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- All Subjects: Microparticles
- All Subjects: Neurology
- Creators: VanAuker, Michael
Description
The combination of immunohistochemical (IHC) stainings and optical microscopy has allowed for the visualization of specific microscopic structures within tissue; however, limitations in light and antibody penetration mitigate the scale on which these images can be taken (Alshammari et al, 2016; Marx, 2014). Tissue clearing, specifically the removal of lipids to improve sample transparency, solves the former weakness well, but does not improve antibody penetration significantly (Chung et al, 2013; Treweek et al, 2015). Therefore, there is a need to equalize the maximum depth that light can pass through a section with the depth at which there is recognizable fluorescence. This is particularly important when staining blood vessels as traditional size limitations exclusively allows for cross sectional visualization. Passive CLARITY Technique (PACT) has been at the forefront of tissue clearing protocols, utilizing an acrylamide hydrogel solution to maintain structure and sodium dodecyl sulfate to wash out lipids (Tomer et al, 2014). PACT is limited in its ability to clear larger sections and is not conducive to IHC antibody diffusion (Treweek et al, 2015). In order to circumvent these drawbacks, CUBIC was developed as an alternative passive protocol, aimed at being scalable to any tissue size (Richardson, 2015; Susaki et al, 2015). This study compared the effectiveness of both protocols in high and low lipid tissues in the context of blood vessel staining efficacy. Upon initial comparison, it became apparent that there was a statistically significant difference in mean DAPI intensity at all depths, up to 200 micrometers, between CUBIC and PACT \u2014 the former showcasing brighter stainings. Moreover, it was found that PACT does not remove erythrocytes from the tissue meaning that their auto-fluorescence is seen during imaging. Therefore, for blood vessel stainings, only CUBIC was optimized and quantitatively analyzed. In both tissue conditions as well as for two stainings, DAPI and fibronectin (FNCT), optimized CUBIC demonstrated a statistically significant difference from standard CUBIC with regards to mean fluorescent intensity.
ContributorsSidhu, Gurpaul Singh (Author) / VanAuker, Michael (Thesis director) / Kodibagkar, Vikram (Committee member) / Harrington Bioengineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
With microspheres growing in popularity as viable systems for targeted drug therapeutics, there exist a host of diseases and pathology induced side effects which could be treated with poly(lactic-co-glycolic acid) [PLGA] microparticle systems [6,10,12]. While PLGA systems are already applied in a wide variety the clinical setting [11], microparticles still have some way to go before they are viable systems for drug delivery. One of the main reasons for this is a lack of fabrication processes and systems which produce monodisperse particles while also being feasible for industrialization [10]. This honors thesis investigates various microparticle fabrication techniques \u2014 two using mechanical agitation and one using fluid dynamics \u2014 with the long term goal of incorporating norepinephrine and adenosine into the particles for metabolic stimulatory purposes. It was found that mechanical agitation processes lead to large values for dispersity and the polydispersity index while fluid dynamics methods have the potential to create more uniform and predictable outcomes. The research concludes by needing further investigation into methods and prototype systems involving fluid dynamics methods; however, these systems yield promising results for fabricating monodisperse particles which have the potential to encapsulate a wide variety of therapeutic drugs.
ContributorsRiley, Levi Louis (Author) / Vernon, Brent (Thesis director) / VanAuker, Michael (Committee member) / Harrington Bioengineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2018-12