Maria Elvira Brocca

Progesterone down-regulates spinal cord inflammatory mediators and increases myelination in experimental autoimmune encephalomyelitis

In mice with experimental autoimmune encephalomyelitis (EAE) pretreatment with progesterone improves clinical signs and decreases the loss of myelin basic protein (MBP) and proteolipid protein (PLP) measured by immunohistochemistry and in situ hybridization. Presently, we analyzed if progesterone effects in the spinal cord of EAE mice involved the decreased transcription of local inflammatory mediators and the increased transcription of myelin proteins and myelin transcription factors. C57Bl/6 female mice were divided into controls, EAE and EAE receiving progesterone (100mg implant) 7 days before EAE induction. Tissues were collected on day 17 post-immunization. Real time PCR technology demonstrated that progesterone blocked the EAE-induced increase of the proinflammatory mediators tumor necrosis factor alpha (TNFα) and its receptor TNFR1, the microglial marker CD11b and toll-like receptor 4 (TLR4) mRNAs, and increased mRNA expression of PLP and MBP, the myelin transcription factors NKx2.2 and Olig1 and enhanced CC1+oligodendrocyte density respect of untreated EAE mice. Immunocytochemistry demonstrated decreased Iba1+microglial cells. Confocal microscopy demonstrated that TNFα colocalized with glial-fibrillary acidic protein+astrocytes and OX-42+microglial cells. Therefore, progesterone treatment improved the clinical signs of EAE, decreased inflammatory glial reactivity and increased myelination. Data suggest that progesterone neuroprotection involves the modulation of transcriptional events in the spinal cord of EAE mice.

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.

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.

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.

Mineralocorticoid receptor associates with pro-inflammatory bias in the hippocampus of spontaneously hypertensive rats

Damage observed in the hippocampus of the adult spontaneously hypertensive rat (SHR) resembles the neuropathology of mineralocorticoid‐induced hypertension, supporting a similar endocrine dysfunction in both entities. In the present study, we tested the hypothesis that increased expression of the hippocampal mineralocorticoid receptor (MR) in SHR animals is associated with a prevalent expression of pro‐inflammatory over anti‐inflammatory factors. Accordingly, in the hippocampus, we measured mRNA expression and immunoreactivity of the MR and glucocorticoid receptor (GR) using a quantitative polymerase chain reaction and histochemistry. We also measured serum‐glucocorticoid‐activated kinase 1 (Sgk1 mRNA), the number and phenotype of Iba1+ microglia, as well as mRNA expression levels of the pro‐inflammatory factors cyclo‐oxygenase 2 (Cox2), Nlrp3 inflammasome and tumour necrosis factor α (Tnfα). Expression of anti‐inflammatory transforming growth factor (Tgf)β mRNA and the NADPH‐diaphorase activity of nitric oxide synthase (NOS) were also determined. The results showed that, in the hippocampus of SHR rats, expression of MR and the number of immunoreactive MR/GR co‐expressing cells were increased compared to Wistar‐Kyoto control animals. Expression of Sgk1, Cox2, Nlrp3 and the number of ramified glia cells positive for Iba1+ were also increased, whereas Tgfβ mRNA expression and the NADPH‐diaphorase activity of NOS were decreased. We propose that, in the SHR hippocampus, increased MR expression causes a bias towards a pro‐inflammatory phenotype characteristic for hypertensive encephalopathy.

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.

Selective oestrogen receptor agonists rescued hippocampus parameters in male spontaneously hypertensive rats

Spontaneously hypertensive rats (SHR) show pronounced hippocampus alterations, including low brain‐derived neurotrophic factor (BDNF) expression, reduced neurogenesis, astrogliosis and increased aromatase expression. These changes are reverted by treatment with 17β‐oestradiol. To determine which oestradiol receptor (ER) type is involved in these neuroprotective effects, we used agonists of the ERα [propylpyrazole triol (PPT)] and the ERβ [diarylpropionitrite (DPN)] given over 2 weeks to 4‐month‐old male SHR. Wistar Kyoto normotensive rats served as controls. Using immunocytochemistry, we determined glial fibrillary protein (GFAP)+ astrocytes in the CA1, CA3 and hilus of the dentate gyrus of the hippocampus, aromatase immunostaining in the hilus, and doublecortin (DCX)+ neuronal progenitors in the inner granular zone of the dentate gyrus. Brain‐derived neurotrophic factor mRNA was also measured in the hippocampus by the quantitative polymerase chain reaction. In SHR, PPT had no effect on blood pressure, decreased astrogliosis, slightly increased BDNF mRNA, had no effect on the number of DCX+ progenitors, and increased aromatase staining. Treatment with DPN decreased blood pressure, decreased astrogliosis, increased BDNF mRNA and DCX+ progenitors, and did not modify aromatase staining. We hypothesise that, although both receptor types may participate in the previously reported beneficial effects of 17β‐oestradiol in SHR, receptor activation with DPN may preferentially facilitate BDNF mRNA expression and neurogenesis. The results of the present study may help in the design of ER‐based neuroprotection for the encephalopathy of hypertension.

