The false-positive responses of analgesic drugs to the intradermal serotonin- and compound 48/80-induced scratches as an animal model of itch.

Intradermal injection of pruritogens such as serotonin, histamine and compound 48/80 into the skin and then, the evaluation of the scratching behavior is the commonly used animal model to advance pruritic research and drug development. However, predictive validity of this model is poorly documented. There is a close interaction between itch and pain sensations with regard to mediation through an anatomically and functionally identical neuronal pathway. One approach is whether the existing animal model of itch differentiates itch or pain to show efficacy of clinically effective analgesic drugs as a back translation. In this study, we explored the effects of different group of analgesic drugs on serotonin and compound 48/80-induced scratching behavior in Balb-C mice. Serotonin (25 µg) and compound 48/80 (100 µg) was injected intradermally in a volume of 50 µl into the rostral part of skin on the back of male mice and scratches were counted for a 30-min observation period. Morphine (1, 3, 10 mg/kg), tramadol (20, 40, 80 mg/kg), cannabinoid agonist CP 55,940 (0.1, 0.3, 1 mg/kg), paracetamol (100, 200, 300 mg/kg) and diclofenac (50, 100, 200 mg/kg) were given intraperitoneally 30 min prior to pruritogen injection. The analgesic drugs dose dependently blocked serotonin and compound 48/80-induced straching behavior with exerting complete inhibition at certain doses. Our data suggests that intradermal pruritogen-induced scratching models may not discriminate pain and itch sensations and give false positive results when standard analgesic drugs are used.

Systemic and spinal administration of FAAH, MAGL inhibitors and dual FAAH/MAGL inhibitors produce antipruritic effect in mice.

The increase of endocannabinoid tonus by inhibiting fatty acid amide hydrolase (FAAH) or monoacylglycerol lipase (MAGL) represents a promising therapeutic approach in a variety of disease to overcome serious central side effects of exocannabinoids. Recent studies reported that systemic administration of FAAH and MAGL inhibitors produce antipruritic action. Dual FAAH/MAGL inhibitors have also been described to get enhanced endocannabinoid therapeutic effect. In this study, we examined and compared dose-related antipruritic effects of systemic (intraperitoneal; ip) or intrathecal (it) administration of selective FAAH inhibitor PF-3845 (5, 10, and 20 mg/kg, i.p.; 1, 5, and 10 µg, i.t.), MAGL inhibitor JZL184 (4, 20, and 40 mg/kg, i.p.; 1, 5, and 10 µg, i.t.) and dual FAAH/MAGL inhibitor JZL195 (2, 5, and 20 mg/kg, i.p.; 1, 5, and 10 µg, i.t.) on serotonin (5-HT)-induced scratching model. Serotonin (25 µg) was injected intradermally in a volume of 50 µl into the rostral part of skin on the back of male Balb-C mice. Both systemic or intrathecal administration of PF-3845, JZL184 or JZL195 produced similar dose-dependent antipruritic effects. Our results suggest that endocannabinoid-degrading enzymes FAAH and MAGL are involved in pruritic process at spinal level. FAAH, MAGL or dual FAAH/MAGL inhibitors have promising antipruritic effects, at least, in part through spinal site of action.

The critical role of spinal 5-HT7 receptors in opioid and non-opioid type stress-induced analgesia.

The opioid and non-opioid types of stress-induced analgesia have been well defined. One of the non-opioid type involve the endocannabinoid system. We previously reported that the spinal serotonin 7 receptor (5-HT7) blockers inhibit both morphine and cannabinoid-induced analgesia, thus we hypothesized that descending serotonergic pathways-spinal 5-HT7 receptor loop might contribute to stress-induced analgesia. Stress-induced analgesia was induced with warm (32°C) or cold (20°C) water swim stress in male Balb-C mice. The effects of intrathecal injection of a selective 5-HT7 receptor antagonist, SB 269970, of the denervation of serotonergic neurons by intrathecal administration of 5,7-dihydroxytryptamine (5,7-DHT) and of lesions of the dorsolateral funiculus on opioid and non-opioid type stress-induced analgesia were evaluated with the tail-flick and hot plate tests. The expression of 5-HT7 receptors mRNA in the dorsal lumbar region of spinal cord were analyzed by RT-PCR following spinal serotonin depletion or dorsolateral funiculus lesion. The effects of the selective 5-HT7 receptor agonists LP 44 and AS 19 were tested on nociception. Intrathecal SB 269970 blocked both opioid and non-opioid type stress-induced analgesia. Dorsolateral funiculus lesion or denervation of the spinal serotonergic neurons resulted in a marked decrease in 5-HT7 receptor expression in the dorsal lumbar spinal cord, accompanied by inhibition of opioid and non-opioid type stress-induced analgesia. However, the systemic or intrathecal LP 44 and AS 19 alone did not produce analgesia in unstressed mice. These results indicate that descending serotonergic pathways and the spinal 5-HT7 receptor loop play a crucial role in mediating both opioid and non-opioid type stress-induced analgesia.

