Abstract:Objective To analyze the neuroimaging changes of tree shrew models of Alzheimer's disease. Methods Nineteen healthy adult female tree shrews were randomly divided into control (5 animals) and model group (14 animals). The model of Alzheimer's disease was induced by intracerebroventricular injection of Aβ_1-40 using a stereotaxic devise and proved successfully by visuospatial congnitive task. The in vivo microstructural changes in the brain of tree shrew AD models and control group (0, 1, 2, 3, 4 weeks) were observed on 1.5T MRI (T₂WI), and on 7.0T MRI (12 week)(T₂WI, DTI). Results Reference memory errors were increased in the model group at 3 or 4 weeks (P<0.05), and so working memory errors (P<0.05) and period of time to perform (P<0.05, P<0.05, P<0.01) from 2 to 4 weeks. Thus the model was proved to be established successfully. T2WI test and DTI test were carried out. Hippocampus atrophy of the model group at 3 and 4 weeks was observed compared with that at 0 or 1 week or 2 weeks on a 1.5T Philips Gyroscan. Compared with the control group, the temporal horn width in the model group was significantly increased (P<0.01) at 12 weeks on a 7.0T Bruker Biospec Scanner. DTI test at 12 weeks showed that ADC of bilateral hippocampus was up-regulated in the model group (P<0.01). In the color coded orientation view, loss of the corpus callosum fibers was obvious in the model group. Conclusions Intracerebroventricular injection of Aβ_1-40 can lead to learing and memory impairment in tree shrews.There are abnomal MRI signal changes in the brain, and the temporal horn width, hypocampal apparent diffusion coefficient (ADC) value and corpus callosum damage may provide reference value for the diagnosis of Alzheimer's disease.

Abstract:Objective To investigate the role of mitochondrial cytochrome c on hepatocyte apoptosis in non-alcoholic fatty liver disease in rabbits and its pathogenesis. Methods Forty Japanese white rabbits were randomly assigned to control group and model group. The model group was divided into three subgroups:4-week, 6-week, and 8-week groups, with 10 rabbits in each group. The model groups were subcutaneously injected with peanut oil (1.2 mL/kg), twice a week for 4 weeks, 6 weeks or 8 weeks. The rabbits of all groups were killed at the right time. Serum samples were collected to detect the serum biochemical index levels. Liver tissue samples were taken for pathological observation using HE staining. The hepatocyte apoptosis index (AI) was measured by flow cytometry, and mitochondrial permeability transition pore (MPTP) was evaluated by ultraviolet spectrophotometry. Immunohistochemistry was employed to detect the hepatic expressions of Bcl-2, Bax, CYT C and caspase-3. Western blot was performed to detect the changes of CYT C and caspase-3 protein expressions. Results The model groups showed hepatic injury and high level of TC, TG, CRP, IL-6 and TNF-α beginning from 4 weeks. With the NAFLD process, the hepatocyte apoptosis index was significantly increased at 4-8 weeks and the MPTP was gradually increased. In the model group, hepatic Bcl-2, Bax, CYT C and caspase-3 expressions were increased steadily with the time passing. Conclusions NAFLD-induced liver damage is associated with apoptosis, and the mitochondrial cytochrome c-mediated apoptotic pathway plays a role in the occurrence of NAFLD.

Abstract:Objective To investigate the effect of dexmedetomidine on the renal function and the expression of tight junction protein ZO-1 and occluding in the kidney of rats with septicopyemia induced by lipopolysaccharide.Methods Thirty adult male SD rats were randomly divided into 3 groups (n=10 each):control group(group C), LPS group (group M), and dexmedetomidine group (group Dex). In the groups Dex and LPS, the rats were infused with saline and lipopolysaccharide (5 mg/kg, i.v.) respectively. In the Dex group, after intravenous injection of lipopolysaccharide (5 mg/kg), the rats were firstly infused the loaded dose of dexmedetomidine (7 μg/kg) for 15 minutes, and then changed to 5 μg/kg·min for 30 minutes. Blood samples were collected at 24 h later for measuring TNF-α, IL-1β, Scr and BUN. The kidney tissues were examined by histopathology. The expression of ZO-1 and occludin was detected by Western blot. Results Compared with the group C, the kidney tissue of group M was extensively damaged with tubular dilatation and inflammation, while reduced in the group Dex. Compared with the group C, the expressions of Scr, BUN, IL-1β and TNF-α were all enhanced in the groups M and Dex (P<0.05), while the inflammatory factors in the group Dex were significantly lower than those of the group M (P<0.05). The Results The Western blot analysis showed that the expressions of protein ZO-1 and occludin in the group Dex were significantly higher than those of the group M (P<0.05). Conclusions Dexmedetomidine can improve the renal function of rats with septicopyemia, inhibit the acute renal injury and inflammation, increase the expression of protein ZO-1, and exert certain protective effect on the kidneys.

