Standing on the shoulders of mice.

While inbred mice have informed most of what we know about the immune system in the modern era, they have clear limitations with respect to their ability to be informative regarding genetic heterogeneity or microbial influences. They have also not been very predictive as models of human disease or vaccination results. Although there are concerted attempts to compensate for these flaws, the rapid rise of human studies, driven by both technical and conceptual advances, promises to fill in these gaps, as well as provide direct information about human diseases and vaccination responses. Work on human immunity has already provided important additional perspectives on basic immunology such as the importance of clonal deletion to self-tolerance, and while many challenges remain, it seems inevitable that "the human model" will continue to inform us about the immune system and even allow for the discovery of new mechanisms.

Effects of excess sodium consumption on arterial function in C57BL/6 mice.

Excess sodium consumption contributes to arterial dysfunction in humans. The C57BL/6 strain of mice has been used to identify mechanisms by which arterial dysfunction occurs after excess sodium consumption. However, there are concerns that C57BL/6 mice have strain-specific resistance to high-sodium (HS) diet-induced hypertension. To address this concern, we performed a meta-analysis to determine if excess sodium consumption in C57BL/6 mice induces arterial dysfunction. Databases were searched for HS versus standard diet studies that measured arterial function [i.e., systolic blood pressure (BP), endothelium-dependent dilation (EDD), and central arterial stiffness] in C57BL/6 mice. A total of 39 studies were included, demonstrating that the HS condition resulted in higher systolic BP than control mice with a mean difference of 9.8 mmHg (95% confidence interval [CI] = [5.6, 14], P < 0.001). Subgroup analysis indicated that the systolic BP was higher in HS compared with the control condition when measured during night compared with daytime with telemetry (P < 0.001). We also identified that the difference in systolic BP between HS and control was ∼2.5-fold higher when administered through drinking water than through food (P < 0.001). A total of 12 studies were included, demonstrating that the HS condition resulted in lower EDD than control with a weighted mean difference of -12.0% (95% CI = [-20.0, -4.1], P = 0.003). It should be noted that there was considerable variability across studies with more than half of the studies showing no effect of the HS condition on systolic BP or EDD. In summary, excess sodium consumption elevates systolic BP and impairs EDD in C57BL/6 mice.NEW & NOTEWORTHY C57BL/6 mice are perceived as resistant to high-sodium diet-induced arterial dysfunction. This meta-analysis demonstrates that excess sodium consumption elevates blood pressure and impairs endothelium-dependent dilation in C57BL/6 mice. Nighttime measurements show more pronounced blood pressure elevation. In addition, sodium administration via drinking water, compared with food, induces a greater blood pressure elevation. These findings may be influenced by outlier studies, as the majority of studies showed no adverse effect of excess sodium consumption on arterial function.

Impact of Experimental Tuberculosis on Fertility of Female BALB/c Mice.

The study revealed no effects of pregnancy and childbirth on the course of tuberculosis in female BALB/c mice after aerosol infection with Mycobacterium tuberculosis. However, we demonstrated a negative effect of tuberculosis infection on the fertility of infected females, which manifested in a longer period from mating to pregnancy and in a smaller litter size. Impaired reproductive function in response to the effect of the systemic infectious process was accompanied by the development of immunosuppression confirmed by an immunological test (delayed-type hypersensitivity to tuberculin) and the formation of genital tract dysbiosis during pregnancy and postpartum period.

Limited bedding nesting paradigm alters maternal behavior and pup's early developmental milestones but did not induce anxiety- or depressive-like behavior in two different inbred mice.

Animal models are crucial to understanding the mechanisms underlying the deleterious consequences of early-life stress. Here, we aimed to examine the effect of the limited bedding nesting (LBN) paradigm on early life development milestones and anxiety- and/or depression-like behavior in adolescent and adult mice from two inbred mice of both sexes. C57BL/6NCrl and BALB/c litters were exposed to the LBN paradigm postnatal day (PND) 2-9. Maternal behavior recording occurred on PND 3-9, and pups were weighed daily and examined to verify the eye-opening on PND 10-22. The male and female offspring underwent evaluation in the open field test, elevated plus-maze, and the forced swimming test during adolescence (PND 45-49) and adulthood (PND 75-79). We found that LBN impaired the maternal behavior patterns of both strain dams, mainly on C57BL/6NCrl strain. Also, LBN delayed the pup's eye-opening time and reduced body weight gain, impacting C57BL/6NCrl pups more. We also found that LBN decreased anxiety-related indices in adolescent and adult male but not female mice of both strains. Furthermore, LBN decreased depression-related indices only adolescent female and male BALB/c and female but not male C57BL/6NCrl mice. These findings reinforce the evidence that the LBN paradigm impairs the maternal behavior pattern and pup's early developmental milestones but does not induce anxiety- or depressive-like behavior outcomes during later life.

