Experimental Animals
Online ISSN : 1881-7122
Print ISSN : 1341-1357
ISSN-L : 0007-5124
Advance online publication
Showing 1-29 articles out of 29 articles from Advance online publication
  • Md. Mehedi HASAN, Shizuka KONISHI, Miyuu TANAKA, Takeshi IZAWA, Jyoji ...
    Type: Original
    Article ID: 21-0132
    Published: 2021
    [Advance publication] Released: October 18, 2021
    JOURNAL OPEN ACCESS ADVANCE PUBLICATION

    Coiled-coil domain containing 85c (Ccdc85c) is a causative gene for genetic hydrocephalus and subcortical heterotopia with frequent brain hemorrhage. In the present study, we examined the expression pattern of CCDC85C protein and intermediate filament proteins, such as nestin, vimentin, GFAP, and cytokeratin AE1/AE3, during lateral ventricle development in rats. CCDC85C was expressed in the neuroepithelial cells of the dorsal lateral ventricle wall, diminishing with development and almost disappearing at postnatal day 20. By immunoelectron microscopy, CCDC85C was localized in the cell-cell junction and apical membrane. The expression of nestin and vimentin was decreased in the wall of the lateral ventricle in manner similar to CCDC85C, but GFAP expression started immediately after birth and became stronger with age. Moreover, cytokeratin expression was found at postnatal day 13 and increased at postnatal day 20 in conjunction with the disappearance of CCDC85C expression. Taken together, CCDC85C is expressed in the cell-cell junctions lining the wall of the lateral ventricle and plays a role in neural development with other intermediate filaments in the embryonic and postnatal periods. Our chronological study will help to relate CCDC85C protein with intermediate filaments to elucidate the detailed role of CCDC85C protein during neurogenesis.

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  • Lixia YAO, Weihua SHAO, Yan CHEN, Suxing WANG, Dai HUANG
    Type: Original
    Article ID: 21-0064
    Published: 2021
    [Advance publication] Released: October 07, 2021
    JOURNAL OPEN ACCESS ADVANCE PUBLICATION

    Endothelial-to-mesenchymal transition (EndMT) is involved in cardiac fibrosis induced by angiotensin II (Ang II). A disintegrin and metalloproteinase 8 (ADAM8), a member of ADAMs family, participates in cell adhesion, proteolysis and various signaling. However, its effects on the development of cardiac fibrosis remain completely unknown. This study aimed to reveal whether ADAM8 aggravates cardiac fibrosis induced by Ang II in vivo and in vitro. The C57BL/6J mice or cardiac endothelial cells were subjected to Ang II infusion to induce fibrosis. The results showed that systolic blood pressure and diastolic blood pressure were significantly increased under Ang II infusion, and ADAM8 was up-regulated. ADAM8 inhibition attenuated Ang II-induced cardiac dysfunction. ADAM8 knockdown suppressed Ang II-induced cardiac fibrosis as evidenced by the down-regulation of CTGF, collagen I, and collagen III. In addition, the endothelial marker (VE-cadherin) was decreased, whilst mesenchymal markers (α-SMA and FSP1) were increased following Ang II infusion. However, ADAM8 repression inhibited Ang II-induced EndMT. Moreover, ADAM8 silencing repressed the activation of TGF-β1/Smad2/Smad3 pathways. Consistent with the results in vivo, we also found the inhibitory effects of ADAM8 inhibition on EndMT in vitro. All data suggest that ADAM8 promotes Ang II-induced cardiac fibrosis and EndMT via activating TGF-β1/Smad2/Smad3 pathways.

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  • Yuzuru IIZUKA, Hyounju KIM, Maki NAKASATOMI, Akiyo MATSUMOTO, Jun SHIM ...
    Type: Original
    Article ID: 21-0109
    Published: 2021
    [Advance publication] Released: September 28, 2021
    JOURNAL OPEN ACCESS ADVANCE PUBLICATION

    Research into the prevention and treatment of age-related metabolic diseases are important in the present-day situation of the aging population. We propose that an elderly diabetic mouse model may be useful to such research as it exhibits deterioration of glucose and lipid metabolism. Although the KK mouse strain is commonly used as a model of moderate obesity and type 2 diabetes, the utility of this strain as an elderly obese and diabetic model mouse for research into aging remains unclear. The present study aimed to investigate age-related changes of glucose and lipid metabolism in male KK mice fed a standard chow diet. We demonstrate that 40 weeks KK mice exhibit age-related dysfunctions, such as development of insulin resistance associated with pancreatic islet hypertrophy and decreased lipolysis in white adipose tissue (WAT) compared with 15 weeks KK mice. However, aging does not appear to cause mitochondrial dysfunction of brown adipose tissue. Unexpectedly, hyperglycemia, potential glucose uptake in insulin-sensitive organs, hepatic lipid accumulation, hypertrophy of adipocytes, and inflammation in epididymal WAT did not worsen but rather compensated in 40 weeks KK mice. Our data indicate that the use of male KK mice as an elderly obese and diabetic mouse model has some limitations and in order to represent a useful elderly obese and diabetic animal model, it may be necessary to induce deterioration of glucose and lipid metabolism in KK mice through breeding with high-sucrose or high-fat diets.

