Morphine Pellets Research Articles

Assessing effects of tamoxifen on tolerance, dependence, and glutamate and glutamine levels in frontal cortex and hippocampus in chronic morphine treatment

Tamoxifen has been shown to reduce glutamate release from presynaptic glutamatergic nerves and reverse tolerance to morphine-induced respiratory depression. Changes in glutamatergic neurotransmission in the central nervous system contribute to morphine tolerance, dependence, and withdrawal. This study, therefore, evaluated effects of tamoxifen on development of analgesic tolerance and dependence, and brain glutamate and glutamine levels in chronic morphine administration. Mice implanted with placebo or morphine pellets were injected with tamoxifen (0.6–2 mg/kg) or vehicle twice daily for 3 days. Nociceptive response was evaluated in the hot plate and tail immersion tests, 4, 48 and 72 h post-implant, and following a challenge dose of morphine (10 mg/kg). Withdrawal signs were determined after naloxone (1 mg/kg) administration. Morphine increased nociceptive threshold which declined over time. At 72 h, acute morphine elicited tolerance to the analgesic effect in the hot plate test in vehicle or tamoxifen administered animals. In the tail immersion test, however, tolerance to morphine analgesia was observed in tamoxifen, but not vehicle, co-administration. Tamoxifen did not reduce withdrawal signs. In contrast to previous reports, glutamate and glutamine levels in the hippocampus and frontal cortex did not change in the morphine-vehicle group. Confirming previous findings, tamoxifen (2 mg/kg) decreased glutamate and glutamine concentrations in the hippocampus in animals with placebo pellets. Both doses of tamoxifen significantly changed glutamate and/or glutamine concentrations in both regions in morphine pellet implanted animals. These results suggest that tamoxifen has no effect on dependence but may facilitate tolerance development to the antinociception, possibly mediated at the spinal level, in chronic morphine administration.

Memantine and SKF82958 but not an enriched environment modulate naloxone-precipitated morphine abstinence syndrome without affecting hippocampal tPA mRNA levels in rats

There is accumulating evidence supporting the involvement of tissue-plasminogen activator (tPA) in the mechanisms underlying the effects of morphine and an enriched environment. This study was designed to investigate possible interactive roles of the glutamatergic and the dopaminergic systems regarding hippocampal tPA in the neurobiology of morphine dependence. For this purpose, Wistar albino rats, housed in either a standard- (SE) or an enriched environment (EE) were implanted subcutaneously with morphine (150 mg base) or placebo pellets. Behavioral and somatic signs of morphine abstinence precipitated by an opioid-receptor antagonist naloxone (1 mg/kg, i.p.) 72 h after the pellet implantation were observed individually for 15 min in all groups. Memantine (10 mg/kg i.p.), an antagonist of N-methyl-D-aspartic acid class of glutamatergic receptor-subtype decreased teeth-chattering, ptosis, diarrhea and the loss of body weight. SKF82958 (1 mg/kg, i.p.), a dopamine D1-receptor agonist decreased jumping and ptosis but increased rearing and loss of body weight. On the other hand, co-administration of SKF82958 with memantine prevented some of their effects that occur when administered alone at the same doses. Furthermore, the EE did not change the intensity of morphine abstinence. The level of hippocampal tPA mRNA was found to be lower in the SE morphine abstinence group than in the placebo group and close to the EE morphine abstinence group, whereas there was no significant alteration of its level in the memantine or SKF82958 groups. These findings suggest that the interaction between the glutamatergic and the dopaminergic systems may be an important component of the neurobiology of morphine dependence, and the role of tPA in this interaction should be further investigated.

The effect of acute topiramate administration on morphine withdrawal syndrome and brain-derived neurotrophic factor in central nervous system

