TRP Channel Cooperation for Nociception: Therapeutic Opportunities.

Chronic pain treatment remains a sore challenge, and in our aging society, the number of patients reporting inadequate pain relief continues to grow. Current treatment options all have their drawbacks, including limited efficacy and the propensity of abuse and addiction; the latter is exemplified by the ongoing opioid crisis. Extensive research in the last few decades has focused on mechanisms underlying chronic pain states, thereby producing attractive opportunities for novel, effective and safe pharmaceutical interventions. Members of the transient receptor potential (TRP) ion channel family represent innovative targets to tackle pain sensation at the root. Three TRP channels, TRPV1, TRPM3, and TRPA1, are of particular interest, as they were identified as sensors of chemical- and heat-induced pain in nociceptor neurons. This review summarizes the knowledge regarding TRP channel-based pain therapies, including the bumpy road of the clinical development of TRPV1 antagonists, the current status of TRPA1 antagonists, and the future potential of targeting TRPM3.

A TRP channel trio mediates acute noxious heat sensing.

Acute pain represents a crucial alarm signal to protect us from injury. Whereas the nociceptive neurons that convey pain signals were described more than a century ago, the molecular sensors that detect noxious thermal or mechanical insults have yet to be fully identified. Here we show that acute noxious heat sensing in mice depends on a triad of transient receptor potential (TRP) ion channels: TRPM3, TRPV1, and TRPA1. We found that robust somatosensory heat responsiveness at the cellular and behavioural levels is observed only if at least one of these TRP channels is functional. However, combined genetic or pharmacological elimination of all three channels largely and selectively prevents heat responses in both isolated sensory neurons and rapidly firing C and Aδ sensory nerve fibres that innervate the skin. Strikingly, Trpv1-/-Trpm3-/-Trpa1-/- triple knockout (TKO) mice lack the acute withdrawal response to noxious heat that is necessary to avoid burn injury, while showing normal nociceptive responses to cold or mechanical stimuli and a preserved preference for moderate temperatures. These findings indicate that the initiation of the acute heat-evoked pain response in sensory nerve endings relies on three functionally redundant TRP channels, representing a fault-tolerant mechanism to avoid burn injury.

Publisher Correction: A TRP channel trio mediates acute noxious heat sensing.

In this Letter, the trace is missing in Fig. 1e. This error has been corrected online.

Protease secretions by the invading blastocyst induce calcium oscillations in endometrial epithelial cells via the protease-activated receptor 2.

BACKGROUND: Early embryo implantation is a complex phenomenon characterized by the presence of an implantation-competent blastocyst and a receptive endometrium. Embryo development and endometrial receptivity must be synchronized and an adequate two-way dialogue between them is necessary for maternal recognition and implantation. Proteases have been described as blastocyst-secreted proteins involved in the hatching process and early implantation events. These enzymes stimulate intracellular calcium signaling pathways in endometrial epithelial cells (EEC). However, the exact molecular players underlying protease-induced calcium signaling, the subsequent downstream signaling pathways and the biological impact of its activation remain elusive. METHODS: To identify gene expression of the receptors and ion channels of interest in human and mouse endometrial epithelial cells, RNA sequencing, RT-qPCR and in situ hybridization experiments were conducted. Calcium microfluorimetric experiments were performed to study their functional expression. RESULTS: We showed that trypsin evoked intracellular calcium oscillations in EEC of mouse and human, and identified the protease-activated receptor 2 (PAR2) as the molecular entity initiating protease-induced calcium responses in EEC. In addition, this study unraveled the molecular players involved in the downstream signaling of PAR2 by showing that depletion and re-filling of intracellular calcium stores occurs via PLC, IP3R and the STIM1/Orai1 complex. Finally, in vitro experiments in the presence of a specific PAR2 agonist evoked an upregulation of the 'Window of implantation' markers in human endometrial epithelial cells. CONCLUSIONS: These findings provide new insights into the blastocyst-derived protease signaling and allocate a key role for PAR2 as maternal sensor for signals released by the developing blastocyst.

Primidone improves symptoms in TRPM3-linked developmental and epileptic encephalopathy with spike-and-wave activation in sleep.

