Laboratory Animal and Comparative Medicine

Mechanism of Intermittent Fasting in Improving Olanzapine-induced Metabolic Disorders in Mice

Han LI, Xiaorui ZHANG, Chengfang ZHANG

2023, 43(1): 3-10. DOI: 10.12300/j.issn.1674-5817.2022.089

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Objective To explore the beneficial role and potential mechanism of intermittent fasting in olanzapine-induced metabolic disorders. Methods C57BL/6J mice were randomly divided into four groups: Saline + ad libitum (Saline+Ad libitum), Saline + intermittent fasting (Saline +IF), olanzapine administration + ad libitum (Olanzapine+ Ad libitum), and olanzapine administration + intermittent fasting (Olanzapine+IF)， with eight mice in each group. The IF group adopted the 5∶2 scheme, that is, fasting on Monday and Thursday every week, and eating freely in the rest of the time. Ad libitum feeding as the control of intermittent fasting, Saline gavage as the control of olanzapine administration. The experiment lasted for 12 weeks. The differences of body mass, liver mass and epididymal adipose tissue mass were compared between the olanzapine-treated group and the control group after IF intervention. The body fat mass, lean body mass, and visceral fat infiltration of mice were analyzed by nuclear magnetic resonance and HE staining, respectively. Furthermore, the levels of fasting blood glucose, insulin, and insulin resistance index (HOMA-IR) in the process of glucose metabolism were also measured by glucose oxidase method and radioimmunoassay, respectively. The effects of IF on H₂O₂ release and the level of cytochrome C mRNA, a marker related to mitochondrial damage, were detected by ELISA and real-time fluorescence quantitative PCR. Results After 12 weeks of treatment, olanzapine induced a significant increase in body mass, body fat, lean body mass and visceral fat infiltration (P<0.05), as well as fasting blood glucose, insulin, and HOMA-IR (P<0.05); however, IF significantly reduced the above indicators (P<0.05). Further studies showed that the release of H₂O₂ and the expression of Cytochrome C mRNA in adipose tissue of mice after intermittent fasting treatment were significantly decreased (P<0.05). Conclusion Intermittent fasting therapy can alleviate olanzapine-induced metabolic disorders in mice. The underlying mechanism may involve the inhibition of oxidative stress level and the maintenance of mitochondrial functions.

Screening of Differentially Expressed Genes in Rat Synovitis by Transcriptome Sequencing and in Vitro Verification of Therapeutic Target of Fraxetin

Ling YANG, Di ZHUANG, Lilun JIN

2023, 43(1): 11-20. DOI: 10.12300/j.issn.1674-5817.2022.100

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Objective Using transcriptome sequencing to screen the differentially expressed genes between the synovial tissue of rats with knee osteoarthritis (KOA) induced by monosodium iodoacetate (MIA) and that of normal rats, and then screen the target of fraxetin in the treatment of synovitis. Methods SD rats were divided into KOA group and the negative control (NC) group. Rat right knee KOA model was prepared by MIA knee joint injection in KOA group and none treatments in NC group. Four weeks after modeling, the right knee synovial tissue of rats in each group was taken for transcriptome sequencing. Then the differential gene expression analysis, GO enrichment analysis, KEGG function enrichment analysis and PPI protein network interaction analysis were performed. The synovial macrophage Raw264.7 cells were divided into the control group, lipopolysaccharide (LPS) intervention group and LPS+60 μmol/L fraxetin intervention group, then RNA-sequencing results were verified by qRT-PCR in the three groups. Results The results of differential gene-expression analysis showed that there were 1 730 up-regulated genes and 1 546 down-regulated genes in the KOA group compared with the control group, among which the significantly up-regulated genes were mmp12, Acod1, Acan, Col2a1, Atp6v0d2 (|log2(FoldChange)|≥1, adjusted P<0.01). KEGG cluster analysis and GO cluster analysis showed that differential genes were mainly involved in the regulation of inflammation and immune metabolism, such as tricarboxylic acid cycle and mitochondrial function. The expressions of Acod1 and Atp6v0d2 in Raw264.7 cells after LPS intervention were significantly higher. Compared with the LPS intervention group, the expression level of Atp6v0d2 in Raw264.7 cells after LPS+fraxetin combined intervention was significantly lower. Conclusion After modeling KOA induced by MIA, macrophage-related genes mmp12, Acod1 and Atp6v0D2, which mediate inflammation and immune metabolism, were highly expressed in the synovial tissue of rats, suggesting that there might be immune metabolism changes mediated by synovial macrophages during the occurrence and development of KOA. The increased expression of Acod1 and Atp6v0d2 in macrophages Raw264.7 after LPS intervention can preliminarily confirm this result. Among them, Atp6v0d2 may be a potential target of fraxetin in the treatment of synovitis, which provides a new idea for KOA treatment.

