Newborn Mouse Pups Research Articles

The gut microbiome, resistome, and mycobiome in preterm newborn infants and mouse pups: lack of lasting effects by antimicrobial therapy or probiotic prophylaxis

BackgroundEnhancing our understanding of the underlying influences of medical interventions on the microbiome, resistome and mycobiome of preterm born infants holds significant potential for advancing infection prevention and treatment strategies. We conducted a prospective quasi-intervention study to better understand how antibiotics, and probiotics, and other medical factors influence the gut development of preterm infants. A controlled neonatal mice model was conducted in parallel, designed to closely reflect and predict exposures. Preterm infants and neonatal mice were stratified into four groups: antibiotics only, probiotics only, antibiotics followed by probiotics, and none of these interventions. Stool samples from both preterm infants and neonatal mice were collected at varying time points and analyzed by 16 S rRNA amplicon sequencing, ITS amplicon sequencing and whole genome shotgun sequencing.ResultsThe human infant microbiomes showed an unexpectedly high degree of heterogeneity. Little impact from medical exposure (antibiotics/probiotics) was observed on the strain patterns, however, Bifidobacterium bifidum was found more abundant after exposure to probiotics, regardless of prior antibiotic administration. Twenty-seven antibiotic resistant genes were identified in the resistome. High intra-variability was evident within the different treatment groups. Lastly, we found significant effects of antibiotics and probiotics on the mycobiome but not on the microbiome and resistome of preterm infants.ConclusionsAlthough our analyses showed transient effects, these results provide positive motivation to continue the research on the effects of medical interventions on the microbiome, resistome and mycobiome of preterm infants.

Modeling Brain Tumors <em>In Vivo</em> Using Electroporation-Based Delivery of Plasmid DNA Representing Patient Mutation Signatures

Tumor models are critical for the preclinical testing of brain tumors in terms of exploring new, more efficacious treatments. With significant interest in immunotherapy, it is even more critical to have a consistent, clinically pertinent, immunocompetent mouse model to examine the tumor and immune cell populations in the brain and their response to treatment. While most preclinical models utilize orthotopic transplantation of established tumor cell lines, the modeling system presented here allows for a "personalized" representation of patient-specific tumor mutations in a gradual, yet effective development from DNA constructs inserted into dividing neural precursor cells (NPCs) in vivo. DNA constructs feature the mosaic analysis with the dual-recombinase-mediated cassette exchange (MADR) method, allowing for single-copy, somatic mutagenesis of driver mutations. Using newborn mouse pups between birth and 3 days old, NPCs are targeted by taking advantage of these dividing cells lining the lateral ventricles. Microinjection of DNA plasmids (e.g., MADR-derived, transposons, CRISPR-directed sgRNA) into the ventricles is followed by electroporation using paddles that surround the rostral region of the head. Upon electrical stimulation, the DNA is taken up into the dividing cells, with the potential of integrating into the genome. The use of this method has successfully been demonstrated in developing both pediatric and adult brain tumors, including the most common malignant brain tumor, glioblastoma. This article discusses and demonstrates the different steps of developing a brain tumor model using this technique, including the procedure of anesthetizing young mouse pups, to microinjection of the plasmid mix, followed by electroporation. With this autochthonous, immunocompetent mouse model, researchers will have the ability to expand preclinical modeling approaches, in efforts to improve and examine efficacious cancer treatment.

