Age- and sex-matched control mice consumed water only. On either withdrawal day 1 or 70, subgroups of animals were subjected a to 1-day behavioral test battery that included the light–dark box shuttle test, marble-burying test, and Porsolt forced swim test. As expected, adolescent mice consumed more alcohol than adults and females consumed more alcohol than males. However, despite binge-like levels of alcohol consumption, we detected relatively few signs of alcohol withdrawal-induced negative affect and there was no correlation between affective behavior and circulating corticosterone levels. We discuss these findings within the context of our published work, highlighting procedural differences that might account for the relatively weak effect of binge-drinking history upon anxiety and depressive-like behavior observed herein.A prior history of excessive drinking induces a negative affective state in both humans and laboratory rodents, the manifestation of which varies with the age of drinking-onset. In adolescent male mice, negative affect incubates over the course of a 30-day alcohol withdrawal period. In contrast, the negative affect exhibited by adult male mice is robust at 1 day withdrawal, but dissipates with the passage of time. As females tend to consume more alcohol than males, we aimed to explore the affective disturbances exhibited by adolescent and adult C57BL/6J mice of both sexes during more protracted alcohol withdrawal and to relate any behavioral changes observed to plasma corticosterone levels as a biochemical index of stress. Male and female, adolescent and adult, mice were subjected to 14 consecutive days of binge alcohol-drinking using a multi-bottle-choice Drinking-in-the-Dark (DID) procedure (5, 10, 20 and 40%  Binge-drinking is the most common form of alcohol abuse amongst adolescents. The National Institute of Alcohol Abuse and Alcoholism (NIAAA) cites that 90% of all underage drinkers within the United States have engaged in binge-drinking behaviors , with anExcessive alcohol experience during adolescence impinges upon neurodevelopment, particularly that within the mesocorticolimbic system regulating motivation, emotion, learning, and memory, as well as volitional control over behavior ,12. ConsGlobally, a sex difference exists with respect to the prevalence of affective disorders, with females being twice as likely as males to be diagnosed with an anxiety-related disorder, irrespective of past or concurrent drug abuse . The resHerein, we examined sex differences in the effects of withdrawal from a 2-week binge-drinking history either during adolescence or adulthood upon anxiety- and depression-like behavior in B6 mice. As in our published work ,24,35, aThis study employed a combination of male and female, adolescent  and adult (PND = 56) C57BL/6J (B6) mice obtained from The Jackson Laboratory  or bred in-house in the Psychology vivarium at the University of California, Santa Barbara (UCSB) from breeder pairs originally obtained from the Jackson Laboratory. Mouse origin was based on practical considerations at the time of testing of the final cohorts of this large-scale study  and was not considered to be a major factor in determining binge-drinking behavior, based off prior work conducted by our laboratory . At PND N = 102) were subjected to 14 consecutive days of binge- drinking using a multi-bottle-choice DID procedure that involved concurrent access to unsweetened ethanol at 5, 10, 20, and 40% (v/v) concentrations [Approximately half of the mice N = 2 were sutrations ,35. On etrations ,41,42,43Immediately after the 2-h alcohol-drinking period on the 10th drinking day, submandibular blood samples were collected from alcohol-drinking mice only. This sampling time-point was selected as the alcohol intake had stabilized, thereby providing a more accurate measure of their average daily alcohol consumption. Additionally, this time-point allowed sufficient recovery time prior to behavioral testing at the 1-day withdrawal time-point. A time-line of all the procedures employed in this study is provided in Headspace gas chromatography is the gold standard for ethanol analysis due to its effectiveness and accuracy in determining levels in various substances, including blood . BACs weTo assay circulating plasma CORT levels, submandibular blood samples (50 μL) were collected from all the mice 24 h before behavioral testing for negative affect (for baseline CORT) at approximately 10:00 h (±30 min) and trunk blood was collected immediately following behavioral testing at approximately 17:00 h (±30 min) to index stressor-induced changes in circulating CORT. For all the animals tested at 1-day withdrawal, the baseline blood samples were collected before mice were habituated to the drinking cages on the final (14th) day of drinking. All the bloods samples were collected in a blood collection tubes lined with lithium heparin  and centrifuged at 10,500 rpm at 4 °C for 20-min to obtain plasma. The extracted plasma sample was kept frozen at −80 °C until assayed. Duplicate samples were analyzed using the DetectX Corticosterone Immunoassay kit K014-H5 . CORT levels were determined following the manufacturer’s recommended instructions.In prior work, male B6 mice with a history of adolescent-onset binge-drinking exhibit no signs of negative affect when tested at one day withdrawal but exhibit robust anxiety- and depressive-like behaviors when tested at 30 days withdrawal ,24. In cTM tracking software , our dependent measures of latency to enter the light side, total time spent in the light side and total number of light entries were recorded over a 5-min period. The boxes were cleaned in-between each use with Rescue Disinfectant Veterinary Wipes . Immediately upon completion of this assay, the animals were transferred back into their home cages and transported to a distinct procedural room for marble-burying testing.The light–dark shuttle-box was used to measure anxiety-like behaviors, with decreased activity in the light-side interpreted as reflecting an anxiety-like phenotype ,48. AnimTM tracking software and the total time spent burying and the latency to start burying was recorded by a blind observer using a stopwatch. At the end of the marble-burying session, the animals were returned to their home cages and transported to a 3rd procedural room for forced swim testing.The marble-burying test is particularly sensitive to the effects of alcohol withdrawal based on our prior work ,41,42,43TM tracking software determined the latency to first immobile episode, total time spent immobile, and the number of immobile episodes. Immobility is defined as the lack of vertical or horizontal displacement of the animal’s center of gravity for at least 5-s. Upon the conclusion of this assay, animals were euthanized by rapid decapitation and trunk blood collected to index stressor-induced increases in plasma CORT.The Porsolt Forced Swim test is commonly employed assay for depressive-like behaviors and their reversal by anti-depressant treatments . Excessit-test comparisons were employed when appropriate. Based on our prior evidence for age-related differences in the effects of binge-drinking upon anxiety-related behavior of male mice, we classified all behavioral ANOVAs rendering an α = 0.05 − 0.10 as a statistical and notable trend [All statistical tests were conducted using the IBM SPSS Statistics software . All graphs were created using the GraphPad Prism software . Previous findings from our laboratory indicate an age-dependent effect of alcohol withdrawal upon anxiety ,24,35. Tle trend ,26,42,43p’s > 0.07; F = 54.48, p < 0.001), which reflected more alcohol consumption in adolescent versus adult mice  = 34.33, p < 0.001) that reflected more alcohol consumption in females versus males  = 0.01, p = 0.91). However, a significant Sex × Drinking interaction was observed for the adult-onset animals tested in early withdrawal  = 7.53, p = 0.01). Deconstruction of this interaction did not reveal any significant group differences; however, a statistical trend in the latency to enter the light side was observed for female mice in that female binge-drinkers exhibited a shorter latency to enter the light side, compared with their water-drinking counterparts (t(26) = 1.97, p = 0.06). In contrast, male binge-drinkers exhibited a longer latency to enter the light side than their water-drinking counterparts (t (25) = 1.92, p = 0.07).A Sex × Drinking ANOVA conducted on the latency to enter the light side of the shuttle-box yielded no significant interaction for the adolescent mice in early withdrawal A  = 0.01, p = 0.93). However, a significant main effect of drinking was noted for these animals  = 6.29, p = 0.02), reflecting a shorter latency to enter the light side for the adolescent binge- versus water-drinking animals. No significant Sex x Drinking interaction or main effects were confirmed for the adult mice in protracted withdrawal  = 1.23, p = 0.27).For adolescent mice in protracted withdrawal, no significant Sex × Drinking interactions were observed C  = 0.93, p = 0.34). However, a significant main effect of Drinking was observed for these animals  = 22.54, p < 0.001), which reflected more time spent in the light-side by the binge-drinkers versus the water controls. For the adult-onset mice tested in early withdrawal, the interaction exhibited a statistical trend  = 3.50, p = 0.07), which reflected more time spent in the light-side by alcohol- versus water-drinking males (t (25) = 1.95, p = 0.06). In contrast, no significant alcohol-related trend was observed for female mice tested in early withdrawal (t-test: p = 0.51).A Sex x Drinking ANOVA indicated no significant interaction for the total time spent in the light side of the shuttle-box by the adolescent-onset mice tested in early withdrawal A  = 3.17, p = 0.08). In adolescent males, binge-drinkers spent significantly more time in the light-side compared to their water-drinking controls (t (20) = 4.13, p = 0.001). In contrast, no effect of Drinking was observed for the adolescent females tested in protracted withdrawal . No significant two-way interaction or trend was observed for the adult-onset mice tested in protracted withdrawal  = 1.84, p = 0.18). However a significant main effect of Drinking was detected in the adult animals  = 8.44, p = 0.01), with adult binge-drinkers spending more time in the light side versus the adult water controls.Analyses of the total time spent in the light side for adolescent mice tested in protracted withdrawal also revealed a Sex x Drinking trend C  = 0.76, p = 0.39) or the adult-onset mice  = 0.09, p = 0.76). However, a trend for a main effect of Sex was observed for the adolescent mice tested in early withdrawal  = 2.98, p = 0.09), that reflected more light entries in female versus male mice.Examination of the total number of light entries in early withdrawal indicated no significant interaction or trends for either the adolescent-onset A  = 0.38, p = 0.54) or for the adult mice  = 0.80, p = 0.38); however a significant main effect of Drinking was noted for the adult-onset mice  = 13.65, p = 0.001) indicating that the binge-drinkers had a higher number of light entries than water-controls.For the animals in protracted withdrawal, no significant interaction or trend was observed for the adolescent mice C  = 1.58, p = 0.22). However, there was a significant main effect of Drinking  = 8.11, p = 0.01), which reflected a shorter latency to start burying in alcohol- versus water-drinking adolescents. For the adult-onset mice, the results of the two-way ANOVA indicated a statistical trend for the interaction  = 3.76, p = 0.06), which reflected a shorter latency to bury in alcohol- versus water-drinking males tested in early withdrawal (t (25) = 2.30, p = 0.03), but no alcohol-related difference in adult females. (p = 0.92).Examination of the latency to start burying for adolescent-onset mice in early withdrawal indicated no significant Sex × Drinking interaction A  = 4.38, p = 0.04). Deconstruction of this interaction along the Sex factor revealed a shorter latency to bury in alcohol- versus water-drinking females (t (16) = 5.29, p < 0.001) and males (t (20) = 4.90, p < 0.001). A two-way interaction was not observed for the adult-onset mice