N‐methyl‐D‐aspartate (NMDA) receptor involvement in central nervous system prostaglandin production during the relapse phase of chronic relapsing experimental autoimmune encephalomyelitis (CR EAE)

Our previous studies have established that major changes in central nervous system (CNS) prostaglandin (PG) levels occur during the relapse phase of chronic relapsing experimental autoimmune encephalomyelitis (CR EAE), an animal model of the human demyelinating disease multiple sclerosis. PG production is controlled through a series of enzymic pathways that, in EAE, are influenced by neuroantigen‐driven autoimmune events. In non‐immune‐based models of CNS disease, endogenous glucocorticoids have been proposed as instigators of PG synthesis via activation of the N‐methyl‐D‐aspartate (NMDA) receptor. Glucocorticoids have an important regulatory role in the pathogenesis EAE and the NMDA receptor is intimately involved in many of the characteristic neuroinflammatory processes that govern the disease. Therefore, the alterations in prostanoid concentrations during the relapse stage of CR EAE may ultimately be governed by glucocorticoid‐induced NMDA receptor activation. The current investigation has examined the proposed glucocorticoid–NMDA receptor link by determining the effects of the receptor antagonist, (+) MK‐801, on CNS PGE ₂ and PGD ₂ levels in Biozzi mice with relapse symptoms of CR EAE. Prostanoid concentrations in the cerebral cortex were not altered by drug administration, and in cerebellar tissues, a vehicle effect negated any drug‐induced changes. However, the level of PGD ₂ in spinal cords from (+) MK‐801‐dosed mice was significantly lower, compared to controls, but PGE ₂ concentrations remained unchanged. The results suggest that glucocorticoid–NMDA receptor‐linked events are not primarily responsible for PG generation in the brain but may influence prostanoid production in discrete areas of the CNS.     

Behavioural disturbance requiring medical referral: A case of anti‐N‐methyl‐D‐aspartate receptor encephalitis in the emergency department

A 17‐year‐old woman presented to the ED with behavioural disturbance and psychotic features. Brief dystonic jerks were noted so she was referred to the medical team. A diagnosis of anti‐N‐methyl‐D‐aspartate receptor encephalitis was made. Immunotherapy was instituted early and the clinical outcome was excellent. It is important to consider this condition in young women presenting with acute behavioural or psychotic symptoms.     

Protective effects of PF‐4708671 against N‐methyl‐d‐aspartic acid‐induced retinal damage in rats

We previously demonstrated that rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), protects against N‐methyl‐d ‐aspartic acid (NMDA)‐induced retinal damage in rats. Rapamycin inhibits mTOR activity, thereby preventing the phosphorylation of ribosomal protein S6, which is a downstream target of S6 kinase. Therefore, we aimed to determine whether PF‐4708671, an inhibitor of S6 kinase, protects against NMDA‐induced retinal injury. Intravitreal injection of NMDA (200 nmol/eye) caused cell loss in the ganglion cell layer and neuroinflammatory responses, such as an increase in the number of CD45‐positive leukocytes and Iba1‐positive microglia. Surprisingly, simultaneous injection of PF‐4708671 (50 nmol/eye) with NMDA significantly attenuated these responses without affecting phosphorylated S6 levels. These results suggest that PF‐4708671 and rapamycin likely protect against NMDA‐induced retinal damage via distinct pathways. The neuroprotective effect of PF‐4708671 is unlikely to be associated with inhibition of the S6 kinase, even though PF‐4708671 is reported to be a S6 kinase inhibitor.     

