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    History, Current Status, Challenges and Opportunities of Laboratory Monkey Industry in China
    SUN Qiang
    Laboratory Animal and Comparative Medicine    2024, 44 (4): 343-356.   DOI: 10.12300/j.issn.1674-5817.2024.112
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    Laboratory animals play a crucial role in foundational scientific research and clinical medicine. Non-human primates (NHP), particularly Macaca mulatta and Macaca fascicularis, have long been highly valued due to their close resemblance to humans. After more than half a century of development, China's NHP laboratory animal industry has gradually transitioned from its early stage of rapid and unregulated growth to a mature stage of standardization and refinement. However, there has been a dramatic surge in global biopharmaceutical research in recent years, leading to a sharp increase in demand for NHP laboratory animals. This surge, coupled with the lack of long-term strategic planning among breeding enterprises, has resulted in severe aging of breeding populations and a significant decline in reproductive capabilities, further widening the supply gap. Under the dual pressures of rising demand and declining supply, the prices of NHP laboratory animals have surged. Although the cyclical downturn in the biopharmaceutical industry in recent years has lowered the demand for NHP laboratory animals to some extent, leading to significant price drops, the prices remain high. At the same time, against the backdrop of high prices, issues such as the accelerating aging of breeding populations, the lower standards for microbial quality control, insufficient genetic quality control, and blind investment in facility construction have emerged within the NHP laboratory animal industry. This report provides a comprehensive review of the history and current status of China's NHP laboratory animal industry, with a focus on laboratory monkeys. It explores the factors shaping the current industry landscape and identifies potential challenges and opportunities facing the industry. It aims to offer insights and references for the future development of China's NHP laboratory animal industry.

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    Statistical Analysis of the Leakage Situation in the Automated Watering System for Mice in Tsinghua University Laboratory Animal Resources Center
    Qianqian TANG, Xiuli ZHANG, Zai CHANG
    Laboratory Animal and Comparative Medicine    2024, 44 (1): 85-91.   DOI: 10.12300/j.issn.1674-5817.2023.132
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    Objective To calculate the leakage rate of the automated watering system in Tsinghua University Laboratory Animal Resources Center, to evaluate the safety of the system, and provide references for selection, maintenance, and management of automated watering systems in animal facilities. Methods This study investigated the automated watering system installed in South and North Barriers of Tsinghua University Laboratory Animal Resources Center (Phase II). Water leakage monitoring was conducted over two periods, one over a period of 3 years and the other over 1.5 years. The occurrence of water leakage events at the two barriers during the monitoring period was statistically analyzed, classifying the causes into four categories: mishandling by personnel, animal behavior, obstruction by foreign objects, and deformation of fittings. The total daily leakage rate due to these causes and the daily leakage rate caused by quality issues, i.e. obstruction by foreign objects and deformation of fittings were calculated. Further analysis and discussion focused on the causes of water leakage and its impact on the facilities. At the same time, the number of caretakers at the end of the monitoring period in the Phase I facility without automated watering system and the Phase II facility with automated watering system were counted. Finally the difference in the number of cages per capita under the two watering systems was calculated. Results A total of 52 water leakage incidents occurred in both areas during the monitoring period, with a total daily leakage rate of 0.000 13%. Among them, 31 were caused by personnel mishandling, accounting for approximately 60% of total leakages. Enhanced training, supervision, inspection, and effective reminder measures could reduce leakage caused by personnel mishandling. There were 2 cases of water leakages caused by animal behavior, 0 leakage due to obstruction by foreign objects, and 19 leakages due to system quality issues, with a daily leakage rate of 0.000 07%. According to the operation data of Tsinghua University Laboratory Animal Resources Center, the average number of cages managed per person in facilities equipped with the automated watering system was 908, compared to 570 cages in facilities without the automated watering system. This represents an approximate 59% increase in the number of cages managed per person with the adoption of the automated watering system. Conclusion The daily leakage rate of the automated watering system in the Tsinghua University Laboratory Animal Resources Center is significantly lower than the theoretical design rate of 0.003%, which demonstrates the system's safety and effectiveness. Additionally, the adoption of an automated watering system can signi?cantly enhance caretaking ef?ciency. While initial investments in the system are required, the subsequent increase in ef?ciency leads to a continuous decrease in labor costs, thereby reducing the total operational expenses of the facility. In the context of modernizing animal facility construction, automated watering systems are becoming an essential consideration in facility design and operation.

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    Establishment of PCR Identification Method for Pig Blood Type
    Jiaoxiang WANG, Yan WANG, Ke HU, Kaixiang XU, Taiyun WEI, Deling JIAO, Heng ZHAO, Hongye ZHAO, HongJiang WEI
    Laboratory Animal and Comparative Medicine    2023, 43 (6): 585-594.   DOI: 10.12300/j.issn.1674-5817.2023.065
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    Objective Xenotransplantation is an effective way to address the shortage of human organ donors, but it faces serious immune rejection reactions, including hyperacute rejection caused by blood type differences. Establishing a stable, convenient, and reliable method for pig blood type identification can quickly screen suitable donor pigs for xenograft research. Methods Banna miniature inbred pigs, Diannan small eared pigs, and Bama Xiang pigs were selected as the research objects. DNA was extracted from the blood, oral buccal mucosa, and fetal fibroblasts of the three strains of pigs using DNA extraction kits. The target fragment of the ABO homologous gene EAA intron 7 in pigs was amplified using PCR method. Blood agglutination reaction was used to detect hemolysis in pig anterior vena cava whole blood after adding anti A and B antibodies. Immunohistochemical method was used to detect the expression level of A antigen in pig heart, liver, spleen, lung, and kidney tissues. Immunofluorescence method was used to detect the expression level of A antigen in pig oral mucosa. By comparing the results of different methods for determining pig blood types, the stability and reliability of the PCR method were verified, and a convenient PCR based pig blood type identification method was established. Results Firstly, the blood PCR results of 69 inbred strains of Banna miniature pigs, 7 Diannan small eared pigs, and 34 Bama Xiang pigs showed 20 AO blood types, 66 AA blood types, and 24 O blood types. The PCR results of fetal fibroblasts from 47 Diannan small eared pigs showed that all 47 fetuses were O blood type. Among them, the oral mucosal PCR results of 8 gene edited cloned pigs were consistent with those of donor fetal fibroblasts, all of which were O blood type. The oral mucosal PCR results of 8 wild-type pigs (2 inbred lines of Banna miniature pigs, 4 Diannan small eared pigs, and 2 Bama Xiang pigs) were consistent with the blood PCR identification results. Then, 11 inbred lines of Banna miniature pigs, 4 Diannan small eared pigs, and 2 Bama Xiang pigs were randomly selected for blood agglutination reaction validation, and the results were consistent with the PCR identification results of both blood samples and oral mucosa samples. Moreover, immuno-histochemical analysis was performed on the heart, liver, lung, kidney, and spleen tissues of one Banna miniature pig inbred line and two Bama Xiang pigs, and the results were consistent with blood PCR identification and blood agglutination reaction results. Finally, oral mucosal samples were collected from 2 inbred strains of Banna miniature pigs and 1 Bama Xiang pig for immunofluorescence detection, and the results were consistent with the blood PCR identification results. Conclusion By collecting fetal cells and oral mucosal samples from live pigs for PCR detection, the blood type of pigs can be accurately and efficiently identified, providing a convenient method for blood type screening of xenograft donor pigs.

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    Guidelines for the Selection of Animal Models and Preclinical Drug Trials for Spontaneous Intracerebral Hemorrhage (2024 Edition)
    Laboratory Animal and Comparative Medicine    2024, 44 (1): 3-30.   DOI: 10.12300/j.issn.1674-5817.2024.001
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    Spontaneous intracerebral hemorrhage (sICH), the most prevalent and lethal subtype of stroke, is characterized by spontaneous hemorrhage in the brain parenchyma. Presently, there are no effective methods for preventing and treating sICH. The existing sICH animal models can be broadly categorized into three classes: (1) induced intracerebral hemorrhage models, including autologous blood injection model, collagenase injection model, microballoon inflation model, and hyperglycemia-induced sICH hematoma expansion model; (2) spontaneous hypertensive intracerebral hemorrhage models mainly include stroke-prone spontaneously hypertensive rats (SHRsp) and stroke-prone renovascular hypertensive rats (RHRsp); (3) gene-modified models encompassing transgenic hypertensive intracerebral hemorrhage, transgenic cerebral amyloid angiopathy, arteriovenous malformation-related, cerebral cavernous malformation-related and collagen-related genetically modified animal models for sICH. These models contribute not only to unraveling the pathogenesis of sICH and exploring preventive or therapeutic interventions, but also serve as invaluable tools for conducting preclinical drug trials to advance novel treatments. This guide comprehensively reviews sICH pathogenesis, delineates the superiority and inferiority of different species of modeling animals, explains the modeling principles and techniques for various sICH animal models, elucidates the technical details of animal model production, summarizes the pathophysiological mechanism simulated by the models and their clinical relevance, outlines the neurobehavioral evaluation methodologies for sICH animal models, compares the advantages and disadvantages of various models, and suggests their applicable research areas. Additionally, it underscores critical considerations in the design of preclinical drug trials for sICH.

