Morphine-tolerant Rats Research Articles

The role of brain catecholamines in morphine analgesic action in morphine tolerant rats.

The role of brain catecholamines in morphine analgesic action in morphine tolerant rats.

Restoration of morphine analgesia in morphine-tolerant rats after the intraventricular administration of 6-hydroxydopamine.

Restoration of morphine analgesia in morphine-tolerant rats after the intraventricular administration of 6-hydroxydopamine.

Effect of 6-hydroxydopamine on catecholamine concentrations and behaviour in the morphine-tolerant rat.

Effect of 6-hydroxydopamine on catecholamine concentrations and behaviour in the morphine-tolerant rat.

On the Possible Role of ERK, p38 and CaMKII in the Regulation of CGRP Expression in Morphine-Tolerant Rats

BackgroundThe neuropeptide, calcitonin gene-related peptide (CGRP) has been proposed to be a regulator of the development of morphine analgesic tolerance and thereby could be a target to reduce the induction of this phenomenon under clinical conditions. However, the mechanisms of CGRP regulation are unclear. We investigated here the possible role of the extracellular signal-regulated protein kinase (ERK), p38 and calcium/calmodulin-dependent protein kinase II (CaMKII) in CGRP regulation following chronic morphine treatment.ResultsA 7-day treatment with morphine (15 μg/day) led to an increase in CGRP contents in the spinal cord dorsal horn (SCDH) and dorsal root ganglion (DRG) and this effect was prevented by the inhibition of the ERK, p38 or CaMKII pathway. The phosphorylation/activation of ERK, p38 and CaMKII was enhanced in the SCDH following chronic morphine while in DRG only the phosphorylation of CaMKII was increased. Moreover, our chronic morphine treatment up-regulated neuronal nitric oxide synthase (nNOS) levels in the SCDH, an effect blocked by the inhibition of the ERK, p38 or CaMKII pathway. The blockade of nNOS activity also suppressed chronic morphine-induced CGRP increases in the DRG and SCDH. Double immunofluorescence studies revealed that nNOS and CaMKII are co-localized in the SCDH and that CaMKII is activated in CGRP-expressing DRG neurons.ConclusionsThe activation of spinal ERK, p38 and CaMKII, alongside nNOS, is involved in chronic morphine-induced CGRP up-regulation in both the DRG and SCDH. Moreover, the stimulation of CaMKII in the DRG likely directly regulates the expression of CGRP associated with morphine analgesic tolerance.

The role of extracellular signal-regulated kinase signal pathway in the development of chronic morphine tolerance in rats

Objective To investigate the role of extracellular signal-regulated kinase(ERK)signal pathway in the spinal cord in the development of chronic morphine tolerance.Methods Thirty healthy male SD rats weighing 300-350 g in which intrathecal(IT)catheters were successfully implanted without complication were randomly divided into 3 groups(n = 10 each): group Ⅰ control(group C);group Ⅱ morphine tolerance(group M)and group Ⅲ morphine tolerance + PD98059(ERK upstream kinase MEK inhibitor)(group P).Morphine tolerance was induced with IT morphine 10 μg twice a day for 7 consecutive days.In group P PD98059 10 μg was injected IT at 30 min before each morphine administration.Tail flick latency(TFL)(the time between the onset of heat stimulus and voluntary tail withdrawal)was measured once a day at 30 min after first IT morphine administration and at 1 day after last IT morphine.Maximum analgesic effect(MPE)was calculated.MPE =(TFL after IT morphine - baseline TFL)/(12 - baseline TFL)× 100%.The animals were sacrificed after last TFL measurement.The L3-5 segment of the spinal cord was isolated for determination of the expression of μ-receptor and phosphorylated ERK 1/2(p-ERK1/2)by Western blot analysis and fluoroimmunoassay.Results Morphine tolerance was induced in group M and M + P.MPE was higher in group P than in group M.The expression of μ-receptor in spinal dorsal horn was significantly lower while the p-ERK1/2 expression was higher in group M than in group C.IT PD98059 significantly up-regulated μ-receptor expression and down-regulated p-ERK expression in group P as compared with group M.Conclusion ERK signal pathway is involved in the development of chronic morphine tolerance in rats. Key words: Morphine; Drug tolerance; Extracellular signal-regulated kinase

