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(Stroke. 1999;30:2448-2455.)
© 1999 American Heart Association, Inc.

Original Contributions

Striatal Outflow of Adenosine, Excitatory Amino Acids, -Aminobutyric Acid, and Taurine in Awake Freely Moving Rats After Middle Cerebral Artery Occlusion

Correlations With Neurological Deficit and Histopathological Damage

Alessia Melani, BSci; Leonardo Pantoni, MD, PhD; Claudia Corsi, PhD; Loria Bianchi, PhD; Angela Monopoli, PhD; Rosalia Bertorelli, PhD; Giancarlo Pepeu, MD Felicita Pedata, PhD

From the Departments of Preclinical and Clinical Pharmacology (A. Melani, C.C., L.B., G.P., F.P.) and Neurological and Psychiatric Sciences (L.P.), University of Florence, and Schering-Plough Research Institute, Milan (A. Monopoli, R.B.), Italy.

Correspondence to Felicita Pedata, Department of Preclinical and Clinical Pharmacology, University of Florence, V.le Pieraccini 6, 50139 Florence, Italy. E-mail pedata{at}server1.pharm.unifi.it

	Abstract

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Background and Purpose—While a number of studies have investigated transmitter outflow in anesthetized animals after middle cerebral artery occlusion (MCAO) performed by craniectomy, studies have never been performed after MCAO induced by intraluminal filament. In addition, it has been reported that after MCAO, infarct volume correlates with functional outcome and with transmitter outflow, although there are no studies that demonstrate a direct correlation between transmitter outflow and functional outcome. The purpose of the present study was to assess excitatory amino acids, -aminobutyric acid, taurine, and adenosine outflow in awake rats after intraluminal MCAO and to determine whether, in the same animal, outflow was correlated with neurological outcome and histological damage.

Methods—Vertical microdialysis probes were placed in the striatum of male Wistar rats. After 24 hours, permanent MCAO was induced by the intraluminal suture technique. The transmitter concentrations in the dialysate were determined by high-performance liquid chromatography. Twenty-four hours after MCAO, neurological deficit and histological outcome were evaluated.

Results—All transmitters significantly increased after MCAO. Twenty-four hours after MCAO, the rats showed a severe sensorimotor deficit and massive ischemic damage in the striatum and in the cortex (9±2% and 25±6% of hemispheric volume, respectively). Significant correlations were found between the efflux of all transmitters, neurological score, and striatal infarct volume.

Conclusions—In this study, for the first time, amino acid and adenosine extracellular concentrations during MCAO by the intraluminal suture technique were determined in awake and freely moving rats, and a significant correlation was found between transmitter outflow and neurological deficit. The evaluation of neurological deficit, histological damage, and transmitter outflow in the same animal may represent a useful approach for studying neuroprotective properties of new drugs/agents against focal ischemia.

Key Words: adenosine • amino acids • cerebral ischemia, focal • middle cerebral artery occlusion • rats

	Introduction

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Several animal models have been used to study cerebral ischemia¹ in an effort to understand its pathophysiology and to identify therapeutic strategies for minimizing the severity of ischemic damage. Whereas global ischemia usually results from neuronal injury within vulnerable brain regions, focal ischemia brings about a localized brain infarction. Focal ischemia induced by middle cerebral artery occlusion (MCAO) in the rat² has gained increasing acceptance as a model for hemispheric infarction in humans.¹ After permanent occlusion, a cortical and striatal infarct with temporal and spatial evolution occurs³ within the vascular territory supplied by the middle cerebral artery (MCA).

The importance of studying the pattern of excitatory amino acid efflux in different models of cerebral ischemia derives from the finding that a massive release of glutamate is considered to play a major role in inducing ischemic and postischemic cell death.⁴ In fact, antagonists of glutamate receptors reduce the ischemic damage occurring in the ischemic penumbra,^{5 6} and inhibitors of glutamate release exhibit cerebroprotective activity against ischemia/reperfusion-evoked injury.⁷

Adenosine, whose tissue level increases dramatically in ischemia as a consequence of energy metabolism failure, exerts a critical role in ischemic brain damage⁸ through still undefined mechanisms. Both preischemic and postischemic acute administration of A₁ adenosine agonists reduce postischemic neuronal loss⁹ and spatial memory loss^{10 11} and facilitate postischemic neurological recovery.¹² Both A_2A agonists and antagonists have shown neuroprotective properties in the hippocampus and cortex within different models of ischemia. While it was suggested that peripheral effects account for the protective effect of A_2A agonists, a decrease in excitatory amino acid outflow is considered a possible mechanism through which A_2A receptor antagonists exert their neuroprotective effects.¹³ Furthermore, preischemic administration of A₃ adenosine agonist was shown to impair postischemic blood flow and to enhance mortality and neuronal damage, while an A₃ antagonist was shown to reverse the effects of the agonist in a global ischemia model.^{14 15}

In this study, using the brain microdialysis technique, we investigated the efflux of excitatory amino acids, -aminobutyric acid (GABA), taurine, and adenosine during permanent focal ischemia induced by the intraluminal MCAO method developed by Zea Longa et al.¹⁶ Although several researchers^{17 18 19 20 21 22} have investigated the release of amino acids and purines on anesthetized animals after inducing MCAO through a craniectomy, this has not yet been done after MCAO induced by the intraluminal suture technique,¹⁶ a much less invasive method. In this study transmitter level estimations were performed in awake and freely moving animals. The study was conducted in the striatum, which represents the "ischemic core," with minimal postocclusion blood flow because this area is supplied exclusively by the lenticulostriate end arteries.²

In this study we established a correlation between neurotransmitter efflux and functional outcome, measured as sensorimotor deficit, and between neurotransmitter efflux and histological damage in the same animals subjected to focal cerebral ischemia.

	Materials and Methods

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Animal Housing and Surgery
Male Wistar rats (Nossan, Italy) weighing 270 to 290 g were used. They were housed in groups of 3 with free access to food and water and kept on a 12-hour light/dark cycle. The guidelines of the European Community for animal experiments were followed. All efforts were made to minimize animal suffering and to reduce the number of animals used.

