The Developing Neurovascular Anatomy of the Embryo: A Technique of Simultaneous Evaluation Using Fluorescent Labeling, Confocal Microscopy, and Three-Dimensional Reconstruction

Abstract

The close spatial relationship between peripheral nerves and blood vessels in the adult is well known. However, evidence supporting the congruent development of these structures in embryos remains anecdotal. Neurovascular relationships also have been shown to be conserved in other vertebrates. This homology suggests that either peripheral nerves or blood vessels, or both, might have fundamental morphogenetic roles during embryologic development. Both peripheral nerves and blood vessels have been independently implicated as etiologic agents in the pathogenesis of congenital disabilities, and several congenital anomalies fit their distribution patterns. This article presents a technique for the simultaneous visualization of peripheral nerves and blood vessels at different stages in the developing embryo. The forelimbs of 310 quail embryos were dissected over a 1-year period. Peripheral nerves were labeled with the neural crest and axon antibody, HNK-1, followed by fluorescein-conjugated secondary antibodies. Blood vessels were labeled by a perfusion technique using the fluorescent dye, dioctadecyl-tetramethylindocarbocyanine. Specimens were processed and imaged in whole-mount with confocal microscopy, and images were reconstructed using three-dimensional modeling software. Both nerves and blood vessels seem to undergo a highly stereotypic sequence of development in the embryonic quail forelimb. Furthermore, the existence of a close spatial relationship between nerves and blood vessels suggests either a high degree of developmental interdependence or shared patterning mechanisms. This technique permits further evaluation of the possible role peripheral nerves and blood vessels might play in the pathogenesis of congenital disabilities and provides a starting point for further studies aimed at elucidating the means by which peripheral nerves and blood vessels are patterned in the forelimb of the avian embryo. (Plast. Reconstr. Surg. 108: 597, 2001.)