**3. Animal Models Provide Mechanistic Insight into Radiation-Induced Salivary Gland Dysfunction**

Preclinical animal models have provided many clues to the underlying mechanisms of radiation-induced salivary gland damage. Previous studies from our labs and others have utilized mouse models of ionizing radiation (IR)-induced salivary gland damage to show that acute hyposalivation detected immediately after IR and before onset of obvious gland damage is associated with aberrant calcium signaling, rapid apoptosis of acinar cells, DNA damage and enhanced reactive oxygen species (ROS) production [1,2,4,8,9,43–48]. Sustained IR-induced salivary dysfunction is additionally impacted by inflammation, neuronal and vascular changes, senescence or dysfunction of adult progenitor cell populations, cytoskeletal rearrangements and replacement of normal parenchyma with fibrotic tissue [1,44,45,49–55] (Figure 1). In mouse and rat models, the events giving rise to early loss of salivary gland function occur within the first 3 days following IR. Thus, we have defined acute time points for animal models as the first 3 days and chronic time points as ≥30 days post-IR. Acute IR-induced hyposalivation in mice is observed within the first few hours following IR with a marked loss of acinar cells, a decrease in saliva flow and altered saliva composition [1,4,56]. Available research investigates chronic hyposalivation anywhere from 30 to 300 days post-IR with fibrosis developing between 4 and 6 months [1,44] and as early as 30 days post-fractionated IR in minipigs [57]. Here, we summarize and discuss the signaling processes involved in IR-induced hyposalivation during both acute and chronic time points post-IR.

**Figure 1.** Timeline of Radiation-Induced Changes in the Rodent Salivary Gland. Following irradiation, rodent models show decreased saliva flow at approximately 3 days and a loss of amylase secretion reported as early as 4 days in rats post-IR [2,56,58]. In the acute phase, immediate DNA damage [6,59], rapid apoptosis of acinar cells [4,6,58], and elevated levels of intracellular calcium [45,46] and reactive oxygen species [45,46,60,61] contribute to acute loss of glandular function following irradiation. This period is also marked by release of ATP, which activates the P2X7 receptor (P2X7R), and P2X7R-dependent release of prostaglandin E2 (PGE2) in murine parotid cells [48]. During the transition phase, loss of apical/basolateral polarity as a result of PKCζ inactivation [55,62,63], increases nuclear Yes-associated protein (Yap) levels [55,64], compensatory proliferation [62,65,66], cellular senescence [60,67,68], and cytoskeletal rearrangements [50,69], which contribute to long-term dysfunction. Changes in innervation and vasculature have been reported as early as 24 h post-IR [53], as well as at chronic time points [54,70]. Though inconsistently reported, fibrosis generally appears between 4 and 6 months following irradiation [1,44,71]. There is little information regarding the effect of irradiation on the immune landscape of the salivary glands in rodent models, although one study indicates changes at 300 days post-IR in mice [54].
