Intrastriatal N-methyl-D-aspartate Injection Research Articles

Neuroprotective effects of xenon: a therapeutic window of opportunity in rats subjected to transient cerebral ischemia

Brain insults are a major cause of acute mortality and chronic morbidity. Given the largely ineffective current therapeutic strategies, the development of new and efficient therapeutic interventions is clearly needed. A series of previous investigations has shown that the noble and anesthetic gas xenon, which has low-affinity antagonistic properties at the N-methyl-D-aspartate (NMDA) receptor, also exhibits potentially neuroprotective properties with no proven adverse side effects. Surprisingly and in contrast with most drugs that are being developed as therapeutic agents, the dose-response neuroprotective effect of xenon has been poorly studied, although this effect could be of major critical importance for its clinical development as a neuroprotectant. Here we show, using ex vivo and in vivo models of excitotoxic insults and transient brain ischemia, that xenon, administered at subanesthetic doses, offers global neuroprotection from reduction of neurotransmitter release induced by ischemia, a critical event known to be involved in excitotoxicity, to reduction of subsequent cell injury and neuronal death. Maximal neuroprotection was obtained with xenon at 50 vol%, a concentration at which xenon further exhibited significant neuroprotective effects in vivo even when administered up to 4 h after intrastriatal NMDA injection and up to at least 2 h after induction of transient brain ischemia.

Selective blockade of metabotropic glutamate receptor subtype 5 is neuroprotective

We have used potent and selective non-competitive antagonists of metabotropic glutamate receptor subtype 5 (mGlu5) —- 2-methyl-6-phenylethynylpyridine (MPEP), [6-methyl-2-(phenylazo)-3-pyridinol] (SIB-1757) and [( E)-2-methyl-6-(2-phenylethenyl)pyridine] (SIB-1893) — to examine whether endogenous activation of this particular metabotropic glutamate receptor subtype contributes to neuronal degeneration. In cortical cultures challenged with N-methyl- d-aspartate (NMDA), all three mGlu5 receptor antagonists were neuroprotective. The effect of MPEP was highly specific because the close analogue, 3-methyl-6-phenylethynylpyridine (iso-MPEP), which did not antagonize heterologously expressed mGlu5 receptors, was devoid of activity on NMDA toxicity. Neuroprotection by mGlu5 receptor antagonists was also observed in cortical cultures challenged with a toxic concentration of β-amyloid peptide. We have also examined the effect of mGlu5 receptor antagonists in in vivo models of excitotoxic degeneration. MPEP and SIB-1893 were neuroprotective against neuronal damage induced by intrastriatal injection of NMDA or quinolinic acid. These results indicate that mGlu5 receptors represent a suitable target for novel neuroprotective agents of potential application in neurodegenerative disorders.

A transforming growth factor-beta antagonist unmasks the neuroprotective role of this endogenous cytokine in excitotoxic and ischemic brain injury.

Various studies describe increased concentrations of transforming growth factor-beta (TGF-beta) in brain tissue after acute brain injury. However, the role of endogenously produced TGF-beta after brain damage to the CNS remains to be clearly established. Here, the authors examine the influence of TGF-beta produced after an episode of cerebral ischemia by injecting a soluble TGF-beta type II receptor fused with the Fc region of a human immunoglobulin (TbetaRIIs-Fc). First, this molecular construct was characterized as a selective antagonist of TGF-beta. Then, the authors tested its ability to reverse the effect of TGF-beta1 on excitotoxic cell death in murine cortical cell cultures. The addition of 1 microg/mL of TbetaRIIs-Fc to the exposure medium antagonized the neuroprotective activity of TGF-beta1 in N-methyl-D-aspartate (NMDA)-induced excitotoxic cell death. These results are consistent with the hypothesis that TGF-beta1 exerts a negative modulatory action on NMDA receptor-mediated excitotoxicity. To determine the role of TGF-beta1 produced in response to brain damage, the authors used a model of an excitotoxic lesion induced by the intrastriatal injection of 75 nmol of NMDA in the presence of 1.5 microg of TbetaRIIs-Fc. The intrastriatal injection of NMDA was demonstrated to induce an early upregulation of the expression of TGF-beta1 mRNA. Furthermore, when added to the excitotoxin, TbetaRIIs-Fc increased (by 2.2-fold, P < 0.05) the lesion size. These observations were strengthened by the fact that an intracortical injection of TbetaRIIs-Fc in rats subjected to a 30-minute reversible cerebral focal ischemia aggravated the volume of infarction. In the group injected with the TGF-beta1 antagonist, a 3.5-fold increase was measured in the infarction size (43.3 +/- 9.5 versus 152.8 +/- 46.3 mm3; P < 0.05). In conclusion, by antagonizing the influence of TGF-beta in brain tissue subjected to excitotoxic or ischemic lesion, the authors markedly exacerbated the resulting extent of necrosis. These results suggest that, in response to such insults, brain tissue responds by the synthesis of a neuroprotective cytokine, TGF-beta1, which is involved in the limitation of the extent of the injury. The pharmacologic potentiation of this endogenous defensive mechanism might represent an alternative and novel strategy for the therapy of hypoxic-ischemic cerebral injury.

