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Mechano-neuromodulation of autonomic pelvic nerves was demonstrated for the first time using a triboelectric neurostimulator integrated with flexible neural clip interface. The detailed stimulation parameters such as current, pulse width, and charges generated by the proposed TENG were investigated for the stimulation of autonomic pelvic nerve. In in vivo experiments, different beats per minute (BPM) were delivered to the nerves to investigate the effect of stimulation frequency on mechano-neurostimulation while monitoring changes in bladder pressure and the occurrence of micturition. Furthermore, different numbers of pulses were applied to stimulate the pelvic nerve. The bladder contractions with micturition were observed when applying more than 50 BPM as well as one pulse. Comparison of stimulation was performed using a commercial stimulator with the similar long-pulse widths as generated by TENGs. In addition, chronic implantation of flexible clip interface was demonstrated with functionality test of the interface. The results demonstrate that this technology may potentially be used for self-powered mechano-neuromodulation for bladder function in the future. Image 1 • Generation of charge-balanced waveforms for autonomic nerve stimulation by the proposed triboelectric neurostimulator. • Bladder modulation by mechano-stimulation of frequency and number of pulses. • Comparison of bladder modulation by a commercial stimulator to verify long pulse width and low amplitude stimulation. • Demonstration of chronic implantation of flexible clip interface on pelvic nerve and the functionality for pelvic nerve stimulation. [ABSTRACT FROM AUTHOR]

Abstract The aims of the present study were to describe, in male rats, the anatomical organization of the major and accessory pelvic ganglia (MPG, AG; respectively), the interrelationship of the pelvic plexus components, and the morphometry of the pelvic postganglionic neurons. Anatomical, histochemical and histological studies were performed in anesthetized adult Wistar male rats. We found that the pelvic plexus consists of intricate neural circuits composed of two MPG, and three pairs of AG (AGI, AGII, AGIII) anatomically interrelated through ipsilateral and contralateral commissural nerves. Around 30 nerves emerge from each MPG and 17 from AGI and AGII. The MPG efferent nerves spread out preganglionic information to several pelvic organs controlling urinary, bowel, reproductive and sexual functions, while AG innervation is more regional, and it is confined to reproductive organs located in the rostral region of the urogenital tract. Both MPG and AG contain nerve fascicles, blood vessels, small intensely fluorescent cells, satellite cells and oval neuronal somata with one to three nucleoli. The soma area of AG neurons is larger than those of MPG neurons (p < 0.005). The MPG contains about 75% of the total pelvic postganglionic neurons. Our findings corroborated previous reports about MPG inputs, and add new information regarding pelvic ganglia efferent branches, AG neurons (number and morphometry), and neural interrelationship between the pelvic plexus components. This information will be useful in designing future studies about the role of pelvic innervation in the physiology and pathophysiology of pelvic functions. Graphical abstract The pelvic plexus in the male rat is composed by 4 bilateral ganglia (major pelvic ganglia, MPG and three accessory ganglia, G I, II and III), two input nerves, ipsilateral and contralateral commissural nerves and efferent nerves. IMG, inferior mesenteric ganglion; HG, hypogastric nerve; PN, pelvic nerve; PD, pudendal nerve; SBPd, sensory branch of the pudendal nerve, DNP, dorsal nerve of the penis, CN, cavernous nerve. Dots represent soma neurons within the pelvic plexus. Unlabelled Image Highlights • The pelvic plexus of the male rat is made of two major ganglia and three pairs of accessory ganglia. • The components of the pelvic plexus are anatomically interrelated through ipsilateral and contralateral commissural nerves. • The accessory ganglia represent around 25% of the postganglionic pelvic innervation. • Efferent nerves of the MPG present a topographical organization. [ABSTRACT FROM AUTHOR]

