Table of Content

28 October 2022, Volume 39 Issue 5

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Effects of Chronic Stress Combined with Solitary Rearing on Behavior, Serum 5-HT and Hippocampal Neurons in Spontaneously Hypertensive Rats

CHEN Lei , LIU Yeqian , ZHAO Hongxia, LI Hong, WU Biru, ZENG Yong, MA Danfeng, CHEN Chunming , LIU Lin , REN Weiqiong , YI Jian

2022, 39(5):  1-6.  DOI: 10. 3969 / j. issn. 1006-6179. 2022. 05. 001

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Objective To observe the effects of chronic unpredictable mild stress ( CUMS) combined with solitary rearing on the behavior, serum 5-HT and neurons in the hippocampus of spontaneously hypertensive rats ( SHR) . Method Twenty SHR rats were randomly divided into SHR group and model group, and the same number of Sprague-Dawley ( SD) rats were set as the control group. Predictive stress (CUMS) combined with solitary rearing was used to intervene for 5 weeks, and the rest of the groups were fed normally. The caudal arterial blood pressure of rats in each group was measured on the 14th day and at the end of modeling, respectively. Open-field experiment and sugar water consumption experiment were performed. Forced swimming experiment was conducted after modeling, 5-HT content in serum was detected by ELISA, and activity and arrangement of hippocampal neurons and the number of neurons’ Nissl bodies were observed by HE staining and Nissl staining. Result Compared with the control group, the blood pressure of SHR group increased significantly ( P < 0. 01) , body weight growth rate increased obviously(P<0. 01) , the level of activity and the number of upright, sugar water consumption rate and forced swimming fixed time, 5-HT content there was no significant difference (P>0. 05) , roundness, hippocampal neurons form order, nissl bodies and more dense; Systolic blood pressure of model group was significantly increased (P< 0. 01) , body weight growth rate was not significantly different ( P > 0. 05) , horizontal activity and upright times, sugar water consumption rate were significantly decreased (P<0. 05, P<0. 01) , forced swimming immobile time was significantly increased (P<0. 01) , 5-HT content was significantly decreased (P<0. 01) . The arrangement of hippocampal neurons was loose and the nite body was apoptotic. Compared with SHR group, systolic blood pressure in model group was not significantly increased (P>0. 05) , body weight growth rate, horizontal activity and upright times, sugar water consumption rate were significantly decreased ( P<0. 01) , the immobile time of forced swimming was significantly increased ( P < 0. 01 ) , 5-HT content was significantly decreased ( P < 0. 01 ) , the arrangement of hippocampal neurons was loose, and Nissl bodies apoptosis. Conclusion Chronic unpredictable stress (CUMS) combined with solitary rearing induced depression-like changes in behavior, neurotransmitters and hippocampal neurons in SHR rats.


Study on the Anti-inflammatory Mechanism of Nigella Glandulifera Freyn Seeds from the View of Network Pharmacology

ZHAO Kaiyue, LIU Mimin, ZENG Li, JIANG Hailun, LI Zhuorong, XU Fang , LIU Rui

2022, 39(5):  7-14.  DOI: 10. 3969 / j. issn. 1006-6179. 2022. 05. 002

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Objective To study the anti-inflammatory mechanism of Nigella glandulifera seeds with the tools of network pharmacology. Method Component targets were predicted using literature searching and the Swiss Target Prediction database. The disease targets were collected using OMIM, DrugBank, Genecard databases. The common targets were analyzed using Venny 2. 1. 0 and STRING software. According to the average degree value, important targets were taken out. A common component-target interaction network was created using the Cytoscape software. The biological function enrichment and signaling pathway enrichment analysis of the important targets were performed through Metascape. Result Fifty-seven components were collected, and 101 important interaction targets of drugs and diseases were predicted. Enrichment of biological functions illustrated 111 cell components, 139 molecular functions, 2 104 biological processes, and 165 signaling pathways. Cell components were mainly membrane components, and the principal molecular functions were focused on the G protein-coupled receptors. Biological processes involved kinases, positive regulation of transferase activity, and regulation of the MAPK cascade. Pathway enrichment predicted that the Th17 cell differentiation pathway referred to IL-6 and STAT3 as key genes were closely related to the anti-inflammatory effects of Nigella glandulifera seeds. Conclusion Given the characteristics of multi-component, multi-target, and multi-pathway of the traditional Chinese medicine, the potential anti-inflammatory mechanism of Nigella glandulifera seeds was analyzed using network pharmacology approach so as to provide the basis for further study. However, the key target and the specific regulatory mechanism need to be further verified.


