Martha C. Rivera-Cervantes

Neuronal death and tumor necrosis factor-α response to glutamate-induced excitotoxicity in the cerebral cortex of neonatal rats

Neuronal death and lactate dehydrogenase (LDH) activity were evaluated in the cerebral cortices of neonatal rats after exposure to monosodium L-glutamate (MSG) to induce neuroexcitotoxicity. A time-response profile for tumor necrosis factor-alpha (TNF-alpha) expression was drawn, with measurements taken every 6 h after the first dose of MSG during the first 8 postnatal days, and at days 10 and 14 after birth. An increase in neuronal loss accompanied by high LDH activity and high TNF-alpha levels was observed at 8 and 10 days. These results indicate that neuronal loss may occur via an apoptosis-like mechanism directed selectively against neurons that express glutamate receptors, mainly the N-methyl-D-aspartate, which it may be strengthen by high TNF-alpha levels through a feedback mechanism to induce cell death via apoptosis.

Role of p38 MAPK and pro-inflammatory cytokines expression in glutamate-induced neuronal death of neonatal rats.

Pro‐inflammatory cytokines TNF‐α, IL‐1β and IL‐6 rises significantly during neuronal damage and activate the signaling p38 MAPK pathway, which is involved in the apoptotic (AP) neuronal death. Systemic administration of glutamate as monosodium salt (MSG) to newborn animals induces neuronal death, however whether neurons die by AP or necrosis through MAPK p38 pathway activation it is unknown. In this study, TNF‐α, IL‐1β and IL‐6 expression levels, AP neuronal death and cellular type that produces TNF‐α was also identified in the cerebral cortex (CC) and striatum (St) of rats at 8, 10, and 14 days of age after neonatal exposure to MSG. TNF‐α production and AP neuronal death was significantly increased in the CC at PD8–10, and in the St in all ages studied by excitotoxicity effect induced with MSG. This effect was completely inhibited by SB203580 (p38 inhibitor) in both regions studied. TNF‐α, IL‐1β and IL‐6 RNAm increased after MSG administration, whereas SB203580 did not modify their expression. These data indicates that neuronal death induced by excitotoxicity appears to be mediated through p38 signaling pathway activated by TNF‐α and their inhibition may have an important neuroprotective role as part of anti‐inflammatory therapeutic strategy.

Selective Estrogen Receptor Modulators Regulate Dendritic Spine Plasticity in the Hippocampus of Male Rats

Some selective estrogen receptor modulators, such as raloxifene and tamoxifen, are neuroprotective and reduce brain inflammation in several experimental models of neurodegeneration. In addition, raloxifene and tamoxifen counteract cognitive deficits caused by gonadal hormone deprivation in male rats. In this study, we have explored whether raloxifene and tamoxifen may regulate the number and geometry of dendritic spines in CA1 pyramidal neurons of the rat hippocampus. Young adult male rats were injected with raloxifene (1 mg/kg), tamoxifen (1 mg/kg), or vehicle and killed 24 h after the injection. Animals treated with raloxifene or tamoxifen showed an increased numerical density of dendritic spines in CA1 pyramidal neurons compared to animals treated with vehicle. Raloxifene and tamoxifen had also specific effects in the morphology of spines. These findings suggest that raloxifene and tamoxifen may influence the processing of information by hippocampal pyramidal neurons by affecting the number and shape of dendritic spines.

Glutamate excitotoxicity activates the MAPK/ERK signaling pathway and induces the survival of rat hippocampal neurons in vivo.

Current knowledge concerning the molecular mechanisms of the cellular response to excitotoxic insults in neurodegenerative diseases is insufficient. Although glutamate (Glu) has been widely studied as the main excitatory neurotransmitter and principal excitotoxic agent, the neuroprotective response enacted by neurons is not yet completely understood. Some of the molecular participants have been revealed, but the signaling pathways involved in this protective response are just beginning to be identified. Here, we demonstrate in vivo that, in response to the cell damage and death induced by Glu excitotoxicity, neurons orchestrate a survival response through the extracellular signal-regulated kinase (ERK) signaling pathway by increasing ERK expression in the rat hippocampal (CA1) region, allowing increased neuronal survival. In addition, this protective response is specifically reversed by U0126, an ERK inhibitor, which promotes cell death only when it is administered together with Glu. Our findings demonstrate that the ERK signaling pathway has a neuroprotective role in the response to Glu-induced excitotoxicity in hippocampal neurons. Therefore, the ERK signaling pathway may be activated as a cellular response to excitotoxic injury to prevent damage and neural loss, representing a novel therapeutic target in the treatment of neurodegenerative diseases.

