Cheng Yuan Lai

Colon mustard oil instillation induced cross-organ reflex sensitization on the pelvic-urethra reflex activity in rats.

ABSTRACT We investigated the participation of cyclin‐dependent kinase‐5 (Cdk5)‐mediated N‐methyl‐d‐aspartate receptor (NMDAR) NR2B subunit phosphorylation in cross‐organ reflex sensitization caused by colon irritation. The external urethral sphincter electromyogram (EUSE) reflex activity evoked by the pelvic afferent nerve test stimulation (TS, 1 stimulation/30 s) and protein expression in the spinal cord and dorsal root ganglion tissue (T13‐L2 and L6‐S2 ipsilateral to the stimulation) in response to colon mustard oil (MO) instillation were tested in anesthetized rats. When compared with a baseline reflex activity with a single action potential evoked by the TS before the administration of test agents, MO instillation into the descending colon sensitized the evoked activity characterized by elongated firing in the reflex activity in association with increased protein levels of Cdk5, PSD95, and phosphorylated NR2B (pNR2B) but not of total NR2B (tNR2B) in the spinal cord tissue. Both cross‐organ reflex sensitization and increments in protein expression were reversed by intra‐colonic pretreatments with ruthenium red (a non‐selective transient receptor potential vanilloid, TRPV, antagonist), capsaizepine (a TRPV1‐selective antagonist), lidocaine (a nerve conduction blocker) as well as by the intra‐thecal pretreatment with APV (a NRMDR antagonist) Co‐101244 (a NR2B‐selective antagonist) and roscovitine (a Cdk5 antagonist). Moreover, compared with the control group, both the increase in pNR2B and the cross‐organ reflex sensitization were attenuated in the si‐RNA of NR2B rats. All these results suggested that Cdk‐dependent NMDAR NR2B subunit phosphorylation mediates the development of cross‐organ pelvic–urethra reflex sensitization caused by acute colon irritation which could possibly underlie the high concurrence of pelvic pain syndrome with irritable bowel syndrome.

Neuroactive steroids inhibit spinal reflex potentiation by selectively enhancing specific spinal GABAA receptor subtypes

ABSTRACT Recently, we demonstrated a spinal GABAA receptor (GABAAR)‐dependent inhibition on the induction of repetitive stimulation‐induced spinal reflex potentiation. However, it remains unclear whether steroid hormones modulate such an inhibition. Here, we show that progesterone is capable of producing GABAARs‐dependent inhibition of the induction of spinal reflex potentiation by actions through neurosteroid metabolites. Progesterone (5 mg/kg, twice daily for 4 days) up‐regulates the expression of GABAAR α2, α3, α4 and δ subunits, and is associated with attenuated repetitive stimulation‐induced spinal reflex activity in ovariectomized rats. These changes were blocked by finasteride (50 mg/kg, twice daily), an antagonist of neurosteroid synthesis from progesterone, but not by the progesterone receptor antagonist, RU486 (100 mg/kg, twice daily). The induction of spinal reflex potentiation was attenuated after a short (30 min) intrathecal treatment with the neurosteroids, allopregnanolone (ALLOP, 10 μM, 10 μL) and 3α,5α‐tetrahydrodeoxycorticosterone (THDOC, 10 μM, 10 μL). Acute intrathecal administration of the GABAAR antagonist, bicuculline (10 μM, 10 μL) reversed the inhibition produced by progesterone, THDOC and allopregnanolone. These results imply that progesterone‐mediated effects on GABAAR expression and neural inhibition are regulated by neurosteroids synthesis rather than progesterone receptor activation.

