Natural Killer Cells Degenerate Intact Sensory Afferents following Nerve Injury.

Sensory axons degenerate following separation from their cell body, but partial injury to peripheral nerves may leave the integrity of damaged axons preserved. We show that an endogenous ligand for the natural killer (NK) cell receptor NKG2D, Retinoic Acid Early 1 (RAE1), is re-expressed in adult dorsal root ganglion neurons following peripheral nerve injury, triggering selective degeneration of injured axons. Infiltration of cytotoxic NK cells into the sciatic nerve by extravasation occurs within 3 days following crush injury. Using a combination of genetic cell ablation and cytokine-antibody complex stimulation, we show that NK cell function correlates with loss of sensation due to degeneration of injured afferents and reduced incidence of post-injury hypersensitivity. This neuro-immune mechanism of selective NK cell-mediated degeneration of damaged but intact sensory axons complements Wallerian degeneration and suggests the therapeutic potential of modulating NK cell function to resolve painful neuropathy through the clearance of partially damaged nerves.

Adipose-derived stem cells decolonize skin Staphylococcus aureus by enhancing phagocytic activity of peripheral blood mononuclear cells in the atopic rats.

Staphylococcus aureus (S. aureus) is known to induce apoptosis of host immune cells and impair phagocytic clearance, thereby being pivotal in the pathogenesis of atopic dermatitis (AD). Adipose-derived stem cells (ASCs) exert therapeutic effects against inflammatory and immune diseases. In the present study, we investigated whether systemic administration of ASCs restores the phagocytic activity of peripheral blood mononuclear cells (PBMCs) and decolonizes cutaneous S. aureus under AD conditions. AD was induced by injecting capsaicin into neonatal rat pups. ASCs were extracted from the subcutaneous adipose tissues of naïve rats and administered to AD rats once a week for a month. Systemic administration of ASCs ameliorated AD-like symptoms, such as dermatitis scores, serum IgE, IFN-γ+/IL-4+ cell ratio, and skin colonization by S. aureus in AD rats. Increased FasL mRNA and annexin V+/7-AAD+ cells in the PBMCs obtained from AD rats were drastically reversed when co-cultured with ASCs. In contrast, both PBMCs and CD163+ cells bearing fluorescent zymosan particles significantly increased in AD rats treated with ASCs. Additionally, the administration of ASCs led to an increase in the mRNA levels of antimicrobial peptides, such as cathelicidin and ß-defensin, in the skin of AD rats. Our results demonstrate that systemic administration of ASCs led to decolonization of S. aureus by attenuating apoptosis of immune cells in addition to restoring phagocytic activity. This contributes to the improvement of skin conditions in AD rats. Therefore, administration of ASCs may be helpful in the treatment of patients with intractable AD.

Common and discrete mechanisms underlying chronic pain and itch: peripheral and central sensitization.

Normally, an obvious antagonism exists between pain and itch. In normal conditions, painful stimuli suppress itch sensation, whereas pain killers often generate itch. Although pain and itch are mediated by separate pathways under normal conditions, most chemicals are not highly specific to one sensation in chronic pathologic conditions. Notably, in patients with neuropathic pain, histamine primarily induces pain rather than itch, while in patients with atopic dermatitis, bradykinin triggers itch rather than pain. Accordingly, repetitive scratching even enhances itch sensation in chronic itch conditions. Physicians often prescribe pain relievers to patients with chronic itch, suggesting common mechanisms underlying chronic pain and itch, especially peripheral and central sensitization. Rather than separating itch and pain, studies should investigate chronic itch and pain including neuropathic and inflammatory conditions. Here, we reviewed chronic sensitization leading to chronic pain and itch at both peripheral and central levels. Studies investigating the connection between pain and itch facilitate the development of new therapeutics against both chronic dysesthesias based on the underlying pathophysiology.

Capsaicin induces atopic dermatitis-like manifestations through dysregulation of proteolytic system and alteration of filaggrin processing in rats.

