Down-regulation of N-methyl D-aspartate receptor in rat-modeled disuse osteopenia

Lack of mechanical stress may result in osteoporosis; however, the underlying mechanisms of disuse osteoporosis remain unclear. It has been indicated that mechanical loading causes extracellular glutamate accumulation in osteoblasts. We hypothesized that the glutamate receptor mediation on bone cells might also be involved in mechanically stimulated osteogenesis. In this study, we investigated the changes of bone formation and the expressions of osteogenic genes and N-methyl D-aspartate (NMDA) receptors, the major glutamate receptors, in disused bones. Rat modeled disuse osteopenia in hind limbs was induced by a 3-week tail suspension in Sprague-Dawley rats. Bone mineral density and trabecular bone volume of distal femurs were measured to verify the osteopenia of disused bones. The mRNA expressions of cbfa1/Runx2, type I collagen, alkaline phosphatase (ALP) and osteocalcin (OC) in bones were measured as osteogenic markers. The influences of mechanical unloading on the expressions of NMDA receptors (NR₁ and NR₂D) in bones were also examined. The effects of NMDA mediation on osteogenesis were tested by a treatment of MK-801, a non-competitive NMDA receptor antagonist, in cultured osteoblasts and bone marrow stroma cells. Our result showed that mRNA expressions of cbfa1/Runx2, type I collagen, ALP and OC were significantly decreased in disused bones. The mRNA and protein expressions of NR₁ and NR₂D were significantly decreased in disused bones; furthermore, immunolocalization of both receptors showed decreases in osteoblasts, but not in osteoclasts. The results from the in vitro study showed that MK-801 inhibited mRNA expression of cbfa1/Runx2 in bone marrow stroma cells and also inhibited those of collagen type I, ALP and OC of osteoblasts in a dose-dependent manner. These results suggest that NMDA receptor mediation may play an important role in transmitting mechanical loading in bones, and decreases of the expressions of NMDA receptors in disused bones, especially in osteoblasts, may contribute to the decrease of osteogenesis.     

MK-801 enhances gabaculine-induced loss of the righting reflex in mice, but not immobility

PURPOSE: gamma-Aminobutyric acid (GABA) and N-methyl-D-aspartate (NMDA) receptors are important targets for anesthetic action at the in vitro cellular level. Gabaculine is a GABA-trans-aminase inhibitor that increases endogenous GABA in the brain, and enhances GABA activity. We have recently shown that unconsciousness is associated with the enhanced GABA activity due to gabaculine, but that immobility is not. MK-801 is a selective NMDA channel blocker. In this study, we examined behaviourally whether gabaculine in combination with MK-801 could produce these components of the general anesthetic state. We further compared the effect of MK-801 with ketamine, another NMDA channel blocker. METHODS: All drugs were administered intraperitoneally to adult male ddY mice. To assess the general anesthetic components, two endpoints were used. One was loss of the righting reflex (LORR; as a measure of unconsciousness) and the other was loss of movement in response to tail-clamp stimulation (as a measure of immobility). RESULTS: Large doses of MK-801 alone (10-50 mg.kg(-1)) induced neither LORR nor immobility in response to noxious stimulation. However, even a small dose (0.2 mgxkg(-1)) significantly enhanced gabaculine-induced LORR (P < 0.05), although gabaculine in combination with MK-801 (0.2-10 mgxkg(-1)) produced no immobility. However, gabaculine plus a subanesthetic dose of ketamine (30 mgxkg(-1)), which acts on NMDA, opioid and nicotinic acetylcholine receptors and neuronal Na(+) channels, suppressed the pain response, but did not achieve a full effect. Ketamine alone dose-dependently produced both LORR and immobility. CONCLUSION: These findings suggest that gabaculine-induced LORR is modulated by blocking NMDA receptors, but that immobility is not mediated through GABA or NMDA receptors.     

