Obesity induces extracellular vesicle release from the endothelium as a contributor to brain damage after cerebral ischemia in rats.

OBJECTIVES: Cerebral ischemia is the most common cause of disability, the second most common cause of dementia, and the fourth most common cause of death in the developed world [Sveinsson OA, Kjartansson O, Valdimarsson EM. Heilablóðþurrð/heiladrep: Faraldsfræði, orsakir og einkenni [Cerebral ischemia/infarction - epidemiology, causes and symptoms]. Laeknabladid. 2014 May;100(5):271-9. Icelandic. doi:10.17992/lbl.2014.05.543]. Obesity has been associated with worse outcomes after ischemia in rats, triggering proinflammatory cytokine production related to the brain microvasculature. The way obesity triggers these effects remains mostly unknown. Therefore, the aim of this study was to elucidate the cellular mechanisms of damage triggered by obesity in the context of cerebral ischemia. METHODS: We used a rat model of obesity induced by a 20% high fructose diet (HFD) and evaluated peripheral alterations in plasma (lipid and cytokine profiles). Then, we performed cerebral ischemia surgery using two-vessel occlusion (2VO) and analyzed neurological/motor performance and glial activation. Next, we treated endothelial cell line cultures with glutamate in vitro to simulate an excitotoxic environment, and we added 20% plasma from obese rats. Subsequently, we isolated EVs released from endothelial cells and treated primary cultures of astrocytes with them. RESULTS: Rats fed a HFD had an increased BMI with dyslipidemia and high levels of proinflammatory cytokines. Glia from the obese rats exhibited altered morphology, suggesting hyperreactivity related to neurological and motor deficits. Plasma from obese rats induced activation of endothelial cells, increasing proinflammatory signals and releasing more EVs. Similarly, these EVs caused an increase in NF-κB and astrocyte cytotoxicity. Together, the results suggest that obesity activates proinflammatory signals in endothelial cells, resulting in the release of EVs that simultaneously contribute to astrocyte activation.

High fructose diet-induced obesity worsens post-ischemic brain injury in the hippocampus of female rats.

Objectives: Cerebral ischemia is caused by a reduction of the blood flow in a specific area in the brain, triggering cellular cascades in the tissue that result in neuronal death. This phenomenon leads to neurological decline in patients with stroke. The extent of the injury after stroke could be related to the condition of obesity. Thus, we aim to analyze the effect of obesity induced by a high fructose diet (HFD) on the brain after cerebral ischemia in rats.Methods: We induced the obesity model in female Wistar rats with 20% fructose in water for 11 weeks. We then performed cerebral ischemia surgery (2-vessel occlusion), carried out the neurological test 6, 24 and 48 h post-ischemia and analyzed the histological markers.Results: The HFD induced an obese phenotype without insulin resistance. The obese rats exhibited worse neurological performance at 6 h post-ischemia and showed neuronal loss and astroglial and microglial immunoreactivity changes in the caudate putamen, motor cortex, amygdala and hippocampus at 48 h post-ischemia. However, the most commonly affected area was the hippocampus, where we found an increase in interleukin 1ß in the blood vessels of the dentate gyrus, a remarkable disruption of MAP-2+ dendrites, a loss of brain-derived neurotrophic factor and the presence of PHF-tau. In conclusion, a HFD induces an obese phenotype and worsens the neuronal loss, inflammation and plasticity impairment in the hippocampus after cerebral ischemia.

Liver X Receptor Agonist Modifies the DNA Methylation Profile of Synapse and Neurogenesis-Related Genes in the Triple Transgenic Mouse Model of Alzheimer's Disease.

The liver X receptor agonist, GW3965, improves cognition in Alzheimer's disease (AD) mouse models. Here, we determined if short-term GW3965 treatment induces changes in the DNA methylation state of the hippocampus, which are associated with cognitive improvement. Twenty-four-month-old triple-transgenic AD (3xTg-AD) mice were treated with GW3965 (50 mg/kg/day for 6 days). DNA methylation state was examined by modified bisulfite conversion and hybridization on Illumina Infinium Methylation BeadChip 450 k arrays. The Morris water maze was used for behavioral analysis. Our results show in addition to improvement in cognition methylation changes in 39 of 13,715 interrogated probes in treated 3xTg-AD mice compared with untreated 3xTg-AD mice. These changes in methylation probes include 29 gene loci. Importantly, changes in methylation status were mainly from synapse-related genes (SYP, SYN1, and DLG3) and neurogenesis-associated genes (HMGB3 and RBBP7). Thus, our results indicate that liver X receptors (LXR) agonist treatment induces rapid changes in DNA methylation, particularly in loci associated with genes involved in neurogenesis and synaptic function. Our results suggest a new potential mechanism to explain the beneficial effect of GW3965.