Significance of Evaluating Hip Joint Development through X-ray in Early Screening of Guide Dogs

DONG Jianyi, LI Changyi, BAI Jing, WANG Fujin, WANG Jingyu, ZHOU Zijuan

2022, 39(5):  15-19.  DOI: 10. 3969 / j. issn. 1006-6179. 2022. 05. 003

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Objective Guide dogs are working dogs that help visually impaired persons travel safely. Method This study collected X-ray films of 160 dogs( male Labradors 86, female Labradors 52; male golden 14,female golden 8) from China Guide Dog Training Centre during the year 2018—2021. The developmental status of hip joint was analyzed by classification of hip dysplasia in dogs of different breeds and genders. Result The hip joint of 130 dogs (81. 25%) developed very well, while 30 dogs (18. 75%) had different degrees of dysplasia. We used Chi-squared Test for data analysis. There was a significant difference between Labrador retrievers and golden retrievers ( χ2=21. 45,P<0. 01) , while the difference between male and female dogs was not significant ( χ2= 0. 09,P > 0. 05). For the 30 dogs with hip dysplasia, there was no significant difference between 2 breeds ( χ2= 1. 53,P > 0. 05). Conclusion X-ray examination can effectively screen the development of hip joint in dogs aged 12-14 months.





Effect of Naoshuantong Capsule on Angiogenesis in Rats with Focal Cerebral Ischemia Reperfusion

GUO Min , LIU Tingting, WU Zheng, TIAN Xin, ZHU Zixin, ZHENG Wenrong, WANG Yufeng , ZHENG Ruifang , XING Jianguo, SUN Fangling, WANG Wen

2022, 39(5):  20-25.  DOI: 10. 3969 / j. issn. 1006-6179. 2022. 05. 004

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Objective To study the effect of Naoshuantong capsule on angiogenesis in rats with focal cerebral ischemia reperfusion. Method Healthy adult male SD rats were induced to develop occlusion of the middle cerebral artery by suture embolus, Then, the rats were randomly divided into the sham group, a model group, and a low-dose Naoshuantong capsule and high-dose Naoshuantong capsule groups ( 1. 05, 2. 62 g / kg, respectively) . Naoshuantong capsule was administered once per day for 7 days, from 24 h after surgery a day. To examine the recovery of neurological function, we measured modified neurological severity score (mNss) method ; the infarction volume was measured by 2, 3, 5-triphenyltetrazolium chloride ( TTC) staining technique; Western blot was used to detect the expression of angiogenesis related proteins in rat cerebral cortex; Angiogenesis was detected by immunofluorescence staining. Result Compared with those in the sham group,the scores of neurological behavior and the cerebral infarction volume ratios in the model group were significantly increased ( P < 0. 001) . Compared with that in the model group,the low and high-dose Naoshuantong capsule groups exhibited a dramatic improvement of the neurological behavior scores ( P < 0. 05 ) , Naoshuantong capsule high-dose groups exhibited a significant reduction in the volume of cerebral infarction ( P < 0. 05 ) . Compared with the model group, the expression of cortical angiogenesis-related proteins vascular endothelial growth factor (VEGF) and basic fibroblast growth factor ( bFGF) in the Naoshuantong capsule low and high-dose groups were significantly increased( VEGF:P< 0. 05,P< 0. 001;bFGF:P < 0. 05,P < 0. 001) ; Compared with the model group, the number of Lectin + / Ki67 + cellls were significantly increased ( P < 0. 05, P < 0. 001 ) . Conclusion Naoshuantong capsule may promote angiogenesis to improve neurological function after ischemia in rats.


