Male B6 Research Articles

Early treatment of a CD137L neutralizing antibody decreases mechanical hypersensitivity and functional recovery time following a sciatic nerve crush

Abstract To investigate the role of CD137L in pain development and nerve regeneration following peripheral nerve injury, we examined pain-like and functional recovery-related behavioral responses following intrathecal injection (IT) of a CD137L neutralizing antibody (αCD137L; daily day 0–7) after sciatic nerve crush (SNC) in male and female B6 mice. Mice were randomly assigned to 1 of 4 groups: no IT, PBS, 0.1 or 0.5μg of αCD137L, and then were evaluated for sensitivity (von Frey, Hargreaves, & cold plantar test), sensory (pin prick assay) and motor (hind limb grip strength, toe spread reflex and toe spacing score) functional recovery before surgery and on post-surgery days 1–77. On post-SNC day 1, all groups showed changes in sensitivity and decreased sensory and functional responses. By day 7, αCD137L treated mice showed reduced mechanical hypersensitivity compared to no IT and PBS-treated controls and were at baseline levels by day 42, about 1–2 weeks earlier than both control groups. All groups showed hyposensitivity to heat stimulus on day 1, but quickly returned to baseline with no group differences. On days 1–35 hyposensitivity to a cold stimulus was also found in all groups, but αCD137Ltreated mice were slightly more sensitive on days 49–77. Grip strength improved in all groups on day 14 without significant group differences. On days 3–14, αCD137L-treated groups had higher toe space scores compared to controls. Finally, sensory recovery (pin prick) in αCD137L-treated groups emerged on day 3, a week earlier than the control groups. Together, our results suggest that CD137L likely contributes to sensory hypersensitivity and functional changes early on following peripheral nervy injury.

Phenotypic variations in transferred progeny due to genotype of surrogate mother.

Does the genotype of the surrogate mother modulate the body composition and immunity of her offspring? C57BL/6J (B6) progenies carried by immunodeficient NOD SCID (NS) mothers had increased adaptive but decreased innate, immune responsiveness in comparison with the same genotype offspring carried by immunocompetent mothers, B6 and BALB/c (C); the B6 progenies carried by the same genotype mothers also showed higher body fat than the others. Differences in the major histocompatibility complex (MHC) genes between mother and foetus is considered as an important factor in prenatal embryo development, whereas the impact of such dissimilarity on the phenotype of the mature progeny is unclear. Transplantation of two-cell mouse embryos into recipient females of the different MHC (H2) genotypes was used as an approach to simulate three variants of the immunogenic mother-foetus interaction: (i) bidirectional immunogenic dialogue between B6 (H2b haplotype) embryos and C (H2d haplotype) surrogate mother; (ii) one-way immunogenic interaction between B6 embryos and immunodeficient NS (H2g7 haplotype) surrogate mother and (iii) reduced immunogenetic dialogue between embryos and surrogate mother of the same H2b haplotype resulting in only a maternal response to HY antigens of male foetuses. Delivered by Caesarean section, pups were fostered by lactating B6 females and weighed after weaning (n = 171). Body mass and composition and innate and adaptive immunity were assessed in selected progeny groups at 9-11 weeks of age. The study was performed on the specific pathogen-free mouse, inbred strains C57BL/6J, NOD SCID and BALB/c. Plasma progesterone in pregnant females was measured by enzyme-linked immunosorbent assay (ELISA). Body composition was determined by magnetic resonance spectroscopy using a low-field NMR spectrometer (EchoMRI, USA). To assess peritoneal macrophage responses (innate immunity) to anthrax, lactate dehydrogenase (LDH) and interleukin-1 (IL-1β) were measured in a culture medium 24 h after the addition of both anthrax-lethal factor and anthrax-protective antigen. To assess adaptive immunity, 9-10 males in experimental groups were infected with Helicobacter hepaticus. Faeces collected 2 and 4 weeks after infection was used for quantitative assessment of the H. hepaticus DNA by real-time polymerase chain reaction. IgA, interferon (IFN-γ), tumour necrosis factor (TNFα), interleukin-17 (IL-17) and interleukin-10 (IL-10) in colon tissue and IgG in serum were determined in samples collected 4 weeks after gavage with H. hepaticus using ELISA. For statistical analyses, ANCOVA, post hoc least significant difference (LSD) test, Student's t-test, Spearman rank correlations and χ2 test were performed. P-value <0.05 was considered as a statistically significant difference. ANCOVA with litter size and age as covariates revealed significant effects of the surrogate mother genotype on body mass and percent of fat in their adult progeny (F2149 = 15.60, P < 0.001 and F2149 = 5.02, P = 0.007, respectively). Adult B6 mice carried by B6 surrogate mothers were characterized by a higher percentage of body fat in comparison with offspring that were carried by NS and C females. In comparison with the male offspring carried by the B6 and C mothers, male B6 progenies carried by immunodeficient NS mothers had a higher humoral immune response (serum IgG) against oral infection with H. hepaticus, but lower in vitro macrophage IL-1β reaction to the anthrax. Four weeks after the infection of offspring, concentrations of serum IgG and colon IL-10 correlated positively with maternal progesterone on Day 4 after embryo transfer and negatively with DNA of H. hepaticus. One-way ANOVA confirmed a statistically significant impact of surrogate mother genotype on adaptive (IgG) and innate (IL-1β) immunity (F2.26 = 26.39, P < 0.001 and F2.27 = 5.89, P = 0.008, respectively). N/A. The main limitation of our study is the number of combinations of mother and foetus interactions, in particular, transfer of only one embryo genotype was used. Also, it is a descriptive study, which requires further analysis of the epigenetic mechanisms of the observed phenotypic effects of surrogate mother genotype. Our experimental data demonstrate that the transfer of inbred embryos to surrogate mothers of the different genotypes is a prospective experimental model for the study of epigenetic effects of the immunogenetic interactions between mother and foetus. The experimental approach tested in our study will be in demand for the development of criteria for choosing surrogate mothers. In particular, immunocompetence of the surrogate mother along with genetic distance of her MHC alleles to the transferred embryos have a significant impact on offspring development. This work was supported by the Russian FPI (6/099/2017), budget projects (0324-2016-0002 and 0324-2018-0016) and implemented using the equipment of the Centre for Genetic Resources of Laboratory Animals at ICG SB RAS, supported by the Ministry of Education and Science of Russia (Unique project identifier RFMEFI62117X0015). The authors report no conflicts of interest.

