Partial cross tolerance to D-Ala2-D-Leu5-enkephalin after chronic spinal morphine infusion

The development of tolerance to morphine and cross tolerance to D-Ala2-D-Leu5-enkephalin (DADL) at spinal cord level to the inhibition of tail flick response was studied in rats tolerant to morphine. The long term intrathecal infusion of morphine sulfate was accomplished by means of an osmotic minipump. Intrathecal infusion of morphine sulfate (2 micrograms/hr) markedly elevated the tail flick latency measured 24 hr after the start of infusion. The increased tail flick latencies gradually decreased during 6 days of intrathecal infusion of morphine sulfate. Tolerance to morphine and DADL was determined by inhibition to the tail flick response after intrathecal administration of cumulative doses of morphine sulfate and DADL. Chronic intrathecal infusion of morphine induced a marked tolerance to morphine but developed only a slight cross tolerance to DADL. The results indicate that there exists two separate types of opioid receptor, mu- and delta-opioid receptor in the spinal cord of rats.     

Lactoferrin enhances opioid-mediated analgesia via nitric oxide in the rat spinal cord

Lactoferrin (LF) is a multifunctional protein that is found in milk, neutrophils, and other biological fluids, and its receptors have also been identified in the central nervous system. Recently, we found that bovine milk-derived LF (BLF) produced analgesia via a mu-opioid receptor-mediated response in the spinal cord. However, the precise mechanism of this analgesic effect remains unclear. In this study, spinally applied BLF produced analgesia that was reversed by coadministration with a nitric oxide (NO) synthase inhibitor, NG-nitro-l-arginine methyl ester, during phases 1 and 2 in the formalin test. Spinal coadministration of a mu-opioid receptor agonist, morphine, with a subeffective dose of BLF produced a much more highly potentiated analgesia than that produced by morphine alone during phases 1 and 2 in the formalin test. This potentiated analgesia by morphine with BLF was reversed by a mu-opioid receptor antagonist, d-Phe-Cys-Tyr-d-Trp-Orn-Thr-NH2, or by NG-nitro-l-arginine methyl ester. In the tail-flick test, continuous spinal infusion of morphine via an osmotic minipump over 6 days resulted in development of tolerance by day 4, but no tolerance of BLF was observed throughout the experiment. These results suggest that BLF acts as an enhancer of the spinal opioidergic system via an NO-mediated mechanism.     

Thalidomide Promotes Morphine Efficacy and Prevents Morphine-Induced Tolerance in Rats with Diabetic Neuropathy

Opioid analgesics have less efficacy in diabetic neuropathy treatment, and tolerance often occurs after chronic usage. Given that thalidomide can potentiate the morphine efficacy in diabetic neuropathy treatment, we investigated the effects of intrathecal administrations of thalidomide on morphine tolerance during the treatment of diabetic neuropathy. We found that intrathecal administrations of thalidomide (25 mg/kg/ml) potentiated the analgesic effects of morphine on mechanical hyperalgesia and prevented the development of morphine tolerance. While this treatment regimen did not alter the protein levels of μ-opioid receptor (MOR) in the spinal cord of diabetic rats, chronic morphine treatment robustly increased MOR binding density in the synaptic plasma membranes fraction, but decreased it in the microsomal fraction. Furthermore, thalidomide was able to reverse the distribution of MOR altered by chronic morphine treatment. Finally, STZ-induced diabetes promoted PKC activation and enhanced TNFα level in the spinal cord, which were attenuated by intrathecal administrations of thalidomide. Taken together, these results suggested that thalidomide may potentiate morphine efficacy on diabetic neuropathy and prevent the development of morphine tolerance by suppressing PKC activation and TNFα level in the spinal cord.     