[A potential neuroprotective and synaptic plasticity mechanism induced by estradiol through PI3K/GSK3beta in cerebral ischaemia]. / Mecanismo potencial de neuroprotección y plasticidadsináptica inducidas por el estradiol a través dePI3K/GSK3beta en la isquemia cerebra.

AIM: To review the basic molecular mechanisms that are triggered by neuroactive steroids related to protection and plasticity, and their possible therapeutic application in cases of cerebral ischaemia. DEVELOPMENT: The term 'neuroprotection' embraces a series of strategies and effects that are aimed at preventing, impeding or delaying anomalies in the functioning of the central nervous system. The neuroactive steroids, and particularly estradiol, have been widely reported owing to their neuroprotective action because they give rise to a wide range of cell signals and generate effects in genes by means of canonical pathways or through non-conventional mechanisms that are involved in neuronal survival, dendritogenesis and synapse remodelling. Thus, neuroactive steroids become an important long-term protective therapeutic alternative due to the fact that such effects converge on neuronal plasticity. CONCLUSIONS: Further work needs to be carried out to study the mechanisms of action of neuroactive steroids, especially the non-conventional ones, which involve proteins such as GSK-3beta and beta-catenin. These proteins are involved in the functions of synaptic plasticity and survival, and play a crucial role in maintaining and recovering the functional integrity of the brain after the appearance of the lesions caused by cerebral ischaemia.

Mecanismo potencial de neuroprotección y plasticidad sináptica inducidas por el estradiol a través de PI3K/GSK3beta en la isquemia cerebral / A potential neuroprotective and synaptic plasticity mechanism induced by estradioll through PI3K/GSK3beta in cerebral ischaemia

Revisar los mecanismos moleculares básicos desencadenados por esteroides neuroactivos relacionadoscon la protección y la plasticidad, y su posible aplicación terapéutica en la isquemia cerebral. Desarrollo. El término ‘neuroprotección’abarca una serie de estrategias y efectos encaminados a prevenir, impedir o retrasar anomalías en el funcionamiento del sistema nervioso central. Los esteroides neuroactivos, en particular el estradiol, se han referenciado ampliamente por su acción neuroprotectora porque originan una gran diversidad de señales celulares y producen efectos génicos por mediode rutas canónicas o a través de mecanismos no convencionales, involucrados en la supervivencia neuronal, la dendritogénesis y la remodelación sináptica, lo que convierte a los esteroides neuroactivos en una importante alternativa terapéuticade protección a largo plazo por su convergencia con dichos efectos en plasticidad neuronal. Conclusión. Es necesario profundizar en el estudio de los mecanismos de acción de los esteroides neuroactivos, en especial sobre los no convencionales, que involucran proteínas tales como la GSK-3beta y la beta-catenina, en las cuales convergen funciones de supervivencia y plasticidadsináptica y que pueden desempeñar un papel crucial en el mantenimiento y la recuperación de la integridad funcional del cerebro frente a las lesiones propias de la isquemia cerebral

To review the basic molecular mechanisms that are triggered by neuroactive steroids related to protection and plasticity, and their possible therapeutic application in cases of cerebral ischaemia. Development. The term ‘neuroprotection’ embraces a series of strategies and effects that are aimed at preventing, impeding or delaying anomalies in the functioning ofthe central nervous system. The neuroactive steroids, and particularly estradiol, have been widely reported owing to their neuroprotective action because they give rise to a wide range of cell signals and generate effects in genes by means ofcanonical pathways or through non-conventional mechanisms that are involved in neuronal survival, dendritogenesis and synapse remodelling. Thus, neuroactive steroids become an important long-term protective therapeutic alternative due to thefact that such effects converge on neuronal plasticity. Conclusions. Further work needs to be carried out to study the mechanisms of action of neuroactive steroids, especially the non-conventional ones, which involve proteins such as GSK-3beta and beta-catenin. These proteins are involved in the functions of synaptic plasticity and survival, and play a crucial role inmaintaining and recovering the functional integrity of the brain after the appearance of the lesions caused by cerebral ischaemia

[Pathophysiology of focal cerebral ischemia: fundamental aspects and its projection on clinical practice]. / Fisiopatología de la isquemia cerebral focal: aspectos básicos y proyección a la clínica.

