Table of Content

25 April 2023, Volume 43 Issue 2

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Animal Models of Human Diseases

Establishment of hKDR^+/+ Humanized and Rag1^-/- Gene Knockout Double Genetically Modified Mouse Model

Susu LIU, Yong WU, Yuan CAO, Haoyang ZHAO, Shijie ZHAI, Xiaowei SUN, Linli LI, Changfa FAN

2023, 43(2):  103-111.  DOI: 10.12300/j.issn.1674-5817.2022.154

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Objective Through improving the potential of vascular endothelial growth factor receptor (VEGFR)-humanized mouse model (hKDR^+/+) with C57BL/6N background to allow the growth of different mouse tumor cell lines, to establish novel tumor-bearing mouse models which can support in vivo tumorigenesis of different mouse tumor cell lines and be used to evaluate drugs targeting VEGFR. Methods Firstly, a method to evaluate the in vivo activity of antibody targeting VEGFR based on the hKDR^+/+ humanized mouse model was established. Recombinant activating gene 1 (Rag1) knockout mice (Rag1^-/-) were established using CRISPR/Cas9 technology. Then these Rag1^-/- mice were crossed with hKDR^+/+ mice to get a double gene modified homozygous hKDR^+/+/Rag1^-/- mouse model by screening. Finally, tumor cell lines derived from different mouse strains were tested on the double gene-modified mouse model to compare the differences in tumor growth. Results Antibodies designed for VEGFR showed significant anti-tumor activity in hKDR^+/+ mice, which significantly reduced tumor volume and weight compared with the PBS group (P<0.01, P<0.05). The number of B cells and T cells in the peripheral blood of Rag1^-/- mice and hKDR^+/+/Rag1^-/- mice decreased (P<0.05, P<0.001). Tumors were observed in hKDR^+/+/Rag1^-/-, Rag1^-/-, wild-type, and hKDR^+/+ mice after 7 d of inoculation of MC38 cells derived from C57BL/6 mice. Tumors were only observed in groups of hKDR^+/+/Rag1^-/- and Rag1^-/- mice, but not in the wild-type and hKDR^+/+ mice after 10 d of inoculation with CT26 cells derived from BALB/c mice. After 3 weeks of inoculation, the tumor volume of hKDR^+/+/Rag1^-/- mice was significantly larger than that of Rag1^-/- mice (P<0.01). Conclusion Rag1 knockout mice were obtained and a novel hKDR^+/+/Rag1^-/- double genes modified mouse model was further screened. The tumor cell lines from different mouse strain origins were more prone to growth in mice with Rag1 gene deficiency. The results suggest that the reduced immune response of hKDR^+/+ humanized mice will improve the capacity of supporting the growth of mouse tumor lines in the model. As a result, more tumor-bearing mouse models may be constructed for the evaluation of drugs targeting VEGFR in this way.

Comparative Study on Different Recovery Periods of the Spermatogenic Dysfunction Mouse Model Induced by Cyclophosphamide

