6N Mice Research Articles

Abstract 591: Absence of Nicotinamide Nucleotide Transhydrogenase Exacerbates Atherosclerosis in Mice

Oxidant stress contributes to endothelial cell injury and inflammation that are hallmarks of early stage atherosclerosis. Emerging evidence implicates mitochondrial reactive oxygen species (ROS) as important contributors to this oxidant stress and differences in mitochondrial function may augment this process. We have shown that variation in mitochondrial function and ROS production associated with ethnicity contributes to endothelial and vascular dysfunction. To model these distinct mitochondrial redox phenotypes we used C57Bl/6N (6N) and C57Bl/6J (6J) mice that also display unique mitochondrial functional properties due to the differential expression nicotinamide nucleotide transhydrogenase (NNT). Mice were treated with adeno-associated virus encoding a gain-of-function form of proprotein convertase subtilisin/kexin type 9 (PCSK9) that leads to hypercholesterolemia, increased LDL levels, and atherosclerosis in mice. PCSK9 treatment and 8 weeks of high fat diet led to increases in plasma lipids in both 6N and 6J mice. However, 6J animals displayed significantly higher levels of fat deposition in the vasculature and increased plaque size in the carotid sinus. 6N mice co-treated with the mitochondria targeted superoxide dismutase mimetic MitoTEMPO for the final 4 weeks of the experiment displayed reduced plasma lipids, but no impact on fat deposition or plaque size was observed. In contrast, MitoTEMPO increased vascular fat deposition and plaque size in 6J mice consistent with a more severe atherosclerotic phenotype. Increased mitochondrial ROS was confirmed by demonstrating elevated vascular superoxide in 6J versus 6N animals and that this difference is exacerbated on high fat diet. MitoTEMPO diminished vascular superoxide production to near baseline levels in both groups, yet increased plaque size and fat deposition in 6J mice suggests a role for hydrogen peroxide in this process. These data indicate that loss of NNT and changes in mitochondrial function increase vascular ROS production and exacerbate lipid deposition and plaque development in the early stages of atherosclerosis.

C57BL/6 Substrains Exhibit Different Responses to Acute Carbon Tetrachloride Exposure: Implications for Work Involving Transgenic Mice.

Biological differences exist between strains of laboratory mice, and it is becoming increasingly evident that there are differences between substrains. In the C57BL/6 mouse, the primary substrains are called 6J and 6N. Previous studies have demonstrated that 6J and 6N mice differ in response to many experimental models of human disease. The aim of our study was to determine if differences exist between 6J and 6N mice in terms of their response to acute carbon tetrachloride (CCl4) exposure. Mice were given CCl4 once and were euthanized 12 to 96 h later. Relative to 6J mice, we found that 6N mice had increased liver injury but more rapid repair. This was because of the increased speed with which necrotic hepatocytes were removed in 6N mice and was directly related to increased recruitment of macrophages to the liver. In parallel, enhanced liver regeneration was observed in 6N relative to 6J mice. Hepatic stellate cell activation occurred earlier in 6N mice, but there was no difference in matrix metabolism between substrains. Taken together, these data demonstrate specific and significant differences in how the C57BL/6 substrains respond to acute CCl4, which has important implications for all mouse studies utilizing this model.

Time-course microarray analysis for identifying candidate genes involved in obesity-associated pathological changes in the mouse colon.

