Abstract:Objective To establish an immunofluorescence assay ( IFA) method of detecting the mammalian orthoreovirus 3 ( Reo3) in animal-derived biological materials and biological products for human use． Methods Reo3-sensitive cells were screened． The virus TCID50 was titrated and antigen slides were prepared according to GB IFA． The best working concentration of IgG-FITC was titrated and the specificity，sensitivity and stability were tested． Results BSC-1 cells were selected as Japanese encephalitis virus ( JEV) -sensitive cells． The TCID50 of the virus was 10 － 5. 8·mL － 1． The best working concentration of the IgG-FITC was 1: 100. There was no cross-reactivity with ectromelia virus and mouse hepatitis virus． The tests of sensitivity and stability showed that the detection sensitivity was 1: 1280 at different time points． The lowest detected virus titer was 10 － 4. 1·mL － 1． Conclusions The established IFA method is good in sensitivity，specificity and stability，and can be used in detecting the Reo3 in animal-derived biological materials and biological products for human use．

Abstract:Objective To establish a ferret model of H3N2 influenza virus infection． Methods Six ferrets were divided into two groups for three each． after anesthetized by ketamine，the ferrets were inoculated intranasally with 500 μL of 106 and 107 TCID50 H3N2 A /Brisbane /10 /07 influenza virus，respectively． Clinical signs were monitored daily，and body temperature was recorded once every day postinfection． Nasal turbinate biopsies were taken every day from day 0 to postinfection day 5 ( d 0 to d． p． i． 5) for real time PCR analysis，the ferrets were euthanized and the lung，liver，slpeen，small intestine and brain tissues were harvested at d． p． i． 5. The lungs were further examined by histopathology．Results For each virus titer tested，transient elevation of body temperature，weight loss，nasal rattling and sneezing were observed in the infected ferrets，but no ferret died． Virus was detected positively in the nasal turbinates and small intestine from all the infected ferrets． The ferrets inoculated with H3N2 virus showed mild interstitial pneumonia in the lung．Conclusions The ferrets were successfully infected with H3N2 influenza virus proved by clinical signs，virus detection，and histological examination． Furthermore，106 TCID50 titer is a more appropriate dose for establishment of a ferret model of flu H3N2 virus infection．

Abstract:Objective To investigate the expression of AT1R in heart and kidney of rats with salt-sensitive hypertension induced by sensory denervation，and to explore their role in the development of salt-sensitive hypertension．Methods Newborn male Wistar rats were given capsaicin subcutaneously to establish the model． After the weaning period，the rats were divided into 4 groups and fed with different salt diets for 4 weeks: control plus normal salt diet ( CONNS) ，control plus high salt diet ( CON-HS) ，capsaicin pretreatment plus normal salt diet ( CAP-NS) ，and capsaicin plus high salt diet ( CAP-HS) ． The rats were killed at 4 weeks after treatment． Immunohistochemistry was used to detect the expression of AT1R protein in the heart and kidney tissues． The expression of AT1R mRNA in heart and kidney was determined by reverse transcription polymerase chain reaction． Results ① The tail-cuff systolic blood pressure was significantly higher in the CAP-HS rats than that in the other three groups at the age of 7 weeks ( P ＜ 0. 01 ) ． ② Immunohistochemistry results showed a significantly higher AT1R protein expression in the CAP-HS group than in the CONNS group ( P ＜ 0. 01) ，and the AT1R protein expression in the kidney and heart tissue was significantly higher in the CONHS group than in the CON-NS group ( P ＜ 0. 05 ) ． ③ RT-PCR results showed that AT1R mRNA expression of the experimental groups CAP-HS and CON-HS was significantly higher than that in the CON-NS group ( P ＜ 0. 01，and P ＜0. 05，respectively) ． Conclusions The over-expression of AT1R protein and AT1R mRNA in the heart and kidney may be associated with the increase of blood pressure in the rats with salt-sensitive hypertension．

