Maternal Separation With Early Weaning Mice Research Articles

Early Life Stress in the Pristane Model of Systemic Lupus Erythematosus increases cardiovascular risk but not kidney disease severity earlier in disease progression

Experiencing Early Life Stress (ELS), such as physical/verbal abuse before the age of 18, has become an increasingly apparent concern in the context of lifelong health. Systemic Lupus Erythematosus (SLE) is one autoimmune condition linked with ELS, predominantly affects women, and has high CVD burden. Exposure to ELS independently increases the risk of CVD and SLE, so we have developed a model to study this gap. The mouse model of ELS, maternal separation with early weaning (MSEW), combined with the pristane model of SLE mimics the accelerated disease (shown by earlier production of autoantibodies, increased vascular stiffness, and vascular dysfunction at 4 months post injection (p.i.) opposed to 7 months p.i.) in ELS. We hypothesized that the accelerated disease and CVD burden in MSEW+SLE mice, as opposed to normally reared (NR)+SLE mice, arose from severe kidney damage and increased blood pressure. MSEW mice were separated from dams for 4 hours/day (postnatal day 2 to 5) and 8 hours/day before weaning at day 17. NR litters were weaned at postnatal day 21. MSEW or NR female mice were randomly administered PBS or pristane through i.p. injection. Urine was collected at 4 months and 7 months p.i. to test for kidney damage markers and telemetry was utilized. MSEW animals develop increased Anti-Smith autoantibodies at 4 months p.i while SLE NR animals had no difference in titer levels compared to NR PBS mice. However, we found no difference in urine NGAL (Neutrophil Gelatinase-Associated Lipocalin) at 4 (p=0.32) or 7 months p.i. (p=0.6) or urine proteinuria 7 months p.i. (p=0.9) between groups. Blood pressure between MSEW+SLE and NR+SLE mice at 4, 5, and 6 months p.i. compared to control mice was not different. At 7 months p.i., SBP in NR+SLE (140.3 ± 10.22, n=4), MSEW+SLE (140.28 ±9.32, n=4) was not statistically greater than the NR/MSEW controls (128.1 ± 3.16, n=5 NR, n=7 MSEW). Ejection fraction in NR+SLE (66.04 ± 3.24, n=3), MSEW+SLE (75.17 ±3.19, n=2) compared to NR/MSEW controls (64.36 ± 2.49, n=5 NR, n=4 MSEW) was slightly changed over time. We see that at 6 months p.i. PWV and adventitial wall thickness is greater in MSEW+SLE (2.89 mm/sec ± 0.11) mice compared to both NR+SLE mice (2.047 mm/sec ± 0.16) and NR control mice (1.49 mm/sec ± 0.12, p=<0.001, F= 26.02), but at seven months PWV is not different between MSEW+SLE (2.94 mm/sec ± 0.13) mice compared to both NR+SLE mice (2.63 mm/sec ± 0.32, p=0.30). The acceleration of autoantibodies and PWV in MSEW+SLE mice, but not kidney or heart function indicates a specific impact on larger vessels and autoimmune cells in SLE. This work furthers the understanding of how ELS accelerates cardiovascular burden in autoimmune diseases and early identifying targets to prevent worsened outcomes in patients exposed to childhood trauma. Crohn’s and Colitis Foundation NIH R25 PROmoTE NIH T32 PRIME F31HL165863-0 NIH P01 HL158500. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract P081: Early Life Stress (ELS) Induces Greater Aortic Pulse Wave Velocity (PWV) Through Reactive Oxygen Species (ROS)

ELS, such as verbal and physical abuse or maltreatment, is linked to a greater risk ofcardiovascular disease (CVD) in adulthood. Aortic stiffness, an independent predictor of CVD,has been suggested as a prognostic tool for early detection and intervention in at-riskpopulations. Several human studies have shown that aortic stiffness (assessed by PWV) isincreased in both adults and adolescents with ELS. ELS mouse models, such as maternalseparation with early weaning (MSEW), show aortic endothelial dysfunction with increasedsuperoxide and systemic inflammation, which are both present in humans with ELS exposureand contribute to aortic stiffness. We hypothesized that MSEW mice develop aortic stiffness in aROS-dependent manner at an early age. The MSEW protocol entails separating pups from thedam 4h/day (postnatal, PD 2 to 5) and 8h/day (PD 6 to 16) followed by weaning at PD17.Normally-reared (NR) litters remain with the dam and are weaned at PD21. Female and maleMSEW and NR mice were treated for 2 weeks with the ROS scavenger, TEMPOL (30mg/kg/day; drinking water) from 8 weeks of age. PWV was assessed prior to and after TEMPOLtreatment. PWV was significantly increased in MSEW mice at baseline (NR: 1.95 mm/sec ±0.15, MSEW: 2.60 mm/sec ± 0.17, p=0.009) in both sexes similarly. PWV was not significantlydifferent between MSEW and NR mice after TEMPOL treatment (NR: 1.78 mm/sec ± 0.06,MSEW: 1.97 mm/sec ± 0.12, p<0.05). MSEW mice had significantly more aorticCD11b + Ly6C + CX3CR1 + inflammatory monocytes compared to NR mice (NR: .9% ± 0.31,MSEW 1.5% ± 0.25, p=0.014) at baseline, however TEMPOL did not affect this. TEMPOLtreatment significantly decreased CD11b + F4/80 + MHCII hi inflammatory resident macrophages inMSEW mice compared to NR mice (Median Fluorescent Intensity; NR: 1,235,274 ± 54413,MSEW : 944,446 ±11728, p=0.007). In conclusion, ELS induces increased aortic stiffness in aROS-specific manner at an early age. Inflammatory resident macrophages may contribute to theELS-specific ROS-dependent mechanism. The mechanisms identified in this work arepotentially powerful tools for preventative therapeutics to reduce CVD burden in ELS-populations.

