MK-801 neurotoxicity in cupric silver-stained sections: Lesion reconstruction by 3-dimensional computer image analysis

Routine histopathologic evaluation of the brain (paraffin embedding, hemato xylin and eosin staining) makes it difficult for an investigator to identif y the overall location and relative extent of lesions as they relate to neu ral substructures. Moreover, it is very difficult to convey this informatio n to others who are less familiar with neuroanatomy. This study combined a 3-dimensional imaging program with a cupric silver stain for neuronal degen eration in order to determine the location and extent of a focal lesion pro duced by MK-801 (dizocilpine maleate), a glutamate receptor antagonist that induces necrosis in a small population of neurons in the cortex of rats. A male Sprague-Dawley rat was treated with a subcutaneous dose of MK-801 (10 mg/kg) and was perfused with fixative through the left ventricle 3 days af ter treatment, a time point known to reveal maximal neurotoxic effects. The brain was embedded in a gelatin matrix, frozen, and serially sectioned at a thickness of 40 mu m. The cupric silver method of de Olmos was used to st ain frozen sections at 320-mu m intervals. Using a color charged-couple dev ice (CCD) camera and a macro lens, a series of 3-dimensional images, which encompassed the entire rostral to caudal extent of the brain, was captured. A computer program was written to define internal and external boundaries in these 2-dimensional images. Then, 3-dimensional reconstructions were gen erated on a Silicon Graphics workstation using IRIS "Explorer." The quality of the 3-dimensional reconstructions allowed fur easy identification of va rious neural substructures while clearly revealing the exact location and e xtent of the resulting necrotic neurons that were positively identified by the cupric silver stain. This 3-dimensional lesion reconstruction method pr ovides a powerful tool fur conveying spatial information about the nature o f neurotoxic lesions in the brain. In addition, it may be used to investiga te further dose-response relationships and the effects of other neurotoxica nts.