Sensitization Of Primary Afferents Research Articles

Erratum: Ishida et al., "In Vivo Calcium Imaging Visualizes Incision-Induced Primary Afferent Sensitization and Its Amelioration by Capsaicin Pretreatment".

Erratum: Ishida et al., "In Vivo Calcium Imaging Visualizes Incision-Induced Primary Afferent Sensitization and Its Amelioration by Capsaicin Pretreatment".

Expression of Glial-Cell-Line-Derived Neurotrophic Factor Family Ligands in Human Intervertebral Discs.

Glial-cell-line-derived neurotrophic factor (GDNF) family ligands (GFLs) contribute to the sensitization of primary afferents and are involved in the pathogenesis of inflammatory pain. The purpose of this preliminary study was to examine the expression of other GFLs (neurturin (NRTN), artemin (ARTN), persephin (PSPN)) and receptors in human IVD cells and tissues exhibiting early and advanced stages of degeneration. Human IVD cells were cultured as a monolayer after isolation from the nucleus pulposus (NP) and anulus fibrosus (AF) tissues. The mRNA expression of NRTN, ARTN, PSPN, and their receptors (GFRA2-GFRA4) was quantified using real-time PCR. Protein expression was evaluated using immunohistochemistry and Western blotting. The expression of NRTN, ARTN, PSPN, and their co-receptors (GFRA2-GFRA4) was identified in human IVD cells at both mRNA and protein levels. A trend was noted wherein the mRNA expression of ARTN, PSPN, and GFRA2 was upregulated by IL-1β treatment in a dose-dependent manner. The percentages of immunopositive cells in the advanced degenerate stage of ARTN, PSPN, and GFRA2 were significantly higher than those in the early degenerate stage. Their expression was enhanced in advanced tissue degeneration, which suggests that GFLs (ARTN and PSPN) may be involved in the pathogenesis of discogenic pain.

Chronic Pelvic Pain Develops in Sensory Neuron Conditional Asic3 Null Mice Subjected to Chemical Injury

Sensitization of primary afferents is an important underlying mechanism for visceral hypersensitivity and pain. We investigated the contribution of acid-sensing ion channels (ASICs) in sensory neurons to the development of pain in a model of chemical-induced cystitis. ASICs are assembled as heterotrimers in bladder sensory neurons; however, the deletion of the ASIC3 subunit results in a loss-of-function phenotype with sensory neurons unable to discharge action potentials in response to acidification. In this study, conditional sensory neuron Asic3 knockout (KO) mice ( Asic3 fl/fl ;Avil-Cre +/- ) and control littermates ( Asic3 fl/fl ;Avil-Cre -/- ) received cyclophosphamide (CYP) (IP 80 mg/kg), or saline, every other day for five days. Experimental observations were made after one day (acute) or 14 days (chronic) after the last dose of CYP. In the acute setting, both control and conditional Asic3 KO mice treated with CYP exhibited an irritated bladder phenotype with a high number of voiding events of small volume. However, voiding activity normalized in both groups within two weeks of receiving CYP. In contrast, conditional Asic3 KO mice treated with CYP developed pelvic allodynia that persisted for at least two weeks, whereas control mice had no pain phenotype. In the chronic setting, no apparent edema or inflammatory cells were observed in bladders of control or conditional Asic3 KO mice, indicating that the pelvic allodynia seen in conditional Asic3 KO mice treated with CYP is likely driven by abnormal functioning of the nervous system and not by inflammation. To assess whether, in the chronic setting, the referred pelvic allodynia seen in conditional Asic3 KO mice treated with CYP is caused by sensitized primary afferents, we examined the firing evoked by sustained suprathreshold electrical stimulation of acutely isolated bladder sensory neurons. Sensory neurons were classified based of the sensitivity of the action potential to tetrodotoxin (TTX), as TTX-resistant (TTX-R) or TTX-sensitive (TTX-S). Strikingly, ~ 40% (11/17) of the bladder sensory neurons with TTX-R action potentials from conditional Asic3 KO mice treated with CYP exhibited aberrant firing (i.e., sensitization) in response to suprathreshold stimulation, compared to 3% (1/33) in control mice. These findings indicate that the pelvic allodynia seen in conditional Asic3 KO mice is driven in part by sensitized bladder afferents. Taken together, our studies support the notion that ASICs operate at the nerve terminals to modulate nociceptor excitability and sensitization. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

In Vivo Calcium Imaging Visualizes Incision-Induced Primary Afferent Sensitization and Its Amelioration by Capsaicin Pretreatment.

