Abstract:Objective It is well known that diethylstilbestrol (DES ) can result in testicular oxidative injury, and one of its mechanisms of action is leading to dysfunction of steroidogenesis. The aim of this study was to investigate the relationship between testicular oxidative injury caused by DES and the key synthetase activities for the synthesis pathway of steroidogenesis and the possible mechanism. Methods Twenty-four 4-wk-old male Wistar albino rats were randomly divided into 4 groups, 6 rats each. Three doses of DES (0.1, 1.0 and 10 μg/kg·d) groups and a vehicle (corn oil) control group, were respectively administered by subcutaneous injection once a day for eight weeks. The rats were sacrificed after 8 weeks treatment and the body weight, testis, epididymis, prostate were weighed, respectively. The testicular tissues were homogenized and the oxidation of MDA and ROS, the activity changes of antioxidases SOD, CAT and GPx, as well as the activities of steroid synthetases 3β-HSD1 and 17β-HSD3 were determined by biochemical measurement. The levels of testosterone and LH in peripheral blood were measured by radioimmunoassay. The intensities of expression of StAR, P450scc, 3β-HSD1, 17β-HSD3-mRNA were detected by PCR. Results In the 10.0 μg/kg dose group, the weights and organ coefficients of testis and prostate were decreased significantly, the oxidation of MDA and ROS was increased distinctly and the activities of SOD, CAT, GPx, 3β-HSD1 and 17β-HSD3 were reduced. The concentration of serum testosterone was decreased in the 10.0 μg/kg dose group. In the 10.0 μg/kg and 1.0 μg/kg dose groups, the decline of LH level presented a dose-dependent manner, and the intensities of immunochemical positive staining for StAR, P450scc, 3β-HSD1 and 17β-HSD3 mRNA were decreased. Conclusions DES exposure results in disturbance of the oxidant/antioxidant balance and decline of testosterone level that induces reproductive impairment in male rats. DES induces reductions of both GPx and 3β-HSD activities which cause the decrease of testosterone synthesis. The expression of P450scc and 3β-HSD-mRNA, which are the key synthetases in biosynthetic pathway of steroidogenesis, are inhibited by DES, and it is speculated that the disturbance of steroidogenic synthesis enzymes may be one of the mechanisms of toxic effects of DES.

Abstract:Objective To investigate the role of basophils in the imbalance of Th1/Th2 response in mouse models of collagen-induced arthritis(CIA). Methods 4-6-weeks old C57/BL6 mice were immunized with collagen at multiple points on the back and foot twice (0 and 3 weeks) to establish a mouse model of collagen-induced arthritis. Blood samples were collected before the first immunization and 1, 3, 6 and 9 weeks after immunization, and cells from lymph nodes were collected. Flow cytometry and ELISA were employed to detect the levels of basophils and IL-4, and the joint swelling was scored. Results Mouse model of CIA was successful established. The ratio of IL-4/IFN-γ of the CIA group was significantly lower than that in the mice before CIA modeling and the control group, indicating a Th2-dominant response. At the same time, the peripheral basophils counting and percentage of IL-4 positive basophils of the CIA group were significantly higher than those of the control group. While, the IL-4/IFN-γ ratio of the CIA group was significantly higher than that of the control group, indicating a Th1-dominant response. The peripheral basophils counting of the CIA group was slightly lower than that of the control group.Conclusions Basophils may participate in the development of CIA in mouse models through affecting the imbalance of Th1/Th2 response.

Abstract:Objective To define the best dose of PMSG/hCG for superovulation and optimize the biological purification system for genetic-modified mice of C57BL/6J background, we injected different dose of PMSG/hCG to promote superovulation, and carried out in vitro fertilization and embryo transplantation. Methods 5-week old female C57BL/6J mice were injected with 4 different doses(5 IU,7.5 IU,10 IU and 15 IU, respectively)of PMSG/hCG. Then the mice were mated with wild type male mice and embryos were obtained by cesarean section. In another approach, oocytes of superovulated mice were fertilized in vitro and then transplanted into surrogate female mice. Results In the cesarean section group, the highest rate of pregnancy was 89.00±19.05% in the 5 IU injection group. But the difference compared with other groups was not statistically significant. In the embryo transplantation group, the average number of oocytes from 10 IU injection group was 30.33±0.89, and the average number of 2-cell embryo was 23.78±0.19, significantly higher than those in the other three groups. Conclusions Administration of 5 IU PMSG/hCG will improve mating efficiency in cesarean section-based purification of C57BL/6J mice. 10 IU PMSG/hCG is the best dose for superovulation of C57BL/6J mice. The number of 2-cell embryos is maximized when 10 IU PMSG/hCG is used in the embryo transplantation.

