Characterizing the Bladder's Response to Onabotulinum Toxin Type A Using a Rat Model.

OBJECTIVES: The aim of this study was to characterize the response of the rat bladder neuromuscular system to intramural injection of onabotulinum toxin type A (BoNT/A) over 9 weeks using in vivo cystometry (CMG) and in vitro contractility (IVC). METHODS: Chronic bladder catheters were implanted in female Sprague-Dawley rats, and either (1) BoNT/A (10 units in 20 µL saline) or (2) saline (20 µL) was injected in 5 × 4 µL doses throughout the bladder wall. At 1, 3, 6, and 9 weeks after injection, conscious restrained CMG was performed. At each time point, 25% of each group (8 BoNT/A and 4 controls) was euthanized and bladders harvested for IVC. We measured IVC in response to electric field stimulation, carbachol, and potassium chloride. RESULTS: In total, 47 animals were included; 31 underwent BoNT/A injection, and 16 received sham (saline). Bladder capacities did not differ significantly between groups for each time point. One week after injection BoNT/A animals exhibited significantly longer bladder contraction durations and lower voiding efficiencies compared with controls. By 3 weeks these values returned to control levels. For BoNT/A animals, contractile response to carbachol stimulation was enhanced at 3 weeks. Otherwise, there were no differences in IVC responses. CONCLUSIONS: One week after BoNT/A injection, prolonged bladder contractions are noted in rats. This may reflect supraspinal compensation for denervation by increasing the duration of efferent drive during voiding. After 3 weeks postinjection, we observed no differences in either CMG or IVC responses suggesting either compensatory efferent sprouting, increased gap junction formation, or loss of BoNT/A effect.

The effects of Ins2(Akita) diabetes and chronic angiotensin II infusion on cystometric properties in mice.

AIMS: Diabetes is associated with both dysfunction of the lower urinary tract (LUT) and overactivity of the renin-angiotensin system (RAS). Although it is well known that the RAS affects normal LUT function, very little is known about RAS effects on the diabetic LUT. Accordingly, we investigated the effects of chronic angiotensin II (AngII) treatment on the LUT in a model of type 1 diabetes. METHODS: Ins2(Akita) diabetic mice (20 weeks old) and their age-matched background controls underwent conscious cystometric evaluation after 4 weeks of chronic AngII treatment (700 ng/kg/min by osmotic pump) or vehicle (saline). RESULTS: Diabetic mice had compensated LUT function with bladder hypertrophy. Specifically, micturition volume, residual volume, and bladder capacity were all increased, while voiding efficiency and pressure generation were unchanged as bladder mass, contraction duration, and phasic urethral function were increased. AngII significantly increased voiding efficiency and peak voiding pressure and decreased phasic frequency irrespective of diabetic state and, in diabetic but not normoglycemic control mice, significantly decreased residual volume and increased contraction duration and nonphasic contraction duration. CONCLUSIONS: The Ins2(Akita) diabetic mice had compensated LUT function at 20 weeks of age. Even under these conditions, AngII had beneficial effects on LUT function, resulting in increased voiding efficiency. Future studies should therefore be conducted to determine whether AngII can rescue the decompensated LUT function occurring in end-stage diabetic uropathy.

A multicenter phase I dose escalation trial to evaluate safety and tolerability of intra-arterial temozolomide for patients with advanced extremity melanoma using normothermic isolated limb infusion.

BACKGROUND: L-phenylalanine mustard (LPAM) has been the standard for use in regional chemotherapy (RC) for unresectable in-transit melanoma. Preclinical data demonstrated that regional temozolomide (TMZ) may be more effective. METHODS: Patients with AJCC Stage IIIB or IIIC extremity melanoma who failed previous LPAM-based RC were treated with TMZ via isolated limb infusion (ILI) according to a modified accelerated titration design. Drug pharmacokinetic (PK) analysis, tumor gene expression, methylation status of the O6-methylguanine methyltransferase (MGMT) promoter, and MGMT expression were evaluated. Primary objectives were to (1) determine dose-limiting toxicities (DLTs) and maximum tolerated dose (MTD) of TMZ via ILI and (2) explore biomarker correlates of response. RESULTS: 28 patients completed treatment over 2.5 years at 3 institutions. 19 patients were treated at the MTD defined as 3,200 mg/m(2) [multiplied by 0.09 (arm), 0.18 (leg)]. Two of five patients had DLTs at the 3,600 mg/m(2) level while only grade 1 (n = 15) and grade 2 (n = 4) clinical toxicities occurred at the MTD. At 3-month post-ILI, 10.5 % (2/19) had CR, 5.3 % (1/19) had PR, 15.8 % (3/19) had SD, and 68.4 % (13/19) had PD. Neither PK parameters of TMZ nor MGMT levels were associated with response or toxicity. CONCLUSION: In this first ever use of intra-arterial TMZ in ILI for melanoma, the MTD was determined. While we could not define a marker for TMZ response, the minimal toxicity of TMZ ILI may allow for repeated treatments to increase the response rate as well as clarify the role of MGMT expression.

