Morphine-tolerant Rats Research Articles

Mechanisms for therapeutic effect of bulleyaconitine A on chronic pain.

Bulleyaconitine A, a diterpenoid alkaloid isolated from Aconitum bulleyanum plants, has been used for the treatment of chronic pain in China since 1985. Clinical studies show that the oral administration of bulleyaconitine A is effective for treating different kinds of chronic pain, including back pain, joint pain, and neuropathic pain with minimal side effect in human patients. The experimental studies have revealed that bulleyaconitine A at therapeutic doses potently inhibits the peripheral sensitization and central sensitization that underlie chronic pain and has no effect on acute pain. Bulleyaconitine A preferably blocks tetrodotoxin-sensitive voltage-gated sodium channels in dorsal root ganglion neurons by inhibition of protein kinase C, and the effect is around 600 times more potent in neuropathic animals than in naïve ones. Bulleyaconitine A at 5 nM inhibits the hypersensitivity of dorsal root ganglion neurons in neuropathic rats but has no effect on excitability of dorsal root ganglion neurons in sham group. Bulleyaconitine A inhibits long-term potentiation at C-fiber synapses in spinal dorsal horn, a synaptic model of pathological pain, preferably in neuropathic pain rats over naïve rats. The following mechanisms may underlie the selective effect of bulleyaconitine A on chronic pain. (1) In neuropathic conditions, protein kinase C and voltage-gated sodium channels in dorsal root ganglion neurons are upregulated, which enhances bulleyaconitine A’s effect. (2) Bulleyaconitine A use-dependently blocks voltage-gated sodium channels and therefore inhibits the ectopic discharges that are important for neuropathic pain. (3) Bulleyaconitine A is shown to inhibit neuropathic pain by the modulation of spinal microglia, which are involved in the chronic pain but not in acute (nociceptive) pain. Moreover, bulleyaconitine A facilitates the anesthetic effect of morphine and inhibits morphine tolerance in rats. Together, bulleyaconitine A is able to inhibit chronic pain by targeting at multiple molecules. Further clinical and experimental studies are needed for evaluating the efficacy of bulleyaconitine A in different forms of chronic pain in patients and for exploring the underlying mechanisms.

Preclinical assessment of utility of M6S for multimodal acute and chronic pain treatment in diabetic neuropathy

AimsPrevious reports from our laboratory have established that morphine-6-O-sulfate (M6S) is a mixed μ/δ opioid receptor (OR) agonist and a potential improved alternative to morphine for treatment of chronic multimodal pain in non-diabetic rats. This study extends the antinociceptive effects of M6S and morphine in STZ-induced diabetic rats. Materials and methodsEffects of morphine and M6S were studied across a range of pain modalities, using hot plate threshold (HPT), pinprick sensitivity threshold (PST) and paw pressure threshold (PPT) tests. Key findingsAcutely, M6S was 3- to 5-fold more potent and 2- to 3-fold more efficacious than morphine in HPT and PST tests. No differences in analgesic drug potency/efficacy were detected in the PPT test. After 7–9days of chronic treatment, tolerance developed to the antinociceptive effects of morphine, but not to M6S, in all three pain tests. Furthermore, morphine-tolerant rats were not cross-tolerant to M6S. The selective δ-OR antagonist, naltrindole, blocked M6S-induced antinociception by 62±3% in the HPT test, 93±5% in the PST test, and 30±17% in the PPT test when examined acutely. SignificanceThese studies provide additional confirmation for the mixed μ/δ activity of M6S and demonstrate potential improved clinical utility for dual μ/δ agonists relative to morphine in treatment of diabetic neuropathy across multiple pain domains.

