The Subdivision of the Spinal Neurons for Detrusor Function / 대한비뇨기과학회지

Purpose: No ideal method for subdividing and assessing changes in neurons of the spinal cord during specific conditions has been established. We attempted to develop a method for subdividing spinal neurons using immunohistochemical and fluorescent staining, which is an important key towards understanding the mechanism of reflex voiding. Materials and Methods: Thirty Sprague-Dawley rats, weighting 200-300g, were divided into five groups. A cystometrogram was performed during saline or acetic acid instillation. We identified the neuronal pathway associated with the detrusor by injecting a pseudorabies virus (PRV) into the detrusor muscle and inspecting the changes in relation to different time sequences. An immunohistochemical staining method was used to stain the fos-protein encoded by the c-fos gene. Immunofluorescent staining was performed to evaluate changes in the neurons in relation to the voiding reflex, and the neurons then subdivided. Results: We confirmed pseudorabies virus (PRV) infection of the cells in the sacral parasympathetic nucleus through immunohistochemical staining two days after injection. On detection of an increase in c-fos positive cells after dividing the c-fos positive area of the L6 and S1 spinal cord into 4 sections, significant increases were observed in the sacral parasympathetic nucleus (SPN) and dorsal commissure (DCM). Double staining was performed to detect the neurons associated with the voiding reflex in the SPN and DCM areas showing overexpression of c-fos. Conclusions: The establishment of a method for detecting morphological changes, and subdividing neurons by immunohistochemical and fluorescent staining, may provide an important key towards understanding the mechanism of various neuromodulations of clinically applied treatments. (Korean J Urol 2005;46:487-494)

Change of Bladder Afferent Neuron after Intravesical Electrical Stimulation / 대한비뇨기과학회지

PURPOSE: There have been reports on the clinical benefits of intravesical electrical stimulation (IVES) in patients with an increased residual urine or reduced bladder capacity. However, studies on the underlying mechanism of IVES have been limited to the A delta afferent and parasympathetic neurons. This study sort to investigate the changes in the expression of the C-fos protein in the thoracolumbar and lumbosacral spinal cord in order to determine the effect of IVES on the C fiber afferent nerve. MATERIALS AND METHODS: Twenty Sprague-Dawley rats were divided into four groups; group 1 (n=5) served as a sham operation, groups 2 (n=5) were instilled with normal saline, groups 3 (n=5) were instilled with 1% acetic acid, and group 4 (n=5) received IVES prior to the administration of 1% acetic acid. The spinal cord was removed at the thoracolumbar (T13-L2) and lumbosacral (L5-S1) levels. Changes in the levels of the C-fos protein in the spinal cord were measured by western-blot analysis. RESULTS: The relative density of the C-fos expression, 2 hours after IVES, was significantly higher, and the fos protein expression induced by IVES was decreased to the sham levels by the 5th day following the IVES. Therefore, it was decided that a 5 day interval between the IVES and the noxious stimulation with acetic acid should be used to avoid the superimposition of C-fos expression. The relative density of the C-fos protein, following noxious stimulation with 1% acetic acid, was significantly higher compared to the saline distension in both the T13-L2 and L5-S1 levels of the spinal cord. However, the IVES prior to the administration of 1% acetic acid significantly decreased the relative density of the C-fos protein at both the T13-L2 and L5-S1 levels of the spinal cord compared to the rats with noxious stimulation alone. CONCLUSIONS: IVES reduces the expression of C-fos, which is mainly conveyed by the unmyelinated C fibers in the noxiously stimulated rats. These results suggest that the bladder C fiber afferent is also involved in modulating the micturition reflex caused by IVES.

What Causes Bladder Fibrosis?: Abnormal Innervation or Abnormal Bladder Dynamics / 대한비뇨기과학회지

PURPOSE: Bladder tissue fibrosis is characterized by the abnormal deposition of connective tissue within different layers of the bladder wall, resulting in "non-compliance". The different etiologies of bladder fibrosis are either neurogenic, which encompasses myelodysplasia and spinal cord injury, or nonneurogenic, due to a bladder outlet obstruction. In this study, bladder fibrosis was examined to see if it was due to the effect of an abnormal innervation or to abnormal bladder dynamics. MATERIALS AND METHODS: Thirty-five male Sprague-Dawley rats (250-300g) were divided into four groups; the normal (n=5), bilateral pelvic nerve transection; denervation (n=10), vesicostomy (n=10) and vesicostomy after denervation (n=10) groups. After 4 weeks, the bladders of the animals in each group were obtained. In the histological study, the collagen to muscle ratio from Masson's trichrome staining, and the elastin density (%) from Van Gieson staining, were determined using a Metamorph ver 4.6r5 image analyzer. The TGF-beta1 and collagen III protein expressions were detected by Western blotting. RESULTS: The collagen to muscle ratios of the normal, vesicostomy, denervation and vesicostomy after denervation groups were; 0.67+/-0.04*, 0.67+/-0.04*, 1.25+/-0.03* and 0.96+/-0.02 (*pvesicostomy after denervation>vesicostomy>normal from the histological study. The levels of TGF-beta1 and of collagen III in the denervation group were higher than those in the vesicostomy after denervation group. The above findings show that pressure and nerve innervation in the bladder both have significant effects on the process of bladder fibrosis. Meanwhile, a vesicostomy could reduce the fibrotic effect caused by denervation, although this is not complete.

Sensory Neuronal Change after Intravesical Electrical Stimulation in Spinailized Rat

The clinical benefits of intravesical electrical stimulation (IVES) in patients with increased residual urine or reduced bladder capacity have been reported. However, studies on the underlying mechanism of IVES has been limited to the A delta afferent and parasympathetic neurons. This study investigated the changes in the calcitonin gene-related peptide (CGRP), substance P (SP), and nitric oxide synthase (NOS) expression in the thoracolumbar and lumbosacral dorsal root ganglia (DRG) of spinalized rats to determine the effect of IVES on the C fiber afferent nerve. Forty Sprague-Dawley rats were divided into normal controls (n=10); IVES treated normal rats (n=10), spinalized rats (n=10), and IVES treated spinalized rats (n=10). IVES was performed for 2 weeks (5 days a week). IVES was started 3 weeks after spinalization in the spinalized animals. All animals had the DRG removed at the thoracolumbar (T13-L2) and lumbosacral (L5-S1) level. Changes in the CGRP, SP and n-NOS levels at the DRG were measured by western-blot analysis. The relative density of the CGRP and SP following spinalization was significantly higher compared to the controls in both the T13-L2 and L5-S1 DRG. However, IVES in the spinalized rat significantly decreased the relative density of the CGRP and SP compared to the rats with spinalization alone. A significant increase in the relative density of n-NOS was detected in the L5-S1 DRG following spinalization. However, the density of n-NOS was significantly lower after IVES in both the T13-L2 and L5-S1 DRGs. In conclusion, IVES significantly reduced the CGRP, SP and n-NOS levels in the DRG of spinalized rats. CGRP, SP and n-NOS are the main factors that contribute to the hyperexcitability of the micturition reflex after spinal cord injury. These results suggest that the bladder C fiber afferent is also involved in modulating the micturition reflex by IVES.