Abstract
Optimum time points for oxygen-glucose deprivation (OGD) and re-oxygenation have been identified to suggest the suitability of PC-12 cells as rapid and sensitive in vitro model of cerebral stroke. Further, the precise role of glucose as one of the limiting factors was ascertained. PC-12 cells were subjected to receive OGD of 1–8 h followed by re-oxygenation for 6 to 96 h in medium having glucose 0–10 mg/ml. Loss of cell viability was assessed using trypan blue dye exclusion and MTT assays. The significant (p < 0.05) reduction in percent viable cell count was started at 2 h of OGD (80.7 ± 2.0) and continued in further OGD periods (3, 4, 5, 6, 7, and 8 h), i.e. 65.7 ± 3.5, 59.7 ± 4.6, 54.3 ± 3.2, 44.7 ± 2.9, 20.3 ± 4.3, 5.7 ± 2.0 of counted cells, respectively. Cells growing in glucose-free medium have shown a gradual (p < 0.001) decrease in cell viability throughout the re-oxygenation. Re-oxygenation of 24 h was found to be first statistically significant time point for all the glucose concentrations. Glucose concentration during re-oxygenation was found to be one of the key factors involved in the growth and proliferation in PC-12 cells. The OGD of 6 h followed by a re-oxygenation period of 24 h with 4–6 mg/ml glucose concentration could be recorded as optimum conditions under our experimental conditions.
The authors are grateful to Director, Industrial Toxicology Research Centre, Lucknow, for his keen interest in the present work. This is a part of the project work financially supported by Indian Council of Medical Research, New Delhi. The technical laboratory assistance by Mr Rajesh Mishra is also acknowledged.