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  • Wataru HIRATA, Taiki TOMODA, Shunsuke YURI, Ayako ISOTANI
    Type: Original
    Article ID: 21-0119
    Published: 2021
    [Advance publication] Released: September 20, 2021
    JOURNAL OPEN ACCESS ADVANCE PUBLICATION

    In mammals, sexual fate is determined by the chromosomes of the male and female gametes during fertilization. Males (XY) or females (XX) are produced when a sperm containing a Y or X-chromosome respectively fertilizes an X-chromosome-containing unfertilized egg. However, sexing of preimplantation stage embryos cannot be conducted visually. To address this, transgenic male mouse models with the ubiquitously expressed green fluorescent protein (GFP) transgene on X- (X-GFP) or Y-chromosomes (Y-GFP) have been established. However, when crossed with wild-type females, sexing of the preimplantation stage embryos by observing the GFP signal is problematic in some cases due to X-inactivation, loss of Y-chromosome (LOY), or loss of transgene fluorescence. In this study, a mouse model with the ubiquitously expressed red fluorescent protein (RFP) transgene on the Y-chromosome was generated since RFP is easily distinguishable from GFP signals. Unfortunately, the ubiquitously expressed tdTomato RFP transgene on the Y-chromosome (Y-RFP) mouse showed the lethal phenotype after birth. No lethal phenotypes were observed when the mitochondrial locating signal N-terminal of tdTomato (mtRFP) was included in the transgene construct. Almost half of the collected fertilized eggs from Y-mtRFP male mice crossed with wild-type females had an RFP signal at the preimplantation stage (E1.5). Therefore, XY eggs were recognized as RFP-positive embryos at the preimplantation stage. Furthermore, 100% sexing was observed at the preimplantation stage using the X-linked GFP/Y-linked RFP male mouse. The established Y-mtRFP mouse models may be used to study sex chromosome related research.

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  • Haruhiko MIYATA, Yuki OYAMA, Yuki KANEDA, Masahito IKAWA
    Type: Original
    Article ID: 21-0082
    Published: 2021
    [Advance publication] Released: September 15, 2021
    JOURNAL OPEN ACCESS ADVANCE PUBLICATION
    Supplementary material

    Kinesin is a molecular motor that moves along microtubules. Testis-enriched kinesin KIF9 (Kinesin family member 9) is localized in the mouse sperm flagellum and is important for normal sperm motility and male fertility; however, it is unclear if the motor domain of KIF9 is involved in these processes. In this study, we substituted threonine of the ATP binding motif in the KIF9 motor domain to asparagine (T100N) in mice using the CRISPR/Cas9 system, which is known to impair kinesin motor activity. T100N mutant mice exhibit reduced sperm motility and male fertility consistent with Kif9 knockout mice. Further, KIF9 was depleted in the spermatozoa of T100N mutant mice although the amounts of KIF9 were comparable between wild-type and T100N mutant testes. These results indicate that the motor domain of KIF9 is essential for its localization in the sperm flagellum.

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  • Masahiko YASUDA, Ritsuki UCHIDA, Yoko KAMAI, Hanako MORITA, Mai TANAKA ...
    Type: Original
    Article ID: 21-0091
    Published: 2021
    [Advance publication] Released: September 13, 2021
    JOURNAL OPEN ACCESS ADVANCE PUBLICATION

    Pneumocystis (P.) carinii is known to cause fatal pneumonia in immunocompromised rats. Cases of P. carinii interstitial pneumonia in immunocompetent rats have been shown histologically to present with perivascular lymphoid cuffs, which have previously been attributed to rat respiratory virus. This study aims to determine the prevalence and pathological characteristics of P. carinii in immunocompetent laboratory rats in experimental facilities in Japan. An epidemiological survey for this agent was performed using PCR to assess 1981 immunocompetent rats from 594 facilities in Japan. We observed that 6 of the 1981 rats (0.30%) from 4 out of 594 facilities (0.67%) were positive for P. carinii without infection of other known pathogens. Gross pulmonary lesions were found in 4 of the 6 affected rats. The lungs of these rats contained scattered dark red/gray foci. Histopathologically, the lungs exhibited interstitial pneumonia with lymphoid perivascular cuffs: Pneumocystis cysts were observed using Grocott's methenamine silver stain. To our knowledge, this report is the first to reveal the prevalence of natural P. carinii infection in immunocompetent laboratory rats in Japan.

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  • Weisong XUE, Yu FU, Haojie ZHANG, Guoping LI, Peihua CAO, Yang LI, Qin ...
    Type: Original
    Article ID: 21-0076
    Published: 2021
    [Advance publication] Released: September 08, 2021
    JOURNAL OPEN ACCESS ADVANCE PUBLICATION

    The current ischemic models of liver failure are difficult and usually time-consuming to produce. The aim of this study was to develop a simplified and reproducible porcine model of acute liver failure for use in preclinical research. Eighteen Bama miniature pigs were randomly divided into Groups A, B, and C. The hepatic artery and common bile duct were ligated in all groups. While the portal vein was completely preserved in Group A, it was narrowed by 1/3 and 1/2 in Groups B and C, respectively. Results of biochemical analyses, encephalopathy scores, and survival times were compared among the groups. Results of hematoxylin-eosin staining, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling, Masson staining, and Ki-67 analyses were recorded. Survival times in Groups B and C were 11.67 ± 1.86 and 2.16 ± 0.75 days, respectively, shorter than that in Group A (>15 days). Following surgery, alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, alkaline phosphatase, total bilirubin, and direct bilirubin levels significantly increased relative to baseline values in all groups (P<0.05). Groups B and C exhibited a significant decrease in encephalopathy scores and a significant increase in ammonia levels, which were negatively correlated with one another. Pathological analysis revealed obvious necrosis of liver cells, which correlated closely with the degree of portal vein constriction. Our simple, highly reproducible model effectively mimics the clinical characteristics of acute liver failure in humans and provides a foundation for further research on artificial liver support system development.

