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HT22 Fare Hipokampal Hücre Hattının Nöronal Farklanma Besiyerine Verdiği Apoptotik Tepkinin Ölçülmesi

Year 2023, Volume: 13 Issue: 3, 375 - 383, 30.09.2023
https://doi.org/10.31832/smj.1264019

Abstract

Amaç: Bu çalışmanın amacı nörobiyoloji çalışmalarında sıkça kullanılmakta olan HT22 fare hipokampal hücre hattının nöronal farklılaşma modeli olarak kullanılabilirliğinin anlaşılması için standart bir farklandırma besiyerine farklı sürelerde verdiği apoptotik cevabın sınanmasıdır.

Gereç ve Yöntemler: HT22 hücrelerinin ekilmesi ve farklandırma dışında kültüre edilmesi için HG-DMEM farklandırma için ise B27+ katkılı NB+ medyumu kullanılmıştır. Kontrol grubu da dâhil olmak üzere toplam 9 farklı grup standart olarak kültüre edilmiştir. Deney sonunda AnnexinV/PI işaretlemesiyle erken/geç apoptozis ve nekrozis oranları akım sitometrik olarak belirlenmiştir. Verilerin normal dağılıma uyup uymadığı Shapiro-Wilks testi ile sınandıktan sonra en uygun testle istatistiksel analiz gerçekleştirilmiştir.

Bulgular: Gruplar arasındaki karşılaştırmada erken apoptozis (P<0,01) ve geç apoptozis (P<0,001) hücre popülasyon oranları açısından istatistiksel olarak anlamlı bir fark tespit edilirken nekrozis açısından anlamlı bir fark olmadığı gözlenmiştir (P=0,064). 48 ve 72 saat farklandırma besiyerinde kültüre edilen grupların büyük çoğunluğunda, sadece HG-DMEM ile veya 24 saat farklandırılan hücrelere kıyasla istatistiksel olarak anlamlı düzeyde (P<0,001) erken apoptozis artışı olduğu görülmüştür. Farklandırma besiyeri ile kültüre edilen grupların çoğunluğunda kontrol grubuna kıyasla anlamlı düzeyde geç apoptozis artışı gözlenirken (P<0,001), farklı süreler farklandırılan gruplar arasında anlamlı bir fark gözlenmemiştir (P>0,05).

Sonuç: Nörodejeneratif hastalıklara ait önemli patobiyolojik özelliklerin in-vitro düzeyde modellenmesinde sıkça kullanılan, fakat nöronal farklılaşma açısından uygunluğu yeterince bilinmeyen HT22 hippokampal hücre hattının, yaygın olarak kullanılan bir farklandırma besiyerine erken apoptozis (kültürleme süresine bağlı) ve geç apoptozis (süreden bağımsız) artışı şeklinde anlamlı tepkiler oluşturduğu anlaşılmıştır. Bulgularımız HT22 hücre hattının farklılaşma çalışmalarında kullanılabilirliği açısından apoptozis düzeyinde önemli ipuçları sunmaktadır.

Supporting Institution

Mersin Üniversitesi Bilimsel Araştırma Projeleri Birimi

Project Number

2018-1-AP5-2895 ve 2020-1-AP5-4104

Thanks

Bu araştırma, makine ve teçhizat alt yapısı, 2018-1-AP5-2895 ve 2020-1-AP5-4104 numaralı Mersin Üniversitesi BAP birimi projelerinin destekleriyle kurulmuş olan Nöroanatomi ve Deneysel Araştırma Laboratuvarında gerçekleştirilmiştir. Akım sitometrik analizler için gerekli hizmet alımı 2020-1-AP5-4104 numaralı BAP projesi tarafından karşılanmıştır. 2018-1-AP5-2895 numaralı güdümlü altyapı projesinin geliştirilmesindeki desteklerinden dolayı başta Prof. Dr. Zeliha KURTOĞLU OLGUNUS olmak üzere Mersin Üniversitesi Tıp Fakültesi Anatomi Anabilim Dalı’nın tüm öğretim elemanlarına ve çalışanlarına teşekkürlerimizi sunarız.

