Neuroinflammation is a physiological protective response in the context of infection and injury. However, neuroinflammation, especially if chronic, may also drive neurodegeneration. Neurodegenerative diseases, such as multiple sclerosis (MS), Alzheimer's disease (AD), Parkinson's disease (PD) and traumatic brain injury (TBI), display inflammatory activation of microglia and astrocytes. Intriguingly, the central nervous system (CNS) is a highly steroidogenic environment synthesizing steroids de novo, as well as metabolizing steroids deriving from the circulation. Neurosteroid synthesis can be substantially affected by neuroinflammation, while, in turn, several steroids, such as 17β-estradiol, dehydroepiandrosterone (DHEA) and allopregnanolone, can regulate neuroinflammatory responses. Here, we review the role of neurosteroids in neuroinflammation in the context of MS, AD, PD and TBI and describe underlying molecular mechanisms. Moreover, we introduce the concept that synthetic neurosteroid analogues could be potentially utilized for the treatment of neurodegenerative diseases in the future.

The subventricular zone (SVZ) is an adult neurogenic niche that contains multipotent stem/progenitor cells that may be a viable target for remyelination in Multiple Sclerosis. In response to demyelination, SVZ progenitors are recruited into myelin lesions. Currently, the effect of inflammation on the endogenous brain stem cell compartment remains poorly characterised. Theiler's murine encephalomyelitis virus (TMEV) induces a demyelinating disease in susceptible SJL/J mice, due to persistence of the virus, which models the chronic progressive form of multiple sclerosis. In contrast, virus is rapidly cleared in TMEV resistant C57BL/6 mice. This viral clearance is critically dependent on infiltration of CD4⁺ and CD8⁺ T cells during the first 3-14 days after infection, through the blood vessels and meninges. This project aimed to characterise the impact of TMEV induced inflammation on SVZ homeostasis. In both strains of mice the most pronounced and consistent inflammation in the CNS was observed in periventricular regions and in particular, the SVZ, which showed targeted infection by the TMEV virus. However, the time course and kinetics for infiltration at 3,7 and 14 days post infection have very different profiles between resistant C57BL/6 and susceptible SJL mice. Using RT-PCR arrays and ELISA I have shown that these differences in T cell infiltration to the SVZ may be due to much higher chemokine and cytokine expression levels in B6 mice. TMEV infection decreases SVZ cell proliferation and results in a loss of neuroblast numbers. Galectin-3 (Gal-3) is a β-galactoside binding protein that is constitutively expressed specifically in the SVZ. Following TMEV infection Gal-3 levels are significantly upregulated in the SVZ, with higher expression in B6 mice compared to SJL mice. Primary SVZ astrocytes secrete extracellular Galectin-3 at much higher levels than cortical astrocytes. Galectin-3 is a pro-inflammatory mediator which upon secretion is able to activate immune and inflammatory signaling events and amplify pro-inflammatory cytokine production. Both SJL and B6 Gal-3^-/- KO mice have decreased expression of CCL2, CCL5 CXCL10 and CCL8 chemokines in the SVZ after TMEV infection. Deletion of Gal-3 prevents the loss of SVZ proliferation and in B6 mice decreases hematopoietic cell infiltration and enhances ectopic neuroblast emigration. These data implicate Galectin- 3 as a novel regulator of the SVZ inflammatory response and may provide a new target for regulating T cell CNS immigration in autoimmune disease.

Stroke is a neurovascular disease that frequently results in decreased motor and cognitive functioning. Obesity is a major risk factor associated with ischemic stroke and is thought to worsen the functional deficits observed after stroke. Previous findings from our laboratory suggest that worse motor deficits in obese animals may be a result from an exacerbated neuroinflammatory response. Most animal studies demonstrate an association between obesity and worse cognitive functioning after stroke. However, the mechanisms are not well studied. This study examines the neuroinflammatory response, ischemic brain tissue damage, and cognitive functioning in diet-induced obese mouse models during the chronic phase after ischemic stroke, defined as weeks after stroke. Our study found an earlier onset of cognitive deficits in obese mice after stroke compared to normal weight mice. We found no differences in the degree of brain damage in obese animals and normal weight animals 11 weeks after stroke, but observed higher levels of microgliosis in obese animals compared to normal weight animals. Due to the limitations of our study, additional studies should be done to assess the severity of cognitive deficits in obese animals compared to normal weight animals in the chronic phase after stroke. Further studies also need to be done to confirm our findings regarding the microglial response and degree of ischemic brain damage during the chronic phase.