A new tridecapeptide with an octaarginine vector has analgesic therapeutic potential and prevents morphine-induced tolerance

A growing body of evidence suggests that peptides may possess analgesic effects without tolerance development. The synthetic tetrapeptide Tyr-d-Arg-Phe-Gly-NH2 was modified with the inclusion of a (d-Arg)8 vector to prevent the action of endopeptidase and to increase the duration of the analgesic action of the tetrapeptide when administered orally. The aim of this study was to estimate the analgesic efficacy of the tetrapeptide with (d-Arg)8 (tridecapeptide, TDP) in experimental models of acute and chronic pain. The analgesic effects of TDP were estimated using a model of acute visceral pain in mice (writhing test) and a model of chronic neuropathic pain (chronic constriction injury (CCI) of the sciatic nerve) in rats. The intravenous administration of morphine (0.32–1mg/kg) and TDP (0.32–1.8mg/kg) produced significant dose-related antinociceptive effects in the writhing test. The potency of TDP after i.g. administration was lower than that after i.v. administration but comparable with that of i.g. morphine. In the CCI model, TDP (0.1, 1 and 10mg/kg, i.g.) induced marked analgesia with repeated administration without any signs of tolerance. The single administration of TDP after morphine treatment (7days) produced a significant analgesic effect in morphine-tolerant rats, indicating the absence of cross-tolerance between these two drugs. The combined administration of TDP and morphine resulted in the reduction of analgesic tolerance to morphine. The absence of cross-tolerance to morphine and the ability to prevent morphine tolerance allows this compound to be a prospective candidate for chronic pain therapy. In order to find the target receptors for TDP, a docking study was performed. It was found that the molecule can bind to the NMDA receptor using electrostatic, hydrogen bonding and hydrophobic interactions.

Effects of extremely low frequency magnetic field on the development of tolerance to the analgesic effect of morphine in rats.

This study examined the effects of low frequency magnetic field (MF) on tolerance to analgesic effect of morphine in rats. Rats were made tolerant to morphine by injecting morphine (10 mg/kg, s) once daily for 8 consecutive days. Rats were simultaneously exposed to an MF (50 Hz at 1, 50, and 100 μT for 30 min) before, immediately, or 30 min after injection of morphine, and also exposed to a 0.5, 6, 12, and 30 Hz at 100 μT for 30 min before injection of morphine. The percentage of maximum possible effect of morphine (% MPE) was measured on the 1st, 4th, and 8th days by hot plate test. We observed that MF radiation (50 Hz at 1 µT and 30 Hz at 100 µT) immediately before and MF radiation (50 Hz at 100 µT) after morphine injection prevented the development of morphine tolerance compared to control. Also, we found that exposure to MF (50 Hz at 1, 50, and 100 µT) 30 min after injection of morphine failed to maintain the analgesic effect of morphine. Our results showed that exposure to low frequency electromagnetic field (30 and 50 Hz) immediately before or after the injection of morphine may be a potential method for treating the development of morphine tolerance in rats. Bioelectromagnetics. 38:618-625, 2017. © 2017 Wiley Periodicals, Inc.

Site-Specific Regulation of P2X7 Receptor Function in Microglia Gates Morphine Analgesic Tolerance.

