Development Of Morphine Tolerance Research Articles

Astaxanthin has a beneficial influence on pain-related symptoms and opioid-induced hyperalgesia in mice with diabetic neuropathy-evidence from behavioral studies.

The treatment of painful diabetic neuropathy is still a clinical problem. The aim of this study was to determine whether astaxanthin, a substance that inhibits mitogen-activated protein kinases, activates nuclear factor erythroid 2-related factor 2 and influences N-methyl-D-aspartate receptor, affects nociceptive transmission in mice with diabetic neuropathy. The studies were performed on streptozotocin-induced mouse diabetic neuropathic pain model. Single intrathecal and intraperitoneal administrations of astaxanthin at various doses were conducted in both males and females. Additionally, repeated twice-daily treatment with astaxanthin (25mg/kg) and morphine (30mg/kg) were performed. Hypersensitivity was evaluated with von Frey and cold plate tests. This behavioral study provides the first evidence that in a mouse model of diabetic neuropathy, single injections of astaxanthin similarly reduce tactile and thermal hypersensitivity in both male and female mice, regardless of the route of administration. Moreover, repeated administration of astaxanthin slightly delays the development of morphine tolerance and significantly suppresses the occurrence of opioid-induced hyperalgesia, although it does not affect blood glucose levels, body weight, or motor coordination. Surprisingly, astaxanthin administered repeatedly produces a better analgesic effect when administered alone than in combination with morphine, and its potency becomes even more pronounced over time. These behavioral results provide a basis for further evaluation of the potential use of astaxanthin in the clinical treatment of diabetic neuropathy and suggest that the multidirectional action of this substance may have positive effects on relieving neuropathic pain in diabetes.

Targeting exosomal double-stranded RNA-TLR3 signaling pathway attenuates morphine tolerance and hyperalgesia

Long-term morphine use leads to tolerance and hyperalgesia in patients with chronic pain, with neuroinflammation playing a key role, but its underlying mechanisms remain elusive. This study determines that repeated intrathecal morphine injections increase double-stranded RNA (dsRNA) production in spinal neurons, due to downregulated adenosine deaminase RNA specific 1 (ADAR1) expression. Lentivirus-induced ADAR1 elevation decreases the high levels of intracellular dsRNA and attenuates morphine tolerance and hyperalgesia. dsRNA is released into cerebrospinal fluid via exosomes (Exos) after repeated morphine injections and is taken up by microglia for TLR3-TRIF-IL-6 signaling activation. Blocking Exos release with GW4869 or inhibition of TLR3 signaling mitigates neuroinflammation, preventing the development of morphine tolerance and hyperalgesia. Intrathecal injection of TLR3 inhibitor alone shows analgesic effects in neuropathic pain, and co-administration with morphine amplifies the analgesic efficacy of morphine. These findings demonstrate that targeting dsRNA-TLR3 signaling to mitigate neuroinflammation could be a promising treatment for morphine tolerance.

Ferroptosis inhibitor ferrostatin-1 attenuates morphine tolerance development in male rats by inhibiting dorsal root ganglion neuronal ferroptosis.

Ferrostatin-1 and liproxstatin-1, both ferroptosis inhibitors, protect cells. Liproxstatin-1 decreases morphine tolerance. Yet, ferrostatin-1's effect on morphine tolerance remains unexplored. This study aimed to evaluate the influence of ferrostatin-1 on the advancement of morphine tolerance and understand the underlying mechanisms in male rats. This experiment involved 36 adult male Wistar albino rats with an average weight ranging from 220 to 260 g. These rats were categorized into six groups: Control, single dose ferrostatin-1, single dose morphine, single dose ferrostatin-1 + morphine, morphine tolerance (twice daily for five days), and ferrostatin-1 + morphine tolerance (twice daily for five days). The antinociceptive action was evaluated using both the hot plate and tail-flick tests. After completing the analgesic tests, tissue samples were gathered from the dorsal root ganglia (DRG) for subsequent analysis. The levels of glutathione, glutathione peroxidase 4 (GPX4), and nuclear factor erythroid 2-related factor 2 (Nrf2), along with the measurements of total oxidant status (TOS) and total antioxidant status (TAS), were assessed in the tissues of the DRG. After tolerance development, the administration of ferrostatin-1 resulted in a significant decrease in morphine tolerance (P < 0.001). Additionally, ferrostatin-1 treatment led to elevated levels of glutathione, GPX4, Nrf2, and TOS (P < 0.001), while simultaneously causing a decrease in TAS levels (P < 0.001). The study found that ferrostatin-1 can reduce morphine tolerance by suppressing ferroptosis and reducing oxidative stress in DRG neurons, suggesting it as a potential therapy for preventing morphine tolerance.

