C3H/HeJ Mice Research Articles

Abstract 8090: Identification Of Rcn2 As A Novel Regulator Of Cytokine Expression

Background- Ath29 is an atherosclerosis susceptibility locus on chromosome 9 identified in an intercross between C57BL/6 (B6) and C3H/HeJ (C3H) apolipoprotein E-deficient (apoE −/− ) mice. This locus was subsequently replicated in two separate intercrosses that developed early or advanced atherosclerotic lesions. The objective of this study was to characterize Ath29 through construction and analysis of a congenic strain and identify underlying candidate genes. Methods and Results- Congenic strain was constructed by introgressing the chromosomal segment harboring Ath29 from C3H.apoE −/− into B6.apoE −/− mice. Congenic mice developed significantly smaller early and advance atherosclerotic lesions than B6.apoE −/− mice. Microarray analysis revealed 317 genes to be differentially expressed in the aorta of congenic mice compared with B6.apoE −/− mice. Pathway analysis of these genes suggested the calcium signaling pathway to be implicated in regulating atherosclerosis susceptibility. Rcn2 is located underneath the linkage peak of Ath29 and involved in calcium signaling. Multiple SNPs between B6 and C3H mice were detected within and surrounding Rcn2 with one SNP falling within an upstream cAMP response element. Immunostaining demonstrated its expression in atherosclerotic lesions. Knockdown of Rcn2 with siRNAs resulted in significant reductions in both baseline and oxidized phospholipid-induced VCAM-1 and MCP-1 expression by endothelial cells. Conclusions- Ath29 is confirmed to be a major atherosclerosis susceptibility locus affecting both early and advanced lesion formation in mice, and Rcn2 is identified as a novel regulator of cytokine expression.

Effects of Withdrawal from Chronic Intermittent Ethanol Vapor on the Level and Circadian Periodicity of Running‐Wheel Activity in C57BL/6J and C3H/HeJ Mice

Alcohol withdrawal is associated with behavioral and chronobiological disturbances that may persist during protracted abstinence. We previously reported that C57BL/6J (B6) mice show marked but temporary reductions in running-wheel activity, and normal free-running circadian rhythms, following a 4-day chronic intermittent ethanol (CIE) vapor exposure (16 hours of ethanol vapor exposure alternating with 8 hours of withdrawal). In the present experiments, we extend these observations in 2 ways: (i) by examining post-CIE locomotor activity in C3H/HeJ (C3H) mice, an inbred strain characterized by high sensitivity to ethanol withdrawal, and (ii) by directly comparing the responses of B6 and C3H mice to a longer-duration CIE protocol. In Experiment 1, C3H mice were exposed to the same 4-day CIE protocol used in our previous study with B6 mice (referred to here as the 1-cycle CIE protocol). In Experiment 2, C3H and B6 mice were exposed to 3 successive 4-day CIE cycles, each separated by 2 days of withdrawal (the 3-cycle CIE protocol). Running-wheel activity was monitored prior to and following CIE, and post-CIE activity was recorded in constant darkness to allow assessment of free-running circadian period and phase. C3H mice displayed pronounced reductions in running-wheel activity that persisted for the duration of the recording period (up to 30 days) following both 1-cycle (Experiment 1) and 3-cycle (Experiment 2) CIE protocols. In contrast, B6 mice showed reductions in locomotor activity that persisted for about 1 week following the 3-cycle CIE protocol, similar to the results of our previous study using a 1-cycle protocol in this strain. Additionally, C3H mice showed significant shortening of free-running period following the 3-cycle, but not the 1-cycle, CIE protocol, while B6 mice showed normal free-running rhythms. These results reveal genetic differences in the persistence of ethanol withdrawal-induced hypo-locomotion. In addition, chronobiological alterations during extended abstinence may depend on both genetic susceptibility and an extended prior withdrawal history. The present data establish a novel experimental model for long-term behavioral and circadian disruptions associated with ethanol withdrawal.

