ANALYTICAL AND QUANTITATIVE CYTOPATHOLOGY AND HISTOPATHOLOGY

1. 197
OBJECTIVE: Ovarian torsion is a gynecologic prob-
lem that presents as acute lower abdominal pain. As a
result, blood flow to the ovaries decreases and ischemia
develops. Many medications have been used to treat
ovarian torsion, including carvacrol. We conducted an
experimental ischemia/reperfusion (torsion-detorsion)
model using rats to observe the effects of carvacrol on
ovarian torsion by immunohistochemical study.
STUDY DESIGN: Thirty-two female Wistar rats were
randomly categorized into 4 groups (8 rats per group):
control group, ischemia group, ischemia/reperfusion
group, and ischemia/reperfusion+carvacrol–treated
group. Ischemia was created by sealing the left ovaries
for 3 hours, followed by 3-hour reperfusion. For the is-
chemia/reperfusion+carvacrol–treated group, 100 mg/kg
carvacrol was administered orally in 2 mL 0.9% NaCl
after the reperfusion. All animals were sacrificed, and
ovarian tissues were dissected for routine paraffin wax
embedding tissue protocol.
RESULTS: Control groups showed normal ovarian his-
tological structures. In the ischemia group, hyperplastic
granulosa cell vascular dilation, severe hemorrhage,
and inflammation were observed. The ischemia/reper-
fusion group showed edema, inflammation, congestion,
degenerated follicles, and cells with pyknotic nuclei. In
the ischemia/reperfusion+carvacrol group, degenerative
changes and vascular pathologies were decreased in
ovarian tissues. Endothelin-1 (ET-1) was expressed in
degenerated follicles and vascular endothelial cells in
the ischemia and ischemia/reperfusion group. Expres-
sion of ET-1 was decreased in follicular cells but nega-
tive in stromal and luteal cells in the ischemia/reper­
fusion+carvacrol group. ADAMTS-5 expression was
positive in degenerated follicles, apoptotic cells, and in-
flammatory cells in the ischemia and ischemia/reper-
fusion groups. In the ischemia/reperfusion+carvacrol
group, corona cells and a few inflammatory cells around
vessels showed positive ADAMTS-5 expression.
CONCLUSION: ET-1 can induce angiogenic devel-
opment with the decrease of degenerative development
and inflammation in the carvacrol group. ADAMTS-5
molecule may be a marker of cellular structure and
extracellular matrix development in different stages of
development of ovarian cells and follicles in ischemia
and oxygen deficiency caused by ischemia reperfusion.
(Anal Quant Cytopathol Histpathol 2020;42:197–
204)
Keywords: ADAMTS-5; carvacrol; detorsion;
endothelin-1; ischemia; ischemia-reperfusion inju-
ry; ovarian torsion; rats, Wistar; reperfusion; reper-
fusion injury; torsion abnormality.
Analytical and Quantitative Cytopathology and Histopathology®
0884-6812/20/4206-0197/$18.00/0 © Science Printers and Publishers, Inc.
Analytical and Quantitative Cytopathology and Histopathology®
Investigation of the Effects of Carvacrol on
Experimental Ischemia/Reperfusion Model of
Rat Ovaries by Immunohistochemistry
Nurullah Peker, M.D., Uğur Değer, M.D., and Fırat Aşır, Ph.D.
From the Departments of Obstetrics and Gynecology and of Histology and Embryology, Faculty of Medicine, Dicle University, Diyar-
bakır; and the Department of Obstetrics and Gynecology, Dicle Memorial Hospital, Diyarbakır, Turkey.
Nurullah Peker is Assistant Professor, Department of Obstetrics and Gynecology, Faculty of Medicine, Dicle University.
Uğur Değer is Physician, Department of Obstetrics and Gynecology, Dicle Memorial Hospital.
Fırat Aşır is Research Assistant, Department of Histology and Embryology, Faculty of Medicine, Dicle University.
Address correspondence to: Fırat Aşır, Ph.D., Department of Histology and Embryology, Faculty of Medicine, Dicle University, 21280
Diyarbakır, Turkey (firatasir@gmail.com).
Financial Disclosure: The authors have no connection to any companies or products mentioned in this article.

