Quantifying perivascular sympathetic innervation: Regional differences in male C57BL/6 mice at 3 and 20 months

Abstract

Perivascular sympathetic innervation density (PSID) is a key determinant of vasomotor responses to sympathetic nerve activity. However, total axonal length (for en passant neurotransmission) per vessel surface area has not been well defined, particularly while preserving 3-dimensional vascular structure. We developed a novel method for quantifying PSID using 3-dimensional anatomical reconstruction and compare a variety of blood vessels in Young (3 months) and Old (20 months) male C57BL/6 mice. Individual vessels were dissected and immunolabeled for tyrosine hydroxylase. The total length of fluorescent axons in defined vessel surface areas was quantified by mapping Z-stack images (magnification = 760×). For Young mice, innervation densities (μm axon length/μm 2 vessel surface area) in mesenteric (0.075 ± 0.002) and femoral (0.080 ± 0.003) arteries were greater ( P < 0.05) than mesenteric veins (0.052 ± 0.002) and gracilis muscle feed arteries (0.040 ± 0.002). Carotid arteries and gracilis muscle veins were not immunoreactive nor were there significant differences in PSID between Young and Old animals. We demonstrate a novel approach to quantify sympathetic innervation of the vasculature while preserving its 3-dimensional structure and document regional variation in PSID that persists with aging in mice. This analytical approach may be used for quantifying PSID in other tissues that have superficial vessels which can be studied in situ or from which embedded vessels can be excised. With appropriate visualization of neuronal projections, it may also be applied to tissues that have other sources of superficial innervation.