Purinergic signaling promotes premature senescence.

Extracellular ATP activates P2 purinergic receptors. Whether purinergic signaling is functionally coupled to cellular senescence is largely unknown. We find that oxidative stress induced release of ATP and caused senescence in human lung fibroblasts. Inhibition of P2 receptors limited oxidative stress-induced senescence, while stimulation with exogenous ATP promoted premature senescence. Pharmacological inhibition of P2Y11 receptor (P2Y11R) inhibited premature senescence induced by either oxidative stress or ATP, while stimulation with a P2Y11R agonist was sufficient to induce cellular senescence. Our data show that both extracellular ATP and a P2Y11R agonist induced calcium (Ca++) release from the endoplasmic reticulum (ER) and that either inhibition of phospholipase C or intracellular Ca++ chelation impaired ATP-induced senescence. We also find that Ca++ that was released from the ER, following ATP-mediated activation of phospholipase C, entered mitochondria in a manner dependent on P2Y11R activation. Once in mitochondria, excessive Ca++ promoted the production of reactive oxygen species in a P2Y11R-dependent fashion, which drove development of premature senescence of lung fibroblasts. Finally, we show that conditioned medium derived from senescent lung fibroblasts, which were induced to senesce through the activation of ATP/P2Y11R-mediated signaling, promoted the proliferation of triple-negative breast cancer cells and their tumorigenic potential by secreting amphiregulin. Our study identifies the existence of a novel purinergic signaling pathway that links extracellular ATP to the development of a protumorigenic premature senescent phenotype in lung fibroblasts that is dependent on P2Y11R activation and ER-to-mitochondria calcium signaling.

Sex differences in lower urinary tract function in mice with or without spinal cord injury.

OBJECTIVES: We examined sex differences of lower urinary tract function and molecular mechanisms in mice with and without spinal cord injury (SCI). METHODS: SCI was induced by Th8-9 spinal cord transection in male and female mice. We evaluated cystometrograms (CMG) and electromyography (EMG) of external urethral sphincter (EUS) at 6 weeks after SCI in spinal intact (SI) and SCI mice. The mRNA levels of Piezo2 and TRPV1 were measured in L6-S1 dorsal root ganglia (DRG). Protein levels of nerve growth factor (NGF) in the bladder mucosa was evaluated using an enzyme-linked immunosorbent assay. RESULTS: Sex differences were found in the EUS behavior during voiding as voiding events in female mice with or without SCI occurred during EUS relaxation periods without EUS bursting activity whereas male mice with or without SCI urinated during EUS bursting activity in EMG recordings. In both sexes, SCI decreased voiding efficiency along with increased tonic EUS activities evident as reduced EUS relaxation time in females and longer active periods of EUS bursting activity in males. mRNA levels of Piezo2 and TRPV1 of DRG in male and female SCI mice were significantly upregulated compared with SI mice. NGF in the bladder mucosa showed a significant increase in male and female SCI mice compared with SI mice. However, there were no significant differences in Piezo2 or TRPV1 levels in DRG or NGF protein levels in the bladder mucosa between male and female SCI mice. CONCLUSIONS: We demonstrated that female and male mice voided during EUS relaxation and EUS bursting activity, respectively. Also, upregulation of TRPV1 and Piezo2 in L6-S1 DRG and NGF in the bladder could be involved in SCI-induced lower urinary tract dysfunction in both sexes of mice.

Role of opioid and ß-adrenergic receptors in bladder underactivity induced by prolonged pudendal nerve stimulation in cats.

