Role of tactile stimulation during the preweaning period on the development of the peripheral sensory sural (SU) nerve in adult artificially reared female rat.

Maternal deprivation, as a result of the artificial rearing (AR) paradigm, disturbs electrophysiological and histological characteristics of the peripheral sensory sural (SU) nerve of infant and adult male rats. Such changes are prevented by providing tactile or social stimulation during isolation. AR also affects the female rat's brain and behavior; however, it is unknown whether this early adverse experience also alters their SU nerve development or if tactile stimulation might prevent these possible developmental effects. To assess these possibilities, the electrophysiological and histological characteristics of the SU nerve from adult diestrus AR female rats that: (i) received no tactile stimulation (AR group), (ii) received tactile stimulation in the anogenital and body area (AR-Tactile group), or (iii) were mother reared (MR group) were determined. We found that the amplitude, but not the area, of the evoked compound action potential response in SU nerves of AR rats was lower than those of SU nerves of MR female rats. Tactile stimulation prevented these effects. Additionally, we found a reduction in the outer diameter and myelin thickness of axons, as well as a large proportion of axons with low myelin thickness in nerves of AR rats compared to the nerves of the MR and AR-Tactile groups of rats; however, tactile stimulation only partially prevented these effects. Our data indicate that maternal deprivation disturbs the development of sensory SU nerves in female rats, whereas tactile stimulation partially prevents the changes generated by AR. Considering that our previous studies have shown more severe effects of AR on male SU nerve development, we suggest that sex-associated factors may be involved in these processes.

Multiparity Reduces Urethral and Vaginal Pressures Following the Bulboglandularis Muscle Stimulation in Rabbits.

Unlike male mammals showing a well-delimited external urethral sphincter, female mammals have urogenital sphincters shaped by muscles like the urethrovaginal sphincter. Childbirth-related injuries affect morphometry and function of urogenital sphincters in women, which frequently underlies pelvic floor disorders, including stress urinary incontinence and pelvic organ prolapse. The bulboglandularis muscle (Bgm) seems to shape a urogenital sphincter in rabbits. We determined herein the effect of multiparity on urethral and vaginal pressures generated by the Bgm stimulation in age-matched nulliparous and multiparous chinchilla-breed rabbits to stimulate the Bgm with trains of ascending frequencies (from 1 to 100 Hz; 4 s duration each). Subsequently, the Bgm was excised, measured in width, and weighed. Significant differences (P ≤ 0.05) were determined with Mann-Whitney U or Student t-tests or repeated measures two-way ANOVA followed by Tukey tests. Spearman's partial coefficients were calculated to investigate the correlation between the highest pressure (urethral or vaginal) and the Bgm width. Multiparity reduced the weight and the width in the Bgm origin and medial regions. Urethral and vaginal pressures increased in response to the electrical stimulation of Bgm with frequencies from 20 to 100 Hz. Multiparas showed significant reductions in both types of pressures. We detected a strong correlation (conditioned by multiparity) between the medial Bgm width and the highest vaginal pressure. Our present findings demonstrate that multiparity impairs the function of Bgm, resulting in diminished urethral and vaginal pressures. Furthermore, the significant narrowness of the Bgm was correlated with the vaginal pressure recorded.

Lumbosacral ventral root avulsion alters reflex activation of bladder, urethra, and perineal muscles during micturition in female rabbits.

AIM: To determine the effect of the lumbosacral ventral root avulsion (VRA) on the reflex activation of bladder, urethra, and activation of perineal muscles during micturition in female rabbits. METHODS: We allocated 14 virgin female rabbits to evaluate, first, the gross anatomy of lumbosacral spinal cord root (n = 5) and, second, to determine the effect of VRA on perineal muscles during micturition (n = 9). We recorded cystometrograms, urethral pressure, and electromyograms of the bulbospongiosus (Bsm) and ischiocavernosus (Ism) muscles before and after the L6-S2 VRA. Standard variables were measured from each recording and analyzed to identify significant differences (P < .05). RESULTS: We found that the L6-S2 VRA affected directly the bladder and urethral function and reduced the duration and the frequency of the bursting of Ism and Bsm muscles during voiding. The Ism and Bsm showed a phasic activation, of different frequencies, during the voiding phase and the L6-S2 VRA inhibited the co-contraction of the Ism and Bsm-bladder-urethra. CONCLUSIONS: The Ism and Bsm are activated at different frequencies to trigger the voiding phase. The L6-S2 VRA affected the activity pattern of both perineal muscles. These modifications affected the bladder and urethra function. It is possible that the restoration of the activation frequency of perineal muscles contributed for an efficient bladder contraction.

