Table of Content

25 August 2018, Volume 38 Issue 4

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Development of Specific Pathogen-free (SPF) Pigs for Prevention and Control of Livestock and Poultry Disease

GAO Cai-xia, XIN Chang, CHEN Hong-yan

2018, 38(4):  245-249.  DOI: 10.3969/j.issn.1674-5817.2018.04.001

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In this paper,we elaborated the importance of specific pathogen-free (SPF) pigs for prevention and control of livestock and poultry disease and the research status at home and abroad,analyzed the existing problems,introduced the common breed methods of SPF pig population,and discussed the application prospect of SPF pigs.The aim of this paper is to provide some theoretical guidance for the development of experimental animals on animal disease prevention and control research.

The Application of Heptamethine Cyanine Dye in Optical Imaging of Hepatocellular Carcinoma Transplantation Model

WANG Qin-zhou, ZHANG Cheng, LI Li, TANG Deng-xu, ZHANG Cai-qin, SHI Chang-hong

2018, 38(4):  250-254.  DOI: 10.3969/j.issn.1674-5817.2018.04.002

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Objective To explore the metabolic characteristics of heptamethine cyanine near-infrared (NIR) fluorescence dye MHI-148 in hepatocellular carcinoma (HCC) xenograft model.Methods Human HCC cell Hep3B was co-cultured with NIR dye MHI-148 solution.The specific accumulation of MHI-148 on tumor cells was observed under fluorescence microscope.Hep3B cells were implanted subcutaneously into nude mice to establish a transplant model.Two weeks later,MHI-148 dye was injected intraperitoneally into tumor bearing nude mice and detected by NIR fluorescence optical imaging.The fluorescence intensity both on the tumor site and the organs of the nude mice was measured at different time point,the tumor fluorescence intensity (tumor,T) and the adjacent normal tissue (background,B) was continuously detected and the maximum T/B value was calculated.Results MHI-148 dyes could be specifically accumulated in HCC cells,and Hep3B cells labeled with green fluoresence protein (GFP) were identified.When MHI-148 was injected into nude mice bearing subcutaneous HCC tumor,the fluorescence intensity per unit area (cm²) of the tumor site reached 8.35×10⁹ after 24 hours,while other organs have less fluorescence aggregation.The ratio of T/B at the site of the tumor gradually increased and reached peak value at 24 hours.Conclusion The hepatocellular carcinoma cells can be specifically recognized by heptamethine cyanine dye MHI-148.The site of subcutaneous HCC tumor can be identified through NIR fluoresence optical imaging.



Comparison of Hepatic Fibrosis Model Induced by Different Concentrations of CCl₄ in Mice

SUN Jia-chang, SUN Wu-yi, LI Xin-ran, PENG Wen-ting, DU Jia-jia, WEI Wei

2018, 38(4):  255-260.  DOI: 10.3969/j.issn.1674-5817.2018.04.003

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Objective To explore a reliable and stable method for establishing hepatic fibrosis model in mice through different dosing level of carbon tetrachloride (CCl₄).Methods Sixty male C57BL/6J mice were randomly divided into control group and model group.The mice in model groups were intraperitoneally injected with 10% CCl₄ at the dosage of 5 mL/kg and 20% CCl₄ at the dosage of 1mL/kg respectively for 8 weeks,twice per week.Mice were sacrificed at the 2nd,4th,6th,8th week after injection.The degree of hepatic fibrosis was evaluated by HE and Masson staining.The changes of alanine aminotransferase (ALT),aspartate aminotransferase (AST) were detected in serum.The oxidative index malondialdehyde (MDA) and antioxidant indices,glutathione (GSH),superoxide dismutase (SOD) were detected in liver homogenate.The protein levels of collagen I and collagen III in liver tissues were determined by Western blotting.Results HE and Masson staining showed that the degree of hepatic fibrosis was aggravated as the development of the model.The expression of collagen was gradually increased,the serum levels of ALT,AST and MDA in liver tissues were significantly increased,while the activities of SOD and GSH were significantly decreased.At the same point of model progress,the degree of hepatic fibrosis of 10% CCl₄ (5 mL/kg) group was more severe than that of 20% (1 mL/kg) group.The levels of ALT,AST and MDA in 10% CCl₄ (5 mL/kg) were significantly higher than 20% CCl₄ (1 mL/kg) group.In addition,the SOD activity and GSH were lower than those of 20% (1 mL/kg) group.Conclusion Both of two methods can induce hepatic fibrosis model,and 10% CCl₄ (5 mL/kg) has advantages such as better effect and shorter modeling time compared with 20% CCl₄ (1 mL/kg),therefor,it is an ideal method for establishment of liver fibrosis model in mice.



