Preservation of salivary flow rates 3 days after single γ-radiation exposure in myr-Akt1 mice.

<p>Four-week old female FVB and myr-Akt1 mice were exposed to 1, 2, or 5 Gy γ-radiation. Three days after irradiation total saliva was collected (over a five minute period) following carbachol injection. Statistical analysis was performed using Student's t-test in Microsoft Excel. Results shown are from ten mice per group in the 1 and 2 Gy doses and four mice per group in the 5 Gy dose. Graphs represent averages and SEM from all mice. Significant differences (p≤0.05) were determined using a two sample t-test comparing FVB to myr-Akt1 and significant differences are marked with an asterisk (*).</p

Reduced apoptosis in myr-Akt1 parotid glands following exposure to ionizing radiation.

<p>In A, four-week old female FVB and myr-Akt1 mice were exposed to 0.5, 1, or 5 Gy γ-radiation and parotid salivary glands were removed 24 hours post-irradiation. Tissues were embedded into paraffin and sections were stained for activated caspase-3. The number of caspase-3 positive cells is graphed as a percentage of the total number of cells per field of view. In B, four-week old female FVB and myr-Akt1 mice were exposed to 1 Gy γ-irradiation and parotid salivary glands were removed at 24, 48, 72 and 96 hours. Tissues were processed for activated caspase-3 immunohistochemistry as described in A. Five fields of view were quantitated for each tissue section and graphed using the averages and SEM from three mice per group. Significant differences (p≤0.05) were determined using a two sample t-test comparing FVB to myr-Akt1 and significant differences are marked with an asterisk (*).</p

Preservation of salivary flow rates 30 days after single γ-radiation exposure by myr-Akt1 or injection with IGF1.

<p>Four-week old female FVB, myr-Akt1 and FVB mice injected with 5 µg recombinant IGF1 were exposed to 1 Gy γ-radiation. IGF1 injections were performed immediately prior to radiation exposure as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004663#pone-0004663-g003" target="_blank">Figure 3</a>. Thirty days after exposure to γ-radiation total saliva was collected following carbachol injection as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004663#pone-0004663-g002" target="_blank">Figure 2</a>. Statistical analysis was performed using multiple comparison testing in the SAS system. Results shown are from ten irradiated FVB mice, ten irradiated myr-Akt1 mice and eight IGF1 plus irradiation mice and graphed using the averages and SEM from all mice. Treatment groups with the same letters are not significantly different from each other.</p

Histological analysis of salivary glands from normal and irradiated salivary glands from untreated and IGF1-treated mice.

<p>A) Normal submandibular salivary gland showing basic structure of acini (arrows) and ducts (asterisk). B) Normal sublingual salivary gland. C) Normal parotid gland. D) Submandibular salivary gland thirty days following exposure to 5 Gy radiation. Note area of focal fibrosis and associated inflammatory cells (asterisk). E) Sublingual salivary gland after thirty days following exposure to 5 Gy radiation. No significant morphological change is seen. F) Parotid salivary gland after exposure to radiation. No Significant morphological change is seen. G) Submandibular gland thirty days following injection with 5 ug IGF1. Note the prominent vacuolization of the glandular acini (Arrows). H) Sublingual gland thirty days following injection with 5 ug IGF1 introduction. Note the prominent vacuolization of the glandular acini (Arrows). I) Parotid gland thirty days following injection with 5 ug IGF1. Note the mildly increased vacuolization of the glandular acini, although less than that observed in either the submandibular or sublingual salivary glands (Arrows). J) Submandibular gland thirty days following injection with 5 ug IGF1 immediately prior exposure to 5 Gy radiation. The left part of the photograph (*) shows atrophy of the acini with mild chronic inflammation. The right side of the picture shows increased vacuolization in the viable acini (arrows). K) Sublingual gland thirty days following injection with 5 ug IGF1 immediately prior to exposure to 5 Gy radiation. Increased vacuolization is noted, however, no significant atrophy is seen. L) Parotid gland thirty days following injection with 5 ug IGF1 immediately prior to exposure to 5 Gy radiation. Note the mildly increased vacuolization with no significant atrophy. For Panels A–C, the magnification is 40×, while the magnification D–L is 20×.</p

Sphere Assay with murine salivary glands.

