Acute ampakines increase voiding function and coordination in a rat model of SCI.

Neurogenic bladder dysfunction causes urological complications and reduces the quality of life in persons with spinal cord injury (SCI). Glutamatergic signaling via AMPA receptors is fundamentally important to the neural circuits controlling bladder voiding. Ampakines are positive allosteric modulators of AMPA receptors that can enhance the function of glutamatergic neural circuits after SCI. We hypothesized that ampakines can acutely stimulate bladder voiding that has been impaired due to thoracic contusion SCI. Adult female Sprague-Dawley rats received a unilateral contusion of the T9 spinal cord (n = 10). Bladder function (cystometry) and coordination with the external urethral sphincter (EUS) were assessed 5 d post-SCI under urethane anesthesia. Data were compared to responses in spinal-intact rats (n = 8). The 'low-impact' ampakine CX1739 (5, 10, or 15 mg/kg) or vehicle (2-hydroxypropyl-beta-cyclodextrin [HPCD]) was administered intravenously. The HPCD vehicle had no discernible impact on voiding. In contrast, following CX1739, the pressure threshold for inducing bladder contraction, voided volume, and the interval between bladder contractions were significantly reduced. These responses occurred in a dose-dependent manner. We conclude that modulating AMPA receptor function using ampakines can rapidly improve bladder-voiding capability at subacute time points following contusion SCI. These results may provide a new and translatable method for therapeutic targeting of bladder dysfunction acutely after SCI.

Hypertension and urologic chronic pelvic pain syndrome: An analysis of MAPP-I data.

BACKGROUND: Urologic chronic pelvic pain syndrome (UCPPS), which includes interstitial cystitis/bladder pain syndrome (IC/BPS) and chronic prostatitis (CP/CPPS), is associated with increased voiding frequency, nocturia, and chronic pelvic pain. The cause of these diseases is unknown and likely involves many different mechanisms. Dysregulated renin-angiotensin-aldosterone-system (RAAS) signaling is a potential pathologic mechanism for IC/BPS and CP/CPPS. Many angiotensin receptor downstream signaling factors, including oxidative stress, fibrosis, mast cell recruitment, and increased inflammatory mediators, are present in the bladders of IC/BPS patients and prostates of CP/CPPS patients. Therefore, we aimed to test the hypothesis that UCPPS patients have dysregulated angiotensin signaling, resulting in increased hypertension compared to controls. Secondly, we evaluated symptom severity in patients with and without hypertension and antihypertensive medication use. METHODS: Data from UCPPS patients (n = 424), fibromyalgia or irritable bowel syndrome (positive controls, n = 200), and healthy controls (n = 415) were obtained from the NIDDK Multidisciplinary Approach to the Study of Chronic Pelvic Pain I (MAPP-I). Diagnosis of hypertension, current antihypertensive medications, pain severity, and urinary symptom severity were analyzed using chi-square test and t-test. RESULTS: The combination of diagnosis and antihypertensive medications use was highest in the UCPPS group (n = 74, 18%), followed by positive (n = 34, 17%) and healthy controls (n = 48, 12%, p = 0.04). There were no differences in symptom severity based on hypertension in UCPPS and CP/CPPS; however, IC/BPS had worse ICSI (p = 0.031), AUA-SI (p = 0.04), and BPI pain severity (0.02). Patients (n = 7) with a hypertension diagnosis not on antihypertensive medications reported the greatest severity of pain and urinary symptoms. CONCLUSION: This pattern of findings suggests that there may be a relationship between hypertension and UCPPS. Treating hypertension among these patients may result in reduced pain and symptom severity. Further investigation on the relationship between hypertension, antihypertensive medication use, and UCPPS and the role of angiotensin signaling in UCPPS conditions is needed.

Acute ampakines increase voiding function and coordination in a rat model of SCI.

