Laboratory Animal and Comparative Medicine

Mice with Liver Fibrosis Based on Acoustic Radiation Force Impulse Imaging

CHEN Gaofeng, GAO Zhiling, LOU Weiwei, HUANG Lingying, JIN Shugen

2020, 40(5): 361. DOI: 10.3969/j.issn.1674-5817.2020.05.001

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Objective Acoustic radiation force impulse imaging (ARFI) was used to explore the characteristics and significance of intrahepatic shear wave velocity of mice with liver fibrosis induced by carbon tetrachloride (CCl4). Methods Six weeks old male C57BL/6J mice were used. The control group mice were given edible oil (0.1 mL/20 g body weight) by gavage once every other day for 8 weeks. In the 4-weeks group, the above-mentioned edible oil was given by gavage once every other day for 4 weeks, then the mixture of 1% CCl4 and edible oil (250 µL/kg) were given by gavage once every other day for 4 weeks. In the 8-weeks group, the mixture of 1% CCl4 and edible oil (250 µL/kg) were given by gavage once every other day for 8 weeks. After 8 weeks of the experiment and another 2 weeks of normal feeding, ARFI was used to detect the intrahepatic shear wave velocity respectively. All the mice were sacrificed and the liver tissues were collected for HE and Sirius red collagen staining, and the pathological changes of liver tissues were observed. Results Compared with the control group, the intrahepatic shear wave velocity of the 4-week group was increased after 2 weeks of normal feeding, and the histological liver fibrosis was formed, while that of the 8-week group was higher than that of the control group after 2 weeks of normal feeding and the liver cirrhosis was formed. However, the intrahepatic shear wave velocity of 4-week group and 8-week group was similar, which was significantly higher than that of after 2 weeks of normal feeding respectively. Conclusion The intrahepatic shear wave velocity of ARFI can reflect the changes of liver fibrosis in mice, but the effect of intrahepatic inflammatory injury on intrahepatic shear wave velocity should be excluded.

Establishment of High Altitude Pulmonary Edema Model in SD Rats by Different Hypoxia Stress Methods

LIN Xue, LEI Youfang, PU Xiaoyan

2020, 40(5): 367. DOI: 10.3969/j.issn.1674-5817.2020.05.002

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Objective To study the effects of different hypoxia stress on the construction of high altitude pulmonary edema (HAPE) model. Methods Total of 60 SD rats were randomly divided into 3 groups: the control group (at an altitude of 400 m), the hypobaric oxygen chamber group (simulating altitude of 6 000 m, and hypoxia stress for 48 h), and the field hypoxia group (at an altitude of 4 200 m, 28 days of hypoxia stress), 20 animals in each group. By detecting lung tissue dry-wet ratio, morphological and pathophysiological characteristics, expressions of key genes including aquaporin-1 (AQP-1) and vascular endothelial growth factor (VEGF), and oxidative stress level, the effect of different hypoxia stress on the establishment of HAPE model in SD rats was compared. Results Compared with the control group, the results of hypobaric oxygen chamber group and the field hypoxia group were as follows: the pulmonary artery pressure of the rats in the two groups increased significantly (P<0.01), while the oxygen partial pressure and oxygen saturation decreased significantly (both P<0.01); the lung tissue moisture content in both groups increased significantly (P<0.01). The lung tissue morphology of the control group was normal under a light microscope and an electron microscope, while the alveolar wall and alveolar septum of the two experimental groups were significantly widened under a light microscope, with a large number of red blood cells and inflammatory cells overflowing, and the obvious edema of alveolar septum was observed under a light microscope. The mRNA and protein levels of AQP-1 in the lung tissues of the two experimental groups were significantly higher than those of the control group, while the mRNA and protein levels of VEGF were significantly lower than those of the control group (all P<0.01). There were low GSH-Px and SOD levels and high MDA level in the serum of the two experimental groups. Conclusion Both the hypobaric oxygen chamber simulating the hypoxia stress at an altitude of 6 000 m for 48 h and the field hypoxia stress at an altitude of 4 200 m for 28 d effectively can construct the HAPE model of SD rats, and the hypobaric oxygen chamber for constructing the SD rat model of HAPE was more dominant.

