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(Stroke. 1995;26:298-304.)
© 1995 American Heart Association, Inc.

Articles

Nitric Oxide Synthase Inhibition and Extracellular Glutamate Concentration After Cerebral Ischemia/Reperfusion

Jing Zhang, MD; Helene Benveniste, MD, PhD; Bruce Klitzman, PhD Claude A. Piantadosi, MD

From the Departments of Medicine (J.Z., C.A.P.), Anesthesiology (H.B.), and Surgery and Cell Biology (B.K.), Duke University Medical Center, Durham, NC.

Correspondence to Jing Zhang, MD, PO Box 3315, Department of Medicine, Duke University Medical Center, Durham, NC 27710.

	Abstract

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Background and Purpose Transient cerebral ischemia in rats results in selective loss of neuronal viability, eg, hippocampal CA1 neurons. The neurochemical variables responsible for this selective vulnerability to ischemia/reperfusion (IR) appear to involve excitatory amino acids. In brain IR, excitatory amino acid toxicity may be modulated by endogenous nitric oxide (NO) gas. To investigate NO in global brain IR, we measured the effects of NO synthase (NOS) inhibition on interstitial excitatory amino acids in rats. Changes in postischemic cerebral blood flow and blood-brain barrier function also were evaluated.

Methods Forebrain ischemia was produced by systemic hypotension and occlusion of both carotid arteries for 15 minutes. Blood flow was restored for 60 minutes by unclamping the carotids and reinfusing with blood. A microdialysis probe was placed into the cortex and hippocampus using a stereotaxic device. Interstitial glutamate concentration was measured during IR with high-performance liquid chromatography. A competitive NOS inhibitor, N-nitro-L-arginine methyl ester (L-NAME), was given intraperitoneally 30 minutes before ischemia in doses of 1, 4, and 20 mg/kg. Changes in cerebral blood flow and blood-brain barrier during IR were determined using laser-Doppler flowmetry and microdialysis with sodium fluorescein.

Results Glutamate in the dialysate during IR increased transiently 10-fold and returned to baseline levels by 30 minutes of reperfusion. Animals treated with L-NAME 30 minutes before ischemia also showed increases in glutamate concentration during ischemia, but glutamate remained elevated during reperfusion. The increase in glutamate concentration during reperfusion caused by L-NAME was prevented by L-arginine. The administration of L-arginine and L-NAME together decreased extracellular glutamate concentration during ischemia. Cerebral blood flow decreased to about 5% of baseline values during ischemia but increased approximately fourfold relative to control values on reperfusion. The hyperemic responses after ischemia were not different between IR groups treated with or without L-NAME. Brain ischemia increased the permeability of the blood-brain barrier to fluorescein; however, this change was attenuated by L-NAME administration at 20 mg/kg.

Conclusions NOS inhibition did not attenuate extracellular glutamate accumulation during ischemia and increased its concentration on reperfusion. The elevated glutamate concentration after IR in L-NAME–treated rats did not appear to be due to either a decrease in cerebral blood flow response after ischemia or increases in local blood-brain barrier permeability. For the most part, the blood-brain barrier was spared in the immediate postischemic period by L-NAME treatment. These data suggest that NO production may oppose synaptic excitatory amino acid accumulation and presumably excitotoxicity during IR.

Key Words: cerebral ischemia • nitric oxide • rats

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Transient ischemia leads to selective and progressive neuronal death in vulnerable brain regions such as the CA1 pyramidal cells in the hippocampus and the small-to-medium neurons of the dorsolateral striatum.^{1 2} The onset of ischemia is followed promptly by synaptic accumulation of excitatory amino acids (EAAs) operating as neurotransmitters via at least three glutamate receptor complexes. These include N-methyl-D-aspartic acid (NMDA), D-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and kainic acid (KA) receptors on neuronal cell membranes.³ Extracellular EAA accumulation during ischemia has been attributed to excessive presynaptic release, as well as to failure in ATP-dependent reuptake by EAA transporters. Glutamate transporters, for instance, have been proposed to contribute substantially to gluta-mate accumulation during ischemia due to cessation of reuptake and transport reversal.⁴

