Abstract:Objective To investigate the pathological evaluation method of intrathalamic neurovirulence test of poliomyelitis vaccine in monkeys. Methods Two hundred and forty healthy rhesus monkeys age 1-3 years, body weight 1.7-4.5 kg, in 12 lots, were used in this study. The monkeys were injected with poliomyelitis vaccine into bilateral thalamus. The animals were sacrificed after observation for 21 days and specimens of the brain and spinal cord were collected. After dehydration, embedding and sectioning, HE and gallocyanin staining were used for pathological observation. The pathological changes were scored, and the severity of the pathological changes were determined by scoring. Results The sections were taken from bilateral cerebellum (needle tracts included), midbrain, pons, medulla oblongata, cervical enlargement and the lumbar enlargement. The pathological changes were evaluated referring to the hemisection scoring method of intraspinal neurovirulence test. Lesions on both sides of the brain stem, cervical enlargement and lumbar enlargement were scored, and the severity of the lesions at these three regions were determined according to the average score. The total incidence rate of specific neuronal pathological changes was 15%. The incidence rates of lesions caused by different types of vaccines were similar. Most of the lesions were minimal, and severe lesions were seen mainly in the type III vaccine tests. Conclusions In intrathalamic neurovirulence test, scoring of the lesions of nervous system by hemisections can objectively reflect the pathological changes in the nervous system. This method is simple and easy to apply, practical, and can facilitate the unification of diagnostic criteria. We would recommend this method to be used in pathological evaluation in intrathalamic neurovirulence test of poliomyelitis vaccine.

Abstract:Objective To assess the potential of whole blood IFN-γ assay for diagnosing mycobacterium in rhesus macaques. Methods Firstly, basic serum IFN-γ concentrations of TST-negative and -positive rhesus macaques were detected. Then, heparinized whole blood from TST-negative and -positive rhesus macaques was incubated with PBS and 200 IU bovine-PPD (tuberculin purified protein derivative) for about 24 h, respectively. The supernatant plasma were harvested and used to determine the concentrations of IFN-γ. The results of plasma IFN-γ concentrations and stimulation index (SI) were used to analyze the diagnostic potential of the whole blood IFN-γ assay. Results The basic serum concentrations of IFN-γ for the TST-positive monkeys were significantly higher than that of the TST-negative macaques, showing a high coefficient of variation. There was no significant effect on the production of IFN-γ in the TST-negative macaques. While significantly elevation of IFN-γ concentrations was found in stimulated plasma of TST-positive macaques (P<0.01). The SI of TST-positive macaques was significantly higher than the TST-negative ones. ROC curve analysis revealed that IFN-γ concentrations and SI could be used as evaluation index of whole blood IFN-γ assay. Conclusions Based on a small sample experiment we have demonstrated that whole blood IFN-γ assay may be one possible auxiliary diagnostic method for tuberculin skin test.

Abstract:Objective To determine the main reproductive performance of six SPF mice and rats preserved in our laboratory, including C57BL/6, BALB/c, NIH, KM, ICR mice and SD rats. Methods Inbred mice mated with full sib and random mating. Meanwhile, closed colony animals were bred by cross circular mating. These indexes of reproductive performance were measured in the six kinds of animals during the first birth to the fourth birth, i.e. the average litter size (ALZ), average baby number reared (ABN) and average weaning number (AWN), and weaning rate (WA) were calculated. Meanwhile,the initial mating age in days, initial bearing age in days, first gestation and intervals were measured. Results The ALZ of inbred mice were 6 to 7. The reproductive indexes were basically stable in the first 3 births, however, the various data were dramatically declined in the fourth birth (P<0.05). The WA of BALB/c mice were 98% to 99%, and that of C57BL/6 mice were 96% to 98%. The ALZ of closed colony animals were 12 to 15, and significantly increased in the second and third births compared with that of the first birth (P<0.05), then was significantly reduced (P<0.05). The WA maintained at 98% to 99% in the NIH, KM and ICR mice. In contrast, The WA of SD rats was slightly lower, reaching 95% to 97%. The initial mating age in days of mice was between 70 d to 80 d. The initial bearing age in days and first gestation of rats were less than that of inbred mice, the initial bearing age in days and first gestation of closed colony rats were 136.3 d and 27.7 d, respectively. The pregnancy interval of inbred mice was between 34 d to 40 d,the pregnancy interval of closed colony mice was between 25.5 d to 28.7 d, the pregnancy interval of rats was between 32.8 d to33.8 d. Conclusions The 6 mouse and rat strains have a higher reproductive performance, and the data obtained in this study provide a basis for commercialized production of mice and rats in southern areas of China, and for establishment of rodent experimental animal resource.

