 37°C for 60 minutes. It was then
placed in a preheated working power stop/wash
buffer for 10 minutes at room temperature and
incubated with Anti-Digoxigenin-Peroxidase for
30 minutes. Each step was separated by careful-
ly washing in PBS. The staining was done with
DAB, and the counterstaining was done in Mayer
hematoxylin.
Immunohistochemical Technique
Formaldehyde-fixed tissue was embedded in par
affin wax for further immunohistochemical exam-
ination. Sections were deparaffinized in absolute
alcohol. The antigen retrieval process was per-
formed twice in citrate buffer solution (pH 6.0),
first for 7 minutes and second for 5 minutes,
boiled in a microwave oven at 700 W. They were
allowed to cool to room temperature for 30 min-
utes and washed twice in distilled water for 5 min-
utes. Endogenous peroxidase activity was blocked
in 0.1% hydrogen peroxide for 50 minutes. Ultra
V block was applied for 10 minutes prior to the
application of primary antibodies TNF-α (catalog
no. ab1793, Abcam, Cambridge, UK, dilution rate:
1/100). Secondary antibody was applied for 20
minutes. Slides were then exposed to streptavidin-
peroxidase for 20 minutes. Chromogen diamino-
benzidine was used. Control slides were prepared
as mentioned above but omitting the primary
antibodies. After counterstaining with hematoxy-
lin and washing in tap water for 8 minutes and in
distilled water for 10 minutes, sections were exam-
ined under a light microscope.
Statistical Analysis
Statistical analysis was performed with IBM SPSS
version 20 (IBM Corp., Released 2011, IBM SPSS
Statistics for Windows, Version 20.0. IBM Corp.,
Armonk, New York, USA). Data were expressed
as mean±SD for each group. To evaluate the dis-
tribution of numerical variables, a normality test
(Shapiro-Wilk test, which is important for consid-
eration at p<0.05) was performed. Kruskal-Wallis
test and Dunn-Bonferroni post-hoc test were used
to compare the groups. P<0.05 was taken as the
level of significance.
Results
Biochemical Results
MDA levels in the testicular tissues of rats in
the torsion/detorsion group were significantly in-
creased as compared to those in the sham group,
while the MDA levels in the torsion/detorsion+
nebivolol group were significantly decreased
as compared to those in the torsion/detorsion
group. Nebivolol treatment prevented the torsion/
detorsion–induced elevation of MDA levels in the
testis. The SOD, CAT, and GSH activities of testic-
ular tissue significantly decreased in the torsion-
detorsion group as compared to those in the sham
group. These values were significantly increased
in the torsion-detorsion+nebivolol group as com-
pared to those in the torsion-detorsion group. The
MDA, SOD, CAT, and GSH levels of the sham,
torsion-detorsion, and torsion-detorsion+nebivolol
groups are shown in Table I.
Apoptotic Findings
Apoptotic cells were identified by TUNEL assay.
In testicular tissue evaluation, the apoptotic index
was normal in terms of spermatogenesis in the
4. Volume 43, Number 2/April 2021 55
Effects of Nebivolol on Ischemia-Reperfusion
control group. In the torsion-detorsion group, a sig-
nificant increase in apoptosis was observed in the
torsion group as compared to in the control group.
There was a significant loss in spermatogenetic
cells (p<0.01). In the nebivolol-treated group, the
apoptotic index reached a value close to that in the
control group, and a decrease in the cells under-
going apoptosis was observed (p>0.05) (Table II)
(Figure 1).
In groups 2 (torsion) and 3 (torsion-detorsion),
significant degenerative changes were observed in
the light microscopic spermatogenetic cells of the
testis. There was a significant decrease in group
4 (torsion-detorsion+nebivolol) as compared to
groups 2 and 3 (Figure 2).
