Von Frey Filament Stimulation Research Articles

Genetic evidence of the function of Phox2a-expressing anterolateral system neurons in the transmission of chronic pain.

The development of the chronic neuropathic pain state often originates at the level of peripheral sensory neurons, whose abnormal function elicits central sensitization and maladaptive plasticity in the nociceptive circuits of the spinal dorsal horn. These changes eventually reach supraspinal areas bringing about cognitive and affective co-morbidities of chronic pain such as anxiety and depression. This transmission presumably relies on the function of spinal projection neurons at the origin of the anterolateral system (AS). However, the identity of these neurons and the extent of their functional contribution remain unknown. Here, we asked these questions in the context of the mouse AS neurons that require the transcription factor Phox2a for their normal target connectivity and function in transmitting acute nociceptive information to the brain. To this end, we examined the effects of a spinal cord-specific loss of Phox2a (Phox2acKO) on the development of central sensitization evoked by the spared nerve injury (SNI) model of chronic pain. We found that SNI-treated Phox2acKO mice developed normal reflexive spinal responses such as mechanical allodynia evidenced by a decreased withdrawal threshold to von Frey filament stimulation and dynamic brush. On the other hand, Phox2acKO attenuated the development of cold but not mechanical hyperalgesia, in behavioral paradigms that require the relay of nociceptive information to the brain. Furthermore, Phox2acKO attenuated anxio-depressive-like behaviors evoked by SNI, measured by performance in the open field test and tail suspension test. Thus, Phox2a AS neurons play a critical role in the generation and maintenance of chronic neuropathic pain.

Trigeminal‐mediated headshaking: A diagnostic challenge

Trigeminal‐mediated headshaking: A diagnostic challenge

IL-33 induces orofacial neuropathic pain through Fyn-dependent phosphorylation of GluN2B in the trigeminal spinal subnucleus caudalis

Orofacial neuropathic pain can cause considerable disruptions in patients' daily lives, especially because of a lack of effective medications as its underlying causative mechanisms are not fully understood. Here, we found neuron-specific expression of the interleukin (IL)-33 receptor in the trigeminal spinal subnucleus caudalis (Vc), distinct from the spinal dorsal horn. Reduction in head withdrawal threshold in response to von Frey filament stimulation of the whisker pad skin was inversely correlated with the upregulation of IL-33 in the Vc after infraorbital nerve injury (IONI). Neutralization of IL-33 in the Vc alleviated mechanical allodynia in the whisker pad skin after IONI; conversely, intracisternal administration of IL-33 elicited mechanical allodynia in the whisker pad skin, which was relieved by GluN2B antagonism. Moreover, IL-33 triggered the potentiation of GluN2B-containing N-methyl-D-aspartate receptor-mediated synaptic currents and phosphorylation of synaptosomal GluN2B in the Vc, whereas IONI-induced GluN2B phosphorylation was inhibited by neutralization of IL-33 in the Vc. IL-33-induced GluN2B phosphorylation was mediated by phosphorylation of Fyn kinase, and inhibition of the Fyn kinase pathway prevented the development of IL-33-induced mechanical allodynia. Our findings provide insights into a new mechanism by which IL-33 directly regulates synaptic transmission and suggest that IL-33 signaling could be a candidate target for therapeutic interventions for orofacial neuropathic pain.

Calcium-independent phospholipase A2 inhibitor produces an analgesic effect in a rat model of neuropathic pain by reducing central sensitization in the dorsal horn

ABSTRACT Objective Phospholipase A2 (PLA2) plays an important role in regulating the production of arachidonic acid and various eicosanoids. The aim of our study was to investigate the analgesic mechanisms of calcium-dependent cytosolic phospholipase A2 and calcium-independent PLA2 (iPLA2) inhibitors in the spinal cord in a rat model of neuropathic pain. Methods Lumbar 5 spinal nerve ligation was performed in male Sprague-Dawley rats to develop a peripheral neuropathic pain model. Paw withdrawal thresholds in response to von Frey filaments, brush, pressure, and pinch were measured. Lumbar wide dynamic range neuronal firing rates and iPLA2 subtype expression were measured by in vivo extracellular recording and double immunofluorescence staining, respectively. Results In our rat models, oral administration of prednisolone, a non-selective PLA2 inhibitor, and intrathecal injection of bromoenolactone, a iPLA2 inhibitor, significantly increased the ipsilateral hindpaw withdrawal thresholds in response to von Frey filament stimulation, but intrathecal injection of arachidonyl trifluoromethyl ketone, a selective cytosolic PLA2 inhibitor, did not show significant changes. In spinal dorsal horn neurons, bromoenolactone reduced neuronal firing rates in response to withdrawal stimulation and spontaneous firing rates in the ipsilateral side of the spinal dorsal horn. In addition, the expression of iPLA2 was co-localized with astrocytes and neurons on the ipsilateral side of the dorsal horn in rats that underwent spinal nerve ligation. Discussion These data suggest that selective iPLA2 inhibitor produce analgesia in neuropathic rats by reducing central sensitization in the dorsal horn.