Abstract:Objective To construct an adenovirus vector expressing small interfering RNA (siRNA) targeting to rat Raf-1 gene and identify its function in cardiomyocytes.Methods The siRNA containing DNA sequence targeting to Raf-1 and its negative control sequence were designed, synthesized, annealed and subcloned into adenoviral shuttle vector pAdTrack-CMV. The recombinant adenovirus vector pAd-siRaf-1 was obtained by homologous recombination with pAdTrack-siRaf-1 linearized by PmeI and pAdeasy-1 in bacteria BJ5183, then transfected into HEK293 cells to package the adenovirus. Cardiomyocytes were infected with the adenovirus pAd-siRaf-1, and the expressions of Raf-1 and NF-κB protein were detected by Western blotting.[³H]-leu incorporation was evaluated by scintillation. The surface area of cardiomyocytes was measured using a HJ2000 image analysis system. Results The adenovirus vectors were verified by enzyme digestion and DNA sequencing. Compared with the Ang Ⅱ group, Raf-1 and NF-κB expression, the surface area and [³H]-leu incorporation of cardiomyocytes were significantly decreased in cardiomyocytes infected with the adenovirus PAd-siRaf-1. Conclusions A recombinant adenovirus vector containing rat siRaf-1 gene is successfully constructed. It can effectively reduce Raf-1 and NF-κB expression and cardiomyocyte hypertrophy induced by Ang Ⅱ.

Abstract:Objective To explore the growth and developmental parameters and behavioral characteristics of rhesus monkeys during the first year of birth and to establish the background data.Methods A total of 18(♂=11, ♀=7) infant rhesus monkeys born from individually caged mothers and with known genetic background and postnatal days were monitored monthly for body weight, body height, head circumference, chest circumference, forelimb length, hind limb length, crown-rump length, tail length and anal-genital distance from postnatal day (PND) 1 to 360, while hematology, blood chemistry and lymphocyte subsets were examined on PND 28, 175 and 360, and finger maze test was carried out on PND 208. Results The body weight showed linear growth with no significant difference between genders (P>0.05). Except for the anal-genital distance of male infants was significantly greater than that of female infants (P<0.01), no significant differences were observed between sexes in other morphological parameters. No significant differences of hematology were seen between genders (P>0.05). Compared with that at PND28, TP and BUN were significantly increased (P<0.01) while ALP decreased with no significant difference (P>0.05) at PND 175 and 360. Compared with that at PND28, CD4⁺ and CD4⁺/CD8⁺ were significantly decreased (P<0.01) while CD8⁺ significantly increased (P<0.01) at PND175 and 360. The number of sessions to solve task 2 in learning test was significantly greater than other tasks with females significantly less than males (P<0.05). The females had higher correct rate than males in the 2-day random memory test (P<0.05). Conclusions Body weight and morphological parameters show a linear growth. The PND. The RBC, HGB, LYMPH, TP, BUN, ALP, CD4⁺, CD8⁺ and CD4⁺/CD8⁺ in hematology, blood chemistry and lymphocyte subsets show relevant changes to the growth and development of organs and systems in infants, which should be highly concerned in drug evaluation. The finger maze test indicates that female infants have better reversal learning and long-term memory than male infants. Background data and behavioral characteristics of infant rhesus monkeys during the first 12 months of birth are established in this study, which provide useful reference and support the evaluation of developmental and reproductive toxicity of drugs in rhesus monkeys.

Abstract:Objective To observe the pathological changes of heart failure caused by transverse aortic constriction in rats. Methods Partial thoracotomy was performed to the second rib and the transverse aortic constriction was performed between the innominate and left carotid arteries to establish a model of heart failure in 24 rats. The same operation was performed on another 8 rats, except for the ligation of the transverse aorta. Echocardiographic assessment, hemodynamic measurement, myocardial histopathological examination and NT-proBNP measurement were performed to the sham group at 12 weeks and model group at 4 weeks, 8 weeks, 12 weeks after the operation. Result At 4 weeks after the operation, NT-proBNP, EF, FS and -dp/dtmax of the model group was significantly increased and LVESV, +dp/dtmax of the model group were significantly decreased (P<0.05). At 8 weeks after the operation, EF and-dp/dtmax of the model group were increased and +dp/dtmax of the model group was significantly decreased (P<0.05). At 12 weeks after the operation, NT-proBNP, EF and +dp/dtmax of the model group were decreased, and LVESV, LVEDV and -dp/dtmax of the model group were increased (P<0.05). The cardiomyocytes became hypertrophic and lined up in disorder at 4 weeks after the operation. Pathologic examination of the myocardial tissue showed connective tissue proliferation and inflammatory cell infiltration at 8 weeks after the operation, and cardiomyocyte apoptosis and collagen fiber deposition at 12 weeks after the operation. Conclusions Transverse aortic constriction induces heart failure in rats. The pathological processes are compensatory hypertrophy at 4 weeks after the operation, initial reaction of decompensation at 8 weeks after the operation, and heart failure at 12 weeks after the operation.

