ANALYTICAL AND QUANTITATIVE CYTOPATHOLOGY AND HISTOPATHOLOGY

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0884-6812/21/4301-0001/$18.00/0 © Science Printers and Publishers, Inc.
Analytical and Quantitative Cytopathology and Histopathology®
A
RTICLES
An Official Periodical of The International Academy of Cytology and the Italian Group of Uropathology
AQCH
ANALYTICAL and
QUANTITATIVE
CYTOPATHOLOGY and
HISTOPATHOLOGY®
OBJECTIVE: Testicular torsion is a common urological
complication mostly affecting adolescents and young
men. In this study we investigated biochemical and
immunohistochemical effects of gallic acid on the damage
induced by testicular torsion-detorsion.
STUDY DESIGN: Forty male rats were divided into
4 groups of 10 animals each: control, torsion, torsion/
detorsion, and torsion/detorsion+gallic acid. Testicles
were removed from the scrotum, and 2.5-hour ischemia
was applied to the left testis by twisting the spermat-
ic cord 720° clockwise. Then 3-hour reperfusion was
allowed for detorsion. Gallic acid 20 mg/kg was orally
administered to the torsion/detorsion+gallic acid group
before reperfusion. Biochemical parameters of testicu-
lar tissue (MDA, SOD, CAT, and GSH levels) were
measured. Testicular tissues were immune-stained with
caspase-3 and TNF-α antibody.
RESULTS: MDA levels in the torsion/detorsion+gallic
acid group were close to those in the control group;
however, it was higher in the torsion/detorsion group
as compared to the control group. Compared to control
group, SOD, CAT, and GSH activities were signifi­
cantly increased in the torsion/detorsion+gallic acid
group. However, those values were decreased in the
torsion-detorsion group. Spermatogenic cells and inter-
stitial cells showed positive caspase-3 expression in the
torsion and torsion-detorsion groups; however, expres-
sion level was decreased in the torsion/detorsion+gallic
acid group. TNF-α expression was observed in degen-
erated spermatogonia, Leydig cells, and macrophages in
Analytical and Quantitative Cytopathology and Histopathology®
Effects of Gallic Acid on Ischemia-Reperfusion
Induced Testicular Injury in a Rat Testicular
Torsion Model
Nilgün Söğütçü, M.D., and Beran Yokuş, Ph.D.
From the Department of Pathology, University of Health Sciences, Diyarbakır Gazi Yaşargil Training and Research Hospital, Diyarbakır;
and the Department of Biochemistry, Faculty of Veterinary Medicine, Dicle University, Diyarbakır, Turkey.
Nilgün Söğütçü is Physician, Department of Pathology, University of Health Sciences, Diyarbakır Gazi Yaşargil Training and Research
Hospital.
Beran Yokuş is Professor, Department of Biochemistry, Faculty of Veterinary Medicine, Dicle University.
Address correspondence to: Nilgün Söğütçü, M.D., Department of Pathology, University of Health Sciences, Diyarbakır Gazi Yaşargil
Training and Research Hospital, Sur, 21200, Diyarbakır, Turkey (nilgunsogutcu@gmail.com).
Financial Disclosure: The authors have no connection to any companies or products mentioned in this article.