The time course of blood brain barrier leakage and its implications on the progression of methamphetamine-induced seizures

The initial goals of these experiments were to determine: 1) if blood-brain barrier (BBB) breakdown was a cause or an effect of METH-induced seizures; 2) all the brain regions where BBB is disrupted as seizures progress; and 3) the correlations between body temperature and vascular leakage and neurodegeneration. A fourth objective was added after initial experimentation to determine if sub-strain differences existed in adult male C57 B6 J (Jackson laboratories, JAX) versus C57 B6N (Charles River, CR) mice involving their susceptibility to BBB breakdown and seizure severity. With the 1st “maximal” intensity myoclonic-tonic seizure (MCT) varying degrees of IgG infiltration across the BBB (≤1 mm2) were prominent in olfactory system (OS) associated regions and in thalamus, hypothalamus and neocortex. IgG infiltration areas in the OS-associated regions of the bed nucleus of the stria terminalis, septum and more medial amygdala nuclei, and the hypothalamus were increased significantly by the time continuous behavioral seizures (CBS) developed. Mice receiving METH that had body temperatures of ≥40 °C had IgG infiltration along with MCT or CBS but peak body temperatures above 40 °C did not significantly increase IgG infiltration. Neurodegeneration seen at ≥6 h was restricted to the OS in both JAX and CR mice and was most prominent in the posteromedial cortical amygdaloid nucleus. Neurodegeneration in the anterior septum (tenia tecta) was seen only in the JAX mice. We hypothesize that METH-induced hypertension and hyperthermia lead to BBB breakdown and other vascular dysfunctions in the OS brain regions resulting in OS hyperexcitation. Excitation of the OS neural network then leads to the development of seizures. These seizures in turn exacerbate the energy depletions and the reactive oxygen stress produced by hyperthermia further damaging the BBB and vascular function. These events form a recurrent cycle that results in ever increasing seizure activity and neurotoxicity.

Effects of sex on ethanol conditioned place preference, activity and variability in C57BL/6J and DBA/2J mice

Previous studies of ethanol drinking in rodents have shown greater intake in females than in males, but the reasons behind this difference are unknown. To address one possible interpretation of the drinking difference, these studies tested the hypothesis that female and male mice differ in sensitivity to the rewarding effects of ethanol using the conditioned place preference (CPP) procedure. To increase the generalizability of the results, sex differences were examined in two inbred mouse strains known to differ in their sensitivity to ethanol reward: C57BL/6J (B6) and DBA/2J (D2). Mice were conditioned in an unbiased CPP procedure using either 1 or 2 g/kg ethanol. To detect possible differences in learning rate, they were tested once at the midpoint of conditioning and again after conditioning ended. As expected, CPP was stronger with 2 g/kg than with 1 g/kg, and D2 mice generally showed stronger CPP than B6 mice. However, there were no sex differences in the rate of CPP acquisition or in CPP magnitude, suggesting no sex difference in ethanol reward sensitivity as indexed by CPP. Nevertheless, there were sex differences in locomotor activity. B6 females were generally more active than B6 males during CPP acquisition whereas D2 females were slightly less active than D2 males during both CPP acquisition and preference testing. Unexpectedly, female mice showed more variability than males in the behavioral measures recorded in these studies, encouraging greater attention to variability in the design, analysis and interpretation of future studies of sex differences in mice.

