Experimental Animals
Online ISSN : 1881-7122
Print ISSN : 1341-1357
ISSN-L : 0007-5124
Advance online publication
Displaying 1-29 of 29 articles from this issue
  • Pengzhi YANG, Jie HE, Changlin WANG, Chi YANG, Fengzeng JIAN
    Article type: Original
    Article ID: 22-0119
    Published: 2022
    Advance online publication: December 09, 2022
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    Spinal cord injury (SCI), characterized by sensory disturbance and motor deficits, is associated with excessive inflammatory cytokine production of microglial cells. Previous studies have demonstrated High mobility group box 2 (HMGB2) as a microglial pro-inflammatory factor in stroke. This present study aims to evaluate the function of HMGB2 in a SCI rat model induced by striking the spinal cord at T9 to T12 using a rod. Our results showed that the levels of HMGB2 were significantly increased in the spinal cord tissues of SCI rats. Besides, HMGB2 downregulation was achieved by receiving an injection of lentivirus encoding HMGB2 shRNA in the spinal cord. Knockdown of HMGB2 suppressed SCI-induced microglial activation and neuroinflammation, as well as alleviated neuronal loss. In addition, we confirmed that HMGB2 silencing lessened lipopolysaccharide (LPS)-induced neuroinflammation in BV-2 cells. Furthermore, our findings demonstrated that HMGB2 knockdown suppressed the canonical nuclear factor of kB (NF-κB) signaling pathway both in vivo and in vitro. Collectively, this study manifested strong anti-inflammatory roles of HMGB2 knockdown on microglia-mediated neuroinflammation and suggested that HMGB2 might serve as a potential target for SCI therapy.

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  • Zongwei LIU, Jiaxue BI, Fang NIU, Hao LIANG, Jibo FAN, Jiajun LI, Duan ...
    Article type: Original
    Article ID: 22-0073
    Published: 2022
    Advance online publication: December 05, 2022
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    A reproducible canine aortic dissection (AD) model is useful for evaluating the performance of novel endovascular treatment devices. Thus, we performed a surgical method for constructing a reproducible Stanford type B AD (TBAD) canine model. Computed tomography angiography was performed 2 h after the modeling procedure to identify the existence of a false lumen, and follow-up imaging was performed 10 d after the procedure using digital subtraction angiography, intravascular ultrasound (IVUS), and color Doppler flow imaging (CDFI) to confirm the stable persistence of the false lumen. The success rate of the modeling operation was 88.8% (16/18). All surviving dogs had distal re-entries (16/16), and the furthest re-entry did not exceed the celiac trunk artery. The number of re-entries in the dogs was 1.50 ± 0.52, and the mean length of the false lumen was 175.37 ± 16.98 mm. IVUS showed that the largest proportion of the false lumen was 84.88 ± 1.27% of the whole lumen. The CDFI showed that the peak systolic velocity in the false lumen (10.89 ± 0.74 cm/s) was significantly slower than that in the true lumen (25.31 ± 1.72 cm/s) (P <0.001). Moreover, the direction of blood flow in the true lumen was consistent, whereas that in the false lumen was disordered. We optimized the traditional surgical method to construct a canine model of TBAD, that improves the success rate of the modeling operation, and designed a novel device to prolong the false lumen. The proposed model have wide implications in evaluating novel endovascular treatment device performance and studying the AD-related hemodynamics.

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  • Yanhui NI, Jingjing CAO, Jing YUAN, Xiaoran NING
    Article type: Original
    Article ID: 22-0103
    Published: 2022
    Advance online publication: December 05, 2022
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    A-kinase anchoring protein 12 (AKAP12) has been identified as an anti-inflammatory and anti-fibrotic regulator in chronic inflammation and cardiovascular disease. However, the potential of AKAP12 in autoimmune disorders, rheumatoid arthritis (RA) and associated cardiac complications remains elusive. Here, a murine model of collagen-induced arthritis (CIA) was successfully induced, followed by adenovirus-mediated AKAP12 short hairpin RNA (shRNA) treatment. AKAP12 silenced mice displayed elevated clinical arthritis scores and significant ankle joint swelling. AKAP12 loss in CIA mice increased inflammatory cell infiltration and cartilage erosion, increased the levels of anti-IIC IgG and inflammatory cytokines IL-1β, IL-6, TNF-α in serum, and upregulated the expression of cartilage-degrading enzymes MMP-1, MMP-3, MMP-13 in synovium, but reduced IL-10. The number of M1 macrophages and the expression of the markers (CCR7, IL-6, TNF-α and iNOS) was enhanced in synovial tissues, while M2 polarized macrophages and the makers (IL-10 and arginase-1) were reduced in response to AKAP12 loss. Moreover, low expression of AKAP12 was detected in the hearts of CIA mice. Loss of AKAP12 results in increased cardiac inflammation and fibrosis. This work suggests that AKAP12 loss aggravates joint inflammation likely through the promotion of M1 macrophage polarization and exacerbates inflammation-caused cardiac fibrosis.