TRPV1 mediates the uterine capsaicin-induced NMDA NR2B-dependent cross-organ reflex sensitization in anesthetized rats

Spinal cord-mediated cross-organ sensitization between the uterus and the lower urinary tract may underlie the high concurrence of obstetrical/gynecological inflammation and chronic pelvic pain syndrome characterized by urogenital pain. However, the neural pathway and the neurotransmitters involved are still unknown. We tested the hypothesis that the excitation of capsaicin-sensitive primary afferent fibers arising from the uterus through the stimulation of transient receptor potential vanilloid 1 (TRPV1) induces cross-organ sensitization on the pelvic-urethra reflex activity. Capsaicin (1-1,000 microM, 0.05 ml) was instilled into the uterus to induce cross-organ reflex sensitization. Activation of capsaicin-sensitive primary afferent fibers by capsaicin instillation into the uterine horn sensitized the pelvic-urethra reflex activity that was reversed by an intrauterine pretreatment with capsaizepine, a TRPV1-selective antagonist. Intrathecal injection of AP5, a glutamatergic N-methyl-D-aspartate (NMDA) antagonist, and Co-101244, an NMDA NR2B-selective antagonist, both abolished the cross-organ reflex sensitization caused by capsaicin instillation. These results demonstrated that TRPV1 plays a crucial role in contributing to the capsaicin-sensitive primary afferent fibers mediating the glutamatergic NMDA-dependent cross-organ sensitization between the uterus and the lower urinary tract when there is a tissue injury.

Estrogen-dependent facilitation on spinal reflex potentiation involves the Cdk5/ERK1/2/NR2B cascade in anesthetized rats

Cyclin-dependent kinase-5 (Cdk5), a proline-directed serine/threonine kinase, may alter pain-related neuronal plasticity by regulating extracellular signal-related kinase-1/2 (ERK1/2) activation. This study investigated whether Cdk5-dependent ERK activation underlies the estrogen-elicited facilitation on the repetitive stimulation-induced spinal reflex potentiaton (SRP) that is presumed to be involved in postinflammatory/neuropathic hyperalgesia and allodynia. Reflex activity of the external urethra sphincter electromyogram evoked by pelvic afferent nerve test stimulation (TS; 1 stimulation/30 s for 10 min) and repetitive stimulation (RS; 1 stimulation/1 s for 10 min) was recorded in anesthetized rats. TS evoked a baseline reflex activity, whereas RS produced SRP. Intrathecal (it) beta-estradiol facilitated the repetitive stimulation-induced SRP that was reversed by pretreatment with the estrogen receptor anatogonist ICI 182,780 (10 nM, 10 microl it), Cdk5 inhibitor roscovitine (100 nM, 10 microl it), ERK inhibitor (U-0126; 100 microM, 10 microl it) and N-methyl-D-aspartate (NMDA) NR2B subunit antagonist (Co-101244; 100 nM, 10 microl it). Moreover, ERalpha (propylpyrazoletriol; 100 nM, 10 microl it) and ERbeta (diarylpropionitrile; 100 microM, 10 microl it) agonists both facilitated the SRP, similar to results with a beta-estradiol injection. In association with the facilitated RS-induced SRP, an intrathecal beta-estradiol injection elicited ERK1/2 and NR2B subunit phosphorylation that were both reversed by intrathecal roscovitine and U-0126. These results indicated that the Cdk/ERK cascade, which is activated by ERalpha and ERbeta, may subsequently phosphorylate the NR2B subunit to develop NMDA-dependent postinflammatory hyperalgesia and allodynia to maintain the protective mechanisms of the body.

Orexin-A modulates glutamatergic NMDA-dependent spinal reflex potentiation via inhibition of NR2B subunit

Glucose-sensitive neurons in the lateral hypothalamic area produce orexin-A (OxA) as well as orexin-B (OxB) and send their axons to the spinal dorsal horn, which predominantly expresses orexin receptor-1 (OX-1), showing a higher sensitivity to OxA. The purpose of the present study was to assess the effects of OxA on the induction of a novel form of activity-dependent reflex potentiation, spinal reflex potentiation (SRP), in the pelvic-urethral reflex activity. External urethra sphincter electromyogram in response to pelvic afferent nerve test stimulation (TS; 1/30 Hz) or repetitive stimulation (RS; 1 Hz) was recorded in anesthetized rats. TS evoked a baseline reflex activity, whereas RS produced SRP, which was abolished by intrathecal OxA (30 nM, 10 mul). Intrathecal SB-408124 (10 muM, 10 mul), an OX-1 antagonist, reversed the abolition on SRP caused by OxA. Although there is, so far, no NR2A- and NR2B-specific agonist available, N-methyl-d-aspartate (NMDA) reversed the abolition on the RS-induced SRP caused by the co-administration of OxA and Co-101244 (30 nM, 10 mul; an NMDA NR2B subunit antagonist), but it did not reverse the abolition by the co-administration of OxA and PPPA (300 nM, 10 mul; an NMDA NR2A subunit antagonist). In conclusion, the activation of descending orexinergic fibers may inhibit the repetitive afferent input-induced central sensitization of pelvic-urethral reflex activity and urethra hyperactivity, indicating that spinal orexinergic neural transmission may be a novel target for the treatment of patients with neuropathetic or postinflammatory pain of pelvic origin.

