Role In Pain Sensation Research Articles

In silico Studies Toward the Discovery of Novel Type-II Inhibitors of TrkA: Pharmacophore-based 3D-QSAR Modeling, Database Screening and Molecular Docking

Tropomysin receptor kinase A (TrkA) is an excellent drug target for its important roles in pain sensation as well as tumour cell growth. Up to now, the discovered TrkA inhibitors belong mostly to type-I class targeting the ATP binding site, while we aim to find type-II inhibitors because they are deemed to have improved kinase selectivity and slower off-rates than their counterparts. The type-II inhibitors can induce TrkA in an inactive DFG-out form and insert in an additional hydrophobic cavity adjacent to the ATP binding pocket. The current article describes efforts to discover novel type-II scaffolds against TrkA via integrating pharmacophore-based 3D-QSAR modeling, database screening and molecular docking. The robustness of the best model, AAHRR.8, was seriously ascertained by the high R2 (0.9027), Q2 (0.7048), low RMSE (0.4016) and SD (0.3635). It was further used as a 3D query to screen against our in-house collection of almost 1.3 million compounds followed by molecular docking simulations using three docking protocols involving Glide SP, Surflex and Glide XP. 12 hits, which mapped well on the best pharmacophore model, displayed good docking score and rational binding mode, were selected as promising selective leads of TrkA. Finally, ADME study was carried out and the results indicated that these 12 compounds own drug-like properties. The 12 hits together with the best 3D-QSAR model will be helpful for future potent TrkA agent development. Keywords: TrkA, Type-II inhibitor, pharmacophore modeling, 3D-QSAR, virtual screening, molecular docking.

The Specification and Maturation of Nociceptive Neurons from Human Embryonic Stem Cells.

Nociceptive neurons play an essential role in pain sensation by transmitting painful stimuli to the central nervous system. However, investigations of nociceptive neuron biology have been hampered by the lack of accessibility of human nociceptive neurons. Here, we describe a system for efficiently guiding human embryonic stem cells into nociceptive neurons by first inducing these cells to the neural lineage. Subsequent addition of retinoic acid and BMP4 at specific time points and concentrations yielded a high population of neural crest progenitor cells (AP2α+, P75+), which further differentiated into nociceptive neurons (TRKA+, Nav1.7+, P2X3+). The overexpression of Neurogenin 1 (Neurog1) promoted the neurons to express genes related to sensory neurons (Peripherin, TrkA) and to further mature into TRPV1+ nociceptive neurons. Importantly, the overexpression of Neurog1 increased the response of these neurons to capsaicin stimulation, a hallmark of mature functional nociceptive neurons. Taken together, this study reveals the important role that Neurog1 plays in generating functional human nociceptive neurons.

Activation of CB1 inhibits NGF-induced sensitization of TRPV1 in adult mouse afferent neurons

Transient receptor potential vanilloid 1 (TRPV1)-containing afferent neurons convey nociceptive signals and play an essential role in pain sensation. Exposure to nerve growth factor (NGF) rapidly increases TRPV1 activity (sensitization). In the present study, we investigated whether treatment with the selective cannabinoid receptor 1 (CB1) agonist arachidonyl-2′-chloroethylamide (ACEA) affects NGF-induced sensitization of TRPV1 in adult mouse dorsal root ganglion (DRG) afferent neurons. We found that CB1, NGF receptor tyrosine kinase A (trkA), and TRPV1 are present in cultured adult mouse small- to medium-sized afferent neurons and treatment with NGF (100ng/ml) for 30min significantly increased the number of neurons that responded to capsaicin (as indicated by increased intracellular Ca2 + concentration). Pretreatment with the CB1 agonist ACEA (10nM) inhibited the NGF-induced response, and this effect of ACEA was reversed by a selective CB1 antagonist. Further, pretreatment with ACEA inhibited NGF-induced phosphorylation of AKT. Blocking PI3 kinase activity also attenuated the NGF-induced increase in the number of neurons that responded to capsaicin. Our results indicate that the analgesic effect of CB1 activation may in part be due to inhibition of NGF-induced sensitization of TRPV1 and also that the effect of CB1 activation is at least partly mediated by attenuation of NGF-induced increased PI3 signaling.

