Anti-hypersensitive effect of angiotensin (1-7) on streptozotocin-induced diabetic neuropathic pain in mice.

BACKGROUND: We have recently reported that the spinal angiotensin (Ang) converting enzyme (ACE)/Ang II/AT1 receptor axis and downstream p38 MAPK phosphorylation are activated in streptozotocin (STZ)-induced diabetic mice and lead to tactile hypersensitivity. Moreover, our previous results suggested that the intrathecal (i.t.) administration of Ang (1-7), an N-terminal fragment of Ang II, may attenuate the Ang II-induced nociceptive behaviour through the inhibition of p38 MAPK phosphorylation via Mas receptors. Here, we investigated whether the i.t. administration of Ang (1-7) can attenuate STZ-induced diabetic neuropathic pain. METHODS: Tactile and thermal hypersensitivities were determined using the von Frey filament and Hargreaves tests, respectively. The protein expression of ACE2, Mas receptors and phospho-p38 MAPK was measured by western blotting. Spinal ACE2 activity was determined using ACE2 activity assay kit. RESULTS: The i.t. administration of Ang (1-7) significantly reduced the tactile and thermal hypersensitivities on day 14 after STZ injection, and these effects were significantly prevented by the Mas receptor antagonist A779. The expression of ACE2 and Mas receptors in the plasma membrane fraction of the lumbar dorsal spinal cord was both significantly decreased in STZ mice. Spinal ACE2 activity was also decreased while p38 MAPK phosphorylation was increased in the lumbar dorsal region of these mice. This phosphorylation was attenuated by the injection of Ang (1-7), whose effect was reversed by A779. CONCLUSIONS: Our data demonstrate that Ang (1-7) attenuates STZ-induced diabetic neuropathic pain and that this occurs through a mechanism involving spinal Mas receptors and he inhibition of p38 MAPK phosphorylation. SIGNIFICANCE: The ACE2/Ang (1-7)/Mas receptor axis was down-regulated in the spinal cord of STZ mice and the i.t. administration of Ang (1-7) attenuated the STZ-induced diabetic neuropathic pain via Mas receptors. Therefore, the activation of this axis could be an effective therapeutic target to alleviate the neuropathic pain in diabetic patients.

Glial pannexin1 contributes to tactile hypersensitivity in a mouse model of orofacial pain.

Drug studies in animal models have implicated pannexin1 (Panx1) in various types of pain, including trigeminal hypersensitivity, neuropathic pain and migraine. However, the tested drugs have limited specificity and efficacy so that direct evidence for Panx1 contribution to pain has been lacking. We here show that tactile hypersensitivity is markedly attenuated by deletion of Panx1 in a mouse model of chronic orofacial pain; in this model, trigeminal ganglion Panx1 expression and function are markedly enhanced. Targeted deletion of Panx1 in GFAP-positive glia or in neurons revealed distinct effects. Panx1 deletion in GFAP-positive glia cells prevented hypersensitivity completely, whereas deletion of neuronal Panx1 reduced baseline sensitivity and the duration of hypersensitivity. In trigeminal ganglia with genetically encoded Ca2+ indicator in GFAP-positive glia or in neurons, both cell populations were found to be hyperactive and hyper-responsive to ATP. These novel findings reveal unique roles for GFAP-positive glial and neuronal Panx1 and describe new chronic pain targets for cell-type specific intervention in this often intractable disease.

Coding and Plasticity in the Mammalian Thermosensory System.

Perception of the thermal environment begins with the activation of peripheral thermosensory neurons innervating the body surface. To understand how temperature is represented in vivo, we used genetically encoded calcium indicators to measure temperature-evoked responses in hundreds of neurons across the trigeminal ganglion. Our results show how warm, hot, and cold stimuli are represented by distinct population responses, uncover unique functional classes of thermosensory neurons mediating heat and cold sensing, and reveal the molecular logic for peripheral warmth sensing. Next, we examined how the peripheral somatosensory system is functionally reorganized to produce altered perception of the thermal environment after injury. We identify fundamental transformations in sensory coding, including the silencing and recruitment of large ensembles of neurons, providing a cellular basis for perceptual changes in temperature sensing, including heat hypersensitivity, persistence of heat perception, cold hyperalgesia, and cold analgesia.