AIMS: To determine the role of opioid and ß-adrenergic receptors in bladder underactivity induced by prolonged pudendal nerve stimulation (PNS). METHODS: In α-chloralose anesthetized cats, 30-min PNS was applied repeatedly for 3-9 times to induce poststimulation or persistent bladder underactivity. Then, naloxone (opioid receptor antagonist, 1 mg/kg, IV) or propranolol (ß-adrenergic receptor antagonist, 3 mg/kg, IV) was given to reverse the bladder underactivity. After the drug treatment, an additional 30-min PNS was applied to counteract the drug effect. Repeated cystometrograms were performed by slowly (1-2 mL/min) infusing the bladder with saline via a urethral catheter to determine the bladder underactivity and the treatment effects. RESULTS: Prolonged (2-4.5 h) PNS induced bladder underactivity evident as a large bladder capacity (169 ± 49% of control) and a reduced amplitude of bladder contraction (59 ± 17% of control). Naloxone fully reversed the bladder underactivity by reducing bladder capacity to 113 ± 58% and increasing the amplitude of bladder contraction to 104 ± 34%. After administration of naloxone an additional 30-min PNS temporarily increased the bladder capacity to the underactive bladder level (193 ± 74%) without changing the amplitude of the bladder contraction. Propranolol had no effect on bladder underactivity. CONCLUSIONS: A tonic enkephalinergic inhibitory mechanism in the CNS plays a critical role in the bladder underactivity induced by prolonged PNS, while the peripheral ß-adrenergic receptor mechanism in the detrusor is not involved. This study provides basic science evidence consistent with the clinical observation that comorbid opioid usage may contribute to voiding dysfunction in patients with Fowler's syndrome.

Pudendal Nerve Block by Adaptively Stepwise Increasing the Intensity of High-Frequency (10 kHz) Biphasic Stimulation.

OBJECTIVE: The purpose of this study is to determine whether adaptively stepwise increasing the intensity of a high-frequency (10 kHz) biphasic stimulation (HFBS) can produce nerve conduction block without generating a large initial response. MATERIALS AND METHODS: In anesthetized cats, three cuff electrodes were implanted on the left pudendal nerve for stimulation or block. The urethral pressure increase induced by pudendal nerve stimulation was used to measure the pudendal nerve block induced by HFBS. RESULTS: HFBS applied suddenly with a large step increase in intensity induced a large (86 ± 16 cmH2O) urethral pressure increase before it blocked pudendal nerve conduction. However, HFBS applied by adaptively stepwise increasing the intensity every 10 to 60 seconds over a long period (33-301 minutes; average 108 ± 35 minutes) with many small intensity increases (0.005-0.1 mA) induced no response or low-amplitude high-frequency urethral pressure changes before it blocked pudendal nerve conduction. The minimal HFBS intensities required by the two different methods to block pudendal nerve conduction are similar. CONCLUSION: This study is important for better understanding the possible mechanisms underlying the HFBS-induced nerve block and provides the possibility of developing a new nerve block method for clinical applications in which an initial large response is a concern.

Model Analysis of Post-Stimulation Block of a Myelinated Axon by Direct Current.

OBJECTIVE: To determine the role of ion concentrations and ion pump activity in conduction block of myelinated axon induced by a long-duration direct current (DC). METHODS: A new axonal conduction model for myelinated axons based on the classical Frankenhaeuser-Huxley (FH) equations is developed that includes ion pump activity and allows the intracellular and extracellular Na+ and K+ concentrations to change with axonal activity. RESULTS: Action potential generation, propagation, and acute DC block occurring within a short period (milliseconds) that do not significantly change the ion concentrations or trigger ion pump activity are successfully simulated by the new model in a similar way as the classical FH model. Different from the classical model, the new model also successfully simulates the post-stimulation block phenomenon, i.e., the axonal conduction block occurring after terminating a long-duration (30 seconds) DC stimulation as observed recently in animal studies. The model reveals a significant K+ accumulation outside the axonal node as the possible mechanism underlying the post-DC block that is slowly reversed by ion pump activity during the post-stimulation period. CONCLUSION: Changes in ion concentrations and ion pump activity play an important role in post-stimulation block induced by long-duration DC stimulation. SIGNIFICANCE: Long-duration stimulation is used clinically for many neuromodulation therapies, but the effects on axonal conduction/block are poorly understood. This new model will be useful for better understanding of the mechanisms underlying long-duration stimulation that changes ion concentrations and triggers ion pump activity.

Treating Lower Urinary Tract Symptoms in Older Adults: Intravesical Options.

