Author ORCID Identifier

https://orcid.org/0000-0002-5708-4677

Date Available

8-2-2021

Year of Publication

2021

Document Type

Doctoral Dissertation

Degree Name

Doctor of Philosophy (PhD)

College

Medicine

Department/School/Program

Neuroscience

Advisor

Dr. Edward D. Hall

Abstract

Traumatic brain injury (TBI) is a leading cause of death and disability in the United States. Each year, an estimated 2.8 million Americans are diagnosed with a TBI due to falling, motor vehicle collisions, gun violence, and sports related concussions. Although inflicted by a single event, the post-traumatic effects of TBI often develop into a life-long disease. Survivors often experience cognitive decline, memory loss, emotional instability, changes in personality, and physical disabilities. A single TBI, and more-so repetitive TBI's, place an individual at a greater risk of developing chronic neurological disorders, such as dementia or Alzheimer’s disease, earlier in life. Additionally, the high costs and long-term care associated with treating TBI also strains families, companies, and the health care system. To develop an effective treatment, the underlying neuropathophysiology of TBI has been well studied for decades. Historically, basic research has been conducted more frequently in males, leaving a gap in knowledge about how females may react to a treatment. This may be a contributing factor as to why all TBI clinical trials have failed, leaving us without a treatment for this disease.

One of the central secondary mechanisms associated with TBI is oxidative stress. Within minutes of the initial mechanical injury, the injured neurons and glial cells begin producing toxic amounts of reactive oxygen/nitrogen species (ROS/RNS), free radicals, undergo apoptosis, and initiate other damaging secondary cascades. Oxidative damage typically peaks around 3 days post injury but may persist for weeks depending on the injury severity. Because of the central role, early onset, and prolonged nature of oxidative stress, it has remained a logical avenue for developing treatments for TBI.

Cells are equipped with an innate antioxidant defense system to battle oxidative stress. The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) is a basic leucine zipper that regulates the expression of several antioxidant proteins within the antioxidant response element (ARE). In healthy cells, Nrf2 is rapidly turned over to maintain a proper redox balance. However, following injury, a rise in oxidative stress, or in the presence of electrophilic species, Nrf2 is shuttled from the cytoplasm to the nucleus to initiate transcription of detoxifying enzymes. Recent understanding of this pathway has led to the development of a class of drugs called “Nrf2 activators”. The pro-electrophilic drug carnosic acid (CA), an extract in the common herb rosemary, has been shown to be an extremely effective Nrf2 activator and antioxidant.

The goal of this dissertation was to describe the innate time course of Nrf2-ARE activity following a single controlled cortical impact injury in male and female mice. Previous work in our lab showed that a single controlled cortical impact (CCI) injury increased markers of oxidative stress and consequently, increased the production of Nrf2 mediated phase II enzymes. A single dose of CA at multiple different time points was able to reduce markers of oxidative stress while boosting the Nrf2 response. The goal of this project was to recapitulate this experiment in male and female mice to illuminate potential iii sex differences in the Nrf2-ARE response to CCI and test the efficacy of CA as a neuroprotective agent.

In Aim 1, mice were sacrificed at 1, 2, 3, 7 days post injury (DPI). Both the injured cortex and hippocampus were analyzed for Nrf2 protein and mRNA the Nrf2-ARE biomarkers HO-1 and NQO1. In Aim 2, mice received the same injury and were given a single 1.0 mg/kg I.P. dose of CA 1 hr post-injury and sacrificed at 1 and 3 DPI and only the cortex was analyzed. We then examined how CA augmented the cellular localization (nuclear vs cytoplasmic) of Nrf2 and transcription of Nrf2 regulated mRNA biomarkers.

Results from Aim 1 detected notable sex-based differences in the innate Nrf2 response. Male mice had greater amounts of HO-1 mRNA, whereas female mice exhibited higher amounts of HO-1 protein. Results from Aim 2 indicate a sex-based difference in the therapeutic action of CA. Nrf2 nuclear localization was increased in males treated with CA, whereas CA treated females had increased cytoplasmic concentrations of Nrf2. Surprisingly, RT-PCR analysis revealed that CA treatment drove down transcription of HO-1, NQO1, and Nrf2 in males. Conversely, CA treatment enhanced transcription of HO-1, NQO1, and decreased Nrf2 in females.

From these results, we can conclude that there are minor regional and temporal differences in the Nrf2-ARE pathway in male and female rodents. Also, the therapeutic mechanism of action associated with CA may be different in males and females given that a single dose had the opposite effect on Nrf2 cellular localization, however, with such modest drug effects, this cannot yet be concluded. Future studies may consider a thorough behavioral and histological analysis following a single dose of CA to measure functional recovery and visualize the neuroprotective effect. Additionally, a pharmacokinetic profile of CA clearance would be beneficial in determining the optimal dosing regimen for each sex.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2021.346

Funding Information

This study was supported by the NIH/NINDS Grant “Nrf2-Antioxidant Response Element Neuroprotection in TBI" (1R01 NS100093) awarded to Dr. Edward D. Hall from 2016-2021. Further support was provided by the NIH Predoctoral Fellowship “Neurobiology of CNS Injury & Repair Training Grant” (5T32 NS077889) sponsored by Dr. Edward D. Hall and Dr. Kathryn E. Saatman and awarded to Jacob A. Dunkerson from 2018-2020.

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