Elsevier

Brain Research

Volume 764, Issues 1–2, 1 August 1997, Pages 133-140
Brain Research

Estrogens attenuate neuronal injury due to hemoglobin, chemical hypoxia, and excitatory amino acids in murine cortical cultures

https://doi.org/10.1016/S0006-8993(97)00437-XGet rights and content

Abstract

A growing body of evidence supports the hypothesis that estrogens may be beneficial in Alzheimer's disease and other neurodegenerative processes. Less is known of their therapeutic potential in acute CNS insults. In this study, we assessed the effect of estrogens in three injury paradigms that may be relevant to CNS hemorrhage, trauma, and ischemia. Supraphysiologic concentrations of 17β-estradiol, estrone, or equilin attenuated neuronal loss due to prolonged exposure to the pro-oxidant hemoglobin, with complete protection at 10 μM. Most of this effect persisted despite concomitant treatment with the antiestrogen ICI 182,780 or the protein synthesis inhibitor cycloheximide. In contrast, the non-estrogenic steroid methylprednisolone, which is currently in clinical use in spinal cord injury, reduced neuronal loss by only about 30%. High concentrations of equilin or estrone also attenuated the submaximal neuronal injury induced by 3.5–4.5 h exposure to the cytochrome oxidase inhibitor sodium azide, with near complete protection at 30 μM. Estrogens had a weaker and somewhat variable effect on pure excitotoxic injury, reducing neuronal loss due to 24 h kainate exposure by about half, and due to 24 h NMDA exposure by 15–65%; similar neuroprotection was provided by the antioxidant 21-aminosteroid U74500A. These results suggest that estrogens may be beneficial in acute CNS injuries associated with oxidative and excitotoxic stress. Investigation of high dose estrogen therapy in in vivo models of CNS hemorrhage, trauma, and ischemia is warranted.

Introduction

Epidemiologic evidence suggests that estrogen replacement therapy is associated with a decreased incidence of Alzheimer's disease in postmenopausal women 25, 29, 40. Although a number of factors may account for this observation, recent studies support the hypothesis that estrogens have a direct protective action on neurons. Estrogens attenuated neuronal death due to amyloid β-peptide, glutamate and other oxidants in a hippocampal cell line [1]and in primary hippocampal cultures [12]. Relatively high concentrations (10−5 M) were required for maximal benefit, but macromolecular synthesis was not; a mechanism that is at least partly independent of nuclear estrogen receptor binding therefore seems likely.

Estrogens have a number of non-genomic effects that may promote the survival of the stressed neuron. Their antioxidant properties have been intensely investigated in atherosclerosis research. In cell free systems, both natural and synthetic estrogens inhibit oxidation of lipids, lipoproteins, and nucleic acids with an efficacy that exceeds that of α-tocopherol 14, 19, 26, 43, 44. Estrogens also directly block vascular and neuronal l-type voltage-gated calcium channels 5, 23, 41. This action may account for their vasodilatory effect, and may attenuate cellular injury associated with calcium influx via these channels. High concentrations (10−5 M) of estrogens decrease the activity of neuronal nitric oxide synthase (nNOS) in brain homogenates via a direct interaction with calmodulin; however, at low concentrations (10−10 M), nNOS activity is enhanced [16]. Other potentially beneficial effects on neurons, which may involve nuclear estrogen receptors, include increased expression of neurotrophins and their high-affinity receptors [45], induction of calcium binding proteins [6], and stabilization of microtubules [10].

Traumatic and ischemic neuronal death is likely a consequence of a complex autodestructive cascade involving multiple injury mechanisms occurring both in series and in parallel 2, 9, 22, 39. Glutamate release, excessive activation of calcium-dependent lipases and proteases, free radical formation, oxidation of cellular constituents, cytoskeletal disruption, and induction of apoptosis are likely key components. Although monotherapy with an agent acting selectively on one mechanism has been protective in experimental models, demonstration of clinical efficacy has been problematic. A therapeutic `cocktail' of several agents acting at different sites may prove more successful, but may be limited by additive toxicities and adverse drug interactions. Furthermore, the rapid administration of several drugs to an unstable patient will likely be logistically difficult if not impossible in the prehospital or emergency department settings. A single agent with several beneficial actions, if clinically tolerated, may be preferable. Studies to date suggest that estrogens may be well suited to such a role. We therefore assessed the efficacy of selected estrogens in injury paradigms that may be relevant to CNS ischemia and trauma. An abstract has already appeared [32].

Section snippets

Cell cultures

Murine cortical cell cultures were prepared as previously described [31]. Under halothane anesthesia, gravid Swiss-Webster mice (15–16 days gestation) were euthanized by cervical dislocation. After removal of brains from embryos, cortices were isolated and incubated in 0.075% acetylated trypsin at 37°C for 1 h. Tissue was then collected by low-speed centrifugation and was resuspended in plating medium consisting of Eagle's minimal essential medium (MEM) supplemented with 5% horse serum, 5%

Estrogens attenuate hemoglobin neurotoxicity

In this culture system, prolonged Hb exposure produces an iron-dependent, oxidative injury to neurons without injuring glia [33]. Concomitant treatment with 17β-estradiol (E2) attenuated Hb neurotoxicity in a concentration-dependent fashion, with complete protection at 10 μM (Fig. 1A). This cytoprotective effect was quantitatively correlated with inhibition of Hb-mediated oxidation of cellular constituents, as determined by the thiobarbituric acid test. Consistent with prior observations [33],

Discussion

The results of this study are in general agreement with recent reports. Behl et al. observed that 17β-estradiol (E2) attenuated the toxicity of amyloid-β peptide, hydrogen peroxide, and glutamate in a hippocampal cell line [1]. In primary rat hippocampal cultures, Goodman et al. reported that E2 and estriol likewise attenuated the neurotoxicity of amyloid-β peptide and glutamate, and also reduced neuronal death induced by iron salts and glucose deprivation [12]. In addition to confirming the

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