ABSTRACT Objectives Nucleus accumbens plays an important role in opioid addiction. Topiramate, increases postsynaptic gamma-aminobutyric acid receptor activity and antagonizes glutamatergic activity. Brain-derived neurotrophic factor (BDNF), which plays a key role in synaptic plasticity, is produced from proBDNF. The aim of this study is to investigate the effects of 100 µM topiramate applied into the lateral ventricle or nucleus accumbens on naloxone-induced morphine withdrawal and the BDNF/proBDNF ratio in the frontal cortex. Methods In the study, 36 adult male Wistar rats weighing 250–350 g were used. Morphine dependence was created with morphine pellets following guide cannula implantations. Withdrawal findings were evaluated in naloxone-induced morphine withdrawal syndrome following topiramate administration, and locomotor activity measurements were performed simultaneously. The brains of sacrificed animals were removed for determination of BDNF/proBDNF ratio. Results Topiramate administered by either route significantly suppressed the number of jumps in morphine withdrawal. Topiramate applied into the nucleus accumbens significantly reduced stereotypical behavior in morphine withdrawal, but did not cause any changes in other locomotor activity behaviors. Topiramate applied into the lateral ventricle significantly decreased the BDNF/proBDNF ratio, whereas administered into the nucleus accumbens significantly increased this ratio. Conclusion The findings of this study indicate that topiramate administered into the lateral ventricle and nucleus accumbens reduces naloxone-induced morphine withdrawal symptoms, stereotypical locomotor activity, and changes the BDNF/proBDNF ratio.

Morphine use induces gastric microbial dysbiosis driving gastric inflammation through TLR2 signalling which is attenuated by proton pump inhibition.

Opioids are the standard drug for pain management; however, their effects on gastric dysfunction are relatively understudied. Opioid users have a higher incidence of gastric pathology leading to increased hospitalization. Herein, we investigated the consequences of morphine use on gastric pathology and the underlying mechanisms. We further investigated the therapeutic benefit of proton pump inhibition to overcome morphine-mediated gastric inflammation. Mice were implanted with 25 mg slow-release morphine and placebo pellets. Gastric microbiome analyses were performed. Gastric damage was assayed. Gastric pH was measured. Germ-free and TLR2KO mice were used to investigate the mechanisms. Gastroprotective studies were performed with the proton pump inhibitor (PPI) omeprazole. Chronic morphine treatment alters gastric microbial composition and induces preferential expansion of pathogenic bacterial communities such as Streptococcus and Pseudomonas. Morphine causes disruption of the gastric mucosal layer, increases apoptosis, and elevates inflammatory cytokines. Moreover, morphine-mediated gastric pathology was significantly attenuated in germ-free mice, and reconstitution of morphine gastric microbiome in germ-free mice resulted gastric inflammation. In addition, morphine-mediated gastric inflammation was attenuated in TLR2KO mice. Morphine causes a decrease in gastric pH, which contributes to gastric dysbiosis leads to gastric inflammation. Omeprazole treatment inhibits gastric acidity, rescuing morphine-induced gastric dysbiosis and preventing inflammation. This study attributes morphine-induced gastric acidity as a driver of gastric dysbiosis and pathology and proposes the therapeutic use of PPI as an inexpensive approach for the clinical management of morphine-associated pathophysiology.

Effect of oxytocin pretreatment on the development of morphine tolerance and dependence in rats

Increased opioid synthesis and release, and enhanced alpha-2 adrenoceptor signaling have been suggested to mediate repeated oxytocin-induced long-lasting effects including elevated pain threshold in rats. This study evaluated whether oxytocin pretreatment would influence development of dependence and tolerance to the nociceptive and body temperature responses to morphine and enhance effects of alpha-2 adrenergic agonist clonidine on nociceptive threshold, body temperature and morphine withdrawal signs. Rats injected subcutaneously with saline or 1 mg/kg oxytocin for 5 days were implanted with placebo or morphine pellets 24 h after the treatment period. Body temperature and nociception were assessed, with nociception determined via by hot plate and tail immersion tests, before and 4, 24 and 48 h after pellet implantation, and following a challenge dose of morphine. Withdrawal signs were determined after naloxone administration. Oxytocin produced analgesia, as evidenced by increased paw withdrawal latency in the hot plate test. Morphine increased body temperature and nociceptive threshold which declined over time. Morphine challenge could not demonstrate tolerance to the body temperature response. Analgesic tolerance was observed in the hot plate test in saline and in both tests in oxytocin pretreated rats. Naloxone-precipitated withdrawal appeared to be less severe in oxytocin pretreatment. Clonidine was ineffective on the withdrawal signs but decreased body temperature and increased tail flick latency in the tail immersion test in oxytocin pretreated animals. These results, while producing evidence for a hyperresponsiveness in alpha-2 adrenoceptors, provide contrasting effects on morphine tolerance and dependence, and their partial mediation by opioidergic and adrenergic activation in repeated oxytocin treatment.

Role of Acetaldehyde in Ethanol Reversal of Tolerance to Morphine-Induced Respiratory Depression in Mice.

