Role of calcitonin gene-related peptide in pain regulation in the parabrachial nucleus of naive rats and rats with neuropathic pain.

Researches have shown that calcitonin gene-related peptide (CGRP) plays a pivotal role in pain modulation. Nociceptive information from the periphery is relayed from parabrachial nucleus (PBN) to brain regions implicated involved in pain. This study investigated the effects and mechanisms of CGRP and CGRP receptors in pain regulation in the PBN of naive and neuropathic pain rats. Chronic sciatic nerve ligation was used to model neuropathic pain, CGRP and CGRP 8-37 were injected into the PBN of the rats, and calcitonin receptor-like receptor (CLR), a main structure of CGRP receptor, was knocked down by lentivirus-coated CLR siRNA. The hot plate test (HPT) and the Randall Selitto Test (RST) was used to determine the latency of the rat hindpaw response. The expression of CLR was detected with RT-PCR and western blotting. We found that intra-PBN injecting of CGRP induced an obvious anti-nociceptive effect in naive and neuropathic pain rats in a dose-dependent manner, the CGRP-induced antinociception was significantly reduced after injection of CGRP 8-37, Moreover, the mRNA and protein levels of CLR, in PBN decreased significantly and the antinociception CGRP-induced was also significantly lower in neuropathic pain rats than that in naive rats. Knockdown CLR in PBN decreased the expression of CLR and the antinociception induced by CGRP was observably decreased. Our results demonstrate that CGRP induced antinociception in PBN of naive or neuropathic pain rats, CGRP receptor mediates this effect. Neuropathic pain induced decreases in the expression of CGRP receptor, as well as in CGRP-induced antinociception in PBN.

Role of mu-opioid receptor in nociceptive modulation in anterior cingulate cortex of rats.

Lots of studies have demonstrated that anterior cingulate cortex plays important roles in the pain perception and pain modulation. The present study explored the role of mu-opioid receptor in nociceptive modulation in anterior cingulate cortex of rats with neuropathic pain. Neuropathic pain model was set up by chronic constriction injury of the left sciatic nerve of rats. The hindpaw withdrawal latency to thermal and mechanical stimulation, by hot plate and Randall Selitto Test respectively, was used to evaluate the rat's responses to noxious stimulation. Results showed that intra-anterior cingulate cortex injection of morphine could induce the antinociception dose-dependently. By intra-anterior cingulate cortex injection of opioid receptor antagonist, the morphine-induced antinociception could be attenuated by naloxone, as well as much significantly by the selective mu-opioid receptor antagonist ß-funaltrexamine, indicating that mu-opioid receptor is involved in the morphine-induced antinociception in anterior cingulate cortex of rats with neuropathic pain. The morphine-induced antinociception was much more decreased in rats with neuropathic pain than that in normal rats, and there was a significant decrease in mu-opioid receptor messenger RNA levels in anterior cingulate cortex of rats with neuropathic pain, indicating that there may be a down-regulation in mu-opioid receptor expression in anterior cingulate cortex of rats with neuropathic pain. To further confirm the role of mu-opioid receptor in morphine-induced antinociception in anterior cingulate cortex, normal rats were received intra-anterior cingulate cortex administration of small interfering RNA targeting mu-opioid receptor and it was found that there was a down-regulation in mu-opioid receptor messenger RNA levels, as well as a down-regulation in mu-opioid receptor expression in anterior cingulate cortex tested by real-time polymerase chain reaction and western blotting. Furthermore, the morphine-induced antinociceptive effect decreased significantly in rats with small interfering RNA targeting mu-opioid receptor, which indicated that knockdown mu-opioid receptor in anterior cingulate cortex could also attenuate morphine-induced antinociceptive effect. These results strongly suggest that mu-opioid receptor plays a significant role in nociceptive modulation in anterior cingulate cortex of rats.

Etelcalcetide, A Novel Calcimimetic, Prevents Vascular Calcification in A Rat Model of Renal Insufficiency with Secondary Hyperparathyroidism.