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  • Masayuki MORI, Jian DAI, Hiroki MIYAHARA, Ying LI, Xiaojing KANG, Kazu ...
    Article type: Original
    Article ID: 22-0122
    Published: 2022
    Advance online publication: November 29, 2022
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    The Matsumoto Eosinophilia Shinshu (MES) is a rat model for hereditary blood eosinophilia. The incidence of eosinophilia is 100% in both female and male MES. The primary cause of the eosinophilia in MES is a loss-of-function mutation in the gene encoding the cytochrome b-245, alpha polypeptide (Cybames mutant allele). CYBA protein is a constituent of the superoxide-generating NADPH oxidase complex, the catalytic subunit of which is either NOX1, NOX2, or NOX4. However, the molecular mechanisms for the loss of CYBA to cause eosinophilia and even which of the three NOX isotypes is causally linked to the disease have been unknown. To resolve the latter issue, we generated F344/N rats knockout for Nox1, Nox2, and Nox4 genes. Also, we bred F344.MES-Cybames congenic rats that have a similar genetic background to the Nox knockout rats. We found that approximately 20% of female F344/N-Nox2em1 rats but none of the males developed blood eosinophilia. Also, we observed that all female F344.MES-Cybames and approximately 50% of male congenic rats developed the disorder. These results revealed that loss of NOX2 is the cause of blood eosinophilia in rats. Meanwhile, the data also indicated that in addition to the loss of NOX2 NADPH oxidase, both the genetic background of F344/N strain and gender influence the development of the disorder. These Nox and Cyba mutant rat strains with different eosinophilia incidences should be useful to elucidate molecular mechanisms and factors involved in the development of the disease.

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  • Takashi INOUE, Terumi YURIMOTO, Fumiko SEKI, Kenya SATO, Erika SASAKI
    Article type: Review
    Article ID: 22-0107
    Published: 2022
    Advance online publication: November 25, 2022
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    The common marmoset, Callithrix jacchus, is increasingly being used as the preferred nonhuman primate (NHP) model in biomedical research. Marmosets share several physiological and biological similarities with humans, as a Simiiformes species, and their use in research programs advances knowledge of several fields. Their unique characteristics, such as small size, high fecundity, and rapid growth, offer additional advances in laboratory settings. This article reviews the developments in experimental disease models using marmosets based on our experience at the Central Institute for Experimental Animals (CIEA) in Japan. The development of genetically modified marmoset models using advanced genome editing technology attracts researchers, particularly in neuroscience-related fields. In parallel, various marmoset models of human diseases induced by surgery or drug administration have contributed to preclinical and translational studies. Among these are models for Parkinson’s disease, induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; spinal cord injury models; a model for type 1 diabetes, induced by the combination of partial pancreatectomy and streptozotocin administration; and a hepatic fibrosis model induced by thioacetamides. The development of these models has been supported by refinements in veterinary care, such as the careful design of anesthetic protocols and better understanding of pathogenic microorganisms. In the second part of this review, we present a compilation of practices currently in use at CIEA that provide optimal animal care and enable safe experimentation.

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  • Kazuki UENO, Shizuya SAIKA, Yuka OKADA, Hiroki IWANISHI, Kentaro SUZUK ...
    Article type: Original
    Article ID: 22-0124
    Published: 2022
    Advance online publication: November 25, 2022
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    Transient Receptor Potential (TRP) ion channels mediate the influx of cations into cells responding to chemical or physical stimuli. TRP vanilloid 1 (TRPV1) regulates cutaneous functions. Its function in cutaneous wound healing, however, has not been clarified. The current study elucidated the role of TRPV1 in cutaneous wound healing of dorsal circular excisional injury using Trpv1-null (KO) and wild type (WT) male/female C57BL/6 mice. Macroscopic observation showed that the remaining cutaneous lesion was significantly larger in KO than that of WT at postoperative days (POD) 7 and 10. Histological analysis showed significantly delayed re-epithelialization in KO at POD7. The number of macrophages in KO and WT similarly returned to the reduced state from POD4 to POD7. Whereas, the number of neutrophils in KO did not significantly return to the reduced state, in contrast to WT. Of note, The H3Cit-labeled NETs (Neutrophil Extracellular Traps) formation of KO was prominently increased both in POD4 and 7. The current results suggest that the loss of TRPV1 induces prolonged neutrophilic inflammation and NETs formation, retarding murine cutaneous wound healing in vivo. This study provides a possible link with TRPV1 and neutrophilic regulation in cutaneous wound healing.

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  • Susumu IWAIDE, Ryohei OBA, Natsumi KOBAYASHI, Tomoaki MURAKAMI
    Article type: Original
    Article ID: 22-0125
    Published: 2022
    Advance online publication: November 23, 2022
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    Amyloid A (AA) amyloidosis is experimentally transmissible in some animal species, such as mice and chickens. While the spleen is important as the initial deposition site in the transmission of AA amyloidosis, it is not essential for establishing the transmission, and its role is not precisely understood. In this study, to clarify why the spleen is the first site of deposition in transmissible AA amyloidosis, we administered amyloid enhancing factor, which is AA fibrils extracted from AA amyloidosis affected mouse to local organs (liver, spleen, kidney, stomach wall, and Peyer’s patches), to tail vain and into peritoneum; then compared the amyloid distribution. Interestingly, initial amyloid deposition was observed at the administration site in each administered organ, not just the spleen. Furthermore, the amount of amyloid deposition in intra-organ administration groups was larger than that of the intravenous or intraperitoneal administration groups. This study indicates that locally exposed AEF initiates in situ amyloid deposition, from which amyloid deposition spreads throughout the body.