Identification of Soat1 as a Quantitative Trait Locus Gene on Mouse Chromosome 1 Contributing to Hyperlipidemia

We previously identified two closely linked quantitative trait loci (QTL) on distal chromosome 1 contributing to major variations in plasma cholesterol and triglyceride levels in an intercross derived from C57BL/6 (B6) and C3H/HeJ (C3H) apolipoprotein E-deficient (apoE−/−) mice. Soat1, encoding sterol o-acyltransferase 1, is a functional candidate gene located underneath the proximal linkage peak. We sequenced the coding region of Soat1 and identified four single nucleotide polymorphisms (SNPs) between B6 and C3H mice. Two of the SNPs resulted in amino-acid substitutions (Ile147Val and His205Tyr). Functional assay revealed an increased enzyme activity of Soat1 in peritoneal macrophages of C3H mice relative to those of B6 mice despite comparable protein expression levels. Allelic variants of Soat1 were associated with variations in plasma cholesterol and triglyceride levels in an intercross between B6.apoE−/− and C3H.apoE−/− mice. Inheritance of the C3H allele resulted in significantly higher plasma lipid levels than inheritance of the B6 allele. Soat1 variants were also significantly linked to major variations in plasma esterified cholesterol levels but not with free cholesterol levels. Trangenic expression of C3H Soat1 in B6.apoE−/− mice resulted in elevations of plasma cholesterol and triglyceride levels. These results indicate that Soat1 is a QTL gene contributing to hyperlipidemia.

Characterization of Ath29, a major mouse atherosclerosis susceptibility locus, and identification of Rcn2 as a novel regulator of cytokine expression

Ath29 is an atherosclerosis susceptibility locus on chromosome 9 identified in an intercross between C57BL/6 (B6) and C3H/HeJ (C3H) apolipoprotein E-deficient (apoE(-/-)) mice. This locus was subsequently replicated in two separate intercrosses that developed early or advanced atherosclerotic lesions. The objective of this study was to characterize Ath29 through construction and analysis of a congenic strain and identify underlying candidate genes. A congenic line was constructed by introgressing the chromosomal segment harboring Ath29 from C3H.apoE(-/-) into B6.apoE(-/-) mice. Congenic mice developed significantly smaller early and advance atherosclerotic lesions than B6.apoE(-/-) mice. Microarray analysis revealed 317 genes to be differentially expressed in the aorta of congenic mice compared with B6.apoE(-/-) mice. Pathway analysis of these genes suggested the Ca(2+) signaling pathway to be implicated in regulating atherosclerosis susceptibility. Rcn2 is located underneath the linkage peak of Ath29 and involved in Ca(2+) signaling. Multiple single-nucleotide polymorphisms between B6 and C3H mice were detected within and surrounding Rcn2 with one single-nucleotide polymorphism falling within an upstream cAMP response element. Immunostaining demonstrated its expression in atherosclerotic lesions. Knockdown of Rcn2 with small interfering RNAs resulted in significant reductions in both baseline and oxidized phospholipid-induced VCAM-1 and monocyte chemoattractant protein-1 expression by endothelial cells. Ath29 is confirmed to be a major atherosclerosis susceptibility locus affecting both early and advanced lesion formation in mice, and Rcn2 is identified as a novel regulator of cytokine expression.

The Cri1 locus is the common genetic cause of susceptibility to Citrobacter rodentium infection in C3H and FVB mouse strains.

Citrobacter rodentium is a natural pathogen of mice that causes intestinal hyperplasia and colitis. Resistant strains such as C57BL/6J (B6) experience a self-limiting disease that peaks between one and two weeks post infection, followed by a clearing of the infection and complete recovery. However, the inbred mouse strains C3H/HeJ (C3), C3H/HeOuJ (C3Ou) and FVB/N (FVB) are highly susceptible to C. rodentium infection and develop more severe symptoms of disease leading to high rates of mortality during infection. We have recently demonstrated through a systematic genetics approach that a single locus on proximal chromosome 15 is responsible for the susceptibility of both C3 and C3Ou mice to C. rodentium infection. We have named the locus Citrobacter rodentium infection 1 (Cri1). Here we show that Cri1 also controls susceptibility to C. rodentium in FVB mice, using a targeted method of genotyping to stratify (B6 x FVB)F2 mice according to their genotype at Cri1. Mice that inherit two copies of the resistant B6 allele have 97% cumulative survival at day 30 post-infection, whereas those that inherit one or two copies of Cri1 from the FVB parent have significantly lower rates of survival (35% and 42%, respectively). These results provide evidence for a common genetic cause of fatal infectious colitis in C3, C3Ou and FVB mice following infection with Citrobacter rodentium.

