A rat model of radiation injury in the mandibular area

Abstract

Background: Radiation technology focuses on delivering the radiation as precisely as possible to the tumor, nonetheless both acute and long-term damage to surrounding normal tissue may develop. Injuries to the surrounding normal tissue after radiotherapy of head and neck cancer are difficult to manage. An animal model is needed to elucidate good treatment modalities. The aim of this study was to establish a rat model where a certain radiation dose gives reproducible tissue reactions in the mandibular area corresponding to injuries obtained in humans.

Method: The left mandible of male Sprague Dawley rats was irradiated by external radiotherapy (single fraction 15 Gy, total dose 75 Gy) every second week five times. Endpoint was six weeks after last radiation treatment, and the test group was compared to non-irradiated controls. Morphological alterations of the soft tissues, bone and tooth formation, as well as alterations of salivation, vascularity and collagen content were assessed. An unpaired, non-parametric Mann-Whitney test was used to compare the statistical differences between the groups.

Results: Analysis of the soft tissues and mandible within the radiation field revealed severe unilateral alopecia and dermatitis of the skin, extensive inflammation of the submandibular gland with loss of serous secretory cells, hyperkeratinization and dense connective fiber bundles of the gingival tissue, and disturbed tooth development with necrosis of the pulp. Production of saliva and the vascularity of the soft tissues were significantly reduced. Furthermore, the collagen fibril diameter was larger and the collagen network denser compared to non-irradiated control rats.

Conclusion: We have established an animal model of radiation injury demonstrating physiological and histological changes corresponding to human radiation injuries, which can be used for future therapeutic evaluations.

Fig. 1

Schematic presentation of the radiation field. The box indicates the area of the head and neck region irradiated, and the cross indicates where the isocenter of radiation is targeted to the mandible

Fig. 2

Salivation six weeks after 5 × 15 Gy radiotherapy. Salivation measured as grams saliva per 15 min in control (n = 11) and irradiated (n = 11) rats. The horizontal line indicates the mean values. *indicates p = 0.0004 by an unpaired, non-parametric Mann–Whitney test

Fig. 3

Changes in salivary gland morphology six weeks after 5 × 15 Gy radiotherapy. a H&E stained section of the left submandibular gland of non-irradiated control rats. The histologic examination primarily demonstrated acini and excretory ducts without any inflammatory reactions. Scale bar indicates 100 μm. b H&E stained section of the left submandibular gland of 5 × 15 Gy irradiated rats. The histologic examination revealed atrophy of the acini, dilated excretory ducts and blood vessels, hemosiderin and chronic inflammation. Scale bar indicates 100 μm

Fig. 4

Changes in mandibular morphology six weeks after 5 × 15 Gy radiotherapy. The non-irradiated control mandible demonstrated darker coloration (left) after five weeks of decalcification with 10 % EDTA compared to the irradiated mandible (right)

Fig. 5

Morphologic changes of gingiva and periodontal membrane six weeks after 5 × 15 Gy radiotherapy. H&E stained sections of the gingiva of non-irradiated control rats (a) and 5 × 15 Gy irradiated rats (b). The histologic examination demonstrates epithelial transformation from slightly parakeratinized in controls to hyperkeratinized and hyperplasic in irradiated rats. This is also evident in the epithelium closest to the tooth. In addition the connective tissue is more fibrous and contains fewer cells after irradiation. Scale bar indicates 200 μm. (c) H&E stained sections of the periodontal membrane of non-irradiated control rats. The periodontal membrane is richly vascularized with fibers organized for proper attachment of tooth to bone. Scale bar indicates 200 μm. (d) H&E stained sections of the periodontal membrane of 5 x15 Gy irradiated rats. The irradiated periodontal membrane demonstrates fibrous connective tissue with few cells, unorganized fibers and infiltration of mononuclear inflammatory cells. Scale bar indicates 200 μm

Fig. 6

Morphologic changes of teeth under development six weeks after 5 × 15 Gy radiotherapy. H&E stained sections of developing teeth of non-irradiated control rats (A, a) and 5 × 15 Gy irradiated rats (B, b). The histologic examination demonstrates disturbed enamel, dentin and cementum formation after irradiation, as seen from the overview section B) compared to A). Scale bar indicates 1 mm. Furthermore, disturbed and unorganized dentin formation and necrosis of odontoblasts and pulp was evident after irradiation, as seen from the close up section b) compared to a). Scale bar indicates 100 μm

Fig. 7

Vascular density six weeks after 5 × 15 Gy radiotherapy. The average vascular density by immunohistochemistry with CD31 in the skin (a), the muscle (b) and the submandibular gland (c) of control (n = 5) and irradiated (n = 5) animals presented as mean ± SD. *indicates p < 0.02 and **indicates p < 0.01 by an unpaired, non-parametric Mann–Whitney test

Fig. 8

Collagen density and diameter six weeks after 5 × 15 Gy radiotherapy. (a) The average fibril density in control (n = 5) and irradiated (n = 5) skin samples from the outer part of the radiation field presented as mean ± SD. (b) The frequency distribution of collagen fibril diameter demonstrates a minor displacement towards larger fibril diameter in the skin after radiotherapy (n = 5) compared to control animals (n = 5)

Fig. 9

Changes in skin morphology six weeks after 5 × 15 Gy radiotherapy. H&E stained section of the skin epidermis and dermis of non-irradiated control rats (a) and 5 × 15 Gy irradiated rats (b). The histologic examination demonstrates alopecia and a dense network of collagen fibers with few cells, increased fibrosis, of the dermis after irradiation. Furthermore, the stratum corneum, the outermost layer of the epidermis, is thicker after irradiation. Scale bar indicates 200 μm