Comparison of intrinsic exercise capacity and response to acute exercise in ICR (Institute of Cancer Research) mice derived from three different lineages.

BACKGROUND: As a laboratory animal resource, the ICR mouse is commonly used in a variety of research fields. However, information on differences in exercise-related characteristics in ICR mice derived from different lineages and the underlying mechanisms remains to be elucidated. In this study, we investigated the intrinsic exercise capacity and a magnitude of response to acute exercise, and sought to identify mechanisms contributing to difference in Korl:ICR (a novel ICR lineage recently established by the National Institute of Food and Drug Safety Evaluation, Korea) and two commercialized ICR lineages derived from different origins (viz., A:ICR mouse from Orient Bio Com, the United States, and B:ICR mouse from Japan SLC Inc., Japan). RESULTS: Results showed that despite no significant difference in body weight and weight-proportioned tissue mass of heart and skeletal muscles among groups, the relatively low intrinsic exercise capacity and exaggerated response to acute exercise were identified in B:ICR comparted with Korl:ICR and A:ICR, as reflected by total work and lactate threshold (LT). Also, the mitochondrial efficiency expressed as the complex 1 and complex 1 + 2 respiratory control ratio (RCR) values for cardiac mitochondrial O2 consumption in B:ICR was significantly lower than that in Korl:ICR with higher level of state 2 respiration by glutamate/malate and UCP3 expression in cardiac muscle. CONCLUSIONS: Taken together, these results indicate that the intrinsic exercise capacity of ICR mouse varies according to lineages, suggesting the role of cardiac mitochondrial coupling efficiency as a possible mechanism that might contribute to differences in the intrinsic exercise capacity and magnitude of response to exercise.

Comparison of response to LPS-induced sepsis in three DBA/2 stocks derived from different sources.

Sepsis, one of the most fatal diseases in the world, is known to culminate in multiple organ failure due to an uncontrolled inflammatory response. Hence, the use of animal models in sepsis research is very important to study complex immune responses. The current study was undertaken to compare commercial stocks with KFDA stocks of DBA/2 mice as an animal model for sepsis study. To compare responses of DBA/2 mice to lipopolysaccharides (LPS)-induced sepsis, we measured altered characteristics of various factors associated with sepsis, including survival curves, organ failure and inflammatory response, in DBA/2Korl stock and two commercial stocks (DBA/2A and DBA/2B). Survival rates after LPS exposure were similar for DBA/2Korl and DBA/2B; however, for times over 20 h, survival rates were reduced and concentration dependent in DBA/2A. In order to evaluate multiple organ failure caused by sepsis, H&E stains were evaluated for liver and spleen tissues obtained in the early (2 h) and later (20 h) stages after exposure to LPS; no significant differences were observed between the three stocks. mRNA and protein levels of proinflammatory cytokines were assessed for evaluating inflammatory reactions, and were found to increase in a dose-dependent manner in most DBA/2 mice after LPS treatment. However, no changes were observed in the mRNA levels of proinflammatory cytokines at 20 h after LPS exposure in the DBA/2A stock. The induction of inflammation-mediated factors by LPS exposure did not induce alterations in the mRNA levels of COX-2 and iNOS in all three DBA/2 stocks. Our results indicate that response of DBA/2Korl to LPS-induced sepsis is similar to the two commercial DBA/2 stocks, thus representing its potential as a useful biological resource established in Korea.

Protective role of fermented mulberry leave extract in LPS­induced inflammation and autophagy of RAW264.7 macrophage cells.