Biasing Gβγ Downstream Signaling with Gallein Inhibits Development of Morphine Tolerance and Potentiates Morphine-Induced Nociception in a Tolerant State.

Opioid analgesics are widely used as a treatment option for pain management and relief. However, the misuse of opioid analgesics has contributed to the current opioid epidemic in the United States. Prescribed opioids such as morphine, codeine, oxycodone, and fentanyl are mu-opioid receptor (MOR) agonists primarily used in the clinic to treat pain or during medical procedures, but development of tolerance limits their utility for treatment of chronic pain. Here we explored the effects of biasing Gβγ signaling on tolerance development after chronic morphine treatment in vivo. We hypothesized that biasing Gβγ signaling with gallein could prevent activation of regulatory signaling pathways that result in tolerance to antinociceptive effects of MOR agonists. Gallein has been shown to bind to Gβγ and inhibit interactions of Gβγ with phospholipase-Cβ3 (PLCβ3) or G-protein-coupled receptor kinase 2 (GRK2) but not G-protein inwardly rectifying potassium (GIRK) channels. In mice, morphine-induced antinociception was evaluated in the 55°C warm water tail withdrawal assay. We used two paradigms for gallein treatment: administration during and after three times-daily morphine administration. Our results show that gallein cotreatment during repeated administration of morphine decreased opioid tolerance development and that gallein treatment in an opioid-tolerant state enhanced the potency of morphine. Mechanistically, our data suggest that PLCβ3 is necessary for potentiating effects of gallein in an opioid-tolerant state but not in preventing the development of tolerance. These studies demonstrate that small molecules that target Gβγ signaling could reduce the need for large doses of opioid analgesics to treat pain by producing an opioid-sparing effect. SIGNIFICANCE STATEMENT: Biasing Gβγ signaling prevents tolerance to repeated morphine administration in vivo and potentiates the antinociceptive effects of morphine in an opioid-tolerant state. Mechanistically, phospholipase-Cβ is necessary for potentiating effects of gallein in an opioid-tolerant state but not in preventing the development of tolerance. This study identifies a novel treatment strategy to decrease the development of tolerance to the analgesic effects of mu-opioid receptor agonists, which are necessary to improve pain treatment and decrease the incidence of opioid use disorder.

Tiam1-mediated maladaptive plasticity underlying morphine tolerance and hyperalgesia.

Opioid pain medications, such as morphine, remain the mainstay for treating severe and chronic pain. Prolonged morphine use, however, triggers analgesic tolerance and hyperalgesia (OIH), which can last for a long period after morphine withdrawal. How morphine induces these detrimental side effects remains unclear. Here, we show that morphine tolerance and OIH are mediated by Tiam1-coordinated synaptic structural and functional plasticity in the spinal nociceptive network. Tiam1 is a Rac1 GTPase guanine nucleotide exchange factor that promotes excitatory synaptogenesis by modulating actin cytoskeletal dynamics. We found that prolonged morphine treatment activated Tiam1 in the spinal dorsal horn and Tiam1 ablation from spinal neurons eliminated morphine antinociceptive tolerance and OIH. At the same time, the pharmacological blockade of Tiam1-Rac1 signalling prevented the development and reserved the established tolerance and OIH. Prolonged morphine treatment increased dendritic spine density and synaptic NMDA receptor activity in spinal dorsal horn neurons, both of which required Tiam1. Furthermore, co-administration of the Tiam1 signalling inhibitor NSC23766 was sufficient to abrogate morphine tolerance in chronic pain management. These findings identify Tiam1-mediated maladaptive plasticity in the spinal nociceptive network as an underlying cause for the development and maintenance of morphine tolerance and OIH and provide a promising therapeutic target to reduce tolerance and prolong morphine use in chronic pain management.

