mRNA In Dorsal Root Ganglia Research Articles

Involvement of the Sodium Channel Nav1.7 in Paclitaxel-induced Peripheral Neuropathy through ERK1/2 Signaling in Rats.

Paclitaxel treatment is a major cause of chemotherapy-induced peripheral neuropathy. The sodium channel Nav1.7 plays a critical role in pain perception. However, whether Nav1.7 in the dorsal root ganglion (DRG) is involved in paclitaxel-induced peripheral neuropathy remains unclear. Thus, our study aimed to evaluate whether Nav1.7 participates in the pathogenesis of paclitaxel-induced neuropathy. Paclitaxel-induced peripheral neuropathy was generated by intraperitoneal administration of paclitaxel on four alternate days. The results showed that DRG mRNA and protein expression levels of Nav1.7 were upregulated between days 7 and 21 after the administration of paclitaxel. Besides, paclitaxel upregulated extracellular signal-regulated kinase (ERK1/2) phosphorylation in DRG. Intrathecal injection of U0126 (a MEK inhibitor) blocking ERK1/2 phosphorylation blunted up-regulation of Nav1.7 in the DRG and correspondingly attenuated hyperalgesia. These results indicated that the sodium channel Nav1.7 in the DRG exerted an important function in paclitaxel-induced neuropathy, which was associated with ERK phosphorylation in neurons.

Systemic Depletion of Nerve Growth Factor Inhibits Disease Progression in a Genetically Engineered Model of Pancreatic Ductal Adenocarcinoma.

In patients with pancreatic ductal adenocarcinoma (PDAC), increased expression of proinflammatory neurotrophic growth factors (eg, nerve growth factor [NGF]) correlates with a poorer prognosis, perineural invasion, and, with regard to NGF, pain severity. We hypothesized that NGF sequestration would reduce inflammation and disease in the KPC mouse model of PDAC. Following biweekly injections of NGF antibody or control immunoglobulin G, beginning at 4 or 8 weeks of age, inflammation and disease stage were assessed using histological, protein expression, and quantitative polymerase chain reaction analyses. In the 8-week anti-NGF group, indicators of neurogenic inflammation in the dorsal root ganglia (substance P and calcitonin gene-related peptide) and spinal cord (glial fibrillary acidic protein) were significantly reduced. In the 4-week anti-NGF group, TRPA1 mRNA in dorsal root ganglia and spinal phosphorylated ERK protein were elevated, but glial fibrillary acidic protein expression was unaffected. In the 8-week anti-NGF group, there was a 40% reduction in the proportion of mice with microscopic perineural invasion, and no macrometastases were observed. Anti-NGF treatment beginning at 4 weeks may increase inflammation and negatively impact disease. Treatment starting at 8 weeks (after disease onset), however, reduces neural inflammation, neural invasion, and metastasis. These data indicate that NGF impacts PDAC progression and metastasis in a temporally dependent manner.

Epigallocatechin-3-gallate attenuates acute and chronic psoriatic itch in mice: Involvement of antioxidant, anti-inflammatory effects and suppression of ERK and Akt signaling pathways

Chronic itch is a distressing symptom of many skin diseases and negatively impacts quality of life. However, there is no medication for most forms of chronic itch, although antihistamines are often used for anti-itch treatment. Epigallocatechin-3-gallate (EGCG), a major green tea polyphenol, exhibits anti-oxidative and anti-inflammatory properties. Our previous studies highlighted a key role of oxidative stress and proinflammatory cytokines in acute and chronic itch. Here, we evaluated the effects of green tea polyphenon 60 and EGCG on acute and chronic itch in mouse models and explored its potential mechanisms. The effects of EGCG were determined by behavioral tests in mouse models of acute and chronic itch, which were induced by compound 48/80, chloroquine (CQ), and 5% imiquimod cream treatment, respectively. We found that systemic or local administration of green tea polyphenon 60 or EGCG significantly alleviated compound 48/80- and chloroquine-induced acute itch in a dose-dependent manner in mice. Incubation of EGCG significantly decreased the accumulation of intracellular reactive oxygen species (ROS) directly induced by compound 48/80 and CQ in cultured ND7-23 cells, a dorsal root ganglia derived cell line. EGCG also attenuated imiquimod-induced chronic psoriatic itch behaviors and skin epidermal hyperplasia in mice. In addition, EGCG inhibited the expression of IL-23 mRNA in skin and TRPV1 mRNA in dorsal root ganglia (DRG). Finally, EGCG remarkably inhibited compound 48/80-induced phosphorylation of extracellular signal-regulated kinase (ERK) and imiquimod-induced p-AKT in the spinal cord of mice, respectively. Collectively, these results indicated EGCG could be a promising strategy for anti-itch therapy.

