L. Pietranera

Neuroprotective effects of estradiol in hippocampal neurons and glia of middle age mice.

During aging the hippocampus experiences structural, molecular, and functional alterations. Protection from age-related disorders is provided by several factors, including estrogens. Since aging defects start at middle age, we studied if 17 beta-estradiol (E(2)) protected the hippocampus at this age period. Middle age (10-12 month old) male C57Bl/6 mice were implanted sc with E(2) (15 microg) or cholesterol pellets. Ten days afterwards they received bromodeoxyuridine (BrdU) 4 and 2h before killing to study cell proliferation in the dentate gyrus (DG). A pronounced depletion of BrdU+cells in the DG was found in cholesterol-treated middle age mice, accompanied by astrocytosis, and by neuronal loss in the hilus. Middle age mice receiving E(2) showed increased number of BrdU+cells while the other parameters were remarkably attenuated. When steroid treatment was prolonged for 2 months to study migration of cells in the granular layer of the DG, cell migration was unaffected by E(2). However, E(2)-treated middle age mice presented higher cell density and increased staining for doublecortin, a marker for differentiating neurons. Thus, from the three basic steps of adult neurogenesis (proliferation, migration, and differentiation), E(2) stimulated progenitor proliferation - even after long exposure to E(2) studied by Ki67 immunocytochemistry - and differentiation towards a neuronal lineage. This result, in conjunction with recovery from other aging indicators as increased deposits of the aging pigment lipofuscin in DG cells, loss of hilar neurons and astrocytosis supports a wide range protection of hippocampal function of middle age mice by estrogenic hormones.

Involvement of brain-derived neurotrophic factor and neurogenesis in oestradiol neuroprotection of the hippocampus of hypertensive rats.

The hippocampus of spontaneously hypertensive rats (SHR) and deoxycorticosterone (DOCA)‐salt hypertensive rats shows decreased cell proliferation and astrogliosis as well as a reduced number of hilar cells. These defects are corrected after administration of 17β‐oestradiol (E2) for 2 weeks. The present work investigated whether E2 treatment of SHR and of hypertensive DOCA‐salt male rats modulated the expression of brain‐derived neurotrophic factor (BDNF), a neurotrophin involved in hippocampal neurogenesis. The neurogenic response to E2 was simultaneously determined by counting the number of doublecortin‐immunopositive immature neurones in the subgranular zone of the dentate gyrus. Both hypertensive models showed decreased expression of BDNF mRNA in the granular zone of the dentate gyrus, without changes in CA1 or CA3 pyramidal cell layers, decreased BDNF protein levels in whole hippocampal tissue, low density of doublecortin (DCX)‐positive immature neurones in the subgranule zone and decreased length of DCX+ neurites in the dentate gyrus. After s.c. implantation of a single E2 pellet for 2 weeks, BDNF mRNA in the dentate gyrus, BDNF protein in whole hippocampus, DCX immunopositive cells and the length of DCX+ neurites were significantly raised in both SHR and DOCA‐salt‐treated rats. These results indicate that: (i) low BDNF expression and deficient neurogenesis distinguished the hippocampus of SHR and DOCA‐salt hypertensive rats and (ii) E2 was able to normalise these biologically important functions in the hippocampus of hypertensive animals.

Abnormalities of the Hippocampus are Similar in Deoxycorticosterone Acetate‐Salt Hypertensive Rats and Spontaneously Hypertensive Rats

Hippocampal neuropathology is a recognised feature of the brain in spontaneously hypertensive rats (SHR), but similar studies are lacking in another model of hypertension, the mineralocorticoid‐salt‐treated rat. The present study aimed to compare changes in hippocampal parameters in 16‐week‐old male SHR (blood pressure approximately 190 mmHg) and their normotensive Wistar‐Kyoto controls, with those of male Sprague‐Dawley rats receiving (i) 10 mg deoxycorticosterone acetate (DOCA) every other day during 3 weeks and drinking 1% NaCl solution (blood pressure approximately 160 mmHg) and normotensive controls treated with (ii) DOCA and drinking water, (iii) drinking water only or (iv) 1% NaCl only. In these experimental groups, we determined: (i) cell proliferation in the dentate gyrus (DG) using the 5‐bromo‐2′‐deoxyuridine‐labelling technique; (ii) the number of glial fibrillary acidic protein (GFAP) positive astrocytes under the CA1, CA3 and DG; (iii) the number of apolipoprotein E (ApoE) positive astrocytes as a marker of potential neuronal damage; and (iv) the number of neurones in the hilus of the DG, taken as representative of neuronal density in other hippocampal subfields. Changes were remarkably similar in both models, indicating a decreased cell proliferation in DG, an increased number of astrocytes immunopositive for GFAP and ApoE and a reduced number of hilar neurones. Although hypertension may be a leading factor for these abnormalities, endocrine mechanisms may be involved, because hypothalamic‐pituitary function, mineralocorticoid receptors and sensitivity to mineralocorticoid treatment are stimulated in SHR, whereas high exogenous mineralocorticoid levels circulate in DOCA‐treated rats. Thus, in addition to the deleterious effects of hypertension, endocrine factors may contribute to the abnormalities of hippocampus in SHR and DOCA‐treated rats.

