Am J Transl Res 2020;12(3):999-1015 www.ajtr.org /ISSN:1943-8141/AJTR0105598 Original Article Long-term effect of extracorporeal shock wave therapy on attenuating radiation-induced chronic cystitis in rat Yen-Ta Chen1,2, Chih-Chao Yang3, Pei-Hsun Sung4,5, Kun-Chen Lin6, John Y Chiang7,8, Chi-Ruei Huang4,5, Kuan-Hui Huang9, Fei-Chi Chuang9, Yi-Ching Chu4,5, Eng-Yen Huang10,11, Hon-Kan Yip4,5,12,13,14 1Division of Urology, Department of Surgery, Kaohsiung Chang Gung Memorial Hospital, College of Medicine, Chang Gung University, Kaohsiung, Taiwan; 2Center for Shockwave Medicine and Tissue Engineering, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan; 3Division of Nephrology, Department of Internal Medicine, Kaohsiung Chang Gung Memorial Hospital, College of Medicine, Chang Gung University, Kaohsiung, Taiwan; 4Division of Cardiology, Department of Internal Medicine, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan; 5Center for Shockwave Medicine and Tissue Engineering, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan; 6Department of Anesthesiology, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, Taiwan; 7Department of Computer Science and Engineering, National Sun Yat-sen University, Kaohsiung, Taiwan; 8Department of Healthcare Administration and Medical Informatics, Kaohsiung Medical University, Kaohsiung, Taiwan; 9Department of Obstetrics and Gynecology, Kaohsiung Chang Gung Memorial Hospital, College of Medicine, Chang Gung University, Kaohsiung, Taiwan; 10Department of Radiation Oncology, Kaohsiung Chang Gung Memorial Hospital, College of Medicine, Chang Gung University, Kaohsiung, Taiwan; 11School of Traditional Chinese Medicine, Chang Gung University College of Medicine, Taoyuan, Taiwan; 12Institute for Translational Research in Biomedicine, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan; 13Department of Medical Research, China Medical University Hospital, China Medical University, Taichung, Taiwan; 14Department of Nursing, Asia University, Taichung, Taiwan Received November 27, 2019; Accepted March 4, 2020; Epub March 15, 2020; Published March 30, 2020 Abstract: Background: This study tested the long-term effect of extracorporeal shock wave (ECSW) therapy on ame- liorating radiotherapy-induced chronic cystitis (CC) in rat. Methods and results: Adult-female SD rats (n = 24) were equally categorized into group 1 (normal control), group 2 (CC induced by radiotherapy with 450 cGy twice with a four-hour interval to the urinary bladder), group 3 [CC with ECSW treatment (0.1 mJ/mm2/120 impulses once every 3 days after radiotherapy)]. Bladder specimens were harvested by day 60 after radiotherapy. By day 60, the degree of detrusor contraction was significantly reduced in group 2 than groups 1 and 3, and significantly reduced in group 3 than in group 1 (P < 0.0001). Number of WBC, occulted blood and bacteria were significantly higher in group 2 than in groups 1 and 3 (P < 0.01), but they showed no difference between the latter two groups (P > 0.3). The protein expressions of oxidative stress (NOX-1/NOX-2/oxidized protein), apoptosis (cleaved-caspase-3/cleaved- PARP), DNA-damaged marker (γ-H2AX), fibrosis (TGF-β/Smad3) and inflammatory signaling (TLR-4/MYD88/Mal/ TRAF6/p-IκBα/p-NFκB/TNF-α/MMP-9/COX-2) were significantly higher in group 2 than in group 1, and were signifi- cantly reduced in group 3 (all P < 0.001). The cellular expressions of inflammatory (CD14+/CD68+/MIF+/MMP-9), immunoreactive (CD4+/CD8+) and cytokeratin (CK17/CK18) biomarkers, and collagen-deposition/fibrotic areas as well as bladder-damaged score/disruption of the bladder mucosa displayed an identical pattern compared to that of oxidative stress among the three groups (all P < 0.0001). Conclusion: The long-term effect of ECSW treatment was reliable on protecting the urinary bladder from radiation-induced CC. Keywords: Radiation, chronic cystitis, inflammation, urinary bladder contractility Introduction on palliative therapy for advanced stage of some solid cancers such as brain tumor, lung Despite state-of-the-art advances in chemo- cancers and urogenital/gynecologic cancers therapy, hormone therapy and target therapy [1-5]. However, a fly in the ointment is that radio- as well as immunotherapy for advanced can- therapy has been found to commonly induce cers, radiotherapy still plays an essential role hemorrhagic cystitis (HC), gross hematuria and Extracorporeal shock wave therapy on radiation-induced chronic cystis late radiation-induced chronic cystitis (CC), No. 2016012804) and performed in accor- which not merely hinder the patient’s quality dance with the Guide for the Care and Use of of life and social activity but also induce psy- Laboratory Animals. chiatric issues, urinary tract infection/sepsis, obstructive uropathy and renal failure as well Animals were housed in an Association for as increase the risk of mortality [6-10]. Assessment and Accreditation of Laboratory Animal Care International (AAALAC; Frederick, During the last decades, plentiful