Experimental Animals
Online ISSN : 1881-7122
Print ISSN : 1341-1357
ISSN-L : 0007-5124
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  • Michito Hamada, Yuki Tsunakawa, Hyojung Jeon, Manoj Kumar Yadav, Sator ...
    2020 Volume 69 Issue 1 Pages 1-10
    Published: 2020
    Released: January 29, 2020
    [Advance publication] Released: October 01, 2019
    JOURNALS OPEN ACCESS

    The transcription factor MafB regulates macrophage differentiation. However, studies on the phenotype of Mafb-deficient macrophages are still limited. Recently, it was shown that the specific expression of MafB permits macrophages to be distinguished from dendritic cells. In addition, MafB has been reported to be involved in various diseases related to macrophages. Studies using macrophage-specific Mafb-deficient mice show that MafB is linked to atherosclerosis, autoimmunity, obesity, and ischemic stroke, all of which exhibit macrophage abnormality. Therefore, MafB is hypothesized to be indispensable for the regulation of macrophages to maintain systemic homeostasis and may serve as an innovative target for treating macrophage-related diseases.

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  • Ai Nishitani, Toru Yoshihara, Miyuu Tanaka, Mitsuru Kuwamura, Masahide ...
    2020 Volume 69 Issue 1 Pages 11-17
    Published: 2020
    Released: January 29, 2020
    [Advance publication] Released: July 09, 2019
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    Hyperpolarization-activated cyclic nucleotide-gated potassium channel 1 (HCN1) contribute to spontaneous rhythmic activity in different tissues, including the heart and brain. Deficiency in HCN1 function is associated with sick sinus syndrome in mice and epilepsy in humans. We recently developed Hcn1-deficient rats and found that they exhibit absence epilepsy. While rearing Hcn1-deficient rats, we noticed loose muscle tension and abnormal gait. We therefore evaluated the muscle strength and motor functions of Hcn1-deficient rats. When subjected to the wire hang test, Hcn1-deficient rats fell down more easily than control F344 rats. Grip strength of Hcn1-deficient rats was significantly smaller than F344 rats. In the inclined plane test, they exhibited a smaller maximum angle. In the rotarod test, the latency to fall was shorter for Hcn1-deficient rats than F344 rats. In the footprint analysis, Hcn1-deficient rats exhibited smaller step length and wider step width than F344 rats. Instead of poor motor coordination ability and muscle weakness, Hcn1-deficient rats exhibited normal electromyograms, muscle histology, and deep tendon reflex. These findings suggest that HCN1 channels contribute to motor coordination and muscle strength, and that the muscle weakness of Hcn1-deficient rats results from the involvement not of the peripheral but of the central nervous system.

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  • Shunya Nakayama, Hiroshi Koie, Miyoko Kato-Tateishi, Chungyu Pai, Yasu ...
    2020 Volume 69 Issue 1 Pages 18-25
    Published: 2020
    Released: January 29, 2020
    [Advance publication] Released: July 12, 2019
    JOURNALS OPEN ACCESS
    Supplementary material

    The demand for monkeys for medical research is increasing, because their ionic mechanism of repolarization is similar to that of humans. The QT interval is the distance between the Q wave and T wave, but this interval is affected by heart rate. Therefore, QT correction methods are commonly used in clinical settings. However, an accurate correction formula for the QT interval in cynomolgus monkeys has not been reported. We assessed snapshot electrocardiograms (ECGs) of 353 ketamine-immobilized monkeys, including aged animals, and contrived a new formula for the corrected QT interval (QTc) as a marker of QT interval prolongation in cynomolgus monkeys. Values for QTc were calculated using the formula [QTc] = [QT] / [RR]n, along with several other formulas commonly used to calculate QTc. We found that the optimal exponent of the QT interval corrected for heart rate, n, was 0.576. The mean value of QTc in healthy monkeys determined using the new formula was 373 ± 31 mm, and there were no significant differences between the sexes. Other ECG parameters were not significantly different between the sexes and there were no age-related effects on QTc. Prolongation of QTc to over 405 ms, as calculated by the new formula, was observed in 50 monkeys with underlying diseases. Additionally, all monkeys with QTc above 440 ms by the new formula had some underlying disease. The results resemble those in humans, suggesting that the new QTc formula could be useful for diagnosis of QT interval prolongation in cynomolgus monkeys.