Mulberry leaves have antioxidant activity and anti­inflammatory effects in several types of cells. However, the efficacy of mulberry leaves fermented with Cordyceps militaris remains unknown. Therefore, the present study aimed to investigate whether the ethanol extracts of mulberry leaves fermented with C. militaris (EMfC) can prevent lipopolysaccharide (LPS)­induced inflammation and autophagy in macrophages. To achieve this, RAW264.7 cells pretreated with three different dose of EMfCs were subsequently stimulated with LPS, and examined for alterations in the regulatory factors of inflammatory responses and key parameters of the autophagy signaling pathway. EMfC treatment inhibited the generation of reactive oxidative species; however, significant activity was observed for 2,2­diphenyl­1­picrylhydrazyl (DPPH) radical scavenging (IC50=579.6703 mg/ml). Most regulatory factors in inflammatory responses were significantly inhibited following treatment with EMfC, without any significant cellular toxicity. EMfC­treated groups exhibited marked suppression of nitrogen oxide (NO) levels, mRNA expression levels of iNOS/COX­2, levels of all inflammatory cytokines (TNF­α, IL­1ß and IL­6) and phosphorylation of MAPK members, as well as recovery of cell cycle progression. Furthermore, similar effects were observed in the LPS­induced autophagy signaling pathway of RAW264.7 cells. The expression levels of microtubule­associated protein 1A/1B­light chain 3 (LC3) and Beclin exhibited a dose­dependent decrease in the EMfC+LPS­treated groups compared with in the Vehicle+LPS­treated group, whereas the phosphorylation of PI3K and mTOR were enhanced in a dose­dependent manner in the same groups. Overall, the results of the present study provide evidence that exposure to EMfC protects against LPS­induced inflammation and autophagy in RAW264.7 cells. These results indicated that EMfC is a potential candidate for treatment of inflammatory diseases.

Synergic Laxative Effects of an Herbal Mixture of Liriope platyphylla, Glycyrrhiza uralensis, and Cinnamomum cassia in Loperamide-Induced Constipation of Sprague Dawley Rats.

Constipation is an acute or chronic illness attributed to various causes, ranging from lifestyle habits to side effects of a disease. To improve the laxative effects of some traditional medicines, herbal mixtures of Liriope platyphylla, Glycyrrhiza uralensis, and Cinnamomum cassia (LGC) were evaluated for their mechanism of action and therapeutic effects in loperamide (Lop)-induced constipated Sprague Dawley rats by examining alterations in excretion parameters, histological structure, mucin secretion, and related protein levels. Food intake and water consumption were constant for all animals. We observed that the Lop+LGC-treated group had significantly greater excretion of stool and urine than was observed in the Lop+Vehicle-treated group. Administration of LGC in the constipation model restored the intestinal transit ratio to normal levels, and increased the number of goblet cells, mucosal layer, and muscle thickness. Mucin secretion was greater in the Lop+LGC-treated group than in the Lop+Vehicle-treated group, and the expression of MUC2 and AQP8 genes were also increased. In addition, reverse transcription polymerase chain reaction and Western blot revealed an increase in the muscarinic acetylcholine receptors (mAChRs) in the Lop+LGC-treated group compared to the Lop+Vehicle-treated group. Furthermore, compared with the Lop+Vehicle-treated group, treatment with LGC reduced the phosphorylation of PKC and PI3K, and expression of Gα protein, but increased levels of IP3. Our results suggest that the traditional herbal mixture of LGC induces a potent laxative effect in Lop-induced constipation through mucosal tissue changes and mucin production. We also demonstrated that the laxative effect of LGC is closely related to the expression of mAChR and its downstream signals, suggesting the possibility of developing a constipation-laxative agent using LGC.

Comparative analysis of dose-dependent neurotoxic response to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in C57BL/6 N mice derived from three different sources.

MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine is commonly used to induce nigrostriatal defects to induce parkinsonism and/or parkinsonian syndrome, to replicate the lesions seen in Parkinson's disease (PD), with use in numerous PD models in mice. It has been suggested that various biological characteristics including strain could result in differing mortality rates, sensitivity to MPTP administration, and reproducibility of lesions in mice, but there is no evidence on the sensitivity of C57BL/6 mice from different origins to MPTP and its associated pathological lesions. In this study, we investigated the magnitude of the dose-dependent response to acute MPTP administration in C57BL/6NKorl mice and two commercialized C57BL/6 stocks derived from the United States and Japan. We measured biological features (body weight, temperature, and composition), nigrostriatal neurotoxic responses (dopamine levels, tyrosine hydroxylase enzymes, and protein carbonylation) and motor function. In results, the three different C57BL/6 stocks exhibited similar overall neurotoxic response and locomotor impairment which increased in a dose-dependent manner with acute MPTP administration (10 mg/kg, 20 mg/kg, and 30 mg/kg, all with external heat support), although some of these differences were not significant. In conclusion, this study provides scientific evidence that C57BL/6NKorl mice can be used as an alternative animal model for practical and targeted PD research.

Comparison of responsiveness to cancer development and anti-cancer drug in three different C57BL/6N stocks.

In our efforts to understand the systemic features of tumors, the importance of animal models is increasing due to the recent growth in the development of immunotherapy and targeted therapies. This has resulted in increased attention towards tumor animal models using C57BL/6N, which are mainly used in immunological studies. In this study, the C57BL/6NKorl stock and two other commercial stocks (C57BL/6NA and C57BL/N6B) are evaluated by comparing the occurrence of tumors using the syngeneic model; furthermore, we compare the response to anti-cancer drugs in the syngeneic model by evaluating survival, growth of tumors, proliferation and molecular biology analysis. In the syngeneic model using LLC (Lewis lung carcinoma) cells, the survival of mice and growth of the tumor showed a better response in the C57BL/6NKorl stock, and was dependent on the cell concentration of the dosing tumor, as compared to the other C57BL/6N stocks. However, the Ki-67 staining showed only little difference in cell proliferation within the tumor tissue each mouse stocks. Comparing the sensitivity to anti-cancer drug by examining changes in growth, volume and weight revealed that cisplatin treatment for tumor-bearing C57BL/6NKorl was more dependent on concentration. The Ki-67 staining, however, showed no difference among the C57BL/6N stocks after cisplatin treatment. The expressions of p27 and p53 tumor suppressor proteins, caspase-3 and Bax showed dose-dependent increase after exposure to cisplatin, whereas the expression of Bcl-2 was reduced in a dose-dependent manner. Furthermore, the expressions of MMP-2 and VEGF involved in metastasis, as well as inflammatory genes IL-1ß, IL-6 and IL-10, showed dose-dependent decrease in tumor tissue after cisplatin exposure. Differences observed among the C57BL/6N stocks were not significant. Taken together, our studies reveal that C57BL/6NKorl has the potential of being a useful biological resource established in Korea, as it does not differ from the two commercially available C57BL/6N stocks when considering response to tumor generation and sensitivity to anti-cancer drugs using the syngeneic tumor model.

Comparison of the anesthetic effects of 2,2,2-tribromoethanol on ICR mice derived from three different sources.

This study was conducted to compare the anesthetic effects of 2,2,2-tribromoethanol (TBE, Avertin®) in ICR mice obtained from three different sources. TBE (2.5%) was intraperitoneally injected at three doses: high-dose group (500 mg/kg), intermediate-dose group (250 mg/kg), and low-dose group (125 mg/kg). Anesthesia time, recovery time, end-tidal peak CO2 (ETCO2), mean arterial blood pressure, heart rate, oxygen saturation (SpO2), body temperature, pH, PCO2, and PO2 of the arterial blood were measured. Stable anesthesia was induced by all doses of TBE and the anesthesia time was maintained exhibited dose dependency. No significant differences in anesthetic duration were found among the three different strains. However, the anesthesia time was longer in female than in male mice, and the duration of anesthesia was significantly longer in female than in male mice in the high-dose group. The recovery time was significantly longer for female than male mice in the intermediate- and high-dose groups. In the ICR strains tested, there were no significant differences in the mean arterial blood pressure, SPO2, arterial blood PCO2, and PO2, which decreased after TBE anesthesia, or in heart rate and ETCO2, which increased after TBE anesthesia. In addition, body temperature, blood biochemical markers, and histopathological changes of the liver, kidney, and lung were not significantly changed by TBE anesthesia. These results suggested that ICR mice from different sources exhibited similar overall responses to a single exposure to TBE anesthesia. In conclusion, TBE is a useful drug that can induce similar anesthetic effects in three different strains of ICR mice.