Inhibition of morphine tolerance by MrgC receptor via modulation of interleukin-1β and matrix metalloproteinase 9 in dorsal root ganglia in rats

Opiate tolerance is a critical issue in pain management. Previous studies show that activation of Mas-related gene (Mrg) C receptor can modulate the development of morphine tolerance. This study was designed to investigate the underlying mechanism(s). Intrathecal (i.t.) administration of morphine (20µg) increased the expression of interleukin-1β (IL-1β) and matrix metalloproteinase-9 (MMP-9) in small- and medium-sized neurons in dorsal root ganglia (DRG). Co-administration of bovine adrenal medulla 8–22 (BAM8-22), a selective MrgC receptor agonist, via i.t. route inhibited the increase of IL-1β and MMP-9 in the DRG. Exposure of DRG cultures to morphine (3.3μM) for 3 or 5 days, but not for 1 day, induced an increase in MMP-9 mRNA expression. The treatment with BAM8-22 (10nM) for 20, 40 or 60min abolished chronic (5 days) morphine-induced increase of MMP-9 mRNA in the cultured DRG. The treatment with BAM8-22 for 1h inhibited chronic morphine-induced increase of MMP-9 and IL-1β mRNA in DRG but these effects were abolished by MrgC receptor antibody. The treatment with BAM8-22 for 24 and 72h respectively inhibited and enhanced morphine-induced expression of MMP-9 and IL-1β mRNA in the cultured DRG. The BAM8-22-induced inhibition and enhancement were abolished by MrgC receptor antibody. The results suggest that the inhibition of IL-1β and MMP-9 expressions in DRG underlain the modulation of morphine tolerance by the acute activation of MrgC receptors. The chronic activation of MrgC receptors can facilitate morphine-induced increase of MMP-9 and IL-1β expressions in DRG.

Effects of duloxetine on pain and walking distance in neuropathic pain models via modulation of the spinal monoamine system.

Approximately 40% of patients with chronic low back pain have a neuropathic component. In this study, we assessed the effects of analgesics on tactile hypersensitivity and walking distance in the rat cauda equina compression (CEC) model of neuropathic low back pain. The effects of analgesics on tactile hypersensitivity were examined using the von Frey test in CEC and partial sciatic nerve ligation (pSNL) models. Effects on walking distance were assessed using a treadmill test. Levels of α2δ1 subunit and ATF-3 mRNA in dorsal-root ganglion (DRG) neurons and those of α2δ1 subunit protein in the spinal cord were determined using quantitative RT-PCR and western blotting, respectively. Histological features were assessed using immunohistological methods. Histological changes indicating nerve damage (increase in ATF-3 mRNA, decrease in NF-200 and an increase in CD68 immunoreactivity) were observed in the CEC model. Duloxetine had analgesic effects in both models and improved walking distance in the CEC model. Pregabalin had analgesic effects in both models; however, the effect was weaker in the CEC model than in the pSNL model. α2δ1 subunit expression in DRG neurons and in the spinal cord was unchanged in the CEC model, but significantly increased in the pSNL model. Indomethacin had no analgesic effect in either model. Intrathecal yohimbine inhibited the effects of duloxetine with significant effects on depression. These findings suggest that the analgesic effects of duloxetine are mainly mediated by the spinal monoamine system, independent of the antidepressant effects of this agent. The findings of this study suggest that duloxetine may be an effective treatment of broad neuropathic pain states, including neuropathic low back pain. The analgesic effects of duloxetine might be mediated by alterations of the descending pain modulatory pathways in the spinal cord, independent of the antidepressant effects.