BackgroundOpioid users regularly consume other drugs such as alcohol (ethanol). Acute administration of ethanol rapidly reverses tolerance to morphine-induced respiratory depression. However, recent research has suggested that the primary metabolite of ethanol, acetaldehyde, may play a key role in mediating the CNS effects seen after ethanol consumption. This research investigated the role of acetaldehyde in ethanol reversal of tolerance to morphine-induced respiratory depression.MethodsTolerance was induced in mice by 6-days implantation of a 75 mg morphine pellet with control mice implanted with a placebo pellet. Tolerance was assessed by acute morphine administration on day 6 and respiration measured by plethysmography. Levels of acetaldehyde were inhibited or enhanced by pre-treatments with the acetaldehyde chelator D-penicillamine and the inhibitor of acetaldehyde dehydrogenase disulfiram respectively.ResultsMorphine pellet implanted mice displayed tolerance to an acute dose of morphine compared to placebo pellet implanted controls. Acute acetaldehyde administration dose-dependently reversed tolerance to morphine respiratory depression. As previously demonstrated, ethanol reversed morphine tolerance, and this was inhibited by D-penicillamine pre-treatment. An acute, low dose of ethanol that did not significantly reverse morphine tolerance was able to do so following disulfiram pre-treatment.ConclusionThese data suggest that acetaldehyde, the primary metabolite of ethanol, is responsible for the reversal of morphine tolerance observed following ethanol administration.

Different Roles of Beclin1 in the Interaction Between Glia and Neurons after Exposure to Morphine and the HIV- Trans-Activator of Transcription (Tat) Protein.

Previously we showed that Beclin1 has a regulatory role in the secretion of inflammatory molecules in glia after exposure to morphine and Tat (an HIV protein). Here we show increased secretion of neuronal growth factors and increased neuronal survival in Beclin1-deficient glia. However, without glia co-culture, neurons deficient in Beclin1 showed greater death and enhanced dendritic beading when compared to wild-type neurons, suggesting that glial-secreted growth factors compensate for the damage reduced autophagy causes neurons. To assess if our ex vivo results correlated with in vivo studies, we used a wild-type (Becn1+/+) and Beclin1-deficient (Becn1+/+) mouse model and intracranially infused the mice with Tat and subcutaneously administered morphine pellets. After morphine implantation, significantly impaired locomotor activities were detected in both Becn1+/+ and Becn1+/-mice, irrespective of Tat infusion. After induction of pain, morphine-induced antinociception was detected. Interestingly, co-exposure to morphine and Tat increased sensitivity to pain in Becn1+/+ mice, but not in similarly treated Becn1+/- mice. Brain homogenates from Becn1+/+ mice exposed to Tat, alone and in combination with morphine, showed increased secretion of pro-inflammatory cytokines and reduced expression of growth factors when compared to similarly treated Becn1+/- mice. Likewise, increased neuronal loss was detected when both Tat and morphine were administered to Becn1+/+ mice, but not in similarly treated Becn1+/- mice. Overall, our findings show that there is a Beclin1-driven interaction between Tat and morphine in glia and neurons. Moreover, reduced glial-Beclin1 may provide a layer of protection to neurons under stressful conditions, such as when exposed to morphine and Tat.

Morphine restores and naloxone-precipitated withdrawal depresses wheel running in rats with hindpaw inflammation

Opioids such as morphine are the most effective treatment for pain, but termination of opioid use can produce severe withdrawal symptoms. The present study models this process by using home cage wheel running to assess well-being as a result of pain, morphine analgesia, and opioid withdrawal. Injection of CFA into the right hindpaw caused a dramatic decrease in wheel running and body weight. Implantation of two morphine pellets (75 mg each) resulted in an increase in body weight on Day 1 of administration and a more gradual restoration of wheel running that was only evident during the dark phase of the circadian cycle on Days 3 and 4 of morphine administration. Continuous morphine administration decreased wheel running during the relatively inactive light phase. These findings are consistent with the clinical goal of pain therapeutics to restore normal activity during the day and facilitate sleep at night. Administration of naloxone (1 mg/kg) on Day 5 of morphine administration depressed wheel running for approximately 4 h and caused an increase in wet dog shakes. Naloxone-precipitated changes were no longer evident 6 h after administration. These findings demonstrate that the use of morphine to treat pain does not protect against opioid withdrawal. Moreover, this study provides additional support for the use of home cage wheel running as a method to assess changes in well-being as a result of pain, analgesia, and opioid withdrawal.