Spinal SGK1/GRASP-1/Rab4 is involved in complete Freund’s adjuvant-induced inflammatory pain via regulating dorsal horn GluR1-containing AMPA receptor trafficking in rats

TOC summary SGK1/GRASP‐1/Rab4 participated in complete Freund’s adjuvant‐induced inflammatory pain via regulating GluR1‐containing AMPAR recycling at the dorsal horn neurons. ABSTRACT The elusiveness of the mechanism underlying pain is a major impediment in developing effective clinical treatments. We examined whether the phosphorylation of spinal serum‐ and glucocorticoid‐inducible kinase 1 (SGK1) and downstream glutamate receptor interacting protein (GRIP)‐associated protein‐1 (GRASP‐1)/Rab4‐dependent GluR1‐containing α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor (AMPAR) recycling play a role in inflammatory pain. After intraplantar injection of complete Freund’s adjuvant (CFA), we assessed thermal hyperalgesia using the Hargreaves test and analyzed dorsal horn samples (L4‐5) using Western blotting, coprecipitation, and immunofluorescence. CFA administration provoked behavioral hyperalgesia along with SGK1 phosphorylation, GluR1 trafficking from the cytosol to the membrane, and phosphorylated SGK1 (pSGK1)‐GRASP‐1, GRASP‐1‐Rab4, and Rab4‐GluR1 coprecipitation in the ipsilateral dorsal horn. In the dorsal horns of hyperalgesic rats, CFA‐enhanced pSGK1 was demonstrated to be colocalized with NeuN, GRASP‐1, Rab4, and GluR1 by immunofluorescence. GSK‐650394 (an SGK1 activation antagonist, 1, 10, and 30 μM, 10 μL/rat, intrathecally) dose‐dependently prevented CFA‐induced pain behavior and the associated SGK1 phosphorylation, GluR1 trafficking, and protein‐protein interactions at 1 day after CFA administration. Intrathecal 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX, an AMPAR antagonist, 1, 3, and 10 μM, 10 μL/rat) attenuated the hyperalgesia and GluR1 trafficking caused by CFA; however, it had no effect on SGK1 phosphorylation. Small interfering RNA targeting Rab4 hindered the CFA‐induced hyperalgesia and the associated GluR1 trafficking and Rab4‐GluR1 coprecipitation. Our results suggest that spinal SGK1 phosphorylation, which subsequently triggers the GRASP‐1/Rab4 cascade, plays a pivotal role in CFA‐induced inflammatory pain by regulating GluR1‐containing AMPAR recycling in the dorsal horn.

Spinal Serum-Inducible and Glucocorticoid-Inducible Kinase 1 Mediates Neuropathic Pain via Kalirin and Downstream PSD-95-Dependent NR2B Phosphorylation in Rats

