Table of Content

28 April 2023, Volume 40 Issue 2

Previous Issue   

Comparison of Four Different Immunosuppression Models Induced by Cyclophosphamide in Mice

YANG Yang, LI Gen, MA Jingwei, ZANG Caixia, BAO Xiuqi, ZHANG Dan

2023, 40(2):  1-9.  DOI: 10. 3969 / j. issn. 1006-6179. 2023. 02. 001

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Objective The aim of present study is to compare the effects of four different modeling method of cyclophosphamide on the immune function of mice. Method Four different ways were employed to develop immunocompromised mouse models by cyclophosphamide, Group A: mice were intraperitoneally injected with cyclophosphamide 80 mg / kg on days 1, 3, and 5, Group B: mice were intraperitoneally injected with cyclophosphamide 60 mg / kg for 5 consecutive days, Group C: mice were intraperitoneally injected with cyclophosphamide 60 mg / kg on days 1, 3, 5, 7, 9, 11 and 13, Group D: mice were intraperitoneally injected with cyclophosphamide 60 mg / kg on days 1, 4, 7, 10 and 13) . The general immunological indexes of mice ( body weight, thymus index, spleen index, whole blood leukocyte and lymphocyte count ) , cellular immunity ( delayed allergic reaction ) and humoral immunity ( determination of half hemolysis value) were observed and compared in the four different models. Result In each experimental group, the relevant detection indexes changed in varying degrees. Compared with the blank control group, the body mass, thymus index, spleen index and WBC count of mice in experimental group A and model group decreased by 19. 15%, 67. 69%, 67. 59% and 83. 87% respectively on the 7th day, while the body mass of mice in model group decreased by 17. 91%, while the thymus index, spleen index and WBC count increased by 7. 14%, 173. 89% and 67. 76% respectively on the 14th day; Compared with the blank control group, the body mass, thymus index and spleen index of mice in experimental group B and model group decreased by 15. 64%, 64. 91% and 64. 60% respectively on the 7th day, and the body mass and thymus index of mice in model group decreased by 17. 86% and 48. 00% respectively on the 14th day, while the spleen index increased by 148. 80%; Compared with the blank control group, the body mass, thymus index, spleen index, WBC count and lymphocyte count of mice in experimental group C and model group decreased by 11. 74%, 82. 61%, 37. 42%, 88. 40% and 91. 44% respectively; Compared with the blank control group, the body mass, thymus index, spleen index, whole blood leukocyte count and lymphocyte count of mice in experimental group D and model group decreased by 10. 83%, 68. 31%, 14. 34%, 66. 15% and 73. 42% respectively. In DTH experiment, the weight difference between left and right ears of immunosuppressive mice decreased by 66. 88%, and in humoral immunity, the serum HC50 of immunosuppressive mice decreased by 74. 52%. In the experiments of group A and B, the relevant detection indexes recovered in varying degrees on the 14th day compared with the 7th day. Meanwhile, the immunosuppressive state of group A mice was more serious in the short term, while the immunosuppressive state of group B mice remained more lasting. Conclusion The above four method could successfully establish immunosuppressive model in mice, and the times, dose, interval days of intraperitoneal injection of cyclophosphamide and the detection endpoint will affect the effect of the immunosuppressive state of mice. So the appropriate model can be selected according to the specific evaluation requirements of drugs.

Comparison of Methods for Constructing of Non-alcoholic Fatty Liver Disease Models Based on ob / ob Mice

ZHANG Rufeng , XIONG Xueyang , LI Xuecheng , NIU Yunchao , WANG Zhiwen , YU Changyuan