Comparative benefits of social housing and buprenorphine on wheel running depressed by morphine withdrawal in rats.

Social support and opioid replacement therapy are commonly used to treat opioid withdrawal. The present study tested the hypothesis that social housing and buprenorphine administration can restore wheel running depressed by morphine withdrawal in rats. Experiment 1 assessed disruptive side effects of buprenorphine and found that administration of low doses (3.2, 10, & 32µg/kg, s.c.) had no impact on voluntary wheel running. Experiment 2 assessed the impact of social housing and acute buprenorphine administration (10µg/kg) on morphine withdrawal. Two 75mg morphine pellets were implanted for 3days to induce dependence. Removal of the morphine pellets caused a decrease in body weight, increase in wet dog shakes, and depression of wheel running during the normally active dark phase of the circadian cycle. Social housing restored wheel running and reduced the number of wet dog shakes but did not affect body weight. Administration of buprenorphine restored wheel running depressed by morphine withdrawal for 2days in individually housed rats and produced time-dependent changes in socially housed rats: Depression of wheel running in the 3h following administration and restoration of running subsequently compared to saline-treated controls. The impact of buprenorphine and social housing to reduce the effect of morphine withdrawal in rats is consistent with the use of opioid substitution therapy and psychotherapy/social support to treat opioid withdrawal in humans. These data provide further validation for the clinical relevance for the use of wheel running to assess spontaneous opioid withdrawal.

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G-protein-biased agonists with reduced β-arrestin-2 activation are being investigated as safer alternatives to clinically-used opioids. β-arrestin-2 has been implicated in the mechanism of opioid-induced antinociceptive tolerance. Opioid-induced analgesic tolerance is classically considered as centrally-mediated, but recent reports implicate nociceptive dorsal root ganglia neurons as critical mediators in this process. Here, we investigated the role of β-arrestin-2 in the mechanism of opioid tolerance in dorsal root ganglia nociceptive neurons using β-arrestin-2 knockout mice and the G-protein-biased μ-opioid receptor agonist, TRV130. Whole-cell current-clamp electrophysiology experiments revealed that 15-18-h overnight exposure to 10 μM morphine in vitro induced acute tolerance in β-arrestin-2 wild-type but not knockout neurons. Furthermore, in wild-type neurons circumventing β-arrestin-2 activation by overnight treatment with 200 nM TRV130 attenuated tolerance. Similarly, acute morphine tolerance in vivo in β-arrestin-2 knockout mice was prevented in the warm-water tail-withdrawal assay. Treatment with 30 mg/kg TRV130 s.c. also inhibited acute antinociceptive tolerance in vivo in wild-type mice. Alternately, in β-arrestin-2 knockout neurons tolerance induced by 7-day in vivo exposure to 50 mg morphine pellet was conserved. Likewise, β-arrestin-2 deletion did not mitigate in vivo antinociceptive tolerance induced by 7-day exposure to 25 mg or 50 mg morphine pellet in both female or male mice, respectively. Consequently, these results indicated that β-arrestin-2 mediates acute but not chronic opioid tolerance in dorsal root ganglia neurons and to antinociception in vivo. This suggests that opioid-induced antinociceptive tolerance may develop even in the absence of β-arrestin-2 activation, and thus significantly affect the clinical utility of biased agonists.

Role of β-arrestin-2 in short- and long-term opioid tolerance in the dorsal root ganglia