2. Ovarian torsion is a clinical complication which
occurs by torsion of the adnexa and which consti-
tutes a surgical emergency, accounting for 2.7% of
all gynecologic emergencies.1 Early diagnosis and
management of ovarian torsion is very import-
ant for woman who desire future fertility. One of
the surgical approaches is salpingo-oophorectomy,
which removes one or both of the ovaries. Recent-
ly, conservative management has been implement-
ed by detorsion of the torsioned segments. During
torsion, ischemia develops in the ovaries due to
reduced blood flow, and lactic acid, hypoxanthine,
and lipid peroxide levels in the tissues increase.2
Restoring torsioned adnexa to the normal position
causes an excess amount of molecular oxygen to
be supplied to the tissues, resulting in some local
and systemic cellular events such as neutrophil in­
filtration, excessive production of reactive oxygen
species (ROS),3,4 and release of nitric oxide (NO).5,6
ROS damages the cellular membrane through the
peroxidation of polyunsaturated fatty acids.7
There are many anti-inflammatory, immuno-
modulatory, and antioxidant free radical scaven-
gers which have been used to prevent such ovar-
ian ischemia/reperfusion injury in tissues.8,9 Car­
vacrol [isopropyl-ortho-cresol, C6H3(OH)(C3H7)]
is a monoterpenic phenolic compound naturally
found in various plants belonging to the family
Lamiaceae.10 Carvacrol has been used as food
or a food additive for the past several decades
and is well investigated in many literatures.11
Numerous studies focused on the therapeutic
potential of carvacrol on different diseases and
have stated that carvacrol has antioxidant,12 anti-
inflammatory,12 anti-tumor,12 bactericidal,13 antitu-
mor,14 and fungicidal activities14 and antimicrobial
properties.15
Endothelins are a family of 21 amino acid pep-
tides involved in physiological functions, with
the 3 forms of ET-1, ET-2, and ET-3.16 Their main
activity is on regulation of blood flow. ET-1 is a
potent endogenous vasoconstrictor which is main-
ly produced in endothelial cells and encoded by
human EDN1 gene.17 ET-1 is expressed in the
cardiovascular system, urinary system, nervous
system, respiratory system, immune system, and
other tissues.16,17 The ADAMTS (a disintegrin and
metalloproteinase with thrombospondin motifs)
family consists of 19 associated proteases that act
in the formation of extracellular matrix.18 Their
functions include collagen processing, cleavage of
the matrix proteoglycans, inhibition of angiogene-
sis, and blood coagulation homoeostasis. They are
also involved in organogenesis, inflammation, and
fertility. ADAMTS-5 is also known as aggrecan-2,
a major component of extracellular matrix and
ADAMTS-5 gene known to be expressed in the
bladder, cervix, esophagus, etc.19 It has been shown
to be anti-tumorigenic in cancer and antiangiogen-
ic in arthritis.20
We investigated the effects of carvacrol on his-
tological changes of ovaries with endothelin-1 and
ADAMTS expression in an ovarian ischemia/re-
perfusion injury model.
Materials and Methods
Study Design
All experimental protocols were conducted at
Dicle University Animal Research Center. All pro-
cedures were approved by the Dicle University
Animal Care and Use Local Committee, with a
record number of 2019/11. Thirty-two female
Wistar rats weighing 230–250 g each were kept
at 24±1°C and 12-hour light/dark cycles with
ad libitum access to water and nutrition. The rats
were randomly assigned to 4 groups (8 rats per
group): control group, ischemia group, ischemia-
reperfusion group, and ischemia/reperfusion+
carvacrol group. Vaginal smears were examined to
follow estrous cycles. 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,
cornified epithelial, and nucleated epithelial) ob­
served in the smear.21
Intramuscular ketamine hydrochloride (50 mg/
kg Ketalar; Eczacibasi, Istanbul, Turkey) and xy-
lazine hydrochloride (10 mg/kg Rompun; Bayer
Türk I
·
laç Ltd, Istanbul, Turkey) were administered
to anesthetize the rats.
A midline abdominal incision of 2.5 cm (laparot-
omy) was performed under sterile conditions for
each rat. The following procedures were applied to
groups after all animals were anesthetized.
Control Group. The ovaries were surgically opened
and then closed, and then blood and ovarian tis-
sues of the animals were taken.
Ischemia Group. The ovaries were surgically opened,
and the left ovaries were sealed for 3 hours to
create ischemia.
Ischemia/Reperfusion Group. The ovaries were sur-
198 Analytical and Quantitative Cytopathology and Histopathology®
Peker et al

3. gically opened, and the left ovaries were sealed
for 3 hours of ischemia. Then, the ovary was un-
sealed, and blood flow was re-allowed for 3 hours
of reperfusion.
Ischemia/Reperfusion+Carvacrol Group. 100 mg/kg
carvacrol dissolved in 2 mL 0.9% NaCl was given
orally after the reperfusion, and tissue specimens
were taken after 3 hours. Then, all animals were
sacrificed with overdose anesthetic, and ovarian
tissues were dissected.