Neurogenic bladder dysfunction causes urological complications and reduces the quality of life in persons with spinal cord injury (SCI). Glutamatergic signaling via AMPA receptors is fundamentally important to the neural circuits controlling bladder voiding. Ampakines are positive allosteric modulators of AMPA receptors that can enhance the function of glutamatergic neural circuits after SCI. We hypothesized that ampakines can acutely stimulate bladder voiding that has been impaired due to thoracic contusion SCI. Adult female Sprague Dawley rats received a unilateral contusion of the T9 spinal cord (n=10). Bladder function (cystometry) and coordination with the external urethral sphincter (EUS) were assessed five days post-SCI under urethane anesthesia. Data were compared to responses in spinal intact rats (n=8). The "low impact" ampakine CX1739 (5, 10, or 15 mg/kg) or vehicle (HPCD) was administered intravenously. The HPCD vehicle had no discernable impact on voiding. In contrast, following CX1739, the pressure threshold for inducing bladder contraction, voided volume, and the interval between bladder contractions were significantly reduced. These responses occurred in a dose-dependent manner. We conclude that modulating AMPA receptor function using ampakines can rapidly improve bladder voiding capability at sub-acute time points following contusion SCI. These results may provide a new and translatable method for therapeutic targeting of bladder dysfunction acutely after SCI.

Optogenetic urothelial cell stimulation induces bladder contractions and pelvic nerve afferent firing.

Urothelial cells, which play an essential role in barrier function, are also thought to play a sensory role in bladder physiology by releasing signaling molecules in response to sensory stimuli that act upon adjacent sensory neurons. However, it is challenging to study this communication due to the overlap in receptor expression and proximity of urothelial cells to sensory neurons. To overcome this challenge, we developed a mouse model where we can directly stimulate urothelial cells using optogenetics. We crossed a uroplakin II (UPK2) cre mouse with a mouse that expresses the light-activated cation channel channelrhodopsin-2 (ChR2) in the presence of cre expression. Optogenetic stimulation of urothelial cells cultured from UPK2-ChR2 mice initiates cellular depolarization and release of ATP. Cystometry recordings demonstrated that optical stimulation of urothelial cells increases bladder pressure and pelvic nerve activity. Increases in bladder pressure persisted, albeit to a lesser extent, when the bladder was excised in an in vitro preparation. The P2X receptor antagonist PPADS significantly reduced optically evoked bladder contractions in vivo and ex vivo. Furthermore, corresponding nerve activity was also inhibited with PPADS. Our data suggest that urothelial cells can initiate robust bladder contractions via sensory nerve signaling or contractions through local signaling mechanisms. These data support a foundation of literature demonstrating communication between sensory neurons and urothelial cells. Importantly, with further use of these optogenetic tools, we hope to scrutinize this signaling mechanism, its importance for normal micturition and nociception, and how it may be altered in pathophysiological conditions.NEW & NOTEWORTHY Urothelial cells play a sensory role in bladder function. However, it has been particularly challenging to study this communication as both sensory neurons and urothelial cells express similar sensory receptors. Here we demonstrate using an optogenetic technique, that specific urothelial stimulation alone resulted in bladder contractions. This approach will have a long-lasting impact on how we study urothelial-to-sensory neuron communication and the changes that occur under disease conditions.

Evaluating the transduction efficiency of systemically delivered AAV vectors in the rat nervous system.

Gene delivery or manipulation with viral vectors is a frequently used tool in basic neuroscience studies. Adeno-associated viruses (AAV) are the most widely used vectors due to their relative safety and long-term efficacy without causing overt immunological complications. Many AAV serotypes have been discovered and engineered that preferentially transduce different populations of neurons. However, efficient targeting of peripheral neurons remains challenging for many researchers, and evaluation of peripheral neuron transduction with AAVs in rats is limited. Here, we aimed to test the efficiency of systemic AAVs to transduce peripheral neurons in rats. We administered AAV9-tdTomato, AAV-PHP.S-tdTomato, or AAV-retro-GFP systemically to neonatal rats via intraperitoneal injection. After 5 weeks, we evaluated expression patterns in peripheral sensory, motor, and autonomic neurons. No significant difference between the serotypes in the transduction of sensory neurons was noted, and all serotypes were more efficient in transducing NF200 + neurons compared to smaller CGRP + neurons. AAV-retro was more efficient at transducing motor neurons compared to other serotypes. Moreover, PHP.S was more efficient at transducing sympathetic neurons, and AAV-retro was more efficient at transducing parasympathetic neurons. These results indicate that specific AAV serotypes target peripheral neuron populations more efficiently than others in the neonatal rat.

