Sex steroid hormones as neuroprotective elements in ischemia models.

Among sex steroid hormones, progesterone and estradiol have a wide diversity of physiological activities that target the nervous system. Not only are they carried by the blood stream, but also they are locally synthesized in the brain and for this reason, estradiol and progesterone are considered 'neurosteroids'. The physiological actions of both hormones range from brain development and neurotransmission to aging, illustrating the importance of a deep understanding of their mechanisms of action. In this review, we summarize key roles that estradiol and progesterone play in the brain. As numerous reports have confirmed a substantial neuroprotective role for estradiol in models of neurodegenerative disease, we focus this review on traumatic brain injury and stroke models. We describe updated data from receptor and signaling events triggered by both hormones, with an emphasis on the mechanisms that have been reported as 'rapid' or 'cytoplasmic actions'. Data showing the therapeutic effects of the hormones, used alone or in combination, are also summarized, with a focus on rodent models of middle cerebral artery occlusion (MCAO). Finally, we draw attention to evidence that neuroprotection by both hormones might be due to a combination of 'cytoplasmic' and 'nuclear' signaling.

Angiotensin II type-2 receptor stimulation induces neuronal VEGF synthesis after cerebral ischemia.

Intense efforts are being undertaken to understand the pathobiology of ischemia and to develop novel and effective treatments. Angiotensin II type 2 receptor (AT2R) is related with a beneficial role in neurodegenerative disorders, including ischemia. However, the underlying molecular mechanism remains elusive. In this study, we have established that AT2R stimulation by C21 compound, a specific AT2R agonist, caused a VEGF upregulation. Using mouse primary cortical neurons exposed to oxygen-glucose deprivation (OGD), we established that this effect was mediated by a mechanism dependent of mTORC1 signaling since mTOR inhibition abolished the C21-induced VEGF upregulation. Also, we have temporally characterized the changes on VEGF levels after ischemia induction in rats using two different approaches: transient and permanent middle cerebral artery occlusion (tMCAO and pMCAO). VEGF levels were permanently augmented after reperfusion (tMCAO) whereas lower levels of VEGF were found after pMCAO, remarkably at 21days. Therefore, C21 compound accelerated the recovery of the neurological status of pMCAO rats, reduced the ischemic damage area and abolished pMCAO-induced VEGF downregulation at 21days. This effect of C21 compound was mainly observed in neurons of the peri-infarct area. Our results suggest that a C21-induced VEGF upregulation may be crucial after an ischemic neuronal insult in both of our experimental approaches. This upregulation was mediated by a mechanism dependent of Akt/mTOR signaling pathway, since mTOR inhibition abolished the VEGF upregulation induced by C21. Considering that VEGF is involved in regenerative processes, we propose that AT2R activation could be used as a potential pharmacological strategy after ischemic stroke.

Stroke and Neuroinflamation: Role of Sexual Hormones.

Inflammatory response in the nervous system, called neuroinflammation, is a common process of several neurodegenerative diseases and brain disorders. To understand the underlying mechanism of this brain response to damage would be interesting to identify new common therapy targets to neurodegenerative processes. Ischemic stroke has an important socioeconomic impact being the second cause of mortality and the first cause of long-term disability in the world. Until now, there is not any pharmacological treatment to reduce the brain damage induced. In this review, we will expose recent evidences about neuroinflammation after stroke in animal models and in human. We summarize the most relevant information about the inflammatory-cellular component: microglia/ astrocytes response and peripheral blood cells infiltration to the brain describing the key adhesion molecules implicated in this process. Also, we review the inflammatory-molecular response including the beneficial/detrimental role of chemokines and cytokines after ischemia. Currently, female sexual hormones (estradiol and progesterone) are considered as neuroprotective agents. We and others laboratories demonstrated anti-inflammatory actions of these hormones after stroke, modulating not only the cellular response (reducing the reactive gliosis), but also the immune response. Here, we will present the current data about the neuroprotective role of estradiol and progesterone after ischemic injury focused in their anti-inflammatory action. Additionally, we will review the recent information about the mechanism of action of both hormones, including different receptors and signaling pathways. Finally, we will discuss the synergistic or antagonic therapeutic effects when they are administered together.

Microsatellite markers reveal strong genetic structure in the endemic Chilean dolphin.

