@article{mishra_gaddameedhi_2023, title={A new role of TRPM8 in circadian rhythm and molecular clock}, volume={1}, ISSN={["1748-1716"]}, url={https://doi.org/10.1111/apha.13934}, DOI={10.1111/apha.13934}, abstractNote={Over the past decades, studies of the TRPM8 channel, a nonselective cation channel, have provided much insight into the fundamental mechanisms of sensory neuron function that lead to the detection of cold sensors. In the current issue of Acta Physiologica, Reimundez et al1 provide evidence of a novel role of TRPM8 in circadian function in mice. The TRPM (transient receptor potential ion channels, where “m” stands for melastatin) is a family of eight different channels, TRPM1TRPM8.2 The TRPM8 is a cold sensor and is also activated by chemical ligands such as menthol and icilin, but recently, their role in maintaining core body temperature has also been recognized.3,4 TRPM8 is expressed in sensory neurons whose axonal afferents innervate peripheral tissues such as skin and oral cavity. Still, it has not been detected in the brain and spinal cord. Besides nerve tissues, TRPM8 is expressed in tissues such as the prostate, bladder, lungs, and urogenital tract. However, other than the sensory nervous system, the functional role of TRPM8 channels is not well understood. In this issue of Acta Physiologica, Reimundez et al1 provide solid experimental evidence for such a novel mechanism— where they demonstrate that TRPM8 might regulate Period 2 (Per2) mRNA levels in central clock (SCN) and peripheral clock (liver and white adipose tissue) and the circadian regulation of core body temperature. Their publication is among the most significant advances in the field of circadian regulation using cold sensors— it will probably set up the stage for several future research activities in which Reimundez and colleagues' ideas will be rigorously dissected and probed in circadian biology research. Previously, Ordás et al5 demonstrated the presence of TRPM8 fibers in the suprachiasmatic nucleus of the hypothalamus (SCN) of the brain, which is the principal circadian pacemaker in mammals and host a range of other physiological processes such as circadian oscillation, autonomic/peripheral and central nervous system function, regulating core body temperature and sleep– wake cycle. Since SCN receives axonal projections mainly from intrinsically photosensitive retinal ganglion cells (ipRGCs) that are responsible for resetting the circadian clock through SCN neuron activity with light as a primary zeitgeber.6 Here, authors used conventional PCR and mouse reporter lines to show the expression of TRPM8 in the inner retina, specifically in the ganglion cell layer (GC) and in the inner nuclear layer (INL). Next, they used the colocalization technique to demonstrate that melanopsin, a marker of ipRGCs7 and using mouse reporter lines, that TRPM8 expressing cells are indeed expressed in a subset of ipRGCs cells. Furthermore, they showed that TRPM8 is also expressed in cholinergic amacrine cells, characterized by releasing two neurotransmitters, GABA and acetylcholine. Injection of the fluorescent anterograde tracer cholera toxin subunit B (CTB594) into both eyes in two TRPM8 reporter lines indicates the existence of ipRGCs expressing TRPM8 and projecting to the SCN (Figure 1). The choroid is an essential high blood flow vascular structure in the eye and is known to regulate ocular and retinal temperature8 and is richly innervated by sensory trigeminal nerve fibers; therefore, the authors next examined the role of TRPM8 in regulation of eye temperature (Teye) using infrared (IR) thermography. Interestingly, they discovered mice lacking TRPM8 started to decline at temperatures below 25°C, and Trpm8−/− mice displayed a significantly lower Teye than WT littermates when the temperature plate was around 15°C. This suggests a function of TRPM8 and ambient temperature in regulating choroid and ciliary body blood flow and, therefore, in controlling internal ocular temperature. The ability of mice to regulate homeostatic temperature control and the expression of TRPM8 in the ipRGCs is indicative but does not directly link that to central clock regulation. The authors performed expression and functional studies to investigate a direct link between TRPM8 and circadian clockwork at the SCN. First, they determined the expression of the Per2 gene, which is one of the essential components of core circadian clocks in the SCN.9 Authors found in the TRPM8 deficient mouse SCN a significant}, journal={ACTA PHYSIOLOGICA}, author={Mishra, Santosh K. and Gaddameedhi, Shobhan}, year={2023}, month={Jan} }
 @article{meneses_gidcumb_marcus_gonzalez_lai_mishra_lascelles_nolan_2023, title={Acute radiotherapy-associated oral pain may promote tumor growth at distant sites}, volume={13}, ISSN={["2234-943X"]}, DOI={10.3389/fonc.2023.1029108}, abstractNote={Introduction Patients developing acute radiotherapy induced dermatitis or oral mucositis commonly experience pain. When severe, this radiotherapy-associated pain (RAP) can necessitate treatment breaks; unfortunately, in a variety of cancers, prolongation of the radiotherapy course has been associated with early cancer relapse and/or death. This is often attributed to accelerated repopulation, but it is unknown whether pain or pain signaling constituents might alter tumor behavior and hasten metastatic disease progression. We studied this by testing the hypothesis that severe acute RAP at one site can hasten tumor growth at a distant site. Methods Mice underwent single fraction tongue irradiation (27 Gy, or 0 Gy “sham” control) to induce severe glossitis. At the time of maximal oral RAP, one of three luciferase-transfected tumor cell lines were injected via tail vein (4T1, B16F10, MOC2; each paired to their syngeneic host: BALB/c or C57BL/6); tumor burden was assessed via in vivo transthoracic bioluminescence imaging and ex vivo pulmonary nodule quantification. Survival was compared using Kaplan-Meier statistics. Results Tongue irradiation and resultant RAP promoted lung tumor growth of 4T1-Luc2 cells in BALB/c mice. This effect was not a result of off-target radiation, nor an artefact of environmental stress caused by standard (subthermoneutral) housing temperatures. RAP did not affect the growth of B16F10-Luc2 cells, however, C57BL/6 mice undergoing tail vein injection of MOC2-Luc2 cells at the time of maximal RAP experienced early lung tumor-attributable death. Lung tumor growth was normalized when RAP was reduced by treatment with resiniferatoxin (300 µg/kg, subcutaneously, once). Discussion This research points towards radiation-induced activation of capsaicin-responsive (TRPV1) neurons as the cause for accelerated growth of tumors at distant (unirradiated) sites.}, journal={FRONTIERS IN ONCOLOGY}, author={Meneses, Constanza S. and Gidcumb, Emily M. and Marcus, Karen L. and Gonzalez, Yarines and Lai, Yen Hao and Mishra, Santosh K. and Lascelles, B. Duncan X. and Nolan, Michael W.}, year={2023}, month={May} }
 @article{minnema_gupta_mishra_lascelles_2022, title={Investigating the Role of Artemin and Its Cognate Receptor, GFR alpha 3, in Osteoarthritis Pain}, volume={16}, ISSN={["1662-453X"]}, url={https://europepmc.org/articles/PMC8829392}, DOI={10.3389/fnins.2022.738976}, abstractNote={Osteoarthritis (OA) associated pain (OA-pain) is a significant global problem. OA-pain limits limb use and mobility and is associated with widespread sensitivity. Therapeutic options are limited, and the available options are often associated with adverse effects. The lack of therapeutic options is partly due to a lack of understanding of clinically relevant underlying neural mechanisms of OA-pain. In previous work in naturally occurring OA-pain in dogs, we identified potential signaling molecules (artemin/GFRα3) that were upregulated. Here, we use multiple approaches, including cellular, mouse genetic, immunological suppression in a mouse model of OA, and clinically relevant measures of sensitivity and limb use to explore the functional role of artemin/GFRα3 signaling in OA-pain. We found the monoiodoacetate (MIA)-induced OA-pain in mice is associated with decreased limb use and hypersensitivity. Exogenous artemin induces mechanical, heat, and cold hypersensitivity, and systemic intraperitoneal anti-artemin monoclonal antibody administration reverses this hypersensitivity and restores limb use in mice with MIA-induced OA-pain. An artemin receptor GFRα3 expression is increased in sensory neurons in the MIA model. Our results provide a molecular basis of arthritis pain linked with artemin/GFRα3 signaling and indicate that further work is warranted to investigate the neuronal plasticity and the pathways that drive pain in OA.}, journal={FRONTIERS IN NEUROSCIENCE}, author={Minnema, Laura and Gupta, Ankita and Mishra, Santosh K. and Lascelles, B. Duncan X.}, year={2022}, month={Jan} }
 @misc{shirolkar_mishra_2022, title={Role of TRP ion channels in pruritus}, volume={768}, ISSN={["1872-7972"]}, DOI={10.1016/j.neulet.2021.136379}, abstractNote={The transient receptor potential (TRP) channel superfamily responds to various physical, chemical, and environmental stimuli including the detection of sensations both harmful and non-harmful. Among these sensations is pruritus, or itch. There are at least 27 different TRP channels and about six of them are involved in pruriception. The function of these six receptors is primarily seen in the skin and the dorsal root ganglia. Identification and biological insights provided by these receptors in pruriception is important for human health as mutations and activations of many of these channels cause discomfort and disease. This review will focus on involvement of TRP channels in pruriception that may render these channels as the targets of many antagonistic topical medications, which may help patients' better cope with the pruritus that results from various cutaneous and systemic diseases.}, journal={NEUROSCIENCE LETTERS}, author={Shirolkar, Parth and Mishra, Santosh K.}, year={2022}, month={Jan} }