Abstract:Objective To analyze the preventive and therapeutic effects of a traditional Chinese medicine Taohe Chengqi decoction on a rat models of hepatic encephalopathy (HE) induced by thioacetamide (TAA).Methods The rat model of HE was established by intraperitoneal injection of TAA,and the effects of Taohe Chengqi decoction were observed by neurobehavioral changes,neurological test,blood ammonia,serum biochemical indexes,and liver and brain pathological examination.Results The rat model of hepatic encephalopathy was successfully established. Low, medium and high dose Taohe Chengqi decoction markedly improved neuro-reflexes and liver and brain pathological damages,and significantly decreased the HE staging and serum biochemical indexes, and the results of high dose group was the best, similar to that of positive drug-treatment group.Conclusions Taohe Chengqi decoction exhibits promising preventive and therapeutic effects on the rat model of TAA-induced hepatic encephalopathy, and these results provide substantial experimetal evidence for its clinical application.

Abstract:Objective To establish a rat model of liver metabolism profile in chronic heart failure (CHF), to explore the dynamics of liver metabolism in CHF from the point of view of metabolism, and to find the characteristic metabolites valuable for the molecular mechanism and management of CHF. Methods Twenty male Wistar rats were assigned to the CHF group to receive aortic coarctation or to the control group to receive sham surgery, and were bred for 24 weeks following surgery. The metabolic profiling of the rat liver tissues was analyzed on a metabonomics research platform. Orthogonal partial least squares-discriminant analysis (OPLS-DA) model and principal component analysis (PCA) model were established for liver tissues of the CHF rats, and the characteristic metabolites were finally derived by data processing with SPSS 19.0 software. Results The PAC and OPLS-DA models were established successfully. Ten characteristic metabolites with significant differences between the CHF and control groups, including lysophosphatidyl choline, lysophosphatidyl ethanolamine, oleic acid, glycocholic acid, and dehydroepiandrosterone sulfate, were screened and identified from the models. Conclusions The metabolic disorders in CHF rats are well fitted to the established metabolic profile models, and these identified characteristic metabolites may provide reference for the pathophysiological molecular mechanism and management, etc., of chronic heart failure.

Abstract:Objective To investigate the effect of glutamine in combination with umbilical cord blood mesenchymal stem cells (MSCs) transplantation on intestinal ischemia-reperfusion injury in rats. Methods Umbilical cord blood mesenchymal stem cells were isolated, and were labeled with CM-DiI fluorescent dye. Eighty Sprague-Dawley rats were randomly divided into normal control group, ischemia reperfusion injury group, glutamine group, MSCs transplantation group and combined group with 15 rats in each group. The control group received saline enema. The injury group was treated with TNBS (ethanol dilution) enema. The glutamine group at 1 h after TNBS received intravenous injection of 0.45 g/kg glutamine. The rats of MSCs transplantation group had tail vein injection of 1 ×10¹⁰/L umbilical cord blood mesenchymal stem cell suspension, and the combined group received intravenous injection of glutamine 0.45 g/kg and 1×10¹⁰/L umbilical cord blood mesenchymal stem cell suspension. ELISA was used to detect the midgut fatty acid binding protein (iFABP), interleukin 6 (IL-6), and superoxide dismutase (SOD) content in the rat serum. The water content of intestinal tissue was detected at 1 h and 3 h after reperfusion in each group. The expressions of NF-kB, Bcl-2 and caspase-3 mRNA and proteins in the rat intestinal epithelial cells after treated with glutamine in combination with MSCs were detected by RT-PCR and Western blot assays. Results The fluorescent tracer method revealed that the transplanted MSCs cells were distributed in the intestinal mucosal lymphoid tissues and glandular epithelial cells, indicating that MSCs might be involved in the repair process of intestinal ischemia-reperfusion injury. The content of serum IFABP and IL-6 in the injured group was significantly higher than that in the control group, while significantly reduced in the glutamine group, MSCs transplantation group and combined group, with the most obvious in the combined group. The content of SOD in the injury group was significantly lower than that in the control group, and significantly increased than that in the glutamine group, MSCs transplantation group, with the most striking in the combined group (P<0.05 for all). The water content of intestinal tissue in the injury group at 1 and 3 hours after reperfusion was significantly higher than that in the control group, significantly lower in the glutamine group, MSCs transplantation group and the combined group, with the most decreased in the combination group, and there was no significant difference between the glutamine group and MSCs transplantation group (P>0.05). Compared with the control group, the caspase-3 and NF-kB mRNA and protein expressions in the intestinal mucosal epithelial cells of the injury group were significantly increased, and the expressions of Bcl-2 mRNA and protein were significantly reduced (P<0.05), the expressions of caspase-3 and NF-kB mRNA and protein were significantly reduced in the glutamine group, MSCs transplantation group and combined group. The expressions of Bcl-2 mRNA and protein were significantly increased (P<0.05), while no significant difference was shown between the glutamine group and MSCs transplantation group (P>0.05), but there was a significant difference between these two groups and the combined group (P<0.05). Conclusions After treated with glutamine and MSCs transplantation, the degree of intestinal ischemia reperfusion injury is obviously reduced in rats. It may be mediated through inhibiting the expression of caspase-3 and NF-kB and promoting the expression of Bcl-2.