AIM: The aim of this study is to review the basic aspects of focal cerebral ischemia as a fundamental element in clinical practice and of neuroprotective strategies. DEVELOPMENT: Ischemia triggers several different responses in nerve tissue which, according to the degree of energetic limitation, can be adaptive or lead to cell death due to necrosis or apoptosis. Establishing these processes is a complex task and the mechanisms involved have still not been fully explained; this is made more difficult by the fact that many of them are simultaneous and also because of the implications they may have, not only in cell death but also in the adaptation of the neurons that suffered ischemic stress and survived. We outline the foundations for understanding the physiopathological phenomena at work in ischemia: neuronal stress and death, and the reaction of the macroglial and microglial cells. This is also illustrated by original images from research into cell response to ischemia at a pre-clinical level in an experimental model of focal cerebral ischemia in rats, evaluated using, for example, hematoxylin-eosin and immunohistochemical techniques for several cell markers. CONCLUSIONS: Cell death in ischemia is a complex phenomenon that can have two different outcomes: necrotic death or apoptotic death. Basic knowledge of the pathophysiology of ischemia and of the response of microglial and macroglial cells is the foundation for elaborating neuroprotective-type strategies, which must not only be oriented towards preventing acute cell death, but also later modes of cell death or strengthening the surviving tissue.

Interactions of estrogens and insulin-like growth factor-I in the brain: implications for neuroprotection.

Data from epidemiological studies suggest that the decline in estrogen following menopause could increase the risk of neurodegenerative diseases. Furthermore, experimental studies on different animal models have shown that estrogen is neuroprotective. The mechanisms involved in the neuroprotective effects of estrogen are still unclear. Anti-oxidant effects, activation of different membrane-associated intracellular signaling pathways, and activation of classical nuclear estrogen receptors (ERs) could contribute to neuroprotection. Interactions with neurotrophins and other growth factors may also be important for the neuroprotective effects of estradiol. In this review we focus on the interaction between insulin-like growth factor-I (IGF-I) and estrogen signaling in the brain and on the implications of this interaction for neuroprotection. During the development of the nervous system, IGF-I promotes the differentiation and survival of specific neuronal populations. In the adult brain, IGF-I is a neuromodulator, regulates synaptic plasticity, is involved in the response of neural tissue to injury and protects neurons against different neurodegenerative stimuli. As an endocrine signal, IGF-I represents a link between the growth and reproductive axes and the interaction between estradiol and IGF-I is of particular physiological relevance for the regulation of growth, sexual maturation and adult neuroendocrine function. There are several potential points of convergence between estradiol and IGF-I receptor (IGF-IR) signaling in the brain. Estrogen activates the mitogen-activated protein kinase (MAPK) pathway and has a synergistic effect with IGF-I on the activation of Akt, a kinase downstream of phosphoinositol-3 kinase. In addition, IGF-IR is necessary for the estradiol induced expression of the anti-apoptotic molecule Bcl-2 in hypothalamic neurons. The interaction of ERs and IGF-IR in the brain may depend on interactions between neural cells expressing ERs with neural cells expressing IGF-IR, or on direct interactions of the signaling pathways of alpha and beta ERs and IGF-IR in the same cell, since most neurons expressing IGF-IR also express at least one of the ER subtypes. In addition, studies on adult ovariectomized rats given intracerebroventricular (i.c.v.) infusions with antagonists for ERs or IGF-IR or with IGF-I have shown that there is a cross-regulation of the expression of ERs and IGF-IR in the brain. The interaction of estradiol and IGF-I and their receptors may be involved in different neural events. In the developing brain, ERs and IGF-IR are interdependent in the promotion of neuronal differentiation. In the adult, ERs and IGF-IR interact in the induction of synaptic plasticity. Furthermore, both in vitro and in vivo studies have shown that there is an interaction between ERs and IGF-IR in the promotion of neuronal survival and in the response of neural tissue to injury, suggesting that a parallel activation or co-activation of ERs and IGF-IR mediates neuroprotection.

