Abstract:ObjrctivePurity of laboratory animals plays a critical role affecting the experimental results. The aim of this study was to validate a high-throughput genotyping platform, valuable in inbred mouse genetic DNA monitoring and strain identification. Methods Multiple polymerase chain reaction and ligase detection reaction (PCR-LDR) genotyping panels were constructed. Forty-five single-nucleotide polymorphism (SNP) on 21 chromosomes were selected as targets, as well as specific primers and probes to these SNP were designed, respectively. A multiple PCR-LDR genotyping protocol was established. ResultsForty-five SNP were successfully genotyped in four panels. The positive detection rate of 3,4 and 45 SNPs were 100%, 90.9% and 36.4%, respectively. All samples collected were homozygous and their genotypes were identified by PCR-LDR. Conclusion The results of this study provide a rapid and high-throughput genotyping approach, which is sufficient for genetic contamination monitoring and strain identification.

Abstract:ObjectiveTo apply reward directed operant conditioning task for learning and memory research in laboratory animals. Methods Sixteen adult male SHR rats and 8 Wistar rats were included in this study. Operant conditioning procedure was used to assess the learning ability of Wistar and SHR rats with magazine training, fixed ratio, signal discrimination and extinction procedure. ResultsSignificant difference was found in magazine training. The SHR rats performed more NP vs. Wistar rats and SHR rats treated with donepezil (P<0.05). There was no significant difference during FR1 schedule. While in higher response ratio FR2 and FR4 schedule, the SHR group showed significantly higher error responses (vs. Wistar rat group, P<0.05), lower rewards eared (vs. Donepezil-treated SHR rat group, P<0.001), and significantly lower correct accuracy rate (vs. Wistar rats and Donepezil-treated SHR rats, P<0.05). Significantly higher error responses were found during signal discrimination task (vs. Wistar rats and Donepezil-treated SHR rats, P <0.05), and lower correct accuracy rate (vs. Wistar rats and Donepezil-treated SHR rats, P<0.05). No significant difference was found during extinction. Neurotransmitter test showed that the hippocampal content of glutamate [(1.0639±0.07086) mg/g], acetylcholine [(2.7760±0.2609)μg/g] and 5-HT [(1.2200±0.1137)μg/g] were significantly lower in the SHR group (vs. Wistar rat group, P<0.05). The content of acetylcholine [(3.9344±0.2747)μg/g] was higher in the Donepezil-treated SHR rats group (vs. Wistar rats,P<0.05；vs. SHR rats, P<0.001). Conclusions Reward directed operant conditioning is a type of positive reinforced conditioning. This task can be used as a new behavior test method for learning and memory study.

Abstract:Objective To examine the effect of different doses of ovalbumin (OVA) on the establishment of an asthmatic mouse model. Methods Ninety-six female specific pathogen-free(SPF)mice in the age of 6 to 8 weeks, were randomly divided into 8 groups:PBS (Controls), 10 μg (A), 20 μg (B), 50 μg (C), 100 μg (D), 200 μg (E), 500 μg (F), and 1000 μg (G) OVA groups. Increasing doses of OVA solution dispensed in PBS containing 1% alum was intraperitoneally injected into the mice exposed to the allergen on days 0,7 and 14, respectively, and the mice were challenged with aerosol inhalation of PBS containing 1% OVA for 7 consecutive days from the 21st to 27th day of intervention. PBS instead of OVA was used for sensitizing and challenging the mice in the control group. Twenty-four hours after the final inhalation and challenge, all the mice were sacrificed for determining the concentration of eosinophils in the bronchoalveolar lavage fluid (BALF), secretory contents of cytokine IL-4 and IL-5, and serum level of antibody IgG2a and IgE by enzyme linked immunosorbent assay (ELISA). Lung tissue samples were taken for pathological assessment of the asthmatic changes and to determine the optimal OVA dose for asthma modeling. ResultsAll the levels of IL-4 and IL-5 in the groups A to G were significantly higher than that of the controls (P<0.01). The level of cytokines was decreased with increasing OVA doses. The eosinophil counts were significantly higher in the groups A to G as compared with that of the control group (P<0.01), and the eosinophil counts were in inverse proportion to the dosage from low to high administration of OVA. Overall serum levels of antibody IgE were significantly elevated in the mice exposed to the allergen in proportion to the controls (P<0.01), and IgE levels were decreased with increasing doses of OVA. The IgG2a titer was increased with increasing doses of OVA. Inflammatory cell infiltration was observed evidently in the pulmonary tissues of the mice exposed to low dose of OVA, yet the pathological changes were not so distinct in the mice receiving high doses of OVA. ConclusionsLow dose of OVA by progressive exposure may result in significant pathological changes in mice with allergic asthma, and that the quality of the established animal models is declined with increasing dosage of OVA. High doses of OVA can lead to immune tolerance in the sensitization protocols.