Multiparity affects conduction properties of pelvic floor nerves in rabbits.

INTRODUCTION: Women often develop pelvic floor dysfunction due to damage to the pelvic musculature during childbirth; however, the effect on pelvic floor nerves function is less understood. This study used adult rabbits to evaluate the electrophysiological and histological characteristics of the bulbospongiosus (Bsn) and pubococcygeus nerves (Pcn) in multiparity. METHODS: Compound nerve action potentials (CNAP) were compared between age-matched nulliparous and multiparous animals and associated to the histological characteristics of myelinated axons from the Bsn and Pcn nerves. The extensor digitorum longus nerve (EDLn) was used as negative control. Data were analyzed with unpaired two-tailed Student's t test or Mann-Whitney U test to determine significant differences between groups. RESULTS: The onset and peak latencies, duration, and conduction velocity of the motor fibers in these pelvic nerves were not significantly different between nulliparous and multiparous animals. However, the peak-to-peak amplitude and area of the CNAP in both Bsn and Pcn were reduced in multiparous rabbits. Histology showed a higher percentage of axons with myelin disorganization caused by multiparity in these pelvic nerves. Together, the data indicate a reduction in the number of functional pelvic axons due to multiparity. As expected, no effect of parity was observed in the EDLn controls. CONCLUSIONS: Present findings demonstrated that multiparity affects myelination and consequently conduction properties in the small pelvic floor nerves.

Early postnatal development of electrophysiological and histological properties of sensory sural nerves in male rats that were maternally deprived and artificially reared: Role of tactile stimulation.

Early adverse experiences disrupt brain development and behavior, but little is known about how such experiences impact on the development of the peripheral nervous system. Recently, we found alterations in the electrophysiological and histological characteristics of the sensory sural (SU) nerve in maternally deprived, artificially reared (AR) adult male rats, as compared with maternally reared (MR) control rats. In the present study, our aim was to characterize the ontogeny of these alterations. Thus, male pups of four postnatal days (PND) were (1) AR group, (2) AR and received daily tactile stimulation to the body and anogenital region (AR-Tactile group); or (3) reared by their mother (MR group). At PND 7, 14, or 21, electrophysiological properties and histological characteristics of the SU nerves were assessed. At PND 7, the electrophysiological properties and most histological parameters of the SU nerve did not differ among MR, AR, and AR-Tactile groups. By contrast, at PND 14 and/or 21, the SU nerve of AR rats showed a lower CAP amplitude and area, and a significant reduction in myelin area and myelin thickness, which were accompanied by a reduction in axon area (day 21 only) compared to the nerves of MR rats. Tactile stimulation (AR-Tactile group) partially prevented most of these alterations. These results suggest that sensory cues from the mother and/or littermates during the first 7-14 PND are relevant for the proper development and function of the adult SU nerve. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 78: 351-362, 2018.

Multiparity modifies contractile properties of pelvic muscles affecting the genesis of vaginal pressure in rabbits.

AIMS: To characterize the contractile properties of the bulbospongiosus (Bsm), isquiocavernosus (Ism), and pubococcygeus muscles (Pcm), and their involvement in the genesis of vaginal pressure in nulliparous and multiparous rabbits. METHODS: Age-matched nulliparous and multiparous rabbits were used to record the isometric contractile responses of each muscle as well as the intravaginal pressure evoked by single square electrical pulses and stimulation trains of ascending frequency. To establish significant differences between groups, two-tail unpaired Student t tests were carried out. The linear correlation between intravaginal pressure and muscle contractile force was analyzed with Pearson correlation tests. For all cases, a P ≤ 0.05 was set as statistically significant. RESULTS: Multiparity decreased the contractile force of Bsm and Ism generated by high-frequency stimulation trains. The normalized force of the Pcm increased when evoked at 1, 4, and 10 Hz while this decreased at higher frequencies (20, 50, and 100 Hz). The contraction of both Bsm and Ism raised particularly the pressure on the perineal vagina while that of the Pcm increased the pressure in the pelvic vagina. Such a functional segregation is still present in multiparous rabbits albeit it was modified. CONCLUSIONS: Multiparity induces changes in the contractile responses of Bsm, Ism, and Pcm, which alterates the vaginal pressure.