Sex steroids and the brain: lessons from animal studies.

Gonadal steroid hormones have multiple effects throughout development on steroid responsive tissues in the brain. The belief that the cellular morphology of the adult brain cannot be modulated or that the synaptic connectivity is "hard-wired" is being rapidly refuted by abundant and growing evidence. Indeed, the brain is capable of undergoing many morphological changes throughout life and gonadal steroids play an important role in many of these processes. Gonadal steroids are implicated in the development of sexually dimorphic structures in the brain, in the control of physiological behaviors and functions and the brain's response to physiological or harmful substances. The effect of sex steroids on neuroprotection and neuroregeneration is an important and expanding area of investigation. Astroglia are targets for estrogen and testosterone and are apparently involved in the actions of sex steroids on the central nervous system. Sex hormones induce changes in the expression of glial fibrillary acidic protein, the growth of astrocytic processes and the extent to which neuronal membranes are covered by astroglial processes. These changes are linked to modifications in the number of synaptic inputs to neurons and suggest that astrocytes may participate in the genesis of gonadal steroid-induced sex differences in synaptic connectivity and synaptic plasticity in the adult brain. Astrocytes and tanycytes may also participate in the cellular effects of sex steroids by releasing neuroactive substances and by regulating the local accumulation of specific growth factors, such as insulin-like growth factor-I, that are involved in estrogen-induced synaptic plasticity and estrogen-mediated neuroendocrine control. Astroglia may also be involved in the regenerative and neuroprotective effects of sex steroids since astroglial activation after brain injury or after peripheral nerve axotomy is regulated by sex hormones.

Insulin-like growth factor I receptors and estrogen receptors colocalize in female rat brain.

Several findings indicate that there is a close interaction between estrogen and insulin-like growth factor I in different brain regions. In adult brain, both estrogen and insulin-like growth factor I have co-ordinated effects in the regulation of neuroendocrine events, synaptic plasticity and neural response to injury. In this study we have qualitatively assessed whether estrogen receptors and insulin-like growth factor I receptor are colocalized in the same cells in the preoptic area, hypothalamus, hippocampus, cerebral cortex and cerebellum of female rat brain using confocal microscopy. Immunoreactivity for estrogen receptors alpha and beta was colocalized with immunoreactivity for insulin-like growth factor I receptor in many neurons from the preoptic area, hypothalamus, hippocampus and cerebral cortex. Furthermore, estrogen receptor beta and insulin-like growth factor I receptor immunoreactivities were colocalized in the Purkinje cells of the cerebellum. Colocalization of estrogen receptor beta and insulin-like growth factor I receptor was also detected in cells with the morphology of astrocytes in all regions assessed. The co-expression of estrogen receptors and insulin-like growth factor I receptor in the same neurons may allow a cross-coupling of their signaling pathways. Furthermore, the colocalization of immunoreactivity for estrogen receptor beta and insulin-like growth factor I receptor in glial cells suggests that glia may also play a role in the interactions of insulin-like growth factor I and estrogen in the rat brain. In conclusion, the co-expression of estrogen receptors and insulin-like growth factor I receptors in the same neural cells suggests that the co-ordinated actions of estrogen and insulin-like growth factor I in the brain may be integrated at the cellular level.

Estradiol and progesterone regulate the expression of insulin-like growth factor-I receptor and insulin-like growth factor binding protein-2 in the hypothalamus of adult female rats.

