Experimental Animals
Online ISSN : 1881-7122
Print ISSN : 1341-1357
ISSN-L : 0007-5124
Volume 72, Issue 3
Displaying 1-13 of 13 articles from this issue
Review: Young Investigator Award
  • Masanori A. Murayama
    2023 Volume 72 Issue 3 Pages 285-293
    Published: 2023
    Released on J-STAGE: August 07, 2023
    Advance online publication: March 06, 2023
    JOURNAL OPEN ACCESS

    Alzheimer’s disease (AD), a progressive neurodegenerative disease characterized by cognitive dysfunction and neuropsychiatric symptoms, is the most prevalent form of dementia among the elderly. Amyloid aggregation, tau hyperphosphorylation, and neural cell loss are the main pathological features. Various hypotheses have been proposed to explain the development of AD. Some therapeutic agents have shown clinical benefits in patients with AD; however, many of these agents have failed. The degree of neural cell loss is associated with the severity of AD. Adult neurogenesis, which governs cognitive and emotional behaviors, occurs in the hippocampus, and some research groups have reported that neural cell transplantation into the hippocampus improves cognitive dysfunction in AD model mice. Based on these clinical findings, stem cell therapy for patients with AD has recently attracted attention. This review provides past and present therapeutic strategies for the management and treatment of AD.

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  • Mônica Horr, Simone Sommerfeld, Murilo V. Silva, Belchiolina B. Fonsec ...
    2023 Volume 72 Issue 3 Pages 294-301
    Published: 2023
    Released on J-STAGE: August 07, 2023
    Advance online publication: January 16, 2023
    JOURNAL OPEN ACCESS
    Supplementary material

    Chicken embryos (CE) are an experimental model used as an important life science research tool worldwide, and then, adequate anesthetic protocols must be adopted to avoid the unjustifiable suffering of animals. Thus, our objective was to evaluate different anesthetic protocols in CEs using an easy inoculation route, the shell membrane (SM). We adopted the heart rate by pulse and the CE movements as a parameter of pain by assessing the vase in the chorioallantoic membrane (CAM) through the shell by a sensor of a multiparametric monitor. CEs were distributed into the following groups: (i) association of ketamine (5 mg/CE), midazolam (0.05 mg/CE) and morphine (0.15 mg/CE); (ii) ketamine (5 mg/CE) and xylazine (0.125 mg/CE); (iii) xylazine (0.0125 mg/CE) and morphine (0.15 mg/CE). The stress method used to test the anesthetic potential of the drugs was high temperature stimulation, keeping the CEs 10 cm from the fire of a Bussen nozzle for 30 s. In this experimental model, associations between different drugs decreased the pulse and the movement, indicating possible sedation. After treatment, the CE’s submitted to the stress method had the heart rate and movements kept low in the groups ketamine-midazolam-morphine and ketamine-xylazine, while the non-drug-treated group increased heart rate. In a group treated with xylazine-morphine, the heart rate did not decrease, but the movement decreased after the stimulus. As the best results were the combinations of ketamine-midazolam-morphine and ketamine-xylazine, we recommend these associations for use in embryos in the final third of embryonic development in experimental protocols and euthanasia.

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  • Sheng Li, Gaoxiang Ouyang, Linhui Yuan, Xiaoxuan Wu, Lijun Zhang
    2023 Volume 72 Issue 3 Pages 302-313
    Published: 2023
    Released on J-STAGE: August 07, 2023
    Advance online publication: January 16, 2023
    JOURNAL OPEN ACCESS

    Diabetic retinopathy (DR), a common complication of diabetes, involves excessive proliferation and inflammation of Muller cells and ultimately leads to vision loss and blindness. SRY-box transcription factor 9 (SOX9) has been reported to be highly expressed in Müller cells in light-induced retinal damage rats, but the functional role of SOX9 in DR remains unclear. To explore this issue, the DR rat model was successfully constructed via injection with streptozotocin (65 mg/kg) and the retinal thicknesses and blood glucose levels were evaluated. Müller cells were treated with 25 mmol/l glucose to create a cell model in vitro. The results indicated that SOX9 expression was significantly increased in DR rat retinas and in Müller cells stimulated with a high glucose (HG) concentration. HG treatment promoted the proliferation and migration capabilities of Müller cells, whereas SOX9 knockdown reversed those behaviors. Moreover, SOX9 knockdown provided protection against an HG-induced inflammatory response, as evidenced by reduced tumor necrosis factor-α, IL-1β, and IL-6 levels in serum and decreased NLRP3 inflammasome activation. Notably, SOX9 acted as a transcription factor that positively regulated thioredoxin-interacting protein (TXNIP), a positive regulator of Müller cells gliosis under HG conditions. A dual-luciferase assay demonstrated that SOX9 could enhance TXNIP expression at the transcriptional level through binding to the promoter of TXNIP. Moreover, TXNIP overexpression restored the effects caused by SOX9 silencing. In conclusion, these findings demonstrate that SOX9 may accelerate the progression of DR by promoting glial cell proliferation, metastasis, and inflammation, which involves the transcriptional regulation of TXNIP, providing new theoretical fundamentals for DR therapy.

