Montelukast prevents testes against ischemia

Turkish Journal of Urology 2014; 40(4): 221-7 • DOI:10.5152/tud.2014.61587
PEDIATRIC UROLOGY
Original Article
Montelukast prevents testes against ischemia-reperfusion injury
through suppression of iNOS expression
Montelukast testiste iskemi-reperfüzyona bağlı oluşan hasarı iNOS ekspresyonunu
baskılayarak önlemektedir
Mesrur Selçuk Sılay1, Hale Toklu2, Ayşim Özağarı3, Mustafa Aydın4, Şermin Tetik5, Göksel Şener2,
Cengiz Miroğlu4, Muammer Kendirci4
ABSTRACT
Objective: To elucidate the mechanism of a possible protective effect of montelukast against testicular
ischemia/reperfusion (I/R) injury.
Material and methods: Fifty-one adult male Wistar-Albino rats were randomly assigned into 6 groups;
sham + saline (S), sham + montelukast (M), I/R + S, I/R + S 30’, I/R + M and I/R + M 30’. Saline or montelukast (10 mg/kg) was intraperitoneally administered 30 minutes prior to (S 30’, M 30’) and during detorsion
(I/R + S, I/R + M) in the I/R groups. The I/R groups underwent 2 hours of ischemia followed by 4 hours
(early-term) of reperfusion in unilateral testes. Half of the rats underwent 24 hours (late-term) of reperfusion
to investigate long-term effects. Testicular tissue samples were examined for biochemical and histopathological parameters. Germ cell apoptosis was evaluated using apoptosis-activating factor 1 (Apaf-1). Inducible nitric oxide synthase (iNOS) activity was analyzed in late-term reperfusion groups. Spermatogenic
functions were assessed for each testis based on the Johnsen criteria.
Department of Urology,
Bezmialem Vakif University
Faculty of Medicine, İstanbul,
Turkey
1
2
Department of Pharmacology,
Marmara University School of
Pharmacy, İstanbul, Turkey
Department of Pathology, Şişli
Etfal Training and Research
Hospital, İstanbul, Turkey
3
Department of Urology, Şişli
Etfal Training and Research
Hospital, İstanbul, Turkey
4
Department of Biochemistry,
Marmara University Faculty of
Medicine, İstanbul, Turkey
5
Submitted:
09.06.2014
Accepted:
21.08.2014
Correspondence:
Mesrur Selçuk Sılay,
Department of Urology,
Bezmialem Vakif University
Faculty of Medicine,
İstanbul, Turkey
Phone: +90 505 645 40 05
E-mail: selcuksilay@gmail.com
©Copyright 2014 by Turkish
Association of Urology
Available online at
www.turkishjournalofurology.com
Results: Unilateral I/R caused a significant increase in serum TNF-α levels in the early-term group compared
to the sham groups. Malondialdehyde levels and myeloperoxidase activity were found to be elevated in the I/R
groups and accompanied with a significant decrease in glutathione levels when compared to the sham groups.
I/R significantly increased iNOS activity and germ cell apoptosis compared to the sham groups. Montelukast
treatment significantly reversed all of these parameters and achieved comparable results with the sham groups.
Finally, spermatogenic indices were similar for the bilateral testes between all groups.
Conclusion: Montelukast exerts protective effects against testicular I/R injury by inhibiting neutrophil activity, reversing the oxidative stress markers, decreasing iNOS activity and attenuating apoptosis.
Key words: Ischemia; montelukast; reperfusion; testis; torsion.
ÖZET
Amaç: Montelukast, sisteinil lökotrien-1 reseptörünü spesifik ve geri dönüşümlü olarak inhibe eden, selektif bir lökotrien D-4 reseptör antagonistidir. Çalışmamızda montelukastın iskemi-reperfüzyon (I/R) sonucu
testiste oluşan oksidatif hasar üzerine olan etkilerini araştırmayı amaçladık.