Demethylation regulation of BDNF gene expression in dorsal root ganglion neurons is implicated in opioid-induced pain hypersensitivity in rats

Repeated administration of morphine may result in opioid-induced hypersensitivity (OIH), which involves altered expression of numerous genes, including brain-derived neurotrophic factor (BDNF) in dorsal root ganglion (DRG) neurons. Yet, it remains unclear how BDNF expression is increased in DRG neurons after repeated morphine treatment. DNA methylation is an important mechanism of epigenetic control of gene expression. In the current study, we hypothesized that the demethylation regulation of certain BDNF gene promoters in DRG neurons may contribute to the development of OIH. Real-time RT-PCR was used to assess changes in the mRNA transcription levels of major BDNF exons including exon I, II, IV, VI, as well as total BDNF mRNA in DRGs from rats after repeated morphine administration. The levels of exon IV and total BDNF mRNA were significantly upregulated by repeated morphine administration, as compared to that in saline control group. Further, ELISA array and immunocytochemistry study revealed a robust upregulation of BDNF protein expression in DRG neurons after repeated morphine exposure. Correspondingly, the methylation levels of BDNF exon IV promoter showed a significant downregulation by morphine treatment. Importantly, intrathecal administration of a BDNF antibody, but not control IgG, significantly inhibited mechanical hypersensitivity that developed in rats after repeated morphine treatment. Conversely, intrathecal administration of an inhibitor of DNA methylation, 5-aza-2′-deoxycytidine (5-aza-dC) markedly upregulated the BDNF protein expression in DRG neurons and enhanced the mechanical allodynia after repeated morphine exposure. Together, our findings suggest that demethylation regulation of BDNF gene promoter may be implicated in the development of OIH through epigenetic control of BDNF expression in DRG neurons.

Radiotherapy Suppresses Bone Cancer Pain through Inhibiting Activation of cAMP Signaling in Rat Dorsal Root Ganglion and Spinal Cord

Radiotherapy is one of the major clinical approaches for treatment of bone cancer pain. Activation of cAMP-PKA signaling pathway plays important roles in bone cancer pain. Here, we examined the effects of radiotherapy on bone cancer pain and accompanying abnormal activation of cAMP-PKA signaling. Female Sprague-Dawley rats were used and received tumor cell implantation (TCI) in rat tibia (TCI cancer pain model). Some of the rats that previously received TCI treatment were treated with X-ray radiation (radiotherapy). Thermal hyperalgesia and mechanical allodynia were measured and used for evaluating level of pain caused by TCI treatment. PKA mRNA expression in dorsal root ganglion (DRG) was detected by RT-PCR. Concentrations of cAMP, IL-1β, and TNF-α as well as PKA activity in DRG and the spinal cord were measured by ELISA. The results showed that radiotherapy significantly suppressed TCI-induced thermal hyperalgesia and mechanical allodynia. The level of PKA mRNA in DRG, cAMP concentration and PKA activity in DRG and in the spinal cord, and concentrations of IL-1β and TNF-α in the spinal cord were significantly reduced by radiotherapy. In addition, radiotherapy also reduced TCI-induced bone loss. These findings suggest that radiotherapy may suppress bone cancer pain through inhibition of activation of cAMP-PKA signaling pathway in DRG and the spinal cord.

Minocycline prevents dynorphin-induced neurotoxicity during neuropathic pain in rats

Despite many advances, our understanding of the involvement of prodynorphin systems in the development of neuropathic pain is not fully understood. Recent studies suggest an important role of neuro–glial interactions in the dynorphin effects associated with neuropathic pain conditions. Our studies show that minocycline reduced prodynorphin mRNA levels that were previously elevated in the spinal and/or dorsal root ganglia (DRG) following sciatic nerve injury. The repeated intrathecal administration of minocycline enhanced the analgesic effects of low-dose dynorphin (0.15 nmol) and U50,488H (25–100 nmol) and prevented the development of flaccid paralysis following high-dose dynorphin administration (15 nmol), suggesting a neuroprotective effect. Minocycline reverts the expression of IL-1β and IL-6 mRNA within the spinal cord and IL-1β mRNA in DRG, which was elevated following intrathecal administration of dynorphin (15 nmol). These results suggest an important role of these proinflammatory cytokines in the development of the neurotoxic effects of dynorphin. Similar to minocycline, a selective inhibitor of MMP-9 (MMP-9 levels are reduced by minocycline) exerts an analgesic effect in behavioral studies, and its administration prevents the occurrence of flaccid paralysis caused by high-dose dynorphin administration (15 nmol). In conclusion, our results underline the importance of neuro–glial interactions as evidenced by the involvement of IL-1β and IL-6 and the minocycline effect in dynorphin-induced toxicity, which suggests that drugs that alter the prodynorphin system could be used to better control neuropathic pain.

