Laboratory Animal and Comparative Medicine

Current Situation and Development Countermeasures of Laboratory Animal Management in Heilongjiang Province

LI Baolong, WANG Yu’e, SI Changde, LI Changwen, LIU Huairan, GUO Shouli, HAN Lingxia, LIU Jingli, LI Yongming, CHEN Hongyan

2021, 41(3): 181-189. DOI: 10.12300/j.issn.1674-5817.2021.043

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As an irreplaceable basic support and strategic biological resource for the development of life science research and medicine, laboratory animals are becoming more and more important. Based on present domestic laboratory animal industry development, this review expounded the development status and management problems of laboratory animals in Heilongjiang Province. This article also provided development countermeasures, such as improving the local laws and regulations pertaining to laboratory animals, strengthening the administrative licensing management, improving production conditions and quality, ensuring transportation and supply, supporting laboratory animal scientific research, promoting talent training, and strengthening biosafety control. These measures are proposed to drive the development of the laboratory animal industry, improve its support and guarantee for the biomedical industry, and make contribution to local economy, society, people's livelihood, and even the development of science and technology.

Status and Development Countermeasures of Laboratory Animal Resource Sharing Service Platform Construction in Shanxi Province

ZHANG Ruihu, WANG Chunfang, SONG Guohua, CHEN Zhaoyang

2021, 41(3): 190-194. DOI: 10.12300/j.issn.1674-5817.2020.207

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The construction of Shanxi Laboratory Animal Resource Sharing Service Platform was summarized in terms of resource development, sharing services and talent cultivation. Combining with the advanced construction experience of developed regions and future development trends, an in-depth analysis of the problems existing in the platform construction was carried out, and the countermeasures and directions were proposed for the construction and development of the platform.

A Brief Interpretation of AVMA Guidelines on Euthanasia of Animals: 2020 Edition

LU Jin, ZHANG Ying, PAN Xueying, WANG Jian, YAN Guofeng, ZHOU Jing, ZHU Lian, CHEN Xuejin, LI Yao, PANG Wanyong

2021, 41(3): 195-206. DOI: 10.12300/j.issn.1674-5817.2021.086

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Overdose of injectable barbiturates or barbituric acid derivatives is the most preferred euthanasia method for the majority of animal species. However, these controlled drugs have limited access, and in this case or in any other scientific concerns, veterinarians may choose alternative, conditional acceptable methods as recommended by American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals. In the newest 2020 Edition, existing euthanasia methods, techniques and agents of euthanasia are updated according to recently released articles. This article summarized the updated content of the new version of the AVMA Guidelines and on this basis, enumerated the most preferred methods of euthanasia, methods acceptable with conditions and unacceptable methods to euthanize different animal species. In addition, principles and precautions related to animal euthanasia were also briefly described.

Effects of Stress on Appetite in Rats Through Hypothalamus and Stomach Ghrelin Pathway

ZHAO Yiling, GAO Pengfei, XIAO Qian, ZHANG Lan, PAN Danqing, XU Yuemei

2021, 41(3): 207-214. DOI: 10.12300/j.issn.1674-5817.2020.160

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Objective To study the molecular mechanism of stress on appetite through hypothalamus and stomach ghrelin pathway. Methods Thirty-two male Wistar rats were randomly divided into a control group and a stress group with 16 rats in each group and 2 rats in each cage. Stress was induced by tail pinch for consecutive 21 days. Food intake and body weight of the rats in each group were recorded. Serum ghrelin concentrations of eight rats in each group were detected by ELISA on day 7 and day 21. Ghrelin protein expression in stomach and hypothalamus as well as Amp-activated protein kinase α2 (AMPKα2) protein expression in hypothalamus of rats were measured by Western blotting. The mRNA levels of ghrelin, growth hormone secretagogue receptor (GHSR) and leptin in stomach as well as ghrelin, AMPKa2, neuropeptide Y (NPY), agouti-related protein (AgRP) in hypothalamus were measured by real-time quantitative PCR. Results On day 7, the total food intake of rats in each cage in the stress group was higher than that in the control group (P < 0.05), and there was no significant difference in total food intake between the two groups on day 21 (P > 0.05). On day 7 and day 21, there was no significant difference in body weight between the two groups (P > 0.05). Stress upregulated the serum levels of acylated ghrelin (P < 0.01), the ghrelin protein expression levels in stomach and hypothalamus (P < 0.05), and the AMPKα2 protein expression level in hypothalamus (P < 0.01). Stress also upregulated the ghrelin and GHSR mRNA expression levels in stomach (P < 0.01), downregulated the leptin mRNA expression level in stomach (P < 0.01), and upregulated the ghrelin, AMPKα2, NPY and AgRP mRNA expression levels in hypothalamus (P < 0.01). Conclusion Tail pinch-induced stress can increase central and peripheral ghrelin levels in rats, and act as a temporary appetite enhancer.