2023, 40(2):  10-16.  DOI: 10. 3969 / j. issn. 1006-6179. 2023. 02. 002

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Objective To compare the characteristics of several modeling method and to find an optimal model for non-alcoholic fatty liver disease on the basis of ob / ob mice. Method Firstly, the second and third exons of leptin gene were knocked out in the background of C57 mice to obtain leptin knockout mice (B-ob / ob mice) , and then HFMCD induction, WD induction, WD combined with carbon tetrachloride induction were performed on the basis of B-ob / ob mice. The levels of ALT, AST, triglyceride and cholesterol in the serum of the mice were analyzed under these induction method; the NAS score of NAFLD was assessed by pathological HE staining; the degree of liver fibrosis was assessed by sirius red staining, and the degree of inflammation and fibrosis in the liver was also quantified by immunohistochemical staining. Result B-ob / ob mice had some fat accumulation and ballooning in the liver under normal dietary conditions without any induction. After 6 weeks of HFMCD induction, liver ALT was significantly elevated and liver fibrosis was increased. 12 weeks after WD induction, HE staining analysis showed some degree of intralobular inflammation in the liver in addition to steatosis and ballooning. After WD combined with carbon tetrachloride induction, HE staining showed that liver steatosis, ballooning and intralobular inflammatory features were more obvious, and liver fibrosis was also significantly increased, immunohistochemical result showed a significant increase in F4 / 80, a marker of hepatic macrophages, and α-SMA, a marker of hepatic stellate cell activation. Conclusion B-ob / ob induced by the combination of WD and CCL4 can mimic the pathological features of NAFLD more comprehensively in a short period of time, and is a better animal model of NASH.


Investigation of Monkeypox Virus Infection in Laboratory Animals in Beijing

LI Xiaobo, WANG Ji, WANG Shujing, WANG Shasha, LI Wei, QIN Xiao, YUE Bingfei, FU Rui

2023, 40(2):  17-21.  DOI: 10. 3969 / j. issn. 1006-6179. 2023. 02. 003

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Objective To investigate monkeypox virus infection in lab animals in Beijing. Method Refer to the guidance of the World Health Organization ( WHO) for laboratory testing of monkeypox virus, a total of 642 animals in Beijing were detected by fluorescent PCR, including 127 monkeys, 215 mice, 80 rats, 40 guinea pigs, 10 hamsters, 85 rabbits and 85 dogs, Pharyngeal swabs were collected from monkeys, cecum was collected from rats, guinea pigs and hamsters, and anal swabs were collected from rabbits and dogs. The collected samples were added with appropriate amount of sterile PBS, The cecum samples were fully homogenized, the swab samples were swirled for 1 min, repeatedly freezethawed at -70 ℃ for 3 times, the supernatant was centrifuged to extract nucleic acid, and the monkeypox virus DNA was detected by fluorescent PCR, and the result were analyzed finally. Result 127 monkeys, 215 mice, 80 rats, 40 guinea pigs, 10 ground mice, 85 rabbits and 85 dogs were all negative for monkeypox virus DNA by fluorescent PCR. Conclusion Monkeypox virus infection has not been found in lab animals in Beijing at present.

CD3 Monoclonal Antibody Induces Humanized Cytokine Storm Mice Model

LIU Chanchan , YAO Yuanyuan , WANG Zhongqing , ZHANG Libo , LI Ting

2023, 40(2):  22-24.  DOI: 10. 3969 / j. issn. 1006-6179. 2023. 02. 004

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Objective Establishing CD3 monoclonal antibody to induce humanized cytokine storm mice model. Method The peripheral blood mononuclear cells (PBMCs) of healthy adults were extracted, and NSG mice aged 10-12 weeks after intravenous injection were randomly divided into 2 groups: (1) PBMCs group (2) OKT3 (CD3 monoclonal antibody, anti-CD3 mAb, 0. 1 mg / mL) + PBMCs. The experimental group was injected with CD3 antibody 0. 5 mg / kg 6 days later, and serum TGF-β, CTLA-4, IL-10, IL-2, INF-γ, IL-7, and IL-6 levels were detected by ELISA 6 hours later. Result Compared with the control group, the use of OKT3 can effectively induce the cytokine storm IL-2 (P<0. 001), INF-γ (P<0. 001), TGF-β (P<0. 001), IL-6 ( P < 0. 001), IL-7 ( P < 0. 001) and CTLA-4 ( P < 0. 001). Conclusion The cytokine storm NSG mouse model constructed this time is conducive to the further study of the disease.