Comparison of scopolamine-induced cognitive impairment responses in three different ICR stocks.

Cognitive impairment responses are important research topics in the study of degenerative brain diseases as well as in understanding of human mental activities. To compare response to scopolamine (SPL)-induced cognitive impairment, we measured altered parameters for learning and memory ability, inflammatory response, oxidative stress, cholinergic dysfunction and neuronal cell damages, in Korl:ICR stock and two commercial breeder stocks (A:ICR and B:ICR) after relevant SPL exposure. In the water maze test, Korl:ICR showed no significant difference in SPL-induced learning and memory impairment compared to the two different ICRs, although escape latency was increased after SPL exposure. Although behavioral assessment using the manual avoidance test revealed reduced latency in all ICR mice after SPL treatment as compared to Vehicle, no differences were observed between the three ICR stocks. To determine cholinergic dysfunction induction by SPL exposure, activity of acetylcholinesterase (AChE) assessed in the three ICR stocks revealed no difference of acetylcholinesterase activity. Furthermore, low levels of superoxide dismutase (SOD) activity and high levels of inflammatory cytokines in SPL-treated group were maintained in all three ICR stocks, although some variations were observed between the SPLtreated groups. Neuronal cell damages induced by SPL showed similar response in all three ICR stocks, as assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, Nissl staining analysis and expression analyses of apoptosis-related proteins. Thus, the results of this study provide strong evidence that Korl:ICR is similar to the other two ICR. Stocks in response to learning and memory capacity.

Use of C57BL/6N mice on the variety of immunological researches.

Inbred mice are an essential animal strain for research as they can improve the reproducibility and reliability of study results. The establishment of new inbred lines is continuing, and new inbred lines are being used in many research fields. C57BL/6 is a mouse laboratory animal that has been developed and used as an inbred strain since early stage of mouse strain development, and, in the 1950s, C57BL/6 was separated into substrains by the Jackson Laboratory (C57BL/6J) and the National Institutes of Health (C57BL/6N). C57BL/6 mice have been used in immunology and antitumor activity studies since the early strain development stage. After the mouse genome was fully described, C57BL/6 mice use in many areas of research has expanded. In particular, immunological characteristics such as those related to cell-mediated immunity and NK cell activity are relatively higher in C57BL/6 mice than in other mice. The C57BL/6NKorl is a stock of C57BL/6N established as part of a localization of experimental animal strategy of the Korean Food and Drug Administration. Based on analysis of single nucleotide polymorphisms (SNPs), C57BL/6NKorl is considered a genetically distinct inbred stock from other C57BL/6N. Various research efforts have been made to describe the characteristics and increase knowledge of the characteristics of C57BL/6Nkorl. The results obtained through these efforts are expected to increase the utilization of C57BL/6Nkorl as a domestic laboratory animal resource and to enhance the reliability of mouse based studies.

Comparative study of the immunological characteristics of three different C57BL/6N mouse substrains.

