Kilohertz alternating current neuromodulation of the pudendal nerves: effects on the anal canal and anal sphincter in rats.

The first two objectives were to establish which stimulation parameters of kilohertz frequency alternating current (KHFAC) neuromodulation influence the effectiveness of pudendal nerve block and its safety. The third aim was to determine whether KHFAC neuromodulation of the pudendal nerve can relax the pelvic musculature, including the anal sphincter. Simulation experiments were conducted to establish which parameters can be adjusted to improve the effectiveness and safety of the nerve block. The outcome measures were block threshold (measure of effectiveness) and block threshold charge per phase (measure of safety). In vivo, the pudendal nerves in 11 male and 2 female anesthetized Sprague Dawley rats were stimulated in the range of 10 Hz to 40 kHz, and the effect on anal pressure was measured. The simulations showed that block threshold and block threshold charge per phase depend on waveform, interphase delay, electrode-to-axon distance, interpolar distance, and electrode array orientation. In vivo, the average anal pressure during unilateral KHFAC stimulation was significantly lower than the average peak anal pressure during low-frequency stimulation (p < 0.001). Stimulation with 20 kHz and 40 kHz (square wave, 10 V amplitude, 50% duty cycle, no interphase delay) induced the largest anal pressure decrease during both unilateral and bilateral stimulation. However, no statistically significant differences were detected between the different frequencies. This study showed that waveform, interphase delay and the alignment of the electrode along the nerve affect the effectiveness and safety of KHFAC stimulation. Additionally, we showed that KHFAC neuromodulation of the pudendal nerves with an electrode array effectively reduces anal pressure in rats.

Androgen receptors in areas of the spinal cord and brainstem: A study in adult male cats.

Sex hormones, including androgens and estrogens, play an important role in autonomic, reproductive and sexual behavior. The areas that are important in these behaviors lie within the spinal cord and brainstem. Relevant dysfunctional behavior in patients with altered androgen availability or androgen receptor sensitivity might be explained by the distribution of androgens and their receptors in the central nervous system. We hypothesize that autonomic dysfunction is correlated with the androgen sensitivity of spinal cord and brainstem areas responsible for autonomic functions. In this study, androgen receptor immunoreactive (AR-IR) nuclei in the spinal cord and brainstem were studied using the androgen receptor antibody PG21 in four uncastrated young adult male cats. A dense distribution of AR-IR nuclei was detected in the superior layers of the dorsal horn, including lamina I. Intensely stained nuclei, but less densely distributed, were found in lamina X and preganglionic sympathetic and parasympathetic cells of the intermediolateral cell column. Areas in the caudal brainstem showing a high density of AR-IR nuclei included the area postrema, the dorsal motor vagus nucleus and the retrotrapezoid nucleus. More cranially, the central linear nucleus in the pons contained a dense distribution of AR-IR nuclei. The mesencephalic periaqueductal gray (PAG) showed a dense distribution of AR-IR nuclei apart from the most central part of the PAG directly adjacent to the ependymal lining. Other areas in the mesencephalon with a dense distribution of AR-IR nuclei were the dorsal raphe nucleus, the retrorubral nucleus, the substantia nigra and the ventral tegmental area of Tsai. It is concluded that AR-IR nuclei are located in specific areas of the central nervous system that are involved in the control of sensory function and autonomic behavior. Furthermore, damage of these AR-IR areas might explain related dysfunction in humans.

The frequency spectrum of bladder non-voiding activity as a trigger-event for conditional stimulation: Closed-loop inhibition of bladder contractions in rats.

AIMS: To test the hypothesis that the frequency of bladder non-voiding contractions (NVCs) can be used as a trigger event for closed-loop conditional inhibition of detrusor contractions via tibial nerve (TN) or dorsal penile nerve (DPN) stimulation. METHODS: In urethane anaesthetized male Wistar rats, the bladder was filled continuously with saline to evoke contractions. To test the plausibility of conditional inhibition via the TN, electrical stimulation was switched on manually when the pressure increased above a threshold of 10 cmH2 0 above the baseline. For testing conditional stimulation via the DPN, the pressure signal was continuously stored and a baseline threshold, the area under the curve (AUC) of the amplitude spectrum in the 0.2-20 Hz range of a 5 s window at the beginning of filling was calculated. When the AUC of subsequent pressure windows superseded the baseline threshold, the DPN was automatically stimulated. RESULTS: TN stimulation failed to inhibit evoked voiding contractions. The NVC frequency spectrum based DPN stimulation successfully inhibited 70% of the evoked contractions and resulted in a 45% increase in bladder capacity (BC). CONCLUSIONS: While, conditional TN stimulation failed to suppress bladder contractions, DPN stimulation, automatically triggered by an increased frequency of bladder non-voiding activity, resulted in bladder inhibition, and a consequential increase in BC. This study demonstrates the plausibility of using the frequency of NVCs as a trigger event for conditional inhibition of detrusor contractions.

