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1.  The Dynamic TRPA1 Channel: A Suitable Pharmacological Pain Target? 
Current pharmaceutical biotechnology  2011;12(10):1689-1697.
Acute pain detection is vital to navigate and survive in one’s environment. Protection and preservation occur because primary afferent nociceptors transduce adverse environmental stimuli into electrical impulses that are transmitted to and interpreted within high levels of the central nervous system. Therefore, it is critical that the molecular mechanisms that convert noxious information into neural signals be identified and their specific functional roles delineated in both acute and chronic pain settings. The Transient Receptor Potential (TRP) channel family member TRP ankyrin 1 (TRPA1) is an excellent candidate molecule to explore and intricately understand how single channel properties can tailor behavioral nociceptive responses. TRPA1 appears to dynamically respond to an amazingly wide range of diverse stimuli that include apparently unrelated modalities such as mechanical, chemical and thermal stimuli that activate somatosensory neurons. How such dissimilar stimuli activate TRPA1, yet result in modality-specific signals to the CNS is unclear. Furthermore, TRPA1 is also involved in persistent to chronic painful states such as inflammation, neuropathic pain, diabetes, fibromyalgia, bronchitis and emphysema. Yet how TRPA1’s role changes from an acute sensor of physical stimuli to its contribution to these diseases that are concomitant with implacable, chronic pain is unknown. TRPA1’s involvement in the nociceptive machinery that relays the adverse stimuli during painful disease states is of considerable interest for drug delivery and design by many pharmaceutical entities. In this review, we will assess the current knowledge base of TRPA1 in acute nociception and persistent inflammatory pain states, and explore its potential as a therapeutic pharmacological target in chronic pervasive conditions such neuropathic pain, persistent inflammation and diabetes.
PMCID: PMC3884818  PMID: 21466445
Mechanotransduction; keratinocyte; cold; inflammation; diabetes; neuropathy; TRPV1; nociceptor
2.  Piezo2 is required for Merkel cell mechanotransduction 
Nature  2014;509(7502):622-626.
Summary
How we sense touch remains fundamentally unknown1,2. The Merkel cell-neurite complex is a gentle touch receptor in the skin that mediates slowly-adapting (SA) responses of Aβ sensory fibers to encode fine details of objects3-6. This mechanoreceptor complex was recognized to play an essential role in sensing gentle touch nearly 50 years ago3,4. However, whether Merkel cells or afferent fibers themselves sense mechanical force is still debated, and the molecular mechanism of mechanotransduction is unknown1,2,7-12. Interestingly, synapse-like junctions are observed between Merkel cells and associated afferents6,13-15, and yet it is unclear if Merkel cells are inherently mechanosensitive or whether they can rapidly transmit such information to the neighboring nerve1,2,16,17. Here we show for the first time that Merkel cells produce touch-sensitive currents in vitro. Piezo2, a mechanically-activated (MA) cation channel, is expressed in Merkel cells. We engineered mice deficient in Piezo2 in the skin, but not in sensory neurons, and show that Merkel cell mechanosensitivity completely depends on Piezo2. In these mice, Merkel cell-neurite complex-mediated SA responses in vivo show reduced static firing rates, and moreover, they display moderately decreased behavioral responses to gentle touch. Our results indicate that Piezo2 is the Merkel cell mechanotransduction channel and provide the first line of evidence that Piezos play a physiological role in mechanosensation in mammals. Furthermore, our data present evidence for a two-receptor site model, where both Merkel cells and innervating afferents act in concert as mechanosensors. The two-receptor system could provide this mechanoreceptor complex with a tuning mechanism to achieve highly sophisticated responses to a given mechanical stimulus15,18,19.
doi:10.1038/nature13251
PMCID: PMC4039622  PMID: 24717433
3.  Spinal Nerve Ligation in Mouse Upregulates TRPV1 Heat Function in Injured IB4 Positive Nociceptors 
Peripheral nerve injury leads to neuropathic pain, but the underlying mechanisms are not clear. The TRPV1 channel expressed by nociceptors is one receptor for noxious heat and inflammatory molecules. Lumbar 4 spinal nerve ligation (SNL) in mice induced persistent heat hyperalgesia 4–10 days following injury. The heat hypersensitivity was completely reversed by the TRPV1 antagonist A-425619. Furthermore, DRG neurons were isolated from the injured L4 ganglia or adjacent L3 ganglia 4–10 days after L4 SNL. Whole-cell patch clamp recordings were performed and heat stimuli (22–50°C/3 sec) were applied to the soma. Neurons were classified by soma size and isolectin-B4 (IB4) binding. Among directly injured L4 neurons, SNL increased the percentage of small-diameter IB4 positive neurons that were heat sensitive from 13% (naive controls) to 56% and conversely, decreased the proportion of small IB4-negative neurons that were heat sensitive from 66% (naive controls) to 34%. There was no change in IB4 binding in neurons from the injured ganglia. Surprisingly, in neurons from the adjacent L3 ganglia, SNL had no effect on the heat responsiveness of either IB4 positive or negative small neurons. Also, SNL had no effect on heat responses in medium-large diameter neurons from either the injured or adjacent ganglia.
