Hypoxia ischemia encephalopathy (HIE) is characterized as a lack of oxygen and blood flow to the brain, and is a leading cause of neonatal mortality and morbidity within the United States. HIE causes immediate cell death and oxidative stress resulting in inflammation, energy failure, and ongoing injury. Though there is a lack of effective therapies for HIE, extracellular vesicles (EVs) have shown incredible potential in attenuating oxidative stress and inflammation. EVs are biological nanoparticles with a lipid membrane containing essential biomolecules. EVs participate in cell-to-cell communication as they travel between membranes of cells within the central nervous system (CNS). Previous studies on adult brain injury models show the potential for EVs to drive neuroprotective and anti-inflammatory processes in the brain. The aim of my project is to evaluate neonatal injury responses to brain-derived EVs (BEVs) following HI injury on ex vivo brain tissues. To mimic an ischemic brain environment, I used an oxygen glucose deprivation (OGD) model to induce hypoxia in neonatal rat brain tissues. I quantified time-dependent changes in the gene expression profiles of brain tissues after BEV treatment by performing RNA extractions and reverse transcription-quantitative polymerase chain reactions (RT-qPCR). This allowed me to compare the expression levels of pro-inflammatory and anti-inflammatory markers to determine the therapeutic efficacy of BEVs on an ischemic model. My results suggested that BEV exposure in OGD-injured models decreased cytotoxicity by encouraging microglia (the brain’s immune cells) to transition from inflammatory to anti-inflammatory phenotypes. Results from the RT-qPCR analysis further suggested that BEVs reduced inflammation through the upregulation of anti-inflammatory cytokines observed in the study. This demonstrates that BEVs play a role in reducing cell death and activating anti-inflammatory pathways in the neonatal brain, providing insight into their potential as a therapeutic tool for future interventions aimed at treating HIE.

Neonatal hypoxic-ischemic encephalopathy (HIE), caused by a lack of blood flow and oxygen to the brain, is a major cause of infant mortality. Primary and secondary energy failure caused by HIE activates microglia, resulting in morphological changes and inflammatory cascades that mediate ongoing pathology. Proinflammatory microglia release cytokines and reactive oxygen species that damage oligodendrocytes, the myelinating cells in the brain that supports neuronal function, thereby causing demyelination of neurons. Previous studies in term-equivalent in vivo ferret models showed that microglia respond to injury and treatments with region-dependent cell morphology changes. However, the effect of combinatorial therapy on microglia and oligodendrocyte in a preterm model is unknown. This project aims to quantify image-based morphological features of microglia and oligodendrocyte in response to neuroinflammation and separate and combinatorial treatments in different brain regions of an in vivo preterm ferret model. Using machine learning supported image processing, I quantified microglia and oligodendrocyte morphology in the healthy control group, injury group of two hours of oxygen-glucose deprivation, and treatment groups of azithromycin (AZ), erythropoietin (Epo), and combined AZ+Epo treatment followed by injury. The machine learning algorithm clusters microglia and oligodendrocytes into distinct shape modes with different morphological parameters, such as perimeter, circularity, and aspect ratio. Perimeter and circularity of both microglia and oligodendrocytes show regional heterogeneity within each shape mode while aspect ratio is homogeneous. Microglia perimeter decreases upon injury in crescent and rod-like shape modes. Epo treatment reverses the decrease to the level of nontreated control, but AZ+Epo treatment only partially reversed the decrease. By quantifying microglia and oligodendrocyte morphological response to neuroinflammation and treatments across regions, I non-destructively assessed therapeutic performance of separate and combinatorial treatments in the preterm ferret model. The assessed performance informs therapeutic choices for preterm populations and have the potential for translating to larger animal models.