Temporal changes in MrgC expression after spinal nerve injury

Mas-related G-protein-coupled receptor subtype C (MrgC) may play an important role in pain sensation. However, the distribution of MrgC receptors in different subpopulations of rodent dorsal root ganglion (DRG) neurons has not been clearly demonstrated owing to a lack of MrgC-selective antibody. It is also unclear whether peripheral nerve injury induces different time-dependent changes in MrgC expression in injured and uninjured DRG neurons. Here we showed that MrgC immunoreactivity is distributed in both IB4-positive (non-peptidergic) and calcitonin gene-related peptide-positive (peptidergic) DRG neurons in mice and rats. Importantly, the MrgC mRNA level and MrgC immunoreactivity were both decreased in the injured L5 DRG compared to corresponding levels in the contralateral (uninjured) DRG in rats on days 14 and 30 after an L5 spinal nerve ligation. In contrast, mRNA and protein levels of MrgC were increased in the adjacent uninjured L4 DRG. Thus, nerve injury may induce temporal changes in MrgC expression that differ between injured and uninjured DRG neurons. In animal behavior tests, chronic constriction injury of the sciatic nerve induced mechanical pain hypersensitivity in wild-type mice and Mrg-clusterΔ−/− mice (Mrg KO). However, the duration of mechanical hypersensitivity was longer in the Mrg KO mice than in their wild-type littermates, indicating that activation of Mrgs may constitute an endogenous mechanism that inhibits the maintenance of neuropathic pain in mice. These findings extend our knowledge about the distribution of MrgC in rodent DRG neurons and the regulation of its expression by nerve injury.

Acid-Sensing Ion Channels Contribute to Neurotoxicity

Acidosis that occurs under pathological conditions not only affects intracellular signaling molecules, but also directly activates a unique family of ligand-gated ion channels: acid-sensing ion channels (ASICs). ASICs are widely expressed throughout the central and peripheral nervous systems and play roles in pain sensation, learning and memory, and fear conditioning. Overactivation of ASICs contributes to neurodegenerative diseases such as ischemic brain/spinal cord injury, multiple sclerosis, Parkinson's disease, and Huntington's disease. Thus, targeting ASICs might be a potential therapeutic strategy for these conditions. This mini-review focuses on the electrophysiology and pharmacology of ASICs and roles of ASICs in neuronal toxicity.

Mas-related G protein-coupled receptor D is coupled to endogenous calcium-activated chloride channel in Xenopus oocytes

Mas-related G protein-coupled receptor D (MrgD) is expressed almost exclusively in nociceptive primary sensory neurons and the neurons located in stratum granulosum of skin. More and more evidence suggest that MrgD plays an important role in pain sensation and/or transduction. Recent studies have demonstrated that the receptor is also involved in itch sensation in both mouse and human. In the present study, we identified a robust inward current in MrgD-expressing Xenopus oocytes by using β-alanine, a putative ligand of MrgD. The currents were sensitive to inhibitor of Ca(2+)-activated chloride channels (CaCCs) and intracellular Ca(2+) chelator, suggesting they were produced by endogenous CaCCs. Furthermore, it was demonstrated that upon the application of phospholipase C (PLC) inhibitor, or antisense oligonucleotides of inositol trisphosphate receptor (IP3R), the β-alanine-induced currents were dramatically depressed. However, protein kinase C inhibitor did not display any visible effect on CaCC currents. In summary, our data suggest that the activation of MrgD promotes the open of endogenous CaCCs via G(q)-PLC-IP3-Ca(2+) pathway. The current findings reveal the functional coupling between MrgD and CaCCs in Xenopus oocytes and also provide a facile model to assay the activity of MrgD.

