| BackgroundThe impairment of anesthetics and sedative drugs on the nervous system has always been a hot research topic in the field of anesthesia. As a new intravenous anesthetic agent, propofol is extensively used in clinical induction and maintenance of anesthesia and sedation in intensive care unit, possesses advantages which include rapidity of onset and recovery, without accumulation, few side effects.Researchers had already found that propofol could block working memory and cause profound amnesia, whereas the report of Lee suggested that single bolus propofol anesthesia show unimpaired learning in adult and aged rats. However, propofol anesthesia not only acts as single bolus clinically, but also can be used as a continuous or intermittent drug for several times or several hours even days within total intravenous anesthesia and ICU. Therefre, to determine wheter propofol impaired learning and memory through single bolus infusion has none pratical significance. The current clinical research has not yet enough evidence to prove that there is a causal relationship between propofol and impairment of learning and mmory, so it is necessary to study the relationship and corresponding mechanism between propofol and nervous toxity through further animal experiments.The discovery of neurogenesis in the brain of adult mammals overturned the long-held dogma that the adult brain has no capacity for generating new neurons. Adult neurognesis, originating from neural progenitor cells(NPCs), has been consistently observed in two regions of the adult brain:the subventricular zone(SVZ) of the lateral ventricle and the subgranular zone(SGZ) of the dentate gyrus in the hippocampus. Adult neurogenesis is composed by neural stem cells proliferation, differentiation, maturation and the integration of new born neurons into the existing neural circuitry, the process of producing new neurons constanly. Hippocampus is an important structure of mammalian limbic system, the internal structure can be divided into three main elements:the dentate gyrus, CA1regionand CA3region. The dentate gyrus is one of the brain regions which generate new neurons of mammalian adult hippocampus, and also an important neural integration network which play a critical role in memory process especially in establishment and application of spatial learning and memory.So far, known anesthetics are thought to work by interacting with ion channels that regulate synaptic transmission and membrane potentials in key regions of the brain and spinal cord. Propofol also acts on synapses, regulates the presynaptic membrane transmitter release and the function of presynaptic and postsynaptic membrane gamma-amino butyric acid(GABA) receptor, thus produces its anesthetic effect by inhibiting central nervous system. But the specific molecular mechanism of propofol remains unknown. Most of the neurotransmitters and ion channels that play important roles in synaptic transmission are proteins. Proteins are not only the most important and direct embodiers and executives in the life activities, but also the target molecules of multiple pathogenic factors and pharmacological actions. Protein modifications, especially phosphorylation and dephosphorylation, play key roles in various cellular functions, such as cell differentiation, cell growth and apoptosis. Studies suggested that abnormal phosphorylation of tau in brain tissue precedes the formation of neurofibrillary tangles in Alzheimer’s disease. Furthermore, increased tau phosphorylation has been reported in animals subjected to isoflurane and desflurane inhalation, which may contribute to the short-term cognitive dysfunction following anesthetic administration. Therefore, detecting the changes in phosphorylation after propofol infusion will be critical to exploring the underlying mechanism of the general anesthetic action of propofol.The brain is a highly interactive entity, in which a number of separate brain areas cooperate to execute biological functions. The thalamus may be considered to be a type of relay system and is believed to act as the switchboard of information between a variety of subcortical areas and the cerebral cortex. Additionally, functional brain imaging also confirmed that the thalamus is the key target for anesthetic action. Studies have shown that the hippocampus is responsible for mental processes such as initial learning and memory as well as for conscious behavior. Wei H et al reported that propofol affects LTD expression in hippocampal CA1dendrites in rats, which was assumed to be the reason for propofol-induced learning and memory deficits. The cerebral cortex is the final target of arousal systems, and the dorsolateral prefrontal cortex, which is one of most important parts of the cerebral cortex, is involved in activities such as emotion recognition, voluntary movements and working memoryas well as the maintenance of the arousal state in mammals.The first part of this paper observed the effect of repeated propofol sedation on spatial learning and memory in rats and newborn neurons in rat hippocampus dentate gyrus by Morris water mazi test, immunofluorescence