Abstract:Objective Mir-153 can negatively regulate the expression of APP and APLP2 protein，the crucial Alzheimer’s disease related genes， and consequently lower the level of their intracellular degradation fragment ( intracellular domains，ICDs) ． Considering the transcriptional activity and pro-apoptotic role of ICDs，the aim of this study was to explore the effect of mir-153 on the expression of GSK-3β，the downstream signaling molecule of the two target genes，and on the ability of cells against damage stress to further identify the role of mir-153 in Alzheimer’s disease．Methods A stably transfected cell line over-expressing mir-153 was developed and mir-153 transgenic mice were generated． Western blot was used to detect the expression of phosphorylated GSK-3β，Tau and their total protein in the cells and mice． The mir-153 stably transfected cells were treated with Aβ42 peptide and H2O2，respectively，to determine the changes of cell viability by MTS and analyze the cell apoptosis by flow cytometry． Results The expression of phosphorylated GSK-3β and it’s total protein were decreased and the phosphorylation of Tau was reduced in the mir-153 stably transfected cells． The expression of phosphorylated GSK-3β and it’s total protein were down-regulated and the level of phosphorylated Tau and its total protein were not significantly changed in the brain of mir-153 transgenic mice． Under the treatment of Aβ42 peptide and H2O2，the viability of mir-153 stably transfected cells were clearly decreased and the apoptosis level of the cells was increased． Conclusion Mir-153 can negatively regulate the expression of GSK-3β，the downstream signaling molecule of its target genes． Over-expressed mir-153 lowers the cellular anti-injury ability．

Abstract:Objective To investigate different behavioral activities among four species of mice in three trials ( open field test，tail suspension test and forced swimming test ) ，and provide a reference in the research on new antidepressant． Methods Different locomotion activity and exploration in a novel environment of C57BL /6，BALB / c，ICR，and KM mice were investigated by open field with an automated video detecting system，and tail suspension test and forced swimming test were performed to determine their state of“behavioral despair”． Results In the open field test，the total duration of movement，speed and movement distance of BALB / c，ICR and KM mice were significantly higher than that of C57BL /6 mice ( P ＜ 0. 05) ，and their number of rearing also was significantly more than that of C57BL /6 mice ( P ＜0. 05) ; the immobility time of C57BL /6 mice was significantly longer than that of the other three species of mice measured by tail suspension test ( P ＜ 0. 05) ，but the fixed time of the four species of mice in the forced swimming test were not significantly different． Conclusions The results of this study demonstrate that among the four mice strains tested，C57BL /6 strain displays less spontaneous activity and less exploration in a novel environment． They are likely to cause behavioral despair under acute stress stimulation． Therefore，the C57BL /6 mice may be probably the strain of choice as an acute stress animal model for studies of depression．

Abstract:Objective To compare the differences of blood physiological and biochemical parameters among the Tibet minipig，Wuzhishan minipig and Bama minipig，bred in Guangzhou area． Methods Using automatic analyzer of blood cells and biochemistry to test 24 blood physiological and biochemical parameters． Results Among the 24 parameters，significant difference exist in Hgb，WBC，MONO% ，HCT% ，GLU and very significant difference exist in LYM% ，NEU% ，MCV，MCH，TP，ALB，BUN，and CREA． The Tibet minipig had the highest Hgb，HCT% ，MCV，MCH，TP，ALB，BUN，CREA． The Wuzhishan minipig had the highest WBC，LYM% ，and the Bama minipig had the highest NEU% ，MONO% ，and GLU． Conclusions The comparison of blood physiological and biochemical parameters among the three types of minipigs provide a theoretical basis for establishing the biological database of minipigs bred in Guangzhou area．

Abstract:Objective To investigate the genetic background features of Macaca fascicularis and to establish the genetic quality monitoring method in Macaca fascicularis． Methods Using microsatellite DNA genetic markers，genetic quality monitoring and DNA polymorphism analysis were conducted in 50 Macaca fascicularis． Results 20 loci with high polymorphism was selected from 100 microsatellite DNA loci，among which allele numbers in Macaca fascicularis were 5-10，and individuals showed a high degree of polymorphism． Observed number of alleles( Na) was 5. 0 ～ 10. 0，effective number of alleles( Ne) was 4. 6118 ～ 8. 3404，gene diversity ( H) was 0. 7832 ～ 0. 8801 and Shannon＇s information index ( I) was 1. 5651 ～ 2. 1592. Conclusions The experiments effectively analyzed the genetic diversity of Macaca fascicularis， and it can provide a theoretical foundation for establishing genetic quality monitoring method with specific microsatellite loci selected in Macaca fascicularis．

