Table of Content

28 October 2019, Volume 36 Issue 05

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Optimization of Chromosome Aberration Test in ICR Mice

2019, 36(05):  1. 

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Objective To perform in vivo chromosome aberration test using ICR mice by administrated with cyclophosphamide (CP)and colchicine (COL)at different time points,and compare the effects of different administration and sampling strategies on the incidence of mouse bone marrow chromosomal aberrations.Method A total of 36 ICR male mice of about 7-8 weeks old were primarily randomly divded into 18 h and 24 h sampling group after the last dosing.Both groups werer further divided into COL 4 mg/kg (4 h before sampling,i.p.)and COL 10 mg/kg (2 h before i.p.)administration groups.In each group,the 0.9% sodium chloride injection was used as the vehicle control,and respectively dosed with 0 mg/kg,10 mg/kg,and 40 mg/kg CP for 3 animals each.The body weights of animals before and after administration during the experiment were recorded.The negative control group and the 10 mg/kg CP group were administered twice at a interval of 24 hours,and the 40 mg/kg CP was administered once.Both femurs of each animals were taken at 18 h or 24 h after the last administration for preparing bone marrow suspension,and COL was intraperitoneally injected 2 h or 4 h before the sampling.Slides with bone marrow samples were prepared and stained with Giemsa for identifying the types of chromosome aberrations,and the average structural aberration cell rate (excluding ctg/csg,AC%),structural aberration cell rate (including ctg/csg,ACG%)rate of cells with multiple aberrations (excluding ctg/csg,MAC%)and polyploid cell rate (PC%)were calculated.Result CP and COL used in this study showed no effect on the body weights of animals,and the administration doses and frequency are feasible.Compared with the vehicle control groups,AC%,AGC%,and MAC% of all CP administered groups were significantly increased (P<0.01)as higher CP dose showed more serious aberrations;AC%,ACG% and MAC% of CP18 h-COL4h group were significantly lower than the CP18 h-COL2h group (P<0.05),suggesting that high doses of COL (10 mg/kg)could produce additional chromosomal damage.Most of the structural aberration types in the vehicle control group were gaps,while chromatid and chromosome breakage were the most dominant aberration types induced by CP.Conclusion It is ideal to take the bone marrow sample 18 hours after administrated with CP and 4 hours after dosed with COL (4 mg/kg)for chromosome aberration test in mice.This study accumulated background data for the exploration of test conditions,and also provided reference for relevant researchers.

Correlation between SNP Loci of SLC1A2 and the Activity Level of Labrador and Golden Retriever

2019, 36(05):  9. 

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Objective To determine the correlation between the SNP loci of SLC1A2 and the activity level of Labrador and Golden Retriever for selection of effective genetic biomarkers.Method Ninety-nine dogs (80 Labrador and 19 Golden)were selected from the Chinese guide dog training center of Dalian.The activity levels of the dogs were evaluated by Passive Test.Venous bloods of the dogs was collected and the genomic DNA was isolated from white blood cells.Three SNP loci of SLC1A2 gene,T129C,T1549G,and T471C,were detected by PCR/sequencing method .Finally,the correlations between activity level and genotypes were analyzed.Result The T129C and T1549G loci of SLC1A2 gene were not polymorphic.The T471C locus had three genotypes:T/T,T/C and C/C.There was no significant correlation between the three genotypes of T471C and the activity level,sex,and breed (P>0.05).There was also no significant correlation between the activity level and the breed.However,there was significant correlation between activity level and sex (P=0.049).Conclusion There is no significant correlation between SNP loci of SLC1A2 gene and activity levels of Labrador and Golden Retriever.

Establishment and Evaluation of a Rat Model of Cognitive Impairment after Traumatic Brain Injury

2019, 36(05):  15. 

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Objective To explore the preparation and evaluation of an animal model of cognitive impairment after Traumatic brain injury.Method Sixty healthy male SD rats and the remaining 48 rats after water maze screening were randomly divided into blank control group,model control group and model group.The electric Cortical Contusion Impactor was used to attack the model and water maze screening was used to determine the cognitive impairment rats after TBI.The changes of vital signs,nerve function and motor function,Morris water maze test,contents of MMP-2,MMP-9,SOD and MDA were observed.Result Compared with the blank group,there were significant differences in vital signs and behavioral indicators (P<0.05),latency of water maze test and space exploration test (P<0.01),serum MMP-2,MMP-9 and MDA levels (P<0.01),and SOD levels (P<0.01).Conclusion The model preparation scheme proposed in this paper can simulate the cognitive impairment after craniocerebral injury,and has good stability,reproducibility,Objective evaluation,and better operation of animal model platform and evaluation system,which is worthy of reference in similar studies.

Effects of Storage Time on Blood Routine Test of Venous Haematocyte Suspension in Mice

2019, 36(05):  21. 