The effects of bilateral bipolar sacral neurostimulation on urinary bladder activity during filling before and after irritation in a rat model.

AIMS: To design an optimal rat model for studying sacral neurostimulation (SNS), and examine the effect of SNS on myogenic non-voiding contractions (NVC) during filling before and after intraluminal irritation. METHODS: Bilateral paired bipolar SNS was performed in 10 female urethane anesthetized Sprague-Dawley rats at the L6-S1 trunks at 10 Hz, 0.1 msec pulse duration and 0.15-0.80 mA (below motor threshold). Transvesical cystometry was performed before and during SNS under conditions of control (saline, N = 10) and irritation [0.25% acetic acid (AA), N = 5]. RESULTS: Functional bladder capacity (FBC) and NVC count were significantly increased during SNS under both control and irritation conditions (P < 0.01 for all). In six instances (four in control, two in irritation), micturition reflexes were completely inhibited by SNS resulting in overflow incontinence. Filling compliance, NVC period and NVC maximum amplitude were not affected by SNS (P > 0.05 for all). Non-parametric two-way analysis of variance for repeated measures revealed increased FBC and NVC count during SNS under both control and irritation conditions (P = 0.004 for both, N = 5 rats). Linear regression analysis of NVC count versus FBC revealed a slope significantly different than zero, independent of control or irritation conditions (slope 33.10 ± 2.43, R(2) = 0.81, P < 0.001). CONCLUSIONS: This model achieved reliable, reversible and robust increases in bladder capacity up to overflow incontinence at stimulations below somatic motor threshold. SNS dramatically increased FBC and reversed AA-induced changes without affecting the character of normal NVC associated with bladder filling. Increased number of NVC is simply a consequence of increased fill time.

Type 1 angiotensin receptors on macrophages ameliorate IL-1 receptor-mediated kidney fibrosis.

In a wide array of kidney diseases, type 1 angiotensin (AT1) receptors are present on the immune cells that infiltrate the renal interstitium. Here, we examined the actions of AT1 receptors on macrophages in progressive renal fibrosis and found that macrophage-specific AT1 receptor deficiency exacerbates kidney fibrosis induced by unilateral ureteral obstruction (UUO). Macrophages isolated from obstructed kidneys of mice lacking AT1 receptors solely on macrophages had heightened expression of proinflammatory M1 cytokines, including IL-1. Evaluation of isolated AT1 receptor-deficient macrophages confirmed the propensity of these cells to produce exaggerated levels of M1 cytokines, which led to more severe renal epithelial cell damage via IL-1 receptor activation in coculture compared with WT macrophages. A murine kidney crosstransplantation concomitant with UUO model revealed that augmentation of renal fibrosis instigated by AT1 receptor-deficient macrophages is mediated by IL-1 receptor stimulation in the kidney. This study indicates that a key role of AT1 receptors on macrophages is to protect the kidney from fibrosis by limiting activation of IL-1 receptors in the kidney.

Enhanced susceptibility to urinary tract infection in the spinal cord-injured host with neurogenic bladder.

