C. difficile intoxicates neurons and pericytes to drive neurogenic inflammation.

Clostridioides difficile infection (CDI) is a major cause of healthcare-associated gastrointestinal infections1,2. The exaggerated colonic inflammation caused by C. difficile toxins such as toxin B (TcdB) damages tissues and promotes C. difficile colonization3-6, but how TcdB causes inflammation is unclear. Here we report that TcdB induces neurogenic inflammation by targeting gut-innervating afferent neurons and pericytes through receptors, including the Frizzled receptors (FZD1, FZD2 and FZD7) in neurons and chondroitin sulfate proteoglycan 4 (CSPG4) in pericytes. TcdB stimulates the secretion of the neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) from neurons and pro-inflammatory cytokines from pericytes. Targeted delivery of the TcdB enzymatic domain, through fusion with a detoxified diphtheria toxin, into peptidergic sensory neurons that express exogeneous diphtheria toxin receptor (an approach we term toxogenetics) is sufficient to induce neurogenic inflammation and recapitulates major colonic histopathology associated with CDI. Conversely, mice lacking SP, CGRP or the SP receptor (neurokinin 1 receptor) show reduced pathology in both models of caecal TcdB injection and CDI. Blocking SP or CGRP signalling reduces tissue damage and C. difficile burden in mice infected with a standard C. difficile strain or with hypervirulent strains expressing the TcdB2 variant. Thus, targeting neurogenic inflammation provides a host-oriented therapeutic approach for treating CDI.

Therapeutic Potential of MRGPRX2 Inhibitors on Mast Cells.

Mast cells (MCs) act as primary effectors in inflammatory and allergic reactions by releasing intracellularly-stored inflammatory mediators in diseases. The two major pathways for MC activation are known to be immunoglobulin E (IgE)-dependent and -independent. Although IgE-dependent signaling is the main pathway to MC activation, IgE-independent pathways have also been found to serve pivotal roles in the pathophysiology of various inflammatory conditions. Recent studies have shown that human and mouse MCs express several regulatory receptors such as toll-like receptors (TLRs), CD48, C300a, and GPCRs, including mas-related GPCR-X2 (MRGPRX2). MRGPRX2 has been reported as a novel GPCR that is expressed in MCs activated by basic secretagogues, neurokinin peptides, host defense antimicrobial peptides, and small molecule compounds (e.g., neuromuscular blocking agents) and leads to MC degranulation and eicosanoids release under in vitro experimental condition. Functional analyses of MRGPRX2 and Mrgprb2 (mouse ortholog) indicate that MRGPRX2 is involved in MC hypersensitivity reactions causing neuroinflammation such as postoperative pain, type 2 inflammation, non-histaminergic itch, and drug-induced anaphylactic-like reactions. In this review, we discuss the roles in innate immunity through functional studies on MRGPRX2-mediated IgE-independent MC activation and also the therapeutic potential of MRGPRX2 inhibitors on allergic and inflammatory diseases.

Evaluation of the Gastroprotective Effect of the Flavonoid Complex from Lychnis chalcedonica L. on the Models of Experimental Ulcerogenesis.

Using rat and mouse models of neurogenic, ethanol-induced, and indometacin-induced damage to the gastric mucosa we demonstrated that course preventive treatment with flavonoid complex from aerial parts of Lychnis chalcedonica L. increased the resistance of gastric mucosa to ulcerogenic factors of different etiology. The gastroprotective effect of the phytocomplex in a dose range of 16-1600 µg/kg was comparable with that of the reference drug plantaglucide and was superior to that of the reference drugs eleutherococcus extract and methyluracil in the therapeutic doses. The antiulcerogenic activity of Lychnis chalcedonica flavonoid complex considerably exceeded activity of Lychnis chalcedonica L. extract demonstrated in our previous experiments.

Neurovascular inflammation in the pathogenesis of brain arteriovenous malformations.