Isolation, Culture and Identification of Cardiac Fibroblasts from Suckling Rat of Naked Mole Rats

ZHANG Chengcai, ZHANG Qianqian, LI Leichen, JIANG Xiaolong, SUN Rui, CUI Shufang

2023, 40(2):  25-29.  DOI: 10. 3969 / j. issn. 1006-6179. 2023. 02. 005

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Objective To establish the method for the isolation, culture and identification of cardiac fibroblasts from naked mole rat suckling mice in vitro. Method Neonatal rat hearts were isolated on ice from naked mole rats born 1 to 3 days after birth. The hearts were cut into 1 mm×1 mm×1 mm pieces with ophthalmic scissors. Cardiac fibroblasts were isolated by digestion with trypsin and type II collagenase successively. Morphological characteristics of cardiac fibroblasts were observed under light microscope, proliferation activity was detected by CCK-8 assay, vimentin expression was detected by Western blot and immunofluorescence assay. Result Cardiac fibroblasts isolated by enzyme digestion were completely adherent in primary culture for 4 h, and the whole 35 mm dish could be covered in 36 h. The proliferation activity of cardiac fibroblasts was the highest after 2 to 3 days of subculture, and the expression of vimentin was confirmed by Western blot and immunofluorescence. Conclusion Cardiac fibroblasts isolated by trypsin and type II collagenase digestion can obtain cardiac fibroblasts with good viability and high purity.


Comparison of Basic Physiological Indexes Between SPF and Conventional Beagle Dogs at Different Growth Time

ZHANG Jinyu, CHEN Jian, HU Jinping, QUAN Fushi, REN Wenzhi

2023, 40(2):  30-34.  DOI: 10. 3969 / j. issn. 1006-6179. 2023. 02. 006

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Objective To analyze the dynamic change of basic physiological indexes between SPF and conventional beagle dogs at different growth time. Method The blood samples of 2-month-old, 6-month-old and 9-month-old were collected between SPF and conventional beagle dogs. Result The MCH and WBC of 6-month-old SPF beagle dogs were different significantly compared with those in conventional beagle dogs. The MCV, MCH and MPV of 9-month-old SPF beagle dogs were significantly lower than those in conventional beagle dogs. With the increase of months, the basic physiological indexes between SPF and conventional beagle dogs began to stabilize. Conclusion Basic physiological indexes of beagle dogs are affected by their different backgrounds.


White Light-induced Retinopathy is Influenced by Multi-factors in Rats

ZHOU Chenghao , CHEN Wenpei , QIN Mohan , CHEN Dixin , GUO Qiuping, LIN Baoqin , SANG Chuanlan

2023, 40(2):  35-41.  DOI: 10. 3969 / j. issn. 1006-6179. 2023. 02. 007

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Objective To asssess the optimal conditions for establishing the white light-induced retinopathy. Method Firstly, SD rats were exposed to white light for the same irradiation time during daytime or nighttime. Secondly, rats of different genders were exposed to light for the same irradiation time. Thirdly, rats were topically administered with or without tropicamide and then exposed to light. Flash eletroretinogram ( FERG) was recorded to evaluate retinal function after light exposure ( LE) . In addition, rats were exposed to light for 4, 8, 12, 16, 20 and 24 h. FERG was recorded at 7, 14 and 21 days after LE. Hematoxylin and eosin staining of retinas was also carried out. Result There were no changes in rats received irradiation during daytime, but retinal function was remarkably decreased in rats exposed to white light during nighttime. No difference was found in retinal function between male and female rats after receiving LE. There was no difference in retinal function between LE+tropicaine group and LE group. Fourteen and twenty-one days after LE, retinal function was significantly reduced by 30% - 50% and outer nuclear layer ( ONL ) of retina was decreased by about 40% in rats exposed to light for 8 - 20 h. However, visual function was almost completely lost, retinal cells in inner nuclear layer and ONL were obviously disappeared, and ONL thickness was even reduced by about 80% in rats received 24 h irradiation. Conclusion A rat model of white light-induced retinal damage was established to evaluate the effect of drugs on retinal functional and structural damage.