Developmental and epileptic encephalopathy with continuous spike-and-wave activation in sleep (CSWS) or DEE-SWAS is an age-dependent disease, often accompanied by a decline in cognitive abilities. Early successful treatment of CSWS is associated with a better cognitive outcome. We retrospectively analyzed the clinical, electrophysiological, radiological, and genetic data of children with DEE-SWAS associated with melastatin-related transient receptor type 3 gene (TRPM3) missense variants. We report two unrelated children with pharmacoresistant DEE-SWAS and developmental delay/regression and different heterozygous de novo missense variants in the TRPM3 gene (NM_001366145.2; c.3397 T > C/p.Ser1133Pro, c.2004G > A/p.Val1002Met). The variant p.Val1002Met (previously known as p.Val990Met or p.Val837Met) and p.Ser1133Pro were recently shown to result in a gain-of-function effect. Based on this finding, previous drug resistance, and the experimentally demonstrated inhibitory effect of primidone on TRPM3, we initiated an individualized therapy with this drug. In both children, developmental regression was stopped, psychomotor development improved, and CSWS was no longer detectable. To our knowledge, this is the first report of a treatment with primidone in TRPM3-associated CSWS. Our results highlight the importance of early genetic diagnosis in patients with epilepsy and the possibility of precision medicine, which should be considered in the future in individuals with a TRPM3-linked DEE-SWAS.

TRPM4 inhibition by meclofenamate suppresses Ca2+-dependent triggered arrhythmias.

AIMS: Cardiac arrhythmias are a major factor in the occurrence of morbidity and sudden death in patients with cardiovascular disease. Disturbances of Ca2+ homeostasis in the heart contribute to the initiation and maintenance of cardiac arrhythmias. Extrasystolic increases in intracellular Ca2+ lead to delayed afterdepolarizations and triggered activity, which can result in heart rhythm abnormalities. It is being suggested that the Ca2+-activated nonselective cation channel TRPM4 is involved in the aetiology of triggered activity, but the exact contribution and in vivo significance are still unclear. METHODS AND RESULTS: In vitro electrophysiological and calcium imaging technique as well as in vivo intracardiac and telemetric electrocardiogram measurements in physiological and pathophysiological conditions were performed. In two distinct Ca2+-dependent proarrhythmic models, freely moving Trpm4-/- mice displayed a reduced burden of cardiac arrhythmias. Looking further into the specific contribution of TRPM4 to the cellular mechanism of arrhythmias, TRPM4 was found to contribute to a long-lasting Ca2+ overload-induced background current, thereby regulating cell excitability in Ca2+ overload conditions. To expand these results, a compound screening revealed meclofenamate as a potent antagonist of TRPM4. In line with the findings from Trpm4-/- mice, 10 µM meclofenamate inhibited the Ca2+ overload-induced background current in ventricular cardiomyocytes and 15 mg/kg meclofenamate suppressed catecholaminergic polymorphic ventricular tachycardia-associated arrhythmias in a TRPM4-dependent manner. CONCLUSION: The presented data establish that TRPM4 represents a novel target in the prevention and treatment of Ca2+-dependent triggered arrhythmias.

ß-Lactam TRPM8 Antagonists Derived from Phe-Phenylalaninol Conjugates: Structure-Activity Relationships and Antiallodynic Activity.

The protein transient receptor potential melastatin type 8 (TRPM8), a non-selective, calcium (Ca2+)-permeable ion channel is implicated in several pathological conditions, including neuropathic pain states. In our previous research endeavors, we have identified ß-lactam derivatives with high hydrophobic character that exhibit potent and selective TRPM8 antagonist activity. This work describes the synthesis of novel derivatives featuring C-terminal amides and diversely substituted N'-terminal monobenzyl groups in an attempt to increase the total polar surface area (TPSA) in this family of compounds. The primary goal was to assess the influence of these substituents on the inhibition of menthol-induced cellular Ca2+ entry, thereby establishing critical structure-activity relationships. While the substitution of the tert-butyl ester by isobutyl amide moieties improved the antagonist activity, none of the N'-monobencyl derivatives, regardless of the substituent on the phenyl ring, achieved the activity of the model dibenzyl compound. The antagonist potency of the most effective compounds was subsequently verified using Patch-Clamp electrophysiology experiments. Furthermore, we evaluated the selectivity of one of these compounds against other members of the transient receptor potential (TRP) ion channel family and some receptors connected to peripheral pain pathways. This compound demonstrated specificity for TRPM8 channels. To better comprehend the potential mode of interaction, we conducted docking experiments to uncover plausible binding sites on the functionally active tetrameric protein. While the four main populated poses are located by the pore zone, a similar location to that described for the N-(3-aminopropyl)-2-[(3-methylphenyl)methoxy]-N-(2-thienylmethyl)-benzamide (AMTB) antagonist cannot be discarded. Finally, in vivo experiments, involving a couple of selected compounds, revealed significant antinociceptive activity within a mice model of cold allodynia induced by oxaliplatin (OXA).

