| 1. BackgroundThere were about1.5million infants and young children to undergo general anesthesia because of the need for various reasons such as surgery or interventional therapy in the world each year. For a long time, if general anesthesia would affect the infant’s mental development has been the focus of attention by family members. The conventional concept is that, as long as there was not exist cerebral hypoxia and other factors at the process of anesthesia, it will not affect the mental development of infants and young children. However, recent studies found that developing neurons can be induced to activate pathways resulting in cell death at the peak of the central nervous system growth and development, when their synaptic development, dendritic branching, and remodeling activities are interfered with. The period of rapid synaptogenesis appears to be the most vulnerable time. The intensity and duration of neurodevelopmental activity vary widely among different species. Neuronal cell death after general anesthesia has recently been demonstrated in neonatal animal models. The possibility of anesthesia-induced neurotoxicity during an uneventful anesthetic procedure in human neonates or infants has led to serious questions about the safety of pediatric anesthesia.Ketamine, a N-methyl-D-aspartate (NMDA) receptor antagonist, is a clinical only significant analgesic effects of intravenous anesthetics, and is the only NMDA receptor antagonist by U.S. FDA approved. It is widely used in surgical anesthesia of infants and young children now. The NMDA receptor is a kind of central nervous system excitatory amino acid ionotropic receptors, with many different allosteric regulatory sites and Ca2+high degree of permeability. In the central nervous system, NMDA receptors are mainly distributed in the cerebral cortex and hippocampus, particularly in the hippocampus. Studies have found that the NMDA receptor is closely related with the central nervous system development to the control of neuronal differentiation, migration and survival, but also learning and memory formation and maintenance play an important role in the process. Many studies have confirmed that it is great significance the NMDA receptor maintain the moderate excitement to the survival of neurons in the critical period of brain development. If the activity of NMDA receptors was changed, it would affect the central nervous system development and function. Blockade of N-methyl-D-aspartate glutamate receptors for only a few hours during late fetal or early neonatal life triggered widespread apoptotic neurodegeneration in the developing rat brain, suggesting that the excitatory neurotransmitter glutamate, acting at NMDA receptors, controls neuronal survival. The severity and distribution regional of apoptosis showed a correlation with age. The5-7d rats after birth was the most serious, and the21d in rats after birth did not show significant neuronal apoptosis, indicating that the neurotoxic effects was time-bound, and the peak period of the rat synapse formation overlap. Ikonomidou et al reported that the NMDA antagonist (MK801and ketamine) caused neurotoxicity in Science magazine. Subsequently, his research team and other researchers confirmed that many anesthetics, including nitrous oxide, isoflurane and ketamine appears to induce neuronal apoptosis, especially juvenile rodent brain apoptosis. However, the exact mechanism of ketamine induced neuronal apoptosis and neurological impairment is unclear. Previous studies have shown that injection of ketamine could cause the NMDA receptor upregulation in the rat hippocampus during the developmental stages, resulting in a wide range of dose-dependent apoptosis and affect long-term learning ability. In addition, As a noncompetitive antagonist of the NMDA receptor, ketamine can act on PCP binding sites of the NMDA receptor, blocking the NMDA receptor coupled calcium channels, reducing calcium influx, reducing the concentration of intracellular calcium, and which can antagonize the regulation of nervous system development by glutamate, aspartate and other excitatory amino acids. Andjus have confirmed that ketamine could reduce the intracellular calcium concentration in neurons. Ketamine reduced the concentration of intracellular calcium and inhibited the activation of mitogen-activated protein kinase, thus inhibiting the proliferation of neural stem cells.Traditional Chinese medical literature recorded that musk has the aromatic resuscitation, refreshing and sedative function. Modern research also showed musk ketone can reduced NMDA receptor expression and reduce the toxicity of excitatory amino acid damage in the model of focal cerebral ischemic injury. Another study found that musk ketone can significantly increase the intake of calcium of dementia in rats, increasing the availability of calcium in the cells of dementia mouse to play the role of anti-dementia. Based on the above studies, we speculate that musk ketone perhaps can reduced NMDA receptor upregulation caused by ketamine and increase intracellular calcium concentration in hippocampal neurons, thereby reducing apoptosis of hippocampal neuronal due to ketamine and improving neurological function. Therefore, we selected postnatal days SD rats to observe the effects of musk ketone on neonatal rat hippocampal neuronal apoptosis and the expression of NMDA receptor after