Spinal Cord Injury Causes Marked Tissue Rearrangement in the Urethra-Experimental Study in the Rat.

Traumatic spinal cord injury (SCI) results in the time-dependent development of urinary impairment due to neurogenic detrusor overactivity (NDO) and detrusor-sphincter-dyssynergia (DSD). This is known to be accompanied by massive changes in the bladder wall. It is presently less clear if the urethra wall also undergoes remodelling. To investigate this issue, female rats were submitted to complete spinal transection at the T8/T9 level and left to recover for 1 week and 4 weeks. To confirm the presence of SCI-induced NDO, bladder function was assessed by cystometry under urethane anesthesia before euthanasia. Spinal intact animals were used as controls. Urethras were collected and processed for further analysis. Following thoracic SCI, time-dependent changes in the urethra wall were observed. Histological assessment revealed marked urethral epithelium reorganization in response to SCI, as evidenced by an increase in epithelial thickness. At the muscular layer, SCI resulted in strong atrophy of the smooth muscle present in the urethral sphincter. Innervation was also affected, as evidenced by a pronounced decrease in the expression of markers of general innervation, particularly those present in sensory and sympathetic nerve fibres. The present data show an evident impact of SCI on the urethra, with significant histological rearrangement, accompanied by sensory and sympathetic denervation. It is likely that these changes will affect urethral function and contribute to SCI-induced urinary dysfunction, and they deserve further investigation.

DU Is Induced by Low Levels of Urinary ATP in a Rat Model of Partial Bladder Outlet Obstruction: The Incidence of Both Events Decreases after Deobstruction.

Objectives: To investigate, in initial phases of bladder outlet obstruction (BOO), the urinary ATP levels, the incidence of detrusor underactivity (DU), and if they change after deobstruction. Methods: Adult female Wistar rats submitted to partial BOO (pBOO) and sham-obstruction were used. Cystometry was performed 3 or 15 days after pBOO and fluid was collected from the urethra for ATP determination. Bladders were harvested for morphological evaluation of the urothelium. DU was defined as the average of voiding contractions (VC) of sham-operated animals, with 3 SD at 15 days after the sham surgery. In another group of animals in which pBOO was relieved at 15 days and bladders were let to recover for 15 days, the incidence of DU and ATP levels were also accessed. The Kruskal-Wallis test was followed by Dunn's multiple comparisons test, and Spearman's correlation test was used. Results: DU was present in 13% and 67% of the bladders at 3 and 15 days after pBOO, respectively, and in 20% of the bladders at 15 days after deobstruction. ATP levels were significantly lower in DU/pBOO versus sham and non-DU/pBOO rats. A strong positive correlation between ATP levels and VC/min was obtained (r = 0.63). DU bladders had extensive areas in which umbrella cells appeared stretched, the width exceeding that presented by sham animals. Conclusions: Low urothelial ATP parallels with a high incidence of DU early after pBOO.

A importância da informação científica na educação para a prevenção de doenças infecciosas virais / The importance of scientific information in education for the prevention of viral infectocontagious diseases / La importancia de la información científica en la educación para la prevención de enfermedades infecciosas virales

Objetivo: orientar alunos do ensino fundamental e médio do CED07-Ceilândia / DF sobre a importância das práticas de higiene em prol da prevenção de doenças infecciosas. Método: o estudo foi desenhado em três fases distintas: aplicação de questionários de higiene pessoal; palestras e workshops práticos sobre patologias humanas; e avaliação do projeto pelos alunos participantes. Resultados: Os resultados mostram que 57% dos alunos compartilham objetos pessoais, um número muito elevado, uma vez que a literatura aponta que existem várias patologias que podem ser adquiridas de objetos individuais. Observou-se também que os alunos não têm o hábito de tirar os sapatos antes de entrar em suas casas. Eles alegaram desconhecer os riscos de contaminação por esse comportamento, mas afirmaram que, após as informações fornecidas pelo projeto, estariam mais atentos a esse fator de contaminação domiciliar. Assim, acredita-se que as práticas educativas e informativas sobre o tema proposto foram relevantes, uma vez que os alunos relataram que aprenderam com as atividades desenvolvidas e estavam dispostos a mudar seu comportamento em relação às práticas de higiene. Conclusão: O estudo também demonstra que tais práticas contribuem para a prevenção de doenças por meio de medidas simples, como a melhoria da higiene pessoal, essencial para a saúde pública, uma vez que muitas doenças graves podem ter reduzido o índice de contaminação apenas com orientações educativas. e práticas de higiene corretas.