technique and laser confocal microscopy to further study the possible neurotoxicity of propofol. In the second part of this paper, we isolated and extracted the phosphoproteins in the thalamus, hippocampus and frontal lobe from the rat experienced propofol anesthesia, and then the proteins that were differentially expressed before and after anesthesia were identified using two-dimensional electrophoresis and mass spectrometry to determine the anesthetic mechanism of propofol.Materials and methods1. SD adult male rats receive intraperitoneal injection of propofol or Intralipid twice a day for7days, respectively. Spatial learning and memory was assessed by Morris water maze test28days after treatment.5-Bromo-2’-deoxyuridine (BrdU) was injected after treatment; numbers of BrdU positive cell in Subgranular zone (SGZ) were checked1,14or28days later treatment. Dendritic complexity of newborn neurons in dentate gyrus were assessed in EGFP transgenic mice14days after treatment by confocal microscopy.2. SD adult male rats receive caudal vein injection of propofol or Intralipid, after20min, all rats were sacrificed by decapitation, and the thalamus, hippocampus and frontal lobes were collected immediately. First, phosphorylated proteins in the three brain regions were extracted, and the proteins that were differentially expressed before and after anesthesia were identified using two-dimensional electrophoresis and mass spectrometry then searching the differentially expressed proteins in the database. Last, the proteins were verificated by Western Blot Analysis.Result1. Propofol sedation exerts significant delay in escape latency and swimming distance of rats after28days’treatment. The residence time of the target area is decreased obviously after5days. Numbers of BrdU positive cell in SGZ did not change one day after treatment, while decreased14and28days after propofol injection. Total dendritic length and branch number of newborn neurons in dentate gyrus decreased14days after propofol injection in transgenic mice; ratios of EGFP cells to DCX positive cells remained consistent.2.21protein spots were found to be significantly altered among the three brain regions——the thalamus, hippocampus and frontal lobe. Sixteen of these phosphoprotein spots were successfully identified using MALDI-TOF MS and the subsequent comparative sequence search in the Mascot database.DicussionHippocampus is the most crucial structure that participates in cognitive function such as learning and memory abilities in central nervous system. The dentate gyrus of the hippocampus is a critical brain region in which new neurons were generated continuely throughout the lifespan. Deng et al reviewed many reports that have produced conflicting results, with some reporting that reduction of neurogenesis impairs hippocampus-dependent learning and memory and others reporting that reduction of neurogenesis has little or no effect. Previous reports also suggested that spatial learning could increase the survival rate of new-born neurons and new neurons proliferation, may possibly decrease the number.Thus, our paper was designed to further investigate the relationship between new neurons and the abilities of spatial learning and memory and how propofol exerts on these. The new neurons developed into mature neurons that possess memory abilities by experiencing neural progenitor cells proliferation and differentiation, then committing to the neuronal lineage, with dendrites extending towards the molecular layer and axons. They mainly existed in the dentate gyrus of hippocampus, and possessed spatial learning capacity after4weeks. Our work found that the behavioral deficits appeared4weeks after treatment when new granule cells were able to participate in the existed circuitry. Simultaneously, we counted the BrdU-positive cell numbers in the subgranular zone of rats in both groups after treatment. The numbers equaled statistically on the next day after treatment, which indicated no influence on the proliferation of adult neural stem cells in hippocampus. The diminished numbers in the propofol sedation group revealed the injured viability of proliferating cells in the subgranular zone during the28days after sedation. These results have clearly shown that repeated propofol impairs adult neurogenesis, especially the survival of neural progenitors, which has been strongly associated to learning, in turn to effect the number of new neurons. Doublecortin (DCX) could be used as a marker of neuronal precursors for the study of neuronal proliferation and migration. We determined to trace the developmental status of newborn neurons that derived from the decreased progenitors, We chose DCX——a reliable marker of newly generated neurons in dentate gyrus of adult hippocampus, then colocalized them with DCX immunostaining. The numbers of DCX-positive cells were reduced14days after propofol sedation, which was consistent with those of BrdU-positive cells. This indicated a consequence of less survival of neuronal progenitors. Moreover, the reductive newborn neurons endured delayed maturation in morphologically. Our results showed that two