Comparative Study of Three Pineal Gland Injury Model in Rats

WANG Danni, SONG Meiqing, YANG Qian, FENG Mali

2021, 41(3): 215-219. DOI: 10.12300/j.issn.1674-5817.2020.156

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Objective To compare pineal gland injury models established using para-chlorophenylalanine (PCPA), electrical stimulation stress, and PCPA plus electrical stimulation, to evaluate the structure and function changes of the pineal gland, and to establish a simple and effective method of pineal gland injury model with short modeling time. Methods Forty rats were adaptively reared for 1 week and randomly divided into blank control, PCPA, electrical stimulation, and PCPA + electrical stimulation groups. The control group was left untreated. The PCPA group was intraperitoneally injected with PCPA 450 mg/kg for 2 consecutive days. The electric stimulation group was given a shuttle box with electric stimulation (voltage 30 V, current 0.8 A, stimulation time 30 s, interval 30 s, 60 cycles) for 5 consecutive days. The rats in the PCPA + electrical stimulation group were given electrical stimulation plus PCPA on the 4th day. The operation was the same as before. The rats in each group were tested for open field activities, elevated plus maze and other behaviors, pentobarbital sodium for sleep and other indicators. ELISA was performed to detect serum melatonin (MT) levels, and the pathological structure of the pineal gland was observed under a microscope. Results The total distance and average speed of the rats in the three experimental groups were significantly lower than those of the rats in the control group (P < 0.01), and the central area time of the PCPA and PCPA + electrical stimulation groups was significantly higher than that of the control group (P < 0.05). The open arm number was significantly higher in the PCPA + electrical stimulation group than that the control group (P < 0.05), and the open arm time ratios of the PCPA and PCPA + electrical stimulation groups were significantly higher than that of the control group (P < 0.05). The latency period to falling asleep in the PCPA group was significantly higher than that in the control group (P < 0.05). The serum MT levels in the three experimental groups were significantly lower than that of the control group (P < 0.01). In the PCPA group, the pineal gland cells were disorderly arranged, nuclear pyknosis was evident, the number of cells was significantly reduced, and vacuolar degeneration was increased. In the electrical stimulation group, the pineal gland cells were disorderly arranged, nuclear pyknosis was evident, the number of cells was slightly reduced, and vacuolar degeneration was slightly increased. In the PCPA + electrical stimulation group, the pineal gland cells were disorderly arranged, nuclear pyknosis was evident, the number of cells was significantly reduced, and vacuolar degeneration was increased. Conclusion Different degrees of damage to the pineal gland in rats can be caused by three different methods of making model, which can provide reference for making different pineal gland injury models.

Establishment of a Xenograft Model of Human Glioma in Rats

TAN Dengxu, WU Pengpeng, ZHAO Yong, SHI Changhong, GE Xu

2021, 41(3): 220-225. DOI: 10.12300/j.issn.1674-5817.2020.094

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Objective To establish a rat xenograft model of human glioma and to provide ideal animal models for clinical and basic research by injecting immunosuppressants. Methods SD rats were divided into rapamycin (Rapa), cyclosporin A (CsA), Rapa+CsA, or negative control groups (n = 5 each) for 3 days before surgery. Human glioma U87-MG cells were injected orthotopically into the rat brains and subcutaneously into the back, and the volume and weight of subcutaneous tumors were monitored regularly. Once the subcutaneous tumors had grown to approximately 600 mm³, the growth of the brain glial tumors in situ was detected by near-infrared fluorescence optical imaging. The rats were sacrificed and tissue samples were isolated, and hematoxylin and eosin and immunohistochemical (IHC) staining were performed to determine the modeling success rate. Results Compared with Rapa or CsA treatment, Rapa+CsA treatment showed an obvious immunosuppressive effect. The success rate of Rapa+CsA treatment model reached to 100%, while that of Rapa or CsA treatment model was only 0% and 40%, respectively. The mean body weight of the rats in the combination group was significantly lower than that in the other groups. A strong near-infrared fluorescent dye signal was detected in the rat brain by optical imaging and was further confirmed to be glioma tissue by histopathological analysis. The IHC staining was strongly positive for the human mitochondrial antibody at the tumor site. Conclusion A rat xenograft model of human glioma can be successfully established by the combined injection of immunosuppressants Rapa+CsA, and the morphology of human glioma can be conformed by pathological analysis.

