DOI: 10.5061/dryad.9w0vt4bsc
title: Data from: Mouse vocal fold permeability in vivo: Effects of novel low-tech injury and instillation methods

description: Objectives: Vocal fold (VF) injury and instillation are emerging
 techniques to study laryngeal pathologies in mouse models. Most approaches
 require high-tech visualization and lengthy anesthesia. Intraperitoneal
 (IP) naphthalene (NAPH) and topical polidocanol (PDOC) are two chemical
 methods of injuring murine lower airways. Oropharyngeal aspiration (OA) is
 used for murine lung instillation. We assessed whether these simple
 low-tech methods could injure and instill mouse VFs, and whether injury
 increased in vivo VF epithelial permeability. Methods: Mouse VFs were
 injured using IP NAPH at 200, 250, 300, or 350 mg/kg, or OA of PDOC at
 0.5% or 2% w/v. Twenty-four hours later, mice received Evans blue (EB) dye
 OA instillation, then were sacrificed after 30 minutes. Coronal larynx
 sections were assessed for VF injury. Permeability was measured by imaging
 EB autofluorescence and quantifying percent positive area and signal
 intensity. VFs were immunostained for basal cells (p63), tight junctions
 (ZO-1), and basement membrane (laminin). Results: VF injury was 100% with
 ≥350 mg/kg NAPH or high-volume 2% PDOC and 0-40% with other treatments. EB
 bound VF lamina propria in up to 100% of mice in a volume-dependent
 manner. Permeability did not differ by injury. Basal cells and tight
 junctions were decreased in injured VFs. Basement membrane was largely
 intact. Conclusions: High-dose NAPH consistently injures mouse VFs. OA
 reliably instills mouse VFs. Uninjured murine VF epithelium is highly
 permeable to low molecular weight dye in vivo. Inherent permeability of
 mouse VFs may facilitate targeted genetic engineering approaches and
 studies of environmental hazards and drug treatments.
description: # Data from: Mouse vocal fold permeability in vivo: Effects of novel
 low-tech injury and instillation methods Dataset DOI:
 [10.5061/dryad.9w0vt4bsc](10.5061/dryad.9w0vt4bsc) ## Description of the
 data and file structure The dataset contains microscopy images and data
 used to generate the graphs and statistical analyses in figures in the
 manuscript. Images (.TIFF) and data files (.xls) can be viewed and/or
 analyzed with a number of free or proprietary software programs. Image
 naming structure: sample#_slide#_tissue_magnification_filter/stain. ###
 Files and variables #### File: Figure_1.zip **Description:**  1. HE
 folder: Images used to create Figure 1A-C and analyzed to generate data
 for Figure 1D. Brightfield images of hematoxylin and eosin
 (H&E)-stained vocal folds, arranged in folders by injury. 2. Figure 1
 data.xls: Data underlying Figure 1D. Columns: injury group (categorical),
 VF sloughing (number of animals per group), no VF sloughing (number of
 animals per group), % with VF sloughing (percent of animals per group).
 #### File: Figure_2-3.zip **Description:**  1. EB IF folder: Images used
 to create Figure 2 and analyzed to generate data for Figure 3. Raw
 immunofluorescent images of Evans blue red autofluorescence in vocal
 folds, arranged in folders by administration of dye or mock dye, then by
 injury. 2. Figure 3 data.xls: Data underlying Figure 3. 1. Tab 1: Data
 underlying Figure 3A. Columns: injury group (categorical), EB fluorescence
 (number of animals per group), no EB fluorescence (number of animals per
 group), % with EB fluorescence (percent of animals per group). 2. Tab 2:
 Experiment 1 background fluorescence in mock-dyed vocal folds. Columns:
 injury (categorical), dye (categorical), animal ID (text), %area 1, 2, and
 3 (percent area of EB fluorescence in vocal folds in 3 technical
 replicates), mean %area (mean of %area 1, 2, and 3), MFI 1, 2, and 3 (mean
 intensity of EB fluorescence (AU) in vocal folds in 3 technical
 replicates), mean MFI (mean of MFI 1, 2, and 3). Last row: grand mean of
 background fluorescent %area and MFI for experiment 1. 3. Tab 3: Data
 underlying Figure 3B-C. Columns: injury (categorical), dye (categorical),
 animal ID (text), %area 1, 2, and 3 (percent area of EB fluorescence in
 vocal folds in 3 technical replicates), mean %area (mean of %area 1, 2,
 and 3), background %area (grand mean of background fluorescent %area from
 tab 2), corrected %area (mean %area - background %area), MFI 1, 2, and 3
 (mean intensity of EB fluorescence (AU) in vocal folds in 3 technical
 replicates), mean MFI (mean of MFI 1, 2, and 3), background MFI (grand
 mean of background fluorescent MFI from tab 2), corrected MFI (mean MFI -
 minus background MFI). #### File: Figure_4.zip **Description:**  1.