Disc degeneration induces a mechano-sensitization of disc afferent nerve fibers that associates with low back pain

We aimed to investigate mechano-sensitivity at the afferent nerve fibers projecting to degenerated intervertebral disc (IVD) and nociceptive behaviour in a rat model of low back pain (LBP). Animal model with LBP was established by lumbar 4/5 IVD puncture and nucleus pulposus aspiration. Invivo single nerve recordings (n=121) were introduced to measure discharge frequency at the afferent nerve fiber innervating the IVD during mechanical stimulations (von Frey filament or intradiscal pressure). Nerve growth factor (NGF) expression levels in the IVD (n=20) were assessed by Western blot. LBP-related behaviour (n=22) was assessed by measuring changes in rearing, mechanical paw-withdrawal threshold, and dynamic weight bearing in a freely walking rat. Inhibitory effect of morphine on the neuronal excitability (n=19) and painful behaviour (n=28) was also assessed. Compared to those with sham or naïve IVD, animal group with degenerated IVD displayed the sensitized neuronal responses and painful behaviour, with hyperexcitability of the afferent nerve fibers in any range of mechanical stimulations (von Frey filament stimulation; 1, 2, and 26g; intradiscal pressure, 1,500-3,000mm Hg), strong upregulation of NGF (200-250 % increase), and LBP-like behaviour such as failure of rearing, front limbs-dependent walking pattern, and hypersensitivity in hind-paws. However, the neuronal hyperexcitability and pain behaviour were attenuated after local (30 μM) or systemic (3mgkg-1) morphine administration. Our study suggests that enhanced mechano-sensitivity at the afferent nerve fiber innervating degenerated IVD is deeply correlated with LBP development, which supports the hypothesis that hyperexcited responses at the nerve fibers represent a decisive source of LBP.

Involvement of the epithelial sodium channel in initiation of mechanically evoked swallows in anaesthetized rats.

Afferents carried by the superior laryngeal nerve play a primary role in the initiation of laryngeal mechanically evoked swallows in anaesthetized rats. Amiloride and its analogues inhibit swallowing evoked by mechanical stimulation, but not swallowing evoked by chemical and electrical stimulation. The epithelial sodium channel is probably involved in the initiation of laryngeal mechanically evoked swallows. The swallowing reflex plays a critical role in airway protection. Because impaired laryngeal mechanosensation is associated with food bolus aspiration, it is important to know how the laryngeal sensory system regulates swallowing initiation. This study was performed to clarify the neuronal mechanism of mechanically evoked swallows. Urethane-anaesthetized Sprague-Dawley male rats were used. A swallow was identified by activation of the suprahyoid and thyrohyoid muscles on electromyography. The swallowing threshold was measured by von Frey filament and electrical stimulation of the larynx. The number of swallows induced by upper airway distension and capsaicin application (0.03nmol, 3μl) to the vocal folds was counted. The effects of topical application (0.3-30nmol, 3μl) of the epithelial sodium channel (ENaC) blocker amiloride and its analogues (benzamil and dimethylamiloride), acid-sensing ion channel (ASIC) inhibitors (mambalgine-1 and diminazene) and gadolinium to the laryngeal mucosa on swallowing initiation were evaluated. A nerve transection study indicated that afferents carried by the superior laryngeal nerve play a primary role in the initiation of laryngeal mechanically evoked swallows. The mechanical threshold of swallowing was increased in a dose-dependent manner by amiloride and its analogues and gadolinium, but not by ASIC inhibitors. The number of swallows by upper airway distension was significantly decreased by benzamil application. However, the initiation of swallows evoked by capsaicin and electrical stimulation was not affected by benzamil application. We speculate that the ENaC is involved in the initiation of laryngeal mechanically evoked swallows.