Etelcalcetide, a novel peptide agonist of the calcium-sensing receptor, prevents vascular calcification in a rat model of renal insufficiency with secondary hyperparathyroidism. Vascular calcification occurs frequently in patients with chronic kidney disease (CKD) and is a consequence of impaired mineral homeostasis and secondary hyperparathyroidism (SHPT). Etelcalcetide substantially lowers parathyroid hormone (PTH) and fibroblast growth factor-23 (FGF23) levels in SHPT patients on hemodialysis. This study compared the effects of etelcalcetide and paricalcitol on vascular calcification in rats with adenine-induced CKD and SHPT. Uremia and SHPT were induced in male Wistar rats fed a diet supplemented with 0.75% adenine for 4 weeks. Rats were injected with vehicle, etelcalcetide, or paricalcitol for 4 weeks from the beginning of adenine diet. Rats fed an adenine-free diet were included as nonuremic controls. Similar reductions in plasma PTH and parathyroid chief cell proliferation were observed in both etelcalcetide- and paricalcitol-treated rats. Serum calcium and phosphorus were significantly lower in etelcalcetide-treated uremic rats and was unchanged in paricalcitol-treated rats. Both serum FGF23 and aortic calcium content were significantly lower in etelcalcetide-treated uremic rats compared with either vehicle- or paricalcitol-treated uremic rats. The degree of aortic calcium content for etelcalcetide-treated rats was similar to that in nonuremic controls and corroborated findings of lack of histologic aortic mineralization in those groups. In conclusion, etelcalcetide and paricalcitol similarly attenuated progression of SHPT in an adenine rat model of CKD. However, etelcalcetide differentially prevented vascular calcification, at least in part, due to reductions in serum FGF23, calcium, and phosphorus levels.

Intravenous Injection of GluR2-3Y Inhibits Repeated Morphine-Primed Reinstatement of Drug Seeking in Rats.

Studies have demonstrated that the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor is essential to drug addiction. In this study, we explored the influence of GluR2-3Y, an interfering peptide to prevent the endocytosis of AMPA receptors containing the GluR2 subunit, on morphine-seeking behavior in the rat self-administration model. After self-administration was established, the rats received intravenous injections of GluR2-3Y during the extinction sessions. There were no significant differences in both active and inactive pokes compared to the control group of rats that received GluR2-3S, indicating that GluR2-3Y has no significant influences on the extinction of morphine self-administration. The other two groups of rats were trained, extinguished, and reinstated by repeated morphine priming (respectively, called Prime 1, Prime 2, and Prime 3). Only one intravenous injection of GluR2-3Y was performed before Prime 1. Compared to the control group, GluR2-3Y did not affect Prime 1, but significantly attenuated the morphine-seeking behavior during repeated morphine-primed reinstatement, indicating an inhibitory after effect of GluR2-3Y on morphine-seeking behavior in rats. The long-term depression (LTD) in the nucleus accumbens (NAc) shell was also assessed. Pretreatment with GluR2-3Y altered the ability of LTD induction to the level of that in the naive group, while pretreatment with GluR2-3S had no effects on LTD. Our results demonstrated that the intravenous injection of GluR2-3Y, to block the endocytosis of AMPA receptors, inhibited the reinstatement of morphine-seeking behavior, which may be induced by modulating the neuronal plasticity in the NAc shell of rats.

Calcitonin gene-related peptide and its receptor plays important role in nociceptive regulation in the arcuate nucleus of hypothalamus of rats with inflammatory pain.

The present study has explored the role of calcitonin gene-related peptide (CGRP) and its receptor in inflammatory pain modulation in arcuate nucleus of hypothalamus (ARC). Our study demonstrated that intra-ARC injection of CGRP induced antinociceptive effects to naïve rats and rats with inflammatory pain, the effect could be inhibited by the selective CGRP receptor antagonist CGRP8-37. Interestingly, the CGRP-induced antinociception effect was decreased in rats with inflammatory pain compared to naïve rats. Similarly, we found that calcitonin receptor like receptor (CLR), a main component of CGRP receptor, had a low decreased expression levels in the ARC regions of rats with inflammatory pain. The CGRP-induced antinociceptive effect was significantly impaired after reducing CLR expression by intra-ARC administration of CLR targeted siRNA. These findings demonstrated that CGRP might play a crucial role in nociceptive modulation in the ARC during inflammatory pain, which was mediated by CGRP receptor in the ARC. This study shed light upon CGRP and its receptor interaction might be valuable strategies for the alleviation of inflammatory pain.

