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.

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.

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.

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.

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.

Participation of peripheral group I and II metabotropic glutamate receptors in the development or maintenance of IL-1beta-induced mechanical allodynia in the orofacial area of conscious rats.

The present study investigated the role of peripheral groups I and II metabotropic glutamate receptors (mGluRs) in interleukin (IL)-1beta-induced mechanical allodynia in the orofacial area of rats. Subcutaneous injection of 10 pg of IL-1beta decreased air-puff thresholds ipsilateral or contralateral to the injection site. The decrease in air-puff thresholds appeared 10 min after the injection of IL-1beta and IL-1beta-induced mechanical allodynia persisted for over 3 h. Pre-treatment with 7-(hydroxyimino) cyclopropa[b] chromen-1a-carboxylate ethyl ester (CPCCOEt) or 2-methyl-6-(phenylethynyl)-pyridine hydrochloride (MPEP), a mGluR1 or mGluR5 antagonist, blocked IL-1beta-induced mechanical allodynia and mirror-image mechanical allodynia produced by a subcutaneous injection of 10 pg of IL-1beta. However, post-treatment with CPCCOEt or MPEP did not affect changes in behavioral responses, which were produced by the IL-1beta injection. Pre-treatment, as well as post-treatment with (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate (APDC), a group II mGluR agonist, blocked either IL-1beta-induced mechanical allodynia or mirror-image mechanical allodynia. The anti-allodynic effects of APDC were abolished by pre-treatment with (2S)-2-amino-2[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid (LY341495), a group II mGluR antagonist. These results indicate that peripheral group II mGluRs are involved in the development and maintenance of IL-1beta-induced mechanical allodynia, while peripheral group I mGluRs are involved in the development of IL-1beta-induced mechanical allodynia. Based on our observations, the peripheral application of group II mGluR agonists may be of therapeutic value in treating inflammatory pain.

Intracellular messengers involved in spontaneous pain, heat hyperalgesia, and mechanical allodynia induced by intrathecal dihydroxyphenylglycine.

We investigated the role of two intracellular second messengers, extracellular signal-regulated protein kinase (ERK) and protein kinase C (PKC), in a model of persistent pain using intrathecal (i.t.) (R,S)-3,5-dihydroxyphenylglycine (DHPG). Spontaneous nociceptive behaviours (SNBs), mechanical allodynia (von Frey thresholds) and heat hyperalgesia (plantar test latencies) induced by DHPG were measured in animals pretreated i.t. with membrane permeable inhibitors of ERK (PD 98059) and PKC (GF 109203X). Spinal administration of PD 98059 dose-dependently reduced SNBs, and attenuated both mechanical allodynia and heat hyperalgesia induced by DHPG. GF 109203X treatment also reduced SNBs and heat hyperalgesia, but did not affect mechanical allodynia induced by DHPG. Neither PD 98059, nor GF 109203X, altered mechanical or thermal thresholds in saline-injected control rats. These results suggest that both ERK and PKC are involved in persistent pain associated with the i.t. administration of DHPG.

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.

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.

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.

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.

Pronociceptive actions of dynorphin maintain chronic neuropathic pain.

Whereas tissue injury increases spinal dynorphin expression, the functional relevance of this upregulation to persistent pain is unknown. Here, mice lacking the prodynorphin gene were studied for sensitivity to non-noxious and noxious stimuli, before and after induction of experimental neuropathic pain. Prodynorphin knock-out (KO) mice had normal responses to acute non-noxious stimuli and a mild increased sensitivity to some noxious stimuli. After spinal nerve ligation (SNL), both wild-type (WT) and KO mice demonstrated decreased thresholds to innocuous mechanical and to noxious thermal stimuli, indicating that dynorphin is not required for initiation of neuropathic pain. However, whereas neuropathic pain was sustained in WT mice, KO mice showed a return to baselines by post-SNL day 10. In WT mice, SNL upregulated lumbar dynorphin content on day 10, but not day 2, after injury. Intrathecal dynorphin antiserum reversed neuropathic pain in WT mice at post-SNL day 10 (when dynorphin was upregulated) but not on post-SNL day 2; intrathecal MK-801 reversed SNL-pain at both times. Opioid (mu, delta, and kappa) receptor density and G-protein activation were not different between WT and KO mice and were unchanged by SNL injury. The observations suggest (1) an early, dynorphin-independent phase of neuropathic pain and a later dynorphin-dependent stage, (2) that upregulated spinal dynorphin is pronociceptive and required for the maintenance of persistent neuropathic pain, and (3) that processes required for the initiation and the maintenance of the neuropathic pain state are distinct. Identification of mechanisms that maintain neuropathic pain appears important for strategies to treat neuropathic pain.