The role of N‐methyl‐d‐aspartate receptor subunit NR2B in spinal cord in cancer pain

Cancer pain is one kind of the most common and severe kinds of chronic pain. No breakthrough regarding the mechanisms and therapeutics of cancer pains has yet been achieved. Based on the well established involvement of the NMDA (N‐methyl‐d ‐aspartate) receptor containing NR2B in inflammatory pain and neuropathic pain and the effective pain relief obtained with ketamine in cancer patients with intractable pain, we supposed that NR2B in the spinal cord was an important factor for cancer pain. In this study, we investigated the possible role of NR2B in the spinal cord using a murine model of bone cancer pain. C3H/HeJ mice were inoculated into the intramedullary space of the right femur with Osteosarcoma NCTC 2472 cells to induce ongoing bone cancer‐related pain behaviors. At day 14 after operation, the expression of NR2B mRNA and NR2B protein in the spinal cord were higher in tumor‐bearing mice compared to the sham mice. Intrathecal administration of 5 and 10 μg of NR2B subunit‐specific NMDA receptor antagonist ifenprodil attenuated cancer‐evoked spontaneous pain, thermal hyperalgesia and mechanical allodynia. These results suggest that NR2B in the spinal cord may participate in bone cancer pain in mice, and ifenprodil may be a useful alternative or adjunct therapy for bone cancer pain. The findings may lead to novel strategies for the treatment of bone cancer pain.     

Peripheral N‐methyl‐d‐aspartate receptors contribute to mechanical hypersensitivity in a rat model of inflammatory temporomandibular joint pain

The aim of this study was to determine whether peripheral N‐methyl‐d ‐aspartate (NMDA) receptors are involved in inflammation‐induced mechanical hypersensitivity of the temporomandibular joint (TMJ) region. We developed a rat model of mechanical sensitivity to Complete FreunD's Adjuvant (CFA; 2μl containing 1μg Mycobacterium tuberculosis)‐induced inflammation of the TMJ and examined changes in sensitivity following injection of NMDA receptor antagonists (dl ‐2‐amino‐5‐phosphonovaleric acid (AP5) or Ifenprodil) with CFA. CFA injected into the TMJ resulted in an increase in mechanical sensitivity relative to pre‐injection that peaked at day 1 and lasted for up to 3 days (n =8, P <0.05). There was no change in mechanical sensitivity in vehicle‐injected rats at any time‐point (n =9). At day 1, there was a significant increase in mechanical sensitivity in animals injected with CFA+vehicle (n =7) relative to those injected with vehicle alone (n =7, P <0.05), and co‐injection of AP5 (n =6) or Ifenprodil (n =7) with CFA blocked this hypersensitivity. Subcutaneous injection of AP5 (n =7) and Ifenprodil (n =5) instead of into the TMJ had no significant effect on CFA‐induced hypersensitivity of the TMJ region. Western blot analysis revealed constitutive expression of the NR1 and NR2B subunits in trigeminal ganglion lysates. Immunohistochemical studies showed that 99% and 28% of trigeminal ganglion neurons that innervated the TMJ contained the NR1 and NR2B subunits respectively. Our findings suggest a role for peripheral NMDA receptors in inflammation‐induced pain of the TMJ region. Targeting peripheral NMDA receptors with peripheral application of NMDA receptor antagonists could provide therapeutic benefit and avoid side effects associated with blockade of NMDA receptors in the central nervous system.     

Depletion of capsaicin sensitive afferents prevents lamina‐dependent increases in spinal N‐methyl‐d‐aspartate receptor subunit 1 expression and phosphorylation associated with thermal hyperalgesia in neuropathic rats