Neuronal nitric oxide synthase and calmodulin-dependent protein kinase IIalpha undergo neurotoxin-induced proteolysis.

Calpain (calcium-activated neutral protease) has been implicated as playing a role of neuronal injury in cerebral ischemia and excitotoxicity. Here we report that, in addition to extreme excitotoxic conditions [N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and kainate challenges], other neurotoxins such as maitotoxin, A23187, and okadaic acid also induce calpain activation, as detected by m-calpain autolytic fragmentation and nonerythroid alpha-spectrin breakdown. Under the same conditions, calmodulin-dependent protein kinase II-alpha (CaMPK-IIalpha) and neuronal nitric oxide synthase (nNOS) are both proteolytically cleaved by calpain. Such fragmentation can be reduced by calpain inhibitors (acetyl-Leu-Leu-Nle-CHO and PD151746). In vitro digestion of protein extract from cortical cultures with purified mu- and m-calpain produced fragmentation patterns for CaMPK-IIalpha and nNOS similar to those produced in situ. Also, several other calpain-sensitive calmodulin-binding proteins (plasma membrane calcium pump, microtubule-associated protein 2, and calcineurin A) and protein kinase C-alpha are also degraded in neurotoxin-treated cultures. Lastly, in a rat pup model of acute excitotoxicity, intrastriatal injection of NMDA resulted in breakdown of CaMPK-IIalpha and nNOS. The degradation of CaMPK-IIalpha, nNOS, and other endogenous calpain substrates may contribute to the neuronal injury associated with various neurotoxins.

3H]L-NG-nitroarginine binding after transient focal ischemia and NMDA-induced excitotoxicity in type I and type III nitric oxide synthase null mice.

We investigated the density and distribution of nitric oxide synthase (NOS) binding by quantitative autoradiography using [3H]L-NG-nitroarginine ([3H]L-NNA) after transient focal ischemia or intrastriatal injection of N-methyl-D-aspartate (NMDA) in wild-type (SV-129 and C57black/6) and type I (neuronal) and type III (endothelial) NOS-deficient mice. The middle cerebral artery (MCA) was occluded by an intraluminal filament for 3 h followed by 10 min to 7 days of reperfusion. Specific [3H]L-NNA binding, observed in the wild-type and type III mutant mouse at baseline, increased by 50-250% in the MCA territory during ischemia and the first 3 h of reperfusion. The density of binding sites (Bmax), but not the dissociation constant (Kd), increased significantly during the ischemic period as did type I NOS mRNA as detected by quantitative reverse transcription polymerase chain reaction. [3H]L-NNA binding after intrastriatal NMDA injection also increased by 20-230%. In the type I NOS-deficient mouse, [3H]L-NNA binding was low and only a very small increase was observed after ischemia or excitotoxicity. Under conditions of this study, [3H]L-NNA did not bind to type II NOS as there was no difference in the distribution or density of [3H]L-NNA binding in the rat spleen obtained after lipopolysaccharide treatment despite induction of NOS type II catalytic activity. Our data suggest that an ischemic/excitotoxic insult up-regulates type I NOS gene expression and [3H]L-NNA binding and that this up-regulation may play a pivotal role in the pathogenesis of ischemic/excitotoxic diseases.

NMDA and kainate induce internucleosomal DNA cleavage associated with both apoptotic and necrotic cell death in the neonatal rat brain.

