Childhood maltreatment is a major risk factor for chronic and severe mental and physical health problems across the lifespan. Increasing evidence supports the hypothesis that maltreatment is associated with epigenetic changes that may subsequently serve as mechanisms of disease. The current review uses a systematic approach to identify and summarize the literature related to childhood maltreatment and alterations in DNA methylation in humans. A total of 100 empirical articles were identified in our systematic review of research published prior to or during March 2020, including studies that focused on candidate genes and studies that leveraged epigenome-wide data in both children and adults. Themes arising from the literature, including consistent and inconsistent patterns of results, are presented. Several directions for future research, including important methodological considerations for future study design, are discussed. Taken together, the literature on childhood maltreatment and DNA methylation underscores the complexity of transactions between the environment and biology across development. Unfortunately, the negative sequelae of childhood maltreatment often begin early in life, and persist across adulthood6, posing risk for premature mortality. Indeed, adults with numerous adverse experiences in childhood die nearly 20 years earlier than those with no early adversity1. Understanding the mechanisms of biological influence is therefore critical to developing innovative targets for intervention to enhance health outcomes among highly vulnerable children with this major adverse exposure.Childhood maltreatment is a highly prevalent public health problem that often has devastating effects on physical and mental health. There are now numerous studies linking childhood maltreatment and other early adversities to nearly all forms of mental illness, as well as chronic medical conditions that cut across multiple organ systems8. DNA methylation is among the most commonly studied epigenetic processes and involves the addition of a methyl group at sites in the DNA where a cytosine nucleotide occurs next to a guanine nucleotide (CpG dinucleotides). Initial work focused on understanding associations of early life stress and DNA methylation examined these processes in rodents and found that low levels of maternal care (licking and grooming and arched-back nursing) was associated with greater methylation of the glucocorticoid receptor (GR) gene in offspring10. Furthermore, this work suggested that methylation was a mechanism of the effect of maternal care on the offspring HPA stress response. Emerging from this groundbreaking work with animal models, some of the earliest studies in humans documenting altered DNA methylation in association with childhood maltreatment focused on the glucocorticoid receptor (GR) gene, NR3C1, which modifies responsiveness of the HPA axis to stress exposure. Our group was the first to demonstrate altered leukocyte DNA methylation of NR3C1 in adults with childhood maltreatment11, associations of NR3C1 methylation with behavior problems and symptoms in children with early adversity12, and maltreatment as a predictor of change in NR3C1 methylation over time13. In 2016, we completed a literature review focused on altered methylation of glucocorticoid signaling genes in association with childhood maltreatment14. Several other recent reviews have also focused on methylation of glucocorticoid signaling genes17, as well as methylation of genes associated with the serotonin system19 in relation to adverse exposures. We expand upon these prior reviews to consider the recent explosion of research in this area that has examined additional genes as well as epigenome-wide effects. Cecil and colleagues recently conducted a systematic review of research focused on childhood maltreatment, specifically experiences of abuse and neglect, and DNA methylation20. The current review provides an important addition to the literature by utilizing a broader conceptualization of childhood maltreatment that includes any experience that involved potential for harm to the child. Our systematic approach to identify and summarize the literature related to childhood maltreatment and other adversities in association with DNA methylation captures research through March, 2020 and includes research focused on candidate genes and studies that leveraged epigenome-wide data in both children and adults.Over the past decade a rapidly growing body of literature has underscored the significant role of epigenetics in the sequelae of childhood maltreatment. Epigenetic processes allow the body to respond to environmental influences by altering gene expression through chemical modifications that regulate chromatin structure and/or DNA accessibility without inducing changes to the DNA sequence21, we conducted a systematic review of human studies investigating the relationship between childhood maltreatment and other adversities and DNA methylation. For the purposes of this review, childhood was defined as events occurring between birth and age 18. Maltreatment was defined as any experience that involved potential for harm to the child, including emotional, sexual, and physical abuse, and emotional and physical neglect. Other adversities included exposure to intimate partner violence or other violence, early parental death or separation, institutional deprivation, and indentured labor. Empirical articles investigating DNA methylation in association with other forms of childhood stress such as low socioeconomic status or famine, without consideration of other adversities, were not included. Studies focusing on experimentally induced stressors were also not included. In addition, because our focus was on childhood maltreatment after birth; we also did not include studies focusing on prenatal stress or substance use and DNA methylation, which have been addressed in other reviews23.In accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelinesStudies were identified by searching PubMed/Medline for empirical articles published in March 2020 or earlier using the following search terms: (((methylation OR epigenetic) AND )), filtered by Humans. Additional searches in PubMed/Medline for studies published during this timeframe were also conducted using the following search terms: (1) emotion regulation AND methylation; (2) abuse AND methylation NOT substance NOT alcohol; (3) trauma AND methylation; (4) early life stress AND methylation; (5) maltreatment AND methylation; (6) adversity AND methylation. Additional articles published during this timeframe identified through other sources  were also included.Our qualitative synthesis included empirical articles that: 1) examined the relationship between childhood maltreatment/adversity and DNA methylation; (2) provided statistical indicators to examine the impact of maltreatment during childhood on DNA methylation; and (3) described maltreatment/adversity that occurred prior to age 18. Empirical articles were excluded if they: (1) did not provide information on childhood maltreatment/adversity and DNA methylation; (2) childhood maltreatment/adversity was not clearly distinguished from adult adversities in study measurement/hypothesis testing; (3) only quality (or quantity) of parental care or support was included as a predictor; (4) were not written in English; (5) were only conducted with animals; and (6) did not use a quantitative approach to summarize research findings . We did not omit articles based on small sample size; this information is included in the tables so that it can be considered when evaluating results. Figure  examinedA total of 100 empirical articles were identified in our systematic review focused on the relationship between childhood maltreatment and DNA methylation. This includes 69 empirical articles focused on candidate genes and 31 empirical articles that leveraged epigenome-wide data. Twenty-eight empirical articles measured DNA methylation in childhood and 72 measured DNA methylation in adulthood. Findings are summarized in Tables NR3C1 which encodes the glucocorticoid receptor (GR)  and FKBP5 which modulates sensitivity of the GR24 .As displayed in Table NR3C1. In our own study of preschoolers, we