Abstract:Objective To study the effects of estrogens (17α-ethynylestradiol and 17β-estradiol) on the ATP-sensitive potassium channel (K_ATP) activities in the enzymatically (collagenase and protease) isolated ICR mouse ventricular cardiomyocytes using inside-out and cell-attached patch clamp techniques. Methods In the inside-out patch configurations, the two estrogens reduced the K_ATP channel activities in a dose-dependent manner at -60 mV holding potential (HP). Results The half-maximal inhibitory concentration (IC50) of the 17α-esthynylestradiol and the 17β-estradiol were 0.3 μmol/L and 0.1 nmol/L, respectively. However, these agents had no effects on the pinacidil- and DNP-induced K_ATP channel activities in the cell-attached patch configuration. However, the inhibitory effect of K_ATP channel activities by the 17-estrogens or the anti-estrogens in the excised inside-out patch configuration were abolished by 1 pmol/L PDBu. Conclusions The results of this study demonstrate that some estrogens decrease the K_ATP channel activity acting on the inside of the isolated cardiomyocytes, and play a protective role to cardiomyocytes.

Abstract:Objective To establish a canine model of intervertebral disc extrusion by surgery and observe the histological and microcirculatory changes of the spinal cord, in order to accumulate data for studies on the pathology and mechanism of treatment for intervertebral disc extrusion. Methods Normal healthy adult dogs were divided randomly into two groups: normal control group and model group. To simulate the intervertebral disc extrusion caused by spinal cord compression, 6Fr double lumen catheter with ballon was inserted into the spinal cord T₁₂-T₁₃ and filled with about 5 mL Iohexol after the exposure of spinal cord L₁ by hemilaminectomy. The spinal cord blood flow (SCBF) at the L1 level before and after compression was measured by laser-Doppler flowmetry. Morphological changes of the compressed spinal cord at 14 days after compression was examined by histopathology.Results The (Texas spinal cord injury score) (TSCIS) scores of the motor function of bilateral hind limbs were highly significantly decreased (P<0.01). The blood flow of spinal cord at the L1 level was significantly decreased (P<0.05) after compression than that before. Compared with the normal control group, the model group showed abnormal vacuolization in the white matter and the number of normal neurons in the ventral horn of gray matter was significantly lower(P<0.01). Conclusions Our findings demonstrate that canine models of intervertebral disc extrusion can be successfully established by balloon catheter compression, showing local impairment of microcirculation and histological changes in the spinal cord. This canine model may provide a useful model for evaluation of therapeutic effects of acupuncture and for mechanism studies.

Abstract:Objective To investigate the subchronic toxicity of a Chinese medicine Danhong injection in dogs. Methods Twenty-four healthy Beagle dogs (body weigh 7-9 kg, male:female=1:1) were used in this study. The test dogs were administered with Danhong injection by intravenous injection once every day for consecutive 6 days/week for thirteen weeks. The Danhong injection was used in 3 doses: 494，247，and 124 mg·kg^-1, repectively. The control dogs received normal saline injection in the same volume. The body weight, blood biochemistry, hematology, viscera relative weight and histopathology were determined for the overall toxicity assessment. Results The dogs administered with 494 g·kg^-1 Danhong injection showed significantly reduced body weight gain, abnormal increase of urea nitrogen (BUN), creatinine (CR) levels, and histopathological changes in the liver. Conclusions Danhong injection (494mg·kg^-1) administered Intravenously has obvious toxicity on the liver and kidney function.