A New Sandwich ELISA Method for Polymyxin-resistant Escherichia coli Based on Synthetic LPS Receptor Peptide

DUAN Yueqing, KE Shuwen, YANG Hua , TANG Biao , LIU Yuehuan

2022, 39(5):  26-30.  DOI: 10. 3969 / j. issn. 1006-6179. 2022. 05. 005

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Objective A small peptide-based sandwich ELISA method was established for the detection of polymyxin E. coli resistance. Method Based on the principle of polymyxin-resistance, and the sequence and spatial structure of lipopolysaccharide ( LPS ) receptor complex in the E. coli. A sandwich ELISIA assay was established, and a detection kit was assembled by coating the 96-well plate with 9 peptides, in which samples were then added to be tested. The carboxyl -terminal labeled 13 peptide and HPR-labeled streptavidin were used as the primary antibody ( working concentration 1 ∶ 1 000) and secondary antibody ( working concentration 1 ∶ 2 000- 10 000) , respectively. Except the laboratory-purified exponential phase polymyxin-resistance positive E. coli samples, field samples ( including soil, river water and manure from breeding farms) , egg yolk powder and experimental animal drinking water were also tested. Result Using the ELISA assay, laboratory-purified exponential bacteria, field samples, egg yolk powder and experimental animal drinking water all could be detected. And, the sensitivity, specificity, precision and repeatability of the kit all achieved the expected goals. Conclusion The sandwich ELISA assay improved the standardization of drug-resistant bacteria detection.

Exploration of Normal Range of Blood Physiological and Biochemical Indexes in Cats and Analysis of Influencing Factors

WANG Zhouxiang, HU Manli , CHEN Yun, DING Yi , ZHAO Deming, HE Zhengming, ZHANG Xin, WAN Juan

2022, 39(5):  31-36.  DOI: 10. 3969 / j. issn. 1006-6179. 2022. 05. 006

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Objective The purpose of this investigation is to explore the range of blood physiological and biochemical indicators of healthy cats ( in good condition without reported disease history) , and the influence of factors including gender and age on these indicators, further to promote the standardization of cats as experimental animals. Method Domestic cats were recruited from Wuhan city as the research subjects, and 75 healthy cats were screened by completely random method . Blood was collected to detect blood cell count and blood biochemical indicators. Then statistical analysis was performed. Result The range of blood cell count indexes and blood biochemical indexes of healthy cats were preliminarily clarified. It was found that the blood cell count and most physiological and biochemical indexes excluding AST ( P< 0. 05) were not significantly influenced by age but not gender of cats. The white blood cell, monocyte, and neutrophil counts had a decrease tendacy with age. Contrarily, hematocrit and average hemoglobin concentration in red blood cells were all increased with age. As the age increasing, the concentration of phosphorus and calcium ions in blood showed a downward trend, while the indicators such as creatinine, cholesterol, globulin and random blood sugar showed an upward trend. Conclusion This study preliminarily explored the normal range of cat’ s blood physiological and biochemical indicators, and found that theses indicators were significantly affected by aging. The result provided a basis for promoting cats as standard experimental animals. Meanwhile, this study suggests that cats of different ages have different nutritional needs, providing theoretical guidance for precision feeding and health care for cats.


Exploration of Bama Pig Ultra-long Time Combined Anesthesia in Complicated Operation of Experimental Surgery

PENG Xu, YIN Hailin

2022, 39(5):  37-44.  DOI: 10. 3969 / j. issn. 1006-6179. 2022. 05. 007

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Objective Explore a combined anesthesia more than 6 hours for Bama pigs and explore the effectiveness of complicated surgical procedures used in experimental surgery. Method Combining four main medicines: Zoletil, Dexdomitor, Isoflurane, Butorphanol to perform single kidney removal and orthotopic transplantation after renal vein occlusion, deep rectal resection and anastomosis, pancreatic resection + spleen removal + gastric perforation anastomosis. Monitor the vital signs of Bama pigs, as well as sedative, analgesic, and muscle relaxation effects during the entire anesthesia process, and also analysis combined with death cases to evaluate the anesthetic effect of the program. Result 1 Bama pig died of hypovolemic shock, the rest of 12 Bama pigs successfully completed more than 6 hours abdominal surgery, no adverse reactions. Heart rate, blood oxygen saturation, mean arterial pressure, body temperature were stable, and both fluctuate within the normal range. Conclusion This anesthesia program can ensure the anesthesia time of the Bama pig exceed 6 hours, and can successfully complete the operations of abdominal organ removal, blood vessel dissection and anastomosis, tissue cutting and suture by surgical robot. During the operation, the anesthesia is stable, the effect of sedation and analgesia is excellent, pigs wake up quickly post-operation. The four main anesthetics are veterinary drugs, and the storage time is longer after the first use, which fully meets the requirements of experimental surgery for extra-long time and complicated operations.