Phenotypic spectrum of the recurrent TRPM3 p.(Val837Met) substitution in seven individuals with global developmental delay and hypotonia.

TRPM3 encodes a transient receptor potential cation channel of the melastatin family, expressed in the central nervous system and in peripheral sensory neurons of the dorsal root ganglia. The recurrent substitution in TRPM3: c.2509G>A, p.(Val837Met) has been associated with syndromic intellectual disability and seizures. In this report, we present the clinical and molecular features of seven previously unreported individuals, identified by exome sequencing, with the recurrent p.(Val837Met) variant and global developmental delay. Other shared clinical features included congenital hypotonia, dysmorphic facial features (broad forehead, deep-set eyes, and down turned mouth), exotropia, and musculoskeletal issues (hip dysplasia, hip dislocation, scoliosis). Seizures were observed in two of seven individuals (febrile seizure in one and generalized tonic-clonic seizures with atonic drops in another), and epileptiform activity was observed in an additional two individuals. This report extends the number of affected individuals to 16 who are heterozygous for the de novo recurrent substitution p.(Val837Met). In contrast with the initial report, epilepsy was not a mandatory feature observed in this series. TRPM3 pathogenic variation should be considered in individuals with global developmental delays, moderate-severe intellectual disability with, or without, childhood-onset epilepsy.

Mapping the expression of transient receptor potential channels across murine placental development.

Transient receptor potential (TRP) channels play prominent roles in ion homeostasis by their ability to control cation influx. Mouse placentation is governed by the processes of trophoblast proliferation, invasion, differentiation, and fusion, all of which require calcium signaling. Although certain TRP channels have been shown to contribute to maternal-fetal transport of magnesium and calcium, a role for TRP channels in specific trophoblast functions has been disregarded. Using qRT-PCR and in situ hybridisation, the spatio-temporal expression pattern of TRP channels in the mouse placenta across gestation (E10.5-E18.5) was assessed. Prominent expression was observed for Trpv2, Trpm6, and Trpm7. Calcium microfluorimetry in primary trophoblast cells isolated at E14.5 of gestation further revealed the functional activity of TRPV2 and TRPM7. Finally, comparing TRP channels expression in mouse trophoblast stem cells (mTSCs) and mouse embryonic stem cells (mESC) confirmed the specific expression of TRPV2 during placental development. Moreover, TRP channel expression was similar in mTSCs compared to primary trophoblasts and validate mTSC as a model to study TRP channels in placental development. Collectivity, our results identify a specific spatio-temporal TRP channel expression pattern in trophoblasts, suggesting a possible involvement in regulating the process of placentation.

TRP channel expression correlates with the epithelial-mesenchymal transition and high-risk endometrial carcinoma.

Transient receptor potential (TRP) channels excel in cellular sensing as they allow rapid ion influx across the plasma membrane in response to a variety of extracellular cues. Recently, a distinct TRP mRNA expression signature was observed in stromal cells (ESC) and epithelial cells (EEC) of the endometrium, a tissue in which cell phenotypic plasticity is essential for normal functioning. However, it is unknown whether TRP channel mRNA expression is subject to the phenotypic switching that occurs during epithelial to mesenchymal transition (EMT) and mesenchymal to epithelial transition (MET), and whether TRP channel mRNA expression is associated with aggressive phenotypes in endometrial cancer (EC). Here, we induced EMT and MET in vitro using in primary EEC and ESC, respectively, and analyzed expression and functionality of TRP channels using RT-qPCR and intracellular Ca2+ imaging. The outcome of these experiments showed a strong association between TRPV2 and TRPC1 mRNA expression and the mesenchymal phenotype, whereas TRPM4 mRNA expression correlated with the epithelial phenotype. In line herewith, increased TRPV2 and TRPC1 mRNA expression levels were observed in both primary and metastatic EC biopsies and in primary EC cells with a high EMT status, indicating an association with an aggressive tumor phenotype. Remarkably, TRPV2 mRNA expression in primary EC biopsies was associated with tumor invasiveness and cancer stage. In contrast, increased TRPM4 mRNA expression was observed in EC biopsies with a low EMT status and less aggressive tumor phenotypes. Taken together, this dataset proved for the first time that TRP channel mRNA expression is strongly linked to cellular phenotypes of the endometrium, and that phenotypic transitions caused by either experimental manipulation or malignancy could alter this expression in a predictable manner. These results implicate that TRP channels are viable biomarkers to identify high-risk EC, and potential targets for EC treatment.