Inhibition of spinal BRD4 alleviates pyroptosis and M1 microglia polarization via STING-IRF3 pathway in morphine-tolerant rats

BackgroundMorphine tolerance has been a challenging medical issue. Neuroinflammation is considered as a critical mechanism for the development of morphine tolerance. Bromodomain-containing protein 4 (BRD4), a key regulator in cell damage and inflammation, participates in the development of chronic pain. However, whether BRD4 is involved in morphine tolerance and the underlying mechanisms remain unknown. MethodsThe morphine-tolerant rat model was established by intrathecal administration of morphine twice daily for 7 days. Behavior test was assessed by a tail-flick latency test. The roles of BRD4, pyroptosis, microglia polarization and related signaling pathways in morphine tolerance were elucidated by Western blot, real-time quantitative polymerase chain reaction, and immunofluorescence. ResultsRepeated morphine administration upregulated BRD4 level, induced pyroptosis, and promoted microglia M1-polarization in spinal cord, accompanied by the release of proinflammatory cytokines, such as TNF-α and IL-1β. JQ-1, a BRD4 antagonist, alleviated the development of morphine tolerance, diminished pyroptosis and induced the switch of microglia from M1 to M2 phenotype. Mechanistically, stimulator of interferon gene (STING)- interferon regulatory factor 3 (IRF3) pathway was activated and the protective effect of JQ-1 against morphine tolerance was at least partially mediated by inhibition of STING-IRF3 pathway. ConclusionThis study demonstrated for the first time that spinal BRD4 contributes to pyroptosis and switch of microglia polarization via STING-IRF3 signaling pathway during the development of morphine tolerance, which extend the understanding of the neuroinflammation mechanism of morphine tolerance and provide an alternative strategy for the precaution against of this medical condition.

Astrocyte PERK and IRE1 Signaling Contributes to Morphine Tolerance and Hyperalgesia through Upregulation of Lipocalin-2 and NLRP3 Inflammasome in the Rodent Spinal Cord.

Endoplasmic reticulum stress plays a crucial role in the pathogenesis of neuroinflammation and chronic pain. This study hypothesized that PRKR-like endoplasmic reticulum kinase (PERK) and inositol-requiring enzyme type 1 (IRE1) regulate lipocalin-2 (LCN2) and Nod-like receptor family pyrin domain containing 3 (NLRP3) expression in astrocytes, thereby contributing to morphine tolerance and hyperalgesia. The study was performed in Sprague-Dawley rats and C57/Bl6 mice of both sexes. The expression of LCN2 and NLRP3 was assessed by Western blotting. The tail-flick, von Frey, and Hargreaves tests were used to evaluate nociceptive behaviors. Chromatin immunoprecipitation was conducted to analyze the binding of activating transcription factor 4 (ATF4) to the promoters of LCN2 and TXNIP. Whole-cell patch-clamp recordings were used to evaluate neuronal excitability. Pharmacologic inhibition of PERK and IRE1 attenuated the development of morphine tolerance and hyperalgesia in male (tail latency on day 7, 8.0 ± 1.13 s in the morphine + GSK2656157 [10 μg] group vs. 5.8 ± 0.65 s in the morphine group; P = 0.04; n = 6 rats/group) and female (tail latency on day 7, 6.0 ± 0.84 s in the morphine + GSK2656157 [10 μg] group vs. 3.1 ± 1.09 s in the morphine group; P = 0.0005; n = 6 rats/group) rats. Activation of PERK and IRE1 upregulated expression of LCN2 and NLRP3 in vivo and in vitro. Chromatin immunoprecipitation analysis showed that ATF4 directly bound to the promoters of the LCN2 and TXNIP. Lipocalin-2 induced neuronal hyperexcitability in the spinal cord and dorsal root ganglia via melanocortin-4 receptor. Astrocyte endoplasmic reticulum stress sensors PERK and IRE1 facilitated morphine tolerance and hyperalgesia through upregulation of LCN2 and NLRP3 in the spinal cord.

