Analyzing the Effects of Radiation on Salivary Glands

Objectives: Patients of head and neck cancer are treated with a combination of radiation, chemotherapy and surgery. Radiotherapy frequently causes permanent loss of the secretory acinar cells and leads to hyposalivation. The available treatments are temporary and palliative. To understand the radiosensitivity of salivary glands, we have investigated how irradiation (IR) leads to tissue hypofunction.
Methods: We used non-irradiated and bilaterally irradiated salivary glands of 6 to 8 week-old female C57BL/6J mice. We collected whole stimulated saliva at 3, 24 and 48 hours (short times), and weekly for 12 weeks after IR. Salivary glands were unilaterally irradiated for histological analysis and quantitative PCR, and stained for MIST1, ZO-1, AQP5, M₃R and gamma H2AX. Acinar and ductal structures were isolated from submandibular gland (SMG), and used to measure [Ca²⁺]_i.
Results: Saliva volume was reduced at 3 hours after IR, recovered by 24 hours, but decreased again by 48 hours. From 1 to 12 weeks after IR, saliva volume decreased in a time-dependent manner. Histological analysis showed no major changes at short times after IR. However, mRNA expression was disrupted. The immunohistochemical staining for gamma H2AX revealed DNA damage was increased, and mRNA expression levels of several inflammation factors were up-regulated at 3 hours. In contrast, M₃r and Aqp5 mRNA expression levels were down-regulated, but recovered by 24 or 48 hours. By 3 months after IR, acinar cells were found in isolated clusters. Notably, calcium imaging revealed that these clusters still showed a change of [Ca²⁺]_i following stimulation with carbachol or ATP.
Conclusions: The short-term response of SMG to IR includes increased DNA damage and expression of inflammatory molecules. Expression levels of several functional proteins are rapidly altered. However, we report that clustered acinar cells surviving at 3 months after IR retain secretory function.