Experimental Study on Establishment of Obesity Model in Rats Induced by High-Calorie Diet

LV Xiaojun, WU Sen, ZHANG Ju, XU Xiaoling, PAN Wangping, LI Hougang, WANG Pinghui, HE Kaiyong

2020, 40(5): 374. DOI: 10.3969/j.issn.1674-5817.2020.05.003

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Objective To observe the effect of high-calorie diet on the establishment of obesity model in rats, and the differences in modeling between two different stocks of rats, and to compare the differences of obesity evaluation methods and obesity-sensitive animals screening methods, providing a reference for the preparation of animal models to study the function of weight loss products. Methods Total of 120 SD (Sprague Dawley) and 120 Wistar male rats were randomly divided into 2 groups (blank control group and model group) by body weight, with 60 rats in each group. The rats in blank control group were given basic diet, and the rats in model group were given improved high-calorie diet. The body weight and body length of the rats were recorded once a week, and the feeds were weighed twice a week to calculate the Lee's index, food intake and food utilization for 6 weeks. After 2 weeks and 6 weeks of feeding, 30 rats in each group were respectively selected to measure the biochemical indicators, and the fat around the kidney and the testis was dissected and weighed to calculate the fat/body weight ratio. The correlations of weight gain and Lee’s index with obesity and obesity sensitivity were analyzed. The correlation between obesity and weight gain after eliminating the last 1/3, 10%, and 20% rats with weight gain after 2 weeks of feeding were compared. Results Compared with blank control group, the body weight, Lee's index, weight gain, food utilization rate, body fat weight and fat/body weight ratio, as well as the serum levels of glucose and triglyceride in the two stocks of rats in model group were increased, while the food intake and the serum level of high density liptein cholesterol (HDL-C) were decreased (all P<0.05). There was no significant difference in each index of model group between SD rats and Wistar rats (P>0.05), but the differences in body length, Lee's index and food utilization rate of the two stocks of rats in blank control group were occasionally appeared during the experiment (all P<0.05). There was significant difference in serum level of HDL-C between SD rats and Wistar rats in the blank group at 2 and 6 weeks of feeding (P<0.05). The correlations of body weight gain with obesity and obesity sensitivity of the two stocks of rats were greater than those of Lee's index with obesity and obesity sensitivity (both P<0.05). When eliminating the last 10% or 20% rats with weight gain, the correlation between weight gain and obesity was better as compared with eliminating last 1/3 rats; the correlation coefficient was the highest when eliminating the last 10%. Conclusions The obesity model of rats induced by the improved high-calorie diet can be successfully established in SD rats and Wistar rats, and there is no significant difference between the two stocks. As an index of obesity evaluation and obesity sensitive rat selection, weight gain is better than Lee's index. Eliminating the last 10% rats with weight gain is useful for selecting the obesity sensitive animals.

Effect of Diosmetin on Acute Lung Injury Induced by Meconium and Its Mechanism in Neonatal Rats

HAO Lixia, LU Rong, MA Xiaxia, FAN Shujuan

2020, 40(5): 384. DOI: 10.3969/j.issn.1674-5817.2020.05.004

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Objective To investigate the therapeutic effect of diosmetin on fetal faecal-induced acute lung injury in neonatal rats. Methods Fourty-eight neonatal male SD rats were enrolled. The rats were injected with 1.5 mL/kg fetal faecal by endotracheal intubation to replicate meconium lung injury model and randomly divided into 4 groups: control group, model group, methylprednil group and diosmetin group for subsequent experiments. Automatic blood gas analyzer was used to detect the arterial oxygenation index. Hematoxylin-eosin (HE) dye was used to detect the degree of lung tissue pathological injury and to evaluate the pathological score of lung injury. The lung tissue was removed and weighed 24 h after the establishment of lung injury model. The levels of inflammatory cytokines including tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β) and interleukin- 6 (IL-6) were detected by enzyme-linked immunosorbent assay. The expressions of NOD-like receptor with pyrin domain 3 (NLRP3) inflammasome, cleaved caspase-1 (c-caspase-1) and IL-1β proteins were detected by Western blotting. Results Compared with the model group, the mean arterial oxygenation index of methylprednil group and diosmetin group decreased significantly (both P<0.05), and the pathological damage degree and lung injury pathological score were significantly reduced (both P<0.05), the levels of inflammatory factors TNF-α, IL-6 and IL-1β as well as the expressions of NLRP3, c-caspase-1 and IL-1β proteins were significantly lower (all P<0.01).Conclusion Diosmetin can down-regulate the levels of NLRP3, c-caspase-1 and IL-1β, as well as reduce the arterial oxygenation index and the levels of inflammatory cytokines, thereby can be used to treat neonatal rats with meconium-induced acute lung injury.