doi:10.1016/j.jpain.2009.09.018
PMCID: PMC2879455  PMID: 20015699
Neuropathic pain; heat; capsaicin; C fiber; patch clamp; hyperalgesia
4.  TRPA1 modulates mechanotransduction in cutaneous sensory neurons 
TRPA1 is expressed by nociceptive neurons of the dorsal root ganglia (DRG) and trigeminal ganglia, but its roles in cold and mechanotransduction are controversial. To determine the contribution of TRPA1 to cold and mechanotransduction in cutaneous primary afferent terminals, we used the ex-vivo skin-nerve preparation from Trpa1+/+, Trpa1+/− or Trpa1−/− adult mouse littermates. Cutaneous fibers from TRPA1-deficient mice showed no deficits in acute cold sensitivity, but they displayed striking deficits in mechanical response properties. C fiber nociceptors from Trpa1−/− mice exhibited action potential firing rates 50% lower than those in wild-type C fibers across a wide range of force intensities. Aδ fiber mechanonociceptors also had reduced firing, but only at high intensity forces (> 100 mN). Surprisingly, the firing rates of low-threshold Aβ and D-hair mechanoreceptive fibers were also altered. TRPA1 protein and mRNA expression was assessed in DRG neurons and cutaneous innervation by using Trpa1 in situ hybridization, an antibody for TRPA1 and an antibody for placental-alkaline-phosphatase (PLAP) in mice where PLAP was substituted for Trpa1. DRG neurons of all sizes expressed Trpa1 mRNA or PLAP immunoreactivity. TRPA1 or PLAP immunolabeling was detected not only on many thin-caliber axons and intraepidermal endings but also on many large-caliber axons as well as lanceolate and Meissner endings. Epidermal and hair follicle keratinocytes also express TRPA1 message and protein. We propose that TRPA1 modulates mechanotransduction via a cell-autonomous mechanism in nociceptor terminals and possibly through a modulatory role in keratinocytes which may interact with sensory terminals to modify their mechanical firing properties.
doi:10.1523/JNEUROSCI.5380-08.2009
PMCID: PMC2744291  PMID: 19369549
Mechanosensory; skin; TRP channel; somatosensory; pain; knockout mice; cold
5.  Spinal muscular atrophy astrocytes exhibit abnormal calcium regulation and reduced growth factor production 
Glia  2013;61(9):1418-1428.
Spinal muscular atrophy is a genetic disorder caused by deletion of the survival motor neuron 1 (SMN1) gene that leads to loss of motor neurons in the spinal cord. Though motor neurons are selectively lost during SMA pathology, selective replacement of SMN in motor neurons does not lead to full rescue in mouse models. Due to the ubiquitous expression of SMN, it is likely that other cell types besides motor neurons are affected by its disruption and therefore may contribute to disease pathology. Here we show that astrocytes in SMAΔ7 mouse spinal cord and from SMA induced pluripotent stem cells (iPSCs) exhibit morphological and cellular changes indicative of activation prior to overt motor neuron loss. Furthermore, our in vitro studies show mis regulation of basal calcium and decreased response to ATP stimulation indicating abnormal astrocyte function. Together, these data show for the first time early disruptions in astrocytes that may contribute to SMA disease pathology.
doi:10.1002/glia.22522
PMCID: PMC3941074  PMID: 23839956
Motor neurons; stem cells; cell autonomous; astrocyte activation
6.  Patients with Sickle Cell Disease Have Increased Sensitivity to Cold and Heat 
American journal of hematology  2012;88(1):37-43.
Sickle Cell Disease (SCD) pain is associated with colder temperatures and touch and described as “cold”, “hot” and “shooting” suggesting hypersensitivity to tactile stimuli. Sickle mice exhibit hypersensitivity to thermal (cold, heat) and mechanical stimuli compared to controls. It is unknown whether humans experience this same hypersensitivity. Thus, we quantified thermal and mechanical sensitivity differences between SCD patients and controls. Our primary hypothesis was that SCD patients will exhibit hypersensitivity to thermal and mechanical stimuli compared to race-matched controls. Our secondary hypothesis was this hypersensitivity will be associated with older and female subjects, and with frequent pain and hemolysis in SCD patients. A total of 55 patients and 57 controls ≥7 years completed quantitative sensory testing. SCD patients detected the sensation of cold and warm temperatures sooner as seen in their significantly lower median cold and heat detection thresholds [29.5°C vs. 28.6°C, p=0.012 and 34.5°C vs. 35.3°C, p=0.02] and experienced cold and heat pain sooner as seen in their significantly lower median cold and heat pain thresholds [21.1°C vs. 14.8°C, p=0.01 and 42.7°C vs. 45.2°C, p=0.04]. We found no mechanical threshold differences. Older age was associated with lower cold, heat, and mechanical pain thresholds in both groups. No association with pain, gender, or hemolysis was found. SCD patients exhibit hypersensitivity to thermal stimuli suggesting peripheral or central sensitization may exist and could contribute to SCD pain.
doi:10.1002/ajh.23341
PMCID: PMC3552380  PMID: 23115062
sickle cell disease; pain; sensitivity
7.  Mechanical sensitization of cutaneous sensory fibers in the spared nerve injury mouse model 
Molecular Pain  2013;9:61.
