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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Morrison,  B., Saatman,  K. E., Meaney,  D. F., and McIntosh,  T. K., 1998, “In Vitro Central Nervous System Models of Mechanically Induced Trauma: A Review,” J. Neurotrauma, 15, pp. 911–928.
Donnelly,  B. R., and Medige,  J., 1997, “Shear Properties of Human Brain Tissue,” ASME J. Biomech. Eng., 119, pp. 423–432.
Galbraith,  J. A., Thibault,  L. E., and Matteson,  D. R., 1993, “Mechanical and Electrical Responses of the Giant Squid Axon to Simple Elongation,” ASME J. Biomech. Eng., 115, pp. 13–22.
Cargill,  R. S., and Thibault,  L. E., 1996, “Acute Alterations in [Ca2+]I in NG108-15 Cells Subjected to High Strain Rate Deformation and Chemical Hypoxia: An in Vitro Model for Neural Trauma,” J. Neurotrauma, 13, pp. 396–407.
Laplaca,  M. C., Lee,  V. M.-Y., and Thibault,  L. E., 1997, “An in Vitro Model of Traumatic Neuronal Injury: Loading Rate-Dependent Changes in Acute Cytosolic Calcium and Lactate Dehydrogenase Release,” J. Neurotrauma, 14, pp. 355–368.
Laplaca,  M. C., and Thibault,  L. E., 1997, “An in Vitro Traumatic Injury Model to Examine the Response of Neurons to a Hydrodynamically Induced Deformation,” Ann. Biomed. Eng., 25, pp. 665–677.
Rzigalinski,  B. A., Weber,  J. T., Willoughby,  K. A., and Ellis,  E. F., 1998, “Intracellular Free Calcium Dynamics in Stretch-Injured Astrocytes,” J. Neurochem., 70, pp. 2377–2385.
Ellis,  E. F., McKinney,  J. S., Willoughby,  K. A., Liang,  S., and Povlishock,  J. T., 1995, “A New Model for Rapid Stretch-Induced Injury of Cells in Culture: Characterization of the Model Using Astrocytes,” J. Neurotrauma, 12, pp. 325–339.
McKinney,  J. S., Willoughby,  K. A., Liang,  S., and Ellis,  E. F., 1996, “Stretch-Induced Injury of Cultured Neuronal, Glial, and Endothelial Cells: Effect of Polyethylene Glycol-Conjugated Superoxide Dismutase,” Stroke, 27, pp. 934–940.
Tavalin,  S. J., Ellis,  E. F., and Satin,  L. S., 1995, “Mechanical Perturbation of Cultured Cortical–Neurons Reveals a Stretch-Induced Delayed Depolarization,” J. Neurophysiol., 74, pp. 2767–2773.
Sall,  J. M., Morehead,  M., Murphy,  S., Goldman,  H., and Walker,  P. D., 1996, “Alterations in CNS Gene Expression in a Rodent Model of Moderate Traumatic Brain Injury Complicated by Acute Alcohol Intoxication,” Exp. Neurol., 139, pp. 257–268.
Raghupathi,  R., and Mcintosh,  T. K., 1996, “Regionally and Temporally Distinct Patterns of Induction of C-Fos, C-Jun, and Junb mRNAs Following Experimental Brain Injury in the Rat,” Brain Res. Mol. Brain Res., 37, pp. 134–144.
Yakovlev,  A. G., and Faden,  A. I., 1995, “Molecular Strategies in CNS Injury,” J. Neurotrauma, 12, pp. 767–777.
Hayes,  R. L., Yang,  K., Raghupathi,  R., and Mcintosh,  T. K., 1995, “Changes in Gene Expression Following Traumatic Brain Injury in the Rat,” J. Neurotrauma, 12, pp. 779–790.
Yang,  K., Perez-Polo,  J. R., Mu,  X. S., Yan,  H. Q., Xue,  J. J., Iwamoto,  Y., Liu,  S. J., Dixon,  C. E., and Hayes,  R. L., 1996, “Increased Expression of Brain-Derived Neurotrophic Factor but Not Neurotrophin-3 mRNA in Rat Brain After Cortical Impact Injury,” J. Neurosci. Res., 44, pp. 157–164.