Neurogenic bladder predisposes to recurrent urinary tract infections (UTI) and renal failure, and susceptibility is commonly ascribed to urinary stasis from elevated residual urine volumes. Escherichia coli UTI was modeled in the spinal cord-injured (SCI) rat with the hypothesis that SCI animals would require fewer bacteria to establish infection, have an exaggerated inflammatory response, and have delayed clearance of infection compared to normal-voiding controls. T10 SCI rats and controls had median infectious doses (ID50) of 10(2) and 10(5) CFU, respectively. Mean residual volumes in the SCI animals did not correlate with susceptibility to initiation of UTI or outcome. In the acute infection, control and SCI rats developed acute cystitis and pyelitis without acute differences in histopathological scores of inflammation. However, in vivo imaging of infected animals revealed persistently higher levels of bacteria in the SCI urine and bladders than were seen for controls over 2 weeks. Likewise, at 2 weeks, acute and chronic inflammatory infiltrates persisted in the bladders and kidneys of SCI rats, whereas inflammation largely resolved within the controls. Together these data demonstrate that SCI rats exhibit delayed clearance of infection and exaggerated inflammatory responses in bladders and kidneys; however, the severity of residual volumes does not predict increased susceptibility to UTI. These studies suggest that host-dependent mechanisms that are discrete from alterations in bladder physiology influence UTI susceptibility with the SCI-neurogenic bladder. This model will allow elucidation of SCI-neurogenic bladder-mediated changes in host response that yield UTI susceptibility and may lead to new preventative and therapeutic options.

Differential vulnerabilities of urethral afferents in diabetes and discovery of a novel urethra-to-urethra reflex.

Urethral reflexes are important regulators of micturition, and impairment of urethral afferent neuronal function may disrupt coordinated bladder and urethral activity, thereby contributing to voiding dysfunction in lower urinary tract disorders. Chemical stimulation by intraurethral irritant solution perfusion was used to determine whether urethral afferent neuronal function is altered in diabetes mellitus (DM). Sprague-Dawley rats were studied 10 wk after streptozotocin injection to induce DM or vehicle alone. Escalating doses of capsaicin (0.1-30 microM) or acetic acid (0.01-1%; AA) were perfused intraurethrally while recording isovolumetric bladder activity, urethral perfusion pressure, and electromyography of the external urethral sphincter (EUS-EMG). Some rats were additionally treated with alpha-bungarotoxin, hexamethonium, or bilateral transection of the sensory branches of the pudendal nerves (PudSNx). Intraurethral capsaicin inhibited bladder contractions in six out of seven control rats but not in any of six DM rats. Low-frequency oscillations (LFOs) of intraurethral pressure were observed in five out of six control rats with capsaicin-induced bladder inhibition. In contrast, intraurethral AA inhibited bladder contractions and enhanced tonic EUS-EMG activity in six out of six control and five out of six DM rats. LFOs occurred in four out of six control and three of five DM rats with AA-induced bladder inhibition. Chemically induced bladder inhibition and LFOs were not prevented by alpha-bungarotoxin but were eliminated by PudSNx and hexamethonium. Finally, LFOs were followed by phasic EUS activity. These findings show that DM affects urethral afferent neurons differentially, compromising those expressing TRPV1 receptors. Urethral smooth muscle LFOs are neurogenically mediated and induce EUS activity, revealing the existence of a hitherto undescribed reflex pathway: a smooth-to-striated muscle urethra-to-urethra reflex.

Sprouting of substance P-expressing primary afferent central terminals and spinal micturition reflex NK1 receptor dependence after spinal cord injury.

The primary afferent neurotransmitter triggering the spinal micturition reflex after complete spinal cord injury (SCI) in the rat is unknown. Substance P detected immunohistochemically in the sacral parasympathetic nucleus was significantly higher in 12 SCI rats than in 12 spinally intact rats (P = 0.008), suggesting substance P as a plausible candidate for the primary afferent neurotransmitter. The effects of the tachykinin NK1 receptor antagonist L-733060 on the spinal micturition reflex were then determined by performing conscious cystometry in an additional 14 intact rats and 14 SCI rats with L-733060 (0.1-100 microg) administered intrathecally at L6-S1. L-733060 was without effect in intact rats, but blocked the spinal micturition reflex in 10 of 14 SCI rats and increased the intermicturition interval in 2 of 4 others at doses ranging from 10 to 100 microg. Both phasic and nonphasic voiding contractions, differentiated according to the presence of phasic external urethral sphincter (EUS) activity, were present in most SCI rats. Both types of contractions were blocked by high doses of L-733060. Interestingly, there was a relative decline in phasic voiding contractions at high doses as well as a decline in contraction amplitude in nonphasic voiding contractions. In other respects, cystometric variables were largely unaffected in either spinally intact or SCI rats. L-733060 did not affect tonic EUS activity at any dose except when the spinal micturition reflex was blocked and tonic activity was consequently lost. These experiments show that tachykinin action at spinal NK1 receptors plays a major role in the spinal micturition reflex in SCI rats.

Voltage-dependent potassium currents of urethral afferent neurons in diabetes mellitus.

