Irina Zabbarova

Botulinum Neurotoxin Serotype A Suppresses Neurotransmitter Release from Afferent as Well as Efferent Nerves in the Urinary Bladder

BACKGROUND Botulinum neurotoxin A (BoNTA), which alleviates overactive bladder symptoms, is thought to act predominantly via the inhibition of transmitter release from parasympathetic nerves. However, actions at other sites such as afferent nerve terminals are possible. OBJECTIVE To evaluate the effects of BoNTA on bladder afferent neuropeptide release and firing. DESIGN, SETTING, AND PARTICIPANTS One side of the bladder of control and chronic (1-2 wk) spinal cord transected (SCT; T(8)-T(9)) adult female mice was injected with BoNTA (0.5 U/5 μl saline). After 48 h, bladders with L(6)-S(2) spinal nerves were prepared for in vitro recordings. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS In bladder preparations, tension and optical mapping of Ca(2+) transients were used to measure intrinsic contractions, those evoked by capsaicin or the electrical stimulation of spinal nerves. Afferent firing was evoked by stretch or intrinsic bladder contractions. The numbers of responding units and firing rates were measured. Animal numbers were used to detect moderate to large between-group differences based on Cohens criteria. Two-way analysis of variance was used to test spatial/temporal differences in Ca(2+) signals as mean plus or minus standard deviation. Differences between data sets were tested with the student t test and skewed data sets with a Mann-Whitney U test (significant when p<0.05). RESULTS AND LIMITATIONS In control and SCT bladders, BoNTA treatment decreased the contractions evoked by electrical stimulation of spinal nerves without altering intrinsic contractions. Afferent firing on untreated sides in response to stretch/intrinsic contractions was increased in SCTs versus controls. On BoNTA-treated sides, afferent firing rates were greatly attenuated in response to mechanical stimulation as were the capsaicin-evoked optical signals mediated by neuropeptide release. CONCLUSIONS SCT caused an increased sensitivity of afferent nerves to mechanical stimulation that was reduced by BoNTA treatment. Increased intrinsic activity after SCT was unaffected by the toxin. Thus BoNTA suppresses neurogenic detrusor overactivity by targeting afferent as well as efferent pathways in the bladder.

Spontaneous Contractions Evoke Afferent Nerve Firing in Mouse Bladders With Detrusor Overactivity

PURPOSE Afferent nerve firing has been linked to spontaneous bladder contractions in a number of lower urinary tract pathologies and it may lead to urgency and incontinence. Using optical mapping, single unit recording and tension measurements we investigated the correlation between afferent nerve firing and spontaneous bladder contractions in spinal cord transected mice. MATERIALS AND METHODS Bladder-nerve preparations (bladder sheets and the associated L6-S2 pelvic nerves) were dissected from normal and spinal cord transected mice showing overactivity on cystometry and opened along the ventral aspect from base to dome. Bladder sheets were mounted horizontally in a temperature regulated chamber to simultaneously record Ca(2+) transients across the mucosal surface, single unit afferent nerve firing and whole bladder tension. RESULTS Single unit afferent fibers were identified by probing their receptive fields. Fibers showed a graded response to von Frey stimulation and a frequency of afferent firing that increased as a function of the degree of stretch. Optical maps of Ca(2+) transients in control bladders demonstrated multiple initiation sites that resulted in high frequency, low amplitude spontaneous contractions. Alternatively in maps of the bladders of spinal cord transected mice Ca(2+) transients arose from 1 or 2 focal sites, resulting in low frequency, high amplitude contractions and concomitant afferent firing. CONCLUSIONS Large amplitude, spontaneous bladder contractions evoke afferent nerve activity, which may contribute to incontinence.

Researching bladder afferents—determining the effects of β3‐adrenergic receptor agonists and botulinum toxin type‐A

A substantial portion of the current research on lower urinary tract dysfunction is focused on afferent mechanisms. The main goals are to define and modulate the signaling pathways by which afferent information is generated, enhanced and conveyed to the central nervous system. Alterations in bladder afferent mechanisms are a potential source of voiding dysfunction and an emerging source for drug targets. Established drug therapies such as muscarinic receptor antagonists, and two emerging therapies, β3‐adrenergic receptor agonists and botulinum toxin type‐A, may act partly through afferent mechanisms. This review focuses on these two new principles and new and established methods for determining their sites of action. It also provides brief information on the innervation of the bladder, afferent receptors and transmitters and how these may communicate with the urothelium, interstitial cells and detrusor smooth muscle to regulate micturition. Peripheral and central mechanisms of afferent sensitization and myogenic mechanisms that lead to detrusor overactivity, overactive bladder symptoms and urgency sensations are also covered. This work is the result from ‘Think Tank’ presentations, and the lengthy discussions that followed, at the 2010 International Consultation on Incontinence Research Society meeting in Bristol, UK. Neurourol. Urodynam. Neurourol. Urodynam. 30:684–691, 2011.

Mechanisms of action of botulinum neurotoxins, β3‐adrenergic receptor agonists, and PDE5 inhibitors in modulating detrusor function in overactive bladders: ICI‐RS 2011

Botulinum neurotoxins type A (BoNT/A), β3‐adrenergic receptor agonists, and phosphodiesterase type 5 (PDE5) inhibitors are promising agents that mitigate lower urinary tract symptoms by attenuating the sensory system. However, whether they act directly on afferent nerves or indirectly through the other cell types is unclear.