Effects of Bevacizumab Injection on the Skin Wound Healing in Cynomolgus Monkeys

Ping YANG, Li CUI, Cheng YU, Zhiyue WEN

2023, 43(1): 21-29. DOI: 10.12300/j.issn.1674-5817.2022.128

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Objective By observing the impact of Bevacizumab injection on the speed of wound healing and the expression of CD34 in cynomolgus monkeys, to verify its delayed wound healing mechanism, and to provide clinical reference for the dosage and frequency of Bevacizumab treatment for oncology surgery patients. Methods Six male cynomolgus monkeys underwent full-thickness skin resection on the back to establish a wound healing evaluation model. Three cynomolgus monkeys were randomly selected as the saline group, and the other three cynomolgus monkeys were allocated to the Bevacizumab treatment group. The monkeys of Bevacizumab group were administrated with Bevacizumab injection (30 mg/kg, i.v.) on day 0, day 4, day 8, and day 11, respectively. The monkeys of saline group were injected with the same volume of saline as the group receiving Bevacizumab. The blood routine test was conducted, and the wound healing of each group of cynomolgus monkeys was observed 28 days after operation. The wound healing rate was analyzed by software Image J, and the severity of the wound was assessed by scoring method. Wound healing skin samples of 6 cynomolgus monkeys were obtained after 4 weeks, and the expression level of CD34 in the wound skin tissues was detected using immunohistochemistry. Results A skin excision wound model has already been established in cynomolgus monkeys. The number of white blood cells and neutrophils in the blood of cynomolgus monkeys increased significantly on the 3^rd day after operation (P<0.05), and the number of white blood cells gradually returned to normal on the 7^th day, while the number of red blood cells, hemoglobin and hematocrit did not change significantly, suggesting that the animals were in good nutritional status. The Bevacizumab injection group wound healing rate was significantly slower than that of the saline group on day 7, and day 28^{(P<0.01, P<0.001). Microvessel density in the wound skin of Bevacizumab group appeared to be significantly lower than that of the saline group (P<0.01). Conclusion The injection of Bevacizumab may delay the wound healing by inhibiting the angiogenesis in the new skin tissue of the cynomolgus monkey. In clinical practice, the timing of giving a tumor patient a bevacizumab injection after surgery should be based on the pros and cons, and the duration, dosage, and frequency of the intervention should be chosen in a rational way.}

Repair Effects of Xiaoyusan New Formula on Cartilage Injury and MMP-13 Expression in Knee Osteoarthritis Model Rabbits

Ruolin CUI, Qing WANG, Ling YANG, Wenchang FENG, Zhiwei LIU, Weiran TENG, Bitao MA, Leyang WANG, Liping QIN, Lilun JIN