Comparative Study of Three Mouse Pain Models

GUO Qiuping, CHEN Dixin, HUANG Jisheng, JIA Dan, NI Qingchun

2023, 40(2):  42-48.  DOI: 10. 3969 / j. issn. 1006-6179. 2023. 02. 008

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Objective Research, comparison, and analysis of three mouse pain animal models. Method we established and compared three different analgesic mouse models ( acetic acid induced writhing model in mice, formaldehyde induced pain model in mice, and acid induced chronic pain model) to analyze sensitivity, pathological characteristics and drug efficacy evaluating ability. Result In the acetic acid pain model, mice showed significant pain responses, which could significantly evaluate the analgesic effect of neurotropin at 10 times dilution of clinical dose; formaldehyde pain model mice can significantly exhibit both phase I and phase II pain responses, and this model can also significantly evaluate the analgesic effect of neurotropin at 10 times dilution of clinical dose; the mouse model of chronic acid pain exhibits stronger ability to evaluate the analgesic effect, which can significantly evaluate the analgesic effect of neurotropin at 60 times dilution of clinical dose. Conclusion By comparing the pain performance and analgesic efficacy evaluation ability of three different pain mechanism models, the acetic acid pain model and formaldehyde pain model in mice did not show significant differences in analgesic ability evaluation. The selection of the two models can mainly be based on the analgesic mechanism of the drug; the chronic pain model is more sensitive to the response of analgesic drugs and can be prioritized for use in the exploration of analgesic drug dosage.

Establishment and Comparative Analysis of Collagen-Induced Arthritis(CIA) and Adjuvant-Induced Arthritis(AA) Rat Models of Rheumatoid Arthritis

LI Rixu, YE Xiaotong, CHEN Jianzhao, DU Qingyun, GUO Qiuping

2023, 40(2):  49-60.  DOI: 10. 3969 / j. issn. 1006-6179. 2023. 02. 009

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Objective To investigate the significance of rheumatoid arthritis (RA) related animal models for researching pathogenesis, influencing factors and treatment of RA. Method We established the CIA and AA rat models of RA to analyse their model stability and repeatability. In addition, we also treated rats in these two model groups with methotrexate and laflumeter to evaluate the drug efficacy evaluating ability of CIA and AA rat models. Result The membrane formation rate of CIA model was earlier than that of AA model. After a certain period of membrane formation ( 14, 28 d) , the two models had a preference for hematology, blood biochemistry and pathology indexes. At the same time, RA symptoms in rats treated with methotrexate or leflunomide showed significant efficacy in both CIA and AA models. Conclusion CIA and AA models rats showed good stability and pathological characteristics of RA. Furthermore, both CIA and AA models manifested high level evaluating abilities of drug efficacy. In conclusion, there are some differences between CIA and AA rat models of RA, such as pathological characteristics, etc. , so the use of rat model needs to be considered according to the specific research aims.

The Role of Neutrophils in Acute Pneumonia Induced by Inhalation Infection of Hypervirulent Klebsiella Penumoniae Clinical Isolate sgh10

QIN Tongtong , ZHAO Yue , SUN Tianqi , ZHANG Lili , ZHOU Dongsheng , YANG Wenhui , LYU Meng , QIU Yefeng

2023, 40(2):  61-67.  DOI: 10. 3969 / j. issn. 1006-6179. 2023. 02. 010

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To explore the key role of neutrophils in the host response after hypervirulent K. pneumoniae sgh10 inhalation infection. Method In vivo elimination model of neutrophils was established by depleting lung tissue neutrophils in mice using monoclonal antibody anti-ly6g ( 10 mg / kg ) , and the model was evaluated by flow cytometry detection and immunofluorescence. Animals were divided into two groups, one injected with mAb and one injected with isotype control Ab at pre-infection, 24 and 48 h post infection to examine mouse survival, lung bacterial load, lung histopathology, cytokines as well as the effect of neutrophil depletion on other immune cells. Reslut The elimination effect of neutrophils was confirmed by flow cytometry and immunofluorescence assays. Depletion of neutrophils in vivo resulted in increased mortality, higher bacterial loads in the lungs, more severe lung injury and higher levels of pro-inflammatory cytokines in mice compared to controls. Furthermore, although neutrophil depleted mice are more susceptible to hypervirulent K. pneumoniae respiratory infection and cause up to 90% mortality, alterations in neutrophils did not significantly affect the levels of several other innate immune cells. Conclusion neutrophils played an important role in the host response of hypervirulent K. pneumoniae infection.

