Role of NADPH oxidase-2 in the progression of the inflammatory response secondary to striatum excitotoxic damage.

BACKGROUND: During excitotoxic damage, neuronal death results from the increase in intracellular calcium, the induction of oxidative stress, and a subsequent inflammatory response. NADPH oxidases (NOX) are relevant sources of reactive oxygen species (ROS) during excitotoxic damage. NADPH oxidase-2 (NOX-2) has been particularly related to neuronal damage and death, as well as to the resolution of the subsequent inflammatory response. As ROS are crucial components of the regulation of inflammatory response, in this work, we evaluated the role of NOX-2 in the progression of inflammation resulting from glutamate-induced excitotoxic damage of the striatum in an in vivo model. METHODS: The striata of wild-type C57BL/6 J and NOX-2 KO mice (gp91Cybbtm1Din/J) were stereotactically injected with monosodium glutamate either alone or in combination with IL-4 or IL-10. The damage was evaluated in histological sections stained with cresyl violet and Fluoro-Jade B. The enzymatic activity of caspase-3 and NOX were also measured. Additionally, the cytokine profile was identified by ELISA and motor activity was verified by the tests of the cylinder, the adhesive tape removal, and the inverted grid. RESULTS: Our results show a neuroprotective effect in mice with a genetic inhibition of NOX-2, which is partially due to a differential response to excitotoxic damage, characterized by the production of anti-inflammatory cytokines. In NOX-2 KO animals, the excitotoxic condition increased the production of interleukin-4, which could contribute to the production of interleukin-10 that decreased neuronal apoptotic death and the magnitude of striatal injury. Treatment with interleukin-4 and interleukin-10 protected from excitotoxic damage in wild-type animals. CONCLUSIONS: The release of proinflammatory cytokines during the excitotoxic event promotes an additional apoptotic death of neurons that survived the initial damage. During the subsequent inflammatory response to excitotoxic damage, ROS generated by NOX-2 play a decisive role in the extension of the lesion and consequently in the severity of the functional compromise, probably by regulating the anti-inflammatory cytokines production.

Recurrent moderate hypoglycemia exacerbates oxidative damage and neuronal death leading to cognitive dysfunction after the hypoglycemic coma.

Moderate recurrent hypoglycemia (RH) is frequent in Type 1 diabetes mellitus (TIDM) patients who are under intensive insulin therapy increasing the risk for severe hypoglycemia (SH). The consequences of RH are not well understood and its repercussions on neuronal damage and cognitive function after a subsequent episode of SH have been poorly investigated. In the current study, we have addressed this question and observed that previous RH during seven consecutive days exacerbated oxidative damage and neuronal death induced by a subsequent episode of SH accompanied by a short period of coma, in the parietal cortex, the striatum and mainly in the hippocampus. These changes correlated with a severe decrease in reduced glutathione content (GSH), and a significant spatial and contextual memory deficit. Administration of the antioxidant, N-acetyl-L-cysteine, (NAC) reduced neuronal death and prevented cognitive impairment. These results demonstrate that previous RH enhances brain vulnerability to acute hypoglycemia and suggests that this effect is mediated by the decline in the antioxidant defense and oxidative damage. The present results highlight the importance of an adequate control of moderate hypoglycemic episodes in TIDM.

Gestational exposure to inorganic arsenic (iAs3+) alters glutamate disposition in the mouse hippocampus and ionotropic glutamate receptor expression leading to memory impairment.

Early life exposure to environmental pollutants and toxic chemicals has been linked to learning and behavioral alterations in children. iAs exposure is associated with different types neurological disorders such as memory and learning impairment. iAs is methylated in the brain by the arsenic III-methyltransferase in a process that requires glutathione (GSH). The xCT-antiporter cell membrane transporter participates in the influx of cystine for GSH synthesis in exchange for glutamate in a 1:1 ratio. In CD-1 mice gestationally exposed to 20 ppm of sodium arsenite in drinking water, we have previously observed up-regulation of xCT in the male mouse hippocampus which caused glutamatergic synapse alterations affecting learning and memory processes. Here, we used the same gestational iAs exposure model to investigate whether the up-regulation of xCT and down-regulation of GLT-1 transporters were associated with higher levels of extracellular glutamate and changes in the expression of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptor, responsible for excitatory fast synaptic transmission. The induction of LTP in the perforant-dentate gyrus pathway (PP-DG) of the hippocampus was also studied, as well as learning and memory formation using the water maze test. Changes in GSH levels were also tested in the hippocampus of animals exposed to iAs. Results showed increased GSH synthesis (p < 0.05), associated with significantly higher extracellular glutamate levels in iAs exposed mice. Exposure was also significantly associated with AMPA subunits down-regulation, deficient LTP induction, and lower excitability of the PP-DG pathway. In addition, animals showed deficient learning and memory in the Morris Water Maze test.

