The effect of interstitial brachytherapy on the general condition was less than SBRT in SD rats

No obvious abnormal symptoms such as hemoptysis and dyspnea were observed in rats treated by both interstitial brachytherapy or SBRT. However, further manifestations including upturned fur, dullness, tight breath, decreased appetite, slow weight gain after 1 week of SBRT, and interstitial brachytherapy showed similar but slighter symptoms. As shown in Fig. 3a, the weight gain was slower in both interstitial brachytherapy and SBRT rat group when compared with the control group, while the increasing rate of interstitial brachytherapy was more rapid than SBRT rat group. Besides, the respiratory rate in both treatment groups was elevated (P < 0.05), in which interstitial brachytherapy was 108/min and SBRT was 125/min, and the respiratory rate in the control group was 85/min at 4 weeks post-treatment (Fig. 3b). Collectively, the SBRT had more obvious effects on body weight, respiratory rate and general condition of rats.

Fig. 3

The alternations of the general condition in two treatment groups with time. a Changes in body weight of rats. b Changes of respiratory rate in rats

The difference in lung volume and CT value of the left and right lungs in SD rats

To further observe the presence of radiation pneumonia, SD rats were anesthetized and underwent chest CT scans at 1, 4, 8 and 16 weeks, respectively. The results showed that the right lung volume of SBRT group was larger than that of interstitial brachytherapy group and control group. However, the right lung volume of SBRT group began to reduce and maintain at the same volume in the first week, which is significantly smaller than interstitial brachytherapy rat group (P < 0.05). As time goes on, no significant change in right lung volume was observed between control group and the interstitial brachytherapy group (P > 0.05). At the 16th week, the right lung volume in the control group was 4.38cm3, 4.27cm3 in the interstitial brachytherapy group and 2.56cm3 in the SBRT group (Fig. 4a). On the contrary, the left lung volume in the SBRT group began to increase over time, and it was larger than 3cm3 in the control group (P < 0.05) (Fig. 4b); besides, left lung volume in the SBRT group was significantly higher than interstitial brachytherapy group at the 8th week (P < 0.05). There was no significant difference in the left lung volume between the control and the interstitial brachytherapy group (P > 0.05).

Fig. 4

The difference of lung volume and CT value of the left and right lungs in SD rats. a Right lung volume checked by chest CT every 4 weeks. b Comparison of left lung volume among three groups. c Comparison of mean CT difference between left and right lungs of rats in three groups. *P < 0.05, **P < 0.01, *** P < 0.001

To identify the tissue types such as fat, effusion or calcification represented in CT, we compared the difference between the right and left lung in three rat groups. The results showed that at the 16th week, the differences between right and left lung CT values were significantly elevated in SBRT group when compared with the control group and the interstitial brachytherapy group (P < 0.05) (Fig. 4c). However, no significant difference of CT values between interstitial brachytherapy and SBRT groups was noticed. These results indicated that SBRT has a certain influence on lung tissue density in rats.

Pathological examination among interstitial brachytherapy, SBRT and control rats

After 1, 4, 8 and 16 weeks of radiotherapy, the gross anatomy of lungs in different rat groups was observed. It was shown that lung tissue in control group was smooth, soft and pink, without congestion and edema. While at the 8th week of the interstitial brachytherapy group, the color of lung tissue was deepened and slightly swollen, besides, a fibrous sclerotic lesion with a radius of about 0.2 cm was observed in the right lower lung. At the 16th week, mild atrophy was observed in the right lung without obvious changes in the shape of the whole lung. In the SBRT group, we found that the color of the right lung was deepened 1 week after treatment, accompanied by visible petechiae in the right middle lung; there were gray and white spots in the irradiation site of the right lung at the 8th week, and the lung tissue was shown as dark red with obvious swelling; at the 16th week, the volume of the right lung was firmly reduced and hard, whereas the volume of the left lung was increased with dark red color (Fig. 5a).

