Elie Beit-Yannai

Oxidative Stress in Closed-Head Injury: Brain Antioxidant Capacity as an Indicator of Functional Outcome

It has been suggested that reactive oxygen species (ROS) play a role in the pathophysiology of brain damage. A number of therapeutic approaches, based on scavenging these radicals, have been attempted both in experimental models and in the clinical setting. In an experimental rat and mouse model of closed-head injury (CHI), we have studied the total tissue nonenzymatic antioxidant capacity to combat ROS. A major mechanism for neutralizing ROS uses endogenous low-molecular weight antioxidants (LMWA). This review deals with the source and nature of ROS in the brain, along with the endogenous defense mechanisms that fight ROS. Special emphasis is placed on LMWA such as ascorbate, urate, tocopherol, lipoic acid, and histidine-related compounds. A novel electrochemical method, using cyclic voltammetry for the determination of total tissue LMWA, is described. The temporal changes in brain LMWA after CHI, as part of the response of the tissue to high ROS levels, and the correlation between the ability of the brain to elevate LMWA and clinical outcome are addressed. We relate to the beneficial effects observed in heat-acclimated rats and the detrimental effects of injury found in apolipoprotein E-deficient mice. Finally, we summarize the effects of cerebroprotective pharmacological agents including the iron chelator desferal, superoxide dismutase, a stable radical from the nitroxide family, and HU-211, a nonpsychotoropic cannabinoid with antioxidant properties. We conclude that ROS play a key role in the pathophysiology of brain injury, and that their neutralization by endogenous or exogenous antioxidants has a protective effect. It is suggested, therefore, that the brain responds to ROS by increasing LMWA, and that the degree of this response is correlated with clinical recovery. The greater the response, the more favorable the outcome.

Diffusion- and T2-WEIGHTED MRI of closed-head injury in rats : A time course study and correlation with histology

Diffusion- and T2-weighted MRI were used to evaluate changes in brain water characteristics following closed-head injury in rats. Images were collected within the first 2 h and at 24 h and 7 days following the traumatic event and then compared with histology. The ratios between the apparent diffusion coefficients (ADCs) of the traumatized tissues and normal brain tissues were significantly different from unity and were found to be 0.79 +/- 0.25 (p < 0.01), 0.49 +/- 0.33 (p < 0.0002), and 3.47 +/- 1.36 (p < 10(-6)) at 1-2 h, 24 h, and 1 week after the trauma, respectively. In severe trauma, areas of hyperintensity which were not apparent on the T2-weighted images could be detected on the diffusion-weighted images within 1-2 h after the trauma. At 24 h following the traumatic event, large areas of hyperintensity are observed in both types of images. One week following the trauma, the ADCs of the traumatized tissues (1.84 +/- 0.69 x 10(-5) cm2/s) are much larger than those of normal brain (0.57 +/- 0.19 x 10(-5) cm2/s) and approach the value of free water. At 7 days, the areas of hyperintensity in the T2-weighted images seem to underestimate the injured areas found by histology. At this time point a good correlation is obtained between the areas of hypointensity observed on the diffusion-weighted images and the infarct areas obtained by histology (r = 0.88).

Cerebroprotective effect of stable nitroxide radicals in closed head injury in the rat

Nitroxide stable radicals are unreactive toward most diamagnetic molecules, but readily undergo one-electron redox reactions with paramagnetic species such as free radicals and transition metals, thus serving as cell permeable antioxidants. The involvement of reactive oxygen species in the pathophysiology of neurotrauma has been well established. The neuroprotective properties of three nitroxides: 2,2,6,6-tetramethylpiperidine-1-N-oxyl (TPO), the hydrophilic analog: TPL, and its reduced form: TPH, were tested in a rat model of closed head injury (CHI). CHI was induced in ether anesthetized rats by a weight drop device and recovery was followed for up to 24 h. The clinical status was evaluated according to a Neurological Severity Score (NSS), at 1 h and 24 h, the difference between these scores, delta NSS, reflecting the extent of recovery. Edema was assessed by measurement of water content at 24 h. The integrity of the blood-brain barrier (BBB) was investigated using Evans Blue extravasation. TPL, TPH and TPO facilitated clinical recovery, the latter causing a more pronounced effect (delta NSS = 7.63 +/- 0.26 in treated rats vs 4.94 +/- 0.48 in control rats, P < 0.001). TPL was found to significantly reduce edema formation (80.13% +/- 0.26 vs 83.65% +/- 0.49, P < 0.001) and to ameliorate BBB disruption (P < 0.001). The therapeutic window of TPL was found to be in the range of 4 h after CHI. The mechanisms underlying the nitroxide neuroprotective activity presumably involve: (a) reoxidation of reduced transition metal ions; (b) a selective radical-radical reaction; and (c) catalytic removal of intracellular and extracellular .O2-. The results indicate that nitroxides could be used in neuroprotective treatment of CHI.