modalities, MD, USA)-approved animal facility in our hospi- including pharmacological agents, surgical in- tal with controlled temperature and light cycles terventions and even hyperbaric oxygen thera- (24°C and 12/12 light cycle). py, have been extensively applied for the HC [11-18]. However, their efficacies are contradic- Rationale of ECSW energy utilized in the pres- tory, highlighting that treatment of radiothera- ent study py-induced HC/CC and the associated compli- cations remains a formidable challenge. Accor- The purpose of this study was to evaluate the dingly, a safe and efficacious treatment alter- long-term effect of ECSW on protecting the uri- native is still eagerly awaited. nary bladder against the radiotherapy. Thus, the study period was designed as a duration of In view of pathophysiology of radiation cystitis 60 days after radiotherapy. Another evaluation that is mainly caused by activation of an in- was that ECSW application should resemble flammatory cascade leading to tissue swelling, the clinical setting of “regularly intermittent mucosa damage, necrosis, and smooth muscle administration” of the medicine for a chronic destruction and finally fibrosis [19-22], era- disease. Along this line of thinking, regularly dication of the inflammatory reactions may intermittent application of ECSW to the urinary prove as an innovative therapeutic strategies bladder was performed for the animals. Based for those of HC/CC patients refractory to con- on these considerations, the safety of ECSW ventional treatment. had to be considered, especially when regular and consistent application of ECSW was uti- Previous studies have established that extra- lized in the present study. According to our pre- corporeal shock wave (ECSW) therapy is able vious in vitro [31] and in vivo [23, 25-30] stud- to inhibit the inflammatory reaction and oxi- ies, we found that the safest and lowest energy dative stress, and augment the angiogenesis [23, 24]. Besides, our studies have displayed of ECSW with promising efficacy was 0.1 mJ/ mm2/120 impulses. This was the reason why that ECSW therapy is not only safe, but has 2 capacities of pro-angiogenesis, anti-ischemia, we utilized the “0.1 mJ/mm /120 impulses anti-fibrosis, anti-inflammation, and pain-allevi- once every 3 days after radiation” (i.e., a total ating effects [25-29]. Based on these afore- of 20 times in the present study). mentioned issues [19-29], we have recently Rationale of radiotherapy dosage (refer to performed a study with a period of 28 days to Figure 1) and duration of study period address the impact of ECSW on reducing the radiation-induced HC/CC in rodent [30]. Our Practically, for achieving greatest efficacy, the results are attractive and promising. However, dosage of radiotherapy for solid cancers of uro- this study also has limitations, such as the opti- genital/gynecologic origin is always quite high mal dosage of radiation and long-term effect of in order to kill the cancer cells that could explain ECSW therapy with appropriate energy on radi- why a lot of side effects are frequently encoun- ation-induced HC were regrettably left unad- tered in those urogenital/gynecologic patients dressed. Accordingly, this study was designed after receiving this therapy [19-22]. On the to answer the limitations of our recent study other hand, for safety, the dosage of radiother- [30]. apy in our previous animal model study [30] Materials and methods was designed as low dosage (i.e., 300 cGy). This, therefore, raises the question that the Ethics and animal care results of our animal model of study [30] might not truthfully reflect the daily clinical practice. All animal procedures were approved by the Accordingly, in the present study, except the Institute of Animal Care and Use Committee at low dose of our previous study (i.e., 300 cGy Kaohsiung Chang Gung Memorial Hospital twice with a four-hour interval to the urinary (Affidavit of Approval of Animal Use Protocol bladder of one animal), we performed a pilot 1000 Am J Transl Res 2020;12(3):999-1015 Extracorporeal shock wave therapy on radiation-induced chronic cystis 1001 Am J Transl Res 2020;12(3):999-1015 Extracorporeal shock wave therapy on radiation-induced chronic cystis Figure 1. Pilot study, urodynamic study of bladder contraction, urine amount and ratio of urine protein to urine creatinine at day 60 after radiotherapy. (A-F) Escala- tion of dosage of radiation energy (i.e., stepwise increase of the radiotherapy dosage) in the pilot study with consistent ECSW application (i.e., 0.1 mJ/mm2/120 impulses/once every 3 days after radiation). We utilized 300 cGy (A, B), 450 cGy (C, D) and 600 cGy (E, F)/twice with 4-h interval for each animal in CC (A, C, E) and CC + ECSW (B, D, F) groups (n = 2/each group). By day 60 after CC induction, the results showed that no detrusor contractility wave was observed in either CC only or in CC + ECSW animals in 600 cGy, only CC + ECSW animals had notable detrusor contractility wave in 450 cGy and 300 cGy.