This article provides an overview of the diagnosis and the treatment of lower urinary tract symptoms in older adults complicated by the neurodegenerative changes in the micturition reflex and further confounded by age-related decline in hepatic and renal clearance raising the propensity of adverse drug reactions. The first-line drug treatment for lower urinary tract symptoms, orally administered antimuscarinics, fails to reach the equilibrium dissociation constant of muscarinic receptors even at their maximum plasma concentration and tends to evoke a half-maximal response at a muscarinic receptor occupancy of just 0.206% in the bladder with a minimal difference from exocrine glands, which raises the adverse drug reaction risk. On the contrary, intravesical antimuscarinics are instilled at concentrations 1000-fold higher than the oral maximum plasma concentration and the equilibrium dissociation constant erects a downhill concentration gradient that drives passive diffusion and achieves a mucosal concentration around ten-fold lower than the instilled concentration for a long-lasting occupation of muscarinic receptors in mucosa and sensory nerves. A high local concentration of antimuscarinics in the bladder triggers alternative mechanisms of action and is supposed to engage retrograde transport to nerve cell bodies for neuroplastic changes that underlie a long-lasting therapeutic effect, while an intrinsically lower systemic uptake of the intravesical route lowers the muscarinic receptor occupancy of exocrine glands to lower the adverse drug reaction relative to the oral route. Thus, the traditional pharmacokinetics and pharmacodynamics of oral treatment are upended by intravesical antimuscarinics to generate a dramatic improvement (~ 76%) noted in a meta-analysis of studies enrolling children with neurogenic lower urinary tract symptoms on the primary endpoint of maximum cystometric bladder capacity as well as the secondary endpoints of filling compliance and uninhibited detrusor contractions. The therapeutic success of intravesical multidose oxybutynin solution or oxybutynin entrapped in the polymer for sustained release in the pediatric population bodes well for patients with lower urinary tract symptoms at the other extreme of the age spectrum. Though generally used to predict oral drug absorption, Lipinski's rule of five can also explain the ten-fold lower systemic uptake from the bladder of positively charged trospium over oxybutynin, a tertiary amine. Chemodenervation by an intradetrusor injection of onabotulinumtoxinA is merited for patients with idiopathic overactive bladder discontinuing oral treatment because of a lack of efficacy. However, age-related peripheral neurodegeneration potentiates the adverse drug reaction risk of urinary retention that motivates the quest of liquid instillation, delivering larger fraction of onabotulinumtoxinA to the mucosa as opposed to muscle by an intradetrusor injection can also probe the neurogenic and myogenic predominance of idiopathic overactive bladder. Overall, the treatment paradigm of lower urinary tract symptoms in older adults should be tailored to individual's overall health status and the risk tolerance for adverse drug reactions.

Effect of high-frequency membrane potential alternation between depolarization and hyperpolarization on dorsal root ganglion neurons of rats.

The purpose of this study was to determine how sensory neurons respond to high-frequency membrane potential alternation between depolarization and hyperpolarization. Membrane currents were recorded from dissociated dorsal root ganglion (DRG) neurons of adult rats using the whole cell patch clamp technique in voltage clamp mode. Stepwise depolarization of the membrane was applied first to determine the threshold membrane potential for inducing an action potential (AP) current. Then, membrane potential alternation between depolarization (to +20 mV) and hyperpolarization (to -110 mV) was applied to the neuron for 10 s at different frequencies (10 Hz to 1 kHz). The tested DRG neurons had APs of either a long duration (>10 ms) or a short duration (<10 ms). Membrane potential alternation at ≥500 Hz completely disrupted the AP generation, disabled the ion channel gating function, and produced membrane current alternating symmetrically across zero. Replacing extracellular sodium with potassium increased the amplitude of the membrane current response and caused the membrane current to be larger during hyperpolarization than during depolarization. These results support the hypothesis that high-frequency biphasic stimulation blocks axonal conduction by driving the potassium channel open constantly. Understanding neural membrane response to high-frequency membrane potential alternation is important to reveal the possible mechanisms underlying axonal conduction block induced by high-frequency biphasic stimulation.

Mechanisms Underlying Poststimulation Block Induced by High-Frequency Biphasic Stimulation.