O Transtorno Bipolar (TB) é uma doença psiquiátrica grave, altamente incapacitante que está associada com diversas comorbidades médicas e altas taxas de suicídio. Embora sua fisiopatologia não esteja completamente elucidada, inúmeros estudos têm mostrado alterações no sistema imune de indivíduos com TB. A resposta crônica destes indivíduos ao estresse parece gerar um aumento da inflamação sistêmica bem como da neuroinflamação. A micróglia ativada devido aos estímulos inflamatórios contínuos deve ocasionar diferentes prejuízos tanto bioquímicos quanto funcionas. Os macrófagos, primeira linha de defesa, são células de característica plástica de extrema importância do sistema imune e podem ser estimulados a polarizar para diferentes formas com liberação de fatores pró e antiinflamatórios, estimulando ou mantendo a homeostase no ambiente agredido de alguma forma. Desta forma, nosso trabalho buscou investigar a resposta fenotípica dos macrófagos contra o meio ambiente pró-inflamatório sistêmico observado no plasma de pacientes bipolares eutímicos, maníacos e depressivos em comparação aos controles. A amostra incluiu 5 controles saudáveis, 8 pacientes bipolares remetidos, 5 pacientes maníacos e 5 pacientes depressivos. As citocinas e quimiocinas de RNAm em células U937 tratadas com plasma mostraram um padrão de expressão diferente relativo entre controles saudáveis e pacientes com TB. As citoquinas inflamatórias tais como IL-1β e TNF-α, em pacientes bipolares maníacos e depressivos demonstram maiores quantidades de IL-1β mRNA do que os pacientes eutímicos e pacientes depressivos induziram maiores quantidades de RNAm de TNF-α do que os pacientes eutímicos em células U937. Já a expressão das quimiocinas CXCL9 e CXCL10 no plasma de pacientes com TB depressivos, demostraram ser de menor expressão significativa no grupo de pacientes maníacos quando comparados a controles e pacientes bipolares eutímicos. Nossos resultados sugerem que as citocinas periféticas devem modular a polarização M1 ou M2 de macrófagos no TB. / Bipolar Disorder (BD) is a severe and highly incapacitating psychiatric disorder which is associated with the presence of medical comorbidities. The progression of BD is related to an important cognitive deficit and also to biological and clinical manifestations that lead to treatment resistance and worse prognosis. Immune disturbances have been widely observed and investigated in BD patients. Chronic inflammatory responses induce neuroinflammation, mainly by pro-inflammatory microglial activation, and result in biochemical and functional impairment. Macrophages are the first line of defense of the immune system and exhibit cell plasticity. As well, microglia represents the resident macrophage of the central nervous system been responsible for its protection. Both cells can be stimulated to polarize into two different phenotypes, mainly pro- and anti-inflammatory, maintaining the homeostasis under physiologic and pathologic conditions. Therefore, we aimed to investigate macrophages phenotypical response when submitted to BD patients plasma in different episodes, which is considered a pro-inflammatory environment, and healthy controls plasma. Subjects included healthy controls (n=5), remitted BD patients (n=8), manic patients (n=5) and depressive patients (n=5). The mRNA expression of chemokynes and cytokines from U937 cells treated with BD patients plasma were different from those submitted to healthy controls plasma. Higher mRNA expression of IL-1β was observed in those cells submitted to manic and depressive BD patients plasma when compared to euthymic patients. Also, depressive BD patients plasma induced higher expression of TNF-α compared to euthymic patients. However, chemokynes expression, such as CXCL9 and CXCL10, were reduced in depressive BD patients. However, chemokynes expression, such as CXCL9 and CXCL10, were reduced in depressive BD patients. Inflammatory cytokines such as IL-1β and TNF-α in bipolar manic and depressive patients demonstrate higher amounts of IL-1β mRNA that euthymic patients and depressive patients induced higher amounts of TNF-α mRNA levels than the patients in euthymic U937. Since the expression of CXCL9 and CXCL10 chemokines in plasma from patients with depressive TB, proved less significant expression in the group of manic patients when compared to controls and euthymic bipolar patients.