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    Construction and Evaluation of End-to-side Anastomosis Model of Autologous Arteriovenous Fistula in Mice
    Xin LIU, Shaobo SHI, Cui ZHANG, Bo YANG, Chuan QU
    Laboratory Animal and Comparative Medicine    2023, 43 (6): 595-603.   DOI: 10.12300/j.issn.1674-5817.2023.093
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    Objective To establish an animal model of autologous arteriovenous fistula in mice and evaluate its effect. Methods The left external jugular vein and common carotid artery of 10 8-week-old male C57BL/6 mice were separated by end-to-side anastomosis of external jugular vein and common carotid artery after anesthesia, and the right jugular vein was exposed without suture as a control, so as to establish an animal model of internal arteriovenous fistula. Doppler ultrasound, HE and Masson staining and immunohistochemical staining were used to observe the hemodynamics, intimal hyperplasia and protein expression of smooth muscle cell proliferation in the outflow vein of the internal arteriovenous fistula and the contralateral control vein, and to evaluate the effect of model construction. Results A total of 10 mice were selected for this study, and 9 mice were successfully modeled, with a success rate of 90%. Ultrasound examinations were performed on the day of surgery, 7 and 14 days after surgery, respectively. The results showed that the flow velocity near the anastomosis was linearly correlated with the diameter of the tube. The higher the flow velocity, the larger the diameter of the tube. There was a positive correlation between peak velocity and lumen diameter (P=0.000 6, R2=0.831 7). After surgery 14 days, HE staining results showed that after autologous arteriovenous fistula molding, the average lumen area of outflow segment vein was significantly decreased (P < 0.000 1), the intima area was significantly increased (P < 0.000 1), the intimal area was significantly increased (P < 0.000 1). On the surgical side of arteriovenous fistula, collagen deposition was significantly increased, and the proportion of Masson-positive regions was significantly increased (P < 0.000 1). Immunohistochemical staining showed that the proportion of collagen 1 positive areas on the surgical side of arteriovenous fistula was significantly upregulated (P < 0.000 1), and α-smooth muscle actin (α-SMA) , proliferating cell nuclear antigen (PCNA) positive cells increased significantly (P < 0.000 1), indicating an increase in local cell proliferation level. Conclusion The established mouse autologous arteriovenous fistula model has the advantages of high success rate, good stability and low cost. The model provides a good carrier for exploring the biological mechanism of intimal hyperplasia in arteriovenous fistulas.

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    Exploration of Laboratory Animal Science Teaching Practice from Perspectives of Curriculum Ideology and Politics
    ZHAO Ya, ZHANG Caiqin, MENG Han, QIN Jing, BAI Bing, ZHAO Yong, GE Xu, SHI Changhong
    Laboratory Animal and Comparative Medicine    2023, 43 (6): 641-646.   DOI: 10.12300/j.issn.1674-5817.2023.109
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    The ideological and political content of the laboratory animal science degree course with the basic task of "cultivating morality and cultivating people" is organically integrated into the teaching system of laboratory animal science. It can have a subtle influence on students' thoughts and behaviors. Combined with the curriculum design and professional characteristics of laboratory animal science, this article discussed the ideological and political elements contained in this course, proposed the forms and methods of integrating ideological and political elements into the curriculum design in each chapter. Additionally, the typical cases and characteristic practices of the organic connection of ideological and political education in the teaching system of laboratory animal science were summarized. Practice has proved that integrating the ideological and political elements into the teaching system of laboratory animal science can enhance teacher's awareness and ability of politics, thus effectively improving the compre-hensive quality of students and enhancing the effectiveness of ideological and political education in laboratory animal science.

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    Explanation and Elaboration for the ARRIVE Guidelines 2.0—Reporting Animal Research and In Vivo Experiments (Ⅳ)
    Xiaying LI, Yonglu TIAN, Xiaoyu LIU, Xuancheng LU, Guoyuan CHEN, Xiao LU, Yu BAI, Jing GAO, Yao LI, Yusheng WEI, Wanyong PANG, Yufeng TAO
    Laboratory Animal and Comparative Medicine    2023, 43 (6): 659-668.   DOI: 10.12300/j.issn.1674-5817.2023.142
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    Improving the reproducibility of biomedical research results is a major challenge.Transparent and accurate reporting of the research process enables readers to 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 fourth part of the article includes the items 1-5 of ARRIVE 2.0 Recommended 11 section, which covers "Abstract" "Background" "Objectives" "Ethical statement" and "Housing and husbandry". 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.

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    Ginkgolide B Promotes Neural Function Recovery of Ischemic Stroke Mice by Regulating Characteristics of Brain T Cells and Their Interactions with Glial Cells
    Jia LIU, Yanrong YE, Yun SHEN, Qiying TANG, Meiqing CHEN, Kehui YI, Shaozhuang CHEN
    Laboratory Animal and Comparative Medicine    2024, 44 (2): 139-148.   DOI: 10.12300/j.issn.1674-5817.2023.121
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    Objective To investigate the regulatory effects of Ginkgolide B on the biological characteristics of brain T cells and their interactions with glial cells during the recovery phase of ischemic stroke in mice. Methods 36 adult C57BL/6 mice were randomly assigned to three groups: sham-operated group (Sham group), control group (PBS group), and Ginkgolide B treatment group (GB group). The Sham group underwent only sham surgeries, whereas the PBS and GB groups were subjected to a middle cerebral artery occlusion (MCAO) model using the filament method, followed by intranasal administration of an equivalent volume of either PBS or Ginkgolide B solution for 14 days post-injury. Neurological function changes were evaluated in all three groups using the rotarod test and a neurological scoring system. On day 15, single-cell sequencing was performed on fresh tissues from the brain injury areas, surrounding cortex, corpus callosum, and striatum of mice in the PBS and GB group to assess the biological characteristics of T cells and their subpopulations, and further explore the interactions and mechanisms among T cells, microglia, and oligodendrocytes. Results Compared with the Sham group, both PBS and GB group exhibited significant improvements in neurological scores and reduced pre-fall motor durations (P < 0.001). Compared with the PBS group, the GB group showed a downward trend in neurological scores and an upward trend in pre-fall motor durations on days 5, 10, and 15 post-ischemic brain injury, with a significant increase in pre-fall motor duration on day 15 (P < 0.05). Compared with the PBS group, the GB group exhibited a significant increase in T cell proliferative activity in the brain 15 days post brain injury (P < 0.05). The number of proliferative T cells and the levels of lipid metabolism were significantly elevated (P < 0.05), and there was a significant increase in extracellular matrix remodeling in all T cells (P < 0.05). Additionally, the interactions between T cells and both microglia and oligodendrocytes, as well as among the microglia themselves and between microglia and oligodendrocytes, were significantly enhanced in the GB group. This was primarily evident in the strengthened interactions between CD74 and macrophage migration inhibitory factor (MIF), as well as colony stimulating factor 1 receptor (CSF1R) and colony stimulating factor 1 (CSF1) (P < 0.05). However, the inflammatory levels of T cells showed no significant differences compared with the PBS group. Conclusion A mouse model of ischemic stroke can be successfully established by MCAO operation. Ginkgolide B may promote neurological recovery post-brain injury in mice by modulating the biological characteristics of T cells within the brain and their interactions with glial cells.

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    Discussion on the Operation, Maintenance and Care Modes of Laboratory Animals Facilities
    Yue HUANG, Ye DONG, Jiale SHU
    Laboratory Animal and Comparative Medicine    2024, 44 (1): 92-96.   DOI: 10.12300/j.issn.1674-5817.2023.078
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    With the rapid development of the biopharmaceutical industry in China, the laboratory animal sector has entered a stage of rapid growth, and the construction of facility operation systems has become increasingly automated and intelligent. Compared to traditional laboratory animal facilities, new facilities require a more specialized technical team for the maintenance of air supply and exhaust systems, air conditioning, automated control, and the entire barrier system. The Lingang Laboratory’s animal facilities accommodate both large and small animals for feeding and experimental purposes. The facility management team has summarized daily maintenance experiences and explored various operational and maintenance modes based on the characteristics of laboratory operation. After analyzing the advantages and disadvantages of three common modes, this paper provides new ideas for the management of these laboratory animal facilities, and offers guidance for peers in choosing the most appropriate professional maintenance mode.

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    Construction of Dmd Gene Mutant Mice and Phenotype Verification in Muscle and Immune Systems
    Min LIANG, Yang GUO, Jinjin WANG, Mengyan ZHU, Jun CHI, Yanjuan CHEN, Chengji WANG, Zhilan YU, Ruling SHEN
    Laboratory Animal and Comparative Medicine    2024, 44 (1): 42-51.   DOI: 10.12300/j.issn.1674-5817.2023.089
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    Objective The aim is to utilize CRISPR/Cas9 gene editing technology to construct Dmd gene mutant mice with a point mutation in exon 23 of the Dmd gene. Subsequently, the phenotypic changes of the mice in muscles and immune systems are analyzed and verified, providing an evaluation model for Duchenne muscular dystrophy and other related diseases. Methods Based on the sequence characteristics of exon 23 of the Dmd gene, small guide RNA (sgRNA) was designed and synthesized. Cas9 mRNA, sgRNA fragments, and oligo donor DNA were microinjected into fertilized eggs of C57BL/6J mice. After transferring the fertilized eggs to surrogate mice, F0 generation mice were born. After mating with F0 generation mice, offspring mice were obtained, and Dmd gene positive mutant (DmdMu/+) mice were obtained after genotype identification. Male hemizygous DmdMu/+(DmdMu/Y) mice were selected for phenotype validation. The body weight of live 3- and 9-month-old mice were recorded. Muscle tension was evaluated through the grid test. Hearts and semitendinosus muscles were collected, and the histopathological changes were observed using HE staining. Further, the expression of Dmd protein in muscle tissue of 9-month-old mice was analyzed by Western blotting. An acute inflammation model was established in DmdMu/Y mice using lipopolysaccharide induction. Peripheral blood from the submandibular vein was collected, and the changes in the proportion of neutrophils and monocytes were detected by flow cytometry. Results The results of genome sequencing and Western blotting confirmed the successful construction of Dmd gene point mutant mice (DmdMu/+ mice). Dmd protein expression was not detected in skeletal muscle and myocardium of DmdMu/+ mice, and it was significantly reduced compared to wild-type C57BL/6J mice (P<0.05). Compared with wild-type mice of the same background, DmdMu/Y mice at 3 and 9 months of age showed significant weight loss (P<0.01) and decreased muscle tension (P<0.05). 9-month-old DmdMu/Y mice exhibited significant pathological changes in skeletal muscle and myocardium, including widening of intermuscular space. Under normal condition, compared with wild-type mice, the proportion of neutrophils and monocytes in the peripheral blood of 3-month-old DmdMu/Y mice was significantly lower than that of wild-type mice (P<0.01). After lipopolysaccharide stimulation, the proportion of neutrophils in peripheral blood of 3-month-old DmdMu/Y mice remained significantly lower compared to that of wild-type mice (P<0.01). The proportion of neutrophils in peripheral blood of 9-month-old DmdMu/Y mice significantly decreased after lipopolysaccharide induction (P<0.01), with a trend of change observed in monocytes between groups. Conclusion The successful construction of the Dmd gene mutant mouse model has confirmed the vital function of Dmd gene in maintaining normal muscle tissue morphology and muscle tone. It preliminarily indicated that Dmd gene deletion could significantly reduce the proportion of neutrophils in peripheral blood, offering a new perspective for the study of immune system alterations in Duchenne muscular dystrophy patients.

