Importance of the brain corticosteroid receptor balance in metaplasticity, cognitive performance and neuro-inflammation.

Bruce McEwen's discovery of receptors for corticosterone in the rat hippocampus introduced higher brain circuits in the neuroendocrinology of stress. Subsequently, these receptors were identified as mineralocorticoid receptors (MRs) that are involved in appraisal processes, choice of coping style, encoding and retrieval. The MR-mediated actions on cognition are complemented by slower actions via glucocorticoid receptors (GRs) on contextualization, rationalization and memory storage of the experience. These sequential phases in cognitive performance depend on synaptic metaplasticity that is regulated by coordinate MR- and GR activation. The receptor activation includes recruitment of coregulators and transcription factors as determinants of context-dependent specificity in steroid action; they can be modulated by genetic variation and (early) experience. Interestingly, inflammatory responses to damage seem to be governed by a similarly balanced MR:GR-mediated action as the initiating, terminating and priming mechanisms involved in stress-adaptation. We conclude with five questions challenging the MR:GR balance hypothesis.

Neurosteroidogenesis and progesterone anti-inflammatory/neuroprotective effects.

Progesterone shows anti-inflammatory and promyelinating effects in mice with experimental autoimmune encephalomyelitis (EAE), a commonly used model for multiple sclerosis (MS). Because neurosteroids have been implicated as protective factors for MS and EAE, we analysed the expression of neurosteroidogenic enzymes in the compromised spinal cord of EAE mice. EAE was induced in female C57Bl6 mice, which were then killed on day 16 after induction. Progesterone was given by pellet implantation 1 week before EAE induction. Untreated EAE mice showed decreased mRNAs for the steroidogenic acute regulatory protein (Star), voltage-dependent anion channel (VDAC), cholesterol side-chain cleavage (P450scc), 5α-reductase, 3α-hydroxysteroid dehydrogenase (3α-HSOR) and aromatase, whereas changes of 3ß-hydroxysteroid dehydrogenase (3ß-HSD) were not significant. mRNA translocator protein (18 kDa) (TSPO) was elevated, concomitantly with a reactive microgliosis. EAE mice also showed abnormal mitochondrial ultrastructure in axons and neuronal bodies, as well as reduced expression of fission and fusion protein mRNAs. Progesterone pretreatment before EAE induction increased Star, VDAC, P450scc, 5α-reductase type I, 3α-HSOR and aromatase mRNAs and did not modify 3ß-HSD. TSPO mRNA was decreased, possibly as a result of reversal of microgliosis. Progesterone pretreatment also improved mitochondrial ultrastructure and increased fission/fusion protein mRNAs. These mitochondrial effects may be part of the progesterone recovery of neurosteroidogenesis. The enzymes 3ß-HSD, 3α-HSOR and 5α-reductase are also responsible for the formation of androgens. Because MS patients and EAE rodents show changes of central androgen levels, it is likely that, together with progestins and oestrogens, neuroandrogens afford neuroprotection for EAE and MS. The data reviewed suggest that enhanced synthesis of neurosteroids contributes in an auto/paracrine manner to reinforce the neuroprotective and anti-inflammatory effects of exogenous progesterone given to EAE mice.

Potential Biomarkers with Plasma Cortisol, Brain-derived Neurotrophic Factor and Nitrites in Patients with Acute Ischemic Stroke.