Early Life Stress Induced Hypothalamic–Pituitary–Adrenal (HPA) Axis Dysfunction Promotes Chronicity of Experimental Colitis in Mice

Inflammatory bowel diseases (IBD) are chronic disorders of the gastrointestinal tract with peak onset during adolescence. The etiology of IBD remains poorly understood, but is believed to involve genetic susceptibility, improper immune regulation, and environmental factors. Psychological stress is one environmental trigger that has been associated with an increased risk of inflammatory diseases. Specifically, previous studies have linked early life stress (ELS) with hypothalamic–pituitary–adrenal (HPA) axis dysfunction that ultimately increases susceptibility for adverse health outcomes later in life. The aim of this study was to determine whether ELS in mice affects susceptibility to, and/or severity of colitis induced subsequent to stress exposure. We hypothesized that ELS would contribute to earlier onset disease and exacerbated colitis. We used an established mouse model of ELS, maternal separation with early weaning (MSEW), where newborn mice are separated from their mothers for 4‐8 hours daily from postnatal day (PD) 2 until early weaning on PD 17. Beginning at PD 28, colitis was induced in MSEW mice and their normally‐reared (NR) counterparts by transient blockade of the interleukin‐10 receptor (IL‐10R). Relative to their NR counterparts, MSEW mice exhibited systemic and colon‐specific deficits of the HPA‐axis endpoint molecule corticosterone, prior to induction of colitis. Following disease onset, NR mice were mostly in remission by 15 days after cessation of IL‐10R blockade, whereas MSEW mice displayed sustained histological pathology with an emphasis on epithelial damage, in addition to consistently elevated colonic tumor necrosis factor (Tnf). Furthermore, pharmacologic induction of HPA‐axis dysfunction via early life exposure (PD 1‐14) to the synthetic glucocorticoid, dexamethasone, mirrors the results we observed in the MSEW model of ELS – both prior to and subsequent to colitis induction. Our results indicate that early life exposure to prolonged stress leads to HPA‐axis dysfunction and colonic corticosterone deficits which may subsequently promote persistent colonic inflammation in susceptible hosts.

Effects of Early Life Stress on the Gut Microbiota of Mice

Exposure to early life stress (ELS) is associated with a greater risk of chronic disease development including depression, cardiovascular disease (CVD), and inflammatory bowel disease (IBD). Changes in the gut microbiota have been linked to IBD and CVD. Rodent models of early life neglect are used to characterize the mechanistic links between ELS exposure and the risk of disease later in life. However, little is understood about ELS exposure and the gut microbiota. We used a mouse model of ELS, maternal separation with early weaning (MSEW) and normally-reared (NR) mice to determine whether the neonate microbiota is altered, and if so, whether the MSEW protocol induces changes in the dam microbiota that are then transmitted to their offspring. MSEW mice (n = 12) were subjected to maternal separation for 4 h per day on postnatal days (PDs) 2–5, and 8 h per day on PDs 6–16, and were subsequently weaned at PD17. NR mice (n = 13) remained undisturbed with dams until weaning on PD21. All mice were maintained on standard chow (NIH-31; NSN 8710-01-005-8438) following weaning. Paired-end Illumina sequencing was conducted on colonic content from PD28 mice and DADA2 was utilized to infer and assign taxonomy to individual amplicon sequence variants (ASVs). The fecal microbiota of dams (n= 5) was also sequenced. Analysis of composition of microbiomes (ANCOM) detected 32 ASVs that had statistically different abundance in MSEW compared to NR mice at PD28. Five of the nine ASVs that increased in MSEW mice were classified as Clostridium senso stricto 1 or Lachnoclostridium, while none the of the 23 ASVs enriched in NR animals belonged to these taxa. Fifty-seven percent (13 of 23) of the ASVs that had greater abundance in NR animals were classified as Lachnospiracea (other than Lachnoclostrium) or Ruminococcaceae. Compared to NR animals, MSEW PD28 mice had reduced phylogenetic diversity (18.7 ± 0.3 compared to 20.3 ± 0.3 in NR; P = 0.003) and Chao1ASV richness (175 ± 8 compared to 198 ± 8 in NR; P = 0.04). Microbial community composition was also altered at PD28 based permutational analysis of variance of Aitchison distance (F(1,24) = 2.29, P = 0.002). The composition, levels of diversity, and abundance of individual ASVs in the microbiota of dams were no different between MSEW and NR cohorts. Thus, the observed microbiota shifts in the young pups are likely driven by endogenous effects of MSEW in the offspring and independent of maternal inheritance. MSEW mice had lower microbial diversity and a reduction in Lachnospiraceae and Ruminococcaceae, which are important fermenters of short-chain fatty acids (SCFAs). Nine different SCFAs were measured in plasma of PD28 mice and MSEW animals had reduced butyrate (P = 0.02) compared to NR controls. Butyrate promote colonic homeostasis as well as regulates metabolic health and CVD risk. Studies are on-going to determine the impact of altered SCFA levels with ELS exposure. This study suggests a direct effect of ELS-exposure on the gut microbiota, which may impact the risk of chronic disease development.

