NGF mediates the neuroprotective effect of the beta(2)-adrenoceptor agonist clenbuterol in vitro and in vivo: evidence from an NGF-antisense study

Previous studies in our laboratory suggested that neuroprotective effects o f the beta(2)-adrenoceptor agonist clenbuterol in vitro and in vivo occurre d due to enhanced synthesis of nerve growth factor. The aim of the present study was to evaluate the effects of a phosphothioated NGF oligodeoxynucleo tide on neuroprotection by clenbuterol in vitro and in vivo. After clenbuterol treatment (1-100 mu M) an increase in nerve growth factor mRNA and protein levels (200-300% of control) was observed in primary cult ures of rat cortical astrocytes. Nerve growth factor antisense oligonucleot ide (0.3-1 mu M for 3 days) reduced the content of nerve growth factor prot ein in the medium of the astrocytes concentration-dependently to 20% of con trol level. Nerve growth factor content in the medium of mixed hippocampal cells was reduced to 55% of sister cultures receiving the vehicle or a rand om control oligonucleotide. In mixed hippocampal cultures pretreated with r andom oligonucleotide (1 mu M, 30 h), clenbuterol (10 mu M) reduced the per centage of damaged neurons after glutamate exposure (0.5 mM, 1 h) to 17%. P retreatment with nerve growth factor antisense oligonucleotide (1 mu M) for 30 h before glutamate incubation blocked the protective effect of clenbute rol. In vivo, clenbuterol (0.01-0.1 mg/kg) reduced the infarct volume in a rat model of permanent focal cerebral ischemia dose-dependently. Nerve grow th factor antisense oligonucleotides injected into the cortical tissue befo re ischemia abolished the cerebroprotective effect of clenbuterol. Our results indicate that the nerve growth factor antisense oligonucleotide presented in this study is a useful tool to investigate the effects of ner ve growth factor knock down. By using the nerve growth factor antisense oli gonucleotide we could demonstrate that nerve growth factor mediated the neu roprotective effects of the beta(2)-adrenoceptor agonist clenbuterol in vit ro and in vivo. (C) 1999 Elsevier Science Ltd. All rights reserved.