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Aims Insular cortex ( IC) is involved in processing the information of pain. The aim of this study was to investigate roles and mechanisms of P2X7 receptors (P2X7Rs) in IC in development of visceral hypersensitivity of adult rats with neonatal maternal deprivation ( NMD). Methods Visceral hypersensitivity was quantified by abdominal withdrawal reflex threshold to colorectal distension ( CRD). Expression of P2X7Rs was determined by qPCR and Western blot. Synaptic transmission in IC was recorded by patch-clamp recording. Results The expression of P2X7Rs and glutamatergic neurotransmission in IC was significantly increased in NMD rats when compared with age-matched controls. Application of Bz ATP (P2X7R agonist) enhanced the frequency of spontaneous excitatory postsynaptic currents ( sEPSC) and miniature excitatory postsynaptic currents ( mEPSC) in IC slices of control rats. Application of BBG (P2X7R antagonist) suppressed the frequencies of sEPSC and mEPSC in IC slices of NMD rats. Microinjection of Bz ATP into right IC significantly decreased CRD threshold in control rats while microinjection of BBG or A438079 into right IC greatly increased CRD threshold in NMD rats. Conclusion Data suggested that the enhanced activities of P2X7Rs in IC, likely through a presynaptic mechanism, contributed to visceral hypersensitivity of adult rats with NMD. [ABSTRACT FROM AUTHOR]

Background: Autism spectrum disorder (ASD) affects many children and juveniles. The pathogenesis of ASD is not well understood. Environmental factors may play important roles in the development of ASD. We examined a possible relationship of inflammatory pain in neonates and the development of ASD in juveniles.Methods: Acute inflammation pain was induced by 5 % formalin (5 μl/day) subcutaneous injection into two hindpaws of postnatal day 3 to 5 (P3-P5) rat pups. Western blot, immunohistochemical, and behavioral examinations were performed at different time points after the insult.Results: Formalin injection caused acute and chronic inflammatory responses including transient local edema, increased levels of inflammatory cytokines, TNF-α, and IL-1β in the blood as well as in the brain, and increased microglia in the brain. One day after the pain insult, there was significant cell death in the cortex and hippocampus. Two weeks later, although the hindpaw local reaction subsided, impaired axonal growth and demyelization were seen in the brain of P21 juvenile rats. The number of bromodeoxyuridine (BrdU) and doublecortin (DCX) double-positive cells in the hippocampal dentate gyrus of P21 rats was significantly lower than that in controls, indicating reduced neurogenesis. In the P21 rat's brain of the formalin group, the expression of autism-related gene neurexin 1 (NRXN1), fragile X mental retardation 1 (FMR1), and oxytocin was significantly downregulated, consistent with the gene alteration in ASD. Juvenile rats in the formalin group showed hyperalgesia, repetitive behaviors, abnormal locomotion, sleep disorder, and distinct deficits in social memory and social activities. These alterations in neuroinflammatory reactions, gene expression, and behaviors were more evident in male than in female rats. Importantly, an anti-inflammation treatment using indomethacin (10 mg/kg, i.p.) at the time of formalin injections suppressed inflammatory responses and neuronal cell death and prevented alterations in ASD-related genes and the development of abnormal behaviors.Conclusions: These novel observations indicate that severe inflammatory pain in neonates and persistent inflammatory reactions may predispose premature infants to development delays and psychiatric disorders including ASD. The prevention of pain stimuli and prompt treatments of inflammation during development appear vitally important in disrupting possible evolution of ASD syndromes. [ABSTRACT FROM AUTHOR]

