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Sabine S Maes,1,2 Stefan Pype,1 Vincent LH Hoffmann,3 Maria Biermans,1 Theo F Meert11CNS Discovery Research, Pain and Neurology, Johnson & Johnson Pharmaceutical Research and Development, a Division of Janssen Pharmaceutica, Beerse, Belgium; 2Department of Anaesthesiology, University Hospital Antwerp, Edegem, Belgium; 3Department of Anesthesiology, Intensive Care and Pain Treatment Center, Amphia Hospital, Breda, The NetherlandsBackground and methods: The role of the endogenous purine nucleoside, adenosine, in nociception is well established. Inhibition of the equilibrative nucleoside transporter (ENT1) prevents adenosine uptake into cells, and could therefore enhance the antinociceptive properties of adenosine. The effects of ENT1 inhibition were studied in two animal models of inflammatory pain. Analgesic activity was assessed in a complete Freund's adjuvant (CFA)-induced and carrageenan-induced mechanical and thermal hyperalgesia model in the guinea pig.Results: Draflazine, dipyridamole, dilazep, lidoflazine, soluflazine, and KF24345 showed efficacy in the CFA thermal hyperalgesia model. Draflazine, the most potent compound in this test, was further characterized in the CFA model of mechanical hyperalgesia and the carrageenan inflammation model of thermal and mechanical hyperalgesia, where it completely reversed the hypersensitivity. The antihyperalgesic effects of draflazine (10 mg/kg, administered subcutaneously) were attenuated by the A1 receptor antagonist, cyclopentyltheophylline (5–40 mg/kg, administered intraperitoneally), by the nonselective adenosine antagonist, caffeine (10–40 mg/kg intraperitoneally), and by the A2 antagonist, DMPX (10 mg/kg administered intraperitoneally).Conclusion: ENT1 inhibition is an effective way of reversing mechanical and thermal inflammatory hyperalgesia in the guinea pig, and these effects are mediated by enhancement of endogenous adenosine levels. Both A1 and A2 adenosine receptor subtypes are likely to be involved.Keywords: adenosine, nucleoside transport inhibition, nociception, inflammation, hyperalgesia

Inflammatory processes may cause depression in subsets of vulnerable individuals. Inflammation-associated behavioral changes are commonly modelled in rodents by administration of bacterial lipopolysaccharide (LPS). However, the time frame in which immune activation and depressive-like behavior occur is not very clear. In this study, we showed that systemic administration of LPS robustly increased circulating levels of corticosterone, leptin, pro- and anti-inflammatory cytokines, and chemokines. Serum concentrations of most analytes peaked within the first 6 h after LPS injection and returned to baseline values by 24 h. Chemokine levels, however, remained elevated for up to 96 h. Using an optimized sucrose preference test (SPT) we showed that sickness behavior was present from 2 to 24 h. LPS-induced anhedonia, as measured by decreased sucrose preference, lasted up to 96 h. To mimic the human situation, where depression develops after chronic inflammation, rats were preexposed to repeated LPS administration or subchronic restraint stress and subsequently challenged with LPS. While these procedures did not increase the duration of anhedonia, our results do indicate that inflammation may cause depressive symptoms such as anhedonia. Using our SPT protocol, more elaborate rodent models can be developed to study the mechanisms underlying inflammation-associated depression in humans.

Clinical observations indicate that activation of the TNF-α system may contribute to the development of inflammation-associated depression. Here, we tested the hypothesis that systemic upregulation of TNF-α induces neuroinflammation and behavioral changes relevant to depression. We report that a single intraperitoneal injection of TNF-α in mice increased serum and brain levels of the proinflammatory mediators TNF-α, IL-6, and MCP-1, in a dose- and time-dependent manner, but not IL-1β. Protein levels of the anti-inflammatory cytokine IL-10 increased in serum but not in the brain. The transient release of immune molecules was followed by glial cell activation as indicated by increased astrocyte activation in bioluminescent Gfap-luc mice and elevated immunoreactivity against the microglial marker Iba1 in the dentate gyrus of TNF-α-challenged mice. Additionally, TNF-α-injected mice were evaluated in a panel of behavioral tests commonly used to study sickness and depressive-like behavior in rodents. Our behavioral data imply that systemic administration of TNF-α induces a strong sickness response characterized by reduced locomotor activity, decreased fluid intake, and body weight loss. Depressive-like behavior could not be separated from sickness at any of the time points studied. Together, these results demonstrate that peripheral TNF-α affects the central nervous system at a neuroimmune and behavioral level.

