Laboratory Animal and Comparative Medicine

A Brief Review on Development and Application of Animal Models of Emerging Infectious Diseases Caused by Three Genus Viruses

LI Feng, LI Shun, REN Xiaonan, ZHOU Xiaohui

2020, 40(3): 173. DOI: 10.3969/j.issn.1674-5817.2020.03.001

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Animal models are key foundation for the research on the pathogenesis of emerging infectious diseases and the development of targeted vaccines and drugs, which play an irreplaceable role in deepening the scientific understanding of new infectious diseases and developing the effective prevention and control measures. In this paper, three kinds of emerging viral infectious pathogens (coronavirus, flavivirus and influenza virus) which are seriously harmful to human life and health in recent years are reviewed. The construction and application of the corresponding experimental animal models are briefly described, and their pros and cons are compared, so as to provide a reference for selecting the appropriate animal models for the further research on pathogenicity mechanism and the studies on vaccines and drugs against those emerging viruses.

Establishment of Bone Metastasis Mouse Models through Injecting Human Lung Cancer Cells into Left Ventricle#br# under Ultrasound Guidance#br#

CHAI Wenjun, SUN Lei, LIU Xiaoli, PAN Hongyu, GUO Tianan, XU Ye, YAN Mingxia

2020, 40(3): 183. DOI: 10.3969/j.issn.1674-5817.2020.03.002

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Objective To establish bone metastasis mouse models of human lung cancer cells injected into the heart under ultrasound guidance. Methods Under ultrasound guidance, human lung cancer A549 and NCI-H1299 cells were injected into the left ventricle of NOD/SCID and BALB/cnu/ nu mice respectively, and the success rate of animal model and the occurrence of bone metastasis were observed by combining with a variety of animal imaging methods such as in vivo imaging,X-ray and Micro-CT. HE staining was used to examine the tumor tissues and bone tissue damages.Results All the animals survived after lung cancer cells were injected into the heart under ultrasound guidance. In vivo imaging showed that luciferase was expressed in the bones of the central axis and limbs of mice. X-ray and Micro-CT reconstructed the three-dimensional bone of mice. There were obvious defects and destruction of bone and periosteum, and the normal bone morphology disappeared, with osteolytic changes. The imaging findings were consistent with the changes of neoplastic bone destruction. Gross necropsy showed that the axial bone and limb joints of the mice were enclosed by tumor tissues, and there were different degrees of bone metastases. Conclusion Ultrasound-guided intracardial injection of lung cancer cell bone metastasis model can effectively improve the success rate of modeling, more accurately simulate the biological characteristics of bone metastasis in clinical lung cancer patients, and provide tools for the study of lung cancer metastasis mechanism, anti-metastasis treatment, and individualized treatment.

Impacts of Okadaic Acid on Neurological Function and Behavior of Zebrafish Larvae#br#

WANG Xue, LIU Kechun, YANG Xueliang, MA Yukui, ZHANG Yun

2020, 40(3): 190. DOI: 10.3969/j.issn.1674-5817.2020.03.003

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Objective To investigate the impacts of okadaic acid (OA) on the neurological function and behavior of zebrafish larvae, and explore the possible application of zebrafish as an animal model in the study of neurodegenerative diseases. Methods Zebrafish larvae at 3 days post fertilization (3 dpf) stage was treated with 0.05 µmol/L, 0.10 µmol/L, and 0.20 µmol/L OA respectively for 7 days. The survival rate of zebrafish larvae was calculated, the swimming track within 30 min was acquired by behaviroral analysis system, and the swimming trajectory, swimming distance and inactive time were recorded. The expression levels of protein phosphatase 2A (PP2A) and nicotinic acetylcholine receptor alpha 7 (α7-nAChR) genes were tested by real-time fluorescent quantitative PCR. Results After 7 days of treatment, the survival rate and swimming trajectory of the zebrafish larvae in 0.05 µmol/L and 0.10 µmol/L OA treatment groups were not significantly different from those in the control group (P>0.05). Zebrafish larvae treated with 0.20 µmol/L OA for 7 days showed inactive swimming, reduced swimming distance and prolonged sluggish time with significant difference as compared with the control group (P<0.05). The expression level of PP2A gene was down-regulated significantly in 0.20 µmol/L OA treatment group (P<0.05), however the expression level of α7-nAChR was not affected (P>0.05). Conclusion The treatment with 0.20 µmol/L OA for 7 days can cause the abnormal neurological function and behavior of zebrafish larvae.