Objective: to guide students of elementary and high-school levels at CED07-Ceilândia/DF on the importance of hygiene practices in favor of preventing against infectious diseases. Method: the study was designed in three distinct phases: application of questionnaires about personal hygiene; lectures and practical workshops on human pathologies; and evaluation of the project by participating students. Results: The results show that 57% of the students share personal items, a considerably high number since the literature points out that there are several pathologies that can be acquired using individual objects. It was also noted that students are not in the habit of removing their shoes before entering their homes. They claimed that they were unaware of the risks of contamination through this behavior, but stated that, after the information provided by the project, they would be more attentive to this home contamination factor. Thus, it is believed that the educational and informational practices on the proposed theme were relevant, as students reported that they learned from the developed activities and were willing to change their behavior regarding hygiene practices. Conclusion: The study also demonstrates that such practices contribute to disease prevention through simple measures, such as better personal hygiene, which is essential for public health, since many serious diseases can have reduced contamination rate only with educational guidelines and correct hygiene practices.

Objetivo: orientar a los estudiantes de primaria y secundaria del CED07-Ceilândia / DF sobre la importancia de las prácticas de higiene a favor de la prevención de enfermedades infecciosas. Método: el estudio se diseñó en tres fases diferenciadas: aplicación de cuestionarios de higiene personal; conferencias y talleres prácticos sobre patologías humanas; y evaluación del proyecto por parte de los estudiantes participantes. Resultados: Los resultados muestran que el 57% de los estudiantes comparten objetos personales, un número muy alto, ya que la literatura señala que existen varias patologías que se pueden adquirir a partir de objetos individuales. También se observó que los estudiantes no tienen la costumbre de quitarse los zapatos antes de ingresar a sus hogares. Afirmaron desconocer los riesgos de contaminación por este comportamiento, pero manifestaron que, luego de la información brindada por el proyecto, estarían más atentos a este factor de contaminación domiciliaria. Así, se cree que las prácticas educativas e informativas sobre el tema propuesto fueron relevantes, ya que los estudiantes informaron que aprendieron de las actividades desarrolladas y estaban dispuestos a cambiar su comportamiento en relación a las prácticas de higiene. Conclusión: El estudio también demuestra que dichas prácticas contribuyen a la prevención de enfermedades a través de medidas simples, como la mejora de la higiene personal, fundamental para la salud pública, ya que muchas enfermedades graves pueden haber reducido la tasa de contaminación solo con pautas educativas. y prácticas de higiene correctas.

Inflammation of peripheral tissues and injury to peripheral nerves induce differing effects in the expression of the calcium-sensitive N-arachydonoylethanolamine-synthesizing enzyme and related molecules in rat primary sensory neurons.

Elevation of intracellular Ca2+ concentration induces the synthesis of N-arachydonoylethanolamine (anandamide) in a subpopulation of primary sensory neurons. N-acylphosphatidylethanolamine phospholipase D (NAPE-PLD) is the only known enzyme that synthesizes anandamide in a Ca2+ -dependent manner. NAPE-PLD mRNA as well as anandamide's main targets, the excitatory transient receptor potential vanilloid type 1 ion channel (TRPV1), the inhibitory cannabinoid type 1 (CB1) receptor, and the main anandamide-hydrolyzing enzyme fatty acid amide hydrolase (FAAH), are all expressed by subpopulations of nociceptive primary sensory neurons. Thus, NAPE-PLD, TRPV1, the CB1 receptor, and FAAH could form an autocrine signaling system that could shape the activity of a major subpopulation of nociceptive primary sensory neurons, contributing to the development of pain. Although the expression patterns of TRPV1, the CB1 receptor, and FAAH have been comprehensively elucidated, little is known about NAPE-PLD expression in primary sensory neurons under physiological and pathological conditions. This study shows that NAPE-PLD is expressed by about one-third of primary sensory neurons, the overwhelming majority of which also express nociceptive markers as well as the CB1 receptor, TRPV1, and FAAH. Inflammation of peripheral tissues and injury to peripheral nerves induce differing but concerted changes in the expression pattern of NAPE-PLD, the CB1 receptor, TRPV1, and FAAH. Together these data indicate the existence of the anatomical basis for an autocrine signaling system in a major proportion of nociceptive primary sensory neurons and that alterations in that autocrine signaling by peripheral pathologies could contribute to the development of both inflammatory and neuropathic pain.