Urethra-to-bladder and urethra-to-urethra reflexes appear to be important for coordination of proper voiding. Diabetes mellitus (DM) is known to result in afferent neuropathy. Neuropathic alterations in electrophysiological properties of urethral afferent neurons may therefore contribute to voiding dysfunction seen in diabetes mellitus. Accordingly, we studied urethral afferent neuronal somata in streptozotocin-induced DM or age-matched vehicle controls by whole-cell patch clamp at 5- or 10-week time points. One week prior to study, Fast Blue was injected into the proximal urethra to label urethral afferent neurons. A previously undescribed diminution of afferent neuronal voltage-dependent potassium currents was a prominent feature of urethral afferent neuropathy in DM, acting to increase neuronal excitability. Thus, unlike bladder afferent neurons, urethral afferent neurons may be hyperexcitable well into DM progression.

Diabetic urethropathy compounds the effects of diabetic cystopathy.

PURPOSE: The effects of short-term and long-term diabetes mellitus on urethral function were investigated to determine the contribution of urethral dysfunction to diabetes mellitus voiding dysfunction. MATERIALS AND METHODS: Isovolumetric bladder pressure, urethral perfusion pressure and external urethral sphincter electromyography were measured in urethane anesthetized, female Sprague-Dawley rats (Charles River Laboratories, Wilmington, Massachusetts) 5 or 10 weeks after streptozotocin induced diabetes mellitus. Urethral responses to serial administration of the skeletal muscle blocker alpha-bungarotoxin, the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine and the alpha-adrenergic agonist L-phenylephrine were determined in diabetes mellitus and age matched controls. RESULTS: Peak bladder pressures and contraction amplitudes were significantly decreased in diabetes mellitus rats. Detrusor-sphincter dyssynergia occurred in approximately 30% of diabetes mellitus rats but never in controls. Alpha-Bungarotoxin caused a greater decrease in baseline urethral perfusion pressure in diabetes mellitus rats than in controls (approximately 40% vs approximately 15%). Bladder contraction associated urethral smooth muscle relaxation amplitudes were significantly less in diabetes mellitus rats than in controls. N(omega)-nitro-L-arginine significantly suppressed urethral relaxation in controls but not in diabetes mellitus rats. L-phenylephrine significantly increased baseline urethral perfusion pressure in diabetes mellitus rats but not in controls. The unassociated conditions of insensitivity to N-nitro-L-arginine and hypersensitivity to L-phenylephrine were more common in 10-week diabetes mellitus rats than in control rats. CONCLUSIONS: Diabetes mellitus induced urethropathy is characterized by external urethral sphincter dysfunction, decreased urethral smooth muscle relaxation and nitric oxide responsiveness, and increased urethral smooth muscle responsiveness to alpha(1)-adrenergic agonists. These changes increase outlet resistance and, thereby, decrease voiding efficiency. This exacerbates voiding dysfunction, creating a vicious cycle of progressive lower urinary tract damage and dysfunction. Early intervention targeting outlet resistance may be indicated.

Effect of 5-hydroxytryptamine1 serotonin receptor agonists on noxiously stimulated micturition in cats with chronic spinal cord injury.

