| Objective:Voltage-gated sodium channels are critical for brain development. To investigate the critical period of voltage-gated sodium channel (VGSC) development in hippocampal CA1neurons of rats, we examined the changes of sodium currents at0-4weeks after birth by an electrophysiology method using whole-cell patch-clamp technique. And then we treated1-week-old rats with3%soveflurane for6hours and studied the effects of soveflurane on sodium currents in hippocampal CA1neurons at1-4weeks of age, to explore the effects of soveflurane on the developing brain and provide supports for the clinic application of sevoflurane in pediatric anesthesia.Methods:The groups of laboratory animals:SD rats were divided into Control groups and Sevoflurane groups according to the ages after birth. Control groups included Ct0(new-born), Ct1(1-week’s old), Ct2(2-week’s old), Ct3(3-week’sold), Ct4(4-week’s old). Sevofiurane groups included Sp1(1-week’s old rats with3%sevoflurane for6hours), Sp2(2-week’s old,1week after sevoflurane exposed), Sp3(3-week’s old,2week after sevoflurane exposed), Sp4(3-week’s old,3week after sevoflurane exposed).1-week-old sevoflurane-exposed rats model preparation:postnatal1week rats were placed in a plexiglass box and exposed to3%sevoflurane6hours.Acute isolation of single pyramidal neurons in hippocampus CA1:rats were anesthetized with arther and decapitated; hippocampus was separated from brain and cut into slices (400-500μm thick) by hand; slices were incubated for1-1.5h at32℃and then digested with pronase XIV for30-40min at32℃; then slices were triturated to release single cells; cells were attached to the bottom of culture dish for30min at25℃.Data record and analysis:sodium currents in hippocampal CA1of five control groups and four sevoflurane groups were recorded using whole-cell patch-clamp technique; the characteristics of sodium channels (current density, steady-state activation, steady-state inactivation, recovery from inactivation) were analyzed using Clampfit10.2and Origin6.0software.Results:1. Changes of VGSC characteristics in hippocampal CA1of control rats at the age of0-4weeksAs rats of control groups grew older, the maximum sodium current density increased. The amplitudes of Ct1, Ct2, Ct3and Ct4compared with Ct0grew by (42.76±4.91)%,(146.80±7.63)%,(208.79±5.28)%and (253.72±5.74)%(n=10, P＜0.05) respectively. The sodium current density in hippocampal CA1between Ct1and Ct2increased more significantly than those of other control groups. The activation curve of sodium channels shifted to the hyperpolarization. The half-activation voltages (mV) of Ct1, Ct2and Ct3respectively were-39.06±0.65,-43.41±0.52,-48.29±0.45(n=10, P＜0.05) and there were no outstanding variation in other control groups. The slope factors of activation curve for each control group did not alter significantly. The inactivation curve and half-inactivation voltage also did not vary markedly. The slope factors of inactivation curve in Ct1and Ct2increased and the values were:5.77±0.56,4.42±0.43(n=10, P＜0.05). The inactivation rate of Ct2was faster than Ct1, and neither Ct0and Ct1groups nor Ct2, Ct3and Ct4groups have marked differences. The recovery from inactivation curve of sodium channels had a hyperpolarizing shift and the recovery time declined during Ct1, Ct2and Ct3.2. Changes of VGSC characteristics in hippocampal CA1of sevoflurane rats at the age of1-4weeksThe sodium current density curve of Sp1was inhibited significantly compared with Ct1and the maximum sodium current density of the two groups were125.58±11.83、63.55±2.18(n=10, P＜0.01), but there was no marked differences between control and sevoflurane groups at age of2-4weeks. Sp1compared with Ct1, the activation curve of sodium channels shifted to the depolarization, both half-activation voltages and slope factors increased. Sevoflurane groups compared with control groups at age of2-4weeks, the activation curve of sodium channels showed no outstanding variation. Sp1compared with Ct1, the inactivation curve of sodium channels shifted to the hyperpolarization, both half-inactivation voltages and slope factors increased. Sevoflurane groups compared with control groups at age of2-4weeks, the inactivation curve of sodium channels showed no outstanding variation. Sevoflurane groups compared with control groups at age of1-3weeks, the recovery from inactivation curve of sodium channel were inhibited and the inhibition declined with age. The recovery from inactivation curve of sodium channels of4-weeks-age between sevoflurane and control groups showed no significant differences.Conclusion:1.1-2weeks after birth is the critical development period. During the period, sodium channels in hippocampal CAl neurons develop faster than other weeks. The sodium current density increases significantly; the activation curve shifts to the hyperpolarization; inactivation rate increases as well as recovery time delays.2. After exposed1-week’s old SD rats to3%soveflurane for6hours, the characteristics of VGSC are inhibited significantly by soveflurane. The current density, activation and inactivation return to normal level at age of2-4weeks. Soveflurane inhibits the characteristics of recovery from inactivation from1-week’s old to3-week’s old, but the inhibition declines with age. At4-week’s old the characteristics of recovery from inactivation can be recovered normally. |
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