Facilitation of spinal morphine analgesia in normal and morphine tolerant animals by neuropeptide SF and related peptides

Neuropeptide FF and related synthetic amidated peptides have been shown to elicit sustained anti-nociceptive responses and potently augment spinal anti-nociceptive actions of spinal morphine in tests of thermal and mechanical nociception. Recent studies have described the occurrence of another octapeptide, neuropeptide SF (NPSF) in the spinal cord and the cerebrospinal fluid and demonstrated its affinity for the NPFF receptors. This study examined the effects of NPSF and two putative precursor peptides, EFW-NPSF and NPAF, on the spinal actions of morphine in normal and opioid tolerant rats using the tailflick and pawpressure tests. In normal rats, NPSF demonstrated weak intrinsic activity but sub-effective doses of the peptide significantly increased the magnitude and duration of spinal morphine anti-nociception in both tests. A low-dose of NPSF also augmented the spinal actions of a delta receptor agonist, deltorphin. The morphine-potentiating effect of NPSF was shared by EFW-NPSF and the octadecapeptide NPAF. In animal rendered tolerant by continuous intrathecal infusion of morphine for 6 days, low dose NPSF itself elicited a significant anti-nociceptive response and potently increased morphine-induced response in both tests. In animals made tolerant by repeated injections of intrathecal morphine, administration of NPSF, EFW-NPSF, and NPAF with morphine reversed the loss of the anti-nociceptive effect and restored the agonist potency. The results demonstrate that in normal animals NPSF and related peptides exert strong potentiating effect on morphine anti-nociception at the spinal level and in tolerant animals these agents can reverse the loss of morphine potency.     

Comparison of analgesic and body temperature responses to intrathecal beta-endorphin and D-Ala2-D-Leu5-enkephalin

The inhibition of tail flick response to radiant heat and body temperature changes after intrathecal administration of β-endorphin (β-EP) and D-Ala²-D-Leu⁵-enkephalin (DADL) were studied in rats. Both opioid peptides caused inhibition of tail flick response. On a molar basis, β-EP was 73% as potent as DADL, but the duration of tail flick inhibition of β-EP was much longer than that of DADL. β-EP induced hyperthermia while DADL did not cause any significant change in body temperature. The tail flick inhibition induced by β-EP (1 nmole) was reversed by 2 mg/kg of naloxone, ip; however, the tail flick inhibition induced by DADL (7 nmole) was not reversed by 2 mg/kg and was incompletely reversed by a higher dose of naloxone one (6 mg/kg, ip). These studies demonstrate the existence of naloxone-resistant opioid receptors in the spinal cord which are sensitive to enkephalin. These results indicate that the opioid receptors involved in the production of opioid responses in the spinal cord are different from those in supraspinal brain areas.     

脊髓蛋白激酶Cα和γ亚型在吗啡依赖和戒断反应中的不同作用

在大鼠吗啡依赖和戒断模型上,采用行为学、免疫组织化学和Western blot方法观察鞘内应用蛋白激酶C(protien kinase C,PKC)抑制剂chelerythrine chloride(CHE)对吗啡依赖大鼠纳洛酮催促成断反应、脊髓Fos蛋白表达和脊髓神经元胞膜和胞浆PKCα、γ表达的影响,以探讨不同亚型PKC在吗啡依赖和戒断反应中的作用。结果表明,鞘内注射CHE能明显减轻吗啡成断症状的评分和吗啡戒断引起的痛觉异常,抑制吗啡成断期间脊髓Fos蛋白表达的增加;吗啡依赖可引起脊髓神经元PKCα和γ表达的上调和转位：吗啡戒断期间存在明显的且可被鞘内注射CHE抑制的PKCα转位,但未观察到明显的PKCγ转位。上述结果表明,脊髓PKC表达上调和转何可能参与吗啡依赖的形成和戒断反应的表达,且PKCα和γ亚型在吗啡依赖和戒断反应中的作用存在差异。     

Proteomic analysis of spinal protein expression in rats exposed to repeated intrathecal morphine injection

