Table of Content

25 August 2021, Volume 41 Issue 4

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40th Anniversary Expert Forum

Construction and Evaluation of Animal Models for Cerebral Ischemia

HU Zhibin, HUANG Ying, DING Yuqiang

2021, 41(4):  271-283.  DOI: 10.12300/j.issn.1674-5817.2021.085

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Cerebral ischemia is a common clinical cerebrovascular disease, accounting for about 80% of all cerebrovascular diseases, and it is mainly caused by insufficient blood supply to the cerebrovascular system. Multiple animal models have currently been used to study the pathogenesis and treatment of cerebral ischemia. In order to provide a reference for the selection and improvement of animal models for exploration of underlying mechanisms, this review highlights multiple animal models on global and focal cerebral ischemia with diagrams, and discusses their advantages and disadvantages. The choice of animals, behavioral evaluations of rodent sensory-motor activities, and histological evaluation progress for cerebral ischemia models are also outlined.



40th Anniversary: Development of Laboratory Animal Sciene Across China

Current Status of Administrative Licensing for Laboratory Animals in Jiangsu Province

CHEN Lin, AI Man, XU Hang, QI Chongyang, SHI Aimin

2021, 41(4):  284-289.  DOI: 10.12300/j.issn.1674-5817.2021.071

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This article investigates the administrative licensing data of laboratory animals in Jiangsu Province, summarizes the administrative licenses for laboratory animals in recent years, analyzes the status of the administrative management of laboratory animals in Jiangsu Province, and proposes corresponding countermeasures to provide a reference for the administration of laboratory animal industry in Jiangsu Province and other regions.



Original Article: Animal Models of Human Diseases

A Comparative Study on Spontaneous Homecage Behaviors of Spinal Cord Injury Mice

YAN Yitong, BAI Fan, JING Yingli, WANG Limiao, LI Zihan, YU Yan

2021, 41(4):  290-298.  DOI: 10.12300/j.issn.1674-5817.2020.221

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Objective To investigate the dysfunction of spontaneous homecage behaviors in mice with spinal cord injury, including exploratory behaviors, voluntary movements, and self-care behaviors. Methods Eight-week female C57BL/6 mice (n = 10) were established into thoracic 10 contusion spinal cord injury models. Other mice (n = 11) were included in the sham group. From the first week to the fifth week after surgery, the mice were observed in homecages at 9 a.m. for 2 hours once a week. The HomeCageScan software was used to analyze the time percentages of 32 daily behaviors. Results Compared with the sham group, the spinal cord injury mice showed significant abnormalities in most aspects of daily behaviors, such as exploratory behaviors, voluntary movements, and self-care behaviors (P < 0.01). Some of these behaviors recovered over time after surgery. At 1-5 weeks after surgery, the spinal cord injury mice exhibited less exploratory behaviors than the sham mice, represented by rearing up and sniffing (P < 0.01), and showed recovery in the 4th week and the 3rd week (P < 0.05). The spinal cord injury mice also exhibited less voluntary movements (P < 0.05), represented by walking slowly and jumping, and showed recovery from the 2nd week and the 5th week, respectively (P < 0.05). The spinal cord injury mice exhibited more self-care behaviors than the sham mice (P < 0.01), represented by twitching and grooming, and showed no significant recovery over time. Conclusion Besides lower limb movement dysfunction, the spinal cord injury mice showed abnormalities in exploratory behaviors, voluntary movements, and self-care behaviors. From 1-5 weeks after injury, the exploratory behaviors and voluntary movements could show different degrees of recovery, while the self-care behaviors showed continuous abnormalities. The automated high-throughput behavior detection system accurately classified and comprehensively recognized dozens of behaviors, which could be used as a more comprehensive and sensitive evaluation index of spinal cord injury disorder.