Effect of Social Isolation on Behavioral Characterization of C57BL / 6 Mice

WU Na, XIAO Fuchuan, ZHANG Minghao, CHEN Bai’ an, LU Jing

2022, 39(5):  45-49.  DOI: 10. 3969 / j. issn. 1006-6179. 2022. 05. 008

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Objective To investigate the effect of social isolation on behavioral characterization of C57BL / 6 mice. Method Sixty 2-month-old C57BL / 6 mice were randomly divided into control group and social isolation group, with 30 mice in each group, half male and half female. Mice in the control group were every 5 / cage, and mice in the social isolation group were every 1 / cage. After 8 weeks, nest building experiment, water maze experiment and open field experiment were carried out. Result Compared with the control group, the score of mice in the social isolation group was significantly lower in the nesting experiment (P<0. 05) . The times of crossing the platform, the times of entering the target quadrant, the percentage of residence time in the target quadrant and the percentage of swimming distance in the target quadrant in the social isolation group were significantly lower in the water maze experiment ( P < 0. 05 ) . The total times of crossing, the total moving distance and the average moving speed increased significantly, and the resting time decreased significantly ( P < 0. 05 ) in the open field experiment. Conclusion The executive ability of daily activities and learning and memory ability of C57BL / 6 mice decreased, and the ability of autonomous movement and inquiry behavior increased after 8 weeks of social isolation.


Detection of Hair Color Genotype in Rats with Spontaneous Congenital Cataract

DONG Min, YOU Jinwei, LIANG Lei, WU Zhenglin, TIAN Xiaoyun

2022, 39(5):  50-52.  DOI: 10. 3969 / j. issn. 1006-6179. 2022. 05. 009

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Objective To determine the homozygosity of hair color gene locus and hair color genotype of newly bred spontaneous congenital cataract ( TSC ) rats. Method The hair color gene was tested by hybridization and backcross test between TSC adult rats of F29 generation and F344 and BN rats with known complex recessive genes. Result The homozygosity of hair color loci A, B, C, D in TSC rats was 100%, and the hair color genotype was AABBcc. Conclusion According to the national standard, the hair color gene of TSC rats has reached homozygosity, which is in line with the inbred line quality standard.


A Comparative Study of Adult Clonorchis Sinensis Parasitized in Experimental Mice and Humans

LIU Liu, ZHANG Fengyu, ZHANG Jing, ZHANG Hao

2022, 39(5):  53-56.  DOI: 10. 3969 / j. issn. 1006-6179. 2022. 05. 010

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Objective To explore the developmental maturity of Clonorchis sinensis in experimental mice, and to understand whether it is different from the naturally infected C. sinensis in humans. Method Eight experimental mice were intragastrically infected with C. sinensis cysticercosis. After 3 weeks of infection, the mouse feces were examined by washing and sedimentation method every other day. After the eggs were detected in the feces, the mice could be killed to obtain the worms. The parasites obtained from mice were stained with acetic magenta, and the specimens were compared with the specimens of C. sinensis obtained from human natural infection to measure the overall and internal structure. Result C. sinensis matured in 23 days in experimental mice. The whole size, oral sucker, abdominal sucker, pharynx, uterus, ovary, seminal vesicle, testis and eggs of C. sinensis adults obtained from mice were smaller than those obtained from humans. Conclusion C. sinensis can mature in mice, but it is smaller than the adult worms obtained in humans.