Recent studies indicate that nitric oxide (NO) is involved in ischemic brain damage.^{5 6} NO is synthesized in the brain from L-arginine by a constitutive NO synthase (NOS). The physiological roles of NO in the central nervous system are complex; it has been implicated in cerebral vasodilation, long-term potentiation, and excitotoxicity.⁷ Mechanisms by which NO could mediate cell damage include inhibition of iron-containing enzymes,⁸ production of toxic peroxynitrite or hydroxyl radicals,⁹ mediation of thiol inactivation and protein ribosylation,¹⁰ and alteration of DNA synthesis leading to cell death.¹¹ Evidence that NO mediates ischemic damage comes from several observations. In primary cultures of rat cerebral cortical neurons, NO released by NOS-containing neurons mediates excitatory neurotoxicity.¹² In vivo, decreased ischemia-induced damage has been reported in focal ischemia models where NO production is reduced.^{13 14} Also, NOS inhibition during focal cerebral ischemia decreased glutamate overflow as well as brain infarct volume.⁵ Other reports, however, have found that NOS inhibition does not alter glutamate-induced neurotoxicity in neuronal cultures of rat hippocampus and that NOS inhibition either before or after stroke does not decrease focal ischemic infarction in rats.^{15 16 17} At present, little is known about the effects of NO in global brain ischemia/reperfusion (IR) except that NOS inhibition does not attenuate or even aggravate the hippocampal lesions induced by forebrain ischemia.^{18 19} We previously observed that NOS inhibition by N-nitro-L-arginine methyl ester (L-NAME), a competitive NOS inhibitor, increased extracellular glutamate levels during reperfusion.²⁰ A protective role of NO production during ischemia also has been suggested in vitro because NO decreases NMDA-mediated increases in intracellular Ca²⁺ in cultured rat forebrain neurons.²¹ Finally, NO may protect neurons by inhibiting the adhesion and activation of blood platelets and neutrophils after brain ischemia.¹⁷

This study was intended to investigate the mechanisms by which L-NAME increases extracellular EAA accumulation during IR. The specificity of L-NAME for NOS inhibition was confirmed by comparing the temporal profile of ischemia-induced glutamate accumulation in the hippocampus and cortex with and without the administration of L-arginine. Factors that might increase glutamate concentration in L-NAME–treated animals during reperfusion also were investigated in this study, including potential toxic effects of L-NAME, vasoconstriction with prolonged brain ischemia/hypoxia due to NOS inhibition, and diffusion of glutamate from blood across a disrupted blood-brain barrier (BBB).

	Materials and Methods

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Male Sprague-Dawley rats (290 to 320 g) anesthetized with sodium pentobarbital (50 to 60 mg/kg IP) were intubated and ventilated mechanically with room air using a small-animal respirator (Edco). The femoral artery and vein were catheterized with PE-50 tubing. Systemic arterial blood pressure was monitored continuously through a pressure transducer (Gould TDN-R) calibrated with a mercury manometer. The signals were displayed on a chart recorder (Gould model 481) and integrated to obtain a mean arterial blood pressure (MABP). The carotid arteries were isolated bilaterally and dissected from the vagus nerve. All rats received 50 U heparin IV before induction of ischemia. Arterial blood samples of 200 µL were drawn periodically to help maintain blood gases at a PaO₂ greater than 80 mm Hg and a PaCO₂ of 35 to 45 mm Hg (IL model 1304, blood gas and pH analyzer). Rectal temperature was maintained close to 37°C by means of a warming plate and an overhead incandescent bulb. Nasopharyngeal temperature was monitored as an indicator of brain temperature with a small thermistor (YSI 400) and kept between 36°C and 37°C.

Reversible forebrain ischemia was produced by occlusion of both carotid arteries with systemic hypotension.²² To induce hypotension, the heparinized rats were bled over 2 to 3 minutes from the venous line to 35 mm Hg. Immediately after blood pressure reached 35 mm Hg, both carotid arteries were occluded using atraumatic aneurysm clamps. A mean pressure of 35 mm Hg was maintained by withdrawing additional blood or reinfusing blood from a syringe. Ischemia was maintained for 15 minutes, and then the cerebral circulation was restored for 60 minutes by reinfusing blood and unclamping the carotid arteries.

For the determination of glutamate concentration, rats were divided into seven experimental groups. These groups included (1) IR+saline (n=5), (2) IR+L-NAME (20 mg/kg, n=5), and (3) IR+L-NAME (20 mg/kg)+L-arginine (40 mg/kg, n=5). Two lower doses of L-NAME were also tested: (4) IR+L-NAME (1 mg/kg, n=3) and (5) IR+L-NAME (4 mg/kg, n=3). In addition, control experiments, including (6) control+L-NAME (20 mg/kg, n=3) and (7) IR+L-arginine (40 mg/kg, n=3) were performed to assess potential independent effects of L-NAME and L-arginine on glutamate concentration. All drugs were given intraperitoneally 30 minutes before ischemia.