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    Interpretation and Elaboration for the ARRIVE Guidelines 2.0—Animal Research: Reporting In Vivo Experiments (V)
    Zhengwen MA, Xiaying LI, Xiaoyu LIU, Yao LI, Jian WANG, Jin LU, Guoyuan CHEN, Xiao LU, Yu BAI, Xuancheng LU, Yonggang LIU, Wanyong PANG, Yufeng TAO
    Laboratory Animal and Comparative Medicine    2024, 44 (1): 105-114.   DOI: 10.12300/j.issn.1674-5817.2023.146
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    Improving the reproducibility of biomedical research results is a major challenge. Transparent and accurate reporting of the research process enables readers to 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 that is applicable to any in vivo animal research report. These guidelines aim to improve the standardization of experimental design, implementation, and reporting, as well as enhance the reliability, repeatability, and clinical translation of animal experimental results. The use of the 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 completeness of the fundamental research review process. At present, the ARRIVE 2.0 guidelines have been widely adopted by international biomedical journals. This article is based on the best practices following the ARRIVE 2.0 guidelines in international journals, and it interprets, explains, and elaborates in Chinese the fifth part of the comprehensive version of the ARRIVE 2.0 guidelines published in PLoS Biology in 2020 (the original text can be found at https://arriveguidelines.org ). This section includes the items 6-11 of Recommended 11 section, covering "Animal Care and Monitoring", "Interpretation/Scientific Implications", "Generalisability/Translation", "Protocol Registration", "Data Access" and "Declaration of Interests". Its aim is to promote a comprehensive understanding and use of the ARRIVE 2.0 guidelines among domestic researchers, to enhance the standardization of experimental animal research and reporting, and to promote high-quality development of experimental animal sciences and comparative medicine research in China.

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    Application of Allograft Endometriosis Rat Model in Pharmaco-dynamic Evaluation of GnRH Agonists
    Ruihua ZHONG, Guoting LI, Wenjie YANG, Xiangjie GUO, Jieyun ZHOU, Yingyi HU, Qicheng NI, Ye YANG, Min ZHANG, Yan ZHU
    Laboratory Animal and Comparative Medicine    2024, 44 (2): 127-138.   DOI: 10.12300/j.issn.1674-5817.2023.150
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    Objective To establish an allogeneic rat model of endometriosis and to evaluate the effects of gonadotropin-releasing hormone (GnRH) agonist GenSci006 on experimental rat endometriosis. Methods Endometrium from SPF grade donor female SD rats were transplanted onto the abdominal wall of recipient female rats to construct an allogeneic endometriosis model. The rats undergoing sham surgery were divided into the sham group. Three weeks later, the length, width and height of the ectopic endometrium were measured, and the volume of the endometrium (V1) was calculated before drug administration. The modeling rats were randomly divided into four groups: model group, triptorelin group (0.25 mg/kg), GenSci006-1 group (0.125 mg/kg) and GenSci006-2 group (0.25 mg/kg). Each group had 16 rats and received a single dose of the corresponding drug. The sham group and model group were administered an equal volume of solvent. Three weeks after administration, ectopic endometrium was measured to calculate the volume V2 and inhibition rate. The effect of GenSci006 on rat uterus and ovarian tissues was assessed by comparing organ coefficients and changes in pathological sections. Enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of serum estradiol (E2), progesterone (P4), follicle stimulating hormone (FSH), and luteinizing hormone (LH). Real-time fluorescent quantitative PCR was used to detect the expression of GnRH receptor (GnRHR) mRNA in the hypothalamus and pituitary. Western blot was used to detect the expression of estradiol receptor alpha (ERα), beta (ERβ) and progesterone receptor (PR) in ectopic endometrium. Results Three weeks after administration, compared with the model group, the body weight of rats in the triptorelin and GenSci006-2 groups significantly increased (P < 0.05), while the volume of ectopic endometrium significantly decreased (P < 0.05). Compared with the sham group, the model group showed no significant changes in uterine and ovarian organ coefficients or endometrial thickness (P > 0.05). Compared with the model group, the uterine organ coefficients and endometrial thickness were significantly reduced in the triptorelin and GenSci006-2 groups (P < 0.05). Compared with the sham group, the serum levels of E2, P4, FSH and LH in the model group showed no significant changes (P > 0.05). Compared with the model group, the ovarian organ coefficient and serum P4 levels of rats in the Triptorelin, GenSci006-1, and GenSci006-2 groups were significantly reduced (P < 0.05), while the serum LH levels of rats in the GenSci006-1 group were significantly increased (P < 0.05). However, there were no significant changes in serum E2 and FSH levels in each group (P > 0.05). Compared with the model group, the expression levels of GnRHR mRNA in the pituitary tissue of rats in the triptorelin and GenSci006-2 groups were significantly downregulated (P < 0.05), with no significantly changes in the hypothalamus (P > 0.05). There were no significant changes in the expression level of GnRHR mRNA in the hypothalamus or the protein levels of ERα, ERβ and PR in the ectopic endometrial tissue in any group (P > 0.05). Conclusion The allogeneic endometriosis rat model is a suitable animal model for screening and evaluating drugs for treating endometriosis. The volume of ectopic endometrium, inhibition rate, uterine and ovarian organ coefficients, and serum E2 levels may serve as indicators for detecting drug efficacy.

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    Study on the Antibody Production Efficiency in Modified Big-BALB/c Mice
    Dan WANG, Xiaolu ZHANG, Yan WANG, Bo FU, Wendong WANG, Jing LIU, Suyin ZHANG, Yihe WU, Deguo WU, Xiaoyan DU, Dawei ZHAN, Xiulin ZHANG, Changlong LI
    Laboratory Animal and Comparative Medicine    2023, 43 (6): 612-618.   DOI: 10.12300/j.issn.1674-5817.2023.035
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    Objective To compare the preparation efficiency of mouse pox and mouse hepatitis antibodies between two substrains of BALB/c and Big-BALB/c (B-BALB/c) mice, and to provide a theoretical basis and reference for the selection of laboratory animals in the preparation of monoclonal antibodies induced in vivo through hybridoma. Methods Individuals weighing more than 5% of the weight of normal animals at 4 weeks of age (the criterion for late selection is more than 10%) were selected from a population of conventionally bred BALB/c mice and bred individually, and a subline of B-BALB/c mice was prepared after 10 generations of selection. A total of 40 BALB/c mice and 40 B-BALB/c mice aged 10 to 11 weeks, half male and half female, were selected and inoculated with the mousepox monoclonal antibody hybridoma cell line G23 or the murine hepatitis monoclonal antibody hybridoma cell line Y15 pre-treated with liquid paraffin, respectively. Mice ascites containing monoclonal antibodies were obtained by in vivo induction. The antibody titer was tested by indirect ELISA. The mice were grouped based on the sub-strains, gender and inoculation type of hybridoma to analyze the ascites production, antibody titer and antibody production, and to evaluate the antibody preparation efficiency of the two BALB/c mouse sub-strains. Results After 10 generations of breeding, the body weight of 10-week-old male and female B-BALB/c mice increased by 22.3% and 12.8%, respectively, compared with BALB/c mice of the same age. Compared with BALB/c mice, B-BALB/c mice had better tolerance and adaptation to secondary ascites collection. Compared with BALB/c mice, the ascites production and antibody titer during the preparation of antibodies in B-BALB/c mice were significantly increased, especially in the hybridoma cell line G23 vaccination group (both P<0.000 1) . After inoculation with the hybridoma cell lines G23 or Y15, the average antibody production of B-BALB/c mice (14.99×104 U and 33.22×104 U) was higher than that of BALB/c mice (5.33×104 U and 19.31×104 U) (both P<0.01). After inoculation with hybridoma cell line G23, the average antibody production per unit body weight of B-BALB/c mice (0.55×104 U/g) was higher than that of BALB/c mice (0.23×104 U/g) (P<0.000 1). And the antibody production per unit body weight of female B-BALB/c or BALB/c mice was higher than that of male B-BALB/c or BALB/c mice (both P<0.01). Conclusion B-BALB/c mice can be used as an alternative to BALB/c mice in the in vivo induction of monoclonal antibody preparation, which can achieve the purpose of reducing the number of experimental animals used, lowering the labor cost, and improving the efficiency of antibody preparation.

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    Preparation Methods and Evaluation Criteria Analysis of Animal Models for Perimenopausal Syndrome
    Tianwei LIANG, Yasheng DENG, Hui HUANG, Na RONG, Xin LIU, Yujie WANG, Jiang LIN
    Laboratory Animal and Comparative Medicine    2024, 44 (1): 74-84.   DOI: 10.12300/j.issn.1674-5817.2023.062
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    Objective To comprehensively analyze the reported preparation methods for animal models of perimenopausal syndrome (PS), to compare the advantages and disadvantages of various preparation elements and detection indexes, so as to provide useful references for the optimization of the relevant animal models as well as the standardization of their application in the efficacy evaluation of new drugs. Methods In this paper, literature research methods were applied using "perimenopausal syndrome" as the subject term. The publication period of the literature was limited to January 2016 to February 2023. Relevant literature on the preparation of PS animal models was retrieved from databases such as China National Knowledge Infrastructure, Wanfang database, and PubMed. After screening the experimental literature that met the inclusion and exclusion criteria, detailed information on experimental animal strains, modeling methods, duration of drug administration, positive drugs, detection indexes and other relevant information were collected. After the above information was standardized, the PS animal model database was established using Excel 2010 software. The model preparation elements and evaluation indexes were summarized systematically, and the statistical results were processed and analyzed using Excel 2010 software. Results A total of 247 articles were screened. SD rats (164 times, 65.86%) and Wistar rats (35 times, 14.06%) were often used to prepare PS animal models. Bilateral ovariectomy (139 times, 53.87%) and natural aging (43 times, 16.80%) were chosen as modeling methods. The ages of rats used for modeling ranged from 7 weeks to 18 months, with 3-month-old rats (22 times, 21.78%) being the most common. The detection indexes were comprehensively evaluated from multiple perspectives, including serum biochemistry, vaginal exfoliated cell smear, histomorphology, general observation, behavioral observation, and organ tissue protein immunoblotting. Western medical evaluation indexes were commonly used to test the successful preparation of models, with vaginal exfoliated cell smears being the most frequently used method (125 times, 85.04%). A model was considered successfully prepared when estrous cycle disorder or irregularity was observed. Some literature also determined modeling success by detecting a significant decrease in serum estradiol levels (5 times, 3.04%). Traditional Chinese medicine (TCM) syndrome evaluation often used a combination of Chinese and Western medical evaluation indexes for comprehensive evaluation, with researchers determining the TCM syndrome through vaginal exfoliated cell smears supplemented by general observation (3 times, 2.04%). Conclusion There are many methods for preparing PS animal models, but there are still significant differences in the selection of animal species, age, criteria for successful modeling, and TCM syndrome evaluation in the related literature.