Rats were anesthetized with chloral hydrate (400 mg/kg IP) and placed in a stereotaxic frame (Kopf). Vertical microdialysis probes were implanted bilaterally into the rat striatum. The microdialysis membranes (AN 69 membrane, Dasco; 220-µm ID and 310-µm OD; molecular weight cutoff >15 000 Da) were 3 mm long. The coordinates used for implantation were 0.7 mm anterior and 3.2 mm lateral to the bregma and 7 mm ventral from the brain surface.²³ The external portion of the probes was fixed to the skull with anchor screws and dental cement. An antibiotic (penicillin G benzathine [Farmitalia], 1 200 000 IU in 8 mL of physiological solution IM) was given to each rat at the end of the operation, after which the rats were housed in plastic cages for complete recovery.

Twenty-four hours later, focal cerebral ischemia was induced by permanent MCAO in the right hemisphere.¹⁶ The animals were anesthetized with 5.0% halothane and spontaneously inhaled 1.0% to 2.0% halothane in air by use of a face mask. They were placed in a supine position on a raised plane to permit the collection of dialysis samples during surgery. Body core temperature was maintained constant at 37°C with a recirculating pad and K module and was monitored via an intrarectal type T thermocouple (Harvard). The surgical procedure to occlude the MCA consisted of insertion of a 4-0 nylon monofilament (Johnson & Johnson) via the external carotid artery into the internal carotid artery to block the origin of the MCA.¹⁶ The sham operation was conducted by inserting the filament into the internal carotid artery and immediately withdrawing it.

Before, during, and after MCAO, dialysis probes (right and left) were perfused continuously with Ringer's solution (147 mmol/L NaCl, 2.3 mmol/L CaCl₂, 4.0 mmol/L KCl; pH 7.0) at a constant flow rate of 3 µL/min by means of a microperfusion pump (CMA/100 microinjection pump, Carnegie Medicine), and dialysis samples were collected every 20 minutes. After a 30-minute stabilization period, dialysis samples were collected for 2 hours before MCAO and until 4 hours after MCAO. MCAO occurred from -20 to 0 minutes (see time on the abscissa of the Figure). At the end of the surgical procedure to occlude the MCA, the neck wound was closed. Anesthesia was discontinued, and the animals were returned to a prone position. Recovery from anesthesia took approximately 15 minutes, after which the animals were allowed free access to food and water. Dialysate samples were collected continuously during surgery and recovery. Time 0 shown in the Figure corresponds to the first dialysate sample after insertion of the filament into the right internal carotid artery. Samples of dialysate were collected from the MCAO right hemisphere and from the contralateral nonischemic left hemisphere.

View larger version (24K): [in this window] [in a new window]   Figure 1. Adenosine, GABA, taurine, glutamate, and aspartate outflow from the rat striatum during permanent MCAO. Data are expressed as percent variation of the spontaneous outflow, which in each rat group is the mean of the first 7 samples collected between -120 and 0 minutes. Each point is the mean±SEM of n rats. •, MCAO hemisphere (n=16); , contralateral hemisphere (n=10); , sham-operated rats (n=4).

Each sample (60 µL) was divided into 2 aliquots (30 µL each) for assaying adenosine and amino acid content in 2 different high-performance liquid chromatography (HPLC) systems.

Adenosine Assay
Adenosine content in the samples was analyzed by HPLC coupled to a spectrofluorometric detector (LC-240, Perkin-Elmer) with a fixed excitation wavelength set at 270 nm and fixed emission wavelength set at 394 nm.²⁴ A Nucleosil C18 column (ID, 4.6 mm; length, 150 mm; Waters) with particle size of 3.5 µm was used. To protect the system from clogging with particulate matter, a Waters In-Line filter with 2-µm pore size was incorporated into the HPLC system upstream from the stationary phase column. The mobile phase was 50 mmol/L acetate buffer (pH 5) with 5% acetonitrile (vol/vol) and 1 mmol/L 1-octanesulfonic acid sodium salt (Eastman Kodak Co), which was pumped at a flow rate of 0.8 mL/min.

Adenosine was detected as a fluorescent derivative (1,N⁶-ethenoadenosine) after derivatization with chloroacetaldehyde. Four microliters of zinc acetate (0.01 mmol/L) was added to 30 µL of each sample. The solution was transferred into glass vials, where 0.18 µL of chloroacetaldehyde (4.5%) was added for each microliter of solution obtained. This solution was kept at 100°C for 20 minutes

The adenosine peaks were identified and quantified by comparing retention time and peak heights with those of known standards run according to the sample procedure. Adenosine was identified by its disappearance after incubation of the sample with 1 U of adenosine deaminase at room temperature for 1 minute. The minimum detectable amount of adenosine was 0.1 pmol.

Amino Acid Assay
Excitatory amino acid, GABA, and taurine analysis was performed with HPLC after derivatization of the amino acids. The equipment (Perkin-Elmer) consisted of a gradient pump series 3B and 650-10S fluorometric detector fitted with a 5-µL flow cell. The excitation wavelength was 340 nm, and the emission wavelength was 455 nm. A Shimadzu C-R4A Chromatopac integrator was connected to the detector. A Nucleosil C18 column (200x4 mm ID), with particle size of 5 µm, was used. The mobile phase consisted of methanol-potassium acetate (0.1 mol/L) brought to pH 5.5 by glacial acetic acid and was run at 1.2 mL/min in 3 linear steps from 25% to 90% methanol (8.5 minutes) followed by an isocratic hold for an additional 3 minutes. Amino acids were derivatized with o-phthalaldehyde as described by Lenda and Sveneby,²⁵ except that the o-phthalaldehyde concentration was 0.3 mg/mL and that 50 mmol/L HCl was used to dissolve the sample in preparation for the chromatography run. The o-phthalaldehyde concentration was increased in comparison to Lenda and Sveneby²⁵ to ensure complete derivatization of the other amino acids. Homoserine was added as an internal standard. One volume (10 µL) of dialysate was mixed with 2 volumes of the derivatization reagent solution in glass capillaries. The contents were mixed and injected after 60 seconds. Standard curves showed a linear relationship between the amount of fluorescence and the quantity of amino acids applied to the column in the range of 0.1 to 100 pmol. Under these experimental conditions, the sensitivity limit (signal-to-noise ratio >3) was 32 fmol.