A novel role for involvement of N-methyl-D-aspartate (NMDA) glutamate receptors in sperm acrosome reaction

Ionotropic glutamate receptors are expressed in mouse and human spermatozoa. However, the possible role of these receptors has not been reported in the sperm acrosome reaction. This study was conducted to demonstrate the function of N-methyl-D-aspartate (NMDA) glutamate receptors in the acrosome reaction of mouse spermatozoa. Epididymal spermatozoa from adult mice were release in a culture medium. The sperm suspension was then divided into six groups: (1) spermatozoa at 0 min, (2) spermatozoa at 60 min (control), (3) spermatozoa treated with NMDA glutamate receptor agonist (L-glutamate, LG), (4) spermatozoa treated with α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainite glutamate receptor agonist (kainic acid), (5) spermatozoa treated with NMDA glutamate receptor antagonist (MK-801)+LG and (6) spermatozoa treated with ethylene glycol tetraacetic acid (EGTA, as a calcium chelator)₊LG. The sperm samples were examined for the acrosome reaction and intracellular calcium concentration. After 60 min, LG but not kainic acid significantly increased both the acrosome reaction and intracellular calcium levels in the spermatozoa compared with the control group. Co-administration of MK-801 or EGTA+LG could significantly reverse the effect of LG in the acrosome reaction and the level of intracellular calcium as compared to the LG group. The possibility that LG induced the acrosome reaction and elevated inter-cellular calcium concentration in mouse spermatozoa and that MK-801 could reverse the effects of LG, may suggest the involvement of NMDA glutamate receptors, at least in the initiation of the acrosome reaction in vitro.     

Remifentanil directly activates human N-methyl-D-aspartate receptors expressed in Xenopus laevis oocytes

BACKGROUND: Clinical studies suggest that intraoperative administration of the clinical remifentanil formulation Ultiva (GlaxoWellcome GmbH & Co, Bad Oldesloe, Germany) increases postoperative pain and postoperative analgesic requirements, but mechanisms remain unclear. N-methyl-D-aspartate (NMDA) receptors are thought to play a major role in development of postoperative pain and opiate tolerance. The authors hypothesized that Ultiva directly stimulates human NMDA receptors. METHODS: To test this hypothesis, the authors expressed human NR1A/NR2A and NR1A/NR2B NMDA receptors in Xenopus laevis oocytes by injection of messenger RNA prepared in vitro. After protein expression, they used a two-electrode voltage clamp to measure currents induced by NMDA receptor agonists and opioids. RESULTS: Noninjected cells were unresponsive to all compounds tested. Glutamate/glycine (1 nM-1 mM each) or Ultiva (0.01 pM-0.1 mM) stimulated NMDA receptors concentration dependently. NR1A/2A EC50 values were 8.0 microM/12 microM for glutamate/glycine and 3.5 nM for Ultiva, and NR1A/2B EC50 values were 3.9 microM/1.9 microM for glutamate/glycine and 0.82 microM for Ultiva. Glycine in combination with Ultiva showed no additive effect compared with Ultiva alone. Ultiva-induced currents were inhibited by MK-801 (pore blocker) but not by 7-CK (glycine antagonist), D-AP5 (glutamate antagonist), or naloxone. Fentanyl (10 microM) did not stimulate NMDA receptors. CONCLUSION: These data indicate that Ultiva but not fentanyl stimulates NMDA receptors of different subunit combinations (NR1A/2A, NR1A/2B). The mechanism seems to be allosteric activation of the NMDA receptor.     