VAMP7 regulates constitutive membrane incorporation of the cold-activated channel TRPM8.

The cation channel TRPM8 plays a central role in the somatosensory system, as a key sensor of innocuously cold temperatures and cooling agents. Although increased functional expression of TRPM8 has been implicated in various forms of pathological cold hypersensitivity, little is known about the cellular and molecular mechanisms that determine TRPM8 abundance at the plasma membrane. Here we demonstrate constitutive transport of TRPM8 towards the plasma membrane in atypical, non-acidic transport vesicles that contain lysosomal-associated membrane protein 1 (LAMP1), and provide evidence that vesicle-associated membrane protein 7 (VAMP7) mediates fusion of these vesicles with the plasma membrane. In line herewith, VAMP7-deficient mice exhibit reduced functional expression of TRPM8 in sensory neurons and concomitant deficits in cold avoidance and icilin-induced cold hypersensitivity. Our results uncover a cellular pathway that controls functional plasma membrane incorporation of a temperature-sensitive TRP channel, and thus regulates thermosensitivity in vivo.

Relationship between transient receptor potential vanilloid-1 expression and the intensity of sensitive skin symptoms.

BACKGROUND: Sensitive skin (SS) is a hyper-reactive condition of the skin secondary to external factors, without objective signs of lesion. Its pathogenesis is still under investigation. Transient receptor potential vanilloid-1 (TRPV1) is a cation channel that responds to low pH and is related to nociception, neurogenic inflammation, and pruritus. AIMS: To determine the expression of TRPV1 in subjects with SS and correlate it with the degree of symptoms and skin pigmentation. PATIENTS/METHODS: We included 31 subjects self-diagnosed as having SS. Colorimetric values were obtained for assessment of skin phototype, and the lactic acid stinging test (LAST) was performed. Two skin biopsies from the nasolabial fold of each volunteer were obtained. Qualitative analysis of TRPV1 was carried out with immunohistochemistry. Quantitative analysis of TRPV1 was carried out with qRT-PCR. RESULTS: LAST was positive in 74% of the subjects, 56% of those having tan and brown skin. Immunohistochemistry staining for TRPV1 was greater in positive subjects (P = 0.03), but showed no correlation with the intensity of symptoms. Positive subjects also had higher TRPV1 mRNA expression compared to negative subjects (P < 0.001). This expression showed a positive correlation with the intensity of referred symptoms (R = 0.75, P < 0.001) and skin pigmentation (R = 0.63, P < 0.001). CONCLUSIONS: TRPV1 expression is upregulated in subjects with sensitive skin, and it correlates with the intensity of the symptoms. Our findings suggest a role for this receptor in the pathogenesis of sensitive skin syndrome.

P2X2 and P2X5 Receptors Mediate Bladder Hyperesthesia in ICC in Female Overactive Bladder.

This study was set to explore the role of P2X2 and P2X5 as the important molecules in sensory afferent of bladder in female overactive bladder (OAB) patients with the bladder hyperesthesia. Sixty-eight OAB patients admitted in Southwest Hospital affiliated to the Third Military Medical University during September, 2011-December, 2012 were selected and included in the experimental group (OAB group) and 30 healthy volunteers during the same period were included as the control group. We recorded voiding diary and urodynamic results, and immunohistochemistry analysis was used to detect P2X2 and P2X5 receptor in interstitial cell of Caja (ICC) in bladder tissue of female OAB patients and healthy volunteers, to tentatively explore the effect of P2X2 and P2X5 in bladder hyperesthesia. Urodynamic study has important diagnostic value in the diagnosis and differential diagnosis of OAB. P2X2 receptor was significantly up-regulated in bladder ICC in OAB group. The blockage of P2X2 receptor could significantly inhibit the contraction of bladder muscle strips, decrease the bladder pressure and the electric discharge of pelvic nerve. PET and urodynamic study showed that micturition desire sense in PAG area of pons in OAB patients was significantly increased compared with the control group. The up-regulation of P2X2 in ICC is an important factor to cause bladder hyperesthesia in OAB patients. PET and urodynamic study indicate that the bladder-originated nervous impulses are important cause of OAB. This study provides a basis for the study of P2X2 receptor in ICC in bladder hyperesthesia of OAB patients.

