Effects of microbiota transplantation and the role of the vagus nerve in gut-brain axis in animals subjected to chronic mild stress.

INTRODUCTION: Currently, there is a growing emphasis on the study of intestinal signaling as an influencer in the pathophysiology of neuropsychiatric diseases, and the gut-brain axis is recognized as a communication route through endocrine, immune, and neural pathways (vagus nerve). Studies have shown that diets that modify the microbiota can reduce stress-related behavior and hypothalamic-pituitary-adrenal axis activation. Investigators have used fecal microbiota transplantation (FMT) approaches to demonstrate that stress-related microbiota composition plays a causal role in behavioral changes. AIM: We hypothesized that FMT may present immunomodulatory, biochemical, endocrine, cognitive, and behavioral benefits in stress situations and that these changes can be mediated via the vagus nerve. METHODS: Animals were subjected to a chronic mild stress (CMS) protocol. In one experiment, animals were divided into five groups: control, control + FMT, control + FMT + CMS, CMS + saline, and CMS + FMT. The animals received FMT, and behavioral tests were performed; cytokine and carbonyl levels were measured. In a second experiment, animals were submitted to vagotomy and divided into two groups: CMS + FMT and CMS + vagotomy + FMT. RESULTS: Animals submitted to the CMS protocol or that received FMT from stressed animals showed behavioral changes and changes in neuroactive substances (increased IL-6 and TNF-α levels and carbonyl proteins). The FMT of healthy donors improved the analyzed parameters. In addition, vagotomy influenced beneficial FMT results, confirmed by behavioral testing and protein carbonyl in the hippocampus. CONCLUSION: Manipulation of the microbiota reversed the behavioral and biochemical changes induced by the CMS protocol, and the vagus nerve influenced the gut-brain axis response.

Modulation of microglial phenotypes improves sepsis-induced hippocampus-dependent cognitive impairments and decreases brain inflammation in an animal model of sepsis.

BACKGROUND: In order to modulate microglial phenotypes in vivo, M1 microglia were depleted by administration of gadolinium chloride and the expression of M2 microglia was induced by IL-4 administration in an animal model of sepsis to better characterize the role of microglial phenotypes in sepsis-induced brain dysfunction. METHODS: Wistar rats were submitted to sham or cecal ligation and perforation (CLP) and treated with IL-4 or GdCl3. Animals were submitted to behavioral tests 10 days after surgery. In a separated cohort of animals at 24 h, 3 and 10 days after surgery, hippocampus was removed and cytokine levels, M1/M2 markers and CKIP-1 levels were determined. RESULTS: Modulation of microglia by IL-4 and GdCl3 was associated with an improvement in long-term cognitive impairment. When treated with IL-4 and GdCl3, the reduction of pro-inflammatory cytokines was apparent in almost all analyzed time points. Additionally, CD11b and iNOS were increased after CLP at all time points, and both IL-4 and GdCl3 treatments were able to reverse this. There was a significant decrease in CD11b gene expression in the CLP+GdCl3 group. IL-4 treatment was able to decrease iNOS expression after sepsis. Furthermore, there was an increase of CKIP-1 in the hippocampus of GdCl3 and IL-4 treated animals 10 days after CLP induction. CONCLUSIONS: GdCl3 and IL-4 are able to manipulate microglial phenotype in an animal models of sepsis, by increasing the polarization toward an M2 phenotype IL-4 and GdCl3 treatment was associated with decreased brain inflammation and functional recovery.

Protective effects of fecal microbiota transplantation in sepsis are independent of the modulation of the intestinal flora.

OBJECTIVE: The aim of this study was to investigate the protective effects of probiotics and fecal transplantation on inflammatory and oxidative parameters in the intestines of two rat models of sepsis. METHODS: Rats were treated with prebiotics, probiotics, or symbiotics and exposed to lipopolysaccharide (LPS) or zymosan after 15 d to induce endotoxemia. Oxidative damage and inflammation were analyzed, and histologic examination of the intestinal tissue was performed. Fecal microbiota transplantation (FMT) was carried out in LPS- and zymosan-induced rat models of sepsis. RESULTS: Supplementation with symbiotics for 15 d effectively reduced the inflammatory parameters compared with supplementation for 7 d. Probiotics, prebiotics, and symbiotics exerted different effects on the evaluated parameters. In general, Lactobacillus rhamnosus and L. casei exerted better local protective effects. Evaluation of the role of the intestinal microbiota through FMT revealed its protective effects irrespective of the previous treatment with probiotics. CONCLUSION: Probiotic strains significantly differ among themselves and exert different effects on the host's health. Symbiotics and FMT could offer additional immunomodulatory benefits to drug therapy, thus serving as a new therapeutic alternative in pediatric patients with sepsis.

Characterization and modulation of microglial phenotypes in an animal model of severe sepsis.