Previous studies have shown that infiltration of capsaicin into the surgical site can prevent incision-induced spontaneous pain like behaviors and heat hyperalgesia. In the present study, we aimed to monitor primary sensory neuron Ca2+ activity in the intact dorsal root ganglia (DRG) using Pirt-GCaMP3 male and female mice pretreated with capsaicin or vehicle before the plantar incision. Intraplantar injection of capsaicin (0.05%) significantly attenuated spontaneous pain, mechanical, and heat hypersensitivity after plantar incision. The Ca2+ response in in vivo DRG and in in situ spinal cord was significantly enhanced in the ipsilateral side compared with contralateral side or naive control. Primary sensory nerve fiber length was significantly decreased in the incision skin area in capsaicin-pretreated animals detected by immunohistochemistry and placental alkaline phosphatase (PLAP) staining. Thus, capsaicin pretreatment attenuates incisional pain by suppressing Ca2+ response because of degeneration of primary sensory nerve fibers in the skin.SIGNIFICANCE STATEMENT Postoperative surgery pain is a major health and economic problem worldwide with ∼235 million major surgical procedures annually. Approximately 50% of these patients report uncontrolled or poorly controlled postoperative pain. However, mechanistic studies of postoperative surgery pain in primary sensory neurons have been limited to in vitro models or small numbers of neurons. Using an innovative, distinctive, and interdisciplinary in vivo populational dorsal root ganglia (DRG) imaging (>1800 neurons/DRG) approach, we revealed increased DRG neuronal Ca2+ activity from postoperative pain mouse model. This indicates widespread DRG primary sensory neuron plasticity. Increased neuronal Ca2+ activity occurs among various sizes of neurons but mostly in small-diameter and medium-diameter nociceptors. Capsaicin pretreatment as a therapeutic option significantly attenuates Ca2+ activity and postoperative pain.

Estrogen augmented visceral pain and colonic neuron modulation in a double-hit model of prenatal and adult stress.

BACKGROUNDChronic stress during pregnancy may increase visceral hyperalgesia of offspring in a sex-dependent way. Combining adult stress in offspring will increase this sensitivity. Based on the evidence implicating estrogen in exacerbating visceral hypersensitivity in female rodents in preclinical models, we predicted that chronic prenatal stress (CPS) + chronic adult stress (CAS) will maximize visceral hyperalgesia; and that estrogen plays an important role in colonic hyperalgesia.AIMThe aim was to illuminate the role of estrogen in colonic hyperalgesia and its underlying mechanisms.METHODSWe established a CPS plus CAS rodent model in which the balloon was used to distend the colorectum. The single-fiber recording in vivo and patch clamp experiments in vitro were used to monitor the colonic neuron’s activity. The reverse transcription-polymerase chain reaction, western blot, and immunofluorescence were used to study the effects of CPS and CAS on colon primary afferent sensitivity. We used ovariectomy and letrozole to reduce estrogen levels of female rats respectively in order to assess the role of estrogen in female-specific enhanced primary afferent sensitization.RESULTSSpontaneous activity and single fiber activity were significantly greater in females than in males. The enhanced sensitization in female rats mainly came from low-threshold neurons. CPS significantly increased single-unit afferent fiber activity in L6-S2 dorsal roots in response. Activity was further enhanced by CAS. In addition, the excitability of colon-projecting dorsal root ganglion (DRG) neurons increased in CPS + CAS rats and was associated with a decrease in transient A-type K+ currents. Compared with ovariectomy, treatment with the aromatase inhibitor letrozole significantly reduced estrogen levels in female rats, confirming the gender difference. Moreover, mice treated with letrozole had decreased colonic DRG neuron excitability. The intrathecal infusion of estrogen increased brain-derived neurotrophic factor (BDNF) protein levels and contributed to the response to visceral pain. Western blotting showed that nerve growth factor protein was upregulated in CPS + CAS mice.CONCLUSIONThis study adds to the evidence that estrogen-dependent sensitization of primary afferent colon neurons is involved in the development of chronic stress-induced visceral hypersensitivity in female rats.