O sistema canabinóide endógeno tem uma importante função modulatória em muitos processos neurobiológicos, incluindo a neuroproteção, a plasticidade neuronal e a neuroinflamação. Neste estudo nós pré-tratamos os camundongos com um agonista inverso do receptor CB1, para avaliarmos se ocorria a neuroproteção através de respostas adaptativas. Camundongos machos adultos (C57BL/6J) de 3 meses de idade foram pré-tratados durantes 4 dias com injeções diearias de 3 mg/Kg de AM251 (agonista inverso CB1) ou com veículo, intraperitonealmente (i.p.). Vinte e quatro horas depois da última injeção de AM251/Veículo, os animais foram injetados com LPS i.p. (500 µg/kg) ou salina. Foram realizados testes comportamentais e testes bioquímicos com estruturas encefálicas. Nossa hipótese era que o pré-tratamento com o agonista inverso de CB1 iria funcionar como um estressor moderado, levando a uma neuroadaptação. Contudo, o pré-tratamento não ativou vias neuroprotetoras, e sim como atuou como um estresse deletério. / The endogenous cannabinoid system seems to play a modulatory function in many neurobiological processes, including neuroprotection, neuronal plasticity and neuroinflammation. In this study we pretreated the mice with an inverse agonist of the receptor CB1, in order to evaluate this neuroprotection through adaptive responses. Three months-old male mice (C57BL/6J) were pretreated during 4 days with daily injections of 3mg/Kg of AM251 (CB1 inverse agonist) or with vehicle, intraperitoneally (i.p.). Twenty-four hours after the last AM251/Vehicle injection, animals were injected i.p. with LPS (500 µg/kg) or saline. Behavioural and bichoemical assays were performed. Our hypothesis was that the pretreatment with the inverse agonist of CB1 was going to act as a moderate stressor, leading to a neuroadaptation. However CB1 pretreatment potentiated or sensitized the pro-inflammatory signaling pathway. Our pre-treatment worked not as a moderate stressor but as a deleterious stressor.

Neuroinflammation is a major driver of secondary brain cell death after ischaemic stroke, seizure activity and traumatic brain injury. In a model of excitotoxic neuroinflammation, striatal injection of a toxic dose of AMPA causes cell death in the striatum after 24 hours. Co-injection of AMPA with the pro-inflammatory cytokine interleukin-1β (IL-1β) leads to additional cortical cell death. Injected alone, IL-1β leads to little or no cell death. It is hypothesised that IL-1β may exacerbate cell death by interfering with blood flow coupling. In the first study, two-dimensional optical imaging spectroscopy was used to measure early changes in the haemodynamic response in the anaesthetised rat barrel cortex before and for 6 hours after injection of vehicle, AMPA, IL-1β, or AMPA+IL-1β. After injection of IL-1β, with or without AMPA, the oxygenated blood flow response to mechanical whisker stimulation approximately halved over the course of 6h. In the second study, to determine whether the IL-1β-dependent changes in blood flow response are reflected by altered cellular activity, local field potentials, multi-unit activity and local tissue oxygenation responses to whisker stimulation were recorded simultaneously from the active barrel before and up to 6h after injection. A similar reduction in the size of the oxygenation response was seen again in the IL-1β- and AMPA+IL-1β-treated groups. Importantly, the level of gamma frequency oscillations at stimulus onset decreased within the first hours after injection of AMPA+IL-1β or IL-1β, suggesting a disruption of the fast-spiking interneuron network in the barrel cortex. These findings, along with histological observations of IL-1β-dependent markers of neuroinflammation, suggest that IL-1β may exacerbate AMPA-induced excitotoxicity by potentiating seizure activity and decoupling the neurovascular response in the cortex.

