Laboratory Animal Science

The Changes of Histopathology and Ultramicrostructure of Skeletal Muscle in Curcumin Pretreatment Rats Under Dry Heat Environment

2018, 35(03): 1.

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Objective To observe the changes of histopathology and ultramicrostrcture of skeletal muscle of curcumin pretreatment rats in the dry-heat environment. Method 200 male rats were randomly devided into 5 groups (n=40):Saline group, solvent group, low dose curcumin pretreatment group, medium dose curcumin pretreatment group, high dose curcumin pretreatment group. All groups were given a isometric gavage for 7 days.At the 8thday,all groups were transferred to the Simulated Climate Cabin for Special Enviroment of Northwest of China with the setting environment: temperature (41±0.5)℃, relative humidity(10±1)%. At the time of 0th min,50th min,100th min,150th min, 10 rats per group were gotten out from the cabin and anesthettized for collecting inferior vena cava blood for the determination of myoglobin, musculi biceps femoris were harvested and fixed in formalin for HE staining and pathological observation by optical microscope. Selected randomly from 5 field of view to measure gray scale of muscle under 10×10 microscope len. At 50th min and 150th min, the 1 mm×1 mm×1 mm tissue of rat musculi biceps femoris of saline group and high dose curcumin pretreatment group were fixed in osmium acid and used for transmission electron microscope observation,respectively. Result 1. The observation result under optical microscope showed: The color of part of muscle fiber of saline group and high dose curcumin pretreatment group fade and pale area gradually increased with the prolonging time in dry heat environment. Saline group fading region gradually fades to white, however, high dose curcumin pretreatment group pales reddish color; In addition, the number of fading muscle fibers gradually increased with time, especially in the saline group.2. Transmission electron microscopy showed that the mitochondria decreased as the main histological change in the dry heat environment, the change in the high dose curcumin pretreatment group was less than that in the saline group. The mitochondrial heap can be seen as a sign of proliferation in the high dose curcumin pretreatment group. 3.There were no significant change in the content of myoglobin in the blood of each group in the dry heat environment (P>0.05). 4.Grey scaning indicated: The greys in saline group and solvent group from were increased from 0 min to 150 min, and the 150 min group significantly higher than that of the 0 min group(P 0.05);in 100 min and 150 min, the greys in high dose and medium dose curcumin groups were lower thant that of saline and solvent group, there were significant differenc(P<0.05). Conclusion Curcumin pretreatment can decrease the number of the red muscle transformed into white muscle in rat skeletal muscle under dry heat environment, and can inhibite the reduce of the mitochondria in muscle fiber, which indicated that curcumin pretreatment can exert protective effects on the stability of the mitochondria in muscle fiber under dry heat environment, we speculate that curumin pretreatment can inhibite the rhabdomyolysis in exertional heat stoke and the secondary multiple organ dysfunction syndrome (MODS).

The Protectvie Effects of Curcumin Pretreatment on the Myocardial of Heat Stroke Rats in Dry Heat Environment

2018, 35(03): 7.