BackgroundObesity is known to increase the risk of colorectal cancer. However, mechanisms underlying the pathogenesis of obesity-induced colorectal cancer are not completely understood. The purposes of this study were to identify differentially expressed genes in the colon of mice with diet-induced obesity and to select candidate genes as early markers of obesity-associated abnormal cell growth in the colon.MethodsC57BL/6N mice were fed normal diet (11% fat energy) or high-fat diet (40% fat energy) and were euthanized at different time points. Genome-wide expression profiles of the colon were determined at 2, 4, 8, and 12 weeks. Cluster analysis was performed using expression data of genes showing log2 fold change of ≥1 or ≤−1 (twofold change), based on time-dependent expression patterns, followed by virtual network analysis.ResultsHigh-fat diet-fed mice showed significant increase in body weight and total visceral fat weight over 12 weeks. Time-course microarray analysis showed that 50, 47, 36, and 411 genes were differentially expressed at 2, 4, 8, and 12 weeks, respectively. Ten cluster profiles representing distinguishable patterns of genes differentially expressed over time were determined. Cluster 4, which consisted of genes showing the most significant alterations in expression in response to high-fat diet over 12 weeks, included Apoa4 (apolipoprotein A-IV), Ppap2b (phosphatidic acid phosphatase type 2B), Cel (carboxyl ester lipase), and Clps (colipase, pancreatic), which interacted strongly with surrounding genes associated with colorectal cancer or obesity.ConclusionsOur data indicate that Apoa4, Ppap2b, Cel, and Clps are candidate early marker genes associated with obesity-related pathological changes in the colon. Genome-wide analyses performed in the present study provide new insights on selecting novel genes that may be associated with the development of diseases of the colon.Electronic supplementary materialThe online version of this article (doi:10.1186/s12263-016-0547-x) contains supplementary material, which is available to authorized users.

A Direct Comparison of Metabolic Responses to High-Fat Diet in C57BL/6J and C57BL/6NJ Mice.

Although nicotinamide nucleotide transhydrogenase (NNT)–deficient C57BL/6J (6J) mice are known to be highly susceptible to diet-induced metabolic disease, this notion stems primarily from comparisons of 6J mice to other inbred strains. To date, very few studies have directly compared metabolic disease susceptibility between NNT-deficient 6J mice and NNT-competent C57BL/6 substrains. In this study, comprehensive profiling of the metabolic response to a high-fat/high-sucrose diet (HFD) were compared across time in 6J and C57BL/6NJ (6N) mice. Given that increased peroxide exposure drives insulin resistance, coupled with the fact that NNT regulates peroxide detoxification, it was hypothesized that 6J mice would experience greater derangements in redox homeostasis/metabolic disease upon HFD exposure. Contrary to this, both lines were found to be highly susceptible to diet-induced metabolic disease, as evidenced by impairments in glucose tolerance as early as 24 h into the HFD. Moreover, various markers of the metabolic syndrome, as well as peroxide stress, were actually blunted, rather than exacerbated, in the 6J mice, likely reflecting compensatory increases in alterative redox-buffering pathways. Together, these data provide evidence that the susceptibility to HFD-induced metabolic disease is similar in the 6J and 6N substrains. Given the numerous genetic variances in the 6J stain, including loss of NNT function, these findings suggest that the 6N substrain is the more logical and representative genetic background model for metabolic studies.

The Relative Gene Expression of GAPDH in Mice Fed with a Short-Term High Fat Diet

The important role of nutrition in health and disease prevention is well recognized. The glyceraldehyde-3dehydrogenase phosphate gene (GAPDH) is considered as a housekeeping gene: it encodes a key enzyme of the glycolysis, and seems to affect the metabolism of fatty acids. The purpose of this work was to study the relative expression of the GAPDH gene in mice subjected to a short-term high fat diet. The expression of the GAPDH gene has been analyzed in the livers of C57BL / 6N mice: 5 control mice and 5 mice subjected to a short term high-fat diet. The quantification of the mRNA has been performed by a real-time PCR using specific primers designated and validated in silico. The data have been analyzed by the REST software. The results showed a significant down expression (p < 0.005) of the GAPDH gene in mice subjected to high-fat diet. This suggests that a lipid diet may have an effect on the expression of the GAPDH gene. Thus the use of GAPDH as a reference gene should be reconsidered notably in gene expression studies.

A Novel Model of Metastatic Conjunctival Melanoma in Immune-Competent Mice.

