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 bacterial species colonizing tooth surfaces, gingival crevices, and oral mucosa^3,4. The epithelial cells lining the gingival tissues are persistently exposed to this dense microbial environment⁵.

Virulence factors secreted by periodontal pathogens (e.g., Porphyromonas gingivalis), including lipopolysaccharides, have been shown to disrupt epithelial barrier function, thereby triggering inflammatory responses in gingival tissues and driving pathological progression^6,7. Inflammatory mediators and mechanical stressors further exacerbate barrier dysfunction, increasing susceptibility to oral diseases⁸. Oral probiotics may help prevent periodontal disease by promoting the stability of the gingival epithelial barrier. Nevertheless, the biological interactions between microorganisms and gingival epithelial cells in modulating barrier function remain largely unelucidated⁹. Thus, the development of reliable methods to assess epithelial barrier function is of paramount importance.

Single-photon laser-scanning microscopy, recognized for its high resolution and established detection system, has proven particularly valuable for examining ex vivo tissue samples¹⁰. Advanced imaging methodologies significantly improve the evaluation of gingival barrier properties. The superficial nature of gingival tissues, with epithelial cells located approximately 100 µm from the surface, makes it ideally suited for such imaging approaches. Our research has established an ex vivo assessment technique utilizing fluorescence-labeled dextrans of varying molecular weights as paracellular permeability tracers. Combined with single-photon laser-scanning microscopy, this method enables accurate quantification of gingival epithelial barrier function, providing insights into the mechanisms of periodontal disease and potential therapeutic interventions.

The protocol established in this study, combining localized gel-based tracer delivery with three-dimensional ex vivo imaging, is specifically optimized to address the unique challenges of assessing the gingival epithelial barrier. First, existing intravenous tracer injection approaches (Table 1) are unable to precisely assess the permeability of localized minute epithelial sites to external agents¹¹. Second, traditional histological approaches require decalcification and sectioning, which disrupt tissue architecture and are labor-intensive and time-consuming^12,13,14. In contrast, our protocol utilizes a highly adhesive gel to achieve effective tracer retention on the complex local surface. Furthermore, through a 3D image, it ensures precise, non-destructive quantification of tracer penetration depth and distribution within the gingival epithelium, thereby fully preserving the spatial architecture of the tissue. Consequently, this method is particularly suitable for high-resolution spatiotemporal assessment of gingival epithelial barrier function within the complex and confined oral environment.

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. gingivalis)-induced mouse periodontitis models. All personnel received comprehensive training in laboratory animal welfare and aseptic technique.

1. Animal handling

1. Throughout the study, house animals under standard specific pathogen-free (SPF) conditions with a 12 h light/dark cycle and provide food and water ad libitum.
2. Preparation and administration of anesthetics and tracers
   1. Maintain sterile conditions throughout the protocol and use tools cleaned with 75% ethanol.
   2. Use male wild-type (WT) C57BL/6J mice in the mature adult age group of 8-10 weeks for the experiment. Divide the mice into three groups: a control group (untreated) and two experimental groups (ligation- and P. gingivalis-induced mouse periodontitis models).
      NOTE: In grouping, mice with similar body weight and week age should be selected to reduce the influence of age and weight on gingival epithelial permeability. Ligation-induced and P. gingivalis-induced mouse periodontitis models represent commonly used mouse models of periodontitis. For detailed modeling protocols, refer to the literature on ligation-induced¹⁵ and P. gingivalis-induced¹⁶ mouse periodontitis models.
   3. Select fluorescent tracers with non-overlapping spectra, such as FITC-labeled dextran (40 kDa; FD40) and rhodamine B-labeled dextran (70 kDa; RD70), to ensure distinct signal detection using corresponding FITC and rhodamine filters.
   4. Dilute the tracers. To follow this protocol, mix FD40 (1 g/L) and RD70 (1 g/L) in a 2% carboxymethylcellulose gel and store at 4 °C.
   5. Intraperitoneally inject 1.25% tribromoethanol (200 mg/kg body weight, 15-17 µL/g body weight) to anesthetize the mice or anesthetize according to the recommended guidelines by the local institution.
      NOTE: Tribromoethanol solutions are light-sensitive. Tert-amyl alcohol is a flammable, volatile, and irritating organic solvent. All procedures involving preparation, handling, and administration must be performed in a chemical fume hood or an extremely well-ventilated area, strictly away from any ignition sources. Personnel must wear appropriate personal protective equipment (PPE), including gloves, chemical splash goggles, and a lab coat. Waste disposal: Waste tribromoethanol solution, tert-amyl alcohol, and all contaminated supplies (e.g., syringes, pipette tips, gloves) must be collected and disposed of as hazardous chemical waste in strict accordance with your institution's Environmental Health and Safety (EHS) regulations and protocols for flammable and toxic chemicals.
3. During anesthesia, maintain the core body temperature at 37 ± 0.5 °C using a feedback-controlled heating pad. Apply eye lubricant to prevent corneal drying.
4. Hold the oral cavity open with a mouth gag and apply a  5 μL to 50 μL mixture of two or more paracellular permeability tracers with different molecular weights topically to the labial gingival surface of the molar region (Figure 1B). Leave for 30 min. If the animal swallows the mixture, reapply it.
5. Isolation of teeth and periodontal tissues
   1. After 30 min of application of permeability tracers, euthanize mice by cervical dislocation. Fix the mice on cardboard, placing them in a supine position, and tape their limbs.
   2. Following fixation, open the oral cavity to isolate the maxillary teeth and periodontal tissues. Gently clear away the surrounding tissues and rinse the specimen thoroughly with PBS.
   3. Place the teeth and periodontal tissues on a glass slide, with modeling clay positioned on both sides to facilitate secure mounting of a coverslip over the sample.