Hicks,  R. R., Numan,  S., Dhillon,  H. S., Prasad,  M. R., and Seroogy,  K. B., 1997, “Alterations in BDNF and NT-3 mRNAs in Rat Hippocampus After Experimental Brain Trauma,” Brain Res. Mol. Brain Res., 48, pp. 401–406.
Yang,  K., Mu,  X. S., Xue,  J. J., Perez-Polo,  J. R., and Hayes,  R. L., 1995, “Regional and Temporal Profiles of C-Fos and Nerve Growth Factor mRNA Expression in Rat Brain After Lateral Cortical Impact Injury,” J. Neurosci. Res., 42, pp. 571–578.
Dekosky,  S. T., Goss,  J. R., Miller,  P. D., Styren,  S. D., Kochanek,  P. M., and Marion,  D., 1994, “Upregulation of Nerve Growth Factor Following Cortical Trauma,” Exp. Neurol., 130, pp. 173–177.
Yakovlev,  A. G., Knoblach,  S. M., Fan,  L., Fox,  G. B., Goodnight,  R., and Faden,  A. I., 1997, “Activation of CPP32-Like Caspases Contributes to Neuronal Apoptosis and Neurological Dysfunction After Traumatic Brain Injury,” J. Neurosci., 17, pp. 7415–7424.
Fan,  L., Young,  P. R., Barone,  F. C., Feuerstein,  G. Z., Smith,  D. H., and Mcintosh,  T. K., 1996, “Experimental Brain Injury Induces Differential Expression of Tumor Necrosis Factor-α mRNA in the CNS,” Brain Res. Mol. Brain Res., 36, pp. 287–291.
Fan,  L., Young,  P. R., Barone,  F. C., Feuerstein,  G. Z., Smith,  D. H., and Mcintosh,  T. K., 1995, “Experimental Brain Injury Induces Expression of Interleukin-1β mRNA in the Rat Brain,” Brain Res. Mol. Brain Res., 30, pp. 125–130.
Clark,  R. S., Chen,  J., Watkins,  S. C., Kochanek,  P. M., Chen,  M., Stetler,  R. A., Loeffert,  J. E., and Graham,  S. H., 1997, “Apoptosis-Suppressor Gene Bcl-2 Expression After Traumatic Brain Injury in Rats,” J. Neurosci., 17, pp. 9172–9182.
Clark,  R. S., Kochanek,  P. M., Chen,  M., Watkins,  S. C., Marion,  D. W., Chen,  J., Hamilton,  R. L., Loeffert,  J. E., and Graham,  S. H., 1999, “Increases in Bcl-2 and Cleavage of Caspase-1 and Caspase-3 in Human Brain After Head Injury,” FASEB J., 13, pp. 813–821.
Morrison,  B., Meaney,  D. F., and Mcintosh,  T. K., 1998, “Mechanical Characterization of an in Vitro Device to Quantitatively Injure Living Brain Tissue,” Ann. Biomed. Eng., 26, pp. 381–390.
Pleasure,  S. J., Selzer,  M. E., and Lee,  V. M.-Y., 1989, “Lamprey Neurofilaments Combine in One Subunit the Features of Each Mammalian NF Triplet Protein But Are Highly Phosphorylated Only in Large Axons,” J. Neurosci., 9, pp. 698–709.
Van Gelder,  R. N., Von Zastrow,  M. E., Yool,  A., Dement,  W. C., Barchas,  J. D., and Eberwine,  J. H., 1990, “Amplified RNA Synthesized From Limited Quantities of Heterogeneous cDNA,” Proc. Natl. Acad. Sci. USA, 87, pp. 1663–1667.
Chow,  N., Cox,  C., Callahan,  L. M., Weimer,  J. M., Guo,  L., and Coleman,  P. D., 1998, “Expression Profiles of Multiple Genes in Single Neurons of Alzheimer’s Disease,” Proc. Natl. Acad. Sci. USA, 95, pp. 9620–9625.
Eberwine,  J., Yeh,  H., Miyashiro,  K., Cao,  Y., Nair,  S., Finnell,  R., Zettel,  M., and Coleman,  P., 1992, “Analysis of Gene Expression in Single Live Neurons,” Proc. Natl. Acad. Sci. USA, 89, pp. 3010–3014.