 @article{mishra_2022, title={The Role of CNTNAP2 in Itch Sensation}, volume={142}, ISSN={["1523-1747"]}, DOI={10.1016/j.jid.2021.07.152}, abstractNote={Itch is a hallmark symptom associated with atopic dermatitis (AD), which is an allergic disorder accentuated by both immunological dysregulation and epidermal barrier defect (Wahlgren, 1991). Autism spectrum disorders (ASDs) are complex neurobehavioral and neurodevelopmental conditions that cause a variety of phenotypes, including impaired social communication, stereotyped behaviors, and altered sensory processing (Dawes et al., 2018; Vahia, 2013). A longitudinal study and a systemic review reveal an association between early AD and subsequent ASD (Lee et al., 2016; Tongo et al., 2015), but how itch is regulated in individuals with ASD remains unknown.}, number={1}, journal={JOURNAL OF INVESTIGATIVE DERMATOLOGY}, author={Mishra, Santosh K.}, year={2022}, month={Jan}, pages={251–253} }
 @misc{fan_mishra_2022, title={The emerging role of neuroimmune interactions in atopic dermatitis and itch}, volume={289}, ISSN={["1742-4658"]}, DOI={10.1111/febs.15860}, abstractNote={Millions of people globally suffer from allergic diseases, and the cases have been rising in the past decades. One of the major manifestations of allergic diseases is itch, which is an unpleasant symptom that triggers the urge to scratch and greatly affects the quality of life. Thus, research on how sensation of itch is detected/transmitted from the contact of the allergen to the nervous system is crucial in mitigating itch. Recent studies have attempted to elucidate the mechanisms of itch in allergic diseases. Here, we aim to review the endogenous mediators released from immune/nonimmune skin cells (that are indirectly involved in the propagation of itch) and the sensory neurons that express receptors for these itch mediators that are associated with direct transmission of itch in cutaneous allergic diseases. As the mechanisms for allergic itch become clearer, new therapeutic approaches to relieve itch are likely to be developed. Recent clinical trials are testing numerous compounds that target the endogenous mediators and their receptors. These studies provide the possibility of more effective itch treatment for allergic diseases.}, number={10}, journal={FEBS JOURNAL}, author={Fan, Jennifer and Mishra, Santosh K.}, year={2022}, month={May}, pages={2723–2735} }
 @article{lai_baumer_meneses_roback_robertson_mishra_lascelles_nolan_2021, title={Irradiation of the Normal Murine Tongue Causes Upregulation and Activation of Transient Receptor Potential (TRP) Ion Channels}, volume={196}, ISSN={["1938-5404"]}, DOI={10.1667/RADE-21-000103.1}, abstractNote={Signal transduction at sensory neurons occurs via transmembrane flux of cations, which is largely governed by the transient receptor potential (TRP) family of ion channels. It is unknown whether TRP channel activation contributes to the pain that accompanies radiation-induced oral mucositis. This study sought to characterize changes in TRP channel expression and function that occur in the locally irradiated tissues and afferent neurons of mice. Female CD-1 mice received single high-dose (27 Gy) tongue irradiation, or sham irradiation. Animals were euthanized either before overt glossitis developed (days 1 and 5 postirradiation), when glossitis was severe (day 11), or after mice had recovered (days 21 and 45). Tongue irradiation caused upregulation of the Trpv1 gene in trigeminal ganglia (TG) neurons. Other TRP genes (Trpv2, Trpv4, Trpa1, Trpm8) and Gfrα3 (which acts upstream of several TRP channels) were also upregulated in TGs and/or tongue tissue, in response to radiation. Ex vivo calcium imaging experiments demonstrated that the proportions of TG neurons responding to histamine (an activator of TRPV1, TRPV4 and TRPA1), TNF-α (an activator of TRPV1, TRPV2 and TRPV4), and capsaicin (a TRPV1 agonist), were increased as early as one day after tongue irradiation; these changes persisted for at least 21 days. In a subsequent experiment, we found that genetic deletion of TRPV1 mitigated weight loss (a surrogate marker of pain severity) in mice with severe glossitis. The results intimate that various TRP channels, and TRPV1 in particular, should be explored as analgesic targets for patients experiencing pain after oral irradiation.}, number={4}, journal={RADIATION RESEARCH}, author={Lai, Yen and Baumer, Wolfgang and Meneses, Constanza and Roback, Donald M. and Robertson, James B. and Mishra, Santosh K. and Lascelles, B. Duncan X. and Nolan, Michael W.}, year={2021}, month={Oct}, pages={331–344} }
 @misc{hashimoto_mishra_olivry_yosipovitch_2021, title={Periostin, an Emerging Player in Itch Sensation}, volume={141}, ISSN={["1523-1747"]}, url={https://doi.org/10.1016/j.jid.2021.03.009}, DOI={10.1016/j.jid.2021.03.009}, abstractNote={Periostin, an extracellular matrix and matricellular protein, binds to several types of integrins that transduce its signals. Its function in allergic inflammation is the establishment of sustained chronic inflammation through an amplification of T helper type 2‒immune responses. In addition, recent studies have shown a significant role of periostin in itch sensation through direct integrin-mediated stimulation of nerve fibers and interaction with immune and nonimmune cells (e.g., macrophages, eosinophils, basophils, and keratinocytes). The objective of this review is to describe the role of periostin in itch induction in human and animal models and its expression in human pruritic conditions.}, number={10}, journal={JOURNAL OF INVESTIGATIVE DERMATOLOGY}, publisher={Elsevier BV}, author={Hashimoto, Takashi and Mishra, Santosh K. and Olivry, Thierry and Yosipovitch, Gil}, year={2021}, month={Oct}, pages={2338–2343} }
 @article{gupta_chiavaccini_minnema_chiu_knazovicky_hash_mishra_lascelles_2021, title={Serum artemin is not correlated with sensitivity within dogs with naturally occurring osteoarthritis pain}, volume={11}, ISSN={["2045-2322"]}, url={https://europepmc.org/articles/PMC7988108}, DOI={10.1038/s41598-021-85976-y}, abstractNote={Osteoarthritis (OA) pain is associated with peripheral and central sensitization in humans and results in widespread increased sensitivity across the body. Sensitization contributes to the OA-associated pain (OAP) state. We recently identified increased levels of an endogenous neurotrophic factor, artemin (ARTN), in dogs with OAP compared to healthy pain-free controls. Circulating ARTN released from damaged tissues in OA, may play a central role in widespread sensitivity and pain. However, the relationship between ARTN and somatosensory sensitivity remains unknown. The study aimed to assess the relationship between serum ARTN concentrations and measures of sensitivity in dogs with OAP using quantitative sensory testing. We hypothesized that there would be a positive association between circulating ARTN and increased sensitivity to mechanical and thermal stimuli in dogs with OAP. We used linear and logistic regression models to assess the relationship between ARTN, sensitization, and pain within a cohort of 43 dogs with spontaneous OAP. Serum ARTN was not associated with the degree of sensitization within dogs with OAP. Further, across dogs with varying OAP severity, we did not find any association between ARTN, and clinical measures of joint pain and disability. Although a relationship between ARTN and joint pain was not ruled out.}, number={1}, journal={SCIENTIFIC REPORTS}, author={Gupta, Ankita and Chiavaccini, Ludovica and Minnema, Laura M. and Chiu, King Wa and Knazovicky, David and Hash, Jonathan A. and Mishra, Santosh K. and Lascelles, B. Duncan X.}, year={2021}, month={Mar} }
 @article{wilzopolski_kietzmann_mishra_stark_baeumer_rossbach_2021, title={TRPV1 and TRPA1 Channels Are Both Involved Downstream of Histamine-Induced Itch}, volume={11}, ISSN={["2218-273X"]}, DOI={10.3390/biom11081166}, abstractNote={Two histamine receptor subtypes (HR), namely H1R and H4R, are involved in the transmission of histamine-induced itch as key components. Although exact downstream signaling mechanisms are still elusive, transient receptor potential (TRP) ion channels play important roles in the sensation of histaminergic and non-histaminergic itch. The aim of this study was to investigate the involvement of TRPV1 and TRPA1 channels in the transmission of histaminergic itch. The potential of TRPV1 and TRPA1 inhibitors to modulate H1R- and H4R-induced signal transmission was tested in a scratching assay in mice in vivo as well as via Ca2+ imaging of murine sensory dorsal root ganglia (DRG) neurons in vitro. TRPV1 inhibition led to a reduction of H1R- and H4R- induced itch, whereas TRPA1 inhibition reduced H4R- but not H1R-induced itch. TRPV1 and TRPA1 inhibition resulted in a reduced Ca2+ influx into sensory neurons in vitro. In conclusion, these results indicate that both channels, TRPV1 and TRPA1, are involved in the transmission of histamine-induced pruritus.}, number={8}, journal={BIOMOLECULES}, author={Wilzopolski, Jenny and Kietzmann, Manfred and Mishra, Santosh K. and Stark, Holger and Baeumer, Wolfgang and Rossbach, Kristine}, year={2021}, month={Aug} }