Sequential alteration of microglia and astrocytes in the rat thalamus following spinal nerve ligation

BackgroundSpinal reactive astrocytes and microglia are known to participate to the initiation and maintenance of neuropathic pain. However, whether reactive astrocytes and microglia in thalamic nuclei that process sensory-discriminative aspects of pain play a role in pain behavior remains poorly investigated. Therefore, the present study evaluated whether the presence of reactive glia (hypertrophy, increased number and upregulation of glial markers) in the ventral posterolateral thalamic nucleus (VPL) correlates with pain symptoms, 14 and 28 days after unilateral L5/L6 spinal nerve ligation (SNL) in rats.MethodsMechanical allodynia and hyperalgesia (von Frey filament stimulation) as well as ambulatory pain (dynamic weight bearing apparatus) were assessed. Levels of nine glial transcripts were determined by quantitative real-time PCR on laser microdissected thalamic nuclei, and levels of proteins were assessed by Western blot. We also studied by immunohistofluorescence the expression of glial markers that label processes (GFAP for astrocytes and iba-1 for microglia) and cell body (S100β for astrocytes and iba-1 for microglia) and quantified the immunostained surface and the number of astrocytes and microglia (conventional counts and optical dissector method of stereological counting).ResultsDifferential, time-dependent responses were observed concerning microglia and astrocytes. Specifically, at day 14, iba-1 immunostained area and number of iba-1 immunopositive cells were decreased in the VPL of SNL as compared to naïve rats. By contrast, at day 28, GFAP-immunostained area was increased in the VPL of SNL as compared to naïve rats while number of GFAP/S100β immunopositive cells remained unchanged. Using quantitative real-time PCR of laser microdissected VPL, we found a sequential increase in mRNA expression of cathepsin S (day 14), fractalkine (day 28), and fractalkine receptor (day 14), three well-known markers of microglial reactivity. Using Western blot, we confirmed an increase in protein expression of fractalkine receptor at day 14.ConclusionsOur results demonstrate a sequential alteration of microglia and astrocytes in the thalamus of animals with lesioned peripheral nerves. Furthermore, our data report unprecedented concomitant molecular signs of microglial activation and morphological signs of microglial decline in the thalamus of these animals.

Mechanical allodynia and enhanced responses to capsaicin are mediated by PI3K in a paclitaxel model of peripheral neuropathy

Paclitaxel chemotherapy treatment often leads to neuropathic pain resistant to available analgesic treatments. Recently spinal Toll-like receptor 4 (TLR4) and the transient receptor potential cation channel subfamily V member 1 (TRPV1) were identified to be involved in the pro-nociceptive effect of paclitaxel. The aim of this study was to investigate the role of phosphatidylinositol 3-kinase (PI3K) and serine/threonine kinases in this process, with the use of their antagonists (wortmannin, LY-294002, and staurosporine). The single paclitaxel administration (8 mg/kg i.p.) in mice induced robust mechanical allodynia measured as a reduced threshold to von Frey filament stimulation and generated reduced tachyphylaxis of capsaicin-evoked responses, recorded as changes in mEPSC frequency in patch-clamp recordings of dorsal horn neurons activity in vitro, for up to eight days. Paclitaxel application also induced increased Akt kinase phosphorylation in rat DRG neurons. All these paclitaxel-induced changes were prevented by the wortmannin in vivo pretreatment. Acute co-application of wortmannin or LY-294002 with paclitaxel in spinal cord slices also attenuated the paclitaxel effect on capsaicin-evoked responses. Staurosporine was effective in the acute in vitro experiments and on the first day after the paclitaxel treatment in vivo, but in contrast to wortmannin, it did not have a significant impact later. Our data suggest that the inhibition of PI3K signaling may help alleviate pathological pain syndromes in the paclitaxel-induced neuropathy.

Specific translocation of protein kinase Cγ to lipid rafts in primary somatosensory cortex is involved in remifentanil-induced hyperalgesia

Objective To determine the involvement of neuron-specific protein kinase C γ isoform(PKCγ) translocation to lipid rafts in primary somatosensory cortex(S1) in remifentanil-induced hyperalgesia. Methods Fifty adult male SD rats weighing 240-260 g (at the age of 8-10 weeks) were randomly assigned to 3 groups: control, the propofol (group P) and the remifentanil group (group R). Intravenous anesthesia with either propofol (group P) or propofol combined with remifentanil(group R) was performed through a 24 G catheter inserted in tail vein for 3 h. The total doses of propofol were 36.0 mg and 26.0 mg in group P and R, respectively. The dose of remifentanil was 19.5 g in group R. Mechanical allodynia in the rats was evaluated by the von Frey filament test, and PKCγ levels in the whole-cell lysates and lipid rafts in the S1 neurons were determined by Western blot. Results ① The threshold of paw-withdrawal response to von Frey filament stimulation in group R at 2 h after stopping drug infusion was significantly lower than that in group P(P<0.05). ② Two hours after stopping drug infusion, the PKCγ protein levels in the lipid rafts of S1 in group R were significantly higher than those in group R(P<0.05). Conclusions These results suggest that increased translocation of PKCγ to lipid raft of S1 neurons may contribute to postoperative pain in rats after propofol-remifentanil-based anesthesia. Key words: Remifentanil; Hyperalgesia; Protein kinase C γisoform; Lipid rafts; Primary somatosensory cortex

