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.

Microglial senescence contributes to female-biased neuroinflammation in the aging mouse hippocampus: implications for Alzheimer's disease.

BACKGROUND: Microglia, the brain's principal immune cells, have been implicated in the pathogenesis of Alzheimer's disease (AD), a condition shown to affect more females than males. Although sex differences in microglial function and transcriptomic programming have been described across development and in disease models of AD, no studies have comprehensively identified the sex divergences that emerge in the aging mouse hippocampus. Further, existing models of AD generally develop pathology (amyloid plaques and tau tangles) early in life and fail to recapitulate the aged brain environment associated with late-onset AD. Here, we examined and compared transcriptomic and translatomic sex effects in young and old murine hippocampal microglia. METHODS: Hippocampal tissue from C57BL6/N and microglial NuTRAP mice of both sexes were collected at young (5-6 month-old [mo]) and old (22-25 mo) ages. Cell sorting and affinity purification techniques were used to isolate the microglial transcriptome and translatome for RNA-sequencing and differential expression analyses. Flow cytometry, qPCR, and imaging approaches were used to confirm the transcriptomic and translatomic findings. RESULTS: There were marginal sex differences identified in the young hippocampal microglia, with most differentially expressed genes (DEGs) restricted to the sex chromosomes. Both sex chromosomally and autosomally encoded sex differences emerged with aging. These sex DEGs identified at old age were primarily female-biased and enriched in senescent and disease-associated microglial signatures. Normalized gene expression values can be accessed through a searchable web interface ( https://neuroepigenomics.omrf.org/ ). Pathway analyses identified upstream regulators induced to a greater extent in females than in males, including inflammatory mediators IFNG, TNF, and IL1B, as well as AD-risk genes TREM2 and APP. CONCLUSIONS: These data suggest that female microglia adopt disease-associated and senescent phenotypes in the aging mouse hippocampus, even in the absence of disease pathology, to a greater extent than males. This sexually divergent microglial phenotype may explain the difference in susceptibility and disease progression in the case of AD pathology. Future studies will need to explore sex differences in microglial heterogeneity in response to AD pathology and determine how sex-specific regulators (i.e., sex chromosomal or hormonal) elicit these sex effects.

Vulnerable newborn types: analysis of subnational, population-based birth cohorts for 541 285 live births in 23 countries, 2000-2021.

OBJECTIVE: To examine prevalence of novel newborn types among 541 285 live births in 23 countries from 2000 to 2021. DESIGN: Descriptive multi-country secondary data analysis. SETTING: Subnational, population-based birth cohort studies (n = 45) in 23 low- and middle-income countries (LMICs) spanning 2000-2021. POPULATION: Liveborn infants. METHODS: Subnational, population-based studies with high-quality birth outcome data from LMICs were invited to join the Vulnerable Newborn Measurement Collaboration. We defined distinct newborn types using gestational age (preterm [PT], term [T]), birthweight for gestational age using INTERGROWTH-21st standards (small for gestational age [SGA], appropriate for gestational age [AGA] or large for gestational age [LGA]), and birthweight (low birthweight, LBW [<2500 g], nonLBW) as ten types (using all three outcomes), six types (by excluding the birthweight categorisation), and four types (by collapsing the AGA and LGA categories). We defined small types as those with at least one classification of LBW, PT or SGA. We presented study characteristics, participant characteristics, data missingness, and prevalence of newborn types by region and study. RESULTS: Among 541 285 live births, 476 939 (88.1%) had non-missing and plausible values for gestational age, birthweight and sex required to construct the newborn types. The median prevalences of ten types across studies were T+AGA+nonLBW (58.0%), T+LGA+nonLBW (3.3%), T+AGA+LBW (0.5%), T+SGA+nonLBW (14.2%), T+SGA+LBW (7.1%), PT+LGA+nonLBW (1.6%), PT+LGA+LBW (0.2%), PT+AGA+nonLBW (3.7%), PT+AGA+LBW (3.6%) and PT+SGA+LBW (1.0%). The median prevalence of small types (six types, 37.6%) varied across studies and within regions and was higher in Southern Asia (52.4%) than in Sub-Saharan Africa (34.9%). CONCLUSIONS: Further investigation is needed to describe the mortality risks associated with newborn types and understand the implications of this framework for local targeting of interventions to prevent adverse pregnancy outcomes in LMICs.

Primary cilia act as mechanosensors during bone healing around an implant.

