Unit of Medical Pharmacy, Department of Pharmacy Practice, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan.
School of Pharmaceutical Sciences, Nagasaki University, Nagasaki, Japan.
J Neurosci Methods. 2019 Mar 1;315:1-5. doi: 10.1016/j.jneumeth.2018.12.020. Epub 2019 Jan 6.
Microdialysis (MD) is conventionally used to measure the in vivo levels of various substances and metabolites in extracellular and cerebrospinal fluid of brain. However, insertion of the MD probe and subsequent perfusion to obtain samples cause damage in the vicinity of the insertion site, raising questions regarding the validity of the measurements.
We used fluorogenic derivatization liquid chromatography-tandem mass spectrometry, that quantifies both high and low abundance proteins, to differentiate the effects of perfusion from the effects of probe insertion on the proteomic profiles of expressed proteins in rat brain.
We found that the expression levels of five proteins were significantly lower in the perfusion group than in the non-perfusion group. Three of these proteins are directly involved in ATP synthesis. In contrast to decreased levels of the three proteins involved in ATP synthesis, ATP assays show that perfusion, following probe insertion, even for a short time (3 h) increased ATP level up to 148% that prior to perfusion, and returned it to normal state (before probe insertion).
There is essentially no information regarding which observed changes are due to probe insertion and which to perfusion.
Our findings partially demonstrate that the influence of whole MD sampling process may not significantly compromise brain function and subsequent analytical results may have physiological equivalence to normal, although energy production is transiently damaged by probe insertion.
微透析（MD）常用于测量脑内细胞外液和脑脊液中各种物质和代谢物的体内水平。然而，插入 MD 探针并随后进行灌注以获取样本会在插入部位附近造成损伤，这引发了对测量结果有效性的质疑。
我们使用荧光衍生化液相色谱-串联质谱法，可定量检测高丰度和低丰度蛋白质，以区分灌注和探针插入对大鼠脑组织中表达蛋白的蛋白质组谱的影响。
我们发现，灌注组中 5 种蛋白质的表达水平明显低于非灌注组。其中 3 种蛋白质直接参与 ATP 合成。与涉及 ATP 合成的 3 种蛋白质的水平降低相反，ATP 测定显示，在探针插入后进行灌注（即使是短时间 3 小时），ATP 水平增加至灌注前的 148%，并恢复至正常状态（探针插入前）。
基本上没有关于哪些观察到的变化是由于探针插入引起的，哪些是由于灌注引起的信息。
我们的发现部分表明，整个 MD 采样过程的影响可能不会显著损害大脑功能，并且随后的分析结果可能具有与正常情况相同的生理等效性，尽管探针插入会短暂损害能量产生。
