Table of Content

25 April 2021, Volume 41 Issue 2

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Development Vision of Laboratory Animal Resources in China Based on Ability Improvement

HE Zhengming

2021, 41(2):  85-90.  DOI: 10.12300/j.issn.1674-5817.2021.042

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Laboratory animal is one of the important basic scientific research conditions to support the science and technology innovation in the fields of life science and biomedicine. The new and rapid development trend of scientific and technological innovation in China calls for strengthening the strategic position of laboratory animal resources, accurately grasping the creation mode of laboratory animal resources, further improving the policy system and operation mechanism conducive to the creation and development of laboratory animal resources, as well as the service platform for the preservation and sharing of laboratory animal resources, aiming to improve the service level of laboratory animal resources for national scientific and technological innovation.

A Modified ALPPS Mice Model Based on the Blockade of Portal Vein by Matrigel

SHEN Hao, CHEN Yao, SHEN Feng, WU Mengchao

2021, 41(2):  91-99.  DOI: 10.12300/j.issn.1674-5817.2020.204

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Objective To construct a modified mice model associating liver partition and portal vein ligation for staged hepatectomy (ALPPS) without relying on microsurgical techniques, and to verify its effectiveness in stimulating liver regeneration. Methods Seventy-five adult C57BL/6 mice were randomly divided into three groups: ALPPS-microsurgery (ALPPS-micro) group, ALPPS-matrigel blocking (ALPPS-matri) group and matrigel blocking control (Control-matri) group. The ALPPS-micro group and the ALPPS-matri group were treated with microscopic ligation and matrigel injection to block the portal vein blood flow, respectively. The Control-matri group served as a control group to verify the effectiveness and safety of this surgery. Data of operation time, intraoperative bleeding and perioperative survival were recorded, and blood and liver samples were collected at different time points after surgery. The effects of different operations on liver regeneration were compared by measuring the ratio of liver weight to body weight (LBR) and detecting the expression levels of proliferation-related molecules Cyclin D1 and Ki-67. Results Compared with the ALPPS-micro group, the ALPPS-matri group had shorter operation time [(19.0±4.6) min vs (37.5±9.3) min, P<0.05], less intraoperative bleeding [(153±39) mL vs (317±124) mL, P<0.05], and lower mortality of Ⅰ stage surgery (4.0% vs 20.0%, P<0.05). The detection results of LBR and the expression levels of proliferation-related molecules suggested that the ALPPS-matri group showed a similar effect on stimulating liver regeneration as the ALPPS-micro group. Conclusion Compared with the traditional microsurgery-based ALPPS model, the modified ALPPS model using matrigel has a similar effect on liver regeneration with shorter operation time, less intraoperative bleeding, and lower perioperative mortality.



Comparison of Different Slicing and Staining Methods of Caudal Intervertebral Discs in Rats

ZHAO Yupeng, ZHOU Pinghui, GUAN Jianzhong

2021, 41(2):  100-107.  DOI: 10.12300/j.issn.1674-5817.2020.108

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Objective To compare the advantages and disadvantages of different slicing and staining methods of caudal intervertebral disc in rats, so as to provide a theoretical basis for clinical diagnosis and experimental research. Methods The caudal intervertebral discs of SD rats were collected and made into frozen sections and paraffin sections. Hematoxylin-eosin staining (HE staining), safranin O-fast green staining, Masson staining, and toluidine blue staining of the caudal intervertebral disc sections, and immunofluorescence staining of typeⅠ collagen (COL-Ⅰ), typeⅡcollagen (COL-Ⅱ) and glycosaminoglycan (GAG) were performed to observe the tissue morphology and protein expression. Results Of the selected staining methods, HE staining of the paraffin sections clearly showed each layer structure of intervertebral discs; Masson staining results showed fibrous annulus tissues and nucleus pulposus tissues; safranin O-fast green staining and toluidine blue staining results showed cartilage layers and subchondral bone structures. In the frozen sections with immunofluorescence staining, COL-Ⅰ was dominant in fibrous annulus, COL-Ⅱwas dominant in nucleus pulposus, and GAG was mainly secreted by nucleus pulposus cells and nucleus pulposus-like cells. Conclusion When observing the layer structure of intervertebral discs, staining techniques such as HE staining and MASSON staining should be chosen, and paraffin sections are better than frozen sections. When observing the expression and distribution of various proteins of intervertebral discs, immunofluorescence should be selected, and frozen sections are superior to paraffin sections.