Establishment of a Rat Model of Granulomatous Lobular Mastitis

ZUO Ximeng, SHI Xiaoguang, LIU Jieli, YANG Zhenrui, GAO Xiang, LAI Rui, ZHAO Ze, WANG Tangshun

2021, 41(4):  299-304.  DOI: 10.12300/j.issn.1674-5817.2020.149

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Objective In this study, an animal model of granulomatous lobular mastitis was constructed using human granulomatous lobular mastitis specimen homogenates and a suspension of complete Freund's adjuvant. Methods Twenty-five rats with a history of maternity were randomly divided into five groups with five rats each: a blank control group, Freund's adjuvant group, normal mammary homogenate group, low-dose lesion homogenate group, and high-dose lesion homogenate group. In the lesion homogenization groups, rats were injected with complete Freund's adjuvant homogenate prepared from granulomatous mastitis patient tissues, and the third and fourth pair of mammary gland injections were administered to the rats at 0.2 mL in the high-dose group and 0.1 mL in the low-dose group. The normal mammary gland homogenate group was injected with normal mammary gland tissue homogenate from patients with granulomatous mastitis. The blank control group was injected with saline. The Freund's adjuvant group was injected with complete Freund's adjuvant. The histomorphological and pathophysiological characteristics of the mammary tissues were observed for 14 days after the completion of modeling, and the effects of the granulomatous lobular mastitis model were compared. Results The morphological changes and physiopathological characteristics of granulomatous lobular mastitis in the high- and low-dose groups were significantly different when compared with the blank control, Freund's adjuvant, and normal breast homogenate groups (P < 0.001). Conclusion Human granulomatous lobular mastitis specimen homogenates and suspensions of complete Freund's adjuvant for granulomatous lobular mastitis can successfully establish an animal model of the disease and lead to insights and new ideas for the etiology of the disease.



Protective Effect of Quercetin on Lipid Metabolism Disorder in Mice Livers Caused by Cadmium

LI Zifa, ZHANG Hao, REN Meng, XU Kaiyong, HU Minghui, ZHOU Miaomiao, WANG Kezhou

2021, 41(4):  305-312.  DOI: 10.12300/j.issn.1674-5817.2021.007

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Objective To investigate the protective effect of quercetin on cadmium-mediated lipid metabolism disorder in livers of mice. Methods C57BL/6J mice were divided into four groups: a control group, a quercetin-treated group (quercetin by intragastrical administration), a cadmium-treated group (cadmium chloride by intragastrical administration), and a cadmium plus quercetin co-treated group (cadmium chloride plus quercetin by intragastrical administration). After the administration, mice weight was measured; the activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in serum, and the contents of total cholesterol (TC) and triglyceride (TG) in both serum and liver were determined; the pathological changes in liver tissues and liver droplets were observed by HE and Oil Red O staining; the expression levels of proteins related to lipogenesis, fatty acid synthase (FAS), and related to lipolysis, lipoprotein lipase (LPL), in liver were detected by Western blotting. Results Compared with the control group, the mice treated with cadmium showed that the weight was significantly decreased (P<0.01), the ALT and AST activities were remarkedly increased in serum (P<0.01), the pathological changes of liver injury were seen obviously, the TC and TG contents were remarkedly elevated in both serum and liver (P<0.01), lipid droplets were greatly accumulated (P<0.01), the FAS expression in liver was up-regulated (P<0.01), while the LPL expression was down-regulated (P<0.01). Quercetin intervention significantly alleviated the abnormal changes of the above indicators. Conclusion Quercetin can protect mice from cadmium-induced liver injury by regulating lipid metabolism disorder in liver.



Effects of Metformin on Cognitive Dysfunction and PI3K/Akt Pathway in Alzheimer's Disease Rats

