Publication: Deneysel Alzheimer hastalığı modelinde reaktif oksijen türleri ve nitrik oksit düzeyleri
Abstract
Yaşlı toplumu etkileyen önemli bir nörodejeneratif hastalık olan Alzheimer hastalığının etiyolojisi henüz tam olarak aydınlatılamamıştır. Ancak, pek çok araştırmada Alzheimer hastalığının serbest radikal oluşumuna neden olduğu ileri sürülmüştür. Öte yandan, sinaptik organizasyonun korunduğu özel bir kültür yöntemi olan organotipik kesit kültürleri canlı beyin kesitlerinin özel filtreler üzerine yerleştirilmesi ile elde edilmektedir Bir bitki alkaloidi olan kolşisin, mikrotübül depolimerizasyonuna neden olarak aksonal transportu inhibe eder. Kolşisinin izomeri olan lumikolşisin ise bu etkiyi gerçekleştirmediğinden, deneysel modellerde kontrol amacıyla kullanılmaktadır. Çalışmamızda, organotipik hipokampal kesit kültürlerinin kolşisin ile muamele edilmesiyle oluşturulan ex vivo Alzheimer hastalığı modelinde, reaktif oksijen türleri (ROT) ile nitrik oksit (NO) düzeyleri ölçüldü. Sıçan beyninden elde edilen canlı hipokampal kesitler 20 gün süreyle kültür ortamında takip edildi. Ardından kültür ortamına kolşisin (10mM) ve lumikolşisin (10mM) eklenerek ROT ve NO düzeyleri kemilüminesans yöntemiyle ölçüldü. Kemilüminesans yöntemi için farklı serbest radikallere duyarlı olan luminol (0. 2 mM) ve lusigenin (0. 2 mM) problarından faydalanıldı. NO ölçümü için luminol-H2O2 sistemi kullanıldı. Hücre hasarının tesbiti kültür ortamlarında laktat dehidrogenaz (LDH) ölçümü ile yapıldı. Ayrıca, kesitler trifeniltetrazolyum klorür (TTC) ile boyanarak hücre hasar oranı saptandı. Bulgularımız, oluşturulan ex vivo Alzheimer hastalığı modelinde ROT ve NO'nun kolşisin grubunda lumikolşisin grubuyla karşılaştırıldığında anlamlı olarak arttığını gösterdi. Kültür ortamlarındaki LDH düzeyleri kolşisin grubunda 89. 3 ± 23. 3 U/mL, lumikolşisin grubunda 56. 2 ± 13. 4 U/mL olarak saptandı. Bunun yanısıra, TTC yöntemiyle belirlenen hücre kaybının % 82. 5 ± 4. 9 olduğu hesaplandı. Kolşisinle oluşturulan deneysel Alzheimer hastalığı modelinde gözlenen serbest radikal düzeylerindeki artış, aksonal dejenerasyonla birlikte mitokondriyal enerji metabolizmasının bozulabileceğini, membran fosfolipitlerinin peroksidasyona açık hale gelebileceğini ve nöronal nitrik oksit sentaz ekspresyonunun artabileceğini düşündürmektedir.. LDH ve TTC ölçümleriyle elde edilen sonuçlarımız serbest radikal artışı bulgularımızı desteklemektedir. Sonuç olarak, organotipik hipokampal kesit kültürlerinin deneysel Alzheimer hastalığı modelleme çalışmalarında kullanılabileceğini gösteren bu çalışmamız, kolşisinle oluşturulan modelde hem serbest radikallerin hem de NO'nun arttığını ve bu artışların Alzheimer hastalığındaki önemini ortaya koymaktadır.
