Publication Date

2018-08-06

Availability

Embargoed

Embargo Period

2020-08-05

Degree Type

Doctoral Essay

Degree Name

Doctor of Philosophy (PHD)

Department

Neuroscience (Medicine)

Date of Defense

2018-06-26

First Committee Member

W. Dalton Dietrich

Second Committee Member

Robert W. Keane

Third Committee Member

Gerhard Dahl

Fourth Committee Member

M. Ross Bullock

Fifth Committee Member

Gregory Conner

Sixth Committee Member

Deborah Shear

Abstract

Inflammasomes are one of the key regulators of interleukin (IL)-1β mediated inflammatory responses after traumatic brain injury. However, the role of inflammasome signaling after penetrating traumatic brain injury (PTBI) has not been determined. Towards this, adult male Sprague-Dawley rats were subjected to sham or penetrating ballistic-like brain injury (PBBI) surgery and sacrificed at various time points. Tissues were assessed by immunoblot analysis for expression of IL-1β, IL-18 and components of the inflammasome: caspase-1, apoptosis-associated speck like protein containing a caspase activation and recruitment domain (ASC), X-linked inhibitor of apoptosis protein (XIAP), NOD-like receptor protein 3 (NLRP3), and gasdermin-D (GSDMD). Specific cell types expressing inflammasome proteins were evaluated immunohistochemically and assessed quantitatively. Cells generated from tissues were assessed by flow cytometry for caspase-1 activity, cell viability, and cluster of differentiation (CD)45 and CD11b expression. After PBBI, expression of IL-1β, IL-18, caspase-1, ASC, XIAP, and NLRP3 peaked around 48 h. The injured cortex from PBBI animals showed pyroptosome formation evidenced by ASC laddering and contained increased expression of GSDMD at 48 h post-injury. ASC-positive immunoreactive neurons within the perilesional cortex were observed at 24 h. At 48 h, ASC expression was significantly increased in active microglia. At 12 weeks post-PBBI, activated microglia continued to express ASC in the cortex near ASC expressing neurons and in the internal capsule. Flow cytometry analysis revealed an increase in activated microglia and infiltrating CD11b+ leukocytes that express caspase-1 activity and undergo pyroptosis 48 h after injury. Treatment with an antibody that neutralizes ASC decreased the number of activated microglia and infiltrating leukocytes undergoing pyroptosis while not decreasing the overall number of these cell types. This is the first report of inflammasome activation and pyroptosis after PBBI. Our results demonstrate cell-specific patterns of inflammasome activation and pyroptosis predominantly in microglia, suggesting a sustained pro-inflammatory state following PTBI that could underlie the long-term sequlae of the injury.

Keywords

Penetrating Traumatic Brain Injury; Penetrating Ballistic-like Brain Injury; Neuroinflammation; Inflammasome; Microglia

Available for download on Wednesday, August 05, 2020

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