Systematic identification of proteins that elicit drug side effects.

Side effect similarities of drugs have recently been employed to predict new drug targets, and networks of side effects and targets have been used to better understand the mechanism of action of drugs. Here, we report a large-scale analysis to systematically predict and characterize proteins that cause drug side effects. We integrated phenotypic data obtained during clinical trials with known drug-target relations to identify overrepresented protein-side effect combinations. Using independent data, we confirm that most of these overrepresentations point to proteins which, when perturbed, cause side effects. Of 1428 side effects studied, 732 were predicted to be predominantly caused by individual proteins, at least 137 of them backed by existing pharmacological or phenotypic data. We prove this concept in vivo by confirming our prediction that activation of the serotonin 7 receptor (HTR7) is responsible for hyperesthesia in mice, which, in turn, can be prevented by a drug that selectively inhibits HTR7. Taken together, we show that a large fraction of complex drug side effects are mediated by individual proteins and create a reference for such relations.

Visceral hypersensitivity in symptomatic diverticular disease and the role of neuropeptides and low grade inflammation.

BACKGROUND: Recurrent abdominal pain is reported by a third of patients with diverticulosis, particularly those with previous episodes of acute diverticulitis. The current understanding of the etiology of this pain is poor. Our aim was to assess visceral sensitivity in patients with diverticular disease and its association with markers of previous inflammation and neuropeptides. METHODS: Patients with asymptomatic and symptomatic diverticular disease underwent a flexible sigmoidoscopy and biopsy followed 5-10 days later by visceral sensitivity testing with barostat-mediated rectal distension. Inflammation was assessed by staining of serotonin (5HT) and CD3 positive cells. mRNA levels of tumor necrosis factor alpha (TNF α) and interleukin-6 (IL-6) were quantitated using RT-PCR. Neuropeptide expression was assessed from percentage area staining with substance P (SP) and mRNA levels of the neurokinin 1 & 2 receptors (NK1 & NK2), and galanin 1 receptor (GALR1). KEY RESULTS: Thirteen asymptomatic and 12 symptomatic patients were recruited. The symptomatic patients had a lower first reported threshold to pain (28.4 mmHg i.q.r 25.0-36.0) than the asymptomatic patients (47 mmHg i.q.r 36.0-52.5, P < 0.001). Symptomatic patients had a higher median overall pain rating for the stimuli than the asymptomatic patients (P < 0.02). Symptomatic patients had greater median relative expression of NK1 and TNF alpha mRNA compared with asymptomatic patients. There was a significant correlation between barostat VAS pain scores and NK 1 expression (Figure 4, r(2) 0.54, P < 0.02). CONCLUSIONS & INFERENCES: Patients with symptomatic diverticular disease exhibit visceral hypersensitivity, and this may be mediated by ongoing low grade inflammation and upregulation of tachykinins.

Hypersensitive glutamate signaling correlates with the development of late-onset behavioral morbidity in diffuse brain-injured circuitry.