PD23-02 LONG LASTING IMPACT OF EARLY LIFE VOIDING DYSFUNCTION ON LOWER URINARY TRACT FUNCTION IN A MURINE MODEL

You have accessJournal of UrologyCME1 Apr 2023PD23-02 LONG LASTING IMPACT OF EARLY LIFE VOIDING DYSFUNCTION ON LOWER URINARY TRACT FUNCTION IN A MURINE MODEL Nao Iguchi, Duncan Wilcox, Anna Malykhina, and Dan Wood Nao IguchiNao Iguchi More articles by this author , Duncan WilcoxDuncan Wilcox More articles by this author , Anna MalykhinaAnna Malykhina More articles by this author , and Dan WoodDan Wood More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003296.02AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: The aim of this study was to investigate long term outcome of voiding dysfunction during early childhood in mice. Despite the association between childhood dysfunctional voiding and lifelong bladder dysfunction with or without renal failure, the underlying mechanism remains poorly understood. Neonatal maternal separation (NMS) disturbs the physiological voiding cycle in mouse pups; which is normally maintained by perigenital-bladder reflex via their mother licking the perineum. METHODS: Newborn mouse pups were divided into control and NMS groups after birth. NMS pups were removed from their dam and housed individually (6 h/day) from postnatal day 2 to 14. Pups in the control group stayed with their dam all the time. Long-term effects of early life voiding dysfunction on LUT were assessed in vivo by void spot assay at 3-, 6-, and 12-months old, and in vitro by detrusor contractility studies using bladder strips at 12 months old. RESULTS: NMS male mice showed urinary frequency with small voids by 1.6 to 2.2-fold through middle-age (12mo≈40yr-old human) (Figure 1). There was a trend towards increased number of small voids in females experienced NMS compared to the control group, however, the change was statistically insignificant (1.2 to 1.5-fold, p>0.05). A significant increase in nerve-mediated (electric field stimulation, EFS) and purinergic agonist (ATP)-induced detrusor contractility was observed in NMS group compared to the control group (1.4-fold, p<0.05). There were no changes in contractility in response to carbachol or substance-P (SP), however, preincubation of the bladder strips with SP induced a 1.6-fold larger decrease in nerve-mediated contractility (TmaxEFS) in NMS group than that in the control group (p<0.005, Figure 2). CONCLUSIONS: Our results provide evidence that the early life disturbance of normal voiding cycle had prolonged impacts on LUT function accompanied with an enhanced involvement of purinergic and peptidergic neurons/signaling in detrusor contractility in later of life. Source of Funding: Ponzio Family Endowment Fund (D.W.) © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e670 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Nao Iguchi More articles by this author Duncan Wilcox More articles by this author Anna Malykhina More articles by this author Dan Wood More articles by this author Expand All Advertisement PDF downloadLoading ...

Effect of Various Preterm Infant Milk Formulas on NEC-Like Gut Injury in Mice.

Formula feeding is an important risk factor for the development of necrotizing enterocolitis in preterm infants. The potential harmful effects of different preterm formulas on the developing intestinal tract remain incompletely understood. Here we demonstrate that feeding newborn mouse pups with various preterm formulas resulted in differing effects on intestinal inflammation, apoptosis, and activation of the pro-inflammatory transcription factor NFκB. 16S rRNA sequencing revealed that each preterm formula resulted in significant gut microbial alterations that were different from dam-fed controls. Formula feeding with EleCare and Similac Special Care caused greater intestinal injury compared to NeoSure. Pre-treatment with Lactobacillus rhamnosus GG ameliorated severity of intestinal injury from EleCare and Similac Special Care. Our findings indicate that not all preterm formulas are the same, and different formulations can have varying effects on intestinal inflammation, apoptosis, and microbiome composition.

Hyperoxia‐Induced Preretinal Membrane Formation and Retinal Detachment in the Mouse

BackgroundRetinal detachment (RD) is a vision‐threatening consequence of several conditions including retinopathy of prematurity (ROP). Numerous studies have established the connection of high oxygen concentration altering retinal development in preterm infants, leading to RD and blindness in later stages. The goal of this study was to develop a reproducible, non‐invasive model mouse of human ROP that could be used to study the cellular events leading to the formation of tractional preretinal membranes and RD.HypothesisPlacing C57BL/6J mice in hyperoxic conditions for one week starting at birth will induce a non‐traumatic sequence of retinopathy leading to tractional RD that mimics the pathophysiology of detachment seen in ROP.MethodsC57BL/6J newborn mice were placed into a hyperoxic environment (65% oxygen saturation) for 7 days then reared in ambient air. Retinas were observed at weeks 4, 5, and 7 via spectral‐domain optical coherence tomography (OCT). Eyes were processed for histology and immunostaining for the myofibroblast marker alpha‐smooth muscle actin (α‐SMA).ResultsFour weeks after birth, 100% of mice placed in hyperoxia exhibited retinal blebs, 91% had developed preretinal membranes, and 9% had RD. At 5 weeks, 100% and 27% had preretinal membranes and RD, respectively. At week 7, the number of mice with RD increased to 33%. Preretinal membranes arose at the surface of the retina above the optic nerve head. RD occurred between the neurosensory retina and the underlying retinal pigment epithelium. Myofibroblasts were detected with the antibody to α‐SMA in the preretinal membranes and above areas of RD.ConclusionThe hyperoxia‐induced retinopathy model was confirmed to induce retinal pathology in newborn mouse pups. Retinal pathology progressed during a 7‐week time period (i.e. retinal blebs to preretinal membranes and folds to retinal detachment). This noninvasive model replicates the oxygen‐induced retinopathy seen in human ROP. The model is useful for investigating the origin of cells that contribute to the formation of tractional retinopathy, especially PRMs and RD. The presence of α‐SMA‐positive cells overlying areas of retinopathy suggests the contractile property of myofibroblasts induces tractional RD.