2023, 43(1): 30-38. DOI: 10.12300/j.issn.1674-5817.2022.099

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Objective To study the effect of Xiaoyusan new formula on the articular cartilage of knee osteoarthritis (KOA) rabbits and its mechanism. Methods A total of 42 New Zealand white rabbits aged 6 months were randomly divided into normal group, model group, ointment of Xiaoyusan group, and ointment of Xiaoyusan new formula group, with 10 rabbits in each group (the other 2 rabbits were used for model validation). Except for the normal group, the right knee joints of all rabbits in the other groups were prepared as KOA models according to the modified Hulth method. After 5 weeks of molding, the rabbits in ointment of Xiaoyusan group, ointment of Xiaoyusan New Formula group were given corresponding ointments for knee arthritis treatment, once a day, each time for 10 hours. After 2-week continuous administration and treatment, the knee joint cartilage of the four groups of rabbits was taken and the cartilage damage of each group was evaluated by Outerbridge grading method. The pathological changes of the cartilage, calcified layer and subchondral bone of the knee joint of rabbits in each group were observed by HE staining method under the light microscope, and the degree of cartilage degeneration was evaluated by Mankin's method. The expression of matrix metalloproteinase-13 (MMP-13) in the cartilage of rabbit knee joint in each group was deteced by immunohistochemistry. Results After the general observation of articular cartilage, the Outerbridge grading showed that the number of high-grade animals in ointment of Xiaoyusan group was reduced compared with the model group (P<0.05), and the number of high-grade animals in ointment of Xiaoyusan new formula group was also reduced (P<0.05) compared with ointment of Xiaoyusan group. HE staining showed that Mankin's scores of articular cartilage in the four groups ranked from high to low: model group (10.82±1.76), ointment of Xiaoyusan group (6.19±1.23), ointment of Xiaoyusan new formula group (2.64±1.18) and normal group (0.28±0.17). The difference among four groups was statistically significant (P<0.05). Immunohistochemical detection showed that the positive rates of MMP-13 expression in rabbit articular cartilage tissues in each group were (67.90±13.94)% of model group, (37.10±19.16)% of ointment of Xiaoyusan group, (13.60±3.10)% of ointment of Xiaoyusan new formula group and (3.20±2.39) % of normal group, ranking from high to low, and the difference among four groups was statistically significant (P<0.05). Conclusion Xiaoyusan new formula can repair articular cartilage degeneration in KOA rabbits and decrease the expression of MMP-13 in cartilage, which may be one of the mechanisms of the treatment.

Taurine Has no Protective Effect on Rat Corneal Endothelial Cells Injured by Benzalkonium Chloride

Jian GE, Jingfen SUN, Yongjie WU

2023, 43(1): 39-43. DOI: 10.12300/j.issn.1674-5817.2022.034

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Objective To observe the protective effect of 2% taurine on corneal endothelial cells injured by benzalkonium chloride in rats. Methods Six piece of corneal endodermis and elastic layer tissue slices were prepared from 6 eyes of 3 SPF SD rats and randomly divided into three groups. The corneal endothelial cells of rats were cultured by tissue block culture for 1 day, then the control group cells were added with 2% taurine solution, while the experimental group cells were added with 2% taurine solution and 0.01% or 0.03% benzalkonium chloride solution. After 1, 2, 4, 5, 6 and 8 days of continuous culture, the growth of corneal endothelial cells in each group was observed under an inverted microscope, and the morphology of endothelial cells was observed under an optical microscope after Wright staining. Results Treated with 0.01% benzalkonium chloride and 2% taurine for 1 day, polygonal endothelial cells appeared on the edge of corneal tissue mass, and the cells were transparent. After 2 days, the number of polygonal cells increased, and there was no fusion growth between cells. After 3 days, the number of polygonal cells decreased and no mitotic signs were observed in endothelial cells. After 4 days, the endothelial nuclei were deeply stained and polygonal cells were rare. After 5 days, the number of endothelial cells decreased, and cell body shrinkage and death occurred. In the experimental group treated with 0.03% benzalammonium chloride and 2% taurine for 1 day, no endothelial cell growth was observed and the cells were sparsely-scattered. In control group, polygonal endothelial cells and a few endothelium-like polygon cells appeared at the edge of tissue blocks after 1 day. After 3 days, the number of polygonal cells at the edge of tissue blocks increased, and there was a phenomenon of gradual fusion growth. After 5 days, the number of endothelial cells increased, and the cells were mostly hexagonal. After 8 days, the endothelial cells formed large sheets, the cell bodies were hexagonal or round, and the nuclei were divided. The growth of corneal endothelial cells in the left and right eyes was uniform, and there was no significant difference in the morphology of the left and right eye endothelial cells in the 0.01% and 0.03% benzalammonium chloride treatment groups and the control group. Conclusion 2% taurine had no protective effect on corneal endothelial cells injured by benzalammonium chloride.

