Peripheral Mechanisms of Dental Pain: The Role of Substance P

Current evidence supports the central role of neuropeptides in the molecular mechanisms underlying dental pain. In particular, substance P, a neuropeptide produced in neuron cell bodies localised in dorsal root and trigeminal ganglia, contributes to the transmission and maintenance of noxious stimuli and inflammatory processes. The major role of substance P in the onset of dental pain and inflammation is increasingly being recognised. Well-grounded experimental and clinical observations have documented an increase in substance P concentration in patients affected by caries, pulpitis, or granulomas and in those undergoing standard orthodontic or orthodontic/dental care procedures. This paper focuses on the role of substance P in the induction and maintenance of inflammation and dental pain, in order to define future lines of research for the evaluation of therapeutic strategies aimed at modulating the complex effects of this mediator in oral tissues.

Related papers:

title: Orthodontic pain: from causes to management--a review.
abstract: Orthodontic pain, the most cited negative effect arising from orthodontic force application, is a major concern for parents, patients, and clinicians. Studies have reported this reaction to be a major deterrent to orthodontic treatment and an important reason for discontinuing treatment. Surprisingly this area, which requires attention in clinical practice as well as in research, is ignored as evidenced by the scarcity of publications on the topic in comparison with other areas of orthodontic research. This review attempts to organize the existing published literature regarding pain, which appears as part of orthodontic mechanotherapy and to address questions that might arise in a clinical setting from the viewpoint of clinicians and patients/parents. It also provides an overview of current management strategies employed for alleviating orthodontic pain.

title: Peripheral mechanisms of odontogenic pain.
abstract: In this article, we review the key basic mechanisms associated with this phenomena and more recently identified mechanisms that are current areas of interest. Although many of these pain mechanisms apply throughout the body, we attempt to describe these mechanisms in the context of trigeminal pain.

title: Hyperalgesia and allodynia: peripheral mechanisms.
abstract: Nociceptive signals are generated by peripheral sensory organs called nociceptors, which are endings of small-diameter nerve fibers responsive to the tissue environment. The myriad chemical mediators capable of activating, sensitizing, or arousing nociceptors include kinins, proinflammatory and anti-inflammatory cytokines, prostanoids, lipooxygenases, the "central immune response mediator" NF-kappaB, neurotrophins and other growth factors, neuropeptides, nitric oxide, histamine, serotonin, proteases, excitatory amino acids, adrenergic amines, and opioids. These mediators may act in combination or at a given time in the inflammatory process, producing subtle changes that result in hyperalgesia or allodynia. We will review the most extensively studied molecular and cellular mechanisms underlying these two clinical abnormalities. The role of the peripheral nervous system in progression of inflammatory joint disease to chronicity is discussed.

title: Signaling Pathways in Sensitization: Toward a Nociceptor Cell Biology
abstract: The electrophysiological properties of peripheral neurons activated by noxious stimuli, the primary afferent nociceptors, have been investigated intensively, and our knowledge about the molecular basis of transducers for noxious stimuli has increased greatly. In contrast, understanding of the intracellular signaling mechanisms regulating nociceptor sensitization downstream of ligand binding to the receptors is still at a relatively nascent stage. After outlining the initiated signaling cascades, we discuss the emerging plasticity within these cascades and the importance of subcellular compartmentalization. In addition, the recently realized importance of functional interactions with the extracellular matrix, cytoskeleton, intracellular organelles such as mitochondria, and sex hormones will be introduced. This burgeoning literature establishes new cellular features crucial for the function of nociceptive neurons and argues that additional focus should be placed on understanding the complex integration of cellular events that make up the "cell biology of pain."

title: Differential TRPV1 and TRPV2 Channel Expression in Dental Pulp
abstract: Hypersensitivity to thermal and mechanical stimuli can occur in painful pulpitis. To explore the neuro-anatomical basis of heat and mechanical sensitivity, we evaluated expression of TRPV1 (heat) and TRPV2 (heat/mechanical) channels in the cell bodies and terminal arborizations of neurons that innervate the dental pulp (DP) and periodontal tissues (PDL). We report that ~50% of trigeminal ganglion (TG) neurons retrogradely labeled from the DP express TRPV2, and this was significantly greater than the general expression of this channel in the TG (15%) and slightly more than what is expressed in the PDL by retrograde labeling (40%). The TRPV1 receptor, however, was less prevalent in neurons innervating the DP than their general expression in the TG (17% vs. 26%) and was more extensively expressed in neurons innervating the PDL (26%). Co-labeling studies showed that 70% of neurons that innervate the DP are myelinated. Approximately 1/3 of the retrogradely labeled neurons from the DP were calcitonin-gene-related-peptide-positive (peptide-expressing), but very few expressed the IB4 marker of non-peptidergic unmyelinated afferents. These findings suggest that the DP has a unique neurochemical innervation with regard to TRP receptor expression, which has significant implications for the mechanisms contributing to odontogenic pain and management strategies.

title: Cellular Mechanisms of Neurogenic Inflammation
abstract: Since the initial observations that stimulation of sensory neurons produces vasodilation, plasma extravasation, and hypersensitivity, much progress has been made in understanding the etiology of neurogenic inflammation. Studies have focused largely on the role of the neuropeptides, substance P and calcitonin gene-related peptide, which are released in the periphery by activation of small diameter sensory neurons. Recent work, however, has begun to emphasize the cellular mechanisms involved in regulating the release of proinflammatory substances from sensory neurons. In this perspective, discussion centers on a number of inflammatory mediators that activate various signal transduction pathways to augment excitability of and transmitter release from sensory neurons. Emphasis is placed on those pathways where multiple lines of evidence support their importance in initiating neurogenic inflammation. Recent studies, however, support the notion that there are novel compounds released during injury that can stimulate or sensitize sensory neurons. Furthermore, only now are intracellular signaling pathways that have been identified in other cell systems being studied in sensory neurons to establish their role in neurogenic inflammation. The challenge remains to ascertain the critical transduction pathways that regulate transmitter release from sensory neurons since this phenomenon triggers neurogenic inflammation. In addition, the cellular mechanisms involved in alterations in neuronal excitability during injury and the cellular pathways that maintain the inflammatory response over time need to be determined. With these advances, we will be able to develop therapeutic interventions to minimize deleterious consequences of neurogenic inflammation.