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    Construction and Evaluation of a Mouse Model with Intestinal Injury by Acute Hypoxic Stress in Plateau
    Jianhua ZHENG, Yunzhi FA, Qiaoyan DONG, Yefeng QIU, Jingqing CHEN
    Laboratory Animal and Comparative Medicine    2024, 44 (1): 31-41.   DOI: 10.12300/j.issn.1674-5817.2023.118
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    Objective By simulating acute hypoxic conditions, an experimental model of intestinal stress injury in plateau mice was established to explore the pathogenic mechanism of acute gastrointestinal diseases in plateau, and to lay foundation for preventive and therapeutic measures. Methods Thirty-six SPF-grade adult male BALB/c mice were randomly divided into four groups: normoxic 24 h, normoxic 72 h, hypoxic 24 h, and hypoxic 72 h, based on body weight using a randomized numerical table method, with nine mice in each group. Mice in the normoxic group were kept in a conventional barrier environment, while those in the hypoxic group were placed in a hypoxic chamber within the barrier environment with oxygen concentration set at 10% to simulate plateau conditions. They were subjected to stress for 24 h and 72 h, respectively, in order to establish a model of intestinal injury induced by acute hypoxia. After modeling, the mice were weighed, anesthetized with 1% pentobarbital sodium, and then euthanized by cervical dislocation. Duodenal and colonic tissues were collected. Histopathological morphology of intestinal tissues was observed after HE staining. Western blotting and immunohistochemistry were used to detect the expression levels of tight junction-related proteins in intestinal tissues. Real-time fluorescence quantitative PCR was performed to measure the expression levels of inflammatory cytokines and chemokines. TUNEL staining was used to assess apoptotic activity of intestinal epithelial cells, thus evaluating intestinal injury-related phenotypes in this model. Results Compared with the normoxic groups, mice in the 24 h and 72 h hypoxia groups showed weight loss, shortened duodenal villi, abnormal crypt structure, and decreased villus/crypt ratio. The colonic mucosa was infiltrated with inflammatory cells and irregular crypt structure. Expression levels of Occludin and zonula occludens-1 (ZO-1) were significantly decreased in duodenal and colonic tissues of mice in the 24 h and 72 h hypoxia groups (P<0.05). The expression of pro-apoptotic protein Bax was significantly up-regulated while expression of anti-apoptotic protein Bcl-2 was significantly down-regulated in duodenal tissues (P<0.05). Apoptotic activity of intestinal epithelial cells was significantly enhanced (P<0.05). In addition, interleukin (IL)-1β, IL-6, monocyte chemoattractant protein-1 (MCP-1), and tumor necrosis factor-α (TNF-α) mRNA levels were significantly increased in duodenal tissues after 24 and 72 h of hypoxic stress(P<0.05). After 24 h of hypoxic stress, there was no significant change in the expression levels of inflammatory cytokines in colon tissues (P>0.05), but after 72 h, the expression levels of pro-inflammatory factors IL-1β, TNF-α, IL-6, MCP-1, and anti-inflammatory factor IL-10 mRNAs significantly increased in colon tissues of mice (P<0.05). Conclusion The usage of a hypoxia chamber to simulate an acute hypoxic environment in plateau can lead to abnormal intestinal tissue structure, intestinal barrier dysfunction, and induce intestinal epithelial cell apoptosis, triggering an intestinal inflammatory response in stress mice. These findings indicate the successful construction of a mouse model for an acute hypoxic stress-induced intestinal injury.

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    Transcriptome Data and Comparative Medical Analysis of COVID-19 Virus Infection
    Tingting FENG, Yitong LI, Yue WU, Jue WANG, Qi KONG
    Laboratory Animal and Comparative Medicine    2024, 44 (1): 62-73.   DOI: 10.12300/j.issn.1674-5817.2023.079
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    Objective To provide more basic information of comparative medicine for the study of biological changes and pathogenesis of COVID-19 by systematical sorting and analyzing the transcriptome data. Methods Following a retrieval strategy, using COVID-19 and SARS-CoV-2 as key words, transcriptome datasets related to COVID-19 from January 2020 to May 2023 were collected from GEO, ArrayExpress and GEN Transcriptome databases. The composition, distribution, and research application of COVID-19 transcriptome data resources were analyzed. Data distribution was visually displayed and correlation analysis was performed. The research applications and limitations of existing COVID-19 transcriptome data were analyzed from the perspectives of clinical medicine and comparative medicine, focusing on clinical-related molecular mechanisms, biomarkers and related immune responses, treatment intervention strategies, etc. The research value and application prospects were discussed. Results A total of 975 sets of COVID-19 transcriptome data were included. Among three databases, datasets from humans accounted for the highest proportion, namely 71.9%, 77.9%, and 90%, respectively. Species other than humans, such as mice, were the main sources of data, with the respiratory and nervous systems having the highest distribution of data. Twenty-seven datasets were associated with clinical significance. Analysis revealed that respiratory tract injury and other related molecular mechanisms were obtained through transcriptome data mining. Biomarkers such as cfDNA could be used as therapeutic targets. The severity of COVID-19 infection was associated with cell changes and disorders represented by M1 macrophages. Comparative medical analysis showed that mice, hamsters, and other animals were susceptible to SARS-CoV-2. Rhesus monkeys and cynomolgus monkeys exhibited infection characteristics highly similar to human. Apart from respiratory symptoms, hamsters also exhibited digestive system symptoms. SARS-CoV-2 can replicate in the respiratory organs of various susceptible animals, the intestines of ferrets and the ears of minks, resulting in interstitial pneumonia, diffuse lung injury and other pathological changes of varying degrees. Based on the differences in immune responses, hamsters can be used for neutralizing antibody reaction research. Conclusion Currently there is a wealth of COVID-19 transcriptome data, but there is a lack of comparative transcriptome research. Transcriptomics can be combined with comparative medicine to further explore the differences in comparative medicine of COVID-19.

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    Construction and Verification of Quality Evaluation Indicator System for Extracorporeal Membrane Oxygenation Animal Experimental Platform
    Shuo WANG, Yunhui LÜ, Xiaokang WANG, Zhenhao ZHANG, Yongchun CUI
    Laboratory Animal and Comparative Medicine    2023, 43 (6): 604-611.   DOI: 10.12300/j.issn.1674-5817.2023.107
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    Objective To establish a standardized and professional service quality evaluation index system for extracorporeal membrane oxygenation (ECMO) animal experimental platform. Methods The literature research and expert consultation were used to establish a factor set for the quality evaluation of ECMO animal experimental platform. Then, experts used the 1/9-9 scale method to compare and score pair-two indicators. Based on the principles of fuzzy analytic hierarchy process and expert scoring results, the ECMO animal experimental platform quality evaluation system was constructed. In order to verify the actual efficacy of this system, a case study was carried out on the ECMO animal experiment platform of FW Animal Experimental Center (FAEC) laboratory. Results A total of 10 experts were included in this study, the questionnaire recovery rate was 100%, the judgment coefficient (Ca) and familiarity coefficient (Cs) were both greater than 0.50, the expert authority was high (Cr>0.80), the validity test was P<0.01, and the coordination was good. The quality evaluation system of ECMO animal experiment platform includes two levels. There are 4 first-level indicators, with professionalism, safety, functionality, and stability ranked from high to low in terms of their weights. There are 15 second-level indicators, and the top 5 weights are personnel's technical expertise, attractiveness of hardware facility, auditability of data, confidentiality capabilities of data, and professionalism in service process. To facilitate the popularization and application of the system, this study also proposed a "star" system to represent the evaluation results of an ECMO animal experimental platform quality. The quality evaluation system established in this study was used to evaluate the FAEC laboratory as a case study, and the evaluation result was five-star. The actual potency value of FAEC laboratory was 0.910, reaching the five-star level, but the average actual appraisal values of "service continuity" and "sufficiency of project completion" were lower than 0.80, which needs to be improved. Conclusion A standardized and professional ECMO animal experimental platform quality evaluation system was established in this study, which would provide a measurable basis for the demander to select the supplier and a method for the supplier to complete the animal experiment of ECMO research and development with high quality.

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    Visual Analysis of Animal Experiments on Traditional Chinese Medicine (TCM) Nursing Technology Based on VOSviewer
    Jinhuan MIAO, Xia XU, Lu ZHOU, Haiyan CHENG, Yan HE
    Laboratory Animal and Comparative Medicine    2023, 43 (6): 626-635.   DOI: 10.12300/j.issn.1674-5817.2023.037
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    Objective Through the visual analysis of animal experimental literature on Traditional Chinese Medicine (TCM) nursing technology, the relevant research hot spots were condensed and the research trends of relevant animal models were discussed, to provide reference for subsequent research. Methods The literature related to animal experiments on Chinese medicine nursing technology was retrieved from Wan Fang database, Chinese Science and Technology Journal Database (VIP), China National Knowledge Infrastructure Engineering Database (CNKI), China Biomedical Literature Database (CBM), PubMed and Web of Science until June 30, 2022. Visual analysis was performed using VOSviewer 1.6.17 software. Results A total of 1 864 articles in Chinese and 126 articles in English were included, with the number of annual publications increasing year by year. The relevant literature involved 18 TCM nursing techniques, with the largest number (426) involving massage having the highest number of articles. It involved 4 496 authors, out of whom 358 were core authors accounting for 7.9% of all authors, and had a total count of 3 706 keywords forming 7 clusters. The research hotspots mainly included massage treatment of inflammatory diseases and analgesic effect, acupoint injection treatment of allergic rhinitis and myocardial ischemia, acupoint application treatment of asthma-related respiratory diseases, and moxibustion treatment of inflammatory diseases. The study of the mechanism of abdominal massage on insulin resistance is the latest research topic among them. Conclusion In recent years, the animal model of abdominal massage has gained increasing popularity in animal experiments of TCM nursing techniques. However, the establishment and application of animal models related to Chinese medicine soaking and Chinese medicine hot ironing have not yet received attention. This area can be explored in the future to further improve the animal experimental research on Chinese medicine nursing technology.