Gereç ve yöntemler: Sekiz haftalık 51 adet erişkin erkek Wistar-Albino sıçan randomize edilerek 6 gruba
ayrıldı; sham + serum fizyolojik (SF), sham + montelukast (M), I/R + SF, I/R + SF 30’, I/R + M ve I/R + M
30’. I/R gruplarında SF veya montelukast, detorsiyondan 30 dakika önce (SF 30’, M 30’) veya detorsiyon
esnasında (I/R + SF, I/R + M) 10 mg/kg intraperitoneal olarak verildi. Dokulardaki iNOS düzeyleri ve apopitoz dereceleri de immünhistokimyasal yöntemlerle belirlendi. Spermatojenik fonksiyonlar Johnsen kriterlerine göre değerlendirilerek her testis için ortalama skorlar hesaplandı. Elde edilen tüm ordinal, numerik
ve nominal veriler, Mann-Whitney U, Dunn testi ve Kruskal Wallis testleriyle yerine göre karşılaştırıldı.
Bulgular: Montelukast tedavisi verilmeyen I/R gruplarında, sham gruplarına kıyasla TNF-α düzeylerinin istatistiksel anlamlı olarak arttığı ve montelukast tedavisinin bu artışı anlamlı şekilde önleyerek sham
gruplarına benzer düzeye getirdiği tespit edildi. Malondialdehit (MDA) ve miyeloperoksidaz (MPO) düzeylerinin, montelukast tedavisi verilmeyen I/R gruplarında sham gruplarına kıyasla anlamlı olarak yükseldiği
saptanırken, bu parametrelerin montelukast tedavisiyle düşürüldüğü belirlendi (p<0,05). Ayrıca glutatyon
(GSH) düzeylerinin, montelukast tedavisi verilmeyen I/R gruplarında sham gruplarına kıyasla anlamlı olarak düştüğü gözlenirken montelukast tedavisinin bu durumu önlediği saptandı (p<0,05). İmmünhistokimyasal olarak değerlendirilen iNOS ekspresyon skorlarının, I/R SF 30’ grubunda sham gruplarına kıyasla
anlamlı olarak arttığı izlenirken, montelukast tedavisinin bu değerleri sham gruplarına benzer düzeylere
indirdiği belirlendi. İmmünhistokimyasal olarak değerlendirilen germ hücre apopitozunun bütün parametrelerinin, montelukast tedavisi verilmeyen I/R gruplarında sham gruplarına kıyasla anlamlı olarak yükseldiği saptanırken, montelukast tedavisinin istatistiksel anlamlı olarak apopitozu önlediği saptandı (p<0,05).
Sonuç: Montelukast, testiste I/R’ye bağlı olarak gelişen hasarı azaltmaktadır. Bu deneysel çalışmanın verileri, montelukast tedavisinin testis torsiyonunda kullanılabilirliğinin araştırılması gerektiğini ortaya koymaktadır.
Anahtar kelimeler: İskemi; montelukast; reperfüzyon; testis; torsiyon.
221
Turkish Journal of Urology 2014; 40(4): 221-7
DOI:10.5152/tud.2014.61587
222
Wistar Albino
n=51
Sham+S
n=9
Sham+M
n=8
I/R+S
n=8
I/R+S 30'
n=8
IR+M
n=9
I/R+M 30'
n=9
S: 6 h
n=4
S: 6 h
n=4
S=6 (2+4) h
n=4
S=6 (2+4) h
n=4
S=6 (2+4) h
n=4
S=6 (2+4) h
n=5
S: 26 h
n=5
S: 26 h
n=4
S: 26 (2+24) h
n=4
S: 26 (2+24) h
n=4
S: 26 (2+24) h
n=5
S: 26 (2+24) h
n=4
Figure 1. The scheme of the study groups
+S: saline; +M: montelukast; S: sacrifice time; R: reperfusion time
*Ischemia period was 2 hours for the I/R groups
Introduction
Testicular torsion is a surgical emergency affecting male
newborns, children and adolescents. The incidence has been
reported as high as 1/4000 in males up to 25 years old.[1]
Early recognition and treatment of the disease is of the utmost
importance to preserve testicular function and save the testis
from orchiectomy. However, even after surgical detorsion of
the testis, the salvage rates are between 42 and 88%, and the
long-term function of these testes are unknown.[2] Therefore,
many pharmaceutical agents are under investigation that can be
used as an adjunct to surgery in order to improve the testicular
salvage rates.[3-8]
Montelukast is a selective antagonist of cysteinyl leukotriene
receptor 1 (CystLT1) and an anti-oxidant used as an antiasthmatic drug in the clinical setting. It has been previously
reported in experimental studies that it preserves the kidney and
the bladder from ischemia/reperfusion damage.[9,10] However,
there has been no appropriately designed study regarding the
effect of montelukast on testicular torsion models. We aimed to
elucidate the exact mechanism of the possible protective effect
of montelukast on testicular ischemia/reperfusion injury in an
experimental model.
