Anthony Norman

Project title: Identification of black carbon in the brain using Raman microscopy.

Degree: MPH | Program: Occupational and Environmental Medicine (OEM) | Project type: Thesis/Dissertation
Completed in: 2021 | Faculty advisor: Coralynn Sack


Introduction: Air pollution is a significant contributor to adverse health outcomes in humans around the world. Particulate matter has been linked to numerous cardiovascular, pulmonary and neurological diseases and associated with increased systemic inflammation and direct cellular toxicity. Earlier studies have suggested three potential routes of entry for particulate matter into the central nervous system: systemic circulation and active transport into cells; leaky capillaries compromising the blood-brain barrier allowing for infiltration of the parenchyma or translocation along the olfactory system. Newer imaging technologies have been developed to allow for direct, non-destructive molecular identification of compounds through excitation of the molecular chemical bond vibrational energies, with minimal preparation. The use of Raman microscopy of multiple varieties, in particular, is quickly expanding throughout the biological sciences. We sought to identify the carbon core material of ultrafine particulate matter in the CNS of mouse and human subjects with known or estimated exposures to diesel exhaust or air pollution, respectively, using Raman microscopic techniques.

Methods: Three groups of tissues were examined. First, mouse alveolar macrophages were exposed to increasing concentrations of diesel exhaust particles (DEP) and imaged with stimulated Raman scattering (SRS) microscopy. Second, a small group of naïve and experimental mice perinatally exposed to diesel exhaust (DE) were sacrificed and their brain tissues directly imaged with SRS along the olfactory bulb, corpus callosum and frontal lobes to identify areas of potential DEP deposition. Third, human brain biopsies from subjects participating in the Adult Changes in Thought (ACT) study were obtained and also imaged with SRS along the olfactory, cerebellar and frontal lobe tissues for signs of black carbon particulate matter to be quantified and compared to estimated individual air pollution exposures.

Results: The macrophage study readily identified the DEP and image analysis suggested a dose-dependent response to the uptake of DEP by the macrophages in a positive, linear fashion, though also with a wide variation in material uptake and extracellular particulate matter seen at most inoculating concentrations. Imaging of the mouse CNS tissues was successful at identifying several large structural features of the parenchyma as well as cellular elements such as red blood cells remaining in the larger blood vessels, as well as likely glial and neuronal cells. Particulate matter was not positively identified between the naïve and DE exposed subjects, however. Imaging of the human brain tissues was complicated by inadequate tissue thickness and the use of a paraffin based embedding media, which created a complex background spectral and fluorescence response. Identification of cellular and parenchymal structures was difficult and no particulate matter was found or distinguished from the background signal intensity.

Discussion: SRS is a useful technique to identify the unique molecular response of DEP from surrounding biological material. The dose response relationship established with in vitro exposure of DEP to alveolar macrophages provides a basis for future, more established studies to build upon. There are numerous potential technical and biological reasons for not being able to identify particles in the mouse CNS tissues, including inadequate resolution of the microscope system, inappropriate specimen preparation or sampling error versus the particulate matter not reaching the deeper tissues of the brain or otherwise being cleared over time. There are numerous similar reasons for being unable to identify the particles in the human biopsy samples, the most likely of which is the inadequate specimen preparation for the SRS microscope. Further study is required to fully determine the potential efficacy of SRS in the process of identifying particulate matter within the CNS and potential correlation to inflammatory induction or similar responses.