Purpose: Low back pain and sciatica are the most common symptoms of patients with lumbar disc herniation (LDH). The pathophysiology of lumbocrural pain and sciatica is not fully understood. The aim of the present study was to define the membrane properties and activities of voltage-gated sodium channels of dorsal root ganglion (DRG) neurons in a rat model of LDH.Methods: LDH was established by transplantation of autologous nucleus pulposus (NP) to lumbar 5 and 6 spinal nerves (L5-L6 DRG) of adult male rats. Mechanical paw withdrawal threshold (PWT) and thermal paw withdrawal latency (PWL) were measured 1 day before and through 35 days after transplantation of NP. Changes in expression of VGSCs were determined by western blotting. L5-L6 DRGs neurons innervating the hindpaw were labeled with DiI and acutely dissociated for measuring excitability and sodium channel currents under whole-cell patch clamp configurations.Results: NP transplantation significantly reduced the PWT and PWL in association with a significant reduction in rheobase and an increase in numbers of action potentials evoked by 2X and 3X rheobase current stimulation. Voltage-gated sodium current density was significantly enhanced in L5-L6 DRG neurons from LDH rats. The inactivation curve showed a leftward shift in LDH rats while activation curve did not significantly alter. However, NP transplantation remarkably enhanced expression of NaV1.7 and NaV1.8 in L5-L6 DRGs but not in T10-12 DRGs.Conclusion: These data suggest that NP application produces pain-related behavior and potentiates sodium current density of DRG neurons, which is most likely mediated by enhanced expression of NaV1.7 and NaV1.8. [ABSTRACT FROM AUTHOR]

Painful diabetic neuropathy is a common complication of diabetes produced by mechanisms that as yet are incompletely defined. The aim of this study was to investigate the roles of nuclear factor-κB (NF-κB) in the regulation of purinergic receptor P2X ligand-gated ion channel 3 (P2X3R) plasticity in dorsal root ganglion (DRG) neurons of rats with painful diabetes. Here, we showed that hindpaw pain hypersensitivity in streptozocin-induced diabetic rats was attenuated by treatment with purinergic receptor antagonist suramin or A-317491. The expression and function of P2X3Rs was markedly enhanced in hindpaw-innervated DRG neurons in diabetic rats. The CpG (cytosine guanine dinucleotide) island in the p2x3r gene promoter region was significantly demethylated, and the expression of DNA methyltransferase 3b was remarkably downregulated in DRGs in diabetic rats. The binding ability of p65 (an active form of NF-κB) with the p2x3r gene promoter region and p65 expression were enhanced significantly in diabetes. The inhibition of p65 signaling using the NF-κB inhibitor pyrrolidine dithiocarbamate or recombinant lentiviral vectors designated as lentiviral vector-p65 small interfering RNA remarkably suppressed P2X3R activities and attenuated diabetic pain hypersensitivity. Insulin treatment significantly attenuated pain hypersensitivity and suppressed the expression of p65 and P2X3Rs. Our findings suggest that the p2x3r gene promoter DNA demethylation and enhanced interaction with p65 contributes to P2X3R sensitization and diabetic pain hypersensitivity. [ABSTRACT FROM AUTHOR]

Chronic visceral pain in patients with irritable bowel syndrome (IBS) has been difficult to treat effectively partially because its pathophysiology is not fully understood. Recent studies show that norepinephrine (NE) plays an important role in the development of visceral hypersensitivity. In this study, we designed to investigate the role of adrenergic signaling in visceral hypersensitivity induced by heterotypical intermittent stress (HIS). Abdominal withdrawal reflex scores (AWRs) used as visceral sensitivity were determined by measuring the visceromoter responses to colorectal distension. Colon-specific dorsal root ganglia neurons (DRGs) were labeled by injection of DiI into the colon wall and were acutely dissociated for whole-cell patch-clamp recordings. Blood plasma level of NE was measured using radioimmunoassay kits. The expression of β2-adrenoceptors was measured by western blotting. We showed that HIS-induced visceral hypersensitivity was attenuated by systemic administration of a β-adrenoceptor antagonist propranolol, in a dose-dependent manner, but not by a α-adrenoceptor antagonist phentolamine. Using specific β–adrenoceptor antagonists, HIS-induced visceral hypersensitivity was alleviated by β2 adrenoceptor antagonist but not by β1- or β3-adrenoceptor antagonist. Administration of a selective β2-adrenoceptor antagonist also normalized hyperexcitability of colon-innervating DRG neurons of HIS rats. Furthermore, administration of β-adrenoceptor antagonist suppressed sustained potassium current density (IK) without any alteration of fast-inactivating potassium current density (IA). Conversely, administration of NE enhanced the neuronal excitability and produced visceral hypersensitivity in healthy control rats, and blocked by β2-adrenoceptor antagonists. In addition, HIS significantly enhanced the NE concentration in the blood plasma but did not change the expression of β2-adrenoceptor in DRGs and the muscularis externa of the colon. The present study might provide a potential molecular target for therapy of visceral hypersensitivity in patents with IBS. [ABSTRACT FROM AUTHOR]

