Estradiol Protects Against Oxygen and Glucose Deprivation in Rat Hippocampal Organotypic Cultures and Activates Akt and Inactivates GSK-3? (original) (raw)
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The Journal of neuroscience : the official journal of the Society for Neuroscience, 2001
The major goal of this study was to compare mechanisms of the neuroprotective potential of 17 beta-estradiol in two models for oxidative stress-independent apoptotic neuronal cell death with that in necrotic neuronal cell death in primary neuronal cultures derived from rat hippocampus, septum, or cortex. Neuronal apoptosis was induced either by staurosporine or ethylcholine aziridinium (AF64A), as models for necrotic cell death glutamate exposure or oxygen-glucose deprivation (OGD) were applied. Long-term (20 hr) pretreatment (0.1 microm 17 beta-estradiol) was neuroprotective in apoptotic neuronal cell death induced by AF64A (40 microm) only in hippocampal and septal neuronal cultures and not in cortical cultures. The neuroprotective effect was blocked by the estrogen antagonists ICI 182,780 and tamoxifen and the phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002. In glutamate and OGD-induced neuronal damage, long-term pretreatment was not effective. In contrast, short-term (1...
Neurochemical Research, 2005
Here we investigated the effects of estradiol replacement in ovariectomized female rats using hippocampal slices exposed to oxygen-glucose deprivation (OGD). OGD induced lactate dehydrogenase (LDH) release to the incubation medium, what was assumed as a parameter of cellular death. In the estradiol-treated group the LDH release was markedly decreased by 23% as compared to the vehicle-treated group. In attempt to study a possible mechanism by which estradiol acts, we investigated some parameters of oxidative stress. In both vehicle-treated and estradiol-treated groups, OGD significantly increased the free radical production by 34% and 16%, respectively, although no significant differences on total antioxidant capacity were observed. Interestingly, estradiol replacement prevented the significant reduction in tryptophan and tyrosine contents caused by OGD observed in vehicle-treated animals. Our results show that estradiol replacement in ovariectomized female rats decreases cellular susceptibility to an ischemic-like injury and suggest a role for the hormone on protein damage prevention.
Journal of Neuroinflammation, 2012
Background: Estradiol has been shown to exert neuroprotective effects in several neurodegenerative conditions, including cerebral ischemia. The presence of this hormone prior to ischemia attenuates the damage associated with such events in a rodent model (middle cerebral artery occlusion (MCAO)), although its therapeutic value when administered post-ischemia has not been assessed. Hence, we evaluated the effects of estradiol treatment after permanent MCAO (pMCAO) was induced in rats, studying the PI3K/AKT/GSK3/β-catenin survival pathway and the activation of SAPK-JNK in two brain areas differently affected by pMCAO: the cortex and hippocampus. In addition, we analyzed the effect of estradiol on the glial response to injury. Methods: Male rats were subjected to pMCAO and estradiol (0.04 mg/kg) was administered 6, 24, and 48 h after surgery. The animals were sacrificed 6 h after the last treatment, and brain damage was evaluated by immunohistochemical quantification of 'reactive gliosis' using antibodies against GFAP and Iba1. In addition, Akt, phospho-Akt Ser473 , phospho-Akt Thr308 , GSK3, phospho-GSK3 Ser21/9 , β-catenin, SAPK-JNK, and pSAPK-JNK Thr183/Tyr185 levels were determined in western blots of the ipsilateral cerebral cortex and hippocampus, and regional differences in neuronal phospho-Akt expression were determined by immunohistochemistry. Results: The increases in the percentage of GFAP-(5.25-fold) and Iba1-(1.8-fold) labeled cells in the cortex and hippocampus indicate that pMCAO induced 'reactive gliosis'. This effect was prevented by post-ischemic estradiol treatment; diminished the number of these cells to those comparable with control animals. pMCAO downregulated the PI3K/AkT/GSK3/β-catenin survival pathway to different extents in the cortex and hippocampus, the activity of which was restored by estradiol treatment more efficiently in the cerebral cortex (the most affected region) than in the hippocampus. No changes in the phosphorylation of SAPK-JNK were observed 54 h after inducing pMCAO, whereas pMCAO did significantly decrease the phospho-Akt Ser473 in neurons, an effect that was reversed by estradiol.