Role of Src kinase in regulating protein kinase C mediated phosphorylation of TRPV1.

BACKGROUND: Transient receptor potential vanilloid-1 (TRPV1), activated by heat, acidic pH, endogenous vanilloids and capsaicin, is essential for thermal hyperalgesia. Under inflammatory conditions, phosphorylation of TRPV1 by protein kinase C (PKC) can sensitize the channel and decrease the activation threshold. Src kinase also phosphorylates TRPV1, promoting channel trafficking to the plasma membrane. These post-translational modifications are important for several chronic pain conditions. This study presents a previously undescribed relationship between Src and PKC phosphorylation of TRPV1, influencing the thermal hypersensitivity associated with TRPV1 activation. METHODS: We assessed TRPV1 channel activity using intracellular calcium imaging and patch-clamp electrophysiology in mouse dorsal root ganglion cultures. Additionally, we used behavioural experiments to evaluate plantar thermal sensitivity following intraplantar injections of activators of known modulators of TRPV1 with and without an Src antagonist. RESULTS: Using calcium imaging and patch-clamp techniques, we demonstrated that pharmacological inhibition of Src kinase or mutation of the Src phosphorylation site on TRPV1 prevented PKC but not PKA-mediated sensitization of TRPV1 in vitro. We found that intraplantar injection of the PKC activator phorbol 12-myristate 13-acetate (PMA) or bradykinin produces thermal hypersensitivity that can be attenuated by pharmacological inhibition of Src. Additionally, complete Freund's Adjuvant (CFA)-induced inflammatory hypersensitivity could also be attenuated by local Src kinase inhibition. CONCLUSIONS: Our data demonstrate that Src phosphorylation is critical for PKC-mediated sensitization of TRPV1. Further, in a model of inflammatory pain, CFA, Src kinase inhibition could reduce thermal hypersensitivity. Targeting of Src kinase may have analgesic benefits in inflammatory pain conditions. SIGNIFICANCE: Src kinase-mediated phosphorylation of TRPV1 is a critical regulator of the PKC-induced sensitization induced by multiple inflammatory mediators. This suggest a new regulatory mechanism governing TRPV1 function and a potential therapeutic target for inflammatory type pain, including cancer pain where Src antagonists are currently utilized.

Characterization of a method to study urodynamics and bladder nociception in male and female mice.

OBJECTIVES: Abdominal electromyogram or visceromotor response (VMR) elicited by bladder distension is a validated as a measure of bladder nociception in mice, however it is not without its limitations. The aim of this study is to address some of these limitations and validate voiding evoked VMR as a measure of bladder nociception mice. METHODS: Using both male and female C57BL/6 mice we assessed the VMR response to cytometry- induced voiding before and after instillation of 0.5% acetic acid into the bladder. We then delivered intravesical lidocaine to confirm the VMR response as nociceptive. VMR and correlative cystometric bladder pressures were analyzed. RESULTS: We found that the VMR can be evoked by continuous fluid infusion into the bladder of both male and female mice. This response is potentiated after bladder injury and can be attenuated by administration of a local anesthetic, providing strong evidence that this method can be used to evaluate bladder nociception. Further, evaluation of cystometric pressure traces obtained during VMR recording revealed that intercontraction intervals were not altered after bladder injury in either male or female mice. However, we did observe a decrease in peak threshold pressures after bladder injury in female mice, which could be rescued by lidocaine administration. CONCLUSIONS: In conclusion, this technique can measure the VMR and bladder nociception associated with voiding in both female and male mice. Although confounds still exist with the use of anesthesia, further exploration of non-anesthetized voiding-evoked VMR is warranted.

A wireless closed-loop system for optogenetic peripheral neuromodulation.