found that childhood maltreatment status and the number of stressful life events was associated with greater NR3C1 methylation25, and NR3C1 methylation mediated effects of maltreatment and stressful life events on child internalizing behavior problems12. Likewise, in 534 school-aged children with low socioeconomic status, children who experienced maltreatment beginning in infancy or early childhood had greater NR3C1 methylation than children who had no maltreatment history26. Interestingly, there were no differences between children who experienced maltreatment that started in the preschool period or later, and those children with no maltreatment history. However, more chronic maltreatment and more types of maltreatment, were associated with greater NR3C1 methylation. Using data from the Bucharest Early Intervention Project, time in institutional care was associated with lower levels of methylation of FKBP5, which modulates sensitivity of the GR, when children were 12 years old27. Lower levels of FKBP5 methylation in association with childhood maltreatment were also observed in our sample of preschoolers28. Another study of Tanzanian children found evidence that maltreatment was associated with hypermethylation of CRH, the gene that encodes the hypothalamic corticotropin releasing hormone (CRH) and its downstream target, POMC, which encodes the pituitary precursor of ACTH29. Taken together, these findings indicate that childhood maltreatment may be associated with altered methylation of genes that regulate the child stress response, suggesting a possible mechanism for the effect of maltreatment and other adversities on poor health outcomes.Most studies of children support the hypothesis that childhood adversity is associated with higher levels of methylation of SLC6A4, which encodes the serotonin receptor, and HTR2A, the gene that encodes the serotonin receptor subtype, 5-HT2A, have been investigated in association with childhood maltreatment and other interpersonal adversities32. Childhood adversity was associated with greater SLC6A4 methylation in two studies32, but lower levels of methylation in association with institutional care27. Using a subsample (n = 785) of the Avon Longitudinal Study of Parents and Children (ALSPAC), Barker et al.33 generated an inflammation-related epigenetic polygenic risk score (i-ePGS). Adversity between birth and 7 years of age was related to higher i-ePGS, which, in turn, was an indirect pathway by which adversity was associated with internalizing behavior problems in later childhood. The receptor gene for the pituitary hormone oxytocin (OXTR), best known for its role in social behavior and attachment, has also been studied in relation to early adversity in children. Fujisawa et al.34 demonstrated greater methylation of OXTR in Japanese children and adolescents with a maltreatment history (n = 44) compared with controls (n = 41), and found that OXTR methylation was negatively associated with gray matter volume in the left orbitofrontal cortex.Epigenetic alterations with childhood maltreatment have been documented in additional candidate genes. Methylation of serotonin signaling genes, including NR3C1 , FKBP5 , SLC6A4 , and OXTR .As displayed in Table NR3C1 methylation observed significant associations of childhood maltreatment and methylation of this gene, but there is considerable variability in the findings. Several studies found increased NR3C1 methylation with childhood maltreatment, but others found no effect, or reduced methylation with maltreatment. Melas and colleagues35 found that childhood adversity was associated with saliva DNA NR3C1 hypermethylation, with childhood parental loss linked to higher methylation near a transcription factor binding site. Two reports analyzed DNA from the Quebec Suicide Brain Bank in relation to childhood maltreatment retrospectively assessed via psychological autopsy, including a group with suicide and childhood maltreatment, a group with suicide but no maltreatment, and a control group. The suicide with maltreatment group had greater hippocampal NR3C1 methylation and reduced NR3C1 expression across exon 1F36, reduced NR3C1 gene expression across exons 1B, 1C, and 1H, and reduced NR3C1 methylation at exon 1H37, in comparison with the other two groups. In contrast, both suicide groups differed from controls in regions of exon 1B, with hypermethylation at two CpG sites and hypomethylation at one CpG site37. In a diverse sample of adults, childhood maltreatment was associated with greater methylation of leukocyte NR3C1, and maltreatment was associated with lower NR3C1 gene expression38. Likewise, in two samples with psychiatric disorders, childhood maltreatment was associated with greater NR3C1 methylation40. In contrast, in a recent study, we found that early adversity, and the number of adverse exposures, was associated with lower NR3C1 methylation in healthy adults41. Other studies found no association of maltreatment and NR3C1 methylation46, but one of these found that NR3C1 methylation moderated effects of maltreatment on cortisol response to the Trier Social Stress Test.Taken together, the majority of studies focused on FKPB5 methylation in adults also demonstrates inconsistent effects. Klengel et al.47 found that childhood maltreatment was associated with lower methylation of FKBP5 in adults with the rs1360780 T risk allele, and this effect was observed in both a subsample of participants from the Grady Trauma Project and a replication sample. This effect was more recently replicated by Tozzi and colleagues48 who also reported that childhood maltreatment was associated with lower FKBP5 methylation among those with the T risk allele of the rs1360780 SNP. In contrast, Harms et al.49 found significant positive associations between stress in childhood and methylation of FKPB5 in young adulthood using a prospective longitudinal design, and a number of studies did not find significant associations among childhood maltreatment and FKBP5 methylation53, or moderation by FKBP5 risk allele51.The literature focused on SLC6A4, the gene for the serotonin transporter. Most studies have found a positive association of childhood maltreatment with methylation of regions of this gene58 although some studies show equivocal or trend-level effects60 or no effect61 of maltreatment on SLC6A4 methylation.Turning to the serotonin system, several studies have examined whether childhood maltreatment is associated with methylation of 62, five studies investigated methylation of the oxytocin-receptor-gene (OXTR) in adulthood; all five reported no overall association of childhood maltreatment with OXTR methylation. A number of these studies did report indirect or moderation effects. In a sample of 309 African American men, childhood adversity had a significant indirect effect on OXTR methylation through socioeconomic instability38. No significant direct effect of childhood adversity on OXTR methylation was observed. Likewise, while Smearman and colleagues63 did not observe simple associations of child abuse history and OXTR methylation when accounting for multiple comparisons in their sample of 393 African American adults, OXTR methylation moderated the association of childhood abuse and psychiatric symptoms.Based on the hypothesis that oxytocin may play a protective role in the biological response to stress and traumaIL-6 promoter, and lower methylation in turn was associated with greater salivary IL-6 in response to the Trier Social Stress Test64. Although this study drew upon a relatively small sample, the findings are consistent with other work demonstrating effects of childhood trauma and maltreatment on inflammatory processes.Several other candidate genes have been examined in individual studies. For example, childhood trauma was associated with lower methylation of the proinflammatory 65.Arising in part out of replication inconsistencies as well as interest in identifying novel variation, the past decade has included an increase in the number of Genome Wide Association Studies (GWAS), which are agnostic by design and interrogate genetic variation across millions of common genetic variants the entire genome. However, a limitation of GWAS is the extremely large samples needed to