OBJECTIVE: To reveal the possible mechanisms underlying poststimulation block induced by high-frequency biphasic stimulation (HFBS). MATERIALS AND METHODS: A new axonal conduction model is developed for unmyelinated axons. This new model is different from the classical axonal conduction model by including both ion concentrations and membrane ion pumps to allow analysis of axonal responses to long-duration stimulation. Using the new model, the post-HFBS block phenomenon reported in animal studies is simulated and analyzed for a wide range of stimulation frequencies (100 Hz-10 kHz). RESULTS: HFBS can significantly change the Na+ and K+ concentrations inside and outside the axon to produce a post-HFBS block of either short-duration (<500 msec) or long-duration (>3 sec) depending on the duration of HFBS. The short-duration block is due to the fast recovery of the Na+ and K+ concentrations outside the axon in periaxonal space by diffusion of ions into and from the large extracellular space, while the long-duration block is due to the slow restoration of the normal Na+ concentration inside the axon by membrane ion pumps. The 100 Hz HFBS requires the minimal electrical energy to achieve the post-HFBS block, while the 10 kHz stimulation is the least effective frequency requiring high intensity and long duration to achieve the block. CONCLUSION: This study reveals two possible ionic mechanisms underlying post-HFBS block of axonal conduction. Understanding these mechanisms is important for improving clinical applications of HFBS block and for developing new nerve block methods employing HFBS.

Temperature Effect on Nerve Conduction Block Induced by High-Frequency (kHz) Biphasic Stimulation.

OBJECTIVES: This study aims to determine temperature effect on nerve conduction block induced by high-frequency (kHz) biphasic stimulation (HFBS). MATERIALS AND METHODS: Frog sciatic nerve-muscle preparation was immersed in Ringer's solution at a temperature of 15 or 20 °C. To induce muscle contractions, a bipolar cuff electrode delivered low-frequency (0.25 Hz) stimulation to the nerve. To induce nerve block, a tripolar cuff electrode was placed distal to the bipolar cuff electrode to deliver HFBS (2 or 10 kHz). A bipolar hook electrode distal to the blocking electrode was used to confirm that the nerve block occurred locally at the site of HFBS. A thread tied onto the foot was attached to a force transducer to measure the muscle contraction force. RESULTS: At 15 °C, both 2- and 10-kHz HFBSs elicited an initial transient muscle contraction and then produced nerve block during the stimulation (ie, acute block), with the 10 kHz having a significantly (p < 0.001) higher acute block threshold (5.9 ± 0.8 mA peak amplitude) than the 2 kHz (1.9 ± 0.3 mA). When the temperature was increased to 20 °C, the acute block threshold for the 10-kHz HFBS was significantly (p < 0.0001) decreased from 5.2 ± 0.3 to 4.4 ± 0.2 mA, whereas the 2-kHz HFBS induced a tonic muscle contraction during the stimulation but elicited nerve block after terminating the 2-kHz HFBS (ie, poststimulation block) with an increased block duration at a higher stimulation intensity. CONCLUSION: Temperature has an important influence on HFBS-induced nerve block. The blocking mechanisms underlying acute and poststimulation nerve blocks are likely to be very different.

Penile Erection Induced by Stimulation of Sacral S1/S2 Spinal Root in Cats.

OBJECTIVE: This study aimed at determining whether stimulation of sacral spinal roots can induce penile erection in cats. MATERIALS AND METHODS: In anesthetized cats, a 20-gauge catheter was inserted into the corpus cavernosum to measure the penile pressure. Stimulus pulses (5-80 Hz, 0.2 ms) were applied through bipolar hook electrodes to sacral ventral roots alone or to combined ventral and dorsal roots of a single S1-S3 segment to induce penile pressure increases and penile erection. RESULTS: Stimulation of the S1 or S2 ventral root at 30 to 40 Hz induced observable penile erection with rigidity and the largest increase (169 ± 11 cmH2O) in penile pressure. Continuous stimulation (10 minutes) of afferent and efferent axons by simultaneous stimulation of the S1 or S2 dorsal and ventral roots at 30 Hz also produced a large increase (190 ± 8 cmH2O) in penile pressure that was sustainable during the entire stimulation period. After a complete spinal cord transection at the T9-T10 level, simultaneous stimulation of the S1 or S2 dorsal and ventral roots induced large (186 ± 9 cmH2O) and sustainable increases in penile pressure. CONCLUSION: This study indicates the possibility to develop a novel neuromodulation device to restore penile erection after spinal cord injury using a minimally invasive surgical approach to insert a lead electrode through the sacral foramen to stimulate a sacral spinal root.

