Small RNAs in the pathogenesis of preeclampsia.

Preeclampsia is a major contributor to maternal and fetal morbidity and mortality. The disorder can be classified into early- and late-onset subtypes, both of which evolve in two stages. The first stage comprises the development of pre-clinical, utero-placental malperfusion. Early and late utero-placental malperfusion have different causes and time courses. Early-onset preeclampsia (20 % of cases) is driven by dysfunctional placentation in the first half of pregnancy. In late-onset preeclampsia (80 % of cases), malperfusion is a consequence of placental compression within the confines of a limited uterine cavity. In both subtypes, the malperfused placenta releases stress signals into the maternal circulation. These stress signals trigger onset of the clinical syndrome (the second stage). Small RNA molecules, which are implicated in cellular stress responses in general, may be involved at different stages. Micro RNAs contribute to abnormal trophoblast invasion, immune dysregulation, angiogenic imbalance, and syncytiotrophoblast-derived extracellular vesicle signalling in preeclampsia. Transfer RNA fragments are placental signals known to be specifically involved in cell stress responses. Disorder-specific differences in small nucleolar RNAs and piwi-interacting RNAs have also been reported. Here, we summarise key small RNA advances in preeclampsia pathogenesis. We propose that existing small RNA classifications are unhelpful and that non-biased assessment of RNA expression, incorporation of non-annotated molecules and consideration of chemical modifications to RNAs may be important in elucidating preeclampsia pathogenesis.

Split Luciferase Molecular Tension Sensors for Bioluminescent Readout of Mechanical Forces in Biological Systems.

The ability of proteins to sense and transmit mechanical forces underlies many biological processes, but characterizing these forces in biological systems remains a challenge. Existing genetically encoded force sensors typically rely on fluorescence or bioluminescence resonance energy transfer (FRET or BRET) to visualize tension. However, these force sensing modules are relatively large, and interpreting measurements requires specialized image analysis and careful control experiments. Here, we report a compact molecular tension sensor that generates a bioluminescent signal in response to tension. This sensor (termed PILATeS) makes use of the split NanoLuc luciferase and consists of the H. sapiens titin I10 domain with the insertion of a 10-15 amino acid tag derived from the C-terminal ß-strand of NanoLuc. Mechanical load across PILATeS mediates exposure of this tag to recruit the complementary split NanoLuc fragment, resulting in force-dependent bioluminescence. We demonstrate the ability of PILATeS to report biologically meaningful forces by visualizing forces at the interface between integrins and extracellular matrix substrates. We further use PILATeS as a genetically encoded sensor of tension experienced by the mechanosensing protein vinculin. We anticipate that PILATeS will provide an accessible means of visualizing molecular-scale forces in biological systems.

Leveraging existing mid-end ultrasound machine for point-of-care intestinal ultrasound in low-resource settings: Prospective, real-world impact on clinical decision-making.

BACKGROUND: Point-of-care ultrasound (POCUS) has transformed inflammatory bowel disease (IBD) management, but the cost to purchase high-end equipment can be prohibitive. AIM: To assess prospectively the feasibility of POCUS using pre-existing mid-end ultrasound equipment without incurring additional cost. METHODS: Consecutive IBD patients underwent POCUS with or without faecal calprotectin (FCP) using a mid-end ultrasound machine. If POCUS with or without FCP could not guide management, we performed additional ileocolonoscopy or cross-sectional imaging. We evaluated the impact of POCUS on IBD management and its correlation with ileocolonoscopy or cross-sectional imaging. We analysed pregnant, paediatric and post-operative patients separately. RESULTS: Among 508 patients with IBD, we analysed 419 (60.4% Crohn's disease [CD]; 61.3% male, age [years]: 36 [18-78]) undergoing 556 POCUS sessions. POCUS with or without FCP independently influenced clinical management in 42.8% of patients with CD and 49.7% with ulcerative colitis (UC). POCUS helped avoid colonoscopy in 51.4% of patients with CD and 51.8% with UC, and cross-sectional imaging in 38.1% of suspected active small bowel CD. In patients with additional diagnostics, POCUS-based decisions remained unchanged in 81.2% with CD and 85% with UC. Sensitivity and specificity of POCUS compared to ileocolonoscopy were 80% and 94.4% for CD and 80.8% and 92.8% for UC, respectively. Sensitivity and specificity compared to cross-sectional imaging were 87.2% and 87.5%, respectively. CONCLUSION: POCUS using existing mid-end ultrasound equipment in low-resource settings influenced IBD clinical decision-making with excellent accuracy, often avoiding colonoscopy and cross-sectional imaging.

