Abstract:ObjectiveTo investigate thrombosis in mouse lung tissue after infection with influenza A virus, and compare the differences between mouse strains with different immune status. MethodsInfluenza H1N1(A/California/7/2009) and H3N2(A/Brisbane/10/07) were used in this infectivity study. Forty BALB/c mice, 50 NOD-Scid mice, 33 Scid mice, 33 BALB/C-nu mice, 33 NOD/LTJ mice and 10 icosl-KO mice were anesthetized by ether inhalation and inoculated intranasally with 0.05 mL suspension of infectious virus H1N1 or H3N2. The pathogenic changes were observed and the virus copy numbers in the lung tissue were detected by real-time PCR. ResultsVarying degrees of interstitial pneumonia were observed in the lung tissue of mice infected with H1N1 and H3N2 viruses. There was thrombosis within the lung tissue in 13 mice of H1N1 group and 6 mice of H3N2 group. The main components of the thrombus were cellulose and platelets. ConclusionsOur results support that thrombosis may occur in the lungs of all studied mouse strains after infection with influenza A (H1N1) and (H3N2) viruses. 

Abstract:Objective This study was aimed to learn spontaneous lesions in the heart of Wistar rats. MethodsA total of 160 male and female matched Wistar rats were studied. Forty rats were sacrificed at 9,2, 8,4 months of age, respectively. Histopathological changes of the heart were observed and analyzed. ResultsNo obvious pathological changes were detected in 9-month old Wistar rats. Mild pathological changes as small foci of necrosis and degeneration were found in the heart in 2.5% (1/40) of the 12-month old rats. The incidence of cardiomyopathy was 57.5%(23/40)and 100%(40/40)in 18- and 24-month old rats, respectively, and there was subendocardial fibrosis in 2.5% (1/40) of the 24-month old rats. The Masson staining showed that mild collagen fibrosis developed firstly at perivascular area and under valve annulus in a few of 9-month old rats, and increased with increasing age.ConclusionsWistar rat can develop heart disease spontaneously. The incidence rises up with increasing age,mainly cardiomyopathy and occasionally subendocardial fibrosis. Collagen fiber deposition may be closely related to the genesis of rat cardiomyopathy.

Abstract:ObjectiveTo establish an animal model of TNNT3((R69H)) mutation transgenic mice. MethodsTransgenic component pEGFP-TNNT3(R69H) was constructed, linearized and purified, then microinjected into fertilized mouse eggs. These eggs were transplanted into pseudopregant mice. The genotype of transgenic founders were identified by PCR and Southern blot. The expression of TNNT3(R69H) transcript in the tissues of the transgenic mice was detected by RT-PCR and Western blot. ResultsTransgenic BDF1 mice were obtained by microinjection. PCR and Southern blot results showed 5 of 40 mice were integrated. ConclusionsTNNT3((R69H)) mutation transgenic mouse models are successfully established by pronuclear microinjection.

Abstract:Objective To study the morphological and functional alterations of the thyroid gland in the second filial generation (F2) of Balb/c mice subjected to different levels of iodine intake. MethodsThe mice were randomly divided into five groups［less iodine group (LI), normal iodine group (NI), 5-fold excessive iodine group (5HI), 10-fold excessive iodine group (10HI), and 50-fold excessive iodine group (50HI)］ and given drinking water containing iodine in different concentrations ［daily intake<0.25 μg (LI), 1.5 μg (NI), 7.5 μg (5HI), 15 μg (10HI), 75 μg(50HI)］. After three months, the mice were mated and their 20-day and 40-day old second filial generations were observed. Stereological parameters including the thickness of the epithelial layer (L), the area of the follicle (SA), the area of follicular lumen (SB), and the ratio of follicular lumen and follicle volume VA) were measured using a photo analytic system to provide a basis for the quantitative evaluation of thyroid gland changes. ResultsThe serum thyroxin T4 level in the 20-day old and 40-day old F2 mice of the LI and 50 HI groups were significantly lower than the normal control group. Histopathological examination revealed that in the NI group, the thyroid gland follicles were of median size, and the most follicular epithelium consisted of a single-layer of cuboidal epithelial cells. In the LI group, there was apparent follicular hyperplasia, and the epithelium was columnar and stratified. In the 50HI group, the epithelium was more flat than that in the NI group, and plenty of colloid was accumulated in the follicular lumen. Assessment of the stereological parameter showed that in the LI group, L and SA were increased, VA was decreased. The data of the 5HI and 10HI groups were similar to those of the NI group. In the 50HI group, L was significantly decreased, and SA, SB and VA were significantly increased.ConclusionsBoth iodine deficiency and excess may cause hypothyroidism in the second filial generation mice. Iodine deficiency produces apparent follicular hyperplasia goiter, and long-term excessive iodine intake may result in colloid cumulative goiter, but the enlargement of thyroid by iodine excess is much less than that by iodine deficiency．The second filial generation mice have a high tolerance of excessive iodine. Obvious morphological changes often occur when the iodine intake excess reaches 50-fold higher than that in normal mice. 