 @article{hoffman_kyriazis_dimitriou_mishra_koch_drosatos_2020, title={B-type natriuretic peptide is upregulated by c-Jun N-terminal kinase and contributes to septic hypotension}, volume={5}, ISSN={["2379-3708"]}, DOI={10.1172/jci.insight.133675}, abstractNote={B-type natriuretic peptide (BNP) is secreted by ventricular cardiomyocytes in response to various types of cardiac stress and has been used as a heart failure marker. In septic patients, increased BNP suggests poor prognosis; however, no causal link has been established. Among various effects, BNP decreases systemic vascular resistance and increases natriuresis that leads to lower blood pressure. We previously observed that JNK inhibition corrects cardiac dysfunction and suppresses cardiac BNP mRNA in endotoxemia. In this study, we investigated the transcriptional mechanism that regulates BNP expression and the involvement of plasma BNP in causing septic hypotension. Our in vitro and in vivo findings confirmed that activation of JNK signaling increases BNP expression in sepsis via direct binding of c-Jun in activating protein–1 (AP-1) regulatory elements of the Nppb promoter. Accordingly, genetic ablation of BNP, as well as treatment with a potentially novel neutralizing anti-BNP monoclonal antibody (19B3) or suppression of its expression via administration of JNK inhibitor SP600125 improved cardiac output, stabilized blood pressure, and improved survival in mice with polymicrobial sepsis. Therefore, inhibition of JNK signaling or BNP in sepsis appears to stabilize blood pressure and improve survival.}, number={8}, journal={JCI INSIGHT}, author={Hoffman, Matthew and Kyriazis, Ioannis D. and Dimitriou, Alexandra and Mishra, Santosh K. and Koch, Walter J. and Drosatos, Konstantinos}, year={2020}, month={Apr} }
 @article{kittaka_debrecht_mishra_2020, title={Differential contribution of sensory transient receptor potential channels in response to the bioactive lipid sphingosine-1-phosphate}, volume={16}, ISSN={["1744-8069"]}, DOI={10.1177/1744806920903515}, abstractNote={Somatosensation encompasses a wide range of sensations including pain, itch, touch, and temperature and is essential for the detection of environmental stimuli, ultimately allowing an organism to escape, communicate, and adapt to its environment. Such sensations are detected by primary sensory neurons whose nerve endings are located in the skin. Compared to external stimuli, mechanisms underlying endogenous stimulation of primary sensory neurons, such as by lipids, are still largely unknown. Here, we focus on one of the endogenous bioactive lipids, sphingosine-1-phosphate (S1P), to investigate the physiological roles of S1P in pain and itch. We showed that S1P-induced calcium responses in sensory neurons through S1P receptors. Transient receptor potential ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1) are nonselective calcium-permeable ion channels that are known to be involved in pain and itch. Neurons that respond to S1P show reduced responsiveness when treated with antagonists that block either TRPA1 or TRPV1 alone or in combination. In addition, using single and double knockout mice (TRPA1; TRPV1; TRPA1/TRPV1) with loss of function of these channels, we demonstrated that both TRP channels are involved in S1P-induced neuronal responses in vitro. Next, we examined the effects of S1P on pain and itch responsiveness in freely behaving mice post-S1P injection into the cheek, neck, and hind paw. Our findings reveal that S1P induces both pain and itch in vivo and that these responses are partially dependent upon the TRPV1, but not TRPA1 channels.}, journal={MOLECULAR PAIN}, author={Kittaka, Hiroki and DeBrecht, Jennifer and Mishra, Santosh K.}, year={2020}, month={Feb} }
 @article{mishra_wheeler_pitake_ding_jiang_fukuyama_paps_ralph_coyne_parkington_et al._2020, title={Periostin Activation of Integrin Receptors on Sensory Neurons Induces Allergic Itch}, volume={31}, ISSN={["2211-1247"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85082772179&partnerID=MN8TOARS}, DOI={10.1016/j.celrep.2020.03.036}, abstractNote={Chronic allergic itch is a common symptom affecting millions of people and animals, but its pathogenesis is not fully explained. Herein, we show that periostin, abundantly expressed in the skin of patients with atopic dermatitis (AD), induces itch in mice, dogs, and monkeys. We identify the integrin αVβ3 expressed on a subset of sensory neurons as the periostin receptor. Using pharmacological and genetic approaches, we inhibited the function of neuronal integrin αVβ3, which significantly reduces periostin-induced itch in mice. Furthermore, we show that the cytokine TSLP, the application of AD-causing MC903 (calcipotriol), and house dust mites all induce periostin secretion. Finally, we establish that the JAK/STAT pathway is a key regulator of periostin secretion in keratinocytes. Altogether, our results identify a TSLP-periostin reciprocal activation loop that links the skin to the spinal cord via peripheral sensory neurons, and we characterize the non-canonical functional role of an integrin in itch.}, number={1}, journal={CELL REPORTS}, author={Mishra, Santosh K. and Wheeler, Joshua J. and Pitake, Saumitra and Ding, Huiping and Jiang, Changyu and Fukuyama, Tomoki and Paps, Judy S. and Ralph, Patrick and Coyne, Jacob and Parkington, Michelle and et al.}, year={2020}, month={Apr} }
 @article{panigrahi_praharaj_kittaka_mridha_black_singh_mercer_bokhoven_torkko_agarwal_et al._2019, title={Exosome proteomic analyses identify inflammatory phenotype and novel biomarkers in African American prostate cancer patients}, volume={8}, ISSN={["2045-7634"]}, DOI={10.1002/cam4.1885}, abstractNote={African American men face a stark prostate cancer (PCa)‐related health disparity, with the highest incidence and mortality rates compared to other races. Additional and innovative measures are warranted to reduce this health disparity. Here, we focused on the identification of a novel serum exosome‐based “protein signature” for potential use in the early detection and better prognosis of PCa in African American men. Nanoparticle tracking analyses showed that compared to healthy individuals, exosome concentration (number/ml) was increased by ~3.2‐fold (P ˂ 0.05) in the sera of African American men with PCa. Mass spectrometry‐based proteomic analysis of serum exosomes identified seven unique and fifty‐five overlapping proteins (up‐ or downregulated) in African Americans with PCa compared to healthy African Americans. Furthermore, ingenuity pathway analyses identified the inflammatory acute‐phase response signaling as the top pathway associated with proteins loaded in exosomes from African American PCa patients. Interestingly, African American PCa E006AA‐hT cells secreted exosomes strongly induced a proinflammatory M2‐phenotype in macrophages and showed calcium response on sensory neurons, suggesting a neuroinflammatory response. Additionally, proteomic analyses showed that the protein Isoform 2 of Filamin A has higher loading (2.6‐fold) in exosomes from African Americans with PCa, but a lesser loading (0.6‐fold) was observed in exosomes from Caucasian men with PCa compared to race‐matched healthy individuals. Interestingly, TCGA and Taylor's dataset as well as IHC analyses of PCa tissue showed a lower Filamin A expression in tissues of PCa patients compared with normal subjects. Overall, these results support the usefulness of serum exosomes to noninvasively detect inflammatory phenotype and to discover novel biomarkers associated with PCa in African American men.}, number={3}, journal={CANCER MEDICINE}, author={Panigrahi, Gati K. and Praharaj, Prakash P. and Kittaka, Hiroki and Mridha, Asit R. and Black, Olen M. and Singh, Rakesh and Mercer, Roger and Bokhoven, Adrie and Torkko, Kathleen C. and Agarwal, Chapla and et al.}, year={2019}, month={Mar}, pages={1110–1123} }
 @article{pitake_middleton_abdus-saboor_mishra_2019, title={Inflammation Induced Sensory Nerve Growth and Pain Hypersensitivity Requires the N-Type Calcium Channel Cav2.2}, volume={13}, ISSN={["1662-453X"]}, DOI={10.3389/fnins.2019.01009}, abstractNote={Voltage-gated calcium channels (VGCCs) are important mediators of pain hypersensitivity during inflammatory states, but their role in sensory nerve growth remains underexplored. Here, we assess the role of the N-type calcium channel Cav2.2 in the complete Freund’s adjuvant (CFA) model of inflammatory pain. We demonstrate with in situ hybridization and immunoblotting, an increase in Cav2.2 expression after hind paw CFA injection in sensory neurons that respond to thermal stimuli, but not in two different mechanosensitive neuronal populations. Further, Cav2.2 upregulation post-CFA correlates with thermal but not mechanical hyperalgesia in behaving mice, and this hypersensitivity is blocked with a specific Cav2.2 inhibitor. Voltage clamp recordings reveal a significant increase in Cav2.2 currents post-CFA, while current clamp analyses demonstrate a significant increase in action potential frequency. Moreover, CFA-induced sensory nerve growth, which involves the extracellular signal-related kinase (ERK1/2) signaling pathway and likely contributes to inflammation-induced hyperalgesia, was blocked with the Cav2.2 inhibitor. Together, this work uncovers a role for Cav2.2 during inflammation, demonstrating that VGCC activity can promote thermal hyperalgesia through both changes in firing rates of sensory neurons as well as promotion of new neurite outgrowth.}, journal={FRONTIERS IN NEUROSCIENCE}, author={Pitake, Saumitra and Middleton, Leah J. and Abdus-Saboor, Ishmail and Mishra, Santosh K.}, year={2019}, month={Sep} }
 @article{wheeler_lascelles_olivry_mishra_2019, title={Itch-associated Neuropeptides and Their Receptor Expression in Dog Dorsal Root Ganglia and Spinal Cord}, volume={99}, ISSN={["1651-2057"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85074372654&partnerID=MN8TOARS}, DOI={10.2340/00015555-3297}, abstractNote={Most canine visits to veterinarians are related to skin diseases with itch being the chief complaint. Historically, several itch-inducing molecules and pathways have been identified in mice, but whether or not these are similar in dogs is not yet known. Herein, we set out to study the expression of pruritogenic neuropeptides, their cognate receptors with a limited functional validation thereof using a multidisciplinary approach. We demonstrated the expression of somatostatin and other major neuropeptides and receptors in canine dorsal root ganglia neurons. Next, we showed that interleukin-31, serotonin, and histamine activate such neurons. Furthermore, we demonstrated the physiological release of somatostatin from dog dorsal root ganglia neurons in response to several endogenous itch mediators. In summary, our results provide the first evidence that dogs use similar pruritogenic pathways to those characterized in mice and we thus identify multiple targets for the future treatment of itch in dogs.}, number={12}, journal={ACTA DERMATO-VENEREOLOGICA}, author={Wheeler, Joshua J. and Lascelles, B. Duncan X. and Olivry, Thierry and Mishra, Santosh K.}, year={2019}, month={Nov}, pages={1131–1135} }