Material and methods
The study was approved by the animal care and use committee
of our institution. Fifty-one male adult Wistar albino rats (300350 g) were randomly assigned into 6 groups:
1.
2.
3.
4.
5.
6.
Sham + saline (S),
Sham + montelukast (M),
Ischemia-reperfusion (I/R) + S.
I/R + S 30’,
I/R + M,
I/R + M 30’.
The scheme of the study protocol is summarized in Figure 1.
Animals were placed in an air-conditioned room with 12-h light
and dark cycles. The temperature (22±2oC) and the humidity
(65-70%) of the room were standardized.
Surgical procedure
Anesthetized (ip. ketamine and xylazine; 75 mg/kg and 10
mg/kg, respectively) rats were positioned in a supine position. Under sterile conditions, the left testis was exteriorized
through a median raphe incision. The gubernaculum was
divided and the left testis was rotated 720° in counterclockwise
direction. Then, the testis was fixed to the scrotum with a 5.0
prolene suture. The incision was closed, and the testis was left
in this torsion position for 2 hours. Afterwards, the torsion was
repaired by opening the incision and counterrotating the testis
to the natural position and joining the gubernacular stumps.
At the time of repair, the testis was examined to ensure that it
had remained in torsion. In addition, the testes were examined
5 minutes after the counter-rotation to evaluate the quality of
detorsion. The testes remained stable within the scrotum until
the time of tissue collection. Rats were decapitated 4 or 24
hours after reperfusion according to the randomization groups.
Then, the trunk blood was collected, and tissue from both testes was removed for analysis.
Sılay et al.
Montelukast prevents testes against ischemia-reperfusion injury through suppression of iNOS expression
Saline or montelukast (10 mg/kg) was intraperitoneally administered 30 minutes prior to (S 30’, M 30’) or during detorsion
(I/R + S, I/R + M) in the I/R groups. The I/R groups underwent
2 hours of ischemia followed by a 4 hour (early-term) reperfusion in the unilateral testes. Moreover, half of the rats underwent
a 24 hour (late-term) reperfusion for further analyses. Testicular
tissue samples were examined for biochemical and histopathological parameters. Germ cell apoptosis was evaluated using
apoptosis-activating factor 1 (Apaf-1). Inducible nitric oxide
synthase (iNOS) activity was analyzed in the late-term reperfusion groups. Spermatogenic functions were assessed for each
testis based on the Johnsen criteria.
Biochemical analyses
Serum TNF-α and IL-1β were quantified using enzyme-linked
immunosorbent assay (ELISA) kits (Biosource International,
Nivelles, Belgium). Tissue samples were homogenized with
ice-cold 150 mM KCl for the determination of malondialdehyde
(MDA) and glutathione (GSH) levels. The MDA levels are
expressed as nmol MDA/g of tissue.[11] GSH was determined
by a spectrophotometric method, and the results are expressed
as µmol GSH/g tissue.[12] Myeloperoxidase (MPO) activity in
tissues was measured by Hillegas method.[13] For this purpose,
tissue samples were homogenized in 50 mM potassium phosphate buffer with a pH of 6.0 and centrifuged at 41,400 g for 10
min. After several standardized procedures, MPO activity was
calculated as U/g tissue.
Histopathological analyses
All of the tissue samples were evaluated by a competent uropathologist (AO) who was aware of the studies’ objectives but
blind to the randomized groups. Histological investigations
were performed in order to determine the tissue damage. Germ
cell apoptosis was evaluated using apoptosis-activating factor 1
(Apaf-1). Inducible nitric oxide synthase (iNOS) activity was
analyzed in late-term reperfusion groups. Spermatogenic functions were evaluated in each testis according to the Johnsen
criteria.[14]
The testicular tissue samples were fixed in Bouin solution and
embedded into paraffin blocks. Then, they were stained with
hematoxylin & eosin, and all the tubules were inspected under
a light microscope. These samples were used to assess the morphological status of the testicular tissues and graded from 1 to
10 according to the maturation degrees.
