EFFECTS OF MODERATE NOISE EXPOSURE ON HEARING FUNCTION IN C57BL/6J MICE

Objective To study characteristics of hearing loss after exposure to moderate noise exposure in C57BL/6J mice. Methods Male C57BL/6J mice with normal hearing at age of 5-6 weeks were chosen for this study. The mice were randomly sclccted to be studied immediately after exposure （Group P0）, or 1 day （Group P1）, 3 days （Group P3）, 7 day （Group P7） or 14 days （P14） after exposure. Their before exposure condition served as the normal control. All mice were exposed to a broad-band white noise at 100 dB SPL for 2 hours, ABR thresholds were used to estimate hearing status at each time point. Results ABR threshold elevation was seen at every tested frequency at P0 （P〈0.01）. Elevation at high-frequencies （16 kHz and 32 kHz） was greater than at lower frequencies （4 kHz and 8 kHz, P〈0.05）. From P1 to P14, ABR thresholds continuously improved, and there was no significant difference between P14 and before exposure （P〉0.05）. Conclusion There is a frequency specific re- sponse to 100 dB SPL broad-band white noise in C57BL/6J mice, with the high-frequency being more susceptible. Hearing loss induced by moderate noise exposure appears reversible in C57BL/6J mice.

A murine model of dengue virus infection in suckling C57BL/6 and BALB/c mice

Dengue is a significant public health concern across tropical and subtropical regions worldwide,principally causing disease in children.Very young children are at increased risk of severe manifestations of dengue infection.The mechanism of dengue disease in this population is not fully understood.In this study,we present a murine model of dengue virus primary infection in suckling C57BL/6 and BALB/c mice in order to investigate disease pathogenesis.Three-day-old C57BL/6 mice intraperitoneally infected with DENV-2 NGC were more susceptible to infection than BALB/c mice,showing increased liver enzymes,extended viremia,dissemination to organs and histological alterations in liver and small intestine.Furthermore,the immune response in DENV-infected C57BL/6 mice exhibited a marked Th1 bias compared to BALB/c mice.These findings highlight the possibility of establishing an immunocompetent mouse model of DENV-2 infection in suckling mice that reproduces certain signs of disease observed in humans and that could be used to further study agerelated mechanisms of dengue pathogenesis.

Anti-leukemic and anti-angiogenic effects of D-Limonene on K562-implanted C57BL/6 mice and the chick chorioallantoic membrane model

Background: D-Limonene, a monoterpene from citrus fruit has been found to have chemopreventive and chemotherapeutic activities in various types of cancers. In this study, we evaluated the in vivo effect of D-Limonene on a K562-induced model of chronic myeloid leukemia(CML) in C57 BL/6 mice.Method: The tail vein injection model of K562 cells in immunocompromised C57 BL/6 mice was developed and evaluated for characteristics of the disease. The mice were treated with D-Limonene and evaluated for haematological parameters. We also evaluated the effect of D-Limonene on angiogenesis using the chick chorioallantoic membrane(CAM) assay.Results: In a complete blood count, a significant dose-dependent reduction in white blood cell, neutrophil and lymphocyte counts, but an elevation in red blood cell count and haemoglobin content was observed with D-Limonene treatment compared to the disease control or untreated group. In the CAM assay, D-Limonene produced a significant dose-dependent reduction in number of blood vessels in treatment groups compared to the vehicle-treated group.Conclusion: These studies suggest promising anti-leukemic and anti-angiogenic effects of D-Limonene in the treatment of CML.

Ultraviolet-attenuated cercariae ofSchistosoma japonicum fail to effectively induce a Th1 response in spite of up-regulating expression of cytotoxicity-related genes in C57BL/6 mice