Abstract:Objective To explore the effect of vasoactive intestinal peptide (VIP) on the content of IL-17A in the brain tissue of rat models of experimental autoimmune encephalomyelitis (EAE). Methods Sixty healthy female Wistar rats were randomly divided into normal control group, EAE control group, low-dose VIP group and high-dose VIP group. Ten healthy guinea pigs were used to prepare anti- IL-17A antibody. Myelin basic protein (MBP)+complete adjuvant (CFA) were used to establish the EAE model. Since the first day of modelling, the low-dose and high-dose VIP groups received intraperitoneal injection of VIP 4 nmol/kg (0.2 mL) and 16 nmol/kg (0.8 mL), respectively, every other day for 10 consecutive days. The normal control group and EAE group were injected with 0.8 mL saline instead of VIP. The incubation period, progression and the peak of neurological dysfunction scores (NDS) of the rats were recorded. The levels of IL-17A in the brain tissue was determined by ELISA assay, and the GFAP⁺astrocyte activation in brain at morbidity peak in the rats was examined using anti-GFAP (glial fibrillary acidic protein) antibodies. Results The incubation period were extended, the progression period was shortened and the peak neuological dysfunction score (NDS) was decreased in the VIP-treated groups, in a dose-response relationship. The cytokine levels of IL-17A and the astrocyte activation degree in brain tissue were reduced in each VIP dose group, in a dose-response relationship. Conclusions VIP exerts therapeutic effect on experimental autoimmune encephalomyelitis through lowering the IL-17A content and inhibition of astrocyte activation in the brain tissue.

Abstract:Objective To explore the possible mechanism of action of telomerase in hepatic precancerous lesions, and the regulatory effect of a Chinese medicine prescription HU Qi Shan (HQS) and its principal drug mistletoe alkali on the telomerase activity in rat liver tissues. Methods Rat model of hepatic precancerous lesions was established by Solt-Farber two-step protocol. The model rats were randomly divided into 5 groups, including the model group, high-dose HQS[8 g/(kg·d)] group, low-dose HQS[4 g/(kg·d)] group, and mistletoe alkali[8 mg/(kg·d)] group. γ-Glutamy-transpeptidase (γ-GT) was analyzed by immunohistochemistry. AFP was detected by immunofluorescence technique.The telomerase activity was detected using a quantitative telomerase detection kit. The expression of NF-κB P65 was detected by immunohistochemistry. The cytoplasmic protein IκB-α was detected by western blotting. Results After treated with HQS and mistletoe alkali, the areas of γ-GT-positive foci and number of AFP-positive cells in the liver tissus were significantly decreased than those of the model group (P<0.05 for both), the telomerase activity was decreased, the number of NF-κB P65-positive cells was also decreased (P<0.05), whereas the intracytoplasmic expression of IκB-α proteins was significantly increased (P<0.05). Conclusions HQS and mistletoe alkali can suppress the telomerase activity. Its possible mechanism may be through inhibition of the over-expressed apoptosis-related genes such as NF-κB P65 and increase the expression of IκB-α decreasing the telomerase activity.

