Lazer Mikrodiseksiyon Tekniği İle Yaralanan Periferik Duyusal Nöronlar Üzerinde Nörotrofin-3 ve Nörotrofin-4/5’in Rejeneratif Etkilerinin Değerlendirilmesi

Abstract

Amaç: Nörotrofin ailesinin önemli üyelerinden NT3 ve NT4/5’in, mikroskop kontrollü lazer mikrodiseksiyon tekniğiyle akson kesisi (aksotomi) oluşturulmuş dorsal kök gangliyon (DRG) nöronlarının hayatta kalma kabiliyetini etkileyip etkilemediğini araştırmak. 

Gereç ve Yöntem: Çalışmada 6-12 haftalık Balb/C ırkı fareler kullanıldı. Aseptik koşullarda DRG’ler çıkartıldı, primer DRG nöron kültürü hazırlandı. Hücre ekiminden 48 saat sonra kültürler, NT3 (50 ng/ml), NT4/5 (50 ng/ml)ve NT3-NT4/5 kombinasyonu ile muamele edildi. Nöritler gövdeden 200 µm uzaklıkta laser mikrodiseksiyon mikroskop sisteminde lazer ışını ile aksotomi edildi. Nöronların ölü-canlı ayrımı için propidyum iyodür (PI) alım testi uygulandı.  Nöronlar zaman aralıklı floresan mikroskobik görüntüleme sistemi ile görüntülendi.

Bulgular: PI, ölü hücrelerin çekirdeğini floresan mikroskobu altında parlak kırmızı gösterdi. Tüm deney grupları,kontrol grubuna göre 24.ve 48. saatlerde aksotomi edilen nöronların hayatta kalma oranlarında önemli artış gerçekleştirdi. En fazla hayatta kalma oranının NT3+NT4 grubunda, sonra sırasıyla NT3, NT4 ve kontrol gruplarında olduğu belirlendi. Hayatta kalma oranları 48. saatte 24. saate göre azalma gösterse de bu azalma istatistiksel olarak önemsizdi.

Sonuç: NT3-NT4/5 kombinasyonu, NT3 (50 ng/ml)ve NT4/5 (50 ng/ml), in vitroaksotomi hasarı modelinde yaralı DRG nöronlarının hayatta kalma oranlarını artırmaktadır. Bu nörotrofik faktörler  periferik sinirlerin mekanik yaralanmalarında terapötik potansiyel taşımaktadır. 

Keywords

• DRG; nöron; aksotomi; NT3; NT4/5

Evaluation of Regenerative Effects of Neurotrophin-3 and Neurotrophin-4/5 on Peripheral Sensory Neurons Injured with Laser Microdissection Technique

Abstract

Objective: To investigate whether of NT3 and NT4/5, important members of the neurotrophin family, affect the survival ability of dorsal root ganglion (DRG) neurons that were created axon injury (axotomy) with a microscope-controlled laser microdissection technique.

Materials and Methods:6-12 week BALB/c mice were used in the study. DRGs were harvested from mice under aseptic conditions and deep anesthesia. Primer DRG neuron culture was prepared. Forty-eight hours after cell culture, the cultures were treated with NT3 (50 ng / ml), NT4 / 5 (50 ng / ml) and NT3-NT4/5 combination. Neurites were axotomized at 200 μm distance from the perikaryon with the microscope-controlled laser beam. A propidium iodide (PI) uptake test was performed to determine the dead-alive distinction of neurons. Neurons were visualized with a time-lapse fluorescent microscopic imaging system. 

Results: PI show the dead cells' nucleus as bright red under the fluorescence microscope. All experimental groups showed a significant increase in axotomized-neurons’ survival rates at 24th and 48th hours according to the control group. It was determined that the highest survival rate was in NT3 + NT4 group, then NT3, NT4 and control groups respectively. Although the survival rates decreased compared to the 24th hour at the 48th hour, this decrease was statistically insignificant. 

Conclusion: Combination of NT3-NT4/5, NT3 (50 ng/ml) and NT4/5 (50 ng/ml) increase the survival rate of DRG neurons in vitro axotomy injury model. These neurotrophic factors have therapeutic potential in the mechanical injury of peripheral nerves. 

