| Objectives To investigate the effect of age on the autophagy andneuroinflammation in hippocampus, as well as that on the cognitive functionin rats undergoing splenectomy; To explore the influence and mechanism ofautophagy activation, anti-neuroinflammation response, and thecombination of these two factors on the cognitive function in aged ratsundergoing splenectomy with the administration of rapamycin orExpendin-4, so as to detect the role of autophagy in the deterioratedcognitive function in aged rats after splenectomy. To investigate the effectsof propofol as well as the electroconvulsive shock (ECS) on the hippocampalautophagy and synaptophysin in depression model rats undergoing ECS, soas to explore the role of autophagy in the protective effect of propofol on thelearning and memory impairment caused by ECS.Methods Part1: Experiment1After adaption to the lab room for1week,36male adult (5~6months) and36aged（24~25months） Sprague-Dawley (SD) rats were randomly assigned to3groups (n=12), respectively:control group (group ac and AC), anesthesia group (group aa and AA), and operation group (group ao and AO). Rats in anesthesia group receivedintraperitoneal injection of the combination of500μg/kg droperidol with20μg/kg fentanyl for general anesthesia; rats in operation group underwentsplenectomy under general anesthesia performed as that in anesthesia; rats incontrol group received intraperitoneal injection of normal saline (NS) withthe same volume at the same time. Morris water maze (MWM) was appliedfor6consecutive days preoperatively and1day postoperatively to evaluatethe cognitive function of rats. After the last performance of MWM,6randomly assigned rats in each group, respectively, were administered toheart perfusion with4%paraformaldehyde to fix brains. The other6ratswere decollated under anesthesia to obtain fresh hippocampus tissue whichthen stored in-80℃. Transmission electron microscopy was applied for thedetection of autophagy activation in hippocampus; immunofluorescentdouble staining was used to detect the co-expression of autophagy makerprotein LC3Ⅱ and neuron marker protein NeuN. Iba1protein wasdetected by immunohistochemistry. Western blot was used to detectautophagic related proteins Beclin1, LC3Ⅱ/Ⅰ and p62and cytokinesIL-1β and IL-6. Experiment260aged（24~25months） SD rats wererandomly assigned to5groups (n=12): control group (group C),splenectomy group (group O), dimethyl sulfoxide (DMSO)+splenectomygroup (group OD), rapamycin+splenectomy group (group OR), rapamycin+3-methyladenine (3-MA)+splenectomy group (group ORM). Rats in group O received lateral ventricle (right) injection of0.9%NS15min beforesplenectomy under the anesthesia performed with the combination ofdroperidol and fentanyl. Rats in group OD, OR and ORM received lateralventricle (right) injection of DMSO2μl+NS3μl, rapamycin2μl（35pmol）+NS3μl, rapamycin2μl（35pmol）+3-MA2μl （600nmol）+NS1μl respectively. Rats in group C received intraperitoneal injection ofNS with the same volume at corresponding time. The aforementionedexperimental assays and indicators were applied. Experiment348aged（24~25months） SD rats were randomly assigned to4groups(n=12):control group (group C), Exendin-4group (group E), splenectomy group(group O), Exendin-4+splenectomy group (group OE). Rats in group OEreceived Exendin-410μg/kg and splenectomy30min later under theanesthesia performed with the combination of droperidol and fentanyl.12hour after the surgery, intraperitoneal injection of Exendin-410μg/kg wasadministered again. Rats in group O received the same treatments with thealteration of Exendin-4to NS with the same volume. Rats in group Ereceived the other same treatments other than the splenectomy. Rats in groupC received intraperitoneal injection of NS with the same volume atcorresponding time other than the splenectomy. The aforementionedexperimental assays and indicators were applied. Part2: Depressionmodel were established with SD rats after stress exposure for28consecutivedays.40model rats were assigned randomly to4groups (n=12): depression model group (group D, exposure to no treatment), ECS group (group E,treated with ECS under sham anesthesia performed with normal saline8ml/kg), propofol group (group P, treated with1week of intraperitonealinjection of propofol80mg/kg), and propofol+ECS group (group PE,treated with ECS under anesthesia performed with intraperitoneal injectionof propofol80mg/kg).Open-field test and sucrose preference test wereapplied to evaluate the depression behavior, and Morris water maze was usedto assess the learning and