Amiloride alleviates morphine tolerance by suppressing ASIC3-dependent neuroinflammation in the spinal cord

BackgroundThe use of morphine in clinical medicine is severely constrained by tolerance. Therefore, it is essential to examine pharmacological therapies that suppress the development of morphine tolerance. Amiloride suppressed the expression of inflammatory cytokines by inhibiting microglial activation. Microglia play a crucial role in the establishment of morphine tolerance. Thus, we anticipated that amiloride might suppress the development of morphine tolerance. During this investigation, we assessed the impact of amiloride on mouse morphine tolerance. MethodsMice received morphine (10 mg/kg, s.c.) twice daily with intrathecally injected amiloride (0.3 μg/5 μl, 1 μg/5 μl, and 3 μg/5 μl) for nine continuous days. To assess morphine tolerance, mice underwent the tail-flick and hot plate tests. BV-2 cells were used to investigate the mechanism of amiloride. By using Western blotting, real-time PCR, and immunofluorescence labeling methods, the levels of acid-sensing ion channels (ASICs), nuclear factor kappa B (NF-kB) p65, p38 mitogen-activated protein kinase (MAPK) proteins, and neuroinflammation-related cytokines were determined. ResultsThe levels of ASIC3 in the spinal cord were considerably increased after long-term morphine administration. Amiloride was found to delay the development of tolerance to chronic morphine assessed via tail-flick and hot plate tests. Amiloride reduced microglial activation and downregulated the cytokines IL-1β and TNF-a by inhibiting ASIC3 in response to morphine. Furthermore, amiloride reduced p38 MAPK phosphorylation and inhibited NF-κB expression. ConclusionsAmiloride effectively reduces chronic morphine tolerance by suppressing microglial activation caused by morphine by inhibiting ASIC3.

μ opioid receptor carboxyl terminal-derived peptide alleviates morphine tolerance by inhibiting β-arrestin2.

The interaction between the μ opioid receptor (MOR) and β-arrestin2 serves as a model for addressing morphine tolerance. A peptide was designed to alleviate morphine tolerance through interfering with the interaction of MOR and β-arrestin2. We developed a peptide derived from MOR. The MOR-TAT-pep peptide was expressed in E. coli Bl21(DE3) and purified. The effects of MOR-TAT-pep in alleviating morphine tolerance was examined through behavior tests. The potential mechanism was detected by Western blotting, Mammalian Two-Hybrid and other techniques. The pretreatment with MOR-TAT-pep prior to morphine usage led to an enhanced analgesic effectiveness of morphine and a significant reduction in the development of morphine tolerance. The peptide directly interacted with β-arrestin2 during morphine treatment and deceased the membrane recruitment of β-arrestin2. MOR-TAT-pep effectively suppressed the increase of β-arrestin2 induced by morphine. The MOR-TAT-pep could alleviate morphine tolerance through inhibition of β-arrestin2.

Toll-like Receptor 2-Melatonin Feedback Loop Regulates the Activation of Spinal NLRP3 Inflammasome in Morphine-Tolerant Rats.

Morphine is amongst the most effective analgesics available for the management of severe pain. However, prolonged morphine treatment leads to analgesic tolerance which limits its clinical usage. Previous studies have demonstrated that melatonin ameliorates morphine tolerance by reducing neuroinflammation. However, little is known about the relationship between Toll like receptor 2 (TLR2) and neuroinflammation in morphine tolerance. The aim of this study was to explore the role of TLR2 in morphine tolerance and its connections with melatonin and Nod-like receptor protein 3 (NLRP3) inflammasome. Sprague-Dawley rats were treated with morphine for 7 days and tail-flick latency test was performed to identify the induction of analgesic tolerance. The roles of TLR2 in microglia activation and morphine tolerance were assessed pharmacologically, and the possible interactions between melatonin, TLR2 and NLRP3 inflammasome were investigated. Morphine tolerance was accompanied by increased TLR2 expression and NLRP3 inflammasome activation in spinal cord. whereas melatonin level was down-regulated. Chronic melatonin administration resulted in a reduced TLR2 expression and NLRP3 inflammasome activation. Moreover, the analgesic effect of morphine was partially restored. Inhibition of TLR2 suppressed the microglia and NLRP3 inflammasome activation, as well as restored the spinal melatonin level while attenuated the development of morphine tolerance. Furthermore, the inhibition of microglia activation ameliorated morphine tolerance via inhibiting TLR2-NLRP3 inflammasome signaling in spinal cord. In this study, we directly demonstrate a TLR2-melatonin negative feedback loop regulating microglia and NLRP3 inflammasome activation during the development of morphine tolerance.