Mechanical loading modulates glutamate receptor subunit expression in bone

The cellular mechanisms coupling mechanical loading with bone remodeling remain unclear. In the CNS, the excitatory amino acid glutamate (Glu) serves as a potent neurotransmitter exerting its effects via various membrane Glu receptors (GluR). Nerves containing Glu exist close to bone cells expressing functional GluRs. Demonstration of a mechanically sensitive glutamate/aspartate transporter protein and the ability of glutamate to stimulate bone resorption in vitro suggest a role for glutamate linking mechanical load and bone remodeling. We used immunohistochemical techniques to identify the expression of N-methyl-d-aspartate acid (NMDA) and non-NMDA (AMPA or kainate) ionotropic GluR subunits on bone cells in vivo. In bone sections from young adult rats, osteoclasts expressed numerous GluR subunits including AMPA (GluR2/3 and GluR4), kainic acid (GluR567) and NMDA (NMDAR2A, NMDAR2B and NMDAR2C) receptor subtypes. Bone lining cells demonstrated immunoexpression for NMDAR2A, NMDAR2B, NMDAR2C, GluR567, GluR23, GluR2 and GluR4 subunits. Immunoexpression was not evident on osteocytes, chondrocytes or vascular channels. To investigate the effects of mechanical loading on GluR expression, we used a Materials Testing System (MTS) to apply 10 N sinusoidal axial compressive loads percutaneously to the right limbs (radius/ulna, tibia/fibula) of rats. Each limb underwent 300-load cycles/day (cycle rate, 1 Hz) for 4 consecutive days. Contralateral, non-loaded limbs served as controls. Mechanically loaded limbs revealed a load-induced loss of immunoexpression for GluR2/3, GluR4, GluR567 and NMDAR2A on osteoclasts and NMDAR2A, NMDAR2B, GluR2/3 and GluR4 on bone lining cells. Both neonatal rabbit and rat osteoclasts were cultured on bone slices to investigate the effect of the NMDA receptor antagonist, MK801, and the AMPA/kainic acid receptor antagonist, NBQX, on osteoclast resorptive activity in vitro. The inhibition of resorptive function seen suggested that both NMDAR and kainic acid receptor function are required for normal osteoclast function. While the exact role of ionotropic GluRs in skeletal tissue remains unclear, the modulation of GluR subunit expression by mechanical loading lends further support for participation of Glu as a mechanical loading effector. These ionotropic receptors appear to be functionally relevant to normal osteoclast resorptive activity.     

Functional characterization of N-methyl-D-aspartic acid-gated channels in bone cells

Our recent identification of glutamate receptors in bone cells suggested a novel means of paracrine communication in the skeleton. To determine whether these receptors are functional, we investigated the effects of the excitatory amino acid, glutamate, and the pharmacological ligand, N-methyl-D-aspartic acid (NMDA), on glutamate-like receptors in the human osteoblastic cell lines MG63 and SaOS-2. Glutamate binds to osteoblasts, with a Kd of approximately 10(-4) mol/L and the NMDA receptor antagonist, D(L)-2-amino-5-phosphonovaleric acid (D-APV), inhibits binding. Using the patch-clamp technique, we measured whole-cell currents before and after addition of L-glutamate or NMDA and investigated the effects of the NMDA channel blockers, dizolcipine maleate (MK801), and Mg2+, and the competitive NMDA receptor antagonist, 3-((R)-2-carboxypiperazin-4-yl)-propyl-1-phosphoric acid (R-CPP), on agonist-induced currents. Both glutamate and NMDA induced significant increases in membrane currents. Application of Mg2+ (200 micromol/L) and MK801 (100 micromol/L) caused a significant decrease in inward currents elicited in response to agonist stimulation. The competitive NMDA receptor antagonist, R-CPP (100 micromol/L), also partially blocked the NMDA-induced currents in MG63 cells. This effect was reversed by addition of further NMDA (100 micromol/L). In Fura-2-loaded osteoblasts, glutamate induced elevation of intracellular free calcium, which was blocked by MK801. These results support the hypothesis that glutamate plays a role in bone cell signaling and suggest a possible role for glutamate agonists/antagonists in the treatment of bone diseases.     

nPKCepsilon and NMDA receptors participate in neuroprotection induced by morphine pretreatment

Morphine pretreatment induces ischemic tolerance in neurons, but it remains uncertain whether novel protein kinase C epsilon isoform (nPKCepsilon) and N-methyl-D-aspartate (NMDA) receptors are involved in this neuroprotection. The present study examined this issue. Hippocampal slices from adult BALB/C mice were incubated with morphine at 0.1-10.0 muM in the presence or absence of various antagonists for 30 minutes and then kept in morphine- and antagonist-free buffer for 30 minutes before being subjected to oxygen-glucose deprivation for 20 minutes. After recovery in oxygenated artificial fluid for 5 hours, assessment of slice injury was done by determination of the intensity of slice stain after they were incubated with 2% 2,3,5-triphenyltetrazolium chloride for 30 minutes and extracted by organic solvent for 24 hours. At designated periods, slices were preserved for immunoblot analysis to observe effects of morphine pretreatment on membrane translocation and total protein expression of nPKCepsilon and phosphorylation of NR1 subunits of NMDA receptors. The neuroprotection induced by morphine pretreatment was partially blocked by chelerythrine (a nonselective PKC blocker), epsilonv(1-2) (a selective nPKCepsilon antagonist), MK-801 (a noncompetitive NMDA receptor blocker), chelerythrine combined with MK-801, and epsilonv(1-2) with MK-801. Morphine pretreatment significantly inhibited nPKCepsilon membrane translocation and phosphorylation of NR1 subunits of NMDA receptors during reperfusion injury. However, epsilonv(1-2) blocked these effects induced by morphine pretreatment. These findings suggested that nPKCepsilon and NMDA receptors might participate in neuroprotection induced by morphine pretreatment, and NMDA receptors might be downstream targets of nPKCepsilon.     