tested in protracted withdrawal  = 0.65, p = 0.43). However, a significant main effect of Drinking Group was apparent that reflected a shorter bury latency in binge-drinking mice versus water-drinking counterparts  = 34.76, p < 0.001).The Sex x Drinking ANOVA for the adolescent mice tested in protracted withdrawal indicated a significant interaction C  = 1.64, p = 0.21). Similarly, the results of the two-way ANOVA for the adult-onset mice in early withdrawal uncovered no significant interactions or trends were detected for the adult-onset mice tested in early withdrawal  = 0.20, p = 0.65). No observable main effects were found for either age group for this variable (p’s > 0.05).A Sex × Drinking ANOVA conducted on the time-spent burying marbles by adolescent-onset mice tested in early withdrawal indicated no significant interactions or noteworthy trends A  = 11.27, p = 0.002) and Drinking Group  = 6.43, p = 0.02), but no significant Sex × Drinking interaction  = 0.02, p = 0.88). Female adolescent-onset mice spent more time burying in protracted withdrawal than males, with binge-drinking mice spending more time burying than water controls  = 1.72, p = 0.20).In protracted withdrawal., analyses of the data for adolescent-onset mice revealed a significant main effect of both Sex  = 0.004, p = 0.95) or adult-onset  = 0.16, p = 0.69) mice in early withdrawal. Similarly, no significant interaction was detected for the adolescent-onset mice in later withdrawal  = 0.10, p = 0.75); although there was a significant main effect of Sex  = 5.10, p = 0.03), which reflected a higher percentage of marble buried in the adolescent females versus males tested during protracted withdrawal. Results for the adult-onset mice tested in later withdrawal yielded a statistical trend for a Sex × Drinking interaction  = 3.44, p = 0.07). This trend reflected more marble-burying in male binge-drinkers versus their water controls (t (19) = 3.61, p = 0.002), while no alcohol-water difference was apparent in the females tested at this time .A Sex × Drinking ANOVA indicated no significant interactions for either the adolescent-onset A  = 0.54, p = 0.47); however, there was a significant main effect of Drinking Group  = 8.06, p = 0.01) that reflected a shorter latency to float in water- versus binge-drinking adolescents. Conversely, a significant Sex x Drinking interaction was detected for the adult-onset mice tested in early withdrawal  = 5.56, p = 0.02). Deconstruction of this interaction along the Sex factor revealed a shorter latency to float in binge-drinking versus water-drinking females (t (26) = 2.38, p = 0.03). In contrast, no significant alcohol effect was noted for males .In early withdrawal, a Sex × Drinking ANOVA conducted on the latency to first float by adolescent-onset mice indicated no significant interaction A  = 0.02, p = 0.88) or adult-onset mice  = 0.04, p = 0.84). However, a significant main effect of Drinking was detected for the adult-onset mice, which reflected a longer latency to float in binge-drinking versus water controls  = 5.56, p = 0.02).In later withdrawal, no significant Sex × Drinking interaction was uncovered for either the adolescent-onset C  = 0.77, p = 0.39), but significant main effects of both Sex  = 8.61, p = 0.01) and Drinking  = 68.36, p < 0.001). Overall, binge-drinking adolescents spent less time immobile versus water-drinking controls, with female mice spending less time immobile than their male counterparts. In contrast, no significant interactions  = 0.46, p = 0.50) or main effects (p’s > 0.05) were observed for the adult-onset mice tested in early withdrawal  = 0.00, p = 1.00) or the adult-onset mice  = 0.86, p = 0.36). However, binge-drinking adult mice did spend significantly less time immobile than their water controls when tested in later withdrawal  = 6.99, p = 0.01).In protracted withdrawal, the two-way ANOVA also failed to reveal a significant interaction for either the adolescent-onset C  = 0.02, p = 0.89), but did show a significant main effect of both Sex  = 4.70, p = 0.04) and Drinking  = 25.35, p < 0.001), with binge-drinking mice exhibiting fewer immobile episodes than water-drinkers and female mice exhibiting less immobility than males. Interestingly, a notable trend for an interaction between Sex and Drinking Group was detected for the adult-onset mice tested in early withdrawal  = 3.34, p = 0.07). However, upon further analyses, no significant water-alcohol differences were observed for either female  or male mice .In line with the data for the time spent immobile A, the dathdrawal B  = 0.57, p = 0.45) or adult-onset mice  = 0.49, p = 0.49). Nevertheless, a significant main effect of Drinking Group for the adult mice showed a higher number of immobile episodes for the water-drinking mice compared the binge-drinking adult mice in protracted withdrawal  = 6.28, p = 0.02).For the animals in protracted withdrawal, the two-way ANOVA failed to determine a significant Sex × Drinking interaction for either adolescent-onset C  = 4.77, p = 0.032). Thus, the interaction was deconstructed along the Withdrawal factor to examine for Sex × Drinking interactions at each withdrawal time-point. This deconstruction revealed no significant interaction for the adolescent mice tested on Withdrawal Day 1 (p = 0.096) or 70 (p = 0.170). The significant Sex × Drinking × Withdrawal interaction was then deconstructed along the Drinking factor to examine for withdrawal-dependent changes in basal CORT. In binge-drinking mice, a significant Sex × Withdrawal interaction was observed  = 6.86, p = 0.012), which reflected a withdrawal-dependent increase in basal CORT in the adolescent-onset female binge-drinkers (t (20) = 3.08, p = 0.006), but not in the other adolescent-onset groups .Analyses of the basal CORT levels of the adolescent-onset mice indicated a significant Sex × Drinking × Withdrawal interaction (drinkers A (t (20)F = 1.79, p = 0.184).In contrast to the adolescent-onset mice, no group differences in basal CORT levels were detected in adult animals B  = 0.54, p = 0.47). However, a main effect of Sex was detected  = 65.33, p < 0.001), which reflected lower stressor-induced CORT levels in females versus males  = 0.703, p = 0.404) and resulted in no significant main effects .A Sex × Withdrawal × Drinking ANOVA indicated no significant interactions for stressor-induced CORT levels in adolescent-onset mice , we did not find any significant correlations between the average alcohol intake of the mice and either basal CORT levels  or stressor-induced increases in CORT levels on the test day . As it might be predicted that alcohol consumption would have a greater impact upon basal and stressor-induced changes in CORT during early versus later withdrawal, we deconstructed the data along the Withdrawal factor for re-analysis. However, we still failed to detect significant relationships between alcohol intake and basal or stressor-induced changes in CORT, even at 1-day withdrawal . Given the sex- and age-related differences in alcohol intake (r (64) = 0.322, p = 0.001). The remainder of the results failed to indicate any significant relationships between alcohol consumption and CORT, even when only the data from withdrawal day 1 were considered (data not shown).When all mice were considered  = 0.211, p = 0.005) and the total time spent marble-burying (r (177) = 0.25, p = 0.001). The total time spent marble-burying was also inversely correlated with stressor-induced CORT (r (177) = −0.18, p = 0.01). However, inspection of p = 0.057). When all mice were considered, no other significant correlations were detected vis-à-vis the average total alcohol intake and our behavioral measures of negative affect (data not shown).When all mice were considered, we did detect significant correlations between basal CORT levels and both the total number of light entries from the light dark box test A  = 0.28, p = 0.01), although a strong positive trend was also observed when only males were considered (r (93) = 0.20, p = 0.055). The relationship also held up when only adults were examined (r (94) = 0.28, p = 0.007; for adolescents, r (85) = 0.20, p = 0.07), arguing that the positive correlation reflected primarily the results of the adult mice. When all mice were considered, stressor-induced CORT levels were inversely related to the time spent burying (r (86) = −0.20, p = 0.07; for males, r(91) = −0.15, p = 0.15) and adolescent mice (r(85) = −0.20, p = 0.07; for adults: r(94) = −0.14, p = 0.17).We also examined how our subject factors of Sex and Age might influence the relationship between basal and stressor-induced changes in CORT and our behavioral measures of negative affect. When all mice were considered, basal CORT levels predicted the time spent burying, a relationship that held only when females were included in the analysis ( burying . While tr (49) = 0.43, p = 0.002), but was inversely related to the extent to which alcohol intake escalated over the course of the 14-day drinking period (r (49) = −0.62, p < 0.0001). Initial alcohol intake also predicted the average alcohol consumption of adolescent mice (r (46) = 0.62, p < 0.0001), but did not reliably predict the extent of escalation (r (46) = −0.17, p = 0.27). Deconstructing the data along the Sex factor did not yield any significant, sex-specific, correlations that were distinct from those observed when both male and female subjects were combined (data not shown).As clear sex- and age-related differences existed with respect to alcohol intake , we condg period B (r (49)ent mice C (r (46)calation D (r (46)N = 95–99), the average total alcohol intake was inversely correlated with the percent of marble buried , with no significant correlations detected regarding the relationship between initial alcohol intake and the escalation of alcohol intake and our three behavioral measures in this assay .Given the failure to detect many alcohol-induced changes in negative affect using omnibus ANOVA approaches, we conducted correlational analyses to determine whether or not binge-drinking history might predict the magnitude of negative affect manifested during alcohol withdrawal as an alternative statistical approach to our dataset. As the marble-burying test yielded results most consistent with our prior reports, in addition to a positive relationship between CORT levels and behavior , we condGiven the age- and sex- related differences in alcohol intake, the data were the deconstructed along these factors for re-analysis of their influence upon the relationship between our drinking measures and anxiety-like behavior in the marble-burying test. Consistent with the data from all mice mentioned above, initial alcohol intake did not predict any aspect of marble-burying behavior when the data was examined as a function of sex, age of drinking onset or withdrawal (data not shown). Curiously, when all female mice were considered, both the average alcohol intake, as well as the escalation of alcohol intake over the 14-day drinking period, predicted lower signs of anxiety-like behavior in the marble-burying test, as indicated by a positive relationship between the drinking measures and the latency to bury  and an iThe goal of the present study was to directly interrogate sex differences in the age-related effects of binge-drinking upon negative affect expressed during early and protracted (70 days) withdrawal. We expected to replicate our prior observations from male mice indicating an interaction between the age of drinking-onset and withdrawal upon negative affect ,41,42,43Adult, male, mice with a 2-week history of binge-drinking under 3- or 4-bottle-choice DID procedures exhibit robust signs of anxiety-like behavior at one day withdrawal that dissipates by 30 days withdrawal ,41,42,43Indeed, some of the results of the present sex difference study do corroborate our earlier findings from studies employing a single sex. Male, adult-onset binge-drinking mice exhibited a longer latency to enter the light-side of shuttle-box on withdrawal Day 1—an effect no longer apparent at 70 days withdrawal B. In theTo the best of our knowledge, only one other published study has attempted to examine directly for sex differences in interactions between the age of binge-drinking onset under DID procedures, alcohol withdrawal and negative affect . In thisWe were also struck by the very few age-related differences in basal anxiety-like behavior exhibited by water-drinking controls during early withdrawal in the present study as our prior work reliably detected higher behavioral indices of anxiety in adolescent versus adult males ,41,42,43Related to this, concurrent testing of male and female subjects may have also mitigated the negative affective state produced by a history of binge-drinking in adults. In support of this notion, exposure to adult female urinary pheromones during elevated plus-maze testing produces a testosterone-driven anxiolytic effect, without impacting locomotor activity in this assay . Such a