Monoclonal antibody targeting mu-opioid receptor attenuates morphine tolerance via enhancing morphine-induced receptor endocytosis

Morphine is a frequently used analgesic that activates the mu-opioid receptor (MOR), which has prominent side effects of tolerance. Although the inefficiency of morphine in inducing the endocytosis of MOR underlies the development of morphine tolerance, currently, there is no effective therapy to treat morphine tolerance. In the current study, we aimed to develop a monoclonal antibody (mAb) precisely targeting MOR and to determine its therapeutic efficacy on morphine tolerance and the underlying molecular mechanisms. We successfully prepared a mAb targeting MOR, named 3A5C7, by hybridoma technique using a strategy of deoxyribonucleic acid immunization combined with cell immunization, and identified it as an immunoglobulin G mAb with high specificity and affinity for MOR and binding ability to antigens with spatial conformation. Treatment of two cell lines, HEK293T and SH-SY5Y, with 3A5C7 enhanced morphine-induced MOR endocytosis via a G protein-coupled receptor kinase 2 (GRK2)/β-arrestin2-dependent mechanism, as demonstrated by immunofluorescence staining, flow cytometry, Western blotting, coimmunoprecipitation, and small interfering ribonucleic acid (siRNA)-based knockdown. This mAb also allowed MOR recycling from cytoplasm to plasma membrane and attenuated morphine-induced phosphorylation of MOR. We established an in vitro morphine tolerance model using differentiated SH-SY5Y cells induced by retinoic acid. Western blot, enzyme-linked immunosorbent assays, and siRNA-based knockdown revealed that 3A5C7 mAb diminished hyperactivation of adenylate cyclase, the in vitro biomarker of morphine tolerance, via the GRK2/β-arrestin2 pathway. Furthermore, in vivo hotplate test demonstrated that chronic intrathecal administration of 3A5C7 significantly alleviated morphine tolerance in mice, and withdrawal jumping test revealed that both chronic and acute 3A5C7 intrathecal administration attenuated morphine dependence. Finally, intrathecal electroporation of silencing short hairpin RNA illustrated that the in vivo anti-tolerance and anti-dependence efficacy of 3A5C7 was mediated by enhanced morphine-induced MOR endocytosis via GRK2/β-arrestin2 pathway. Collectively, our study provided a therapeutic mAb, 3A5C7, targeting MOR to treat morphine tolerance, mediated by enhancing morphine-induced MOR endocytosis. The mAb 3A5C7 demonstrates promising translational value to treat clinical morphine tolerance.

Dexmedetomidine, an α2 agonist, increases the morphine analgesic effect and decreases morphine tolerance development by suppressing oxidative stress and TNF/IL-1 signalling pathway in rats.

The aim of the present study is to examine the possible effect de dexmedetomidine on the development of morphine tolerance in rats including nociception, morphine analgesia, apoptosis, oxidative stress, and tumour necrosis factor (TNF)/ interleukin-1 (IL-1) pathways. In this study, 36 Wistar Albino (225-245 g) rats were used. Animals were divided into 6 groups: saline (S), 20 mcg/kg dexmedetomidine (D), 5 mg/kg morphine (M), M + D, morphine tolerance (MT), and MT + D. The analgesic effect was measured with hot plate and tail-flick analgesia tests. After the analgesia tests, the dorsal root ganglia (DRG) tissues were excised. Oxidative stress parameters [total antioxidant status (TAS), total oxidant status (TOS)], TNF, IL-1 and apoptosis enzymes (Caspase-3, Caspase-9), were measured in DRG tissues. Dexmedetomidine showed an antinociceptive effect when given alone (p < 0.05 to p < 0.001). In addition, dexmedetomidine increased the analgesic effect of morphine (p < 0.001), and also decreased the tolerance to morphine at a significant level (p < 0.01 to p < 0.001). Moreover, it decreased oxidative stress (p < 0.001) and TNF/IL-1 levels when given as an additional drug of single-dose morphine and morphine tolerance group (p < 0.001). Furthermore, dexmedetomidine decreased Caspase-3 and Caspase-9 levels after tolerance development (p < 0.001). Dexmedetomidine has antinociceptive properties, and it increases the analgesic effect of morphine and also prevents tolerance development. These effects probably occur by the modulation of oxidative stress, inflammation and apoptosis.