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  • In Gul KIM, Hana CHO, Jun Jae CHOI, Jung-Woog SHIN, Eun-Jae CHUNG
    Type: Original
    Article ID: 20-0192
    Published: 2021
    [Advance publication] Released: September 01, 2021
    JOURNAL OPEN ACCESS ADVANCE PUBLICATION

    The gastrostomy technique is essential for esophageal reconstruction using a scaffold. To date, there are no established methods to supply nutrients through a gastrostomy tube in rats. The purpose of this study was to analyze the feasibility of a newly modified gastrostomy technique for non-oral nutrition in an adult rat model. We modified the gastrostomy technique for adult rats in a few different ways. (1) The external opening for food injection was made at the midpoint between the ears to prevent damage due to self-harm behaviour. (2) An imbedded subcutaneous tunnel was created between the internal and external openings of the gastrostomy. We compared the efficacy and safety between groups with a T-tube for biliary drainage (TT group, n=14) and a conventional silicone Foley catheter (FC group, n=7) as optimal gastrostomy tubes for in a rat model. We also evaluated the feasibility of the heparin cap connector at the end of gastrostomy tube to control food supply in the TT group (with a cap, n=7; without a cap, n=7). No mortality was observed in the TT group with a cap, whereas most rats in the FC group died within 2 weeks after the procedure. Weight loss decreased significantly in the TT group with a cap compared with all the other groups. The appearance and attitude scores were significantly better in the TT group with a cap. In addition, histologic analysis showed that the TT group a cap showed a marked decrease over time in tissue fibrosis and macrophages compared with the other experimental groups. Therefore, gastrostomy using a silicone T-tube plugged with a cap proved to be a stable and effective option for non-oral feeding in an adult rat model.

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  • Hwan KIM, Junpil BANG, Seung Ho BAEK, Jae-Hak PARK
    Type: Original
    Article ID: 21-0026
    Published: 2021
    [Advance publication] Released: August 30, 2021
    JOURNAL OPEN ACCESS ADVANCE PUBLICATION

    Pathogens can affect physiological and immunological reactions in immunocompromised animals and genetically engineered mice. Specifically, murine norovirus (MNV), Helicobacter, and intestinal protozoa are prevalent in rodent laboratory facilities worldwide. In this study, microbiological test results of the soiled bedding of sentinel mice showed the prevalence of MNV (50.9%, 28/55), Helicobacter hepaticus (29.1%, 16/55), Trichomonas spp. (14.5%, 8/55), and Entamoeba spp. (32.7%, 18/55). No single infections were detected as all cases were confirmed to have complex infections with two or four pathogens. In previous studies, the success rate of the cross-fostering method was not perfect; therefore, in this study, the entire mouse strain of the SPF rodent facility was rederived using embryo transfer. For up to three years, we confirmed that the results were negative with regular health surveillance tests. Embryo transfer was, thus, determined to be an effective method for the rederivation of specific pathogen free (SPF) barrier mouse facilities. This is the report for the effectiveness of embryo transfer as an example of successful microbiological clean-up of a mouse colony with multiple infections in an entire SPF mouse facility and embryo transfer may be useful for rederiving.

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  • Gaku WAGAI, Masao TOGAO, Jun OTSUKA, Yuki OHTA-TAKADA, Shoichi KADO, K ...
    Type: Original
    Article ID: 21-0073
    Published: 2021
    [Advance publication] Released: August 09, 2021
    JOURNAL OPEN ACCESS ADVANCE PUBLICATION

    An incident reporting system (IRS) prevents possible adverse events by collecting and analyzing incidents that occur. However, few studies are available regarding IRSs in the laboratory animal field. This study aimed to develop an incident severity classification for laboratory animals (ISCLA) to evaluate the usefulness of the IRS in laboratory animal facilities. Twenty-three incidents reported from March 2019 to February 2020 on our IRS were retrospectively reviewed. Three of the 23 incidents failed to obtain some experimental data. Two of these incidents were harmless to animals, but the other caused the animals moderate distress. In addition, two of the three incidents made animals unsuitable for experiments. Since the inconsistent impact of incidents on animals and experiments prevented the comparison of the severity of individual incidents, we developed the ISCLA. According to the ISCLA, the above three incidents were classified into Category 3b and 4a. The others were classified into Category 0 (n=5), 1 (n=6), 2 (n=3), and 3a (n=6) in ascending order of severity. No incident was classified into Category 4b and 5. Furthermore, incidents occurring in the animal housing area were more severe than those occurring in the supporting area (P=0.002). This study showed that incident occurrences had characteristics that were not visible from individual incidents alone. Moreover, the ISCLA was considered useful when conducting the IRS and taking improvement measures in laboratory animal facilities.

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  • Guangquan HU, Xiaojie DING, Feng GAO, Jiehua LI
    Type: Original
    Article ID: 21-0063
    Published: 2021
    [Advance publication] Released: August 04, 2021
    JOURNAL OPEN ACCESS ADVANCE PUBLICATION