Partial Agonistic Actions of Sex Hormone Steroids on TRPM3 Function.

Sex hormone steroidal drugs were reported to have modulating actions on the ion channel TRPM3. Pregnenolone sulphate (PS) presents the most potent known endogenous chemical agonist of TRPM3 and affects several gating modes of the channel. These includes a synergistic action of PS and high temperatures on channel opening and the PS-induced opening of a noncanonical pore in the presence of other TRPM3 modulators. Moreover, human TRPM3 variants associated with neurodevelopmental disease exhibit an increased sensitivity for PS. However, other steroidal sex hormones were reported to influence TRPM3 functions with activating or inhibiting capacity. Here, we aimed to answer how DHEAS, estradiol, progesterone and testosterone act on the various modes of TRPM3 function in the wild-type channel and two-channel variants associated with human disease. By means of calcium imaging and whole-cell patch clamp experiments, we revealed that all four drugs are weak TRPM3 agonists that share a common steroidal interaction site. Furthermore, they exhibit increased activity on TRPM3 at physiological temperatures and in channels that carry disease-associated mutations. Finally, all steroids are able to open the noncanonical pore in wild-type and DHEAS also in mutant TRPM3. Collectively, our data provide new valuable insights in TRPM3 gating, structure-function relationships and ligand sensitivity.

The Agonist Action of Alkylphenols on TRPA1 Relates to Their Effects on Membrane Lipid Order: Implications for TRPA1-Mediated Chemosensation.

The Transient Receptor Potential Ankyrin 1 cation channel (TRPA1) is a broadly-tuned chemosensor expressed in nociceptive neurons. Multiple TRPA1 agonists are chemically unrelated non-electrophilic compounds, for which the mechanisms of channel activation remain unknown. Here, we assess the hypothesis that such chemicals activate TRPA1 by inducing mechanical perturbations in the plasma membrane. We characterized the activation of mouse TRPA1 by non-electrophilic alkylphenols (APs) of different carbon chain lengths in the para position of the aromatic ring. Having discarded oxidative stress and the action of electrophilic mediators as activation mechanisms, we determined whether APs induce mechanical perturbations in the plasma membrane using dyes whose fluorescence properties change upon alteration of the lipid environment. APs activated TRPA1, with potency increasing with their lipophilicity. APs increased the generalized polarization of Laurdan fluorescence and the anisotropy of the fluorescence of 1,6-diphenyl-1,3,5-hexatriene (DPH), also according to their lipophilicity. Thus, the potency of APs for TRPA1 activation is an increasing function of their ability to induce lipid order and membrane rigidity. These results support the hypothesis that TRPA1 senses non-electrophilic compounds by detecting the mechanical alterations they produce in the plasma membrane. This may explain how structurally unrelated non-reactive compounds induce TRPA1 activation and support the role of TRPA1 as an unspecific sensor of potentially noxious compounds.

TRPM3 Is Expressed in Afferent Bladder Neurons and Is Upregulated during Bladder Inflammation.

The cation channel TRPM3 is activated by heat and the neurosteroid pregnenolone sulfate. TRPM3 is expressed on sensory neurons innervating the skin, where together with TRPV1 and TRPA1, it functions as one of three redundant sensors of acute heat. Moreover, functional upregulation of TRPM3 during inflammation contributes to heat hyperalgesia. The role of TRPM3 in sensory neurons innervating internal organs such as the bladder is currently unclear. Here, using retrograde labeling and single-molecule fluorescent RNA in situ hybridization, we demonstrate expression of mRNA encoding TRPM3 in a large subset of dorsal root ganglion (DRG) neurons innervating the mouse bladder, and confirm TRPM3 channel functionality in these neurons using Fura-2-based calcium imaging. After induction of cystitis by injection of cyclophosphamide, we observed a robust increase of the functional responses to agonists of TRPM3, TRPV1, and TRPA1 in bladder-innervating DRG neurons. Cystometry and voided spot analysis in control and cyclophosphamide-treated animals did not reveal differences between wild type and TRPM3-deficient mice, indicating that TRPM3 is not critical for normal voiding. We conclude that TRPM3 is functionally expressed in a large proportion of sensory bladder afferent, but its role in bladder sensation remains to be established.