 EB-dyed folder: Images used to create Figure 4A-F. Raw immunofluorescent
 images of Evans blue red autofluorescence in Evans blue-dyed esophagus,
 lungs, and trachea, arranged in folders by tissue. 2. Mock-dyed folder:
 Images used to create Figure 4G-L. Raw immunofluorescent images of Evans
 blue red autofluorescence in mock-dyed esophagus, lungs, and trachea,
 arranged in folders by tissue. #### File: Figure_5.zip **Description:** 
 1. EB IF folder: Images analyzed to generate data for Figure 5. Raw
 immunofluorescent images of Evans blue red autofluorescence in vocal
 folds, arranged in folders by administration of dye or mock dye and by
 volume. 2. Figure 5 data.xls: Data underlying Figure 5. 1. Tab 1: Data
 underlying Figure 5A. Columns: dye volume group (categorical),
 autofluorescence (number of animals per group), no autofluroescence
 (number of animals per group), % with autofluorescence (percent of animals
 per group). 2. Tab 2: Experiment 1 background fluorescence in mock-dyed
 vocal folds. Columns: injury (categorical), dye (categorical), animal ID
 (text), %area 1, 2, and 3 (percent area of EB fluorescence in vocal folds
 in 3 technical replicates), mean %area (mean of %area 1, 2, and 3), MFI 1,
 2, and 3 (mean intensity of EB fluorescence (AU) in vocal folds in 3
 technical replicates), mean MFI (mean of MFI 1, 2, and 3). Last row: grand
 mean of background fluorescent %area and MFI for experiment 1. 3. Tab
 3: Experiment 2 background fluorescence in mock-dyed vocal folds. Columns:
 injury (categorical), dye (categorical), animal ID (text), %area 1, 2, and
 3 (percent area of EB fluorescence in vocal folds in 3 technical
 replicates), mean %area (mean of %area 1, 2, and 3), MFI 1, 2, and 3 (mean
 intensity of EB fluorescence (AU) in vocal folds in 3 technical
 replicates), mean MFI (mean of MFI 1, 2, and 3). Last row: grand mean of
 background fluorescent %area and MFI for experiment 2. 4. Tab 4: Data
 underlying Figure 5B-C. Columns: experiment (categorical), dye
 (categorical), dye volume (categorical), animal ID (text), %area 1, 2, and
 3 (percent area of EB fluorescence in vocal folds in 3 technical
 replicates), mean %area (mean of %area 1, 2, and 3), background %area
 (grand mean of background fluorescent %area from tab 2 or 3), corrected
 %area (mean %area - background %area), MFI 1, 2, and 3 (mean intensity of
 EB fluorescence (AU) in vocal folds in 3 technical replicates), mean MFI
 (mean of MFI 1, 2, and 3), background MFI (grand mean of background
 fluorescent MFI from tab 2 or 3), corrected MFI (mean MFI - minus
 background MFI). #### File: Figure_6.zip **Description:**  1. HE
 folder: Images used to create Figure 6A and analyzed to generate data for
 Figure 6B. Brightfield images of H&E-stained vocal folds, arranged in
 folders by injury. 2. IF folder: Images analyzed to generate data for
 Figure 6C-D. Raw immunofluorescent images of Evans blue red
 autofluorescence in vocal folds, arranged in folders by administration of
 dye or mock dye, then by injury. 3. Figure 6 data.xls: Data underlying
 Figure 6B-D. 1. Tab 1: Data underlying Figure 6B. Columns: injury group
 (categorical), VF sloughing (number of animals per group), no VF sloughing
 (number of animals per group), % with VF sloughing (percent of animals per
 group). 2. Tab 2: Experiment 3 background fluorescence in mock-dyed vocal
 folds. Columns: injury (categorical), dye (categorical), animal ID (text),
 %area 1, 2, and 3 (percent area of EB fluorescence in vocal folds in 3
