Enhanced stress response in rats that suffered acute neuroinflammation induced by neuraminidase three months before

Microglial cells are protagonists in neuroinflammatory processes and their activation is a notorious feature of such events. In acute inflammation, microglial cells return to their basal surveillant state in few days. However, sometimes they evolve towards a primed state, characterized by hypersensitivity to new stimuli and an exacerbated response which may jeopardize brain functions. Because the hypothalamus is a pivotal hub for neuroendocrine and autonomic functions, we have been exploring evidences of microglial priming in this region and its consequences. We used a model of acute ventricular neuroinflammation consisting in the intracerebroventricular (ICV) injection of neuraminidase (NA). This enzyme is found in the cover of neurotropic bacteria and viruses, e.g. influenza, mumps or measles viruses, thus mimicking a brain infection. Three months after inducing neuroinflammation with NA to rats, an acute stressor was applied to investigate the activation of the hypothalamic-pituitary-adrenal (HPA) axis and the stress response elicited, as well as the inflammatory activation of hypothalamic microglial cells. The acute stressor was forced swimming for 6 minutes. Afterwards, blood samples were retrieved to determine corticosterone levels by ELISA, and the brains extracted to analyze microglial cells in histological sections by immunohistochemistry with IBA1 and inflammatory markers by qPCR. Stressed rats previously injected with NA had increased levels of corticosterone compared with control rats that were equally stressed but had been ICV injected with saline. Also, qPCR studies in hypothalamic tissue revealed that NA treated rats presented an increased expression of the genes for the inflammasome protein NLR family pyring domain containing 3 (NLRP3) and the microglial marker IBA1. Concomitantly, the morphological analysis of microglial cells located in the paraventricular nucleus (PVN) showed a morphological bias towards a slightly activated state in microglia...Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Anxiety and mild microglial activation in the amygdala two weeks after NA-induced neuroinflammation

A single injection of neuraminidase (NA) within the cerebral ventricles (ICV) triggers an acute neuroinflammation. Neurological complications or behavioral alterations have been associated to neuroinflammation. While some of these symptoms decline with time along with inflammation, the possibility of long-term sequelae should be considered. Thus, we aimed to explore if NA-induced neuroinflammation provokes behavioral or neurological disturbances at medium (2 weeks) and long (10 weeks) term. Rats were ICV injected with NA or saline. First, neurological alterations of the sensorimotor reflexes were not found, suggesting that NA does not cause disturbances in major brain functions. While the open field test revealed normal locomotor capacity in the animals injected with NA, however the evaluation of specific behaviors (rearing and rearing with support) pointed out an increased anxiety state 2 weeks after NA administration, but not at long term (10 weeks). A histological study of brain areas related to emotions (amygdala) and stress response (hypothalamic PVN) revealed no significant differences in the number of microglia or astrocytes. Nevertheless, the morphological analysis of microglial cells demonstrated that, in the amygdala of NA injected rats, microglia presented a morphology consistent with a slightly activated state. Such morphological change, which was evident 2 weeks after NA injection, was virtually reverted 10 weeks post-ICV. These results point out that NA injected ICV may cause anxiety in the medium term (while not affecting other functions like sensorimotor functions or the locomotor capacity), a behavioral alteration that is transient and that concurs with a mild inflammation, evidenced by the overexpression of certain genes and, more notably, by the morphological bias of microglial cells located in the amygdala towards an activated profile.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Neuraminidase-induced neuroinflammation causes anxiety and microgliosis in the amygdala

An intracerebroventricular (ICV) injection of neuraminidase (NA) within the lateral ventricles originates an acute event of neuroinflammation, which is solved to a great extent after two weeks. Recently, neurological problems or behavioral alterations have been associated with neuroinflammation. Although the majority of them fade along with inflammation resolution, the possibility of long-term sequelae should be taken into consideration. Thus, we aimed to explore if NA-induced neuroinflammation provokes behavioral or neurological disturbances at medium (2 weeks) and long (10 weeks) term. Initially, rats were ICV injected with NA or saline. Two or 10 weeks later they were made to perform a series of neurological tests and behavioral evaluations (open field test). The neuroinflammation status of the brain was studied by immunohistochemistry and qPCR. While no neurological alterations were found, the open field test revealed an increased anxiety state 2 weeks after NA administration, which was not observed after 10 weeks. In accordance with this behavioral findings, an overexpression of the molecular pattern receptor TLR4 was revealed by qPCR in hypothalamic tissue in NA treated animals after 2 weeks of ICV, but not after 10 weeks. Moreover, histological studies showed a microgliosis in the amygdala of NA injected rats 2 weeks post-ICV, as well as a slightly activated state evidenced by morphometric parameters of these cells. These histological findings were not present 10 weeks after the ICV injection. These results suggest that NA-induced neuroinflammation might cause anxiety, with no neurological manifestations, in the medium term, along with a mild microglial activation in amygdala. Such symptoms seem to revert, as they were not detected 10 weeks after NA administration.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Role of the LPA1 receptor in mood and emotional regulation

