In Vivo Calibration of Microdialysis Using Infusion of Stable-Isotope Labeled Neurotransmitters
In Vivo Calibration of Microdialysis Using Infusion of Stable-Isotope Labeled Neurotransmitters
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DOI：
10.1021/cn300199m
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发表时间：
2013-05-01
影响因子：
5
通讯作者：
Kennedy, Robert T.
中科院分区：
文献类型：
--
作者：
Hershey, Neil D.;Kennedy, Robert T.
微透析探针的体内校准需要解释测得的浓度。最流行的体内校准方法是无净流量（NNF），它需要注入几种浓度的神经递质，以确定体内回收率（提取分数或E-d）和细胞外浓度。报道了一种以谷氨酸（GLU）和多巴胺（DA）为模型物质的神经递质微透析在体校正新方法。通过微透析探针灌注C-13（6）-DA和C-13（5）-GLU作为内部校准物。使用液相色谱和质谱，可以区分C-13-形式的内源性形式的每种神经递质。通过测量输注过程中C-13形式的损失直接计算E-d。所测量的内源性C-12形式的神经递质可以校正E-d，以给出体内校准的细胞外浓度。稳定同位素标记（SIL）神经递质的后透析得到了E-d和细胞外C-13（5）-GLU和C-13（6）-DA浓度，与无净通量测量结果相匹配;然而，由于不需要额外的测量来获得校准，因此这些值在一小部分时间内获得。当通过SIL后透析测量时，在GLU和DA的摄取抑制期间E-d降低。由于E-d是直接测量在每个微透析部分，它是可能的，以监测E-d的变化，在瞬态条件下全身注射摄取抑制剂。结果表明，DA和GLU浓度被低估了50%，如果不校正E-d在摄取抑制。SIL后透析提供了与NNF相当的信息，但时间和动物使用量大大减少。
In vivo calibration of microdialysis probes is required for interpreting measured concentrations. The most popular method of in vivo calibration is no-net-flux (NNF), which requires infusing several concentrations of neurotransmitters to determine in vivo recoveries (extraction fraction or E-d) and extracellular concentrations. A new method for in vivo calibration of microdialysis of neurotransmitters using glutamate (GLU) and dopamine (DA) as model analytes is reported. C-13(6)-DA and C-13(5)-GLU were perfused through microdialysis probes as internal calibrators. Using liquid chromatography with mass spectrometry, it was possible to distinguish the C-13-forms from the endogenous forms of each neurotransmitter. E-d was directly calculated by measuring the loss of the C-13-forms during infusion. The measured endogenous C-12 forms of the neurotransmitters could be corrected for E-d to give calibrated extracellular concentrations in vivo. Retrodialysis of stable-isotope-labeled (SIL) neurotransmitters gave E-d and extracellular concentrations of C-13(5)-GLU and C-13(6)-DA that matched no-net-flux measurements; however, the values were obtained in a fraction of time because no added measurements were required to obtain the calibration. E-d was reduced during uptake inhibition for GLU and DA when measured by SIL retrodialysis. Because E-d is directly measured at each microdialysis fraction, it was possible to monitor changes in E-d under transient conditions created by systemic injection of uptake inhibitors. The results show that DA and GLU concentrations are underestimated by as much as 50% if not corrected for E-d during uptake inhibition. SIL retrodialysis provides equivalent information to NNF at much reduced time and animal use.