References

  • Kaynaklar 1. Urbán N, Guillemot F. Neurogenesis in the embryonic and adult brain: same regulators, different roles. Front Cell Neurosci. 2014;8(NOV). doi:10.3389/FNCEL.2014.00396
  • 2. Eriksson PS, Perfilieva E, Björk-Eriksson T, et al. Neurogenesis in the adult human hippocampus. Nat Med 1998 411. 1998;4(11):1313-1317. doi:10.1038/3305
  • 3. Moreno-Jiménez EP, Flor-García M, Terreros-Roncal J, et al. Adult hippocampal neurogenesis is abundant in neurologically healthy subjects and drops sharply in patients with Alzheimer’s disease. Nat Med 2019 254. 2019;25(4):554-560. doi:10.1038/s41591-019-0375-9
  • 4. Lipp HP, Bonfanti L. Adult Neurogenesis in Mammals: Variations and Confusions. Brain Behav Evol. 2016;87(3):205-221. doi:10.1159/000446905
  • 5. Kempermann G. Environmental enrichment, new neurons and the neurobiology of individuality. Nat Rev Neurosci 2019 204. 2019;20(4):235-245. doi:10.1038/s41583-019-0120-x
  • 6. Lim DA, Alvarez-Buylla A. The Adult Ventricular–Subventricular Zone (V-SVZ) and Olfactory Bulb (OB) Neurogenesis. Cold Spring Harb Perspect Biol. 2016;8(5):a018820. doi:10.1101/CSHPERSPECT.A018820
  • 7. Aimone JB, Li Y, Lee SW, Clemenson GD, Deng W, Gage FH. Regulation and function of adult neurogenesis: from genes to cognition. Physiol Rev. 2014;94(4):991-1026. doi:10.1152/PHYSREV.00004.2014/ASSET/IMAGES/LARGE/Z9J0041427010007.
  • 8. Welberg L. Adult neurogenesis is altered in neurodegenerative disease. Nat Neurosci 2021 2412. 2021;24(12):1640-1640. doi:10.1038/s41593-021-00978-3
  • 9. Gordon J, Amini S. General Overview of Neuronal Cell Culture. Methods Mol Biol. 2021;2311:1-8. doi:10.1007/978-1-0716-1437-2_1/COVER
  • 10. Darbinian N. Cultured Cell Line Models of Neuronal Differentiation: NT2, PC12, and SK-N-MC. Methods Mol Biol. 2021;2311:25-38. doi:10.1007/978-1-0716-1437-2_3/COVER
  • 11. Wang C, Cai X, Hu W, et al. Investigation of the neuroprotective effects of crocin via antioxidant activities in HT22 cells and in mice with Alzheimer’s disease. Int J Mol Med. 2019;43(2):956-966. doi:10.3892/IJMM.2018.4032/HTML
  • 12. Park JS, Park JH, Kim KY. Neuroprotective effects of myristargenol A against glutamate-induced apoptotic HT22 cell death. RSC Adv. 2019;9(54):31247-31254. doi:10.1039/C9RA05408A
  • 13. Davis JB, Maher P. Protein kinase C activation inhibits glutamate-induced cytotoxicity in a neuronal cell line. Brain Res. 1994;652(1):169-173. doi:10.1016/0006-8993(94)90334-4
  • 14. Ishikawa M, Aoyama T, Shibata S, et al. miRNA-Based Rapid Differentiation of Purified Neurons from hPSCs Advancestowards Quick Screening for Neuronal Disease Phenotypes In Vitro. Cells. 2020;9(3). doi:10.3390/CELLS9030532
  • 15. Bratton DL, Fadok VA, Richter DA, Kailey JM, Guthrie LA, Henson PM. Appearance of Phosphatidylserine on Apoptotic Cells Requires Calcium-mediated Nonspecific Flip-Flop and Is Enhanced by Loss of the Aminophospholipid Translocase. J Biol Chem. 1997;272(42):26159-26165. doi:10.1074/JBC.272.42.26159
  • 16. Zhang G, Gurtu V, Kain SR, Yan G. Early Detection of Apoptosis Using a Fluorescent Conjugate of Annexin V. https://doi.org/102144/97233pf01. 2018;23(3):525-531. doi:10.2144/97233PF01
  • 17. Elmore S. Apoptosis: A Review of Programmed Cell Death. Toxicol Pathol. 2007;35(4):495. doi:10.1080/01926230701320337
  • 18. Brauchle E, Thude S, Brucker SY, Schenke-Layland K. Cell death stages in single apoptotic and necrotic cells monitored by Raman microspectroscopy. Sci Rep. 2014;4. doi:10.1038/SREP04698
  • 19. Malatesta P, Hartfuss E, Götz M. Isolation of radial glial cells by fluorescent-activated cell sorting reveals a neuronal lineage. Development. 2000;127(24):5253-5263. doi:10.1242/DEV.127.24.5253
  • 20. Miyata T, Kawaguchi A, Okano H, Ogawa M. Asymmetric Inheritance of Radial Glial Fibers by Cortical Neurons. Neuron. 2001;31(5):727-741. doi:10.1016/S0896-6273(01)00420-2
  • 21. Hsieh J. Orchestrating transcriptional control of adult neurogenesis. Genes Dev. 2012;26(10):1010-1021. doi:10.1101/GAD.187336.112
  • 22. Sierra A, Encinas JM, Deudero JJP, et al. Microglia Shape Adult Hippocampal Neurogenesis through Apoptosis-Coupled Phagocytosis. Cell Stem Cell. 2010;7(4):483-495. doi:10.1016/J.STEM.2010.08.014
  • 23. Liu J, Li L, Suo WZ. HT22 hippocampal neuronal cell line possesses functional cholinergic properties. Life Sci. 2009;84(9-10):267-271. doi:10.1016/j.lfs.2008.12.008
  • 24. He M, Liu J, Cheng S, Xing Y, Suo WZ. Differentiation renders susceptibility to excitotoxicity in HT22 neurons. Neural Regen Res. 2013;8(14):1297. doi:10.3969/J.ISSN.1673-5374.2013.14.006
  • 25. Lim J, Bang Y, Kim KM, Choi HJ. Differentiated HT22 cells as a novel model for in vitro screening of serotonin reuptake inhibitors. Front Pharmacol. 2023;13:5432. doi:10.3389/FPHAR.2022.1062650/BIBTEX
  • 26. Zhao Z, Lu R, Zhang B, et al. Differentiation of HT22 neurons induces expression of NMDA receptor that mediates homocysteine cytotoxicity. http://dx.doi.org/101179/1743132811Y0000000057. 2013;34(1):38-43. doi:10.1179/1743132811Y.0000000057