 @article{huang_polgár_solinski_mishra_tseng_iwagaki_boyle_dickie_kriegbaum_wildner_et al._2018, title={Author Correction: Circuit dissection of the role of somatostatin in itch and pain}, volume={21}, ISSN={1097-6256 1546-1726}, url={http://dx.doi.org/10.1038/S41593-018-0149-6}, DOI={10.1038/S41593-018-0149-6}, abstractNote={In the version of this article initially published online, the labels were switched for the right-hand pair of bars in Fig. 4e. The left one of the two should be Chloroquine + veh, the right one Chloroquine + CNO. The error has been corrected in the print, HTML and PDF versions of the article.}, number={6}, journal={Nature Neuroscience}, publisher={Springer Science and Business Media LLC}, author={Huang, Jing and Polgár, Erika and Solinski, Hans Jürgen and Mishra, Santosh K. and Tseng, Pang-Yen and Iwagaki, Noboru and Boyle, Kieran A. and Dickie, Allen C. and Kriegbaum, Mette C. and Wildner, Hendrik and et al.}, year={2018}, month={Apr}, pages={894–894} }
 @article{huang_polgar_solinski_mishra_tseng_iwagaki_boyle_dickie_kriegbaum_wildner_et al._2018, title={Circuit dissection of the role of somatostatin in itch and pain}, volume={21}, ISSN={["1546-1726"]}, DOI={10.1038/s41593-018-0119-z}, abstractNote={Stimuli that elicit itch are detected by sensory neurons that innervate the skin. This information is processed by the spinal cord; however, the way in which this occurs is still poorly understood. Here we investigated the neuronal pathways for itch neurotransmission, particularly the contribution of the neuropeptide somatostatin. We find that in the periphery, somatostatin is exclusively expressed in Nppb+ neurons, and we demonstrate that Nppb+somatostatin+ cells function as pruriceptors. Employing chemogenetics, pharmacology and cell-specific ablation methods, we demonstrate that somatostatin potentiates itch by inhibiting inhibitory dynorphin neurons, which results in disinhibition of GRPR+ neurons. Furthermore, elimination of somatostatin from primary afferents and/or from spinal interneurons demonstrates differential involvement of the peptide released from these sources in itch and pain. Our results define the neural circuit underlying somatostatin-induced itch and characterize a contrasting antinociceptive role for the peptide. Huang et al. demonstrate that somatostatin (Sst)-expressing primary afferents are pruriceptors. In spinal cord, they show that Sst potentiates itch by disinhibition involving dynorphin-expressing spinal neurons and that Sst also suppresses pain.}, number={5}, journal={NATURE NEUROSCIENCE}, author={Huang, Jing and Polgar, Erika and Solinski, Hans Jurgen and Mishra, Santosh K. and Tseng, Pang-Yen and Iwagaki, Noboru and Boyle, Kieran A. and Dickie, Allen C. and Kriegbaum, Mette C. and Wildner, Hendrik and et al.}, year={2018}, month={May}, pages={707-+} }
 @article{wilzopolski_rossbach_mishra_baumer_kietzmann_2018, title={Histamine H4 receptor mediates itch through TRPV1 and TRPA1 ion channels}, volume={41}, journal={Journal of Veterinary Pharmacology and Therapeutics}, author={Wilzopolski, J. and Rossbach, K. and Mishra, S. K. and Baumer, W. and Kietzmann, M.}, year={2018}, pages={48–48} }
 @article{pandey_zhang_mishra_adikaram_harris_kahler_loshakov_sholevar_genis_kittock_et al._2017, title={A central role for R7bp in the regulation of itch sensation}, volume={158}, ISSN={["1872-6623"]}, DOI={10.1097/j.pain.0000000000000860}, abstractNote={Abstract Itch is a protective sensation producing a desire to scratch. Pathologic itch can be a chronic symptom of illnesses such as uremia, cholestatic liver disease, neuropathies and dermatitis, however current therapeutic options are limited. Many types of cell surface receptors, including those present on cells in the skin, on sensory neurons and on neurons in the spinal cord, have been implicated in itch signaling. The role of G protein signaling in the regulation of pruriception is poorly understood. We identify here 2 G protein signaling components whose mutation impairs itch sensation. R7bp (a.k.a. Rgs7bp) is a palmitoylated membrane anchoring protein expressed in neurons that facilitates G&agr;i/o -directed GTPase activating protein activity mediated by the G&bgr;5/R7-RGS complex. Knockout of R7bp diminishes scratching responses to multiple cutaneously applied and intrathecally-administered pruritogens in mice. Knock-in to mice of a GTPase activating protein-insensitive mutant of G&agr;o (Gnao1 G184S/+) produces a similar pruriceptive phenotype. The pruriceptive defect in R7bp knockout mice was rescued in double knockout mice also lacking Oprk1, encoding the G protein-coupled kappa-opioid receptor whose activation is known to inhibit itch sensation. In a model of atopic dermatitis (eczema), R7bp knockout mice showed diminished scratching behavior and enhanced sensitivity to kappa opioid agonists. Taken together, our results indicate that R7bp is a key regulator of itch sensation and suggest the potential targeting of R7bp-dependent GTPase activating protein activity as a novel therapeutic strategy for pathological itch.}, number={5}, journal={PAIN}, author={Pandey, Mritunjay and Zhang, Jian-Hua and Mishra, Santosh K. and Adikaram, Poorni R. and Harris, Benjamin and Kahler, John F. and Loshakov, Anna and Sholevar, Roxanne and Genis, Allison and Kittock, Claire and et al.}, year={2017}, month={May}, pages={931–944} }
 @misc{fukuyama_ganchingco_mishra_olivry_rzagalinski_volmer_baeumer_2017, title={Janus kinase inhibitors display broad anti-itch properties: A possible link through the TRPV1 receptor}, volume={140}, ISSN={["1097-6825"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85013487374&partnerID=MN8TOARS}, DOI={10.1016/j.jaci.2016.12.960}, abstractNote={Janus kinase (JAK) inhibitors are being proposed for the treatment of cancer and inflammatory diseases, such as atopic dermatitis. Their mechanism of action, especially that to reduce itch, remains speculative. The JAK inhibitor oclacitinib is currently approved for the treatment of lesions and pruritus in dogs with atopic dermatitis,1Cosgrove S.B. Wren J.A. Cleaver D.M. Martin D.D. Walsh K.F. Harfst J.A. et al.Efficacy and safety of oclacitinib for the control of pruritus and associated skin lesions in dogs with canine allergic dermatitis.Vet Dermatol. 2013; 24 (479-e114)Crossref Scopus (73) Google Scholar, 2Cosgrove S.B. Wren J.A. Cleaver D.M. Walsh K.F. Follis S.I. King V.I. et al.A blinded, randomized, placebo-controlled trial of the efficacy and safety of the Janus kinase inhibitor oclacitinib (Apoquel(R)) in client-owned dogs with atopic dermatitis.Vet Dermatol. 2013; 24 (e141-2): 587-597Crossref PubMed Scopus (93) Google Scholar whereas tofacitinib is under clinical development for the treatment of the homologous human disease (https://www.clinicaltrials.gov/ct2/show/NCT02001181). In rodent and canine models of allergic dermatitis and in human patients with psoriasis and atopic dermatitis, the antipruritic effect of JAK inhibitors is rapidly visible.1Cosgrove S.B. Wren J.A. Cleaver D.M. Martin D.D. Walsh K.F. Harfst J.A. et al.Efficacy and safety of oclacitinib for the control of pruritus and associated skin lesions in dogs with canine allergic dermatitis.Vet Dermatol. 2013; 24 (479-e114)Crossref Scopus (73) Google Scholar, 2Cosgrove S.B. Wren J.A. Cleaver D.M. Walsh K.F. Follis S.I. King V.I. et al.A blinded, randomized, placebo-controlled trial of the efficacy and safety of the Janus kinase inhibitor oclacitinib (Apoquel(R)) in client-owned dogs with atopic dermatitis.Vet Dermatol. 2013; 24 (e141-2): 587-597Crossref PubMed Scopus (93) Google Scholar, 3Fukuyama T. Ehling S. Cook E. Baumer W. Topically administered Janus-kinase inhibitors tofacitinib and oclacitinib display impressive antipruritic and anti-inflammatory responses in a model of allergic dermatitis.J Pharmacol Exp Ther. 2015; 354: 394-405Crossref PubMed Scopus (17) Google Scholar We suspect that the proposed mechanism of action on pruritus (ie, a reduction of signal transduction after binding of pruritogenic cytokines, such as IL-31, to their receptors) might not be the only explanation for the fast onset and sustained antipruritic action of JAK inhibitors. To identify additional mechanisms of action of the JAK inhibitors oclacitinib and tofacitinib, we used both in vitro (dorsal root ganglion [DRG] neuronal culture) and in vivo approaches in itch mouse models that are known to be dependent and independent of JAK signaling pathways. In this study we observed that the JAK inhibitors oclacitinib and tofacitinib exhibit broad anti-itch properties that appear linked to their inhibition of transient receptor potential cation channel subfamily V member 1 (TRPV1), a receptor known to play an important role in the transmission of both pain and itch.4Tóth B.I. Szallasi A. Bíró T. Transient receptor potential channels and itch: how deep should we scratch?.Handb Exp Pharmacol. 