Time Is a Critical Factor When Evaluating Oligonucleotide Therapeutics in hERG Assays.

In accord with International Conference on Harmonization S7B guidelines, an in vitro human ether-a-go-go-related gene (hERG) assay is one component of an integrated risk assessment for delayed ventricular repolarization. Function of hERG could be affected by direct (acute) mechanisms, or by indirect (chronic) mechanisms. Some approved oligonucleotide therapeutics had submitted hERG data to regulatory agents, which were all collected with the same protocol used for small-molecule testing (incubation time <20 min; acute), however, oligonucleotides have unique mechanisms and time courses of action (indirect). To reframe the hERG testing strategy for silencing RNA (siRNA), an investigation was performed to assess the time course for siRNA-mediated inhibition of hERG function and gene expression. Commercially available siRNAs of hERG were evaluated in a stable hERG-expressed cell line by whole-cell voltage clamp using automated electrophysiology and polymerase chain reaction. In the acute hERG study, no effects were observed after treatment with 100 nM siRNA for 20 min. The chronic effects of 100 nM siRNAs on hERG function were evaluated and recorded over 8-48 h following transfection. At 8 h there was no significant effect, whereas 77% reduction was observed at 48 h. Measurement of hERG mRNA levels demonstrated a 79% and 93% decrease of hERG mRNA at 8 and 48 h, respectively, consistent with inhibition of hERG transcription. The results indicate that an anti-hERG siRNA requires a long exposure time (48 h) in the hERG assay to produce a maximal reduction in hERG current; short exposures (20 min-8 h) had no effect. These findings imply that off-target profiling of novel oligonucleotides could benefit from using hERG protocol with long incubation times to de-risk potential off-target (indirect) effects on the hERG channel. This hERG assay modification may be important to consider if the findings are used to support an integrated nonclinical-clinical risk assessment for QTc (the duration of the QT interval adjusted for heart rate) prolongation.

A Novel CaMKII Inhibitory Peptide Blocks Relapse to Morphine Seeking by Influencing Synaptic Plasticity in the Nucleus Accumbens Shell.

Drugs of abuse cause enduring functional disorders in the brain reward circuits, leading to cravings and compulsive behavior. Although people may rehabilitate by detoxification, there is a high risk of relapse. Therefore, it is crucial to illuminate the mechanisms of relapse and explore the therapeutic strategies for prevention. In this research, by using an animal model of morphine self-administration in rats and a whole-cell patch-clamp in brain slices, we found changes in synaptic plasticity in the nucleus accumbens (NAc) shell were involved in the relapse to morphine-seeking behavior. Compared to the controls, the amplitude of long-term depression (LTD) induced in the medium spiny neurons increased after morphine self-administration was established, recovered after the behavior was extinguished, and increased again during the relapse induced by morphine priming. Intravenous injection of MA, a new peptide obtained by modifying Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitor "myr-AIP", decreased CaMKII activity in the NAc shell and blocked the reinstatement of morphine-seeking behavior without influence on the locomotor activity. Moreover, LTD was absent in the NAc shell of the MA-pretreated rats, whereas it was robust in the saline controls in which morphine-seeking behavior was reinstated. These results indicate that CaMKII regulates morphine-seeking behavior through its involvement in the change of synaptic plasticity in the NAc shell during the relapse, and MA may be of great value in the clinical treatment of relapse to opioid seeking.

Role of MEK-ERK pathway in morphine-induced conditioned place preference in ventral tegmental area of rats.

