Combinational Antioxidant Therapy to Prevent the Development of PTE

Principal Investigator: SHEKH-AHMAD, TAWFEEQ

Proposal Number: EP210060

Award Number: W81XWH-22-1-0677

Period of Performance: 7/1/2022 - 6/30/2025

PUBLIC ABSTRACT

Head trauma, or traumatic brain injury, is the worldwide leading cause of disability and death among children and young adults, as well as a growing problem in the elderly. Traumatic brain injury can result in the death of nerve cells and changes in brain tissue that may eventually lead to the development of epilepsy (chronic seizures), termed post-traumatic epilepsy. This has not only acute but also long-term detrimental consequences affecting proper thinking and memory, increasing the risks of developing disabilities and comorbidities in patients’ later lives, affecting their life span. In recent years, traumatic brain injury has been recognized as a significant and growing problem among military personnel and Veterans who are highly exposed to head injury and are at high risk for developing post-traumatic epilepsy. Despite enormous scientific efforts to explore the mechanisms underlying long-term consequences of traumatic brain injury and to develop therapies that can preserve brain tissue, no specific therapeutic options are yet available for patients. Importantly, post-traumatic epilepsy is one of the only brain diseases in which people at risk can be identified, and yet there is no prophylactic treatment to prevent the development of the disease in those at risk. Moreover, currently there are no diagnostic markers available to predict patients at risk to develop epilepsy following head injury. Recent studies indicate that free radicals, unstable atoms that can damage cells, also play a part in aging and cancer, are key players in such cell and tissue damage. Excessive free radical production starts within hours and continues over days after injury. Therefore, blocking the processes that contribute to the free radical’s production and increasing the antioxidant defenses of nerve cells may be effective in preventing the consequence damage following brain injury, including the development of epilepsy.

We have some compelling early evidence that free radical-induced brain damage can be prevented by blocking a specific source of free radicals within the brain and by increasing the nerve cells’ ability to mop up these free radicals. Therefore, a group of epilepsy researcher, traumatic brain injury researchers and imaging scientists joined forces and formed a research group to investigate the role of oxidative stress following traumatic brain injury in experimental animals. The goals of the proposed research are to develop novel biomarkers (that can distinguish healthy subjects from diseased ones) and therapeutic options by comparing the two approaches and a combination of both, to determine the most effective way of preventing cell death and tissue damage following brain injury induced in rats.

For this purpose, the research group will use a unique telemetry system, which allows video-and-wireless electrocorticography recording enabling monitoring of both behavior and brain activity, and novel brain imaging technology, enabling depiction of subtle changes in brain vascular function. These technologies will be applied in combination with state-of-the-art cognitive tests and histological analysis. Rats will be studied for a very long term (12 months) post-injury in order to provide a comprehensive understanding of the mechanisms leading to the development of epilepsy in an attempt to apply the new knowledge for developing novel means for early prediction of the disease as well as means to suppress the damage along the way. The pharmacological drugs applied in this study are in advanced clinical investigations for indications other than epilepsy and will be tested as a therapy to prevent the development of epilepsy and memory problems following weight drop in animals, a condition that mimics focal brain injury. Vascular dysfunction will be assessed with our novel translational magnetic resonance imaging (MRI) technology, already U.S. Food and Drug Administration (FDA)-approved, thus translation to the clinical scenario may be relatively in the short to medium term (2-5 years).

In addition, 1 year of video-electrocorticography recording and all the acquired MRI and vessel function maps will be openly accessible to the global neuroscience community and will thus tremendously ease future research on acute and chronic sequels post-brain injury. Therefore, the results of our study are expected to generate a significant public health impact since the discovery of oxidative stress mechanisms using new approach will result in novel therapeutic options and innovative diagnostic biomarkers, which are currently unavailable.