Implantation of the microdialysis probe was performed as described by Benveniste et al.²³ A CMA/12 microdialysis probe (3-mm tip; OD, 500 µm) was attached to a micromanipulator on a Kopf stereotaxic device and placed into the left hippocampus using coordinates of anterior-posterior (AP), -3.4 mm; medial-lateral (ML), +2.2 mm; and dorsal-ventral (DV), -3.0 mm. The probe was perfused continuously with Krebs-Ringer bicarbonate buffer at 5µL/min using a CMA/100 microinjection pump (CMA/microdialysis AB). Samples were discarded during the first 120 minutes and collected every 15 minutes thereafter (75 µL per sample) using a CMA/140 microfraction collector (Carnegie Medicin AB). The pH of the perfusate was 7.4 in all experiments. This pH was higher than it is in brain tissue during ischemia and may have affected some of the measured parameters, eg, glutamate concentration or NOS activity. Any effects of pH should have been constant among the groups of animals undergoing IR and IR+L-NAME treatment.

Measurements of glutamate were performed using high-performance liquid chromatography with electrochemical detection as described by Donzanti and Yamamoto,²⁴ with modifications. Dialysate was derivitized with o-phthaldialdehyde and eluted isocratically using a buffer of 0.1 mol/L Na₂HPO₄, 0.13 mmol/L Na₂EDTA, and 28% methanol (pH 6.4). Glutamate levels were quantified with a Coulochem electrochemical detector (ESA model 5100A) with the detector set at +0.6 V. The guard cell was set at a reducing potential of -0.4 V.

In separate experiments, a 3-mm² burr hole was made over the left side of the skull using the same coordinates as for the microdialysis experiments. A fiber-optic probe of 0.8 mm in diameter was placed onto the surface of the intact rat brain with a micromanipulator; the dura was opened, and the brain surface was bathed in normal saline to prevent drying effects. After a 60-minute equilibration period, cerebral blood flow (CBF) was measured with the flow probe coupled to a BMP² laser-Doppler blood perfusion monitor (Vasamedics, Inc). CBF was measured in rats experiencing IR with or without L-NAME pretreatment (20 mg/kg, n=6 for each group). This flow probe measures the velocity and number of red blood cells flowing through tissue microvessels. Laser-Doppler flowmetry correlates well with flow measured by other techniques such as hydrogen clearance.²⁵ Most of the CBF signal measured by this probe was derived from the first 2 mm of tissue beneath the device, as shown by in vitro measurements using slices of rat brain (data not shown). The microdialysis probe sampled tissue volume from just beneath the cortex to approximately 3 mm beneath the brain surface.

Assessment of BBB function in the region of the microdialysis probe was performed using a modification of the fluorescein method of Gulati et al.²⁶ A microdialysis probe was inserted into the hippocampus as described above. The rats were given 0.3 mL of 10% sodium fluorescein IV 15 minutes before brain ischemia (120 minutes after the probe insertion) followed by a constant infusion (0.4 mL/h) using a syringe pump (Sage Instruments, model 351). Fluorescence was quantified in both dialysate and blood samples drawn every 15 minutes using a Turner fluorometer (model 112) with an excitation wavelength range of 315 to 385 nm and an emission range of 460 to 540 nm. All samples were mixed with 10 mmol/L tris(hydroxymethyl)aminomethane buffer to bring the pH to 8, since the intensity of fluorescein is pH dependent. The data were expressed as a fraction of the initial fluorescence values in cerebrospinal fluid and blood, and changes in the ratios were plotted over time. These experiments were carried out in control rats (n=3) and rats undergoing IR or IR with L-NAME at 20 mg/kg (n=6 for each group).

Fluorescein sodium (Funduscein-10) was purchased from Coopervision. L-NAME was obtained from Sigma Chemical Co. Sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, sodium phosphate, potassium phosphate, Trizma base, glutamate, o-phthaldialdehyde, and L-arginine were also purchased from Sigma Chemical Co.