Repeated administration of morphine for treating severe chronic pain may lead to neuroadaptive changes in the spinal cord that are thought to underlie molecular mechanisms of the development of morphine tolerance and physical dependence. Here, we employed a 2-D gel-based proteomic technique to detect the global changes of the spinal cord protein expression in rats that had developed morphine tolerance. Morphine tolerance at the spinal cord level was induced by repeated intrathecal injections of morphine (20 microg/10 microL) twice daily for 5 days and evaluated by measurements of paw withdrawal latencies and maximal possible analgesic effect at day 5. After behavioral tests, the lumbar enlargement segments of spinal cord were harvested and proteins resolved by 2-DE. We found that eight proteins were significantly up-regulated or down-regulated in spinal cord after morphine tolerance development, including proteins involved in targeting and trafficking of the glutamate receptors and opioid receptors, proteins involved in oxidative stress, and cytoskeletal proteins, some of which were confirmed by Western blot analysis. Morphine-induced expressional changes of these proteins in the spinal cord might be involved in the central mechanisms that underlie the development of morphine tolerance. It is very likely that these identified proteins may serve as potential molecular targets for prevention of the development of morphine tolerance and physical dependence.     

An interaction of opioids and galanin in dorsal horn of the spinal cord in mononeuropathic rats

The present study was performed in rats with experimentally induced mononeuropathy after common sciatic nerve ligation. The hind-paw withdrawal latencies to thermal and mechanical stimulation were increased significantly after intrathecal injection of 3 nmol of galanin. The increased hind-paw response latencies induced by galanin were attenuated by following intrathecal injection of 22 nmol, but not 11 or 2.75 nmol of the opioid receptor antagonist naloxone. Further, the increased hind-paw response latencies induced by galanin were prevented by following intrathecal injection of 10 nmol of mu-opioid receptor antagonist, beta-funaltrexamine (beta-FNA), but not by 10 nmol of delta-opioid receptor antagonist, natrindole or 10 nmol of kappa-opioid receptor antagonist, nor-binaltorphimine (nor-BNI). Intrathecal 10 nmol of beta-FNA alone had no significant effects on the hind-paw withdrawal responses. These results demonstrate the existence of a specific interaction between galanin and opioids in the transmission of presumed nociceptive information in the spinal cord of mononeuropathic rats. This interaction involves the activation of mu-opioid receptor.     

Involvement of spinal metabotropic glutamate receptor 5 in the development of tolerance to morphine-induced antinociception

It is well known that prolonged exposure to morphine results in tolerance to morphine-induced antinociception. In the present study, we found that either intrathecal (i.t.) or subcutaneous (s.c.) injection of the selective metabotropic glutamate receptor 5 (mGluR5) antagonist, methyl-6-(phenylethynyl)-pyridine hydrochloride (MPEP), attenuated the development of tolerance to morphine-induced antinociception. Using the receptor binding assay, we found here that the number of mGluR5 in the mouse spinal cord was significantly increased by repeated treatment with morphine. Furthermore, repeated treatment with morphine produced a significant increase in the level of mGluR5 immunoreactivity in the dorsal horn of the mouse spinal cord. Double-labeling experiments showed that the increased mGluR5 was predominantly expressed in the neurons and sparsely expressed in the processes of astrocytes following repeated treatment with morphine. Consistent with these results, the response of Ca2+ to the selective group I mGluR agonist, 3,5-dihydroxyphenylglycine (DHPG), in cultured spinal cord neurons was potently enhanced by 3 days of in vitro treatment with morphine. These findings support the idea that the increased mGluR5 following repeated treatment with morphine leads to enhanced neuronal excitability and synaptic transmission in the dorsal horn of the spinal cord and, in turn, suppresses the morphine-induced antinociception in mice.     

Antinociceptive effects of intrathecally administered human beta-endorphin in the rat and cat

Rats chronically implanted with intrathecal catheters displayed a dose-dependent increase in the hot-plate and tail-flick response latencies following the injection of human beta-endorphin into the lumbar spinal subarachnoid space through the indwelling catheter. beta-Endorphin was approximately 25 times more potent than morphine on a molar basis. Matching morphine and beta-endorphin doses such that approximately equal submaximal submaximal effects occurred, it was observed that the antinociception produced by beta-endorphin lasted approximately three times longer than that produced by morphine. Experiments with intrathecal injection of beta-endorphin into the spinal subarachnoid space of cats fitted with intrathecal catheters also revealed a potent antinociceptive effect which was completely antagonized by naloxone. In the rats, naloxone administered systemically in doses of 10--100 microgram/kg produced a parallel shift in the dose-response curves of both nociceptive measures suggesting a competitive antagonism. Using a dose ratio analysis, an in vivo pA2 of 7.1 for naloxone was obtained. These data and those derived from previous work based on the pA2 suggest that the interaction of morphine, certain pentapeptides, and beta-endorphin is the same with regard to the spinal opiate receptor population mediating behaviorally defined analgesia.     