Protective effects of estradiol in the brain of rats with genetic or mineralocorticoid-induced hypertension

Abnormalities of hippocampus and hypothalamus are commonly observed in rats with genetic (SHR) or mineralocorticoid/salt-induced hypertension. In the hippocampus, changes include decreased cell proliferation in the dentate gyrus (DG), astrogliosis and decreased neuronal density in the hilus, whereas in the hypothalamus expression of arginine vasopressin (AVP) is markedly elevated. Here, we report that estradiol treatment overturns these abnormalities. We used 16-week-old male SHR with blood pressure (BP) approximately 190 mmHg and their normotensive Wistar-Kyoto (WKY) controls, and male Sprague-Dawley rats made hypertensive by administration of 10mg deoxycorticosterone acetate (DOCA) every other day plus 1% NaCl as drinking fluid for 4 weeks (BP approximately 160 mmHg). Controls received oil vehicle plus 1% NaCl only. Half of the animals in each group were implanted s.c. with a single estradiol benzoate pellet weighing 14 mg for 2 weeks. Estradiol-treated SHR and DOCA-salt rats showed, in comparison to their respective steroid-free groups: (a) enhanced proliferation in the DG measured by bromodeoxyuridine incorporation; (b) decreased number of glial fibrillary acidic protein (GFAP) immunopositive astrocytes; (c) increased density of neurons in the hilus of the DG, and (d) decreased hypothalamic AVP mRNA expression. These results indicate that neuronal and glial alterations of hypertensive models are plastic events reversible by steroid treatment. The estradiol protective effects may be of pharmacological interest to attenuate the consequences of hypertensive encephalopathy.

Increased aromatase expression in the hippocampus of spontaneously hypertensive rats: Effects of estradiol administration

There is high incidence of hippocampal abnormalities in spontaneously hypertensive rats (SHR), including decreased neurogenesis in the dentate gyrus, astrogliosis, low expression of brain derived neurotrophic factor and decreased neuronal density in the hilar region, respect of normotensive Wistar Kyoto rats (WKY). Estradiol treatment given for 2 weeks normalized the faulty hippocampal parameters of SHR, without having effects on WKY rats. The present work studied the potential role of local estrogen biosynthesis in the hippocampus of SHR and WKY, by measuring the expression of aromatase, the key enzyme responsible for estrogen biosynthesis and involved in neuroprotection. We used 4 month old male SHR and WKY, half of which received a single sc pellet of 12 mg estradiol benzoate and the remaining half a cholesterol implant. Hippocampi were dissected and processed for aromatase mRNA expression using real time PCR. A second batch of animals was processed for aromatase and glial fibrillary acidic protein (GFAP) immunocytochemistry. Basal level of aromatase mRNA was higher in SHR respect of WKY. Following estradiol treatment, aromatase mRNA was further increased in the SHR group only. In the hilus of the dentate gyrus of cholesterol-implanted SHR, we found aromatase immunoreactive cell processes and fibers more strongly stained respect of WKY rats. Estradiol treatment of SHR further increased the length of immunoreactive processes and fibers in the hilar region and also increased aromatase immunoreactivity in the CA1 but not the CA3 pyramidal cell region. WKY rats were spared from the estradiol effect. Double-labelling experiments showed that aromatase+ processes and fibers of the hilus of SHR-treated rats did no colocalize with GFAP+ astrocyte cell bodies or processes. In conclusion, basal and estradiol-stimulated aromatase expression was enhanced in hypertensive rat hippocampus. A combination of exogenous estrogens and those locally synthesized may better alleviate hypertensive encephalopathy.