A new utilization area for hyperbaric oxygen? Improving quality of stored blood.

BACKGROUND/AIM: Since blood bags have the ability for diffusion of gases, we investigated whether hyperbaric oxygen (HBO) exposure affects several vital parameters of stored blood. MATERIALS AND METHODS: Bloods obtained from the same persons were used as both control and HBO groups and stored in pediatric bags with citrate-phosphate-dextrose solution. HBO administration was performed at 2.5 atm for 90 min, started 1 day after blood collection and repeated every 2 days for a total of 10 times. The study was terminated on the 21st day. Complete blood count, glucose, pH, and osmotic fragility values were measured every week. RESULTS: Glucose and pH levels decreased in stored blood. In the HBO-exposed group, these decreases were less than in the control. In addition, mean corpuscular and platelet volumes tended to increase during storing process, but with HBO, these indexes remained lower, near physiologic levels. Another interesting finding of the study was the relative stable osmotic fragility ratio in the HBO group compared to the control blood. CONCLUSION: HBO exposure has positive effects on pH, stability of erythrocytes, and energy source (glucose) of the medium. Thus, we concluded that HBO may be a useful application for life and quality of stored blood.

Simultaneous determination of cyclosporine A, tacrolimus, sirolimus, and everolimus in whole-blood samples by LC-MS/MS.

OBJECTIVES: Cyclosporine A (CyA), tacrolimus (TRL), sirolimus (SIR), and everolimus (RAD) are immunosuppressive drugs frequently used in organ transplantation. Our aim was to confirm a robust sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for determination of CyA, TRL, SIR, and RAD in whole-blood samples. MATERIALS AND METHODS: We used an integrated online solid-phase extraction-LC-MS/MS system and atmospheric pressure ionization tandem mass spectrometry (API-MS/MS) in the multiple reaction monitoring (MRM) detection mode. CyA, TRL, SIR, and RAD were simultaneously analyzed in whole blood treated with precipitation reagent taken from transplant patients. RESULTS: System performance parameters were suitable for using this method as a high-throughput technique in clinical practice. The high concentration of one analyte in the sample did not affect the concentration of other analytes. Total analytical time was 2.5 min, and retention times of all analytes were shorter than 2 minutes. CONCLUSION: This LC-MS/MS method can be preferable for therapeutic drug monitoring of these immunosuppressive drugs (CyA, TRL, SRL, and RAD) in whole blood. Sample preparation was too short and simple in this method, and it permits robust, rapid, sensitive, selective, and simultaneous determination of these drugs.

Osteopenia in children with cerebral palsy can be treated with oral alendronate.

PURPOSE: Cerebral palsy is one of the most common reasons of osteopenia in childhood. Patients have a significantly decreased bone mineral density, and painful fractures with minor traumas are common. Biphosphonates in the treatment of childhood osteoporosis are increasingly being used. This study aimed to evaluate the efficacy of oral alendronate treatment in children with cerebral palsy. METHODS: Twenty-six children (16 boys and 10 girls) aged 3 to 17 years who had quadriplegic cerebral palsy and osteopenia were included in the study. The patients received alendronate (1 mg/kg/week), calcium (600 mg/day), and vitamin D(3) (400 U/day) over a year. A complete blood count, kidney and liver functional tests, plasma calcium, phosphate and alkaline phosphatase levels, and lumbar vertebral bone mineral density were measured before and after treatment. RESULTS: Compared with pretreatment values, bone mineral density, serum calcium, and phosphate levels of the patients statistically increased and alkaline phosphatase levels decreased after treatment. No patient needed to interrupt treatment because of side effects. CONCLUSIONS: Oral alendronate at a dose of 1 mg/kg/week for the treatment of osteopenia in children with cerebral palsy was found to be safe and effective.