Conjunctival melanoma (CM) is an ocular surface tumor that can lead to fatal metastases. Patients developing, tumor-associated lymphangiogenesis have a significantly increased risk of metastatic disease, because tumor spread primarily occurs via lymphatic vessels to the draining lymph node. Here, we describe a novel immune-competent mouse model of CM that displays tumor-associated lymphangiogenesis with development of metastatic tumors. C57BL/6N mice received C57BL/6N-derived dermal melanoma cells (hepatocyte growth factor [HGF] cyclin dependent kinase-4 [Cdk4]+) or B16F10 via subconjunctival injection. A clinical score quantified primary tumor growth and metastases were identified by macroscopic examination of the draining lymph nodes, lung, and spleen. Confirmation of tumors and metastases was achieved by immunohistochemical staining for markers of pigmented cells (tyrosinase related protein-2 [TRP2]) and S-100, and of cell proliferation (Ki67). The intra- and peritumoral CD31+ blood and lymphatic vessel endothelium hyaluronan receptor-1 (LYVE-1)+ lymphatic vessels were quantified immunohistochemically. All mice rapidly developed aggressive TRP2+, S100+, and Ki67+ CM. Metastatic tumors were found in the lymph node (9%) and lung (6%) of HGF-Cdk4(R24C)-treated mice and in the spleen (8%) and lung (17%) of B16F10-treated mice. The amount of peri- and intratumoral blood vessels was significantly increased compared with lymphatic vessels. This CM model in immune-competent animals offers new possibilities to study the pathobiology of tumor growth, invasion, and mechanisms of metastatic tumor spread, and provides a robust model to explore new immune-based and antilymphangiogenic treatment modalities of this malignancy.

Evaluation of Left Ventricle Function by Regional Fractional Area Change (RFAC) in a Mouse Model of Myocardial Infarction Secondary to Valsartan Treatment.

AimLeft ventricle (LV) regional fractional area change (RFAC) measured by cardiac magnetic resonance (CMR) allows the non-invasive localization and quantification of the degree of myocardial infarction (MI), and could be applied to assess the effectiveness of pharmacological or regenerative therapies. Here we investigate the ability of RFAC to identify regional dysfunction and discriminate the effect of pharmacological treatment with valsartan, a selective antagonist of angiotensin II type 1 receptor, in a model of MI.Methods and ResultsC57BL/6N mice, undergoing coronary artery ligation, were divided into two groups: untreated (MI) or treated with valsartan (MI+Val). Sham-operated mice were used as a control. Cardiac dimensions and function were assessed at baseline, 24 hours, 1 and 4 weeks post surgery by CMR and echocardiography. At sacrifice histology and whole-genome gene expression profiling were performed. RFAC was able to detect significant differences between treatment groups whereas the global ejection fraction was not. RFAC showed greater loss of regional contraction in remote non-infarcted myocardium in MI group than in MI+Val group. Consistently, in the same region MI+Val mice showed reduced myocyte hypertrophy, fibroblast proliferation, and fibrosis and modulation of target genes; in addition, left atrium volumes, appendage length and duct contraction were preserved.ConclusionIn this study, RFAC effectively estimated the degree of systolic dysfunction and discriminated the regions preserved by pharmacological treatment. RFAC index is a promising tool to monitor changes in LV contraction and to assess the effectiveness of therapeutic regimens in clinical settings.

Potential Biomarkers for Radiation-Induced Renal Toxicity following 177Lu-Octreotate Administration in Mice.