Leptin Receptor (Lepr) Is a Negative Modulator of Bone Mechanosensitivity and Genetic Variations in Lepr May Contribute to the Differential Osteogenic Response to Mechanical Stimulation in the C57BL/6J and C3H/HeJ Pair of Mouse Strains

This study investigated the role of leptin receptor (Lepr) signaling in determining the bone mechanosensitivity and also evaluated whether differences in the Lepr signaling may contribute to the differential osteogenic response of the C57BL/6J (B6) and C3H/HeJ (C3H) pair of mouse strains to mechanical stimuli. This study shows that a loading strain of ∼2,500 με, which was insufficient to produce a bone formation response in B6 mice, significantly increased bone formation parameters in leptin-deficient ob(-)/ob(-) mice and that a loading strain of ∼3,000 με also yielded greater osteogenic responses in Lepr-deficient db(-)/db(-) mice than in wild-type littermates. In vitro, a 30-min steady shear stress increased [(3)H]thymidine incorporation and Erk1/2 phosphorylation in ob(-)/ob(-) osteoblasts and db(-)/db(-) osteoblasts much greater than those in corresponding wild-type osteoblasts. The siRNA-mediated suppression of Lepr expression in B6 osteoblasts enhanced (but in osteoblasts of C3H (the mouse strain with poor bone mechanosensitivity) restored) their anabolic responses to shear stress. The Lepr signaling (leptin-induced Jak2/Stat3 phosphorylation) in C3H osteoblasts was higher than that in B6 osteoblasts. One of the three single nucleotide polymorphisms in the C3H Lepr coding region yielded an I359V substitution near the leptin binding region, suggesting that genetic variation of Lepr may contribute to a dysfunctional Lepr signaling in C3H osteoblasts. In conclusion, Lepr signaling is a negative modulator of bone mechanosensitivity. Genetic variations in Lepr, which result in a dysfunctional Lepr signaling in C3H mice, may contribute to the poor osteogenic response to loading in C3H mice.

Differential Gene Expression in High and Low active Animals.

It is well established that physical activity levels are significantly controlled by genetic factors; however, the specific genes and genetic mechanisms involved in the regulation of physical activity is unknown. PURPOSE: To compare the gene expression pattern of several potential candidate genes in both nucleus accumbens (brain) and gastrocnemius/soleus (G/S muscle) between two differentially active inbred strains of mice. METHODS: From previous experiments, nucleus acumbens and G/S tissue were used from 11 high active C57L/J (LJ) mice (8 male, 3 female) and 13 low active C3H/HeJ (C3H) mice (8 male, 5 female). As we had seen previously, while there was no difference in the weight of the mice between strains (LJ=23.7±2.6 gms, avg±SD vs. C3H=23.3±3.6 gms), over 21 days of wheel running activity, the LJ ran farther (11.1±3.0 km/day vs. 1.6±2.1 km/day), longer (367.2±56.3 min/day vs. 92.3±112.7 min/day), and faster (30.3±5.9 m/min vs. 14.6±2.9 m/min) than the C3H mice proving that the LJ mice had higher daily activity than the C3H mice. Standard quantitative RT-PCR techniques developed in our lab were used to determine potential differential expression of previously suggested candidate genes: dopamine receptor 2 (Drd2), nescient helix-loop-helix 2 (Nhlh2), alpha actinin 2 (Actn2), alpha actinin 3 (Actn3), melanocortin-4-receptor (Mc4r), aromatase (Cyp19a1), leptin receptor (Lepr), myostatin (Mstn), 3'-phosphoadenosine 5'-phosphosulfate synthase 2 (Papss2), and glucose transporter 4 (Slc2a4). All expression data was log transformed for normality purposes and the values were compared using one-way ANOVAs between strains within each tissue with the alpha value set = 0.05. RESULTS: Of the comparisons in the brain tissue, Lepr (p=0.02) and Mstn (p=0.0001) were expressed at higher levels in the LJ mice. In the G/S tissue, Nhlh2 (p=0.05) and Slc2a4 (p=0.0005) were both expressed at higher levels in the LJ mice. CONCLUSIONS: Our data suggest that Lepr and Mstn in the nucleus accumbens and Nhlh2 and Slc2a4 in the G/S are differentially expressed in high and low active animals. While this data does not establish a role for any of these genes in activity regulation, it does show that there is an association between these genes and activity; an association that further research should delineate. Supported by NIH R01AR050085 and the Huffines' Institute.