Long-term exposure to repetitive hyperbaric oxygen results in cumulative oxidative stress in rat lung tissue.

CONTEXT: Despite its known benefits, hyperbaric oxygen (HBO) is also reported to enhance the production of reactive oxygen species and can cause oxidative stress in several tissues. Previous studies had shown that HBO-induced oxidative stress is directly proportional to both its exposure pressure and duration. Nevertheless, these studies were usually performed with single-session HBO exposure but its clinical use commonly depends on long-term exposure periods. OBJECTIVE: To clarify the oxidative effect of long-term repetitive HBO in the lung tissue of rats. MATERIALS AND METHODS: Male Sprague-Dawley rats were divided into six study groups exposed to consecutive HBO sessions (2.8 atm/90 min) for 5, 10, 15, 20, 30, and 40 days. Animals were sacrificed 24 h after the last HBO session. An additional control group was set to obtain normal data. Lung malondialdehyde (MDA) and carbonylated protein (PCC) levels were determined as measures of oxidative stress along with the activities of the antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase. RESULTS: None of the measured parameters showed any changes among the groups exposed to 5-15 HBO sessions. However, MDA, PCC, and SOD were found to be significantly increased in the 20 to 40 session groups. DISCUSSION AND CONCLUSION: These results indicate that repetitive treatment with HBO may cause oxidative stress in critical tissues including the lung. Although HBO-mediated free radicals are accepted to be responsible for the benefits of this therapeutic modality, especially in cases with prolonged exposure, possible injurious effects of supranormal values of bio-oxidative products need to be considered.

Systemic and spinal administration of etanercept, a tumor necrosis factor alpha inhibitor, blocks tactile allodynia in diabetic mice.

Painful diabetic neuropathy is one of the most common forms of neuropathic pain syndromes. Tumor necrosis factor alpha (TNF-alpha) is a proinflammatory cytokine that has been implicated as a key pain mediator in the development and maintenance of neuropathic pain conditions. Recent studies showed that endogenous TNF-alpha production was also accelerated in neural tissues and spinal cord under chronic hyperglycemia. Thus, in this study, we investigated whether pharmacological inhibition of TNF-alpha by etanercept, a TNF-alpha antagonist, could block behavioral sign of diabetic neuropathic pain. Diabetes was induced by streptozotocin (STZ) (200 mg/kg, i.p.) in Balb-c mice and behavioral tests were performed between 45 and 60 days after STZ administration. Mechanical and thermal sensitivities were measured by a series of calibrated Von Frey filaments and hot plate test, respectively. Etanercept was given by either intravenous (i.v.), intrathecal (i.th.) or intraplantar (i.pl.) routes to the diabetic mice. Tactile allodynia, but not thermal hyperalgesia, developed in diabetic mice. Both i.v. (1, 10 and 20 mg/kg) or i.th. (1, 5 and 10 µg/mouse) treatments with etanercept produced dose dependent reversal of tactile allodynia in diabetic mice. However, etanercept was found to be inactive against allodynia when given i.pl. (1, 5 and 10 µg/mouse). Our results suggest that etanercept has promising effects on diabetic neuropathic pain with antiallodynic effects when given systemically or intrathecally.

Systemic cannabinoids produce CB1-mediated antinociception by activation of descending serotonergic pathways that act upon spinal 5-HT(7) and 5-HT(2A) receptors.