The kidneys are one of the main dose-limiting organs in peptide receptor radionuclide therapy and due to large inter-individual variations in renal toxicity, biomarkers are urgently needed in order to optimize therapy and reduce renal tissue damage. The aim of this study was to investigate the transcriptional, functional, and morphological effects on renal tissue after 177Lu-octreotate administration in normal mice, and to identify biomarkers for radiation induced renal toxicity.MethodsC57BL/6N mice were i.v. injected with 0, 30, 60, 90, 120, or 150 MBq 177Lu-octreotate (0, 16, 29, 40, 48, and 54 Gy to the kidneys). At 4, 8, and 12 months after administration, radiation-induced effects were evaluated in relation to (a) global transcriptional variations in kidney tissues, (b) morphological changes in the kidneys, (c) changes in white and red blood cell count as well as blood levels of urea, and (d) changes in renal function using 99mTc-DTPA/99mTc-DMSA scintigraphy.ResultsIn general, the highest number of differentially regulated transcripts was observed at 12 months after administration. The Cdkn1a, C3, Dbp, Lcn2, and Per2 genes displayed a distinct dose-dependent regulation, with increased expression level with increasing absorbed dose. Ifng, Tnf, and Il1B were identified as primary up-stream regulators of the recurrently regulated transcripts. Furthermore, previously proposed biomarkers for kidney injury and radiation damage were also observed. The functional investigation revealed reduced excretion of 99mTc-DTPA after 150 MBq, an increased uptake of 99mTc-DMSA at all dose levels compared with the controls, and markedly increased urea level in blood after 150 MBq at 12 months.ConclusionDistinct dose-response relationships were found for several of the regulated transcripts. The Cdkn1a, Dbp, Lcn2, and Per2 genes are proposed as biomarkers for 177Lu-octreotate exposure of kidney. Correlations to functional and morphological effects further confirm applicability of these genes as markers of radiation damage in kidney tissue.

Effects of quercetin derivatives from mulberry leaves: Improved gene expression related hepatic lipid and glucose metabolism in short-term high-fat fed mice

BACKGROUND/OBJECTIVESMulberry leaves contain quercetin derivatives, which have the effects of reducing obesity and improving lipid and glucose metabolism in mice with obesity. It is not clear whether or not mulberry leaves can directly affect metabolic disorders, in the presence of obesity, because of the interaction between obesity and metabolic disorders. The aim of the current study was to assess the direct action of quercetin derivatives on metabolic disorders in non-obese conditions in short-term high-fat diet fed mice.MATERIALS/METHODSC57BL/6N mice were fed a high-fat diet, supplemented with either 0% (control), 1%, or 3% mulberry leaf powder (Mul) or 1% catechin powder for five days. Anthropometric parameters and blood biochemistry were determined, and hepatic gene expression associated with lipid and glucose metabolism was analyzed.RESULTSBody and white fat weights did not differ among the four groups. Plasma triglycerides, total cholesterol, and free fatty acids in the 1%, 3% Mul and catechin groups did not differ significantly from those of the controls, however, plasma glucose and 8-isoprostane levels were significantly reduced. Liver gene expression of gp91phox, a main component of NADPH oxidase, was significantly down-regulated, and PPAR-α, related to β-oxidation, was significantly up-regulated. FAS and GPAT, involved in lipid metabolism, were significantly down-regulated, and Ehhadh was significantly up-regulated. Glucose-metabolism related genes, L-PK and G6Pase, were significantly down-regulated, while GK was significantly up-regulated in the two Mul groups compared to the control group.CONCLUSIONSOur results suggest that the Mul quercetin derivatives can directly improve lipid and glucose metabolism by reducing oxidative stress and enhancing β-oxidation. The 1% Mul and 1% catechin groups had similar levels of polyphenol compound intake (0.4 × 10-5 vs 0.4 × 10-5 mole/5 days) and exhibited similar effects, but neither showed dose-dependent effects on lipid and glucose metabolism or oxidative stress.

A comparative analysis of C57BL/6J and 6N substrains; chemokine/cytokine expression and susceptibility to laser-induced choroidal neovascularization