G-protein-biased agonists with reduced β-arrestin-2 activation are being investigated as safer alternatives to clinically-used opioids. β-arrestin-2 has been implicated in the mechanism of opioid-induced antinociceptive tolerance. Opioid-induced analgesic tolerance is classically considered as centrally-mediated, but recent reports implicate nociceptive dorsal root ganglia neurons as critical mediators in this process. Here, we investigated the role of β-arrestin-2 in the mechanism of opioid tolerance in dorsal root ganglia nociceptive neurons using β-arrestin-2 knockout mice and the G-protein-biased μ-opioid receptor agonist, TRV130. Whole-cell current-clamp electrophysiology experiments revealed that 15-18-h overnight exposure to 10 μM morphine in vitro induced acute tolerance in β-arrestin-2 wild-type but not knockout neurons. Furthermore, in wild-type neurons circumventing β-arrestin-2 activation by overnight treatment with 200 nM TRV130 attenuated tolerance. Similarly, acute morphine tolerance in vivo in β-arrestin-2 knockout mice was prevented in the warm-water tail-withdrawal assay. Treatment with 30 mg/kg TRV130 s.c. also inhibited acute antinociceptive tolerance in vivo in wild-type mice. Alternately, in β-arrestin-2 knockout neurons tolerance induced by 7-day in vivo exposure to 50 mg morphine pellet was conserved. Likewise, β-arrestin-2 deletion did not mitigate in vivo antinociceptive tolerance induced by 7-day exposure to 25 mg or 50 mg morphine pellet in both female or male mice, respectively. Consequently, these results indicated that β-arrestin-2 mediates acute but not chronic opioid tolerance in dorsal root ganglia neurons and to antinociception in vivo. This suggests that opioid-induced antinociceptive tolerance may develop even in the absence of β-arrestin-2 activation, and thus significantly affect the clinical utility of biased agonists.

Social housing promotes recovery of wheel running depressed by inflammatory pain and morphine withdrawal in male rats

The increased use of opioids to treat pain has led to a dramatic increase in opioid abuse. Our previous data indicate that pain may facilitate the development of opioid abuse by increasing the magnitude and duration of opioid withdrawal. The present study tested the hypothesis that social housing would facilitate recovery of activity depressed by pain and opioid withdrawal. Male Sprague Dawley rats were housed either in pairs or alone and then moved to a cage with a running wheel for 6 h daily to assess pain- and opioid withdrawal-induced depression of wheel running. Rats were implanted with two morphine (75 mg each) or placebo pellets to induce opioid dependence and were simultaneously injected with Complete Freund’s Adjuvant or saline into the right hind paw to induce persistent inflammatory pain. Hind paw inflammation depressed wheel running whether rats were implanted with a morphine or placebo pellet. Pair-housed rats showed greater recovery of wheel running than individually housed rats. Spontaneous morphine withdrawal precipitated by removal of the morphine pellets caused a reduction in wheel running that was greater in rats with hind paw inflammation compared to pain free rats. Social housing facilitated recovery from withdrawal in rats with hind paw inflammation, but slowed recovery in pain free rats. These data suggest that social housing facilitates recovery by reducing pain both before and during opioid withdrawal. Our findings are consistent with previous studies showing that social buffering reduces pain-evoked responses.

Sex-specific transgenerational effects of morphine exposure on reward and affective behaviors

Current estimates indicate that millions of people in the United States abuse opioid drugs, which may also affect their offspring. To determine whether parental exposure to morphine alters reward and affective behaviors in subsequent generations we exposed male and female C57BL/6NTac mice to morphine (75 mg) or placebo pellets for 4 weeks. Naïve mice were used as mating partners to create subsequent generations (F1 and F2). Adult male and female F1 and F2 mice were tested in the morphine conditioned place preference paradigm (CPP), marble burying (MB), acoustic startle response (ASR), and open field tests (OFT). Paternal morphine exposure resulted in significantly attenuated preference scores amongst F1 male offspring, but significantly higher preference scores amongst F1 female offspring at the lowest CPP dose tested (5 mg/kg). In contrast, maternal exposure to morphine did not affect morphine reward in the F1 generation; however, the F2 male offspring of morphine-exposed F0 females displayed significantly higher CPP preference scores. Preference scores in F2 females were not affected by F0 male or female morphine exposure. Sex-specific alterations in affective behaviors were observed only in the offspring of F0 males exposed to morphine with F1 males spending less time in the center of the open field and F1 females spending more time in the center of the open field. One generation later, affective behaviors were no longer altered in F2 males but F2 females from the F0 male morphine exposure buried more marbles in the MB test. In summary, early exposure to morphine in males and females causes lineage-specific inheritance of reward and affective behaviors.

Positive Allosteric Modulation of CB1 Cannabinoid Receptor Signaling Enhances Morphine Antinociception and Attenuates Morphine Tolerance Without Enhancing Morphine- Induced Dependence or Reward.