A major goal of research on drug addiction is to develop the effective treatments to deal with the long-term behavioral disorders especially reinstatement induced by the addictive drugs such as opiates, cocaine, and cannabinoid. The molecular mechanisms underlying these substance-related disorders remain unclear so far. Here we used the model of morphine-induced conditioned place preference (CPP) in rats to mimic the progress of drug-taking, withdrawal and relapse in human. The tissue of ventral tegmental area (VTA), one of the most important brain structures associated with abused drug-related disorders, was taken and two-dimensional electrophoresis (2-DE) was performed to analyze and compare the changes of protein expression patterns during the different stages of morphine-induced CPP. First, we found that there were 80 proteins identified to be changed in the process of morphine-induced CPP. Furthermore, as the mitogen-activated protein kinase kinase 1 (MAPKK1) was increased significantly in the stages of establishment and reinstatement, we confirmed the change of activated extracellular signal-regulated kinase (ERK) by Western blotting in VTA tissue and cultured cell. The results demonstrated that the activated MEK-ERK pathway by chronic morphine treatment in VTA was involved in morphine-induced reinstatement. Moreover, inhibition of MEK-ERK pathway by infusion the MEK inhibitor U0126 in VTA blocked the establishment of morphine-induced CPP. The present study found significant changes in a group of protein expressions in VTA during morphine-induced CPP and further confirmed the role of MEK-ERK cell signaling pathway of VTA in morphine addiction.

Single-cell microinjection technology in cell biology.

Single-cell microinjection has been successfully used to deliver exogenous proteins, cDNA constructs, peptides, drugs and particles into transfection-challenged cells. With precisely controlled delivery dosage and timing, microinjection has been used in many studies of primary cultured cells, transgenic animal production, in vitro fertilization and RNA inference. This review discusses the advantages and limits of microinjection as a mechanical delivery method and its applications to attached and suspended cells.

Role of Calcitonin Gene-Related Peptide in Nociceptive Modulationin Anterior Cingulate Cortex of Naïve Rats and Rats With Inflammatory Pain.

It is known that calcitonin gene-related peptide (CGRP) plays a key role in pain modulation in the brain. There are high expressions of CGRP and CGRP receptor in anterior cingulate cortex (ACC), an important brain structure in pain modulation. The present study explored the role and mechanisms of CGRP and CGRP receptor in nociceptive modulation in ACC in naïve rats and inflammatory rats. Administration of different does of CGRP in ACC induced significant antinociception in a dose-dependent manner in both naïve rats and rats with inflammatory pain. The CGRP-induced antinociception was attenuated by injection of the CGRP receptor antagonist CGRP8-37 in ACC. Interestingly, both CGRP-induced antinociception and CGRP receptor expression decreased in ACC in rats with inflammatory pain compared with naïve rats. Knockdown of CGRP receptor in ACC by siRNA targeting to CGRP receptor attenuated both the CGRP receptor expression and the CGRP-induced antinociception significantly in rats. These findings demonstrate that CGRP and CGRP receptor participate in nociceptive modulation in ACC in rats, inhibiting CGRP receptor expression induces decrease in CGRP-induced antinociception in ACC.

Microinjection as a tool of mechanical delivery.

Microinjection to single cells has been widely used in the studies of transduction-challenged cells, transgenic animal production, and in vitro fertilization to mechanically transfer DNAs, RNA interferences, sperms, proteins, peptides, and drugs. The advantages of microinjection include the precision of delivery dosage and timing, high efficiency of transduction as well as low cytotoxicity. However, manual microinjection is labor intensive and time consuming, which limits the application of this technique to large number of cells in a sample. New cell culture matrix ensuring all cells grow in a desired position and orientation is needed for application of high throughput automatic injection systems, which will significantly increase injection speed, cell survival, and success rates.

Small molecules with potent osteogenic-inducing activity in osteoblast cells.