Abstract:Objective To investigate the numbers of corpus luteum and ovarian follicles and compare the levels of serum prolactin (PRL), luteinizing hormone (LH), follicle-stimulating hormone (FSH) and estradiol (E₂) in different phases of estrus cycle in female gerbils. Methods Consecutively taking vaginal smears of the gerbils and directly examined under light microscope to distinguish the four phases of the estrus cycle. Hematoxylin and eosin staining was used to histological examination of the gerbil ovaries, and to detect the levels of serum PRL, LH, FSH and E₂ by ELISA assay during estrus cycle. Results The proportion of cornified vaginal exfolliated cells could be the basis to distinguish four phases respectively: proestrus, oestrus, metoestrus, and dioestrus. Moreover, there were no significant differences between the numbers of ovarian follicles in different phases of estrus cycle. The numbers of corpus luteum in preoestrus were significantly lower than that in the other phases of estrus cycle (P<0.05). The levels of serum PRL and LH were increasing constantly from preoestrus to dioestrus, and both reached a peak at dioestrus (P<0.05). The levels of serum FSH and E₂ both peaked at preoestrus, and were significantly higher than those at oestrus, metoestrus and dioestrus (P<0.05). Conclusions There are no significant differences between the numbers of ovarian follicles in different phases of estrus cycle. Gonadotropin, prolactin and estradiol paly important roles in the regulation of estrous cycle. The phases during which surges of FSH and E₂ occur in Mongolian gerbils are similar to those of rats and mice, while the PRL and LH are different. Our findings provide further reference to the study of reproductive physiology of Mongolian gerbils.

Abstract:Objective To understand the histological characteristics of the major endocrine organs of tree shrew, and provide a normal histological atlas of endocrine organs of tree shrew. Methods Ten artificially fed healthy tree shrews were killed and dissected after anesthesia. The thyroid, parathyroid, adrenal and pituitary glands were observed by gross inspection and samples were taken for routine histological examination with HE staining. Results (1) The thyroid gland was pale yellow, located on both sides of the 2-4 tracheal rings. The thyroid gland was plate-shaped, its surface was covered with a thin fibrous capsule. The thyroid parenchyma was divided into several lobules by stretched capsule membrane. Follicular and parafollicular cells were distributed in the lobules, and red colloid was present in follicular cavity. (2) Each side had one parathyroid, located on the cranial or the outer surface of the middle part of the thyroid gland, and was slightly covered by thyroid. The gland was round or oval, and its parenchyma was made up of the principal cells and eosinophil cells, and acinar structure appeared in the parenchyma. (3) The adrenal glands were oval, yellow color, located in the renal hili, and linked to the kidneys. They were surrounded by a thin capsule. The parenchyma was divided into cortex and medulla. The cortex was divided into zona glomerulosa, zona fasciculata and zona reticularis from outside to inside. The zona glomerulosa was the thickest layer and the zona fasciculata was the thinnest. The medulla cells formed clumps or mesh, with central vein in the central part. (4) The pituitary gland was located in the sella turcica, with no recessus hypophysis. The pituitary gland was composed of the adenohypophysis and neurohypophysis. Its surface was covered with a connective tissue capsule. The pituitary gland was divided into distal part, middle part and pars tuberalis. neurohypophysis was made up of neural and pars infundibularis. Conclusions The histological atlas of endocrine organs in the tree shrew is established, which is close to that of the primate animals in the morphology, and provide histological evidence for the study of tree shrew endocrine organs and disorders, as well as the animal model of human diseases.

Abstract:Objective To study the extracranial scalp electroencephalography (EEG) and intracranial electrocorticography (ECoG) of closed colony New Zealand white rabbits. Methods To record the extracranial scalp EEG and intracranial ECoG of closed colony New Zealand white rabbits, and to compare and analyze the results of those two scanning methods. Results EEG was characteristic of 9-12 c/s α wave and 16-20 c/s β wave with an amplitude of 30-100 μV as the basic rhythm. ECoG showed 10-12 c/s α wave and 16-20 c/s β wave with an amplitude of 200-300 μV as the basic rhythm. Anesthesia could attenuate the electrocerebral activity, cause brain tissue hypoxia, and induce δ wave and slow θ wave in ECoG. Conclusions EEG method is a simple, non-invasive and convenient operation, and can be made in rabbits without anesthesia. The recorded EEG waveform is highly consistent with that of ECoG, and may be used as an alternative to the traditional ECoG in neurofunctional studies.