title: Neuropeptides in dental pulp: the silent protagonists.
abstract: Dental pulp is a soft mesenchymal tissue densely innervated by afferent (sensory) fibers, sympathetic fibers, and parasympathetic fibers. This complexity in pulp innervation has motivated numerous investigations regarding how these 3 major neuronal systems regulate pulp physiology and pathology. Most of this research is focused on neuropeptides and their role in regulating pulpal blood flow and the development of neurogenic inflammation. These neuropeptides include substance P, calcitonin gene-related peptide, neurokinin A, neuropeptide Y, and vasoactive intestinal polypeptide among others. The purpose of this article is to review recent advances in neuropeptide research on dental pulp, including their role in pulp physiology, their release in response to common dental procedures, and their plasticity in response to extensive pulp and dentin injuries. Special attention will be given to neuropeptide interactions with pulp and immune cells via receptors, including studies regarding receptor identification, characterization, mechanisms of action, and their effects in the development of neurogenic inflammation leading to pulp necrosis. Their role in the growth and expansion of periapical lesions will also be discussed. Because centrally released neuropeptides are involved in the development of dental pain, the pain mechanisms of the pulpodentin complex and the effectiveness of present and future pharmacologic therapies for the control of dental pain will be reviewed, including receptor antagonists currently under research. Finally, potential clinical therapies will be proposed, particularly aimed to manipulate neuropeptide expression or blocking their receptors, to modulate a variety of biologic mechanisms, which preliminary results have shown optimistic results.

title: The role of peptides in central sensitization.
abstract: Peptides released in the spinal cord from the central terminals of nociceptors contribute to the persistent hyperalgesia that defines the clinical experience of chronic pain. Using substance P (SP) and calcitonin gene-related peptide (CGRP) as examples, this review addresses the multiple mechanisms through which peptidergic neurotransmission contributes to the development and maintenance of chronic pain. Activation of CGRP receptors on terminals of primary afferent neurons facilitates transmitter release and receptors on spinal neurons increases glutamate activation of AMPA receptors. Both effects are mediated by cAMP-dependent mechanisms. Substance P activates neurokinin receptors (3 subtypes) which couple to phospholipase C and the generation of the intracellular messengers whose downstream effects include depolarizing the membrane and facilitating the function of AMPA and NMDA receptors. Activation of neurokinin-1 receptors also increases the synthesis of prostaglandins whereas activation of neurokinin-3 receptors increases the synthesis of nitric oxide. Both products act as retrograde messengers across synapses and facilitate nociceptive signaling in the spinal cord. Whereas these cellular effects of CGRP and SP at the level of the spinal cord contribute to the development of increased synaptic strength between nociceptors and spinal neurons in the pathway for pain, the different intracellular signaling pathways also activate different transcription factors. The activated transcription factors initiate changes in the expression of genes that contribute to long-term changes in the excitability of spinal and maintain hyperalgesia.

title: Tachykinins and calcitonin gene-related peptide (CGRP) as co-transmitters released from peripheral endings of sensory nerves
abstract: 2. Tachykinins and CGRP in primary afferent neurons 2.1. Synthesis and expression of tachykinins 2.2. Svnthesis and exnression of CGRP 2.3. Co-localization of tachykinins and CGRP in primary afferent neurons and regulation of their expression 2.4. Axonal transport to nerve endings 2.5. Correlation between tissue levels of sensory neuropeptides and the efferent function of primary afferent neurons 2.6. Plasticity of tachykinin and CGRP expression in primary afferent neurons 2.6.1. Interactions between sympathetic nerves and primary afferent neurons 2.6.2. Nerve growth factor (NGF) and expression of sensory neuropeptides 2.6.3. Changes in response to nerve injury 2.64. Changes in relation to peripheral target 2.65. Changes during inflammation 3. Release of tachykinins and CGRP from peripheral endings of primary afferent neurons 3.1. General consid!erations 3.2. Multiple modes of sensory neuropeptide release 4. Biological actions of tachykinins and CGRP 4.1. %ological actions of tachykinins 4.1.1. Bioloaical actions of CGRP 5. Receptors and receptor antagonists 5.1. Tachykinin receptors and receptor antagonists 5.2. CGRP receptors and receptor antagonists 6. Neuroeffector roles of tachykinins and CGRP released from primary afferent neurons 6.1. Cardiovascular system 6.1.1. Heart 6.1.2. Blood vessels and blood flow 6.1.3. Plasma protein extravasation 6.2. Respiratory system 6.2.1. Airway motility 6.2.2. Airway secretions 6.2.3. Pathophysiological implications 6.3. Gastrointestinal system 6.3.1. Motility 6.3.2. Gastric acid secretion, blood flow and ulcers 6.3.3. 6.3.3 Ion transport 6.3.4. Gallbladder and biliary tract 6.3.5. Liver 6.3.6. Pancreas 6.4. Genitourinary system 6.4.1. Local motor responses in the renal pelvis and ureter 6.4.2. Local motor responses in the urinary bladder and urethra 6.4.3. Plasma protein extravasation in the urinary tract 6.4.4. Pathophysiological implications 6.45. Genital organs 6.5. Skin 6.5.1. Pathoph:ysiological implications 6.6. Eye 6.7. Skeletal muscle 6.8. Joints and bone 6.9. Dura mater 6.10. Lymphoid organs and immunocytes 6.11. Mast cells 7. Mechanisms of inactivation of released peptides 4 5

title: Intracellular Signaling in Primary Sensory Neurons and Persistent Pain
abstract: During evolution, living organisms develop a specialized apparatus called nociceptors to sense their environment and avoid hazardous situations. Intense stimulation of high threshold C- and Aδ-fibers of nociceptive primary sensory neurons will elicit pain, which is acute and protective under normal conditions. A further evolution of the early pain system results in the development of nociceptor sensitization under injury or disease conditions, leading to enhanced pain states. This sensitization in the peripheral nervous system is also called peripheral sensitization, as compared to its counterpart, central sensitization. Inflammatory mediators such as proinflammatory cytokines (TNF-α, IL-1β), PGE2, bradykinin, and NGF increase the sensitivity and excitability of nociceptors by enhancing the activity of pronociceptive receptors and ion channels (e.g., TRPV1 and Nav1.8). We will review the evidence demonstrating that activation of multiple intracellular signal pathways such as MAPK pathways in primary sensory neurons results in the induction and maintenance of peripheral sensitization and produces persistent pain. Targeting the critical signaling pathways in the periphery will tackle pain at the source.