Comparative Analysis on Rat Models of Parkinson's Disease Established by Injecting 6- hydroxy Dopamine with Different Sites

LI Min, CHANG Xue-hui, ZHANG Liang-zhi, LEI Zhen, LUO Shen

2018, 38(4):  261-266.  DOI: 10.3969/j.issn.1674-5817.2018.04.004

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Objective To explore the way of establishment of stable and effective Parkinson's Disease(PD) rats model with 6-hydroxyl dopamine (6-OHDA).Methods Firstly,SD rats were randomly divided into four groups,the one spot via midbrain group,the double spots via midbrain group,the one spot-twice injections via striatum group and control group.And 6-OHDA was injected into the brain of rats respectively according to different groups.Then observed the behavioral changes of rats respectively in the first week,second week,fourth week and eighth week,and tissue of brain was obtained after observation of behavioral changes in the eighth week,and then pathological examination was performed by HE staining and immunohistochemistry.Finally,tyrosine hydroxylase (TH) positive cells were analyzed with biosens digital imaging system.Results ① The success rate of PD rats of the one spot via midbrain group was 72.7%,the double spots via midbrain group was 60%,the one spot-twice injections via striatum group was 47.6%,and control group was 0%.The success rates of different groups were statistically significant (P<0.05).②The average revolving number of successful PD model of the one spot via midbrain group in the first week was 7.3±0.2 r/min,and increased than the first week in the second week (P<0.05),increased than the second week in the fourth week (P<0.05),and increased than the fourth week in the eighth week,but there was no statistically significant (P>0.05).There was the similar behavioral change trend in the double spots via midbrain group and the one spot-twice injections via striatum group,but there was no statistical significance at the same time among different groups (P>0.05).③ After analysis of TH positive cells of the damaged side of PD rats with biosens digital imaging system,the average integral optical density (IOD) could be obtained,and the IOD of the one spot via midbrain group,the double spots via midbrain group and the one spot-twice injections via striatum group was significantly smaller than that of the control group (P<0.01),and the IOD of the one spot via midbrain group was smaller than that of the double spots via midbrain group and the one spot-twice injections via striatum group (P<0.05).Conclusions The results suggest that after injection of 6-OHDA into the substantia nigra pars compact singly,the success rate of PD rats is higher,the PD rats is more stable and the mortality rate is lower.



