Antioxidants and free radical scavengers for the treatment of stroke, traumatic brain injury and aging.

The overproduction of reactive oxygen species (ROS) and reactive nitrogen species (RNS) is a common underlying mechanism of many neuropathologies, as they have been shown to damage various cellular components, including proteins, lipids and DNA. Free radicals, especially superoxide (O(2)*-), and non-radicals, such as hydrogen peroxide (H(2)O(2)), can be generated in quantities large enough to overwhelm endogenous protective enzyme systems, such as superoxide dismutase (SOD) and reduced glutathione (GSH). Here we review the mechanisms of ROS and RNS production, and their roles in ischemia, traumatic brain injury and aging. In particular, we discuss several acute and chronic pharmacological therapies that have been extensively studied in order to reduce ROS/RNS loads in cells and the subsequent oxidative stress, so-called "free-radical scavengers." Although the overall aim has been to counteract the detrimental effects of ROS/RNS in these pathologies, success has been limited, especially in human clinical studies. This review highlights some of the recent successes and failures in animal and human studies by attempting to link a compound's chemical structure with its efficacy as a free radical scavenger. In particular, we demonstrate how antioxidants derived from natural products, as well as long-term dietary alterations, may prove to be effective scavengers of ROS and RNS.

Localization of electrogenic Na/bicarbonate cotransporter NBCe1 variants in rat brain.

The activity of HCO(3)(-) transporters contributes to the acid-base environment of the nervous system. In the present study, we used in situ hybridization, immunoblotting, immunohistochemistry, and immunogold electron microscopy to localize electrogenic Na/bicarbonate cotransporter NBCe1 splice variants (-A, -B, and -C) in rat brain. The in situ hybridization data are consistent with NBCe1-B and -C, but not -A, being the predominant NBCe1 variants in brain, particularly in the cerebellum, hippocampus, piriform cortex, and olfactory bulb. An antisense probe to the B and C variants strongly labeled granule neurons in the dentate gyrus of the hippocampus, and cells in the granule layer and Purkinje layer (e.g. Bergmann glia) of the cerebellum. Weaker labeling was observed in the pyramidal layer of the hippocampus and in astrocytes throughout the brain. Similar, but weaker labeling was obtained with an antisense probe to the A and B variants. In immunoblot studies, antibodies to the A and B variants (alphaA/B) and C variant (alphaC) labeled approximately 130-kDa proteins in various brain regions. From immunohistochemistry data, both alphaA/B and alphaC exhibited diffuse labeling throughout brain, but alphaA/B labeling was more intracellular and punctate. Based on co-localization studies with antibodies to neuronal or astrocytic markers, alphaA/B labeled neurons in the pyramidal layer and dentate gyrus of the hippocampus, as well as cortex. alphaC labeled glia surrounding neurons (and possibly neurons) in the neuropil of the Purkinje cell layer of the cerebellum, the pyramidal cell layer and dentate gyrus of the hippocampus, and the cortex. According to electron microscopy data from the cerebellum, alphaA/B primarily labeled neurons intracellularly and alphaC labeled astrocytes at the plasma membrane. In summary, the B and C variants are the predominant NBCe1 variants in rat brain and exhibit different localization profiles.

Two distinct signaling pathways regulate peroxynitrite-induced apoptosis in PC12 cells.

The mechanisms of peroxynitrite-induced apoptosis are not fully understood. We report here that peroxynitrite-induced apoptosis of PC12 cells requires the simultaneous activation of p38 and JNK MAP kinase, which in turn activates the intrinsic apoptotic pathway, as evidenced by Bax translocation to the mitochondria, cytochrome c release to the cytoplasm and activation of caspases, leading to cell death. Peroxynitrite induces inactivation of the Akt pathway. Furthermore, overexpression of constitutively active Akt inhibits both peroxynitrite-induced Bax translocation and cell death. Peroxynitrite-induced death was prevented by overexpression of Bcl-2 and by cyclosporin A, implicating the involvement of the intrinsic apoptotic pathway. Selective inhibition of mixed lineage kinase (MLK), p38 or JNK does not attenuate the decrease in Akt phosphorylation showing that inactivation of the Akt pathway occurs independently of the MLK/MAPK pathway. Together, these results reveal that peroxynitrite-induced activation of the intrinsic apoptotic pathway involves interactions with the MLK/MAPK and Akt signaling pathways.

Exposure to prenatal nicotine transiently increases neuronal nicotinic receptor subunit alpha7, alpha4 and beta2 messenger RNAs in the postnatal rat brain.

