Estefania Acaz-Fonseca

Sex differences in the inflammatory response of primary astrocytes to lipopolysaccharide

BackgroundNumerous neurological and psychiatric disorders show sex differences in incidence, age of onset, symptomatology or outcome. Astrocytes, one of the glial cell types of the brain, show sex differences in number, differentiation and function. Since astrocytes are involved in the response of neural tissue to injury and inflammation, these cells may participate in the generation of sex differences in the response of the brain to pathological insults. To explore this hypothesis, we have examined whether male and female astrocytes show a different response to an inflammatory challenge and whether perinatal testosterone influences this response.MethodsCortical astrocyte cultures were prepared from postnatal day 1 (one day after birth) male or female CD1 mice pups. In addition, cortical astrocyte cultures were also prepared from female pups that were injected at birth with 100 μg of testosterone propionate or vehicle. Cultures were treated for 5 hours with medium containing lipopolysaccharide (LPS) or with control medium. The mRNA levels of IL6, interferon-inducible protein 10 (IP10), TNFα, IL1β, Toll-like receptor 4 (TLR4), steroidogenic acute regulatory protein and translocator protein were assessed by quantitative real-time polymerase chain reaction. Statistical significance was assessed by unpaired t-test or by one-way analysis of variance followed by the Tukey post hoc test.ResultsThe mRNA levels of IL6, TNFα and IL1β after LPS treatment were significantly higher in astrocytes derived from male or androgenized females compared to astrocytes derived from control or vehicle-injected females. In contrast, IP10 mRNA levels after LPS treatment were higher in astrocytes derived from control or vehicle-injected females than in those obtained from males or androgenized females. The different response of male and female astrocytes to LPS was due neither to differences in the basal expression of the inflammatory molecules nor to differences in the expression of the LPS receptor TLR4. In contrast, the different inflammatory response was associated with increased mRNA levels of translocator protein, a key steroidogenic regulator, in female astrocytes that were treated with LPS.ConclusionsMale and female cortical astrocytes respond differentially to an inflammatory challenge and this may be predetermined by perinatal testosterone exposure.

17β-Oestradiol Anti-Inflammatory Effects in Primary Astrocytes Require Oestrogen Receptor β-Mediated Neuroglobin Up-Regulation

Neuroglobin (Ngb), so named after its initial discovery in brain neurones, has received great attention as a result of its neuroprotective effects both in vitro and in vivo. Recently, we demonstrated that, in neurones, Ngb is a 17β‐oestradiol (E2) inducible protein that is pivotal for hormone‐induced anti‐apoptotic effects against H2O2 toxicity. The involvement of Ngb in other brain cell populations, as well as in other neuroprotective effects of E2, is completely unknown at present. We demonstrate Ngb immunoreactivity in reactive astrocytes located in the proximity of a penetrating cortical injury in vivo and the involvement of Ngb in the E2‐mediated anti‐inflammatory effect in primary cortical astrocytes. Upon binding to oestrogen receptor (ER)β, E2 enhances Ngb levels in a dose‐dependent manner. Although with a lesser degree than E2, the pro‐inflammatory stimulation with lipopolysaccharide (LPS) also induces the increase of Ngb protein levels via nuclear factor‐(NF)κB signal(s). Moreover, a negative cross‐talk between ER subtypes and NFκB signal(s) has been demonstrated. In particular, ERα‐activated signals prevent the NFκB‐mediated Ngb increase, whereas LPS impairs the ERβ‐induced up‐regulation of Ngb. Therefore, the co‐expression of both ERα and ERβ is pivotal for mediating E2‐induced Ngb expression in the presence of NFκB‐activated signals. Interestingly, Ngb silencing prevents the effect of E2 on the expression of inflammatory markers (i.e. interleukin 6 and interferon γ‐inducible protein 10). Ngb can be regarded as a key mediator of the different protective effects of E2 in the brain, including protection against oxidative stress and the control of inflammation, both of which are at the root of several neurodegenerative diseases.

Role of astrocytes in the neuroprotective actions of 17β-estradiol and selective estrogen receptor modulators.

Neuroprotective actions of 17β-estradiol (estradiol) are in part mediated by direct actions on neurons. Astrocytes, which play an essential role in the maintenance of the homeostasis of neural tissue, express estrogen receptors and are also involved in the neuroprotective actions of estradiol in the brain. Estradiol controls gliosis and regulates neuroinflammation, edema and glutamate transport acting on astrocytes. In addition, the hormone regulates the release of neurotrophic factors and other neuroprotective molecules by astrocytes. In addition, reactive astrocytes are a local source of neuroprotective estradiol for the injured brain. Since estradiol therapy is not free from peripheral risks, alternatives for the hormone have been explored. Some selective estrogen receptor modulators (SERMs), which are already in use in clinical practice for the treatment of breast cancer, osteoporosis or menopausal symptoms, exert similar actions to estradiol on astrocytes. Therefore, SERMs represent therapeutic alternatives to estradiol for the activation of astroglia-mediated neuroprotective mechanisms.