WANG Baiqiao, LIN Xiaoru, HAN Min, LIU Yu, TANG Chaoling

2021, 41(4):  313-320.  DOI: 10.12300/j.issn.1674-5817.2020.185

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Objective To investigate the effects of metformin (Met) on the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt) pathway and cognitive dysfunction in Alzheimer's disease (AD) rats. Methods Fifty Sprague Dawley rats were selected and divided into the following groups: normal, AD model (AD group), Met low (50 mg/kg), Met medium (100 mg/kg) and Met high (200 mg/kg) according to the random number table method. Each group included 10 rats. With the exception of the normal group, AD models were established by injecting 10 μL streptozotocin (STZ, 3 mg/kg) into the bilateral ventricles. After the model was successfully established, Met at the corresponding dose was administered by gavage. The normal and AD groups were administered the same amount of normal saline by gavage. The medicine was administered once a dayfor 14 days. After the last administration, the Morris water maze test was used to detect the spatial cognitive function of rats. The rats were sacrificed, and the hippocampal tissues were collected. The contents of β-amyloid protein 42 (Aβ42) and phosphorylated tau protein (p-tau) in hippocampus were detected by enzyme-linked immunosorbent assay (ELISA). The pathological changes of neurons in hippocampus were observed by HE staining. The detection of PI3K positive expression was performed by immunohistochemistry. Western blots were used to detect the relative expression levels of PI3K, phosphorylated Akt (p-Akt), insulin receptor substrate-1 (IRS-1), and glycogen synthase kinase-3 (GSK-3) in hippocampus. Results Compared with the normal group, there were pathological damages, such as disordered arrangement and decrease of neurons in hippocampus of rats in the AD group. The number of times the platform was crossed, the ratio of swimming distance to the total distance in the original platform quadrant, the ratio of swimming time to the total time, and the protein expressions of PI3K, p-Akt and IRS-1 in hippocampus of the AD group were significantly low (P < 0.05). The average escape latency, the contents of Aβ42 and p-tau, and the protein expression of GSK-3 in hippocampus were high (P < 0.05). Compared with the AD group, the pathological damages of hippocampal tissue in low, medium, and high dose Met rats were alleviated. The number of times the platform was crossed, the ratio of swimming distance to the total distance in the original platform quadrant, the ratio of swimming time to the total time, and the protein expressions of PI3K, p-Akt and IRS-1 in hippocampus of the AD group were significantly high (P < 0.05). The average escape latency, the contents of Aβ42 and p-tau, and the protein expression of GSK-3 in hippocampus were low (P < 0.05). Moreover, the above indexes were dose-dependent in the Met groups. Conclusion Met can activate the PI3K/Akt pathway, decrease the levels of p-tau and Aβ42 in hippocampus, and improve cognitive impairment in AD rats.



A Modified Method of β-Aminopropionitrile Combined with Angiotensin Ⅱ to Establish an Aortic Dissection Model in Mice

CHENG Lingxia, CHEN Lin, YANG Fan, LIU Ying, CHEN Muhu, HU Yingchun, SONG Qitai, ZHONG Wu

2021, 41(4):  321-326.  DOI: 10.12300/j.issn.1674-5817.2020.200

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Objective To explore an improved method for constructing a mouse model of aortic dissection (AD). Methods AD was induced by the simultaneous administration of β-aminopropionitrile (BAPN) with angiotensin Ⅱ (Ang-Ⅱ) for 16 days in mice. One hundred 6-week-old male C57B1/6J mice were randomly divided into BAPN (BAPN 0.1 g•kg^-1•d^-1 + 0.9% saline), BAPN+Ang-Ⅱ (BAPN 0.1 g•kg^-1•d^-1 + Ang-Ⅱ), and control groups. BAPN (0.1 g•kg^-1•d^-1), freshly prepared and dissolved in drinking water, was administered to all mice in the BAPN and BAPN + Ang-Ⅱ groups for 16 days. Mice in the BAPN+Ang-Ⅱ group were treated with an additional subcutaneous injection of Ang-Ⅱ for 16 days. The concentration of Ang-Ⅱ was halved from 1.5 mg•kg-1•d-1 every week until it was set at 0.375 mg•kg^-1•d^-1 on day 15, and then maintained. The daily water intake and body weight were recorded from the beginning until the end of the study. If a mouse died during the experiment, an autopsy was performed to analyze the cause of death. On day 17, the mice were sacrificed, and each aorta was harvested. The formation of the aortic false lumen was observed pathologically using hematoxylin and eosin (HE) staining. Results Compared with the control group, the amount of water consumed and body weight in the BAPN+Ang-Ⅱ and BAPN groups were significantly reduced (P < 0.05). In the control group, the incidence of dissection and mortality was 0.0%. In the BAPN group, the incidence of AD and the mortality was 7.5% and 2.5%, respectively. In the BAPN+Ang-Ⅱ group, the incidence of AD and the mortality was 80.0% and 30.0%, respectively. Interestingly, the deaths in the BAPN+Ang-Ⅱ group occurred frequently at 1-3 days after dose adjustment, and 83.3% of the mice died a few minutes after the operation. Conclusion This modified method for constructing an AD mouse model induced by BAPN drinking combined with Ang-Ⅱ subcutaneous injection was replicable, regular, effective, cheap, and quick.



