X-radiation does not affect the dental pulp: a morphological study in rats

Introduction: Radiotherapy is one of the methods used as a treatment for malignant tumors in the head and neck region and it can cause tissue damage in the irradiated areas. In head and neck radiotherapy, teeth are often included within the irradiation area and, consequently, the dental pulp; which receives high doses of radiation. Objective: To evaluate the effects of ionizing radiation on the pulp tissue of rat teeth. Methodology: A double-blind experimental assay with 35 Albinus Wistar rats divided into seven groups was performed; one control group, three groups irradiated with 15 Gy, and three groups irradiated with 25 Gy. The irradiated groups were submitted to a single dose of radiation and sacrificed 24 hours, 7 days, and 22 days after irradiation, respectively. The samples were evaluated for the morphological presence of inflammatory infiltrate, edema, necrosis, fibrosis, and degeneration of blood vessels. Statistical analysis was performed using the Kruskal-Wallis and Dunn tests with p < 0.05. Results: Hyaline degeneration of the pulp blood vessels in the irradiated teeth was statistically significant in all irradiated groups. Inflammatory infiltrate, edema, necrosis or fibrosis was not observed. Conclusion: A single X-radiation dose is not able to affect the dental pulp connective tissue in the long term with no clinical damage.


Introduction
Head and neck squamous cell carcinoma (SCC) is one of the most common types of cancer in many parts of the world and it is associated with significant morbidity due to late diagnosis and, consequently, the established modality treatment 1 . Currently, the combination of surgery, radiotherapy, and chemotherapy is recommended for better local and regional control of tumor growth 2 . However, head and neck radiotherapy is associated with a series of acute and chronic toxicities on the tissues of the oral cavity due to the involvement of both the tumor component and the normal anatomical structures of the maxillofacial region in the primary radiation field [3][4][5] . In this context, teeth are also commonly present in the irradiated area during the treatment of head and neck tumors, and consequently; the tooth pulp, a loose connective tissue rich in vessels and nerves, receives high doses of radiation 2-6 .
The effects of ionizing radiation, such as transient or permanent changes in microcirculation and pulp innervation related to changes in pulp sensitivity have been previously reported 6 . In addition, some recent studies have also suggested the possible radiogenic destruction of the pulp as an important event for the etiology of caries related to radiation 6 . However, the absence of morphological changes in the pulp tissue as a direct result of radiotherapy has also been pointed out 7 .
Therefore, due to the lack of a consensus in the literature and contradictory results 2,7-14 , we aim to evaluate the effects of ionizing radiation on the pulp tissue of rat teeth using an animal model to verify the direct effects of radiation therapy on the dental pulp morphology exclusively.

Material and methods
The research project was approved by the Ethics Committee on the Use of Animals of the State University of Campinas, Brazil with protocol no. 2998-1. All procedures performed in the present study involving animals were in accordance with the ethical standards of the institution or practice at which the study was conducted. A sample of 35 male 12-week-old Albinus Wistar rats were kept in polycarbonate cages in groups of five animals in controlled temperature and humidity environments with periods of 12 hours clarity and 12 hours darkness with the appropriate feed and water ad libitum. The animals were divided into seven groups (n = 5). Group A was not irradiated and constituted the control group. Groups B, C, and D received a single dose of 15 Gy of X-radiation and groups E, F, and G received a single dose of 25 Gy of X-radiation 15 . Prior to irradiation, we intramuscularly administered anesthesia with 80 mg/kg of Ketamine Hydrochloride (Dopalen®, Agribrands do Brasil Ltda., Paulínia, SP, Brazil) and 8 mg/kg of Xylasine Hydrochloride (Rompum®, Bayer SA, São Paulo, SP, Brazil).
Animals in the irradiated groups B, C, and D received a single dose of 15 Gy of X-radiation from a linear patient accelerator (Clinac 6/100®, Varian Medical Systems, Palo Alto, CA, USA) with 6 MeVat a distance focal length of 100 cm. Animals in groups E, F, and G underwent the same procedure, but received a single dose of 25 Gy. The beam was collimated to restrict irradiation to the head and neck region (area of 15 X 30 cm). No secondary collimation was used. The animal irradiation protocol was adapted as from Ramos-Perez et al. 15 . Animals in the control group and in the irradiated groups were sacrificed by anesthetic deepening at 24 hours, 7 days, and 22 days after irradiation. Animals in the control group were sacrificed together with the first irradiated group.
The mandibles were dissected and stored in 10% buffered formalin solution for at least one month in identified containers. The right hemi-mandibles were processed and embedded in Paraplast Plus: Tissue Embedding Medium (McCormick™ Scientific, Leica Biosystems, St. Louis, MO, USA), and subsequently sectioned in cross sections of 7 μm thickness using a Leica RM 2155 microtome (Leica, Wetzlar, Germany). Six slides were prepared with three slices of 5 μm each, totaling 42 slides, which were stained with hematoxylin and eosin, and assembled in Canada balsam. The most representative cut showing the three lower molar teeth with the coronal and root portion and without processing artifacts was chosen for analysis.
The slides were morphologically analyzed in an optical microscope (Carl Zeiss Microscopy GmbH, Axio Lab, Jena, Germany) by two oral pathologists (MRV and ILF) who evaluated the presence of the following pathological changes: inflammatory infiltrate, edema, necrosis, fibrosis, and degeneration of blood vessels 2,16 . The investigators were blinded to the origin of the slides, either belonging to the control or irradiated groups. Qualitative and quantitative descriptive statistical analyses of the morphological changes and nonparametric tests of Kruskal-Wallis and Dunn were performed. The Kappa (k) test was used to assess inter-observer and intra-observer agreement using SPSS for Windows (SPSS IncVersion 21.0, Chicago, IL, USA) 17 .