The fast-growing field of bioelectronic medicine aims to develop engineered systems that can relieve clinical conditions by stimulating the peripheral nervous system1-5. This type of technology relies largely on electrical stimulation to provide neuromodulation of organ function or pain. One example is sacral nerve stimulation to treat overactive bladder, urinary incontinence and interstitial cystitis (also known as bladder pain syndrome)4,6,7. Conventional, continuous stimulation protocols, however, can cause discomfort and pain, particularly when treating symptoms that can be intermittent (for example, sudden urinary urgency)8. Direct physical coupling of electrodes to the nerve can lead to injury and inflammation9-11. Furthermore, typical therapeutic stimulators target large nerve bundles that innervate multiple structures, resulting in a lack of organ specificity. Here we introduce a miniaturized bio-optoelectronic implant that avoids these limitations by using (1) an optical stimulation interface that exploits microscale inorganic light-emitting diodes to activate opsins; (2) a soft, high-precision biophysical sensor system that allows continuous measurements of organ function; and (3) a control module and data analytics approach that enables coordinated, closed-loop operation of the system to eliminate pathological behaviours as they occur in real-time. In the example reported here, a soft strain gauge yields real-time information on bladder function in a rat model. Data algorithms identify pathological behaviour, and automated, closed-loop optogenetic neuromodulation of bladder sensory afferents normalizes bladder function. This all-optical scheme for neuromodulation offers chronic stability and the potential to stimulate specific cell types.

Macrophage angiotensin II type 2 receptor triggers neuropathic pain.

Peripheral nerve damage initiates a complex series of structural and cellular processes that culminate in chronic neuropathic pain. The recent success of a type 2 angiotensin II (Ang II) receptor (AT2R) antagonist in a phase II clinical trial for the treatment of postherpetic neuralgia suggests angiotensin signaling is involved in neuropathic pain. However, transcriptome analysis indicates a lack of AT2R gene (Agtr2) expression in human and rodent sensory ganglia, raising questions regarding the tissue/cell target underlying the analgesic effect of AT2R antagonism. We show that selective antagonism of AT2R attenuates neuropathic but not inflammatory mechanical and cold pain hypersensitivity behaviors in mice. Agtr2-expressing macrophages (MΦs) constitute the predominant immune cells that infiltrate the site of nerve injury. Interestingly, neuropathic mechanical and cold pain hypersensitivity can be attenuated by chemogenetic depletion of peripheral MΦs and AT2R-null hematopoietic cell transplantation. Our study identifies AT2R on peripheral MΦs as a critical trigger for pain sensitization at the site of nerve injury, and therefore proposes a translatable peripheral mechanism underlying chronic neuropathic pain.

Angiotensin II Triggers Peripheral Macrophage-to-Sensory Neuron Redox Crosstalk to Elicit Pain.

Injury, inflammation, and nerve damage initiate a wide variety of cellular and molecular processes that culminate in hyperexcitation of sensory nerves, which underlies chronic inflammatory and neuropathic pain. Using behavioral readouts of pain hypersensitivity induced by angiotensin II (Ang II) injection into mouse hindpaws, our study shows that activation of the type 2 Ang II receptor (AT2R) and the cell-damage-sensing ion channel TRPA1 are required for peripheral mechanical pain sensitization induced by Ang II in male and female mice. However, we show that AT2R is not expressed in mouse and human dorsal root ganglia (DRG) sensory neurons. Instead, expression/activation of AT2R on peripheral/skin macrophages (MΦs) constitutes a critical trigger of mouse and human DRG sensory neuron excitation. Ang II-induced peripheral mechanical pain hypersensitivity can be attenuated by chemogenetic depletion of peripheral MΦs. Furthermore, AT2R activation in MΦs triggers production of reactive oxygen/nitrogen species, which trans-activate TRPA1 on mouse and human DRG sensory neurons via cysteine modification of the channel. Our study thus identifies a translatable immune cell-to-sensory neuron signaling crosstalk underlying peripheral nociceptor sensitization. This form of cell-to-cell signaling represents a critical peripheral mechanism for chronic pain and thus identifies multiple druggable analgesic targets.SIGNIFICANCE STATEMENT Pain is a widespread health problem that is undermanaged by currently available analgesics. Findings from a recent clinical trial on a type II angiotensin II receptor (AT2R) antagonist showed effective analgesia for neuropathic pain. AT2R antagonists have been shown to reduce neuropathy-, inflammation- and bone cancer-associated pain in rodents. We report that activation of AT2R in macrophages (MΦs) that infiltrate the site of injury, but not in sensory neurons, triggers an intercellular redox communication with sensory neurons via activation of the cell damage/pain-sensing ion channel TRPA1. This MΦ-to-sensory neuron crosstalk results in peripheral pain sensitization. Our findings provide an evidence-based mechanism underlying the analgesic action of AT2R antagonists, which could accelerate the development of efficacious non-opioid analgesic drugs for multiple pain conditions.