Gonadal hormones interact with insulin-like growthfactor-I (IGF-I) to regulate synaptic plasticity during the estrous cycle in the rat mediobasal hypothalamus. It has been proposed that tanycytes, specialized glial cells lining the ventral region of the third ventricle, may regulate the availability of IGF-I to hypothalamic neurons. IGF-I levels in tanycytes fluctuate during the estrous cycle. Furthermore, estrogen administration to ovariectomized rats increases IGF-I levels in tanycytes, while progesterone, injected simultaneously with estrogen, blocks the estrogen-induced increase of IGF-I levels in tanycytes. To test whether hormonal regulation of IGF-I receptor (IGF-IR) and IGF binding protein-2 (IGFBP-2) may be involved in the accumulation of IGF-I in tanycytes, we assessed the effect of ovarian hormones on the levels of these molecules in the mediobasal hypothalamus of adult female rats. Ovariectomized animals were treated with either oil, estrogen, progesterone, or estrogen and progesterone simultaneously and then killed 6 or 24 h later. Some neurons, some astrocytes, and many tanycytes in the mediobasal hypothalamus were found by confocal microscopy to be immunoreactive for IGF-IR. IGFBP-2 immunoreactivity was restricted almost exclusively to tanycytes and ependymal cells and was colocalized with IGF-IR immunoreactivity in tanycytes. By electron microscope immunocytochemistry using colloidal gold labeling, IGF-IR and IGFBP-2 immunoreactivities were observed in the microvilli of tanycytes in the lumen of the third ventricle. IGF-IR and IGFBP-2 immunoreactive levels on the apical surface of tanycytes were significantly decreased by the administration of progesterone, either alone or in the presence of estradiol. IGF-IR levels in the mediobasal hypothalamus, measured by Western blotting, were not significantly affected by the separate administration of estradiol or progesterone to ovariectomized rats. However, the simultaneous administration of both hormones resulted in a marked decrease in IGF-IR protein levels. Estradiol administration to ovariectomized rats increased IGFBP-2 immunoreactive levels in the hypothalamus. While progesterone did not significantly affect IGFBP-2 expression, the simultaneous injection of estradiol and progesterone resulted in a marked decrease in IGFBP-2 protein levels. The effect of estradiol on IGFBP-2 was observed both in protein and mRNA levels, suggesting a transcriptional regulation. However, the simultaneous administration of progesterone and estradiol had different effects on IGF-IR protein and IGF-IR mRNA levels, as well as on IGFBP-2 protein and IGFBP-2 mRNA levels, suggesting a postranscriptional action. These findings indicate that estradiol and progesterone regulate the expression of IGF-IR and IGFBP-2 in the mediobasal hypothalamus of adult female rats. Regulation of the hypothalamic IGF-I system by ovarian hormones may be physiologically relevant for neuroendocrine regulation and for synaptic plasticity during the estrous cycle. These results do not support the hypothesis that estrogen-induced accumulation of IGF-I by tanycytes is mediated by the hormonal regulation of IGF-IR. However, estrogen-induced up-regulation of IGFBP-2 and progesterone-induced down-regulation of IGF-IR and IGFBP-2 levels in the apical plasma membrane of tanycytes may be involved in the fluctuation of IGF-I levels in the mediobasal hypothalamus during the estrous cycle.

Estrogen receptors and insulin-like growth factor-I receptors mediate estrogen-dependent synaptic plasticity.

Previous studies have shown that estradiol induces a transient disconnection of axo-somatic inhibitory synapses in the hypothalamic arcuate nucleus of adult ovariectomized rats. The synaptic disconnection is accompanied by an increase in the levels of insulin-like growth factor-I (IGF-I) in the arcuate nucleus, suggesting that IGF-I signaling may be involved in the estrogen-induced synaptic plasticity. The role of estrogen receptors and IGF-I receptors in the synaptic changes has been studied by assessing the number of axo-somatic synapses in ovariectomized rats treated with intracerebroventricular administration of the estrogen receptor antagonist ICI 182,780 and the IGF-I receptor antagonist JBI to ovariectomized rats. Estradiol administration resulted in a significant decrease in the number of axo-somatic synapses on arcuate neurons in control ovariectomized rats. Both the estrogen receptor antagonist and the IGF-I receptor antagonist blocked the estrogen-induced synaptic decrease. This finding suggest that estrogen-induced synaptic plasticity in the arcuate nucleus is dependent on the activation of both estrogen receptors and IGF-I receptors.

Insulin-like growth factor-I receptors and estrogen receptors interact in the promotion of neuronal survival and neuroprotection.

Several in vitro and in vivo studies have shown that estrogen has neuroprotective properties. The neuroprotective effects of estrogen are probably exerted through several mechanisms. It is established that estrogen can provide neuroprotection by actions that are independent of estrogen receptor activation. In addition, in several experimental models, activation of estrogen receptors appears to be indispensable for neuroprotection. This review focuses on neuroprotection mediated by estrogen receptors. The interaction of estrogen with growth factor receptor signaling to induce neuroprotection is discussed. Evidence is presented that estrogen receptors and insulin-like growth factor-1 receptors interact in the promotion of neuronal survival and neuroprotection.