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(Stroke. 1996;27:1060-1065.)
© 1996 American Heart Association, Inc.

Articles

Neuroexcitatory Amino Acids and Their Relation to Infarct Size and Neurological Deficit in Ischemic Stroke

José Castillo, MD; Antoni Dávalos, MD; Javier Naveiro, MD Manuel Noya, MD

From the Department of Neurology, Hospital General de Galicia, Clínico Universitario, Santiago de Compostela; and the Section of Neurology, Hospital Doctor Josep Trueta, Girona (A.D.), Spain.

Correspondence to José Castillo, MD, Department of Neurology, Hospital General de Galicia, Santiago de Compostela, Spain.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Background and Purpose The participation of excitatory amino acids (EAAs) in the pathogenesis of ischemic neuronal lesion has been experimentally demonstrated, but clinical experience is scarce. Our objective was to examine EAA levels during the acute phase of cerebral infarction in relation to infarct size and intensity of neurological deficit.

Methods Using high-performance liquid chromatography, we determined the glutamate, aspartate, taurine, and glycine concentrations in the plasma and cerebrospinal fluid (CSF) of 128 patients with ischemic cerebral infarction confirmed by CT and 43 control subjects. Blood and CSF samples were obtained on admission within the first 24 hours from symptom onset. The severity of the neurological deficit was assessed with the Canadian Stroke Scale immediately after these tests and at 48 hours after inclusion in the study. Infarct volume was determined in a second CT performed between the 4th and 7th day after the patient's inclusion.

Results The concentration of plasmatic glutamate was 121.39±80.89 µmol/L in the control group and 163.71±103.13 µmol/L in the patient group (P=.015); in CSF it was 3.46±1.20 µmol/L in control subjects and 6.55±4.65 µmol/L in patients (P<.0001). The concentration of glycine in plasma was 158.02±32.15 µmol/L in control subjects and 189.37±74.04 µmol/L in patients (P=.007); in CSF it was 6.18±2.28 µmol/L in control subjects and 11.23±6.96 µmol/L in patients (P<.0001). The concentrations of glutamate in plasma and in CSF were significantly higher in patients with large cerebral infarcts and in those with cortical infarcts. Levels of glutamate and glycine in plasma and CSF were significantly higher in patients with a higher degree of neurological deficit.

Conclusions Our results support the excitotoxic activity of glutamate and glycine in patients with cerebral infarction.

Key Words: blood • cerebral infarction • excitatory amino acids • glutamates

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Experimental studies suggest the participation of EAAs in the pathogenesis of ischemic neuronal lesion.^{1 2 3 4 5 6 7 8 9} The accumulation of EAAs in extracellular spaces is due to an increase in their release as well as a decrease in their reuptake.⁷ Both alterations are secondary to cerebral ischemia, which is responsible for the energy failure of the ion exchange system and for the depolarization of the membrane.¹⁰ The neurotoxicity of glutamate and the other EAAs is the result of an excessive activation of the postsynaptic NMDA receptors, which start the massive inflow of ionic calcium, and of the AMPA receptors, which facilitate the incorporation of sodium into the cell.^{4 11} Both ions give rise to edema and neuronal necrosis. Another argument in favor of the glutamate excitotoxicity hypothesis is the efficacy of glutamate antagonists, which minimize the toxic effects of this amino acid and decrease infarct size in experimental models.^{11 12 13 14 15}

The considerable body of experience with animal models contrasts with an absence of clinical studies analyzing the neurotoxicity of glutamate during the acute phase of cerebral infarction; thus, the aim of our study was to examine EAA levels during the acute phase of cerebral infarction in relation to infarct size and intensity of neurological deficit.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
The study included 128 patients (76 men and 52 women; mean age, 68.01±10.79 years; age range, 38 to 89 years) selected from among 556 stroke patients admitted consecutively between October 1992 and September 1994. The study protocol was approved by the Ethics Committee of the Hospital General de Galicia. Inclusion criteria were the following: (1) hemispheric ischemic stroke; (2) first episode of stroke; (3) admission within 24 hours after the onset of symptoms (or of sleep when the patient woke with stroke); (4) persistence of neurological deficit on admission; (5) absence of treated psychiatric illness or serious disease; (6) no treatment with antiepileptics, sympathomimetic drugs, or calcium antagonists; and (7) informed consent. The following were causes for exclusion: an inclusion delay of more than 24 hours (178 subjects), ongoing treatment with drugs not permitted in the study (147 subjects), unavailability of CSF samples (124 subjects), neurological deficit recovered before inclusion (53 subjects), cancer or serious diseases (53 subjects), mass effect on CT (35 subjects), refusal to participate or transfer to another hospital (16 subjects), inclusion in a stroke trial (14 subjects), or death within the first 24 hours before the initial evaluation (7 subjects). In all patients, diagnosis was confirmed by CT. The final diagnosis yielded atherothrombotic infarction in 57 patients (44.5%), embolic infarction in 41 patients (32.0%), lacunar infarction in 16 patients (12.5%), and infarction of undetermined origin in 14 patients (10.9%). Based on the extension and topography of the infarct on CT, the patients were grouped as follows: total anterior circulation infarcts (10 patients), partial anterior circulation infarcts (73 patients), lacunar infarcts (16 patients), and hemispheric posterior circulation infarcts (18 patients)¹⁶ ; in 6 patients the infarct was not observed on the second CT. Of the patients with partial anterior circulation infarcts, 28 presented exclusively with a cortical infarct and 39 with a deep infarct.

