Buffers and solutions 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.