Misini Bashkim, Freinbichler Wolfhardt, Colivicchi Maria Alessandra, Bisilimi Kemajl, Linert Wolfgang, Tipton Keith F, Della Corte Laura
Institute for Applied Synthetic Chemistry, Vienna University of Technology, Getreidemarkt 9/163-AC, A-1060 Vienna, Austria.
Dipartimento di Neuroscienze, Psicologia, Area del Farmaco e Salute del Bambino (NEUROFARBA), Università degli Studi di Firenze, Viale G. Pieraccini 6, 50139 Firenze, Italy.
J Neurosci Methods. 2015 Aug 15;251:1-6. doi: 10.1016/j.jneumeth.2015.04.014. Epub 2015 May 13.
Terephthalate (TA(2-)), which reacts with highly reactive oxygen species (hROS) to form the fluorophor 2-hydroxy terephthalic acid (OH-TA) with a high selectivity, has been used for determining hROS formation during in vivo microdialysis. Previously this involved collecting fractions of the microdialysate and determining the OH-TA formed after HPLC (the batch method).
This work reports the development and validation of a procedure for continuously determining hROS formation during microdialysis. TA(2-) was added to the artificial cerebrospinal fluid (aCSF) perfusing medium to trap hROS. OH-TA formation was detected in real time with a sensitive fluorescence detector equipped with a capillary flow cell that was coupled directly to the effluent stream of the microdialysis system.
The behaviour of the system was assessed by comparison with the batch method and using a well-characterized animal model of excitotoxic damage, based on the application of high concentrations (1mM and 500μM) of the non-NMDA glutamate receptor agonist kainate (KA) to the neostriatum. Data for the evoked release of taurine were also determined in these samples. No temporal difference between hROS and taurine release could be detected.
COMPARISON WITH EXISTING METHOD(S): The flow method had a comparable sensitivity of hROS detection to the batch method. It was simpler, cheaper and less time-consuming than the batch method.
This direct system is convenient and technically undemanding. It should be useful for the rapid assessment of the hROS responses to neurotoxins and other compounds in microdialysis experiments in vivo.
对苯二甲酸盐（TA(2-)）可与高活性氧物种（hROS）发生反应，以高选择性形成荧光团2-羟基对苯二甲酸（OH-TA），已被用于在体内微透析过程中测定hROS的生成。此前这涉及收集微透析液的各个部分，并测定经高效液相色谱（HPLC）后形成的OH-TA（分批法）。
本研究报告了一种在微透析过程中连续测定hROS生成的方法的开发与验证。将TA(2-)添加到灌注人工脑脊液（aCSF）的介质中以捕获hROS。使用配备有毛细管流通池的灵敏荧光检测器实时检测OH-TA的形成，该流通池直接连接到微透析系统的流出液流。
通过与分批法进行比较，并使用基于向新纹状体施加高浓度（1mM和500μM）非NMDA谷氨酸受体激动剂海藻酸（KA）的特征明确的兴奋性毒性损伤动物模型，对该系统的性能进行了评估。还测定了这些样品中牛磺酸诱发释放的数据。未检测到hROS释放和牛磺酸释放之间的时间差异。
流动法对hROS检测的灵敏度与分批法相当。它比分批法更简单、更便宜且耗时更少。
这种直接系统方便且技术要求不高。它应有助于在体内微透析实验中快速评估hROS对神经毒素和其他化合物的反应。