Chitin-Derived AVR-48 Prevents Experimental Bronchopulmonary Dysplasia (BPD) and BPD-Associated Pulmonary Hypertension in Newborn Mice.

Bronchopulmonary dysplasia (BPD) is the most common complication of prematurity and a key contributor to the large health care burden associated with prematurity, longer hospital stays, higher hospital costs, and frequent re-hospitalizations of affected patients through the first year of life and increased resource utilization throughout childhood. This disease is associated with abnormal pulmonary function that may lead to BPD-associated pulmonary hypertension (PH), a major contributor to neonatal mortality and morbidity. In the absence of any definitive treatment options, this life-threatening disease is associated with high resource utilization during and after neonatal intensive care unit (NICU) stay. The goal of this study was to test the safety and efficacy of a small molecule derivative of chitin, AVR-48, as prophylactic therapy for preventing experimental BPD in a mouse model. Two doses of AVR-48 were delivered either intranasally (0.11 mg/kg), intraperitoneally (10 mg/kg), or intravenously (IV) (10 mg/kg) to newborn mouse pups on postnatal day (P)2 and P4. The outcomes were assessed by measuring total inflammatory cells in the broncho-alveolar lavage fluid (BALF), chord length, septal thickness, and radial alveolar counts of the alveoli, Fulton’s Index (for PH), cell proliferation and cell death by immunostaining, and markers of inflammation by Western blotting and ELISA. The bioavailability and safety of the drug were assessed by pharmacokinetic and toxicity studies in both neonatal mice and rat pups (P3-P5). Following AVR-48 treatment, alveolar simplification was improved, as evident from chord length, septal thickness, and radial alveolar counts; total inflammatory cells were decreased in the BALF; Fulton’s Index was decreased and lung inflammation and cell death were decreased, while angiogenesis and cell proliferation were increased. AVR-48 was found to be safe and the no-observed-adverse-effect level (NOAEL) in rat pups was determined to be 100 mg/kg when delivered via IV dosing with a 20-fold safety margin. With no reported toxicity and with a shorter half-life, AVR-48 is able to reverse the worsening cardiopulmonary phenotype of experimental BPD and BPD-PH, compared to controls, thus positioning it as a future drug candidate.

Early-life adversity selectively interrupts the dendritic differentiation of dorsolateral striatal neurons in male mice.

The effects of early-life adversity (ELA) on dendritic differentiation of striatal neurons were investigated in the dorsal striatum including the dorsomedial striatum and dorsolateral striatum (DMS and DLS, respectively). An animal model of ELA was created by changing the growth environment of newborn mouse pups by giving limited bedding and nesting materials from postnatal day 2 to day 9 (P2-P9). One week after the stress paradigm (P16), the dendritic branches and spines of striatal spiny neurons as well as the synapses represented by postsynaptic density protein-95 (PSD-95) in DMS and DLS were stereologically analyzed. Adverse experience in early life selectively affected the spiny neurons in DLS, leading to abundant proximal dendritic branches and an increased number of filopodia-like protrusions, but a reduced number of dendritic spines. The selective effects of stress on neurons in DLS were further identified by reduced expression of PSD-95, including a reduced optical density of PSD-95 immunoreactivity and fewer individual PSD-95 immunoreactive synapses in this region. Notably, stress in early life affected either D1 or D2 dopamine receptor-expressing DLS neurons. These findings suggest that adverse early-life experience delayed the maturation of dendritic spines on neurons in the dorsolateral striatum. Altered dendritic differentiation provoked by stress in early life may contribute critically to the formation of proper neuronal circuits in the dorsal striatum and, therefore, affect striatum-dependent habitual behavior and emotional function later in life.