Atopic dermatitis (AD) is a complex disease featuring pruritic skin inflammation. Many animal models have been developed. In a rat model, subcutaneous capsaicin injection within 48 hours after birth induces AD-like skin manifestations of dermatitis and scratching behaviour 3 weeks after the injection. When 2- to 4-week-old rats were injected with capsaicin, the lag period was shortened, and the severity of skin manifestations was significantly reduced, suggesting influences of postnatal development. Lgr6 is an epidermal stem cell marker that is normally restricted to the isthmus area of hair follicles at postnatal 2 weeks. Lgr6 persisted in the interfollicular epidermis of capsaicin-injected rats beyond 3 weeks after birth, indicating that capsaicin-induced skin manifestations were influenced by postnatal epidermal development. Capsaicin injection induced alteration of proteolytic processing of filaggrin and corneodesmosin, suggesting epidermal barrier dysfunction. Inappropriate degradation of matriptase was observed. Degrees of proteolysis of these proteins were corelated with the severity of manifestations, suggesting that inappropriate proteolysis might be a possible cause of the skin manifestations. These results strongly suggest that capsaicin may dysregulate the protease system, resulting in alteration of profilaggrin and corneodesmosin proteolysis and skin manifestations. These events may be influenced by postnatal epidermal development.

Oxytocin produces thermal analgesia via vasopressin-1a receptor by modulating TRPV1 and potassium conductance in the dorsal root ganglion neurons.

Recent studies have provided several lines of evidence that peripheral administration of oxytocin induces analgesia in human and rodents. However, the exact underlying mechanism of analgesia still remains elusive. In the present study, we aimed to identify which receptor could mediate the analgesic effect of intraperitoneal injection of oxytocin and its cellular mechanisms in thermal pain behavior. We found that oxytocin-induced analgesia could be reversed by d(CH2)5[Tyr(Me)2,Dab5] AVP, a vasopressin-1a (V1a) receptor antagonist, but not by desGly-NH2-d(CH2)5[DTyr2, Thr4]OVT, an oxytocin receptor antagonist. Single cell RT-PCR analysis revealed that V1a receptor, compared to oxytocin, vasopressin-1b and vasopressin-2 receptors, was more profoundly expressed in dorsal root ganglion (DRG) neurons and the expression of V1a receptor was predominant in transient receptor potential vanilloid 1 (TRPV1)-expressing DRG neurons. Fura-2 based calcium imaging experiments showed that capsaicin-induced calcium transient was significantly inhibited by oxytocin and that such inhibition was reversed by V1a receptor antagonist. Additionally, whole cell patch clamp recording demonstrated that oxytocin significantly increased potassium conductance via V1a receptor in DRG neurons. Taken together, our findings suggest that analgesic effects produced by peripheral administration of oxytocin were attributable to the activation of V1a receptor, resulting in reduction of TRPV1 activity and enhancement of potassium conductance in DRG neurons.

Brief Isolation Changes Nociceptive Behaviors and Compromises Drug Tests in Mice.

Herding with a litter is known to comfort rodents, whereas isolation and grouping with noncagemates provoke stress. The effects of stress induced by isolation and grouping with noncagemates on pain responses, and their underlying mechanisms remain elusive. We assessed the effect of isolation, a common condition during behavioral tests, and of grouping on defecation and pain behaviors of mice. Fecal pellets were counted 2 hours after exposure to the test chamber. It is significantly more in the isolated mice than in the grouped mice. Hindpaw withdrawal threshold and withdrawal latency were adopted as the indicatives of mechanical and thermal pain sensitivities, respectively. Interestingly, isolated mice showed higher pain thresholds than mice grouping with cagemates, and even those with noncagemates, indicating analgesic effects. Such effects were reduced by intrathecal injection of 0.01 mg/kg of naloxone (opioid receptor antagonist), atosiban (oxytocin and vasopressin receptor antagonist), and ketanserin (5-HT receptor antagonist). Intraperitoneal delivery of 1 mg/kg of naloxone and atosiban, but not ketanserin, also alleviated the isolation-induced analgesic effects. In contrast, these drugs at the same dose had no significant effect on the mice grouping with cagemates. In addition, the effect of morphine on thermal pain was more robust in the mice grouping with cagemates than in the isolated mice. These data demonstrated that brief isolation caused analgesia, mediated by endogenous opioidergic, oxytocinergic, and serotonergic pathways. These results indicate that isolation during pain behavioral tests can affect pain responses and the efficacy of drugs; thus, nociception tests should be conducted in grouping.

