Introduction and validation of a new semi-automated method to determine sympathetic fiber density in target tissues
Introduction and validation of a new semi-automated method to determine sympathetic fiber density in target tissues
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DOI：
10.1371/journal.pone.0217475
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发表时间：
2019-05-29
期刊：
影响因子：
3.7
通讯作者：
Pongratz, Georg
中科院分区：
文献类型：
--
作者：
Bleck, Dennis;Ma, Li;Pongratz, Georg
近年来，交感神经纤维在慢性炎症中的作用越来越明显。炎症发作时，受累组织的交感神经活动增加。然而，交感神经纤维在慢性炎症组织中基本缺失。显然，炎症部位的交感神经与免疫系统之间存在着非常动态的关系，因此，一种快速简便的量化靶器官神经纤维密度的方法对回答潜在的研究问题具有重要价值。目前，神经纤维密度要么是通过繁琐的人工计数来确定的，这并不适合高通量的方法，要么是通过昂贵的自动化过程，依赖于专门的软件和高端显微镜设备。通常，酪氨酸羟化酶（TH）被用作交感神经纤维的标记物。为了克服目前的定量瓶颈，采用一种经济高效的替代方法，建立了一个自动化的过程，并与传统的人工计数th阳性交感神经纤维的方法进行了比较。由于TH不仅在交感神经纤维上表达，而且在许多产生儿茶酚胺的细胞中也表达，因此自动测定纤维密度的先决条件是可靠地区分细胞和纤维。因此，使用神经纤维中表达的外周蛋白作为次要标记物进行额外染色。使用这种新方法，我们研究了syndecan-3敲除（SDC3KO）小鼠系的脾脏，并证明了手动计数和自动计数在SNS纤维密度上的结果相同(手动计数：野生型：22.57 +/-11.72纤维/ mm(2)；Ko: 31.95 +/-18.85纤维每毫米(2)；P = 0.05；自动计数：野生型：31.6 +/-18.98纤维每毫米(2)；Ko: 45.49 +/-19.65纤维/ mm(2)；P = 0.02)。综上所述，该方法简便易行，可作为一种高通量、可靠、快速测定靶组织中SNS神经纤维密度的方法。
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 tissue. 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. 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 distinct between cells and fibers. Therefore, an additional staining using peripherin exclusively expressed in nervous fibers as a secondary marker was established. Using this novel approach, we studied the spleens from a syndecan-3 knockout (SDC3KO) mouse line, and demonstrated equal results on SNS fiber density for both manual and automated counts (Manual counts: wildtype: 22.57 +/-11.72 fibers per mm(2); ko: 31.95 +/-18.85 fibers per mm(2); p = 0.05; Automated counts: wildtype: 31.6 +/-18.98 fibers per mm(2); ko: 45.49 +/-19.65 fibers per mm(2); p = 0.02). In conclusion, this new and simple method can be used as a high-throughput approach to reliably and quickly estimate SNS nerve fiber density in target tissues.