Abstract:Objective To explore the relationship between protein levels of SAMHD1 expression with viral load of SIV in the SIVmac239-infected CEMx174 cells.Methods CEMx174 cells were infected with SIVmac239, and the cells and supernatant were collected daily for analysis. The viral loads and the SAMHD1 mRNA level were detected by real-time RT-PCR and the level of P27 and SAMHD1 proteins were detected by Western blotting. Results The mRNA level of intracellular SAMHD1 gene was increased and SAMHD1 protein decreased gradually when CEMx174 cells were infected with SIVmac239. Conclusions There is a positive correlation between viral load in the supernatant and the mRNA level of SAMHD1, and a negative correlation between the increase of P27 protein and the decrease of SAMHD1 protein in the SIVmac239-infected CEMx174 cells.

Abstract:Objective To develop a more convenient and stable method for assessment of drug sensitivity of prostate cancer based on real-time cell analysis system as a reference for clinical treatment. Methods Human prostate cancer VCaP, DU145, PC-3, PC-3M-2B4 and PC-3M-IE8 cells were chosen to detect the sensitivity to three drugs, docetaxel, cabazitaxel and abiraterone acetate. Serial dilutions of the three drugs were used to treat the cell culture for 24 hours. The drug-induced effects on the cell lines after an incubation of 24 hours were recorded by the real-time cell analysis system to determine the half maximal inhibitory concentration (IC50). Results Docetaxel showd IC50 of 8.81 nmol/L, 11.61 nmol/L, 1.78 nmol/L, 1.44 nmol/L, 8.69 nmol/L for VCaP, DU145, PC-3, PC-3M-2B4, PC-3M-IE8 cells, respectively. Cabazitaxel showed IC50 of 3.73 nmol/L, 3.96 nmol/L, 10.41 nmol/L, 5.43 nmol/L, and 7.37 nmol/L, respectively, for the five cell lines. Abiraterone acetate showed IC50 of 8.34 μmol/L, 8.60 μmol/L, 24.20 μmol/L, 8.59 μmol/L, and 13.21 μmol/L for the five cell lines. Conclusions PC-3M-2B4 and DU145, VCaP and PC-3 cells can be used as control for docetaxel, cabazitaxel and abiraterone acetate to establish cell models for the drug screening in vitro and to provide reference for clinical applications.

Abstract:Objective To decrease the p53 gene expression at cellular and animal levels in marmoset using RNA interference technique. Methods The shRNA interference sequences were designed and inserted into the adeno-associated virus vector plasmid after bioinformatics analysis. The plasmids were transfected into African green monkey kidney cos-7 cells. The suppression of p53 mRNA was detected by real-time PCR, and the changes of p53 protein expression were detected by Western bolt. The adeno-associated virus-8 was injected through the hind leg vein. The changes of p53 protein expression in the liver tissue was detected by Western blot and immunohistochemistry. Results We screened two RNA interference effective arget sequences. The expression of p53 mRNA was suppressed (82.7±8.1)% and (80.7±7.5)%, respectively (P<0.05), and the expression of p53 protein was decreased (77.3±11.5)% and (73.7±10.7)%, respectively (P<0.05). The two marmosets after virus infection showed that there were virus distributions in the liver, testes, and neck detected by in vivo fluorescence imaging. The expression of p53 in the marmoset liver was detected by western blot, immunohistochemistry analysis showing no obvious changes. Conclusions In the present study, the decrease of P53 gene expression at cellular level is achieved, however, the liver P53 protein in the marmoset liver is not significantly changes. Further optimization of the way of infection is needed in the future.

Abstract:Objective To investigate the accumulation of mutations and single nucleotide polymorphisms (SNPs) in the displacement loop (D-loop) of mitochondrial DNA (mtDNA) might be associated with cancer risk and disease outcome. Methods We obtained cancerous and noncancerous liver tissues from 49 HBV-related HCC patients at the Fourth Hospital of Hebei Medical University. mtDNA of the liver tissues was extracted with Mitochondrial DNA Extraction Kit. Mutation and polymorphism were confirmed by repeated analysis. We assessed the prediction power of D-loop SNPs in hepatocellular carcinoma (HCC) patients. Results No mutation in these HCC patients had prediction power for post-operational survival, whereas one SNP site (nucleotide 150 C/T) was identified by the log-rank test for statistically significant prediction of HCC survival. In an overall multivariate analysis, allele 150 was identified as an independent predictor of HCC outcome. The length of survival of patients with allele 150C was significantly shorter than that of patients with allele 150T (relative risk, 0.246; 95% CI, 0.070-0.861; P=0.028). Conclusions The analysis of genetic polymorphisms in the mitochondrial D-loop helps to identify patient subgroups at high risk of a poor disease outcome.

