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

doi:10.1016/S0197-0186(99)00032-7

Neurochemistry International

Volume 35, Issue 1, July 1999, Pages 47-57

https://doi.org/10.1016/S0197-0186(99)00032-7 Get rights and content

Abstract

Previous studies in our laboratory suggested that neuroprotective effects of the β₂-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 μM) 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 μM 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 μM, 30 h), clenbuterol (10 μM) reduced the percentage of damaged neurons after glutamate exposure (0.5 mM, 1 h) to 17%. Pretreatment with nerve growth factor antisense oligonucleotide (1 μM) 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 β₂-adrenoceptor agonist clenbuterol in vitro and in vivo.

Introduction

The survival of neurons from various brain regions have been shown to depend on neurotrophic factors, both during development and in adulthood. A variety of such factors has been identified over recent years, among them the neurotrophins nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4/5 (NT-4/5) (Lewin and Barde, 1996). Some of these factors prevented neuronal cell death in animal models of neuropathy, motoneural disorders and cerebral ischemia (Gao et al., 1997, Hefti, 1997, Lindsay, 1996, Yuen and Mobley, 1995). Thus, they are promising candidates for the therapy of neurodegenerative diseases. The delivery of neurotrophic factors to the CNS is one of the large obstacles against their therapeutic usage, since these proteins are not able to cross the blood-brain-barrier. The induction of growth factor synthesis in the brain by systemically administered lipophilic drugs, such as the β₂-adrenergic agonist clenbuterol, would be an elegant way to overcome these problems of application (Carswell, 1993, Krieglstein et al., 1998, Mocchetti, 1991). In our laboratory, clenbuterol has been shown to protect neurons from excitotoxic damage in mixed primary cultures of hippocampal neurons (Semkova et al., 1996b). The neuroprotective effect of clenbuterol was blocked by NGF-antibodies suggesting a mechanism mediated by NGF (Semkova et al., 1996b). It has been proposed that astrocytes released the neurotrophin after β₂-adrenoceptor stimulation in the mixed hippocampal cells used in the previous study. Astrocytes have been widely discussed as a possible source of neuroprotective factors that support neuronal survival under lesioning conditions (Lu et al., 1991, Mantyh et al., 1995, Nieto-Sampedro et al., 1982, Rudge, 1993).

In rat brain tissue, the stimulation of central β₂-receptors induced the synthesis of NGF- and bFGF-mRNA (Follesa and Mocchetti, 1993, Hayes et al., 1995). Furthermore, the protective effects of clenbuterol against ischemic damage in rat brain tissue were accompanied by increased expression of NGF mRNA (Semkova et al., 1996a). These findings suggested a neuroprotective potency of β₂-adrenergic drugs against neuronal damage in vivo that was mediated by induction of NGF. To further clarify this issue, it would be necessary to block the expression of NGF. Antisense oligodeoxynucleotides (ODN) have been used to knock down the expression of specific proteins. Therefore, antisense ODN are useful tools to investigate the role of specific proteins in vitro and in vivo (Schlingensiepen et al., 1997, Wahlestedt, 1994, Wahlestedt et al., 1993). However, before investigating the role of a specific protein by using antisense-technology, it is necessary to characterize the efficacy and the specificity of the antisense ODN and, furthermore, to exclude toxic effects on the system investigated in vitro or in vivo. To our knowledge, there is no report about an antisense ODN directed against rat NGF expression.

Here, we report about the effects of a newly designed NGF antisense ODN on the expression of NGF in primary cultures of mixed hippocampal cells and astrocytes. We demonstrated that the neuroprotective effect of clenbuterol against glutamate-induced excitototoxicity was blocked by antisense mediated NGF knock down. Furthermore, we injected the NGF antisense oligonucleotide into cortical tissue to find out whether NGF mediated the protective effect of clenbuterol against ischemic brain damage in vivo.

Section snippets

Animals

Neonatal (P1–2) pups from Fischer 344 rats (Charles River, Sulzfeld, Germany) were used for preparing cultured cells. Male Long Evans rats (Møllegaard, Denmark) were used for ischemia experiments. The animals were maintained under controlled light and environmental conditions (12:12 h dark/light cycle, 23±1°C, 55% relative humidity) and had free access to food (Altromin, Lage, Germany) and water.

Cell culture agents and other substances

Eagle’s minimum essential medium (MEM) (containing 2 mM l-glutamine, 28 mM of glucose and 22 mM of

Stimulation of NGF mRNA and NGF protein synthesis in cultured astrocytes by clenbuterol

NGF mRNA levels were determined after exposure of rat cortical astrocytes to clenbuterol (1–100 μM) for 4 h by RT-PCR. Fig. 1a represents a photograph of the RT-PCR products visualized after electrophoresis in a 2% agarose gel containing ethidium bromide. The bands showing the expression of NGF mRNA in the samples are located between 300 and 500 bp of the size standard (expected size 391 bp). Clenbuterol (1–100 μM) increased the expression of NGF mRNA in astrocytes after 3 h of incubation. In

Discussion

Investigating the NGF antisense oligonucleotide effect in the present study, we could demonstrate that the neuroprotective effect of the β₂-adrenergic drug clenbuterol against glutamate-induced damage of cultured hippocampal neurons was mediated by NGF. Moreover, the NGF antisense blocked the protective effect of clenbuterol in a model of focal cerebral ischemia in the rat, suggesting a mechanism mediated by NGF.

These findings are in line with our previous results showing that clenbuterol

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

Abstract

Introduction

Section snippets

Animals

Cell culture agents and other substances

Stimulation of NGF mRNA and NGF protein synthesis in cultured astrocytes by clenbuterol

Discussion

Experimental Neurobiology

Experimental Neurology

Cell

Brain Research

European Journal Pharmacology

Brain Research Bulletin

Trends in Pharmacological Sciences

Molecular Medicine Today

Basic principles of antisense therapeutics