Anatomical organization and somatic axonal components of the lumbosacral nerves in female rabbits.

AIM: To determine the anatomical organization and somatic axonal components of the lumbosacral nerves in female rabbits. METHODS: Chinchilla adult anesthetized female rabbits were used. Anatomical, electrophysiological, and histological studies were performed. RESULTS: L7, S1, and some fibers from S2 and S3 form the lumbosacral trunk, which gives origin to the sciatic nerve and innervation to the gluteal region. From S2 to S3 originates the pudendal nerve, whose branches innervates the striated anal and urethra sphincters, as well as the bulbospongiosus, ischiocavernosus, and constrictor vulvae muscles. The sensory field of the pudendal nerve is ∼1800 mm2 and is localized in the clitoral sheath and perineal and perigenital skin. The organization of the pudendal nerve varies between individuals, three patterns were identified, and one of them was present in 50% of the animals. From S3 emerge the pelvic nerve, which anastomoses to form a plexus localized between the vagina and the rectum. The innervation of the pelvic floor originates from S3 to S4 fibers. CONCLUSIONS: Most of the sacral spinal nerves of rabbit are mixed, carrying sensory, and motor information. Sacral nerves innervate the hind limbs, pelvic viscera, clitoris, perineal muscles, inguinal and anal glands and perineal, perigenital, and rump skin. The detailed description of the sacral nerves organization, topography, and axonal components further the knowledge of the innervation in pelvic and perinal structures of the female rabbit. This information will be useful in future studies about the physiology and physiopathology of urinary, fecal, reproductive, and sexual functions.

Activity of the external urethral sphincter evoked by genital stimulation in male rats.

AIMS: To determine whether the external urethral sphincter (EUS) fasciculi of male rats respond to the mechanical stimulation of genital structures and to characterize the pattern of the electromyographic (EMG) activity of the three regions of the EUS: the cranial (CrEUS), the medial (MeEUS) and the caudal (CaEUS). METHODS: Electromyographic signals were recorded from the CrEUS, MeEUS and CaEUS regions of the male rat's EUS, before, during and after the mechanical stimulation of the urogenital structures. RESULTS: The CrEUS, MeEUS and CaEUS regions responded when brushing and squeezing the foreskin and glans as well as to penile and prostatic urethral distension. The CaEUS EMG amplitude (P < 0.01) and frequency (P < 0.05) were lower in comparison to the CrEUS and MeEUS responses to the mechanical stimulation. In addition, the CaEUS was characterized by a short or no afterdischarge. In contrast, the CrEUS and MeEUS responded by presenting a long discharge after the penile or prostatic urethral distension. CONCLUSIONS: The activity of the EUS is modulated by both, cutaneous and visceral genitourinary stimuli, with motor units being activated by mechanoreceptors located in the foreskin, glans, bladder, and urethra. The CrEUS, MeEUS and CaEUS have differential EMG patterns, indicating that the EUS consists of three anatomically and functionally different regions. Precise coordination in the muscular activity of these regions may be crucial for the control of male expulsive urethral functions, i.e., during voiding and ejaculation. Neurourol. Urodynam. 35:914-919, 2016. © 2015 Wiley Periodicals, Inc.

Anatomic and functional properties of bulboglandularis striated muscle support its contribution as sphincter in female rabbit micturition.

PURPOSE: To determine anatomic and functional properties of the bulboglandularis muscle (Bgm) for clarifying its role in micturition in female rabbits. MATERIAL AND METHODS: Virgin female rabbits were used to describe the gross anatomy and innervation of the Bgm, to determine the effect of the Bgm contraction on urethral pressure, and to evaluate the Bgm activity during the induced-micturition. Both electromyogram and cystometrogram activity were simultaneously recorded in urethane-anesthetized rabbits. Bladder function was assessed measuring standard urodynamic variables before and after blocking the Bgm activity for approaching its contribution to micturition. The relevance of the Bgm activation for micturition was approached applying lidocaine injections. RESULTS: The Bgm was composed of circularly oriented striated fibers enveloping distal urethra and pelvic vagina. Both the venous plexus and urethra were comprised by the Bgm contraction induced by electrical stimulation. The Bgm showed bursts of tonic activity at the storage phase of micturition that gradually decreased until turning off as the onset of the voiding phase. The voided volume, the voiding efficiency, the threshold pressure, and the maximal pressure were decreased after lidocaine injection. Contrastingly, the threshold volume, the residual volume, the voiding duration, and the urethral resistance at voiding were increased. CONCLUSIONS: Present anatomical and physiological findings support that the Bgm acts as a sphincter during micturition of female rabbits. Neurourol. Urodynam. 35:689-695, 2016. © 2015 Wiley Periodicals, Inc.