The Zinc-Finger Domain Containing Protein ZC4H2 Interacts with TRPV4, Enhancing Channel Activity and Turnover at the Plasma Membrane.

The Ca2+-permeable Transient Receptor Potential channel vanilloid subfamily member 4 (TRPV4) is involved in a broad range of physiological processes, including the regulation of systemic osmotic pressure, bone resorption, vascular tone, and bladder function. Mutations in the TRPV4 gene are the cause of a spectrum of inherited diseases (or TRPV4-pathies), which include skeletal dysplasias, arthropathies, and neuropathies. There is little understanding of the pathophysiological mechanisms underlying these variable disease phenotypes, but it has been hypothesized that disease-causing mutations affect interaction with regulatory proteins. Here, we performed a mammalian protein-protein interaction trap (MAPPIT) screen to identify proteins that interact with the cytosolic N terminus of human TRPV4, a region containing the majority of disease-causing mutations. We discovered the zinc-finger domain-containing protein ZC4H2 as a TRPV4-interacting protein. In heterologous expression experiments, we found that ZC4H2 increases both the basal activity of human TRPV4 as well as Ca2+ responses evoked by ligands or hypotonic cell swelling. Using total internal reflection fluorescence (TIRF) microscopy, we further showed that ZC4H2 accelerates TRPV4 turnover at the plasma membrane. Overall, these data demonstrate that ZC4H2 is a positive modulator of TRPV4, and suggest a link between TRPV4 and ZC4H2-associated rare disorders, which have several neuromuscular symptoms in common with TRPV4-pathies.

Mimicking Sampson's Retrograde Menstrual Theory in Rats: A New Rat Model for Ongoing Endometriosis-Associated Pain.

Endometriosis is a prevalent gynecologic disease, defined by dysfunctional endometrium-like lesions outside of the uterine cavity. These lesions are presumably established via retrograde menstruation, i.e., endometrial tissue that flows backwards during menses into the abdomen and deposits on the organs. As ongoing pain is one of the main pain symptoms of patients, an animal model that illuminates this problem is highly anticipated. In the present study, we developed and validated a rat model for ongoing endometriosis-associated pain. First, menstrual endometrial tissue was successfully generated in donor rats, as validated by gross examination, histology and qPCR. Next, endometriosis was induced in recipient animals by intraperitoneal injection of menstrual tissue. This resulted in neuro-angiogenesis as well as established endometriosis lesions, which were similar to their human counterparts, since epithelial and stromal cells were observed. Furthermore, significant differences were noted between control and endometriosis animals concerning bodyweight and posture changes, indicating the presence of ongoing pain in animals with endometriosis. In summary, a rat model for endometriosis was established that reliably mimics the human pathophysiology of endometriosis and in which signs of ongoing pain were detected, thus providing a new research tool for therapy development.

Store-independent coupling between the Secretory Pathway Ca2+ transport ATPase SPCA1 and Orai1 in Golgi stress and Hailey-Hailey disease.

The Secretory Pathway Ca2+ ATPases SPCA1 and SPCA2 transport Ca2+ and Mn2+ into the Golgi and Secretory Pathway. SPCA2 mediates store-independent Ca2+ entry (SICE) via STIM1-independent activation of Orai1, inducing constitutive Ca2+ influx in mammary epithelial cells during lactation. Here, we show that like SPCA2, also the overexpression of the ubiquitous SPCA1 induces cytosolic Ca2+ influx, which is abolished by Orai1 knockdown and occurs independently of STIM1. This process elevates the Ca2+ concentration in the cytosol and in the non-endoplasmic reticulum (ER) stores, pointing to a functional coupling between Orai1 and SPCA1. In agreement with this, we demonstrate via Total Internal Reflection Fluorescence microscopy that Orai1 and SPCA1a co-localize near the plasma membrane. Interestingly, SPCA1 overexpression also induces Golgi swelling, which coincides with translocation of the transcription factor TFE3 to the nucleus, a marker of Golgi stress. The induction of Golgi stress depends on a combination of SPCA1 activity and SICE, suggesting a role for the increased Ca2+ level in the non-ER stores. Finally, we tested whether impaired SPCA1a/Orai1 coupling may be implicated in the skin disorder Hailey-Hailey disease (HHD), which is caused by SPCA1 loss-of-function. We identified HHD-associated SPCA1a mutations that impair either the Ca2+ transport function, Orai1 activation, or both, while all mutations affect the Ca2+ content of the non-ER stores. Thus, the functional coupling between SPCA1 and Orai1 increases cytosolic and intraluminal Ca2+ levels, representing a novel mechanism of SICE that may be affected in HHD.