Abstract:ObjectiveTo determine whether the SPF hamsters transfered from building 204 to isolator workshop were microbially polluted using different trasport boxes, and select qualified boxes for laboratory animal transportation.MethodsUsing triptic soy agar (TSA) culture dishes, simulating laboratory animal transportation process, and compare the results of settling plates in different boxes. ResultsThe microorganisms in individually ventilated cages cages (IVC) could be controlled bellow 14 cfu/4 h, and the morphology was the same with the colony in SPF building, therefore, were accord with standard. But in the conventional transport boxes, the colony were above 70 cfu/4 h, which failed to meet the standards.Conclusions SPF hamsters are not microbially polluted during the transport from building 204 to the isolator workshop using IVC cages. IVC cages are much better than conventional trasport boxes and should be used in laboratory animal transportation. 

Abstract:Objective Comparing the methods for total RNA isolation from ferret tissues with Trizol, RNeasy mini kit, modified RNeasy mini kit1 and modified RNeasy mini kit2 in removing SYBR Green real time PCR inhibitors for diagnosis of influenza H3N2 virus infection. MethodsComparing the total RNA quality isolated by methods Trizol, RNeasy mini kit, modified RNeasy mini kit1 and modified RNeasy mini kit2, respectively, and also the results of assay of SYBR Green real time PCR for the influenza virus H3N2 in tissue total RNA. ResultsThe quality of RNA isolated by the above four methods were measured using a Nano drop 2.0 UV spectrophotometer. A260/280 of the RNA isolated by RNeasy mini kit was below 1.8, and the other three were between 1.8 and 2.1. For A260/230, only the RNA isolated by Trizol was about 2.0, the other three were below 2.0. RNA isolated by Trizol ran in the 1.5% agrose gel electrophoresis showed three bands, 28 s, 18 s, and 5 s, while from the same tissues isolated by RNeasy mini kit showed three bands and genomic DNA, 28 s and 18 s. As for modified RNeasy mini kit1 and modified RNeasy mini kit2, there were only two bands 28 s and 18 s. Judging form the melting curve of SYBR Green real time PCR, the templates obtained from both turbinates and lung or intestine by the four methods were normal with the same wave crest position for samples and standard. Though the melting curve of tissues was specific, the products of SYBR Green real time PCR were different:the product by Trizol method was a single specific band on the 1.5% agrose gel electrophoresis. By RNeasy mini kit, modified RNeasy mini kit1 and modified RNeasy mini kit2, the products were multi-bands. The products of turbinate samples were all a single specific band by the four different methods. ConclusionsFor turbinate swab samples, the four methods are all right for virus quantification, but for the ferret tissues including lung and intestine, the classical Trizol method is as creditable for as SYBR Green real time PCR.

Abstract:Objective To get Bordetella bronchiseptic (Bb)expressing recombinant protein pertactin(PRN)and establish an indirect ELISA for detection of rabbit Bordetella bronchiseptic antibodies. MethodsAccording to a pair of designed specific primers, PRN gene of rabbit Bordetella bronchiseptica was amplified by PCR and inserted into prokaryotic expression vector pGEX-4T-1. The recombinant expression plasmids were transfected into BL21(DE3) strain. The optimal concentration of coated antigen recombinant protein PRN and serum dilution was determined to develop the ELISA technique. ResultsPRN gene of Bordetellabronchiseptica was successfully cloned and expressed. The SDS- PAGE and Western blot analysis unfolded the excellent immunogenicity of the recombinant proteins which were used as coating antigens to develop the ELISA method for Bb-specific antibody diagnosis. The peridium consistency of the recombinant protein PRN determined in this experiment was 500 ng /mL, and the optimal testing serum dilution was 1:40. ConclusionsThe PRN indirect ELISA developed in this study offers a simple and practical way for monitoring antibody of Bb, and provides much information for laying a basis for development of a Bb diagnosis kit. 