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  • Shizuka Konishi, Natsuki Tanaka, Tomoji Mashimo, Takashi Yamamoto, Tet ...
    2020 Volume 69 Issue 1 Pages 26-33
    Published: 2020
    Released: January 29, 2020
    [Advance publication] Released: July 23, 2019
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    Spontaneous hhy mice show hydrocephalus and subcortical heterotopia, and a mutation in the Ccdc85c gene has been identified. To contribute to the comparison of the role of Ccdc85c in different species, we established a Ccdc85c KO rat and investigated its pathological phenotypes. Ccdc85c KO rats were produced by genomic engineering using transcription activator-like effector nuclease (TALEN). The KO rats had an approximately 350-bp deletion in Ccdc85c and lacked CCDC85C protein expression. The KO rats showed non-obstructive hydrocephalus, subcortical heterotopia, and intracranial hemorrhage. The KO rats had many pathological characteristics similar to those in hhy mice. These results indicate that CCDC85C plays an important role in cerebral development in rats, and the function of CCDC85C in the cerebrum are similar in rats and mice.

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  • Yongxia Cheng, Jingchao Li, Chong Wang, Heran Yang, Ying Wang, Tao Zha ...
    2020 Volume 69 Issue 1 Pages 34-44
    Published: 2020
    Released: January 29, 2020
    [Advance publication] Released: July 29, 2019
    JOURNALS OPEN ACCESS

    Diabetic cardiomyopathy (DCM) is one of the cardiovascular complications of diabetes mellitus independent of hypertension, coronary disease, and other heart diseases. The development of DCM is multifactorial and hard to detect at an early stage. Long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (Malat1) is emerging as a regulator of DCM, the underlying mechanism of its role in DCM has not been elaborated yet. In this study, we established a mouse DCM model via streptozocin injection as evidenced by cell hypertrophy and cell apoptosis of myocardial tissue, and found that Malat1 expression was upregulated in the myocardium in DCM mice. Meanwhile, elevated expression of pro-apoptotic factors p53, p21, cleaved caspase 3, cleaved caspase 9 and BAX, and down-regulation of anti-apoptotic BCL-2 were observed in DCM myocardium. We further investigated the effect of Malat1 on cardiomyocytes under high glucose condition by silencing Malat1 with its specific short-hairpin RNA. Like in vivo, expression of Malat1 in cardiomyocytes was notably raised, remarkable cell apoptosis and changes in apoptosis-related factors were also observed following high glucose treatment. Besides, we validated that Malat1 acted as a sponge of miR-181a-5p. Inhibition of miR-181a-5p could, at least partially, abolish Malat1 knockdown-induced alteration in cardiomyocytes. In addition, p53, a critical regulator of apoptosis, was validated to be a downstream target of miR-181a-5p. In summary, our findings reveal that Malat1 knockdown attenuates high glucose-induced cardiomyocyte apoptosis via releasing miR-181a-5p, and this mechanism may provide us with new diagnosis target of DCM.

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  • Bing Chen, Chong-Yang Qi, Li Chen, Meng-Jun Dai, Ya-You Miao, Rui Chen ...
    2020 Volume 69 Issue 1 Pages 45-53
    Published: 2020
    Released: January 29, 2020
    [Advance publication] Released: August 08, 2019
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    Supplementary material

    Ip3r1 encodes an inositol 1,4,5-trisphosphate-responsive calcium channel. Mutations in the IP3R1 gene in humans may cause Gillespie syndrome (GS) typically presents as fixed dilated pupils in affected infants, which was referred to as iris hypoplasia. However, there is no report of mice with Ip3r1 heterozygous mutations showing dilated pupils. Here, we report a new Ip3r1 allele with short-term dilated pupil phenotype derived from an N-ethyl-N-nitrosourea (ENU) mutagenesis screen. This allele carries a G5927A transition mutation in Ip3r1 gene (NM_010585), which is predicted to result in a C1976Y amino acid change in the open reading frame of IP3R1 (NP_034715). We named this novel Ip3r1 allele Ip3r1C1976Y. Histology and pharmacological tests show that the dilated pupil phenotype is a mydriasis caused by the functional defect in the iris constrictor muscles in Ip3r1C1976Y. The dilated pupil phenotype in Ip3r1C1976Y was referred to as mydriasis and excluding iris hypoplasia. IHC analysis revealed increased expression of BIP protein, the master regulator of unfolded protein response (UPR) signaling, in Ip3r1C1976Y mice that did not recover. This study is the first report of an Ip3r1 mutation being associated with the mydriasis phenotype. Ip3r1C1976Y mice represent a self-healing model that may be used to study the therapeutic approach for Ip3r1-related diseases.