Immunohistochemical analyses were performed for assessing
apoptosis and iNOS levels. Apaf-1 (Apoptosis protease activating factor-1, neomarkers, rabbit polyclonal, prediluted) was
used for incubation over the night. Additionally, 30 minutes of
incubation was performed with iNOS (Neomarkers, rabbit polyclonal, prediluted). The sections underwent a standard washing
process. The immunohistochemical staining results were evaluated according to a previously described system.[15] Accordingly,
for both Apaf-1 and iNOS, the intracytoplasmic staining density and the ratio of positive cells were determined. Expression
scores were calculated by multiplying those two components.
Statistical analyses
All of the variables were analyzed using Mann-Whitney U,
Dunn and Kruskal Wallis tests (SPSS 13.0, Inc., Chicago, IL,
USA). The values are provided as the mean±standard deviation
of the mean (SD). A p value of <0.05 was considered statistically significant. In multiple comparisons, a z value of z >1.96
was considered statistically significant.
Results
At the assigned times following torsion and detorsion (T/D),
all of the histopathological and molecular parameters were
compared among the groups. Unilateral I/R caused a significant
increase in serum TNF-α levels in the early-term group when
compared to the sham groups (p<0.05). Montelukast treatment
significantly decreased these levels and achieved comparable
results with the sham groups. However, IL-1ss levels were
found to be comparable among all groups (p>0.05).
The oxidative stress markers, including the malondialdehyde
(MDA) level and myeloperoxidase (MPO) activity, detected in
the tissue samples were found to be elevated in the I/R groups
and accompanied by a significant decrease in the glutathione
level compared to the sham groups (p<0.001). The results
remained constant for the early and late term reperfusion
groups. Montelukast treatment reversed these parameters and
brought them to the same levels as the sham groups (p>0.05).
Figure 2 shows the comparison of the oxidative stress markers
between the 6 groups.
The immunohistochemical analyses of the ischemic testes in
the late-term group indicated that I/R significantly increased
the iNOS expression levels when compared to sham groups
(p<0.05). Moreover, the montelukast treatment decreased these
levels and achieved comparable results to the sham groups
(p>0.05). Figure 3 demonstrates the immunohistochemical
staining with iNOS of all of the groups. Accordingly, iNOS
staining was significantly predominant in the I/R groups compared to the sham groups. Montelukast treatment significantly
decreased the iNOS staining density and generality.
The other immunohistochemical analyses in the early-term
group for germ cell apoptosis demonstrated that I/R significantly increased all of the apoptotic markers in the ischemic
testes compared to the sham groups, and this effect was significantly decreased with the administration of montelukast
223
Turkish Journal of Urology 2014; 40(4): 221-7
DOI:10.5152/tud.2014.61587
224
2
15
75
***
***
***
+
**
+
+
25
0
GSH (µmol/g)
50
+
10
MPO (U/g)
MDA (nmol/g)
+
++
5
I/R+S I/R+S 30' I/R+M I/R+M 30’
**
1
0
0
Sham+S Sham+M
**
Sham+S
Sham+M
I/R+S
I/R+S 30' I/R+M
I/R+M 30’
Sham+S
Sham+M
I/R+S
I/R+S 30' I/R+M
I/R+M 30’
Figure 2. Comparison of the study groups for the different oxidative stress markers
MDA: malondialdehyde; MPO: myeloperoxidase; GSH: glutathione
*p<0.05, **p<0.01, ***p<0.001, comparison with the sham groups
+p<0.05, ++p<0.01, +++p<0.001, comparison of the treatment groups with the I/R groups
a
b
c
d
e
f
Figure 3. Immunohistochemical staining of the seminiferous tubules with iNOS. x40
a: Sham + SF; b: Sham + M; c: I/R + SF; d: I/R + SF 30’; e: I/R + M, f: I/R + M 30’
The bold stained cytoplasm of the cells represents the iNOS activity. The untreated I/R groups (c, d) have predominant staining compared to the sham
groups. This indicates that the montelukast treatment significantly decreases the iNOS staining
Sılay et al.