TECHNICAL ABSTRACT

Background: Traumatic brain injury (TBI) has been identified as a major cause of death and disability in both civilian life and military personnel. Secondary consequences include impaired cognition and emergence of acute and late seizures, termed post-traumatic epilepsy (PTE). There is a growing evidence that excessive reactive oxygen species (ROS) production, oxidative stress, and neuroinflammation contribute to TBI-induced brain damage, and consequently to the development of PTE. Clinical trials of unspecific antioxidant therapy remained unsuccessful, warranting more specific intervention, such as targeting ROS production by inhibiting NADPH oxidase (NOX) enzymes, identified as main source of ROS production following TBI. However, non-specific NOX inhibition has been associated with suppression of physiological signaling and off-target effects. Additional promising antioxidant intervention is to upregulate the intrinsic antioxidant defenses of cells, through the activation of Nrf2 pathway, identified as an endogenous key regulator of antioxidant defense.

Hypothesis and Objectives: Our working hypothesis is that identifying and targeting the main specific NOX isoform that contributes to ROS generation following TBI model will decrease ROS-mediated secondary injury and cell death, while avoiding the systemic suppression of physiological NOX signaling associated with nonselective NOX inhibitors. We further hypothesize that targeted antioxidant intervention, based on both upregulating the intrinsic antioxidant defenses of cells via activation of Nrf2, and inhibiting ROS production via selective-NOX inhibition, which may even aggravate primary injury as it can also orchestrate neuroinflammation, is the most effective mean at attenuating the secondary injury induce by TBI, preventing epileptogenesis and also development of PTE and its comorbidities.

Specific Aims: The specific aims of this project are (1) to study the cellular and molecular mechanisms underlying oxidative stress and NOX activity in PTE; (2) to investigate the therapeutic potential of Nrf2 activation and NOX inhibition (alone and as a combination therapy) to prevent/suppress the development of epileptic seizures in the acute and chronic phase following TBI; (3) to identify short- and long-term PTE comorbidities, blood-brain barrier (BBB) dysfunction and brain damage at several time points post-injury; (4) and to identify new diagnostic biomarkers to detect patients at risk for PTE.

Research Strategy: First, we will determine the time course of NOX activity as well as the most effective treatment to counteract the excessive NOX-mediated ROS production in a weight drop closed head injury (CHI) rat model. We then will determine if either each treatment alone (Nrf2 activation and NOX inhibition) or combination of both is superior in reducing oxidative stress, neuroinflammation, and brain damage in animals subjected to CHI. The most effective interventional treatment determined from these experiments will be then tested for its therapeutic effect on preventing the development of PTE following moderate and severe TBI by applying video-wireless-electrocorticography recording that allows continuous recordings (24/7, for a period of months) in freely moving animals in at several time points (up to 12 months post-injury). Moreover, the extent of damage and cognitive decline will be validated by applying wide battery of behavioral tests and delayed contrast magnetic resonance imaging (MRI) for depicting subtle BBB dysfunction early/late post-injury. Finally, we will combine different analysis platforms for studying several biomarkers including systemic and brain oxidative stress and neuroinflammation biomarkers, as well as electroencephalography and BBB biomarkers in overpowered sample size to predict favorable/unfavorable outcomes for up to 1 year following CHI model of TBI.

Innovation and Impact: TBI is specifically prevalent in young men in the military and is the leading cause of death and disability in combat injuries. In addition, Veterans are specifically prone to long-term sequelae of TBI. Therefore, research on diagnosing and treating PTE is highly relevant to military health. The incidence of TBI is growing worldwide with no available specific therapies. We have recently reported that our proposed antioxidant treatment strategy prevented the development of epilepsy in 70% of animals following status epilepticus, and can also modify the severity of epilepsy. We here aim to implement this strategy in an animal model of TBI that recapitulate brain injury in civilian and military setting. We also aim to identify diagnostics biomarkers for the early prediction of patients at risk for development of PTE, of whom early and appropriate intervention could prevent, or at least modify the severity of epilepsy and its comorbidities. Therefore, the proposed study is highly relevant to military health and fully addresses the vision of Epilepsy Research Program Idea Development Award to improving the health, care, and well-being of all military Service Members, Veterans, and civilian communities who suffer from PTE.