Effect of tibial nerve stimulation on bladder afferent nerve activity in a rat detrusor overactivity model.

OBJECTIVES: To study the post-stimulation effect of tibial nerve stimulation on rat bladder afferent activity, and urodynamic parameters in normal and acetic acid-induced detrusor overactivity conditions. METHODS: In urethane anesthetized male Wistar rats, the tibial nerve was stimulated for 30 min at 5 Hz, pulse width 200 µs and amplitude approximately threefold the threshold to induce a slight toe movement. The post-stimulation effect was studied by measuring afferent nerve activity of postganglionic pelvic nerve branches and various urodynamic parameters under two different conditions: (i) in physiological saline filling experiments (simulating normal bladder condition); and (ii) in acetic acid irritated bladders (simulating detrusor overactivity). RESULTS: After 30 min of tibial nerve stimulation in saline filling experiments, the bladder capacity, threshold pressure and afferent nerve activity were not significantly different from the prestimulation measurements. The instillation of 0.5% acetic acid significantly reduced the bladder capacity and increased the afferent nerve activity. Tibial nerve stimulation significantly improved the bladder capacity and suppressed the afferent nerve activity compared with prestimulation acetic acid measurements. CONCLUSIONS: Tibial nerve stimulation is able to significantly restore the bladder capacity by inhibiting afferent nerve activity in chemically irritated rat bladders. The present study provides important basic electrophysiological evidence to substantiate the clinical use of tibial nerve stimulation for treatment of symptoms related to detrusor overactivity.

Frequency analysis of urinary bladder pre-voiding activity in normal and overactive rat detrusor.

AIMS: To test the hypothesis that voiding in anesthetized rats is preceded by recurrent changes in the pattern of bladder pressure. To explore the use of frequency analysis as an analytical tool for automatically detecting these changes and to provide quantitative data on bladder pre-voiding activity. METHODS: We developed an algorithm, based on frequency analysis, to study bladder pressure during the filling phase in anesthetized rats. Two applications of the algorithm were tested: (i) as a predictor of a voiding contraction with alarms generated which would make conditional nerve stimulation to prevent incontinence possible; and (ii) as a new index to quantify rapid pressure transients in normal and overactive detrusor conditions (i.e., induced by acetic acid instillation into the bladder). RESULTS: The results show that a very high percentage (∼90%) of the alarms was generated by the algorithm within 100 sec before voiding. The index of rapid transients and the bladder volume before voiding were respectively ∼13% less and ∼42% less in acetic acid. CONCLUSIONS: We have shown that a simple algorithm, based on frequency analysis of bladder pressure, can be used to predict voiding and to provide quantitative data on non-voiding bladder activity and its changes due to pathology. Although the results refer to anesthetized rats, they are promising and warrant further urodynamic investigation to identify if similar patterns occur in non-anesthetized rats and in humans.

Longitudinal leak point pressure measurements in rats using a modified port à cath system.

Stress urinary incontinence, defined as involuntary loss of urine secondary to an increase in abdominal pressure, represents one of the most significant urological problems. Several animal models to simulate stress urinary incontinence have been presented, including methods to quantify leak point pressure. We have modified an existing leak point pressure procedure to longitudinally quantify stress urinary incontinence in rats by introducing the port à cath system. Reproducible leak point pressure measurements were carried out over a period of more than 40 days at different bladder volumes. Leak point pressure neither showed a significant relationship with the number of times anesthesia was applied nor a significant change over time. The port à cath system provided a reliable, sensitive device for longitudinal urinary bladder pressure measurements in animals with an implanted bladder catheter. This set-up therefore enables the evaluation of bladder pressure in different models for stress urinary incontinence, such as vaginal distention or pudendal transection over long periods of time within the same animal.

Androgen receptors in the forebrain: A study in adult male cats.