Injection of N-methyl-D-aspartate (NMDA) or kainate in the striatum of 7-day-old rats induced massive cell loss in the ipsilateral striatum, hippocampus and inner cortical layers. In order to examine whether apoptosis contributes to cell death in this model of excitotoxic injury we examined the progression of internucleosomal DNA fragmentation and changes in cellular ultrastructure. Agarose gel electrophoresis of DNA extracted from the ipsilateral striatum, cerebral cortex and hippocampus clearly showed breakdown of DNA into oligonucleosome-sized fragments, indicative of apoptosis, 12 h post-NMDA injection. In addition, an increase between 12 and 24 h was observed as well as a continuous presence 5 days later. Kainate induced a similar time course of oligonucleosomal DNA fragmentation, but the intensity of the ethidium bromide stained bands was less compared with that observed for NMDA. DNA fragmentation was not detected in animals intrastriatally injected with Tris-HCl or in animals treated with MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohept-5,10-imine hydrogen maleate, 1 mg/kg] 30 min after NMDA injection. MK-801 had no effect on DNA fragmentation induced by kainate. In addition to agarose gel electrophoresis, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labelling (TUNEL) was used for detection of DNA fragmentation in sections. A gradual increase in the density of both apoptotic and non-apoptotic TUNEL nuclei was found in the anterior cingulate (ACC) and retrosplenial (RSC) areas of the cortex, the striatum, and the CA1 area and dentate gyrus of the hippocampus over the first 24 h post-NMDA or kainate injection. In the contralateral hemisphere hardly any TUNEL nuclei were present and their density was comparable with that in animals injected with vehicle only. In the ipsilateral mammillary nucleus (MN), which showed no signs of acute cell swelling after intrastriatal injection with NMDA, internucleosomal DNA fragmentation was found 24 and 48 h after intrastriatal NMDA injection. Here, the density of TUNEL cells with apoptotic morphology was high at 12 and 24 h post-NMDA injection but returned to control levels by 5 days. Electron microscopy showed cells with a clearly apoptotic morphology in the ACC and RSC and in the MN 24 h after NMDA injection. In the CA1 area of the hippocampus a necrotic, rather than an apoptotic, ultrastructure prevailed, indicating that the TUNEL method stained both apoptotic and necrotic cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Early Evolution and Recovery from Excitotoxic Injury in the Neonatal Rat Brain: A Study Combining Magnetic Resonance Imaging, Electrical Impedance, and Histology

We explored the therapeutic potentials of two N-methyl-D-aspartate (NMDA) receptor antagonists in vivo using different techniques. NMDA injected into the striatum of neonatal rats (20 nmol/0.5 microliters) induced a rapid increase in the diffusion-weighted (DW) image intensity, spreading over a large part of the ipsilateral hemisphere. Subcutaneous injection of the NMDA receptor antagonist MK-801 (1 mg/kg) or D-(E)-4-(3-phosphono-2-prop-enyl)-2-piperazine-carboxylic acid (D-CPPene; 1.5 mg/kg) reversed both the volume and the grading of the NMDA-induced hyperintensity of DW images, the reversal by MK-801 being more rapid than that by D-CPPene. In the cerebral cortex, there was an inverse relationship between changes in DW image intensity and the size of the extracellular space, assessed by electrical impedance measurements. The reduction of the hyperintense volume in DW images 1 or 2 h after MK-801 or D-CPPene treatment of NMDA-injected animals depended on the type of antagonist used and on the interval between intrastriatal NMDA injection and antagonist treatment. The reduction was 95% when MK-801 was given with a delay of 90 min and decreased to 20% when it was given at 360 min. With D-CPPene, the reduction was 80% after a delay of 30 min and virtually absent when it was administered at 360 min. Quantitative analysis showed significant correlations between the residual hyperintense volume 1 or 2 h after MK-801 or D-CPPene treatment and the final lesion volume, assessed from either T2-weighted images (R = 0.89, p < 0.001) or histology (R = 0.80, p < 0.001) 5 days after the insult. This study illustrates the sensitivity of DW magnetic resonance imaging to monitor in vivo early events after an excitotoxic insult and the effect of putative protective drugs that may counteract the resulting damage.

