Amelioration of morphine withdrawal syndrome by systemic and intranasal administration of mesenchymal stem cell-derived secretome in preclinical models of morphine dependence.

BACKGROUND: Morphine is an opiate commonly used in the treatment of moderate to severe pain. However, prolonged administration can lead to physical dependence and strong withdrawal symptoms upon cessation of morphine use. These symptoms can include anxiety, irritability, increased heart rate, and muscle cramps, which strongly promote morphine use relapse. The morphine-induced increases in neuroinflammation, brain oxidative stress, and alteration of glutamate levels in the hippocampus and nucleus accumbens have been associated with morphine dependence and a higher severity of withdrawal symptoms. Due to its rich content in potent anti-inflammatory and antioxidant factors, secretome derived from human mesenchymal stem cells (hMSCs) is proposed as a preclinical therapeutic tool for the treatment of this complex neurological condition associated with neuroinflammation and brain oxidative stress. METHODS: Two animal models of morphine dependence were used to evaluate the therapeutic efficacy of hMSC-derived secretome in reducing morphine withdrawal signs. In the first model, rats were implanted subcutaneously with mini-pumps which released morphine at a concentration of 10 mg/kg/day for seven days. Three days after pump implantation, animals were treated with a simultaneous intravenous and intranasal administration of hMSC-derived secretome or vehicle, and withdrawal signs were precipitated on day seven by i.p. naloxone administration. In this model, brain alterations associated with withdrawal were also analyzed before withdrawal precipitation. In the second animal model, rats voluntarily consuming morphine for three weeks were intravenously and intranasally treated with hMSC-derived secretome or vehicle, and withdrawal signs were induced by morphine deprivation. RESULTS: In both animal models secretome administration induced a significant reduction of withdrawal signs, as shown by a reduction in a combined withdrawal score. Secretome administration also promoted a reduction in morphine-induced neuroinflammation in the hippocampus and nucleus accumbens, while no changes were observed in extracellular glutamate levels in the nucleus accumbens. CONCLUSION: Data presented from two animal models of morphine dependence suggest that administration of secretome derived from hMSCs reduces the development of opioid withdrawal signs, which correlates with a reduction in neuroinflammation in the hippocampus and nucleus accumbens.

Systemic administration of N-acetylcysteine during the extinction period and on the reinstatement day decreased the maintenance of morphine rewarding properties in the rats.

Many animal studies and early clinical trials suggested that N-acetylcysteine (NAC) may benefit addiction treatment. The present study tried to evaluate whether chronic administration of systemic NAC during the extinction period and acute administration of systemic NAC on the reinstatement day could reduce the maintenance of the morphine rewarding properties in the conditioned place preference (CPP) paradigm in the rats. Ninety-six adult male Wistar rats (190-220 g) were examined with morphine (7 mg/kg; sc) and saline (1 mL/kg; sc) during the 3-day conditioning phase in the CPP paradigm. After the acquisition of morphine CPP, different doses of NAC were daily administered during the extinction period (5, 10, 25, and 50 mg/kg; ip), or 30 min before the CPP test on the reinstatement day (2, 5, 10, 25, and 50 mg/kg; ip). Conditioning score and locomotor activity were recorded by the video tracking system and Ethovision software after acquisition on the post-conditioning day, the extinction period, and reinstatement day. Daily NAC administration in high doses (25 and 50 mg/kg; ip) reduced extinction-responding compared with the vehicle-control group during the extinction period. Although a single injection of NAC in doses 10, 25, 50 mg/kg decreased the reinstatement of morphine-induced CPP, two lower doses (2 and 5 mg/kg) could not significantly reduce the CPP scores. These are the first data suggesting that NAC's application during the extinction period could attenuate the morphine reward-associated behaviors in the rats. Moreover, NAC could inhibit the reinstatement of morphine CPP, which adds to the growing appreciation that the NAC may have potential therapeutic use in combating morphine dependence. It can be consistent with the hypothesis of the involvement of the glutamatergic system in the pathophysiology of addiction.

Peptide Mimetic of BDNF Loop 4 Blocks Behavioral Signs of Morphine Withdrawal Syndrome and Prevents the Increase in ΔFosB Level in the Striatum of Rats.