Abstract:Objective The aim of this study was to observe the anesthetic effect of xylazine hydrochloride Injection on Beagle dogs. Methods Anaesthetizing 30 healthy Beagles with xylazine hydrochloride injection, some physiological indexes of the dogs, such as body temperature (T), respiration (R), heart rate (P), and blood pressure (systolic pressure SBP, diastolic pressure DBP), were monitored. Results After using xylazine hydrochloride injection, the body temperature, respiration, heart rate, blood pressure, and peripheral vascular resistance were decreased in the Beagles. Conclusions Xylazine hydrochloride injection can keep a stable induction period, and has advantages including powerful and quick effect, simple method and operation, safe and insignificant toxicity and side effects,and has a better effect of anaesthesia.

Abstract:Objective To study the toxicity of fosthiazate feeding for 90 days in rats, and to determine the maximal non-effective dose of fosthiazate, in order to provide the reference dose for safety in production and chronic toxicity experiment. Methods A total of 80 SD rats (half female and half male) were randomly divided into 4 groups, respectively: 0.8 mg/kg·bw·d group, 4.0 mg/kg·bw·d group, 20.0 mg/kg·bw·d group, and normal control group. The rats were sacrificed to determine the indices including serum biochemical parameters, body weight, routine urine test and organ coefficients after the end of the experiment, and the results were statistically analyzed. Results In the high dose group, the body weight gain was slowed in male and female rats. The TG and CHE in the high dose group of male rats and the TP, ALB, CREA, GLU, and CHE in the high dose group of female rats were significantly lower than those of normal control group. The ALP in the high dose group of female rats was higher than that of the normal control group. The positive rates of BIL, SG, and PRO in both male and female rats had significant differences compared with those of normal control group. The organ coefficients of brain, lung, kidney, adrenal, and testis of male rats, and the organ coefficients of brain, lung, and kidney of female rats in the high dose group were significantly higher than those of the normal control group. The ovaries and uterus in the female rats of high dose group were significantly lower than those of normal control group(P<0.05，P<0.01). Conclusions The oral dose of fosthiazate at 4.0 mg/kg·bw·d fed for 90 days and above cause toxic effects on rats, and its maximal non-effect dose of long-term intake of low-dose fosthiazate on rats is 4 mg/kg·bw·d.

Abstract:Objective To improve the accuracy of detection through analyzing the phenotypes of P. pneumotropica isolates in laboratory animals in Beijing area. Methods 306 suspicious P. pneumotropica strains were identified by biochemical identification and 16S rDNA sequencing. Then, to obtain the phylogenetic relationships combined with colony characteristics on blood agar plates and biochemical characteristics of 53 biotypes. Results BD Phoenix 100 automated bacterial identification system and 16S rDNA sequencing identified P. pneumotropica positive rate of 306 isolates were 164/306 and 227/306, respectively. There were 140 phenotypes in 227 true-positive strains, of which 106 were biotype Heyl and 23 were biotype Jawetz.Conclusions In the samples of laboratory animals in Beijing area, P. pneumotropica infection mainly are of biotype Heyl, and less is of biotype Jawetz. The phenotypes are diverse and widely distributed.

Abstract:Objective To establish an indirect immunofluorescence assay for detection of murine norovirus (MNV). Methods Mouse leukaemic monocyte macrophage cell line RAW264.7 cells were infected with MNV-1 and cultured for 36 hours to collect the virus and uninfected cells, and to make antigen glass slides. BALB/c mice were gavaged with MNV-1 (10⁷TCID50) and infected sera were collected as positive control. The serum was 1:10 diluted and used for measuring MNV antibody by immunofluorescence assay (IFA). 80 serum samples were tested using the two methods, IFA and ELISA, and the discrepant samples were validated by Western blotting. Results RAW264.7 cells were infected with MNV-1 for 36-48 h, showing an infection rate of 60% of the cells, and the cells infected for 36 h were preferred. IFA method was used to detect the serum with MNV-1 infection and showed that the antibody content was gradually increased at one week after infection, reaching a maximum antibody concentration at 4 weeks after infection, and maintained a stable level later. The mouse serum at four weeks after MNV-1infection was used as positive quality control. Among the 80 serum samples, 27 positive and 53 negative cases were detected by IFA method, and 32 positive and 48 negative cases were detected by ELISA. The five discrepant samples were verified by Western blotting, resulted in 3 positive and 2 negative cases. The coincidence rate of IFA was 96.0% and that of ELISA methods was 97.5%. Conclusions Basically, immunofluorescence assay can be used to detect the MNV-1 infection in mice, although false negative result may occur occasionally. IFA and ELISA detection can be selected as initial screening measures, and use Western blot assay to verify the discrepant samples.