Application of Immunocompetent HBV cccDNA Mouse Model in the Efficacy Evaluation of Matrine and Artemisinin on Inhibiting HBV

WU Yue, LÜ Xiaoqin, LIU Yang, XIANG Xia, ZHAO Zhonghua, XU Ruqing, PAN Shuohan, HE Mingzhong, ZHANG Huatang, LAI Guoqi

2021, 41(2):  108-115.  DOI: 10.12300/j.issn.1674-5817.2021.016

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Objective To evaluate the effects of matrine and artemisinin on immunocompetent hepatitis B virus (HBV) infected mouse model, and to lay a foundation for the application of the model. Methods HBV covalently closed circular DNA (HBV cccDNA) was synthesized in vitro. Male CBA/CaJ mice aged 8 to 10 weeks were injected with HBV cccDNA at high pressure through tail vein, and then intraperitoneally injected with matrine or artemisinin in acute or chronic infection periods, respectively. Quantitative PCR was used to detect HBV DNA and cccDNA in serum or liver tissues of mice. Enzyme-linked immunoadsorption assay (ELISA) was used to measure the content of HBV surface antigen (HBsAg) in serum. Immunohistochemistry was used to determine HBsAg and HBV core antigen (HBcAg) in liver tissues. Results In HBV acute infection period, matrine and artemisinin injection at week 4 significantly inhibited the secretion of HBsAg in serum of mice (both P<0.05), and reduced the expressions of HBV DNA (both P<0.01). In HBV chronic infection period, matrine and artemisinin had no significant inhibitory effects on HBsAg in serum and liver tissues (all P>0.05), but the decrease of HBsAg in serum in the group administered with matrine or artemisinin was significantly higher than that in the normal saline control group (both P<0.05). Moreover, matrine decreased the copy number of HBV DNA in serum and liver tissues (P<0.05 and P<0.01, respectively) and the copy number of HBV cccDNA in liver tissues (P<0.05). Artemisinin only inhibited the copy number of HBV DNA in serum of mice (P<0.01), but had no significant effect on HBV DNA and HBV cccDNA in liver tissues (both P>0.05). In addition, matrine had no significant effect on HBcAg in liver tissues (P>0.05), but artemisinin did the opposite (P<0.05). Conclusion The immunocompetent HBV cccDNA mouse model can be used to evaluate the efficacy of drugs for the treatment of acute and chronic hepatitis B.



Construction and Evaluation of Skin Photoaging Mouse Model

KONG Yue, GUO Yan

2021, 41(2):  116-121.  DOI: 10.12300/j.issn.1674-5817.2020.191

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Objective To establish a mouse model of skin photoaging induced by ultraviolet B (UVB) radiation, and to provide a reference for the study of skin photoaging. Methods Thirty male KM mice were divided into a control group and a model group. The control group received normal light, while the model group was subcutaneously injected with D-galactose combined with narrow-band UVB daily irradiation for 40 min at a dose of 120 mJ/cm2 for 40 d. The skin appearance of mice with fur on the back being removed was observed. Skin tissue pathology was observed after HE staining and Masson staining. Biochemical indexes of skin tissue homogenate including glutathione peroxidase (GSH-PX), superoxide dismutase (SOD), hydroxyproline (HYP), and malondialdehyde (MDA) were measured by enzymatic analysis. The expressions of matrix metalloproteinase-1 (MMP-1), c-fos, and c-jun were determined by Western blotting. Results The skin of the model group was darker, looser, drier, more rough, and deeper than that of the control group. Pathological observation revealed that the epidermis thickened and the dermis fibers were reduced, broken, and disorderly arranged in the model group. In the model group, the activities of GSH-PX and SOD decreased, the content of MDA increased, the content of HYP decreased, and the expression levels of MMP-1, c-fos and c-jun of the model group increased (all P<0.01). Conclusion The skin damage of mice induced by subcutaneous injection of D-galactose combined with UVB irradiation is consistent with photoaging, so it is an effective mouse model of skin photoaging.