Attenuated Strain of Mycobacterium tuberculosis BN: Characteristics.

We evaluated the vaccine properties of a novel attenuated strain of M. tuberculosis BN (Mtb BN) and its impact on the gut microbiota in inbred female mice in comparison with a virulent strain Mtb H37Rv and a vaccine strain BCG. The Mtb BN strain demonstrated the highest anti-tuberculosis vaccine effect in I/St mice highly susceptible to tuberculosis infection and the same effect as BCG in mice of the recombinant strain B6.I-100 and in ß2 microglobulin gene knockout mice. No adverse effects of the new Mtb BN strain on the gut microbiota of BALB/c mice were revealed. The virulent strain Mtb H37Rv and the vaccine strain BCG decreased the main indicators of normocenosis (Bifidobacterium spp., Bifidobacterium animalis subsp. lactis, Akkermansia, and Erysipelotrichaceae) and led to disappearance of Clostridium perfingens, E. coli, Pseudomonas spp., which contributed to reduction of species diversity and the development of dysbiosis.

Feeding role of mouse embryonic fibroblast cells is influenced by genetic background, cell passage and day of isolation.

Mouse embryonic fibroblast (MEF) cells are commonly used as feeder cells to maintain the pluripotent state of stem cells. MEFs produce growth factors and provide adhesion molecules and extracellular matrix (ECM) compounds for cellular binding. In the present study, we compared the expression levels of Fgf2, Bmp4, ActivinA, Lif and Tgfb1 genes at the mRNA level and the level of Fgf2 protein secretion and Lif cytokine secretion at passages one, three and five of MEFs isolated from 13.5-day-old and 15.5-day-old embryos of NMRI and C57BL/6 mice using real-time PCR and enzyme-linked immunosorbent assay. We observed differences in the expression levels of the studied genes and secretion of the two growth factors in the three passages of MEFs isolated from 13.5-day-old and 15.5-day-old embryos, respectively. These differences were also observed between the NMRI and C57BL/6 strains. The results of this study suggested that researchers should use mice embryos that have different genetic backgrounds and ages, in addition to different MEF passages, when producing MEFs based on the application and type of their study.

Population and subspecies diversity at mouse centromere satellites.

BACKGROUND: Mammalian centromeres are satellite-rich chromatin domains that execute conserved roles in kinetochore assembly and chromosome segregation. Centromere satellites evolve rapidly between species, but little is known about population-level diversity across these loci. RESULTS: We developed a k-mer based method to quantify centromere copy number and sequence variation from whole genome sequencing data. We applied this method to diverse inbred and wild house mouse (Mus musculus) genomes to profile diversity across the core centromere (minor) satellite and the pericentromeric (major) satellite repeat. We show that minor satellite copy number varies more than 10-fold among inbred mouse strains, whereas major satellite copy numbers span a 3-fold range. In contrast to widely held assumptions about the homogeneity of mouse centromere repeats, we uncover marked satellite sequence heterogeneity within single genomes, with diversity levels across the minor satellite exceeding those at the major satellite. Analyses in wild-caught mice implicate subspecies and population origin as significant determinants of variation in satellite copy number and satellite heterogeneity. Intriguingly, we also find that wild-caught mice harbor dramatically reduced minor satellite copy number and elevated satellite sequence heterogeneity compared to inbred strains, suggesting that inbreeding may reshape centromere architecture in pronounced ways. CONCLUSION: Taken together, our results highlight the power of k-mer based approaches for probing variation across repetitive regions, provide an initial portrait of centromere variation across Mus musculus, and lay the groundwork for future functional studies on the consequences of natural genetic variation at these essential chromatin domains.