Activity of compound GSB-106, a low-molecular mimetic of loop 4 of the brain neurotrophic factor (BDNF), was studied in experimental morphine withdrawal syndrome simulated in outbred rats. Single and subchronic (5 intraperitoneal injections) administration of GSB-106 in a dose of 0.1 mg/kg significantly reduced the total index of morphine withdrawal syndrome by 55.2 and 45.6%, respectively. GSB-106 reduced the severity of some behavioral signs (piloerection, gnashing of teeth, wet-dog shaking, and runaway attempts), but had no effect on mechanical allodynia formed in the rats with dependence. Subchronic treatment with GSB-106 prevented the increase in the content of ΔFosB (product of early response gene) in the striatum induced by morphine withdrawal. The results confirmed the concept on the involvement of neurotrophins, specifically BDNF and its analogs, in the mechanisms associated with the formation of opiate dependence.

Effect of Essential Oil of Zhumeria majdae on Morphine Tolerance and Dependence in Mice.

OBJECTIVE: To evaluate the effects of Zhumeria majdae essential oil (ZMEO) on morphine dependence and tolerance in mice. METHODS: ZMEO (10, 20, and 40 mg/kg) and clonidine (0.1 mg/kg) as the positive control were injected intraperitoneally (i.p.). The effect of ZMEO and clonidine on the dependence were evaluated by counting the number of jumps induced by naloxone (5 mg/kg) while the tolerance was evaluated by the tail-flick test. RESULTS: ZMEO at the dose of 10 mg/kg during the development period led to a significant inhibition of morphine tolerance (P<0.01), while it led to reduced morphine dependence with the doses of 20 and 40 mg/kg. ZMEO at two dose levels of 20 and 40 mg/kg indicated significant antinociceptive activity (P>0.01), and significantly reduced the withdrawal signs (number of jumps) of mice (P>0.01). CONCLUSIONS: ZMEO had significant effects on morphine tolerance and dependence. The linalool rich essential oil of Z. majdae plays a major role in the reduction of tolerance and dependence induced by morphine.

Oleoyl alanine (HU595): a stable monomethylated oleoyl glycine interferes with acute naloxone precipitated morphine withdrawal in male rats.

RATIONALE: Oleoyl glycine, a little studied fatty acid amide similar in structure to anandamide, interferes with nicotine addiction in mice and acute naloxone-precipitated morphine withdrawal (MWD) in rats. Because endogenous oleoyl glycine is subject to rapid enzymatic deactivation, we evaluated the potential of more stable analogs to interfere with opiate withdrawal. OBJECTIVES: The potential of monomethylated oleoyl glycine (oleoyl alanine, HU595) to interfere with somatic and aversive effects of acute naloxone-precipitated MWD, its duration, and mechanism of action was assessed in male Sprague Dawley rats. The potential of dimethylated oleoyl glycine (HU596) to interfere with the aversive effects of naloxone-precipitated MWD was also investigated. RESULTS: Oleoyl alanine (HU595) interfered with somatic and aversive effects produced by naloxone-precipitated MWD at equivalent doses (1 and 5 mg/kg, i.p.) as we have reported for oleoyl glycine; however, oleoyl alanine produced a longer lasting (60 min) interference, yet did not produce rewarding or aversive effects on its own and did not modify locomotor activity. HU596 was not effective. The interference with aversive effects of naloxone-precipitated MWD by oleoyl alanine was prevented by both a PPARα antagonist and a CB1 receptor antagonist. Accordingly, the compound was found to inhibit FAAH and activate PPARα in vitro. Finally, oleoyl alanine also reduced acute naloxone-precipitated MWD anhedonia, as measured by decreased saccharin preference. CONCLUSIONS: Oleoyl alanine (also an endogenous fatty acid) may be a more stable and effective treatment for opiate withdrawal than oleoyl glycine.

Acute naloxone-precipitated morphine withdrawal elicits nausea-like somatic behaviors in rats in a manner suppressed by N-oleoylglycine.

