Effects Of Ketamine On Excitatory Neurotransmitters Levels In Rat Relevant Brain Area

Objective:1. To discover the best methods of extraction, derivatization and chromatographic condition, and select the appropriate internal standard. To establish the method of simultaneous quantitative analysis of excitatory neurotransmitters (Glu, Asp, Gly, Leu) in rat brain.2. Using the established method, to detective the content of Glu, Asp, Gly and Leu in the prefrontal cortex, hippocampus, amygdale and mammillary bodies of the rat that injected with ketamine.3. The experimental data were statistically analyzed to clarify the effects of ketamine on excitatory neurotransmitters levels in rat relevant brain area.4. To discover the relationship between the role of ketamine's pathophysiological and the changes of excitatory amino acids in related rat brain area.Methords:1. The excitatory neurotransmitters in the rat fronttoparietal cortex, hippocampus, amygdale and mammillary bodies were quantited simultaneously using high-performance liqid chromatography. Glu, Asp, Gly and Leu in the rat brain were extracted ultrasonically with the mixture of ultrapure water and ethanol (4:1, v/v). Norleucine was used as internal standard. The extract and norlecine were derivated using the mixed solvent of acetonitrile, triethylamine and cyanogen isothiocyanate acid phenethyl ester (8:1:1, v/v/v). 0.1mol/L sodium acetate solution (added glacial acetic acid to adjust pH to 5.4) and acetonitrile were used as mobile phase to gradient elution. The retention times were engaged to qualitae and the internal standard working curves were efficient to quantitative.2. The fifty-four health male Sprague-Dawley rats were randomly divided into control group (n = 6), saline control group (n= 6) and experimental groups. And the experimental groups were divided into 7 groups, which would be continuous injected with ketamine 3d,5d,7d,9d, 11d, 13d and 15d (n=6, in each group). All groups of rats were numbered respectively. All rats of saline control group and experimental groups were intraperitoneal injected, and were killed at the moment of 15min after the last administration. According to the map of rat brain stereotaxic, the measured brain areas of all experimental rats were separated rapidly and the excitatory neurotransmitters were quantitated using the HPLC after treated. Experimental data were analyzed statistically with SPSS software.Results:1. For excitatory neurotransmitters such as Asp, Glu, Gly and Leu, the established method of analysis had good linear relationships in the concentration ranges of 0.1 to 200μg/mL, and the correlation coefficientsγare near to 0.9998. The minimum detection limits of Asp, Glu, Gly, and Leu were 40.5ng/mL,4ng/mL,47.6ng/mL and 8.4ng/mL, respectively. The recoveries of these four amino acids were higher than 93.7%. The RSDs of intra-day precisions and inter-day precisions were lower than 3% and 10%, respectively.2. In the prefrontal cortex, compared with the control group, the content of Asp, Glu and Leu in a decrease after injection of ketamine, Asp dropped to the lowest value in the 5d, and had significantly different form 3d to 15d group (p<0.05); Glu dropped to the minimum value in the 13d, and had significantly different form 3d to 15d group (p<0.01); Leu dropped to the minimum value in the 7d, and had significantly different in the 5d,7d,9d,13d and 15d group (p<0.05); the content of Gly had both increase and decrease after injectione of ketamine, Gly rose to the highest value in the 9d, when the lowest value was in the 5d, and had significant difference form 3d to 15d group (p<0.05). When compared with the saline group, we found that Asp, Glu and Leu content had both increase and reduce after injection of ketamine, Asp rose to the highest value in the 3d, and had significant difference form 3d to 13d group(p <0.01); Glu rose to the highest value in the 3d, and had significant difference in the 3d,7d,9d, 11d and 13d group(p<0.05); Leu rose to the highest value in the 13d, and the content of Leu had a balance in the 13d and 15 group,3d,5d,7d,9d,13d and 15d had significant differenc(p<0.05); Gly in a rise after injection of ketamine, it rose to the highest value in the 9d, and had significant difference from 3d to 13d group (p<0.01).3. In the hippocampus, compared with the control group, the content of Asp and Glu in a decrease after injection of ketamine, Asp dropped to the minimum value in the 5d, and had