Over 30 Years of DiI Use for Human Neuroanatomical Tract Tracing: A Scoping Review

Abstract

In the present study, we conducted a scoping review to provide an overview of the existing literature on the carbocyanine dye DiI, in human neuroanatomical tract tracing. The PubMed, Scopus, and Web of Science databases were systematically searched. We identified 61 studies published during the last three decades. While studies incorporated specimens across human life from the embryonic stage onwards, the majority of studies focused on adult human tissue. Studies that utilized peripheral nervous system (PNS) tissue were a minority, with the majority of studies focusing on the central nervous system (CNS). The most common topic of interest in previous tract tracing investigations was the connectivity of the visual pathway. DiI crystals were more commonly applied. Nevertheless, several studies utilized DiI in a paste or dissolved form. The maximum tracing distance and tracing speed achieved was, respectively, 70 mm and 1 mm/h. We identified studies that focused on optimizing tracing efficacy by varying parameters such as fixation, incubation temperature, dye re-application, or the application of electric fields. Additional studies aimed at broadening the scope of DiI use by assessing the utility of archival tissue and compatibility of tissue clearing in DiI applications. A combination of DiI tracing and immunohistochemistry in double-labeling studies have been shown to provide the means for assessing connectivity of phenotypically defined human CNS and PNS neuronal populations.

Keywords: DiI; axonal tracing; carbocyanine dyes; neuroanatomical tracing; neuroanatomy; post-mortem human brain.

Figure 1

PRISMA-ScR flowchart for study selection.

Figure 2

Basic methodological steps and their variations reported in previous studies. Regarding tissue preparation, fixed or unfixed tissues have been used in the form of whole organs or tissue blocks/sections. Various modes of dye application have been utilized ranging from crystal/powder to solution/paste and dye-covered glass beads. Incubation of specimens, preferably in a dark environment, has been reported to take place at temperatures ranging between 4 and 45 °C. The duration of incubation also varied widely between a few hours and years. After incubation, the specimens were sectioned and DiI was observed using fluorescence microscopy in the presence or absence of counterstaining. [Images used/adapted with permission (Creative Commons CC-BY license): [47,85,86,87].