Functional Assessment of Mouse Gingival Epithelial Barrier Using Fluorescent Tracers

Summary

The goal of this protocol is to provide a standardized approach for quantitatively assessing the integrity of the murine gingival epithelial barrier by topically applying fluorescence-labeled dextrans of varying molecular sizes on the gingival surface in vivo, followed by ex vivo imaging using a single-photon microscopy system.

Abstract

The gingival epithelial barrier serves as a critical frontline defense in oral immunity and homeostasis. An intact epithelial barrier effectively prevents the penetration of harmful substances from the oral environment, whereas its disruption is closely associated with the pathogenesis of conditions such as periodontitis and oral lichen planus. Here, we established a topical application-based method to assess the integrity of the gingival epithelial barrier in mice. First, a mixture of fluorescently labeled dextrans with different molecular weights (40 or 70 kDa) in a 2% sodium carboxymethylcellulose was applied topically to the labial gingival surface of the molar region. After a 30 min incubation period, the maxilla was dissected and fixed in a customized holder for immediate imaging using a single-photon microscopy system. This approach enables visualization and quantification of tracers of different sizes across the epithelial layers, providing a functional readout of gingival epithelial barrier integrity applicable to studies of oral inflammation, wound healing, and disease modeling.

Introduction

The gingival epithelial barrier constitutes a critical defensive interface in the oral cavity, preserving tissue integrity by selectively regulating substance transport while preventing microbial invasion¹. Composed of stratified squamous epithelium interconnected by tight junction complexes, this dynamic structure precisely controls paracellular permeability to restrict the non-selective penetration of microorganisms, macromolecules, and solutes². Multiple factors can compromise the integrity of the gingival barrier. The oral cavity hosts the second most diverse microbial community in the human body, with over 700 bacte....

Protocol

All experimental procedures were approved by the Ethics Committee of Animal Research, Peking University Health Science Center, and complied with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996). During the process, the experimental animals were carefully treated to minimize their pain and discomfort.

The details of each work step for in vivo permeability assessment in mouse gingival epithelia are described below (Figure 1A). Here, we provided an example of detecting epithelial barrier function in both ligation- and Porphyromonas gingivalis (P. ....

Results

According to the standardized protocol, mount the maxillary teeth and periodontal tissues (including gingiva, periodontal ligament, alveolar bone, and cementum) of a mouse onto a custom-made holder (Figure 1C). Microscopic examination revealed green (FD40) and red (FD70) fluorescence signals in the gingiva, demonstrating tracer permeation across the epithelial barrier. After identifying the region of interest under the ocular lens, images were acquired using the software. In the control grou.......

Discussion

The gingival epithelium serves as an essential physical barrier against bacterial invasion, while oral microorganisms exhibit dual regulatory capacity through either disrupting barrier integrity with virulence factors or promoting its maintenance. These biological interactions ultimately determine tissue homeostasis or disease progression^17,18. Building on this foundation, we developed an innovative methodological approach to further investigate these mechanisms........

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
40 kDa FITC-labeled dextran	Sigma Aldrich	78331
70 kDa rhodamine B-labeled dextran	Sigma Aldrich	R9379
Blunt tissue separation nickel	Bejinghuabo Company	NZW28
carboxymethylcellulose	MREDA	M049898
Disposable sterile syringe	Zhiyu Company		1 mL
ImageJ software	National Institutes of Health
Microtubes	Axygen	MCT-150-C	1.5 mL
NIS-Elements AR Image Software	Nikon
Phosphate buffered saline 1x	Servicebio	G4207-500
Single photon microscope Nikon A1 MP+	Nikon
Tissue scissors	Bejinghuabo Company	M286-05