The coupling of the spinal postsynaptic density-95 (PSD-95) with the glutamatergic N-methyl-d-aspartate receptor NR2B subunit and the subsequent NR2B phosphorylation contribute to pain-related plasticity. Increasing evidence reveals that kalirin, a Rho-guanine nucleotide exchange factor, modulates PSD-95-NR2B-dependent neuroplasticity. Our laboratory recently demonstrated that serum-inducible and glucocorticoid-inducible kinase 1 (SGK1) participates in inflammation-associated pain hypersensitivity by modulating spinal glutamatergic neurotransmission. Because kalirin is one of the proteins in PSD that is highly phosphorylated by various kinases, we tested whether kalirin could be a downstream target of spinal SGK1 that participates in neuropathic pain development via regulation of the PSD-95–NR2B coupling-dependent phosphorylation of NR2B. We observed that spinal nerve ligation (SNL, L5) in male Sprague Dawley rats resulted in behavioral allodynia, which was associated with phosphorylated SGK1 (pSGK1), kalirin, and phosphorylated NR2B (pNR2B) expression and an increase in pSGK1–kalirin–PSD-95–pNR2B coprecipitation in the ipsilateral dorsal horn (L4–L5). SNL-enhanced kalirin immunofluorescence was coincident with pSGK1, PSD-95, and pNR2B immunoreactivity. Small-interfering RNA (siRNA) that targeted spinal kalirin mRNA expression (10 μg, 10 μl; i.t.) reduced SNL-induced allodynia, kalirin and pNR2B expression, as well as kalirin–PSD-95 and PSD-95–pNR2B coupling and costaining without affecting SGK1 phosphorylation. Daily administration of GSK-650394 (an SGK1 antagonist; 100 nm, 10 μl, i.t.) not only exhibited effects similar to the kalirin mRNA-targeting siRNA but also attenuated pSGK1–kalirin costaining and SGK1–kalirin coupling. We suggest that nerve injury could induce spinal SGK1 phosphorylation that subsequently interacts with and upregulates kalirin to participate in neuropathic pain development via PSD-95–NR2B coupling-dependent NR2B phosphorylation.

VPS26A–SNX27 Interaction-Dependent mGluR5 Recycling in Dorsal Horn Neurons Mediates Neuropathic Pain in Rats

Retromer, which crucially contributes to endosomal sorting machinery through the retrieval and recycling of signaling receptors away from degradation, has been identified as a critical element for glutamatergic-receptor-dependent neural plasticity at excitatory synapses. We observed it accompanied by behavioral allodynia; neuropathic injury time-dependently enhanced VPS26A and SNX27 expression; VPS26A–SNX27 coprecipitation; and VPS26A-positive, SNX27-positive, and VPS26A–SNX27 double-labeled immunoreactivity in the dorsal horn of Sprague Dawley rats that were all sufficiently ameliorated through the focal knock-down of spinal VPS26A expression. Although the knock-down of spinal SNX27 expression exhibited similar effects, spinal nerve ligation (SNL)-enhanced VPS26A expression remained unaffected. Moreover, SNL also increased membrane-bound and total mGluR5 abundance, VPS26A-bound SNX27 and mGluR5 and mGluR5-bound VPS26A and SNX27 coprecipitation, and mGluR5-positive and VPS26A/SNX27/mGluR5 triple-labeled immunoreactivity in the dorsal horn, and these effects were all attenuated through the focal knock-down of spinal VPS26A and SNX27 expression. Although administration with MPEP adequately ameliorated SNL-associated allodynia, mGluR5 expression, and membrane insertion, SNL-enhanced VPS26A and SNX27 expression were unaffected. Together, these results suggested a role of spinal VPS26A–SNX27-dependent mGluR5 recycling in the development of neuropathic pain. This is the first study that links retromer-associated sorting machinery with the spinal plasticity underlying pain hypersensitivity and proposes the possible pathophysiological relevance of endocytic recycling in pain pathophysiology through the modification of glutamatergic mGluR5 recycling. SIGNIFICANCE STATEMENT VPS26A–SNX27-dependent mGluR5 recycling plays a role in the development of neuropathic pain. The regulation of the VPS26A–SNX27 interaction that modifies mGluR5 trafficking and expression in the dorsal horn provides a novel therapeutic strategy for pain relief.

Protein Kinase A–dependent Spinal α-Amino-3-hydroxy-5-methyl-4-isoxazoleproprionate–receptor Trafficking Mediates Capsaicin-induced Colon-Urethra Cross-organ Reflex Sensitization