Religion (p = 0.023), information (p < 0.001) and motivation (p < 0.001) were significant predictors of attitude. School , education of father (p = 0.001), information (p = 0.026) and behavioral skill (p = 0.019) were significant predictors of practice. There is a need to focus on both school-based and community-based health education intervention to address the poor knowledge, attitude, and practice among adolescent girls for a healthier future.Addressing the gap in knowledge, attitude, and practice among adolescent girls are important as malnutrition has a negative effect on their future generation. This study aimed to determine the knowledge, attitude, and practice of adolescent girls towards reducing malnutrition in Maiduguri Metropolitan Council, Borno State, Nigeria. This was a school-based cross-sectional study conducted among 612 adolescent girls (10 to 19 years old). KoBo collect toolbox was used for the data collection between 3 June and 31 July 2019. Multivariable logistic regression was used to identify predictors of knowledge, attitude, and practice towards reducing malnutrition. The majority of respondents  had poor knowledge and attitude towards reducing malnutrition respectively, 278 (49.5%) had poor practice towards reducing malnutrition. Schooling (GGSS;  Adolescents are young individuals that are between the ages of 10 and 19 years’ old; this is a period of transition from childhood to adulthood and also a critical phase of physical growth and development . In NigeNigerian demographic and health survey 2013 reveals the prevalence of malnutrition among adolescent girl to be about 23%, similar to a study in Ibadan which showed that 23% of adolescent girls are malnourished . StudiesThe prevalence of nutritional knowledge was found to be 8.3% in Sokoto state . InadequThe use of theory helps in identifying individual features and surrounding environment, to determine characteristics, belief, and personal values associated with various health behavior that is capable of changing the attitude and behavior of an adolescent. Information, motivation, behavioral skill (MB) has three constructs that interact with each other theoretically, resulting from changing in behavior. The Information–Motivation–Behavioral Skills Model (IMB) model has been widely used and has proven to be a useful model in improving healthy practices, as shown in There are several gaps in knowledge, attitude, and practice among adolescent girls towards malnutrition, serving as barriers to adolescent health. Many countries and regions have limited information on the health needs, magnitude, and burden of malnutrition among these age groups . This isTherefore, to be able to overcome the emerging issue of malnutrition among adolescent girls, this study was conducted to determine knowledge, attitude, and practice of adolescent girls towards reducing malnutrition in Maiduguri Metropolitan Council, Borno state, Nigeria using information, motivation, behavioral skill model.The study location was Maiduguri, the Borno state capital, Nigeria, usually called Yerwa by its citizens. It is one of the 27 local government council of Borno state, situated at latitude 11.85, longitude 13.16, altitude 30 m and 325 m above sea level with a land area of about 69,435 square kilometers, operating on the West African time zone ,24. The p1 (0.47) and p2 (0.02) [It was a school-based cross-sectional study conducted between 3 June and 31 July 2019 among adolescent girls aged 10 to 19 years’ old in six secondary schools in MMC, Borno state, Nigeria. Two proportion formula for sample size by Lemeshow and Lwanga was used to determine the sample size , with 0.2 (0.02) . The calA two-stage random sampling technique was used. First stage six out of the eight secondary schools in Maiduguri Metropolitan Council (MMC) were randomly selected using a random number generator, where all schools were given equal chances of participation. The second stage, the schools had six arms, junior secondary 1–3 (JSS 1–3) for the junior set and senior secondary 1–3 (SS 1–3) for the senior set. Two classes were randomly selected in each grade level/arm, and then finally, individuals from these classes were randomly selected. Six hundred and twelve adolescent girls were randomly selected; the number of adolescent girls from the six selected schools was determined using a proportionate sampling technique. Finally, adolescent girls aged 10 to 19 years were selected.The instrument for data collection used was questionnaire through the respondent’s interview by enumerators using KoBo collect toolbox. A total of ten enumerators who were university graduate were trained for the data collection. Each interview lasted for 20 min per respondents, and the ten enumerators interviewed a total of 50 respondents per day. KoBo Toolbox used was an open and free data entry tool developed by Harvard Humanitarian initiative with support from various organizations like Brigham and Women’s Hospital, USAID . The appThe section is composed of fifteen questions to provide information on age, class, ethnicity, religion, place of residence, household size, household income, head of household, age of the father, father’s education, father’s occupation, age of mother, mother’s education, mother’s occupation, and family type.This section consists of 28 questions on malnutrition. The options were “true”, “false”, or “don’t know”. Each correct response (true) was score one (1) point, and each wrong response  or “don’t know” was scored zero (0). The total knowledge score was 28. Knowledge items were categorized into scores ≥ 50% (14 to 28) were considered good knowledge, while <50% (14) were considered poor knowledge [This section consists of 17 statement on malnutrition. Attitude statement was rated on a five (5) point Likert scale (1 to 5), that ranged from Strongly disagree “1”, disagree “2”, don’t know “3”, agree “4”, and strongly agree “5”. Mean score for attitude statement was used to categorized attitude into two categories. Scores < than the mean score was considered a negative attitude while scores > the mean scores were considered positive attitude .This section consists of two subsections, food consumption of 12 food groups and their meal frequency in seven days: Food consumption had a total score of 12 and meal frequency had a total score of 35. The total practice score was 47. The practice score was categorized into two groups. Scores below the median score were considered poor practice, while scores above the median score were considered as good practice .This section consists of 16 statements from the IMB constructs. Information on malnutrition includes seven statements. The options for information were on a five (5) point Likert scale ranging from strongly disagree to strongly agree (1 to 5). Total information score ranges from 7 to 35. Information was categorized into two groups. Respondents who scored < the mean were considered poor information on malnutrition, while those above the mean were considered as having good information on malnutrition.Motivation on malnutrition consisted of 4 statements. The options were on a five (5) point Likert scale ranging from strongly disagree to strongly agree (1 to 5). Total motivation score ranges from 4 to 20, and motivation was categorized into two-level. Respondents who scored < the median score were considered as having poor motivation on malnutrition, while above the median score were considered as having good motivation on malnutrition.Behavioral skills on malnutrition consisted of 5 statements. The options were on a five (5) point Likert scale ranging from very hard to very easy. Very hard “1”, hard “2”, don’t know “3”, easy “4”, and very easy “5”. Total behavioral skill score ranges from 5 to 25. Behavioral skills were categorized into two groups. Scores < the median were considered poor behavioral skills on malnutrition, while those above the median score were considered as having good behavioral on malnutrition.p < 0.25 in simple logistic regression were tested in multiple logistic regression. p < 0.05 was considered statistically significant. Y = α + β1X1 + β2X2 +…… βNXN, where Y is the knowledge, attitude, practice; B is the slope of the line, X is the independent variable, and a is the intercept.Data collected were transferred from the cloud storage of KoBo collect toolbox to statistical package for social sciences  software version 25 for analysis. Mean, and standard deviation was used to describe normally distributed continuous data, median and interquartile range was used to describe not normally distributed continuous data, frequencies and percentages were used to describe categorical data. Chi-square was used to test the association between each categorical independent variable with knowledge, attitude, and practice. Logistic regression was used to determine the predictors of knowledge, attitude, and practice of adolescent girls towards malnutrition. Factors with p < 0.05), RMSEA < 0.08, CFI, NFI, IFI, and TLI value close to 0.9 or greater, parsimonious fit of <5.0 indicated good fit [Spearman Rho correlation was used to analyze the association between the three IMB construct, further evaluation of IMB construct was done using structural equation model (SEM), where knowledge, attitude, and practice was explained by the three constructs . The overall model fit was examined via standard indices , normed fit index (NFI), incremental fit index (IFI), Tucker Lewis index (TLI), parsimonious fit, and root mean square error of approximation (RMSEA)). Chi-square  located in the same school compound, in Maiduguri Metropolitan Council, Borno State, Nigeria. The Cronbach’s alpha coefficient for knowledge was 0.784, attitude 0.751. practice 0.801, information 0.761, motivation 0.844, and behavioral skills 0.878 were found to be acceptable greater than 0.7 . RespondContent validity of the questionnaire was assessed by six experts , five frEthical approval was obtained from the Jawatankuasa Etika Universiti Putra Malaysia UPM UPM/TNCPI/RMC/JKEUPM/1.4.18.2 (JKEUPM). The study was registered with the Pan African Clinical Trials Registry (PACTR201905528313816). The approval letter was obtained from the ministry of education Maiduguri, Borno State, Nigeria and an official letter of cooperation from the ministry of education Maiduguri was also written to each selected school. Signed informed consent was obtained from participants, and their parents/guardian in this study since the study population were adolescent girls younger than 18 years old.A total of 612 respondents were randomly selected based on the inclusion criteria of the study. Among the eligible respondents, 562 consented and were interviewed. The response rate was 92%.According to this study, 509 (90.6%) are unaware that one of the causes of malnutrition is not eating enough food, 521 (92.7%) do not know that meat is a source of protein, 545 (97%) are not aware that lack of iron in the diet can cause anemia, 533 (94.5%) did not agree that iodine deficiency can be caused by eating or preparing foods with salt that is not iodized, and 511 (90.9%) disagree that calcium is a mineral that makes the bones strong and healthy. The median and (IQR) for knowledge score was 9 (6). Majority of respondents 459 (81.7%) had poor knowledge of malnutrition.The medium (IQR) for food consumption was 4 (2), medium (IQR) for meal frequency was 25 (9). Median IQR for practice was 29.5 (9). 