Expression of cleaved SNAP-25 after bladder wall injection of onabotulinumtoxina or abobotulinumtoxina: A comparative study in the mice.

AIMS: To study the expression of cleaved synaptosomal associated protein of 25 kDa (cSNAP-25) in the bladder wall injected with onabotulinumtoxinA (onabotA) or abobotulinumtoxinA (abobotA) and compare the relative potency of these two brands. METHODS: One injection of 0.5 U of onabotA or abobotA diluted in 2 µl of saline was carried out in the bladder dome of adult female mice, whose bladders were exposed by laparotomy. Three days later bladders were collected, divided in five segments (dome, upper, middle and lower body, and trigone) and each one was sectioned and immunoreacted against cSNAP-25, the end product of botulinum toxin type A (BoNT/A) activity. From each of the five segments one section was taken at random and the number of cSNAP-25 immunoreactive (IR) fibers was determined. RESULTS: Each injection resulted in the cleavage of SNAP-25 in all bladder sections, including those of the more distant segment from the injection point. The average number of cSNAP-25 positive fibers was higher in the onabotA, 341 ± 301, than in the abobotA-treated mice, 208 ± 152 (P = 0.003). The number of cSNAP-25 IR fibers varied three to five-fold between animals of each experimental group. CONCLUSIONS: These findings confirm that, when injected in the bladder wall, in the same unit amount and same volume, onabotA is 1.6 times more potent to cleave SNAP-25 than abobotA. The conversion ratio suggested by these experiments is 1:1.6 between onabotA and abobotA. Each injection, although preformed in the same way, may induce substantially different amounts of cSNAP-25. Neurourol. Urodynam. 36:86-90, 2017. © 2015 Wiley Periodicals, Inc.

Spared nerve injury model to study orofacial pain.

BACKGROUND & OBJECTIVES: There are many difficulties in generating and testing orofacial pain in animal models. Thus, only a few and limited models that mimic the human condition are available. The aim of the present research was to develop a new model of trigeminal pain by using a spared nerve injury (SNI) surgical approach in the rat face (SNI-face). METHODS: Under anaesthesia, a small incision was made in the infraorbital region of adult male Wistar rats. Three of the main infraorbital nerve branches were tightly ligated and a 2 mm segment distal to the ligation was resected. Control rats were sham-operated by exposing the nerves. Chemical hyperalgesia was evaluated 15 days after the surgery by analyzing the time spent in face grooming activity and the number of head withdrawals in response to the orofacial formalin test. RESULTS: SNI-face rats presented a significant increase of the formalin-induced pain-related behaviours evaluated both in the acute and tonic phases (expected biphasic pattern), in comparison to sham controls. INTERPRETATION & CONCLUSIONS: The SNI-face model in the rat appears to be a valid approach to evaluate experimental trigeminal pain. Ongoing studies will test the usefulness of this model to evaluate therapeutic strategies for the treatment of orofacial pain.

New insights from immunohistochemistry for the characterization of epidural scar tissue.

BACKGROUND: The association between epidural fibrosis and recurrent symptoms after lumbar spine surgery remains a matter of debate in scientific literature and the underlying pathophysiological mechanism has not been clearly elucidated. OBJECTIVE: To investigate the presence of nerve fibers and the expression of osteopontin in epidural fibrous tissue after lumbar surgery in humans. STUDY DESIGN: Laboratory study of human tissue samples. METHODS: Twenty-four patients with persistent or recurrent low back and/or leg pain after lumbar spine surgery, in whom no relevant findings were present on magnetic resonance imaging (MRI) besides epidural scar tissue, were submitted to epiduroscopy. Biopsy samples of epidural scar tissue resting in the posterior epidural and periradicular space were obtained from 15 patients, using an endoscopic grasping forceps, in locations where the stimulation with the tip of a Fogarty consistently reproduced pain. Biopsy samples were processed for examination under optical and transmission electron microscopes and under a fluorescence microscope after incubation in primary antibodies against beta3-tubulin or against osteopontin. RESULTS: Optical and transmission electron microscopy revealed a homogeneous fibrous tissue rich in collagen and lacking nerve fibers. No immunofluorescence was present in any of the samples immunoreacted against beta3-tubulin. In the samples immunoreacted against osteopontin, a punctate signal was detected around the collagen fibers. LIMITATIONS: Being a human study, there was no control group, so it is not possible to determine the contribution of osteopontin in the formation of epidural fibrosis and its relation to the patients' symptoms. Additional animal studies are needed to investigate these issues. CONCLUSION: Rather than direct stimulation of nociceptors in the epidural scar tissue, other factors should relate epidural fibrosis and recurrent symptoms after lumbar spine surgery. Osteopontin seems to play a role in the formation of epidural fibrosis.