Mechanism of Hypertonic Saline to Reduce Cerebral Edema in Rats with Cerebral Hemorrhage by Protecting Blood-brain Barrier

DIWU Feihu, ZHAO Zhijing, DENG Yiheng

2021, 41(4):  327-332.  DOI: 10.12300/j.issn.1674-5817.2020.140

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Objective To investigate the mechanism of hypertonic saline (HS) treatment to reduce cerebral edema in intracerebral hemorrhage (ICH) rats by. Methods Sixty male SD rats were randomly divided into control, model, and treatment groups. An ICH rat model was established by injecting fetal bovine serum (FBS) into the brain parenchyma of rats. The rats in the treatment group were injected with 0.3 mL/h 10% HS via the tail vein for 48 h. The mortality of the rats was compared between the groups. Brain tissues were collected to observe the morphological and pathological changes, detect the water content differences in the brain, and measure the ratio of matrix metalloproteinase-9 (MMP-9) to tissue inhibitor of metalloproteinases-1 (TIMP-1) and the expression of related tight junction proteins, including claudins, occludin, and zonula occludens-1 (ZO-1). Results The mortality and water content of the brain tissues in the model group were significantly higher than those in the treatment group (all P < 0.05). The naked eye observation showed that the midline of the brain in the model group shifted from the lesion side to the contralateral side, while the midline of the brain in the treatment group did not. The ratio of MMP-9/TIMP-1 in the brain tissue of rats in the treatment group was significantly lower than that in the model group, while the tight junction proteins of claudin-5, occludin, and ZO-1 in the brain tissue of rats in the treatment group were higher than those in the model group (all P < 0.05). Conclusion HS can maintain the structural integrity of the blood-brain barrier by reducing the ratio of MMP-9/TIMP-1, stably and tightly connecting the expression of claudin-5, occludin, and ZO-1, thereby improving the severity of brain edema after ICH and reducing the mortality of experimental model rats.



Laboratory Animals and Safety Evaluation

Chronic Toxicity Study on Deinococcus Radiodurans R1 in Rats

XU Qin, WANG Wei, DONG Xiang, LI Jianying, SHI Wenhui, LIU Xiaolu, LIU Aizhong, MA Na, SONG Laiyang, LIU Jiangwei

2021, 41(4):  333-342.  DOI: 10.12300/j.issn.1674-5817.2020.213

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Objective To observe the chronic toxicity of Deinococcus radiodurans R1 (DRR1) in SD rats.Methods SD rats were divided randomly into 5 groups: 10⁹/mL live DRR1 group (LDRH), 10⁷/mL live DRR1 group (LDRL), 10⁹/mL DRR1 broken products group (BDRH), 10⁷/mL DRR1 broken products group (BDRL), and agar medium including 0.5% tryptone, 0.1% glucose and 0.3% yeast agar medium as control group (TGY), and 16 rats in each group with half males and half females. The rats were gavaged once a day for consecutive 30 days,and then 8 rats in each group were randomly selected, and anaesthetized intraperitoneally with 3% pentobarbital sodium 0.15 mL/100 g body weight, the blood and organ samples were collected to analyze blood routine, serum electrolyte, serum biochemical indexes and organ coefficients. The heart, liver, kidney and lung of LDRH and BDRH group rats were fixed with 10% neutral formaldehyde (V/V), stained with Hematoxylin-eosin and examined by optical microscope to analyze the toxicity of DRR1. The delayed toxicity effect of DRR1 was performed on the 14 ^th day after drug withdrawal, by the same way as above groups. The behavior, food intake and body weight of rats were observed after administration and recovery period.Results After 30 days of administration with different concentrations of DRR1 living bacteria and its fragments, at the point of drug withdrawal 0 d and 14 d, no abnormal movement and respiration were observed, no abnormal phenomena such as slow reaction, pupil change, eyeball protrusion and skin color change were observed; no abnormal secretion was found in mouth, ear, nose and eye corner; no abnormallities were found in urination and defecation and stool morphology. Compared with TGY group, the food intake, body weight and organ coefficient of the DRR1 groups had no significant changes. The number of white blood cells (WBC) in LDRH, decreased significantly on the 30 ^th day of administration (P < 0.05), and returned to the TGY group level after 14 days of drug withdrawal. The number of red blood cells, hematocrit and hemoglobin had no significant changes, the number of lymphocytes, granulocytes, monocytes and their percentages had no significant changes, and the number of platelets had no significant changes. The serum alanine aminotransferase (ALT), alkaline phosphatase (ALP), total protein (TP), uric acid (UA), urea (UREA), blood glucose (Glu), phosphorus (P) and magnesium (Mg) were not significantly changed after drug withdrawal. The levels of aspartate aminotransferase (AST), creatine kinase (CK), creatine kinase MB (CK-MB) and lactate dehydrogenase (LDH) in LDRH group were significantly increased (P < 0.05). After 14 days of recovery, AST and CK decreased to the level of TGY, CK-MB was still higher than that of TGY group (P < 0.05); LDH in the recovery period was significantly higher than that 0 h after drug withdrawal (P < 0.05), no significant change compared with recovery period TGY group. There was no significant change in serum ion concentration (P > 0.05) on the 30th day of administration. On the 14th day after drug withdrawal, Cl^- and Ca²⁺ decreased, Na⁺ and pH increased (P < 0.05) in LDRH group. No significant pathological changes were found in heart, liver, lung and kidney of LDRH and BDRH group.Conclusion DRR1 live bacteria and its bacterial fragments are practically non-toxic on rats. This study provides experimental data for the application of DRR1.