Objective： To better understand the reason that Schistosoma japonicurn （S. japonicum） ultraviolet （UV）- radiated cercariae could not induce high level of protection in C57BL/6 mice. Methods： Microarray technology was performed to investigate the gene transcription profile in skin draining lymph nodes （sdLNs） at 1 w after exposure to attenuated cercariae （AC） or normal cercariae （NC） of S. japonicum in C57BL/6 mice. The expressions of some representative genes were further confirmed by real-time PCR. Subsequently, the expressions of Th1/Th2 cytokine genes, cytotoxicity-related genes, as well as co-stimulator genes in spleens from AC-vaccinated and NC- infected mice were analyzed by real-time PCR at w 3 and 6 post-exposure. Results： The gene expressions of Th1 cytokines, including interferon-y （IFN-γ）, interleukin （IL）-12 and tumor necrosis factor-α （TNF-α） in the sdLNs were significantly lower in AC-vaccinated mice than in NC-infected mice. Furthermore, the gene expressions of Th1- and Th2- cytokines, including IFN-γ, IL-12, TNF-α, IL-4 and IL-10, in the spleens from AC-vaccinated mice showed little changes at w 3 and 6 post-vaccination. In addition, cytotoxicity-related molecules including granzyme A, granzyme B, granzyme K, perforin 1 and Fas L were up-regulated from the early stage of vaccination, and peaked at the 3rd w after vaccination with UV-AC. Conclusion： UV-AC of S. japonicum could not ef- fectively induce a Thl response in C57BL/6 mice, which may be an explanation for the low protection against parasite challenge, and the role played by up-regulated expression of cytotoxicity-related genes in mice needs to be further investigated.

Differences in action potential propagation speed and axon initial segment plasticity between neurons from Sprague-Dawley rats and C57BL/6 mice

Action potentials(APs)in neurons are generated at the axon initial segment(AIS).AP dynamics,including initiation and propagation,are intimately associated with neuronal excitability and neurotransmitter release kinetics.Most learning and memory studies at the single-neuron level have relied on the use of animal models,most notably rodents.Here,we studied AP initiation and propagation in cultured hippocampal neurons from Sprague-Dawley(SD)rats and C57BL/6(C57)mice with genetically encoded voltage indicator(GEVI)-based voltage imaging.Our data showed that APs traveled bidirectionally in neurons from both species;forward-propagating APs(fpAPs)had a different speed than backpropagating APs(bpAPs).Additionally,we observed distinct AP propagation characteristics in AISs emerging from the somatic envelope compared to those originating from dendrites.Compared with rat neurons,mouse neurons exhibited higher bpAP speed and lower fpAP speed,more distally located ankyrin G(AnkG)in AISs,and longer Nav1.2 lengths in AISs.Moreover,during AIS plasticity,AnkG and Nav1.2 showed distal shifts in location and shorter lengths of labeled AISs in rat neurons;in mouse neurons,however,they showed a longer AnkG-labeled length and more distal Nav1.2 location.Our findings suggest that hippocampal neurons in SD rats and C57 mice may have different AP propagation speeds,different AnkG and Nav1.2 patterns in the AIS,and different AIS plasticity properties,indicating that comparisons between these species must be carefully considered.

Different sources of MSCs on pulmonary fibrosis in C57BL/6 mice

Since stem cell therapy is the most effective treatment in the field of tissue reparation and reconstitution,the present study aimed to explore the different sources of mesenchymal stem cells(MSCs)on the different effects of pulmonary fibrosis-related cytokines in C57BL/6 mice.For reaching this goal,we isolated MSCs from umbilical cord blood and placenta and used for stem cell therapy in a mouse model of pulmonary fibrosis model.The pulmonary fibrosis model was done by injecting bleomycin into the trachea of C57BL/6 mice.Then we assessed the degree of pulmonary fibrosis in each mouse lung tissue at weeks 1,2,3,and 4.In addition,flow cytometry was used to evaluate the frequency of CD73,CD90,CD106,CD34,CD45,CD14 cells at the mononuclear cell level;and western blotting assays revealed the expression of IκB-α.Our results showed that stem cell therapy by placenta-derived MSC had a lower level of CD34,CD45,CD14 cells at the mononuclear cell level,and that improved pulmonary fibrosis at both molecular and pathological levels.In addition,western blotting assays revealed that the expression of IκB-αwas down-regulated in MSC-treated animals.In addition,placenta-derived MSC was the most effective in improving pulmonary fibrosis in comparison to other sources.This study suggests that MSC might be a novel therapeutic approach in pulmonary fibrosis due to an enhanced anti-inflammatory effect.Also,MSC modification by gene editing could enhance their therapeutic effect in mouse pulmonary fibrosis.