Gonadal hormones and the control of reactive gliosis

Astrocytes and microglia respond to central nervous system (CNS) injury with changes in morphology, proliferation, migration and expression of inflammatory regulators. This phenomenon is known as reactive gliosis. Activation of astrocytes and microglia after acute neural insults, such as stroke or traumatic CNS injury, is considered to be an adaptive response that contributes to minimize neuronal damage. However, reactive gliosis may amplify CNS damage under chronic neurodegenerative conditions. Progesterone, estradiol and testosterone have been shown to control reactive gliosis in different models of CNS injury, modifying the number of reactive astrocytes and reactive microglia and the expression of anti-inflammatory and proinflammatory mediators. The actions of gonadal hormones on reactive gliosis involve different mechanisms, including the modulation of the activity of steroid receptors, such as estrogen receptors α and β, the regulation of nuclear factor-κB mediated transcription of inflammatory molecules and the recruitment of the transcriptional corepressor c-terminal binding protein to proinflammatory promoters. In addition, the Parkinsons disease related gene parkin and the endocannabinoid system also participate in the regulation of reactive gliosis by estradiol. The control exerted by gonadal hormones on reactive gliosis may affect the response of neural tissue to trauma and neurodegeneration and may contribute to sex differences in the manifestation of neurodegenerative diseases. However, the precise functional consequences of the regulation of reactive gliosis by gonadal hormones under acute and chronic neurodegenerative conditions are still not fully clarified.

Regulation of astroglia by gonadal steroid hormones under physiological and pathological conditions.

In the last years there has been a considerable advance in the knowledge on the regulation of astrocytes by sex steroids under physiological and pathological conditions. By the activation of a variety of nuclear and membrane receptors, sex steroid hormones regulate the functions of astrocytes and their communication with other cell types in the central nervous system. Under physiological conditions astrocytes participate in the neuroendocrine and behavioral actions of gonadal steroids, as well as in the hormonal control of brain tissue homeostasis. Under pathological conditions astrocytes mediate, at least partially, the neuroprotective effects of gonadal steroid hormones; given that sex steroids modulate reactive astrogliosis and reduce the release of pro-inflammatory molecules by these cells. Given the side effects that sex steroids may have when administered systemically, a number of synthetic agonists of the receptors for gonadal steroid hormones in the nervous system have been developed, and may be considered for clinical use after brain injury as potential enhancers of the neuroprotective astrocytic functions.

Sex differences in glia reactivity after cortical brain injury

Several brain disorders associated with neuroinflammation show sex differences in their incidence, onset, progression and/or outcome. The different regulation of the neuroinflammatory response in males and females could underlie these sex differences. In this study, we have explored whether reactive gliosis after a penetrating cortical injury exhibits sex differences. Males presented a higher density of Iba1 immunoreactive cells in the proximity of the wound (0–220 μm) than females. This sex difference was due to a higher number of Iba1 immunoreactive cells with nonreactive morphology. In addition microglia/macrophages in that region expressed arginase‐1, marker of alternatively activated microglia, and the neuroprotective protein Neuroglobin, in a greater proportion in males than in females. No sex differences were found in the number of astrocytes around the lesion. However, the percentage of astrocytes expressing chemokine (C‐C motif) ligand 2 (CCL2), involved in recruitment of immune cells and gliosis regulation, was higher in males. Males also presented a significantly higher density of neurons in the lesion edge than females. These findings indicate that male and female mice have different neuroinflammatory responses after a cortical stab wound injury and suggest that sex differences in reactive gliosis may contribute to sex differences in neuroinflammatory diseases. GLIA 2015;63:1966–1981

Changes in cannabinoid receptors, aquaporin 4 and vimentin expression after traumatic brain injury in adolescent male mice. Association with edema and neurological deficit.