Crino,  P. B., Trojanowski,  J. Q., Dichter,  M. A., and Eberwine,  J., 1996, “Embryonic Neuronal Markers in Tuberous Sclerosis: Single-Cell Molecular Pathology,” Proc. Natl. Acad. Sci. USA, 93, pp. 14152–14157.
Mackler,  S. A., and Eberwine,  J. H., 1993, “Diversity of Glutamate Receptor Subunit mRNA Expression Within Live Hippocampal CA1 Neurons,” Mol. Pharmacol., 44, pp. 308–315.
Derisi,  J. L., Iyer,  V. R., and Brown,  P. O., 1997, “Exploring the Metabolic and Genetic Control of Gene Expression on a Genomic Scale,” Science, 278, pp. 680–686.
Hummel,  T. J., and Sligo,  J., 1971, “Empirical Comparison of Univariate and Multivariate Analysis of Variance Procedures,” Psychol. Bull., 76, pp. 49–57.
Stevens, J., 1986, “Multiple Regression,” in Applied Multivariate Statistics for the Social Sciences, Lawrence Erlbaum Associates, Hillsdale, NJ, pp. 51–112.
Koh,  J. Y., and Choi,  D. W., 1987, “Quantitative Determination of Glutamate Mediated Cortical Neuronal Injury in Cell Culture By Lactate Dehydrogenase Efflux Assay,” J. Neurosci. Methods, 20, pp. 83–90.
Pauwels,  P. J., Van Assouw,  H. P., Leysen,  J. E., and Janssen,  P. A., 1989, “Ca2+-Mediated Neuronal Death in Rat Brain Neuronal Cultures by Veratridine: Protection by Flunarizine,” Mol. Pharmacol., 36, pp. 525–531.
Lyall,  F., Deehan,  M. R., Greer,  I. A., Boswell,  F., Brown,  W. C., and McInnes,  G. T., 1994, “Mechanical Stretch Increases Proto-Oncogene Expression and Phosphoinositide Turnover in Vascular Smooth Muscle Cells,” J. Hypertens., 12, pp. 1139–1145.
Mason,  D. J., Suva,  L. J., Genever,  P. G., Patton,  A. J., Steuckle,  S., Hillam,  R. A., and Skerry,  T. M., 1997, “Mechanically Regulated Expression of a Neural Glutamate Transporter in Bone: a Role for Excitatory Amino Acids As Osteotropic Agents?” Bone, 20, pp. 199–205.
Allen,  S. P., Wade,  S. S., and Prewitt,  R. L., 1997, “Myogenic Tone Attenuates Pressure-Induced Gene Expression in Isolated Small Arteries,” Hypertension, 30, pp. 203–208.
Sumpio,  B. E., Chang,  R., Xu,  W. J., Wang,  X. J., and Du,  W., 1997, “Regulation of Tpa in Endothelial Cells Exposed to Cyclic Strain: Role of CRE, AP-2, and SSRE Binding Sites,” Am. J. Phys., 273, pp. C1441–C1448.
Malek,  A. M., Gibbons,  G. H., Dzau,  V. J., and Izumo,  S., 1993, “Fluid Shear Stress Differentially Modulates Expression of Genes Encoding Basic Fibroblast Growth Factor and Platelet-Derived Growth Factor B Chain in Vascular Endothelium,” J. Clin. Invest., 92, pp. 2013–2021.
Zhang,  Z., Xiao,  Z., and Diamond,  S. L., 1999, “Shear Stress Induction of C-Type Natriuretic Peptide (CNP) in Endothelial Cell Is Independent of NO Autocrine Signaling,” Ann. Biomed. Eng., 27, pp. 419–426.
Conti,  A. C., Raghupathi,  R., Trojanowski,  J. Q., and Mcintosh,  T. K., 1998, “Experimental Brain Injury Induces Regionally Distinct Apoptosis During the Acute and Delayed Post-Traumatic Period,” J. Neurosci., 18, pp. 5663–5672.
Chao,  D. T., and Korsmeyer,  S. J., 1998, “BCL-2 Family: Regulators of Cell Death,” Annu. Rev. Immunol., 16, pp. 395–419.


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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