Saturated lipids decrease mitofusin 2 leading to endoplasmic reticulum stress activation and insulin resistance in hypothalamic cells.

Endoplasmic reticulum (ER) and mitochondria dysfunction contribute to insulin resistance generation during obesity and diabetes. ER and mitochondria interact through Mitofusin 2 (MTF2), which anchors in the outer mitochondrial and ER membranes regulating energy metabolism. Ablation of MTF2 leads to ER stress activation and insulin resistance. Here we determine whether lipotoxic insult induced by saturated lipids decreases MTF2 expression leading to ER stress response in hypothalamus and its effects on insulin sensitivity using in vitro and in vivo models. We found that lipotoxic stimulation induced by palmitic acid, but not the monounsaturated palmitoleic acid, decreases MTF2 protein levels in hypothalamic mHypoA-CLU192 cells. Also, palmitic acid incubation activates ER stress response evidenced by increase in the protein levels of GRP78/BIP marker at later stage than MTF2 downregulation. Additionally, we found that MTF2 alterations induced by palmitic, but not palmitoleic, stimulation exacerbate insulin resistance in hypothalamic cells. Insulin resistance induced by palmitic acid is prevented by pre-incubation of the anti-inflammatory and the ER stress release reagents, sodium salicylate and 4 phenylbutirate, respectively. Finally, we demonstrated that lipotoxic insult induced by high fat feeding to mice decreases MTF2 proteins levels in arcuate nucleus of hypothalamus. Our data indicate that saturated lipids modulate MTF2 expression in hypothalamus coordinating the ER stress response and the susceptibility to insulin resistance.

Protection of hypoglycemia-induced neuronal death by ß-hydroxybutyrate involves the preservation of energy levels and decreased production of reactive oxygen species.

Glucose is the main energy substrate in brain but in certain circumstances such as prolonged fasting and the suckling period alternative substrates can be used such as the ketone bodies (KB), beta-hydroxybutyrate (BHB), and acetoacetate. It has been shown that KB prevent neuronal death induced during energy limiting conditions and excitotoxicity. The protective effect of KB has been mainly attributed to the improvement of mitochondrial function. In the present study, we have investigated the protective effect of D-BHB against neuronal death induced by severe noncoma hypoglycemia in the rat in vivo and by glucose deprivation (GD) in cortical cultures. Results show that systemic administration of D-BHB reduces reactive oxygen species (ROS) production in distinct cortical areas and subregions of the hippocampus and efficiently prevents neuronal death in the cortex of hypoglycemic animals. In vitro results show that D-BHB stimulates ATP production and reduces ROS levels, while the nonphysiologic isomer of BHB, L-BHB, has no effect on energy production but reduces ROS levels. Data suggest that protection by BHB, not only results from its metabolic action but is also related to its capability to reduce ROS, rendering this KB as a suitable candidate for the treatment of ischemic and traumatic injury.

Activation of NOX2 by the stimulation of ionotropic and metabotropic glutamate receptors contributes to glutamate neurotoxicity in vivo through the production of reactive oxygen species and calpain activation.

Prolonged activation of glutamate receptors leads to excitotoxicity. Several processes such as reactive oxygen species (ROS) production and activation of the calcium-dependent protease, calpain, contribute to glutamate-induced damage. It has been suggested that the ROS-producing enzyme, NADPH oxidase (NOX), plays a role in excitotoxicity. Studies have reported NOX activation after NMDA receptor stimulation during excitotoxic damage, but the role of non-NMDA and metabotropic receptors is unknown. We evaluated the roles of different glutamate receptor subtypes on NOX activation and neuronal death induced by the intrastriatal administration of glutamate in mice. In wild-type mice, NOX2 immunoreactivity in neurons and microglia was stimulated by glutamate administration, and it progressively increased as microglia became activated; calpain activity was also induced. By contrast, mice lacking NOX2 were less vulnerable to excitotoxicity, and there was reduced ROS production and protein nitrosylation, microglial reactivity, and calpain activation. These results suggest that NOX2 is stimulated by glutamate in neurons and reactive microglia through the activation of ionotropic and metabotropic receptors. Neuronal damage involves ROS production by NOX2, which, in turn, contributes to calpain activation.