Depression is a debilitating psychiatric condition characterized by anhedonia and behavioural despair among others symptoms. Despite the high prevalence and devastating impact of depression, underlying neurobiological mechanisms of mood disorders are still not well known. Regardless its complexity, central features of this disease can be modelled in rodents in order to better understand the potential mechanisms underlying. On the other hand, the lack of LPA1 receptor compromises the morphological and functional integrity of the limbic circuit and the neurogenesis in hippocampus, induces cognitive alterations on hippocampal-dependent tasks and dysfunctional coping of chronic stress, provokes exaggerated endocrine responses to emotional stimuli and impairs adaptation of the hypothalamic-pituitary-adrenal axis after chronic stress. Factors, which all have been related with depression. Here, we sought to establish the involvement of the LPA1 receptor in regulation of mood and emotion. To this end, in wild-type and maLPA1-null mice active coping responses to stress were examined using the forced swimming test (FST). To assess hedonic behaviour saccharine preference test and female urine sniffing test were used. Our data indicated that the absence of the LPA1 receptor significantly affected to coping strategies. Thus, while null mice displayed less immobility than wt in FST, exhibited more climbing and less swimming behaviour, responses that could be interpreted as an emotional over-reaction (i.e., a panic-like response) to stress situations. Concerning hedonic behaviour, the lack of the LPA1 receptor diminished saccharin preference and female urine sniffing time. Overall, these data supports the role of LPA1 receptor in mood and emotional regulation. Specially, the lack of this receptor induced emotional dysregulation and anhedonic behaviour, a core symptom of depression.Universidad de Málaga, Campus de Excelencia Andalucía Tech. Andalusian Regional Ministries of Economy, Innovation, Science and Employment (SEJ-1863; CTS643) and of Health (PI-0234-2013; Nicolas Monardes Programme), MINECO (PSI2013-44901-P) and National Institute of Health Carlos III (Sara Borrel)

Microglial and neurogenic alterations in hypothalamus due to acute stress

This study was supported by FEDER/Ministerio de Ciencia, Innovación y Universidades – Agencia Estatal de Investigación from Spain (PSI2017-83408-P to Pedraza C.), and Ministerio de Educación, Cultura y Deporte from Spain (FPU16/05308 to Nieto-Quero A).Microglial cells are an important glial population known to be involved in several biological processes such as stress response. These cells engage an activated state following a stress insult that may lead to nervous tissue damage, including new cell generation impairment. This has been widely studied in regions with notable neurogenesis such as de hippocampus, however, the effect in other regions with fewer yet relevant neurogenesis remains partially unknown. One of them is the hypothalamus, a key vegetative control center playing an important role in stress response. Moreover, most of the stress models studied concern neuroinflammatory and neurogenic changes due to a chronic stressor but not a single stress event. Given the repercussion of these processes alone, it would be interesting to elucidate the relationship between microglial response, hypothalamic neurogenesis, and acute stress. This project focuses on studying acute stressed C57BL/6J mice, both at the histological and molecular level. An intense stressor combining water immersion and movement restriction was performed. Using immunohistochemical and molecular analysis with Luminex, we could analyze microglial distribution and morphology, neurogenesis, and inflammatory environment in the hypothalamic parenchyma (paraventromedial, ventromedial and arcuate nucleus).Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Effects on the hippocampal microglia after acute treatment of a psychological stressor associated with depressive-like behaviours

