| BackgroundDuring the past three decades, a line of data shows that inhalational anesthetics induced not only general anesthesia but also neuroprotection and neurotoxicity, which draws much attention to anesthetist. Why and how these drugs produced so complicated and conflicted effects is largely unknown. Many ion channels, receptors and neurotransmitters are indicated to be central targets of volatile anesthetics by studies in vivo and in vitro. But how to initiate and end the general anesthesia by these molecular is still not clear. As to the neurotoxicity of volatile anesthetics, most of the proofs come from animal studies and the mechanisms are limited to focusing on apoptosis.CaMK Ⅱ, an important protein kinase in the brain, is expressed most abundantly in neurons, and is involved in regulating many aspects of neuronal function, including neurotransmitter synthesis and release, modulation of ion channel activity, cellular transport, cellular morphology and neurite extension, long-term plasticity, learning, memory consolidation, etc. Many of these functions are related to general anesthesia. However, whether CaMKⅡ are involved in modulation on anesthesia is not clear. Recent studies showed that CaMK II played important roles in some neuronal pathology including cerebral ischemia injury, Alzheimer’s disease, Parkinson’s disease, etc, which lead to much attention payed on CaMKⅡ as new drug targets for neuroprotection or myocardiac protection.Our recent studies show that a reversible inhibition of CaMKⅡ phosphorylation occurs during inhalational anesthesia-recovery cycle. According to our results and data from previous reports, our hypothesis is:CaMK Ⅱ phosphorylation might be involved in the modulation on general anesthesia and neurotoxicity induced by volatile anesthetics. So this study is aimed to investigate the effect of CaMK Ⅱ phosphorylation on general anesthesia and neurotoxicity induced by isoflurane. The results will be benefit to clarify the machanism of general anesthesia and carry out clinical anesthesia practice in a more safe and reasonable way.Experiment Ⅰ:The effect of CaMKⅡ phosphorylation on the course of isoflurane inhalational anesthesiaObjective To explore the effect of CaMK Ⅱ phosphorylation on the course of isoflurane inhalational anesthesia. Methods SD rats were randomized to divided into Control group (vehicle), myrAIP group (CaMKⅡ inhibitor,1,5, lOuM) and CaM group (CaMKⅡagonist,1.25,5, lOug)(each group, n=10). Anesthesia was induced by isoflurane inhalation and maintained for30min with1MAC isoflurane40min after the drugs were administrated by i.c.v. The time of LORR disappearance and recovery was recorded. The expression of pCaMK Ⅱ in the brain was analyzed by western blot at30min after the induction of anesthesia.Results Compared to control group, the recovery time of LORR induced by isoflurane anesthesia becomes more longer by inhibitor of CaMK Ⅱ (P<0.05); however, both the disappearance and recovery time of LORR becomes more shorter by agonist of CaMK Ⅱ(P<0.05). A significant decrease on the level of CaMK Ⅱ phosphorylation in the brain occurred40min after administration of inhibitor of CaMK Ⅱ and30min after isoflurane anesthesia(P0.05).Conclusion A reversible alteration of CaMK Ⅱ phosphorylation occurred during isoflurane anesthesia-recovery cycle. Modulation on the CaMK Ⅱ phosphorylation in brain might change the anesthesia-recovery course.Experiment II:Isoflurane explosure induced neurotoxicity in primary culture cortical neuronsObjective To explore the neurotoxicity induced by explosure of isoflurane. Methods Cortical neurons from rat fetus at embryonic days16-18were separated and cultured in vitra for7-10days and then received explosure by vehicle and isoflurane(0.32,0.64,0.96mM) for6h respectively(n=10,each). The MTT and LDH were detected and the apoptosis neurons were detected with TUNEL staining. Results:Compared to control group, a significant decrease of the MTT and increase of LDH and apoptosis neurons were induced by isoflurane explosure at three doses(P<0.05) but without dose-response effect(P>0.05). Conclusion: Explosure of isoflurane induced neurotoxicity by increasing apoptosis.Experiment Ⅲ:Effect of CaMK Ⅱ phosphorylation on neurotoxicity induced by isoflurane explosure in primary culture cortical neuronsObjective To explore the effect of CaMK Ⅱ phosphorylation on the neurotoxicity induced by explosure of isoflurane. Methods Cortical neurons were separated and cultured in vitro for7-10days and then received explosure of isoflurane (0.64mM, for6h), inhibitor of CaMK Ⅱ (myrAIP;1,5,10uM) or agonist of CaMK Ⅱ (CaM;1,5,10ug) respectively (n=10,each). The MTT and LDH were detected and the apoptosis neurons were detected with TUNEL staining. Results Compared to control group, myrAIP caused more decrease of the MTT and increase of LDH and apoptosis neurons than that induced by isoflurane explosure, whereas, CaM partially reverse these effect with less decrease of the MTT and increase of LDH and apoptosis neurons than that induced by isoflurane explosure. Conclusion Inhibition of CaMK Ⅱ phosphorylation deteriorate the neurotoxicity induced by isoflurane explosure whereas activation of CaMK Ⅱ phosphorylation partially reverse the effect, indicating explosure of isoflurane induced neurotoxicity by modulation of CaMK Ⅱ phosphorylation on apoptosis.Summary1. Modulation on the level of CaMK Ⅱ phosphorylation in brain might change isoflurane inhalational anesthesia-recovery course, indicating CaMK Ⅱ phosphorylation might be part of mechanism of general anesthesia.2. Explosure of isoflurane produced neurotoxicity in primary cortical culture neurons.3. Inhibition of CaMK Ⅱ phosphorylation deterious neurotoxicity of isoflurane, whereas increase in CaMK Ⅱ phosphorylation relief the neurotoxicity as demonstrated by high apoptosis or low apoptosis respectively, which indicating isoflurane induced neurotoxicity by modulation of CaMK Ⅱ phosphorylation on apoptosis. |
PDF Full Text Request