Genes Within the MHC Region Have a Dramatic Influence on Radiation-Enhanced Atherosclerosis in Mice

C3H/HeJ (C3H) mice develop much smaller atherosclerotic lesions than C57BL/6 (B6) mice when deficient in apolipoprotein E (apoE⁻(/)⁻) or fed an atherogenic diet. The 2 strains differ in H2 haplotypes, with B6 having H2(b) and C3H having H2(k). C3.SW-H2(b)/SnJ (C3.SW) is a congenic strain of C3H/HeJ in which H2(k) is replaced with H2(b). We performed bone marrow transplantation and found that atherosclerosis-resistant C3.SW.apoE⁻(/)⁻ mice reconstituted with bone marrow from either C3.SW.apoE⁻(/)⁻ or B6.apoE⁻(/)⁻ mice after lethal irradiation had significantly larger atherosclerotic lesions than B6.apoE⁻(/)⁻ mice receiving identical treatments and much larger lesions than C3H.apoE⁻(/)⁻ mice reconstituted with syngeneic bone marrow. For syngeneic transplantation, C3.SW.apoE⁻(/)⁻ mice exhibited a 21-fold increase in lesion size over C3H.apoE⁻(/)⁻ mice (152 800±21 937 versus 7060±2290 μm²/section) and a near 4-fold increase over B6.apoE⁻(/)⁻ mice (40 529±4675 μm²/section). C3.SW.apoE⁻(/)⁻ mice reconstituted with syngeneic marrow exhibited enhanced lesion formation relative to those reconstituted with B6 marrow (152 800±21 937 versus 107 000±9374 μm²/section; P=0.067). Sublethal irradiation led to a 6-fold increase of lesion size in C3.SW.apoE⁻(/)⁻ mice (9795±2804 versus 1550±607 μm²/section; P=0.008). Wild-type C3.SW mice reconstituted with apoE(+/+) or apoE⁻(/)⁻ bone marrow had significantly larger atherosclerotic lesions than C3H mice receiving identical treatments on an atherogenic diet. These results indicate that gene(s) within the H2 region have a dramatic impact on radiation-enhanced atherosclerosis, and their effect is conveyed partially through bone marrow-derived cells.

Collagen fibril morphology and mechanical properties of the Achilles tendon in two inbred mouse strains

The relationship between collagen fibril morphology and the functional behavior of tendon tissue has been investigated in numerous experimental studies. Several of these studies suggest that larger fibril radius is a primary determinant of higher tendon stiffness and strength; others have shown that factors apart from fibril radius (such as fibril-fibril interactions) may be critical to improved tendon strength. In the present study, we investigate these factors in two inbred mouse strains that are widely used in skeletal structure-function research: C57BL/6J (B6) and C3H/HeJ (C3H). The aim was to establish a quantitative baseline that will allow one to assess how regulation of tendon extracellular matrix architecture affects tensile mechanical properties. We specifically focused on collagen fibril structure and glycosaminoglycan (GAG) content--the two primary constituents of tendon by dry weight--and their potential functional interactions. For this purpose, Achilles tendons from both groups were tested to failure in tension. Tendon collagen morphology was analyzed from transmission electron microscopy images of tendon sections perpendicular to the longitudinal axis. Our results showed that the two inbred strains are macroscopically similar, but C3H mice have a higher elastic modulus (P < 0.05). Structurally, C3H mice showed a larger collagen fibril radius compared to B6 mice (96 +/- 7 nm and 80 +/- 10 nm respectively). Tendons from C3H mice also showed smaller specific fibril surface (0.015 +/- 0.001 nm nm(-2) vs. 0.017 +/- 0.003 nm nm(-2) in the B6 tendons, P < 0.05), and accordingly a lower concentration of GAGs (0.60 +/- 0.07 microg mg(-1) vs. 0.83 +/- 0.11 microg mg(-1), P < 0.05). As in other studies of tendon structure and function, larger collagen fibril radius appears to be associated with stiffer tendon, but this functional difference could also be attributed to reduced potential surface area exchange between fibrils and the surrounding proteoglycan-rich matrix, in which the hydrophilic GAG side chains may promote inter-fibril sliding. This study provides an architectural and functional baseline for a comparative murine model that can be used to investigate the genetic regulation of tendon biomechanics.

Mechanical loading of mouse caudal vertebrae increases trabecular and cortical bone mass-dependence on dose and genotype