Inbred mice, a systematically developed homogeneous animal, have been developed to maintain experimental reproducibility and to minimize experimental variables in animal-based studies. In particular, C57BL/6 mice are frequently used in experiments into immunology and antitumor activity experiments. This study was compared the immunological characteristics of C57BL/6NKorl, a Korean developed experimental animal resource, with those of two other C57BL/6N substrains. Mouse body, thymus, and spleen weights in C57BL/6NKorl were not significantly different from those of the other two C57BL/6N substrains. Among the three substrains, there was no difference in the distribution of T and B cells, which are lymphocytes involved in adaptive immunity, and no difference in NK cells related to innate immunity. Results for macrophages and granulocytes, which have roles in innate immunity, were similar in all three substrains. In order to investigate the expressions of major histocompatibility complex (MHC) molecules and allogenic antigens, splenocytes were separated from obtained spleen and analyzed by using flow cytometry. MHC class I and II molecules, which are important during self/non-self-discrimination, were similar in the three substrains. In addition, expression of alloantigen involved in allografts showed similar results in the three substrain. Thus, the results of this study provide strong evidence that C57BL/6NKorl is immunologically similar to two other C57BL/6N substrains.

Comparison of humoral and cell-mediated immunity in three different C57BL/6N mouse substrains.

Adaptive immunity is a type of immune response mediated by T and B cells, and is important response for immune response amplification and memory. In this study, the adaptive immunologic properties of C57BL/6NKorl substrain were compared with those of two other C57BL/6N substrains. There were no significant differences between the C57BL/6NKorl and the two other C57BL/6N substrains in the histological structures of the thymus and spleen, which are immunologic organs containing T cell and B cells. In addition, flow cytometric analysis did not reveal any significant differences in the distribution of T and B cell populations of the three substrains. To evaluate cell-mediated immunity of T cells in the three different substrains, we treated isolated T cells from spleen with Con A. The T cells of C57BL/6NKorl showed Con A-dependent proliferation of T cells at lower cell number than those in T cells from the other two C57BL/6N substrains. B cell-mediated humoral immune responses were not significant different among the three substrains. Thus, the results of this study provide evidence that C57BL/6NKorl mice are similar to those two other C57BL/6N substrains in humoral immunity, but C57BL/6NKorl has stronger response in cell mediated immunity.

Comparative analysis of basal locomotor activity-related metabolic phenotypes between C57BL/6 mice and ICR mice substrains derived from three different sources.

Animal model, as an indispensable tool for scientific purposes of biomedical researches and clinical application, is a commonly used in various researches. Regarding to this, it is necessary to establish the metabolic phenotype of animal model to minimize spurious interpretations and ensure a level of accuracy and reliability adequate for experimental research. However, the metabolic phenotype-related analysis within rodent strains derived from different source is nonexistent, especially in C57BL/6Korl mice and Korl:ICR mice (a domestic mouse strain). To compare the physiological and metabolic phenotypes over a period of time, we utilized the C57BL/6 mice (C57BL/6Korl, A:C57BL/6, and B:C57BL/6) and ICR mice (Korl:ICR, A:ICR, and B:ICR) derived from three different sources. Our data showed that average body weight, body temperature, food intake, and water consumption have a similar tendency among the C57BL/6 and ICR groups, except for the higher body weight of C57BL/6Korl mice over a period of time. Moreover, some significant differences was observed in adipose tissue mass and adipocyte size among the groups, with a higher tendency of C57BL/6Korl mice and Korl:ICR mice. Most importantly, resting metabolic rate (RMR) serves as an approximation of the metabolic phenotype showed no significant difference among the groups of C57BL/6 mice and ICR mice, except for the lower oxygen uptake of C57BL/6Korl mice compare to the A:C57BL/6 mice. Taken together, our data suggest that C57BL/6 mice and ICR mice derived from three different sources have an overall similar feature of physiological and metabolic phenotypes.

Comparative study of fertilization rates of C57BL/6NKorl and C57BL/6N mice obtained from two other sources.