Increased Expression of the Mineralocorticoid Receptor in the Brain of Spontaneously Hypertensive Rats

The mineralocorticoid receptor (MR) has been considered as both neuroprotective and damaging to the function of the central nervous system. MR may be also involved in central regulation of blood pressure. In the present study, we compared the expression of MR and the glucocorticoid receptor (GR) in the hippocampus and hypothalamus of 16‐week‐old spontaneously hypertensive rats (SHR) and normotensive control Wistar Kyoto (WKY) rats. In the hippocampus, MR expression was studied by in situ hybridization (ISH), quantitative polymerase chain reaction (PCR) and immunohistochemistry, whereas GR expression was analysed using the latter two procedures. Hypertensive animals showed an increased expression of MR mRNA in the whole hippocampus according to qPCR data and also in CA3 by ISH. Immunocytochemical staining for MR of the dorsal hippocampus, however, did not reveal differences between SHR and WKY rats. SHR showed elevated hypothalamic MR mRNA by qPCR, as well as an increased number of MR immunopositive cells in the magnocellular paraventricular region, compared to WKY rats. By contrast, expression levels of GR mRNA or protein in the hippocampus and hypothalamus of SHR were similar to those of WKY rats. Furthermore, we investigated the role of MR in the hypertensive rats by i.c.v. injection of the MR antagonist RU‐2831. This compound produced a significant drop in blood pressure for SHR. In conclusion, MR expression is increased in the hippocampus and hypothalamus of SHR. We suggest that pathological MR overdrive may take responsibility for up‐regulation of blood pressure and the encephalopathy of hypertension.

Estradiol increases dendritic length and spine density in CA1 neurons of the hippocampus of spontaneously hypertensive rats: a Golgi impregnation study.

Increased neuronal vulnerability has been described in the brain of spontaneously hypertensive rats (SHR), models of primary hypertension. Previous data indicate that estradiol treatment corrects several dysfunctions of the hippocampus and hypothalamus of SHR. Considering this evidence we analyzed the dendritic arborization and spine density of the CA1 subfield in SHR and Wistar-Kyoto (WKY) normotensive rats with and without estradiol treatment. Five month old male SHR and WKY rats received single estradiol or cholesterol pellets (sham treatment) for 2 weeks. A substantial rise of circulating estradiol (>25 fold) and testicular atrophy was present in all estradiol-receiving rats. In both SHR and WKY rats, estradiol decreased blood pressure by ~20 mm Hg; however, a moderate hypertension persisted in SHR (164 mm Hg). Using a modified Golgi impregnation technique, apical and basal dendrites of the CA1 subfield were subjected to Sholl analysis. Spine density was also statistically analyzed. Apical dendritic length was significantly lower in SHR compared to WKY rats (p<0.01), whereas estradiol treatment increased dendritic length in the SHR group only (SHR vs SHR+estradiol; p<0.01). Apical dendritic length plotted against the shell distances 20-100, 120-200 and 220-300 μm, revealed that changes were more pronounced in the range 120-200 μm between SHR vs. WKY rats (p<0.05) and SHR vs. SHR+estradiol (p<0.05). Instead, basal dendrites were not significantly modified by hypertension or steroid treatment. Spine density of apical dendrites was lower in SHR than WKY (p<0.05) and was up-regulated in the SHR+estradiol group compared to the SHR group (p<0.001). Similar changes were obtained for basal dendritic spines. These data suggest that changes of neuronal processes in SHR are plastic events restorable by estradiol treatment. In conjunction with previous results, the present data reveal new targets of estradiol neuroprotection in the brain of hypertensive rats.

Progestin Regulation of Galanin and Prolactin Gene Expression in Oestrogen-Induced Pituitary Tumours

Galanin is a peptide widely distributed in the hypothalamic‐pituitary axis. In the female rat pituitary, galanin is mainly present in lactotrophs, where it regulates their secretion and proliferation. Galanin expression is increased in oestrogen‐induced prolactinomas, and it has been proposed that oestrogen effects on lactotroph function and proliferation could be mediated by galanin. Previous studies from our laboratory demonstrated that the synthetic progestin levonorgestrel antagonizes pituitary tumorigenesis of rats given oestrogen, reducing the number of proliferating cells and increasing cell death by nonapoptotic mechanism(s). To elucidate the role of galanin in levonorgestrel effects on the tumours, we examined galanin and prolactin mRNA and peptide expression in prolactinomas of rats receiving the progestin. Levonorgestrel reduced the pituitary weight and serum prolactin concentrations in oestrogen‐treated rats. Galanin mRNA expression (determined by in situ hybridization), and the number of galanin expressing cells (determined by immunocytochemistry) were also reduced by the progestin in tumour‐bearing rats. However, neither prolactin mRNA content, nor the number of prolactin‐expressing cells, were modified by levonorgestrel treatment of oestrogen‐receiving rats. The present study suggests that levonorgestrel controls pituitary growth by diminishing galanin expression. In contrast, changes in serum prolactin concentration seem to be more related to the reduction in tumour size, since the reduction in galanin expression was not large enough to regulate prolactin mRNA expression or the percentage of lactotrophs.