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Objective To investigate the protective effects of curcumin pretreatment on myocardial injury of heat stroke rats in the dry heat environment. Method Fifty SPF 6-8 weeks old male SD rats were randomly divided into five groups (n=10):normal temperature control group (NC group), dry heat control group (DHC group), 50 mg/kg curcumin pretreatmet group (L-cur group), 100 mg/kg curcumin pretreatment group (M-cur group) and 200 mg/kg curcumin pretreatment group (H-cur group).The NC and DHC group were given a gavage of normal saline, while curcumin pretreatment group were given a gavage of curcumin with different concentration once a day for 7 consecutive days. At 8th day, all except for NC group were transferred to the climate cabin (The Simulated Climate Cabin for Special Environment of Northwest of China) with the conditions of (41±0.5) °C temperature, (10±1) % relative humidity. At the time of 150th minute since the experiment began, the rats were in heat stroke states, And same done to Control group.The changes of serum myocardial enzyme CK(phosphocreatine kinase),CK-MB(creatine kinase) and LDH(lactate dehydrogenase) were detected by automatic biochemical detector.The pathological changes of myocardium were observed by HE staining.Ultrastructural changes of myocardium were observed by electron microscopy. Result The serum myocardial enzymes CK, CK-MB and LDH in the DH group were significantly higher than those in the NC group (P<0.01).Curcumin intervention group CK, CK-MB and LDH in curcumin pretreatment groups were all decreased with the increase of curcumin concentration,and they had significant differert (P<0.01).HE staining result suggest:The myocardial tissue in the dry heat control group increased with the time of thermal exposure, and the bleeding gradually increased.The curcumin pretreatment group reduced the hyperemia and hemorrhage of myocardial tissue, especially in the high concentration group.The result of the electron microscope show that: Myocardial cell mitochondria swelled, structure of mitochondrial cristae appeared disorder, and partial myofilament fractured.Some of the mitochondria arranged irregularly, myocardial cell injury gradually increased with the thermal exposure time;The mitochondrial structure of myocardial cells in curcumin pretreatment groups were more complete, with clear cytoplasmic membrane and a large number of nucleoli,the Z line were clearer, the arrangement were more regular, the intervention effects were obvious. Conclusion Curcumin preteatment had protective effect on the myocardial injury of the heat stroke rats in the dry heat environment, and the effects appeared dose dependent.

Establishment of Orthotopic Transplantation Model of Liver Cancer and Imaging Evaluation

2018, 35(03): 12.

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Object A patient-derived orthotopic xenograft PDOX model was established to study the application of near-infrared fluorescence (NIRF) in vivo imaging and PET/CT in model evaluation. Method The PDX model of hepatocellular carcinoma was established by subcutaneously injecting clinical fresh hepatocellular carcinoma (HCC) specimens into severely immunodeficient NPG mice. The PDX model was evaluated by histomorphometric observation, STR genotyping, and immunohistochemistry. PDX models were implanted in nude mice to establish PDOX models by orthotopic liver transplantation. Near-infrared fluorescent dye IR-783 was injected to detect the occurrence of tumors by in vivo imaging of small animals. 18F-FDG was injected through the tail vein and observed by small animals PET/CT to confirm liver orthotopic tumor growth. Result The STR genotyping result showed that the human characteristics, histomorphological observation and immunohistochemical detection of PDX tumors indicated that PDX tumors retained the pathological features of primary tumors; detection of liver tumors was detected by near-infrared fluorescence in vivo imaging; PET/CT was available. The enrichment of 18F-FDG molecular probes in the mouse liver was clearly observed. Conclusion The PDOX model of hepatocellular carcinoma was established successfully. The model can be evaluated by living animal imaging and PET/CT imaging technology. It provides a good animal model for the treatment and pathogenesis of hepatocellular carcinoma.

Study on Damage Protection Methods Induced by High-energy Xenon Light on Ocular Tissue

2018, 35(03): 17.

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Object To evaluate the protection method induced by high-energy xenon light on oclular tissue. Method Sixty SD rats were randomly divided into six groups: positive control group; surgical drapes group; thin white plastic sheet group; thin brown plastic sheet group; thick brown plastic sheet group and negative control group (n=10). The rats were fixed in special fixation-machine and irradiated by high-energy xenon light for 5 min (5 d, twice/d). Fissure lamps, full-field retinal electrography (ffERG), coherent optical tomography (OCT), fundus photograph and pathological examination (HE staining) were used to evaluate ocular tissue damage. Result The staining score of corneal fluorescein sodium in positive control group and surgical drapes group were greater than that of the control group (P<0.05). The amplification of b wave (dark-adaptation 3.0 and light-adaptation 3.0 response) in positive control group and surgical draping group were significantly decreasing than the negative control group (P<0.05) and the peak-time of b wave were increasing than the negative control group (P<0.05). The amplification of ∑OPs1-4wave in positive control group and surgical draping group were decreasing than the negative control group (P<0.05). OCT found that the thickness of outer nuclear layer (ONL) in positive control group and surgical draping group were thinner than the negative control group (P<0.05), which was accordance with HE staining result . There was no obvious abnormality observed with fundus photograph in each group. Conclusion Surgical drapes group, thin white plastic sheet group, thin brown plastic sheet group and thick brown plastic sheet group have a certain protective effect on high-energy xenon light and surgical drapes group is weak.

