Avaliação da oscilação circadiana da microbiota intestinal cultivável em ratos Wistar macho

Relevância: O intestino de mamíferos possui populações microbianas abundantes, com grande diversidade taxonômica e que sofre influência de diversos processos no organismo. Um dos fatores que influenciam este microbioma são variações no ciclo claro-escuro. Novas publicações, utilizando metagenômica, sugerem que a luz possui uma importante função na constituição de comunidades microbianas, apresentando variações em sua abundância, de forma circadiana. Entretanto, este método molecular não distingue microorganismos viáveis de não viáveis. Estudos que avaliem esta variação utilizando métodos que quantifiquem microrganismos metabolicamente ativos (e.g, cultura microbiana) são escassos. Objetivo: Compreender a oscilação circadiana do microbioma intestinal de ratos Wistar macho através de análise de cultura microbiana de diferentes tecidos do trato gastrointestinal (i.e., ceco e reto) e fezes. Métodos: Durante um período de 24h, 3 animais foram eutanasiados, a cada 6 horas (n = 12), sendo o zeitgeber time zero (marcador de passagem de tempo; ZT0) correspondendo às 7 horas da manhã. Imediatamente após a eutanásia, foi feito a dissecção dos segmentos intestinais. Para possibilitar o plaqueamento nos meios de cultura (i.e., Brain Heart Infusion Agar/BHI, Mitis Salivarius Agar/MS, Sabouraud Agar/SA e Brucella Agar/BA), todas as amostras foram diluídas em tampão fosfato-salino (PBS). Os meios foram armazenados em condições gasosas com diferentes tensões de oxigênio/nitrogênio e em temperaturas específicas (entre 26ºC e 37ºC, dependendo do meio de cultura) para possibilitar o cultivo microbiano. Então, realizou-se a contagem de Unidades Formadoras de Colônia por ml (UFC/ml). As análises estatísticas foram feitas utilizando o teste de Kruskal-Wallis, seguido pelo teste de múltiplas comparações de Dunn. Resultados: No meio Brucella, a contagem de UFC/ml nas fezes foi significativamente maior no ZT0, seguido dos ZT6, ZT18 e ZT12 (p = 0,0156). Em relação aos meios de cultura, nas amostras do meio BHI, foram contadas mais UFC/ml em fezes (FZ) do que em ceco (CE) e mais em ceco do que em reto (RT) no ZT6 (p = 0,0321) e no ZT12 (p = 0,0036). Nos meios Sabouraud (p = 0,0214) e Mitis Salivarius (p = 0,0107), foi observado o mesmo padrão (i.e., FZ>CE>RT) no ZT0. Conclusões: Nós demonstramos que, a cada 6 horas, é possível observar variação nas contagens de UFC pelo método de cultura microbiana, abrangendo a oscilação diurna de bactérias anaeróbias metabolicamente ativas. Além desta variação nos horários avaliados, também foram apresentadas diferenças quantitativas de comunidades em diferentes tecidos. Estudos futuros devem ser realizados objetivando a complementaridade de métodos moleculares e culturas microbianas, visando um melhor entendimento mecanístico e fisiológico do microbioma intestinal. Apoio Financeiro: FIPE/HCPA, CNPq, CAPES, FAPERGS.Relevance: The intestine of mammals has abundant microbial populations, with great taxonomic diversity and which is influenced by several processes in the organism. One of the factors that influence this microbiome is variations in the light-dark cycle. New studies, using metagenomics, suggest that light plays an important role in the constitution of microbial communities, with variations in its abundance, in a circadian way. However, this molecular method does not distinguish viable from non-viable microorganisms. Studies that evaluate this variation utilizing methods to quantify metabolically active microorganisms (e.g, microbial culture) are scarce. Objective: To understand the circadian oscillation of the gut microbiome of male Wistar rats through analysis of the microbial culture of different tissues of the gastrointestinal tract (i.e., cecum and rectum) and feces. Methods: During a 24h period, three animals were euthanized, every 6 hours (n = 12), with zeitgeber time zero (time pass marker; ZT) corresponding to 7 am. Immediately after euthanasia, the intestinal segments were dissected. To enable plating in the culture media (i.e., Brain Heart Infusion Agar/BHI, Mitis Salivarius Agar/MS, Sabouraud Agar/SA, and Brucella Agar/BA), all samples were diluted in phosphate buffered saline (PBS). The media were stored in gaseous conditions with different tensions of oxygen/nitrogen and at specific temperatures (between 26ºC e 37ºC, depending on the culture medium) to enable microbial cultivation. Then, the Colony Forming Units per ml (CFU/ml) count was performed. Statistical analyzes were performed using the Kruskal-Wallis test, followed by Dunn's multiple comparison test. In the Brucella medium, the CFU/ml count in feces was significantly higher in ZT0, followed by ZT6, ZT18 and ZT12 (p = 0.0156). Results: Regarding the culture media, in the BHI samples, more CFU/ml was counted more in feces (FZ) than in cecum (CE) and more in cecum than in rectum (RT) in ZT6 (p = 0.0321) and in the ZT12 (p = 0.0036). In Sabouraud (p = 0.0214) and Mitis Salivarius (p = 0.0107), the same pattern (i.e., FZ> CE> RT) was observed in ZT0. Conclusion: We demonstrated that every 6 hours, it is possible to observe variation in CFU counts by the microbial culture method, covering the daytime oscillation of metabolically active anaerobic bacteria. In addition to this variation in the evaluated times, quantitative differences in communities in different tissues were also presented. Future studies should be carried out focusing on the complementarity of molecular methods and microbial cultures, aiming at a better mechanistic and physiological understanding of the intestinal microbiome. Financial support: FIPE/HCPA, CNPq, CAPES