Active learning streamlines development of high performance catalysts for higher alcohol synthesis.

Developing efficient catalysts for syngas-based higher alcohol synthesis (HAS) remains a formidable research challenge. The chain growth and CO insertion requirements demand multicomponent materials, whose complex reaction dynamics and extensive chemical space defy catalyst design norms. We present an alternative strategy by integrating active learning into experimental workflows, exemplified via the FeCoCuZr catalyst family. Our data-aided framework streamlines navigation of the extensive composition and reaction condition space in 86 experiments, offering >90% reduction in environmental footprint and costs over traditional programs. It identifies the Fe65Co19Cu5Zr11 catalyst with optimized reaction conditions to attain higher alcohol productivities of 1.1 gHA h-1 gcat-1 under stable operation for 150 h on stream, a 5-fold improvement over typically reported yields. Characterization reveals catalytic properties linked to superior activities despite moderate higher alcohol selectivities. To better reflect catalyst demands, we devise multi-objective optimization to maximize higher alcohol productivity while minimizing undesired CO2 and CH4 selectivities. An intrinsic trade-off between these metrics is uncovered, identifying Pareto-optimal catalysts not readily discernible by human experts. Finally, based on feature-importance analysis, we formulate data-informed guidelines to develop performance-specific FeCoCuZr systems. This approach goes beyond existing HAS catalyst design strategies, is adaptable to broader catalytic transformations, and fosters laboratory sustainability.

Sunlight-mediated removal of endocrine disruptors from wastewater using trimetallic core-shell Ag-TeO2@ZnO nanocomposites.

The current work emphasizes the preparation of trimetallic core-shell Ag-TeO2@ZnO nanocomposites (NCs) by thermo-mechanical method for the efficient photocatalytic degradation of 2,4-Dichlorophenol and ß-naphthol pollutants. FE-SEM shows that Ag and TeO2nanoparticles are deposited on the surface of ZnO nanotubes. The band gap of pristine ZnO NPs and 5 wt% Ag-TeO2@ZnO nanocomposites are found to be 3.16 and 2.96 eV, respectively. The calculated specific surface area (SBET) of pristine ZnO NPs and 5 wt% Ag-TeO2@ZnO nanocomposites are 40.47 and 45.66 m2g-1respectively, confirming that Ag and TeO2nanoparticles contribute to increasing in surface area of pure ZnO. The synthesised nanocomposite showed excellent photocatalytic performance for the degradation of ß -naphthol (95.6%) in 40 min at the concentration of (0.6 mg ml-1) and 2,4-DCP (99.6%) in 180 min (0.4 mg ml-1) under natural sunlight. Cyclic Voltammetry and Electrochemical Impedance Spectroscopy were carried out to study the electrochemical properties. The determination of reactive oxygen species (ROS) confirmed that the degradation of the pollutants by 5 wt% Ag-TeO2@ZnO NCs was due to the formation of superoxide radicals. Electron paramagnetic resonance revealed the presence of sharp signals in pure ZnO nanoparticles at g ∼1.95 and oxygen vacancy peak at g ∼2.01 in 5 wt% Ag-TeO2@ZnO NCs. To study the mechanism behind the degradation of pollutants, Scavenger test using histidine and ascorbic acid (ROS scavengers) was performed. The synthesised nanocomposites are highly stable and showed enhanced efficiency up to three cycles, confirming their reusability as a photocatalyst.