Understanding genetic differentiation and speciation processes in marine species with high dispersal capabilities is challenging. The Chilean dolphin, Cephalorhynchus eutropia, is the only endemic cetacean of Chile and is found in two different coastal habitats: a northern habitat with exposed coastlines, bays and estuaries from Valparaíso (33°02'S) to Chiloé (42°00'S), and a southern habitat with highly fragmented inshore coastline, channels and fjords between Chiloé and Navarino Island (55°14'S). With the aim of evaluating the potential existence of conservation units for this species, we analyzed the genetic diversity and population structure of the Chilean dolphin along its entire range. We genotyped 21 dinucleotide microsatellites for 53 skin samples collected between 1998 and 2012 (swab: n = 8, biopsy: n = 38, entanglement n = 7). Bayesian clustering and spatial model analyses identified two genetically distinct populations corresponding to the northern and southern habitats. Genetic diversity levels were similar in the two populations (He: 0.42 v/s 0.45 for southern and northern populations, respectively), while effective size population was higher in the southern area (Ne: 101 v/s 39). Genetic differentiation between these two populations was high and significant (FST = 0.15 and RST = 0.19), indicating little or no current gene flow. Because of the absence of evident geographical barriers between the northern and southern populations, we propose that genetic differentiation may reflect ecological adaptation to the different habitat conditions and resource uses. Therefore, the two genetic populations of this endemic and Near Threatened species should be considered as different conservation units with independent management strategies.

Estradiol and Progesterone Administration After pMCAO Stimulates the Neurological Recovery and Reduces the Detrimental Effect of Ischemia Mainly in Hippocampus.

Epidemiological studies have suggested a differential response, males versus female, in stroke incidence and prognosis. These divergences in brain response after damage are based mostly on hormonal differences. To date, estradiol and progesterone administered independently have demonstrated neuroprotection after ischemia in animal models. Nonetheless, contradictory results were revealed using a combined administration. In order to evaluate the effects of combinatorial treatment administered after ischemia induction, we used two different approaches: in vivo and in vitro models. Male rats which underwent permanent middle cerebral artery occlusion were treated with a combination of estradiol/progesterone at 6, 24 and 48 h after injury and sacrificed at 54 h post-ischemia. The rat brains were evaluated for reactive gliosis, NeuN-positive neurons, levels of synapse-associated proteins and activity levels of PI3K/Akt/GSK3/ß-catenin survival pathway. Also, primary cortical neurons were subjected to oxygen and glucose deprivation for 17 h and returned to a normal environment in the presence of estradiol or estradiol/progesterone. Cell viability was evaluated, and activity levels of the PI3K/Akt/GSK3/ß-catenin pathway. Our results indicate that some beneficial effects of estradiol were abolished in the presence of progesterone, particularly in the cerebral cortex (core). However, the combinatorial treatment showed positive effects in the hippocampus.

ATP-P2X7 Receptor Modulates Axon Initial Segment Composition and Function in Physiological Conditions and Brain Injury.

Axon properties, including action potential initiation and modulation, depend on both AIS integrity and the regulation of ion channel expression in the AIS. Alteration of the axon initial segment (AIS) has been implicated in neurodegenerative, psychiatric, and brain trauma diseases, thus identification of the physiological mechanisms that regulate the AIS is required to understand and circumvent AIS alterations in pathological conditions. Here, we show that the purinergic P2X7 receptor and its agonist, adenosine triphosphate (ATP), modulate both structural proteins and ion channel density at the AIS in cultured neurons and brain slices. In cultured hippocampal neurons, an increment of extracellular ATP concentration or P2X7-green fluorescent protein (GFP) expression reduced the density of ankyrin G and voltage-gated sodium channels at the AIS. This effect is mediated by P2X7-regulated calcium influx and calpain activation, and impaired by P2X7 inhibition with Brilliant Blue G (BBG), or P2X7 suppression. Electrophysiological studies in brain slices showed that P2X7-GFP transfection decreased both sodium current amplitude and intrinsic neuronal excitability, while P2X7 inhibition had the opposite effect. Finally, inhibition of P2X7 with BBG prevented AIS disruption after ischemia/reperfusion in rats. In conclusion, our study demonstrates an involvement of P2X7 receptors in the regulation of AIS mediated neuronal excitability in physiological and pathological conditions.