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Objective To investigate the effect of storage time on blood routine test of venous haematocyte suspension in mice．Method Twenty adult ICR mice and 20 C57BL/6 J mice were randomly selected,which were adopted venous blood.Collection each of quantitative venous blood added in diluent which were adequate for quantitative blood cell analytical instrument to be mixed to make venous haematocyte suspensions．Then detected by trace blood determination on 0 min,15 min,30 min,1 h,2 h,4 h,6 h,8 h in the MEK-6410C full-automatic blood cell analyzer．Result The change trend of blood routine data in ICR mice and C57BL/6 J mice is basically the same．Compared with 0 min,RBC decreased significantly at 2 h,4 h,6 h and 8 h(P0.05).Conclusion By trace blood determination,the blood routine test of mice is done as soon as possible after the haematocyte suspension is made．The RBC is better to be measured in 1 h,HCT,MCV related to RBC were tested should not be more than 30 min.The PLT is better to be measured in 2 h,PCT,MPV related to PLT were tested should not be more than 1 h,The data of WBC and the content of HGB are stable and should be completed within 8 h.

Establishment of the Rat Model of Ovarian Hyperstimulation Syndrome

2019, 36(05):  25. 

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Objective To explore and evaluate the method for the establishment of OHSS model in rats,and to provide an ideal model for study of OHSS mechanism and drug screening.Method Totally 60 Wistar and SD rats,3-5 weeks old,were randomly grouped and injected with different doses of pregnant mare serum gonadotropin (PMSG)and human chorionic gonadotropin (hCG)to establish the OHSS model.The model was evaluated by general pathology examination,measurement of viscera index,and concentration of progesterone,estrogen and VEGF in the peripheral blood.Result Among the 4 groups,SD rats that are 3 weeks old showed a greater degree of ovarian enlargement,vascular permeability enhancement,serum VEGF level increasement and other OHSS typical changes.It’s more suitable for the establishment of OHSS rat model.Conclusion A more suitable model was obtained and can lay the foundation for further study of OHSS mechanism and drugs screening.

Optimization of Mouse Embryo Transfer Technology and Purification of Cytomegalovirus

2019, 36(05):  31. 

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Objective A mouse cytomegalovirus purification method was established to obtain mice without MCMV infection.Method The purification conditions were optimized by using different hormone,different estrous cycle injection hormone,receptor mouse strain,different transplantation method ,different embryo transfer,and the number of transplanted embryos.The donor mice were purified by the optimized method of embryo transfer purification.Result The result showed that the number of embryos produced by SIGMA was over 17 at 10 IU.The number of embryos available for ovulatory superovulation is the largest,and the number of embryos available for superovulation is about 23.The offspring of C57 male mice and ICR female mice were the recipient mice,with a litter size of 44.5%.The effect of tubal transplantation is better than that of uterus transplantation,with a birth rate of 40.64%.The pregnancy rate of transplanted 2 cell embryos was 80%,better than that of single cells and 8 cells.When the recipient mice transplanted 24 embryos,the litter size reached 43.75%.Mice without MCMV infection were purified by embryo transfer.Conclusion A purification method of murine cytomegalovirus was established to provide reliable protection for the population of mice without MCMV infection.

Establishment and Application of Real-time Fluorescent Quantitative PCR Method for Detection of Bovine Herpesvirus Type 1

2019, 36(05):  35. 

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Objective To establish a real-time fluorescent quantitative PCR method for detection of Bovine herpesvirus type 1 (BHV-1)in Bovine origin samples.Method The primers and TaqMan probe were designed and synthesized according to the published BHV-1 specific sequences of gB gene.Q-PCR method is established.Then carries on the specificity,sensitivity,repeatability and stability of this method were tested.The method is used to detect 181 Bovine origin samples.Result The developed Q-PCR method was no cross reaction with Bovine parainfluenza virus type 3 (BPIV3),Bovine viral diarrhea virus type 1(BVDV1),herpes virus type 1 (HSV-1),Felid herpesvirus 1 (FHV-1),and Pig pseudo rabies virus (PRV);sensitivity was 1×101 copies/μL;Coefficient of variation(CV)was less than 5%;There were 6 positive reaction detected in the 181 Bovine origin samples.Conclusion The developed PCR method is good in specificity,sensitivity,repeatability and stability and may be used for rapid quantitative detection the BHV-1 in Bovine origin samples.

Study of Blood Index of Young SD Rats in Different Stages

2019, 36(05):  42. 