the ketamine anesthesia, and the Morris water maze test to study the changes in their spatial learning ability. Subsequently, in vitro cell culture, we observed the impact of musk ketone of different doses on hippocampal neuronal apoptosis and morphology by ketamine treatment, and observed the intracellular calcium concentration changes by the confocal laser in order to identify the underlying mechanism. It may provide a theoretical basis for further clinical studies.2. MethodsFirst, we selected the postnatal7d SD rats which were divided into control group and ketamine group. The rats in ketamine group suffered intraperitoneal injection of20mg/kg ketamine interval of90min, for a total of five times. The rats in control group suffered in the volume of the corresponding time points intraperitoneal injection of normal saline. The rats were collected blood through left ventricular for blood gas analysis.Then,105rats were randomly divided into five groups (n=21). Control group: normal saline; ketamine group:intraperitoneal injection of ketamine20mg/kg total of5times90minutes apart; low dose muscone group:intraperitoneal injection of musk ketone0.5mg/kg, intraperitoneal injection of ketamine20mg/kg5interval of90minutes; dose of musk ketone group:intraperitoneal injection of musk ketone lmg/kg, intraperitoneal injection of ketamine20mg/kg,5times90minutes apart; high doses of musk ketone group:intraperitoneal injection of muscone2mg/kg intraperitoneal injection of ketamine20mg/kg,5times90minutes apart. From each group12rats were perfused for neural apoptosis detection and NMDA receptor immune staining;3to take the the protein westernblot detection of NMDA-2B. The other30rats were fed to28d for the Morris water maze test.In vitro cultured hippocampal neurons for5days were identified, and were randomly divided into blank control group:normal training, no treatment, but the medium was changed at the same time with the experimental group; ketamine group: cultured cells by adding medium diluted into1000μmol/l ketamine; low doses of musk ketone group:the cultured cells with culture diluted into1000μmol/l ketamine and0.125g/l musk ketone; the dose musk ketone group:cultured cells by adding medium diluted into1000umol/l ketamineand0.25g/l musk ketone; high doses muscone group:the cultured cells with culture medium was diluted1000μmol/l of ketamine and0.5g/l musk ketone. Application of DAPI for number of apoptotic cells, MTT assay for cell activity, the image analysis software for measuring the total length of dendritic branches and a dendritic branch number respectively. In addition, we used laser scanning confocal technique to observe the impact of musk ketone on the intracellular calcium concentration in hippocampal neurons. 3. Results3.1Blood gas analysisThe pH value of the control group and ketamine group rats were7.43±0.05and7.41±0.01respectively; PO2were86.00mmHg±8.66mmHg and47.05mmHg±2.87mmHg; SO2were96.50%±1.50%and96.25%±0.43%respectively. Compared with the control group, the pH value of the ketamine group, PO2and SO2were not statistically different. The PCO2control group and ketamine group rats were40.73mmHg±3.73mmHg and47.05mmHg±2.87mmHg. Compared with the control group, PCO2in the ketamine group rats increased slightly, but no significant difference (P=0.059).3.2Neurons apoptosis in hippocampal CA3areaCompared with the control group, rats ketamine group, the number of apoptotic cells density was significantly increased (P<0.05). Compared with the ketamine group, the number density of low doses of musk ketone apoptotic cells decreased, but not statistically significant (P=0.093), the dose of musk ketone group the number of apoptotic cells density was significantly lower, the difference was statistically significant (P=0.028), high doses muscone group there was no significant difference (P=0.612).3.3NMDA-2B receptor of HippocampusThe gray value of NMDA-2B receptors in Ketamine group (1.10±0.04) was significantly higher (0.79±0.05). Compared with the ketamine group, low dose (0.98±0.50), medium dose (0.82±0.14) and high dose (0.65±0.03), musk ketone group are significantly reduced, the difference was statistically significant (P<0.05).3.4Morris water mazeThere was a statistically significant difference of the escape latency from the2d. The escape latency of rats in ketamine group was significantly longer than the control group. Compared with the ketamine group, the rats of each dose of musk ketone group escape latency differences were not statistically significant. Compared with the control group, the percentage of the target quadrant distance of ketamine in rats significantly shortened purpose quadrant time was significantly shorter, the difference was statistically significant (P<0.05). Compared with the ketamine group, the percentage of the target quadrant distance and purpose of quadrant time of rats in any dose of musk ketone were not statistically different (P>0.05).3.5The effect of Musk ketone on neuronal apoptosis after ketamine treatmentCompared with normal control group, the ketamine group, the number of apoptotic cells increased significantly (P<0.05). Apoptosis in the ketamine group compared with the low dose and middle dose of musk ketone group were significantly reduced, no significant difference in the high dose group.3.6MTT method for the determination of cell activityCompared with the control group, After ketamine