Publication: Nörodejeneratif hastalıkların patogenezinde serbest radikallerin rolünün incelenmesi
Abstract
Nörodejeneratif hastalıklarda nöronal hücre ölümünün etiyolojisi henüz tamamen aydınlatılamamıştır. Santral sinir sisteminde görülen ve farklı hücre popülasyonlarının kaybına neden olan mekanizmalar arasında eksitotoksisite, mitokondriyal enerji metabolizması bozuklukları, artan hücre içi kalsiyum düzeyleri ve serbest radikaller yer almaktadır. Son yıllarda yapılan çalışmalarda bu mekanizmaların birlikte etkin olabileceğine ilişkin hipotezler mevcuttur. Çalışmamızda deneysel olarak üç farklı nörodejeneratif hastalık modeli oluşturularak, serbest radikal düzeyleri kemilüminesans yöntemiyle ölçüldü. Deneysel epilepsi modelinde, ekstrasellüler K+ düzeyi arttırılarak veya NMDA uyarısı gerçekleştirilerek, in vitro olarak spontan epileptiform aktivite oluşturuldu. Alzheimer modelinde, organotipik kesit kültürleri kolşisin ve lumikolşisin ile indüklenerek, aksonal transport inhibe edilmiştir. In vivo Huntington hastalığı modelinde 3-nitropropiyonik asit (3-NPA) sıçanlara sistemik olarak uygulandı. Her üç modelde de beyin kesitlerinden salınan serbest radikaller kemilüminesans yöntemiyle ölçüldü. Bunun dışında hücre hasar belirteci olarak laktat dehidrogenaz (LDH) ve trifeniltetrazolyum klorür (TTC) spektrofotometrik olarak tayin edildi. Ayrıca agaroz jel elektroforezi ve TUNEL ile apoptoz oluşumu incelendi. Çalışmamızın sonucunda deneysel epilepsi, Alzheimer hastalığı ve Huntington hastalığı modellerinde serbest radikallerin arttığını saptadık. Sonuçlarımız LDH ve TTC ölçümleriyle uyumlu bulundu. Huntington hastalığı modelinde 3-NPA'nın sistemik uygulanmasıyla, agaroz jel elektroforezinde 'ladder' tarzında DNA kırıklarına, TUNEL ile de apoptotik hücrelere rastlandı. Sonuç olarak, çalışmamızda nörodejeneratif hastalıkların patogenezinde serbest radikallerin arttığını, bu artışta eksitotoksisitenin yanısıra, mitokondriyal enerji metabolizmasındaki bozuklukların ve artan hücre içi kalsiyumunun da katkıda bulunduğunu dolaylı olarak gösterdik. Deneysel Huntington hastalığı modelinde apoptotik hücre ölümünün serbest radikal oluşumuyla uyumlu olduğunu saptadık. Deneysel epilepsi, Alzheimer hastalığı ve Huntington hastalığı modellerinde, artan serbest radikallere uygun antioksidanların kullanılarak nörodejeneratif etkinin azaltılabileceğini düşünmekteyiz. Bu doğrultuda çalışmaların geliştirilmesi önerilmektedir. THE ROLE OF FREE RADICALS IN THE PATHOGENESIS OF NEURODEGENERATIVE DISEASES
The etiology of neuronal death in neurodegenerative diseases remains undetermined. Death of specific cell populations of central nervous system depends on excitotoxicity, disturbance of energy metabolism, increased intracellular calcium levels and free radical generation. In our study, free radical generation was investigated in three different experimental neurodegenerative disease models. In the experimental epilepsy model, increase in extracellular K+ levels and NMDA stimulation resulted in spontaneus epileptiform activity in vitro. Alzheimer's disease model was performed with colchicine, an axonal transport inhibitor. Organotypic slice cultures were treated with colchicine and lumicolchicine. In the in vivo Huntington's disease model, 3-nitropropionic acid (3-NPA) was systemically given to rats. In all these models brain slices were taken and free radical levels were detected by the chemiluminescence method. Lactate dehydrogenase (LDH) activity and triphenyltetrazolium chloride (TTC) method were used for the determination of cell loss in the experimental models. Agarose gel electrophoresis and TUNEL method were performed for the estimation of apoptosis. Free radical generation was increased in our experimental epilepsy, Alzheimer's disease and Huntington's disease models. Increaseing free radicals were correlated with LDH and TTC measurements. Systemic 3-NPA injections in Huntington's disease model were resulted with 'DNA laddering' in agarose gel electrophoresis and TUNEL positive apoptotic cells in striatum. In conclusion, we have shown that free radicals are important in the pathogenesis of neurodegenerative disease. Excitotoxicity, mitochondrial energy depletion and increased calcium levels accompanied free radical generation. We can conclude that, antioxidant therapy strategies can be developed for free radical generation in experimental epilepsy, Alzheimer's disease and Huntington's disease models.
The etiology of neuronal death in neurodegenerative diseases remains undetermined. Death of specific cell populations of central nervous system depends on excitotoxicity, disturbance of energy metabolism, increased intracellular calcium levels and free radical generation. In our study, free radical generation was investigated in three different experimental neurodegenerative disease models. In the experimental epilepsy model, increase in extracellular K+ levels and NMDA stimulation resulted in spontaneus epileptiform activity in vitro. Alzheimer's disease model was performed with colchicine, an axonal transport inhibitor. Organotypic slice cultures were treated with colchicine and lumicolchicine. In the in vivo Huntington's disease model, 3-nitropropionic acid (3-NPA) was systemically given to rats. In all these models brain slices were taken and free radical levels were detected by the chemiluminescence method. Lactate dehydrogenase (LDH) activity and triphenyltetrazolium chloride (TTC) method were used for the determination of cell loss in the experimental models. Agarose gel electrophoresis and TUNEL method were performed for the estimation of apoptosis. Free radical generation was increased in our experimental epilepsy, Alzheimer's disease and Huntington's disease models. Increaseing free radicals were correlated with LDH and TTC measurements. Systemic 3-NPA injections in Huntington's disease model were resulted with 'DNA laddering' in agarose gel electrophoresis and TUNEL positive apoptotic cells in striatum. In conclusion, we have shown that free radicals are important in the pathogenesis of neurodegenerative disease. Excitotoxicity, mitochondrial energy depletion and increased calcium levels accompanied free radical generation. We can conclude that, antioxidant therapy strategies can be developed for free radical generation in experimental epilepsy, Alzheimer's disease and Huntington's disease models.