The study aimed at investigating perivascular co-transmitters of vasomotor control in the auricle. Another aim was to provide three-dimensional arterial maps of the auricle, as a proxy of periarterial autonomic innervation. Twelve paired human auricles were used to visualize the arteries following Spalteholz clearing and μ-CT-based reconstruction.

Perivascular innervation staining was conducted using anti-tyrosine hydroxylase (TH), anti-neuropeptide Y (NPY), anti-vasoactive intestinal peptide (VIP) and anti-choline acetyltransferase (ChAT). Auricular vasomotor responses are considered to be signs of clinical conditions including a migraine. The mechanisms of auricular vasomotor control are still debatable. 

Auricular Vasculature

The combined Spalteholz technique and μ-CT revealed a highly consistent arrangement of the auricular vasculature. The superficial temporal (STA) and posterior auricular artery (PAA) supply the helical rim arcade and arcade, with the STA mainly forming the superior and the PAA forming the middle and inferior auricular artery.

Co-existence of sympathetic NPY+ and TH+ terminals mediating vasoconstriction, and VIP+ and ACh+ indicating cholinergic vasodilatation, was found in the perivascular zone. The presence of both sympathetic vasoconstriction and cholinergic co-innervation for active vasodilatation was shown in the perivascular auricular region.

Assuming that the highly-consistent vasculature gives way to these terminals, this periarterial innervation may be found spread out across the helix.

Histology and Immunohistochemistry

Immediately after removal two auricles were immersion-fixed in 4% paraformaldehyde (PFA) with phosphate buffer saline (PBS; 0.1 M; pH 7.4) for six weeks. One ear was cut into radial segments as shown. Planes were chosen with the external acoustic meatus as an axis. For cryoprotection, segments were immersed in 30% sucrose in PBS with 0.1% sodium azide. 30 µm thick cryosections were made on a cryomicrotome Zeiss Hyrax S30 with freezing unit Zeiss hyrax KS34; sections were collected in PBS with 0.1% sodium azide.

Microscopy

Tissue sections were imaged using a Keyence research microscope (BZ9000, Keyence, Neu-Isenburg, Germany). Photoshop CS2 (Adobe Systems, Mountain View, CA, USA) was used to process the images with minimal alterations to color, saturation, contrast, and background.

This study, for the first time, presents data on the periarterial autonomic innervation of the human auricle in conjunction with a 3D visualization of the vascular tree, showing the proximity of autonomic nerve fibers to the perivascular beds of the helical arteries. It used a combination of the Spalteholz technique and μ-CT to depict auricular vascularity in detail in combination with histology and immunohistochemistry.

Human Ear

Though the autonomic innervation of the human ear has been investigated with both functional and surgical methodology, this was to date not the case for the periarterial autonomic innervation.This study gave evidence for ChAT+ and VIP+ nerve terminals along with NPY+ and TH+ terminals at the human auricular helix, indicating the innervation of active vasoconstriction and -dilatation.

These terminals were found in the perivascular zone, formed by terminal branches of the superficial temporal artery and posterior auricular artery. The 3D reconstruction from μ-CT of ears showed a highly consistent pattern of anterior auricular branches, potentially forming the pathways for the perivascular autonomic nerve system.