The severity of neurological deficit was assessed with the CSS on admission and 48 hours after inclusion. Evaluation with the CSS was performed by the same physician in all cases. The CSS evaluates level of consciousness; aphasia; orientation; facial paresis; and power in arm, hand, and leg on a scale from 1.5 to 10.¹⁷ All the CT examinations were performed on a CT Systec 3000 plus (GEC) scanner with a 512x512-matrix display. A cerebral CT was performed on all patients on admission; a second CT was completed between the 4th and 7th day after the patient's inclusion in the study. In the second examination, we determined infarct volume using the appropriate software. Five patients died before the second CT, and 15 patients (11.7%) died during hospitalization.

A blood sample was taken on arrival at the hospital 8.7±5.8 hours (range, 1.5 to 23.0 hours) after the onset of symptoms. After informed consent was obtained and the cerebral CT was completed, a spinal tap was performed on all patients within the first 24 hours after the onset of symptoms. The interval between the times of the blood sample and the CSF sample was 1.2±0.7 hours. Samples of blood and CSF were obtained from 43 control subjects without neurological disorders subjected to epidural anesthesia (26 men and 17 women; mean age, 56.04±17.51 years; age range, 19 to 81 years).

Blood was extracted by cubital venous puncture and collected in glass tubes with EDTA-K3; it was centrifuged at 3000g for 15 minutes, and the supernatant was conserved at -80°C until it was processed. The CSF was centrifuged at 2000g for 10 minutes, and the supernatant was stored at -80°C. Glutamate, aspartate, taurine, and glycine were quantified in an LKB/4151 Alpha Plus autoanalyzer by the cation exchange chromatography method.^{18 19} A 500-µL sample of plasma or CSF was mixed with 250 µL of sulfosalicylic acid at 10% to deproteinize it. After centrifugation and filtration through a membrane with a pore diameter of 0.22 µm, the supernatant was aspirated and a 1.66-µmol/L solution of norleucine was added at a 5:1 ratio as an internal control. A 200-µL fraction of this solution was introduced into a cathode ion exchange column (Reference 4418520). Buffers with different pH (LKB-43101191, Lithium Chemical Kit) were pumped along the column, maintaining exact temperature control. The eluate from the column was mixed with ninhydrin, the mixture was passed through a photometer, and final concentration, expressed in micromoles per liter, was calculated with the use of a computerized system.

Results are expressed as mean±1 SD. Student's t test or ANOVA was used for comparison of two groups or more than two groups, respectively. Statistical significance among quantitative variables for the same population was determined with a linear regression analysis. To identify variables that might have had an influence on the CSS score, a logistic regression analysis was performed; CSS scores were divided into two categories (5 points=0; <5 points=1). Independent variables were used as continuous variables. Values of P<.05 were considered statistically significant.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Table 1 shows the EAA concentrations. Glutamate and glycine in plasma, and particularly in CSF, reached higher levels in patients than in control subjects. Taurine level in CSF and aspartate level in plasma were significantly higher in the control group. The correlation between the levels of EAAs in plasma and in CSF was statistically significant for glutamate (Pearson coefficient=.727, P<.0001), taurine (Pearson coefficient=.274, P=.000), and glycine (Pearson coefficient=.489, P<.0001).

View this table: [in this window] [in a new window]   Table 1. Excitatory Amino Acid Levels on Admission in Control Subjects and Patients

The correlation between EAA levels and infarct volume showed a moderate statistical significance. Levels of glutamate in plasma and in CSF were higher in patients with larger infarcts. In contrast, levels of taurine in plasma and in CSF were more elevated in patients with smaller infarcts (Figs 1 and 2).

View larger version (23K): [in this window] [in a new window]   Figure 1. Correlations between EAA levels in plasma and cerebral infarct size. Coef indicates coefficient.