Post-ischemic estradiol treatment reduced glial response and triggers distinct cortical and hippocampal signaling in a rat model of cerebral ischemia.

BACKGROUND: Estradiol has been shown to exert neuroprotective effects in several neurodegenerative conditions, including cerebral ischemia. The presence of this hormone prior to ischemia attenuates the damage associated with such events in a rodent model (middle cerebral artery occlusion (MCAO)), although its therapeutic value when administered post-ischemia has not been assessed. Hence, we evaluated the effects of estradiol treatment after permanent MCAO (pMCAO) was induced in rats, studying the PI3K/AKT/GSK3/ß-catenin survival pathway and the activation of SAPK-JNK in two brain areas differently affected by pMCAO: the cortex and hippocampus. In addition, we analyzed the effect of estradiol on the glial response to injury. METHODS: Male rats were subjected to pMCAO and estradiol (0.04 mg/kg) was administered 6, 24, and 48 h after surgery. The animals were sacrificed 6 h after the last treatment, and brain damage was evaluated by immunohistochemical quantification of 'reactive gliosis' using antibodies against GFAP and Iba1. In addition, Akt, phospho-Akt(Ser473), phospho-Akt(Thr308), GSK3, phospho-GSK3(Ser21/9), ß-catenin, SAPK-JNK, and pSAPK-JNK(Thr183/Tyr185) levels were determined in western blots of the ipsilateral cerebral cortex and hippocampus, and regional differences in neuronal phospho-Akt expression were determined by immunohistochemistry. RESULTS: The increases in the percentage of GFAP- (5.25-fold) and Iba1- (1.8-fold) labeled cells in the cortex and hippocampus indicate that pMCAO induced 'reactive gliosis'. This effect was prevented by post-ischemic estradiol treatment; diminished the number of these cells to those comparable with control animals. pMCAO down-regulated the PI3K/AkT/GSK3/ß-catenin survival pathway to different extents in the cortex and hippocampus, the activity of which was restored by estradiol treatment more efficiently in the cerebral cortex (the most affected region) than in the hippocampus. No changes in the phosphorylation of SAPK-JNK were observed 54 h after inducing pMCAO, whereas pMCAO did significantly decrease the phospho-Akt(Ser473) in neurons, an effect that was reversed by estradiol. CONCLUSION: The present study demonstrates that post-pMCAO estradiol treatment attenuates ischemic injury in both neurons and glia, events in which the PI3K/AKT/GSK3/ß-catenin pathway is at least partly involved. These findings indicate that estradiol is a potentially useful treatment to enhance recovery after human ischemic stroke.

Signaling through the leukocyte integrin LFA-1 in T cells induces a transient activation of Rac-1 that is regulated by Vav and PI3K/Akt-1.

Integrin LFA-1 is a receptor that is able to transmit multiple intracellular signals in leukocytes. Herein we show that LFA-1 induces a potent and transient increase in the activity of the small GTPase Rac-1 in T cells. Maximal Rac-1 activity peaked 10-15 min after LFA-1 stimulation and rapidly declined to basal levels at longer times. We have identified Vav, a guanine nucleotide exchange factor for Rac-1, and PI3K/Akt, as regulators of the activation and inactivation phases of the activity of Rac-1, respectively, in the context of LFA-1 signaling based on the following experimental evidence: (i) LFA-1 induced activation of Vav and PI3K/Akt with kinetics consistent with a regulatory role for these molecules on Rac-1, (ii) overexpression of a constitutively active Vav mutant induces activation of Rac independently of LFA-1 stimulation whereas overexpression of a dominant-negative Vav mutant blocks LFA-1-mediated Rac activation, (iii) pharmacological inhibition of PI3K/Akt prevented the fall in the activity of Rac-1 after its initial activation but had no effect on Vav activity, and (iv) overexpression of a dominant-negative or a constitutively active Akt-1 induced or inhibited, respectively, Rac-1 activity. Finally, we show that T cells with a sustained Rac activity have impaired capacity to elongate onto ICAM-1. These results demonstrate that down-regulation of the activity of this GTPase is a requirement for the regulation of T cell morphology and motility and highlight the importance of temporal regulation of the signaling triggered from this integrin.