Dietary Effect of Squalene on Lipid Metabolism of Obese/Diabetes KK-<i>A^y</i>Mice and Wild-Type C57BL/6J Mice

The purpose of this study was to evaluate the influence of squalene (SQ) on plasma and hepatic lipid levels of obese/diabetic KK-Ay mice and wild-type C57BL/6J mice. SQ supplementation significantly increased the HDL cholesterol of KK-Ay mice, which was paralleled with no significant difference in the total and non-HDL cholesterol levels. The increase in HDL cholesterol was also found in the plasma of normal C57BL/6J mice, but the difference was not significant. SQ administration significantly increased neutral lipids (NL) in the liver of KK-Ay mice, while no significant difference was observed in the polar lipids and the total cholesterol levels. The increase in NL was primarily due to the increase in TAG. However, the cholesterol level significantly increased due to SQ intake in the liver of C57BL/6J mice, while no significant difference was found in other lipid levels. The present study suggests that SQ may effectively increase HDL cholesterol level, an important anti-atherosclerotic factor, especially in subjects with metabolic disorders.

Characterization of Endophytes Isolated from <i>Eclipta prostrate</i>and Roles in Regulating the Gut Microbiota of C57BL/6J Mice

Eclipta prostrata has been extensively studied for its promoting effect on human health. Previous study proved that E. prostrata possessed anti-osteoporosis function in SAMP6 mice via gut microbiota (GM). Endophytes are widespread in plants, especially in Chinese herbal medicines. However, little is known regarding the endophytes of E. prostrata. In this study, we focus on screening and identifying the endophytes from plant E. prostrata and exploring their roles in modulating GM. According to biochemical, physiological tests and 16S rDNA sequence analysis, ten endophytes were characterized in different locations of plant E. prostrata belonging to Lactococcus, Bacillus, Enterococcus, Exiguobacterium and Pantoea. The antimicrobial activity of endophytes EP01-10 was investigated via the oxford cup method. Furthermore, the acid and bile salt resistant ability of EP01-10 was detected to explore their survival ability in gastrointestinal tract. Results indicated that strains of Lactococcus, Bacillus and Exiguobacterium (EP01, 03 and 05) showed strong antimicrobial activity against Escherichia coli and Staphylococcus aureus and strains of Lactococcus, Bacillus and Enterococcus (EP01, 02, 04, 08 and 10) have the ability to survive in the gastrointestinal tract. 16S rRNA sequencing of GM in C57BL/6J mice was performed for diversity and structure analysis responded to the administration of E. prostrata extract. E. prostrata extract acted on improving the microflora diversity, particularly in increasing the abundance of Lactococcus significantly. Thus, as an endophyte, Lactococcus plays an important role in E. prostrata modulating intestinal tract. Our study suggested that endophytes of Chinese herbal medicines might be a novel target for the treatment of diseases by regulating the GM.

Expression ofα2-Na/K-ATPase in C57BL/6J Mice Inner Ear and Its Relationship with Age-related Hearing Loss

K^(+)cycling in the cochlea is critical to maintain hearing.Many sodium-potassium pumps are proved to participate in K^(+)cycling,such as Na/K-ATPase.Theα2-Na/K-ATPase is an important isoform of Na/K-ATPase.The expression ofα2-Na/K-ATPase in the cochlea is not clear.In this study,we used C57BL/6 mice as a model of presbycusis and implemented immunohistochemistry staining and quantitative real time-PCR,and theα2-Na/K-ATPase expression pattern was confirmed in the inner ear.It was foundα2-Na/K-ATPase was expressed widely in cochlea and its mRNA and protein expression was gradually reduced with aging(4-,14-,26-and 48-weeks old mice).We suspected that,the down-regulation ofα2-Na/K-ATPase expression might be associated with the remodeling of K^(+)cycling,degeneration of morphological structure and decrease of hearing function in aging C57 mice.In conclusion,we speculated that the reduction ofα2-Na/K-ATPase might play an important role in the pathogenesis of age-related hearing loss.

