Abstract:As a model organism, zebrafish have many advantages over other animal models and is suitable for studies on establishment of human disease model and mechanism. In zebrafish, there are two phases of endocrine formation during early development, which are directed by concomitant activity of many signaling pathways. Zebrafish pancreas possess similar cell structure with that of other animals, which can express various endocrine hormones including insulin. The main organs required for metabolic control, such as the pancreas, islet, and insulin sensitive tissue (muscle, liver) are conserved in zebrafish, and the mechanisms of glucose regulation in zebrafish is similar to that seen in mammalian models. These render it an excellent model to study glucose metabolism. Hyperglycemia in zebrafish model can be induced by administration of the diabetogenic drug, streptozotocin (STZ), alternatively immersion of the fish in glucose solution and water, or disturbing of signaling pathways associated with glucose metabolism. Glucose levels in adult zebrafish blood or embryo tissue and phenotype of retinal cell layers or retinal vasculature are the commonly used measurement organs in zebrafish diabetic models.

Abstract:Objective To compare the characteristics of ulcer wound healing in current commonly used C57BL/6J-db/db mouse models of spontaneous gene mutation-induced type 2 diabetes and in C57BL/6J mice with diabetes induced by streptozotocin (STZ), and to provide a basis for related experimental studies on diabetic ulcer in animal models. Methods To establish the mouse models of diabetic ulcer wound, observe the healing time and calculate the wound healing rate at 0, 3, 5, 7, 10, 14 days. Tissue samples were collected at days 7 and 14. HE and Masson staining, and immunohistochemistry (CD31 and PCNA) were used to observe the pathological changes of the wound tissues. Gene expressions of collagen-Ⅲα, fibronectin and α-SMA were detected by fluorescent quantitative analysis. Results The wound healing time of db/db mice was significantly delayed compared with the STZ mice, which was extended from 16.6±0.8 d to 20.2±1.3 d (P<0.001). Compared with the STZ group, the growth of granulation tissue in the db/db group was slow, the length of newly formed epithelium was insufficient, the collagen deposition was disordered, and the wound healing was poor. At 7 days, the expression of CD31 and PCNA was significantly lower in the db/db group (P<0.01), and at 7 and 14 days, the increase of collagen-Ⅲα and α-SMA genes up-regulation was significantly lower in the db/db group than in the STZ group. Conclusions Both the two types of diabetic mice show delayed wound healing. However, compared with the STZ-induced diabetic mice, the gene mutation db/db mice are more suitable for studies of diabetic ulcer wound healing as regarding the extent of the delay and the degree of difficulty of wound healing.

Abstract:Objective To observe the biological characteristics of db/db mice, and to provide the basis for application of db/db mice in experimental research. Methods Spontaneous type 2 diabetes BKS.Cg-Dock7^m+/+Lepr^db/JNju mice and wild type mice of the same age were used in this study. Their fasting blood glucose was determined at 8,12,16, 20 and 24 weeks of age, the body weight was recorded at 10, 12,16, 20 and 24 weeks of age, the levels of serum insulin, total cholesterol and triglyceride were measured, the organ weight and liver coefficient were determined, and the liver and pancreas were taken for pathological examination at 24 weeks of age. Results The db/db mice maintained a high level of fasting blood glucose and body weight. Levels of serum insulin, total cholesterol, and triglyceride were significantly higher than the wild type mice. Compared with wild type mice, liver weight and kidney weight were also significantly increased. Obvious pathological changes of liver and pancreas were observed in 24-week old db/db mice. Conclusions db/db mouse has obvious characteristics of type 2 diabetes, such as hyperglycemia, hyperlipidemia, and hyperinsulinemia, can maintain stable levels of high blood glucose,and is an ideal animal model for experimental study of type 2 diabetes mellitus.