Pentraxin 3 (PTX3) is an acute phase protein which regulates peripheral inflammationand it has been suggested to have neuroprotective properties. Inflammation iscommonly associated with poor outcome during diverse central nervous system (CNS)disorders, but the role of PTX3 in brain inflammation is completely unknown. Westudied the role of PTX3 in brain inflammation and repair after stroke, a CNS disorderwhich is the third cause of death worldwide. To induce ischaemic stroke, we used themiddle cerebral artery occlusion (MCAo) model and found that the pro-inflammatorycytokine interleukin (IL)-1 was the inducer of PTX3 expression in the brain. Theanalysis of markers of inflammation and repair up to 14 days after MCAo in wild typeand PTX3 knockout (KO) mice revealed that, in general, lack of PTX3 has a negativeeffect on recovery after MCAo. PTX3 KO mice had delayed oedema resolution,defective glial scar, impaired microglial proliferation and reduced angiogenesis andneurogenesis. Therefore, PTX3 emerges as a target for stroke recovery and possiblyother CNS inflammatory diseases. PTX3 was, however, not involved in remoteneurodegeneration in the substantia nigra (SN) (an area of the brain remote butconnected with the area affected by the stroke), but we observed that remoteinflammation preceded remote neuronal death in the SN. Therefore, prevention ofremote inflammation may help prevent remote neurodegeneration in the SN afterstroke. This could have long term implications in SN neurodegeneration, which is akey pathological feature of Parkinson´s disease.

Stroke is a leading cause of death and disability worldwide. Although rapid recognition and prompt treatment have dropped mortality rates, most stroke survivors are left with permanent disability. Approximately 87% of all strokes result from the thromboembolic occlusion of the cerebrovasculature (ischemic strokes). Potential stroke therapeutics have included anti-inflammatory drugs, as well as many other targets with the goal of mitigating the acute and chronic inflammatory responses typically seen in an ischemic stroke. While these approaches have had great success in preclinical studies, their clinical translation has been less successful. Master inflammatory cytokines, such as IL-1, are of particular interest. IL-1’s isoforms, IL-1α and IL-1β, were long thought to have similar function. While IL-1β has been extensively studied in stroke, the role of IL-1α during post stroke inflammation has been overlooked. Because IL-1 inhibitors have been unsuccessful in clinical application, we reasoned that IL-1α may provide previously unknown benefits to the brain after injury. We hypothesized that IL-1α could be protective or even accelerate reparative processes in the brain such as producing new blood vessels (angiogenesis) or neurons (neurogenesis). To test that IL-1α is protective after stroke, we tested IL-1α’s protective effects on primary cortical neurons in in vitro models of stroke. We showed that IL-1α was directly protective on primary cortical neurons in a dose-dependent fashion. We then performed mouse middle cerebral artery occlusion stroke studies to determine the safety of giving IL-1α in vivo. These studies showed that administering IL-1α acutely was neuroprotective. However, intravenous (IV) administration of IL-1α resulted in transient, hemodynamic changes following drug delivery. To minimize these systemic effects, we administered IL-1α intra-arterially (IA) directly into the stroke affected brain tissue, allowing us to significantly lower the concentration of administered IL-1α. In comparison to IV, IA IL-1α showed greater histological protection from ischemic injury as well as improved functional recovery following stroke, all without systemic side effects. To test that IL-1α could aid in neurorepair following stroke, we tested IL-1α’s ability to help damaged blood vessels repair in vitro. We found that IL-1α significantly increased brain endothelial cell activation, proliferation, migration, and capillary formation. We tested IL-1α’s proangiogenic properties in vivo by administering IL-1α three days following stroke. Delayed administration allowed us to separate IL-1α’s acute neuroprotective effects from potential subacute angiogenic effects. We found that mice receiving IL-1α performed significantly better on behavioral tests and also showed greater vascularization within the penumbra two weeks following stroke. We also found that IL-1α treated animals showed more endothelial activation than vehicle treated animals. Finally, our studies showed that IL-1α treated animals showed increased early-phase neurogenesis with evidence of increased proliferation at the subventricular zone suggesting that IL-1α’s beneficial effects are even more far-reaching than previously thought. In conclusion, our experiments suggest that the inflammatory cytokine IL-1α is neuroprotective and neuroreparative in experimental ischemic stroke and worthy of further study as a novel stroke therapy.