We aim to characterize the kinetics of early and late microglial phenotypes after systemic inflammation in an animal model of severe sepsis and the effects of minocycline on these phenotypes. Rats were subjected to CLP, and some animals were treated with minocycline (10 ug/kg) by i.c.v. administration. Animals were killed 24 hours, 5, 10 and 30 days after sepsis induction, and serum and hippocampus were collected for subsequent analyses. Real-time PCR was performed for M1 and M2 markers. TNF-α, IL-1ß, IL-6, IL-10, CCL-22 and nitrite/nitrate levels were measured. Immunofluorescence for IBA-1, CD11b and arginase was also performed. We demonstrated that early after sepsis, there was a preponderant up-regulation of M1 markers, and this was not switched to M2 phenotype markers later on. We found that up-regulation of both M1 and M2 markers co-existed up to 30 days after sepsis induction. In addition, minocycline induced a down-regulation, predominantly, of M1 markers. Our results suggest early activation of M1 microglia that is followed by an overlap of both M1 and M2 phenotypes and that the beneficial effects of minocycline on sepsis-associated brain dysfunction may be related to its effects predominantly on the M1 phenotype.

The impact of age on long-term behavioral and neurochemical parameters in an animal model of severe sepsis.

This study aimed to evaluate behavioral and neurochemical parameters in adult (180-day-old) and young (60-day-old) rats subjected to sepsis. Sepsis was induced by cecal ligation and perforation (CLP). Thirty days after surgery, behavioral tests were performed, and the ß-amyloid content, oxidative damage, and cytokine levels were measured in the hippocampus and prefrontal cortex. In both adult and young rats, sepsis impaired the inhibitory avoidance task performance and increased immobility time in the forced swimming test. However, the adult septic rats had a higher immobility time compared to the young rats. Both sepsis and aging induced brain inflammation and oxidative damage and increased Aß content. Sepsis along with aging had additive effects on hippocampal interleukin-1 levels and prefrontal carbonyl levels. Taken together, our results suggest that age has a minor influence on brain inflammation and behavioral alterations observed in septic rats.

Microglial Cells Depletion Increases Inflammation and Modifies Microglial Phenotypes in an Animal Model of Severe Sepsis.

Sepsis-associated encephalopathy is highly prevalent and has impact both in early and late morbidity and mortality. The mechanisms by which sepsis induces brain dysfunction include neuroinflammation, disrupted blood-brain barrier, oxidative stress, and microglial activation, but the cellular and molecular mechanisms involved in these events are not completely understood. Our objective was to determine the effects of microglial depletion in the early systemic and brain inflammatory response and its impact in phenotypes expression in an animal model of sepsis. Animals were subjected to CLP, and depletion of microglial cells was accomplished by administration of (Lipo)-encapsulated clodronate and microglial repopulation by doxycycline. Clod-lip treatment was effective in decreasing microglia density in the hippocampus of animals. Pro-inflammatory cytokines were increased in the CLP+PBS, and liposomes administration increased even further these cytokines mainly 7 days, suggesting that microglial depletion exacerbates both local and systemic inflammation. In contrast, repopulation with doxycycline was able to revert the cytokine levels in both serum and cerebral structures on day 7 and 14 after repopulation. There were no differences in the correlation between M1 and M2 markers by real-time PCR, but immunohistochemistry showed significant increase in CD11b expression in CLP+PBS with greater expression in CLP + liposomes in the hippocampus. These results suggest that the depletion of microglia during severe sepsis development could be associated with early exacerbation of brain and systemic inflammation and repopulation is able to revert this condition, once a rapid neurological recovery is noticed until 7 days after sepsis.

The protective effects of fecal microbiota transplantation in an experimental model of necrotizing enterocolitis.

BACKGROUND: Necrotizing enterocolitis (NEC) is a serious disease that affects premature neonates, causing high mortality. In the search for new options of treatment it was investigated whether fecal microbiota transplantation (FMT) decreased the inflammatory response during NEC development in experimental model. METHODS: Wistar rats were used and divided as follows: naïve, control (NEC induction), FMT-before (transplantation of microbiota before insult) and FMT-after (microbiota transplantation after insult). The microbiota transplantation was performed by administering a feces solution obtained from an adult donor rat. The induction of enterocolitis involves feeding by artificial formula, hypothermia, hypoxia and endotoxin administration. MPO activity, TNF-α, IL-1ß and IL-6 levels, oxidative and nitrosative damage and the grade of intestinal mucosa lesion were analyzed. RESULTS: The control group had a significant increase of inflammatory and oxidative parameters when compared to naive animals. Both FMT-before and after decreased all inflammatory and oxidative damage parameters when compared to control group. This was also true to the intestinal mucosa damage. CONCLUSION: FMT administered just before or after NEC induction improved gut and systemic inflammation, and gut oxidative damage and intestinal injury.