Investigation on Biological Characteristics and Aging Phenotype of SHJH ^hr Mice

Huiqing TANG, Shufu CHANG, Zhifeng YU, Lei ZHANG, Xiaoqian TAN, Wei QU, Liang LI, Zhen QIAN, Jianzhong GU, Ping XU

2023, 43(1): 44-52. DOI: 10.12300/j.issn.1674-5817.2022.069

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Objective To measure and analyze biological characteristics and aging phenotype of SHJH ^hr mice and provide basic data for the application of the mouse model in aging mechanisms research and antiaging drug development. Methods With ICR mice of the same age as control group, the body mass growth data of SHJH ^hr mice at the age of 3 to 16 weeks, the reproduction ability of 1 to 4 fetuses and the life cycle of SHJH ^hr mice were measured. Blood routine (30 items) and serum biochemical indexes (25 items) of 6-week-old SHJH ^hr mice were measured. The venous blood of 8-week-old SHJH ^hr mice was collected for flow cytometry analysis to determine the content of immune cells. The aging bone structure of the cancellous bone and bone mineral density of SHJH ^hr mice aged 4, 8 and 26 weeks were measured by micro-CT. Histopathological changes of bone and joint of 8-week-old mice were observed. Results Compared with ICR mice, the female and male body mass of SHJH ^hr mice were significantly lower at the age of 16 weeks (P < 0.05), and the reproductive performance of female mice was low (P < 0.01) or did not have normal reproductive capacity. The shortest survival time of SHJH ^hr mice was 57 weeks and the longest was 71 weeks, which was shorter than those of normal ICR mice, showing obvious rapid aging phenomenon. At the same time, some physiological and biochemical indexes of blood and pathological changes of bone and cartilage tissues also showed the accelerated aging and abnormality of animal physiological functions. Conclusion SHJH ^hr mice have some biological characteristics of rapid aging as well as some physiological and pathological changes caused by aging.

Analysis on Ileum and Colon Microflora of SPF Male SD Rats based on High-throughput Sequencing

Qin XU, Yan NI, Wenhui SHI, Jianying LI, Jiangwei LIU, Hongqiong ZHAO, Xinming XU

2023, 43(1): 53-60. DOI: 10.12300/j.issn.1674-5817.2022.103

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Objective To analyze the microflora structure and abundance of ileum and colon of SPF male SD rats by high-throughput sequencing technique. Methods The ileal and colonic lumen contents of 30 SPF male SD rats were collected, the total bacterial DNA in the contents was extracted and amplified by PCR. The V3-V4 region of bacterial 16S rRNA in the samples was sequenced using Illumina NovaSeq sequencing platform. Then the species structure and abundance of the intestinal flora were analyzed at the phylum and genus level based on validated data. The diversity and differences between ileal and colonic flora were analyzed using the QIIME software (amplicon) analysis tool. The Tax4Fun program was applied to predict the dominant gene enrichment pathways of ileal and colonic flora. Results At the phylum level, the dominant microflora in the ileum of male SD rats were Firmicutes and Actinobacteria, accounting for more than 98%. The dominant colonic microbial community were mainly Firmicutes and Bacteroidetes, accounting for more than 95%. At the genus level, Lactobacillus and Alistipes were the predominant bacteria in the ileum. The colon was dominated by Lactobacillus and Romboutsia. In terms of flora diversity, the alpha diversity of colonic flora including richness index (Chao1 index) and diversity index (Shannon index) were significantly higher than those of ileum (P < 0.01), and the structural variability of the composition of its flora species was smaller than that of ileum; the ileal flora with significant structural variability were mainly of the phylum Firmicutes, Romboutsia, Peptostreptococcaceae, while the colonic flora had significant structural differences in Bacteroidales. In terms of flora function, the ileal flora dominant genes were significantly enriched in lipid-like metabolism, polyketide metabolism, membrane transport, biodegradation and other pathways, while the colonic flora dominant genes were significantly enriched in glycan biosynthesis metabolism, energy metabolism, biosynthesis of cofactor and vitamins and other products, and other pathways. Conclusion There are significant differences in structure and abundance between the ileal and colonic flora of SPF male SD rats, and the abundance and diversity of colonic flora are higher than those of ileal flora.