title: Substance P
abstract: Substance P is considered to be an important neuropeptide in nociceptive processes. Although substance P was described more than 60 years ago, there is still controversy about its exact role in nociception. This article reviews the current knowledge about the function of substance P in pain. Special emphasis is put on how to use this knowledge in the development of new ways to treat pain.

title: NK1, NK2, NK3 and CGRP1 receptors identified in rat oral soft tissues, and in bone and dental hard tissue cells
abstract: The distribution of the tachykinin receptors neurokinin-1 (NK1), neurokinin-2 (NK2) and neurokinin-3 (NK3), and the calcitonin gene-related peptide-1 (CGRP1) receptor were examined in rat teeth and tooth-supporting tissues by immunohistochemical methods and light and confocal microscopy. Western blot analysis was performed to identify the NK1- and the CGRP1-receptor proteins in the dental pulp. The results showed that odontoblasts and ameloblasts, cementoblasts and cementocytes, osteoblasts and osteocytes are all supported with the tachykinin receptors NK1 and NK2, but a distinct, graded cellular labeling pattern was demonstrated. The ameloblasts were also positive for CGRP1 receptor. Blood vessels in oral tissues expressed the tachykinin receptors NK1, NK2 and NK3, and the CGRP1 receptor. Both gingival and Malassez epithelium were abundantly supplied by NK2 receptor. Pulpal and periodontal fibroblasts demonstrated NK1 and NK2 receptors. Western blot analysis identified both the NK1- and the CGRP1-receptor proteins in the dental pulp. These results clearly indicate that the neuropeptides substance P, neurokinin A, neurokinin B and CGRP, released from sensory axons upon stimulation, directly modulate the function of the different types of bone and dental hard tissue cells, and regulate functions of blood vessels, fibroblasts and epithelial cells in oral tissues.

title: Substance P Sensitizes P2X3 in Nociceptive Trigeminal Neurons
abstract: Peripheral inflammation produces pain hypersensitivity by sensitizing nociceptors. Potentiation of P2X3 receptor activity in nociceptors may play an important role in this peripheral sensitization. However, we do not fully understand how P2X3 activity is elevated in inflammation. Thus, we investigated whether P2X3 activity in trigeminal nociceptive neurons is regulated by the neurokinin-1 (NK-1) receptor that is activated by an inflammatory mediator, substance P. Single-cell RT-PCR and immunohistochemistry revealed that NK-1 in nociceptive neurons was mainly co-expressed with P2X3. Ca2+ imaging and whole-cell patch-clamp recordings indicated that both substance P and Sar-substance P, a selective NK-1 agonist, significantly potentiated α,β-meATP-induced currents and [Ca2+]i responses in nociceptive neurons. These potentiating effects were completely blocked by GR82334, a specific NK-1 antagonist. Our results demonstrate that substance P sensitizes P2X3 receptor through the activation of NK-1, thus warranting these receptors as possible targets for pain therapy in the orofacial region.  α,β-methylene adenosine 5′-triphosphate (ATP), α,β-meATP; neurokinin-1, NK-1; single-cell reverse-transcription polymerase chain-reaction, single-cell RT-PCR;  Sar-substance P.

title: Substance P enhances NF-kappaB transactivation and chemokine response in murine macrophages via ERK1/2 and p38 MAPK signaling pathways.
abstract: The neuropeptide substance P (SP), as a major mediator of neuroimmunomodulatory activity, modulates diverse functions of immune cells, including macrophages. In the current study, we focused on the yet uncertain role of SP in enhancing the inducible/inflammatory chemokine response of macrophages and the signaling mechanism involved. We studied the effect on the murine monocyte/macrophage cell line RAW 264.7 as well as isolated primary macrophages. Our data show that SP, at nanomolar concentrations, elicited selective chemokine production from murine macrophages. Among the chemokines examined, macrophage inflammatory protein-2 and monocyte chemoattractant protein-1 are two major chemokines that were synthesized by macrophages in response to SP. Furthermore, SP treatment strongly induced the classic pathway of IkappaB-dependent NF-kappaB activation and enhanced DNA binding as well as transactivation activity of the transcription factor. SP-evoked transcriptional induction of chemokines was specific, since it was blocked by treatment with selective neurokinin-1 receptor antagonists. Moreover, SP stimulation of macrophages activated the ERK1/2 and p38 MAPK but not JNKs. Blockade of these two MAPK pathways with specific inhibitors abolished SP-elicited nuclear translocation of phosphorylated NF-kappaB p65 and NF-kappaB-driven chemokine production, suggesting that the two MAPKs lie in the signaling pathways leading to the chemokine response. Collectively, our data demonstrate that SP enhances selective inflammatory chemokine production by murine macrophages via ERK/p38 MAPK-mediated NF-kappaB activation.

title: Effects of the bisphosphonate ibandronate on hyperalgesia, substance P, and cytokine levels in a rat model of persistent inflammatory pain
abstract: The anti‐inflammatory and analgesic properties of different bisphosphonates have been demonstrated in both animal and human studies. Ibandronate is a third‐generation bisphosphonate effective in managing different types of bone pain. In this study we investigated its effects in a standard pre‐clinical model of inflammatory pain. We evaluated the effects of a single injection of different doses (0.5, 1.0, and 2.0mg/kg i.p.) of ibandronate on inflammatory oedema and cutaneous hyperalgesia produced by the intraplantar injection of complete Freund's adjuvant (CFA) in the rat hind‐paw. In addition, we measured the effects of this drug (1.0mg/kg i.p.) on hind‐paw levels of different pro‐inflammatory mediators (PGE‐2, SP, TNF‐α, and IL‐1β). We also measured the levels of SP protein and of its mRNA in the ipsilateral dorsal root ganglia (DRG). Ibandronate proved able to reduce the inflammatory oedema, the hyperalgesia to mechanical stimulation, and the levels of SP in the inflamed tissue as measured 3 and 7 days following CFA‐injection. This drug significantly reduced the levels of TNF‐α and IL‐1β only on day 7. On the other hand, the levels of PGE‐2 in the inflamed hind‐paw were unaffected by the administration of this bisphosphonate. Finally, ibandronate blocked the overexpression of SP mRNA in DRG induced by CFA‐injection in the hind‐paw.