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  • Taichi Noda, Ayumu Taira, Hina Shinohara, Kimi Araki
    2023 Volume 72 Issue 3 Pages 314-323
    Published: 2023
    Released on J-STAGE: August 07, 2023
    Advance online publication: January 30, 2023
    JOURNAL OPEN ACCESS
    Supplementary material

    Spermatozoa released from the testis acquire fertilizing ability by translocating thorough the epididymis. Further, accessory gland secretions ejaculated into the female reproductive tract along with spermatozoa are also required to ensure male fecundity, such as the maintenance of proper sperm count and inhibition of premature sperm capacitation in the uterus. Here, we focus on a testis-enriched gene “Aldoart2”, an epididymis-enriched gene “Serpina16”, and seminal vesicle-enriched genes “Aoc1l3” and “Pate14” which were thought to be important for male fertility based on the previous studies. We independently deleted almost the entire protein-coding sequence of these genes in mice using CRISPR/Cas9. There were no overt defects in the histology and the sperm morphology and motility of any knockout (KO) mice. Further, Aoc1l3 and Pate14 KO males were able to form copulatory plugs. Finally, female mice that mated with these KO males delivered pups at a comparable level with the control males. Given our data, we demonstrated that the four genes predominantly expressed in the testis, epididymis, or seminal vesicle are independently dispensable for male fertility.

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  • Jianqiang Wei, Jing Yin, Ying Cui, Kaijie Wang, Mingyan Hong, Jianzhon ...
    2023 Volume 72 Issue 3 Pages 324-335
    Published: 2023
    Released on J-STAGE: August 07, 2023
    Advance online publication: February 03, 2023
    JOURNAL OPEN ACCESS

    Intracerebral hemorrhage (ICH) is an incurable neurological disease. Microglia activation and its related inflammation contribute to ICH-associated brain damage. FERM domain containing kindlin 1 (FERMT1) is an integrin-binding protein that participates in microglia-associated inflammation, but its role in ICH is unclear. An ICH model was constructed by injecting 50 µl of autologous blood into the bregma of rats. FERMT1 siRNA was injected into the right ventricle of the rat for knockdown of FERMT1. A significant striatal hematoma was observed in ICH rats. FERMT1 knockdown reduced the water content of brain tissue, alleviated brain hematoma and improved behavioral function in ICH rats. FERMT1 knockdown reduced microglia activity, inhibited NLR family pyrin domain containing 3 (NLRP3) inflammasome activity and decreased the expression of inflammatory factors including IL-1β and IL-18 in the peri-hematoma tissues. BV2 microglial cells were transfected with FERMT1 siRNA and incubated with 60 µM Hemin for 24 h. Activation of NLRP3 inflammasome induced by hemin were reduced in microglia when FERMT1 was knocked down, leading to decreased production of inflammatory factors IL-1β and IL-18. In addition, knockdown of FERMT1 prevented the activation of nuclear factor kappa B (NF-κB) signaling pathway in vivo and in vitro. Our findings suggested that down-regulation of FERMT1 attenuated microglial inflammation and brain damage induced by ICH via NLRP3/NF-κB pathway. FERMT1 is a key regulator of inflammatory damage in rats after ICH.