Etanercept Restores the Antinociceptive Effect of Morphine and Suppresses Spinal Neuroinflammation in Morphine-Tolerant Rats

In the present study we examined the effect of the tumor necrosis factor (TNF)-α antagonist etanercept on the antinociceptive effect of morphine in morphine-tolerant rats. Male Wistar rats were implanted with 2 intrathecal catheters, and 1 was connected to a mini-osmotic pump for either morphine (15 μg/h) or saline (1 μL/h) infusion for 5 days. On day 5, either etanercept (5 μg, 25 μg, and 50 μg/10 μL) or saline (10 μL) was injected via the other catheter after morphine infusion was discontinued. Three hours later, morphine (15 μg/10 μL, intrathecally) was given and tail-flick latency was measured to evaluate the antinociceptive effect of morphine. Rats were then killed and their spinal cords were removed for quantitative real-time polymerase chain reaction and immunohistochemistry to measure proinflammatory cytokines expression. We found that acute etanercept (50 μg) treatment preserved a significant antinociceptive effect of morphine in morphine-tolerant rats. In addition, the expression of TNFα mRNA was increased by 2.5-fold, interleukin (IL)-1β mRNA increased by 13-fold and IL-6 mRNA by 111-fold in the dorsal spinal cord of morphine-tolerant rats. The increase in TNFα, IL-1β, and IL-6 mRNA expression was blocked by 50 μg etanercept pretreatment. The immunohistochemistry analysis revealed that 50 μg etanercept suppressed proinflammatory cytokines expression and neuroinflammation in the microglia. The present study demonstrates that etanercept restores the antinociceptive effect of morphine in morphine-tolerant rats by inhibition of proinflammatory cytokine TNF-α, IL-1β, and IL-6 expression and spinal neuroinflammation. The results suggest that etanercept could also be an adjuvant therapy for morphine tolerance, which extends the effectiveness of opioids in clinical pain management.

Purinergic P2X Receptor Regulates N -Methyl-d-aspartate Receptor Expression and Synaptic Excitatory Amino Acid Concentration in Morphine-tolerant Rats

The present study examined the effect of P2X receptor antagonist 2',3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP) on morphine tolerance in rats. Male Wistar rats were implanted with two intrathecal catheters with or without a microdialysis probe, then received a continuous intrathecal infusion of saline (control) or morphine (tolerance induction) for 5 days. Long-term morphine infusion induced antinociceptive tolerance and up-regulated N-methyl-d-aspartate receptor subunits NR1 and NR2B expression in both total lysate and synaptosome fraction of the spinal cord dorsal horn. TNP-ATP (50 μg) treatment potentiated the antinociceptive effect of morphine, with a 5.5-fold leftward shift of the morphine dose-response curve in morphine-tolerant rats, and this was associated with reversal of the up-regulated NR1 and NR2B subunits in the synaptosome fraction. NR1/NR2B-specific antagonist ifenprodil treatment produced a similar effect as TNP-ATP; it also potentiated the antinociceptive effect of morphine. On day 5, morphine challenge resulted in a significant increase in aspartate and glutamate concentration in the cerebrospinal fluid dialysates of morphine-tolerant rats, and this effect was reversed by TNP-ATP treatment. Moreover, the amount of immunoprecipitated postsynaptic density-95/NR1/NR2B complex was increased in morphine-tolerant rats, and this was prevented by the TNP-ATP treatment. The findings suggest that attenuation of morphine tolerance by TNP-ATP is attributed to down-regulation of N-methyl-d-aspartate receptor subunits NR1 and NR2B expression in the synaptosomal membrane and inhibition of excitatory amino acids release in morphine-tolerant rats. The TNP-ATP regulation on the N-methyl-d-aspartate receptor expression may be involved in a loss of scaffolding proteins postsynaptic density-95.