PURPOSE: The serotonin 5-hydroxytryptamine(1A/7) receptor agonist (R)-8-OH-DPAT (8-hydroxy-2-(di-n-propylamino)tetralin) (Sigma) and the 5-hydroxytryptamine(1A/1B/1D) agonist GR-46611 (3-[3-(2-dimethylaminoethyl)-1H-indol-5-yl]-N-(4-methoxybenzyl)acrylamide) (Tocris Cookson, Ellisville, Missouri) inhibit bladder activity during saline infusion into the bladder of cats with chronic spinal cord injury (saline infused, spinal cord injured cats), suggesting an effect on mechanosensitive bladder afferent C fibers or their targets. We investigated the effects of (R)-8-OH-DPAT and GR-46611 on bladder activity in chronic spinal cord injured cats during infusion of dilute acetic acid into the bladder to stimulate chemosensitive bladder afferent C fibers (acid infused, spinal cord injured cats). MATERIALS AND METHODS: Chloralose anesthetized, spinal cord injured cats were catheterized through the bladder dome for filling cystometry during 0.5% acetic acid infusion. Dose-response curves for (R)-8-OH-DPAT (0.3 to 30 microg/kg intravenously) or GR-46611 (0.03 to 300 microg/kg intravenously) were followed by the 5-hydroxytryptamine(1A) antagonist WAY-100635 (N-tert-butyl-3-(4-(2-methoxyphenyl)-piperazin-1-yl)-2-phenylpropanamide) (Sigma) (300 microg/kg). Threshold volume, bladder capacity, residual volume, micturition volume and arterial pressure were measured and external urethral sphincter electromyogram was recorded. RESULTS: Acid infused, spinal cord injured cats responded to (R)-8-OH-DPAT but not to GR-46611 with dose dependent increases in threshold volume, capacity and residual volume (significant above 3 microg/kg). Effects of (R)-8-OH-DPAT were largely reversed by WAY-100635. Neither (R)-8-OH-DPAT nor GR-46611 augmented external urethral sphincter electromyogram activity. CONCLUSIONS: Based on differences in the response to GR-46611 in saline vs acid infused, spinal cord injured animals it is tempting to speculate that 2 distinct populations of bladder afferent C fibers (1 chemosensitive and 1 mechanosensitive) can initiate spinal bladder reflexes. Because 5-hydroxytryptamine(1A) receptor agonists increased bladder capacity under saline or acid infused conditions, they are promising candidates for decreasing bladder hyperactivity and increasing bladder capacity in patients with chronic spinal cord injury.

Mechanism of origin of conduction disturbances in aging human atrial bundles: experimental and model study.

BACKGROUND: Aging is associated with a significant increase in atrial tachyarrhythmias, especially atrial fibrillation. A macroscopic repolarization gradient created artificially by a stimulus at one site before a premature stimulus from a second site is widely considered to be part of the experimental protocol necessary for the initiation of such arrhythmias in the laboratory. How such gradients occur naturally in aging atrial tissue is unknown. OBJECTIVE: The objective of this study was to determine if the pattern of cellular connectivity in aging human atrial bundles produces a mechanism for variable early premature responses. METHODS: Extracellular and intracellular potentials were recorded after control and premature stimuli at a single site in aging human atrial bundles. We also measured cellular geometry, the distribution of connexins, and the distribution of collagenous septa. A model of the atrial bundles was constructed based on the morphological results. Action potential propagation and the sodium current were analyzed after premature stimuli in the model. RESULTS: Similar extracellular potential waveform responses occurred after early premature stimuli in the aging bundles and in the model. Variable premature conduction patterns were accounted for by the single model of aging atrial structure. A major feature of the model results was that the conduction events and the magnitude of the sodium current at multiple sites were very sensitive to small changes in the location and the timing of premature stimuli. CONCLUSION: In aging human atrial bundles stimulated from only a single site, premature stimuli induce variable arrhythmogenic conduction responses. The generation of these responses is greatly enhanced by remodeling of cellular connectivity during aging. The results provide insight into sodium current structural interactions as a general mechanism of arrhythmogenic atrial responses to premature stimuli.

Activation of the external urethral sphincter central pattern generator by a 5-HT(1A) receptor agonist in rats with chronic spinal cord injury.

We recently demonstrated that treatment with the 5-HT(1A/7) receptor agonist [(R)-(+)-8-hydroxy-2-di-n-propylamino]tetralin (8-OH-DPAT) increases bladder capacity in chloralose-anesthetized female cats with chronic spinal cord injury. In the current study, we investigated the effects of 8-OH-DPAT on bladder capacity and external urethral sphincter (EUS) activity in urethane-anesthetized female rats (initial body mass 175-200 g) with chronic spinal cord injury (transsection at T10). Cystometric study took place 8-12 wk posttranssection. Intravesical pressure was monitored in urethane-anesthetized rats with a transvesical catheter, and EUS activity was assessed electromyographically. Spinal cord injury disrupts phasic activity of the EUS, resulting in decreased voiding efficiency and increased residual volume. 8-OH-DPAT induced a dose-dependent decrease in bladder capacity (the opposite of its effect in chronic spinal cord-injured cats) with an increase in micturition volume and decrease in residual volume resulting from improvement in voiding efficiency. The unexpected improvement in voiding efficiency can be explained by the 8-OH-DPAT-induced emergence of phasic EUS relaxation. Phasic EUS relaxation was also altered by 8-OH-DPAT in spinally intact rats, whereas the 5-HT(1A) receptor antagonist N-tert-butyl-3-[4-(2-methoxyphenyl)-piperazin-1-yl]-2-phenylpropanamide (WAY-100635), on its own, was without effect. It remains to be determined when phasic relaxation is restored after spinal cord injury, and indeed whether it is ever truly lost or is only temporarily separated from excitatory input.