Abstract:ObjectiveTo establish an animal model of hepatic encephalopathy (HE). MethodsUsing carbon tetrachloride-alcohol in drinking water to establish a rat model of chronic liver failure-caused hepatic encephalopathy. The behavior activities were observed by maze test, blood biochemistry was measured, and liver and brain morphology was examined by electron microscopy.Results1) Under the naked eyes, the livers of model rats were enlarged in general, their surface was rough, with dark red coarse particles. In the normal group the livers were of normal appearance. There were lots of fibosis, hepatocyte lysis, diffuse necrosis and widespread hepatocyte ballooning degeneration in the model group. 2)Compared with the control group, the model group showed that the escape latency was significantly longer in the Morris water maze test at the ninth week. Took the incubation period extended beyond the normal x +2.5 s as standard, there were 14/20 (55%) rats having a longer latency in Morris water maze test in this experiment. 3) In the HE model group, the blood ammonia concentration was significantly higher than that in the control group,with a significant difference between the two groups (P <0.01). 4) In the normal group ,the EEG was α-wave dominant and without distinct abnormalities, but changes of hepatic encephalopathy appeared in the model group. ConclusionsA rather ideal rat model of liver cirrhosis acompanied with hepatic encephalopathy (HE) has been established. The HE changes can be more easily detected by Morris water maze test in the rats.

Abstract:ObjectiveTo express bovine interferon-γ (BovIFN-γ) in E.coli, and preliminarily identify its biological activity. MethodsAccording to the gene sequence in GenBank to construct the BovIFN-γ gene, and use it as the template, to amplify the BovIFN-γ gene by polymerase chain reaction (PCR). To insert the BovIFN-γ gene into vector PET-28a, transform it into E.coli BL21-competent cells, induce it with IPTG to express BovIFN-γ, and analyze it by Western blot. Using the NI-NTA affinity chromatography and electroelution to purify the recombination protein, and detect the protein antigenicity by commercial BovIFN-γ kit. ResultsThe recombinant plasmid PET-28a-BIFN-γ was successfully constructed, and BovIFN-γ was efficiently expressed in E.coli. The protein expression accounted for 32% of the total soluble bacterial proteins. The expression products mainly existed in soluble form in the cell lysate supernatant, and the proteion could be discriminated by BovIFN-γmonoclonal antibody. The recombinant protein purified by NI-NTA affinity chromatography had higher antigen activity than that by electroelution. ConclusionsThe soluble recombinant BovIFN-γ protein is successfully expressed in E.coli, and the protein can react with BovIFN-γ monoclonal antibody. The purified recombinant BovIFN-γ protein has good antigenicity.

Abstract:ObjectiveTo explore the effect of cigarette smoke exposure on the expression of 4-hydroxynonenal (4-HNE) and gamma-glutamylcysteine synthetase(γ-GCS)in bronchoalveolar lavage fluid (BALF) and peripheral blood of rats. Methods Thirty male Wistar rats were randomly divided into three groups:non-smoking group, 6-weeks smoking group and 12-weeks smoking group. 4-HNE and γ-GCS in BALF and peripheral blood were quantitatively analyzed by avidin-biotin complex enzyme-linked immunosorbent assay (ABC-ELISA). Results(1)The expressions of 4-HNE in BALF and peripheral blood of rats in the 6-weeks smoking group(21.376±2.80; 18.333±2.585)and the 12-weeks smoking group(28.634±2.999；22.754±3.882)were significantly higher than those in the non-smoking group(15.917±1.584；12.305±1.822)(all P<0.01), and those of the 12-weeks smoking group were significantly higher than those of the 6-weeks smoking group (all P<0.01).(2)The expression of γ-GCS in BALF and peripheral blood of rats in the 6-weeks smoking group(15.802±1.646; 16.221±1.577)were significantly higher than those in the non-smoking group(13.525±2.311；11.847±1.171)(P<0.05； P<0.01), and those of the 12-weeks smoking group(11.527±1.606；9.015±1.569)were significantly lower than those of the non-smoking group (P<0.05；P<0.01).(3)The level of 4-HNE in BALF and peripheral blood in the 6-weeks smoking group was positively related with that of γ-GCS (r=0.764,r=0.674), and the level of 4-HNE in BALF of the 12-weeks smoking group was negatively related with γ-GCS(r=-0.777)(all P<0.05), but there was no significant correlation between the 4-HNE and γ-GCS in peripheral blood of the 12-weeks smoking group. Conclusions Cigarette smoke exposure can upregulate the expression of 4-HNE and γ-GCS in BALF and peripheral blood in rats. As smoking is prolonged, the expression of γ-GCS is gradually reduced, which indicates that 4-HNE and γ-GCS may play an important role in the oxidation/antioxidant imbalance in chronic obstructive pulmonary disease.

