Annually approximately 1.5 million Americans suffer from a traumatic brain injury (TBI) increasing the risk of developing a further neurological complication later in life [1-3]. The molecular drivers of the subsequent ensuing pathologies after the initial injury event are vast and include signaling processes that may contribute to neurodegenerative diseases such as Alzheimer’s Disease (AD). One such molecular signaling pathway that may link TBI to AD is necroptosis. Necroptosis is an atypical mode of cell death compared with traditional apoptosis, both of which have been demonstrated to be present post-TBI [4-6]. Necroptosis is initiated by tissue necrosis factor (TNF) signaling through the RIPK1/RIPK3/MLKL pathway, leading to cell failure and subsequent death. Prior studies in rodent TBI models report necroptotic activity acutely after injury, within 48 hours. Here, the study objective was to recapitulate prior data and characterize MLKL and RIPK1 cortical expression post-TBI with our lab’s controlled cortical impact mouse model. Using standard immunohistochemistry approaches, it was determined that the tissue sections acquired by prior lab members were of poor quality to conduct robust MLKL and RIPK1 immunostaining assessment. Therefore, the thesis focused on presenting the staining method completed. The discussion also expanded on expected results from these studies regarding the spatial distribution necroptotic signaling in this TBI model.

Traumatic brain injury involves a primary mechanical injury that is followed by a secondary<br/>inflammatory cascade. The inflammatory cascade in the CNS releases cytokines which are<br/>associated with leukocytosis and a systemic immune response. Acute changes to peripheral<br/>immune cell populations post-TBI include a 4.5-fold increase of neutrophils 3 hours post-injury,<br/>and 2.7-fold or higher increase of monocytes 24 hours post-injury. Flow Cytometry is a<br/>technique that integrates fluidics, optics, and electronics to characterize cells based on their light<br/>scatter and antigen expression via monoclonal antibodies conjugated to fluorochromes. Flow<br/>cytometry is a valuable tool in cell characterization however the standard technique for data<br/>analysis, manual gating, is associated with inefficiency, subjectivity, and irreproducibility.<br/>Unsupervised analysis that uses algorithms packaged as plug-ins for flow cytometry analysis<br/>software has been discussed as a solution to the limits of manual gating and as an alternative<br/>method of data visualization and exploration. This investigation evaluated the use of tSNE<br/>(dimensionality reduction algorithm) and FlowSOM (population clustering algorithm)<br/>unsupervised flow cytometry analysis of immune cell population changes in female mice that<br/>have been exposed to a LPS-induced systemic inflammatory challenge, results were compared to<br/>those of manual gating. Flow cytometry data was obtained from blood samples taken prior to and<br/>24 hours after LPS injection. Unsupervised analysis was able to identify populations of<br/>neutrophils and pro-inflammatory/anti-inflammatory monocytes, it also identified several more<br/>populations however further inquiry with a more specific fluorescent panel would be required to<br/>establish the specificity and validity of these populations. Unsupervised analysis with tSNE and<br/>FlowSOM demonstrated the efficient and intuitive nature of the technique, however it also<br/>illustrated the importance of the investigator in preparing data and modulating plug-in settings.

Traumatic brain injury (TBI) is a widespread health issue that affects approximately 1.7 million lives per year. The effects of TBI go past the incident of primary injury, as chronic damage can follow for years and cause irreversible neurodegeneration. A potential strategy for repair that has been studied is cell transplantation, as neural stem cells improve neurological function. While promising, neural stem cell transplantation presents challenges due to a relatively low survival rate post-implantation and issues with determining the optimal method of transplantation. Shear-thinning hydrogels are a type of hydrogel whose linkages break when under shear stress, exhibiting viscous flow, but reform and recover upon relaxation. Such properties allow them to be easily injected for minimally invasive delivery, while also shielding encapsulated cells from high shear forces, which would normally degrade the function and viability of such cells. As such, it is salient to research whether shear-thinning hydrogels are feasible candidates in neural cell transplantation applications for neuroregenerative medicine. In this honors thesis, shear-thinning hydrogels were formed through guest-host interactions of adamantane modified HA (guest ad-HA) and beta-cyclodextrin modified HA (host CD-HA). The purpose of the study was to characterize the injection force profile of different weight percentages of the HA shear-thinning hydrogel. The break force and average glide force were also compared between the differing weight percentages. By understanding the force exerted on the hydrogel when being injected, we could characterize how neural cells may respond to encapsulation and injection within HA shear-thinning hydrogels. We identified that 5% weight HA hydrogel required greater injection force than 4% weight HA hydrogel to be fully delivered. Such contexts are valuable, as this implies that higher weight percentage gels impart higher shear forces on encapsulated cells than lower weight gels. Further study is required to optimize our injection force system’s sensitivity and to investigate if cell encapsulation increases the force required for injection.