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  • Chenyang GU, Jiale LIU, Yajing LI, Qiankun ZHANG, Chaoqun LIN, Jiajun ...
    Article type: Original
    Article ID: 22-0131
    Published: 2022
    Advance online publication: November 23, 2022
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    The middle cerebral artery occlusion model (MCAO) is one of the most common stroke models in neuroscience research. The establishment of the mouse MCAO model in terms of animal survival depends on anesthesia, which is an important part of the entire surgical process. The 7-day survival rate of the MCAO model under isoflurane (ISO) anesthesia (35%) was lower than ketamine/xylazine (KX) anesthesia (70%), which demonstrated that the success rate of the MCAO model under KX anesthesia would be significantly higher than that under ISO anesthesia. As confirmed by TTC staining and MRI, the cerebral infarction area of mice successfully modeled under ISO anesthesia was significantly smaller than that of KX anesthesia. The diameter of cerebral blood vessels under ISO anesthesia was significantly larger than that under KX, and the blood perfusion volume was also significantly increased in the same area. ISO has proven to delay the coagulation time and affect the activation of coagulation factors. ISO anesthesia may cause bleeding, vasodilation, respiratory depression, and other phenomena that affect the success rate and death of diseased animal models. In conclusion, compared with ISO anesthesia, KX anesthesia is a safer and more suitable method for the establishment of a mouse MCAO model. The data will inform safer and more detailed anesthesia recommendations for the establishment of animal models of vascular-related major injury diseases.

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  • Lulu SHI, Mingzhe ZOU, Xingxing ZHOU, Songhua WANG, Wei MENG, Zhou LAN
    Article type: Original
    Article ID: 22-0115
    Published: 2022
    Advance online publication: October 31, 2022
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    A comparative study was conducted to determine whether hesperetin and pectolinarigenin could lower total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL), and high-density lipoprotein cholesterol (HDL) in a high-fat diet (HFD)-induced high lipid model in Golden Syrian hamsters. 48 Golden Syrian hamsters (8 weeks old) were fed with a HFD for 15 days. HFD induced significant increases in plasma TC, TG, LDL, and HDL. Then, these high lipid hamsters were divided into four groups and were administered with 0.5% sodium carboxymethyl cellulose (CMC-Na), hesperetin (100 mg/kg/day), pectolinarigenin (100 mg/kg/day) or atorvastatin (1.0 mg/kg/day), for 7 weeks. It was found that pectolinarigenin treatment resulted in significant reductions in body weight, adiposity index, serum levels of TC, TG and hepatic TC, TG and free fatty acid compared to those in control hamsters with hyperlipidemia (P<0.05). However, hesperetin treatment only caused reductions in plasma TC and hepatic TG levels. Besides, the hamsters treated with pectolinarigenin showed a relatively normal hepatic architecture compared to the hepatic steatosis shown in the control group. Moreover, the expressions of fatty-acid synthase (Fasn) and solute carrier family 27 member 1 (Slc27a1) involved in lipid biosynthesis, were suppressed in the pectolinarigenin-treated groups, and the expression of carnitine palmitoyltransferase 1A (Cpt1a) involved in fatty acid oxidation was increased in the pectolinarigenin-treated group. Taken together, these results suggest pectolinarigenin exerts stronger protective effects against hyperlipidemia and hepatic steatosis than hesperetin, which may involve the inhibition of lipid uptake and biosynthesis.

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  • Yulan TANG, Chen WANG, Mark Joseph M. DESAMERO, Mun Keong KOK, James K ...
    Article type: Original
    Article ID: 22-0092
    Published: 2022
    Advance online publication: October 28, 2022
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    Although hair loss is not a horrible disease, it sometimes reduces the patients’ quality of life (QOL) and increases their mental stress. Currently, there is no effective treatment for hair loss. It is known that honeybee propolis has various biological activities, including stimulating the proliferation of hair matrix keratinocytes. However, little is known with the hair promoting activity of stingless bee propolis. Hence, this study investigates the hair growth-promoting activity of Philippines stingless bee propolis extract and the underlying a molecular mechanism of promoting hair growth. For the evaluation of hair growth stimulating activity, 99.5% ethanolic extract of Philippines stingless bee propolis is examined using the simple shaving model in C57BL/6N mice. Melaninization of dorsal skin and histological analysis of hair follicles (HFs) revealed that propolis promotes hair growth by stimulating HFs development. The expression of mRNA (Wnt3a, Ctnnb1/β-catenin, Lef1, and Bmp2) and protein (WNT3A and β-catenin) of selected Wnt/β-catenin associated genes explains Philippines stingless bee propolis promoting HFs development by activating Wnt/β-catenin signaling pathway. These results suggest that the treatment of propolis strongly promotes hair growth by stimulating the development of HFs via activation of Wnt/β-catenin signaling pathway. This further indicates the potential of Philippines stingless bee propolis as a novel promising agricultural product for hair growth.

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  • Celine Swee May KHOO, Tomohiro HATAKENAKA, Nahoko MATSUKI, Seiya MINAG ...
    Article type: Original
    Article ID: 22-0078
    Published: 2022
    Advance online publication: October 27, 2022
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    The metastasis of various cancers is promoted by hyperglycemia. In contrast, melanoma and colorectal cancer seemed to be exceptional. We confirmed that the metastasis of melanoma B16-F10 could be suppressed by hyperglycemia. It was attractive from the prognostic point of view of the prevention of metastasis, though the problem of the risk of diabetes remained. Then, the effect of moderate hyperglycemic condition was investigated using a pre-diabetic model mouse (GKKO mouse). The metastasis of B16-F10 cells to liver was focused and the number and volume of metastatic colonies in liver were analyzed. The medians of the number of metastatic colonies in GKKO mice were 0.57-fold (p=0.06) compared to control mice. Analysis of macrophage markers revealed upregulation of CD86, a tumor-suppressive M1-type marker, and downregulation of CD206, a tumor-promotive M2-type marker. A tendency of upregulation of Cxcl10, a pro-inflammatory cytokine was also observed. Regarding cellular activities of B16-F10, migration activity and invasion activity were reduced by moderate hyperglycemia. In conclusion, metastasis of B16-F10 cells to liver could be suppressed by moderate hyperglycemia without the risk of diabetes. This information should contribute to dietary planning during prognosis.