The data are expressed in absolute values (µmol/L) or as percent changes of the basal outflow, which is the mean of the first 7 samples in MCAO and sham-operated rat groups. The mean of transmitter outflow during the ischemic period was calculated as the mean of the percent changes from 0 to 240 minutes and is indicated as mean percent outflow.

In Vitro Recovery Experiments
To evaluate the recovery of adenosine and amino acids through the dialysis membrane, in vitro experiments were performed. Dialysis probes were immersed in Ringer's solution, at room temperature, containing known concentrations of adenosine and amino acids. The probes were perfused with Ringer's solution at 3 µL/min, and samples were collected every 20 minutes. The recovery rates were as follows: 6.2±0.8% for adenosine, 7.7±2.5% for glutamate, 23.6±8.4% for aspartate, 14.6±5.7% for GABA, and 5.9±2.2% for taurine.

The concentration values reported in this article were not corrected for recovery.

Neurological Test
Neurological evaluation of motor sensory functions was performed before probe implantation and 24 hours after MCAO. The examiners did not have knowledge of the procedure that the rat had undergone. Furthermore, 2 observers consecutively and independently performed the neurological examination of each rat. Adherence to a predetermined time excluded behavioral changes based on circadian rhythm. The neurological examination consisted of all 6 tests as developed and described by Garcia et al.²⁶ The feasibility and reliability of the test have been described by Pantoni et al.²⁷ The score assigned to each rat at the completion of the evaluation equals the sum of all 6 test scores: (1) spontaneous activity; (2) symmetry in the movement of 4 limbs; (3) forepaw outstretching; (4) climbing; (5) body proprioception; and (6) response to vibrissae touch. Of the 6 tests, 3 have a minimum score of 1 and 3 have a minimum score of 0, and all 6 tests have a maximum score of 3. Thus, the final minimum score is 3 and the maximum is 18.

Histological Analysis
Twenty-four hours after MCAO, the rats were anesthetized with chloral hydrate (administered intraperitoneally) and decapitated. The brains were rapidly removed and fixed with Liquid of Carnoy (6:3:1 absolute ethanol, chloroform, and glacial acetic acid) and then embedded in paraffin after dehydration in different concentrations of ethanol and xylene. Each brain was cut into 7-µm-thick sections and stained with acetate cresyl violet (1%).

The lesioned area of the brain (evaluated as pallid area) was measured with the use of an image analysis system (Image ProPlus, Image & Computer, Rho). We calculated striatal and cortical damage in cubic millimeters, expressed as percentage of the volume of the ipsilateral hemisphere.

Statistical Analysis
Statistically significant differences in neurotransmitter release among MCAO hemisphere, contralateral nonischemic hemisphere, and sham-operated rats were evaluated by 1-way ANOVA followed by the post hoc Fisher's test. Differences between the neurological scores 24 hours before and 24 hours after occlusion were evaluated by paired Student's t test. Correlations between neurotransmitter release and neurological deficit and ischemic brain damage were calculated by linear regression analysis.

Differences were considered statistically significant at P<0.05.

	Results

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Animal Survival Rate
No rats were lost after microdialysis surgery, but the mortality 24 hours after MCAO was 33%. All sham-operated rats were alive 24 hours after operation.

Transmitter Outflow After Permanent MCAO
The extracellular concentrations of adenosine, glutamate, aspartate, GABA, and taurine in the rat striatum before occlusion are reported in Table 1.

View this table: [in this window] [in a new window]   Table 1. Basal Extracellular Concentration of Adenosine, Glutamate, Aspartate, GABA, and Taurine in Rat Striatum

The Figure shows the time course, during a 6-hour collection period, of glutamate, aspartate, adenosine, GABA, and taurine outflow in the MCAO hemisphere, the contralateral nonischemic hemisphere, and sham-operated rats. In the contralateral hemisphere and sham-operated rats, the extracellular levels of all transmitters remained stable over the 6-hour collection period. MCAO (time 0) led to an increase in all transmitters. Adenosine and taurine levels appeared to increase more promptly than glutamate, aspartate, and GABA. All substances reached a maximum increase (evaluated as peak value over preischemic basal levels) between 80 and 160 minutes after MCAO. The maximum increase was 40-fold for GABA, 15-fold for adenosine, 7-fold for taurine, 6-fold for glutamate, and 2-fold for aspartate. Between 2 and 3 hours after occlusion, adenosine, glutamate, and aspartate levels began to decrease but remained at a sustained level until 240 minutes, while GABA and taurine levels remained high until the end of the experiment.

Table 2 shows the mean percent outflow of transmitters between 0 and 240 minutes in the MCAO hemisphere, contralateral hemisphere, and sham-operated rats. The differences among the levels in MCAO hemisphere and contralateral hemisphere and sham-operated rats were statistically significant for all transmitters. No statistically significant differences were found between contralateral hemisphere and sham-operated rats.

View this table: [in this window] [in a new window]   Table 2. Effect of MCAO on Transmitter Outflow

Ischemic Brain Damage
Histological assessment of ischemic damage was performed 24 hours after MCAO in 8 MCAO rats and 4 sham-operated rats. Damage was found only in the vascular territories supplied by the MCA, striatum, and cortex.^{3 26} Of 8 animals, 2 did not suffer damage, 1 had a little striatal damage, and 5 had definite cortical and striatal damage. In the 6 rats that underwent damage, the volume of the damage was 20.8±5.0 mm³ in the striatum and 58.1±16.3 mm³ in the cortex. The mean percentage of ischemic damage, calculated in relation to the volume of the ipsilateral hemisphere, was 9.1±1.7% in the striatum and 25.2±5.9% in the cortex. When one considers that the striatum volume is approximately one third as large as the cortex volume,²⁸ the extent of damage in the striatum was similar to that in the cortex. The volume of the ipsilateral hemisphere (mean±SEM, 218.9±11.5 mm³) was 11% higher than the volume of the contralateral hemisphere (mean±SEM, 196.2±7.8 mm³). The difference was statistically significant (P<0.03, paired Student's t test). The increase in volume of the ipsilateral side was presumably due to edema after MCAO. No ischemic damage was found in the contralateral hemisphere and in the sham-operated rats.

A statistically significant correlation between the striatal damage and the striatal efflux of all transmitters was found (Table 3).