Regional Anesthesia and Pain

PURPOSE: Not all bone cancer pain can be effectively treated with current therapies. In the present study, the effects of ip administration of alpha-2 agonists (dexmedetomidine and clonidine), N-methyl-D-aspartate (NMDA) antagonists (MK-801 and ketamine), an N-acetylaspartylglutamate peptidase inhibitor (ZJ-43), and morphine were examined in a mouse bone cancer pain model. METHODS: A bone cancer pain model was produced by injection of murine sarcoma cells into the medullary cavity of the distal femur. To estimate the level of bone cancer pain, the number of pain-related behaviours induced by repeated applications of a von Frey monofilament (0.166 g) to the site of tumour cells implantation was counted. Drugs were administered two weeks after the implantation. RESULTS: Morphine produced a significant analgesic effect (P < 0.001). The alpha-2 agonists produced analgesic effects (P < 0.001) with an efficacy similar to that of morphine, but only at doses that produced severe sedation. MK-801 had only limited analgesic effects, while ketamine produced an analgesic effect (P < 0.001) with the same efficacy as morphine. ZJ-43 (100 mg.kg(-1)) had a significant analgesic effect (P < 0.05) and the effect of ZJ-43 was antagonized by the selective group II metabotropic glutamate receptor (mGluR) antagonist. CONCLUSION: These data suggest that alpha-2 agonists produce an analgesic effect only at a sedative dose and that ketamine, but not MK-801, is associated with an analgesic response without overt side effects. The effect of ZJ-43 is mediated by activating group II mGluRs.     

Pharmacologic studies of the neuroprotective actions of a glutamate antagonist in ischemia.

Ischemia may increase glutamate release, which can lead to neuronal damage. The therapeutic value of drugs that antagonize glutamate's effects are being investigated in CNS ischemia. This study examined the efficacy of a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten- 5,10-imine hydrogen maleate], in reducing ischemic injury. We explored the limits of this therapy and different properties of MK-801 that might be involved in its neuroprotective actions. Two focal CNS ischemia models were used, a multiple cerebral embolic model (MCEM) and a rabbit spinal cord ischemia model (RSCIM). When animals were treated 5 minutes after the onset of injury, MK-801 was effective in reducing ischemic damage in both models. However, when treatment was delayed 10 minutes after the ischemic insult in the MCEM, no neuroprotection was observed even when the MK-801 dose was increased eightfold. We also did not find a beneficial effect of MK-801 pretreatment with a dose that was one tenth of the effective dose in the RSCIM. Studies using the (-) MK-801 isomer showed that MK-801 neuroprotection exhibited stereoselectivity. The contribution of anticonvulsant activity and sedation to MK-801's neuroprotective actions was examined indirectly using phenytoin and midazolam, respectively. Neither drug was effective in reducing ischemic injury in the MCEM. This suggests that MK-801's neuroprotective efficacy in ischemia is mediated through its NMDA receptor antagonist activity independent of its anticonvulsant or sedative properties. These results support the hypothesis that excessive NMDA receptor excitation may be involved in ischemic neuronal damage.     