Discussion
Testicular torsion is ischemic damage in tissue,
and detorsion is reperfusion injury. In the rat mod
el with testicular torsion, oxidative stress has been
shown to cause adverse effects within minutes
and hours after reperfusion has been created. It
has been shown that antioxidant enzyme activi-
ties decrease and ROS production increases after
testicular torsion.13 Testicular damage caused by
ischemia followed by reperfusion is assumed to be
partly a result of lipid peroxidation, which in turn
leads to membrane damage.14
MDA is the product of lipid peroxidation and is
the parameter that determines the increased free
Table I Comparison of MDA, SOD, CAT, and GSH Activities in All Groups
Multiple comparisons
for groups
Mean Kruskal-Wallis (Dunn-Bonferroni test)
Parameter Group Mean±SD rank test value (p<0.05)
MDA (1) Control 7.36±0.36 12.68 29.806 (3)
(2) Torsion 9.66±0.76 24.34 p=0 (4)
(3) Torsion/detorsion 14.22±0.55 34.52 (1)(4)
(4) Torsion/detorsion+nebivolol 6.25±0.45 10.42 (2)(3)
SOD (1) Control 3.12±0.31 29.86 30.412 (2)(3)
(2) Torsion 1.67±0.32 13.34 p=0 (1)(4)
(3) Torsion/detorsion 1.28±0.16 7.66 (1)(4)
(4) Torsion/detorsion+nebivolol 3.26±0.42 31.02 (2)(3)
CAT (1) Control 0.039±0.006 30.95 31.886 (2)(3)
(2) Torsion 0.025±0.004 15.95 p=0 (1)(4)
(3) Torsion/detorsion 0.018±0.002 5.48 (1)(4)
(4) Torsion/detorsion+nebivolol 0.039±0.003 28.24 (2)(3)
GSH (1) Control 358.8±8.76 29.36 29.786 (2)(3)
(2) Torsion 301.3±32.8 8.78 p=0 (1)(4)
(3) Torsion/detorsion 312.8±2.91 12.68 (1)(4)
(4) Torsion/detorsion+nebivolol 362.3±6.12 31.26 (2)(3)
Table II TUNEL Assay Results with Apoptotic Distribution
Between Groups in Germ Cells
Mini- Maxi-
Group Mean±SD mum mum p Value
Control 2.24±1.01 0.78 3.68
Torsion 11.85±2.12 7.70 17.78 <0.01*
Torsion/detorsion
27.34±3.84
21.60
27.93
<0.01*
Torsion/detorsion+
nebivolol 3.43±1.29 1.54 4.30 <0.01**
Columns with different superscripts are significantly different (*p<0.01 as
compared to control group, **p<0.01 as compared to control and
torsion-applied groups). Figure 1 TUNEL assay results of the groups.
5. 56 Analytical and Quantitative Cytopathology and Histopathology®
Taş et al
radical formation in ischemia-reperfusion tissue
damage.15 SOD is an oxygen radical scavenging
enzyme that protects cells against damage caused
by ROS.16 Reduced glutathione is one of the free
radical scavengers that helps restore the physi-
ological structure of the cell membrane. It is an
important factor for detoxification of oxygen me-
tabolites, mostly hydrogen peroxide and lipid hy
droperoxide.17
Apoptosis is characterized by marked biochem-
ical and morphological changes, such as DNA
fragmentation, plasma membrane bubbling, and
cell volume shrinkage. Hypoxia-ischemia induces
apoptotic and necrotic cell death, partly due to
permanent changes in cellular energy homeosta-
sis.18 In studies related to testicular torsion, 3 hours
of 720° testicular torsion has been shown to be 3
times higher than the number of apoptotic germ
cells per sectional area, and a detectable increase
in apoptosis 4 hours after torsion repair.18,19
Nebivolol is a drug with nitric oxide stimu-
lation, antioxidant properties, and endothelium-
based vasodilator effects. Considering the roles of
antioxidant defense mechanisms in cardiovascu-
lar diseases,20 it has been reported that in the light
of endothelial dysfunction and high oxidative
Figure 2 TUNEL assay for all groups. (A) Control group: TUNEL negative expression in spermatogenetic cells and Sertoli cells in
seminiferous tubules (black arrow). TUNEL staining, Bar=50 µm. (B) Torsion group: spermatogenetic cells (blue arrow) from the
basement membrane of the seminiferous tubules to the lumen, and the apoptosis and TUNEL positive reaction in the Sertoli cells (yellow
arrow), TUNEL positive expression (red arrow) in the intertubular area. TUNEL staining, Bar=50 µm. (C) Torsion/detorsion group:
apoptotic increase in spermatogenetic (blue arrow) and Sertoli cells (yellow arrow) along the basement membrane, positive TUNEL
expression in the intertubular area, positive TUNEL reaction (red arrow). TUNEL staining, Bar=50 µm. (D) Torsion/detorsion+nebivolol
group: positive reaction with spermatogenetic cells in the seminiferous tubules (blue arrow) and a decrease in apoptotic appearance in
Sertoli cells (yellow arrow), negative TUNEL reaction in Leydig cells in the intertubular area. TUNEL staining, Bar=50 µm.