2015; 226: 89-133Crossref PubMed Scopus (20) Google Scholar To illustrate the involvement of TRPV1 in the anti-itch properties of JAK inhibitors, we used 3 different approaches: (1) in vitro calcium imaging in DRG neurons; (2) in vivo methods with a mouse model of itch and one of capsaicin-induced pain; and (3) TRPV1-transfected AD293 cells. We used murine primary sensory neurons and compared the responses evoked by several types of pruritogens that are dependent and independent of JAK signaling pathways. We examined the activation of small-diameter DRG neurons (mostly TRPV1-expressing ones) with or without exposure to oclacitinib or tofacitinib using Fura-2 ratiometric Ca2+ imaging. In vitro treatment with these 2 JAK inhibitors resulted in a significant reduction in the response of DRG neurons to the following stimuli: IL-31, TNF-α, histamine, compound 48/80, chloroquine, protease-activated receptor 2 (PAR-2) activating peptide (SLIGRL-NH2), capsaicin, and a low-pH solution (Fig 1, A). However, the response of DRG neurons to serotonin (5-HT) and allyl isothiocyanate (AITC) was not significantly changed compared with that in the vehicle-only control group after treatment with these JAK inhibitors (Fig 1, A and B). To examine whether the JAK inhibitors also reduced the itch response in vivo, we induced scratching responses in mice with all the compounds previously tested on the primary DRG sensory neurons. We confirmed in vivo a similar inhibitory pattern after oral administration of the JAK inhibitors compared with the vehicle control group, whereas there was no change in itch behavior after 5-HT (Fig 1, C). It has been shown above that these 2 JAK inhibitors reduced the itch induced by JAK-dependent cytokines involved in sensory perception, such as IL-31.3Fukuyama T. Ehling S. Cook E. Baumer W. Topically administered Janus-kinase inhibitors tofacitinib and oclacitinib display impressive antipruritic and anti-inflammatory responses in a model of allergic dermatitis.J Pharmacol Exp Ther. 2015; 354: 394-405Crossref PubMed Scopus (17) Google Scholar However, our results indicated a broader action than previously demonstrated with the reduction of itch induced by JAK-independent compounds that included TNF-α, histamine, compound 48/80, chloroquine, and SLIGRL-NH2. Similarly, we also observed a reduction of pain (manifested by wiping behavior) induced by capsaicin, the latter being a specific agonist for TRPV1. In support of this proposed novel mechanism of JAK inhibitors, a strikingly similar pattern of reduced scratching behavior to histamine, compound 48/80, and PAR-2 has similarly been shown by a TRPV1 antagonist.5Yun J.W. Seo J.A. Jang W.H. Koh H.J. Bae I.H. Park Y.H. et al.Antipruritic effects of TRPV1 antagonist in murine atopic dermatitis and itching models.J Invest Dermatol. 2011; 131: 1576-1579Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar Chloroquine interacts not only with TRPV1 but also with the other TRP ion channels.6Than J.Y. Li L. Hasan R. Zhang X. Excitation and modulation of TRPA1, TRPV1, and TRPM8 channel-expressing sensory neurons by the pruritogen chloroquine.J Biol Chem. 2013; 288: 12818-12827Crossref PubMed Scopus (68) Google Scholar Additionally, IL-31–induced itch is known to be dependent on both TRPV1- and transient receptor potential cation channel subfamily A member 1 (TRPA1)-expressing neurons.7Cevikbas F. Wang X. Akiyama T. Kempkes C. Savinko T. Antal A. et al.A sensory neuron-expressed IL-31 receptor mediates T helper cell-dependent itch: involvement of TRPV1 and TRPA1.J Allergy Clin Immunol. 2014; 133: 448-460Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar Interestingly, we did not see any effect of the tested JAK inhibitors on TRPA1-associated sensory perception induced by AITC and 5-HT (Fig 1, A and C).8Liu B. Escalera J. Balakrishna S. Fan L. Caceres A.I. Robinson E. et al.TRPA1 controls inflammation and pruritogen responses in allergic contact dermatitis.FASEB J. 2013; 27: 3549-3563Crossref PubMed Scopus (165) Google Scholar After these observations, we hypothesized that the broad antipruritic effect of oclacitinib and tofacitinib could be related to their direct inhibition of TRPV1 rather than only JAK inhibition. To confirm the direct association between TRPV1 and JAK inhibitors, we examined the in vitro and in vivo responses to IL-31, TNF-α, histamine, compound 48/80, chloroquine, SLIGRL-NH2, and capsaicin in TRPV1-knockout mice. Our inhibitory results are similar to those previously published,5Yun J.W. Seo J.A. Jang W.H. Koh H.J. Bae I.H. Park Y.H. et al.Antipruritic effects of TRPV1 antagonist in murine atopic dermatitis and itching models.J Invest Dermatol. 2011; 131: 1576-1579Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 6Than J.Y. Li L. Hasan R. Zhang X. Excitation and modulation of TRPA1, TRPV1, and TRPM8 channel-expressing sensory neurons by the pruritogen chloroquine.J Biol Chem. 2013; 288: 12818-12827Crossref PubMed Scopus (68) Google Scholar, 7Cevikbas F. Wang X. Akiyama T. Kempkes C. Savinko T. Antal A. et al.A sensory neuron-expressed IL-31 receptor mediates T helper cell-dependent itch: involvement of TRPV1 and TRPA1.J Allergy Clin Immunol. 2014; 133: 448-460Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar and the inhibition pattern mimics that of the JAK inhibitors (Fig 2, A and B). To test the effects of the JAK inhibitors in a more clinically relevant chronic itch setting, we used the toluene-2,4,-diisocyanate (TDI)–induced mouse model of chronic allergic itch. First, we observed a significant increase in expression of TRPV1 receptor–positive neurons in the DRG neurons of chronically challenged mice (Fig 2, C and D). Second, TDI-challenged mice topically treated with tofacitinib during the challenge phase showed a significant decrease in itch behavior (Fig 2, E). To further demonstrate a direct interaction of JAK inhibitors with TRPV1, we performed Ca2+ imaging on TRPV1-transfected AD293 cells and studied the effects of oclacitinib and tofacitinib on capsaicin-induced calcium influx. The 2 JAK inhibitors significantly reduced the response in TRPV1-transfected cells to capsaicin stimulation (see Fig E1, A, in this article's Online Repository at www.jacionline.org). Finally, we conducted matrix-assisted laser desorption/ionization (MALDI)–ultra-high-resolution mass spectrometric experiments on TRPV1 immunoprecipitates isolated from the TRPV1-transfected AD293 cells (see Fig E1, B). We detected higher concentrations of JAK inhibitors in TRPV1-transfected AD293 cells compared with those in vehicle-treated and mock-transfected AD293 cells (see Fig E1, C and D), although there were still some traces of JAK inhibitors in mock-transfected gel (see Fig E1, C). Because a small Ca2+ influx to a higher concentration of capsaicin was also found in mock-transfected cells, a low level of TRPV1 expression was likely present in wild-type cells. Future studies will include radioligand experiments to demonstrate direct binding to TRPV1. The inhibition of itch by JAK inhibitors has been reported to be both fast and sustained in dogs and human patients with psoriasis.1Cosgrove S.B. Wren J.A. Cleaver D.M. Martin D.D. Walsh K.F. Harfst J.A. et al.Efficacy and safety of oclacitinib for the control of pruritus and associated skin lesions in dogs with canine allergic dermatitis.Vet Dermatol. 2013; 24 (479-e114)Crossref Scopus (73) Google Scholar, 2Cosgrove S.B. Wren J.A. Cleaver D.M. Walsh K.F. Follis S.I. King V.I. et al.A blinded, randomized, placebo-controlled trial of the efficacy and safety of the Janus kinase inhibitor oclacitinib (Apoquel(R)) in client-owned dogs with atopic dermatitis.Vet Dermatol. 2013; 24 (e141-2): 587-597Crossref PubMed Scopus (93) Google Scholar, 9Bushmakin A.G. Mamolo C. Cappelleri J.C. Stewart M. The relationship between pruritus and the clinical signs of psoriasis in patients receiving tofacitinib.J Dermatolog Treat. 2015; 26: 19-22Crossref PubMed Scopus (31) Google Scholar However, this is difficult to explain by just the reduction of itch signaling by a few cytokines, such as IL-31. This direct inhibitory action on TRPV1 is likely the missing link, explaining the broad inhibitory action of the JAK inhibitors oclacitinib and tofacitinib. In summary, our results indicate a much more comprehensive relief of itch by JAK inhibitors, which is mediated through TRPV1 receptors and not just by inhibiting the signal transduction of itch-inducing cytokines. In addition to itch inhibition, the reduced response to capsaicin by JAK inhibitors indicates an extension of possible benefit in attenuating pain transduction through TRPV1, as already documented in patients with rheumatoid arthritis. Our study provides a novel mechanism for the antipruritic role of JAK inhibitors through TRPV1 receptor channels. We appreciate the technical support provided by Jenny Wilzopolski. Tofacitinib (CP-690550, C16H20N6O.C6H8O7) and 5-HT were purchased from Tocris (Minneapolis, Minn), and oclacitinib (PF-03394197, C15H23N5O2S) was purchased from Adooq Bioscience (Irvine, Calif) or Zoetis (Florham Park, NJ). Capsaicin, chloroquine, compound 48/80, histamine dihydrochloride, potassium chloride, poly-l-lysine, laminin, and TDI were obtained from Sigma (St Louis, Mo). AITC, collagenase, Dynabeads Protein G, Fura-2–acetoxymethyl ester, methylcellulose, PBS, NuPAGE LDS Sample Buffer, NuPAGE Sample Reducing Agent, Tris/Glycine/SDS buffer, PAR-2–activating peptide (SLIGRL-NH2), Tween 20, and Tween 80 were from Thermo Fisher Scientific (Waltham, Mass). Dulbecco modified Eagle medium, dispase, and Ca2+- and Mg2+-free HBSS were from Mediatech (Manassas, Va). Recombinant mouse IL-31 and TNF-α were obtained from PeproTech (Rocky Hill, NJ). FuGENE HD Transfection Reagent was from Promega (Madison, Wis). Mini-PROTEAN TGX Stain-Free Precast Gels were from Bio-Rad Laboratories (Richmond, Calif). VR1 antibody (P-19) was obtained from Santa Cruz Biotechnology (Santa Cruz, Calif). Rabbit TRPV1 antibody and mouse TUJ-1 antibody were obtained from Chemicon (Billerica, Mass). Female BALB/cAnN mice (7 weeks old) were purchased from Charles River Laboratories (Raleigh, NC). Female C57BL/6 wild-type and TRPV1 KO mice (age 6 weeks) were purchased from Jackson Laboratory (Bar Harbor, Me). They were housed in groups of 4 mice per cage under controlled lighting (12-hour light-dark cycle), temperature (22°C ± 3°C), humidity (55% ± 15%), and ventilation (at least 10 complete fresh-air changes per hour). Standard rodent chow and water were available ad libitum. All aspects of the current study were conducted in accordance with the Animal Care and Use Program of the North Carolina State University (IACUC protocol no. 13-111-B). Mice were killed by means of CO2 asphyxiation, and DRGs along the whole vertebral column were excised. Isolated DRGs were enzymatically digested in dispase (2.5 U/mL) and collagenase (2.5 mg/mL) dissolved in HBSS at 37°C. Neurons were dissociated by means of mechanical trituration with fire-polished Pasteur pipettes. Cells were washed in DRG medium (Dulbecco modified Eagle medium with l-glutamine, 10% heat-inactivated FCS, and 1% penicillin-streptomycin) and resuspended in 200 to 300 μL of DRG medium, and 20 μL was placed onto poly-l-lysine (0.1 mg/mL)– and laminin (0.1 mg/mL)–coated coverslips. Cells were incubated at 37°C in a 5% CO2 atmosphere for 2 hours and then flooded with 1 mL of DRG medium and further incubated at 37°C in a 5% CO2 atmosphere. Measurements were performed within the next 24 hours. FuGENE HD Transfection Reagent was used to transiently transfect AD293 cells with the TRPV1-pEAK8 plasmid. The functionality of TRPV1-transfected AD293 cells was validated by measuring capsaicin-evoked cytosolic calcium mobilization 48 hours after transfection. Calcium imaging was performed on cultured mouse DRG neurons and TRPV1-transfected AD293 cells, as described previously,E1Rossbach K. Nassenstein C. Gschwandtner M. Schnell D. Sander K. Seifert R. et al.Histamine H1, H3 and H4 receptors are involved in pruritus.Neuroscience. 