Integrated Evaluation of Central Nervous System Lesions: Stains for Neurons, Astrocytes, and Microglia Reveal the Spatial and Temporal Features of MK-801-induced Neuronal Necrosis in the Rat Cerebral Cortex

Abstract

Routinely processed, hematoxylin and eosin (H&E)-stained slides are typically used to assess the morphologic integrity of the central nervous system in neurotoxicity safety studies. However, the value of special stains for improving neuropathologic evaluations during the assessment of neurotoxicity has been emphasized in the neuroscience literature and by regulatory agencies. The primary objective of the present study was to characterize the spatial and temporal changes in neurons, astrocytes, and microglia after dizocilpine maleate (MK-801)-induced focal neuronal necrosis in the posterior cingulate/retrosplenial (PC/RS) cortex of the rat. A secondary objective was to evaluate the application of special stains and a novel sectioning procedure for detecting neurotoxicity. Sixty adult male Sprague-Dawley rats were treated with sterile water vehicle or 10 mg/kg MK-801 and perfused through the left ventricle (pumped at 65 mm Hg pressure) with 10% neutral buffered formalin or 4% paraformaldehyde at 4 hr and on days 1, 3, 7, 14, and 28 after treatment. For light microscopic evaluation, brain sections were stained with H&E, a special cupric-silver (CS) stain that selectively impregnates degenerating neurons and makes them readily evident, glial fibrillary acidic protein (GFAP) immunohistochemistry for astrocytes, and Griffonia simplicifolia isolectin B4(GSA) histochemistry for microglia. Brains perfusion-fixed with 4% paraformaldehyde were prepared for CS staining with a novel frozen-sectioning procedure for multiple embedding in a composite gelatin block. In H&E sections from treated rats, necrotic nerve cell bodies were observed in PC/RS cortical layers 3 and 4 on days 1, 3, 7, and 14, but not on day 28. These necrotic neurons required high magnification for detection (x20 objective, x10 ocular). In contrast, degenerating neurons selectively stained with CS were observed in the same location as necrotic neurons seen with H&E but at low magnification (x2 objective, x10 ocular). Cupric-silver staining showed details not seen with H&E, including dendritic and axonal degeneration with progressive fragmentation. Beginning on day 3, GFAP immunohistochemistry revealed hypertrophic astrocytes in a diffuse pattern throughout the region of cell body necrosis, a change that persisted throughout the study. However, GSA lectin histochemistry identified a few reactive microglia on day 1 in a multifocal pattern throughout the region of cell body necrosis. Reactive microglia were observed on days 3, 7, and 14, but not on day 28. Glial changes observed with H&E staining were limited to an increase in the cellularity of glial cell nuclei in the area of neuronal necrosis. This study provides a comprehensive and integrated view of the temporal changes occurring in neurons, astrocytes, and microglia during acute neurotoxic injury. Moreover, advantages for using new staining and sectioning methodologies to enhance the toxicologic evaluation of the central nervous system are demonstrated.

Hyperphosphorylated tau and neurofilament and cytoskeletal disruptions in mice overexpressing human p25, an activator of cdk5

“…Trimmed tissues were dehydrated and embedded as above, and sections (6 m) were stained with the modified Bielschowsky stain (21,22).…”

Methods to identify and characterize developmental neurotoxicity for human health risk assessment. II: neuropathology.

“…These procedures have been used for the identification of apoptotic neurons during the early postnatal period (22) as well as for neurotoxicity evaluations (23,24). To date, silver degeneration stains have been applied mostly in adult neurotoxicity studies (25,26), but their potential for adding to the understanding of developmental neurotoxicity is beginning to be explored. For example, silver degeneration stains have been used recently to show that ethanol and other drugs that antagonize the N -methyl-D-aspartate receptor (the major excitatory receptor in the brain) dramatically increase the rate of apoptosis during the early postnatal period in rat pups (27,28).…”

“…Many researchers consider an increased level of GFAP a definitive marker of brain injury, including damage induced by neurotoxicants. GFAP content can be evaluated biochemically or immunohistochemically in tissue sections (26,42,43). Enzyme-linked immunosorbent assays for GFAP performed in developmental neurotoxicity studies have revealed alterations in GFAP content in response to such neurotoxicants as polychlorinated biphenyls, methylmercury, methylazoxymethanol, 6-hydroxydopamine, tributyltin, and triethyltin (37)(38)(39)(40)(41)(42)(43)(44)(45)(46).…”

“…Cytokine production and antigen presentation are associated with this activation and transformation. Changes in microglial morphology and immunology correlate with neurotoxicity in adult rats (26,50). The nature of these glial reactions may vary with different injurious stimuli.…”

Distribution and Severity of Neuropathology in β-Mannosidase-Deficient Mice is Strain Dependent

“…Following removal from the skull, brains were placed in 4% paraformaldehyde for 24 h, and then transferred to cacodylate storage buffer. Brains were embedded in a 5Â5 array in a gelatin matrix using MultiBrain Technology (NeuroScience Associates, Knoxville, TN) as reported in previous studies (Fix et al 1996;Ong et al 2001). The block of embedded brains was frozen and sectioned coronally at 30 mm beginning at the olfactory bulb and proceeding to the medulla.…”