Brain arteriovenous malformations (bAVM) arise as congenital or sporadic focal lesions with a significant risk for intracerebral hemorrhage (ICH). A wide range of interindividual differences is present in the onset, progression, and severity of bAVM. A growing body of gene expression and polymorphism-based research studies support the involvement of localized inflammation in bAVM disease progression and rupture. In this review article, we analyze the altered responses of neural, vascular, and immune cell types that contribute to the inflammatory process, which exacerbates the pathophysiological progression of vascular dysmorphogenesis in bAVM lesions. The cumulative effect of inflammation in bAVM development is orchestrated by various genetic moderators and inflammatory mediators. We also discuss the potential therapies for the treatment of brain AVM by targeting the inflammatory processes and mediators. Elucidating the precise role of inflammation in the bAVM growth and hemorrhage would open novel avenues for noninvasive and effectual causal therapy that may complement the current therapeutic strategies.

Pathogenesis of pelvic pain syndrome associated with endometriosis in patients resistant to surgical treatment.

AIM: Endometriosis is one of the most common gynecological diseases diagnosed in almost 70% of patients with chronic pelvic pain (CPP). However, a quarter of women with pelvic pain is diagnosed with external genital endometriosis (EGE) during laparoscopy. A special group is represented by patients with PP that did not stop after the removal of endometrial foci. The mechanisms of the pathogenesis of the formation of pain syndrome are not completely explored yet. According to several authors, a significant role in the pathogenesis of pelvic pain recurrence after surgical treatment of EGE is played by active neuroangiogenesis, both in ectopic and eutopic endometrium. The aim of the study was to expand the understanding of the pathogenesis of pelvic pain that did not stop (recurrence) after surgical treatment of external genital endometriosis. MATERIAL AND METHODS: The study involved 2 stages. At the first stage (algological), data from B&B, NRS and VRS algological questionnaires, which were completed by patients with recurrent PP after surgical treatment of EGE, were analyzed (n = 130, aged 18-45 years old, average age 32.5 ± 7.6 years). All women were operated on for EGE no later than 3-6 months after assessing the patients by the algological questionnaires; they did not receive drug therapy after surgical treatment and sought medical attention for recurrent pelvic pain. Materials for the study of the endometrium were obtained by the pipelle biopsymethod. The control group was formed from a number of women with EGE without PP, who applied for surgical treatment of infertility (n = 30). RESULTS: The results of the study have shown that the basis of pathogenesis of pelvic pain recurrence in patients who did not receive medical therapy after surgical treatment of EGE is the activation of neuro-angiogenesis processes and reduction of apoptosis. The results show a statistically significant 1.6 times increasing expression of NGF in eutopic endometrium (57.9 ± 2.5 vs 35.3 ± 2.1% of patients with the silent form of the gene and its receptor NTRK1 1.8 times (2.78 ± 0.25 versus 1.56 ± 0.21.e. respectively). Conclusion: The pathogenesis of pelvic pain in patients who did not receive medical therapy after surgical treatment of endometriosis compared to no pain form of the disease is the activation of the processes of neurogenesis in the eutopic endometrium.

Alarmins and c-Jun N-Terminal Kinase (JNK) Signaling in Neuroinflammation.

Neuroinflammation is involved in the progression or secondary injury of multiple brain conditions, including stroke and neurodegenerative diseases. Alarmins, also known as damage-associated molecular patterns, are released in the presence of neuroinflammation and in the acute phase of ischemia. Defensins, cathelicidin, high-mobility group box protein 1, S100 proteins, heat shock proteins, nucleic acids, histones, nucleosomes, and monosodium urate microcrystals are thought to be alarmins. They are released from damaged or dying cells and activate the innate immune system by interacting with pattern recognition receptors. Being principal sterile inflammation triggering agents, alarmins are considered biomarkers and therapeutic targets. They are recognized by host cells and prime the innate immune system toward cell death and distress. In stroke, alarmins act as mediators initiating the inflammatory response after the release from the cellular components of the infarct core and penumbra. Increased c-Jun N-terminal kinase (JNK) phosphorylation may be involved in the mechanism of stress-induced release of alarmins. Putative crosstalk between the alarmin-associated pathways and JNK signaling seems to be inherently interwoven. This review outlines the role of alarmins/JNK-signaling in cerebral neurovascular inflammation and summarizes the complex response of cells to alarmins. Emerging anti-JNK and anti-alarmin drug treatment strategies are discussed.