Up-regulation of Μ-opioid receptors in the spinal cord of morphine-tolerant rats

Though morphine remains the most powerful drug for treating pain, its effectiveness is limited by the development of tolerance and dependence. The mechanism underlying development of tolerance to morphine is still poorly understood. One of the factors could be an alteration in the number of Μ-receptors within specific parts of the nervous system. However, reports on changes in the Μ-opioid receptor density in the spinal cord after chronic morphine administration are conflicting. Most of the studies have used subcutaneously implanted morphine pellets to produce tolerance. However, it does not simulate clinical conditions, where it is more common to administer morphine at intervals, either by injections or orally. In the present study, rats were made tolerant to morphine by injecting increasing doses of morphine (10-50 mg/kg, subcutaneously) for five days.In vitro tissue autoradiography for localization of Μ-receptor in the spinal cord was done using [³H]-DAMGO. As compared to the spinal cord of control rats, the spinal cord of tolerant rats showed an 18.8% increase or up-regulation in the density of Μ-receptors in the superficial layers of the dorsal horn. This up-regulation of Μ-receptors after morphine tolerance suggests that a fraction of the receptors have been rendered desensitized, which in turn could lead to tolerance     

Chronic opioid potentiates presynaptic but impairs postsynaptic N-methyl-D-aspartic acid receptor activity in spinal cords: implications for opioid hyperalgesia and tolerance

Opioids are the most effective analgesics for the treatment of moderate to severe pain. However, chronic opioid treatment can cause both hyperalgesia and analgesic tolerance, which limit their clinical efficacy. In this study, we determined the role of pre- and postsynaptic NMDA receptors (NMDARs) in controlling increased glutamatergic input in the spinal cord induced by chronic systemic morphine administration. Whole-cell voltage clamp recordings of excitatory postsynaptic currents (EPSCs) were performed on dorsal horn neurons in rat spinal cord slices. Chronic morphine significantly increased the amplitude of monosynaptic EPSCs evoked from the dorsal root and the frequency of spontaneous EPSCs, and these changes were largely attenuated by blocking NMDARs and by inhibiting PKC, but not PKA. Also, blocking NR2A- or NR2B-containing NMDARs significantly reduced the frequency of spontaneous EPSCs and the amplitude of evoked EPSCs in morphine-treated rats. Strikingly, morphine treatment largely decreased the amplitude of evoked NMDAR-EPSCs and NMDAR currents of dorsal horn neurons elicited by puff NMDA application. The reduction in postsynaptic NMDAR currents caused by morphine was prevented by resiniferatoxin pretreatment to ablate TRPV1-expressing primary afferents. Furthermore, intrathecal injection of the NMDAR antagonist significantly attenuated the development of analgesic tolerance and the reduction in nociceptive thresholds induced by chronic morphine. Collectively, our findings indicate that chronic opioid treatment potentiates presynaptic, but impairs postsynaptic, NMDAR activity in the spinal cord. PKC-mediated increases in NMDAR activity at nociceptive primary afferent terminals in the spinal cord contribute critically to the development of opioid hyperalgesia and analgesic tolerance.     