Dextran Sulphate of Sodium-induced colitis in mice: antihyperalgesic effects of ethanolic extract of Citrus reticulata and potential damage to the central nervous system.

Citrus species are widely related to antihyperalgesic and anti-inflammatory effects. The aim of this study was to investigate if treatment with ethanolic extract from peels of mature Citrus reticulata Blanco causes antihyperalgesic effects on the referred mechanical hyperalgesia in a model of dextran sulphate of sodium (DSS)-induced colitis in mice, as well as the possible oxidative damage in different regions of the brain induced by its inflammatory reaction. Antihyperalgesia (30 to 300 mg/kg) was investigated by behavioral response (frequency of response to von Frey filament stimulation) in Swiss mice, while damage to central nervous system was investigated through techniques that evaluated oxidative stress using male black C57 BL6 mice (n=8). Treatment of the animals with the extract (100 mg/kg) from days 3 to 5 after colitis induction reduced referred the mechanical hyperalgesia (32.6 ± 5.1) in relation to the control group (57.4 ± 2.0). Levels of lipid peroxidation or carbonyl proteins were augmented in colitis-induced animals in relation to the disease group. These results indicated an antihyperalgesic effect of the studied extract and a potential impairment of the central nervous system functioning caused by inflammation during colitis, which could be related to mental disorders observed in patients suffering of this pathology.

Mechanical Allodynia Assessment in a Murine Neuropathic Pain Model.

Experimental animal models are unique tools (i) to study pain transmission and pathophysiology of neuropathic pain, (ii) to identify novel molecular targets and (iii) to test the potential analgesic effect of specific molecules. The chronic constriction injury (CCI) model of neuropathic pain is the first model of post-traumatic painful peripheral neuropathy, originally developed by Bennett and Xie in the late 1980s. The chronic constriction is performed in the sciatic nerve and induces a partial denervation involving myelinated afferent axons and unmyelinated axons. Damage to unmyelinated axons is much more severe than myelinated afferents. As the model induces a partial denervation, it is very useful for the analysis of pain behaviours. Stimulation of the hind paw, a target of the sciatic nerve, induces pain which can be quantitated. Thus, mechanical allodynia is usually assessed 7, 14 and 21 days after CCI of the sciatic nerve by measuring the hind paw withdrawal response to von Frey filament stimulation. Here, we describe in detail the protocol allowing a reliable and reproducible CCI model in mice. Overall, researchers most commonly use this surgical model to discover more efficacious drugs for the pharmacological control of chronic pain states.

The characteristics of peripheral neuropathy in type 2 diabetic rats induced by low dose STZ

Objective To prepare the rat model of type 2 diabetes mellitus (T2DM), and to observe the characteristics of peripheral neuropathy. Methods High fat and high sugar diets were fed for 8 weeks to induce insulin resistance and then low dose streptozotocin (STZ) was injected intraperitoneally to induce type 2 diabetes mellitus models in Sprague Dawley rats. Blood glucose and serum insulin levels continuous were monitored. Tactile allodynia in response to von Frey (VF) filament stimulation of the plantar hind paws and paw withdrawal thermal latency (PWTL) to plantar test were used as the criterion for diabetic neuropathy. Instruments AD was used to detect nerve conduction velocity (NCV) of sciatic nerve in rat and the morphological and pathological changes of sciatic nerve were detected by electron microscope. Results The characteristics of T2DM rats by peripheral neuropathy in this method were that 50% force withdrawal threshold and PWTL were measured. Both values of diabetic rats were decreased from the day of STZ injection until 4 weeks after STZ injection, and then increased 8 weeks after STZ injection (50% force withdrawal threshold values, (11.8±0.8)g, (8.4±0.7)g and (16.2±1.4)g; PWTL (10.2±0.9)s, (8.3±1.2)s and (13.2±1.0)s. These results indicated that tactile sensation changed from hypersensitive to hyposensitive. Compared to the NC group, the sciatic nerve motor and sensory conduction velocity were significantly decreased at 4 and 8 weeks in DM group, respectively. Compared to DM group at 4 weeks, the sciatic nerve motor and sensory conduction velocities were further decreased in the DM group at 8 weeks. Conclusively, sciatic nerve showed obvious demyelination and axonal collapse. Conclusions T2DM rat model was successfully induced by high fat and sugar diet combined with small dose of STZ injection. The rat model has typical pathological change of peripheral nerve. It might provide a particularly advantageous tool for investigations of diabetes and its chronic complications. Key words: Diabetes mellitus, experimental/CO; Peripheral nervous system diseases/CO; Streptozocin/AD

