Author(s):
Machlusil Husna, Rumman Karimah, Hidayat Sujuti, Aulanni’am Aulanni’am
Email(s):
machlusilhusna.fk@ub.ac.id
DOI:
10.52711/0974-360X.2026.00599 
Address:
Machlusil Husna1, Rumman Karimah2, Hidayat Sujuti3, Aulanni’am Aulanni’am4
1Department of Neurology, Faculty of Medicine Universitas Brawijaya, Saiful Anwar General Hospital, Malang, East Java, Indonesia.
2 Faculty of Medicine Universitas Brawijaya, Saiful Anwar General Hospital, Malang, East Java, Indonesia.
3Department of Ophthalmology, Faculty of Medicine Universitas Brawijaya, Saiful Anwar General Hospital, Malang, East Java, Indonesia.
4Department of Biochemistry, Laboratory of Biochemistry, Faculty of Sciences, Universitas Brawijaya, Malang, East Java, Indonesia.
*Corresponding Author
Published In:
Volume - 19,
Issue - 9,
Year - 2026
ABSTRACT:
Background: Most in vitro epilepsy models are based on measuring epileptiform activity, whether in slice or neuron culture. Traditional electrophysiological techniques are not yet able to measure neuronal activity in cells that are less electrically active. Significant intracellular calcium fluctuations occur in neurons, which can resemble neuronal activity and appear alongside neuronal depolarization. Calcium oscillations correlate with a burst of neuronal action potentials, depicting epileptiform activity. An alternative approach to measure neuronal depolarization is by observing the intracellular calcium fluctuation, which can be visualized by calcium fluorescence. The SH-SY5Y cell line is easy to handle and can be differentiated into a stable neuronal culture. This culture can be used to evaluate neuronal death or injury in degenerative disease, cerebral ischemia, and epilepsy. However, the in vitro epilepsy model using SH-SY5Y cells remains very limited, with variable treatment duration and dose. However, the in vitro epilepsy model using SH-SY5Y cells remains very limited, with variable treatment duration and dose. This research aimed to determine the optimal dose of kainic acid for SH-SY5Y cultures as an in vitro epilepsy model by observing intracellular calcium fluctuations. METHOD: Cell line SH-SY5Y culture was carried out until 80% confluency, then the cells were observed for the next 3 days until intervention. The cell cultures were divided into five groups: a control group and an epilepsy model group, each comprising 4 subgroups with various kainic acid doses (4µM, 8µM, 16µM, and 20µM). Every group consists of 5 samples. Kainic acid was administered for 48 hours, and we then performed a calcium assay to detect intracellular calcium flux fluctuations using Fluo-4. Observation was done with confocal laser scanning microscopy periodically for 52 seconds with a 13-second interval. RESULT: Intracellular calcium fluorescence was highest with a kainic acid dose of 20µM compared to other groups at all times of observation, followed by 4 µM, 8µM, the control group, and 16µM. Calcium fluorescence at doses of 4µM was higher than at doses of 8µM and 16µM. Kainic acid doses were positively correlated with the increase of intracellular calcium fluorescence, with the closest correlation found at a dose of 20µM. CONCLUSION: The SH-Sy5Y cell line can be used as an in vitro epilepsy model. A kainic acid concentration of 20µM appeared to be optimal for inducing intracellular calcium fluctuations characteristic of epileptiform activity. Further research with a narrower time interval and a longer observation duration is needed; a millisecond time interval and a few minutes of observation are recommended to achieve better calcium fluctuation results.
Cite this article:
Machlusil Husna, Rumman Karimah, Hidayat Sujuti, Aulanni’am Aulanni’am. SH-SH5Y-based In vitro Epilepsy Model: Analysis of Intracellular Calcium Imaging Across Different Doses of Kainic Acid. Research Journal Pharmacy and Technology. 2026;19(9):4295-1. doi: 10.52711/0974-360X.2026.00599
Cite(Electronic):
Machlusil Husna, Rumman Karimah, Hidayat Sujuti, Aulanni’am Aulanni’am. SH-SH5Y-based In vitro Epilepsy Model: Analysis of Intracellular Calcium Imaging Across Different Doses of Kainic Acid. Research Journal Pharmacy and Technology. 2026;19(9):4295-1. doi: 10.52711/0974-360X.2026.00599 Available on: https://www.rjptonline.org/AbstractView.aspx?PID=2026-19-9-45
REFERENCES:
1. Avoli M, Jefferys JGR. Models of drug-induced epileptiform synchronization in vitro. J Neurosci Methods. 2016; 260(1959): 26–32.
