Gereç ve Yöntemler: 40 adet Wistar albino rat kullanılarak 4 grup oluşturuldu. Grup 1'de (kontrol grubu) (n=10) herhangi bir ilaç verilmedi ve herhangi bir işlem yapılmadı. Grup 2'de (cisplatin grubu) (n=10) sadece 7,5 mg/kg cisplatin verildi. Grup 3'te (edaravon grubu) (n=10) sadece 1 mg/kg edaravon verildi. Grup 4'e (cisplatin+edaravon grubu) (n=10) 7,5 mg/kg cisplatin ve 1 mg/kg edaravon verildi. Kan malondialdehid (MDA) ve nitrik oksit (NO) düzeyleri çalışıldı. Tüm gruplarda akciğerler çıkarıldı. Doku hasarı ışık mikroskobu kullanılarak değerlendirildi. Ayrıca değerlendirme için immünohistokimya kullanıldı.

Bulgular: Cisplatin grubunda MDA ve NO düzeyleri diğer gruplara göre daha yüksek bulundu (p<0.05). Histopatolojik hasar cisplatin grubunda diğer gruplara göre daha belirgindi ve aradaki fark istatistiksel olarak anlamlıydı (p<0.05). CD68 ve bcl 2 ile immün boyama, sisplatin grubunda immün ekspresyonun cisplatin+edaravon grubuna göre anlamlı derecede yüksek olduğunu gösterdi.

Sonuç: Kısa dönem bulgularımıza göre edaravon, cisplatin kaynaklı akciğer hasarının önlenmesinde etkilidir.

Numerous drugs and molecules such as simvastatin, quercetin, and silymarin have been introduced to reverse the cisplatin induced cell toxicity^6-8. These chemical agents protect the cell by using enzymatic and non-enzymatic systems. Edaravone is a free radical scavenger and has been utilized for years for treatment of ischemic events such as cerebral infarct and stroke⁹. Edaravone, mainly is an antioxidant, decreases lipid peroxidation by inhibiting lipooxyenase. In addition, edaravone contributes to the neutralization of free radicals by increasing electron transport¹⁰. For all these reasons, we thought that edaravone may be effective in preventing cisplatin-induced lung injury. To the best of our knowledge, there is currently no other study investigating the protective effect of edaravone on cisplatin-induced lung injury.

Forty adult Wistar albino rats were used for the study. The animals were taken from the Department of Erciyes University Animal Experiments. The study was carried out in Erciyes University Faculty of Medicine, Department of Histology-Embryology. The rats were fed with free access to the food and water and treated to a heat between 20 and 22 ͦ C and to a 12 hour sunlight during the day. All the animals were randomly allocated into the 4 groups. The instructions at the website of www.randomization.com were followed.

4 groups were created. In group 1 (n=10, control group), nothing was given. In group 2 (n=10, cisplatin group), single dose 7,5 mg/kg cisplatin was administered. In group 3 (n=10, edaravone group) single dose 1 mg/kg edaravone was given. In group 4 (n=10, cisplatin+ edaravone group), single dose 7.5 mg/kg cisplatin and 1 mg/kg edaravone were administered. Ketamine hydrochloride (50 mg/kg, Ketalar, Eczacibasi, Istanbul, Turkey) and xylazin hydrochloride (5 mg/kg, Rompun, Bayer, Leverkusen, Germany) were given via intraperitoneal route for anesthesia. Blood samples were yielded by puncturing the cardiac tissue. Lung tissue was extirpated and then, sacrification procedure was executed by applying cervical dislocation.

Tissue samples were fixed in 10% formaldehyde solution. Then dehydration and parafin blocking processes were performed, respectively. The tissues were cut with a thickness of 5 micrometers and stained with hematoxylin and eosin (H&E) after deparaffinization and rehydration. The specimens were then examined and photographed by a single clinician by using light microscopy (Olympus® Inc. Tokyo, Japan). A modified semi-quantitative scoring was performed for the microscopic evaluation of the lung damage and four categories, Grade 0: None (0%) 1: Minimal (0-5%) 2: Mild (5-25%) 3: Moderate ( 25-50%) 4: Severe (more than 50%) were defined (11). To grade the damage to the lung; neutrophyle infiltration, congestion, narrowing of alveolar spaces and inflammation were utilized as the parameters of the scoring system.

CD 68 and Bcl 2 Measurement: Expressions of macrophage CD 68 and Bcl 2 were fulfilled by immunohistochemically. Expression levels were graded using the 0-3+ range. (0: no staining, 1: less than 10% nuclear staining, 2: 10-30% nuclear staining, 3: more than 30% nuclear staining)¹¹.

