| Research Abstract |
We have accomplished two projects supported by this grant. First, we examined the relationship between mitochondrial membrane potential after OGD (oxygen-glucose deprivation) and death modes, apoptosis and necrosis Second, we examined mitochondrial membrane potential during reoxygenation after oxygen-glucose deprivation. The first project suggested physiological protective mechanism of neuron from toxic effect of free radical during early reperfusion phase. Therefore, we moved to mitochondrial mechanism involved in active neuronal suicide, apoptosis because active neuronal death executed by neuron itself seems to be a main cause of neuronal damage after ischemia.
1.The relationship between mitochondrial membrane potential and death modes
We employed oxygen glucose depletion (OGD) in neuronal cell culture to reproduce ischemic condition and examined the relationship between MMP, consumption of high-energy phosphate and subsequent death mode during OGD.
MMP hyperpolarized (normalized JC-1 f
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luorescence 1.98 +/- 0.11, mean +/- SD, control=1.0) at 30 min reoxygenation following 30min OGD, while MMP turned to depolarize (normalized JC-1 fluorescence 0.58 +/- 0.10) at 30 min reoxygenation following 90min OGD. The result of MMP following 600GD varied (normalized JC-1 fluorescence 1.12 +/- 0.13). Most neurons were viable (76%) at 3 hrs following 30min OGD, while most neuron was dead (72%) at 3 hrs following 90 min OGD. Most neurons (66%) were TUNEL positive at 24 hours following 30 min OGD. Only a few neurons (12%) were TUNEL positive following 90 min OGD. Cytochrome c became diffuse in cytoplasma at 24 hrs of reoxygenation following 30 min OGD and 90 min OGD, and was already diffuse even at 3 hrs following 90 min OGD. The intracellular ATP content was 8.1±6.6% and 3.2±1.9% after 30 min OGD and 30 min reoxygenation following 30 min OGD, respectively ; 60 min OGD did not significantly change these levels (7.1±5.8%, 2.6±0.5%).
Mitochondria membrane potential during reoxygenation following OGD depends on the length of OGD. Long OGD results in depolarization, while shorter OGD induces hyperpolarization. Hyperpolarization after OGD did not accompany ATP production. This observation suggests the inhibition of electron reentry into an inner membrane during reoxygenation and the disturbance of FoF1-ATP synthase. Neurons are still viable during hyperpolarization, but this hyperpolarization seems to link the subsequent manifestation of apoptosis. The dissipation of mitochondrial membrane potential seems to be a consequence of severe energy deficit and results in necrosis. Hyperpolarization of mitochondria seems to reflect disturbance of ATP production, and may be a process to switch on apoptotic cascade.
2.The effect of propofol on consumption of ATP after OGD and subsequent neuronal death
Primary hippocampal cell culture was incubated with oxygen-glucose deprivation for 30 min (30OGD) or 90 min (90OGD). Propofol was added to the culture at a concentration of 0.1μM (Pro0.1) or 1.0μM (Pro1.0). ATP content was assayed using the luciferin-luciferase reaction. Neuronal viability and appearance of apoptosis was assessed. ATP content was decreased after OGD (0.276±0.115μM/μg (control), 0.172±0.125μM/μg (30OGD), and 0.096±0.092μM/μg (90OGD)). Propofol did not alter ATP content. MMP was hyperpolarized after 30OGD (1.26±0.23 (vehicle), 1.29±0.13 (Pro0.1), and 1.18±0.06 (Pro1.0)) but approached depolarization after 90OGD (0.77±0.04 (vehicle), 0.89±0.04 (Pro0.1), and 1.03±0.15 (Pro1.0)). Viability of cells decreased from 91.8±2.0% (vehicle), 88.6±4.6% (Pro0.1), and 84.5±4.1% (Pro1.0) in control conditions to 31.7±18.5% (vehicle), 43.6±25.0% (Pro0.1), and 56.9±20.2% (Pro1.0) after 90OGD. At 24 h after OGD, TUNEL-positive cells were increased to 34.5±6.2% (vehicle), 26.7±7.9% (Pro0.1), and 30.4±7.1% (Pro1.0) in the 30OGD group. No pharmacological effect of propofol on the incidence of apoptosis was found. Propofol inhibited acute neuronal death, but did not prevent hyperporalization and subsequent apoptosis. Propofol induces a moratorium on neuronal death, during which pharmacological intervention might be able to prevent cell death. Less
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