Luciana Pietranera

Progesterone neuroprotection in the Wobbler mouse, a genetic model of spinal cord motor neuron disease

Motor neuron degeneration characterizes the spinal cord of patients with amyotrophic lateral sclerosis and the Wobbler mouse mutant. Considering that progesterone (PROG) provides neuroprotection in experimental ischemia and injury, its potential role in neurodegeneration was studied in the murine model. Two-month-old symptomatic Wobbler mice were left untreated or received sc a 20-mg PROG implant for 15 days. Both light and electron microscopy of Wobbler mice spinal cord showed severely affected motor neurons with profuse cytoplasmic vacuolation of the endoplasmic reticulum and/or Golgi apparatus and ruptured mitochondria with damaged cristae, a profile indicative of a type II cytoplasmic form of cell death. In contrast to untreated mice, neuropathology was less severe in Wobbler mice receiving PROG; including a reduction of vacuolation and of the number of vacuolated cells and better conservation of the mitochondrial ultrastructure. In biochemical studies, we determined the mRNA for the alpha3 subunit of Na,K-ATPase, a neuronal enzyme controlling ion fluxes, neurotransmission, membrane potential, and nutrient uptake. In untreated Wobbler mice, mRNA levels in motor neurons were reduced by half compared to controls, whereas PROG treatment of Wobbler mice restored the expression of alpha3 subunit Na,K-ATPase mRNA. Therefore, PROG was able to rescue motor neurons from degeneration, based on recovery of histopathological abnormalities and of mRNA levels of the sodium pump. However, because the gene mutation in Wobbler mice is still unknown, further studies are needed to unveil the action of PROG and the mechanism of neuronal death in this genetic model of neurodegeneration.

Changes in Fos Expression in Various Brain Regions during Deoxycorticosterone Acetate Treatment: Relation to Salt Appetite, Vasopressin mRNA and the Mineralocorticoid Receptor

Salt appetite, a conditioning factor for hypertension and cardiovascular diseases, is produced when high doses of mineralocorticoids are given to experimental animals. A commonly used procedure to identify neuronal activation is to determine the number of Fos-immunoreactive cells. In rats with established salt appetite after 8 days of deoxycorticosterone acetate (DOCA) treatment, Fos-positive cells were studied in seven brain areas. Significant increases in Fos activity were recorded in the paraventricular (PVN) and supraoptic (SON) nuclei, median preoptic nucleus (MnPO), organum vasculosum of the lamina terminalis (OVLT), preoptic area (POA), bed nucleus of the stria terminalis (BNST) and amygdala (AMYG). In most of these areas, increased Fos expression was also observed early (2 h) after a single DOCA injection, well before salt appetite develops. Using a mineralocorticoid receptor (MR) antibody, we studied whether Fos-active regions also expressed MR. MR-positive cells were found in the OVLT, MnPO, AMYG and BNST, but not in the POA, PVN and SON. In the PVN and SON, nevertheless, prolonged or single DOCA treatment increased expression of mRNA for arginine vasopressin (AVP). The present demonstration of Fos activation, in conjunction with differential expression of MR and stimulation of AVP mRNA, suggests that a neuroanatomical pathway comprising the AMYG, osmosensitive brain regions and magnocellular nuclei becomes activated during DOCA effects on salt appetite. It is recognized, however, that DOCA effects may also depend on mechanisms and brain structures other than those considered in the present investigation. Since some Fos-positive regions were devoid of MR, a comprehensive view of DOCA-induced salt appetite should consider nongenomic pathways of steroid action, including the role of reduced DOC metabolites binding to GABAergic membrane receptors.

Mineralocorticoid Treatment Upregulates the Hypothalamic Vasopressinergic System of Spontaneously Hypertensive Rats

Mineralocorticoid effects in the brain include the control of cardiovascular functions, induction of salt appetite, interaction with the vasoactive neuropeptides arginine vasopressin (AVP) and angiotensin II and development or aggravation of hypertension. In this regard, mineralocorticoids may play a pathogenic role in rats with a genetic form of hypertension (spontaneously hypertensive rats, SHR). Our objective was to compare the response of the hypothalamic vasopressinergic system to mineralocorticoid administration in SHR and control Wistar-Kyoto (WKY) rats. Sixteen-week-old male SHR showing a systolic blood pressure of 190 ± 5 mm Hg and normotensive WKY rats (130 ± 5 mm Hg) were treated subcutaneously with oil vehicle or a single 10-mg dose of deoxycorticosterone acetate (DOCA). After 2 h, rats were sacrificed and brains prepared for immunocytochemistry of Fos and vasopressin V1a receptor (V1aR) and for non-isotopic in situ hybridization of AVP mRNA. In the basal state, SHR demonstrated a higher number of AVP mRNA- and V1aR-immunopositive cells in the magnocellular division of the paraventricular hypothalamic nucleus (PVN) than WKY rats. After DOCA injection, SHR responded with a significant increase in both parameters with respect to vehicle-injected SHR. In WKY rats, DOCA was without effect on AVP mRNA although it increased the number of V1aR-positive cells. Changes in the number of Fos-positive nuclei were measured in the PVN, median preoptic nucleus (MnPO) and organum vasculosum of the lamina terminalis (OVLT), a circumventricular region showing anatomical connections with the PVN. In vehicle-injected rats, the PVN of SHR showed a higher number of Fos-positive nuclei than in WKY rats, whereas after DOCA treatment, a significant increment occurred in the OVLT but not in the PVN or MnPO of the SHR group only. These data suggest that the enhanced response of the vasopressinergic system to mineralocorticoids may contribute to the abnormal blood pressure of SHR.