Phosphorylation of the N‐methyl‐d ‐aspartate (NMDA) receptor NR1 subunit (pNR1) in the spinal cord is associated with increased neuronal responsiveness, which underlies the process of central sensitization. Because of the importance of NR1 in central sensitization, the first goal of this study was to examine both time‐ and lamina‐dependent changes in spinal NR1 and pNR1 expression in a chronic constriction injury (CCI) model of neuropathic pain. Increased excitability of capsaicin sensitive primary afferents (CSPAs), which express TRPV1 receptors, also contributes to central sensitization. Thus, we next examined whether the depletion of CSPAs with resiniferatoxin (RTX) modified the change of spinal NR1 and pNR1 expression induced by CCI. Experimental rats were euthanized at 1, 3, 7, 14, and 28 days post‐CCI surgery and spinal cords processed for NR1 or pNR1 immunostaining. The number of NR1 or pNR1‐immunoreactive neurons was significantly increased in all lamina (I–VI) of the ipsilateral L4/L5 dorsal horn from 1 or 7 days post‐CCI, respectively. Pretreatment with RTX (0.3mg/kg, s.c. in the scruff of the neck or intraplantar) 2 days prior to CCI completely prevented induction of thermal hyperalgesia, but not mechanical allodynia in neuropathic rats. Interestingly, RTX treatment significantly attenuated the CCI‐induced upregulation of NR1 and pNR1 in spinal laminae I–II and V–VI, but not laminae III–IV as compared with that of vehicle‐treated CCI rats. These findings demonstrate that the increased expression of NR1 and pNR1 in spinal laminae I–II and V–VI is dependent on activation of CSPAs, which ultimately contribute to the development of thermal hyperalgesia in neuropathic rats.     

Phosphorylation of spinal N‐methyl‐d‐aspartate receptor NR1 subunits by extracellular signal‐regulated kinase in dorsal horn neurons and microglia contributes to diabetes‐induced painful neuropathy

The N‐methyl‐d ‐aspartate receptor (NMDAR) contributes to central sensitization in the spinal cord, a phenomenon which comprises various pathophysiological mechanisms responsible for neuropathic pain‐like signs in animal models. NMDAR function is modulated by post‐translational modifications including phosphorylation, and this is proposed to underlie its involvement in the production of pain hypersensitivity. As in diabetic patients, streptozotocin‐induced diabetic rats exhibit or not somatic mechanical hyperalgesia; these rats were named DH and DNH respectively. At three weeks of diabetes, we present evidence that somatic mechanical hyperalgesia was correlated with an enhanced phosphorylation of the NMDAR NR1 subunit (pNR1) in the rat spinal cord. This increase was not found in normal and DNH rats, suggesting that this regulation was specific to hyperalgesia. Double immunofluorescence studies revealed that the numbers of pNR1‐immunoreactive neurons and microglial cells were significantly increased in all laminae (I—II and III—VI) of the dorsal horn from DH animals. Western‐blots analysis showed no change in NR1 protein levels, whatever the behavioural and glycemic status of the animals. Chronic intrathecal treatment (5μg/rat/day for 7 days) by U0126 and MK801, which blocked MEK (an upstream kinase of extracellular signal‐regulated protein kinase: ERK) and the NMDAR respectively, simultaneously suppressed somatic mechanical hyperalgesia developed by diabetic rats and decreased pNR1. These results indicate for the first time that increased expression of pNR1 is regulated by ERK and the NMDAR via a feedforward mechanism in spinal neurons and microglia and represents one mechanism involved in central sensitization and somatic mechanical hyperalgesia after diabetes.     

Cannabidiol reverses the mCPP‐induced increase in marble‐burying behavior

Cannabidiol (CBD), one of the main components of Cannabis sp., presents clinical and preclinical anxiolytic properties. Recent results using the marble‐burying test (MBT) suggest that CBD can also induce anticompulsive‐like effects. Meta‐chloro‐phenyl‐piperazine (mCPP) is a nonspecific serotonergic agonist (acting mainly at 5HT1A, 5HT2C and 5HT1D receptors) reported to increase symptoms in OCD patients and block the anticompulsive‐like effect of serotonin reuptake inhibitors (SRIs) in animal models. The aim of this study was to investigate the interference of CBD on mCPP effects in repetitive burying. Administration of mCPP showed dual effects in the MBT, increasing the number of buried marbles at lower (0.1 mg/kg) while decreasing it at higher doses (1 mg/kg), an effect not related to a general increase in anxiety‐like behavior. As found previously, CBD (30 mg/kg) and the positive control fluoxetine (FLX; 10 mg/kg) decreased burying behavior without changing general exploratory activity. A similar effect was found when subeffective doses of CBD (15 mg/kg) and FLX (3 mg/kg) were administered together. These subeffective doses alone were also able to block mCPP‐induced repetitive burying. The results, in addition to reinforcing a possible anticompulsive effect of CBD, also suggest that mCPP‐induced repetitive burying could be a useful test for the screening of compounds with presumed anticompulsive properties.     