Protective effect of estrogens on the brain of rats with essential and endocrine hypertension.

Abstract Estrogen neuroprotection has been shown in pathological conditions damaging the hippocampus, such as trauma, aging, neurodegeneration, excitotoxicity, oxidative stress, hypoglycemia, amyloid-β peptide exposure and ischemia. Hypertensive encephalopathy also targets the hippocampus; therefore, hypertension seems an appropriate circumstance to evaluate steroid neuroprotection. Two experimental models of hypertension, spontaneously hypertensive rats (SHR) and deoxycorticosterone (DOCA)-salt hypertensive rats, develop hippocampal abnormalities, which include decreased neurogenesis in the dentate gyrus, astrogliosis, low expression of brain-derived neurotrophic factor (BDNF) and decreased number of neurons in the hilar region, with respect of their normotensive strains Wistar Kyoto (WKY) and Sprague-Dawley rats. After estradiol was given for 2 weeks to SHR and DOCA-treated rats, both hypertensive models normalized their faulty hippocampal parameters. Thus, estradiol treatment positively modulated neurogenesis in the dentate gyrus of the hippocampus, according to bromodeoxyuridine incorporation and doublecortin immunocytochemistry, decreased reactive astrogliosis, increased BDNF mRNA and protein expression in the dentate gyrus and increased neuronal number in the hilar region of the dentate gyrus. A role of local estrogen biosynthesis is suggested in SHR, because basal aromatase mRNA in the hippocampus and immunoreactive aromatase protein in cell processes of the dentate gyrus were highly expressed in these rats. Estradiol further stimulated aromatase-related parameters in SHR but not in WKY. These observations strongly support that a combination of exogenous estrogens to those locally synthesized might better alleviate hypertensive encephalopathy. These studies broaden estrogen neuroprotective functions to the hippocampus of hypertensive rat models.

Mineralocorticoid Receptors, Neuroinflammation and Hypertensive Encephalopathy

Worldwide, raised blood pressure is estimated to affect 35–40% of the adult population and is a main conditioning factor for cardiovascular diseases and stroke. Animal models of hypertension have provided great advances concerning the pathophysiology of human hypertension, as already shown for the deoxycorticosterone-salt treated rat, the Dahl-salt sensitive rat, the Zucker obese rat and the spontaneously hypertensive rat (SHR). SHR has been widely used to study abnormalities of the brain in chronic hypertension. This review summarises present and past evidence that in the SHR, hypertension causes hippocampal tissue damage which triggers a pro-inflammatory feedforward cascade affecting this vulnerable brain region. The cascade is driven by mineralocorticoid receptor (MR) activation responding to endogenous corticosterone rather than aldosterone. Increased MR expression is a generalised feature of the SHR which seems to support first the rise in blood pressure. Then oxidative stress caused by vasculopathy and hypoxia further increases MR activation in hippocampal neurons and glia cells, activates microglia activation and pro-inflammatory mediators, and down-regulates anti-inflammatory factors. In contrast to MR, involvement of the glucocorticoid receptor (GR) in SHR is less certain. GR showed normal expression levels and blockage with an antagonist failed to reduce blood pressure of SHR. The findings support the concept that MR:GR imbalance caused by vasculopathy causes a switch in MR function towards a proverbial “death” receptor.

Non-reproductive Functions of Aromatase in the Central Nervous System Under Physiological and Pathological Conditions

The modulation of brain function and behavior by steroid hormones was classically associated with their secretion by peripheral endocrine glands. The discovery that the brain expresses the enzyme aromatase, which produces estradiol from testosterone, expanded this traditional concept. One of the best-studied roles of brain estradiol synthesis is the control of reproductive behavior. In addition, there is increasing evidence that estradiol from neural origin is also involved in a variety of non-reproductive functions. These include the regulation of neurogenesis, neuronal development, synaptic transmission, and plasticity in brain regions not directly related with the control of reproduction. Central aromatase is also involved in the modulation of cognition, mood, and non-reproductive behaviors. Furthermore, under pathological conditions aromatase is upregulated in the central nervous system. This upregulation represents a neuroprotective and likely also a reparative response by increasing local estradiol levels in order to maintain the homeostasis of the neural tissue. In this paper, we review the non-reproductive functions of neural aromatase and neural-derived estradiol under physiological and pathological conditions. We also consider the existence of sex differences in the role of the enzyme in both contexts.