Changes Of ERK1/2 And PKC Activities In The Brain And Their Possible Roles In General Anesthesia

General anesthetics have been applied to clinical practice for more than 160 years. However, it is still not fully understood how these general anesthetics induce such complex clinical effects as unconsciousness, amnesia, immobility, etc. which areas and what molecules they are targeted at in the central nervous system. For a long time, scientists have been making unremitting efforts to unveil these mysteries at different levels including integral individual, cell, molecule, etc. Though some reasons are still unknown to us, it is certain that general anesthetics cause the effects of unconsciousness, amnesia and immobility by activating the central nervous system (CNS).Intracellular signal transduction molecules distribute extensively and regulate many physical functions of cells. Extracellular signal regulated kinase (ERK1/2) is an important intracellular signal regulated molecule, which is highly expressed in the nervous system and quickly gets phosphorylated when stimulated. It has previously been found that isoflurane or sevoflurane could potentialize or attenuate the phosphorylation of ERK1/2 in some areas or nuclei in the mouse brain. Besides, the alteration was very consistent with the anesthesia-emergence process. These findings imply that the phosphorylation of ERK1/2 may closely relate to the mechanism of general anesthesia. For the first part of the study, based on our early research into pERK1/2 changes induced by inhalational anesthetics, we further observed the influence of propofol and ketamine anesthesia on pERK1/2 so as to find the similarities and differences between inhalational anesthetics and intravenous anesthetics, and detected the targets of general anesthetics in the central nervous system. In part two, we investigated whether EW and central amygdaloid nucleus with significant pERK1/2 changes induced by three general anesthetics had an effect on general anesthesia by immunofluorescence and brain nuclei lesion. Protein kinase C (PKC) is another intracellular signal regulated kinase, which regulates many important physical functions in cells. Many researches in vitro have found that PKC may be a potential target molecule of general anesthesia. As a result, in the third part, to clarify the action of PKC in general anesthesia, we studied the effects of chelerythrine-an inhibitor of protein kinase C on sevoflurane anesthesia in mice. SynapsinⅠis a synaptic vesicle related protein, many of whose sites are phosphorylated by protein kinases. It can modulate the release of neurotransmitter through phosphorylation and dephosphorylation. Likewise, do general anesthetics affect the release of neurotransmitter by regulating the phosphorylation of synapsinⅠ? Correspondingly, in part four, we observed the influence of sevoflurane anesthesia on phosphorylated synapsinⅠin the sensation /motor cortex of the mouse brain for the purpose of exploring the effects of general anesthesia on synaptic transmission.Part 1 Effects of intravenous anesthetics on the alteration of pERK1/2 expression in the mouse brain 1. Comparison of pERK1/2 expression changes in the mouse brain under the anesthesia of propofol, ketamine and sevofluraneSixty adult male BALB/c mice were randomly divided into groups: the normal control and the anesthesia groups undergoing propofol, ketamine and sevoflurane, respectively. Mice in the control group were sacrificed by cervical dislocation after 5min oxygen uptake. Mice in the anesthesia groups received propofol or ketamine 100mg/kg ip or sevoflurane 1 MAC for 30 min, and then were sacrificed by cervical dislocation at anesthesia for 5 min, emergence from anesthesia for 5min or 1 h. Brains were taken out for the determination of phosphorylated ERK1/2 expression by immunohistochemical staining.As was shown, the areas where pERK1/2 positive cells changed in the process of propofol, ketamine and sevoflurane anesthesia were almost the same. The areas of pERK1/2 depression were mainly involved in hippocampus and cerebral cortex, while the areas of pERK1/2 activation were mainly some subcortical nuclei. The number of pERK1/2-immunoreactive cells in such areas as sensorimotor cortex, cingulate cortex, piriform cortex, perirhinal cortex and hippocampus significantly decreased during the anesthesia of propofol, ketamine and sevoflurane (p<0.05), while in some subcortical nuclei including central amygdaloid nucleus, lateral parabrachial nucleus, hypothalamic paraventricular nucleus, medullary ventrolateral reticular nucleus, supraoptic nucleus and Edinger–Westphal nucleus (EW), the positive cells markedly increased during anesthesia, compared with the control group (p<0.01). Except hippocampus and hypothalamic paraventricular nucleus in propofol and ketamine anesthesia, the expression of pERK1/2 in other brain areas/nuclei restored to the level of the control group 1h after the animals emerged from the anesthesia (p>0.05).Moreover, the subcortical nuclei displaying pERK1/2 expression changes induced by these anesthetics were partly different. In lateral habenular nucleus, pERK1/2 positive cells increased markedly in the propofol group. In thalamic paraventricular nucleus, there were more pERK1/2 positive cells under ketamine anesthesia. Importantly, pERK1/2 positive cells in ventrolateral portion of ventromedial hypothalamic nucleus increased under propofol and ketamine anesthesia, but stayed unchanged in the sevoflurane group.2. Relationship between pERK1/2 and GABAergic neuronsThe wholly-sectioned mouse brains from the control and 5 min propofol anesthesia were prepared for double labeling of pERK1/2 and GAD67. Three mice were randomly selected from each group (only a set of brain sections per mouse).In consequence, few colocalized cells of pERK1/2 and GAD67 were found in the mouse brain.Part 2 Influence of EW or central amygadala nucleus on general anesthesia1. Relationship between pERK1/2 and urocortin neuronsThe midbrain sections (40μm thick) from the mice in 5min propofol, ketamine and sevoflurane anesthesia (3 mice each group) were taken out for double labeling of pERK1/2 and urocortin.The brain sections showed there were a lot of pERK1/2 and urocortin colocolized neurons in EW nuclei, which implied that the neurons of urocortin may participate in the process of general anesthesia.2. Effects of leisioned EW or central amygdaloid nucleus on general anesthesiaSprague-Dawley male rats weighing 280-300g were leisioned at EW and central amygdaloid nucleus by electricity. 