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  • Kazutoshi NISHIJIMA, Ryoichi SAITO, Tamio OHNO, Shin TANAKA
    Article type: Original
    Article ID: 22-0110
    Published: 2022
    Advance online publication: October 27, 2022
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    To understand effects of aging and reproductive history in the bones of common marmosets (Callithrix jacchus), mandibles from 79 males and 66 females were analyzed. Dry bone specimen was prepared from dissected mandible, and analyzed using a dual-energy x-ray absorptiometry (DXA) measurement system in terms of bone weight (BnW), bone area (AREA), bone mineral content (BMC), bone mineral density (BMD, ratio of BMC to AREA) and bone mineral ratio (BMR, ratio of BMC to BnW). The mandible bones became porous and thicker with age. The age-related changes in BnW, AREA and BMC showed inflection points at around 1.5-2 Y and 13-15 Y. The period before 1.5-2 Y corresponds to the growth phase, the period between the inflection points is the aging phase, followed by senescence after the second inflection point. BMD increased until 1.5-2 Y and gradually decreased thereafter in males, with a more dramatic decrease in females, probably because of pregnancy and lactation. BMR was stable after reaching its peak by 1 Y, unlike the other parameters we analyzed. BMD of parous female tended to be lower than that of nulliparous female aged 2-5 Y. This study identified some of the particular effects of aging and reproductive history on characteristics of mandible bones in common marmoset.

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  • Jing-Huan QIU, Li ZHANG, Ke-Xin LI, Qiu-Hong ZHANG, Ke-Rui FAN, Kun CH ...
    Article type: Original
    Article ID: 22-0104
    Published: 2022
    Advance online publication: October 26, 2022
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    Itaconate, produced by aconitate decarboxylase 1 (ACOD1) which is encoded by immune-responsive gene 1 (Irg1), is one of the metabolites derived from the tricarboxylic acid cycle. It has been reported that exogenous itaconate plays an anti-inflammatory role in the progression of multiple diseases, including activated macrophage, ischemia-reperfusion Injury, and acute lung injury. However, the role and specific mechanism of endogenous itaconate in endotoxemia-induced acute lung injury (ALI) remain unclear. The animal model of ALI in WT and Irg1–/– mice was constructed by LPS intraperitoneal injection. Ultrahigh performance liquid chromatography-tandem mass spectroscopy (UPLC-MS/MS) analysis was performed to measure the quantity of endogenous itaconate. The protective effect of itaconate was investigated by the behavioral assessment and levels of inflammatory cytokines. Acute lung injury was assessed by H&E staining, BALF, and Evans blue leakage. Western blotting was used to detect the IRG1 expression and autophagic protein in the lung. We demonstrated that IRG1 was highly expressed in ALI, and endogenous itaconate was produced simultaneously and up to 100 times. Using Irg1–/– mice, we found that endogenous itaconate was likely to exert an anti-inflammatory effect by activating NRF2 and promoting autophagy. Furthermore, Autophagy was restrained by LPS but enhanced by 4-octyl itaconate (4-OI) pretreatment. Our study illustrated the deficiency of IRG1/Itaconate aggravates ALI and the IRG1/Itaconate pathway protects against ALI. The protective mechanisms could be related to facilitate autophagy. Such findings may provide a theoretical foundation for endotoxemia-induced ALI treatment.

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  • Koichiro KAWAGUCHI, Azusa ASAI, Ryuta MIKAWA, Noboru OGISO, Masataka S ...
    Article type: Original
    Article ID: 22-0109
    Published: 2022
    Advance online publication: October 26, 2022
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    Aging is an extremely complex biological process, and various models, from unicellular organisms to mammals, have been used in its research. The mouse is the most widely used model for studying human aging and diseases due to its high homology and well-established strategies for genetic manipulation. In spite of these advantages, the maximum lifespan of laboratory mice is nearly three years, which makes it time-consuming to obtain animals of the desired age. To avoid these issues and efficiently conduct aging research, the National Center for Geriatrics and Gerontology operates the “Aging Farm”, a system that supplies aging animals in response to researchers’ requests. In the present study, as part of the Aging Farm project, we examined changes in the physiological functions of and gene expression in the lung tissues of aging-grown animals as they aged. A decline in the physiological function of the lungs was already apparent before 6 months of age and continued until at least 1 year of age. On the other hand, gene expression profiling by RNA sequencing showed small changes in the early stages of aging, but more pronounced changes at 12 and 24 months of age than at 3 months of age. Age-related lung tissue changes are considered to be involved in the pathogenesis of various chronic respiratory diseases, and the characterization of aging animals will ensure the quality of the Aging Farm as a resource for aging research.

