Dysregulation of mTOR Signaling after Brain Ischemia.

In this review, we provide recent data on the role of mTOR kinase in the brain under physiological conditions and after damage, with a particular focus on cerebral ischemia. We cover the upstream and downstream pathways that regulate the activation state of mTOR complexes. Furthermore, we summarize recent advances in our understanding of mTORC1 and mTORC2 status in ischemia-hypoxia at tissue and cellular levels and analyze the existing evidence related to two types of neural cells, namely glia and neurons. Finally, we discuss the potential use of mTORC1 and mTORC2 as therapeutic targets after stroke.

Focal cerebral ischemia induces changes in oligodendrocytic tau isoforms in the damaged area.

Ischemic stroke is a major cause of death and the first leading cause of long-term disability worldwide. The only therapeutic strategy available to date is reperfusion and not all the patients are suitable for this treatment. Blood flow blockage or reduction leads to considerable brain damage, affecting both gray and white matter. The detrimental effects of ischemia have been studied extensively in the former but not in the latter. Previous reports indicate that preservation of white matter integrity reduces deleterious effect of ischemia on the brain. Oligodendrocytes are sensitive to ischemic damage, however, some reports demonstrate that oligodendrogenesis occurs after ischemia. These glial cells have a complex cytoskeletal network, including tau, that plays a key role to proper myelination. 4R-Tau/3R-Tau, which differ in the presence/absence of Exon 10, are found in oligodendrocytes; but the precise role of each isoform is not understood. Using permanent middle cerebral artery occlusion model and immunofluorescence, we demonstrate that cerebral ischemia induces an increase in 3R-Tau versus 4R-Tau in oligodendrocytes in the damaged area. In addition, cellular distribution of Tau undergoes a change after ischemia, with some oligodendrocytic processes showing positive staining for 3R-Tau. This occurs simultaneously with the amelioration of neurological damage in ischemic rats. We propose that ischemia triggers an endogenous mechanism involving 3R-Tau, that induces colonization of the ischemic damaged area by oligodendrocytes in an attempt to myelinate-injured axons. Understanding the molecular mechanism of this phenomenon could pave the way for the design of therapeutic strategies that exploit glial cells for the treatment of ischemia.

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.

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.

The dynamics of oligodendrocyte populations following permanent ischemia promotes long-term spontaneous remyelination of damaged area.

Stroke is a major public health concern, with limited clinically approved interventions available to enhance sensorimotor recovery beyond reperfusion. Remarkably, spontaneous recovery is observed in certain stroke patients, suggesting the existence of a brain self-repair mechanism not yet fully understood. In a rat model of permanent cerebral ischemia, we described an increase in oligodendrocytes expressing 3RTau in damaged area. Considering that restoration of myelin integrity ameliorates symptoms in many neurodegenerative diseases, here we hypothesize that this cellular response could trigger remyelination. Our results revealed after ischemia an early recruitment of OPCs to damaged area, followed by their differentiation into 3RTau+ pre-myelinating cells and subsequent into remyelinating oligodendrocytes. Using rat brain slices and mouse primary culture we confirmed the presence of 3RTau in pre-myelinating and a subset of mature oligodendrocytes. The myelin status analysis confirmed long-term remyelination in the damaged area. Postmortem samples from stroke subjects showed a reduction in oligodendrocytes, 3RTau+ cells, and myelin complexity in subcortical white matter. In conclusion, the dynamics of oligodendrocyte populations after ischemia reveals a spontaneous brain self-repair mechanism which restores the functionality of neuronal circuits long-term by remyelination of damaged area. This is evidenced by the improvement of sensorimotor functions in ischemic rats. A deep understanding of this mechanism could be valuable in the search for alternative oligodendrocyte-based, therapeutic interventions to reduce the effects of stroke.

Pharmacological inhibition of mTORC1 reduces neural death and damage volume after MCAO by modulating microglial reactivity.

Ischemic stroke is a sudden and acute disease characterized by neuronal death, increment of reactive gliosis (reactive microglia and astrocytes), and a severe inflammatory process. Neuroinflammation is an early event after cerebral ischemia, with microglia playing a leading role. Reactive microglia involve functional and morphological changes that drive a wide variety of phenotypes. In this context, deciphering the molecular mechanisms underlying such reactive microglial is essential to devise strategies to protect neurons and maintain certain brain functions affected by early neuroinflammation after ischemia. Here, we studied the role of mammalian target of rapamycin (mTOR) activity in the microglial response using a murine model of cerebral ischemia in the acute phase. We also determined the therapeutic relevance of the pharmacological administration of rapamycin, a mTOR inhibitor, before and after ischemic injury. Our data show that rapamycin, administered before or after brain ischemia induction, reduced the volume of brain damage and neuronal loss by attenuating the microglial response. Therefore, our findings indicate that the pharmacological inhibition of mTORC1 in the acute phase of ischemia may provide an alternative strategy to reduce neuronal damage through attenuation of the associated neuroinflammation.

Genetic Analysis as a Tool to Improve the Monitoring of Stranded Cetaceans in Chile.

Cetacean strandings are a valuable source of information for several studies from species richness to conservation and management. During the examination of strandings, taxonomic and sex identification might be hindered for several reasons. Molecular techniques are valuable tools to obtain that missing information. This study evaluates how gene fragment amplification protocols can support the records of strandings done in the field in Chile by identifying, corroborating, or correcting the identification of the species and sex of the recorded individuals. Through a collaboration between a scientific laboratory and government institution in Chile, 63 samples were analyzed. Thirty-nine samples were successfully identified to the species level. In total, 17 species of six families were detected, including six species of conservation interest. Of the 39 samples, 29 corresponded to corroborations of field identifications. Seven corresponded to unidentified samples and three to corrected misidentifications, adding up to 28% of the identified samples. Sex was successfully identified for 58 of the 63 individuals. Twenty were corroborations, 34 were previously unidentified, and four were corrections. Applying this method improves the stranding database of Chile and provides new data for future management and conservation tasks.