BACKGROUND: Acute Ischemic Stroke (AIS) represents an economic challenge for health systems all over the globe. Changes of neuroactive steroids have been found in different neurological diseases. We have previously demonstrated that old patients with AIS show changes of plasma cortisol and estradiol concentrations, in that increased steroid levels are associated with a deterioration of neurological status and a worse cognitive decline. OBJECTIVE: The present study assessed in patients with AIS if changes of behavior, Brain-Derived Neurotrophic Factor (BDNF) and Nitrites (NO-2) bear a relationship with the degree of hypercortisolism. METHODS: We recruited patients hospitalized within the first 24 hours of AIS. Subjects were divided into two groups, each one composed of 40 control subjects and 40 AIS patients, including men and women. The neurological condition was assessed using the National Institute of Health Stroke Scale (NIHSS) and the cognitive status with the Montreal Cognitive Assessment (MoCA). The emotional status was evaluated using the Montgomery-Asberg Depression Rating Scale (MADRS), whereas the Modified Rankin Scale (MRS) was used to determine the functional condition. BDNF and NO-2 plasma levels were measured by ELISA and the Griess reaction method, respectively. RESULTS: We found that in AIS patients, increased plasma cortisol was negatively correlated with plasma BDNF and NO-2 levels, neurological condition, cognition, functional responses and emotional status, suggesting a relationship between the declines of clinical, behavioral and blood parameters with stress-induced cortisol elevation. CONCLUSION: Nitrites and BDNF may represent potential biomarkers for cortisol negative effects on the area of cerebral ischemia and penumbra, potentiating ischemic cell damage.

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)ß 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 Tgfß 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.

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 17ß-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 ERß [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 17ß-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.

The progesterone receptor agonist Nestorone holds back proinflammatory mediators and neuropathology in the wobbler mouse model of motoneuron degeneration.

Wobbler mutant mice suffer from progressive motoneuron degeneration and glial cell reactivity in the spinal cord. To prevent development of these abnormalities, we employed Nestorone, a high-affinity progesterone receptor agonist endowed with neuroprotective, promyelinating and anti-inflammatory activities in experimental brain ischemia, preventing neuroinflammation and chemical degeneration. Five-month-old Wobbler mice (wr-/wr-) received s.c. injections of 200µg/day/mouse of Nestorone in vegetable oil or vehicle for 10days. Control NFR/NFR mice (background strain for Wobbler) received vehicle only. Vehicle-treated Wobblers showed typical spinal cord abnormalities, such as vacuolated motoneurons, decreased immunoreactive choline-acetyltransferase, decreased expression of glutamine synthase (GS), increased glial fibrillary acidic protein-positive (GFAP) astrogliosis and curved digits in forelimbs. These cell-specific abnormalities were normalized in Nestorone-treated Wobblers. In addition, vehicle-treated Wobblers showed Iba1+ microgliosis, high expression of the microglial marker CD11b mRNA and up-regulation of the proinflammatory markers TNFα and iNOS mRNAs. In Nestorone-treated Wobblers, Iba1+ microgliosis subsided, whereas CD11b, TNFα and iNOS mRNAs were down-regulated. NFκB mRNA was increased in Wobbler spinal cord and decreased by Nestorone, whereas expression of its inhibitor IκBα was increased in Nestorone-treated Wobblers compared to control mice and vehicle-treated Wobblers. In conclusion, our results showed that Nestorone restraining effects on proinflammatory mediators, microgliosis and astrogliosis may support neurons in their resistance against degenerative processes.

Progesterone and allopregnanolone in the central nervous system: response to injury and implication for neuroprotection.

Progesterone is a well-known steroid hormone, synthesized by ovaries and placenta in females, and by adrenal glands in both males and females. Several tissues are targets of progesterone and the nervous system is a major one. Progesterone is also locally synthesized by the nervous system and qualifies, therefore, as a neurosteroid. In addition, the nervous system has the capacity to bio-convert progesterone into its active metabolite allopregnanolone. The enzymes required for progesterone and allopregnanolone synthesis are widely distributed in brain and spinal cord. Increased local biosynthesis of pregnenolone, progesterone and 5α-dihydroprogesterone may be a part of an endogenous neuroprotective mechanism in response to nervous system injuries. Progesterone and allopregnanolone neuroprotective effects have been widely recognized. Multiple receptors or associated proteins may contribute to the progesterone effects: classical nuclear receptors (PR), membrane progesterone receptor component 1 (PGRMC1), membrane progesterone receptors (mPR), and γ-aminobutyric acid type A (GABAA) receptors after conversion to allopregnanolone. In this review, we will succinctly describe progesterone and allopregnanolone biosynthetic pathways and enzyme distribution in brain and spinal cord. Then, we will summarize our work on progesterone receptor distribution and cellular expression in brain and spinal cord; neurosteroid stimulation after nervous system injuries (spinal cord injury, traumatic brain injury, and stroke); and on progesterone and allopregnanolone neuroprotective effects in different experimental models including stroke and spinal cord injury. We will discuss in detail the neuroprotective effects of progesterone on the nervous system via PR, and of allopregnanolone via its modulation of GABAA receptors.

