Table of Content

28 February 2019, Volume 36 Issue 01

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Analysis of Proficiency Testing Results of Laboratory Animals’ Genetic Quality during 2011—2017

2019, 36(01):  1. 

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Objective Proficiency testing (PT) during 2011—2017 were evaluated in order to find problems of laboratories and to understand the change of the testing capacity of laboratories. Method According to ISO/IEC 17043, six PT were carried on and the change of the testing capacity was analyzed. The capacity of all the laboratories was evaluated. Result Totally 57 laboratories participated in the six PT. The average satisfaction rate was 82.5%. The satisfaction rate of six PT was 60.0%, 80.0%, 90.0%, 80.0%, 90.9% and 81.8%, respectively. Conclusion PT can help laboratories to increase their testing capacity and strengthen the system construction of laboratory animals’ quality monitoring.

Histopathological Study on Skin Wound Healing of Minipig after Deep Second Degree Burn

2019, 36(01):  5. 

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Objective To establish the model of porcine deep second degree burn and observe the histopathological changes of the skin at different time points. Method Use the constant tempera turescaler to prepare minipig deep second degree burn. The morphological changes of the wound were observed and the healing rate was measured on the 1st, 3rd, 7th, 14th, 21st and 28th day, respectively. According to the rule of skin tissue healing and the operability of the experiment, scalded tissue was sampled on the 1st, 14th and 28th day, respectively. The histopathological changes of the skin were observed at different time points by HE, Masson staining and immunohistochemical staining with anti-CD31 antibody on the 1st, 14th and 28th day. Result Postburn skin tissue has accumulated into the deep dermis and is confirmed to reach the standard deep second degree burn by using the different methods. On the 14th and 28th day of the postburn, different degrees of tissue repair is performed on the damaged skin respectively. On the 28th day of deep second degree burn, the skin has basically healed, but the skin tissue has not yet fully matured. In addition to the relatively mature layers of the epidermis, the dermis has not yet fully formed the scar tissue.Conclusion The healing rate of male pigs is faster than female pigs.

Establish Standard Biological Indicators for Normal SD Rats in GLP Barrier Environment Animal Laboratory

2019, 36(01):  10. 

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Objective Establish the normal biological indicators of normal Sprague Dawley (SD) rats for long term toxicity test in barrier environment animal laboratory of center for safety evaluation of traditional Chinese medicine(GLP), to provide references for the future researchers. Method Breeding normally ten batches of normal SD rats in long term toxicity test, measured weight gains and food intakes once a week, measured organ indexes, blood and biochemical indicators at the end of 3, 6 months and recovery period. Result The result show that the food intakes of male SD rats were higher, increase linearly in body weights,higher than that in females, the organ indexes were declined in male rats. It showed that the growth and development,efficiency of feed utilization of male rats was faster than that in female rats. There were no significant changes in blood and biochemical indexes. Conclusion These normal data indicators can provide reference indexes for future GLP barrier environmental laboratory rat test.

Effect of Long Term Administration of Fumikang Vaginal Suppository on Hematology and Blood Biochemical Indexes in Rabbits

2019, 36(01):  17. 

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Objective To observe the effect of long term administration of Fumikang vaginal suppository on hematology and blood biochemical indexes in rabbits，to provide the scientific basis for its clinical safe use．Method Rabbits were vaginal administrated Fumikang vaginal suppository at dose of 8.586， 2.856，0.952 g/kg respectively，once a day for 6 months．By the end of the test and 4 weeks after drug withdrawal，the hematology and blood biochemical examination were tested．Result Compared with excipient control group，the hemoglobin and hematocrit in 8.586 g/kg dose group were significantly increased(P0.05).In each group，the blood biochemical parameters for those administration for 6 months and 4 weeks after stopping medication and excipient control groups showed no significant difference. Conclusion There was no long-term toxicity on the hematologic and blood biochemical indicators in 2.856 g/kg of Fumikang vaginal suppository.The safe dose of Fumikang vaginal suppository given to rabbit is over 2.865 g/kg, it is equivalently 20 times of clinical dosage(according to 60 kg body weight of adult).

Influence of the Shape of the Testing Environment on Novel Object Recognition Test in Mice

2019, 36(01):  22. 

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Object To investigate the effects of the result of novel object recognition tests in different experimental conditions with ICR mice. Method Divided 40 ICR mice(half male and female)into 4 groups randomly, the square female group (the female mice group working in the square box), the square male group, the round female group and the round male group. According to the same experimental process of novel object recognition, the exploration time of mice in each group was measured. Result There was no significant differences between the square female group and the square male group, the round female group and the round male group in the total exploration time and the discrimination ratio of novel object (P>0.05). Compared the square group(the square female group+the square male group) with the round group (the round female group + the round male group), there was no significantly differences in the discrimination ratio of novel object (P>0.05), but the total exploration time of the round group was significantly longer than that of square group (P<0.01). Conclusion In the experiment of novel objects, the sex of mice did not affect the evaluation of learning and memory ability of mice, and the choice of round behavior box for experiments could better evaluate the learning and memory ability of mice.

