Perbamon Prcg. Neuro-Psgcho~harmwoL & Biol. Psychiat. 1995. Vol. 19. pp. 943-953

Copyright 0 1995 Elsevier Science Ltd

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LIDOCAINE REDUCES THE HYPOXIA-INDUCED RELEASE OF AN EXCITATORY AMINO ACID ANALOG FROM RAT STRIATU

SLICES IN SUPERFUSION

LEOPOLD0 DIAZ*, ARIEL GOMEZ* AND GONZALO BUSTOS#

*Departamento de Ciencias Neurologicas, Facultad de Medicina, Campus Oriente, Universidad de Chile, Santiago, Chile, and #Laboratorio de Farmacologia-Bioquimica, Facultad de Ciencias Biol6gicas,

Pontificia Universidad Catolica de Chile, Santiago, Chile

(Final form, December 1994)

Abstract

Diaz Leopoldo, Ariel Gomez and Gonzalo Bustos. Lidocaine reduces the hypoxia-induced release of an excitatory amino acid analog from rat striatal slices in superfusion. Prog. Neuro-Psychopharmacol. & Biol. Psychiat. 1995, 19(S): 943-953.

Lidocaine has been extensively investigated as a potential neuroprotective drug against ischemia- induced neurodegeneration without reaching any satisfactory conclusion. The present work evaluates the effect of lidocaine -17 pM- on the hypoxia-induced release of tritiated D-aspartate from rat striatal slices in superfusion. Hypoxia resulted in a significant increase in the amount of D-aspartate released from striatal slices preloaded with the tritiated excitatory amino acid analog. The addition of lidocaine to the superfusion solution resulted in a drastic reduction in the amount of D-aspartate release evoked by hypoxia, rendering it close to normal values.

Keywords: cerebral hypoxia, excitatory amino acids, excitotoxicity, lidocaine.

Abbreviations: Central Nervous System (CNS), Krebs ringer phosphate (Krp), trichloroacetic acid (TCA).

Introduction

Cerebral hypoxia-ischemia is a major cause of CNS injury throughout the whole life of the individuals. It represents the physiopathological basis of several clinical entities including neonatal asphyxia, cerebral infarction, post cardiopulmonary arrest encephalopathy and focal cerebral ischemia either associated to vasospasm or to traumatic brain injury.

The discouraging results obtained with several therapeutical strategies in cerebral ischemia have led to nihilistic postures regarding the pharmacological management of this entity (Biller and Love, 1991) and/or the significance of experimental research in this field (Millikan, 1992). Several neuroprotective drugs have been investigated by different groups regarding their ability to minimize the effects of hypoxia- ischemia in the most vulnerable brain areas. These drugs, when effective, seem to exert their

943

944 L. Diaz et al.

neuroprotective effect through a variety of molecular mechanisms.

Lidocaine, a local anesthetic which acts as a voltage-dependent sodium channel blocker, has been extensively investigated as a potential neuroprotective drug. In spite of publications that either confirm (Evans and Kobrine, 1987; Evans et al., 1989; Shokunbi et al., 1990) or disprove (Highaghy et al., 1987; Sutherland et al., 1989) the idea that lidocaine could be useful as a neuroprotective agent, it is widely used in some clinical centers when intracranial pressure is elevated. It has been postulated that the neuroprotective effect of lidocaine could be attributed to one or more of the following pharmacological effects of this drug: a) reduction in the intracranial pressure; b) reduction in neuronal ATP consumption; c) reduction in the cerebral metabolical rate of oxygen; d) reduction in the release of excitatory amino acids (Hall and Murdoch, 1990). Some of the above mentioned mechanisms have also been advocated to explain the neuroprotective effects of other sodium channel blockers such as phenytoin, lamotrigine and riluzone (Artru and Michenfelder, 1981; Boxer et al., 1990; Lipton, 1993).

It has been suggested that the main mechanism responsible for the rise of extracellular concentration of excitatory amino acids in cerebral hypoxia-ischemia is a passive, calcium-independent, sodium- dependent, carrier-mediated release of the neurotransmitter from the cytosolic pool (Sanchez-Prieto and Gonzalez, 1988; Adam-Vizi, 1992; Levi and Raitieri, 1993). The mechanism underlying this passive efflux of excitatory amino acids would be a reverse function of the high affinity, sodium-dependent, neurotransmitter uptake system, due to a reversal of the normal sodium transmembrane gradient associated to the neuronal energetic failure (Hansen, 1985; Taylor, 1993).