Antioxidant capacity contributes to protection of ketone bodies against oxidative damage induced during hypoglycemic conditions.

Ketone bodies play a key role in mammalian energy metabolism during the suckling period. Normally ketone bodies' blood concentration during adulthood is very low, although it can rise during starvation, an exogenous infusion or a ketogenic diet. Whenever ketone bodies' levels increase, their oxidation in the brain rises. For this reason they have been used as protective molecules against refractory epilepsy and in experimental models of ischemia and excitotoxicity. The mechanisms underlying the protective effect of these compounds are not completely understood. Here, we studied a possible antioxidant capacity of ketone bodies and whether it contributes to the protection against oxidative damage induced during hypoglycemia. We report for the first time the scavenging capacity of the ketone bodies, acetoacetate (AcAc) and both the physiological and non-physiological isomers of beta-hydroxybutyrate (D- and L-BHB, respectively), for diverse reactive oxygen species (ROS). Hydroxyl radicals (.OH) were effectively scavenged by D- and L-BHB. In addition, the three ketone bodies were able to reduce cell death and ROS production induced by the glycolysis inhibitor, iodoacetate (IOA), while only D-BHB and AcAc prevented neuronal ATP decline. Finally, in an in vivo model of insulin-induced hypoglycemia, the administration of D- or L-BHB, but not of AcAc, was able to prevent the hypoglycemia-induced increase in lipid peroxidation in the rat hippocampus. Our data suggest that the antioxidant capacity contributes to protection of ketone bodies against oxidative damage in in vitro and in vivo models associated with free radical production and energy impairment.

Contribution of NMDA and non-NMDA receptors to in vivo glutamate-induced calpain activation in the rat striatum. Relation to neuronal damage.

Glutamate, the major excitatory neurotransmitter, can cause the death of neurons by a mechanism known as excitotoxicity. This is a calcium-dependent process and activation of the NMDA receptor subtype contributes mainly to neuronal damage, due to its high permeability to calcium. Activation of calpain, a calcium-dependent cysteine protease, has been implicated in necrotic excitotoxic neuronal death. We have investigated the contribution of NMDA and non-NMDA ionotropic receptors to calpain activation and neuronal death induced by the acute administration of glutamate into the rat striatum. Calpain activity was assessed by the cleavage of the cytoskeletal protein, alpha-spectrin. Caspase-3 activity was also studied because glutamate can also lead to apoptosis. Results show no caspase-3 activity, but a strong calpain activation involving both NMDA and non-NMDA receptors. Although neuronal damage is mediated mainly by the NMDA receptor subtype, it can not be attributed solely to calpain activity.

D-beta-hydroxybutyrate prevents glutamate-mediated lipoperoxidation and neuronal damage elicited during glycolysis inhibition in vivo.

Excitotoxic neuronal death mediated by over-activation of glutamate receptors has been implicated in ischemia, hypoglycemia and some neurodegenerative diseases. It involves oxidative stress and is highly facilitated during impairment of energy metabolism. We have shown previously that in vivo systemic glycolysis inhibition with iodoacetate (IOA), exacerbates glutamate excitotoxicity. We have now investigated whether this effect involves oxidative damage to membrane lipids, as evaluated by the presence of thiobarbituric acid-reactive substances. We have also tested whether the ketone body, D-beta-hydroxybutyrate (D-BHB), prevents lipoperoxidation and tissue damage. Results show that glutamate intrastriatal injection in control rats transiently enhances lipoperoxidation, while in IOA-treated animals increased lipoperoxidation is sustained. Treatment with D-BHB significantly reduces striatal lesions and lipoperoxidation. Vitamin E also reduced neuronal damage and lipoperoxidation. Results suggest that glycolysis impairment favors a pro-oxidant condition and situates oxidative damage as an important mediator of in vivo induced excitotoxicity. Results provide evidence for the neuroprotective effect of D-BHB against glutamate toxicity.

Role of oxidative stress on beta-amyloid neurotoxicity elicited during impairment of energy metabolism in the hippocampus: protection by antioxidants.