The NMDA antagonist MK-801 improved metabolic recovery after 10 minutes global cerebral ischaemia in rats measured with 31 phosphorous magnetic resonance spectroscopy

Summary The blockade of postsynaptic receptors for excitatory amino acids is a promising new field for the possible treatment of cerebral ischaemia. The most important receptor seems to be the N-methyl-D-aspartate (NMDA) subtype of the glutamate receptors and MK-801 is a potent non-competitive antagonist to the NMDA receptor.31P NMR Spectroscopy was used to measure the recovery of intracellular pH and the high energy phosphates Phosphocreatine (PCr) and ATP after ten minutes of temporary global cerebral ischaemia in the rat. Cerebral ischaemia was obtained by combining bilateral carotid ligation and systemic hypotension (2 vessel occlusion model).Two intervention groups with intravenous injection of MK-801 in doses of 0.25mg/kg and 0.5mg/kg 15 minutes before onset of ischaemia were compared to a control group. Both intervention groups showed a more rapid recovery of PCr and ATP than the control group, but there were no significant differences for intracellular pH.     

Remifentanil Directly Activates Human N-methyl-d-aspartate Receptors Expressed in Xenopus laevis Oocytes

Background: Clinical studies suggest that intraoperative administration of the clinical remifentanil formulation Ultiva(R) (GlaxoWellcome GmbH & Co, Bad Oldesloe, Germany) increases postoperative pain and postoperative analgesic requirements, but mechanisms remain unclear. N-methyl-d-aspartate (NMDA) receptors are thought to play a major role in development of postoperative pain and opiate tolerance. The authors hypothesized that Ultiva(R) directly stimulates human NMDA receptors.

Methods: To test this hypothesis, the authors expressed human NR1A/NR2A and NR1A/NR2B NMDA receptors in Xenopus laevis oocytes by injection of messenger RNA prepared in vitro. After protein expression, they used a two-electrode voltage clamp to measure currents induced by NMDA receptor agonists and opioids.

Results: Noninjected cells were unresponsive to all compounds tested. Glutamate/glycine (1 nm-1 mm each) or Ultiva(R) (0.01 pm-0.1 mm) stimulated NMDA receptors concentration dependently. NR1A/2A EC50 values were 8.0 [mu]M/12 [mu]M for glutamate/glycine and 3.5 nM for Ultiva(R), and NR1A/2B EC50 values were 3.9 [mu]M/1.9 [mu]M for glutamate/glycine and 0.82 [mu]M for Ultiva(R). Glycine in combination with Ultiva(R) showed no additive effect compared with Ultiva(R) alone. Ultiva(R)-induced currents were inhibited by MK-801 (pore blocker) but not by 7-CK (glycine antagonist), D-AP5 (glutamate antagonist), or naloxone. Fentanyl (10 [mu]M) did not stimulate NMDA receptors.     

Contrasting roles of the N-methyl-D-aspartate receptor in the production of immobilization by conventional and aromatic anesthetics

We hypothesized that N-methyl-d-aspartate (NMDA) receptors mediate some or all of the capacity of inhaled anesthetics to prevent movement in the face of noxious stimulation, and that this capacity to prevent movement correlates directly with the in vitro capacity of such anesthetics to block the NMDA receptor. To test this hypothesis, we measured the effect of IV infusion of the NMDA blockers dizocilpine (MK-801) and (R)-4-(3-phosphonopropyl) piperazine-2-carboxylic acid (CPP) to decrease the MAC (the minimum alveolar concentration of anesthetic that prevents movement in 50% of subjects given a noxious stimulation) of 8 conventional anesthetics (cyclopropane, desflurane, enflurane, halothane, isoflurane, nitrous oxide, sevoflurane, and xenon) and 8 aromatic compounds (benzene, fluorobenzene, o-difluorobenzene, p-difluorobenzene, 1,2,4-trifluorobenzene, 1,3,5-trifluorobenzene, pentafluorobenzene, and hexafluorobenzene) and, for comparison, etomidate. We postulated that MK-801 or CPP infusions would decrease MAC in inverse proportion to the in vitro capacity of these anesthetics to block the NMDA receptor. This notion proved correct for the aromatic inhaled anesthetics, but not for the conventional anesthetics. At the greatest infusion of MK-801 (32 microg x kg(-1) x min(-1)) the MACs of conventional anesthetics decreased by 59.4 +/- 3.4% (mean +/- sd) and at 8 microg x kg(-1) x min(-1) by 45.5 +/- 4.2%, a decrease not significantly different from a 51.4 +/- 19.0% decrease produced in the EC50 for etomidate, an anesthetic that acts solely by enhancing gamma-amino butyric acid (GABA) receptors. We conclude that some aromatic anesthetics may produce immobility in the face of noxious stimulation by blocking the action of glutamate on NMDA receptors but that conventional inhaled anesthetics do not.     