Age-related macular degeneration (AMD) is the most prevalent cause of blindness in the elderly. To study potential underlying mechanisms of AMD, animal models are utilized, focusing mostly on mice. Recently, genomic and phenotypic differences between the so-called control substrains, C57BL/6J and C57BL/6N, have been described in models of ocular and non-ocular diseases. In particular, the rd8 mutation of the Crb1 gene present in the C57BL/6N has been shown to impact certain ocular phenotypes and appears to augment phenotypes generally associated with inflammation. Here, we investigated angiogenic factor and cytokine expression using pathway arrays as well as the susceptibility to laser-induced choroidal neovascularization (CNV), a model of wet AMD, in the two substrains. Age-matched 3-month-old C57BL/6J and C57BL/6N animals differed in gene expression levels for angiogenic factors and cytokines, with 6N animals expressing higher levels of inflammatory markers than 6Js. Yet laser-induced CNV was comparable in size between the two substrains. This lack of difference in CNV size was correlated with a gene expression profile that was comparable between the two substrains, due to the fact that the degree of change in gene expression of inflammatory markers after CNV was blunted in 6N mice. In summary, significant gene expression differences exist between C57BL/6J and C57BL/6N animals, reinforcing the notion that appropriate litter-mate controls or genetic background controls need to be used. Contrary to our expectation, CNV was not augmented in 6N animals, suggesting that low chronic inflammation in the RPE might provide a level of pre-conditioning and protection against stress.

Nicotinic agonist-induced improvement of vigilance in mice in the 5-choice continuous performance test

Impaired attentional processing is prevalent in numerous neuropsychiatric disorders and may negatively impact other cognitive and functional domains. Nicotine – a nonspecific nicotinic acetylcholine receptor (nAChR) agonist – improves vigilance in healthy subjects and schizophrenia patients as measured by continuous performance tests (CPTs), but the nAChR mediating this effect remains unclear. Here we examine the effects of: (a) nicotine; (b) the selective α7 nAChR agonist PNU 282987; and (c) the selective α4β2 nAChR agonist ABT-418 alone and in combination with scopolamine-induced disruption of mouse 5-choice (5C-)CPT performance. This task requires the inhibition of responses to non-target stimuli as well as active responses to target stimuli, consistent with human CPTs.C57BL/6N mice were trained to perform the 5C-CPT. Drug effects were examined in extended session and variable stimulus-duration challenges of performance. Acute drug effects on scopolamine-induced disruption in performance were also investigated.Nicotine and ABT-418 subtly but significantly improved performance of normal mice and attenuated scopolamine-induced disruptions in the 5C-CPT. PNU 282–987 had no effects on performance.The similarity of nicotine and ABT-418 effects provides support for an α4β2 nAChR mechanism of action for nicotine-induced improvement in attention/vigilance. Moreover, the data provide pharmacological predictive validation for the 5C-CPT because nicotine improved and scopolamine disrupted normal performance of the task, consistent with healthy humans in the CPT. Future studies using more selective agonists may result in more robust improvements in performance.

The Volatile Anesthetic Isoflurane Prevents Ventilator-Induced Lung Injury via Phosphoinositide 3-Kinase/Akt Signaling in Mice

Mechanical ventilation leads to ventilator-induced lung injury in animals, and can contribute to acute lung injury/acute respiratory distress syndrome in humans. Acute lung injury/acute respiratory distress syndrome currently causes an unacceptably high rate of morbidity and mortality among critically ill patients. Volatile anesthetics have been shown to exert anti-inflammatory and organ-protective effects in vivo. We investigated the effects of the volatile anesthetic isoflurane on lung injury during mechanical ventilation. C57BL/6N mice were ventilated with a tidal volume of 12 mL/kg body weight for 6 hours in the absence or presence of isoflurane, and, in a second series, with or without the specific phosphoinositide 3-kinase/Akt inhibitor LY294002. Lung injury was determined by comparative histology, and by the isolation of bronchoalveolar lavage for differential cell counting and analysis of cytokine levels using enzyme-linked immunosorbent assays. Lung homogenates were analyzed for protein expression by Western blotting. Mechanical ventilation caused increases in alveolar wall thickening, cellular infiltration, and an elevated ventilator-induced lung injury score. Neutrophil influx and cytokine (i.e., interleukin-1β, and macrophage inflammatory protein-2) release were enhanced in the bronchoalveolar lavage of ventilated mice. The expression levels of the stress proteins hemeoxygenase-1 and heat shock protein-70 were elevated in lung tissue homogenates. Isoflurane ventilation significantly reduced lung damage, inflammation, and stress protein expression. In contrast, phosphorylation of Akt protein was substantially increased during mechanical ventilation with isoflurane. Inhibition of phosphoinositide 3-kinase/Akt signaling before mechanical ventilation completely reversed the lung-protective effects of isoflurane treatment in vivo. Inhalation of isoflurane during mechanical ventilation protects against lung injury by preventing proinflammatory responses. This protection is mediated via phosphoinositide 3-kinase/Akt signaling.