Measurement and Analysis of Conservation and Reproductive Performance in Four SPF Rats

ZHU Wanyue, ZUO Qin, LIANG Chunnan, LIU Zuomin

2022, 39(5):  57-61.  DOI: 10. 3969 / j. issn. 1006-6179. 2022. 05. 011

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Objective To determine the propagation level of four SPF rats ( SD, Wistar, F344 and BN) stored in national rodent experimental animal repository. Method The closure of the closure using the recording parallel line mating method using the cycle mating method.  The average number of vegetation and the size of the four-day tires of the four kinds of spine rats. The average number of vibe and the number of daily. And recorded the initial mating age, the first production age, the initial production and intervening of the tires. Result The average number of ventricles in SD rats was 12 to 13, the first 2-breeding level was basically maintained relatively stable, the result of the detection of the three, the four testers was reduced in the first 1-25. The residual rate of SD rats was 98. 2%; the initial production of the SD rats was 84, 110. 7, 26. 7 d, the tires of the tires of 26 d to and 31 d. Wartar rats were 10-11, the average number of the first 2 tires, the average number of vibe average and the level of the system is relatively stable, 3,4 tires detection result showed that the first and second tension showed the trend of the production of the various types of tires (P<0. 05) ; the initial mating age of the Wistar rats was 98. 3%; the initial production age of the Wistar rats was 91, 118. 5, 27. 5 d, respectively, the tires were 25 to 30 d. F344 rats first to four quarters of the average number of 8, the first three trials of each product, the average number of vibes and the basis of the basic maintenance of the relatively stable, the fourth tone detection index than the front three trials showed a significant decline in phenomenon ( P < 0. 05) ; variance analysis result also showed a significant difference in the number of hunting of the enemy (P<0. 05) ; the first mid-term age of F344 rats was 98. 4%; the first production age of the F344 ratio was 92. 7, 122. 7, 30 d, the tires of the various times of the tires were 27 degrels and 40 d. BN rats first to four quarters of the average number of 5 to the average number of four-day, the average number of vibe average and the number of daily system is relatively stable, and the ratio of BN rats was 98. 9%; the initial mating age of the BN rats was 82. 8, 124. 6, 41. 8 d, respectively, and the tires of the tires of the tires of 33-37 d. Conclusion The proportion of 4 kinds of SPF rats in the resource library was significantly higher than the proportion of the proportion of the breeding levels of the scale production and national rodents of the national rodents.

Construction and Characterization of OmpA Genes Deletion Mutant of Rodentibacter heylii

XING Jin, FENG Yufang , ZHANG Xueqing, GAO Qiang, ZHAO Deming , YUE Bingfei

2022, 39(5):  62-68.  DOI: 10. 3969 / j. issn. 1006-6179. 2022. 05. 012

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Objective To explore the role of OmpA gene in the pathogenicity of Rodentibacter heylii. Method The homologous recombination technique was used to construct the OmpA gene deletion strain of R. heylii. Its growth characteristics, genetic stability, biochemical characteristics, mass spectrometry characteristics, biological development and effects on mouse lung type Ⅱ epithelial cell line ( MLE-12 cells ) Adhesion; intraperitoneal injection of the OmpA gene-deficient strain to verify its pathogenicity to mice. Result The OmpA gene deletion strain of R. heylii was successfully constructed. The gene deletion strain could be inherited stably. Compared with the wild-type strain, the biochemical characteristics did not change, the mass spectrogram and growth curve were basically the same, and the biofilm formation ability was significantly enhanced ( P<0. 01) ; Adhesion to MLE-12 cells decreased from 43. 9% to 18. 3% (P<0. 001) ; Intraperitoneal challenge to mice, the lethality rate decreased from 100% to 20% ( P < 0. 01 ) . The pathogenicity of the gene deletion strain to mice decreased significantly. Conclusion The deletion of the OmpA gene significantly weakened the pathogenicity of R. heylii, confirming that OmpA plays an important role in the infection process of R. heylii.


Establishment of Loop-mediated Isothermal Amplification Method for Aspiculuris Tetraptera

HUANG Jian, FENG Yufang, XING Jin, WEI Jie, LI Xiaobo, YUE Bingfei

2022, 39(5):  69-71.  DOI: 10. 3969 / j. issn. 1006-6179. 2022. 05. 013

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Objective To establish a rapid and efficient loop mediated isothermal amplification method for aspiculuris tetraptera. Method The LMAP primers were designed according to the published COI sequence of aspiculuris tetraptera, and Giardia lamblia and water were used as controls for specificity evaluation. The genomic DNA of aspiculuris tetraptera was diluted in series to verify its sensitivity. LAMP products were identified by fluorescent color and agarose gel electrophoresis. Result The LAMP product is colored by fluorescence and the result is green, and the brown is negative. After electrophoresis, the LAMP products of aspiculuris tetraptera showed characteristic ladder-like bands. There was no band in the control group. Conclusion A simple method for detecting aspiculuris tetraptera by LAMP assay was established.