In Vivo Luminal Measurement of Distension-evoked Urothelial ATP Release in Rodents.

ATP, released from the urothelium in response to bladder distension, is thought to play a significant sensory role in the control of micturition. Therefore, accurate measurement of urothelial ATP release in a physiological setting is an important first step in studying the mechanisms that control purinergic signaling in the urinary bladder. Existing techniques to study mechanically evoked urothelial ATP release utilize cultured cells plated on flexible supports or bladder tissue pinned into Ussing chambers; however, each of these techniques does not fully emulate conditions in the intact bladder. Therefore, an experimental setup was developed to directly measure ATP concentrations in the lumen of the rodent urinary bladder. In this setup, the bladders of anesthetized rodents are perfused through catheters in both the dome of the bladder and via the external urethral orifice. Pressure in the bladder is increased by capping the urethral catheter while perfusing sterile fluid into the bladder through the dome. Measurement of intravesical pressure is achieved using a pressure transducer attached to the bladder dome catheter, akin to the setup used for cystometry. Once the desired pressure is reached, the urethral catheter's cap is removed, and fluid collected for ATP quantification by luciferin-luciferase assay. Through this experimental setup, the mechanisms controlling both mechanical and chemical stimulation of urothelial ATP release can be interrogated by including various agonists or antagonists into the perfusate or by comparing results between wildtype and genetically modified animals.

Vaginal Lubrication and Pressure Increase Induced by Pudendal Nerve Stimulation in Cats.

BACKGROUND: Vaginal lubrication and contractions are among the top difficulties affecting sexual intercourse in women after spinal cord injury. AIM: This study aimed at determining if pudendal nerve stimulation (PNS) can improve vaginal lubrication and induce increases in vaginal pressure. METHODS: In anesthetized cats, a small piece of cotton was inserted into the vagina for 10 minutes with or without PNS to measure vaginal wetness by the weight increase of the vaginal cotton. Then, a small balloon catheter was inserted into the vagina to measure the pressure increase induced by PNS. Intensity response of the vagina to PNS (30 Hz, 0.2 ms, 5 seconds) was determined at 1-4 times of intensity threshold (T) for PNS to induce an observable vaginal pressure increase. Frequency response was determined at 2T intensity in a range of PNS frequencies (5-50 Hz). Finally, fatigue in vaginal pressure was determined by applying PNS (30 Hz, 2T) either continuously or intermittently (5 seconds on and 5 seconds off) for 4 minutes. OUTCOMES: The effectiveness of PNS in increasing vaginal wetness and pressure is evaluated. RESULTS: PNS significantly (P = .0327) increased the measurement of vaginal wetness from 15.8 ± 3.8 mg during control without stimulation to 32.4 ± 4.7 mg after stimulation. Vaginal pressure increased as PNS intensity or frequency increased. PNS (30 Hz, 2T) induced vaginal pressure increase ≥80% of the maximal response. Intermittent PNS induced significantly (P = .0354) smaller fatigue (45.6 ± 3.7%) in vaginal pressure than continuous PNS (69.1 ± 3.0%) during the 4-minute stimulation. CLINICAL TRANSLATION: This study raises the possibility of developing a novel pudendal neuromodulation device to improve female sexual function after spinal cord injury. STRENGTHS & LIMITATIONS: This study provides preclinical data supporting the development of a novel pudendal neuromodulation device. The limitation includes the lack of chemical analysis of the vaginal secretion. CONCLUSION: PNS can improve vaginal lubrication and induce increases in vaginal pressure. Chen J, Zhong Y, Wang J, et al. Vaginal Lubrication and Pressure Increase Induced by Pudendal Nerve Stimulation in Cats. J Sex Med 2022;19:1517-1523.

Intracellular sodium concentration and membrane potential oscillation in axonal conduction block induced by high-frequency biphasic stimulation.

