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- All Subjects: Traumatic Brain Injury
- Creators: Kleim, Jeffrey
- Creators: Aftab, Umar
Description
Traumatic brain injury (TBI) is a leading cause of disability worldwide with 1.7 million TBIs reported annually in the United States. Broadly, TBI can be classified into focal injury, associated with cerebral contusion, and diffuse injury, a widespread injury pathology. TBI results in a host of pathological alterations and may lead to a transient blood-brain-barrier (BBB) breakdown. Although the BBB dysfunction after TBI may provide a window for therapeutic delivery, the current drug delivery approaches remains largely inefficient due to rapid clearance, inactivation and degradation. One potential strategy to address the current therapeutic limitations is to employ nanoparticle (NP)-based technology to archive greater efficacy and reduced clearance compared to standard drug administration. However, NP application for TBI is challenging not only due to the transient temporal resolution of the BBB breakdown, but also due to the heterogeneous (focal/diffuse) aspect of the disease itself. Furthermore, recent literature suggests sex of the animal influences neuroinflammation/outcome after TBI; yet, the influence of sex on BBB integrity following TBI and subsequent NP delivery has not been previously investigated. The overarching hypothesis for this thesis is that TBI-induced compromised BBB and leaky vasculature will enable delivery of systemically injected NPs to the injury penumbra. This study specifically explored the feasibility and the temporal accumulation of NPs in preclinical mouse models of focal and diffuse TBI. Key findings from these studies include the following. (1) After focal TBI, NPs ranging from 20-500nm exhibited peak accumulation within the injury penumbra acutely (1h) post-injury. (2) A smaller delayed peak of NP accumulation (40nm) was observed sub-acutely (3d) after focal brain injury. (3) Mild diffuse TBI simulated with a mild closed head injury model did not display any measurable NP accumulation after 1h post-injury. (4) In contrast, a moderate diffuse model (fluid percussion injury) demonstrated peak accumulation at 3h post-injury with up to 500 nm size NPs accumulating in cortical tissue. (5) Robust NP accumulation (40nm) was found in female mice compared to the males at 24h and 3d following focal brain injury. Taken together, these results demonstrate the potential for NP delivery at acute and sub-acute time points after TBI by exploiting the compromised BBB. Results also reveal a potential sex dependent component of BBB disruption leading to altered NP accumulation. The applications of this research are far-reaching ranging from theranostic delivery to personalized NP delivery for effective therapeutic outcome.
ContributorsBharadwaj, Vimala Nagabhushana (Author) / Stabenfeldt, Sarah E (Thesis advisor) / Kodibagkar, Vikram D (Thesis advisor) / Kleim, Jeffrey (Committee member) / Tian, Yanqing (Committee member) / Lifshitz, Jonathan (Committee member) / Anderson, Trent R (Committee member) / Arizona State University (Publisher)
Created2018
Description
Traumatic brain injury (TBI) affects an estimated 1.7 million people in the United States each year and is a leading cause of death and disability for children and young adults in industrialized countries. Unfortunately, the molecular and cellular mechanisms of injury progression have yet to be fully elucidated. Consequently, this complexity impacts the development of accurate diagnosis and treatment options. Biomarkers, objective signatures of injury, can inform and facilitate development of sensitive and specific theranostic devices. Discovery techniques that take advantage of mining the temporal complexity of TBI are critical for the identification of high specificity biomarkers.
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.
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.
ContributorsMartinez, Briana Isabella (Author) / Stabenfeldt, Sarah E (Thesis advisor) / Lifshitz, Jonathan (Committee member) / Sierks, Michael (Committee member) / Kleim, Jeffrey (Committee member) / Arizona State University (Publisher)
Created2020
Description
Traumatic brain injury (TBI)—sudden impact or acceleration trauma to the head—is a major cause of death and disability worldwide and is particularly amplified in pediatric cases. TBI is the leading cause of mortality and morbidity in children and adolescents. Adolescence is a critical time where the brain undergoes cognitive development and brain injury-induced disruptions to these processes can lead to life-long debilitating morbidities. The aim of this study was to determine if exercising spatial and contextual memory circuits using a novel rehabilitation strategy called Peg Forest Rehabilitation (PFR) could mitigate the onset of injury-induced cognitive deficits in juvenile rats subjected to diffuse TBI. The PFR aims to synthesize neuroplasticity-based enrichment to improve cognitive outcomes after TBI. We hypothesized that PFR treatment would mitigate the onset of brain injury-induced cognitive deficits and reduce neuroinflammation. Juvenile male Sprague-Dawley rats (post-natal day 35) were subjected to diffuse traumatic brain injury via midline fluid percussion injury or a control surgery. One-week post-injury, rats were exposed to PFR or cage control exploration (15 min/day). PFR allowed free navigation through random configuration of the peg-filled arena for 10 days over 2 weeks. Control rats remained in home cages in the center of the arena with the peg-board removed for 15 min/day/10 days. One-week post-rehabilitation (one-month post-injury), cognitive performance was assessed for short-term (novel object recognition; NOR), long-term (novel location recognition; NLR), and working (temporal order recognition; TOR) memory performance, calculated as a discrimination index between novel and familiar objects. Tissue was collected for immunohistochemistry and stained for ionized calcium binding proteins (Iba-1) to visualize microglia morphology, and somatostatin. PFR attenuated TBI-induced deficits on the NOR task, where the TBI-PFR treatment group spent significantly more time with the novel object compared with the familiar (*p=0.0046). Regardless of rehabilitation, brain-injured rats had hyper-ramified microglia in the hypothalamus indicated by longer branch lengths and more endpoints per cell compared with uninjured shams. Analysis of somatostatin data is ongoing. In this study, passive, intermittent PFR that involved dynamic, novel spatial navigation, prevented TBI-induced cognitive impairment in adolescent rats. Spatial navigation training may have clinical efficacy and should be further investigated.
ContributorsAftab, Umar (Author) / Rowe, Rachel K. (Thesis director) / Newbern, Jason M. (Thesis director) / Ortiz, J. Bryce (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05