| Objective In recent years,both scientific research and clinical practice have found that ketamine influences cognitive function.Patients undergone anesthesia and surgery,developing symptoms such as changes in personality,deficit of social skills and cognitive dysfunction,are considered as suffering from postoperative cognitive dysfunction(POCD).Moreover,ketamine abuse is also a threatening social problem because there are lots of reports about cognitive impairment of abusers.It is currently believed that ketamine has a wide and varied impact on cognitive function through different mechanisms.Among them,ketamine can cause an increase in extracellular glutamate concentration,leading excitatory/inhibitory imbalance and glutamate excitotoxicity through blockade of NMDA receptors.Studies have shown that metabotropic glutamate receptors(m Glu Rs)play an important role in glutamate transmission,and it is suggested that m Glu Rs be a therapeutic target for cognitive impairment in schizophrenia cause they participate in the pathophysiological process of schizophrenia.Although exogenous agonists of m Glu R2/3s have achieved remarkable success in animal experiments,they are not as effective as expected during clinical trials.However,researchers applied an inhibitor of 2-(phosphonomethy1)·pentanedioic acid(2-PMPA)in a stroke model of transient middle cerebral artery embolization,to suppress the hydrolysis of N-acetylaspartylglutamate(NAAG),a peptide defined as an endogenous agonist m Glu R3 in the brain.It was shown that the post-truamatic release of glutamate was significantly reduced,suggesting that NAAG has a neuroprotective potential.To date,it is rarely reported whether 2-PMPA can prevent general anesthetic toxicity or postoperative cognitive dysfunction.This study was divided into two parts of experiments.In the first part,we used ketamine to create a model of cognitive impairment in early adult mice and applied different drugs to modulate m Glu R2/3s so as to investigate how m Glu R2/3s influenced changes in hippocampal transmitters and in behaviors induced by ketamine.In the second part,the potential mechanisms of therapeutic application of 2-PMPA were explored through western blot and immunohistochemistry.Methods Female 6~8-week-old C57BL/6 mice weighing 20±5g,were randomized into 6 groups: Control group(given the same volume of saline),Ketamine group(given Ketamine at dosage of 30 mg/kg,repeated every 30 mins once a day,three times a day),MK801 group(given MK801 at dosage of 1 mg/kg once a day),LY354740 group(given LY354740 at a dosage of 10 mg/kg 30 mins before first injection of ketamine,once a day),LY341495 group(given LY41495 at a dosage of 0.5 mg/kg 30 mins before first injection of ketamine,once a day)and 2-PMPA group(given 2-PMPA at a dosage of 50 mg/kg 5 mins after every injection of ketamine,three times a day).All administrations were done intraperitoneally and lasted for 14 days.(1)10 minutes after the end of the first-day administration,5 mice from each group were sacrificed by cervical dislocation and fresh hippocampal tissues were rapidly harvested for evaluating changes in levels of glutamate brought by acute treatments;(2)24 hours after the end of the last administration,5 mice from each group were rapidly harvested for evaluating changes in levels of both glutamate and GABA brought by chronic treatments;(3)Behavioral tests such as open field test(OFT),elevated plus maze test(EPMT)and water maze test(WMT)were applied to investigate the changes in emotion,learning and memory functions of mice;(4)5 brains respectively from group Control,Ketamine,and 2-PMPA were prepared into paraffin sections and stained via DAB staining to observe the expression of GAD67 and GFAP positive cells in hippocampus.(5)The protein expression of EAAT2,GAD67,v Glu T1 and x CT in the hippocampus was detected via Western blot among group Control,Ketamine,and 2-PMPA.Results(1)At the end of the first day of injection(D1)10 min after injection,compared with the control group(C group),the glutamate content increased significantly among Ketamine group(K group),MK801 group(M group)and LY341495 group(LY34 group)in the hippocampus(P<0.01).Compared with K group,the glutamate content was significant lower in both 2-PMPA group(PM group)and LY354740 group(LY35 group)(P<0.01);glutamate content of LY34 group was significantly higher(P<0.05).(2)At the end of the last injection(D14),compared with group C,the content of glutamate in hippocampus of group K and LY34 increased significantly(P<0.05),and the content of glutamate in hippocampus of PM group,LY35 group and M group changed 24 hours later.There was no statistical difference(P>0.05);GABA content in hippocampus of K group,LY34 group and M group was significantly decreased(P<0.05);there was no significant difference in GABA content in hippocampus between PM group and LY35 group.