Gene knockdown with lentiviral vector‐mediated intrathecal RNA interference of protein kinase C gamma reverses chronic morphine tolerance in rats

Although morphine is a widely used opioid analgesic, morphine tolerance (MT) has limited the use of the drug because it creates the necessity for high doses. Protein kinase C (PKC), especially the PKCγ isoform, is considered to play a key role in the development of MT. Because RNA interference provides a powerful method for the investigation of gene function, and lentiviral delivery systems have been approved for human use, this present study examined rats tolerant to morphine to determine whether an intrathecal injection of a lentiviral vector of PKCγ short hairpin RNA (LV-shPKCγ) down-regulated the expression of the PKCγ gene and reversed MT. MT was induced by intrathecal morphine (10 µg b.i.d.) for six consecutive days. A lentiviral-mediated short hairpin RNA (shRNA) system was synthesized to deliver the PKCγ shRNAs to the spinal cord of the rats with MT. Mechanical and thermal paw withdrawal threshold were assessed to determine the analgesic effects of morphine. Expression of PKCγ mRNA and protein was determined by reverse transcriptase-polymerase chain reaction and western blotting analysis, respectively. The chronic administration of morphine induced a stabilized analgesic tolerance. A single injection of LV-shPKCγ significantly reversed morphine antinociceptive tolerance. Compared to the control group, PKCγ mRNA and protein levels were dramatically down-regulated in the LV-shPKCγ group. A single injection of LV-shPKCγ reversed MT by reducing the expression of PKCγ in the spinal cord. These findings indicate that the use of LV-shPKCγ might be a potential strategy for therapy in MT.

Role of expression of protein kinase C in spinal dorsal horn in metabotropic glutamate receptor 5's participation in development of morphine tolerance in rats

Objective To evaluate the role of the expression of protein kinase Cγ and Cα in spinal dorsal horn in the metabotropic glutamate receptor subtype 5 (mGluR5)'s participation in the development of morphine tolerance in rats. Methods Thirty-two male SD rats in which the intrathecal (IT) catheter was successfully placed were randomly divided into 4 groups (n = 8 each): control group (group C), morphine tolerance group (group M), antisense oligodeoxynucleotide (ODN) plus morphine group (group ANT) and mismatch ODN plus morphine group (group MIS). Rats in group M received 0.9% normal saline (NS) 5 μl twice a day for 8 days, and IT morphine 15 μg was injected simultaneously at 6, 7 and 8 days twice a day. Rats in group ANT and MIS received IT antisense and mismatch ODN 30 nmol (in 0.9% NS 5 μl) twice a day for 8 days respectively and IT morphine 15 μg was injected simultaneously at 6, 7 and 8 days twice a day. Rats in group C received NS instead twice a day for 8 days. Paw-withdrawl threshold (PWT) to thermal and mechanical stimulation was measured at 6, 7 and 8 days after ODN injection (T1-3). The animals were killed on 9th day (T4) and the lumbar segment of the spinal cord was removed for determination of the expression of mGluR5, PKCα and PKCγ mRNA (by RT-PCR), PKCαand PKCγ ( by Western blot). Results PWT to thermal and mechanical stimulation was significantly higher at T1.2in group M and MIS and at T1.3 in group ANT, the expression of mGluR5 mRNA, PKCα and PKCγ was significantly higher at T4 in group M and MIS, and mGluR5 mRNA expression was significantly lower at T4 in group ANT than in group C ( P ＜ 0.05). PWT to thermal and mechanical stimulation was significantly higher at T2.3, while the expression of mGluR5 mRNA, PKCα and PKCγ lower at T4 in group ANT than in group M ( P ＜ 0.05). Conclusion The expression of protein kinase C in spinal dorsal horn plays an important role in the mGluR5's participation in the development of morphine tolerance in rats. Key words: Protein kinase C; Receptors,glutamate; Morphine; Drug tolerance; Spinal cord

Changes in expression of vanilioid receptor subtype 1 in dorsal root ganglion and effect of electroacupuncture on morphine tolerance in rats with inflammatory pain and morphine tolerance