Abstract:Objective To investigate the anesthesia management and effect of cardiopulmonary bypass ( CPB) on minipigs． Methods Thirty-four laboratory minipigs were divided into two groups: the cardiac arrest group ( 18 pigs) underwent tricuspid valve replacement with autologous pericardium under cardiopulmonary bypass，and the parallel group ( 16 pigs) receiving the tricuspid valve replacement with autologous pericardium and CPB in parallel． The dosages of anesthetics and vasoactive drugs，maintenance times of basic anesthesia and general anesthesia，recovery time after anesthesia，survival rates at 3 days and 1 week and hemodynamics of each period were recorded，and the effects of basic anesthesia and general anesthesia were evaluated． Results All the 34 minipigs underwent surgery under general anesthesia smoothly with stable hemodynamics in each period，with only one pig died 3 days after the surgery． The survival rate was 97． 1% ． The anesthesia effect was satisfactory． Conclusions Rational and combined adoption of anesthetics and vasoactive drugs，careful observation and swift and right treatments are key points of successful cardiac surgery anesthesia under cardiopulmonary bypass on minipigs．

Abstract:Objective Using an improved method to extract high quality total RNA from mouse epididymal adipose tissue，to clone and analyze the full-length cDNA encoding perilipin． Methods According to the specification of reagent，the extraction procedure of total RNA from adipose tissue was modified to extract high quality total RNA from C57BL /6J mouse epididymal adipose tissue． The cDNA encoding perilipin was amplified by RT-PCR using the total RNA ． The PCR product was cloned into pMD18-T easy vector and then transformed into E． coli JM109． The recombinant plasmid was identified with restriction enzyme digestion analysis and nucleotide sequencing． Results Using the improved method we successfully extracted high quality total RNA． The recombinant pMD18-T easy vector had a complete open reading frame of perilipin and shared 100% homology with the sequence of mRNA for perilipin reported in GenBank． Conclusions The improved extraction method of total RNA from fat tissue is feasible． The cDNA of perilipin is successfully cloned，which will be helpful for the further research on its biological function．

Abstract:Objective To investigate the post-UCO ( unilateral carotid occlusion) ischemic symptoms and possible correlations of anterior and posterior communicating arteries ( ACoA and PCoA) patterns of the circle of Willis between parents and their progeny in gerbils and to selectively breed an ischemia-prone gerbil group． Methods We used 398 gerbils from five generations to develop focal cerebral ischemia after UCO，and compared the UCO ischemic symptoms and possible correlations of ACoA and PCoA patterns between parents and their progeny． The progeny which are from the parents with incomplete Willis circle were mated to selectively breed an ischemia-prone group． Results It was shown that if the parents had complete ACoAs，their progeny also had a complete ACoAs，and there were significant differences when the parents’ACoAs were incomplete: in 60. 4% of offsprings the type of ACoA was consistent with that of their mother and in 48. 2% consistent with that of their father． After selectively breeding for five generations，the proportion of UCO ischemia increased from 40% in the F1 animals to 75% in the F5 animals． Conclusions Variations in the circle of Willis are of genetic nature and we have successfully established an ischemia-prone gerbil group．

Abstract:Objective To Investigate the pathogenetic mechanism of nonalcoholic fatty liver disease ( NAFLD) in gerbils，the changes in the lipid metabolism，liver function and antioxidation in its development caused by high fat diet．Methods One hundred and twenty gerbils were divided randomly into 2 groups: the model group fed with high fat diet and control group fed with normal diet． Ten gerbils were killed at 1，2，4，6，8 and 16 weeks after the start of diet，respectively． The pathological changes of the liver were examined，and the liver index，serum CHO，TG，LDL-c，HDL-c，GOP，GPT，the liver SOD，GSH-PX，CAT，FFA were determined． Results In the model group，simple fatty liver was observed at 2 weeks，mild inflammation in the hepatic portal areas at 6 weeks，focal perisinosodal fibrosis/ pericellular fibrosis of acini band at 8 weeks，and moderate liver fibrosis at 16 weeks after the start of diet． CHO，HDL-C，LDL-C and FAA were obviously elevated ( P ﹤ 0. 05，P ﹤ 0. 01) at 1，2，4，6，8，16 weeks，TG was raised at 1，2，4 weeks( P ﹤ 0. 05，P ﹤ 0. 01 ) ，GOT and GPT were Significantly increased after 16 weeks ( P ﹤ 0. 01 ) ，GSH-PX、CAT and SOD were Significantly decreased after 8 weeks( P ﹤ 0. 05，P ﹤ 0. 01 ) ． Conclusions Gerbil models of simple fatty liver can be developed at 2 weeks after high fat diet feeding，and moderate liver fibrosis after 16 weeks． Lipid metabolic disorders and oxidative stress play different roles in the development of nonalcoholic fatty liver disease．

