The blood-brain barrier (BBB) is regulated by factors that can be secreted by multiple cell types, including astrocytes, that maintain the BBB in health and promote repair after injury. However, astrocyte contributions to the BBB are largely assumed from transplantation studies in which astrocyte progenitor grafts conferred BBB-like properties to tissues that normally lack a BBB. To determine if astrocytes contribute an essential and non-redundant function in maintaining the healthy BBB, I genetically ablated a small number of astrocytes using a conditional, tamoxifen-inducible mouse model. Within 2 hours after induction, I observed sparse astrocyte death in the cortex and leakage of the small molecule Cadaverine and large plasma protein fibrinogen, which are normally contained by a functional BBB. Vessels within regions of ablated astrocytes showed reduced expression of the tight junction protein zonula occludens-1, indicating impairment of the physical barrier formed between endothelial cells. Cadaverine leakage persisted for weeks, a feature I also found in mice after mild concussive traumatic brain injury (cTBI), thus highlighting the potential for revealing astrocyte roles in post-injury repair. Unlike the genetic ablation model, astrocytes within Cadaverine leakage areas did not undergo cell death after cTBI and instead downregulated homeostatic proteins. Our preliminary results show this atypical phenotype appearing 10 minutes after cTBI, along with severe vessel rupture, BBB leakage, and disruption of endfoot and basement membrane proteins. This damage persists for months, suggesting that the BBB fails to repair in these areas. Our results provide direct in-vivo evidence for essential astrocyte roles in the maintenance of the healthy BBB. Maintenance and/or repair fail after mild concussive cTBI, possibly contributing to irreversible progression to neurodegenerative diseases. / Doctor of Philosophy / The blood-brain barrier (BBB) is a unique property of blood vessels in the Central Nervous System (CNS) different from other vessels in the body. The physically tight barrier of the BBB is formed by tight junction proteins between endothelial cells and limits paracellular diffusion. The metabolic barrier is formed by concentrations of glucose transporters that promote transport of essential nutrients to the brain. Lastly, a transport barrier limits the passage of molecules and cells across the endothelial cell layer, preventing the entry of non-essential molecules, including pathogens and immune cells found in the blood. The BBB is thought to be induced and maintained by factors secreted by nearby cells in the brain. Among these cells are astrocytes, a type of glial cell that nearly completely cover blood vessels with their processes called endfeet. This strategic positioning led the field to assume that astrocytes are responsible for generating the unique properties of the BBB. Yet little direct evidence exists to support this conclusion, and newer evidence calls into question if astrocytes are even needed for BBB functions. To test this, I used a genetic mouse model to induce death of small numbers of astrocytes in adult mice. This caused leakage of blood contents of various sizes into the brain. In addition, the tight junction proteins responsible for forming the physical BBB were disrupted. These effects remained for weeks, a feature I also found after mild concussive traumatic brain injury (cTBI). This suggests that astrocytes may have an additional function in repairing the injured BBB. Our results demonstrate an essential role for astrocytes in the maintenance of the healthy adult BBB. Maintenance and/or its repair fail after cTBI, possibly contributing to the cascade into irreversible progression to neurodegenerative diseases.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/112807 |
| Date | 14 June 2021 |
| Creators | Heithoff, Benjamin Patrick |
| Contributors | Biological Sciences, Robel, Stefanie, Fox, Michael A., Olsen, Michelle Lynne, Cimini, Daniela |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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