In diffuse brain-injured rats, robust sensory sensitivity to manual whisker stimulation develops over 1 month post-injury, comparable to agitation expressed by brain-injured individuals with overstimulation. In the rat, whisker somatosensation relies on thalamocortical glutamatergic relays between the ventral posterior medial (VPM) thalamus and barrel fields of somatosensory cortex (S1BF). Using novel glutamate-selective microelectrode arrays coupled to amperometry, we test the hypothesis that disrupted glutamatergic neurotransmission underlies the whisker sensory sensitivity associated with diffuse brain injury. We report hypersensitive glutamate neurotransmission that parallels and correlates with the development of post-traumatic sensory sensitivity. Hypersensitivity is demonstrated by significant 110% increases in VPM extracellular glutamate levels, and 100% increase in potassium-evoked glutamate release in the VPM and S1BF, with no change in glutamate clearance. Further, evoked glutamate release showed 50% greater sensitivity to a calcium channel antagonist in brain-injured over uninjured VPM. In conjunction with no changes in glutamate transporter gene expression and exogenous glutamate clearance efficiency, these data support a presynaptic origin for enduring post-traumatic circuit alterations. In the anatomically-distinct whisker circuit, the injury-induced functional alterations correlate with the development of late-onset behavioral morbidity. Effective therapies to modulate presynaptic glutamate function in diffuse-injured circuits may translate into improvements in essential brain function and behavioral performance in other brain-injured circuits in rodents and in humans.

Hallux interfalándico patológico e incidencia de los distintos grados de onicocriptosis / Pathologic interphalangeal hallux and the incidence of the different stafes of onycochryptosis

El objetivo de este estudio es demostrar la correlación existente entre la patología de Onicocriptosis y la presencia de Hallux interfalángico patológico. Algunos conceptos claves que debemos manejar son el de Hallux Abductus interfalángico del primer dedo y Onicocriptosis, cuya etiología es muy variada. La muestra del estudio está formada por un total de 312 pies pertenecientes a 156 sujetos, de los cuales son 189 mujeres y 123 hombres. El tipo de estudio diseñado es un estudio transversal, analítico, observacional y de caso y control. Los resultados del valor medio de HIF del grupo de estudio son de 17,39“. El valor medio de HIF del grupo control es de 13, 47“. Nuestros resultados muestran que en el grupo de estudio el 73,71% presentan valores de HIF por encima del valor de 13,47“. El valor de HIF no influye en el mayor o menor grado de afectación de onicocriptosis. La mayoría de los pacientes, casi el 60% presentan onicocriptosis de grade 1. Y la mayoría de los casos presentan afectación de los bordes tibial y peroneal. La afectación es mayor en mujeres que en hombres siendo de más del doble para la afectación del borde tibial y peroneal. Como conclusiones de este estudio podemos extraer que existe un alto grado de correlación entre la presencia de HIF patológico y Onicocriptosis. Los pacientes con onicocriptosis tienen un ángulo interfalángico significativamente superior a aquellos que no lo padecen. Los grados de Onicocriptosis más frecuentes son los estadios 1° y 2°. E1 valor medio del H.I.F de este estudio es de 13,47“. No existen resullados significativos para la variables edad y sexo de la muestra. El valor dc HIF no influye en el mayor 0 menor grado de afectación de onicocriptosis (AU)

The aim of this study is to prove the possible relation between onycochryptosis and the pathologic interphalangeal Hallux. Some of the key concepts that we must take into account are interphalangeal Hallux Abductus of the first toe and onycochryptosis, whose etiology is very wide. The sample of this study is 312 feet belonging to 156 subjects, of which 189 are women and 123 men. We conducted a transverse, analytical, observational, case-control study. The mean value of the PIH of the study group is 17,39“ whereas, the mean of the control group is 13, 47". Our results show that in the study group, 73,71% present a PIH value higher than 13,47”. PIH value does not influence the major or minor degree of affectation of onycochryptosis. Most of the patients, almost 60% present onycochryptosis of stage 1. And the majority of the cases present affectation of the tibial and peroneal edges. The affectation is higher in women than men, being more than twice the affectation of the tibial and peroneal edge. As conclusions, we can state that there is a high degree of correlation between the presence of PIH and onycochryptosis. Patients with onycochryptosis have an interphalangeal angle significantly superior to those who do not suffer this condition. The most frequent degrees of onycochryptosis are stage 1 and 2. The mean value of the PIH of this study is 13,47“. We could not find significant results related to age and sex. PIH Value does not influence the major or minor degree of affectation (AU)

Key role for spinal dorsal horn microglial kinin B1 receptor in early diabetic pain neuropathy.