Histological Characteristics of the Kidney in Mongolian Gerbils of Different Ages

Lingqun LU, Honggang GUO, Qiaojuan SHI, Fangwei DAI, Xiaofeng CHU

2023, 43(1): 61-66. DOI: 10.12300/j.issn.1674-5817.2022.130

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Objective To explore the histological characteristics of the kidney in Mongolian gerbils of different ages. Methods Ten Mongolian gerbils of 2, 6, and 12 months old (half male and half female) were selected. After euthanasia, the kidneys were taken and paraffin sections were made. After HE, MASSON and PAS staining, the structural differences of different parts of the kidney tissue in gerbils of different ages were observed by digital scanning, and the relevant data of the kidney tissue were measured by image analysis software. Results The number of proximal convoluted tubules was more than that of distal convoluted tubules in the renal cortex and outer medulla of gerbils. With age, the glomerular density decreased, the glomerular diameter increased, the basement membrane of renal tubules thickened, and the fibrous components between renal tubules increased. Conclusion The histological structure of Mongolian gerbil's kidney varies with age, which may be related to glomerulosclerosis and parenchymal cell reduction. The specific mechanism needs further study.

A Preliminary Study on the Domestication and Breeding Methods of Experimental Cats

Yongping HE, Meixian XIE, Zhihui PANG, Qingxiu LI, Huahong HE, Namin ZHANG, Wei LI

2023, 43(1): 67-72. DOI: 10.12300/j.issn.1674-5817.2022.118

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Objective Focused on the laboratory animal domestication and breeding of domestic cats, to explore the feeding management methods and breeding techniques of experimental cats. Methods Seven Chinese garden cats from three litters were introduced from the rural suburbs of Guangzhou, and a breeding seed colony was established. The cats were domesticated in captivity, bred, closed breeding and transmission according to the feeding and management methods of laboratory animal. The population reproduction, the number of pregnancies per year, the litter season, the birth and weaning quality of the cats, and the survival rate of weaning were statistically collected. Results The young breeding cats were able to adapt to the cage feeding management. In the transmission breeding and the expanded breeding colony, the number of female cats pregnant with one, two or three litters a year accounted for 63.2%, 26.3% and 10.5%, respectively. The proportions of litters born from the 1st to the 4th quarters were 20.7%, 20.7%, 27.6%, and 31.0%. A total of 29 pregnancies and 101 kittens were got from 19 female cats, with an average of (3.5±1.33) kittens per litter. The birth weights of female and male cats were (89.31±13.69) g and (93.47±15.12) g, respectively. Sixty-seven kittens survived from weaning. The average survival rate was 60.86%, and the weaning weights of female and male cats were (361.62±82.77) g and (376.0±91.71) g, respectively. Conclusion Domestic Chinese garden cats can adapt to laboratory animal feeding and breeding rules, and have strong fertility. They can normally pregnant and breeding throughout the year. The kittens grow to 5-6 months of age can meet the weight requirements for the examination of pharmaceutical hypotensive substances, and can be used as experimental cats for pharmaceutical examination with clear origin.

