Clenbuterol protects mouse cerebral cortex and rat hippocampus from ischemic damage and attenuates glutamate neurotoxicity in cultured hippocampal neurons by induction of NGF.
It has been shown previously that clenbuterol, a beta 2-adrenergic receptor agonist, enhances NGF synthesis in adult rat brain. Since NGF is able to protect neurons against damage, we tried to find out whether clenbuterol can rescue cultured hippocampal neurons from excitotoxic damage by induction of NGF. The neuroprotective activity of clenbuterol on neurons in the vulnerable CA1 subfield of the hippocampus was tested in a rat model of transient forebrain ischemia. Additionally, in the mouse model of focal cerebral ischemia the ability of clenbuterol to reduce the infarct size was examined. Exposure of mixed neuronal/glial hippocampal cultures to clenbuterol (1 to 100 microM) enhanced significantly the content of NGF measured in the culture medium by two-site ELISA. The excitotoxic injury was induced in the same type of cells after 14 days in vitro by exposure to 1 mM L-glutamate for 1 h in serum-free medium. NGF itself (0.15 to 100 ng/ml) added to the growth medium 4 h before until 18 h after induction of injury (the point of glutamate-toxicity measurement), protected hippocampal neurons from excitotoxic damage. Clenbuterol (1 to 100 microM) provided similar neuroprotection as NGF under the same experimental conditions. The neuroprotective activity of clenbuterol (100 microM) against glutamate-induced damage in hippocampal cultures was blocked by anti-NGF monoclonal antibodies (0.5 microgram/ml) added to the medium during the clenbuterol exposure, demonstrating that the neuronal rescue is mediated by NGF. Propranolol, a beta-adrenergic receptor antagonist (10 microM) added 20 min before and kept in the medium during exposure of the cultures to clenbuterol (1 microM) reversed the neuroprotective activity, suggesting that the induction of NGF and neuroprotection caused by clenbuterol are mediated via beta-adrenergic receptor activation. The capacity of clenbuterol to protect hippocampal neurons was also demonstrated in vivo in a rat model of transient forebrain ischemia. Clenbuterol (4 x 1 mg/kg) administered intraperitoneally increased the number of viable neurons in CA1 subfield of the rat hippocampus. Furthermore, clenbuterol (0.3 and 1 mg/kg, i.p. and 1 mg/kg, s.c.) reduced significantly the infarct area on the mouse brain surface after occlusion of the middle cerebral artery. The present data demonstrate that clenbuterol induces NGF synthesis in cultured hippocampal cells and protects hippocampal neurons from excitotoxic damage. The neuroprotective activity of clenbuterol is also demonstrated in vivo in two rodent models of cerebral ischemia. The results offer strong evidence that the neuroprotective activity of clenbuterol is caused by activation of beta-adrenergic receptors and the subsequent increased expression of NGF.
Semkova
I
I
Institut für Pharmakologie und Toxikologie, Fachbereich Pharmazie und Lebensmittelchemie, Philipps-Universität, Marburg, Germany.
Schilling
M
M
Henrich-Noack
P
P
Rami
A
A
Krieglstein
J
J
D000818:Animals; D000831:Animals, Newborn; D002545:Brain Ischemia / Q000188:drug therapy; D002478:Cells, Cultured / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D002540:Cerebral Cortex / Q000098:blood supply / Q000187:drug effects*; D002976:Clenbuterol / Q000494:pharmacology*; D018698:Glutamic Acid / Q000494:pharmacology; D006624:Hippocampus / Q000098:blood supply / Q000166:cytology* / Q000187:drug effects; D007124:Immunoenzyme Techniques; D007150:Immunohistochemistry; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D009414:Nerve Growth Factors / Q000032:analysis / Q000096:biosynthesis*; D009474:Neurons / Q000737:chemistry / Q000166:cytology / Q000187:drug effects; D018696:Neuroprotective Agents / Q000494:pharmacology*; D009498:Neurotoxins / Q000494:pharmacology; D051381:Rats; D011916:Rats, Inbred F344; D017208:Rats, Wistar; D015427:Reperfusion Injury / Q000188:drug therapy*
D009414:Nerve Growth Factors; D018696:Neuroprotective Agents; D009498:Neurotoxins; D018698:Glutamic Acid; D002976:Clenbuterol