Temporal evolution of NMDA-induced excitoxicity in the neonatal rat brain measured with 1H nuclear magnetic resonance imaging

The aim of this study is to characterize the evolution of excitotoxic damage in neonatal rat brain by diffusion-weighted and T 2-weighted magnetic resonance imaging. Results are compared with histological findings. Magnetic resonance imaging was performed at various times (15 min, 24 h, 3 days and 5 days) after intrastriatal microinjection of N-methyl- d-aspartate (NMDA) at postnatal day 8. The transverse relaxation time (T 2) and apparent diffusion coefficient of water were determined. The results show an acute reduction of the apparent diffusion coefficient, reflected by an ipsilateral hyperintensity in the diffusion-weighted images, within 15 min after intrastriatal NMDA injection. At this time no changes in the T 2-weighted images were apparent. The volume of the hyperintensity was relatively large with a radius of approximately 2 mm and coincided with histological signs of pronounced karyo-dendritic swelling. Subcutaneous administration of MK-801 25 min after the intracerebral NMDA injection readily reversed the hyperintensity and resulted in complete protection as verified by histology. Areas with increased T 2 values were observed 1 day after NMDA microinjection and corresponded to regions with obvious cell necrosis. Five days after NMDA injection the lesion was evident using both diffusion- and T 2-weighted images and coincided with an overt lesion comprising areas of cell loss and dilatation of the ipsilateral ventricle. In conclusion, this study illustrates the possibility of using diffusion-weighted imaging as a tool to monitor efficacy of treatment strategies at an early stage of excitotoxic injury.

Delayed administration of basic fibroblast growth factor protects against N-methyl-D-aspartate neurotoxicity in neonatal rats

Neuroprotective effects of basic fibroblast growth factor (bFGF) (200 μ/kg i.p.) were examined at different time points following intrastriatal injection of N-methyl-D-aspartate (NMDA) in seven-day-old rats. When administered 0.5 h before intrastriatal NMDA injection, 200 μ/kg of bFGF significantly reduced NMDA-induced lesion by 47% as compared with vehicle treatment. Posttreatment with the same dose of bFGF at 0, 0.5 or 1 h after NMDA injection also significantly reduced those lesions by 45, 35 or 26%, respectively, while no neuroprotective effects was observed when administered at 2 or 4 h after NMDA injection.

Basic Fibroblast Growth Factor Protects against Hypoxia-Ischemia and NMDA Neurotoxicity in Neonatal Rats

Basic fibroblast growth factor (bFGF) is a polypeptide that promotes neuronal survival and blocks excitatory amino acid (EAA) neurotoxicity in vitro at very low concentrations. In the present study, we examined whether systemically administered bFGF could prevent neuronal damage induced by either EAAs or hypoxia-ischemia in vivo. Neuroprotective effects were examined in a neonatal model of hypoxia-ischemia (unilateral ligation of the carotid artery followed by exposure to 8% oxygen for 1.5 h) and following intrastriatal injection of N-methyl-D-aspartate (NMDA) in 7-day-old rats. Intraperitoneal administration of a single dose of bFGF (50-300 micrograms/kg) 30 min before intrastriatal injection of NMDA showed a dose-dependent neuroprotective effect. Repeated doses of bFGF (100 micrograms/kg) both before and after intrastriatal NMDA injection produced a much greater significant protective effect than a single dose administered prior to the injection. Intraperitoneal injection of single dose of 100 micrograms/kg of bFGF 30 min before hypoxia-ischemia reduced neuronal damage by 38% (p = 0.14), while administration of bFGF at a dose of 100 micrograms/kg i.p. three times, 30 min before and 0 and 30 min after hypoxia-ischemia, significantly reduced neuronal damage by 64% (p = 0.004). Systemic administration of bFGF did not change body temperature for up to 3 h. These results show that systemic administration of bFGF can exert neuroprotective effects against both NMDA-induced excitotoxicity and hypoxia-ischemia in vivo.

The polyamine synthesis inhibitor α-difluoromethylornithine is neuroprotective against N-methyl-D-aspartate-induced brain damage in vivo

We examined the ability of α-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, the rate limiting enzyme for polyamine biosynthesis, to protect the brain of the perinatal rat from N-methyl-D-aspartate (NMDA)-induced brain damage. Treatment of the rat pups with DFMO administered either by i.p. injection (500 mg/kg × 2) or through the milk of the mother (2% solution in mother's drinking water) significantly reduced, by 48 and 62%, respectively, the brain damage produced by intrastriatal NMDA injection. We conclude that activation of polyamine synthesis may mediate part of the neurotoxic action of NMDA.

Protection Against N-methyl-D-aspartate Receptor-Mediated Neuronal Degeneration In Rat Brain by 7-chlorokynurenate and 3-amino-1-hydroxypyrrolid-2-one, Antagonists at The Allosteric Site for Glycine.

