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MK-801 Neurotoxicity in Cupric Silver-Stained Sections: Lesion Reconstruction by 3-Dimensional Computer Image Analysis

The Procter & Gamble Company, Miami Valley Laboratories, Cincinnati, Ohio 45253

Susan R. Stitzel

The Procter & Gamble Company, Miami Valley Laboratories, Cincinnati, Ohio 45253

Gregg M. Ridder

The Procter & Gamble Company, Miami Valley Laboratories, Cincinnati, Ohio 45253

Robert C. Switzer

Neuroscience Associates, Knoxville, Tennessee 37922

Routine histopathologic evaluation of the brain (paraffin embedding, hematoxylin and eosin staining) makes it difficult for an investigator to identify the overall location and relative extent of lesions as they relate to neural substructures. Moreover, it is very difficult to convey this information to others who are less familiar with neuroanatomy. This study combined a 3-dimensional imaging program with a cupric silver stain for neuronal degeneration in order to determine the location and extent of a focal lesion produced 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 after 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 µm. The cupric silver method of de Olmos was used to stain frozen sections at 320-µm intervals. Using a color charged-couple device (CCD) camera and a macro lens, a series of 2-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 generated on a Silicon Graphics workstation using IRIS "Explorer." The quality of the 3-dimensional reconstructions allowed for easy identification of various neural substructures while clearly revealing the exact location and extent of the resulting necrotic neurons that were positively identified by the cupric silver stain. This 3-dimensional lesion reconstruction method provides a powerful tool for conveying spatial information about the nature of neurotoxic lesions in the brain. In addition, it may be used to investigate further dose-response relationships and the effects of other neurotoxicants.

Key Words: Brain lesion • dizocilpine maleate • neuronal degeneration • neuropathology • neurotoxicant • N-methyl-D-aspartate (NMDA) antagonist

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