Secondary antibodies used.

The ductus arteriosus is a muscular artery connecting the pulmonary trunk directly to the aorta in fetal circulation in order to by-pass the fluid filled lungs. Post-natally, this vessel is speculated to undergo obliteration, fibrosis and ultimately metamorphosize into a band of ligament, thereby changing name from the ductus arteriosus to the ligamentum arteriosum (LA). Earlier studies into the innervation of the ductus arteriosus reported innervation from the left aortic and vagus nerves. However, information of what becomes of the innervation is scanty and contradictory. I hypothesized that; this fetal shunt still receives innervation even in post-uterine life. To test this, LA of human, pig, and wild-type mice were studied using double-immunofluorescence labeling using antibodies directed against structural and general neuronal marker proteins (Smooth muscle actin and Protein gene product 9.5 (PGP 9.5, respectively). Additionally, TEM studies were performed on mouse LA. Results from the present study demonstrates an extensive innervation of the LA in animals (mice and pigs) and in senescent humans validated by two independent methods, i.e., immunolabeling with antibody directed against PGP 9.5 and TEM. Intense immunoreactivity was clearly visible in samples subjected to PGP-immunolabeling. TEM revealed the presence of nerve terminals with about 30% of all nerve terminals observed less than 1 μm away from smooth muscle cells within the LA. This clearly differs from elastic arteries, where the distance between autonomic terminals and smooth muscle cells is rarely less than 1 μm. Conceivably, these results imply that the so- called LA receives innervation representative of that present within the ductus arteriosus during fetal life. This provides the first reliable study of innervation of the LA and makes room for further investigation into the neurochemistry of this innervation. This is crucial as the presence of nerve terminals may play a role in vessel compliance or impedance of the two great vessels related to this structure. The substances released by these fibers may also have an influence on cells and tissues in the immediate microenvironment of this structure.

动脉导管（ductus arteriosus）是一条肌性动脉，在胎儿循环中直接连接肺动脉干与主动脉，以绕过处于充盈状态的肺脏。出生后，该血管被认为会经历闭塞、纤维化过程，最终转变成韧带索状结构，其命名也由动脉导管（ductus arteriosus）变更为动脉韧带（ligamentum arteriosum，LA）。既往针对动脉导管神经支配的研究显示，其神经纤维来自左主动脉神经与迷走神经。然而，关于该神经支配在出生后的转归情况，现有资料不仅匮乏，且存在相互矛盾之处。本研究提出如下假说：即使在出生后的个体中，这一胎儿时期的分流血管仍接受神经支配。为验证该假说，我们对人类、猪及野生型小鼠的动脉韧带（LA）开展研究：采用分别靶向结构标记蛋白与通用神经元标记蛋白的抗体（分别为平滑肌肌动蛋白（Smooth muscle actin）与蛋白基因产物9.5（Protein gene product 9.5，PGP 9.5））进行双重免疫荧光标记。此外，本研究还利用透射电子显微镜（Transmission Electron Microscopy，TEM）对小鼠的动脉韧带（LA）样本进行了观察。本研究结果通过两种独立方法得到验证：针对PGP 9.5的抗体免疫标记与TEM观察。结果显示，动物（小鼠与猪）及老年人类的动脉韧带（LA）均存在广泛的神经支配。PGP免疫标记样本中可观察到清晰的强免疫反应信号。TEM结果表明，动脉韧带（LA）内约30%的神经末梢与平滑肌细胞之间的距离不足1微米；这一特征与弹性动脉显著不同——弹性动脉中自主神经末梢与平滑肌细胞的间距极少小于1微米。据此可推断，所谓的动脉韧带（LA）所接受的神经支配，与胎儿时期动脉导管内的神经支配特征相一致。本研究首次对动脉韧带（LA）的神经支配开展了系统性可靠研究，为进一步探索该神经支配的神经化学机制提供了研究空间。鉴于神经末梢的存在可能对与该结构相关的两大血管的血管顺应性或阻抗产生影响，且此类神经纤维释放的物质或对该结构局部微环境中的细胞与组织发挥作用，因此本研究具有重要的科学价值。