Effects of 17ß-estradiol on the cytoarchitecture of pyramidal CA1 neurons in normoglycemic and diabetic male spontaneously hypertensive rats.

Previous work has shown a reduction of apical dendritic length and spine density in neurons from the CA1 hippocampus subfield of spontaneously hypertensive rats (SHRs). These abnormalities are prevented by treatment for 2 weeks with 17ß-estradiol. In view of the fact that diabetes and hypertension are comorbid diseases, we have now studied the effect of Streptozotocin-induced diabetes on the dendritic tree and spines of CA1 hippocampus neurons, and also compared the regulation of these parameters by 17ß-estradiol in diabetic and normoglycemic SHR. Twenty-week-old male SHR received i.v. 40-mg/kg Streptozotocin or vehicle and studied 1 month afterward. A group of normoglycemic and hyperglycemic SHR also received s.c. a single 17ß-estradiol pellet or vehicle for 2weeks. Hippocampus sections were impregnated with silver nitrate following a modified Golgi's method and the arbor of CA1 pyramidal neurons analyzed by Sholl's method. 17ß-Estradiol treatment of normoglycemic SHR reversed the reduced length of apical dendrites, the low spine density and additionally decreased blood pressure (BP). Diabetic SHR showed increased length of apical and basal dendrites but reduced spine density compared to normoglycemic SHR. Diabetes also decreased BP of SHR. Treatment with 17ß-estradiol of diabetic SHR enhanced dendritic length, increased dendritic spine density and further decreased BP. Thus, changes of cytoarchitecture of CA1 neurons due to 17ß-estradiol treatment of normoglycemic SHR persisted after diabetes induction. A decrease of BP may also contribute to the central effects of 17ß-estradiol in SHR diabetic rats.

Expression and cellular localization of the classical progesterone receptor in healthy and amyotrophic lateral sclerosis affected spinal cord.

BACKGROUND AND PURPOSE: Previous studies have suggested that elevated progesterone levels are associated with a slower disease course in amyotrophic lateral sclerosis (ALS). Given that the effects of progesterone are mediated in part by the classical progesterone receptor (PR), the expression and cellular localization of the A and B isoforms (PR-A and PR-B, respectively) of the PR in control (neuropathologically normal) and ALS-affected spinal cord (SC) were examined. METHODS: Semi-quantitative RT-PCR, immunohistochemistry and immunofluorescence analyses of the cervical and lumbar SC of post-mortem ALS patients (n = 19) and control subjects (n = 10) were performed. Primers and antibodies used allowed the detection of both PR-A and PR-B isoforms together (PR-A+B) or PR-B isoform alone. RESULTS: Lumbar PR-A+B and cervical PR-B mRNA expression were significantly higher in ALS than controls. In both ALS and controls, PR-A+B immunoreactivity (IR) was occasionally detected in motor neurons. In contrast, PR-A+B IR was prominent in axonal processes and vessels. This was more evident in nerve roots and large arteries in ALS compared with controls. Colocalization of PR-A+B with markers of neurons, axonal processes and vascular endothelium was also observed. CONCLUSIONS: Evidence that both PR-A and PR-B isoforms are expressed in the human SC is provided, with some regional variation in isoform expression between ALS and controls. The IR was more prominent in nerve roots and large arteries in ALS, suggesting a potential role in the degenerative process.

