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Development of μ-Low-Flow-Push–Pull Perfusion Probes for Ex Vivo Sampling from Mouse Hippocampal Tissue Slices
ACS Chemical Neuroscience ( IF 5 ) Pub Date : 2017-11-10 00:00:00 , DOI: 10.1021/acschemneuro.7b00277
Marissa R. Cabay 1 , Alyssa McRay 1 , David E. Featherstone 1 , Scott A. Shippy 1
Affiliation  

This work demonstrates a reduced tip μ-low-flow-push–pull perfusion technique for ex vivo sampling of the extracellular space of mouse hippocampal brain slices. Concentric fused-silica capillary probes are pulled by an in-house gravity puller with a butane flame producing probe tips averaging an overall outer diameter of 30.3 ± 8 μm. The 10–30 nL/min perfusion flow rate through the probe generates an average recovery of 90%. Sampling was performed with mouse brain tissue slices to characterize basal neurotransmitter content in this model system. Samples were collected from hippocampal tissue slices at a volume of 200 nL per sample. Sample arginine, histamine, lysine, glycine, glutamate, and aspartate content was quantified by micellar electrokinetic chromatography with LED-induced fluorescence detection. Primary amine content was sampled over several hours to determine evidence for tissue damage and loss of extracellular content from the tissue slice. Overall, all amino acid concentrations trended lower as an effect of time relative to tissue slicing. There were significant concentration decreases seen for histamine, lysine, and aspartate between time points 0–2 and 2–6 h (p < 0.05) relative to tissue slicing. Analysis of averaged sampling experiments does not appear to reveal significant probe-insertion-related amino acid changes. The work presented shows the applicability of an 80% reduction of probe tip size relative to previous designs for the collection of extracellular content from thin tissue slices.

中文翻译:

小鼠海马组织切片活体取样的μ-低流量-推拉式灌注探针的开发

这项工作证明了减少的尖端μ-低流量-推拉式灌注技术在体外的应用小鼠海马脑片细胞外空间的采样。同心熔融石英毛细管探针由内部重力拉拔器牵拉,丁烷产生火焰的探针尖端的平均外径为30.3±8μm。通过探头的10–30 nL / min灌注流速可产生90%的平均回收率。用小鼠脑组织切片进行采样以表征该模型系统中基础神经递质的含量。从海马组织切片收集样品,每个样品的体积为200 nL。样品精氨酸,组胺,赖氨酸,甘氨酸,谷氨酸和天冬氨酸的含量通过胶束电动色谱和LED诱导的荧光检测进行定量。在数小时内对伯胺含量进行采样,以确定组织损伤和组织切片中细胞外含量损失的证据。总体而言,相对于组织切片,所有氨基酸浓度随时间的变化趋向于降低。组胺,赖氨酸和天门冬氨酸的浓度在0–2和2–6小时之间显着降低(p <0.05)相对于组织切片。对平均采样实验的分析似乎并未显示出明显的探针插入相关的氨基酸变化。提出的工作表明,相对于以前的设计,从薄组织切片中收集细胞外内容物,探针尖端尺寸减小了80%的适用性。
更新日期:2017-11-11
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