278 (49.5%) of respondents had a poor practice towards reducing malnutrition as presented in The sources of information of majority of respondents were school teacher 36.1%, media 6.6%, family 26.2%, friends 13.5%, social media 1.6% (WhatsApp and Facebook), clinic 8.9%, health worker 4.1%, and health education program 3.0% as shown in p < 0.001) were significantly associated with knowledge towards reducing malnutrition. Information (p < 0.001), motivation (p < 0.001) and behavioral skills on malnutrition (p = 0.024) were significantly associated with the attitude towards reducing malnutrition. Schools (p < 0.001), monthly income (p = 0.044), education of father (p = 0.009), head of household (p = 0.035), information (p = 0.035), and behavioral skills on malnutrition (p < 0.001) were significantly associated with practice towards reducing malnutrition.The bivariate analysis shows that schools , respectively.School, age of adolescent, class, ethnicity, religion, place of residence, monthly income, and education of father with p < 0.25 were included in multivariable logistic regression for predicting attitude towards reducing malnutrition. Religion, information, and motivation on malnutrition were significant predictors of attitude towards reducing malnutrition. Respondents in Islamic religion were less likely to have good attitude towards reducing malnutrition as compared to Christians . Respondents with good information on malnutrition were more likely to have a good attitude towards reducing malnutrition as compared to those with poor information on malnutrition . Respondents with good motivation on malnutrition were more likely to have a good attitude towards reducing malnutrition as compared to those with poor motivation on malnutrition School, age of adolescent, class, ethnicity, religion, monthly income, head of household, education of father, occupation of father, age of mother, education of mother, occupation of mother, information, motivation, and behavioral skills with p < 0.25 were included in multivariable logistic regression for predicting practice towards reducing malnutrition. School, education of father, information and behavioral skills on malnutrition were significant predictors of practice towards reducing malnutrition. Respondents from GGSS, Government Girls College (GGC), Shehu garbai secondary school, bulabulin, zajeri day secondary were more likely to have good practice towards reducing malnutrition as compared to those from Yerwa . Respondents whose fathers had informal education were less likely to have good practice towards reducing malnutrition as compared to those whose fathers had no education . Respondents with good information on malnutrition were more likely to have good practice towards reducing malnutrition as compared to those with information AOR = 1.595, 95% CI: 1.058–2.404, p = 0.026). Respondents with good behavioral skills on malnutrition were more likely to have good practice towards reducing malnutrition as compared to those with poor behavioral skills AOR = 1.572, 95% CI: 1.077–2.296, p = 0.019) as presented in School, ethnicity, religion, place of residence, monthly income, head of household, age of father, education of father, age of mother, education of mother, occupation of mother, family type, information, motivation, and behavioral skill with p = 0.018), motivation was statistically associated with behavioral skills (p < 0.001), while behavioral skills were statistically associated with information (p = 0.018) and motivation (p < 0.001).IMB model shows the association between its construct. Information was statistically associated with behavioral skills (p = 0.018), knowledge (p = 0.002), and attitude (p < 0.001). Motivation had a statistically significant association with behavioral skills (p < 0.001), knowledge (p < 0.001), attitude (p < 0.001) and practice (p = 0.001). More so, behavioral skills reveal statistically significant association with information (p = 0.018), motivation (p < 0.001), attitude (p < 0.001) and practice (p < 0.001).IMB also shows statistically significant association with knowledge, attitude, and practice towards reducing malnutrition. Information had a statistically significant association with behavioral skills (p = 0.002), motivation and knowledge (p < 0.001), motivation and practice (p = 0.016), behavioral skill and practice (p < 0.001), information and attitude (p < 0.001), motivation and attitude (p = 0.004) lastly, behavioral skills and attitude (p = 0.014).Structural equation model based on the IMB construct and knowledge, attitude, and practice showed statistical association in all the paths. Information and knowledge , majority 309 (55.0%) had household size ≥9 members, majority of fathers 206 (37.2%) had tertiary education while the majority of mothers 187 (33.6%) had secondary school education, trading/business was the occupation of the majority of fathers 288 (52.7%) and mothers 232 (42.4%), while 510  had poor knowledge and attitude towards reducing malnutrition respectively, 278 (49.5%) had poor practice towards reducing malnutrition. A higher number of respondents  had poor information, motivation, and behavioral skill towards reducing malnutrition. The majority of the respondent’s source of information was the schoolteacher. The school was a predictor of knowledge towards reducing malnutrition. Religion, information, and motivation were predictors of attitude towards reducing malnutrition. School, education of father, information, and behavioral skills were predictors of practice towards reducing malnutrition.The majority of respondents in this study were in their middle adolescent stage 280 (49.8%) this is in line with a study conducted in northwest Ethiopia, 2017, Ibadan Nigeria 2014, and Southwest Nigeria 2017, who reported majority 402 (52.3%), 68 (73.1%), and 180 (71.7%) respectively of adolescent girls were in middle adolescent stage ,38. The This study revealed that the respondents have poor knowledge towards reducing malnutrition 451 80.2%) which is contrary to studies elsewhere in Southwest Ethiopia, Nairobi city, Kenya, Palestine, and Darab city, Fars province, Iran where 201 44.2%), 32.7%, 52.4%, and 38.5% of the respondents had poor knowledge, respectively ,41,42. F.2%, 32.70.2% whicAdolescent girls in this study have poor knowledge of definition, forms, causes, consequences, and preventive measures of malnutrition, micronutrient deficiencies like iron, iodine, vitamin A, and calcium. Poor knowledge towards malnutrition in this study may be influenced by the type of schools the adolescent girls attend since the study was carried out in government secondary schools where the majority of respondents were from the middle and lower-class families. Also, poor knowledge may be contributed to by the lack of exposure of the girls to varieties of nutritional health education in the schools as all attention in the northeast of Nigeria has been geared towards under-five children and pregnant and lactating mothers, overlooking the adolescents who are the future mothers.Majority of respondents 322 (57.3%) in this study had a poor attitude towards reducing malnutrition which is in line with the study conducted in Sulawesi, Indonesia where 55.5% of respondents had a negative attitude towards reducing malnutrition . The pooThis study further revealed an association between schools and knowledge of adolescent girls towards reducing malnutrition. This shows that being in school increases the chance of having good knowledge. In this study, information, motivation, and behavioral skill (IMB) on malnutrition were associated with the attitude towards reducing malnutrition. These imply that the use of IMB theory in studies might help adolescent girls in holding holistic perception of their behavior. The purpose of the IMB model recommends that well informed, motivated individuals who have the required behavioral skills among adolescents were found to be more likely to maintain healthy behavior resulting to positive health outcome ,49. TherMultivariate logistic regression analysis in this study hypothesized that school was a predictor of knowledge towards reducing malnutrition. Religion, information, and motivation were predictors of attitude towards reducing malnutrition. School, education of father, information, and behavioral skills were predictors of practice towards reducing malnutrition. Our results indicate that adolescent girls whose fathers had no education were less likely to have good practice towards reducing malnutrition, which could be associated with inaccessibility to nutrition-related information on the consequences of malnutrition.The study further examines whether the IMB model can be applied to explain the level of knowledge, attitude, and practices towards reducing malnutrition among adolescent girls. The study reveals a statistically significant association between information, motivation, behavioral skills with knowledge, attitude, and practice. This indicates that adequate and comprehensive information, motivation and behavioral skill would improve knowledge, attitude, and practice of adolescent girls. The authors hypothesized that a higher level of information, motivation and behavioral skills would be significantly associated with improved knowledge, attitude, and practice.The findings further reveal that information on its own is not sufficient to bring about the required behavioral change. Still, when combined with the appropriate motivation, it helps in equipping the individual with the necessary skills and competence to have the desired behavior for prevention and promotion purposes. Subsequently, this will lead to a good behavioral outcome. This is similar to studies conducted on adherence to medicine which reveals that information was not significant with medication adherence . StudiesThe adolescent stage is a unique point of intervention in their life cycle, that will not only address the issue of malnutrition in this phase of their life but will also tackle malnutrition beyond the 1000 day among future offspring’s. The finding from this study reveals a great lack of knowledge, attitude, and practice towards reducing malnutrition. Without active intervention, adolescent girls are at risk of malnutrition, and other associated health problem. Therefore, the findings from this study can play an essential role in designing and implementing appropriate interventions to improve the health outcome among adolescent girls. To the best of our knowledge, this is the first time the IMB model was evaluated among adolescents towards reducing malnutrition in Nigeria. Importantly, the model shows the association between the IMB constructs, which are essential to improving knowledge, attitude, and practice among adolescent girls. Adequate nutritional needs remain throughout life, enhancing the quality of life.The study population were adolescent girls from six secondary schools. This does not represent adolescent girls attending private schools, the school dropped out and those not attending any school at all in Maiduguri metropolitan council, who may be at risk of malnutrition due to inadequate knowledge, attitude, and practice towards reducing malnutrition. The use of self-reported questionnaire may also be seen as a limitation.Ignorance about malnutrition, macro and micronutrient prevailed among adolescent girls. Access to knowledge, attitude, and practice towards malnutrition was poor for adolescent girls. Increasing knowledge on malnutrition and its related consequences may improve the attitude and practice of adolescent girls towards reducing malnutrition and a healthy lifestyle. Findings from this study have identified the gaps in knowledge, attitude, and practice towards reducing malnutrition among adolescent girls. The study suggests that there is a need to provide a comprehensive and sound health education intervention on malnutrition, for the present and future health benefits of adolescent girls. When adolescent girls become healthy at this phase, they will grow to be healthy adults and in turn, have a healthy and productive offspring. Future studies to inform the effective development and implementation of a theory-based intervention for adolescent girls enrolled in schools and out of school will go a long way, leading to a healthy life as adolescent girls, adults, and also to healthier offspring that will translate into a healthier community.


