Kulak Çınlaması Oluşturulmuş Sıçanların Koklear Çekirdeklerinde Bazı İyon Kanalı Ekspresyonlarının İncelenmesi

Yıl 2024, Sayı: 22, 293 - 307, 30.04.2024

Yasemin Üstündağ , Gürsel Dinç , Ramazan Bal

https://doi.org/10.38079/igusabder.1400747

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

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

Kaynakça

• 1. Wilson JP, Sutton GJ. Acoustical correlates of tonal tinnitus. CIBA Foundation Symposium. 1981;85:82-10. doi: 10.1002/9780470720677.ch6.
• 2. Heller AJ. Classification and epidemiology of tinnitus. The Otolaryngologic Clinics of North America. 2003;36:239–248.
• 3. Bauer CA, Brozoski TJ. Tinnitus: Theories, Mechanisms, and Treatments. In: Schacht J, Popper AN, Fay RR, eds. Auditory Trauma, Protection and Repair. New York: Springer Science+Business Media. LLC; 2008:101-125.
• 4. Kizawa K, Kitahara T, Horii A, et al. Behavioral assessment and identification of a molecular marker in a salicylate-induced tinnitus in rats. Neuroscience. 2010;165:1323-1332.
• 5. Holmes S, Padgham ND. “Ringing in the ears’’: narrative review of tinnitus and ıts ımpact. Biological Research for Nursing. 2011;13(1):97-108.
• 6. Heffner HE, Hefner RS. Behavioural Test For Tinnitus in Animals. In: Eggermont JJ, Zeng FG, Popper AN, Fay RR, eds. Tinnitus. Springer Handbook of Auditory Reasearh. Newyork: Springer Science+Bussiness Media. 2012;21-58.
• 7. Estes WK, Skinner BF. Some quantitative properties of anxiety. J Exp Psychol. 1941;29:390-400.
• 8. Jastreboff PJ, Brennan JF, Sasaki CT. An animal model for tinnitus. Laryngoscope. 1988;98(3):280-286.
• 9. Jastreboff PJ, Brennan JF. Evaluating the loudness of phantom auditory perception (tinnitus) in rats. Audiology. 1994;33:202-217.
• 10. Jastreboff PJ, Sasaki CT. An animal model of tinnitus: a decade of development. Am J Otol. 1994;15:19–27.
• 11. Penner MJ, Jastreboff PJ. Tinnitus: Psychophysical Observations in Humans and An Animal Model. In: Clinical aspects of hearing. Newyork: Van De Springer; 1996:258-304.
• 12. Bauer CA, Brozoski TJ, Rojas R, et al. Behavioral model of chronic tinnitus in rats. Otolaryngol Head Neck Surg. 1999;121:457-462.
• 13. Kaltenbach JA, Afman CE. Hyperactivity in the dorsal cochlear nucleus after intense sound exposure and its resemblance to tone-evoked activity: a physiological model for tinnitus. Hear Res. 2000;140:165-172.
• 14. Bauer CA, Brozoski TJ. Assessing tinnitus and prospective tinnitus therapeutics using a psychophysical animal model. J Assoc Res Otolaryngol. 2001;2:54–64.
• 15. Brozoski TJ, Bauer CA, Caspary DM. Elevated fusiform cell activity in the dorsal cochlear nucleus of chinchillas with psychophysical evidence of tinnitus. J Neurosci. 2002;22:2383–2390.
• 16. Jastreboff PJ, Brennan JF, Coleman JK, et al. Phantom auditory sensation in rats: an animal model for tinnitus. Behav Neurosci. 1988;102(6):811-822.
• 17. Pilati N, Large C, Forsythe ID, et al. Acoustic over-exposure triggers burst firing in dorsal cochlear nucleus fusiform cells. Hearing Research. 2012;283:98-106.
• 18. Shore S, Wu C. Mechanisms of noise-induced tinnitus: Insights from cellular studies. Neuron. 2019;103(1):8–20. doi: 10.1016/j.neuron.2019.05.008.