Drug Resistance of Mouse Somatic Cells to Rifampicin in Experimental Tuberculosis.

We have demonstrated that long-term exposure of intact mice to rifampicin (6 months) induces resistance to this drug, which manifested in inability of rifampicin to suppress the growth of Mycobacterium tuberculosis in the lungs and spleen during subsequent infection. It the same time, isoniazid is still effective in these mice. In this case, the phenomenon of somatic resistance to rifampicin in mice was observed if the treatment was started in a short period (within 4 days) after infection with M. tuberculosis. If the interval between infection and rifampicin administration was longer (3 weeks), the resistance disappeared.

Efficacy of BCG Vaccination Depends on Host Genetics.

We studied the effectiveness of anti-tuberculosis vaccination with BCG in mice of inbred strains and F1 hybrids (highly resistant to tuberculosis infection) that represent a wide range of genetically determined differences in susceptibility to infection with virulent Mycobacterium tuberculosis. The greatest relative effect was found in susceptible mice, with the exception of highly susceptible I/St mice that were practically not protected by vaccination. Despite significant effect of vaccination in inbred mice, their resistance to M. tuberculosis infection did not exceed that of non-vaccinated highly resistant F1 hybrids.

Effects of Azadirachta indica seed kernel extracts on early erythrocytic schizogony of Plasmodium berghei and pro-inflammatory response in inbred mice.

BACKGROUND: Medicinal plant research may contribute to develop new pharmacological control tools for vector borne diseases, such as malaria. METHODS: The effects of methanol extracts (ME) obtained from seed kernel of ripe and unripe Azadirachta indica fruits were studied on erythrocytic proliferation of the rodent malaria parasite Plasmodium berghei strain ANKA and on mice pro-inflammatory response, as evaluated by measuring the matrix-metalloproteinase-9 (MMP-9) and tumour necrosis factor (TNF) plasma levels, in two mouse strains (C57BL/6 and BALB/c) which are considered as prototypical of Th1 and Th2 immune response, respectively. RESULTS: ME obtained from seed kernel of unripe Azadirachta indica fruits decreased by about 30% the proportion of erythrocytes infected with the malaria parasite in C57BL/6 mice in the 4 days suppressive test. In this treatment group, MMP-9 and TNF levels were notably higher than those measured in the same mouse strain treated with the anti-malarial drug artesunate, Azadirachta indica kernel extracts from ripe fruits or solvent. In BALB/c mice, treatment with kernel extracts did not influence parasitaemia. MMP-9 and TNF levels measured in this mouse strain were notably lower than those recorded in C57BL/6 mice and did not vary among treatment groups. CONCLUSIONS: The effects of the ME on the parasite-host interactions appeared to be mouse strain-dependent, but also related to the ripening stage of the neem fruits, as only the unripe fruit seed kernel extracts displayed appreciable bioactivity.

Unstable FoxP3+ T regulatory cells in NZW mice.

Regulatory T (Treg) cells that express the transcription factor FoxP3 play a key role in self-tolerance and the control of inflammation. In mice and humans, there is a wide interindividual range in Treg frequency, but little is known about the underlying genetic or epigenetic mechanisms. We explored this issue in inbred strains of mice, with a special focus on the low proportion of Treg cells found in NZW mice. Mixed bone marrow chimera experiments showed this paucity to be intrinsic to NZW Treg cells, a dearth that could be tied to poor stability of the Treg pool and of FoxP3 expression. This instability was not a consequence of differential epigenetic marks, because Treg-specific CpG hypomethylation profiles at the Foxp3 locus were similar in all strains tested. It was also unrelated to the high expression of IFN signature genes in NZW, as shown by intercross to mice with an Ifnar1 knockout. NZW Tregs were less sensitive to limiting doses of trophic cytokines, IL-2 and -33, for population homeostasis and for maintenance of FoxP3 expression. Gene-expression profiles highlighted specific differences in the transcriptome of NZW Tregs compared with those of other strains, but no single defect could obviously account for the instability. Rather, NZW Tregs showed a general up-regulation of transcripts normally repressed in Treg cells, and we speculate that this network-level bias may account for NZW Treg instability.

Changes in Microbiota and Development of Nonspecific Inflammation of Genitals in Female C57Bl/6 Mice after Aerosol Infection with Mycobacterium tuberculosis.