title: Potent NK1 antagonism by SR-140333 reduces rat colonic secretory response to immunocyte activation.
abstract: The potent neurokinin receptor 1 (NK1) antagonist SR-140333 has previously been shown to reduce castor oil-induced secretion in animal models. The importance of tachykinins in neuroimmune control of secretion and the effect of SR-140333 on key points in this pathway were elucidated in the present study to determine the type of intestinal dysfunction best targeted by this antagonist. Rat colonic secretion and substance P (SP) release were determined in vitro with the use of Ussing chamber and enzyme immunoassay techniques. NK1 receptors played a secretory role as receptor agonists stimulated secretion and SR-140333 antagonized the response to SP response (pK(b) = 9.2). Sensory fiber stimulation released SP and evoked a large secretion that was reduced by 69% in the presence of SR-140333 (10 nM). Likewise, mastocytes also released SP. The subsequent secretory response was reduced by 43% in the presence of SR-140333 (50 nM). SP was also released from granulocytes; however, this did not cause secretion. Functional NK3 receptors were present in the colon as senktide stimulated secretion, an effect that was increased during stress. We conclude that NK3 receptors may play a role in stress-related disorders, whereas NK1 receptors are more important in mast cell/afferent-mediated secretion.

title: The effect of different vasoconstrictors and local anesthetic solutions on substance P expression in human dental pulp.
abstract: The purpose of this study was to quantify the effect of the infiltration injection of different vasoconstrictor and anesthetic solutions on substance P (SP) expression in healthy human dental pulp. Thirty pulp samples were obtained from healthy upper premolars in which extraction was indicated for orthodontic reasons and were randomly assigned into three groups of 10 samples each: 2% lidocaine with 1:80,000 epinephrine (Lido group), 3% Prilocaine with 1:200000 felypressin (Prilo group); and 4% Prilocaine without vasoconstrictor (Prilo-no-VC group). All teeth were extracted 10 minutes after anesthetic application. Pulp samples were processed and SP was measured by radioimmunoassay. SP expression for the Lido, Prilo, and Prilo-no-VC groups were 616.49, 663.76, and 760.79 pmol/mg pulp tissue, respectively. Analysis of variance showed statistically significant differences between groups (p = 0.001). Tukey Honestly Significant Difference (HSD) post hoc tests showed significant statistical differences between the Prilo-no-VC group and the Lido group (p < 0.01) and between the Prilo-no-VC group and the Prilo group (p < 0.05). It can be concluded that infiltration injection of local anesthetics with vasoconstrictor attenuate SP expression in human dental pulp.

title: Target finding of pain nerve fibers: Neural growth mechanisms in the tooth pulp
abstract: The tooth pulp has a dense sensory innervation which, upon stimulation, conveys sensory signals perceived as pain. This innervation, which originates from the trigeminal ganglion, is established through a series of regulated steps during development, and represents an interesting example of tissue targeting by pain-specific nerves. We have investigated various potentially neurotrophic and neurorepulsive influences during this process. The dental papilla/pulp appears to secrete neurite growth inhibitory molecular factors at early stages, which prevent nerve fibers from entering the tissue at what appears to be inappropriate timepoints. Later, a shift from repulsive to attractive factors apparently takes place, and nerve fibers then enter the tooth. When nerve fibers have invaded the dental mesenchyme, a complicated interplay of secreted and membrane-bound factors probably directs the nerve terminals to appropriate sites. Laminin-8 (alpha4beta1gamma1, Lm-411), which is produced by pulpal cells, emerges as an important candidate molecule in this context. Insights into the interactions between the dental pulp nerve fibers and their environment may become important in the search for novel ways to ameliorate pain in the tooth, as well as at other sites.

title: The effect of tooth bleaching on substance P expression in human dental pulp.
abstract: The purpose of this study was to quantify the effect of tooth bleaching on substance P (SP) expression in healthy human dental pulp. Forty pulp samples were obtained from healthy premolars in which extraction was indicated for orthodontic reasons. Thirty of these premolars were assigned into three different tooth-bleaching protocols: group 1 (n = 10): Opalescence Xtra Boost (Ultradent Products, South Jordan, UT) (38% H(2)O(2)) for 15 minutes; group 2 (n = 10): Lase Peroxide (DMC, Brazil) (35% H(2)O(2)) activated with infrared laser diode (Biolux; BioArt, Brazil) for 3 minutes, and group 3 (n = 10): Zoom! Whitening System (Discuss Dental, Culver City, CA) (25% H(2)O(2)) light activated for 20 minutes. The remaining 10 healthy premolars serve as a control group. Teeth were anesthetized immediately after bleaching and were extracted 10 minutes later. All pulp samples were processed and SP was measured by radioimmunoassay. Greater SP expression was found in the Zoom! Whitening System, followed by the Lase Peroxide group, Opalescence Xtra Boost, and the lower SP values were for the control group. Analysis of variance showed statistically significant differences between groups (p = 0.0001). Tukey HSD post hoc tests showed significant differences in the light (p < 0.01) and laser (p < 0.05) activated bleaching systems when compared with control values. It can be concluded that light- and laser-activated tooth-bleaching systems increase SP expression in human dental pulp significantly higher than normal values.