Parathyroid Hormone-Related Peptide Elicits Peripheral TRPV1-dependent Mechanical Hypersensitivity.

Bone metastasis in breast, prostate and lung cancers often leads to chronic pain, which is poorly managed by existing analgesics. The neurobiological mechanisms that underlie chronic pain associated with bone-metastasized cancers are not well understood, but sensitization of peripheral nociceptors by tumor microenvironment factors has been demonstrated to be important. Parathyroid hormone-related peptide (PTHrP) is highly expressed in bone-metastasized breast and prostate cancers, and is critical to growth and proliferation of these tumors in the bone tumor microenvironment. Previous studies have suggested that PTHrP could sensitize nociceptive sensory neurons, resulting in peripheral pain hypersensitivity. In this study, we found that PTHrP induces both heat and mechanical hypersensitivity, that are dependent on the pain-transducing transient receptor potential channel family vanilloid, member-1 (TRPV1), but not the mechano-transducing TRPV4 and TRPA1 ion channels. Functional ratiometric Ca2+ imaging and voltage-clamp electrophysiological analysis of cultured mouse DRG neurons show significant potentiation of TRPV1, but not TRPA1 or TRPV4 channel activation by PTHrP. Interestingly, PTHrP exposure led to the slow and sustained activation of TRPV1, in the absence of any exogenous channel agonist, and is dependent on the expression of the type-1 parathyroid hormone receptor (PTH1), as well as on downstream phosphorylation of the channel by protein kinase C (PKC). Accordingly, local administration of specific small-molecule antagonists of TRPV1 to mouse hindpaws after the development of PTHrP-induced mechanical hypersensitivity led to its significant attenuation. Collectively, our findings suggest that PTHrP/PTH1-mediated flow activation of TRPV1 channel contributes at least in part to the development and maintenance of peripheral mechanical pain hypersensitivity, and could therefore constitute a mechanism for nociceptor sensitization in the context of metastatic bone cancer pain.

Optogenetic silencing of nociceptive primary afferents reduces evoked and ongoing bladder pain.

Patients with interstitial cystitis/bladder pain syndrome (IC/BPS) suffer from chronic pain that severely affects quality of life. Although the underlying pathophysiology is not well understood, inhibition of bladder sensory afferents temporarily relieves pain. Here, we explored the possibility that optogenetic inhibition of nociceptive sensory afferents could be used to modulate bladder pain. The light-activated inhibitory proton pump Archaerhodopsin (Arch) was expressed under control of the sensory neuron-specific sodium channel (sns) gene to selectively silence these neurons. Optically silencing nociceptive sensory afferents significantly blunted the evoked visceromotor response to bladder distension and led to small but significant changes in bladder function. To study of the role of nociceptive sensory afferents in freely behaving mice, we developed a fully implantable, flexible, wirelessly powered optoelectronic system for the long-term manipulation of bladder afferent expressed opsins. We found that optogenetic inhibition of nociceptive sensory afferents reduced both ongoing pain and evoked cutaneous hypersensitivity in the context of cystitis, but had no effect in uninjured, naïve mice. These results suggest that selective optogenetic silencing of nociceptive bladder afferents may represent a potential future therapeutic strategy for the treatment of bladder pain.

Nociceptive TRP Channels: Sensory Detectors and Transducers in Multiple Pain Pathologies.

Specialized receptors belonging to the transient receptor potential (TRP) family of ligand-gated ion channels constitute the critical detectors and transducers of pain-causing stimuli. Nociceptive TRP channels are predominantly expressed by distinct subsets of sensory neurons of the peripheral nervous system. Several of these TRP channels are also expressed in neurons of the central nervous system, and in non-neuronal cells that communicate with sensory nerves. Nociceptive TRPs are activated by specific physico-chemical stimuli to provide the excitatory trigger in neurons. In addition, decades of research has identified a large number of immune and neuromodulators as mediators of nociceptive TRP channel activation during injury, inflammatory and other pathological conditions. These findings have led to aggressive targeting of TRP channels for the development of new-generation analgesics. This review summarizes the complex activation and/or modulation of nociceptive TRP channels under pathophysiological conditions, and how these changes underlie acute and chronic pain conditions. Furthermore, development of small-molecule antagonists for several TRP channels as analgesics, and the positive and negative outcomes of these drugs in clinical trials are discussed. Understanding the diverse functional and modulatory properties of nociceptive TRP channels is critical to function-based drug targeting for the development of evidence-based and efficacious new generation analgesics.