There is mounting evidence underscoring a role for the urothelium in urinary bladder sensation. Previous functional studies have identified bladder primary afferents with mechanosensitive properties suggesting urothelial innervation and/or communication. The current study identifies a group of urothelium-innervating afferent neurons in rat, and characterizes and compares the properties of these and non-urothelial afferent neuron populations. Lumbosacral (LS) primary afferent neurons were retrogradely labeled using intraparenchymal (IPar) microinjection or intravesical (IVes) infusion of tracer into the bladder. Using these techniques, separate populations of neurons were differentiated by dorsal root ganglion (DRG) somata labeling and dye distribution within the bladder. IPar- and IVes-labeled neurons accounted for 85.0% and 14.4% of labeled L6-S1 neurons (P < .001), respectively, with only 0.6% of neurons labeled by both techniques. Following IVes labeling, dye was contained only within the periurothelial bladder region in contrast to non-urothelial distribution of dye after IPar labeling. Electrophysiological characterization by in situ patch-clamp recordings from whole-mount DRG preparations indicated no significant difference in passive or active membrane properties of IPar and IVes DRG neurons. However, calcium imaging of isolated neurons indicates that a greater proportion of IPar- than IVes-labeled neurons express functional TRPA1 (45.7% versus 25.6%, respectively; P < .05). This study demonstrates that two anatomically distinct groups of LS bladder afferents can be identified in rat. Further studies of urothelial afferents and the phenotypic differences between non-/urothelial afferents may have important implications for normal and pathophysiological bladder sensory processing. [ABSTRACT FROM AUTHOR]

Abstract: In the present study, we characterized the evoked electrical activity from T13 to S2 dorsal roots (DRs) during glass probe-stimulation of the vagina–cervix–uterus junction (VCUJ) of female Wistar rats. The results showed that gentle stimulation of VCUJ evoked high-amplitude electrical activity in L3 and L6 DRs. Hypogastric or pelvic nerve transection failed to abolish this activity. L6–S1 spinal trunk transection abolished the high-amplitude electrical activity evoked in L6 DR, while transection of the lumbosacral trunk blocked the high-amplitude electrical activity evoked in L3 DR. These data suggest that during copulation, penile intromission likely activates the low-threshold sensory receptors of the VCUJ, thereby evoking sensory neural activity that enters the spinal cord via L3 and L6 dorsal roots, whose axons travel through the lumbosacral trunk and pudendal nerve. [Copyright &y& Elsevier]

Abstract: Introduction: The pelvic, hypogastric and pudendal nerves carry sensory information from the urinary bladder. The pelvic nerve is reported to be the most important one of these afferent nerves in producing urinary bladder sensation. The primary types of mechanoreceptors in the bladder can be divided into stretch-sensitive and stretch-insensitive units. The former is considered to be more important in producing bladder sensation. However, little is known about the precise receptive field properties of the stretch-insensitive mechanoreceptors in the urinary bladder. Therefore, in this study, we systematically investigated the receptive field characteristics of the pelvic single unit innervating the rat bladder wall. Materials and methods: Functional single unit recordings were made from the pelvic nerve afferent filaments of anesthetized rats. A von Frey device was used for quantitative mechanical stimulation of the bladder surface. In addition, electrical stimulation was used for estimating conduction velocities of the nerve fibers in the receptive field. Results: The threshold value for mechanical stimulation was statistically lower at the caudal portion of the bladder body (sites IV and V) than the other bladder sites. The bladder neck (site I) had the highest mechanical stimulation threshold value for the bladder stretch-insensitive mechanoreceptors. In most cases, the pelvic nerve had bilateral receptive fields. The majority of the pelvic nerve afferents had conduction velocities in the slow A-δ or C fiber range. Mechanical stimulation threshold values were higher in males than in females in a portion of the bladder sites. Discussion: The pelvic stretch-insensitive bladder mechanoreceptors are 1) higher threshold at the bladder base, 2) contain large bilateral receptive fields and 3) demonstrate relatively slow conduction velocities. These characteristics indicate a non-uniform distribution of stretch-insensitive mechanoreceptors in the rat urinary bladder wall. [Copyright &y& Elsevier]

