Development of Model Systems to Assess the Role of Estrogens in Neuroprotection





Darlene C. Deecher and Istvan Merchenthaler

Women's Health Research Institute, Wyeth Research, Collegeville, PA



It is now known that estrogens modulate far more diverse functions than just the reproductive axis. Prior focus of estrogen action has been on reproduction, therefore, our understanding of estrogen action in the brain is limited. Within the last ten years, investigators have determined that estrogens can act as neurotrophins and that the brain is highly plastic and hormone- dependent. Understanding the normal brain function and the effects of estrogen depletion on the neural circuitry will be important in determining the level of influence estrogen plays in brain dysfunction. Hence, the importance of defining a strategy to identify neuroprotective compounds as well as elucidate estrogen regulated genes and mechanisms of action are imperative to understanding the role of estrogens in the brain. Steps taken to develop neuroprotective models are to identify cell lines that are estrogen responsive, characterize their estrogen receptor content and develop in vivo models that support estrogen mediated neuroprotection in the brain.
An immortalized mouse hippocampal neuronal cell line, HT22, was characterized for estrogen receptor content and evaluated for estrogen responsive activity. The estrogen receptor (ER) content was determined using radioligand binding, Western blot analysis, RT- PCR and immunocytochemistry. A glutamate toxicity bioassay was developed using this cell line to profile the activity of various estrogens. The ERalpha selective compounds were active in this cell line indicating that ERalpha is likely involved in the neuroprotection. The estrogen antagonist, ICI-182780, blocked the neuroprotection of estrogens under glutamate challenge. Verification of estrogen neuroprotection in the HT22 cells was done using rat primary cortical and hippocampal neurons. The neuroprotective effects of estrogens were also tested in two animal models. The effect on permanent focal ischemia was tested in rats and mice following the unilateral occlusion of the middle cerebral artery (MCAO). The effect of estrogens on global ischemia was tested in gerbils following the temporary occlusion of the common carotid arteries. MCAO-induced lesions in the ipsilateral cerebral cortex were prevented with estrogen pretreatment. In vivo autoradiography clearly depicted estrogen receptors (ER) in the cerebral cortex of lesioned animals but not in control littermates. Estrogen protected ERbeta knockout (KO) mice but not ERalphaKO mice, confirming that the ischemic lesion upregulates the expression of ERalpha and the neuroprotection by estrogen in the cerebral cortex is mediated via ERalpha. Estrogen pretreatment also protected the CA1 region of the hippocampus from ischemic injury in the gerbil model. The presence of ER binding sites detected by in vivo autoradiography in the CA1 region of the hippocampus provided clear evidence for a direct action of estrogens in this brain region. In summary, the ERalpha specific receptor appears to be involved in these models of neuroprotection.




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