| Objective:Sevoflurane is commonly used in pediatric anesthesia. Whether early sevoflurane exposure could inhibit on brain development has become the focus of the clinic. Repetitive transcranial magnetic stimulation(r TMS), as a noninvasive technique for modulating neuronal activity, has been introduced into clinical application recently. The aim of this study is to investigate the early brain development, how early sevoflurane exposure inhibits brain development from the view of L-type Ca2+ channel activity, whether and how r TMS regulates L-type Ca2+ channel activity. This study provides the support for the clinical applications of neonatal sevoflurane exposure and r TMS. Methods:Our experiment focus on the early development of L L-type Ca2+ channel activity, the effect of early sevoflurane exposure on the L-type Ca2+ channel and r TMS regulates L-type Ca2+ channel activity.1. Animals and groupsAll SD rats were randomly divided into three groups: con group(con 1w, con 2w, con 3w, con 4w, con 5w), sevo group(sevo 1w, sevo 2w, sevo 3w, sevo 4w, sevo 5w) and r TMS group(r TMS 2w, r TMS 3w, r TMS 4w, r TMS 5w).2. Sevoflurane exposure7 days SD rats were placed in a plexiglass box for 6 h, in which the concentrate of sevoflurane is 3%. The rats in control group were treated with 100% oxygen for 6h.3. Low-frequency r TMS applicationAfter early sevoflurane exposure, the rats were treated with 1Hz low-frequency r TMS for 7 days, 400 stimuli every day.4. Preparation of CA1 pyramidal neurons of hippocampal slicesThe rats were decapitated under diethyl ether anesthesia, and the brain was rapidly removed and immersed in chilled(0-4°C) artificial cerebrospinal fluid(ACSF) for 1-2 min. Then the brain was sliced(400 um) in ice-cold ACSF using a vibrating tissue slicer. Slices were incubated in ACSF solution constantly oxygenated with 95%O2+5%CO2 at room temperature(25-26°C) and allowed to recovery at least 1h in a storage chamber.5. Data analysisThe whole-cell patch clamp technique was applied to record L-type Ca2+ channel currents in all groups, and the L-type Ca2+ channel property for all groups at 1w, 2w, 3w, 4w and 5w after birth were analyzed using Clampfit 10. One-way analysis of variance was applied to examine the statistical significance. Result:1. L-type Ca2+ channel properties in the developing CA1 pyramidal neurons fromcontrol groups.The maximal L-type Ca2+ channel current density significantly increased between 2w and 3w groups(n=10, P<0.01), the L-type Ca2+ channel current density in con 2w and con 3w(p A/p F):-6.75±0.44,-12.58±0.71. The peak amplitude of maximal L-type Ca2+ channel current density showed no markedly difference between con 1w and con 2w, con 3w and con 4w, con 4w and con 5w(n=10, P>0.05).Compared to con 1w, the half-activation potential of L-type Ca2+ channel in con 2w decreased(n=10, P<0.05). Compared to con 2w, the half-activation potential of L-type Ca2+ channel in con 3w decreased(n=10, P<0.05). There was no significant difference in the half-activation potential of L-type Ca2+ channel between con 3w and con 4w,con 4w and con 5w(n=10, P>0.05)。There was no significant difference in k values between con 1w and con 2w,con 2w and con 3w,con 3w and con 4w,con 4w and con 5w(n=10, P>0.05).There was no significant difference in the half-inactivation potential and k values of L-type Ca2+ channel between con 1w and con 2w,con 2w and con 3w,con 3w and con 4w,con 4w and con 5w(n=10, P>0.05).2. Effects of early sevoflurane exposure on L-type Ca2+ channel activity in CA1 pyramidal neuronsCompared to the con 1w, L-type Ca2+ channel current density in sevo 1w decreased by 39.28%(n=10, P<0.01). Compared to