Establishment of Atopic Dermatitis Mouse Model

JI Lian, MA Tie, DI Zheng-hong, Liu Dong-yan

2018, 38(4):  267-271.  DOI: 10.3969/j.issn.1674-5817.2018.04.005

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Objective To explore a novel method for atopic dermatitis (AD) model in mouse induced by ovalbumin (OVA).Method The 6-weeks BALB/c female mice were subcutaneously injected with 0.5 mL of 200 µg/mL OVA solution on 1st day and subcutaneously injected with 0.5 mL of 100 µg/mL OVA solution on 6th day to induce systemic sensitization again.On 18th day and 39th day,1.5 cm² sterile gauze with 50 µL,1 000 µg/mL OVA solution were attached on the back of the mice and replaced every day for consecutive 7 days for the second local sensitization.On 46th day,the pathological changes in mouse skins were observed to judge whether mouse AD model was successfully established and the rates of being model and the mortality rate was calculated.Result The dermatitis changes,such as scab,flushing,desquamation,and so on,were shown in AD model mice.Also,the epidermal cells were thickened and the dermis was infiltrated with lymphocytes in AD model mice.The total serum IgE level in the AD model mice [(6 296.17±1 021.28) ng/Lsignificantly increased as compared with blank control mice [(1 280.97±501.24) ng/L](P<0.01) and the mice injected with saline [(1 327.57±629.71) ng/L(P<0.01).The rates of AD model and the mortality were respectively 94% and 6%.Conclusion Our study introduces a simple and stable method for mouse AD model induced by OVA.



Serological and Viral Replication of Murine Norovirus in ICR Mice with Different Routes of Artificial Infection

LUO Yin-zhu, ZHANG Yu, YUAN Wen, HE Li-fang, HUANG Bi-hong, WU Rui-ke, Min Fan-gui, WANG Jing, PAN Jin-chun, HUANG Ren

2018, 38(4):  272-277.  DOI: 10.3969/j.issn.1674-5817.2018.04.006

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Objective To investigate the serum antibody levels and viral replication of murine norovirus (MNV)in mice by artificial infecting in a variety of ways.Method ICR mice were inoculated with MNV (4 ×10⁴ copies/µL) respectively through intravenous combining with intraperitoneally (iv+ip group),drinking water (dw group),intragastrical injection (ig group).Heart,liver,spleen,lung,kidney,brain,gastrointestinal contents samples and serum were taken on 0,3,6,14,25,34,51 day postinfection (dpi) (2~3 mice each time).qRT-PCR and ELISA method were applied to detect virus RNA and serum antibody.Results Asymptomatic infection has been identified in all mice.The specific antibody can be first detected 25 dpi in three groups.The level of antibodies was the highest in the ig group in early times,then antibody levels of the iv+ip group rose faster 51 dpi than the other groups.MNV RNA can be detected 3 dpi in the gastrointestinal tract,spleen and brain.Different viral load in each organization among groups.The viral load of cecum contents (3~ 51 d),spleen (6 d) and liver (14 d) viral load in the iv+ip group is higher than the other two groups.Conclusion The effects of different inoculation methods on the antibody level and virus replication of MNV were significant.The effect of infection combined intravenous with intraperitoneally come out to be good,which can be used as MNV effective inoculation for animal model.Serum antibody ELISA detection combined with cecum contents nucleic acid detection is advantageous to the MNV early monitoring.



Study on Indicators of Chronic Fatigue Syndrome Model in Rat

RAO Zi-liang, FENG Xue-xuan, HUANG Xiao-hong, LI Li-si, LI Jin-feng, KUANG Shao-song, WANG Gang

2018, 38(4):  278-283.  DOI: 10.3969/j.issn.1674-5817.2018.04.007

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Objective To make a rat model of chronic fatigue syndrome (CFS) by overcrowding and other stresses,and to test the related indicators.Methods The rats were stimulated by different stress factors,such as irregular crowding,noise and diurnal reversal for 64 days.The mental status,coat and skin color were observed.Weight and food intake were measured every week.Blood routine,immunoglobulin G (IgG) and blood lactate were measured regularly.Results After modeling,rat independent activity decreased.Mental fatigue,skin loosed,body hair withered,and semi-quantitative score value increased.Body weight and food intake decreased.White blood cell,red blood cell,hemoglobin and erythrocyte specific volume decreased.Inactive time after forced swimming,blood lactic acid and IgG increased.Conclusion The rat models of CFS were successfully constructed by overcrowding,noise and sleep disorders.Semi-quantitative scores,body weight,food intake,white blood cell,red blood cell,hemoglobin,erythrocyte specific volume,inactive time after forced swimming,blood lactic acid and IgG can be used as reference indicators.