Influence of astragalosides and Panax notoginseng saponins compatibility on MMP-9 and TIMP-1 after cerebral ischemia-reperfusion in mice

To investigate the effect of astragalosides (AST) and Panax notoginseng saponins (PNS) compatibility on the expression of matrix metalloproteinases-9 (MMP-9) and tissue inhibitor of metal11oproteinase-1 (TIMP-1) after cerebral ischemia/reperfusion (I/R) injury in mice, to probe into its anti-ischemic brain injury protection mechanism. C57BL/6N mice were randomly divided into sham-operation group, model group, AST and PNS compatibility of high, medium and low-dose group, AST group, PNS group and edaravone group. Cerebral ischemia-reperfusion injury were prepared by bilateral common carotid artery ligation for 20 min followed by 24 hours reperfusion after administration for 4 days. Pathomorphism was detected with HE staining and the expression of MMP-9 and TIMP-1 protein in brain was detected by western-blot. The neuronal survival rate in the drug groups was significantly higher than the control group (P < 0.01), and the effect of the-middle dose compatibility group was more obvious. Factorial analysis manifested that AST110 mg x kg(-1) and PNS115 mg x kg(-1) compatibility had a synergistic interaction (P < 0.01). The expression of MMP-9 protein in the drug groups was lower than the model group significantly (P < 0.01 or P < 0.05), but the expression of TIMP-1 protein was higher than the model group significantly (P < 0.01 or P < 0.05), and the effect of the-middle dose compatibility group was more obvious, the two drugs had the stacking interaction (P < 0.05). AST110 mg x kg(-1) and PNS115 mg x kg(-1) compatibility has a synergistic effect against ischemia-reperfusion injury in mice by accommodating MMP-9/TIMP-1 probably.

A novel model of CCl4-induced cirrhosis with ascites in the mouse

The current approaches to study the molecular mechanisms involved in the pathophysiology of liver diseases often rely on the use of transgenic mice. However, experimental models of decompensated cirrhosis have not been clearly established in mice. Thus, we aimed to set an efficient and well-tolerated protocol to induce cirrhosis in mice able to progress up to the ascitic stage. C57BL/6N mice received CCl(4) subcutaneously, intraperitoneally or by inhalation. In the latter group, gaseous CCl(4) was administered according to three different schedules: increasing exposure times, twice weekly (traditional protocol; TP), short inhalation cycles, twice or three times weekly. Portal hypertension, sodium retention, and ascites developed in all groups between 11 and 15 weeks. Mortality reached 70% in the TP group, but it was only 0-10% with all other protocols. All the inhalation groups had significantly more ascites at sacrifice than those receiving CCl(4) subcutaneously and intraperitoneally. Extensive abdominal adhesions and evidence of enhanced hepatic inflammation, as suggested by the increased gene expression of pro-inflammatory cytokines in liver tissue, were found in the intraperitoneal group, while large granulomas at the injection site and marked neutrophil infiltration of lungs developed in the subcutaneous group. No extra-hepatic damage could be detected in mice inhaling CCl(4). The use of short cycles of CCl(4) inhalation represents a novel, safe, and effective method to induce decompensated cirrhosis in mice. Intraperitoneal CCl(4) leads instead to abdominal adhesions precluding a correct evaluation of ascites, while subcutaneous CCl(4) causes an unwanted systemic inflammatory response.