Pathophysiological Role of TLR4 in Chronic Relapsing Itch Induced by Subcutaneous Capsaicin Injection in Neonatal Rats.

Despite the high prevalence of chronic dermatitis and the accompanied intractable itch, therapeutics that specifically target itching have low efficacy. Increasing evidence suggests that TLRs contribute to immune activation and neural sensitization; however, their roles in chronic itch remain elusive. Here, we show that the RBL-2H3 mast cell line expresses TLR4 and that treatment with a TLR4 antagonist opposes the LPS dependent increase in mRNA levels of Th2 and innate cytokines. The pathological role of TLR4 activation in itching was studied in neonate rats that developed chronic itch due to neuronal damage after receiving subcutaneous capsaicin injections. Treatment with a TLR4 antagonist protected these rats with chronic itch against scratching behavior and chronic dermatitis. TLR4 antagonist treatment also restored the density of cutaneous nerve fibers and inhibited the histopathological changes that are associated with mast cell activation after capsaicin injection. Additionally, the expression of IL-1ß, IL-4, IL-5, IL-10, and IL-13 mRNA in the lesional skin decreased after TLR4 antagonist treatment. Based on these data, we propose that inhibiting TLR4 alleviated itch in a rat model of chronic relapsing itch, and the reduction in the itch was associated with TLR4 signaling in mast cells and nerve fibers.

Membrane-delimited coupling of TRPV1 and mGluR5 on presynaptic terminals of nociceptive neurons.

Transient receptor potential vanilloid subtype 1 (TRPV1) and metabotropic glutamate receptor 5 (mGluR5) located on peripheral sensory terminals have been shown to play critical roles in the transduction and modulation of pain sensation. To date, however, very little is known regarding the significance of functional expression of mGluR5 and TRPV1 on the central terminals of sensory neurons in the dorsal horn of the spinal cord. Here we show that TRPV1 on central presynaptic terminals is coupled to mGluR5 in a membrane-delimited manner, thereby contributing to the modulation of nociceptive synaptic transmission in the substantia gelatinosa neurons of the spinal cord. Further, our results demonstrate that TRPV1 is involved in the pain behaviors induced by spinal mGluR5 activation, and diacylglycerol produced by the activation of mGluR5 mediates functional coupling of mGluR5 and TRPV1 on the presynaptic terminals. Thus, mGluR5-TRPV1 coupling on the central presynaptic terminals of nociceptive neurons may be an important mechanism underlying central sensitization under pathological pain conditions.

Analgesic effect of highly reversible ω-conotoxin FVIA on N type Ca2+ channels.

BACKGROUND: N-type Ca2+ channels (Ca(v)2.2) play an important role in the transmission of pain signals to the central nervous system. ω-Conotoxin (CTx)-MVIIA, also called ziconotide (Prialt®), effectively alleviates pain, without causing addiction, by blocking the pores of these channels. Unfortunately, CTx-MVIIA has a narrow therapeutic window and produces serious side effects due to the poor reversibility of its binding to the channel. It would thus be desirable to identify new analgesic blockers with binding characteristics that lead to fewer adverse side effects. RESULTS: Here we identify a new CTx, FVIA, from the Korean Conus Fulmen and describe its effects on pain responses and blood pressure. The inhibitory effect of CTx-FVIA on N-type Ca2+ channel currents was dose-dependent and similar to that of CTx-MVIIA. However, the two conopeptides exhibited markedly different degrees of reversibility after block. CTx-FVIA effectively and dose-dependently reduced nociceptive behavior in the formalin test and in neuropathic pain models, and reduced mechanical and thermal allodynia in the tail nerve injury rat model. CTx-FVIA (10 ng) also showed significant analgesic effects on writhing in mouse neurotransmitter- and cytokine-induced pain models, though it had no effect on acute thermal pain and interferon-γ induced pain. Interestingly, although both CTx-FVIA and CTx-MVIIA depressed arterial blood pressure immediately after administration, pressure recovered faster and to a greater degree after CTx-FVIA administration. CONCLUSIONS: The analgesic potency of CTx-FVIA and its greater reversibility could represent advantages over CTx-MVIIA for the treatment of refractory pain and contribute to the design of an analgesic with high potency and low side effects.