Effect of Tetramethylpyrazine on Extracellular Regulated Protein Kinase Pathway in Collagen-induced Arthritis Rats

LIANG Jinfeng, LIU Wenhua, YAN Hong, DENG Shaojie

2020, 40(5): 391. DOI: 10.3969/j.issn.1674-5817.2020.05.005

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Objective To investigate the effects of tetramethylpyrazine (TMP) on the extracellular regulated protein kinase (ERK) pathway in collagen-induced arthritis rats. Methods Thirty male SD rats were randomly divided into control group, model group and TMP group, with 10 rats in each group. The rats in the model group and the TMP group were modeled with collagen-induced arthritis. After the successful modeling, the TMP group was treated daily with TMP (0.1 g/kg) by intragastric administration, for continuous 28 d. The model group and the normal group were given the same volume of physiological saline. The volume changes of the hind paws in the three groups were measured weekly, and the inflammatory cell infiltration of each group was observed by HE staining. The levels of tumor necrosis factor α (TNF-α), angiopoietin 1 (Ang-1), interleukin 1β (IL-1β) and vascular endothelial growth factor (VEGF) in synovial tissue and serum of rats were detected by realtime PCR and ELISA, respectively. The expressions of ERK pathway-related proteins in synovial tissues were detected by Western blotting. Results From the second week after modeling, the hind paw volume in the model group was higher than that in the control group (P<0.05), and the one in the TMP group was lower than that in the model group (P<0.05). The expression levels of inflammatory factors TNF-α, Ang-1 and IL-1β in the model group and the TMP group were higher than those in the control group (P<0.05), but the ones in the TMP group were lower than those in the model group (P<0.05). The level of VEGF in the synovial tissue of rats in the TMP group was lower than that of the model group (P<0.05). The expression of p-ERK1/2 protein in the model group and the TMP group was increased (P<0.05), and the expression level of p-ERK1/2 protein in the TMP group was lower than that of the model group (P<0.05). Conclusion TMP can effectively regulate the levels of TNF-α and IL-1β in serum and synovial tissue of rats with collagen-induced arthritis and alleviate the symptoms of joint swelling by inhibiting the ERK pathway.

Changes of Retinal Morphology and Expressions of RhoA and ROCK-2 in Ocular Ischemic Syndrome Rats

AN Xin, LIU Lanjing, BU Jianping

2020, 40(5): 397. DOI: 10.3969/j.issn.1674-5817.2020.05.006

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Objective To set up an ideal rat model for ocular ischemic syndrome (OIS), and to investigate the differences in the retinal morphology and the expressions of Ras-related monomer guanosine triphosphate kinase A (RhoA) and Rho-associated coiled coil forming protein kinase-2 (ROCK-2) in rat retina. Methods Twenty BN rats were randomly divided into two groups: the sham group and OIS model group (by bilateral carotid artery occlusion, BCCAO). Overall retinal thickness and retinal cell density were evaluated by histological analysis three months after modeling, respectively. The protein expressions of RhoA and ROCK-2 were also examined by immunohistochemical staining and Western blotting. Results Compared with the sham group, the number of retinal ganglion cells (RGC) was diminished in the model group (P<0.01). The significant decrease of thickness was found in the total retina, inner plexiform layer (IPL), inner nuclear layer (INL) and outer plexiform layer (OPL)(P<0.05); however, no change was evident in the outer nuclear layers (ONL) (P>0.05). The expressions of RhoA and ROCK-2 in the model group were significantly increased as compared with the sham group by immunohistochemical staining and Western blotting analysis (the former P<0.01, the latter P<0.05). Conclusion RhoA and ROCK-2 are significantly expressed in the OIS model group, which maybe provide new ideas for the treatment of this disease.

Effects of Sodium Oxalate on Autophagy and Apoptosis of Cholangiocarcinoma Cells#br#