Circulating gonadal and adrenal steroids in amyotrophic lateral sclerosis: possible markers of susceptibility and outcome.

Although changes of circulating steroids have been reported in patients with sporadic amyotrophic lateral sclerosis (ALS), a full comparison of the adrenal and gonadal steroid profile between control subjects and ALS patients is lacking. Considering that respiratory failure is the most frequent cause of death in ALS, we looked into whether a relationship emerged between circulating steroids and respiratory parameters. Serum levels of adrenal and gonadal steroids were measured in 52 age- and gender-matched subjects (28 ALS and 24 controls) using radioimmunoassay techniques. We also evaluated respiratory parameters in ALS patients, including forced vital capacity (FVC), maximal inspiratory pressure (MIP), and maximal expiratory pressure (MEP). We found increased levels of testosterone in female ALS patients compared to healthy female subjects. Furthermore, control subjects showed a significant decline of testosterone, dehydroepiandrosterone and its sulfate, and a borderline decline of progesterone with increasing age. Instead, testosterone did not decline with increasing age in ALS patients. We also found that the dehydroepiandrosterone sulfate/cortisol ratio was positively associated with FVC, MIP, and MEP. Moreover, ALS patients showing higher testosterone levels and lower progesterone/free testosterone ratio presented a more rapid worsening of the monthly FVC. In conclusion, first our study revealed a differential steroid profile with age and gender in ALS patients relative to controls. Second, we demonstrated an association between some steroids and their ratios with respiratory function and disease progression. Thus, we hypothesize that the endogenous steroid profile could be a marker of susceptibility and prognosis in ALS patients.

17α-Oestradiol-induced neuroprotection in the brain of spontaneously hypertensive rats.

17ß-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, 17ß-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.

Progesterone reduces the expression of spinal cyclooxygenase-2 and inducible nitric oxide synthase and prevents allodynia in a rat model of central neuropathic pain.

BACKGROUND: Spinal cord injury (SCI) results in the development of chronic pain that is refractory to conventional treatment. Progesterone, a neuroprotective steroid, may offer a promising perspective in pain modulation after central injury. Here, we explore the impact of progesterone administration on the post-injury inflammatory cascade involving the enzymes cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) at the spinal cord level. We also analyse pain behaviours, the profile of glial cell activation, and IκB-α mRNA levels, as an index of NF-κB transactivation. METHODS: We used biochemical, immunohistochemical and molecular techniques, as well as behavioural studies, to investigate the effects of progesterone in a well-characterized model of central neuropathic pain. RESULTS: Injured animals receiving progesterone presented reduced mRNA levels of the proinflammatory enzymes, as well as decreased COX-2 activity and nitrite levels, as compared to vehicle-treated injured rats. Further, animals receiving the steroid exhibited lower levels of IκB-α mRNA, suggesting decreased NF-κB transactivation. Progesterone administration also attenuated the injury-induced increase in the number of glial fibrillary acidic protein and OX-42 positive cells both at early and late time points after injury, and prevented the development of mechanical and thermal allodynia. Further, when injured rats received early progesterone administration for a critical period of time after injury, they did not display allodynic behaviours even after the treatment had stopped. CONCLUSIONS: Our results suggest that progesterone, by modulating early neuroinflammatory events triggered after SCI, may represent a useful strategy to prevent the development of central chronic pain.

Revisiting the roles of progesterone and allopregnanolone in the nervous system: resurgence of the progesterone receptors.