View this table: [in this window] [in a new window]   Table 3. Correlations (r2) Between Transmitter Efflux and Neurological Deficit and Histopathological Outcome 24 Hours After MCAO

Neurological Evaluation
The neurological score was evaluated in sham-operated and in MCAO rat groups before probe implantation and 24 hours after occlusion. Microdialysis surgery for implantation of the bilateral probes did not modify the motor sensory functions as evaluated before sample collection was started.

In the sham-operated rats (n=4), the neurological score (mean±SEM, 18±0) did not change after the operation. In the MCAO group (n=16), the neurological score (mean±SEM, 10±1) was reduced by 44% (P<10^-7, paired Student's t test) 24 hours after MCAO. The neurological score (mean±SEM, 11.0±0.6) determined in 5 rats 48 hours after MCAO was not significantly different from that obtained after 24 hours.

A statistically significant correlation between the neurological score and the striatal efflux of all transmitters was found (Table 3), as well as between the neurological score and the striatal damage (r²=0.45; n=12; P<0.016).

	Discussion

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Advantages of the Intraluminal Approach of MCAO in Combination With Striatal Microdialysis
The intraluminal approach of MCAO used in this study does not require a craniectomy with the associated operative trauma and, in combination with brain microdialysis, allows the determination of transmitter extracellular concentrations in awake and freely moving animals 24 hours after microdialysis probe implantation, when cerebral metabolism and cerebral blood flow are normalized.²⁹ This is an advantage over studies in which amino acid and purine outflows are measured after MCAO on the same day as probe implantation and on anesthetized animals, since it has been shown that adenosine extracellular concentration is 20 times lower 24 hours after probe implantation than it is 2 hours after.³⁰ Moreover, in the present study the extracellular concentrations of glutamate and aspartate are 3-fold lower and those of GABA and taurine 10-fold lower than they are 2 hours after probe implantation.³¹

The increase in amino acid transmitters and adenosine found in our experiments confirms the increase observed by several authors in both the cortex^{17 19 20 21 32} and striatum^{17 18 22} of anesthetized animals after MCAO. In agreement with them, we found that GABA shows the largest outflow increase of the amino acids. However, in our experiments, using the same ventromedial placement of the microdialysis probe in the striatum, the changes in excitatory neurotransmitters and GABA outflow are smaller than those occurring after permanent MCAO performed by craniectomy and proximal to the lenticulostriate end arteries^{17 18} ; this procedure, besides involving the cortex, largely involves the striatum, as in our experiments.

These observations indicate that one advantage of the procedure used in the present experiments is that the effects of ischemia are not compounded by those of microdialysis probe implantation. Furthermore, the intraluminal suture technique allows the MCA to be blocked at its origin and thus provokes definite striatal damage²⁶ and ensuing sensorimotor deficit. Thus, this technique allows assessment of the relationship of transmitter outflow to sensorimotor deficit and to histological damage.

Neurotransmitter Changes After MCAO
Our results show that MCAO is followed by an increase in all assayed transmitters from the occluded but not from the contralateral hemisphere. In fact, after MCAO, cerebral blood flow is only slightly affected³³ or not affected³⁴ in the striatum and cortex of the hemisphere contralateral to that occluded by the intraluminal filament.

In agreement with previous observations in the cortex,^{19 20} our results show that the increase in adenosine efflux soon after MCAO has a slightly higher intensity than that of glutamate and aspartate. This might be explained by the observation¹⁹ that the blood flow threshold for the increase in purine release is approximately 25 mL/100 g per minute, while that for glutamate elevation is approximately 20 mL/100 g per minute.

Adenosine reaches its peak between 60 and 120 minutes after MCAO, estimated as a maximal adenosine concentration of 0.2 µmol/L. When this value is corrected for microdialysis membrane recovery, it is estimated as 3.1 µmol/L. Adenosine concentrations of 24 to 40 µmol/L (values corrected for membrane recovery) have been monitored by microdialysis after induction of global ischemia in gerbil and rat.^{35 36} However, these values were reached on the first day after microdialysis tube implantation, when the estimated adenosine extracellular concentration at rest is significantly higher than that found in experiments conducted 24 hours after surgery,³⁰ as discussed in the previous paragraph. The adenosine concentration estimated in the present experiments under normoxic conditions (13 nmol/L; value corrected for recovery, 210 nmol/L) is sufficient to stimulate both A₁ and A_2A receptors.³⁷ At the 3.1-µmol/L concentration, adenosine may also stimulate A₃ adenosine receptors. Estimation of adenosine concentration appears to be important when one considers that stimulation of A₁, A_2A, and A₃ adenosine receptors may modify the outcome of the ischemic episode.^{13 38} Between 2 and 3 hours after permanent MCAO, following peak efflux, adenosine levels tend to decrease but are still significantly elevated 240 minutes after MCAO. A decrease in cortical¹⁹ and striatal¹⁸ adenosine levels after permanent MCAO has already been reported. After transient global ischemia,³⁹ glutamate and aspartate levels rapidly normalize on reperfusion. In our model of permanent MCAO, glutamate and aspartate, despite a tendency to decrease, exhibit a sustained efflux until the end of the experiment.

A reciprocal relationship between adenosine and excitatory amino acids has been demonstrated in the striatum of young rats in vivo: excitatory amino acids stimulate the outflow of adenosine,^{30 40} and adenosine, through A₁ receptors, reduces the outflow of excitatory amino acids under normoxic³¹ and ischemic conditions.^{41 42} Such regulation may be useful to control neuronal excitability and/or neurotoxicity. On the other hand, a stimulatory role of A_2A receptors has been shown on the excitatory amino acid outflow measured in vivo from the ischemic cortex⁴³ and from the normoxic striatum.^{44 45} Thus, estimation of the time course of adenosine and excitatory amino acid efflux during ischemia might help in understanding the mechanism of action of purinergic or aminoacidergic drugs endowed with neuroprotective activity against ischemia.