2011; 190: 89-102Crossref PubMed Scopus (117) Google Scholar with minor modifications. Cells were first loaded with 2 μmol/L Fura-2–acetoxymethyl ester for 30 minutes at 37°C in a 5% CO2 atmosphere protected from light. The coverslips were mounted on a perfusion block and viewed through an inverted fluorescence microscope (TE200; Nikon, Melville, NY). Fluorescence was excited by UV light at 340 and 380 nm alternately, and the emitted light was collected every 100 ms by using a camera attached to a Lambda LS lamp and a Lambda optical filter changer. Cells were incubated in Locke buffer (pH 7.4; 136 mmol/L NaCl, 5.4 mmol/L KCl, 2.9 mmol/L CaCl2, 2.5 mmol/L MgCl2, 10.9 mmol/L D-glucose, 0.6 mmol/L NaHPO4, and 14.3 mmol/L NaHCO3) during imaging. Solutions were delivered through a temperature-controlled perfusion system at a flow rate of 3 mL/min. Recombinant mouse IL-31 (1 μg/mL), recombinant mouse TNF-α (1 μg/mL), histamine dihydrochloride (1 mmol/L), compound 48/80 (1 mmol/L), the PAR-2 agonist SLIGRL-NH2 (1 mmol/L), chloroquine (100 μmol/L), 5-HT (1 mmol/L), AITC (10 μmol/L), capsaicin (1 μmol/L), and low-pH solution (pH 4.0) were delivered. At the end of the experiment, the viability of the neurons was confirmed by an increase in Ca2+ evoked by an application of 150 mmol/L potassium chloride. Cells were judged to be responsive if the ratio value increased by greater than 10% of the resting level after chemical application. DRG neurons and TRPV1-transfected AD293 cells were exposed to 0.1% dimethyl sulfoxide (vehicle) and each JAK inhibitor (oclacitinib or tofacitinib at 1 μmol/L) in Locke buffer just before (approximately 1 minute) the stimulus exposure. The selected concentrations were adapted from previous reports.E2Fukuyama T. Ehling S. Cook E. Baumer W. Topically administered Janus-kinase inhibitors tofacitinib and oclacitinib display impressive antipruritic and anti-inflammatory responses in a model of allergic dermatitis.J Pharmacol Exp Ther. 2015; 354: 394-405Crossref PubMed Scopus (69) Google Scholar, E3Heine A. Held S.A. Daecke S.N. Wallner S. Yajnanarayana S.P. Kurts C. et al.The JAK-inhibitor ruxolitinib impairs dendritic cell function in vitro and in vivo.Blood. 2013; 122: 1192-1202Crossref PubMed Scopus (255) Google Scholar, E4Kubo S. Yamaoka K. Kondo M. Yamagata K. Zhao J. Iwata S. et al.The JAK inhibitor, tofacitinib, reduces the T cell stimulatory capacity of human monocyte-derived dendritic cells.Ann Rheum Dis. 2014; 73: 2192-2198Crossref PubMed Scopus (110) Google Scholar This concentration (1 μmol/L) did not induce any cell toxicity. The hair on the thoracic region of the backs and cheeks of the mice was shaved with a hair clipper a day before the experiment. The JAK inhibitors oclacitinib or tofacitinib were administered orally 30 minutes before injection of the pruritogen. Each JAK inhibitor was diluted in a 0.5% methylcellulose/0.25% Tween 20 solution to doses: tofacitinib, 30 mg/kg; oclacitinib, 45 mg/kg. The oral doses of tofacitinib and oclacitinib used in this study were selected based on our previously published study.E2Fukuyama T. Ehling S. Cook E. Baumer W. Topically administered Janus-kinase inhibitors tofacitinib and oclacitinib display impressive antipruritic and anti-inflammatory responses in a model of allergic dermatitis.J Pharmacol Exp Ther. 2015; 354: 394-405Crossref PubMed Scopus (69) Google Scholar Recombinant mouse IL-31, 1 μg per injection site; recombinant mouse TNF-α, 1 μg per injection site; histamine dihydrochloride, 50 μg per injection site; compound 48/80, 20 μg per injection site; and chloroquine, 50 μg or 200 μg per injection site (note the higher concentration corresponds to former studies in which no difference between wild-type and TRPV1−/− mice was observedE5Kim S. Barry D.M. Liu X.Y. Yin S. Munanairi A. Meng Q.T. et al.Facilitation of TRPV4 by TRPV1 is required for itch transmission in some sensory neuron populations.Sci Signal. 2016; 9: ra71Crossref PubMed Scopus (54) Google Scholar), SLIGRL-NH2 (50 μg per injection site) and 5-HT (10 μg per injection site) were injected once intradermally in a volume of 50 μL into the shaved part of the back 30 minutes after administration of the JAK inhibitor. Capsaicin (10 μg per injection site) was injected intradermally in a volume of 10 μL into the shaved part of the cheek 30 minutes after administration of the JAK inhibitor. Scratching or wiping behavior was evaluated for 30 minutes immediately after injection of the pruritogen or capsaicin. A TDI-induced itch model was generated based on the method described previously,E2Fukuyama T. Ehling S. Cook E. Baumer W. Topically administered Janus-kinase inhibitors tofacitinib and oclacitinib display impressive antipruritic and anti-inflammatory responses in a model of allergic dermatitis.J Pharmacol Exp Ther. 2015; 354: 394-405Crossref PubMed Scopus (69) Google Scholar with the difference that the allergic itch was chronic. Hair was removed from the abdominal regions of female BALB/c mice with a depilatory cream the day before the first sensitization (day 1). On day 1, the shaved abdominal region was stripped 10 times with adhesive tape just before sensitization with 50 μL of 5% TDI. The same sensitization without tape stripping was performed on days 2 and 3. To induce a chronic model, the allergic reaction was boosted repeatedly for the next 6 weeks through application of 50 μL of 0.5% TDI onto the shaved neck (once weekly). A week after the last TDI boost, either vehicle (1:7 acetone/dimethyl sulfoxide) or tofacitinib (0.5%) was topically administered 30 minutes before TDI challenge to neck skin (n = 6 for each group). The tofacitinib concentration was selected according to a former study performed by our group.E2Fukuyama T. Ehling S. Cook E. Baumer W. Topically administered Janus-kinase inhibitors tofacitinib and oclacitinib display impressive antipruritic and anti-inflammatory responses in a model of allergic dermatitis.J Pharmacol Exp Ther. 2015; 354: 394-405Crossref PubMed Scopus (69) Google Scholar Scratching bouts were monitored for 1 hour immediately after 0.5% TDI challenge. Immunohistochemistry was performed on DRG sections (12 μm) from untreated and TDI-treated mice. Rabbit TRPV1 (1:500 dilution) and mouse TUJ-1 (1:500 dilution) antibodies were used. Total numbers of neurons expressing the TRPV1 receptor were counted for untreated and treated samples and were normalized to the TUJ-1 as a neuron marker. Either mock- or TRPV1-transfected AD293 cells were incubated for 10 minutes with oclacitinib (10 μmol/L) or tofacitinib (10 μmol/L) and then thoroughly washed with PBS, solubilized, and immunoprecipitated with Dynabeads Protein G and VR1 antibody (P-19). SDS-PAGE of the eluted sample was performed with NuPAGE LDS Sample Buffer, NuPAGE Sample Reducing Agent, and gel electrophoresis on Mini-PROTEAN TGX Stain-Free Precast 10% Gel. Precipitates from the gel were analyzed with MALDI–ultra-high-resolution Fourier-transform ion cyclotron resonance (FTICR) mass spectrometry using α-cyanohydroxycinnamic acid as a matrix substance after cutting the respective bands from the gel and extracting them with ethyl acetate to visualize the presence of tofacitinib or oclacitinib interacting with TRPV1.E6Fukuyama T. Tschernig T. Qi Y. Volmer D.A. Baumer W. Aggression behaviour induced by oral administration of the Janus-kinase inhibitor tofacitinib, but not oclacitinib, under stressful conditions.Eur J Pharmacol. 2015; 764: 278-282Crossref PubMed Scopus (19) Google Scholar, E7Qi Y. Hainz N. Tschernig T. Meier C. Volmer D.A. Differential distribution of probenecid as detected by on-tissue mass spectrometry.Cell Tissue Res. 2015; 360: 427-429Crossref PubMed Scopus (5) Google Scholar Signals for the drugs were acquired with mass accuracies of less than 1 ppm, and results were confirmed with tandem mass spectrometry by using authentic standards. All data were expressed as means ± 1 SDs. Statistical significance of the difference between the vehicle control and treated groups were estimated at the 5% and 1% levels of probability. The Student t test was used to test the significance of differences between mean values between 2 groups. Comparisons for more than 3 groups were carried out by using a 1-way ANOVA, followed by the Dunnett multiple comparison test. Comparisons of proportions were made with the Fisher exact test. Data were analyzed with Prism 4 software (GraphPad Software, San Diego, Calif).}, number={1}, journal={JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY}, author={Fukuyama, Tomoki and Ganchingco, Joy Rachel and Mishra, Santosh K. and Olivry, Thierry and Rzagalinski, Ignacy and Volmer, Dietrich A. and Baeumer, Wolfgang}, year={2017}, month={Jul}, pages={306-+} }
