Semkova I, Schilling M, Henrich-Noack P, Rami A, Krieglstein J
Institut für Pharmakologie und Toxikologie, Fachbereich Pharmazie und Lebensmittelchemie, Philipps-Universität, Marburg, Germany.
Brain Res. 1996 Apr 22;717(1-2):44-54. doi: 10.1016/0006-8993(95)01567-1.
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.
先前已经表明，β2 - 肾上腺素能受体激动剂克伦特罗可增强成年大鼠脑中NGF的合成。由于NGF能够保护神经元免受损伤，我们试图弄清楚克伦特罗是否能通过诱导NGF来挽救培养的海马神经元免受兴奋性毒性损伤。在短暂性前脑缺血的大鼠模型中测试了克伦特罗对海马易损CA1亚区神经元的神经保护活性。此外，在局灶性脑缺血的小鼠模型中，研究了克伦特罗减小梗死面积的能力。将混合的海马神经元/胶质细胞培养物暴露于克伦特罗（1至100微摩尔），通过双位点ELISA法显著提高了培养基中测得的NGF含量。体外培养14天后，在无血清培养基中暴露于1毫摩尔L - 谷氨酸1小时，在同一类型细胞中诱导兴奋性毒性损伤。在损伤诱导前4小时直至损伤诱导后18小时（谷氨酸毒性测量时间点），向生长培养基中添加NGF本身（0.15至100纳克/毫升），可保护海马神经元免受兴奋性毒性损伤。在相同实验条件下，克伦特罗（1至100微摩尔）提供了与NGF相似的神经保护作用。在克伦特罗暴露期间向培养基中添加抗NGF单克隆抗体（0.5微克/毫升），可阻断克伦特罗（100微摩尔）对海马培养物中谷氨酸诱导损伤的神经保护活性，表明神经元的挽救是由NGF介导的。在培养物暴露于克伦特罗（1微摩尔）前20分钟添加β - 肾上腺素能受体拮抗剂普萘洛尔（10微摩尔）并在暴露期间保持在培养基中，可逆转神经保护活性，表明克伦特罗诱导的NGF和神经保护作用是通过β - 肾上腺素能受体激活介导的。克伦特罗保护海马神经元的能力在短暂性前脑缺血的大鼠模型体内也得到了证实。腹腔注射克伦特罗（4×1毫克/千克）增加了大鼠海马CA1亚区存活神经元的数量。此外，克伦特罗（0.3和1毫克/千克，腹腔注射；1毫克/千克，皮下注射）在大脑中动脉闭塞后显著减小了小鼠脑表面的梗死面积。目前的数据表明，克伦特罗在培养的海马细胞中诱导NGF合成，并保护海马神经元免受兴奋性毒性损伤。克伦特罗的神经保护活性在两种脑缺血啮齿动物模型体内也得到了证实。结果提供了有力证据，表明克伦特罗的神经保护活性是由β - 肾上腺素能受体激活以及随后NGF表达增加引起的。