Objective To investigate the changes in expression of vanilloid receptor subtype 1 (VR1) in dorsal root ganglion (DRG) and effect of electroacupuncture (EA)on morphine tolerance in rats with inflammatory pain (IP) and morphine tolerance. Methods Thirty 8 month old male SD rats in which intrathecal (IT) catheters were successfully implanted without complication were randomly divided into 6 groups ( n = 5 each): group A IP + normal saline (NS) 10 μl IT twice a day × 7 days;group B intact rats + morphine 10 μg/kg(10 μl )IT twice a day × 7 days;group C IP + morphine 10 μg/kg(10 μl) IT once;group DIP + morphine 10μg/kg(10 μl) IT twice a day × 7 days;group E and F IP + EA (frequency 2/15 Hz) + morphine 10μg/kg(10 μl) IT twice a day × 7 days. IP was induced by injecting complete Freund's adjuvant (CFA) into the anlle joint of the left hindlimb. IT morphine or NS was started on the 4th day after induction of IP. EA of Yanglingquan and Zusanli lasting 30 min was performed once a day after first IT administration of morphine for 7 days. Paw withdrawal latency (PWL) to a thermal nociceptive stimulus was measured before induction of IP (baseline), at 1 day before and 1-7days of consecutive IT administration. The animals were sacrificed after last PWL measurement. The DRGs of the lumbar segment (L4-6) were removed for determination of VR1 expression in total and membrane protein using Western blot analysis. Results There was no significant difference in the baseline PWL measured before induction of IP among the 6 groups. Morphine tolerance developed in group B and D but did not develop in group E and F.The expression of VR1 in total and membrane protein of DRG was highest in group D and was significantly lower in group E than in group F. Conclusion VR1 in DRG is involved in the development of morphine tolerance. EA can inhibit morphine tolerance by down-regulating the expression of VR1. Key words: Drug tolerance; Morphine; Inflammation; Electroacupuncture; TRPV cation channels

Role of glucocorticoid receptor in induction of neuronal apoptosis in spinal dorsal horn in chronic morphine tolerant rats

Objective To investigate the role of glucocorticoid receptor (GR) in the induction of neuronal apoptosis in spinal dorsal horn in chronic morphine tolerant rats. Methods Twenty healthy male SD rats weighing 300-350 g in which intrathecal (IT) catheter was successfully implanted without complication were randomized into 4 groups ( n = 5 each): group Ⅰ control ( group C);group Ⅱ morphine ( group M );group Ⅲ morphine +RU38486 (GR antagonist) (group MR) and group Ⅳ morphine + dexamethasone (GR agonist) (group MD).Normal saline 10 μl, morphine 10 μg, morphine 10 μg + RU38486 2 μg and morphine 10μg + dexamethasone 4 μg were injected IT twice a day at 8:00 and 20:00 for6 consecutive days in group C, M, MR and MD respectively. Tail flick latency (TFL) to a thermal nociceptive stimulus was measured every day at 30 min after IT administration in the morning (8:00). Hyperalgesia was considered to be a sign of morphine tolerance. The animals were killed at 7 days after IT drug administration. The L3-5 segment of the spinal cord was isolated for determination of neuronal apoptosis in spinal dorsal horn by TUNEL staining. Apoptotic index was calculated ( the number of apoptotic neurons/the total number of neurons × 100% ). Results TFL was significantly prolonged at day 1 and 3 of IT morphine 10 μg twice a day and returned to baseline at day 5 and 7 indicating morphine tolerance. RU38486 inhibited while dexamethasone enhanced morphine tolerance. IT morphine significantly increased the number of apoptotic neurons in spinal dorsal horn. Morphine-induced neuronal apoptosis was decreased by IT RU38486 and increased by IT dexamethasone. Conclusion Glucocorticoid receptors may be involved in morphine tolerance by inducing neuronal apoptosis in spinal dorsal horn. Key words: Glucocorticoids; Morphine; Drug tolerance; Spinal cord; Apoptosis

