Neonatal Sevoflurane Exposure Inhibits Activity Of Ca²⁺ Channels For Hippocampal Pyramidal Neurons In Rats During Developing Period

Objective:Sevoflurane is a kind of inhalation anesthetics and has been widely used in pediatric and obstetric anesthesia. The widespread use of sevoflurane in infants has made its effects on brain development a hot issue for clinical anesthesia. Voltage-gated Ca2+channels (VGCCs) have a key function in neurons survival and regulating neuronal excitability, VGCCs are essential for brain development. The aim of this study is to explore the key point of development of VGCCs for hippocampal CA1pyramidal neurons in rats during the period of rapid brain development using whole-cell patch clamp technique. Furthermore, we investigated the effects of neonatal sevoflurane exposure on activity of VGCCs for hippocampal CA1pyramidal neurons in rats during the period of rapid brain development, exploring the inhibition of sevoflurane on VGCCs and different inhibition induced by different concentration of sevoflurane. That will provide support of experimental study for clinical application of sevoflurane.Methods:1. Experimental animal1-week-old Sprague-Dawley rats (n=45) were randomly assigned to3groups: control group,2.1%sevoflurane (Sevo1group and3%sevoflurane (Sevo2) group. According to the age of rats, every group was divided into5subgroups:week1group (1w), week2group (2w), week3group (3w), week4group (4w), week5group (5w).2. Anesthesia treatment1-week-old rats were placed into a home-made anesthetic induction chamber to receive gas exposure. The rats in control group breathed independently100%oxygen with a gas flow of4L/min for6h. Rats in2.1%sevoflurane group and3%sevoflurane group were respectively and continuously exposed to2.1%and3%sevoflurane for6h using oxygen as gas carrier with a gas flow of4L/min.3. Preparation of isolated hippocampal CA1pyramidal neuronsRat was decapitated, the brain was rapidly removed, the hippocampus was then separated out and manually cut into400-600um thick slices. The slices were placed into ACSF to incubate for1h at32℃. The CA1region of hippocampus was dissected out for enzymolysis for20-45minutes. The slices were dissociated into cell suspension for recording.4. Electrophysiological recording and data analysisCa2+currents of hippocampal CA1pyramidal neurons in every group was recorded by whole-cell patch clamp recording, processed by Clampfit10.3, Origin6.0and analyzed statistically by SPSS18.Results:1. Properties of VGCCs for hippocampal CA1pyramidal neurons in rats of control group during the period of rapid brain developmentThe maximal current density (pA/pF) of VGCCs for hippocampal CA1pyramidal neurons in rats of control group during1-5weeks were:Con1w,9.56±0.63; Con2w,10.84±0.24; Con3w,14.08±0.93; Con4w,15.00±0.84; Con5w,17.22±0.59。With the increases of rat age, the maximal current density of VGCCs increased gradually. The biggest growth occurred between Con2w and Con3w was (29.89±0.58)%(P<0.01).With the increment of rat age from1-week-old to5-week-old, VGCCs activated at more hyperpolarized voltages. There was a significant difference in the values of half-activation potential (V1/2) of VGCCs between Con1w and Con2w (P<0.05), but not significant difference between Con2w and Con3w, Con3w and Con4w, Con4w and Con5w (P>0.05).The VGCCs inactivated at more hyperpolarized voltages when rats growing older. No statistical difference was detected between Con1w and Con2w, Con2w and Con3w. as well as Con3w and Con4w, Con4w and Con5w in the values of half-inactivation potential (V1/2)(P>0.05).2. Properties of VGCCs for hippocampal CA1pyramidal neurons in rats of sevoflurane group during the period of rapid brain developmentCompared to control group, sevoflurane significantly decreased the current density of VGCCs. The maximal current density in Sevo11w, Sevo12w and Sevo21w, Sevo22w groups were below the level of Con1w group. At week1, the maximal current density was decreased by62%(P<0.01),28%(P<0.01) in Sevo2and Sevo1groups. At week2, the maximal current density was decreased by43%(P<0.01),22%(P<0.01) in Sevo2and Sevo1groups.The steady-state activation curve was significantly shifted to the depolarization direction in Sevo21w and Sevo22w groups. And the half-activation potential (V1/2)(P<0.05) and activation slope factor (k)(P<0.01) increased in Sevo21w and Sevo22w groups as compared with the control group. No difference could be found in Sevo23w, Sevo24w and Sevo25w groups (P>0.05). There was no statistical difference between Sevo1and Con groups (P>0.05).Sevoflurane shifted the steady-state inactivation curve to the depolarization direction and induced significant increase in the half-inactivation potential (V1/2) in Sevo21w, Sevo22w and Sevo23w groups (P<0.01). There was no difference in Sevo24w and Sevo25w groups (P>0.05). That significant changes induced by sevoflurane occurred in Sevo11w, Sevo12w (P<0.05) but not Sevo13w, Sevo14w and Sevo15w groups (P>0.05).Conclusion:1. From1w to5w, the current density of VGCCs increased gradually. There was a significant difference between2w and3w. That suggest that the critical period of development for VGCCs may occurs between2w and3w in normal rat.2. Sevoflurane persistently inhibits activity of VGCCs for hippocampal pyramidal neurons in rats during developing period. With the increment of rat age, that inhibition decreased gradually. VGCCs recovered to the level of normal development.3. The inhibition induced by3%sevoflurane is more than that of2.1%sevoflurane. Therefore, sevoflurane with diffenert concentration cause different inhibition on activity of VGCCs.