Most in vivo studies addressing the skeletal responses of mice to mechanical loading have targeted cortical bone. To investigate trabecular bone responses also we have developed a caudal vertebral axial compression device (CVAD) that transmits mechanical loads to compress the fifth caudal vertebra via stainless steel pins inserted into the forth and sixth caudal vertebral bodies. Here, we used the CVAD in C57BL/6 (B6) and C3H/Hej (C3H) female mice (15weeks of age) to investigate whether the effect of regular bouts of mechanical stimulation on bone modeling and bone mass was dependent on dose and genotype. A combined micro-computed tomographic and dynamic histomorphometric analysis was carried out at the end of a 4-week loading regimen (3,000 cycles, 10 Hz, 3 xweek) for load amplitudes of 0N, 2N, 4N and 8N. Significant increases in trabecular bone mass of 9 and 21% for loads of 4N and 8N, respectively, were observed in B6 mice. A significant increase of 10% in trabecular bone mass occurred for a load of 8N in the C3H strain. For other loads, no significant increases were detected. Both mouse strains exhibited substantial increases in trabecular bone formation rates for all loads, B6: 111% (2N), 86% (4N), 164% (8N), C3H: 41% (2N), 38% (4N), 141% (8N). Significant decreases in osteoclast number of 146 and 93% for a load of 8N were detected in B6 and C3H mice, respectively. These findings demonstrate that the effect of loading on the structural and functional parameters of bone is dose and genotype dependent. The caudal vertebral loading model established here is proposed for further studies addressing the molecular processes involved in the skeletal responses to mechanical stimuli.

DYNAMICS OF MICRONUCLEI IN MOUSE SKIN FIBROBLASTS AFTER GAMMA IRRADIATION

Micronuclei (MN) were assessed in dermal fibroblasts from C3H HeJ and C57 Bl6 mice (6-10 mo of age) irradiated in vivo as a potential method of biodosimetry. Radiation-induced MN [per 1,000 binucleated (BN) cells], assessed in fibroblasts obtained 1 d post-irradiation, increased in a dose-dependent manner in the range of 1 - 10 Gy per single dose. Analysis at 1 wk post irradiation showed some attenuation of MN response in C3H HeJ male mice, suggesting partial recovery of DNA damage. This was not observed in C57 Bl6 mice. Monomicronucleated cells predominated in unirradiated fibroblasts, whereas in irradiated fibroblasts multimicronucleated cells predominated at dose levels above about 5 Gy (more than 1,000 MN per 1,000 BN cells). Modeling of the data indicate that assaying total MN in dermal fibroblasts from samples of irradiated skin taken up to 1 wk after irradiation can provide biodosimetric information (with an accuracy of 1-2 Gy) for doses up to at least 6 Gy with lesser accuracy at higher doses. Percentage of multimicronucleated cells may be useful as a substitute of total MN at the higher doses, but total percentage of micronucleated cells is of limited value as a substitute.

Indications of IL1β, IL18 and VEGF cascade in maintenance of homeostasis following transient pulmonary inflammation due to carbon nanoparticle (CNP) challenge in mice

Aims: Carbonaceous nanoparticles possess an emerging source of human exposure. The pulmonary inflammation caused by deposited nanoparticles is a key event for their adverse health effects. Epidemiological studies suggest that individuals with favourable lung physiology are at lower risk for particulate matter associated respiratory diseases. We selected a mouse strain C3H/HeJ (C3) with robust lung physiology and exposed it to moderately toxic carbon nanoparticles (CNP) to study the elicited pulmonary inflammation and its resolution. Methods: 5µg, 20µg and 50µg CNP were intratracheally (i.t.) instilled in C3 mice. Inflammation was assessed using histology, broncheoalveolar lavage (BAL) analysis and by a panel of 62 protein markers to analyze the molecular response. Results: 24h after instillation of CNP, C3 mice exhibited a typical dose response, with the lowest dose (5µg) representing the 'no effect level' as reflected by polymorphonuclear leucocyte (PMN) influx, and BAL/lung concentrations of pro-inflammatory cytokines and proteins. Accordingly time course assessment of the inflammatory response was performed after 3 and 7days with 20µg CNP instilled animals. Compared to 24h, BAL PMN counts were significantly decreased at day 3 and by day 7 complete resolution of PMN influx was observed. We have identified protein markers related to acute response and also for time course response in lung and BAL. After complete resolution of PMN influx on day7, protein concentrations of FGA, CRP, APCS, CCL5, CCL9, FGF2, CCL11, vWF, XCL1, CXCL5, THPO, VEGFA, IL1B, IL18, CSF1, EDN1, CCL3, CCL4, F3 and CD40L were significantly elevated in the lung and BAL. These factors are involved in a closely regulated molecular cascade with IL1β/IL18at the upstream and FGF2/EDN1/VEGF at the downstream. BAL-protein concentration and histological analysis did not reveal any evidence of tissue injury in 20µg CNP instilled animals. Conclusion: Considering the role of VEGF, FGF2 and EDN1 in lung development and morphogenesis together with the lack of any evident tissue damage we suggest a protective/homeostatic machinery is activated to counter the CNP challenge as a precautionary measure.