In order to get some insight into the possible effect of lidocaine on the release of excitatory amino acids evoked by hypoxia, the authors assessed the effect of this drug on the release of a tritiated, nonmetabolizable excitatory amino acid analog from superfused striatal slices under hypoxic conditions in vitro.

Methods

The experimental model is a modification of that one used previously by Abarca and Bustos (1985) and consists in the assessment of the release of D-[2,3-3H]-aspartic acid from striatal slices in a superfusion system under aerobic and anaerobic conditions, with or without the addition of lidocaine to the superfusion solution.

Striatal slices of adult [250 g] male Sprague Dawley rats were obtained according to a previously described procedure (Alger et al., 1984). Briefly, the rats were killed by decapitation, the brain was quickly removed and the striatum was dissected at 4°C. Striatal slices were prepared using a Sorvall tissue slicer, by chopping at 0.2 mm intervals in the coronal plane. Then, the slices were preincubated at 37°C in 4 ml Krp solution.

D-[2,3-3H]-aspartic acid was purchased from New England Nuclear Corp. [final concentration 0.074 umol/ml, sp. act. 1 mCi/ml]. Lidocaine was obtained from Laboratorio Beta [solution for clinical use, concentration 20 mg/ml]

Three experimental groups were defined:

Lidocaine reduces hypoxk-induced excitatory amino acid release 945

-1: Release of D-[2,3-3H]-aspartic acid under aerobic conditions (n= 5). Release of D-[2,3-3H]-aspartic acid under hypoxic conditions (n= 5). Group 2: Release of D-[2,3-3H]-aspartic acid under hypoxic conditions, with addition of lidocaine Group 3:

(n= 5).

The Krp solution used in these experiments had the following composition: 128 mM NaCl, 4.8 mM KCl, 0.75 mM CaCl2, 1.2 mM MgSO4, 16 mM Na2HP04, 16 mM glucose [pH 7.41. Other modifications of the Krp solution are described in the text.

Slices were incubated for 30 min at 37°C in 2 ml of a Krp solution [pH 7.41 saturated with 95% oxygen and containing D-[2,3-3H]-aspartic acid [final concentration 0.2 @I].

At the end of the incubation period, the slices were rapidly separated from the incubation medium through a 5.0 ml lucite chamber with a nylon mesh bottom. The slices were then transferred to a superfusion chamber and superfused with oxygenated and prewarmed to 37°C Krp solution. A continuous flow of 3 ml/min was maintained by means of a peristaltic pump. An initial period of 30 mm was allowed to elapse before the hypoxia-induced release began to be assessed.

For the assessment of the release of D-[2,3-3H]-aspartic acid under aerobic conditions, 20 samples of one min each were collected under the conditions described above.

For the assessment of the release of D-[2,3-3H]-aspartic acid under hypoxic conditions, 5 samples of one min each were collected under aerobic conditions [the same conditions described above]. Then, the super-fusion solution was changed to a Krp solution, pH 7.4, prewarmed to 37°C and saturated with nitrogen. An additional flow of nitrogen [0.5 l/mm] was delivered directly to the superfusion chamber in order to assure a rapid substitution of the intra-chamber atmosphere for an hypoxic environment (Dfaz et al., 1994). 10 samples of one mm each were collected. Finally, 5 samples of one min each were collected, again under aerobic conditions.

For the assessment of the effect of lidocaine on the release of D-[2,3-3H]-aspartic acid evoked by hypoxia, the same experimental procedure described above was employed with the only difference that lidocaine [final concentration 17 pM] was added to the nitrogen-saturated Krp solution. This concentration was chosen because it has proven to be neuroprotective in several in vivo experimental models of hypoxia-ischemia (Evans et al., 1984; Diaz et al., 1993).