Abstract: Marked urinary bladder distension, hydroureters, severe bilateral hydronephrosis, and perirenal fluid accumulation were identified ultrasonographically in two rams from the same farm but separated by 24 months’ interval. Both rams were observed to urinate but the flow rate of urine was estimated to be less than normal and the duration was reduced to around 5–10s when around 20–30s is considered normal. There was normal tail and rectal muscle tone, and normal perineal and pelvic limb reflexes. A presumptive diagnosis of pelvic nerve dysfunction causing detrusor atony and bladder distension with secondary pressure-induced hydronephrosis is suggested but unproven. Detailed dissection at necrospy found no evidence of urethral obstruction. [Copyright &y& Elsevier]

Abstract: The bladder and distal colon are innervated by lumbar splanchnic (LSN) and pelvic nerves (PN) whose axons arise from dorsal root ganglia (DRG) neurons at thoracolumbar (TL) and lumbosacral (LS) spinal levels, respectively. In an attempt to understand the molecular basis of differences between LSN and PN mechanosensitive afferents, we analyzed the gene expression of two potentially counteracting ion channel groups involved in mechanosensation, transient receptor potential channels (TRPV1 and TRPA1) and mechanosensitive two pore-domain K+ (K2P) channels (TREK-1, TREK-2 and TRAAK), in TL and LS DRG neurons innervating mouse bladder or distal colon. The proportion of TRPV1-expressing cells (41∼61%) did not differ between TL and LS neurons innervating bladder or colon. TRPA1 was seldom detected in bladder LS neurons whereas it was expressed in 64∼66% of bladder TL, colon TL and colon LS neurons. Coexpression of TRPV1 and TRPA1 was frequent. TREK-1-expressing cells were more prevalent in LS than TL ganglia in both bladder- and colon-DRG neurons. All three K2P channels were detected more frequently in TRPV1-positive neurons in TL ganglia. More than half of TL neurons expressing only TRPA1 were devoid of any of the three K2P channels, whereas all TL neurons expressing both TRPA1 and TRPV1 expressed at least one of the K2P channels. These results reveal clear differences between LSN and PN sensory pathways in TRPA1 and TREK-1 gene expression and in the gene expression of K2P channels in TRPV1-expressing neurons. This study further documents heterogeneity of visceral afferents based on combinations of the five channels examined. [Copyright &y& Elsevier]

Abstract: The effect of genital stimulation, either by vaginocervical stimulation (VCS) using a calibrated vaginal probe combined with manual flank stimulation (FS), or by mounts performed by the male, on the hypothalamus and preoptic area concentration of the progesterone receptors A (PR-A) and B (PR-B) was assessed in ovariectomized (ovx) estrogen-primed rats. VCS/FS or stimulation provided by male mounts, even without intromission, significantly decreased PR-B concentration in the hypoythalamus. Down regulation of PR produced by genital stimulation was quantitatively similar to that elicited by progesterone (P) administration. Bilateral or unilateral transection of the pelvic or the pudendal nerves prevented down regulation elicited by VCS/FS. Repeated VCS/FS elicited lordosis behavior in most ovx estrogen primed rats, but the lordosis intensity was lower than that observed in response to P. P administered to ovx estrogen primed rats, induced sequential inhibition, i.e., failure to display estrous behavior in response to a second P injection (24h after the initial P injection). VCS/FS failed to elicit sequential inhibition, since rats responded with normal estrous behavior to the second injection of P. This suggests that down regulation by VCS, by contrast with P, failed to inhibit the subpopulation of PR involved in the facilitation of estrous behavior by P. [Copyright &y& Elsevier]

Introduction: The colon and rectum are regulated by the autonomic nervous system (ANS). Abnormalities of the ANS are associated with diseases of the colon and rectum while its modulation is a putative mechanism for sacral nerve stimulation. The purpose of this study is to establish a rat model elucidating the role of the efferent ANS on rectal motility.Materials and Methods: Rectal motility following transection or stimulation of parasympathetic pelvic nerves (PN) or sympathetic hypogastric nerves (HGN) was measured with rectal strain gauge transducers and quantified as a motility index (MI). Colonic transit was measured 24 hours after transection by calculating the geometric center (GC) of distribution of (51)CrResults and Discussion: Transection of PN and HGN decreased MI to 518 +/- 185 g*s (p < 0.05) and increased MI to 5,029 +/- 1,954 g*s (p < 0.05), respectively, compared to sham (975 +/- 243 g*s). Sectioning of PN and HGN decreased transit with GC = 4.9 +/- 0.2 (p < 0.05) and increased transit with GC = 8.1 +/- 0.7 (p < 0.02), respectively, compared to sham (GC = 5.8 +/- 0.3). Stimulation of PN and HGN increased MI to 831 +/- 157% (p < 0.01) and decreased MI to 251 +/- 24% (p < 0.05), respectively.Conclusion: Rectal motility is significantly altered by sectioning or stimulating either HGN or PN. This model may be useful in studying how sacral nerve stimulation exerts its effects and provide insight into the maladies of colonic motility. [ABSTRACT FROM AUTHOR]