the con 2w, L-type Ca2+ channel current density in sevo 2w decreased by 29.86%(n=10, P<0.05). Compared to the con 3w, L-type Ca2+ channel current density in sevo 3w decreased by 18.40%(n=10, P<0.05). No significant difference for L-type Ca2+ channel current density was observed between sevo 4w and con 4w, sevo 5w and con5 w groups(n=10, P>0.05).Compared to the con 1w, the half-activation potential of L-type Ca2+ channel in sevo 1w increased(n=10, P<0.01). Compared to the con 2w, the half-activation potential of L-type Ca2+ channel in sevo 2w increased(n=10, P<0.05). No significant difference was observed for half-activation potential of L-type Ca2+ channel between sevo 3w and con 3w,sevo 4w and con 4w,sevo 5w and con 5w(n=10, P>0.05). No significant difference was observed for k values between sevo 1w and con 1w,sevo 2w and con 2w, sevo 3w and con 3w,sevo 4w and con 4w,sevo 5w and con 5w(n=10, P>0.05).Compared to the con 1w, the half-inactivation potential of L-type Ca2+ channel in sevo 1w decreased(n=10, P<0.01). Compared to the con 2w, the half-inactivation potential of L-type Ca2+ channel in sevo 2w decreased(n=10, P<0.05). Compared to the con 3w, the half-inactivation potential of L-type Ca2+ channel in sevo 3w decreased(n=10, P<0.05). No significant difference was observed for half-inactivation potential of L-type Ca2+ channel between sevo 4w and con 4w,sevo 5w and con 5w(n=10, P>0.05. Compared to the con 1w, k value in sevo 1w decreased(n=10, P<0.05). No significant difference was observed for k values between sevo 2w and con 2w,sevo 3w and con 3w,sevo 4w and con 4w,sevo 5w and con 5w(n=10, P>0.05).3. r TMS improves the L-type Ca2+ channel activity in CA1 pyramidal neuronsThe peak L-type Ca2+ channel current density(p A/p F) in r TMS 2w, r TMS 3w:-6.40±0.64,-12.16±0.55. Compared to the sevo 2w, the L-type Ca2+ channel current density increased in r TMS 2w(n=10, P<0.05). Compared to the sevo 3w, the L-type Ca2+ channel current density increased in r TMS 3w(n=10, P<0.05). There is no significant difference for L-type Ca2+ channel current density between r TMS 4w and sevo 4w, r TMS 5w and sevo 5w(n=10, P>0.05).Compared to the sevo 2w, the half-activation potential of L-type Ca2+ channel decreased in r TMS 2w(n=10, P<0.05). There was no significant difference for half-activation potential between r TMS 3w and sevo 3w,r TMS 4w and sevo 4w,r TMS 5w and sevo 5w(n=10, P>0.05). There was no significant difference for k values between r TMS 2w and sevo 2w,r TMS 3w and sevo 3w,r TMS 4w and sevo 4w,r TMS 5w and sevo 5w(n=10, P>0.05).Compared to the sevo 2w, the half-inactivation potential of L-type Ca2+ channel increased in r TMS 2w(n=10, P<0.05). There was no significant difference for half-inactivation potential r TMS 3w and sevo 3w,r TMS 4w and sevo 4w,r TMS 5w and sevo 5w(n=10, P>0.05). There was no significant difference for k values between r TMS 2w and sevo 2w,r TMS 3w and sevo 3w,r TMS 4w and sevo 4w,r TMS 5w and sevo 5w(n=10, P>0.05). Conclusion:1. The peak amplitude of L-type Ca2+ channel currents significantly increased with the growth of rats, especially between 2w and 3w, suggesting that the period of 2w and 3w were critical for the development of L-type Ca2+ channel.2. Early sevoflurane exposure inhibited L-type Ca2+ channel activity in CA1 pyramidal neurons of hippocampus. The sevoflurane significantly inhibited L-type Ca2+ channel activity at 1w, 2w and 3w.3. r TMS increased the L-type Ca2+ channel currents, made the steady-state activation and inactivation curves to the normal level, suggested that r TMS regulated L-type Ca2+ channel activity. |
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