Age-associated oxidative stress has been implicated in neuronal damage linked with Alzheimer's disease (AD). In addition to the role of beta-amyloid peptide (Abeta) in the pathogenesis of AD, reduced glucose oxidative metabolism and decreased mitochondrial activity have been suggested as associated factors. However, the relationship between Abeta toxicity, metabolic impairment, and oxidative stress is far from being understood. In vivo neurotoxicity of Abeta25-35 peptide has been conflicting. However, in previous studies, we have shown that Abeta25-35 consistently induces synaptic toxicity and neuronal death in the hippocampus in vivo, when administered during moderate glycolytic or mitochondrial inhibition. In the present study, we have investigated whether enhancement of Abeta neurotoxicity during these conditions involves oxidative stress. Results show increased lipoperoxidation (LPO) when Abeta is administered in the hippocampus of rats previously treated with the glycolysis inhibitor, iodoacetate. Neuronal damage and LPO are efficiently prevented by vitamin E, while the spin trapper, alpha-phenyl-N-tert-butyl nitrone, shows partial protection. Abeta stimulates LPO in synaptosomes, but toxicity is only observed in the presence of metabolic inhibitors. Damage and LPO are efficiently prevented by vitamin E. The present results suggest an interaction between oxidative stress and metabolic impairment in the Abeta neurotoxic cascade.

beta-Amyloid neurotoxicity is exacerbated during glycolysis inhibition and mitochondrial impairment in the rat hippocampus in vivo and in isolated nerve terminals: implications for Alzheimer's disease.

Senile plaques composed mainly by beta-amyloid (Abeta) protein are one of the pathological hallmarks of Alzheimer's disease (AD). In vitro, Abeta and its active fragment 25-35 have been shown either to be directly neurotoxic or to exacerbate the damaging effect of other neurotoxic insults. However, the attempts to replicate Abeta neurotoxicity in vivo have yielded conflicting results. One of the most consistent alterations in AD is a reduced resting glucose utilization. Important evidence suggests that impairment of brain energy metabolism can lead to neuronal damage or facilitate the deleterious effects of some neurotoxic agents. In the present study we have investigated the influence of glycolysis inhibition induced by iodoacetate, and mitochondrial impairment induced by 3-nitropropionic acid (3-NP), in the toxicity of Abeta. We have studied Abeta neurotoxicity during energy deficiency both in vivo in the dentate gyrus of the hippocampal formation and in presynaptic terminals isolated from neocortex and hippocampus. Results show that during metabolic inhibition an enhanced vulnerability of hippocampal neurons to Abeta peptide toxicity occurs, probably resulting from decreased glucose metabolism and mitochondrial ATP production. Synaptosomal response to energy impairment and Abeta toxicity was evaluated by the MTT assay. Results suggest that synapses may be particularly sensitive to metabolic perturbation, which in turn exacerbates Abeta toxicity. The present data provide experimental support to the hypothesis that certain risk factors such as metabolic dysfunction and amyloid accumulation may interact to exacerbate AD, and that metabolic substrates such as pyruvate may play a role as a therapeutic tool.

Efectos de la DL-3-hidroxi, 3-etil, 3-fenil propionamida (HEPP) sobre un nuevo modelo de status epilepticus focal: el síndrome de abstinencia al GABA (SAG) / Efects of DL-3-hydroxy, 3-ethil, 3-phenyl propionamide (HEPP) on a new model of focal status epilepticus: the gaba withdrawal syndrome

El síndrome de Abstinencia al GABA (SAG), es un nuevo modelo de epileptogénesis focal en el que la actividad paroxística se induce mediante la interrupción de infusiones intracorticales de GABA. En estudios preliminares hemos observado que esta actividad muestra una extraordinaria resistencia a los anticonvulsivantes clásicos. En ratas que mostraban un SAG inducido por la suspención de infusiones localizadas de GABA (50 µg ml por hora en 24 horas), se administró el nuevo anticonvulsivante HEPP (50 mg/kg ip) 60 minutos después del inicio del SAG. La HEPP produjo una disminución significativa de la frecuencia de descarga en 8 de 10 ratas, con efecto máximo aproximadamante a los 30 min después de la inyección y una duración del efecto de cerca de 90 min. Cuando la HEPP se administró a un nuevo grupo de ratas (n = 5) al segundo día del SAG, el compuesto no produjo efectos. A pesar de que se ignora aún el mecanismo de acción de la HEPP, el SAG representa un modelo útil para la evaluación de nuevos anticonvulsivantes