Serotonin (5-HT) plays an important role in the descending control of pain. We evaluated the role of descending serotonergic pathways and spinal 5-HT7 and 5-HT(2A) receptors in comparison to that of 5-HT(1A) and 5-HT3 receptors in the antinociceptive effects of systemically administered cannabinoids. Antinociceptive effects were evaluated by radiant heat tail-flick and hot plate tests in Balb-C mice. The selective CB1 receptor agonist, ACEA; a mixed CB1 and CB2 receptor agonist, WIN 55,212-2; and a selective CB2 receptor agonist, GW405833, were given systemically to induce antinociception. Spinal 5-HT was depleted with intrathecal (i.th.) injection of 5,7-dihydroxytryptamine (5,7-DHT). Bilateral surgical lesions of the dorsolateral funiculus were performed. Selective 5-HT7, 5-HT(2A), 5-HT(1A) and 5-HT3 antagonists-SB-269970, ketanserin, WAY 100635 and ondansetron, respectively-were administered i.th. Risperidone, an atypical antipsychotic displaying 5-HT(2A) antagonism, also irreversibly binds to and inactivates the 5-HT7 receptors. Thus, we also injected risperidone i.th. to elucidate the role of spinal 5-HT7 and 5-HT(2A) receptors in cannabinoid-mediated antinociception. WIN 55,212-2 and ACEA produced dose-dependent antinociception, which were reversed by selective CB1 receptor antagonist rimonabant. GW405833 did not produce any antinociception. The antinociceptive effects of WIN 55,212-2 and ACEA were totally absent in spinal 5-HT depleted and dorsolateral funiculus lesioned mice. I.th. administration of SB-269970, ketanserin, and risperidone, but not WAY 100635 or ondansetron, blocked both WIN 55,212-2- and ACEA-induced antinociception. These findings suggest that systemically administered cannabinoids interact with descending serotonergic pathways via CB1-mediated mechanisms and exert a central antinociceptive effect involving spinal 5-HT7 and 5-HT(2A) receptors.

The additive antinociceptive interaction between WIN 55,212-2, a cannabinoid agonist, and ketorolac.

Combinations of nonsteroidal antiinflammatory drugs (NSAIDs) and opioids are widespread in the management of pain, allowing better analgesia with reduced side effects. Cannabinoids are promising analgesic drugs that have pharmacological properties similar to those of opioids. However, the beneficial effects of cannabinoids for pain treatment are counterbalanced by their psychotomimetic side effects. We designed the present study to evaluate the antinociceptive interaction between cannabinoids and NSAIDs in mice, using the acetic acid-induced writhing test and tail-flick test. Interactions were analyzed using isobolographic analysis. WIN 55,212-2, a cannabinoid agonist, and the NSAID ketorolac, either alone or in combination, produced dose-dependent antinociception in the writhing test. Isobolographic analysis showed additive interactions between WIN 55,212-2 and ketorolac when they were coadministered systemically. Ketorolac is inactive in the radiant heat tail-flick test in which WIN 55,212-2 was active. Ketorolac did not influence WIN 55,212-2-induced antinociception in the tail-flick test. This study demonstrated an additive antinociceptive interaction between WIN 55,212-2 and ketorolac in an inflammatory visceral pain model. The combination of cannabinoids and NSAIDs may have utility in the pharmacotherapy of pain.

Lack of cross-tolerance to the antinociceptive effects of systemic and topical cannabinoids in morphine-tolerant mice.

Opioids and cannabinoids produce antinociception through activity at spinal, supraspinal and peripheral sites. Tolerance to the antinociceptive effects of both the opioids and the cannabinoids develop when these agents are administered chronically. Although mutual potentiation of antinociceptive effects have been reported between opioids and cannabinoids, the development of antinociceptive cross-tolerance between these systems has not been demonstrated consistently. In the present investigation, we explored the possibility of antinociceptive cross-tolerance between systemic or topical morphine and systemic or topical cannabinoids in mice. Mice were made tolerant to morphine either by the subcutaneous (s.c.) implantation of a morphine pellet or repeated topical administration and then challenged with the mixed CB(1) and CB(2) receptor agonist WIN 55, 212-2 given s.c. or topically. Antinociception was indicated by increased tail-flick latencies to noxious radiant heat. Implantation with morphine pellets did not attenuate the antinociceptive potency of systemic or topical WIN 55,212-2. Moreover, twice-daily topical administration of morphine did not attenuate the antinociceptive potency of WIN 55,212-2 applied topically. These observations suggest that opioids and cannabinoids produce antinociception through mechanisms that are independent of each other at either the systemic or peripheral levels.

Topical cannabinoid enhances topical morphine antinociception.