Montelukast prevents testes against ischemia-reperfusion injury through suppression of iNOS expression
225
a
b
c
d
e
f
Figure 4. Immunohistochemical staining of seminiferous tubules with Apaf-1. x40
a: Sham + SF; b: Sham + M; c: I/R + SF; d: I/R + SF 30’; e: I/R + M, f: I/R + M 30’
The areas with arrows indicate the brown-stained cell cytoplasm stained with Apaf-1 and indicate apoptosis
(p<0.05). Figure 4 demonstrates the immunohistochemical
staining of Apaf-1 of all groups. Accordingly, Apaf-1 staining is
significantly predominant in I/R groups when compared to the
sham groups. Montelukast treatment significantly decreases the
Apaf-1 staining density and generality. Finally, similar results
were achieved for the bilateral testes of all of the groups regarding spermatogenic indices, as determined by the Johnsen criteria
(p>0.05).
Discussion
Our results demonstrate that montelukast has protective effects
on testicular tissue after I/R injury. In the study groups in
which montelukast was not administered, the oxidative stress
markers both in the blood and tissue were significantly worse.
Additionally, an elevation of iNOS activity and germ cell
apoptosis were also detected. Montelukast reversed all of these
harmful effects and reduced the testicular damage related to I/R
injury.
Testicular torsion is a surgical emergency affecting young men
and adolescents. The incidence is reported as 1/158 in males
under 24 years old.[1,2] Immediate detorsion should be performed
in order to prevent testicular damage. However, even after a successful surgery, atrophy may occur, and this may lead to infertility. Therefore, it is important to develop treatment strategies that
decrease testicular damage and prevent oxidative stress. There
are several reported anti-oxidative treatment options, but the
main concern is the balance between the risk and the benefits.
These medications, including trimetazidine, erythropoietin, taurine and curcumin, have been shown to decrease the apoptotic
index and free radicals within the testicular tissue after I/R.[5-8]
However, due to extensive side effects, none of them are currently in clinical use, and the trials are considered experimental.
Montelukast specifically inhibits the CystLT1 receptor and is a
selective leukotriene D-4 antagonist. It is in clinical use as an
anti-asthmatic drug due to its effects decreasing eosinophilic
inflammation in the lungs. It has also been demonstrated that
montelukast has inhibitory effects on neutrophil migration and
has positive effects on the oxidative system. Therefore, in some
organs, the effects of montelukast have been previously studied.
In one study, the investigators demonstrated that montelukast
increases the viability of skin flaps when compared to the control group.[16] In another trial focusing on the liver, it was demonstrated that montelukast reversed the histopathological altera-
Turkish Journal of Urology 2014; 40(4): 221-7
DOI:10.5152/tud.2014.61587
226
a
b
c
Figure 5. Hematoxylin & eosin staining of seminiferous tubules. x20
a: Sham + SF; b: I/R + SF; c: I/R + M
The areas with the arrows indicate the neutrophilic accumulation into the interstitial area
tions and protected from the tissue from damage due to I/R injury.
[17]
The results were similar both in kidney and bladder tissues,
and the protective effects of montelukast have been proven.[9,10]
Montelukast has also been studied previously in testicular torsion
models.[18,19] However in both of these models, there were severe
methodological errors, and the mechanism of montelukast had
not yet been explored. Therefore, in our study, we aimed to demonstrate the effect of montelukast by assessing not only apoptosis
but also several other parameters, including iNOS activity, neutrophil migration, spermatogenetic functions and oxidative stress
markers, both in the blood and tissue samples.
NO is the end product of NOS, and it is physiologically found
in low concentrations in tissues. In the case of I/R, its concentration dramatically increases, and after combining with oxygen,
and peroxynitrite is formed. Finally, it causes DNA damage and
apoptosis. Many studies have shown that testicular iNOS levels
increase after I/R, which is one of the major causes of tissue
damage.[20,21] It has been previously demonstrated using Western
blotting that the iNOS levels are significantly increased 24
hours after I/R.[21] In that study, the investigators noted that the
iNOS levels were not detectable before 24 hours. Therefore, we
evaluated the iNOS levels in rats that were sacrificed 24 hours
after the detorsion period. Finally, we demonstrated that I/R
significantly increased the iNOS expression levels compared
to the sham groups. In addition, the montelukast treatment
decreased the iNOS levels and achieved comparable results to
the sham groups. This evaluation is an important parameter in
understanding the mechanism for the protective effect of montelukast in testicular torsion. Our study is the first to evaluate this
parameter after montelukast administration.
