A novel method for oxygen glucose deprivation model in organotypic spinal cord slices

作者:Liu, Jing-Jie; Ding, Xiao-Yan; Xiang, Li; Zhao, Feng; Huang, Sheng-Li*
来源:Brain Research Bulletin, 2017, 135: 163-169.
DOI:10.1016/j.brainresbull.2017.10.010

摘要

This study aimed to establish a model to closely mimic spinal cord hypoxic-ischemic injury with high production and high reproducibility. Fourteen-day cultured organotypic spinal cord slices were divided into 4 groups: control (Ctrl), oxygen glucose deprived for 30 min (OGD 30 min), OGD 60 min, and OGD 120 min. The Ctrl slices were incubated with 1 ml propidium iodide (PI) solution (5 mu g/ml) for 30 min. The OGD groups were incubated with 1 ml glucose-free DMEM/F12 medium and 5 mu l PI solution (1 mg/ml) for 30 min, 60 min and 120 min, respectively. Positive control slice was fixed by 4% paraformaldehyde for 20 min. The culture medium in each group was then collected and the Lactate Dehydrogenase (LDH) level in the medium was tested using Multi-Analyte ELISArray kits. Structure and refraction of the spinal cord slices were observed by light microscope. Fluorescence intensity of PI was examined by fluorescence microscopy and was tested by IPP Software. Morphology of astrocytes was observed by immunofluorescence histochemistry. Caspase 3 and caspase 3 active in different groups were tested by Western blot. In the OGD groups, the refraction of spinal cord slices decreased and the structure was unclear. The changes of refraction and strticture in the OGD 120 min group were similar to that in the positive control slice. Astrocyte morphology changed significantly. With the increase of OGD time, processes became thick and twisted, and nuclear condensations became more apparent. Obvious changes in morphology were observed in the OGD 60 min group, and normal morphology disappeared in the OGD 120 min group. Fluorescence intensity of PI increased along with the extension of OGD time. The difference was significant between 30 min and 60 min, but not significant between 60 min and 120 min. The intensity at OGD 120 min was close to that in the positive control. Compare with the Ctrl group, the OGD groups had significantly higher LDH levels and caspase 3 active/caspase 3 ratios. The values increased with the extension of OGD time and reached peak at 120 min. The increase was significant between 30 min and 60 min, but not significant between 60 min and 120 min. Organotypic spinal cord slices cultured in glucose-free medium and anaerobic incubator could mimic hypoxia-ischemia of the spinal cord perfectly; 60 min could be the best duration for OGD. This technique might be a simple and efficient method to obtain in vitro model for spinal cord hypoxic-ischemic injury in sufficient number and with high quality.