<p>Representative microscope images of spheres grown from submandibular (A) and Parotid (B) glands from 10-week old mice.</p

Label-Retaining Cells in the Adult Murine Salivary Glands Possess Characteristics of Adult Progenitor Cells

<div><p>Radiotherapy is the primary treatment for patients with head and neck cancer, which account for roughly 500,000 annual cases worldwide. Dysfunction of the salivary glands and associated conditions like xerostomia and dysphagia are often developed by these patients, greatly diminishing their life quality. Current preventative and palliative care fail to deliver an improvement in the quality of life, thus accentuating the need for regenerative therapies. In this study, a model of label retaining cells (LRCs) in murine salivary glands was developed, in which LRCs demonstrated proliferative potential and possessed markers of putative salivary progenitors. Mice were labeled with 5-Ethynyl-2′-deoxyuridine (EdU) at postnatal day 10 and chased for 8 weeks. Tissue sections from salivary glands obtained at the end of chase demonstrated co-localization between LRCs and the salivary progenitor markers keratin 5 and keratin 14, as well as <i>kit</i> mRNA, indicating that LRCs encompass a heterogeneous population of salivary progenitors. Proliferative potential of LRCs was demonstrated by a sphere assay, in which LRCs were found in primary and secondary spheres and they co-localized with the proliferation marker Ki67 throughout sphere formation. Surprisingly, LRCs were shown to be radio-resistant and evade apoptosis following radiation treatment. The clinical significance of these findings lie in the potential of this model to study the mechanisms that prevent salivary progenitors from maintaining homeostasis upon exposure to radiation, which will in turn facilitate the development of regenerative therapies for salivary gland dysfunction.</p></div

Molecular markers in salivary gland LRCs.

<p>Representative images of parotid and submandibular glands of 10-day old and 10-week old animals stained for Keratin 14 (A–D), Keratin 5 (E–L), Smooth Muscle alpha Actin (M–P). Q–T) Fluorescence in Situ Hybridization for kit mRNA. EdU LRCs are shown in green, DAPI in blue, and all other markers in red. White arrowheads point at co-localization of each marker with the LRCs in the acinar compartment. Yellow arrowheads point at co-localization of each marker with the LRCs in the ductal compartment. Full size images of every panel are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107893#pone.0107893.s002" target="_blank">Figure S2</a>.</p

Differentiation of Salivary gland Spheres.

<p>A–B) Amylase staining (red) of parotid-derived spheres at days 2–3 in culture. C–D) Confocal images at Z = 0.5 um and Z = 6 um of double staining for amylase (red) and EdU (green) at day 4. Areas in yellow dashed squares are shown in C’ and D’. White arrow points at an amylase-positive cell with traces of EdU. Glands were obtained from mice at 10 weeks of age. E–G) Double immunofluorescence staining for Amylase (red) and EdU (Green) of parotid gland of 10-week old mice. White arrowhead points at LRCs in the acinar compartment; yellow arrowhead points at LRCs in ductal structures.</p

Effect of radiation on salivary gland LRCs.

<p>A) Experimental setup. A single 5 Gy dose of radiation was given at week 4 to EdU-pulsed animals (n = 7). Tissue was collected at week 10. Representative images of EdU staining of parotid (B) and submandibular (C) glands are shown for irradiated animals and untreated controls. EdU positive cells were quantified manually per individual compartment for both treatments and expressed as percentage of LRCs per compartment for both glands (D–E). P values were obtained with 2-sided unpaired t-test per compartment (n = 7 for irradiated groups, n = 12 for parotid untreated control group and n = 6 for submandibular untreated control group). F) Experimental setup. 5 Gy dose of radiation treatment was given 24-hours prior tissue collection to EdU-pulsed animals (n = 3). G–J) Immunofluorescence staining of Activated Caspase-3 (red) and EdU (green) in parotid and submandibular glands. No co-localization is observed.</p

Label retaining assay in murine salivary glands.

<p>A) Label Retaining Assay. At 10 days after birth (P10), FVB mice were pulsed with 4 doses of EdU (100 mg/kg) or BrdU (30 mg/kg) 12 hours apart. Tissue was collected from 10-week old animals. B) LRCs from 3 mice were manually quantified per salivary gland compartment (acinar and ductal). Data are expressed as percentage from the total of cells in each individual compartment for both parotid and submandibular glands. C) Comparison of labeling efficiency between EdU and BrdU. Data are shown as percentage of LRCs per individual compartment. A 2-sided unpaired T-test was performed for analysis (n = 5 per group). D–E) Representative images of parotid and submandibular glands of BrdU-pulsed animals. LRCs in the acinar compartment (a) are shown with white arrowheads. LRCs in ductal compartment (d) are pointed with black arrows. Example of ductal compartment is delineated by dashed line and pointed with black arrowhead F–K) Representative fluorescent images of salivary glands from EdU-pulsed animals. EdU LRCs are shown in green and DAPI in blue.</p