Synthesis and bradykinin inhibitory activity of novel non-peptide compounds, and evaluation of in vivo analgesic activity.

A series of novel non-peptide diamide compounds was synthesized and evaluated as antibradykinin agents by utilizing guinea-pig ileum smooth muscle. Among the final compounds, (Z)-4-(4-(bis(4-fluorophenyl)methyl)piperazin-1-yl)-4-oxo-N-(4-phenylbutan-2-yl)but-2-enamide showed most favorable bradykinin inhibitory activity and demonstrated analgesic efficacies in the rat models of inflammatory and neuropathic pain.

Synthesis and T-type calcium channel blocking activity of novel diphenylpiperazine compounds, and evaluation of in vivo analgesic activity.

Novel diphenylpiperazine derivatives were synthesized and evaluated for their inhibitory activity against T-type calcium channel by whole-cell patch clamp recordings on HEK293 cells. Among the test compounds, 2 and 3d were effective in decreasing the response to formalin in both the first and second phases and demonstrated antiallodynic effects in a rat model of neuropathic pain.

Postconditioning protects skeletal muscle from ischemia-reperfusion injury.

Ischemia-reperfusion (I/R) injury caused by abrupt restoration of the circulation after prolonged ischemic insult induces significant morbidity after reconstructive microsurgery. The authors investigated whether a postconditioning (post-con) procedure attenuated skeletal muscle I/R injury and protected muscular function. Three hours of complete ischemia was induced by occluding the muscular branches of rat extensor digitorum longus (EDL) muscle. The post-con procedure was started at the end of ischemia and involved six cycles of 15 seconds of reperfusion followed by 15 seconds of re-occlusion (3 minutes of total intervention) prior to initiating unlimited reperfusion. EDL muscle contractilities were compared with those of normal sides (no ischemic exposure), and experimental group results were also compared with control group results (3 hours of ischemia followed by full reperfusion without post-con) at 3 hours and 5 days postreperfusion. Muscle wet weights, myeloperoxidase (MPO) activities, and histological results were also evaluated. The muscle contractilities in the post-con group were significantly preserved at both 3 hours and 5 days postreperfusion as compared with ischemic controls. Decreased inflammatory cell infiltration, MPO activity, and wet weight of postconditioned EDL muscle suggested that post-con attenuated acute inflammatory reactions induced by I/R. This study demonstrates that post-con provides effective functional protection to skeletal muscles from I/R injury.

T-type Ca2+ channels as therapeutic targets in the nervous system.

Low-voltage-activated calcium channels, also known as T-type calcium channels, are widely expressed in various types of neurons. In contrast to high-voltage-activated calcium channels which can be activated by a strong depolarization of membrane potential, T-type channels can be activated by a weak depolarization near the resting membrane potential once deinactivated by hyperpolarization, and therefore can regulate the excitability and electroresponsiveness of neurons under physiological conditions near resting states. Recently, the molecular diversity and functional multiplicity of T-type channels have been demonstrated through molecular genetic studies coupled with physiological and behavioral analysis. Understanding the functional consequences of modulation of each subtype of these channels in vivo could point to the right direction for developing therapeutic tools for relevant diseases.

Complete Freund's adjuvant-induced intervertebral discitis as an animal model for discogenic low back pain.

