Department of Chemistry, University of Pittsburgh, Pittsburgh, PA 15260, United States.
ACS Chem Neurosci. 2013 May 15;4(5):838-48. doi: 10.1021/cn400082d. Epub 2013 Apr 30.
We demonstrate here a method that perfuses a small region of an organotypic hippocampal culture with a solution containing an enzyme substrate, a neuropeptide. Perfusate containing hydrolysis products is continually collected and subsequently analyzed for the products of the enzymatic degradation of the peptide substrate. The driving force for perfusion is an electric field. The fused silica capillaries used as "push" and "pull" or "source" and "collection" capillaries have a ζ-potential that is negative and greater in magnitude than the tissue's ζ-potential. Thus, depending on the magnitudes of particular dimensions, the electroosmotic flow in the capillaries augments the fluid velocity in the tissue. The flow rate is not directly measured; however, we determine it using a finite-element approach. We have determined the collection efficiency of the system using an all d-amino acid internal standard. The flow rates are low, in the nL/min range, and adjustable by controlling the current or voltage in the system. The collection efficiency of the d-amino acid peptide internal standard is variable, increasing with increased current and thus electroosmotic flow rate. The collection efficiency can be rationalized in the context of a Peclet number. Electroosmotic push-pull perfusion of the neuropeptide galanin (gal1-29) through the extracellular space of an organotypic hippocampal culture results in its hydrolysis by ectopeptidase reactions occurring in the extracellular space. The products of hydrolysis were identified by MALDI-MS. Experiments at two levels of current (8-12 μA and 19-40 μA) show that the probability of seeing hydrolysis products (apparently from aminopeptidases) is greater in the Cornu Ammonis area 3 (CA3) than in the Cornu Ammonis area 1 (CA1) in the higher current experiments. In the lower current experiments, shorter peptide products of aminopeptidases (gal13-29 to gal20-19) are seen with greater frequency in CA3 than in CA1 but there is no statistically significant difference for longer peptides (gal3-29 to gal12-29).
我们在此展示了一种方法，可将含有酶底物和神经肽的溶液灌注到器官型海马培养物的小区域中。含有水解产物的灌流液不断被收集，并随后对肽底物的酶降解产物进行分析。灌注的驱动力是电场。用作“推送”和“抽吸”或“源”和“收集”毛细管的熔融石英毛细管的 ζ 电位为负，且绝对值大于组织的 ζ 电位。因此，根据特定尺寸的大小，毛细管中的电渗流会增加组织中的流体速度。流量率不是直接测量的；但是，我们使用有限元方法确定了它。我们使用全 d-氨基酸内标确定了系统的收集效率。流量率很低，在 nL/min 范围内，可以通过控制系统中的电流或电压来调节。d-氨基酸肽内标物的收集效率是可变的，随着电流的增加而增加，因此电渗流速率也随之增加。在 Peclet 数的背景下，可以对收集效率进行合理化。通过电渗推送-抽吸方法将神经肽甘丙肽（gal1-29）穿过器官型海马培养物的细胞外空间进行灌注，导致其在细胞外空间中通过外肽酶反应进行水解。水解产物通过 MALDI-MS 进行鉴定。在两个电流水平（8-12 μA 和 19-40 μA）下进行的实验表明，在较高电流实验中，在 CA3 区（CA3）中观察到水解产物（显然来自氨肽酶）的可能性大于在 CA1 区（CA1）中；在较低电流实验中，较短的氨肽酶肽产物（gal13-29 至 gal20-19）在 CA3 中比在 CA1 中更频繁地出现，但对于较长的肽（gal3-29 至 gal12-29）没有统计学上的显著差异。