Immunofluorescent images are a common way to analyze cell response in the presence of brain disease. Microglia - the brain's immune cells - have a range of functional states dependent on their local environment to keep the brain environment healthy. Microglia are typically stained and viewed with immunofluorescent imaging to study the brain's immune response. Microglial functionality and microglia morphology (shape) are highly correlated [5]. By taking and quantifying images of microglia in healthy and diseased brains, we can gain insights into their functional state and their local environment. In addition, most fundamental research about microglia involves the use of animal models, where many species are used to model brain disease. However, limited research directly compares microglia response in one species to another. Previously, research within the Nance Lab has focused on quantifying rat microglial features such as area, perimeter, or circularity [3]. Here, we developed a method to quantify features of microglia, with a focus on microglial branching – the arm-like protrusions from the cell body expanding upon previous work by adding additional branching features to the quantification pipeline to look at the number and length of branches around each cell, which gives us information on the functional state of the cell. We investigated the species-dependent effect on the microglial shape by analyzing images of cells obtained from the neonatal human-term equivalent rat (postnatal day 10, P10), ferret (P21), and mouse (P12). We see qualitative differences in morphology, such as more extensive branching in the rat compared to the ferret. Our ongoing work aims to quantify feature differences in microglia between the rat and ferret and expand to other species.

Hypoxic Ischemic Encephalopathy (HIE) is an injury to a newborn that can occur during pregnancy or the birthing process. HIE is caused by a restriction of blood flow to the brain which leads to inflammation and neuronal death. A myriad of symptoms and disorders including developmental delays and cerebral palsy can result from HIE. The widespread treatment for HIE among clinicians is therapeutic hypothermia; however, this treatment method can only reduce the severity of the injury, is not a curative procedure, and is only effective in a small percentage of babies with HIE. One possible solution to the lack of effective HIE therapies is a nanoparticle-based therapeutic used to decrease inflammatory responses in the brain following HIE. Prior work in the Nance Lab has shown a polymer nanoparticle can specifically target microglial cells in a pro-inflammatory state in an injured brain. In this study, I have successfully loaded the antioxidant N-Acetyl-Cysteine (NAC) into the polymer nanoparticle platform. Using an ex vivo model of neuroinflammation, I displayed that polymer nanoparticles loaded with NAC can be harnessed to change microglia in a proinflammatory state to a more anti-inflammatory phenotype. This nanoparticle treatment was also combined with a priming dose of azithromycin (AZ) prior to NAC-encapsulated nanoparticle administration to further reduce the rate of microglial inflammation in the injured brain. The reduction of microglial inflammation and increase in anti-inflammatory microglial phenotype presence caused by these two therapeutics provides a platform that can be tested in preclinical models of HIE, with the long-term goal to improve the quality of life for children and families affected by HIE.

Hypoxic-ischemic encephalopathy (HIE), resulting from a lack of blood and oxygen flow to the brain, is the leading cause of morbidity and mortality in newborns, and currently has no cure. Our lab is investigating curcumin for use as a neuroprotectant agent, as it has anti-inflammatory, antioxidant, and antiapoptotic effects. Our current studies have been focused on further improving the drug encapsulation of curcumin in a polymeric nanoparticle platform, as well as methods to increase long-term shelf-life stability of the nanoparticle therapeutics. Previous research from our lab has successfully loaded curcumin into poly(ethylene glycol)-poly(lactic-co-glycolic acid) (PEG-PLGA) nanoparticles as a delivery vehicle. PEG-PLGA is an FDA approved, biodegradable polymer platform that allows for improved drug delivery efficiency, controlled and sustained drug release, and improved penetration and diffusion in the brain. We have shown that curcumin loaded PEG-PLGA nanoparticles have resulted in significant neuroprotection when used as a treatment for hypoxic-ischemic neonatal rats (term equivalent to human). In order to progress towards scale-up and clinical translation of the therapeutic, I have tested variations to the formulation method at every step of the formulation process, including changes in PEG-PLGA molecular weight ratios, surfactants, and organic solvents used. I have assessed the impacts of each formulation parameter on colloidal stability and drug loading, with the aim to create a scalable, stable platform that can retain drug delivery and drug activity properties during distribution and shelf-life storage. I have identified that nanoparticle drop size, surfactant type and concentration, and freezing protectant (cryoprotectant) have the biggest impact on drug loading and stability. Improving the stability is the first step in making the therapeutic more accessible, cheaper, and easier to transport for a larger impact.