ANALYTICAL AND QUANTITATIVE CYTOPATHOLOGY AND HISTOPATHOLOGY

1. 8
Analytical and Quantitative Cytopathology and Histopathology®
0884-6812/20/4201-0008/$18.00/0 © Science Printers and Publishers, Inc.
Analytical and Quantitative Cytopathology and Histopathology®
OBJECTIVE: To investigate the effect of allopurinol on
an experimentally induced ovarian ischemia-reperfusion
model.
STUDY DESIGN: Female rats in the estrous cycle
(n=32) were divided into sham, ischemia, ischemia-
reperfusion, and ischemia-reperfusion+allopurinol–
treated groups. In the sham group the ovaries were
opened and closed. In the ischemia group the ovaries were
sealed for 2-hour ischemia. In the ischemia-reperfusion
group, after ischemia, 2.5 hours of reperfusion was done.
In the ischemia-reperfusion+allopurinol group, 3 hours
after ischemia-reperfusion, 50 mg/kg allopurinol was
administered.
RESULTS: In the allopurinol-administered group, MDA
levels were decreased. GSH values were decreased in the
ischemia and ischemia-reperfusion group but increased
in the allopurinol-treated group as compared to the con-
trol group. Caspase-3 expression was positive in enlarged
corpus luteum cells. sFlt-1 expression was positive in
vascular endothelial cells between preantral and antral
follicles and some macrophages but negative in granular
cells. In the ischemia group, sFlt-1 expression was pos-
itive in degenerative preantral and antral follicle cells,
endothelial cells, and intense inflammatory cells. In the
ischemia-reperfusion group, increased sFlt-1 expression
was observed in luteal cells of the corpus luteum, vas-
cular endothelial, and inflammatory cells. In the isch-
emia-reperfusion+allopurinol group, granular cells and
corpus luteum cells showed decreased sFlt-1 expression,
while being positive in vascular endothelial cells.
CONCLUSION: Allopurinol inhibits development of
apoptosis and reduces oxidative load in the ischemia-
reperfusion stage, thus protecting the ovary from dam-
age. (Anal Quant Cytopathol Histpathol 2020;42:
8–16)
Keywords: allopurinol, Caspase-3, FLT1 protein,
fms-like tyrosine kinase-1, ischemia, ischemia-
reperfusion injury, ovarian diseases, ovarian ische­
mia, ovarian torsion, ovary, oxidative stress, rats,
reperfusion injury, sFlt-1, vascular endothelial
growth factor receptor-1.
Ovarian torsion is one of the most important
gynecological conditions, with an incidence of 3%
worldwide. It is defined as the twist of the ovary
around a center line consisting of the infundibu­
lopelvic and tubo-ovarian ligament.1 Ovarian tor-
Protective Effect of Allopurinol on
Experimental Ovarian Ischemia-Reperfusion
Injury Model of Rats
Engin Yurtçu, M.D., Cihan Toğrul, M.D., and Engin Deveci, Ph.D.
From the Department of Obstetric and Gynecology, Karabük University Medical School, Karabük; the Department of Obstetric and
Gynecology, Hitit University Medical School, Çorum; and the Department of Histology and Embryology, Dicle University Medical
School, Diyarbakır, Turkey.
Engin Yurtçu is Assistant Professor, Department of Obstetric and Gynecology, Karabük University Medical School.
Cihan Toğrul is Associate Professor, Department of Obstetric and Gynecology, Hitit University Medical School.
Engin Deveci is Professor, Department of Histology and Embryology, Dicle University Medical School.
Address correspondence to: Engin Deveci, Ph.D., Department of Histology and Embryology, Dicle University Medical School, Univer-
sity Street, Diyarbakır 21280, Turkey (engindeveci64@hotmail.com).
Financial Disclosure: The authors have no connection to any companies or products mentioned in this article.