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    Advantages of Ciona intestinalis as a Model Organism and Its Applications
    Ruiqi LI, Han DUAN, Luo GAN, Yuan ZHENG, Wen YANG
    Laboratory Animal and Comparative Medicine    2024, 44 (2): 162-179.   DOI: 10.12300/j.issn.1674-5817.2023.159
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    With the continuous development of experimental biology, the limitations of commonly utilized model organisms are becoming increasingly apparent. Discrepancies between research conducted on laboratory animals and humans significantly impede the translational application of findings derived from animal experiments. This review introduces ascidian Ciona intestinalis as a novel model organism, an invertebrate that is evolutionarily closest to vertebrates and is a sister group to vertebrates. The review summarizes recent research progress on Ciona intestinalis in various fields to illustrate the significant advantages and promising application prospects of it as a model organism. The research progress outlined in the review mainly encompasses: (1) The whole-genome sequencing of Ciona intestinalis has been determined and numerous related databases have been established. Various embryonic gene editing technologies have been successfully applied, making it an animal model easy to manipulate genetically and study the functions and interactions of target genes visually. (2) In the field of neurobiology, Ciona intestinalis boasts a central nervous system structure similar to that of vertebrates and possesses numerous homologous neuropeptides and hormone molecules. These features grant it an edge in exploring the mechanisms and functional evolution of endocrine and neuroendocrine-related molecules. Additionally, the sensitivity and habituation of its larvae to light stimulation provide an avenue for exploring mechanisms related to behavioral plasticity. (3) In the field of immunology, Ciona intestinalis possesses a mature innate immune system and has evolved precursor genes to the adaptive immune system, with a relatively simple coding of immune-related genes. These features make it an exemplary model organism for immunological studies. (4) In the field of developmental biology, many studies have focused on the notochord development process in Ciona intestinalis and the regulatory mechanisms of gene expression within it, indicating common evolutionary developmental strategies among chordates. Additionally, insights into its heart development also significantly enhance our comprehension on the genetic network of human heart development. (5) In medical research, the ability of Ciona intestinalis to regenerate its neural complex and siphon, as well as the resilience of its heart to recover contractile function from substantial damage, renders it a valuable animal model for the study of regeneration and heart injury. It also has unique advantages as a research model for Alzheimer's disease and new drug development. Furthermore, its brief five-month lifespan facilitates the observation and recording of the entire aging process and the exploration of the effects of various factors on aging. In summary, this review aims to demonstrate that Ciona intestinalis stands out as a model organism with unique attributes and is expected to play a significant role in a wider range of scientific research areas.

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    Research on the Virulence Identification and Preservation Methods of Desert-type Leishmania donovani Strains
    Lifu LIAO, Yun LUO, Shen SHI, Yimei XU
    Laboratory Animal and Comparative Medicine    2023, 43 (6): 619-625.   DOI: 10.12300/j.issn.1674-5817.2023.034
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    Objective To determine the virulence of desert-type Leishmania donovani strains through animal infection experiments and to explore preservation methods for maintaining their pathogenicity. Methods The isolated strain was cultured in vitro for 7, 30, 36, 44, 60, 90, and 150 days, respectively, and inoculated into Lagurus lagurus (L.lagurus) with the dose of 2.6×105 per animal by intraperitoneal injection. The spleen coefficient, infection rate, and antibody positive rate of the inoculated animals were detected at day 60 after infection. The desert-type Leishmania donovani strain was further inoculated with Cricetulus migratorius (C.migratorius) and L. lagurus, respectively, for passaging and preservation. The survival time of two kinds of animals and pathogenicity change of the stain in their bodies were compared. Results After inoculation of desert-type Leishmania donovani strains cultured in vitro for 7-150 days, the spleen coefficient of inoculated L.lagurus gradually increased from 1% on day 7 to 2.2% on day 30, which was more than 10 times of the normal spleen coefficient. Additionally, on day 60, the spleen coefficient remained 3 times higher than the normal value. The infection rate and antibody positive rate decreased from 80% on day 7 to 0% on day 60. At 90 days, there were no significant differences between the infected groups and the control group, and all the observed indexes were within the normal range. The survival time of L.lagurus infected with the in vivo passage strain ranged from 1 to 13 months, and half of the infected individuals died within 4 months. In contrast, C.migratorius had a survival time ranging from 5 to 31 months, and half of the infected individuals died within an average of 13.7 months. There was a significant difference in the average time of death between the two groups (t=0.000 1, P<0.001), but no significant difference in spleen coefficient (t=0.990, P>0.05). This strain exhibited equal virulence in both animals and remained virulent for up to 4 years after continuous passage. Conclusion With the prolonged culture time, the virulence of the strain decreases gradually. At 90 d, it has no pathogenicity to L. lagurus. Long-term in vitro culture fails to preserve it's pathogenicity to L.lagurus. Only in vivo inoculation can maintain the virulence of this strain.

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    Rescue Technology and Its Application of Endangered Gene-Edited Mice
    Yongqiang NIE, Zhaoxia WANG
    Laboratory Animal and Comparative Medicine    2023, 43 (6): 636-640.   DOI: 10.12300/j.issn.1674-5817.2023.080
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    Gene-edited mice are the most ideal laboratory animals for studying human gene functions, exploring disease mechanisms, and developing new drugs. Strain resulting from low fertility, aging, illness, etc. can cause irreversible losses to scientific research, so strain rescues of genetically engineering mice require different measures accordingly. Meanwhile, cost control is another key point when a specific technology is applied. First of all, when the only remaining gene-edited mouse in reproductive age suddenly dies, the dead male mouse can be rescued by in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI), while the female mouse can be saved by ovarian transplantation, etc. Secondly, due to aging or diseases, mice can be saved through IVF-embryo transfer (ET) and unilateral epididymal tail assisted reproduction. Thirdly, round sperm injection (ROSI) and ovarian transplantation can be used to save endangered mice before sexual maturity with poor life status. This paper reviews rescue techniques of common endangered mice and their applications, which provides a reference for relevant practitioners to better maintain gene-edited mouse strains.

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    Pathologic Diagnosis of a Pituicytoma in a Han-Wistar Rat
    Minbo HOU, Tiantian CUI, Naying SU, Miaomiao ZHANG, Yongmin JIAO, Jianyan YAN, Xijie WANG, Ohira TOKO
    Laboratory Animal and Comparative Medicine    2023, 43 (6): 654-658.   DOI: 10.12300/j.issn.1674-5817.2023.064
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    A case of pituicytoma was observed in a Han-Wistar rat from the control group of a 2-year carcinogenicity study. No obvious abnormality were found in clinical observation and necropsy. Hematoxylin and Eosin (HE) staining results showed that nodular hyperplasia in the pars nervosa of the pituitary, which was well demarcated and compressed the adjacent normal tissue. The tumor cells were similar to the glial cells with round or oval nuclei, cytoplasm rich in eosinophilic or vacuole. The tumor cells differentiated well, with no obvious cell pleomorphism and visible mitotic figures. Some tumor cells were arranged in a pseudorosette formation. Immunohistochemical staining (IHC) analysis confirmed positive expression of Glial fibrillary acidic protein (GFAP) and S-100 protein. The tumor was diagnosed as the spontaneous benign pituicytoma combining the HE and IHC staining results.

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    Investigation on Current Biosafety Management Status in Laboratory Animal Institutions in Hunan Province
    Meitong LIU, Zhang CHEN, Zhaoqiang ZHANG, Di FAN, Zhan HU, Hailing MA
    Laboratory Animal and Comparative Medicine    2024, 44 (2): 202-208.   DOI: 10.12300/j.issn.1674-5817.2023.129
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    Objective This study aimed to investigate the current status of biosafety management in laboratory animal institutions in Hunan Province, identify management shortcomings, and provide targeted recommendations for improvement. Methods A questionnaire survey was conducted in July 2023 among 40 laboratory animal institutions in Hunan Province, covering topics such as the establishment of laboratory management system and its overall operation status, emergency management for incidents, medical waste disposal, animal transportation, personnel practitioner management, environmental disinfection, and equipment maintenance. The awareness of biosafety, animal bite incidents, and vaccination history was surveyed from at least one animal feeder or experimental operation practitioner from each institution, totaling 50 practitioners. Through literature review and comparative analysis, this paper analyzed the current status of biosafety management in production, transportation, and use of laboratory animals in Hunan Province from aspects such as biosafety management systems, personnel training, disinfection of environmental facilities and waste disposal, and provided recommendations. Results Among the 40 laboratory animal institutions in Hunan Province,39 institutions (97.5%) had established laboratory management systems, 37 (92.5%) had emergency response plans for incidents of to transport laboratory animals, 15 (37.5%) had complete records of emergency drills for laboratory animal biosafety, and 39 (97.5%) had established protocols for carcass and waste management. Moreover, 26 institutions (65.0%) had laboratory biosafety risk assessment reports. 4 out of 8 (50.0%) production units had emergency plans for animal transportation, with 7 (87.5%) using special vehicles to transport laboratory animals. 2 (4.0%) of the 50 practitioners were unaware of emergency response plans, 6 participants (12.0%) did not consistently wear personal protective equipment, and 38 participants (76.0%) had been bitten or scratched by laboratory animals. The results of the survey showed that emergency management, transportation management and personnel management of laboratory animal incidents still need improvement in Hunan Province. Relevant functional departments should strengthen supervision and management efforts, establish local regulations for the transportation management of laboratory animals, ensure strict compliance with relevant documents, conduct emergency drills in all units, and strengthen training for practitioners. Conclusion While daily management practices in the 40 laboratory animal institutions in Hunan Province are generally satisfactory, there remains a need for further improvement in emergency management, transportation management, personnel management, etc. Gradually enhancing the level of laboratory animal safety management is essential to collectively safeguard public health safety.