Paraffin Wax Embedding Protocol and
Immunohistochemical Staining
The protocol is described in the study by Obut
and Oglak.22 Tissues were fixed with zinc-formalin
solution (catalog no. Z2902, Sigma-Aldrich, St.
Louis, Missouri, USA) and washed under tap
water for 5 minutes. Tissues were passed through
ascending alcohol series for about 24 hours. To
remove alcohol, tissues were washed with xylene
2×30 minutes and incubated within paraffin wax.
Tissues were embedded in paraffin wax, and 4–6
µm sections were cut with a microtome (catalog
no. RM2265, Leica, Wetzlar, Germany). Sections
were incubated at 60°C for 3–4 hours. After being
deparaffinized within xylene for 2×30 minutes,
sections were brought to distilled water. Some
of the sections were stained with routine hema-
toxylin and eosin, and the rest were soaked in
PBS for 3×5 minutes. Hydrogen peroxide solu-
tion (catalog no. TA-015-HP, Thermo Fisher, Fre-
mont, California, USA) was dropped on sections
for 20 minutes. After washing in PBS for 3×5 min-
utes, ultra V Block (catalog no. TA-015-UB, Ther-
mo Fisher) was applied to sections for 8 minutes.
Sections were incubated with primary antibod-
ies anti-ADAMTS 5 (catalog no. ab41037, Abcam,
Cambridge, UK; dilution rate: 1/100) and anti-
endothelin 1 (catalog no. ab117757, Abcam; dilu-
tion rate: 1/200) at +4°C overnight. The next day,
sections were left at room temperature for 30–60
minutes. Following the PBS step, sections were
washed with biotiny­
lated secondary antibody (cat-
alog no. TP-015-BN, Thermo Fisher) for 14 min-
utes. Streptavidin-peroxidase (catalog no. TS-015-
HR, Thermo Fisher) was dropped onto sections
for 15 minutes. Clear-ing with PBS, DAB (catalog
no. TA-001-HCX, Thermo Fisher) was used as
chromogen and reaction was stopped by PBS.
Sections were counterstained with Harris hema-
toxylin and mounted with Entellan (catalog no.
107961, Sigma-Aldrich). Slides were analyzed with
Zeiss Imager A2 and photomicrographed.
Results
Figure 1 shows ovarian sections stained with
hematoxylin-eosin. In the control group, the ger­
minal layer, primordial follicles, secondary fol-
licles, tertiary follicles, and corpus luteum were
observed to be normal. Granulosa cells were poly­
gonal with eosinophilic cytoplasm and chromatin-
rich nuclei. The stromal area contained irregu-
lar connective tissue with vascular structures and
hemorrhage. In the ischemia group, granulosa
cells were hyperplastic and apoptotic. Blood ves-
sels were dilated, with a high level of hemor-
rhage and inflammation. In the ischemia/reper-
fusion group, preantral and antral follicles were
degenerated with apoptotic follicular cells and
pyknotic nuclei. There was vascular congestion,
edema, and inflammation around the follicles. In
the ischemia/reperfusion+carvacrol group, the
germinal layer was normal and degenerative
changes were decreased in the preantral and an-
tral follicles. Congestion in the small blood vessels
was observed. Inflammation around the vessels
was also diminished.
Figure 2 shows ovarian sections immunostained
with endothelin-1 antibody. In the control group,
some macrophages and stromal cells and endothe-
lial cells of the artery and veins showed positive
endothelin-1 expression. In the ischemia group,
endothelin-1 expression was observed in an intense
inflammatory region adjacent to the antral follicle,
degenerated endothelial cells, inflammatory cells,
and stromal cells. In the ischemia/reperfusion
group, endothelin-1 expression was increased in
degenerated cells of the preantral and antral folli-
cles and hyperplastic endothelial cells of overdi-
lated blood vessels. In the ischemia/reperfusion+
carvacrol group, endothelin-1 expression was pos-
itive in some cells of preantral and antral follicles
and endothelial cells of small capillary vessels but
negative in stromal cells and luteal cells.
Figure 3 shows ovarian sections immunostained
with ADAMTS-5 antibody. In the control group
some granulosa and theca cells of antral follicle
and fibroblasts expressed ADAMTS-5. In the is-
chemia group, ADAMTS-5 expression was ob­
served in degenerated preantral and antral follicles,
apoptotic cells, and inflammatory cells around
the vessels. In the ischemia/reperfusion group,
degenerated theca cells of antral follicle and in-
Volume 42, Number 6/December 2020 199
Effects of Carvacrol on Ischemia/Reperfusion

4. flammatory cells around the vessels showed pos-
itive ADAMTS-5 expression. In the ischemia/
reperfusion+carvacrol group, corona cells of pre-
antral and antral follicles and a few inflammatory
cells around vessels expressed ADAMTS-5.