Mechanisms underlying enhanced P2X receptor-mediated responses in the neuropathic pain state.

P2X3 and P2X2/3 receptors in dorsal root ganglia (DRG) appear to participate in producing nociceptive responses after nerve injury. However, the mechanisms underlying the receptor-mediated nociception in the neuropathic state remain unclear. Using spared nerve injury (SNI) rats, we found that allodynic and nocifensive (flinch) behavioral responses developed after injury can be reversed by P2X receptor antagonists, indicating an involvement of P2X receptors. Immunocytochemical studies revealed that P2X3 receptors are expressed in small and medium but rarely in large DRG neurons of both normal and SNI rats. Thus, contrary to the conventional view that only large A beta cells mediate allodynia, small and medium cells are intimately involved in P2X3 receptor-mediated allodynia. Measuring ATP levels in the subcutaneous space of the rat paw, we showed that ATP release does not change after SNI. On the other hand, the P2X receptor agonist, alpha beta-methylene ATP produces 3.5-fold larger flinch responses at a 8.0-fold lower dose. Thus, sensitization of P2X3 receptors rather than a change in ATP release is responsible for the neuropathic pain behaviors. We further demonstrated that sensitization of P2X3 receptors arises from an increase in receptor function. ATP-induced P2X3 receptor-mediated currents in DRG neurons is 2.5-fold larger after SNI. The expression of P2X3 receptors on the cell membrane is significantly enhanced while the total expression of P2X3 receptors remained unchanged. Thus, the enhancement of trafficking of P2X3 receptors is likely an important mechanism contributing to the increase in receptor function after nerve injury.

Excitatory amino acid concentrations increase in the spinal cord dorsal horn after repeated intramuscular injection of acidic saline.

Chronic muscle pain is common and often difficult to treat. In this study, we further characterize a model of chronic muscle pain induced by repeated intramuscular injection of acidic saline. Two injections of acid into muscle separated by 5 days result in secondary mechanical hyperalgesia that lasts for up to 4 weeks. Blockade of spinal NMDA receptors prior to the second injection intramuscular acid injection delays the onset of hyperalgesia, where as the maintenance phase of hyperalgesia, evaluated 1 week after the second intramuscular injection, is dependent on activation of spinal AMPA/kainate and NMDA receptors. In order to determine if behavioral hyperalgesia and glutamate receptor involvement are associated with increased concentrations of excitatory amino acids (EAA), we utilized microdialysis to evaluate extracellular glutamate and aspartate concentrations in the spinal dorsal horn during the first and second intramuscular acid injections, and 1 week after the development of mechanical hyperalgesia. The second intramuscular injection evoked a calcium-dependent increase in both spinal glutamate and aspartate concentrations. Glutamate concentrations within the dorsal horn were also increased 1 week after the second acid injection. Our data suggest increased release of spinal EAAs in the dorsal horn contributes to the development and maintenance of hyperalgesia.

Minocycline attenuates mechanical allodynia and proinflammatory cytokine expression in rat models of pain facilitation.

Activated glial cells (microglia and astroglia) in the spinal cord play a major role in mediating enhanced pain states by releasing proinflammatory cytokines and other substances thought to facilitate pain transmission. In the present study, we report that intrathecal administration of minocycline, a selective inhibitor of microglial cell activation, inhibits low threshold mechanical allodynia, as measured by the von Frey test, in two models of pain facilitation. In a rat model of neuropathic pain induced by sciatic nerve inflammation (sciatic inflammatory neuropathy, SIN), minocycline delayed the induction of allodynia in both acute and persistent paradigms. Moreover, minocycline was able to attenuate established SIN-induced allodynia 1 day, but not 1 week later, suggesting a limited role of microglial activation in more perseverative pain states. Our data are consistent with a crucial role for microglial cells in initiating, rather than maintaining, enhanced pain responses. In a model of spinal immune activation by intrathecal HIV-1 gp120, we show that the anti-allodynic effects of minocycline are associated with decreased microglial activation, attenuated mRNA expression of interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), IL-1beta-converting enzyme, TNF-alpha-converting enzyme, IL-1 receptor antagonist and IL-10 in lumbar dorsal spinal cord, and reduced IL-1beta and TNF-alpha levels in the CSF. In contrast, no significant effects of minocycline were observed on gp120-induced IL-6 and cyclooxygenase-2 expression in spinal cord or CSF IL-6 levels. Taken together these data highlight the importance of microglial activation in the development of exaggerated pain states.