CNTF-STAT3-IL-6 Axis Mediates Neuroinflammatory Cascade across Schwann Cell-Neuron-Microglia.

Neuroinflammation is a crucial mechanism in many neurological disorders. Injury to the peripheral sensory nerves leads to a neuroinflammatory response in the somatosensory pathway, from dorsal root ganglia (DRG) to the spinal cord, contributing to neuropathic pain. How the immune reaction is initiated peripherally and propagated to the spinal cord remains less clear. Here, we find that ciliary neurotrophic factor (CNTF), highly expressed in Schwann cells, mediates neuroinflammatory response through the activating signal transducer and activator of transcription 3 (STAT3) and inducing interleukin 6 (IL-6) in sensory neurons. Cntf deficiency attenuates neuroinflammation in DRG and the spinal cord with alleviated pain post-injury. Recombinant CNTF applied to the sensory nerves recapitulates neuroinflammation in the DRG and spinal cord, with consequent pain development. We delineate the CNTF-STAT3-IL-6 axis in mediating the onset and progression of the inflammatory cascade from the periphery to the spinal cord with therapeutic implications for neuropathic pain.

Cholinergic leukocytes in sepsis and at the neuroimmune junction in the spleen.

The spleen is a key participant in the pathophysiology of sepsis and inflammatory disease. Many splenocytes exhibit a cholinergic phenotype, but our knowledge regarding their cholinergic biology and how they are affected by sepsis is incomplete. We evaluated effects of acute sepsis on the spleen using the cecal ligation and puncture (CLP) model in C57BL/6 and ChATBAC-eGFP mice. Quantification of cholinergic gene expression showed that choline acetyltransferase and vesicular acetylcholine transporter (VAChT) are present and that VAChT is upregulated in sepsis, suggesting increased capacity for release of acetylcholine (ACh). High affinity choline transporter is not expressed but organic acid transporters are, providing additional mechanisms for release. Flow cytometry studies identified subpopulations of cholinergic T and B cells as well as monocytes/macrophages. Neither abundance nor GFP intensity of cholinergic T cells changed in sepsis, suggesting that ACh synthetic capacity was not altered. Spleens have low acetylcholinesterase activity, and the enzyme is localized primarily in red pulp, characteristics expected to favor cholinergic signaling. For cellular studies, ACh was quantified by mass spectroscopy using d4-ACh internal standard. Isolated splenocytes from male mice contain more ACh than females, suggesting the potential for gender-dependent differences in cholinergic immune function. Isolated splenocytes exhibit basal ACh release, which can be increased by isoproterenol (4 and 24 h) or by T cell activation with antibodies to CD3 and CD28 (24 h). Collectively, these data support the concept that sepsis enhances cholinergic function in the spleen and that release of ACh can be triggered by stimuli via different mechanisms.

Evidence for neurogenic inflammation in lichen planopilaris and frontal fibrosing alopecia pathogenic mechanism.

Lichen planopilaris (LPP) and frontal fibrosing alopecia (FFA) are lymphocytic scarring alopecias affecting primarily the scalp. Although both diseases may share some clinical and histopathological features, in the last decade, FFA has become an "epidemic" particularly in Europe, North and South America with unique clinical manifestations compared to LPP, thus, raising the idea that this disease may have a different pathogenesis. Symptoms such as scalp burning, pruritus or pain are usually present in both diseases, suggesting a possible role for nerves and neuropeptides in the pathogenesis of both diseases. Based on some previous studies, neuropeptides, such as substance P (SP) and calcitonin gene-related peptide (CGRP), have been associated with lipid metabolism and many chronic inflammatory disorders. In this study, we asked if these neuropeptides are associated with LPP and FFA scalp lesions. Alteration in the expression of SP and CGRP in affected and unaffected scalp skin from patients with both diseases was found with examination of sections using immunohistochemical techniques and confocal microscopy. We then quantitatively assessed and compared SP and CGRP expression from control, LPP and FFA scalp biopsies. Although LPP and FFA share similar histopathologic findings, opposite results were found in affected and unaffected scalp in the ELISA tests, suggesting that these diseases may have different pathogenic mechanisms. We also found presence of histopathological inflammation irrespective of evident clinical lesions, which raises the possibility that both diseases may be more generalized processes affecting the scalp.