Establishment of Real-time Fluorescence Quantitative PCR for Detecting Stranscriptional-level of XDH/XO Gene in Wistar Rats

2019, 36(01):  26. 

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Objective To establish Quantitative Real-time PCR (RT-qPCR) method for detecting the transcription level of xanthine dehydrogenase/oxidase (XDH/XO) gene in Wistar rats，to quantitatively detect XDH/XO gene at the transcriptional level. Method Total RNA was extracted from the liver tissue of Wistar rats. The cDNA was obtained by reverse transcription, and diluted to 5 concentration gradients with 10 dilution factors. RT-qPCR was performed using the designed primer sequences and reference genes to obtain XDH/XO gene standard curve. Furthermore, changes in the transcription level of XDH/XO gene in the animal model of hyperuricemia in Wistar rats were examined. Result The XDH/XO gene standard curve obtained by RT-qPCR method has a single dissolution peak and R2 is close to 1. It can detect the change of XDH/XO gene transcription level in animal model of high uric acid. Conclusion RT-qPCR is a quantitative and accurate method for detecting the XDH/XO gene transcription level in Wistar rats. It can be applied to the pathogenesis of hyperuricemia and new drug research.

Comparation of Six Nucleic Acid Extraction Kits in the Extraction Efficiecy of Hepatitis Virus Nucleic Acid in Fecal Samples

2019, 36(01):  31. 

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Objective In this study, six commercial kits were used to extract mouse hepatitis virus(MHV)nucleic acid from faeces, and screen for an extraction method with higher efficiency and shorter time. Method Fecal samples infected with MHV were collected immediately and treated with liquid nitrogen grinding method , magnetic beads homogenization and PBS dissolves centrifugation method , respectively. RNA of above samples were then extracted with the same nucleic acid extraction kit to identify the best pretreatment method ; Based on which, the RNA was extracted using six kits and reverse transcribed into cDNA followed with TaqMan real-time PCR, respectively. Ct value of real-time PCR combined with sequencing and serum antibody ELISA test were used to evaluate and analyze the extraction efficiency of six kits. Result For the fecal sample pretreatment medthod, with liquid nitrogen grinding method had a significantly higher extraction efficiency than that with the magnetic bead homogenization method .For the RNA extraction efficiency among the six kits, TIANGEN DP422 concentration: (549.70±52.38) ng/μL， Ct value: (24.51±0.10) showed the highest extraction efficiency, TIANGEN SD101 concentration:(274.13±6.87)ng/μL；Ct value： (2.39±0.017) and QIAGEN 52904 concentration:(288.13±15.11)ng/μL；Ct value:(2.40±0.012) displayed the shortest extraction time, and XS VRL concentration:(348.80±15.85)ng/μL，Ct value:(24.70±0.13) turned out to be the most convenient. Conclusion Liquid nitrogen extraction method is more efficient in fecal sample pretreatment, and TIANGEN DP422 extraction kit with the highest MHV RNA extraction efficiency is recommended for the molecular biological detection of MHV in feces.

Improvement and Evaluation of Necrotizing Enterocolitis Models in Neonatal Rats

2019, 36(01):  36. 

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Objective The present study aimed to modify and evaluate necrotizing enterocolitis (NEC) models widely used in rats, and to determine the better method . Method Ninety rat pups were divided into five groups: Group A as control (n=10). Group B to E (n=20, respectively), rats were raised with simulated milk and subjected to hypoxia, cold stress, LPS with different ways and doses (Group B p.o. LPS 5 mg/kg; Group C p.o.LPS 10 mg/kg; Group D i.p. LPS 2.0 mg/kg; Group E i.p. LPS 5 mg/kg) per 12hours for 72hours. Followed by observing and recording body weight, survival time and sample collection, gross examination, HE staining, NEC injury scored, TNF-α levels in the intestine and kidneys were detected. Result We found that Group A rats were normal, but rats from groups B-E exhibited NEC clinical characteristics, but to varying degrees in different groups. The Group D (i.p.LPS 2.0 mg/kg) has comparatively lower mortality rate, no- NEC-associated death rate, higher morbidity rate and moderate TNF-α level in intestine and kidneys and NEC injury score when compared to other groups. Conclusion The modified method that through combination of formula feeding, hypoxia, cold stress, and low-dose LPS intraperitoneal injection is an optimal strategy of modeling rat pup NEC.

Preparation and Evaluation of Chronic Myocardial Ischemia Model Induced by Different Doses of Isoproterenol in Mice

2019, 36(01):  41. 