Measuring the Apoptotic Response of the HT22 Mouse Hippocampal Cell Line to Neuronal Differentiation Medium

Year 2023, Volume: 13 Issue: 3, 375 - 383, 30.09.2023
https://doi.org/10.31832/smj.1264019

Abstract

Objective: The aim of this study is to test the apoptotic response of the HT22 mouse hippocampal cell line, which is frequently used in neurobiology studies, to a standard differentiation medium at different times in order to understand its usability as a neuronal differentiation model.

Methods: For seeding and culturing HT22 cells aside the differentiation, HG-DMEM and for differentiation B27+ supplemented NB+ medium were used respectively. A total of 9 different groups, including the control group, were cultured in standard cell culture environment. At the end of the experiment, the rates of early/late apoptosis and necrosis were determined with flow cytometric analysis of AnnexinV/PI labeled HT22 cells. After testing whether the raw data fit the normal distribution or not by employing Shapiro-Wilks test, further statistical analyzes were performed with the most appropriate test parametric or non-parametric tests.

Results: Statistically significant differences were found in terms of early apoptosis (P<0,01) and late apoptosis (P<0,001) cell population rates, while there was no significant difference of necrosis (P=0,064). Significant (P<0,001) increase in early apoptosis was evident in the majority of groups cultured differentiation medium for 48 or 72 hours, compared to the cells cultured with only HG-DMEM or differentiation media for 24 hours. While a significant increase in late apoptosis was observed in the majority of the groups cultured with the differentiation medium compared to the control group (P<0.001), no significant difference was observed between the groups differentiated for different durations (P>0,05).