• 19. Han KH, Cho H, Han KR, et al. Role of microRNA-375-3p-mediated regulation in tinnitus. International Journal of Molecular Medicine. 2021;48:136.
• 20. Guitton MJ. Salicylate induces tinnitus through activation cochlear NMDA receptors. Journal of Neuroscience. 2003;23:3944-3952.
• 21. Li S, Kalappa BI, Tzounopoulos T. Noise-induced plasticity of KCNQ2/3 and HCN channels underlies vulnerability and resilience to tinnitus. eLife. 2015;4:e07242. doi: 10.7554/eLife.07242.
• 22. Puel JL, Guitton MJ. Salicylate-induced tinnitus: molecular mechanisms and modulation by anxiety. Prog Brain Res. 2007;166:41-146. doi: 10.1016/S0079-6123(07)66012-9.
• 23. Brozoski TJ, Bauer CA. The effect of dorsal cochlear nucleus ablation on tinnitus in rats. Hearing Research. 2005;206:227–236.
• 24. Shah MM. Cortical HCN channels: function, trafficking and plasticity. J Physiol. 2014;592(13):2711–2719.
• 25. Chang X, Wang J, Jiang H, et al. Hyperpolarization-activated cyclic nucleotide-gated channels: an emerging role in neurodegenerative diseases. Front Mol Neurosci. 2019;12:141. doi: 10.3389/fnmol.2019.00141.
• 26. Kaupp UB, Seifert R. Molecular diversity of pacemaker ion channels. Annu Rev Physiol. 2001;63:235-257.
• 27. Dribben WH, Eisenman LN, Mennerick S. Magnesium induces neuronal apoptosis by suppressing excitability. Cell Death and Disease. 2010;1:e63. doi: 10.1038/cddis.2010.39
• 28. Zou ZG, Rios FJ, Montezano AC, et al. TRPM7, Magnesium, and signaling. Int J Mol Sci. 2019;20:1877. doi: 10.3390/ijms20081877.
• 29. Bal R, Ustundag Y, Bulut F, et al. Flufenamic acid prevents behavioral manifestations of salicylate-induced tinnitus in the rat. Arch Med Sci. 2016;12(1):208–215.
• 30. Qaswal AB. Magnesium ions depolarize the neuronal membrane via quantum tunneling through the closed channels. Quantum Rep. 2020;2:57–63. doi: 10.3390/quantum2010005.
• 31. Olah ME, Jackson MF, Li H, et al. Ca2+-dependent induction of TRPM2 currents in hippocampal neurons. J Physiol. 2009;587:965-979.
• 32. Jiang LH, Yang W, Zou J, Beech DJ. TRPM2 channel properties, functions and therapeutic potentials. Expert Opinion on Therapeutic Targets. 2010;9:973-988.
• 33. Dehmel S, Pradhan S, Koehler S, et al. Noise overexposure alters long-term somatosensory-auditory processing in the dorsal cochlear nucleus possible basis for tinnitus-related hyperactivity? Journal of Neuroscience. 2012;32(5):1660-1671. doi: 10.1523/JNEUROSCI.4608-11.2012.
• 34. Wang J, Ou SW, Wang YJ. Distribution and function of voltage-gated sodium channels in the nervous system. CHANNELS. 2017;11(6):534–554.
• 35. Liu Y, Li X. Effects of salicylate on voltage-gated sodium channels in rat inferior colliculus neurons. Hear Res. 2004;193(1-2):68-74. doi: 10.1016/j.heares.2004.03.006.
• 36. Yin M, Xia C, Cong W, et al. Aberrant expression of Nav1.6 in the cochlear nucleus correlates with salicylate-induced tinnitus in rats. Biochemical and Biophysical Research Communications. 2020;526:786-792.
• 37. Fryatt AG, Mulheran M, Egerton J, et al. Ototrauma induces sodium channel plasticity in auditory afferent neurons. Molecular and Cellular Neuroscience. 2011;48:51-61. doi: 10.1016/j.mcn.2011.06.005.