Effects Of Docosahexaenoic Acid And Tenuigenin On Learning And Memory Impairment Induced By Repeated Propofol Anesthesia In Young Rats

Part One Effects of Docosahexaenoic Acid on Learning and Memory Impairment Induced by Repeated Propofol Anesthesia in young RatsObjective The aim of this study was to investigate the effects of docosahexaenoic acid (DHA) on the learning and memory ability in young rats exposed to propofol and its underlying mechanisms. It maybe provide an improved solution to the side effects of propofol in clinical use.Methods Sixty SD rats were randomly divided into six groups:saline control group (group A), solvent control group (group B), propofol group (group C), low dose DHA+propofol group (group D), medium dose DHA+propofol group (group E), high dose DHA+propofol group (group F). The animals in group A received 0.9% 10 ml/kg saline by intraperitoneal injection once daily for consecutive 5 days. The animals in group B received 10 mg/kg Intralipid by intraperitoneal injection once daily for consecutive 5 days. The rats in experimental group C, group D, group E and group F were administered 75 mg/kg propofol by intraperitoneal injection once daily for 5 consecutive days. Rats in experiment group D, group E and group F also received a single oral dose of 0.3,1, and 3 g/kg DHA, respectively, for 10 consecutive days prior to propofol exposure. Hippocampus-dependent spatial learning and memory were assessed via Morris water maze (MWM) testing. The rats were trained on 5 consecutive days via 4 consecutive trials each day, which were in a pool. Each rat was trained to find a hidden circular platform. The rat was allowed a maximum of 120 seconds to reach the platform, and if it could not find the platform within 120 seconds, it would be placed on the platform for 30 seconds. It will have a rest for sixty seconds.Time to reach the platform were recorded. A probe trial was performed to assess memory 24 h after the last training session. During the probe trial the hidden platform was removed from the tank and the rats were allowed to swim freely for 120 seconds. Latency to find maze platform (sec) and number of platform crossings were recorded. At 24 h after the Morris water maze task was performed, hippocampus were obtained, which was immediately divided into two portions. One portion of tissue was used for the determination of the the amino acid neurotransmitters, and another portion was kept at -80℃ for subsequent biochemical assays. The levels of glutamate (Glu) and γ-aminobutyric acid (GABA) were determined by high performance liquid chromatography (HPLC) with fluorescent detection as described previously. The contents of Glu, GABA were quantified by comparison with the standard curves for each amino acid. The supernatant was collected and quantitatively assayed for the activities of SOD and GSH-Px, and the levels of MDA and BDNF. The detection of these indicators used ELISA kits according to the manufacturers’instructions.Results During the experiment, the activity of rats in propofol group was reduced and spirits atrophy, the activity of rats in other groups also reduced in some degree. Administration of the propofol in group C led to a significant elevation of latency to find maze platform compared with group A (P<0.01). Compared to the group C, group D, group E and group F’s latency to find maze platform were reduced, and the differences of group E (P<0.05) and group F (P<0.01) were statistically significant. Obviously, with the increase of dosage of DHA, the latency to find maze platform of group D, group E and group F were reduced gradually. These suggested that treatment with DHA could effectively enhance spatial learning and memory of young rats and the effect of DHA showed apparent dose-dependent. The content of Glu (P>0.05), GABA (P>0.05), and ratio of Glu/GABA (P>0.05) were not significantly different between group A and group B. As compared with group A, the content of Glu (P<0.01) and the ratio of Glu/GABA (P<0.01) in group C significantly increased and its GABA content decreased (P<0.05). Application of DHA in group D, group E (P<0.05) and group F (P<0.01) led to a decreased in the content of Glu and the ratio of Glu/GABA from those in group C and an increase of GABA level can be seen in group D, group E (P<0.05) and group F (P<0.05) compared with group C. With the increase of dosage of DHA, apparent dose-dependent effect of DHA can be seen. These results indicated that DHA significantly alleviated the learning and memory impairment induced by propofol anesthesia via balancing the levels of Glu and GABA. SOD and GSH-Px activity were significantly decreased by 35.1%(P<0.01) and 39.8%(P<0.01), respectively compared with the control group. DHA treatment exhibited a marked decrease in SOD and GSH-Px activity in group E (P<0.05) and group F (P<0.01) and with the increase of the dose, the inhibitory effects of DHA to the activity of SOD and GSH-Px were more obvious. However, there was no any significant in SOD activity or GSH-Px activity between group A and group B. The BDNF level in the rats of group C were decreased by 34.7%(P<0.05) compared to those in group A. Treatment with DHA of group D, group E, group F resulted in a significant increase in the level of BDNF, which remarkably increased by 11.3%, 33.9%(P<0.05),47.8%(P<0.01), respectively compared with group C. There was no statistical significant differences in the BDNF level between the group A and group B (P>0.05). Similarly, with the increase of the dose, the effect of DHA showed apparent dose-dependent. It showed that the MDA level of propofol-exposed rats was significantly higher than those of control rats (P<0.01). And DHA treatment could decrease the MDA activity