    Myocardial infarction (MI) is a severe coronary artery disease resulted from substantial and sustained ischemia. Abnormal upregulation of calcium and integrin binding protein 1 (CIB1) has been found in several cardiovascular diseases. In this study, we established a mouse model of MI by permanent ligation of the left anterior descending coronary artery. CIB1 was upregulated in the heart of MI mice. Notably, CIB1 knockdown by intramuscular injection of lentivirus-mediated short hairpin RNA (shRNA) targeting Cib1 improved cardiac function and attenuated myocardial hypertrophy and infarct area in MI mice. MI-induced upregulation of α-SMA, vimentin, Collagen I, and Collagen III, which resulted in collagen production and myocardial fibrosis, were regressed by CIB1 silencing. In vitro, cardiac fibroblasts (CFs) isolated from mice were subjected to angiotensin II (Ang II) treatment. Inhibition of CIB1 downregulated the expression of α-SMA, vimentin, Collagen I, and Collagen III in Ang II-treated CFs. Moreover, CIB1 knockdown inhibited Ang II-induced phosphorylation of PI3K-p85 and Akt in CFs. The effect of CIB1 knockdown on Ang II-induced cellular injury was comparable to that of LY294002, a specific inhibitor of the PI3K/Akt pathway. We demonstrated that MI-induced cardiac hypertrophy, myocardial fibrosis, and cardiac dysfunction might be attributed to the upregulation of CIB1 in MI mice. Downregulation of CIB1 alleviated myocardial fibrosis and cardiac dysfunction by decreasing the expression of α-SMA, vimentin, Collagen I, and Collagen III via inhibiting the PI3K/Akt pathway. Therefore, CIB1 may be a potential target for MI treatment.

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  • Jihong HE, Jian MAO, Lei HOU, Shimin JIN, Xiaodong WANG, Zhaoqi DING, ...
    Type: Original
    Article ID: 21-0028
    Published: 2021
    [Advance publication] Released: August 03, 2021
    JOURNAL OPEN ACCESS ADVANCE PUBLICATION

    Minocycline is a type of tetracycline antibiotic with broad-spectrum antibacterial activity that has been demonstrated to protect the brain against a series of central nervous system diseases. However, the precise mechanisms of these neuroprotective actions remain unknown. In the present study, we found that minocycline treatment significantly reduced HT22 cell apoptosis in a mechanical cell injury model. In addition, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining confirmed the neuroprotective effects of minocycline in vivo through the inhibition of apoptosis in a rat model of controlled cortical impact (CCI) brain injury. The western blotting analysis revealed that minocycline treatment significantly downregulated the pro-apoptotic proteins BAX and cleaved caspase-3 and upregulated the anti-apoptotic protein BCL-2. Furthermore, the beam-walking test showed that the administration of minocycline ameliorated traumatic brain injury (TBI)-induced deficits in motor function. Taken together, these findings suggested that minocycline attenuated neuronal apoptosis and improved motor function following TBI.

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  • Shuzo KANEKO, Joichi USUI, Masahiro HAGIWARA, Tatsuya SHIMIZU, Ryota I ...
    Type: Original
    Article ID: 21-0054
    Published: 2021
    [Advance publication] Released: July 28, 2021
    JOURNAL OPEN ACCESS ADVANCE PUBLICATION

    Focal segmental glomerulosclerosis (FSGS) is a major renal complication of human mitochondrial disease. However, its pathogenesis has not been fully explained. In this study, we focused on the glomerular injury of mito-miceΔ and investigated the pathogenesis of their renal involvement. We analyzed biochemical data and histology in mito-miceΔ. The proteinuria began to show in some mito-miceΔ with around 80% of mitochondrial DNA deletion, then proteinuria developed dependent with higher mitochondrial DNA deletion, more than 90% deletion. Mito-miceΔ with proteinuria histologically revealed FSGS. Immunohistochemistry demonstrated extensive distal tubular casts due to abundant glomerular proteinuria. Additionally, the loss of podocyte-related protein and podocyte’s number were found. Therefore, the podocyte injuries and its depletion had a temporal relationship with the development of proteinuria. This study suggested mitochondrial DNA deletion-dependent podocyte injuries as the pathogenesis of renal involvement in mito-miceΔ. The podocytes are the main target of mitochondrial dysfunction originated from the accumulation of mitochondrial DNA abnormality in the kidney.

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  • Yue YU, Shu-Ming YE, De-Yun LIU, Li-Qi YANG
    Type: Original
    Article ID: 21-0056
    Published: 2021
    [Advance publication] Released: July 20, 2021
    JOURNAL OPEN ACCESS ADVANCE PUBLICATION

    Non-alcoholic fatty liver disease (NAFLD) is a complication of childhood obesity and an oxidative stress-related multisystem disease. A mitochondria-targeting hydrogen sulfide (H2S) donor AP39 has antioxidant property, while the mechanism underlying the function of AP39 on pediatric NAFLD remains undefined. Here, 3-week-old SD rats were received a high-fat diet (HFD) feeding and injected with AP39 (0.05 or 0.1 mg/kg/day) via the tail vein for up to 7 weeks. AP39 reduced weight gain of HFD rats and improved HFD-caused liver injury, as evidenced by reduced liver index, improved liver pathological damage, decreased NAFLD activity score, as well as low ALT and AST activities. AP39 also reduced serum TC, TG, LDL-C concentrations but increased HDL-C. Moreover, AP39 prevented ROS generation, reduced MDA content and increased GSH level and SOD activity. Furthermore, AP39 increased H2S level, protected mitochondrial DNA (mtDNA), reduced mitochondrial swelling, and restored mitochondrial membrane potential (MMP) alteration. Notably, AP39 diminished HIF-1 mRNA and protein level, possibly indicating the alleviation in mitochondrial damage. In short, AP39 protects against HFD-induced liver injury in young rats probably through attenuating lipid accumulation, oxidative stress and mitochondrial dysfunction.