Serotonin Facilitates Peripheral Pain Sensitivity in a Manner That Depends on the Nonproton Ligand Sensing Domain of ASIC3 Channel

Tissue acidosis and inflammatory mediators play critical roles in inflammatory pain. Extracellular acidosis activates acid-sensing ion channels (ASICs), which have emerged as key sensors for extracellular protons in the central and peripheral nervous systems and play key roles in pain sensation and transmission. Additionally, inflammatory mediators, such as serotonin (5-HT), are known to enhance pain sensation. However, functional interactions among protons, inflammatory mediators, and ASICs in pain sensation are poorly understood. In the present study, we show that 5-HT, a classical pro-inflammatory mediator, specifically enhances the proton-evoked sustained, but not transient, currents mediated by homomeric ASIC3 channels and heteromeric ASIC3/1a and ASIC3/1b channels. Unexpectedly, the effect of 5-HT on ASIC3 channels does not involve activation of 5-HT receptors, but is mediated via a functional interaction between 5-HT and ASIC3 channels. We further show that the effect of 5-HT on ASIC3 channels depends on the newly identified nonproton ligand sensing domain. Finally, coapplication of 5-HT and acid significantly increased pain-related behaviors as assayed by the paw-licking test in mice, which was largely attenuated in ASIC3 knock-out mice, and inhibited by the nonselective ASIC inhibitor amiloride. Together, these data identify ASIC3 channels as an unexpected molecular target for acute actions of 5-HT in inflammatory pain sensation and reveal an important role of ASIC3 channels in regulating inflammatory pain via coincident detection of extracellular protons and inflammatory mediators.

Increasing TNF levels solely in the rat hippocampus produces persistent pain-like symptoms

The manifestation of chronic, neuropathic pain includes elevated levels of the cytokine tumor necrosis factor–alpha (TNF). Previously, we have shown that the hippocampus, an area of the brain most notable for its role in learning and memory formation, plays a fundamental role in pain sensation. Using an animal model of peripheral neuropathic pain, we have demonstrated that intracerebroventricular infusion of a TNF antibody adjacent to the hippocampus completely alleviated pain. Furthermore, intracerebroventricular infusion of rTNF adjacent to the hippocampus induced pain behavior in naïve animals similar to that expressed during a model of neuropathic pain. These data support our premise that enhanced production of hippocampal-TNF is integral in pain sensation. In the present study, TNF gene expression was induced exclusively in the hippocampus, eliciting increased local bioactive TNF levels, and animals were assessed for pain behaviors. Male Sprague-Dawley rats received stereotaxic injection of gold nanorod (GNR)-complexed cDNA (control or TNF) plasmids (nanoplasmidexes), and pain responses (i.e., thermal hyperalgesia and mechanical allodynia) were measured. Animals receiving hippocampal microinjection of TNF nanoplasmidexes developed thermal hyperalgesia bilaterally. Sensitivity to mechanical stimulation also developed bilaterally in the rat hind paws. In support of these behavioral findings, immunoreactive staining for TNF, bioactive levels of TNF, and levels of TNF mRNA per polymerase chain reaction analysis were assessed in several brain regions and found to be increased only in the hippocampus. These findings indicate that the specific elevation of TNF in the hippocampus is not a consequence of pain, but in fact induces these behaviors/symptoms.