Abstract:Objective To investigate the effect of acupuncture combined with human amniotic mesenchymal stem cells (HAMCs) transplantation on the osteoporosis in ovariectomized rats. Methods Forty-five healthy female non-pregnant Wistar rats were randomly divided into sham operated group, model group and experimental group (acupuncture+HAMC transplantation), 15 rats in each group. At 7 days after the surgical wound healing, the rats received acupuncture every day and intravenous injection of HAMCs suspension (except the sham operated group) once a week for consecutive 12 weeks. The body weight was measured every week. 24 hours after the last intervention, the rats were killed and specimens were collected. Specimens of the mid-, proximal- and distal femur were taken to measure the bone mineral density using a dual energy X-ray absorptiometer. At 12 weeks after the intervention, changes of the expression of serum 25-hydroxy-vitamin D (25-HVD), C-terminal peptide of type I collagen (CTX-I), bone specific alkaline phosphatase (BAP) and human tartrate-resistant acid phosphatase 5b (TRACP 5b) were determined by ELISA, the biomechanical properties of the removed femur was measured, and the expression of transforming growth factor-β (TGF-β) in the vertebrae was assessed by RT-PCR assay. Results Body weight of rats in the sham operation group was increased gradually in accordance with the natural growth of animals, that of the model group was significantly higher than that of the sham operated group since two months after modeling, and the body weight of the experimental group was similar to that of the sham operated group. The bone mineral density and calcium content of the model group were significantly decreased compared with that of the sham operated group (P<0.05), and the bone mineral density and bone calcium content of the experimental group were significantly higher than that of the model group (P<0.05). The ELISA showed that the expressions of serum 25-HVD, CTX I, BAP, and TRAP5b in the model group were significantly lower than that of the sham operated group, and those of the experimental group were significantly higher than that of the model group (P<0.05). Measurement of biomechanical properties showed that the limit load, limit stress and elastic modulus of the femur of the sham operated group were significantly higher than that of the model group (P<0.05), and the limit load and elastic modulus of the experimental group were significantly higher than that of the sham operated group (P<0.05). T-PCR showed that the expression of TGF-β1 m-RNA in the experimental group was significantly up-regulated than that of the sham operated and model groups (P<0.05). Conclusions Acupuncture combined with human amniotic mesenchymal stem cell (HAMCs) transplantation has a synergistic effect on the treatment of osteoporosis, and can improve the conditions in ovariectomized rats.

Abstract:Objective To analyze and evaluate the population genetic quality of 3 subbreeds of China Agricultural University miniature pigs in Beijing. Method According to the local standard DB11/T828.3-2011, 25 pairs of microsatellite primers were used in 3 subbreeds of China Agricultural University miniature pigs, and software Popgen32 was used to process the data. Results 24 microsatellite loci shared 130,122 and 138 alleles in the China Agricultural University miniature pigs I, II, III, respectively. The average heterozygosity was 0.6759, 0.5967 and 0.6779, respectively, while the average polymorphism information content (PIC) was 0.6344, 0.5540 and 0.6403, respectively. The genetic distance between China Agricultural University miniature pig II and III was 0.4251, while the genetic distance between China Agricultural University miniature pig I and II was 0.2084. Conclusions In the 3 subbreeds, China Agricultural University miniature pigs II and III have genetic stability and genetic diversity, and both of which satisfy with the genetic characteristics of closed colony laboratory animal.

Abstract:Objective This study aimed to establish a reliable primary culture protocol for preparing murine spleen-derived mesenchymal stem cells (MSCs) by tissue explant culture. Methods Healthy mouse spleens were crushed by syringe handle to harvest spleen mesenchymal tissues. Then the tiny pieces of spleen tissue were digested by collagenase II before seeded into culture flasks. The morphological characteristics of spleen tissue-derived cells were observed under the inverted microscope. Further, the surface antigen profile of the cells was analyzed by flow cytometry (FACS). The cells were induced to differentiate into osteoblasts and adipocytes. Results The murine spleen-derived MSCs exhibited a spindle-shaped appearance. The FACS results showed that the spleen-derived MSCs highly expressed CD29, CD44, CD105 and Sca-1, but weakly expressed CD11b, CD34, CD45 and Ia. In addition, the spleen-derived MSCs steadily differentiated into osteoblasts and adipocytes in the induction medium.Conclusions A method of primary culture of murine spleen-derived MSCs by explant culture is successfully established. The harvested MSCs exhibit high purity and cell proliferation ability, and provide a reliable cell model for related researches.

Abstract:Objective To investigate the status of Helicobacter pylori and "Helicobacter macacae" infection in rhesus and cynomolgus monkeys in China. Methods With the use of 16S rRNA specific primers for Helicobacter spp and Helicobacter pylori (HP) from published literatures, and new 16S rRNA specific primers designed for "Helicobacter macacae" (HM), we investigated the infection status of these two Helicobacter spps in both of 45 rhesus and 90 cynomolgus monkeys by qPCR or conventional PCR on stool samples. Results All three primer sets for 16S rRNA exhibited excellently sensitivity and specificity. Both the infection rates of HP and HM were 100% among 45 young adult rhesus monkeys. The infection rate of HP and HM in 90 young adult cynomolgus monkeys were 100% and 97.8%, respectively. Conclusions Helicobacter pylori and "Helicobacter macacae" are present in almost every artificially bred adult rhesus and cynomolgus individuals which may adversely affect the health of laboratory monkeys and the accuracy of related animal experiments.