2. Morris G, Jiruska P, Jefferys JGR, Powell AD. A new approach of modified submerged patch clamp recording reveals interneuronal dynamics during epileptiform oscillations. Front Neurosci. 2016; 10(NOV).
3. Wickham J, Ledri M, Bengzon J, Jespersen B, Pinborg LH, Englund E, et al. Inhibition of epileptiform activity by neuropeptide Y in brain tissue from drug-resistant temporal lobe epilepsy patients. Sci Rep. 2019 Dec 1; 9(1).
4. Morris G, Leite M, Kullmann DM, Pavlov X, Schorge S, Lignani G. Activity clamp provides insights into paradoxical effects of the anti-seizure drug carbamazepine. Journal of Neuroscience. 2017 May 31; 37(22): 5484–95.
5. Jimbo Y. MEA-based recording of neuronal activity in vitro. Arch Ital Biol. 2007; 145(3–4): 289–97.
6. Jäckel D, Bakkum DJ, Russell TL, Müller J, Radivojevic M, Frey U, et al. Combination of High-density Microelectrode Array and Patch Clamp Recordings to Enable Studies of Multisynaptic Integration. Sci Rep. 2017; 7(1): 1–17.
7. de Melo Reis RA, Freitas HR, de Mello FG. Cell Calcium Imaging as a Reliable Method to Study Neuron–Glial Circuits. Vol. 14, Frontiers in Neuroscience. Frontiers Media S.A.; 2020.
8. Lopez-Suarez L, Awabdh S Al, Coumoul X, Chauvet C. The SH-SY5Y human neuroblastoma cell line, a relevant in vitro cell model for investigating neurotoxicology in humans: Focus on organic pollutants. Vol. 92, NeuroToxicology. Elsevier B.V.; 2022. p. 131–55.
9. Kumari N, Jeyabalan S, Rajangam J, Gopinathan N, Ramakrishnan SR, Reddy VJ. Neuroprotective Potential of Total Extract of Ulva Lactuca: An In vitro study. Res J Pharm Technol. 2023; 16(12): 5948–53.
10. Karthika S, Kannappan N, Suriyaprakash T. Effect of Medicinal plants on amyloid β1-42 Intoxicated SH-SY5Y cell Lines-As Neuroprotective Evaluation. Res J Pharm Technol. 2020; 13(7): 3351–5.
11. Rusina E, Bernard C, Williamson A. The kainic acid models of temporal lobe epilepsy. Vol. 8, eNeuro. Society for Neuroscience; 2021.
12. Pacico N, Meur AM. New In Vitro Phenotypic Assay for Epilepsy : Fluorescent Measurement of Synchronized Neuronal Calcium Oscillations. PLoS One. 2014; 9(1).
13. Lafayette W. Kainic Acid-induced F-344 Rat model of Mesial Temporal Lobe Epilepsy : Gene Expression and Canonical Pathways. Toxicol Pathol. 2009; 37: 776–89.
14. Zhang XM, Zhu J. Kainic Acid-Induced Neurotoxicity: Targeting Glial Responses and Glia-Derived Cytokines. Curr Neuropharmacol. 2011; 9(2): 388–98.
15. Zhang X, Qiao Z, Liu N, Gao L, Wei L, Liu A, et al. Stereotypical patterns of epileptiform calcium signal in hippocampal CA1, CA3, dentate gyrus and entorhinal cortex in freely moving mice. Sci Rep. 2019; 9(1): 1–9.
16. Vargas JR, Takahashi DK, Thomson KE, Wilcox KS. The expression of kainate receptor subunits in hippocampal astrocytes after experimentally induced status epilepticus. J Neuropathol Exp Neurol. 2013; 72(10): 919–32.
17. Jafari N, He L, Khalil CB, Yeh HJ, Harris NG, Stern JM, et al. Intrinsic brain network stability during kainic acid-induced epileptogenesis. Epilepsia Open. 2025 Apr 1;10(2):508–20.