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  • Kenan Çağrı Tümer, Haydar Özdemİr, Hatice Eröksüz
    2020 Volume 69 Issue 1 Pages 54-61
    Published: 2020
    Released: January 29, 2020
    [Advance publication] Released: August 29, 2019
    JOURNALS OPEN ACCESS

    Sepsis is a potentially life-threatening condition, and it is frequently complicated by myocardial damage. Data on myocardial damage in rabbit caecal ligation and puncture (CLP) models are limited, although numerous animal models have been used to study sepsis-associated myocardial damage. This study aimed to investigate the effect of CLP on cardiac muscle by measuring serum cardiac troponin I (cTnI) concentrations and by detecting both histopathological changes and cTnI immunoreactivity in cardiomyocytes in rabbits. After CLP was performed in rabbits, blood samples were taken from the jugular vein at 0, 4, 8, and 12 h for haematological and biochemical analyses. At the end of the experiment, all of the rabbits were euthanised to examine the histopathological changes and the cTnI immunoreactivity in cardiac muscle tissue. No changes in serum cTnI concentration were observed in the experimental group (EG) or control group (CG) at 0 and 4 h. In EG, the mean serum cTnI concentrations were 0.230 ± 0.209 and 1.177 ± 0.971 ng/ml at 8 and 12 h, respectively. In CG, the mean serum cTnI concentrations were 0.032 ± 0.014 and 0.031 ± 0.021 ng/ml at 8 and 12 h, respectively. Moreover, cytoplasmic cTnI immunoreactivity decreased in EG compared with that in CG (P<0.01). The results demonstrated that CLP induced a systemic inflammatory response and caused myocardial damage in rabbits.

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  • Jincheol Seo, Hyeon-Gu Yeo, Junghyung Park, Jinyoung Won, Keonwoo Kim, ...
    2020 Volume 69 Issue 1 Pages 62-69
    Published: 2020
    Released: January 29, 2020
    [Advance publication] Released: September 03, 2019
    JOURNALS OPEN ACCESS
    Supplementary material

    Pigs are often selected for large animal models including for neuroscience and behavioral research, because their anatomy and biochemistry are similar to those of humans. However, behavioral assessments, in combination with objective long-term monitoring, is difficult. In this study, we introduced an automated video tracking system which was previously used in rodent studies, for use with pig models. Locomotor behaviors (total distance, number of zone transitions, and velocity) were evaluated and their changes were validated by different 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration methods and dosing regimens. Three minipigs (23–29 kg) received subcutaneous or intravenous MPTP, either 1 or 3 times per week. Immediately after MPTP injection, the minipigs remained in a corner and exhibited reduced trajectory. In addition, the total distance travelled, number of zone transitions, and velocity were greatly reduced at every MPTP administration in all the minipigs, accompanying to increased resting time. However, the MPTP-induced symptoms were reversed when MPTP administration was terminated. In conclusion, this automated video-tracking system was able to monitor long-term locomotor activity and differentiate detailed alterations in large animals. It has the advantages of being easy to use, higher resolution, less effort, and more delicate tracking. Additionally, as our method can be applied to the animals’ home pen, no habituation is needed.

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  • Andrzej Gugołek, Janusz Strychalski, Jerzy Juśkiewicz, Ewa Żary-Sikors ...
    2020 Volume 69 Issue 1 Pages 70-79
    Published: 2020
    Released: January 29, 2020
    [Advance publication] Released: September 17, 2019
    JOURNALS OPEN ACCESS