Child malnutrition (undernutrition) and adult non-communicable diseases (NCDs) are major global public health problems. While convincing evidence links prenatal malnutrition with increased risk of NCDs, less is known about the long-term sequelae of malnutrition in childhood. We therefore examined evidence of associations between postnatal malnutrition, encompassing documented severe childhood malnutrition in low/middle-income countries (LMICs) or famine exposure, and later-life cardiometabolic NCDs.Our peer-reviewed search strategy focused on ‘severe childhood malnutrition’, ‘LMICs’, ‘famine’, and ‘cardiometabolic NCDs’ to identify studies in Medline, Embase, Global Health, and the Cumulative Index to Nursing and Allied Health Literature (CINAHL) databases. We synthesised results narratively and assessed study quality with the UK National Institute for Health and Care Excellence checklist.We identified 57 studies of cardiometabolic NCD outcomes in survivors of documented severe childhood malnutrition in LMICs (n=14) and historical famines (n=43). Exposure to severe malnutrition or famine in childhood was consistently associated with increased risk of cardiovascular disease (7/8 studies), hypertension (8/11), impaired glucose metabolism (15/24) and metabolic syndrome (6/6) in later life. Evidence for effects on lipid metabolism , obesity  and other outcomes was less consistent. Sex-specific differences were observed in some cohorts, with women consistently at higher risk of glucose metabolism disorders and metabolic syndrome.Severe malnutrition or famine during childhood is associated with increased risk of cardiometabolic NCDs, suggesting that developmental plasticity extends beyond prenatal life. Severe malnutrition in childhood thus has serious implications not only for acute morbidity and mortality but also for survivors’ long-term health. Heterogeneity across studies, confounding by prenatal malnutrition, and age effects in famine studies preclude firm conclusions on causality. Research to improve understanding of mechanisms linking postnatal malnutrition and NCDs is needed to inform policy and programming to improve the lifelong health of severe malnutrition survivors. Many countries face a large ‘double burden’ of malnutrition: high prevalence of child undernutrition combined with a growing epidemic of overweight/obesity and associated cardiometabolic non-communicable diseases (NCDs).Convincing evidence for the Developmental Origins of Health and Disease hypothesis links prenatal malnutrition with increased long-term NCD risk, but less is known about the effects of severe malnutrition in childhood on NCD risk.Our review identified 57 studies examining NCD outcomes among survivors of historical famines (n=43) and severe childhood malnutrition (n=14).Severe malnutrition and famine exposure in childhood were consistently associated with increased risk of cardiovascular disease, impaired glucose metabolism, and metabolic syndrome (MetS). Some sex-specific effects were observed, with famine-exposed women at higher risk of glucose metabolism disorders and MetS.Heterogeneity across studies, uncontrolled confounding by prenatal malnutrition, and inadequate statistical adjustment for age effects in some famine studies were key limitations.Preventing and treating severe malnutrition in children are not only important in their own right but also play a potentially important role in preventing NCDs. This is especially important in the context of impending global hunger related to the COVID-19 pandemic.Further research into mechanisms linking severe malnutrition in childhood with NCDs is needed to inform policy, programming, and patient management strategies that support long-term health in survivors of early-life malnutrition.Severe malnutrition (undernutrition) in childhood and adult non-communicable diseases (NCDs) are two of the world’s most urgent public health problems.4At present, efforts to address severe childhood malnutrition are focused on community-based management with ready-to-use therapeutic foods along with inpatient treatment of complicated cases to prevent short-term mortality.9 10et al in 2017, no systematic review examining evidence on NCD outcomes following severe malnutrition or famine exposure in childhood currently exists in the literature.While a narrative review examining evidence of differences in cardiometabolic risk between marasmus and kwashiorkor survivors was conducted by Boyne This review follows the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines.A peer-reviewed search strategy focused around ‘severe childhood malnutrition, ‘LMICs’, ‘famine’, and ‘cardiometabolic NCDs’ was used to identify studies in Medline, Embase, Global Health, and CINAHL databases .Exposure: exposure definitions included two closely related groups: (1) documented severe malnutrition in childhood defined according to standard classifications based on low weight-for-height, low weight-for-age (WFA), low mid-upper arm circumference (MUAC), or nutritional oedema, or (2) famine conditions defined by severe food insecurity in the local area or country setting. Although severe stunting is a form of severe malnutrition, we excluded studies that considered stunted children alone as the association between stunting and increased NCD risk has been described elsewhere.13Comparators: a comparison group unexposed to documented severe malnutrition or famine in childhood was preferred but not required.Outcomes: a range of cardiometabolic NCD outcomes  was considered if they were based on an objective clinical outcome measured at least 1 year after exposure to severe malnutrition or famine. All study designs were eligible. Grey literature and unpublished studies were excluded.Studies were screened for inclusion by a single author (KG) using a two-step process. First, potentially relevant studies were identified by screening titles and abstracts against the eligibility criteria. The full-text articles of identified studies were then reassessed to confirm their suitability for inclusion.–) few or no checklist criteria have been fulfilled and the conclusions are likely or very likely to alter.A risk-of-bias assessment at study level was conducted using the appraisal checklist for quantitative studies reporting correlations and associations from the UK National Institute for Health and Care Excellence. This 16-item checklist facilitates assessment of a study’s internal and external validity (EV) based on key aspects of study design, including characteristics of study participants, definition of independent variables, outcomes assessed, and analytical methods.Data were extracted using a standardised Microsoft Excel (2016) template that was piloted and adapted during the review process. The data extracted included: publication year, study design, study population, exposure definition, time since exposure, control group characteristics, outcomes, analytical methods, and key findings.Due to the wide range of included NCD-related outcomes, meta-analysis was impossible. Therefore, a narrative synthesis was carried out for each outcome with a focus on any differential effects of exposure between subgroups where data allowed . Effect sizes for similar outcomes were compared across studies to identify areas of agreement or inconsistency in the results. Data from studies of famine survivors and documented severe childhood malnutrition were analysed separately to account for the different nature of the exposures.Neither patients nor the public were involved in this research.The search yielded 2765 articles after removing duplicates. Three articles identified by senior authors were included for a total of 2768 articles for screening by title and abstract, which resulted in 73 articles for full-text appraisal. Another 24 articles were then excluded as they did not meet inclusion criteria. Eight eligible articles were identified from hand-searching reference lists for a total of 57 included articles .A total of 57 articles published between 1968 and 2019 were included, with 31 (54%) published between 2015 and 2019. Among these studies, 14 (25%) examined NCD outcomes among survivors of documented severe childhood malnutrition in LMICs and 43 (75%) studied famine survivors. Famine studies were predominantly retrospective cohorts , followed by prospective cohorts  and cross-sectional studies . Studies of documented severe malnutrition survivors were prospective cohorts  or case-control studies .The studies represent findings from 15 countries; however, most were conducted among survivors of the Great Chinese Famine , whereas sample sizes varied between 62 and 105 374 for famine studies . For two of the famine studies, the number of cases and controls was not reported, and so the total number of participants was taken as the sample size.20–22et al (2001), childhood WFA z-scores for all participants were analysed against NCD outcomes regardless of whether they classified as malnourished.25Various criteria were used to define severe malnutrition exposure, with admission to nutritional rehabilitation units based on WHO, Wellcome and Gomez classifications being the most common. Three studies used clinical diagnosis of marasmus or kwashiorkor without precise definitions,p-values can be found in Famine exposure was most frequently defined according to participants’ birthdates and residency in famine-affected areas during childhood. However, five studies used individual self-reports of famine exposure.10.1136/bmjgh-2020-003161.supp2Supplementary data10.1136/bmjgh-2020-003161.supp3Supplementary dataIncluded studies examined the impact of severe childhood malnutrition or famine exposure on one or more cardiometabolic NCD outcomes, including: glucose metabolism, cardiovascular disease (CVD), dyslipidaemia, non-alcoholic fatty liver disease (NAFLD), blood markers of cardiometabolic disease , physical capacity, metabolic syndrome, chronic kidney disease, epigenetic profile, telomere length, thyroid function, and anthropometry. Outcomes were assessed between 2 and 70 years post exposure in studies of documented severe malnutrition and 28–70 years post exposure in famine studies.Because many of the studies presented in Famine exposure during childhood and adolescence was associated with increased risk of CVD  in 7/8 studies from the UK, China, Russia and the Netherlands,31 33There was also consistent evidence of positive association between famine exposure and elevated blood pressure (BP) and hypertension in 8/11 studies from China, Nigeria and Russia.32 33Cardiovascular outcomes examined in studies of severe childhood malnutrition survivors largely focused on BP. These studies found mixed effects on BP outcomes, with one Jamaican study finding higher diastolic blood pressure (dBP) , but no differences in sBP, in survivors compared with controls at ~30 years, while a Mexican study found lower dBP (p=0.001) and sBP (p<0.0001) in survivors at ~20 years.25The single study that