Jingwei MA, Gen LI, Yang YANG, Caixia ZANG, Xiuqi BAO, Dan ZHANG

2023, 43(2):  112-123.  DOI: 10.12300/j.issn.1674-5817.2022.167

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Objective To compare and evaluate the improvement degree of spermatogenic dysfunction mice at different recovery periods after cyclophosphamide modeling. Methods Forty-eight male ICR mice aged 4-5 weeks with the body weight of approximately 18-20 g were randomly divided into three control groups and three model groups, with 8 mice in each group. Each mouse of three model groups was intraperitoneally injected with 60 mg/kg cyclophosphamide continuously from the 1^st to 7^th day of the experiment, while each mouse of three control groups was intraperitoneally injected with the corresponding volume of normal saline. Then these mice were continued to be fed for another 7, 14 and 21 days after cyclophosphamide injection, respectively. A corresponding control group was set for each model group. The mice in each group were sacrificed after blood collection through orbital veins at corresponding time points. Testis, epididymis and seminal vesicle were taken and weighed, and their reproductive organ indexes were calculated. Histopathological changes of testis and epididymis were compared after HE staining.Sperm quality analysis was used to determine sperm-related indexes. Serum reproductive hormone content, testicular oxidative stress level and testicular signature enzyme activity were detected by ELISA and related kits.Results Compared with the control group, on the 7^th, 14^th and 21^st day after cyclophosphamide treatment, the testicular index of mice in the model group decreased significantly (P<0.01). The epididymis index decreased significantly on the 7^th and 14^th day, and the seminal vesicle index decreased obviously on the 7^th and 21^st day (P<0.05). And the histopathological damage of testis and epididymis of the model group gradually alleviated over time. On the 7^th and 14^th day after cyclophosphamide treatment, the sperm count of the model group declined remarkably (P<0.01), the serum testosterone (T) level reduced (P<0.05), the malonaldehyde (MDA) content of testis increased significantly (P<0.01), the content of reduced glutathione (GSH) and superoxide dismutase (SOD) decreased obviously (P<0.05),the lactic dehydrogenase (LDH) activity of testis reduced obviously (P<0.05), the gamma-glutamyl transpeptidase (γ-GT) activity increased significantly (P<0.05), the latter two of which are important testicular signature enzymes. Therein on the 7^th day after cyclophosphamide treatment, the sperm motility decreased significantly (P<0.001), the rate of sperm malformation increased obviously (P<0.05), the serum levels of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) increased notably (P<0.01). Nevertheless on the 21^st day after cyclophosphamide treatment, the sperm-related indexes, the content of serum reproductive hormone, the level of testicular oxidative stress and the activity of testicular signature enzyme did not change significantly (P＞0.05). Conclusion The reproductive toxicity in mice was more apparent on the 7^th day after intraperitoneal injection with 60 mg/kg cyclophosphamide for seven days, at which time the more desirable spermatogenic dysfunction model of mice could be established. However, with the prolongation of the recovery period, the indexes of spermatogenic dysfunction in mice gradually recovered and approached the normal level on the 21^st day after cyclophosphamide treatment.

Physiological Indexes and Histopathology Analysis of Sodium Iodate-Induced Retinitis Pigmentosa in Rats

Ying TAN, Wenping LIAO, Qilong GAO, Yong LI, Xinhui SHI, Jingkun WANG

2023, 43(2):  124-135.  DOI: 10.12300/j.issn.1674-5817.2022.149

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Objective To evaluate the effects of intraperitoneal injection of sodium iodate on the physiological indexes and retinal histopathological characteristics of SD rats comprehensively. Methods A total of 64 rats were randomly divided into negative control group and model group, half male and half female. The model group was intraperitoneally injected with 6 mg/mL sodium iodate once at the dose of 10 mL/kg and the negative control group was injected with 10 mL/kg 0.9% NaCl once. The body weight of all surviving rats was detected on the day of injection (0 day) and the 2nd, 6th, 9th, 13th, 16th, 20th, 23rd, 27th, 29th, 36th, 43rd, 50th and 57th days after injection. On the 3rd, 7th, 21st, 28th, 41st and 62nd days after injection, the rats were randomly selected (12 rats in each group on the 28th day, and 4 rats in each group at other time points, those in each group were half male and half female) for serum biochemical indexes detection. The organs were dissected and weighed, and then the main organs and tissues were stained with HE, and the eyes were stained with HE and TUNEL. Blood routine indexes were detected on the 28th and 62nd day after injection. Results After injection of sodium iodate, 88% of the rats in the model group had transient loose stools. During the observation period, the body weight of the rats increased slightly and was more obvious in male rats. On the 28th day after injection, compared with the negative control group, the red blood cell volume (RDW) of female rats and blood urea nitrogen (BUN), reticulocyte count (Retic#) and reticulocyte percentage (Retic%) of male rats in the model group increased significantly (all P＜0.05). The white blood cell (WBC), red blood cell (RBC), hemoglobin (HGB) and hematocrit (HCT) of male and female rats showed decreasing trends, but there were no significant differences between the two groups (all P >0.05). The thymus weight and coefficient of male rats in the model group were smaller than those in the negative control group except for the 7th day after injection, but there were no significant differences between the two groups (all P ＞0.05). Histopathological examination showed that the retina of the model group gradually developed from wavy changes to abnormal electrocardiogram (ECG)-like changes, with disordered arrangement of each layer, focal thinning of the outer nuclear layer, and apoptosis of the outer nuclear layer of the retina. The incidence of lesions, lesion score and the number of apoptotic cells in the model group were significantly higher than or more than those in the negative control group at the same time, and the difference between the groups on the 28th day was statistically significant (all P＜0.01). Conclusion In addition to retinal degeneration in rats, intraperitoneal injection of sodium iodate also had a certain degree of influence on serum biochemical and blood routine indexes, and also showed a slight slow growth of body weight and transient changes in fecal traits. Therefore, when using this model to evaluate drug safety, the effects of modeling reagents on animals should be paid to attention.

