Increasing evidence supports a positive effect of low intensity extracorporeal shock wave therapy (Li-ESWT) on erectile dysfunction, probably by promoting angiogenesis as suggested by studies in animal models. There is currently scarce data on the impact of Li-ESWT on the bladder. In a rat model of diabetic underactive bladder, Li-ESWT could enhance muscle contractile activity of the bladder and urethra, significantly restored bladder morphology, possibly via an increase in smooth muscle actin expression in the bladder wall and recovery of neuronal integrity and innervation (1). Further to these findings, we used a streptozotocin-induced diabetic rat model to investigate the molecular effect of Li-ESWT on the diabetic bladder. In order to do this, we studied the changes in genes associated with mechanosensation, inflammation/ischemia, and contractility. Our hypothesis was that Li-ESWT may reverse molecular changes induced by diabetes in the bladder.

All animal experiments were carried out in accordance with the European Directive 2010/63/EEC, approved by the local Veterinary Directorate and the local University Ethics Committee. 
Fifteen adult male Wistar albino rats were randomized into 3 groups: a group of diabetic rats without treatment (DM, n=5), a group of diabetic rats treated with Li-ESWT (DM-ESWT; n=5) and a control group (Control bladder CB; n=5). Diabetes mellitus (DM) type II was induced by injection of a single intraperitoneal dose of streptozotocin (60 mg/kg). Twenty days after the induction of DM, each rat in group DM-ESWT received ESWT treatment. The rats were housed under a 12:12-h light/dark cycle, with food and water ad libitum. 
Treatment with Li-ESWT was applied as previously described (2). Briefly, rats were anaesthetized with IM injection of a mixture of ketamine (70 mg/kg of body weight) and xylazine (6 mg/kg of body weight) and placed in a supine position. The ultrasound gel was applied on the skin of the shaved lower abdomen and a specially designed shock wave probe for small animals was used (Omnispec ED1000 standard system, Medispec Ltd, Yehud, Israel) (3) to deliver a total of 300 shockwave pulses at an energy energy flux density of 0.09mJ/mm2 at 2Hz in contact with the skin on the dorsal surface of the bladder. This procedure was repeated three times per week for two weeks (delivering a total of 1800 shockwave pulses), followed by a two-week washout period, as previously described (2).
Following the experimental period, rats were sacrificed, their bladders harvested and total RNA was extracted from each tissue with the NucleoSpin® RNA Kit (Macherey-Nagel™) according to the manufacturer’s instructions. cDNA was synthesized using oligodT and PrimeScriptTM 1st strand cDNA Synthesis kit (Takara).  Quantitative Real Time Polymerase Chain Reaction (qRT-PCR) was performed in order to analyze the expression pattern of the Transient Receptor Potential Vanilloid 1 (TRPV1) (Rn00583117_m1), interleukin 1β (IL1b) (Rn00580432_m1) and the muscarinic receptors M1, M2 and M3 (Chrm1, Chrm2 and Chrm3 respectively) (Rn00589936_s1, Rn02532311_s1, and Rn00560986_s1) as well as the endogenous housekeeping gene GAPDH (Rn01775763_g1) and the and the ΔΔCt was calculated. Data were analyzed using SPSS software, and expressed as mean±standard deviation from the mean. Multiple groups were compared using one-way ANOVA.

The expression of the TRPV1, IL1b, and Chrm2 genes was significantly different between the three groups (p=0,002, 0,000 and 0,011, respectively). The expression of all genes appeared to be increased in the DM group when compared to CB group, but was statistically significant only for the TRPV1 and IL1b genes (p=0,002 and 0,000, respectively). Treatment with Li-ESWT significantly reduced the expression of genes IL1b and Chrm2 (p=0,001 and 0,011, respectively), whereas a tendency for reduced expression was noted for TRPV1 (p=0,069) as well.
However the expression of Chrm1and Chrm3 genes was not significantly different between the three groups of animals (0,084 and 0,109 respectively). In DM group there was a weak, but not significant increase of Chrm1 and Chrm3 expression (0,172 and 0,141 respectively) compared to CB group. Finally Li-ESWT treatment induced no significant decrease of Chrm1 and Chrm3 expression (0,149 and 0,344 respectively) compared to DM group.

Results suggest a dysregulation of receptor genes associated with afferent-related mechanosensation, inflammation/ischemia, and contractility in diabetes, in support of previous reports and hypotheses on the pathophysiological mechanisms leading to diabetic bladder dysfunction. Bladder ischemia is thought to be involved in the pathogenesis of afferent dysfunction in both conditions of detrusor overactivity and underactivity. In animal models of chronic bladder ischemia, upregulation of oxidative stress and proinflammatory markers in the bladder wall may lead to increased afferent activity and bladder overactivity. Peripheral afferent dysfunction as well as oxidative stress and changes in muscarinic receptor activity as a consequence of chronic bladder ischemia have been proposed to contribute to the pathophysiology of detrusor underactivity. Both conditions may be found in DM, either separately at different time-points on the progress of the disease, or simultaneously. Li-ESWT appears to partly restore the expression of receptors towards normalization, suggesting a possible mechanism of action for this treatment in bladder functional disease states. Functional experiments are needed to confirm the importance of our findings

In this rat model, diabetes appears to affect the bladder at a molecular level by increasing the expression of receptors related to mechanosensation, inflammation/ischemia, and contractility. Treatment of the diabetic rat bladder with Li-ESWT could restore the expression of IL1b, TRPV1 and Chrm2 receptor genes towards normalization, suggesting a therapeutic potential of this treatment modality for diabetic cystopathy.

Wang HS, Oh BS, Wang B, Ruan Y, Zhou J, Banie L, et al. Low-intensity extracorporeal shockwave therapy ameliorates diabetic underactive bladder in streptozotocin-induced diabetic rats. BJU Int. 2018;122(3):490-500
Sokolakis I, Dimitriadis F, Psalla D, Karakiulakis G, Kalyvianakis D, Hatzichristou D. Effects of low-intensity shock wave therapy (LiST) on the erectile tissue of naturally aged rats. Int J Impot Res. 2018 Aug 17
Assaly-Kaddoum R, Giuliano F, Laurin M, Gorny D, Kergoat M, Bernabe J, et al. Low Intensity Extracorporeal Shock Wave Therapy Improves Erectile Function in a Model of Type II Diabetes Independently of NO/cGMP Pathway. J Urol. 2016;196(3):950-6