indices of dendritic complexity, total length and numbers of branches, were weakened by propofol sedation. Studies have demonstrated that even minor alterations in dendritic structure can have a marked impact on the biophysical properties of neurons and dramatically affect the manner in which neurons transmit information. Actually, dendritic complexity has been strong related to hippocampus-dependent learning in several models, including enriched environment, voluntary exercises and short photoperiods. During neuronal development, dendritic complexity is a marker indicating the extent of maturation, and is able to suggest future ability of learning. Subsequently, the propofol sedation-induced decline of adult neurogenesis, both in quantity and morphology, can be inferred to render the behavioral deficits later.The study revealed toxic effects of propofol on learning and adult neurogenesis. But the role of propofol was not impairment of neural stem cells proliferation but influence the survivl and dendrite structure of newborn neurons. Propofol has potential neurotoxicity,but the specific mechanism remains unclear.Hence, we screened the differentially expressed phosphoprotein profiles of the thalamus, hippocampus, frontal lobe in reponse to propofol by using the proteomic analyses. To determin the possible mechanism of propofol through bioinformatics analysis and western blot confirmation. The identified differentially expressed phosphorylated proteins were subjected to bioinformatics analysis. Six proteins, including keratin18, gelsolin, tubulin2c chain, macrophage-capping protein? actin and apolipoprotein E were identified. These proteins form cytoskeletal structures or participate in stabilizing the cytoskeletal structure. Alterations in cytoskeletal architecture can result in changes in ion channels, which was first found in neurocytes, and account for the occurrence and development of brain-related disorders.Tau proteins are type II MAPs that are abundant in neurons in the central nervous system, and their phosphorylation state can be modulated by a specific set of phosphatases and phosphokinases, which play a vital role in maintaining neuronal function and development. Hyperphosphorylation of tau is assumed to be involved in the neuropathogenesis of several types of dementia, such as Alzheimer’s disease and postoperative cognitive dysfunction. Apolipoprotein E also plays a vital role in the pathogenesis of Alzheimer’s disease, in which apolipoprotein E is found in the amyloid plaques and neurofibrillary tangles characteristic of the disease. The deposition of monomers and polymers of hyperphosphorylated tau protein in the brain of transgenic mice expressing apolipoprotein E4(C112R) suggested that apolipoprotein E affected tau phosphorylation. We speculate that the postoperative cognitive dysfunction induced by anesthetic propofol may result from alterations in serum apolipoprotein E levels. Nevertheless, the interwoven relationship between these factors and whether apolipoprotein E phosphorylation is involved in this process must be further explored.Bioinformatic analysis indicated that gelsolin and keratin18exhibit responses to ethanol. Ethanol can weaken our body’s reactions to outside stimulation, which is similar to the behavioral changes that emerge after propofol anesthesia. Amino acid neurotransmitter receptors play an important role in alcohol dependence. Ethanol is both a gamma-aminobutyric acid (GABA) receptor agonist and an N-methyl-D-aspartate (NMDA) receptor antagonist, which results in degenerative alterations of the nervous system during brain development by inhibiting ERK phosphorylation. The effect of ethanol on brain stem cell apoptosis during brain development is similar to that of propofo. Glutamic acid (Glu) serves as the main neurotransmitter for the input and output as well as the intrinsic circuitry of the hippocampus, which appears to be the brain region that is most sensitive to alcohol damage and is the target of the anesthetic effect of propofol. In summary, the targets of and the transmitters affected by general anesthetic agents are similar to those of alcohol. Furthermore, studies have shown that ethanol was able to induce the dephosphorylation of keratin18in the liver and kidney. In our study, keratin18was also dephosphorylated in the hippocampus of rats that received propofol administration compared with the control rats. We speculated that the proteins responsive to ethanol may also contribute to the anesthetic effect of propofol. However, the specific mechanisms require further study.In conclusion,16differentially expressed phosphorylated proteins in the thalamus, hippocampus and frontal lobes were found using proteomics methods in this study. Additionally, bioinformatics were also used to analyze the common characteristics of the differentially expressed proteins to study the underlying mechanisms of the general anesthetic action. These experimental data will definitely provide meaningful references for the clarification of the mechanism of action of the general anesthetic propofol. |
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