1. Histochemical Study Fluoro Jade-B (FJB) staining  FJB selectively labels degenerating neurons on rat brain tissue.  The number of fluorescent neurons is proportional to the number of degenerating neurons.  The more effective the neuroprotective ability of GCEE, the lower the number of fluorescent neurons. Procedure MTT Assay  MTT is converted into formazan crystals by the activity of mitochondria in living cells.  The concentration of formazan crystal is proportional to the number of viable cells.  Tert-butyl hydroperoxide (tBHP), an oxidizing agent, induces oxidative stress in cells.  The lower the number of viable cells, the more cytotoxic effects of tBHP.  Traumatic brain injury (TBI) can initiate secondary injuries, such as the excessive formation of reactive oxygen species (ROS) and reactive nitrogen species (RNS), resulting in oxidative stress in which cellular components begin to lose their structures and functions (oxidative damage), ultimately leading to neuronal cell death (neurodegeneration), which is frequently seen in TBI 1.  Glutathione (GSH) is the most abundant antioxidant synthesized in the brain and plays an important role in scavenging ROS/RNS 2. GSH is synthesized through a two- step process. Its synthesis is regulated by negative feedback in which the activity of γ-glutamylcysteine synthetase is inhibited by final product, GSH. (Fig. 1)  γ-glutamylcysteine ethyl ester (GCEE) is a GSH precursor. It overcomes negative feedback and increases the levels of GSH in the brain 3. (Fig. 2) Jooyoung Cho 1, Fathima Shazna Thowfeik 2, Edward J. Merino 2, Mark E. Bardgett 3, Pa trick Sullivan 4, Andrea Sebastian 4, Tanea T. Reed 1 1 Eastern Kentucky University, Department of Chemistry, Richmond, KY 40475, 2 University of Cincinnati, Department of Chemistry, Cincinnati, OH 45221 3 Northern Kentucky University, Department of Psychology, Newport, KY 41099, and 4 University of Kentucky, Spinal Cord and Brain Injury Research Center, Lexington, KY 40506 Figure 1. Mechanism of glutathione synthesis Figure 2. Structure of GCEE Figure 3. FJB positive neurons in TBI models Saline 60GCEE 60 GCEE 30Saline 30 Figure 4. The number of FJB positive neurons in TBI models Figure 5. Cytotoxicity of tBHP on astrocytes Figure 6. Protective effect of GCEE on ROS production Tissue Preparation Cell Culture Materials and Methods Dichlorofluorescein (DCF) assay  Non-fluorescent dichlorofluorescein diacetate (H 2 DCFDA ) is converted into highly fluorescent DCF by intracellular ROS.  The levels of fluorescence are proportional to the levels of intracellular ROS.  The more effective the ability of GCEE to decrease intracellular ROS levels, the lower the relative fluorescence. Background  Cortical contusion injury (CCI) simulated moderate TBI in GCEE and Saline groups.  Fig. 3 indicates that a significant increase of FJB positive neurons in CCI induced groups, while relatively less density and distribution of FJB positive neurons in GCEE treatment groups after CCI compared to saline groups.  Fig. 4 indicates that TBI increased the number of degenerating neurons, while treatment with 150 mg/kg GCEE 30 min after TBI did not significantly increase the number of degenerating neurons.  Fig. 5 indicates a significant decrease of cell viability when astrocytes were exposed to 200 µM tBHP for 60 min.  Fig. 6 indicates a statistically significant decrease in intracellular ROS levels when astrocytes were treated with 750µM GCEE for 60 min during oxidative stress. Acknowledgements  Eastern Kentucky University, Department of Chemistry  National Institutes of Health (NINDS) Grant #: 1R15NS072870-01A1 References 1.Mazzeo, A.T., et. al. Exp Neurol, 2009. 218(2): p. 363-70. 2.Bains, M., et al. Biochim Biophys Acta, 2012. 1822(5): p. 675-84. 3. Drake, J., et al. J Neurosci Res, 2002. 68(6): p. 776-84. 4. https://www.invitrogen.com/site/us/en/home.html(accessed August 5, 2013) 5. Lok, J., et al. J Neurochem, 2011. 118(2): p. 248-55  Histochemical study shows a potential neuroprotective effect of GCEE on moderate TBI.  Cell study shows the capability of GCEE to decrease intracellular ROS levels in astrocytes during oxidative stress.  Overall, GCEE can have potent therapeutic effects in oxidative stress-associated neurodegeneration following TBI. Conclusions Discussion Cell Study Rat primary cortical astrocytes (Invitrogen, Carlsbad, CA, USA) were grown in DMEM culture medium supplemented with 15% FBS at 37 ⁰ C with 5% CO2 When cells were 100% confluent, cells were transferred into 24 well plates After 48 hours, experiments were performed CraniotomyCCITreatment Naïve-- ShamX-- SalineXX-- GCEEXX 150 mg/kg 30 min /60 min after injury Future Works  Investigation into regional distribution of degenerating neurons and the effects of GCEE following TBI.  Before performing MTT assay, use the media without phenol red dye for tBHP or GCEE treatment to prevent interference with reagents.  Co-culture of neurons and astrocytes to investigate the benefit of GCEE addition on neuroprotection in this system. * * * P < 0.05 * * *