Prolonged exposure to bradykinin and prostaglandin E2 increases TRPV1 mRNA but does not alter TRPV1 and TRPV1b protein expression in cultured rat primary sensory neurons.

Sensitisation of the capsaicin receptor, transient receptor potential vanilloid type 1 (TRPV1) ion channel in nociceptive primary sensory neurons (PSN) underlies the development of inflammatory heat hyperalgesia. Removal of the negative-dominant splice variant of the TRPV1 molecule, TRPV1b from TRPV1/TRPV1b heterotetrameric channels, which should be associated with changes in the expression of TRPV1 and TRPV1b transcripts and proteins, has been suggested to contribute to that sensitisation. Respective reverse-transcriptase polymerase chain reaction (RT-PCR) and Western-blotting revealed that both TRPV1 and TRPV1b mRNA, and their encoded proteins are expressed in rat cultured PSN. Sequencing of the RT-PCR products showed that TRPV1b mRNA lacks the entire exon 7. Further, growing PSN for 2 days in the presence of 10µM bradykinin (BK) and 10µM prostaglandin E2 (PGE2) significantly increases TRPV1 responsiveness and TRPV1 mRNA expression, without producing any changes in TRPV1b mRNA, and TRPV1 and TRPV1b protein expression. These data challenge the hypothesis that alterations in the composition of the TRPV1 ion channel contributes to the sensitisation.

Characterisation of cannabinoid 1 receptor expression in the perikarya, and peripheral and spinal processes of primary sensory neurons.

The cannabinoid 1 (CB1) receptor is expressed by a sub-population of primary sensory neurons. However, data on the neurochemical identity of the CB1 receptor-expressing cells, and CB1 receptor expression by the peripheral and central terminals of these neurons are inconsistent and limited. We characterised CB1 receptor expression in dorsal root ganglia (DRG) and spinal cord at the lumbar 4-5 level, as well as in the urinary bladder and glabrous skin of the hindpaw. About 1/3 of DRG neurons exhibited immunopositivity for the CB1 receptor, the majority of which showed positivity for the nociceptive markers calcitonin gene-related peptide (CGRP) or/and Griffonia (bandeiraea) simplicifolia IB4 isolectin-binding. Virtually all CB1 receptor-immunostained fibres showed immunopositivity for CGRP in the skin, while very few did in the urinary bladder. No CB1 receptor-immunopositive nerve fibres were IB4 positive in either peripheral tissue. Spinal laminae I and II-outer showed the highest density of CB1 receptor-immunopositive punctae, the majority of which showed positivity for CGRP or/and IB4 binding. These data indicate that a major sub-population of nociceptive primary sensory neurons expresses CB1 receptors that are transported to both peripheral and central terminals of these cells. Therefore, the present data suggest that manipulation of endogenous CB1 receptor agonist levels in these areas may significantly reduce nociceptive input into the spinal cord.

Transient receptor potential vanilloid 1 mediates nerve growth factor-induced bladder hyperactivity and noxious input.