HE Dawei, HE Aolin, ZHOU Minghui, YU Yaping, ZHANG Minghua

2020, 40(5): 403. DOI: 10.3969/j.issn.1674-5817.2020.05.007

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Objective To observe the effects of sodium oxalate, an inhibitor of lactate dehydrogenase A (LDHA), on autophagy and apoptosis of cholangiocarcinoma cell line Hucct-1, and to explore the related mechanisms. Methods Hucct-1 cells were treated with different concentrations of sodium oxalate. And LDHA siRNA was transfected into Hucct-1 cells by LipofectAMINE 2000 and Opti-MEM medium. Then the cell proliferation was detected by CCK8 assay, the apoptosis was detected by flow cytometry, and the cell migration was detected by scratch healing test. Meanwhile, the expressions of autophagy-related mRNAs and proteins were detected respectively by RT-PCR and Western blotting. And the lactic acid detection kit was used to detect lactate content in cells. Results Sodium oxalate inhibited the proliferation of Hucct-1 cells in a dose-dependent manner, its half-inhibitory concentration increased was 50 mmol/L. After Hucct-1 cells were treated with 50 mmol/L sodium oxalate or transfected with LDHA siRNA, the mRNA and protein expression levels of LDHA were decreased (both P<0.01), and the intracellular lactic acid content was decreased (P<0.01). In the meantime, the apoptosis rate was increased (P<0.01), and the cell migration distance was decreased (P<0.01). And the expression levels of autophagy-related gene 7 (ATG7), Beclin-1 and microtubule-associated protein 1 light chain 3Ⅱ (LC-3Ⅱ) were increased (all P<0.01). In addition, the expression of P62 protein was significantly decreased (P<0.01). Conclusion Sodium oxalate can promote autophagy and induce apoptosis by inhibiting the expression of LDHA in Hucct-1 cells.

Effects of Modified Wuzhuyu Decoction on Blood Pressure and Vascular Endothelial Function in Rats with Hypertension Induced by High Sugar and Fat Diet and Alcohol Drinking

WU Haojie, ZHOU Ruitang, NI Ning

2020, 40(5): 410. DOI: 10.3969/j.issn.1674-5817.2020.05.008

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Objective To investigate the effects of modified Wuzhuyu (Tetradium ruticarpum) decoction on blood pressure and vascular endothelial function in rats with hypertension induced by high sugar and fat diet and alcohol drinking. Methods One hundred SPF male SD rats were selected, 20 were used as control group, and 80 were used for establishing hypertension model induced by high sugar and fat diet and gradient alcohol drinking. The successfully induced model rats were randomly divided into 4 groups with 20 rats in each: hypertension model group, modified Wuzhuyu decoction low-dose treatment group, middle-dose treatment group, and high-dose treatment group. The rats of treatment group were oral gavaged daily with different concentrations of modified Wuzhuyu decoction, and the normal control group and hypertension model group were oral gavaged with distillation respectively. The systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MBP), and heart rate (HR) were detected before the experiment, after the successful modeling (before administration), and after treatment. The serum intercellular adhesion molecule-1 (ICAM-1), endothelin-1 (ET-1), nitric oxide (NO), and endothelial nitric oxide synthase (eNOS) were detected by ELISA. Results Compared with the control group, the SBP, DBP, and MBP in each experimental group were significantly increased after modeling (before administration) (P<0.05). Compared with the hypertension model group, the SBP, DBP, and MBP in the medium-dose and high-dose treatment groups decreased significantly after treatment (P<0.05). There was no significant difference in HR between the groups of rats before and after treatment (P>0.05). After treatment, compared with the control group, the levels of ICAM-1 and ET-1 in each experimental group increased significantly, and the levels of NO and eNOS decreased significantly (P<0.05). Compared with the hypertension model group, the levels of ICAM-1 and ET-1 in the medium-dose and high-dose treatment groups were significantly increased, while the levels of NO and eNOS were significantly reduced (P<0.05). Conclusion Medium-dose and high-dose modified Wuzhuyu decoction has obvious antihypertensive effect on hypertensive rats induced by high sugar and fat diet and alcohol drinking .

Preliminary Study on Heart Tolerance to 60Coγ-ray Radiation in Naked Mole Rats#br#

LI Yu, LIU Yinhang, CHEN Chao

2020, 40(5): 415. DOI: 10.3969/j.issn.1674-5817.2020.05.009

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Objective To observe and analyze the changes of cardiac morphology and biochemical indexes in naked mole rats after 60Coγ-ray radiation, and to preliminarily study the characteristics of 60Coγ-ray radiation tolerance in naked mole rats heart. Methods The naked mole rats were generally radiated by the single 10 Gy 60Coγ-ray, and then their hearts were collected after 7 days and 14 days respectively. After paraffin section and hematoxylin eosin (HE) staining, the microstructure of naked mole rat heart was observed under an optical microscope, and the pathological analysis was carried out. The malondialdehyde (MDA), total antioxidant capacity (T-AOC), glutathione (GSH) and oxidized glutathione (GSSG) contents in heart tissues were detected by biochemical test kit. Results Microscopic examination showed that there was no obvious atrophy, edema, necrosis and fibrosis in the heart tissues of naked mole rats after irradiation. According to the biochemical test, the content of MDA in heart tissues increased significantly after irradiation (P<0.01), and the T-AOC, GSH and GSSG contents decreased significantly with time (all P<0.05). Conclusion The heart of naked mole rats can tolerate 60Coγ-ray to some extent, but the specific mechanism of tolerance is not clear.