RATIONALE: Acute naloxone-precipitated morphine withdrawal (MWD) produces a conditioned place aversion (CPA) in rats even after one or two exposures to high-dose (20 mg/kg, sc) morphine followed 24-h later by naloxone (1 mg/kg, sc). However, the somatic withdrawal reactions produced by acute naloxone-precipitated MWD in rats have not been investigated. A recently discovered fatty acid amide, N-oleoylglycine (OlGly), which has been suggested to act as a fatty acid amide hydrolase (FAAH) inhibitor and as a peroxisome proliferator-activated receptor alpha (PPARα) agonist, was previously shown to interfere with a naloxone-precipitated MWD-induced CPA in rats. OBJECTIVES: The aims of these studies were to examine the somatic withdrawal responses produced by acute naloxone-precipitated MWD and determine whether OlGly can also interfere with these responses. RESULTS: Here, we report that following two exposures to morphine (20 mg/kg, sc) each followed by naloxone (1 mg/kg, sc) 24 h later, rats display nausea-like somatic reactions of lying flattened on belly, abdominal contractions and diarrhea, and display increased mouthing movements and loss of body weight. OlGly (5 mg/kg, ip) interfered with naloxone-precipitated MWD-induced abdominal contractions, lying on belly, diarrhea and mouthing movements in male Sprague-Dawley rats, by both a cannabinoid 1 (CB1) and a PPARα mechanism of action. Since these withdrawal reactions are symptomatic of nausea, we evaluated the potential of OlGly to interfere with lithium chloride (LiCl)-induced and MWD-induced conditioned gaping in rats, a selective measure of nausea; the suppression of MWD-induced gaping reactions by OlGly was both CB1 and PPARα mediated. CONCLUSION: These results suggest that the aversive effects of acute naloxone-precipitated MWD reflect nausea, which is suppressed by OlGly.

Effect of Yulangsan Polysaccharide on the Reinstatement of Morphine-Induced Conditioned Place Preference in Sprague-Dawley Rats.

We previously reported that Yulangsan polysaccharide (YLSP), which was isolated from the root of Millettia pulchra Kurz, attenuates withdrawal symptoms of morphine dependence by regulating the nitric oxide pathway and modulating monoaminergic neurotransmitters. In this study, we investigated the effects and mechanism of YLSP on the reinstatement of morphine-induced conditioned place preference (CPP) in rats. A CPP procedure was employed to assess the behavior of rats, and indicators of serum and four brain regions (nucleus accumbens, ventral tegmental area, hippocampus and prefrontal cortex) were determined to explore its underlying mechanism. YLSP inhibited priming morphine-induced reinstatement of CPP in a dose-dependent manner. YLSP markedly reduced nitric oxide and nitric oxide synthase levels in the brain. Moreover, YLSP significantly decreased the dopamine and norepinephrine levels in the serum and brain. Furthermore, YLSP significantly decreased cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) concentrations, inhibited the expression of dopamine D1 receptors and cAMP response element binding protein mRNA, and improved the expression of dopamine D2 receptor mRNA in the four brain regions. Our findings indicated that YLSP could inhibit the reinstatement of morphine-induced CPP possibly by modulating the NO-cGMP and D1R-cAMP signaling pathways.

Thalidomide attenuates development of morphine dependence in mice by inhibiting PI3K/Akt and nitric oxide signaling pathways.

Morphine dependence and the subsequent withdrawal syndrome restrict its clinical use in management of chronic pain. The precise mechanism for the development of dependence is still elusive. Thalidomide is a glutamic acid derivative, recently has been reconsidered for its clinical use due to elucidation of different clinical effects. Phosphoinositide 3-kinase (PI3K) is an intracellular transducer enzyme which activates Akt which in turns increases the level of nitric oxide. It is well established that elevated levels of nitric oxide has a pivotal role in the development of morphine dependence. In the present study, we aimed to explore the effect of thalidomide on the development of morphine dependence targeting PI3K/Akt (PKB) and nitric oxide (NO) pathways. Male NMRI mice and human glioblastoma T98G cell line were used to study the effect of thalidomide on morphine dependence. In both models the consequent effect of thalidomide on PI3K/Akt and/or NO signaling in morphine dependence was determined. Thalidomide alone or in combination with PI3K inhibitor, Akt inhibitor or nitric oxide synthase (NOS) inhibitors significantly reduced naloxone induced withdrawal signs in morphine dependent mice. Also, the levels of nitrite in hippocampus of morphine dependent mice were significantly reduced by thalidomide in compared to vehicle treated morphine dependent mice. In T98G human glioblastoma cells, thalidomide alone or in combination with PI3K and Akt inhibitors significantly reduced iNOS expression in comparison to the morphine treated cells. Also, morphine-induced p-Akt was suppressed when T98G cells were pretreated with thalidomide. Our results suggest that morphine induces Akt, which has a crucial role in the induction of NOS activity, leading to morphine dependence. Moreover, these data indicate that thalidomide attenuates the development of morphine dependence in vivo and in vitro by inhibition of PI3K/Akt and nitric oxide signaling pathways.