The decrease in adenosine levels to near preischemic values, despite ongoing ischemia, may be due to degradation of adenosine by adenosine deaminase.^{18 19} The activity of this enzyme is maximal when adenosine concentrations reach a high value.⁴⁶ The decrease might also be attributed to reconversion of adenosine to AMP, ADP, and ATP by adenosine kinase. This metabolic step is very efficacious in regulating adenosine concentrations^{40 47} and may be explained by a gradual, limited recovery in blood flow.^{21 48}

The partial recovery in blood flow^{21 48} and ATP synthesis 3 hours after MCAO could partly restore the excitatory amino acid uptake mechanism in the penumbral portion of the ischemic area. A different factor may be an increase in glutamate metabolism by astrocytes, observed after ischemia in vivo.⁴⁹

Unlike the excitatory amino acids, the levels of the inhibitory amino acids GABA and taurine remained high after permanent MCAO, in agreement with the results of Shirotani et al,²² and are still high 4 hours after MCAO. Two mechanisms may be invoked to explain the long-lasting increase in GABA levels. First, the uptake mechanisms of glutamate and GABA are located in different cells: glutamate is removed from the extracellular space mostly by glial uptake,⁵⁰ whereas GABA is removed primarily by neuronal uptake.⁵¹ Thus, according to our results, in the first hours after MCAO, the neuronal uptake systems appear to be more severely compromised than the glial uptake systems, although necrosis of both neurons and astrocytes has been observed within 48 to 72 hours after ischemia.^{52 53} Second, an activation of glutamic acid decarboxylase, shown during ischemia/reperfusion, leading to rapid conversion of cellular glutamate into GABA,⁵⁴ may increase the level of the latter neurotransmitter at the expense of the first. No satisfactory explanation can be offered for the persistent increase in taurine levels. In view of intense and persistent GABA outflow after MCAO, it should be noted that, although GABA is the principal mediator of synaptic inhibition, GABA_A receptors, when intensively activated, excite rather than inhibit neurons.⁵⁵ On the other hand, evidence of adenosine regulation of GABA release is controversial. Adenosine, through A₁ receptors, does not modify GABA outflow from either normoxic or ischemic striatum in vivo,^{31 42} but it depresses the ischemia-evoked GABA outflow from the cerebral cortex.⁵⁶ Through A_2A receptors, adenosine inhibits the K⁺-evoked GABA output from striatal terminals^{57 58} and increases electrically evoked GABA outflow from striatal micropunches⁵⁹ or pallidum in vivo.⁶⁰

Relationships Between Transmitter Outflow and Neuropathological Damage
In our experiments, we report a significant correlation between the levels of glutamate, aspartate, GABA, taurine, and adenosine in the dialysate, measured for 4 hours after occlusion, and the neurological deficit, evaluated 24 hours after occlusion. Thus, it can be assumed that the increase in striatal transmitter outflow is predictive of motor impairment. A significant correlation also exists between the outflow of all transmitters and the extent of ischemic damage, calculated 24 hours after occlusion. Histologically, ischemic damage is defined by an area of pallor which, according to Garcia et al,²⁶ between 12 and 24 hours after MCAO, has a time course similar to that indicated by the number of necrotic neurons. When one considers that in our experiments the dialysis probe site is included within the infarct area, the relationship found suggests that the extent of striatal areas of neuronal injury after 24 hours can be predicted from the increase in striatal transmitter outflow.

Finally, in our experiments, ischemic damage is associated with a relevant functional deficit that, 24 hours after occlusion, has the same neurological score as that reported by Garcia et al.²⁶ The correlation we found between striatal ischemic damage and neurological score confirms the correlation previously found by Garcia et al²⁶ between the number of necrotic neurons in the cortex and neurological score on the first day after MCAO.

In conclusion, amino acid and adenosine extracellular concentrations during focal ischemia, induced by MCAO performed by the intraluminal suture technique, were determined in awake and freely moving rats. For the first time a significant correlation between transmitter outflow and neurological deficit is described. The evaluation of neurological deficit, histological damage, and transmitter outflow in the same animal can provide more support to the assessment of neuroprotective properties of drugs against focal ischemia and relate it to neurochemical changes.

	Acknowledgments

 
This study was supported by grants from the University of Florence and the National Program of Research on Drugs of the Ministero dell'Università e della Ricerca Scientifica e Tecnologica, Italy. We are grateful to C.L. Susini for technical assistance.

Received May 3, 1999; revision received August 2, 1999; accepted August 5, 1999.

	References

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
1. Ginsberg MD, Busto R. Rodent models of cerebral ischemia. Stroke. 1989;20:1627–1642.[Abstract/Free Full Text]

2. Tyson GW, Teasdale GM, Graham DI, McCulloch J. Focal cerebral ischemia in the rat: topography of hemodynamic and histopathological changes. Ann Neurol. 1984;15:559–567.[Medline] [Order article via Infotrieve]

3. Garcia JH, Liu KF, Ho KL. Neuronal necrosis after middle cerebral artery occlusion in Wistar rats progresses at different time intervals in the caudoputamen and the cortex. Stroke. 1995;26:636–643.[Abstract/Free Full Text]

4. Choi DW, Rothman SM. The role of glutamate neurotoxicity in hypoxic-ischemic neuronal death. Ann Rev Neurosci. 1990;13:171–182.[Medline] [Order article via Infotrieve]

5. Morely P, Hogan MJ, Hakim AM. Calcium-mediated mechanisms of ischemic injury and protection. Brain Pathol. 1994;4:37–47.[Medline] [Order article via Infotrieve]

6. Hossmann K-A. Glutamate-mediated injury in focal cerebral ischemia: the excitation hypothesis revisited. Brain Pathol. 1994;4:22–36.

7. Leach MJ, Swan JH, Eisenthal D, Dopson M, Nobbs M. BW619C89, a glutamate release inhibitor, protects against focal cerebral ischemia damage. Stroke. 1993;24:1063–1067.[Abstract/Free Full Text]

8. Rudolphi KA, Schubert P, Parkinson FE, Fredholm BB. Neuroprotective role of adenosine in cerebral ischemia. Trends Pharmacol Sci. 1992;13:439–445.[Medline] [Order article via Infotrieve]

9. Evans MC, Swan JH, Meldrum BS. An adenosine analogue, 2-chloro-adenosine, protects against long term development of ischemic cell loss in the rat hippocampus. Neurosci Lett. 1987;83:287–292.[Medline] [Order article via Infotrieve]

10. Von Lubitz DKJE, Beenhakker M, Lin RC-S, Carter MF, Paul IA, Bischofberger N, Jacobson KA. Reduction of postischemic brain damage and memory deficits following treatment with the selective adenosine A₁ receptor agonist. Eur J Pharmacol. 1996;302:43–48.[Medline] [Order article via Infotrieve]

11. Von Lubitz DKJE, Carter MF, Beenhakker M, Lin RC-S, Jacobson KA. Adenosine: a prototherapeutic concept in neurodegeneration. Ann N Y Acad Sci. 1996;765:163.[Medline] [Order article via Infotrieve]

12. Phillis JW, O'Regan MH. Deoxycoformycin antagonizes ischemia-induced neuronal degeneration. Brain Res Bull. 1988;22:537–540.