Inhibition of Activity of GABA Transporter GAT1 byδ-Opioid Receptor

Analgesia is a well-documented effect of acupuncture. A critical role in pain sensation plays the nervous system, including the GABAergic system and opioid receptor (OR) activation. Here we investigated regulation of GABA transporter GAT1 by δOR in rats and in Xenopus oocytes. Synaptosomes of brain from rats chronically exposed to opiates exhibited reduced GABA uptake, indicating that GABA transport might be regulated by opioid receptors. For further investigation we have expressed GAT1 of mouse brain together with mouse δOR and μOR in Xenopus oocytes. The function of GAT1 was analyzed in terms of Na+-dependent [3H]GABA uptake as well as GAT1-mediated currents. Coexpression of δOR led to reduced number of fully functional GAT1 transporters, reduced substrate translocation, and GAT1-mediated current. Activation of δOR further reduced the rate of GABA uptake as well as GAT1-mediated current. Coexpression of μOR, as well as μOR activation, affected neither the number of transporters, nor rate of GABA uptake, nor GAT1-mediated current. Inhibition of GAT1-mediated current by activation of δOR was confirmed in whole-cell patch-clamp experiments on rat brain slices of periaqueductal gray. We conclude that inhibition of GAT1 function will strengthen the inhibitory action of the GABAergic system and hence may contribute to acupuncture-induced analgesia.

A Novel Toxin that Targets Acid-Sensing Ion Channels to Produce Pain

Natural products have served as invaluable keys to unlocking molecular underpinnings of pain sensation. In particular, plant-derived irritants, such as capsaicin, menthol, mustard oil, have proven essential for identifying and/or characterizing excitatory TRP ion channels that play major roles in pain sensation. Animals also possess chemical defense mechanisms to inflict pain in predators. To more fully exploit the rich pharmacopeia of animal toxins, we established an unbiased screen to discover venoms capable of activating specific subpopulations of somatosensory neurons, with the goal of developing novel probes and identifying important physiological targets for pain research and therapeutics. We show that venom from the Texas coral snake - whose bite produces excruciating pain - contains a novel toxin that potently and persistently activates a subset of somatosensory neurons. First, we use biochemical and molecular methods to identify a unique two-component toxin that form a high affinity heteromeric complex. Second, we use electrophysiological and genetic approaches to identify acid-sensing ion channels (ASICs) as the molecular target. Furthermore, behavioral experiments show that toxin-evoked activation of ASIC channels elicits pain by recruiting a canonical population of primary afferent nociceptors that detect thermal and inflammatory pain.

An Itch To Be Scratched

The description of itch (formally known as pruritus) as an "unpleasant sensation that elicits the desire or reflex to scratch" (Ikoma et al., 2006) is immediately familiar. Research in the field of pruritoception has added to our understanding of this area of sensory neurobiology as it pertains to both normal and pathological conditions. In particular, much progress has been made on the mechanisms and circuits of itch, which we review here.

Amitriptyline inhibits currents and decreases the mRNA expression of voltage-gated sodium channels in cultured rat cortical neurons

Amitriptyline (AMI) is widely used for migraine prophylaxis, but its mechanism is undetermined. Cortical spreading depression (CSD), reflected by alterations in calcium, sodium, and Na +-K +ATP channels, has been implicated in migraine and as a headache trigger. Evidences indicate that mutations in sodium and calcium channels are involved in migraine. Sodium channels are critical for the electrical excitability of sensory neurons and play a key role in pain sensation by controlling afferent impulse discharge. To investigate the mechanism underlying AMI effectiveness for migraine prophylaxis, we studied the effects of AMI on voltage-gated sodium channels in cultured rat cortical neurons by the whole-cell patch clamp recording and real-time reverse transcription polymerase chain reaction (RT-PCR). We found that AMI blocked sodium channel current ( I Na) in a concentration-dependent, not voltage-dependent manner. AMI also altered the activation and steady-state inactivation of I Na toward hyperpolarization. Results of real-time RT-PCR indicated that AMI inhibited the expression for sodium channels in a concentration-dependent manner; inhibition of expression of the Na V1.1 and Na V1.6 sodium channels was more than that of Na V1.2. From these results, we speculate that AMI may reduce CSD by inhibiting I Na and mRNA expression of sodium channels. This may contribute to strategies for migraine prophylaxis.