Activation of the cAMP-PKA signaling pathway in rat dorsal root ganglion and spinal cord contributes toward induction and maintenance of bone cancer pain.

The objective of this study was to explore the role of cyclic adenosine monophosphate-protein kinase A (cAMP-PKA) signaling in the development of bone cancer pain in rats. Female Sprague-Dawley rats (N=48) were divided randomly into four groups: sham (n=8), tumor cell implantation (TCI) (n=16), TCI+saline (n=8), and TCI+PKA inhibitor (n=16). Bone cancer-induced pain behaviors - thermal hyperalgesia and mechanical allodynia - were tested at postoperative days -3, -1, 1, 3, 5, 7, 10, and 14. A PKA inhibitor, Rp-cAMPS (1 mmol/l/20 μl), was injected intrathecally on postoperative days 3, 4, and 5 (early phase) or 7, 8, and 9 postoperative days (late phase). The expression of PKA mRNA in dorsal root ganglia (DRG) was detected by reverse transcription-PCR. The concentration of cAMP and activity of PKA in DRG and spinal cord were measured by enzyme-linked immunosorbent assay. TCI treatment induced significant pain behaviors, manifested as thermal hyperalgesia and mechanical allodynia. Spinal administration of the PKA inhibitor Rp-cAMPS during the early phase and late phase significantly delayed or reversed, respectively, TCI-induced thermal hyperalgesia and mechanical allodynia. TCI treatment also led to obvious tumor growth and bone destruction. The level of PKA mRNA in the DRG, as well as the concentration of cAMP and the activity of PKA, in both the DRG and spinal cord were significantly increased after TCI treatment (P<0.01). We conclude that the inhibition of the cAMP-PKA signaling pathway may reduce bone cancer pain.

Role of sodium channels in recovery of sciatic nerve‐stretch injury in rats

To elucidate the mechanism of functional recovery after gradual nerve-stretch injury, we used rats in which the femur length was increased by 15 mm at 1.5 mm/day. We performed electrophysiology, mRNA analysis of tetrodotoxin-resistant voltage-gated sodium channels (TTX-R VGSCs) in dorsal root ganglia, and histology of unmyelinated sciatic nerve fibers and examined pain thresholds at 1, 10, 20, and 30 days after cessation of lengthening. Electrophysiology revealed conduction block after cessation that recovered after 30 days. TTX-R VGSC levels decreased immediately after cessation but were restored after 10 (Nav1.9) or 20 (Nav1.8) days. Histology revealed that injured unmyelinated nerve fibers regenerate 30 days after cessation. Pain threshold decreased gradually during lengthening but had not recovered to the control group level after 30 days. Early restoration of TTX-R VGSC mRNA in dorsal root ganglia preceded functional recovery of stretched nerves before regeneration of injured unmyelinated nerve fibers.

B-Type Natriuretic Peptide is Neither Itch-Specific Nor Functions Upstream of the GRP-GRPR Signaling Pathway