Reduced dosing and liability in methadone maintenance treatment by targeting oestrogen signal for morphine addiction.

Methadone maintenance treatment (MMT) is the major tapering therapy for morphine addictive patients. There have gender differences reported in response to MMT. This study discovered that the estrogen-response element single nucleotide polymorphism (ERE-SNP; rs16974799, C/T) of cytochrome 2B6 gene (cyp2b6; methadone catabolic enzyme) responded differently to MMT dosing. Oestradiol was associated with high MMT dosing, high enantiomer (R- or S-) of 2-ethylidene-1,5-dimethyl-3,3-dipheny-pyrrolidine (EDDP; methadone metabolite) to methadone ratio and increased drug-seeking behaviour, implicating oestradiol-CYP-EDDP/methadone axis decreasing MMT efficacy. In mouse model, oestrogen mitigates methadone antinociceptive response, facilitates methadone catabolism and up-regulates methadone-associated metabolizing enzymes. Oestrogen also ablates chronic methadone administration-induced rewarding response. Mechanism dissection revealed the CC genotype of CYP2B6-ERE-SNP exerts higher ERE sequence alignment score, higher estrogenic response as compared to TT genotype. At last, preclinical study via targeting estrogen signal that tamoxifen (TMX; selective estrogen receptor modulator, SERM) could facilitate the tolerance phase rewarding response of methadone. Strikingly, TMX also reduces tapering/abstinence phases methadone liability in mice. In conclusion, this study demonstrates altering methadone metabolism through targeting estrogen signals might be able to free morphine addictive patients from the addiction of opioid replacement therapy. Therefore, the add-on therapy clinical trial introducing SERM in MMT regimen is suggested.

Simvastatin prevents morphine-induced tolerance and dependence in mice.

BACKGROUND: Tolerance to analgesic effects of opioids and dependence to them are main concerns in the treatment of chronic pain conditions, limiting clinical application of these drugs. This study aimed to evaluate the effect of simvastatin on the morphine-induced tolerance and dependence in mice. MATERIAL AND METHODS: For this purpose, mice were treated with either daily morphine (20 mg/kg, s.c.) alone, or in combination with simvastatin (2.5, 5 and 10mg/kg, i.p.), for 9 continuous days. Antinociceptive effect of morphine was assessed through measuring latency time withdrawal of paw exposed to thermal stimulus, in the hot plate test. Naloxone-precipitated morphine withdrawal (5mg/kg, i.p.), was used for dependence evaluation. Changes in brain gene expression levels of induced nitric oxide synthase (iNOS), astroglia marker, glial fibrillary acidic protein (GFAP), ionized calcium-binding protein (Iba1) a microglia activation marker, a pro-inflammatory mediator and tumor necrosis alpha (TNF-α) were measured after withdrawal by real-time polymerase chain reaction (RT-PCR). RESULTS: Behavioral tests indicated that latency time increased after morphine treatment in the hot plate test. However, this effect decreased on day 7, demonstrating tolerance to antinociceptive effect of morphine. Reduced anti-nociceptive effect of morphine was returned in animals treated with simvastatin (5 and 10mg/kg) in combination with morphine. Simvastatin (5 and 10mg/kg) attenuated morphine dependence as indicated by a less severe antagonist-precipitated withdrawal syndrome. Administration of naloxone was associated with the increased expression of TNF-α, GFAP, Iba1 and iNOS in the brain samples of morphine dependent mice, while the nine days treatment with both 5 and 10mg/kg simvastatin reduced such changes. CONCLUSION: The obtained results showed that the protective effects of simvastatin against both tolerance to nociceptive effects of morphine as well as withdrawal-induced behavioral profile are meaningful. Inhibition of glia activity as well as antioxidant effects of pharmaceutical simvastatin further proves its neuroprotective property.

