DOI
https://doi.org/10.25772/EK84-EB90
Author ORCID Identifier
https://orcid.org/0000-0001-7337-4271
Defense Date
2023
Document Type
Dissertation
Degree Name
Doctor of Philosophy
Department
Neuroscience
First Advisor
Audrey Lafrenaye, Ph.D.
Second Advisor
Jeffrey Dupree, Ph.D.
Third Advisor
Carmen Sato-Bigbee, Ph.D.
Fourth Advisor
Dong Sun, M.D., Ph.D.
Fifth Advisor
Phillip Gerk, Pharm.D., Ph.D.
Sixth Advisor
Gretchen Neigh, Ph.D.
Abstract
Traumatic brain injury (TBI) is a mechanical insult to the head that leads to brain damage and, in turn, causes long-term sensory, motor, cognitive, and affective dysfunction. Diffuse pathologies seen following such injury are associated with these life-altering outcomes that impact the daily lives of TBI survivors. The diffuse pathology that this body of work focuses on is neuronal membrane disruption; it is characterized by increased permeability of the neuron’s plasma membrane. Moreover, our lab had previously found that membrane disruption is exacerbated with intracranial pressure (ICP) elevation. We set out to measure the duration of membrane disruption following injury with ICP elevation. Still, we needed to define the duration of membrane disruption after TBI as the lone insult. The histological findings indicate that membrane disruption occurs in a biphasic trajectory, sub-acute (6h to 3d) and late (2w-4w). Interestingly, not all of these membrane disrupted neurons are labeled with the neuronal marker NeuN. These cells were included in the quantification, which revealed that membrane disrupted NeuN- cells increase at 2w. We followed this lead with NeuN protein quantification, glial assessments, cell death evaluations and analysis of the total cell population. NeuN protein levels were not changing, and they were not glia either. We interrogated the tissue for cell death using a DNA damage sensitive assay as well as quantification of the total cell population to evaluate for cell loss; cells are not dying and overall NeuN negative population is not changing. However, there is a consistent population of NeuN negative cells in the lateral neocortex of rats. Following this study, we wanted to identify this NeuN- membrane disrupted subpopulation. We posited the NeuN negative membrane disrupted subpopulation as adapting an immature profile; we used immature/early markers, Doublecortin and cFos in cellular and molecular analyses. Overall, we found that this NeuN- membrane disrupted subpopulation could not be identified in an immature state using those markers. Simultaneously, there is no cohesive molecular mechanism for late membrane disruption. Previous data from our lab exhibited Cathepsin B, a lysosomal protease relocalized 2 into the cytosol 6h post-injury. We set out to further elaborate the dynamics of Cathepsin B following TBI from 6h-4w. Cathepsin B protein levels and activity in the lateral neocortex did not change after injury at any time compared to shams. Cathepsin B binding partners Bak, Bcl-XL and AIF levels were also quantified and no differences were found after injury. However, the histological studies of Cathepsin B reveal significant Cathepsin B relocalization from the lysosomes in membrane disrupted neurons. Additionally, Cathepsin B relocalized and membrane disrupted neurons demonstrated cellular and nuclear atrophy. Therefore, with Cathepsin B relocalization coinciding with late membrane disruption, it was proposed as a candidate involved in membrane disruption. Using Cathepsin B inhibitor, CA-074Me, we could evaluate Cathepsin B’s role in membrane disruption and we also wanted to scrutinize the impact of Cathepsin B inhibition on ICP elevation. Through this pharmacological approach, we found that there were some changes in Cathepsin B and AIF proteins that were not significantly different. In addition, the vehicle, 10% DMSO, used to prepare the CA-074Me was found to have some ameliorative effects in membrane disruption, though the groups given the drug had more membrane disruption than the vehicle. Cathepsin B localization with CA-074Me is more localized in lysosomes in non- disrupted neurons, however, has the opposing effect in membrane disrupted neurons . This study also had somatosensory sensitivity increase among the injured and more so in injury/ ICP elevation animals. When given CA-074Me, injured/ICP elevated animals exhibit sensitivity lowered to injured levels. Taken all together, this work highlights the dynamic nature of membrane permeability following injury may have detrimental or ameliorative effects, as well as avenues for remediating secondary sequelae following TBI.
Rights
© Martina L. Hernandez
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
9-21-2023