ABSTRACT
We describe a novel procedure for organotypic cultivation of free-floating brain sections of postnatal rats with a modified roller technique. Three hundred to 350-microm-thick sections of hippocampus are cultured for 13-15 days at 35.5 degrees C in 10-15 ml of feeding medium in 50-100 ml bottles under constant rotation on a horizontal high-speed mini-roller (60 rpm). Histological analysis (paraffin sections, Nissl Cresyl Violet and Hematoxylin/Eosin staining) demonstrates good survival of neuronal and glial cells and complete preservation of the neuronal organization of cultivated hippocampus with minimal central necrosis. This novel protocol permits not only survival and development of long-term three-dimensional organotypic postnatal brain tissue but also allows simultaneous cultivation of any number of brain sections in one bottle (up to 50 and even more) and therefore is useful for high throughput study of neurocytotoxic and hypoxic/ischemic neuronal damage with subsequent histological, immunocytochemical, biochemical, and molecular analysis.