Quality Control of Laboratory Animals

Analysis of Antibody Changes in Laboratory Mice Infected with Murine Norovirus

LI Xiaobo, FU Rui, WANG Shujing, LI Wei, WANG Shasha, HUANG Zongwen, QIN Xiao, WANG Ji, YUE Bingfei

2021, 41(4):  343-350.  DOI: 10.12300/j.issn.1674-5817.2020.127

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Objective To study the changes of total IgG antibodies and neutralizing antibodies in KM, BALB/c, NIH, C57BL/6J and BALB/c-nu mice infected with murine norovirus (MNV). Methods Five strains of KM, BALB/c, NIH, C57BL/6J and BALB/c-nu mice were collected. Each strain was divided into control group and infection group. The infection group was inoculated with MNV by gavage, and the control group was inoculated with the same volume of normal saline. Blood and serum were collected from 1 to 9 weeks after infection, total IgG antibody level (mean absorbance) was determined by ELISA, and neutralizing antibody titer (half protective dose, PD₅₀) was determined by RAW264.7 cytopathic method. T-test was used to compare the mean absorbance difference between the infection group and the control group at each time point; One way ANOVA was used to compare the total antibody and neutralizing antibody titer of each strain of the infection group, and to analyze the antibody changes of each strain of mice. Results T-test showed that the mean absorbance of total IgG antibody in KM, BALB/c, NIH, C57BL/6J and BALB/c-nu mice infected on the day 21, 35, 14, 14 and 28 was significantly higher than that of the control group (P < 0.01). To compared the total IgG antibody level of each strain of mice, the results showed NIH>C57BL/6J>KM>BALB/c>BALB/c-nu. The total IgG antibody levels of BALB/c and BALB/c-nu mice were significantly lower than that of NIH and C57BL/6J mice (P < 0.05); The antibody level of NIH, C57BL/6J, KM and BALB/c mice increased rapidly at the 6th week of infection, and NIH mice entered the plateau stage at the 49th day. The IgG antibody of the other three strains was still on the rise during the experimental period, and the overall antibody level of BALB-/c-nu mice was the lowest. The overall neutralizing antibody titers of each strain of mice were compared, showing that C57BL/6J>NIH>KM>BALB/c>BALB/c-nu, and the mean values of lgPD₅₀ were 3.881, 3.819, 3.746, 3.146, and 2.338, respectively. Analysis of variance showed that neutralizing antibody titer of BALB/c and BALB/c-nu mice was significantly lower than that of C57BL/6J, NIH and KM mice (P < 0.01). Except for BALB/c-nu mice, the neutralizing antibody of the other four strains increased rapidly at the second week of infection, and the neutralizing antibody of BALB/c-nu mice was always at a low level. Conclusion The humoral immune response of NIH, C57BL/6J and KM mice infected with MNV is stronger, and that of BALB/c and BALB/c-nu mice is weaker.