Patients with long-standing diabetes frequently demonstrate gastric hypersensitivity with an unknown mechanism. The present study was designed to investigate roles for nuclear factor-κB (NF-κB) and the endogenous H2S-producing enzyme cystathionine-β-synthetase (CBS) signaling pathways by examining cbs gene methylation status in adult rats with diabetes. Intraperitoneal injection of streptozotocin (STZ) produced gastric hypersensitivity in female rats in response to gastric balloon distention. Treatment with the CBS inhibitor aminooxyacetic acid significantly attenuated STZ-induced gastric hypersensitivity in a dose-dependent fashion. Aminooxyacetic acid treatment also reversed hyperexcitability of gastric-specific dorsal root ganglion (DRG) neurons labeled by the dye Dil in diabetic rats. Conversely, the H2S donor NaHS enhanced neuronal excitability of gastric DRG neurons. Expression of CBS and p65 were markedly enhanced in gastric DRGs in diabetic rats. Blockade of NF-κB signaling using pyrrolidine dithiocarbamate reversed the upregulation of CBS expression. Interestingly, STZ treatment led to a significant demethylation of CpG islands in the cbs gene promoter region, as determined by methylation-specific PCR and bisulfite sequencing. STZ treatment also remarkably downregulated the expression of DNA methyltransferase 3a and 3b. More importantly, STZ treatment significantly enhanced the ability of cbs to bind DNA at the p65 consensus site, as shown by chromatin immunoprecipitation assays. Our findings suggest that upregulation of cbs expression is attributed to cbs promoter DNA demethylation and p65 activation and that the enhanced interaction of the cbs gene and p65 contributes to gastric hypersensitivity in diabetes. This finding may guide the development and evaluation of new treatment modalities for patients with diabetic gastric hypersensitivity. [ABSTRACT FROM AUTHOR]

Aims To investigate whether electroacupuncture ( EA) produced analgesic effect and whether nuclear factor kappa B ( NF-κ B) and cystathionine β synthase ( CBS) involved in EA-mediated analgesia in painful diabetic neuropathy in rats. Methods Diabetes was induced by an intraperitoneal injection of streptozotocin ( STZ) in adult female rats. Mechanical pain threshold was measured by von Frey filaments. EA was applied at acupoint Zu- San- Li ( ST-36) in both hindlimbs. Western blot analysis was employed to detect changes in protein levels of NF-κB and CBS in spinal dorsal root ganglion ( DRGs). Results Mechanical allodynia was developed 2 weeks after STZ injection and lasted for another 4 weeks. STZ injection significantly enhanced expression of p65 and CBS in lumbar L4-6 DRGs when compared with age-matched controls. EA markedly attenuated mechanical allodynia. Importantly, EA treatment remarkably inhibited p65 and CBS expression in DRGs. Additionally, intrathecal injection of the p65 antagonist pyrrolidine dithiocarbamate attenuated mechanical allodynia and markedly inhibited CBS expression in DRGs in STZ rats. Conclusions These data indicate that EA produced an analgesic effect, which might be mediated at least in a part by inhibition of NF-κB signaling pathway in primary sensory neurons in rats with diabetes. [ABSTRACT FROM AUTHOR]