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Objective The aim of this study is to collect physiological indicators of young SD rats at different developmental stages and establish relevant background data,to provide reference for the safety evaluation of pediatric drugs.Method The blood samples of about 200 young SD rats (3,6,8,12 and 16 weeks)were collected,and the corresponding indexes were determined by automatic blood cell counting instrument,biochemical analyzer.Result From the result of each index parameter,the blood indexes of 6-week-old rats were different from those of 16-week-old rats in RBC,HGB,TP,BUN,etc.The blood indexes of 12-week-old rats had no difference from those of 16-week-old rats in WBC,HGB and BUN.Conclusion The result show that the blood indexes of young SD rats at different stages have a certain time correlation.Establishing background data of different stages has a certain guiding significance for statistical analysis and discussion of blood indexes of young SD rats.

Efficacy of COX Inhibitors on Lumbar Disc Herniation in Rats

2019, 36(05):  47. 

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Objective To study the effect of cyclooxygenase inhibitor Fenbid on lumbar disc herniation.Method Rats were modeled by oppressing the dorsal root ganglion and transplanting the autologous tail nucleus pulposus to the lumbar spine.The COX inhibitor Fenbid was administered at a dose of 0.051 g/kg for 28 days.Animals were scored for motor function,mechanical stimulation retraction tests,animal thigh circumference measurements,and gait measurements.After 28 days of dosing,lumbar vertebrae (L4-L6)and their nerves were fixed with 10% neutral formalin and histopathological examinations were performed.The serum and nerve root tissue under pressure were measured for inflammatory factor concentrations.Result The COX inhibitor,Fenbid,can improve the animals' motor function score,50% mechanical paw withdrawal threshold,thigh circumference ratio,sciatic nerve function index (SFI),the pathological changes of dorsal root ganglion and intervertebral disc,reduce the levels of inflammatory factors (PGE2,COX-2,NO,IL-1β,IL-6)at the serum and lesion sites.Conclusion COX inhibitors improve the symptoms of lumbar disc herniation in rats.

Study on Periodic Lesions in Mice with Chronic Liver Injury

2019, 36(05):  54. 

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Objective To observe changes in liver damage induced by carbon tetrachloride (CCl4)induced chronic liver disease in mice.Method One week after normal feeding in normal BALB/c mice,0.5% CCl4 solution (10 μL/g,one time every three days for 10 weeks,lasting 10 weeks)was intraperitoneally injected.After 2,4,6,8,and 10 weeks of injection,the serum ALT and AST were detected,HE and Masson staining after liver fixation,observation of liver structure,cell morphology and fibrosis degree in mice.Result When CCl4 solution was injected for 2 weeks,the liver cell damage was mainly denatured with a large number of inflammatory cells infiltration and a small amount of collagen deposition,and ALT and AST were slightly elevated;At 4 weeks,hepatocyte injury was mainly necrosis (P<0.01),and the number of hepatocytes (P<0.01)and inflammatory cell infiltration decreased (P<0.01).Collagen deposition developed into stripe collagen fiber deposition,and ALT and AST increased significantly(P<0.01);At 6 weeks,necrotic hepatocytes were significantly reduced (P<0.01),the number of hepatocytes increased (P<0.01),the infiltration of inflammatory cells was further reduced (P<0.01),collagen fiber deposition was transformed to collagen deposition (P<0.05),ALT and AST were basically normal(P<0.01));At 8 weeks,the degeneration of hepatocyte was increased again,with a large number of inflammatory cell infiltration (P<0.01),and a slight increase in ALT and AST;At 10 weeks,hepatocyte injury was mainly necrosis (P<0.01).The number of hepatocytes (P<0.01)and inflammatory cell infiltration decreased (P<0.05).Collagen deposition developed into stripe collagen fiber deposition,and ALT and AST increased significantly (P<0.01).Conclusion The chronic liver injury model of mice induced by 0.5%CCl4 solution has obvious and regular liver injury cycle changes,and the degeneration stage,necrotic stage and remission stage of hepatocyte appear in turn.

Establishment of Real-time Fluorescent Quantitative PCR to Detect Various Mycoplasma Commonly Found in Laboratory Animal

2019, 36(05):  60. 

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Objective This study aimed at developing a new real-time fluorescent quantitative PCR method for detecting various Mycoplasma commonly found in laboratory animal.Method In this study,a pair of primers and a Taqman probe were designed based on conserved region of 16 s rRNA gene from various Mycoplasma which sensitive to mice and rats,and the amplification efficiency,sensitivity,specificity and repetition of this method were evaluated with standard plasmid.Result The result showed the standard curve had a good linear relationship (R2=0.9972),and the amplification efficiency was 98.11%.The lowest detection limitation of this method was 5 copies/μL,which was about 100 times higher than the conventional PCR.And this real-time fluorescent quantitatiuve PCR method had a strongly specificity for distinguishing the other 8 pathogenic strains.Moreover,it showed the excellent reproducibility with the coefficient of variation of Intra-assay and inter-assay less than 2%.Using this real-time fluorescent quantitative PCR and conventional PCR to detect a batch of clinical samples,the result showed that the positive rate of samples from mice was 3.33% (2/60)and 1.67%(1/60)respecyively,and the positive rate of both method of samples from cells was 5% (3/60).Conclusion In summary,the real-time fluorescent quantitative PCR method developed in this study was fast,specific and sensitive,and could be applied in detection for Mycoplasma of laboratory animal.