Expression of an N-Methyl-D-Aspartate-Type Receptor by Human and Rat Osteoblasts and Osteoclasts Suggests a Novel Glutamate Signaling Pathway in Bone

Signaling between the various types of cells found in bone is responsible for controlling the activity of osteoblasts and osteoclasts, and therefore the regulation of bone mass. Our identification of a neuronal glutamate transporter in osteoblasts and osteocytes suggests the possibility that bone cells may use the excitatory amino acid glutamate as a signaling molecule. In these studies we report the expression of different subtypes of glutamate receptors in osteoblasts and osteoclasts in vitro and in vivo. We have identified expression in human and rat bone cells of N-methyl-D-aspartate receptor-1 (NMDAR-1) and 2D subunits and PSD-95, the NMDA receptor clustering protein associated with signaling in the central nervous system. In situ hybridization and immunohistochemistry localized NMDAR-1 expression to osteoblasts and osteoclasts in human tissue sections. These findings strengthen the suggestion that glutamate is involved in signaling between bone cells.     

Effects of the N-methyl-D-aspartate receptor blocker MK-801 on neurologic function after experimental brain injury

Pharmacologic inhibition of excitatory amino acid (EAA) neurotransmission attenuates cell death in models of global and focal ischemia and hypoglycemia and improves neurologic outcome after experimental traumatic spinal cord injury. The present study examined the effects of the noncompetitive N-methyl-D-aspartate (NMDA) receptor blocker MK-801 on cardiovascular and neurologic function after experimental fluid-percussion (FP) brain injury in the rat. Animals received either an intravenous bolus of MK-801 (1 mg/kg) or saline (equal volume) 15 min prior to FP brain injury or 15 min following FP brain injury. MK-801 pretreatment significantly improved postinjury cardiovascular variables and attenuated postinjury neurologic dysfunction. Postinjury treatment with MK-801 also significantly improved cardiovascular variables, but had little effect on postinjury neurologic scores. These results suggest that EAA neurotransmitters may be involved in the pathophysiological sequelae of traumatic brain injury and that noncompetitive blockade of the NMDA receptor prior to brain injury may reduce EAA-induced damage and limit neurologic dysfunction.     

NBQX treatment improves mitochondrial function and reduces oxidative events after spinal cord injury

The purpose of this study was to examine the effects of inhibiting ionotropic glutamate receptor subtypes on measures of oxidative stress events at acute times following traumatic spinal cord injury (SCI). Rats received a moderate contusion injury and 15 min later were treated with one of two doses of 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzol[f]quinoxaline-7-sulfonamide disodium (NBQX), MK-801, or the appropriate vehicle. At 4 h following injury, spinal cords were removed and a crude synaptosomal preparation obtained to examine mitochondrial function using the MTT assay, as well as measures of reactive oxygen species (ROS), lipid peroxidation, and glutamate and glucose uptake. We report here that intraspinal treatment with either 15 or 30 nmol of NBQX improves mitochondrial function and reduces the levels of ROS and lipid peroxidation products. In contrast, MK-801, given intravenously at doses of 1.0 or 5.0 mg/kg, was without effect on these same measures. Neither drug treatment had an effect on glutamate or glucose uptake, both of which are reduced at acute times following SCI. Previous studies have documented that drugs acting on non-N-methyl-D-aspartate (NMDA) receptors exhibit greater efficacy compared to NMDA receptor antagonists on recovery of function and tissue sparing following traumatic spinal cord injury. The results of this study provide a potential mechanism by which blockade of the non-NMDA ionotropic receptors exhibit positive effects following traumatic SCI.     