Application of Virtual Reality in the Behavior Analysis of Laboratory Animal

Chaochao ZHANG, Xuesong TIAN

2023, 43(1): 73-78. DOI: 10.12300/j.issn.1674-5817.2022.078

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Virtual reality (VR) is an emerging technology for computer scene simulation that has become a reliable tool for animal behavior analysis in recent years. The behavior of an animal is crucial for its survival. Animal behavior analysis provides useful information for scientific progress in genetics, ecology, neuroscience, economics, and robotics. Animal behavior analysis can be divided into open-loop studies, in which the stimulus is independent of the animal's response to the stimulus, and closed-loop studies, in which the stimulus is adjusted according to the real-time movement of the animal. In this paper, we have discussed the origin, concept, and applications of animal behavior analysis, and reviewed the shortcomings of open-loop research on animal behavior represented by visual stimulation using traditional technology. Then taking visual, olfactory, tactile, and auditory stimulation as examples, we analyzed that the animal behavior analysis system using VR technology could combine precise experimental control with the complex behavior of laboratory animals, and complete the closed-loop research that could not be achieved using the experimental methods of the real physical world. The prospect of VR in developing superior simulations and virtual animals in the future was also discussed.

Safety Management Countermeasures Based on the Status of Laboratory Animal Administrative Licensing in Jiangsu Province

Chongyang QI, Lin CHEN, Man AI, Hang XU, Housen ZHANG

2023, 43(1): 79-85. DOI: 10.12300/j.issn.1674-5817.2022.083

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With the rapid development of life science, laboratory animals play an important role in many fields, such as biomedicine. In recent years, the national reforms “to delegate power, streamline administration and optimize government services” continues deepen, the laboratory animals administrative departments have paid more attention to interim and post-event supervision, in which the safety management of laboratory animals is the key and challenging point in the supervision process. In this paper, the information regarding licenses for laboratory animals in Jiangsu, facility areas, animal production and usage, employees, and other information the past three years were introduced. Combined with legal administrative management in recent years, the potential safety risks in the production and use of laboratory animals were analyzed, such as risks in management systems, employees, facility operation, animal quality control, accidental injury and zoonosis, animal transportation, and waste disposal. Additionally, the current construction, daily management services, and the list of interim and post-event supervision of the laboratory animal safety management system were introduced. Based on this discussion, we proposed relevant safety management countermeasures and suggestions and provided support for further improvements to laboratory animal safety management.

Introduction to the International Guide for Animal Research Reporting ARRIVE 2.0, and Its Implementation Plan in the Journal

Junyan ZHANG, Xiaoyu LIU, Yao LI, Guoyuan CHEN, Xiao LU, Yu BAI, Xuancheng LU, Wanyong PANG, Baojin WU

2023, 43(1): 86-94. DOI: 10.12300/j.issn.1674-5817.2023.014

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Animal experiments play an important role in the process of biomedical research, and is a necessary way to transform basic medicine into clinical medicine. The standardization of animal experimental studies and reports determines the reliability and reproducibility of research results, and is also the key to transforming the results of animal experiments into clinical trials. In view of how to design and implement animal experiments, write animal experiment reports, and publish relevant academic papers in a more standardized way, LACM (Laboratory Animal and Comparative Medicine) has launched a new column of comparative medical research and reporting standards from 2023, focusing on the introduction and interpretation of international general norms related to laboratory animal and comparative medicine, such as ARRIVE 2.0 guidelines (Animal Research: Reporting of In Vivo Experiments). This article focuses on the development and application, basic content and priority of ARRIVE 2.0, as well as the scheme of implementing ARRIVE 2.0 guidelines in international biomedical journals, and explains the current situation and future plans of LACM following ARRIVE 2.0 guidelines. The research and report of animal experimental medicine following the ARRIVE 2.0 guidelines and other international norms is one of the important driving forces to promote the high-quality development of experimental animal science and biomedicine in China, and also a powerful means to implement the 3R principle and improve the welfare of laboratory animals. Through this article, we hope the majority of scientific researchers and editors will attach great importance and actively implement these international standards.

Scientific Innovation Teams in China

2023, 43(1): 99-100. DOI: 10.12300/j.issn.1674-5817.2023.011

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