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Mitochondrial dysfunction in long-term neuronal cultures mimics changes with aging.

Dong W, Cheng S, Huang F, Fan W, Chen Y, Shi H, He H - Med. Sci. Monit. (2011)

Bottom Line: The levels of cellular senescence were evaluated by cytochemical staining of senescence-associated β-galactosidase (SA-β-Gal) at DIV 5, 10, 15, 20, 25 and 30.In addition, we investigated the changes in mitochondrial membrane potential (Δψm) and intracellular reactive oxygen species (ROS) generation of hippocampal neurons by flow cytometry at different ages.The proportion of the senescent cells steadily increased with age in neuron cultures.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrated Traditional Chinese and Western Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou, China.

ABSTRACT

Background: Aging is a highly complex process that affects various tissues and systems in the body. Senescent changes are relatively more prevalent and severe in the postmitotic cells. Mitochondria play an important role in the aging process. Recently, cell cultures have been widely used as an in vitro model to study aging. The present study was designed to investigate mitochondrial dysfunction associated with aging in a long-term cell culture system.

Material/methods: Rat hippocampal neurons were maintained in culture in serum-free medium for 30 days in vitro (DIV). The morphology and development of hippocampal neurons was observed by phase contrast microscope. The levels of cellular senescence were evaluated by cytochemical staining of senescence-associated β-galactosidase (SA-β-Gal) at DIV 5, 10, 15, 20, 25 and 30. In addition, we investigated the changes in mitochondrial membrane potential (Δψm) and intracellular reactive oxygen species (ROS) generation of hippocampal neurons by flow cytometry at different ages.

Results: The proportion of the senescent cells steadily increased with age in neuron cultures. Δψm decreased gradually with age in long-term culture, while ROS generation increased.

Conclusions: This study indicates an age-related decrease in mitochondrial function in long-term hippocampal neuronal culture and suggests that DIV 25 neurons could possibly serve as a platform for the future study of anti-aging from the perspective of mitochondrial function.

Show MeSH
Phase contrast photomicrographs of live rat primary hippocampal neurons in culture plated at 310 cells/mm2. A-F show neurons of ages 5, 10, 15, 20, 25 and 30 DIV, respectively. Scale bar =100 μm.
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f1-medscimonit-17-4-br91: Phase contrast photomicrographs of live rat primary hippocampal neurons in culture plated at 310 cells/mm2. A-F show neurons of ages 5, 10, 15, 20, 25 and 30 DIV, respectively. Scale bar =100 μm.

Mentions: Dissociated cells from the hippocampus of newborn rat brain were plated at a cell density of 310 cells/mm2. Within the first 2 days after plating, cell density decreased to 260 cells/mm2. Once stabilized, the number of surviving neurons remained constant up to 25 days and then started to decline. Photomicrographs of rat hippocampal neurons at different ages in culture are shown in Figure 1. Hippocampal neurons at DIV 5 extended prominent neurite (Figure 1A). The first 15 days was a period of maturation of young neurons in culture, during which they formed an extensive network of processes and increased the size of their cell bodies (Figure 1A–C). Mature cells exhibited an increase in the size of the cell bodies, thick dendritic processes, and an extremely dense and extensive network (Figure 1C, D). Neurons with vacuolated soma and beaded or fragmented neurites were observed after DIV 20, and then the number of neurons decreased after DIV 25 (Figure 1E, F).


Mitochondrial dysfunction in long-term neuronal cultures mimics changes with aging.

Dong W, Cheng S, Huang F, Fan W, Chen Y, Shi H, He H - Med. Sci. Monit. (2011)

Phase contrast photomicrographs of live rat primary hippocampal neurons in culture plated at 310 cells/mm2. A-F show neurons of ages 5, 10, 15, 20, 25 and 30 DIV, respectively. Scale bar =100 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3539510&req=5

f1-medscimonit-17-4-br91: Phase contrast photomicrographs of live rat primary hippocampal neurons in culture plated at 310 cells/mm2. A-F show neurons of ages 5, 10, 15, 20, 25 and 30 DIV, respectively. Scale bar =100 μm.
Mentions: Dissociated cells from the hippocampus of newborn rat brain were plated at a cell density of 310 cells/mm2. Within the first 2 days after plating, cell density decreased to 260 cells/mm2. Once stabilized, the number of surviving neurons remained constant up to 25 days and then started to decline. Photomicrographs of rat hippocampal neurons at different ages in culture are shown in Figure 1. Hippocampal neurons at DIV 5 extended prominent neurite (Figure 1A). The first 15 days was a period of maturation of young neurons in culture, during which they formed an extensive network of processes and increased the size of their cell bodies (Figure 1A–C). Mature cells exhibited an increase in the size of the cell bodies, thick dendritic processes, and an extremely dense and extensive network (Figure 1C, D). Neurons with vacuolated soma and beaded or fragmented neurites were observed after DIV 20, and then the number of neurons decreased after DIV 25 (Figure 1E, F).

Bottom Line: The levels of cellular senescence were evaluated by cytochemical staining of senescence-associated β-galactosidase (SA-β-Gal) at DIV 5, 10, 15, 20, 25 and 30.In addition, we investigated the changes in mitochondrial membrane potential (Δψm) and intracellular reactive oxygen species (ROS) generation of hippocampal neurons by flow cytometry at different ages.The proportion of the senescent cells steadily increased with age in neuron cultures.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrated Traditional Chinese and Western Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou, China.

ABSTRACT

Background: Aging is a highly complex process that affects various tissues and systems in the body. Senescent changes are relatively more prevalent and severe in the postmitotic cells. Mitochondria play an important role in the aging process. Recently, cell cultures have been widely used as an in vitro model to study aging. The present study was designed to investigate mitochondrial dysfunction associated with aging in a long-term cell culture system.

Material/methods: Rat hippocampal neurons were maintained in culture in serum-free medium for 30 days in vitro (DIV). The morphology and development of hippocampal neurons was observed by phase contrast microscope. The levels of cellular senescence were evaluated by cytochemical staining of senescence-associated β-galactosidase (SA-β-Gal) at DIV 5, 10, 15, 20, 25 and 30. In addition, we investigated the changes in mitochondrial membrane potential (Δψm) and intracellular reactive oxygen species (ROS) generation of hippocampal neurons by flow cytometry at different ages.

Results: The proportion of the senescent cells steadily increased with age in neuron cultures. Δψm decreased gradually with age in long-term culture, while ROS generation increased.

Conclusions: This study indicates an age-related decrease in mitochondrial function in long-term hippocampal neuronal culture and suggests that DIV 25 neurons could possibly serve as a platform for the future study of anti-aging from the perspective of mitochondrial function.

Show MeSH