吗啡耐受增加福尔马林致痛大鼠脊髓Fos和NADPH-d阳性神经元的表达

运用Ｆｏｓ免疫组织化学、ＮＡＤＰＨ－ｄ组织化学及Ｆｏｓ／ＮＡＤＰＨ－ｄ双标技术,研究了吗啡耐受对福尔马林致痛大鼠脊髓Ｆｏｓ、ＮＡＤＰＨ－ｄ阳性及Ｆｏｓ／ＮＡＤＰＨ－ｄ双标神经元表达的影响。结果观察到：在非吗啡耐受大鼠,福尔马林诱发的Ｆｏｓ－ｌｉｋｅ ｉｍｍｕｎｏｒｅａｃｔｉｖｉｔｙ（Ｆｏｓ－ＬＩ）主要分布在同侧脊髓背角浅层和颈部,急性静注吗啡可减少Ｆｏｓ－ＬＩ表达;长时间应用吗啡导致福尔马林诱发的     

Serum IL-10 involved in morphine tolerance development during adjuvant-induced arthritis

Opioid receptors play an important role in modulation of hyperalgesia in inflamed tissues, but chronic morphine application induces such side effects as tolerance. There is near communications between cytokines and mu opioid receptor expression. This study was aimed to assess the role of serum IL-10 in morphine tolerance development during adjuvant-induced arthritis. Adjuvant arthritis (AA) was induced on day 0 by single injection of Complete Freund’s Adjuvant (CFA) into the rats’ hindpaw. Hyperalgesia, edema, and spinal mu opioid receptor (mOR) variations were assessed on 0, 7, 14, and 21 days of the study. For assessment of the morphine tolerance development, morphine effective dose (4 mg/kg) was administered from the 14th day after CFA injection and continued until the morphine post-dose paw withdrawal latency (PWL); it did not significantly differ from the baseline. For assessment of the effects of IL-10 on tolerance induction, a neutralizing dose (ND50) of anti-IL-10 was administered daily during different stages of the study. AA induction in the right hindpaw of rats resulted in unilateral inflammation and hyperalgesia within 21 days of the study. Anti-IL-10 antibody administration in the AA rats induced marked elevation of hyperalgesia compared to the AA control group. Our data also indicated that morphine effective anti-hyperalgesic dose significantly decreased in the AA rats compared to the control group, which this symptom was aligned with spinal mu opioid receptor (mOR) expression increase during AA. Moreover, there was a significant difference in morphine tolerance induction between the AA and control rats, and our results also demonstrated that IL-10 played an important role in tolerance-induction process. It can be concluded that morphine tolerance slowly progressed when administered morphine effective dose was reduced during AA chronic inflammation. On the other hand, it seems that increased level of serum IL-10 may affect morphine tolerance development during inflammation.     

Dynorphin: potent analgesic effect in spinal cord of the rat

Evidence is presented to show a strong and long-lasting analgesic effect after injection of dynorphin into the subarachnoid space of the spinal cord of the rat. Calculating on a molar basis dynorphin was 6-10 times more potent than morphine and 65-100 times more potent than morphiceptin, the specific mu receptor agonist. Dynorphin analgesia was completely reversed by intrathecal injection of anti-dynorphin IgG and partially reversed by naloxone. Acute tolerance to morphine analgesia did not affect the occurrence of dynorphin analgesia. Evidence from different lines of approach suggest that dynorphin may bind with kappa receptors in the spinal cord to exert its analgesic effect.     

Morphine tolerance in arthritic rats and serotonergic system

To understand whether chronic inflammation alters the development of morphine tolerance, the tail-flick test was used to evaluate the analgesic effect of morphine (75 mg tablet, s.c.) in the arthritic rats at the day 9-12 after the inoculation with Freund's adjuvant. Spinal cord monoamines and amino acid neurotransmitters were concomitantly measured. Chronic inflammation attenuated the antinociceptive effect of morphine as tolerance developed faster in the arthritic rats compared to the vehicle-treated controls. In addition, ratio of 5-hydroxyindole-3-acetic acid/5-hydroxytryptamine (5-HIAA/5-HT) increased in the lumbar spinal cord of arthritic rats without any change in the concentrations of norepinephrine, glutamate, aspartate or GABA. Interestingly, increased serotonin turnover in the spinal cord was observed in both control and arthritic rats 24 hours after morphine treatment. Overall, the results suggest a significant role of serotonin up-regulation in the spinal cord during chronic pain and the development of morphine tolerance.     