Opioid analgesics represent a critical treatment for chronic pain in the analgesic ladder of the World Health Organization. However, their use can result in a number of unwanted side-effects including incomplete efficacy, constipation, physical dependence, and overdose liability. Cannabinoids enhance the pain-relieving effects of opioids in preclinical studies and dampen unwanted side-effects resulting from excessive opioid intake. We recently reported that a CB1 positive allosteric modulator (PAM) exhibits antinociceptive efficacy in models of pathological pain and lacks the adverse side effects of direct CB1 receptor activation. In the present study, we evaluated whether a CB1 PAM would enhance morphine’s therapeutic efficacy in an animal model of chemotherapy-induced neuropathic pain and characterized its impact on unwanted side-effects associated with chronic opioid administration. In paclitaxel-treated mice, both the CB1 PAM GAT211 and the opioid analgesic morphine reduced paclitaxel-induced behavioral hypersensitivities to mechanical and cold stimulation in a dose-dependent manner. Isobolographic analysis revealed that combinations of GAT211 and morphine resulted in anti-allodynic synergism. In paclitaxel-treated mice, a sub-threshold dose of GAT211 prevented the development of tolerance to the anti-allodynic effects of morphine over 20 days of once daily dosing. However, GAT211 did not reliably alter somatic withdrawal signs (i.e., jumps, paw tremors) in morphine-dependent neuropathic mice challenged with naloxone. In otherwise naïve mice, GAT211 also prolonged antinociceptive efficacy of morphine in the tail-flick test and reduced the overall right-ward shift in the ED50 for morphine to produce antinociception in the tail-flick test, consistent with attenuation of morphine tolerance. Pretreatment with GAT211 did not alter somatic signs of μ opioid receptor dependence in mice rendered dependent upon morphine via subcutaneous implantation of a morphine pellet. Moreover, GAT211 did not reliably alter μ-opioid receptor-mediated reward as measured by conditioned place preference to morphine. Our results suggest that a CB1 PAM may be beneficial in enhancing and prolonging the therapeutic properties of opioids while potentially sparing unwanted side-effects (e.g., tolerance) that occur with repeated opioid treatment.

Melanin-concentrating hormone in rat nucleus accumbens or lateral hypothalamus differentially impacts morphine and food seeking behaviors.

Identifying neural substrates that are differentially affected by drugs of abuse and natural rewards is key to finding a target for an efficacious treatment for substance abuse. Melanin-concentrating hormone is a polypeptide with an inhibitory effect on the mesolimbic dopamine system. Here we test the hypothesis that melanin-concentrating hormone in the lateral hypothalamus and nucleus accumbens shell is differentially involved in the regulation of morphine and food-rewarded behaviors. Male Sprague-Dawley rats were trained with morphine (5.0 mg/kg, subcutaneously) or food pellets (standard chow, 10-14 g) to induce a conditioned place preference, immediately followed by extinction training. Melanin-concentrating hormone (1.0 µg/side) or saline was infused into the nucleus accumbens shell or lateral hypothalamus before the reinstatement primed by morphine or food, and locomotor activity was simultaneously monitored. As the comparison, melanin-concentrating hormone was also microinjected into the nucleus accumbens shell or lateral hypothalamus before the expression of food or morphine-induced conditioned place preference. Microinfusion of melanin-concentrating hormone into the nucleus accumbens shell (but not into the lateral hypothalamus) prevented the reinstatement of morphine conditioned place preference but had no effect on the reinstatement of food conditioned place preference. In contrast, microinfusion of melanin-concentrating hormone into the lateral hypothalamus (but not in the nucleus accumbens shell) inhibited the reinstatement of food conditioned place preference but had no effect on the reinstatement of morphine conditioned place preference. These results suggest a clear double dissociation of melanin-concentrating hormone in morphine/food rewarding behaviors and melanin-concentrating hormone in the nucleus accumbens shell. Melanin-concentrating hormone could be a potential target for therapeutic intervention for morphine abuse without affecting natural rewards.

Experimental Colitis Enhances the Rate of Antinociceptive Tolerance to Morphine via Peripheral Opioid Receptors.

