|
Sign In to gain access to subscriptions and/or personal tools.
|
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
Andrew S. Fix
The Procter & Gamble Co., Miami Valley Laboratories, Cincinnati, Ohio 45253
Joseph F. Ross
The Procter & Gamble Co., Miami Valley Laboratories, Cincinnati, Ohio 45253
Susan R. Stitzel
The Procter & Gamble Co., Miami Valley Laboratories, Cincinnati, Ohio 45253
Robert C. Switzer
Neuroscience Associates, Knoxville, Tennessee 37922
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, X 10 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.
Key Words: Brain • cupric-silver stain • dizocilpine maleate • glial fibrillary acidic protein (GFAP) • GFAP immunohistochemistry • lectin histochemistry • neuropathology • neurotoxicity • risk assessment • toxicity
Toxicologic Pathology, Vol. 24, No. 3,
291-304 (1996)
DOI: 10.1177/019262339602400305
 CiteULike  Complore  Connotea  Del.icio.us  Digg  Reddit  Technorati  Twitter What's this?
This article has been cited by other articles:
|
|
|
|
 
J. P. Morrison, T.-V. Ton, J. B. Collins, R. C. Switzer, P. B. Little, D. L. Morgan, and R. C. Sills
Gene Expression Studies Reveal That DNA Damage, Vascular Perturbation, and Inflammation Contribute to the Pathogenesis of Carbonyl Sulfide Neurotoxicity
Toxicol Pathol,
June 1, 2009;
37(4):
502 - 511.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. Bolon, D. C. Anthony, M. Butt, D. Dorman, M. V. Green, P. B. Little, W. M. Valentine, D. Weinstock, J. Yan, and R. C. Sills
"Current Pathology Techniques" Symposium Review: Advances and Issues in Neuropathology
Toxicol Pathol,
October 1, 2008;
36(6):
871 - 889.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. Bolon, R. Garman, K. Jensen, G. Krinke, B. Stuart, and An Ad Hoc Working Group of the STP Scientific and
A 'Best Practices' Approach to Neuropathologic Assessment in Developmental Neurotoxicity Testing--for Today
Toxicol Pathol,
April 1, 2006;
34(3):
296 - 313.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Z. Cheng, S. Z. Guo, A. J. Lipton, and D. Gozal
Domoic Acid Lesions in Nucleus of the Solitary Tract: Time-Dependent Recovery of Hypoxic Ventilatory Response and Peripheral Afferent Axonal Plasticity
J. Neurosci.,
April 15, 2002;
22(8):
3215 - 3226.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. Bolon
Comparative and Correlative Neuroanatomy for the Toxicologic Pathologist
Toxicol Pathol,
January 1, 2000;
28(1):
6 - 27.
[Abstract]
[PDF]
|
|
|
|
|
|
|
R. C. Switzer
Application of Silver Degeneration Stains for Neurotoxicity Testing
Toxicol Pathol,
January 1, 2000;
28(1):
70 - 83.
[Abstract]
[PDF]
|
|
|
|
|
|
|
A. S. Fix, S. R. Stitzel, G. M. Ridder, and R. C. Switzer
MK-801 Neurotoxicity in Cupric Silver-Stained Sections: Lesion Reconstruction by 3-Dimensional Computer Image Analysis
Toxicol Pathol,
January 1, 2000;
28(1):
84 - 90.
[Abstract]
[PDF]
|
|
|
|
|
|
|
L. C. Schmued and K. J. Hopkins
Fluoro-Jade: Novel Fluorochromes for Detecting Toxicant-Induced Neuronal Degeneration
Toxicol Pathol,
January 1, 2000;
28(1):
91 - 99.
[Abstract]
[PDF]
|
|
|
|
|
|
|
J.F. Ross
ECOs, FOBs, and UFOs: Making Sense of Observational Data
Toxicol Pathol,
January 1, 2000;
28(1):
132 - 136.
[Abstract]
[PDF]
|
|
|
|
|
|
|
J. M. Siegel, R. Nienhuis, S. Gulyani, S. Ouyang, M. F. Wu, E. Mignot, R. C. Switzer, G. McMurry, and M. Cornford
Neuronal Degeneration in Canine Narcolepsy
J. Neurosci.,
January 1, 1999;
19(1):
248 - 257.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. A. Collins, J.-y. Zou, and E. J. Neafsey
Brain damage due to episodic alcohol exposure in vivo and in vitro: furosemide neuroprotection implicates edema-based mechanism
FASEB J,
February 1, 1998;
12(2):
221 - 230.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
A. Radovsky, L. Katz, U. Ebmeyer, and P. Safar
Ischemic Neurons in Rat Brains After 6, 8, or 10 Minutes of Transient Hypoxic Ischemia
Toxicol Pathol,
September 1, 1997;
25(5):
500 - 505.
[Abstract]
[PDF]
|
|
|
|
|
|
|
T. A. Bertram
Editorial: Neurotoxicity: A Field of Increasing Importance for the Toxicologic Pathologist
Toxicol Pathol,
May 1, 1996;
24(3):
384 - 385.
[PDF]
|
|
|
|
|
|
|
M. K. Ahlijanian, N. X. Barrezueta, R. D. Williams, A. Jakowski, K. P. Kowsz, S. McCarthy, T. Coskran, A. Carlo, P. A. Seymour, J. E. Burkhardt, et al.
Hyperphosphorylated tau and neurofilament and cytoskeletal disruptions in mice overexpressing human p25, an activator of cdk5
PNAS,
March 14, 2000;
97(6):
2910 - 2915.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