Progesterone is commonly considered as a female reproductive hormone and is well-known for its role in pregnancy. It is less well appreciated that progesterone and its metabolite allopregnanolone are also male hormones, as they are produced in both sexes by the adrenal glands. In addition, they are synthesized within the nervous system. Progesterone and allopregnanolone are associated with adaptation to stress, and increased production of progesterone within the brain may be part of the response of neural cells to injury. Progesterone receptors (PR) are widely distributed throughout the brain, but their study has been mainly limited to the hypothalamus and reproductive functions, and the extra-hypothalamic receptors have been neglected. This lack of information about brain functions of PR is unexpected, as the protective and trophic effects of progesterone are much investigated, and as the therapeutic potential of progesterone as a neuroprotective and promyelinating agent is currently being assessed in clinical trials. The little attention devoted to the brain functions of PR may relate to the widely accepted assumption that non-reproductive actions of progesterone may be mainly mediated by allopregnanolone, which does not bind to PR, but acts as a potent positive modulator of γ-aminobutyric acid type A (GABA(A) receptors. The aim of this review is to critically discuss effects of progesterone on the nervous system via PR, and of allopregnanolone via its modulation of GABA(A) receptors, with main focus on the brain.

Progesterone protective effects in neurodegeneration and neuroinflammation.

Progesterone is a neuroprotective, promyelinating and anti-inflammatory factor for the nervous system. Here, we review the effects of progesterone in models of motoneurone degeneration and neuroinflammation. In neurodegeneration of the Wobbler mouse, a subset of spinal cord motoneurones showed increased activity of nitric oxide synthase (NOS), increased intramitochondrial NOS, decreased activity of respiratory chain complexes, and decreased activity and protein expression of Mn-superoxide dismutase type 2 (MnSOD2). Clinically, Wobblers suffered several degrees of motor impairment. Progesterone treatment restored the expression of neuronal markers, decreased the activity of NOS and enhanced complex I respiratory activity and MnSOD2. Long-term treatment with progesterone increased muscle strength, biceps weight and survival. Collectively, these data suggest that progesterone prevented neurodegeneration. To study the effects of progesterone in neuroinflammation, we employed mice with experimental autoimmune encephalomyelitis (EAE). EAE mice spinal cord showed increased mRNA levels of the inflammatory mediators tumour necrosis factor (TNF)α and its receptor TNFR1, the microglial marker CD11b, inducible NOS and the toll-like receptor 4. Progesterone pretreatment of EAE mice blocked the proinflammatory mediators, decreased Iba1+ microglial cells and attenuated clinical signs of EAE. Therefore, reactive glial cells became targets of progesterone anti-inflammatory effects. These results represent a starting point for testing the usefulness of neuroactive steroids in neurological disorders.

Progesterone attenuates several hippocampal abnormalities of the Wobbler mouse.

It is now recognised that progesterone plays a protective role for diseases of the central nervous system. In the Wobbler mouse, a model of motoneurone degeneration, progesterone treatment prevents spinal cord neuropathology and clinical progression of the disease. However, neuropathological and functional abnormalities have also been discovered in the brain of Wobbler mice and patients with amyotrophic lateral sclerosis. The present study examined the hippocampus of control and afflicted Wobbler mice and the changes in response to progesterone treatment. Mice received either a single progesterone implant (20 mg for 18 days). We found that the hippocampal pathology of the untreated Wobblers involved a decreased expression of brain-derived neurotrophic factor (BDNF) mRNA, decreased astrogliosis in the stratum lucidum, stratum radiatum and stratum lacunosum-moleculare, decreased doublecortin (DCX)-positive neuroblasts in the subgranular zone of the dentate gyrus and a decreased density of GABA immunoreactive hippocampal interneurones and granule cells of the dentate gyrus. Although progesterone did not change the normal parameters of control mice, it attenuated several hippocampal abnormalities in Wobblers. Thus, progesterone increased hippocampal BDNF mRNA expression, decreased glial fibrillary acidic protein-positive astrocytes and increased the number of GABAergic interneurones and granule cells. The number of DCX expressing neuroblasts and immature neurones remained impaired in both progesterone-treated and untreated Wobblers. In conclusion, progesterone treatment exerted beneficial effects on some aspects of hippocampal neuropathology, suggesting its neuroprotective role in the brain, in agreement with previous data obtained in the spinal cord of Wobbler mice.