BACKGROUND: Although numerous animal models for low back pain associated with intervertebral disk (IVD) degeneration have been proposed, insufficient data have been provided to make any conclusions regarding pain. Our aim in this study was to determine the reliability of complete Freund's adjuvant (CFA) injection into the rat spine as an animal model representing human discogenic pain. METHODS: We studied IVD degenerative changes with pain development after a 10-microL CFA injection into the L5-6 IVD of adult rats using behavioral, histologic, and biochemical studies. Serial histologic changes were analyzed to detect degenerative changes. Expression of calcitonin gene-related peptide (CGRP), prostaglandin E (PGE), and inducible nitric oxide synthase (iNOS) were determined using immunohistochemistry or real-time polymerase chain reaction as support data for pain development. In addition, CGRP immunoreactivity (ir) at the IVD was considered indirect evidence of neural ingrowth into the IVD. RESULTS: There was a significant increase of the hindpaw withdrawal response in the CFA group until 7 wk postoperatively (P < 0.05). Histologic analyses revealed progressive degenerative changes of the disks without any damage in adjacent structures, including nerve roots. In the CGRP-ir staining study, the bilateral dorsal horns and IVD had positive ir after intradiscal CFA injection. CGRP mRNA expression was increased in the dorsal root ganglion (DRG) at 2 and 4 wk, whereas PGE and iNOS mRNAs were markedly increased at 2 wk. The increment of CGRP expression was higher in allodynic rats compared with nonallodynic rats. CONCLUSION: Intradiscal CFA injection led to chronic disk degeneration with allodynia, which was suggested by pain behavior and expression of pain-related mediators. The increment of CGRP, PGE, and iNOS also suggest pain-related signal processing between the IVD and the neural pathway in this animal model. This animal model may be useful for future research related to the pathophysiology and development of novel treatment for spine-related pain.

Developmental characteristics of neuropathic pain induced by peripheral nerve injury of rats during neonatal period.

To gain an insight into the developmental characteristics of neuropathic pain induced by peripheral nerve injury during neonatal period, we employed three groups of rats suffering from peripheral nerve injury at different postnatal times, and compared the onset time, severity and persistency of neuropathic pain behaviors, such as mechanical and cold allodynia. The first group (P0 group) was subjected to partial injury of tail-innervating nerves within 24 h after birth, the second group (P10 group) underwent nerve injury at postnatal day (P) 10, and the third group (P60 group) was subjected to injury at P60. Although mechanical allodynia was readily detectable in the P60 group even 1 day after nerve injury, the signs of neuropathic pain were observed from 6 or 8 weeks after nerve injury in the P0 or P10 groups, respectively. Compared with the P60 group, the P0 group showed more robust mechanical and cold allodynia, whereas the P10 group exhibited rather milder pains. In addition, while the P0 and P60 groups showed long-lasting signs of mechanical allodynia, the P10 group exhibited shorter persistency. These results indicate that peripheral nerve injury during neonatal period leads to neuropathic pain with distinct developmental characteristics later in life.

Attenuated neuropathic pain in Cav3.1 null mice.

To assess the role of alpha(1G) T-type Ca2+ channels in neuropathic pain after L5 spinal nerve ligation, we examined behavioral pain susceptibility in mice lacking CaV3.1 (alpha1G(-/-)), the gene encoding the pore-forming units of these channels. Reduced spontaneous pain responses and an increased threshold for paw withdrawal in response to mechanical stimulation were observed in these mice. The alpha1G(-/-) mice also showed attenuated thermal hyperalgesia in response to both low-(IR30) and high-intensity (IR60) infrared stimulation. Our results reveal the importance of alpha(1G) T-type Ca2+ channels in the development of neuropathic pain, and suggest that selective modulation of alpha1G subtype channels may provide a novel approach to the treatment of allodynia and hyperalgesia.

Intrathecal injection of the sigma(1) receptor antagonist BD1047 blocks both mechanical allodynia and increases in spinal NR1 expression during the induction phase of rodent neuropathic pain.

BACKGROUND: Selective blockade of spinal sigma(1) receptors (Sig-1R) suppresses nociceptive behaviors in the mouse formalin test. The current study was designed to verify whether intrathecal Sig-1R antagonists can also suppress chronic neuropathic pain. METHODS: Neuropathic pain was produced by chronic constriction injury (CCI) of the right sciatic nerve in rats. The Sig-1R antagonist BD1047 was administered intrathecally twice daily from postoperative days 0 to 5 (induction phase of neuropathic pain) or from days 15 to 20 (maintenance phase). Western blot and immunohistochemistry were performed to determine changes in Sig-1R expression and to examine the effect of BD1047 on N-methyl-D-aspartate receptor subunit 1 expression and phosphorylation in spinal cord dorsal horn from neuropathic rats. RESULTS: BD1047 administered on postoperative days 0-5 significantly attenuated CCI-induced mechanical allodynia, but not thermal hyperalgesia, and this suppression was blocked by intrathecal administration of the Sig-1R agonist PRE084. In contrast, BD1047 treatment during the maintenance phase of neuropathic pain had no effect on mechanical allodynia. Sig-1R expression significantly increased in the ipsilateral spinal cord dorsal horn from days 1 to 3 after CCI. Importantly, BD1047 (30 nmol) administered intrathecally during the induction, but not the maintenance phase, blocked the CCI-induced increase in N-methyl-D-aspartate receptor subunit 1 expression and phosphorylation. CONCLUSIONS: These results demonstrate that spinal Sig-1Rs play a critical role in both the induction of mechanical allodynia and the activation of spinal N-methyl-d-aspartate receptors in CCI rats and suggest a potential therapeutic role for the use of Sig-1R antagonists in the clinical management of neuropathic pain.

