| ObjectiveBrain injury could be seen frequently in forensic and clinic practice. The molecular mechanisms of secondary neuron injury due to brain injury are still largely unknown, so it is very important to set up suitable experiment models. For traumatic brain injury in vivo models, the injury area and degree are hardly controlled, even the mechanical force is same. The cultured neurons are even and can be well controlled in injury area and degree. The neuron samples are very convenient in observation and detection after injury, especially in studies for brain injury mechanisms.We successfully established the cultured cortical neurons injury model in vitro by oxygen-glucose deprivation. To illuminate the function of calpainl on secondary neuron injury after brain injury, the expression of calpainl and its substrate map2 was studied by immunofluorescence and western blot techniques using the OGD model.Materials and MethodCortical neurons were derived from P1 to 2 Wistar rats. The isolated cortices were minced and digested with 0.25%trypsin for 3min at 37℃. Enzymatic digestion was terminated by mixing the suspension with DMEM/F12 supplemented with 10%horse serum. Cells were then centrifuged at 1500rpm for 7min. The cells were resuspended in DMEM/F12 with 15%horse serum and 1%B27 and then passed through nylon mesh. The cells were plated onto poly-L-lysine-coated 6-well plates (for immunofluorescence staining, cells were plated on poly-L-lysine-coated glass coverslips on the bottom of 6-well plates) at a density of 2×106cells/well, and incubated at 37℃in a humidified incubator with 5%CO2 and 95%air. To prevent proliferation of nonneuronal cells, 10μmol/L cytosine arabinoside was added at 48h after plating.Experiments were performed at the 7th day after initial plating. The neurons were divided into five groups randomly, one control group and four injury groups. For injury groups, the neurons were washed once with low glucose DMEM and then exposed to oxygen-glucose deprivation DMEM containing low glucose and 0.5mmol/L sodium dithionite for 30min. After OGD, the cultures were replaced with standard medium and reincubated for 1h,6h,12h and 24h, respectively. For control group, the medium was changed normally. The level of calpainl and its substrate map2 in neurons was detected by double immunofluorescence staining and Western blot.ResultsWe successfully established the cultured cortical neurons injury model in vitro by oxygen-glucose deprivation, the neurons had rounded or elliptical cell bodies with long intact dendrites in normal groups. The neurons remained round shape with short dendrites at 1h post-OGD, while wrinkled with little dendrites at 6h and 12h post-OGD. The neurons partly recovered and some dendrites appeared again at 24h post-OGD. The western blot of calpainl and map2 showed molecular sizes of 75kDa and 70kDa, respectively. After OGD, the expression of calpainl and map2 revealed a time-dependent changes. The calpainl protein level increased at 1h post-OGD, and reached a peak at 6h and 12h. At 24h post-OGD, calpainl still kept at a high level, compared with control group. The map2 protein level decreased slightly at 1h post-OGD and continually decreased at 6h and 12h, the level increased at 24h post-OGD, compared with 6h and 12h groups, but lower than control group.Conclusions1. We successfully established the cultured cortical neurons injury model by oxygen-glucose deprivation.2. The expression of calpainl and its substrate map2 after OGD injury revealed a time-dependent changes.3. The time-dependent changes of calpainl and map2 may be used as markers for the age determination of brain injury.
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