Conclusion: HT22 hippocampal cell line, which is frequently used in in-vitro modeling of important pathobiological features of neurodegenerative diseases, but poorly understood for the suitability in neuronal differentiation, has a markedly increased response to a widely used differentiation medium in terms of early apoptosis (culturing duration dependent) and late apoptosis (regardless of duration). Our findings provide important clues at the level of apoptosis in terms of the usability of the HT22 cell line in differentiation studies.

Project Number

2018-1-AP5-2895 ve 2020-1-AP5-4104

References

  • Kaynaklar 1. Urbán N, Guillemot F. Neurogenesis in the embryonic and adult brain: same regulators, different roles. Front Cell Neurosci. 2014;8(NOV). doi:10.3389/FNCEL.2014.00396
  • 2. Eriksson PS, Perfilieva E, Björk-Eriksson T, et al. Neurogenesis in the adult human hippocampus. Nat Med 1998 411. 1998;4(11):1313-1317. doi:10.1038/3305
  • 3. Moreno-Jiménez EP, Flor-García M, Terreros-Roncal J, et al. Adult hippocampal neurogenesis is abundant in neurologically healthy subjects and drops sharply in patients with Alzheimer’s disease. Nat Med 2019 254. 2019;25(4):554-560. doi:10.1038/s41591-019-0375-9
  • 4. Lipp HP, Bonfanti L. Adult Neurogenesis in Mammals: Variations and Confusions. Brain Behav Evol. 2016;87(3):205-221. doi:10.1159/000446905
  • 5. Kempermann G. Environmental enrichment, new neurons and the neurobiology of individuality. Nat Rev Neurosci 2019 204. 2019;20(4):235-245. doi:10.1038/s41583-019-0120-x
  • 6. Lim DA, Alvarez-Buylla A. The Adult Ventricular–Subventricular Zone (V-SVZ) and Olfactory Bulb (OB) Neurogenesis. Cold Spring Harb Perspect Biol. 2016;8(5):a018820. doi:10.1101/CSHPERSPECT.A018820
  • 7. Aimone JB, Li Y, Lee SW, Clemenson GD, Deng W, Gage FH. Regulation and function of adult neurogenesis: from genes to cognition. Physiol Rev. 2014;94(4):991-1026. doi:10.1152/PHYSREV.00004.2014/ASSET/IMAGES/LARGE/Z9J0041427010007.
  • 8. Welberg L. Adult neurogenesis is altered in neurodegenerative disease. Nat Neurosci 2021 2412. 2021;24(12):1640-1640. doi:10.1038/s41593-021-00978-3
  • 9. Gordon J, Amini S. General Overview of Neuronal Cell Culture. Methods Mol Biol. 2021;2311:1-8. doi:10.1007/978-1-0716-1437-2_1/COVER
  • 10. Darbinian N. Cultured Cell Line Models of Neuronal Differentiation: NT2, PC12, and SK-N-MC. Methods Mol Biol. 2021;2311:25-38. doi:10.1007/978-1-0716-1437-2_3/COVER
  • 11. Wang C, Cai X, Hu W, et al. Investigation of the neuroprotective effects of crocin via antioxidant activities in HT22 cells and in mice with Alzheimer’s disease. Int J Mol Med. 2019;43(2):956-966. doi:10.3892/IJMM.2018.4032/HTML
  • 12. Park JS, Park JH, Kim KY. Neuroprotective effects of myristargenol A against glutamate-induced apoptotic HT22 cell death. RSC Adv. 2019;9(54):31247-31254. doi:10.1039/C9RA05408A
  • 13. Davis JB, Maher P. Protein kinase C activation inhibits glutamate-induced cytotoxicity in a neuronal cell line. Brain Res. 1994;652(1):169-173. doi:10.1016/0006-8993(94)90334-4