Establishment of a Standardized MuSK Myasthenia Gravis Mouse Model

2019, 36(05):  66. 

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Objective To Model a muscle-specific receptor tyrosine kinase antibody (MuSK-Ab)-positive myasthenia gravis mouse model according to international standardization tablets.Method Female C57BL/6 mice (40 mice)aged 6-8 weeks were conditioned for 1 week and randomly modeled.On the first day of modeling,immunization antigens were injected at four locations (two hind legs and two shoulders)(50 μL per site,containing 12.5μg of mouse peptide,CFA complete Freund’s adjuvant);modeling days 30 and 50 Immune antigens were injected at four locations (two hind legs and two shoulders)and the onset was scored daily after day 70.Result The model success rate is 50%.The score is 1 point for 1 mouse,2 points for 3,3 points for 5,4 for 1.Conclusion The preparation of the MuSK-Ab mouse model according to international standardization is helpful for in-depth study of myasthenia gravis.

Study of the Effects of Puhuasan on Chronic Renal Failure in Experimental Rats

2019, 36(05):  69. 

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Objective To study the effects of Puhuasan on chronic renal failure in rats.Method The rats were induced into chronic renal failure (CRF)by Platt,the Serum indexes like creatinine(Scr)、Urea nitrogen(BUN)、calcium(Ca)、phosphorus(P)、Parathyroid hormone(PTH)and middle molecule substence(MMS)were tested.Result The result showed that the function of renal were improved,high phosphorus and low calcium levels were adjusted,PTH and MMS were inhibited by Puhuasan.Conclusion Puhuasan is one of effective medicine to cure uremia,it’s function may be connected with improving the clinical symptoms and reducing toxin in CRF.

Analysis of Stability for Samples of Peptidase 3 in the Kidney Homogenate of Mice

2019, 36(05):  73. 

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Objective To investigate the samples stability of peptidase 3 in order to understand the storage condition and transportation condition of samples and to guarantee the samples to be qualified for the demand of proficiency test.Method On the basis of the national standard GB/T 14927.1—2008,samples were prepared,stability test were carried on 4 temperatures(-20 ℃，4 ℃，room temperature and 37 ℃)and 9 time points (1 d、2 d、3 d、4 d、5 d、6 d、7 d、14 d and 28 d).Result Peptidase 3 samples can be preserved only in 1 day at 37 ℃.At 4 ℃、room temperature and -20 ℃,Peptidase 3 samples can be stably preserved during 28 days.Conclusion The preservation and transportation conditions of the peptidase 3 samples must be controlled strictly during proficiency test.

Establishment of a Full-thickness Skin Grafting Model for Wistar Rats

2019, 36(05):  77. 

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Objective A method of establishing full-thickness skin graft model of Wistar rat was introduced to improve the success rate of Wistar skin grafting.Method Twelve clean Wistar rats,male,3 month old,and weight 300-330 g,were used to establish a full-thickness skin grafting model by weighing and anaesthesia,skin preparation and labeling,rat fixation and disinfection,cutting and pruning all thick skin slices,full thickness skin graft,and taking measures to avoid gnawing and scratching of rats.Result In the 12 Wistar rats,all the skin grafts on the lateral chest survived and no skin infection and necrosis occurred.After the operation,the rats were able to move freely and fed for 1-2 months without any animal death.Conclusion The experimental method established in this experiment is simple and effective,and provides a specific experimental method for relevant research,which is worthy of popularization and application．

Progress on Molecular Biology of Mouse Norovirus

2019, 36(05):  81. 

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This article elaborated the molecular biology and replication of mouse norovirus(MNV),It provides a theoretical basis for the research of prevention and control of MNV in mice.

Caorbon Dioxide for Euthanasia to Mice and Rats

2019, 36(05):  85. 

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Rats and mice are currently the most widely used laboratory animals,and their euthanasia method have been heatedly debated.The carbon dioxide euthanasia method has the advantages of simple use,low risk and low cost.In order to make the implementation of carbon dioxide euthanasia more in line with the requirements of experimental animal welfare ethics,the application of this technology should fully consider the pressure,pain and other reactions of experimental animals.This paper reviews the evaluation system of euthanasia by inhaling carbon dioxide,the combined use of carbon dioxide and other anesthetics,and the specific implementation method of carbon dioxide euthanasia in mice and rats.The purpose of this review is to provide useful technical references to caorbon dioxide for euthanasia to mice and rats