Analysis of Immunological Characteristics of BABL/c Mutant Haired Mice

2018, 35(03): 25.

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Objective To investigate whether there is a mutation difference between the immunological indexes of BALB/c mutant mice and the normal BALB/c mice.Method Six weeks old BALB/c mutant curly and normal BALB/c mice each 20 (male and female) were selected respectively．Then 6 blood routine indexes were detected using automatic blood cell analyzer；4 antibody and complement indexes were detected using automatic biochemical analyzer；and the ELISA was used to detect serum cytokines TNF-α、 IL-2、IL-4；the result of the two groups were compared.Result The female and group result of NE、NE#、MO、and IL-4 between BALB/c mutant curly mice and normal mice have significant differences(P<0.05，P<0.01)； the male and group result of IgG and C3 between BALB/c mutant curly mice and normal mice have significant differences(P<0.01)；the sex and group result of IgM between BALB/c mutant curly mice and normal mice have significant differences (P<0.05，P<0.01)；the female result of LY between BALB/c mutant curly mice and normal mice have significant differences(P<0.05)．Conclusion There are differences in immunological parameters between BALB/c mutant curly mice and normal BALB/c mice，suggesting that the mutant gene may have a certain effect on the immune system of mice．

Established and Evaluated an Animal Model of Microbial Aerosol and Noxious gas Co-exposure by Respiratory Inhalation

2018, 35(03): 29.

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Objective Establish the respiratory system injury model of rat by inhalation formaldehyde combining with microbial aerosol. Method Thirty one wistar rat (180±10 g,8 w) were divided into four groups, separately growth under normal condition (group CO28), exposure under(2.5±0.2)ppm formaldehyde (group FA28), exposure under(1×107)cfu/m3 PAO1 (group PAO), and exposure under (3.4±0.3) mg/m3 formaldehyde combining with PAO1 aerosol (group FA&PAO). The animal morphology injury of lung tissue was observed by HE staining, the apoptosis of lung epithelial cells was observed by Tunnel chromogenic in situ detection. The peripheral blood were collected to examination IgM、IL-1β、IL-8、TNF-α、CC10 concentration and SOD activity. Result Morphological observation showed that the lung tissue of three experimental groups was changed with alveolar septum widened, interstitial edema and inflammatory cell infiltration and the most obvious changes were detected in co-exposure groups. Tunnel tests also showed that more apoptotic cells were detected in the lung epithelium cell in co-exposure groups. In contrast to those of normal control groups, after inhalation of microbial aerosol, rat’s serum IgM、 TNF-α、IL-1β、IL-8 levels increased significantly, with more significantly increasing in co-exposure groups(P0.05). Conclusion Formaldehyde combining with microbial aerosol co-exposure induced synergistic damage of rat lung tissues and destruction the scavenging ability of lung tissue to heterogeneity pollutants.

Feasibility Study of Adenovirus Mediated BMP-4 Gene Transfection in Rabbit SMSCs to Cartilage

2018, 35(03): 34.