Distribution and immunohistochemical characterization of primary afferent neurons innervating the levator ani muscle of the female squirrel monkey.

OBJECTIVE: This study was undertaken to examine the neurofilament and neurochemical composition of subpopulations of primary afferent neurons innervating the levator ani muscle by combining retrograde tracing and triple labeling immunofluorescence in the female squirrel monkey. STUDY DESIGN: Cholera toxin B subunit (CTB) was injected unilaterally into the levator ani muscle of 3 monkeys to identify primary sensory neurons in the dorsal root ganglia (DRG) and their central projections in the spinal cord. L7-S2 DRG were processed for dual or triple labeling immunofluorescence 3 days after injection to examine labeling of the 200 kD neurofilament marker RT97 (a marker of myelinated neurons), calcitonin gene-related peptide (CGRP; a marker of peptidergic neurons), isolectin B4 (IB4; a marker of small, unmyelinated neurons), and nerve growth factor receptor (TrkA) in CTB-positive neurons. RESULTS: RT97-negative (C-fiber) neurons were more numerous (74% of total CTB-labeled neurons) and smaller in size than RT97-positive (A-fiber) afferent neurons (26% of CTB-labeled neurons). IB4 labeling was almost exclusively found in RT97-negative afferent neurons. Approximately 43% of all CTB-labeled DRG neurons expressed CGRP, and the majority of these were small. The distribution and sizes of CTB-labeled TrkA-positive DRG neurons were similar to those of CTB-labeled CGRP-positive DRG neurons. CONCLUSION: The levator ani muscle is innervated by 3 major subpopulations of primary afferent neurons consisting of cells with large, neurofilament-rich soma and A fibers (putative proprioceptive neurons) and those with small, peptidergic or nonpeptidergic, neurofilament-poor soma and C fibers (putative nociceptive, mechanoreceptive, ergoreceptive, and thermoreceptive neurons). Future investigation is needed to elucidate the relationship between primary sensory neuron subpopulations and changes in neuropeptide and neurotrophin expression on experimental levator ani nerve damage, childbirth, and aging.

Immunohistochemical evidence for the interaction between levator ani and pudendal motor neurons in the coordination of pelvic floor and visceral activity in the squirrel monkey.

OBJECTIVE: The purpose of this study was to characterize the spinal distribution of afferent and efferent pathways that innervate the levator ani (LA) muscle in the female squirrel monkey. STUDY DESIGN: Cholera toxin B (CTB) was injected unilaterally into the LA muscle of 5 monkeys to identify primary sensory neurons in the dorsal root ganglia (DRG) and motor neurons in the spinal cord that contribute fibers to the LA nerve. Fluoro-Gold (FG) was injected into the external anal sphincter of 2 of these animals to label pudendal motor neurons (1 of these animals underwent unilateral LA neurectomy before CTB injection). Spinal cord and DRG were processed for immunofluorescence 3 to 7 days after injections. RESULTS: Retrograde transport of CTB from the LA muscle labeled primary afferent neurons in the ipsilateral DRG, their central projections, and motor neurons in the medial portion of the ipsilateral ventral horn of the spinal cord (L7-S2 segments). Injection of FG into the external anal sphincter labeled cells in Onuf's nucleus, primarily in L7. Importantly, CTB-labeled LA motor neurons were virtually absent in Onuf's nucleus, where all pudendal motor neurons are located. CTB-labeled processes were observed within Onuf's nucleus, adjacent to FG-labeled pudendal motor neurons, and appeared to derive from dendrites of LA motor neurons that project into Onuf's nucleus. CONCLUSION: The LA muscle has a distinct innervation with very little or no contribution from the pudendal nerve. The intriguing labeling of LA neural elements within a nucleus that innervates the external urethral and anal sphincters (involved in pelvic visceral control) may represent a neuroanatomic substrate for physiologic integration of spinal and supraspinal inputs for the coordination of pelvic floor and visceral activity.

Effects of alpha 1-adrenergic receptor subtype selective antagonists on lower urinary tract function in rats with bladder outlet obstruction.