Estradiol enhances Akt activation in cortical explant cultures following neuronal injury
Molecular Brain Research, 2002
We have previously demonstrated that estradiol reduces cell death in cortical explant cultures following injury induced by metabolic inhibition in a receptor-dependent fashion. In this study, we examined whether cell death involves apoptosis and assessed the potential mediators of estradiol's actions. Cortical explant cultures were generated from postnatal day 3 rat pups. On day 7 in vitro, explants were injured by exposure to 1 mM 2-DG / 2 mM KCN for 2 h to model the metabolic inhibition observed during ischemia. Explants were fixed in 4% paraformaldehyde at 2, 6, 10 and 24 h following the injury period and 18-mm thick sections were cut on a cryostat and stained with cresyl violet to assess cell death. The same sections were also labeled by TUNEL to determine whether cell death occurred by apoptosis. Other sections were used for immunohistochemistry to determine whether cells that stained positive for activated caspase 3 were also immunopositive for NeuN, a neuronal marker, or GFAP, an astrocyte marker. Protein was extracted for Western blot analysis from a separate set of explants collected at 0, 0.5, 1, 2 and 4 h following the conclusion of the injury. Estradiol treatment significantly reduced the number of cells undergoing apoptotic cell death as indicated by nuclear condensation visualized by cresyl violet staining (P,0.05). TUNEL staining revealed that the majority of pyknotic and fragmented nuclei were also TUNEL positive. Furthermore, caspase 3 activation appeared to be restricted to neurons. To examine a possible mechanism by which estradiol prevents apoptosis, we examined the level of activation of Akt kinase, which mediates antiapoptotic signals. Potential activation was measured by phosphorylation of Akt at Ser473 by Western blot analysis. In the absence of estradiol, pAkt levels were significantly increased at 2 h following the termination of injury. Explants that were pretreated with estradiol exhibited elevated levels of pAkt at 1 h following injury. Treatment with ICI 182,780 prevented the effect of estradiol. These studies suggest that estradiol prevents injury-induced apoptosis and that Akt activation may mediate these protective effects.
Neurochemical Research, 2006
The molecular basis of estrogen-mediated neuroprotection against brain ischemia remains unclear. In the present study, we investigated changes in expression of estrogen receptors (ERs) a and b and excitatory amino acid transporters (EAAT) 1 and 2 in rat organotypic hippocampal slice cultures treated with estradiol and subsequently exposed to oxygen-glucose deprivation (OGD). Pretreatment with 17b-estradiol (10 nM) for 7 days protected the CA1 area of hippocampus against OGD (60 min), reducing cellular injury by 46% compared to the vehicle control group. Levels of ERa protein were significantly reduced by 20% after OGD in both vehicle-and estradiol-treated cultures, whereas ERb was significantly up-regulated by 25% in the estradiol-treated cultures. In contrast, EAAT1 and EAAT2 levels were unchanged in response to estradiol treatment in this model of OGD. These findings suggest that estrogen-induced neuroprotection against ischemia might involve regulation of ERb and, consequently, of the genes influenced by this receptor.
Neurochemistry International, 2015
Disturbance in blood circulation is associated with numerous pathological conditions characterized by cognitive decline and neurodegeneration. Activation of pro-apoptotic signaling previously detected in the synaptosomal fraction may underlie neurodegeneration in the prefrontal cortex of rats submitted to permanent bilateral common carotid arteries occlusion (two-vessel occlusion, 2VO). 17β-Estradiol (E) exerts potent neuroprotective effects in the brain affecting, among other, ischemia-induced pathological changes. As most significant changes in rats submitted to 2VO were observed on 7th day following the insult, of interest was to examine whether 7 day treatment with low dose of E (33.3 μg/kg/day) prevents formerly reported neurodegeneration and may represent additional therapy during the early postischemic period. Role of E treatment on apoptotic pathway was monitored on Bcl-2 family members, cytochrome c, caspase 3 and PARP protein level in the synaptosomal (P2) fraction of the prefrontal cortex. Furthermore, changes of these proteins were examined in the cytosolic, mitochondrial and nuclear fraction, with the emphasis on potential involvement of extracellular signal-regulated kinases (ERK) and protein kinase B (Akt) activation and their role in nuclear translocation of transcriptional nuclear factor kappa B (NF-kB) associated with alteration of Bax and Bcl-2 gene expression. The extent of cellular damage was determined using DNA fragmentation and Fluoro-Jade B staining. The absence of activation of apoptotic cascade both in the P2 and cell accompanied with decreased DNA fragmentation and number of degenerating neurons clearly indicates that E treatment ensures the efficient protection against ischemic insult. Moreover, E-mediated modulation of pro-apoptotic signaling in the cortical cellular fractions involves cooperative activation of ERK and Akt, which may be implicated in the observed prevention of neurodegenerative changes.