18. Rosalia Cannarsa, Daniela Landuzzi, Chiara Cavina, Sanzio Candeletti, Patrizia Romualdi. Kainic Acid Down-regulates NOP Receptor Density and Gene Expression in Human Neuroblastoma SH-SY5Y Cells. Journal of Molecular Neuroscience. 2008; Feb 20; 35: 171–7.
19. Nampoothiri M, Reddy ND, John J, Kumar N, Kutty Nampurath G, Rao Chamallamudi M. Insulin blocks glutamate-induced neurotoxicity in differentiated SH-SY5Y neuronal cells. Behavioural Neurology. 2014;2014.
20. Cetindag Ciltas A, Gundogdu S, Yulak F. Levetiracetam Protects Against Glutamate-Induced Excitotoxicity in SH-SY5Y Cell Line. International Journal of Nature and Life Sciences. 2022 Dec 1; 6(2): 142–50.
21. Mzezewa R, Lotila J, Kiiski H, Vinogradov A, Kapucu FE, Peltola J, et al. A kainic acid-induced seizure model in human pluripotent stem cell-derived cortical neurons for studying the role of IL-6 in the functional activity. Stem Cell Res. 2022 Apr 1;60.
22. Zanker J, Hüser D, Savy A, Lázaro-Petri S, Hammer EM, Schwarzer C, et al. Evaluation of the SH-SY5Y cell line as an in vitro model for potency testing of a neuropeptide-expressing AAV vector. Front Mol Neurosci. 2023; 16.
23. Karthika S., Kannappan N, Suriyaprakash TNK. Effect of Medicinal Plants on Amyloid β1-42 Intoxicated SH-SY5Y Cell Lines - As Neuroprotective Evaluation. Res J Pharm Technol. 2020;7(13):3351–5.
24. Ovey IS, Ozsimsek A, Velioglu HA, Altay O, Mardinoglu A, Yulug B. EGb 761 reduces Ca2+ influx and apoptosis after pentylenetetrazole treatment in a neuroblastoma cell line. Front Cell Neurosci. 2023; 17.
25. Tang Q, Li T, Chen K, Deng X, Zhang Q, Tang H, et al. PS-NPs Induced Neurotoxic Effects in SHSY-5Y Cells via Autophagy Activation and Mitochondrial Dysfunction. Brain Sci. 2022 Jul 1; 12(7).
26. Taskiran AS, Ergul M. The modulator action of thiamine against pentylenetetrazole-induced seizures, apoptosis, nitric oxide, and oxidative stress in rats and SH-SY5Y neuronal cell line. Chem Biol Interact. 2021 May 1;340.
27. Than A, Tan Y, Ong WY, Farooqui AA, Chen P. Kainate receptors mediate regulated exocytosis of secretory phospholipase A 2 in SH-SY5Y neuroblastoma cells. Neurosignals. 2012 Mar; 20(2): 72–85.
28. Costanza M, Ciotti A, Consonni A, Cipelletti B, Cattalini A, Cagnoli C, et al. CNS autoimmune response in the MAM/pilocarpine rat model of epileptogenic cortical malformation. Proc Natl Acad Sci U S A. 2024; 121(17).
29. Xiang J, Jiang Y. Regulation of Cu-Zn superoxide dismutase on SCN2A in SH-SY5Y cells as a potential therapy for temporal lobe epilepsy. Mol Med Rep. 2014; 9(18): 16–22.
30. Steinlein OK. Calcium signaling and epilepsy. Cell Tissue Res. 2014; 357(2): 385–93.
31. Arena A, Zimmer TS, van Scheppingen J, Korotkov A, Anink JJ, Mühlebner A, et al. Oxidative stress and inflammation in a spectrum of epileptogenic cortical malformations: molecular insights into their interdependence. Brain Pathology. 2019 May 1; 29(3): 351–65.
32. Zhang Y, Hu X, Zou LQ. Flavonoids as therapeutic agents for epilepsy: unveiling anti-inflammatory and antioxidant pathways for novel treatments. Vol. 15, Frontiers in Pharmacology. Frontiers Media SA; 2024.
33. Carrese AM, Vitale R, Turco M, Masola V, Aniello F, Vitale E, et al. Sustained Depolarization Induces Gene Expression Pattern Changes Related to Synaptic Plasticity in a Human Cholinergic Cellular Model. Mol Neurobiol. 2025 Jan 1;62(1):935–45.
