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Breaking Biological Barriers with a Toothbrush

K. Amano^1,, K. Miyake^2,, J.L. Borke³, and P.L. McNeil^1,2,*

¹ Department of Cellular Biology and Anatomy,
² Institute of Molecular Medicine and Genetics, and
³ Department of Oral Biology and Maxillofacial Pathology, Medical College of Georgia, Augusta, GA 30912-2000, USA

View larger version (63K): [in this window] [in a new window]   Figure 1. Cell wounding in the gingiva and tongue induced by brushing. (A) Confocal fluorescence image of epithelium and underlying connective tissue from the undisturbed (not brushed) maxillary gingiva. Epithelial cells labeled with the cell wound marker, fluorescein-labeled dextran, are not observed. (B) Confocal fluorescence image of epithelium and underlying connective tissue from the brushed, mandibular gingiva (taken 15 min after brushing). Epithelial cells labeled with fluorescein-labeled dextran are observed in all strata. (C) Confocal fluorescence image of epithelium and underlying connective tissue from the brushed mandibular gingiva (taken 3 hrs after brushing). Epithelial cells labeled with fluorescein-labeled dextran are observed in all strata. (D) Skeletal muscle of a tongue, which was not brushed. Fluorescein-labeled dextran is observed in the connective tissues surrounding the individual myocytes. The myocytes themselves are not labeled intracellularly with the fluorescein-labeled dextran. (E) Skeletal muscle of a tongue 15 min after brushing. Many of the myocytes display strong cytoplasm labeling with fluorescein-labeled dextran. (F) Skeletal muscle of a tongue 3 hrs after brushing. Again, many of the myocytes are strongly labeled with fluorescein-labeled dextran. (G) Quantitative analysis of fluorescein-labeled dextran labeling of epithelial cells. The average fluorescence intensity resulting from fluorescein-labeled dextran staining of cells was measured over randomly assigned domains of the epithelium of the undisturbed (not brushed) maxillary gingiva and brushed mandibular gingiva at two intervals after brushing, 15 min and 3 hrs. Significantly more fluorescence was measured at both time points in the brushed mandibular gingiva. Error bar = SEM; asterisk indicates a p value of < 0.001 (n = 3 animals, 5 sections each, 18–20 images each section), when paired with the undisturbed (not brushed) control, Mann-Whitney test. (H) Quantitative analysis of fluorescein-labeled dextran labeling of skeletal muscle cells. The average fluorescence intensity of individual myocytes was measured in undisturbed and brushed (15 min, 3 hrs) tongues. Error bar = SEM. Asterisk indicates a p value of < 0.001 (n = 3 animals, 5 sections each, 18–20 images each section) when paired with the undisturbed (not brushed) control, Mann-Whitney test. Magnification bar = 25 µm.

View larger version (45K): [in this window] [in a new window]   Figure 2. Induction of c-fos expression by brushing. Paired differential interference contrast (DIC) and fluorescence (c-fos) confocal images are shown. (A,B) In the undisturbed maxillary gingiva epithelium, very few nuclei labeled with an antibody against c-fos are observed in either the epithelium or connective tissue. (C,D) In the undisturbed tongue, very few c-fos-positive nuclei are observed in the cells of the epithelium, connective tissue, or skeletal muscle. (E,F) In the brushed mandibular gingiva, many c-fos-positive nuclei are observed in both the epithelium and connective tissue. (G,H) In the brushed tongue, many c-fos-positive nuclei are observed in cells of the epithelium, connective tissue, and skeletal muscle. (I) Quantitative analysis of c-fos expression as a function of brushing. The number of c-fos-positive cells counted in standardized measurement areas (200 µm X 50 µm) over the gingival epithelium (Gingiva) and over tongue skeletal muscle (Tongue) are indicated for undisturbed tissue (Undisturbed) and brushed tissue (Brushed). Error bar = SEM; asterisk denotes a p value of < 0.001 (n = 3 animals, 5 sections each, 6 images each section) when paired with the undisturbed (not brushed) control, Mann-Whitney test. Magnification bar = 20 µm.

View larger version (38K): [in this window] [in a new window]   Figure 3. Serum albumin as an alternative probe for wounding. (A) Undisturbed maxillary gingiva was immunostained with an antibody directed against mouse serum albumin. As was the case when fluorescein-labeled dextran was used as a cell wound probe, very little intracellular labeling of epithelial cells with this antibody is observed. (B) Brushed mandibular gingiva immunostained for serum albumin. In contrast to undisturbed gingiva, brushed gingiva displayed heavy labeling of surface epithelial cells. (C) Undisturbed tongue skeletal muscle was immunostained for serum albumin. Positively stained skeletal muscle fibers were rarely observed. (D) Brushed tongue was immunostained for serum albumin. Positively stained myofibers were abundant. (E) The same section of undisturbed gingiva was immunostained with an antibody against c-fos. As was the case in fluorescein-labeled dextran-treated tissues, very few positively stained nuclei were observed. (F) Corresponding immunostaining for c-fos. Numerous c-fos-positive nuclei were observed in brushed epithelium. (G) Corresponding immunostaining for c-fos. Myofiber nuclei, positively stained for c-fos, were not observed. (H) Corresponding immunostaining for c-fos. Positively stained myofiber nuclei were abundant. Magnification bar = 20 µm.

View larger version (66K): [in this window] [in a new window]   Figure 4. Brushing induced penetration from the oral cavity of fluorescein-labeled dextran into underlying connective tissue. Paired DIC (DIC) and fluorescein dextran-derived fluorescence (FDx) images are shown. (A,B) The maxillary gingival epithelium was bathed for 1 min in fluorescein-labeled dextran but was not otherwise perturbed. No penetration of the fluorescein-labeled dextran beneath the outermost strata of the epithelium is observed. (C,D) The tongue was bathed in fluorescein-labeled dextran, but was not otherwise disturbed. As in the case of undisturbed gingival epithelium, no penetration of fluorescein-labeled dextran was observed beneath the most superficial strata of the epithelium. (E,F) The mandibular gingiva was bathed for 1 min in fluorescein-labeled dextran and then brushed for another min. Penetration of fluorescein-labeled dextran between epithelial cells and into the underlying connective tissue is not observed. (G,H) The tongue was bathed in fluorescein-labeled dextran and then brushed. Enhanced penetration of the fluorescein-labeled dextran probe was not induced by brushing of this epithelium. Magnification bar = 50 µm.