Background
The spared nerve injury (SNI) model of neuropathic pain produces robust and reproducible behavioral mechanical hypersensitivity. Although this rodent model of neuropathic pain has been well established and widely used, peripheral mechanisms underlying this phenotype remain incompletely understood. Here we investigated the role of cutaneous sensory fibers in the maintenance of mechanical hyperalgesia in mice post-SNI.
Findings
SNI produced robust, long-lasting behavioral mechanical hypersensitivity compared to sham and naïve controls beginning by post-operative day (POD) 1 and continuing through at least POD 180. We performed teased fiber recordings on single cutaneous fibers from the spared sural nerve using ex vivo skin-nerve preparations. Recordings were made between POD 16–42 after SNI or sham surgery. Aδ-mechanoreceptors (AM) and C fibers, many of which are nociceptors, from SNI mice fired significantly more action potentials in response to suprathreshold mechanical stimulation than did fibers from either sham or naïve control mice. However, there was no increase in spontaneous activity.
Conclusions
To our knowledge, this is the first study evaluating the contribution of primary afferent fibers in the SNI model. These data suggest that enhanced suprathreshold firing in AM and C fibers may play a role in the marked, persistent mechanical hypersensitivity observed in this model. These results may provide insight into mechanisms underlying neuropathic pain in humans.
doi:10.1186/1744-8069-9-61
PMCID: PMC3906996  PMID: 24286165
Neuropathic; Nociceptor; Sensory neuron; C fiber; A fiber; Hyperalgesia; Mechanotransduction
8.  Opportunities for Live Cell FT-Infrared Imaging: Macromolecule Identification with 2D and 3D Localization 
Infrared (IR) spectromicroscopy, or chemical imaging, is an evolving technique that is poised to make significant contributions in the fields of biology and medicine. Recent developments in sources, detectors, measurement techniques and speciman holders have now made diffraction-limited Fourier transform infrared (FTIR) imaging of cellular chemistry in living cells a reality. The availability of bright, broadband IR sources and large area, pixelated detectors facilitate live cell imaging, which requires rapid measurements using non-destructive probes. In this work, we review advances in the field of FTIR spectromicroscopy that have contributed to live-cell two and three-dimensional IR imaging, and discuss several key examples that highlight the utility of this technique for studying the structure and chemistry of living cells.
doi:10.3390/ijms141122753
PMCID: PMC3856089  PMID: 24256815
infrared spectromicroscopy; flow cell; raster scanning; microtomography; transflection; scattering; deconvolution; Thalassiosira weissflogii; sensory neurons
9.  TRPV1, but not TRPA1, in primary sensory neurons contributes to cutaneous incision-mediated hypersensitivity 
Molecular Pain  2013;9:9.
Background
Mechanisms underlying postoperative pain remain poorly understood. In rodents, skin-only incisions induce mechanical and heat hypersensitivity similar to levels observed with skin plus deep incisions. Therefore, cutaneous injury might drive the majority of postoperative pain. TRPA1 and TRPV1 channels are known to mediate inflammatory and nerve injury pain, making them key targets for pain therapeutics. These channels are also expressed extensively in cutaneous nerve fibers. Therefore, we investigated whether TRPA1 and TRPV1 contribute to mechanical and heat hypersensitivity following skin-only surgical incision.
Results
Behavioral responses to mechanical and heat stimulation were compared between skin-incised and uninjured, sham control groups. Elevated mechanical responsiveness occurred 1 day post skin-incision regardless of genetic ablation or pharmacological inhibition of TRPA1. To determine whether functional changes in TRPA1 occur at the level of sensory neuron somata, we evaluated cytoplasmic calcium changes in sensory neurons isolated from ipsilateral lumbar 3–5 DRGs of skin-only incised and sham wild type (WT) mice during stimulation with the TRPA1 agonist cinnamaldehyde. There were no changes in the percentage of neurons responding to cinnamaldehyde or in their response amplitudes. Likewise, the subpopulation of DRG somata retrogradely labeled specifically from the incised region of the plantar hind paw showed no functional up-regulation of TRPA1 after skin-only incision. Next, we conducted behavior tests for heat sensitivity and found that heat hypersensitivity peaked at day 1 post skin-only incision. Skin incision-induced heat hypersensitivity was significantly decreased in TRPV1-deficient mice. In addition, we conducted calcium imaging with the TRPV1 agonist capsaicin. DRG neurons from WT mice exhibited sensitization to TRPV1 activation, as more neurons (66%) from skin-incised mice responded to capsaicin compared to controls (46%), and the sensitization occurred specifically in isolectin B4 (IB4)-positive neurons where 80% of incised neurons responded to capsaicin compared to just 44% of controls.