To flourish or perish: evolutionary TRiPs into the sensory biology of plant-herbivore interactions.

The interactions between plants and their herbivores are highly complex systems generating on one side an extraordinary diversity of plant protection mechanisms and on the other side sophisticated consumer feeding strategies. Herbivores have evolved complex, integrative sensory systems that allow them to distinguish between food sources having mere bad flavors from the actually toxic ones. These systems are based on the senses of taste, olfaction and somatosensation in the oral and nasal cavities, and on post-ingestive chemosensory mechanisms. The potential ability of plant defensive chemical traits to induce tissue damage in foragers is mainly encoded in the latter through chemesthetic sensations such as burning, pain, itch, irritation, tingling, and numbness, all of which induce innate aversive behavioral responses. Here, we discuss the involvement of transient receptor potential (TRP) channels in the chemosensory mechanisms that are at the core of complex and fascinating plant-herbivore ecological networks. We review how "sensory" TRPs are activated by a myriad of plant-derived compounds, leading to cation influx, membrane depolarization, and excitation of sensory nerve fibers of the oronasal cavities in mammals and bitter-sensing cells in insects. We also illustrate how TRP channel expression patterns and functionalities vary between species, leading to intriguing evolutionary adaptations to the specific habitats and life cycles of individual organisms.

Peripheral thermosensation in mammals.

Our ability to perceive temperature is crucial: it enables us to swiftly react to noxiously cold or hot objects and helps us to maintain a constant body temperature. Sensory nerve endings, upon depolarization by temperature-gated ion channels, convey electrical signals from the periphery to the CNS, eliciting a sense of temperature. In the past two decades, we have witnessed important advances in our understanding of mammalian thermosensation, with the identification and animal-model assessment of candidate molecular thermosensors - such as types of transient receptor potential (TRP) cation channels - involved in peripheral thermosensation. Ongoing research aims to understand how these miniature thermometers operate at the cellular and molecular level, and how they can be pharmacologically targeted to treat pain without disturbing vital thermoregulatory processes.

Functional and molecular characterisation of the bilateral pelvic nerve crush injury rat model for neurogenic detrusor underactivity.

OBJECTIVES: To create a rat model for neurogenic detrusor underactivity (DU) by bilateral pelvic nerve crush injury (BPNI) and to study temporal changes in detrusor contractility and morphology. MATERIALS AND METHODS: Male Sprague-Dawley rats were subjected to BPNI or sham surgery and evaluated at 1, 3 and 9 weeks after surgery. Bladder function was determined in vivo by awake cystometry, micturition pattern analysis, and 24-h urine collection. Bladders were harvested for in vitro pharmacological investigation by isometric tension recording. Bladders and major pelvic ganglia were investigated by quantitative reverse transcription-polymerase chain reaction and histochemistry. RESULTS: Overflow incontinence was observed at 1 week after BPNI. At 3 and 9 weeks after BPNI, rats showed a bladder phenotype characteristic for DU with increased post-void residual urine volumes, reduced voiding efficiencies, and lower maximum pressures. In isolated bladder strips, contractile responses to KCl, carbachol, and α,ß-methylene adenosine 5'-triphosphate (α,ß-mATP) were preserved. On the other hand, neural-induced contractility was reduced after BPNI, in line with reduced expression of protein gene product 9.5 and choline acetyltransferase in the major pelvic ganglion at 1 week after BPNI. The bladder-to-body weight ratio and detrusor thickness increased after BPNI, indicating detrusor hypertrophy to compensate for the reduced neural input. CONCLUSIONS: BPNI induces a rat model for neurogenic DU. In this model, the detrusor maintains its contractility but denervation of the detrusor was observed.