Study on Gene Cloning and Preliminary Function of Junction Adhesion Molecule A in Tree Shrew#br#

LI Xiaofei, SUN Xiaomei, WANG Wenguang, KUANG Dexuan, LU Caixia, TONG Pinfen, HAN Yuanyuan, LI Na, DAI Jiejie

2020, 40(3): 196. DOI: 10.3969/j.issn.1674-5817.2020.03.004

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Objective To explore the mechanism of junctional adhesion molecule A (JAM-A) as a reovirus receptor to invade the primary lung cells of tree shrew. Methods Total RNA was extracted from normal tree shrew tissues. JAM-A gene sequences were obtained by reverse transcription-PCR and rapid-amplification of cDNA ends. Translation of amino acids encoded by JAM-A gene were analyzed by using Unipro UGENE software. The phylogenetic tree was constructed by MEGA 6.0 software using the maximum likelihood method. Using real-time fluorescent quantitative-PCR to detect the relative expression level of JAM-A in 25 tissues and blood of tree shrew. The specific antibody blocking treatment of the receptor was performed on the primary tree shrew alveolar epithelial cells, and the fluorescence intensity of the virus antigen after reovirus infection was determined. Results The full-length cDNA sequences of JAM-A gene with 2 962 bp were obtained. Amino acid alignment and phylogenetic tree analysis showed that the genetic relationship of JAM-A gene in tree shrew was more closer to human than that of rodent. JAM-A molecule was widely expressed in the peripheral tissues of tree shrews, with the more higher expression levels in the respiratory and digestive tract. The integrated absorbance of the immunofluorescence of the virus antigen after the specific antibody was applied to the cells was significantly decreased (P<0.000 1). Conclusion The whole sequence of JAM-A gene was retrieved and analyzed for the first time, and the JAM-A molecule was verified to be the main receptor for reovirus to invade tree shrew, suggesting that tree shrew may be used as a new animal model of reovirus.

Metabonomics Study of Spontaneous Diabetic Model Mice on Whole Plant Protein Formula Feed#br#

LI Leibin, XU Jia, FANG Yuanshu, JIANG Zhengqian, QIU Yinger, WANG Wenqian, ZHAO Xianzhe

2020, 40(3): 204. DOI: 10.3969/j.issn.1674-5817.2020.03.005

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Objective To assess the influence on C57BL/6J mice and diabetic model db/db, ob/ob mice by feeding with whole plant protein formula diet. Methods Fifty male and 50 female C57BL/6J mice were divided into normal fish protein-based diet control group and whole plant protein formula diet experiment group, 20 female db/db and 20 female ob/ob mice were randomly divided into normal diabetic model diet group and whole plant protein formula diabetic model diet group, respectively. All the mice were 4 weeks old and the period of experiment was lasted for 16 weeks. During the experiment, the daily situation and every 4 weeks blood glucose changes in each group of mice were observed. After 16 weeks, the urine samples were used for studying metabonomics, the blood samples of each group were collected to test blood biochemistry, and the part of free gossypol target organs like liver, kidney, and pancreas were taken for histological observation. Results No discernible difference was observed in body weight, blood glucose and histological changes between the experiment group and the control group, respectively. A series of metabonomics studies revealed some differences between the experiment group and the control group in the urine, in which the concentrations of leucine, isoleucine, tyrosine and threonine were different. Conclusion Through the study of nutriology, toxicology and metabonomics, it is preliminarily believed that the replacement of fish protein by degossypolled cottonseed protein in laboratory animal diet is safe.