Free Radical Biology and Medicine, 2012
17β-estradiol (E2) has been shown to protect against ischemic brain injury, yet its targets and the mechanisms are unclear. E2 may exert multiple regulatory actions on astrocytes that may greatly contribute to its ability to protect the brain. Mitochondria are recognized to play central roles in the development of injury during ischemia. Increasing evidence indicates that mitochondrial mechanisms are critically involved in E2-mediated protection. In this study, the effect of E2 and the role of mitochondria were evaluated in primary cultures of astrocytes subjected to an ischemialike condition of oxygen-glucose deprivation (OGD)/reperfusion. We showed that E2 treatment significantly protects against OGD/reperfusion-induced cell death as determined by cell viability, apoptosis and lactate dehydrogenase leakage. The protective effects of E2 on astrocytic survival were blocked by an estrogen receptor (ER) antagonist (ICI 182,780), and were mimicked by an estrogen receptor (ER) agonist selective for ERα (PPT), but not by an ER agonist selective for ERβ (DPN). OGD/reperfusion provoked mitochondria dysfunction as manifested by an increase of cellular reactive oxygen species production, loss of mitochondrial membrane potential and depletion of ATP. E2 pretreatment significantly inhibited OGD/reperfusion-induced mitochondrial dysfunction, and this effect was also blocked by ICI 182,780. Therefore, we concluded that E2 provides direct protection to astrocytes from ischemic injury by an ER-dependent mechanism, highlighting an important role for ERα. Estrogen protects against mitochondria dysfunction at the early phase of ischemic injury. However, overall implications for protection against brain ischemia and its complex sequelae await further exploration.
Neuroscience Letters, 2004
It is well-established that 17-estradiol (17-E 2 ) confers neuroprotection to male and female rats exposed to focal cerebral ischemia, while less is known about the effects of the hormone under conditions of transient global ischemia. Since translocation of cytochrome c from the mitochondria to the cytosol is a critical step in apoptotic cell death after cerebral ischemia, we have investigated whether 17-E 2 interferes with such mechanism to exert neuroprotection. Global ischemia, induced in male Wistar rats by 5-min 4 vessel occlusion (4VO), resulted in a significant increase of cytosolic cytochrome c (cyt-c) levels as detected by Western blotting at 6 h after reperfusion. 17-E 2 (0.2 mg/kg, i.p.) given 1 h before ischemia minimized cytochrome c translocation and the latter effect was partially reversed by tamoxifen (0.25 mg/kg, i.p.). Bilateral cell counting revealed that delayed hippocampal damage typically caused by 4VO was abolished by 17-E 2 and this was partially reversed by tamoxifen in the CA3 subregion, but not in CA1/CA2 or CA4. These findings provide the original observation that 17-E 2 reduces delayed hippocampal damage caused by 4VO in male rats and blocks cytochrome c translocation during the early stages of neuronal death, thus providing an important mechanism involved in estrogen-mediated neuroprotection.
Frontiers in Pharmacology, 2018
Perinatal asphyxia (PA) remains as one of the most important causes of shortterm mortality, psychiatric and neurological disorders in children, without an effective treatment. In previous studies we have observed that the expression of different neurodegenerative markers increases in CA1 hippocampal area of 4-months-old male rats born by cesarean section and exposed for 19 min to PA. We have also shown that a late treatment with 17β estradiol (daily dose of 250 µg/kg for 3 days) was able to revert the brain alterations observed in those animals. Based on these previous results, the main aim of the present study was to explore the mechanism by which the estrogenic treatment is involved in the reversion of the chronic neurodegenerative conditions induced by PA. We demonstrated that estradiol treatment of adult PA exposed animals induced an increase in estrogen receptor (ER) α and insulin-like growth factor receptor (IGF-1R) protein levels, an activation of the phosphatidylinositol 3-kinase/Akt/glycogen synthase kinase 3 beta/β-catenin signaling pathway and an increase in Bcl-2/Bax ratio in the hippocampus in comparison to PA exposed animals treated with vehicle. Taking together, our data suggest that the interaction between ERα and IGF-IR, with the subsequent downstream activation, underlies the beneficial effects of estradiol observed in late treatment of PA.
Hormones and Behavior, 2006
Exogenous administration of estrogen has been shown to significantly reduce ischemia-induced neuronal degeneration. However, the longterm impact of such treatment on neuronal protection and functional recovery remain largely unknown. The present study assessed the effects of a 15-day pretreatment with 17β-estradiol on memory deficits and neuronal damage up to 6 months following a 10-min global ischemia in rats. Four groups of ovariectomized female rats [sham-operated and ischemic rats receiving a 15-day pretreatment of either the vehicle or 17βestradiol (100 μg/kg)] were tested. The 8-arm radial maze and object recognition tests served to evaluate the impact of 17β-estradiol treatment on ischemia-induced spatial and recognition memory impairments, respectively. Testing in the radial maze was initiated at two distinct time intervals following reperfusion (7 and 120 days) to evaluate changes in memory functions over time. Our findings revealed long-lasting neuroprotective effects of 17β-estradiol treatment on hippocampal CA1 pyramidal cells in ovariectomized ischemic rats (43.5% greater neuronal survival than observed in vehicletreated ischemic animals). Importantly, this neuronal protection translated into significant improvements of recognition and spatial memory functions in estradiol-treated ischemic rats.