Improvement of Detection Method of Pasteurella pneumotropica and Its Medical Elimination in Mice

ZHANG Tao, CUI Can, MA Guanzhong, ZHANG Aihua

2021, 41(4):  351-357.  DOI: 10.12300/j.issn.1674-5817.2020.197

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Objective To modify the method for sensitive detection of Pasteurella pneumotropica in mice in routine monitoring, and to explore the feasibility of medical elimination in Pasteurella pneumotropica infected mice.Methods The Pasteurella pneumotropica were detected by routine procedure and modified method that using fluid medium before inoculation. The detection rates of the two methods were compared in statistics. The sensitivity of Pasteurella pneumotropica isolated from local facility to 12 antibiotics was studied by antimicrobial disk sensitivity test. Forty-eight female C57BL/6 mice were randomly divided into 6 groups, with 8 in each group. All the groups were set to different concentration of enrofloxacin continuously for 6 weeks to find out the safe dosage. Mice were infected with local Pasteurella pneumotropica strain, the Pasteurella pneumotropica positive mice were administered enrofloxacin in drinking water at different doses to evaluate the effect of Pasteurella pneumotropica clearance results.Results The detection rates of Pasteurella pneumotropica by routine procedure and improved method were 1.3% and 15.1%, respectively. Pasteurella pneumotropica was sensitive to enrofloxacin, cefoxitin and cefadrazole etc. The mice were safe to enrofloxacin within a daily dosage of 300 mg/kg. The Pasteurella pneumotropica was not detected from positive mice treated with 85 mg/kg enrofloxacin after 2 weeks, and kept negative after withdrawl for 6 weeks.Conclusion Using fluid medium before inoculation can improve the positive detection rate of Pasteurella pneumotropica, which is simple, convenient, and suitable for daily health monitoring of animal facility. Oral administration of enrofloxacin more than a dosage of 85 mg·kg^–1·d^–1 can elimilate Pasteurella pneumotropica from the positive mice safely and effectively, and be an alternative contingency plan for the elimination of Pasteurella pneumotropica in mice.



Laboratory AnimalWelfare and Ethics

Necessity of an Acclimatization Period in a Home Cage Experiment of Mice

YIN Yuan, LU Lu, WANG Yun, YANG Jing, FU Heling

2021, 41(4):  358-362.  DOI: 10.12300/j.issn.1674-5817.2020.154

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Objective To investigate whether an acclimatization period should be introduced into the home cage experiment for the detection of metabolic function in mice, and how to judge and set its length. Methods Data from 31 home cage experiments in 2016-2017 were collected, and each experiment was set up with a 3-day (72 hours) acclimatization period. Data such as water consumption, food intake, heat production, and body weight were recorded. The data were analyzed using 24-hour interval analysis to determine the acclimatization effect. Results Approximately 12% of the 356 mice drank less than 1 mL at the end of 96 hours. There were significant differences in water consumption, food intake, heat production rate, and body weight between 24-48 hours and 48-72 hours, but no significant differences were detected between 48-72 hours and 72-96 hours, except in food intake. Independent verification of the food intake of each experiment showed that there was a significant correlation between food intake adaptability and weight.Conclusion Approximately 12% of the mice were unable to learn or adapt to the new water bottle after the 72-hour acclimatization period, therefore, researchers should consider increasing the sample size of the experiment to eliminate invalid data. If food intake is not the focus of a study, the 48-hour acclimatization is sufficient to obtain reliable results.

Laboratory Animals Industry and Information Service

Data Analysis and Countermeasures of Laboratory Animal E-Commerce Platform

WEN Jinyin, CHEN Yan, LI Huiping

2021, 41(4):  363-368.  DOI: 10.12300/j.issn.1674-5817.2021-017

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The study takes the laboratory animal e-commerce platform as the analysis object, pays attention to data characteristics of product category and data indicators such as the number of visits, click-through rates, conversion rates, and search rates during platform operation. We put forward countermeasures and suggestions for related issues: Further clarify and standardize product categories, improve search engine functions, enhance personalized services, strengthen supervision mechanisms, etc., and provide important reference and guidance for the sustainable operation and development of the laboratory animal e-commerce platform.