Aim: Investigate whether juvenile exercise could induce a long‐term muscle memory, boosting the effects of exercise in adults.Methods: We devised a 5‐week climbing exercise scheme with food reward administered to male juvenile rats (post‐natal week 4–9). Subsequently, the animals were subjected to 10 weeks of detraining (week 9–19) without climbing and finally retraining during week 19–21.Results: The juvenile exercise increased fiber cross‐sectional area (fCSA) by 21% (p = 0.0035), boosted nuclear accretion by 13% (p = 0.057), and reduced intraperitoneal fat content by 28% (p = 0.007) and body weight by 9% (p = 0.001). During detraining, the fCSA became similar in the animals that had been climbing compared to naive controls, but the elevated number of myonuclei induced by the climbing were maintained (15%, p = 0.033). When the naive rats were subjected to 2 weeks of adult exercise there was little effect on fCSA, while the previously trained rats displayed an increase of 19% (p = 0.0007). Similarly, when the rats were subjected to unilateral surgical overload in lieu of the adult climbing exercise, the increase in fCSA was 20% (p = 0.0039) in the climbing group, while there was no significant increase in naive rats when comparing to the contralateral leg.Conclusion: This demonstrates that juvenile exercise can establish a muscle memory boosting the effects of adult exercise. The juvenile climbing exercise with food reward also led to leaner animals with lower body weight. These differences were to some extent maintained throughout the adult detraining period in spite of all animals being fed ad libitum, indicating a form of body weight memory.

Cytokines are mediators of inflammation that could lead to fibrosis. The aim was to monitor cytokine levels in saliva and serum after locally fractionated radiotherapy of the head and neck in mice and investigate associations with salivary gland fibrosis and hyposalivation. C57BL/6 mice were randomized to sham or X-ray irradiation of 66 Gy in 10 fractions over 5 days. Blood and saliva were collected on days −7, 5, 35, 80, and 105 following cytokine analysis. The harvested submandibular salivary gland was assessed for the presence of fibrosis. Decision tree regression analysis was used to investigate whether cytokine levels could predict late endpoints in terms of hyposalivation or fibrosis. Significant formation of fibrosis in gland tissue and reduced saliva production was found after irradiation. The pro-inflammatory cytokines IL-1α, TNF, TIMP1, G-CSF, KC, and MIP-1α showed increased levels in saliva in irradiated mice and a strong correlation with late endpoints. The decision tree analysis largely separated controls from irradiated animals, with IL-1α being the strongest predictor. Pro-inflammatory cytokines in saliva, but not in serum, were associated with late endpoints. This indicates that cytokine expression in saliva is a good biomarker for local salivary gland damage with IL-1α as the strongest single predictor.

Extracellular vesicles (EVs) are membrane-bound particles released from cells, and their cargo can alter the function of recipient cells. EVs from X-irradiated cells have been shown to play a likely role in non-targeted effects. However, EVs derived from proton irradiated cells have not yet been studied. We aimed to investigate the proteome of EVs and their cell of origin after proton or X-irradiation. The EVs were derived from a human oral squamous cell carcinoma (OSCC) cell line exposed to 0, 4, or 8 Gy from either protons or X-rays. The EVs and irradiated OSCC cells underwent liquid chromatography–mass spectrometry for protein identification. Interestingly, we found different protein profiles both in the EVs and in the OSCC cells after proton irradiation compared to X-irradiation. In the EVs, we found that protons cause a downregulation of proteins involved in cell growth and DNA damage response compared to X-rays. In the OSCC cells, proton and X-irradiation induced dissimilar cell death pathways and distinct DNA damage repair systems. These results are of potential importance for understanding how non-targeted effects in normal tissue can be limited and for future implementation of proton therapy in the clinic.