Novel charged sodium and calcium channel inhibitor active against neurogenic inflammation.

Voltage-dependent sodium and calcium channels in pain-initiating nociceptor neurons are attractive targets for new analgesics. We made a permanently charged cationic derivative of an N-type calcium channel-inhibitor. Unlike cationic derivatives of local anesthetic sodium channel blockers like QX-314, this cationic compound inhibited N-type calcium channels more effectively with extracellular than intracellular application. Surprisingly, the compound is also a highly effective sodium channel inhibitor when applied extracellularly, producing more potent inhibition than lidocaine or bupivacaine. The charged inhibitor produced potent and long-lasting analgesia in mouse models of incisional wound and inflammatory pain, inhibited release of the neuropeptide calcitonin gene-related peptide (CGRP) from dorsal root ganglion neurons, and reduced inflammation in a mouse model of allergic asthma, which has a strong neurogenic component. The results show that some cationic molecules applied extracellularly can powerfully inhibit both sodium channels and calcium channels, thereby blocking both nociceptor excitability and pro-inflammatory peptide release.

Nerve sprouting and neurogenic inflammation characterize the neurogenic detrusor overactive bladder of patients no longer responsive to drug therapies.

Urothelium and Lamina Propria (LP) are considered an integrate sensory system which is able to control the detrusor activity. Complete supra-sacral spinal cord lesions cause Neurogenic Detrusor Overactivity (NDO) whose main symptoms are urgency and incontinence. NDO therapy at first consists in anti-muscarinic drugs; secondly, in intra-vesical injection of botulinum toxin. However, with time, all the patients become insensitive to the drugs and decide for cystoplastic surgery. With the aim to get deeper in both NDO and drug's efficacy lack pathogenesis, we investigated the innervation, muscular and connective changes in NDO bladders after surgery by using morphological and quantitative methodologies. Bladder innervation showed a significant global loss associated with an increase in the nerve endings located in the upper LP where a neurogenic inflammation was also present. Smooth muscle cells (SMC) anomalies and fibrosis were found in the detrusor. The increased innervation in the ULP is suggestive for a sprouting and could condition NDO evolution and drug efficacy length. Denervation might cause the SMC anomalies responsible for the detrusor altered contractile activity and intra-cellular traffic and favour the appearance of fibrosis. Inflammation might accelerate these damages. From the clinical point of view, an early anti-inflammatory treatment could positively influence the disease fate.

OTUB1 inhibits CNS autoimmunity by preventing IFN-γ-induced hyperactivation of astrocytes.

Astrocytes are critical regulators of neuroinflammation in multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). Growing evidence indicates that ubiquitination of signaling molecules is an important cell-intrinsic mechanism governing astrocyte function during MS and EAE Here, we identified an upregulation of the deubiquitinase OTU domain, ubiquitin aldehyde binding 1 (OTUB1) in astrocytes during MS and EAE Mice with astrocyte-specific OTUB1 ablation developed more severe EAE due to increased leukocyte accumulation, proinflammatory gene transcription, and demyelination in the spinal cord as compared to control mice. OTUB1-deficient astrocytes were hyperactivated in response to IFN-γ, a fingerprint cytokine of encephalitogenic T cells, and produced more proinflammatory cytokines and chemokines than control astrocytes. Mechanistically, OTUB1 inhibited IFN-γ-induced Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling by K48 deubiquitination and stabilization of the JAK2 inhibitor suppressor of cytokine signaling 1 (SOCS1). Thus, astrocyte-specific OTUB1 is a critical inhibitor of neuroinflammation in CNS autoimmunity.

Auricular vagus nerve stimulation protects against postoperative cognitive dysfunction by attenuating neuroinflammation and neurodegeneration in aged rats.