BACKGROUND: The pro-nociceptive kinin B1 receptor (B1R) is upregulated on sensory C-fibres, astrocytes and microglia in the spinal cord of streptozotocin (STZ)-diabetic rat. This study aims at defining the role of microglial kinin B1R in diabetic pain neuropathy. METHODS: Sprague-Dawley rats were made diabetic with STZ (65 mg/kg, i.p.), and 4 days later, two specific inhibitors of microglial cells (fluorocitrate, 1 nmol, i.t.; minocycline, 10 mg/kg, i.p.) were administered to assess the impact on thermal hyperalgesia, allodynia and mRNA expression (qRT-PCR) of B1R and pro-inflammatory markers. Spinal B1R binding sites ((125I)-HPP-desArg10-Hoe 140) were also measured by quantitative autoradiography. Inhibition of microglia was confirmed by confocal microscopy with the specific marker Iba-1. Effects of intrathecal and/or systemic administration of B1R agonist (des-Arg9-BK) and antagonists (SSR240612 and R-715) were measured on neuropathic pain manifestations. RESULTS: STZ-diabetic rats displayed significant tactile and cold allodynia compared with control rats. Intrathecal or peripheral blockade of B1R or inhibition of microglia reversed time-dependently tactile and cold allodynia in diabetic rats without affecting basal values in control rats. Microglia inhibition also abolished thermal hyperalgesia and the enhanced allodynia induced by intrathecal des-Arg9-BK without affecting hyperglycemia in STZ rats. The enhanced mRNA expression (B1R, IL-1beta, TNF-alpha, TRPV1) and Iba-1 immunoreactivity in the STZ spinal cord were normalized by fluorocitrate or minocycline, yet B1R binding sites were reduced by 38%. CONCLUSION: The upregulation of kinin B1R in spinal dorsal horn microglia by pro-inflammatory cytokines is proposed as a crucial mechanism in early pain neuropathy in STZ-diabetic rats.

Allodynia in migraine: frequent random association or unavoidable consequence?

Allodynia, the perception of pain induced by a non-painful stimulus, is frequently associated with migraine, especially when chronic, and mainly in the aura subtype. Among migraineurs, allodynia is thought to be caused by the headache and the activation of nociceptors with the development of central sensitization in subjects with an altered regulation of the central nociceptive pathway. The persistence of pain sensation seems to be able to induce central sensitization in the caudal nucleus of the trigeminal nerve by lowering the neuronal pain threshold. Different pathogenetic mechanisms may be involved and genetic, environmental and psychological elements should be considered. The complaint of allodynia is more frequent during the headache attack (acute allodynia) than in-between attacks (interictal allodynia). Acute allodynia is generally referred to the painful region but may diffuse to other areas, cephalic or even extracephalic. Extracephalic allodynia could not be mediated by nucleus caudalis as its neurons do not express whole-body receptive fields. The likely mechanism is thalamic sensitization. This symptom must be carefully assessed because it may be as annoying and limiting in daily activities as pain itself, and because its presence seems to reduce the efficacy of drugs used for migraine attacks. Instrumental measures may be applied, and clinical questionnaires to assess the presence of allodynic symptoms have also been developed and validated. All these aspects will be discussed.

TRPV1 receptor in expression of opioid-induced hyperalgesia.