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  • Zhaohong GENG, Yuchan YUAN, Dan HE, Hewang LEE, Hongyan WANG, Nan NIU, ...
    Article type: Original
    Article ID: 22-0054
    Published: 2022
    Advance online publication: October 25, 2022
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    Pyruvate dehydrogenase complex (PDH) is an important complex of three enzymes that transforms pyruvate into acetyl-CoA, subsequently entering the tricarboxylic acid (TCA) cycle to produce ATP and electron donors. As a key regulator of energy and metabolic homeostasis, PDH is considered a potential therapeutic target of many diseases. On the other hand, the relationship between PDH and obesity is not clear. In this study, peripheral blood of Pdha1fl/flLyz2-Cre and C57BL/6 mice fed a high-fat diet (HFD) was collected and subjected to extensive transcriptome sequencing. Differentially expressed genes (DEGs) were identified. Enrichment of functions and signaling pathways analyses were performed based on Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to verify the genes selected from RNA sequencing (RNA-seq). Eventually, we found that Pdha1fl/flLyz2-Cre mice were more susceptible to HFD-induced obesity. A total of 302 up-regulated genes and 30 down-regulated genes were screened that were differentially expressed between Pdha1fl/flLyz2-Cre mice fed the HFD and the control groups. Furthermore, we verified that significant transcriptional changes in the genes Sgstm1, Ncoa4, Rraga, Slc3a2, Usp15, Gabarapl2, Wipi1, Sh3glb1, Mtmr3,and Cd36 were consistent with the results obtained from RNA-seq analysis. In summary, this study preliminarily established that there is a close relationship between Pdha1 and obesity and revealed the possible downstream pathways and target genes involved, laying a good foundation for the further study of Pdha1 function in the future.

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  • Qiushi LI, Xuying LIU, Ruixian XING, Rubo SUI
    Article type: Original
    Article ID: 22-0060
    Published: 2022
    Advance online publication: October 14, 2022
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    Stroke, a type of acute cerebrovascular disease, is a global disease with high mortality. Neuronal ischemia and hypoxia are closely related to occurrence and development of cognitive impairment. Transmembrane p24 trafficking protein 10 (TMED10) as a transmembrane protein involves in vesicle protein transport in the secretory pathways. However, the function and mechanism of TMED10 on ischemic stroke and cognitive impairments remain unclear. In current study, TMED10 was highly expressed in cerebral ischemic penumbra of middle cerebral artery occlusion (MCAO) mouse model. Downregulation of TMED10 suppressed cell survival and facilitated apoptosis in primary cortical neurons, which were grown under oxygen glucose deprivation/reoxygenation (OGD/R) condition. Upregulation of TMED10 protected neurons form apoptosis induced by OGD/R. Further research indicated that the decrease of TMED10 resulted in neuronal mitochondrial injury through increasing reactive oxygen species (ROS) production. Meanwhile, TMED10 reduction induced neuronal apoptosis and mitochondrial damage through activating the c-Jun N-terminal kinase (JNK) pathway. Moreover, the knockdown of TMED10 increased cerebral infarction area, aggravated neuronal injury and promoted neuronal apoptosis through activating the JNK pathway in the cerebral ischemic penumbra of MCAO mouse model. Additionally, Morris water maze test verified that the severity of cognitive impairment increased with the decline of TMED10. Collectively, this study reveals that TMED10 inhibits mitochondrial damage, and protects neurons from apoptosis in MCAO-induced ischemic stroke and cognitive impairment via blocking the JNK pathway.

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  • Yuki AOKI, Shunsuke KAKO, Ken MIYAZAWA, Masako TABUCHI, Fumika KIMURA, ...
    Article type: Original
    Article ID: 22-0099
    Published: 2022
    Advance online publication: October 18, 2022
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    The risk of relapse is associated with orthodontic treatment and is a major problem. Discussions about the risk of relapse have been made for a long time. However, the detailed mechanism of relapse has not yet been elucidated, although basic research has been conducted to clarify this mechanism. This study aimed to evaluate relapse following orthodontic treatment in mice (C57BL/6) tested via the coil spring method of tooth movement for 21 days and mechanical retention for 7 days after completion of tooth movement. During the experiment, relapse was observed and evaluated over 7 days. At the end of the orthodontic tooth movement, the average distance was 259.6 (± 10.9) μm, and tooth movement was observed in all mice. No significant difference was found in the distance at the end of the experimental tooth movement nor at the end of the 7-day mechanical retention. The distance at the start of relapse observation was 258.6 (± 10.4) μm, whereas that at the end was 155.4 (± 12.4) μm, indicating that the distance decreased significantly. Relative to the total relapse distance over the 7-day period, 45.7 (± 4.3)% of the relapse was observed on Day 0–1. We established a relapse observation mouse model, which allowed us to develop an effective and reproducible method for optimal evaluation of relapse. Our findings clarified that most of the relapse occurs within 7 days at the initial observation stage.

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  • Yuki YOSHIMURA, Kazuomi NAKAMURA, Misako SENO, Misa MOCHIZUKI, Kenji K ...
    Article type: Original
    Article ID: 22-0077
    Published: 2022
    Advance online publication: October 11, 2022
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    c-Fos is a useful marker gene of neuron activation for neuroscience and physiology research. The mechanism and function of neural networks have been elucidated using c-Fos reporter knock-in (KI) mice, but the small size of the mice makes it difficult to perform surgical procedures on specific brain regions. On the other hand, there is a large amount of accumulated data on behavioral studies using rats. Thus, the generation of c-Fos reporter rat is expected, but it is difficult to generate gene-modified rats. Furthermore, c-Fos gene abnormality is expected to be severe in rats, as shown in homozygous of c-Fos knockout (KO) mouse, but such analysis has rarely been performed and is not certain. This study generated c-Fos-deficient rats using CRISPR/Cas, with 1067 bp deletion including exon 1 of the c-Fos gene. Homozygous c-Fos KO rats had growth latency and the same tooth and bone abnormality as homozygous c-Fos KO mice but not heterozygous c-Fos KO rats. Therefore, the c-Fos gene in rats is expected to have the same function as that in mice, and the generation of c-Fos reporter KI rats is further anticipated.