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.

Pharmacological modulation of eicosanoid levels and hyperalgesia in yeast-induced inflammation.

Injection of brewer's yeast into the rat paw results in edema and a subsequent hyperalgesia. The edema was accompanied by an increase in 5-lipoxygenase products, and the hyperalgesia coincided with the formation of both cyclooxygenase and 5-lipoxygenase products. When administered perorally, indomethacin inhibited cyclooxygenase product formation, phenidone inhibited 5-lipoxygenase product formation, and 3-amino-1-(m-[trifluoromethyl]-phenyl)-2-pyrazoline (BW 755C) inhibited formation of products of both pathways. These compounds were also effective analgesic agents. The correlation of these effects with the suppression of hyperalgesia suggests the participation of products from both cyclooxygenase and 5-lipoxygenase pathways in the mediation of hyperalgesia.

Antinociceptive effect of intrathecally administered antiserum against calcitonin gene-related peptide on thermal and mechanical noxious stimuli in experimental hyperalgesic rats.

We compared the effects of intrathecal administration of antiserum against calcitonin gene-related peptide (CGRP) between thermo- and mechano-nociceptive responses, using experimental hyperalgesic rats. An intrathecal administration of anti-CGRP antiserum, but not antiserum absorbed by synthetic CGRP, normalized either adjuvant- or carrageenin-induced hyperalgesia both in the paw radiant heat and the paw pressure tests, with little effect on non-hyperalgesic paws. These results suggest that endogenous CGRP, probably present in primary afferents, promotes both thermo- and mechano-nociceptive transmission in the spinal dorsal horn, at least in the hyperalgesic states with inflammations.

Differential activation of spinal cord dynorphin and enkephalin neurons during hyperalgesia: evidence using cDNA hybridization.

A unilateral experimental inflammation of the hindlimb produces hyperalgesia to both mechanical and radiant thermal stimuli that is rapid in onset. During this period, parameters of dynorphin biosynthesis are elevated to a much greater degree than those of the enkephalin system. An increase in the content of the peptide dynorphin A(1-8) occurs in the spinal cord segments that receive sensory input from the affected limb. This is accompanied by a rapid (within 24 h) and pronounced increase in the levels of mRNA coding for the dynorphin protein precursor. Maximum elevations (6- to 8-fold) of preprodynorphin mRNA are observed between days 2 and 5 subsequent to the induction of inflammation. Compared to the increase in mRNA, the increase in dynorphin A(1-8) peptide was appreciably delayed and proportionately less; maximal increases in peptide (3-fold) were seen at day 5 of inflammation. Dorsal spinal cord preproenkephalin mRNA is elevated to a lesser degree (50-80%). However, the increase in preproenkephalin mRNA is apparently not enough to yield a measurable increase in the proenkephalin-derived peptide met5-enkephalin-Arg6-Gly7-Leu8, the levels of which showed no significant change during the 14-day inflammatory period. These data suggest the active participation of opioid neurons, especially those containing dynorphin, at the spinal level, in the modulation of sensory afferent input during peripheral inflammatory pain states.

Intrathecal injection of cAMP response element binding protein (CREB) antisense oligonucleotide attenuates tactile allodynia caused by partial sciatic nerve ligation.

The transcription factor cAMP responsive element binding protein (CREB) is important in regulating immediate-early genes and some late-effector genes involved in neuroplasticity in response to peripheral injury and stressful insults. Partial nerve injury elicited neuropathic pain is accompanied by increased phosphorylation of CREB in the ipsilateral spinal cord dorsal horn (Ma and Quirion, Pain 93 (2001) 295; Miletic et al., Pain 99 (2002) 493). The aim of this study is to determine whether increased phosphorylation of CREB in the dorsal horn contributes to the pathogenesis of neuropathic pain. Three weeks following partial sciatic nerve ligation (PSNL), daily intrathecal injection of antisense CREB oligodeoxynucleotide (ODN, 20 microg/day) for 5 days significantly attenuated tactile allodynia. The attenuation lasted for more than 4 days. Total CREB and phosphorylated CREB in both ipsilateral and contralateral dorsal horn neurons were dramatically reduced in antisense ODN injected PSNL rats 1 week after injection. The extent of reduction of total CREB and phosphorylated CREB containing cells in the dorsal horn ipsilateral to injury was greater than in the contralateral dorsal horn. These data suggest that phosphorylation of CREB is an important contributing event in the central plasticity of nerve injury and in the pathogenesis of neuropathic pain.