Minoxidil Cannot Be Used To Target Lysyl Hydroxylases during Postnatal Mouse Lung Development: A Cautionary Note.

The lysyl hydroxylases (procollagen-lysine 5-dioxygenases) PLOD1, PLOD2, and PLOD3 have been proposed as pathogenic mediators of stunted lung development in bronchopulmonary dysplasia (BPD), a common complication of preterm birth. In affected infants, pulmonary oxygen toxicity stunts lung development. Mice lacking Plod1 exhibit 15% mortality, and mice lacking Plod2 or Plod3 exhibit embryonic lethality. Therefore, to address any pathogenic role of lysyl hydroxylases in stunted lung development associated with BPD, minoxidil was administered to newborn mice in an oxygen toxicity-based BPD animal model. Minoxidil, which has attracted much interest in the management of systemic hypertension and androgenetic alopecia, can also be used to reduce lysyl hydroxylase activity in cultured cells. An in vivo pilot dosing study established 50 mg⋅kg-1⋅day-1 as the maximum possible minoxidil dose for intraperitoneal administration in newborn mouse pups. When administered at 50 mg⋅kg-1⋅day-1 to newborn mouse pups, minoxidil was detected in the lungs but did not impact lysine hydroxylation, collagen crosslinking, or lysyl hydroxylase expression in the lungs. Consistent with no impact on mouse lung extracellular matrix structures, minoxidil administration did not alter the course of normal or stunted lung development in newborn mice. At doses of up to 50 mg⋅kg⋅day-1, pharmacologically active concentrations of minoxidil were not achieved in neonatal mouse lung tissue; thus, minoxidil cannot be used to attenuate lysyl hydroxylase expression or activity during mouse lung development. These data also highlight the need for new and specific lysyl hydroxylase inhibitors. SIGNIFICANCE STATEMENT: Extracellular matrix crosslinking is mediated by lysyl hydroxylases, which generate hydroxylated lysyl residues in procollagen peptides. Deregulated collagen crosslinking is a pathogenic component of a spectrum of diseases, and thus, there is interest in validating lysyl hydroxylases as pathogenic mediators of disease and potential "druggable" targets. Minoxidil, administered at the maximum possible dose, did not inhibit lysyl hydroxylation in newborn mouse lungs, suggesting that minoxidil was unlikely to be of use in studies that pharmacologically target lysyl hydroxylation in vivo.

Impact of litter size on survival, growth and lung alveolarization of newborn mouse pups

BackgroundLung alveolarization, the development of the alveoli, is disturbed in preterm infants with bronchopulmonary dysplasia (BPD), the most common complication of preterm birth. Animal models based on oxygen toxicity to the developing mouse lung are used to understand the mechanisms of stunted alveolarization in BPD, and to develop new medical management strategies for affected infants. The toxicity of genetic and pharmacological interventions, together with maternal cannibalism, reduce mouse litter sizes in experimental studies. The impact of litter size on normal and stunted lung alveolarization is unknown, but may influence data interpretation. The aim of the study was to assess the impact of litter size on normal and oxygen-stunted lung alveolarization in mice. MethodsBPD was experimentally modelled in newborn C57BL/6J mice by exposure to 85% O2 in the inspired air for the first 14 days of post-natal life. Perturbations to mouse lung architecture were assessed by design-based stereology, in which the alveolar density, total number of alveoli, gas-exchange surface area, and the septal thickness were estimated. ResultsLitter sizes of a single mouse were not viable to post-natal day 14. Normal lung alveolarization was comparable in mouse pups in litters of 2, 4, 6, and 8 pups per litter. Hyperoxia was equally effective at stunting lung alveolarization in mouse pups in litters of 2, 4, 6, and 8 pups per litter. ConclusionsStudies on normal lung alveolarization as well as alveolarization stunted by oxygen toxicity can be undertaken in mouse litters as small as two pups, and as large as eight pups. There is no evidence to suggest that data cannot be compared within and between litters of two to eight mouse pups.

Lrrk2 alleles modulate inflammation during microbial infection of mice in a sex-dependent manner.