Conclusions
Our data suggest that enhanced TRPA1 function does not mediate the mechanical hypersensitivity that follows skin-only surgical incision. However, the heat hypersensitivity is dependent on TRPV1, and functional up-regulation of TRPV1 in IB4-binding DRG neurons may mediate the heat hypersensitivity after skin incision injury.
doi:10.1186/1744-8069-9-9
PMCID: PMC3602024  PMID: 23497345
TRPV1; TRPA1; Postoperative pain; Sensory neuron; Keratinocyte; Mechanical; Heat; Skin; Cutaneous; Calcium imaging
10.  TRPA1 Is Functionally Expressed Primarily by IB4-Binding, Non-Peptidergic Mouse and Rat Sensory Neurons 
PLoS ONE  2012;7(10):e47988.
Subpopulations of somatosensory neurons are characterized by functional properties and expression of receptor proteins and surface markers. CGRP expression and IB4-binding are commonly used to define peptidergic and non-peptidergic subpopulations. TRPA1 is a polymodal, plasma membrane ion channel that contributes to mechanical and cold hypersensitivity during tissue injury, making it a key target for pain therapeutics. Some studies have shown that TRPA1 is predominantly expressed by peptidergic sensory neurons, but others indicate that TRPA1 is expressed extensively within non-peptidergic, IB4-binding neurons. We used FURA-2 calcium imaging to define the functional distribution of TRPA1 among peptidergic and non-peptidergic adult mouse (C57BL/6J) DRG neurons. Approximately 80% of all small-diameter (<27 µm) neurons from lumbar 1–6 DRGs that responded to TRPA1 agonists allyl isothiocyanate (AITC; 79%) or cinnamaldehyde (84%) were IB4-positive. Retrograde labeling via plantar hind paw injection of WGA-Alexafluor594 showed similarly that most (81%) cutaneous neurons responding to TRPA1 agonists were IB4-positive. Additionally, we cultured DRG neurons from a novel CGRP-GFP mouse where GFP expression is driven by the CGRPα promoter, enabling identification of CGRP-expressing live neurons. Interestingly, 78% of TRPA1-responsive neurons were CGRP-negative. Co-labeling with IB4 revealed that the majority (66%) of TRPA1 agonist responders were IB4-positive but CGRP-negative. Among TRPA1-null DRGs, few small neurons (2–4%) responded to either TRPA1 agonist, indicating that both cinnamaldehyde and AITC specifically target TRPA1. Additionally, few large neurons (≥27 µm diameter) responded to AITC (6%) or cinnamaldehyde (4%), confirming that most large-diameter somata lack functional TRPA1. Comparison of mouse and rat DRGs showed that the majority of TRPA1-responsive neurons in both species were IB4-positive. Together, these data demonstrate that TRPA1 is functionally expressed primarily in the IB4-positive, CGRP-negative subpopulation of small lumbar DRG neurons from rodents. Thus, IB4 binding is a better indicator than neuropeptides for TRPA1 expression.
doi:10.1371/journal.pone.0047988
PMCID: PMC3485059  PMID: 23133534
11.  Prostaglandin metabolite induces inhibition of TRPA1 and channel-dependent nociception 
Molecular Pain  2012;8:75.
Background
The Transient Receptor Potential (TRP) ion channel TRPA1 is a key player in pain pathways. Irritant chemicals activate ion channel TRPA1 via covalent modification of N-terminal cysteines. We and others have shown that 15-Deoxy-Δ12, 14-prostaglandin J2 (15d-PGJ2) similarly activates TRPA1 and causes channel-dependent nociception. Paradoxically, 15d-PGJ2 can also be anti-nociceptive in several pain models. Here we hypothesized that activation and subsequent desensitization of TRPA1 in dorsal root ganglion (DRG) neurons underlies the anti-nociceptive property of 15d-PGJ2. To investigate this, we utilized a battery of behavioral assays and intracellular Ca2+ imaging in DRG neurons to test if pre-treatment with 15d-PGJ2 inhibited TRPA1 to subsequent stimulation.
Results
Intraplantar pre-injection of 15d-PGJ2, in contrast to mustard oil (AITC), attenuated acute nocifensive responses to subsequent injections of 15d-PGJ2 and AITC, but not capsaicin (CAP). Intraplantar 15d-PGJ2—administered after the induction of inflammation—reduced mechanical hypersensitivity in the Complete Freund’s Adjuvant (CFA) model for up to 2 h post-injection. The 15d-PGJ2-mediated reduction in mechanical hypersensitivity is dependent on TRPA1, as this effect was absent in TRPA1 knockout mice. Ca2+ imaging studies of DRG neurons demonstrated that 15d-PGJ2 pre-exposure reduced the magnitude and number of neuronal responses to AITC, but not CAP. AITC responses were not reduced when neurons were pre-exposed to 15d-PGJ2 combined with HC-030031 (TRPA1 antagonist), demonstrating that inhibitory effects of 15d-PGJ2 depend on TRPA1 activation. Single daily doses of 15d-PGJ2, administered during the course of 4 days in the CFA model, effectively reversed mechanical hypersensitivity without apparent tolerance or toxicity.