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    Integrative Analysis of Omics Data in Animal Models of Coronavirus Infection
    WU Yue, LI Lu, ZHANG Yang, WANG Jue, FENG Tingting, LI Yitong, WANG Kai, KONG Qi
    Laboratory Animal and Comparative Medicine    2024, 44 (4): 357-373.   DOI: 10.12300/j.issn.1674-5817.2024.008
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    Objective This study analyzes the omics data resources in human-infecting coronavirus animal models collected from various public databases, focusing on data distribution, dataset quantity, data types, species, strains, and research content. It aims to enhance our understanding of biological characteristics and pathogenic mechanisms of coronaviruses, thereby providing a solid foundation for devising effective therapeutic strategies and preventive measures. Methods Query strategies, including specific virus names, time ranges, and inclusion and exclusion criteria, were defined to retrieve data from major public omics databases such as GEO and ArrayExpress. Secondary filtering was performed based on different field types to obtain a more accurate data list. An omics data text database was established for bibliometric analysis. Co-occurrence networks were constructed for the analysis of the correlation strengths between different research themes, technical methods, and involved species. The cell types, organs, and biological pathways involved in studies were examined to further elucidate the pathogenic interplay between pathogens and hosts. Results About twenty public databases containing coronavirus-related omics data were identified, with a primary focus on novel coronavirus infection. Commonly used species include humans, mice, hamsters, and monkeys, while the commonly used virus strains are Wuhan-Hu-1 and USA-WA1/2020. Lung tissues are primarily used in animal models such as mice, macaques, and ferrets, while airway epithelial cells and Calu-3 cells are predominantly employed in human-related studies. Expression profiling data indicate that gene pathways involved in inflammation, cytokine response, complement pathway, cell damage, proliferation, and differentiation are significantly upregulated after infection. Proteomics studies reveal significant changes in phosphoproteome, ubiquitinome, and total proteome of patient samples at different infection stages. Specific protein categories, including viral receptors and proteases, transcription factors, cytokines, proteins associated with coagulation system, angiogenesis-related proteins, and fibrosis markers, show alterations after coronavirus infection. In addition, metabolomics data suggest that phosphocholine, phosphoethanolamine, arachidonic acid, and oleic acid could serve as potential metabolic markers. Epigenomics research indicates m6A methylation plays a role in SARS-CoV-2 replication, infection, and transmission, affecting host cell-virus interactions. Among these, N, S, and non-structural proteins 2 and 3 exhibit the most significant ubiquitination. Trends in microbiomics research suggest that microbial communities in the gut and wastewater are emerging as new research focuses. Conclusion The data types of coronavirus omics are diverse, with a wide variety of models and cell types used. The selection of species and technical methods for modelling varies based on the characteristics of different viruses. Multi-omics data from animal models of coronavirus infection can reveal key interactions between hosts and pathogens, identifying biomarkers and potential therapeutic targets, and provide valuable information for a deeper understanding of biological characteristics and infection mechanisms of coronaviruses.

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    Construction and Evaluation of Theranostic Near-infrared Fluorescent Probe for Targeting Inflammatory Brain Edema
    Jing QIN, Yong ZHAO, Caiqin ZHANG, Bing BAI, Changhong SHI
    Laboratory Animal and Comparative Medicine    2024, 44 (3): 243-250.   DOI: 10.12300/j.issn.1674-5817.2023.166
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    Objective A novel compound based on near-infrared fluorescence (NIRF) probe was prepared to achieve dynamic monitoring of an inflammatory brain edema model in mice and real-time evaluation of therapeutic effects through in vivo imaging. Methods The NIRF probe IR-783 was chemically linked with clinical brain edema therapeutic drug furosemide (FSM) to obtain the new compound, IR-783-FSM. The ultraviolet fluorescence properties of the compound were evaluated using an ultraviolet spectrophotometer. The uptake of the compound by mouse macrophage cells RAW 264.7 was detected with in vitro cellular experiments. Its cytotoxicity was evaluated through CCK8 assays. A brain edema model was established in BALB/c mice via intraperitoneal injection of lipopolysaccharide (LPS), confirmed by HE staining and dry-wet weight methods for brain tissues. The mice in the brain edema model were divided into control group, IR-783, and IR-783-FSM treatment groups, receiving intraperitoneal injections of PBS, IR-783, and IR-783-FSM, respectively. Real-time in vivo fluorescence imaging was then performed. The mice in each group were euthanized after 10 hours. Ex vivo brain imaging and dry-wet weight measurements were performed to observe the NIRF imaging characteristics and therapeutic effects of IR-783-FSM on brain edema model. Results The newly synthesized compound, IR-783-FSM, retained the excellent near-infrared fluorescence characteristics of IR-783. It could target mouse macrophages with an IC50 of 48.82 μmol/L. A brain edema model could be successfully constructed with intraperitoneal injection of LPS, with significantly higher brain tissue water content compared to the control group (P<0.01). In vivo imaging showed that IR-783-FSM had a significantly stronger fluorescence signal in the brain edema model than IR-783. Compared to the control group, the brain water content was significantly reduced in the 2, 5, and 8 mmol/L IR-783-FSM treatment groups (P<0.01). Conclusion The newly synthesized NIRF probe IR-783-FSM facilitates dynamic monitoring of brain edema and real-time evaluation of therapeutic effects.

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    Discussion on the Construction and Operational Experience of Intelligent Control Systems for Laboratory Animal Facilities: Shenzhen Institute for Drug Control as an Example
    Long ZENG, Junpeng LI, Xiaowei WANG, Ning CHEN, Ping WANG, Meirong QIN
    Laboratory Animal and Comparative Medicine    2024, 44 (2): 220-226.   DOI: 10.12300/j.issn.1674-5817.2023.162
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    Intelligent control systems can effectively assist in the construction and management of laboratory animal facilities, improving operational efficiency, ensuring the reliability of animal experimental results, and significantly saving human resources. The intelligent control system for laboratory animal facilities at Shenzhen Institute for Drug Control was completed in April 2021. It includes an intelligent management platform and an information management system for animal laboratories. The intelligent management platform regulates room environment parameters such as temperature, humidity, and pressure through building equipment management system, controlling devices such as the Venturi valve, electric air valve, electric water valve, and steam humidification valve. At the same time, various environmental parameters are monitored online through the environmental monitoring system. The laboratory’s intelligence is further enhanced by systems such as automatic lighting control, full HD video monitoring, automatic access control and door system, independent ventilation and feeding, automatic cleaning, automatic exhaust gas treatment, centralized gas supply, and real-time instrument parameter monitoring. The information management system for animal laboratories integrates inspection, instrument and equipment, personnel, documents, standard substances, reagents, inspection standards, books, records, scientific research management, relevant applications, quality management, and query statistics. For animal experimentation, a management module has been developed to achieve a comprehensive digitization of animal management. Furthermore, real-time collection and recording of data such as balance calibration, sample quality, and animal weight are facilitated through electronic experimental recording. In summary, the Animal Laboratory of Shenzhen Institute for Drug Control has extensively utilized intelligent systems to achieve real-time online control and monitoring, improve efficiency, ensure high-quality facility operation, and meet standard requirements. Smooth execution of all inspection and research activities has been achieved over the past three years. This paper provides insights into the construction, management, and operation of laboratory animal facilities at Shenzhen Institute for Drug Control, offering guidance for the implementation of intelligent control in similar facilities across China.

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    Advances in Comparative Medical Research on Anatomy and Histological Structure of Intervertebral Discs in Humans and Other Animals
    Li ZHANG, Yu KUANG, Lingxia HAN
    Laboratory Animal and Comparative Medicine    2024, 44 (2): 192-201.   DOI: 10.12300/j.issn.1674-5817.2023.141
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    The 2023 China Health Report on Spine Degeneration noted a significant increase in lumbar surgery among patients under 35 years old in recent years, indicating a trend towards younger onset of cervical and lumbar diseases. Lumbar intervertebral disc herniation has become a major concern, making the study of disc degeneration pathogenesis and treatment methods clinically significant. At present, human intervertebral disc diseases are primarily diagnosed through imaging due to the challenges of obtaining tissue samples from the spine. Therefore, experimental animals have emerged as alternative research subjects because they are cost-effective, have short experimental cycles, and are easily accessible. Given the structural and physiological differences between human and other animal intervertebral discs, comparing their anatomy and histological characteristics forms the foundation of research into human disc degeneration. The purpose of this paper is to collect and review relevant studies on anatomical and histological structures of intervertebral discs in different animals and conduct a comparative analysis from four aspects, namely, intervertebral disc height, lumbar disc geometry, lumbar disc cartilaginous endplate characteristics, and extracellular matrix components. The results show that humans, kangaroos, sheep, pigs, and rats exhibit similar relative heights between the sixth and seventh cervical vertebrae. Mice possess lumbar disc geometries most akin to humans. Compared to other animals, humans have the thickest cartilaginous endplates and the lowest cell densities. The collagen within the fibrous annulus differs most notably in pigs compared to humans, while water content in the nucleus pulposus is consistent across pigs, sheep, rabbits, rats, and humans. Additionally, this paper describes the commonalities and discrepancies in disc degeneration manifestations between humans and animals, and summarizes modeling methods for disc degeneration in different experimental animals. Ultimately, the aims of this paper is to provide fundamental data for selecting suitable experimental animal models for the study of intervertebral disc degeneration.

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    Advances in Research on Pathological and Molecular Mechanism of Hyperuricemic Nephropathy Based on Animal Models
    Fangqi BAO, Haiye TU, Mingsun FANG, Qian ZHANG, Minli CHEN
    Laboratory Animal and Comparative Medicine    2024, 44 (2): 180-191.   DOI: 10.12300/j.issn.1674-5817.2023.123
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    Uric acid (UA), the final product of human purine metabolism, can cause hyperuricemia (HUA) when excessively accumulated. HUA is closely linked to chronic kidney diseases (CKD) and is considered an independent risk factor. Hyperuricemic nephropathy, a form of CKD induced by HUA, has seen significant advances in understanding through research into the pathogenic roles of uric acid and the development of HUA animal models. Although progress has been made in understanding the pathophysiological mechanisms by which UA induces CKD, much remains to be learned about its pathological molecular mechanisms. New approaches in animal modeling or the selection of model animals may potentially lead to significant breakthroughs in research on hyperuricemia as well as related CKD. This paper reviews the research progress on the molecular mechanisms of hyperuricemic nephropathy, focusing on oxidative stress, inflammation, autophagy, fibrosis, and gut microbiota. Oxidative stress is induced by uric acid intracellularly through xanthine oxidase, NADPH oxidases, and mitochondria, leading to cellular damage. In terms of inflammation, uric acid crystals can activate the NLRP3 inflammasome, triggering an inflammatory cascade. The role of free uric acid as a pro-inflammatory agent, however, remains controversial. Depending on the study conducted, autophagy has been found to either alleviate or exacerbate inflammation induced by uric acid. Fibrosis, particularly through epithelial-mesenchymal transition (EMT), is a major mechanism by which uric acid causes glomerulosclerosis and tubulointerstitial fibrosis. Extensive research has explored various signaling pathways involved in uric acid-induced EMT. Beneficial gut microbiota protect the kidneys by synthesizing short-chain fatty acids, reducing urea’s enterohepatic circulation, and decreasing uric acid production. This paper aims to enhance understanding of the complex relationships between HUA and CKD, serving as a reference for further research and new drug development.