Dexmedetomidina, un agonista de α2, incrementa el efecto analgésico de la morfina y reduce el desarrollo de tolerancia a la morfina suprimiendo el estrés oxidativo y la vía de señalización de TNF/IL-1 en ratas

BackgroundThe aim of the present study is to examine the possible effect de dexmedetomidine on the development of morphine tolerance in rats including nociception, morphine analgesia, apoptosis, oxidative stress, and tumour necrosis factor (TNF)/ interleukin-1 (IL-1) pathways. Materials and methodsIn this study, 36 Wistar Albino (225–245 g) rats were used. Animals were divided into 6 groups: saline (S), 20 mcg/kg dexmedetomidine (D), 5 mg/kg morphine (M), M + D, morphine tolerance (MT), and MT + D. The analgesic effect was measured with hot plate and tail-flick analgesia tests. After the analgesia tests, the dorsal root ganglia (DRG) tissues were excised. Oxidative stress parameters [total antioxidant status (TAS), total oxidant status (TOS)], TNF, IL-1 and apoptosis enzymes (Caspase-3, Caspase-9), were measured in DRG tissues. ResultsDexmedetomidine showed an antinociceptive effect when given alone (p < 0.05 to p < 0.001). In addition, dexmedetomidine increased the analgesic effect of morphine (p < 0.001), and also decreased the tolerance to morphine at a significant level (p < 0.01 to p < 0.001). Moreover, it decreased oxidative stress (p < 0.001) and TNF/IL-1 levels when given as an additional drug of single-dose morphine and morphine tolerance group (p < 0.001). Furthermore, dexmedetomidine decreased Caspase-3 and Caspase-9 levels after tolerance development (p < 0.001). ConclusiónDexmedetomidine has antinociceptive properties, and it increases the analgesic effect of morphine and also prevents tolerance development. These effects probably occur by the modulation of oxidative stress, inflammation and apoptosis.

Preclinical evidence for the use of the atypical antipsychotic, brexpiprazole, for opioid use disorder

Opioid addiction is characterized by adaptations in the mesolimbic dopamine system that occur during chronic opioid use. Alterations in dopaminergic transmission contribute to pathological drug-seeking behavior and other symptoms associated with opioid withdrawal following drug discontinuation, making drug abstinence challenging and contributing to high rates of relapse among those suffering from substance use disorder. Recently, the use of dopamine partial agonists has been proposed as a potential strategy to restore dopaminergic signalling during drug withdrawal, while avoiding the adverse side effects associated with stronger modulators of dopaminergic transmission. We investigated the effects of the atypical antipsychotic brexpiprazole, which is a partial agonist at dopamine D2 and D3 receptors, in a mouse model of opioid dependence. The development of opioid dependence in mice is characterized by locomotor sensitization, analgesic tolerance, opioid-induced hyperalgesia, and drug-seeking behavior. We set up four paradigms to model the effects of brexpiprazole on each of these adaptations that occur during chronic opioid use in male and female C57BL/6J mice. Concomitant treatment of brexpiprazole during chronic morphine administration attenuated the development of locomotor sensitization. Brexpiprazole treatment abolished morphine place preference and blocked reinstatement of this behavior following extinction. Brexpiprazole treatment did not alter morphine analgesia, nor did it impact the development of morphine tolerance. However, brexpiprazole treatment did prevent the expression of opioid-induced hyperalgesia in a tail-withdrawal assay, while failing to improve somatic withdrawal symptoms. Altogether, these results provide preclinical evidence for the efficacy of brexpiprazole as a modulator of dopamine-dependent behaviors during opioid use and withdrawal.