Conclusions
Taken together, our data support the hypothesis that 15d-PGJ2 induces activation followed by persistent inhibition of TRPA1 channels in DRG sensory neurons in vitro and in vivo. Moreover, we demonstrate novel evidence that 15d-PGJ2 is analgesic in mouse models of pain via a TRPA1-dependent mechanism. Collectively, our studies support that TRPA1 agonists may be useful as pain therapeutics.
doi:10.1186/1744-8069-8-75
PMCID: PMC3526547  PMID: 23013719
TRPA1; 15d-PGJ2; Mustard oil; Negative modulation; Mechanical hypersensitivity
12.  Sickle cell mice exhibit mechanical allodynia and enhanced responsiveness in light touch cutaneous mechanoreceptors 
Molecular Pain  2012;8:62.
Background
Sickle cell disease (SCD) is associated with both acute vaso-occlusive painful events as well as chronic pain syndromes, including heightened sensitivity to touch. We have previously shown that mice with severe SCD (HbSS mice; express 100% human sickle hemoglobin in red blood cells; RBCs) have sensitized nociceptors, which contribute to increased mechanical sensitivity. Yet, the hypersensitivity in these neural populations alone may not fully explain the mechanical allodynia phenotype in mouse and humans.
Findings
Using the Light Touch Behavioral Assay, we found HbSS mice exhibited increased responses to repeated application of both innocuous punctate and dynamic force compared to control HbAA mice (100% normal human hemoglobin). HbSS mice exhibited a 2-fold increase in percent response to a 0.7mN von Frey monofilament when compared to control HbAA mice. Moreover, HbSS mice exhibited a 1.7-fold increase in percent response to the dynamic light touch “puffed” cotton swab stimulus. We further investigated the mechanisms that drive this behavioral phenotype by focusing on the cutaneous sensory neurons that primarily transduce innocuous, light touch. Low threshold cutaneous afferents from HbSS mice exhibited sensitization to mechanical stimuli that manifested as an increase in the number of evoked action potentials to suprathreshold force. Rapidly adapting (RA) Aβ and Aδ D-hair fibers showed the greatest sensitization, each with a 75% increase in suprathreshold firing compared to controls. Slowly adapting (SA) Aβ afferents had a 25% increase in suprathreshold firing compared to HbAA controls.
Conclusions
These novel findings demonstrate mice with severe SCD exhibit mechanical allodynia to both punctate and dynamic light touch and suggest that this behavioral phenotype may be mediated in part by the sensitization of light touch cutaneous afferent fibers to suprathreshold force. These findings indicate that Aβ fibers can be sensitized to mechanical force and should potentially be examined for sensitization in other tissue injury and disease models.
doi:10.1186/1744-8069-8-62
PMCID: PMC3495672  PMID: 22963123
Primary afferents; Allodynia; Pain; Nociception; Anxiety; Open field test
13.  TRPA1 Mediates Mechanical Sensitization in Nociceptors during Inflammation 
PLoS ONE  2012;7(8):e43597.
Inflammation is a part of the body’s natural response to tissue injury which initiates the healing process. Unfortunately, inflammation is frequently painful and leads to hypersensitivity to mechanical stimuli, which is difficult to treat clinically. While it is well established that altered sensory processing in the spinal cord contributes to mechanical hypersensitivity (central sensitization), it is still debated whether primary afferent neurons become sensitized to mechanical stimuli after tissue inflammation. We induced inflammation in C57BL/6 mice via intraplantar injection of Complete Freund’s Adjuvant. Cutaneous C fibers exhibited increased action potential firing to suprathreshold mechanical stimuli. We found that abnormal responses to intense mechanical stimuli were completely suppressed by acute incubation of the receptive terminals with the TRPA1 inhibitor, HC-030031. Further, elevated responses were predominantly exhibited by a specific subgroup of C fibers, which we determined to be C-Mechano Cold sensitive fibers. Thus, in the presence of HC-030031, C fiber mechanical responses in inflamed mice were not different than responses in saline-injected controls. We also demonstrate that injection of the HC-030031 compound into the hind paw of inflamed mice alleviates behavioral mechanical hyperalgesia without affecting heat hyperalgesia. Further, we pharmacologically anesthetized the TRPA1-expressing fibers in vivo by co-injecting the membrane-impermeable sodium channel inhibitor QX-314 and the TRPA1 agonist cinnamaldehyde into the hind paw. This approach also alleviated behavioral mechanical hyperalgesia in inflamed mice but left heat hypersensitivity intact. Our findings indicate that C-Mechano Cold sensitive fibers exhibit enhanced firing to suprathreshold mechanical stimuli in a TRPA1-dependent manner during inflammation, and that input from these fibers drives mechanical hyperalgesia in inflamed mice.
doi:10.1371/journal.pone.0043597
PMCID: PMC3426543  PMID: 22927999
14.  Pain tests provoke modality-specific cardiovascular responses in awake, unrestrained rats 
Pain  2010;152(2):274-284.