Neuroprotection and sex steroid hormones: evidence of estradiol-mediated protection in hypertensive encephalopathy.

Besides their effects on reproduction, estrogens exert neuroprotective effects for brain diseases. Thus, estrogens ameliorate the negative aspects of aging and age-associated diseases in the nervous system, including hypertension. Within the brain, the hippocampus is sensitive to the effects of hypertension, as exemplified in a genetic model, the spontaneously hypertensive rat (SHR). In the dentate gyrus of the hippocampus, SHR present decreased neurogenesis, astrogliosis, low expression of brain derived neurotrophic factor (BDNF), decreased number of neurons in the hilus and increased basal levels of the estrogen-synthesizing enzyme aromatase, with respect to the Wistar Kyoto (WKY) normotensive strain. In the hypothalamus, SHR show increased expression of the hypertensinogenic peptide arginine vasopressin (AVP) and its V1b receptor. From the therapeutic point of view, it was highly rewarding that estradiol treatment decreased blood pressure and attenuated brain abnormalities of SHR, rendering hypertension a suitable model to test estrogen neuroprotection. When estradiol treatment was given for 2 weeks, SHR normalized their faulty brain parameters. This was shown by the enhancement of neurogenesis in the dentate gyrus, according to increased bromodeoxyuridine incorporation and doublecortin labeling, decreased reactive astrogliosis, increased BDNF mRNA and protein expression in the dentate gyrus, increased neuronal number in the hilus of the dentate gyrus and a further hyperexpression of aromatase. The presence of estradiol receptors in hippocampus and hypothalamus suggests the possibility of direct effects of estradiol on brain cells. Successful neuroprotection produced by estradiol in hypertensive rats should encourage the treatment with non-feminizing estrogens and estrogen receptor modulators for age-associated diseases.

17α-Oestradiol-Induced Neuroprotection in the Brain of Spontaneously Hypertensive Rats

17β‐oestradiol is a powerful neuroprotective factor for the brain abnormalities of spontaneously hypertensive rats (SHR). 17α‐Oestradiol, a nonfeminising isomer showing low affinity for oestrogen receptors, is also endowed with neuroprotective effects in vivo and in vitro. We therefore investigated whether treatment with 17α‐oestradiol prevented pathological changes of the hippocampus and hypothalamus of SHR. We used 20‐week‐old male SHR with a blood pressure of approximately 170 mmHg receiving s.c. a single 800 μg pellet of 17α‐oestradiol dissolved in cholesterol or vehicle only for 2 weeks Normotensive Wistar–Kyoto (WKY) rats were used as controls. 17α‐Oestradiol did not modify blood pressure, serum prolactin, 17β‐oestradiol levels or the weight of the testis and pituitary of SHR. In the brain, we analysed steroid effects on hippocampus Ki67+ proliferating cells, doublecortin (DCX) positive neuroblasts, glial fibrillary acidic protein (GFAP)+ astrocyte density, aromatase immunostaining and brain‐derived neurotrophic factor (BDNF) mRNA. In the hypothalamus, we determined arginine vasopressin (AVP) mRNA. Treatment of SHR with 17α‐oestradiol enhanced the number of Ki67+ in the subgranular zone and DCX+ cells in the inner granule cell layer of the dentate gyrus, increased BDNF mRNA in the CA1 region and gyrus dentatus, decreased GFAP+ astrogliosis in the CA1 subfield, and decreased hypothalamic AVP mRNA. Aromatase expression was unmodified. By contrast to SHR, normotensive WKY rats were unresponsive to 17α‐oestradiol. These data indicate a role for 17α‐oestradiol as a protective factor for the treatment of hypertensive encephalopathy. Furthermore, 17α‐oestradiol is weakly oestrogenic in the periphery and can be used in males.