  • 14. Ishikawa M, Aoyama T, Shibata S, et al. miRNA-Based Rapid Differentiation of Purified Neurons from hPSCs Advancestowards Quick Screening for Neuronal Disease Phenotypes In Vitro. Cells. 2020;9(3). doi:10.3390/CELLS9030532
  • 15. Bratton DL, Fadok VA, Richter DA, Kailey JM, Guthrie LA, Henson PM. Appearance of Phosphatidylserine on Apoptotic Cells Requires Calcium-mediated Nonspecific Flip-Flop and Is Enhanced by Loss of the Aminophospholipid Translocase. J Biol Chem. 1997;272(42):26159-26165. doi:10.1074/JBC.272.42.26159
  • 16. Zhang G, Gurtu V, Kain SR, Yan G. Early Detection of Apoptosis Using a Fluorescent Conjugate of Annexin V. https://doi.org/102144/97233pf01. 2018;23(3):525-531. doi:10.2144/97233PF01
  • 17. Elmore S. Apoptosis: A Review of Programmed Cell Death. Toxicol Pathol. 2007;35(4):495. doi:10.1080/01926230701320337
  • 18. Brauchle E, Thude S, Brucker SY, Schenke-Layland K. Cell death stages in single apoptotic and necrotic cells monitored by Raman microspectroscopy. Sci Rep. 2014;4. doi:10.1038/SREP04698
  • 19. Malatesta P, Hartfuss E, Götz M. Isolation of radial glial cells by fluorescent-activated cell sorting reveals a neuronal lineage. Development. 2000;127(24):5253-5263. doi:10.1242/DEV.127.24.5253
  • 20. Miyata T, Kawaguchi A, Okano H, Ogawa M. Asymmetric Inheritance of Radial Glial Fibers by Cortical Neurons. Neuron. 2001;31(5):727-741. doi:10.1016/S0896-6273(01)00420-2
  • 21. Hsieh J. Orchestrating transcriptional control of adult neurogenesis. Genes Dev. 2012;26(10):1010-1021. doi:10.1101/GAD.187336.112
  • 22. Sierra A, Encinas JM, Deudero JJP, et al. Microglia Shape Adult Hippocampal Neurogenesis through Apoptosis-Coupled Phagocytosis. Cell Stem Cell. 2010;7(4):483-495. doi:10.1016/J.STEM.2010.08.014
  • 23. Liu J, Li L, Suo WZ. HT22 hippocampal neuronal cell line possesses functional cholinergic properties. Life Sci. 2009;84(9-10):267-271. doi:10.1016/j.lfs.2008.12.008
  • 24. He M, Liu J, Cheng S, Xing Y, Suo WZ. Differentiation renders susceptibility to excitotoxicity in HT22 neurons. Neural Regen Res. 2013;8(14):1297. doi:10.3969/J.ISSN.1673-5374.2013.14.006
  • 25. Lim J, Bang Y, Kim KM, Choi HJ. Differentiated HT22 cells as a novel model for in vitro screening of serotonin reuptake inhibitors. Front Pharmacol. 2023;13:5432. doi:10.3389/FPHAR.2022.1062650/BIBTEX
  • 26. Zhao Z, Lu R, Zhang B, et al. Differentiation of HT22 neurons induces expression of NMDA receptor that mediates homocysteine cytotoxicity. http://dx.doi.org/101179/1743132811Y0000000057. 2013;34(1):38-43. doi:10.1179/1743132811Y.0000000057
There are 26 citations in total.

Details

Primary Language Turkish
Subjects Health Care Administration
Journal Section Articles
Authors

Ayla Batu Öztürk 0000-0003-3221-4292

Derya Yetkin 0000-0002-1452-5655

Nail Can Öztürk 0000-0001-9459-2120

Project Number 2018-1-AP5-2895 ve 2020-1-AP5-4104
Publication Date September 30, 2023
Submission Date March 12, 2023
Published in Issue Year 2023 Volume: 13 Issue: 3

Cite

AMA Batu Öztürk A, Yetkin D, Öztürk NC. HT22 Fare Hipokampal Hücre Hattının Nöronal Farklanma Besiyerine Verdiği Apoptotik Tepkinin Ölçülmesi. Sakarya Tıp Dergisi. September 2023;13(3):375-383. doi:10.31832/smj.1264019

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