UNLABELLED: Opiates are currently the mainstay for treatment of moderate to severe pain. However, prolonged administration of opiates has been reported to elicit hyperalgesia in animals, and examples of opiate-induced hyperalgesia have been reported in humans as well. Despite the potential clinical significance of such opiate-induced actions, the mechanisms of opiate-induced hypersensitivity remain unknown. The transient receptor potential vanilloid1 (TRPV1) receptor, a molecular sensor of noxious heat, acts as an integrator of multiple forms of noxious stimuli and plays an important role in the development of inflammation-induced hyperalgesia. Because animals treated with opiates show thermal hyperalgesia, we examined the possible role of TRPV1 receptors in the development of morphine-induced hyperalgesia using TRPV1 wild-type (WT) and knock-out (KO) mice and with administration of a TRPV1 antagonist in mice and rats. Administration of morphine by subcutaneous implantation of morphine pellets elicited both thermal and tactile hypersensitivity in TRPV1 WT mice but not in TRPV1 KO mice. Moreover, oral administration of a TRPV1 antagonist reversed both thermal and tactile hypersensitivity induced by sustained morphine administration in mice and rats. Immunohistochemical analyses indicate that sustained morphine administration modestly increases TRPV1 labeling in the dorsal root ganglia. In addition, sustained morphine increased flinching and plasma extravasation after peripheral stimulation with capsaicin, suggesting an increase in TRPV1 receptor function in the periphery in morphine-treated animals. Collectively, our data indicate that the TRPV1 receptor is an essential peripheral mechanism in expression of morphine-induced hyperalgesia. PERSPECTIVE: Opioid-induced hyperalgesia possibly limits the usefulness of opioids, emphasizing the value of alternative methods of pain control. We demonstrate that TRPV1 channels play an important role in peripheral mechanisms of opioid-induced hyperalgesia. Such information may lead to the discovery of analgesics lacking such adaptations and improving treatment of chronic pain.

Serotonin 5-HT2A receptor involvement and Fos expression at the spinal level in vincristine-induced neuropathy in the rat.

We recently showed that peripheral and spinal 5-HT2A receptors (5-HT2AR) are involved in a rodent model of neuropathy induced by a nucleoside analogue reverse transcriptase inhibitor. In this paper, we show that 5-HT2AR are also involved in neuropathy induced by an anti-neoplasic drug, vincristine. Vincristine-treated rats (0.1mg/kg, daily i.p. administration for two 5-day cycles) developed thermal allodynia and mechanical hypersensitivity, which decreased in a dose-related manner after epidural injection a 5-HT2A receptor antagonist. Moreover, 5-HT2A-/- mice did not develop vincristine-induced neuropathy contrarily to their 5-HT2A+/+ littermates. In vincristine-treated rats, the number of nociceptive dorsal root ganglion cells expressing the 5-HT2AR was increased by 38%, and 5-HT2AR immunolabelling was enhanced in layers I-IV of the dorsal horn. At the EM level, a 76.3% increase in the density of 5-HT2AR immunopositive axon terminals within superficial layers of the dorsal horn was noted after vincristine treatment. Immunocytochemical study of Fos expression in vincristine-treated rats revealed a significant increase in the number of Fos-positive neurons not only in regions where nociceptive fibres terminate superficial (I-II) and deep layers (V-VI) of the spinal cord, but also in intermediate layers, suggesting that Abeta fibres could be involved in the spinal sensitization observed in this model. Double labelling experiments showed that Fos-positive neurons were endowed with 5-HT2AR immunolabelling in the dorsal horn of vincristine-treated rats. These data provide support to the idea that, in vincristine-induced neuropathy, 5-HT2AR are involved in the sensitization of peripheral nociceptors and spinal nociceptive processing.

Extracellular signal-regulated kinases mediate melittin-induced hypersensitivity of spinal neurons to chemical and thermal but not mechanical stimuli.

Subcutaneous melittin injection causes central plasticity at the spinal level in wide-dynamic-range (WDR) neurons, which are hypersensitive to various nociceptive stimuli. Previous behavioral studies demonstrated that the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase 1/2(ERK1/2), p38 MAPK, and c-Jun N-terminal kinase are involved in both peripheral and spinal processing of melittin-induced nociception and hypersensitivity. Yet the functional roles of the three MAPKs vary among different stimulus modalities, and must be further studied at the cellular level in vivo. In this report, extracellular single unit recordings were performed to investigate whether activation of ERK1/2 in the primary injury site of melittin is essential to the establishment of a spinally sensitized state. Localized peripheral administration of a single dose of the MEK inhibitor U0126 (1 µg/10 µl) significantly suppressed neuronal hyper-responsiveness to thermal stimulus and chemical (melittin)-induced tonic firing of WDR neurons after full establishment of a spinally sensitized state. However, U0126 failed to affect mechanical hypersensitivity to both noxious and non-noxious stimuli. Melittin-induced enhancement of thermal hypersensitivity was also greatly inhibited by a single dose of capsazepine, a thermal nociceptor (TRPV1) blocker. These results suggest that activation of the ERK signaling pathway in the periphery is likely necessary for maintenance of a spinally sensitized state; activation of ERK1/2 in the primary injury site may regulate TRPV1, leading to dorsal horn hypersensitivity to thermal and chemical stimuli. ERK signaling pathways are not likely to be associated with melittin-induced dorsal horn hypersensitivity to mechanical stimuli.