Abstract:Objective To establish a neonatal rat model of global brain hypoxic-ischemic injury. Methods Timed pregnant Sprague-Dawley rat dams were decapitated on the expected day of delivery (gestation day 21), hemostats were used to occlude the four vessels of cornu uteri for five minutes, then the pups were delivered by cesarean section and introduced to the surrogate dams for cross foster. Results7 rat pups died within 3 days after born in 91 rat pups delivered by Delayed C-section (death rate was 7.7%). The neuroethology of rat pups were evaluated by behavioral tests such as righting reflex test (P2d), trapeze test and inclined plane test (P14d). At the last, 21-day-old rat pups were killed by decapitation and the brains collected for HE staining. Delayed C-section group was suggested to have significant difference with the control groups on both behavioral tests and HE staining pictures. Conclusion Delayed C-section delivery with cross fostered by surrogate dams can be an easy method to make a neonatal rat model of global brain hypoxic-ischemic damage for long-term experiments.

Abstract:Objective To study the characteristics of expression of TRIF (TIR-domain-containing adapter-inducing interferon-β, TRIF) innate immune signaling factors in hepatic cells and its protein expression pattern in hepatic fibrosis in rats, and to explore the relationship between the important signal downstream of TOLL-like receptor TRIF and pathogenetic mechanism of hepatic fibrosis. Methods To generate rat models of hepatic fibrosis by subcutaneous injection of carbon tetrachloride together with low protein and high fat diet and alcohol water drinking. Thirty two SD rats were randomly divided into control group and model group. Intracardiac saline and formaldehyde fixative perfusion was performed in some rats and liver tissue samples were taken for histopathological and immunohistochemical examination. Other rats were killed by direct decapitation and fresh liver tissue samples were taken for electron microscopic examination and Western blot assay. Results The HE staining showed that normal rat liver displaying regular lobular structure without inflammatory cell infiltration, but the model rat livers showed distinct disordered lobular structure, decreased amount of hepatocytes and the presence of hepatic fobrosis. Electron microscopic observation revealed deposition of collagen fibers and cytolysis of hepatocytes, karyolysis of Kuppfer cells, cytolysis of sinusoidal endothelial cells and a large number of abnormal cells infiltration. Immunohistochemistry showed a high expression of TRIF in endothelial and stellate cells, mainly, in the cell nuclei, but also could be seen in the cytoplasm. Compared with the normal control group, the TRIF protein expression was significantly increased in the model rat livers (P<0.01), consistent with that observed by histopathology. ConclusionsTRIF expression is significantly increased in liver fibrosis, indicating an apparent activation of Toll-like receptor and a series of immunological responses via the downstream signal transduction pathways, producing a series of in vivo immune responses. It is preliminarily confirmed that the innate immune signal factor TRIF plays an important role in the pathogenesis of hepatic fibrosis.

Abstract:ObjectiveTo establish a mouse infection model for evaluating the virulence of Vibrio parahaemolyticus. Methods 4-5-week old female Balb/c mice were monitored for symptoms and lethality after being infected intraperitoneally with Vibrio parahaemolyticus bacterial suspension. ResultsThe survival rate of mice after i.p. infection with a highly virulent RIMD2210633 strain (10⁷CFU) cultivated in a high salt medium (2% NaCl) was 20-30%, while that with environmental non-virulent S251 strain was 100%. ConclusionsWe have successfully established a mouse infection model using the RIMD2210633 strain. This mouse model is suitable for evaluating the Vibrio parahaemolyticus virulence, and is used to compare the virulence of highly virulent and environmental non-virulent strains cultured in medium at different salt concentrations.

Abstract:Objective To study the effects of blockade of CD40-CD40L pathway on the survival of xenogeneic skin graft. Methods The extracellular domain of human CD40 ligand (CD40L) was cloned by RT-PCR, and thereby a skin-specific transgene expression vector for the soluble CD40L molecule (sCD40L) was constructed by keratine 14 (K14) promoter. Using the sCD40L skin-specific transgene construct, transgenic mice were generated by pronuclear microinjection. ResultsThe sCD40L fragment with expected size and sequence was correctly cloned and its skin-specific expression vector correctly constructed. By microinjection, embryo transfer, and PCR screening test, one of 49 G0 mice had amplified specific bands, which was absent in the negative controls. ConclusionWe have successfully established a sCD40L transgenic positive mouse model.

