This study was designed to investigate the protective effect of adipose derived mesenchymal stem cells (AdMSCs) against radiation-induced bladder injury (RIBI). Female rats were divided into 4 groups: (a) controls, consisting of nontreated rats; (b) radiation-treated rats; (c) radiation-treated rats receiving AdMSCs; and (d) radiation-treated rats receiving AdMSCs conditioned medium. AdMSCs or AdMSCs conditioned medium was injected into the muscular layer of bladder 24 h after radiation. Twelve weeks after radiation, urinary bladder tissue was collected for histological assessment and enzyme-linked immunosorbent assay (ELISA) after metabolic cage investigation. At the 1 w, 4 w, and 8 w time points following cells injection, 3 randomly selected rats in RC group and AdMSCs group were sacrificed to track injected AdMSCs. Metabolic cage investigation revealed that AdMSCs showed protective effect for radiation-induced bladder dysfunction. The histological and ELISA results indicated that the fibrosis and inflammation within the bladder were ameliorated by AdMSCs. AdMSCs conditioned medium showed similar effects in preventing radiation-induced bladder dysfunction. In addition, histological data indicated a time-dependent decrease in the number of AdMSCs in the bladder following injection. AdMSCs prevented radiation induced bladder dysfunction and histological changes. Paracrine effect might be involved in the protective effects of AdMSCs for RIBI.
Pelvic radiotherapy is one of the standard therapies for a wide variety of tumors localized in the rectum, uterus/cervix, prostate, and urinary bladder. In spite of recent advances that improve range and direction of radiation, radiotherapy is unavoidably associated with normal tissue side effects. One of the organs that are frequently affected by pelvic radiotherapy is the urinary bladder [
Mesenchymal stem cells (MSCs), firstly identified in bone marrow, have been demonstrated to be pluripotent cells with great potential to differentiate into various mesodermal lineages [
Adipose tissue represents an abundant and accessible source of mesenchymal stem cells (AdMSCs) [
This study was therefore designed to explore the effects of AdMSCs in improving bladder function in a rat model of RIBI. Also, the possible mechanism involved in AdMSCs improving bladder function of RIBI was explored. To our knowledge, this is the first study to explore the feasibility of using AdMSCs to rescue radiation-induced bladder injury.
Sixty-two female Sprague-Dawley rats (10 weeks old) were used in our study. X-ray was delivered to the bladder area of the rats to induce RIBI animal model. Radiated rats were randomly divided into three groups: radiation control group (RC,
For animals, the rats were anesthetized with ketamine (100 mg/kg) and midazolam (5 mg/kg). A lead shield with a 25 mm × 15 mm window was used to limit the radiation to the bladder according to our previously used protocol [
AdMSCs were isolated and cultured as described previously [
To prepare AdMSCs conditioned medium, 1 × 106 AdMSCs were seeded in FBS supplemented DMEM medium on 6-well dish. The culture medium was changed into serum-free DMED medium. Twenty-four hours later, the medium was collected and centrifuged at 3,000 ×g for 5 min. The supernatant was collected and concentrated.
Briefly, 1 × 105 AdMSCs were harvested and suspended in 500
After anesthetization, a skin incision was made to expose the bladder and either 800
According to a previously described protocol [
Middle parts of bladder tissues were fixed in 4% paraformaldehyde in PBS at 4°C overnight, after which the tissue was transferred to 30% sucrose in PBS at 4°C overnight. The tissues were embedded in Optimal Cutting Temperature (OCT) and stored at −80°C.
For immunofluorescence staining, sections were incubated with 3% goat serum for 30 min at room temperature. Bladder sections were then incubated with primary antibodies overnight at 4°C, followed by Alex594-conjugated goat anti-rabbit IgG (Invitrogen, Carlsbad, CA, USA, 1 : 200) for 1 h at room temperature. The primary antibodies used in the present study was rabbit anti-CD31 (Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1 : 200). EdU staining was performed as described before.
For Masson’s trichrome staining, the slides were stained using commercial available kits (Jiancheng, Nanjing, China) following the protocols provided by the manufacturer.
For tissue specimens, data were averaged on 6 sections from each bladder. Images were captured on a Nikon microscope with a Spot RT color digital camera, and digital histomorphometric analysis was performed using Image-Pro Plus 6.0 software (Media Cybernetics, Silver Spring, MD, USA).
Protein concentration of tumor necrosis factor-
Results are expressed as mean ± standard deviation and data were analyzed by one-way analysis of variance (ANOVA) to compare among multiple groups followed by post hoc comparisons with the least significant difference test using Prism 4 (GraphPad Software, San Diego, CA, USA). A
The cultured AdMSCs demonstrated a spindle-shaped morphology (Figure
Morphology, flow cytometric analysis of AdMSCs. (a) Phase-contrast image of AdMSCs. (b) Flow cytometric analysis showed that cultured AdMSCs expressed CD29, CD44, but not CD31 or CD45. Black lines indicate isotype control while red lines indicate positively stained cells of each marker.
AdMSCs were labeled with EdU before bladder injection. Histological data indicated a time-dependent decrease in the number of AdMSCs in the bladder following injection. Twelve weeks after injection, very limited number of AdMSCs could be observed within the bladder (Figure
Tracking of injected AdMSCs in bladder tissue. (a) Up: representative images of EdU positive cells within the bladder wall at different time points after cell injection. The white bar indicates 200
As shown in Figure
Results of metabolic cage investigation.
The increased number of blood vessels was observed in the submucosa in treated rats (Figures
Histological analysis of bladder tissue. (a) Representative images of blood vessels in the submucosa layer. The white arrows indicate blood vessels while the white bar indicates 200
Results of the expressions of TNF-
RIRI associated symptoms such as increased urinary frequency, urgency, and dysuria appear early after the treatment and resolve within a few weeks after the end of radiotherapy [
AdMSCs have attracted rising interest for regenerative medicine due to sufficient adipose sources and easy isolation [
BmMSCs have been demonstrated to be effective in restoring radiation-impaired bladder function [
Recently, increasing evidence indicates the role of paracrine effect of AdMSCs in promoting tissue repair. AdMSCs are capable of producing different types of cytokines, which have trophic effects on cytoprotection, cell survival, and immunomodulation [
Our study has some limitations. First, we only assessed some cytokines in the bladder tissues after the treatment. Further studies designed to detect a wider panel of cytokines are needed. Second, although paracrine effect of AdMSCs is shown to be one of the possible mechanisms, it is still unknown what the exact contents are and how these are responsible for the improved function and preserved structure. Further studies aimed at identifying the key factors released by AdMSCs are also needed.
In conclusion, administration of AdMSCs is effective in improving bladder function and preserving bladder microstructure of RIBI. Paracrine effect of AdMSCs is responsible for the functional improvement and structural preservation.
The authors declare that there is no conflict of interests regarding the publication of this paper.
Xuefeng Qiu and Shiwei Zhang contributed equally to this work.
This study was supported by Grants from Natural Science Foundation of Jiangsu Province (BK20150112), China Postdoctoral Science Foundation (2014M551562), and National Natural Science Foundation of China (81302542).