Variants in the leucine-rich repeat kinase-2 (LRRK2) gene are associated with Parkinson's disease, leprosy, and Crohn's disease, three disorders with inflammation as an important component. Because of its high expression in granulocytes and CD68-positive cells, LRRK2 may have a function in innate immunity. We tested this hypothesis in two ways. First, adult mice were intravenously inoculated with Salmonella typhimurium, resulting in sepsis. Second, newborn mouse pups were intranasally infected with reovirus (serotype 3 Dearing), which induced encephalitis. In both mouse models, wild-type Lrrk2 expression was protective and showed a sex effect, with female Lrrk2-deficient animals not controlling infection as well as males. Mice expressing Lrrk2 carrying the Parkinson's disease-linked p.G2019S mutation controlled infection better, with reduced bacterial growth and longer animal survival during sepsis. This gain-of-function effect conferred by the p.G2019S mutation was mediated by myeloid cells and was abolished in animals expressing a kinase-dead Lrrk2 variant, p.D1994S. Mouse pups with reovirus-induced encephalitis that expressed the p.G2019S Lrrk2 mutation showed increased mortality despite lower viral titers. The p.G2019S mutant Lrrk2 augmented immune cell chemotaxis and generated more reactive oxygen species during virulent infection. Reovirus-infected brains from mice expressing the p.G2019S mutant Lrrk2 contained higher concentrations of α-synuclein. Animals expressing one or two p.D1994S Lrrk2 alleles showed lower mortality from reovirus-induced encephalitis. Thus, Lrrk2 alleles may alter the course of microbial infections by modulating inflammation, and this may be dependent on the sex and genotype of the host as well as the type of pathogen.

Effect of maternal yokukansan treatment on somatosensory map formation in the cerebral cortex of newborn mouse pups

ABSTRACTAimThe effectiveness of traditional Japanese herbal medicine (kampo) is gradually attracting worldwide attention. Although yokukansan has been shown to be clinically effective in suppressing maternal post‐partum depression, the safety of maternal yokukansan treatment in newborn infants has not been fully established.MethodsHerein we examined the effect of maternal yokukansan treatment on neuronal circuit formation using somatosensory maps in the cerebral cortex of the newborn mouse brain.ResultsSomatosensory maps in newborn mouse pups were formed normally at postnatal day (P)5, even though yokukansan was given to lactating mouse dams. Paroxetine injected into pups strongly inhibited somatosensory map formation in mouse pups at P5, whereas paroxetine injected into lactating dams did not inhibit somatosensory map formation in pups. Yokukansan also did not affect the timing of somatosensory map formation in mouse pups at P3 and P4.ConclusionMaternal yokukansan treatment does not have apparent adverse effects on somatosensory map formation in the brain of mouse pups.

Transmission of microRNA antimiRs to mouse offspring via the maternal-placental-fetal unit.

The emergence of microRNA as regulators of organogenesis and tissue differentiation has stimulated interest in the ablation of microRNA expression and function during discrete periods of development. To this end, inducible, conditional modulation of microRNA expression with doxycycline-based tetracycline-controlled transactivator and tamoxifen-based estrogen receptor systems has found widespread use. However, the induction agents and components of genome recombination systems negatively impact pregnancy, parturition, and postnatal development; thereby limiting the use of these technologies between late gestation and the early postnatal period. MicroRNA inhibitor (antimiR) administration also represents a means of neutralizing microRNA function in vitro and in vivo. To date, these studies have used direct (parenteral) administration of antimiRs to experimental animals. As an extension of this approach, an alternative means of regulating microRNA expression and function is described here: the maternal–placental–fetal transmission of antimiRs. When administered to pregnant dams, antimiRs were detected in offspring and resulted in a pronounced and persistent reduction in detectable steady-state free microRNA levels in the heart, kidney, liver, lungs, and brain. This effect was comparable to direct injection of newborn mouse pups with antimiRs, although maternal delivery resulted in fewer off-target effects. Furthermore, depletion of steady-state microRNA levels via the maternal route resulted in concomitant increases in steady-state levels of selected microRNA targets. This novel methodology permits the temporal regulation of microRNA function during late gestation and in neonates, without recourse to conventional approaches that rely on doxycycline and tamoxifen, which may confound studies on developmental processes.