Prolonged Increase in T-Cell Receptor (TCR) Variant Fractions of Spleen T Lymphocytes in Pregnant Mice after γ Irradiation

To investigate the relationship between the radiation-induced increase of T-cell receptor (TCR) defective variant fractions and physiological status such as pregnancy, C57BL/ 6N mice were irradiated with 3 Gy of gamma rays at various days of gestation, just before and just after pregnancy. While the highest level of variant fractions in spleen T lymphocytes appeared at 9 days postirradiation and resolved fairly rapidly for nonpregnant mice, the increased variant fractions for pregnant mice irradiated at 16.5 days of gestation reached a plateau at 14 days postirradiation and remained at high levels until 28 days after irradiation. Therefore, variant fractions 28 days postirradiation were measured to determine the overall effect of radiation on the kinetics of TCR variant fractions during gestation. There was no significant difference in the baseline TCR variant fraction between unirradiated nonpregnant and pregnant mice. TCR variant fractions after irradiation were about twofold higher in pregnant mice (from 10.5 days of gestation until delivery) than those in nonpregnant mice. Both gamma radiation and pregnancy caused a decrease in the proportion of naïve T-cell subsets and an increase in TCR variant fractions of naïve T cells. In addition, the prolonged postirradiation increase in the TCR variant fractions of pregnant mice was associated with an increase in serum progesterone level. Differences between pregnant and nonpregnant mice in the kinetics of postirradiation restoration of T-cell systems may be involved in producing the differences in residual TCR variant fractions of these mice.

Adipose Tissue Proadipogenic Redox Changes in Obesity

The role of inflammation and oxidative stress in the development of obesity and associated metabolic disorders is under debate. We investigated the redox metabolism in a non-diabetic obesity model, i.e. 11-week-old obese Zucker rats. Antioxidant enzyme activities, lipophilic antioxidant (alpha-tocopherol, coenzymes Q) and hydrophilic antioxidant (glutathione, vitamin C) contents and their redox state (% oxidized form), were studied in inguinal white fat and compared with blood and liver. The adipose tissues of obese animals showed a specific higher content of hydrophilic molecules in a lower redox state than those of lean animals, which were associated with lower lipophilic molecule content and lipid peroxidation. Conversely and as expected, glutathione content decreased and its redox state increased in adipose tissues of rats subjected to lipopolysaccharide-induced systemic oxidative stress. In these in vivo models, oxidative stress and obesity thus had opposite effects on adipose tissue redox state. Moreover, the increase in glutathione content and the decrease of its redox state by antioxidant treatment promoted in vitro the accumulation of triglycerides in preadipocytes. Taken together and contrary to the emergent view, our results suggest that obesity is associated with an intracellular reduced redox state that promotes on its own the development of a deleterious proadipogenic process.

Alternative therapy of earth elements increases the chondroprotective effects of chondroitin sulfate in mice

The administration of mineral sulphur water is an alternative experimental approach for the treatment of rheumatic diseases, such as osteoarthritis (OA), that cause the degeneration of bone and cartilage and sufferance to the patients. Chondroitin sulfate (CS) is a symptomatic slow acting nutropeucital agent currently used in molecular therapy of OA. Therefore, we have studied the role and efficacy of the selective soil paste from the mineral sulphur enriched spring (mud)-therapy alone or in combination with CS in the treatment of OA. The study was performed on 40 C57 Black 6N mice, an experimental model which spontaneously develop an osteoarthritic process. The animals were divided in 4 groups and were treated with the single agents or with the combination. After 30 days of treatment all the mice were sacrificed and right knees and blood were collected. It was found that CS determined a reduction of radiological and histological features of chondrodegeneration and that mud-therapy increased the effects of CS in the animal group treated with the combination. However, the effects of thermal therapy alone were not statistically significant. Since OA is characterized by an increase of the production of nitric oxide (NO) by chondrocytes in extracellular matrix with its consequent elevation in serum and synovial fluid, we have evaluated the effects of the treatments on serum NO levels. CS alone induced a statistically significant reduction of NO serum levels (90+/-13 micromM vs 219+/-60 microM of control group, P<0.05) while mud-therapy alone induced a not statistically significant reduction of serum NO (170+/-62 microM, P>0.05). However, the latter strongly potentiated the decrease of serum NO induced by CS (31+/-1.5 microM) with a high statistical significance if compared to both the control group (P<0.01) and the CS-treated group (P<0.05). In conclusion, this study demonstrates that mud-therapy with sulphur mineral water could represent an important phase of the therapeutic strategy of OA. This experimental strategy could integrate and potentiate the standard pharmacological tools. Moreover, we have set a valid experimental in vivo model for the study of the thermal effects on the development of OA.