Abstract:Objective To study the serum metabonomic changes in abnormal Savda rat model of type 2 diabetes mellitus (T2DM). Methods Sprague Dawley (SD) rats were subjected to abnormal Savda-producing conditions followed by induction of T2DM. Plasma samples were collected from each test group and analyzed by ¹H nuclear magnetic resonance (NMR) spectroscopy. The spectral profiles were analyzed by multivariate analysis using orthogonal projection to latent structurs discriminant analysis (OPLS-DA). Results The content of glucose and lactic acid in plasma of the rats with abnormal Savda syndrome was decreased, while the contents of VLDL, LDL and carnitine increased obviously. For abnormal Savda rat model with T2DM, the concentration of amino acids (alanine, valine, isoleucine, leucine, tyrosine, glutamic acid, phenylalanine, methyl-histidine, glycine), and lactic acid, pyruvic acid, glycoprotein, citric acid, creatinine, β-hydroxybutyric acid, acetoacetic acid, acetic acid, acetate, creatine, carnitine, scyllo-inositol, VLDL, LDL were decreased, and glucose and taurine concentrations were increased significantly. Conclusions Protein metabolism, phenylalanine and tyrosine metabolic pathway, glycolytic pathway and the lipid metabolic pathway may be considered as biomarkers of abnormal Savda rat model of T2DM.

Abstract:Objective To explore the effect and mechanism of aerobic exercise on islet β-cells in type 2 diabetic rats. Methods Thirty healthy SPF 8-week old Wistar rats were randomly divided into control group (C, n=10), diabetic control groups (DMC) and diabetic exercise (DME) groups, 10 rats in each group, among which 7 successful rat models were used in the experiment. The diabetic rat model was established by high fat and sugar diet and i.p. injection of streptozotocin in a dose of 50 mg/kg. The rats of group DME were forced to perform 20 m/min running for 30 min, once a day, 6 days in a week, for 8 weeks. Other rats were allowed free movement. At the end of experiment, serum glucose and insulin were measured and homeostasis model assessment (HOMA) was calculated, and pancreatic tissue samples were collected for histopathological examination. The morphology and structure of pancreatic islets were observed under a digital microscope, the perimeter and area of islets were analyzed by image analysis, and shape factor of islets was calculated. The insulin content, glucokinase and ultramicro-ATPase activity in the pancreatic homogenate were determined. Results In the DME group, the perimeter and area of islets were significantly higher than the DMC group (P<0.05), but still lower than the control group. The shape factor was significantly increased, the cell hypertrophy, vacuolization and nuclear pyknosis were markedly alleviated than those in the DMC group, the insulin content, glucokinase and the trace total ATP activity in the DME group were significantly higher than those in the DMC group (P<0.05), and the SF and HOMA were significantly changed. Conclusions Aerobic exercise can reduce the blood glucose level, improve the morphology of islets and β-cells in the type 2 diabetic rats. It may be due to increase of the activity of glucose kinase and ATP-synthase, and increased insulin sensitivity in the pancreas.

Abstract:Objective To investigate the improving effects of Shenshao decoction on myocardial structure and function in diabetic cardiomyopathy,and its effect on expression of TLR4/MyD88-dependent pathway signal in diabetic cardiomyopathy. Methods Diabetes mellitus was induced in 8-week-old male Wistar rats by a single intraperitoneal injection of streptozotocin. The changes of plasma myocardial enzyme (CK, LDH) and high sensitive C reactive protein (hsCRP) were measured. Cardiac function was measured by left ventricular intubation. Hematoxylin and eosin staining and electron microscopy were used to observe the changes of myocardial morphology and ultrastructure in rats. Expression of Toll-like receptor 4(TLR4), myeloid differentiation protein 88(MyD88), and nuclear factor-κB P65(NF-κB P65) were tested by immunohistochemistry. Results After 6 weeks of treatment, the left ventricular diastolic and systolic functions were obviously improved;the degrees of myocardial fibers and mitochondrial damage were obviously relieved;the content of CK, LDH and hsCRP decreased (P<0.05), and hsCPR was positively correlated with CK and LDH (r=0.823,r=0.819,P<0.05).The expressions of TLR4, MyD88, NF-κB P65 were significantly decreased (P<0.05).There was no significant difference in the above mentioned indicators between the control group and control+Shenshao decoction group (P>0.05). Conclusions Shenshao decoction can reduce the myocardial injury in diabetic cardiomyopathy and improve cardiac diastolic and systolic functions.The mechanism may be related to attenuated inflammation by TLR4/MyD88 dependent signaling pathway.