Tolerance to the analgesic effects of opioids is a major problem in chronic pain management. Microglia are implicated in opioid tolerance, but the core mechanisms regulating their response to opioids remain obscure. By selectively ablating microglia in the spinal cord using a saporin-conjugated antibody to Mac1, we demonstrate a causal role for microglia in the development, but not maintenance, of morphine tolerance in male rats. Increased P2X7 receptor (P2X7R) activity is a cardinal feature of microglial activation, and in this study we found that morphine potentiates P2X7R-mediated Ca2+ responses in resident spinal microglia acutely isolated from morphine tolerant rats. The increased P2X7R function was blocked in cultured microglia by PP2, a Src family protein tyrosine kinase inhibitor. We identified Src family kinase activation mediated by μ-receptors as a key mechanistic step required for morphine potentiation of P2X7R function. Furthermore, we show by site-directed mutagenesis that tyrosine (Y382-384) within the P2X7R C-terminus is differentially modulated by repeated morphine treatment and has no bearing on normal P2X7R function. Intrathecal administration of a palmitoylated peptide corresponding to the Y382-384 site suppressed morphine-induced microglial reactivity and preserved the antinociceptive effects of morphine in male rats. Thus, site-specific regulation of P2X7R function mediated by Y382-384 is a novel cellular determinant of the microglial response to morphine that critically underlies the development of morphine analgesic tolerance.SIGNIFICANCE STATEMENT Controlling pain is one of the most difficult challenges in medicine and its management is a requirement of a large diversity of illnesses. Although morphine and other opioids offer dramatic and impressive relief of pain, their impact is truncated by loss of efficacy (analgesic tolerance). Understanding why this occurs and how to prevent it are of critical importance in improving pain therapies. We uncovered a novel site (Y382-384) within the P2X7 receptor that can be targeted to blunt the development of morphine analgesic tolerance, without affecting normal P2X7 receptor function. Our findings provide a critical missing mechanistic piece, site-specific modulation by Y382-384, that unifies P2X7R function to the activation of spinal microglia and the development of morphine tolerance.

Evaluation of Analgesia, Tolerance, and the Mechanism of Action of Morphine-6-O-Sulfate Across Multiple Pain Modalities in Sprague-Dawley Rats.

Morphine-6-O-sulfate (M6S) is a mixed μ/δ-opioid receptor (OR) agonist and potential alternative to morphine for treatment of chronic multimodal pain. To provide more support for this hypothesis, the antinociceptive effects of M6S and morphine were compared in tests that access a range of pain modalities, including hot plate threshold (HPT), pinprick sensitivity threshold (PST) and paw pressure threshold tests. Acutely, M6S was 2- to 3-fold more potent than morphine in HPT and PST tests, specifically, derived from best-fit analysis of dose-response relationships of morphine/M6S half-effective dose (ED50) ratios (lower, upper 95% confidence interval [CI]) were 2.8 (2.0-5.8) in HPT and 2.2 (2.1, 2.4) in PST tests. No differences in analgesic drug potencies were detected in the PPT test (morphine/M6S ED50 ratio 1.2 (95% CI, 0.8-1.4). After 7 to 9 days of chronic treatment, tolerance developed to the antinociceptive effects of morphine, but not to M6S, in all 3 pain tests. Morphine-tolerant rats were not crosstolerant to M6S. The antinociceptive effects of M6S were not sensitive to κ-OR antagonists. However, the δ-OR antagonist, naltrindole, blocked M6S-induced antinociception by 55% ± 4% (95% CI, 39-75) in the HPT test, 94% ± 4% (95% CI, 84-105) in the PST test, and 5% ± 17% (95% CI, -47 to 59) or 51% ± 14% (95% CI, 14-84; 6 rats per each group) in the paw pressure threshold test when examined acutely or after 7 days of chronic treatment, respectively. Activity via δ-ORs thus appears to be an important determinant of M6S action. M6S also exhibited favorable antinociceptive and tolerance profiles compared with morphine in 3 different antinociceptive assays, indicating that M6S may serve as a useful alternative for rotation in morphine-tolerant subjects.

Spinal CX3CL1/CX3CR1 May Not Directly Participate in the Development of Morphine Tolerance in Rats.

CX3CL1 (fractalkine), the sole member of chemokine CX3C family, is implicated in inflammatory and neuropathic pain via activating its receptor CX3CR1 on neural cells in spinal cord. However, it has not been fully elucidated whether CX3CL1 or CX3CR1 contributes to the development of morphine tolerance. In this study, we found that chronic morphine exposure did not alter the expressions of CX3CL1 and CX3CR1 in spinal cord. And neither exogenous CX3CL1 nor CX3CR1 inhibitor could affect the development of morphine tolerance. The cellular localizations of spinal CX3CL1 and CX3CR1 changed from neuron and microglia, respectively, to all the neural cells during the development of morphine tolerance. A microarray profiling revealed that 15 members of chemokine family excluding CX3CL1 and CX3CR1 were up-regulated in morphine-treated rats. Our study provides evidence that spinal CX3CL1 and CX3CR1 may not be involved in the development of morphine tolerance directly.