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Objective To study the feasibility of adenovirus-mediated BMP-4 transfected rabbit synovial mesenchymal stem cells (SMSCs) with green fluorescent protein (EGFP) gene into chondrocytes. Method The rabbit knee joint capsule was taken, and the type I collagenase was used to digest and separate the SMSCs. The surface markers were detected by flow cytometry. The recombinant adenovirus (Ad-BMP-4) containing BMP-4 gene and EGFP gene was transfected into the SMSCs by different multiplicity of infection (50,100,200,300,400,500 MOI respectively) Efficiency, find the most suitable transfection conditions, and test the cell growth curve under different transfection conditions. The EGFP-labeled cells were observed by ELISA to determine if the transfected SMSCs entered the chondrocyte differentiation lineage. Result The morphology of SMSCs obtained after isolation and purification showed the proper structure of mesenchymal stem cells. The surface markers highly expressed CD45 and CD90, and basically did not express CD34 and CD45, which was consistent with many features of SMSCs. The transfection efficiencies were 54.3%, 72.2%, 77.8%, 85%, 86.6%, 89.3% after transfection with MOI of 50,100,200,300,400,500, respectively, and the fluorescence microscopy was more and more High green fluorescence expression, MOI=300 for transfection suitable conditions. The result of ELISA showed that BMP-4 was highly expressed, and cartilage genes such as SOX9, COLⅡ and AGC protein were also highly expressed. Conclusion This study shows that recombinant adenovirus transfected rabbit SMSCs, MOI amount of 300 can quickly and efficiently express BMP-4 and activate other cartilage-related genes, prompting SMSCs to chondrocytes.

Correlation of Pancreatic Carcinoma Patients’ Clinical Features and PDX Models

2018, 35(03): 34.

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Objective To explore the influence factors of pancreatic carcinoma patient-derived xenograft (PDX) model and improve the utilization rate of clinical samples. Method Sixty-two patients’ fresh pancreatic carcinoma specimens were transplanted subcutaneously into nude mice to establish PDX models. Correlation of the patients’ clinical feature and PDX models were analysed in order to figure out the efficiency of modelling. The hereditary consistency of the primary tumors and xenograft tumor was detected by STR genotyping, and the similarity of tumor tissue structure were analysed by histopathological features and immunohistochemical examination. Result Total 29 cases PDX models were established stabely, and total success rate reached to 47%(29/62). The patients’ clinical features did not show apparent correlation with the success rate of PDX models (P>0.05). There were more significant correlation between primary tumors’ size and PDX models (P<0.05), especially for lymphatic metastasis (P<0.01). The stabe consistency of primary tumors and transplanted tumors was confirmed by STR genotyping, histopathological features and CA19-9 expression. Conclusion We have successfully established 29 casespancreatic carcinoma PDX model. Primary tumors’ size and lymphatic metastasis were the key influence factors on success rate of PDX models.

MCP Inhibited Bladder Tumor Growth by Downregulation of Galectin-3

2018, 35(03): 41.

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Modified citrus pectin (MCP) is a carbohydrate enriched complex, which has been implicated in cancer treatment and prevention.However, the effects of MCP on urinary bladder cancer (UBC) are unknown. In this study, MCP was first tested in T24 and J82 human UBC cells and showed the inhibition of cell viability. oral administration of MCP to the T24 xenograft-bearing nude mice inhibited the tumor growth significantly (P<0.05). Quantification analysis of immunohistochemistry staining for Ki67 and cleaved Caspase-3 confirmed the decrease of proliferation index (P<0.05) in 700 mg/kg MCP-fed UBC xenografts，and its potiential target galectin-3 was downregulated.MCP may become an attractive agent, as a natural dietary fiber, for prevention and therapy of UBCs.

Construction of an Animal Model of Ocular Damage Caused by Long-term Microgravity Exposure

2018, 35(03): 52.