Molecular Mechanisms of TRPV1 Channel Activation

Transient Receptor Potential (TRP) cation channels participate in various fundamental processes in cell- and organism-physiology in unicellular eukaryotes, invertebrates and vertebrates. Interestingly, many TRP channels function as detectors of sensory stimuli. The TRPV1 (vanilloid 1) channel serves as an integrator of noxious chemical and physical stimuli known to cause irritation and pain, such as elevated temperatures, acids, and irritant chemical compounds, and its activation has been linked to acute nociceptive pain and neurogenic inflammation. The mechanisms by which the channel detects incoming stimuli, how the sensing domains are coupled to channel gating and how these processes are connected to specific structural regions in the channel are not fully understood, but valuable information is available. Many sites in- volved in agonist detection have been characterized and gating models that describe many features of the channel's behav- ior have been put forward. Structural and functional information indicates TRP channels are similar to voltage-activated potassium channels, with a tetrameric organization and six-transmembrane-region subunits, a pore domain with multi-ion binding properties and an intracellular S6 gate that seems to be the point of convergence of the many activation modalities leading to the opening of the ion conduction pathway. Furthermore, TRPV1 expression is altered in various disease states and TRPV1 gene polymorphism was speculated to play a role in pain sensation. The complex activation and regulation of TRPV1 may have important implications for drug development.

Light Control of Cell Excitability Using a Photochromic Blocker for Voltage-Gated Ion Channels

Rendering proteins light sensitive gives the opportunity to control various cellular functions with great spatial and temporal precision. Native proteins can be photosensitized using photochromic ligands (PCLs), small light sensitive molecules that can be reversibly converted between active and inactive forms with light. We engineered a new PCL for voltage-gated ion channels, QAQ, which consists of an Azobenzene photoswitch flanked by two Quaternary ammoniums. Azobenzenes rapidly photoisomerize between cis and trans configurations upon 380 or 500 nm light irradiation, respectively. Quaternary ammoniums are internal blockers for voltage-gated sodium, potassium and calcium channels. Our studies show that intracellular QAQ blocks indiscriminitely those three types of ion channels in its trans state, while photoisomerization to the cis configuration relieves block.QAQ is a permanently charged, membrane-impermeable molecule that needs hydrophilic pathways to reach its site of action. Loading QAQ into neurons through a patch pipette can be used to control excitability of sub-cellular domains in a single cell. More importantly, QAQ can be loaded into multiple cells at a time, passing through open TRPV1 or P2X7 receptors, two non-selective cation channels that show pore-dilation upon prolonged activation. Experiments in HEK cells and hippocampal neurons show that QAQ can enter cells only upon expression AND activation of TRPV1 or P2X7R. QAQ can also be loaded into cells naturally expressing those channels, like the sensory neurons of the dorsal root ganglia that play a crucial role in pain sensation. QAQ acts like a local anesthetic, silencing sensory neurons by blocking voltage-gated cation channels. In this context, light can then be used to restore nociception. QAQ is therefore a very promising PCL molecule with both scientific and clinical applications.

Direct Single Injection of p38 Mitogen-Activated Protein Kinase Inhibitor Does Not Affect Calcitonin Gene-Related Peptide Expression in Dorsal Root Ganglion Neurons Innervating Punctured Discs in Rats