Lipidomic Analysis of Dynamic Eicosanoid Responses during the Induction and Resolution of Lyme Arthritis

Eicosanoids and other bioactive lipid mediators are indispensable regulators of biological processes, as demonstrated by the numerous inflammatory diseases resulting from their dysregulation, including cancer, hyperalgesia, atherosclerosis, and arthritis. Despite their importance, a robust strategy comparable with gene or protein array technology for comprehensively analyzing the eicosanoid metabolome has not been forthcoming. We have developed liquid chromatography-tandem mass spectrometry methodology that quantitatively and comprehensively analyzes the eicosanoid metabolome and utilized this approach to characterize eicosanoid production during experimental Lyme arthritis in mice infected with the bacterium Borrelia burgdorferi. Eicosanoids were extracted throughout infection from the joints of Lyme arthritis-resistant and -susceptible mice and subjected to lipidomic profiling. We identified temporal and quantitative differences between these mouse strains in the production of eicosanoids, which correlated with differences in arthritis development. The eicosanoid biosynthetic enzyme cyclooxygenase (COX)-2 has been implicated in the regulation of Lyme arthritis pathology, and subsequent lipidomic profiling of B. burgdorferi-infected COX-2(-/-) mice identified reductions not only in COX-2 products but, surprisingly, also significant off-target reductions in 5-lipoxygenase metabolites. Our results demonstrate the utility of a comprehensive lipidomic approach for identifying potential contributors to disease pathology and may facilitate the development of more precisely targeted treatment strategies.

Quantitative Trait Locus Analysis of Neointimal Formation in an Intercross Between C57BL/6 and C3H/HeJ Apolipoprotein E–Deficient Mice

Inbred mouse strains C57BL/6J (B6) and C3H/HeJ (C3H) exhibit marked differences in neointimal formation after arterial injury when deficient in apolipoprotein E (apoE(-/-)) and fed a Western diet. Quantitative trait locus (QTL) analysis was performed on an intercross between B6.apoE(-/-) and C3H.apoE(-/-) mice to determine genetic factors contributing to the phenotype. Female B6.apoE(-/-) mice were crossed with male C3H.apoE(-/-) mice to generate F(1)s, which were intercrossed to generate 204 male F(2) progeny. At 10 weeks of age, F(2)s underwent endothelium denudation injury to the left common carotid artery. Mice were fed a Western diet for 1 week before and 4 weeks after injury and analyzed for neointimal lesion size, plasma lipid and MCP-1 levels. One significant QTL, named Nih1 (61cM, LOD score: 5.02), on chromosome 12 and a suggestive locus on chromosome 13 (35cM, LOD: 2.67) were identified to influence lesion size. One significant QTL on distal chromosome 1 accounted for major variations in plasma non-HDL cholesterol and triglyceride levels. Four suggestive QTLs on chromosomes 1, 2, and 3 were detected for circulating MCP-1 levels. No correlations were observed between neointimal lesion size and plasma lipid levels or between lesion size and plasma MCP-1 levels. Neointimal formation is controlled by genetic factors independent of those affecting plasma lipid levels and circulating MCP-1 levels in the B6 and C3H mouse model.

Variable Effects of Caloric Restriction on Metabolic and Breathing Regulation Between Two Inbred Mouse Strains

The World Health Organization reports that ~800 million people worldwide are undernourished, corresponding to a daily mortality rate of 25,000 people. While altered metabolic and breathing characteristics occur with severe caloric restriction (CR), the mechanisms are unclear. We hypothesize that a robust genetic difference between C3H/HeJ (C3) and C57BL/6 (B6) mice lead to variation in metabolic and breathing regulation with CR. C3 and B6 mice (n = 8 per strain) were fed a diet equal to 33% of the daily food intake for 21 days. Using plethysmography, breathing characteristics were measured weekly on room air, and following acute hypercapnic (5% CO2) and hypoxic (10% O2) challenges. While the decline in body weight through 21 days of CR was similar in C3 vs. B6 strains (~40%), reductions in body temperature (10.0±1.8 vs. 2.8±1.0°C) and O2 consumption (1.36±0.32 vs. ‐0.04±0.16 ml/hr/g) were significantly (P &lt; 0.01) lower in C3 relative to B6 mice. Minute ventilation (VE) on room air remained unchanged with CR in both strains, but hypercapnic and hypoxic VE were depressed incrementally though 14 days. By day 21 of CR, hypercapnic and hypoxic VE recovered in B6 but not in C3 mice. In conclusion, genetic factors determine variation in metabolic and breathing regulation during CR, allowing B6 mice to maintain indices of homeostasis closer to control values than C3 mice.