The tyrosine kinases of Src family play an important role in the central sensitization following peripheral inflammation. However, whether the Src family in the arcuate nucleus (ARC) of mediobasal hypothalamus is involved in central sensitization remains unknown. The aim of this study was to investigate the role and mechanisms of tyrosine kinases of Src family in N-methyl-d-aspartate (NMDA) receptor activity in the ARC following peripheral inflammation. Peripheral inflammation was induced by unilateral injection of complete Freund's adjuvant (CFA) into rat hindpaw. The neuronal activities of the ARC were recorded using electrophysiological field recording from the in vitro mediobasal hypothalamic slices from control and CFA rats. Expression of total and phosphorylated Src and NR2B subunit protein was analyzed by Western blot and immuoprecipitation. Our results showed that CFA injection resulted in an increase in mechanical and thermal sensitivity, which was partially blocked by neonatal monosodium glutamate treatment. CFA injection also enhanced spontaneous firings of ARC neurons, which were reversed by the NMDA receptor NR2B subunit specific antagonist Ro25–6981 and by PP2, an Src family tyrosine kinase inhibitor. In addition, peripheral inflammation enhanced Src phosphorylation and NMDA receptor NR2B subunit phosphorylation without alteration of total NR2B subunit expression in the ARC. Peripheral inflammation also increased the association of NR2B protein with p-Src protein in the ARC. Administration of PP2 blocked the upregulation of NR2B phosphorylation induced by CFA injection. Taken together, our present results suggest that the arcuate Src activation-induced tyrosine phosphorylation of NR2B NMDA subunit may contribute to inflammatory pain. [ABSTRACT FROM AUTHOR]

The TRPM8 channel is a principal cold transducer that is expressed on some primary afferents of the somatic and cranial sensory systems. However, it is uncertain whether TRPM8-expressing afferent neurons have the ability to convey innocuous and noxious cold stimuli with sensory discrimination between the two sub-modalities. Using rat dorsal root ganglion (DRG) neurons and the patch-clamp recording technique, we characterized membrane and action potential properties of TRPM8-expressing DRG neurons at 24°C and 10°C. TRPM8-expressing neurons could be classified into TTX-sensitive (TTXs/TRPM8) and TTX-resistant (TTXr/TRPM8) subtypes based on the sensitivity to tetrodotoxin (TTX) block of their action potentials. These two subtypes of cold-sensing cells displayed different membrane and action potential properties. Voltage-activated inward Na+ currents were highly susceptible to cooling temperature and abolished by ~95% at 10°C in TTXs/TRPM8 DRG neurons, but remained substantially large at 10°C in TTXr/TRPM8 cells. In both TTXs/TRPM8 and TTXr/TRPM8 cells, voltage-activated outward K+ currents were substantially inhibited at 10°C, and the cooling-sensitive outward currents resembled A-type K+ currents. TTXs/TRPM8 neurons and TTXr/TRPM8 neurons were shown to fire action potentials at innocuous and noxious cold temperatures respectively, demonstrating sensory discrimination between innocuous and noxious cold by the two subpopulations of cold-sensing DRG neurons. The effects of cooling temperatures on voltage-gated Na+ channels and A-type K+ currents are likely to be contributing factors to sensory discrimination of cold by TTXs/TRPM8 and TTXr/TRPM8 afferent neurons. [ABSTRACT FROM AUTHOR]