Bifunctional opioid/nociceptin hybrid KGNOP1 effectively attenuates pain-related behaviour in a rat model of neuropathy

A bifunctional peptide containing an opioid and nociceptin receptor-binding pharmacophore, H-Dmt-D-Arg-Aba-β-Ala-Arg-Tyr-Tyr-Arg-Ile-Lys-NH2 (KGNOP1), was tested for its analgesic properties when administered intrathecally in naïve and chronic constriction injury (CCI)-exposed rats with neuropathy-like symptoms. KGNOP1 significantly increased the acute pain threshold, as measured by the tail-flick test, and also increased the threshold of a painful reaction to mechanical and thermal stimuli in CCI-exposed rats. Both of the effects could be blocked by pre-administration of [Nphe1]-Nociceptin (1-13)-NH2 (NPhe) or naloxone, antagonists for nociceptin and opioid receptors, respectively. This led us to conclude that KGNOP1 acts as a dual opioid and nociceptin receptor agonist in vivo. The analgesic effect of KGNOP1 proved to be more powerful than clinical drugs such as morphine and buprenorphine. Repeated daily intrathecal injections of KGNOP1 led to the development of analgesic tolerance, with the antiallodynic action being completely abolished on day 6. Nevertheless, the development of tolerance to the antihyperalgesic effect was delayed in comparison to morphine, which lost its efficacy as measured by the cold plate test after 3days of daily intrathecal administration, whereas KGNOP1 was efficient up to day 6. A single intrathecal injection of morphine to KGNOP1-tolerant rats did not raise the pain threshold in any of the behavioural tests; in contrast, a single intrathecal dose of KGNOP1 significantly suppressed allodynia and hyperalgesia in morphine-tolerant rats.

MiR-219-5p targets CaMKIIγ to attenuate morphine tolerance in rats

Morphine tolerance is a clinical challenge in pain management. Emerging evidence suggests that microRNA (miRNA) plays a regulatory role in the development of morphine tolerance. miR-219-5p (miR-219) targets calmodulin-dependent protein kinase II γ (CaMKIIγ) to activate central pain sensitization via N-methyl-D-aspartate (NMDA) receptor. Therefore, we hypothesized that miR-219-5p attenuates morphine tolerance by targeting CaMKIIγ. We found that the expression of miR-219-5p was decreased significantly after chronic morphine treatment. Overexpression of miR-219-5p by lentivirus injection prevents the development of morphine tolerance. CaMKIIγ, the target gene of miR-219-5p was downregulated by overexpression of miR-219-5p both in vivo and in vitro. Furthermore, we found that lentiviral-mediated miR-219-5p decreased the expression of NMDA receptor subunit 1 (NR1), leading to attenuation of morphine tolerance. Overall, the data demonstrate that miR-219-5p plays a crucial role in alleviating morphine tolerance by inhibiting the CaMKII/NMDA receptor pathway. Overexpression of miR-219-5p may be a potential strategy to ameliorate morphine tolerance.

Involvement of chemokine CXCL11 in the development of morphine tolerance in rats with cancer-induced bone pain.