Helicobacter hepaticus Infection Promotes High Fat Diet-induced Non-alcoholic Fatty Liver Disease in Mice

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

2021, 41(2):  122-130.  DOI: 10.12300/j.issn.1674-5817.2020.181

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Objective To investigate the effect of Helicobacter hepaticus (H.h) on non-alcoholic fatty liver disease induced by a high fat diet (HFD). Methods Twenty 6-week-old male BALB/c mice were randomly divided into four groups: control group, H.h group, HFD group and H.h+HFD group. The H.h+HFD group was fed a HFD after H.h infection. After 12 weeks of feeding, the levels of triglyceride (TG), alanine transaminase (ALT) and aspartate aminotransferase (AST) were measured. Besides, the liver index was calculated, and oil red O staining, Sirius red staining, hematoxylin-eosin staining, real-time fluorescence quantitative PCR (RT-PCR) and immunohistochemistry were conducted to evaluate liver pathological changes in the liver samples. Results The serum levels of TG, ALT and AST in the H.h+HFD group were significantly higher than those in the control group, H.h group and HFD group (all P<0.05). The livers of mice in the H.h+HFD group showed multiple lesions, fat accumulation, ballooning degeneration and necrosis, collagen fiber deposition. The livers of mice in the H.h group exhibited inflammatory cell aggregation and a small amount of collagen fiber deposition, whereas the livers of mice in the HFD group showed lipid droplets accumulation, steatosis and a small amount of collagen fiber deposition. Moreover, the mRNA levels of interleukin (IL)-6, IL-1?, transforming growth factor-? (TGF-?) and tumor necrosis factor-〈 (TNF-〈) were significantly elevated in the livers of mice in the H.h+HFD group compared to the other three groups (all P<0.05). The expressions of alpha-smooth muscle actin (〈-SMA) and collagen typeⅠ(CollagenⅠ) were significantly increased in the H.h+HFD group. Conclusion H.h infection can promote non-alcoholic fatty liver disease induced by feeding HFD to mice.



Jejunal Nerve Regulation and Histomorphological Changes in Rats with Early Type 1 Diabetes

WANG Man, ZHI Minghua, GENG Xufang, TONG Miaomiao, LIANG Hongyu, ZHAO Ding

2021, 41(2):  131-137.  DOI: 10.12300/j.issn.1674-5817.2020.065

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Objective To investigate functional changes of nitrergic and cholinergic nerves in the jejunum smooth muscle, and to observe jejunal histomorphology in rats with early type 1 diabetes. Methods Male Sprague Dawley (SD) rats were randomly divided into a normal control group and a diabetic model group. Rats in the diabetic model group were administered 60 mg/kg streptozotocin (STZ) by intraperitoneal injection. After 4 weeks, neurogenic contractile responses were induced by electric field stimulation, and functional changes in nitrergic and cholinergic nerves were observed in the isolated jejunum of diabetic rats. Histomorphological changes in the jejunum were observed by hematoxylin and eosin staining of paraffin-embedded tissue sections and immunohistochemistry. Results Compared with that in the normal rats, the jejunal intermuscular cholinergic nerve rats with early diabetes was damaged, whereas the nitrogenous nerve did not exhibit a such lesions. The villi of the jejunum were disrupted and disorderly, the muscularis was thickened, goblet cell numbers were decreased, and the ratio of villus length to intestinal crypt depth was diminished in diabetic rats. Conclusion In the early stage (4 weeks) of STZ-induced type 1 diabetes in rats, the neurological function and morphology of jejunal tissues were significantly changed. It suggests that the intestine is a sensitive organ in diabetes and has important clinical implications for the early prevention and detection of diabetes.



Therapeutic Effect of Wuling Powder on Guizhou Mini-pigs of Hyperlipidemia Model

ZHANG Hui, WU Yanjun, YAO Jin, LU Taofeng, WANG Guoqi, ZHAO Hai, YAO Gang, WU Shuguang

