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.
Time-Lapse Visualization of Microglia Cell Processes using Fluorescent Miniature (Miniscope) Imaging
One approach to develop new tools for stem cell transplants may be to look towards the endogenous repair response for inspiration. Specifically, activated cell types surrounding the injury secrete the chemokine stromal cell-derived factor-1α (SDF-1α), which has been shown to play a critical role in recruiting endogenous neural progenitor/stem cells (NPSCs) to the site of injury. Therefore, it was hypothesized that improving NPSC response to SDF-1α may be a viable mechanism for improving NPSC transplant retention and migration into the surrounding host tissue. To this end, work presented here has 1. identified critical extracellular signals that mediate the NPSC response to SDF-1α, 2. incorporated these findings into the development of a transplantation platform that increases NPSC responsiveness to SDF-1α and 3. observed increased NPSC responsiveness to local exogenous SDF-1α signaling following transplantation within our novel system. Future work will include studies investigating NSPC response to endogenous, injury-induced SDF-1α and the application of this work to understanding differences between stem cell sources and their implications in cell therapies.
To determine if Egr3 alters Htr2a transcription under stress, I examined messenger ribonucleic acid (mRNA) levels of these two genes in wildtype (WT) and Egr3 -/- mice after 6hrs of sleep deprivation (SD). I found both genes are increased in WT mice after SD compared with controls. In addition, Egr3 is required for Htr2a induction because SD fails to induce Htr2a expression in Egr3 -/- mice. Next, I performed chromatin immunoprecipitation (ChIP) to determine if EGR3 binds to Htr2a promoter in vivo. I found a significant increase of EGR3 binding to Htr2a distal promoter 2hrs after seizure. To determine the functionality of this binding, I co-transfected the CMV- EGR3 vector or CMV- vector alone with the Htr2a distal promoter reporter clone. I found overexpression of EGR3 activates the Htr2a distal promoter-driven luciferase gene. Although the ChIP assay shows no direct binding of EGR3 to Htr2a proximal promoter, I found EGR3 overexpression activates Htr2a proximal promoter-driven luciferase gene. These findings suggest that EGR3 regulates Htr2a probably through both direct and indirect ways.
Over-the-counter (OTC) analgesics, omega-3 fatty acids, specialized pro-resolving mediators (SPMs), and remote ischemic conditioning (RIC) were administered before or following midline fluid percussion injury. Behavioral, histological, and molecular analyses were used to assess treatment effects on functional outcome and secondary injury progression. Acute administration of common OTC analgesics had little effect on post-injury outcome in mice. Dietary supplementation with omega-3 fatty acid docosahexaenoic acid (DHA) prior to or following diffuse TBI significantly reduced injury-induced sensory sensitivity and markers of neuroinflammation with no effect on spatial learning. Intraperitoneal administration of omega-3 fatty acid-derived SPM resolvin E1 significantly increased post-injury sleep and suppressed microglial activation. Aspirin-triggered (AT) resolvin D1 administration improved both motor and cognitive outcome following diffuse TBI. RIC treatment in mice demonstrated little effect on functional outcome from diffuse TBI. Untargeted proteomic analysis of plasma samples from RIC-treated mice was used to identify candidate molecular correlates of RIC. Identification of these candidates represents a vital first step in elucidating the neuroprotective mechanisms underlying RIC. The overall findings suggest that omega-3 fatty acid supplementation, SPM administration, and RIC may serve as effective practical therapies to reduce the somatic, cognitive, and neurological burden of diffuse TBI felt by millions of Americans.
Domain antibody fragment (dAb) phage display, a powerful screening technique to uncover protein-protein interactions, has been applied to biomarker discovery in various cancers and more recently, neurological conditions such as Alzheimer’s Disease and stroke. The small size of dAbs (12-15 kDa) and ability to screen against brain vasculature make them ideal for interacting with the neural milieu in vivo. Despite these characteristics, implementation of dAb phage display to elucidate temporal mechanisms of TBI has yet to reach its full potential.
My dissertation employs a unique target identification pipeline that entails in vivo dAb phage display and next generation sequencing (NGS) analysis to screen for temporal biomarkers of TBI. Using a mouse model of controlled cortical impact (CCI) injury, targeting motifs were designed based on the heavy complementarity determining region (HCDR3) structure of dAbs with preferential binding to acute (1 day) and subacute (7 days) post-injury timepoints. Bioreactivity for these two constructs was validated via immunohistochemistry. Further, immunoprecipitation-mass spectrometry analysis identified temporally distinct candidate biological targets in brain tissue lysate.
The pipeline of phage display followed by NGS analysis demonstrated a unique approach to discover motifs that are sensitive to the heterogeneous and diverse pathology caused by neural injury. This strategy successfully achieves 1) target motif identification for TBI at distinct timepoints and 2) characterization of their spatiotemporal specificity.
As many as 20–55% of patients with a history of traumatic brain injury (TBI) experience chronic endocrine dysfunction, leading to impaired quality of life, impaired rehabilitation efforts and lowered life expectancy. Endocrine dysfunction after TBI is thought to result from acceleration–deceleration forces to the brain within the skull, creating enduring hypothalamic and pituitary neuropathology, and subsequent hypothalamic–pituitary endocrine (HPE) dysfunction. These experiments were designed to test the hypothesis that a single diffuse TBI results in chronic dysfunction of corticosterone (CORT), a glucocorticoid released in response to stress and testosterone. We used a rodent model of diffuse TBI induced by midline fluid percussion injury (mFPI). At 2months postinjury compared with uninjured control animals, circulating levels of CORT were evaluated at rest, under restraint stress and in response to dexamethasone, a synthetic glucocorticoid commonly used to test HPE axis regulation. Testosterone was evaluated at rest. Further, we assessed changes in injury-induced neuron morphology (Golgi stain), neuropathology (silver stain) and activated astrocytes (GFAP) in the paraventricular nucleus (PVN) of the hypothalamus. Resting plasma CORT levels were decreased at 2months postinjury and there was a blunted CORT increase in response to restraint induced stress. No changes in testosterone were measured. These changes in CORT were observed concomitantly with altered complexity of neuron processes in the PVN over time, devoid of neuropathology or astrocytosis. Results provide evidence that a single moderate diffuse TBI leads to changes in CORT function, which can contribute to the persistence of symptoms related to endocrine dysfunction. Future experiments aim to evaluate additional HP-related hormones and endocrine circuit pathology following diffuse TBI.