STUDY DESIGN.: Immunohistological analysis of punctured disc after application of a p38 MAP kinase inhibitor. OBJECTIVE.: To examine effect of direct application on dorsal root ganglion (DRG) neurons innervating damaged rat discs. SUMMARY OF BACKGROUND DATA.: Degeneration of lumbar discs is one cause of low back pain. Pathogenesis may involve sensory nerve ingrowth into disc inner layers; tumor necrosis factor-alpha (TNF-alpha) is thought to be a major inducer of ingrowth. Because p38 mitogen-activated protein kinase (p38) upregulates TNF-alpha expression and may play a crucial role in pain sensation, we investigated the effect of one injection of inhibitor on expression of the pain-related neuropeptide calcitonin gene-related peptide (CGRP). METHODS.: The neuro-tracer fluoro-gold was applied to the surfaces of L4/5 discs to label the innervating DRG neurons (n = 30). Of 30 rats, 10 were controls, whereas the other 20 were the experimental model (i.e., discs were punctured with 23-gauge needle). P38 specific inhibitor or saline was applied simultaneously (n = 10 each, Puncture + inhibitor and puncture + saline groups). Fourteen days postsurgery, DRGs from L1 to L6 were harvested, sectioned, and immunostained for CGRP. Proportion of CGRP-immunoreactive DRG neurons was evaluated in all groups. RESULTS.: Fluoro-gold-labeled neurons innervating the L4/5 disc were distributed throughout L1 to L6 DRGs in all groups. Proportions of labeled neurons positive for CGRP were 15.2% +/- 8% (controls), 27.2% +/- 10% (puncture + saline), and 25.2% +/- 8% (puncture + inhibitor). Proportion of immunoreactive neurons was significantly increased in the puncture groups compared with controls. However, there was no significant difference between the 2 puncture groups (P > 0.1). CONCLUSION.: In this model, CGRP was upregulated in DRG neurons innervating the damaged disc. However, a direct single application of p38 inhibitor did not suppress CGRP expression in innervating DRG neurons. Future research with p38 inhibitor in this model should evaluate multiple or systemic administration of inhibitor.

Trk kinase inhibitors as new treatments for cancer and pain

Background: Tropomyosin-related kinases (Trks) are a family of receptor tyrosine kinases activated by neurotrophins. Trks play important roles in pain sensation as well as tumour cell growth and survival signaling. Thus, inhibitors of Trk receptor kinases might provide targeted treatments for pain and cancer. Objective: This paper reviews those patent applications since 2002 claiming small-molecule inhibitors of Trk receptor kinases. Methods: Primary literature and patents were searched with SciFinder and Google Scholar. Patents were selected based on their relevance to Trks and were evaluated and representative compounds were listed as examples. Results/conclusion: Several series of Trk inhibitors with excellent in vitro potencies have been reported and a number of compounds have gone into the clinic. It should be noted that few of these inhibitors are Trk selective, demonstrating that targeting Trk kinases for treatment of pain and/or cancer offers a promising but also challenging approach.

Biophysical Properties of Human Nav1.7 Splice Variants and Their Regulation by Protein Kinase A

The sodium channel Na(v)1.7 is preferentially expressed in nociceptive neurons and is believed to play a crucial role in pain sensation. Four alternative splice variants are expressed in human dorsal root ganglion neurons, two of which differ in exon 5 by two amino acids in the S3 segment of domain I (exons 5A and 5N). Two others differ in exon 11 by the presence (11L) or absence (11S) of an 11 amino acid sequence in the loop between domains I and II, an important region for PKA regulation. In the present study, we used the whole cell configuration of the patch-clamp technique to investigate the biophysical properties and 8-bromo-cyclic adenosine monophosphate (8Br-cAMP) modulation of these splice variants expressed in tsA201 cells in the presence of the beta(1)-subunit. The alternative splicing of Na(v)1.7 had no effect on most of the biophysical properties of this channel, including activation, inactivation, and recovery from inactivation. However, development of inactivation experiments revealed that the isoform containing exon 5A had slower kinetics of inactivation for negative potentials than that of the variant containing exon 5N. This difference was associated with higher ramp current amplitudes for isoforms containing exon 5A. Moreover, 8Br-cAMP-mediated phosphorylation induced a negative shift of the activation curve of variants containing exon 11S, whereas inactivation properties were unchanged. Isoforms with exon 11L were not modulated by 8Br-cAMP-induced phosphorylation. We conclude that alternative splicing of human Na(v)1.7 can specifically modulate the biophysical properties and cAMP-mediated regulation of this channel. Changing the proportions of these variants may thus influence neuronal excitability and pain sensation.

Tetrodotoxin: a brief history.