Opioids and cannabinoids produce antinociception through both spinal and supraspinal action. Both opioids and cannabinoids also have important peripheral action. Many previous studies indicate that systemically administered cannabinoids enhance antinociceptive properties of opioids. Experiments were conducted to test the hypothesis that topical cannabinoids would enhance the topical antinociceptive effects of morphine. Antinociception was measured in the radiant tail-flick test after immersion of the tail of mice into a solution of dimethyl sulfoxide (DMSO) containing WIN 55, 212-2, a cannabinoid agonist and morphine, an opioid agonist. Morphine and WIN 55, 212-2 produce time dependent topical analgesic effects limited to the portion of the tail exposed to drugs. WIN 55, 212-2 had a potency lower than that of morphine. The topical antinociceptive effects of WIN 55, 212-2 were blocked by systemic pretreatment of cannabinoid CB1 receptor selective antagonist, AM 251. This suggests that topical antinociceptive effects of WIN 55, 212-2 involve CB1 receptors. Combination of topical WIN 55, 212-2 with topical morphine yielded significantly greater analgesic effects than that of topical morphine alone. The ability of the CB1 receptor antagonist AM 251 to antagonize the enhancement of antinociception of morphine by WIN 55, 212-2 indicates that WIN 55, 212-2 acts through a CB1 receptor to enhance the potency of topical morphine. Additionally, spinally administered ineffective doses of WIN 55, 212-2 potentiated the antinociceptive effects of topical morphine. These results demonstrate an antinociceptive interaction between topical opioids with topical, and spinal cannabinoids. These observations are significant in using of topical combination of cannabinoid and morphine in the management of pain.

L-type and T-type calcium channel blockade potentiate the analgesic effects of morphine and selective mu opioid agonist, but not to selective delta and kappa agonist at the level of the spinal cord in mice.

We examined the effects of amlodipine, a selective L-type voltage dependent Ca(2+) channel (VDCC) blocker, and mibefradil, a selective T-type VDCC blocker on the antinociceptive effects of morphine, and mu, delta and kappa opioid receptor selective agonist-induced antinociception at the spinal level. Intrathecally administered amlodipine and mibefradil potentiated morphine and [D-Ala(2), N mePhe(4), Gly-ol(5)] enkephalin (DAMGO)-induced antinociception by shifting their dose response curves to the left. However, intrathecally administered amlodipine and mibefradil did not affect [D-Pen(2), D-Pen(5)]enkephalin (DPDPE) and [trans-(+/-)-3,4-dichloro-N-methyl-N-[2-(1-pyrolidinyl)cyclohexyl] benzene acetamide (U-50, 488H)-induced antinociception. These data indicate that L-type and T-type VDCC blockers synergistically potentiate the analgesic effects of mu opioid receptor agonists, but not delta and kappa opioid receptor agonists, at the spinal level. Additionally, these data suggest that there is an important functional interaction between L-type and/ or T-type VDCC and mu opioid receptors in the process of analgesia.

The interaction between IL-1beta and morphine: possible mechanism of the deficiency of morphine-induced analgesia in diabetic mice.

It is known that diabetic mice are less sensitive to the analgesic effect of morphine. Some factor(s) derived from mononuclear cells, e.g. interleukin-1beta (IL-1beta), may be responsible for the diminished analgesic effect of morphine in diabetic mice. Therefore, we examined direct effects of IL-1beta, intracerebroventricularly (i.c.v.), on morphine-induced analgesia, subcutaneously (s.c.), in diabetic and control mice by using the tail-flick test. Morphine at doses of 1, 2 and 5 mg/kg (s.c.) produced dose-dependent analgesia in diabetic and control mice but diabetic mice were less sensitive to the analgesic effect of morphine when compared to the controls. IL-1beta at a dose of 0.1 ng/mouse produced analgesia in control mice but not in diabetics, whereas IL-1beta at a dose of 10 ng/mouse produced a hyperalgesic effect both in diabetic and control mice. IL-1beta at a dose of 1 ng/mouse has neither an analgesic nor a hyperalgesic effect in control and diabetic mice. Administration of a neutral (neither analgesic nor hyperalgesic) dose of IL-1beta, 1 ng/mouse (i.c.v.), just prior to administration of morphine (s.c.) abolished the analgesic effect of morphine at doses of 1, 2 and 5 mg/kg in control mice and the analgesic effect of morphine became similar to that in diabetics. The diminished analgesic effect of morphine in diabetes was attenuated further with IL-1beta at a dose of 1 ng/mouse (i.c.v.). These results suggest that the decreased analgesic effect of morphine in diabetes may be related to IL-1beta.