OBJECTIVES: To explore the role of transient receptor potential vanilloid 1 (TRPV1) in the excitatory effects of chronic administration of nerve growth factor (NGF) on bladder-generated sensory input and reflex activity. To explore new therapeutic targets for bladder dysfunction. MATERIALS AND METHODS: Wild-type (WT) and TRPV1 knockout (KO) mice received daily intraperitoneal injections of NGF (1 µg/10 g) or saline for a period of 4 days, during which time thermal sensitivity was evaluated daily. On the 5th day, mice were anaesthetized and cystometries were performed. The frequency, amplitude and area under the curve (AUC) of bladder reflex contractions were determined. c-Fos expression was evaluated on L6 spinal cord sections of WT and TRPV1 KO mice treated with saline or chronic NGF by immunohistochemistry. TrkA receptor staining intensity was determined in L6 spinal cord sections and respective dorsal root ganglia of WT and TRPV1 KO mice. RESULTS: Repeated administration of NGF induced thermal hypersensitivity in WT but not in TRPV1 KO mice. The frequency of bladder contractions of saline-treated WT and TRPV1 KO mice was similar, the values respectively being 0.45 ± 0.12/min and 0.46 ± 0.16/min. Treatment with NGF enhanced bladder reflex activity in WT mice to 1.23 ± 0.41/min (P < 0.05). In NGF-treated KO mice, the frequency of bladder contractions was 0.60 ± 0.05/min. Irrespective of treatment, no differences were observed in the amplitude of bladder contractions of WT and TRPV1 KO mice. The AUC was significantly increased in NGF-treated WT-mice, when compared with saline-treated WT-mice. No changes were found in AUC of saline-treated and NGF-treated TRPV1 KO mice. Chronic administration of NGF resulted in a significant increase of spinal c-Fos expression in WT mice (P < 0.05 vs KO animals), but not in TRPV1 KO animals. TrkA expression was similar in WT and TRPV1 KO mice. CONCLUSIONS: NGF-induced bladder overactivity and noxious input depend on the interaction of NGF with TRPV1. The lack of bladder overactivity in TRPV1 KO mice treated with NGF does not represent loss of TrkA expression. TRPV1 is essential for NGF-driven bladder dysfunction and represents a bottleneck target in bladder pathologies associated with NGF up-regulation.

Spread of onabotulinumtoxinA after bladder injection. Experimental study using the distribution of cleaved SNAP-25 as the marker of the toxin action.

BACKGROUND: OnabotulinumtoxinA (Onabot/A) has been used to treat detrusor overactivity disorders. The treatment is based on several injections of toxin throughout the bladder wall. However, injection protocols are not well established among clinicians, varying in dose and dilution. OBJECTIVE: Study the distribution and neurochemistry of cleaved synaptosome-associated protein of 25 kDa (cSNAP-25) after Onabot/A administration in the guinea pig bladder. In addition, we analyzed which factor, dose or volume, contributes more to the diffusion of the toxin. DESIGN, SETTING, AND PARTICIPANTS: Guinea pig bladders were treated with Onabot/A via intramural injection or an instillation. MEASUREMENTS: Bladder cryostat sections were processed for single or dual immunohistochemistry staining with antibodies against cSNAP-25, vesicular acetylcholine transporter, tyrosine hydroxylase, and calcitonin gene-related peptide. Different administration methods and doses were analyzed. Statistical analysis was performed using the chi-square test for colocalization studies after multiple injections and the t test for the evaluation of affected fibers after a single injection. RESULTS AND LIMITATIONS: cSNAP-25 immunoreactive fibers were abundant throughout the bladder tissue in the mucosa and muscular layer. Double labeling showed that parasympathetic fibers are more affected than sympathetic or sensory. A single Onabot/A injection is more effective if diluted in a higher volume. Onabot/A instillation in the bladder does not cleave SNAP-25 protein. CONCLUSIONS: A single Onabot/A injection spreads the neurotoxin activity to the opposite side of the guinea pig bladder. This action is more evident when high saline volumes are used to dissolve Onabot/A. The toxin cleaves the SNAP-25 protein mainly in cholinergic but also in adrenergic and sensory fibers. In contrast with intramural injection, instillation of Onabot/A does not cleave SNAP-25 in nerve fibers.

Effect of onabotulinumtoxinA on intramural parasympathetic ganglia: an experimental study in the guinea pig bladder.

