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TECHNICAL PAPERS

Dynamic Mechanical Stretch of Organotypic Brain Slice Cultures Induces Differential Genomic Expression: Relationship to Mechanical Parameters

[+] Author and Article Information
Barclay Morrison

Departments of Neurosurgery and Bioengineering, University of Pennsylvania, Philadelphia, PA 19104Veterans Administration Medical Center, Philadelphia, PA 19104

David F. Meaney, Susan S. Margulies

Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104Veterans Administration Medical Center, Philadelphia, PA 19104

Tracy K. McIntosh

Departments of Neurosurgery and Bioengineering, University of Pennsylvania, Philadelphia, PA 19104Veterans Administration Medical Center, Philadelphia, PA 19104

J Biomech Eng 122(3), 224-230 (Feb 06, 2000) (7 pages) doi:10.1115/1.429650 History: Received December 08, 1999; Revised February 06, 2000
Copyright © 2000 by ASME
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References

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Figures

Grahic Jump Location
An annotated picture of the Mechanical Stretch Device demonstrates its major components. A laser displacement transducer (LDT), mounted on a three-axis manipulator, measures the dynamic displacement of the membrane, allowing for calculation of the peak membrane strain. The organotypic brain slice cultures are grown in custom-built wells on a silicone membrane that is deformed by an applied vacuum as is depicted in the exploded view illustration.
Grahic Jump Location
(A) A schematic representation of the deformation of an organotypic brain slice culture during the dynamic stretch. (B) Representative data traces including displacement, strain, and strain rate acquired from the laser displacement transducer during the dynamic displacement of the culture substrate, i.e., the silicone membrane. This particular event was terminated in approximately 40 ms with a strain of approximately 0.35 and a strain rate of approximately 25 s−1.
Grahic Jump Location
Photomicrographs of organotypic brain slice cultures after 18 days in vitro demonstrating the presence of various cell types. (A) A transverse section cut through the culture and stained with H & E demonstrates that the cultures remain several cell layers thick (approximately 275 μm) with cells dispersed throughout the thickness. The lower surface was adhered to the membrane and the upper surface was bathed in media. All other sections shown (B–D) were cut in a plane parallel to the membrane. (B) Cells stained for GFAP demonstrated a typical astrocytic morphology of a starlike shape with short, thick processes. (C) Neurons stained for NF-M throughout the cultures as well as in anatomically defined structures such as the CA3 region of the hippocampus. Neurons in this region demonstrated the typical morphology of pyramidal neurons with large nuclei and apical processes. (D) Oligodendrocytes stained for CNP and were concentrated within white matter tracts such as the alveus. These cells were typically smaller than other cells and closely apposed to adjacent cells in a linear fashion. Scale bar represents 50 μm in all panels.
Grahic Jump Location
LDH release was measured at 6, 24, and 48 hours after stretch or sham stretch and calculated as a percentage of total releasable LDH. At each time point, stretched cultures released significantly more LDH than did control cultures, indicating that dynamic substrate strain resulted in membrane damage. Both control and stretched cultures demonstrated a basal level of LDH release that is normally observed in primary cultures 3435. For the control group n=5, and for the stretched group n=6. Values are presented as mean ±SEM.
Grahic Jump Location
Stretch of organotypic brain slice cultures affected the expression of several genes. Twenty-four hours after stretch the expression of bcl-2 (p<0.001), CREB (p<0.001), and GAD65(p<0.01) was significantly decreased, whereas that of BDNF (p<0.02), NGF (p<0.02), and TrkA (p<0.05) was significantly increased over expression in control cultures. For the control group n=7, and for the stretched group n=41. Values are presented as mean ±SEM.

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