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  • Wachiryah Thong-asa, Kanthaporn Puenpha, Thannaporn Lairaksa, Siriwiph ...
    2023 Volume 72 Issue 3 Pages 336-345
    Published: 2023
    Released on J-STAGE: August 07, 2023
    Advance online publication: February 08, 2023
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    Cerebral ischemia reperfusion (IR) injury as found in stroke is a complex and heterogeneous disorder and closely related to disability and death. Today, nutraceuticals and protective therapy to increase neuronal integrity and prevent pathological complication are common. We investigated the neuroprotective effect of betanin against cerebral IR injury in mice. Forty male institute of cancer research (ICR) mice were divided into Sham-veh, IR-veh, IR-Bet50 and IR-Bet100 groups. After 2 weeks of oral administration of normal saline (vehicle; veh) or 50 mg/kg or 100 mg/kg of betanin (Bet), mice were subjected to IR induction using 30-min bilateral common carotid artery occlusion, followed by 24 h of reperfusion. Brain infarction, oxidative status, cortical and hippocampal neurons and white matter pathologies were evaluated. Results showed that IR significantly increases brain infarction, Cornus Ammonis 1 (CA1) hippocampal and corpus callosum (CC) and internal capsule (IC) white matter degeneration (P<0.05). Brain oxidative status revealed significant elevation of malondialdehyde (MDA) together with a significant decrease in catalase (CAT) activity, induced by IR (P<0.05). Pretreatment with betanin 100 mg/kg led to a significant reduction in brain infarction and MDA, CA1 hippocampus, CC and IC white matter degeneration. Betanin also led to a significant increase in CAT activity (P<0.05), with enhancing effect on reduced glutathione levels (GSH, P<0.05). The present study revealed the neuroprotective efficacy of betanin against IR injury in mice’s brains, including its inhibition of lipid peroxidation, and boosting of GSH and CAT activity.

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  • Ce Xu, Jiahui Han, Di Jia, Jimin Cai, Jianming Yuan, Xin Ge
    2023 Volume 72 Issue 3 Pages 346-355
    Published: 2023
    Released on J-STAGE: August 07, 2023
    Advance online publication: March 02, 2023
    JOURNAL OPEN ACCESS

    An increasing number of studies have suggested that oxidative stress and inflammation play momentous roles in acute pulmonary embolism (APE). Honokiol, a bioactive biphenolic phytochemical substance, is known for its strong anti-oxidative and anti-inflammatory effects, and it served as an activator of sirtuin3 (SIRT3) in the present study. The purposes of the study were to explore the effects of honokiol on APE rats and investigate whether the function of honokiol is mediated by SIRT3 activation. In the study, the rats received a right femoral vein injection of dextran gel G-50 particles (12 mg/kg) to establish the APE model and were subsequently administered honokiol and/or a selective SIRT3 inhibitor 3-(1H-1,2,3-triazol-4-yl)pyridine (3-TYP; 5 mg/kg) intraperitoneally. The results showed that SIRT3 activation by honokiol attenuated the loss in lung function, ameliorated the inflammatory response and oxidative damage, and inhibited apoptosis in lung tissues of the rats with APE but that this was reversed by 3-TYP. In addition, we found that the AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway might be activated by honokiol but restrained by 3-TYP. These results indicated that honokiol was capable of suppressing the adverse effects of APE and that this was diminished by SIRT3 suppression, implying that activation of SIRT3 might serve as a therapeutic method for APE.

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  • Long Yang, Chunxue Wu, Ying Cui, Shimin Dong
    2023 Volume 72 Issue 3 Pages 356-366
    Published: 2023
    Released on J-STAGE: August 07, 2023
    Advance online publication: March 13, 2023
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    Myocardial cell damage is associated with apoptosis and excessive inflammatory response in sepsis. Histone deacetylases (HDACs) are implicated in the progression of heart diseases. This study aims to explore the role of histone deacetylase 9 (HDAC9) in sepsis-induced myocardial injury. Lipopolysaccharide (LPS)-induced Sprague Dawley rats and cardiomyocyte line H9C2 were used as models in vivo and in vitro. The results showed that HDAC9 was significantly upregulated after LPS stimulation, and HDAC9 knockdown remarkably improved cardiac function, as evidenced by decreased left ventricular internal diameter end diastole (LVEDD) and left ventricular internal diameter end systole (LVESD), and increased fractional shortening (FS)% and ejection fraction (EF)%. In addition, HDAC9 silencing alleviated release of inflammatory cytokines (tumor necrosis factor-α (TNF-α), IL-6 and IL-1β) and cardiomyocyte apoptosis in vivo and in vitro. Furthermore, HDAC9 inhibition was proved to suppress nuclear factor-kappa B (NF-κB) activation with reducing the levels of p-IκBα and p-p65, and p65 nuclear translocation. Additionally, interaction between miR-214-3p and HDAC9 was determined through bioinformatics analysis, RT-qPCR, western blot and dual luciferase reporter assay. Our data revealed that miR-214-3p directly targeted the 3’UTR of HDAC9. Our findings demonstrate that HDAC9 suppression ameliorates LPS-induced cardiac dysfunction by inhibiting the NF-κB signaling pathway and presents a promising therapeutic agent for the treatment of LPS-stimulated myocardial injury.