8-OH-DPAT Prevents Morphine-Induced Apoptosis in Rat Dorsal Raphe Nucleus

Previously, we found that activation of serotonin 1A (5-HT1A) receptors in the dorsal raphe nucleus (DRN) decreased the development of tolerance to the analgesic effect of morphine. It has been indicated that tolerance to the analgesic effect of morphine is associated with apoptosis in the central nervous system. In this investigation we attempted to evaluate the effect of 8-OH-DPAT (8-hydroxy-2-[di-n-propylamino]tetralin), a specific 5-HT1A receptor agonist, on morphine-induced tolerance and apoptosis in rat DRN. Nociception was assessed using a hotplate apparatus. The terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) method was used to analyze apoptosis. Tolerance to the analgesic effect of morphine was complete by 10 days after morphine administration (5 mg/kg/d, i.p.), whereas a significant analgesic effect was observed through the 10th day in 8-OH-DPAT-treated animals. Furthermore, the results showed that the number of TUNEL positive cells had been increased in morphine-tolerant rats (control group: morphine, i.p. + saline, intra-DRN) in comparison with the saline-treated animals. The results also indicated that 8-OH-DPAT (2, 4, and 8 μg/rat/d) attenuated the number of apoptotic cells in the DRN in comparison with the control group. However, 8-OH-DPAT (8 μg/rat/d, intra-DRN) failed to reduce morphine-induced apoptosis in the presence of the 5-HT1A receptor antagonist, NAN-190 (6 μg/rat/d, intra-DRN). We found that intra-DRN injection of a specific 5-HT1A receptor agonist attenuated morphine-induced apoptosis in rat DRN, which may have a key role in morphine tolerance.

Changes in expression of CGRP, SP and BDNF in dorsal root ganglion and effects of electroacupuncture on morphine tolerance in rats with chronic inflammatory pain and morphine tolerance

Objective To investigate the changes in the expression of calcitonin gene-related peptide (CGRP), substance P (SP) and brain-derived neurotrophic factor (BDNF) in dorsal root ganglion (DRG) and the effect of electroacupuncture (EA) on morphine tolerance in rats with chronic inflammatory pain (CIP) and morphine tolerance. Methods Twenty-five 8-month-old male SD rats weighing 230-250 g in which intrathecal (IT)catheters were successfully implanted without complications were randomly divided into 5 groups ( n = 5 each):groupA CIP + normal saline (NS) 10 μl IT twice a day for 7 consecutive days; group B CIP + morphine 10 μg/kg ( 10 μl) IT twice for the first day only; group C CIP + morphine 10 μg/kg ( 10 μl) IT twice a day for 7 consecutive days; group D CIP + EA (intensity 2 mA, frequency 2 Hz, wave length 0.6 ms) + morphine 10 μ g/kg (10 μl) IT twice a day for7 consecutive days; group E CIP + EA (intensity 2 mA, frequency 15 Hz,wave length 0.4 ms) + morphine 10μg/kg (10 μl) IT twice a day for7 consecutive days. CIP was induced by injecting complete Freund's adjuvant (CFA) into the ankle joint of the left hindlimb. IT morphine or NS was started on the 4th day after induction of CIP. EA of Yanglingquan and Zusanli lasting 30 min was performed once a day after first IT administration of morphine for 7 days. Paw withdrawal latency (PWL) to a thermal nociceptive stimulus was measured before induction of CIP, 1 day before (baseline) and at day 1-7 after administration (T0-8) . The animals were sacrificed after the last PWL measurement. The DRGs of the lumbar segment (L4-6) were removed for determination of CGRP, SP and BDNF mRNA expression using RT-PCR. Results PWL was significantly shorter at T1 than at T0 in all groups, and at T3-8 than at T2 in group B-E, while longer at T2 than at T1 in group B-E ( P ＜0.05). PWL was significantly longer in group B-E and CGRP, SP and BDNF mRNA expression higher in group C than in group A ( P ＜ 0.05). PWL was significantly longer in group C-E than in group B ( P ＜ 0.05). PWL was significantly longer and CGRP, SP and BDNF mRNA expression lower in group D and E than in group C ( P ＜0.05). PWL was significantly lower and CGRP, SP and BDNF mRNA expression higher in group E than in group D ( P ＜ 0.05). Conclusion Up-regulation of the expression of CGRP, SP and BDNF mRNA in DRG is involved in the devepopment of morphine tolerance. EA can inhibit the devepopment of morphine tolerance by inhibiting the expression of CGRP2 SP and BDNF mRNA. Key words: Drug tolerance; Morphine; Inflammation; Electroacupuncture; Calcitonin gene-related peptide; Substance P; Brain-derived neurotrophic factor