Comparative study of alpha‐ and beta‐pinene effect on PTZ‐induced convulsions in mice

Convulsions occur in response to a loss of balance between excitatory and inhibitory neurotransmitters, and the treatment for this condition consists in restore such lost balance. Many anticonvulsant drugs present side effects which may limit their use. This fact has stimulated the search for new sources of treatment from aromatic plants. Many monoterpenes commonly present in essential oils are known because of their anticonvulsant properties. The anticonvulsant effect of α‐ and β‐pinene, two structural isomers, is still little studied. Thus, the present work evaluated the anticonvulsant effect of α‐ and β‐pinene in pentylenetetrazole‐induced convulsions model. Initially, the oral LD₅₀ for α‐ and β‐pinene was estimated. Following the oral administration, a mild sedation was observed and no deaths were recorded; the LD₅₀ estimated for both monoterpenes was greater than 2 000 mg/kg, p.o. Further, animals were orally treated with α‐pinene (100, 200 and 400 mg/kg), β‐pinene (100, 200 and 400 mg/kg) and the equimolar mixture of α‐ and β‐pinene (400 mg/kg) and subjected to the pentylenetetrazole‐induced convulsions model. In this model, only the dose of 400 mg/kg of the compounds was able to significantly decrease the seizure intensity. The latency of first convulsion was significantly increased by the mixture of α‐ and β‐pinene (400 mg/kg). In addition, β‐pinene and the mixture of the two monoterpenes, both at a dose of 400 mg/kg, significantly increased the time of death of animals. The treatment with β‐pinene and the equimolar mixture of the two monoterpenes significantly reduced hippocampal nitrite level and striatal content of dopamine (DA) and norepinephrine (NE). Taken together, the results suggest that α‐pinene appears to be devoid of anticonvulsant action. This fact, however, seems to be dependent on the chemical structure of the compound, since pretreatment with the β‐pinene increased the time of death pf PTZ‐treated mice, which seems to depend on the ability of the compound to reduce nitrite concentration and NE and DA content, during the pentylenetetrazole‐induced seizure.     

Parachlorophenylalanine‐induced 5‐HT depletion alters behavioral and brain neurotransmitters levels in 6‐Hz psychomotor seizure model in mice

The present study was designed to investigate the role of serotonin and other neurotransmitters namely dopamine (DA), histamine, nor‐epinephrine (NE), glutamate, and γ‐aminobutyric acid (GABA) in the 6‐Hz‐induced psychomotor seizures in Swiss albino mice. Parachlorophenylalanine (PCPA, 300 mg/kg/day, i.p for 3 days)‐treated mice were given 6‐Hz stimulation. Sodium valproate (SVP) (200 mg/kg/day, p.o for 3 days) was used as a reference antiepileptic drug. The behavioral changes induced by 6 Hz including increased rearing and grooming, Straub's tail, behavioral arrest, stun position were amplified by PCPA. The 6‐Hz‐induced seizures were accompanied by reduced brain 5‐HT, DA, NE, histamine, GABA, and enhanced glutamate levels. PCPA facilitated further reduction of endogenous 5‐HT and DA levels but not NE, histamine, GABA, and glutamate levels. Pre‐ and post‐treatment with SVP protected the mice from 6‐Hz seizures and attenuated PCPA‐induced changes in the levels of 5‐HT and DA in the mice brain suggesting the protective effect of SVP in the pharmacoresistant model of epilepsy involving mainly serotonergic mechanism. However, the study also suggests modulation of other neurotransmitters both in 6‐Hz psychomotor seizures and in the action of SVP against such seizures.     