examined cardiovascular structure and function found reduced left ventricular outflow tract, stroke volume, cardiac output, and pulse wave velocity, together with increased systemic vascular resistance in survivors versus controls.43The evidence shows a positive association between childhood famine exposure and impaired glucose metabolism, with 2/15 studies finding increased risk of hyperglycaemia and 7/15 showing increased diabetes risk.31 45 46 48 49Increased risk of hyperglycaemia in famine-exposed women was found in two Chinese studies. Both found similarly increased risk as a result of early childhood famine exposure  after childhood famine exposure,31Of the remaining six studies, five reported null findings and one reported a negative relationship between famine exposure and impaired glucose metabolism in adulthood. Studies from Bangladesh, Nigeria, China and Russia found no association between childhood famine exposure and impaired glucose metabolism when outcomes were assessed between ~30 and 80 years.26The evidence indicates that severe childhood malnutrition is associated with impaired glucose metabolism in survivors, with 6/9 studies reporting a positive association with diabetes, reduced insulin sensitivity, or glucose intolerance.15 16The remaining 3/9 studies found no differences in glucose metabolism between severe malnutrition survivors and controls; however, these studies assessed outcomes in children, adolescents, and lean adults on a low-fat diet, respectively, which may have led to underestimation of the long-term effects of severe malnutrition.9 24 25Of eight studies that reported on lipid metabolism outcomes, four reported no difference in lipid profiles between famine-exposed participants and controls.56Three studies of severe malnutrition survivors examined lipid metabolism, with two finding no differences between the lipid profiles of controls and survivors at ~9 years  and ~20 years (Mexico), respectively.20All five studies of metabolic syndrome (MetS) in Chinese famine survivors showed an increased risk in participants exposed between 0 and 9 years.63Evidence on obesity-related outcomes in famine survivors showed mixed effects. Four Chinese studies reported increased BMI, obesity or overweight among those exposed to famine between 0 and 9 years and followed up in late adulthood.17 37 41In contrast with famine survivors, evidence on anthropometric outcomes in severe malnutrition survivors indicates that they remain thinner than unexposed controls through childhood to adulthood. Of six studies, five reported that survivors had lower BMI, WFA, MUAC or WAZ than controls when measured as older children or adults.20 65Results for outcomes with ≤3 studies reporting results can be found in 10.1136/bmjgh-2020-003161.supp4Supplementary dataWe found evidence to support the hypothesis that exposure to severe malnutrition or famine during childhood increases long-term risk of cardiometabolic NCDs. The evidence was strongest for an association with CVD , impaired glucose metabolism  and MetS, while evidence for effects on lipid metabolism and obesity risk was less consistent. Where increased risk of NCDs in exposed groups was reported as an OR, effect sizes ranged from 1.11 to 5.50. Overall, these results suggest that childhood malnutrition may have a clinically, as well as statistically, significant effect on NCD risk in some survivors. Sex-specific differences were observed in some cohorts, with famine-exposed women at higher risk of glucose metabolism disorders and MetS than their male counterparts.Current literature suggests that developmental plasticity extends beyond prenatal life and that severe malnutrition in childhood exerts independent effects on NCD risk. Due to heterogeneity in exposure age across studies, it is difficult to conclude whether morbidity risk is higher among children exposed at specific ages. However, it appears that the window of plasticity could remain open beyond the first 1000 days of life, which is the focus of much current child health policy and programming.et al (2017) by systematically identifying new evidence for an effect of severe malnutrition and famine exposure in childhood on NCD risk.Our results build on a narrative review of differences in cardiometabolic risk between marasmus and kwashiorkor survivors from Boyne There is little mechanistic evidence linking severe malnutrition or famine in childhood and long-term NCD risk, with most studies speculating on mechanisms or extrapolating from findings on prenatal malnutrition. Commonly proposed mechanisms include:There is strong evidence that periods of rapid postnatal weight gain increase obesity and CVD risk later in life, with observational and intervention studies showing that accelerated early growth is associated with later body fatness as well as increased BP, cholesterolaemia and insulin resistance.73–75Endocrine changes caused by malnutrition may influence body composition in adult survivors and affect their NCD risk.78 79Animal studies have shown that postnatal malnutrition negatively impacts pancreatic β-cell function.80Exposure to stressors in utero and during childhood may alter the set-point of the HPA axis as an adaptation to cope with an anticipated high-stress environment in later life.83While literature on epigenetic effects of early-life malnutrition on NCD risk largely focuses on the antenatal period, postnatal malnutrition may also cause epigenetic changes that contribute to future cardiometabolic disease risk.Women exposed to famine in childhood appear to be at higher risk of glucose metabolism disorders and MetS than famine-exposed men. This finding is supported by a recent meta-analysis examining the effect of early-life famine exposure on risk of MetS in adulthood that included 39 studies (n=81 504). Compared with a non-exposed group, early-life famine exposure significantly increased the risk of MetS in women only.90We acknowledge several limitations. Due to our broad scope, there was extensive clinical and methodological heterogeneity between studies, rendering it difficult to directly compare study findings. As all studies were observational, associations cannot be interpreted as causal. This makes it difficult to disentangle the effects of fetal and postnatal malnutrition. Few studies controlled for effects of foetal malnutrition as the proxy measure of birth weight was rarely available; however, four studies showed independent effects of postnatal malnutrition on NCD risk after controlling for birth weight.91 92Another limitation is the small sample size in some studies; thus, the children included may not represent the wider affected/at-risk population and chance observations are possible. We also note the risk of selection and information bias. Because severe childhood malnutrition is associated with high mortality, participants may represent the healthiest survivors and any effects observed in these populations may underestimate long-term health impacts of severe malnutrition.Finally, we recognise the currently limited data sources on this topic; although we identified an important number of individual papers, many were from the same famine event and some had high risk of bias. However, even when studies with higher risk of bias were discounted from our analysis, we found that most studies from the Great Chinese Famine, Dutch Hunger Winter and Siege of Leningrad (the famine events for which there were >2 available studies) showed a positive association between famine exposure and NCD outcomes. Therefore, our conclusions regarding the potential relationship between famine exposure and NCDs remain valid and all studies were included in the results so as to present a complete overview of available literature. We hope that future research in different settings might add further weight to our findings.Our review has several strengths. To our knowledge, this is the only systematic review on this topic. It examines a highly topical subject; COVID-19 threatens to trigger food crises and famines worldwide, putting great numbers of children at risk of severe malnutrition.80 81If postnatal malnutrition influences long-term NCD risk as our results suggest, this is of global public health significance given the growing NCD epidemic in LMICs where there remains a large burden of severe childhood malnutrition.With over 80% of premature NCD-related deaths occurring in LMICs, where health systems already struggle with this massive disease burden, the need for interventions to prevent NCDs among severe malnutrition survivors is urgent. Our review highlights the limited evidence relevant to LMIC contexts, where severe malnutrition remains a threat to public health. Many of the highest quality studies we identified were conducted in settings where famine events were short lived and therefore may underestimate the effects of severe malnutrition in some contemporary contexts where malnutrition is endemic. There is a need for more high-quality studies in a wider range of contemporary LMIC settings to explore the potential links between severe malnutrition and NCDs.There is reasonable strength and consistency in the findings and biological plausibility to indicate an association between severe malnutrition or famine exposure and increased NCD risk. However, the precise mechanisms underlying this association remain largely unclear. For instance, how do long-term outcomes differ by duration, intensity, and age at which a child experiences malnutrition and what are the biological processes leading to long-term adverse effects. Research in this area will be essential to inform policy and programming around prevention and management strategies for severe childhood malnutrition that promote long-term health in survivors as well as strategies to mitigate NCD risk among famine survivors. Recent predictions of major hunger following the COVID-19 pandemic make our review particularly timely.Our review indicates that severe malnutrition or famine exposure in childhood is associated with increased NCD risk later in life. The evidence on CVD, impaired glucose metabolism and MetS consistently shows deleterious effects of postnatal malnutrition on these chronic disease outcomes. Evidence for effects on lipid metabolism and obesity risk is less consistent.Given that many countries with large burdens of child malnutrition also face NCD epidemics, understanding associations between severe childhood malnutrition and chronic diseases has major implications for preventing long-term morbidity and mortality. Increased global hunger resulting from the COVID-19 pandemic makes this link more important than ever. Efforts must be made to prevent and appropriately treat child malnutrition: not only to avoid short-term mortality, but to avoid escalating an already overwhelming NCD burden in decades to come. Better evidence is required from contemporary LMIC contexts where severe malnutrition may be inflicting long-lasting damage on public health.


