Bleck Dennis, Erdene-Byambadoo Lkham, Brinks Ralph, Schneider Matthias, Pongratz Georg
Hiller Research Center Rheumatology at University Hospital Düsseldorf, Medical Faculty, Heinrich-Heine-University, Düsseldorf, Germany.
Bio Protoc. 2019 Dec 20;9(24):e3454. doi: 10.21769/BioProtoc.3454.
In recent years, the role of sympathetic nervous fibers in chronic inflammation has become increasingly evident. At the onset of inflammation, sympathetic activity is increased in the affected tissue. However, sympathetic fibers are largely absent from chronically inflamed tissues. Apparently, there is a very dynamic relationship between sympathetic innervation and the immune system in areas of inflammation, and hence a rapid and easy method for quantification of nerve fiber density of target organs is of great value to answer potential research questions. Sympathetic nerve ends lie in close proximity to immune cells in lymphoid tissues and lymphoid cells are equipped with catecholamine receptors. Catecholamines such as dopamine and adrenaline are secreted by sympathetic nervous fibers and can influence immune cell activity directly. Thereby the sympathetic nervous system immediately participates in the regulation of inflammation. Changes in innervation density could therefore indicate dysregulation of inflammatory processes. Currently, nervous fiber densities are either determined by tedious manual counting, which is not suitable for high throughput approaches, or by expensive automated processes relying on specialized software and high-end microscopy equipment. Usually, tyrosine hydroxylase (TH) is used as the marker for sympathetic fibers. In order to overcome the current quantification bottleneck with a cost-efficient alternative, an automated process was established and compared to the classic manual approach of counting TH-positive sympathetic fibers. Since TH is not exclusively expressed on sympathetic fibers, but also in a number of catecholamine-producing cells, a prerequisite for automated determination of fiber densities is to reliably distinguish between cells and fibers. Therefore, an additional stain using peripherin which is exclusively expressed in nervous fibers as a secondary marker was established. This new and simple method can be used as a high-throughput approach to reliably and quickly estimate sympathetic nervous system (SNS) nerve fiber density in target tissues.
近年来，交感神经纤维在慢性炎症中的作用日益明显。在炎症发作时，受影响组织中的交感神经活动会增强。然而，慢性炎症组织中大多不存在交感神经纤维。显然，在炎症区域，交感神经支配与免疫系统之间存在非常动态的关系，因此，一种快速简便的定量目标器官神经纤维密度的方法对于回答潜在的研究问题具有重要价值。交感神经末梢位于淋巴组织中与免疫细胞紧密相邻的位置，并且淋巴细胞配备有儿茶酚胺受体。多巴胺和肾上腺素等儿茶酚胺由交感神经纤维分泌，可直接影响免疫细胞活性。由此，交感神经系统立即参与炎症调节。神经支配密度的变化因此可能表明炎症过程的失调。目前，神经纤维密度要么通过繁琐的手动计数来确定，这不适用于高通量方法，要么通过依赖专门软件和高端显微镜设备的昂贵自动化过程来确定。通常，酪氨酸羟化酶（TH）用作交感神经纤维的标志物。为了用一种经济高效的替代方法克服当前的定量瓶颈，建立了一种自动化方法，并与经典的手动计数TH阳性交感神经纤维的方法进行了比较。由于TH并非仅在交感神经纤维上表达，还存在于一些产生儿茶酚胺的细胞中，因此自动化测定纤维密度的一个前提是可靠地区分细胞和纤维。因此，建立了一种额外的染色方法，使用仅在神经纤维中表达的外周蛋白作为二级标志物。这种新的简单方法可作为一种高通量方法，用于可靠且快速地估计目标组织中交感神经系统（SNS）神经纤维密度。