Statistical analyses were performed using a STATVIEW 512+ (Brain Power, Inc) computer software package. All data were entered into a computer spreadsheet and analyzed by repeated-measurement ANOVA and Scheffé's F test. A value of P<.05 was considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Physiological Variables
Table 1 shows the effects of the IR protocol and administration of L-NAME on MABP in the rats. A significant increase in MABP was seen only after L-NAME treatment at 20 mg/kg with and without IR. This effect was abolished when L-arginine at 40 mg/kg was given with L-NAME. L-Arginine administration alone did not significantly affect the MABP. Arterial PO₂, PCO₂, and pH values were in the normal ranges and showed no significant changes during the experiments among any groups (see Table 2).

View this table: [in this window] [in a new window]   Table 1. Effects of Ischemia/Reperfusion and L-NAME Administration on Mean Arterial Blood Pressure

View this table: [in this window] [in a new window]   Table 2. Changes in Blood Gas and Arterial pH Levels

Changes in Extracellular Glutamate Concentration
Implantation of the microdialysis probe did not affect the glutamate profile substantially in the control rats, but transient cerebral ischemia led to a 10-fold increase in glutamate concentration in the perfusion medium. The glutamate level in the perfusate gradually returned to baseline values by 30 minutes of reperfusion. The magnitude and temporal profile of ischemia-induced changes in glutamate were similar to our previous results.²⁰ Of note, glutamate was collected primarily from the hippocampus with contributions from cerebral cortex and corpus callosum.

When IR experiments were performed in rats pretreated with L-NAME, glutamate accumulation during ischemia was similar to that in untreated animals experiencing ischemia. The glutamate concentrations, however, were much higher in the IR+L-NAME groups during reperfusion (Fig 1). This effect of L-NAME on glutamate concentration occurred at every dose, even as low as 1 mg/kg (Table 3). L-NAME treatment without ischemia tended to increase the basal glutamate level, although these basal changes were not statistically different from those in the control rats. This may have been due to the normally low basal glutamate levels and to the small numbers of animals in these groups (Fig 1 and Table 3).

View larger version (22K): [in this window] [in a new window]   Figure 1. Graph shows effects of the nitric oxide synthase inhibitor N-nitro-L-arginine (L-NAME) and L-arginine on extracellular glutamate concentration during brain ischemia and reperfusion. Glutamate concentrations in L-NAME–treated animals were similar to those of untreated animals during brain ischemia, but glutamate concentrations remained high during reperfusion. This effect of L-NAME was reversed by L-arginine. Data (µmol/L) are mean±SEM. IR indicates ischemia/reperfusion; Arg, L-arginine. *P<.05 for IR+L-NAME (n=5) vs IR (n = 5); +P<.05 for IR+L-NAME+Arg (40 mg/kg, n=5) vs IR.

View this table: [in this window] [in a new window]   Table 3. Effect of L-NAME Dose on Glutamate Concentration During Ischemia/Reperfusion

To confirm the specificity of L-NAME for the NO pathway, L-arginine was given with L-NAME to determine if the effects of L-NAME were reversible and attributable to NOS inhibition. In these experiments, the increase in glutamate concentration during reperfusion after 20 mg/kg of L-NAME was reversed by L-arginine administered at a dose of 40 mg/kg (Fig 1). L-Arginine also decreased glutamate concentration during ischemia when it was given with L-NAME. There was also a tendency for glutamate to decrease during ischemia when L-arginine was given without L-NAME, although the change from control was not statistically significant in three experiments (data not shown).

Effect of L-NAME on Cerebral Blood Flow
CBF was measured to determine whether L-NAME treatment at 20 mg/kg decreased postischemic hyperemia, thereby contributing to the enhanced glutamate accumulation during reperfusion. In control IR experiments, CBF values were between 10 and 15 laser-Doppler units before carotid occlusion. During carotid occlusion, CBF decreased to about 5% of baseline values. After reperfusion, CBF increased approximately fourfold relative to control values (Fig 2). In L-NAME–treated animals (20 mg/kg), despite an increase in MABP, CBF was approximately 75% of the baseline value before ischemia. This result is similar to values reported previously.²⁷ Nevertheless, the hyperemic response after IR was not different among groups of animals treated with or without L-NAME.

View larger version (20K): [in this window] [in a new window]   Figure 2. Graph shows changes in cerebral blood flow during brain ischemia and reperfusion with and without N-nitro-L-arginine (L-NAME) administration. The hyperemic responses after ischemia were not different between ischemia/reperfusion (IR) only and IR with L-NAME pretreatment (20 mg/kg). Data are mean±SEM; n=6 for each group. LD indicates laser-Doppler.