2. Volume 42, Number 1/February 2020 9
Allopurinol in Ovarian Ischemia-Reperfusion Injury
sion can be seen at any stage of a woman’s life,
but it occurs most often during the premenarchal
or reproductive years. Therefore, early diagnosis
is very important for the woman’s reproductive
health. Once ovarian torsion is suspected, surgery
or detorsion is the mainstay for diagnosis and
treatment.2 Ovarian torsion is associated with a
reduction in venous return, ovarian enlargement,
edema, and interstitial hemorrhage. Ovarian ne-
crosis can occur when the arterial flow is dis­
rupted.3 Detorsion causes more severe damage
as compared to the damage caused by the tor-
sion in that tissue. Although reperfusion proce-
dure has been reported to be a method of treat­
ment aimed at providing normal function of
the ovary and preventing possible infertility, the
ischemia-reperfusion procedure has been demon­
strated to result in infertility in rats.4,5
After the ischemic/hypoxic period, when re-
versed, visceral organs are perfused with oxygen­
ated blood, which leads to the generation of reac-
tive oxygen species (ROS). These products cause
secondary cell damage, leading to cell death by
both apoptosis and necrosis.6 Detorsion of the
twisted ovary recovers the vascular supply and
leads to preservation of the affected ovary. How­
ever, ischemia-reperfusion injury is the main prob-
lem for the preserved ovary. Torsion causes is-
chemia, hypoxic damage, increasing tissue lactic
acid, as well as hypoxanthine and lipid peroxide
levels.7,8 Following detorsion and reperfusion of an
ischemic organ, additional injury occurs through
neutrophil infiltration and excessive ROS produc­
tion. The structures most sensitive to ROS are
membrane lipids. Lipid oxidation results in the
production of toxic substances, including malon-
dialdehyde (MDA).9 Several studies have reported
that anti-inflammatory and antioxidant free rad­
ical scavengers can prevent ischemia-reperfusion
injury in the ovary. The ischemia-reperfusion mod­
el leads to the release of various free radicals, which
are the most important mediators of oxidative tis­
sue injury and consequential organ dysfunction.
Ischemia-reperfusion leads to oxidative stress
and deleterious effects on cellular function. Cells
may die during this ischemic period, which is
known as necrosis.10 An excessive inflammatory
response is clearly recognized as a key mecha-
nism of injury during reperfusion. Various che­
mokines and metabolites are investigated asso-
ciated with ischemia-reperfusion–induced tissue
injury. Given these findings, it has been hypoth­
esized that pharmacological strategies that limit
neutrophil recruitment may also limit the damage
induced by reperfusion.11 Therefore, several anti-
inflammatory and antioxidant agents have been
used to prevent ischemia-reperfusion injury in tis­
sue.8 Allopurinol (4-hydroxypyrazole[3,4-d]pyri-
midine), used in the present study, is a free radi-
cal scavenger used for several diseases12 such as
hyperuricemia,13 gout,14 and inflammation,12 and
it is also used for myocardial protection during
cardiac or aortic surgery or post-ischemic reperfu-
sion.15 Allopurinol is a xanthine oxidase inhibitor
which was demonstrated in previous studies to
have protective effects against ischemia16 via block­
ing purine breakdown. Research has been per­
formed on allopurinol treatment against ischemia-
reperfusion organ damage.
Caspases are a family of genes maintaining
homeostasis through regulating cell death and in-
flammation. They participate in ordered processes
such as apoptosis and inflammation. Caspases
are classified according to their roles in apop­
tosis; caspase-3 acts as an executioner caspase.17
Glamoclija et al18 examined caspase-3 expression
in granulosa cells from human ovarian tissue and
preovulatory human ovarian follicles to deter-
mine whether caspase-3 activation plays a role in
the formation of apoptotic cell death. They also
analyzed the relationship between apoptotic mor-
phological features and DNA fragmentation in
granulosa cells of ovarian follicles.
Soluble fms-like tyrosine kinase–1 (sFlt-1) is a
transmembrane glycoprotein with tyrosine kinase
activity and is a potent inhibitor of vascular en­
dothelial growth factor. Soluble FLT1 is produced
by a variety of tissues such as the placenta, en­
dothelial cells, and peripheral blood mononucle-
ar cells.19 Recently, several studies have demon-
strated proliferative suppression of sFlt-1 which
caused apoptosis in endothelial cell lines.20
This study investigated the effect of allopurinol
on experimentally induced ovarian torsion/detor-
sion ischemia-reperfusion injury in rats using bio­
chemical, histopathologic, and immunohistochemi-
cal methods.