C57BL/6 and DBA/1 Mice Differ in Their Response to Supplementation with 1,25D and Paricalcitol

Active vitamin D (1,25D) is a fat-soluble vitamin that is mainly produced in the skin by the conversion of 7-dehydrocholesterol under ultraviolet light stimulation.Its role in calcium homeostasis,bone growth, and prevention of rickets and osteomalacia has been known for over two hundred years.Its

Establishment of Germ-line Competent C57BL/6J Embryonic Stem Cell Lines

Objective To establish C57BL/6J embryonic stem （ES） cell lines with potential germ- line contribution Methods ES cells were isolated from blastocyst inner cell mass of C5 7BL/6J mice, and cultured for 15 passages, and then injected into blastococels of ICR mice blastocysts to establish chimeric mice. Results Three ES cell lines （mC57ES1,mC57ES3, mC57ES7） derived from the inner cell mass of C57BL/6J mice blastocysts were established. They were characteristic of undifferentiated state, including normal XY karyotype, expression of a specific cell surface marker “stage-specific embryonic antigen-I” and alkaline phosphatase in continuous passage. When injected into immunodeficient mice, mC57ES1 cells consistently differentiated into derivatives of all three embryonic germ layers. When mC57ES1 cells were transferred into ICR mice blastocysts, 4 chimeric mice have been obtained. One male of them revealed successful germ-line transmission. Conclussion We have obtained C57BL/6J ES cell lines with a potential germ-line contribution, which can be used to generate transgenic and gene knock-out mice.

Behavioral study of an aging model induced by different doses of D-galactose in C57BL／6J mice

Rodents chronically injected with D-galactose (D-gal have been used as an animal model for brain aging or antiaging pharmacology research. However, the dose of D-gal used to induce behavioral impairment has been reported in a wide range. Besides, very few studies used the C57BL inbred mouse strain to construct this model, which is one of the

Downregulation of Inwardly Rectifying Potassium Channel 5.1 Expression in C57BL/6J Cochlear Lateral Wall

Age-related hearing loss（AHL） is one of the most common sensory disorders among elderly persons. The inwardly rectifying potassium channel 5.1（Kir5.1） plays a vital role in regulating cochlear K~＋ circulation which is necessary for normal hearing. The distribution of Kir5.1 in C57BL/6J mice cochleae, and the relationship between the expression of Kir5.1 and the etiology of AHL were investigated. Forty C57BL/6J mice were randomly divided into four groups at 4, 12, 24 and 52 weeks of age respectively. The location of Kir5.1 was detected by immunofluorescence technique. The m RNA and protein expression of Kir5.1 was evaluated in mice cochleae using real-time polymerase-chain reactions（RT-PCR） and Western blotting respectively. Kir5.1 was detected in the type Ⅱ and Ⅳfibrocytes of the spiral ligament in the cochlear lateral wall of C57BL/6J mice. The expression levels of Kir5.1 m RNA and protein in the cochleae of aging C57BL/6J mice were down-regulated. It was suggested that the age-related decreased expression of Kir5.1 in the lateral wall of C57BL/6J mice was associated with hearing loss. Our results indicated that Kir5.1 may play an important role in the pathogenesis of AHL.

Paraquat induces selective dopaminergic nigrostriatal degeneration in aging C57BL/6 mice