Abstract:ObjectiveThe aim of this study was to observe the effect of postural changes on autonomic nervous control of heart in Beagle dogs. Methods Electrocardiogram (ECG) was recorded in static posture (lying, standing, sitting and hanging) and moving posture (walking) from conscious and unrestrained female Beagle dogs using a non-invasive telemetry system, and the autonomic nervous function was investigated by power spectral analysis of heart rate variability (HRV). ResultsIn static postures, the RR interval, SDNN, RMSSD, pNNabs (50), total power (TP), very low frequency (VLF) power and normalized high frequency (HF) power were significantly higher than those in moving posture (P<0.05, P<0.01), while the heart rate, normalized LF and the LF to HF ratio were significantly lowered (P<0.05, P<0.01). Conclusions The parasympathetic nervous activity is dominant in static state of different postures, while sympathetic nervous activity is dominant in moving state. Postural changes can influence the heart rate and inevitably affect the cardiac autonomic nervous control. It depends on the intensity of the vagus nerve activity, and leads to the disturbance of LF/HF balance. 

Abstract:ObjectiveTo explore the process and characteristics of fever induced by dry yeast, 2,4-dinitrophenol, lipopolysaccharide (LPS), and bacterial endotoxin ( standard substance) in SD rats, and to compare the influence of different concentrations of exogenous pyrogens on the fever process.Methods To establish rat fever models induced by dry yeast (2 g/kg, 1 g/kg), and 2,4-dinitrophenol(30 mg/kg, 15 mg/kg), LPS (100 μg/kg, 20 μg/kg), and bacterial endotoxin (120 EU/kg, 60 EU/kg), record the fever values at different time points, draw the average fever curves, and compare their fever characteristics.ResultsIn the febrile rats induced by subcutaneous injection of dry yeast suspension, temperature began to rise after 2-3 h, reached to the peak value after 6-7 h, and lasted for 20 h. In the fibrile rats induced by subcutaneous injection of 2,4-dinitrophenol solution, the temperature began to rise after 20 min, reached to the peak value after 1-1.5 h, and lasted for 3-5 h. In the febrile rats induced by intraperitoneal injection of LPS or bacterial endotoxin ( standard substance), temperature began to rise after 30 min, then the fever curves were biphasic or triphasic, and lasted for 5 h to 8 h.Conclusions Different exogenous pyrogens at different concentrations cause different fever process and characteristics in SD rats. In antipyretic experiments, suitable fever models should be appropriately selected based on the nasture of the tested drug and according to the types of fever process and characteristics of the used animal models. 

Abstract:ObjectiveTo compare the effects of two combined anesthesia on cardiac catheterization in minipigs.Methods Twelve minipigs were anesthetized with either pentobarbital sodium and ketamine (group Ⅰ) or propofol and ketamine (group Ⅱ). Vital signs were observed and anesthetic effects were evaluated during the perioperative period. ResultsAll animals were alive. The response time and recovery time were 7.6±2.4 vs. 2.4±1.4 min, 30.8±8.8 vs. 16.5±2.8 min, group Ⅰ vs. group Ⅱ, P＜0.05. There were no significant differences in the heart rate and respiratory rate. Propofol and ketamine anesthesia got a better anaesthetic effect. Conclusions Both the two ways of anesthesia can be applied in large animal experiments. Propofol combined with katamine anesthesia can achieve a better anaesthetic effect and earlier recovery, and appears to be a satisfactory method for pigs during surgical operation.