Several reports have demonstrated that the exact mechanism
for apoptosis in germ cells after torsion is extensive neutrophil
migration and the subsequent elevation of ROS. In our study,
the montelukast administration 30 minutes prior to detorsion
and during detorsion significantly decreased the migration of
neutrophils to the testis, as shown in Figure 5. We believe that
this inhibition is the major cause for the decrease in apoptosis
in germ cells.
Spermatogenesis is among the other parameters that are negatively affected after testicular torsion. There is no consensus on
the period for the worsening of this parameter, and many studies
have demonstrated various results depending on the Johnsen
criteria. This period is subject to change, and the range is as
wide as between 2 hours and 3 months.[22]
In our study, we evaluated the spermatogenetic functions histopathologically using the Johnsen criteria. Spermatogenesis was
evaluated in both testes in the groups sacrificed 24 hours after
detorsion. However, the results did not demonstrate any significant difference among the groups, and the Johnsen scores were
similar. After evaluating the literature, this is an expected finding that may be attributed to the timing of the sacrifice, which
was a relatively short period after detorsion.
In this study, aimed to elucidate the possible protective effect
of montelukast in unilateral testicular T/D, and the following
results were found: testicular torsion/detorsion increased the
inflammatory mediators in venous blood samples and resulted
in significantly increased iNOS activity in the unilateral testis.
Additionally, I/R caused an imbalance in oxidative mechanisms,
Sılay et al.
Montelukast prevents testes against ischemia-reperfusion injury through suppression of iNOS expression
resulting in germ cell apoptosis in the ischemic testis. All of the
parameters above can be potentially reversed with the administration of systemic montelukast. Montelukast exerts protective
effects against testicular I/R injury via inhibiting neutrophil activity,
decreasing the iNOS levels and attenuating apoptosis. Further investigations are needed to prove that montelukast is a useful therapeutic
adjunct to surgical repair in cases of human testicular torsion.
Ethics Committee Approval: Ethics committee approval was
received for this study from the ethics committee of Şişli Etfal
Training and Research Hospital.
Peer-review: Externally peer-reviewed.
Author Contributions: Concept - M.S.S.; Design - M.S.S.,
M.K., H.T.; Supervision - C.M., G.Ş., M.K.; Funding - M.S.S.,
H.T.; Materials - H.T., G.Ş., Ş.T.; Data Collection and/or
Processing - M.S.S., M.A., A.Ö.; Analysis and/or Interpretation
- M.S.S., H.T., Ş.T., M.A., A.Ö.; Literature Review - M.S.S.,
M.K., G.Ş.; Writer - M.S.S., H.T.; Critical Review - C.M., G.Ş.,
A.Ö.; Other - M.A., Ş.T., A.Ö.
Conflict of Interest: No conflict of interest was declared by
the authors.
Financial Disclosure: The authors declared that this study has
received no financial support.
Etik Komite Onayı: Bu çalışma için etik komite onayı Şişli
Etfal Eğitim ve Araştırma Hastanesi’nden alınmıştır.
Hakem değerlendirmesi: Dış bağımsız.
Yazar Katkıları: Fikir - M.S.S.; Tasarım - M.S.S., M.K., H.T.;
Denetleme - C.M., G.Ş., M.K.; Kaynaklar - M.S.S., H.T.; Malzemeler
- H.T., G.Ş., Ş.T.; Veri toplanması ve/veya işlemesi - M.S.S., M.A.,
A.Ö.; Analiz ve/veya yorum - M.S.S., H.T., Ş.T., M.A., A.Ö.;
Literatür taraması - M.S.S., M.K., G.Ş.; Yazıyı yazan - M.S.S., H.T.;
Eleştirel İnceleme - C.M., G.Ş., A.Ö.; Diğer - M.A., Ş.T., A.Ö.
Çıkar Çatışması: Yazarlar çıkar çatışması bildirmemişlerdir.
Finansal Destek: Yazarlar bu çalışma için finansal destek
almadıklarını beyan etmişlerdir.
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