detect effects after adjusting for multiple testing. Similar to GWAS approaches, epigenome-wide association studies interrogate markers across the entire genome. Consideration of the biology of DNA methylation as well as optimization of power has led to the development of new frameworks for analyzing multiple markers, including examination of CpG sites that are in close proximity to one another , examination of DNA methylation in biological pathways known to be involved in the condition, and application of analytic tools that use a ranking approach rather than relying on p values. Researchers have also developed quality control standards and approaches for addressing both concerns with Type I error as well as population stratification that may arise in diverse samples70 as well as with age-related development such as menopause72. Several analytical methods have been developed to measure epigenetic age as indicated by epigenome-wide methylation profiles77. This emerging area of research has the potential to provide exciting evidence for understanding the impact of early adversity on molecular age.Given that biological processes function as part of a larger interrelated system, research has increasingly focused on how either systems or patterns of alterations in methylation may be important in efforts to understand epigenetic modifications. Studies have begun to explore DNA methylation as a measure of molecular aging, with evidence that this epigenetic “clock” is associated with age-related disorders and mortality78 found differential whole-genome methylation in children with a maltreatment history relative to children with no maltreatment. Maltreated children had higher levels of methylation at sites where methylation was generally low, and lower methylation at sites where methylation was generally high, compared to non-maltreated children. Using data from a subsample (n = 774) of the ALSPAC, Dunn et al.79 examined associations among childhood adversity and epigenome-wide methylation in children at 7 years of age. Thirty-eight differentially methylated CpG sites were identified in association with early adversity, and the developmental timing of adversity was the most salient predictor of methylation. Neither the simple presen-ce/absence of adversity, recency of adversity, nor accumulation of adverse experiences was associated with altered methylation.As displayed in Table 80. Furthermore, threat-related early adversity exerted a significant indirect effect on depressive symptoms through accelerated DNA methylation age, suggesting that this measure of early adversity is clinically relevant to psychiatric outcomes. This is consistent with Jovanovic et al.81 that demonstrated that violence exposure was associated with greater DNA methylation age acceleration in African American children who were 6–13 years of age.Studies of early adversity and epigenetic age are just emerging in samples of children. Very recently in a sample of 247 children who were 8–16 years of age, threat-related early adversity (including abuse and violence exposure), but not deprivation (including neglect), was associated with accelerated DNA methylation age82 identified nine differentially methylated regions (DMRs) in the genome that replicated across the two cohorts and were associated with childhood adversity. No individual CpG sites in their epigenome-wide analysis replicated across the cohorts. O’Donnell et al.83 used a different methodological approach to describe variation in DNA methylation, and found that childhood maltreatment was associated with variation in methylation at 27 years of age utilizing principal components scores to describe variation in methylation. Lutz et al.84 examined genome-wide DNA methylation and gene expression in postmortem brain samples of adults with a history of depression who died by suicide. This study found that child abuse history was associated with differential methylation specifically in oligodendrocytes in the cingulate cortex, as well as expression of myelin-related genes.As displayed in Table 85 used the ALSPAC and the MRC National Survey of Health and Development to examine associations of childhood psychosocial adversity, including abuse and neglect, and DNA methylation age acceleration. Childhood sexual abuse was associated with methylation age acceleration in ALSPAC, and this effect remained significant when controlling for socioeconomic position. Data regarding sexual abuse was not available in the MRC survey, however Tamman et al.86 also found that childhood sexual abuse was associated with increased DNA methylation age. Neither individual adversity types nor a cumulative measure of adversity were associated with methylation age acceleration in ALSPAC and the MRC National Survey of Health and Development85. Han et al.87 also reported a positive association between a history of childhood trauma and epigenetic aging in adults with MDD. DNA methylation age was also examined in the sample of 27 year olds described above, with analyses revealing no differences in epigenetic age as a function of childhood adversity83. Although there have been more studies focused on childhood adversity and accelerated epigenetic age in adults, few studies overall have been completed.Investigations using epigenome-wide data to explore effects of early adversity on accelerated epigenetic aging have shown mixed outcomes. Lawn et al.This systematic review examined associations of childhood maltreatment and DNA methylation in children and adults. One hundred empirical articles focused on humans were identified. These studies included both candidate gene and epigenome-wide approaches. Strengths of the literature included: (1) rigorous approaches to measure childhood maltreatment in studies focused on DNA methylation in children, including record review methods; 2) several racially and ethnically diverse samples in the child studies; (3) diverse and innovative approaches to measuring epigenome-wide effects of maltreatment, including exploration of how early adversity may lead to epigenetic age acceleration; and (4) several replication studies focused on childhood maltreatment and methylation of glucocorticoid signaling genes in children and adults. Collectively, these studies provide evidence that childhood maltreatment and other adversities are associated with DNA methylation. Genes and pathways observed to have altered methylation in relation to childhood maltreatment, and the expected epigenetic pathways from maltreatment to health and mental health outcomes, are displayed in Fig.  several NR3C1 and FKBP5. Most studies in children demonstrated increased NR3C1 methylation with maltreatment. In adults, several studies documented greater NR3C1 methylation in those exposed to childhood maltreatment, whereas others demonstrated no associations of maltreatment and NR3C1 methylation. Furthermore, in our own recent work we found lower NR3C1 methylation in association with childhood adversity41, and other studies have identified CpG sites in NR3C1 that are hypomethylated in association with maltreatment or suicide37 and post-traumatic stress disorder (PTSD)89. For FKBP5, Klengel and colleagues first published findings of an interaction of childhood maltreatment and FKBP5 genotype such that maltreatment was associated with lower FKBP5 methylation in adults with the T risk allele47. Lower FKBP5 methylation with maltreatment—although not interacting with genotype—has been seen in the two studies with published data in children. The maltreatment by genotype interaction predicting lower FKBP5 methylation in adults was recently replicated48, but other studies have not observed an effect of maltreatment, or an interaction of maltreatment and genotype, and one study of young adults found that stressful life events in childhood were associated with greater FKBP5 methylation, which in turn mediated effects of early stress on prefrontal brain activity49.Studies of childhood adversity and DNA methylation often focused on methylation of candidate genes that regulate glucocorticoid signaling including NR3C1 gene promoter are generally very low, and this may also limit the ability to reliably detect group differences. However, there is evidence that NR3C1 methylation