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MRM Protocols Assist Brain Studies

By Eddy Ball
October 2006

Kennita Johnson
Principal Investigator Kennita Johnson. (Photo courtesy of Steve McCaw)

brain structure
With imaging, researchers could look at internal structures of the brain (hippocampus highlighted) from different angles. Shown are a ventral (left) and a dorsal view (right) of the same normal whole brain. (Image courtesy of NIEHS Laboratory of Experimental Pathology)

In a recent study published in the September issue of the journal NeuroToxicology, an interdisciplinary team of NIEHS scientists in the Laboratory of Experimental Pathology demonstrated new applications for laboratory protocols that can expand the potential of developmental neurotoxicity testing. According to the researchers, the use of magnetic resonance microscopy (MRM) in combination with the Cavalieri method of volumetric estimation shows good agreement with conventional methods, which utilize linear measurements on stained histologic sections. The protocol also can help investigators utilize time and personnel more efficiently and will provide a more thorough examination of the whole brain with the ability to obtain three-dimensional volumetric measurements of important brain structures. These features will permit additional insights from studies on the effects of exposure to environmental agents on the developing rat brain.

Because it is non-invasive and examines the intact organ in place, MRM is more adaptable than conventional histological methods, giving it potential for use in longitudinal studies of live animals and ultimately in human studies. In addition, the sensitivity of three-dimensional MRM may allow scientists to identify exposures at lower doses than those which cause gross neuropathology and readily detect effects of exposure not easily determined by the limited tissue sampling of conventional histology. Researchers can also use MRM as an adjunct to conventional histology, since it does not destroy the organ and offers a unique dataset that is easily stored and accessed for further interpretation. "The data is entirely digital," explained lead researcher Kennita Johnson, a post-doctoral biomedical engineer at NIEHS. "We can share it by e-mail or put it on a server."

Along with Johnson, neuroscientist Jill Marcus and pathologist Robert Maronpot collaborated with specialists from two area labs, MRPath, Inc., and Integrated Laboratory Systems, Inc., in the study. The team studied first-time pregnant female Sprague-Dawley rats that were injected with either methylazoxymethanol acetate (MAM) or saline during their pregnancies. A compound found in seeds from the tropical plant Cycad, MAM is a known neurotoxin, DNA methylating agent and carcinogen with documented effects on rat brain.

At 23 and 60 days following birth, investigators imaged rat brains to measure changes in form, volume and size. They used a small animal MR imaging system to scan intact brains and then performed conventional histologic examination of stained slices to identify histologic changes. To determine volume of the whole brains, the researchers initially used the gold-standard water displacement method before examining MRM data with the more efficient Cavalieri method, a mathematical model that can be easily used with computer software to make rapid estimations of volume.

Study findings were consistent with earlier research into the effects of MAM. The compound had no long-term effect on body weight of control and dose animals. The researchers did find a statistically significant difference in fixed brain weight, whole brain weight and morphology, and whole brain volume between control and dose animals. Examining the MRM whole-brain scans, researchers were able to detect a trend toward reduction in size of the hippocampus and cerebrum in the high dose animals.

The researchers concluded that their protocol offered several additional benefits over conventional methods. Three-dimensional MR images made possible a more complete morphological assessment using both linear and volumetric measurements by revealing microscopic changes that impact brain function before cell death or lesions become apparent. MRM overcame problems with precise orientation inherent in histologic sectioning that can confound measurements of brain structures. MRM enabled researchers to obtain three dimensional images throughout the brain in contrast to time-consuming discrete histological slices. Using the Cavalieri method with MRM, scientists can obtain an unbiased estimation of substructure volumes of any size or shape, eliminating the process of exhaustively tracing brain substructures.

Citation: Johnson K, Ryan L, Davis J, Elmore A, Guenther B, Marcus J, Maronpot RR. 2006. Application of magnetic resonance imaging in developmental neurotoxicity testing: A pilot study. Neurotoxicology 27(5): 846-851.

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