View larger version (22K): [in this window] [in a new window]   Figure 2. Correlations between EAA levels in CSF and cerebral infarct size. Coef indicates coefficient.

Patients with total anterior circulation infarcts showed higher concentrations of glutamate in CSF. Taurine levels in plasma and in CSF were significantly lower in patients with the most extensive infarcts (Table 2). In patients with partial anterior circulation infarcts, the concentration of glutamate was higher in those with a cortical rather than deep localization, independently of infarct volume (plasmatic glutamate, 194.8±116.9 versus 126.4±94.1 µmol/L, P=.010; CSF glutamate, 8.2±5.3 versus 5.0±4.5 µmol/L, P=.0001).

View this table: [in this window] [in a new window]   Table 2. EAAs, Size, and Topography of Cerebral Infarcts

EAA concentrations in plasma and CSF were similar in the 43 patients with a CSS score on admission of <5 points and in the 85 patients with a score 5 points. At 48 hours, the CSS scores were <5 points in 51 patients and 5 points in 77 patients. The most severely affected patients showed significantly higher plasma levels of glutamate (216.3±126.4 versus 128.4±64.9 µmol/L, P<.0001) and glycine (218.2±74.1 versus 170.8±68.2 µmol/L, P<.0001). Aspartate (11.5±2.4 versus 13.5±3.6 µmol/L, P<.0001) and taurine levels in plasma (135.1±51.7 versus 173.9±66.1 µmol/L, P<.0001) were higher in those with a lower degree of neurological deficit. In CSF, the most severely affected patients showed higher levels of glutamate (9.1±5.6 versus 5.1±3.2 µmol/L, P<.0001) and glycine (13.3±4.7 versus 10.1±7.8 µmol/L, P=.012) and lower levels of taurine (4.5±2.4 versus 6.6±2.8 µmol/L, P<.0001); the aspartate concentrations were similar in the two groups (3.1±0.8 versus 3.2±1.2 µmol/L, P=.489).

Multiple logistic regression analysis with the CSS score at 48 hours as dependent variable demonstrated that elevated glutamate levels in plasma and glycine in CSF were associated with a high severity of neurological deficit (Table 3). Increased levels of aspartate in plasma and aspartate and taurine in CSF were associated with a better neurological condition at 48 hours.

View this table: [in this window] [in a new window]   Table 3. Logistic Regression Analysis, With CSS Score at 48 Hours as Dependent Variable (0, 5 Points; 1, <5 Points)

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
The idea of the neurotoxicity of certain amino acids was introduced by Olney and Sharpe.²⁰ Subsequently, Meldrum²¹ and Rothman and Olney⁴ postulated that this mechanism was involved in the pathogenesis of ischemic cellular lesion.

Glutamate is the most abundant amino acid in the central nervous system and, like the other excitatory amino acids, is an important excitatory neurotransmitter.²² Glutamate is stored in the presynaptic vesicles and released in response to a depolarization of the presynaptic neuronal membrane.¹⁰ It carries out its activity by stimulating receptors located on the postsynaptic membranes of virtually all the neurons of the central nervous system.²³

The excitotoxic hypothesis of ischemic cerebral injury postulates that the decline in cerebral perfusion to below critical values gives rise to an accumulation of glutamate and other EAAs within and around the ischemic zone. In animal models of focal cerebral ischemia, an elevation of the levels of glutamate,^{1 2 6 8 9 24 25} aspartate,^{2 8 9 24} taurine,^{24 26} and glycine^{8 9 24} has been demonstrated. According to this hypothesis, these EAAs increase the stimulation of NMDA receptors, previously deblocked by depolarization of the membrane; sodium and calcium enter into the cell, initiating a cascade of biochemical changes such as mitochondrial lesions, proteolysis of microfilaments, breakage of membrane phospholipids, formation of free radicals, and cell death.^{4 27 28}

At the core of ischemic injury, glutamate is released at very high concentrations, approximately 80 times the baseline level.²⁹ The neurotoxicity of the EAAs is irrelevant in this central area of serious cell injury¹¹ ; however, in the peripheral penumbral tissues, although the concentration reached by the EAAs is lower,⁸ their neurotoxic effects are of greater importance.¹¹ Glutamate release stimulates a depolarization in the periphery of cerebral ischemia, similar to a propagated depression, which is responsible for the increase in cerebral injury.^{8 30 31}

Clinical experience with EAAs in cerebral ischemia is scant. Using microdialysis techniques, Bullock et al³² found elevated concentrations of glutamate and aspartate in a patient with a massive cerebral infarct, and Kanthan et al³³ showed elevated glutamate concentrations immediately after ischemia in five patients subjected to surgical resections for untreatable epilepsy, concentrations that reached levels 100 times greater than basal measurements. Persson and Hillered³⁴ showed extracellular elevations in glutamate, aspartate, and taurine levels in patients with subarachnoid hemorrhage and vasospasm, and Kashiwagi et al³⁵ obtained similar results in the CSF of patients with subarachnoid hemorrhage.