A chemical screen of 45,000 compounds from a diverse collection led to the identification of two series of small molecules with potent osteogenic activity in mouse MC3T3-E1 osteoblast cells. The first chemical group was characterized by an amino benzothiazole core (AMG0892 series) and the second group by a naphthyl amide core (AMG0309 series). Using alkaline phosphatase (ALP), osteocalcin (OCL) and calcium as markers of osteoblast differentiation and mineralization, both chemical series showed EC(50)s in the 0.01-0.2 microM range and were consistent for all three markers. Compounds inhibited cell proliferation, had no effect on apoptosis and showed evidence for CREB pathway activity. The present compounds represent some of the most potent osteogenic small molecules reported to date and provide new tools for elucidating signaling mechanisms in osteoblasts.

The role of intracellular amyloid beta in Alzheimer's disease.

Extracellular amyloid beta (Abeta) that confers neurotoxicity and modulates synaptic plasticity and memory function has been central to the amyloid hypothesis of Alzheimer's disease (AD) pathology. Like many other misfolded proteins identified in neurodegenerative disorders, Abeta also accumulates inside the AD neurons. This intracellular Abeta affects a variety of cellular physiology from protein degradation, axonal transport, autophagy to apoptosis, further documenting the role of Abeta in AD. Therapeutics targeting intracellular Abeta could be effective treatment for AD.

Morphine: a protective or destructive role in neurons?

Morphine has received intensive research interest for a long time. However, until recently, the protective versus destructive roles of morphine in the neuronal system have not been studied. There is evidence suggesting that morphine induces apoptotic cell death in neuronal and glial cells, whereas controversial studies support a neuroprotective role for morphine. The exact mechanisms for both protective and destructive pathways are not clear and are still under investigation. Improved understanding of morphine neuroprotection and neurotoxicity will be helpful to control morphine side effects in medical applications and to identify new targets for potential therapies and prevention strategies to opioid addiction.

Involvement of ORL1 receptor and ERK kinase in the orphanin FQ-induced nociception in the nucleus accumbens of rats.

Nociceptin/orphanin FQ (N/OFQ) is a heptadecapeptide, which has been identified as an endogenous ligand of the opioid receptor-like (ORL1) receptor. The present study investigated the nociceptive effect of intra-nucleus accumbens (intra-NAc) injection of OFQ, and the involvement of ERK pathway in such effect. Intra-NAc injection of OFQ (0.1, 0.5, 1 nmol) dose-dependently decreased the nociceptive thresholds on the hindpaw withdrawal response to thermal and mechanical stimulation in rats. Moreover, the intra-NAc injection of OFQ-induced decreases in HWLs were antagonized by intra-NAc injection of (Nphe(1))nociceptin(1-13)NH(2), an antagonist of ORL1 receptor, in a dose-dependent way. Furthermore, the OFQ-induced nociception could be attenuated by pretreatment with the mitogen-activated protein kinase/ERK kinase (MEK) inhibitor 1,4-diamino-2,3-dicyano-1,4-bis(2-aminopheylthio)butadiene (U0126). Our results demonstrate that OFQ induces nociceptive effects in NAc. The effect was blocked by the antagonist (Nphe(1))nociceptin(1-13)NH(2) and attenuated by U0126, suggesting that the activation of ERK pathways is involved in the OFQ-induced nociceptive effect in the NAc of rats.

Potential protection of curcumin against hypoxia-induced decreases in beta-III tubulin content in rat prefrontal cortical neurons.

The central nervous system (CNS) is highly dependent on adequate supply of oxygen and is sensitive to hypoxia. It is known that hypoxia induces injuries on the brain tissue and the neuronal activity. Curcumin, a yellow pigment obtained from the rhizome of C. longa Linn., has been regarded as a multi-functional drug with antioxidative activity. In the present study, we first demonstrated a significant decrease in the content of beta-III tubulin protein in rat prefrontal cortex (PFC) tissues induced by repeated hypoxia, but not in rat cerebellum tissue. These suggest a relatively higher sensitivity and probably a higher vulnerability of rat PFC tissue to hypoxia in vivo. We reconfirmed the effect of hypoxia to primary cultured neurons from rat PFC and found a significant decrease in the contents of beta-III tubulin protein after chronic exposure to hypoxia. Moreover, we demonstrated that the hypoxia-induced decrease in beta-III tubulin protein content could be restored by curcumin, suggesting a potential protection of curcumin against hypoxia-induced decreases in beta-III tubulin content in rat PFC neurons.

