Investigation of Some Ion Channel Expressions in Cochlear Nucleus of Tinnitus Induced Rats

Öz

Aim: The aim of this study is to gain a better understanding of how certain ion channels play a role in the molecular mechanisms of salicylate- and noise-induced tinnitus. Method: The present study was conducted on thirty-two, 4-month-old, male Wistar Albino rats. Rats were equally divided into four groups; two experimental groups and two control groups. The assessment of tinnitus was based on a behavioral test which was modified from the conditional suppression method. Tinnitus was induced by sodium salicylate administration and noise exposure in rats in which the suppression ratios were zero (0). All animals in both experimental and control groups were decapitated in deep anaesthesia for 2 h after salicylate or saline administration and noise exposure, consecutively. Tissues from the left and right cochlear nucleus were dissected immediately in ice-cold RNA later (Invitrogen). Before reverse transcription, the RNA pools were arranged. Quantitative changes in HCN1, HCN2, HCN4, SCN1A, SCN2A1, SCN3A, TRPM2, TRPM7 and GAPDH mRNA expressions in the cochlear nucleus in both experimental and control groups were examined by quantitative real-time PCR method. Statistical data were analysed using the SPSS 21 program (Version 21.0, SPSS Inc., Chicago, IL, USA) with the Kruskal-Wallis and Mann-Whitney U tests. Results: Fold changes in the expression levels of SCNA1, SCN2A1, SCN3A, TRPM2, TRPM7, CACNA1B, HCN1, HCN2 and HCN4 genes in both salicylate-induces tinnitus (SAT) and noise-induced tinnitus (NT) groups compared with the control group. According to these data, it is seen that the mRNA levels of all genes are lower in the cochlear nucleus area of the rats in both SAT and NT groups than in the control group. Considering each of these genes in NT group: SCNA1, SCN3A, TRPM7 genes slightly decreased; SCN2A1, TRPM2, HCN1 and HCN4 genes slightly increased compared with the SAT group. For HCN2 gene fold changes were nearly the same in the NT and SAT groups. Conclusion: The findings of this study suggest that tinnitus generation may be closely related to alterations in several key ion channel families activity including voltage-gated calcium channels, hyperpolarization-activated cyclic nucleotide–gated (HCN) channels, transient receptor potential (TRP) channels, voltage-gated sodium channels within the CN, specifically in response to salicylate-induced and noise-induced tinnitus models.

Anahtar Kelimeler

• Ion channels
• qPCR
• rat
• tinnitus

Proje Numarası

FÜBAP VF.11.12

Kulak Çınlaması Oluşturulmuş Sıçanların Koklear Çekirdeklerinde Bazı İyon Kanalı Ekspresyonlarının İncelenmesi

Öz

Amaç: Bu çalışmanın amacı, belirli iyon kanallarının salisilat ve gürültü ile indüklenen tinnitusun moleküler mekanizmalarında nasıl bir rol oynadığını daha iyi anlamaktır. Yöntem: Çalışma, 32 tane 4 aylık erkek Wistar Albino sıçanlar üzerinde gerçekleştirilmiştir. Sıçanlar, iki deney ve iki kontrol grubu olmak üzere dört gruba eşit olarak bölünmüştür. Tinnitus değerlendirmesi, koşullu baskılama yönteminden modifiye edilen bir davranış testine dayanmaktadır. Tinnitus, baskılama oranları sıfır (0) olan sıçanlarda sodyum salisilat uygulaması ve gürültü maruziyeti ile indüklenmiştir. Tüm deney ve kontrol gruplarındaki hayvanlar, salisilat veya salin uygulamasından ve ardışık gürültü maruziyetinden 2 saat sonra derin anestezi altında dekapite edilmiştir. Sol ve sağ koklear çekirdek dokuları hemen soğuk RNA later (Invitrogen) içersinde diseke edilmiştir. Ters transkripsiyondan önce, RNA havuzları düzenlenmiştir. Her iki deney ve kontrol grubundaki koklear çekirdekte HCN1, HCN2, HCN4, SCN1A, SCN2A1, SCN3A, TRPM2, TRPM7 ve GAPDH mRNA ekspresyonlarındaki niceliksel değişiklikler, nicel gerçek zamanlı PCR yöntemi ile incelenmiştir. İstatistiksel veriler, Kruskal-Wallis ve Mann-Whitney U testleri ile SPSS 21.0 programı (Version 21.0, SPSS Inc., Chicago, IL, USA) kullanılarak analiz edilmiştir. Bulgular: SCNA1, SCN2A1, SCN3A, TRPM2, TRPM7, CACNA1B, HCN1, HCN2 ve HCN4 genlerinin ekspresyon düzeylerindeki katlamalı değişiklikler, hem salisilat ile indüklenen tinnitus (SAT) hem de gürültü ile indüklenen tinnitus (NT) grupları ile kontrol grubu arasında karşılaştırıldı. Bu verilere göre, tüm genlerin mRNA seviyelerinin, hem SAT hem de NT gruplarındaki sıçanların koklear çekirdek alanında kontrol grubundan daha düşük olduğu görülmektedir. NT grubundaki her bir bu genleri dikkate alındığında: SCNA1, SCN3A, TRPM7 genleri hafifçe azalmış, SCN2A1, TRPM2, HCN1 ve HCN4 genleri SAT grubu ile karşılaştırıldığında hafifçe artmıştır. HCN2 geni için katlanma değişiklikleri NT ve SAT gruplarında neredeyse aynıdır. Sonuç: Bu çalışmanın bulguları, tinnitus oluşumunun salisilatla indüklenmiş ve gürültüyle indüklenmiş tinnitus modellerine yanıt olarak, özellikle CN içindeki voltajlı kalsiyum kanalları, hiperpolarizasyon-aktive siklik nükleotid-gated (HCN) kanalları, geçici reseptör potansiyeli (TRP) kanalları, voltajlı sodyum kanalları gibi birkaç önemli iyon kanalı ailesinin aktivitesindeki değişikliklerle yakından ilişkili olabileceğini önermektedir.

Anahtar Kelimeler

• İyon kanalları
• qPCR
• sıçan
• tinnitus

Destekleyen Kurum

Fırat Üniversitesi BAP

Proje Numarası

FÜBAP VF.11.12

Kaynakça

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