Effects of Mesenchymal Stem Cells Treatment on Radiation-Induced Proctitis in Rats

Abstract

Purpose: There are no effective treatment methods with which to control complications of radiation proctitis with fistula or recurrent bleeding following radiation treatment for prostate, cervical, or rectal cancer. Mesenchymal stem cells (MSCs) can induce immune modification, resulting in tissue repair and regeneration. Therefore, we used a rat model of radiation-induced proctitis and observed the effects of using human placenta-derived (PD) and adipose tissue-derived (AD) MSCs.

Materials and methods: Female Sprague Dawley rats were irradiated at the pelvic area with 25 Gy. We injected 1×10⁶ cells of human PD-MSCs, human AD-MSCs, human foreskin fibroblasts, and control media into the rectal submucosa following irradiation. We sacrificed rats for pathologic evaluation.

Results: Fibrosis on the rectum was reduced in both MSC groups, compared to the control group. Mucosal Ki-67 indices of both MSC injected groups were higher than those in the control group. Although caspase-3 positive cells in the mucosa gradually increased and decreased in the control group, those in both MSC injected groups increased rapidly and decreased thereafter.

Conclusion: We demonstrated the effects of regional MSC injection treatment for radiation-induced proctitis in rats. MSC injection reduced fibrosis and increased proliferation in rat mucosa. Human AD-MSCs and PD-MSCs had similar effectiveness.

Keywords: Proctitis; mesenchymal stem cells; radiotherapy; rat model.

Fig. 1. Histologic changes in the rat rectum following 2 weeks of irradiation. Mucosal atrophy and submucosal fibrosis are noted. (A) 20 Gy irradiated group. (B) 23 Gy irradiated group. (C) 25 Gy irradiated groups (left; H&E stain, right; Masson trichrome stain).

Fig. 2. Injection of human placental-derived or adipose tissue-derived mesenchymal stem cells. Following general anesthesia with inhalation, (A) the external side of the rectum was exposed after partial incision of the skin and muscle layer. (B) Stem cells were injected with a 1-cc syringe and a 31-gauge needle.

Fig. 3. Characteristics of AD-MSCs and PD-MSCs. (A) AD-MSC. (B) PD-MSC. They all had DNA XY genes and showed CD 44 (+), CD 73 (+), CD 105 (+), CD 34 (-), CD 31 (-), and CD 45 (-). AD-MSC, adipose tissue-derived mesenchymal stem cell; PD-MSC, placenta-derived mesenchymal stem cell.

Fig. 4. Weight changes according to time. Compared to control group weights, the weights of the AD-MSC group, PD-MSC group, HHF group, and radiation control group were significantly lower. ^*p<0.001. AD-MSC, adipose tissue-derived mesenchymal stem cell; PD-MSC, placenta-derived mesenchymal stem cell; HFF, human foreskin fibroblast.

Fig. 5. Histologic images of the rectum on the second- and fourth weeks following injection of AD-MSCs (H&E stain and Masson trichrome stain, ×100). Cell injection sites (yellow circle) are found at the submucosal layer. AD-MSC, adipose tissue-derived mesenchymal stem cell.

Fig. 6. Rectal tissue image obtained with Living Image Software (Xenogen^®). Signals from the injected cells (AD-MSC and PD-MSC) were found on week 2 (A) and week 4 (B). AD-MSC, adipose tissue-derived mesenchymal stem cell; PD-MSC, placenta-derived mesenchymal stem cell.

Fig. 7. Image of fluorescence in situ hybridization for human Y chromosome in the rectal tissue of rats on week 2 following irradiation. Y chromosomes (white arrows) were found. MSC, mesenchymal stem cell.

Fig. 8. Images of fibrosis with Masson trichrome stain. Area fractions of fibrosis in mesenchymal stem cell injection groups were significantly lower than those in the control or HFF groups on week 4. ^*,**p<0.01. AD, adipose tissue-derived; PD, placenta-derived; HFF, human foreskin fibroblast.

Fig. 9. Ki-67 proliferation index values of the rectal mucosa on weeks 2 and 4 following irradiation. ^*,**p<0.01. AD, adipose tissue-derived; PD, placenta-derived; HFF, human foreskin fibroblast.

Fig. 10. Caspase-3 positive cells in each rectal mucosal gland following stem cells injection in each group. ^*p<0.05. AD, adipose tissue-derived; PD, placenta-derived.