Background Radiotherapy of head and neck cancer may cause detrimental late side effects such as fibrosis and hyposalivation. Our aim was to investigate late radiation-induced cellular and molecular changes of the salivary glands after fractionated irradiation to the head and neck in a murine model. Methods 12-week-old female C57BL/6J mice were irradiated with X-rays to a total dose of 66 Gy, given in 10 fractions over 5 days. The radiation field covered the oral cavity and major salivary glands. Salivary gland function was assessed by collecting saliva at baseline and at various time points after irradiation. The submandibular (SMG), sublingual (SLG), and parotid glands (PG) were dissected at day 105. Using different staining techniques, morphological, cellular, and molecular changes were investigated in the salivary glands. Results Saliva production was significantly reduced in irradiated compared to control mice at day 35, 80, and 105. We observed a significant decrease in total gland area and a significant increase in fibrotic area in irradiated compared to control SMG at day 105. Atrophy of acinar cells was observed in all irradiated SMG and SLG. Increased amount of chronic inflammatory cells, increased cell proliferation and altered expression of apoptotic markers were found in atrophic areas of irradiated glands. Conclusion Acinar and duct cells in irradiated salivary glands show increased cell proliferation and altered expression of apoptotic markers, proposing an attempt to overcome or withstand tissue damage caused by irradiation. This suggests a potential for regeneration of salivary glands after radiation therapy.

Background and purpose: Radiotherapy of head and neck cancer may cause detrimental late side effects such as fibrosis and hyposalivation. We investigated mouse salivary glands after fractionated irradiation, with the aim to elucidate cellular plasticity and potential regeneration. Methods: 12-week-old female C57BL/6JRj mice were irradiated with X-rays to a total dose of 66 Gy, given in 10 fractions over 5 days. The radiation field covered the oral cavity and major salivary glands. The submandibular (SMG), sublingual (SLG), and parotid glands (PG) were dissected at day 100 after finishing irradiation. Using different histological staining techniques, morphological, cellular, and molecular changes were investigated in irradiated and control SMG, SLG, and PG. Results: Atrophy of acinar cells was observed in irradiated SMG and SLG, but not in PG. Surprisingly, the acinar atrophy was confined to one region of each irradiated gland. These atrophic regions showed distinct cellular and molecular compositions compared to non-atrophic regions and control glands. Increased expression of the ductal cell markers keratin 5 and 19 (K5 and K19), along with increased percentages of proliferating myofibroblasts, fibroblasts, leukocytes, and acinar cells (Nkcc1+) were observed in the atrophic regions compared to controls. In addition, some of the K19+ and K5+ duct-like cells also co-expressed Nkcc1. Interpretation: Through a detailed histological assessment of the cellular and molecular changes in the major salivary glands of irradiated mice, we observed signs of cellular plasticity where ductal cells adopt an acinar cell phenotype upon irradiation. This suggests a regenerative potential of salivary glands after irradiation.

Radiotherapy of head and neck cancer is known to cause both early and late-occurring toxicities. To better appraise normal tissue responses and their dependence on treatment parameters such as radiation field and type, as well as dose and fractionation scheme, a preclinical model with relevant endpoints is required. 12-week old female C57BL/6J mice were irradiated with 100 or 180 kV X-rays to total doses ranging from 30 to 85 Gy, given in 10 fractions over 5 days. The radiation field covered the oral cavity, swallowing structures, and salivary glands. Monte Carlo simulations were employed to estimate tissue dose distribution. The follow-up period was 35 days, in order to study the early radiation-induced effects. Baseline and post irradiation investigations included macroscopic and microscopic examinations of the skin, lips, salivary glands, and oral mucosa. Saliva sampling was performed to assess the salivary gland function following radiation exposure. A dose dependent radiation dermatitis in the skin was observed for doses above 30 Gy. Oral mucositis in the tongue appeared as ulcerations on the ventral surface of the tongue for doses of 75-85 Gy. The irradiated mice showed significantly reduced saliva production compared to controls. In summary, a preclinical model to investigate a broad panel of normal tissue responses following fractionated irradiation of the head and neck region was established. The optimal dose to study early radiation-induced effects was found to be around 75 Gy, as this was the highest tolerated dose that gave acute effects similar to what is observed in cancer patients.