Abstract:Objective To establish the CTRP4 transgenic mouse model and investigate the function of the novel adipocytokine CTRP4. Methods CTRP4 overexpressing vector in pCAGGS was firstly constructed and then microinjected into zygote to establish the founder transgenic mice. F1 heterozygotes were generated by founder mice mating with wildtype mice, and the CTRP4 transgenic homozygotes were generated by F1 littermates. The genotype was confirmed by PCR and test cross method. The expression level of CTRP4 in transgenic mice was detected by western blot. Result The human CTRP4 transgenic homozygote mice line was established, and the expression level of CTRP4 was confirmed raletively high in detected tissues including heart, liver, brain and kidney. Conclusion The human CTRP4 transgenic mice was successfully established.

Abstract:Objective To construct Cchl1a3 gene R528H knock-in mouse model related to hypokalemic periodic paralysis.Methods ES cells were transfected with Cchl1a3-Konckin targeting vector linearized by Not I digestion, selected in the medium containing both G418 and ganciclovoir.Resistant clones were screened by PCR and further confirmed by DNA sequencing for correct homologous recombinants.Chimera mice were obtained by routing microinjection of homologous recombined ES cells into blastocysts. Heterozygous mice were obtained by mating. Through heterozygous mice with FLP mice mating, removal of neo gene heterozygous mice were established and identified with the PCR and DNA sequencing. After mating, homozygous offspring were constructed and observed.Results ES cells were successfully transfected with targeting vector. It was confirmed that 9 resistant clones happened right homologous recombination by PCR and DNA sequencing. 7 chimera mice were obtained by microinjection. After breeding the chimeric mice, heterozygous mice were mated FLP mice to obtain 9 heterozygous mice removal of neo gene, the finally obtained 15 homozygous mice with Flp-deleted neo gene. In the developmental stage of sexual maturity, the spirit of the mice, restaurants and activities in good condition, but the gradual emergence of hair removal at 4 months of age, skin ulceration and even death. Conclusions We successfully constructed Cchl1a3 gene R528H mutation homozygous mice. And it laid a foundation for the study of human CACNA1S gene function and to clarify the molecular mechanism of hypokalemic periodic paralysis.

Abstract:Objective To evaluate the anatomic localization and size of acute necrotic myocardium in the ischemic-reperfused rat hearts using ^99mTC-Glucarate and microSPECT/CT. Methods The ischemic-reperfused (IR) rat heart models were established by ligating left anterior descending coronary artery for 60 min. ^99mTC-Glucarate was intravenously injected into the rats 24 hours after IR operations. Images were acquired 30 min after administration of ^99mTC-Glucarate using microSPECT/CT. Anatomic localization and size of acute necrotic myocardium were analyzed with microSPECT/CT imaging,and these results were compared to those determined by triphenyltetrazolium chloride (TTC) staining. Results The microSPECT/CT images showed hot spot accumulations of ^99mTC-Glucarate in IR hearts (the heart-to-liver ratio was 1.90±0.33), not in controls (P < 0.05). The anatomic localization of ^99mTC-Glucarate-labeled necrotic myocardium were in correspondence with TTC staining results. The hot spot size was related significantly to necrotic myocardial size determined by TTC staining (R²=0.964). Conclusions The localization and size of acute necrotic myocardium can be assessed by non-invasive microSPECT/CT imaging with ^99mTc-Glucarate.

Abstract:Objective To provide a good evaluation for research the hyperthyroidism drugs, two kinds of hyperthyroidism animal models were copied, and compared the similarities and differences of their formability and sustainability. Methods Euthyrox-induced model: The rat was taken Euthyrox (50μg·100g^-1 rat weight) for 21 d. And then the blood and urine were collected in 1 d, 7 d and 14 d after last time oral administration of Euthyrox;Yersiniaenterocolitica-induced model: the rats were injected Yersiniaenterocolitica(YE) in 0、5、10、15、20 (IOCV). And then the blood and urine were collected in 5d, 15d and 25d after last IOCV of YE. Results Euthyrox-induced model: T3, T4, FT3, FT4 were increased significantly, but returned normal 1 week later in the model group, PCA showed that model group deviated from the blank group, but recovered 2 weeks later; Yersiniaenterocolitica-induced model: T3 and T4 were increased significantly in 15 and 25 d, TSH significant raised in 25 d, the model group deviated from the blank group in PCA, and didn't return to the blank group in 15d or 25d. Conclusion Euthyrox-induced modelhas good formability but recovered quickly, which is suitable for prevention of drug delivery on efficacy evaluation; Yersiniaenterocolitica-induced model with good formability and sustainability could be used for treatment of drug delivery on efficacy evaluation.

