Low-Intensity Extracorporeal Shock Wave Therapy Promotes Bladder Regeneration and Improves Overactive Bladder Induced by Ovarian Hormone Deficiency from Rat Animal Model to Human Clinical Trial

Abstract

Postmenopausal women with ovary hormone deficiency (OHD) are subject to overactive bladder (OAB) symptoms. The present study attempted to elucidate whether low-intensity extracorporeal shock wave therapy (LiESWT) alters bladder angiogenesis, decreases inflammatory response, and ameliorates bladder hyperactivity to influence bladder function in OHD-induced OAB in human clinical trial and rat model. The ovariectomized (OVX) for 12 months Sprague-Dawley rat model mimicking the physiological condition of menopause was utilized to induce OAB and assess the potential therapeutic mechanism of LiESWT (0.12 mJ/mm2, 300 pulses, and 3 pulses/second). The randomized, single-blinded clinical trial was enrolled 58 participants to investigate the therapeutic efficacy of LiESWT (0.25 mJ/mm2, 3000 pulses, 3 pulses/second) on postmenopausal women with OAB. The results revealed that 8 weeks' LiESWT inhibited interstitial fibrosis, promoted cell proliferation, enhanced angiogenesis protein expression, and elevated the protein phosphorylation of ErK1/2, P38, and Akt, leading to decreased urinary frequency, nocturia, urgency, urgency incontinence, and post-voided residual urine volume, but increased voided urine volume and the maximal flow rate of postmenopausal participants. In conclusion, LiESWT attenuated inflammatory responses, increased angiogenesis, and promoted proliferation and differentiation, thereby improved OAB symptoms, thereafter promoting social activity and the quality of life of postmenopausal participants.

Keywords: bladder angiogenesis; low-intensity extracorporeal shock wave therapy; ovarian hormone deficiency; overactive bladder.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1

Urodynamic analysis of bladder cystometric parameters and voiding behavior shown in an OVX induced OAB of rat model. (A) Cystometry recordings of micturition pressure, voiding volume, and frequency, including voiding contraction (arrows) and non-voiding contraction (asterisks). (B) Tracing analysis of 24-h voiding behavior by metabolic cage. The OVX group significantly increased bladder maturation pressure, voiding contraction, non-voiding contraction, and micturition frequency, whereas LiESWT treatment significantly improved bladder voiding pattern and capacity.

Figure 2

Histopathological examination for bladder damage was shown by Masson’s trichrome staining, immunostaining, and Western blots. (A–D, A′–D′) The bladder pathological features of the sham group (A,A′), the OVX group (B,B′), the OVX + SW4 group (C,C′), and the OVX + SW8 group (D,D′). Masson’s trichrome stain showed the navy blue-stained nucleus, green-stained collagen, and red-stained smooth muscle were highlighted. In the sham group (A,A′), there were three to five layers of UL and only sparse collagen (yellow arrow) distributed in the SL. In the OVX group (B,B′), the morphology was characterized by thinner layer of UL (black arrows), and much collagen accumulation and interstitial fibrosis in the SL (yellow arrows). In contrast, the pathological features of the OVX + SW4 group (C,C′) and the OVX + SW8 group (D,D′) showed improved OVX-associated bladder damages by increasing a thicker layer of urothelium (black arrows) and reducing interstitial fibrosis (yellow arrows) compared with the OVX group. Particularly, there are many mononuclear cell groups gathered into sphere (yellow arrowheads) and much collagen accumulation (yellow arrows) around the sphere in the SL of the OVX + SW8 group (D,D′). (E–H) The distribution of cell-adhesion marker E-cadherin by immunostaining was shown. In the sham group (E), the E-cadherin staining was found in intercellular junctions of urothelium. On the contrary, there was less E-cadherin staining expression in the thin urothelium of the OVX group (F), but the immunostaining of the OVX + SW4 group (G) and the OVX + SW8 group (H) was enhanced the staining in the UL. (I,J) Western blot was evaluated the protein levels of bladder inflammation (TGF-ß1 and COX-2), interstitial fibrosis (fibronectin and type I collagen), and urothelial structure (E-cadherin and uroplakin III). Both the inflammatory and fibrosis markers (TGF-ß1, fibronectin, type I collagen, and COX-2) were significantly increased in the OVX group compared to the sham group. However, the proteins noticeably decreased in the OVX + SW4 group and the OVX + SW8 group compared to the OVX group except TGF-ß1 expression in the OVX + SW4 group. Note: CK, cytokeratin; UL, urothelial layer; UBL, urothelial basal layer; SL, suburothelial layer; UPKIII, uroplakin III; TGF-ß1, transforming growth factor ß 1. Data were expressed as mean ± SD for n = 6, * p < 0.05; ** p < 0.01 versus the sham group. †p < 0.05; ††p < 0.01 versus the OVX group.