Background: Intracellular redistribution of &agr;-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate receptors (AMPARs) is known to be induced by natural painful stimulation. We tested the hypothesis that that protein kinase A (PKA)-dependent AMPAR trafficking underlies the development of N-methyl-d-aspartate receptor–mediated cross-organ sensitization in vivo. Methods: We recorded urethra reflex activity and analyzed immunoblotting of lumbosacral (L6-S2) dorsal horn (DH) tissue obtained from animal preparations after intrathecal 8-bromo-cyclic adenosine monophosphate injection or intracolonic instillation with 8-methyl-N-vanillyl-trans-6-nonenamide (capsaicin). Results: Intrathecal 8-bromo-cyclic adenosine monophosphate (300 &mgr;M, 10 &mgr;l) induced reflex potentiation (81.85 ± 22.21 spikes/stimulation) and increased the number of AMPAR Glu receptor 1 subunits in the membrane fraction of DH (1.8-fold increase vs. control). This process was prevented by pretreatment with the PKA inhibitor N-[2- ((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide(10 &mgr;M, 10 &mgr;l, 2.7 ± 0.8 [mean ± SE] spikes/stimulation) and human thyroid A kinase–anchoring protein (10 &mgr;M, 10 &mgr;l, 11.5 ± 4.8 spikes/stimulation), an inhibitor of PKA and PKA-A kinase–anchoring protein interactions. Intracolonic capsaicin instillation sensitized the urethra reflex (137.2 ± 62.4 spikes/stimulation) and, relative to control, simultaneously provoked an increase (2.9-fold) in the membrane fraction and a decrease (0.9-fold) in the cytosolic fraction of Glu receptor 1 subunits in DH. Inhibition of PKA activity and disruption of PKA-A kinase–anchoring protein interaction in the DH (2.0 ± 0.6 and 16.7 ± 2.8 spikes/stimulation, respectively) are sufficient to prevent capsaicin-dependent reflex sensitization and AMPAR trafficking in the membrane fraction (0.6- and 0.5-fold increase capsaicin). Conclusion: Delivery of AMPAR-containing Glu receptor 1 subunits to the membranes of lumbosacral DH neurons through a PKA-dependent pathway contributes to noxious stimulation-induced synaptic strengthening, which plays roles in colon-urethra reflex sensitization.

Modulation of Nerve Injury–induced HDAC4 Cytoplasmic Retention Contributes to Neuropathic Pain in Rats

Background:The histone deacetylases (HDACs) have been implicated in pain hypersensitivity. This study investigated the potential involvement of an HDAC4-related mechanism in the spinal nerve ligation (SNL)-induced nociceptive hypersensitivity. Methods:The left L5 to L6 spinal nerves of 627 adult male Sprague–Dawley rats were surgically ligated. The withdrawal threshold of hind paws and the abundances, cellular location, and interactions of proteins in the dorsal horn were assayed before and after surgery. The 14-3-3&bgr;-targeting small-interfering RNA, a serum- and glucocorticoid-inducible kinase 1 (SGK1) antagonist, or an HDAC inhibitor was spinally injected to elucidate the role of 14-3-3&bgr;, SGK1, and HDAC4. Results:Without affecting the HDAC4 level, SNL provoked SGK1 phosphorylation (mean ± SEM from 0.24 ± 0.02 to 0.78 ± 0.06 at day 7, n = 6), HDAC4 phosphorylation (from 0.38 ± 0.03 to 0.72 ± 0.06 at day 7, n = 6), 14-3-3&bgr; expression (from 0.53 ± 0.09 to 0.88 ± 0.09 at day 7, n = 6), cytoplasmic HDAC4 retention (from 1.18 ± 0.16 to 1.92 ± 0.11 at day 7, n = 6), and HDAC4-14-3-3&bgr; coupling (approximately 2.4-fold) in the ipsilateral dorsal horn in association with behavioral allodynia. Knockdown of spinal 14-3-3&bgr; expression prevented the SNL-provoked HDAC4 retention (from 1.89 ± 0.15 to 1.32 ± 0.08 at day 7, n = 6), HDAC4-14-3-3&bgr; coupling (approximately 0.6-fold above SNL 7D), and behavioral allodynia (from 0.16 ± 0.3 to 6 ± 1.78 at day 7, n = 7), but not SGK1 (from 0.78 ± 0.06 to 0.71 ± 0.04 at day 7, n = 6) or HDAC4 (from 0.75 ± 0.15 to 0.68 ± 0.11 at day 7, n = 6) phosphorylation. Conclusion:Neuropathic pain maintenance involves the spinal SGK1 activation–dependent HDAC4 phosphorylation and its subsequent association with 14-3-3&bgr; that promotes cytoplasmic HDAC4 retention in dorsal horn neurons.