Effects of neuroprotective cocktails on hippocampal neuron death in an in vitro model of cerebral ischemia

Cocktails of neuroprotectants acting at different parts of the ischemic injury cascade may have advantages over single agents. This study investigated, singly and in combination, the neuroprotective efficacy of an energy substrate (3.5 mM fructose 1,6-bisphosphate, FBP), an antagonist of NMDA receptors (1 and 10 microM MK-801), a free-radical scavenger (100 microM ascorbate), an adenosine A1 receptor agonist (10 microM 2-chloroadenosine), and an inhibitor of neurotransmission (2% isoflurane). These agents were evaluated for their ability to prevent loss and morphologic damage of CA1 neurons in rat hippocampal slices when these agents were administered during 30 minutes in vitro ischemia (combined oxygen/glucose deprivation at 37 degrees C) followed by 5 hours of recovery. Ten microM MK-801, alone or in combination with the other compounds, prevented loss of CA1 neurons and preserved their histologic appearance. Isoflurane, which prevents glutamate receptor-dependent cell death in this model, was also protective. Protection against neuron loss was also found when a subtherapeutic concentration of MK-801 (1 microM) was combined with 2-chloroadenosine (which indirectly causes NMDA receptor suppression), but not FBP or ascorbate. The authors conclude that in this model, the strategy of antagonizing NMDA receptors appears more protective than fructose-1,6-bisphosphate, 2-chloroadenosine or ascorbate.     

Neuropathological endpoints in experimental stroke pharmacotherapy: The importance of both early and late evaluation

Summary This study adresses the issue of endpoint selection in the evaluation of neuroprotective drugs in experimental focal ischaemia. Previous work with the permanent middle cerebral artery (MCA) occlusion model in the rat has demonstrated that the ischaemic lesion does not acquire its final appearance until at least 28 days after the ictus. Therefore, the effect of the NMDA receptor blocker MK-801 (dizocilpine maleate) was evaluated both early (3 days) and late (28 days) after MCA occlusion to determine if the previously reported protective effect of a single post-ischaemic dose of MK801 found in acute experiments remained after 28 days.Mk-801 (0.5mg/kg, i.v.) or isotonic saline was randomly given to rats 30 min after MCA occlusion. Infarct volume and volume of ipsilateral and contralateral hemispheres were estimated from camera lucida drawings of 8 defined coronal histological sections of the brain. As expected, a 40% (p<0.05) reduction of infarct size was found in MK-801 treated rats after 3 days. In animals evaluated 28 days after MCA occlusion, no significant difference in infarct size, total tissue loss (infarct volume + ipsilateral hemisphere atrophy) or remaining non-infarcted tissue (contralateral hemisphere — total tissue loss) was seen between the MK-801 and placebo treated rats.The results suggest that the single dose treatment with MK-801 postponed the evolution of the infarct, which at 3 days after MCA occlusion is still in progress, possibly by ameliorating oedema formation. It remains to be shown if a multiple dose treatment with NMDA receptor antagonists improves the final neuropathological outcome after experimental stroke. The study illustrates the importance of including a late endpoint when evaluating the efficacy of neuroprotective stroke therapy.     

The NMDA Type Glutamate Receptors Expressed by Primary Rat Osteoblasts Have the Same Electrophysiological Characteristics as Neuronal Receptors

Cells of mammalian bone express glutamate receptors. Functional N-methyl-D-aspartate (NMDA) receptors have been demonstrated in human, osteoblastic MG-63 cells, but currents in these cells, unlike those of mammalian neurons, are blocked by Mg(2+) in a voltage-insensitive manner. Differences between the characteristics of NMDA currents in bone cells and in neurons may reflect molecular variation of the receptors or associated molecules, with implications for the role(s) of glutamate in these different tissues and for targeting of ligands/antagonists. To determine whether NMDA receptors in primary bone cells are functional, and whether the currents carried by these receptors resemble those of MG-63 cells or those of mammalian neurons, we have applied the whole cell patch clamp technique to primary cultures of rat osteoblasts. In 0-Mg(2+) saline, 25% of cells showed a slowly developing inward current in response to bath perfusion with 1 mM or 100 microM NMDA. Antibodies against NMDA receptors stained approximately 26% of cells. When NMDA was applied by rapid superfusion, kinetics of the currents were similar to those of neuronal NMDA currents, reaching a peak within 20-30 ms. 1 mM Mg(2+) reduced current amplitude at negative holding potentials and caused the I-V relationship of the currents to adopt a 'J' shape rather than the linear relationship seen in the absence of added Mg(2+). Co-application of glycine (20 microM) with NMDA increased current amplitude by only 18%, suggesting that glycine is released from cells within the cultures. Currents were blocked by (+)-MK-801 and DL-2-amino-5-phosphonovaleric acid. Fluorimetric monitoring of [Ca(2+)](i) using fura-2 showed that, in Mg(2+)-free medium, NMDA caused a sustained rise in [Ca(2+)](i) that could be reversed by subsequent application of MK-801. We conclude that rat femoral osteoblasts express functional NMDA receptors and that these receptors differ from those previously identified in MG-63 cells. NMDA receptors of primary osteoblasts show a 'classical' voltage-sensitive Mg(2+) block, similar to that seen in neuronal NMDA receptors, and will therefore function as detectors of coincident receptor activation and membrane depolarization.     