Tetrodotoxin (TTX), contained in puffer, has become an extremely popular chemical tool in the physiological and pharmacological laboratories since our discovery of its channel blocking action in the early 1960s. This brief review describes the history of discovery of TTX action on sodium channels, and represents a story primarily of my own work. TTX inhibits voltage-gated sodium channels in a highly potent and selective manner without effects on any other receptor and ion channel systems. TTX blocks the sodium channel only from outside of the nerve membrane, and is due to binding to the selectivity filter resulting in prevention of sodium ion flow. It does not impairs the channel gating mechanism. More recently, the TTX-resistant sodium channels have been discovered in the nervous system and received much attention because of their role in pain sensation. TTX is now known to be produced not by puffer but by bacteria, and reaches various species of animals via food chain.

MrgD Activation Inhibits KCNQ/M-Currents and Contributes to Enhanced Neuronal Excitability

The recently identified Mas-related gene (Mrg) family of G-protein-coupled receptors is expressed almost exclusively in dorsal root ganglion (DRG) neurons. The expression of one family member, MrgD, is even further confined to IB4+, nonpeptidergic, small-diameter nociceptors. Although the functional consequences of MrgD activation are not known, this expression profile provides intriguing potential for a role in pain sensation or modulation. In a recombinant cell line, we first assessed the functional significance of MrgD activation by coexpressing MrgD with the KCNQ2/3 potassium channel, a channel implicated in pain. Whole-cell voltage-clamp recordings revealed that bath application of the ligand for MrgD, beta-alanine, resulted in robust inhibition of KCNQ2/3 activity. Pharmacological blockade of G(i/o) and phospholipase C signaling revealed a partial and complete block of the response, respectively. We extended these observations to dissociated DRG neuron cultures by examining MrgD modulation of M-currents (carried primarily by KCNQ2/3). Here too, beta-alanine-induced activation of endogenous MrgD inhibited M-currents, but primarily via a pertussis toxin-sensitive pathway. Finally, we assessed the consequence of beta-alanine-induced activation of MrgD in phasic neurons. Phasic neurons that fired a single action potential (AP) before beta-alanine application fired multiple APs during beta-alanine exposure. In sum, we provide evidence for a novel interaction between MrgD and KCNQ/M-type potassium channels that contributes to an increase in excitability of DRG neurons and thus may enhance the signaling of primary afferent nociceptive neurons.

Characteristics of Sensory Dorsal Root Ganglia Neurons Innervating the Lumbar Vertebral Body in Rats

Characteristics of sensory dorsal root ganglia (DRG) neurons innervating the L5 vertebral body were investigated in rats by using a retrograde neurotransport method, lectin affinity- and immuno-histochemistry to further elucidate the causes of diffuse pain suffered by some elderly patients in their back, lateral trunk, and iliac crest, after lumbar osteoporotic vertebral fracture. We used calcitonin gene-related peptide (CGRP) as a marker of small peptide-containing neurons and the glycoprotein binding the isolectin from Griffonia simplicifolia (IB4) as a marker of small non-peptide-containing neurons. Neurons innervating the L5 vertebral bodies, retrogradely labeled with fluoro-gold (FG), were distributed throughout DRGs from T13 to L6. The proportion of CGRP-immunoreactive (IR) FG-labeled neurons was 32%. The proportion of IB4-binding FG-labeled neurons was significantly smaller, at 4%. Other neurons that were non-CGRP-IR and non-IB4-binding were mostly large neurons, and they may transmit proprioception from vertebral bodies. Most neurons transmitting pain are CGRP-IR peptide-containing neurons. They may have a more significant role in pain sensation in the vertebral bodies as peptidergic DRG neurons. This article shows that vertebral bodies are innervated by CGRP-IR neurons. CGRP-IR neurons may play a role in pain sensation through peptidergic DRG neurons. These findings contribute to an understanding of pain associated with the vertebral body such as tumor, infection, or osteoporotic fracture.