Study On DA Concentration And D₂DRmRNA Expression In Brain Of Ketamine Abused Rats

Objective To study the content changes of monoamine neurotransmitter dopamine (DA) and dopamine receptor ii mRNA (D₂DRmRNA) in rat brain, which were given different doses of ketamine through i.p injection for a long time, and the correlation with ketamine in blood. Meanwhile the changes of two indexes and abstinence reaction were also investigated after drug withdrawal for the relationship research between ketamine addiction and DA, D₂DRmRNA.Methods Wistar male rats were used for modeling ketamine abusing circumstances, to build animal model given different doses of ketamine through i.p injection for a long time. According to administration dose, 120 rats were divided into four large groups randomly, 0mg·kg^-1 (physiologic saline control group), 10mg·kg^-1, 30mg·kg^-1 and 60mg·kg^-1. The experimental groups were given hydrochloride of ketamine through i.p injection everyday, and control group was given physiologic saline. These four groups were divided into five small groups(n=6),and were executed at 45mins, one week, two weeks, three weeks after drug administration and the third day after three weeks drug withdrawal, respectively. The heart blood and brain of rats were collected for the detection of ketamine, DA and D₂DRmRNA. Meanwhile the locomotor activity 30mins after the injection and abstinence reaction were also investigated. 1. Ketamine analysis method. After adding SKF525A, serum samples were alkalized and extracted with ethyl acetate. Analysis was performed with a GC equipped with a NPD and a GC/MS. Qualitative analysis was based on retention time in the chromatographic system coupled with the ion fragmentation spectrum in the mass spectrometer. Quantitative analysis was based on an internal standard method. 2. DA analysis method. Brain samples added perchloric acid were homogenated and centrifuged and the supernatant were analyzed by high performance liquid chromatography with electrochemical detection (HPLC-ECD) for the concentration of DA. 3. D₂DRmRNA analysis method. Analysis was performed with real time PCR. 4. Statistical processing. The concentration changes of major indexes at five time point and four dose levels were measured by factorial experiment statistical method. And the correlation between DA, D₂DRmRNA and ketamine were also analyzed.Results 1. The linear range for ketamine in rats blood detected by GC and GC/MS was 0.2～40μg·mL^-1.The limit of detection was 0.01μg·mL^-1 and analytical recovery was 91.33%～92.88%,The inter- and intra-day relative standard deviations of precisions were less than 10 % and 15% respectively. The linear range for DA and its metabolites in rats brain detected by HPLC-ECD was 25～300ng·mL^-1.The limit of detection was 0.02 ng·mL^-1and analytical recovery was 88.65～113.84%.The inter- and intra-day relative standard deviations of precisions were less than 10 % and 15% respectively. In the amplification curve of D₂DRmRNA from real time PCR, there are distinct deflection point and flat baseline, and at the same time the exponential phase curves were steep and straight.2. The content of ketamine in rats blood were increased gradually with the extension of drug administration time, and still could be detected at the third day after three weeks drug withdrawal.3. At the same administration time, the contents of DA and D₂DRmRNA have significant difference between the three doses group(P<0.05). Compared with the control group, the concentrations of DA in ketamine administration group were higher, and the order was 30mg·kg^-1>10mg·kg^-1>60mg·kg^-1 >control group(P<0.05);the expressive level of D₂DRmRNA were decreased as the multiple of control group, and the order was 30mg·kg^-1<10mg·kg^-1<60 mg·kg^-1< control group(P<0.05).4. The concentrations of DA and D₂DRmRNA in rat brains have significant correlation with ketamine in the groups of 10mg·kg^-1 and 30mg·kg^-1. The correlation coefficient(r) was 0.752 and -0.806, respectively. However, this trend didn't exist in the 60mg·kg^-1group.5. At the same dose, the 10mg·kg^-1 and 30mg·kg^-1group rats have more DA than the control group at 45mins, one week, two weeks, three weeks after drug administration, which shows a increased tendency(P<0.05). The expressive level of D₂DRmRNA were lower than the control group, and shows a decreased tendency(P<0.05). At the third day after three weeks drug withdrawal, although, the concentrations of DA were fewer than the two weeks and three weeks group, it was still higher than the control group(P<0.05). The expressive level of D₂DRmRNA was higher than the two weeks and three weeks group, but still lower than the control group(P<0.05) .Conclusion 1. The GC and GC/MS methods for the determination of ketamine, the HPLC-ECD method for DA and the RT-PCR method for D₂DRmRNA were established. These methods was proved to be simple, sensitive and reproducible and could be applied to analyze the detection of ketamine, DA and D₂DRmRNA.2. The content of ketamine could increase gradually with continuous administration, and still could be detected at the third day after three weeks drug withdrawal. It indicates that ketamine might accumulate in body. Or it might cause damage of liver and kidney, and make the half-live of ketamine in body extended.3. Compared with the control group, more DA and less D₂DRmRNA were detected in rat's brains given ketamine. The reason might be that ketamine could make the content of DA increased through rising the releasing of DA or blocking its degradation. Besides, there is a possibility that ketamine could accumulate in body. Therefore, the concentration of DA was able to increase. Meanwhile, D₂DR was stimulated continually by superabundant DA so that the expression of its mRNA was reduced.4. The concentrations of DA and D₂DRmRNA in rat brains have significant correlation with ketamine at 10mg·kg^-1～30mg·kg^-1, and shows a increased and decreased tendency with the extension of drug administration time, respectively. In the 60mg·kg-1group, however, the concentrations of DA and D₂DRmRNA in rat brains have not significant correlation with ketamine, and also don't show an increased and decreased tendency, but kept stable. The reason of showing up this phenomenon might be that ketamine at higher dose could have a more lulling effect on the central nerves so that the excitatory effect on the limbic system was weakened.5. According to the theory of MLDS participating in the addiction and the hypothesis of reward deficiency syndrome, combined with the content changes of DA and its receptor D₂DRmRNA in rat brain given ketamine, and the changes after drug withdrawal, we may draw a conclusion that DA and D₂DRmRNA might be associated with the ketamine addiction.