Changes in hippocampal NMDA-R subunit composition induced by exposure of neonatal rats to l-glutamate

Overactivation of NMDA‐Rs may mediate excitotoxic cell death associated with epileptic seizures, and hypoxic–ischemic conditions. We assessed whether repeated subcutaneous administration of l‐glutamate to neonatal rats affects the subunit composition of NMDA‐Rs. Accordingly, cortical and hippocampal tissue from 14‐day‐old rats was analyzed by Western blotting and RT‐PCR to quantify the protein and mRNA expression of different NMDA‐R subunits. In addition, tissue sections were Nissl stained to assess the cell damage in this tissue. Early exposure of neonatal rats to l‐glutamate differentially affects the expression of mRNA transcripts for NMDA‐R subunits in the cerebral cortex and hippocampus. In the cerebral cortex, a decrease in NR2B subunit mRNA expression was observed, as well as a loss of NR1 and NR2A protein. By contrast, neonatal l‐glutamate administration augmented the transcripts encoding the NR1, NR2B, and NR2C subunits in the hippocampal formation. The expression of mRNA encoding the NR2A subunit was not affected by neonatal l‐glutamate administration in either of the brain regions examined. This differential expression of NMDA‐R subunits following neonatal exposure to l‐glutamate may represent an adaptive response of the glutamate receptors to overactivation in order to reduce the effect of high l‐glutamate during the early period of life when the animal is more vulnerable to excitotoxicity.

NKCC1 and KCC2 protein expression is sexually dimorphic in the hippocampus and entorhinal cortex of neonatal rats

Seizure susceptibility appears to be greater in males than females during the early developmental stages of the brain when the gamma-aminobutyric acid (GABA), acting through its GABA-A receptor, predominantly produces neuronal depolarization. GABA-mediated excitation has been observed when the NKCC1 (chloride importer) expression level is higher than KCC2 (chloride exporter). In this study, the relative protein expression of NKCC1 and KCC2 over β-actin was evaluated in the hippocampus and entorhinal cortex of male and female rats during postnatal days (PND) 1, 3, 5, 7, 9, 11, 13 and 15 using Western blotting assays. For both cerebral regions in the females, the NKCC1/β-actin expression ratio was constant during all evaluated ages, whereas the KCC2/β-actin expression ratio increased gradually until reaching a maximal level at PND9 that was nearly three- and ten-fold higher in the hippocampus and entorhinal cortex, respectively, compared with the initial level. In males, the NKCC1/β-actin expression ratio was constant during the first week, peaking almost three-fold higher than the initial level at PND9 in the hippocampus and at PND11 in the entorhinal cortex and then returning to the initial values at PND13, whereas the KCC2/β-actin expression ratio increased gradually to reach a maximal and steady level at PND5, which were nearly two- and four-fold higher in the hippocampus and entorhinal cortex, respectively, compared with the intial level. In conclusion, the NKCC1/β-actin and KCC2/β-actin expression ratios displayed a specific expression profile for each gender and cerebral region, which could be related with the differences in seizure susceptibility observed between genders.

P38 MAPK Inhibition Protects Against Glutamate Neurotoxicity and Modifies NMDA and AMPA Receptor Subunit Expression

NMDA and AMPA receptors are thought to be responsible for Ca++ influx during glutamate-induced excitotoxicity and, therefore, hippocampal neuronal death. We assessed whether excitotoxicity induced by neonatal treatment with monosodium glutamate in rats at postnatal age of 1, 3, 5, and 7 modifies the hippocampal expression of the NMDAR subunit NR1 and the AMPAR subunits GluR1/GluR2 at postnatal days 8, 10, 12, and 14. We also assessed the involvement of MAPK signaling by using the p38 inhibitor SB203580. Our results showed that monosodium glutamate induces neuronal death and alters the expression of the subunits evaluated in the hippocampus at all ages studied, which could be prevented by SB203580 treatment.Furthermore, expression of the NRSF gene silencing factor also increased in response to excitotoxicity, suggesting a relationship in suppressing GluR2-expression, which was regulated by the p38-MAPK pathway inhibitor SB203580. This result suggests that selectively blocking the pro-death signaling pathway may reduce neuronal death in some neurodegenerative diseases in which these neurotoxic processes are present and produce major clinical benefits in the treatment of these pathologies.