Abstract:Objective To study the effect of different doses of monocrotaline in combination with isopropylarterenol on the hemodynamic resonse, heart index and right heart hypertrophy index in rats. Methods Sixty-four healthy adult SD rats, male:female=1:1, body weight 200-250 g, were randomly divided into blank control group (n=16) and three model groups (n=16 in each group). The high dose model group (n=16) received i.p. injection of monocrotaline 80 mg/kg once and epinephrine 10 mg/kg once daily for one week. The moderate dose group received i.p. injection of monocrotaline 55 mg/kg once and epinephrine 8 mg/kg once daily for one week. The low dose group received i.p. injection of monocrotaline 30 mg/kg once and epinephrine 3 mg/kg once daily for one week. The rats were fed for 6 weeks, and then pulmonary artery pressure and right ventricular pressure were tested and heart index and right ventricular hypertrophy index were determined. Results Compared with the control group, the mean pulmonary artery pressure and right ventricular systolic blood pressure in the low dose monocrotaline group were not significantly changed, but significantly changed in the moderate dose monocrotaline group (P<0.05). The heart index and right ventricular hypertrophy index in the low dose monocrotaline group were not significantly changed, but in the moderate dose monocrotaline group, the heart index was significantly reduced (P<0.01) and the right ventricular hypertrophy index was significantly increased (P<0.05). Conclusions The use of a single injection of 55 mg/kg monocrotaline in combination with continuous injection of 8 mg/kg isopropylarterenol once daily for one week can ensure the survival rate of rats, and the successful formation of pulmonary artery hypertension, leading to heart weakness.

Abstract:Objective To establish a detection method of Salmonella in laboratory animals based on a high-throughput sequencing technology, and to apply it in detection of Salmonella in laboratory animals. Methods DNA samples were extracted from mouse feces. Universal primers for 16S rDNA, 23S rDNA, 16S-23S rDNA, 23S-5S rDNA region, gyrB preferred area were designed, respectively. Each primer was tested and analyzed to determine the best amplification conditions and build a database. Forty-two samples of Salmonella were assayed by Illumina high-throughput sequencing technology and evaluated the specificity and stability of this method. Results The species preferred region of Salmonella was gyrB gene region. The primers for gyrB gene were FP5'-AACCACCGCAATCAGACCTT3' and FP5'-AGCCACGAAACCTTCACYA-3'. The primers were optimized and determined, through a high-throughput sequencing, and the sequence analysis detected very small amount of Salmonella in the 42 samples, indicating that this detection method is stable, highly sensitive, and the limit of detection reached to 0-10² CFU. Conclusions We have established a complete detection system for detection of Salmonella in laboratory animals based on a high-throughput sequencing technology, This system can detect trace amounts of Salmonella in laboratory animals, and this detection method is stable and highly sensitive, which can be also used in detection of other kinds of pathogenic microorganism in laboratory animals.

Abstract:Objective To introduce a simple fixing method for tail vein injection in mice. Methods Twenty tumor-bearing male BALB/c nude mice were used in this study. Tail vein injection was performed to these mice by two laboratory technicians A and B, respectively. The injection time and success rate were recorded and analyzed. Results Mouse tail vein injection was successfully completed by the two technicians with the cage lid pressing method. Conclusions Cage lid pressing method is a simple method for tail vein injection in mice, especially provides a more efficient method for those special form of mice.

Abstract:Objective To provide a device and an effective method for tail vein injection in mice. Methods Doing the tail vein injection in mice with the self-designed device which is consisted of constant temperature part, lighting part and holding part. The difficulty and time of injection with and without the device were compared. Results It was faster and more accurate to perform the tail vein injection in mice with this self-designed device. Conclusion Using this self-designed device can significantly improve the efficiency and save the injection time.

Abstract:The development of laboratory animal science plays a supporting role in the biomedical field, pharmacy and other fields. It is an important symbol to measure the level of science and technology in each countries in the world. The quality of employees directly affects the speed and quality of the development of laboratory animal industry. The purpose of this standard is to regulate the requirements of animal practitioners, to strengthen the training and qualification certification, and to promote the quality of employees.