Urine output is an early and strong predictor of acute kidney injury and associated mortality: a systematic literature review of 50 clinical studies.

BACKGROUND: Although the present diagnosis of acute kidney injury (AKI) involves measurement of acute increases in serum creatinine (SC) and reduced urine output (UO), measurement of UO is underutilized for diagnosis of AKI in clinical practice. The purpose of this investigation was to conduct a systematic literature review of published studies that evaluate both UO and SC in the detection of AKI to better understand incidence, healthcare resource use, and mortality in relation to these diagnostic measures and how these outcomes may vary by population subtype. METHODS: The systematic literature review was performed following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) checklist. Data were extracted from comparative studies focused on the diagnostic accuracy of UO and SC, relevant clinical outcomes, and resource usage. Quality and validity were assessed using the National Institute for Health and Care Excellence (NICE) single technology appraisal quality checklist for randomized controlled trials and the Newcastle-Ottawa Quality Assessment Scale for observational studies. RESULTS: A total of 1729 publications were screened, with 50 studies eligible for inclusion. A majority of studies (76%) used the Kidney Disease: Improving Global Outcomes (KDIGO) criteria to classify AKI and focused on the comparison of UO alone versus SC alone, while few studies analyzed a diagnosis of AKI based on the presence of both UO and SC, or the presence of at least one of UO or SC indicators. Of the included studies, 33% analyzed patients treated for cardiovascular diseases and 30% analyzed patients treated in a general intensive care unit. The use of UO criteria was more often associated with increased incidence of AKI (36%), than was the application of SC criteria (21%), which was consistent across the subgroup analyses performed. Furthermore, the use of UO criteria was associated with an earlier diagnosis of AKI (2.4-46.0 h). Both diagnostic modalities accurately predicted risk of AKI-related mortality. CONCLUSIONS: Evidence suggests that the inclusion of UO criteria provides substantial diagnostic and prognostic value to the detection of AKI.

Long-term impact of COVID-19 hospitalisation among individuals with pre-existing airway diseases in the UK: a multicentre, longitudinal cohort study - PHOSP-COVID.

Background: The long-term outcomes of COVID-19 hospitalisation in individuals with pre-existing airway diseases are unknown. Methods: Adult participants hospitalised for confirmed or clinically suspected COVID-19 and discharged between 5 March 2020 and 31 March 2021 were recruited to the Post-hospitalisation COVID-19 (PHOSP-COVID) study. Participants attended research visits at 5 months and 1 year post discharge. Clinical characteristics, perceived recovery, burden of symptoms and health-related quality of life (HRQoL) of individuals with pre-existing airway disease (i.e., asthma, COPD or bronchiectasis) were compared to the non-airways group. Results: A total of 615 out of 2697 (22.8%) participants had a history of pre-existing airway diseases (72.0% diagnosed with asthma, 22.9% COPD and 5.1% bronchiectasis). At 1 year, the airways group participants were less likely to feel fully recovered (20.4% versus 33.2%, p<0.001), had higher burden of anxiety (29.1% versus 22.0%, p=0.002), depression (31.2% versus 24.7%, p=0.006), higher percentage of impaired mobility using short physical performance battery ≤10 (57.4% versus 45.2%, p<0.001) and 27% had a new disability (assessed by the Washington Group Short Set on Functioning) versus 16.6%, p=0.014. HRQoL assessed using EQ-5D-5L Utility Index was lower in the airways group (mean±SD 0.64±0.27 versus 0.73±0.25, p<0.001). Burden of breathlessness, fatigue and cough measured using a study-specific tool was higher in the airways group. Conclusion: Individuals with pre-existing airway diseases hospitalised due to COVID-19 were less likely to feel fully recovered, had lower physiological performance measurements, more burden of symptoms and reduced HRQoL up to 1 year post-hospital discharge.

Serum autotaxin level: a promising diagnostic biomarker in differentiating Graves' disease and thyroiditis.