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  • Weijie XU, Xiang HUANG, Wei LI, Gang QIAN, Beiye ZHOU, Xiaofei WANG
    Article type: Original
    Article ID: 22-0023
    Published: 2022
    Advance online publication: October 03, 2022
    JOURNAL OPEN ACCESS ADVANCE PUBLICATION

    Carbon monoxide (CO) has been reported to exhibit a therapeutic effect in lipopolysaccharide (LPS)-induced acute lung injury (ALI). However, the precise mechanism by which CO confers protection against ALI remains unclear. Pyroptosis has been recently proposed to play an essential role in the initiation and progression of ALI. Thus, we investigated whether pyroptosis is involved in the protection of CO against ALI and its underlying mechanism. First, an LPS-induced ALI mouse model was established. To determine the role of pyroptosis, we evaluated histological changes and the expression levels of cleaved caspase-11, N-gasdermin D (GSDMD), and IL-1β in lung tissues, which are the indicators of pyroptosis. Inhalation of CO exhibited protective effects on LPS-induced ALI by decreasing TNF-α and IL-10 expression and ameliorating pathological changes in lung tissue. In vitro, CO significantly reduced the expression of cleaved caspase-11, N-GSDMD, IL-1β, and IL-18. In addition, it increased nuclear factor E2-related factor 2 (NRF-2) expression in a time-dependent manner in RAW cells and decreased N-GSDMD expression. The expression of cleaved GSDMD and release of LDH were increased after treatment with a specific NRF-2 inhibitor, ML385, indicating that NRF-2 mediates the inhibition of pyroptosis by CO. Taken together, these results demonstrated that CO upregulated NRF-2 to inhibit pyroptosis and subsequently ameliorated LPS-induced ALI.

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  • Tingmei WU, Min LI, Li TIAN, Peilin CONG, Xinwei HUANG, Huanghui WU, Q ...
    Article type: Original
    Article ID: 22-0053
    Published: 2022
    Advance online publication: September 22, 2022
    JOURNAL OPEN ACCESS ADVANCE PUBLICATION

    Aging is one of the greatest risk factors for postoperative cognitive dysfunction (POCD), also known as perioperative neurocognitive disorder (PND). Animal models of PND are usually induced in mice over 18 months of age, which imposes expensive economic and time costs for PND-related studies. Sleep disorders, including sleep fragmentation, are reported to aggravate memory impairment in neurocognitive-related diseases such as Alzheimer’s disease (AD). Therefore the aim of the present study was to explore whether a PND model could be constructed in younger mice with the help of fragmented sleep. We found that fragmented sleep followed by laparotomy under isoflurane anesthesia could stably induce PND in 15-month-old mice. To determine whether the neurocognitive decline in this model could be salvaged by clinical treatments, we administered repetitive transcranial magnetic stimulation (rTMS) to the model mice before anesthesia and surgery. We found that 10 days of high-frequency rTMS (HF-rTMS) could improve spatial learning and memory deficits in this modified PND model. We are the first to successfully construct a PND model in younger mice, which is more economical, that can be used as an alternative model for future PND studies.

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  • Hoang Trung HIEU, Miyuu TANAKA, Mitsuru KUWAMURA, Tomoji MASHIMO, Tada ...
    Article type: Original
    Article ID: 22-0086
    Published: 2022
    Advance online publication: September 19, 2022
    JOURNAL OPEN ACCESS ADVANCE PUBLICATION

    Rodent coat color genes have been studied as a bioresource to understand developmental and cellular processes. The Downunder rat is a fancy variety with a marking on its belly that runs from the neck to the breech and appears to mirror the dorsal hooded marking. Here, we established a congenic strain carrying the Downunder (Du) gene in an F344 genetic background. In addition to the ventral marking, Du/+ rats exhibit anophthalmia or microphthalmia with incomplete penetrance. Du/Du embryos die in the early stages of organogenesis. Genetic linkage analysis mapped the Du gene to rat chromosome 3 and haplotype mapping with congenic rats localized the Du locus to a 3.9-Mb region. The Du locus includes two functional genes, glycosyltransferase-like domain-containing 1 (Gtdc1) and zinc finger E-box binding homeobox 2 (Zeb2). Although we found no functional variation within any of Zeb2’s exons or intron-exon boundaries, Zeb2 mRNA levels were significantly lower in Du/+ rats compared with wild-type rats. It is known that melanocyte-specific Zeb2 deletion results in the congenital loss of hair pigmentation in mice. Taken together, our results indicate that the Du mutation exerts pleiotropic effects on hair pigmentation, eye morphology, and development. Moreover, the Zeb2 gene is a strong candidate for the Du mutation.