BackgroundA recent study by Mishra and Hoon identified B-type natriuretic peptide (BNP) as an important peptide for itch transmission and proposed that BNP activates spinal natriuretic peptide receptor-A (NPRA) expressing neurons, which release gastrin releasing peptide (GRP) to activate GRP receptor (GRPR) expressing neurons to relay itch information from the periphery to the brain (Science 340:968–971, 2013). A central premise for the validity of this novel pathway is the absence of GRP in the dorsal root ganglion (DRG) neurons. To this end, they showed that Grp mRNA in DRG neurons is either absent or barely detectable and claimed that BNP but not GRP is a major neurotransmitter for itch in pruriceptors. They showed that NPRA immunostaining is perfectly co-localized with Grp-eGFP in the spinal cord, and a few acute pain behaviors in Nppb -/- mice were tested. They claimed that BNP is an itch-selective peptide that acts as the first station of a dedicated neuronal pathway comprising a GRP-GRPR cascade for itch. However, our studies, along with the others, do not support their claims.FindingsWe were unable to reproduce the immunostaining of BNP and NPRA as shown by Mishra and Hoon. By contrast, we were able to detect Grp mRNA in DRGs using in situ hybridization and real time RT-PCR. We show that the expression pattern of Grp mRNA is comparable to that of GRP protein in DRGs. Pharmacological and genetic blockade of GRP-GRPR signaling does not significantly affect intrathecal BNP-induced scratching behavior. We show that BNP inhibits inflammatory pain and morphine analgesia.ConclusionsAccumulating evidence demonstrates that GRP is a key neurotransmitter in pruriceptors for mediating histamine-independent itch. BNP-NPRA signaling is involved in both itch and pain and does not function upstream of the GRP-GRPR dedicated neuronal pathway. The site of BNP action in itch and pain and its relationship with GRP remain to be clarified.

Mu Opioid Receptors on Primary Afferent Nav1.8 Neurons Contribute to Opiate-Induced Analgesia: Insight from Conditional Knockout Mice

Opiates are powerful drugs to treat severe pain, and act via mu opioid receptors distributed throughout the nervous system. Their clinical use is hampered by centrally-mediated adverse effects, including nausea or respiratory depression. Here we used a genetic approach to investigate the potential of peripheral mu opioid receptors as targets for pain treatment. We generated conditional knockout (cKO) mice in which mu opioid receptors are deleted specifically in primary afferent Nav1.8-positive neurons. Mutant animals were compared to controls for acute nociception, inflammatory pain, opiate-induced analgesia and constipation. There was a 76% decrease of mu receptor-positive neurons and a 60% reduction of mu-receptor mRNA in dorsal root ganglia of cKO mice. Mutant mice showed normal responses to heat, mechanical, visceral and chemical stimuli, as well as unchanged morphine antinociception and tolerance to antinociception in models of acute pain. Inflammatory pain developed similarly in cKO and controls mice after Complete Freund’s Adjuvant. In the inflammation model, however, opiate-induced (morphine, fentanyl and loperamide) analgesia was reduced in mutant mice as compared to controls, and abolished at low doses. Morphine-induced constipation remained intact in cKO mice. We therefore genetically demonstrate for the first time that mu opioid receptors partly mediate opiate analgesia at the level of Nav1.8-positive sensory neurons. In our study, this mechanism operates under conditions of inflammatory pain, but not nociception. Previous pharmacology suggests that peripheral opiates may be clinically useful, and our data further demonstrate that Nav1.8 neuron-associated mu opioid receptors are feasible targets to alleviate some forms of persistent pain.

TRPV1 in GABAergic Interneurons Mediates Neuropathic Mechanical Allodynia and Disinhibition of the Nociceptive Circuitry in the Spinal Cord

Neuropathic pain and allodynia may arise from sensitization of central circuits. We report a mechanism of disinhibition-based central sensitization resulting from long-term depression (LTD) of GABAergic interneurons as a consequence of TRPV1 activation in the spinal cord. Intrathecal administration of TRPV1 agonists led to mechanical allodynia that was not dependent on peripheral TRPV1 neurons. TRPV1 was functionally expressed in GABAergic spinal interneurons and activation of spinal TRPV1 resulted in LTD of excitatory inputs and a reduction of inhibitory signaling to spinothalamic tract (STT) projection neurons. Mechanical hypersensitivity after peripheral nerve injury was attenuated in TRPV1(-/-) mice but not in mice lacking TRPV1-expressing peripheral neurons. Mechanical pain was reversed by a spinally applied TRPV1 antagonist while avoiding the hyperthermic side effect of systemic treatment. Our results demonstrate that spinal TRPV1 plays a critical role as a synaptic regulator and suggest the utility of central nervous system-specific TRPV1 antagonists for treating neuropathic pain.