Overview of Studies in Animal Models of Schizophrenia

Ling HU, Zhibin HU, Yunqing HU, Yuqiang DING

2023, 43(2):  145-155.  DOI: 10.12300/j.issn.1674-5817.2022.174

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Schizophrenia (SCZ) is a highly destructive and complex psychiatric disorder illness, accompanied by a variety of positive and negative symptoms along with cognitive impairment, which brings a heavy social burden. Elucidation of the pathogenesis and therapeutic development is challenging because the complex interplay between genetic risk factors and environmental factors in essential neurodevelopmental processes. Therefore, preparing appropriate animal models can help people better understanding the neurobiological basis of SCZ and provide theoretical basis for finding new treatments. In order to provide reference for the application and improvement of SCZ animal models, this commentary reviewed several main modeling methods for animal models of SCZ, including neurodevelopmental models, drug-induced animal models, and genetic models, and the behavioral evaluation, histological analysis and possible molecular mechanisms of SCZ animal models were also outlined.

Advances in Animal Aging Models

Danyang YIN, Yi HU, Rengfei SHI

2023, 43(2):  156-162.  DOI: 10.12300/j.issn.1674-5817.2022.094

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With the increasing severity of global aging, aging-related issues have become the hotspot in the field of health. In recent years, animal aging models have been widely developed and applied, which is of great significance in the study of aging mechanism. Animals with short life span, such as Caenorhabditis Elegans and Drosophila Melanogaster, have natural advantages in the study of aging. Various rat and mouse aging models have been used in aging studies. In recent years, new animal aging models have been developed, such as the African turquoise killifish. The authors reviewed main animal models used in the study of aging, and analyzed the establishment methods, evaluation indexes, advantages and disadvantages of each model in order to provide reference for related research.

Recent Advances of Animal Models of Renal Interstitial Fibrosis

Can LAI, Lele LI, Tala HU, Yan MENG

2023, 43(2):  163-172.  DOI: 10.12300/j.issn.1674-5817.2022.171

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Renal interstitial fibrosis is a common pathway in the progression of many renal diseases. Whether it is chronic kidney disease or acute kidney injury that cannot be fully recovered, the progression process mostly enters end-stage renal failure after renal interstitial fibrosis. The animal model of renal interstitial fibrosis is an important research tool for exploring the pathogenesis of renal interstitial fibrosis and new diagnostic and treatment methods. Different animal models have their own characteristics. Researchers can establish different models based on their own experience and experimental purposes, and carry out scientific research on this basis to provide more new methods for the prevention and treatment of kidney diseases. The authors focused on several common animal models of renal interstitial fibrosis to provide the reference for related researchers, including surgical models induced by unilateral ureteral obstruction, ischemia-reperfusion injury, 5/6 nephrectomy, and microembolization; chemical models induced by cyclosporine A, adriamycin, aristolochic acid, mercuric chloride（HgCl₂）, gentamicin, cisplatin, and adenine; transgenic hybridization and kidney injury molecule 1 (KIM-1) induced transgenic modification model; composite model induced by bilateral ischemia-reperfusion injury (BIRI) combined with gentamicin, unilateral nephrectomy combined with angiotensin II (Ang II), and unilateral ischemia-reperfusion injury (UIRI) combined with pLVX-shTNC plasmid.

Research Progress of Animal Models of Stress Cardiomyopathy

Haosheng WU, Hang SU, Chao ZHU, Wenhui WANG, Shengbing WU, Shuai CUI, Meiqi ZHOU

2023, 43(2):  173-179.  DOI: 10.12300/j.issn.1674-5817.2022.102

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Stress cardiomyopathy (SC) is an acute cardiomyopathy caused by intense mental stimulation or physical stress. Currently, few interventions are effective in reducing mortality, improving prognosis, or preventing recurrence in acute or chronic stages of SC. Therefore, it is particularly important to establish a reliable and effective SC animal model to study the pathogenesis and prevention of SC. In this paper, the preparation methods and evaluation indexes of SC models at home and abroad were reviewed, including the operation details of catecholamine hormone injection method, binding method, binding combined with electroshock method, binding combined with dehydration method, vagus nerve electrical stimulation and other modeling methods, as well as evaluation indexes such as open field test, cardiac ultrasound, electrocardiogram and ELISA, striving to provide useful reference for model selection and establishment of SC research.