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  • Jinxia Zhu, Hongxiu Chen, Jianjiao Cui, Xiaorui Zhang, Guangwei Liu
    2023 Volume 72 Issue 3 Pages 367-378
    Published: 2023
    Released on J-STAGE: August 07, 2023
    Advance online publication: March 13, 2023
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    Autoimmune hepatitis (AIH) is a kind of autoimmune disease mediated by T cells, and its incidence is gradually increasing in the world. Oroxylin A (OA) is one of the major bioactive flavonoids that has been reported to inhibit inflammatory. Here, an AIH model of mouse was induced by Concanavalin A (Con A). It found that serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were decreased in mice with the treatment of OA. Hematoxylin-eosin staining showed that the liver injury was attenuated by OA, and TUNEL staining indicated that the cells apoptosis of liver was weakened in mice with OA treatment. ELISA analysis of cytokines and chemokines suggested that OA reduced the expression of IL-6, IL-17A, chemokine ligand 2 (CCL2), C-X-C motif chemokine ligand 1 (CXCL1) and CXCL10, but promoted the expression of IL-10 and TGF-β in mice. The mRNA levels of Il-17a in liver and spleen tissues were also significantly decreased, on the contrary, the mRNA levels of Il-10 in liver and spleen tissues were increased. The proportion of Treg/Th17 detected by flow cytometry revealed that OA promoted the differentiation of Treg and inhibited the differentiation of Th17 both in the liver and spleen. The results of this study demonstrated the inhibitory effects of OA on AIH-induced liver injury and the inflammatory response of AIH, and revealed that OA affected the balance of Treg/Th17 and shifted the balance toward Treg differentiation. It provided new potential drugs for the prevention of AIH.

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  • Xiaoxiang Wang, Lan Yu, Yao Chen, Xing Xiong, Hongmei Ran
    2023 Volume 72 Issue 3 Pages 379-388
    Published: 2023
    Released on J-STAGE: August 07, 2023
    Advance online publication: March 23, 2023
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    Pancreatic fibrosis (PF) is a hallmark of chronic pancreatitis (CP), but its molecular mechanism remains unclear. This study was conducted to explore the role of Kruppel-like factor 4 (KLF4) in PF in CP mice. The CP mouse model was established using caerulein. After KLF4 interference, pathological changes in pancreatic tissues and fibrosis degree were observed by hematoxylin-eosin staining and Masson staining, and levels of Collagen I, Collagen III, and alpha-smooth muscle actin, inflammatory cytokines, KLF4, signal transducer and activator of transcription 5A (STAT5) in pancreatic tissues were measured by enzyme-linked immunosorbent assay, quantitative real-time polymerase chain reaction, Western blot assay, and immunofluorescence. The enrichment of KLF4 on the STAT5 promoter and the binding of KLF4 to the STAT5 promoter were analyzed. The rescue experiments were performed by co-injection of sh-STAT5 and sh-KLF4 to confirm the regulatory mechanism of KLF4. KLF4 was upregulated in CP mice. Inhibition of KLF4 effectively attenuated pancreatic inflammation and PF in mice. KLF4 was enriched on the STAT5 promoter and enhanced the transcriptional and protein levels of STAT5. Overexpression of STAT5 reversed the inhibitory role of silencing KLF4 in PF. In summary, KLF4 promoted the transcription and expression of STAT5, which further facilitated PF in CP mice.

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  • Yuqing Cheng, Shuangzhe Lin, Tianyi Ren, Jianbin Zhang, Yingying Shi, ...
    2023 Volume 72 Issue 3 Pages 389-401
    Published: 2023
    Released on J-STAGE: August 07, 2023
    Advance online publication: April 05, 2023
    JOURNAL OPEN ACCESS
    Supplementary material