Nociception modulates heart rate (HR) and mean arterial pressure (MAP), suggesting their use as indicators of pain in animals. We explored this with telemetric recording in unrestrained control and neuropathic (spinal nerve ligation) rats. Plantar stimulation was performed emulating techniques commonly used to measure pain, specifically brush stroke, von Frey fiber application, noxious pin stimulation, acetone for cooling, and radiant heating, while recording MAP, HR, and specific evoked somatomotor behaviors (none; simple withdrawal; or sustained lifting, shaking and grooming representing hyperalgesia). Pin produced elevations in both HR and MAP, and greater responses accompanied hyperalgesia behavior compared to simple withdrawal. Von Frey stimulation depressed MAP, and increased HR only when stimulation produced hyperalgesia behavior, suggesting that minimal nociception occurs without this behavior. Brush increased MAP even when no movement was evoked. Cold elevated both HR and MAP whether or not there was withdrawal, but MAP increased more when withdrawal was triggered. Heating consistently depressed HR and MAP, independent of behavior. Other than a greater HR response to pin in animals made hyperalgesic by injury, cardiovascular events evoked by stimulation did not differ between control and neuropathic animals. We conclude that a) thermoregulation rather than pain may dominate responses to heat and cooling stimuli; b) brush and cooling stimuli may be perceived and produce cardiovascular activation without nocifensive withdrawal; c) sensations that produce hyperalgesia behavior are accompanied by greater cardiovascular activation than those producing simple withdrawal; and d) von Frey stimulation lacks cardiovascular evidence of nociception except when hyperalgesia behavior is evoked.
doi:10.1016/j.pain.2010.09.010
PMCID: PMC3022106  PMID: 20943317
Neuropathic pain; animal pain tests; nociception; pain models; rat; cardiovascular effects of pain
15.  Physiological basis of tingling paresthesia evoked by hydroxy-α-sanshool 
Hydroxy-α-sanshool, the active ingredient in plants of the prickly ash plant family, induces robust tingling paresthesia by activating a subset of somatosensory neurons. However, the subtypes and physiological function of sanshool-sensitive neurons remain unknown. Here we use the ex vivo skin-nerve preparation to examine the pattern and intensity with which the sensory terminals of cutaneous neurons respond to hydroxy-α-sanshool. We found that sanshool excites virtually all D-hair afferents, a distinct subset of ultra-sensitive light touch receptors in the skin, and targets novel populations of Aβ and C-fiber nerve afferents. Thus, sanshool provides a novel pharmacological tool for discriminating functional subtypes of cutaneous mechanoreceptors. The identification of sanshool-sensitive fibers represents an essential first step in identifying the cellular and molecular mechanisms underlying tingling paresthesia that accompanies peripheral neuropathy and injury.
doi:10.1523/JNEUROSCI.4666-09.2010
PMCID: PMC2852189  PMID: 20335471
somatosensory; pain; TRPV; mechanosensory; potassium channels; nociceptor; cutaneous
16.  TRPA1 Mediates Mechanical Currents in the Plasma Membrane of Mouse Sensory Neurons 
PLoS ONE  2010;5(8):e12177.
Mechanosensitive channels serve as essential sensors for cells to interact with their environment. The identity of mechanosensitive channels that underlie somatosensory touch transduction is still a mystery. One promising mechanotransduction candidate is the Transient Receptor Potential Ankyrin 1 (TRPA1) ion channel. To determine the role of TRPA1 in the generation of mechanically-sensitive currents, we used dorsal root ganglion (DRG) neuron cultures from adult mice and applied rapid focal mechanical stimulation (indentation) to the soma membrane. Small neurons (diameter <27 µm) were studied because TRPA1 is functionally present in these neurons which largely give rise to C-fiber afferents in vivo. Small neurons were classified by isolectin B4 binding.
Mechanically-activated inward currents were classified into two subtypes: Slowly Adapting and Transient. First, significantly more IB4 negative neurons (84%) responded to mechanical stimulation than IB4 positive neurons (54%). Second, 89% of Slowly Adapting currents were present in IB4 negative neurons whereas only 11% were found in IB4 positive neurons. Third, Slowly Adapting currents were completely absent in IB4 negative neurons from TRPA1−/− mice. Consistent with this, Slowly Adapting currents were abolished in wild type IB4 negative neurons stimulated in the presence of a TRPA1 antagonist, HC-030031. In addition, the amplitude of Transient mechanically-activated currents in IB4 positive neurons from TRPA1−/− mice was reduced by over 60% compared to TRPA1+/+ controls; however, a similar reduction did not occur in wild-type neurons treated with HC-030031. Transfection of TRPA1 in HEK293 cells did not significantly alter the proportion or magnitude of mechanically-activated currents in HEK293 cells, indicating that TRPA1 alone is not sufficient to confer mechanical sensitivity.
