n-tert-butyl-(2-sulfophenyl)nitrone and Brain-Injuries

Infiltration of T lymphocytes is a key feature in transplant rejection and in several autoimmune disorders, but the role of T lymphocytes in traumatic brain injury (TBI) is largely unknown. Here we studied trafficking of immune cells in the brain after experimental TBI. We found that scavenging of reactive oxygen species (ROS) at the endothelial level dramatically reduced the infiltration of activated T lymphocytes. Immune cell infiltration was studied 12 h to 7 days after controlled cortical contusion in rats by ex vivo propagation of T lymphocytes (TcR+, CD8+), neutrophils (MPO+), and macrophages/microglia (ED-1+) from biopsies taken from injured cortex and analyzed by flow cytometry, as well as by quantitative immunohistochemistry. T lymphocyte and neutrophil infiltration peaked at 24 h and macrophages/microglia at 7 days post-injury. Pretreatment with 2-sulfophenyl-N-tert-butyl nitrone (S-PBN) produced a dramatic reduction of TcR+ T lymphocytes and a significantly smaller attenuation of neutrophil infiltration at 24 h post-injury, but did not affect CD8+ T lymphocytes or macrophages/microglia. S-PBN significantly reduced the expression of the endothelial adhesion molecules ICAM-1 and VCAM at 24 h for following TBI. We conclude that ROS inhibition at the endothelial level influenced T lymphocyte and neutrophil infiltration following TBI. We submit that the reduction of T lymphocyte infiltration is a key feature in improving TBI outcome after S-PBN treatment. Our data suggest that targeting T lymphocyte trafficking to the injured brain at the microvascular level is a novel concept of neuroprotection in TBI and warrants further exploration.

Topics: Animals; Benzenesulfonates; Brain Injuries; Cell Movement; Intercellular Adhesion Molecule-1; Macrophages; Male; Microglia; Neutrophil Infiltration; Rats; Rats, Sprague-Dawley; T-Lymphocytes; Vascular Cell Adhesion Molecule-1

Nitric oxide (NO) and oxygen free radicals are implicated in the pathophysiology of traumatic brain injury (TBI). Peroxynitrite formation from NO and superoxide contributes to secondary neuronal injury but the neuroprotective effects of nitric oxide synthase (NOS)-inhibitors have been contradictory. This study was undertaken to examine whether PTtic administration of the (NOS)-inhibitor N-nitro-l-arginine methyl ester (L-NAME), and a combination of L-NAME and the nitrone radical scavenger 2-sulfo-phenyl-N-tert-butyl nitrone (S-PBN) favorable affects neuronal injury in a model of TBI.. A weight-drop model of TBI was used. The animals received L-NAME, S-PBN or a combination of the drugs 15 minutes prothrombin time (PT) and sacrificed after 24 hours or six days. NOS activity was measured by the conversion of L-[U-C]arginine to L-[U-C]citrulline. Peroxynitrite formation, cellular apoptosis, neuronal degeneration and survival were assessed by nitrotyrosine-, TUNEL-, Fluoro-Jade- and NeuN-stainings.. eNOS and nNOS activity was significantly reduced in animals that received L-NAME alone or the combination with S-PBN. iNOS activity or iNOS immunoreactivity was not affected. All treatments significantly reduced neuronal degeneration and nitrotyrosine immunoreactivity at 24 hours and increased neuronal survival at six days PT. No differences were detected between L-NAME and L-NAME + S-PBN groups.. NO from NOS contributes to secondary neuronal injury in this TBI-model. PTtic treatment does not inhibit early beneficial NO-related effects. L-NAME and S-PBN limit peroxynitrite formation, promoting neuronal survival. The combination of L-NAME and S-PBN was neuroprotective; surprisingly no additive effects were found on nitrotyrosine formation, apoptosis or neuronal survival.

Topics: Animals; Apoptosis; Benzenesulfonates; Brain; Brain Injuries; Cell Survival; Drug Combinations; Enzyme Inhibitors; Fluoresceins; Fluorescent Dyes; Immunohistochemistry; In Situ Nick-End Labeling; Male; Nerve Degeneration; Neurons; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Organic Chemicals; Rats; Rats, Sprague-Dawley; Tyrosine

Changes in regional cerebral blood flow (rCBF) and glucose metabolism are commonly associated with traumatic brain injury (TBI). Reactive oxygen species (ROS) have been implicated as key contributors to the secondary injury process after TBI. Here, pretreatment with the nitrone radical scavengers (alpha-phenyl-N-tert-butyl nitrone (PBN) or its sulfonated analogue sodium 2-sulfophenyl-N-tert-butyl nitrone (S-PBN) were used as tools to study the effects of ROS on rCBF and glucose metabolism after moderate (2.4-2.6 atm) lateral fluid percussion injury (FPI) in rats. S-PBN has a half-life in plasma of 9 min and does not penetrate the blood-brain barrier (BBB). In contrast, PBN has a half-life of 3 h and readily penetrates the BBB. Regional cerebral blood flow (rCBF) and glucose metabolism was estimated by using (99m)Tc-HMPAO and [(18)F]Fluoro-2-deoxyglucose (FDG) autoradiography, respectively, at 42 min (n = 37) and 12 h (n = 34) after the injury. Regions of interest were the parietal cortex and hippocampus bilaterally. As expected, FPI produced an early (42-min) hypoperfusion in ipsilateral cortex and an increase in glucose metabolism in both cortex and hippocampus, giving way to a state of hypoperfusion and decreased glucose metabolism at 12 h postinjury. On the contralateral side, a hypoperfusion in the cortex and hippocampus was seen at 12 h only, but no significant changes in glucose metabolism. Both S-PBN and PBN attenuated the trauma-induced changes in rCBF and glucose metabolism. Thus, the early improvement in rCBF and glucose metabolism correlates with and may partly mediate the improved functional and morphological outcome after TBI in nitrone-treated rats.