Postoperative cognitive dysfunction (POCD) has been increasingly recognized as a significant complication after surgery, especially in senior patients. Vagus nerve stimulation (VNS) reportedly provides beneficial effects against various brain disorders, supporting a hypothesis of its protective role in POCD. However, direct stimulation of the vagus nerve is invasive, as it requires a surgical incision in the neck. Thus, we employed a non-invasive VNS method by stimulating the dermatome in the external ear, which is innervated by the vagus nerve (auricular vagus nerve stimulation; aVNS) and sought to investigate the efficacy of this method in treating surgery-induced cognitive dysfunction in an aged rat model of POCD. We observed that the treatment of aVNS alleviated postoperative memory impairment after exploratory laparotomy surgery, as demonstrated by the shorter swimming latency and distance in Morris water maze tests. Moreover, aVNS also reduced postoperative apoptosis in the hippocampus of the aged rats. Concomitant with these beneficial effects, we found that treatment with aVNS attenuated postoperative neuroinflammation (i.e., the protein level of interleukin-1ß and tumor necrosis factor-α, along with the nuclear protein expression of NF-κB) and Alzheimer's-related pathology (tau phosphorylation at AT-8 and Ser396, as well as the levels of Aß40 and Aß42) in the hippocampus of the aged rats. In conclusion, our study is the first to reveal the neuroprotective effect of aVNS against POCD. This effect might be attributed to the inhibition of neuroinflammation and Alzheimer's-related pathology. This study suggests non-invasive aVNS may serve as a promising method for clinical treatment of POCD.

Nogo-A promotes inflammatory heat hyperalgesia by maintaining TRPV-1 function in the rat dorsal root ganglion neuron.

Nogo-A is a key inhibitory molecule of axon regeneration in oligodendrocytes. However, little is known about its role in adult neurons. In this study, we showed an important function of Nogo-A on regulation of inflammatory pain in dorsal root ganglion (DRG) neurons. In adult rats with complete Freund's adjuvant (CFA) hind paw inflammation, DRG neurons showed a significant increase in Nogo-A expression. Disruption of Nogo-A signaling with Nogo-66 receptor antagonist peptide, Nogo-A blocking antibody, Nogo-A short hairpin RNA, or Nogo-A gene knockout attenuated CFA-induced inflammatory heat hyperalgesia. Moreover, disruption of Nogo-A signaling suppressed the function and expression in DRG neurons of the transient receptor potential vanilloid subfamily member (TRPV)-1 channel, which is known to be the endogenous transducer of noxious heat during inflammation. These effects were accompanied with a reduction in LIM domain kinase (LIMK)/cofilin phosphorylation and actin polymerization. Similar disruption of actin filament architecture by direct action of Latrunculin A reduced the TRPV-1 activity and up-regulation of TRPV-1 protein caused by CFA. We conclude that Nogo-A plays an essential role in the development of inflammatory heat hyperalgesia, partly through maintaining TRPV-1 function via activation of the LIMK/cofilin pathway, which regulates actin filament dynamics. These findings support a therapeutic potential of modulating Nogo-A signaling in pain management.-Hu, F., Liu, H.-C., Su, D.-Q., Chen, H.-J., Chan, S.-O., Wang, Y., Wang, J. Nogo-A promotes inflammatory heat hyperalgesia by maintaining TRPV-1 function in the rat dorsal root ganglion neuron.

Roles of inflammation, neurogenic inflammation, and neuroinflammation in pain.

Inflammation is the body's response to injury and infection, involving a complex biological response of the somatosensory, immune, autonomic, and vascular systems. Inflammatory mediators such as prostaglandin, proinflammatory cytokines, and chemokines induce pain via direct activation of nociceptors, the primary sensory neurons that detect noxious stimuli. Neurogenic inflammation is triggered by nerve activation and results in neuropeptide release and rapid plasma extravasation and edema, contributing to pain conditions such as headache. Neuroinflammation is a localized inflammation in the peripheral nervous system (PNS) and central nervous system (CNS). A characteristic feature of neuroinflammation is the activation of glial cells in dorsal root ganglia, spinal cord, and brain which leads to the production of proinflammatory cytokines and chemokines in the PNS and CNS that drives peripheral sensitization and central sensitization. Here, we discuss the distinct roles of inflammation, neurogenic inflammation, and neuroinflammation in the regulation of different types of pain conditions, with a special focus on neuroinflammation in postoperative pain and opioid-induced hyperalgesia.