13. Ongini E, Schubert P. Neuroprotection induced by stimulating A₁ or blocking A_2A adenosine receptors: an apparent paradox. Drug Devel Res. 1998;45:387–393.

14. Von Lubitz DKJE, Lin RC-S, Popik P, Carter MF, Jacobson KA. Adenosine A₃ receptor stimulation and cerebral ischemia. Eur J Pharmacol. 1994;263:59–67.[Medline] [Order article via Infotrieve]

15. Von Lubitz DKJE. Adenosine and cerebral ischemia: therapeutic future or death of a brave concept? Eur J Pharmacol. 1999;365:9–25.[Medline] [Order article via Infotrieve]

16. Zea Longa E, Weinstein PR, Carlson S, Cummins R. Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke. 1989;20:84–91.[Abstract/Free Full Text]

17. Butcher SP, Bulloch R, Graham DI, McCulloch J. Correlation between amino acid release and neuropathologic outcome in rat brain following middle cerebral artery occlusion. Stroke. 1990;21:1727–1733.[Abstract/Free Full Text]

18. Hillered L, Hallström A, Segersvärd S, Persson L, Ungerstedt U. Dynamics of extracellular metabolites in the striatum after middle cerebral artery occlusion in the rat monitored by intracerebral microdialysis. J Cereb Blood Flow Metab. 1989;9:607–616.[Medline] [Order article via Infotrieve]

19. Matsumoto K, Graf R, Rosner G, Shimada N, Heiss WD. Flow thresholds for extracellular purine catabolite elevation in cat focal ischemia. Brain Res. 1992;579:309–314.[Medline] [Order article via Infotrieve]

20. Phillis JW, Smith-Barbour M, O'Regan MH, Perkins LM. Amino acid and purine release in rat brain following temporary middle cerebral artery occlusion. Neurochem Res. 1994;19:1125–1130.[Medline] [Order article via Infotrieve]

21. Takagi K, Ginsberg MD, Globus MY-T, Dietrich WD, Martinez E, Kraydieh S, Busto R. Changes in amino acid neurotransmitters and cerebral blood flow in the ischemic penumbra region following middle cerebral artery occlusion in the rat: correlation with histopathology. J Cereb Blood Flow Metab. 1993;13:575–585.[Medline] [Order article via Infotrieve]

22. Shirotani T, Shima K, Chigasaki H. In vivo studies of extracellular metabolites in the striatum after middle cerebral artery occlusion in stroke-prone spontaneously hypertensive rats. Stroke. 1995;26:878–884.[Abstract/Free Full Text]

23. Paxinos G, Watson G, eds. The Rat Brain in Stereotaxic Coordinates. New York, NY: Academic Press: 1982.

24. Pedata F, Latini S, Pazzagli M, Pepeu G. Adenosine outflow from hippocampal slices evoked by ischemic-like conditions: effect of the excitatory amino acid antagonists. Drug Devel Res. 1993;28:395–398.

25. Lenda K, Sveneby G. Rapid high performance liquid chromatographic determination of amino acids in synaptosomal extracts. J Chromatogr B. 1980;198:516–519.

26. Garcia HJ, Wagner S, Liu K-F, Hu X-J. Neurological deficit and extent of neuronal necrosis attributable to middle cerebral artery occlusion in rats: statistical validation. Stroke. 1995;26:627–634.[Abstract/Free Full Text]

27. Pantoni L, Bartolini L, Pracucci G, Inzitari D. Interrater agreement on a simple neurological score in rats. Stroke. 1998;28:871–872.

28. Lyden PD, Lonzo LM, Nunez SY, Dockstader T, Mathieu-Costello O, Zivin JA. Effect of ischemic cerebral volume changes on behavior. Behav Brain Res. 1997;87:59–67.[Medline] [Order article via Infotrieve]

29. Benveniste H, Drejer J, Schousboe A, Diemer NH. Regional cerebral glucose phosphorylation and blood flow after insertion of a microdialysis fiber through the dorsal hippocampus in the rat. J Neurochem. 1987;49:729–734.[Medline] [Order article via Infotrieve]

30. Pazzagli M, Pedata F, Pepeu G. Effect of K⁺ depolarization, tetrodotoxin, and NMDA receptor inhibition on extracellular adenosine levels in rat striatum. Eur J Pharmacol. 1993;234:61–65.[Medline] [Order article via Infotrieve]

31. Corsi C, Pazzagli M, Bianchi L, Della Corte L, Pepeu G, Pedata F. In vivo amino acid release from the striatum of aging rats: adenosine modulation. Neurobiol Aging. 1997;18:243–250.[Medline] [Order article via Infotrieve]

32. Matsumoto K, Lo EH, Pierce AR, Halpern EF, Newcomb R. Secondary elevation of extracellular neurotransmitter amino acids in the reperfusion phase following focal cerebral ischemia. J Cereb Blood Flow Metab. 1996;16:114–124.[Medline] [Order article via Infotrieve]

33. Laing RJ, Jakubowski J, Laing RW. Middle cerebral artery occlusion without craniectomy in rats: which method works best? Stroke. 1993;24:294–298.[Abstract/Free Full Text]

34. Nagasawa H, Kogure K. Correlation between cerebral blood flow and histologic changes in a new rat model of middle cerebral artery occlusion. Stroke. 1989;20:1037–1043.[Abstract/Free Full Text]

35. Dux E, Fastbom J, Ungersted U, Rudolphi KA, Fredholm BB. Protective effect of adenosine and a novel xanthine derivative propentophylline on the cell damage after bilateral carotid occlusion in the gerbil hippocampus. Brain Res. 1990;516:248–256.[Medline] [Order article via Infotrieve]