Blood-Brain Barrier Function
Local measurements of BBB function during IR are summarized in Fig 3. These data are expressed as the ratio between fluorescence signals in the cerebrospinal fluid and the blood; all values are normalized to preischemia control values. The absolute values for fluorescein concentration in blood versus dialysate were approximately 1000 to 1. The microdialysis procedure also yielded stable values for the integrity of the BBB for the period of the experiments as shown in Fig 3. Brain ischemia increased the permeability of the BBB to sodium fluorescein beginning during ischemia and continuing through early reperfusion. L-NAME at 20 mg/kg prevented the change in BBB integrity after reperfusion (P<.05).

View larger version (35K): [in this window] [in a new window]   Figure 3. Bar graph shows effects of ischemia/reperfusion (IR) and N-nitro-L-arginine (L-NAME) administration on blood-brain barrier (BBB) function. Brain ischemia increased the permeability of the BBB to sodium fluorescein beginning during ischemia and continuing through early reperfusion. L-NAME at 20 mg/kg prevented the change in BBB function after reperfusion. Data are mean±SEM. (The ratio between fluorescence in cerebrospinal fluid [CSF] and blood was obtained after dividing the absolute value by the corresponding initial baseline value.) *P<.05 for IR (n=6) vs control (n=3); P<.05 for IR+L-NAME (n=6) vs IR.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
IR in rat brain produced a 10-fold increase in glutamate concentration in the perfusion medium during ischemia, which gradually returned to baseline by 30 minutes of reperfusion. The magnitude and temporal profile of ischemia-induced changes in glutamate were similar to the results reported previously by Benveniste.²⁸ Calculations of the concentration of extracellular glutamate yield values of up to 1 mmol/L, taking into account the shrinkage of extracellular space of 50% of the normal value during ischemia, the tortuosity factor, and the in vitro recovery efficiency of the probe.²⁹ This increased extracellular glutamate level, coupled with increased intracellular calcium concentration, enhanced production of oxygen radicals, and other deleterious processes, is a major factor in inducing cell death after brain ischemia.

Inhibition of NOS by L-NAME did not block glutamate accumulation during global IR, as was reported for a focal ischemic model in which NOS inhibition decreased striatal glutamate concentration.⁵ Our result in global brain ischemia is not surprising, since NO generation is likely to be very low because of the extremely limited oxygen delivery (CBF of approximately 5%) and NMDA receptor activity in this model. NO production is oxygen dependent³⁰ and is stimulated by NMDA activation.³¹ As a result, both hypoxia and NMDA antagonism inhibit NOS activity.^{30 31} NMDA activity during forebrain ischemia may be limited by several events, such as extracellular acidification³² and zinc release.³³ Both NMDA antagonists and NOS inhibitors can decrease infarct size in focal brain ischemia,^{13 14 32 34} but this effect has not been found in global brain ischemia.^{18 19 35} It may be argued, however, that AMPA/KA receptor activity also stimulates NO production.³⁶ One study speculated that production of NO during global ischemia is related to the extent of the ischemic insult.³⁷ Such NO production could be due to activation of AMPA/KA receptors, but blockade of NOS does not prevent KA-elicited neuronal death in cultures of cerebellar granule cells.³⁸

Potentiation of glutamate release during reperfusion was detected even when L-NAME was administered at 1 mg/kg 30 minutes before ischemia. The mechanisms for this dramatic postischemic increase in glutamate concentration are unclear. Prado et al³⁹ reported that L-NAME at 30 mg/kg limited restoration of regional cortical perfusion after occlusion of the carotid arteries. This effect could be associated with both NOS inhibition and muscarinic receptor antagonism because high doses of L-NAME oppose acetylcholine-induced relaxation of smooth muscle.⁴⁰ In our model, an L-NAME–mediated decrease in CBF was not found at a dose of 20 mg/kg. The discrepancy may be related to the difference in drug dose as well as to the severity of brain ischemia. The model of Prado et al was different from ours in that the measured CBF was higher during ischemia (40% of control instead of <5% in our model). It is possible, although unlikely, that L-NAME preferentially compromised reperfusion in deeper brain structures such as hippocampus, which went undetected by surface flowmetry. Changes in composition of the dialysate originated from both the hippocampus and the cortex. Therefore, preferential prolonged ischemia of deep hippocampus cannot adequately explain the increase in glutamate concentration on reperfusion (Fig 1) because the pronounced cortical hyperemia detected around more than half of the dialysis probe during reperfusion should have been sufficient to attenuate overall glutamate accumulation.