Prospective Follow-up of Adolescents with and at Risk for Depression::Protocol and Methods of the Identifying Depression Early in Adolescence Risk Stratified Cohort (IDEA-RiSCo) Longitudinal Assessments

Objective: To present the protocol and methods for the prospective longitudinal assessments — including clinical and digital phenotyping approaches — of the Identifying Depression Early in Adolescence Risk Stratified Cohort (IDEA-RiSCo) study, which comprises Brazilian adolescents stratified at baseline by risk of developing depression or presence of depression. Method: Of 7,720 screened adolescents aged 14-16, we recruited 150 participants (75 boys, 75 girls) based on a composite risk score: 50 with low risk for developing depression (LR), 50 with high risk for developing depression (HR), and 50 with an active untreated major depressive episode (MDD). Three annual follow-up assessments were conducted, involving clinical measures (parent and adolescent-reported questionnaires and psychiatrist assessments), active and passive data sensing via smartphones, and neurobiological measures (neuroimaging and biological material samples). Retention rates were 96% (Wave 1), 94% (Wave 2), and 88% (Wave 3), with no significant differences by sex or group (p &gt; 0.05). Participants highlighted their familiarity with the research team and assessment process as a motivator for sustained engagement.Discussion: This protocol relied on novel aspects, such as the use of a WhatsApp bot, which is particularly pertinent for low-to-middle-income countries, and the collection of information from diverse sources in a longitudinal design, encompassing clinical data, self-reports, parental reports, GPS data, and ecological momentary assessments. The study engaged adolescents over an extensive period and demonstrated the feasibility of conducting a prospective follow-up study with a risk-enriched cohort of adolescents in a middle-income country, integrating mobile technology with traditional methodologies to enhance longitudinal data collection. <br/

Effective recommendations towards healthy routines to preserve mental health during the COVID-19 pandemic

Objective: To assess the adherence to a set of evidence-based recommendations to support mental health during the coronavirus disease 2019 (COVID-19) pandemic and its association with depressive and anxiety symptoms. Methods: A team of health workers and researchers prepared the recommendations, formatted into three volumes (1: COVID-19 prevention; 2: Healthy habits; 3: Biological clock and sleep). Participants were randomized to receive only Volume 1 (control), Volumes 1 and 2, Volumes 1 and 3, or all volumes. We used a convenience sample of Portuguese-speaking participants over age 18 years. An online survey consisting of sociodemographic and behavioral questionnaires and mental health instruments (Patient Health Questionnaire-9 [PHQ-9] and Generalized Anxiety Disorder-7 [GAD-7]) was administered. At 14 and 28 days later, participants were invited to complete follow-up surveys, which also included questions regarding adherence to the recommendations. A total of 409 participants completed the study – mostly young adult women holding university degrees. Results: The set of recommendations contained in Volumes 2 and 3 was effective in protecting mental health, as suggested by significant associations of adherence with PHQ-9 and GAD-7 scores (reflecting anxiety and depression symptoms, respectively). Conclusion: The recommendations developed in this study could be useful to prevent negative mental health effects in the context of the pandemic and beyond