Allodynia and hyperalgesia in diabetic rats are mediated by GABA and depletion of spinal potassium-chloride co-transporters.

Diabetic rats show behavioral indices of painful neuropathy that may model the human condition. Hyperalgesia during the formalin test in diabetic rats is accompanied by the apparently paradoxical decrease in spinal release of excitatory neurotransmitters and increase in the inhibitory neurotransmitter GABA. Decreased expression of the potassium-chloride co-transporter, KCC2, in the spinal cord promotes excitatory properties of GABA. We therefore measured spinal KCC2 expression and explored the role of the GABA(A) receptor in rats with painful diabetic neuropathy. KCC2 protein levels were significantly reduced in the spinal cord of diabetic rats, while levels of NKCC1 and the GABA(A) receptor were unchanged. Spinal delivery of the GABA(A) receptor antagonist bicuculline reduced formalin-evoked flinching in diabetic rats and also dose-dependently alleviated tactile allodynia. GABA(A) receptor-mediated rate-dependent depression of the spinal H reflex was absent in the spinal cord of diabetic rats. Control rats treated with the KCC2 blocker DIOA, mimicked diabetes by showing increased formalin-evoked flinching and diminished rate- dependent depression. The ability of bicuculline to alleviate allodynia and formalin-evoked hyperalgesia in diabetic rats is consistent with a reversal of the properties of GABA predicted by reduced spinal KCC2 and suggests that reduced KCC2 expression and increased GABA release contribute to spinally mediated hyperalgesia in diabetes.

Comparison of icilin- and cold-evoked responses of spinal neurones, and their modulation of mechanical activity, in a model of neuropathic pain.

Cold allodynia is a poorly understood symptom of neuropathic pain. Two members of the transient receptor potential (TRP) family of proteins, TRPM8 and TRPA1, may contribute to cold somatosensation. The aim of the present study was to investigate the usefulness of icilin as a pharmacological tool to study primary afferent fibre responses to cold stimuli and to determine whether there are differences in the responses of spinal neurones to cooling of peripheral receptive fields in control versus neuropathic rats. The effects of icilin, a TRPM8 and TRPA1 agonist, on intracellular Ca(2+) ([Ca(2+)](i)) responses of small diameter adult dorsal root ganglia (DRG) neurones were determined. Icilin (10 nM-10 microM) produced a concentration-related increase in [Ca(2+)](i) in DRG neurones, which was attenuated by the non-selective TRP channel antagonist ruthenium red (10 microM). In vivo electrophysiology in naïve, sham-operated and SNL rats demonstrated that application of ice to receptive fields evoked firing of wide dynamic range (WDR) neurones, which was significantly greater in SNL rats than naïve and sham-operated rats. Intraplantar injection of icilin did not evoke firing of WDR neurones in naïve, sham-operated or SNL rats but inhibited mechanically-evoked responses of WDR neurones in naïve and sham-operated rats, whilst facilitating mechanically-evoked responses in SNL rats. Icilin increased both innocuous (sham-operated and SNL rats) and noxious (SNL rats) receptive field sizes of WDR neurones. Our data suggests that icilin modulates the mechanosensitivity of dorsal horn neurones. The differing effects of ice and icilin on dorsal horn neurones indicate different mechanisms of action.

Toll-like receptor 3 contributes to spinal glial activation and tactile allodynia after nerve injury.