Differential Effects of Niacin on High-Fat Diet–Induced Adipose Tissue Inflammation and Nonalcoholic Fatty Liver Disease in C57BL6/J and B6129SF2/J Mice

Pharmacological doses of niacin improve adipose tissue (AT) inflammation and nonalcoholic fatty liver disease (NAFLD) in rodents chronically fed a high-fat diet (HFD). However, recent mouse studies have demonstrated significant metabolic changes in different strains of mice fed a HFD. Therefore, the aim of this study was to assess the effect of niacin on both AT inflammation and liver steatosis in two mouse strains, C57BL6/J (B6) and B6129SF2/J (B6129), under HFD feeding. Thirty-two male B6 and 32 male B6129 mice were randomized into four groups: Chow/Vehicle (CV), Chow/Niacin (CN), HFD/Vehicle (HV), and HFD/Niacin (HN). They were fed either a chow (10% fat) or HFD (60% fat) for 20 weeks. Niacin (360 mg/kg/day) or vehicle was added to the drinking water from week 5 until the end of the study. As expected, HFD-fed mice gained more weight than chow-fed mice in both strains, with no difference in weight gain between strains. Crown-like structure (CLS) number, a hallmark of AT inflammation, was increased in HV mice of both strains compared to CV mice. In addition, HV B6 mice had higher CLS number than HV B6129 mice. In B6129 mice, niacin decreased CLS number in HN compared to HV mice, while this decrease was not observed in B6 mice. Liver weight to body weight (L/B) ratio, liver triglyceride (TG) content and NASH score was increased in HV compared to CV B6 mice. In contrast, in B6129 mice, only NASH score was increased in HV compared to CV controls. Niacin had no impact on L/B ratio, TG content, or NASH score in B6 mice. However, in B6129 mice, niacin increased all three parameters in HN compared to HV mice. In conclusion, there are strain differential effects on AT inflammation and NAFLD induced by HFD feeding. Interestingly, liver steatosis is significantly increased in HFD-fed B6129 mice treated with niacin. This increase is potentially due to methyl deficiency, as niacin is a potent hepatic methyl consumer and 129 mice are more sensitive to methyl deficiency. Disclosure H. Fang: None. E. Graff: None. Z. Li: None. R.L. Judd: None.

Sex and strain dependent differences in mucosal immunology and microbiota composition in mice

BackgroundA dysbiosis in the intestinal microbiome plays a role in the pathogenesis of several immunological diseases. These diseases often show a sex bias, suggesting sex differences in immune responses and in the intestinal microbiome. We hypothesized that sex differences in immune responses are associated with sex differences in microbiota composition.MethodsFecal microbiota composition (MITchip), mRNA expression in intestinal tissue (microarray), and immune cell populations in mesenteric lymph nodes (MLNs) were studied in male and female mice of two mouse strains (C57B1/6OlaHsd and Balb/cOlaHsd). Transcriptomics and microbiota data were combined to identify bacterial species which may potentially be related to sex-specific differences in intestinal immune related genes.ResultsWe found clear sex differences in intestinal microbiota species, diversity, and richness in healthy mice. However, the nature of the sex effects appeared to be determined by the mouse strain as different bacterial species were enriched in males and females of the two strains. For example, Lactobacillus plantarum and Bacteroides distasonis were enriched in B6 females as compared to B6 males, while Bifidobacterium was enriched BALB/c females as compared to BALB/c males. The strain-dependent sex effects were also observed in the expression of immunological genes in the colon. We found that the abundance of various bacteria (e.g., Clostridium leptum et rel.) which were enriched in B6 females positively correlated with the expression of several genes (e.g., Il-2rb, Ccr3, and Cd80) which could be related to immunological functions, such as inflammatory responses and migration of leukocytes. The abundance of several bacteria (e.g., Faecalibacterium prausnitzii et rel. and Coprobacillus et rel.- Clostridium ramosum et rel.) which were enriched in BALB/c males positively correlated to the expression of several genes (e.g., Apoe, Il-1b, and Stat4) related to several immunological functions, such as proliferation and quantity of lymphocytes. The net result was the same, since both mouse strains showed similar sex induced differences in immune cell populations in the MLNs.ConclusionsOur data suggests a correlation between microbiota and intestinal immune populations in a sex and strain-specific way. These findings may contribute to the development of more sex and genetic specific treatments for intestinal-related disorders.