Background Paraquat （PQ; 1,1 ＇-dimethyl-4,4＇-bipyridinium）, a widely used herbicide that is structurally similar to the known dopaminergic neurotoxicant MPTP （1-methyl-l, 2, 3,6-tetrahydropyridine ）, has been suggested as a potential etiologic factor for the development of Parkinson＇ s disease （PD）. Aging is an accepted risk factor for idiopathic Parkinson＇ s disease. The aim of this study was to test the hypothesis that paraquat could induce PD-like nigrostriatal dopaminergic degeneration in aging C57BL/6 mice. Methods Senile male C57BL/6 mice were intraperitoneally injected with either saline or PQ at 2-day intervals for a total of 10 doses. Locomotor activity and performance on the pole test were measured 7 days after the last injection and animals were sacrificed one day later. Level of dopamine （DA） and its metabolites levels in the striatum were measured by high-performance liquid chromatography with an electrochemical detector （ HPLCECD）, and numbers of tyrosine hydroxylase （TH） positive neurons were estimated using immunohistochemistry. Results Locomotor activities were significantly decreased and the behavioral performance on the pole test were significantly impaired in the PQ treated group. Level of DA and its metabolites levels in the striatum were declined by 8 days after the last injection. Immunohistochemical analyses showed that PQ was associated with a reduction in numbers of tyrosine hydroxylase positive neurons. Conclusions Long-term repeated exposes to PQ can selectively impair the nigrostriatal dopaminergic system of senile mice, suggesting that PQ could play an important role in the pathogenesis of Parkinson＇ s disease （PD）. Our results also validate a novel model of PD induced by exposure to a toxic environmental agent.

Transglutaminase 3 expression in C57BL/6J mouse embryo epidermis and the correlation with its differentiation

Epidermal-type transglutaminase 3 (TGM3) is involved in the cross-linking of structural proteins to form the cornifiedenvelope in the epidermis. In the present study, we detected the expression of TGM3 in the mouse embryo using RT-PCR.TGM3 mRNA is weakly presented from E11.5 to E14.5 and increases significantly from E15.5 to birth. Then wedetermined the spatial and temporal expression pattern of TGM3 in the skin and other organs by in situ hybridization. Wefound a deprivation of TGM3 in skin at E11.5, while a rich supply in periderm cells and a weak expression in basal cellsfrom E12.5 to E14.5. From the period of E15.5 to E16.5, after keratinization in the epidermis, TGM3 was expressed inthe granular and cornified layers. The electron microscopic observation of the C57BL/6J mouse limb bud skin develop-ment provided several morphological evidences for the epidermal differentiation. The above findings suggest that theexpression of TGM3 plays a important role in the epidermis differentiation in embryogenesis.

Axonal damage in myelin oligodendrocyte glycoprotein peptide-induced experimental autoimmune encephalomyelitis in a C57BL/6 mouse model may be not secondary to inflammatory demyelination

The present study established a chronic experimental autoimmune encephalomyelitis model in C57BL/6 mice induced by myelin oligodendrocyte glycoprotein peptides and complete Freund＇s adjuvant. Onset latency was 12 days, with an incidence rate of 100%. Neuropathological characteristics included perivascular inflammatory cell infiltration, demyelination, neuronal degeneration, and axonal damage within cerebral and myelic white matter. Electron microscopy revealed swollen mitochondria, complete organ disappearance, and fused or broken myelin sheath structure, which were accompanied by myelin sheath reconstruction. Moreover, axonal damage was not consistent with demyelination distribution, and severity of axonal damage did not correlate with demyelination. Results suggested that axonal damage in an experimental autoimmune encephalomyelitis model is not secondary to inflammatory demyelination.

Changes of the intestinal endocrine cells in the C57BL/6 mouse after implantation of murine lung carcinoma (3LL): An immunohistochemical quantitative study

AIM: To study the distributions and frequencies of intestinal endocrine cells in the C57BL/6 mouse with immunohistochemical method using seven types of specific antisera against chromogranin A (CGA), serotonin,somatostatin, glucagons, gastrin, cholecystokinin (CCK)-8 and human pancreatic polypeptide (hPP) after abdominal subcutaneous implantation of murine lung carcinoma (3LL).METHODS: The experimental animals were divided into two groups, one is non-implanted Sham and the other is 3LL-implanted group. Samples were collected from six regions of intestinal tract at 28th d after implantation of 3LL cells (1×105 cell/mouse).RESULTS: In this study, five types of immunoreactive (IR) cells were identified except for gastrin and hPP. The regional distributions of the intestinal endocrine cells in the 3LL-implanted group were similar to those of the non-implanted Sham. However, significant decreases of IR cells were detected in 3LL-implanted group compared to those of non-implanted Sham. CGA- and serotonin-IR cells significantly decreased in 3LL-implanted groups compared to that of non-implanted Sham. Somatostatin-IR cells in the jejunum and ileum and CCK-8-IR cells in the jejunum of 3LL-implanted groups significantly decreased compared to that of non-implanted Sham. In addition,glucagon-IR cells were restricted to the ileum and colon of non-implanted Sham.CONCLUSION: Implantation of tumor cell mass (3LL)induced severe quantifiable changes of intestinal endocrine cell density and the abnormality in density of intestinal endocrine cells may contribute to the development of gastrointestinal symptoms such as anorexia and indigestion, frequently encountered in patients with cancer.