2021, 41(2):  138-142.  DOI: 10.12300/j.issn.1674-5817.2020.188

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Objective To study the therapeutic effect of Wuling powder on hyperlipidemia induced by a high-fat diet in Guizhou mini-pigs. Methods Twelve healthy Guizhou mini-pigs were selected, 4 pigs fed a standard diet (normal group), and 8 pigs fed a high-fat diet for 24 weeks to establish a hyperlipidemia model. Further, the model pigs were divided into model group and Wuling powder treatment group, and fed a high-fat diet for 8 weeks. Wuling powder (0.55 g/kg) was added to the diet of the treatment group every day. Body weight, body size, abdominal girth, serum lipid parameters, and main syndromes and signs were measured and recorded. Results After the animal model was established successfully, body weight and body mass index (BMI) of the model group were found to be higher than that of the normal group, although the difference was not significant (P>0.05). The levels of the following main blood lipid parameters: total cholesterol (TC), triglyceride (TG), and low-density lipoprotein cholesterol (LDL-C) increased significantly (all P<0.05) in the model group, but high-density lipoprotein cholesterol (HDL-C) level decreased significantly (P<0.05) as compared with the normal group. The pigs in the model group showed obesity, abdominal enlargement, hair thinning, tiredness and fatigue, and light or soft stool, which are syndroms of hyperlipidemia. After Wuling powder treatment, body weight and BMI of the mini-pigs in the treatment group were lower than those in the model group (P>0.05); the comprehensive score of traditional Chinese medicine syndrome was significantly reduced, in addition to the TC, TG, and LDL-C levels (P<0.05); HDL-C levels were significantly increased (P<0.05). Conclusion The hyperlipidemia model is successfully established in Guizhou mini-pigs, and lipid metabolism disorder can be improved to a certain degree after intervention treatment with Wuling powder.



Establishment of a Resistance Training Model for Rats

LIN Haiqi, WANG Zhen, LIN Wentao

2021, 41(2):  143-147.  DOI: 10.12300/j.issn.1674-5817.2020.089

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Objective This study discusses the construction of a resistance training model for rats using a treadmill. Methods A total of 40 male SD rats aged 19 months were selected to determine the conditions for the establishment of a resistance training model. To this end, the angle and speed of the treadmill were measured, the changes of the rat tail load and lactic acid levels in their blood were tested. Results Rats underwent resistance training on the treadmill at an incline of 35°and a running speed of 15 m/min, the exercise regimen significantly improved. After exercising for 8-15 s, the average blood lactic acid concentration was 3.8-5.2 mmol/L. According to the energy metabolism and blood lactic acid half-time response, the resistance training of rats was performed as one training session for 15 s and 30 s intervals. After 4 training sessions in each group, the blood lactic acid concentration was relatively stable. The blood lactic acid concentration was 4-5 mmol/L after resistance training for 3 groups per day and 3 min intervals between groups. Conclusion A resistance training model in rats is established based on the incline, speed of the rat treadmill, and the tail load of rats.



Isolation and Culture of Spinal Microvascular Endothelial Cells of Tree Shrews and Experimental Study on Infection with Enterovirus 71

SHI Meiyan, WANG Xuan, WANG Wenguang, RUAN Leiying, DAI Jiejie

2021, 41(2):  148-154.  DOI: 10.12300/j.issn.1674-5817.2020.208

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Objective To establish an effective method of isolation and primary culture method for microvascular endothelial cells derived from the spinal cord of tree shrews in vitro. Enterovirus 71 (EV71) was used to infect these cells to explore its infectious characteristics and provide a reference for the study of the mechanism of EV71 induced damage to the central nervous system. Methods The spinal cord tissues were digested twice with type Ⅱ collagenase, dispase, and DNaseⅠ, then the microvascular endothelial cells were obtained. EV71 was used to infect tree shrew spinal microvascular endothelial cells, and the virus titer at different time points was measured. The expression of EV71 in the infected cells was detected by immunofluorescence assay to determine the infectivity of EV71 to the spinal cord microvascular endothelial cells of tree shrews. Results The microvascular endothelial cells were typically branched and beaded, and the passage cells obtained after puromycin purification and culture were mainly irregular polygonal cells. The cellular immunofluorescence results showed that CD31 and vWF expressions were positive. The spinal microvascular endothelial cells of tree shrews were infected with EV71 at a multiplicity of infection of 1, the cells showed typical cytopathic appearance, and the virus titer was approximately 3.2×10⁶ TCID₅₀/mL. This proved that EV71 infected and proliferated in the tree shrew spinal cord microvascular endothelial cells, and within 48 h, the viral load in the supernatant increased linearly, reaching its peak at 12 h. The indirect immunofluorescence method detected virus particles in the cytoplasm of the cells 12 h after infection. Conclusions The isolation and purification methods of spinal cord microvascular endothelial cells from tree shrews are successfully established, and the infectivity to the obtained cells and proliferation of EV71 in the cells are confirmed, providing a basis for the study of the mechanism of EV71 invading the central nervous system.