Potential protection of green tea polyphenols against ultraviolet irradiation-induced injury on rat cortical neurons.

The present study was performed to investigate the possible protective effects of green tea polyphenols against ultraviolet (UV)-C light irradiation-induced cell death in the cultured rat cortical neurons. We found that UV-C light irradiation induced marked cell death tested by 3-(4,5-Dimethylthiazole-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) and TdT-mediated biotin-dUTP nicked-end labeling (TUNEL) assay. Protective effects of green tea polyphenols on UV-C light irradiation-induced apoptosis in cortical neurons were demonstrated by testing the content of Bax, which is involved in cell death. The expression of active Bax in cultured rat cortical neurons was inhibited significantly by green tea polyphenols compared to UV irradiation group tested by the immunoprecipitation assay and Western blot assay. However, there were no significant changes in the contents of total Bax after treatment with green tea polyphenols in UV-C light-irradiated rat cortical neurons. Our results demonstrated that the green tea polyphenols inhibited the active Bax expression, suggesting a neuroprotective effect of green tea polyphenols against the UV-C light irradiation-induced injury on cortical neurons.

Prolonged morphine application modulates Bax and Hsp70 levels in primary rat neurons.

Morphine has been used for pain treatment with a long history. Some data suggest that morphine is toxic to neurons and induces apoptosis, while other evidence shows that morphine could have beneficial effects against cell death. To determine how morphine affects pro-apoptotic protein Bax and molecular chaperone Hsp70, different concentrations of morphine were examined. Our results show that prolonged morphine administration for 5 days at 1microM concentration protects against serum deprivation induced cell death in rat primary neurons. Morphine treatment decreases Bax and Hsp70 levels in cultured rat primary neurons, suggesting morphine may have a protective role in staurosporine and serum deprivation induced cytotoxicity.

Involvement of galanin in nociceptive regulation in the arcuate nucleus of hypothalamus in rats with mononeuropathy.

The hindpaw withdrawal latencies (HWLs) to noxious thermal and mechanical stimulation increased significantly after intra-hypothalamic arcuate nucleus (ARC) injection of galanin in mononeuropathic rats, while intra-ARC injection of the putative antagonist of galanin receptors markedly reduced the HWLs. The number of galaninergic neurons in the ARC increased in rats with mononeuropathy than that in normal rats. The results demonstrated that both endogenous and exogenous galanin were involved in the regulation of nociception in the ARC of rats with peripheral nerve injury.

Involvement of opioid receptors in oxytocin-induced antinociception in the nucleus accumbens of rats.

UNLABELLED: Antinociceptive effects of oxytocin have been demonstrated in mice, rats, dogs, and humans. It has been shown that oxytocin receptors and fibers with oxytocin were distributed in the nucleus accumbens (NAc) of rats. The present study was performed to investigate the regulating role of oxytocin in nociception in the NAc of rats. Intra-NAc administration of oxytocin-induced dose-dependent increases in the hindpaw withdrawal latency (HWL) to noxious thermal and mechanical stimulation in rats, indicating that oxytocin has antinociceptive effects in the NAc of rats. Furthermore, the oxytocin-induced antinociceptive effects were attenuated by intra-NAc administration of the opioid-receptor antagonist naloxone, suggesting that the endogenous opioid system is involved in the oxytocin-induced antinociception in the NAc. Moreover, the oxytocin-induced antinociception was attenuated by intra-NAc injection of the kappa-receptor antagonist nor-binaltorphimine (nor-BNI) and the mu-receptor antagonist beta-funaltrexamine, but not by the delta-receptor antagonist naltrindole, demonstrating the involvements of mu- and kappa-receptors, but not delta-receptor, in the oxytocin-induced antinociception in the NAc of rats. PERSPECTIVE: This article supplements the evidence that oxytocin regulates nociception in the central nervous system. It presents additional material for clinical application of oxytocin as an analgesia drug.