Background & Aims: Chronic stress exacerbates or causes relapse of symptoms such as abdominal pain and cramping in patients with irritable bowel syndrome. We investigated whether chronic stress increases plasma norepinephrine and sensitizes colon-specific dorsal root ganglion (DRG) neurons by increasing expression of nerve growth factor (NGF) in the colon wall. Methods: Heterotypic chronic stress (HeCS) was applied to male Wistar rats and neurologic and molecular responses were analyzed. Tissues were analyzed for NGF expression. Results: HeCS significantly increased visceromoter response to colorectal distension; expression of NGF increased in colonic muscularis externa and mucosa/submucosa. Rheobase decreased, resting membrane potential was depolarized, and electrogenesis of action potentials increased in colon-specific thoracolumbar DRG neurons. Luminal administration of resiniferatoxin in distal colon, systemic administration of anti-NGF antibody, or inhibition of the NGF receptor trkA by k252a or antisense oligonucleotides in thoracolumbar DRG blocked the chronic stress-induced visceral hypersensitivity to colorectal distension. Blockade of α1/α2- and β1/β2-adrenergic receptors prevented the stress-induced visceral hypersensitivity and increased expression of NGF in the colon wall. HeCS did not induce any inflammatory response in the colon wall. Conclusions: The peripheral stress mediator norepinephrine induces visceral hypersensitivity to colorectal distension in response to HeCS by increasing the expression of NGF in the colon wall, which sensitizes primary afferents in the absence of an inflammatory response. [Copyright &y& Elsevier]

Highlights • Behavioral analysis demonstrated that the CPSP animals displayed a significant decrease in mechanical allodynia threshold. • A significant increase in P2 × 4 receptors expression in microglia in thalamic peri-lesion tissues post-hemorrhage. • The mechanical allodynia in rats with CPSP were reversed by blocking P2 × 4 receptors. • A significant alleviation of mechanical allodynia was achieved following the administration of adrenergic antidepressants and antiepileptics. • A significant decrease in P2 × 4 receptor expression after treatment with adrenergic antidepressants and antiepileptics. Abstract Stroke is the leading cause of disability and death in the world. Central post-stroke pain (CPSP), a central neuropathic pain syndrome occurring after cerebral stroke, is a serious problem. But on account of the lack of reliable animal models, the mechanisms underlying CPSP remains poorly understood. To better understand of the pathophysiological basis of CPSP, we developed and characterized a new rat model of CPSP. This model is based on a hemorrhagic stroke lesion with intra-thalamic autologous blood (ITAB) injection in the ventral posterolateral nucleus of the thalamus. Behavioral analysis demonstrated that the animals displayed a significant decrease in mechanical allodynia threshold. We found a significant increase in P2 × 4 receptor expression in microglia in thalamic peri-lesion tissues post-hemorrhage. The mechanical allodynia in rats with CPSP were reversed by blocking P2 × 4 receptors. A significant alleviation of mechanical allodynia was achieved following the administration of adrenergic antidepressants and antiepileptics. Meanwhile, we found a significant decrease in P2 × 4 receptor expression after treatment with these drugs. Taken together, our results suggest that targeting P2 × 4 receptor may be effective in the treatment of CPSP. [ABSTRACT FROM AUTHOR]

Nuclear factor-kappa B (NF-κB) signaling is implicated in both cancer development and inflammation processes. However, the roles and mechanisms of NF-κB signaling in the development of cancer-induced pain (CIP) remain unknown. This study was designed to investigate the roles of the p65 subunit of NF-κB in regulation of the purinergic receptor (P2X3R) plasticity in dorsal root ganglion (DRG) of CIP rats. We showed here that tumor cell injection produced mechanical and thermal hyperalgesia, and an enhanced body weight-bearing difference, which was correlated with an upregulation of p65 and P2X3R expression in lumber DRGs and a potentiation of ATP-evoked responses of tibia-innervating DRG neurons. Inhibition of NF-κB signaling using p65 inhibitor pyrrolidine dithiocarbamate, BAY-11-7082, or lentiviral-p65 short-hairpin RNA significantly attenuated CIP and reversed the activities of P2X3R. Interestingly, tumor cell injection led to a significant demethylation of CpG island in p2x3r gene promoter and enhanced ability of p65 to bind the promoter of p2x3r gene. Our findings suggest that upregulation of P2X3R expression was mediated by the enhanced binding capability of p65 with demethylated promoter of p2x3r gene, thus contributing to CIP. NF-κBp65 might be a potential target for treating CIP, a neuropathic pain generated by tumor cell-induced injury to nerves that innervate the skin. [ABSTRACT FROM AUTHOR]