Experimental Study of Shegan Zhike Capsules on LPS-induced Acute Lung Injury in Rats

BAO Yulong, ZHU Jinghe, FAN Yinglan, WU Xiaolin, LIU He, LI Guoxin

2023, 40(2):  68-72.  DOI: 10. 3969 / j. issn. 1006-6179. 2023. 02. 011

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Objective To investigate the effect of Shegan Zhike Capsule on lipopolysaccharide ( LPS) - induced acute lung injury (ALI) in rats. Method 70 rats were randomly divided into 7 groups according to their body weights, namely, blank group, model group, low, medium and high dose groups of Shegan Zhike capsule (30. 00 mg, 60. 00 mg,120. 00 mg extract / kg) ,Zhongyang and Xiyang groups, with 10 rats in each group. They were administered for 7 consecutive days. On the fifth day of the experiment, 1 h after the administration by gavage, except for the blank group, rats in other groups were instilled with LPS (5 mg / kg) intratracheally to make Ali rat models, The blank control group was infused with the same dose of normal saline. 48 hours after model establishment, blood was collected from abdominal aorta of rats to measure IL-1β、TNF-α content. The whole lung was taken and weighed to calculate the lung index. The left lung tissue of rats was taken to measure the wet / dry mass ratio of the lung. The right middle lobe of lung was taken for pathological observation. Result Compared with the model control group, The contents of IL-1β and TNF-αin the low, medium and high dose groups ( 30. 00 mg, 60. 00 mg, 120. 00 mg extract / kg) of Shegan Zhike capsule were significantly decreased (P<0. 01) ; Compared with the model control group, the lung index and lung wet / dry mass ratio of rats in the high dose group of Shegan Zhike capsule were significantly reduced ( P< 0. 01,P< 0. 05) ;Compared with the blank control group, the alveolar space of the model control group was widened, the alveolar cavity exudation, and the degree of alveolar damage and destruction were significantly increased ( P<0. 01,P<0. 05) . Conclusion Shegan Zhike capsule has a protective effect on lipopolysaccharide induced acute lung injury in rats, and its mechanism may be related to reducing pulmonary edema and inhibiting the synthesis and release of inflammatory factors.

Design and Use of Self-made Acupuncture and Moxibustion Fixation Device for Rats in Supine Position

LYU Jiaqi, TANG Kunpeng, YAN Liping, MA Mengna, REN Huashan, WEN Tan

2023, 40(2):  73-76.  DOI: 10. 3969 / j. issn. 1006-6179. 2023. 02. 012

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Objective In order to facilitate the acupuncture and moxibustion experiment in rats, a supine position fixing device which is suitable for the acupuncture and moxibustion operation of acupoints in abdomen and lower limbs of rats was designed on the basis of following the 3R principles. Method Discarded coke bottles were collected and processed into the fixation device according to the predesigned program. By using the natural instince of rats like seeking holes,the rat were bound in supine posture bottle, then the bound rat were stably placed between two fixed bottom plates. The acupoints of the abdomen and lower limbs were fully exposed, and the rats were treated with electroacupuncture. It was compared between the device fixation group and the traditional restraint fixation group from the aspects of the number of operator, the operation time, and the different states of rat undergoing fixation and treatment. Result Compared with the restraint fixation, the fixation of the rat by this device can be completed by only one person, and the fixation time of a single rat is significantly shortened (P<0. 01) . The various conditions of the rat during the fixation and treatment are obvious improve. Conclusion The materials of this device are easy to obtain and easy to modify. Compared with other fixation methods, it has the advantages of simple operation, reliable fixation, saving manpower and time, etc. It is a more applicable method for acupuncture and moxibustion operation on the abdomen and lower limbs of rats, and it provides a new safe and reliable choice for rat fixation in the experiment and is worthy to promote.