Case Report of Geniposide Induced Bilirubin Pigmentation in Sprague-Dawley Rat

ZHOU Fei, WANG Hao-an, CHEN Tao, QIU Shuang, CHEN Ke, CEN Xiao-bo

2018, 38(4):  284-287.  DOI: 10.3969/j.issn.1674-5817.2018.04.008

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Objective Fine pigment granules in livers and kidneys of SD rat induced by geniposide were differentially diagnosed in this report.Methods A gross observation was performed on liver and kidney of SD rats which were administrated geniposide by oral gavage,and routine HE staining slides for microscopic analysis was carried out.In addition,the blood biochemical and urine index in clinical pathology were analyzed,and special staining was used for differential diagnosis.Result Compared to the control group,livers and kidneys of SD rats in each dose group were found discoloration of gray green,as well as the serum TB and GGT was increased significantly (P<0.05).The contents of urine URO and BIL in dose group were increased with dose-relation.Microscopically,liver and kidneys in all dose group animals were found tiny brown-yellow pigment droplets which were test material related.These pigment particles were positive for Fouchet staining but negative for Perls blue.Conclusion In this case,the bilirubin pigmentation was definitely diagnosed by using clinical pathology data combination with histopathological examination.In addition,the relationship between histopathologic changes of livers and kidneys and clinical pathology was explained.This can provide theory and method on pigmentation differential diagnosis for future toxicologic pathology work.



Detection of Main Organ Weights, Blood Physiological,Biochemical and Immunologcal Indexes in SCID Mice

YUAN Shui-juan, SHI Guo-jun, TANG Hui-qing, SHI Mei-lian

2018, 38(4):  288-292.  DOI: 10.3969/j.issn.1674-5817.2018.04.009

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Objective To determinate the main organ weights,blood physiological and biochemical indexes,immunoglobulins,complements and immune cells data of SCID mice.Methods Twenty SCID mice at the age of 4,8 and 16 weeks old respectively,with half male and female,were selected for detecting the main organ weight,blood physiology and biochemistry indexes,immunoglobulins and complements data.Twenty SCID mice at the age of 8 weeks old,with half male and female,were selected for detecting T cells and subgroups,B cells,and NK cells.Results The thymus weight of SCID mice was independent of gender and age,and there was no significant difference.The levels of red blood cell,lymphocyte,IgG,IgA and IgM in the 4-week-old SCID mice were significantly greater than those in 8 weeks and 16 weeks.T cells,B cells,and NK cells,separated and sampled from spleen,are no significant difference in gender.CD3+ was the smallest proportion in total immune cells,and B220+ accounted for the largest proportion.Conclusions SCID mice are immunodeficiency animals of T and B cells comprised.The blood physiological and biochemical data of this experiment were basically consistent with the data of SCID mice abroad.It shows that the biological characteristics,such as heredity character,physiology biochemistry of this strain have not changed since the introduction.

Studies on Effects of Different Cornu Cervi Pantotrichum on Bone Marrow Mesenchymal Stem Cells Based on Principal Component Analysis

QU Lei-ming, LI Feng, GONG Wei, LIU Wei

2018, 38(4):  293-298.  DOI: 10.3969/j.issn.1674-5817.2018.04.010

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Objective To systematically evaluate the similarity and discrepancy of eight different Cornu Cervi Pantotrichum by principal component analysis (PCA) which could accelerate the proliferation of the bone marrow mesenchymal stem cells cultured in vitro and their differentiation toward osteoblasts.Methods Seventy-two female SD rats were divided into nine groups randomly,one normal group given water and eight administrated group.Cornu Cervi Pantotrichum was classified by growth and processes.Bone marrow mesenchymal stem cells (BMSCs) were exposed to different culture medium containing different Cornu Cervi Pantotrichum serum.The activities of osteoblastic markers such as alkaline phosphatase(ALP),bone gla protein(BGP),bone morphogenetic protein 2(BMP-2) were detected by ELISA.Cell proliferation was measured by MTT assay.The principal component analysis was applied to study characteristic of velvet antler based on the indexes mentioned above.Results PCA analysis of absorbance value(A) and ALP can distinguish wax slices from blood slices and bone slices.The accuracy of the discriminant analysis was 100% for A and ALP.Conclusion This method can be used to evaluate the quality of Cornu Cervi Pantotrichum.