Figure 3

LiESWT improved OVX-induced pathological alteration and urothelial junction-associated protein expression. The expressions of proliferating and differential markers (Ki67, CK14 and CD44) and urothelial tight junction proteins (Claudin-4 and ZO-1) were assessed by immunofluorescence evaluation (A–H) and Western blot (I,J). (A–D) The staining of proliferation marker Ki67 was less distribution in the bladder tissues of the sham group (A), the OVX group (B) and the OVX + SW4 group (C). On the contrary, the Ki67 immunostaining was obviously expressed in the UBL and the sphere of SL in the OVX + SW8 group (D). (E–H) Double-labeled analysis of Claudin-4 (fluorescein isothiocyanate; green, upper panels) and CK14 (rhodamine; red, lower panels) was widely distributed in the UL of the sham group (E). The co-staining was restricted to the thin and disrupted urothelium in OVX group (F). However, the labeling of the OVX + SW8 group (H) was markedly expressed in the UBL and the sphere of SL compared to the OVX group (F) and the OVX + SW4 group (G). Nuclear DNA was labeled with DAPI (blue). (I, J) The protein levels of Ki67, CK14, CD44, Claudin-4, and ZO-1 were investigated by Western blotting analysis. The protein levels noticeably enhanced in the OVX + SW8 group compared to the OVX group. Note: CK, cytokeratin; UL, urothelial layer; UBL, urothelial basal layer; SL, suburothelial layer. Values are means ± SD for n = 6. * p < 0.05; ** p < 0.01 versus the sham group. ††p < 0.01 versus the OVX group.

Figure 4

Proposed potential mechanism of regulating angiogenic remodeling triggered by LiESWT. The angiogenesis markers were analyzed by immunostaining (A–D) and Western blots (E–H). (A–D) The α-SMA immunostaining widely distributed in smooth muscle of microvessels beneath UBL and vessels in the SL and muscular layer in the sham group (A). In the OVX group (B), the α-SMA expression was reduced in the SL compared to the sham group. However, the expression levels in the OVX + SW4 group (C) and the OVX + SW8 group (D) were increased in the SL and beneath UBL. (E,G) The levels of angiogenesis-associated proteins, including Laminin, Integrin-α6 (Laminin receptor), α-SMA, CD31, VEGF, VEGF-R1, and VEGF-R2 (VEGF receptor), were quantified by Western blots. Furthermore, the protein level in the OVX group was much lower than the sham group, except Integrin-α6. However, the expressions of angiogenesis markers were obviously increased in the OVX + SW4 group and the OVX + SW8 group compared with the OVX group. Therefore, LiESWT altered bladder angiogenic remodeling. Note: UL, urothelial layer; UBL, urothelial basal layer; SL, suburothelial layer; VEGF, vascular endothelial growth factor. (F,H): Proposed potential mechanism involved in regulating angiogenic remodeling triggered by LiESWT. The signaling-related kinases for angiogenic response, including Erk1/2, p-Erk1/2, P38, p-P38, C-Jun, p-C-Jun, Akt, and p-Akt, were quantified by Western blots. The expression levels of both Erk1/2 and Akt proteins were significantly declined in the OVX group, but the expression level of C-Jun was promoted as compared with the sham group. Besides, LiESWT treatment significantly promoted the phosphorylation of Erk1/2, P38, and Akt in the bladder as compared with the OVX group, whereas it reduced the phosphorylation of C-Jun. Therefore, LiESWT enhanced angiogenesis through the phosphorylation of P38, Erk1/2, and Akt. Values are means ± SD for n = 6. * p < 0.05; ** p < 0.01 versus the sham group. †p < 0.05; ††p < 0.01 versus the OVX group.