PURPOSE: We investigated whether onabotulinumtoxinA injected in the bladder would affect preganglionic parasympathetic nerve endings in intramural ganglia. MATERIALS AND METHODS: Guinea pig bladders were injected with 5 U of botulinum toxin. At 24 hours bladders were collected and processed for immunohistochemistry using tyrosine hydroxylase, and intact and cleaved SNAP-25. To identify the different populations of affected fibers coursing the ganglia we performed double immunoreactions for cleaved SNAP-25 and VAChT, TH or CGRP. RESULTS: VAChT immunoreactive fibers were identified in axons and varicosities of presynaptic to postganglionic parasympathetic neurons. Those fibers were also immunoreactive to SV2 and SNAP-25. The rare CGRP and TH immunoreactive fibers coursing in the ganglia did not express SV2 or SNAP-25. After onabotulinumtoxinA injection the cleaved form of SNAP-25 was abundantly expressed in parasympathetic fibers. CONCLUSIONS: Botulinum toxin injection in the bladder wall affects preganglionic parasympathetic nerve terminals. This could contribute to the strong effect of botulinum toxin on bladder smooth muscle activity.

Expression of apoptosis-regulating genes in the rat prostate following botulinum toxin type A injection.

BACKGROUND: Onabotulinumtoxin A (OnabotA) injection has been investigated as a novel treatment for benign prostatic enlargement caused by benign prostatic hyperplasia. An OnabotA-induced volume reduction caused by sympathetic fibers impairment has been proposed as a potential mechanism of action. Our aim was to investigate the expression of apoptosis-regulating proteins in the rat prostate following OnabotA intraprostatic injection. METHODS: Adult Wistar rats were injected in the ventral lobes of the prostate with 10 U of OnabotA or saline. A set of OnabotA-injected animals was further treated with 0.5 mg/kg of phenylephrine (PHE) subcutaneously daily. All animals were sacrificed after 1 week and had their prostates harvested. Immunohistochemical staining was performed for Bax, Bcl-xL and caspase-3 proteins and visualized by the avidin-biotin method. The optical density of the glandular cells was also determined, with measurement of differences between average optical densities for each group. RESULTS: Saline-treated animals showed intense epithelial staining for Bcl-xL and a faint labelling for both Bax and Caspase-3. OnabotA-treated rats showed a reduced epithelial staining of Bcl-xL and a consistently increased Bax and Caspase-3 staining when compared with saline-treated animals. PHE-treated animals showed a stronger Bcl-xL staining and reduced staining of both Bax and Caspase-3 when compared to the OnabotA group. Mean signal intensity measurements for each immunoreaction confirmed a significant decrease of the signal intensity for Bcl-xL and a significant increase of the signal intensity for Bax and Caspase 3 in OnabotA-injected animals when compared with the control group. In OnabotA+PHE treated animals mean signal intensity for Bcl-xL, Bax and Caspase 3 immunoreactions was identical to that of the control animals. CONCLUSIONS: These results support the hypothesis that OnabotA activates apoptotic pathways in the rat prostate through a mechanism that involves sympathetic outflow impairment.

Rat detrusor overactivity induced by chronic spinalization can be abolished by a transient receptor potential vanilloid 1 (TRPV1) antagonist.

PURPOSE: To evaluate the effect of a transient receptor potential vanilloid 1 (TRPV1) antagonist GRC 6211 on neurogenic detrusor overactivity (NDO) of spinal origin. MATERIALS AND METHODS: Cystometries under urethane anaesthesia were obtained in 14 chronic spinalized rats to confirm NDO. Two groups were created. In the first one (n=10), GRC 6211 (0.01, 0.1 and 1mg/kg weight) was administered via the duodenum in cumulative doses and cystometries performed 150 min after the administration of each dose of the drug. In the second group (n=4), used as control, the animals were submitted to cystometries during 12 hours, without administration of GRC 6211. Frequency and amplitude of bladder contractions were recorded in both groups. RESULTS: The mean (±SDev) bladder detrusor muscle contraction frequency of spinalized rats was 0.7±0.27 contractions/min. GRC 6211 produced a significant dose-dependent effect, with the frequency diminished to 0.53±0.23, 0.40±0.20 and 0.20±0.13 contractions/min, respectively. The mean (± SDev) amplitude of bladder contractions was 48.4±4.4 cmH(2)O. After administration of 0.01 mg/kg, 0.1mg/kg and 1mg/kg of GRC 6211, the amplitude decreased to 47.1±4.3, 45.6±5.6 and 40.2±4.1 cm H(2)O respectively. The effect was significant at 0.1 and 1mg/kg doses. Cystometries performed in the control group of spinalized rats showed no evidence of detrusor fatigue caused by the urethane anaesthesia and long duration of the experiment. CONCLUSION: TRPV1 antagonists may be very effective in reducing NDO of spinal origin. This finding may have profound implications for the pathogenesis and future treatment options of patients with spinal NDO.