Korean Chungtaejeon tea extract attenuates weight gain in C57BL/6J-Lep ob/ob mice and regulates adipogenesis and lipolysis in 3T3-L1 adipocytes

OBJECTIVE： Traditional Korean Chungtaejeon （CTJ） tea is a type of fermented tea, which has received increasing attention in recent years because of its purported health benefits. The present study was designed to investigate the effect and mechanism of CTJ tea extract on body weight gain using C57BL/6J-Lep ob/ob mice and 3T3-L1 adipocytes, respectively. METHODS： The effects of CTJ on cell viability, lipid accumulation, and expression of protein and mRNA were measured in 3T3-L1 adipocytes by using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, oil red O staining, Western blotting, and reverse transcriptase-polymerase chain reaction analyses. C57BL6J-Lep ob/ob mice were administered with CTJ （200 or 400 mg/kg body weight） for ten weeks. Then, body weight, food intake, total cholesterol, and triglyceride were measured in ob/ob mice. RESULTS： CTJ tea extract treated at 250 wg/mL （CTJ250） significantly suppressed lipid accumulation in the differentiated 3T3-L1 adipocytes. Likewise, CTJ250 significantly decreased the protein expression of peroxisome proliferator-activated receptor Y （PPARy）, CCJ^u~,T/enhancer-binding protein a, and adipocyte lipid- binding protein, and regulated the mRNA expression of PPART, sterol regulatory element-binding protein- lc gene, fatty acid synthase, adipocyte lipid-binding protein, hormone-sensitive lipase, carnitine palmitoyl transferase 1, cluster of differentiation 36, and acetyl-CoA carboxylase in the differentiated 3T3-L1 adipocytes. Mice administered with CTJ showed dose-dependent decrease in body weight gain, starting from week 4 of the experiment. CTJ tea extract administered at 400 mg/kg body weight significantly decreased fat mass, food efficacy ratio, and levels of plasma triglyceride and total cholesterol. CONCLUSION： CTJ attenuated weight gain in ob/ob mice and regulated the activity of the molecules involved in adipogenesis and lipolysis in 3T3-L1 adipocytes. CTJ is a potentially valuable herbal therapy for the prevention of obesity and/or obesity-related disorders.

Mouse-adapted SARS-CoV-2 replicates efficiently in the upper and lower respiratory tract of BALB/c and C57BL/6J mice

Dear Editor,As of June,2020,more than ten million cases of COVID-19 have been reported worldwide.The causative pathogen of the disease is a novel coronavirus named severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)(World Health Organization,2020).Animal infection models are important to characterize the infection,pathogenesis,and immunology of SARS・CoV・2,as well as for the development of medications and vaccines against COVID-19.Mice are particularly attractive animal models for their identical genetic background,reliable reproducibility,well characterized biology,and the huge availability of research reagents and knockout animals.Models in inbreed mice such as BALB/c and C57BL/6J(C57),which are widely used in research,are highly desired.

C57BL/6小鼠微小三毛滴虫的观察与种系发育分析

为鉴定从某外单位引进的C57BL/6小鼠粪样检测中发现的虫体种类,本试验对虫体进行形态学观察,并进行种系发育分析。取C57BL/6小鼠粪样涂片和瑞士染色后进行显微镜观察,提取粪样总DNA,根据三毛滴虫的16S rRNA进行PCR扩增后测序,运用MEGA11进行种系发育分析。显微镜观察发现,虫体符合微小三毛滴虫的形态学特征,测序发现微小三毛滴虫的基因序列与鼠三毛滴虫高度同源。