Microarray analysis of rat hippocampus exposed to excitotoxicity: Reversal Na+/Ca2+ exchanger NCX3 is overexpressed in glial cells

Multiple factors are involved in the glutamate‐induced excitotoxicity phenomenon, such as overload of ionotropic and metabotropic receptors, excess Ca2+ influx, nitric oxide synthase activation, oxidative damage due to increase in free radicals, and release of endogenous polyamine, among others. In order to attempt a more integrated approach to address this issue, we established, by microarray analysis, the hippocampus gene expression profiles under glutamate‐induced excitotoxicity conditions. Increased gene expression is mainly related to excitotoxicity (CaMKII, glypican 2, GFAP, NCX3, IL‐2, and Gmeb2) or with cell damage response (dynactin and Ecel1). Several genes that augmented their expression are related to glutamatergic system modulation, in particular with NMDA receptor modulation and calcium homeostasis (IL‐2, CaMKII, acrosin, Gmeb2, hAChE, Slc83a, and SP1 factor). Conversely, among genes that diminished their expression, we found the Syngap 1, which is downregulated by CaMKII, and the MHC II, which is downregulated by glutamate. Changes observed in gene expression induced by monosodium glutamate (MSG) neonatal treatment in the hippocampus are consistent with the activation of the mechanisms that modulate NMDA receptor function as well as with the implementation of plastic response to cell damage and intracellular calcium homeostasis. Regarding this aspect, we report here that NCX3/Slc8a3, a Na+/Ca2+ membrane exchanger, is highly expressed in astrocytes, both in vitro and in vivo, in response to glutamate‐induced excitotoxicity. Hence, the results of this analysis present a broad view of the expression profile elicited by MSG neonatal treatment, and lead us to suggest the possible molecular pathways of action and reaction involved under this experimental model of excitotoxicity.

Excitotoxicity Triggered by Neonatal Monosodium Glutamate Treatment and Blood–Brain Barrier Function

It is likely that monosodium glutamate (MSG) is the excitotoxin that has been most commonly employed to characterize the process of excitotoxicity and to improve understanding of the ways that this process is related to several pathological conditions of the central nervous system. Excitotoxicity triggered by neonatal MSG treatment produces a significant pathophysiological impact on adulthood, which could be due to modifications in the blood-brain barrier (BBB) permeability and vice versa. This mini-review analyzes this topic through brief descriptions about excitotoxicity, BBB structure and function, role of the BBB in the regulation of Glu extracellular levels, conditions that promote breakdown of the BBB, and modifications induced by neonatal MSG treatment that could alter the behavior of the BBB. In conclusion, additional studies to better characterize the effects of neonatal MSG treatment on excitatory amino acids transporters, ionic exchangers, and efflux transporters, as well as the role of the signaling pathways mediated by erythropoietin and vascular endothelial growth factor in the cellular elements of the BBB, should be performed to identify the mechanisms underlying the increase in neurovascular permeability associated with excitotoxicity observed in several diseases and studied using neonatal MSG treatment.

Activity increase in EpoR and Epo expression by intranasal recombinant human erythropoietin (rhEpo) administration in ischemic hippocampi of adult rats.

Erythropoietin in the nervous system is a potential neuroprotective factor for cerebral ischemic damage due to specific-binding to the erythropoietin receptor, which is associated with survival mechanisms. However, the role of its receptor is unclear. Thus, this work assessed whether a low dose (500UI/Kg) of intranasal recombinant human erythropoietin administered 3h after ischemia induced changes in the activation of its receptor at the Tyr456-phosphorylated site in ischemic hippocampi in rats. The results showed that recombinant human erythropoietin after injury maintained cell survival and was associated with an increase in receptor phosphorylation at the Tyr456 site as an initial signaling step, which correlated with a neuroprotective effect.