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    Effects of Puerarin on Bone Density in Rats and Mice: A Meta-analysis
    Jinhua HU, Jingjie HAN, Min JIN, Bin HU, Yuefen LOU
    Laboratory Animal and Comparative Medicine    2024, 44 (2): 149-161.   DOI: 10.12300/j.issn.1674-5817.2023.127
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    Objective To evaluate the effects of puerarin on bone density in rats and mice through a meta-analysis. Methods The databases, including CNKI, SinoMed, Wanfang data, VIP, PubMed, EMBase, Web of Science, the Cochrane Library, and Scopus from their inception to November 6, 2023, were searched for literature on the effects of puerarin treatment on bone density in rats and mice. Inclusion criteria for the literature were randomized controlled trials with a placebo or blank control group; the subject animals were rats or mice; the intervention was puerarin; and the results included bone density measurements. Exclusion criteria included combination therapy with puerarin; lack of original research data; unpublished studies; and using mandible as the measurement site for bone density. Risks of bias were assessed using SYRCLE's RoB tool. Data analysis was conducted with Stata 16.0 and Rev Man 5.3 software. Results After applying the inclusion and exclusion criteria, a total of 429 records were identified and 42 articles covering 41 studies were ultimately included. 925 animals were involved and the data analysis results indicated that puerarin improved bone density in rats and mice compared to the control group: femur [37 studies, n=824, standardized mean difference (SMD)=2.12, 95% confidence interval (CI)=1.69-2.54, P < 0.000 1], lumbar spine (13 studies, n=271, SMD=2.25, 95% CI=1.49-3.01, P < 0.000 1), tibia (4 studies, n=95, SMD=0.94, 95% CI=0.05-1.83, P=0.04), and the whole body (4 studies, n=94, SMD=1.89, 95% CI=0.50-3.29, P=0.008), with all inter-group differences in bone density being statistically significant. Conclusion Puerarin can improve bone density in rats and mice. This study provides a valuable reference for clinical studies on the prevention and treatment of osteoporosis with puerarin.

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    Three Dimensions of Animal Experiment Ethics: Analysis Based on Value of Life, Animal Welfare, and Risk Prevention
    ZHAO Yong
    Laboratory Animal and Comparative Medicine    2024, 44 (4): 445-454.   DOI: 10.12300/j.issn.1674-5817.2024.108
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    Ethical review of animal experiments is based on factors such as the necessity of the experiment, its scientific validity, the professional competence of the experimenters, and research conditions, to determine the ethical permissibility of an animal experiment. Attitudes towards laboratory animals and animal experiments vary significantly due to differences in cultural traditions, religious beliefs, personality traits, and roles within the experimentation process. How animal experiment ethics can advance in tandem with the advancements of life sciences, while consistently providing forward-looking guidance and safeguarding fundamental ethics, is a subject that requires continuous exploration, practice, and research. Ensuring the welfare of laboratory animals has now become a widely accepted ethical consensus. However, attitudes and principles towards different types of animal experiments, and the methods to genuinely and effectively ensure the welfare quality of animals during live animal experiments, should be central to animal experiment ethics. Based on the deep integration of biotechnology in the field of laboratory animals, this paper considers the contribution of animal experimentation to the development of scientific ethical concepts. It systematically elucidates the special value of life in laboratory animals and their relationship with biosafety and ecological safety. It also outlines welfare quality assessment methods for different species of laboratory animals, demonstrating that the philosophical ideology of the value of life is the core of animal experiment ethics. The quality and significance of animal experiments determine the ethical level at which the value of life in laboratory animals is realized, and animal welfare technologies provide a robust ethical guarantee for animal experiments. The close integration of ethical theories with life sciences in laboratory animals is an objective requirement for animal experiment ethics. The value of life, animal welfare, and risk prevention collectively form the core elements of ethical review in animal experiments, serving as fundamental factors in improving the quality of ethical reviews and avoiding ethical deviations.

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    Screening of Hypoxia-Sensitive and Hypoxia-Tolerant Wistar Rats and Preliminary Exploration of Hypoxia Sensitivity in Their G 1 Generation
    XIAO Pan, WANG Hongyi, LU Lu, ZHANG Mei, CHEN Keming, SHEN Dongshuai, NIU Tingxian
    Laboratory Animal and Comparative Medicine    2024, 44 (4): 374-383.   DOI: 10.12300/j.issn.1674-5817.2024.017
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    Objective By screening and passaging G0 generation Wistar rats, we obtained hypoxia-sensitive and hypoxia-tolerant G1 generation rats, and then the differences in hypoxia sensitivity among these rats were preliminarily explored. Methods 200 Wistar rats (half male and half female) were selected as G0 generation and placed in a controlled oxygen concentration system. The hypoxia tolerance time, which refers to the time from placement to near death, was recorded for the G0 generation rats at an oxygen volume fraction of 3%. 30 rats (half male and half female) with the shortest hypoxia tolerance time were selected for mating and passage to obtain G1 generation hypoxia-sensitive rats. Similarly, 30 rats (half male and half female) with the longest hypoxia tolerance time were selected for mating and passage to obtain G1 generation hypoxia-tolerant rats. An additional 24 standard Wistar rats were randomly divided into two groups: a control group and a model group, with 12 rats in each group (half male and half female). The control group was kept in a normoxic environment, while the model group, along with the G1 generation hypoxia-sensitive rats (G1 sensitive group) and G1 generation hypoxia-tolerant rats (G1 tolerant group), were placed in a hypobaric hypoxia chamber (simulating an altitude of 5 000 m). After 12 hours, various indicators, including blood gas, complete blood count, blood biochemistry, pathological sections, and hypoxia-related genes were detected or observed to compare the differences in hypoxia sensitivity among the 4 groups. Results Compared with the G0 generation standard rats, the hypoxia tolerance time of G1 generation rats was significantly prolonged (P<0.01). Compared with the model group, the oxygen saturation (SatO2) in G1 tolerant group was significantly higher (P<0.05). In the G1 sensitive group, the levels of white blood cell (WBC) count, neutrophil (NEUT) count, hemoglobin (HGB) concentration, hematocrit (HCT), red blood cell distribution width (RDW), platelet (PLT), and creatinine (Cr) significantly increased (P<0.05 or P<0.01), while actual bicarbonate (AB) content significantly decreased (P<0.05), and the brain and lung coefficients were significantly elevated (P<0.05). In addition, pathological section results showed that the brain and lung tissues in the model group, G1 sensitive group, and G1 tolerant group all suffered from significant damage, with no evident differences in the gene expression levels of hypoxia-inducible factor-1 α (HIF-1α) and vascular endothelial growth factor A (VEGFA) in brain tissues among the three groups (P>0.05). Conclusion Compared with standard rats, G1 generation hypoxia-sensitive/tolerant rats exhibit good signs of hypoxia sensitivity/tolerance traits, but further screening and passage are still needed to purify them.

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    Diagnosis of a Primary Pleomorphic Liposarcoma in a Tree Shrew( Tupaia belangeri subsp . yaoshanensis
    Zhuxin LI, Liang LIANG, Yingying CAO, Shanshan ZHAI, Yinhan DAI, Xia HE, Junyu TAO, Jing LENG, Haibo TANG
    Laboratory Animal and Comparative Medicine    2023, 43 (6): 647-653.   DOI: 10.12300/j.issn.1674-5817.2023.058
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    A large spontaneous mass on the dorsal abdomen near the hip joint was found in an aged female Tupaia belangeri subsp. yaoshanensis after 5 years of routine feeding. The tumor in the diseased tree shrew was huge, with an intact surface and no ulceration; however, it caused inconvenience in movement while maintaining a good mental state. After inhalation anesthesia with isoflurane (2%-4%), the tree shrew was euthanized after the tree shrew entered deep anesthesia. Anatomical dissection of the tumor, the tumor boundary was unclear and infiltrated into surrounding tissues. HE staining showed that small focal pleomorphism and large areas of adipocytes were seen in the tumor tissue. The pleomorphic sarcoma area was mainly composed of atypical epithelioid cells with easily visible nuclear divisions. The size of the adipocytes was significantly different, and more pleomorphic adipocytes were seen. The cell volume was large, the nucleus was deeply stained and deformed, the edge was impressed, and the cytoplasm was seen with multiple vesicular lipid droplets. Immunohistochemical results showed that the tumor cells were positive for Vimentin, the small focal polymorphic adipocyte nucleus was positive for S-100, and Ki-67 exhibited a higher proportion of positivity. Combined with HE staining and immunohistochemical results, the spontaneous tumor in this tree shrew was comprehensively diagnosed as pleomorphic liposarcoma.