Funding: This study was supported by FEDER/Ministerio de Ciencia, Innovación y Universidades – Agencia Estatal de Investigación from Spain (PSI2017-83408-P to C. Pedraza), and Ministerio de Universidades from Spain (FPU16/05308 to A. Nieto-Quero and FPU19/03629 to M.I. Infantes-López).Stressful life events may have a negative impact on mental health compromising people's well-being, so knowing the neurobiological changes that occur after psychosocial stressors can have an impact on overall health. However, the neurobiological mechanisms responsible for the negative effects are not known in detail, and the initial changes that take place after the initiation of a stress protocol are much less well understood. Hippocampus constitutes a target structure of the adverse effects of stress. Among the possible mechanisms involved, the response of microglia to stress is receiving increasing interest. For this reason, after 1 and 24 hours of submitted C57BL/6J mice to acute and intense stress procedure denominated WIRS (water immersion restraint stress), the microglial response were analysed using a set of morphofunctional parameters. Then, the levels of the cytokines: IL-6, IFN-gamma and TNF-alpha cytokine were measured. Furthermore, a complementary proteomic analysis based on the principle of mass spectrometry was carried out. Results reveal that acute stress increased the number of microglia and induced microglial morphofunctional changes. Regarding cytokines, acute stress only increased IL-6 levels, which remained elevated at 24 h. Proteomic analysis, over time (in 24 h post-stress), showed an increase in proteins associated with the intracellular calcium metabolism. These findings suggest a neuroinflammatory response after acute stress observed at one hour after the application of the WIRS protocol and maintained at least 24 hours after the end of the stressor.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

Social defeat stress induces microglial alterations and impaired cell survival in the hypothalamus according to behavioral phenotype

Stress is the main environmental cause for depression, known to cause brain immune alterations. As major brain immune cells, microglia undergo transcriptional and, consequently, morphological changes that result in tissue damage, including new cell generation impairment. Even so, few brain regions have been thoroughly studied, excluding key regulators as the hypothalamus, in which this process remains partially unknown. Moreover, there is a poor understanding in physiology related to behavioral outcome. Therefore, it would be interesting to study the relationship between microglia and cell proliferation in stressed mice while controlling for behavior. Here, we used the social defeat stress (SDS) paradigm as a depression-inducing protocol in 8-week-old male C57BL/6J mice for 10 consecutive days. Intruder mice behavior was analyzed to assess depression using behavioral tests and K-means clustering. By immunohistochemical and imaging procedures, microglial morphology, and distribution, as well as cell survival, were analyzed in the hypothalamic paraventricular, ventromedial and arcuate nucleus. Finally, statistical mediation analysis was conducted to evaluate the relationship among variables. Results show mice response to SDS was different, being half the mice resilient and half sensitive to depressive-like symptoms. Microglial morphological activation was enhanced in the ventromedial and arcuate nucleus, especially in stress sensitive animals. Similar results were observed in cell survival, which was particularly affected in sensitive mice. Strikingly, these cell survival changes were statistically mediated by microglial activation. As a conclusion, hypothalamic regions were found to respond differently to stress, accordingly to behavioral outcome, in terms of microglial activation and subsequent decrease in cell survival.This study was supported by FEDER/Ministerio de Ciencia, Innovación y Universidades – Agencia Estatal de Investigación from Spain (PSI2017-83408-P to Pedraza C.), FEDER/Junta de Andalucía from Spain (UMA20-FEDERJA-112 to Pedraza C. and Pérez- Martín M), and Ministerio de Educación, Cultura y Deporte from Spain (FPU19/03629 to Infantes-López MI and FPU16/05308 to Nieto-Quero A). Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Effects of chronic stress on hippocampal microglia and neurogenesis of mice under social defeat stress.

Introduction: Chronic stress is the main environmental factor in the aetiology of depression and it is known that this type of stress may cause alterations in brain regions such as the hippocampus. Nevertheless, changes in a cellular basis are still a subject of study. Objective: The analysis of microglial cells and immature neurons in the dentate gyrus (DG) of stressed mice. Methods: C57BL/6J mice were subjected to Social Defeat Stress model (SDS), consisting of 6 days of social isolation prior to 10 days of stressor. The DG was analysed using immunohistochemistry techniques against Iba1 (microglia) and DCX (immature neurons) following image analysis (ImageJ) to obtain morphological and distribution data of microglial somas. Furthermore, hippocampal neurogenesis was assessed through stereological quantification of DCX+ cells (Visiopharm). Results: An increase in soma size under chronic stress conditions was shown, as well as a less circular and more ameboid soma. These changes were observed mainly in the infrapyramidal blade of the DG. According to microglial cells distribution parameters, the granular cell layer (GCL) was the region which presented the highest microglial density under SDS. Regarding hippocampal neurogenesis, a decrease in the number of DCX+ Type 2-3 cells was observed in the whole DG. Conclusion: All these results o!er a more profound insight of stress changes at a cellular level and could contribute to a better understanding of neurobiological basis in pathologies such as depression. Projects: PSI2017-83408-P (MINECO) and P20 00460 (Consejería de Conocimiento, Investigación y Universidades, Junta de Andalucía).University of Málaga and the projects PID2020-117464RB-I00 (Ministerio de Ciencia e Innovación, Spain) to Pedraza C. and Pérez-Martin M., PSI2017-83408-P (FEDER/Ministerio de Ciencia, Innovación y Universidades, Spain) to Pedraza C., UMA20- FEDERJA-112 (FEDER/Junta de Andalucía) to Pedraza C. and Pérez-Martin M. and P20-00460 (Consejería de Conocimiento, Investigación y Universidades, Junta de Andalucía) to Pedraza C. Predoctoral Fellowship: FPU16/05308 to Nieto-Quero A. and FPU19/03629 to Infantes-López MI., Ayuda A.2 para Contrato Predoctoral Del I Plan Propio de Investigación, Transferencia y Divulgación Científica de la Universidad de Málaga, Convocatoria 2021 to Munoz-Martin J. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