Protective effects of atorvastatin against morphine-induced tolerance and dependence in mice.

BACKGROUND: In this study, we evaluated the effects of atorvastatin, a lipid-lowering medication on morphine-induced tolerance and dependence in mice. METHODS: Tolerance was induced by subcutaneous administration of morphine (20mg/kg) to animals, twice a day for 9days. Atorvastatin was given at the doses of 5, 10 and 20mg/kg, 30min before each morphine administration, once daily for 9days. Hot plate test was employed to assess antinociceptive effect of morphine on days 1, 3, 5, 7 and 9. Dependence was evaluated by naloxone-precipitated withdrawal syndrome. We attempted to verify withdrawal regulation of induced nitric oxide synthase (iNOS), astroglia marker, glial fibrillary acidic protein (GFAP), ionized calcium-binding protein (Iba1), a microglia activation marker, a pro-inflammatory mediator, tumor necrosis alpha (TNF-α) and immune receptor, toll like receptor 4 (TLR-4) genes by real-time polymerase chain reaction (RT-PCR). Lipid peroxidation was estimated by assessing malondialdehyde (MDA) content in the spinal cord of animals. RESULTS: Tolerance to antinociceptive effects of morphine was observed on days 7 and 9. Decrease in morphine-induced antinociception was reversed by concomitant intraperitoneal administration of atorvastatin (10 and 20mg/kg). Atorvastatin (10 and 20mg/kg) mitigated naloxone-induced withdrawal parameters. Brain expression levels of TNF-α, GFAP, Iba1 and iNOS increased in morphine withdrawn animals which were attenuated by nine days treatment with atorvastatin. Increased MDA was also normalized in withdrawn animals received atorvastatin. CONCLUSION: Atorvastatin exhibits meaningful protective effects against both tolerance to antinociceptive effects of morphine and withdrawal-induced behavioral profile. Neuroprotective effects of atorvastatin is further supported via inhibition of glia activity and antioxidant effects.

Bergenin decreases the morphine-induced physical dependence via antioxidative activity in mice.

Oxidative stress plays a role in the development of physical dependence induced by morphine. Bergenin, a polyphenol found in many Asian, African, and South American medicinal plants, is a potent antinarcotic agent with wide spectrum of pharmacological activities including antioxidant action. In the present study, we observed that bergenin decreased the development of physical dependence induced by morphine in mice and the antioxidant activity of bergenin plays a role in the antinarcotic effects through adapting to morphine-induced oxidative stress in the brain. The naloxone-precipitated withdrawal symptom (jumping frequency) was significantly ameliorated (50% of control group) by administration of bergenin (20 mg/kg) in morphine-treated mice. Furthermore, morphine-induced down-regulation of glutathione (GSH) contents was reversed by bergenin administration in the frontal cortex and liver. Bergenin had no effects on the increased levels of nfr2-dependent antioxidant enzyme HO1 and NQO1 in the frontal cortex, striatum, and liver of morphine-treated mice. However, the morphine-induced increase in nrf2 nuclear translocation in the frontal cortex and striatum was inhibited by bergenin treatment. These results suggest that bergenin has a potential antinarcotic effect via regulation of GSH contents and oxidative stress.

Gap junction blockers: a potential approach to attenuate morphine withdrawal symptoms.

BACKGROUND: The exact mechanisms of morphine-induced dependence and withdrawal symptoms remain unclear. In order to identify an agent that can prevent withdrawal syndrome, many studies have been performed. This study was aimed to evaluate the effect of gap junction blockers; carbenoxolone (CBX) or mefloquine (MFQ); on morphine withdrawal symptoms in male rat. Adult male Wistar rats (225 - 275 g) were selected randomly and divided into 10 groups. All groups underwent stereotaxic surgery and in order to induce dependency, morphine was administered subcutaneously) Sc) at an interval of 12 hours for nine continuous days. On the ninth day of the experiment, animals received vehicle or CBX (100, 400, 600 µg/10 µl/rat, icv) or MFQ (50, 100 and 200 µg/10 µl/rat, icv) after the last saline or morphine (Sc) injection. Morphine withdrawal symptoms were precipitated by naloxone hydrochloride 10 min after the treatments. The withdrawal signs including: jumping, rearing, genital grooming, abdomen writhing, wet dog shake and stool weight, were recorded for 60 minutes. RESULTS: Results showed that CBX and MFQ decreased all withdrawal signs; and the analysis indicated that they could attenuate the total withdrawal scores significantly. CONCLUSION: Taking together it is concluded that gap junction blockers prevented naloxone-precipitated withdrawal symptoms.

