Simulated vaginal delivery causes transients vaginal smooth muscle hypersensitivity and urethral sphincter dysfunction.

BACKGROUND: Although pelvic floor dysfunction (PFD) has a multifactorial etiology, pregnancy and childbirth are considered crucial events predisposing to urinary incontinence as well as pelvic organ prolapse, which are highly prevalent. Rats are the most frequently used animal model and pudendal nerve crush (PNC) and vaginal distension (VD) are often used to mimic vaginal delivery. OBJECTIVE: To document the time course of events after simulated vaginal delivery (SVD) on the urethral sphincter and the vaginal smooth muscle layer. MATERIALS AND METHODS: Virgin female Sprague-Dawley rats were subjected to SVD (PNC + VD) or sham surgery and evaluated at 7, 14, 21, and 42 days after the injury. Urethral function was determined in vivo by microultrasound during cystometry and vaginal smooth muscle layer was harvested for in vitro pharmacologic investigation by isometric tension recording. Furthermore, vaginal and urethral samples were investigated by immunohistochemistry and real-time quantitative polymerase chain reaction. RESULTS: Microultrasound showed no bursting of the urethral sphincter in the SVD group at 7 days with a functional recovery starting at 14 days, and normal bursting at 21 and 42 days. Vaginal smooth muscle showed higher sensitivity to carbachol at 14 and 21 days after injury; however, at 42 days, its sensitivity decreased when compared with sham. CONCLUSION: SVD induces urethral dysfunction and a shift in vaginal smooth muscle contractile responses to carbachol.

Quantitative analysis of motor evoked potentials in the neonatal lamb.

Evoking motor potentials are an objective assessment method for neuromotor function, yet this was to our knowledge never done in neonatal lambs. There is neither a method for standardized quantification of motor evoked potentials (MEPs). We first aimed to evaluate the feasibility of MEP recording in neonatal lambs and test its validity. Second we aimed to develop an algorithm for its quantification and test its reliability since manual input is required. We recorded myogenic MEPs after transcranial motor cortex stimulation in 6 lambs aged 1-2 days. MEPs were also measured in one lamb undergoing Neuro-Muscular Blockade (NMB) and another undergoing lumbar spinal cord (SC) transection, both serving as controls. We computed 5 parameters using a custom-made algorithm: motor threshold, latency, area-under-the-curve, peak-to-peak amplitude and duration. Intra- and inter-observer reliability was analyzed. MEPs could be easily recorded, disappearing after NMB and SC transection. The algorithm allowed for analysis, hence physiologic readings of the parameters in all 4 limbs of all lambs were obtained. Our method was shown to have high intra- and inter-observer ( ≥70%) reliability for latency, area-under-the-curve and peak-to-peak amplitude. These results suggest that standardized MEP recording and analysis in neonatal lambs is feasible, and can reliably assess neuromotor function.

Amniotic Fluid Derived Stem Cells with a Renal Progenitor Phenotype Inhibit Interstitial Fibrosis in Renal Ischemia and Reperfusion Injury in Rats.

OBJECTIVES: Mesenchymal stem cells derived from human amniotic fluid (hAFSCs) are a promising source for cellular therapy, especially for renal disorders, as a subpopulation is derived from the fetal urinary tract. The purpose of this study was to evaluate if hAFSCs with a renal progenitor phenotype demonstrate a nephroprotective effect in acute ischemia reperfusion (I/R) model and prevent late stage fibrosis. METHODS: A total of 45 male 12-wk-old Wistar rats were divided into three equal groups;: rats subjected to I/R injury and treated with Chang Medium, rats subjected to I/R injury and treated with hAFSCs and sham-operated animals. In the first part of this study, hAFSCs that highly expressed CD24, CD117, SIX2 and PAX2 were isolated and characterized. In the second part, renal I/R injury was induced in male rats and cellular treatment was performed 6 hours later via arterial injection. Functional and histological analyses were performed 24 hours, 48 hours and 2 months after treatment using serum creatinine, urine protein to creatinine ratio, inflammatory and regeneration markers and histomorphometric analysis of the kidney. Statistical analysis was performed by analysis of variance followed by the Tukey's test for multiple comparisons or by nonparametric Kruskal-Wallis followed by Dunn. Statistical significance level was defined as p <0.05. RESULTS: hAFSCs treatment resulted in significantly reduced serum creatinine level at 24 hours, less tubular necrosis, less hyaline cast formation, higher proliferation index, less inflammatory cell infiltration and less myofibroblasts at 48 h. The treated group had less fibrosis and proteinuria at 2 months after injury. CONCLUSION: hAFSCs contain a renal progenitor cell subpopulation that has a nephroprotective effect when delivered intra-arterially in rats with renal I/R injury, and reduces interstitial fibrosis on long term follow-up.

A surgical technique for homogenous renal distribution of substances in rats.

Intra-arterial injection of mesenchymal stem cells has been proven to result in a superior nephroprotection compared to intravenous injection. This avoids initial passage through filter organs such as the lung, liver and spleen. The aim of the present study was to investigate whether suprarenal aortic delivery results in a homogenous distribution to both kidneys. Chinese ink was used to evaluate the renal distribution pattern for the comparison of two retrograde intra-aortic injection methods. In the first, the aorta caudal to the renal branches was temporarily clamped and Chinese ink was injected at the level of the renal arteries. In the second, a distal aortic clamp was combined with alternated clamping of the contralateral arteries. Immediately after injection, kidneys were harvested for histological analysis. Amniotic fluid stem cells labeled with LacZ were injected in the aorta by alternated clamping of the renal arteries in order to track the cells in a rat ischemia/reperfusion model. Without renal artery clamping, intra-aortic administration resulted in a delivery of the ink into the right kidney, whereas administration with alternated clamping of the contralateral renal artery, together with distal aortic artery clamping, resulted in a more homogenous distribution of the ink in both kidneys. Moreover, LacZ-positive cells were found in both kidneys after 6 h of injection. In conclusion, the retrograde administration of Chinese ink in two steps is a fast and reproducible technique, which results in a more homogenous distribution of the stain in both kidneys than a single administration combined by only clamping the aorta.