Application of Analytic Hierarchy Process in Biosafety Management of Laboratory Animals

XU Qing-hua, TONG Lin

2018, 38(4):  299-303.  DOI: 10.3969/j.issn.1674-5817.2018.04.011

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Objective To determinate the weights coefficient of the comprehensive evaluation indexes in biosafety management of laboratory animals objectively and scientifically.Method The analytic hierarchy process (AHP) was used to define the weight coefficients in 11 synthetic evaluation indexes.Result The most important weight coefficient (0.2157) were that facility environment and health certificate of laboratory animals the consistent ratio (CR) is 0.0209<0.1.Conclusions The weight coefficients in 11 synthetic evaluation indexes have no logical confusion,indicated that this index system and the weight coefficients of indexes could be applied in biosafety management in different units and laboratories.

Investigation on Several Heavy Metals and Harmful Chemicals in Different Beddings

ZHAO Wei-bo, MIN Fan-gui, PAN Jin-chun, MEI Yi-duo, HUANG Hai-yan, HUANG Ren

2018, 38(4):  304-306.  DOI: 10.3969/j.issn.1674-5817.2018.04.012

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Objective To determine and compare the levels of several heavy metals and harmful chemicals in 10 kinds of laboratory animal bedding materials come from experimental animal unit in Guangdong,so as to provide users with reference.Methods The pollution levels of 5 kinds of heavy metals and 3 kinds of harmful chemicals were detected according to the current methods.Results Aflatoxin B1 was detected in all the samples,but it did not exceed the standard requirements.The lead content in 3 beddings exceeded the standard requirements,and the heavy metal chromium pollution was detected in 9 bedddings.Conclusion The results can be used as reference for the purchase and use of bedding,and can also provide reference for the establishment of local standards.

Case Report on Pathomorphological Changes of Kidney in Spontaneous Nephroblastoma Occurring in SD Rat

QIU Bo, WANG Yan, HU Jian-ting

2018, 38(4):  314-317.  DOI: 10.3969/j.issn.1674-5817.2018.04.015

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In a 26-week repeated dose toxicity study with SD rats,one case of nephroblastoma is observed in the vehicle control group (0.9% saline) after the administration.The gross morphology shows that the right abdomen inflated and the whole right kidney is a mass.Some areas of the mass is gray or black brown.The mass is larger,irregular shape,pseudocystic fibrosis can be seen on the surface of the mass.The texture of the mass is both hard and soft.Left kidney is normal.HE staining shows that large mass involved most of the kidney and are surrounded by a pseudocapsule of compressed renal parenchyma.The mass in the rat is characterized by a triphasic pattern of blastemal,epithelial and stromal cells.Organoid epithelial differentiation into glomeruloid and epithelial-lined tubules further characterizes nephroblastoma.The above results are in line with the diagnostic features of nephroblastoma.



Introduction to Biological Characteristics of Pneumocystis carinii

FENG Jie, LIN Jin-xing, GAO Cheng

2018, 38(4):  320-324.  DOI: 10.3969/j.issn.1674-5817.2018.04.017

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Pneumocystis carinii is a zoonosis that is opportunistic pathogenic and worldwide distributed.It can infect a wide range of mammal species,endanger human and animal health.In this paper,the research progresses of classification and detection methods were reviewed.Further,the related contents of Pneumocystis carinii in national standards of laboratory animals were introduced and suggestions were proposed.