Leishmania infantum in naturally infected dogs.In canine visceral leishmaniasis (CVL), lymphopenia, and the disorganization of lymphoid organs such as spleen and lymph nodes have been demonstrated. However, the involvement of thymus in CVL has not been evaluated so far. Herein, we investigated whether the thymus can be colonized by Leishmania and sequenced. Samples of spleen, skin and popliteal lymph nodes were collected and submitted to immunohistochemistry and parasitological culture followed by multilocus enzyme electrophoresis.Thymus were obtained from 16 of 58 dogs and samples of this organ were submitted to immunohistochemistry for laminin and fibronectin detection, histopathology, in situ hybridization and polymerase chain reaction (PCR) targeting the gene ITS‐1 for L. infantum was identified in all dogs. DNA and amastigote forms of Leishmania were detected in the thymus from 16 dogs by PCR and in eight by immunohistochemistry. Besides thymus, parasites were detected in spleen, lymph nodes, and skin. A granulomatous or pyogranulomatous thymitis was observed in eight dogs associated to intact amastigotes forms of this parasite. Fibronectin deposition in thymus was higher in dogs with more clinical signs.L. infantum, which may generate inflammatory reactions leading to alterations in thymic microarchitecture.These results demonstrate that the thymus of dogs can be parasitized by  L. infantum. Infection generates inflammatory reactions leading to thymic alterations. Thymic extracellular matrix alterations are associated with disease worsening in canine visceral leishmaniasisThe thymus of dogs can be parasitized by  The thymus is a primary lymphoid organ that plays a role in T‐cell homeostasis maintenance, and its product can dictate the immunological profile of individuals.+ and CD8+ surface molecules. Another extremely important function of the thymus is the positive and negative selection of T cells. In this process, macrophages at the corticomedullary junction remove, by phagocytosis, T lymphocytes that recognize both self‐major histocompatibility complex molecules and self‐antigens.L. infantum in the thymus of naturally infected dogs, and to describe the inflammatory infiltrate, the microarchitecture alterations and the deposition of extracellular matrix components (laminin and fibronectin) in this organ associated with parasitism. Herein, we observed infection by L. infantum in the thymus of all studied dogs, as well as an association between thymic microarchitecture alterations and disease‐worsening.Although the association between parasitism and histological lesions has been frequently described in the spleen and lymph node of dogs infected by 22.1L. infantum‐naturally infected dogs that were destined for euthanasia as recommended by the Brazilian Ministry of Health polices. All dog owners had provided formal written consent. The samples were collected during necropsies conducted by veterinarians from the Laboratório de Pesquisa Clínica em Dermatozoonoses em Animais Domésticos (LAPCLIN‐DERMZO–INI/FIOCRUZ). This study was approved by the Ethics Committee on Animal Use of the Oswaldo Cruz Foundation  and performed according to Brazilian Law 11794/08 and the Brazilian Society of Laboratory Animal Science.The animals included in this study were 2.2L. infantum antibodies by a rapid immunochromatographic test  and by enzyme‐linked immunosorbent assay (Bio‐Manguinhos). The tests were performed by public health services responsible for the VL surveillance and control program of the state of Rio de Janeiro, with the permission of the owners.A descriptive study was conducted using a nonprobabilistic sample of dogs from Barra Mansa (22°32′25.19″S and 44°10′35.33″W), Rio de Janeiro State, Brazil, a VL endemic area.The seropositive dogs were sent by the local authorities  to be euthanized at Instituto Nacional de Infectologia Evandro Chagas (INI), Fundação Oswaldo Cruz, Brazil. Euthanasia procedure was performed according to recommendations of the Brazilian Ministry of Health for the control of VL. From the 58 animals necropsied, we were able to recover enough thymus samples from only 16 dogs, which were included in the study.2.3The animals were submitted to a physical examination that consisted of the inspection of the skin, ocular, and nasal mucosae, as well as palpation of the superficial lymph nodes and organs. The following clinical signs were considered compatible with VL: low body score, cachexia, apathy, pale mucosae, regional or generalized lymphadenomegaly, splenomegaly, hepatomegaly, skin ulcers, furfuraceous desquamation of skin, onychogryphosis, local or generalized alopecia, and keratoconjunctivitis.2.4Leishmania species identification.After physical examination, the dogs were euthanized using 1.0 mL/kg of intravenous Thiopental 1.0% (Thiopentax Cristália). After the detection of an absence of corneal reflex induced by deep anesthesia, 10 mL of intravenous potassium chloride 19.1% (Isofarma) were administered. At necropsy, thymus samples were fixed in 10% neutral formalin and processed routinely for embedding in paraffin for immunohistochemistry, in situ hybridization (ISH), histopathology, and Polymerase chain reaction PCR. Additionally, samples of spleen, skin and popliteal lymph nodes were collected aseptically, immersed in sterile saline, and submitted to parasitological culture followed by multilocus enzyme electrophoresis (MLEE) for 2.5Leishmania promastigotes, isolated from tissues, were further identified at the species level by MLEE using five enzymatic systems according to protocols previously described.The samples were seeded onto biphasic NNN/Schneider's Insect Medium  containing 10% fetal bovine serum and incubated at 26 ± 28°C2.62), the medullary area (mm2) of tissue and the cortex:medulla ratio were analyzed using the ImageJ software (NIH).The sections (5‐µm‐thick) were stained with hematoxylin and eosin and examined by light microscopy . The cortical area , and intense (more than 10 parasitized macrophages).For the evaluation of the parasite load in the thymus, macrophages parasitized with amastigote forms of 2.8After deparaffinization, rehydration, blocking of endogenous peroxidase, antigen retrieval, blockade of nonspecific protein binding , the specimens were incubated with the primary antibodies directed to laminin and fibronectin (Abcam), followed by incubation with anti‐rabbit secondary antibody (Abcam) and streptavidin‐horseradish peroxidase. Aminoethylcarbazole  was used as the substrate–chromogen system and the slides were counterstained with Mayer's hematoxylin (Sigma‐Aldrich Co). The slides were examined under a light microscope (Nikon). Laminin and fibronectin deposits were quantified using the ImageJ software (NIH) and we analyzed at least 10 pictures/microscopy field. The results were expressed as area fraction (representative of the percentage of the stained area).2.9Leishmania present in the thymus were obtained by PCR the internal transcribed spacer‐1 (ITS‐1) gene fragmentLeishmania sp but not sequenced. The primer sequences are described in Table The Phred/Phrap/Consed script and aligned with MEGA 7 software . A search using the BLAST algorithm (http://ncbi.gov) was carried out to confirm the species.After amplification, the products were purified with MinElute 96 UF PCR purification kit, Qiagen. The purified products were sequenced using the same primers with BigDye Terminator v3.1 Cycle Sequencing Kit  according to the manufacturer's specifications, in 96‐well plates using a concentration of 3.2 μM of each primer (forward and reverse) and 1.6 μL of the purified PCR product. The reactions were performed by Fiocruz sequencing platform . The obtained sequences were analyzed using 2.10Leishmania species was not identified by PCR. In these samples, ISH was used to identify amastigote forms of L. infantum by detecting a specific nucleic acid sequence of this parasite. For this purpose, an L. infantum‐specific oligonucleotide probe labeled with digoxigenin that targets the minicircle kinetoplast DNA gene of the parasiteISH was used only in thymus samples in which the 2.112) had normal distribution (P > .05) as indicated by Kolmogorov‐Smirnov test in SPSS software. The construction of the Gaussian mixture  was performed in excel and SPSS 24 software. A table with average, median, standard deviation, and maximum and minimum value of the number of amastigotes/mm2 was constructed. The normal distribution and the division into groups according to the intensity of the parasite load , and intense (more than 10 parasitized macrophages). All variables   of the 16 evaluated dogs were positive for re Table . The par3.4Leishmania amastigote forms, a granulomatous or pyogranulomatous thymitis was observed: in six dogs  and two dogs (#6 and 15), respectively. The granulomatous inflammatory infiltrate was poorly delimited and composed mainly by macrophages with few lymphocytes and plasma cells. The pyogranulomatous inflammatory infiltrate was poorly delimited and composed mainly by macrophages and neutrophils with few lymphocytes and plasma cells. The distribution of the inflammatory infiltrate was multifocal or diffuse and it affected cortical and medullar regions. In one dog (#6), the interlobular trabeculae were thickened due to fibrosis and intense pyogranulomatous inflammatory infiltrate associated with many amastigote forms of Leishmania within macrophages .In the thymus of eight dogs positive for 3.52 (mean ± SEM: 2.5 ± 0.35). We did not observe differences in cortex:medulla ratio when groups based on age, clinical signs or parasite load were compared (P > .05)     and from 1.18 to 19.20 area fraction (mean ± SEM: 9.06 ± 1.66), respectively CD8(+) cells to peripheral lymphoid organs, where they acquired an activated phenotype similar to activated effector or memory T cells. They suggested that, in this model, these cells escaped from the negative selection process, and some of them became potentially autoimmune.Leishmania infection could modify the regular functionality of thymus. In fact, lymphopenia, splenomegaly, and lymphadenopathy have been described in canine visceral leishmaniasis (CVL), as well as microarchitecture disruption of both spleen and lymph nodes.The thymic microenvironment is formed mainly by fibronectin and laminin.Leishmania and consequent tissue damage observed in the thymus, the maturation of lymphocytes could be altered in these thymic islands and might contribute to the immunopathology of secondary lymphoid organs in CVL. Although the reminiscent thymus maintains thymocyte maturation over the whole life of the animal, the effect of thymic infection in the course of leishmaniasis would be more severe in pups, since there is intense thymus activity and immunological maturation in young animals. Thus, studies focused on the thymus of young dogs would better‐clarify this hypothesis.In our study, the animals were in the adult phase and we observed involution of thymus in all of them. In dogs, the complete development of thymus occurs at 2 months of age, entering into a process of involution after 6 months of age, but maintaining its activity during the animal's life and acting as a modulator of other lymphoid organs.Leishmania persistence. In fact, we previously observed F4/80+ cells colocalizing with parasites in the thymus of L. infantum‐infected mice.Regarding the alterations observed in thymic extracellular matrix compounds, fibronectin interconnecting segment domain can be recognized by receptors expressed in macrophages favoring the invasion of parasites and reducing the activation of macrophages.The higher expression of extracellular matrix molecules such as laminin, collagen, and fibronectin in animals with more clinical signs has been demonstrated in lymphoid organs such as spleen and lymph node, and also in the liver.The lack of correlation between fibronectin expression and cortex/medulla ratio can be explained by the large variability in measurements of these areas due to the age differences of the evaluated dogs. There are several factors that may influence the thymus histological patterns: diabetes,L. infantum, which may generate inflammatory reactions leading to alterations in thymic microarchitecture. These alterations probably compromise the maturation of T lymphocytes in the thymus favoring the failure of the immune response in infected dogs.Taken together, these results demonstrate that the thymus of dogs can be parasitized by The authors declare that there are no conflict of interests.RCM, FNM, and RP conceived and designed the research; AVAS, TLS, FBF, AAVMJ, LCF, CPBF, and PC performed the experiments; AVAS, TLS, RP, RCM, and FNM prepared the figures and wrote the manuscript; AVAS, TLS, RCM, and FNM analyzed the data; AVAS, T.L.S, RP, RCM, and FNM edited the manuscript; and AVAS, TLS, FBF, AAVMJ, LCF, CPBF, PC, RP, RCM, and FNM revised and approved the final manuscript.