Leak K+ channel mRNAs in dorsal root ganglia: Relation to inflammation and spontaneous pain behaviour

Two pore domain potassium (K2P) channels (KCNKx.x) cause K+leak currents and are major contributors to resting membrane potential. Their roles in dorsal root ganglion (DRG) neurons normally, and in pathological pain models, are poorly understood. Therefore, we examined mRNA levels for 10 K2P channels in L4 and L5 rat DRGs normally, and 1day and 4days after unilateral cutaneous inflammation, induced by intradermal complete Freund's adjuvant (CFA) injections. Spontaneous foot lifting (SFL) duration (spontaneous pain behaviour) was measured in 1day and 4day rats <1h before DRG harvest. mRNA levels for KCNK channels and Kv1.4 relative to GAPDH (n=4–6 rats/group) were determined with real-time RT-PCR. This study is the first to demonstrate expression of THIK1, THIK2 and TWIK2 mRNA in DRGs. Abundance in normal DRGs was, in descending order:Kv1.4>TRESK(KCNK18)>TRAAK(KCNK4)>TREK2(KCNK10)=TWIK2(KCNK6)>TREK1 (KCNK2)=THIK2(KCNK12)>TASK1(KCNK3)>TASK2(KCNK5)>THIK1(KCNK13)=TASK3(KCNK9).During inflammation, the main differences from normal in DRG mRNA levels were bilateral, suggesting systemic regulation, although some channels showed evidence of ipsilateral modulation. By 1day, bilateral K2P mRNA levels had decreased (THIK1) or increased (TASK1, THIK2) but by 4days they were consistently decreased (TASK2, TASK3) or tended to decrease (excluding TRAAK). The decreased TASK2 mRNA was mirrored by decreased protein (TASK2-immunoreactivity) at 4days. Ipsilateral mRNA levels at 4days compared with 1day were lower (TRESK, TASK1, TASK3, TASK2 and THIK2) or higher (THIK1). Ipsilateral SFL duration during inflammation was positively correlated with ipsilateral TASK1 and TASK3 mRNAs, and contralateral TASK1, TRESK and TASK2 mRNAs. Thus changes in K2P mRNA levels occurred during inflammation and for 4 K2P channels were associated with spontaneous pain behaviour (SFL). K2P channels and their altered expression are therefore associated with inflammation-induced pain.

Effect of sympathetic nerve on the expression of substance P mRNA in dorsal root ganglion of chronically compressed brachial plexus

Objective To investigate the effect of different function of sympathetic nerve on the pain of peripheral nerve chronic compression. Methods Forty-eight male Sprague-Dawley rats were made into lower trunk chronic compression models and divided into 6 groups (A1,B1,C1,A2,B2,C2) with 8 rats per group. The C8T1 dorsal root ganglions of the compressed sides of group A1 (control group), B1 (sympathetic block group)and C1 (de-sympathetic group) were harvested 3 months after compression surgery. The compressed lower trunks of group A2 (control group), B2(sympathetic block group)and C2(de-sympathetic group)were decompressed 3 months after compression surgery and bred for another month and then the C8T1 dorsal root ganglions of the compressed sides were harvested. The levels of substance P mRNA in the C8T1 dorsal root ganglions were tested with RT-PCR technique. Results the mean relative levels of substance P mRNA of group A1, B1 and C1 were (3.620 ± 0.830) × 10-2, (2.945 ± 0.724) × 10-2, (2.239 ± 0.734) × 10-2, respectively, with a significant difference (P = 0.006) and those of group A2, B2 and C2 were (3.163 ± 1.026) × 10-2, (2.355 ± 0.680) × 10-2,(1.487 ± 0.802) × 10-2, the difference among which was statistically significant (P = 0.003). Conclusion The pain of peripheral nerve chronic compression is affected by sympathetic function. The more lower the sympathetic function is, the more light the pain is. Sympathetic blockage or resection helps to relieve the pain of peripheral nerve compression disease after being decompressed. Key words: Sympathetic nerve; Substance P; Chronic compression; Pain

Altered expression of P2X3 in vagal and spinal afferents following esophagitis in rats