A bacosides containing Bacopa monnieri extract alleviates allodynia and hyperalgesia in the chronic constriction injury model of neuropathic pain in rats

BackgroundThe current therapy of neuropathic pain is inadequate and is limited by the extent of pain relief and the occurrence of dose dependant side effects. Insufficient control of pain with conventional medications prompts the use of complementary and alternative medicine therapies by patients with neuropathic pain. This study therefore investigated a standardized methanolic extract of Bacopa monnieri, a widely reputed nootropic plant, for prospective antinociceptive effect in the chronic constriction injury (CCI) model of neuropathic pain.MethodsPlacement of four loose ligatures around the sciatic nerve produced partial denervation of the hindpaw in rats. Bacopa monnieri (40 and 80 mg/kg, p.o) and the positive control, gabapentin (75 mg/kg, i.p), were administered daily after CCI or sham surgery and the behavioral paradigms of static- and dynamic-allodynia (paw withdrawal threshold to von Frey filament stimulation [PWT] and paw withdrawal latency to light-brushing [PWL]), cold-allodynia (paw withdrawal duration [PWD] to acetone), heat- (PWL to heat-stimulus) and punctate-hyperalgesia (PWD to pin-prick) were assessed on days 3, 7, 14 and 21.ResultsCCI consistently generated static- (days 3–21), dynamic- (days 14–21) and cold-allodynia (days 3–21) plus heat- and mechano-hyperalgesia (days 3–21). The tested doses of Bacopa monnieri significantly attenuated the CCI-induced allodynia and hyperalgesia, exemplified by increased PWT (days 7–21), PWL to light brushing (days 14–21) and heat (days 7–21) as well as decreased PWD to pin prick and cold stimuli (days 3–21). The extract also counterbalanced the CCI-induced aberrations in the nociceptive behaviors by increasing the pain threshold to that of pre-surgery baseline. Gabapentin also afforded analogous beneficial behavioral profile but of higher magnitude.ConclusionsOur findings suggest that Bacopa monnieri can be used as adjuvant therapy for neuropathic pain conditions afflicted with allodynia and hyperalgesia.

Regulation of transient receptor potential cation channel subfamily V1 protein synthesis by the phosphoinositide 3-kinase/Akt pathway in colonic hypersensitivity

The transient receptor potential cation channel subfamily V member 1 (TRPV1), also known as the capsaicin receptor or vanilloid receptor 1 (VR1), is expressed in nociceptive neurons in the dorsal root ganglia (DRG) and participates in the transmission of pain. The present study investigated the underlying molecular mechanisms by which TRPV1 was regulated by nerve growth factor (NGF) signaling pathways in colonic hypersensitivity in response to colitis. We found that during colitis TRPV1 protein levels were significantly increased in specifically labeled colonic afferent neurons in both L1 and S1 DRGs. TRPV1 protein up-regulation in DRG was also enhanced by NGF treatment. We then found that TRPV1 protein up-regulation in DRG was regulated by activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway both in vivo and in vitro. Suppression of endogenous PI3K/Akt activity during colitis or NGF treatment with a specific PI3K inhibitor LY294002 reduced TRPV1 protein production in DRG neurons, and also reduced colitis-evoked TRPV1-mediated visceral hypersensitivity tested by hyper-responsiveness to colorectal distention (CRD) and von Frey filament stimulation of abdomen. Further studies showed that TRPV1 mRNA levels in the DRG were not regulated by either colitis or NGF. We then found that an up-regulation of the protein synthesis pathway was involved by which both colitis and NGF caused a PI3K-dependent increase in the phosphorylation level of eukaryotic translation initiation factor 4E-binding protein (4E-BP)1. These results suggest a novel mechanism in colonic hypersensitivity which involves PI3K/Akt-mediated TRPV1 protein, not mRNA, up-regulation in primary afferent neurons, likely through activation of the protein synthesis pathways.