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  • Kazuhisa KISHI, Momo GOTO, Yoshiharu TSURU, Masatoshi HORI
    Article type: Original
    Article ID: 22-0097
    Published: 2022
    Advance online publication: September 15, 2022
    JOURNAL OPEN ACCESS ADVANCE PUBLICATION

    Tracking metabolic changes in skeletal muscle and bone using animal models of diabetes mellitus (DM) provides important insights for the management of DM complications. In this study, we aimed to establish a method for monitoring changes in body composition characteristics, such as fat mass, skeletal muscle mass (lean mass), bone mineral density, and bone mineral content, during DM progression using a dual-energy X-ray absorptiometry (DXA) system in a mouse model of streptozotocin (STZ)-induced type 1 DM. In the DM model, STZ administration resulted in increased blood glucose levels, increased water and food intake, and decreased body weight. Serum insulin levels were significantly decreased on day 30 of STZ administration. The DXA analysis revealed significant and persistent decreases in fat mass, lower limb skeletal muscle mass, and bone mineral content in DM mice. We measured tibialis anterior (TA) muscle weight and performed a quantitative analysis of tibial microstructure by micro-computed tomography imaging in DM mice. The TA muscle weight of DM mice was significantly lower than that of control mice. In addition, the trabecular bone volume fraction, trabecular thickness, trabecular number, and cortical thickness were significantly decreased in DM mice. Pearson's product-moment correlation coefficient analysis showed a high correlation between the DXA-measured and actual body composition. In conclusion, longitudinal measurement of body composition changes using a DXA system may be useful for monitoring abnormalities in muscle and bone metabolism in animal models of metabolic diseases such as DM mice.

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  • Ren WEI, Xiaojing CHANG, Zhongyin WU, Chen DUAN, Jiang XIONG, Wei GUO
    Article type: Original
    Article ID: 22-0020
    Published: 2022
    Advance online publication: September 03, 2022
    JOURNAL OPEN ACCESS ADVANCE PUBLICATION

    Previous abdominal aortic aneurysm (AAA) animal modeling methodologies were either expensive or complicated. Here, we developed a novel AAA model which was simple to set up and generated minimal calcification. Twenty-four rats were divided randomly into four groups. Groups 1, 2 and 3 underwent surgery in which 15% hydrochloric acid (HCl) was applied periarterially to the abdominal aorta for 5 minutes, followed by sacrifice 1 week (group 1), 2 weeks (group 2), and 4 weeks (group 3) after surgery. The maximum aortic diameter (MAD) was measured at surgery and before animal sacrifice. Rats in group 4 were sham-treated. The MADs in group 1, 2 and 3 showed significant dilation compared with group 4, with a mean dilation rate of 33.8% in the first week after surgery. Histopathological examination revealed infiltration of macrophages into the adventitia, obvious apoptosis of smooth muscle cells, and rupture and collapse of the elastic fibers. Furthermore, no calcification was observed in the dilated aorta. The mRNA expression levels of inflammatory factors were at least two-fold higher in group 1 than in group 4, indicating significant inflammatory response in the progression of AAA information. In conclusion, periarterial application of 15% HCl is a convenient and reliable model to create an abdominal aortic aneurysm in rats, and the potential development mechanism may be related to the proinflammatory effects of HCl.

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  • Masaki WATANABE, Koki HIURA, Hayato SASAKI, Tadashi OKAMURA, Nobuya SA ...
    Article type: Original
    Article ID: 22-0057
    Published: 2022
    Advance online publication: September 03, 2022
    JOURNAL OPEN ACCESS ADVANCE PUBLICATION

    Animal models of podocytopathy and chronic kidney diseases (CKD) help elucidate these pathologies. Adriamycin (ADR)-induced nephropathy is a common rodent model of podocytopathy. BALB/c mice are sensitive to ADR, whereas C57BL/6 (B6) mice, the most commonly used strain, are resistant to ADR. Therefore, mouse strains with the B6 genetic background cannot be used as an ADR nephropathy model. We previously generated DNA-dependent protein kinase catalytic subunit (Prkdc) mutant B6 mice (B6-PrkdcR2140C) carrying the R2140C mutation that causes ADR nephropathy. However, whether ADR nephropathy in the novel strain progresses to CKD after ADR administration has not been evaluated. Therefore, we examined whether the B6-PrkdcR2140C mice develop CKD after ADR administration. We also evaluated whether differences existed in the genetic background in ADR nephropathy by comparing the B6-PrkdcR2140C mice with BALB/c mice. Our findings demonstrated that B6-PrkdcR2140C progresses to CKD and is resistant to nephropathy compared with the BALB/c mice. The B6-PrkdcR2140C and BALB/c mice differed in the expression of genes related to inflammatory mediators, and further analysis is required to identify factors that contribute to resistance to nephropathy.

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  • Takashi KURAMOTO
    Article type: Review
    Article ID: 22-0089
    Published: 2022
    Advance online publication: September 03, 2022
    JOURNAL OPEN ACCESS ADVANCE PUBLICATION

    The laboratory rat (Rattus norvegicus) is a key model organism for biomedical research. Rats can be subjected to strict genetic and environmental controls. The rat’s large body size is suitable for both surgical operations and repeated measurements of physiological parameters. These advantages have led to the development of numerous rat models for genetic diseases. Forward genetics is a proven approach for identifying the causative genes of these disease models but requires genome resources including genetic markers and genome sequences. Over the last few decades, rat genome resources have been developed and deposited in bioresource centers, which have enabled us to perform positional cloning in rats. To date, more than 100 disease-related genes have been identified by positional cloning. Since some disease models are more accessible in rats than mice, the identification of causative genes in these models has sometimes led to the discovery of novel functions of genes. As before, various mutant rats are also expected to be discovered and developed as disease models in the future. Thus, the forward genetics continues to be an important approach to find genes involved in disease phenotypes in rats. In this review, I provide an overview the development of rat genome resources and describe examples of positional cloning in rats in which novel gene functions have been identified.