BACKGROUND: Recent studies have suggested that serum autotaxin (ATX) may be a promising diagnostic biomarker in differentiating between Graves' disease (GD) and thyroiditis, as well as serving as a monitoring biomarker for GD. This study will evaluate the use of serum ATX as a diagnostic biomarker in these conditions. METHODS: In this prospective interventional study, blood samples were collected from the patients who met both inclusion and exclusion criteria, and serum ATX levels were measured by using the MyBioSource human Autotaxin ELISA kit. RESULTS: A total of 32 patients were enrolled, of which 18.8% were newly diagnosed with GD, 21.9% were thyroiditis, and 59.3% were on treatment for GD. Serum autotaxin antigen was significantly higher in GD patients than in thyroiditis (603.3217 ± 444.24 v/s 214.74 ± 55.91, P = <.005). Serum ATX measurement successfully discriminated GD patients from thyroiditis (AUC = 0.952, 95%CI: 0.00-1.00) with an optimal cutoff value of ≥257.20 ng/L (sensitivity = 100 and specificity = 81.71). Monitoring the efficacy of serum ATX was analyzed and showed a significant difference. CONCLUSION: The serum ATX was higher in subjects with GD as compared to thyroiditis, and ATX levels were found to be decreased during the treatment period. In conclusion, serum ATX can be used as a diagnostic and monitoring biomarker in GD.

Facilitators and Barriers for Private Health Sector Engagement for TB Care in India: A Systematic Review and Meta-Synthesis of Qualitative Research.

INTRODUCTION: Private sector engagement is recognized as one of the most critical interventions to achieve the End TB goals in India. We conducted a systematic review and a meta-synthesis of qualitative studies to identify the barriers and facilitators for private sector engagement in TB care in India. METHODS: A systematic search in electronic databases was done. We assessed the methodological limitations of individual studies, synthesized the evidence using thematic analysis, and assessed our confidence in each finding. RESULTS: Of the 19 eligible articles included for the qualitative synthesis, 31.5% (6/19) were conducted in northern states of India. Included studies had details from 31 focus group discussions and 303 in-depth interviews conducted among various stakeholders. The synthesis revealed that barriers to engaging the private sector were lack of coordination mechanisms, lack of the National TB Elimination Program (NTEP) staff capacity to deal with the private sector, lack of private practitioners' knowledge on various programmatic aspects, and perceived complexity of the data exchange mechanism. The private sector felt that NTEP was not sensitive to the patient's confidentiality and demanded too much patient data. The private sector considered nonfinancial incentives like recognition, feedback, involving them in planning, and giving them equal status in partnership as powerful enablers for their engagement in TB care. CONCLUSION: Factors related to the context in which the engagement occurs, the architecture of the engagement, and interaction among the actors contribute to barriers to engaging the private sector for TB care in India. Strengthening policies to protect patient confidentiality, using behavior change communication to NTEP program managers, providing managerial and soft-skill training to NTEP staff, promoting nonfinancial incentives to private providers, establishing a coordination mechanism between the sectors, and simplifying the data exchange mechanisms need to be done to further strengthen the private-sector engagement.

Prognostic implications of immunohistochemistry in patients with endometrial cancer.

Various histological cell types, high histological grade, extensive myometrial invasion, and the presence of lymphovascular involvement are recognized as risk factors for disease development. Individuals carrying mutations in MutL homolog 1 (MLH1), MutS homolog 2 (MSH2), MutS homolog 6 (MSH6), or postmeiotic segregation increased 2 (PMS2) genes face an increased susceptibility to both endometrial and colorectal malignancies, with a lifetime risk ranging from 40% to 60%. This research aimed to investigate the prevalence of specific immunohistochemical (IHC) markers and microsatellite instability in endometrial carcinomas and explore potential associations with patient characteristics and clinical outcomes. Out of 58 patients with comprehensive follow-up data, a subgroup of 21 cases underwent rigorous IHC evaluation, involving estrogen receptor (ER), progesterone receptor (PR), Ki67, MLH1, MSH2, MSH6, PMS2, and p53 markers. Statistical analysis, employing the χ² (chi-squared) test, was conducted to assess the connection between individual IHC markers and clinical outcomes, with particular emphasis on the influence of radiation, chemotherapy, or brachytherapy treatment, as well as the occurrence of recurrence or mortality. Notably, significant correlations were observed in cases where MSH2 and MSH6 exhibited positive results, indicating their association with the use of chemotherapy and brachytherapy. However, the analysis pertaining to International Federation of Gynecology and Obstetrics (FIGO) stage or tumor grade did not reveal any statistically significant relationships with these parameters.