Objectives: Preclinical abuse liability assessment of novel clinical CNS-active candidates involves several tests, addressing different aspects characteristic for abuse potential, which are considered predictive for substance abuse of these candidates, thus ensuring an appropriate translational approach. To demonstrate how such a strategy could work, a known drug of abuse, methylphenidate was evaluated in a full rodent test battery, comprising four test models, and in accordance with the requirements of the FDA, ICH and EMA guidelines., Methods: Methylphenidate was tested orally at 2.5, 5 or 10mg/kg for its physical dependence potential in a repeated dose non-precipitated withdrawal test, for its drug profiling in a drug discrimination learning procedure (single escalating doses), and for its reinforcing properties in a conditioned place preference test (alternate dosing days) and an intravenous self-administration procedure (0.05 to 1mg/kg/IV infusion during 5 daily 1-h test sessions). The stimulant d-amphetamine served as positive control and was administered subcutaneously at 0.8mg/kg in the first three test models. In the intravenous self-administration procedure rats were habituated to intravenously self-administer d-amphetamine at 0.06mg/kg/IV infusion prior to methylphenidate substitution., Results: Cessation of subchronic dosing up to 10mg/kg methylphenidate led to sustained or even exacerbated effects on locomotion and behavior, body temperature, body weight, food consumption, and alteration of the diurnal rhythm during withdrawal. Clear generalization to d-amphetamine was obtained in the drug discrimination test at 5 and 10mg/kg. Distinct reinforcing properties were present in the conditioned place preference test at 10mg/kg and in the intravenous self-administration study from 0.05mg/kg/IV infusion onwards. The maximum plasma exposure after oral administration of methylphenidate over the dose ranges tested in the present rat studies covered at least 1.9-fold to 18.9-fold the recommended human therapeutic exposure of 10ng/ml, a plasma level that is considered representative of the human efficacious methylphenidate dose. The ratio Cmax Hu/rat calculated from the intravenous self-administration data ranged from 14.9 to 576.5. Consequently the regulatory requirements, stating that preclinical drug abuse liability studies should include high doses that produce plasma levels that are multiples of the therapeutic dose were fulfilled (FDA, EMA, ICH)., Discussion: The presented preclinical models, implemented within a drug development environment, were considered highly predictive to assess the abuse potential of methylphenidate, and in accordance with the regulatory requirements of drug licensing authorities in terms of appropriate methods, dose selection and subsequent plasma exposure., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Substantial evidence indicates an association between clinical depression and altered immune function. Systemic administration of bacterial lipopolysaccharide (LPS) is commonly used to study inflammation-associated behavioral changes in rodents. In these experiments, we tested the hypothesis that peripheral immune activation leads to neuroinflammation and depressive-like behavior in mice. We report that systemic administration of LPS induced astrocyte activation in transgenic GFAP-luc mice and increased immunoreactivity against the microglial marker ionized calcium-binding adapter molecule 1 in the dentate gyrus of wild-type mice. Furthermore, LPS treatment caused a strong but transient increase in cytokine levels in the serum and brain. In addition to studying LPS-induced neuroinflammation, we tested whether sickness could be separated from depressive-like behavior by evaluating LPS-treated mice in a panel of behavioral paradigms. Our behavioral data indicate that systemic LPS administration caused sickness and mild depressive-like behavior. However, due to the overlapping time course and mild effects on depression-related behavior per se, it was not possible to separate sickness from depressive-like behavior in the present rodent model.