title: Quantitative analysis of substance P, neurokinin A and calcitonin gene-related peptide in gingival crevicular fluid associated with painful human teeth.
abstract: The aim of this study was to investigate the presence of substance P (SP), neurokinin A (NKA) and calcitonin gene-related peptide (CGRP) in the gingival crevicular fluid of teeth diagnosed with pain of pulpal origin compared with clinically healthy teeth, and to detect any changes in the levels of these neuropeptides in gingival crevicular fluid after removal of the pulp from the painful teeth. Gingival crevicular fluid was collected at baseline from one interproximal site at a painful and a non-painful contralateral tooth from 54 adult patients. Sampling was repeated after 1 wk in a subset of 21 subjects. Samples were analysed for SP, NKA, and CGRP using radioimmunoassay. The mean levels of SP and NKA were significantly higher in gingival crevicular fluid from painful teeth compared with non-painful teeth. The level of SP in the GCF of painful teeth fell significantly 1 wk after pulpectomy. In contralateral teeth, there were no significant differences in the levels of SP and NKA after 1 wk. It is concluded that SP and NKA are present in significantly greater amounts in the GCF of painful teeth compared with healthy teeth.

title: Substance P expression in human tooth pulp in relation to caries and pain experience.
abstract: The neuropeptide substance P (SP) is found within a subpopulation of nociceptive afferent nerve fibres and has been shown to be upregulated in a variety of sites following peripheral inflammation. The aim of this study was to investigate the expression of SP within human teeth, both in health and disease, and to seek a correlation between reported pain history and SP expression within pulpal nerves. Coronal pulps were removed from 62 permanent mandibular molars with a known pain history. Teeth were categorised as intact, moderately or grossly carious. Using indirect immunofluorescence, sections were double-labelled for the general neuronal marker protein gene product 9.5 (PGP 9.5) and for SP. Image analysis was then used to quantify the percentage area of PGP 9.5-labelled tissue which was also labelled for SP. Throughout the pulp, the expression of SP was found to be significantly increased with the progression of caries. Furthermore, SP expression was significantly greater in grossly carious painful specimens than in grossly carious asymptomatic specimens. These data would suggest that the expression of SP within pulpal nerves undergoes dynamic changes following caries, which may have an important clinical significance in terms of inflammation and pain experience.

title: Tissue levels of immunoreactive substance P are increased in patients with irreversible pulpitis.
abstract: Odontogenic pain often involves inflammation of dental pulp tissue. Dental pulp is highly innervated with a subpopulation of sensory neurons containing neuropeptides. Substance P, released from afferent fibers (e.g. nociceptors) is associated with the development of neurogenic inflammation. In this study, we tested the hypothesis that irreversible pulpitis is associated with increased activity of peptidergic neurons, as measured by increased pulpal levels of immunoreactive substance P (iSP). We determined in vivo pulpal levels of immunoreactive substance P in human teeth with a diagnosis of normal pulp or irreversible pulpitis using CMA/20 microdialysis probes inserted into vital pulps of 24 teeth from 21 patients. Probes were perfused with a modified Locke-Ringer's buffer and immunoreactive substance P levels in the dialysate were measured using a radioimmunoassay. Mean extracellular levels of immunoreactive substance P were significantly higher (>8-fold) in teeth diagnosed with irreversible pulpitis than immunoreactive substance P levels in dental pulp diagnosed as normal (147.7 +/- 34.0 pM versus 18.2 +/- 6.2 pM). These observations suggest that biochemical measures of inflammatory mediators exhibit significant change during irreversible pulpitis and may contribute to clinical signs and symptoms.

title: Immunocytochemical localization of the neurokinin 1 receptor in rat dental pulp.
abstract: The dentin-pulp complex is a peripheral end-organ supplied by dense sensory nerve fibers. Substance P, a representative neuropeptide widely distributed in the dental pulp, has been reported to play roles in pain transmission and the amplification of inflammation. We analyzed here the expression of the neurokinin 1 (NK1) receptor, preferentially activated by substance P, using immunocytochemistry in rat dental pulp at both the light and electron microscopic levels. Conspicuous NK1 receptor immunoreactivity was found in the odontoblasts; immunolabelings were present at their plasma membrane and endosomal structures, especially in their cytoplasmic processes. Immunoreactions for NK1 receptor were also detectable in a part of the nerve terminals associated with the cytoplasmic processes of the odontoblasts. Furthermore, the endothelial cells of capillaries and post-capillary venules and the fibroblasts were labeled with the NK1 receptor in the subodontoblast layer. These findings suggest that pulpal cells and nerve fibers are targets for substance P that mediate multiple functions, including a vasoactive function and the regulation of vascular permeability as well as the modulation of pain transmission.

title: Substance P receptor expression in healthy and inflamed human pulp tissue.
abstract: To use radioreceptor analysis for comparing substance P (SP) receptor expression in human pulp tissue samples collected from teeth having a clinical diagnosis of acute irreversible pulpitis, healthy pulps and teeth with induced inflammation.



Five pulp samples were obtained from teeth having a clinical diagnosis of acute irreversible pulpitis. Another 10 pulp samples were obtained from healthy premolars where extraction was indicated for orthodontic purposes. In five of these premolars inflammation was induced prior to pulp collection. All of the samples were processed and labelled with 125I-SP. Binding sites were identified by 125I-SP and standard SP competition assays. Kruskal-Wallis and Mann-Whitney (post-hoc) tests were used to establish statistically significant differences between the groups.



Substance P receptor expression was found in all human pulp tissue samples. Most receptors were found in the group of pulps from teeth having a clinical diagnosis of acute irreversible pulpitis, followed by the group of pulps having induced inflammation. The least number of receptors was expressed in the group of healthy pulps. Statistical analysis revealed significant differences between the group of healthy pulp and both inflamed pulp groups (P < 0.01).