Objective.A new axonal conduction model was used to analyze the interaction between intracellular sodium concentration and membrane potential oscillation in axonal conduction block induced by high-frequency (kHz) biphasic stimulation (HFBS).Approach.The model includes intracellular and extracellular sodium and potassium concentrations and ion pumps. First, the HFBS (1 kHz, 5.4 mA) was applied for a duration (59.4 s) long enough to produce an axonal conduction block after terminating the stimulation, i.e. a post-stimulation block. Then, the intensity of HFBS was reduced to a lower level for 4 s to determine if the axonal conduction block could be maintained.Main results.The block duration was shortened from 1363 ms to 5 ms as the reduced HFBS intensity was increased from 0 mA to 4.1 mA. The block was maintained for the entire tested period (4000 ms) if the reduced intensity was above 4.2 mA. At the low intensity (<4.2 mA) the membrane potential oscillation disrupted the post-stimulation block caused by the increased intracellular sodium concentration, while at the high intensity (>4.2 mA) the membrane potential oscillation was strong enough to maintain the block and further increased the intracellular sodium concentration.Significance.This study indicates a possibility to develop a new nerve block method to reduce the HFBS intensity, which can extend the battery life for an implantable nerve stimulator in clinical applications to block pain of peripheral origin.

Sacral neuromodulation of bladder underactivity induced by prolonged pudendal afferent firing in cats.

This study examined the effect of sacral neuromodulation on persistent bladder underactivity induced by prolonged pudendal nerve stimulation (PudNS). In 10 α-chloralose-anesthetized cats, repetitive application of 30-min PudNS induced bladder underactivity evident as an increase in bladder capacity during a cystometrogram (CMG). S1 or S2 dorsal root stimulation (15 or 30 Hz) at 1 or 1.5 times threshold intensity (T) for inducing reflex hindlimb movement (S1) or anal sphincter twitch (S2) was applied during a CMG to determine if the stimulation can reverse the bladder underactivity. Persistent (>3 h) bladder underactivity consisting of a significant increase in bladder capacity to 163.1 ± 11.3% of control was induced after repetitive (1-10 times) application of 30-min PudNS. S2 but not S1 dorsal root stimulation at 15 Hz and 1 T intensity reversed the PudNS-induced bladder underactivity by significantly reducing the large bladder capacity to 124.3 ± 12.9% of control. Other stimulation parameters were not effective. After the induction of persistent underactivity, recordings of reflex bladder activity under isovolumetric conditions revealed that S2 dorsal root stimulation consistently induced the largest bladder contraction at 15 Hz and 1 T when compared with other frequencies (5-40 Hz) or intensities (0.25-1.5 T). This study provides basic science evidence consistent with the hypothesis that abnormal pudendal afferent activity contributes to the bladder underactivity in Fowler's syndrome and that sacral neuromodulation treats this disorder by reversing the bladder inhibition induced by pudendal nerve afferent activity.

Superficial peroneal neuromodulation of nonobstructive urinary retention induced by prolonged pudendal afferent activity in cats.

The purpose of this study is to determine whether superficial peroneal nerve stimulation (SPNS) can improve nonobstructive urinary retention (NOUR) induced by prolonged pudendal nerve stimulation (PNS). In this exploratory acute study using eight cats under anesthesia, PNS and SPNS were applied by nerve cuff electrodes. Skin surface electrodes were also used for SPNS. A double lumen catheter was inserted via the bladder dome for bladder infusion and pressure measurement and to allow voiding without a physical urethral outlet obstruction. The voided and postvoid residual (PVR) volumes were also recorded. NOUR induced by repetitive (4-13 times) application of 30-min PNS significantly (P < 0.05) reduced voiding efficiency by 49.5 ± 16.8% of control (78.3 ± 7.9%), with a large PVR volume at 208.2 ± 82.6% of control bladder capacity. SPNS (1 Hz, 0.2 ms) at 1.5-2 times threshold intensity (T) for inducing posterior thigh muscle contractions was applied either continuously (SPNSc) or intermittently (SPNSi) during cystometrograms to improve the PNS-induced NOUR. SPNSc and SPNSi applied by nerve cuff electrodes significantly (P < 0.05) increased voiding efficiency to 74.5 ± 18.9% and 67.0 ± 15.3%, respectively, and reduced PVR volume to 54.5 ± 39.0% and 88.3 ± 56.0%, respectively. SPNSc and SPNSi applied noninvasively by skin surface electrodes also improved NOUR similar to the stimulation applied by a cuff electrode. This study indicates that abnormal pudendal afferent activity could be a pathophysiological cause for the NOUR occurring in Fowler's syndrome and a noninvasive superficial peroneal neuromodulation therapy might be developed to treat NOUR in patients with Fowler's syndrome.