Traumatic brain injury (TBI) incidence rises during adolescence because during this critical neurodevelopmental period some risky behaviors increase. The purpose of this study was to assess the contribution of cannabinoid receptors (CB1 and CB2), blood brain barrier proteins (AQP4) and astrogliosis markers (vimentin) to neurological deficit and brain edema formation in a TBI weight drop model in adolescent male mice. These molecules were selected since they are known to change shortly after lesion. Here we extended their study in three different timepoints after TBI, including short (24h), early mid-term (72h) and late mid-term (two weeks). Our results showed that TBI induced an increase in brain edema up to 72 h after lesion that was directly associated with neurological deficit. Neurological deficit appeared 24 h after TBI and was completely recovered two weeks after trauma. CB1 receptor expression decreased after TBI and was negatively correlated with edema formation and behavioral impairments. CB2 receptor increased after injury and was associated with high neurological deficit whereas no correlation with edema was found. AQP4 increased after TBI and was positively correlated with edema and neurological impairments as occurred with vimentin expression in the same manner. The results suggest that CB1 and CB2 differ in the mechanisms to resolve TBI and also that some of their neuroprotective effects related to the control of reactive astrogliosis may be due to the regulation of AQP4 expression on the end-feet of astrocytes.

Correlation of brain levels of progesterone and dehydroepiandrosterone with neurological recovery after traumatic brain injury in female mice.

Traumatic brain injury (TBI) is an important cause of disability in humans. Neuroactive steroids, such as progesterone and dehydroepiandrosterone (DHEA), are neuroprotective in TBI models. However in order to design potential neuroprotective strategies based on neuroactive steroids it is important to determine whether its brain levels are altered by TBI. In this study we have used a weight-drop model of TBI in young adult female mice to determine the levels of neuroactive steroids in the brain and plasma at 24h, 72 h and 2 weeks after injury. We have also analyzed whether the levels of neuroactive steroids after TBI correlated with the neurological score of the animals. TBI caused neurological deficit detectable at 24 and 72 h, which recovered by 2 weeks after injury. Brain levels of progesterone, tetrahydroprogesterone (THP), isopregnanolone and 17β-estradiol were decreased 24h, 72 h and 2 weeks after TBI. DHEA and brain testosterone levels presented a transient decrease at 24h after lesion. Brain levels of progesterone and DHEA showed a positive correlation with neurological recovery. Plasma analyses showed that progesterone was decreased 72 h after lesion but, in contrast with brain progesterone, its levels did not correlate with neurological deficit. These findings indicate that TBI alters the levels of neuroactive steroids in the brain with independence of its plasma levels and suggest that the pharmacological increase in the brain of the levels of progesterone and DHEA may result in the improvement of neurological recovery after TBI.

CB2 cannabinoid receptor is involved in the anti-inflammatory effects of leptin in a model of traumatic brain injury

BACKGROUND AND PURPOSE The rates for traumatic brain injury (TBI) have risen in the last decade. Studies in animal models and clinical trials have not yet resulted in an effective treatment for TBI. Leptin, a 16kDa peptidic hormone is mainly known as a regulator of energy balance and has been shown to exert neuroprotective effects in different models of brain pathology. In this study, we have assessed whether leptin exerts protective actions in a TBI mouse model. In addition, the possible implication of CB2 cannabinoid receptor in leptin actions has been explored, since it is known that the endocannabinoid system interacts with leptin and actively participates in brain recovery after lesions. METHODS Swiss (CD1) male mice were subjected to weigh-drop model for TBI. Prior to the lesion, mice were injected with an antagonist of CB2 receptor (AM630) or the vehicle and immediately after TBI, they received leptin or vehicle treatment. Data were analyzed using a two-way ANOVA or the non-parametric test Kruskal-Wallis when appropriate. For correlation analyses, Spearmans rho test, followed by linear regression test, was used. RESULTS TBI induced a neurological deficit, which was improved by leptin treatment. Leptin recovered several parameters affected by TBI, including the expression of cannabinoid receptors, axonal injury marker and neuroinflammatory components. The effects of leptin were prevented or reduced when it was administered in combination with the CB2 receptor antagonist, AM630. CONCLUSIONS AND IMPLICATIONS Since some of the beneficial effects of leptin were not evident in the presence of AM630, our results suggest that CB2 receptor might be involved in the full expression of the neuroprotective effects of the hormone. These findings open new avenues for the study of leptin as a therapeutic treatment for TBI and enhance the importance of CB2 receptor in TBI pathophysiology and recovery.

Estradiol meets notch signaling in developing neurons.

The transmembrane receptor Notch, a master developmental regulator, controls gliogenesis, neurogenesis, and neurite development in the nervous system. Estradiol, acting as a hormonal signal or as a neurosteroid, also regulates these developmental processes. Here we review recent evidence indicating that estradiol and Notch signaling interact in developing hippocampal neurons by a mechanism involving the putative membrane receptor G protein-coupled receptor 30. This interaction is relevant for the control of neuronal differentiation, since the downregulation of Notch signaling by estradiol results in the upregulation of neurogenin 3, which in turn promotes dendritogenesis.