Pathogenesis of abdominal pain in bowel obstruction: role of mechanical stress-induced upregulation of nerve growth factor in gut smooth muscle cells.

Abdominal pain is one of the major symptoms in bowel obstruction (BO); its cellular mechanisms remain incompletely understood. We tested the hypothesis that mechanical stress in obstruction upregulates expression of nociception mediator nerve growth factor (NGF) in gut smooth muscle cells (SMCs), and NGF sensitizes primary sensory nerve to contribute to pain in BO. Partial colon obstruction was induced with a silicon band implanted in the distal bowel of Sprague-Dawley rats. Colon-projecting sensory neurons in the dorsal root ganglia (T13 to L2) were identified for patch-clamp and gene expression studies. Referred visceral sensitivity was assessed by measuring withdrawal response to stimulation by von Frey filaments in the lower abdomen. Membrane excitability of colon-projecting dorsal root ganglia neurons was significantly enhanced, and the withdrawal response to von Frey filament stimulation markedly increased in BO rats. The expression of NGF mRNA and protein was increased in a time-dependent manner (day 1-day 7) in colonic SMC but not in mucosa/submucosa of the obstructed colon. Mechanical stretch in vitro caused robust NGF mRNA and protein expression in colonic SMC. Treatment with anti-NGF antibody attenuated colon neuron hyperexcitability and referred hypersensitivity in BO rats. Obstruction led to significant increases of tetrodotoxin-resistant Na currents and mRNA expression of Nav1.8 but not Nav1.6 and Nav1.7 in colon neurons; these changes were abolished by anti-NGF treatment. In conclusion, mechanical stress-induced upregulation of NGF in colon SMC underlies the visceral hypersensitivity in BO through increased gene expression and activity of tetrodotoxin-resistant Na channels in sensory neurons.

Transgenic Mice Expressing MCP-1 by the Urothelium Demonstrate Bladder Hypersensitivity, Pelvic Pain and Voiding Dysfunction: A Multidisciplinary Approach to the Study of Chronic Pelvic Pain Research Network Animal Model Study.

Monocyte chemoattractant protein-1 (MCP-1) is one of the key chemokines that play important roles in diverse inflammatory and chronic pain conditions. Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic and debilitating inflammatory condition of the urinary bladder characterized by the hallmark symptoms of pelvic pain and voiding dysfunction. To facilitate IC/BPS research, we used transgenic technology to develop a novel urothelial MCP-1 secretion mouse model (URO-MCP-1). A transgene consisting of the uroplakin II gene promoter and the mouse MCP-1 coding sequence with a secretory element was constructed and microinjected. URO-MCP-1 mice were found to express MCP-1 mRNA in the bladder epithelium and MCP-1 protein in the urine, and developed bladder inflammation 24 hours after intravesical administration of a single sub-noxious dose of lipopolysaccharide (LPS). The inflamed bladders of URO-MCP-1 mice exhibited elevated mRNAs for interleukin (IL)-1ß, IL-6, substance P precursor, and nerve growth factor as well as increased macrophage infiltration. In parallel with these phenotypic changes, URO-MCP-1 mice manifested significant functional changes at days 1 and 3 after cystitis induction. These functional changes included pelvic pain as measured by von Frey filament stimulation and voiding dysfunction (increased urinary frequency, reduced average volume voided per micturition, and reduced maximum volume voided per micturition) as measured by micturition cages. Micturition changes remained evident at day 7 after cystitis induction, although these changes were not statistically significant. Control wild-type C57BL/6 mice manifested no clear changes in histological, biochemical and behavioral features after similar cystitis induction with LPS. Taken together, our results indicate that URO-MCP-1 mice are hypersensitive to bladder irritants such as LPS and develop pelvic pain and voiding dysfunction upon cystitis induction, providing a novel model for IC/BPS research.