Morphine is viewed as one of the classical treatments for intractable pain, but its role is limited by side effects, including analgesic tolerance. A few chemokines have been reported to be engaged in the mechanisms of morphine tolerance. However, the exact roles of CXC chemokine 11 (CXCL11) in chronic morphine tolerance remain unknown. In this study, Walker 256 mammary gland carcinoma cells were inoculated into the tibia of rats to provoke cancer-induced bone pain. Then, morphine was intrathecally administered twice daily for seven consecutive days to induce drug tolerance. We found that the level of CXCL11 in lumbar spinal cord was increased during the development of morphine tolerance in cancer-induced bone pain rats. Meanwhile, CXCL11 was co-localized with markers of astrocytes and neurons in the spinal cord. Inhibition of CXCL11 by neutralizing antibodies could remarkably attenuate the degree of morphine tolerance and decrease the activation of astrocytes. Moreover, blocking astrocyte activation by d, l-Fluorocitric acid could distinctly alleviate morphine tolerance and reduce the expression of CXCL11. Finally, morphine stimulation could induce the release of CXCL11 by cultured astrocytes and neurons invitro. In summary, our results provide evidence that spinal CXCL11 plays a powerful modulatory role in the development of morphine tolerance through cross-talking between astrocytes and neurons. Read the Review series "Pain".

Effects of extremely low frequency electromagnetic fields on morphine analgesia and tolerance in rats.

Several studies have demonstrated that the electromagnetic fields produce analgesic activity. The aim of this study was to investigate the effects of extremely low frequency (ELF) electromagnetic fields (EMF) on morphine analgesia and tolerance in rats. In the study, 78 adult male Wistar albino rats (approximately 240 ± 12 g) were used. The application of 50 Hz magnetic field, each day the same times for 30 minutes for 15 days, and a total of four times every 15 minute intervals. To constitute morphine tolerance, high dose of morphine (50 mg/kg) were administered for 3 days in rats and tolerance was evaluated on day 4. Prior to analgesia tests, the effective dose (5 mg/kg) of morphine was injected into rats. In the statistical analyzes of the data, analysis of variance (two-way ANOVA) was used and the multiple comparison determined by Tukey tests. The maximum analgesic effect of the 5 mT magnetic field was determined on 7 days. Administration of morphine (5 mg/kg) in rats exposed to a magnetic field, the analgesic effect was significantly higher compared to the morphine group (p < 0.05). Morphine tolerant animals exposed to a magnetic field, the analgesic effect was found significantly higher than morphine tolerant group rats (p < 0.05). Analgesia test data demonstrated that application of ELF-EMFs to rats increases the morphine analgesia and reduces morphine tolerance.

Monocyte chemoattractant protein-1 contributes to morphine tolerance in rats with cancer-induced bone pain

Cancer-induced bone pain can severely compromise the life quality of patients, while tolerance limits the use of opioids in the treatment of cancer pain. Monocyte chemoattractant protein-1 (MCP-1) is known to contribute to neuropathic pain. However, the role of spinal MCP-1 in the development of morphine tolerance in patients with cancer-induced bone pain remains unclear. The aim of the present study was to investigate the role of spinal MCP-1 in morphine tolerance in bone cancer pain rats (MTBP rats). Bone cancer pain was induced by intramedullary injection of Walker 256 cells into the tibia of the rats, while morphine tolerance was induced by continuous intrathecal injection of morphine over a period of 9 days. In addition, anti-MCP-1 antibodies were intrathecally injected to rats in various groups in order to investigate the association of MCP-1 with mechanical and heat hyperalgesia using the paw withdrawal threshold (PWT) and thermal withdrawal latency (TWL) tests, respectively. Furthermore, MCP-1 and CCR2 expression levels were measured using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis, and CCR2 expression levels were measured using RT-qPCR. The results indicated that MCP-1 and CCR2 expression levels were significantly increased in the spinal cord of MTBP rats. Intrathecal administration of anti-MCP-1 neutralizing antibodies was observed to attenuate the mechanical and thermal allodynia in MTBP rats. Therefore, the upregulation of spinal MCP-1 and CCR2 expression levels may contribute to the development of mechanical allodynia in MTBP rats. In conclusion, MCP-1/CCR2 signaling may serve a crucial role in morphine tolerance development in rats suffering from cancer-induced bone pain.

MiR-365 targets β-arrestin 2 to reverse morphine tolerance in rats.