PURPOSE: Antagonists of alpha 1-adrenergic receptors (alpha 1ARs) relieve obstructive and irritative symptoms in patients with bladder outlet obstruction. However, to our knowledge mechanisms underlying the relief of irritative symptoms remain unknown. Because bladder alpha 1dARs are up-regulated in some rats with bladder outlet obstruction, we investigated the effect of the alpha 1aAR antagonist 5-methyl urapidil (5MU) vs the alpha 1a/alpha 1dAR antagonist tamsulosin on urinary frequency in obstructed rats. MATERIALS AND METHODS: Baseline frequency was measured using a chronic micturition recording system and then obstruction (40 rats) or sham obstruction surgery (11 rats) was performed. After 6 weeks frequency was reassessed, followed by subcutaneous implantation of osmotic pumps to deliver 5MU, tamsulosin or vehicle for 1 week. Upon the completion of drug treatment urinary frequency was again measured and the pressor response to the alpha 1AR agonist phenylephrine was documented. RESULTS: Obstructed bladder mass was an average of 4.9 times greater than bladder mass in sham operated rats (p <0.001). Urinary frequency was elevated in obstructed rats with a bladder mass of greater than 500 mg vs all rats with a bladder mass of under 255 mg (p = 0.01). Of rats with a bladder mass of greater than 500 mg frequency was decreased in those treated with tamsulosin (p = 0.03) but not in those treated with 5MU. Tamsulosin and 5MU inhibited the pressor response to phenylephrine. CONCLUSIONS: Urinary frequency is increased in rats with a bladder mass of greater than 500 mg. The combined alpha 1a/alpha 1dAR antagonist tamsulosin decreases urinary frequency more than the alpha 1aAR selective antagonist 5MU. This finding supports the hypothesis that the alpha 1dAR is important for mediating irritative symptoms.

Inhibition of bladder activity by 5-hydroxytryptamine1 serotonin receptor agonists in cats with chronic spinal cord injury.

The serotonin (5-hydroxytryptamine1A) 5-HT1A receptor agonist 8-OH-DPAT [(R)- (+)-8-hydroxy-2-(di-n-propylamino)tetralin] inhibits bladder activity under nociceptive but not innocuous conditions in cats with an intact spinal cord, suggestive of an effect on primary afferent C fibers or their targets. Because C fibers play a key role in reflex micturition in chronic spinal cord injury (SCI), we investigated the effect of 8-OH-DPAT on micturition in SCI cats. We also investigated GR-46611 (3-[3-(2-dimethylaminoethyl)-1H-indol-5-yl]-N-(4-methoxybenzyl)acrylamide), which has agonist activity predominantly at 5-HT1B and 5-HT1D receptors but also at the 5-HT1A receptor. Chloralose-anesthetized cats were catheterized through the bladder dome for saline-filling cystometry. Dose-response curves for i.v. 8-OH-DPAT (0.3-30 microg/kg) and GR-46611 (0.03-300 microg/kg) were followed in three cases each by 5-HT1A antagonist WAY-100635 [N-tert-butyl-3-(4-(2-methoxyphenyl)-piperazin-1-yl)-2-phenylpropanamide] at 300 microg/kg. Threshold volume, capacity, residual volume, micturition volume, and arterial pressure were measured. Intact cats showed few significant changes in cystometric variables. SCI cats responded to both 8-OH-DPAT and GR-46611 with dose-dependent increases in threshold volume, capacity, and residual volume, significant at > or =10 microg/kg for 8-OH-DPAT and at > or =3 microg/kg for GR-46611. Effects of 8-OH-DPAT but not GR-46611 were largely reversed by WAY-100635. Both 5-HT1A and 5-HT1B/1D agonists may offer a promising means of reducing bladder hyperactivity and increasing bladder capacity in patients with chronic SCI.

Induction of bladder sphincter dyssynergia by kappa-2 opioid receptor agonists in the female rat.