At the end of the experiment, the slices were recovered and homogenized in 4 ml of TCA [ 15% w/v] to extract the remaining radioactivity. Tissue and samples radioactivity were assayed by liquid scintillation counting using a Nuclear Chicago Scintillation counter [counting efficiency= 30%]. The results were expressed as fraction of total radioactivity= [radioactivity in the superfusion samples of min. “n”/radioactivity in the tissue in the mm “n- 1”] * 100.

Data Analvsis

The arcsine transformation was applied to the data and statistical analysis using the Tukey-Kramer pos- test was performed (Zar, 1984).

Results

The incubation of striatal slices in aerobic conditions for 30 mm. at 37°C in the presence of 0.2 pM of the labeled amino acid resulted in a significant accumulation in the slices of the radioactive tracer. Spontaneous release of D-[2,3-3H]-aspartic acid under aerobic conditions was 0.23% (mean f (3, n= 5). The present results are similar to those reported by Arqueros et al. (1985).

946 L. Diaz el al.

Effect of Hvooxia on the Release of D-As~artic Acid

The nitrogen-induced anaerobiosis resulted in a marked and sustained elevation of the amount of D- [2,3-3H]-aspartic acid released from the striatal slices. The time-course of the release showed that the larger proportion of the radiolabeled tracer was released during the first minute of the nitrogen stimulation period. The increase in the amount of the radiolabeled amino acid release reached statistical significance -

when compared to spontaneous release- [p I 0.05, Tukey-Kramer test] during the first 9 minutes of

anaerobiosis. This difference was even more pronounced [p 2 0.00 1, Tukey-Kramer test] during the first three minutes of induced hypoxia (Table 1, Fig. 1).

Table I

Effects of Hypoxia and Hypoxia Plus Lidocaine on the Release of Tritiated D-Asp&c Acid from Rat Striatal Slices.

Amount of tritiated D-aspartate release (% of total radioactivity released)

min Aerobic Anaerobic Anaerobic+lidocaine

6 0.23% + 0.09% 7 0.26% f 0.06% 8 0.21% + 0.09% 9 0.26% zt 0.13% 10 0.25% f 0.08% 11 0.22% f 0.08% 12 0.24% rt 0.09% 13 0.20% f 0.07% 14 0.23% f 0.09% 15 0.23% f 0.11%

16 0.18% zt 0.06% 17 0.19% f 0.04% 18 0.20% + 0.07% 19 0.23% f 0.13% 20 0.18% i 0.06%

0.37% f 0.13% 0.25% zt 0.11% 0.23% +. 0.10% 0.25% f 0.13% 0.23% f 0.10%

0.51% + 0.21% 0.47% + 0.3 1% 0.40% -c 0.25% 0.34% + 0.20% 0.39% f 0.33% begin hypoxia 8.24% f 4.28% (**) 2.32% c 1.36% (**) 1.40% + 0.54% (**) 0.82% f 0.31% (*) 0.73% f 0.35% (*) 0.59% f 0.34% (*) 0.54% + 0.29% (*) 0.53% f 0.24% (**) 0.53% + 0.27% (*) 0.46% + 0.37% end hypoxia 0.59% f 0.79% 0.30% + 0.27% 0.26% f 0.21% 0.30% + 0.25% 0.26% f 0.17%

0.30% f 0.06% 0.26% + 0.10% 0.23% + 0.08% 0.21% f 0.08% 0.21% f 0.09% begin hypoxia 1.19% f 0.92% (#I#) 0.49% f 0.17% (##) 0.34% f 0.19% (#I#) 0.31% f 0.18% (#) 0.27% f 0.12% (#) 0.3 1% f 0.09% (#) 0.25% f 0.10% (#) 0.17% f 0.04% (#) 0.18% + 0.03% (#) 0.17% f 0.05% end hypoxia 0.28% rt 0.08% 0.18% + 0.02% 0.17% f 0.02% 0.17% f 0.02% 0.18% + 0.03%

(**): p 50.001, (*): p < 0.050, compared with basal release.

(##): p I 0.001, (#): p I 0.050, compared with hipoxia-induced release. (Tukey-Kramer test). Each value represents the mean of 5 assays f standard deviation.

Lidocaine reduces hypoxia-induced excitatory amino acid release 947

10

8

8

0

W of total radioactivity released

Krp-NP / Krp-NP-lidocalne

x

6 10 16 20 26

Superfueion time (min.)