Materials and Methods
Experimental Design
All procedures performed in this experiment were
approved by the Ethics Committee for the Treat­
ment of Experimental Animals (Dicle University
Faculty of Medicine, Turkey). Female Wistar rats

3. 10 Analytical and Quantitative Cytopathology and Histopathology®
Yurtçu et al
(250–280 g) were maintained under 22±1°C and
12-hour light/dark cycles with water ad libi-
tum and free access to standard pellet feed. Rats
were randomly divided into 4 groups as fol-
lows: sham group (n=8), ischemia group (n=8),
ischemia-reperfusion group (n=8), and ischemia-
reperfusion+allopurinol–treated group (n=8).
Estrous cycles were evaluated by daily vagi-
nal smear. Vaginal cells were left to air dry after
being smeared on a lumen, and then the stage of
the estrous cycle was determined from the cell
types (presence or absence of leukocytes, corni-
fied epithelial, and nucleated epithelial) observed
in the smear.21
Surgical Procedure
Anxiety of the rats was high when preparing for
the surgical procedure; therefore, intramuscular
ketamine hydrochloride (50 mg/kg) and xylazine
hydrochloride (10 mg/kg) were administered to
each rat for anesthesia. In all of the groups, a mid-
line abdominal incision of 2.5 cm (laparotomy) was
performed under sterile conditions.
Sham Group. Under anesthesia the ovaries were
surgically opened and then closed. The animals
were sacrificed with overdose anesthetic, and
blood and ovarian tissues of the animals were
taken.
Ischemia Group. Under anesthesia the ovaries were
surgically opened and the left ovary was sealed for
ischemia.
Ischemia-Reperfusion Group. After 2.5 hours of is-
chemia, blood flow was re-allowed for 2 hours
of reperfusion. The animals were sacrificed with
overdose anesthetic, and blood and ovarian tissues
were taken.
Ischemia-Reperfusion and Allopurinol-Treated Group.
Under anesthesia the ovaries were surgically
opened, and ischemia was performed to the left
ovary for 2.5 hours. Then reperfusion was per-
formed for 2.5 hours. Allopurinol solution was
injected intraperitoneally at a concentration of 50
mg/kg of body weight, and 3 hours after the
reperfusion tissue samples were taken. For allo-
purinol treatment, allopurinol in powder form
(Sigma-Aldrich, St. Louis, Missouri, USA) was dis-
solved in saline, and 2 M NaOH was added to
generate a final pH of approximately 10.5.22 The
animals were sacrificed with overdose anesthetic,
and blood and ovarian tissues of the animals were
taken.
Malondialdehyde (MDA) and Glutathione
Peroxidase (GSH-Px) Assays
MDA levels and GSH-Px activities were deter-
mined in the ovary of each rat, and the average
values of each group were calculated. Each ovary
sample was prepared as a 10% homogenate (ac-
cording to weight) in 0.9% saline using a homog­
enizer on ice. Then, the homogenate was cen­
trifuged at 2000 rpm for 10 minutes, and the
supernatant was collected. MDA levels were de-
termined using the double heating method of
Draper and Hadley.23 The GSH-Px activity was
measured by the method of Paglia and Valentine.24
An enzymatic reaction was initiated by the addi-
tion of hydrogen peroxide (H2O2) to a tube that
contained reduced nicotinamide adenine dinu­
cleotide phosphate, reduced glutathione, sodium
azide, and glutathione reductase. The change in
absorbance at 340 nm was monitored by spectro­
photometry. Data were expressed as U/g protein.
Histopathological Analysis
The ovarium samples were fixed with neutral buf-
fered 10% formalin solution. After preservation,
ovarium samples were directly dehydrated in a
graded series of ethanol and embedded into par­
affin wax. Five-mm sections were cut with micro-
tome (Rotary Microtome RM 2265, Leica Biosys­
tems, Germany) and mounted on the coated slides.
Sections were passed through xylol, then descen-
ding alcohols, and brought to distilled water for
2 minutes. Sections were stained in hematoxylin
for 5 minutes and then washed in tap water for
10 minutes. After staining with eosin for 5 min-
utes, slides were passed through ascending alco-
hol, and then in xylol for 15 minutes. Slides were
mounted with Entellan and examined under light
microscope.