Role of oxidative stress and inducible nitric oxide synthase in morphine-induced tolerance and dependence in mice. Effect of alpha-lipoic acid.

In this study, the possible role of oxidative stress and nitric oxide (NO) synthase isoforms in the development of morphine tolerance and dependence, and effect of alpha-lipoic acid on these parameters were investigated in mice. The development of morphine tolerance as measured by the hot plate test and dependence, as assessed by naloxone-precipitated withdrawal manifestations, produced an increase in brain glutamate and malondialdehyde (MDA) levels and NO production as well as a decrease in brain intracellular reduced glutathione (GSH) level and glutathione peroxidase (GSH-Px) activity. Also, the development of these syndromes increased inducible but not neuronal NO synthase mRNA and protein expressions in mice brain. Co-administration of alpha-lipoic acid (α-LA) inhibited the development of morphine tolerance and dependence, their associated biochemical alterations, except elevation of brain glutamate level, and their associated increase in brain inducible NO synthase mRNA and protein expressions. The inhibitory effect of α-LA on morphine-induced tolerance and dependence and on naloxone-induced biochemical alterations in morphine-dependent mice was enhanced by concurrent administration of the NMDA receptor antagonist, dizocilpine, the antioxidant, N-acetylcysteine or the selective inducible NO synthase inhibitor, aminoguanidine. On the other hand, this inhibitory effect of α-LA was not changed by concurrent administration of the selective neuronal NO synthase inhibitor, 7-nitroindazole but antagonized by concurrent administration of the NO precursor, L-arginine. These results suggest that α-LA through inhibition of morphine-induced oxidative stress and increase in the expression and activity of inducible NO synthase in the brain can attenuate the development of morphine tolerance and dependence.

Inhibition of brain oxidative stress and inducible nitric oxide synthase expression by thymoquinone attenuates the development of morphine tolerance and dependence in mice.

In this study, the effect of thymoquinone on morphine-induced tolerance and dependence in mice was investigated. Repeated administration of thymoquinone along with morphine attenuated the development of morphine tolerance, as measured by the hot plate test, and dependence, as assessed by naloxone-precipitated withdrawal manifestations. Concurrently, morphine-induced progressive increase in brain malondialdehyde (MDA) level and nitric oxide (NO) production as well as progressive decrease in brain intracellular reduced glutathione (GSH) level and glutathione peroxidase (GSH-Px) activity were inhibited by co-administration of thymoquinone. Morphine-induced progressive increase in brain glutamate level was not inhibited by concomitant administration of thymoquinone. Similarly, co-administration of thymoquinone inhibited naloxone-induced increase in brain MDA level, NO overproduction and decrease in brain intracellular GSH level and GSH-Px activities but it did not inhibit naloxone-induced elevation of brain glutamate level in morphine-dependent mice. The inhibitory effect of thymoquinone on morphine-induced tolerance and dependence on naloxone-induced biochemical alterations in morphine-dependent mice was enhanced by concurrent i.p. administration of the NMDA receptor antagonist, dizocilpine, the antioxidant, N-acetylcysteine or the NO synthase inhibitor, L-N (G)-nitroarginine methyl ester. On the other hand, this inhibitory effect of thymoquinone was antagonized by concurrent i.p. administration of NO precursor, L-arginine. In addition, concomitant administration of thymoquinone inhibited morphine tolerance and dependence-induced increase in inducible but not in neuronal NO synthase mRNA expression in mice brain. These results demonstrate that inhibition of morphine-induced oxidative stress, increase in the expression of brain inducible NO synthase and NO overproduction by thymoquinone can attenuate the development of morphine tolerance and dependence.

Involvement of noradrenergic transmission in the PVN on CREB activation, TORC1 levels, and pituitary-adrenal axis activity during morphine withdrawal.