Fig 1. Release of tritiated D-aspartate fom striatal slices precharged with the tracer in different experimental conditions. Each line represents the mean value of 5 assays: [aerobic, -*-I, [hypoxia, -.-I [hypoxia plus lidocaine, -+-I. Standard deviations have been ommited as they are shown in Table 1.

Effect

The addition of lidocaine [17 yM] drastically reduced the nitrogen-induced release of D-[2,3-3H]- aspartic acid, rendering it close to basal values (Table 1, Fig.1). In comparison to the nitrogen-induced release, the reduction in the amount of the labeled amino acid release when lidocaine was added to the superfusion solution reached statistical significance [p 5 0.05, Tukey-Kramer test] during the first 9

minutes of induced hypoxia, this difference being more pronounced [p 5 0.001, Tukey-Kramer test] during the first three minutes.

In order to detect any eventual change in the pH of the superfusion solutions during the experiments that could affect the release of the radiolabeled amino acid, the pH of the solutions was measured at the end of the experiments. The values did not change when compared to the initial pH of the superfusion solutions.

948 L. Diaz et al.

Effect of Positive Pressure on the Release of D-asuartic Acid

The experimental procedure employed resulted in a rise of the intra-chamber pressure of l-3 cm water. The eventual effect of this mild intra-chamber positive pressure on the release of D-[2,3-3H]-aspartic acid was ruled out by an additional experimental protocol. Briefly, the same experimental design employed for the assessment of the nitrogen-induced release of the tracer was employed, but the superfusion solutions were saturated only with oxygen and the intra-chamber nitrogen flow was substituted by an oxygen flow. No statistically significant differences [Tukey-Kramer test] in the amount of tracer released were observed when compared to the spontaneous [aerobic, normobaric] release of D-[2,3-3H]-aspartic acid from the striatal slices.

Discussion

Excitatorv Amino Acids and Hynoxia-Induced Neuronai Iniurv

There is an increasing amount of evidence linking the pathological release of excitatory amino acids and the consequent abnormal stimulation of excitatory amino acid receptors as a common final pathway leading to neuronal death in cerebral hypoxia-ischemia as well as in CNS trauma, seizures and several neurodegenerative disorders (Rothman and Olney, 1987; Siegal et al., 1990; Engelsen et al., 1990; Coyle and Puttfarcken, 1993). The density of glutamatergic innervation in some brain areas (Cotman et al., 1987; Greenamyre tal al., 1987) and /or the concentration of some calcium-binding proteins [parvalbumin, calbindin D-281 in some neuronal populations [Sloviter, 19891 may explain the “selective vulnerability” to hypoxia-ischemia, hypoglycemia and status epilepticus of discrete brain areas such as hippocampus, striatum and cerebellar cortex.

The rise in extracellular concentrations of excitatory amino acids in cerebral hypoxia-ischemia has been well documented by microdialysis techniques in animal models (Benveniste et al., 1984) as well as in humans (Persson and Hillered, 1992). A reduction in the extracellular space volume has been demonstrated in cerebral ischemia (Hansen and Olsen, 1980) probably due to cytotoxic edema of the neurons -secondary to the rise in intracellular concentrations of sodium, chloride and water as a consequence of the energetic failure and the stimulation of excitatory amino acid receptors- and a glial edema secondary to a massive recapture of glutamate and sodium (Kempski et al., 1989; Torp et al., 1991). However this reduction of the extracellular space is not sufficient to explain the rise of extracellular concentrations of excitatory amino acids, which has been postulated to be due to a failure of the presynaptic high-affinity, sodium-dependent neurotransmitter reuptake system and/or to a calcium- independent release of excitatory amino acids from the nerve terminals (Hauptman et al., 1984; Silverstein et al., 1986; Kanai et al., 1993). Both events would be directly related to changes in transmembrane gradients of sodium and potassium as a consequence of the global energetic failure (Kass and Lipton, 1982).