Early life neuroimmune interactions modulate neonatal nociceptive priming

Young children perceive nociceptive stimuli and experience pain differently than adults, but the underlying mechanisms that cause these differential effects are unknown. The result of this uncertainty can lead to incomplete pain management for these patients. Further, a vulnerable period occurs during early life whereby aversive stimuli can be biologically encoded possibly through altered neuroimmune and endocrine interactions that influence sensory responsiveness later in life. We evaluated the role of neuroimmune and endocrine interactions in early life, and explored the effects on “neonatal priming.” We assessed pain-like behaviors, sensory neuron response properties with ex vivo electrophysiology, and dorsal root ganglion (DRG) and macrophage specific gene expression under normal and/or genetically altered conditions in both uninjured mice and in animals that experienced surgical hindpaw incisions. We first found that sensory neuron deletion of the receptor for the endocrine signaling molecule growth hormone (GH), induced pain-like behaviors in neonates. Acute peripheral sensitization after incision injury was prevented by local GH treatment, and macrophage ablation of GH receptor also prevented incision-related effects. Macrophages appear to sequester injury-site GH, which releases a tonic inhibition on the sensory neurons and drives primary afferent sensitization. Manipulations in GH signaling or ablating neonatal macrophages was found to be critical for the development of neonatal priming. New ATAC-seq data indicates differences in chromatin accessibility in immature macrophages that may underlie how early life neuroimmune interactions modulate neonatal priming effects. Results demonstrate that the periphery has unique modulators of neonatal nociception that often require distinct immune cell and endocrine signaling activities that influence sensory neuron function. We are currently investigating how altering this process may affect neonatal priming through epigenetic modifications in macrophages. R01NS105715.

Early Life Nociception is Influenced by Peripheral Growth Hormone Signaling.

A number of cellular systems work in concert to modulate nociceptive processing in the periphery, but the mechanisms that regulate neonatal nociception may be distinct compared with adults. Our previous work indicated a relationship between neonatal hypersensitivity and growth hormone (GH) signaling. Here, we explored the peripheral mechanisms by which GH modulated neonatal nociception under normal and injury conditions (incision) in male and female mice. We found that GH receptor (GHr) signaling in primary afferents maintains a tonic inhibition of peripheral hypersensitivity. After injury, a macrophage dependent displacement of injury-site GH was found to modulate neuronal transcription at least in part via serum response factor (SRF) regulation. A single GH injection into the injured hindpaw muscle effectively restored available GH signaling to neurons and prevented acute pain-like behaviors, primary afferent sensitization, and neuronal gene expression changes. GH treatment also inhibited long-term somatosensory changes observed after repeated peripheral insult. Results may indicate a novel mechanism of neonatal nociception.SIGNIFICANCE STATEMENT Although it is noted that mechanisms of pain development in early life are unique compared with adults, little research focuses on neonatal-specific peripheral mechanisms of nociception. This gap is evident in the lack of specialized care for infants following an injury including surgeries. This report evaluates how distinct cellular systems in the periphery including the endocrine, immune and nervous systems work together to modulate neonatal-specific nociception. We uncovered a novel mechanism by which muscle injury induces a macrophage-dependent sequestration of peripheral growth hormone (GH) that effectively removes its normal tonic inhibition of neonatal nociceptors to promote acute pain-like behaviors. Results indicate a possible new strategy for treatment of neonatal postsurgical pain.

Expression of artemin and GFRα3 in an animal model of migraine: possible role in the pathogenesis of this disorder.