Extracellular vesicle-mediated delivery of anti-miR-106b inhibits morphine-induced primary ciliogenesis in the brain

Repeated use of opioids such as morphine causes changes in theshape and signal transduction pathways of various braincells,including astrocytes and neurons, resulting in alterations in brain functioning and ultimately leading to opioid use disorder. We previously demonstrated that extracellular vesicle(EV)-induced primary ciliogenesis contributes to the development ofmorphine tolerance. Herein, we aimed to investigate theunderlying mechanisms and potential EV-mediated therapeutic approach to inhibit morphine-mediated primaryciliogenesis. We demonstrated that miRNA cargo inmorphine-stimulated-astrocyte-derived EVs (morphine-ADEVs) mediated morphine-induced primary ciliogenesis in astrocytes. CEP97 is a target of miR-106b and is a negative regulator of primary ciliogenesis. Intranasal delivery of ADEVs loaded with anti-miR-106b decreased the expression of miR-106b in astrocytes, inhibited primary ciliogenesis, and prevented the development of tolerance in morphine-administered mice. Furthermore, we confirmed primary ciliogenesis in the astrocytes of opioid abusers. miR-106b-5p in morphine-ADEVs induces primary ciliogenesis via targeting CEP97. Intranasal delivery of ADEVs loaded with anti-miR-106b ameliorates morphine-mediated primary ciliogenesis and prevents morphine tolerance. Our findings bring new insights into the mechanisms underlying primary cilium-mediated morphine tolerance and pave the way for developing ADEV-mediated small RNA delivery strategies for preventing substance use disorders.

EFFECT OF THİAMİNE ON MORPHİNE ANALGESİA AND TOLERANCE İN RATS

Absract: The latest research have demonstrated that inflammation, oxidative stress and apoptosis plays a majör role in morphine analgesia and tolerance development. This search goals to examine the possible role of thiamine use on oxidative stress, inflammation and apoptosis in the development of morphine analgesia and morphine tolerance in rats. &#x0D; Methods: Thirty-six male Wistar rats were used in this study. The rats were severed into six groups: saline, 100 mg/kg thiamine, 5 mg/kg morphine, thiamine + morphine, morphine tolerance and thiamine + morphine tolerance. The resulting analgesic effect was measured by hot plate and tail movement analgesia tests. TAS and TOS, inflammation parameters, and apoptosis protein levels of the dorsal root ganglion tissues sample were measured using an ELISA kit. &#x0D; Results: When thiamine was given alone, it did not show anti-nociceptive effect (p&gt;0.05). In addition, thiamine enhanced the analgesic effect of morphine (p &lt; 0.05) and also significantly reduced tolerance to morphine (p &lt; 0.05). However, it reduced TOS when administered with a single dose of morphine and tolerance induction (p &lt; 0.05). In addition, thiamine reduced apoptosis protein levels after tolerance development (p &lt; 0.05). &#x0D; Conclusion: Consequently, these results may attain by reducing TOS, inflammation, and apoptosis.

Molecular identification of bulbospinal ON neurons by GPER, which drives pain and morphine tolerance.

The rostral ventromedial medulla (RVM) exerts bidirectional descending modulation of pain attributable to the activity of electrophysiologically identified pronociceptive ON and antinociceptive OFF neurons. Here, we report that GABAergic ON neurons specifically express G protein-coupled estrogen receptor (GPER). GPER+ neurons exhibited characteristic ON-like responses upon peripheral nociceptive stimulation. Optogenetic activation of GPER+ neurons facilitated, but their ablation abrogated, pain. Furthermore, activation of GPER caused depolarization of ON cells, potentiated pain, and ameliorated morphine analgesia through desensitizing μ-type opioid receptor-mediated (MOR-mediated) activation of potassium currents. In contrast, genetic ablation or pharmacological blockade of GPER attenuated pain, enhanced morphine analgesia, and delayed the development of morphine tolerance in diverse preclinical pain models. Our data strongly indicate that GPER is a marker for GABAergic ON cells and illuminate the mechanisms underlying hormonal regulation of pain and analgesia, thus highlighting GPER as a promising target for the treatment of pain and opioid tolerance.