Early life stress in mice promotes chronicity of experimental colitis

Abstract Inflammatory bowel diseases (IBD) are chronic disorders of the gastrointestinal tract with peak onset during adolescence. The etiology of IBD remains poorly understood, but is believed to involve genetic susceptibility, improper immune regulation, and environmental factors. Psychological stress is one environmental trigger that has been associated with an increased risk of inflammatory diseases. Specifically, previous studies have linked early life stress (ELS) with hypothalamic–pituitary–adrenal (HPA) axis dysfunction leading to adverse health outcomes later in life. Our goal was to determine whether ELS in mice affects susceptibility to, and severity of, colitis – induced subsequent to stress exposure. We used an established mouse model of ELS, maternal separation with early weaning (MSEW), in which newborn mice are separated from their mothers for 4–8 hours daily from day of life (DOL) 2 until early weaning on DOL 17. Then, starting on DOL 28, colitis was induced in MSEW mice and their normally-reared (NR) counterparts by transient blockade of the interleukin-10 receptor (IL-10R). Prior to induction of colitis, MSEW mice showed systemic and colon-specific depletion of corticosterone relative to NR mice. In addition, MSEW mice with active colitis harbored significantly reduced numbers of colonic Foxp3+ T cells compared to colitic NR mice. Finally, whereas NR mice were mostly in remission by 15 days following cessation of IL-10R blockade, MSEW mice showed consistently elevated Tnf in the proximal colon and sustained epithelial damage. Our results suggest that ELS-mediated HPA-axis dysfunction causes hypo-corticosteronism, which may subsequently promote prolonged colonic inflammation in susceptible hosts.

Early life stress induces dysregulation of the heme pathway in adult mice.

Early life stress (ELS) is associated with cardiovascular disease (CVD) risk in adulthood, but the underlying vascular mechanisms are poorly understood. Increased hemoglobin and heme have recently been implicated to mediate endothelial dysfunction in several vascular diseases. Chronic physiological stress is associated with alterations in the heme pathway that have been well‐described in the literature. However, very little is known about the heme pathway with exposure to ELS or chronic psychosocial stress. Utilizing a mouse model of ELS, maternal separation with early weaning (MSEW), we previously reported that MSEW induces endothelial dysfunction via increased superoxide production. We reasoned that heme dysregulation may be one of the culprits induced by MSEW and sustained throughout adulthood; thus, we hypothesized that MSEW induces heme dysfunction. We investigated whether circulating levels of heme, a circulating pro‐oxidant mediator, are increased by MSEW and examined the role of the heme metabolic pathway and heme homeostasis in this process. We found that circulating levels of heme are increased in mice exposed to MSEW and that plasma from MSEW mice stimulated higher superoxide production in cultured mouse aortic endothelial cells (MAECs) compared to plasma from normally reared mice. The heme scavenger hemopexin blunted this enhanced superoxide production. Splenic haptoglobin abundance was significantly lower and hemoglobin levels per red blood cell were significantly higher in MSEW versus control mice. These findings lead us to propose that ELS induces increased circulating heme through dysregulation of the haptoglobin‐hemoglobin system representing a mechanistic link between ELS and CVD risk in adulthood.

Early-life stress exacerbates the effects of WIN55,212-2 and modulates the cannabinoid receptor type 1 expression

Early-life stress induces an abnormal brain development and increases the risk of psychiatric diseases, including depression, anxiety and substance use disorders. We have developed a reliable model for maternal neglect, named maternal separation with early weaning (MSEW) in CD1 mice. In the present study, we evaluated the long-term effects on anxiety-like behaviours, nociception as well as the Iba1-positive microglial cells in this model in comparison to standard nest (SN) mice. Moreover, we investigated whether MSEW alters the cannabinoid agonist WIN55,212-2 effects regarding reward, spatial and emotional memories, tolerance to different cannabinoid responses, and physical dependence. Adult male offspring of MSEW group showed impaired responses on spatial and emotional memories after a repeated WIN55,212-2 treatment. These behavioural impairments were associated with an increase in basolateral amygdala and hippocampal CB1-expressing fibres and higher number of CB1-containing cells in cerebellum. Additionally, MSEW promotes a higher number of Iba1-positive microglial cells in basolateral amygdala and cerebellum. As for the cannabinoid-induced effects, rearing conditions did not influence the rewarding effects of WIN55,212-2 in the conditioned place preference paradigm. However, MSEW mice showed a delay in the development of tolerance to the cannabinoid effects. Moreover, CB1-positive fibres were reduced in limbic areas in MSEW mice after cannabinoid withdrawal precipitated with the CB1 antagonist SR141617A. These findings support that early-life stress promotes behavioural and molecular changes in the sensitivity to cannabinoids, which are mediated by alterations in CB1 signalling in limbic areas and it induces an increased Iba1-microglial marker which could interfere in emotional memories formation.