2. Single-photon microscope setup

1. Carefully position the dissected mouse teeth and periodontal tissues onto the custom-designed sample holder, ensuring stable placement for imaging.
2. Acquire images using an upright single-photon microscope equipped with a 25×/NA 1.0 water-immersion objective, focusing on the mounted support region beneath the teeth and periodontal tissues.
   NOTE: Fluorescence signals should be visible immediately after removing the tracer gel.

3. Maxillary teeth and periodontal tissues imaging

1. Begin imaging promptly after selecting the region of interest on the buccal surface of the second molar.
2. Acquire Z-stack images with a step size of 1 µm, starting from the maxilla gingival surface to a depth of 100-200 µm, for three-dimensional (3D) reconstruction.
3. Perform Z-series imaging to evaluate epithelial barrier permeability in the gingiva. Configure the software parameters in the referenced software as follows:
   1. In the File menu, create a new folder and assign it a descriptive name for the dataset.
   2. Under the Acquisition tab, set XY format to 512 × 512 with a resolution of 0.99 µm/pixel.
   3. Set the Zoom factor to 1.
   4. Set excitation wavelengths to 488 nm (FD40) and 561 nm (RD70), with emission windows of 500-550 nm and 570-620 nm, respectively.
      NOTE: Gain values were set to 50 (488 nm) and 60 (561 nm), respectively.
   5. For Z-series and 3D imaging, select ND Acquisition mode. Set Z-step to 1 µm (adjust as needed).
4. Once parameters are configured, select Live mode to visualize the gingival area in both individual and merged fluorescence channels within the imaging window.
5. Click Run Now to start acquisition after parameter configuration.

4. Permeability measurement

1. Divide the fluorescence intensity of FD40 and RD70 into different channels. No background subtraction is required; merge the Z-axis slices. Then, use ImageJ software to calculate the fluorescence intensity to evaluate the permeability and indicate the epithelial barrier function.
   1. Click File and select Open to open the maxilla picture taken under the single-photon microscope.
   2. Go to the Image menu and change the image type to 8-bit format for analysis.
      NOTE: Images captured by fluorescence microscopes and confocal systems are generally in RGB format and cannot be used directly for fluorescence intensity measurement. They need to be split into individual channels or monochrome images should be directly imported.
   3. Open the Analyze menu, choose Set Measurements, and check Area, Mean, and IntDen. Confirm by clicking OK.
   4. Select the channel to be analyzed and adjust the threshold: Go to Image | Adjust | Threshold. The system will automatically apply a default value and algorithm, typically using Red to indicate selected regions.
      NOTE: As fluorescent images typically feature black backgrounds, also select the Dark Background option.
   5. Set measurement parameters: Go to Analyze and select Set Measurements. In the dialog box, check Mean gray value and Limit to threshold. Finally, click OK.
      NOTE: It is recommended to check Display label so the image name appears in the results table, making it easier to identify the experimental group.
   6. Perform measurement: Select the Freehand Selections tool. Use the cursor to manually and precisely trace the outline of the fluorescent signal region on the gingival tissue side. Click Analyze | Measure. The results table will appear, where IntDen represents the fluorescence intensity.
2. Copy the results to a spreadsheet for calculations and import for graphing.
3. Calculation of Relative Fluorescence Intensity: First, calculate the mean fluorescence intensity of the control group. Then, divide the fluorescence intensity value of each experimental group by the control group mean to obtain the relative fluorescence intensity.

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