Steroid protection in aging and age-associated diseases

Neuroactive steroids are secretory products of peripheral endocrine glands that modulate a variety of brain functions. A close relationship between neuroactive steroid structure and function becomes most evident under pathological circumstances. On one side, overproduction of glucocorticoid and mineralocorticoid neuroactive steroids may be detrimental to the hippocampus, which is enriched in glucocorticoid receptors (GR) and mineralocorticoid receptors (MR). Thus, a dysfunction of the adrenocortical system in aging and age-associated diseases (diabetes, hypertension) is able to cause hippocampal damage. Whereas aging and uncontrolled diabetes show a predominant GR overdrive, a MR overdrive characterizes hypertensive animals. Some abnormalities commonly found in the hippocampus of aging, diabetic and hypertensive animals include decreased neurogenesis, astrogliosis and neuronal loss in the hilus of the dentate gyrus (DG). On the other side, and in contrast to adrenal gland-derived steroids, estrogens qualify as hippocampal neuroprotectants. Given to middle-age mice, estrogens stimulated proliferation and differentiation of newborn cells in the DG, decreased astrogliosis and increased hilar neuronal number. Similar estrogen effects were obtained in mice with streptozotocin-induced diabetes and in spontaneously hypertensive rats (SHR). The results suggest that in aging and age-associated diseases, adrenocortical steroid overdrive sensitizes the hippocampus to the pathological milieu imposed by a pre-existing degeneration or illness. In this setting, estradiol neuroprotection rescues hippocampal parameters previously altered by the pathological environment.

Estrogens and Neuroendocrine Hypothalamic-Pituitary-Adrenal Axis Function

The function of the HPA axis is subject to regulation by many factors, which achieve relevance under normal and pathological conditions. In the case of aging, this period of life is associated with disturbances of the HPA axis and signs of hippocampal vulnerability. We examined 20-month-old male rats, in which abnormalities of the HPA axis included altered response to stress, reduced effectiveness of the steroid negative feedback and low expression of hippocampal glucocorticoid receptors (GR). Estrogen treatment of aging rats normalized the response to stress, restored the dexamethasone inhibition of the stress response and increased GR density in defined hippocampal areas. Although estrogens could influence the hippocampus of aging animals directly, their effects could be also mediated by estrogen-sensitive forebrain cholinergic neurons projecting to the hippocampus. Additionally, estrogens normalized the deficient granule cell proliferation that aging mice present in the dentate gyrus, and attenuated several markers of hippocampal aging, such as astrocytosis, high lipofucsin content and neuronal loss in the hilus of the dentate gyrus. These effects may be important for the regulation of the HPA axis, in the context that hippocampal function as a whole was normalized by estrogen action. Therefore, estrogens are powerful neuroprotectants in cases of hippocampal dysfunction, and as part of this effect, they contribute to stabilize the function of the HPA axis.

Sexual dimorphism in diethylstilbestrol-induced prolactin pituitary tumors in F344 rats.

Female F344 rats treated chronically with diethylstilbestrol (DES) develop prolactin (PRL)-producing pituitary tumors. These tumors are larger in female than in male rats. To investigate gender differences in DES-induced pituitary tumor formation, we employed female and male rats and neonatally androgenized females, which received 100 µg of testosterone propionate (TP) after birth. At 3 months of age, all rats were deprived of their gonads and divided into control and DES-treated groups. Forty days after beginning treatment, control pituitary weight and serum PRL were similar in gonadectomized males (GDX), ovariectomized females (OVX) and androgenized-ovariectomized females (OVX + TP), but weight of DES-induced tumors was 2.5-fold higher and serum PRL 5.6-fold higher in OVX + DES than in GDX + DES or OXV + TP + DES (p < 0.001). At the pituitary level, nuclear estrogen receptors (NE2R) amounted to >100 fmol/mg DNA in all rats receiving DES. However, NE2R were lower in OVX + DES (101.3 ± 9.0 fmol/mg DNA) than in GDX + DES (174.6 ± 16.8; p < 0.05) and in OXV + DES + TP (150.3 ± 27.7; p < 0.05). A similar profile was found for cytosolic progestin receptors. Using electron microscopy (EM), hyperplasia/hypertrophy of lactotropes was found in all DES-stimulated pituitaries. However, tumors of OVX + DES rats were enriched in hyperstimulated typical lactotropes, i.e., cells with high rate of hormonal synthesis, processing and secretion. Instead, tumors from GDX + DES and OVX + TP + DES rats were a mixture of typical and atypical lactotropes, i.e. a cell subpopulation with refractory secretory response and a few gonadotropes. In agreement with these data, immunoreactive pituitary PRL was lower in OVX + DES than in OVX + TP + DES and GDX + DES groups. Thus, differences in the sensitivity to DES, serum and tumor PRL, NE2R and progestin receptors between estrogenized female rats on one side and male and TP-androgenized females on the other, may by due in part to heterogeneity of cell populations. Our data further suggest that neonatal hypothalamic exposure to androgens, as in normal males or androgenized females with masculinization of hypothalamic centers, may condition the response to DES stimulation later in life.

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.

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.