Histopathological features for ovarian injury
were follicular cell degeneration (granulosa cells),
vascular occlusion, hemorrhage, and inflamma-
tion (neutrophil infiltration). Each sample was
scored for each feature using a scale of 0 to 4
(0=none, 1=mild, 2=moderate, 3=severe, and 4=
most severe). Ovary sections were blindly ana-
lyzed by the same histopathologist. Histopathol­
ogical tissue injury scores were determined as
explained above.

4. Volume 42, Number 1/February 2020 11
Allopurinol in Ovarian Ischemia-Reperfusion Injury
Immunohistochemical Methods
Sections prepared from paraffin blocks mounted
on slides and antigen retrieval process for tis­
sues was performed in citrate buffer solution (pH
6.0) twice (5 minutes and 3 minutes, distinctly)
in a microwave oven at 700 W. The sections were
left to cool at room temperature for 20 minutes
and washed in distilled water twice for 4 min­
utes. Endogenous peroxidase activity was blocked
in 10% hydrogen peroxide solution for 7 min­
utes. Ultra V block (Histostain-Plus Kit, 1754084A,
Novex Life Technologies, Frederick, Maryland,
USA) was applied for 8 minutes prior to the
application of primary antibody (Caspase-3 Anti-
body Kit, Santa Cruz Biotechnology, USA) and
left overnight. Secondary antibody (Histostain-
Plus Kit) was applied for 20 minutes. The sections
were then exposed to streptavidin-peroxidase
for 20 minutes. Diaminobenzidine (DAB-Plus Sub­
strate Kit, 1636518A, Novex Life Technologies)
was used as a chromogen. After being counter­
stained with hematoxylin and washed in tap
water for 3 minutes and in distilled water for
2×3 min, the slides were mounted. Sections were
examined under light microscope (Carl Zeiss
Imager A2, Germany).
Statistical Analysis
Statistical analyses were performed with SPSS
(IBM SPSS Statistics for Windows, Version 22.0,
Released 2013, IBM Corp., Armonk, New York,
USA). Descriptive statistics were presented as
median (min-max) and mean±standard deviation
values. The significance of the difference among
more than two groups was evaluated by using
the Kruskal-Wallis test since data did not meet
the assumptions of the parametric test ANOVA.
Post-hoc tests with Bonferroni correction were
used to determine which groups differed with
pairwise comparison. A value of p<0.05 was con-
sidered as statistically significant.
Results
We evaluated biochemical, histopathological, and
immunohistochemical parameters to determine
the efficacy of allopurinol on ischemia and reper-
fusion injury of rat ovaries. Results are shown in
Table I. When we compared the groups in terms
of MDA levels, a statistically significant differ­
ence was found (p<0.05); especially in the ische-
mia group and ischemia-reperfusion groups, MDA
values were increased as compared to the control
group. In the allopurinol-treated group we ob-
served that MDA values were decreased. Gluta­
thione values were decreased in the ischemia and
ischemia-reperfusion groups as compared to those
in the control group. On the contrary, in the
allopurinol-treated group glutathione values were
increased.
There was a statistically significant difference
between the groups in terms of histopathologic
scoring. The median histopathology score of the
ischemia-reperfusion group was higher than
that of the sham group (p<0.001). The histologic
score was found to be lower in the ischemia-
reperfusion+allopurinol group as compared with
the ischemia-reperfusion group (Figure 1).
In our study, degeneration of coronal and gran­
ular cells, degeneration of collagen fibrils, dilation
of blood vessels, and intense obstruction in the
antral follicle in the ischemia group and inflam­
mation and necrotic cells (yellow arrow) in the in­
terfollicular area were observed (Figure 2B). In the
ischemia-reperfusion group, degenerative changes
and apoptosis in the cells surrounding the oocyte
cell nucleus within the antral follicle, inflamma-
tion in the stromal region, and dilation and ob-
struction in the blood vessels were also record-
ed (Figure 2C). Histopathological sections of the
group treated with ischemia-reperfusion+allopu-
rinol revealed that some antral follicle granulosa
cells showed hypertrophy and mild thickening
of the connective tissue sheath around the follicle
(Figure 2D).