Research Progress on Influenza A Virus and Nervous System Disease of Human and Experimental Animals

Xiangrong DING, Shurui HUO, Jiejie DAI

2023, 43(2):  180-185.  DOI: 10.12300/j.issn.1674-5817.2022.142

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Influenza is a highly contagious disease that mainly affects the respiratory system and often leads to lung complications. Also it can cause a variety of very rare and serious neurological complications, including Guillain-Barre syndrome, transverse myelitis, meningoencephalitis and others. In recent years, neurological complications caused by influenza A virus have been reported in many countries and regions, and gradually attracted international attention. However, the pathogenesis of this complication remains unclear, and there are few related cases and animal experimental studies,and no specific treatment. Therefore, the authors summarized the study of neurological complications caused by influenza A virus in human and laboratory animals, in order to have a comprehensive understanding of the neurological diseases caused by influenza A virus.

Research Progress on Neuroprotective Effects and Mechanisms of Glucagon-like Peptide 1 Analogues in Alzheimer's Disease

Chenghan MEI, Beibei CHEN

2023, 43(2):  186-193.  DOI: 10.12300/j.issn.1674-5817.2022.136

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Glucagon-like peptide 1 (GLP-1) is a kind of incretin produced in the intestinal with multiple pharmacological effects, which can stimulate insulin secretion effectively. Various GLP-1 analogues have been widely used in the treatment of type 2 diabetes mellitus. Alzheimer's disease (AD) is closely related to type 2 diabetes mellitus, with some common pathological features, such as insulin resistance, and epidemiological studies also showed that patients with type 2 diabetes mellitus have an increased risk of developing AD. GLP-1 analogues have shown beneficial effects in both preclinical animal research and clinical trials of AD. Therefore, the authors summarized the main characteristics of GLP-1 and AD, and analyzed the mechanisms of GLP-1 in preclinical AD studies of animal models. GLP-1 readily crosses the blood-brain barrier and exerts its neuroprotective effects by binding to and activating the widely distributed GLP-1 receptors (GLP-1Rs) in the brain, affecting multiple physiological and pathological processes including glucose metabolism, neuroinflammation, mitochondrial function, and cell proliferation. Insulin resistance and inflammation are key common pathways in AD and type 2 diabetes. GLP-1 may exert its neuroprotective effects by improving mitochondrial function and glycolysis, reducing oxidative stress levels, exerting anti-inflammatory and anti-apoptotic effects, inducing neurogenesis, and inhibiting glial cell proliferation. This paper maybe provide the reference for further study of GLP-1 analogues in AD, hoping to open new therapy venues for AD patients.

Progress in Experimental Animals Abroad

Analysis on the Development Status of Laboratory Animals in Japan

Huan GOU, Xinying AN, Yujia TONG, Yan WANG, Shuang YANG

2023, 43(2):  194-204.  DOI: 10.12300/j.issn.1674-5817.2022.141

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Experimental animals have made important contributions to human medical research and life and health. It is known that the development of laboratory animal science in Japan has been relatively rapid in the past few decades, providing important support for the development of the world's experimental animal field. Therefore, it is of great significance to understand the management mode and resource storage situation of Japanese experimental animals, analyze the advantages of Japanese experimental animal development, and propose suggestions to strengthen the high-quality development of experimental animals in China. Through literature research, the authors first analyzed the management system of experimental animals in Japan, including regulations and policies, research funding management, experimental animal management, talent cultivation, and standard and normative systems. Then, the current status of experimental animal research in Japan was summarized, including experimental animal resources, major research institutions, and production enterprises. On this basis, it was found that the field of experimental animal research in Japan currently exhibits characteristics such as a complete policy system, flexible management methods, rich resource reserves, and large-scale industrial development. Finally, by comparing the existing problems in China, suggestions for the development of experimental animal technology in China are proposed: (1) drawing on the legal management method of experimental animals in Japan, strengthening and improving the legislation and management model of experimental animals in China; (2) increaseing investment in scientific research funds, playing the role of research institutions, societies and industries, and promoting the incremental construction and industrial development of experimental animal resources.