Fluid accumulation syndrome in sepsis and septic shock: pathophysiology, relevance and treatment-a comprehensive review.

In this review, we aimed to comprehensively summarize current literature on pathophysiology, relevance, diagnosis and treatment of fluid accumulation in patients with sepsis/septic shock. Fluid accumulation syndrome (FAS) is defined as fluid accumulation (any degree, expressed as percentage from baseline body weight) with new onset organ-failure. Over the years, many studies have described the negative impact of FAS on clinically relevant outcomes. While the relationship between FAS and ICU outcomes is well described, uncertainty exists regarding its diagnosis, monitoring and treatment. A stepwise approach is suggested to prevent and treat FAS in patients with septic shock, including minimizing fluid intake (e.g., by limiting intravenous fluid administration and employing de-escalation whenever possible), limiting sodium and chloride administration, and maximizing fluid output (e.g., with diuretics, or renal replacement therapy). Current literature implies the need for a multi-tier, multi-modal approach to de-resuscitation, combining a restrictive fluid management regime with a standardized early active de-resuscitation, maintenance fluid reduction (avoiding fluid creep) and potentially using physical measures such as compression stockings.Trial registration: Not applicable.

Novel Characterization of Socioecological Determinants of Health in Rural Alabama.

INTRODUCTION: Socioecological determinants of health (SEDOHs) influence disparities in surgical outcomes. However, SEDOHs are challenging to measure, limiting our ability to address disparities. Using a validated survey (SEDOH-88), we assessed SEDOHs in three rural communities in Alabama. We hypothesized that SEDOHs would vary significantly across sites but measuring them would be acceptable and feasible. MATERIALS AND METHODS: This was a retrospective review of a prospectively maintained database involving surgical patients who completed the SEDOH-88 and a secondary survey assessing it's acceptability or feasibility from August 2021 to July 2023. Included patients underwent endoscopic, minimally invasive, or open surgery at three rural hospitals: Demopolis (DM), Alexander City (AC), and Greenville (GV). RESULTS: The 107 participants comprised 48 (44.9%) from DM, 27 (25.2%) from AC, and 32 (29.9%) from GV, respectively. The median age was 64 y, and 65.6% were female. When comparing DM to AC and GV by individual factors, DM had the largest Black population (78.7 versus 22.2 versus 48.3%, P < 0.001) and more often required help reading hospital materials (20.5 versus 3.7 versus 10.3%, P = 0.007). When comparing DM to AC and GV by structural and environmental factors, DM had more Medicaid enrollees (27.3 versus 3.7 versus 6.9%, P = 0.033) and lacked fresh produce (18.2 versus 25.9 versus 39.3%, P = 0.033) and internet access (63.6 versus 100.0 versus 86.2%, P < 0.001). The SEDOH-88 had an overall 90.9% positive acceptability and feasibility score. CONCLUSIONS: SEDOHs varied significantly across rural communities regarding individual (race or health literacy), structural (insurance), and environmental-level factors (nutritious food or internet access). The high acceptability and feasibility of the SEDOH-88 shows it's potential utility in identifying targets for future disparity-reducing interventions.

An intranasal nanoparticle STING agonist protects against respiratory viruses in animal models.