2. the torsion and torsion-detorsion groups. In the torsion/
detorsion+gallic acid group, TNF-α expression was ob­
served in some interstitial region rather than in cells of
seminiferous tubules.
CONCLUSION: Gallic acid treatment could be an
alternative therapy in testicular ischemia-reperfusion
injury to decrease inflammation, germ cell degeneration,
and apoptosis. (Anal Quant Cytopathol Histpathol
2021;43:1–7)
Keywords: detorsion; ischemia-reperfusion injury;
oxidative stress; rats, Wistar; reperfusion injury;
spermatic cord torsion; spermatogenesis; testic-
ular torsion; testis; torsion abnormality; torsion-
detorsion.
Testicular torsion decreases the blood flow to the
testis and leads to ischemia in tissues. The extent
of testicular tissue damage is related to both the
degree of twisting and the rapidity of surgical
intervention to counter-rotate both the testis and
spermatic cord to reestablish normal blood flow
to the organ.1,2 Previous studies using a rat model
of testicular torsion have demonstrated that a
1-hour, 720° rotation of the testis followed by reper-
fusion results in the permanent loss of spermato-
genesis.3
Testes are very sensitive to free radical damage,
therefore torsion and detorsion injury is a subject of
interest. Various therapeutic agents have been used
on testicular injury induced by torsion-detorsion,
such as antioxidants, phytotherapeutics, modula-
tors of inflammation, and vasodilators.4,5
Gallic acid (3,4,5-trihydroxybenzoic acid) is a
polyhydroxy phenolic compound found in grapes,
green tea, lemon, and other natural sources.6 Gal-
lic acid is an antioxidant and is formed as a free
molecule or sometimes as part of a tannin mol-
ecule. It has been documented to possess sever-
al biological activities such as antibacterial, anti-
inflammatory, antifungal, and antiviral.7,8
Caspase-3 is used as an apoptosis marker in
many molecular techniques. Zymogen procaspase-
3 is localized in cytosol, mitochondria, and nuclei.
The subcellular location of procaspase-3 has been
reported to be cytosolic and mitochondrial in the
liver.9 Various caspase substrates are involved in
the regulation of DNA structure, repair, and repli-
cation. Caspase-3 substrate cleavage was observed
under oxidative stress in different pathological
conditions.10
Tumor necrosis factor–α (TNF-α) is a cyto-
kine produced by various immune cells, includ-
ing macrophages/monocytes. TNF-α plays a role
in many cellular pathways, such as inflamma-
tion, proliferation, and apoptosis. TNF-α has been
suggested as a central player in inflammatory cell
activation and recruitment and has been impli-
cated in the development of many chronic inflam­
matory diseases.11
The aim of this study is to investigate the bio-
chemical and immunohistochemical effects of gal-
lic acid against damage after testicular torsion-
detorsion.
Materials and Methods
All experimental protocols were approved by the
Dicle University Faculty of Medicine Animal Care
and Use Ethics Committee. The study was com-
posed of 40 male Wistar albino rats weighing 200–
250 g each. The rats were kept in separate cages
at 22±1°C with 12/12 hour light/dark cycles. They
were provided water ad libitum and free access
to standard feed pellets. In this study all surgi-
cal procedures were performed under anesthesia
with intramuscular injection of 50 mg/kg keta-
mine hydroxide (Ketalar; Pfizer, Turkey) and 10
mg/kg xylazine (Rompun; Bayer, Germany). All
opera­
tions were performed under sterile condi-
tions. Animals were divided into 4 groups (10 ani-
mals per group) as described below.
Study Groups
Control Group. The animals did not undergo any
surgical operation and were sacrificed at the end of
the experiment.
Torsion Group. Ischemia injury was performed by
torsion of the left testis, with a 720° twisting of the
spermatic cord to produce a total occlusion of the
testis for 2.5 hours.
Torsion/Detorsion Group. The left testicles were
reperfused for 3 hours after 2.5 hours of ischemia.
Torsion/Detorsion+Gallic Acid Group. The left tes-
ticles were twisted clockwise 720°. The rats were
administered 20 mg/kg gallic acid by oral gavage
60 minutes before ischemia.
Surgical Procedure
The rats were anesthetized under aseptic condi-
tions by intramuscular injection of 50 mg/kg ke-
tamine hydroxide and 10 mg/kg xylazine. The
2 Analytical and Quantitative Cytopathology and Histopathology®
Söğütçü and Yokuş

3. tunica was removed with the help of a forceps to
make the vaginal testicular tissue visible. A scro-
tal pocket was created to place the testicles back
into the scrotum after twisting. The left testicle
was rotated 720° clockwise for 2.5 hours around
the longitudinal axis of the spermatic cord to
create torsion. To prevent deterioration, testis dar-
tos and testicular tunica albuginea were fixed in
the scrotal pocket by passing a 4/0 nontraumat­
ic absorbable suture. Detorsion was treated for 3
hours after torsion application.
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.12 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.13 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.14
Statistical Analysis
The data obtained in the study were expressed as
arithmetic mean±standard deviation.
Statistical analyses were made using the IBM
SPSS Statistics for Windows program (IBM Corp.,
Armonk, New York). Kruskal-Wallis test and
Dunn-Bonferroni post-hoc tests were used in com-
parison of the groups. P<0.05 was taken as the
significance level.
Results
The MDA levels in the testicular tissues of the
rats in the torsion/detorsion group were statisti­
cally significantly increased as compared to the
control group. As seen in Table I, the MDA level
in gallic acid treatment was close to that of the
controls. SOD, CAT, and GSH activities in the tes­
ticular tissue of the rats in the torsion-detorsion
group decreased significantly as compared to the
control group; however, those values were sig-
nificantly increased in the group treated with gal-
lic acid. The MDA, SOD, CAT, and GSH levels
of the control, torsion, torsion/detorsion, and tor-
sion/detorsion+gallic acid groups are shown in Ta-
ble I.
Seminiferous tubular diameter was measured
from approximately 10 different areas. Testicu-
lar tubular diameters of the torsion and torsion-
detorsion groups were decreased as compared to
that of the control group. Diameter of tubules in
the gallic acid group was close to that of the control
group (Table I).
Figure 1 shows the comparison of caspase-3
immunostaining of sections of testicular tissues
in all groups, and Figure 2 shows the comparison
of TNF-α immunostaining of sections of testicular
tissues in all groups.
Discussion
Surgery is the basic approach in the treatment of
testicular torsion, which is considered an urgent
pathological event. Permanent tissue damage may
occur depending on the twisting time. Previous
studies with a rat model of testicular torsion have
demonstrated that a 2-hour, 720° rotation of the
testes followed by reperfusion causes a signifi-
cant increase in testicular lipid peroxidation prod-
ucts, nitric oxide content, and myeloperoxidase
activity.12
Turner et al13 suggested that at least 1 hour of
720° testicular torsion results in impaired sper­
matogenesis and apoptosis in the ischemic testis
of the rat. Cellular ischemia causes an increase
in intracellular hypoxanthine and xanthine and
an increase in intracellular calcium as a result of
adenosine triphosphate breakdown.14 As a result,
it disrupts DNA and protein function. It causes
lipid oxidation of both the cell and mitochondrial
membranes, leading to testicular dysfunction and
cell death.15
MDA is the product of lipid peroxidation and is
the parameter that determines the increased free
radical formation in ischemia-reperfusion tissue
damage. 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 physio­
logical structure of the cell membrane. It is an
important factor for detoxification of oxygen me-
tabolites and is mostly hydrogen peroxide and
lipid hydroperoxide.17 ROS increase in oxidative
stress leads to lipid peroxidation, protein dena-
turation, DNA damage, inflammation, cell prolif-
eration, cell dysfunction, and apoptosis in the cell
membrane.18 In this study, biochemical findings
Volume 43, Number 1/February 2021 3
Gallic Acid and Ischemia-Reperfusion Testicular Injury