The primary cilium is an organelle that senses cues in a cell's local environment. Some of these cues constitute molecular signals; here, we investigate the extent to which primary cilia can also sense mechanical stimuli. We used a conditional approach to delete Kif3a in pre-osteoblasts and then employed a motion device that generated a spatial distribution of strain around an intra-osseous implant positioned in the mouse tibia. We correlated interfacial strain fields with cell behaviors ranging from proliferation through all stages of osteogenic differentiation. We found that peri-implant cells in the Col1Cre;Kif3a(fl/fl) mice were unable to proliferate in response to a mechanical stimulus, failed to deposit and then orient collagen fibers to the strain fields caused by implant displacement, and failed to differentiate into bone-forming osteoblasts. Collectively, these data demonstrate that the lack of a functioning primary cilium blunts the normal response of a cell to a defined mechanical stimulus. The ability to manipulate the genetic background of peri-implant cells within the context of a whole, living tissue provides a rare opportunity to explore mechanotransduction from a multi-scale perspective.

An objective scoring system for laparoscopic nephrectomy.

BACKGROUND AND PURPOSE: The current first-line recommended modality for nephrectomy is by the laparoscopic approach. This is one of the most frequent laparoscopic interventions conducted in urology. From a skills acquisition and delivery perspective in minimally invasive urologic surgery, there is a paucity of objective scoring systems for advanced laparoscopic urologic procedures. We developed a system of direct observation with structured criteria to evaluate the surgical conduction of laparoscopic nephrectomy (LN). We tested the application and preliminary validity of the scoring system. METHODS: Sixty cases of prerecorded LN performed in four teaching hospitals were each analyzed by four mentors. Each mentor scored each case based on a 100-point scoring systemthat comprised 20 key steps for LN (each step ranging 0 to 5). Steps included port placement and safety checks in addition to the actual case. In addition, a negative marking system based on a 50-point index scoring system was deployed such that technically unsound techniques were penalized. The sum of the two resulted in the final score. The final scores independently submitted for each recorded case were analyzed and compared. The system was then used to predict the experience of a surgeon for 10 pilot cases. The cases included a mix of five fellows and five experienced laparoscopic urologic surgeons. The cases were blinded to the independent assessors. A further 20 cases involving 10 cases performed by a trainee who sufficiently completed training (as deemed by the recent award of a certificate of specialist training in urology) vs one who is not ready were reviewed. RESULTS: There was no significant difference in the scores submitted by each of the four mentors for each of the cases observed. There was a strong correlation between overall score and seniority/experience of the performing surgeon of each case; ie, it was able to predict whether an experienced surgeon or laparoscopic fellow performed the case. It was able to predict accurately between a trainee who sufficiently completed training vs one who is "not ready." CONCLUSION: The scoring system was a reliable tool for assessing the performance of LN and accurately predicts the level of experience of the surgeon. This system could be a useful supplementary tool for assessing the baseline skill and progress of trainees.

Heavy metals distribution in an Iowa suburban landscape.

This study investigated the degree to which human activities through urbanization influence heavy metal concentrations in a suburban landscape in Ankeny, IA. Residential areas from different years in nine time periods of development were identified from aerial photos. Soil cores were collected from the center of the front yard of 10 randomly selected homes. Cores were subdivided into 0- to 5-, 5- to 10-, and 10- to 20-cm increments from a composite of five cores. The soils were analyzed for organic C, pH, and total Cd, Co, Cr, Cu, Ni, Pb, and Zn. Results showed that organic C increased and pH decreased with time, and that there was a general decreasing trend in heavy metal concentrations from the pre-1939 period until 1983-1990, after which there was a sharp increase in the concentrations of most of the metals. The mean Cu concentration ranged from 21 mg kg(-1) for the pre-1939 time period of development to 14.9 mg kg(-1) for the recent period of development (2003-2005). Nickel concentrations increased significantly with depth with means of 21.3 mg kg(-1) at depth 0 to 5 cm, 22.5 mg kg(-1) at depth 5 to 10 cm, and 23.0 mg kg(-1) at depth 10 to 20 cm. The concentrations of heavy metals were significantly intercorrelated, except Zn, suggesting their coexistence as mineral constituents or common contamination source. The concentrations of Cu and Pb in some locations could be due to anthropogenic inputs or higher organic matter content in soils adjacent to older homes. There appears to have been a source that caused an increase in Cd, Cr, Co, Cu, Pb, and Ni concentrations in soil adjacent to homes built between 1983 and 1990.

Newborn care: the effect of a traditional illness, asram, in Ghana.