Tamoxifen dosing for Cre-mediated recombination in experimental bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD) is the most common complication of preterm birth characterized by blunted post-natal lung development. BPD can be modelled in mice by exposure of newborn mouse pups to elevated oxygen levels. Little is known about the mechanisms of perturbed lung development associated with BPD. The advent of transgenic mice, where genetic rearrangements can be induced in particular cell-types at particular time-points during organogenesis, have great potential to explore the pathogenic mechanisms at play during arrested lung development. Many inducible, conditional transgenic technologies available rely on the application of the estrogen-receptor modulator, tamoxifen. While tamoxifen is well-tolerated and has been widely employed in adult mice, or in healthy developing mice; tamoxifen is not well-tolerated in combination with hyperoxia, in the most widely-used mouse model of BPD. To address this, we set out to establish a safe and effective tamoxifen dosing regimen that can be used in newborn mouse pups subjected to injurious stimuli, such as exposure to elevated levels of environmental oxygen. Our data reveal that a single intraperitoneal dose of tamoxifen of 0.2mg applied to newborn mouse pups in 10μl Miglyol vehicle was adequate to successfully drive Cre recombinase-mediated genome rearrangements by the fifth day of life, in a murine model of BPD. The number of recombined cells was comparable to that observed in regular tamoxifen administration protocols. These findings will be useful to investigators where tamoxifen dosing is problematic in the background of injurious stimuli and mouse models of human and veterinary disease.

Do Laboratory Mouse Females that Lose Their Litters Behave Differently around Parturition?

Efficiency in laboratory mouse breeding is hampered by poor reproductive performance, including the loss of entire litters shortly after birth. However, the underlying mechanisms are not yet fully understood and establishing the cause of death in laboratory mouse pups can be complicated. Newborn mouse pups are generally hidden in nests, dead pups are often eaten by the female, and the widespread practice of leaving periparturient females undisturbed complicates inspection, which may delay the discovery of pup loss. In order to efficiently prevent problems with litter loss, it is important to find key factors for survival. We investigated differences in periparturient behavior between female laboratory mice whose pups survived until weaning and females whose entire litters were lost. Video recordings of 82 primiparous females of the C57BL/6 strain or knockouts with C57BL/6 background were used. The mice were observed from 24 h before until 24 h after parturition and female behaviors coded using a pre-established ethogram. The relationship between behavior and survival was analyzed using logistic models, where litter survival was regressed on the proportion of 30-s observations with at least one occurrence of the behavior. We found that females with surviving litters performed more nest building behavior during the last 24 h before parturition (p = 0.004) and spent less time outside the nest during the entire observation period (p = 0.001). Increased litter survival was also associated with more passive maternal behaviors and the female ignoring still pups less. Females that lost their litters performed more parturition-related behaviors, suggesting prolonged labor. The results indicate that maternal behavior plays a significant role in laboratory mouse pup survival. Complications at parturition also contribute to litter mortality.

Essentiality of arachidonic acid intake in murine early development

We previously reported the importance of long-chain polyunsaturated fatty acid (LC-PUFA (>C20)) intake, including arachidonic acid (ARA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), for growth. This follow-up study focuses on ARA using a novel artificial rearing model during the lactation period in delta-6-desaturase knockout (D6D-KO) mice. Newborn D6D-KO male mouse pups were separated from dams within 48 hours and fed artificial milks containing 18-C essential fatty acids (EFAs) (16–17% LA, 3.8–4.1% ALA) with or without 1.2% ARA. After weaning, mice were maintained on similar diets: 15% LA, 2.3–2.4% ALA with or without 1.9% ARA. As a reference group, new born wild type (WT) male mouse pups were maintained by artificial milk and diet containing LA and ALA without ARA. Aspects of brain function were measured behaviorally (motor activity and rota-rod test) when mice were age 9 weeks. Body weight in the KO-Cont group was significantly lower (approximately 30%) than in the WT-Cont group, but this decrease was ameliorated by providing ARA in the KO-ARA group. The motor activity and coordination in the KO-Cont group decreased markedly compared to the WT-Cont group. The KO-ARA group had a tendency toward deteriorated motor coordination, although the motor activity was significantly enhanced compared to the KO-Cont group. In KO-ARA group brains, the level of ARA was increased and DHA decreased compared to WT-Cont. These results suggest that intake of LA and ALA only is insufficient to support healthy growth, and that ARA is also required, at least during the lactation period. These findings also suggested that continued intake of relatively high levels of ARA and without supplemental DHA during development led to an increased motor activity above that of WT animals. These studies indicate that both ARA dose and proper combination with DHA must be delineated to define optimal growth and behavioral function.