at exons 1F, 1B, and 1C, is inversely associated with GR gene expression37. Since GR mediates negative feedback to the HPA axis, higher methylation—and lower GR gene expression—is expected in conditions with elevated cortisol and there is some evidence for this91. But variable findings for methylation of NR3C1 and FKBP5 are perhaps not surprising given that maltreatment, trauma, and stress-related psychiatric disorders have been linked to both exaggerated and diminished basal and provoked cortisol concentrations, which may in part depend on measured or unmeasured individual and trauma characteristics93. Moreover, many genes and gene products regulate HPA axis function, and complex interactions among participant characteristics (including age and genetic ancestry), genotype , and stress exposure may best characterize these effects.Many studies in adults utilized samples with chronic disease and mental health diagnoses, and did not exclude participants with consistent medication use, which may have influenced the findings. Overall levels of methylation of the 94. Although the potential risk for statistical artifacts provide important cautions to investigations exploring interaction effects, interactions and conditional effects are known to occur in biological systems and may be appropriate to examine in large samples and/or using stratification or model invariance strategies. Methylation of serotonin signaling genes was also examined in multiple studies. Most studies of adults and some work in children found elevated methylation of SLC6A4 with maltreatment, but there are also reports of lower SLC6A4 methylation at some CpG sites with adversity, or no difference between adversity groups. Differences in epigenetic findings in SLC6A4 as well as other candidate genes may also be related to inconsistencies in the ways that the number of differentially methylated regions are assessed. For example, SLC6A4 has 81 CpG sites with studies adopting a range of approaches to assessing this large region, including selection of target sites based on the literature, multiple testing of sites, and strategies for binning methylation at multiple sites95. Given evidence for ancestry-related differences in methylation, differences across studies related to ancestry may also potentially contribute to apparent inconsistencies96.Interaction effects may also characterize methylation of oxytocin system genes, given findings of indirect or moderation effects in the absence of simple effects83 for an example of integration of genotype and epigenetic influences), interactions among systems of genes, and differences in aspects of the phenotyping and heterogeneity of different studies. Due to traditional approaches to corrections for multiple testing, studies that analyze methylation of multiple markers across the genome tend to require large samples which are often heterogeneous and may have less intensive phenotyping and measurement of maltreatment and other relevant exposures. Promising markers identified with epigenome-wide approaches show some consistency in terms of their roles in neural cell development , signaling and apoptosis (LINGO3 and 2NPFF2), neural influences on movement (ALS2), neuroinflammation (ITGB1), and some tentative evidence for immune markers (CXCL1).Studies using agnostic approaches to exploring multiple markers across the epigenome also suggested that maltreatment is associated with variation in methylation, though these epigenome-wide studies have generally failed to identify commonly studied candidate genes, leading to questions about candidate approaches. Several reasons for these inconsistencies have been advanced, including large effect sizes needed to detect an effect after multiple testing corrections, biological considerations such as the potential combinatory impact of genotype and DNA methylation  may accelerate aging. A number of adult and child studies find age acceleration among individuals exposed to maltreatment . Childhood maltreatment and other adverse exposures alter epigenome-wide profiles, thereby likely contributing to chronic disease and physical agingStudies of maltreatment and methylation in children were often characterized by careful measurement of adverse exposures using structured record review techniques and in-depth interview methods, and they often utilized prospective and longitudinal designs. In children, DNA methylation was most frequently measured in saliva or buccal epithelial cells, with fewer studies measuring methylation in blood. Many of the samples were racially and ethnically diverse, and these studies covered the full developmental spectrum, from early childhood through adolescence. In contrast, nearly all the adult studies drew upon retrospective reports of childhood maltreatment. Utilization of prospective longitudinal designs and record review techniques would address a significant gap in the literature. Furthermore, in contrast to studies of maltreatment and methylation of candidate genes in children, most adult studies measured methylation in leukocyte DNA.79. More research is needed to understand how early adversity at each developmental epoch may be associated with differences in epigenetic marks. Moreover, longitudinal research is needed to explore whether epigenetic changes secondary to early adversity may “reset” during later developmental periods or perhaps may be impacted by later experiences, which either attenuate or compound early adversity. Retrospective research with adults who report childhood adversity has numerous benefits in terms of cost- and time-effectiveness; however, reliance on retrospective reports of childhood adversity present important methodological challenges related to recalling the timing of childhood events, ability to recall very early life events, and systematic recall biases, introducing measurement limitations that are difficult to overcome103. Many studies use the Childhood Trauma Questionnaire104 or other self-report measures. Some self-report measures, such as the Traumatic Life Events Questionnaire105 and the Maltreatment and Abuse Chronology of Exposure106, include trauma characteristics such as the age or frequency of occurrence, which may be important determinants of epigenetic and phenotypic outcomes. Future research may also benefit from interview approaches that can help with retrospective recall such as participant-tailored anchoring.Moving beyond simple effects of maltreatment on DNA methylation, several studies highlighted the importance of the developmental timing of exposure to adversity49 assessed stress when children were 9–13 years old and then methylation approximately 10 years later. O’Donnell et al.83 assessed childhood maltreatment in the first 15 years of life and methylation at age 27. Future research should capitalize on existing studies of children that utilized rigorous measures of the environment as they develop over time to expand the literature in adults. Likewise, very few studies drew upon repeated assessments of DNA methylation over time. In our own research, we demonstrated that childhood maltreatment and other adversities are associated with change in methylation of glucocorticoid signaling genes over time in early childhood107. Future research should further examine maltreatment as a predictor of change in methylation across development.Although many adult studies reviewed here relied on recall, much can be learned from the few studies that capitalized on longitudinal cohorts. For example, Harms et al.NR3C1 in late childhood and adolescence108. Smoking and depression in pregnancy have also been associated with altered methylation of placental stress-related genes, such as NR3C1 and HSD11B2, which encodes the enzyme that inactivates cortisol112. Associations of childhood maltreatment and DNA methylation may be partially accounted for by prenatal environmental factors. Conversely, maltreatment may exert a unique and independent effect on DNA methylation above and beyond prenatal exposures. Barker et al.33 found that child adversity between birth and 7 years of age was associated with an inflammation-related epigenetic polygenic risk score (i-ePGS) at age 7, but there was no association of prenatal adversity and the