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Objective To prepare chronic myocardial ischemia models in mice and determine the best molding dose, and to discuss the evaluation indexes of the model which could provide reference for the preparation of related models and the evaluation of their efficacy. Method Three model groups of different doses were treated with subcutaneous injection of isoproterenol (32 mg/kg, 16 mg/kg, 8 mg/kg) for three consecutive days, after 24 hours, 8 days and 15 days ECG were recorded. On the eighth day and the fifteenth day, markers of myocardial injury in the blood were detected and the morphological changes of myocardium were observed. Result The ECG, markers of myocardial injury and pathology were all markedly changed in the group of 32 mg/kg isoproterenol(P<0.05 or P<0.01), and the mortality rate was in the acceptable range. The result could be used for evaluating the efficacy of the medicine for long-term treatment of chronic myocardial ischemia.The mortality rate of the group of 16 mg/kg isoproterenol was low, and indexes of ECG and markers of myocardial injury were better than that of the group of 32 mg/kg isoproterenol, and the dose did not cause irreversible pathological damage to the heart muscle. Conclusion The result showed that the chronic myocardial ischemia models in mice can be established by using two different molding dose according to different test expectation.

Study of Intestinal Flora in Rat and Crucian Carp

2019, 36(01):  48. 

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Objective The distribution of intestinal microflora in different parts of animals，intestine which living environment are quite different. Method With taking the contents of different intestinal segments of mice and crucian carp, extracting and culturing with different selective medium, and then carring out the plate count. Result It was found that the proportion of Lactobacillus in the intestine of rats was higher than that of crucian carp, and the proportion of other colonies (Bifidobacterium, Enterococcus, Escherichia coli) was significantly smaller than that of crucian carp. The distribution of intestinal anaerobic Bifidobacterium in rats and crucian carp increase in the downward direction along the gut.The magnitude of aerobic Enterococcus in the intestine of rats and crucian carp was almost same. Conclusion The inestinal microbiological information of different animals can preovide some experimental basis for the study of pathology and toxicology.

Study on Antitumor Effect of Recombinant Human Interferon Endothelial Peptide Fusion Protein

2019, 36(01):  52. 

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Objective To investigate the antitumor effect of I30 peptide, and to provide experimental result for the further development of I30 peptide. Method Using NIH mouse, 4 mice were injected intraperitoneally with H22 tumor strain, and all mice were sacrificed at 1 week, and ascites was extracted. Another 40 mice were injected with 0.2 mL ascitic fluid, and the cell concentration was 1×106个/mL. At 4 h after inoculation mice were treated in different groups. The mice were divided into 4 groups randomly, IFN group, subcutaneous injection of interferon 9×105 IU/mL; I30 high dose group, subcutaneous injection of 80 μg/I30; I30 low dose group, subcutaneous injection of 30 μg/I30; negative control group, subcutaneous injection of normal saline. Mice were continuous administrated for 16 d. After 2 h, all animals were sacrificed, and the subcutaneous tumor mass was dissected. Result The treatment group had obvious therapeutic effect, and the difference was significant (P<0.05). Conclusion I30 has anti-tumor effect.

Improvement of Learning and Memory Ability of BXT on Aging Rats and it’s Molecular Mechanism

2019, 36(01):  55. 

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Objective Using D-galactose-induced aging rats as animal model to study the effect of Banxia Xiexin Decoction (BXT) on aged rat’s learning and memory ability and the expression of Ach, NE, DA, 5-HT receptor mRNA in the hippocampus of the brain,explore the mechanism of BXT to improve the aged rat’s learning and memory ability. Method According to Rat escape latency by Y water maze test,and D- galactose injection to establish aging rat model;Then the animals divided into Aging control group, Huperzine A group, Strong control group, BXT low dose group, BXT medium dose group, BXT high dose group,intragastric administration by different group.After 6 weeks of treatment, the learning and memory abilities of rats were tested by Y-type water maze again,and detect NE, DA, 5-TH receptor mRNA and antibody expression, observe the pathologic changes of hippocampus, therefore explore the molecular mechanism of BXT on aging rat’s learning and memory ability. Result The mean escape latency in the Y-type water maze test was shortened in BXT-treated group. The HE staining of the hippocampus showed that the damage of the hippocampus neurons in the high dose BXT group was improved compared with the model control group, but there was no significant improvement in the immunohistochemical result in medium and low dose BXT group. The result of immunohistochemistry showed that the BXT high dose group had more chromosomes than the model control group, and the cells were darker. Conclusion This study found that BXT can improve aging rat’s learning and memory ability,especially the high-dose BXT group.The possible machanism is to increase the aging model hippocampus expression of CHRM1, DRD2, HTR1a, ADRA2a mRNA.

Experimental Study of Choroidal Neovascularization in Brown Norway Rats Induced by 532 nm Laser at Different Power

2019, 36(01):  62. 