Absence of CD137L decreases mechanical hypersensitivity and functional recovery time following sciatic nerve crush

Abstract CD137 Ligand (CD137L), a tumor necrosis factor superfamily molecule, is involved in immune responses such as migration, proliferation and production of cytokines. To investigate the involvement of CD137L in pain development and nerve regeneration following peripheral nerve injury, we examined various pain-like and functional recovery-related behavioral responses in CD137L knockout (KO) mice and wild type (WT) B6 mice following sciatic nerve crush (SNC). Male and female B6 WT and CD137LKO mice underwent a sham or SNC surgery and were evaluated for hypersensitivity (von Frey, Hargreaves &amp; cold water tail withdrawal tests), sensory (pin prick assay) and motor (hind limb grip strength, toe spread reflex and toe spacing score) functional recovery. Behaviors were assessed before surgery and on post-surgery days 1–77. On post-SNC Day 1, both KO and WT mice showed increased hypersensitivity and decreased sensory and functional responses. By Day 7, CD137LKO mice showed reduced mechanical hypersensitivity compared to WT and were responding at sham levels by Day 42, whereas WT controls reached sham levels by Days 56–63. Lack of CD137L did not alter warm or cold thermal hypersensitivity. Grip strength improved in both strains beginning on Day 7, but CD137LKO mice reached sham levels faster than WT controls (Day 17 vs. Day 70). No differences were found on toe spread reflex and toe spacing scores. Finally, pin prick scores revealed that sensory recovery in both strains of mice emerged on Day 7, but the CD137LKO mice responded similar to shams by Day 28, one week earlier than WT. Together, these results suggest that CD137L mediates the sensory hypersensitivity and impedes the restoration of sensory and motor functions following peripheral nerve injury.

Altered gastrointestinal motility involving autoantibodies in the experimental autoimmune encephalomyelitis model of multiple sclerosis.

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system that, in addition to motor, sensory, and cognitive symptoms, also causes constipation, which is poorly understood. Here, we characterize gastrointestinal (GI) dysmotility in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS and evaluate whether autoantibodies target the enteric nervous system (ENS) and cause dysmotility. EAE was induced in male SJL and B6 mice. GI motility was assessed in vivo and ex vivo in wild type (WT) and B cell-deficient mice. MS and EAE serum was used to survey potential targets in the ENS and changes in the ENS structure were characterized using immunohistochemistry. EAE mice developed accelerated gastric emptying and delayed whole GI transit with reduced colonic motility. Fecal water content was reduced, and colonic migrating myoelectrical complexes (CMMC) and slow waves were less frequent. Colons from EAE mice exhibited decreased GFAP levels in glia. Sera from MS patients and from EAE mice targeted ENS neurons and glia. B-cell deficiency in EAE protected against colonic dysmotility. Consistent with symptoms experienced in MS, we demonstrate that EAE mice widely exhibit features of GI dysmotility that persisted in the absence of extrinsic innervation, suggesting direct involvement of ENS neurocircuitry. The absence of GI dysmotility in B cell-deficient mice with EAE together with EAE and MS serum immunoreactivity against ENS targets suggests that MS could be classified among other diseases known to induce autoimmune GI dysmotility.

Multi‐Omic Approach to Identifying Altered Metabolic and Signaling Pathways in the Mouse Heart with Chronic Exposure to Atypical Antipsychotic Medications

Atypical (AA) antipsychotic drugs, including Risperidone (RIS) and Olanzapine (OLAN), cause cardiac arrhythmia and significant endocrine and metabolic risk factors for cardiovascular disease, including obesity, insulin resistance, hyperglycemia, and dyslipidemia. AA drugs have complex pharmacology, antagonizing G‐protein coupled receptors including: dopamine, serotonin, α‐adrenergic and muscarinic receptors. However, the underlying pharmacology linking cardiovascular morbidities to AA therapy is unknown. We hypothesize that AAs may alter cardiac energy metabolism and present early gene expression markers in the pathogenesis to cardiovascular disease. In this study we aim to investigate the mechanisms by which AAs may promote cardiometabolic dysfunction. Male B6 mice were dosed daily with either 5 mg/kg OLAN or 1 mg/kg RIS or vehicle control (V) for 4 weeks and chronic effects of AAs were assessed in whole heart ventricles by RNAseq, miRNAseq and Proteomic analysis. Results suggest changes in transcriptomic and proteomic profiles and in the mouse heart with chronic exposure to AAs relating to mitochondrial dysfunction, lipid oxidation and metabolism, contractility, cardiac hypertrophy and PPAR signaling. Further pathway analysis of these data sets using Ingenuity Pathway Analysis revealed altered canonical pathways and disease and function that are consistent between the RIS and OLAN treated groups compared to V. To further investigate the effect of AA treatment on cardiac mitochondrial function, cardiac mitochondria were isolated from female B6 mice dosed 4 weeks daily with V or 0.75 mg/kg RIS and oxygen consumption studies were performed on the XF24 extracellular flux analyzer (Agilent) to measure various respiratory states. Isolated mitochondria were well coupled and showed a RCR (OCRADP/OCROligo) of ~ 3. RIS treatment led to an increase in State II (Succinate) and State III (phosphorylating) respiration compared to V. In conclusion, our data demonstrate chronic effects of AAs on cardiac mitochondrial function and bioenergetics and indicators of cardiac function.Support or Funding InformationThis work was funded in part by NIH grant DK095413 awarded to KH and NIH grant AR067858 awarded to KJM.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