VEGF signaling mediates bladder neuroplasticity and inflammation in response to BCG.

BACKGROUND: This work tests the hypothesis that increased levels of vascular endothelial growth factor (VEGF) observed during bladder inflammation modulates nerve plasticity. METHODS: Chronic inflammation was induced by intravesical instillations of Bacillus Calmette-Guérin (BCG) into the urinary bladder and the density of nerves expressing the transient receptor potential vanilloid subfamily 1 (TRPV1) or pan-neuronal marker PGP9.5 was used to quantify alterations in peripheral nerve plasticity. Some mice were treated with B20, a VEGF neutralizing antibody to reduce the participation of VEGF. Additional mice were treated systemically with antibodies engineered to specifically block the binding of VEGF to NRP1 (anti-NRP1B) and NRP2 (NRP2B), or the binding of semaphorins to NRP1 (anti-NRP1 A) to diminish activity of axon guidance molecules such as neuropilins (NRPs) and semaphorins (SEMAs). To confirm that VEGF is capable of inducing inflammation and neuronal plasticity, another group of mice was instilled with recombinant VEGF165 or VEGF121 into the urinary bladder. RESULTS: The major finding of this work was that chronic BCG instillation resulted in inflammation and an overwhelming increase in both PGP9.5 and TRPV1 immunoreactivity, primarily in the sub-urothelium of the urinary bladder. Treatment of mice with anti-VEGF neutralizing antibody (B20) abolished the effect of BCG on inflammation and nerve density.NRP1A and NRP1B antibodies, known to reduce BCG-induced inflammation, failed to block BCG-induced increase in nerve fibers. However, the NRP2B antibody dramatically potentiated the effects of BCG in increasing PGP9.5-, TRPV1-, substance P (SP)-, and calcitonin gene-related peptide (CGRP)-immunoreactivity (IR). Finally, instillation of VEGF121 or VEGF165 into the mouse bladder recapitulated the effects of BCG and resulted in a significant inflammation and increase in nerve density. CONCLUSIONS: For the first time, evidence is being presented supporting that chronic BCG instillation into the mouse bladder promotes a significant increase in peripheral nerve density that was mimicked by VEGF instillation. Effects of BCG were abolished by pre-treatment with neutralizing VEGF antibody. The present results implicate the VEGF pathway as a key modulator of inflammation and nerve plasticity, introduces a new animal model for investigation of VEGF-induced nerve plasticity, and suggests putative mechanisms underlying this phenomenon.

Severe burn injury induces a characteristic activation of extracellular signal-regulated kinase 1/2 in spinal dorsal horn neurons.

We have studied scalding-type burn injury-induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2) in the spinal dorsal horn, which is a recognised marker for spinal nociceptive processing. At 5min after severe scalding injury to mouse hind-paw, a substantial number of phosphorylated ERK1/2 (pERK1/2) immunopositive neurons were found in the ipsilateral dorsal horn. At 1h post-injury, the number of pERK1/2-labelled neurons remained substantially the same. However, at 3h post-injury, a further increase in the number of labelled neurons was found on the ipsilateral side, while a remarkable increase in the number of labelled neurons on the contralateral side resulted in there being no significant difference between the extent of the labelling on both sides. By 6h post-injury, the number of labelled neurons was reduced on both sides without there being significant difference between the two sides. A similar pattern of severe scalding injury-induced activation of ERK1/2 in spinal dorsal horn neurons over the same time-course was found in mice which lacked the transient receptor potential type 1 receptor (TRPV1) except that the extent to which ERK1/2 was activated in the ipsilateral dorsal horn at 5 min post-injury was significantly greater in wild-type animals when compared to TRPV1 null animals. This difference in activation of ERK1/2 in spinal dorsal horn neurons was abolished within 1h after injury, demonstrating that TRPV1 is not essential for the maintenance of ongoing spinal nociceptive processing in inflammatory pain conditions in mouse resulting from at least certain types of severe burn injury.

Modulation of urinary bladder innervation: TRPV1 and botulinum toxin A.