4578 Early life stress promotes chronicity of experimental colitis

OBJECTIVES/GOALS: The overall goal of this study was to determine the effect of early life stress (ELS) on the intestinal CD4+ T cell immune compartment, at homeostasis and after induction of experimental Inflammatory Bowel Disease (IBD). METHODS/STUDY POPULATION: We used a mouse model of ELS, maternal separation with early weaning (MSEW). We used IL-10 reporter mice to enable analysis of IL-10-producing cells. Mice were examined on postnatal day 28 to determine the impact of ELS on gut regulatory T cells. Plasma levels of corticosterone (rodent stress response hormone) was determined by ELISA. Colitis was induced in MSEW and normal rear (NR) mice via intraperitoneal injection of α-IL-10R every 5 days until day 15. Mice were euthanized on days 20 and 30. Colonic tissue sections were stained for histological analysis. Remaining tissue was further processed for flow cytometric analysis of CD4+ T cells and innate lymphoid cells. RESULTS/ANTICIPATED RESULTS: Plasma corticosterone was elevated in MSEW mice compared to their NR counterparts at 4 weeks of age. We observed that the MSEW stress protocol does not affect the baseline colonic CD4+ T cell or innate lymphoid cell populations. There was a reduction in the intestinal CD4+ T cells and regulatory T cells on day 20 in α-IL-10R MSEW mice compared to NR counterparts. This difference disappeared by day 30. Histological scoring showed no difference in disease severity between α-IL-10R treated MSEW and NR mice on day 20. However, on day 30, when α-IL-10R NR mice are recovering from colitis, MSEW mice showed persistent histological inflammation, mainly attributable to sustained epithelial damage. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results suggest that ELS prolongs intestinal inflammation and impairs epithelial repair. Future studies will focus on elucidating the mechanisms responsible for ELS-dependent impairment of mucosal repair in experimental colitis.

Maternal separation increases cocaine intake through a mechanism involving plasticity in glutamate signalling.

Early-life stress (ELS) is associated with negative consequences, including maladaptive long-lasting brain effects. These alterations seem to increase the likelihood of developing substance use disorders. However, the molecular consequences of ELS are poorly understood. In the present study, we tested the impact of ELS induced by maternal separation with early weaning (MSEW) in CD1 male mice at different phases of cocaine self-administration (SA). We also investigated the subsequent alterations on GluR2, GluR1, cAMP response element-binding (CREB), and CREB-phosphorylation (pCREB) in ventral tegmental area (VTA) and nucleus accumbens (NAc) induced by both MSEW and cocaine SA. Our results show that MSEW animals expressed a higher cocaine intake, an increased vulnerability to the acquisition of cocaine SA, and incapacity to extinguish cocaine SA behaviour. MSEW mice showed decreased GluR2 and increased GluR1 and pCREB in NAc. Also, results displayed reduction of basal levels of GluR1 and CREB and an elevation of GluR1/GluR2 ratio in the VTA. Such results hint at an enhanced glutamatergic function in NAc and increased excitability of VTA DA neurons in maternally separated mice. Altogether, our results suggest that MSEW induces molecular alterations in the brain areas related to reward processing, increasing the vulnerability to depression and cocaine-seeking behaviour.

Early Life Stress in Mice Alters Microbial Composition

Early‐life stress (ELS) increases the risk of developing cardiovascular disease (CVD) and other chronic inflammatory diseases including inflammatory bowel disease (IBD). Early‐life perturbations in the gut microbiota that enhance a pro‐inflammatory state or reduce microbial metabolites that are important for regulating metabolic homeostasis may predispose children to developing IBD or CVD later in life. Previous investigations of ELS and the gut microbiota have not focused on early stages of gut microbial colonization and community development. We used a mouse model of ELS, maternal separation with early weaning (MSEW), and hypothesized that MSEW would lead to changes in the gut microbiota indicative of a pro‐inflammatory community composition. MSEW mice were subjected to maternal separation for 4 h per day on postnatal days (PDs) 2–5, and 8 h per day on PDs 6–16, and were subsequently weaned at PD 17. Normally reared (NR) mice remained undisturbed with dams until weaning on PD 21. All mice were maintained on standard chow (NIH‐31; NSN 8710‐01‐005‐8438) following weaning. We examined colonic microbiota composition in the MSEW and NR groups via Illumina sequencing of the 16s rRNA gene at PD 10 (n = 9 NR; 14 MSEW) during the separation protocol, and PD 28 (n = 13 NR; 12 MSEW) subsequent to weaning. Fecal microbiota of dams (n = 6) were also sequenced to evaluate potential maternal contribution of microbiota to their pups. Cardiac norepinephrine (NE) levels confirmed a stress phenotype; male MSEW mice had significantly greater NE at PD 28 (P = 0.01, 3.54 ± 0.38 NE/protein (ng/mg), n = 5) compared to NR male mice (2.06 ± 0.38 NE/protein (ng/mg), n = 4). Differences in microbial composition and diversity were not evident between MSEW and NR mice at PD 10 but were significant at PD 28. There were no changes in the microbial composition of dams when they were assigned to MSEW or NR (P = 0.55), providing evidence that microbiota differences detected at PD 28 were not inherited from their mothers. The microbiota of MSEW mice at PD 28 displayed reduced phylogenetic diversity (P = 0.03), a reflection of microbial functional trait diversity. Linear discriminant analysis (LDA) effect size (LEfSe) was used to identify microbial taxa with differential abundances between MSEW and NR mice at PD 28. MSEW mice were enriched in the bacterial families Lactobacillaceae (P = 0.011) and Peptostreptococcaceae (P = 0.017). Microbial communities of NR mice were enriched in the short‐chain fatty acid (SCFA) fermenting families Ruminococcaceae (P = 0.011) and Bacteroidaceae (P = 0.004). Microbial‐produced SCFAs promote colonic homeostasis as well as regulate metabolic health and CVD risk. Further studies are on‐going to determine SCFAs in MSEW and NR mice. In summary, MSEW mice had lower relative abundances of SCFA fermenting taxa and were enriched in taxa, Peptostreptococcacea and Lactobacillaceae, that promote inflammation and degradation of the colonic mucus barrier in mice.Support or Funding InformationFunding SourcesThis work was supported by National Institute of Health Institutional Research and Academic Career Development Award (to KMK), T32 DK007545 and PhRMA Translational Medicine and Therapeutics Postdoctoral Fellowship (to KCM), and a Research Initiatives Award from the Crohn’s and Colitis Foundation (to CLM and JSP).