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  • Jianhua SUN, Qi ZHANG, Xiaokun LIU, Xiaoming SHANG
    Type: Original
    Article ID: 21-0060
    Published: 2021
    [Advance publication] Released: July 08, 2021
    JOURNAL OPEN ACCESS ADVANCE PUBLICATION

    Myocardial infarction (MI) is a common cardiovascular disease with high morbidity and mortality. In this study, we explored the role of interferon-induced protein with tetratricopeptide repeats 3 (IFIT3) in MI. MI was induced by ligation of the left anterior descending coronary artery. Lentivirus-mediated RNA interference of IFIT3 expression was performed by tail vein injection 72 hours before MI modeling. Cardiac injury indexes and inflammatory response were examined 3 days after MI. Cardiac function indexes, infarct size, and cardiac fibrosis were assessed 4 weeks after MI. IFIT3 expression was upregulated in myocardial tissues at both 3 days and 4 weeks after MI. Knockdown of IFIT3 significantly relieved the myocardial injury, as evidenced by the decrease in serum levels of cTnI and CK-MB. In addition, IFIT3 knockdown significantly reduced the number of CD68+ macrophages and the levels of interleukin-1β, interleukin-6, and tumor necrosis factor-α, indicating that the inflammatory response was relieved. Moreover, IFIT3 silencing also significantly improved cardiac function and reduced infarct size, myocardial fibrosis, and collagen content in mice with MI. Mechanically, the present study showed that the activation of the MAPK pathway was observed in myocardial tissues of MI mice, which was blocked by IFIT3 knockdown, as indicated by the decreased phosphorylation of JNK, p-38, and ERK. Collectively, our results revealed the role of IFIT3 in the inflammatory response and myocardial fibrosis after MI, indicating that IFIT3 might be a potential target for MI treatment.

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  • Shengming ZHANG, Ping LI, Minglong XIN, Xianglan JIN, Longguo ZHAO, Yo ...
    Type: Original
    Article ID: 21-0067
    Published: 2021
    [Advance publication] Released: July 05, 2021
    JOURNAL OPEN ACCESS ADVANCE PUBLICATION

    Exposure to chronic psychosocial stress is a risk factor for various pulmonary diseases. In view of the essential role of dipeptidyl peptidase 4 (DPP4) in animal and human lung pathobiology, we investigated the role of DPP4 in stress-related lung injury in mice. Eight-week-old male mice were randomly divided into a non-stress group and a 2-week immobilization stress group. Non-stress control mice were left undisturbed. The mice subjected to immobilized stress were randomly assigned to the vehicle or the DPP4 inhibitor anagliptin for 2 weeks. Chronic stress reduced subcutaneous and inguinal adipose volumes and increased blood DPP4 levels. The stressed mice showed increased levels in the lungs of genes and/or proteins related to oxidative stress (p67phox, p47phox, p22phox and gp91phox), inflammation (monocyte chemoattractant protein-1, vascular cell adhesion molecule-1, and intracellular adhesion molecule-1), apoptosis (caspase-3, -8, -9), senescence (p16INK4A, p21, and p53) and proteolysis (matrix metalloproteinase-2 to -9, cathepsin S/K, and tissue inhibitor of matrix metalloproteinase-1 and -2), and reduced levels of eNOS, Sirt1, and Bcl-2 proteins; and these effects were reversed by genetic and pharmacological inhibitions of DPP4. We then exposed human umbilical vein endothelial cells in vitro to hydrogen peroxide; anagliptin treatment was also observed to mitigate oxidative and inflammatory molecules in this setting. Anagliptin can improve lung injury in stressed mice, possibly by mitigating vascular inflammation, oxidative stress production, and proteolysis. DPP4 may become a new therapeutic target for chronic psychological stress-related lung disease in humans and animals.

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  • Na AHN, Sangho ROH, Jaehak PARK
    Type: Original
    Article ID: 21-0066
    Published: 2021
    [Advance publication] Released: June 30, 2021
    JOURNAL OPEN ACCESS ADVANCE PUBLICATION

    The Institutional Animal Care and Use Committee (IACUC) of Seoul National University (SNU) plays a key role in monitoring and managing the humane use of animals in scientific research. Here, as one of the pioneers of the IACUC in Korea, we reported SNU-IACUC operations and activities including committee establishment and legal formulation, protocol review, and post-approval monitoring of protocols, which the IACUC has undertaken in the last decade. In addition, legal regulations and improvements were also discussed, and encompassed the limited number of committee members and the single IACUC policy in Korea. As of December, 2020, amendments are on the table at the National Assembly. We also emphasized the independent nature of the IACUC in protecting activities, including approval and monitoring animal experiments, and its public role in narrowing the knowledge gap between society and scientists. Thus, the aim of this report is to help society and scientists understand the operations of the SNU-IACUC and its role in animal welfare.

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  • Haruna TAMANO, Haruna TOKORO, Daichi MURAKAMI, Ryo FURUHATA, Satoko NA ...
    Type: Original
    Article ID: 21-0044
    Published: 2021
    [Advance publication] Released: June 29, 2021
    JOURNAL OPEN ACCESS ADVANCE PUBLICATION

    Ninjin-yoei-to (NYT), a Kampo medicine, has ameliorative effects on cognitive dysfunction via enhancing cholinergic neuron activity. To explore an efficacy of NYT administration for prevention and cure of Alzheimer’s disease, here we examined the effect of NYT on amyloid β1-42 (Aβ1-42)-induced neurodegeneration in the dentate gyrus. A diet containing 3% NYT was administered to mice for 2 weeks and human Aβ1-42 was intracerebroventricularly injected. Neurodegeneration in the dentate granule cell layer of the hippocampus, which was determined 2 weeks after the injection, was rescued by administration of the diet for 4 weeks. Aβ staining (uptake) was not modified in the dentate granule cell layer by pre-administration of the diet for 2 weeks, while Aβ1-42-induced increase in intracellular Zn2+ was reduced, suggesting that pre-administration of NYT prior to Aβ injection is effective for reducing Aβ1-42-induced Zn2+ toxicity in the dentate gyrus. As a matter of fact, Aβ1-42-induced neurodegeneration in the dentate gyrus was rescued by pre-administration of NYT. Interestingly, the level of metallothioneins, intracellular Zn2+-binding proteins, which can capture Zn2+ from Zn-Aβ1-42 complexes, was elevated in the dentate granule cell layer by pre-administration of NYT. The present study suggests that pre-administration of NYT prevents Aβ1-42-mediated neurodegeneration in the dentate gyurs by induced synthesis of metallothioneins, which reduces intracellular Zn2+ toxicity induced by Aβ1-42.