Quality Control of Laboratory Animals

Prevalence of Mouse Norovirus in Experimental Mice in Beijing

Fangni LIU, Junping LU, Yuehua KE, Changjun WANG, Jinpeng GUO

2023, 43(2):  205-212.  DOI: 10.12300/j.issn.1674-5817.2022.126

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Objective To investigate the infection of mouse norovirus (MNV) in experimental mice raised under natural conditions from 19 biological companies in Beijing. Methods The mice used in this study were randomly selected from mice produced by 19 companies, and 14 mice of each strain and batch were combined into one cage, totaling 1 396 cages of 19 544 mice. The fecal samples from BALB/c, C57BL/6, ICR, KM, and BALB/c-nude mice were collected. TaqMan probe fluorescence quantitative PCR method was used to detect MNV infection of mice with MNV-1 primer, and whether the mice were infected with MNV was determined according to cycle threshold (Ct value). The chi-square test was used to analyze the difference of positive rate among the fecal samples from the five types of mice. The Ct values of the positive samples were statistically described; the non-parametric test was used to analyze the differences in Ct values among the five types of mice. Results A total of 1 396 fecal samples were collected. The positive rates of fecal MNV detection in BALB/c, C57BL/6, ICR, KM, and BALB/c-nude mice were 17.65%, 39.33%, 10.57%, 18.32% and 27.4%, respectively. According to the chi-square test results, the positive rate of fecal in C57BL/6 mice was higher than that in BALB/c, ICR, and KM mice (all P<0.05), and the positive rate of BALB/c-nude mice was higher than that in ICR and BALB/c mice (P<0.001, P＜0.05) . The viral load of BALB/c-nude or C57BL/6 mice was generally greater than that of KM mice (P<0.05). Conclusion MNV-1 primers can be applied to the detection of MNV infection in mice. The positive rate of MNV in five types of experimental mice in Beijing ranges from 10% to 40%, among which C57BL/6 mice and BALB/c-nude mice have higher positive rates of MNV than the others.

Guidelines for Comparative Medical Research and Reporting

Explanation and Elaboration for the ARRIVE Guidelines 2.0—Reporting Animal Research and In Vivo Experiments (Ⅰ)

Jian WANG, Jin LU, Zhengwen MA, Guoyuan CHEN, Xiao LU, Yu BAI, Xiaoyu LIU, Xuancheng LU, Jing GAO, Yao LI, Wanyong Pang

2023, 43(2):  213-224.  DOI: 10.12300/j.issn.1674-5817.2023.043

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Improving the reproducibility of biomedical research results is a major challenge. Researchers reporting their research process transparently and accurately can help readers evaluate the reliability of the research results and further explore the experiment by repeating it or building upon its findings. The ARRIVE 2.0 guidelines, released in 2019 by the UK National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs), provide a checklist applicable to any in vivo animal research report. These guidelines aim to improve the standardization of experimental design, implementation, and reporting, as well as the reliability, repeatability, and clinical translatability of animal experimental results. The use of ARRIVE 2.0 guidelines not only enriches the details of animal experimental research reports, ensuring that information on animal experimental results is fully evaluated and utilized, but also enables readers to understand the content expressed by the author accurately and clearly, promoting the transparency and integrity of the fundamental research review process. At present, the ARRIVE 2.0 guidelines have been widely adopted by international biomedical journals. this article is a Chinese translation based on the best practices of international journals following the ARRIVE 2.0 guidelines in international journals, specifically for the complete interpretation of the ARRIVE 2.0 guidelines published in the PLoS Biology journal in 2020 (original text can be found at https://arriveguidelines.org). The first part of the article includes the preface and the "Key 10" section, which covers "study design" "sample size" and "inclusion and exclusion criteria". Its aim is to promote the full understanding and use of the ARRIVE 2.0 guidelines by domestic researchers, enhance the standardization of experimental animal research and reporting, and promote the high-quality development of experimental animal technology and comparative medicine research in China.