Abstract: Subfertility and severe pelvic pains are symptoms associated with endometriosis (ENDO), a common condition among women that is characterized by the growth of the uterine endometrium on the surface of organs within the pelvic region and abdominal cavity. The contribution of the CNS to symptoms associated with ENDO is not known. In the present study, the preoptic area (POA) of the hypothalamus was investigated, as this region of the forebrain is known to play an important role in the neuroendocrine control of the reproductive cycle, mating behavior, and antinociception. Female rats were either induced for ENDO by autotransplantation of uterine tissue (n =20) or uterine fat for surgical sham controls (n =11). Terminal extracellular electrophysiological recordings (urethane anesthesia) were conducted in the POA six weeks post-ENDO induction when the rats were in either the proestrus or metestrus stages of their estrous cycle. Significant differences were found between the ENDO versus SHAM groups of animals for the proportion of inhibitory responses as well as the percentage of neurons responding to stimulation of the abdominal branches of the vagus, which innervates portions of the female reproductive tract, including the ovaries. The endometriotic cysts were found to be significantly larger in proestrus rats (stage when hormones are elevated). These data demonstrate that the responses of POA neurons are influenced by the presence of endometriotic cysts in the abdominal cavity. Since the POA is known to be part of the neural circuitries that mediate nociception and fertility, any deviation from its normal activity under ENDO conditions could contribute to the constellation of symptoms that ensue. [Copyright &y& Elsevier]

Penile erection is a vascular event controlled by the autonomic nervous system. The spinal cord contains the autonomic preganglionic neurons that innervate the penile erectile tissue and the pudendal motoneurons that innervate the perineal striated muscles. Sympathetic pathways are anti-erectile, sacral parasympathetic pathways are pro-erectile, and contraction of the perineal striated muscles upon activity of the pudendal nerves improves penile rigidity. Spinal neurons controlling erection are activated by information from peripheral and supraspinal origin. Both peripheral and supraspinal information is capable of either eliciting erection or modulating or inhibiting an erection already present. Sensory information from the genitals is a potent activator of pro-erectile spinal neurons and elicits reflexive erections. Some pre-motor neurons of the medulla, pons and diencephalon project directly onto spinal sympathetic, parasympathetic and pudendal motoneurons. They receive in turn sensory information from the genitals. These spinal projecting pathways release a variety of neurotransmitters, including biogenic amines (serotonin, dopamine, noradrenaline, and adrenaline) and peptides that, through interactions with many receptor subtypes, exert complex effects on the spinal network that controls penile erection. Some supraspinal structures (e.g. the paraventricular nucleus and the medial preoptic area of the hypothalamus, the medial amygdala), whose roles in erection have been demonstrated in animal models, may not project directly onto spinal pro-erectile neurons. They are nevertheless prone to regulate penile erection in more integrated and coordinated responses of the body, as those occurring during sexual behavior. The application of basic and clinical research data to treatment options for erectile dysfunction has recently proved successful. Pro-erectile effects of phosphodiesterase type 5 inhibitors, acting in the penis, and of melanocortin agonists, acting in the brain, illustrate these recent developments. [Copyright &y& Elsevier]