Abstract:Objective To establish a stable and fast method for primary culture of mouse cardiomyocytes. Methods Dishes were coated with polylysine firstly. A two-step approach was used to isolate and digest mouse cardiomyocytes cells (0.25% trypsin in 4℃ overnight and 0.5 mg/mL to 1.0 mg/mL collagenase +5 mg/mL albumin collagen digestion liquid in 37℃ for short-time digestion), then the cardiomyocytes were purified through differential adhesion for 70 min and 5-bromodeoxyuridine (BrdU). The cell morphology was observed under an inverted microscope. The survival rate of cardiacmyocytes was detected by trypan-blue staining and their purity was identified by α-actinin immunofluorescence staining. Results The cardiomyocytes were in good shape and pulsed spontaneously. The survival rate of the cardiomyocytes reached 98% and the purity was 95%. Conclusions This method described in this study is an ideal method for primary culture of mouse cardiomyocytes with a high survival rate and high purity.

Abstract:Diarrhea is a common intestinal symptom in macaque. The corresponding intestinal lesions of macaque are mainly described at autopsy but less observed by colonoscopy. The aim of this study was to develop a colonoscopic technique and to obtain endoscopic images of the entire colon in macaques. Eight healthy adult macaques (5 males and 3 females) without diarrhea for 2 months, were fed Glauber's salt through nasogastric tubes. The colon cleanliness was well matched to the endoscopic observation of macaque colon. The procedure took 10-20 min for each animal. There was no obvious abnormality in the colon of four animals except some slight differences of mucosal structure from that of human beings. Small pieces of erosion and ulcer in the colons were observed in four macaques which presented mild diarrhea for less than 1 day, while a severe stenosis was observed in one of those four macaques. No animal died during and one week after the endoscopic procedure. Colonoscopy may safely performed in macaques. The images taken by colonoscopy may be important to establish diagnosis and treatment of colitis in macaques in time and to evaluate the efficacy of drug intervention as well. This technique is also helpful to provide qualified macaques for scientific researches.

Abstract:Objective To evaluate the feasibility and safety of transjugular liver biopsy(TJLB)by using the LABS 200 liver access and biopsy set (Cook Inc, USA).Methods Five minipigs were operated though TJLB puncture under the imaging guidance. The liver biopsies were analyzed by histological examination. Results Technical success of TJLB was achieved in all the 5 minipigs. No procedure-related complications occurred, and sufficient amount of specimen for histological examination was obtained in all cases.Conclusions Our preliminary results indicate that transjugular liver biopsy with the use of Cook LABS 200 liver access and biopsy set is clinically safe and feasible, and provide technical support for its clinical application.

Abstract:Objective To establish an easier and better method for primary culture of rabbit fibroblast-like synoviocytes(FLS) by modified tissue cell culture. Method Healthy rabbit joint synovial layers were cut into small pieces and siphoned off water attached on the tissue with sterile filter paper and then cultured. The growth status and morphology were observed. The growth curve of the 3^rd passage cells was measured by CCK-8 assay, and the expression of vimentin was detected by immunocytochemistry. Results The FLS cultured in vitro exhibited a spindle-shaped appearance and could rapidly expand. The cell growth curve was typical of S type, and the cells highly expressed vimentin. Conclusions Primary culture of rabbit FLS by the improved explant culture method is successfully established. The method is simple and highly efficient. It provideds a new method for isolation of FLS.

Abstract:Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) characterized by demyelination and inflammation lesions. MS predominantly affects young adults with a high incidence of disability. However, the exact pathogenesis of MS is still not clear. Studies found that microglia polarization tending to pro-inflammatory M1-like state during the onset of MS, causing the M1/M2 ratio imbalance, forming pro-inflammatory microenvironment state, and which further leading to nervous tissue damage ultimately. Microglia polarization may be considered as the initiator of pathologic alterations by releasing pro-inflammatory cytokines and secondarily trigger the initial microglia response. Given the pivotal role of imbalanced microglia polarization in MS initiation, a critical review of microglia polarization is presented here, in order to elucidate the pathogenesis of MS and highlight the noteworthy candidate therapeutic targets for clinic treatment.

Abstract:According to relevant national laws and regulations, practitioner training was included into laboratory animal science teaching reform. By adjusting the training content and teaching method and use of animal models of typical human diseases, the transformation of training mode was realized and improved. By the assessment of basic theory in combination with practical operation, the thinking ability and hands-on skill of the practitioners are much improved. Through classroom instruction, experimental teaching, quality assessment and tracking survey, the evaluating process of the training quality of training teaching is performed. Therefore, the teaching reform of the laboratory animal science based on the training of practitioners is established.