PURPOSE: The nonselective kappa opioid receptor agonist ethylketocyclazocine suppresses external urethral sphincter (EUS) reflexes in cats. We examined the role of spinal kappa-opioid receptor subtypes in the control of EUS function in rats using selective kappa-1 (U-50,488) or kappa-2 (GR-89,696) opiate receptor agonists. MATERIALS AND METHODS: Urethane anesthetized female rats were catheterized through the bladder dome for cystometry. EUS function was assessed electromyographically. Drugs were administered intrathecally or intravenously. RESULTS: Micturition in rats is accompanied at different times by tonic (continuous) EUS spike activity and by phasic bursts of spikes separated by pauses. GR-89,696 (0.05 to 5 microg intrathecally) caused a dose dependent decrease in the number of bursts per micturition without affecting spike frequency within individual bursts or during periods of tonic activity. It resulted in decreased voiding efficiency and at high doses dyssynergia and overflow incontinence. The nonselective opiate receptor antagonist naloxone (1 mg/kg intravenously) blocked GR-89,696 effects. U-50,488H (0.05 to 15 microg intrathecally) caused no change in cystometric parameters or in EUS-electromyography. CONCLUSIONS: Efficient voiding in rats depends on a spinal pattern generator causing EUS motor neuron firing to occur in bursts, resulting in rapid urethral contraction and relaxation. Intrathecal kappa-2-opiate receptor agonists suppress this pattern generator, decreasing the number of bursts occurring during each micturition without decreasing motor neuron spike frequency during individual bursts or during tonic spike activity associated with urethral closure. Resultant dyssynergia leads to decreased voiding efficiency. The relevance of kappa-2 opioid receptors should be explored in higher species, especially regarding spinal cord injury induced dyssynergia.

Cell size and communication: role in structural and electrical development and remodeling of the heart.

With the advent of new information about alterations of cardiac gap junctions in disease conditions associated with arrhythmias, there have been major advances in the genetic and metabolic manipulation of gap junctions. In contrast, in naturally occurring cardiac preparations, little is known about cell-to-cell transmission and the subcellular events of propagation or about structural mechanisms that may affect conduction events at this small size scale. Therefore, the aim of this article is to review results that produce the following unifying picture: changes in cardiac conduction due to remodeling cardiac morphology ultimately are limited to changes in three morphologic parameters: (1) cell geometry (size and shape), (2) gap junctions (distribution and conductivity), and (3) interstitial space (size and distribution). In this article, we consider changes in conduction that result from the remodeling of cell size and gap junction distribution that occurs with developmental ventricular hypertrophy from birth to maturity. We then go on to changes in longitudinal and transverse propagation in aging human atrial bundles that are produced by remodeling interstitial space due to deposition of collagenous septa. At present, experimental limitations in naturally occurring preparations prevent measurement of the conductance of individual gap junctional plaques, as well as the delays in conduction associated with cell-to-cell transmission. Therefore, we consider the development of mathematical electrical models based on documented cardiac microstructure to gain insight into the role of specific morphologic parameters in generating the changes in anisotropic propagation that we measured in the tissue preparations. A major antiarrhythmic implication of the results is that an "indirect" therapeutic target is interstitial collagen, because regulation of its deposition and turnover to prevent or alter microfibrosis can enhance side-to-side electrical coupling between small groups of cells in aging atrial bundles.

Innervation of the levator ani and coccygeus muscles of the female rat.

In humans, the pelvic floor skeletal muscles support the viscera. Damage to innervation of these muscles during parturition may contribute to pelvic organ prolapse and urinary incontinence. Unfortunately, animal models that are suitable for studying parturition-induced pelvic floor neuropathy and its treatment are rare. The present study describes the intrapelvic skeletal muscles (i.e., the iliocaudalis, pubocaudalis, and coccygeus) and their innervation in the rat to assess its usefulness as a model for studies of pelvic floor nerve damage and repair. Dissection of rat intrapelvic skeletal muscles demonstrated a general similarity with human pelvic floor muscles. Innervation of the iliocaudalis and pubocaudalis muscles (which together constitute the levator ani muscles) was provided by a nerve (the "levator ani nerve") that entered the pelvic cavity alongside the pelvic nerve, and then branched and penetrated the ventromedial (i.e., intrapelvic) surface of these muscles. Innervation of the rat coccygeus muscle (the "coccygeal nerve") was derived from two adjacent branches of the L6-S1 trunk that penetrated the muscle on its rostral edge. Acetylcholinesterase staining revealed a single motor endplate zone in each muscle, closely adjacent to the point of nerve penetration. Transection of the levator ani or coccygeal nerves (with a 2-week survival time) reduced muscle mass and myocyte diameter in the iliocaudalis and pubocaudalis or coccygeus muscles, respectively. The pudendal nerve did not innervate the intrapelvic skeletal muscles. We conclude that the intrapelvic skeletal muscles in the rat are similar to those described in our previous studies of humans and that they have a distinct innervation with no contribution from the pudendal nerve.