i-ePGS. Future research should disentangle prenatal exposures and adversities experienced in childhood to better understand effects of maltreatment on DNA methylation in both childhood and adulthood.Importantly, the majority of studies focused on exposure to childhood maltreatment without consideration of the prenatal environment. Prenatal exposures exert epigenetic effects on several biological systems, particularly the development of the child stress response. For example, intimate partner violence in pregnancy is associated with increased 115. For example, Braithwaite et al.116 found that maternal depressive symptoms in pregnancy were associated with greater methylation of NR3C1 in male, but not female, infants. Stroud et al.109 found that the moderating effects of placental HSD11B2 methylation on links between prenatal major depressive disorder and infant cortisol response emerged most strongly for newborn daughters, whereas direct and moderating effects of SLC6A4 gene expression were evident only for sons109. In one of the few studies identified in this review that examined sex differences, Cicchetti et al.78 found that boys and girls showed different directions of the effect of maltreatment on methylation of ALDH2, a gene that encodes a key enzyme in the metabolism of alcohol. Sex differences were also observed in the effect of the developmental timing of adversity. As we have shown in a meta-analysis with ADCYAP1R1, the adenylate cyclase activating receptor gene associated with PTSD, depending on the functional outcomes of the gene(s), sex and developmental differences are reasonable to expect117. Future work on maltreatment and DNA methylation should consider the role of child sex to ensure that important moderation effects are not being overlooked.Very few studies examined sex differences in the effects of childhood maltreatment on DNA methylation. Studies focused on in utero stress exposure often find sex differences in epigenetic pathways and observed outcomes in both preclinical and human modelsFKBP5 methylation over time in preschoolers with early adversity107. More recently, Vinkers et al.118 observed changes in methylation among soldiers successfully treated for PTSD such that changes in methylation were observed among soldiers who had reductions in PTSD symptoms. Taken together, this work provides initial evidence that psychosocial interventions exert influence on the epigenome.An additional future direction is to move from association to causal inference. Maltreatment is often confounded with additional measures of adversity, including poverty and other sociodemographic factors, as well as personality factors, and potentially genetic factors. Many studies reviewed utilized statistical control for potential confounding factors; however, true random assignment designs are not ethical in humans. Thus, synthesizing across preclinical studies (where random assignment is possible) and human association studies will be critical. Additionally, innovative designs in human studies, including random assignment to interventions to reduce maltreatment, and control groups that are demographically and psychosocially matched to maltreatment groups, may allow the field to move closer to causal inference. Research considering intervention effects on change in methylation over time may also address this gap in knowledge. Indeed, we found that service utilization was associated with increases in 121 and primate research has identified correspondence in DNA methylation profiles in the brain and blood122. Similar gene expression was found in the cerebellum and peripheral blood mononuclear cells (PBMCs) across 4000 unique genes123. Correspondence between DNA methylation in PBMCs and postmortem brain tissue was identified with respect to a marker of reward and stress-induced responses124. Recent research showed good correspondence of DNA methylation in blood and saliva125. Studies of methylation age have also shown consistency across tissue types75. Interestingly, there is some evidence126 that saliva samples may be more strongly associated with brain methylation than blood samples, although correlations of methylation in the brain with saliva, blood, and buccal cell DNA were all observed to be high and the strength of these associations appear to depend on the genomic region of interest127. These studies have provided helpful signals regarding the utility of blood, brain, saliva, and buccal cells, and Epigenomic Roadmap datasets now provide some insights into the ways that peripheral methylation profiles may map on to those in brain tissue129. Nonetheless, cell type heterogeneity remains a significant challenge for epigenetic research that has been addressed using a number of strategies such as cell counts, cell separation and examination of single cells, and analytic strategies that account for cell type133.DNA methylation has been measured in several tissue types, including blood, saliva, and buccal cells. Yet, the majority of studies focused on DNA methylation in children utilized saliva and buccal cells, and fewer examined methylation in blood. Although some researchers have questioned the value of peripheral markers in research aimed at understanding psychobehavioral outcomes believed to be related to central brain processes, with some researchers suggesting that psychiatric epigenetic research should be limited to brain tissue, a number of studies point to the value of peripheral indicators. Research has shown reasonable concordance between gene-expression signals in blood and brain137. Research is needed to carefully document reliability considerations for proper assessment of reproducibility; interested readers are directed to a thorough discussion of reliability, replication, and reproducibility in DNA methylation measurement (see ref. 134). In addition to the importance of clear site level documentation of findings in publications, suggestions for addressing reliability concerns in ongoing research include analytic approaches such as dropping CpG sites with low observed variability, replicating findings with procedures such as pyrosequencing, and integrating preanalysis reliability metrics137. Focusing the analyses on regions with more variability across participants reduces the likelihood of false positives, and also reduces the number of comparisons, thereby increasing power. It is also important to note that methylation arrays assess only CpG sites; this yields fewer sites than whole-genome bisulfite sequencing approaches, and some research suggests some important variability occurs outside of these CpG islands138. Standards in this field continue to be refined, and researchers should follow these developments closely. At a minimum, researchers should implement strategies to address quality control , cell heterogeneity, and sample ancestry141.Another concern involves the technology and analytic strategies used to interrogate genome methylation. Power to detect true differences and false positives are both major concerns, so large samples sizes and replication samples are required, but very large studies may not be able to provide rich data on maltreatment. Researchers have also described the importance of considering reliability of BeadChip technology, the most frequently applied technology for interrogating the genome, as a function of: (a) sample type, with lower reliability and replicability in dried blood spots, (b) tissue type, with some probes demonstrating greater cross-tissue concordance, (c) platform , and (d) observed variability at each site, with lower reliability at sites with less variabilityOur systematic review examined associations of maltreatment and other interpersonal adversities during childhood with DNA methylation in children and adults. We identified 100 studies, including studies utilizing both candidate gene approaches and epigenome-wide analyses. Several strengths of the literature were identified, as well as directions for future research. Key challenges facing the field and associated recommendations are described in Fig. 65. Researchers have also begun to explore ways that methodological approaches may improve understanding of epigenetic factors and adversity outcomes such as repeated measurement, sampling