Abstract:ObjectiveTo observe the ultrastructural changes in retinal neurons in APP transgenic mice. MethodsWe make comparison between six 10-month-old APP transgenic mice and 6 non-transgenic female mice of the same background and age. All mice were perfusion sacrificed. The whole right eyes were taken and processed for electron microscopic observation of changes in the retinal neurons. ResultsCompared with the control group, the APP transgenic mice group showed evidently pathological changes in neurons of each retina layers. The outer segment structure of cones and rods was blurring, there was cell pyknosis in the outer nuclear layer, cells were dried-up, chromatin was condensed in nuclear layer cells, ganglion cells showed disintegrated plasma membrane, cytoplasmic vacuoles and degenerative organelles, and mitochodria were severely swollen. ConclusionsThere are obviously pathological changes in the retinal neurons in APP transgenic mice.

Abstract:ObjectiveTo study the effect of collagen cross-linking induced by riboflavin and ultraviolet A (UVA) on biomechanical properties of the sclera in guinea pigs. Methods The changes of biomechanical properties of the sclera induced by riboflavin and ultraviolet A (UVA)-induced collagen crosslinking were examined in ten guinea pigs and compared with those of ten non-treated guinea pigs. Histological and ultrastructural changes of the sclera were examined to evaluate the side-effects. ResultsAt one month after the UVA plus riboflavin treatment, the ultimate stress increased by 147%, elastic modulus increased by 193%, ultimate strain reduced by 21.9% in the equatorial sclera and significantly changed by 108%, 191%, 40.42% in the posterior sclera, respectively. Light microscopy showed no pathological alterations. Transmission electron microscopy showed active hyperplasia of scleral fibroblasts in the crosslinking area of the sclera in the guinea pigs of the experimental group. ConclusionsThe method using UVA plus riboflavin to induce scleral collagen cross-linking shows effect to improve the biomechanical properties and to enhance the tension and strength of the sclera. Collagen cross-linking may become a new approach of clinical treatment for pathologic myopia.

Abstract:ObjectiveTo investigate the effectiveness and feasibility of a method to establish a guinea pig model of myopia induced by flickering light (FL) stimulation, and further observe the abnormal changes in this process. Methods Twenty-four 2-week-old guinea pigs were randomly assigned to FL group, non-flicker indoor light group, and daylight group (n=8 for each). Animals in the FL group were raised under 600 lux illumination with a duty cycle of 50% at a flash rate of 0.5 Hz. Animals in the non-flicker light group and normal group were reared under 600 lux illumination and natural daylight, respectively. Refraction, axial length, and radius of curvature were measured before and after 2,4, 6,8, 0,2 weeks treatment. After the collection of fundus photographs,histopathological changes were examined by light and electron microscopy. ResultsThe FL group developed rapidly towards myopia throughout experimental period.Significant difference was noted for refraction in all the 3 groups at 12th week, compared with the non-flicker light group. Eyes in the FL group were approximately -5.4±1.5 D more myopic with an increase in axial length by 0.74±0.18 mm. Compared with the daylight group, eyes in the FL group were approximately -6.6±1.5 D more myopic with an increase in axial length by 0.86±0.24 mm. As to the FL group, tessellated fundus appeared more prevalent, and the photoreceptor layer was more likely to develop a disordered outer segment, which contained much deciduous disc membranes. ConclusionsGuinea pigs develop accelerated ocular growth and refractive myopic changes by flickering light. Such abnormal light perception ultimately influences the development of retinal photoreceptor cells.

Abstract:ObjectiveTo compare three induction methods of VX2 carcinoma in rabbits. MethodsThirty New Zealand rabbits were randomly divided into 3 groups:impaction embedding group, modified impaction embedding group and percutaneous puncture implantation group. The VX2 models were established separately and CT evaluation was carried out at 1,2, and 3 weeks after tumor implantation. All the animals were sacrificed and tissues were harvested 3 weeks after implantation to compare the success rate and the metastasis rate in the abdominal wall and other organs. ResultsThe success rate was 93% (28/30) and both the two failed cases were in the percutaneous puncture implantation group. Relative to the other groups, the abdominal wall metastasis rate of the modified impaction embedding group is obviously lower. ConclusionsThe modified impaction embedding technique is a promising novel technique in preparation of VX2 hepatoma in rabbits with a high success rate and low abdominal wall metastasis rate.