Glyoxal-induced exacerbation of pruritus and dermatitis is associated with staphylococcus aureus colonization in the skin of a rat model of atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is a highly pruritic, chronic inflammatory skin disease associated with hyperreactivity to environmental triggers. Among those, outdoor air pollutants such as particulate matter (PM) have been reported to aggravate pre-existing AD. However, underlying mechanisms of air pollution-induced aggravation of AD have hardly been studied. OBJECTIVE: To investigate the molecular mechanisms by which glyoxal, a PM-forming organic compound, exacerbates the symptoms of AD induced by neonatal capsaicin treatment. METHODS: Naïve and AD rats had been exposed to either fresh air or vaporized glyoxal for 5 weeks (2 h/day and 5 days/week) since one week of age. Pruritus and dermatitis were measured every week. The skin and blood were collected and immunological traits such as Staphylococcus aureus skin colonization, production of antimicrobial peptides and immunoglobulin, and mRNA expression of inflammatory cytokines were analyzed. RESULTS: Exposure to glyoxal aggravated pruritus and dermatitis in AD rats, but did not induce any symptoms in naïve rats. Staphylococcus aureus skin colonization was increased in the skin of both naïve and AD rats. Expression of antimicrobial peptides such as LL-37 and ß-defensin-2 was also increased by exposure to glyoxal in the skin of both naïve and AD rats. The mRNA expression of Th1-related cytokines was elevated on exposure to glyoxal. However, serum immunoglobulin production was not significantly changed by exposure to glyoxal. CONCLUSION: In AD rats, exposure to glyoxal exacerbated pruritus and cutaneous inflammation, which was associated with increased colonization of S. aureus and subsequent immunological alterations in the skin.

Induction of total insensitivity to capsaicin and hypersensitivity to garlic extract in human by decreased expression of TRPV1.

TRPV1 is a cation channel which is activated by temperature (> or =42 degrees C) and capsaicin. In the present study, we found a person with total insensitivity to capsaicin and attempted to unravel its causes. The expression levels of TRPV1 protein and mRNA in the cells of the person's buccal mucosa were less than half of those in a normal subject. Sequential analysis of mRNA and genomic DNA revealed several point mutations mostly in the second intron of the person's TRPV1. Interestingly, the subject showed hypersensitivity to garlic extract, but TRPA1 (allicin receptor) level was normal. These results suggest that the decreased expression of TRPV1 may be related to a functional knock out in capsaicin sensation and hypersensitivity to allicin in humans.

Effects of Exposure of Formaldehyde to a Rat Model of Atopic Dermatitis Induced by Neonatal Capsaicin Treatment.

Atopic dermatitis is chronically relapsing pruritic eczema and prevails around the world especially in developed countries. Complex interactions between genetic and environmental factors are known to play an important role in the pathophysiology of atopic dermatitis. However, we still lack a detailed picture of the pathogenesis of this disease. Thus, it is of importance to develop appropriate animal models for elucidating the progression of atopic dermatitis. Moreover, investigating the effect of environmental factors such as air pollutants on atopic dermatitis expands understanding of the disease. Here, we describe a method for inducing atopic dermatitis in rats with neonatal capsaicin treatment and a protocol for exposure of a constant concentration of formaldehyde to rats to reveal effects on the development of atopic dermatitis in infantile and adolescent periods. These protocols have been successfully applied to several experiments and can be used for other substances.