36. Hagberg H, Andersson P, Lacarewicz J, Jacobson I, Butcher S, Sandberg M. Extracellular adenosine, inosine, hypoxanthine, and xanthine in relation to tissue nucleotides and purines in rat striatum during transient ischemia. J Neurochem. 1987;49:227–231.[Medline] [Order article via Infotrieve]

37. Fredholm BB, Abbracchio MP, Burnstock G, Daly JW, Harden TK, Jacobson KA, Leff P, Williams M. Nomenclature and classification of purinoceptors. Pharmacol Rev. 1994;46:143–156.[Medline] [Order article via Infotrieve]

38. Jacobson KA. Adenosine A₃ receptors: novel ligand and paradoxical effects. Trends Pharmacol Sci. 1998;19:184–191.[Medline] [Order article via Infotrieve]

39. Globus MY-T, Busto R, Dietrich WD, Martinez E, Valdés I, Ginsberg MD. Effect of ischemia on the in vivo release of striatal dopamine, glutamate and GABA studied by intracerebral microdialysis. J Neurochem. 1988;51:1455–1464.[Medline] [Order article via Infotrieve]

40. Pazzagli M, Corsi C, Fratti S, Pedata F, Pepeu G. Regulation of extracellular adenosine levels in the striatum of aging rats. Brain Res. 1995;684:103–106.[Medline] [Order article via Infotrieve]

41. Sciotti VM, Roche FM, Grabb MC, Van Wylen DGL. Adenosine receptor blockade augments interstitial fluid levels of excitatory amino acids during cerebral ischemia. J Cereb Blood Flow Metab. 1992;12:646–655.[Medline] [Order article via Infotrieve]

42. Goda H, Ooboshi H, Ibayashi S, Sadoshima S, Fujishima M. Modulation of ischemia-evoked release of excitatory and inhibitory amino acids by adenosine A₁ receptor agonist. Eur J Pharmacol. 1998;357:149–155.[Medline] [Order article via Infotrieve]

43. O'Regan MH, Simpson LM, Perkins LM, Phillis JW. The selective A₂ adenosine receptor agonist CGS 21680 enhances excitatory transmitter amino acid release from the ischemic rat cerebral cortex. Neurosci Lett. 1992;138:169–172.[Medline] [Order article via Infotrieve]

44. Popoli P, Betto P, Reggio R, Ricciarello G. Adenosine A_2A receptor stimulation enhances striatal extracellular glutamate levels in rats. Eur J Pharmacol. 1995;287:215–217.[Medline] [Order article via Infotrieve]

45. Corsi C, Melani A, Bianchi L, Pepeu G, Pedata F. Striatal A_2A adenosine receptors differentially regulate spontaneous and K⁺-evoked glutamate release in vivo in young and aged rats. Neuroreport. 1999;10:687–691.[Medline] [Order article via Infotrieve]

46. Meghji P. Adenosine production and metabolism. In: Stone T, ed. Adenosine in the Nervous System. London, England: Academic Press; 1991:25–42.

47. White TD. Potentiation of excitatory amino acid-evoked adenosine release from rat cortex by inhibitors of adenosine kinase and adenosine deaminase and by cortex by acadesine. Eur J Pharmacol. 1996;303:27–38.[Medline] [Order article via Infotrieve]

48. Kita H, Shima K, Iwata M, Chigasaki H. Local cerebral blood flow, glucose content and glucose utilization in focal cerebral ischemia in spontaneously hypertensive stroke-prone rats. In: Avezaat CJJ, van Eijndhoven JHM, Maas AIR, Tans JThJ, eds. Intracranial Pressure. Berlin, Germany: Springer-Verlag; 1993;38:182–185.

49. Petito CK, Chung MC, Verkhovsky LM, Cooper AJL. Brain glutamine synthetase increases following cerebral ischemia in the rat. Brain Res. 1992;569:275–280.[Medline] [Order article via Infotrieve]

50. Schousboe A, Larsson OM, Drejer J, Krogsgaard-Larsen P, Hertz L. Uptake and release of glutamate, glutamine and GABA in cultured neurons and astrocytes. In: Hertz L, Kvamme E, McGeer EG, Schousboe A, eds. Glutamine, Glutamate and GABA in the Central Nervous System. New York, NY; Liss; 1983:297–315.

51. Larsson OM, Thorbek P, Krogsgaard-Larsen P, Schousboe A. Effect of homo-b-proline and other heterocyclic GABA analogues on GABA uptake in neurons and astroglial cells and GABA receptor binding. J Neurochem. 1981;37:1509–1516.[Medline] [Order article via Infotrieve]

52. Garcia JH, Yoshida H, Chen H, Li Y, Zhang ZG, Lian J, Chen S, Chopp M. Progression from ischemic injury to infarct following middle cerebral artery occlusion in the rat. Am J Pathol. 1993;142:623–663.[Abstract]

53. Garcia JK, Liu K-F, Yoshida Y, Chen S, Lian J. Influx of leukocytes and platelets in an evolving brain infarct (Wistar rats). Am J Pathol. 1994;144:188–199.[Abstract]

54. Erecinska M, Nelson D, Wilson DF, Silver IA. Neurotransmitter amino acids in the CNS, I: regional changes in amino acid levels in rat brain during ischemia and reperfusion. Brain Res. 1984;304:9–22.[Medline] [Order article via Infotrieve]

55. Staley KJ, Soldo BL, Proctor WR. Ionic mechanisms of neuronal excitation by inhibitory GABA_A receptors. Science. 1995;269:977–981.[Abstract/Free Full Text]

56. O' Regan MH, Simpson RE, Perkins LM, Phillis JW. Adenosine receptor agonists inhibit the release of gamma-aminobutyric acid (GABA) from the ischemic rat cerebral cortex. Brain Res. 1992;582:22–26.[Medline] [Order article via Infotrieve]

57. Kurokawa M, Kirk IP, Kirkpatrick KA, Kase H, Richardson PJ. Inhibition by KF17837 of adenosine A_2A receptor-mediated modulation of striatal GABA and ACh release. Br J Pharmacol. 1994;113:43–48.[Medline] [Order article via Infotrieve]