Abstract:Objective To establish an simple and effective animal model of multiple organ dysfunction syndrome ( MODS) induced by lipopolysaccharide ( LPS) in sheep and observe the changes of blood gas，blood routine test and blood biochemistry． Methods Twelve sheep ( 6 males and 6 femals) were randomized into group 1 and group 2，six sheep in each group． After anesthesia，3 μg / kg LPS was infused intravenously within 30 minutes in the group 1 and 6 μg / kg LPS within 60 minutes in the group 2． The blood gas，blood routine test and blood biochemistry were measured at different time points． Results Two sheep in the group 1 ( 33. 3% ) and three sheep in the group 2 ( 50. 0% ) died after the intravenous injection of LPS． The oxygenation index was significantly decreased ( P ＜ 0. 05 or P ＜ 0. 01 ) ，aspartic transaminase ( AST) ，alanine aminotransferase ( ALT) ，total bilirubin ( TBIL) and creatinine ( CRE) were significantly increased ( P ＜0. 05 or P ＜ 0. 01 ) at different time points. The number of leukocytes was significantly decreased within 2 hours after intravenous injection of LPS，then markedly increased ( P ＜ 0. 05 or P ＜ 0. 01) ． Conclusions A sheep model of MODS has been successfully established by two doses of LPS． Significant changes of the indexes of blood gas，blood routine test and biochemistry can be seen in those model sheep．

Abstract:Objective To use the upper extremity precision movement analysis system in evaluation of the effect of human bone marrow mesenehymal stem cells ( hBMSC) in the treatment of non-human primate models of photochemically induced stroke． Methods Eighteen adult male cynomolgus monkeys were used in this study． Training the animals for 2 months with mMAP and collect the basic data before the surgery． The infarction models were induced photochemically．the18 monkeys were divided into three groups at 4 weeks after the surgery． The control group consisted of 6 animals receiving physiological saline in a volume of 250 μL at 3 mm away from the outer edge of the infarction． The high-dose and low-dose groups received hBMSC with the cell number of 5 × 106 and 1 × 106 cells/ animal，respectively，at the same site and volume for the control group． Behavioral data were collected at 1，3 days，1，2，3，4 weeks after surgery and 1，3days，1，2，3，4，5，6，8 weeks after hBMSC transplantation． A primate neurological deficit scale was used to assess the neurological deficit at the same time． Results All 18 animals showed focal neurological signs corresponding to the targeted infarct site after the surgery． The time at that the animals picked up the food was much slower than that before the surgery ( P ＜ 0. 01) ． The score was 32. 3 ± 5. 13 at the first post-operatively evaluation． The average score declined but never fully recovered after 4 weeks． From the 3rd day after transplantation，there were significant differences between the control and both low and high dose-groups with the movement analysis system ( P ＜ 0. 05) ． From the 2nd week after transplantation，there were significant differences between the control and both low and high dose-groups in neurological deficit scores ( P ＜0. 05) ． There was no significant difference between the high-dose and low-dose groups． Conclusions The precision movement analysis system used in this study reflects neurological function objectively and accurately，and can be used to measure the neuronal loss in vivo and to evaluate the effects of therapeutic strategies involving neural or stem cell transplantation in the establishment of animal stroke models and evaluation of drug efficacy studies．