Abstract: Background: The pathogenesis of pain in irritable bowel syndrome (IBS) is poorly understood and treatment remains difficult. We have previously reported that TTX-resistant (TTX-R) sodium channels in colon-specific dorsal root ganglion (DRG) neurons were sensitized and the expression of the endogenous hydrogen sulfide producing enzyme cystathionine β-synthetase (CBS) was upregulated in a rat model of visceral hypersensitivity induced by neonatal maternal deprivation (NMD). However, the detailed molecular mechanism for activation of sodium channels remains unknown. This study was designed to examine roles for CBS–H2S signaling in sensitization of sodium channels in a previously validated rat model of IBS. Methods: Neonatal male rats (postnatal days 2–15) were exposed to a 3hour period of daily maternal separation with temperature maintained at ~33°C. Colon-specific dorsal root ganglion (DRG) neurons were labeled with DiI and acutely dissociated for measuring excitability and sodium channel current under whole-cell patch clamp configurations. The expression of NaV1.8 was analyzed by Western blot and Immunofluorescence study. The endogenous H2S producing enzyme CBS antagonist was injected intraperitoneally. Results: We showed that CBS was colocalized with NaV1.8 in colon-specific DRG neurons pre-labeled with DiI. Pretreatment of O-(Carboxymethyl) hydroxylamine hemihydrochloride (AOAA), an inhibitor of CBS, significantly reduced expression of NaV1.8 in NMD rats. AOAA treatment also inhibited the TTX-R sodium current density, right-shifted the V1/2 of activation curve, and reversed hyperexcitability of colon-specific DRG neurons in NMD rats. Conversely, addition of NaHS, a donor of H2S, greatly enhanced TTX-R sodium current density, left shifted the activation curve and enhanced excitability of colon DRG neurons in age-matched healthy rats. Furthermore, application of H-89, an inhibitor of protein kinase A, markedly attenuated the potentiation of TTX-R sodium current density by NaHS. Conclusion: These data suggest that sensitization of sodium channels of colon DRG neurons in NMD rats is most likely mediated by CBS–H2S signaling, thus identifying a potential target for treatment for chronic visceral pain in patients with IBS. [Copyright &y& Elsevier]

Abstract: Hydrogen sulfide (H2S), an endogenous gas molecule synthesized by cystathionine-β-synthetase (CBS), is involved in inflammation and nociceptive signaling. However, the molecular and epigenetic mechanisms of CBS-H2S signaling in peripheral nociceptive processing remain unknown. We demonstrated that peripheral inflammation induced by intraplantar injection of complete Freund adjuvant significantly up-regulated expression of CBS at both protein and mRNA levels in rat dorsal root ganglia (DRG). The CBS inhibitors hydroxylamine and aminooxyacetic acid attenuated mechanical hyperalgesia in a dose-dependent manner and reversed hyperexcitability of DRG neurons in inflamed rats. Intraplantar administration of NaHS (its addition mimics CBS production of H2S) or l-cysteine in healthy rats elicited mechanical hyperalgesia. Application of NaHS in vitro enhanced excitability and tetrodotoxin (TTX)-resistant sodium current of DRG neurons from healthy rats, which was attenuated by pretreatment of protein kinase A inhibitor H89. Methylation-specific PCR and bisulfite sequencing demonstrated that promoter region of cbs gene was less methylated in DRG samples from inflamed rats than that from controls. Peripheral inflammation did not alter expression of DNA methyltransferase 3a and 3b, the 2 major enzymes for DNA methylation, but led to a significant up-regulation of methyl-binding domain protein 4 and growth arrest and DNA damage inducible protein 45α, the enzymes involved in active DNA demethylation. Our findings suggest that epigenetic regulation of CBS expression may contribute to inflammatory hyperalgesia. H2S seems to increase TTX-resistant sodium channel current, which may be mediated by protein kinase A pathway, thus identifying a potential therapeutic target for the treatment of chronic pain. [Copyright &y& Elsevier]