BackgroundNeurotrophic factors have been implicated in hyperalgesia and peripheral levels of these molecules are altered in migraine pathophysiology. Artemin, a vasculature-derived neurotrophic factor, contributes to pain modulation and trigeminal primary afferent sensitization through binding its selective receptor GFRα3. The distribution of artemin and GFRα3 in the dura mater raises an anatomy supports that they may be involved in migraine. In this study we evaluated the expression of artemin and GFRα3 in an animal migraine model that may be relevant for migraine.MethodsIn this study, using a rat migraine model by administration of nitroglycerin (NTG), we investigated the expression of artemin in the dura mater and GFRα3 in the trigeminal ganglia (TG) by means of quantitative reverse transcription-polymerase chain reaction, western blot and immunofluorescence labeling.ResultsArtemin immunoreactivity was found in the smooth muscle cells of dural vasculature and GFRα3 was present in cytoplasm of TG neurons. The mRNA levels of artemin and GFRα3 were significantly elevated after NTG treatment at 2 and 4 h respectively (P < 0.05). The expression of artemin protein was increased at 4 h and continually up to 8 h in the dura mater following NTG administration (P < 0.05). The expression of GFRα3 protein was elevated at 4 h and continually up to 10 h in the TG following NTG administration (P < 0.05).ConclusionThe findings suggest that artemin and GFRα3 play an important role in the pathogenesis of migraine and may represent potential therapeutic targets for the treatment of migraine.

Responses of the human maternal HPA axis to obesity and stress: Implications for offspring behaviour

development, events that increase intestinal permeability, allowing, consequently, the access of luminal antigens to the mucosa. Food components, microorganisms, and toxins can stimulate local responses by activating immune cells and/or directly sensitizing nerve endings. Primary afferent sensitization could affect responsiveness to visceral stimuli and modify neuropeptide release form nerves, consequently leading to neurogenic inflammation. In fact, mast cell proximity to nerves and its correlation with abdominal pain have been identified in IBS patients. Moreover, the presence of higher amount of corticotropin-releasing hormone in mucosal eosinophils in IBS, as compared to healthy controls, in correlation with psychological stress has been identified, enabling local pain signaling and barrier alterations upon eosinophil degranulation. B lymphocytes and plasma cells are also part of the inflammatory infiltrate and display an activated profile in association with major clinical symptoms in IBS. In this presentation Iwill review themain mechanisms associated with pain signaling in IBS with a special focus on stress-induced activation of the mucosal immune system.

Itch and pain differences and commonalities.

Pain and itch are generally regarded antagonistic as painful stimuli such as scratching suppresses itch. Moreover, inhibition of pain processing by opioids generates itch further supporting their opposing role. Separate specific pathways for itch and pain processing have been uncovered, and several molecular markers have been established in mice that identify neurons involved in the processing of histaminergic and non-histaminergic itch on primary afferent and spinal level. These results are in agreement with the specificity theory for itch and might suggest that pain and itch should be investigated separately on the level of neurons, mediators, and mechanisms. However, in addition to broadly overlapping mediators of itch and pain, there is also evidence for overlapping functions in primary afferents: nociceptive primary afferents can provoke itch when activated very locally in the epidermis, and sensitization of both nociceptors and pruriceptors has been found following local nerve growth factor application in volunteers. Thus, also mechanisms that underlie the development of chronic itch and pain including spontaneous activity and sensitization of primary afferents as well as spinal cord sensitization may well overlap to a great extent. Rather than separating itch and pain, research concepts should therefore address the common mechanisms. Such an approach appears most appropriate for clinical conditions of neuropathic itch and pain and also chronic inflammatory conditions. While itch researchers can benefit from the large body of information of the pain field, pain researchers will find behavioral readouts of spontaneous itch much simpler than those for spontaneous pain in animals and the skin as source of the pruritic activity much more accessible even in patients.