Effect of Prostacyclin E1 on Hepatic Fiber in Rabbits after Embolization with Lipiodol #br#

LI Lei, LI Xiangli, HU Huimin

2020, 40(3): 212. DOI: 10.3969/j.issn.1674-5817.2020.03.006

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Objective To investigate the effect of prostacyclin E1 (PGE1) on the expressions of Smad2 and Smad4 mRNA in rabbits with hepatic fibrosis after embolization with lipiodol. Methods Thirty-six adult rabbits were randomly divided into sham operation group, model group and administration group. After one week acclimation, all the rabbits in the three groups were intubated under anesthesia, and hepatic artery embolization with iodized oil was simulated. Then, 1 mL normal saline was injected into the right hepatic artery in the sham operation group, 1 mL iodized oil and 0.2 mL PGE1 were injected into the right hepatic artery in the administration group, and 1 mL iodized oil was injected through the right hepatic artery in the model group. On the 2nd, 4th, 6th and 8th day after the operation, the serum indices of each group were detected by ELISA. On the 8th day after the operation, all the rabbits were euthanized, and their liver tissues were taken out for pathological examination. The mRNA levels of Smad2 and Smad4 in the liver tissues of each group were detected by RT-PCR. Results There was no significant change in the serological indices of the rabbits in the sham operation group on the 2nd, 4th, 6th and 8th day after operation. Compared with the sham operation group, the serological indices of the administration group and the model group increased significantly with time (P<0.05). Compared with the model group, the serological indices of each time point in the administration group decreased significantly (P<0.05). On the 8th day after operation, there was no obvious fibrosis in the sham operation group, while there were different degrees of fibrosis in the pathological specimens of the administration group and the model group, and the fibrosis in the administration group was more obvious than that in the model group (P<0.05). The expression levels of Smad2 and Smad4 mRNA in the liver tissues of rabbits in the administration group and the model group were significantly higher than those in the sham operation group (P<0.05). The expressions of Smad2 and Smad4 mRNA in the liver tissues of the administration group were significantly lower than those of the model group (P<0.05). Conclusion PGE1 can improve the liver fibrosis and decrease the expressions of Smad2 and Smad4 mRNA in liver tissues, which has a positive effect on the protection of liver injury.

Protective Effect of Camel Milk on DSS-induced Acute Colitis in Mice

CUI Changwan, YU Miao, WANG Shuang, WU Si, SUN Zhengrong

2020, 40(3): 218. DOI: 10.3969/j.issn.1674-5817.2020.03.007

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Objective To investigate the effect of camel milk on the acute colitis induced by dextran sodium sulfate (DSS) in mice. Methods Male C57BL/6 mice (6-8 weeks) were randomly divided into 2 groups: the mice were treated by intragastric administration of double distilled water as the control group, and the experimental group were given with camel milk. After 14 d, 3% DSS was used to induce acute colitis in mice. Flow cytometry was used to analyze the proportion of immune cells. Cytokines were measured by enzyme-linked immunosorbent assay (ELISA). Results Compared to the control group, the mice with camel milk treatment showed slow weight loss (P<0.05), improval survival rate, decline in histological score (P<0.05). In the experimental group, the percentage of interferon-γ (IFN-γ) positively expressed T lymphocytes (CD4+IFN-γ+) increased (P<0.05), while the percentage of interleukin-17 (IL-17) positively expressed T lymphocytes (CD4+IL-17+) decreased(P<0.05). The expression levels of IL-17 and tumor necrosis factor-α (TNF-α) were reduced(P<0.05), and the expression of IFN-γ was increased in the cell culture supernatant of the experimental group (P<0.05). Conclusion Camel milk can relieve the progression of acute colitis induced by DSS by regulating the differentiation of T cells and the secretion of cytokines.

