Defense Date

2020

Document Type

Thesis

Degree Name

Master of Science

Department

Anatomy & Neurobiology

First Advisor

Dr. Andrew Ottens

Second Advisor

Dr. Jeffery Dupree

Third Advisor

Dr. Kurt Hauser

Abstract

Extra-pulmonary effects of nanoparticle inhalation have been well documented; however, mechanisms for conveying peripheral bioactivity remain elusive. Recent studies point to indirect molecular mediators shed into the circulation. Protein and lipid alternatives to cytokines or chemokines have been proposed, yet the diversity among responses suggests a broader assemblage of factors. Recently we have proposed proteolytic fragment peptides as an additional source of bioactivity, which interestingly were significantly associated with exosomal proteins after enrichment analysis. Here we tested whether modeled exposure to multi-walled carbon nanotubes (MWCNT) could alter the composition and bioactivity of circulating exosomes. Male C57BL/6 mice were exposed by oropharyngeal aspiration to 10 or 40 µg MWCNT-7 or vehicle dispersion media (0.6 mg/mL mouse albumin and 0.01 mg/mL DPPC) with serum collected 4 h after. Serum-exosomes were isolated using size exclusion chromatography, with purity affirmed by Western blot, electron microscopy and particle size assays. A dose-dependent changes in the quantity and size of circulating exosomes was observed with MWCNT exposure. Subsequent proteomic mass spectrometry revealed that MWCNT treatment significantly altered serum exosome protein cargo, with particular increases in focal adhesion proteins and decreases in protease inhibitors, which together are consistent with altered signaling. Following up on earlier work that showed MWCNT exposure impaired the blood-brain barrier and promoted neuroinflammatory glial responses, we treated primary astrocyte cultures with the exosomal fraction. GFAP immunofluorescence showed a dose-dependent astrocyte reactivity, with thicker processes and increased GFAP staining. Overall, results here demonstrated that MWCNT exposure has a significant effect on circulating exosomes that altered their trafficked cargo and bioactivity. Consistent with barrier disruption and neuroinflammation in the brains of exposed animals, MWCNT-altered serum-exosomes promoted an astrogliosis phenotype that supports their involvement in driving neurological outcomes after nanoparticle exposure.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

12-4-2020

Available for download on Wednesday, December 03, 2025

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