The persisting interest around neurotoxins such as vanilloids and botulinum toxin (BoNT) derives from their marked effect on detrusor overactivity refractory to conventional antimuscarinic treatments. In addition, both are administered by intravesical route. This offers three potential advantages. First, intravesical therapy is an easy way to provide high concentrations of pharmacological agents in the bladder tissue without causing unsuitable levels in other organs. Second, drugs effective on the bladder, but inappropriate for systemic administration, can be safely used as it is the case of vanilloids and BoNT. Third, the effects of one single treatment might be extremely longlasting, contributing to render these therapies highly attractive to patients despite the fact that the reasons to the prolonged effect are still incompletely understood. Attractive as it may be, intravesical pharmacological therapy should still be considered as a second-line treatment in patients refractory to conventional oral antimuscarinic therapy or who do not tolerate its systemic side effects. However, the increasing off-label use of these neurotoxins justifies a reappraisal of their pharmacological properties.

Distribution of the high-affinity binding site and intracellular target of botulinum toxin type A in the human bladder.

BACKGROUND: Botulinum toxin type A (BoNTA) has been successfully used in the treatment of refractory detrusor overactivity. The toxin is internalized after binding a high-affinity receptor, synaptic vesicle protein 2 (SV2), which is exposed in the cell membrane during the exocytosis process. In the cytoplasm, BoNTA cleaves specific sites of synaptosomal-associated protein 25 (SNAP-25), preventing the assembly of the synaptic fusion complex SNARE and blocking exocytosis. OBJECTIVE: In the present work, the distribution of SV2 and SNAP-25 was first investigated in human bladders. The neurochemistry of BoNTA-sensitive structures was then investigated using markers for parasympathetic, sympathetic, and sensory fibers. DESIGN, SETTING, AND PARTICIPANTS: Human bladders were obtained from cadaveric organ donors (age range: 19-74 yr). MEASUREMENTS: Bladder sections were processed for single or dual immunofluorescence staining with antibodies against SV2, SNAP-25, ß-3 tubulin, vesicular acetylcholine transporter, tyrosine hydroxilase, and calcitonin gene-related peptide. RESULTS AND LIMITATIONS: SV2 and SNAP-25 immunoreactive fibers were distributed throughout the suburothelium and muscular layer. Double labeling showed extensive colocalization of both proteins in nerve fibers. SV2 is more expressed in parasympathetic fibers than in sympathetic or sensory fibers. No expression was found in urothelial or muscular cells. Because only normal bladders were used, this distribution should be applied with caution to pathologic bladders. CONCLUSIONS: SV2 and SNAP-25 colocalize abundantly throughout the urinary bladder. SV2 is more abundant in cholinergic, parasympathetic fibers. These nerves are suggested to be the main target for BoNTA action in the human urinary bladder.

Functional transient receptor potential vanilloid 1 is expressed in human urothelial cells.

PURPOSE: We investigated the expression and functional status of TRPV1 receptor in human urothelial cells. MATERIAL AND METHODS: Human urothelium was cultured and TRPV1 receptor expression was studied by immunocytochemistry and reverse transcriptase-polymerase chain reaction. The influence of inflammatory mediators on TRPV1 mRNA levels was also studied. Functional assays (cobalt uptake measurements and whole cell voltage clamp records) were used to study the response of urothelial cells to capsaicin, temperature, low pH and inflammatory mediators. Capsaicin induced adenosine triphosphate release from urothelial cells was assessed by bioluminescence. RESULTS: TRPV1 protein and mRNA were detected in human urothelial cells and mRNA more than tripled in the presence of inflammatory mediators. Nerve growth factor treatment alone did not affect TRPV1 mRNA expression. Capsaicin (100 nM and 1 microM) and heat (41C and 45C) evoked cobalt uptake and inflammatory mediators lowered the temperature threshold for TRPV1 activation to 37C. Capsaicin (1 microM) induced TRPV1 desensitization to further applications of the agonist. In whole cell patch clamp experiments 1 microM capsaicin and a heat ramp from 37C to 50C caused inward currents. The same concentration of capsaicin induced the release of about 7 fmol adenosine triphosphate per mg. CONCLUSIONS: TRPV1 receptors expressed by human urothelial cells respond to capsaicin and thermal stimuli. Capsaicin evoked release of adenosine triphosphate suggests that human urothelial TRPV1 is involved in the afferent branch of the micturition reflex. Inflammatory mediators decrease the TRPV1 thermal threshold of activation to body temperature and increase its expression. This finding may be relevant for symptoms associated with cystitis.