Alzheimer's disease is an important neurodegenerative disease which affects the aging population. The etiology of Alzheimer's disease is still uncertain. However, a number of studies show that Alzheimer's disease is associated with free radical generation. On the other hand, organotypic slice culture technique is a specific method with the characteristics of synaptic organization. In this method, freshly isolated brain slices are carefully transferred to sterile membrane units and kept in a tissue culture incubator. Colchicine is a plant alkaloid which blocks axonal transport by microtubule depolymerization. Lumicolchicine, an isomer of colchicines, fails to mimic this effect and is used for control groups. In this study, we have determined the levels of reactive oxygen species (ROS) and nitric oxide (NO) in an experimental model of Alzheimer's disease which was induced by adding colchicine to organotypic hippocampal slice cultures. Freshly isolated hippocampal slices were incubated in a culture flask for 20 days. At the end of this time, colchicine (10mM) or lumicochicine (10mM) was added to the culture medium and chemiluminescence measurements were made using different probes for ROS and NO. Luminol (0. 2 mM) and lucigenin (0. 2 mM) were used as chemiluminescence probes for the detection of different free radical species. NO measurements were performed by luminol-H2O2 system. Lactate dehydrogenase (LDH) activity in culture medium and triphenyltetrazolium chloride (TTC) staining of hippocampal slices were used for determining neuronal cell loss. In our experimental Alzheimer's disease model, increased ROS and NO levels were determined in the colchicine group as compared with the lumicolchicine group. LDH activity in the culture medium was 89. 3 ± 23. 3 U/mL in the colchicine group and 56. 2 ± 13. 4 U/mL in lumicolchicine group. The neuronal cell loss was found to be 82. 5 ± 4. 9 % with the TTC method. In our experimental Alzheimer's disease model, we have shown increased free radical generation following axonal degeneration. Therefore, we conclude that colchicine can inhibit the mitochondrial energy metabolism, induce membrane phospholipid peroxidation and expression of neuronal nitric oxide synthase. Our results with LDH activity and TTC measurements supported the free radical generation in this system. In conclusion, treatment of organotypic hippocampal slice cultures with colchicine, which results in increased levels of ROS and NO, can be used to model Alzheimer's disease.
Alzheimer's disease is an important neurodegenerative disease which affects the aging population. The etiology of Alzheimer's disease is still uncertain. However, a number of studies show that Alzheimer's disease is associated with free radical generation. On the other hand, organotypic slice culture technique is a specific method with the characteristics of synaptic organization. In this method, freshly isolated brain slices are carefully transferred to sterile membrane units and kept in a tissue culture incubator. Colchicine is a plant alkaloid which blocks axonal transport by microtubule depolymerization. Lumicolchicine, an isomer of colchicines, fails to mimic this effect and is used for control groups. In this study, we have determined the levels of reactive oxygen species (ROS) and nitric oxide (NO) in an experimental model of Alzheimer's disease which was induced by adding colchicine to organotypic hippocampal slice cultures. Freshly isolated hippocampal slices were incubated in a culture flask for 20 days. At the end of this time, colchicine (10mM) or lumicochicine (10mM) was added to the culture medium and chemiluminescence measurements were made using different probes for ROS and NO. Luminol (0. 2 mM) and lucigenin (0. 2 mM) were used as chemiluminescence probes for the detection of different free radical species. NO measurements were performed by luminol-H2O2 system. Lactate dehydrogenase (LDH) activity in culture medium and triphenyltetrazolium chloride (TTC) staining of hippocampal slices were used for determining neuronal cell loss. In our experimental Alzheimer's disease model, increased ROS and NO levels were determined in the colchicine group as compared with the lumicolchicine group. LDH activity in the culture medium was 89. 3 ± 23. 3 U/mL in the colchicine group and 56. 2 ± 13. 4 U/mL in lumicolchicine group. The neuronal cell loss was found to be 82. 5 ± 4. 9 % with the TTC method. In our experimental Alzheimer's disease model, we have shown increased free radical generation following axonal degeneration. Therefore, we conclude that colchicine can inhibit the mitochondrial energy metabolism, induce membrane phospholipid peroxidation and expression of neuronal nitric oxide synthase. Our results with LDH activity and TTC measurements supported the free radical generation in this system. In conclusion, treatment of organotypic hippocampal slice cultures with colchicine, which results in increased levels of ROS and NO, can be used to model Alzheimer's disease.