C57BL/6N is the most widely used inbred mouse strain applied in a wide variety of research areas including cancer, cardiovascular biology, developmental biology, diabetes and obesity, genetics, immunology, neurobiology, and sensorineural research. To compare the fertilization rates of C57BL/6NKorl mice with two commercial C57BL/6N stocks, differences in reproductive organ structures, sperm and egg numbers, fertilization rates, and embryo development rates among C57BL/6NKorl (Korea FDA source), C57BL/6NA (USA source), and C57BL/6NB (Japan source) mice were determined. Among the stocks, no significant differences were detected in organ weight and histological structure of male and female reproductive organs, although body weight was higher in C57BL/6NKorl mice than that in the other groups. The concentration and morphology of sperm and eggs in C57BL/6NKorl mice were similar to those of C57BL/6NA and C57BL/6NB mice. Furthermore, the three stocks had similar in vitro fertilization and embryo development rates, although these rates tended to be higher in C57BL/6NB mice. Pup body weight was higher in C57BL/6NKorl and C57BL/6NB mice than that in C57BL/6NA mice. The results of the present study suggest that C57BL/6NKorl, C57BL/6NA, and C57BL/6NB mice obtained from three different sources have similar fertilization and embryo development rates, although there were slight differences in the magnitude of their responses rates.

Erratum: Comparative study of the immunological characteristics of three different C57BL/6N mouse substrains.

[This corrects the article on p. 124 in vol. 33, PMID: 28747978.].

Erratum: Comparison of humoral and cell-mediated immunity in three different C57BL/6N mouse substrains.

[This corrects the article on p. 132 in vol. 33, PMID: 28747979.].

Erratum: Comparative analysis of basal locomotor activity-related metabolic phenotypes between C57BL/6 mice and ICR mice substrains derived from three different sources.

[This corrects the article on p. 140 in vol. 33, PMID: 28747980.].

Erratum: Comparative study of fertilization rates of C57BL/6NKorl and C57BL/6N mice obtained from two other sources.

[This corrects the article on p. 179 in vol. 33, PMID: 28747985.].

Interleukin-18-mediated interferon-gamma secretion is regulated by thymosin beta 4 in human NK cells.

Thymosin beta 4 (Tß4) is the major G-actin sequestering molecule and is abundant in lymphoid tissues. However, it is not clear what regulates Tß4 expression and what its function is on natural killer (NK) cells. We investigated whether interleukin-18 (IL-18) has a role in Tß4 expression and if enhanced Tß4 influences IL-18-mediated interferon-gamma (IFN-γ) secretion. In this study, recombinant human IL-18 (rhIL-18) enhanced the endogenous level of Tß4 through p38MAPK and JNK signaling pathway in the human NK cell line, NK-92MI. Overexpression of endogeneous Tß4 stimulated IFN-γ expression and secretion. Additionally, pretreatment with an inhibitor for Tß4 decreased IL-18-enhanced IFN-γ secretion, and transfection with Tß4-specific short hairpin RNA resulted in reduction of IFN-γ production in primary NK cells as well as in the human NK cell line. Taken together, these data indicated that Tß4 is regulated by IL-18 and is involved in IL-18-enhanced IFN-γ secretion in NK cells.

Hypoxia-induced IL-18 increases hypoxia-inducible factor-1alpha expression through a Rac1-dependent NF-kappaB pathway.

Interleukin-18 (IL-18) plays pivotal roles in linking inflammatory immune responses and tumor progression and metastasis, yet the manner in which this occurs remains to be sufficiently clarified. Here we report that hypoxia induces the transcription and secretion of IL-18, which subsequently induces the expression of hypoxia-inducible factor-1alpha (HIF-1alpha). Mechanistically, IL-18 induces HIF-1alpha through the activity of the GTPase Rac1, which inducibly associates with the IL-18 receptor beta (IL-18Rbeta) subunit, via a PI3K-AKT-NF-kappaB-dependent pathway. Importantly, the knockdown of the IL-18Rbeta subunit inhibited IL-18-driven tumor cell metastasis. Collectively, these findings demonstrate a feed-forward pathway in HIF-1alpha-mediated tumor progression, in which the induction of IL-18 by hypoxia or inflammatory cells augments the expression of both HIF-1alpha and tumor cell metastasis.