When the control group caspase-3 activity was
examined, caspase-3 expression in oocyte cells and
granular cells in the preantral and antral follicles
was negative; however, it was positive in stro-
mal cells between some fibrils around the follicle
(Figure 3A). In the ischemia group, degenerated
granular cells in the antral follicle, luteal cells in
the corpus luteum, and intense inflammatory cells
in the stromal region showed positive expres-
sion of caspase-3 (Figure 3B). In the ischemia-
reperfusion group, the expression of caspase-3
was positive in oocyte, granular, stromal cells,
and theca cells in the mature antral follicle (Fig-
ure 3C). In the ischemia-reperfusion+allopuri­
nol group, caspase-3 negative expression was ob-
served in granulosa cells and stromal cells in the
antral follicle. Caspase-3 expression was positive
in the enlarged corpus luteum cells (Figure 3D).
When the control group sFlt-1 activity was ex-
amined, sFlt-1 expression was positive in the vas-

5. 12 Analytical and Quantitative Cytopathology and Histopathology®
Yurtçu et al
Table I Parameters of Granular Cell Degeneration, Vascular Dilation and Congestion, Inflammation, Caspase-3 Expression, sFlt-1
Expression, GSH, and MDA in Control, Ischemia, Ischemia-Reperfusion, and Ischemia-Reperfusion+Allopurinol–Treated Groups
Multiple
comparisons
Mean
Kruskal-Wallis for groups
Parameter Group N Mean±SD rank test value (p<0.05)
Granular cell degeneration (1) Control 8 0.62±0.51 8.0 25.339 (2)(3)
(2) Ischemia 8 3.62±0.51 25.6 p=0 (1)(4)
(3) I/R 8 3.25±0.70 23.3 (1)(4)
(4) I/R+allopurinol 8 0.75±0.46 9.0 (2)(3)
Vascular dilation and congestion (1) Control 8 0.25±0.46 7.3 25.583 (2)(3)
(2) Ischemia 8 3.75±0.46 26.7 p=0 (1)(3)(4)
(3) I/R 8 3.00±0.75 22.1 (1)(2)(4)
(4) I/R+allopurinol 8 0.62±0.74 9.7 (2)(3)
Inflammation (1) Control 8 0.62±0.51 9.5 25.084 (2)(3)
(2) Ischemia 8 3.50±0.75 24.3 p=0 (1)(4)
(3) I/R 8 3.62±0.51 24.6 (1)(4)
(4) I/R+allopurinol 8 0.37±0.51 7.5 (2)(3)
Caspase-3 expression (1) Control 8 0.87±0.64 8.5 24.983 (2)(3)
(2) Ischemia 8 3.75±0.46 26.0 p=0 (1)(4)
(3) I/R 8 3.37±0.51 23.0 (1)(4)
(4) I/R+allopurinol 8 0.87±0.83 8.4 (2)(3)
sFlt-1 expression (1) Control 8 3.00±0.75 13.5 1.797
(2) Ischemia 8 3.37±0.51 18.1 p=0.616
(3) I/R 8 3.37±0.51 18.1
(4) I/R+allopurinol 8 3.25±0.46 16.2
GSH (1) Control 8 10.22±1.28 26.88 27.028 (2)(3)
(2) Ischemia 8 3.56±0.92 4.63 p=0 (1)(4)
(3) I/R 8 5.94±0.59 12.38 (1)(4)
(4) I/R+allopurinol 8 8.97±0.70 22.13 (2)(3)
MDA (1) Control 8 2.76±0.29 7.06 25.096 (2)(3)
(2) Ischemia 8 6.02±0.76 27.31 p=0 (1)(4)
(3) I/R 8 5.05±0.63 21.69 (1)(4)
(4) I/R+allopurinol 8 3.03±0.50 9.94 (2)(3)
GSH = glutathione, I/R = ischemia-reperfusion, MDA = malondialdehyde.
Figure 1
Graphical illustration of Table
I. All parameters (granular cell
degeneration, vascular dilation
and congestion, inflammation,
Caspase-3 expression, sFlt-1
expression, GSH, and MDA)
are shown individually for
each group.