The expression and functional responses of P2X receptors in bladder and cutaneous sensory neurons of adult rats and mice have been studied using immunohistochemistry and patch clamp techniques. Cell bodies of bladder pelvic afferents were identified in L6 and S1 dorsal root ganglia (DRG), following Fast Blue injection into the muscle wall of the urinary bladder. Similarly, cutaneous sensory neurons were identified in L3 and L4 DRG, following Fast Blue injection into the saphenous nerve innervating the skin. Bladder sensory neurons contained only weak to moderate P2X3-immunoreactivity (IR), in contrast to strong P2X3-IR observed in a sub-population of cutaneous afferents. Whole-cell patch-clamp recordings revealed that approximately 90% of bladder afferent neurons responded to αβ-methylene ATP (αβmeATP) and ATP (30 μM) with persistent currents, which were inhibited by 2′,3′-O-trinitrophenyl-ATP (TNP-ATP) (0.3 μM) to 6.4±1.9% and 8.0±2.6% of control, respectively (n=8). The remaining bladder sensory neurons demonstrated biphasic, transient or no response to P2X agonists. In contrast, only 24% of cutaneous afferent neurons gave persistent currents to αβmeATP (30 μM), with 66% of cells giving transient or biphasic currents and the remaining 10% being non-responsive. Our results suggest that, in contrast to DRG neurons in general, bladder sensory neurons projecting via pelvic nerves express predominantly P2X2/3 heteromeric receptors, which are likely to mediate the important roles of ATP as a signaling molecule of urinary bladder filling and nociception. [Copyright &y& Elsevier]

Reflex plasticity between pelvic afferent nerve (PAN) and pudendal efferent nerve (PEN), as well as external-urethral sphinctor (EUS) activity was examined in anesthetized rats. A progressive increase in the number of evoked action potentials per stimulus occurred in PEN and EUS activity when PAN was repetitively stimulated (1 Hz). This potentiation in pelvic-pudendal reflex (PPR) activity induced by repetitive stimulation was abolished by APV (D-2-amino-5-phosphonoraleric acid, i.t. 100 μM, 2–5 μl) and attenuated by the NBQX (2, 3-dihydroxy-6-nitro-7-sulfamoyl-benzo (F) quinoxaline, i.t. 20 μM, 2–5 μl) but was not affected by the presence of bicuculline (i.t. 10 μM, 2–5 μl). The duration of contraction wave of intra-urethral pressure (IUP) elicited by a single electric shock was elongated by potentiated PPR, while the peak pressure was not affected. Both intrathecal application of glutamate (i.t. 0.1 mM, 2–5 μl) and NMDA (N-methyl-D-aspartic acid, i.t. 0.1 mM, 2–5 μl) induced spontaneous repetitive (0.31±0.02 Hz) burst discharges in PEN and EUSE and produced small contraction wave in IUP, which is similar to the high frequency oscillation phase during a voiding cycle of urinary bladder in rats. All these results demonstrate that repetitive stimulation of PAN can induce a distinct and long-lasting modulation in PPR activity and this change may be physiologically relevant in urinary continence. [Copyright &y& Elsevier]

Sexually experienced Wistar male rats were used to investigate (a) urine voiding in the presence of nearby estrous females and the control of such voiding by (b) steroid hormones and (c) peripheral nerves supplying the genitourinary system. The first experiment showed that males always have a low rate of urine voiding that is significantly increased when a receptive female is around. Thus, it is suggested that an airborne scent from the female stimulates the olfactory system of males, triggering urine emission to transmit sex-related messages, i.e., male rats display the well-known urine-marking behavior of mammals. The number of urine marks and sniffing to females decreased after castration, and were restored after exogenous treatment with testosterone or estradiol. The proposed hypothesis is that airborne scents from the female activate the aromatization process in nuclei of the olfactory pathway of the male, evoking a cascade of neuronal responses that finish in urine marking. Peripheral nerves supplying the genitourinary system are the viscerocutaneous branch of the pelvic nerve (Vc) and the hypogastric (Hg). Data showed that both nerves are important for the central control of urine storage and voiding. Transection of Vc almost blocked urine marking, while Hg lesion increased the number of marks. Thus, it is discussed that Vc is the most important nerve in charge of voiding the bladder, and that Hg is important for continence. [Copyright &y& Elsevier]