Effect of Helicobacter hepaticus Infection on Chronic Colitis Induced by Dextran Sulfate Sodium

WU Zhihao, SHEN Chen, YIN Jun, ZHU Liqi, ZHANG Quan

2021, 41(3): 226-231. DOI: 10.12300/j.issn.1674-5817.2020.136

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Objective To investigate the effect of Helicobacter hepaticus (H.h) infection on chronic colitis induced by dextran sulfate sodium (DSS). Methods Forty male interleukin (IL)-10^-/- mice were randomly divided into control, H.h, DSS, and H.h + DSS groups. Mice in the H.h group and H.h + DSS group were infected with H.h by gavage. Five days later, the mice in DSS group and H.h + DSS group received two 7-day cycles of a 2% DSS drink with a 5-day withdrawal time between them, while the other groups received sterilized water. At the end of the experiment, all groups of mice were sacrificed and the colon of each mouse was excised, the colon length was measured, and the colon divided into three parts for histopathological examination, cytokine mRNA transcription detection, and phosphorylation detection of signal transducer and activator of transcription 3 (STAT3). Results The pathological results showed that lymphocytes infiltration, loss of epithelium, degree of fibrosis, and crypt atrophy were more obvious in the H.h + DSS group than those in the DSS group. In addition, the transcription levels of the cytokines IL-6 and tumor necrosis factor-α (TNF-α) were upregulated, while STAT3 was activated. Conclusion H.h infection can aggravate DSS-induced chronic colitis in mice.

Thyroid Function of Chronic Intermittent Hypoxia Rat Model

TIAN Hui, GAO Long, WANG Zehui

2021, 41(3): 232-237. DOI: 10.12300/j.issn.1674-5817.2020.153

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Objective To establish a chronic intermittent hypoxia (CIH) rat model to explore the correlation of obstructive sleep apnea-hypopnea syndrome (OSAHS) with thyroid function and its mechanism. Methods SD rats were divided into control group, CIH group, and reoxygenation group (RH group). The CIH animal model was established using a hypoxic chamber and was identified. The expression levels of thyroid stimulating hormone (TRH), thyroid stimulating hormone (TSH), triiodothyronine (T3), and thyroxine (T4) in the serum of each group of rats were detected by radioimmunoassay. The pathological changes in the thyroid tissue in each group were observed by hematoxylin and eosin (HE) staining and toluidine blue staining. Changes in the ultrastructure of the thyroid tissue in rats were observed by electron microscopy. Results The CIH and RH rats had blood oxygen saturations of 70%-92%, and arterial oxygen partial pressures of 60.7-80.1 mmHg, which were close to the pathophysiology of OSAHS, therefore, the CIH animal model was successfully established. Compared with the expression levels of TRH, TSH, T3, and T4 in the serum of rats in the control group, those of rats in the CIH group were significantly reduced (all P < 0.05), while the thyroid mass was significantly increased (P < 0.01). Compared with the expression levels of TRH, TSH, T3, and T4 in the serum of the CIH group rats, those in the serum of the RH group rats increased significantly (all P < 0.05), while the thyroid mass was decreased (P < 0.05). The HE and toluidine blue staining results showed that the thyroid tissue of the control group had intact thyroid follicles, whereas the thyroid tissue of the CIH group showed normal follicular structure loss, some follicular cells with vacuoles, and a dark basal nucleus. The thyroid tissue of the RH group showed normal follicular structure, but some follicular cells were still vacuolar with dark nuclei. Electron microscopy showed that the thyroid tissue of the control group rats had normal follicular cells, while the follicular cells in the thyroid tissue of the CIH group showed nuclear contraction, increased heterochromatin, and matrix loss. The thyroid tissue of the RH group had complete follicular cell structure. Conclusion CIH can cause thyroid dysregulation in rats, and thyroid function partially returns to normal once hypoxia is resolved.