Determination and Comparative Analysis of Hematological Indexes and Percentage of Lymphocyte Subsets in Several Immunodeficient Mice

TAN Xiaoqian, QU Wei, LI Liang, QIAN Zhen, GU Jianzhong, XU Ping

2021, 41(2):  155-160.  DOI: 10.12300/j.issn.1674-5817.2020.123

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Objective To determine and compare the hematological indexes (blood biochemical and physiological indexes) and the percentage of lymphocyte subsets in immunodeficient mice, providing basic data for tumor and immunology research applications. Methods (1) A total of 4 immunodeficient mouse strains, BALB/cA-nude, SCID, NOD-SCID, and NPSG, were included. Twenty mice (6-week-old, half male and half female) were analyzed. Blood biochemical (25 items) and physiological indexes (30 items) were determined and compared with those of BALB/cA-nude mice of the same age. (2) Four strains of immunodeficient mice, 4 mice (6-week-old, half male and half female) were randomly selected and analyzed with flow cytometry. Their symphocyte subsets were compared with those of BALB/cA-nude mice of the same age. Results (1) The white blood cell (WBC) count, lymphocyte (LYMPH) count, lymphocyte percentage (LYMPH%), and neutrophil count of immunodeficient mice were lower than those of BALB/cA mice (P<0.01). In contrast, the neutrophil percentage (NEUT%) and reticulocyte count were higher than those of BALB/cA mice (P<0.05, P<0.01). Furthermore, the levels of WBC, LYMPH and LYMPH% of SCID, NOD-SCID, and NPSG mice were significantly lower than those of BALB/cA-nude mice, while NEUT% was higher than that of BALB/cA-nude mice (P<0.05). (2) The blood glucose and triglyceride levels in NOD-SCID and NPSG mice were lower than those in BALB/cA mice (P<0.01), while uric acid level was higher than that of BALB/cA mice (P<0.05). (3) BALB/cA mice had no T cells and low activity of B cells and natural killer (NK) cells. SCID and NOD-SCID mice had no T cells and lower activity of B cells and NK cells than BALB/cA-nude mice. NPSG mice had no T, B, and NK cells. Conclusion The blood biochemical and physiological indexes and the percentage of blood lymphocyte subsets in BALB/cA-nude, SCID, NOD-SCID and NPSG mice are consistent with the characteristics of corresponding immunodeficient mouse strains.



Application of Liquid Desiccant Environmental Control Equipment in Laboratory Animal Facilities

ZHANG Ting, CHEN Liangliang, LI Haixiang, HUANG Fazhou, CAI Wukai

2021, 41(2):  161-165.  DOI: 10.12300/j.issn.1674-5817.2020.199

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In recent years, liquid desiccant environmental control equipment has been gradually applied in the laboratory animal facility settings. This study measured and analyzed the application of liquid desiccant environmental control equipment in laboratory animal facilities and compared it with traditional systems for environmental control, power distribution, energy consumption, exhaust air treatment, installation conditions, use and maintenance, in order to provide a reference for promoting the application of liquid desiccant environmental control equipment in laboratory animal facilities.



Important Literature Review for the 40th Anniversary of Laboratory Animal and Comparative Medicine

GAO Cheng

2021, 41(2):  169-173.  DOI: 10.12300/j.issn.1674-5817.2020.047

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Classification and Research Progress on Dendritic Cell Subsets in Mice

RUAN Leiying, SUN Xiaomei

2021, 41(2):  174-180.  DOI: 10.12300/j.issn.1674-5817.2020.052

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Mouse, a representative rodent laboratory animal, has always been a powerful tool for studying dendritic cell (DC) systems. Our current knowledge of DCs has been largely derived from studies of murine models, and according to surface markers, cell development, and migratory characteristics, DCs can be categorized with respect to their subsets. To date, there is no uniform classification scheme for mouse DC subsets, which has greatly hindered research involving DCs. Thus, we believe that basic studies to define murine DC subsets are still needed, and it is crucial to seek scientific, effective, and recognized methods to establish categorical definitions. This review proposed a schema incorporating the classification, phenotypes, and characteristics of murine DC subsets, and compares them with human DC counterparts. The aim is to clarify the classification and characteristics of the current status of DC subsets in mice and summarize the existing challenges. This would be beneficial for gaining an in-depth understanding of the function of DC subsets and would provide novel research directions for DC subset categorizations.