ISOLATION OF PRIMARY ASTROCYTES FROM HUMAN BRAIN TISSUE AND ASSESSMENT OF PROTOTYPICAL INFLAMMATORY RESPONSES FOR NEURODEGENERATIVE RESEARCH

Date

2014-03

Authors

Borgmann, Kathleen R.
Tang, Lin
Ghorpade, Anuja

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Abstract

Purpose (a): A common link in CNS disease is inflammation and the contribution of astrocyte inflammatory responses to neurodegeneration remains a focus of investigation. Non-human glial models may be limited in providing data that extrapolate directly to human neurodegenerative diseases, thus much remains to be learned in the genetically relevant context of primary human astroglial cultures. Methods (b): Here we describe the isolation and purification of primary human astrocytes from fetal brain in detail. We expand this protocol to include the assessment of astrocyte responses to inflammation through changes in cell morphology and expression of astrocyte specific markers, mitochondrial pore opening and activity, proinflammatory chemokine secretion and glutamate uptake. Results (c): Pure cultures were uniform in size and shape, and at least 95% positive for astrocyte markers. Mitochondrial pore staining revealed punctate calcein staining, which was decreased during inflammation. Upon treatment with a prototypical mediator of astrocyte inflammatory responses, interleukin (IL)-1beta, astrocyte processes became constricted; indicating a reactive astrocytic state, chemokine secretion increased significantly and the ability of astrocytes to clear glutamate was significantly impaired. Untreated cultures that demonstrated reactive phenotypes or those that failed to attain reactive states upon IL-1beta-treatment were excluded. Conclusions (d): These parameters established a framework to assess the overall purity, health, responsiveness to inflammation and thus the suitability of the culture for experimental use of primary human astrocyte cultures for neurodegenerative research.

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