Opioids are highly effective analgesics, however, their therapeutic use is limited by adverse effects that include respiratory depression, dependence, and tolerance. Inflammation has been implicated as a significant driver for the development of tolerance to opioids. Recent studies show that chronic morphine in mice results in gut microbial dysbiosis and inflammation in the colon. In the present study, we examined whether colonic inflammation results in tolerance to the antinociceptive effects of morphine. Colonic inflammation was induced in mice by intrarectal administration of 2,4,6-trinitro-benzene sulfonic acid. The development of antinociceptive tolerance was determined by warm-water tail-immersion assay in mice implanted with 25-, 50-, or 75-mg morphine pellet. Colonic inflammation significantly enhanced the rate at which tolerance developed in each cohort of chronic morphine-treated mice. At the lowest dose of morphine pellet (25 mg), antinociceptive tolerance only developed in the presence of colonic inflammation, whereas in 50- and 75-mg pelleted mice, tolerance developed faster in the inflamed animals than in the noninflamed mice. The enhanced antinociceptive tolerance was attenuated with daily administration of peripheral opioid receptor antagonist, 6β-N-heterocyclic-substituted naltrexamine derivative [17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(4'pyridyl)acetamido]morphinan (NAP)], irrespective of colonic inflammation. Collectively, these findings show that the rate of tolerance to morphine antinociception is exaggerated in the presence of colonic inflammation, and tolerance is prevented by a peripheral μ-opioid receptor antagonist. These studies suggest a peripheral component to the development of antinociceptive tolerance to opioids. Furthermore, peripherally selective opioid antagonists may be useful adjuncts in opioid-based pain management. SIGNIFICANCE STATEMENT: This study supports the notion that inflammation influences the development of antinociceptive tolerance to chronic morphine exposure. We found that, in the presence of colonic inflammation, the rate of development of tolerance to the antinociceptive effects of morphine increased. We also found that treatment with a peripheral opioid receptor antagonist prevented morphine antinociceptive tolerance. Increasing opioid intake during an inflammatory state would result in decreased analgesia and enhanced analgesic tolerance, which puts patients with inflammatory bowel diseases, inflammatory joint diseases, and sickle cell anemia at risk for heavy opioid use.

The Effect of Colonic Inflammation on Morphine Induced Antinociceptive Tolerance

Opioids are one of the most widely prescribed classes of drugs for the treatment of moderate to severe pain. Opioids are prescribed for abdominal pain, a major symptom associated with Crohn's Disease. Recent studies have reported that continued opioid use in patients with Crohn's Disease is associated with increased mortality rate, duration and severity of disease, as well as infection. However, it is not clear if this association is causal. We have previously demonstrated that chronic use of morphine, in addition to the development of antinociceptive tolerance, also causes the disruption of the intestinal barrier function resulting in bacterial translocation from the gut lumen into the peritoneal organs, thus inducing an inflammatory response in the colon. Therefore, we tested if inflammation in the colon, as in Crohn's disease, affects the rate of antinociceptive tolerance development to morphine. To test this hypothesis, we induced colonic inflammation in Swiss Webster male mice using 2,4,6‐trinitro‐benzene sulfonic acid (TNBS) or vehicle intrarectally and then surgically implanted 25 mg, 50 mg (2 × 25 mg), or 75 mg slow release morphine pellets or placebo pellets subcutaneously on the dorsum. Tail immersion assay was performed on day 3, 4, and 5 post treatment and colons were extracted from the animals to measure mRNA expression of Interleukin 1 beta (IL‐1β) and tumor necrosis factor alpha (TNF‐α) by real‐time polymerase chain reaction (RT‐PCR). A morphine (10 mg/kg) challenge was used to test for the development of antinociceptive tolerance at various times in morphine pelleted with (n= 7) or without TNBS (n= 5). In TNBS + morphine pelleted mice, tolerance developed by day 5 in 25 mg morphine pelleted mice, by day 4 in 50 mg (2 × 25 mg) morphine pelleted mice, and as early as day 2 in 75 mg morphine pelleted mice. In the absence of TNBS‐induced inflammation, tolerance to morphine did not develop to either 25 mg or 50 mg morphine pellets up to day 5, and at day 4 in 75 mg morphine pellets. Furthermore, IL‐1β production was significantly increased in the morphine and placebo pelleted treated animals with TNBS from day 1 compared to the vehicle treated groups. In summary, chronic morphine administration in the presence of colonic inflammation leads to an increased rate and extent of antinociceptive tolerance development.Support or Funding InformationAgency: NIDA Agency Number: 5R01DA036975‐04This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Long-term morphine delivery via slow release morphine pellets or osmotic pumps: Plasma concentration, analgesia, and naloxone-precipitated withdrawal