Infusion of the "mononuclear fraction" of human cord blood cells (HUCBC), which is composed of immature blood cells and hematopoietic progenitors, is known to reduce neurobehavioral deficits in rats subject to middle cerebral artery occlusion (MCAO). When MCAO rats are infused with 106 cells 24 hours after the induction of the stroke, their motor function improves. To extend these findings, we first examined the behavioral recovery of MCAO rats in the presence of increasing doses of HUCBC. The recovery in behavioral performance seen with measurements of spontaneous activity and motor deficits, depended on the amount of cells delivered, with 106 HUCBC being the threshold for significant behavioral recovery. Measurements of the ischemic volume revealed an inverse relationship between HUCBC dose and damage volume, which reached significance at the higher HUCBC doses (107 and 3-5x107 cells). Moreover, investigation of the distribution of the intravenously injected cells showed that HUCBC were localized to the injured brain hemisphere and the spleen. Given these findings, we hypothesized that there may be a role of HUCBC in the modulation of the peripheral or brain-localized immune response that is normally evoked after stroke. Results on the effect of HUCBC infusion on splenocytes indicated that HUCBC treatment prevented the alterations in splenocyte type (CD8+ depletion) and function (T-cell suppression) induced by stroke. In particular, examination of cytokine production from splenocyte cultures of HUCBC-treated MCAO rats revealed increased production of IL-10 and decreased production of IFNgamma relative to MCAO rats. Microglia (immunostained with a CD11b antibody) and B cells (identified with the B220 cell marker) that were increased after MCAO were dramatically decreased after HUCBC treatment. Proinflammatory cytokines such as TNF-alpha, IL-1beta and IL-2 were upregulated after MCAO surgery and their expression was abrogated after HUCBC infusion. All these findings indicate that the action of HUCBC may be specifically related to the modulation of the stroke-induced inflammatory response, providing a possible mechanism by which cord blood cells have been repeatedly reported to promote functional recovery from ischemic injury.

Clinical and preclinical studies revealed that heart failure induces depression. Injury of myocardial tissue initiates an inflammation cascade that extends to the CNS and might contribute to depression following myocardial infarction (MI). Sex differences were noticed in the progression of heart failure and depression in clinical studies. We hypothesized that depression-like behavior induced by HF post-MI is influenced by sex through modulation of inflammation pathway. First, we evaluated sex differences in depression-like behavior in male and female rats at 8-10 weeks post-MI, as well as circulating cytokines, the extent of inflammation in the PFC, PVN, and amygdala, and mBDNF levels in the PFC and amygdala that are affected by neuroinflammation. Then we evaluated the effect of ovariectomy (OVX) and whether estrogen replacement with 17β-estradiol (E2) prevents post-MI induced depression-like behavior through inhibiting neuroinflammation. Thirdly we evaluated the role of inflammation for cardiac dysfunction and development of depression-like behavior in OVX female rats post-MI by oral treatment with pentoxifylline (PTX). Male rats developed depression-like behavior by ten weeks post-MI but not females as assessed by sucrose preference test (SPT) and forced swim test (FST). Both developed similar cardiac dysfunction and a comparable increase in plasma and PVN cytokine levels, but cytokine levels increased and mBDNF levels decreased only in the PFC of male rats post-MI. OVX per se decreased sucrose consumption and induced passive behavior assessed by SPT and FST, respectively.The combination of MI and OVX aggravated depression-like behvaiour. E2 treatment prevented the development of mild depression-like behavior in OVX and severe symptoms in OVX female rats post-MI. E2 had no effect on cardiac dysfunction in OVX female rats at 10 weeks post-MI. Despite the similar increase in circulating cytokines in OVX ± E2 at 10 weeks post-MI, E2 decreased the proinflammatory cytokines and increased IL-10 (anti-inflammatory cytokine) in the PFC. Finally, we evaluated the role of neuroinflammation in depression-like behavior in OVX female rats post-MI through inhibiting cytokine production by administering oral PTX. PTX prevented depression-like behavior in OVX female rats post-MI and reduced cytokines levels in plasma, PVN and PFC. However, PTX did not affect the progression of cardiac dysfunction at 10 weeks post-MI. Sex determines the development of depression-like behavior in HF post-MI and neuroinflammation appears to play a critical pathway that is affected by sex and can be inhibited by hormonal replacement or anti-inflammatory agents.