handling, OD values decreased significantly, the difference was statistically significant (P<0.05). Compared with the ketamine group, the OD value of low-dose and middle dose group was significantly increased, the difference was statistically significant (P<0.05), but high doses of musk ketone group was no significant difference (P>0.05).3.7Muscone on the intracellular calcium concentration in hippocampal neuronsAdded0.25g/1musk ketone, fluorescence intensity was significantly enhanced, and the difference was statistically significant (P<0.05).3.8The effect of muscone on hippocampal neurons morphologyCompared with the control group, the ketamine group, the hippocampal neurons of a dendritic branch number and dendritic branching, the total length significantly reduce the difference was statistically significant (P<0.05). Compared with the ketamine group, low dose, medium dose and high dose musk ketone group of hippocampal neurons of a dendritic branch number and dendritic branching, the total length of no significant change was no significant difference (P>0.05).4. DiscussionThis study found that pH value, oxygen partial pressure and oxygen saturation has no significant change after repeated injection of20mg/kg ketamine in neonatal rat. Although Carbon dioxide partial pressure has slightly higher, but there was no significant difference from the statistical results. Therefore, we suggest that the repeated injection of20mg/kg ketamine will not result in neonatal rats with hypoxia and carbon dioxide accumulation. The animal model can be used for the experimental study of the evaluation of ketamine on neuronal development impact.To determine that the effect of musk ketone on hippocampal neurons after ketamine anesthesia, we selected the newborn7d rats, repeated injection of20mg/kg ketamine a total of five times, interval90min. The results showed that the NMDA-2B receptor expression of rats hippocampus in ketamine group was increased significantly. The rats with the pre-injection of musk ketone can be reflected in the expression of NMD A receptor up-regulated in a dose-dependent decreased expression. During apoptosis research, we found that hippocampal neuronal apoptosis of rats in ketamine group was significantly increased; in doses of musk ketone can be reduced ketamine-induced neuronal apoptosis. However, higher doses of musk ketone instead give rise to apoptosis. Thus, we speculate that ketamine caused by NMDA receptor upregulation may not be the only mechanism to lead to neuron excitotoxicity, musk ketone enables NMDA receptor downregulation, thereby reducing apoptosis, but higher doses of musk ketone not only failed to play a better protection, but the increase in apoptotic cells specific for unknown reasons.Subsequently, in the Morris water maze test, we found that repeated injection of ketamine can extend the rat water maze escape latency, shorten the time to explore, which suggest spatial learning and memory abilities of the rats were declined. Various doses of musk ketone could not improve the decline in the ability of learning and memory.We believe that the main reasons for the neurons in a large number of apoptosis and NMDA receptor expression enhanced ketamine led to the decline of cognitive function in rats. A certain dose of musk ketone can reduce neuronal apoptosis and reduce the expression of NMDA receptors, but does not improve ketamine-induced learning and memory function decline. This indicates that the number of neuronal apoptosis in the critical period of neural development may affect their adult learning and memory, but the ability of the learning and memory as well as by many other factors.To explore the mechanism of musk ketone on the different effects of ketamine anesthesia in hippocampal neuronal development, we conducted in vitro experiments.0.25g/l musk ketone to the hippocampal neurons, showing that the intracellular Ca2+concentration rapidly significantly increased, and accordingly, we speculate, musk ketone has been able to reduce the ketamine-induced apoptosis of hippocampal neurons, in addition to contrast can reduce the expression of NMDA receptor upregulation outside to promote calcium influx, increasing available intracellular calcium, thereby activating the normal downstream signaling pathways may be part of the reason. However, excessive doses of musk ketone may promote excessive increase in intracellular calcium ion concentration of intracellular calcium, and it will promote the apoptosis of hippocampal neurons. Ketamine can cause damage to hippocampal neuron dendritic development, the formation of the neural network, thus affecting their adult learning and memory function. Musk ketone did not improve the toxic effects of ketamine on dendritic development, so the rats in musk ketone group could not improve learning and memory function.5. ConclusionMuscone can reduce ketamine-induced apoptosis of hippocampal neurons by reducing ketamine-induced NMDA receptor upregulation. Muscone can promote calcium influx, and reduce the apoptosis of hippocampal neurons in a certain dose range; too high doses of musk ketone can cause increased apoptosis in neurons. Muscone had no effect on ketamine-induced dendritic development toxic effects, and thus various doses of muscone could not improve neonatal rats with adult learning and memory function. |
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