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. 2019 Jun 25;52(3):45-58.
doi: 10.1267/ahc.19006. Epub 2019 Jun 19.

Long-term Pilocarpine Treatment Improves Salivary Flow in Irradiated Mice

Affiliations

Long-term Pilocarpine Treatment Improves Salivary Flow in Irradiated Mice

Akie Taniguchi et al. Acta Histochem Cytochem. .

Abstract

Radiation therapy for head and neck cancer frequently causes salivary gland dysfunction. Pilocarpine is a clinically approved and effective drug that induces saliva secretion, thereby keeping the oral mucosa moist and reducing discomfort in patients, but the effect is transient. We expected that this drug also has beneficial long-term effects that maintain the integrity of salivary glands by reducing, for instance, apoptosis. Here, we examined the effects of long-term pilocarpine administration in irradiated mice. The results indicated that long-term pilocarpine administration significantly improved salivary flow in irradiated mice, suggesting the potential beneficial effects of long-term administration. To elucidate the underlying mechanism, we analyzed the histology, apoptosis, and proliferation of acinar cells, and the expression of functional membrane proteins such as transmembrane member 16A, aquaporin-5, and Na-K-Cl cotransporter. Long-term pilocarpine treatment seemed to decrease irradiation-induced apoptosis, although the change was not statistically significant. The present results indicated that long-term administration of pilocarpine has beneficial effects on salivary flow in irradiated mice, and suggested that long-term administration possibly decreases apoptosis in irradiated salivary glands.

Keywords: apoptosis; pilocarpine; radiation; salivary glands; xerostomia.

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Conflict of interest statement

VThe authors declare that there are no conflicts of interest.

Figures

Fig. 1.
Fig. 1.
Animal groups and administration procedure. Animals were divided into three groups. CTR: control group. IRD: irradiated group. IRD+Pilo: irradiated and pilocarpine-administered group. Pilocarpine administration was initiated 5 days before irradiation. Salivary flow was measured at 30 and 63 days after irradiation. Tissue sampling was performed at 65 days after irradiation.
Fig. 2.
Fig. 2.
Salivary flow. Pilocarpine-stimulated salivary flow per body weight at 30 days (A) and 63 days (B) after irradiation. Data are expressed as dot and box plots.
Fig. 3.
Fig. 3.
Hematoxylin-eosin staining. Typical images of the parotid (A, B, and C) and submandibular (D, E, and F) glands from each group are shown. Some of acini and ducts are indicated by “a” and “d”, respectively. Mononuclear infiltrates and increased interstitial fibers in increased interstitial spaces (asterisks) are indicated by arrows and arrowheads, respectively, in B and C.
Fig. 4.
Fig. 4.
Enzyme immunohistochemistry for cleaved caspase-3. Typical images of the parotid (A, B, and C) and submandibular (D, E, and F) glands from each group are shown. Arrows indicate positive acinar cells. Arrowheads indicate abnormally enlarged parotid acinar cells with enlarged nuclei.
Fig. 5.
Fig. 5.
Enzyme immunohistochemistry for Ki67. Typical images of the parotid (A, B, and C) and submandibular (D, E, and F) glands from each group are shown. Arrows indicate positive acinar cells. Arrowhead indicates abnormally enlarged parotid acinar cells with enlarged nuclei.
Fig. 6.
Fig. 6.
Immunofluorescence analysis of TMEM16A. Typical fluorescence images of the parotid (A, B, and C) and submandibular (D, E, and F) glands from each group are shown. Each image was captured under identical conditions. Insets show higher magnification views of a section of individual images. Arrowheads indicate nonspecific labeling in ducts (see Fig. 12)
Fig. 7.
Fig. 7.
Immunofluorescence analysis of AQP5. Typical fluorescence images of the parotid (A, B, and C) and submandibular (D, E, and F) glands from each group are shown. Each image was captured under identical conditions. Insets show higher magnification views of a section of individual images.
Fig. 8.
Fig. 8.
Immunofluorescence analysis of NKCC1. Typical fluorescence images of the parotid (A, B, and C) and submandibular (D, E, and F) glands from each group are shown. Each image was captured under identical conditions. Insets show higher magnification views of a section of individual images.
Fig. 9.
Fig. 9.
Immunofluorescence localization of AQP5 and NKCC1. Double labeling with guinea pig anti-AQP5 (AffGPTM41, green) and rabbit anti-NKCC1 (magenta) in the CTR parotid gland. AQP5 is localized chiefly to the apical membrane, including intercellular secretory canaliculi, and weakly to the basolateral membrane, whereas NKCC1 is chiefly localized to the basolateral membrane.
Fig. 10.
Fig. 10.
Immunofluorescence localization of TMEM16A, AQP5, and NKCC1 in abnormal cells. Sections from IRD parotid glands were fluorescently labeled. Fluorescence images of TMEM16A, AQP5, and NKCC1 shown in green are merged with those for DAPI in magenta. Abnormally enlarged parotid acinar cells with enlarged nuclei are indicated by arrows.
Fig. 11.
Fig. 11.
Specificity of antibodies confirmed by immunoblotting and immunofluorescence. The position of Novex sharp pre-stained protein standards (kDa) is marked on the right of each blot (A, F, K, and L). Bands near the expected size of each target protein are indicated by arrows in each blot (A, F, K, and L). A. Immunoblotting of control HeLa and cisplatin-treated HeLa cell homogenates with cleaved caspase-3 antibody. Positive bands are only seen in cisplatin-treated HeLa cell homogenates. B–E. Immunofluorescence images of control and cisplatin-treated HeLa cells incubated with cleaved caspase-3 antibody (B and D) and the corresponding Nomarski differential interference-contrast images (C and E). Specific labeling is only seen in cisplatin-treated, apoptosis-induced, HeLa cells. F. Immunoblotting of HeLa cell homogenates with Ki67 antibody. G–J. Immunofluorescence images of HeLa cells incubated with (G) or without (I) Ki67 antibody and the corresponding Nomarski differential interference-contrast images (H and J). K, L. Immunoblotting of mouse parotid gland homogenates with TMEM16A (K) or NKCC1 (L) antibodies.
Fig. 12.
Fig. 12.
Histochemical controls for immunolabeling. A, B. As a control for cleaved caspase-3 and Ki67 staining, sections from IRD parotid (A) and submandibular (B) glands were incubated with blocking solution instead of primary antibody solution. C, D. As a control for AQP5 staining, sections were incubated with antibody solution preabsorbed with an antigen peptide. E, F. As a control for TMEM16A and NKCC1 staining, sections were incubated with blocking solution instead of primary antibody solutions. Arrows indicate nonspecific labeling in ducts.

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