4. showed that compared with the control group,
MDA and the oxidative stress level increased,
while levels of SOD, CAT, and GSH decreased in
the torsion and detorsion groups (Table I). The
presence of high levels of MDA in testicular tis-
sues of torsion and detorsion rats has been con­
sidered an index for the status of lipid peroxida-
tion and oxidative stress.
Gallic acid, a polyphenolic acid, is found in
various plants with antioxidant, antifungal, anti-
viral, and antiallergic activities. It has been noted
that gallic acid can induce vascular contraction re-
sponses and reduce vascular pressure in the tho­
racic aorta. Gallic acid can be added to foods to
clean free oxygen molecules and aid in lipid per­
oxidation through the hydrolysis of tannic acid.19
A number of experimental evidences were report-
ed for the use of different components in testicular
torsion-detorsion injury treatment.20 These studies
showed that those components prevent or reduce
testicular damage following reperfusion, showing
that the primary cause of testicular damage was
generated by ROS production, mitochondrial cal­
cium overload, and cellular apoptosis.
Apoptotic cell death has been reported to play
an important role in the limitation of the tes-
ticular germ cell population following testicular
ischemia-reperfusion injury and is associated with
male infertility. Spermatogenic cell homeostasis
after testicular ischemia-reperfusion has been re-
ported to depend on the balance between cell pro-
duction, germ cell differentiation, and programmed
cell death from a constantly renewed testicular stem
cell population.21
4 Analytical and Quantitative Cytopathology and Histopathology®
Söğütçü and Yokuş
Table I Different Parameters of the Experimental Groups with Mean±Standard Deviation
Multiple comparisons
for groups
Kruskal-Wallis (Dunn-Bonferroni test)
Parameter Group Mean±SD Mean rank test value (p<0.05)
MDA (1) Control 6.28±0.49 8.10 33.770 (2)(3)
(2) Torsion 8.78±0.77 25.50 p=0 (1)
(3) Torsion/detorsion 13.43±0.47 35.50 (1)(4)
(4) Torsion/detorsion+gallic acid 6.72±0.40 12.90 (3)
SOD (1) Control 3.96±0.46 33.90 33.487 (2)(3)
(2) Torsion 2.10±0.36 14.90 p=0 (1)
(3) Torsion/detorsion 1.52±0.20 6.20 (1)(4)
(4) Torsion/detorsion+gallic acid 3.43±0.33 27.00 (3)
CAT (1) Control 0.04±0.004 31.45 33.109 (2)(3)
(2) Torsion 0.02±0.003 15.50 p=0 (1)(4)
(3) Torsion/detorsion 0.01±0.002 5.50 (1)(4)
(4) Torsion/detorsion+gallic acid 0.04±0.004 29.55 (2)(3)
GSH (1) Control 361.43±12.73 30.00 29.634 (2)(3)
(2) Torsion 310.31±6.72 12.00 p=0 (1)(4)
(3) Torsion/detorsion 306.27±9.33 9.00 (1)(4)
(4) Torsion/detorsion+gallic acid 364.01±7.65 31.00 (2)(3)
Diameter of (1) Control 305.83±11.11 32.05 29.416 (2)(3)
seminiferous (2) Torsion 260.08±13.92 11.20 p=0 (1)(4)
tubule (3) Torsion/detorsion 255.15±7.11 9.90 (1)(4)
(4) Torsion/detorsion+gallic acid 301.08±9.67 28.85 (2)(3)
Caspase-3 (1) Control 0.50±0.70 6.15 31.288 (2)(3)
(2) Torsion 3.10±0.56 27.00 p=0 (1)
(3) Torsion/detorsion 3.60±0.51 32.30 (1)(4)
(4) Torsion/detorsion+gallic acid 2.10±0.56 16.55 (3)
TNF-α (1) Control 1.20±0.63 8.70 32.453 (2)(3)
(2) Torsion 3.20±0.42 28.00 p=0 (1)(4)
(3) Torsion/detorsion 3.70±0.48 33.00 (1)(4)
(4) Torsion/detorsion+gallic acid 1.60±0.69 12.30 (2)(3)