Abstract:Objective To establish a mouse's cross-territory ear flap that enables chronic, in-vivo observation of the change of vascular morphology. Methods 30 ICR mice, weighing 25～40 g, were used for this study. Commercial depilatory cream was used to first remove the hair of the mice, after which the vascular pattern in the ears was investigated. According to the observation of the vascular pattern in the mouse's ear, the eye scissors were used the sever the outer 2/3 of the base of the ear, in which process a ear's flap based on a vascular pedicle but crossed three vascular territories was created. After the creation of the flap, the mice were placed on an automatic controlled movable machine with the ear's flaps spread over a customized Plexiglas. Then the flaps were photographed under the stereoscope (×25) at the following time points: 1,2,3,5,7,10,14,21,30 d. the necrosis of the flap, and the morphological change of the vessels within the flap were analyzed. Results The ICR mouse's ear was supplied three angiosomes, which were respectively named as the cephalic, median and caudal angiosomes from inside out. Five days after the flap's creating, necrotic rate of(15±7)% was developed. The choke vessels between the medial and median angiosomes expanded rapidly in diameter, reaching the plateau 10d after flap creation, resulting the dilated choke veins and arteries at their peak being 3.9±0.5 and 3.5±0.7, respectively, than their initial sizes. The diameter of the choke veins began to shrink at approximately 10d, stabilizing after 21d. The diameter of the choke arteries plateaued and stabilized at around 10d. Conclusion ① after harvest of extended flap, the dilation of veins seemed to passive, whereas the dilation of arteries seemed to active; ② the number of the choke vessels between the dynamic and potential territories that are involved in dilation and extent of the dilation are much smaller than that of the choke vessels between the anatomic and dynamic territories;③ the mouse's ear flap is an excellent model of further study of mechanism underlining the dilation of choke vessels and for the screening of vasoactive drugs that augment the survival of the large flap.

Abstract:Objective To measure the circadian rhythm of consecutive monitoring electrocardiogram(ECG), blood pressure, respiration, activities in Bama minipigs by telemetry technology. Methods Six 6-month-old Bama minipigs were implanted with VAP port in Superficial femoral artery, after recovery 7 days, 24 hours ECG, blood pressure, activity and respiratory parameters were recorded and analyzed in the EMAK noninvasive telemetry system. Results The ECG, blood pressure, respiration and activities in Bama minipigs showed circadian rhythm changes, Bama minipigs daytime heart rate was significantly higher than the nighttime heart rate (P< 0.01), and daytime PR interval, QRS interval and QT interval was significantly lower than the nighttime (P< 0.05, P< 0.01), daytime mean heart rate was 76.22 beats/min, nighttime mean heart rate was 67.03 night beats/min; daytime mean PR interval, QRS interval and QT interval were 109.97 ms, 42.72 ms and 380.37 ms, nighttime mean PR interval, QRS interval and QT interval were 112.32ms, 44.01ms and 389.24ms. Bama minipigs daytime systolic blood pressure, diastolic blood pressure and mean blood pressure were significantly higher than the nighttime (P< 0.01), daytime mean systolic blood pressure, diastolic blood pressure and mean blood pressure were 129.57 mmHg, 96.75 mmHg and 111.73 mmHg, nighttime mean systolic blood pressure, diastolic blood pressure, mean blood pressure were 122.81 mmHg, 92.65 mmHg and 106.19 mmHg, and the nighttime reduction rate of systolic blood pressure, diastolic blood pressure and mean blood pressure were 19.89%, 19.05% and 19.35%. In addition, Bama minipigs daytime activity and respiratory rate were significantly higher than the nighttime (P< 0.01). Conclusion It's Feasible to measure the circadian rhythm of electrocardiogram(ECG), blood pressure, activity and respiratory parameters in Bama minipigs by telemetry technology, and can truly shows those physiological parameters of the Bama minipigs within 24h, to provide a reference for pharmacology and toxicology research by using Bama minipigs.

