Effects of chemical ischemia in cerebral cortex slices. Focus on nitric oxide.
Superfused rat cerebral cortex slices were submitted to a continuous electrical (5 Hz) stimulation and treated with sodium azide (1-10 mM) in the presence of 2 mM 2-deoxyglucose ("chemical ischemia"). Presynaptic cholinergic activity, evaluated as acetylcholine release, was inhibited depending on the sodium azide concentrations and on the length of application (5-30 min). Following a 5-min treatment with 10 mM sodium azide, acetylcholine release was reduced to 45+/-2.3%; simultaneously, there was a 15- and 10-fold increase in glutamate and nitric oxide effluxes, respectively. After restoring normal superfusion conditions, acetylcholine release recovered to 70+/-3.1% of the controls; the N-methyl-D-aspartate receptor antagonist MK-801 (10 microM) as well as the nitric oxide scavengers, haemoglobin (20 microM) and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-l-oxyl-3-oxide (150 microM), improved the recovery in presynaptic activity, showing that both glutamate and nitric oxide play detrimental roles in chemical ischemia. On the other hand, the post-ischemic recovery was worsened by the guanylylcyclase inhibitor 1H-[l,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (10 microM), suggesting that the activation of such a pathway plays a neuroprotective role and that the nitric oxide-induced harmful effects depend on different mechanisms. Chemical ischemia-evoked nitric oxide efflux partly derived from its calcium-dependent endogenous synthesis, since both the intracellular calcium chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (1 mM), and the nitric oxide synthase inhibitor, N(omega)-nitro-L-arginine methyl ester (100 microM), substantially prevented sodium azide effects. Nitric oxide efflux was only weakly reduced by MK-801 and was not modified by either the L-type calcium channel blocker, nifedipine (10 microM) or the N-type calcium channel blocker omega-conotoxin (0.5 microM), thus suggesting a prevailing intracellular calcium-dependence of nitric oxide production, although a partial extracellular calcium source cannot be ruled out. These findings show that sodium azide plus 2-deoxyglucose treatment is a useful protocol to induce brain ischemia in vitro and underline the involvement of nitric oxide in the complex events following the ischemic insult.
Cavallini
S
S
Department of Clinical and Experimental Medicine, Section of Pharmacology, University of Ferrara, Via Fossato di Mortara 17, 44100 Ferrara, Italy.
Marti
M
M
Marino
S
S
Selvatici
R
R
Beani
L
L
Bianchi
C
C
Siniscalchi
A
A
D000109:Acetylcholine / Q000378:metabolism; D000818:Animals; D000963:Antimetabolites / Q000494:pharmacology; D002545:Brain Ischemia / Q000139:chemically induced* / Q000378:metabolism*; D002374:Catalase / Q000378:metabolism; D006152:Cyclic GMP / Q000502:physiology; D003847:Deoxyglucose / Q000494:pharmacology; D004734:Energy Metabolism / Q000187:drug effects; D018698:Glutamic Acid / Q000378:metabolism; D006162:Guanylate Cyclase / Q000378:metabolism; D066298:In Vitro Techniques; D008297:Male; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D019810:Sodium Azide / Q000378:metabolism / Q000494:pharmacology; D014665:Vasodilator Agents / Q000494:pharmacology
D000963:Antimetabolites; D014665:Vasodilator Agents; D009569:Nitric Oxide; D018698:Glutamic Acid; D019810:Sodium Azide; D003847:Deoxyglucose; D002374:Catalase; D019001:Nitric Oxide Synthase; D006162:Guanylate Cyclase; D006152:Cyclic GMP; D000109:Acetylcholine