Pathway focused protein profiling indicates differential function for IL-1B, -18 and VEGF during initiation and resolution of lung inflammation evoked by carbon nanoparticle exposure in mice

BackgroundCarbonaceous nanoparticles possess an emerging source of human exposure due to the massive release of combustion products and the ongoing revolution in nanotechnology. Pulmonary inflammation caused by deposited nanoparticles is central for their adverse health effects. Epidemiological studies suggest that individuals with favourable lung physiology are at lower risk for particulate matter associated respiratory diseases probably due to efficient control of inflammation and repair process. Therefore we selected a mouse strain C3H/HeJ (C3) with robust lung physiology and exposed it to moderately toxic carbon nanoparticles (CNP) to study the elicited pulmonary inflammation and its resolution.Methods5 μg, 20 μg and 50 μg CNP were intratracheally (i.t.) instilled in C3 mice to identify the optimal dose for subsequent time course studies. Pulmonary inflammation was assessed using histology, bronchoalveolar lavage (BAL) analysis and by a panel of 62 protein markers.Results1 day after instillation of CNP, C3 mice exhibited a typical dose response, with the lowest dose (5 μg) representing the 'no effect level' as reflected by polymorphonuclear leucocyte (PMN), and BAL/lung concentrations of pro-inflammatory proteins. Histological analysis and BAL-protein concentration did not reveal any evidence of tissue injury in 20 μg CNP instilled animals. Accordingly time course assessment of the inflammatory response was performed after 3 and 7 days with this dose (20 μg). Compared to day 1, BAL PMN counts were significantly decreased at day 3 and completely returned to normal by day 7. We have identified protein markers related to the acute response and also to the time dependent response in lung and BAL. After complete resolution of PMN influx on day 7, we detected elevated concentrations of 20 markers that included IL1B, IL18, FGF2, EDN1, and VEGF in lung and/or BAL. Biological pathway analysis revealed these factors to be involved in a closely regulated molecular cascade with IL1B/IL18 as upstream and FGF2/EDN1/VEGF as downstream molecules.ConclusionConsidering the role of VEGF, FGF2 and EDN1 in lung development and morphogenesis together with the lack of any evident tissue damage we suggest a protective/homeostatic machinery to be associated in lungs of stable organisms to counter the CNP challenge as a precautionary measure.

Prenatal activation of 5‐HT2A receptor induces expression of 5‐HT1B receptor in phrenic motoneurons and alters the organization of their premotor network in newborn mice

In newborn mice of the control [C3H/HeJ (C3H)] and monoamine oxidase A-deficient (Tg8) strains, in which levels of endogenous serotonin (5-HT) were drastically increased, we investigated how 5-HT system dysregulation affected the maturation of phrenic motoneurons (PhMns), which innervate the diaphragm. First, using immunocytochemistry and confocal microscopy, we observed a 5-HT(2A) receptor (5-HT(2A)-R) expression in PhMns of both C3H and Tg8 neonates at the somatic and dendritic levels, whereas 5-HT(1B) receptor (5-HT(1B)-R) expression was observed only in Tg8 PhMns at the somatic level. We investigated the interactions between 5-HT(2A)-R and 5-HT(1B)-R during maturation by treating pregnant C3H mice with a 5-HT(2A)-R agonist (2,5-dimethoxy-4-iodoamphetamine hydrochloride). This pharmacological overactivation of 5-HT(2A)-R induced a somatic expression of 5-HT(1B)-R in PhMns of their progeny. Conversely, treatment of pregnant Tg8 mice with a 5-HT(2A)-R antagonist (ketanserin) decreased the 5-HT(1B)-R density in PhMns of their progeny. Second, using retrograde transneuronal tracing with rabies virus injected into the diaphragm of Tg8 and C3H neonates, we studied the organization of the premotor network driving PhMns. The interneuronal network monosynaptically connected to PhMns was much more extensive in Tg8 than in C3H neonates. However, treatment of pregnant C3H mice with 2,5-dimethoxy-4-iodoamphetamine hydrochloride switched the premotoneuronal network of their progeny from a C3H- to a Tg8-like pattern. These results show that a prenatal 5-HT excess affects, via the overactivation of 5-HT(2A)-R, the expression of 5-HT(1B)-R in PhMns and the organization of their premotor network.