    Chinchillas are herbivores, but wild chinchillas may occasionally consume animal-based foods. The aim of this study was to determine the effect of fish meal (FM) and mealworm meal (MWM) included in complete pelleted diets on nutrient digestibility and gastrointestinal function in chinchillas. The experiment was performed on 24 male, divided into three groups, n=8. Control group (C) was fed a diet containing 10% soybean meal (SBM). In the experimental group FM, chinchillas received a diet containing 3% fish meal, and the diet administered to the experimental group MWM was supplemented with 4% dried mealworm larvae meal. The nutrient digestibility of diets was determined. At the end of the experiment animals were euthanized and their digestive tracts were removed to analyze gut activity. FM group animals were characterized by lower crude fat digestibility, whereas both alternative protein sources improved the digestibility of acid detergent fiber (ADF). A considerable increase in the activity of cecal intracellular and extracellular bacterial enzymes (in particular β-glucosidase, β-galactosidase and β-xylosidase) was noted in the FM group, which however did not increase the concentrations of short-chain fatty acids (SCFA). The inclusion of MWM in chinchilla diets shifted the bacterial fermentation site from the cecum (lowest SCFA pool) to the colon (highest SCFA pool), thus enabling to derive additional energy from less digestible dietary components. In conclusion, chinchilla diets can be supplemented with small amounts of animal protein such as fish meal and dried mealworm larvae meal.

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  • Yiwei Feng, Wei Xia, Ketong Ji, Yongjing Lai, Qingyuan Feng, Honglin C ...
    2020 Volume 69 Issue 1 Pages 80-91
    Published: 2020
    Released: January 29, 2020
    [Advance publication] Released: September 17, 2019
    JOURNALS OPEN ACCESS

    Systematic classification and determination of various cells in normal peripheral blood of artificially feeding Tupaia belangeri chinensis of different ages and genders and evaluation of the effectiveness of an automatic blood cell classification counter for measuring tree shrew blood cells. Child, young and adult tree shrews (forty for each group) were randomly selected, half male and half female. After the animals were stable, the peripheral blood of each group was collected through the femoral vein, and the morphology of various blood cells of the tree shrew was observed and classified by the manual microscopic counting method and by an automatic blood cell classification counter. The Reference intervals of the normal peripheral blood cell absolute count, cell diameter and white blood cell percentage in tree shrews of different ages and genders has been calculated. White blood cell count and neutrophil relative count increased with age, while lymphocyte relative count decreased. The white blood cell count, neutrophil relative count, and lymphocyte relative count in the child group, as well as lymphocyte relative count in the young group, significantly differed according to gender (P<0.05), and the differences in other indicators were not significant. The Bland-Altman plot and the Passing-Bablok scattergram showed that the change trend of each indicator was consistent but exhibited large systematic differences between methods. Differences in peripheral blood cells exist among different age groups and different genders. An automatic blood cell classification counter is not suitable for the absolute count of blood cells in the tree shrew.

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  • Luca Bordoni, Eugenio Gutiérrez Jiménez, Søren Nielsen, Leif Østergaar ...
    2020 Volume 69 Issue 1 Pages 92-103
    Published: 2020
    Released: January 29, 2020
    [Advance publication] Released: September 18, 2019
    JOURNALS OPEN ACCESS

    The most used experimental mouse model of hyponatremia and elevated intracranial pressure (ICP) is intraperitoneal injection of water in combination with antidiuretics. This model of water intoxication (WI) results in extreme pathological changes and death within 1 h. To improve preclinical studies of the pathophysiology of elevated ICP, we characterized diuresis, cardiovascular parameters, blood ionogram and effects of antidiuretics in this model. We subsequently developed a new mouse model with mild hyponatremia and sustained increased ICP. To investigate the classical protocol (severe WI), C57BL/6mice were anesthetized and received an intraperitoneal injection of 20% body weight of MilliQ water with or without 0.4 µg·kg−1 desmopressin acetate (dDAVP). Corresponding Sham groups were also studied. In the new WI protocol (mild WI), 10% body weight of a solution containing 6.5 mM NaHCO3, 1.125 mM KCl and 29.75 mM NaCl was intraperitoneally injected. By severe WI, ICP and mean arterial pressure increased until brain stem herniation occurred (23 ± 3 min after injection). The cardiovascular effects were accelerated by dDAVP. Severe WI induced a halt to urine production irrespective of the use of dDAVP. Following the new mild WI protocol, ICP also increased but was sustained at a pathologically high level without inducing herniation. Mean arterial pressure and urine production were not affected during mild WI. In conclusion, the new mild WI protocol is a superior experimental model to study the pathophysiological effects of elevated ICP induced by water intoxication.