Results
Both intra-observer and inter-observer reproducibility to repeated measures showed a perfect concordance of k = 1. No degeneration was observed in pulp blood vessels of the control group (Figure 1). Similar hyaline degeneration of pulp blood vessels was observed in 100% of the irradiated animals (Groups B, C, D, E, F, and G) at both doses of 15 Gy and 25 Gy at sacrifice (24 hours, 7 days, and 22 days post irradiation, except G group; Figures 2 and 3, Table 1). Animals in Group G did not survive more than 10 days and findings were considered inside this period. Hyperemic blood vessels were found less frequently in the control animals (p < 0.05; Figure 1, Table 2).
No significant inflammatory infiltrate, edema, necrosis or fibrosis were observed, irrespective of dose and sacrifice time, with no difference between the control and irradiated groups (Table 1).

Discussion
The negative response to the pulp sensitivity test may be indicative of necrosis or decreased pulp blood flow with ionizing radiation as an inductive agent 6,11,12,16,18,19 . However, studies demonstrating direct effects of ionizing radiation on the dental pulp are still scarce in the English literature 2 .
It is known radiation can induce changes in the dental pulp, a delicate connective tissue with moderate sensitivity to radiation 16 . In the present study, hyaline degeneration of the pulp blood vessels was observed. This event is a consequence of endothelial injury originating from a leakage of plasma proteins through damaged endothelial cells in the vascular wall 16 . This alteration, when extensive, contributes to the obliteration of capillary lumens, and causes an impaired blood supply which results in localized malnutrition, decreased oxygenation, and, loss of pulp sensitivity 16 . However, the clinical significance of hyaline degeneration is not stated in the literature. X-radiation does not affect the dental pulp: a morphological study in rats These vascular changes were observed in the coronal and root pulp teeth of irradiated rats but not in animals of the control group. Previous studies have already seen pulp changes such as thickening of blood vessel walls, occasional degeneration of connective tissue and odontoblasts, and subtle changes in the walls of the pulp blood vessels 8,12 . Nevertheless, in another similar study with rats, inflammation and pulp hyalinization were not observed with the presence of vascular congestion 2 . It should be noted the radiation dose in this study was lower with 12 Gy and 18 Gy, and that different experimental designs may generate contradictory results.
In view of our findings, we believe hyalinization of blood vessels can cause decreasing blood flow and, consequently, transient loss of pulp sensitivity as an immediate event after radiotherapy 12 . Thus, patients submitted to head and neck radiotherapy might present a higher number of teeth with a negative response to the pulp sensitivity test 6,11,18,19 . However, around 75% of the blood flow can be reestablished in the irradiated field within one year after radiotherapy. Therefore, the decrease in pulp sensitivity can be considered as a speculated transient event due to changes in the microcirculation with no late clinical effects 6,12,15,20-22 . In the present study, vascular alteration was found at 24 hours, 7 days, and 22 days after irradiation, and long-term studies to investigate the progressive events may further illuminate radiation effects on pulp patterns. Meanwhile, it should be noted that animal models have presented the limitation of experimental design due to the need for prior anesthesia of the animals for irradiation and follow-up. This condition renders the animals more susceptible to death. Moreover, the teeth of research animals were healthy, and this may be one reason why no inflammatory changes were observed in the pulp in this and in other studies 9,10,16,17 . Also, the hyperemic vessels were clearly seen in the control group; a frequent and completely reversible event at initial pulp aggression with no quite relevant clinical findings. Interestingly, inflammatory changes, edema, necrosis or fibrosis were not triggered by radiation in the dental pulp. Histological sections were meticulous investigated under optical microscopy.

Conclusion
Despite the interest of the clinical context to the field; the pathological and clinical significance of hyaline degeneration is still obscured. Nonetheless, the absence of necrosis areas or inflammatory infiltrate could be considered the major data brought by the study because this morphological maintenance suggests a single X-radiation dose is not able to affect the dental pulp connective tissue in the long term.