Abstract:Objective To define dynamic changes of viral loads and antibody responses in ICR mice naturally infected with hepatitis virus in an MHV contaminative facility. Method A total 50 ICR were housed by different "dirty bedding" exposure.Antigen and antibody was detected after 2，4，8，14，21，28，35，42，56 and 84 days. Result Mouse hepatitis virus (MHV) was detected in lung after 2 days, and positive rate is 20% (1/5). MHV was detected in lung, liver, cecal and feces during 4 and 56 days. The positive rate was 0/5 in lung, liver, cecal and feces on 84 days after experiments. Antibody positive rates were 100% during 8 and 84 days. Conclusion Serological method can be used as the main method for the diagnosis of the daily supervision, and antigen detection method can only be applied to early diagnosis.

Abstract:Objective To study the effect of low-level 84 disinfection solution on reproductive physiology of mice. Methods 72 KM mice were randomly divided into three groups: 0.5% 84 disinfection solution group (group A), 2.0% 84 disinfection solution group (group B) and saline solution group (group C). 12 males and 12 females in each group. Corresponding medicine was given once a day by intragastric gavage for 9 weeks. The estrous cycle of female mice was observed by exfoliative cytology of vagina. After 9 weeks, The sperm abnormality test was performed, Serum samples were taken for sex hormone test by ELISA, and the histological change of testis (or ovary), heart, liver, lung and kidney were observed. Results Compared with group C, No significant difference was observed in the weight variation and serum sex hormone level in group A and B (P> 0.05). The difference of the estrous cycle of female mice was not significant (P> 0.05). No histopathological abnormality was observed. Compared with group C, There was no significant difference in the rate of sperm abnormality in group A (P> 0.05), but the rate in group B was significantly increased (P<0.05). Conclusion 0.5% 84 disinfection solution has no significant effect on reproductive physiology of mice, The concentration of disinfection solution must be strictly controlled when used.

Abstract:Objective To establish a simple, fast and economic total DNA extraction method to serve the rapid identification of model mouse genotype in large number of mice. Methods Three methods, i.e. phenol extraction, isopropyl alcohol precipitation and mouse ear boiling methods were used to extract the total DNA from ten C57-rasmodel mice. The purity and yield of DNA obtained by the three methods were compared, and polymerase chain reaction (PCR) assay was used to compare the efficacy of the three extraction methods. Results Among the three methods, phenol extraction was the best and isopropyl alcohol precipitation was the poorest in DNA yield. In terms of DNA purity, the phenol extraction was the best and the mouse ear boiling method was the poorest. All the three methods could be used to extract the total DNA from mice serving as template of PCR reaction for the mouse genotype identification. The time consumption of three methods are 12.5 hr,13 hr and 0.18 hr. Mouse ear boiling method was significantly lower than that of the other two methods (P< 0.01 ),.The obtained total DNA can be stored at conventional-20℃ for 7 days and 30 days later still can be used as a template for PCR reaction. Conclusions Among the three methods studied, the mouse ear boiling method is simple and with the lowest cost, so it is feasible for total DNA extraction in scaled genotyping experiments.

Abstract:Objective To diagnosis tumor transplanted nude mice Strongyloidiasis. Methods Postmortem microscopic examination of the tumor transplanted nude mice detected Strongyloides stercoralis for morphological identification and double polymerase chain reaction (PCR) assay for molecular diagnosis of Strongyloides stercoralis infection in tumor transplanted nude mice. Results Presence of numerous S. stercoralis in autopsy in tumor transplanted nude mice samples preliminary determined the diagnosis of strongyloidiasis. Confirmed diagnosis of Strongyloides stercoralis infection by double PCR detection of specific DNA in tumor transplanted nude mice samples. Conclusion The most important clue to prevent such serious consequences is early diagnosis. Tumor transplanted recipients and donors should be screened for parasitic infections including strongyloidiasis. To the authors' knowledge, this study is the first extensive report on diagnosis tumor transplanted nude mice Strongyloidiasis.