Lack of Effect of Intrathecally Administered N-methyl-D-aspartate Receptor Antagonists in a Rat Model for Postoperative Pain

Background: Evidence from experiments by others indicates an important role for excitatory amino acids activating spinal n-methyl-d-aspartate (NMDA) receptors in models of persistent pain. The purpose of this study was to examine the effect of intrathecal (+)-5-methyl-10,11-dihydro-5H-dibenzo(a,d)cyclohepten-5,10-imine (MK-801), a noncompetitive NMDA receptor antagonist, 2-amino-5-phosphonovaleric acid (AP5), a competitive NMDA receptor antagonist, and N-G-nitro-L-arginine methyl ester (L-Name), a nitric oxide synthase inhibitor, on pain behaviors in a rat model of postoperative pain.

Methods: Rats with intrathecal catheters were anesthetized and underwent a plantar incision. Withdrawal threshold to punctate stimulation applied adjacent to the wound, response frequency to application of a nonpunctate stimulus applied directly to the wound, and nonevoked pain behaviors were measured before and after intrathecal administration of MK-801 or AP5. The effect of intrathecal L-Name on mechanical hyperalgesia was also examined.

Results: Mechanical hyperalgesia increased and was persistent after plantar incision and was not decreased by intrathecal administration of 4, 14, or 40 nmol MK-801 or 10 nmol AP5. Only the greatest dose of AP5, 30 nmol, caused a small decrease in punctate and nonpunctate hyperalgesia. Intrathecal L-Name had no effect. Neither intrathecal MK-801 nor intrathecal AP5 affected nonevoked pain behaviors. The greatest doses caused motor deficits.     

Effect of pretreatment with intrathecal excitatory amino acid receptor antagonists on the development of pain behavior caused by plantar incision

BACKGROUND: Drugs that block spinal excitatory amino acid receptor activation may prevent pain after surgery. The authors previously studied the effect of excitatory amino acid receptor antagonists after incision. In the present study, we examined the role of N-methyl-d-aspartate (NMDA), non-NMDA, and metabotropic glutamate receptors (mGluRs) on the development of pain behavior after plantar incision. METHODS: Rats with lumbar intrathecal catheters were anesthetized with halothane. Fifteen minutes before an incision was made, drug [40 nmol MK-801; 20 nmol NBQX; or 200 nmol (+)-MCPG] or vehicle was injected intrathecally followed by an infusion of the same drug for 75 min. Withdrawal thresholds to calibrated von Frey filaments applied adjacent to the wound and response frequencies to a blunt mechanical stimulus applied directly to the wound were measured before incision and 1, 2, 4, and 6 h after incision and then once daily for 6 days. RESULTS: Preincision treatments with antagonists against the NMDA (MK-801) and group I and II metabotropic receptors [(+)-MCPG] did not inhibit the development of mechanical hyperalgesia caused by incision. Preincision treatment with the non-NMDA receptor antagonist NBQX increased withdrawal thresholds at 1 and 2 h and on postoperative day 1 compared with the vehicle group; response frequencies were reduced 1 and 2 h after incision and on postoperative day 2 (P < 0.05). In an additional group, postincision treatment with NBQX was similar to preincision treatment. CONCLUSION: Spinal NMDA and mGluR antagonists may not be useful for preventing postsurgical pain. Spinal non-NMDA receptor antagonists reduced pain behaviors, but a preventive effect using preincision treatment was not apparent.