Induction of intestinal tumors and lymphomas in C57BL/6N mice by a food-borne carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine.

2‐Amino‐l‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP) is the most abundant heterocyclic amine contained in cooked meat and fish. Although PhIP has been demonstrated to induce various types of tumors in rats, lymphomas predominated in mice using the CDF1 strain. To investigate the carcinogenic activity of PhIP on other organs in mice with a different genetic background, PhIP was administered to C57BL/6N mice. After a 40‐week administration of 300 ppm of PhIP in a high‐fat diet followed by continuous feeding with a high fat diet, C57BL/6N mice developed adenomas and adenocarcinomas in the small intestine, the incidences being 52% in males and 68% in females at weeks 95 and 70, respectively. Lymphomas of B‐cell origin also developed in both sexes as frequently as in the CDF1 strain, incidences being 48% in males and 32% in females. Although the incidence in PhIP‐treated female mice did not differ from that in the control mice, lymphomas developed significantly earlier in the PhIP‐treated mice. The present study demonstrated that the intestinal tract is another potential target of PhIP‐induced carcinogenesis in mice, and that the carcinogenic activity of PhIP could be affected by the genetic background of the animals.

Strain and substrain differences in context- and tone-dependent fear conditioning of inbred mice

The performance of C57BL/6J (6J), C57BL/6N (6N), DBA/2J (2J) and DBA/2N (2N) mice in context- and tone-dependent fear conditioning was determined 24 h after fear conditioning to evaluate and compare different behavioral measures as indices of emotional learning. Freezing, the change in activity and the size of the explored area were evaluated as behavioral parameters indicating fear. Additionally, the heart rate (HR) increase elicited by tone presentation was evaluated as an autonomic indicator of fear. During the context-dependent memory test, freezing was high only in 6J and 6N mice, whereas a drop of activity and a reduced exploratory area was measured in all strains. During the tone-dependent memory test, high freezing, low activity, reduced exploratory area and a strong HR increase were demonstrated only in 6N and 6J mice, whereas behavioral and HR changes of 2J and 2N mice were always low. In extinction tests, context- and tone-dependent freezing of 6J mice decayed significantly faster than the freezing of 6N mice, whereas in both substrains the conditioned tachycardia to tone extinguished similarly in the home cage. The data demonstrate that monitoring of additional behavioral measures besides freezing and autonomic measures is necessary to interpret differences in associative learning performance of mouse strains that could be related to a differential expression of fear.

Metastatic phenotype in hybrid cells derived from B16 melanoma

To determine whether or not the metastatic phenotype of mouse melanoma B16 is dominant, two derivative cell lines of B16, high metastatic F10 and low metastatic F1 were hybridized by somatic cell fusion. Prior to fusion, F10 and F1 cells were transfected with plasmid pKO neo or pSV2 hph, which confers resistance to G-418 or hygromycin B, respectively. Hybrid clones were isolated by dual resistance to G-418 and hygromycin B. The seven isolated near-tetraploid hybrid clones were examined for metastatic phenotypes. Hybrid cells were injected into the tail vein of syngeneic C57BL 6N mice and the number of metastatic nodules in the lung was counted 21 days later. Four hybrid clones had high metastatic phenotypes while one clone showed an intermediate metastatic phenotype and two low metastatic phenotypes. Most of the hybrid clones retained modally stable chromosome compositions, whereas one of the two low metastatic clones lost chromosomes during in vitro and in vivo proliferations. These results suggest that the high metastatic ability is genetically dominant in B16 melanoma cells and low metastatic hybrid cells may be the result of a loss of a gene(s) responsible for acquisition of the metastatic phenotype in the process of chromosomal rearrangement.