across tissues, twin/family approaches, and psychological/pharmacological treatments. Epigenetic approaches are also perhaps best understood in combination with thorough assessments of genotype, epigenome, and gene expression. Many factors related to the type and timing of adversity, availability and quality of buffers to adversity, and new events and time since early adversity are likely critically important influences on negative outcomes of adversity. Ongoing recognition of the complexity of biological and environmental factors as well as phenotypic nuances is critically needed—apparent inconsistences do not necessarily negate findings from either of the divergent studies but rather may point to important considerations for ongoing research and theoretical conceptualizations.In less than two decades, there has been an explosion of research in childhood maltreatment and epigenetics. Mirroring trends observed in genetic research, studies have increasingly focused on sampling methylation across the genome. As with genetic research, advantages of this approach include opportunities to identify key mechanisms that would otherwise have been overlooked. However, this approach generally requires larger samples to account for multiple testing which in turn has been criticized for less nuanced phenotyping of samples permitted in smaller cohorts. Some of these concerns may be somewhat addressed through collaborations such as efforts undertaken by the Psychiatric Genetics Consortium working groups, particularly when care is taken to balance genomic inflation/deflation and to address concerns that can arise with diverse samples interrogated on differing platforms


Recent advances in cell engineering strategies such as reprogramming of somatic cells into induced pluripotent stem cells and direct cell fate conversion have impressively demonstrated the feasibility to manipulate cell identities by the overexpression of cell fate-determining transcription factors. These strategies are now increasingly implemented for transcription factor-guided differentiation of neural precursors and forward programming of pluripotent stem cells toward specific neural subtypes. This review covers major achievements, pros and cons, as well as future prospects of transcription factor-based cell fate specification and the applicability of these approaches for the generation of donor cells for brain repair.Traditionally,  Classically, they involve extrinsic factors such as morphogens to guide the differentiation process toward a specific cell fate, thereby mimicking regionalization processes during nervous system development. This approach has led to significant advances, for instance, for the generation of midbrain dopamine neurons for the treatment of PD , which are not yet committed to the neural lineage, was shown to be feasible with bHLH TFs. A milestone in the field of neuronal forward programming was reached in 2011, when the groups of Marius Wernig and Thomas Südhof reported that combined overexpression of the TFs Ascl1, Brn2, and Myt1l not only suffices to transdifferentiate mouse fibroblasts into neurons  but alsontiation . Severalntiation  and humantiation  toward antiation , and 11 ntiation . In humantiation .in vitro studies employing overexpression of Ngn1 in mouse ESCs, transduced cells underwent morphological rearrangements forming neurite-like structures already within the first 72 h and became electrophysiologically excitable as early as 4 days after transgene induction , ZEB1, ISL1, TLX3, and POU4F1  are upregulated already in this early phase of reprogramming, whilst inhibitors of neurogenesis as for instance REST and HES1 are repressed. Concomitant with the dynamics of mRNA regulation, the expression of the pluripotency-associated miRNA cluster 302/367 decreases, whereas the abundance of neuronal miRNAs such as miR124, miR96, and miR9 increases upon differentiation  exit of the original cell fate, (ii) repression of alternative lineage decisions, and (iii) activation of a pro-neuronal transcriptional program.When thinking of forward programming as a tool to produce neural cell types for brain repair, it is particularly relevant to thoroughly characterize the exact phenotype of the obtained cells. Already CUX1 , and found that the induced GABAergic neurons express markers reminiscent of derivatives of the medial ganglionic eminence, but not alternative birthplaces such as the lateral ganglionic eminence, caudal ganglionic eminence or the preoptic area. As somatostatin- and parvalbumin-positive inhibitory interneurons appear to play a particular role in several neurological and neuropsychiatric diseases, the generation of these subtypes would be highly desirable. in vivo.Ascl1 – which is expressed in more ventral regions of the telencephalon in vivo  – is aloin vivo . Howeverin vivo counterparts and found that although the morphogen-driven dopaminergic differentiation recapitulated key developmental stages of embryonic dopaminergic lineage specification, the gene expression profile of in vitro generated populations still differed from that of native midbrain dopamine neurons. The question remains whether forward programming can further improve the authenticity of specialized dopaminergic neuron subpopulations.Representing a prime target of PD, midbrain dopamine neurons are a particularly attractive donor cell population for neural repair. However, the efficacy of neuroregeneration seems to heavily depend on the fidelity of neuronal subtype specification. This was recently exemplified by Already beginning in the end of last century, several labs reported that overexpression of the mesencephalic TF Nurr1 in primary adult rat hippocampal NPCs  and primAldh2, it is only upon co-expression that they synergistically induce more advanced markers such as Dat and Tyrp1  and the efficiency of the concomitant growth factor regimen . Thus, the results of these two studies stress the context-dependency of TF-based forward programming. This is further nicely exemplified by the fact that NFIA was recently demonstrated to act as a gliogenic switch in iPSC-derived NPCs, too, facilitating the fast generation of astrocytes in combination with glia-promoting, LIF-containing medium. Continued overexpression of this TF, however, inhibited astrogenesis from NPCs, probably by inducing premature G1 cell cycle arrest . Interestingly, using this system, the authors could generate proliferative OPCs, which terminally differentiated into almost pure cultures of oligodendroglial cells expressing characteristic markers such as CNP and PLP upon mitogen withdrawal  NPC stages, as they might provide additional phenotype-instructing differentiation cues, which could otherwise only be delivered by combining several TFs upstream of the lineage-relevant signaling pathways. This was nicely exemplified by the group of Johan Ericson and Thomas Perlmann, who showed that overexpression of Lmx1a in ESC-derived mouse NPCs suffices to instruct dopaminergic neuron differentiation when combined with Shh and Fgf8 treatment, whereas caudalized NPCs only adopt a dopaminergic phenotype if the three midbrain-associated TFs Lmx1a, En1, and Otx2 are overexpressed in combination . On the in vitro is to co-culture forward programmed neurons with glial cells. Notably, the species from which the glial cells are retrieved might influence their maturation-promoting effect: In a recent study, culturing human glutamatergic neurons  on mouse astrocytes was found to be superior to co-culture with rat astrocytes, whereas co-culture with human astrocytes did not support neuronal survival beyond 4 weeks. Interestingly, GABAergic neurons did not show this selective response to glial co-culture  cell fate conversion. Besides generating precious insights into how cell fates are established and controlled by transcriptional and epigenetic regulation, one major asset of these approaches is that they can provide new donor sources for brain repair. Yet, a number of issues need to be addressed more deeply before forward programming can be implemented in a clinical setting.LF, KL, and OB contributed to the writing and editing of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Therapeutic approaches have been done