&J&v 0 f In Vitro Brain Hypoxia Models

Brain slices have been extensively used as a tool for research in cerebral hypoxia-ischemia (Fujiwara et al., 1987; Fujiwara et al., 1992; Leblond and Krnjevic, 1989). Some authors have criticized the experimental findings obtained with this methodology based on the severe metabolical abnormalities occurring in slices under “normal [aerobic]” conditions (Lipton an Whittingham, 1984). Recent measurements of intra-slice oxygen pressures have demonstrated that previous reported abnormalities of the metabolical status of brain slices could be attributable to the existence of anoxic areas in the depth of the slice in spite of an adequate external oxygen supply (Jiang et al., 1991). Another methodological objection to the brain slice method could be that the superfusate would “wash” the extracellular space thus avoiding the local accumulation of potentially deleterious [lactate, potassium, arachidonic acid, glycine,

Lidocaine reduces hypoxia-induced excitatory amino acid release 949

nitric oxide] or neuroprotective [adenosine] substances, which could result in striking differences between in vitro and in vivo findings (Krnjevic and Leblond, 1989). In spite of these arguments, this methodology provides the unique opportunity to manipulate several relevant parameters such as: extracellular pH, temperature, ionic [sodium, potassium, calcium] concentrations, extracellular glucose supply, degree of oxygenation, drug concentrations, etc.

Some authors have questioned the validity of release studies that involve preloading the tissue with radiolabeled neurotransmitters and then assessing the efflux of radioactivity (Nicholls, 1988; Wilkinson and Nicholls, 1989). Specifically, the validity of D-[2,3-3H]-aspartic acid as a marker of the release of excitatory amino acids has been a matter of debate, considering that this analog would not label the vesicular pool of the neurotransmitter because the only high affinity uptake system demonstrated in purified synaptic vesicles obtained from glutamatergic nerve terminals is specific for glutamate (Naito and Ueda 1985). However, there is some experimental evidence supporting the calcium-dependent release of D-[2,3-3H]-aspartic acid, the existence of subpopulations of aspartatergic neurons has been demonstrated in the cerebral cortex of the rat and the colocalization and corelease of glutamate and aspartate from nerve terminals has also been demonstrated (Szerb, 1988; Giuffrida and Rustioni, 1989). In the particular physiopathological situation of hipoxia-induced release of excitatory amino acids, these considerations am of less importance because the main source of the neurotransmitter released would be the cytosolic pool, which is adequately labeled with D-[2,3-3H]-aspartic acid.

Lidocaine as a Neuroarotective Drug

Lidocaine significantly reduces the hypoxia-induced release of D-[2,3-3H]-aspartic acid. Although the neuroprotective effect of lidocaine has been previously addressed to this mechanism (Meldrum, 1985; Rasool et al. 1990), the evidence offered was indirect. The possible mechanism underlying this effect of lidocaine could be the blockade of voltage-dependent sodium channels at the glutamatergic nerve terminals, thus preventing the energy failure-induced inversion of the sodium transmembrane gradient that would lead to a reversal function of the high affinity sodium-dependent carrier. Although a direct action of lidocaine blocking the phencyclidine site of the ionophore associated to the NMDA subtype receptor -a property shared with tetrodotoxin and other local anesthetics- cannot be ruled out, this hypothesis is less plausible because the concentrations required for this effect would be significantly higher (Vignon et al., 1982).

It is concluded that lidocaine is effective as neuroprotective agent against hypoxia-ischemia induced neurodegeneration. Among the possible mechanisms that could explain the neuroprotective effects of this drug, the fact that lidocaine reduces the hypoxia-induced release of excitatory amino acids from nerve terminals should be included. Elevation of the extracellular concentrations of these neurotransmltters in hypoxia-ischemia is crucial for the subsequent abusive stimulation of the postsynaptic receptors. However, it should not be forgotten that lidocaine has side effects such as systemic arterial hypotension that could mask or hinder their neuroprotective actions.

Acknowledgments

The present work is dedicated to the memory of the late Professor Dr. Reinaldo Poblete, Director (1975-1993) of the Instituto de Neurocirugia e Investigaciones Cerebrales, Santiago-Chile.

The help of Dr. Enrique Morgado in the statistical analysis of the data and his critical comments are gratefully acknowledged.

950 L. Diw el ai.

This work was partially supported by FONDECYT Grant N” 744190 (Dr. G. Bustos) and a Grant from the Sandoz Foundation for Gerontological Research (Drs. L. Diaz and A. Gomez).

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