5 days after the operation, these rats were anesthetized by propofol, ketamine and sevoflurane, with the time of loss of righting reflex (LORR) and the time of recovery of righting reflex recorded. Then, the rats were sacrificed, perfused and fixed. Brains were taken out for hematoxylin– eosin (HE) staining. The leision groups and the failure groups were formed according to the result of HE staining. At the same time, twelve normal rats were anesthetized as a blank.Our studies demonstrated that when the central amygdaloid nucleus were leisioned, the time of loss of righting reflex of propofol and ketamine anesthesia groups showed no significant differences compared with that of the control group (P>0.05). The time of recovery of righting reflex of the propofol anesthesia group was shorter than that of the control group but no statistical significance was found between the ketamine group and the control group. The time of loss of righting reflex and recovery of righting reflex displayed no significant changes in the sevoflurane anesthesia group.In EW-leisioned rats, compared with that of the control group, the time of loss of righting reflex was markedly prolonged in the propofol group (P<0.01) but the ketamine group revealed no significant differences. The time of recovery of righting reflex was shorter in the ketamine group while showed no significant changes in the propofol group. There existed no statistical significance of the time of loss of righting reflex and recovery of righting reflex between the sevoflurane group and the control group.Part 3 Influence of protein kinase C on sevoflurane anesthesia75 male BALB/c mice were randomly divided into five groups: the control group and four chelerythrine (CHE) groups (15 mice each group). Animals in the control group were intraperitoneally injected with 5% DMSO25ml/kg and those in the four CHE groups were administered chelerythrine 2.5, 5, 10 and 20mg/kg intraperitoneal injection. 45 minutes later, three animals were chosen from each group for western blotting analysis of pPKCγin cerebral cortices and hippocampi. Time of loss of righting reflex (LORR) and emergence of clamp-tail reflex under sevoflurane anesthesia was recorded for the rest.Consequently, as was shown by Western blotting, the expression of pPKCγafter chelerythrine ip in the mouse sensorimotor cortices was weakened compared with that in the control group. The time of LORR was shorter and the time of recovery of righting reflex was longer in the sevoflurane group after chelerythrine 5, 10 and 20mg/kg ip (p<0.01).Part 4 Influence of sevoflurane anesthesia on the phosphorylation of synapsinⅠ20 male BALB/c mice were randomly divided into the control group and the 30 min sevoflurane anesthesia group (10 mice each group). Mice were anethetized by 1 MAC sevoflurane for 30 min. Then, mice were sacrificed by cervical dislocation for the analysis of phosphorylated synapsin I (pSYNⅠ) by western blotting or immunohistochemistry.Immunohistochemistry showed the expression of pSYNⅠin the sevoflurane anesthesia group decreased in sensorimotor cortex, compared with that in the control group (P<0.01), but the pSYNⅠO.D/ Actin O.D had no significance (P>0.05).Conclusion:1. The areas where pERK1/2 positive cells changed were almost the same in the process of propofol, ketamine and sevoflurane anesthesia. Our studies proved pERK1/2 depression mainly arose in hippocampus and cerebral cortices, such as sensorimotor cortex, cingulate cortex, piriform cortex and perirhinal cortex. These areas may relate to the loss of sensation or cognition induced by anesthesia, while pERK1/2 activation largely took place in the subcortical nuclei including central amygdaloid nucleus, hypothalamic paraventricular nucleus, supraoptic nucleus, Edinger–Westphal nucleus, etc. However, the parts of pERK1/2 activation in these subcortical nuclei were not completely the same. It is worthwile to make a further research how these subcortical nuclei act in the process of general anesthesia.2. Few colocalized pERK1/2 and GAD67 positive cells have been found on the brain sections of the control and propofol anesthesia. So we are justified in believing the neurons of pERK1/2 activation or depression under general anesthesia are not GABAergic neurons.3. A lot of colocalized pERK1/2 and urocortin positive neurons have been spotted in EW nucleus of the mouse midbrain under propofol, ketamine and sevoflurane anesthesia. It proves that pERK1/2 activation in EW nucleus relates to stress regulation under general anesthesia.4. When the central amygdaloid nucleus or EW nucleus were leisioned in SD rats, the time of loss of righting reflex or recorvey of righting reflex changed little, which manifests that the two nuclei do not play crucial roles in the mechanism of unconsciousness induced by general anesthetics.5. When the mice were injected with chelerythrine intraperitoneal, the time of loss of right reflex was shortened, while the time of recovery of righting reflex was prolonged under sevoflurane anesthesia. So we conclude that the effects of general anethesia can be enhanced by inhibiting PKC activities.6. Compared with the control group, the expression of phosphorylated synapsin I (pSYNⅠ) decreased in the sensorimotor cortex under sevoflurane anesthesia. The finding suggests it may be a mode of general anesthesia that the release of neurotransmitter was decreased by means of the attenuation of phosphorylated synapsin I.