The genomic footprint of whaling and isolation in fin whale populations.

Twentieth century industrial whaling pushed several species to the brink of extinction, with fin whales being the most impacted. However, a small, resident population in the Gulf of California was not targeted by whaling. Here, we analyzed 50 whole-genomes from the Eastern North Pacific (ENP) and Gulf of California (GOC) fin whale populations to investigate their demographic history and the genomic effects of natural and human-induced bottlenecks. We show that the two populations diverged ~16,000 years ago, after which the ENP population expanded and then suffered a 99% reduction in effective size during the whaling period. In contrast, the GOC population remained small and isolated, receiving less than one migrant per generation. However, this low level of migration has been crucial for maintaining its viability. Our study exposes the severity of whaling, emphasizes the importance of migration, and demonstrates the use of genome-based analyses and simulations to inform conservation strategies.

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.

Glial Cells as Therapeutic Approaches in Brain Ischemia-Reperfusion Injury.

Ischemic stroke is the second cause of mortality and the first cause of long-term disability constituting a serious socioeconomic burden worldwide. Approved treatments include thrombectomy and rtPA intravenous administration, which, despite their efficacy in some cases, are not suitable for a great proportion of patients. Glial cell-related therapies are progressively overcoming inefficient neuron-centered approaches in the preclinical phase. Exploiting the ability of microglia to naturally switch between detrimental and protective phenotypes represents a promising therapeutic treatment, in a similar way to what happens with astrocytes. However, the duality present in many of the roles of these cells upon ischemia poses a notorious difficulty in disentangling the precise pathways to target. Still, promoting M2/A2 microglia/astrocyte protective phenotypes and inhibiting M1/A1 neurotoxic profiles is globally rendering promising results in different in vivo models of stroke. On the other hand, described oligodendrogenesis after brain ischemia seems to be strictly beneficial, although these cells are the less studied players in the stroke paradigm and negative effects could be described for oligodendrocytes in the next years. Here, we review recent advances in understanding the precise role of mentioned glial cell types in the main pathological events of ischemic stroke, including inflammation, blood brain barrier integrity, excitotoxicity, reactive oxygen species management, metabolic support, and neurogenesis, among others, with a special attention to tested therapeutic approaches.

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.

[Hospitals and healthcare in Leon province at the end of the Ancien Régime]. / Hospitalidad y asistencia en la provincia de León a finales del Antiguo Régimen (1728-1896).

This work is structured in two parts. First, we examine the major features of the healthcare system of the province of León around the mid-18th century, focusing on its territorial distribution, the scope of its responsibilities and its type of administration. In the second part, we study San Antonio Abad Hospital, the most important hospital in the province at that time, describing the sociological profile of its patients according to their sex, marital status, age and place of origin.

Largest baleen whale mass mortality during strong El Niño event is likely related to harmful toxic algal bloom.

While large mass mortality events (MMEs) are well known for toothed whales, they have been rare in baleen whales due to their less gregarious behavior. Although in most cases the cause of mortality has not been conclusively identified, some baleen whale mortality events have been linked to bio-oceanographic conditions, such as harmful algal blooms (HABs). In Southern Chile, HABs can be triggered by the ocean-atmosphere phenomenon El Niño. The frequency of the strongest El Niño events is increasing due to climate change. In March 2015, by far the largest reported mass mortality of baleen whales took place in a gulf in Southern Chile. Here, we show that the synchronous death of at least 343, primarily sei whales can be attributed to HABs during a building El Niño. Although considered an oceanic species, the sei whales died while feeding near to shore in previously unknown large aggregations. This provides evidence of new feeding grounds for the species. The combination of older and newer remains of whales in the same area indicate that MMEs have occurred more than once in recent years. Large HABs and reports of marine mammal MMEs along the Northeast Pacific coast may indicate similar processes in both hemispheres. Increasing MMEs through HABs may become a serious concern in the conservation of endangered whale species.

Ancient female philopatry, asymmetric male gene flow, and synchronous population expansion support the influence of climatic oscillations on the evolution of South American sea lion (Otaria flavescens).

The South American sea lion (Otaria flavescens) is widely distributed along the southern Atlantic and Pacific coasts of South America with a history of significant commercial exploitation. We aimed to evaluate the population genetic structure and the evolutionary history of South American sea lion along its distribution by analyses of mitochondrial DNA (mtDNA) and 10 nuclear microsatellites loci. We analyzed 147 sequences of mtDNA control region and genotyped 111 individuals of South American sea lion for 10 microsatellite loci, representing six populations (Peru, Northern Chile, Southern Chile, Uruguay (Brazil), Argentina and Falkland (Malvinas) Islands) and covering the entire distribution of the species. The mtDNA phylogeny shows that haplotypes from the two oceans comprise two very divergent clades as observed in previous studies, suggesting a long period (>1 million years) of low inter-oceanic female gene flow. Bayesian analysis of bi-parental genetic diversity supports significant (but less pronounced than mitochondrial) genetic structure between Pacific and Atlantic populations, although also suggested some inter-oceanic gene flow mediated by males. Higher male migration rates were found in the intra-oceanic population comparisons, supporting very high female philopatry in the species. Demographic analyses showed that populations from both oceans went through a large population expansion ~10,000 years ago, suggesting a very similar influence of historical environmental factors, such as the last glacial cycle, on both regions. Our results support the proposition that the Pacific and Atlantic populations of the South American sea lion should be considered distinct evolutionarily significant units, with at least two managements units in each ocean.

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.

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.

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.

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.