Induction of thermal and mechanical hypersensitivity by parathyroid hormone-related peptide through upregulation of TRPV1 function and trafficking.

The neurobiological mechanisms underlying chronic pain associated with cancers are not well understood. It has been hypothesized that factors specifically elevated in the tumor microenvironment sensitize adjacent nociceptive afferents. We show that parathyroid hormone-related peptide (PTHrP), which is found at elevated levels in the tumor microenvironment of advanced breast and prostate cancers, is a critical modulator of sensory neurons. Intraplantar injection of PTHrP led to the development of thermal and mechanical hypersensitivity in both male and female mice, which were absent in mice lacking functional transient receptor potential vanilloid-1 (TRPV1). The PTHrP treatment of cultured mouse sensory neurons enhanced action potential firing, and increased TRPV1 activation, which was dependent on protein kinase C (PKC) activity. Parathyroid hormone-related peptide induced robust potentiation of TRPV1 activation and enhancement of neuronal firing at mild acidic pH that is relevant to acidic tumor microenvironment. We also observed an increase in plasma membrane TRPV1 protein levels after exposure to PTHrP, leading to upregulation in the proportion of TRPV1-responsive neurons, which was dependent on the activity of PKC and Src kinases. Furthermore, co-injection of PKC or Src inhibitors attenuated PTHrP-induced thermal but not mechanical hypersensitivity. Altogether, our results suggest that PTHrP and mild acidic conditions could induce constitutive pathological activation of sensory neurons through upregulation of TRPV1 function and trafficking, which could serve as a mechanism for peripheral sensitization of nociceptive afferents in the tumor microenvironment.

Distinct modifications in Kv2.1 channel via chemokine receptor CXCR4 regulate neuronal survival-death dynamics.

The chemokine stromal cell-derived factor-1α (SDF-1α) has multiple effects on neuronal activity, survival, and death under conditions that generate a proinflammatory microenvironment within the brain, via signaling through C-X-C-type chemokine receptor 4 (CXCR4), although the underlying cellular/molecular mechanisms are unclear. Using rat hippocampal neurons, we investigated distinct modifications in the voltage-gated K⁺ (Kv) channel Kv2.1 in response to short- and long-term SDF-1α/CXCR4-mediated signaling as an underlying mechanism for CXCR4-dependent regulation of neuronal survival and death. Acute exposure of neurons to SDF-1α led to dynamic dephosphorylation and altered localization of Kv2.1 channel, resulting in enhanced voltage-dependent activation of Kv2.1-based delayed-rectifier Kv currents (I(DR)). These changes were dependent on CXCR4- and/or NMDA receptor-mediated activation of calcineurin and provide neuroprotection. However, prolonged SDF-1α treatment leads to CXCR4-mediated activation of p38 mitogen-activated protein kinase, resulting in phosphorylation of Kv2.1 at S800 and enhanced surface trafficking of the channel protein, resulting in increased I(DR)/Kv2.1 current density. This, in combination with sustained dephosphorylation-induced enhancement of the voltage-dependent activation of I(DR)/Kv2.1, predisposed neurons to excessive K⁺ efflux, a vital step for the neuronal apoptotic program. Such apoptotic death was dependent on CXCR4 and Kv2.1 function and was absent in cells expressing the Kv2.1-S800A mutant channel. Furthermore, similar modifications in Kv2.1 and CXCR4/Kv2.1-dependent apoptosis were observed following treatment of neurons with the human immunodeficiency virus-1 (HIV-1) glycoprotein gp120. Therefore, distinct modifications in Kv2.1 in response to short- and long-term CXCR4-mediated signaling could provide a basis for neuroprotection or apoptosis in neuropathologies, such as neuroinflammation, stroke, brain tumors, and HIV-associated neurodegeneration.