Androgens and their receptors are present throughout the body. Various structures such as muscles, genitals, and prostate express androgen receptors. The central nervous system also expresses androgen receptors. Androgens cross the blood-brain barrier to reach these central areas. In the central nervous system, androgens are involved in multiple functions. The current study investigated in which forebrain areas androgens are expressed in the male cat. Androgen receptor immunoreactive (AR-IR) nuclei were plotted and the results were quantified with a Heidelberg Topaz II + scanner and Linocolor 5.0 software. The density and intensity of the labeled cells were the main outcomes of interest. The analysis revealed a dense distribution of AR-IR nuclei in the preoptic area, periventricular complex of the hypothalamus, posterior hypothalamic area, ventromedial hypothalamic, parvocellular hypothalamic, infundibular, and supramammillary nucleus. Numerous AR-IR cells were also observed in the dorsal division of the anterior olfactory nucleus, lateral septal nucleus, medial and lateral divisions of the bed nucleus of the stria terminalis, lateral olfactory tract nucleus, anterior amygdaloid area, and the central and medial amygdaloid nuclei. AR-IR nuclei were predominantly observed in areas involved in autonomic and neuroendocrinergic responses which are important for many physiological processes and behaviors.

Urethane anesthesia in acute lower urinary tract studies in the male rat.

Urethane is a widely used anesthetic in animal lower urinary tract research. Our objective was to investigate the quality of anesthesia and the correlation between bladder (voiding) contractions, micturition pressure, bladder capacity and urethane dosage and body weight. Urethane was given subcutaneously and/or intraperitoneally (1.0-1.2 g/kg). The bladder was filled with saline and the bladder pressure was recorded continuously. Animals in which the subcutaneous/intraperitoneal ratio was higher needed less urethane. Heavier animals needed less extra urethane. In animals, in which no bladder contractions could be evoked, the total amount of urethane given was similar to that in those that did show contractions. In the animals that did void, the bladder never emptied completely and residual volumes remained. There was no relationship between animal weight or total amount of urethane and mean capacity. Anesthesia lasted up till 14 h, during which bladder (voiding) contractions could be recorded. Considering all results, we conclude that urethane is a well suited anesthetic for acute lower urinary tract physiological research in the intact rat.

Inhibitory effects of tibial nerve stimulation on bladder neurophysiology in rats.

Tibial nerve stimulation (TNS) is a form of peripheral neuromodulation which has been found effective in treating overactive bladder symptoms, with lesser side effects than first line pharmacotherapy. Despite its widespread clinical use, the underlying mechanism of action is not fully understood. Our aim was to study its effect on the bladder neurophysiology and the trigger mechanism of voiding in the overactive detrusor, simulated by acetic acid (AA) instillation. In urethane anaesthetized male Wistar rats, the tibial nerve was stimulated for 30 min at 5 Hz, pulse width 200 µs and amplitude approximately three times the threshold to induce a slight toe movement. The pressure at which a voiding contraction was triggered (pthres) did not change significantly between the pre- and post-TNS measurements in AA induced detrusor overactivity. It was found that TNS significantly reversed the effects of AA irritation by increasing the bladder compliance and the bladder volume at pthres, as well as suppressed the threshold afferent nerve activity. The slope of the linear relationship between pressure and the afferent activity increased after AA instillation and decreased significantly after stimulation. In addition to its well-known central inhibitory mechanisms, this study has demonstrated that TNS improves bladder storage capacity by delaying the onset of voiding, via an inhibitory effect on the bladder afferent signaling at the peripheral level.

Neurophysiological modeling of bladder afferent activity in the rat overactive bladder model.

The overactive bladder (OAB) is a syndrome-based urinary dysfunction characterized by "urgency, with or without urge incontinence, usually with frequency and nocturia". Earlier we developed a mathematical model of bladder nerve activity during voiding in anesthetized rats and found that the nerve activity in the relaxation phase of voiding contractions was all afferent. In the present study, we applied this mathematical model to an acetic acid (AA) rat model of bladder overactivity to study the sensitivity of afferent fibers in intact nerves to bladder pressure and volume changes. The afferent activity in the filling phase and the slope, i.e., the sensitivity of the afferent fibers to pressure changes in the post-void relaxation phase, were found to be significantly higher in AA than in saline measurements, while the offset (nerve activity at pressure ~0) and maximum pressure were comparable. We have thus shown, for the first time, that the sensitivity of afferent fibers in the OAB can be studied without cutting nerves or preparation of single fibers. We conclude that bladder overactivity induced by AA in rats is neurogenic in origin and is caused by increased sensitivity of afferent sensors in the bladder wall.

Afferent bladder nerve activity in the rat: a mechanism for starting and stopping voiding contractions.

The objective of this work was to study the relation between afferent bladder nerve activity and bladder mechanics and the mechanisms that initiate and terminate bladder contractions. Bladder nerve activity, pressure and volume were recorded during the micturition cycle in the rat. The highest correlation was found between afferent nerve activity and stress (pressure x volume). Afferent nerve activity depended linearly on stress within 6%, and both slope and offset were independent of the bladder-filling rate. The levels of afferent bladder nerve activity at the onset and cessation of efferent firing to the bladder were highly reproducible with coefficients of variation of