Pathological Observation of Spontaneous Tumor in Aged SD Rats

ZHOU Xiaoli, ZHANG Qian, GAO Zheng, QIAN Zhiyong

2020, 40(5): 420. DOI: 10.3969/j.issn.1674-5817.2020.05.010

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Objective To observe the occurrence of spontaneous tumors in SD rats, and to provide the background data for carrying out new drug induction trials. Methods In a carcinogenicity test, 120 normal SD rats (SPF-grade) at 4 weeks of age were divided into male and female with the same number. All rats were observed for one week before the experiment. After 104 weeks of conventional feeding, the sedation was performed. The dead rats during the experiment and the sacrificed rats were dissected. The organs such as heart, lung, liver, spleen, kidney, adrenal gland, thymus, brain, testis and epididymis (or uterus) were weighed, and the types and incidence of spontaneous tumors in each organ were observed by an optical microscope after routine pathology. Results A total of 113 SD rats were observed (7 rats were lost during the test), the tumor-bearing rats accounted for 66.4% (75/113), of which the benign tumors accounted for 48.7% (55/113), and the malignant tumors accounted for 17.7% (20/113). Tumors occurred in 49.1% (28/57) of male rats, of which 50.9% (26/57) were benign tumors, and 3.5% (2/57) were malignant tumors; the benign tumors were mainly pituitary adenomas (36.8%) and adrenal medulla tumor (7.0%), the malignant tumors were mainly thyroid adenocarcinoma (1.8%) and fibrosarcoma (1.8%). Female rats had tumors of 83.9% (47/56), of which the benign tumors accounted for 51.8% (29/56), and the malignant tumors were 32.1% (18/56); the benign tumors were mainly pituitary adenomas (50.0%), breast fibrous glands tumors (12.5%) and breast adenomas (12.5%), the malignant tumors were mainly breast adenocarcinoma (25.0%) and fibrosarcoma (3.6%). Conclusion The incidence of spontaneous tumors was higher in more than 2 years old SD rats, and the incidence of benign tumors was higher than that of malignant tumors, which may be related to aging, tumors and individual differences.

Exploration of Phospho-HER2 Immunohistochemistry Staining on Frozen Tumor Tissues

FAN Shuqiong, ZHENG Li

2020, 40(5): 426. DOI: 10.3969/j.issn.1674-5817.2020.05.011

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Objective To investigate the optimized fixation and storage conditions for the immunohistochemistry detection of phospho-human epidermal growth factor receptor 2 (HER2) on frozen sections. Methods Xenograft model was established by subcutaneously injecting human breast cancer cell BT474 into the abdomen of BALB/c-nu/nu mice. The mice were sacrificed when the tumor volume up to 300 mm3. The tumor samples were quickly frozen, sectioned, fixed in different reagents and stored for different time period. Phospho-HER2 immunohistochemistry was performed to evaluate the change of protein expression under these conditions. Results Good cell morphology was kept by the fixation of frozen section in 50% formalin for 5 minutes. The expression of phospho-HER2 protein was stable within 12 weeks after fixation when the tumor tissues were stored in -20 ℃. Conclusion The immunohistochemistry staining on frozen tissues takes less time and can be a supplement or replacement of traditional staining on formalin-fixed and paraffin-embedded sections.

Application of Zebrafish as A Model Animal in Research of Human Eye Diseases

DI Yanan, ZHU Liying, QIAN Wen, PAN Wei

2020, 40(5): 440. DOI: 10.3969/j.issn.1674-5817.2020.05.014

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As an excellent animal model, zebrafish (Danio rerio) has been widely used in the fields of developmental biology, genetics, toxicology and so on. Because it is great similar to human in aspect of eye morphology, anatomical structure, gene expression and function, and has advantages on rapid development, sperm reception way, embryo transparency and easy observation, zebrafish has been increasingly used in the study of eye development and related diseases such as eye defect, glaucoma, cataract, rod cone degeneration amd diabetic retinopathy. This article will review the eye development and structure of zebrafish as well as the advantages and disadvantages of zebrafish as a model for the study of eye diseases, which will help to have a more comprehensive understanding of the application value of zebrafish in the research of human eye diseases.

Progress in Pathogenesis and Immune Response of Enterovirus 71#br#

SHI Meiyan, LU Caixia, DAI Jiejie

2020, 40(5): 449. DOI: 10.3969/j.issn.1674-5817.2020.05.015

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