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An In Vitro Trauma Model to Study Rodent and Human Astrocyte Reactivity

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Astrocytes

Part of the book series: Methods in Molecular Biology ((MIMB,volume 814))

Abstract

Protocols are presented describing a unique in vitro injury model and how to culture and mature mouse, rat, and human astrocytes for its use. This injury model produces widespread injury and astrocyte reactivity that enable quantitative measurements of morphological, biochemical, and functional changes in rodent and human reactive astrocytes. To investigate structural and molecular mechanisms of reactivity in vitro, cultured astrocytes need to be purified and then in vitro “matured” to reach a highly differentiated state. This is achieved by culturing astrocytes on deformable collagen-coated membranes in the presence of adult-derived horse serum (HS), followed by its stepwise withdrawal. These in vitro matured, process-bearing, quiescent astrocytes are then subjected to mechanical stretch injury by an abrupt pressure pulse from a pressure control device that briefly deforms the culture well bottom. This inflicts a measured reproducible, widespread strain that induces reactivity and injury in rodent and human astrocytes. Cross-species comparisons are possible because mouse, rat, and human astrocytes are grown using essentially the same in vitro treatment regimen. Human astrocytes from fetal cerebral cortex are compared to those derived from cortical biopsies of epilepsy patients (ages 1–12 years old), with regard to growth, purity, and differentiation. This opens a unique opportunity for future studies on glial biology, maturation, and pathology of human astrocytes. Prototypical astrocyte proteins including GFAP, S100, aquaporin4, glutamate transporters, and tenascin are expressed in mouse, rat, and human in vitro matured astrocyte. Upon pressure-stretching, rodent and human astrocytes undergo dynamic morphological, gene expression, and protein changes that are characteristic for trauma-induced reactive astrogliosis.

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Abbreviations

bFGF:

Basic fibroblast growth factor

CIC II:

Second-generation cell injury controller

CM:

Conditioned medium

CNS:

Central nervous system

Conc.:

Concentration

EGF:

Epidermal growth factor

FBS:

Fetal bovine serum

HS:

Horse serum

Min:

Minutes

RT:

Room temperature

SD:

Standard deviation

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Acknowledgments

Excellent technical assistance has been provided by Cristina Costales, Eunice Kwon, and Ana Fernandez. We greatly appreciate the excellent cooperation of the surgeons Dr. Gary Mathern, Dept. of Neurosurgery and his team, My Huynh and Dr. Julia Chang as well as Dr. Angela Chen, Dept. of OBGYN for providing brain biopsy and human fetal tissue. Special gratitude goes to patients and their parents for providing full consent in donating tissue for research purposes. We acknowledge the efficient assistance in deidentification and transfer of the specimen by Dr. Sarah Dry, Dept. of Pathology and Dr. Harry Vinters Dept. of Neuropathology. We further like to thank Dr. Michael Sofroniew and Rose Korsak, Dept. of Neurobiology as well as Dr. Campagnoni, and his assistants Vance Handley and Vilma Spreuer, Semel Institute, for providing mouse pups and Dr. Jean deVellis, Semel Institute, IDDRC, for providing cell culture and imaging facilities. Thank goes to Drs. Mary Hamby and Ariane Sharif, for their helpful comments to the manuscript.

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  1. Semel Institute for Neuroscience and Human Behavior, UCLA, Los Angeles, CA, USA

    Ina-Beate Wanner

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  1. Ina-Beate Wanner
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Correspondence to Ina-Beate Wanner .

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  1. Dept. Molecular & Experimental, Scripps Research Institute, N. Torrey Pines Road 10550, La Jolla, 92037, California, USA

    Richard Milner

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Wanner, IB. (2012). An In Vitro Trauma Model to Study Rodent and Human Astrocyte Reactivity. In: Milner, R. (eds) Astrocytes. Methods in Molecular Biology, vol 814. Humana Press. https://doi.org/10.1007/978-1-61779-452-0_14

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