S13-3. Glial dysfunction and eplileptogenicity

Temporal lobe epilepsy (TLE) is the most common type of drug-resistant epilepsy, partly characterized by hippocampus sclerosis. Recent accumulating evidence show that changes in glial cells are obvious in TEL, but an involvement of glial changes in TLE is still a matter of debate. Here, we show that status epilepticus (SE) induced activation of microglia, followed by induction of “epileptogenic astrocytes” in the hippocampus. Pilocarpine (Pilo) was used to induce SE in male adult B6 mice. Morphological and functional changes in glia were assessed by immunohistochemical analysis and Ca2+ imaging in the hippocampal slices, respectively. Pilo increased a fast and transient microglial activation (1–3 days after SE), which was followed by sustained activation of astrocytes in the hippocampus (7–28 days after SW). Twenty-eight days after SE, the mice showed susceptibility to Pilo, suggesting induction of epileptogenicity. At this time point, reactive astrocytes displayed excess Ca2+ excitability, reduction of which resulted in inhibition of epileotogenicity. Taken together, after SE, astrocytes become “epileptogenic astrocytes”, whose aberrant Ca2+ excitability should be a cause of epileptogenicity.

V1b Receptor Antagonist SSR149415 and Naltrexone Synergistically Decrease Excessive Alcohol Drinking in Male and Female Mice.

A recent clinical trial found that pharmacological blockade of V1b receptors reduces alcohol relapse in alcohol-dependent patients. SSR149415 is a selective V1b receptor antagonist that has potential for development as an alcohol dependency treatment. In this study, we investigated whether SSR149415 alone or in combination with the mu-opioid receptor (MOP-r) antagonist naltrexone (NTN) would alter excessive alcohol drinking in mice. Both sexes of C57BL/6J (B6) mice were subjected to a chronic intermittent access (IA) drinking paradigm (2-bottle choice, 24-hour access every other day) for 3weeks. Sucrose and saccharin drinking were used as controls for alcohol-specific drug effects. Neuronal proopiomelanocortin (POMC) enhancer (nPE) knockout mice with hypothalamic-specific loss of POMC (including beta-endorphin, the main endogenous ligand of MOP-r) were used as a genetic control for the effects of NTN. Acute administration of SSR149415 (1 to 30mg/kg) reduced alcohol intake and preference in a dose-dependent manner in both male and female B6 mice after IA. To investigate potential synergistic effects between NTN and SSR149415, we tested 6 different combination doses of SSR149415 and NTN, and found that a combination of SSR149415 (3mg/kg) and NTN (1mg/kg) reduced alcohol intake profoundly at doses lower than the individual effective doses in both sexes of B6 mice. We confirmed the effect of SSR149415 on reducing alcohol intake in nPE-/- male mice, consistent with independent mechanisms by which SSR149415 and NTN decrease alcohol drinking. The combination of V1b antagonist SSR149415 with NTN at individual subthreshold doses shows potential in alcoholism treatment, possibly with less adverse effects.

Preliminary pharmacokinetics of tramadol hydrochloride after administration via different routes in male and female B6 mice

Objective1) To determine the pharmacokinetics of tramadol hydrochloride and its active metabolite, O-desmethyltramadol (M1), after administration through different routes in female and male C57Bl/6 mice; 2) to evaluate the stability of tramadol solutions; and 3) to identify a suitable dose regimen for prospective clinical analgesia in B6 mice. Study designProspective, randomized, blinded, parallel design. AnimalsA total of 18 male and 18 female C57Bl/6 mice (20–30 g). MethodsMice were administered 25 mg kg−1 tramadol as a bolus [intravenously (IV), intraperitoneally (IP), subcutaneously (SQ), orally per gavage (OSgavage)] over 25 hours [orally in drinking water (OSwater) or Syrspend SF (OSSyrsp)]. Venous blood was sampled at six predetermined time points over 4 to 31 hours, depending on administration route, to determine tramadol and M1 plasma concentrations (liquid chromatography and tandem mass spectrometry detection). Pharmacokinetic parameters were described using a noncompartmental model. The stability of tramadol in water (acidified and untreated) and Syrspend SF (0.20 mg mL−1) at ambient conditions for 1 week was evaluated. ResultsAfter all administration routes, Cmax was >100 ng mL−1 for tramadol and >40 ng mL−1 for M1 (reported analgesic ranges in man) followed by short half-lives (2–6 hours). The mean tramadol plasma concentration after self-administration remained >100 ng mL−1 throughout consumption time. M1 was found in the OSSyrs group only at 7 hours, whereas it was detectable in OSwater throughout administration. Tramadol had low oral bioavailability (26%). Short-lasting side effects were observed only after IV administration. Water and Syrspend SF solutions were stable for 1 week. Conclusions and clinical relevance1) At the dose administered, high plasma concentrations of tramadol and M1 were obtained, with half-life depending on the administration route. 2) Plasma levels were stable over self-consumption time. 3) Solutions were stable for 1 week at ambient conditions.