on the basis of disease severity with the avoidance of culprit and cross-reacting NSAIDs, and when indicated, aspirin desensitization is an effective treatment option. Biologic approaches targeting Type 2 cytokines are emerging as potential therapeutic options. Here, we summarize the up-to-date evidence of pathophysiologic mechanisms and diagnosis/management approaches to the patients with NERD with its phenotypic classification.Nonsteroidal antiinflammatory drug (NSAID)-exacerbated respiratory disease (NERD) is characterized by moderate-to-severe asthma and a higher prevalence of chronic rhinosinusitis/nasal polyps, but is a highly heterogeneous disorder with various clinical manifestations. Two major pathogenic mechanisms are: (1) overproduction of cysteinyl leukotrienes with dysregulation of arachidonic acid metabolism and (2) increased type 2 eosinophilic inflammation affected by genetic mechanisms. Aspirin challenge is the gold standard to diagnose NERD, whereas reliable  Aspirin  and nonsteroidal antiinflammatory drugs (NSAIDs) are the most commonly prescribed drugs in the world ; howevervia orally, bronchially or nasally route) and supported by potential biomarkers  nasal polyps (NPs) with persistent eosinophilic inflammation in the upper and lower airways  as well omarkers . In addiiagnosis . This re4 metabolite levels, the frequency of asthma exacerbation, medication requirements (high-dose ICS-LABA or systemic corticosteroids) and asthma severity, suggesting that stratified strategies according to subtype classification may help achieve better clinical outcomes in the management of NERD.NERD is characterized by chronic type 2 inflammation in the upper and lower airways; therefore, patients suffer from chronic persistent asthmatic symptoms and CRS with/without NPs, which are exacerbated by ASA/NSAID exposure and refractory to conventional medical or surgical treatment. Some patients are accompanied by cutaneous symptoms such as urticaria, angioedema, flushing or gastrointestinal symptoms . PreviouThe major upper and lower airway symptoms of NERD are mediated by increased levels of CysLTs with dysregulation of arachidonic acid (AA) metabolism and intense type 2/eosinophilic inflammation .4, via 5-lipoxygenase [5-LO] and LTC4 synthase [LTC4S]), prostaglandin (PG) pathway  and thromboxanes (TBX) A2 by PG synthase and TBX synthase (2 is a major finding in NERD (4) mainly derived from various inflammatory cells, including neutrophils, monocytes, and basophils, eosinophils and mast cells, which further increases after ASA/NSAID exposure compared to asthmatic patients with ASA/NSAID tolerance (ATA). Moreover, the increased expression of 5-LO and LTC4S was noted in NERD patients with overproduction of CysLTs; increased CysLTs bind to CysLT receptor 1/2, subsequently inducing bronchoconstriction and amplifying inflammatory signal pathways (2/PGD2 play a major role in the pathogenesis of NERD. Increased PGD2 (released from mast cells and eosinophils) binds to prostanoid receptors to induce bronchoconstriction  receptors  in NERD patients (2 (the first unstable precursors of PG products from AA metabolism) without COX-1 (2 and its receptor levels could contribute to CysLTs overproduction in NERD patients. Lipoxin (LX) A4 and its epimer (15-epi-LXA4) are also called as the ASA-triggered lipoxins, and have antiinflammatory effects in airway inflammation (2) is expressed on human neutrophils, eosinophils, macrophages, T cells, ILCs (ILC2 and NK cells) and epithelial cells of the respiratory tract. After binding their receptors, it leads to the restoration of epithelial barrier function and resolution of allergic inflammation through down-regulation of chemotaxis and cell activation  and increased levels of the pro-inflammatory CysLTs , enhancing type 2 inflammation via interacting with epithelial cells and autocrine functions of eosinophils in the asthmatic airway  and CysLTs overproduction in which increased CysLTs contributes to driving type 2 inflammatory responses . The keyc airway . There hc airway , 2) incrc airway , 3) incrc airway , leadingc airway .Recent studies suggested that the activation of neutrophils may be related to the severity of airway inflammation in NERD , althougHLA DPB1*0301 has been regarded as a strong genetic marker and replicated in the 2 ethnic groups Polish and Korean populations  which were associated with the phenotypes of NERD  lead to amplify the biological activity of CysLTs, the SNPs of prostanoid receptor genes  were associated with the development of NERD (CCR3 −520 T>C), chemoattractant receptor molecular expressed in Th2 cells (CRTH2 −466 T>C) and IL5R (-5993 G>A), were reported (Many genetic studies have focused on CysLTs-related and eosinophil activating genes (major pathogenic mechanisms) according to single nucleotide polymorphisms (SNPs) and genome-wide association studies (GWASs) .  is suggested to be the most reliable biomarker for the diagnosis of NERD. Several studies demonstrated that patients with NERD had higher baseline concentrations of urinary LTE4 as well as greater increase after aspirin/NSAID exposure than in patients with ATA, suggesting that urine LTE4 level could be used as a clinical diagnostic test  is essential. ASA desensitization can be beneficial for NERD patients when indicated.Treatment strategies for asthma should follow stepwise management guidelines with maintaining inhaled corticosteroids with or without long-acting beta 2 agonists, leukotriene modifier drugs and/or biologic agents on the basis of disease severity and rescue medications . BecauseInitial treatment for CRS includes intranasal corticosteroids with intranasal saline irrigation. Intranasal corticosteroids have shown to be highly effective in reducing nasal inflammation and in shrinking NPs, which are recommended as a first-line treatment in patients with CRSwNP . BecauseDespite the heterogeneity of NERD, therapeutic approaches have been proposed according to symptom severity. However, these different phenotypes contribute to the variability in response to treatment. A recent study found that clinical severity and courses differ among the 4 subtypes of NERD, which affect antiasthmatic medications required . SubtypeASA desensitization is an effective treatment option when standard medical treatments are not effective or daily ASA/NSAIDs therapy is required for other medical conditions, such as coronary artery disease or chronic inflammatory disease . MultiplThe emergence of biologics in the management of asthma and CRSwNP has represented potential and promising therapy for NERD. New biologics targeting type 2 cytokines, such as IL-4, IL-5 and IL-13 as well as IgE, have been reported in clinical trials, which could reduce asthma exacerbation and oral corticosteroid use, and improve lung function . In addi4 and PGD2 metabolites, in patients with NERD and lead to the development of ASA tolerance with a reduction in urinary LTE4 concentrations during oral ASA challenge  are essential in the management of NERD. In phenotypic clusters of NERD, subtype 4 patients (NERD with urticaria) would need omalizumab as an effective option, which can inhibit activated basophils and mast cells, the key elements of NERD and urticaria ; subtypeDietary intervention may be beneficial for controlling symptoms in patients with NERD. Some studies demonstrated that restricting dietary salicylates, including fruits, vegetables, berries, herbs, and spices, improves nasal and asthmatic symptoms, which can be explained by the known contribution of salicylates in the pathogenesis . A previPatients with NERD present with a variety of clinical features affected by chronic type 2 eosinophilic inflammation with the overproduction of CysLTs in the upper and lower airways. Although NERD tend to be associated with severe asthma and CRSwNP, an improved understanding of clinical features and underlying pathogenesis of NERD will aid in diagnostic evaluations and new therapeutic strategies for improving clinical outcomes. With the increasing recognition of phenotypic heterogeneity of NERD, efforts are needed to establish precision medicine strategies tailored to individual phenotypes/endotypes with potential biomarkers.The clinical features, diagnosis and treatment of NERD were described by S-DW and the pathophysiologic mechanisms including molecular genetic mechanisms were described by QL. This article was written under supervision of H-SP. She, as corresponding author, performed the overall design and review of this article.This study was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute, funded by the Ministry of Health & Welfare, Republic of Korea (H116C0992).