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Sex differences in the incidence, clinical manifestation, disease course, and prognosis of neurological diseases, such as autism spectrum disorders or Alzheimer's disease, have been reported. Obesity has been postulated as a risk factor for cognitive decline and Alzheimer's disease and, during pregnancy, increases the risk of autism spectrum disorders in the offspring. Obesity is associated with increased serum and brain levels of free fatty acids, such as palmitic acid, which activate microglial cells triggering a potent inflammatory cascade. In this study, we have determined the effect of palmitic acid in the inflammatory profile, motility, and phagocytosis of primary male and female microglia, both in basal conditions and in the presence of a pro-inflammatory stimulus (interferon-γ). Male microglia in vitro showed higher migration than female microglia under basal and stimulated conditions. In contrast, female microglia had higher basal and stimulated phagocytic activity than male microglia. Palmitic acid did not affect basal migration or phagocytosis, but abolished the migration and phagocytic activity of male and female microglia in response to interferon-γ. These findings extend previous observations of sex differences in microglia and suggest that palmitic acid impairs the protective responses of these cells.

Astrocyte-neuron cross-talk is an essential component of the mechanisms involved in the neuroendocrine and neuroprotective actions of estradiol. Astrocytes express estrogen receptors, show morphological and functional modifications in response to estradiol and participate in the hormonal regulation of synaptic plasticity and neuroendocrine events. In addition, estradiol interferes with the activation of astrocytes under pathological conditions, modulating the release of neurotrophic factors and inflammatory molecules by these cells. Furthermore, under neurodegenerative conditions, astrocytes synthesize estradiol, which acts as a local neuroprotectant. The actions of estradiol on astrocytes can be imitated by selective estrogen receptor modulators. Some of these molecules, which are free of the peripheral risks associated with estrogen therapy, exert estradiol-like anti-inflammatory actions on astrocytes and are potential therapeutic candidates for the control of reactive astrogliosis.

Astrocytes react to brain injury triggering neuroinflammatory processes that determine the degree of neuronal damage. However, the signaling events associated to astrocyte activation remain largely undefined. The nuclear factor of activated T-cells (NFAT) is a transcription factor family implicated in activation of immune cells. We previously characterized the expression of NFAT isoforms in cultured astrocytes, and NFAT activation in response to mechanical lesion. Here we analyze NFATc3 in two mouse models of inflammatory brain damage: hippocampal excitotoxicity induced by intracerebral kainic acid (KA) injection and cortical mechanical lesion. Immunofluorescence results demonstrated that NFATc3 is specifically induced in a subset of reactive astrocytes, and not in microglia or neurons. In KA-treated brains, NFATc3 expression is transient and NFATc3-positive astrocytes concentrate around damaged neurons in areas CA3 and CA1. Complementary Western blot and RT-PCR analysis revealed an NFAT-dependent induction of RCAN1-4 and COX-2 in hippocampus as soon as 6 h after KA exposure, indicating that NFAT activation precedes NFATc3 over-expression. Moreover, activation of NFAT by ATP increased NFATc3 mRNA levels in astrocyte cultures, suggesting that NFATc3 expression is controlled through an auto-regulatory loop. Meanwhile, stab wound enhanced NFATc3 expression specifically in a subclass of reactive astrocytes confined within the proximal layer of the glial scar, and GFAP immunoreactivity was attenuated in NFATc3-expressing astrocytes. In conclusion, our work establishes NFATc3 as a marker of activation for a specific population of astrocytes in response to brain damage, which may have consequences for neuronal survival.

Previous studies have shown that the neuroprotective hormone, testosterone, administered immediately after neural injury, reduces reactive astrogliosis. In this study we have assessed the effect of early and late therapy with testosterone or its metabolites, oestradiol and dihydrotestosterone, on reactive astroglia and reactive microglia after a stab wound brain injury in orchidectomized Wistar rats. Animals received daily s.c. injections of testosterone, oestradiol or dihydrotestosterone on days 0-2 or on days 5-7 after injury. The number of vimentin immunoreactive astrocytes and the volume fraction of major histocompatibility complex-II (MHC-II) immunoreactive microglia were estimated in the hippocampus in the lateral border of the wound. Both early and delayed administration of testosterone or oestradiol, but not dihydrotestosterone, resulted in a significant decrease in the number of vimentin-immunoreactive astrocytes. The volume fraction of MHC-II immunoreactive microglia was significantly decreased in the animals that received testosterone or oestradiol in both early and delayed treatments and in animals that received early dihydrotestosterone administration. Thus, both early and delayed administration of testosterone reduces reactive astroglia and reactive microglia and these effects may be at least in part mediated by oestradiol, while dihydrotestosterone may mediate part of the early effects of testosterone on reactive microglia. In conclusion, testosterone controls reactive gliosis and its metabolites, oestradiol and dihydrotestosterone, may be involved in this hormonal effect. The regulation of gliosis may be part of the neuroprotective mechanism of testosterone.