Influences of placental growth factor on mouse retinal vascular development.

Placental growth factor (PGF) is important for wound-healing and vascular collaterogenesis. PGF deficiency is associated with preeclampsia, a hypertensive disease of human pregnancy. Offspring born to preeclamptic mothers display cognitive impairments and brain vascular and neurostructural deviations. Low PGF production during development may contribute to alterations in offspring cerebrovascular beds. Retina is a readily accessible part of the central nervous system with a well-described pattern of vascular development in mice. Impacts of PGF deficiency were addressed during mouse retinal vascularization. Retinal vessels were compared between Pgf-/- and congenic C57BL/6 (B6) mice. PGF deficiency altered neonatal retinal vascularization patterns. Some anatomic alterations persisted into adulthood, particularly in males. Greater arterial wall collagen IV expression was found in adult Pgf-/- females. Pregnancy (studied in adult females at gestational days 11.5 or 18.5) induced subtle changes upon the mother's retinal vasculature but these pregnancy-induced changes did not differ between genotypes. Significant sex-related differences occurred between adult male and female B6 although sexually dimorphic retinal vascular differences were absent in B6 neonates. Overall, PGF has a role in retinal vascular angiogenesis and vessel organization during development but does not affect retinal vessel adaptations in adult females during pregnancy. Developmental Dynamics 246:700-712, 2017. © 2017 Wiley Periodicals, Inc.

Obesity and fatty liver are prevented by inhibition of the aryl hydrocarbon receptor in both female and male mice

Inhibition of the aryl hydrocarbon receptor (AHR) prevents Western diet–induced obesity and fatty liver in C57Bl/6J (B6) male mice. The AHR is a ligand-activated nuclear receptor that regulates genes involved in xenobiotic metabolism and T-cell differentiation. Here, we tested the hypothesis that AHR antagonism would also prevent obesity and fatty liver in female mice and that B6 mice (higher-affinity AHR) and congenic B6.D2 mice (lower-affinity AHR) would differentially respond to AHR inhibition. Female and male adult B6 and B6.D2 mice were fed control and Western diets with and without α-naphthoflavone (NF), an AHR inhibitor. A nonlinear mixed-model analysis was developed to project asymptote body mass. We found that obesity, adiposity, and liver steatosis were reduced to near control levels in all female and male B6 and B6.D2 experimental groups fed Western diet with NF. However, differences were noted in that female B6.D2 vs B6 mice on Western diet became more obese; and in general, female mice compared with male mice had a greater fat mass to body mass ratio, were less responsive to NF, and had reduced liver steatosis and hepatomegaly. We report that male mice fed Western diet containing NF or CH-223191, another AHR inhibitor, caused reduced mRNA levels of several liver genes involved in metabolism, including Cyp1b1 and Scd1, offering evidence for a possible mechanism by which the AHR regulates obesity. In conclusion, although there are some sex- and Ahr allelic-dependent differences, AHR inhibition prevents obesity and liver steatosis in both males and females regardless of the ligand-binding capacity of the AHR. We also present evidence consistent with the notion that an AHR-CYP1B1-SCD1 axis is involved in obesity, providing potentially convenient and effective targets for treatment.

An immunogenic phenotype in paternal antigen‐specific CD8+ T cells at embryo implantation elicits later fetal loss in mice

Central to pregnancy success is a state of T cell tolerance to paternal antigens, which is initiated at conception. The role and regulation of specific phenotypes of CD8+ T cells in mediating pregnancy tolerance is not clear. This study aimed to investigate the impact on pregnancy outcome of altering the cytokine environment during maternal CD8+ T cell priming in early pregnancy. Transgenic Act-mOVA male mice were mated to C57BL/6 (B6) females to generate fetuses expressing ovalbumin (OVA) as a model paternal antigen. OVA-reactive CD8+ OT-I T cells were activated in vitro with OVA in the presence of either transforming growth factor-β1 (TGFB1) plus interleukin-10 (IL10), or IL2, to mimic normal or dysregulated uterine conditions, respectively, and transferred into pregnant mice on gestational day 3.5. OT-I T cells activated with TGFB1 and IL10, like naive OT-I T cells, did not alter embryo implantation or fetal viability. In contrast, OT-I T cells activated with IL2 caused extensive fetal loss manifesting in mid-gestation. IL2-activated OT-I T cells expressed less FOXP3 and higher interferon-γ (IFNG) than cells activated with TGFB1 and IL10. Fetal loss did not occur in females mated with B6 males, demonstrating the antigen specificity of fetal loss, and was not abrogated by maternal genetic C1q deficiency indicating a mechanism independent of antibody-mediated cytotoxicity. These data indicate that alternative phenotypes generated in maternal CD8+ T cells at the time of priming with paternal antigens can impact pregnancy outcome, such that inappropriate activation of CD8+ T cells before implantation is capable of causing antigen-specific fetal loss later in pregnancy.