This study determined the effects of prenatal nicotine exposure (2 mg/kg/day) in Sprague Dawley CD rats via subcutaneously implanted osmotic minipumps, during gestational days 7-21, on postnatal levels of neuronal nicotinic receptor alpha4, alpha7 and beta2 subunit messenger RNAs. Northern analysis of postnatal day 1, 7, 14 and 28 hippocampal/septal and cortical total RNA using alpha-[32P]dCTP-labeled alpha4, alpha7 and beta2 complementary DNA probes identified a single (5.7-kb) alpha7 messenger RNA, three (2.4-, 3.8- and 8.0-kb) alpha4 messenger RNAs and four (3.7-, 5.0-, 7.5- and 10.0-kb) beta2 messenger RNAs. In comparison to prenatal saline, prenatal nicotine produced several significantly higher messenger RNA levels (cortical: 5.7-kb alpha7, 2.4-, 3.8- and 8.0-kb alpha4, 10.0-kb beta2; hippocampal/septal: 2.4- and 8.0-kb alpha4); these increases occurred predominantly on, but were not restricted to, postnatal day 14. Effects of nicotine were generally resolved by postnatal day 28. Collapsing the data across sex and age, a significant treatment effect indicated that hippocampal/septal and cortical 8.0-kb alpha4 messenger RNA levels and 10.0-kb beta2 messenger RNA levels were significantly higher following prenatal nicotine exposure. This is the first study indicating that prenatal nicotine produces alterations in developing postnatal rat neuronal nicotinic receptor messenger RNA levels, possibly by premature stimulation of neuronal nicotinic receptors. These results further implicate the teratogenic potential of nicotine in postnatal neuronal development.

Postnatal developmental regulation of neuronal nicotinic receptor subunit alpha 7 and multiple alpha 4 and beta 2 mRNA species in the rat.

This study examined the postnatal development of neuronal nicotinic receptor (nAChR) alpha 7, alpha 4 and beta 2 subunit mRNA in the Sprague Dawley rat brain. The hippocampus, septum and cortex were removed on postnatal day 1 (P1), P7, P14, or P28 and analyzed by sex. Northern analysis of cortical and pooled hippocampal and septal total RNA with 32P-alpha-dCTP-labeled alpha 7, alpha 4 (recognizing alpha 4.1 and alpha 4.2 mRNA), and beta 2 nAChR cDNA probes identified three (2.4, 3.8 and 8.0 kb) alpha 4, four (3.5, 5.0, 7.5 and 10.0 kb) beta 2 and a single 5.7 kb alpha 7 mRNA species. Cortical alpha 4 mRNA peaked on P14 and remained high on P28, whereas hippocampal/septal alpha 4 mRNA was higher on P7 and P14 than on P1 and P28. Expression of cortical and hippocampal/septal beta 2 mRNAs decreased on P7, followed by a dramatic peak on P14. alpha 7 mRNA peaked on P7. Throughout development, 2.4 kb alpha 4 mRNA was more intense than 3.8 kb alpha 4 mRNA, whereas 5.0 kb beta 2 mRNA was the most intense cortical and hippocampal/septal beta 2 mRNA species. The alpha 4.1-specific cDNA probe detected similar-sized alpha 4 bands as the pan-specific alpha 4 cDNA probe, therefore precluding the identification of any band as alpha 4.2-specific. These results suggest that postnatal expression of alpha 4 and alpha 7 but not beta 2 mRNAs is brain region-specific, and that the contribution of multiple nAChR subunit mRNA species in development may vary.

Prenatal nicotine sex-dependently alters agonist-induced locomotion and stereotypy.

This study examined the effects of prenatal nicotine exposure (2 mg/kg/day) via s.c. osmotic minipumps, gestational days 7-22, on nicotine- and lobeline-induced locomotor activity and stereotypy in 14-day-old rat pups. Prenatal nicotine exposure increased fetal mortality and produced decreases in weight gain apparent after weaning, but did not affect acquisition of developmental milestones. Compared to male pups prenatally exposed to saline, those prenatally exposed to nicotine and challenged with nicotine (1 mg/kg, i.p.) exhibited significantly greater locomotor activity, whereas a lobeline challenge (1 mg/kg, s.c.) produced significantly greater stereotypy. No effects of prenatal exposure were observed on locomotor activity or stereotypy in females. Results suggest that 1) central control of motor function may be more vulnerable to prenatal nicotine in males, and 2) nicotine and lobeline possess distinct pharmacological profiles.