The ovarian hormone progesterone is neuroprotective in some animal models of neurodegeneration. Progesterone actions in the brain may partly be mediated by the locally produced metabolites 5α-dihydroprogesterone and 3α,5α-tetrahydroprogesterone. The neuroprotective effects of these two metabolites of progesterone were assessed in this study. Ovariectomized Wistar rats were injected with kainic acid, to induce excitotoxic neuronal death in the hippocampus, and with different doses of 5α-dihydroprogesterone and 3α,5α-tetrahydroprogesterone. The number of surviving neurones in the hilus of the dentate gyrus of the hippocampus was assessed with the optical dlisector method. The administration of kainic acid resulted in a significant decrease in the number of hilar neurones and in the induction of vimentin expression in reactive astrocytes, a sign of neural damage. Low doses of 5α-dihydroprogesterone (0.25 and 0.5 mg/kg body weight, b.w.) prevented the loss of hilar neurones and the appearance of vimentin immunoreactivity in astrocytes. Higher doses (1-2 mg/kg b.w.) were not neuroprotective. By contrast, low doses of 3α,5α-tetrahydroprogesterone (0.25-1 mg/kg b.w.) were unable to protect the hilus from kainic acid while higher doses (2-4 mg/kg b.w.) were protective. The different optimal neuroprotective doses of 5α-dihydroprogesterone and 3α, 5α-tetrahydroprogesterone suggest that these two steroids may protect neurones using different mechanisms. The neuroprotective effects of 3α, 5α-tetrahydroprogesterone may be exerted by the inhibition of neuronal activity via the GABAA receptor. This latter possibility is supported by the observation that 3β,5α-tetrahydroprogesterone, an isomer of 3α,5α-tetrahydroprogesterone that does not bind to GABAA receptor, was not neuroprotective. In summary, our findings suggest that progesterone neuroprotective effects may be, at least in part, mediated by its reduced metabolites 5α-dihydroprogesterone and 3α,5α-tetrahydroprogesterone.

The enzyme aromatase catalyzes the conversion of testosterone and other C19 steroids to estradiol. Under normal circumstances, the expression of aromatase in the central nervous system of mammals is restricted to neurons. However, the expression of the enzyme is induced in astrocytes in vitro by stressful stimuli. Furthermore, different types of brain injury induce in vivo the expression of aromatase in reactive astrocytes. The expression of aromatase by reactive astrocytes is neuroprotective, because the pharmacological inhibition of the enzyme in the brain exacerbates neuronal death after different forms of mild neurodegenerative stimuli that do not significantly affect neuronal survival under control conditions. These findings indicate that the induction of aromatase in reactive astrocytes, and the consecutive increase in the local production of estradiol in the brain at injured sites, may be an endogenous neural response to reduce the extent of neurodegenerative damage.

Pregnenolone and dehydroepiandrosterone (DHEA) are sex hormone precursors and neuroprotective steroids. Effects of pregnenolone and DHEA may be in part mediated by their conversion to testosterone and by the consecutive conversion of testosterone to estradiol by the enzyme aromatase. This enzyme is induced in reactive astrocytes after different forms of neurodegenerative lesions and the resultant local production of estradiol in the brain has been shown to be neuroprotective. The participation of aromatase in the neuroprotective effect of pregnenolone and DHEA has been assessed in this study. The protective effect of different doses (12.5, 25, 50, and 100 mg/kg) of pregnenolone or DHEA, against systemic kainic acid (7 mg/kg b.w.), was assessed on hippocampal hilar neurons in gonadectomized Wistar male rats. To determine whether the neuroprotective effect of pregnenolone and DHEA was dependent on their conversion to estradiol, the aromatase inhibitor fadrozole (4.16 mg/mL) was administered using subcutaneous osmotic minipumps. The number of Nissl-stained neurons in the hilus of the dentate gyrus of the hippocampal formation was estimated by the optical disector method. The administration of kainic acid resulted in a significant decrease in the number of hilar neurons compared to rats injected with vehicles. Pregnenolone and DHEA showed a dose-dependent protective effect of hilar neurons against kainic acid. The administration of the aromatase inhibitor fadrozole blocked the neuroprotective effect of pregnenolone and DHEA. These findings suggest that estradiol formation by aromatase mediates neuroprotective effects of pregnenolone and DHEA against excitotoxic-induced neuronal death in the hippocampus. © 2003 Wiley Periodicals, Inc.

Estrogen receptors are direct regulators of transcription that function by binding to specific DNA sequences in promoters of target genes. The two cloned forms of estrogen receptors, α and β, are expressed in the central nervous system by different neuronal populations. Astrocytes in vitro are also reported to express estrogen receptor α; however, this expression has not been confirmed in the rat brain in vivo. The apparent absence of estrogen receptors in glia in vivo contrasts with the well-known effects of this hormone on astrocytes of different brain areas, including the hippocampal formation. In this study, the expression of estrogen receptors in the hippocampal formation of adult male rats has been assessed by confocal microscopy. Estrogen receptor α-immunoreactivity was localized in neuronal nuclei in the pyramidal cell layer of CA1-CA3 fields. Estrogen receptor β-immunoreactivity was observed in the perikarya, apical dendrites, and cell nuclei of pyramidal neurons in CA1 and CA2. Furthermore, estrogen receptor β-immunoreactive glia were observed in CA1, CA2, CA3, and in the hilus of the dentate gyrus of male and female rats. Estrogen receptor β-immunoreactivity was localized in glial processes and perikarya and, in some cases, in glial cell nuclei. Double immunocytochemical labeling of estrogen receptor β and the specific astroglial marker glial fibrillary acidic protein revealed that estrogen receptor β-immunoreactive glial cells were astrocytes. Estrogen receptor α was not co-localized with glial fibrillary acidic protein. The presence of estrogen receptor β in astrocytes of adult male and female rats demonstrates a possible mechanism by which estrogen can directly modulate gene expression in these cells. GLIA 26:260–267, 1999. © 1999 Wiley-Liss, Inc.