 technical replicates), mean %area (mean of %area 1, 2, and 3), MFI 1, 2,
 and 3 (mean intensity of EB fluorescence (AU) in vocal folds in 3
 technical replicates), mean MFI (mean of MFI 1, 2, and 3). Last row: grand
 mean of background fluorescent %area and MFI for experiment 3. 3. Tab 3:
 Data underlying Figure 6C-D. Columns: injury (categorical), dye
 (categorical), animal ID (text), %area 1, 2, and 3 (percent area of EB
 fluorescence in vocal folds in 3 technical replicates), mean %area (mean
 of %area 1, 2, and 3), background %area (grand mean of background
 fluorescent %area from tab 2), corrected %area (mean %area - background
 %area), MFI 1, 2, and 3 (mean intensity of EB fluorescence (AU) in vocal
 folds in 3 technical replicates), mean MFI (mean of MFI 1, 2, and 3),
 background MFI (grand mean of background fluorescent MFI from tab 2),
 corrected MFI (mean MFI - minus background MFI). #### File: Figure_7.zip
 **Description:**  1. Folder p63 K14 IF: Images used to create Figure 7A-C
 and  analyzed to generate data for Figure 7D and H. Raw immunofluorescent
 images of vocal folds stained for p63 (green), cytokeratin 14 (K14; red),
 and DAPI (blue), arranged in folders by experiment, then by injury. Select
 images thresholded to remove background signal are also included. 2.
 Figure 7 data.xls: Data underlying Figure 7D and H. Columns: experiment
 (categorical), injury (categorical), animal ID (text), DAPI+ epithelial
 cells (number of cells in vocal fold epithelium with blue fluorescent
 nuclear signal), p63+ epithelial cells (number of cells in vocal fold
 epithelium with green fluorescent nuclear signal), %p63+ epithelial cells
 (percent of DAPI+ cells in vocal fold epithelium that are p63+).  3.
 Folder ZO-1 Laminin IF: Images used to create Figure 7E-G. Raw
 immunofluorescent images of vocal folds stained for zonula occludens 1
 (ZO-1, green), laminin (red), and DAPI (blue), arranged in folders by
 experiment, then by injury. Select images thresholded to remove background
 signal are also included. #### File: Supporting_Information.zip
 **Description:**  1. Supporting Information data.xls: Data underlying
 Table S1 and Figures S3 and S4. 1. Tab 1: Data underlying Table S2.
 Repeated measurements for 20% of quantitative data are presented. Columns:
 animal ID (text), measurement 1 (number), and measurement 2 (number),
 presented for raw % area (mean percent area of EB fluorescence in vocal
 folds averaged from 3 technical replicates), raw MFI (mean fluorescence
 intensity (AU) of EB fluorescence in vocal folds averaged from 3 technical
 replicates), p63+ epithelial cells (number of cells in vocal fold
 epithelium with green fluorescent nuclear signal), and total epithelial
 cells (number of cells in vocal fold epithelium with blue fluorescent
 nuclear signal).  2. Tab 2: Data underlying Figures S3A and S4A. Columns:
 experiment (categorical), injury (categorical), animal ID (text), sex
 (categorical), age in weeks (numerical), VF sloughing (categorical). 3.
 Tab 3: Data underlying Figures S3B-C and S4B-D. Columns: experiment
 (categorical), dye volume in ul (numerical), animal ID (text), sex
 (categorical), age in weeks (numerical), corrected %area (mean %area -
 background %area), corrected MFI (mean MFI - background MFI), total VF
 area (area of the analyzed region of interest comprising the medial
 thyroarytenoid muscle and overlying vocal fold lamina propria and
 epithelium).