6. Volume 42, Number 1/February 2020 13
Allopurinol in Ovarian Ischemia-Reperfusion Injury
cular endothelial cells between the preantral and
antral follicles and some macrophage cells in the
stroma, but negative sFlt-1 expression was seen in
granular cells (Figure 4A). In the ischemia group,
Figure 2
(A) Control group. Normal
appearance of ovarian
follicularis. H-E staining,
Bar=50 µm. (B) Ischemia
group. Inflammation and
necrotic cells (yellow arrow)
in the interfollicular area.
H-E staining, Bar=50 µm.
(C) Ischemia-reperfusion
group. Degeneration and
apoptosis in antral follicle
cells (black arrow). Dilation
and congestion in the blood
vessels (yellow arrow). H-E
staining, Bar=50 µm.
(D) Ischemia-reperfusion+
allopurinol group.
Hypertrophy in some
granulosa cells (red arrow).
H-E staining, Bar=50 µm.
Figure 3
(A) Control group. Negative
caspase-3 expression in oocyte
cells and granular cells in the
preantral and antral follicles
(arrow). Caspase-3
immunostain, 50 µm.
(B) Ischemia group. Positive
caspase-3 expression in the
antral follicle, luteal cells in
the corpus luteum (arrow), and
intense inflammatory cells in
the stromal region. Caspase-3
immunostain, Bar=50 µm.
(C) Ischemia-reperfusion
group. Positive caspase-3
expression in oocyte cells
(yellow arrow), and granular,
stromal cells, and theca cells
in the mature antral follicle.
Caspase-3 immunostain,
Bar=50 µm. (D) Ischemia-
reperfusion+allopurinol group.
Positive caspase-3 expression
in the enlarged corpus luteum
cells (red arrow). Caspase-3
immunostain, Bar=50 µm.

7. 14 Analytical and Quantitative Cytopathology and Histopathology®
Yurtçu et al
the expression of sFlt-1 was positive in degen-
erative preantral and antral follicle cells, endo-
thelial cells of dilated blood vessels, and intense
inflammatory cells (Figure 4B). In the ischemia-
reperfusion group, increased sFlt-1 expression was
observed in luteal cells of the corpus luteum,
vascular endothelial, and inflammatory cells (Fig-
ure 4C). In the ischemia-reperfusion+allopurinol
group, granular cells and cells in the corpus lute-
um showed decreased sFlt-1 expression, whereas
sFlt-1 expression was positive in endothelial cells
of regular blood vessels (Figure 4D).
Discussion
Ovaries have many pivotal roles in mammals,
including coordination of the development of
secondary sexual characteristics, stimulation and
management of ovulation, and supporting success-
ful uterine implantation and the early phase of
pregnancy. These functions are performed through
complex mechanisms under stable oxygen supply
and consumption conditions, which require nor-
mal microcirculation.10,25,26 Additionally, the pres­
ence of ROS and antioxidants in the female repro­
ductive tract has been demonstrated by various
animal and human studies.27 Oxidative damage
associated with ischemia-reperfusion may impair
such ovarian functions, from oocyte maturation
to fertilization, and this is supported by the his-
tological changes observed in the present study.
Malondialdehyde (MDA) is the basic product of
poly­
unsaturated fatty acid peroxidation and is
quite a toxic molecule. Therefore, it is used to
determine in vivo and in vitro oxidative stress
levels.28 Ischemia-reperfusion injury leads to the
production of excess amounts of highly reactive
molecules that cause damage to lipids, proteins,
and DNA as a result of a series of toxic events.29
MDA, which is a marker of oxidative damage with
the emergence of lipid peroxidation, increased in
ischemia reperfusion and impaired cell membrane
permeability. Therefore, it causes degenerative
change of the tissue, inflammation, and apoptosis.
In our study, while MDA level was high in both
the ischemia and the ischemia-reperfusion groups,
it was decreased in the ischemia-reperfusion+
allopurinol group. Glutathione is one of the most
important indicators of antioxidant capacity; it
protects the tissues against damage caused by
oxidative stress. In the study performed by Yapca
et al,30 it was determined that glutathione concen-
trations of ovarian tissue decreased significantly in
Figure 4
(A) Control group. Positive
sFlt-1 expression in the vascu-
lar endothelial cells between
the preantral and antral
follicles and some macrophage
cells in the stroma, negative
sFlt-1 expression in granular
cells (yellow arrow). sFlt-1
immunostaining, 50 µm.