    Metabolic-associated fatty liver disease (MAFLD) and alcoholic hepatitis (AH) are among the most prevalent liver diseases worldwide, and their coexistence is common in clinical practice. However, currently established models of MAFLD-AH coexistence do not fully replicate their pathological characteristics and require sophisticated experimental techniques. Therefore, we aimed to develop an easily replicable model that mimics obesity-induced MAFLD-AH in patients. Our goal was to establish a murine model that replicates MAFLD and AH coexistence, resulting in significant liver injury and inflammation. To this end, we administered a single ethanol gavage dose to ob/ob mice on a chow diet. The administration of a single dose of ethanol led to elevated serum transaminase levels, increased liver steatosis, and apoptosis in ob/ob mice. Furthermore, ethanol binge caused a significant increase in oxidative stress in ob/ob mice, as measured via 4-hydroxynonenal. Importantly, the single dose of ethanol also markedly exacerbated liver neutrophil infiltration and upregulated the hepatic mRNA expression of several chemokines and neutrophil-related proteins, including Cxcl1, Cxcl2, and Lcn2. Whole-liver transcriptomic analysis revealed that ethanol-induced changes in gene expression profile shared similar features with AH and MAFLD. In ob/ob mice, a single dose of ethanol binge caused significant liver injury and neutrophil infiltration. This easy-to-replicate murine model successfully mimics the pathological and clinical features of patients with coexisting MAFLD and AH and closely resembles the transcriptional regulation seen in human disease.

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  • Yuyo Ka, Ryoji Ito, Ryoko Nozu, Kayo Tomiyama, Masami Ueno, Tomoyuki O ...
    2023 Volume 72 Issue 3 Pages 402-412
    Published: 2023
    Released on J-STAGE: August 07, 2023
    Advance online publication: April 04, 2023
    JOURNAL OPEN ACCESS
    Supplementary material

    Humanized mice are widely used to study the human immune system in vivo and investigate therapeutic targets for various human diseases. Immunodeficient NOD/Shi-scid-IL2rγnull (NOG) mice transferred with human hematopoietic stem cells are a useful model for studying human immune systems and analyzing engrafted human immune cells. The gut microbiota plays a significant role in the development and function of immune cells and the maintenance of immune homeostasis; however, there is currently no available animal model that has been reconstituted with human gut microbiota and immune systems in vivo. In this study, we established a new model of CD34+ cell-transferred humanized germ-free NOG mice using an aseptic method. Flow cytometric analysis revealed that the germ-free humanized mice exhibited a lower level of human CD3+ T cells than the SPF humanized mice. Additionally, we found that the human CD3+ T cells slightly increased after transplanting human gut microbiota into the germ-free humanized mice, suggesting that the human microbiota supports T cell proliferation or maintenance in humanized mice colonized by the gut microbiota. Consequently, the dual-humanized mice may be useful for investigating the physiological role of the gut microbiota in human immunity in vivo and for application as a new humanized mouse model in cancer immunology.

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  • Xiao-Po Liu, Jia-Qi Li, Ruo-Yu Li, Guo-Long Cao, Yun-Bo Feng, Wei Zhan ...
    2023 Volume 72 Issue 3 Pages 413-424
    Published: 2023
    Released on J-STAGE: August 07, 2023
    Advance online publication: April 05, 2023
    JOURNAL OPEN ACCESS

    The imbalance of bone resorption and bone formation causes osteoporosis (OP), a common skeletal disorder. Decreased osteogenic activity was found in the bone marrow cultures from N-acetylglucosaminyl transferase V (MGAT5)-deficient mice. We hypothesized that MGAT5 was associated with osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and involved in the pathological mechanisms of osteoporosis. To test this hypothesis, the mRNA and protein expression levels of MGAT5 were determined in bone tissues of ovariectomized (OVX) mice, a well-established OP model, and the role of MGAT5 in osteogenic activity was investigated in murine BMSCs. As expected, being accompanied by the loss of bone mass density and osteogenic markers (runt-related transcription factor 2, osteocalcin and osterix), a reduced expression of MGAT5 in vertebrae and femur tissues were found in OP mice. In vitro, knockdown of Mgat5 inhibited the osteogenic differentiation potential of BMSCs, as evidenced by the decreased expressions of osteogenic markers and less alkaline phosphatase and alizarin red S staining. Mechanically, knockdown of Mgat5 suppressed the nuclear translocation of β-catenin, thereby downregulating the expressions of downstream genes c-myc and axis inhibition protein 2, which were also associated with osteogenic differentiation. In addition, Mgat5 knockdown inhibited bone morphogenetic protein (BMP)/transforming growth factor (TGF)-β signaling pathway. In conclusion, MGAT5 may modulate the osteogenic differentiation of BMSCs via the β-catenin, BMP type 2 (BMP2) and TGF-β signals and involved in the process of OP.

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