Infectious process even at the initial stage after aerosol infection with Mycobacterium tuberculosis induced rapid changes in vaginal microbiota in mice. Rapid decrease in both the quantity and diversity of microbiota was noted, and then, partial recovery of normal flora was observed. Changes in vaginal microbiota was detected as soon as in 3-7 days after lung infection, while inflammatory changes appeared by day 35. At the early stage of infection, no signs of inflammation were observed, neither M. tuberculosis nor its DNA were detected in mouse genital organs.

Pituitary tumor-transforming gene 1 regulates the patterning of retinal mosaics.

Neurons are commonly organized as regular arrays within a structure, and their patterning is achieved by minimizing the proximity between like-type cells, but molecular mechanisms regulating this process have, until recently, been unexplored. We performed a forward genetic screen using recombinant inbred (RI) strains derived from two parental A/J and C57BL/6J mouse strains to identify genomic loci controlling spacing of cholinergic amacrine cells, which is a subclass of retinal interneuron. We found conspicuous variation in mosaic regularity across these strains and mapped a sizeable proportion of that variation to a locus on chromosome 11 that was subsequently validated with a chromosome substitution strain. Using a bioinformatics approach to narrow the list of potential candidate genes, we identified pituitary tumor-transforming gene 1 (Pttg1) as the most promising. Expression of Pttg1 was significantly different between the two parental strains and correlated with mosaic regularity across the RI strains. We identified a seven-nucleotide deletion in the Pttg1 promoter in the C57BL/6J mouse strain and confirmed a direct role for this motif in modulating Pttg1 expression. Analysis of Pttg1 KO mice revealed a reduction in the mosaic regularity of cholinergic amacrine cells, as well as horizontal cells, but not in two other retinal cell types. Together, these results implicate Pttg1 in the regulation of homotypic spacing between specific types of retinal neurons. The genetic variant identified creates a binding motif for the transcriptional activator protein 1 complex, which may be instrumental in driving differential expression of downstream processes that participate in neuronal spacing.

Using the Tools of Behavioral Neuroscience to Determine the Identity of Different Mouse Strains in a Laboratory Course.

Understanding the neural mechanisms underlying behavior depends on our ability to define and to measure these behaviors in the model animal. We describe an upper-level course which provides students with hands-on experience in the methods of behavioral neuroscience. There are many well-established behavioral tests which are relatively easy for students to conduct that can be used to determine the performance of animals in such tasks as anxiety, motor performance and memory. Laboratory mice bred specifically to exhibit particular behavioral characteristics are readily available from vendors along with well documented behavioral profiles for these strains. We used two albino strains CD1 and BALBc as our model animals. Students were given the task of identifying the strains based on the results of a battery of behavioral tests but were not given information about the mice. These two strains were chosen for their clear differences particularly in tests of anxiety. Students conducted elevated plus maze and zero maze tests, open field test, light-dark exploratory task, rotarod, balance beam test, spatial or novel object learning. Students were able to correctly identify the two strains by comparing their own data with the published literature in the field. The course structure encouraged students to work in teams to design protocols, and then to collect and explore data. Students were enthusiastic about the hands-on laboratory experience and were able to demonstrate an appreciation for and understanding of these methods in behavioral neuroscience.

Low ambient temperature during early postnatal development fails to cause a permanent induction of brown adipocytes.

The brown adipocyte phenotype (BAP) in white adipose tissue (WAT) is transiently induced in adult mammals in response to reduced ambient temperature. Since it is unknown whether a cold challenge can permanently induce brown adipocytes (BAs), we reared C57BL/6J (B6) and AxB8/PgJ (AxB8) mice at 17 or 29°C from birth to weaning, to assess the BAP in young and adult mice. Energy balance measurements showed that 17°C reduced fat mass in the preweaning mice by increasing energy expenditure and suppressed diet-induced obesity in adults. Microarray analysis of global gene expression of inguinal fat (ING) from 10-day-old (D) mice indicates that expression at 17°C vs. 29°C was not different. Between 10 and 21 days of age, the BAP was induced coincident with morphologic remodeling of ING and marked changes in expression of neural development genes (e.g., Akap 12 and Ngfr). Analyses of Ucp1 mRNA and protein showed that 17°C transiently increased the BAP in ING from 21D mice; however, BAs were unexpectedly present in mice reared at 29°C. The involution of the BAP in WAT occurred after weaning in mice reared at 23°C. Therefore, the capacity to stimulate thermogenically competent BAs in WAT is set by a temperature-independent, genetically controlled program between birth and weaning.