Changes of C-reactive Protein and Inflammatory Factors in Juema Pig Model with High Altitude Multiple Organ Dysfunction Syndrome

XIAO Pan, NIU Tingxian, LUO Xiaohong, WANG Hongyi, GUO Xiaoyu, LU Lu, FENG Xiaoming

2021, 41(3): 238-243. DOI: 10.12300/j.issn.1674-5817.2020.119

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Objective To investigate the changes of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1α) and C-reactive protein (CRP) in the development of inflammation in the Juma pig odel with high altitude multiple organ dysfunction syndrome (H-MODS). Methods Three doses (0.25, 0.35, and 0.50 mg/kg) of endotoxin lipopolysaccharide (LPS) were used to induce the H-MODS model (group B, C and D), and normal saline was used as control (group A). The serum levels of TNF-α, IL-1β and CRP were detected at different time points. Results The expression levels of TNF-α and IL-1β increased sharply after LPS infusion to the pigs, and reached the highest level at 6 h; the levels at 3-12 h of the pigs treated with LPS were increased compared with the level at 0 h of the pigs with the same treatment and the level of compared group A at the same time point (P < 0.05 or P < 0.01), and then decreased gradually. The expression levels of TNF-α at 24 h in groups B and D were significantly higher than that in the same group at 0 h and group A at the same time point (P < 0.05), and the expression level of TNF-α was significantly higher in group B than that in group A at 72 h (P < 0.05). The expression level of IL-1β at 24 h in group D was higher than that at 0 h and that in group A at the same time point (P < 0.05), and then decreased gradually. Compared with 0 h and group A, the expression levels of IL-1α in group D decreased significantly at 48 h and 72 h (P < 0.05 or P < 0.01). After intravenous infusion of LPS, the expression levels of CRP in groups B, C and D began to increase, and reached the maximum at 24-48 h; the expression levels of CRP in the other groups at 6-72 h were significantly higher than those in group A and at 0 h (P < 0.05 or P < 0.01). Conclusion Severe inflammatory reaction occurs during the development of H-MODS, and the expressions of inflammatory factors TNF-α and IL-1β increase rapidly first and then decrease sharply under immune regulation, displaying large fluctuations in short time until achieving an equilibrium; the CRP expression changes slowly throughout the inflammatory reaction in vivo.

Effects of Matrine on Th1/Th2 Balance and Related Pathways in Rats with Experimental Autoimmune Thyroiditis

YAO Ting, GAO Yuan, ZENG Fangxin

2021, 41(3): 244-251. DOI: 10.12300/j.issn.1674-5817.2020.086

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Objective To investigate the effects of matrine on inflammation, helper T lymphocytes (Th)1/Th2, and monocyte chemoattractant protein-1 (MCP-1)/cell surface chemokine receptor 2(CCR2) signaling in rats with experimental autoimmune thyroiditis (EAT). Methods Seventy-five Lewis rats were divided into control, model, and low-, medium-, and high-dose matrine groups using the random number table method (n = 15 each). The EAT rat model was induced by immunization with porcine thyroglobulin (pTG). The low-, medium-, and high-dose groups were intragastrically administered 0.2, 0.4, and 0.6 g/kg matrine, respectively, at a volume of 2 mL. Rats in the control and model groups were given 2 mL of normal saline, once every other day for 4 weeks. The histomorphological changes in the thyroid were observed by hematoxylin and eosin (HE) staining. Serum levels of thyroid stimulating hormone (TSH), anti-thyroglobulin antibody (TGAb), thyroid peroxidase antibody (TPOAb), cytokine interferon-γ (IFN-γ), and interleukin (IL)-12, IL-4, and IL-10 were detected by ELISA. Results Compared with the TSH, TGAb, TPOAb, IFN-γ, IL-12, IFN-γ/IL-4, IL-12/IL-10, nuclear factor-kappa B p65 (NF-κBp65), MCP-1, and CCR2 protein levels in the control group, those in the model group were significantly increased, and the levels of IL-4 and IL-10 were significantly decreased (P < 0.05). After matrine treatment, TSH, TGAb, TPOAb, IFN-γ, IL-12, IFN-γ/IL-4, IL-12/IL-10, NF-κBp65, MCP-1, and CCR2 protein levels were significantly decreased, and IL-4 and IL-10 levels were significantly increased (P < 0.05); the differences were statistically significant (P < 0.05). Conclusion Matrine can inhibit the production of thyroid antibodies, downregulate Th1 cytokines, and upregulate Th2 cytokines, thus reducing the Th1/Th2 ratio and inhibiting the MCP-1/CCR2 signaling axis, thus playing a protective role in EAT.