Abstract P1116: Early Life Stress Induces HDAC-Dependent Expression of Pro-Oxidant Genes in the Aorta

Childhood adversity or early life stress (ELS) is an understudied risk factor for cardiovascular disease (CVD). The mouse ELS model, maternal separation with early weaning (MSEW), generates robust effects on anxiety as well as immunological consequences in adulthood. Previous studies from our laboratory showed that MSEW induces a pro-oxidant vascular phenotype in adulthood. Early in life or during development, epigenetic mechanisms such as the cellular balance of histone acetylation and deacetylation (HDACs) critically influences gene expression. We hypothesized that MSEW induces HDAC-dependent expression of pro-oxidant genes in the vasculature. Thoracic aortae were isolated from male adult (12 wk) normally-reared (NR) or MSEW mice. Histone 3 lysine acetylation (H3K27) status was found to be significantly decreased in aorta from MSEW compared to NR mice, indicating MSEW induced increased HDAC activity. The MSEW-induced increased in aortic NADPH oxidase (NOX) 2, NOX4, and toll-like receptor (TLR) 4 expression and was significantly blunted with ex vivo trichostatin A (TSA) treatment with no changes in NR mice (relative mRNA; NOX2: MSEW+VEH 1.55±0.12 vs MSEW+TSA 1.24±0.16, NOX4: MSEW+VEH 1.68±0.14 vs MSEW+TSA 1.25±0.07, TLR4: MSEW+VEH 1.65±0.16 vs MSEW+TSA 0.79±0.09, N=4-8, p<0.05). Further, MSEW-induced endothelial dysfunction was reversed with ex vivo TSA treatment with no changes observed in NR mice (E max NR+VEH 86.3±3.5, NR+TSA 82.8±6.3, MSEW+VEH 64.1±8.2 vs MSEW+TSA 85.9±1.7, N=8-12, p<0.05). Reports show that HDAC9 is associated with increased risk for vascular diseases. Aorta from MSEW mice had a significant increase in HDAC9 abundance compared to NR mice (rel. mRNA; NR: 1.02±0.1 vs MSEW: 1.67±0.2, N=6, p<0.05). To determine whether HDAC9 directly induces NOX2, NOX4, and/or TLR4 expression, we utilized HDAC9 over-expressing (HDAC9-OE) mice. Aortic tissue from HDAC9-OE mice revealed a 10-fold NOX4 and 13-fold TLR4 increase, with no change in NOX2 expression. Our results indicate that HDACs, especially HDAC9, are ELS-sensitive mediator(s) of pro-oxidant gene expression. We conclude, that activation of HDAC-dependent pro-oxidant genes, specifically NOX4 and TLR4 pathways, contribute to increased CVD risk following ELS.

Perineuronal Nets, Inhibitory Interneurons, and Anxiety-Related Ventral Hippocampal Neuronal Oscillations Are Altered by Early Life Adversity

BackgroundIn humans, accumulated adverse experiences during childhood increase the risk of anxiety disorders and attention-deficit/hyperactivity disorder. In rodents, the ventral hippocampus (vHIP) is associated with anxiety regulation, and lesions in this region alter both anxiety-like behavior and activity levels. Neuronal oscillations in the vHIP of the theta frequency range (4–12 Hz) have been implicated in anxious states and derive in part from the activity of inhibitory interneurons in the hippocampus, some of which are enwrapped with perineuronal nets (PNNs), extracellular matrix structures known to regulate plasticity. We sought to investigate the associations among early life stress–induced anxiety and hyperactivity with vHIP neuronal oscillations, inhibitory interneurons, and PNNs in mice. MethodsWe used repeated maternal separation with early weaning (MSEW) to model accumulated early life adversity in mouse offspring and studied the underlying cellular and electrophysiological changes in the vHIP that are associated with excessive anxiety and hyperactivity. ResultsWe found increased anxiety-like behavior and activity levels in MSEW adult males, along with increased theta power and enhanced theta–gamma coupling in the vHIP. MSEW mice showed reduced intensity of parvalbumin as well as increased PNN intensity around parvalbumin-positive interneurons in the vHIP. We further observed that MSEW increased orthodenticle homeobox protein 2, a transcription factor promoting PNN development, in the choroid plexus, where it is produced, as well as in parvalbumin-positive interneurons, where it is sequestered. ConclusionsThese findings raise the possibility of causal links among parvalbumin-positive interneurons, PNNs, orthodenticle homeobox protein 2, and MSEW-induced anxiety and hyperactivity.

