Hypothesis / aims of study
Overactive bladder (OAB) has been treated with medications and invasive treatments. However, there is a strong demand for non-invasive treatment for refractory OAB. In these days, low-energy shockwave therapy (LESW) is known to induce various biological effects such as angiogenesis, anti-inflammation, nerve regeneration, cell proliferation, and alteration of membrane permeability. The aims of this study were to evaluate whether LESW improves ischemic-induced OAB in a rat model (AI model) and to investigate its therapeutic mechanisms.
Study design, materials and methods
Sixteen-week-old male Sprague-Dawley rats were randomly divided into three groups: the AI, AI-SW and control groups. The AI and AI-SW groups underwent endothelial artery injury and received a high cholesterol diet. In the AI-SW group, LESW was shot onto the abdominal wall once a week from 4 to 7 weeks after AI surgery (20-23 weeks of age). A shockwave generator DUOLITH SD1 was used with an intensity of 0.25 mJ/mm2 (total energy flux density), a frequency of 3 Hz, and 1800 shots. At 24 weeks of age (8 weeks after AI), a conscious cystometry was performed followed by measuring blood flow of the bladder using the laser speckle contrast imaging (n=8 for each group). Blood flow was normalized by blood pressure simultaneously monitored from left internal carotid artery. The bladder was harvested in the early phase (24 hours after the first LESW: 20 weeks of age) and in the chronic phase (24 weeks of age) for molecular analyses and histological evaluations.
Results
Voiding interval was significantly shorter in the AI group (mean ± SEM: 5.1 ± 0.8 min) than the control group (17.3 ± 3.0 min), while significant improvement was observed in the AI-SW group (14.9 ± 3.3 min). Blood flow of the bladder significantly increased in the AI-SW group than in the AI at three points of saline infusion: 0, 0.5, and 1.0 ml.
Microarray analysis showed higher gene expression of soluble guanylate cyclase (GC) alpha and beta in the AI-SW group than in the AI group in the chronic phase. Polymerase chain reaction (PCR) and Western blotting revealed that gene/protein expression of GC alpha was significantly higher in the AI-SW and control groups than in the AI group. mRNA of vascular endothelial growth factor (VEGF) was highly expressed in the early phase after LESW, followed by increased protein expression (P=0.069) in the chronic phase. Enzyme-linked immunosorbent assay (ELISA) demonstrated a significant elevation of cyclic guanosine monophosphate (cGMP) in the bladder of the AI-SW group.
Histological examinations showed rich vascularity in the suburothelium with positive stain of VEGF and GC alpha/beta in the AI-SW group, in contrast to poor vascularity in the thinned suburothelium of the AI group.
Interpretation of results
LESW is considered to stimulate mechanosensors on cell membranes. Previous In-vitro studies revealed that LESW upregulated VEGF and enhance nitric oxide (NO) production via activation of eNOS in human umbilical vein endothelial cells (HUVECs). In-vivo studies of LESW demonstrated that angiogenesis involving VEGF improved various pathophysiological conditions such as cardiac dysfunction and erectile dysfunction.
Possible mechanisms of therapeutic effect of LESW on OAB:
1. Recovered blood flow due to angiogenesis may decrease oxidative stress and inflammatory cytokines, which can ameliorate OAB in the pathophysiology of chronic pelvic ischemia.
2. GC is known to be degraded under hypoxia. Improved blood flow due to angiogenesis may restore GC in the bladder, resulting in elevation of cGMP. (LESW may also stimulate GC-cGMP pathway via activation of eNOS.) Elevated cGMP relaxes vascular smooth muscle (vasodilation) and sphincter/detrusor smooth muscle and inhibits afferent nerve activities.
As limitations, we do not yet investigate upstream mediators (e.g. NO, eNOS and Akt) and several genes related to anti-inflammation and neurogenesis which were indicated by microarray analysis.