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  • Kanako TAKAHASHI, Takeru SHIMA, Mariko SOYA, Leandro Kansuke OHAROMARI ...
    Type: Original
    Article ID: 21-0057
    Published: 2021
    [Advance publication] Released: June 28, 2021
    JOURNAL OPEN ACCESS ADVANCE PUBLICATION

    In animal experiments aimed at extrapolation to humans, it is essential to ensure the reproducibility of experiments and universality between animals and humans. However, among animals with the same generic name but from different breeders, which is to say different stocks, even resting physiological conditions, such as genetics, do not coincide, and, therefore, exercise capacity and physiological responses may also vary. To address this issue, we examined the differences in exercise capacity and exercise-induced metabolic and endocrine responses among stocks of Wistar rats using an established treadmill running model for rodents, which mimics physiological responses in humans. Wistar rats from four breeders were acclimated to treadmill running and then had a catheter inserted into their external jugular veins. Subsequently, the rats were subjected to an incremental treadmill running test (IRT). We found that there were significant differences in the exercise capacity among Wistar rats from different breeders. Additionally, the dynamics of blood lactate, glucose, and adrenocorticotropic hormone levels during the IRT were found to vary among the Wistar rats from different breeders; only one stock showed human-type exercise-induced physiological responses. These results indicate that Wistar rats could have different capacities for and physiological responses to the same exercise depending on their stocks. Thus, the selection of the stock of experimental animals may affect the validity of the results when verifying exercise effects.

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  • Zhenzhu LIU, Jing LI, Shu LIN, Yuhang WU, Dan HE, Peng QU
    Type: Original
    Article ID: 21-0002
    Published: 2021
    [Advance publication] Released: June 22, 2021
    JOURNAL OPEN ACCESS ADVANCE PUBLICATION

    PI3K is a downstream target of multiple cell-surface receptors, which acts as a crucial modulator of both cell polarization and survival. PI3K/AKT signaling pathway is commonly involved in cancer, atherosclerosis, and other diseases. However, its role in cardiovascular diseases, especially in atherosclerosis, remains to be further investigated. To determine the effect of PI3K/AKT signaling pathway on cellular inflammatory response and oxidative stress, PI3K inhibitor (GDC0941) and AKT inhibitor (MK2206) were used. First, THP-1 cells were incubated with ox‐LDL (100 µg/ml) to establish an in vitro atherosclerosis model. The inflammatory factors and foam cell formation were then evaluated to ascertain and compare the effects of PI3K and AKT inhibition. ApoE–/– mice fed a high-fat diet were used to assess the roles of PI3K and AKT in aortic plaque formation. Our results showed that the inhibition of PI3K or AKT could suppress the activation of NLRP3, decreased the expression levels of p-p65/p65 and reduced the production of MitoROS in THP-1 cells. Inhibition of PI3K or AKT could also reduced atherosclerosis lesion and plaque area, and decreased the levels of NLRP3 and IL-1β in ApoE–/– mice. The effect of PI3K inhibition was more significant than AKT. Therefore, PI3K inhibition can retard the progress of atherosclerosis. Besides, there may be other AKT-independent pathways that regulate the formation of atherosclerosis.

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  • Mutsuki KURAOKA, Yoshitsugu AOKI, Shin’ichi TAKEDA
    Type: Review
    Article ID: 21-0072
    Published: 2021
    [Advance publication] Released: June 16, 2021
    JOURNAL OPEN ACCESS ADVANCE PUBLICATION

    Duchenne muscular dystrophy (DMD) is an X-linked lethal muscle disorder characterized by primary muscle degeneration. Therapeutic strategies for DMD have been extensively explored, and some are in the stage of human clinical trials. Along with the development of new therapies, sensitive outcome measures are needed to monitor the effects of new treatments. Therefore, we investigated outcome measures such as biomarkers and motor function evaluation in a dystrophic model of beagle dogs, canine X-linked muscular dystrophy in Japan (CXMDJ). Osteopontin (OPN), a myogenic inflammatory cytokine, was explored as a potential biomarker in dystrophic dogs over the disease course. The serum OPN levels of CXMDJ dystrophic dogs were elevated, even in the early disease phase, and this could be related to the presence of regenerating muscle fibers; as such, OPN would be a promising biomarker for muscle regeneration. Next, accelerometry, which is an efficient method to quantify performance in validated tasks, was used to evaluate motor function longitudinally in dystrophic dogs. We measured three-axis acceleration and angular velocity with wireless hybrid sensors during gait evaluations. Multiple parameters of acceleration and angular velocity showed notedly lower values in dystrophic dogs compared with wild-type dogs, even at the onset of muscle weakness. These parameters accordingly decreased with exacerbation of clinical manifestations along with the disease course. Multiple parameters also indicated gait abnormalities in dystrophic dogs, such as a waddling gait. These outcome measures could be applicable in clinical trials of patients with DMD or other muscle disorders.