AimsSlow-release morphine sulfate pellets and osmotic pumps are common routes of chronic morphine delivery in mouse models, but direct comparisons of these drug delivery systems are lacking. In this study, we assessed the efficacy of slow-release pellets versus osmotic pumps in delivering morphine to adult mice. Main methodsMale C57BL/6NCr mice (8weeksold) were implanted subcutaneously with slow-release pellets (25mg morphine sulfate) or osmotic pumps (64mg/mL, 1.0μL/h). Plasma morphine concentrations were quantified via LC-MS/MS, analgesic efficacy was determined by tail flick assay, and dependence was assessed with naloxone-precipitated withdrawal behaviors (jumping) and physiological effects (excretion, weight loss). Key findingsMorphine pellets delivered significantly higher plasma drug concentrations compared to osmotic pumps, which were limited by the solubility of the morphine sulfate and pump volume/flow rate. Within 96h post-implantation, plasma morphine concentrations were indistinguishable in pellet vs. pump-treated samples. While osmotic pump did not have an antinociceptive effect in the tail flick assay, pumps and pellets induced comparable dependence symptoms (naloxone-precipitated jumping behavior) from 24–72h post-implantation. SignificanceIn this study, we compared slow-release morphine pellets to osmotic minipumps for morphine delivery in mice. We found that osmotic pumps and subcutaneous morphine sulfate pellets yielded significantly different pharmacokinetics over a 7-day period, and as a result significantly different antinociceptive efficacy. Nonetheless, both delivery methods induced dependence as measured by naloxone-precipitated withdrawal.

Effects of Injection of Gamma-Aminobutyric Acid Agonists into the Nucleus Accumbens on Naloxone-Induced Morphine Withdrawal

Aims: This study was to investigate the effects of local administration of gamma-aminobutyric acid (GABA) agonists into the nucleus accumbens (NAc) on naloxone-induced morphine withdrawal symptoms. Methods: Bilateral guide cannulas were stereotaxically implanted in the shell or core regions of the NAc of Sprague-Dawley rats. After a recovery period, 3 morphine pellets, each consisting of 75 mg morphine base, were placed subcutaneously on the first and third days of the study with the rats under mild ether anaesthesia. The GABA agonists, baclofen hydrochloride or muscimol hydrobromide, were injected into the NAc, and morphine withdrawal was induced by naloxone on the fifth day. Results: Administration of baclofen to the shell or core regions of the NAc of Sprague-Dawley rats led to statistically significant decreases in both behavioural and locomotor activity parameters during the morphine withdrawal period, compared to the control group. However, there were no statistically significant changes in locomotor activity or withdrawal behavioural parameters, with the exception of wet dog shakes, between control and muscimol-treated groups. Conclusion: These findings show that GABAergic conduction in the NAc is effective on the morphine withdrawal symptoms, and that both the shell and core regions of the NAc are associated with this effect.

Early morphine exposure reduced viral burden in the periphery and induced regional difference in brains of LP-BM5 MAIDS model

Abstract Intravenous opioid use contributes to 11% of HIV infections in the U.S.A. Morphine suppresses the immune system while exacerbating the progression of HIV. To investigate morphine effects on HIV-associated neurocognitive disorders (HAND), we used the LP-BM5 infection murine AIDS (MAIDS) model. Previously, we found that morphine exposure at 7 wks post-infection (p.i.) enhanced cognitive impairment associated with MAIDS, increased hippocampal viral loads, and decreased CCL5 expression in examined brain regions, without affecting peripheral immunodeficiency. Because many HIV+ patients are exposed to opioids before infection, we examined these parameters after exposing the animals to morphine before and immediately after infection. Male mice were implanted with two placebo or 25mg morphine pellets for 6 days, with infection occurring on post-pellet implantation day 3. At 7 wks p.i., cognitive impairment was assessed by the spontaneous alternation T-maze task. At wk 8, blood, spleen and brain regions (hippocampus, striatum and frontal lobe) were harvested. LP-BM5 induced splenomegaly and higher levels of serum IgG2a and IgM, regardless of morphine treatment. Morphine significantly decreased body weight-normalized spleen weight. Morphine did not alter viral BM5Def RNA in spleen or any brain region examined. Morphine appeared to reduce LP-BM5-induced CCL5 expression in the striatum and frontal lobe. LP-BM5 alone appeared to increase the expression of both IFN-α and IFN-β in the frontal lobe only. Infection or morphine exposure did not affect animals’ performance in the T-maze task. Together with our previous results, these results suggest that timing of morphine exposure can alter the modulation and progression of MAIDS and HAND.