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    Application and Comparison of Different Anesthetic Ventilation Methods in Minimally Invasive Thoracic Surgery Training
    Yishu LIU, Shanmin ZHAO, Liping CAI
    Laboratory Animal and Comparative Medicine    2024, 44 (1): 97-104.   DOI: 10.12300/j.issn.1674-5817.2023.135
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    Objective To compare the effectiveness and safety of one-lung ventilation and small tidal volume two-lung ventilation anesthesia methods in the training of minimally invasive thoracic surgery on experimental pigs. Methods Forty experimental pigs undergoing robotic thoracic surgery were randomly divided into two groups: two-lung ventilation group (n=20) and one-lung ventilation group (n=20). The two-lung ventilation group underwent single-lumen tracheal intubation, utilizing a small tidal volume with a fast respiratory rate combined with carbon dioxide pneumothorax for anesthesia ventilation during the operation. The one-lung ventilation group received one-lung ventilation using a double-lumen bronchial catheter placed under fiberoptic bronchoscopic guidance. The anesthesia implementation indexes from the two groups were compared, including the values of vital signs such as operative heart rate (HR), noninvasive mean blood pressure (MAP), end-tidal carbon dioxide (ETCO2), and oxygen saturation (SpO2), as well as the assessment of surgical training performance. Results The intubation success rate for animals in both groups was 100%, with no intraoperative deaths. The intubation completion time was significantly shorter in the two-lung ventilation group compared to the one-lung ventilation group (P < 0.001). Within each group, SpO2 levels were significantly higher in the two-lung ventilation group at 30 minutes after the start of thoracic surgery (T1) and at surgery completion (T3) compared to 60 minutes after the start of surgery (T2) (P<0.05). ETCO2, HR, and MAP were significantly higher at T2 and T3 compared to T1 (P<0.05). In the one-lung ventilation group, SpO2 levels were significantly higher at T1 and T3 compared to T2 (P<0.05), while ETCO2 levels gradually increasing over time (P<0.05). In the between-group comparisons at the same time points, SpO2 levels of the two-lung ventilation group were significantly higher than those of the one-lung ventilation group at all time points (T1, T2, T3) (P<0.05). Conclusion Both one-lung ventilation and two-lung ventilation anesthesia methods are effective and safe for use in surgical training, with controllable effects on intraoperative animal vital signs and minimal impact on surgical operation training, meeting the needs of robotic thoracic surgery training. One-lung ventilation provides a better experience during pneumonectomy procedures, while small tidal volume two-lung ventilation is easier to implement and does not require additional equipment purchase, making it a feasible supplemental anesthesia option for thoracoscopic surgery on experimental pigs.

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    Establishment and Evaluation of Mouse Model of Pregnancy Pain-depression Comorbidity Induced by Chronic Unpredictable Stress, Complete Freund's Adjuvant and Formalin
    Yisu ZHANG, Xinru LIU, Ruojie WU, Rui LIU, Hong OUYANG, Xiaohong LI
    Laboratory Animal and Comparative Medicine    2024, 44 (3): 259-269.   DOI: 10.12300/j.issn.1674-5817.2024.005
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    Objective To establish a mouse model of pregnancy pain-depression comorbidity induced by chronic unpredictable stress (CUS), complete Freund's adjuvant (CFA), and formalin, and to systematically evaluate the associated phenotypes and preliminarily explore the pathological basis of the comorbidity. Methods Eight-week-old C57BL/6J female mice were randomly strarified divided into a control group (no intervention before pregnancy) and a CUS model group (CUS intervention before pregnancy) based on sucrose preference test (SPT) data. After completing the CUS treatment, female and male mice were paired and mated. Pain was induced by injecting 50% CFA and 5% formalin in the right hind foot during pregnancy to create a model of pregnancy pain-depression comorbidity. The experiment was divided into 8 subgroups: control-blank group, CUS-blank group, control-CFA group, CUS-CFA group, control-formalin group, CUS-formalin group, control-CFA+formalin group, and CUS-CFA+formalin group, with 10 mice in each group. The mice in each group were subject to behavioral tests, including the SPT, forced swimming test, tail suspension test, and open field test before and after CUS intervention, during pregnancy, and after delivery. Pain sensitivity changes were measured using mechanical allodynia and thermal hyperalgesia tests. Mice were then euthanized. Levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in hippocampus, as well as cortisol and adrenocorticotropic hormone (ACTH) in serum, were detected by enzyme-linked immunosorbent assay (ELISA). Results Compared with the control-blank group, the CUS-blank group showed a significant depression-like behavior with reduced pain threshold (P<0.001). The control-CFA+formalin group showed a decrease in pain threshold after both CFA injection and formalin injection (P<0.01). Compared with the control-blank and control-formalin groups, the pain threshold was significantly lower in the CUS-formalin group (P<0.01), with a sequential decrease among the three. Compared with the control-blank and control-CFA groups, the pain threshold was significantly lower in the CUS-CFA group (P<0.001), with a sequential decrease among the three. Compared with the control-blank and control-CFA+formalin groups, the mechanical pain threshold of mice in the CUS-CFA+formalin group was significantly lower (P<0.001) and the thermal radiation tolerance time was shorter (P<0.01), both with sequential decreases among the three. Compared with the control-CFA+formalin and the CUS-blank groups, the CUS-CFA+formalin group had a significantly lower percentage of sucrose preference (P<0.001), longer immobility time during the forced swimming test (P<0.001) and tail suspension test (P<0.001), reduced central exploration time in the open field test (P<0.001), reduced total exploration distance (P<0.001), and reduced percentage of distance traveled for central exploration (P<0.001). Compared with the control-CFA+formalin and CUS-blank groups, the serum cortisol and ACTH levels of the CUS-CFA+formalin group were significantly higher (P<0.01), and the levels of IL-6 and TNF-α in the hippocampus were higher (P<0.05). Conclusion The combination of CUS+CFA+formalin injections is an ideal method for establishing a C57BL/6J mouse model of pregnancy pain-depression comorbidity. The behavioral changes in model mice may be attributed to the regulation of inflammatory response in hippocampus and hormone levels in the hypothalamic-pituitary-adrenal (HPA) axis.

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    Metabolomics Analysis of Tupaia belangeri Breast Tumor Model
    Xi FANG, Qingqing AO, Chunhong LI, Yiqiang OUYANG, Songchao GUO, Bing HU
    Laboratory Animal and Comparative Medicine    2024, 44 (1): 52-61.   DOI: 10.12300/j.issn.1674-5817.2023.094
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    Objective To explore the metabolic changes during the development of Tupaia belangeri breast tumors, to investigate the close relationship between the changes of serum metabolic substances and the occurrence and progression of tumors, and to screen for biomarkers reflecting the progression of breast tumors. Methods Breast tumors in Tupaia belangeri were induced by orally administering 7,12-dimethylbenzoanthracene (DMBA) three times, with a 15-day interval between each administration, along with a high-fat and high-sugar diet. The DMBA-induced breast cancer group and the DMBA-inducedwithout breast cancer group were compared with the control group. Untargeted determination of serum metabolites was performed using gas chromatography-time-of-flight mass spectrometry (GC-TOFMS) in DMBA-induced Tupaia belangeri with breast cancer, DMBA-induced without breast cancer and the control group. Multidimensional statistical analysis including unsupervised principal component analysis (PCA), and orthogonal partial least squares analysis (OPLS-DA) were conducted. Furthermore, t-test was used for intergroup differential comparison. Differential metabolites were screened under VIP>1 and P<0.05 conditions, and significantly changing differential metabolites were identified using the HMDB online database. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database was utilized to enrich metabolic-related gene regulatory pathways. Results The incidence of breast tumors was 40% in DMBA-induced Tupaia belangeri. Compared with the control group, 30 metabolic differential products were detected in the serum of the group with breast cancer, with 18 down-regulated and 12 up-regulated (VIP>1, P<0.05). KEGG pathway analysis revealed significant changes in four metabolic pathways: glutamate metabolism, glyceride metabolism, citric acid cycle, and alanine metabolism. Compared with the group without breast cancer, 18 metabolic differential products were detected, with 7 down-regulated and 11 up-regulated (VIP>1, P<0.05). KEGG pathway analysis revealed significant changes in the citric acid cycle and glutamate metabolism. Compared with the control group, 31 metabolic differential products were detected in the serum of the groups without breast cancer, with 14 down-regulated and 17 up-regulated (VIP>1, P<0.05). KEGG pathway analysis revealed significant changes in three metabolic pathways: glutamate metabolism, glyceride metabolism, and citric acid cycle. Conclusion Metabolomics analysis can reveal the characteristics of changes in metabolites in the serum of breast tumors. The results suggest that glutamate metabolism, glyceride metabolism, citric acid cycle, and alanine metabolism pathways are associated with the occurrence and development of DMBA-induced breast tumors in Tupaia belangeri. It provides a foundation for further research into the biological mechanism of breast cancer.

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    Effects of Different Pellet Feed Hardness on Growth and Reproduction, Feed Utilization Rate, and Environmental Dust in Laboratory Mice
    Dong WU, Rui SHI, Peishan LUO, Ling'en LI, Xijing SHENG, Mengyang WANG, Lu NI, Sujuan WANG, Huixin YANG, Jing ZHAO
    Laboratory Animal and Comparative Medicine    2024, 44 (3): 313-320.   DOI: 10.12300/j.issn.1674-5817.2023.138
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    Objective To study the effects of different pellet feed hardness on the growth and reproduction, feed utilization rate, and environmental dust in laboratory mice. Methods One hundred of fifty 50 3-week-old SPF-grade C57BL/6JGpt and 150 ICR laboratory mice were randomly divided into three groups, with an equal number of males and females. They were fed diets with different hardness of 18.62 kg, 23.15 kg, and 27.89 kg. Body weight, feed utilization rate, and dust levels in cages were recorded and calculated for mice aged 3-10 weeks. Forty-five 6-week-old male mice and ninety 4-week-old female mice from each strain were randomly divided into three groups and fed pellet feeds with three different hardness levels. After 2 weeks of adaptation to the same hardness feed, the mice were paired at a 1∶2 male-to-female ratio and monitored for reproductive data for 3 months. Results At the age of 4 weeks, the body weight of male C57BL/6JGpt mice in 23.15 kg group was significantly higher than that in the 18.62 kg and 27.89 kg groups (P<0.01), and the body weight of females in the 18.62 kg group was significantly higher than that in the 27.89 kg group (P<0.05). There was no significant difference in body weight among ICR mice aged 3-10 weeks across different feed hardness groups (P>0.05). For both strains, feed utilization rate for males was higher than that for females across different feed hardness groups at all weeks of age (P<0.01). Compared to the 27.89 kg group, both the 18.62 kg and 23.15 kg groups showed a significant increase in the 50-mesh dust levels in cages for both strains aged 4-8 weeks (except for 7-week-old C57BL/6JGpt mice) (P<0.05). For both C57BL/6JGpt and ICR mice, there was no significant difference in basic reproductive performance such as interval between the first litter and the monthly production index among the three feed hardness groups during the experimental period (P>0.05). However, the monthly production index of C57BL/6JGpt mice first increased and then decreased with the increase of feed hardness, while that of ICR mice increased with increasing feed hardness, though these differences were not statistically significant (P>0.05). Conclusion Different strains and genders had different tolerance to feed hardness. C57BL/6JGpt mice are more adapted to lower hardness feeds, while ICR mice are better suited to slightly higher hardness feeds.

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