Abstract PR149

Background & Objectives: Increased oxidative stress has been linked to anesthetics-induced nerve injury in a diabetic neuropathy (DN) rat model. The present study was aimed to investigate the effects of diphenyleneiodonium chloride (DPI), an NADPH oxidase (NOX) inhibitor on bupivacaine-induced sciatic nerve injury in DN rats. Materials & Methods: A rat DN model was established by high fat diet (HFD) feeding and streptozotocin (STZ) injection, and confirmed by blood glucose test, mechanical allodynia in response to von Frey (VF) filament stimulation, Paw withdrawal thermal latency (PWTL) test and nerve conduction velocity (NCV). Right sciatic nerve block was performed using 0.2 mL 0.5% bupivacaine. DPI (1mg/kg) was injected subcutaneously at 24 h and 30 min before nerve block. After 24 h of nerve block, nerve conduction function, sciatic nerve injury, levels of lipid peroxides and hydroperoxides, and expression of NOX2 and NOX4 was evaluated. Results: A rat model of DN was successfully established. Compared with the control group, the sensory and motor recovery time, and the value of VF and PWTL significantly raised, NCV of sciatic nerve significantly declined, and sciatic nerve injury was more severe in bupivacaine-treated DN group. DPI treatment significantly improved nerve function and injury. Compared with the bupivacaine-treated DN group, the levels of lipid peroxides and hydrperoxide, and the protein express of NOX2 and NOX4 significantly reduced in DPI group. Conclusion: Subcutaneous injection of DPI can effectively protect against the functional and neurohistological damage of sciatic nerves induced by bupivacaine in an HFD/STZ-induced DN model. Disclosure of Interest: None declared

MP72-05 TOLL-LIKE RECEPTOR ACTIVATION IS ASSOCIATED WITH BLADDER INFLAMMATION, PELVIC PAIN AND VOIDING DYSFUNCTION IN A TRANSGENIC INTERSTITIAL CYSTITIS/BLADDER PAIN SYNDROME-LIKE ANIMAL MODEL: A MAPP RESEARCH NETWORK ANIMAL MODEL STUDY

You have accessJournal of UrologyInfections/Inflammation/Cystic Disease of the Genitourinary Tract: Interstitial Cystitis1 Apr 2016MP72-05 TOLL-LIKE RECEPTOR ACTIVATION IS ASSOCIATED WITH BLADDER INFLAMMATION, PELVIC PAIN AND VOIDING DYSFUNCTION IN A TRANSGENIC INTERSTITIAL CYSTITIS/BLADDER PAIN SYNDROME-LIKE ANIMAL MODEL: A MAPP RESEARCH NETWORK ANIMAL MODEL STUDY Yaoqin Wang, Suming Xu, Michael O'Donnell, Susan Lutgendorf, Pengchao Li, Lang Feng, Catherine Bradley, Bradley Erickson, Vincent Magnotta, Karl Kreder, and Yi Luo Yaoqin WangYaoqin Wang More articles by this author , Suming XuSuming Xu More articles by this author , Michael O'DonnellMichael O'Donnell More articles by this author , Susan LutgendorfSusan Lutgendorf More articles by this author , Pengchao LiPengchao Li More articles by this author , Lang FengLang Feng More articles by this author , Catherine BradleyCatherine Bradley More articles by this author , Bradley EricksonBradley Erickson More articles by this author , Vincent MagnottaVincent Magnotta More articles by this author , Karl KrederKarl Kreder More articles by this author , and Yi LuoYi Luo More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.1618AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Toll-like receptor (TLR) activation has been demonstrated in several chronic pain conditions but has not been well explored in IC/BPS. We previously showed that peripheral blood mononuclear cells from IC/BPS patients produced elevated proinflammatory cytokines in response to TLR2/4 stimulations, indicating the presence of altered peripheral TLR activation in IC/BPS. Here we used our validated transgenic autoimmune cystitis model (URO-OVA) that reproduces many clinical features of IC/BPS to further investigate whether central and peripheral TLR activations are associated with bladder inflammation, pelvic pain and voiding dysfunction seen in human IC/BPS. METHODS URO-OVA mice (n=8) were induced for cystitis by adoptive transfer of antigen specific CD8+ T cells. At day 7 after cystitis induction mice were analyzed for pelvic pain (von Frey filament stimulation), voiding dysfunction (micturition cages) and bladder inflammation (H&E staining). Splenocytes were prepared and stimulated with escalating doses of Pam3CSK4 (a TLR2 agonist) or lipopolysaccharide (LPS, a TLR4 agonist) ranging 10-5-102 µg/ml for 24 hrs, followed by ELISA analysis of proinflammatory cytokines in the conditioned culture supernatants. Lumbar spinal cords were processed for total RNA extraction and RT-PCR analysis of mRNAs for proinflammatory cytokines, TLR and glial activation markers. URO-OVA mice (n=8) not induced for cystitis were analyzed in parallel. RESULTS After cystitis induction URO-OVA mice developed profound bladder histopathology, increased pelvic responses to von Frey filament stimulation (p<0.05 for all applied forces ranging 0.04-4 g), increased urinary frequency (control 4.86±0.634 vs. cystitis 9.0±0.447 voids, p<0.001), and decreased average volume voided per micturition (control 0.26±0.036 vs. cystitis 0.14±0.016 g, p=0.022). These changes were associated with increased splenocyte production of IL-1ß, IL-6 and TNF-a in response to TLR2/4 stimulations. Elevated expressions of IL-6, TNF-a, TLR4 and CD11b mRNAs in the lumbar spinal cords were observed in cystitis-induced mice, which was also associated with phenotypic and functional changes. CONCLUSIONS Central and peripheral TLR activations are associated with bladder inflammation, pelvic pain and voiding dysfunction in URO-OVA mice, providing a potential mechanistic insight for human IC/BPS. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e953-e954 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Yaoqin Wang More articles by this author Suming Xu More articles by this author Michael O'Donnell More articles by this author Susan Lutgendorf More articles by this author Pengchao Li More articles by this author Lang Feng More articles by this author Catherine Bradley More articles by this author Bradley Erickson More articles by this author Vincent Magnotta More articles by this author Karl Kreder More articles by this author Yi Luo More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Protease-Activated Receptor 4 Induces Bladder Pain through High Mobility Group Box-1.

