Rat model of long term effects after fractionated (2Gy/day) 60Gy irradiation of rat liver

Background:The application of radiotherapy in liver tumours is limited by supposed sensitivity of liver tissue (RILD). However liver focused application of a high single dose irradiation (HDI) on healthy liver has mild acute consequences. Consequences of fractionated irradiation (FI) of the rat liver have not yet been studied.

Materials and Methods: Rats were exposed to a liver-focused (by CT-scan) fractionated irradiation (total dose of 60Gy, 2Gy/day and 5 days/week) or high single dose irradiation (25Gy) on liver tissue. Irradiated animals were sacrificed at 1, 3, 6, 12, 24, and 48 and 96h (25Gy) at 3 months after beginning the experiments (single dose and fractionated dose). Blood cells, serum, liver were preserved after sacrifing the animals. Tissue samples were used for immunohistochemical studies, for protein and RNA isolation. Sera were used to monitor tissue damage. Results: All animals were alive 3 months after fractionated delivery or high single dose irradiation. Animals treated with FI or HDI reached the maximal weight 15 and 9 weeks after the irradiation. Although liver weight was reduced, liver weight/body weight ratio in irradiated animals was not significantly different compared to that of control animals. Three months after FI and HDI, macroscopic evaluation of the liver showed homogenously diffuse multiple small “scars” on the surface of the organ when compared with controls. At time of sacrifice, fractionated liver irradiation induced a significant increase of alcaline phosphatase (AP), whereas single dose irradiation induced a significant increase of LDH (lactatdehydrogenase) and of AST (aspartate aminotransferase). Microscopic examination (HE staining) of the tissue showed no obvious changes. Immunohistology showed the presence of an increased number of neutrophils distributed through the liver parenchyma after FI and HDI. The number of bile ducts (CK-19 positivity) was increased in the FI livers while no difference compared to controls was found in the liver of single dose irradiated animals. Although an increased number of granulocytes (elastase positive cells) was detected in the vessel wall of the portal field, proteins of the “priovisional clot” (fibronectin, fibrin and VWF) were not increasingly deposited. No signs of fibrosis were found in the “inflamed” areas at three months after irradiation.

Conclusions: Selective high single doses or fractionated irradiation of rat liver are well tolerated. The mechanisms underlying long-term microscopic changes such as presence of granulocytes in liver parenchyma in both models and of increased biliary ducts after fractionated irradiation have to be elucidated. The lack of ”provisional clot” formation in the inflamed areas after liver irradiation could be determined by an effective counteraction between MMPs and TIMPs, which gene-expression increased just 3 hours after single dose irradiation but not three months after FI or HDI.