Early life stress induces vascular expression of pro‐oxidant, proinflammatory genes in adulthood in an HDAC9‐dependent manner

Early life stress (ELS) has been correlated with an increased risk of developing cardiovascular disease (CVD) in adulthood; however, mechanisms linking ELS and CVD risk are unknown. Toll‐like receptor (TLR) activation, specifically TLR4, has been implicated as a key link between inflammation and CVD. TLR4 signaling can activated NADPH oxidases (NOXs) that produce superoxide to increase CVD risk. Further, multiple studies have found that NF‐kB activation, an important downstream step in the TLR4 signaling pathway, requires the activation of certain NOX isoforms, including NOX2 and NOX4. We utilized a mouse model of maternal separation with early weaning (MSEW) to investigate the effect of ELS on the expression and activation of genes encoding these pro‐oxidant, proinflammatory proteins. MSEW mice were separated 4hr/day during postnatal day (PD)2–5, 8 hr/day during PD6‐16, and weaned at PD17. Normally‐reared control mice were left undisturbed and weaned at PD21. Experiments were conducted on 3–6 month old male mice. We have previously shown that MSEW induces NOX‐dependent superoxide production and vascular endothelial dysfunction. We also found increased gene expression of NOX2, NOX4, and TLR4 in the aorta of MSEW mice. Recent immunohistochemical studies have also revealed increased aortic TLR4 protein expression in MSEW mice (n=5/group). Due to similar patterns of expression of genes involved in the TLR4 signaling pathway, we further hypothesized that a common upstream transcription factor could be a driver of these ELS‐induced gene changes. We focused this study on histone deacetylases (HDACs), as numerous studies have implicated HDACs in the modulation of proinflammatory responses and the progression of a variety of CVDs. Interestingly, we found that MSEW induced aortic HDAC9 protein expression, exclusive of other HDAC isoforms. A 3‐hr ex vivo pretreatment with trichostatin A (TSA), a pan‐inhibitor of HDACs, improved vascular endothelial function in MSEW mice (n=5/group, p=0.047). Further, ex vivo TSA treatment led to a trend of decreased expression of NOX2, NOX4, and TLR4 genes (n=4–8/group). These results suggest that ELS leads to HDAC9‐dependent pro‐oxidative and proinflammatory gene expression that increase CVD risk in adulthood. They also further emphasize the role of TLR4 signaling on superoxide production and vascular endothelial function. Future studies aim to clarify the role of TLR4 signaling in these phenotypes, as well as the effect of increased HDAC9 expression on the transcriptional regulation of TLR4 and other genes that could confer CVD risk in adulthood.Support or Funding InformationThis research was funded by PO1HL69999 and the UAB PREP Program.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Maternal separation increases alcohol-drinking behaviour and reduces endocannabinoid levels in the mouse striatum and prefrontal cortex

Childhood adversity is associated with an increased risk of mood, anxiety and substance use disorders. Maternal separation is a reliable rodent model of early life adversity that leads to depression-like symptoms, which may increase the vulnerability to alcohol consumption during adolescence. However, the specific alterations in the pattern of alcohol consumption induced by maternal separation and the underlying molecular mechanisms are still unclear. The purpose of this study is to evaluate the long-term effects of maternal separation with early weaning (MSEW) on emotional and social behaviour, alcohol rewarding properties, and alcohol consumption, abstinence and relapse in adolescent male C57BL/6 mice. In addition, endocannabinoid and monoamine levels were analysed in discrete brain areas. Results showed that MSEW mice presented emotional alterations related to depressive-like behaviour and modified endocannabinoid levels in the striatum and the prefrontal cortex. MSEW mice also showed impairments in alcohol-induced conditioned place preference and higher alcohol intake in a model of binge drinking. Moreover, MSEW animals displayed a higher propensity to relapse in the two-bottle choice paradigm following a period of alcohol abstinence associated with reduced monoamine levels in the striatum. Such results indicate that exposure to early life stress increased the vulnerability to alcohol binge-drinking during adolescence, which may be partially explained by decreased sensitivity to alcohol rewarding properties and the ability to potentiate alcohol intake following a period of abstinence.

Abstract 042: Evidence of Angiotensin II (AngII)-dependent Obesity-induced Hypertension in Female Mice Exposed to Postnatal Neglect

Previously, we have shown that female mice subjected to maternal separation with early weaning (MSEW), a model of postnatal neglect, display exacerbated diet-induced obesity and high blood pressure (BP) compared with control mice. Female MSEW mice show activated renin-angiotensin system components, including increased plasma renin activity and adipose tissue-derived angiotensinogen secretion. The goal of this study was to test whether augmented obesity-induced hypertension in female MSEW mice is AngII-dependent. Mouse MSEW was achieved by repeated, daily separations from the dam and 4-day early weaning. Normally reared controls (C) were weaned at postnatal day 21. Each experimental group of female weanlings was comprised of 6 mice each and derived from 3 different litters, that were placed on high fat diet (HFD, 60% kcal from fat). After 18 weeks, mice were implanted with radiotelemetry for BP measurement. At week 20, average 24-hr systolic blood pressure (SBP) was 134±2 mmHg in MSEW mice and 126±2 in C (P<0.05). No significant changes in mean arterial pressure, diastolic blood pressure or heart rate were observed between groups. Next, we also determined the BP sensitivity to the acute administration of AngII (1, 10 and 50 ug/kg, s.c.). AngII-induced BP changes, assessed by BP area under the curve, were similar between MSEW and C mice at all doses (50 ug/kg dose:145±10 vs. 132±15 mmHgx30 min, respectively). Chronic enalapril treatment (2.5 mg/kg/day, drinking water, 7 days) was conducted to block endogenous AngII synthesis. Enalapril reduced SBP 15±2 mmHg in MSEW mice but only 6±1 mmHg in C mice (p<0.05). The BP response to acute AngII doses increased similarly in MSEW and C enalapril-treated mice, (50 ug/kg dose: 200±13 vs. 207±22 mmHgx30 min, respectively). In addition, BP and HR responses to acute injections (i.p) of mecamylamine (5 mg/kg), propranolol (5 mg/kg) or atropine (1 mg/kg) were similar between untreated MSEW and C mice, suggesting that exacerbated BP in female MSEW mice is independent of sympathetic or parasympathetic dysfunction. Taken together, these data provide evidence that increased BP in female MSEW mice results from elevated circulating AngII rather than enhanced AngII sensitivity or sympathetic nerve activity.