Peculiarities of the Inflammatory Process in the Reproductive Organs of C57Bl/6 Female Mice with Experimental Tuberculosis.

Intravenous infection of C57Bl/6 female mice with M. tuberculosis H37Rv led to involvement of the lungs and dissemination of the tuberculous infection to the abdominal and pelvic organs. M. tuberculosis were detected in the lungs and spleen in 14, 35, and 90 days and in the uterine horns in 90 days after infection. Morphological analysis of organs showed successive development of exudative necrotic tuberculosis of the lungs, acute and chronic nonspecific inflammation in the reproductive organs (vagina, uterus, and uterine horns). The inflammatory process in the reproductive organs was associated with the development of anaerobic dysbiosis, that was most pronounced in 35 days after infection. Antituberculous therapy was followed by reduction of M. tuberculosis count in the lungs and spleen in 60 and 90 days after infection, eliminatation of M. tuberculosis in the uterine horns, arrest of nonspecific inflammation in female reproductive organs, recovery of the balance between aerobic and anaerobic microflora, and development of candidiasis of the urogenital mucosa.

Mitochondrial gene polymorphisms that protect mice from colitis.

BACKGROUND & AIMS: Dysregulated energy homeostasis in the intestinal mucosa frequently is observed in patients with ulcerative colitis (UC). Intestinal tissues from these patients have reduced activity of the mitochondrial oxidative phosphorylation (OXPHOS) complex, so mitochondrial dysfunction could contribute to the pathogenesis of UC. However, little is known about the mechanisms by which OXPHOS activity could be altered. We used conplastic mice, which have identical nuclear but different mitochondrial genomes, to investigate activities of the OXPHOS complex. METHODS: Colitis was induced in C57BL/6J wild-type (B6.B6) and 3 strains of conplastic mice (B6.NZB, B6.NOD, and B6.AKR) by administration of dextran sodium sulfate or rectal application of trinitrobenzene sulfonate. Colon tissues were collected and analyzed by histopathology, immunohistochemical analysis, and immunoblot analysis; we also measured mucosal levels of adenosine triphosphate (ATP) and reactive oxygen species, OXPHOS complex activity, and epithelial cell proliferation and apoptosis. RESULTS: We identified mice with increased mucosal OXPHOS complex activities and levels of ATP. These mice developed less-severe colitis after administration of dextran sodium sulfate or trinitrobenzene sulfonate than mice with lower mucosal levels of ATP. Colon tissues from these mice also had increased enterocyte proliferation and transcription factor nuclear factor-κB activity, which have been shown to protect the mucosal barrier-defects in these processes have been associated with inflammatory bowel disease. CONCLUSIONS: Variants in mitochondrial DNA that increase mucosal levels of ATP protect mice from colitis. Increasing mitochondrial ATP synthesis in intestinal epithelial cells could be a therapeutic approach for UC.

Role of nucleus accumbens dopamine receptor subtypes in the learning and expression of alcohol-seeking behavior.

These studies examined the roles of dopamine D1- and D2-like receptors within the nucleus accumbens (Acb) in the acquisition and expression of ethanol-induced (2g/kg) conditioned place preference (CPP) in adult male DBA/2J mice. Bilateral intra-Acb infusions of the D1-like dopamine receptor antagonist SCH23390 (0.05, 0.5µg/side) or the D2-like dopamine receptor antagonist raclopride (0.5-5.0µg/side) were administered 30min before each ethanol conditioning trial (acquisition studies) or before preference tests (expression studies). CPP was conditioned to tactile cues using an unbiased apparatus and procedure. Intra-Acb infusion of SCH23390 prevented CPP acquisition, whereas intra-Acb infusion of raclopride did not. Intra-Acb infusion of both antagonists, however, dose-dependently reduced ethanol-stimulated locomotor activity during conditioning. In contrast, intra-Acb antagonist infusion had no effect on ethanol CPP expression, suggesting that dopamine's role in the Acb is limited to neurobiological processes engaged during the learning of the relationship between contextual cues and ethanol reward. Control experiments showed that intra-Acb injection of SCH23390 alone produced no place conditioning and did not interfere with the acquisition of conditioned place aversion induced by lithium chloride, suggesting that the antagonist's effect on ethanol CPP was not due to a more general detrimental effect on associative learning. Overall, these data suggest that D1-like (but not D2-like) dopamine Acb receptors play an important role in the learning of context-ethanol associations, either by modulating the magnitude of ethanol reward or the rate of learning about ethanol reward.