The potential role of unregulated autonomous bladder micromotions in urinary storage and voiding dysfunction; overactive bladder and detrusor underactivity

The isolated bladder shows autonomous micromotions, which increase with bladder distension, generate sensory nerve activity, and are altered in models of urinary dysfunction. Intravesical pressure resulting from autonomous activity putatively reflects three key variables; the extent of micromotion initiation, distances over which micromotions propagate, and overall bladder tone. In vivo, these variables are subordinate to the efferent drive of the central nervous system. In the micturition cycle storage phase, efferent inhibition keeps autonomous activity generally at a low level, where it may signal ‘state of fullness’, whilst maintaining compliance. In the voiding phase, mass efferent excitation elicits generalised contraction (global motility initiation). In lower urinary tract dysfunction, efferent control of the bladder can be impaired, for example due to peripheral ‘patchy’ denervation. In this case, loss of efferent inhibition may enable unregulated micromotility, and afferent stimulation, predisposing to urinary urgency. If denervation is relatively slight, the detrimental impact on voiding may be low, as the adjacent innervated areas may be able to initiate micromotility synchronous with the efferent nerve drive, so that even denervated areas can contribute to the voiding contraction. This would become increasingly inefficient the more severe the denervation, such that ability of triggered micromotility to propagate sufficiently to engage the denervated areas in voiding declines, so the voiding contraction increasingly develops the characteristics of underactivity. In summary, reduced peripheral coverage by the dual efferent innervation (inhibitory and excitatory) impairs regulation of micromotility initiation and propagation, potentially allowing emergence of overactive bladder and, with progression, detrusor underactivity.

Mitochondrial targeting of radioprotectants using peptidyl conjugates

Ionizing radiation activates a mitochondrial nitric oxide synthase, leading to inhibition of the respiratory chain, generation of excess superoxide, peroxynitrite production and nitrosative damage. We have measured the radioprotective effects of a nitric oxide synthase antagonist (AMT) versus a free radical scavenger (4-amino-TEMPO) using electrochemical detection of nitric oxide and peroxynitrite. To enhance their efficacy, we have conjugated these compounds to peptides and peptide isosteres--derived from the antibiotic gramicidin S--that target the mitochondria. The targeting ability of these peptidyl conjugates was measured using quantitative mass spectrometry.

Targeted delivery of radioprotective agents to mitochondria.

Adverse effects of ionizing radiation are mediated through reactive oxygen and nitrogen species. Mitochondria are the principal source of these species in the cell and play an important role in irradiation-induced apoptosis. The use of free radical scavengers and nitric oxide synthase inhibitors has proven to protect normal tissues and, in some cases, to sensitize tumor tissues to radiation damage. Dual molecules that combine radical-scavenging and NOS-inhibitory functions may be particularly effective. Drugging strategies that target mitochondria can enhance the effectiveness of such agents, in comparison to systemic administration, and circumvent side effects.

Sophisticated models and methods for studying neurogenic bladder dysfunction

To describe how the use of new and established animal models and methods can generate vital and far reaching experimental data in the study of mechanism underlying neurogenic bladder overactivity.

Do we understand any more about bladder interstitial cells?—ICI‐RS 2013

To present a brief review on discussions from “Do we understand any more about lower urinary tract interstitial cells?” session at the 2013 International Consultation on Incontinence‐Research Society (ICI‐RS) meeting in Bristol, UK.

Fgfr2 is integral for bladder mesenchyme patterning and function

While urothelial signals, including sonic hedgehog (Shh), drive bladder mesenchyme differentiation, it is unclear which pathways within the mesenchyme are critical for its development. Studies have shown that fibroblast growth factor receptor (Fgfr)2 is necessary for kidney and ureter mesenchymal development. The objective of the present study was to determine the role of Fgfr2 in the bladder mesenchyme. We used Tbx18cre mice to delete Fgfr2 in the bladder mesenchyme (Fgfr2(BM-/-)). We performed three-dimensional reconstructions, quantitative real-time PCR, in situ hybridization, immunolabeling, ELISAs, immunoblot analysis, void stain on paper, ex vivo bladder sheet assays, and in vivo decerebrated cystometry. Compared with control bladders, embryonic day 16.5 (E16.5) Fgfr2(BM-/-) bladders had thin muscle layers with less α-smooth muscle actin and thickened lamina propria with increased collagen type Ia and IIIa that intruded into the muscle. The reciprocal changes in mutant layer thicknesses appeared partly due to a cell fate switch. From postnatal days 1 to 30, Fgfr2(BM-/-) bladders demonstrated progressive muscle loss and increased collagen expression. Postnatal Fgfr2(BM-/-) bladder sheets exhibited decreased agonist-mediated contractility and increased passive stretch tension versus control bladder sheets. Cystometry revealed high baseline and threshold pressures and shortened intercontractile intervals in Fgfr2(BM-/-) versus control bladders. Mechanistically, whereas Shh expression appeared normal, mRNA and protein readouts of hedgehog activity were increased in E16.5 Fgfr2(BM-/-) versus control bladders. Moreover, E16.5 Fgfr2(BM-/-) bladders exhibited higher levels of Cdo and Boc, hedgehog coreceptors that enhance sensitivity to Shh, compared with control bladders. In conclusion, loss of Fgfr2 in the bladder mesenchyme leads to abnormal bladder morphology and decreased compliance and contractility.