*3.6. Neuronal and Vascular Changes*

Alternative pathways that may be influencing IR-induced hyposalivation include damage to non-epithelial tissue within the salivary gland, such as neurons or vasculature. Importantly, parasympathetic neurons have been suggested to play a role in salivary gland regeneration post-IR damage. During embryonal development, SMGs that receive IR exposure exhibit increased epithelial and neuronal cell apoptosis at 24 and 72 h post-IR, respectively [53]. Neurturin (NRTN) is essential for parasympathetic neuronal development and survival, including in murine salivary glands [53,79,80]. Delivery of human NRTN by adenovirus serotype 5 vector (AdNRTN) to murine SMGs 24 h prior to IR (5 Gy) preserved function at 60 days post-IR [81]. Additionally, NRTN delivery by adeno-associated virus serotype 2 (AAV2) in CH3 mice and minipigs prior to IR improved saliva flow rate at 300 days and 16 weeks, respectively [54]. Treatment with NRTN has been shown to enhance parasympathetic innervation and reduce epithelial apoptosis post-IR, consistent with increased end bud formation within SMGs, supporting a potential regenerative role for neurotrophic signaling in the repair of IR-induced salivary gland damage [53]. Rats administered 18 Gy IR exhibit reduced levels of the neurotrophic factors brain-derived neurotrophic factor (BDNF) and NTRN as well as decreased levels of the neurotrophic factor receptor, GRFα2, acetylcholinesterase and neurofilament staining in SMGs, which could be reversed with α-lipoic acid treatment [70]. In minipigs following 20 Gy IR, there is a reduction in levels of BDNF, NTRN, acetylcholinesterase and the acetylcholine receptor, Chrm1, indicative of decreased parasympathetic innervation, responses that can be reversed with intraglandular adenoviral delivery of Shh at 4 weeks post-IR [52].

As for the vasculature in salivary glands, endothelial cell death occurs 4 h after 15 Gy IR, measured as an increase in caspase-3 cleavage in platelet endothelial cell adhesion molecule (CD31) positive cells, which correlates with an overall reduction in microvessel content in salivary gland tissue sections, responses modulated by treatment with the ROS scavenger Tempol for 10 min prior to IR in mice [82]. Minipigs receiving 20 Gy IR exhibit reduced blood flow and CD31 and vascular endothelial growth factor (VEGF) levels in parotid glands 20 weeks post-IR, indicative of the microvascular damage induced by IR [52]. At 90 days following 15 Gy IR, mice show an increase in blood vessel dilation in SMGs that coincides with a reduction in total capillary volume and diminished salivary function. Interestingly, co-treatment of mice with FMS-like tyrosine kinase-3 ligand (Flt-3L), stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) (i.e., F/S/G treatment) one month following IR promoted increased endothelial cell division, capillary content and endothelial nitric oxide synthase and endoglin expression due to bone-marrow-derived immune cell recruitment and activation of endothelial cells by F/S/G, which correlated with increased acinar cell number and saliva flow at day

90 [83]. Overall, these data suggest that repair of acute and chronic IR-induced damage to salivary glands likely requires contributions from neuronal and vascular cells.