High-frequency stimulation induces axonal conduction block without generating initial action potentials.

The purpose of this modeling study is to develop a novel method to block nerve conduction by high frequency biphasic stimulation (HFBS) without generating initial action potentials. An axonal conduction model including both ion concentrations and membrane ion pumps is used to analyze the axonal response to 1 kHz HFBS. The intensity of HFBS is increased in multiple steps while maintaining the intensity at a sub-threshold level to avoid generating an action potential. Axonal conduction block by HFBS is defined as the failure of action potential propagation at the site of HFBS. The simulation analysis shows that step-increases in sub-threshold intensity during HFBS can successfully block axonal conduction without generating an initial response because the excitation threshold of the axon can be gradually increased by the sub-threshold HFBS. The mechanisms underlying the increase in excitation threshold involve changes in intracellular and extracellular sodium and potassium concentration, change in the resting potential, partial inactivation of the sodium channel and partial activation of the potassium channel by HFBS. When the excitation threshold reaches a sufficient level, an acute block occurs first and after additional sub-threshold HFBS it is followed by a post-stimulation block. This study indicates that step-increases in sub-threshold HFBS intensity induces a gradual increase in axonal excitation threshold that may allow HFBS to block nerve conduction without generating an initial response. If this finding is proven to be true in human, it will significantly impact clinical applications of HFBS to treat chronic pain.

Defecation induced by stimulation of sacral S2 spinal root in cats.

The aim of this study was to determine whether stimulation of sacral spinal nerve roots can induce defecation in cats. In anesthetized cats, bipolar hook electrodes were placed on the S1-S3 dorsal and/or ventral roots. Stimulus pulses (1-50 Hz, 0.2 ms) were applied to an individual S1-S3 root to induce proximal/distal colon contractions and defecation. Balloon catheters were inserted into the proximal and distal colon to measure contraction pressure. Glass marbles were inserted into the rectum to demonstrate defecation by videotaping the elimination of marbles. Stimulation of the S2 ventral root at 7 Hz induced significantly (P < 0.05) larger contractions (32 ± 9 cmH2O) in both proximal and distal colon than stimulation of the S1 or S3 ventral root. Intermittent (5 times) stimulation (1 min on and 1 min off) of both dorsal and ventral S2 roots at 7 Hz produced reproducible colon contractions without fatigue, whereas continuous stimulation of 5-min duration caused significant fatigue in colon contractions. Stimulation (7 Hz) of both dorsal and ventral S2 roots together successfully induced defecation that eliminated 1 or 2 marbles from the rectum. This study indicates the possibility to develop a novel neuromodulation device to restore defecation function after spinal cord injury using a minimally invasive surgical approach to insert a lead electrode via the sacral foramen to stimulate a sacral spinal root.NEW & NOTEWORTHY This study in cats determined the optimal stimulation parameters and the spinal segment for sacral spinal root stimulation to induce colon contraction. The results have significant implications for design of a novel neuromodulation device to restore defecation function after spinal cord injury (SCI) and for optimizing sacral neuromodulation parameters to treat non-SCI people with chronic constipation.

Low pressure voiding induced by stimulation and 1 kHz post-stimulation block of the pudendal nerves in cats.