Abstract:Objective To establish a reverse transcription nested polymerase chain reaction (RT-nPCR ) assay for detection of tree shrews orthoreovirus (TRV). Methods Three strains of TRV were respectively isolated from fresh feces of three tree shrews that came from the same field at different times. We designed and synthesized two pairs of MRV L1 gene nested primers and established the system of RT-nPCR. The TRV RNA was extracted and reversely transcribed to cDNA as a template for nested-PCR amplification. The developed RT-nPCR was optimized. The specificity and sensitivity were tested. Finally, the RT-nPCR was used to detect TRV in 25 tree shrew samples. Results Taking the genomic RNA of TRV as template, the RT-nPCR was able to amplify a specific fragment band targeting the L1 gene, while there were no target bands in the normal cell control, (Wa strain rotavirus, hepatitis A virus, and herpes simplex virus). The RNA of TRV was diluted by 1:10 to 1:10⁹. Each dilution sample was analyzed by the RT-nPCR. The minimum detectable concentration of RNA was 0.01 pg/μL. The results of RT-nPCR detection showed that 4 of the 15 tree shrews were TRV-positive in the survival group, and 10 of 10 tree shrews were TRV-positive in the death group. Conclusions The RT-nRCR assay established in this study is accurate, specific and sensitive. Therefore, it can be used for routine detection of TRV in quality assurance testing.

Abstract:Objective To establish an effective PCR assay for detection of Bartonella, and application of this assay in tree shrew. Methods Sequence of Bartonella was obtained from NCBI Genbank. Three pairs of primers were designed based on this sequence. One pair of primers was determined through amplifying the major strains in China. Sixty tree shrew blood samples were tested with this PCR assay. The positive amplified fragments were sequenced to verify the reliability of this method. Results A PCR method for detection of Bartonella is successfully established, with a high specificity and the sensitivity was of 2.0×10^-5 μg/mL. Among the tested 60 blood samples, 15 positive cases were detected. Sequencing of the samples confirmed a 25% infection rate of Bartonella in the tree shrews, well consistent with the amplification results, and verified the applicability of this detection method. Conclusions The establishment of this method provides the basis for detection of Bartonella in tree shrew.

Abstract:The present situation, efficacy, problem and strategy of laboratory animal license management in Guangdong Province were reviewed in this article. The laboratory animal license management has been implemented for 10 years and showed good results. Now there are comprehensive management of quality, facilities, personnel, SOP, and unified item bank, and making standard of laboratory animals in Guangdong Province. The legislation of Guangdong laboratory animal management regulations is completed. Guangdong laboratory animal license is increased year by year. The distribution of industry and area is expanded. Through analysis of problems and search for strategy, Guangdong laboratory animal management will be further developed.

Abstract:Objective To explore the use of integrated two methods in vitro in prediction of eye irritation caused by cosmetics.Methods Chorioallantoic membrane vascular assay (CAMVA), bovine corneal opacity and permeability (BCOP) and Draize rabbit eye irritation test were used to determine the predictive potential of eye irritation of 60 kinds of cosmetics. Results CAMVA method was able to distinguish 41 non-irritant samples and 18 irritant samples. BCOP method was able to predict 35 non-irritant samples, 21 mild-moderate irritant samples and 4 severe irritant samples. Combination of CAMVA and BCOP methods could obviously improve the identification ability of irritation, and the classification consistency with Draize rabbit eye irritation testing reached 98.3%. Conclusions The integrated test strategy combined BCOP with CAMVA can be used to appropriately predict ocular irritation of cosmetics, with a prediction range covering non-irritant to severe irritant samples.