Background: Diabetic neuropathy is a complication of diabetes mellitus that develops in about 50% of people with diabetes. Despite its widespread occurrence and devastating effects, this complication is still not fully understood, and there is no treatment available to prevent its development., Methods: In this study, immunocytochemistry for activating transcription factor 3, a marker for cell injury, was used to investigate the stress response in dorsal root ganglion neurons in both in vitro and ex vivo models of diabetic neuropathy., Results: Our findings showed increased activating transcription factor 3 expression in hyperglycemic culture conditions and in dorsal root ganglion neurons isolated from diabetic rats. Glial cell line-derived neurotrophic factor, a substance with known neuroprotective properties, was able to reduce diabetes mellitus-induced neuronal stress in vitro, while gabapentin and carbamazepine, currently used to treat neuropathic pain, showed only limited effects., Conclusion: Growth factors may have a therapeutic benefit as neurotrophic agents in the treatment of diabetic peripheral neuropathy, but gabapentin and carbamazepine have no direct protective effect on sensory neurons. This research also indicates that immunocytochemistry for activating transcription factor 3 is a valuable tool for evaluation of pharmacological substances in dorsal root ganglion cultures.

Phosphodiesterases (PDEs) form a family of enzymes involved in the hydrolysis of cyclic adenosine and guanosine monophosphate (cAMP and cGMP). PDE10A is a member of this family that is almost exclusively expressed in the striatum. Increasing cAMP/cGMP levels via inhibition of PDE10A is under consideration as a novel therapeutic avenue in the discovery of antipsychotics. Papaverine has been used as a pharmacological tool to establish the possible clinical use of PDE10A inhibitors as antipsychotics. Papaverine is known to increase cAMP levels in striatum and to decrease blood pressure, body temperature and locomotor activity after systemic administration. In this study, the effects of papaverine are compared to those of a more specific PDE10A inhibitor MP10. Papaverine raised striatal cAMP levels with hypothermia, hypoactivity and decreased cardiovascular responses. The more selective MP10 had significantly less effects on body temperature and cardiovascular functions, but reduced locomotor activity to a similar extend as papaverine.

Angiogenesis is an important issue in cancer research and opioids are often used to treat pain in cancer patients. Therefore it is important to know if the use of opioids is associated with an aberrant stimulation of tumor growth triggered by the stimulation of angiogenesis in cancer patients. Some studies in the literature have suggested the presence of the μ3 opioid receptor, known as the receptor for many opioids, on endothelial cells, which are key players in the process of angiogenesis. In this study we used endothelial cells known to express the μ3 opioid receptor (MOR3), to evaluate the effects of morphine on angiogenesis. We first investigated the effect of morphine on the proliferation of endothelial cells. We showed that morphine is able to stimulate vascular endothelial cell proliferation in vitro. This effect of morphine is mediated by the mitogen-activated protein kinase (MAPK) pathway as pre-treatment with PD98059 inhibited this excessive proliferation. Because previous studies indicated nitric oxide (NO) as a downstream messenger we investigated the role of NO in the aberrant proliferation of endothelial cells. Our data could not confirm these findings using intracellular NO measurements and quantitative fluorescence microscopy. The potential use and pitfalls of opioids in cancer patients is discussed in light of these negative findings.

Background: Many animal models can be used to study the underlying pathophysiological mechanisms of neuropathic pain. Most of these models rely on a partial denervation of the limb of the animal by ligating a selected nerve. In this study, we performed nerve lesions on three peripheral nerves supplying the plantar side of the rat hindpaw by differentially transecting the saphenous, the tibial, and the sural nerves alone or in paired combinations., Methods: The development of neuropathic pain symptoms at three different anatomical areas (medial, central, and lateral) of the glabrous skin of the hindpaw was evaluated by sensory testing over a 12-wk period. Mechanical hyperalgesia (pinprick), cold allodynia (acetone), and abnormalities of hindpaw posture were continuously present in animals with tibial and tibial and saphenous nerve transection., Results: Transection of the tibial and sural nerves induced cold allodynia and moderate mechanical hyperalgesia. Transection of the sural, the saphenous, or both nerves simultaneously induced no signs of specific neuropathic pain behavior and no abnormalities in posture of the affected hindpaw were noted after adequate stimulation., Conclusions: The overlapping innervation of nerve distribution can complicate the interpretation of nerve ligation studies of peripheral neuropathies.

