배양조건에 의한 일과성 저산소상태 후 신경세포회복의 차이

초록

Objective: The transverse hippocampal slice is one of the most commonly studied in vitro models of mammalian brain physiology. However, despite its broad usage, there has been no standardization of slice preparation techniques or recording conditions. It is well known that variations in recording conditions can result in profoundly different effects on neuronal responses. Evoked field potentials, recorded extracellularly, were used to investigate the effects of variations in hippocampal slice preparation protocols on hypoxia responses of CA1 neurons. Material & Methods: Before hypoxic injury, hippocampal slices were incubated for 4 hours. During the incubation period, the slices were placed in an incubation chamber (21℃) for recovery from preparation injury and then transferred to a recording chamber (34℃) for further recovery and baseline electric recording with current stimulation (0.1 Hz). Various time periods in the incubation chamber and recording chamber were applied to each experimental group (group 1 = 60 min:180 min, group 2 = 90 min:150 min, group 3 = 180 min:60 min, time in incubation chamber:time in recording chamber) before 10 minutes of hypoxia produced by replacing 95% O₂ + 5% CO₂ mixed gas with 95% N₂ + 5% CO₂ gas. Calcium and magnesium ions and several drugs affecting glutamate receptors were also studied. Recoveries from hypoxic injury of hippocampal slices were estimated by percent recovery of population spike (PS). Statistical analyses were performed using paired t-test. Results: The percent recovery of PS after 10 minutes of hypoxia was considerably enhanced by increasing the period of current stimulation during the incubation period before hypoxic injury. Temperature effects were also studied (group 4), but no statistically significant differences were observed. Low magnesium ion concentration of artificial CSF (Mg-free aCSF) during incubation enhanced the recovery of PS, whereas low calcium (calcium-free) and high magnesium ion concentration (2 mM) reduced the recovery after hypoxic injury. L-glutamate (100 μM) and AP-5 (50 μM) had no effect on the recovery of PS, but CNQX (10 μM) in artificial CSF during incubation markedly enhanced PS recovery. Co-treatment with AP-5 (50 μM), CNQX (10 μM), and high magnesium concentration (2 mM) enhanced PS recovery immediately after hypoxic injury, but the effect decayed rapidly thereafter and lost statistical significance. Conclusions: Judging from the above results, the conditions of baseline recording are important in observing the recovery of the population spike after hypoxia, and the time and conditions should be controlled more strictly to obtain reliable results.

키워드