AIMS: To explore the role of a traditional illness of the newborn, asram, in care-seeking in rural Ghana. METHODS: Data are from formative research into newborn care which included collecting qualitative data from 14 villages in Brong Ahafo region of Ghana through 25 birth narratives, 30 in-depth interviews and two focus groups with recently delivered/pregnant women, 20 in-depth interviews and six focus groups with birth attendants/grandmothers, 12 in-depth interviews and two focus groups with husbands, and six in-depth interviews with asram healers. RESULTS: The study confirmed that asram is characterised by symptoms which include green/black veins, a big head and the newborn growing lean. However, a complex classification of 14 types of asram covering a wide array of symptoms was identified. Asram was perceived as a common illness which cannot be treated at health facilities and to which many danger signs in the newborn are attributed, and thus it affects care-seeking. Asram treatment includes frequent cold herbal baths and air-drying; however, oral treatments and preventive bathing are also used. Any modification of asram treatment was reported to require the sanction of a healer. CONCLUSION: Understanding traditional illnesses as a potential barrier to newborn care-seeking is essential for designing care-seeking interventions. An asram diagnosis can prevent sick newborns being taken to health facilities and traditional treatment exposes them to the risk of hypothermia.

How did formative research inform the development of a home-based neonatal care intervention in rural Ghana?

Formative research is often used to inform intervention design, but the design process is rarely reported. This study describes how an integrated home visit intervention for newborns in Ghana was designed. As a first step in the design process, the known intervention parameters were listed, information required to refine the intervention was then identified and a formative research strategy designed. The strategy included synthesizing available data, collecting data on newborn care practices in homes and facilities, on barriers and facilitators to adopting desired behaviors and on practical issues such as whom to include in the intervention. The data were used to develop an intervention plan through workshops with national and international stakeholders and experts. The intervention plan was operationalized by district level committees. This included developing work plans, a creative brief for the materials and completing a community volunteer inventory. The intervention was then piloted and the intervention materials were finalized. The design process took over a year and was iterative. Throughout the process, literature was reviewed to identify the best practice. The intervention focuses on birth preparedness, using treated bednets in pregnancy, early and exclusive breastfeeding, thermal care, special care for small babies and prompt care seeking for newborns with danger signs. The need for a problem-solving approach was identified to help ensure behavior change. A subset of behaviors were already being performed adequately, or were the focus of other interventions, but were important to reinforce in the visits. These include attending antenatal care and care seeking for danger signs in pregnancy. On the basis of the intervention content, the timing of newborn deaths and the acceptability of visits, two antenatal and three visits in the first week of life (days 1, 3 and 7) were planned. Several household members were identified to include in the visits as they were involved in newborn care or they made financial decisions. Birth attendants and health workers were often the locus of control for immediate newborn care, and sensitization activities were designed to improve their practices and to help ensure that families received consistent messages. An existing cadre of community volunteers was identified to deliver the intervention-these volunteers were already trusted and accepted by the community, already visited pregnant women at home and had the time and commitment to deliver the intervention. A supervision and remuneration system was developed through discussions with the volunteers and at the planning workshops. The need for community entry activities was identified to garner community support for the intervention, to encourage self-identification of pregnant and delivered women and to motivate the volunteer through community recognition. Formative research is an essential step in helping to ensure the development of an effective, appropriate and sustainable intervention.

Predictors of growth from 1 to 18 months among breast-fed Ghanaian infants.

OBJECTIVE: To examine factors associated with the physical growth of breast-fed Ghanaian infants during the first 18 months of life. DESIGN: A community-based longitudinal study. SETTING: The study was carried out in Techiman, a district capital and major food trading center in the Brong Ahafo region of Ghana. SUBJECTS: One-month old infants (n=216) with birth weight >/= 2.5 kg were recruited from Maternal and Child Health Centers. METHOD: From 6 to 12 months, infants were provided with one of four types of nutritionally enhanced complementary foods. Anthropometric assessments were completed monthly from 1 to 12 months and every other month from 12 to 18 months. Information was collected on household characteristics, morbidity from common infections and dietary intakes. Blood samples were collected at 6 and 12 months to assess iron, zinc, riboflavin and vitamin A status. Multiple regression analysis was used to examine factors associated with growth during the age intervals of 1-6, 4-6, 6-12 and 12-18 months as well as size attained at 12 and 18 months. RESULTS: Prevalence of diarrhea and fever were negatively associated with growth during the first year of life. No significant relationship was found between respiratory illness (defined as cough or purulent nasal discharge) and growth. With the exception of dietary zinc intake, dietary variables were generally not significantly associated with growth. Maternal education was positively associated with growth during most of the age intervals. CONCLUSION: These findings suggest that interventions to reduce morbidity and improve the education of girls may benefit children's growth in this population. SPONSORSHIP: Nestle Foundation; Rockefeller Foundation African Dissertation Internship Award; Fulbright Scholarship. European Journal of Clinical Nutrition (2000) 54, 41-49

Predictors of micronutrient status among six- to twelve-month-old breast-fed Ghanaian infants.

