Neurocyte protective agent
A nerve cell protection, central nervous system technology, applied in nervous system diseases, material inspection products, cardiovascular system diseases, etc.
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Embodiment 1
[0277] Embodiment 1: OGD test
[0278] In this example, a model in which ischemia-like cell damage was induced by applying a load to rat primary cultured cerebral cortical neurons by oxygen-glucose deprivation was used. To study whether donepezil hydrochloride has neuronal protective effect on the above-mentioned ischemic injury.
[0279]In this example, the primary cultured nerve cells were obtained from the cerebral cortex of rat fetuses (17-19 days old viviparous). The culture medium used contained 10% fetal bovine serum (Gibco BRL), 10% horse serum (Gibco BRL), 5 μg / mL insulin (Sigma), 30 nM sodium selenite (Sigma), 100 μM putrescine in DMEM (Gibco BRL). (Sigma), 20 nM progesterone (Sigma), 15 nM biotin (Sigma), 100 units / mL penicillin (GibcoBRL), 100 μg / mL streptomycin (Gibco BRL), 8 mM glucose and 1 mM pyruvate (Sigma) (reference Scholtz et al., 1988), the cells were cultured at 37°C and 5% CO2. After culturing for 7 days or more, OGD treatment was performed. OGD tre...
Embodiment 2
[0288] Embodiment 2: excitotoxicity test
[0289] In this example, the cytoprotective effect of donepezil against NMDA toxicity was investigated. A model in which NMDA stimulation was applied to primary cultured rat cerebral cortex neurons to induce cell damage was used. 100 μM NMDA was added to the rat primary cultured neurons obtained as in Example 1, and the amount of LDH present in the culture solution was measured 9 hours later.
[0290] In this example, the primary cultured nerve cells were obtained from the cerebral cortex of rat fetuses (17-19 days old viviparous). As a culture solution, glucose (1 g / L), penicillin / streptomycin (100 units / mL), and 10% FCS were added to MEM (manufactured by Invitrogen). Media exchange was performed every 2-3 days. When expressing KA toxicity, a neurobasal medium (manufactured by Invitrogen) supplemented with B-27 and 0.25 mM glutamine was used as the medium. Cells at 37°C, 5% CO 2 cultivated in the environment. After culturing for...
Embodiment 3
[0292] Embodiment 3: excitotoxicity test
[0293] Kainic acid is known to enhance nerve cell death induced by β-amyloid (Aβ), which is one of the causative factors of Alzheimer's disease. In Examples, a model in which cell damage was induced by administering kainic acid to primary cultured rat cerebral cortical neurons was used.
[0294] In this example, the primary cultured nerve cells were obtained from the cerebral cortex of rat fetuses (17-19 days old viviparous). cells at 37°C, 5% CO 2 cultivated in the environment. After culturing for more than 7 days, add NMDA or kainic acid to the culture medium, at 37°C, 5% CO 2 Incubate overnight. Donepezil was added to the cells 24 hours before the application of kainic acid stimulation. Then, the amount of LDH present in the culture solution was measured 24 hours after the addition of kainic acid as an index of neuroprotective effect.
[0295] The results of this example are as Figure 6 shown. Depend on Figure 6 It was f...
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