Abstract:ObjectiveTo study the dynamic changes of becteria and the dust particles in barrier animal facilities. MethodsTo measure the quantity of falling bacteria and dust particles of larger than or equal 0.3 μm in diameter at different times and in the different functional areas of the barrier animal facilities. ResultsThe quantity of falling bacteria and dust particles were significantly increased in barrier animal room after breeding operation, and was the lowest after spray disinfection. There was a high quantity of bacteria and dust at 00∶00 in the mice and rats breeding room, but was a valley value in the rabbits breeding room at the same time. The quantity of falling bacteria and dust particles was significantly increased in the clean and semi-clean corridor at working time, and was kept a relatively stable level at non-working times. ConclusionsThere are regular changes of the quantity of falling bacteria and dust particles in barrier animal facilities, and these changes are related with animal species, spray disinfection, mobility of persons and materials and operation of animal care.

Abstract:ObjectiveTo establish an in vitro model of caloric restriction (CR) with human neuroblastoma cell line SH-SY5Y cells and observe the effects of different energy supplies on the growth of SH-SY5Y cells. MethodsHuman neuroblastoma cell line SH-SY5Y cells were cultured in different media with glucose at different concentrations:low glucose group (2 g/L), normal glucose group (3.15 g/L) and high glucose group (4.5 g/L). MTT metabolic rate, cell growth curve and LDH leakage rate were measured to observe the cell growth status of different groups. ResultsCompared with the normal control group, the cells of the high glucose group had shorter cytoplasmic processes and shrank cell bodies, showed a little bit lower MTT metabolic rate, higher LDH leakage rate and poorer growth status, while the cultured cells of the low glucose group had more extensive processes, greatly lower MTT metabolic rate and lower LDH leakage rate, and the cells grew slower but showed good morphology. ConclusionsCulture at high glucose concentration is injurious to cells and induces a higher metabolic rate, so the cells are inclined to be senescent and to die. Culture at low glucose concentration in an allowable range of caloric restriction is protective to the cells, unharmful to the cells, and favorable for cell growth and prolongs the cell survival.

Abstract:ObjectiveTo observe the morphological characteristics of rat submandibular gland cells (RSMGs) co-cultured with silk fibroin-chitosan, (SFCs) by inverted microscopy, scanning electron microscopy (SEM), fluorescence microscopy and laser scanning confocal microscopy (LSCM), and provide technical support for observation and evaluation of cell growth on three-dimensional (3D) scaffolds. MethodsThe submandibular glands were excised from SD rats at age of 0 to 8-days and RSMGs were primarily cultured, purified and subcultured. The origin of cells were identified by immunocytochemistry (CK8 and amylase). Then RSMGs were chosen for the further studies when cells reached 75% to 80% confluence. Meanwhile SFCs blend membranes (5 mm x 5 mm x 2 mm) were prepared. RSMGs cultured on the scaffolds were monitored for 3,5 to 7 days. The morphological appearance of the seeded scaffolds was observed by inverted microscopy, SEM, fluorescence microscopy and LSCM. ResultsLiving cells that were seeded on SFCs medium of 3D scaffolds could be observed directly under the invert microscope. SEM showed the surface ultrastructure of the complex growth situation more clearly. After fluorescent staining, cells anchoring on scaffolds were observed by fluorescence microscopy and LSCM. The fluorescent signals of cell nuclei were evenly distributed in the pores of scaffolds. Through layer scanning and 3D LSCM reconstruction technique, images of thick specimens could be obtained. By rotating the images, 3D profile or whole structure could be observed from different angles and more accurate information of positioning could be obtained. ConclusionsAll four kinds of microscopic techniques can be used in examining the co-cultivation of RSMGs and SFCs. Images can be observed more distinctly through 3D LSCM reconstruction technique combined with fluorescent staining technique. It has a wide application value in this research field.

Abstract:According to the elaborating of laboratory animal import and export relevant laws and regulations, this article analyzed the import and export of animal problems in the management process, proposed the solving measures, and provided the theory and practice to the workers engaged in laboratory animals issues.