Morphine tolerance is a challenging clinical problem that limits its clinical application in pain treatment. Non-coding microRNAs (miRNAs) modulate gene expression in a post transcriptional manner, and their dysregulation causes various diseases. However, the significance of miRNAs in morphine tolerance is still poorly understood. In the present study, we hypothesized that microRNA-365 (miR-365) is a key functional small RNA that reverses morphine tolerance through regulation of β-arrestin 2 in rats. Here, microarray analysis and quantitative real-time PCR showed that miR-365 was robustly decreased in the spinal cord after chronic morphine administration. In situ hybridization and immunochemistry double staining showed that miR-365 was expressed in neurons of the spinal cord. We identified β-arrestin 2 as the target gene of miR-365 by bioinformatics analysis and luciferase reporter assay. The data showed that overexpression of miR-365 prevented and reversed established morphine tolerance, and increased expression of miR-365 caused a decrease in expression of β-arrestin 2 protein. miR-365 downregulation is involved in the development and maintenance of morphine tolerance through regulation of β-arrestin 2, and miR-365 upregulation provides a promising and novel approach for treatment of morphine tolerance.

Upregulation of (C-X-C motif) Ligand 13 (CXCL13) Attenuates Morphine Analgesia in Rats with Cancer-Induced Bone Pain.

BackgroundThe aim of this study was to investigate the role of chemokine (C-X-C motif) ligand 13 (CXCL13) in morphine tolerance in rats with cancer-induced bone pain (CIBP).Material/MethodsWe established a rat CIBP model and a rat CIBP-morphine tolerance (BM) model. BM rats were intrathecally administered rmCXCL13, neutralizing anti-CXCL13, and normal saline, while the control group rats underwent a sham operation and were injected with normal saline. The morphine analgesia was assessed by measuring mechanical withdrawal threshold (MWT) and mechanical withdrawal duration (MWD) at various time points. The co-expressions of CXCL13 and NeuN were measured by immunofluorescence double-staining. CXCL13 protein and mRNA expressions were detected by Western blot and quantitative real-time polymerase chain reaction (RT-qPCR), respectively.ResultsCompared to the sham-operation (S) group, the BM group showed obviously decreased MWT and increased MWD on Day 9 after CIBP, but obviously increased MWT and decreased MWD on Day 3 after morphine administration; subsequently, the MWT was decreased and MWD was increased (all P<0.05). In comparison with the S+saline group, increased MWT and decreased MWD were observed in BM rats on Day 3 after anti-CXCL13 administration, and obviously decreased MWT and increased MWD were found in BM rats on Day 3 after rmCXCL13 administration (all P<0.05).ConclusionsUp-regulated CXCL13 has a negative role in morphine analgesia in relief of CIBP, which may provide a new target for the management of CIBP.

Establishing an animal model for National Acupuncture Detoxification Association (NADA) auricular acupuncture protocol

The use of opioids in the treatment of chronic pain has increased dramatically in the past few decades making them one of the most commonly prescribed medications in the US. However, long-term use of opioids is limited by development of tolerance (decreased antinociceptive efficacy) and opioid-induced hyperalgesia − paradoxical sensitization to noxious (hyperalgesia) and non-noxious (allodynia) stimuli. Novel adjunctive therapies are needed to increase the efficacy and prolong the duration of action of opioids in chronic pain treatment.Acupuncture is often used as an adjunct therapy for the treatment of symptoms induced by non-clinical use of opioids. The National Acupuncture Detoxification Association (NADA) auricular acupuncture protocol is the most common form of acupuncture treatment for substance abuse. The standardized, easy to use and virtually painless procedure make it an attractive complementary treatment option for patients suffering from opioid-induced adverse effects. Clinical trials designed to test the efficacy of the NADA protocol yielded contradictory results. The mechanism by which NADA acupuncture could serve as a successful treatment remains unknown. Therefore, establishing an animal model of NADA acupuncture can provide a tool for investigating the efficacy and cellular mechanisms of NADA treatment.Previous studies have shown that repeated morphine administration in rodents can produce locomotor sensitization and reduce analgesic potency of a challenge dose of morphine, indicating development of morphine tolerance. Here we show that NADA acupuncture treatment can both reduce morphine-induced locomotor sensitization and prevent the development of morphine tolerance in rats, thus validating a new model for NADA acupuncture studies. Our data provides support for evidence-based use of NADA acupuncture as a new adjunctive approach that can potentially improve the side-effect profile of morphine and other prescription opioids.