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  • Daiji Kiyozumi, Masashi Mori, Mayo Kodani, Masahito Ikawa
    2020 Volume 69 Issue 1 Pages 104-109
    Published: 2020
    Released: January 29, 2020
    [Advance publication] Released: September 26, 2019
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    QBRICK, FRAS1, and FREM2 compose a family of extracellular matrix proteins characterized by twelve consecutive CSPG repeats and single or multiple Calx-β motifs. Dysfunction of these proteins have been associated with Fraser syndrome, which is characterized by malformation of skin, eyes, digits, and kidneys. FREM3 is another member of the 12-CSPG protein family. However, it remains unknown whether genetic dysfunction of FREM3 also causes Fraser syndrome or another developmental disorder. Here we investigated a Frem3 mutant mouse line generated by CRISPR/Cas9-mediated genome editing. The FREM3 mutant homozygotes were born at the expected Mendelian ratio and did not possess any defects characteristic of Fraser syndrome. These results indicate that the dysfunction of FREM3 is not associated with Fraser syndrome.

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  • Zhihai Shi, Huijie Xing, Chunli Qi, Meixia Fang, Jiangnan Fu, Xingwang ...
    2020 Volume 69 Issue 1 Pages 110-118
    Published: 2020
    Released: January 29, 2020
    [Advance publication] Released: September 26, 2019
    JOURNALS OPEN ACCESS

    There has been a lack of suitable fatty liver models and characterization techniques for histopathological evaluation of alcoholic fatty liver (AFL). This work aimed to exploit an magnetic resonance imaging (MRI) technique for characterizing an alcohol-induced fatty liver model established in tree shrews (Tupaia belangeri chinese). The animals were treated with 15% alcohol for two weeks instead of drinking water to induce AFL. Blood alanine aminotransferase (ALT), aspartate aminotransferase (AST), alcohol, and liver malondialdehyde (MDA) concentrations were determined, and the histopathology of the liver was checked by hematoxylin & eosin (HE) and Oil red O staining on day 0 and on the 4th, 7th and 14th days after alcohol feeding. MRI was used to trace the histopathological changes in the liver of tree shrews in real time. Compared with the control group, the levels of ALT, AST, and MDA significantly increased in the alcohol-induced group and were positively correlated with the induction time. HE and Oil red O staining revealed that a moderate fatty lesion occurred in the liver on the 4th day and that a serious AFL was successfully induced on the 14th day. MRI further confirmed the formation of AFL. MRI, as noninvasive examination technique, provides an alternative tool for accurate characterization of AFL in live subjects. It is comparable to HE or Oil red O staining for histopathological examination, but is more suitable by virtue of its high flexibility and compliance. The AFL model of tree shrews combined with MRI characterization can work as a platform for studying fatty liver diseases and medications for their treatment.

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  • Hiroya Konno, Tomomichi Ishizaka, Katsuyoshi Chiba, Kazuhiko Mori
    2020 Volume 69 Issue 1 Pages 119-126
    Published: 2020
    Released: January 29, 2020
    [Advance publication] Released: October 22, 2019
    JOURNALS OPEN ACCESS

    Measurement of the renal resistive index (RRI) is one of the standard diagnostic procedures for assessing kidney disability clinically. This method is expected to be used for the same purpose in many kinds of animals, including monkeys utilized in conventional toxicology studies. To establish a practical RRI measurement procedure in cynomolgus monkeys (Macaca fascicularis), RRI was measured by ultrasonography in the spine position in conscious and ketamine-immobilized monkeys. The RRI of conscious monkeys and ketamine-immobilized monkeys could be measured consistently without excessive abdominal or thoracic movement. Consequently, the variability of the RRI in conscious monkeys was comparable to that in ketamine-anesthetized monkeys. No sex difference in RRI was noted between the two conditions. The mean values and SD of the RRI of 48 healthy monkeys (n=24/sex) were 0.55 ± 0.07 and 0.50 ± 0.05, under conscious and ketamine-immobilized conditions, respectively. The RRI of ketamine-immobilized monkeys was significantly lower than that of conscious monkeys, correlating with the decreased blood pressure and heart rate. In a monkey model of cisplatin-induced acute renal injury, which was characterized histopathologically by minimal to mild renal tubular necrosis and regeneration, the RRI was increased beyond the cut off value (mean + 2SD, 0.68) associated with the progression of renal pathogenesis. The present results suggest that ultrasonographic measurement of the RRI in conscious monkeys would be a useful tool in conventional toxicology studies evaluating drug-induced renal injury.

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