memory function of rats. Western-blot was used todetect the expression of Beclin-1and LC3Ⅱ/Ⅰand p62, and ELISA wasapplied to assess the expression of synaptophysin.Results Part1: Experiment1Before surgery, aged ratspossessed deteriorated cognitive function compared with adult rats; surgeryresulted in impairment of the space learning and memory function as well ascognitive flexibility, more serious deterioration was observed in aged rats;autolysosome were shown in the hippocampal neuron of the adult and agedrats in transmission electron microscopy; immunofluorescent doublestaining showed the co-expression of LC3Ⅱ and NeuN, and LC3Ⅱwerefound expressed in cytoplasm. The base expression levels of Beclin-1andLC3Ⅱ/Ⅰ in the hippocampus of adult rats before surgery were higher thanthat in aged rats; surgery led to the up-regulated expression level of theBeclin1and LC3Ⅱ/Ⅰin both adult and aged rats; oposite expressionpattern was found in the p62expression; and a cross-effect between age and surgery on the Beclin1and LC3Ⅱ/Ⅰand p62expression were shown; thebase expression levels of Iba1、IL-1β and IL-6were higher in aged ratscompared with the adult rats; surgery increased the expression of Iba1, IL-1βand IL-6, and this stimulative effect was magnified in aged rats.Experiment2The splenectomy resulted in up-regulated expression ofBeclin1and LC3Ⅱ/Ⅰ, down-regulated expression of p62, increase in theexpression of Iba1, IL-1β and IL-6in the hippocampus, besides the cognitivefunction impairment in aged rats. Rapamycin caused further increase in theexpression of Beclin1and LC3Ⅱ/Ⅰas well as the expression levels of Iba1,IL-1β and IL-6, and decrease in p62expression as well in the hippocampus,besides alleviating the cognitive function impairment.3-MA reversed totallythe effect of rapamycin on the expression of Beclin1, LC3Ⅱ/Ⅰand p62,mitigated its inhibitory effect on the expression of Iba1, IL-1β and IL-6inthe hippocampus as well as the protective effect on postoperative cognitivefunction. Experiment3The splenectomy resulted in up-regulatedexpression of Beclin1and LC3Ⅱ/Ⅰ, down-regulated expression of p62,increase in the expression of Iba1, IL-1βand IL-6in the hippocampus,besides the cognitive function impairment in aged rats. Exendin-4decreasedthe relatively elevated blood glucose levels induced by the splenectomy12hand24h after surgery, resulted in further increase in the Beclin1and LC3Ⅱ/Ⅰ expression, further decrease in p62expression as well as the theexpression of Iba1、IL-1β and IL-6in the hippocampus, in addition mitigated the postoperative cognitive function impairment. Part2: Rats in group Eand that in group PE showed higher open-field test scores as well as sucrosepreference percentage compared with rats in group D. Longer escape latency,shorter space exploration time, higher level of expression of Beclin1, LC3Ⅱ/Ⅰ, and synaptophysin were revealed in rats in group E. Compared withgroup E, rats in group PE possessed shorter escape latency, longer spaceexploration time, lower expression level of Beclin1, LC3Ⅱ/Ⅰ, andsynaptophysin. Change of p62was opposite to Beclin1and LC3Ⅱ/Ⅰ.Conclusions （1）Surgery leading to more obvious cognitive functiondeterioration in aged rats than in adult rats is related with the insufficientactivation of autophagy in hippocampus as well as the magnifiedneuroinflammation by the age factor in aged rats after surgery.(2) Activatingthe autophagy in hippocampus could alleviate the cognitive functionimpairment in aged rats; administration of rapamycin before surgery couldprotect the postoperative cognitive function which be induced partly byinhibiting the neuroinflammation in the hippocampus.(3) The deteriorationin postoperative cognitive function is related with the surgery inducedincrease in blood glucose and the neuroinflammation in the hippocampus aswell as the interaction between these two factors on autophagy; Exendin-4shows protective effect on the postoperative cognitive function viastabilizing the postoperative blood glucose, activating autophagy, andmitigating the neuroinflammation in the hippocampus.(4) ECS inducing autophagy and overexpression of synaptophysin in hippocampus results incognition decline after ECS. Propofol can inhibit excessive ECS-inducedautophagy and overexpression of synaptophysin in hippocampus, thusprotecting learning and memory functions in depressed rats after ECS. Theinhibition effect of propofol on the overexpression of synaptophysin mayresult from its inhibitory effects on the excessive induction of autophagy. |
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