Melatonin reverses morphine tolerance by inhibiting microglia activation and HSP27 expression

AimsMelatonin has been reported to attenuate opioid tolerance. In this study, we explored the possible mechanism of melatonin in diminishing morphine tolerance. Main methodsTwo intrathecal (i.t.) catheters were implanted in male Wistar rats for drug delivery. One was linked to a mini-osmotic pump for morphine or saline infusion. On the seventh day, 50μg of melatonin or vehicle was injected through the other catheter instantly after discontinuation of morphine or saline infusion; 3h later, 15μg of morphine or saline was injected. The antinociceptive response was then measured using the tail-flick test every 30min for 120min. Key findingsThe results showed that chronic morphine infusion elicited antinociceptive tolerance and upregulated heat shock protein 27 (HSP27) expression in the dorsal horn of the rat spinal cord. Melatonin pretreatment partially restored morphine's antinociceptive effect in morphine-tolerant rats and reversed morphine-induced HSP27 upregulation. In addition, chronic morphine infusion induced microglial cell activation and was reversed by melatonin treatment. SignificanceThe present study provides evidence that melatonin, acting via inhibiting morphine-induced neuroinflammation, can be useful as a therapeutic adjuvant for patients under long-term opioid treatment for pain relief.

D-serine in the midbrain periaqueductal gray contributes to morphine tolerance in rats.

BackgroundThe N-methyl-D-aspartate subtype of glutamate receptor plays a critical role in morphine tolerance. D-serine, a co-agonist of N-methyl-D-aspartate receptor, participates in many physiological and pathophysiological processes via regulating N-methyl-D-aspartate receptor activation. The purinergic P2X7 receptor activation can induce the D-serine release in the central nervous system. This study aimed to investigate the role of the ventrolateral midbrain periaqueductal gray D-serine in the mechanism of morphine tolerance in rats. The development of morphine tolerance was induced in normal adult male Sprague–Dawley rats through subcutaneous injection of morphine (10 mg/kg). The analgesic effect of morphine (5 mg/kg, i.p.) was assessed by measuring mechanical withdrawal thresholds in rats with an electronic von Frey anesthesiometer. The D-serine concentration and serine racemase expression levels in the ventrolateral midbrain periaqueductal gray were evaluated through enzyme-linked immunosorbent assay and Western blot analysis, respectively. The effects of intra-ventrolateral midbrain periaqueductal gray injections of the D-serine degrading enzyme D-amino acid oxidase and antisense oligodeoxynucleotide targeting the P2X7 receptor on chronic morphine-treated rats were also explored.ResultsWe found that repeated morphine administrations decreased the antinociceptive potency of morphine evidenced by the percent changes in mechanical pain threshold in rats. By contrast, the D-serine contents and the expression levels of the serine racemase protein were upregulated in the ventrolateral midbrain periaqueductal gray in morphine-tolerant rats. The development of morphine tolerance was markedly alleviated by intra-ventrolateral midbrain periaqueductal gray injections of D-amino acid oxidase or antisense oligodeoxynucleotide targeting the P2X7 receptor.ConclusionsOur data indicate that the development of antinociceptive tolerance to morphine is partially mediated by ventrolateral midbrain periaqueductal gray D-serine content, and the activation of the ventrolateral midbrain periaqueductal gray P2X7 receptor is an essential prelude to D-serine release. These results suggest that a cascade involving P2X7 receptor–D-serine–N-methyl-D-aspartate receptor mediated signaling pathway in the supraspinal mechanism of morphine tolerance.