The role of pelvic and perineal striated muscles in urethral function during micturition in female rabbits.

AIM: To evaluate the role of pelvic and perineal striated muscles on urethral function during micturition. METHODS: Pubococcygeus, or both bulbospongiosus and ischiocavernosus muscles were electrically stimulated during the voiding phase of micturition, and bladder and urethral pressure were simultaneously recorded in urethane anesthetized female rabbits. Bladder and urethral function were assessed measuring urodynamic and urethral pressure variables obtained before and during the stimulation of muscles. Two-tail paired t-tests were carried out in order to determine significant differences (P < 0.05) between groups. RESULTS: Electric stimulation of the pubococcygeus during voiding decreased voiding efficiency and voided volume, whereas residual volume, the duration of voiding, the interval between bladder contraction and urethral resistance increased. Simultaneously, there was an increase in maximum urethral pressure, as well as an increase in the pressure to return to baseline and in the pressure required to close the urethra. Electrical stimulation of bulbospongiosus and ischiocavernosus muscles increased voiding efficiency, voiding duration, and the maximal pressure in bladder. Meanwhile, the maximal urethral pressure, the time related to the rise of urethral pressure, and the urethral pressure required to close the urethra decreased. CONCLUSIONS: The stimulation of pelvic and perineal muscles have opposing roles in urethral function during micturition. Pubococcygeus muscles facilitate urethral closure, while they inhibit bladder contraction. In contrast, bulbospongiosus and ischiocavernosus muscles prevent urethral contraction while they promote bladder contraction.

Análisis de los parámetros del eyaculado en la rata Wistar de laboratorio: descripción de la técnica / Analysis of the parameters of the ejaculate in the laboratory Wistar rat: technical description

The number of spermatozoa, weight and size of the seminal plug has been quantified in the ejaculate of the laboratory rat and sperm viability, sperm mobility and sperm concentration in samples obtained from the epididymis. The reference values for the rat ejaculate have not been determined maybe due to the difficulty to obtain it directly from the male. Nevertheless, an approach can be obtained analyzing the ejaculate collected from the inseminated female. The objective of this study was to evaluate and propose the values of the macroscospic and microscopic parameters of the semen and seminal plug obtained from the female. The seminal analysis of the Wistar rats was performed adapting the methods used for other species. After one ejaculatory series, semen contained in the uterine horns was obtained, as well as the seminal plug from the vagina. In more than one hundred ejaculates, 94.54% were off-white with 2.35 ± 0.06 mm of viscosity and 8.13 ± 0.02 of pH. The sperm concentration was 16.3 ± 0.59 millions of spermatozoa per mL, 0.72 ± 0.01 mobility index, 64.4 ± 0.7% viability and 99.11 ± 0.20% normal morphology. From 99% of seminal plugs, 92.7% were hardened, they weighed 115.63 ± 1.54 mg, measured 12.41 ± 0.13 and 5.31 ± 0.05 mm of length and width respectively, and volume 87.70 ± 1.74 mm3 . In conclusion, the used method to obtain and to evaluate the parameters of the ejaculate is reliable, for that reason, it is suggested that the results obtained could be considered as indicative values for semen and seminal plug for the Wistar rat.

Se ha cuantificado en el eyaculado de la rata de laboratorio, el número de espermatozoides, peso y tamaño del tapón seminal, y en muestras obtenidas de epidídimo, la viabilidad, movilidad y concentración espermáticas. Los valores de referencia para el eyaculado de la rata no se han determinado quizá por la dificultad para obtenerlo directamente del macho. No obstante, puede obtenerse una aproximación al analizar el eyaculado recolectado de una hembra recién inseminada. El objetivo de este estudio fue evaluar y proponer los valores de los parámetros macroscópicos y microscópicos del semen y tapón seminal obtenidos de la hembra. El análisis del semen de ratas Wistar se realizó adecuando los métodos usados para otras especies. Después de una serie eyaculatoria, se obtuvo el semen contenido en los cuernos uterinos y el tapón seminal de la vagina. En más de 100 eyaculados, 94.54% fueron blanquecinos, con 2.35 ± 0.06 mm de viscosidad y 8.13 ± 0.02 de pH. La concentración espermática fue de 16.3 ± 0.59 millones de espermatozoides por mL, 0.72 ± 0.01 de índice de movilidad, 64.4 ± 0.7% de viabilidad y 99.11 ± 0.20% de morfología normal. Del 99% de los tapones seminales, 92.7% fueron endurecidos, pesaron 115.63 ± 1.54 mg, midieron 12.41 ± 0.13 y 5.31 ± 0.05 mm de largo y ancho, respectivamente, y de volumen 87.70 ± 1.74 mm³. En conclusión, el método utilizado para obtener y evaluar los parámetros del eyaculado es confiable, por ello se sugiere que los resultados obtenidos podrían considerarse como valores indicativos para el semen y tapón seminal de la rata Wistar.