Abstract:ObjectiveTo established a mouse model of multi-drug resistant (MDR) colon carcinoma and initially explore the mechanism of drug resistance.MethodsThe mouse models were generated by subcutaneous inoculation of drug-sensitive colon cancer HCT-8 cells and drug-resistant colon cancer HCT-8/VCR cells in mice. Their drug-resistance properties were assessed by vincristine (VCR) and cyclophosphamide (CTX) in vivo. The expression levels of multi-drug resistance gene 1 (P-gp/MDR1) and multi-drug resistance-associated protein 1 (MRP1) were detected by real-time PCR and Western blotting in tumor tissues of the mouse models.ResultsThe speed of tumor growth was not significantly different between the MDR and sensitive mouse models. There was a more intense drug-resistance in the MDR mouse models than in the sensitive mouse models. Real-time PCR and Western blotting showed that the expression level of P-gp/MDR1 in the MDR mouse models was enhanced, but the MRP1 expression did not show significant differences. ConclusionsA mouse model of MDR colon carcinoma has been successfully established. The mechanism of MDR in the mouse models is related to the overexpression of P-gp/MDR1 gene. 

Abstract:ObjectiveTo investigate the presence of Bartonella in rhesus monkeys and to analyze the gltA gene sequence.MethodsBartonella were isolated from 16 rhesus monkeys coming from Fujian province, isolated with Guy''s culture-medium. According to the reported complete nucleotide sequence of gltA gene in NCBI, one pairs of primers were designed and synthesized. The gltA gene was amplified from the lysate of Bartonella colony by PCR. Then the bands were cloned and sequenced. ResultsBartonella pathogen was successfully isolated from 3 rhesus monkeys. The sequencing of gltA showed that the isolated Bartonella belonged to Bartonella quintana, with a homology of 98%.ConclusionBartonella pathogen is existed in rhesus mokey coming from Fujian province. The prevalence and spreading of Bartonella infection may happen between mouse and primate in the epidemic area.

Abstract:ObjectiveTo investigate the effects of forsythoside (FS) on immune regulatory mechanism of proliferation and secretion of NO and TNF-α of mouse spleen T and B lymphocytes in vitro. MethodsMouse spleen and splenocytes were prepared by aseptic separation and the cells were suspended with RPMI 1640 and cultured in RPMI 1640 containing 10% fetal bovine serum in vitro and stimulant concanavalin (ConA) and lipopolysaccharide (LPS). Different concentrations of 0,0 and 160 μg/mL FS were added separately in culture medium at different times. MTT was used to assay the changes of optical absorbance of T and B lymphocytes. ELISA and Griess methods were used to detect the TNF-α and NO levels. ResultsProliferation and survival rate of ConA-induced T lymphocytes treated with FS at low and medium concentration were significantly increased at 24 h and 48 h, and cell transformation was inhibited after FS induction for 72 h. LPS-induced splenic B cell proliferation and survival were significantly improved by FS at low concentration after 24 h. NO secretion of T and B lymphocytes was enhanced by FS at low and medium concentrations. TNF-α secretion of FS-treated B and T lymphocytes treated with FS at medium concentration was promoted, but TNF-α secretion of T lymphocytes was inhibited by FS at high concentration. In addition, FS had a significant effect on proliferation of splenic lymphocytes in vitro from mouse injected with cyclophosphamide, but no significant effect on the lymphocyte NO secretion. ConclusionOur findings suggest that FS may regulate immune cell function via lymphocyte proliferation and cytokine secretion.

Abstract:ObjectiveTo investigate the effects of different surgical procedures on the prognosis of experimental animals with VX2 tumor invading the inferior vena cava (IVC). Methods1. A rabbit model of VX2 tumor invading IVC was established and confirmed by ultrasound examination. 2. The rabbits were randomly divided into three groups, 12 rabbits in each group:Group A had complete excision of the tumor and IVC, group B had complete excision of the IVC and tumor followed by IVC reconstruction with an artificial vascular graft, and group C had separation and removal of the tumor. All the removed IVC were sent for pathological examination. The rabbits had ultrasound examination of the IVC. The collateral circulation and IVC blood flow was observed by digital subtraction angiography (DSA) after the IVC blockage. The natural death time of the rabbits was observed and autopsy was performed. Results1. Ultrasound examination showed that all VX2 tumors grew successfully in the animal models (2.75±0.91 cm²). The lumen of the IVC was not narrowed (diameter 2.53±0.23 mm). 2. DSA showed that the medium and long term patency of the artificial grafts in replacement of infrarenal IVC was low. In the experimental group, communicating branches were gradually enlarged and superficial veins in the abdominal wall were gradually dilated after the surgery. 3. The experimental groups had a longer survival time than the control group (P1<0.001<0.05, P2=0.036<0.05). The group A had a significantly longer survival time than the group B (P3=0.04<0.05).There was no significant difference in distant metastasis between the groups A and B,but significantly lower than that in the control group. ConclusionsThere is a better survival time and survival rate in the rabbits after complete resection of the tumor invading the inferior vena cava. Rabbits with artificial IVC reconstruction have a low medium and long term patency, a higher perioperative mortality, and a shorter survival time than the animals without IVC reconstruction.

