Bound Volume Number

2

Document Type

Honors Capstone Project

Date of Submission

Spring 5-1-2015

Capstone Advisor

Dr. Mary Lou Vallano

Honors Reader

Dr. John Russell

Capstone Major

Biology

Capstone College

Arts and Science

Audio/Visual Component

no

Keywords

hemorrhagic stroke, hemin, microglia

Capstone Prize Winner

no

Won Capstone Funding

no

Honors Categories

Social Sciences

Subject Categories

Cellular and Molecular Physiology | Medical Physiology | Other Neuroscience and Neurobiology

Abstract

An in vitro model of intracerebral hemorrhage was established to examine the protective versus cytotoxic roles of microglia in the context of mild versus severe injury. Co-cultures of microglia, astrocytes, and granule neurons were prepared from the cerebellar cortex of neonatal rats, and grown in standard medium containing fetal bovine serum or, in some cases, a serum-free chemically defined medium. To mimic hemorrhagic stroke, co-cultures grown for 7-8 days in vitro were challenged with two different concentrations of the toxic blood product hemin, corresponding to a mild versus a severe brain bleed. Immunocyto-chemical, real-time RT-PCR, iron deposition, and cell survival assays were performed to assess the effects of hemin, with emphasis on the functional effector states of microglia. In cultures grown in serum-containing medium, the lower concentration of hemin (20 mM) induced significant expression of the protective enzyme, heme oxygenase 1 (HO-1). Consistent with this, iron deposition was localized to the microglia in the cultures. Only a modest induction of the inflammatory molecule TNF-a, and no significant cell death was observed 24 hrs after hemin addition. In contrast, the higher concentration of hemin (100 mM) induced greater expression of HO-1, and iron deposition was detected in all three cell types in the cultures. Moreover, significant and robust induction of the inflammatory molecules TNF-a, COX-2, iNOS, and significant neuronal death were observed. These results suggest that microglia are protective and serve to effectively catabolize hemin when exposed to 20 mM hemin. However, increasing the hemin concentration to 100 mM appears to exceed the capacity of the microglia to effectively catabolize the hemin load, resulting in an inflammatory and cytotoxic environment. Serum proteins, in particular albumin and hemopexin, can bind blood products such as hemin, and slowly release them to be processed by microglia after a brain bleed. To test the possible protective effect of albumin, co-cultures grown in a chemically defined medium lacking serum were exposed to hemin as described above, with or without co-addition of an equivalent amount of albumin. In these cultures, significant neuronal death was observed in response to 20 mM hemin, with essentially total cell death in response to 100 mM hemin. Addition of albumin to the cultures significantly decreased the amount of neuronal death in response to both concentrations of hemin. These data suggest that addition of agents that bind toxic blood products may serve as protective agents after hemorrhagic stroke. In future studies, this in vitro model should prove valuable in characterizing the signaling pathways underlying distinct functional effector states of microglia, and developing strategies to preserve the protective phenotype.

Creative Commons License

Creative Commons Attribution 3.0 License
This work is licensed under a Creative Commons Attribution 3.0 License.

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