Anxious phenotype potentiates damage in peripheral organs of animals submitted to sepsis / Fenótipo ansioso potencializa danos em órgãos periféricos de animais submetidos à sepse

Introduction: Anxiety and sepsis are important public health problems that present high morbidity, mortality and significant economic repercussions. The present study investigated the presence of oxidative damage in peripheral organs in two lines of animals that are bred for high and low freezing responses to contextual cues that are previously associated with foot shock (Carioca High-conditioned Freezing [CHF] and Carioca Low-conditioned Freezing [CLF]) associated to sepsis. Methods: Animals were subject to sepsis by the cecal ligation and perforation (CLP) or sham operated. 24 hours and 10 days after sepsis animals were euthanized and removed adrenal, kidney, lung, serum, heart for the determination of carbonyl protein levels and adrenal for check weight this structure. Results: Sepsis increased oxidative damage in different systemic organs, included serum. There wasn't a significant increase in protein carbonyls in heart and kidney. Anxious phenotype potentiates this damage. Conclusion: These findings suggest that an anxious phenotype plus sepsis may induce more pronounced organs damage, and promote more alterations in the HPA axis. These findings may help to explain, at least in part, the common point of the mechanisms involved with the pathophysiology of sepsis and anxiety.

Introdução: Ansiedade e sepse são importantes problemas de saúde pública que apresentam alta morbidade, mortalidade e repercussões econômicas significativas. O presente estudo investigou a presença de dano oxidativo em órgãos periféricos em duas linhagens de animais criados para respostas de alta (CHF) e baixa (CLF) ansiedade associado a sepse. Métodos: Os animais foram submetidos a sham (controle) ou sepse por ligação e perfuração cecal (CLP). 24 horas e 10 dias após a sepse os animais foram eutanasiados e estruturas foram removidas: adrenal, rim, pulmão, soro e coração para a determinação dos níveis de proteínas carboniladas e adrenal para verificação do peso dessa estrutura. Resultados: A sepse aumentou o dano oxidativo em diferentes órgãos sistêmicos, incluindo o soro. Não houve um aumento significativo de proteínas carbonilas no coração e nos rins. Fenótipo ansioso potencializa esse dano. Conclusão: Esses achados sugerem que um fenótipo ansioso associado a sepse pode induzir dano mais pronunciado aos órgãos e promover mais alterações no eixo HPA. Esses achados podem ajudar a explicar, pelo menos em parte, o ponto comum dos mecanismos envolvidos na fisiopatologia da sepse e da ansiedade.

The additive effect of aging on sepsis-induced cognitive impairment and neuroinflammation.

Systemic inflammation is emerging as a significant driver of cognitive decline in the aged and vulnerable brain. In sepsis survivors animals low-grade brain inflammation occurs, suggesting that sepsis is able to induce in microglia a primed-like state. The purpose of this study is to analyze the role of sepsis-induced brain inflammation in the progression of the physiological process of brain aging. Wistar rats 2month-old were subjected to sepsis and 60 and 90days after were submitted to the new object recognition test and brain was removed to the determination of cytokines, myeloperoxidase (MPO) activity, amyloid-beta peptide (Aß) and immunohistochemistry markers of microglial activation. In the hippocampus, from 60 to 90days there was an increase in TNF-α and IL-1ß levels in septic animals. This also occurred to the levels of IL-1ß and IL-6 in the prefrontal cortex. This was associated with persistent increased in microglial activation and Aß levels. In conclusion, neuroinflammation is persistent after sepsis and this could burst the usual inflammation that occurs during brain aging.

Resveratrol and fish oil reduce catecholamine-induced mortality in obese rats: role of oxidative stress in the myocardium and aorta.

The exact mechanisms of the relationship between obesity and cardiovascular events are not yet fully understood; however, oxidative stress may be involved. Thus, the aim of the present study was to evaluate the effects of resveratrol and fish oil on catecholamine-induced mortality in obese rats. To begin with, rats were divided into five groups: (1) lean, (2) obese, (3) obese supplemented with resveratrol, (4) obese supplemented with fish oil and (5) obese supplemented with resveratrol and fish oil (n 18 rats per group), for 2 months. After supplementation, the groups were subdivided as with (n 10) and without (n 8) cardiovascular catecholaminergic stress after isoproterenol (60 mg/kg) injection. At 24 h later, the survival rate was analysed. The obese group showed lower survival rates (10 %) when compared with the lean group (70 %). On the other hand, resveratrol (50 %) and fish oil (40 %) increased the survival rate of obese rats (χ(2) test, P= 0·019). Biochemical analyses of the myocardium and aorta revealed that obese rats had higher levels of superoxide and oxidative damage to lipids and protein. This was associated with reduced superoxide dismutase and glutathione peroxidase activity in both the myocardium and aorta. The supplementation increased antioxidant enzyme activities and reduced oxidative damage. We also evaluated the nuclear factor-erythroid 2 p45-related factor 2 (Nrf2)/Kelch-like ECH-associated protein 1 antioxidant pathway. Nrf2 protein levels that were reduced in obese rats were increased by the antioxidant treatment. Taken together, these results showed that resveratrol and fish oil reduce catecholamine-induced mortality in obese rats, partly through the reduction of oxidative stress.