We have demonstrated an elevation of glutamate and glycine levels during the acute phase of cerebral infarction. Differences from the control values were more significant when the amino acids were determined in CSF. The good correlation between EAA levels in CSF and in plasma suggests a good diffusion through the blood-brain barrier. Thus, glutamate determination in plasma may be sufficient for evaluating its changes during cerebral ischemia. Under normal circumstances, glutamate does not cross the blood-brain barrier³⁶ ; thus, its increase in plasma depends on alterations in the blood-brain barrier during cerebral ischemia. However, the percentage of CSF glutamate that crosses the blood-brain barrier is difficult to determine, given that unlike other neurotransmitters, this amino acid has an important additional role as a metabolic intermediary in the Krebs cycle and in -aminobutyric acid synthesis. Our results are in agreement with those obtained in experimental pathology.^{8 9 24 26 37 38} Glutamate is considered to be the main agent responsible for neurotoxicity in cerebral ischemia^{9 11} ; glycine potentiates the inflow of glutamate-dependent calcium into the cells.^{4 8 39}

The concentrations of aspartate in CSF and of taurine in plasma were similar in patients and control subjects. Aspartate levels in plasma and taurine levels in CSF were significantly lower in the patient group. Although the excitotoxicity mechanisms of aspartate and taurine are not well known, the concentrations of both amino acids have been shown to be high in animal models of focal cerebral ischemia^{24 26 40 41} and during the vasospasm that accompanies subarachnoid hemorrhage.^{34 35} Another hypothesis suggests that taurine may act more as a neuromodulator than as a neuroexciter in the central nervous system.⁴²

The clearance of the EAA during cerebral ischemia may explain the differences between clinical and experimental findings. The increase in EAAs is detectable in the extracellular space soon after experimental cerebral ischemia^{8 9 25 41} and in human models of ischemia simulated during surgery,³³ but it is very transitory for aspartate.^{9 41} Nevertheless, glutamate levels remain high for a longer time,^{9 25 32 41} and glycine levels progressively increase during ischemia.⁹ Conditions of chronic excitotoxicity, as described in permanent experimental focal ischemia⁴³ and in many patients with traumatic brain injury probably due to prolonged and fluctuating ischemia,⁴⁴ may explain the persistence in the elevated levels of glutamate and glycine but not of the other EAAs. Indirect evidence for the persistence of glutamate excitotoxicity is supported by the late effectiveness that some NMDA receptor inhibitors have demonstrated in the treatment of experimental cerebral ischemia.^{44 45}

Although in this study only a slight correlation was demonstrated between infarct volume and the levels of glutamate in plasma and glutamate and glycine in CSF, experimental studies have shown a clear correlation.^{8 46 47} The concentration of glutamate in plasma and in CSF was lower in patients with infarcts corresponding to the posterior region, but this is not surprising given that these were smaller infarcts. Nevertheless, in infarcts in the anterior region, the concentrations of glutamate in plasma and in CSF were significantly higher in infarcts with a cortical topography than in those with a deep topography, even though their volumes were similar. The greater elevation of glutamate levels in cortical infarcts coincides with experimental findings that demonstrate a higher content of glutamate in certain regions of the brain, such as the cerebral cortex and the hippocampus,^{10 48} and an increased release of glutamate during focal ischemia induced in the same areas.^{8 9 41} In addition, the concentrations of glutamate and glycine in plasma and CSF were significantly higher in patients with a greater extent of neurological damage, as assessed by the CSS 48 hours after admission, possibly as a result of the greater volume of these infarcts.

Although the difficulty of extrapolating the findings obtained in animal experimentation to the human clinic is considerable,⁴⁹ our results can be globally superimposed on those demonstrated in models of focal cerebral ischemia, and they support the excitotoxic activity of glutamate and glycine in patients with cerebral infarction.

	Selected Abbreviations and Acronyms

 

AMPA = -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid CSF = cerebrospinal fluid CSS = Canadian Stroke Scale EAA(s) = excitatory amino acid(s) NMDA = N-methyl-D-aspartate

	Acknowledgments

 
This study was supported by a grant from Xunta de Galicia (investigation project XUGA 20802B93).

Received December 21, 1995; revision received February 23, 1996; accepted February 26, 1996.

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