Topics: Animals; Benzenesulfonates; Brain Injuries; Cerebrovascular Circulation; Cyclic N-Oxides; Fluorodeoxyglucose F18; Free Radical Scavengers; Glucose; Hippocampus; Male; Neuroprotective Agents; Nitrogen Oxides; Radionuclide Imaging; Radiopharmaceuticals; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Technetium Tc 99m Exametazime

In previous studies, the authors showed that the nitrone radical scavenger alpha-phenyl-N- tert -butyl nitrone (PBN) and its sulfo-derivative, 2-sulfo-phenyl-N- tert -butyl nitrone (S-PBN), attenuated cognitive disturbance and reduced tissue damage after traumatic brain injury (TBI) in rats. In the current study, the production of reactive oxygen species (ROS) after TBI was monitored with microdialysis and the 4-hydroxybenzoic acid (4-HBA) trapping method. A single dose of PBN (30 mg/kg) or an equimolar dose of S-PBN (47 mg/kg) was administered intravenously 30 minutes before a controlled cortical contusion injury in rats. Plasma and brain tissue drug concentrations were analyzed at the end of the microdialysis experiment (3 hours after injury) and, in a separate experiment with S-PBN, at 30 and 60 minutes after injury. Traumatic brain injury caused a significant increase in ROS formation that lasted for 60 minutes after the injury as evidenced by increased 3,4-dihydroxybenzoic acid (3,4-DHBA) concentrations in the dialysate. PBN and S-PBN equally and significantly attenuated the posttraumatic increase in 3,4-DHBA formation. High PBN concentrations were found bilaterally in brain tissue up to 3 hours after injury. In contrast, S-PBN was rapidly cleared from the circulation and was not detectable in brain at 30 minutes after injury or at any later time point. The results suggest that scavenging of ROS after TBI may contribute to the neuroprotective properties observed with nitrone spin-trapping agents. S-PBN, which remained undetectable even in traumatized brain tissue, reduced ROS production to the same extent as PBN that readily crossed the blood-brain barrier. This finding supports an important role for ROS production at the blood-endothelial interface in TBI.

Topics: Animals; Benzenesulfonates; Blood-Brain Barrier; Brain Injuries; Cyclic N-Oxides; Free Radical Scavengers; Hydroxybenzoates; Male; Microdialysis; Neuroprotective Agents; Nitrogen Oxides; Parabens; Parietal Lobe; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species

Brain homogenate was used as a model system to study antioxidant properties of several natural and synthetic antioxidants under oxidative stress. Oxidative stress was induced by Fe/ascorbate system and lipid peroxidation as well as protein modification were studied. Thiobarbituric acid reactive substances (TBARS) were used as a marker of lipid peroxidation. The preventive effect concerning lipid peroxidation decreased in the order: buthylated hydroxytoluene (BHT) (3.5), stobadine (ST) (35), serotonin (54), trolox (98), U 74389G (160), melatonin (3100), (the numbers in the brackets represent IC50 in micromol/l). Methylprednisolone had no effect, and spin traps interfered with TBARS determination. Concerning creatine kinase (CK) activity as a selected marker of oxidative modification of proteins, the preventive effect of antioxidants (30 micromol/l) decreased in the order: BHT (30), trolox (75), stobadine (ST) (77), alpha-phenyl-N-tert-buthylnitrone (PBN) (87), sodium salt of N-tert-buthyl-C-(phenyl-2-sulfone) nitrone (SPBN) (90), (the numbers in the brackets represent the loss of CK activity in percentages, when 100% was the loss of CK activity in the absence of any antioxidant). The nonglucocorticoid steroid U 74389G, methylprednisolone and serotonin had no preventive effects, while melatonin had antioxidant effect only in a higher concentration (1 mmol/l).

Topics: Animals; Antioxidants; Benzenesulfonates; Brain; Brain Injuries; Butylated Hydroxytoluene; Carbolines; Chromans; Creatine Kinase; Cyclic N-Oxides; Inhibitory Concentration 50; Lipid Peroxidation; Male; Melatonin; Methylprednisolone; Models, Chemical; Neuroprotective Agents; Nitrogen Oxides; Oxidative Stress; Oxygen; Pregnatrienes; Rats; Rats, Wistar; Serotonin; Thiobarbituric Acid Reactive Substances