Interleukin-6 (IL-6) is a pleiotropic cytokine, signaling intracellularly via its unique membrane-bound receptor IL-6R and gp130. In peripheral nerve injury models, IL-6 and IL-6R are increased at the injured nerve and the respective dorsal root ganglion. IL-6 is increased at the ipsilateral dorsal and ventral horn of the spinal cord. IL-6 is known to affect neuronal survival, differentiation and regeneration. It is involved in synaptic plasticity and hyperexitability and induces the synthesis or release of other substances with known neuroprotective or neuromodulatory effects. In this study, intrathecal administration of recombinant rat IL-6 to rats with a chronic constriction injury of the sciatic nerve, induced a logarithmic dose-dependent increase in cold allodynia with a threshold of 10 pg IL-6 and a maximal effect at 100 ng IL-6. Intrathecal administration of saline or denaturated IL-6 was without effect. In rats with a chronic constriction injury, systemic administered IL-6 did not induce a hyperalgesic effect, illustrating that IL-6 acts at the level of the dorsal root ganglion or the spinal cord. Intraplantar injection of 100 ng IL-6 in the operated hind paw resulted in an increased cold allodynia. This study demonstrates that the sensitivity to exogenous intrathecal or peripheral IL-6 increases in rats with a mononeuropathy.

Unlabelled: Application of four loose ligatures to the sciatic nerve of a rat (chronic constriction injury [CCI]) induces clear hypersensitivity to non-noxious stimulation and chemical irritants. However, in this study, an injection of formalin in the hind paw of a rat with CCI-induced mononeuropathy resulted in an ipsilateral decreased flinching and licking or biting behavior in both phases of the formalin testing. The effect was independent of the formalin concentration used. This altered behavior was accompanied with smaller plasma levels of adrenocorticotrope hormone and corticosterone compared with sham and non-operated animals. Formalin injection in the contralateral nonligated hind paw of CCI rats also reduced the licking or biting behavior as compared with sham-operated and non-operated control animals only in the second phase of the formalin test. Thus, CCI reduces the pain reactivity and hypothalamic-pituitary-adrenal-axis activation to ipsilateral and contralateral formalin injection. Further research should investigate whether the decreased pain reactivity by CCI is situated at the peripheral, spinal, or supraspinal level or is result of changes in the stress reactivity and coping strategies., Implications: We evaluated the changes in the behavioral reactions and the hormonal effects of a noxious chemical stimulus, i.e., formalin injection in animals with previously induced chronic constriction injury to the sciatic nerve. The effect in animals injected at the ipsilateral and contralateral site, sham-operated and controls, were compared.

Implications: That IL-6 is an interesting target in the study of pain is underscored by its biomolecular properties, its localization after experimental pain, and its modulating effect on pain after administration.

Surgery is followed by an acute-phase response, including hypothalamo-pituitary-adrenal (HPA)-axis activation and fever. Considering its physiological properties and its behaviour in plasma after stress and surgery, the pro-inflammatory cytokine interleukin (IL)-6 is a putative candidate in eliciting these stress-related symptoms. However, evidence for this hypothesis is lacking. Rats subjected to individual psychological stress for 1h were injected intraperitoneally with saline or 3.33 microg per 100g rat neutralizing antibodies against rat IL-6. Thereafter, the single-housed rats were anaesthetized for 25 min, with or without undergoing a laparotomy. Intermittently, oesophageal temperatures were measured at defined time points. A parallel group of rats undergoing the same study protocol were decapitated, at time points when body temperatures differed, to obtain blood for measurement of plasma adrenocorticotropic hormone and corticosterone. Individual housing resulted in hyperthermia. Antibodies against IL-6 accelerated normalization of body temperature after individualizing stress, limited postoperative hyperthermia after laparotomy, but accentuated hyperthermia after anaesthesia alone. Antibody administration was not able to significantly influence the plasma hormone levels during any experiment. The present study indicates that IL-6 is a thermoregulatory factor during psychological, anaesthesiological and surgical stress, but the cytokine does not participate in HPA-axis activation until 6h after anaesthesia or surgery. A dose-finding study with antibodies against IL-6 ought to further identify the degree of contribution of IL-6 to perioperative thermoregulation.