Activation of p38 mitogen-activated protein kinase in the dorsal root ganglion contributes to pain hypersensitivity after plantar incision

BackgroundThe phosphorylation of p38 mitogen-activated protein kinase (MAPK) in the dorsal root ganglion (DRG) promotes primary afferent sensitization. The role of p38MAPK signaling in the DRG in the pathogenesis of plantar incision hyperalgesia has not been investigated. ResultsLevels of phosphorylated p38MAPK (p-p38MAPK) obviously increased in the DRG after plantar incision. Unmyelinated and myelinated DRG neurons that express p-p38MAPK contained small to medium cell bodies, suggesting that p-p38MAPK expression is induced in neurons with C- and Aδ-fibers. The p-p38MAPK inhibitors FR167653 or SB203580 inhibited incision-induced mechanical hypersensitivity and spontaneous pain behavior. The systemic administration of tumor necrosis factor-α (TNF-α) inhibitor prevented subsequent incision-induced activation of p38MAPK in the DRG and alleviated mechanical hypersensitivity after the incision. Conclusionsp38MAPK signaling in the DRG plays a crucial role in the development of primary afferent sensitization and pain behavior caused by plantar incision.

Inflammation-Induced Changes in BKCa Currents in Cutaneous Dorsal Root Ganglion Neurons from the Adult Rat

BackgroundInflammation-induced sensitization of primary afferents is associated with a decrease in K+ current. However, the type of K+ current and basis for the decrease varies as a function of target of innervation. Because glabrous skin of the rat hindpaw is used often to assess changes in nociception in models of persistent pain, the purpose of the present study was to determine the type and extent to which K+ currents contribute to the inflammation-induced sensitization of cutaneous afferents. Acutely dissociated retrogradely labeled cutaneous dorsal root ganglion neurons from naïve and inflamed (3 days post complete Freund’s adjuvant injection) rats were studied with whole cell and perforated patch techniques.ResultsInflammation-induced sensitization of small diameter cutaneous neurons was associated with an increase in action potential duration and rate of decay of the afterhyperpolarization. However, no changes in voltage-gated K+ currents were detected. In contrast, Ca2+ modulated iberiotoxin sensitive and paxilline sensitive K+ (BKCa) currents were significantly smaller in small diameter IB4+ neurons. This decrease in current was not associated with a detectable change in total protein levels of the BKCa channel α or β subunits. Single cell PCR analysis revealed a significant change in the pattern of expression of α subunit splice variants and β subunits that were consistent, at least in part, with inflammation-induced changes in the biophysical properties of BKCa currents in cutaneous neurons.ConclusionsResults of this study provide additional support for the conclusion that it may be possible, if not necessary to selectively treat pain arising from specific body regions. Because a decrease in BKCa current appears to contribute to the inflammation-induced sensitization of cutaneous afferents, BKCa channel openers may be effective for the treatment of inflammatory pain.

A Peripheral Adrenoceptor-mediated Sympathetic Mechanism Can Transform Stress-induced Analgesia into Hyperalgesia

Stress has paradoxical effects on pain, causing stress-induced analgesia but also exacerbating pain via poorly understood mechanisms. Adrenergic neurotransmission is integral in pathways that regulate the response to both pain and stress. Hyperalgesia is often associated with enhanced adrenergic sensitivity of primary afferents, but sympathetic nervous system outflow has not been demonstrated to exacerbate pain perception after stress. Rats or C57/BL6 wild-type mice treated with α-2 receptor antagonists or α-2A receptor knockout mice were exposed to ultrasonic noise stress or footshock stress and subsequently tested for hotplate paw withdrawal latencies. The sensory sensitivity of α-2A knockout mice to electrical and chemical stimuli was tested neurophysiologically and behaviorally. The effects of sympatholytic treatments were investigated. Noise and footshock stressors induced thermal hyperalgesia in rats pretreated systemically with α-2 antagonists. Wild-type mice pretreated with α-2 antagonists and α-2A knockout mice also exhibited thermal hyperalgesia induced by noise stress. Local spinal or intraplantar injection of an α-2 antagonist counteracted stress-induced analgesia without causing hyperalgesia. The α-2A knockout mice had decreased thresholds for peripheral sensitization with sulprostone and for windup of the dorsal horn neuronal response to repetitive electrical stimuli. Stress-induced hyperalgesia was abolished and the sensitization was attenuated by sympathectomy or systemic administration of an α-1-adrenergic antagonist. Sympathetic postganglionic nerves can enhance pain sensation via a peripheral α-1-adrenoceptor mechanism when sympathetic outflow is disinhibited. The net effect of stress on pain sensation reflects a balance between descending spinal inhibition and sympathetic outflow that can shift toward pain facilitation when central and peripheral α-2-adrenoceptor inhibitory mechanisms are attenuated.