Observation and Analysis of Pathological Changes of Spleen in Rodent Laboratory Animal Pathological Examination

ZHAO Mengyang, YANG Lifeng, ZHAO Deming, ZHOU Xiangmei

2022, 39(5):  72-74.  DOI: 10. 3969 / j. issn. 1006-6179. 2022. 05. 014

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Objective In order to provide suggestions for future laboratory animal inspection and feeding, we observed the pathological changes of spleen in rodent laboratory animals in Beijing in October 2020, and analyzed the possible causes of pathological changes. Method After euthanasia, the spleens of the animals were taken and sectioned for HE staining. Pathological observation and diagnosis were made by light microscope. Result A total of 360 rodents ( including 170 mice, 80 rats, 100 guinea pigs and 10 hamsters) were tested. Among them, 10 SPF ICR mice and 10 SPF SD rats from different institutions showed pathological changes, such as the decrease of white pulp cells, the increase of extramedullary hematopoiesis and macrophages of red pulp, and so on. Conclusion The pathological changes of spleen in sampling examination may be related to the age of laboratory animals, long-term exposure to certain toxic substances, autoimmune deficiency, infection or tumors. Therefore, in the sampling inspection of the quality of laboratory animals, institutions should avoid sending young or old animals for inspection, and at the same time further improve the feeding and management system to ensure the health of laboratory animals.





Application Research on the Establishment of Experimental Animal Management Mode under the Guidance of Blockchain Technology

LI Yunze, CHEN Hong

2022, 39(5):  75-78.  DOI: 10. 3969 / j. issn. 1006-6179. 2022. 05. 015

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Objective Application of blockchain technology improve the management mode of experimental animals. Method We tried to apply blockchain technology in the management of experimental animals, including distributed text technology, asymmetric encryption and authorization technology, consensus mechanism and smart contract, etc. , to improve the management level of experimental animals, and carried out feasibility evaluation through analysis and discussion. Result Process management of experimental animals using blockchain technology can effectively improve efficiency and save labor and economic costs. At the same time enhance security, conducive to national supervision. Conclusion The use of blockchain technology can effectively improve the management level of experimental animals, establish a new management mode, and provide reference for the management of experimental animals.





Application of Germ-free Animals in Gut Microbiome Research

ZHU Xi, TAN Xiang, WU Zhifeng, YANG Yapeng, CHENG Wei, WEI Hong

2022, 39(5):  79-83.  DOI: 10. 3969 / j. issn. 1006-6179. 2022. 05. 016

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The gut microbes are numerous and variety. As a “ functional organ” , gut microbes play an important role in nutrition metabolism, immune response and gut protection. The research on complex gut microbes relies on germ-free animal models, and the development of germ-free animal technology has push on the research of gut microbes in turn. They promote each other and develop in coordination, which not only helps us to understand intestinal function microbiome deeply, but also promotes the germ-free animal technology system to be more mature. In view of the close relationship between them, this review focuses on the application of germ-free animal in livestock microbiome and nutritional microbiome, as well as briefly discusses the establishment of germ-free animal system and the limiting factors of it, which has set the direction for promoting the close cooperation and common development of germ-free animal technology and microbiome.


Potential Impact of Feed Differences on Animal Research

LI Zhiping, ZHAO Deming, ZHOU Xiangmei, YANG Lifeng, QIAO Jian

2022, 39(5):  84-88.  DOI: 10. 3969 / j. issn. 1006-6179. 2022. 05. 017

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Feed plays a central role in the physiological development of laboratory animals. High-quality animal feed can optimize growth, maintenance, and reproduction in most species. However, feeds are highly variable, and differences in feed quality and composition may affect animal / population health, and may introduce complex variables in animal-based studies, thereby affecting the reproducibility and reproducibility of studies. This article summarizes the sources of experimental animal feed differences, discusses how feed as an external factor in animal experiments affects animal health, research design, and reproducibility of experimental result, and on this basis, provides reference for how to mitigate these effects.