Anti-morphine antibody contributes to the development of morphine tolerance in rats

Previous studies have shown that tolerance to the antinociceptive effect of morphine develops after a prolonged exposure, but its mechanisms remain unclear. In the present study, we examined whether anti-morphine antibody produced by chronic morphine exposure would contribute to the development of morphine antinociceptive tolerance in rats. Our results showed that anti-morphine antibody was present in rats rendered tolerance to antinociception after intrathecal morphine exposure for seven consecutive days. Superfusion of anti-morphine antibody onto spinal cord slice dose-dependently produced an inward excitatory current in spinal cord dorsal horn neurons using whole-cell patch-clamp recording, which surpassed morphine-induced outward inhibiting current. Co-administration of morphine with a monoclonal antibody (2.4G 2) against Fc receptors for seven days significantly attenuated the production of anti-morphine antibody as well as the behavioral manifestation of morphine tolerance in same rats. These results indicate that anti-morphine antibody produced by morphine exposure may contribute to the development of morphine tolerance possibly through counteracting the inhibitory morphine effect on spinal cord dorsal horn neurons.

Co-administration of ultra-low dose naloxone attenuates morphine tolerance in rats via attenuation of NMDA receptor neurotransmission and suppression of neuroinflammation in the spinal cords

Although mechanisms underlying ultra-low dose naloxone-induced analgesia have been proposed, possible interactions with glutamatergic transmission and glial cell activation have not been addressed. In the present study, we examined the effect of ultra-low dose naloxone on spinal glutamatergic transmission and glial cell activity in rats chronically infused with morphine. In male Wistar rats, intrathecal morphine infusion (15 μg/h) for 5 days induced (1) antinociceptive tolerance, (2) downregulation of glutamate transporters (GTs) GLT-1, GLAST, and EAAC1, (3) increasing of NMDA receptor (NMDAR) NR1 subunit expression and phosphorylation, (4) upregulation of protein kinase C gamma (PKCγ) expression, and (5) glial cell activation. On day 5, morphine challenge (15 μg/10 μl) caused a significant increase in the concentration of the excitatory amino acids (EAAs) aspartate and glutamate in the spinal CSF dialysates of morphine-tolerant rats. Intrathecal co-infusion of ultra-low dose naloxone (15 pg/h) with morphine attenuated tolerance development, reversed GTs expression, inhibited the NMDAR NR1 subunit expression and phosphorylation, and PKCγ expression, inhibited glial cell activation, and suppressed the morphine-evoked EAAs release. These effects may result in preservation of the antinociceptive effect of acute morphine challenge in chronic morphine-infused rats. Ultra-low dose naloxone infusion alone did not produce an antinociceptive effect. These findings demonstrated that attenuation of glutamatergic transmission and neuroinflammation by ultra-low dose naloxone co-infusion preserves the lasting antinociceptive effect of morphine in rats chronically infused with morphine.

Attenuation of dynamin‐dependent internalization decreases antinociception during the expression of morphine tolerance

