Research Scientist Walter Reed Army Institute of Research Olney, Maryland
Abstract Text:
Background: Oxidative stress is a pathological state that occurs when the generation of cellular free radicals outbalances the elimination of these radicals through the innate antioxidants system. The brain is uniquely susceptible to oxidative stress due to its high metabolic demand, and because brain tissue contains high levels of redox-susceptible polyunsaturated fatty acids and redox transition metals, making it an ideal target for free radical’s attack. 3-nitrotyrosine (3-NT) and protein carbonyl (PC) are hallmark indicators of free radical-mediated protein modification during oxidative stress. Although, the mitochondrial dysfunction is a pivotal target for neuroprotection strategies for traumatic brain injury (TBI), the comprehensive time-course evaluation of mitochondrial oxidative stress and antioxidants are lacking in the pre-clinical penetrating TBI (PTBI). The recently published mitochondrial bioenergetics time-course analyses in PTBI model revealed that the injury core and perilesional regions (i.e., frontal cortex + striatum) displayed biphasic energy crisis response, which initiated immediately at 30 minutes after trauma and extended up to 2 weeks post-injury period (J. Neurotrauma 2021, PMID: 33544034). The preliminary observations on the oxidative profile measured at the acute injury phase discovered the elevated oxidative stress responses together with decreased antioxidants following PTBI. For the interest of future evaluation of mitochondria targeted neuroprotection drugs, we conducted a full time-course evaluation of oxidative stress and antioxidant markers following PTBI in rats.
Methods: Anesthetized adult male Sprague-Dawley rats were subjected to either 10% unilateral penetrating traumatic brain injury (PTBI) or sham craniectomy. Time-course examination of oxidative stress and antioxidants parameters were conducted at 30 min, 3h, 6h, 24h, 3d, 7d and 14d post-PTBI (n=6 animals / group X 7 time-points). From the ipsilateral hemisphere of PTBI and Sham injured brains, the frontal cortex and striatum regions were dissected, and immediately mitochondrial fractions were isolated at respective time points and stored at -800C for assay. The oxidative stress parameters, such as protein oxidation markers 3-NT and PC, were measured. Antioxidants such as glutathione, peroxiredoxins (PRX-3), thioredoxins (TRX), nicotinamide adenine dinucleotide phosphate (NADPH), superoxide dismutase (SOD) and catalase (CAT) expression were quantified. Additionally, the glutathione (GSH) content was extracted from fresh homogenates, cytosol, and mitochondrial sub-fractions samples.
Results: In general, the PTBI mitochondrial time-courses of 3-NT and PC displayed significantly increased (14-53%, vs. Sham) protein oxidation adducts during the first 3d time-points post-injury. All antioxidant contents in the PTBI group were significantly decreased during the 30min to 7d post-injury period. In particular, the mitochondrial expression of TRX and PRX-3 were significantly decreased (20-70%, vs. Sham) up to 7d, where the lowest content was observed at 3d post-injury. GSH was decreased (20-80%, vs. Sham) in all fractions of the PTBI group up to 7d post-injury. Additionally, both SOD and NADPH contents displayed similar decreased trends (20-50%, vs. Sham), whereas the CAT content exhibited a biphasic increase (45-75%, vs. Sham) up to 7d post-injury. Overall, we observed a maximum imbalance of oxidative stress and antioxidant markers during the first week following PTBI.
Conclusion: The current results highlight the mitochondria-centric acute pathophysiological responses following penetrating TBI, which may be used as novel theragnostic and prognostic indicators for TBI. The time-course data on mitochondrial oxidative stress and antioxidants following PTBI concluded that these oxidative damage-associated responses were apparent instantly and remain elevated during the first week of acute brain trauma, and such secondary injury responses should be mitigated immediately by targeted mitochondrial therapeutics.
Keywords: Mitochondria, Oxidative stress, Antioxidants, Time-course, Penetrating Traumatic like Brain Injury (PTBI), Drug Interventions.