Protective effect of N,N’‐dimethylthiourea against stress‐induced gastric mucosal lesions in rats

In the present study, we examined the protective effect of N,N’‐dimethylthiourea (DMTU), a scavenger of hydroxyl radical (), against water‐immersion restraint stress (WIRS)‐induced gastric mucosal lesions in rats. When male Wistar rats fasted for 24 h were exposed to WIRS for 3 h, gastric mucosal lesions occurred with increases in the levels of gastric mucosal myeloperoxidase (MPO), an index of tissue neutrophil infiltration, pro‐inflammatory cytokines (tumor necrosis factor alpha and interleukin 1beta), lipid peroxide (LPO), and nitrite/nitrate (NOx), an index of nitric oxide synthesis, and decreases in the levels of gastric mucosal nonprotein SH and vitamin C and gastric adherent mucus. DMTU (1, 2.5, or 5 mmol/kg) administered orally at 0.5 h before the onset of WIRS reduced the severity of gastric mucosal lesions with attenuation of the changes in the levels of gastric mucosal MPO, pro‐inflammatory cytokines, LPO, NOx, nonprotein SH, and vitamin C and gastric adherent mucus found at 3 h after the onset of WIRS in a dose‐dependent manner. Serum levels of corticosterone and glucose, which are indices of stress responses, increased in rats exposed to WIRS for 3 h, but DMTU pre‐administered at any dose had no effect on these increases. These results indicate that DMTU protects against WIRS‐induced gastric mucosal lesions in rats by exerting its antioxidant action including scavenging and its anti‐inflammatory action without affecting the stress response.     

N‐methyl‐d‐aspartate receptor‐mediated modulations of the anti‐allodynic effects of 5‐HT1B/1D receptor stimulation in a rat model of trigeminal neuropathic pain

Previous studies showed that triptans and other 5‐HT_1B/1D‐receptor agonists attenuate hyper‐responsiveness to mechanical stimulation of the face in a rat model of trigeminal neuropathic pain, probably by activating 5‐HT_1B/1D‐receptors on primary afferent nociceptive fibers. We now tested whether blockade of post‐synaptic receptors for the excitatory amino acid glutamate released by these fibers would increase this action. We thus evaluated whether (±)1‐hydroxy‐3‐aminopyrrolidine‐2‐one (HA‐966), an antagonist at the glycine/d ‐serine site of N‐methyl‐d ‐aspartate (NMDA)‐receptors, would potentiate the anti‐allodynic action of dihydroergotamine and zolmitriptan in rats with chronic constriction injury to the infraorbital nerve (CCI‐ION). Complementary studies were performed with other NMDA‐receptor ligands and in rats with chronic constriction injury to the sciatic nerve (CCI‐SN) for comparison. Injury was produced by loose ligatures of the nerves. Responsiveness to mechanical stimulation (vibrissae or hindpaw territories) with von Frey filaments was used to evaluate allodynia 2 weeks after nerve ligature. Rats received NMDA‐receptor ligands or saline 20 min before dihydroergotamine (25–100μg/kg, i.v.) or zolmitriptan (25–100μg/kg, s.c.). HA‐966 (2.5 mg/kg, s.c.), inactive on its own, enhanced the anti‐allodynic effects of dihydroergotamine (eightfold increase) and zolmitriptan (threefold increase) in CCI‐ION rats, but these drugs exerted no effects in allodynic CCI‐SN rats. NMDA‐receptor blockade by memantine (5 mg/kg, i.p.) also enhanced, whereas activation at glycine/NMDA site by d ‐cycloserine (3 mg/kg, i.p.) reduced the anti‐allodynic properties of zolmitriptan in CCI‐ION rats. Combined administration of NMDA‐receptor antagonist and 5‐HT_1B/1D‐receptor agonist may be a promising approach for alleviating trigeminal neuropathic pain.     