7-Chlorokynurenate (7-Cl KYNA) and 3-amino-1-hydroxypyrrolid-2-one (HA-966), two selective antagonists of the glycine site on the N-methyl-D-aspartate (NMDA) receptor, have been used to assess the involvement of this site in the neurodegeneration resulting from injection of excitotoxins in the rat brain. In the rat striatum, reductions in the enzymes choline acetyltransferase (CAT) and glutamate decarboxylase (GAD), occurring 7 days after a unilateral, intrastriatal injection of quinolinate (200 nmol), were prevented in a dose-dependent manner by intrastriatal administration of 7-Cl KYNA (10 - 50 nmol) and HA-966 (200 - 500 nmol) 1 h after the excitotoxin. In the rat hippocampus, degeneration of pyramidal and granule neurons caused by direct injection of quinolinate (60 nmol) was completely prevented by 7-Cl KYNA (50 nmol) and partially by HA-966 (500 nmol) injected intrahippocampally 1 h after the excitotoxin. In the rat striatum, 7-Cl KYNA (50 nmol) and HA-966 (500 nmol) also reduced neurotoxicity caused by intrastriatal injection of NMDA (200 nmol), but not that caused by the 'non-NMDA' receptor agonists DL-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) or kainate. The time course of protective effects of 7-Cl KYNA and HA-966 in the striatum was similar to that previously observed with the uncompetitive NMDA receptor antagonist MK-801, indicating that activation of the glycine site contributes to the delayed degeneration of neurons which occurs over the first 5 h following quinolinate injection. The neuroprotective effects of both 7-Cl KYNA and HA-966 in the rat striatum appear to be mediated via the glycine site on the NMDA receptor as they were completely reversed by D-serine, but not L-serine. These results indicate that activation of the glycine site is essential for the expression of the delayed degeneration of neurons resulting from intracerebral injection of an NMDA receptor agonist, a process which bears similarities to the delayed neurodegeneration which results from a period of cerebral ischaemia.

Quantitative assessment of neuroprotection against NMDA-induced brain injury

In immature rodent brain, unilateral intrastriatal injections of selected excitatory amino acid (EAA) receptor agonists, such as N-methyl- d-aspartate (NMDA), produce prominent ipsilateral forebrain lesions. In Postnatal Day (PND) 7 rats that receive a right intrastriatal injection of NMDA (25 nmol) and are sacrificed 5 days later, there is a considerable and consistent reduction in the weight of the injected cerebral hemisphere relative to that of the contralateral side (−28.5 ± 1.9%, n = 6). In animals treated with specific NMDA receptor antagonists, the severity of NMDA-induced damage is markedly reduced. We have previously reported that the efficacy of potential neuroprotective drugs in limiting NMDA-induced lesions can be assessed quantitatively by comparison of hemisphere weights after a unilateral NMDA injection. In this study, we compared three quantitative methods to evaluate the severity of NMDA-induced brain injury and the degree of neuroprotection provided by NMDA receptor antagonists. We characterized the severity of brain injury resulting from intrastriatal injections of 1–50 nmol NMDA in PND 7 rats sacrificed on PND 12 by (i) comparison of cerebral hemisphere weights; (ii) assay of the activity of the cholinergic neuronal marker, choline acetyltransferase (ChAT) activity; and (iii) measurement of regional brain cross-sectional areas. The severity of the resulting brain injury as assessed by comparison of hemisphere weights increased linearly with the amount of NMDA injected into the striatum up to 25 nmol NMDA. The magnitude of injury was highly correlated with the degree of reduction in ChAT activity ( r 2 = 0.97). Quantification of neuroprotection against NMDA toxicity by measurement of cerebral hemisphere weight disparities was highly correlated with comparisons of hemisphere and striatal cross-sectional areas ( r 2 = 0.98). The potency and efficacy of the dissociative anesthetic ketamine was easily distinguished from those of two other NMDA receptor antagonists (MK-801, (+)-5-methyl-10,11-dihydro-5H-dibenzo[ a,d]cyclohepten-5,10-immine maleate and CPP, 3-((±)-2-carboxypiperazine-4-yl)-propyl-1-phosphonic acid) using this model. These data demonstrate that this model can be used to accurately quantitate NMDA-induced brain injury and evaluate neuro-protective properties of glutamate antagonists in vivo.