Early life stress in male mice induces superoxide production and endothelial dysfunction in adulthood.

Early life stress (ELS) is a risk for cardiovascular disease in adulthood although very little mechanistic insight is available. Because oxidative stress and endothelial dysfunction are major contributors to cardiovascular risk, we hypothesized that ELS induces endothelial dysfunction in adult male mice via increased superoxide production. Studies employed a mouse model of ELS, maternal separation with early weaning (MSEW), in which litters were separated from the dam for 4 h/day [postnatal days (PD) 2-5] and 8 h/day (PD6-16), and weaned at PD17. Control litters remained undisturbed until weaning at PD21. When compared with control mice, thoracic aortic rings from adult male MSEW mice displayed significant endothelial dysfunction that was reversed by the superoxide scavenger, polyethylene glycol-superoxide dismutase (PEG-SOD). PEG-SOD-inhibitable superoxide production by aortae from MSEW mice was significantly greater than observed in control aortae, although unaffected by nitric oxide synthase inhibition, suggesting that uncoupled nitric oxide synthase was not responsible for the accelerated superoxide production. Aortic SOD expression, plasma SOD activity, and total antioxidant activity were similar in MSEW and control mice, indicating unaltered antioxidant capacity in MSEW mice. Increased expression of the NADPH oxidase subunits, NOX2 and NOX4, was evident in the aortae of MSEW mice. Moreover, endothelial dysfunction and superoxide production in MSEW mice was reversed with the NADPH oxidase inhibitor, apocynin, indicating increased NADPH oxidase-dependent superoxide production and endothelial dysfunction. The finding that MSEW induces superoxide production and endothelial dysfunction in adult mice may provide a mechanistic link between ELS and adult cardiovascular disease risk.

Abstract 360: Early Life Stress Increases Hematocrit and Endothelin-1 in Mice

In humans, early life stress (ELS) is an independent risk factor for adult cardiovascular disease (CVD). We have shown that mice subjected to ELS by maternal separation with early weaning (MSEW), develop vascular endothelial dysfunction in adulthood. A marker of endothelial dysfunction and CVD is high hematocrit, an abnormally elevated level of circulating red blood cells. Hematocrit is largely regulated by erythropoietin (EPO), a protein that is released predominantly from the kidney under conditions of hypoxia. We hypothesized that MSEW increases circulating EPO and hematocrit in adult male mice. We used the MSEW model in C57BL6J mice to study the mechanisms of ELS-mediated alteration in hematocrit. MSEW litters were subjected to maternal separation 4h/day (postnatal day (PD) 2-5) and 8h/day (PD6-16), and weaned at PD17. Control (CON) litters were undisturbed until weaning at PD21. At 13 weeks of age, blood was collected from CON and MSEW male mice by cardiac puncture and lung tissue was excised. Hematocrit of MSEW mice was significantly higher than CON mice (46.2 ± 0.03 vs 43.3 ± 0.03%, p = 0.004). Plasma EPO, as measured by ELISA, was elevated in MSEW mice, however not significantly (112.89 ± 51.32 vs 61.62 ± 20.73 pg/ml, p = 0.06). We further hypothesized that MSEW enhances circulating endothelin-1 (ET-1) levels, a vasoactive peptide regulated by hypoxia and EPO. We found that plasma ET-1 was significantly increased in MSEW mice compared to CON (1.55 ± 0.41 vs 1.26 ± 0.23 pg/ml, p = 0.02). Endothelin receptor type A and B density and binding in lung, as measured by radioligand binding, was not different between groups, suggesting that increased circulating ET-1 in MSEW mice was not due to decreased ET-1 clearance in the lungs and most likely is due to increased production of ET-1. Taken together, our data suggest that MSEW-induced endothelial dysfunction may be mediated by an interplay of increased circulating red blood cells and elevated ET-1 production. Further studies are necessary to determine the exact role of these factors in this phenomenon.

Early life stress induces circulating factor(s) promoting endothelial dysfunction (1085.3)

In humans, early life stress (ELS) increases cardiovascular disease (CVD) risk and elevates circulating pro‐oxidant factors; however the relationship between ELS‐induced CVD and circulating pro‐oxidant factors is not known. In a mouse model of ELS, maternal separation with early weaning (MSEW), we previously showed that MSEW induces adult endothelial dysfunction. We hypothesized that MSEW promotes endothelial dysfunction by increasing circulating factors that increase superoxide (O2‐) production and reduce NO bioavailability in endothelial cells. MSEW litters underwent maternal separation during postnatal days (PD) 2‐16 for 4‐8h/day, and weaning at PD17. Control (CON) litters remained undisturbed and were weaned at PD21. To determine the impact of MSEW‐induced circulating factors on endothelial function, we incubated Mouse Aortic Endothelial Cells (MAECs) with plasma of CON or MSEW mice (25 ul plasma/ml media; 24 hr). MSEW plasma‐treated MAECs had increased O2‐ levels compared to CON (dihydroethidium‐HPLC; CON = 25.7 + 9.7 vs MSEW = 107.0 + 18.5 pmol/mg pr/hr; p < 0.001). Apocynin (300 μM; NADPH oxidase inhibitor) abolished this effect. However, no change in NO bioavailability (nitrite determination by HPLC) was found. In conclusion, MSEW may promote cardiovascular disease risk by inducing circulating factors that enhance endothelial O2‐ production in an NADPH oxidase‐dependent manner.Grant Funding Source: Supported by NIH grants 1F32 HL116145; P01 HL69999