Purinergic P2X(3) receptors are predominantly expressed in small diameter primary afferent neurons and activation of these receptors by adenosine triphosphate is reported to play an important role in nociceptive signaling. The objective of this study was to investigate the expression of P2X(3) receptors in spinal and vagal sensory neurons and esophageal tissues following esophagitis in rats. Two groups of rats were used including 7 days fundus-ligated (7D-ligated) esophagitis and sham-operated controls. Esophagitis was produced by ligating the fundus and partial obstruction of pylorus that initiated reflux of gastric contents. The sham-operated rats underwent midline incision without surgical manipulation of the stomach. Expressions of P2X(3) receptors in thoracic dorsal root ganglia (DRGs), nodose ganglia (NGs), and esophageal tissues were evaluated by RT-PCR, western blot and immunohistochemistry. Esophageal neurons were identified by retrograde transport of Fast Blue from the esophagus. There were no significant differences in P2X(3) mRNA expressions in DRGs (T1-T3) and NGs between 7D-ligated and sham-operated rats. However, there was an upregulation of P2X(3) mRNA in DRGs (T6-T12) and in the esophageal muscle. At protein level, P2X(3) exhibited significant upregulation both in DRGs and in NGs of rats having chronic esophagitis. Immunohistochemical analysis exhibited a significant increase in P2X(3) and TRPV1 co-expression in DRGs and NGs in 7D-ligated rats compared to sham-operated rats. The present findings suggest that chronic esophagitis results in upregulation of P2X(3) and its co-localization with TRPV1 receptor in vagal and spinal afferents. Changes in P2X(3) expression in vagal and spinal sensory neurons may contribute to esophageal hypersensitivity following acid reflux-induced esophagitis.

Estrogen altered visceromotor reflex and P2X 3 mRNA expression in a rat model of colitis

P2X 3 and P2X 2/3 receptors are expressed in peripheral tissues and dorsal root ganglia (DRG) and participate in peripheral pain. However, the mechanisms underlying P2X receptor-mediated nociception at different ovarial hormone levels has not been examined. In this study, 24 female rats were randomly divided into sham-operated (sham), ovariectomized (OVX), estrogen-treated, and estrogen–progesterone-treated groups with colitis. In each group, the visceromotor reflex (VMR) to colorectal distension was tested and the DRG were harvested for a real-time PCR analysis of P2X 3 and P2X 2 receptor mRNA. In OVX rats with colitis we found that the VMR to colorectal distension and P2X 3 receptor mRNA in DRG were both significantly decreased. Estrogen replacement reversed the decrease. However, neither the VMR nor the P2X 3 mRNA level in DRG from OVX colitis rats was reversed by the complex of estrogen and progesterone. Patch-clamp recording showed that in colitis rats, estradiol rapidly potentiated the sustained and transient currents evoked by ATP to 336 ± 49% and 122 ± 12% of controls, respectively, in a subpopulation of DRG neurons, which were blocked by ICI 182, 780, an antagonist of the estrogen receptor. Whereas progesterone rapidly inhibited the transient currents induced by ATP to 67 ± 10% of control and had no effect on the sustained currents evoked by the same agonist. These results indicate that P2X 3 receptors are likely to be an important contributor to the altered colonic functions in colitis rats, where the underlying mechanisms are closely related to endogenous estrogen modulation.

Involvement of increased expression of transient receptor potential melastatin 8 in oxaliplatin-induced cold allodynia in mice

Oxaliplatin is a chemotherapy drug and induces peripheral neuropathy which is aggravated by exposure to cold, the mechanism of which is unclear. In the present study, we investigated in mice whether transient receptor potential melastatin 8 (TRPM8), which is activated by cooling temperature, would be involved in cold allodynia induced by oxaliplatin. Mice were given an intraperitoneal injection of oxaliplatin. Acetone was applied to hind paw for cooling stimulation, and the time spent for licking to the hind paw was measured. The expression of TRPM8 mRNA in dorsal root ganglion was determined by the RT-PCR method. An injection of oxaliplatin induced cold allodynia, which peaked on day 3 after injection and did not disappear even on day 25. Peak cold allodynia was inhibited by capsazepine, a blocker of both TRPM8 and heat-activated TRPV1, but not by 5′-iodoresiniferatoxin, a TRPV1 blocker. Oxaliplatin increased wet-dog shake and jumping behaviors evoked by the TRPM8 agonist icilin. An injection of oxaliplatin increased the expression level of TRPM8 mRNA at day 3 after injection and the expression was decreased to the near-normal level on days 10 and 25. These results suggest that cold allodynia induced by oxaliplatin is at least partly due to the increased expression of TRPM8 in the primary afferents.