Substance P receptor expression in human pulp tissue is significantly increased during inflammatory phenomena such as acute irreversible pulpitis.

title: Production of Tumor Necrosis Factor-alpha, Interleukin 1-beta, Interleukin 2, and Interleukin 6 by rat leukocyte subpopulations after exposure to Substance P
abstract: The interaction between components of the nervous system and multiple target cells in the cutaneous immune system has been receiving increasing attention. Recently, the involvement of neuropeptides has been demonstrated to play an important role in the inflammatory cascade. Neuropeptides such as Substance P are released by cutaneous neurons and modulate the function of immunocompetent and inflammatory cells as well as epithelial and endothelial cells. Substance P has been shown to function as a mediator for cell proliferation, cytokine production, and as an upregulator of various cell surface receptors. In this study, we show the effect of Substance P on the production of Tumor Necrosis Factor-alpha, Interleukin 1-beta, Interleukin 2, and Interleukin 6 by T-lymphocytes, macrophages and neutrophils. These data demonstrate that pathophysiological levels of Substance P induce production of cytokines in all three cell populations tested. Interestingly, T-cells demonstrated the highest percentage of cells expressing all four cytokines. In contrast, macrophages and neutrophils produced the highest absolute levels of cytokines. The elucidation of mediating mechanisms of Substance P activation of leukocytes is crucial to the understanding of the cutaneous inflammatory cascade and involvement of the peripheral nervous system on the immune system. These findings suggest that Substance P participates in the complex network of mediators that regulate cutaneous inflammation and potentially the rate of wound healing.

title: Role of substance P and calcitonin gene-related peptide in the regulation of interleukin-8 and monocyte chemotactic protein-1 expression in human dental pulp.
abstract: To determine whether leucocyte infiltration during neurogenic inflammation in the pulp is regulated by neuropeptides via inducing the release of proinflammatory chemokines interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) from human dental pulp.



Cultured primary pulp cells and pulp tissue explants were stimulated with substance P (SP) and/or calcitonin gene-related peptide (CGRP). IL-8 or MCP-1, secreted from cultured cells or produced in pulp explants, was analysed by enzyme-linked immunosorbent assay.



Substance P induced IL-8 secretion from cultured pulp cells (approximately threefold increase over control, P < 0.05) and from pulp tissue explants (two- to three fold). SP only minimally to moderately induced MCP-1 (approximately two fold) in cultured pulp cells. While MCP-1 induction in cultured pulp cells was detected after 24 h of SP stimulation, no induction was observed in pulp tissue. CGRP did not induce IL-8, but moderately increased MCP-1 production (approximately three fold) in cultured pulp cells. There was no synergistic induction of MCP-1 by SP plus CGRP stimulation of pulp cells.



Substance P is a stronger inducer of IL-8 production in dental pulp than CGRP. IL-8 is more strongly induced than MCP-1 by SP, suggesting a more important role for IL-8 than MCP-1 in leucocyte infiltration during neurogenic inflammation in dental pulp.

title: Involvement of substance P, mast cells, TNF-alpha and ICAM-1 in the infiltration of inflammatory cells in human periapical granulomas.
abstract: Substance P (SP), a potent proinflammatory peptide present in sensory neurons, is believed to be a major mediator of neurogenic inflammation. The aim of this study was to examine the localization and involvement of SP, mast cells and tumor necrosis factor-alpha (TNF-alpha)-positive cells in human periapical granulomas.



Sections from seven periapical granulomas were stained using a variety of immunohistochemical methods.



SP-immunoreactive nerve fibers, mast cells and TNF-alpha-positive cells were found localized in the vicinity of blood vessels in all the samples of periapical granulomas. The vascular endothelial cells stained positively for E-selectin and intercellular adhesion molecule-1. SP, TNF-alpha-positive cells and E-selectin could not be detected in clinically healthy periodontal ligament, and served as a negative control.



Our findings suggest that SP, mast cells, TNF-alpha-positive cells and E-selectin may modulate the pathogenesis of apical periodontitis and may be responsible for stimulating the formation of granuloma with the resorption of periapical bone.

title: The effect of cavity preparation on substance P expression in human dental pulp.
abstract: Substance P (SP) plays an important role during neurogenic inflammation of dental pulp. The purpose of this study was to use a radioimmunoassay for determining the effect of cavity preparation on SP expression in healthy human dental pulp. Ten pulp samples were obtained from healthy premolars where extraction was indicated for orthodontic reasons. Deep cavity preparation (<1 mm remaining dentine thickness) was performed before extraction in five of these bicuspids. All samples were processed and I-SP labeled; SP was quantified by competition assay. The results revealed SP expression in all human pulp samples. Mann-Whitney's U test revealed statistically significant higher expression in pulp from teeth where cavity preparation had been performed compared to control values (p < 0.05). These findings suggest that SP is released during common dental procedures (such as cavity preparation) and its expression may have an important clinical significance in terms of experiencing inflammation and pain.

title: The Damon System and release of substance P in gingival crevicular fluid during orthodontic tooth movement in adults.
abstract: Metabolism by peptidases plays an important role in modulating the levels of biologically active neuropeptides. One of these neuropeptides, substance P (SP), a component of gingival crevicular fluid (GCF), may exponentiate the inflammatory process during orthodontic tooth movement. The aim of this study was to investigate the GCF levels of SP in patients using different bracket systems. Subjects were 10 patients (four males, six females; mean age, 25.1 ± 4.4 years) undergoing orthodontic movement (leveling) in the maxilla. Conventional brackets were placed on the left side, while the teeth on the right received self-ligating brackets. The teeth on the mandibular left side without any orthodontic attachments served as controls. GCF was sampled at 0, 1, 24, and 168 hours after initiation of treatment. Prevention of plaque-induced inflammation allowed assessment of the dynamics of mechanically stimulated SP levels in the GCF, which was determined using commercially enzyme-linked immunoabsorbent assay (ELISA) kits. GCF levels of SP for the Damon System sites were significantly lower than for the teeth with conventional brackets at 24 hours. This result indicates that the Damon System inhibited an increase in the amount of SP in the GCF. Thus, the Damon System is useful to reduce the inflammation and pain resulting from orthodontic forces.

title: The effect of three different rotary instrumentation systems on substance P and calcitonin gene-related peptide expression in human periodontal ligament.
abstract: The purpose of this study was to quantify the effect of three different rotary root canal preparation systems on substance P and calcitonin gene-related peptide expression in healthy human periodontal ligament.



Fifty periodontal ligament samples were obtained from healthy premolars in which extraction was indicated for orthodontic reasons. Before extraction, 40 of these premolars were equally divided into four groups, and root canals were prepared using four different systems: the ProTaper Universal rotary system, the RaCe rotary system, the Mtwo rotary system, and the hand instrumentation technique. The remaining 10 healthy premolars that were extracted without treatment served as a negative control group. All periodontal ligament samples were processed, and SP and CGRP were measured by radioimmunoassay.