Sex-specific regulation of CNS autoimmunity by the signal lymphocytic activation molecule (Slam) locus

Abstract Multiple sclerosis (MS) is a demyelinating inflammatory disease of the central nervous system (CNS). Epidemiological studies have documented a 3–6 fold increase in MS incidence and prevalence in females over the last half-century, while remaining relatively stable in males. This establishes that gene×environment×sex interactions contribute to MS susceptibility. Genome-wide association studies (GWAS) of MS patients identified multiple candidate genes, including a locus containing the signal lymphocytic activation molecule (SLAM) family of receptors, a region rich in immune-relevant genes that is highly conserved between humans and mice. There are two major Slam haplotypes segregating in laboratory mice. Slam haplotype-1 is present in C57BL/6 (B6) mice and haplotype-2 is expressed in most other common inbred laboratory strains, including 129 mice. The congenic B6.129c1 mouse possesses the 129-derived Slam haplotype-2 locus on the B6 background, which allows for assessing the potential contribution of SLAM family genes to MS susceptibility using the myelin oligodendrocyte (MOG) model of experimental autoimmune encephalomyelitis (EAE). We show that male B6.129c1 congenic mice demonstrate significant protection against MOG-EAE, but only in males. Furthermore, draining lymph nodes from immunized B6.129c1 males, but not females, had increased Foxp3+ regulatory T cells (Treg) and IL-10 levels compared with B6, suggesting that SLAM/Slam haplotypes regulate autoimmunity by modulating the generation of immunoregulatory cells in a sex-specific manner. Taken together, our results support a role for the SLAM locus is MS pathogenesis, and reveal a novel sexual dimorphism in the genetic control of this autoimmune disease

Metabolomic Analysis of Microdialysis Samples from the Frontal Association Cortex (FrA) of C57BL/6J (B6) Mouse during Isoflurane Anesthesia

Preliminary data have begun to characterize the metabolome in mouse FrA during sleep (FASEB J 30.1, 2016). Anesthesia and sleep are different states of consciousness that share a number of trait similarities. This ongoing study is quantifying the similarities and differences in the FrA metabolome during sleep and anesthesia. Microdialysis samples have been collected from the FrA of adult, male B6 mice (n=12) anesthetized with isoflurane. All experiments have adhered to FASEB policies on humane use of animals in research. The samples are being analyzed using ultra‐performance liquid chromatography coupled with high‐resolution mass spectrometry on an Exactive Plus Orbitrap instrument. An untargeted metabolomics method is being used to identify brain metabolites (Anal Chem 82:3212, 2010). To date we have analyzed 15 samples from 3 mice during isoflurane (1.1%) anesthesia. The results revealed approximately 90 known molecules in each 25 μL microdialysis sample. In FrA, 44 analytes were common to states of sleep and anesthesia, including myo‐inositol, tryptophan, homovanillic acid, uracil, and pyruvate. State‐specific differences in metabolomic measures included 37 metabolites present during anesthesia that were not observed during sleep, and absence of 6 metabolites during anesthesia that were found during sleep. Known metabolites common to both sleep and anesthesia were analyzed by partial least squares discriminant analysis (PLS‐DA). Variable importance projection scores calculated from PLS‐DA analyses highlighted 20 of the 44 metabolites present during sleep and anesthesia that contributed significantly to the observed separation in the PLS‐DA. Histological comparisons of FrA microdialysis sites during sleep and anesthesia are pending. These emerging results support the interpretation that metabolomic analyses provide a powerful approach for developing a neurochemical cartography of lower level phenotypes putatively regulating states of consciousness.Support or Funding InformationNIH grants HL65252 &amp; Kavli Foundation (RL and HAB); MH099231 &amp; 1P01NS083514 (CC and GT)

Paternal preconception alcohol exposure imparts intergenerational alcohol-related behaviors to male offspring on a pure C57BL/6J background.