Antihyperalgesic and analgesic properties of the N‐methyl‐d‐aspartate (NMDA) receptor antagonist neramexane in a human surrogate model of neurogenic hyperalgesia

NMDA‐receptors are a major target in the prevention and treatment of hyperalgesic pain states in neuropathic pain. However, previous studies revealed equivocal results depending on study design and efficacy parameters. We tested the analgesic (generalized reduction of generation and processing of nociceptive signalling) and anti‐hyperalgesic (prevention of central sensitization) properties of the NMDA‐receptor antagonist neramexane and the potassium channel opener flupirtine in the intradermal capsaicin injection model. Furthermore, we tested the effect on pain summation (wind up). Eighteen healthy subjects received either a single dose of neramexane (40mg p.o.), flupirtine (100mg) or placebo in a double‐blind, randomized, cross‐over study. Pain evoked by intradermal capsaicin injection as well as pain evoked by pinpricks was significantly reduced by neramexane (?22% to ?30% vs. placebo) in the non‐sensitized skin indicating a marked analgesic effect. Moreover, dynamic mechanical allodynia (pain to light touch) was also significantly attenuated by neramexane (?28% vs. placebo). However, static secondary hyperalgesia to pinprick stimuli after capsaicin injection was not significantly reduced (?9% vs. placebo). Flupirtine showed no analgesic or anti‐hyperalgesic effect. Mechanically‐evoked wind up of pain sensation was not affected by any treatment. The results suggests that in a human surrogate model of neurogenic hyperalgesia a single low‐dose of neramexane had a marked analgesic effect in the sensitized and in the non‐sensitized state and thus may be a useful drug to treat the enhanced pain sensitivity in neuropathic pain patients. Its efficacy may be based on analgesia rather than anti‐hyperalgesia or anti‐windup. In contrast, flupirtine showed neither an analgesic nor an anti‐hyperalgesic effect at a dose used for the treatment of postoperative pain.     

Neuropharmacological effects of lipoic acid and ubiquinone on δ‐aminolevulinic dehydratase,Na+, K+‐ATPase,and Mg2+‐ATPase activities in rat hippocampus after pilocarpine‐induced seizures

In this study, we investigated the effects of lipoic acid (LA) in the hippocampus oxidative stress caused by pilocarpine‐induced seizures in adult rats. Wistar rats were treated with 0.9% saline (i.p., control group), LA (10 mg/kg, i.p., LA group), ubiquinone [20 mg/kg, i.p., ubiquinone (UQ) group], pilocarpine (400 mg/kg, i.p., P400 group), and the association of LA (10 mg/kg, i.p.) plus pilocarpine (400 mg/kg, i.p.) or UQ (20 mg/kg, i.p.) plus pilocarpine (400 mg/kg, i.p.), 30 min before of administration of P400 (LA plus P400 group and UQ plus P400 group, respectively). After the treatments, all groups were observed for 1 h. The enzyme activities (δ‐aminolevulinic dehydratase (δ‐ALA‐D), Mg²⁺‐ATPase, and Na⁺, K⁺‐ATPase) were measured using spectrophotometric methods, and the results compared to values obtained from saline and pilocarpine‐treated animals. Protective effects of LA and UQ were also evaluated on the same parameters. We reported here for the first time that Na⁺, K⁺‐ATPase and δ‐ALA‐D activities inhibition and Mg²⁺‐ATPase stimulation in the pilocarpine model are probably attributed to the oxidative stress caused by seizures in the rat hippocampus. The addition of the antioxidants LA and UQ may reverses the previously mentioned Na⁺, K⁺‐ATPase and δ‐ALA‐D inhibitions and Mg²⁺‐ATPase stimulation. Conclusions: The oxidative stress plays an important signaling role in pilocarpine‐induced seizures, and antioxidant drugs might be considered as therapeutical tools in this pathology.