Analysis on Air Distribution by Computational Fluid Dynamics Simulation in Two Types of Laboratory Animal Barrier Facilities

ZHOU Bin, ZHAO Yong, WANG Yabing, LIU Jihong

2021, 41(3): 252-258. DOI: 10.12300/j.issn.1674-5817.2021.077

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Taking the removal efficiencies of waste heat, ammonia, and carbon dioxide, and the mean age of air as the evaluation indicators of air distribution, the pros and cons of air distribution in two different types of animal barriers, an experimental facility and a breeding facility, were analyzed at various air change rates and exhaust outlet positions by computational fluid dynamics (CFD) numerical simulation technology. The results of selected indicators showed that ceiling air supply plus bottom exhaust outlet were suitable for the laboratory animal barrier facilities. In addition, air change rate in high-density breeding facilities should be adjusted according to the room temperature.

Analysis on Carriage of Streptococcus pneumoniae in Captive Rhesus Macaques

LIU Yu, LI Yanyan, ZHANG Wei, YANG Fengmei, LIU Quan, LI Yongjie, JIN Weihua, DUAN Suqin, WANG Junbin, CHEN Lixiong, XU Hongjie, ZHAO Yuan, HE Zhanlong

2021, 41(3): 259-265. DOI: 10.12300/j.issn.1674-5817.2021.034

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Objective To explore the natural infection status of Streptococcus pneumoniae in captive bred rhesus macaques, and to provide reference for the clinical diagnosis and treatment of Streptococcus pneumoniae infection. Methods Twenty infant monkeys, 30 adult monkeys and 30 elderly monkeys were randomly selected from a rhesus macaques population, and the nasal swabs and pharynx swabs were collected and screened for Streptococcus pneumoniae simultaneously by bacterial culture and real-time fluorescent quantitative PCR. The mother monkeys of the positive infant monkeys were screened for Streptococcus pneumoniae. Gram staining method, Optochin drug sensitivity test, bile solubility test, inulin fermentation test and PCR amplification and sequencing were used to further identification. Results The positive rate of rhesus macaques carrying Streptococcus pneumoniae was 20% (4/20), and the mother macaques of the four positive infant macaques tested negative. The positive rate was 0% in both adult and elderly rhesus monkeys. The morphological observation results of the bacterial culture showed that the bacteria appeared gray and raised, and produced a surrounding green zone of hemolysis with a flattened center, indicating typical Streptococcus pneumoniae colony characteristics. The results of Gram dyeing, bile dissolution test, and inulin fermentation test were all positive, and the bacteria were sensitive to Optochin. The above results showed that the four strains isolated from the infant monkeys’ nasopharyngeal swabs were Streptococcus pneumoniae. Conclusion The investigation findings show that Streptococcus pneumoniae is mainly found in the noses and throats of 20% infant monkeys among rhesus macaques, and no vertical transmission from mother to child is found.

Application of Sandwich Teaching Method Based on Internet Interactive Platform in Teaching of Medical Experimental Zoology

WEI Sheng, GENG Xiwen, XU Kaiyong, HU Minghui, ZHOU Miaomiao, LI Zifa

2021, 41(3): 266-270. DOI: 10.12300/j.issn.1674-5817.2020.114

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Medical experimental zoology is a supporting discipline for biomedical research with strong practicality and applicability. Adapting the talent training strategy of medical colleges, cultivating practical and innovative talents, reforming the teaching mode, and improving students' practical abilities are problems that urgently require solutions in the current course of medical experimental zoology. This article aims to explore the teaching advantages and related concepts of the internet interactive platform + sandwich teaching method in the course of medical experimental zoology to improve its teaching quality.

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