Mu-opioid receptors are involved in the tolerance to nicotine antinociception

Several studies have shown the participation of the endogenous opioid system on the antinociceptive effects and addictive properties of nicotine. The aim of the present study was to explore the involvement of the mu-opioid receptors in the development of tolerance to nicotine antinociception. Chronic treatment of C57BL/6 mice with nicotine (5 mg/kg s.c., three times daily during 12 days) resulted in tolerance to its antinociceptive responses in the tail-immersion test. We investigated the possible existence of adaptive changes in the expression and/or functional activity of mu-opioid receptors in these tolerant mice by using autoradiography of [(3)H]D-Ala(2)-MePhe(4)-Gly-ol(5) enkephalin ([(3)H]DAMGO) binding and DAMGO-stimulated guanosine [(35)S]5'-(gamma-thio)-triphosphate ([(35)S]GTPgammaS) binding. The density of mu-opioid receptors in the spinal cord was not modified in nicotine-tolerant mice, whereas a decrease was found in the caudate-putamen, as well as in the core and the shell of the nucleus accumbens. However, the functional activity of these receptors was significantly increased in the spinal cord as a consequence of nicotine treatment. To further investigate the role of mu-opioid receptors in the tolerance to nicotine-induced antinociception, we evaluated this response in C57BL/6 mu-opioid receptor knockout mice. Chronic nicotine treatment produced tolerance in both wild-type and knockout animals, but tolerance developed faster in mice lacking mu-opioid receptors. These results indicate that mu-opioid receptors play an important role in the development of tolerance to nicotine antinociceptive effects.     

High and low frequency electroacupuncture analgesia are mediated by different types of opioid receptors at spinal level: a cross tolerance study

Previous studies have shown that rats subjected to low or high frequency electroacupuncture (EA) stimulation release enkephalins or dynorphins respectively to produce analgesia. This conclusion was tested in the present study by using cross tolerance technique for further analysing their receptor mechanisms. The main results were as follows: (1) In rats subjected to 2 Hz EA for 6 h, there was a gradual decrease in the analgesic effect, leading to a state of tolerance to 2 Hz EA analgesia. These rats, however, still responded to 100 Hz EA. Likewise, rats made tolerant to 100 Hz EA were still effective to 2 Hz EA stimulation, showing not significant cross tolerance between 2 Hz and 100 Hz EA analgesia. (2) Rats made-tolerant to 100 Hz EA analgesia showed a diminished response to intrathecal dynorphin A (1-13), a kappa agonist, whereas the analgesic effect of the delta agonist [D-Pen2, D-pen5] enkephalin (DPDPE) remained intact. (3) Rats made tolerant to 2 Hz EA analgesia showed a cross tolerance to DPDPE, but not to dynorphin A (1-13). Results obtained from aforementioned cross tolerance studies suggest that 2 Hz and 100 Hz EA analgesia are mediated by delta and kappa opioid receptors, respectively, at the spinal cord of the rat.     

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.

     

NO参与介导吗啡戒断大鼠脊髓神经元敏感化

运用Fos免疫组织化学、NADPH－d组织化学、F/NADPH－d双标、鞘内注射和反义寡核苷酸技术,观察吗啡戒断大鼠脊髓神经元活动变化及NO在其中的作用,结果发现：非吗啡依赖大鼠急性应用纳洛酮和吗啡依赖大鼠脊髓水平Fos-LI和NADPH－d阳性神经元表达与对照组相比无明显变化,二者也无Fos／NADPH－d双标神经元表达;吗啡依赖纳洛酮催促戒断大鼠脊髓Fos-LI、NADPH－d阳性神经元、纤维和终末表达明显增加,且出现Fos／NADPH－d双标神经元表达。Fos-LI和Fos／NADPH－d双标神经元呈现双侧脊髓全层分布,NADPH－d阳性神经元、纤维和终末主要位于双侧脊髓背角浅层。鞘内注射NOS抑制剂L－NA和nNOS反义寡核苷酸均明显降低吗啡依赖大鼠纳洛酮催促戒断症状评分,减少吗啡戒断大鼠脊髓Fos-LI表达。上述结果提示：NO参与介导吗啡戒断大鼠脊髓神经元敏感化。