Abstract:Objective To establish and evaluate a rat model presenting symptoms of arthritis-hypertension disease (AHD). Methods A total of forty healthy 5-6 week-old male SD rats were used in this study. Hypertension was induced by constriction of the left renal artery by two kidney one clip (2K1C) with a 0.25 mm silver clamp, and AHD model was established by injecting 0.1 mL complete Freund adjuvant to the left hind paw. Tail artery pressure was measured with a non-invasive blood pressure measurement system. The degree of swelling in the non-inflammatory joint of rats was measured with a paw volume measuring instrument, the arthritis index and incidence of inflammation were evaluated. The rats were sacrificed on the 35^th day. The thoracic aorta, ankle joint and spleen tissues were examined by pathology using HE staining. Results The joint of AHD model rat was significantly swollen, extensive synovial cell hyperplasia, inflammatory cell infiltration, vascular pannus formation, and bone and cartilage destruction. The number of germinal centers in spleen was increased, and a large number of lymphocyte infiltration, diffuse proliferation of white pulp, and red pulp infiltration were present. The arthritis index, incidence of inflammation and histopathological scores of the joint and spleen were significantly higher than adjuvant arthritis (AA) rats; meanwhile, the blood pressure of AHD model rat was significantly increased, the thickness and cross-sectional area of thoracic aorta were significantly increased, while the lumen diameter was significantly reduced. The blood pressure and vascular injury were significantly increased or aggravated compared with the HT rats. Conclusions A rat model of arthritis-hypertension disease is successfully established by using complete Freund adjuvant intradermal injection to the footpad and surgery to narrow the left renal artery.

Abstract:Objective To determine the localization of iRhom2 and its mutant proteins of Uncv mice in Vero cells by PKH26 combined with Hoechst 33258 staining. Methods The cell membrane was stained with PKH26, and the nuclei were stained with Hoechst 33258 dye, and observed by laser scanning confocal microscopy. Results It was found that wild iRhom2 was distributed in the cytoplasm, and its iRhom2^mut was present both in cytoplasm and cell nuclei. Conclusions The results of our study suggest that a deletion in N-terminal of iRhom2 affects its subcellular localization.

Abstract:Objective To analyze the genetic quality of 24 domestic inbred strains mice using microsatellite loci panel. Methods Previously selected 30 microsatellite loci of mouse with high polymorphism and more allele numbers were used to synthesize corresponding fluorescently-labeled primers. Then the genomic DNA samples of each mouse were amplified by PCR and the products were analyzed by STR scanning to genotype the inbred strains of mice. Results Out of the 24 inbred strains, 15 inbred strains showed the same genotype within one strain at 30 loci. Among different strains, microsatellite loci indicated polymorphism which could be used to distinguish different strains. However, the rest 9 strains demonstrated polymorphism within strains. Conclusions Our stuoly provides a useful microsatellite panel to detect genetic quality of inbred mice and distinguish different strains with the optimized microsatellite loci.

Abstract:Objective To optimize the preparation of air bag materials for abdominal withdrawal reflex test. Methods To measure the diameters of different pressue-air bags by digital vernier caliper and X ray imaging. Using the pain threshold (3 points) as a criterion to test the pressure values of different air bags in the rats with corresponding visceral pain sensitivity. Results With the increasing pressure, the changes of the diameter of round latex balloon were uniform and gentle, and could meet the pressure range used in the experiment (20~80 mmHg). The threshold of visceral pain in rats was near the nociceptive stimulus value,and would not cause irreversible damage to the rat intestinal tract. Conclusions Air bag made from round latrex ballon is an ideal tool to be used in abdominal withdrawal reflex test in rats.

Abstract:Objective To explore a reliable method to establish a rat model of hyperuricemia associated with abnormal uric acid excretion, and to lay the foundation for the study of pathogenesis of uric acid excretion disorder and the optimization of the treatment plan. Methods The models were established respectively by potasium oxonate(300 mg/kg) with pyrazinamide (300 mg/kg) or ethambutol(250 mg/kg). Continuous dosing for 1, 3 and 5 weeks, to determine the content of uric acid in rat blood, urine, and stool, the function of liver and kidney was detected and pathological examination was performed. Results The blood uric acid in the potasium oxonate and ethambutol group was increased first and then decreased, while in the potasium oxonate and pyrazinamide group were increased steadily and the excretion of uric acid in urine was stable during the continuous administration. The two methods showed no harmful effect on the liver and kidney function. Conclusions A stable rat model of hyperuricemia associated with uric acid excretion disorder can be effectively established by potassium oxonate and pyrazinamide, exhibiting similar manifestations of clinical hyperuricemia and uric acid excretion disorder.

Abstract:Objective To establish a patient-derived xenografts (PDX) mouse model of liver cancer (LC) and to explore its role in precision medicine. Methods PDX model was established by subcutaneous implantation of tumor tissues in NCG mice. The morphological structure of tumor tissue was exaimed using HE staining. Fifteen BALB/c nude mice were subcutaneously inoculated with tumor cell suspension from the PDX models. The xenograft mice were randomly divided into 5-fluorouracil (5-FU) group, sorafenib group and negative control group. The tumor volume and body weight of the tumor-bearing mice were measured regularly, the tumor inhibition rate was calculated and the curative effect was evaluated. Results The success rate was 33.3% (6/18) in the establishment of liver cancer PDX mouse model, and the model well retained the characteristics of the primary tumor. In one case of PDX mouse model, the tumor inhibition rates of 5-FU and sorafenib group were 63.7% and 29.6%, with a statistically significant differece between them (P<0.05), and there was no significant difference between the sorafenib group and negative control group, consistent with clinical observation. Conclusions The PDX mouse model of liver cancer can maintain the histological structure of primary tumor, and can be applied to precision medicine for patients with liver cancer.

Abstract:Objective To verify the feasibility of Cas9 microinjection in frozen-thawed pronuclear embryos, based on the model of pronuclear embryos of C57BL/6J mice by in vitro fertilization. Methods After fertilized mouse pronuclear embryos cultured in vitro, one-cell and 2-cell embryos were frozen using EFS20/40 cryopreservation tube. The next day recovered and then cultured. The recovery rate and survival rate of the one-cell and 2-cell embryos were compared. The frozen-thawed and fresh pronuclear embryos were injected with Cas9 mixture and injection buffer into the cytoplasm, and then cultivated to 2-cell embryos,and the survival rate and development rate of the 2-cell embryos were compared. Results The recovery rate of frozen-thawed one-cell embryos was 92.5%, the survival rate was 92.8%, the recovery rate of 2-cell embryos was 90.5% and the survival rate was 95.8%, showing no significant difference. Furthermore, the survival rate of fresh one-cell embryos after Cas9 injection was 92.7%, the survival rate of one-cell embryos of the blank group was 97.5%. While the survival rate of Cas9 injected frozen-thawed one-cell embryos was 82.6%, and that of the blank group was 92%,showing a significant difference between the frozen-thawed injected group and other groups(P<0.05).The development rate of 2-cell embryos after Cas9 injection was not significantly different. Conclusions Frozen-thawed pronuclear embryos can be used for Cas9 microinjection.

Abstract:Objective To improve the orotracheal intubation verifying technique and reduce the complication of backflow in rat experiment. Methods A new position evaluation of anti-backflow device was designed and made of safety IV catheter and closed IV catheter system. 60 adult male Sprague Dawley rats 216±20 g were randomly assigned to two groups:group A (n=40) for verifying placement, group B (n=20) for anti-backflow test. Group A was further divided into group A1 using self-designed positioning device, group A2 using aerosol, group A3 taking cotton fiber for positioning judgment. The group B was divided into two subgroups, B1 and B2, counting escaped bubbles as a means of positioning observation, the difference is that group B1 using frustum of a cone shape anti-backflow device, while the group B2 using common airway tube. Routine endotracheal intubation was performed to observe and record the time of positioning, the location of exhalation phase, and the length of inspiratory phase countercurrent water column. The group A1 further performed tracheotomy under direct vision clearly to confirm the anatomic positioning status. Results During the exhalation cycle,three or more bubbles were observed to escape continuously, indicating that the intubation tube was properly placed and open in the airway. Positioning time:It took 1.75±1.02 respiratory cycles in group A1,3.30±0.95 respiratory cycles in group A2 and 4.10±0.99 respiratory cycles in group A3 to complete the assessment the positioning status. There was no statistically significant difference between groups A2 and A3 (P>0.05). The time needed for group A1 was significantly shorter than that of groups A2 and A3 (P<0.01). The longest countercurrent water column length in group B1 was 3.23±0.53 cm, and 8.48±1.01 cm in the group B2. Conclusions The new designed anti-backflow positioning evaluation device is a simple and convenient appliance to evaluate the location of orotracheal intubation in rat experiment. It can effectively improve the positioning efficiency and has practical application value.

Abstract:Objective In order to study the biological characteristics of macrophages and provide the materials to study the survival mechanism of intracellular parasites, we conducted this study to establish a high-purity alveolar macrophage isolation and culture method. Methods Goat lungs were lavaged with normal saline in sterile environment several times, and cells were collected and then goat alveolar macrophages were purified by density gradient centrifugation using peripheral blood mononuclear cells (PBMC) solution. The isolated goat alveolar macrophages were cultured in cell culture medium containing 10% fetal bovine serum and cell morphology was observed under an inverted microscope every day,and the phagocytic activity of the cells was detected by chicken red blood cell phagocytosis test. Flow cytometry was used to detect CD14, a characteristic monocyte-macrophage surface marker. Results The adherent cells were characterized by typical macrophage morphology, pseudopodia and protrusions, showing round and irregular shape, rich cytoplasm, and large cell body. Of the cultured macrophages, 54.5% could phagocytize chicken erythrocytes and showed good phagocytic activity. After one month of in vitro culture, 93.7% of the cells were able to express CD14 antigen, which had a macrophage-specific immunophenotype. Conclusions The alveolar macrophages obtained in this study have high purity and good bioactivity, thus provide a cell model for studying the immune mechanism of intracellular parasites.

Abstract:Objective The aim of this study is to establish a multiplex polymerase chain raction (PCR) to identify of four kinds of laboratory animal pathogens:Pasteurella multocida, Bordetella bronchiseptica, Mycoplasma pneumoniae and Klebsiella pneumoniae. Methods Specific primers were designed based on GenBank data. The multiplex PCR system was established through optimization of multiple PCR and detection of its specificity and sensitivity. This technique was used to test artificially infected samples and tracheal secretions of experimental animals (rat, mouse, guinea pig, rabbit, hamster), and comparing the detection results by this method and traditional detection test. Results Target bands of Pasteurella multocida (356 bp), Bordetella bronchiseptica (237 bp), Mycoplasma pneumoniae (266 bp), and Klebsiella pneumoniae (142 bp) were obtained, with a detection sensitivity of Klebsiella pneumoniae of 10 pg, and that of Pasteurella multocida, Bordetella bronchiseptica and Mycoplasma pneumoniae of 1 pg by this newly developed multiplex PCR assay. No target bands were observed from the non-specific pathogens of artificially infected samples. The tracheal secretions taken from 45 experimental animals (mice and rabbits) were tested with this new PCR assay, among which 15 cases of Klebsiella pneumonia and 9 cases of Pasteurella multocida were detected as positive, while all the results of traditional method and serological test were negative. Conclusions A simple, rapid, specific and highly sensitive multiplex PCR system has been successfully established.It is valuable for detection of Pasteurella multocida, Bordetella bronchiseptica, Mycoplasma pneumoniae, and Klebsiella pneumoniae in laboratory animals.

Abstract:Hypoxia/ischemia is a common clinical pathophysiological process and cause of death,and it is a common problem in extreme conditions such as high altitude,astronautics and diving.Hamartin is a kind of effective endogenous neuroprotectant and could increase cell tolerance to acute hypoxia or ischemia, thus,is of significance to research.The role of hamartin in hypoxia/ischemia has been a research focus of many scientists.Elucidating the related protective effect and its DNA methylation on hypoxia/ischemia can not only reduce injury,but also lay a basic for further studying the role of hamartin and its DNA methylation in other pathophysiological processes and provide theoretical guidance for the following clinical study.In this paper,we review the structure,mechanism and role of hamartin and the effect of its DNA methylation on hypoxia/ischemia.

Abstract:Abdominal withdrawal reflex (AWR) test, which evaluates the behavioral response of animals through expanding the intestinal tract, is the most widely used method to assess animal's visceral sensitivity in recent years. However, as AWR experimental operation involves many factors, it's hard to find a commonly recognized operating procedure. Through review of literature of AWR experimental measuring methods, this article summarizes the basic steps of AWR operation. Meanwhile, it scrutinizes the differences of various measuring methods in relevant factors, including expansion method and airbag method, with special operating suggestions to them. Hopefully, this article will provide an option to future researchers who will use this method to evaluate animal's visceral sensitivity, and provide a necessary foundation for the standardization of the experimental operation.

Abstract:Objective Laboratory animal practitionres need further management through technical and capacity training. Methods Relying on the Laboratory Animal Center of University and its highly qualified faculty, a high-quality training base for laboratory animal practitioners has been set up. Results Based on the relevant laws and regulations about experimental animals, appropriate training outlines, different training books and courses were prepared and organized for different trainees. Focusing on the detailed and exact experiments, training base has put emphasis on the practical ability and operational skills. Conclusions How to regulate the training for laboratory animal practitioner by means of base training is discussed and tried out in this paper.

Abstract:Objective To compare the license issuance and status of administration of laboratory animals in Jiangsu and Zhejiang provinces in the last five years, and to study the status of laboratory animal license management and industry development level in the two provinces. Methods Taking a combination of current status investigation, literature search, information collection and comparative analysis, to study the similarities and differences in the annual issuance of laboratory animal licenses, level of environmental facility, type of institution division, regional distribution of institutions, etc. in the two provinces over the last five years. Results Over the past five years, the total number of the issued laboratory animal licenses and approved institutions in Jiangsu province are significantly more than in Zhejiang province. The proportion of barrier environmental facilities in Jiangsu is slightly higher than that in Zhejiang. Laboratory animal institutions cover most areas of the provinces, relatively concentrated in the capital cities and economically developed cities. The business type institutions account for the highest percentage in the two provinces. Animal species in the two provinces are approximately the same. Conclusions The industrial structure of the laboratory animals in Jiangsu province is more perfect than that in Zhejiang province. Industrialization and socialization get a higher degree in Jiangsu province. The range of regular species of the laboratory animals needs to be further expanded. The competent departments need further optimize and promote license management system of the laboratory animals.