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Objective To induce the choroidal neovascularization model of Brown Norwegian rats with different power of 532 nm laser,we would explore the highest fluorescein leakage rate by the best laser power. Method Forty-five Norwegian rats were randomly divided into three experimental groups: 15 rats in each group. The laser power were 400 mW, 300 mW, 200 mW, the spot diameter was 100 μm, the exposure time was 100 ms, and each eye was lasered 10～12 points. The experimental datas were collected from the 1st～4th week after lasering Brown Norway. The experimental datas were statistically analyzed by fundus fluorescein angiography and optical coherence tomography. Result The retina of the fundus showed that the retina had edema due to the destruction of the vitreous membrane after fluorescence induction. FFA showed that the the leakage rate of the laser power of 200 mW compared with 300 mW and 400 mW at the 1st to 4th was statistically significant (P0.05), and the leakage rate at the 2nd and 4th weeks was statistically significant (P0.05).But there was significant difference between the 1st and 3rd week of 200 mW laser power(P0.05). Optical coherence tomography showed retinal was disordered, then retinal begained to form choroidal neovascularization.The choroidal neovascularization was ultimately stabilized,and part of the retinal structure atrophy thinning. Conclusion 200 mW, 300 mW and 400 mW laser power of 532 nm laser can induce the formation of choroidal neovascularization model by Brown Norway. The formation rate of 300 mW laser power is the highest.The choroidal neovascularization begins to occur after the 1st week, reaching the peak in the 3rd week, and the choroidal neovascularization tends to be stable at the 4rth week.

Preliminary Study on Artificial Rearing of Lactating Mice

2019, 36(01):  68. 

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Objective This study aimed at developing a new experimental model for the artifically rearing of neonatal mice to lay the foundation for further study of the nutritional needs and growth of mice. Method Five pregnant BALB/c mice of 10—12 gestation days were kept until deliver. Total 27 neonatal mice were immediately randomized into two groups at postnatal day 3, one for maternal rearing and another for artificial rearing to postnatal day 14. Result 100% of the artificial rearing mice survived at 10 days, 92.9% at 11 days, 78.6% at 12 days, 50% and 35.7% at 13 days and 14 days, respectively. However, significant differences of body weight were found between the maternal reared mice and the artificial reared mice at postanal day 10 (3.02±0.67 vs. 9.00±0.70，P<0.01). Conclusion The new formula and nipple at least succesfully achieved the artifical rearing of neonatal mice to some extent, however, further study was needed to improve the survival rate and increase the body mass of artificially fed mice during lactation.

Brief Introduction to High Fat Diet-Induced Rat Non-alcoholic Fatty Liver Model and Discussion on Technical Points

2019, 36(01):  73. 

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Dietary factors are the most important environmental factors leading to the occurrence of non-alcoholic fatty liver disease. The non-alcoholic fatty liver model induced by high-fat diet has the characteristics of being close to the clinical and stable model, so it is widely used in non-alcoholic fatty liver research. This article briefly introduced the model of fatty liver in rats commonly used in the non-alcoholic fatty liver inducing model, and discussed the technical points and experience of the non-alcoholic fatty liver model induced by high-fat diet.

Pathological Changes of Radiation Pneumonia in Kunming Mice

2019, 36(01):  77. 

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Objective To clarify development of the pathological process of radiation pneumonia. Method Using 6MV X-ray, of which the cumulative dose is 20Gy, irradiates the full chest once. The hematoxylin-eosin staining was conducted respectively on the 7th day, 14th day and 28th day after irradiation for pathological study. In addition, the Image-Pro plus analysis system was applied to quantitatively analyze thickness of alveolar walls, and surface mass density of alveolar spaces and interstitial tissue. Result Alveolar atrophy, alveolar septum widening and inflammatory cell infiltration were observed along with the development process. The collapsed or consolidated alveolus and mild pulmonary fibrosis were observed after 28 days. Furthermore, the differences of thickness of alveolar walls and surface mass density of alveolar spaces and interstitial tissue were very significant. Conclusion Radiation pneumonia model in Kunming Mouse could be replicated successfully by the mentioned method, of which the pathological changes are in accordance with the clinical course.

Advances in Detection of Rat Parvovirus

2019, 36(01):  80. 

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Rat parvovirus(RPV)is a DNA virus which contains variety of serotypes and can be divided into different strains. Both experimental rats and wild rodents can be naturally infected with RPV, but clinical disease and histological lesions have been observed different in rat infected with different strains. The RPV can have appreciable deleterious effects on research by contaminating rat tissues or organs and then result ing in changes in physiological and pharmacological parameters of rat. Therefore, it is critical to identify the infected experimental rat to minimize the impact on research. This paper aims to systematically expound the research of rat parvovirus detection technology, compare different method, and put forward problems and its prospects to provide reference of the experimental animal quality control.