CircRalgapa1 facilitates morphine tolerance via miR-873a-5p/A20 axis in mice

Morphine tolerance (MT) caused by long-term use of morphine is a major medical problem. The underlying molecular mechanisms of morphine tolerance remain unclear. Here, we establish the morphine tolerance model in mice and verify whether a novel circRNA, circRalgapa1 is involved in morphine tolerance and its specific molecular mechanism. We show that the expression of circRalgapa1 in the spinal cord is significantly down-expressed in the spinal cord of morphine-tolerant mice. CircRalgapa1 is mainly located in the neuronal cytoplasm and co-localizes with miR-873a-5p. Mechanically, circRalgapa1 acts as competing endogenous RNAs (ceRNAs) to regulate the inhibitory of miR-873a-5p on A20 (also known as tumor necrosis factor α-induced protein 3, TNFAIP3). Functionally, overexpression of circRalgapa1 by intrathecal injection of adeno-associated virus (AAV- circRalgapa1) attenuated the formation of morphine tolerance and partially reversed the development of morphine tolerance. Moreover, overexpression of miR-873a-5p blocked the effect of AAV-circRalgapa1 on alleviating morphine tolerance in mice. In conclusion, chronic morphine administration-mediated down-regulation of circRalgapa1 in the spinal cord contributes to morphine tolerance via miR-873a-5p/A20 axis in mice. Overexpression of circRalgapa1 may be a promising RNA-based therapy for morphine tolerance.

Peripheral sensory neuron CB2 cannabinoid receptors are necessary for both CB2-mediated antinociceptive efficacy and sparing of morphine tolerance in a mouse model of anti-retroviral toxic neuropathy

Painful peripheral neuropathy is a common neurological complication associated with human immunodeficiency virus (HIV) infection and anti-retroviral therapy. We characterized the impact of two CB2 cannabinoid agonists (AM1710 and LY2828360 – ligands differing in signaling bias and CNS penetration) on neuropathic nociception induced by the antiretroviral agent Zalcitabine (2',3'-dideoxycytidine; ddC). We also used a conditional knockout approach to identify cell types mediating CB2 agonist-induced antinociceptive efficacy and sparing of morphine tolerance. AM1710 and LY2828360 alleviated ddC-induced neuropathic nociception in mice of both sexes. These benefits were absent in global CB2 knockout mice, which exhibited robust morphine antinociception. Like morphine, AM1710 blunted ddC-induced increases in proinflammatory cytokine (IL-1β, TNF-α) and chemokine (CCL2) mRNA expression levels. We generated advillinCre/+;CB2f/f conditional knockout mice to ascertain the role of CB2 localized to primary sensory neurons in CB2-mediated therapeutic effects. Antinociceptive efficacy of both AM1710 and LY2828360, but not reference analgesics, were absent in advillinCre/+;CB2f/f mice, which exhibited robust ddC-induced neuropathy. In ddC-treated CB2f/f mice, LY2828360 suppressed development of morphine tolerance and reversed established morphine tolerance, albeit with greater efficacy in male compared to female mice. LY2828360 failed to block or reverse morphine tolerance in advillinCre/+;CB2f/f mice. The present studies indicate that CB2 activation may alleviate HIV-associated antiretroviral neuropathy and identify a previously unreported mechanism through which CB2 activation produces antinociceptive efficacy. Our results also provide the first evidence that a CB2 agonist can reverse established morphine tolerance and demonstrate that CB2 localized to peripheral sensory neurons mediates the opioid tolerance sparing efficacy of CB2 agonists.

Receptor Transporter Protein 4 (RTP4) in the Hypothalamus Is Involved in the Development of Antinociceptive Tolerance to Morphine.

Receptor transporter protein 4 (RTP4), one of the receptor chaperone proteins, contributes to the maturation and membrane trafficking of opioid receptor heteromers consisting of mu (MOPr) and delta (DOPr) opioid receptors (MOPr-DOPr). Although MOPr-DOPr is known to mediate the development of morphine tolerance, the extent to which RTP4 plays a role in this process has not been elucidated. Given that RTP4 can be upregulated by repeated administration of morphine, especially in the hypothalamus, here we investigated the effect of hypothalamus-selective ablation of RTP4 on the development of antinociceptive tolerance to morphine. In this study, we generated RTP4flox mice and selectively knocked-out RTP4 using local injection of adeno-associated virus expressing Cre recombinase (AAV-Cre) into the hypothalamus. The AAV-Cre injection partially, but significantly, decreased the level of RTP4 expression, and suppressed the development of antinociceptive tolerance to morphine. Next, we examined the mechanism of regulation of RTP4 and found that, in neuronal cells, Rtp4 induction is via Gi and MAPK activation, while, in microglial cells, the induction is via Toll-like receptor 4. Together, these studies highlight the role of MOR activity in regulating RTP4, which, in turn, plays an important role in modulating morphine effects in vivo.