| INTRODUCTION: Minocycline is a second-generation semisynthetic tetracycline antibiotics, and possesses anti-inflammatory effects that are completely distinct from its antimicrobial action. Minocycline can be rapidly and completely absorbed, and has an excellent tissue penetration into the brain and cerebrospinal fluid by passing through the blood-brain barrier. Recent studies have shown that minocycline has remarkable neuroprotective effects in animal models of cerebral ischaemia, traumatic brain injury, Huntington's and Parkinson's disease. Minocycline is able to provide neuroprotection against global ischemia in gerbils and focal brain ischemia in rats with a wide therapeutic window. In previous studies, several neuroprotection mechanisms of minocycline have been reported, such as inhibition of microglial activation. However, there are so many pathways being activated during brain ischemia, and it is unclear by what minocycline shows its neuroprotection. On the other hand, glutamate is the primary endogenous excitatory amino acid in brain, and plays an important role in brain injuries via its receptors, including NMDA and AMPA recetors. Released glutamate produces excitotoxicity and induces injury as one of pathologic processes in cerebral ischaemia, and NMDA receptor is involved in the excitotoxicity in early ischemic injuries of neurons by inducing proliferation and activation of microglial cells, and production of iNOS and ICE. Microglial activation and excitotoxic neuronal death can be inhibited by minocycline, which may be one mechanism of the neuroprotection of minocycline.However, whether minocycline directly or indirectly inhibits NMDA receptor-mediated excitoxicity in the ischemic injury is still unclear.OBJECTIVES:(1) We wanted to establish an in vitro ischemic model induced by oxygen glucose deprivation (OGD) in rat primary neurons.(2) We further determined the property of protective effect of minocycline on OGD- and NMDA-induced injuries in rat primary neurons and hippocampal slices. Our purpose was to clarify the relationship between minocycline effect and NMDA receptor activation.(3) To explore the therapeutic window, we observed the effect of minocycline given before or after different time of OGD treatment.METHODS:(1) Induction of OGD injury. Primary cortical neurons were prepared from brains of newborn rats; the neurons at 10-12 days in vitro were used. The neurons were exposed to OGD in a glucose-free balanced salt buffer using the hypoxia chamber (temperature: 37癈, atmosphere: 95% N2 and 5% CO2) to induced injury. The injuries of neurons were evaluated by morphological changes of neurons and neuron viability (MTT assay). The positive control drugs, nerve growth factor (NGF) and edaravone, were used to evaluate the effectiveness of OGD model.(2) Observation of the effect of minocycline on OGD- and NMDA-induced injuries. Minocycline was added to the culture media to observe the effect on neuron morphological changes and decreased viability induced by OGD or NMDA. MK-801, an NMDA receptor antagonist, was used as a control. Minocycline was also added at different time points before and after OGD to observe its therapeutic windows.(3) Observation of the effect of minocycline on light transmittance change induced by OGD or NMDA in rat hippocampal slices. Rat hippocampal sliced were treated by OGD or NMDA for 10 min and their light transmittance (LT) was measured in the absence or presence of minocycline or MK-801.Data were expressed as X s. The differences between treatments were analyzed byone-way ANOVA using SPSS for Window 10.0.RESULTS:(1) Induction of OGD injury. Forty-five min after OGD, the pO2 in the medium reduced from (-152 5) mmHg (control) to (29 1) mmHg (n = 5,P< 0.01), and 5 h after OGD treatment from (161 4) mmHg (control) to (28 ?2) mmHg (n = 5,P< 0.01). Glucose concentration was below measurable limit after OGD, and was (1.63 ?0.27) g/L in the control (n = 5). The experiment conditions met OGD necessity. When OGD du...
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