Mu‐opioid receptor (MOP) internalization has been proposed as a cellular mechanism of morphine tolerance. In order to test this hypothesis in vivo, the fluorescent MOP agonist, DERM‐A594, was microinjected into the periaqueductal gray (vlPAG). Rats were pretreated with either saline (0.4 μl) or morphine (5 μg/0.4 μl) 2X/day for 2 days. On day 3, rats were given either a saline or morphine challenge 30 min prior to DERM‐A594 (300 ng/0.4 μl). Intracellular DERM‐A594 intensity was similar in all groups except for a significant decrease in DERM‐A594 label in morphine tolerant rats given a morphine challenge (F (3, 34) = 3.899, p<0.05). These results suggest that repeated morphine administration increases morphine‐induced internalization of MOPs. To further test this idea, a myr‐dynamin inhibitory peptide (Dyn+) and a scrambled peptide (Dyn−) were microinjected into the vlPAG prior to microinjection of DERM‐A594. Dyn+ reduced DERM‐A594 antinociception compared to Dyn− (15 ± 3 s vs. 33 ± 5 s). Dyn+ also reduced morphine antinociception in both saline (D50 = 8.1 μg vs. Dyn− = 3.8 μg) and morphine pretreated rats (D50 = 10.7 μg vs. Dyn− = 5.8 μg). A comparable shift in D50 values in saline and morphine pretreated rats suggests that Dyn+ attenuates antinociception similarly in both groups. Thus, these studies demonstrate that internalization is important for MOP signaling in the vlPAG. Supported by DA023318(TM), DA015498(MM).

Spinal antinociception of synthetic omega-conotoxin SO-3, a selective N-type neuronal voltage-sensitive calcium channel blocker, and its effects on morphine analgesia in chemical stimulus tests in rodent

SO-3, a novel Omega-superfamily conotoxin derived from Conus striatus, selectively inhibits N-type neuronal voltage-sensitive calcium channels. In current study, antinociception of SO-3 compared with MVIIA or morphine and its effects on morphine analgesia were investigated in rodent chemical stimulus tests after acute or repeated intrathecal administration. In mice acetic acid writhing test, similar to MVIIA, SO-3 caused dose- and time-dependent spinal antinociception with ED(50) of 0.25 microg/kg and t(1/2) of 4h, which was more potent and longer-acting than morphine. In rat formalin test after intrathecal bolus injection, SO-3 produced dose- and time-dependent antinociception by suppressing acute (ED(50), 1.79 microg/kg) and tonic phases (ED(50), 0.41 microg/kg), which was similar to MVIIA and approximately 10-fold potency and twice longer-acting of morphine in blocking tonic phase responses. After repeated intrathecal injections twice daily for 5 consecutive days, SO-3 produced analgesia without loss of potency whereas morphine produced analgesia tolerance in rat formalin test; further, SO-3 still produced potent analgesia in morphine-tolerant rats. SO-3 co-administered with morphine left-shift the dose-response curve of morphine in mice acetic acid writhing test and significantly potentiated morphine analgesia in rat formalin test. No changes in motor function were seen in mice or rats receiving antinociceptive doses of SO-3 whereas MVIIA caused motor dysfunction at doses of 1.0-2.0 microg/kg in rats. This study showed that (1) novel SO-3 produced potent and long-acting spinal antinociception without observable motor dysfunction, (2) SO-3 significantly potentiated morphine analgesia, (3) After repeated intrathecal administration, SO-3 produced neither tolerance nor cross-tolerance to morphine analgesia.

The kappa-opioid receptor is upregulated in the spinal cord and locus ceruleus but downregulated in the dorsal root ganglia of morphine tolerant rats

As a non-selective agonist of opioid receptors, morphine can also act on the kappa-opioid receptor (KOR) when activating the mu-opioid receptor (MOR) and delta-opioid receptor (DOR). Although previous findings indicate that KOR plays an important role in morphine analgesia and antinociceptive tolerance, the reasons for the paradoxical functions of KOR in analgesia and anti-analgesia responses are still unclear. The aim of this study was to explore the role of the KOR in morphine analgesia and antinociceptive tolerance. As such, the changes in KOR expression in different regions of the nervous system in morphine tolerant rats were examined. We were able to attain morphine tolerance in rats via subcutaneous injection of morphine (10 mg/kg) twice daily for 7-consecutive days. Competitive real-time PCR, immunohistochemistry, and Western blot analyses were used to assess KOR expression in related regions of the nervous system, including the thalamus, hypothalamus, hippocampus, locus ceruleus (LC), periaqueductal gray (PAG), lumber-sacral spinal cord, and dorsal root ganglia (DRG). The expression of KOR increased in the locus ceruleus and spinal cord, but was significantly decreased in the DRG of morphine tolerant rats ( P < 0.05). No other significant changes in KOR expression were observed in the other regions. Consequently, we propose that the locus ceruleus and spinal cord are likely the dominant CNS regions and the DRG is the main peripheral site in which chronic morphine exerts its effect on KOR. Prolonged morphine administration induces inconsistent changes of KOR in the central and peripheral nervous system.

