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Title: 1425 - 3D-Imaging of Whole Neuronal and Vascular Networks in Intact Dental Pulp

Authors:

Cristiane França (Presenter)
Oregon Health and Science University

Rachel Riggers, Oregon Health & Science University
Agnieszka Balkowiec, Oregon Health & Science University
John Muschler, Oregon Health & Science University
Luiz Bertassoni, Oregon Health and Science University

Abstract:

Objectives: Despite current understanding of the site-specific organization of tissues constituting the dental pulp, direct visualization of their interactions at the whole-organ level and with sub-cellular-resolution has remained impossible. The development of CLARITY (Clear Lipid-exchanged Acrylamide Tissue hYdrogel) microscopy, a novel method for 3D volumetric imaging of whole organs with high resolution, has enabled successful imaging of intact tissues with preserved anatomic structures down to the level of single synapses. Here, we report for the first time, CLARITY-cleared imaging of intact whole dental pulp immunostained for neurons and blood vessels.

Methods: Dental pulp from extracted teeth (n=4) were fixed for 3 days in bis-acrylamide, acrylamide and paraformaldehyde hydrogel blends at 4oC and polymerized under vacuum (3h, 37oC). Next, tissues were maintained in sodium dodecyl sulfate/boric acid (37oC) for 8 wks, with one solution change/week. After optical transparency was achieved, tissues were immunostained for PGP9.5, a marker for peripheral neurons, CD31 a marker for vascular capillaries, and DAPI. Samples were imaged using a Zeiss LightsheetZ.1 microscope, and images were reconstructed using Arivis vision4D for high resolution 3D volumetric renderings of the whole dental pulp from the apex to the crown.

Results: In the pulp core, images showed well-defined, intricate neurovascular bundles with discernible neurons innervating long blood vessels from apex to crown. 3D-movie reconstructions suggested that the dental pulp innervation is at least twice as dense as the vasculature, and that the blood vessels occupy peripheral areas relative to nerves, which are more homogeneously distributed throughout the root, except in the so-called cell-free zone.

Conclusions: Dental pulps subjected to CLARITY microscopy and 3D high-resolution imaging revealed novel evidence of the 3D distribution and density of the pulp neurovasculature, and can be an outstanding tool to study the intricacies of dental tissues in the context of disease and treatment methods.

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This abstract is based on research that was funded entirely or partially by an outside source:
This work was supported by funding from the NIDCR-NIH (R01DE026170 to LEB), the Medical Research Foundation of Oregon (MRF to LEB) and the OHSU Fellowship for Diversity and Inclusion in Research (OHSU-OFDIR to CMF)

Disclosure Statement:
The submitter must disclose the names of the organizations with which any author have a relationship, the nature of the relationship, and the clinical or research area involved. The following is submitted: None

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