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Objection To explore the method of constructing an animal model of ocular damage caused by long-term microgravity exposure. Method Twenty four Sprague-Dawley rats were randomly divided into two model groups (M4 and M8, n=6) and two normal control groups (N4 and N8, n=6). Rats of the M groups were tail-suspended with 30°degree, with the control rats living ad libitum. After 4 and 8 weeks, all rats were subjected for exanimation of retinal function and morphology. Specifically, electroretinogram (ERG) and visual-evoked potential (VEP) were conducted to assess the influence of microgravity on retinal function. Optical Coherence Tomography (OCT) were performed to observe the alive structure of retinas, and paraffin section of retinas were done to examine the retinal histology. In addition, the fundus vasculature was observed by animal fundus imaging system. Result No obvious changes were found in retinal function and structure after four weeks of tail-suspension. However, surprise came out when rats were examined under microgravity for eight weeks. Amplitudes of ERG from tail-suspended rats declined[(686.60±86.20) vs. (158.80±23.69) μV, P<0.05], with the latency of P1-wave of VEP prolonged [(89.83±4.98) vs. (96.67±4.41) ms, P<0.05]. The ONL reflected from OCT and retinal histology became thinner in the tail-suspended rats (35.13 ± 1.02) vs. (20.32±1.92) μm, P<0.05]. Fundus photography shows increased blood flow in choroid membrane. Conclusion Eight weeks tail-suspended SD rats well simulate the effects of long-term microgravity on eyes. Therefore, it could be one of the best animal model of ocular damage caused by long-term microgravity exposure.

Establishment of Asthma Animal Models in Damp Environment

2018, 35(03): 59.

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Objective Studying the different effect of high humidity environment on healthy mice and asthma mice; establishing models of high humidity injury animals and asthma animals in high humidity environment. Method Female BALB/c mice were placed in artificial climate box to simulate a high humidity environment. The mice were divided into control group (group A), asthma group (group B), high humidity group (group C) and high humidity asthma group(group D). Group A was fed under normal temperature and normal humidity. The B group was fed under normal temperature and normal humidity and sensitized and stimulated by OVA. The C group was fed under normal temperature and high humidity. The D group was raised was fed under normal temperature and high humidity and sensitized and stimulated by ovalbumin (OVA). Result Compared with B, C, D group and A group, there are different degrees of pulpy stool, dark fur, irascible behavior, weight loss, abnormal blood routine index (P<0.05). The immune inflammatory response, the proportion of eosinophils in alveolar lavage fluid, and pathological changes in lung in group B and D were significantly higher than those in group A and C (P<0.05).The inflammatory response in group D was higher than that in group B (P<0.05). Conclusion The high humidity environment can induce or aggravate the symptoms of asthma. The animal model of asthma in high humidity environment can lay the foundation for the study of the occurrence and development of asthma in the high humidity environment.

P53 Knockout in Mouse Primary Ovarian Epithelial Cells and Biological Characteristic Changes

2018, 35(03): 64.

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Objective To establish a TP53 gene knockout cell line using CRISPR/Cas9 system in mouse primary ovarian epithelial cells(MPOE cells), and study the effect of TP53 gene knock out on cell proliferation, cell cycle progression, colony formation,migration and invasion.Method The LentiCRISPRv2-sgRNA TP53 gene knockout plasmid was constructed and packaged in 293FT cell. The supernatant was collected，and infected the MPOE cells after filtered．The positive clones were selected by puromycin. PCR, Western blot and Immunofluorescence were used to detect TP53 knockout cell lines．Cell proliferation, cell cycle progression, colony formation, migration and invasion were examined by MTT, flow cytometry analysis, colony formation assay and transwell assay, respectively. Result TP53 knockout MPOE cell line was generated in which cell proliferation and colony formation were increased，DNA synthesis was elevated, moreover, cell migration and invasion were increased. Conclusion The TP53 gene stable knockout cell line of MPOE was successfully generated by CRISPR/Cas9 system and the biological characteristics changed obviously．

Effect of General Anesthesia on Fasting Blood Glucose in Bama Miniature Pigs

2018, 35(03): 70.

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Objective Investigating the effect of general anesthesia on fasting blood glucose in type 2 diabetic Bama miniature pigs induced by high glucose and high fat diet. Method Sixteen head of Bama miniature pigs were randomly divided into 2 groups, eight heads in each group, the control group was normal feed group, and the experimental group was feed by high glucose and high fat diet. Fasting blood glucose was measured before and after general anesthesia in sixteen Bama miniature pigs. The blood glucose values before and after anesthesia were statistically analyzed in the group and between groups. Result The difference of fasting blood glucose between the two groups was significant before and after the anesthesia, and the fasting blood glucose levels in the high fat and high fat diet group increased significantly compared with the control group. Conclusion After general anesthesia, the fasting blood glucose levels of Bama miniature pigs increased significantly, and the fasting blood glucose increased significantly in the type 2 diabetic induced by high glucose and high fat dietcompared with the control group.