Experimental and clinical findings have shown that administration of adrenoceptor antagonists alleviated different aspects of drug withdrawal and dependence. The present study tested the hypothesis that changes in CREB activation and phosphorylated TORC1 levels in the hypothalamic paraventricular nucleus (PVN) after naloxone-precipitated morphine withdrawal as well as the HPA axis activity arises from α(1)- and/or ß-adrenoceptor activation. The effects of morphine dependence and withdrawal on CREB phosphorylation (pCREB), phosphorylated TORC1 (pTORC1), and HPA axis response were measured by Western-blot, immunohistochemistry and radioimmunoassay in rats pretreated with prazosin (α(1)-adrenoceptor antagonist) or propranolol (ß-adrenoceptor antagonist). In addition, the effects of morphine withdrawal on MHPG (the main NA metabolite at the central nervous system) and NA content and turnover were evaluated by HPLC. We found an increase in MHPG and NA turnover in morphine-withdrawn rats, which were accompanied by increased pCREB immunoreactivity and plasma corticosterone concentrations. Levels of the inactive form of TORC1 (pTORC1) were decreased during withdrawal. Prazosin but not propranolol blocked the rise in pCREB level and the decrease in pTORC1 immunoreactivity. In addition, the HPA axis response to morphine withdrawal was attenuated in prazosin-pretreated rats. Present results suggest that, during acute morphine withdrawal, NA may control the HPA axis activity through CREB activation at the PVN level. We concluded that the combined increase in CREB phosphorylation and decrease in pTORC1 levels might represent, in part, two of the mechanisms of CREB activation at the PVN during morphine withdrawal.

RSK2 signaling in medial habenula contributes to acute morphine analgesia.

It has been established that mu opioid receptors activate the ERK1/2 signaling cascade both in vitro and in vivo. The Ser/Thr kinase RSK2 is a direct downstream effector of ERK1/2 and has a role in cellular signaling, cell survival growth, and differentiation; however, its role in biological processes in vivo is less well known. Here we determined whether RSK2 contributes to mu-mediated signaling in vivo. Knockout mice for the rsk2 gene were tested for main morphine effects, including analgesia, tolerance to analgesia, locomotor activation, and sensitization to this effect, as well as morphine withdrawal. The deletion of RSK2 reduced acute morphine analgesia in the tail immersion test, indicating a role for this kinase in mu receptor-mediated nociceptive processing. All other morphine effects and adaptations to chronic morphine were unchanged. Because the mu opioid receptor and RSK2 both show high density in the habenula, we specifically downregulated RSK2 in this brain metastructure using an adeno-associated-virally mediated shRNA approach. Remarkably, morphine analgesia was significantly reduced, as observed in the total knockout animals. Together, these data indicate that RSK2 has a role in nociception, and strongly suggest that a mu opioid receptor-RSK2 signaling mechanism contributes to morphine analgesia at the level of habenula. This study opens novel perspectives for both our understanding of opioid analgesia, and the identification of signaling pathways operating in the habenular complex.

Role of melatonin in the prevention of morphine-induced hyperalgesia and spinal glial activation in rats: protein kinase C pathway involved.

PURPOSES: Morphine can induce tolerance and hyperalgesia after long-term administration. Glial activation is believed to cause and maintain a state of morphine-induced pain hypersensitivity. The present study examines the effect of melatonin on tolerance, hyperalgesia, and reactive gliosis induced by morphine in rats. METHODS: The study examines the effect of melatonin on morphine-induced hyperalgesia using tail-flick test. Immunohistochemistry and Western blot was performed to investigate the expression of glial fibrillary acidic protein (GFAP) indicative of spinal glial activity. This study also measures protein kinase C (PKC) activity and cyclic adenosine monophosphate (cAMP) levels in spinal cords to investigate the mechanisms which melatonin involved. RESULTS: When coadministered intragastrically (i.g.) with morphine, melatonin in doses of 50 or 100 mg/kg significantly prevented hyperalgesia after termination of morphine. Immunohistochemistry and Western blot with GFAP revealed that melatonin significantly decreased morphine-induced over-expression of GFAP in spinal cord (p < .05). By measuring PKC activity and cAMP levels, the upregulated PKC activity and cAMP levels induced by morphine were significantly inhibited by melatonin. CONCLUSIONS: Melatonin can prevent morphine-withdrawal-induced hyperalgesia and glial reactivity. This effect of melatonin after morphine administration may mediated by inhibiting PKC activity and cAMP upregulation.