Keywords

• DRG; neuron; axotomy; NT3; NT4/5

References

1. 1. Kandel ER, Schwartz JH, Jessell TM, Siegelbaum S, Hudspeth AJ. Principles of neural science. 5th ed. New York, NY ; London: McGraw-Hill; 2013. l, 1709 p. p.2. Bradbury EJ, McMahon SB, Ramer MS. Keeping in touch: sensory neurone regeneration in the CNS. Trends Pharmacol Sci. 2000;21(10):389-94.3. Tuffaha SH, Budihardjo JD, Sarhane KA, Khusheim M, Song D, Broyles JM, et al. Growth Hormone Therapy Accelerates Axonal Regeneration, Promotes Motor Reinnervation, and Reduces Muscle Atrophy following Peripheral Nerve Injury. Plast Reconstr Surg. 2016;137(6):1771-80.4. Gordon T. The role of neurotrophic factors in nerve regeneration. Neurosurg Focus. 2009;26(2):E3.5. Lu B, Pang PT, Woo NH. The yin and yang of neurotrophin action. Nat Rev Neurosci. 2005;6(8):603-14.6. Xiao N, Le QT. Neurotrophic Factors and Their Potential Applications in Tissue Regeneration. Arch Immunol Ther Ex. 2016;64(2):89-99.7. Ernfors P, Lee KF, Kucera J, Jaenisch R. Lack of neurotrophin-3 leads to deficiencies in the peripheral nervous system and loss of limb proprioceptive afferents. Cell. 1994;77(4):503-12.8. Funakoshi H, Frisen J, Barbany G, Timmusk T, Zachrisson O, Verge VM, et al. Differential expression of mRNAs for neurotrophins and their receptors after axotomy of the sciatic nerve. J Cell Biol. 1993;123(2):455-65.9. Berkemeier LR, Winslow JW, Kaplan DR, Nikolics K, Goeddel DV, Rosenthal A. Neurotrophin-5 - a Novel Neurotrophic Factor That Activates Trk and Trkb. Neuron. 1991;7(5):857-66.10. Üstün R, Oğuz EK. Degenerative effect of Ankaferd Blood Stopper® on mice peripheral sensory neurons in vitro. Folia Neuropathologica. 2018;56(1):67-74.11. Ustun R, Oguz EK, Seker A, Korkaya H. Thymoquinone protects DRG neurons from axotomy-induced cell death. Neurol Res. 2018:1-8.12. Cengiz N, Ozturk G, Erdogan E, Him A, Oguz EK. Consequences of neurite transection in vitro. J Neurotrauma. 2012;29(15):2465-74.13. Ozturk G, Cengiz N, Erdogan E, Him A, Oguz EK, Yenidunya E, et al. Two distinct types of dying back axonal degeneration in vitro. Neuropathol Appl Neurobiol. 2013;39(4):362-76.14. Helmrich A, Barnes D. Animal cell culture equipment and techniques. Methods Cell Biol. 1998;57:3-17.15. Merten OW. Introduction to animal cell culture technology-past, present and future. Cytotechnology. 2006;50(1-3):1-7.16. Geuna S. The sciatic nerve injury model in pre-clinical research. Journal of Neuroscience Methods. 2015;243:39-46.17. Tzekov R, Quezada A, Gautier M, Biggins D, Frances C, Mouzon B, et al. Repetitive mild traumatic brain injury causes optic nerve and retinal damage in a mouse model. J Neuropathol Exp Neurol. 2014;73(4):345-61.18. Geuna S, Raimondo S, Fregnan F, Haastert-Talini K, Grothe C. In vitro models for peripheral nerve regeneration. Eur J Neurosci. 2016;43(3):287-96.19. Huang EJ, Reichardt LF. Neurotrophins: roles in neuronal development and function. Annu Rev Neurosci. 2001;24:677-736.20. Takeda M, Suzuki Y, Obara N, Tsunekawa H. Immunohistochemical detection of neurotrophin-3 and-4, and their receptors in mouse taste bud cells. Arch Histol Cytol. 2005;68(5):393-403.21. Maisonpierre PC, Belluscio L, Squinto S, Ip NY, Furth ME, Lindsay RM, et al. Neurotrophin-3: a neurotrophic factor related to NGF and BDNF. Science. 1990;247(4949 Pt 1):1446-51.22. Hohn A, Leibrock J, Bailey K, Barde YA. Identification and characterization of a novel member of the nerve growth factor/brain-derived neurotrophic factor family. Nature. 1990;344(6264):339-41.23. Li L, Oppenheim RW, Lei M, Houenou LJ. Neurotrophic agents prevent motoneuron death following sciatic nerve section in the neonatal mouse. J Neurobiol. 1994;25(7):759-66.24. Hyman C, Juhasz M, Jackson C, Wright P, Ip NY, Lindsay RM. Overlapping and Distinct Actions of the Neurotrophins Bdnf, Nt-3, and Nt-4/5 on Cultured Dopaminergic and Gabaergic Neurons of the Ventral Mesencephalon. Journal of Neuroscience. 1994;14(1):335-47.25. Lingor P, Unsicker K, Krieglstein K. GDNF and NT-4 protect midbrain dopaminergic neurons from toxic damage by iron and nitric oxide. Exp Neurol. 2000;163(1):55-62.