Exercise with angina and cramp?

Exercise with angina and cramp?

AF‐353, a novel, potent and orally bioavailable P2X3/P2X2/3 receptor antagonist

Purinoceptors containing the P2X3 subunit (P2X3 homotrimeric and P2X2/3 heterotrimeric) are members of the P2X family of ion channels gated by ATP and may participate in primary afferent sensitization in a variety of pain-related diseases. The current work describes the in vitro pharmacological characteristics of AF-353, a novel, orally bioavailable, highly potent and selective P2X3/P2X2/3 receptor antagonist. The antagonistic potencies (pIC(50)) of AF-353 for rat and human P2X3 and human P2X2/3 receptors were determined using methods of radioligand binding, intracellular calcium flux and whole cell voltage-clamp electrophysiology. The pIC(50) estimates for these receptors ranged from 7.3 to 8.5, while concentrations 300-fold higher had little or no effect on other P2X channels or on an assortment of receptors, enzymes and transporter proteins. In contrast to A-317491 and TNP-ATP, competition binding and intracellular calcium flux experiments suggested that AF-353 inhibits activation by ATP in a non-competitive fashion. Favourable pharmacokinetic parameters were observed in rat, with good oral bioavailability (%F = 32.9), reasonable half-life (t(1/2) = 1.63 h) and plasma-free fraction (98.2% protein bound). The combination of a favourable pharmacokinetic profile with the antagonist potency and selectivity for P2X3 and P2X2/3 receptors suggests that AF-353 is an excellent in vivo tool compound for study of these channels in animal models and demonstrates the feasibility of identifying and optimizing molecules into potential clinical candidates, and, ultimately, into a novel class of therapeutics for the treatment of pain-related disorders.

A Rapamycin-Sensitive Signaling Pathway Is Essential for the Full Expression of Persistent Pain States

Translational control through the mammalian target of rapamycin (mTOR) is critical for synaptic plasticity, cell growth, and axon guidance. Recently, it was also shown that mTOR signaling was essential for the maintenance of the sensitivity of subsets of adult sensory neurons. Here, we show that persistent pain states, but not acute pain behavior, are substantially alleviated by centrally administered rapamycin, an inhibitor of the mTOR pathway. We demonstrate that rapamycin modulates nociception by acting on subsets of primary afferents and superficial dorsal horn neurons to reduce both primary afferent sensitivity and central plasticity. We found that the active form of mTOR is present in a subpopulation of myelinated dorsal root axons, but rarely in unmyelinated C-fibers, and heavily expressed in the dorsal horn by lamina I/III projection neurons that are known to mediate the induction and maintenance of pain states. Intrathecal injections of rapamycin inhibited the activation of downstream targets of mTOR in dorsal horn and dorsal roots and reduced the thermal sensitivity of A-fibers. Moreover, in vitro studies showed that rapamycin increased the electrical activation threshold of Adelta-fibers in dorsal roots. Together, our results imply that central rapamycin reduces neuropathic pain by acting both on an mTOR-positive subset of A-nociceptors and lamina I projection neurons and suggest a new pharmacological route for therapeutic intervention in persistent pain states.

W1728 Activation of MAPK and TRPV1 Contribute to Primary Afferent Sensitization in a Rat Model of Chronic Visceral Hypersensitivity

W1728 Activation of MAPK and TRPV1 Contribute to Primary Afferent Sensitization in a Rat Model of Chronic Visceral Hypersensitivity

Role of sodium ferulate in the nociceptive sensory facilitation of neuropathic pain injury mediated by P2X 3 receptor