Respiratory viral infections cause morbidity and mortality worldwide. Despite the success of vaccines, vaccination efficacy is weakened by the rapid emergence of viral variants with immunoevasive properties. The development of an off-the-shelf, effective, and safe therapy against respiratory viral infections is thus desirable. Here, we develop NanoSTING, a nanoparticle formulation of the endogenous STING agonist, 2'-3' cGAMP, to function as an immune activator and demonstrate its safety in mice and rats. A single intranasal dose of NanoSTING protects against pathogenic strains of SARS-CoV-2 (alpha and delta VOC) in hamsters. In transmission experiments, NanoSTING reduces the transmission of SARS-CoV-2 Omicron VOC to naïve hamsters. NanoSTING also protects against oseltamivir-sensitive and oseltamivir-resistant strains of influenza in mice. Mechanistically, NanoSTING upregulates locoregional interferon-dependent and interferon-independent pathways in mice, hamsters, as well as non-human primates. Our results thus implicate NanoSTING as a broad-spectrum immune activator for controlling respiratory virus infection.

Periendoscopic Care Continuum in Acute Cholangitis Caused By Common Bile Duct Stone.

Acute cholangitis (AC) is a biliary tract infection with in-hospital mortality rates reaching up to 14.7%. The underlying condition is biliary obstruction caused by benign and malignant etiologies, as well as bacteriobilia, with commom bile duct (CBD) stone being one of the most common causes. Currently, the diagnosis is validated using Tokyo Guidelines 2018 criteria. Acute cholangitis due to CBD stone should be managed in a comprehensive manner, i.e., periendoscopic care continuum, consisting of pre-endoscopic care, endoscopic management, and post-endoscopic care. Pre-endoscopic care is primarily comprised of supportive therapy, antibiotic administration, optimal timing of endoscopic retrograde cholangiopancreatography (ERCP), pre-ERCP preparation, and informed consent. Endoscopic management is biliary decompression with stone extraction facilitated via ERCP procedure. Selective biliary cannulation should be performed meticulously. Bile aspiration and minimal bile duct contrast injection should be done to minimize the worsening of biliary infection. Endoscopic biliary sphincterotomy, endoscopic papillary balloon dilatation, and/or endoscopic papillary large balloon dilatation are all safe procedures that can be used in AC. Special precautions must be undertaken in critical and severe acute cholangitis patients who may not tolerate bleeding, in whom endoscopic biliary sphincterotomy may be postponed to decrease the risk of bleeding, and biliary decompression may be only attempted without CBD stone extraction. Nasobiliary tubes and plastic biliary stents are equally effective and safe for patients who have only undergone biliary decompression. In post-endoscopic care, management of adverse events and observation of therapy response are mandatory.

Predator in proximity: how does a large carnivore respond to anthropogenic pressures at fine-scales? Implications for interface area management.

Background: Driven by habitat loss and fragmentation, large carnivores are increasingly navigating human-dominated landscapes, where their activity is restricted and their behaviour altered. This movement, however, raises significant concerns and costs for people living nearby. While intricately linked, studies often isolate human and carnivore impacts, hindering effective management efforts. Hence, in this study, we brought these two into a common framework, focusing on an interface area between the critical tiger habitat and the human-dominated multiple-use buffer area of a central Indian protected area. Methods: We employed a fine-scale camera trap survey complemented by GPS-collar movement data to understand spatio-temporal activity patterns and adjustments of tigers in response to anthropogenic pressures. We used an occupancy framework to evaluate space use, Bayesian circular GLMs to model temporal activity, and home range and step length analyses to assess the movement patterns of tigers. Further, we used predation-risk models to understand conflict patterns as a function of tiger presence and other habitat variables. Results: Despite disturbance, a high proportion of the sampled area was occupied by 17 unique tigers (ψ = 0.76; CI [0.73-0.92]). The distance to villages (ß ± SE = 0.63 ± 0.21) and the relative abundance of large-bodied wild prey (ß ± SE = 0.72 ± 0.37) emerged as key predictors of tiger space use probability, indicating a preference for wild prey by tigers, while human influences constrained their habitat utilisation. Distance to villages was also identified as the most significant predictor of the tigers' temporal activity (µ ± σ = 3.03 ± 0.06 rad) that exhibited higher nocturnality near villages. A total of 11% of tiger home ranges were within village boundaries, accompanied by faster movement in these areas (displacement 40-82% higher). Livestock depredation probability by tigers increased with proximity to villages (P = 0.002) and highway (P = 0.003). Although tiger space use probability (P = 0.056) and wild prey abundance (P = 0.134) were non-significant at the 0.05 threshold, their presence in the best-fit predation-risk model suggests their contextual relevance for understanding conflict risk. The results highlight the importance of appropriately managing livestock near human infrastructures to effectively mitigate conflict. Conclusions: Shared space of carnivores and humans requires dynamic site-specific actions grounded in evidence-based decision-making. This study emphasises the importance of concurrently addressing the intricate interactions between humans and large carnivores, particularly the latter's behavioural adaptations and role in conflict dynamics. Such an integrated approach is essential to unravel cause-effect relationships and promote effective interface management in human-dominated landscapes.