These parallel genetic and pharmacological data demonstrate that TRPA1 mediates the Slowly Adapting mechanically-activated currents in small-diameter IB4 negative neurons from adult mice. The TRPA1 protein may also contribute to a complex that mediates Transient mechanically-activated currents in small IB4 positive C fiber type neurons.
doi:10.1371/journal.pone.0012177
PMCID: PMC2922334  PMID: 20808441
17.  Neurotrophin-3 significantly reduces sodium channel expression linked to neuropathic pain states 
Experimental neurology  2008;213(2):303-314.
Neuropathic pain resulting from chronic constriction injury (CCI) is critically linked to sensitization of peripheral nociceptors. Voltage gated sodium channels are major contributors to this state and their expression can be upregulated by nerve growth factor (NGF). We have previously demonstrated that neurotrophin-3 (NT-3) acts antagonistically to NGF in modulation of aspects of CCI-induced changes in trkA-associated nociceptor phenotype and thermal hyperalgesia. Thus, we hypothesized that exposure of neurons to increased levels of NT-3 would reduce expression of Nav1.8 and Nav1.9 in DRG neurons subject to CCI. In adult male rats, Nav1.8 and Nav1.9 mRNAs are expressed at high levels in predominantly small to medium size neurons. One week following CCI, there is reduced incidence of neurons expressing detectable Nav1.8 and Nav1.9 mRNA, but without a significant decline in mean level of neuronal expression, and similar findings observed immunohistochemically. There is also increased accumulation/redistribution of channel protein in the nerve most apparent proximal to the first constriction site. Intrathecal infusion of NT-3 significantly attenuates neuronal expression of Nav1.8 and Nav1.9 mRNA contralateral and most notably, ipsilateral to CCI, with a similar impact on relative protein expression at the level of the neuron and constricted nerve. We also observe reduced expression of the common neurotrophin receptor p75 in response to CCI that is not reversed by NT-3 in small to medium sized neurons and may confer an enhanced ability of NT-3 to signal via trkA, as has been previously shown in other cell types. These findings are consistent with an analgesic role for NT-3.
doi:10.1016/j.expneurol.2008.06.002
PMCID: PMC2751854  PMID: 18601922
Nav1.8; Nav1.9; DRG; sciatic nerve; CCI; nociceptor; nerve growth factor
18.  CONSTITUTIVE ACTIVITY AT THE CANNABINOID CB1 RECEPTOR IS REQUIRED FOR BEHAVIORAL RESPONSE TO NOXIOUS CHEMICAL STIMULATION OF TRPV1: ANTINOCICEPTIVE ACTIONS OF CB1 INVERSE AGONISTS 
The potential modulation of TRPV1 nociceptive activity by the CB1 receptor was investigated here using CB1 wildtype (WT) and knock-out (KO) mice as well as selective CB1 inverse agonists. No significant differences were detected in baseline thermal thresholds of ICR, CB1WT or CB1KO mice. Intraplantar capsaicin produced dose- and time-related paw flinch responses in ICR and CB1WT mice and induced plasma extravasation yet minimal responses were seen in CB1KO animals with no apparent differences in TRPV1 channel expression. Capsaicin-evoked CGRP release from spinal cord tissue and capsaicin-evoked action potentials on isolated skin-nerve preparation were significantly decreased in CB1KO mice. Pretreatment with intraplantar galanin and bradykinin, compounds known to sensitize TRPV1 receptors, restored capsaicin-induced flinching in CB1KO mice. The possibility that constitutive activity at the CB1 receptor is required to maintain the TRPV1 receptor in a “sensitized” state was tested using CB1 inverse agonists. The CB1 inverse agonists elicited concentration-related inhibition of capsaicin-induced calcium influx in F-11 cells and produced dose-related inhibition of capsaicin-induced flinching in ICR mice. These data suggest that constitutive activity at the CB1 receptor maintains the TRPV1 channel in a sensitized state responsive to noxious chemical stimuli. Treatment with CB1 inverse agonists may promote desensitization of the channel resulting in antinociceptive actions against chemical stimulus modalities. These studies propose possible therapeutic exploitation of a novel mechanism providing pain relief by CB1 inverse agonists.
doi:10.1523/JNEUROSCI.3322-08.2008
PMCID: PMC2744288  PMID: 18987195
TRPV1; CB1; Capsaicin; PIP2; phospholipase C; knockout mouse
19.  Species and strain differences in rodent sciatic nerve anatomy: Implications for studies of neuropathic pain 
Pain  2008;136(1-2):188-201.
