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Updated: Aug 30, 2026
Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species in Live Rat Cortical Neurons
Published on: May 23, 2011
Mitochondrial hyperpolarization after transient oxygen-glucose deprivation and subsequent apoptosis in cultured rat
Takehiko Iijima1, Tatsuya Mishima, Kimio Akagawa
1Department of Anesthesiology, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka City 181-8611, Tokyo, Japan. iijmt@kyorin-u.ac.jp
Abstract:
Mitochondrial membrane potential (MMP) regulates the production of high-energy phosphate and apoptotic cascade, both occurring after ischemic impact. The timed profile of MMP differing from grading ischemic impact has to be determined. Primary rat hippocampal cultures were exposed to oxygen-glucose deprivation (OGD) for 30, 60, and 90 min and then were reoxygenated. MMP was expressed as a voltage-dependent dye, JC-1 fluorescence, under confocal microscopy. Cell viability was assessed by calcein AM and ethidium homodimer, each at 3 hours and 24 hours after 30, 60, and 90 min of OGD. The appearance of apoptosis was also evaluated by the TUNEL method at 24 hours. Hyperpolarization of MMP (2.31+/-0.94 normalized JC-1 fluorescence ratio between red and green) was observed during reoxygenation after 30 min OGD, while 60 min OGD induced depolarization (0.66+/-0.22, Valinomycin (potassium ionophore)-induced depolarization: 0.53+/-0.19). The fluorescence of mitochondria became weak after 90 min OGD. Most of the neurons were shrunken after 90 min and neurons were TUNEL-positive 24 hours after 30 min OGD, although most neurons were viable at 3 hours. A longer period of OGD induced necrosis, and most neurons remained viable after only 3 hours. Our data present that the short (30 min) OGD induced hyperpolarization of MMP during reoxygenation, while a longer OGD (60 or 90 min) induced depolarization and acute necrosis. Neurons were still viable even during hyperpolarization of mitochondria, but this hyperpolarization appears to be linked to subsequent apoptotic change.
Insights
Short oxygen-glucose deprivation (OGD) in neurons causes mitochondrial membrane potential (MMP) hyperpolarization, linked to later apoptosis. Longer OGD durations induce depolarization and necrosis, impacting cell viability.
Area of Science:
- Neuroscience
- Cell Biology
- Biochemistry
Background:
- Mitochondrial membrane potential (MMP) is crucial for cellular energy production and apoptosis.
- Ischemic events, like oxygen-glucose deprivation (OGD), significantly impact neuronal MMP.
- Understanding the time-dependent changes in MMP following ischemia is vital for neuroprotection strategies.
Purpose of the Study:
- To investigate the time-dependent profile of MMP changes in rat hippocampal neurons after varying durations of OGD.
- To correlate MMP alterations with subsequent cell viability and apoptosis.
- To elucidate the role of MMP hyperpolarization and depolarization in neuronal injury.
Main Methods:
- Primary rat hippocampal cultures were subjected to OGD for 30, 60, or 90 minutes, followed by reoxygenation.
- MMP was measured using JC-1 fluorescence, a voltage-dependent dye, analyzed via confocal microscopy.
- Cell viability was assessed using calcein AM and ethidium homodimer staining.
- Apoptosis was evaluated using the TUNEL assay.
Main Results:
- Short OGD (30 min) induced MMP hyperpolarization during reoxygenation, with neurons remaining viable initially but showing later signs of apoptosis.
- Prolonged OGD (60 and 90 min) led to MMP depolarization and significant neuronal shrinkage.
- Necrosis was observed after longer OGD periods, while neurons generally remained viable at 3 hours post-OGD, irrespective of duration.
Conclusions:
- Neuronal MMP exhibits distinct responses to varying OGD durations, with hyperpolarization after short OGD and depolarization after prolonged OGD.
- MMP hyperpolarization, while not immediately detrimental to cell viability, is associated with subsequent apoptotic cell death.
- The study highlights the critical role of MMP dynamics in determining neuronal fate after ischemic insults, differentiating between apoptotic and necrotic pathways.