58. Mori A, Shindou T, Ichimura M, Nonaka H, Kase H. The role of adenosine A_2A receptors in regulating GABAergic synaptic transmission in striatal medium spiny neurons. J Neurosci. 1996;16:605–611.[Abstract/Free Full Text]

59. Mayfield RD, Larson G, Orona RA, Zahniser NR. Opposing actions of adenosine A_2A and dopamine D₂ receptor activation on GABA release in the basal ganglia: evidence for an A_2A/D₂ receptor interaction in globus pallidus. Synapse. 1996;22:132–138.[Medline] [Order article via Infotrieve]

60. Ferré S, O'Connor WT, Fuxe K, Ungerstedt U. The striatopallidal neuron: a main locus for adenosine-dopamine interactions in the brain. J Neurosci. 1993;13:5402–5406.[Abstract]

Editorial Comment

Correlations With Neurological Deficit and Histopathological Damage

Dag K. J. E. von Lubitz, MD, Guest Editor

Emergency Medicine Research Laboratories, Department of Emergency Medicine, University of Michigan Health System, Ann Arbor, Michigan

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
The article by Melani et al represents yet another excursion into the field of neurotransmitter and neuromodulator release during or after cerebral ischemia. Yet, while the field is old, the study offers a number of new and important insights on the temporal relationships governing the release of adenosine versus the outflow of other transmitters, correlation of postischemic levels of excitatory neurotransmitters with the ensuing damage, and, probably most significantly, with the extent of postischemic neurological deficit. Seen in such context, the work of Melani et al offers laboratory corroboration of recent clinical studies demonstrating a close relationship between extracellular concentration of excitatory amino acids and the extent of cerebral damage and subsequent recovery following either stroke or head injury.^1–4 Yet, the paper also reminds of a number of still-unresolved issues requiring a substantial research effort before their definitive settlement is at hand. One of these issues concerns the mechanism(s) involved in the ischemia-induced increase in neurotoxic excitatory amino acid concentration (ie, release from the presynaptic terminals and reversal of glutamate transporters versus altered metabolism).⁵ Clearly, that issue alone has a significant bearing on the future course of the therapeutic strategies targeted at the manipulation of neutrotransmitter release.⁶ The functional role of inhibitory neurotransmitters and neuromodulators (GABA, taurine, adenosine), whose large quantities are liberated during and after ischemia, poses another persistently unclear element highlighted by Melani et al.

There is a substantial amount of direct experimental evidence showing that activation of both GABA and adenosine (primarily A₁) receptors induces a wide spectrum of processes whose cumulative result is the reduction of ischemia-induced brain damage.^7–9 As clearly demonstrated by Melani et al (and, earlier, also by Matsumoto et al¹⁰), adenosine is released before glutamate, although the duration of its maximal elevation is significantly shorter than that of either GABA or taurine. One could, therefore, construe that the temporal sequence of events centered on the release of these inhibitory agents might indicate the presence of an endogenous cerebroprotective complex whose activation serves to limit the extent of injury. Studies employing agonists and antagonists of adenosine A₁ and GABA receptors give credence to such a concept.^7,9 Yet, as indicated by the presented data (and, in their discussion, also by the authors themselves), the nature of the processes constituting the endogenous "defensive retaliation"¹¹ complex of the brain against a potentially lethal incident appears to be less than straightforward. More importantly, the presented data indicate that the temporal (and very likely spatial) interplay of excitatory and inhibitory mechanisms activated by ischemia offers a challenging field for further research and a very promising arena for the development of new concepts of therapeutic interventions.¹²

Received May 3, 1999; revision received August 2, 1999; accepted August 5, 1999.

	References

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
1. Koura SS, Doppenberg EM, Marmarou A, Choi S, Young HF, Bullock R. Relationship between excitatory amino acid release and outcome after severe human head injury. Acta Neurochir Suppl (Wien).. 1998;71:244–246.[Medline] [Order article via Infotrieve]

2. Mendelowitsch A, Sekhar LN, Wright DC, Nadel A, Miyashita H, Richardson R, Kent M, Shuaib A. An increase in extracellular glutamate is a sensitive method of detecting ischaemic neuronal damage during cranial base and cerebrovascular surgery. Acta Neurochir (Wien).. 1998;140:349–355.[Medline] [Order article via Infotrieve]

3. Enblad P, Valtysson J, Andersson J, Lilja A, Valind S, Antoni G, Langstrom B, Hillered L, Persson L. Simultaneous intracerebra: microdialysis and positron emission tomography in the detection of ischaemia in patients with subarachnoid hemorrhage. J Cereb Blood Flow Metab.. 1996;16:637–644.[Medline] [Order article via Infotrieve]

4. Bullock R, Zauner A, Woodward J, Young HF. Massive persistent release of excitatory amino acids following human occlusive stroke. Stroke.. 1995;26:2187–2189.[Abstract/Free Full Text]

5. Obrenovitch TP. Origins of glutamate release in ischemia. Acta Neurochir Suppl (Wien).. 1966;66:50–55.

6. Obrenovitch TP, Richards DA. Extracellular neurotransmitter changes in cerebral ischemia. Cerebrovasc Brain Metab Rev.. 1995;7:1–54.[Medline] [Order article via Infotrieve]

7. Lyden PD. GABA and neuroprotection. Int Rev Neurobiol.. 1997;40:233–258.[Medline] [Order article via Infotrieve]

8. Deckert J, Gleiter CH. Adenosine: an endogenous neuroprotective metabolite and neuromodulator, J Neural Transm Suppl.. 1994;43:23–31.[Medline] [Order article via Infotrieve]

9. von Lubitz D.K.J.E. Adenosine and cerebral ischemia: therapeutic future or death of a brave concept? Eur J Pharmacol.. 1999;37:85–102.

10. Matsumoto K, Graf R, Rosner G, Schimada N, Weiss WD. Flow threshold for extracellular purine catabolite elevation in rat focal ischemia. Brain Res.. 1992;579:309–314.

11. Newby AC. Adenosine as a retaliatory metabolite. Trends Biol Sci.. 1984;9:42–44.

12. Shuaib A, Kanthan R. Amplification of inhibitory mechanisms in cerebral ischemia: an alternative approach to neuronal protection. Histol Histopathol.. 1997;12:185–194.[Medline] [Order article via Infotrieve]

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