While alcohol use disorder (AUD) is a highly heritable condition, the basis of AUD in families with a history of alcoholism is difficult to explain by genetic variation alone. Emerging evidence suggests that parental experience prior to conception can affect inheritance of complex behaviors in offspring via non-genomic (epigenetic) mechanisms. For instance, male C57BL/6J (B6) mice exposed to chronic intermittent vapor ethanol (CIE) prior to mating with Strain 129S1/SvImJ ethanol-naïve females produce male offspring with reduced ethanol-drinking preference, increased ethanol sensitivity, and increased brain-derived neurotrophic factor (BDNF) expression in the ventral tegmental area (VTA). In the present study, we tested the hypothesis that these intergenerational effects of paternal CIE are reproducible in male offspring on an inbred B6 background. To this end, B6 males were exposed to 6 weeks of CIE (or room air as a control) before mating with ethanol-naïve B6 females to produce ethanol (E)-sired and control (C)-sired male and female offspring. We observed a sex-specific effect, as E-sired males exhibited decreased two-bottle free-choice ethanol-drinking preference, increased sensitivity to the anxiolytic effects of ethanol, and increased VTA BDNF expression; no differences were observed in female offspring. These findings confirm and extend our previous results by demonstrating that the effects of paternal preconception ethanol are reproducible using genetically identical, inbred B6 animals.

411 - Pulmonary Myeloperoxidase Activity Worsens Insulin Resistance in Obese Mice Exposed to Bacterial Endotoxin

Neutrophilic inflammation (NI) is a poorly known process occurring in the lung of obese subjects exposed to indoor airborne pollutants that may worsen many obesity-associated metabolic abnormalities, including insulin resistance (IR). Indeed, obese patients exposed to indoor-airborne pollutants show worse IR than those unexposed, however the reasons for this disparity are partially known. Bacterial lipopolysaccharide (LPS) is a stressor that when administrated by intratracheal instillation (ITI) causes NI in the lung. At sites of NI, myeloperoxidase (MPO) oxidizes chloride anions to hypochlorous acid/hypochlorite (HOCl/OCl − ) which can damage the lung, increase systemic oxidative stress/inflammation and thus worsen IR. Hydrazide 4-aminobenzoic acid (ABAH) is a fairly-specific inhibitor of MPO. Taurine is a non-cell-permeable scavenger of HOCl. Herein we hypothesized that inhibition of oxidative processes mediated by MPO in the obese lung can reduce IR in obesity. To test this hypothesis we used male B6 mice which were fed a high-chicken-fat diet and fructose (obese) and a low-fat diet and tap-water (control). During the last week of diet and on a daily basis both groups of mice were ITI with either PBS (vehicle), ABAH (10 µmol/mouse) or taurine (5nmol/mouse). The last day-of-treatment animals were ITI with 2.5 µg LPS/mouse or PBS alone; and 6 h later an intraperitoneal glucose tolerance test was performed. A total of 6 groups, for both control and obese mice, were compared (PBS, LPS, ABAH, ABAH+LPS, Tau and Tau+LPS). Compared to control, LPS treatment to obese mice increased more lung MPO activity, chlorotyrosine, nitrotyrosine, TNF-α; but reduced insulin sensitivity. These differences between control and obese mice upon treatment with LPS were abrogated when animals were pre-treated with either ABAH or taurine. We conclude that MPO-driven oxidative modifications in the lung of obese animals are responsible for worsening IR when and may provide a therapeutic target to reduce IR in obese subjects exposed to indoor-airborne pollutants. Supported by PROICO 2-3214 & PICT-2014-3369 (to DCR), PROICO 10-0414 (To SEGM) and PIP2015-2017-112215-0100603CO (To DCR, SEA & SEGM).

Interferon-gamma deficiency protects against aging-related goblet cell loss.

Aging is a well-recognized risk factor for dry eye. Interferon-gamma (IFN-γ) has been implicated in conjunctival keratinization and goblet cell loss in dry eye. We investigated the role of IFN-γ in age-related dry eye by evaluating young (8 weeks) and aged (15 months; 15M) C57BL/6 (B6) and IFN-γKO mice. Age effects on the conjunctiva and cornea epithelium were assessed with PAS staining and corneal staining, respectively. Expression of T cell-related cytokines (IL-17A, IFN-γ), chemokines (CXCL10 and CCL20), in the ocular surface epithelium was evaluated by real time PCR. A significant decrease in filled goblet cells was noted in 15M B6 mice and this was significantly lower than age and sex-matched IFN-γKO mice. Aged male B6 had significantly higher IFN-γ, and CXCL10 mRNA in their conjunctiva than female B6 mice. Aged IFN-γKO females had significantly higher IL-17A mRNA in conjunctiva than IFN-γKO males and B6 mice. Corneal barrier dysfunction was observed in 15M female B6 and aged IFN-γKO mice of both sexes; however it was significantly higher in IFN-γKO compared to B6 mice. While there was a significant increase in IL 17A, and CCL20 in corneas of aged female B6 and IFN-γKO mice compared to males, these changes were more evident in aged female IFN-γKO group.Partial resistance of IFN-γKO mice to aging-induced goblet cell loss indicates IFN-γ is involved in the age-related decline in conjunctival goblet cells. Increased corneal IL-17A expression paralleled corneal barrier disruption in aging female of both strains. IFN-γ appears to suppress IL-17A on the ocular surface.