Fig. 5

Overall view, microscopic view and percentage of the inflammation area in the normal area of the lung in the three groups. a Overall view of lung tissues in control, interstitial brachytherapy and SBRT groups. b Microscopic view of lung tissues in control, interstitial brachytherapy and SBRT groups, × 400. c Percentage of the inflammation area in control, interstitial brachytherapy and SBRT groups every 4 weeks. *P < 0.05, **P < 0.01, *** P < 0.001

The 40 × imaging results of HE staining were represented in Fig. 5b, the lung tissue structure in the control rat group was normal, the bronchial ciliary epithelial cells were orderly arranged, and no exfoliated cells were observed, the alveolar epithelial cells were normal and full without obvious degeneration and necrosis, no proliferation of fibrous tissue and inflammatory cell infiltration in the lung interstitial, and no other special pathological changes were noticed. In the interstitial brachytherapy group, the lung tissue structure was relatively normal, but there was thickened alveolar septum at varying degrees, accompanied by alveolar epithelium and fibrous tissue proliferation, no obvious pathological changes were observed in other structures. In the SBRT group, the alveolar septum was obviously thickened, and the blood vessels were reduced. There were more fibrocyte and epithelial cell proliferation, and some macrophages and foam cells have been detected in the proliferative area.

We further performed the inflammatory infiltration analysis at a magnificent 400 × by Image J software. The results showed that the percentage of right pneumonitis in the normal area in the SBRT group was slightly larger than that of interstitial brachytherapy group without a significant difference at week 1 (P > 0.05). However, the difference became significant at week 4, 8 and 16 (P < 0.05). The pneumonitis lesion area in the SBRT group gradually increased with time, and was stable at 23%, while the area in the interstitial brachytherapy group remained stable at 15%. Thus, we indicated that the degree of lung injury was more severe after SBRT, and the degree of lung injury of these two types of radiation did not gradually ameliorate over time (Fig. 5c).

Serum cytokines among interstitial brachytherapy, SBRT and control rats

As shown in Fig. 6a, the IFN-γ expression was increased with time in the interstitial brachytherapy group, and reached its peak at week 16. The IFN-γ expression in interstitial brachytherapy group was significantly different from that in the SBRT group at week 1, 4, 8 and 16 (P < 0.05). Besides, the expressions of IL-2, IL-6 and IL-10 in SBRT group were significantly higher than that of interstitial brachytherapy group, and they represented a declining tendency with time (Fig. 6b-d). As shown in Fig. 6e, the TGF-β expression in interstitial brachytherapy group reached its peak with the increase of time from week 1 to week 16, and it was significantly lower than SBRT group (P < 0.05), indicating the presence of more fibrosis-promoting cytokines and heavier pulmonary fibrosis in rats of SBRT group.

Fig. 6

The concentration changes of inflammatory cytokines, antifibrotic cytokines and profibrotic cytokines in rat serum were detected by ELISA. The serum IFN-γ (a), IL-2 (b), IL-6 (c), IL-10 (d) and TGF-β (e) expressions (pg/ml) in each group were measured by ELISA every 4 weeks. *P < 0.05, **P < 0.01, *** P < 0.001

The effect of interstitial brachytherapy and SBRT on pulmonary ventilation/perfusion

CT perfusion imaging was conducted to assess the impact of two treatment approaches on pulmonary ventilation/perfusion (V/P). As shown in Fig. 7a, the V/P of the right lung in SBRT group was poor, while the V/P of the left lung was better. In the interstitial brachytherapy group, no obvious difference of V/P in the right and left lung was noticed. Further quantitative SPECT/CT results suggested that the V/P imaging area (%) of right middle lung was significantly larger in interstitial brachytherapy group when compared with SBRT group, while the V/P imaging area (%) of left middle and lower lung was significantly smaller in the SBRT group compared with interstitial brachytherapy group (all P < 0.05) (Fig. 7b, c).

Fig. 7

Lung perfusion imaging, right lung perfusion and left lung perfusion examinations. a Pulmonary perfusion imaging of SD rats at 16 weeks in interstitial brachytherapy and SBRT groups. Right (b) and left lung V/P imaging area (%) (c) in interstitial brachytherapy and SBRT groups. *P < 0.05, **P < 0.01, *** P < 0.001

The difference in mortality in interstitial brachytherapy and SBRT rat groups

After the two different treatments, we found the presence of fatal acute radiation pneumonia developed in 3.2 days after SBRT, manifesting crouching, hair erection and loss, listlessness, shortness of breath, food refusal, reduced activity, and death, with a mortality rate of 16.7% (Table 1). Pathological biopsy was performed immediately, as shown in Fig. 8, a large amount of blood congestion was observed in pulmonary vessels and interstitial under a light microscope, accompanied by the thickened alveolar septum, alveolar epithelial cells and elastic fibers proliferation, and inflammatory cells infiltration such as lymphocytes and neutrophils. A small amount of protein-like substance exudates in the lung interstitial. There were no dead rats in the control group and interstitial radiotherapy group.

Table 1 Comparison of mortality rate between two groups (n, %)Fig. 8

Pathological sections of dead rats