Abstract 335: Vascular Histone Deacetylase Mediates Early Life Stress-Induced Endothelial Dysfunction

Chromatin remodeling is an important factor in the etiology of vascular pathologies. Also, early life stress (ELS) is linked to increased risk of vascular disease in adults. We used maternal separation with early weaning (MSEW) to study mechanisms of ELS-mediated adult vascular dysfunction in male C57BL/6J mice. Litters were subjected to maternal separation 4h/day (postnatal day (PD) 2-5) and 8h/day (PD6-16), and weaned at PD17. Control (CON) litters were undisturbed until weaning at PD21. Subsequent experiments were performed at 12 weeks old. MSEW blunted aortic ACh-mediated vasorelaxation (MSEW: 68% vs CON: 90%, p=0.01), while SNP-induced vasorelaxation was similar in CON and MSEW aortae. Apocynin (300 μM) and superoxide dismutase (100 U/mL) normalized MSEW-induced endothelial dysfunction. We hypothesize that ELS induces aortic endothelial dysfunction by increasing NADPH oxidase expression and/or decreasing nitric oxide synthase 3 (NOS3) expression. Aortic protein expression of NADPH oxidase subunit p67 was elevated in MSEW mice (45% increase from CON, n=11, p=0.02). NOS3 protein expression and NOS3 serine 1177 phosphorylation was not different between groups, indicating that NOS3 activation by phosphorylation does not contribute to ELS-induced endothelial dysfunction. We further hypothesize that chromatin modification mediates ELS-induced endothelial dysfunction. Aortic mRNA expressions of 84 chromatin modification enzymes (methyltransferases, demethylases, acetyltransferases, deacetylases) were assessed by qRT-PCR. Only histone deacetylase (HDAC) 1, 6 and 9 mRNA levels were significantly upregulated in MSEW aortae compared to CON (17%, 29% and 67% increase, respectively, p<0.05). However, only HDAC 9 protein expression was elevated in MSEW aortae (2 fold increase from CON, n=6, p=0.01). Accordingly, histone 3 lysine acetylation was slightly decreased in MSEW aortae (16% decrease from CON, n=6, p = 0.06). Pretreatment of aortae with an HDAC inhibitor, trichostatin A (TSA), normalized ACh-induced vasorelaxation in MSEW mice (MSEW: 68% vs MSEW + TSA: 88%, p=0.02), while not affecting ACh-induced vasorelaxation in CON mice. We conclude that ELS induces endothelial dysfunction, most likely, through an HDAC 9-mediated pathway.

Abstract 57: Histone Deacetylase Inhibition Attenuates Early Life Stress-Induced Endothelial Dysfunction

Epidemiological studies show that early life stress (ELS) is linked to cardiovascular disease in adulthood. We used a model of maternal separation with early weaning (MSEW) to study the mechanisms of ELS-mediated adult vascular dysfunction in male C57BL/6J mice. MSEW litters were subjected to maternal separation 4h/day (postnatal day (PD) 2 to 5) and 8h/day (PD6 to 16), and weaned at PD17. Control (CON) litters were undisturbed until weaning at PD21. All subsequent experiments were performed in adult mice (12 weeks old). We hypothesized that MSEW increases vascular inflammation and endothelial dysfunction in male mice. Systolic blood pressure (tail-cuff) of MSEW mice was not different from CON mice (109.3 + 10.9 vs 116.7 + 20.8 mmHg, respectively). Circulating soluble intercellular adhesion molecule (CON: 333.5 + 19.4 vs MSEW: 406.2 + 23.1 ng/ml; p = 0.03) and macrophage colony stimulating factor (CON: 737.4 + 19.6 vs MSEW: 945.3 + 65.4 pg/ml; p = 0.01) were elevated by MSEW. Also, aortic adventitial macrophage infiltration was increased in mice exposed to MSEW (F4/80 immunostaining; CON: 2.8 + 2.3 vs MSEW: 7.0 + 2.2 cells/mm 2 ; p = 0.05). We performed wire myography on thoracic aortae to determine vasorelaxation with cumulative concentration-response curve to acetylcholine (ACh; 1 X 10 -9 M to 3 X 10 -5 M) and sodium nitroprusside (SNP; 1 X 10 -10 M to 3 X 10 -5 M). MSEW induced blunted ACh-mediated vasorelaxation (MSEW: 67.6 + 5.8 vs CON: 89.9 + 2.7 % of phenylephrine constriction (% of PE), p = 0.01), while SNP-induced vasorelaxation was similar in CON and MSEW mice. We further hypothesized that MSEW-induced endothelial dysfunction is mediated via increased histone deacetylase (HDAC) expression. Real-time quantitative PCR revealed upregulation of HDAC 1, 6 and 9 in aortae of MSEW mice (1.28 + 0.12, 1.28 + 0.18 and 1.65 + 0.05 fold change from CON, respectively, p < 0.05). Pretreatment with trichostatin A (TSA), an HDAC inhibitor, normalized ACh-induced vasorelaxation in aortae of MSEW mice (MSEW: 67.6 + 5.8 vs MSEW + TSA: 88.44 + 3.2 % of PE, p = 0.02), while not affecting ACh-induced vasorelaxation in aortae from CON mice (CON: 89.9 + 2.7 vs CON + TSA: 90.3 + 4.5 % of PE). We conclude that ELS induces blood pressure-independent endothelial dysfunction through an HDAC-mediated pathway.