Effect of Acupuncture on Bone Metabolism and Bone Quality of Ovariectomized Osteoporosis Rats in Youth

YAN Guoqiang , LIU Lili , WANG Chao , ZHAO Danhui

2023, 40(2):  77-81.  DOI: 10. 3969 / j. issn. 1006-6179. 2023. 02. 013

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Objective To observe the effects of acupuncture on bone metabolism and bone quality in ovariectomized osteoporosis model rats, and to explore the mechanism of acupuncture in treating osteoporosis in youth. Method Forty female SD rats were randomly divided into a normal group, a sham operation group, a model group and a acupuncture group, with 10 rats in each group. The model group and acupuncture group used the ovary to prepare the rat model of osteoporosis in youth. The model group did not carry out intervention treatment. The acupuncture group was given electroacupuncture to stimulate “ Shenshu” , “ Zusanli” and “Xuanzhong” at the 2nd week after operation, once a day, for 8 consecutive weeks. The contents of serum alkaline phosphatase ( ALP ) and osteocalcin ( OC) were detected by enzyme-linked immunosorbent assay ( ELISA ) ; Femoral bone mineral density ( BMD ) and bone morphological parameters were measured by micro CT. Result After 8 weeks of intervention, compared with the normal group and sham operation groups, ALP and OC increased in the model and acupuncture groups, BMD, BV / TV and Tb. Th decreases,and Tb. Sp increased (P < 0. 05) , indicating the success of osteoporosis modeling. Compared with the model group, ALP and OC in the acupuncture group decreased, BMD、 BV / TV and Tb. Th increases, and Tb. Sp decreased ( P < 0. 05 ) , indicating that acupuncture can improve bone mineral density and bone microstructure. Conclusion Acupuncture has the effect of preventing osteoporosis in youth, and its mechanism may be related to the promotion of bone formation and repair, the increase of bone density, and the enhancement of the structural strength of bone trabeculae.

Thoughts on Pain Management and Animal Welfare in the Construction of Medical Experimental Animal Models

OU Langlin, WANG Zhaosi, MEI Jian, ZHU Xiangrui, MA Cui

2023, 40(2):  82-88.  DOI: 10. 3969 / j. issn. 1006-6179. 2023. 02. 014

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Animal experiments based on animal models are necessary means in basic medical research, drug development and evaluation of medical devices, and play a very important role in the development of medicine and even modern science and technology. In recent years, the continuous development of animal experiments has brought many new challenges. More attention should be paid to the welfare of experimental animals in biomedical research than ever before. This paper aims to strengthen the depth control of pain management and animal welfare of basic medical postgraduates and other animal experiment practitioners while ensuring the experimental result. Through the comparative analysis of animal models of diseases involved in new coronavirus research and tumor research, the important position of pain management in animal welfare is highlighted. The related contents of animal welfare and pain management in the construction of common medical experimental animal models in China at the present stage are summarized and suggestions are put forward.

Establishment Methods of Traumatic Hemorrhage Model in Rabbits

CAO Lingli, XU Weigong

2023, 40(2):  89-93.  DOI: 10. 3969 / j. issn. 1006-6179. 2023. 02. 015

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Objective To summarize the method of establishing traumatic hemorrhage model in rabbit. Method By consulting the domestic and foreign literatures on rabbit traumatic hemorrhage model, selection of anesthetics and dosage, modeling process, and evaluation indicators of hemostatic effects were analyzed. Result The common rabbit traumatic hemorrhage models include auricular arteriovenous traumatic models, surface skin traumatic models, femoral artery traumatic models, and Visceral organs models such as liver and spleen. When determining the anesthetics and doses, types of hemorrhage model, operation time and medicinal properties should be comprehensively considered. When considering model establishment process and evaluation indicator, We can improve it on the basis of basic operation procedures according to concrete conditions. Conclusion The standard method of establishing rabbit traumatic hemorrhage model plays a key role in the scientific evaluation of the hemostatic materials. This reviewr provides a reference for the establishment of rabbit traumatic hemorrhage model and the determination of evaluation indicator.

Analysis on the Development Status of Organoid Technology

MA Liansu, LI Junnan

2023, 40(2):  94-100.  DOI: 10. 3969 / j. issn. 1006-6179. 2023. 02. 016

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The development of organoid has a history of over 100 years. With the proposed concept of organoids-on-ohips(OOC) , it is possible to construct the microphysiological system of human organs on chip. Organoid technology is expected to be the most potential alternative method, and has become a technical focus competing in the field of medicine and health worldwide. In this article we introduce classification, manufacturing and application of organoids and OOC, and analyse the policies, research, development and industrial status quo in China and developed countries/ regions such as Europe and the United States. Basing on the conclusion, we insight the bottleneck problem of organoid technology, and look forward to the future development trend.