Stroke is the leading cause of long-term disability in the United States. Although acute revascularization therapies can be used to abort or reduce stroke burden, there are currently no drugs that improve recovery after a stroke has happened. The inflammatory response is a promising target for such therapies as it occurs in the days and weeks after a stroke and can be both detrimental and beneficial. A major unanswered question is how the injured brain modulates immune responses, and if there are molecular pathways that can be utilized to exert beneficial or limit detrimental effects on functional recovery via modulating the overall immune response. Astrocytes are a key component of the brain's injury response - so-called "reactive astrocytes" are ubiquitous after brain injury. They are also increasingly recognized as key components of the brain's innate immune system. We propose to ask if Transforming Growth Factor Beta (TGF?) signaling in astrocytes modulates inflammation after stroke because it is a master regulator of immune responses. TGF? can resolve immune responses after injury and drive immune cell phenotypes towards less inflammatory states. Our preliminary experiments show that TGF? signaling is increased in the brain after stroke, persists for weeks, and occurs in reactive astrocytes. To test if TGF?'s function in reactive astrocytes mirror its role in other types of immune cells we constructed mice in which TGF? signaling is decreased only in astrocytes. We have found that primary astrocytes from these mice exhibit a more "pro-inflammatory" phenotype after oxygen- glucose deprivation, and the mice themselves demonstrate increased inflammatory responses after stroke. Based on this data we hypothesize that after stroke, TGF? signaling (1) occurs in reactive astrocytes, (2) limits the inflammatory response, and (3) improves functional recovery. We plan to test our hypothesis in three Specific Aims.
In Aim 1 we will use reporter mice and immunohistochemistry to determine patterns of TGF? signaling after stroke. We hypothesize that there are increased responses to TGF-? for weeks after stroke, and that reactive astrocytes are responding to TGF? after stroke.
In Aim 2 we will test the function of astrocytic TGF? signaling in the neuroinflammatory response to ischemia, using genetic and pharmacological approaches and in vivo and in vitro experiments to target TGF? signaling in astrocytes. We hypothesize that astrocytic TGF? signaling drives resolution of the immune response to stroke.
In Aim 3 we will use a genetic mouse model to ask if stroke-induced astrocytic TGF? signaling is beneficial or detrimental for functional recovery. We predict that astrocytic TGF? signaling improves recovery from stroke. With the completion of the proposed experiments we will have defined the length and cell specificity of TGF? responses after stroke. We will gain insight into how astrocytes influence the immune response to stroke, and into the functional diversity of reactive astrocytes. Our findings may lead to therapies that will target the brain's immune responses and benefit patients who present for medical care in the days after stroke.

Public Health Relevance

Stroke is the third leading cause of death in the US, and a leading cause of disability, and there are currently no drugs that improve recovery after stroke. Neuroinflammation affects many processes important for recovery from stroke and modulating neuroinflammation is therefore likely to be a way we can improve recovery from stroke. In this application we propose to study the effects of a master regulator of neuroinflammation, transforming growth factor beta, to determine how its effects in astrocytes can be manipulated to increase successful recovery from stroke.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01NS067132-04
Application #
8656157
Study Section
Clinical Neuroimmunology and Brain Tumors Study Section (CNBT)
Program Officer
Bosetti, Francesca
Project Start
Project End
Budget Start
Budget End
Support Year
4
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Stanford University
Department
Neurology
Type
Schools of Medicine
DUNS #
City
Stanford
State
CA
Country
United States
Zip Code
94304
Doyle, Kristian P; Buckwalter, Marion S (2014) A mouse model of permanent focal ischemia: distal middle cerebral artery occlusion. Methods Mol Biol 1135:103-10
Cekanaviciute, Egle; Fathali, Nancy; Doyle, Kristian P et al. (2014) Astrocytic transforming growth factor-beta signaling reduces subacute neuroinflammation after stroke in mice. Glia 62:1227-40
Cekanaviciute, Egle; Dietrich, Hans K; Axtell, Robert C et al. (2014) Astrocytic TGF-? signaling limits inflammation and reduces neuronal damage during central nervous system Toxoplasma infection. J Immunol 193:139-49