Inflammasomes: An Emerging Mechanism Translating Environmental Toxicant Exposure Into Neuroinflammation in Parkinson's Disease.

Evidence indicates that complex gene-environment interactions underlie the incidence and progression of Parkinson's disease (PD). Neuroinflammation is a well-characterized feature of PD widely believed to exacerbate the neurodegenerative process. Environmental toxicants associated with PD, such as pesticides and heavy metals, can cause cellular damage and stress potentially triggering an inflammatory response. Toxicant exposure can cause stress and damage to cells by impairing mitochondrial function, deregulating lysosomal function, and enhancing the spread of misfolded proteins. These stress-associated mechanisms produce sterile triggers such as reactive oxygen species (ROS) along with a variety of proteinaceous insults that are well documented in PD. These associations provide a compelling rationale for analysis of sterile inflammatory mechanisms that may link environmental exposure to neuroinflammation and PD progression. Intracellular inflammasomes are cytosolic assemblies of proteins that contain pattern recognition receptors, and a growing body of evidence implicates the association between inflammasome activation and neurodegenerative disease. Characterization of how inflammasomes may function in PD is a high priority because the majority of PD cases are sporadic, supporting the widely held belief that environmental exposure is a major factor in disease initiation and progression. Inflammasomes may represent a common mechanism that helps to explain the strong association between exposure and PD by mechanistically linking environmental toxicant-driven cellular stress with neuroinflammation and ultimately cell death.

Expression of acetyl-histone H3 and acetyl-histone H4 in dorsal root ganglion and spinal dorsal horn in rat chronic pain models.

AIMS: Histone acetylation and deacetylation are two histone posttranslational modifications that are usually controlled by histone acetyltransferases (HATs) and histone deacetylases (HDACs). Although HATs or HDACs Inhibitors could relieve pain hypersensitivities in chronic pain animal models, it is not clear on the expression of global histone acetylation in the dorsal root ganglion (DRG) or spinal dorsal horn in chronic pain conditions. MAIN METHODS: A spinal nerve ligation (SNL)-induced neuropathic pain model and a complete Freund's adjuvant (CFA)-induced inflammatory pain model in rats were used to examine the expression of total acetyl-histone H3 (AcH3) and total acetyl-histone H4 (AcH4) by immunofluorescence or western blot. KEY FINDINGS: AcH3 and AcH4 not only localized in neuronal nuclei, but also in nuclei of glial cells in the DRG. Unilateral SNL induced the increase of AcH3 and AcH4 expression in the injured lumbar 5 (L5) DRG, but not in the uninjured L5 DRG or the spinal dorsal horn, while unilateral intraplantar injection of CFA increased AcH3 and AcH4 expression in the ipsilateral L4/5 spinal dorsal horn, but not in the L4/5 DRG. SIGNIFICANCE: These results provide morphological evidence for global histone acetylation expression in the DRG and spinal cord and indicate the differential expression in the DRG and spinal dorsal horn in different chronic pain models. More precise epigenetic mechanisms of histone acetylation on the target genes need to be revealed.

The role of neutrophil granule proteins in neuroinflammation and Alzheimer's disease.

Neutrophils are the innate immune system's first line of defense. Neutrophils play a critical role in protecting the host against infectious pathogens, resolving sterile injuries, and mediating inflammatory responses. The granules of neutrophils and their constituent proteins are central to these functions. Although neutrophils may exert a protective role upon acute inflammatory conditions or insults, continued activity of neutrophils in chronic inflammatory diseases can contribute to tissue damage. Neutrophil granule proteins are involved in a number of chronic inflammatory conditions and diseases. However, the functions of these proteins in neuroinflammation and chronic neuroinflammatory diseases, including Alzheimer's disease (AD), remain to be elucidated. In this review, we discuss recent findings from our lab and others that suggest possible functions for neutrophils and the neutrophil granule proteins, CAP37, neutrophil elastase, and cathepsin G, in neuroinflammation, with an emphasis on AD. These findings reveal that neutrophil granule proteins may exert both neuroprotective and neurotoxic effects. Further research should determine whether neutrophil granule proteins are valid targets for therapeutic interventions in chronic neuroinflammatory diseases.