Changes of Biological Reducing Activity in Rat Brain following Closed Head Injury: A Cyclic Voltammetry Study in Normal and Heat-Acclimated Rats

Reactive oxygen species (ROS) are normally generated in the brain during metabolism, and their production is enhanced by various insults. Low molecular weight antioxidants (LMWA) are one of the defense mechanisms of the living cell against ROS. The reducing capacity of brain tissue (total LMWA) was measured by cyclic voltammetry (CV), which records biological oxidation potential specific to the type of scavenger(s) present and anodic current intensity (Ia), which depends on scavenger concentration. In the present study, the reducing capacity of rat brain following closed head injury (CHI) was measured. In addition, CV of heat-acclimated traumatized rats was used to correlate endogenous cerebroprotection after CHI with LMWA activity. Sham-injured rat brains displayed two anodic potentials: at 350 ± 50 mV (Ia = 0.75 ± 0.06 μA/mg protein) and at 750 ± 50 mV (Ia = 1.00 ± 0.05 μA/mg protein). Following CHI, the anodic waves appeared at the same potentials as in the sham animals. However, within 5 min of CHI, the total reducing capacity was transiently decreased by 40% (p < 0.01). A second dip was detected at 24 h (60%, p < 0.005). By 48 h and at 7 days, the Ia levels normalized. The acclimated rats displayed anodic potentials identical to those of normothermic rats. However, the Ia of both potentials was lower (60% of control, p < 0.001). The Ia profile after CHI was the direct opposite of the normothermic Ia profile: no immediate decrease of Ia and an increase from 4 h and up to 7 days (40–50%, p < 0.001). We suggest that the lowered levels of LMWA in the post-CHI period reflect their consumption due to overproduction of free radicals. The augmented concentration of LMWA found in the brain of the heat-acclimated rats suggests that these rats are better able to cope with these harmful radicals, resulting in a more favorable outcome following CHI.

Overall low molecular weight antioxidant activity of biological fluids and tissues by cyclic voltammetry

Publisher Summary This chapter describes cyclic voltammetry method that is designed to evaluate the total antioxidant activity of lipophilic and hydrophilic low molecular weight antioxidants (LMWA) without specific determination of the various compounds. Evaluation of antioxidant activity using the cyclic voltammetry approach has several advantages over other methods. Cyclic voltammetry measurements allow the evaluation of the antioxidant status of both water- and lipid-soluble LMWA. The outcome of such measurements can be used for the assessment of the overall antioxidant activity that is derived from the reducing LMWA without measuring specific compounds. Examination of cyclic voltammograms indicates the types of the various compounds responsible for the antioxidant activity of a biological sample and their total concentration. Preparation of samples for such evaluation is simple and does not require advanced procedures. The measurement itself is rapid and the results can be analyzed immediately.

Antioxidants attenuate acute toxicity of tumor necrosis factor-alpha induced by brain injury in rat.

Tumor necrosis factor-alpha alpha (TNF-alpha) and reactive oxygen species (ROS) are produced in the brain after traumatic injury and have deleterious effects. In a rat model of closed head injury (CHI), the synthetic antioxidant from the nitroxide family, Tempol, improved recovery and protected the blood-brain barrier. Similar protection was found after CHI in heat-acclimated rats, in which the endogenous antioxidants have been shown to be elevated after CHI. The present study examined the relationship between TNF-alpha and ROS after CHI, namely, whether after CHI, antioxidants that afforded cerebroprotection also attenuated brain levels of TNF-alpha. Three groups of rats were subjected to CHI: (1) control, nontreated, (2) Tempol-treated, and (3) heat-acclimated (30 days at 34 degrees C). Four hours after injury (time for peak production of TNF-alpha), the activity of TNF-alpha was measured. Although clinical recovery was facilitated in rats of the two treated groups, TNF-alpha activity was as high as in the traumatized, untreated rats. Moreover, direct injection of TNF-alpha into mouse brain induced disruption of the blood-brain barrier, indicating its acute harmful effect. This toxic effect was attenuated by before and after treatment with Tempol. Our results support the hypothesis that in vivo antioxidants neutralize TNF-alpha toxicity, probably by interfering with activation of the transcription factor NF-kappa-B.

Mechanism of Brain Protection by Nitroxide Radicals in Experimental Model of Closed-Head Injury

Reactive oxygen-derived species were previously implicated in mediation of post-traumatic brain damage; however, the efficacy of traditional antioxidants in preventing/reversing the damage is sometimes limited. The present work focused on the mechanisms underlying the neuroprotective activity of cell permeable, nontoxic, antioxidants, namely stable nitroxide radicals in an experimental model of rat closed-head injury. Brain damage was induced by the weight-drop method and the clinical status was evaluated according to a neurological severity score at 1 h and 24 h, where the difference between these scores reflects the extent of recovery. The metal chelator deferoxamine as well as three nitroxide derivatives, differing in hydrophilicity and charge, and one hydroxylamine (a reduced nitroxide) facilitated the clinical recovery and decreased the brain edema. The nitroxides, but neither the hydroxylamine nor deferoxamine, protected the integrity of the blood-brain barrier. Superoxide dismutase also improved the clinical recovery but did not affect brain edema or the blood-brain barrier. The results suggest that by switching back and forth between themselves, the nitroxide and hydroxylamine act catalytically as self-replenishing antioxidants, and protect brain tissue by terminating radical-chain reactions, oxidizing deleterious metal ions, and by removal of intracellular superoxide.

Neuroprotection against oxidative stress by serum from heat acclimated rats

Exposure of PC12 cells, to 1% serum derived from normothermic (CON) rats resulted in 79% cell death. Sister cultures treated with 1% serum derived from heat acclimated (ACC) rats, were neuroprotected and expressed a significant reduction in cell death. In PC12 cells exposed to a free radical generator causing an oxidative stress, 90% cell death was measured in CON serum treated cultures, while ACC serum treated cultures were neuroprotected. Xanthine oxidase activity and uric acid (UA) levels were lower in ACC serum compared to CON. Addition of UA to both sera abolished the difference in cell viability, and toxicity of ACC serum reached that of CON. These findings suggest a causal relationship between the lower levels of UA in ACC and the neuroprotective effect observed. The present study proposes heat acclimation as an experimental and/or clinical tool for the achievement of neuroprotection.