Optimization of kidney function in cardiac surgery patients with intra-abdominal hypertension: expert opinion.

Cardiac surgery-associated acute kidney injury (CSA-AKI) affects up to 42% of cardiac surgery patients. CSA-AKI is multifactorial, with low abdominal perfusion pressure often overlooked. Abdominal perfusion pressure is calculated as mean arterial pressure minus intra-abdominal pressure (IAP). IAH decreases cardiac output and compresses the renal vasculature and renal parenchyma. Recent studies have highlighted the frequent occurrence of IAH in cardiac surgery patients and have linked the role of low perfusion pressure to the occurrence of AKI. This review and expert opinion illustrate current evidence on the pathophysiology, diagnosis, and therapy of IAH and ACS in the context of AKI.

Flow Cytometry Analysis of Microglial Phenotypes in the Murine Brain During Aging and Disease.

Microglia, the brain's primary resident immune cell, exists in various phenotypic states depending on intrinsic and extrinsic signaling. Distinguishing between these phenotypes can offer valuable biological insights into neurodevelopmental and neurodegenerative processes. Recent advances in single-cell transcriptomic profiling have allowed for increased granularity and better separation of distinct microglial states. While techniques such as immunofluorescence and single-cell RNA sequencing (scRNA-seq) are available to differentiate microglial phenotypes and functions, these methods present notable limitations, including challenging quantification methods, high cost, and advanced analytical techniques. This protocol addresses these limitations by presenting an optimized cell preparation procedure that prevents ex vivo activation and a flow cytometry panel to distinguish four distinct microglial states from murine brain tissue. Following cell preparation, fluorescent antibodies were applied to label 1) homeostatic, 2) disease-associated (DAM), 3) interferon response (IRM), and 4) lipid-droplet accumulating (LDAM) microglia, based on gene markers identified in previous scRNA-Seq studies. Stained cells were analyzed by flow cytometry to assess phenotypic distribution as a function of age and sex. A key advantage of this procedure is its adaptability, allowing the panel provided to be enhanced using additional markers with an appropriate cell analyzer (i.e., Cytek Aurora 5 laser spectral flow cytometer) and interrogating different brain regions or disease models. Additionally, this protocol does not require microglial cell sorting, resulting in a relatively quick and straightforward experiment. Ultimately, this protocol can compare the distribution of microglial phenotypic states between various experimental groups, such as disease state or age, with a lower cost and higher throughput than scRNA-seq. Key features • Analysis of microglial phenotypes from murine brain without the need for cell sorting, imaging, or scRNA-seq. • This protocol can distinguish between homeostatic, disease-associated (DAM), lipid-droplet accumulating (LDAM), and interferon response (IRM) microglia from any murine brain region and/or disease model of interest. • This protocol can be modified to incorporate additional markers of interest or dyes when using a cell analyzer capable of multiple color detections.