Microglia are polarized to M1 type in high-anxiety inbred mice in response to lipopolysaccharide challenge.

Immune activation in the brain has been shown to contribute to neurodevelopmental and pathological progression of mental disorders, and microglia play a central role in these processes. But how genetic predisposition and environmental risk factors may act in combination to affect microglial activation and the underlying molecular mechanisms are largely unclear. In this work, we studied the inflammatory profile of microglia across four inbred strains of mice with different anxiety traits: C57BL/6J, FVB/N, DBA/2J, and 129S2/Sv. Importantly, we found that a high-anxiety strain, naïve DBA/2J mice, had significantly more M1 (MHCII(+)CD206(-))-polarized microglia, whereas another high-anxiety strain, naïve 129S2/Sv mice, expressed significantly more activated (MHCII(+)) perivascular macrophages than the other strains. After a systemic LPS challenge, polarization to M1 microglia in DBA/2J and 129S2/Sv mice was even more prominent than in C57BL/6J and FVB/N mice, and was correlated with their anxiety-like behaviors. Macrophage M1/M2 polarization in the spleen showed a similar pattern in DBA/2J and 129S2/Sv mice in response to LPS stimulation. Furthermore, DBA/2J mice expressed higher mRNA levels of Il1b, Il6, and Tnf, and higher Nos2/Arg1 ratio but lower Chi3l3 level in the hypothalamus before and after LPS stimulation, respectively. In comparison, 129S1/Sv, a sibling line of 129S2/Sv, expressed significantly higher levels of other immune-related genes in the brain. We further discovered a group of myeloid transcription factors that may underpin the strain-specific differences in microglial activation. We conclude that proinflammatory microglial activation reflects anxiety traits in mice, especially after a peripheral innate immune challenge. Our work sheds new light in understanding the potential molecular mechanisms of stress-induced microglial activation and polarization.

Repeated ethanol exposure during early and late adolescence: double dissociation of effects on waiting and choice impulsivity.

BACKGROUND: A strong association exists between impulsivity and binge drinking, and between adolescent alcohol exposure and alcohol abuse in humans. To understand the extent to which early-life alcohol exposure contributes to increased impulsivity, we developed an animal model of binge drinking using 2 strains of mice, C57BL/6J (B6) and DBA2/J (D2), that differ in both motor impulsivity and alcohol drinking. METHODS: Mice were treated with 2 g/kg ethanol (EtOH) during their early (intermittent ethanol exposure [IEE]_Early; postnatal day [PND]30 to 45) or late (IEE_Late; PND45 to 60) adolescence or with saline (control group [CON]) throughout the adolescence period. To determine the consequences IEE on waiting impulsivity and attentional function, the number of premature responses and omissions, respectively, were evaluated in adulthood using the 5-choice serial reaction time task (5-CSRTT). To examine the effects of IEE on choice impulsivity, risky decision making was assessed in adulthood using a mouse version of the Iowa Gambling Task (mIGT). Additionally, the acute effects of EtOH in adulthood on waiting impulsivity and choice preference were investigated. RESULTS: We provide experimental evidence that IEE during late, but not early, adolescence disrupts waiting impulsivity and attentional abilities in the 5-CSRTT. In contrast, IEE during early, but not late, adolescence altered risky decision making in the mIGT. D2 mice consistently showed lower premature responding than B6 mice in both the mIGT and the 5-CSRTT, but greater risky decision making on the mIGT. IEE and CON mice showed similar responsiveness to the acute EtOH effects on premature responding, but increased risky choices only in B6_IEE_Early mice. CONCLUSIONS: Our observations suggest a direct effect of IEE during adolescence on waiting and choice impulsivity and attention later in life.