Research Scientist Walter Reed Army Institute of Research Olney, Maryland
Abstract Text: Traumatic Brain Injury (TBI) is a major health concern in both military and civilian sectors worldwide, often leading to death and disability. Mitochondrial dysfunction is a commonality among various severity of TBI that leads to secondary brain injury progression. Studies from experimental TBI support a key role for mitochondria-centered pathological cascade that includes impaired energy, calcium (Ca2+) and redox homeostasis following acute TBI. Oxidative stress represents the imbalance between reactive oxygen and nitrogen species (ROS-RNS) and antioxidants. Such imbalance may lead to irreversible oxidative modifications of membrane components, loss of mitochondrial membrane integrity and apoptosis. To gauge the acute penetrating injury effect and mechanism, the current study evaluates the brain mitochondrial oxidative stress, Ca2+ homeostasis, mitochondrial membrane integrity and apoptosis parameters after 24h penetrating TBI (PTBI).
Methods Using a pre-clinical model of PTBI, anesthetized adult male rats were subjected to either 10% unilateral PTBI or uninjured Sham craniectomy (n=6/group). Animals were euthanized at 24h post-PTBI, and brain mitochondria were isolated from the injury core and perilesional area that includes the frontal cortex and striatum. The oxidative stress parameters such as real-time ROS-RNS production, protein adducts formation (3-nitrotyrosine, protein carbonyl), and product of lipid peroxidation (4-hydroxynonenal) were measured. Similarly, antioxidants such as glutathione (GSH), peroxiredoxins (PRX-3), thioredoxins (TRX), reduced nicotinamide adenine dinucleotide phosphate (NADPH), superoxide dismutase (SOD) and catalase (CAT) were quantified using Western blot. Additionally, Ca2+ homeostasis parameters such as Ca2+ load capacity, mitochondrial permeability transition pore (mPTP), and Ca2+ induced mitochondrial swelling was measured by spectrophotometric method. Both outer and inner mitochondrial membrane integrity markers such as voltage-dependent anion channels (VDAC), cytochrome c (Cyt C), and apoptosis markers such as B-cell lymphoma-2 (Bcl-2), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were quantified.
Results: Following 24h post-injury, significantly elevated ROS-RNS related markers, including 4-hydroxynonenal (4-HNE), 3-nitrotyrosine (3-NT) and protein carbonyls (PC) were observed in the PTBI group compared to Sham. Mitochondrial antioxidants GSH, PRX-3, TRX, NADPH, and SOD levels were significantly decreased, whereas CAT level increased in PTBI vs. Sham. The PTBI mitochondria also displayed significant loss of Ca2+ homeostasis, early opening of mPTP, and increased Ca2+- induced mitochondrial swelling compared to Sham. Electron microscopy data indicated increased swelling and loss of membrane integrity in PTBI mitochondria compared to Sham. Both outer and inner mitochondrial membrane integrity markers, such as VDAC and Cyt C levels, were significantly decreased in PTBI vs. Sham. Release of Cyt C and VDAC can exacerbate apoptotic cell death, lower anti-apoptotic protein BCL-2, and spiked apoptotic inducer protein GAPDH levels in our result support such a phenomenon.
Conclusion: Overall, the PTBI group showed increased oxidative stress, decreased antioxidants, decreased Ca2+ load capacity and early mPTP opening at 24h post-injury. The significant loss of mitochondrial membrane integrity and elevated apoptosis indicators may contribute to non-salvageable acute-phase effects following PTBI. Collectively, our results suggest that early therapeutic intervention is crucial for preserving mitochondrial functions during the acute phase of PTBI.