34. Fernandez-Perez EJ, Muñoz B, Bascuñan DA, Peters C, Riffo-Lepe NO, Espinoza MP, et al. Synaptic dysregulation and hyperexcitability induced by intracellular amyloid beta oligomers. Aging Cell. 2021 Sep 1;20(9).
35. Xiong QJ, Hu ZL, Wu PF, Ni L, Deng ZF, Wu WN, et al. Acid-sensing ion channels contribute to the increase in vesicular release from SH-SY5Y cells stimulated by extracellular protons. Am J Physiol Cell Physiol. 2012 Aug 15;303(4).
36. Hotka M, Kubista H. The paroxysmal depolarization shift in epilepsy research. Vol. 107, International Journal of Biochemistry and Cell Biology. Elsevier Ltd, 2019. p. 77–81.
37. Kubista H, Boehm S, Hotka M. The paroxysmal depolarization shift: Reconsidering its role in epilepsy, epileptogenesis and beyond. Vol. 20, International Journal of Molecular Sciences. MDPI AG; 2019.
38. Laryushkin DP, Maiorov SA, Zinchenko VP, Gaidin SG, Kosenkov AM. Role of L-type voltage-gated calcium channels in epileptiform activity of neurons. Int J Mol Sci. 2021;22(19).
39. Rubi L, Schandl U, Lagler M, Geier P, Spies D, Gupta K Das, et al. Raised activity of L-type calcium channels renders neurons prone to form paroxysmal depolarization shifts. Neuromolecular Med. 2013 Sep;15(3):476–92.
40. Zinchenko VP, Teplov IY, Tyurin F V., Malibayeva AE, Kairat BK, Tuleukhanov ST. The Effect of Intracellular Calcium Buffer Bapta on Epileptiform Activity of Hippocampal Neurons. Int J Mol Sci. 2025 Aug 1; 26(15).
41. Cox LA (Tony). Biological mechanisms of non-linear dose-response for respirable mineral fibers. Toxicol Appl Pharmacol. 2018 Dec 15;361:137–44.
42. Cerella C, Diederich M, Ghibelli L. The dual role of calcium as messenger and stressor in cell damage, death, and survival. Int J Cell Biol. 2010; 2010.
43. Chamaa F, Darwish B, Arnaout R, Nahas Z, Al-Chaer ED, Saadé NE, et al. Sustained Activation of the Anterior Thalamic Neurons with Low Doses of Kainic Acid Boosts Hippocampal Neurogenesis. Cells. 2022 Nov 1;11(21).
44. Zepeda A, Aguilar-Arredondo A, Michel G, Ramos-Languren LE, Escobar ML, Arias C. Functional recovery of the dentate gyrus after a focal lesion is accompanied by structural reorganization in the adult rat. Brain Struct Funct. 2013 Mar; 218(2): 437–53.
45. Dawe GB, Musgaard M, Andrews ED, Daniels BA, Aurousseau MRP, Biggin PC, et al. Defining the structural relationship between kainate-receptor deactivation and desensitization. Nat Struct Mol Biol. 2013 Sep; 20(9): 1054–61.
46. Kamiya Haruyuki, Ozawa Seiji. Kainate receptor-mediated inhibition of presynaptic Ca2+ influx and EPSP in area CA1 of the rat hippocampus. Journal of Physiology. 1998; 3(509): 833–45.
47. Rodríguez-Chávez V, Flores-Soto E, Molina-Salinas G, Martínez-Razo LD, Montaño LM, Cerbón M. Prolactin reduces the kainic acid-induced increase in intracellular Ca2+ concentration, leading to neuroprotection of hippocampal neurons. Neurosci Lett. 2023 Jul 27; 810.
48. Arundine M, Tymianski M. Molecular mechanisms of calcium-dependent neurodegeneration in excitotoxicity. Cell Calcium. 2003; 34(4–5): 325–37.
49. Gade D, Meshram A, Nagrecha D, Jadhav Y, Pawar P. Inimical effects of Monosodium Glutamate on various systems. Asian J Research Chem. 2024; 17(3): 174–8.
50. Kumaravel P, Prabhakaran A, Melchias G, Edward A, Natarajan P, Sathishkumar S. Monosodium glutamate exposure alters histopathological and biochemical variables in rats under constant light. Asian J Res Pharm Sci. 2014; 4(1): 1–4.