Distribution of membrane progesterone receptor alpha in the male mouse and rat brain and its regulation after traumatic brain injury.

Progesterone has been shown to exert pleiotropic actions in the brain of both male and females. In particular, after traumatic brain injury (TBI), progesterone has important neuroprotective effects. In addition to intracellular progesterone receptors, membrane receptors of the hormone such as membrane progesterone receptor (mPR) may also be involved in neuroprotection. Three mPR subtypes (mPRα, mPRß, and mPRγ) have been described and mPRα is best characterized pharmacologically. In the present study we investigated the distribution, cellular localization and the regulation of mPRα in male mouse and rat brain. We showed by reverse transcription-PCR that mPRα is expressed at similar levels in the male and female mouse brain suggesting that its expression may not be influenced by steroid levels. Treatment of males by estradiol or progesterone did not modify the level of expression of mPRα as shown by Western blot analysis. In situ hybridization and immunohistochemistry analysis showed a wide expression of mPRα in particular in the olfactory bulb, striatum, cortex, thalamus, hypothalamus, septum, hippocampus and cerebellum. Double immunofluorescence and confocal microscopy analysis showed that mPRα is expressed by neurons but not by oligodendrocytes and astrocytes. In the rat brain, the distribution of mPRα was similar to that observed in mouse brain; and after TBI, mPRα expression was induced in oligodendrocytes, astrocytes and reactive microglia. The wide neuroanatomical distribution of mPRα suggests that this receptor may play a role beyond neuroendocrine and reproductive functions. However, in the absence of injury its role might be restricted to neurons. The induction of mPRα after TBI in microglia, astrocytes and oligodendrocytes, points to a potential role in mediating the modulatory effects of progesterone in inflammation, ion and water homeostasis and myelin repair in the injured brain.

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.

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.

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.

Progesterone effects on neuronal brain-derived neurotrophic factor and glial cells during progression of Wobbler mouse neurodegeneration.

Previous results have shown a depletion of brain-derived neurotrophic factor (BDNF) mRNA in the degenerating motoneurons from clinically afflicted Wobbler mice, whereas progesterone treatment reverts this depletion. We now compared progesterone regulation of BDNF in motoneurons and oligodendrocytes of Wobbler mice at the progressive (EP, 1-3 months), symptomatic (SYM, 5-8 months old), and late stages (LS, 12-13 months). As controls we used NFR/NFR mice. Controls and Wobbler mice of different ages remained untreated or received a 20 mg progesterone pellet during 18 days. BDNF mRNA was determined in the ventral, intermediolateral, and dorsal gray matter by film autoradiography and in motoneurons using in situ hybridization. A depletion of BDNF mRNA already occurred at the EP stage of Wobblers, but progesterone was inactive at this period. In contrast, progesterone upregulated the low levels of BDNF mRNA in SYM Wobblers in the three gray matter regions analyzed. Progesterone also increased BDNF mRNA in LS Wobblers, according to grain counting procedures. BDNF protein analyzed by enzyme-linked immunosorbent assay (ELISA) in ventral horns or immunostaining of motoneurons was normal in steroid-naive SYM Wobblers. BDNF protein was decreased by progesterone, suggesting increased anterograde transport and/or release of neuronal BDNF. Wobbler mice also showed depletion of CC1-immunopositive oligodendrocytes, whereas progesterone treatment enhanced the density of BDNF+ and CC1+ oligodendrocytes in EP, SYM, and LS Wobblers. Our results suggest that BDNF could be involved in progesterone effects on motoneurons at the SYM and LS periods, whereas effects on oligodendrocytes occurred at all stages of the Wobbler disease. These steroid actions may be important to arrest the ongoing neurodegeneration.