significantly different form 3d to 15d group (p<0.05); Glu dropped to the minimum value in the 15d, and had a significantly different form 3d to 15d group (p<0.01); the content of Gly dropped to the lowest value in the 5d, and rose to the highest value in the 13d, and had significantly different form 5d to 15d group (p<0.05); the content of Leu had both increase and decrease after injection of ketamine, and the content of Leu had a balance between 13d and 15d, Leu dropped to the minimum value in the 7d group, and rose to the highest value in the 13d, and had asignificantly different in the 3d,5d,7d, 11d and 15d group (p<0.05). When compared with the saline group, we found that the content of Asp, Glu, Gly and Leu had both increase and reduce after injection of ketamine, both Asp and Glu rose to the highest value in the 3d, Asp had significant difference form 3d to 13d group (p<0.01), Glu had significant difference in the 3d,7d,9d, 11d,13d and 15d group (p<0.05); both Gly and Leu rose to the highest value in the 13d, Gly had significant difference form 3d to 13d group (p<0.01), Leu had significant difference in the 3d,7d,9d,13d and 15d group (p<0.01).4. In the amygdala, compared with the control group, the content of Asp and Glu in a decrease after injection of ketamine, Asp dropped to the lowest value in the 5d, and had significantly different form 3d to 13d group (p<0.01); Glu dropped to the lowest value in the 13d, and had significantly different form 3d to 15d group (p<0.01); the content of Gly and Leu had both increase and decrease after injection of ketamine, Gly rose to the highest value in the 13d, and dropped to the lowest value in the 5d, and had significantly different form 3d to 15d group (p<0.05); Leu had a significantly different in the 3d,5d,7d,11d andl3d group (p<0.01). When compared with the saline group, we found that the content of Asp and Glu had a reduce after injection of ketamine, Asp dropped to the lowest value in the 5d, and had significantly different form 3d,5d,7d,11d and 13d group (p<0.05); Glu dropped to the lowest value in the 13d, and had significant difference form 3d to 15d group (p<0.01); the content of Gly had both increase and decrease after injection of ketamine, it rose to the highest value in the 13d, and had significantly different in the 3d,5d,7d, 11d,13d and 15d group (p<0.05); the content of Leu had a increase after injection of ketamine, it rose to the highest value in the 13d, and had significant difference form 3d to 15d group (p<0.05).5. In the mammillary bodies, compared with the control group, the content of Asp, Glu and Leu in a decrease after injection of ketamine, Asp and Leu dropped to the minimum value in the 11d, Glu dropped to the minimum value in the 13d, Leu had a significantly different form 3d to 11d group(p<0.01), and both Asp and Glu had a significantly different form 3d to 15d group (p<0.01); the content of Gly had both increase and decrease after injection of ketamine, Gly rose to the highest value in the 9d, and dropped to the minimum value in the 11d, and had significantly different form 3d,7d,9d, 11d and 15d group (p<0.05). When compared with the saline group, we found that the content of Asp, Glu and Leu had both increase and reduce after injection of ketamine, Asp and Glu rose to the highest value in the 3d, Asp had significant difference in the 3d and 11d group (p<0.05), Glu had significant difference from 3d to 15d group (p<0.01); Leu rose to the highest value in the 15d, and had significant difference in the 5d,9d, 11d,13d and 15d group (p<0.01); the content of Gly had a increase after injection of ketamine, it rose to the highest value in the 9d, and had significant difference in the 5d,7d,9d, 13d and 15d group (p<0.05).Conclusion:1. Established a high sensitivity, high selectivity, high recovery rate, simple and efficent quantitative analysis method of Glu, Asp, Gly, Leu in rat related brain regions.2. Ketamine had different influence to excitability neurotransmitters in rat fronttoparietal cortex, hippocampus, amygdale and mammillary bodies. The content of Asp, Glu and Leu changed significantly in related rat brain. When compared with the control group, the content of Asp and Glu had a reduce; the content of Gly and Leu had both increase and decrease, but the change of Gly was small, and the content of Leu was low in the rat brain, the reason that led to this change possibly was that ketamine acted on NMDA receptors and NMDA receptors caused.