Unlabelled: Inotropic glutamate receptors in the spinal cord (N-methyl-D-aspartic acid [NMDA], alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid [AMPA], and kainate receptors) seem to play a key role in acute pain transmission and the neuronal plasticity in chronic pain states. Vocalization responses produced by activation of these receptors on the pain pathways can be quantified semiautomatically and thus could be used as a research tool. We studied vocalization responses induced by intrathecal administration of various agonists acting at the glutamate receptors in normal rats and in the presence of peripheral inflammation and a chronic constriction injury model of neuropathic pain. The nonselective endogenous agonist, glutamate, and the NMDA receptor glycine site agonist D-serine did not produce vocalization, whereas selective agonists acting at AMPA, NMDA, and kainate receptors produced dose-related vocalization responses. The vocalization response evoked by the administration of AMPA was significantly increased in the neuropathic pain model. In conclusion, spinal administration of ionotropic glutamate receptor agonists produce short-lasting, dose-related vocalization responses that can be used as a basic research and screening tool for analgesic studies. However, peripheral inflammation or nerve injury did not substantially alter vocalization responses overall, possibly indicating that the vocalization test is not a good tool for studying the role of excitatory amino acids in these pathological pain conditions., Implications: Vocalization responses evoked by spinal administration of ionotropic glutamate receptor agonists can be used for experimental analgesic studies. However, pathological pain models did not substantially alter vocalization responses, possibly indicating that this test is not suitable for studying the role of spinal excitatory amino acids in central sensitization.

A new, non-invasive method for measuring reserpine-induced tremor in rodents is described here. The test procedure is based on the piezo-electric principle and was evaluated using the tremorogenic compound reserpine and the stereotypies-inducing drug apomorphine. Whereas for reserpine an orderly and dose-related increase in activity was observed, no such effect was detected with apomorphine. In order to further evaluate the test procedure, studies on the antagonism of reserpine-induced tremor were also performed. Results from these studies indicated that the DA-agonist lisuride, but not the S2-antagonist ritanserin, were able to antagonize the reserpine-induced tremor in a dose-related manner.

Background: Epidural administration of an aqueous suspension of n-butyl-p-aminobenzoate (BAB) to humans results in long-lasting sensory blockade without motor block. The dose-response of BAB administered epidurally and intrathecally as a solution was studied in rats to define the local anesthetic properties in an established animal model., Methods: The time course of changes in tail withdrawal latency and motor function were determined in rats after epidural or intrathecal administration of solutions of BAB or bupivacaine. The dose-response relation was determined and median effective dose values were calculated., Results: After epidural and intrathecal administration of BAB solutions, the onset and duration of the antinociceptive action were comparable to bupivacaine. Median effective dose values for tail-withdrawal latency of 6 s or more were significantly greater for BAB. After both routes of administration, BAB clearly affected motor function., Conclusions: When administered epidurally and intrathecally as a solution, BAB is a local anesthetic of relative low potency with onset and duration of action comparable to those of bupivacaine. These findings suggest that the long-lasting action obtained after applying BAB suspension results from the slow dissolution (continuous release) of the solid BAB deposited in the epidural space.

A growing number of studies have implicated the hypothalamic-pituitary-adrenal (HPA) axis in acute and chronic alcoholization and in ethanol withdrawal. In order to study the ethanol/HPA axis interaction during alcohol withdrawal, we performed experiments using adrenalectomized (ADX) male rats alcoholized by a chronic pulmonary alcoholization procedure. Eight hours after the 3 weeks of the alcoholization procedure, the rats were evaluated for a tremor activity. In order to reduce the great variability of the withdrawal tremors, we estimated the supersensitivity of the withdrawn rats to the tremorogenic compound harmine. We also studied the effect of a hydrocortisone treatment given in the drinking bottle during the alcoholization procedure on the harmine-induced tremors of ADX and sham rats. Alcohol withdrawal resulted in increased tremor response to 10 mg/kg harmine, and a protective effect of adrenalectomy on this effect was observed. Hydrocortisone administration to ADX or sham rats did not affect the tremor profile of the alcohol withdrawn rats.