Blockade of nitric oxide overproduction and oxidative stress by Nigella sativa oil attenuates morphine-induced tolerance and dependence in mice.

The effects of Nigella sativa oil on morphine-induced tolerance and dependence in mice and possible mechanism(s) of these effects were investigated, for the first time, in this study. Repeated administration of Nigella sativa oil (4 ml/kg, p.o.) along with morphine (5 mg/kg, s.c.) attenuated the development of tolerance, as measured by the hot plate test, and dependence, as assessed by naloxone (5 mg/kg, i.p.)-precipitated withdrawal manifestations. Concomitantly, nitric oxide overproduction and increase in brain malondialdehyde level induced by repeated administration of morphine to mice or by administration of naloxone to morphine-dependent mice were inhibited by co-administration of the oil. Also, the decrease in brain intracellular reduced glutathione level and glutathione peroxidase activity induced by both treatments were inhibited by co-administration of the oil. The increase in brain glutamate level induced by both treatments was not inhibited by concurrent administration of the oil. The inhibitory effect of the oil on morphine-induced tolerance and dependence and on naloxone-induced biochemical alterations in morphine-dependent mice was enhanced by concurrent i.p. administration of the NMDA receptor antagonist, dizocilpine (0.25 mg/kg). Similarly, concurrent i.p. administration of the NO synthase inhibitors; L-N (G)-nitroarginine methyl ester (10 mg/kg), aminoguanidine (20 mg/kg) and 7-nitroindazole (25 mg/kg) or the antioxidant, N-acetylcysteine (50 mg/kg) enhanced this inhibitory effect of the oil. On the other hand, this effect was antagonized by concurrent i.p. administration of the nitric oxide precursor, L-arginine (300 mg/kg). These results provide evidence that Nigella sativa oil, through inhibition of morphine-induced NO overproduction and oxidative stress, appears to have a therapeutic potential in opioid tolerance and dependence.

Enhancement of tolerance development to morphine in rats prenatally exposed to morphine, methadone, and buprenorphine.

BACKGROUND: Abuse of addictive substances is a serious problem that has a significant impact on areas such as health, the economy, and public safety. Heroin use among young women of reproductive age has drawn much attention around the world. However, there is a lack of information on effects of prenatal exposure to opioids on their offspring. In this study, an animal model was established to study effects of prenatal exposure to opioids on offspring. METHODS: Female pregnant Sprague-Dawley rats were sub-grouped to receive (1) vehicle, (2) 2-4 mg/kg morphine (1 mg/kg increment per week), (3) 7 mg/kg methadone, and (4) 3 mg/kg buprenorphine, subcutaneously, once or twice a day from E3 to E20. The experiments were conducted on animals 8-12 weeks old and with body weight between 250 and 350 g. RESULTS: Results showed that prenatal exposure to buprenorphine caused higher mortality than other tested substance groups. Although we observed a significantly lower increase in body weight in all of the opioid-administered dams, the birth weight of the offspring was not altered in all treated groups. Moreover, no obvious behavioral abnormality or body-weight difference was noted during the growing period (8-12 weeks) in all offspring. When the male offspring received morphine injection twice a day for 4 days, the prenatally opioid-exposed rats more quickly developed a tolerance to morphine (as shown by the tail-flick tests), most notably the prenatally buprenorphine-exposed offspring. However, the tolerance development to methadone or buprenorphine was not different in offspring exposed prenatally to methadone or buprenorphine, respectively, when compared with that of the vehicle controlled group. Similar results were also obtained in the female animals. CONCLUSIONS: Animals prenatally exposed to morphine, methadone, or buprenorphine developed tolerance to morphine faster than their controlled mates. In our animal model, prenatal exposure to buprenorphine also resulted in higher mortality and much less sensitivity to morphine-induced antinociception than prenatal exposure to morphine or methadone. This indicates that buprenorphine in higher doses may not be an ideal maintenance drug for treating pregnant women. This study provides a reference in selecting doses for clinical usage in treating pregnant heroin addicts.