Potentiation of capsaicin-induced neurogenic inflammation by 5-HT7 receptors in the rat hind paw: Involvement of calcitonin gen-related peptide.

A decrease in the activation threshold of primary sensory neurons to transient receptor potential V1 (TRPV1) stimulation by serotonin 5-HT7 receptors has been reported but no confirmation if this might translate into facilitation of neurogenic inflammation has been provided. We analysed the modulation of capsaicin (CAP)-induced neurogenic inflammation in the rat hind paw by the selective 5-HT7 receptor agonist, LP-44, and the involvement of calcitonin gen-related peptide (CGRP) in this effect. Animals received intra-plantar injections (30 µL) of vehicle, CAP (0.05%, 0.1% and 0.2%), LP-44 (7.5 and 15 nmol) and the combination of LP-44 + CAP; then, the time course of the inflammatory responses was measured. The effect of the 5-HT7 receptor antagonist, SB-269970 (3 mg/kg, s.c.), on responses produced by LP-44 alone and combined with CAP was tested. As expected, CAP produced concentration- and time-dependent inflammatory responses in the hind paw. Interestingly, LP-44 by itself also produced inflammation in a concentration- and time-dependent manner, and magnified CAP-induced responses. Systemic pre-treatment with SB-269970 significantly blunted LP-44 (15 nmol)-induced inflammation as well as magnified inflammatory responses produced by the combination of LP-44 (7.5 and 15 nmol) + CAP (0.1%) thus confirming the involvement of 5-HT7 receptors. Finally, the non-peptide CGRP receptor antagonist, BIBN4096 (3 mg/kg, s.c.), strongly inhibited the potentiated inflammatory responses induced by LP-44 (7.5 and 15 nmol) + CAP (0.1%) thus substantiating their neurogenic nature. Thus, sensitization of CAP-sensitive primary sensory neurons by 5-HT7 receptors may result in facilitation of neurogenic inflammation involving CGRP in the rat hind paw.

Psychophysical and vasomotor evidence for interdependency of TRPA1 and TRPV1-evoked nociceptive responses in human skin: an experimental study.

The TRPA1 and TRPV1 receptors are important pharmaceutical targets for antipruritic and analgesic therapy. Obtaining further knowledge on their roles and interrelationship in humans is therefore crucial. Preclinical results are contradictory concerning coexpression and functional interdependency of TRPV1 and TRPA1, but no human evidence exists. This human experimental study investigated whether functional responses from the subpopulation of TRPA1 nociceptors could be evoked after defunctionalization of TRPV1 nociceptors by cutaneous application of high-concentration capsaicin. Two quadratic areas on each forearm were randomized to pretreatment with an 8% topical capsaicin patch or vehicle for 24 hours. Subsequently, areas were provoked by transdermal 1% topical capsaicin (TRPV1 agonist) or 10% topical allyl isothiocyanate ("AITC," a TRPA1 agonist), delivered by 12 mm Finn chambers. Evoked pain intensities were recorded during pretreatments and chemical provocations. Quantitative sensory tests were performed before and after provocations to assess changes of heat pain sensitivity. Imaging of vasomotor responses was used to assess neurogenic inflammation after the chemical provocations. In the capsaicin-pretreated areas, both the subsequent 1% capsaicin- and 10% AITC-provoked pain was inhibited by 92.9 ± 2.5% and 86.9 ± 5.0% (both: P < 0.001), respectively. The capsaicin-ablated skin areas showed significant heat hypoalgesia at baseline (P < 0.001) as well as heat antihyperalgesia, and inhibition of neurogenic inflammation evoked by both 1% capsaicin and 10% AITC provocations (both: P < 0.001). Ablation of cutaneous capsaicin-sensitive afferents caused consistent and equal inhibition of both TRPV1- and TRPA1-provoked responses assessed psychophysically and by imaging of vasomotor responses. This study suggests that TRPA1 nociceptive responses in human skin strongly depend on intact capsaicin-sensitive, TRPV1 fibers.