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Concomitant increases in the extracellular concentrations of excitatory and inhibitory amino acids in the rat hippocampus during forebrain ischemia D. Lekieffre, J. Callebert, M. Plotkine and R.G. Boulu Laboratoire de Pharmacologie, FacultO des Sciences Pharmaceutiques et Biologiques. b)fiversitO Rein; Descartes. Pari.s (France: (Received 22 July 1991 : Revised version received 16 December 1991; Accepted 16 December 1991 ) Key words: Microdialysis; Hippocampus; Amino acid; Forebrain ischemia: Rat The extracellular concentrations of aspartate, glutamate, glutamine, taurine and y-aminobutyric acid in the hippocampus were determined during and after forebrain ischemia (4-vessel model) in the unanaesthetized rat. lschemia led to a large increase in both inhibitory (taurine and y°aminobutyric acid) and excitatory amino acids (aspartate, glutamate). These results suggest that in this model, as previously proposed in other models of ischemia, the large increase of inhibitory amino acids could counterbalance the excitotoxicity due to aspartate and glutamatc.

There is considerable evidence that high concentrations of glutamate (Glu) are neurotoxic [21] and that the overstimulation of Glu receptors due to a massive release of Glu contributes to the great vulnerability of hippocampal neurons during cerebral ischemia. A major part of the excitatory innervation of the hippocampus is glutamatergic [31] and the hippocampus has a high density of Glu receptors [19]. Microdialysis experiments have shown that the extracellular Glu concentration is substantially increased following experimental forebrain [3, 4, 10] or focal ischemia [11]. Lastly, removal of excitatory glutamatergic afferents to the hippocampus protects this structure from ischemic damage [12]. However, to our knowledge, no data are available on the release of Glu in the '4-vessel model' in unanaesthetized rats. As previous experiments have demonstrated that the release of excitatory amino acids (EAA) during ischemia is associated with a release of inhibitory amino acids (IAA) [3, 10], this study was carried out to examine changes in hippocampal extracellular concentrations of these two groups of amino acids (AA) in the '4-vessel model' of ischemia in the unanaesthetized rat. Male Wistar rats weighing 250-280 g were anaesthetized with ether and prepared for 4-vessel occlusion [26]. Both vertebral arteries were permanently occluded by electrocauterisation within the alar foraminae of the

Correspondence: D. Lekieffre, Laboratoire de Pharmacologie, 4. avenue de l'Observatoire, 75006 Paris, France.

first cervical vertebra. At the same time, both common carotid arteries were isolated and atraumatic arterial clamps were placed around each one. The following day, a microdialysis probe was implanted and 3 h later cerebral ischemia was initiated in the unanaesthetized rat by clamping each carotid artery for either 15 or 30 rain. Carotid clamping resulted in a loss of righting reflex within 60 s and the loss lasted the entire period of ischemia. The dialysis fibre was implanted by the technique of Ungerstedt et al. [32] under chloral hydrate anaesthesia (300 mg/kg/10 ml, i.p.). Except for a 4-mm long section in each hemisphere, corresponding to the hippocampus, the fibre (Hospal AN 69) was coated with Epoxy glue to make it impermeable. The fibre was inserted horizontally into the dorsal hippocampus in the frontal plane 3.5 mm behind the bregma and 3.6 mm below the surface of the skull [22]. Preliminary experiments showed that AA concentrations were stabilized after 2 h of perfusion. In the definitive experiment, dialysates were collected 2 h after initiating fibre perfusion with Ringer's solution (147 mM NaCI, 4 mM KC1, 2.4 mM CaC12; flow rate: 2.5 ~l/min). Two consecutive 20-min samples of perfusate were collected before ischemia to determine basal concentrations. Samples were collected at 15-min intervals during ischemia and at 20-min intervals after ischemia. All samples were frozen at -40°C until analysis. Rectal temperature was kept constant at 37°C by means of a heating lamp. The rat was sacrificed at the end of the experiment and the brain was removed and frozen at -40°C.

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Cryostat sections were cut to ensure the correct implantation of the fibre. Aspartate (Asp), Glu, glutamine (Gln), glycine (Gly), taurine (Tau), phosphoethanolamine (PEA) and y-aminobutyric acid (GABA) concentrations were determined by HPLC with fluorescence detection and automated pre-column derivatization with o-phtaldialdehyde modified from Lindroth and Mopper [16]. A second group of rats was used for histological examination, they were subjected to 15 min (n=9) or 30 min (n=10) ischemia. Seventy-two hours after ischemia, the rats were anaesthetized with pentobarbital (60 mg/kg, i.p.) and perfused with FAM (formaldehyde, acetic acid, methanol, 1/1/8) via the ascending aorta. The brains were left in situ [\~r 24 h at -4°C, removed and kept in FAM

for one week. They were then embedded in paraffin and 7-/am coronal sections were cut and stained with Luxol fast blue and Cresyl violet. Hippocampal damage was evaluated at a level 5.7 mm anterior to the interaural line [22]. The CA1 sector was arbitrarily divided into 3 subsegments (a, b and c, according to Von Lubitz et al. [33]) and surviving pyramidal cells were counted under the light microscope (x 400) for a total of 3 grid lengths (3 x 250/am) of each subsegment. All values are means +_ S.E.M. Amino acid concentrations (/aM) were compared using Student's t-test. The surviving cells were evaluated by ANOVA and Dunnett test. The basal concentrations of Asp, Glu, Gln, Tau, PEA and GABA in the dialysates before 15- and 30-rain Glutamate

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