| BackgroundPostoperative cognitive dysfunction (POCD) is one of the common complications of central nervous system after anesthesia and surgery. In spite of many risk factors, increasing age (≥60) is the only definitely significant risk factor for the development of POCD. It is generally believed that, anesthetics may be one of the main factors inducing POCD. Propofol (2,6-diisopropylphenol) has the advantage of fast induction, better-quality recovery as well as amnesia and is widely used for induction and maintenance of general anesthesia as well as for procedural sedation(e.g. painless gastroscopy). Ketamine, which can produce analgesia, is as a noncompetitive antagonist to the phencyclidine site of the N-methylmethyl-D-aspartate (NMDA) receptor for the excitatory neurotransmitter glutamate. However, NMDA receptor-mediated long term potential (LTP) is believed to induce memory. Increasing evidence indicate that propofol or ketamine can produced amnesia and behavior disorder at anesthetic doses, but the precise molecular mechanisms underlying the cognitive impairment are poorly understood so far. Cognitive functions are known to involve multiple brain areas, complex processes and variable cellular components. It is demonstrated that hippocampus is one of the most important brain areas that participating in cognitive functions such as memory and learning. Many factors impairing hippocampus can cause cognitive dysfunction.Proteins are the most important biomacromolecules that directly embody and execute the function of life activities. They are also the target molecules that many causative factors and drugs bind and produce effects. Therefore, it is most significant to deeply explore the protein composition and activities pattern so as to elucidate the physiological and pathological process. The new method of proteomics analysis used to investigate changes in abundance of proteins has the advantage that high-capacity screening of proteins and integrally studying the differentially expressed protein profile and the interactions between proteins. Recently, proteomic tools have been used to explore the anesthetic effects, mostly focused on the inhalation anesthetics, and the results were enlightening. However, there is few study devoted to assess the effect of propofol on the cognitive function by proteomics analysis. Objective1. To observe the effects of propofol/or ketamine on cognitive function in aged rats at the 1st day and 7th day after termination of propofol/or ketamine anesthesia through behavioral experiments;2. To study the differentially expressed protein profile in aged rat hippocampus by proteomics analysis and bioinformatics tools, preliminarily understand the possible alternation of hippocampus proteins'network in post-propofol/or post-ketamine anesthesia was preliminary, try to explore the hippocampus protein mostly related to cognitive function and thus integrally deeply investigate the molecular mechanism of cognitive impairment after propofol/or ketamine anesthesia.3. To roughly investigate the value of S-100βand neuron specific enolase (NSE) in reflecting POCD after propofol/or ketamine anesthesia.MethodsMale Wistar rats aged 20 months weighing 560-610g were randomly divided into 2 groups:control group(C) and propofol group(P)/ketamine group(K). The rats in Group P or Group K received intraperitoneal(IP) propofol 60mg/kg(6ml/kg) or ketamine 80mg/kg(8ml/kg) firstly and were boosted half of the initial dose when righting reflex emerged, whereas the rats in Group C received equal volume normal saline instead of propofol/ketamine in same way. One day after termination of drug administration, the rats were assessed with Morris water maze Test 4 times a day for 7 consecutive days and Step-down Test (1st day and 7th day, respectively). The rats were killed by decapitation after the test was finished. The blood samples in each group were collected to test the concentrations of S100βand NSE by ELISA and the hippocampus was isolated immediately. Proteins extracted from rat hippocampus were quantified by the 2D Quant Protein Assay Kit and 150μg protein solution was loaded in an IPG gel strip for 2-dimensional electrophoresis (2-DE) analysis.2-DE gels were silver stained to colour the isolated protein spots. Gel images were scanned and input into the gel image analyzing software ImageMaster 2-D Platinum(V5.0) to match the gels of two groups, compare protein expression difference and screen for the differentially expressed protein spots. The differential expressed protein spots were cut off and digested by trypsin for matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) analysis to obtain corresponding peptide mass fingerprint (PMF) identities. Each protein were identified and confirmed by checking the biological information provided by MS and location of the corresponding protein spot in gel images. In addition, identification of MS results was also performed by MALDI-TOF/TOF