Greater SP and CGRP expression were found in the ProTaper Universal group followed by the hand instrumentation group, the RaCe, and the Mtwo groups. The lower SP and CGRP values were for the negative control group. The Kruskal-Wallis test showed statistically significant differences between groups (p < 0.0001). Post hoc Least Significant Difference (LSD) tests showed statistically significant differences in SP and CGRP expression between the negative control group and all the other groups except the Mtwo group. Hand instrumentation also showed statistically significant differences with all the other groups, except the ProTaper Universal group. Differences between the three rotary systems were also statistically significant.



SP and CGRP expression in periodontal ligament increases when teeth are prepared with ProTaper Universal and RaCe rotary instrumentation systems as well as with hand instrumentation. Mtwo maintains SP and CGRP levels.

title: The effect of dentine-bonding agents on substance P release in human dental pulp.
abstract: To quantify the effect of dentine-bonding agents on Substance P (SP) release in healthy human dental pulp tissue.



Forty pulp samples were obtained from healthy pre-molars where extraction was indicated for orthodontic reasons. In thirty of these pre-molars, a standardized Class V cavity preparation was performed, and teeth were divided equally into three groups: (i) Unetched-cavity control group: Class V cavities only; (ii) Experimental Group I: 'One-step' self-etch bonding agent was placed in the cavity; and (iii) Experimental Group II: 'Two-step' total-etch bonding agent was placed in the cavity. The remaining ten healthy pre-molars where extracted without treatment and served as an intact-teeth control group. SP was measured by radioimmunoassay.



Greater SP release was found in the 'one-step' bonding agent group, followed by the 'two-step' bonding agent group and the unetched-cavity control group. The lower SP values were for the intact-teeth control group. anova showed statistically significant differences between groups (P = 0.0001). Tukey HSD post hoc tests showed statistically significant differences in SP release between the intact-teeth control group and the three other groups (P < 0.01) and between the unetched-cavity control group and the 'one-step' bonding agent group (P < 0.05). No significant difference was found between the 'two-step' bonding agent and the unetched-cavity control group.



Dentine-bonding agents placed over Class V cavity preparations increased SP release. One-step dentine-bonding agents increased SP release most.

title: Substance P stimulates release of RANKL via COX-2 expression in human dental pulp cells
abstract: Our previous study found that substance P (SP), a sensory neuropeptide, was expressed in the dental pulp of rats during experimental tooth movement. We examined the effects of SP on the production of prostaglandin (PG) E2 and the receptor activator of nuclear factor- B ligand (RANKL) by human dental pulp fi broblast-like (HDPF) cells.
  SP was added to cultured HDPF cells at concentrations ranging from of 10−4 to 10−12 mol/L. PGE2 and soluble RANKL (sRANKL) levels were determined using enzyme-linked immunosorbent assay kits. Gene expression was confi rmed by RT-PCR analysis. Pit formation assays using dentin slices were carried out to examine the effect of SP on osteoclastogenesis.
 Results: The levels of PGE2 and sRANKL increased in the presence of SP, though the increases were greater in the experimental groups in both a time- and concentration-dependent manner, and the increase of RANKL was partially mediated by PGE2 . The gene expression of cyclooxygenase (COX)-2 and RANKL was up-regulated, and conditioned medium samples obtained from HDPF cells treated with SP induced bone resorption.
 Conclusions: SP stimulated the production of PGE2 and RANKL, and promoted bone resorption. Therefore, SP may be involved in pulpal inflammation and root resorption during orthodontic tooth movement.

title: Relationship between substance P and interleukin-1beta in gingival crevicular fluid during orthodontic tooth movement in adults.
abstract: Metabolism by peptidases plays an important role in modulating the levels of biologically-active neuropeptides, while that of substance P (SP), a component of gingival crevicular fluid (GCF), may potentiate the inflammatory process in orthodontic tooth movement. The aim of this study was two-fold: (1) to investigate GCF levels of SP and interleukin-1beta (IL-1beta) during human orthodontic tooth movement, and (2) to determine the correlation coefficients between SP and IL-1beta levels in the GCF. The subjects were 3 males, with a mean age of 21.3 +/- 2.8 years old, and 6 females, with a mean age of 23.1 +/- 2.4 years, undergoing orthodontic movement of a single tooth, with the contralateral tooth used as the control. GCF was sampled at the control and treatment (compression) sites before and 1, 4, 8, 24, 72, 120, and 168 hours after initiation of orthodontic treatment. Prevention of plaque-induced inflammation allowed assessment of the dynamics of mechanically stimulated SP and IL-1beta levels in the GCF, which were determined using enzyme-linked immunosorbent assay (ELISA) kits. GCF levels of SP and IL-1beta for the treated teeth were significantly higher (P < 0.001) than for the corresponding control teeth from 8 to 72 hours, and peaked at 24 hours. These results show that the amounts of SP and IL-1beta in GCF increase with orthodontic tooth movement, and indicate that such increases may be involved in inflammation in response to mechanical stress.

title: Perception of pain during orthodontic treatment with fixed appliances.
abstract: The aims of this study were to investigate the initial time at which pain occurs after insertion of two initial wires of different sizes, the duration of the pain, the areas affected within the mouth, the level of self-medication, the effect of this pain on daily life, and whether gender is important in the perception of pain. The study group consisted of 109 patients (52 boys, 57 girls) with a mean chronological age of 13.6 years for boys and 14.7 years for girls. Insertion of either a 0.014 or 0.016 inch wire was by random selection. Following insertion of the archwires, a questionnaire comprising a total of 49 questions was given to the patients. They described the time of initial pain in the first question, answered the next 24 questions as 'yes' or 'no', and used a visual analogue scale for the final 24 questions. No significant differences were found in terms of gender, in the perception period of initial pain as regards the areas affected within the mouth or the effect of pain on daily living when the 0.014 and 0.016 inch wire groups were compared at 6 hours, 1, 2, 3, 4, 5, 6 and 7 days. At 24 hours, which was found to be statistically significant, more pain relief was used in the 0.014 inch archwire group. The results show that in both groups, initial pain was perceived at 2 hours, peaked at 24 hours and had decreased by day 3.

