Intranasal Erythropoietin Protects CA1 Hippocampal Cells, Modulated by Specific Time Pattern Molecular Changes After Ischemic Damage in Rats.

Erythropoietin, a multitarget molecule exhibited neuroprotective properties, especially against cerebral ischemia. However, little effort has been made to determinate both the administration pathway and doses that diminishes neuronal damage. In this study, we investigate the effect on CA1 region of different intranasal doses of rHuEPO (500, 1000 and 2500 IU/kg) applied in distinct post-damage times (1, 6, and 24 h) against ischemic cellular damage. Furthermore, most effective dose and time were used to evaluate gen and protein expression changes in 3 key molecules (EPO, EPOR, and ßcR). We established that CA1-region present histopathological damage in this ischemia model and that rHuEPO protects cells against damage, particularly at 1000 IU dose. Molecular data shows that EPO and EPOR gene expression are upregulated in a short term after damage treatment with rHuEPO (1 h); oppositely, BcR is upregulated in ischemic and Isc + EPO. Protein expression data displays no changes on EPO expression in evaluated times after treatment, but a tendency to increase 24 h after damage; in the opposite way, EPOR is upregulated significantly 6 h after treatment and this effect last until 24 h. So, our data suggest that a single intranasal dose of rHuEPO (1 h post-injury) provides histological neurorestoration in CA1 hippocampal region, even if we did not observe a dose-dependent dose effect, the medium dose evaluated (1000 UI/kg of b.w.) was more effective and sufficient for induces molecular changes that provides a platform for neuroprotection.

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.

HIF-1α expression in the hippocampus and peripheral macrophages after glutamate-induced excitotoxicity.

Hypoxia-inducible factor-1 alpha (HIF-1α) is a master transcription factor that regulates the response to hypoxia and ischemia and induces the expression of various genes, including vascular endothelial growth factor (VEGF) and erythropoietin (EPO). This study shows the systemic response of increased HIF-1α, EPO, and VEGF mRNA and protein. In addition, VEGF expression was increased in neurons and over-expressed in glial cells in a model of neuroexcitotoxicity in the hippocampus, in which rats were neonatally exposed to high glutamate concentrations. Simultaneous increases in HIF-1α, EPO and VEGF mRNA in peritoneal macrophages were also observed. Our study is consistent with the hypothesis that these genes exert a protective effect in response to neurotoxicity.

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.

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

Pro-inflammatory cytokines TNF-alpha, IL-1beta 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-alpha, IL-1beta and IL-6 expression levels, AP neuronal death and cellular type that produces TNF-alpha 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-alpha 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-alpha, IL-1beta 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-alpha and their inhibition may have an important neuroprotective role as part of anti-inflammatory therapeutic strategy.

Proinflammatory cytokines and apoptosis following glutamate-induced excitotoxicity mediated by p38 MAPK in the hippocampus of neonatal rats.

The proinflammatory cytokines TNF-alpha, IL-1beta, and IL-6 rise during neuronal damage and activate the apoptotic mitogen-activated protein kinase p38. We studied apoptosis, the levels of TNF-alpha, IL-1beta, and IL-6, and the cell type producing TNF-alpha in rats at 8, 10, and 14 days of age after neonatal exposure to glutamate, which induces neuronal damage. TNF-alpha production was significantly increased by glutamate, but inhibited by SB203580 (a p38 inhibitor). TNF-alpha, IL-1beta, and IL-6 mRNA levels increased, but SB203580 did not modify their expression. Thus, the p38 signaling pathway influences the expression of inflammatory genes and its inhibition may offer anti-inflammatory therapy.

NMDA and AMPA receptor expression and cortical neuronal death are associated with p38 in glutamate-induced excitotoxicity in vivo.

Early overstimulation of ionotropic glutamate receptors (iGluRs), such as the N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors, produces excitotoxicity in several brain regions. The molecular composition of those receptors and their regulation by intracellular signaling systems could be determinants in the development of progressive neurodegenerative mechanisms in the central nervous system (CNS). Studies of p38 mitogen-activated protein kinase (MAPK) activation, morphologic changes including cell number, and the expression of the NR1 and GluR2 subunits, by reverse transcriptase-PCR were evaluated at early postnatal ages (postnatal day [PD]8-14) in cerebral cortex of rats treated with monosodium glutamate (MSG; 4 mg/g body weight) administered subcutaneously on PD1, 3, 5, and 7. An important increase in p38 activity at PD8 and loss of cortical cell number were observed from PD8-14 in animals treated with MSG, together with significant morphologic changes characterized by cell shrinkage, nuclear hyperchromatism, and cytoplasmic vacuolation. These morphologic changes were prevented by SB203580, an inhibitor of p38 signaling, at PD8-14. No change in cerebral cortex thickness was observed among experimental or control rats. A significant increase in NR1 subunit expression was observed in response to MSG from PD8-14. GluR2 expression increased from PD8-12, but at PD14, its expression was reduced to 54% with respect to controls. SB203580 prevented alone the decreased in GluR2 expression induced by MSG. These results suggest that initial neuronal death (at PD8 and 10) in cerebral cortex may be due to an excessive Ca2+ influx through NMDA receptors, whereas the further damage process could be mediated by AMPA receptors through p38 signaling. This could represent a determinant mechanism to decide whether nerve cells survive or die.

Neuronal death and tumor necrosis factor-alpha 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.