Discussion
Torsion performed by the rotation of the ovary
about its own axis results in decreased arterial
blood flow and obstruction of venous and lym-
phatic drainage. As a result of ischemia, ovarian
size increases, hemorrhage, and venous conges-
tion occur. A decrease in blood flow causes an
increase in lactic acid, hypoxanthine, and lipid
peroxide levels in ovarian tissue.23 It triggers hy-
poxanthine and xanthine oxidase to be effective
in ischemic tissue that develops with deteriora-
tion in ovary and oxygen. Free oxygen radicals
which damage tissues are induced. Production of
increased free oxygen radicals causes peroxida-
tion of the cell membrane and mitochondrial lip-
ids, leading to further damage to the ischemic
tissue. Thus, the damage is increased more after
detorsion.24 Akdemir et al found that ischemia/
reperfusion injury caused follicular degeneration,
vascular congestion, edema, hemorrhage, and in­
flammatory cell infiltration. They observed that
follicular degeneration was mainly in the primor-
dial and developing follicles and in the corpus lu-
teum.25 Eser et al observed that ovarian sections
contained severe edema, severe vascular conges-
tion, severe hemorrhage, and severe polymorpho-
200 Analytical and Quantitative Cytopathology and Histopathology®
Peker et al
Figure 1 Ovarian sections stained with hematoxylin-eosin. (A) In the control group, the germinal layer, primordial follicles, secondary
follicles, tertiary follicles, and corpus luteum were observed to be normal. Granulosa cells were polygonal with eosinophilic cytoplasm
and chromatin-rich nuclei. The stromal area contained irregular connective tissue with vascular structures and hemorrhage. (B) In the
ischemia group, granulosa cells were hyperplastic and apoptotic. Blood vessels were dilated (red arrow) with a high level of hemorrhage
(red star) and inflammation. (C) In the ischemia/reperfusion group, preantral and antral follicles were degenerated with apoptotic follicular
cells and pyknotic nuclei. There was vascular congestion, edema, and inflammation around the follicles. (D) In the ischemia/reperfusion+
carvacrol group, the germinal layer was normal and degenerative changes were decreased in the preantral and antral follicles. Congestion
in the small blood vessels was observed. Inflammation around the vessels was also diminished.

5. nuclear leukocyte infiltration in an ischemia/reper-
fusion model of the ovary.26 Our hematoxylin-eosin
staining of ovarian sections is shown in Figure 1.
We observed hyperplastic and apoptotic granu-
losa cells in the ischemia group. There was hem-
orrhage and inflammation (Figure 1B). In the
ischemia/reperfusion group, degenerated prean-
tral and antral follicles and apoptotic follicular
cells with pyknotic nuclei were clear. Vascular
congestion, edema, and inflammation were seen
in the stromal area (Figure 1C). In the ischemia/
reperfusion+carvacrol–treated group, degenera-
tive changes and inflammation were decreased
(Figure 1D). Our histopathological results were
consistent with those reported in the literature.
Carvacrol is a major component of the essential
oils of certain aromatic plants and has attracted
much attention due to its biological properties in
the treatment of human diseases.27 Carvacrol ex-
hibits strong antioxidant and hydrophobic proper-
ties associated with its substituted aromatic ring,
as well as hydrophilic properties associated with
its phenolic OH group, which have previously been
associated with antioxidant, anti-inflammatory,
antibacterial, antifungal, antiprotozoal, anticarci­
nogenic, antidiabetic, antinociceptive, cardiopro-
tective, and neuroprotective effects.28-30 In a study
it was reported that carvacrol has protective prop-
erties in reducing oxidative stress and increasing
the antioxidative effect on ethanol-induced neu-
Volume 42, Number 6/December 2020 201
Effects of Carvacrol on Ischemia/Reperfusion
Figure 2 Ovarian sections immunostained with endothelin-1 antibody. (A) In the control group, some macrophages and stromal cells
and endothelial cells of the artery and veins showed positive endothelin-1 expression. (B) In the ischemia group, endothelin-1 expression
was observed in an intense inflammatory region adjacent to the antral follicle, degenerated endothelial cells, inflammatory cells, and
stromal cells (black arrows). (C) In the ischemia/reperfusion group, endothelin-1 expression was increased in degenerated cells of the
preantral and antral follicles and hyperplastic endothelial cells of over-dilated blood vessels (black arrows). (D) In the ischemia/
reperfusion+carvacrol group, endothelin-1 expression was positive in some cells of preantral and antral follicles and endothelial cells of
small capillary vessels (black arrows) but negative in stromal cells and luteal cells (yellow arrows).