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  • Junyan CHEN, Naoki FUJITA, Tae TAKEDA, Wataru HANYU, Hirohide TAKATANI ...
    Article type: Original
    Article ID: 22-0026
    Published: 2022
    Advance online publication: August 15, 2022
    JOURNAL OPEN ACCESS ADVANCE PUBLICATION

    Spinal cord injury (SCI) is a common neurological disorder in dogs. A secondary injury that occurs in the acute phase causes expansion of inflammation, resulting in lesion extension and further loss of function. Mesenchymal stem cells (MSCs) have trophic effects and the ability to migrate toward injured tissues; therefore, MSC-based therapy is considered promising for the treatment of canine SCI. We recently reported that bone marrow peri-adipocyte cells (BM-PACs) can be obtained from canine bone marrow and have stem cell potential superior to that of conventional bone marrow MSCs (BMMSCs). However, their therapeutic potential for SCI have been still unknow. Here, we first evaluated the ability of BM-PACs to secrete hepatocyte growth factor (HGF) and their migration ability toward inflammatory milieu in vitro. BM-PACs can secrete HGF in response to pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α and interleukin (IL)-1β, and exhibit migration ability toward these cytokines. Next, BM-PACs were intravenously administered into nude mice with acute SCI to analyze the homing ability and therapeutic effects of HGF secreted by BM-PACs. BM-PACs homed to the injured spinal cord, where the HGF expression level increased 7 days after administration. Intravenous administration of BM-PACs induced functional recovery and pathological improvement, indicated by less demyelinating area, more preserved axons, and less glial scar formation compared with the mice only received vehicle. These findings suggest that the intravenous administration of BM-PACs can be a novel therapeutic intervention for acute canine SCI.

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  • Nagi FUJII, Yuta NAKATA, Yoko KATO
    Article type: Original
    Article ID: 22-0043
    Published: 2022
    Advance online publication: August 15, 2022
    JOURNAL OPEN ACCESS ADVANCE PUBLICATION

    It is well known that the survivability of gametes of postmortem carcass was decreased as time passes after death. In this study, it was examined whether cytoplasmic replacement rescues the survivability of germinal vesicle stage (GV) oocytes of postmortem carcass in the mouse. Reactive oxygen species (ROS) levels and mitochondria numbers in GV oocytes of the dead mice stored at 4 degrees were significantly impaired after 44 h postmortem compared to the control (0h). However, when kayoplasts of GV oocytes of postmortem carcass was transferred to recipient ooplasts (GV transfer), proportion of in vitro maturation (IVM), normal spindle morphology, in vitro and in vivo developmental ability after IVF of reconstituted oocytes was improved. Moreover, secondary follicle oocytes of postmortem carcass were developed, matured and fertilized in vitro and developed to go to term, when GV transfer was conducted at the GV phase. Thus, transfer of GV karyoplast recovered from postmortem carcass, which viability was decreased, into fresh GV recipient ooplasm, rescues survivability of reconstituted oocytes. It suggested the effective use of oocytes of dead animals in the mouse and this achievement must apply to other rare animal species, especially animals under control by human.

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  • Kai KANG, Lukuan CUI, Qian ZHANG, Shijun GAO
    Article type: Original
    Article ID: 22-0009
    Published: 2022
    Advance online publication: August 05, 2022
    JOURNAL OPEN ACCESS ADVANCE PUBLICATION

    Although many surgical or non-operative therapies have been developed to treat Achilles tendon injuries, the prognosis of which is often unsatisfactory. Recently, biologic approaches using multipotent stem cells like tendon-derived stem cells (TDSCs) pose a possible treatment option. To evaluate whether the Leucine rich repeat containing 32 (Lrrc32) affects the tenogenic differentiation of TDSCs and thus promotes Achilles tendon healing. TDSCs were infected with the recombinant Lrrc32-overexpressing lentivirus (LV-Lrrc32) and then locally injected into the injured site of rat. Four weeks after surgery, the Achilles tendon tissue (~0.5 cm) around the injured area was harvested for analysis. Pathological results showed that Lrrc32-overexpressing TDSCs significantly improved the morphological changes of the injured tendons. Specifically, the increased collagen-I expression and hydroxyproline content in extracellular matrix, and more orderly arrangement of the regenerated collagen fibers were observed in the Lrrc32 overexpression group. Moreover, 4 weeks after injection of Lrrc32-overexpressing TDSCs, the expression of tenocyte-related genes such as tenomodulin (Tnmd), scleraxis (Scx) and decorin (Dcn) were upregulated in the area of the healing tendon. These findings indicated that Lrrc32 promoted the tenogenic differentiation of TDSCs in vivo. Additionally, Lrrc32 overexpression also increased the expression of TGF-β1 and p-SMAD2/3, suggesting that the beneficial effects of Lrrc32 on tendon repair might be associated with the expression of TGF-β1 and p-SMAD2/3. Our findings collectively revealed that Lrrc32-overexpressed TDSCs promoted tendon healing more effectively than TDSCs alone.

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  • Gisele Henrique Cardoso MARTINS, Juliete PALANDI, Vitória Helena Kuhn ...
    Article type: Review
    Article ID: 19-0140
    Published: 2020
    Advance online publication: 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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