Differential expression of MU [μ] Opioid receptors and N-type calcium channels in the amygdala of morphine tolerant rat: An autoradiographic & immunohistochemistry study

Introduction: Recent studies have shown that amygdala plays a pivotal role in the emotional processing of pain. Opioids like morphine relieve pain by suppressing both the sensory and emotional components of pain. On chronic morphine administration, tolerance develops decreasing its analgesic effect. The underlying changes in the amygdala during morphine tolerance are not definitely known. Mu opioid receptors are binding sites for morphine while N-type voltage-gated calcium channels (VGCCs) directly control the release of neurotransmitters from presynaptic nerve endings. Aims & Objectives: The present study was designed to investigate the status of mu receptors and N-type VGCCs in the amygdala of morphine tolerant rat by autoradiography and immunohistochemistry respectively. Material & Methods: Wistar rats were made morphine tolerant by repeated administration of morphine for 5 days. Later, the rats were euthanized and cryostat sections of the brain were cut at the region of the amygdala. For autoradiographic localization, the sections were incubated with [3H] labeled DAMGO. For immunohistochemistry, the sections were exposed to primary antibody against N-type VGCCs. Results: Using image analysis software, a significant down-regulation of mu receptors and an up-regulation of N-VGCCs were observed after tolerance. This differential change in expression would indicate a less effective antinociceptive action of morphine through / though higher neurotransmitter release after tolerance. Conclusion: In conclusion, the study suggests that morphine tolerance could be associated with altered functional state of the amygdala. Keywords: Amygdala, Mu-opioid receptor, Voltage-gated calcium channel, Autoradiography, Immunohistochemistry

Predicting Neuroinflammation in Morphine Tolerance for Tolerance Therapy from Immunostaining Images of Rat Spinal Cord.

Long-term morphine treatment leads to tolerance which attenuates analgesic effect and hampers clinical utilization. Recent studies have sought to reveal the mechanism of opioid receptors and neuroinflammation by observing morphological changes of cells in the rat spinal cord. This work proposes a high-content screening (HCS) based computational method, HCS-Morph, for predicting neuroinflammation in morphine tolerance to facilitate the development of tolerance therapy using immunostaining images for astrocytes, microglia, and neurons in the spinal cord. HCS-Morph first extracts numerous HCS-based features of cellular phenotypes. Next, an inheritable bi-objective genetic algorithm is used to identify a minimal set of features by maximizing the prediction accuracy of neuroinflammation. Finally, a mathematic model using a support vector machine with the identified features is established to predict drug-treated images to assess the effects of tolerance therapy. The dataset consists of 15 saline controls (1 μl/h), 15 morphine-tolerant rats (15 μg/h), and 10 rats receiving a co-infusion of morphine (15 μg/h) and gabapentin (15 μg/h, Sigma). The three individual models of astrocytes, microglia, and neurons for predicting neuroinflammation yielded respective Jackknife test accuracies of 96.67%, 90.00%, and 86.67% on the 30 rats, and respective independent test accuracies of 100%, 90%, and 60% on the 10 co-infused rats. The experimental results suggest that neuroinflammation activity expresses more predominantly in astrocytes and microglia than in neuron cells. The set of features for predicting neuroinflammation from images of astrocytes comprises mean cell intensity, total cell area, and second-order geometric moment (relating to cell distribution), relevant to cell communication, cell extension, and cell migration, respectively. The present investigation provides the first evidence for the role of gabapentin in the attenuation of morphine tolerance from phenotypic changes of astrocytes and microglia. Based on neuroinflammation prediction, the proposed computer-aided image diagnosis system can greatly facilitate the development of tolerance therapy with anti-inflammatory drugs.

Role of neurokinin-1 receptor antagonist in attenuating morphine tolerance in rats

Role of neurokinin-1 receptor antagonist in attenuating morphine tolerance in rats