| General anesthetics have been applied into clinical practice for more than 160 years. However, until now, it is still not fully understood how these general anesthetics induce their complex clinical effects including unconsciousness, amnesia, immobility and which areas and what molecules they are targeted at in the central nervous system. This situation leads to a limitation for the security and controllability of the clinical application of general anesthesia and the development of novel drugs.It is well known that general anesthetics induce the effect of unconsciousness, amnesia, immobility, etc, by activating on the central nerve system (CNS). It is believed that the sedation and amnesia effect of inhaled anesthetics may be related to forecortex, parietal cortex and hippocampus, while immobility may be related to spinal cord and lower brain stem. Thus, for the mechanism of general anesthesia, especially for inhalation anesthesia, multiple-target-hypothesis becomes more favorable in contrast to one-target-theory such as lipid theory, ion channel theory, receptor theory and gene theory, etc. A large amount of receptors and molecular have been reported to be involved in the production of general anesthesia, but what role they play and how they interact among these molecules is still unclear. Intracellular signal transduction system, which can be actived quickly to transform the information from extramembrane to the intramembrane, even to nucleus, has drawn much attention as to the study on the mechanism of general anesthesia. In this study, the alternation of action of some signal transduction moleculars under sevoflurane anesthesia was investigated to explore the central mechanism of inhalation anesthesia.ObjectiveAlternations of the expression of phosphorylated ERK1/2, CREB, PKCγduring sevoflurane anesthesia-emergence course were investigated to clarify the role of these signal transduction molecules in mechanism of general anesthesia and to provide basis for the targets of inhaled anesthetics.MethodsPart 11. Male BALB/c mice at 8 weeks of age were randomly divided into five groups: group Sevo-1 and Sevo-2, undergoing sevoflurane anesthesia for 5min and 1h respectively; group E-1 and E-2, receiving sevoflurane anesthesia for 1h and emerged from anesthesia for 2min or 1h, respectively; Animals in group Con, were left untreated. At the end of treatments, the brain of all animals were taken out and processed for determination of phosphorylated ERK1/2 expression by Western blotting and the immunohistochemical staining.2. Twelve Male BALB/c mice were randomly divided into two groups: control (Con) and Sevo-1h which was exposured under sevoflurane anesthesia for 1h. At the end of treatments, artery blood samples were collected through left ventricular puncture following opening of the thoracic cavity for the blood gas analysis and assays of electrolytes.3. Eighteen Male BALB/c mice were randomly divided into 3 groups: Con, DMSO and SL327. Animals in the latter two groups were given an intraperitoneal injection of 25%DMSO,6 ml·kg-1 and SL327,30ml·kg-1, respectively, 1h before sevoflurane anesthesia. All the mice were anesthetized with 4% sevoflurane and maintained for 30min under 1.0 MAC sevoflurane. After recording the time of loss of righting reflex (LORR) and emergence of clamp-tail reflex, the animal was decapitated and processed for determination of phosphorylated ERK1/2 expressions by Western blotting.Part 2BALB/c mice at 8 weeks of age were randomly divided into five groups: Con, Sevo-1 and Sevo-2, E-1 and E-2. The treatments were as same as that in part 1. At the end of treatments, the brain of all animals was taken out and processed for Western blotting for determination of phosphorylated CREB expressions.Part 31. Eight normal BALB/c mice were sacrificed by cervical dislocation and fixed with perfusion. The brains were sectioned and immunohistochemically stained for pPKCγ.2. Forty-eight Male BALB/c mice at 8 weeks of age were randomly divided into four groups: group Sevo-1 and Sevo-2, receiving sevoflurane anesthesia for 5 min and 1h, respectively; E-1h, sevoflurane anesthesia for 1h and emergence for 1h; Con, normal animals without being treated. At the end of treatments, the brain of all animals were taken out and processed for Western blotting and the immunohistochemical staining for determination of phosphorylated PKCγexpressions.ResultsPart 11. As shown by Western blotting, the pERK expressions in the mice sensory cortex and hippcampus were decreased after sevoflurane inhalation and restored to the level of control after emergence from anesthesia (E-1, E-2); whereas the expression of pERK in brainstem was increased after sevoflurane inhalation and gradually restored but not back to the level of control after stopping of inhalation. For the immunohistochemistry staining, except LPBD, SO and PVN, the numbers of pERK1/2-like immunoreactive (pERK1/2-ir) neurons in lateral reticular nucleus (LRt), solitary tract nucleus (Sol), Edinger-Westphal nucleus (EW) and dorsal part of lateral parabrachial nucleus (LPBD), supraoptic nucleus (SO), paraventricular thalamic nucleus (PVN), central amygdaloid nucleus (Ce), bed nucleus of the stria terminalis (BST) were significantly increased during sevoflurane anesthesia and then restored to the level of control after emergence compared to the control group in which fewer pERK1/2-ir cells were observed in these nuclei; whereas the numbers of pERK1/2-ir neurons in ventrolateral periaqueductal gray (VLPAG), paraventricular thalamic nucleus (PV), arcuate hypothalamic nucleus (Arc), hippocampus (Hip), prelimbic cortex (PreL), cingulate cortex (Cg), motor cortex (M) and sensory cortex (S), where numerous pERK1/2-ir cells were seen in control group, were significantly decreased during sevoflurane anesthesia and restored to the level of control group again at 2min or 1h after stopping of sevoflurane inhalation.2. There is no statistic difference between control and Sevo-1h in each index of blood gas analysis and electrolytes examination.3. The time of LORR and the time of emergence of clamp-tail reflex were shorter than that in control after injection of SL327 but not DMSO. Western blotting showed that the pERK expressions in the mice sensory/motor cortex and hippcampus were significantly decreased in SL327 group compared to the control.Part 2There is no significant difference in the expression of pCREB in sensory cortex, hippcampus and brain stem among different groups.Part 31. Phosphorylated PKCγ-like immunoreactivities were widely distributed in discreted area in the mouse brain. The positive products were mainly located in neurons, both perikaria and processes. Strong immunoreactivity staining for pPKCγwas observed in the pyramidal neurons in CA1 of hippocampus, Purkinje cells in cerebellum, the neurons in layer V of neocortex, the deep layers of prelimbic and insular cortex, granular layer of prepiriform cortex, basolateral amygdaloid nucleus, lateral amygdaloid nucleus, as well as fornix and pyramidal tract. In contrast, neurons in most nuclei of rostral thalamus, medial and dorsolateral geniculate nuclei, superficial area of dorsal cochlear nucleus, nucleus of spinal trigeminal tract and gigantocellular reticular nucleus were weakly stained. Few glial cells were faintly stained.2. Results of Western blotting showed that the pPKCγexpression in the mice hippcampus was increased after sevoflurane inhalation and down to the level of control after emergence (E-1h). The numbers of pPKCγ-like immunoreactive neurons (pPKCγ-ir) were significantly increased during sevoflurane anesthesia in basolateral amygdaloid nucleus where fewer pPKCγ-ir cells were found in control group, but not down to the level of control after emergence. On the contrary, the positive staining of pPKCγwas significantly decreased during sevoflurane anesthesia in pyramidal tract and fornix where strong pPKCγ-ir staining was found in control group, but restored to the level of control after emergence.Conclusion1. A significant alternation of expression of pERK1/2 in some brain areas/nuclei in mice was observed during sevoflurane anesthesia and emergence process. As to our knowledge, the increase of the expression of pERK1/2 in certain nuclei, such as LPBD and PVN, may be involved in anesthetics stress; the decrease of the expression of pERK1/2 in other brain areas, such as motor cortex, sensory coxtex and hippocampus, may be involved in the loss of consciousness, sensorium and cognition. During anesthesia and emergence course, only the phosphorylation level of ERK, but not the expression of total ERK1/2, was changed, indicating that general anesthesia probably induces activation of ERK pathway but not the synthesis of protein de novo.2. The time of induction and emergence (as shown by LORR and clamp-tail reflex) under sevoflurane anesthesia was shorten after the pathway of MEK-ERK was blocked with intraperitoneal injection of SL327, indicating that ERK perhaps played different roles in different course of general anesthesia course. The precise mechanisms need to be further studied.3. There was no significant difference in expression of pCREB among different groups during anesthesia and emergence course, showing that CREB perhaps wasn't involved in the intracellular events of anesthesia, or, it has not be activated during the time observe.4. The expression of pPKCγin neural fiber tracts was decreased during sevoflurane anesthesia and emergence course, indicating that general anesthesia probably suppressed the action of pPKCγin axons. Further studies were needed to clarify whether general anesthesia affect the axonal transport or the conduction of firings on the fibers.5. The alternation of the expression of phosphorylated ERK1/2 during sevoflurane anesthesia was similar to that in isoflurane anesthesia in our previous studies, indicating that common mechanisms may be shared by different volatile anesthetics, such as activation of phosphorylated ERK1/2.
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