Effects of Polysaccharide from Pleurotus eryngii Bacterial Chaffs on Spleen Index，Spleen Lymphocyte Proliferation and Immune Factor of Mice

2018, 35(03): 74.

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Objective To observe the role of polysaccharide from pleurotus eryngii bacterial chaffs on spleen index，spleen lymphocyte proliferation and immune factor of mice. Method The ultrasonic extraction of polysaccharide from pleurotus eryngii bacterial chaffs were mixed with the basic feed, which was made into low dose (200 mg/kg), medium dose (400 mg/kg) and high dose (800 mg/kg) feed. Forty eight KM mice were randomly divided into four group, including blank group, low dose group, medium dose group and high dose group. After 60 days of continuous feeding, the index of spleen, the proliferation of splenic lymphocytes and the content of IFN-γ, TNF-α and IL-6 were measured to study the immune regulation effect of polysaccharide on mice. Result Compared three kinds of polysaccharide groups with blank control group,the low dose group could significantly increase the spleen index (P<0.05),the medium dose group could significantly increase the spleen index and spleen lymphocyte proliferation in mice (P<0.05).In addition, it could increase the content of TNF-α and IL-6 (P<0.05); High-dose group could significantly increase the content of IFN-γ (P<0.05) and which also could significantly increased the spleen index,the spleen lymphocyte proliferation, the content of TNF-α and IL-6 in mice (P<0.01). Conclusion The polysaccharide from pleurotus eryngii bacterial chaffs play important role on regulating spleen index，spleen lymphocyte proliferation and Immune factor in mice.

Establishment and Application of an Animal Model through Deep Venous Puncture Catheterization (DVPC) in Beagles

2018, 35(03): 79.

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Objective To establish an animal model through DVPC in beagles and to discuss its field of application. Method Eight adult beagles were randomly divided into the group of femoral vein catheterization (FVC) and the group of jugular vein catheterization (JVC), each group with four beagles. After anesthesia with a mixture of 1.5%-3% (v/v) isoflurane and oxygen, the beagles were fixed with a supine position in the operating table.The femoral vein and jugular were positioned to carry out DVPC and the catheters were fastened. Result DVPC for eight beagles was successful and the anesthetic effect was good. Compared with JVC, FVC operates more conveniently with shorter operating time, easier nursing and fewer complications. Conclusion The establishment of DVPC in beagles makes it possible for the application of blood perfusion and purification to laboratory animals. Meanwhile, it facilitates relevant clinical basic research. It can also be applied by vets to poisoned dogs for blood perfusion or purification and also to shock dogs for quick and mass fluid infusion.

Design and Research on the Information Management System for the Animal Experiment in Medicine Universities

2018, 35(03): 81.

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To promote the standardization of teaching and research in medicine universities,we will improve the management of teaching and research funding and the overall quality and efficiency, to adapt the development of experimental animals discipline and enhance the welfare ethics level of experimental animals,to improve the work efficiency of animal experiment and service quality of scientific research，we study designed an information management system for the animal experiment in medicine universities.Its functions advenced and operation method convenient and good prospect of promotion application.To provide a new way for experimental animals standardized management and standardization of experiments on animals in medicine universities,so as to promote the scientific and standardization of teaching and scientific research.

Research Progress of Laboratory Animal Models of Diabetic Neuropathy

2018, 35(03): 86.

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Diabetic nephropathy (DN) is one of the major chronic complications of diabetes mellitus, and is the main cause of end-stage renal disease. Ideal diabetic nephropathy animal models are important to clarify the pathogenesis of DN, and helpful for supplying effective prevention strategy. The current animal models of DN mainly include the induced, spontaneous, and genetically modified diabetic nephropathy models. This paper reviewed the progress of laboratory animal models of diabetic nephropathy.

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