Pathologic Change of Spontaneous Mucinous Adenocarcinoma of the Ileum in a Sprague-Dawley Rat#br#

YING Yong, XIA Yujie, TANG Liansheng, JIA Chanyuan, ZHANG Haijing, HU Jianting

2020, 40(3): 223. DOI: 10.3969/j.issn.1674-5817.2020.03.008

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A control Sprague-Dawley male rat from the control group of a repeated dose toxicity study had a mucinous adenocarcinoma in the ileum. The gross morphology showed that 3 nodules in the serosal layer of ileum were observed, with the size of 1.4 cm 1.3 cm, 0.7 cm × 0.5 cm and 0.4 cm × 0.3 cm, respectively. HE staining showed that the neoplasm was primarily composed of cuboidal or columnar and goblet cells which were arranged in a glandular form. Alcian blue-PAS staining revealed the expression of mucin in neoplastic cells and in the lumen. The above results are in line with the diagnostic features of mucinous adenocarcinoma in the ileum.

Construction and Application of an Improved Skull Defect Model in Rats

ZHANG Yawen, ZHU Guangxu, LI Yazhe, LI Hao, WANG Shaoye, ZHANG Haoyun, WANG Fengbin

2020, 40(3): 227. DOI: 10.3969/j.issn.1674-5817.2020.03.009

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Objective To explore the construction of an ideal skull defect animal model in rats, and to improve the success rate of skull defect animal model. Methods Forty adult male SD rats were randomly divided into the control group and experimental group. In the control group, the traditional surgery method of skull defect model was used, only using a hollow drill and the force was perpendicular to the skull surface to drill the full-thickness defect of the skull. The modified surgical method was used in the experimental group rats, by using both hollow and solid drills, and changing the direction of the drill's force at different stages in the skull drilling process. The drilling time, existence of intraoperative complications such as periosteum, dura mater, brain tissue injury and bleeding, as well as the fixed position and osseointegration of postoperative alumina bioceramical materials implanted were compared, and the modeling success rate was compared after 8 weeks between the two groups. Results Compared with the control group, the drilling time of the experimental group was shortened (P<0.01), the intraoperative complications such as periosteum, dura mater, brain tissue injury and bleeding were significantly reduced (P<0.05), and there was no displacement and loosening of alumina bioceramical materials after operation (P<0.05), and the material was well bonded to the skull, and the success rate of modeling was significantly increased (P<0.05). Conclusion The modified method for skull defect model in rats has the advantages of short drilling time and less complications. It can greatly improve the success rate of model establishment and can effectively evaluate the bone repair performance of orthopedic implant materials in vivo.

Alleviation Effect of Formononetin on Osteoporosis in Type 2 Diabetes Mellitus Complicated with Diabetic Bone Disease#br#

HOU Wei, WAN Dun

2020, 40(3): 232. DOI: 10.3969/j.issn.1674-5817.2020.03.010

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Objective To investigate whether the formononetin has a mitigating effect on osteoporosis in type 2 diabetic rats with diabetic bone disease. Methods A model of type 2 diabetes mellitus was established by combining Streptozotocin (STZ)+ high-fat diet. High, medium and low dosages (100 mg/kg, 50 mg/kg, 20 mg/kg) of formononetin were administered by intragastric administration for 2 weeks, followed by detection of rats in each group. The bone mineral density, serum testosterone (T) and estradiol (E2) levels, as well as the mRNA expressions of osteocalcin (OC) in femur and CYP24A1 in kidney of rats were measured. Results Compared with the control group, the bone mineral density, serum T and E2 levels, femoral OC, and renal CYP24A1 mRNA expression decreased in the type 2 diabetes group, while the formononetin could reverse the above phenomenon, and the high dosage group had the most positive effect. Conclusion Formononetin can alleviate osteoporosis in type 2 diabetic rats with diabetic bone disease.