Pain is the significant presenting symptom in Interstitial Cystitis/Painful Bladder Syndrome (IC/PBS). Activation of urothelial protease activated receptor 4 (PAR4) causes pain through release of urothelial macrophage migration inhibitory factor (MIF). High Mobility Group Box-1 (HMGB1), a chromatin-binding protein, mediates bladder pain (but not inflammation) in an experimental model (cyclophosphamide) of cystitis. To determine if PAR4-induced bladder hypersensitivity depends on HMGB1 downstream, we tested whether: 1) bladder PAR4 stimulation affected urothelial HMGB1 release; 2) blocking MIF inhibited urothelial HMGB1 release; and 3) blocking HMGB1 prevented PAR4-induced bladder hypersensitivity. HMGB1 release was examined in immortalized human urothelial cultures (UROtsa) exposed to PAR4-activating peptide (PAR4-AP; 100 μM; 2 hours) or scrambled control peptide. Female C57BL/6 mice, pretreated with a HMGB1 inhibitor (glycyrrhizin: 50 mg/kg; ip) or vehicle, received intravesical PAR4-AP or a control peptide (100 μM; 1 hour) to determine 1) HMGB1 levels at 1 hour in the intravesical fluid (released HMGB1) and urothelium, and 2) abdominal hypersensitivity to von Frey filament stimulation 24 hours later. We also tested mice pretreated with a MIF blocker (ISO-1: 20 mg/kg; ip) to determine whether MIF mediated PAR4-induced urothelial HMGB1 release. PAR4-AP triggered HMGB1 release from human (in vitro) and mice (in vivo) urothelial cells. Intravesical PAR4 activation elicited abdominal hypersensitivity in mice that was prevented by blocking HMGB1. MIF inhibition prevented PAR4-mediated HMGB1 release from mouse urothelium. Urothelial MIF and HGMB1 represent novel targets for therapeutic intervention in bladder pain conditions.

The inhibitory effect of locally injected dexmedetomidine on carrageenan-induced nociception in rats

Recent studies showed that the administration of dexmedetomidine relieved hyperalgesia in the presence of neuropathic pain. These findings have led to the hypothesis that the local administration of dexmedetomidine is useful for relieving acute inflammatory nociception, such as postoperative pain. Thus, we evaluated the inhibitory effect of locally injected dexmedetomidine on acute inflammatory nociception. Acute inflammatory nociception was induced by an intraplantar injection of 1% carrageenan into the hindpaws of rats, and dexmedetomidine was also injected combined with carrageenan. The paw withdrawal threshold based on von Frey filament stimulation was measured until 12h after injection. We compared the area under the time-curve (AUC) between carrageenan and carrageenan with dexmedetomidine. To clarify that the action of dexmedetomidine was via α2-adrenoceptors, we evaluated the effect of yohimbine, a selective antagonist of α2-adrenoceptors, on the anti-nociception of dexmedetomidine. As the results, the intraplantar injection of carrageenan with over 10μM dexmedetomidine significantly increased AUC, compared to that with only carrageenan injection. This effect of dexmedetomidine was reversed by the addition of yohimbine to carrageenan and dexmedetomidine. These results demonstrated that the locally injected dexmedetomidine was effective against carrageenan-induced inflammatory nociception via α2-adrenoceptors. The findings suggest that the local injection of dexmedetomidine is useful for relieving local acute inflammatory nociception.