Abstract:Objective To insure the health of experiment mice, since 2009 our lab tool laboratory rodents from Beijing area to investigate the health status of laboratory rodents. We mainly use the pathologic method to investigate experiment mice. Methods The specimens we took from the experimental animals were mainly hearts, livers, spleens, lungs, kidneys, small intestines and large intestines. Specimens were immediately fixed by formalin. HE staining sections, PAS staining sections, oil red "O" staining frozen sections were observed under light microscope. Results According to the pathological examination there are different degree of pathological changes among the organs of laboratory rodents, such as hepatic and kidney. Conclusion Combined with the result of microbial detection, the laboratory rodents are not infectious with Pathogenic microorganism, such as bacteria, virus and parasite. And they have no harm to human beings, other animals and the surrounding environment. But small parts of laboratory rodents show certain extents pathological anomaly in clinically.

Abstract:Objective To study on the anti-hepatic fibrosis effects of salvianolic acid A injection (SAA), and further to provide the theoretical basis for the clinical application. Methods Using CCl₄ induced hepatocyte injury in vitro, the hepatocyte viability, the levels of ALT, AST and LDH in cell culture supernatants and the levels of SOD and MDA in cell lysates were detected. In addition, the hepatic fibrosis rat model was made by subcutaneous injection of CCl₄, the serum LN, HA, SOD and MDA levels were detected and the pathological changes in liver tissue were also observed. Results Compared with model group, the hepatocyte viability in SAA high or low dose group and Vit E group were significantly increased (P< 0.01), and the activities of ALT, AST and LDH in SAA high dose group were significantly lowed (P< 0.01). The activity of SOD in SAA high dose group and Vit E group was significantly increased (P< 0.05), while MDA content was decreased (P< 0.05). Vivo test showed that the levels of serum LN and HA in SAA high dose group were significant lower than those of hepatic fibrosis rat model group (P< 0.05). Moreover, the activity of SOD in SAA high or low dose group was significantly increased (P< 0.05,P< 0.01), while MDA content was lowed (P< 0.05,P< 0.01), and can improve the pathological of liver tissues. Conclusions SAA injection can anti-lipid peroxidation and thereby protect hepatocyte and reduce hepatic fibrosis.

Abstract:The prevention of myocardial and brain dysfunction induced by positive acceleration(+Gz) exposure is the focus in the field of aerospace medicine research topic. The characteristics and mechanisms that +Gz exposure caused damages to vital organs such as heart and brain remain to be further elucidated. The research literature about +Gz acceleration exposure-induced heart and brain injuries in experimental animals and its mechanisms at home and abroad was reviewed in this paper.

Abstract:Middle cerebral artery occlusion model with suture-occluded method in rats is widely regarded as the standard animal model of focal cerebral ischemia. In the process of preparation, it is easy to be influenced by multiple factors; among them the successful operation plays a key role. This article summarized from several aspects, such as the anatomic locations of the major arteries in the rat's neck, where to perform the incision, which side to perform the operation and where to insert the suture.

Abstract:The genetically modified mice, as a helpful model, have been widely used in life scientific research. However, several new issues appeared subsequently with the wide application of the genetically modified mice. Here, we mainly discussed and analyzed the problems in the management and usage of genetically modified mice, which underlies the foundation for establishing management practice of the genetically modified mice.

Abstract:Objective To develop an instrument that detects the endurance of an animal hanging on a bar during medical and pharmaceutical scientific experiments. Methods One pair of opto sensors were used for signal conversion. A solenoid valve device was used for water level. A 51-Series microcontroller was used to control the experimental setup and record the results. Results We have developed a microcontroller-based experimental instrument that can automatically detects the endurance of an animal hanging on a bar during medical and pharmaceutical scientific experiments. Our experimental results with 28 mice indicated that the applicability of the instrument is good. Conclusion The instrument provides an innovative tool for detecting the endurance of an animal.

Abstract:This paper discusses the definition, classification, selection, monitoring and evaluation of animal biosafety isolation device. Evaluation order of animal biosafety isolation device follows animal survival needs-biosafety needs-animal welfare requirements.