Antagonism of the discriminative stimulus properties of 10 mg/kg cocaine was studied in rats by use of the dopamine D1 antagonist SCH 23390 and the D2 antagonist haloperidol. Whereas SCH 23390 and haloperidol were by themselves unable to antagonize the cueing properties of cocaine, the combination of both dopamine antagonists resulted in a complete blockade of the cocaine cue. In the presence of a fixed dose of 0.01 and 0.04 mg/kg haloperidol, the ED50's (it is the effective dose in 50% of the animals) of SCH 23390 for cocaine antagonism were 0.043 and 0.012 mg/kg, respectively. Similarly, the ED50's of haloperidol in combination with 0.01 and 0.04 mg/kg SCH 23390 were 0.021 and 0.024 mg/kg. The combined treatment of haloperidol and SCH 23390 resulted in strong response-rate reductions. At all combination regimens resulting in a complete blockade of the cocaine cue, response rate was reduced to less than 20% of the control values. These results indicate that the cueing properties of cocaine are both dopamine D1- and D2-mediated and that a combined antagonism of both receptor subtypes can lead to a complete antagonism of the cueing properties of cocaine which is associated with severe attenuation of response rate.

Background: Data on the analgesic properties of alpha 2 agonists and their interactions with opioids are conflicting. Experiments were conducted in rats to evaluate whether various available alpha 2 agonists can potentiate the analgesic properties of opioids and to determine the route of administration needed for optimal interaction., Methods: The tail-withdrawal reaction test was used as an analgesia assay. In separate experiments, the interactions between systemic (subcutaneous, intravenous, and intraperitoneal) and spinally (epidural and intrathecal) administered alpha 2 agonists and opioids were evaluated. The antagonism of medetomidine plus fentanyl with naloxone and/or idazoxan also was studied., Results: All alpha 2 agonists tested, when injected subcutaneously with fentanyl, potentiated the opioid-induced analgesia. In terms of a reduction of the amount of fentanyl needed to produce a deep surgical analgesia (tail-withdrawal reaction latency > or = 10 s) over more than 2 h, the relative order of potency of the alpha 2 agonists tested was: medetomidine > dexmedetomidine > xylazine > clonidine > detomidine. For some of these alpha 2 agonists there was a biphasic effect: at the larger doses tested, a reduction in the fentanyl potentiation was present. The potentiation of the opioid activity with alpha 2 agonists was also demonstrated in terms of a longer duration of analgesia after combined treatment with fixed doses of opioids. The interaction between the alpha 2 agonists and the opioids remained present when a more profound criterion of analgesia (tail-withdrawal reaction latency > or = 30 s) was used. Furthermore, the interactions between the alpha 2 agonists tested and fentanyl or sufentanil appeared to be independent of the route of administration. Potentiations were observed after simultaneous subcutaneous injections of both groups of compounds, after the combination of intraperitoneal (alpha 2 agonist) plus subcutaneous (opioid), intravenous (alpha 2 agonist) plus epidural (opioid) and simultaneous epidural or intrathecal administrations. With regard to antagonism of the analgesic activity of combined treatment with alpha 2 agonists plus opioids, naloxone provided total antagonism, whereas the alpha 2 antagonist idazoxan overcame only the alpha 2 agonist-induced potentiation of fentanyl., Conclusions: The tested alpha 2 agonists can potentiate the analgesic properties of opioids, but they have no intrinsic antinociceptive effects on spinal reflexes on their own. The potentiating activities of the alpha 2 agonists could be measured both in terms of a reduction of the amount of opioid needed to reach a particular level of analgesia and in terms of a longer duration of analgesia with a fixed dose of the opioid. The potentiations were observed with various alpha 2 agonists and opioids and appeared independent of the routes of administration.