In vivo, cerebrospinal fluid molecules are regulating the transition of the immature fetal human brain into a mature adult brain. By culturing hiPSC-3D neural aggregates in human cerebrospinal fluid (hCSF) obtained from healthy adult individuals, we demonstrate that hCSF rapidly triggers neurogenesis, gliogenesis, synapse formation, neurite outgrowth, suppresses proliferation of residing neural stem cells, and results in the formation of synchronously active neuronal circuits in vitro within 3 days. Thus, a physiologically relevant and adult brain-like milieu triggers maturation of hiPSC-3D neural aggregates into highly functional neuronal circuits in vitro. The approach presented here opens a new avenue to identify novel physiological factors for the improvement of hiPSC neural in vitro models.Human induced pluripotent stem cell (hiPSC)-derived   •Human CSF triggers rapidly multiple maturation processes in human 3D neural models•Human CSF triggers human neurogenesis and suppresses neural stem cell proliferation•Human CSF triggers human astrocyte development, neurite growth, and synapse formation•Human CSF triggers the maturation of neurons into highly functional neuronal circuits in vitro.In this article, Izsak and colleagues show that human cerebrospinal fluid triggers rapid maturation of human iPSC-3D neural aggregates indicated by neural stem cell quiescence, enhanced neurite growth, synapse development, neuronal as well as astrocyte differentiation, and formation of highly functional neuronal circuits  Human cerebrospinal fluid (hCSF) flows within the ventricular system, central canal, and subarachnoid space, and surrounds the central nervous system. Molecules within the CSF regulate cellular processes within the embryonic and adult brain .in vitro models to obtain insights into brain development and human neuronal function. For decades, DMEM and neurobasal culture media have been used as the cell culture environment for human- and animal-derived neurons in vitro. in vitro -derived neural cells are used as in vitro . A compain vitro models  and limited neuronal maturation are common phenomena described for 2D and 3D hiPSC-derived neural o models . Applicao models . Howeverin vitro model.In the present study, we analyzed the functional and cellular impact of hCSF on hiPSC-3D neural aggregates (3D NAs) comprised of cortical neurons, glial cells, and residing proliferative neural cells . For thiWe applied the commonly used “dual-SMAD-inhibition” protocol for neural differentiation of hiPSC into cortical NSCs  Figure A. hiPSC-Immunocytochemistry and confocal imaging show that adherently growing 3D NAs consist of astroglial cells and cortical neurons C and S1.in vitro model.Since 3D NAs are comprised of highly functional neurons and astroglial cells, however retain immature properties, such as ongoing proliferation, we used adherently growing hiPSC-3D NAs to describe the influence of hCSF on a complex human 3D neural First, we studied whether neuronal network activity improved when 3D NAs were cultured in hCSF. Before application of individual hCSF samples, 3D NAs were cultured on six-well MEAs and were maintained in BP-based medium for 2 to 3 weeks D. AccordTo exclude that the handling of hCSF samples, i.e., freezing and thawing of the samples, had an impact on the effect of hCSF on human neuronal network function described here, we tested freshly collected hCSF on 3D NA cultures on six-well MEAs. Forty minutes after CSF isolation, pre-warmed hCSF samples were applied on 3D NAs, which showed asynchronous, partially synchronous, or highly synchronous activity when cultured in BP-based culture medium. Identical to frozen samples , the freNext, we aimed to prove the feasibility of culturing 3D NAs in hCSF over a long time period and evaluated if hCSF-induced improved network activity further increased over time. For this purpose, we cultured hiPSC-derived networks for 4 weeks in hCSF and performed MEA recordings every day . After hCSF treatment, 3D NA cultures showed robust attachment to MEA recording electrode array fields, and no detachment of 3D NAs was observed A. As desWe assessed whether short-term hCSF treatment causes long-lasting changes in neuronal network function of hiPSC neurons. For this purpose, we applied BP-based medium to 3D NA cultures that had been cultured for 72 h in hCSF A, i. As Complementary applied phase-contrast imaging showed that adherently growing 3D NAs cultured in BP-based cultivation medium move and grow over time . In contin vitro and in vivo  and inhibitory post-synaptic currents  B. Three  in vivo , and it  in vivo D, ii, anWhen injecting currents at stepwise increments with the cell in current clamp, 13 out of 20 and 12 out of 18 neurons showed evoked responses, i.e., action potentials (APs), for BP and hCSF, respectively F; B, iiiNext, we applied immunofluorescent staining and fluorescent confocal imaging to obtain a more detailed morphological and cellular insight into the properties of neural cells in 3D NAs cultured in hCSF or BP-based medium.+ neurite net around 3D NAs cultured for 3 days in hCSF, or in BP-based medium , we applied hCSF from healthy adult individuals to evaluate, if a physiologically relevant and adult brain-like milieu triggers neural maturation processes and enhances neuronal functionality in 3D NAs with immature properties. We demonstrated that hCSF induces several maturation processes, including neurogenesis, electrophysiological maturation of neurons, gliogenesis, and synapse and neurite net formation, which ultimately leads to a rapid formation of synchronously active neuronal circuits in 3D NA cultures. Since patch-clamp and MEA recordings demonstrate the improved electrophysiological function of neurons and enhanced neuronal circuit activity, which persists after removal of hCSF, we conclude that hCSF-induced maturation processes are responsible for this enhanced functionality on single neuronal and neuronal circuit level.In an hiPSC-derived 3D NA Astrocytes promote synapse formation, neurite growth, and neuronal electrophysiological function . Here, w+ astrocytes  in vitro when exposed to an appropriate in vitro milieu. Since astrocytes represent a heterogeneous group that differs in its capacity to enhance the neuronal properties and astrocyte development requires a diverse set of signaling cues, future studies will help to understand the mechanism of hCSF-induced astrocyte development in human NSCs and to identify novel human gliogenic signaling cues.Several studies have shown that the differentiation of hiPSCs into human astrocytes requires 3 to 6 months e.g.,  and earltrocytes . In contin vitro models, e.g., brain organoids.As a control condition, we used current, state-of-the art culture medium, the BP medium supplemented with commonly used differentiation factors, e.g., BDNF, GDNF, DAPT. However, these culture conditions are not sufficient to achieve morphological and functional properties in a human 3D neural model as described here by using hCSF. From a neurodevelopmental aspect it is interesting that hCSF not only causes astrocyte development, but induces neuronal differentiation, neuronal maturation, and suppresses proliferation. Thus, adult hCSF contains currently unknown factors that might be necessary to promote neurogenesis and suppression of proliferation to achieve terminal neural maturation in other 3D human neural in vitro neurons in primary neuronal cultures  is commonly used to create a gliogenic milieu in vitro  were cultured and differentiated into cortical NSCs as described elsewhere . Within Two to five hiPSC-3D NAs were seeded as a 5-μL drop directly on PDL/laminin-coated electrode arrays of six-well PEDOT-CNT MEAs. After 1 h, BP medium with supplements was added. Half medium exchanges were performed twice a week. Baseline recordings have been performed in BP medium with supplements before the application of either hCSF or fresh culture medium. Data were recorded and analyzed using MC_Rack software (Multi Channel Systems), SPANNER software suite , and custom-built MATLAB software .in vitro. Calculations and data analysis were performed in custom-made IGOR Pro 8  software.Five to ten hiPSC-3D NAs were seeded on PDL/laminin-coated coverslips and cultured with BP medium with supplements. Half medium exchanges were performed twice a week, 14–20 days after differentiation. For the experiment, coverslips were mounted under a differential interference microscope (Nikon E600FN) together with a CCD camera (Sony XC-73CE) to visually identify the cells. Cells were perfused (2–3 mL/min) with artificial CSF. The micropipette was filled with an intracellular solution. The data were collected with a sampling frequency of 10 kHz and filtered at 3 kHz using an EPC-9 amplifier . Whole-cell recordings were carried out at 32°C and all recordings were performed between the second (14 days) and third weeks (21 days) For further details and details about hCSF sample collection, immunocytochemistry, image acquisition, and analyses, as well as statistical analysis see For statistical analysis either matched one-way ANOVA with Dunnett correction (baseline compared with indicated group) or Tukey correction (comparison between groups) were applied; two-way, unpaired t test was applied to calculate indicated p values. All presented data show mean value ± SD, n refers to the number of individual cultures treated with hCSF or BP medium and N refers to the number of individual experiments. For statistical analysis, GraphPad Prism 8.0 software was used.J.I. performed the experiments, analyzed the data, and prepared the figures. H.S. performed patch-clamp experiments. S.T. developed data analysis programs and critically revised the manuscript. E.H. critically revised the manuscript. S.I. conceived the study, performed part of the experiments and wrote the manuscript.S.I. holds a position at Cellectricon. Cellectricon were not involved in the study, and all experiments and data analyses were conducted at the Sahlgrenska Academy at the University of Gothenburg.S.T. is founder of Result Medical GmbH, Düsseldorf, Germany.The other authors declare no conflict of interest.