 @article{goswami_mishra_maric_kaszas_gonnella_clokie_kominsky_gross_keller_mannes_et al._2014, title={Molecular Signatures of Mouse TRPV1-Lineage Neurons Revealed by RNA-Seq Transcriptome Analysis}, volume={15}, ISSN={1526-5900}, url={http://dx.doi.org/10.1016/j.jpain.2014.09.010}, DOI={10.1016/j.jpain.2014.09.010}, abstractNote={Disorders of pain neural systems are frequently chronic and, when recalcitrant to treatment, can severely degrade the quality of life. The pain pathway begins with sensory neurons in dorsal root or trigeminal ganglia, and the neuronal subpopulations that express the transient receptor potential cation channel, subfamily V, member 1 (TRPV1) ion channel transduce sensations of painful heat and inflammation and play a fundamental role in clinical pain arising from cancer and arthritis. In the present study, we elucidate the complete transcriptomes of neurons from the TRPV1 lineage and a non-TRPV1 neuroglial population in sensory ganglia through the combined application of next-gen deep RNA-Seq, genetic neuronal labeling with fluorescence-activated cell sorting, or neuron-selective chemoablation. RNA-Seq accurately quantitates gene expression, a difficult parameter to determine with most other methods, especially for very low and very high expressed genes. Differentially expressed genes are present at every level of cellular function from the nucleus to the plasma membrane. We identified many ligand receptor pairs in the TRPV1 population, suggesting that autonomous presynaptic regulation may be a major regulatory mechanism in nociceptive neurons. The data define, in a quantitative, cell population-specific fashion, the molecular signature of a distinct and clinically important group of pain-sensing neurons and provide an overall framework for understanding the transcriptome of TRPV1 nociceptive neurons.Next-gen RNA-Seq, combined with molecular genetics, provides a comprehensive and quantitative measurement of transcripts in TRPV1 lineage neurons and a contrasting transcriptome from non-TRPV1 neurons and cells. The transcriptome highlights previously unrecognized protein families, identifies multiple molecular circuits for excitatory or inhibitory autocrine and paracrine signaling, and suggests new combinatorial approaches to pain control.}, number={12}, journal={The Journal of Pain}, publisher={Elsevier BV}, author={Goswami, Samridhi C. and Mishra, Santosh K. and Maric, Dragan and Kaszas, Krisztian and Gonnella, Gian Luigi and Clokie, Samuel J. and Kominsky, Hal D. and Gross, Jacklyn R. and Keller, Jason M. and Mannes, Andrew J. and et al.}, year={2014}, month={Dec}, pages={1338–1359} }
 @article{shukla_zheng_mishra_amin_steiner_grant_kesavapany_pant_2013, title={A truncated peptide from p35, a Cdk5 activator, prevents Alzheimer's disease phenotypes in model mice}, volume={27}, ISSN={0892-6638 1530-6860}, url={http://dx.doi.org/10.1096/fj.12-217497}, DOI={10.1096/fj.12-217497}, abstractNote={Alzheimer's disease (AD), one of the leading neurodegenerative disorders of older adults, which causes major socioeconomic burdens globally, lacks effective therapeutics without significant side effects. Besides the hallmark pathology of amyloid plaques and neurofibrillary tangles (NFTs), it has been reported that cyclin‐dependent kinase 5 (Cdk5), a critical neuronal kinase, is hyperactivated in AD brains and is, in part, responsible for the above pathology. Here we show that a modified truncated 24‐aa peptide (TFP5), derived from the Cdk5 activator p35, penetrates the blood‐brain barrier after intraperitoneal injections, inhibits abnormal Cdk5 hyperactivity, and significantly rescues AD pathology (up to 70–80%) in 5XFAD AD model mice. The mutant mice, injected with TFP5 exhibit behavioral rescue, whereas no rescue was observed in mutant mice injected with either saline or scrambled peptide. However, TFP5 does not inhibit cell cycle Cdks or normal Cdk5/p35 activity, and thereby has no toxic side effects (even at 200 mg/kg), a common problem in most current therapeutics for AD. In addition, treated mice displayed decreased inflammation, amyloid plaques, NFTs, cell death, and an extended life by 2 mo. These results suggest TFP5 as a potential therapeutic, toxicity‐free candidate for AD.—Shukla, V., Zheng, Y.‐L., Mishra, S. K., Amin, N. D., Steiner, J., Grant, P., Kesavapany, S., Pant, H. C. A truncated peptide from p35, a Cdk5 activator, prevents Alzheimer's disease phenotypes in model mice. FASEB J. 27, 174–186 (2013). www.fasebj.org}, number={1}, journal={The FASEB Journal}, publisher={FASEB}, author={Shukla, Varsha and Zheng, Ya-Li and Mishra, Santosh K. and Amin, Niranjana D. and Steiner, Joseph and Grant, Philip and Kesavapany, Sashi and Pant, Harish C.}, year={2013}, month={Jan}, pages={174–186} }
 @article{mishra_hoon_2013, title={The Cells and Circuitry for Itch Responses in Mice}, volume={340}, ISSN={0036-8075 1095-9203}, url={http://dx.doi.org/10.1126/science.1233765}, DOI={10.1126/science.1233765}, abstractNote={The Master Switch for Itch? Recently, gastrin-releasing peptide (GRP) has been implicated as the primary neurotransmitter between itch-sensitive nerve fibers and downstream neurons in the spinal cord. However, Mishra and Hoon (p. 968) challenge this view, provide evidence that natriuretic polypeptide b (Nppb) is the central itch neurotransmitter, and suggest that GRP is released by second-order neurons in the spinal dorsal horn that express the Nppb receptor and are excited by Nppb. Natriuretic polypeptide b is the primary neurotransmitter in TRPV1-expressing, itch-sensitive afferent nerve fibers. Itch is triggered by somatosensory neurons expressing the ion channel TRPV1 (transient receptor potential cation channel subfamily V member 1), but the mechanisms underlying this nociceptive response remain poorly understood. Here, we show that the neuropeptide natriuretic polypeptide b (Nppb) is expressed in a subset of TRPV1 neurons and found that Nppb−/− mice selectively lose almost all behavioral responses to itch-inducing agents. Nppb triggered potent scratching when injected intrathecally in wild-type and Nppb−/− mice, showing that this neuropeptide evokes itch when released from somatosensory neurons. Itch responses were blocked by toxin-mediated ablation of Nppb-receptor–expressing cells, but a second neuropeptide, gastrin-releasing peptide, still induced strong responses in the toxin-treated animals. Thus, our results define the primary pruriceptive neurons, characterize Nppb as an itch-selective neuropeptide, and reveal the next two stages of this dedicated neuronal pathway.}, number={6135}, journal={Science}, publisher={American Association for the Advancement of Science (AAAS)}, author={Mishra, S. K. and Hoon, M. A.}, year={2013}, month={May}, pages={968–971} }
 @article{pogorzala_mishra_hoon_2013, title={The Cellular Code for Mammalian Thermosensation}, volume={33}, ISSN={0270-6474 1529-2401}, url={http://dx.doi.org/10.1523/jneurosci.5788-12.2013}, DOI={10.1523/jneurosci.5788-12.2013}, abstractNote={Mammalian somatosenory neurons respond to thermal stimuli and allow animals to reliably discriminate hot from cold and to select their preferred environments. Previously, we generated mice that are completely insensitive to temperatures from noxious cold to painful heat (−5 to 55°C) by ablating several different classes of nociceptor early in development. In the present study, we have adopted a selective ablation strategy in adult mice to study this phenotype and have demonstrated that separate populations of molecularly defined neurons respond to hot and cold. TRPV1-expressing neurons are responsible for all behavioral responses to temperatures between 40 and 50°C, whereas TRPM8 neurons are required for cold aversion. We also show that more extreme cold and heat activate additional populations of nociceptors, including cells expressing Mrgprd. Therefore, although eliminating Mrgprd neurons alone does not affect behavioral responses to temperature, when combined with ablation of TRPV1 or TRPM8 cells, it significantly decreases responses to extreme heat and cold, respectively. Ablation of TRPM8 neurons distorts responses to preferred temperatures, suggesting that the pleasant thermal sensation of warmth may in fact just reflect reduced aversive input from TRPM8 and TRPV1 neurons. As predicted by this hypothesis, mice lacking both classes of thermosensor exhibited neither aversive nor attractive responses to temperatures between 10 and 50°C. Our results provide a simple cellular basis for mammalian thermosensation whereby two molecularly defined classes of sensory neurons detect and encode both attractive and aversive cues.}, number={13}, journal={Journal of Neuroscience}, publisher={Society for Neuroscience}, author={Pogorzala, L. A. and Mishra, S. K. and Hoon, M. A.}, year={2013}, month={Mar}, pages={5533–5541} }
 @article{mishra_holzman_hoon_2012, title={A Nociceptive Signaling Role for Neuromedin B}, volume={32}, ISSN={0270-6474 1529-2401}, url={http://dx.doi.org/10.1523/jneurosci.1533-12.2012}, DOI={10.1523/jneurosci.1533-12.2012}, abstractNote={Here we used an array-based differential screen to uncover the expression of the neuropeptide neuromedin B (NMB) in the trigeminal ganglia of mice. Double-labeling experiments reveal NMB is expressed in a subset of sensory neurons that colabel with calcitonin gene-related peptide and TRPV1 suggestive of a role for NMB in nociception. Indeed, administration of NMB antagonist greatly attenuates edema and nerve sensitization following stimulation of peripheral nerves with mustard oil, demonstrating that NMB contributes to neurogenic inflammation. Moreover, direct injection of NMB causes local swelling and nociceptive sensitization. Interestingly, we also find that the receptor for NMB is expressed in interneurons in the superficial layers of the dorsal horn. We used NMB-saporin to specifically eliminate NMBR-expressing neurons and determined they are required in responses to noxious heat, but not for reaction to mechanical and pruritic stimuli. Thus, NMB may be a neurotransmitter that is selectively involved in the perception of thermal stimuli.}, number={25}, journal={Journal of Neuroscience}, publisher={Society for Neuroscience}, author={Mishra, S. K. and Holzman, S. and Hoon, M. A.}, year={2012}, month={Jun}, pages={8686–8695} }
