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Irradiation of the head & neck (H&N) region may cause a variety of normal tissue damages, and we have established a preclinical mouse model that allows for studying several endpoints (Juvkam et al., 2022). The aim of the present work was to investigate the systemic responses in terms of cytokine expression in serum and saliva and correlate them to macro- and microscopically observed effects.

C57BL/6 female 12-week old mice were randomised to sham treatment or high-dose irradiation of 75 Gy in 10 fractions over 5 days. The radiation field covered the oral cavity, swallowing structures, and salivary glands. Baseline and post-RT investigations included micro- and macroscopic examinations of the skin, oral mucosa and salivary gland as well as blood and saliva collection. Cytokine analysis of serum and saliva was performed with the 12-Plex Luminex Mouse Discovery Assay kit (cat. no.: LXSAMSM-12, Bio-Techne Ltd, Abingdon, UK) and recorded on a Luminex IS 200 instrument (Bio-Rad, Hercules, CA, USA). Cytokines were assessed at baseline and at day 35, 80, and 105 post treatment.

The analysis detected the following cytokines and chemokines in serum and saliva samples: IL-1α, IL-6, KC, MIP-1α, G-CSF, TNF, IL-12 and TIMP-1. Increased levels of IL-1α in saliva was found on day 35 and 105 after onset of irradiation compared to controls. IL-1a has been reported to activate inflammatory processes as well as to contribute to fibrosis (Cooper et al., 1995). TIMP-1, which has been associated with fibrosis (Ulrich et al., 2003), was also upregulated in saliva samples in the irradiated group on day 35, 80 and 105. In fact, increased fibrotic areas were observed in submandibular glands in the irradiated mice on day 105. TNF was also increased in serum and saliva samples from irradiated group on day 35 and 105. TNF has previously been linked to xerostomia and oral mucositis in H&N cancer patients (Russo et al., 2016). There was a tendency towards an increased level of G-CSF in serum on day 35 in irradiated group. G-CSF is reported to reduce the severity of mucositis in patients after chemotherapy by stimulating the stem cells proliferation (Cooper et al., 1995) which might be a sign of regeneration in oral mucosa. Oral mucositis was indeed found in the irradiated cohort.

The cytokine profiles of serum and saliva samples from mice were analyzed following fractionated radiation treatment of H&N in the search for biomarkers specific for radiation-induced effects. To our knowledge, no one has previously performed cytokine analyses in mouse saliva after irradiation. Rather early after irradiation (day 35), increased levels of salivary cytokines involved in fibrosis were detected (IL-1α, TIMP-1), and fibrosis was demonstrated in the salivary glands (day 105). Also, a cytokine involved in stem cell proliferation could point to healing of oral mucositis (G-CSF). The findings may shed light on underlying mechanisms leading to the manifestation of early and late side effects.