5. Caspase proteins serve as cysteine protease
enzymes in apoptosis, of which caspase-3 is an
activated cell death protease that catalyzes the
specific cleavage of many critical cellular proteins.
Caspase-3 is essential in apoptosis for chromatin
condensation and DNA fragmentation for the dis-
sociation of the cell and formation of apoptotic
bodies.22 In the torsion group, caspase-3 expres-
sion was positive in seminiferous tubules and the
intertubular area. The expression was significantly
more dense in the spermatogenic cells and inter-
stitial cells. In the torsion/detorsion+gallic acid
group, cells with high mitotic activity and nega-
tive caspase-3 expression were observed (Figure 1).
TNF-α, which encourages the caspase cascade
and cell apoptosis, is a proinflammatory pleiotropic
cytokine.23 It is known that burn injuries prompt
the activation of inflammatory pathways and trig-
ger the release of various cytokines. Experimental
evidence suggested that it would be beneficial
to observe proinflammatory biomarkers, such as
TNF-α, in such conditions.24,25 In our study, TNF-α
expression was positive in degenerative sper-
matogonia, Sertoli cells, Leydig cells, and intersti-
tial macrophages due to increased inflammation
in the torsion/detorsion group. Torsion/detor-
sion injury induced apoptotic cell formation with
increased TNF-α signals. In the gallic acid–treated
group it was observed that a new process started
in cell structuring with the decrease of TNF-α ac­
tivity in degenerative spermatocytes, Sertoli cells,
and Leydig cells, with decreased inflammation in
seminiferous tubule cells (Figure 2).
Oxidative stress by testicular torsion may cause
male infertility, and we think that gallic acid ther-
apy before ischemia may be an alternative solution
to decrease inflammation, germ cell degeneration,
and apoptosis.
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Volume 43, Number 1/February 2021 5
Gallic Acid and Ischemia-Reperfusion Testicular Injury
Figure 1
Caspase-3 immunostaining of
sections of testicular tissues in
all groups. (A) Control group:
caspase-3 expression was
negative in spermatogenic
cells and interstitial cells.
(B) Torsion group: caspase-3
expression was positive in
the majority of cells through
the spermatogenic cell line,
Leydig cells, and connective
tissue cells in the intertubular
area. (C) Torsion/detorsion
group: significant increase
in caspase-3 activity was
observed in degenerated cells
and in spermatid, Leydig,
and other interstitial cells. (D)
Torsion/detorsion+gallic acid
group: spermatogenic cells
mainly showed negative caspase-3 expression in the the seminiferous tubules; however, caspase-3 expression was positive in some
spermatid cells. High mitotic activity was observed in spermatogonia with negative caspase-3 expression.

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6 Analytical and Quantitative Cytopathology and Histopathology®
Söğütçü and Yokuş
Figure 2
TNF-α immunostaining of
sections of testicular tissues
in all groups. (A) Control
group: TNF-α expression was
negative in spermatogenic
cells, Leydig cells, and other
intertubular cells. (B) Torsion
group: TNF-α expression was
positive in degenerated
spermatogonia and in
interstitial Leydig and
macrophage cells. (C) Torsion/
detorsion group: increased
TNF-α expression was
observed in apoptotic cells,
interstitial cells, and in the
basement membrane of the
seminiferous tubules.
(D) Torsion/detorsion+gallic
acid group: while negative
TNF-α expression was observed in spermatogenic cells and spermatid cells in seminiferous tubules, TNF-α expression was positive in
some macrophage cells and endothelial cells in the intertubular area.

7. Volume 43, Number 1/February 2021 7
Gallic Acid and Ischemia-Reperfusion Testicular Injury
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