MS. Bioinformatics analysis through Gene Ontology (GO) software was further used to summarize the subcellular distribution, molecular function and biological processes of the differentially-expressed identified proteins. The expression changes of selected protein were also assayed using Western-blot to verify the reliability of 2-DE results.ResultsPropofol fraction1. Behavioral experiments:(1) In the step-down test, aged rats in Group P showed significantly learning and memory impairment at the 1st day after propofol anesthesia compared with those in Group C. For post-propofol anesthesia aged rats, the latency, total time of electric shock and error-number in the learning phase increased significantly (p<0.01). The total time of electric shock and error-number displayed similar trend in the memory phase, while the latency decreased significantly (p<0.01). There were no differences between two groups at the 7th day after propofol anesthesia with respect to each index as above.(2) In Morris water maze, the escape latency and total swimming distance to find the submerged platform in Group P at the 1st day after propofol anesthesia were significantly longer than that of Group C (p<0.05 or 0.01). In the probe test, the two groups were comparable with respect to both time and distance of aged rats spent in target quadrant as well as number of crossing over the position.2.2-DE and Proteomics ananlysis: Compared with Group C,21 or 15 differentially expressed proteins were detected respectively on the 1st day or 7th day after propofol anesthesia, among which 17 or 10 proteins were successfully identified with MALDI-TOF MS. Gene Ontology(GO) analysis revealed that the numbers of identified proteins which mainly distributed in cell component were:11 or 5 (organelle); 12 or 3 (cytosol); 3 or 3 (nucleus); 7 or 0 (cytoskeleton); 8 or 1 (membrane), respectively. Functionally, most finally identified proteins were involved in catalytic activities (12,5 respective); binding activities (9,4); protein folding(2,0); structural molecule(5,1); chaperone(1,0) and antioxidant(1,0). According to biological process category,6 proteins were involved in metabolic process respectively; 10 or 2 proteins were involved in biological regulation; 10 or 1 were involved in developmental process; 1 protein was involved in stimulus-response on the 1st day after propofol anesthesia while none was on the 7th day. Change patterns of 2 selected proteins in 2-DE were identical with those in Western blot.3. Two groups were comparable with respect to both the concentrations of S100βprotein and NSE.Ketamine fraction1. Behavioral experiments: The results of step-down test and Morris water maze test in ketamine fraction displayed a similar trend with propofol fraction.2.2-DE and Proteomics ananlysis: Compared with Group C,26 or 15 differentially expressed proteins were detected respectively on the 1st day or 7th day after propofol anesthesia, among which 21 or 8 proteins were successfully identified with MALDI-TOF MS. Gene Ontology(GO) analysis revealed that the numbers of identified proteins which mainly distributed in cell component were:14 or 6 (organelle); 13 or 2 (cytosol); 5 or 2 (nucleus); 8 or 2(membrane); 5 or 1 (cytoskeleton), respectively. Functionally, most finally identified proteins were involved in catalytic activities (16,6 respective); binding activities (13,4); protein folding(1,0); structural molecule(4,1); tansport(1,1) and chaperone(1,0). According to biological process category,11 or 5 proteins were involved in metabolic process; 10 or 4 proteins were involved in biological regulation; 15 or 4 proteins were involved in developmental process; 6 proteins were involved in stimulus-response on the 1st day after propofol anesthesia while none was on the 7th day. Change patterns of 2 selected proteins in 2-DE were identical with those in Western blot.3. Two groups were comparable with respect to both the concentrations of S100βprotein and NSE.ConclusionPropofol or ketamine can cause the cognitive dysfunction in aging rats on the 1st day after anesthesia whereas has no effect on that of 7th day. The mechanism may involved in the multiple differentially expressed proteins of hippacampus in the 1st day which induced by propofol or ketamine anesthesia. The differentially expressed proteins show multidimensional network relationship with each other. Multiple processes and variable functions in rat hippocampus are involved which impair the synapse plasticity and memory, including glycolysis and energy metabolism, protein synthesis and protein qulity control, cytoskeleton morphology and dynamics, synaptic vesicle transport and recycle, transmitter release, antioxidation, Ca2+ homeostasis, signal transduction and apoptosis. NSE and S100βprotein, serving as biochemical markers, appear to be of limited value in detecting POCD after propofol or ketamine anesthesia. |
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