in hippocampus of rats in group D, group E (P<0.05) and group F (P<0.01) compared to the group C. These indicate DHA treatment can reduce the oxidative stress induced by propofol repeated anesthesia.Conclusion DHA treatment improved learning and memory impairment by balancing the levels of Glu and GABA, decreasing oxidative damage and increasing BDNF level in young rats with repeated propofol anesthesia. DHA treatment showed dose-dependent. Based on our results, we suggest that DHA could be effectively used for postperative therapy of learning and memory dysfunction induced by repeated propofol anesthesia, although more studies should be conducted to support this hypothesis.Part Two Effects of Tenuigenin on Learning and Memory Impairment Induced by Repeated Propofol Anesthesia in young RatsObjective The aim of this study is to investigate the effect of tenuigenin on the learning and memory impairment induced by repeated propofol anesthesia in rat and the possible mechanisms. It maybe provide another improved solution to the side effects of propofol in clinical use.Methods Sixty SD rats were randomly divided into six groups:saline control group (group A), solvent control group (group B), propofol group (group C), low dose tenuigenin+propofol group (group D), medium dose tenuigenin+propofol group (group E), high dose tenuigenin+propofol group (group F). The animals in group A received 0.9% 10 ml/kg saline by intraperitoneal injection once daily for consecutive 5 days. The animals in group B received 10 mg/kg Intralipid by intraperitoneal injection once daily for consecutive 5 days. The rats in experimental group C, group D, group E and group F were administered 75 mg/kg propofol by intraperitoneal injection once daily for 5 consecutive days. Rats in experiment group D, group E and group F also received a single oral dose of 50,200, and 500mg/kg/day tenuigenin, respectively, for 10 consecutive days prior to propofol exposure. Morris water maze test was carried out from day 16 to day 20 after given propofol or Intralipid, saline. All rats were not given any drug on day 21 before testing. During the experiment, we observed the daily performance of all rats every day, recorded the weight every week and compared the weight changes in different groups. Morris water maze test was used to evaluate the ability of learning and memory. Brain coefficient was observed after anatomy and histopathological analysis was performed to analysis the hippocampus. The levels of MDA, SOD and GSH were detected to assess the degree of cell damages. The DNA was extracted from hippocampal cells of each group to perform the fragmented detection. Western blot was used to determine the expression of the apoptosis related proteins Bcl-2 and Caspase-3.Results During the experiment, the activity of rats in group C was reduced and spirits atrophy, the activity of rats in other group also reduced in some degree. The weight has no significant changes compared with the group A in three weeks. However, the group C showed slowly increasing trend. Group C and different doses tenuigenin treated groups showed significant longer escape latencies and significant decrease in the numbers of crossing over a platform position compared with the control group (P<0.05). However, treated with tenuigenin significantly alleviated the changes compared with the group A (P<0.05).The indexes of oxidative damage have significantly changes compared with the group A (P<0.05), indicating propofol can significantly induce the damage of brain cells. The brain coefficient in the propofol groups was decreased compared with the group A, indicating brain cell atrophy was occurred. However, tenuigenin administration could significantly increase in some degree compared with the group C.By histopathological examination revealed, group A and solvent control group B hippocampal have clear organizational structure, complete nerve cell morphology, tightly packed around without edema; stained uniform, Nucleus intact, prominent nucleoli bluish purple; cytoplasmic staining clear.Propofol rat nerve cells in the hippocampus tissue atrophy, the number was significantly reduced, there is stain phenomenon, irregular arrangement; some nuclei dissolve condensation, the nucleolus is not obvious. Perinuclear vacuoles large number of substances; uneven cytoplasmic staining; peripheral nerve cells scattered in a large number of inflammatory cells. Each dose group of the tenuigenin compared with the propofol group, hippocampal tissue has improved, showing a small amount of nerve cell morphology shrinkage, chromatin uniform, less structured arrangement; nuclei increase in size, some obvious nucleoli, cytoplasmic staining is still clear; visible scattered in small amount of inflammatory cells.DNA electrophoresis in the group A and group B are single band, suggesting the structure of DNA was intact. However, DNA electrophoresis of the hippocampal cells demonstrated significant ladder band, revealing DNA breaks. Tenuigenin treatment also showed ladder band, but the symptom was not significant compared with the group C. The results of ELISA demonstrated that the expression of Bcl-2 was significantly downregulated and also activated the expression of Caspase-3 in the group C compared with the group A(P<0.05).However, tenuigenin administration significantly attenuated the changes (P<0.05).Conclusion The results demonstrated that repeated propofol anesthesia could induce the learning and memory dysfunction, and tenuigenin showed protective effects on the learning and memory impairment induced by repeated propofol anesthesia. Tenuigenin alleviated the hippocampal damage by regulating expression of Bcl-2 and Caspase-3,attenuated the cell apoptosis.