Estrogen Elicits Dorsal Root Ganglion Axon Sprouting via a Renin-Angiotensin System

Many painful conditions occur more frequently in women, and estrogen is a predisposing factor. Estrogen may contribute to some pain syndromes by enhancing axon outgrowth by sensory dorsal root ganglion (DRG) neurons. The objective of the present study was to define mechanisms by which estrogen elicits axon sprouting. The estrogen receptor-alpha agonist propyl pyrazole triol induced neurite outgrowth from cultured neonatal DRG neurons, whereas the estrogen receptor-beta agonist diarylpropionitrile was ineffective. 17beta-Estradiol (E2) elicited sprouting from peripherin-positive unmyelinated neurons, but not larger NF200-positive myelinated neurons. Microarray analysis showed that E2 up-regulates angiotensin II (ANGII) receptor type 2 (AT2) mRNA in vitro, and studies in adult rats confirmed increased DRG mRNA and protein in vivo. AT2 plays a central role in E2-induced axon sprouting because AT2 blockade by PD123,319 eliminated estrogen-mediated sprouting in vitro. We assessed whether AT2 may be responding to locally synthesized ANGII. DRG from adult rats expressed mRNA for renin, angiotensinogen, and angiotensin converting enzyme (ACE), and protein products were present and occasionally colocalized within neurons and other DRG cells. We determined if locally synthesized ANGII plays a role in estrogen-mediated sprouting by blocking its formation using the ACE inhibitor enalapril. ACE inhibition prevented estrogen-induced neuritogenesis. These findings support the hypothesis that estrogen promotes DRG nociceptor axon sprouting by up-regulating the AT2 receptor, and that locally synthesized ANGII can induce axon formation. Therefore, estrogen may contribute to some pain syndromes by enhancing the pro-neuritogenic effects of AT2 activation by ANGII.

P75NTR expression in rat urinary bladder sensory neurons and spinal cord with cyclophosphamide‐induced cystitis

A role for nerve growth factor (NGF) in contributing to increased voiding frequency and altered sensation from the urinary bladder has been suggested. Previous studies have examined the expression and regulation of tyrosine kinase receptors (Trks) in micturition reflexes with urinary bladder inflammation. The present studies examine the expression and regulation of another receptor known to bind NGF, p75(NTR), after various durations of bladder inflammation induced by cyclophosphamide (CYP). CYP-induced cystitis increased (P < or = 0.001) p75(NTR) expression in the superficial lateral and medial dorsal horn in L1-L2 and L6-S1 spinal segments. The number of p75(NTR)-immunoreactive (-IR) cells in the lumbosacral dorsal root ganglia (DRG) also increased (P < or = 0.05) with CYP-induced cystitis (acute, intermediate, and chronic). Quantitative, real-time polymerase chain reaction also demonstrated significant increases (P < or = 0.01) in p75(NTR) mRNA in DRG with intermediate and chronic CYP-induced cystitis. Retrograde dye-tracing techniques with Fastblue were used to identify presumptive bladder afferent cells in the lumbosacral DRG. In bladder afferent cells in DRG, p75(NTR)-IR was also increased (P < or = 0.01) with cystitis. In addition to increases in p75(NTR)-IR in DRG cell bodies, increases (P < or = 0.001) in pericellular (encircling DRG cells) p75(NTR)-IR in DRG also increased. Confocal analyses demonstrated that pericellular p75(NTR)-IR was not colocalized with the glial marker, glial fibrillary acidic protein (GFAP). These studies demonstrate that p75(NTR) expression in micturition reflexes is present constitutively and modified by bladder inflammation. The functional significance of p75(NTR) expression in micturition reflexes remains to be determined.