6. ron damage.31 Suo et al32 reported that apopto-
sis in liver ischemia/reperfusion injury was sup-
pressed by carvacrol treatment and that carvac-
rol could reduce ischemia/reperfusion–induced
liver damage by anti-oxidative and anti-apoptotic
effects.
The role of endothelins in the follicle is stated to
be on regulation of steroidogenesis, but it may also
be the stimulation of DNA synthesis by granulosa
cell growth and EDN1.33 Endothelin-1 immunore-
activity was found to be present in the granulosa
cells of the follicles in 2/3 of the large antral fol-
licles as the antrum develops. Endothelin-1 was
reported to inhibit the secretion of LH-induced,
FSH-induced, and hCG-induced progesterone by
granulosa cells.34,35 Kalani et al36 stated that ET-1
may contribute to the development of endothelial
dysfunction and increase the production of reac-
tive oxygen species. Kowala et al37 showed that
ET-1 induced activation and accumulation of mac-
rophages. Endothelin-1 immunostained ovarian
sections are shown in Figure 2. In the ischemia
group, endothelin-1 expression was observed in
degenerated endothelial cells, inflammatory cells,
and stromal cells (Figure 2b). In the ischemia/
reperfusion group, increased endothelin-1 expres-
sion was observed in degenerated follicles and
vascular endothelial cells (Figure 2c). In the ische-
mia/reperfusion+carvacrol group, endothelin-1
expression was slight in follicular cells and endo-
202 Analytical and Quantitative Cytopathology and Histopathology®
Peker et al
Figure 3 Ovarian sections immunostained with ADAMTS-5 antibody. (A) In the control group some granulosa and theca cells of antral
follicle and fibroblasts expressed ADAMTS-5. (B) In the ischemia group, ADAMTS-5 expression was observed in degenerated preantral
and antral follicles, apoptotic cells, and inflammatory cells around vessels (black arrows). (C) In the ischemia/reperfusion group,
degenerated theca cells of antral follicle and inflammatory cells around vessels showed positive ADAMTS-5 expression (black arrows).
(D) In the ischemia/reperfusion+carvacrol group, corona cells of preantral and antral follicles and a few inflammatory cells around vessels
expressed ADAMTS-5 (black arrow).

7. thelial cells but negative in stromal cells and luteal
cells (Figure 2d).
ADAMTS has critical roles in the degradation of
versicans, expansion of COC formation, ovulation,
and angiogenesis. For successful ovulation, the
formation of the cumulus oocyte complex (COC)
and the rupture of the ovarian surface epithelium
should be regular. The synthesis of basic extra-
cellular matrix components such as versican and
hyaluronic acid (HA) increases as COC forma-
tion occurs. Versican, a substrate of ADAMTS-1,
ADAMTS-4, and ADAMTS-5, has been implicat-
ed in the remodeling of the extracellular matrix,
the movement of cumulus cells, and the main-
tenance of structural and functional integrity of
the matrix.38 In a study ADAMTS-1, ADAMTS-4,
and ADAMTS-5 were expressed by the granulosa
cells of small follicles. ADAMTS-5 plays an im­
portant role in cumulus cells collected from the
ovaries, ovarian cells containing ovulation cone at
the follicle tear site, and in newly formed corpus
luteum cells. ADAMTS-5 is expressed in gran-
ulosa cells at specific follicular developmental
stages. ADAMTS-5 signal in the adult ovary has
been shown to be localized in granulosa cells of
antral follicles showing signs of atresia.39 Ovar-
ian sections stained with ADAMTS-5 antibody
are shown in Figure 3. In the ischemia group,
ADAMTS-5 expression was seen in degenerated
follicles and in apoptotic and inflammatory cells
(Figure 3b). In the ischemia/reperfusion group,
ADAMTS-5 expression was positive in degener-
ated theca cells and inflammatory cells (Figure
3c). In the ischemia/reperfusion+carvacrol group,
corona cells and a few inflammatory cells around
vessels showed positive ADAMTS-5 expression
(Figure 3d).
In conclusion, we think that ET-1 can induce
angiogenic development with the decrease of de­
generative development and inflammation in the
carvacrol group. ET-1 is important for the devel­
opment of endothelial dysfunction and inflamma-
tion increase and macrophage activity. We suggest
that the ADAMTS-5 molecule may be a marker
of cellular structure and extracellular matrix de-
velopment in different stages of development of
ovarian cells and follicles in ischemia and oxygen
deficiency caused by ischemia reperfusion.
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