Protocol for Primary Microglial Culture Preparation.

Primary microglia, in either mono-culture or co-culture with neurons or astrocytes, are a powerful tool for studying mechanisms underlying microglial inflammatory responses and cell type-specific interactions in the central nervous system (CNS). This protocol provides the details of how to prepare high purity primary microglia from newborn mouse pups. The overall steps include brain cell dissociation, mixed glial cell culture, and microglia isolation.

Standardisation of oxygen exposure in the development of mouse models for bronchopulmonary dysplasia.

ABSTRACTProgress in developing new therapies for bronchopulmonary dysplasia (BPD) is sometimes complicated by the lack of a standardised animal model. Our objective was to develop a robust hyperoxia-based mouse model of BPD that recapitulated the pathological perturbations to lung structure noted in infants with BPD. Newborn mouse pups were exposed to a varying fraction of oxygen in the inspired air (FiO2) and a varying window of hyperoxia exposure, after which lung structure was assessed by design-based stereology with systemic uniform random sampling. The efficacy of a candidate therapeutic intervention using parenteral nutrition was evaluated to demonstrate the utility of the standardised BPD model for drug discovery. An FiO2 of 0.85 for the first 14 days of life decreased total alveoli number and concomitantly increased alveolar septal wall thickness, which are two key histopathological characteristics of BPD. A reduction in FiO2 to 0.60 or 0.40 also caused a decrease in the total alveoli number, but the septal wall thickness was not impacted. Neither a decreasing oxygen gradient (from FiO2 0.85 to 0.21 over the first 14 days of life) nor an oscillation in FiO2 (between 0.85 and 0.40 on a 24 h:24 h cycle) had an appreciable impact on lung development. The risk of missing beneficial effects of therapeutic interventions at FiO2 0.85, using parenteral nutrition as an intervention in the model, was also noted, highlighting the utility of lower FiO2 in selected studies, and underscoring the need to tailor the model employed to the experimental intervention. Thus, a state-of-the-art BPD animal model that recapitulates the two histopathological hallmark perturbations to lung architecture associated with BPD is described. The model presented here, where injurious stimuli have been systematically evaluated, provides a most promising approach for the development of new strategies to drive postnatal lung maturation in affected infants.

Abstract 3299: The role of H3K9 acetylation and tri-methylation in stem cell radiosensitivity

Abstract Radiation therapy is often associated with normal tissue injury as an undesired side effect. This is promoted by the depletion of normal stem cells and a decreased regenerative capacity of the affected organs. To reduce normal tissue injury during radiotherapy protective treatments have to be developed that improve stem cell survival without protecting tumor cells. In this study we investigate the acetylation and methylation of histone 3 at lysine 9 (H3K9), that is responsive to DNA damage and uniquely regulated in stem cells, as potential target for the development of radioprotective therapeutics. We analyzed stem cells in mouse tissue niches in vivo, murine embryonic stem cells in culture and neural stem cells freshly isolated from newborn mouse pups. We investigate repair factors, histone modifications and apoptosis pathways by immunofluorescence staining, western blotting and flow cytometry after X-ray irradiation or micro-irradiation of individual cells. H3K9 modifying enzymes are targeted with inhibitors, activators, RNA interference or overexpression by plasmid transfection and the effect on stem cells’ radiosensitivity evaluated. We show that stem cells are highly radio-sensitive and have reduced DNA repair with low activation of DNA damage factors like ATM. We screened for stem cell specific levels of histone modifications that are responsive to DNA damage and discovered that stem cells of adult tissue niches like brain and testis as well as murine embryonic and neuronal stem cells show very high levels of H3K9ac, while H3K9me3 levels are decreased. In tumor cells H3K9 acetylation (H3K9ac) was shown to be reduced after damage induction, while H3K9 tri-methylation (H3K9me3) is increased to fully activate the repair factor ATM. We hypothesize that in stem cells, an inept transition between H3K9 acetylation and tri-methylation prevents robust activation of ATM leading to high radiosensitivity. In agreement with this hypothesis, we find reduced down-regulation of H3K9ac in stem cells at the DNA damage sites. To transiently improve the H3K9ac/me3 switch in stem cells in order to enhance activation of DNA repair factors and stem cell survival after irradiation, we aim to transiently reduce H3K9 acetylation by targeting the responsible acetyltransferases and deacetylases. We show expression levels of H3K9 modifying enzymes in stem cells before and after irradiation as well as preliminary results of the effect of down- or up-regulation of H3K9 modifying enzymes on stem cell radiosensitivity. We conclude that elevated H3K9 acetylation and reduced H3K9 tri-methylation levels in stem cells and their responsible H3K9 modifying enzymes are potential targets to improve ATM activation and stem cell survival after irradiation. Citation Format: Barbara Meyer, Keith M. Jacobs, Suyash Raj, Cheri L. Zobel, Dennis E. Hallahan, Girdhar G. Sharma. The role of H3K9 acetylation and tri-methylation in stem cell radiosensitivity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3299. doi:10.1158/1538-7445.AM2015-3299