L-NAME at a dose of 130 mg/kg has been reported to alter BBB morphology.²⁷ This finding raises the possibility that L-NAME facilitates glutamate diffusion into cerebrospinal fluid from plasma, where glutamate concentrations are higher normally.⁴¹ In this forebrain ischemia model, IR-induced BBB disruption in the hippocampus was similar to that seen in a previous study⁴² ; however, BBB integrity was spared for the most part after brain ischemia in animals receiving L-NAME at 20 mg/kg. Although measurement of fluorescein passage is not a direct quantification of glutamate diffusion, the two compounds share important properties such as low molecular weight and a negative charge. The fluorescein data are consistent with the conclusion that excess glutamate did not come from the plasma. It is possible that L-NAME increased glutamate concentration in IR by stimulating glutamate release, decreasing its reuptake or directly damaging neurons and glia leading to glutamate efflux. However, glycine and glutamine did not increase in proportion to glutamate in L-NAME–treated animals even at doses of 20 mg/kg, suggesting that the glutamate did not directly leak from dying neurons (data not shown). Finally, NOS inhibition also appears to decrease EAA overflow in brain slices.⁴³ Hence, other NO-related processes during IR probably explain the L-NAME effect on glutamate level after global IR.

The relationships between NO and reactive oxygen species should be considered in assessing the effects of L-NAME on glutamate concentration in IR. Forebrain ischemia increases reactive oxygen species production and lipid peroxidation,⁴⁴ although the sources of these reactive oxygen species are still uncertain.^{22 44 45 46 47} The brain is susceptible to oxidative damage,⁴⁶ and protection from ischemia by antioxidants can be demonstrated after middle cerebral artery occlusion in rats.⁴⁸ NO reacts with superoxide rapidly in the aqueous phase to form peroxynitrite. Peroxynitrite is stable in alkaline solutions but decays rapidly once protonated to NO₂ and a hydroxyl radical–like species.⁶ In the present study, L-NAME protected the BBB from IR damage, which is consistent with a previous finding that NOS inhibition attenuated cold-induced brain edema⁴⁹ and is consistent with the hypothesis that peroxynitrite mediates vasogenic edema.

Inhibition of NOS, possibly beneficial in decreasing peroxynitrite-mediated damage,⁹ may also disturb the concentration balance between superoxide and NO, thereby increasing the flux of hydrogen peroxide. Hydrogen peroxide can enhance glutamate release from hippocampal slices⁵⁰ and decrease EAA reuptake in cell cultures.⁵¹ The reuptake process is a possible target, since astrocytes are under the influence of NO released by neurons.⁵² Hypothetically, the production of superoxide and/or hydrogen peroxide during reperfusion, enhanced by NOS inhibition, could block glutamate reuptake by changing the redox status of glutamate transporters. Furthermore, production of NO in the brain may be a defensive response to decrease EAA toxicity in global brain ischemia. This concept is suggested by data showing that NO decreases the affinity of glutamate for rat brain synaptic membranes⁵³ and that NO attenuates NMDA-mediated Ca²⁺ influx.²¹ This hypothesis also agrees with results showing that NOS inhibition aggravates hippocampal lesions induced by forebrain ischemia.^{18 19} The reversal of L-NAME–mediated glutamate accumulation by L-arginine supports the specificity of L-NAME for the NO pathway and agrees with this hypothesis. Remarkably, administration of L-arginine and L-NAME decreases glutamate concentration during ischemia. This result, although presently unexplained, coincides with a recent finding that L-arginine decreases the size of infarcts induced in focal brain ischemia.⁵⁴

In conclusion, inhibition of NOS does not prevent ischemia-induced glutamate accumulation but increases its level during reperfusion. This increase in glutamate concentration can be reversed when L-arginine is given with L-NAME before brain ischemia. More importantly, administration of both drugs reduces glutamate level during ischemia. Further studies are needed to investigate the mechanisms involved and to determine whether scavengers of oxygen-based radicals modify glutamate accumulation in vivo when L-NAME is given. Also, specific studies will be needed to investigate the potential endothelial protection offered by inhibition of NOS after brain IR.

	Acknowledgments

 
This work was supported by research funds from the Veteran's Administration and by National Institutes of Health grant HL-42444. The authors thank Craig Marshall for his excellent technical assistance.

Received May 19, 1994; revision received August 11, 1994; accepted November 9, 1994.

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