Change in the expressionof phosphorylated synapsin I in spinal dorsal hornfollowing chronic morphine tolerance in rats

：Objective To investigatethe change in the expression of phosphorylated synapsin Ⅰ in the spinal dorsal horn following chronic morphine tolerance in rats.MethodsForty-five 1-2 month old male SD rats weighing 150-180 g were randomly divided into 5groups(n = 9 each): group Ⅰ sham operation(group S);groupⅡ normal saline(group NS);group Ⅲ morphine(group M);group Ⅳ Ketamine(group K)and groupⅤ M+K.An intrathecal(IT)catheter was placed in thesubarachnoid space at L3,4 interspace in group Ⅱ-Ⅴ.Animals showing motor orsensory dysfunction after surgery were excluded.Three days after surgery NS 40 μl,morphine20μg,ketamine 30 μg and morphine 20 μg+ketamine 30 μgwere injected IT twice a day for 7 consecutive days in group Ⅱ,Ⅲ,Ⅳ and Ⅴ respectively.50% paw withdrawal threshold(PWT)to mechanicalstimulus and paw withdrawal latency(PWL)to noxious thermal stimulus were measuredbefore(T_0,baseline),on the 1st,3rd,5th and 7th day of IT drug administration(T_(1-4))andat 1 day after IT drug administration(T_5).The animals were sacrificed after last painthreshold measurement.The lumbar segment(L_(3-6))was removed for determination of theexpression of phosphorylated synapsin Ⅰprotein.ResultsIn group M,PWT to mechanical stimulus was increased at T_(1,2),returned to the baselinelevel at T,and decreased at T_(4,5),and PWL to noxious thermal stimulus was prolonged atT_(1-3),returned to the baseline level at T_4 and shortened at T_5 as compared to thebaseline value at T_0indicating successful establishment of morphine tolerance.In groupM+K,PWT to mechanical stimulus was increased and PWL to noxious thermal stimulus wasprolonged at T_(1-5) as compared to the baseline value,indicating inhibition of morphinetolerance by ketamine.The expression of phospberylatod synapsin(Ser 603)was significantlyhigher in group M and M+K than in group S,and was significantly lower in group M+K than ingroup M.Conclusion The phosphorylated synapsin Ⅰ in the spinal dorsal horn may be involved in thedevelopment of chronic morphine tolerance which is partly mediated by NMDA receptor. Key words: Morphine; Drug tolerance; Spinal cord; Synaptophysin

Modulation of Morphine-induced Antinociception in Acute and Chronic Opioid Treatment by Ibudilast

Opioid analgesics are effective in relieving chronic pain, but they have serious adverse effects, including development of tolerance and dependence. Ibudilast, an inhibitor of glial activation and cyclic nucleotide phosphodiesterases, has shown potential in the treatment of neuropathic pain and opioid withdrawal. Because glial cell activation could also be involved in the development of opioid tolerance in rats, the authors studied the antinociceptive effects of ibudilast and morphine in different models of coadministration. Antinociception was assessed using male Sprague- Dawley rats in hot plate and tail-flick tests. The effects of ibudilast on acute morphine-induced antinociception, induction of morphine tolerance, and established morphine tolerance were studied. Systemic ibudilast produced modest dose-related antinociception and decreased locomotor activity at the studied doses of 2.5-22.5 mg/kg. The highest tested dose of 22.5 mg/kg produced 52% of the maximum possible effect in the tail-flick test. It had an additive antinociceptive effect when combined with systemic morphine. Coadministration of ibudilast with morphine did not attenuate the development of morphine tolerance. However, in morphine-tolerant rats, ibudilast partly restored morphine-induced antinociception. Ibudilast produces modest antinociception, and it is effective in restoring but not in preventing morphine tolerance. The mechanisms of the effects of ibudilast should be better understood before it is considered for clinical use.