Cerebrolysin® protects neurons from ischemia-induced loss of microtubule — associated protein 2
Microtubule — associated protein 2 (MAP2) was studied in cultured neurons from chick embryo hemispheres after neuronal damage caused by cytotoxic ischemia. Cortical cells were pretreated with 0, 10, 20, 40 or 80µ1 Cerebrolysin® per ml media. After 8 days in vitro (DIV), ischemia was induced by a 60 minutes incubation with 1 mM L-glutamate. After this toxic stress, cells were allowed to recover from ischemia for 48 hours in a medium again supplemented with Cerebrolysin® in the appropriate concentration. For immunoblot analysis cortical cells were lysed in 1% SDS and the supernatants were assayed for protein content by the method of Lowry. Proteins were then separated and transferred to nitrocellulose (0.5 A for 2h). Western blots were blocked with low fat milk and incubated with the primary monoclonal antibody (Chemicon) reacting with all forms of MAP2 (Map2a, Map2b, Map2c) for 12–14 hours at 4°C. Incubation with the horseradish peroxidase — conjugated secondary antibody lasted for 2h. For detection we used a nonradioactive ECL (enhanced chemiluminescence) system (Amersham).