Department of Chemistry, University of Illinois Chicago, Chicago, Illinois 60607, United States.
Anal Chem. 2022 Mar 8;94(9):3767-3773. doi: 10.1021/acs.analchem.1c03819. Epub 2022 Feb 24.
The development of methods to generate quantitative chemical content information from precise tissue locations is needed to understand fundamental cellular and tissue physiology. This work describes a method to perfuse the extracellular fluid of fly brains using μ-low-flow push-pull perfusion (μLFPP) for quantitative chemical content determinations. Miniaturization of push-pull perfusion probe designs allowed the development of methods for probe tip placement into and sampling from the fruit fly's brain. Perfusate analysis identified and quantified arginine, octopamine, histidine, taurine, glycine, glutamate, and aspartate. The perfusate data did not exhibit any statistical differences based on sex. The perfusate analysis was compared to hemolymph samples to confirm probe placement in fly brain tissues. The appearance of probe placement into the brain space was confirmed with the following observations. Hemolymph and perfusate samples were found to contain analytes unique to each sample type. Quantitated levels of perfusate were not a simple dilution of hemolymph content. Further, the discovery of perfusates with composition similar to both hemolymph and brain perfusate when damage was intentionally inflicted supports the observation that perfusates are distinct from hemolymph. The analysis of perfusate collected for greater than an hour of sampling exhibits the possibility of monitoring applications. Altogether, this work demonstrates the viability of performing μ-low-flow push-pull perfusion for studies of fly brain tissues to identify and quantitate neurotransmitter content.
需要开发从精确的组织位置生成定量化学内容信息的方法，以了解基本的细胞和组织生理学。这项工作描述了一种使用微低流量推挽灌流 (μLFPP) 从果蝇大脑中灌流细胞外液以进行定量化学内容测定的方法。推挽灌流探针设计的小型化允许开发将探针尖端放置到并从果蝇大脑中采样的方法。灌流液分析鉴定和定量了精氨酸、章鱼胺、组氨酸、牛磺酸、甘氨酸、谷氨酸和天冬氨酸。灌流液数据没有表现出基于性别的任何统计学差异。将灌流液分析与血淋巴样本进行比较，以确认探针在果蝇脑组织中的位置。通过以下观察可以确认探针放置到脑空间中的位置。发现血淋巴和灌流液样本都含有每种样本类型特有的分析物。灌流液的定量水平不是血淋巴含量的简单稀释。此外，当故意造成损伤时，发现灌流液的组成与血淋巴和脑灌流液相似，这支持了灌流液与血淋巴不同的观察结果。对采集超过一小时的灌流液进行分析表明了监测应用的可能性。总的来说，这项工作证明了使用微低流量推挽灌流进行果蝇脑组织研究以鉴定和定量神经递质含量的可行性。