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Significance: Cerebrovascular reactivity (CVR), defined as the ability of the cerebral vasculature to dilate or constrict in response to a vasoactive stimulus, is an important indicator of the brain’s vascular health. However, mechanisms of cerebrovascular dysregulation are poorly understood, and no effective treatment strategies for impaired CVR exist. Preclinical murine models provide an excellent platform for interrogating mechanisms underlying CVR dysregulation and determining novel therapeutics that restore impaired CVR. However, quantification of CVR in mice is challenging.Aim: We present means of assessing CVR in awake mice using intraperitoneal injection of acetazolamide (ACZ) combined with continuous monitoring of cerebral blood flow.Approach: Measurements of cerebral blood flow were made with a minimally invasive diffuse correlation spectroscopy sensor that was secured to an optical window glued to the intact skull. Two source–detector separations (3 and 4.5 mm) per hemisphere were used to probe different depths. CVR was quantified as the relative increase in blood flow due to ACZ. CVR was assessed once daily for 5 days in 5 mice.Results: We found that CVR and the response half-time were remarkably similar across hemispheres and across 3- versus 4.5-mm separations, suggesting a homogenous, whole brain response to ACZ. Mean(std) intra- and intermouse coefficients of variations were 15(9)% and 19(10)%, respectively, for global CVR and 24(15)% and 27(11)%, respectively, for global response half-time.Conclusion: In sum, we report a repeatable method of measuring CVR in free-behaving mice which can be used to screen for impairments with disease and to track changes in CVR with therapeutic interventions. However, quantification of CVR in mice is challenging. Common techniques to assess CVR include intravenous injection of the potent vasodilator acetazolamide (ACZ) and induction of hypercapnia via inhalation of a high-concentration  carbon dioxide gas mixture. For both of these techniques, cerebral blood flow is monitored before and after intervention, and CVR is defined as the relative percent change in blood flow from preintervention levels normalized to either the dose of ACZ or to the change in arterial partial pressure of carbon dioxide. Although these assessments are routinely used in humans, delivery of the vasoactive stimulus and reliable monitoring of the stimulus response is more challenging in mice. In the case of ACZ, although intravenous tail vein injection is possible, longitudinal assessment of CVR with this approach is challenging because repeated tail vein injections without the use of a catheter cause inconsistent drug delivery and increase risk of infection and vein collapse.Herein, we present a novel means of assessing CVR in mice that overcomes several of these technical limitations and enables longitudinal monitoring of CVR in awake, free behaving animals. In this approach, mice are given ACZ intraperitoneally, and the cerebral blood flow response is continuously monitored with diffuse correlation spectroscopy (DCS) using a minimally invasive sensor that is secured to the intact skull. Given the relative ease of both intraperitoneal injections and assessment of blood flow with DCS, multiple longitudinal measurements of CVR can easily be acquired. In this work, we characterize average CVR in healthy mice, and we quantify the spatial and temporal heterogeneity of the CVR response.2Five 3- to 4-month-old male C57BL/6 mice were used. Measurements of resting state cerebral blood flow and CVR were assessed once daily for 5 days at the same time each day. All animal procedures were approved by Emory University Institutional Animal Care and Use Committee and followed the NIH Guidelines for the Care and Use of Laboratory Animals.2.1To enable continuous monitoring of cerebral blood flow with DCS, a laser cut clear acrylic optical window  was secured to the intact skull at least 3 days prior to the start of the study. To implant the window, mice were anesthetized with 1% to 2% isoflurane, a midline incision was made that extended from between the ears to behind the eyes, fascia was removed, and a thin , an array of four photon counting avalanche photodiodes , and a hardware autocorrelator board . Thus to minimize experiment duration, blood flow was monitored for 10-min post-ACZ injection, after which time the optical sensor was removed, and the animal was returned to its cage.To assess CVR, the DCS sensor was secured to the optical window, and the mice were placed in an empty cage. Blood flow was monitored continuously (1 Hz) throughout the duration of the experiment. After a 10-min baseline period, mice were given an intraperitoneal injection of To quantify CVR, Here njection . We also2.3Paired Wilcoxon signed-rank tests were used to determine whether baseline BFI, CVR, and 3A total of 25 measurements were made in 5 mice across 5 consecutive days. Five measurements were discarded: two were discarded due to abrupt movement during intraperitoneal injections, and three were discarded in mouse 4 due to poor sensor contact with the optical window as a result of dried dental cement on the window surface.Resting state blood flow showed significant temporal variation over the 10-min baseline period, as seen in the representative timeseries in Across all mice, baseline BFI at the 3-mm source–detector separation % , Table 24Herein, we present an approach to estimate CVR in awake, free-behaving mice via intraperitoneal injection of ACZ coupled with minimally invasive assessment of cerebral blood flow with DCS. ACZ acts to inhibit carbonic anhydrase, an enzyme that aids in the transport of carbon dioxide from the tissues to the lungs. This inhibition increases local carbon dioxide levels by slowing carbon dioxide transport from the tissue to the blood stream, causing an increase in blood flow.,,,,To characterize this new technique, we quantified CVR in multiple mice across several days. We found that CVR and the response half-time were remarkably similar across hemispheres and across source–detector separations  and 5, sThis approach also enabled us to characterize resting-state blood flow in awake, free-behaving mice. Significant temporal variability . Three out of five discarded datasets were rejected from a single mouse due to poor sensor contact with the optical window as a result of dried dental dement on the window surface. Future studies should employ careful visual inspection of the window surface during surgery to remove any surface imperfections that prohibit proper sensor contact with the window. The remaining two discarded datasets were rejected due to abrupt movement during ACZ injection that caused the needle (and thus the ACZ solution) to become displaced from its intended target of the intraperitoneal cavity. This motion occurred in two separate mice toward the end of the study (days 4 and 5) and was possibly caused by skin soreness after multiple days of repeated injections. If so, a topical analgesic may mitigate this issue when repeated injections are warranted.5This study reports a method for longitudinal assessments of cerebral blood flow and CVR in awake, free-behaving mice which can be easily adapted to assess cerebrovascular impairment in various disease states. This approach overcomes several key challenges of vascular reactivity assessment in mice by removing the need for a repeated tail vein injection, femoral arterial catheter, intubation, or the confounding influence of anesthesia. Future work can utilize this method to screen for cerebrovascular impairments in murine models of disease and to track changes in vascular reactivity with therapeutic intervention.Click here for additional data file.Click here for additional data file.