NGF mediates the neuroprotective effect of the β2-adrenoceptor agonist clenbuterol in vitro and in vivo:: evidence from an NGF-antisense study

Previous studies in our laboratory suggested that neuroprotective effects of the beta(2)-adrenoceptor agonist clenbuterol in vitro and in vivo occurred due to enhanced synthesis of nerve growth factor. The aim of the present study was to evaluate the effects of a phosphothioated NGF oligodeoxynucleotide 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 cultures of rat cortical astrocytes. Nerve growth factor antisense oligonucleotide (0.3-1 mu M for 3 days) reduced the content of nerve growth factor protein in the medium of the astrocytes concentration-dependently to 20% of control level. Nerve growth factor content in the medium of mixed hippocampal cells was reduced to 55% of sister cultures receiving the vehicle or a random control oligonucleotide. In mixed hippocampal cultures pretreated with random oligonucleotide (1 mu M, 30 h), clenbuterol (10 mu M) reduced the percentage of damaged neurons after glutamate exposure (0.5 mM, 1 h) to 17%. Pretreatment with nerve growth factor antisense oligonucleotide (1 mu M) for 30 h before glutamate incubation blocked the protective effect of clenbuterol. 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 growth factor antisense oligonucleotides injected into the cortical tissue before 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 nerve growth factor knock down. By using the nerve growth factor antisense oligonucleotide we could demonstrate that nerve growth factor mediated the neuroprotective effects of the beta(2)-adrenoceptor agonist clenbuterol in vitro and in vivo. (C) 1999 Elsevier Science Ltd. All rights reserved.