Sensory and somatomotor components of the "sensory branch" of the pudendal nerve in the male rat.

Surgical microscopy and electrophysiological techniques were used to describe in the adult male rat the peripheral distribution of the sensory branch of the pudendal nerve (SBPdn) and its sensory and somatomotor axonal components. Gross and histological features of the urethralis muscle were also determined. We propose to name the SBPdn branches according to the corresponding target structure. We found branches to the urethral diverticulum, major pelvic ganglion, corpus cavernosus of the penis, urethralis muscle, preputial glands, corpus spongiosus of the penis, foreskin and glans penis. Under the pubic bone three anastomotic branches form a bridge-like structure we called pelvic plexus. Through electrophysiological studies it was determined that the SBPdn carries efferent fibers to the striated urethralis muscle, as well as afferent axons from the glans penis and foreskin. We concluded that the SBPdn of the male rat has sensory and somatomotor components. This nerve is mixed and not exclusively sensory as was previously described. The finding of the anatomic relation to the major pelvic ganglion implies nerve communication with the autonomic nervous system. The functions of the somatomotor component and nerves to the urethral diverticulum, cavernosus bodies and preputial glands of the SBPdn remain to be determined. According to its targets, this innervation could contribute in the control of genitourinary functions such as voiding, erection, ejaculation and urinary marking.

Pattern of sensory innervation of the perineal skin in the female rat.

Here we describe the nerves innervating the perineal skin together with their sensory fields in the adult female rat. Electrophysiological recording showed that the lumbosacral and L6-S1 trunks, in part by way of the sacral plexus, transmit sensory information from the perineal skin via four nerves: the viscerocutaneous branch of the pelvic nerve innervating the skin at the midline between the vaginal opening and anus, the sensory branch of the pudendal nerve innervating the clitoral sheath, the distal perineal branch of the pudendal nerve innervating a broad area of skin adjacent to the vaginal opening and anus, and the proximal perineal branch of the sacral plexus innervating a broad area of skin adjacent to the clitoris and vaginal opening. The sensory fields of three of these nerves overlapped to some degree: the viscerocutaneous branch of the pelvic and the distal perineal branch of the pudendal nerves at the midline skin between the vaginal opening and the anus, and the distal perineal branch of the pudendal nerve and the proximal perineal branch of the sacral plexus at the skin lateral to the vaginal opening. Such overlap might provide a safeguard helping to ensure that somatosensory input from the perineal region important for triggering reproductive and nonreproductive reflexes reaches the CNS.

An anatomical and electrophysiological study of the genitofemoral nerve and some of its targets in the male rat.

Anatomical descriptions of the genitofemoral nerve (GFn) innervating the lower pelvic area are contradictory. Here we re-examine its origin and innervation by its various branches of principal target organs in the male rat. Using gross dissection, electrophysiological techniques and retrograde tracing of motoneurones with horseradish peroxidase, we confirm that the GFn originates from lumbar spinal nerves 1 and 2, and that at the level of the common iliac artery it divides into a lateral femoral and a medial genital branch. In contrast to previous studies, we report that the genital and not the femoral branch innervates the abdominal-inguinal skin, and not only the genital but also the femoral branch innervates the cremaster muscle (Cm) surrounding the testes. Motoneurones innervating the Cm proper are located in the ventral nucleus of L1 and L2, and those innervating the muscular transition region of the rostral Cm are located in the ventral nucleus in L1 and the ventrolateral nucleus in L2. The GFn may contribute to male reproductive performance by transmitting cutaneous information during copulation and, via contraction of the Cm to promote ejaculation, the protective displacement of the testes into the abdominal cavity during fighting and as a sperm-protecting thermoregulatory measure.