Abstract:Objective To establish a mouse model of lung adenocarcinoma induced by N-ethyl-N-nitrosourea (ENU) in mice and to characterize its tumor pathology. Methods BABL/c mice were used to establish the lung cancer model. On the 17^th day of pregnancy, the parental female mice received intraperitoneally ENU or buffer injection, respectively. The 32-week old offsprings were sacrificed and entire lungs were harvested and processed for histopathological observation of HE stained semiserial sections. The pathological features of the lung tumors were assessed. ResultsMultiple tumors in offspring''s lungs were induced transplacentally by a single injection of ENU. The tumors were at different development stages of adenoma or adenocarcinoma. The adenocarcinomas were classified as bronchioloalveolar-like adenocarcinoma (female:5/6; male:4/6) and adenocarcinoma of various differentiation (female:4/6; male:5/6). The frequency of cancer development was 5 out of 6 mice in both sexes. The frequency of tumors which developed into malignant tumors was 16 out of 43 tumors in female and 12 out of 31 tumors in male mice. ConclusionsWe have successfully established a mouse model of lung adenocarcinoma in mouse offsprings by a single injection of ENU in pregnant female mice. The heterogeneous presentation of tumor pathology indicates the complex molecular mechanisms of its carcinogenesis.

Abstract:ObjectiveThe aim of this study was to explore the effects over time of lactic acid (LA) on IκBα phosphorylation and expressions of nuclear factor-kappa B p65 protein, tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) mRNA in the NF-κB signaling pathway in rat intestinal mucosal microvascular endothelial cells stimulated by lipopolysaccharide (LPS), and then to assess the optimal action time of LA and its regulating site in the NF-κB signaling pathways.MethodsIκBα, phosphorylated IκBα (p-IκBα) and NF-κB p65 proteins were monitored by Western blot analysis, and the TNF-α and IL-6 mRNA was analyzed using real-time PCR. ResultsLA treatment reduced TNF-α and IL-6 mRNA expression in LPS-stimulated RIMMVECs, with the greatest effect after 24 h and 3 h. The highest inhibitory effect of LA on IκBα phosphorylation and NF-κB transcriptional activity was during 4 to 8 h. The site of action of LA was IκBα phosphorylation in the signaling pathways. ConclusionsThe results of this study suggest that LA inhibits downstream inflammatory cytokine expression through inhibiting IκBα phosphorylation and blocking activation of NF-κB, thus playing a key anti-inflammatory role in the intestinal mucosal microvascular endothelial cells in rats.

Abstract:ObjectiveTo investigate the effect of micro-plasma radio-frequency on the ultrastructure of skin collagen and the level of hydroxyproline in ginea pigs.MethodsFifteen guinea pigs were enrolled in this study. One side of their dorsal skin was exposed to 60 W/10 kJ radiofrequency plasma treatment and the other side was taken as control. Samples were taken immediately, one week and one month after the micro-plasma treatment and examined by light and transmission electron microscopy (TEM), and the level of hydroxyproline content by hydroxyproline assay kit. Results Immediately after the 60 W/10 kJ plasma treatment, the epidermis showed focal lesions, obviously homogenization and complete vaporization loss or degeneration and necrosis of the superficial and middle-level layer derma, and a large area of collagen homogenization. Special staining demonstrated that the micro-plasma beam mainly affected the dermal collagen fibers, forming focal collagen fiber agglutination and degeneration. One week after the micro-plasma treatment, the superficial dermal collagen fibers gradually became into an orderly dense arrangement. One month after the micro-plasma treatment, the superficial collagen fibers showed markedly compact and dense arrangement. TEM showed that the epidermal cells were relatively complete, with normal intercellular structures. But the dermal collagen fibers lost their original normal structure and dermal cells showed disintegration and numerous apoptosis. A small amount of apoptosis remained but collagen structure gradually restored after one month. The level of hydroxyproline after one week was higher than that of before plasma treatment, but the difference was not significant (P>0.05). The level of hydroxyproline after one month was significantly higher than that before plasma treatment, with a statistically significant difference (P<0.05). ConclusionsThe novel micro-plasma radio-frequency technique has obvious stimulating effect on skin collagen. Its main tissue target is dermal collagen tissue. It can stimulate neo-collagen hyperplasia in the skin to a certain degree.