The goal of this study is to induce low-pressure voiding by stimulation and bilateral 1 kHz post-stimulation block of the pudendal nerves. In anesthetized cats, wire hook electrodes were placed on the left and/or right pudendal nerves. Stimulus pulses (30 Hz, 0.2 ms) were applied to one pudendal nerve to induce a reflex bladder contraction and to produce contractions of the external urethral sphincter (EUS). High frequency (1 kHz) biphasic stimulation was applied to block axonal conduction in both pudendal nerves and block EUS activity. In 4 cats, a catheter was inserted into the distal urethra to perfuse and measure the back pressure caused by the EUS contraction. In another 5 cats, a catheter was inserted into the bladder dome and the urethra was left open to allow voiding. The 1 kHz stimulation (30-60 s, 0.5-5 mA) delivered via a wire hook electrode completely blocked pudendal nerve conduction for ≥2 min after terminating the stimulation, i.e., a post-stimulation block. The block gradually disappeared in 6-18 min. The block duration increased with increasing amplitude or duration of the 1 kHz stimulation. Without the 1 kHz block, 30 Hz stimulation alone induced high-pressure (90 cmH2O) voiding. When combined with the 1 kHz block, the 30 Hz stimulation induced low-pressure (≤50 cmH2O) voiding with a high voiding efficiency (80%). In summary, a minimally invasive surgical approach might be developed to restore voiding function after spinal cord injury by stimulation and block of the pudendal nerves using lead electrodes.

Functional roles for PIEZO1 and PIEZO2 in urothelial mechanotransduction and lower urinary tract interoception.

The mechanisms that link visceral mechanosensation to the perception of internal organ status (i.e., interoception) remain elusive. In response to bladder filling, the urothelium releases ATP, which is hypothesized to stimulate voiding function by communicating the degree of bladder fullness to subjacent tissues, including afferent nerve fibers. To determine if PIEZO channels function as mechanosensors in these events, we generated conditional urothelial Piezo1-, Piezo2-, and dual Piezo1/2-knockout (KO) mice. While functional PIEZO1 channels were expressed in all urothelial cell layers, Piezo1-KO mice had a limited phenotype. Piezo2 expression was limited to a small subset of superficial umbrella cells, yet male Piezo2-KO mice exhibited incontinence (i.e., leakage) when their voiding behavior was monitored during their active dark phase. Dual Piezo1/2-KO mice had the most affected phenotype, characterized by decreased urothelial responses to mechanical stimulation, diminished ATP release, bladder hypoactivity in anesthetized Piezo1/2-KO females but not males, and urinary incontinence in both male and female Piezo1/2-KO mice during their dark phase but not inactive light one. Our studies reveal that the urothelium functions in a sex- and circadian rhythm-dependent manner to link urothelial PIEZO1/2 channel-driven mechanotransduction to normal voiding function and behavior, and in the absence of these signals, bladder dysfunction ensues.

TRP Channel Agonists Activate Different Afferent Neuromodulatory Mechanisms in Guinea Pig Urinary Bladder.

Activation of TRP channels expressed in urinary bladder afferent nerves and urothelium releases neurotransmitters that influence bladder function. Experiments were undertaken to examine the mechanisms underlying effects of TRPA1 (allyl isothiocyanate, AITC), TRPV1 (capsaicin, CAPS), and TRPC (oleoyl-2-acetyl-sn-glycerol, OAG) agonists on guinea pig bladder activity. Effects of these agonists were compared with effects of nitro-oleic acid (OA-NO2), an electrophilic nitro-fatty acid, known to activate TRPV1, TRPA1 or TRPC channels in sensory neurons. AITC (100 µM) increased (231%) area of spontaneous bladder contractions (SBCs) an effect reduced by a TRPA1 antagonist (HC3-03001, HC3, 10 µM) and reversed to inhibition by indomethacin (INDO, 500 nM) a cyclooxygenase inhibitor. The post-INDO inhibitory effect of AITC was mimicked (39% depression) by calcitonin gene-related peptide (CGRP, 100 nM) and blocked by a CGRP antagonist (BIBN, 25 µM). CAPS (1 µM) suppressed SBCs by 30% in 81% of strips, an effect blocked by a TRPV1 antagonist (diarylpiperazine, 1 µM) or BIBN. SBCs were suppressed by OA-NO2 (30 µM, 21% in 77% of strips) or by OAG (50 µM, 30%) an effect blocked by BIBN. OA-NO2 effects were not altered by HC3 or diarylpiperazine. OA-NO2 also induced excitation in 23% of bladder strips. These observations raise the possibility that guinea pig bladder is innervated by at least two types of afferent nerves: [1] Type A express TRPA1 receptors that induce the release of prostaglandins and excite the detrusor, [2] Type B express TRPV1, TRPA1 and TRPC receptors and release CGRP that inhibits the detrusor.