2023, Vol. 27 ›› Issue (11): 1733-1738

Establishment of an in vitro model of H2O2-induced neuronal senescence

Fu Xinya1, Ma Jinjin1, Li Meimei1, Wang Xingran1, Xie Jile2, Saijilafu1   

Abstract: BACKGROUND: Aging leads to loss of neuronal morphology and function, which becomes the most important risk factor for neurodegenerative diseases. Current research on the mechanism of neuronal aging is still incompleted, and establishing an in vitro aging model will promote relevant research. As a small number of mature mammalian neurons, dorsal root ganglion is easy to isolate and culture in vitro, and therefore, it is a suitable target for establishing the in vitro aging model.
OBJECTIVE: To explore suitable conditions for inducing neuronal senescence and establish an in vitro model of H2O2-induced neuronal senescence.
METHODS: The dorsal root ganglia were obtained from 1-month-old ICR mice and cultured in vitro with 10, 20, and 50 μmol/L H2O2 for 1 and 2 hours. The appropriate conditions for inducing cell senescence were screened through detecting the activity of β-galactosidase and cell survival rate of neurons. These induction conditions were further identified by detecting the expression level of autophagy-related protein SQSTM1/p62, the content of reactive oxygen species, and the axon growth ability of neurons. Consequently, the in vitro aging model was successfully established in neurons.
RESULTS AND CONCLUSION: By detecting the activity of β-galactosidase and the survival rate of neurons, culture with 10 μmol/L H2O2 for 2 hours was selected as suitable induction conditions. Under these suitable conditions, the expression of SQSTM1/p62 was significantly down-regulated (P < 0.01), consistent with the characteristics of cell senescence; the production of reactive oxygen species increased significantly (P < 0.01), indicating impaired function of mitochondria and emergence of senescent cells; the growth ability of neuronal axons was significantly weakened (P < 0.001), conforming to the characteristics of neuron aging. In conclusion, culture with 10 μmol/L H2O2 for 2 hours can be successfully used for the establishment of neuronal aging models in vitro.
Key words: neuron, senescence, H2O2, cell survival rate, senescence marker, β-galactosidase, SQSTM1/p62, reactive oxygen species