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  • Chang LIU, Liangshan WANG, Xianpei WANG, Xiaotong HOU
    Type: Original
    Article ID: 20-0191
    Published: 2021
    [Advance publication] Released: June 15, 2021
    JOURNAL OPEN ACCESS ADVANCE PUBLICATION

    The neonatal mice myocardial infarction (MI) has been established as one of the heart regeneration models. However, the role of inflammation in this model is still unclear. We sought to systematically evaluate this model and explore the role of inflammation in it. Postnatal day 1(P1) or day 7 (P7) mice were conducted LAD ligation. Cardiac damage, repair, and regeneration were examined by histology and echocardiography. Inflammation was detected by heart section HE staining and tissue qPCR. Dexamethasone (Dex) was used to inhibit inflammation and its effects on heart regeneration were evaluated. Two days after P1 mice MI, cardiomyocytes in ischemia area died and heart function decreased. Then surrounding cardiomyocytes proliferated to repair the injury. At day 28 after MI, hearts were almost fully regenerated with a little fibrosis existed. In contrary, P7 mice MI resulted in thinning and fibrosis of the ventricular wall. Inflammation was induced by LAD ligation after P1 mice MI and dynamic changed during the process. Inhibition of inflammation by Dex impaired heart regeneration. These demonstrated that cardiomyocytes death and heart regeneration occurred in this model and inflammation might play a crucial role in it. Modulating inflammation may provide a promising therapeutic strategy to support heart regeneration.

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  • Nicole Gee-Zhi TEE, Sze-Jie LOO, Li-Ping SU, Zhong-Hao TAO, Fu GUI, Ju ...
    Type: Original
    Article ID: 21-0050
    Published: 2021
    [Advance publication] Released: June 15, 2021
    JOURNAL OPEN ACCESS ADVANCE PUBLICATION

    Transverse aortic constriction (TAC) has been widely used to study cardiac hypertrophy, fibrosis, diastolic dysfunction, and heart failure in rodents. Few studies have been reported in preclinical animal models. The similar physiology and anatomy between non-human primates (NHPs) and humans make NHPs valuable models for disease modeling and testing of drugs and devices. In the current study, we aimed to establish a TAC model in NHPs and characterize the structural and functional profiles of the heart after TAC. A non-absorbable suture was placed around the aorta between the brachiocephalic artery and left common carotid artery to create TAC. NHPs were divided into 2 groups according to pressure gradient (PG): the Mild Group (PG=31.01 ± 12.40 mmHg, n=3) and the Moderate Group (PG=53.00 ± 9.37 mmHg, n=4). At 4 weeks after TAC, animals in both TAC groups developed cardiac hypertrophy: enlarged myocytes and increased wall thickness of the left ventricular (LV) anterior wall. Although both TAC groups had normal systolic function that was similar to a Sham Group, the Moderate Group showed diastolic dysfunction that was associated with more severe cardiac fibrosis, as evidenced by a reduced A wave velocity, large E wave velocity/A wave velocity ratio, and short isovolumic relaxation time corrected by heart rate. Furthermore, no LV arrhythmia was observed in either animal group after TAC. A diastolic dysfunction model with cardiac hypertrophy and fibrosis was successfully developed in NHPs.

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  • Dan LI, Yonghou ZHAO, Peng BAI, Yan LI, Siqi WAN, Xi ZHU, Mengyu LIU
    Type: Original
    Article ID: 21-0034
    Published: 2021
    [Advance publication] Released: June 10, 2021
    JOURNAL OPEN ACCESS ADVANCE PUBLICATION

    Intracerebral hemorrhage (ICH) is the most devastating subtype of stroke with high morbidity and mortality. The previous study has confirmed the therapeutic effect of Baihui (DU20)-penetrating-Qubin (GB7) acupuncture on ICH, while the related mechanism is left to be revealed. The aim of this study was to investigate the relevant mechanisms. ICH rat models were established utilizing the autologous blood injection method and the beneficial effect was found after DU20-penetrating-GB7 acupuncture along with decreased miR-34a-5p levels in the perihemorrhagic penumbra. Inversely, upregulating miR-34a-5p expression inhibited microglia M2 polarization while accelerated M1 polarization through targeting Krüppel-like factor 4 (Klf4), and thereby diminished the protective effect of DU20-penetrating-GB7 acupuncture on ICH. The results suggested the therapeutic effect of DU20-penetrating-GB7 acupuncture on ICH might be attributed to its modulation on microglia polarization through miR-34a-5p/Klf4 signaling.

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  • Jianhua WU, Lizhen WU, Li ZHANG, Huanhuan XU, Min WANG, Lin WANG, Jie ...
    Type: Original
    Article ID: 20-0195
    Published: 2021
    [Advance publication] Released: June 07, 2021
    JOURNAL OPEN ACCESS ADVANCE PUBLICATION