title: Orthodontic anchoring techniques and its influence on pain, discomfort, and jaw function--a randomized controlled trial.
abstract: The aim of this trial was to evaluate and compare perceived pain, discomfort, and jaw function impairment between orthodontic treatments combined with skeletal anchorage and treatment using conventional anchorage with headgear or transpalatal bar. A total of 120 adolescent patients in order to start orthodontic treatment were consecutively recruited and randomized into three groups with different anchorage. Group A underwent installation of a skeletal anchorage (Onplant or Orthosystem implant), group B received headgear, and group C a transpalatal bar. Questionnaires were used to assess pain intensity, discomfort, analgesic consumption, and jaw function impairment from baseline to the end of treatment. Pain scores overall peaked on day 2 and were almost back to baseline on day 7. The site with the highest pain scores during treatment was incisors in contact but with no differences between groups. Pain intensity from molars was significantly less in the skeletal anchorage group A compared to the transpalatal bar group C the first 4 days in treatment and with no sign differences compared to headgear. The results confirm that there were very few significant differences between patients' perceptions of skeletal and conventional anchorage systems during orthodontic treatment. Consequently, these new appliances were well accepted by the patients in a long time perspective and can thus be recommended.

title: Effects of local anesthesia on substance P and CGRP content of the human dental pulp.
abstract: The aim of the study was to determine immunoreactive Substance P (iSP) and immunoreactive calcitonin gene-related peptide (iCGRP) content in the human dental pulp and whether local anesthesia has an effect on the neuropeptide content. Dental pulps were obtained from patients, who underwent surgical extraction of all 4 impacted wisdom teeth under general isoflurane gas anesthesia. There was a very high interindividual variation in tissue content, with small variation in levels found in teeth from the same patient. Pulps obtained from lower teeth without local anesthesia contained an average of 131 +/- 62 fmol/mg protein of iCGRP and 15 +/- 9 fmol/mg iSP (n = 10). With additional mandibular block anesthesia the values were 194 +/- 71 fmol/mg iCGRP (statistically significant, p = 0.0356, Mann-Whitney-rank-sum-test) and 12 +/- 3.6 fmol/mg iSP. The results suggest that local anesthesia attenuates neuropeptide release in the human dental pulp during surgical extraction.

title: Neuropeptide and kinin antagonists.
abstract: Neuropeptides and kinins are important messengers in the nervous system and--on the basis of their anatomical localisation and the effects produced when the substances themselves are administered, to animals or to human subjects-a significant number of them have been suggested to have a role in pain and inflammation. Experiments in gene deletion (knock-out or null mutant) mice and parallel experiments with pharmacological receptor antagonists in a variety of species have strengthened the evidence that a number of peptides, notably substance P and calcitonin gene-related peptide (CGRP), and the kinins have a pathophysiological role in nociception. Clinical studies with non-peptide pharmacological antagonists are now in progress to determine if blocking the action of these peptides might have utility in the treatment of pain.

title: Potential therapeutic targets for neurokinin-1 receptor antagonists.
abstract: The peptide substance P and its tachykinin receptor, neurokinin-1 (NK1), have been the focus of considerable research for their role in a variety of both central and peripheral diseases. Recent preclinical data, as well as relevant clinical findings, support the potential therapeutic value of NK1 receptor antagonists in centrally mediated disease states, including anxiety and depression. In addition, a separate body of literature supports the use of NK1 receptor antagonists as inhibitors of centrally mediated emetic and cough responses. The role of NK1 receptor antagonists as analgesic agents with potential to treat migraine headache has also been investigated. NK1 receptors are also found in a number of peripheral regions, including the bladder, gastrointestinal tract and bone marrow. Preclinical models have been employed to address the potential therapeutic uses for NK1 receptor antagonists in diseases associated with inflammatory responses, including asthma, irritable bowel syndrome and cystitis of the bladder. Finally, other more recent publications suggest a role for NK1 receptor antagonists as tumour suppressants and haematopoietic agents. These applications for NK1 receptor antagonists are discussed in this review.

title: Emerging Peripheral Receptor Targets for Deep-tissue Craniofacial Pain Therapies
abstract: While effective therapies are available for some types of craniofacial pain, treatments for deep-tissue craniofacial pain such as temporomandibular disorders are less efficacious. Several ion channels and receptors which are prominent in craniofacial nociceptive mechanisms have been identified on trigeminal primary afferent neurons. Many of these receptors and channels exhibit unusual distributions compared with extracranial regions. For example, expression of the ATP receptor P2X3 is strongly implicated in nociception and is more abundant on trigeminal primary afferent neurons than analogous extracranial neurons, making them potentially productive targets specifically for craniofacial pain therapies. The initial part of this review therefore focuses on P2X3 as a potential therapeutic target to treat deep-tissue craniofacial pain. In the trigeminal ganglion, P2X3 receptors are often co-expressed with the nociceptive neuropeptides CGRP and SP. Therefore, we discuss the role of CGRP and SP in deep-tissue craniofacial pain and suggest that neuropeptide antagonists, which have shown promise for the treatment of migraine, may have wider therapeutic potential, including the treatment of deep-tissue craniofacial pain. P2X3, TRPV1, and ASIC3 are often co-expressed in trigeminal neurons, implying the formation of functional complexes that allow craniofacial nociceptive neurons to respond synergistically to altered ATP and pH in pain. Future therapeutics for craniofacial pain thus might be more efficacious if targeted at combinations of P2X3, CGRP, TRPV1, and ASIC3.

title: Effects of nimesulide on pain and on synovial fluid concentrations of substance P, interleukin‐6 and interleukin‐8 in patients with knee osteoarthritis: comparison with celecoxib
abstract: This study was designed to investigate the analgesic effects of nimesulide and celecoxib in patients with knee osteoarthritis (OA). In patients with joint effusion, the effects of these non‐steroidal anti‐inflammatory drugs (NSAIDs) on synovial fluid concentrations of substance P (SP), interleukin (IL)‐6 and IL‐8 also were evaluated.