6. Volume 43, Number 2/April 2021 57
Effects of Nebivolol on Ischemia-Reperfusion
stress, the ideal drug to be used in treatment can
increase nitric oxide production and reduce oxi-
dative stress in vascular tissues.21 Gandhi et al22 in
2008 showed that nebivolol has a protective effect
against renal ischemia/reperfusion damage due
to its beta 1 adrenoreceptor blocking activity and
apoptotic and anti-inflammatory properties.
Heeba et al23 demonstrated the oxidative stress-
reducing effect of nebivolol and the antioxidant
and anti-apoptotic effects of nebivolol with cere-
bral ischemia-reperfusion injury. In a rat model of
testicular IR, we have recently shown that 24
hours after IR germ cell apoptosis in the contra-
lateral testis increased significantly and that the
extent of apoptosis increases with the duration of
the ischemia.24
In TUNEL dyeing technique, it has been stated
that DNA yarn breaks can be induced not only
by apoptosis, but also by necrosis.25 Although is-
chemia confirms that apoptosis and necrosis be-
gin, reperfusion accelerates apoptosis. One study
confirmed that there was an increase in the apop-
totic index compared to the control group in the
IR group, but TUNEL-positive cells were higher
in the group with 720° ischemia for 2 hours and
sequential reperfusion for 1 hour.26
In this study, the number of apoptotic index
increased in spermatogenetic cells in the torsion
Figure 3 TNF-α immune staining of all groups. (A) Control group: TNF-α expression was observed mildly in spermatogenetic cells and
Sertoli cells in seminiferous tubules, Leydig cells in the intertubular space, connective tissue cells, and endothelial cells in the blood
vessel. (B) Torsion group: TNF-α expression was positive in the spermotogenic cells (blue arrrow), spermium in the lumen as well as in
the Sertoli cells. TNF-α expression was increased in Sertoli cells. TNF-α expression was observed intensely in inflammatory cells around
the blood vessels and Leydig cells in the intertubular area (red arrow). (C) Torsion/detorsion group: an increase in TNF-α expression was
observed in spermatogenetic cells and Leydig cells (arrow). (D) Torsion/detorsion+nebivolol group: spermatogenetic cells (red arrow) and
few Sertoli cells (yellow arrow) in the basal membrane of some seminiferous cells have positive TNF-α expression, while negative TNF-α
expression has been observed in the majority of tubular cells and negative TNF-α expression in the intertubular area (red arrow). TNF-α
immunostaining, Bar=50 µm.
7. 58 Analytical and Quantitative Cytopathology and Histopathology®
Taş et al
and detorsion groups. It has increased significant
ly, especially to the detorsion phase. After the ap-
plication of nebivolol, a decrease in the number
of apoptotic indexes was observed in the cells
(Tables I–II) (Figures 1–2).
In our study it was seen that after torsion and
torsion-detorsion, TNF-α may alter the cytokine
secretion profile of Sertoli cells and may affect
Sertoli cell functions and spermatogenesis with
increased inflammation (Figure 3).
It has been thought that there is an increase in
TNF-α expression after reperfusion of the testi-
cle, which may be responsible for stimulating a
stress-related kinase signaling pathway leading to
neutrophil uptake from the testicular vasculature.
This study has shown that unilateral testicu-
lar torsion-detorsion causes testicular damage to
the ipsilateral testis as evidenced by biochemical
and apoptotic changes in the tissues and retains
the morphological structure in the testes after
nebivolol administration, after testicular torsion-
detorsion damage, and decreased apoptotic index
and inflammation.
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