Toll-like receptors (TLRs) play an essential role in innate immune responses and in the initiation of adaptive immune responses. Microglia, the resident innate immune cells in the CNS, express TLRs. In this study, we show that TLR3 is crucial for spinal cord glial activation and tactile allodynia after peripheral nerve injury. Intrathecal administration of TLR3 antisense oligodeoxynucleotide suppressed nerve injury-induced tactile allodynia, and decreased the phosphorylation of p38 mitogen-activated protein kinase, but not extracellular signal-regulated protein kinases 1/2, in spinal glial cells. Antisense knockdown of TLR3 also attenuated the activation of spinal microglia, but not astrocytes, caused by nerve injury. Furthermore, down-regulation of TLR3 inhibited nerve injury-induced up-regulation of spinal pro-inflammatory cytokines, such as interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha. Conversely, intrathecal injection of the TLR3 agonist polyinosine-polycytidylic acid induced behavioral, morphological, and biochemical changes similar to those observed after nerve injury. Indeed, TLR3-deficient mice did not develop tactile allodynia after nerve injury or polyinosine-polycytidylic acid injection. Our results indicate that TLR3 has a substantial role in the activation of spinal glial cells and the development of tactile allodynia after nerve injury. Thus, blocking TLR3 in the spinal glial cells might provide a fruitful strategy for treating neuropathic pain.

A role for G protein-coupled receptor kinase 2 in mechanical allodynia.

Inflammation and nerve injury can both induce mechanical allodynia via mechanisms involving the production of pro-inflammatory cytokines and increased neuronal activity. Many neurotransmitters involved in pain signal via G protein-coupled receptors (GPCRs). GPCR kinase (GRK)2 is a member of the GRK family that regulates agonist-induced desensitization and signalling of GPCRs. Low intracellular GRK2 levels are associated with increased receptor signalling. The aim of this study was to investigate whether mechanical allodynia is associated with decreased spinal cord GRK2 expression and whether reduced GRK2 increases inflammation-induced mechanical allodynia. Mechanical allodynia was induced in rats by chronic constriction injury of the sciatic nerve. After 2 weeks, neuronal GRK2 expression was decreased bilaterally in the superficial layers of the lumbar spinal cord dorsal horn. Moreover, interleukin-1beta significantly reduced GRK2 expression ex vivo in spinal cord slices. To investigate whether reduced GRK2 potentiates inflammation-induced mechanical allodynia, we used GRK2(+/-) animals expressing decreased GRK2. At baseline, the threshold for mechanical stimulation did not differ between GRK2(+/-) and wild-type mice. However, GRK2(+/-) animals were more sensitive to mechanical stimulation than wild-type animals after intraplantar lambda-carrageenan injection. We propose cytokine-induced down-regulation of spinal cord neuronal GRK2 expression as a novel mechanism that contributes to increased neuronal signalling in mechanical allodynia.

Anti-allodynic effects of peripheral delta opioid receptors in neuropathic pain.

The analgesic effects of local administration of opioid agonists into peripheral tissues in alleviating pain have been well documented in both clinical and preclinical studies, although few studies have examined their effects in neuropathic pain. In this study, we investigated the anti-allodynic effects of peripherally acting delta opioid receptor (DOR) agonists in a rat model of neuropathic pain. Peripheral nerve injury (PNI) produced a time-dependent decrease in mechanical withdrawal thresholds that was attenuated by local administration into the hind paw of either morphine or the DOR agonist deltorphin II. Using Western blotting techniques, no change in DOR protein expression was detected in DRG ipsilateral to the site of injury compared to contralateral. However, an up-regulation of DOR protein was found in neuropathic DRG compared to sham, suggesting that there may be a bilateral increase in the expression of DOR following PNI. Results obtained from immunohistochemical studies confirmed up-regulation in small and large DRG neurons in neuropathic compared to sham animals. Additionally, there was an increase in DOR protein within the ipsilateral sciatic nerve of neuropathic animals compared to sham and contralateral neuropathic conditions indicating the occurrence of receptor trafficking to the site of injury. Taken together, our findings suggest that functional peripheral DORs are present in sensory neurons following PNI and validate the development of selective DOR agonists for alleviating neuropathic pain.