Optimization of PCR Detection Method for Pasteurella pneumophila in Mice and Its Preliminary Application in Different Sample Treatment

PENG Lina, PAN Yajun, ZHANG Man, GU Jeffrey Jianan, XU Wangjie

2020, 40(3): 236. DOI: 10.3969/j.issn.1674-5817.2020.03.011

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Objective To establish a real-time, rapid, sensitive and simple PCR method for the detection of Pasteurella pneumophila in living laboratory animals. Methods By comparing different sample collection and processing methods, optimizing the sample processing process, and simplifying the method of DNA extraction, PCR amplification of Jawetz type and Heyl type of Pasteurella pneumophila was performed after rapid PCR template acquisition and combined with DNA sequencing for identification. Results Among the 4 sampling methods, the oral sample had the best detection effect. In 2 methods of DNA extraction, the bacterial genomic DNA could be obtained rapidly by culturing and boiling the samples for 1 min, which was more effective than the kit extraction method. Conclusion Rapid extraction of bacterial genomic DNA from the oral samples of experimental animals by incubation and boiling for 1 min as PCR template can significantly improve the positive detection rate of Pasteurella pneumophila.

Observation of LumianningⅡ or Ketamine Combined with Propofol in Experimental Pigs Undergoing Natural Orifice Transluminal Endoscopic Surgery#br#

HUANG Peidi, LI Haiwen, ZHENG Huantian, LI Jian

2020, 40(3): 242. DOI: 10.3969/j.issn.1674-5817.2020.03.012

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Objective To observe the anesthetic effect of different induced anaesthetics LumianningⅡ or ketamine combined with propofol for peroral natural orifice transluminal endoscopic surgery across the stomach wall and into the abdomen for bracket placement to remove gallstone with gallbladder preservation in experimental pigs. Methods Sixteen Bama miniature pigs were randomly divided into two groups. The anesthesia was induced in the pigs with Lumianning II (group A) or ketamine (group B), and maintained with propofol during the operation. The vital signs, intraoperative conditions, anesthesia induction time, surgical time, recovery time and propofol dosage in the two groups were compared. Results The anesthesia of two groups went smoothly. For the two groups, the differences of the average heart rate, average respiratory rate, average blood oxygen saturation, the average systolic blood pressure and the average diastolic blood pressure before, during and after surgery were not statistically significant (all P＞0.05). The average body temperature during surgery was lower than that before and after surgery, the difference was statistically significant (P＜0.05). The incidence of mild body movements in group A was 25.0%, and that in group B was 37.5%. Three experimental pigs in group A showed convulsions during the operation, but none in group B. The difference was not statistically significant (P＞0.05). The average induction anesthesia time, average operation time, average recovery time and average total dosage of propofol between group A and group B were not statistically significant (all P＞0.05). Conclusion LumianningⅡ- or ketamine-induced anesthesia and propofol maintenance can be used in the experimental pigs to achieve safe anesthetic effect during the natural orifice transluminal endoscopic surgery approaching for gallstone removal. The ketamine group has shorter induction time with no convulsions. This study can be a value of reference for clinical practice.

Establishment of a Method for Detecting Acute Eye Irritation of Disinfectant Using Porcine Cornea#br#

Establishment of a Method for Detecting Acute Eye Irritation of Disinfectant Using Porcine Cornea

2020, 40(3): 248. DOI: 10.3969/j.issn.1674-5817.2020.03.013

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Objective To establish an in vitro test method for acute eye irritation of disinfection products by using porcine cornea. Methods In vitro detection conditions on porcine corneal system were investigated and optimizated, which used ethanol and imidazole as positive control. Results It was confirmed by liquid and solid disinfectants that after the exposure of ethanol for 10 min and incubation for 80 min, or the imidazole exposure for 3 h and incubation for 70 min, the in vitro stimulation score was within the standard range of bovine corneal opacity and permeability (BCOP) corresponding positives, and the test system was established. Conclusion A method for detecting acute eye irritation of disinfectants is initially established by using pig eyes instead of bovine eyes, which can provide a reference for the safety evaluation of disinfection products.

Strategies to Prevent and Eliminate Infetion of Corynebacterium bovis from Laboratory Animal Facility#br#

LI Ming

2020, 40(3): 252. DOI: 10.3969/j.issn.1674-5817.2020.03.014

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