This study describes the factors associated with hemoglobin and plasma ferritin, zinc and retinol concentrations and erythrocyte riboflavin status among 208 Ghanaian infants who participated in a complementary feeding intervention trial from 6 to 12 mo of age. Anthropometric, morbidity and dietary data were collected regularly from 1 to 12 mo; blood samples were collected at 6 and 12 mo. The prevalence of low micronutrient status at 6 and 12 mo, respectively, was as follows: hemoglobin <100 g/L, 30 and 34%; plasma ferritin <12 microg/L, 17 and 43%; plasma zinc <10.7 micromol/L, 4 and 6%; plasma retinol <0.7 micromol/L, 26 and 26%; erythrocyte riboflavin <200 umol/L of packed red cells, 14 and 10%. Multiple regression was used to identify factors significantly associated with micronutrient status. From 6 to 12 mo, fever prevalence was associated with a decrease in hemoglobin, but an increase in erythrocyte riboflavin concentrations, and diarrhea prevalence was related to a decrease in plasma retinol. Seasonal differences were evident for most of the indicators of micronutrient status, and elevated C-reactive protein levels (indicative of recent infection) were related to lower hemoglobin, retinol and zinc concentrations but higher ferritin and erythrocyte riboflavin concentrations. Weight at birth or at 1 mo of age was positively related to iron, zinc and vitamin A status, but a more rapid weight gain was associated with depletion of iron stores. Socioeconomic status was related to higher hemoglobin, riboflavin and zinc concentrations. The feeding of a micronutrient-fortified food was positively associated with plasma ferritin and retinol concentrations at 12 mo. These results suggest that prenatal factors, socioeconomic status, dietary intake and morbidity all influence infant micronutrient status, and that fortification of complementary foods is one potential avenue for preventing deficiencies.

A randomized, community-based trial of the effects of improved, centrally processed complementary foods on growth and micronutrient status of Ghanaian infants from 6 to 12 mo of age.

BACKGROUND: Koko, a fermented maize porridge used as the primary complementary food in Ghana, has been implicated in the high prevalence of child malnutrition. Weanimix, a cereal-legume blend developed by the United Nations Children's Fund and the Ghanaian government, has been promoted as an alternative. OBJECTIVE: We evaluated the effect of feeding Weanimix and 3 other locally formulated, centrally processed complementary foods on the nutritional status of 208 breast-fed infants. DESIGN: Infants were randomly assigned to receive 1 of 4 foods from 6 to 12 mo of age: Weanimix (W), Weanimix plus vitamins and minerals (WM), Weanimix plus fish powder (WF), and koko plus fish powder (KF). Dietary and anthropometric data were collected regularly. Blood was collected at 6 and 12 mo of age to assess iron, zinc, vitamin A, and riboflavin status. Before and after the intervention, cross-sectional data on the anthropometric status of infants not included in the intervention (NI; n = 464) were collected. RESULTS: There were no significant differences between intervention groups in weight or length gain or in hemoglobin, hematocrit, transferrin saturation, plasma zinc, or erythrocyte riboflavin values between 6 and 12 mo of age. From 9 to 12 mo of age, z scores were lower in NI infants than in the combined intervention groups [at 12 mo: -1.71 +/- 0.90 compared with -1.19 +/- 0.93 for weight and -1.27 +/- 1.02 compared with -0.63 +/- 0.84 for length (P < 0.001 for both), respectively]. The percentage of infants with low ferritin values increased significantly between 6 and 12 mo of age in groups W, WF, and KF but not in group WM. Change in plasma retinol between 6 and 12 mo of age was significantly greater in group WM than in the other 3 groups combined (0.14 +/- 0.3 compared with -0.04 +/- 0.3 micromol/L, P = 0. 003). CONCLUSIONS: All 4 foods improved growth relative to the NI group. Infants fed WM had better iron stores and vitamin A status than those fed nonfortified foods.