Hindlimb pain models developed in rats have been transposed to mice, but assumed sciatic nerve neuroanatomic similarities have not been examined. We compared sciatic nerve structural organization in mouse strains (C57BL/6J, DBA/2J, and B6129PF2/J) and rat strains (Wistar, Brown Norway, and Sprague–Dawley). Dissection and retrograde labeling showed mouse sciatic nerve origins predominantly from the third lumbar (L3) and L4 spinal nerves, unlike the L4 and L5 in rats. Proportionate contributions by each level differed significantly between strains in both mice and rats. Whereas all rats had six lumbar vertebrae, variable patterns in mice included mostly five vertebrae in DBA/2J, mostly six vertebrae in C57BL/6J, and a mix in B6129PF2/J. Mice with a short lumbar vertebral column showed a rostral shift in relative contributions to the sciatic nerve by L3 and L4. Ligation of the mouse L4 nerve created hyperalgesia similar to that in rats after L5 ligation, and motor changes were similar after mouse L4 and rat L5 ligation (foot cupping) and after mouse L3 and rat L4 ligation (flexion weakness). Thus, mouse L3 and L4 neural segments are anatomically and functionally homologous with rat L4 and L5 segments. Neuronal changes after distal injury or inflammation should be sought in the mouse L3 and L4 ganglia, and the spinal nerve ligation model in mice should involve ligation of the L4 nerve while L3 remains intact. Strain-dependent variability in segmental contributions to the sciatic nerve may account in part for genetic differences in pain behavior after spinal nerve ligation.
doi:10.1016/j.pain.2008.01.016
PMCID: PMC2700063  PMID: 18316160
Neuropathic pain; Dorsal root ganglion; Pain models; Spinal nerve Ligation; Sciatic nerve anatomy
20.  Roles of transient receptor potential channels in pain 
Brain research reviews  2008;60(1):2-23.
Pain perception begins with the activation of primary sensory nociceptors. Over the past decade, flourishing research has revealed that members of the Transient Receptor Potential (TRP) ion channel family are fundamental molecules that detect noxious stimuli and transduce a diverse range of physical and chemical energy into action potentials in somatosensory nociceptors. Here we highlight the roles of TRP vanilloid 1 (TRPV1), TRP melastatin 8 (TRPM8) and TRP ankyrin 1 (TRPA1) in the activation of nociceptors by heat and cold environmental stimuli, mechanical force, and by chemicals including exogenous plant and environmental compounds as well as endogenous inflammatory molecules. The contribution of these channels to pain and somatosensation is discussed at levels ranging from whole animal behavior to molecular modulation by intracellular signaling proteins. An emerging theme is that TRP channels are not simple ion channel transducers of one or two stimuli, but instead serve multidimensional roles in signaling sensory stimuli that are exceptionally diverse in modality and in their environmental milieu.
doi:10.1016/j.brainresrev.2008.12.018
PMCID: PMC2683630  PMID: 19203589
TRPA1; TRPV1; TRPM8; Cold; Mechanotransduction; Nociceptor; Sensory neuron; Cutaneous; Inflammation; Prostaglandin; NGF; Artemin; Bradykinin; Kinase; Scaffold protein
21.  Pharmacological blockade of TRPA1 inhibits mechanical firing in nociceptors 
Molecular Pain  2009;5:19.
Background
TRPA1 has been implicated in both chemo- and mechanosensation. Recent work demonstrates that inhibiting TRPA1 function reduces mechanical hypersensitivity produced by inflammation. Furthermore, a broad range of chemical irritants require functional TRPA1 to exert their effects. In this study we use the ex-vivo skin-nerve preparation to directly determine the contribution of TRPA1 to mechanical- and chemical-evoked responses at the level of the primary afferent terminal.
Results
Acute application of HC-030031, a selective TRPA1 antagonist, inhibited all formalin responses in rat C fibers but had no effect on TRPV1 function, assessed by capsaicin responsiveness. Genetic ablation experiments corroborated the pharmacological findings as C fibers from wild type mice responded to both formalin and capsaicin, but fibers from their TRPA1-deficient littermates responded only to capsaicin. HC-030031 markedly reduced the mechanically-evoked action potential firing in rat and wild type mouse C fibers, particularly at high-intensity forces, but had no effect on the mechanical responsiveness of Aδ fiber nociceptors. Furthermore, HC-030031 had no effect on mechanically-evoked firing in C fibers from TRPA1-deficient mice, indicating that HC-030031 inhibits mechanically-evoked firing via a TRPA1-dependent mechanism.
Conclusion
Our data show that acute pharmacological blockade of TRPA1 at the cutaneous receptive field inhibits formalin-evoked activation and markedly reduces mechanically-evoked action potential firing in C fibers. Thus, functional TRPA1 at sensory afferent terminals in skin is required for their responsiveness to both noxious chemical and mechanical stimuli.
doi:10.1186/1744-8069-5-19
PMCID: PMC2681449  PMID: 19383149

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