Neuropathic pain usually is persistent and no effective treatment. ATP plays an important role in the initiation of pain. P2X 3 receptors are localized in the dorsal root ganglion (DRG) neurons and activated by extracellular ATP. Sodium ferulate (SF) is an active principle from Chinese herbal medicine and has anti-inflammatory activities. This study observed the effects of SF on the nociceptive facilitation of the primary sensory afferent after chronic constriction injury (CCI) mediated by P2X 3 receptor. In this study, the content of ATP in DRG neurons was measured by high-performance liquid chromatography (HPLC). P2X 3 agonist-activated currents in DRG neurons was recorded by the whole-cell patch-clamp skill. The expression of P2X 3 mRNA in DRG neurons was analyzed by in situ hybridization. The ATP content of DRG was increased after CCI. In CCI rats treated with SF, the content of ATP in DRG neurons was reduced. SF decreased the increment of P2X 3 agonist-activated currents and P2X 3 mRNA expression in DRG neurons during CCI. SF may inhibit the initiation of pain and primary afferent sensitization mediated by P2X 3 receptor during CCI.

Sensitization of primary afferents to mechanical and heat stimuli after incision in a novel in vitro mouse glabrous skin-nerve preparation

In this study, we recorded activity from afferent fibers innervating the mouse plantar skin, the same region evaluated in pain behavior experiments. We compared responses of afferents from incised and unincised hind paw skin. The plantar skin together with attached medial and lateral plantar nerves was dissected until they could be completely removed intact and placed in an organ bath chamber continuously perfused with oxygenated Kreb’s solution with the temperature maintained at 32 °C. Afferent nerve activities to feedback-controlled mechanical and heat stimuli and cooling were recorded. Eighty-five single Aδ- and C-fiber afferents were recorded, 42 from control and the remainder from incised animals. A greater proportion of C-fibers (11/34) from incised skin had spontaneous activity than in the unincised preparation (2/32). The mechanical thresholds of both Aδ- and C-fiber units were not different between control and incised groups but the responses to suprathreshold mechanical stimulation were increased in low threshold Aδ- and C-fibers. The greatest change in heat sensitivity was apparent when multi-fiber total activity was measured; threshold was reduced, total spikes were greater and the peak discharge frequency was increased. In summary, feedback-controlled stimulation identified mechanical sensitization after incision in an in vitro preparation. Few fibers were excited by cooling. Heat sensitization of primary afferents was more prominent when activities of unclassified afferents are included. The preparation allows us to study afferent function of the same tissue that is examined for in vivo pain behavior assays in mice.

Involvement of Peripheral Purinoceptors in Sympathetic Modulation of Capsaicin-Induced Sensitization of Primary Afferent Fibers

Purinoceptors are distributed in primary afferent terminals, where transmission of nociceptive information is modulated by these receptors. In the present study, we evaluated whether the activation or blockade of purinoceptors of subtypes P2X and P2Y in the periphery affected the sensitization of primary afferents induced by intradermal injection of capsaicin (CAP) and examined their role in sympathetic modulation of sensitization of primary nociceptive afferents. Afferent activity was recorded from single Adelta- and C-primary afferent fibers in the tibial nerve in anesthetized rats. Peripheral pretreatment with alpha,beta-methylene adenosine 5'-triphosphate (alpha,beta-meATP), a P2X-selective receptor agonist, could potentiate the CAP-induced enhancement of responses of Adelta- and C-primary afferent nociceptive fibers to mechanical stimuli in sympathetically intact rats. After sympathetic denervation, the enhanced responses of both Adelta- and C-fibers after CAP injection were dramatically reduced. However, this reduction could be restored when P2X receptors were activated by alpha,beta-meATP. A blockade of P2X receptors by pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid could significantly reduce the CAP-induced sensitization of Adelta- and C-fibers. Pretreatment with uridine 5'-triphosphate, a P2Y-selective receptor agonist, did not significantly affect or restore the CAP-induced sensitization of Adelta- and C-fibers under sympathetically intact or sympathectomized conditions. Our study supports the view that ATP plays a role in modulation of primary afferent nociceptor sensitivity mainly by P2X receptors. Combined with our previous study, our data also provide further evidence that the sensitization of primary afferent nociceptors is subject to sympathetic modulation by activation of P2X as well as alpha(1)-adrenergic receptors.