 @article{shukla_mishra_pant_2011, title={Oxidative stress in neurodegeneration}, volume={2011}, DOI={10.1155/2011/572634}, abstractNote={It has been demonstrated that oxidative stress has a ubiquitous role in neurodegenerative diseases. Major source of oxidative stress due to reactive oxygen species (ROS) is related to mitochondria as an endogenous source. Although there is ample evidence from tissues of patients with neurodegenerative disorders of morphological, biochemical, and molecular abnormalities in mitochondria, it is still not very clear whether the oxidative stress itself contributes to the onset of neurodegeneration or it is part of the neurodegenerative process as secondary manifestation. This paper begins with an overview of how oxidative stress occurs, discussing various oxidants and antioxidants, and role of oxidative stress in diseases in general. It highlights the role of oxidative stress in neurodegenerative diseases like Alzheimer's, Parkinson's, and Huntington's diseases and amyotrophic lateral sclerosis. The last part of the paper describes the role of oxidative stress causing deregulation of cyclin-dependent kinase 5 (Cdk5) hyperactivity associated with neurodegeneration.}, journal={Advances in Pharmacological Sciences}, author={Shukla, V. and Mishra, S.K. and Pant, H.C.}, year={2011}, pages={1–13} }
 @article{kaszas_keller_coddou_mishra_hoon_stojilkovic_jacobson_iadarola_2011, title={Small Molecule Positive Allosteric Modulation of TRPV1 Activation by Vanilloids and Acidic pH}, volume={340}, ISSN={1521-0103}, url={http://dx.doi.org/10.1124/jpet.111.183053}, DOI={10.1124/jpet.111.183053}, abstractNote={Transient receptor potential cation channel subfamily V member 1 (TRPV1) is a high-conductance, nonselective cation channel strongly expressed in nociceptive primary afferent neurons of the peripheral nervous system and functions as a multimodal nociceptor gated by temperatures greater than 43°C, protons, and small-molecule vanilloid ligands such as capsaicin. The ability to respond to heat, low pH, vanilloids, and endovanilloids and altered sensitivity and expression in experimental inflammatory and neuropathic pain models made TRPV1 a major target for the development of novel, nonopioid analgesics and resulted in the discovery of potent antagonists. In human clinical trials, observations of hyperthermia and the potential for thermal damage by suppressing the ability to sense noxious heat suggested that full-scale blockade of TRPV1 function can be counterproductive and subtler pharmacological approaches are necessary. Here we show that the dihydropyridine derivative 4,5-diethyl-3-(2-methoxyethylthio)-2-methyl-6-phenyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate (MRS1477) behaves as a positive allosteric modulator of both proton and vanilloid activation of TRPV1. Under inflammatory-mimetic conditions of low pH (6.0) and protein kinase C phosphorylation, addition of MRS1477 further increased sensitivity of already sensitized TPRV1 toward capsaicin. MRS1477 does not affect inhibition by capsazepine or ruthenium red and remains effective in potentiating activation by pH in the presence of an orthosteric vanilloid antagonist. These results indicate a distinct site on TRPV1 for positive allosteric modulation that may bind endogenous compounds or novel pharmacological agents. Positive modulation of TRPV1 sensitivity suggests that it may be possible to produce a selective analgesia through calcium overload restricted to highly active nociceptive nerve endings at sites of tissue damage and inflammation.}, number={1}, journal={Journal of Pharmacology and Experimental Therapeutics}, publisher={American Society for Pharmacology & Experimental Therapeutics (ASPET)}, author={Kaszas, K. and Keller, J. M. and Coddou, C. and Mishra, S. K. and Hoon, M. A. and Stojilkovic, S. and Jacobson, K. A. and Iadarola, M. J.}, year={2011}, month={Oct}, pages={152–160} }
 @article{mishra_hoon_2011, title={TRPV1 lineage neurons and their functional role in thermal nociception}, volume={71}, ISSN={0168-0102}, DOI={10.1016/j.neures.2011.07.341}, journal={Neuroscience Research}, publisher={Elsevier BV}, author={Mishra, Santosh and Hoon, Mark}, year={2011}, month={Sep}, pages={e80} }
 @article{mishra_hoon_2010, title={Ablation of TrpV1 neurons reveals their selective role in thermal pain sensation}, volume={43}, ISSN={1044-7431}, url={http://dx.doi.org/10.1016/j.mcn.2009.10.006}, DOI={10.1016/j.mcn.2009.10.006}, abstractNote={Here we make use of neural ablation to investigate the properties of the TrpV1-expressing neurons in the trigeminal and dorsal root ganglia of mice. Resiniferotoxin (RTX), a potent TrpV1 agonist, administered either by direct injection in the ganglion or intrathecally killed approximately 70% of TrpV1 cells and resulted in modest thermal analgesia. Interestingly, after carageenan injection in the hind paw, the analgesic effects of RTX were dramatically increased with mice now paradoxically showing far less response to heat applied at sites of inflammation. This additional carageenan and RTX-induced analgesia was transient, lasting less than 2 days, and likely resulted from deafferentation of remaining TrpV1 neurons. Remarkably, although RTX affected sensitivity to heat, mechanical sensitivity (both of normal and inflamed tissue) was completely unaltered by toxin-mediated silencing of the TrpV1 sensory input. Thus, our data demonstrate that TrpV1 neurons are selectively tuned nociceptors that mediate responses to thermal but not mechanical pain and insinuate a labeled line model for somatosensory coding.}, number={1}, journal={Molecular and Cellular Neuroscience}, publisher={Elsevier BV}, author={Mishra, Santosh K. and Hoon, Mark A.}, year={2010}, month={Jan}, pages={157–163} }
 @article{mishra_tisel_orestes_bhangoo_hoon_2010, title={TRPV1-lineage neurons are required for thermal sensation}, volume={30}, ISSN={0261-4189}, url={http://dx.doi.org/10.1038/emboj.2010.325}, DOI={10.1038/emboj.2010.325}, abstractNote={The ion-channel TRPV1 is believed to be a major sensor of noxious heat, but surprisingly animals lacking TRPV1 still display marked responses to elevated temperature. In this study, we explored the role of TRPV1-expressing neurons in somatosensation by generating mice wherein this lineage of cells was selectively labelled or ablated. Our data show that TRPV1 is an embryonic marker of many nociceptors including all TRPV1- and TRPM8-neurons as well as many Mrg-expressing neurons. Mutant mice lacking these cells are completely insensitive to hot or cold but in marked contrast retain normal touch and mechanical pain sensation. These animals also exhibit defective body temperature control and lose both itch and pain reactions to potent chemical mediators. Together with previous cell ablation studies, our results define and delimit the roles of TRPV1- and TRPM8-neurons in thermosensation, thermoregulation and nociception, thus significantly extending the concept of labelled lines in somatosensory coding.}, number={3}, journal={The EMBO Journal}, publisher={Wiley}, author={Mishra, Santosh K and Tisel, Sarah M and Orestes, Peihan and Bhangoo, Sonia K and Hoon, Mark A}, year={2010}, month={Dec}, pages={582–593} }
 @article{mishra_braun_shukla_füllgrabe_schomerus_korf_gachet_ikehara_sevigny_robson_et al._2006, title={Extracellular nucleotide signaling in adult neural stem cells: synergism with growth factor-mediated cellular proliferation}, volume={133}, ISSN={0950-1991 1477-9129}, url={http://dx.doi.org/10.1242/dev.02233}, DOI={10.1242/dev.02233}, abstractNote={We have previously shown that the extracellular nucleoside triphosphate-hydrolyzing enzyme NTPDase2 is highly expressed in situ by stem/progenitor cells of the two neurogenic regions of the adult murine brain: the subventricular zone (type B cells) and the dentate gyrus of the hippocampus (residual radial glia). We explored the possibility that adult multipotent neural stem cells express nucleotide receptors and investigated their functional properties in vitro. Neurospheres cultured from the adult mouse SVZ in the presence of epidermal growth factor and fibroblast growth factor 2 expressed the ecto-nucleotidases NTPDase2 and the tissue non-specific isoform of alkaline phosphatase, hydrolyzing extracellular ATP to adenosine. ATP, ADP and, to a lesser extent, UTP evoked rapid Ca2+ transients in neurospheres that were exclusively mediated by the metabotropic P2Y1 and P2Y2 nucleotide receptors. In addition, agonists of these receptors and low concentrations of adenosine augmented cell proliferation in the presence of growth factors. Neurosphere cell proliferation was attenuated after application of the P2Y1-receptor antagonist MRS2179 and in neurospheres from P2Y1-receptor knockout mice. In situ hybridization identified P2Y1-receptor mRNA in clusters of SVZ cells. Our results infer nucleotide receptor-mediated synergism that augments growth factor-mediated cell proliferation. Together with the in situ data, this supports the notion that extracellular nucleotides contribute to the control of adult neurogenesis.}, number={4}, journal={Development}, publisher={The Company of Biologists}, author={Mishra, S.K. and Braun, N. and Shukla, V. and Füllgrabe, M. and Schomerus, C. and Korf, H.W. and Gachet, C. and Ikehara, Y. and Sevigny, J. and Robson, S.C. and et al.}, year={2006}, month={Feb}, pages={675–684} }