Biochemistry: The MDA kit (Cat. No: E0156Ra, Bioassay Technology Laboratory, Türkiye) was studied by using ELİSA method and their amounts were determined as ng/ml at 450 nm in the ELISA reader. The NO levels were detected by using the NO kit (Cat. No: E0703Ra, Bioassay Technology Laboratory, Türkiye and the measurement was explained as μmol/ L at 450 nm in the ELISA reader.

Statistical Analysis: Statistical Package for the Social Sciences (18.00 SPSS Inc., Chicago, IL) was used for statistical analyses. Kruskal Wallis test and Mann-Whitney U test were used for analyses. p-value <0.05 was considered as significant.

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Table 1: Nitric Oxide (NO), Malondialdehyde (MDA) measurements in serum samples of the groups

There was no difference among the groups in relation with the macroscopic features. Total tissue damage scores were demonstrated in Table 2. The histopathological tissue injury was found to be significantly higher in the cisplatin group than the cisplatin+ edaravone group (p<0.05). Also, macrophage CD 68 and bcl 2 expressions were significantly higher in the cisplatin group than in the cisplatin+resveratrol group (p<0.05) (Table 3).

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Table 2: Distribution of histological damage according to the groups

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Table 3: Distribution of immunhistochemical damage according to the groups

The morphology of the lung tissue was detected as normal in the control group (Figure 1A). Exposure to the cisplatin led to an increase in the neutrophyle infiltration, congestion, narrowing of alveolar spaces. Moreover, inflammation was prominently seen in the cisplatin group (Figure 1B). In the edaravone group, the appearance and microscopy of the lung tissue was similar to the control group (Figure 1C). In the cisplatin+edaravone group, addition of edaravone improved the congestion, and inflammation (Figure 1D). In the cisplatin group, the expression of the CD 68 and bcl 2 were more common in the cisplatin group than in the cisplatin+ edaravone group (Figure 2 and 3).

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Figure 1: Light microscopic appearance of lung tissue by using Hematoxylin & Eosin staining. A. Control group, B. Cisplatin group, C. Edaravone group, D. Cisplatin + edaravone group.

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Figure 2: Immunohistochemical staining with CD 68 dye. A. Control group, B. Cisplatin group, C. Edaravone group, D. Cisplatin + edaravone group

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Figure 3: Immunohistochemical staining with bcl 2 dye. A. Control group, B. Cisplatin group, C. Edaravone group, D. Cisplatin + edaravone group

Antineoplastic drugs such as cisplatin have been used in the treatment of cancers of ovarium, lung and solid organs¹². The improvement and combination of cancer therapy led to an increase in survival rates. This has made it important to prevent cisplatin-related toxicity. The toxicity of the cisplatin is dose dependent. Borovskaya et al reported that the dose and duration of cisplatin were the major contributed factors in terms of the toxicity of cisplatin¹³. Cisplatin behaves as a direct cellular toxin, increases the activity of lipooxygenase and finally causes an increase in the levels of free radicals and reactive oxygen species¹⁴.

Since oxidative stres has been introduced an important cause of cisplatin toxicity many antioxidants were used to reverse the adverse effects due to cisplatin¹⁵. Kara et al.¹⁶ reported that an antioxidant chemical, resveratrol, reduces free radicals and decreases the harmful effect of oxidative stres. Abe et al.¹⁷ reported that edaravone reversed the harmful effects due to liver ischemia. Later, Kara et al.¹⁸ reported that edaravone could be an effective agent in the short-term treatment and prevention of ovarian ischemia and reperfusion damage. Theories about the mechanism of action of edaravone are relation with neutralizing free radicals, scavenging ROS, inhibiting lipid peroxidation, and detoxifying hydroxyl radicals¹⁹. In the present study, intraperitoneal administration of edaravone decreased the MDA and NO levels and enhanced the morphology and microscopic appearance of the lung tissue. The improvement in the morphology and structural characteristics was prominent in the cisplatin+edaravone group.

In this study, a promising free-radical scavenger, edaravone, was assessed on cisplatin induced lung injury. Edaravone led to an improvement both histogically and biochemically. The levels of MDA and NO significantly decreased. Also, histopathological damage due to cisplatin was reduced with edaravone treatment.

In conclusion, according to our short-term findings, edaravone seems to reverse lung injury due to cisplatin. However, large prospective, randomized trials are required.

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