(B) Ischemia group. Positive
sFlt-1 expression in degenera-
tive preantral and antral folli-
cle cells (yellow arrow). sFlt-1
immunostaining, 50 µm. (C)
Ischemia-reperfusion group.
An increase sFlt-1 expression
in luteal cells of the corpus
luteum, vascular endothelial
and inflammatory cells (red
arrow). sFlt-1 immunostaining,
50 µm. (D) Ischemia-
reperfusion+allopurinol group.
Positive sFlt-1 expression was
positive in endothelial cells
(yellow arrow). sFlt-1
immunostaining, 50 µm.

8. Volume 42, Number 1/February 2020 15
Allopurinol in Ovarian Ischemia-Reperfusion Injury
ischemia-reperfusion–treated rats. In the study of
Aksak Karamese et al31 it was reported that gluta­
thione levels were significantly suppressed when
3 hours of ischemia was followed by the same
period of reperfusion. In our study glutathione
values were decreased in the ischemia and the
ischemia-reperfusion groups, but glutathione val-
ues were increased with allopurinol administra-
tion similar to the control group. There are many
studies in the literature about the improvement
of ischemia-reperfusion injury. These studies dem-
onstrated that agents with antioxidant or anti-
inflammatory activities may be beneficial in re­
ducing ovarian ischemia-reperfusion injury and
revealed the beneficial effect of controlled reper-
fusion in the prevention of ovarian tissue dam-
age. Although there are many studies in the lit­
erature, ischemia-reperfusion damage continues to
be a serious problem clinically. Essentially, early
diagnosis and treatment of ovarian torsion play
an important role in providing urgent protection
against life-threatening complications from ische-
mia and preventing future infertility.32
Allopurinol, with its antioxidant properties, in-
hibits the production of superoxide anions such as
O2 and H2O2 as xanthine oxidase inhibitors. Allo­
purinol is a potential treatment for a range of con-
ditions including chronic heart failure ischemia-
reperfusion injury, vascular disease, chronic kid-
ney disease, and diabetes.19 Previous studies have
shown that ROS production during organ trans­
plantation leads to irreparable damage to tissue,
whereas allopurinol can reduce the production
of free radicals while reducing damage.33,34 In
a study by Soylu Karapinar et al,35 vascular con­
gestion, edema, hemorrhage, and inflammatory
cell infiltration were determined in the ovarian
tissue due to 3-hour ischemia or 3-hour ischemia/
3-hour reperfusion. Ischemia and reperfusion of
ovarian tissue blood flow was negatively affected,
and that caused significant oxidative stress and
finally raised histological damage of the ovary.
Our results of histologic parameters showed
that treatment with allopurinol in the ischemia-
reperfusion group of rats ameliorated the devel­
opment of ischemia and reperfusion ovarian tis-
sue injury (Table I) (Figure 1).
Many studies have shown that oxidative stress
and excessive inflammatory products, depending
on their densities in ischemia-reperfusion injuries,
cause either reversible cell damage or irrever-
sible, lethal cell damage, such as apoptosis and
necrosis.36 Sapmaz et al37 found that the number
of apoptotic cells increased significantly in the
ovaries after ischemia-reperfusion. They detected
TUNEL-positive granulosa cells only in medium
or large ovarian follicles. They also reported that
ischemia-reperfusion injury does not reduce the
ovarian germ cell pool but instead leads to oocyte
maturation problems due to loss of some inter-
nal factors mediated by granulosa cell death. The
number of apoptotic cells positive in the ischemia
and ischemia-reperfusion groups in the ovary was
statistically higher as compared to in the con­
trol group. Caspase-3 expression was found to be
increased. sFlt-1 is secreted from endothelial cells
into their immediate extracellular space as well
as into the general circulation and reduces the
bio­
availability of VEGF by binding and sequester­
ing this growth factor.38 We observed that in the
ischemia-reperfusion+allopurinol–treated group,
granular cells and cells in the corpus luteum
showed decreased sFlt-1 expression.
Allopurinol has been shown to inhibit the de-
velopment of apoptosis and to reduce the oxida-
tive load at the ischemia-reperfusion stage and
protects cellular damage in the ovary. It may be
effective in regulating angiogenesis by inducing
vascular endothelial growth.
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