Figure 5

Analysis of postmenopausal participants with OAB symptoms. (A) The percentage of postmenopausal participants with OAB symptoms. OAB consisted of daytime frequency, nocturia urgency with or without urgency incontinence. (B) The therapeutic efficacy of LiESWT improved the OAB symptoms evaluated by 3-day urinary diary. The changes in daytime frequency, nocturia and urgency by 3-day urinary diary record at W4, W8, F1, F3, and F6 compared with W0. The mean times of daytime frequency, nocturia and urgency were noticeably decreased after LiESWT treatment. W0: the baseline, W4: 4-week of LiESWT treatment, W8: 8-week of LiESWT treatment, F1: 1-month follow-up, F3: 3-month follow-up, F6: 6-month follow-up. * p < 0.05; ** p < 0.01 as compared with W0.

Figure 6

Improvement in OAB symptoms and life bothersome questionnaire scores after LiESWT treatment. (A,C) The therapeutic effect of LiESWT was analyzed by the OAB symptoms and life bothersome questionnaires, including OABSS, ICIQ-SF, UDI-6, and IIQ-7. LiESWT treatment significantly reduced in the scores of OABSS, ICIQ-SF, UDI-6, and IIQ-7 as compared with the sham group. (B,D) Improvement of OABSS sub-scores for OAB symptoms after LiESWT treatment, including daytime frequency, nocturia, urgency, and urgency incontinence. LiESWT improved OAB symptoms and the QoL. Note: OABSS, overactive bladder symptom scores. ICIQ-SF: international consultation on incontinence questionnaire-short form, UDI-6: urogenital distress inventory-6, IIQ-7: incontinence impact questionnaire-7, W0: the baseline, W4: 4 weeks of LiESWT treatment, W8: 8 weeks of LiESWT treatment, F1: 1-month follow-up, F3: 3-month follow-up. The blue or orange font denotes the p-value before and after 4 weeks treatment in the sham group or in the LiESWT-treated group, respectively. The purple font indicates the p-value between the sham group and the LiESWT-treated group at the W0 and W4. * p < 0.05; ** p < 0.01 as compared with W0.

Figure 7

A study diagram proposed for the potential effect of LiESWT on improving the OAB symptoms. Part I: Rat animal experiment: LiESWT (0.12 mJ/mm2, 300 impulses, 3 Hz, and once/week) for OVX-induced OAB rat was placed on the lower abdomen. The effect of LiESWT enhanced anti-inflammation, promoted cell proliferation, and altered angiogenesis through VEGF/VEGF-R/MAPK (P38 and Erk1/2) pathways and Laminin/Integrin-α6/Akt pathways to improve OAB-induced by OHD. Part II: Human clinical trial: LiESWT (0.25 mJ/mm2, 3000 pulses, 3 Hz, and once/week) for postmenopausal OAB participants was placed on the lower abdomen with two fingers apart from the pubis. The results revealed that LiESWT improved bladder function and ameliorated the OAB symptoms after 8-week LiESWT. Note: OVX, ovariectomy; OHD, ovary hormone deficiency; OAB, overactive bladder; LiESWT, low-intensity extracorporeal shock wave therapy; CK, cytokeratin; VEGF, vascular endothelial growth factor; TGF-ß1, transforming growth factor ß 1.