    Inflammatory allergic reaction is the main cause of allergic rhinitis (AR). Previous studies indicated that miR-224-5p was downregulated in the nasal mucosa of patients with AR, while the function of miR-224-5p in AR remains unclear. To explore this issue, AR mouse model was established using ovalbumin (OVA). For treatment group, lentivirus (LV)-miR-224-5p or its control was intranasally administrated to AR mice. miR-224-5p expression was detected by reverse transcription-quantitative PCR, followed by assessing the immunoglobulin E (IgE) level. Pathological alterations in nasal mucosa were detected using Hematoxylin-Eosin staining and Sirius red staining, followed by assessing the levels of inflammatory cells and factors. The NLRP3 inflammasome and TLR4/MyD88/NF-κB pathway were measured by Western blot, and then the relationship between miR-224-5p and toll-like receptor 4 (TLR4) was verified. The results showed that miR-224-5p was significantly decreased in nasal mucosa of AR mice. AR mice exhibited increased sneezing and nasal rubbing events, IgE level in serum, and pathological alterations in nasal mucosa, while overexpression of miR-224-5p markedly attenuated these changes. The levels of inflammatory cells in nasal lavage fluid and pro-inflammatory factors in serum and nasal mucosa were significantly increased in AR mice, which were reduced by miR-224-5p overexpression. Of note, LV-miR-224-5p treatment remarkably suppressed the activations of NLRP3 inflammasome and the TLR4/MyD88/NF-κB pathway in AR mice. Furthermore, miR-224-5p could bind to 3’-untranslated region (3’-UTR) of TLR4 and negatively regulate TLR4 level. Overall, we conclude that miR-224-5p may relieve AR by negatively regulating TLR4/MyD88/NF-κB pathway, indicating that miR-224-5p may be a promising target for AR treatment.

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  • Bing CHEN, Hong-Ling WANG, Rui CHEN, Li CHEN, Shun YANG, Yi WANG, Zhen ...
    Type: Original
    Article ID: 21-0005
    Published: 2021
    [Advance publication] Released: June 02, 2021
    JOURNAL OPEN ACCESS ADVANCE PUBLICATION

    In this study, we describe an N-ethyl-N-nitrosourea-induced mouse model with a corneal opacity phenotype that was associated with “eye open at birth” (EOB). Histological and immunohistochemistry staining analysis showed abnormal differentiation of the corneal epithelial cells in the mutant mice. The EOB phenotype was dominantly inherited on a C57BL/6 (B6) background. This allele carries a T941A substitution in exon 4 that leads to an L314Q amino acid change in the open reading frame of MAP3K1 (MEEK1). We named this novel Map3k1 allele Map3k1L314Q. Phalloidin staining of F-actin was reduced in the mutant epithelial leading edge cells, which is indicative of abnormality in epithelial cell migration. Interestingly enough, not only p-c-Jun and p-JNK but also c-Jun levels were decreased in the mutant epithelial leading edge cells. This study identifies a novel mouse Map3k1 allele causing EOB phenotype and the EOB phenotype in Map3k1L314Q mousemay be associated with the reduced level of p-JNK and c-Jun.

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  • Dina VILLANUEVA-GARCÍA, Daniel MOTA-ROJAS, Agatha MIRANDA-CORTÉS, Dani ...
    Type: Review
    Article ID: 20-0185
    Published: 2021
    [Advance publication] Released: May 27, 2021
    JOURNAL OPEN ACCESS ADVANCE PUBLICATION

    Objective. The aim of this review is to analyze the cardiorespiratory and tissue-protective effects of caffeine in animal models. Peer-reviewed literature published between 1975 and 2021 was retrieved from CAB Abstracts, PubMed, ISI Web of Knowledge, and Scopus. Extracted data were analyzed to address the mechanism of action of caffeine on cardiorespiratory parameters (heart rate and rhythm), vasopressor effects, and some indices of respiratory function; we close this review by discussing the existing debate on the research carried out on the effects of caffeine on tissue protection. Adenosine acts through specific receptors and is a negative inotropic andchronotropic agent. Blockage of its cardiac receptors can cause tachycardia (with arrhythmogenic potential) due to the intense activity of β1 receptors. In terms of tissue protection, caffeine produces inhibition of hyperoxia-induced pulmonary inflammation by decreasing proinflammatory cytokine expression in animal models. Conclusion. The protection that caffeine provides to tissues is not limited to the CNS, as studies have demonstrated that it generates attenuation of inflammatory effects in pulmonary tissue, where it inhibits the effects of some pro-inflammatory cytokines and prevents functional and structural changes.

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  • Shilpa PRABHAKAR, Sevda LULE, Cintia Carla DA HORA, Xandra O. BREAKEFI ...
    Type: Original
    Article ID: 20-0186
    Published: 2021
    [Advance publication] Released: May 25, 2021
    JOURNAL OPEN ACCESS ADVANCE PUBLICATION

    Adeno-associated virus (AAV)-based gene therapy is gaining popularity owing to its excellent safety profile and effective therapeutic outcomes in a number of diseases. Intravenous (IV) injection of AAV into the tail vein, facial vein and retro-orbital (RO) venous sinus have all been useful strategies to infuse the viral vector systemically. However, tail vein injection is technically challenging in juvenile mice, and injection at young ages (≤postnatal day-(P)21) is essentially impossible. The temporal or facial vein is localized anterior to the ear bud and is markedly visible in the first couple of days postnatally. However, this method is age-dependent and requires a dissecting microscope. Retro-orbital injection (ROI), on the other hand, is suitable for all murine ages, including newborn and older mice, and is relatively less stressful to animals compared to tail vein injection. Although many reports have shown ROI as an effective route of AAV delivery, herein we aim to highlight and summarize the methods and benefits of ROI. To capture the full spectrum of transduction efficiency mediated by ROI, we transduced the editing-dependent reporter mice (Ai9 Cre reporter mice) with the AAV9 vector, which targets a wide range of peripheral tissues with exceptional brain tropism. We also provide a comprehensive description of the ROI technique to facilitate viral vector administration without complications.

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  • Gisele Henrique Cardoso MARTINS, Juliete PALANDI, Vitória Helena Kuhn ...
    Type: Review
    Article ID: 19-0140
    Published: 2020
    [Advance publication] Released: March 23, 2020
    JOURNAL OPEN ACCESS ADVANCE PUBLICATION
    This article released online on March 23, 2020 as advance publication was withdrawn from consideration for publication in Experimental Animals at author’s request.
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