Abstract:Objective To evaluate the feasibility of conventional intra-arterial angiography， intra-venous angiography，transventricular angiography and angiography through the central artery of the left ear in assessment the of elatase-induced saccular aneurysm in rabbits． Methods Ten New Zealand white rabbits underwent surgical operation to develop a model of elastase-induced saccular aneurysms at the origin of the right common carotid artery． Each rabbit underwent conventional intra-arterial digital subtraction angiography， intra-venous digital substraction angiography，transventricular angiography and angiography through the central artery of the left ear at 3 weeks after the modeling procedure． Using an external sizing reference，the diameter of aneurysm，including the width，height and neck，measured．The data obtained by different angiographic imaging were compared by one-way repeated measures ANOVA． Results The aneurysm morphology in the tested animals was clearly displayed by conventional intra-arterial angiography，intra-venous digital substraction angiography，transventricular angiography and angiography through the central artery of the left ear in rabbits，respectively． There were no significant differences in the width，height and neck of aneurysms measured by different angiographic methods ( P = 0. 646，0. 427 and 0. 625，respectively) ． Conclusions The different angiographies can clearly show the size of elastase-induced saccular aneurysms in rabbits，and an appropriate angiographic method can b selected according to different experimental aims and conditons．

Abstract:Objective To study the spontaneous occurrence of hypophyseal cyst in beagle dogs and establish the backgroud information of experimental animals for good laboratory practice ( GLP) ． Methods Sixty beagle dogs ( male∶female = 1∶ 1) aged 8-10 months were used in this study． The incidence of hypophyseal cyst was recorded and analyzed，and their histopathological features were examined by histopathology． Results Almost all the cysts were seen in the pars distalis hypophysis，and the wall was covered by a squamous or cuboidal epithelium． The total incidence rate of hypophyseal cyst was 23. 3% ． The incidence rate in females was 13. 3% and in males was 33. 3% ． Conclusions It is suggested that spontaneous occurrence of hypophyseal cyst in Beagle dogs should be monitored so as to provide correct histopathological backgroud information of the dogs for drug safety evaluation．

Abstract:Objective To observe the repairing effects of allogeneic bone marrow mesenchymal stem cells ( MSCs) on jejunal radiation damage in rats． Methods Rat MSCs were isolated，purified and cultured in vitro． The model of ileal radiation damage was made． Sixty rats were randomly divided into normal control group，model group，and MSCs group( n= 12) ． The enrichment of MSCs in the jejunum were observed under laser scanning confocal microscope． Pathological changes of the jejunum were examined． Results The structure of normal rat jejunal mucosa was clear，with deep crypts and glands． In the model group at 7 days after irradiation，the epithelial cells were necrotic and the crypts were almost completely destroyed． But mucosal necrosis was less severe， the mucosa got thickening， and mitotic activity was significantly increased in the treatment group，compared with that of model group at 30 days after irradiation． Conclusions The model of jejunal radiation damage is successfully established． MSCs can promote the regeneration and repairment of the jejunal radiation damages

Abstract:Objective To establish a mouse model of acute infection with M． tuberculosis suitable for pharmacodynamic evaluation of anti-TB drug candidates． Methods Female C57BL /6J mice were infected with intravenous injection of 3 × 105 CFU M． tuberculosis H37Rv． Bacterial load and pathological changes in the spleen and lung were analyzed at 1 d，1 w，2 w，3 w，4 w，6 w and 8 w post-infection． Results The bacteria load in the spleen in increased during the first 3 weeks，and maintained at a high level during the following 5 weeks． The bacteria load in the lung increased during the following 8 weeks after infection． Granuloma formation could be detected in the lung and spleen at 3 weeks after infection，and pathological injury became more severe at 6-8 weeks． Conclusions Mice hematogenously infected with a high dose of M． tuberculosis show an acute infectious condition． The mouse model of acute TB infectioon can be used in evaluation of anti-TB drug candidates．

Abstract:Objective To establish a new SNP genotyping method using Pfu DNA polymerase extend phosphorothioate-modified primers in a single tube bi-directional allele specific amplification ( SB-ASA) ． Methods For rat dbSNP loci rs8149053， we designed two outer and two inner allele-specific primers that were 3 ’-terminal phosphorothioate modified． Pfu DNA polymerase was used to specifically amplify the genome template and the SNP genotype was determined by different target segment DNA eletrophoresis． Results Different length segments represented different genotypes that were validated by sequencing． Conclusions Pfu polymerase genotyping assay is a rapid and specific method for detecting the genotype of the known SNP in a single PCR reaction．

Abstract:The standards of bacteriological quality monitoring of laboratory animals are very important index to evaluate the quality of laboratory animals． Based on the analysis of the standards of microbiological quality monitoring of laboratory animals in developed countries，in this paper，we will discuss some current problems such as test item，test methods，test frequency and sampling method and make suggestions for bacteriological quality monitoring of laboratory animals in China．