WNT signaling in microglia and the glioma microenvironment.

WNT signaling in microglia and the glioma microenvironment Gunnar Schulte WNT signaling is important during embryonic development and organogenesis having specific roles in the development of the CNS such as regulation of neural tube formation, axon guidance and CNS stem cell regulation. Our work has recently established a role of WNT signaling in the regulation of the brain’s macrophages, the microglia and thus WNTs emerge as novel regulators of CNS inflammatory responses. First of all, it appeared that b-catenin levels are elevated in microglia in Alzheimer disease (AD) brains as well as microglia cells in AD mouse models. Employing in vitro studies of primary mouse microglia isolated from newborn mouse pups indicated that both WNT-3A and WNT-5A induce diverse signaling routes in microglia leading to differential proinflammatory modulation of the cells. Interestingly, the net effect of WNT stimulation on the inflammatory potential of mouse microglia is context dependent. While WNTs increase inflammatory markers when giving to microglia alone, they are able to act in an anti-inflammatory manner when microglia are activated by prestimulation with lipopolysaccharides. Our findings thereby indicate that WNTs act on microglia as a homeostatic regulator, further underlined by yet unpublished data that suggest that WNT-5A is elevated in human glioma associated with a distinct inflammatory signature of the tumor as well as a substantial invasion of microglia.

Ionizing Radiation Perturbs Cell Cycle Progression of Neural Precursors in the Subventricular Zone Without Affecting Their Long-Term Self-Renewal

Damage to normal human brain cells from exposure to ionizing radiation may occur during the course of radiotherapy or from accidental exposure. Delayed effects may complicate the immediate effects resulting in neurodegeneration and cognitive decline. We examined cellular and molecular changes associated with exposure of neural stem/progenitor cells (NSPs) to 137Cs γ-ray doses in the range of 0 to 8 Gy. Subventricular zone NSPs isolated from newborn mouse pups were analyzed for proliferation, self-renewal, and differentiation, shortly after irradiation. Strikingly, there was no apparent increase in the fraction of dying cells after irradiation, and the number of single cells that formed neurospheres showed no significant change from control. Upon differentiation, irradiated neural precursors did not differ in their ability to generate neurons, astrocytes, and oligodendrocytes. By contrast, progression of NSPs through the cell cycle decreased dramatically after exposure to 8 Gy (p < .001). Mice at postnatal day 10 were exposed to 8 Gy of γ rays delivered to the whole body and NSPs of the subventricular zone were analyzed using a four-color flow cytometry panel combined with ethynyl deoxyuridine incorporation. Similar flow cytometric analyses were performed on NSPs cultured as neurospheres. These studies revealed that neither the percentage of neural stem cells nor their proliferation was affected. By contrast, γ-irradiation decreased the proliferation of two classes of multipotent cells and increased the proliferation of a specific glial-restricted precursor. Altogether, these results support the conclusion that primitive neural precursors are radioresistant, but their proliferation is slowed down as a consequence of γ-ray exposure.