Genetic Variation in Mouse Femoral Tissue-Level Mineral Content Underlies Differences in Whole Bone Mechanical Properties

A/J mice, as compared to C57BL/6J (B6) mice, have a significantly greater total femoral mineral (ash) content which correlates with an increased femoral stiffness (resistance to deformation), but also with an increased brittleness (catastrophic failure). To determine if this whole bone variation in mineral content is indicative of significant mineral and/or matrix variation at the tissue level, femora from 16-week-old female A/J and B6 mice were isolated, embedded in PMMA, sectioned and mounted on barium fluoride infrared windows for FTIRI analyses. In addition, preliminary studies of femora from C3H/HeJ (C3H) mice were conducted, since they have an ash content intermediate to A/J and B6. Mean values for mineral-to-matrix ratio were significantly different for A/J (8.4 ± 0.8) and B6 (7.5 ± 0.4), as were values for collagen cross-link maturity (1.8 ± 0.05 and 3.2 ± 0.1, respectively). C3H mice appeared to have a mineral-to-matrix ratio intermediate of A/J and B6, and cross-link maturity greater than both A/J and B6. B6 femora had similar carbonate-to-amide ratios, carbonate-to-mineral ratios and acid phosphate levels. Thus, whole bone differences in mineral content are concurrent with tissue-level variation in mineral content and collagen maturity in inbred mice. The greater stiffness and brittleness of A/J femora are likely due to differential biological control (osteoblast activity) of the amount of mineral.

Alveolar Process Anabolic Activity in C3H/HeJ and C57BL/6J Inbred Mice

The purpose of this study was to characterize bone activity of the alveolar process in C3H/HeJ (C3H) and C57BL/6J (B6) inbred mice. Based on observations in other animal species, we hypothesized that the bone-formation rate/bone surface (BFR/BS) is greater in the alveolar process compared to the body of the mandible and that the bone anabolic activity is greater in the alveolar process of the mandible than in the maxilla. We also examined the alveolar process of C3H and B6 mice for the presence of secondary osteons. Jaws from 17-week-old C3H and B6 female mice (N = 15/group) were harvested. Histomorphometric parameters were evaluated in sections from the alveolar process, each of which included at least one molar root. In C3H and B6 mice, BFR/BS was not significantly different (P >0.05) between the alveolar process and the body of the mandible. In C3H mice, BFR/BS was significantly greater (P = 0.05) in the mandible compared to the maxilla. BFR/bone volume (BV) was not significantly different (P >0.05) between C3H mandible and maxilla. In the B6 inbred mouse, BFR/BS and BFR/bone volume (BV) were not significantly different (P >0.05) between jaws. After analyzing 165 bone sections, we identified 25 secondary osteons. The surface anabolic activity was not different between the body and the alveolar process of the mandible. The surface activity was greater in the C3H mandible than in the maxilla. Although secondary osteonal bone remodeling existed in the C3H and B6 alveolar bone, this process was not a consistent finding.

32 wk old C3H/HeJ mice actively respond to mechanical loading

Numerous studies indicate that C3H/HeJ (C3H) mice are mildly responsive to mechanical loading compared to C57BL/6J (C57) mice. Guided by data indicating high baseline periosteal osteoblast activity in 16 wk C3H mice, we speculated that simply allowing the C3H mice to age until basal periosteal bone formation was equivalent to that of 16 wk C57 mice would restore mechanoresponsiveness in C3H mice. We tested this hypothesis by subjecting the right tibiae of 32 wk old C3H mice and 16 wk old C57 mice to low magnitude rest-inserted loading (peak strain: 1235 μɛ) and then exposing the right tibiae of 32 wk C3H mice to low (1085 μɛ) or moderate (1875 μɛ) magnitude cyclic loading. The osteoblastic response to loading on the endocortical and periosteal surfaces was evaluated via dynamic histomorphometry. At 32 wk of age, C3H mice responded to low magnitude rest-inserted loading with significantly elevated periosteal mineralizing surface, mineral apposition rate and bone formation compared to unloaded contralateral bones. Surprisingly, the periosteal bone formation induced by low magnitude rest-inserted loading in C3H mice exceeded that induced in 16 wk C57 mice. At 32 wk of age, C3H mice also demonstrated an elevated response to increased magnitudes of cyclic loading. We conclude that a high level of basal osteoblast function in 16 wk C3H mice appears to overwhelm the ability of the tissue to respond to an otherwise anabolic mechanical loading stimulus. However, when basal surface osteoblast activity is equivalent to that of 16 wk C57 mice, C3H mice demonstrate a clear ability to respond to either rest-inserted or cyclic loading.