Differential effects of cyclosporin and etanercept treatment on various pathologic parameters in a murine model of irradiation-induced mucositis

Abstract

Background: Radiation therapy is the most prescribed treatment for many oncologic indications. One of its common side effects is mucositis with hallmark apoptosis in the intestinal crypt and diarrhea.

Objective: We investigated the potential beneficial effects of etanercept and cyclosporin treatment during radiation exposure. The effects of these drugs on intestinal apoptosis, long-term weight loss, diarrhea severity, and survival were examined.

Methods: For acute observation studies, animals pretreated with phosphate buffer saline (PBS) vehicle, either etanercept, or cyclosporin were challenged with either 1 Gy or 13 Gy irradiation and sacrificed 6 hours later. The animals' small intestines were then harvested for histologic analysis. For chronic survival studies, 14.5 Gy irradiation was applied. Etanercept or cyclosporin treatments were given 15 minutes before the irradiation, followed by daily administration.

Results: At 6 hours postirradiation the maximum apoptotic index observed in the small intestine was ∼25% for both 1 Gy and 13 Gy irradiation. Etanercept and cyclosporin pretreatment had no effect on the irradiation-induced apoptosis. During chronic observation, the rate of weight loss was similar in all test groups. At 7 days postirradiation, the weight loss in phosphate buffered saline-treated control, etanercept, and cyclosporin groups reached a maximum at 19%, 24%, and 31.8%, respectively. The weight lost in the cyclosporin group was significantly higher than in the control group. Neither treatment reduced the severity of diarrhea, but cyclosporin increased the survival rate. Sixty percent of cyclosporin-treated animals survived compared with 27% in the PBS-treated control group and 47% in the etanercept-treated group. Serum tumor necrosis factor-α levels, a biomarker for both etanercept's mechanism of action and treatment efficacy, was inhibited by etanercept throughout the study, but cyclosporin only showed an inhibitory effect at 48 hours postirradiation.

Conclusions: Our study demonstrates that cyclosporin increases the survival rate of irradiated animals without affecting parameters such as intestinal histology, weight loss, and diarrhea severity.

Keywords: cyclosporin; etanercept; inflammation; irradiation; mucositis.

Figure 1

(A) Cartoon illustrating cell positional frequency plots. Cell position 1 represents the cells located at the base of the crypt. The positional number increases in an ascending order away from the base. 50 crypt lengths were analyzed for each mouse and the frequency of apoptotic cells at each cell position was plotted. (B) Micrograph of intestinal crypt showing apoptotic cells (400× magnification). The sections were prepared as outlined in the Method section, and stained with hematoxylin and eosin stain. Apoptotic cells take on a rounded appearance (white arrows), and are frequently found near the base of the crypt. (C) Mitotic cells are often rare or difficult to capture due to the short duration of this portion of the cell cycle. This micrograph was taken at 630× magnification to show the detail of a mitotic cell (yellow arrow).

Figure 2

Cell positional frequency plots of apoptotic index after exposure to irradiation. The base line apoptotic index of the naïve animals (○) is also shown. (A) The frequency of apoptosis in all treatment groups was significantly elevated over naïve animals in positions 1–14 (P ≤ 0.05). Etanercept (■) and cyclosporin (50 and 100 mg/kg) (▴, ♦) treatment had no effect on the frequency of crypt cell apoptosis after 1 Gy irradiation. Etanercept (25 mg/kg) and cyclosporin were administered just before the irradiation. Apoptosis at cell positions 15–21 was generally not observed. (B) 13 Gy irradiation significantly increased the number of apoptotic cells over naïve animals in positions 1–11 for the phosphate buffered PBS-treated group, positions 1–12 for the 25 mg/kg etanercept group, and positions 1–13 for the cyclosporin groups. Etanercept (25 mg/kg) (■) and cyclosporin (50 and 100 mg/kg) (▴, ♦) given before the irradiation had no effect on the apoptotic index. The pattern of apoptosis at the 13-Gy irradiation level was similar to the 1-Gy level. There was no significant difference in the frequency of crypt cell apoptosis in the PBS-treated animals caused by 1 Gy and 13 Gy irradiation.

Figure 3

Effects of etanercept (■) and cyclosporin (▴) treatment on weight loss after 14.5-Gy partial body irradiation. The animals received daily dosing of either treatment. The percent weight loss in the cyclosporin-treated animals were significantly higher than the PBS-treated animals throughout the study. There was no statistical difference in the weight loss between the PBS-treated group and etanercept-treated animals. The weight loss in all groups reached a plateau after 7 days. *P ≤ 0.05.

Figure 4

Effects of cyclosporin and etanercept treatment on the diarrhea severity of the 14.5-Gy irradiated animals. The cumulative diarrhea score increased exponentially 7 days following irradiation. The severity of diarrhea in the PBS-treated and etanercept-treated animals plateau after 15 days. Etanercept had no effect on diarrhea severity. Cyclosporin treatment slightly aggravated the diarrhea in the irradiated animals.

Figure 5

Effects of cyclosporin and etanercept treatment on the survival of 14.5-Gy irradiated animals. Fifteen animals were assigned to each treatment group at the beginning of the study. Death of the animals was first observed at Day 5 postirradiation. Daily treatment with cyclosporin significantly increased the survival rate of the animals at the end of the study. The number of surviving animals in the etanercept-treated group was also higher than the PBS-treated animals. But this difference was not statistically significant.

Figure 6

Effects of etanercept and cyclosporin treatment on circulating TNF-α level. Irradiation upregulated serum tumor necrosis factor-alpha (TNF-α) level from baseline within 12 hours. This high level was maintained throughout the experimental period. Etanercept treatment consistently downregulated this increase. Cyclosporin only significantly downregulated the circulating TNF-α level at the 48 hours time point. *P ≤ 0.05.