3D-Imaging of Whole Neuronal and Vascular Networks of the Human Dental Pulp via CLARITY and Light Sheet Microscopy

Cristiane Miranda França,Peter Manning Lococo,Anibal Diogenes,Luiz Eduardo Bertassoni,Matthias Widbiller,John L Muschler,Rachelle Riggers

doi:10.1038/s41598-019-47221-5

Abstract

Direct visualization of the spatial relationships of the dental pulp tissue at the whole-organ has remained challenging. CLARITY (Clear Lipid-exchanged Acrylamide Tissue hYdrogel) is a tissue clearing method that has enabled successful 3-dimensional (3D) imaging of intact tissues with high-resolution and preserved anatomic structures. We used CLARITY to study the whole human dental pulp with emphasis on the neurovascular components. Dental pulps from sound teeth were CLARITY-cleared, immunostained for PGP9.5 and CD31, as markers for peripheral neurons and blood vessels, respectively, and imaged with light sheet microscopy. Visualization of the whole dental pulp innervation and vasculature was achieved. Innervation comprised 40% of the dental pulp volume and the vasculature another 40%. Marked innervation morphological differences between uni- and multiradicular teeth were found, also distinct neurovascular interplays. Quantification of the neural and vascular structures distribution, diameter and area showed that blood vessels in the capillary size range was twice as high as that of nerve fibers. In conclusion whole CLARITY-cleared dental pulp samples revealed 3D-morphological neurovascular interactions that could not be visualized with standard microscopy. This represents an outstanding tool to study the molecular and structural intricacies of whole dental tissues in the context of disease and treatment methods.

Highlights

Www.nature.com/scientificreports of the vascular network architecture in dental pulp
CLARITY-cleared tissues can be immunolabelled with a wide range of markers, and imaged using standard microscopy methods or using light sheet microscopy which allows for faster processing times and increased imaging depth[17,21,22,23]
The hydrogel pre-polymer was thermally polymerized by incubation at 37 °C for 3 h inside a vacuum chamber filled with nitrogen gas (Fig. 1B) forming a meshwork of hydrogel covalently linked to native proteins, small molecules and nucleic acids

Summary

Introduction

Www.nature.com/scientificreports of the vascular network architecture in dental pulp. This finding characterizes a distinctive pattern of splitting for peripheral and core nerve bundles between uni- and multiradicular teeth, which is only possible to track if a root section of at least 5 mm in length is imaged, analyzed with 3D rotations and further examined layer by layer with XY and XZ slices, as enabled by CLARITY and light sheet microscopy, but not possible with standard microscopy techniques.

Results

Conclusion