Do changes in microglial status underlie neurogenesis impairments and depressive-like behaviours induced by psychological stress? A systematic review in animal models

Stress may have a negative effect on mental health and is the primary environmental risk factor in the aetiology of depression. Nevertheless, the neurobiological mechanisms underlying this mood disorder remain poorly characterized. The hippocampus is a target structure of the adverse effects of stress, and hippocampal neuro- genesis plays a crucial role. However, we do not know the mechanisms by which stress impacts neurogenesis. Recent studies indicate that changes in neuroinflammation, primarily via microglial cells, may play an essential role in this process. However, the relationship between stress, microglial changes, and alterations in neurogenesis and their involvement in the development of depression is poorly characterized. For this reason, this systematic review aims to synthesise and evaluate current studies that have investigated the relationship between these variables. Taken together, the revised data, although not entirely conclusive, seem to suggest that microglial changes induced by psychological stress regulate neurogenesis and in turn may be responsible for the devel- opment of depressive-like behaviours, but other factors that influence these stressful experiences should not be dismissed.We would like to thank the University of Malaga/CBUA for the contribution to the payment of the open access charge. This study was supported by FEDER/Ministerio de Ciencia, Innovacion ´ y Universidades – Agencia Estatal de Investigacion ´ (PSI 2017-83408-P) to C.P. and Ministerio de Universidades (FPU16/05308 to AN-Q). PC-P has been funded by the research project PSI 2017-83408- P as technician (CI-19-082)

Cognitive impairment in a murine model of experimental autoimmune encephalomyelitis with relapsing-remitting course

Multiple sclerosis (MS) is a neuroinflammatory disorder characterized by demyelination and progressive axonal loss that affects the central nervous system. In addition of physical disability and the neurodegenerative process, MS associates with co-morbid behavioral, neuropsychiatric and cognitive impairment, including learning and memory deficits. The study of cognitive impairment in the currently most suitable experimental animal model of MS, experimental autoimmune encephalomyelitis (EAE), constitutes a very valuable tool to translate ultimately into clinical a better diagnosis and more effective treatment protocols. In our study, we analyzed the behavioral profile of a murine model of EAE induced by myelin oligodendrocyte glycoprotein peptide (MOG35-55) which develops a relapsing-remitting course. In the early neuroinflammatory phase of the disease, i.e. 19-21 days post immunization (dpi), EAE mice exhibited deficits in motor coordination/skill learning (Rotarod test), and spatial working memory (spontaneous alternation in Y-maze), as well as depressive symptoms (tail suspension test) and anxiety-like behavior (elevated plus-maze). EAE mice did not yet show object recognition memory impairments, suggesting that reference memory was not altered in this phase. However, from 33-35 dpi until late phases (49-52 dpi), independently of clinical score, EAE mice exhibited a memory decline showing lower discrimination index in the object recognition test. EAE late phase was also characterized by motor coordination and spatial working memory impairments as well as higher anxiety-like behavior. Overall, these data demonstrates a differential pattern of gradual cognitive dysfunctions during the relapsing-remitting EAE course that could help to understand the development of progressive cognitive decline in MS patients. Funding: Andalusian Regional Ministries of Economy, Innovation, Science and Employment (SEJ-1863; CTS643) and of Health (PI-0234-2013; Nicola´s Monardes Programme).Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech