Abstract:Objective To compare the effects of two approaches of SIVmac239 infection in endothelial cells, directly by cell-free virus or by T cell-mediated infection, to explore the predominant approach of SIVmac239 infection in endothelial cells, and finally lay a theoretical foundation of the molecular mechanism of SIV viral disruption of the blood-brain barrier. Methods We adopted two distinct ways to infect endothelial cells. One was to directly infect the endothelial cells by cell-free virus, another was by co-culture of infected CEMx174 cells with the endothelial cells. The degree of infection in the endothelial cells was evaluated by nested-PCR, indirect immunofluorescence assay, Western blotting and ELISA assays. Results The provirus DNA was found in the endothelial cells infected in either way. However, the endothelial cells infected by co-culture with CEMx174 cells had a significantly higher amount of provirus DNA and higher expression of SIV P27 in the cells and in the supernatant than those in the ECs directly infected by cell-free virus. Conclusions The way of T cell-mediated infection substantially enhances the capacity of SIV infection of endothelial cells than the direct infection by cell-free virus. It indicates that the T cell-mediated infection may serve as the principal route of SIV disruption of the blood-brain barrier.

Abstract:Objective To study the pathological features of two huge spontaneous tumors in Wistar and GK rats.Methods Forty Wistar rats and 40 GK rats were included in this study. Among those rats, two huge spontaneous tumors were observed in a Wistar rat at 14 months of age and in a GK rat at 22 months of age. The growth and survival status of the tumor-bearing rats were recorded. The tumors were surgically removed, and their pathological features were examined using HE and immunohistochemical staining (vimentin, CK19, α-SMA, CD31, CD34, S-100, NF及Ki-67). Results Both the two tumors were completely resected by surgery without much difficulties, and both host rats survived well after the operation. The weight of the two huge tumors was 502 g and 119 g, which corresponding to 64% and 24% of the body weight of their host rats, respectively. The tumors surface had a complete capsule, with a clear boundary separating from the normal surrounding tissues, and no vascular pedicle structure was found. According to the results of immunohistochemical staining, both the two tumors were diagnosed as benign fibroma. Conclusion This type of huge spontaneous tumors is benign fibroma. Besides the impact on the activities of the rats, the tumors have no significant impact on the living conditions in the hosts.

Abstract:Objective To select a simple, stable and reliable mouse model of alcoholic liver disease. Methods The mouse models of alcoholic liver disease were induced by oral gavage ethanol or Lierber-DeCarli ethanol liquid diet for 8 weeks. The food intake and body weight were recorded. Pathological changes were examined using HE staining. Liver injury was assessed by the activities of serum ALT, AST, AKP and γ-GT, and serum and hepatic TC and TG. Results After modeling, both models showed significantly increased activities of serum ALT, AST, AKP, and contents of serum and hepatic TG (P<0.05), indicating the successful development of alcoholic steatohepatitis. However, oral ethanol gavage led to body weight loss and weak mental state. Ethanol liquid diet less affected the body weight and mental state. Ethanol liquid diet enhanced liver to-body weight ratio and serum TC, but oral gavage of ethanol did not. The changes of serum ALT, AST, serum and hepatic TG, and hepatic steatosis in the ethanol liquid diet models were more severe than those in the oral gavage ethanol models, suggesting that Lierber-DeCarli ethanol liquid diet led to more serious liver injury than oral gavage ethanol. Conclusions Lierber-DeCarli ethanol liquid diet model is better than oral gavage ethanol model, and is more suitable for studies on mechanisms and evaluation of hepato-protective drugs for alcoholic liver disease.

Abstract:Objective To observe the effects of cyclosporin A on the expression of phosphorylated AKT in hepatocytes, and to investigate the mechanism of insulin resistance caused by cyclosporin A. Methods This study included two parts. 1. In vitro experiment: Human hepatocyte HL77022 cell line was cultured at different concentrations of cyclosporin A (0.1 μmol/L, 1 μmol/L, 5 μmol/L) for 48 hours. The expressions of phosphorylated AKT (P-AKT) in HL77022 cells were measured by Western blot assay. 2. Rat in vivo experiment: SD rats were randomly divided into 2 groups. The rats in the control group were administrated with distilled water 1 mL/Kg/d. The rats in the cyclosporine group were administrated with cyclosporine 25 mg/Kg/d. The total experiment time was 5 months. The levels of fasting blood glucose and insulin were tested at the end of the experiment. The insulin resistance index and insulin sensitivity index were calculated. The expression of P-AKT in the rat hepatocytes was measured by immunohistochemistry. Results 1. Each group of the HL7702 cells treated by CsA (0.1 μmol/L, 1 μmol/L, 5 μmol/L) showed a significantly decreased expression of P-AKT (P<0.05, P<0.01, and P<0.01). 2. After 5 months of therapy, the fasting blood glucose level of rats in the cyclosporine group was 9.28 mmol/L, indicating that cyclosporine-induced diabetic rat models were established. The insulin sensitivity index in the cyclosporine group was lower than that in the control group (P<0.05). The expression of P-AKT in liver in the cyclosporine group was significantly lower than that in the control group (P<0.05). Conclusions Therapeutic dose of cyclosporine has hyperglycemic effects on rats. Cyclosporine can reduce the expression of phosphorylated AKT in hepatic tissue in rats and also decrease the expression of P-AKT in human hepatocyte HL77022 cells, which indicate that cyclosporine may cause insulin resistance by interfering PI3K/AKT signal transduction pathway.

Abstract:Objective To investigate the repeated dose toxicity of doxorubicin liposome injection and doxorubicin injection in rats. Methods Ninety SD rats (body weight 180-220 g, male:female=1:1) were divided into 3 groups (30 rats in each group), and were administered intravenously with physiological saline, doxorubicin liposome injection (1 mg·kg^-1) and doxorubicin injection (1 mg·kg^-1), respectively, once every three days for thirteen times. The body weight,blood biochemistry, hematology, organ coefficient and histopathology were analyzed for the overall toxicity assessment. Results The rats administered with doxorubicin liposome injection (1 mg/kg) showed hair loss and skin ulcer, significantly reduced growth of body weight, increased levels of urea nitrogen (BUN), alanine aminotransferase (ALT), blood platelet (PLT), and kidney and heart coefficients, decreased thymus and testicular coefficients, myofibrillar rupture and lysis, and partial loss of cell nuclei, hyaline casts in the renal convoluted tubules, interstitial edema and loss of spermatogenic cells in the testicular tubules. Compared with the doxorubicin liposome injection group, similar abnormal changes were also observed in the doxorubicin injection group, but the hair loss and skin ulcer were milder and the heart and kidney toxicities were severer. Conclusions Compared with doxorubicin injection, the doxorubicin liposome injection causes milder heart and kidney toxicity but more serious skin toxicity.

Abstract:Objective To investigate the effect of different doses of microbe-derived antioxidant on sleep and antioxidant ability in mice. Methods Sixty male Kunming mice with similar body weight were randomly divided into 4 groups.The control group received normal saline, and the experimental groups received microbe-derived antioxidant in a dose of 0.5 g/kg bw, 1.0 g/kg bw or 1.5 g/kg bw once per day, respectively. The experiment period was 30 days. At the end of experiment, the mice of each group were intraperitoneally injected sodium pentobarbital to induce sleep. The mice fall sleep was judged by righting reflex. After the test of sleep, blood was taken for detection of serum antioxidant ability. Results Compared with the low dose and high dose groups, the middle dose group showed a significantly prolonged sodium pentobarbital-induced sleeping time (P<0.05). Compared with the control group, low and high dose groups, the middle dose group had highly significantly increased GSH-Px activity (P<0.01) and significantly increased content of SOD. Under these conditions, the middle dose group reduced both the contents of MDA and 8-ISO-PGF2α (P<0.05) compared with the control group. Conclusions Our results suggest that microbe-derived antioxidant exerts effect on sleep and antioxidant ability in rats. Supplement of 1.0 g/kg bw/d shows the most significant effects.

Abstract:Objective To establish the baseline data of body weight, main organ weights, hematological and biochemical indexes in SPF congenital cataract mice. Methods Body weight, main organs weights, hematological and biochemical indexes of the congenital cataract mice were determined at 28 days and 56 days of age, respectively. Normal KM mice in the same age were taken as control. Results There were no statistically significant differences in all indexes of the mice at 28 days of age. Compared with the 56-day old normal KM mice: (1) Statistically significant differences were found in the body weight, and weights of the heart, liver, spleen, lung, kidney and testis (P<0.05 or P<0.01; (2) Statistically significant differences were found in hematological indexes WBC, PLT, MPV, LYMP, PDW for female mice and MPV, PDW for male mice (P<0.01); (3) Among the biochemical indexes, there were also statistically significant differences in UREA, ALP, TP, UA, TG, GLU for female and ALT, ALP, TP, ALB, UA, GLU for male mice. Conclusions There are statistical differences in the body weight, main organ weights, hematological indexes and biochemical indexes between the congenital cataract mice and normal KM mice at 56 days of age. These results may provide a useful reference for future research.

Abstract:Objective To construct Fndc5 knockout mouse models and provide animal models for related studies in the future. Methods Indels were introduced into FNIII domain of Fndc5 gene by TALEN technology in mice, and genotypes were identified by sequencing. To set stable genetic system by pairing. Then at mRNA and DNA levels identified the genetype of born mice. At the same time the body weight and blood glucose of the mice at different ages were analyzed. Finally the Fndc5 expression in the kidney, liver, brain, muscles, heart and other organs was determined by qPCR. Results Four different Fndc5-KO lines were generated. The body weight and blood glucose of the mice at different ages showed no significant differences. Finally high Fndc5 expressions in the muscles, heart and other organs were determined. Conclusions We Have for the first time successfully generated Fndc5 knockout (KO) mouse model using TALEN mediated DNA targeting technique, and performed preliminary analysis. This Fndc5 knockout (KO) mouse model provides a novel tool for further studies on the in vivo function of FNDC5.

Abstract:Objective To investigate the effects of simvastatin on the bone loss and deterioration of bone quality with different intervention programs. Methods Thirty two 3-month-old female Sprague-Dawley (SD) rats were randomized into 4 groups of 8 animals in each: All rats but those in the sham group (A) received bilateral ovariectomy, and treated by vehicle (B), simvastatin (5 mg/kg/day) at first half period (C) or at latter half period (D). The rats in group C received simvastatin by a gavage after the OVX operation immediately, and stopped at 10 weeks after OVX. The rats in group D began to receive simvastatin treatment from 10 weeks after OVX and ended at 20 weeks after OVX. At week 20, all rats were sacrificed and the concentrations of serum PINP and ICTP were detected by ELISA, L3 vertabra was used to detect the bone mineral density, L4 vertebra was used to analyze the maximum loading and elastic modulus by compression test, and the microstructure of the L5 vertebra was detected by micro-computed tomography. Results 1. ELISA analysis: the concentrations of serum P1NP and ICTP in the groups A, B and C were significantly higher than that of group A (P<0.05). 2. BMD test showed that the rats in group B had significantly lower BMD than the other 3 groups (P<0.05), while the BMD of groups C and D were markedly lower than that of group A (P<0.05). 3. Biomechanical test: the maximum load and elastic modulus of L4 vertebrae in the group B were significantly lower than the other 3 groups (P<0.05), and those of the groups C and D were markedly lower than that in the group A (P<0.05). 4. micro-CT: BV/TV and Tb.N in the sham operated rats were significantly higher than those of the other 3 groups, while the opposite trends was found in Tb.Sp (P<0.05), and the Tb.Sp in the groups C and D were significantly lower than that of group B (P<0.05). Conclusions Our data demonstrate that bone loss and deterioration of bone micro-structure and biomechanical properties occurred at 20 weeks after ovariectomy, both the first-half-period and latter-half-period treatment by simvastatin may partially prevent these changes, but can not restore the BMD to normal level.

Abstract:Objective The purpose of this study was to compare two types of myocardial infarction (MI) models in Beagle dogs. Methods 30 dogs were divided randomly into three groups (n=10). ① The sham-operated group underwent pericardiotomy but without coronary artery ligation. The other two model groups were made under video-assisted thoracoscopy (VATS), where the left anterior descending coronary artery was closed by titanium clips: ② The direct vision group: the minimally invasive closure of the artery was performed under direct vision. In this group, the thoracoscopic operation was performed through a 3.0 cm small incision opened at the margin of the left third rib. ③ The thoracoscopic group: the video-assisted thoracoscope was inserted into the chest through a 1.0 cm exploratory hole in the midline of the third rib, and the surgical instruments were inserted through two 0.5 cm operating holes at the para-sternum line of the third rib and midclavicular line of the fourth rib. Electrocardiogram (ECG) was recorded and the levels of serum creatine kinase-MB (CK-MB) and troponin I (cTnI) were measured after modeling. The heart tissue samples were examined by histology using HE staining. The success rate of model establishment, durations from skin incision to chest closing and the wound healing were recorded. Results Compared with the sham-operated group, changes of myocardial infarction were observed in the two model groups (ECG S-T segment elevation, increased serum CK-MB and cTnI levels, myocardial ischemia and fibrosis, and reduced amount of cardiomyocytes). The survival rate was 90% in both of the two model groups. The operating time was shorter in the minimally invasive surgery under direct vision group, and the wound healing time was shorter in the thoracoscopic group. Conclusions The myocardial infarction models generated by minimally invasive surgery have less trauma and low mortality in the dogs. This model is suitable for investigation of pathophysiological mechanism associated with myocardial infarction.

Abstract:Objective To detect and analyze the HBV-related indexes in the urine of HBV transgenic mice and further understand the biological characteristics of transgenic mice, and to clarify the tissue sources of HBV-related indexes. Methods HBV-related indexes in the urine of transgenic mice were tested using enzyme-linked immune sorbent assay (ELISA) and fluorescence quantitative PCR (real-time RCR). The tissue sources were confirmed by several experiments, i.e. hydrodynamic transfection of mice, RNA interference to inhibit HBV-expression in the transgenic mice, and to infect normal mice with HBV-positive serum from patients. Results Expression of HBsAg, HBeAg and HBV-DNA was present in the urine of transgenic mice, of which the HBsAg expression level was high (6674±619.8 IU/mL), but lower than that in the serum (16470±2704 IU/mL). The level of HBsAg expression in the urine of male mice was higher than that in female mice. The level of HBeAg expression in the urine was lower and the HBeAg positive rate of urine was higher than that of blood, and the levels of HBeAg expression showed significant inter-individual and inter-sexual differences. HBV-DNA level reached 10³-10⁵ copy/mL in the urine, but no related antibody expression was detected. The experiments such as hydrodynamic infection test indicated that the HBV-related indexes in the urine are derived from replication in the kidneys rather than secreted from the liver, entered into the blood circulation, and discharged from the urine. The kidneys are an independent expression site of HBV. Conclusions The expression of HBV-related indexes is present in the urine of transgenic mice and it is a long-term expression along with the age in months, of which the expression levels of HBsAg and HBV-DNA are rather high and stable. HBsAg titer in the urine of the male mice is higher than that of female mice. HBeAg expression level in the male mice is more stable compared with that in female mice. No expressions of various kinds of antibodies have been found in the urine. The kidneys are an independent expression site of HBV.

Abstract:Objective To simulate the process of hypoxic-ischemic brain injury at high altitude in a simulated cabin with plateau low pressure environment, and to prepare a rat model of cerebral injuries at different high altitudes. Method Thirty-two 0-day-old neonatal SD rats were divided into four groups, namely group A (control group) and three test groups:group B (2000 m group), group C (4000 m group), and group D (6000 m group). The rats of control group were reared in a barrier environment. The rats of test groups were placed in a simulated cabin with plateau low pressure environment, and to prepare neonatal cerebral ischemia-hypoxia model by sport activities. The sport movements were carried out in the cabin in a swimming groove 60 min/d, and not less than 20 hours a day at high altitude low pressure environment. Zea Longa 5 point scale standard was used to determine the behavioral scores at the 3th 7th 11th 15th days, and samples were collected on the 15^th day to observe red blood cell morphology using HE and 2,3,5-triphenyltetrazolium chloride (TTC) staining and ultrastructure by scanning electron microscopy.Result (1) The neurological scores of the test groups A, B, C were significantly different from that of the control group (P<0.05), and the scores of test group D and control group were very significantly different (P<0.01). (2) The histopathological examination using HE staining showed inflammatory cell infiltration in all rats of the test groups, and the extent of inflammatory cell infiltration was positively correlated with the increase of altitude. (3) The histopathology with TTC staining revealed prominent ischemia in the cerebral cortex of rats in the plateau hypoxic environment. (4) Scanning electron microscopy showed that the rat erythrocytes were cap-like in the group B, irregular in the group C, and zigzag shape in the group D. Conclusions In this study, a rat model of neonatal hypoxic-ischemic encephalopathy (HIE) is successfully established by hypoxic cabin combined with sport activity. This model is stable, reliable, more closely mimicking the pathogenesis and clinical manifestation of neonatal HIE than models prepared with other methods, therefore, may be used in related research.

Abstract:Objective To acquire the prevalence and molecular identification data on Syphacia muris and provide reference for the revision of national standard. Methods 923 batches of 5199 SPF animals (including one batch of 5 monkeys, 3 batches of 25 mini-pigs, 28 batches of 55 rabbits, 13 batches of 248 hamsters, 37 batches of 198 guinea pigs, 93 batches of 459 rats, 742 batches of 4179 mice, 5 batches of 25 chickens and one batch of 5 ducks) and 145 batches of 1389 clean animals (including one batch of 3 rabbits, 4 batches of 31 hamsters, 16 batches of 157 guinea pigs, 32 batches of 268 rats and 92 batches of 930 mice) came from 50 different manufactures in China. Direct microscopy real-time dynamic video recording techniques in combination with morphological identification method were applied to screen the Syphacia muris infestation. A multiple polymerase chain reaction (multiple-PCR) testing of the isolate based on amplification of the conserved portions of the Syphacia muris internal transcribed spacer (ITS), 28S ribosomal RNA (28S rRNA), NADH dehydrogenase subunits 1 (nad1) and cytochrome c oxidase subunit 1 (cox1) genes, and the molecular sequencing of the multiple-PCR amplicons was used to confirm the Syphacia muris infection. Results Syphacia muris eggs, larvae and adults were detected by using direct microscopy real-time dynamic video recording technique. Syphacia muris were detected based on the morphology and size of ovum, larvae, and female and male adult worms. Multiple-PCR and sequencing were performed to identify ITS, 28S rRNA, nad1 and cox1 genes of DNA extracted from the single egg, larva and adult parasite Syphacia muris. This approach allowed the specific identification with no amplicon being amplified from heterogeneous DNA samples, and sequencing confirmed the identity of the amplified sequences. Molecular characterization by multiple-PCR amplification and sequencing of the ITS, 28S rRNA, nad1 and cox1 genes demonstrated the presence of Syphacia muris. Multiple-PCR followed by sequencing confirmed 285 of 5199 SPF and 135 of 1389 clean animal samples classified as positive by using direct microscopy real-time dynamic video recording technique in the study as containing Syphacia muris-specific DNA. Comparison of the partial sequences of the ITS, 28S rRNA, nad1 and cox1 genes revealed 100% similarity amongst Syphacia muris from different animals. The prevalence of Syphacia muris infection in SPF and clean animals were 5.5% (285/5199) and 9.7% (135/1389), respectively. Conclusions Direct microscopy real-time dynamic video recording technique, multiple-PCR and sequencing can be used to rapidly detect and accurately identify Syphacia muris. The zoonotic nature of Syphacia muris can be regard as a public health alter, hence the good quality control of animal has an important role in protecting human health and safeguarding people safety. This is the first molecular identification and infection investigation of Syphacia muris in SPF and clean animals in China.

Abstract:Objective To investigate the key factors for PV Loop evaluation in rats and to improve this method. To provide examples of cardiac function measurements obtained from normal rats and from rats with cardiac hypertrophy induced by transverse aortic constriction (TAC). To establish a more reliable method for rats heart measurements. Methods Rats underwent left ventricular catheterization through the right carotid artery. Through adjustments of the position of the pressure-volume conductance catheter, the optimal PV Loops and a number of cardiac functional parameters were acquired. The key influencing factors, calibration of volume, position of the catheter in the left ventricle (LV) and suspension of ventilator were assessed. Results 1. The real volume of left ventricle were acquired by injecting appropriate volume of hypertonic saline through jugular vein, which deducted the parallel conductivities of ventricular wall. 2. To get better PV loops, it's important to adjust the position of the catheter in the left ventricle until all of the pressure and volume sensors were located in the ventricle as well as out of touch with the ventricular wall. 3. Suspension the ventilator during the test is conducive to stable and reasonable data acquisition. We further assessed the cardiac functions of healthy rats and rats with cardiac hypertrophy with this improved method, which showed better performances. Conclusions This study we have evaluated the influences of calibration of volume, position of the catheter in the left ventricle (LV) and ventilator on measurements of rats PV loops, and further improved this method. Moreover, we have validated this method with measurements of cardiac functions of normal rats and cardiac hypertrophic rats.

Abstract:To summarize and introduce the available methods of establishing rabbit models of VX2 nasopharyngeal carcinoma (NPC), and to explore the improvements at each stage in the preparation of the rabbit models, in order to provide a favorable animal model for future experimental research.

Abstract:Pulmonary fibrosis can severely disrupt lung functions, but its etiology and pathogenesis remain unclear. Animal models of lung fibrosis play an important role in investigation of the mechanism by which pulmonary fibrosis develops. This review summarizes the characteristics, advantages, and disadvantages of widely used and newly established animal models of lung fibrosis.