(P>0.05);Compared with K group,glutamate content in LY35 hippocampus tissue was significantly decreased(P<0.05);GABA content in PM group and LY35 hippocampus tissue was significantly increased(P<0.05).(3)OFT: Compared with group C,the total movement distance of group M mice in the open field was significantly increased(P<0.05).There was no significant difference in the total movement distance of mice in each group(P>0.05).Compared with group C,the number of entry into the central area of group K and group LY34 was significantly decreased(P<0.05),the number of entry into the central area of group M was increased(P<0.05),and there was no significant difference among the other groups(P>0.05).Compared with group K,the number of LY35 group entering the central area increased significantly(P<0.05).Activity time in the central area: compared with the C group,the activity time in the central area of the K group was significantly reduced(P<0.05),and there was no significant difference between the other groups(P>0.05).Compared with group K,the number of excretion in PM group and LY35 group was significantly reduced,and the difference was statistically significant(P<0.05).EPMT: Compared with group C,there was no significant difference in total movement distance,total arm entry frequency,and open/close arm entry frequency ratio(P>0.05);compared with group K,LY35 group and M group total arm Frequency increased significantly,the difference was statistically significant(P<0.05).WMT: There was no statistical difference in escape latency on the last day of the training period(T0-5)reaching the platform(P>0.05),and in the orientation course experiment(T1-1 to T1-4 for 4 days),compared with the C group.The escape latency was significantly prolonged in group K,group M and group LY34(P>0.01).Compared with group K,the escape latency of group PM and group LY35 was significantly shorter(P<0.01); the space exploration experiment(T1-5),and C Compared with group K,group M and group LY34,the latency of the original platform was significantly prolonged(P>0.01);compared with group K,the latent period of the original platform was significantly shortened in the PM group(P<0.01).The latency of the original platform position was significantly shortened(P<0.05).There was no statistical difference in the latency of the original platform in the LY34 group(P>0.05).Compared with the C group,the number of crossings of the original platform in the M group was significantly reduced(P<0.05).P>0.01),the number of crossings of the original platform position in the K group and the LY34 group was significantly reduced(both P>0.05),and the differences among the other groups were not statistically significant(P>0.05).(4)Compared with group C,the number of GAD67 and GFAP positive cells in hippocampus of group K was significantly lower(P<0.01,P<0.05),and the number of GAD67 positive cells in PM group was significantly lower(P<0.05);compared with group K,the number of GAD67 and GFAP positive cells in the hippocampus of the PM group increased(P<0.05).(5)Compared with group C,the expression of x CT and v Glu T1 in hippocampus of group K was significantly up-regulated,while the expression of EAAT2 and GAD67 was significantly down-regulated.There was no significant change in the expression of each protein in hippocampus of PM group.Conclusion(1)Exposure to ketamine can induce an increase in glutamate concentration in the hippocampus of mice,and chronic repeated exposure will lead to imbalance of glutamate/GABA ratio,resulting in an excitatory/inhibitory imbalance,leading to expression of mood disorders and spatial learning and memory deficits in mice;(2)Group II metabolic glutamate receptors participate in the glutamate signaling pathway,and their agonism can reduce glutamate release,reverse the excitation/inhibition imbalance,improve ketamine-induced mood disorders and spatial learning and memory deficits;(3)Chronic ketamine exposure can induce decreased expression of GAD67-positive cells in the hippocampus of C57 mice,decrease GAD67 expression and affect GABA synthesis,lead to or exacerbate excitatory/inhibitory imbalances,and show cognitive dysfunction.2-PMPA inhibits NAAG hydrolysis,regulates glutamate over-release,reduces excitotoxicity,reverses excitation/inhibition imbalance,and improves cognitive function;(4)Chronic ketamine exposure induces an extracellular glutamate status in the hippocampus,which may be related to decreased expression of astrocytes,decreased expression of EAAT2 and up-regulation of glutamate/cystine reverse transporter in the glutamate reuptake system.Stimulation of upregulation of v Glu T1 results in imbalance of glutamate homeostasis.Application of 2-PMPA reduced the degradation of NAAG in the hippocampus,stimulated the m Glu3-type receptors,prevented the structure and functional disorders of the glutamate reuptake system,and alleviated the imbalance of glutamate homeostasis. |
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