Table of Content

28 August 2024, Volume 41 Issue 4

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Establishment of Hypoxic-ischemic Encephalopathy Model in Full-term Infants

SHEN Tianfeng , GAN Na, XIE Yuqing , HE Chuxin, CHEN Shujing, WU Fangying

2024, 41(4):  1-7.  DOI: 10.3969/j.issn.1006-6179.2024.04.001

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To establish a simple and stable rat model of full-term infants with the whole
hypoxic-ischemic brain damage (HIBD) in full-term infants and to explore the evaluation method of the
model. Method Eighty-two 10-day-old SD rats were randomly divided into sham operation group
( Sham, n = 22) and model group (HIBD, n = 60) . The whole brain HIBD rat model of full-term infants
was prepared by ligating the bilateral common carotid arteries of 10-day-old SD rats for 1. 5 hours and
revascularization after 30 minutes of hypoxia. The sham operation group did not receive ischemia and
hypoxia treatment. ( 1) Pathological related detection: 3 days after operation, TTC staining, HE and
Nissl staining, hippocampal electron microscopy were used to observe the pathological changes of brain
tissue. Brain water content was measured by brain dry-wet ratio method to evaluate brain edema. ( 2)
Behavioral evaluation: Convulsion assessment was performed 12 hours after surgery, Longa score was
performed 3 days after surgery, suspension test was performed 10 days after surgery, and Morris water maze test was performed 3 weeks after surgery. HIBD was graded according to the postoperative general
condition and behavioral experiment result. Result Compared with the sham operation group, TTC
staining showed bilateral white infarcts in the HIBD group. HE and Nissl staining showed disordered
arrangement and necrosis of neurons in hippocampus. Electron microscopy showed neuronal cell edema,
mitochondrial vacuolization, and increased autophagic lysosomes in the hippocampus. Brain water content
increased significantly (P<0. 01) . Compared with the sham operation group, rats in the HIBD group had
seizures with different degrees of motor nerve dysfunction in the early stage, and developmental delay and
decreased learning and cognitive ability in the long term ( P < 0. 01 ) . Conclusion The pathological
model of moderate to severe neonatal rat HIBD can be reproduced by ligation of bilateral common carotid
arteries, hypoxia and recanalization in 10-day-old neonatal rats.





Establishment and Application of Fluorescent Quantitative PCR Assay for

Laboratory Animal Toxoplasma gondii

LUO Tingyu, LI Kaili, LI Changwen, CHEN Hongyan, XIA Changyou, GAO Caixia

2024, 41(4):  8-14.  DOI: 10.3969/j.issn.1006-6179.2024.04.002

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To establish and apply TaqMan probe real-time fluorescent quantitative PCR
method for laboratory animal Toxoplasma gondii. Method By comparing the sequences of various strains
of Toxoplasma gondii published by NCBI, the conservative region of 529 bp repetitive sequence was
selected to design primers and probe, and a fluorescence quantitative PCR method was established.
Subsequently, the specificity and sensitivity of the method was confirmed. Plasmid standards were


obtained from 106 to 102copies/ μL in 10-fold series dilution, and 3 parallels were detected for each

concentration and repeated 3 times. The coefficient of variation intra-assay and inter-assay was calculated
to evaluate the reproducibility and stability of the method. 14 canine tissue samples and 66 porcine whole
blood samples were detected by the established quantitative PCR method and the PCR method
recommended by national standard to evaluate the capability of this method in practical application.
Result The established quantitative PCR method showed a linear relationship between Ct value and
plasmid concentration in the range of 108~ 101copies/ μL, the standard curve Slope was - 3. 285, R
2 value was 1, and the amplification efficiency was 101. 663%. The lowest detectable concentration of
Toxoplasma gondii was 101copies/ μL. There was no cross reaction with other 14 common swine
pathogens. Replicates were detected at 106 to 102 copies/ μL plasmid standards, and the coefficient of
variation intra-assay and inter-assay was less than 1. 21% and 0. 62%, respectively. The established
quantitative PCR method and the national standard PCR method were used to detect the positive rate of
14 canine tissue samples and 66 porcine whole blood samples. The result showed that the positive rate of
the established quantitative PCR method and the national standard PCR method were 10% ( 8 / 80) and
7. 5% ( 6 / 80 ) , respectively, and the positive coincidence rate reached 100%. Conclusion The
established Toxoplasma gondii FQ-PCR method can effectively detect Toxoplasma gondii. It provides a
good method for daily monitoring of toxoplasma gondii in laboratory animals.

Study on the Difference of Brain Injury in Rats with#br# Necrotizing Enterocolitis at Different Ages#br#

LI Huayuan , LU Mengxue , SUN Ying , WANG Jing , LU Yanyun , GUO Yu , ZHANG Dandan , BAO Rong

2024, 41(4):  15-21.  DOI: 10.3969/j.issn.1006-6179.2024.04.003

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Three models of necrotizing enterocolitis ( NEC) in rats of different ages were
established to study the differences of brain injury. Method Sixty newborn SD rats were divided into 6
groups: 1-day-old control group, 1 day old model group, 3 day old control group, 3 day old model
group, 5 day old control group and 5 day old model group, with 10 rats in each group. NEC models were
established in all model groups by artificial feeding, hypoxia, cold stimulation and lipopolysaccharide
( LPS) induction. Rats in the control group were fed with breast milk in normal environment. During the
modeling period, the activity, abdominal distension and diarrhea of rats were observed, and the weight
changes were monitored. After the rats were killed, the overall morphology of intestinal tissue was observed. HE staining was used to observe the pathological changes of small intestine tissue, and the
injury score was performed. Nissl staining was used to observe the number of nissl bodies in brain tissue.
TUNEL staining was used to observe the level of apoptosis in brain tissue. Result Compared with the
control group at the same age, the rats in the model group had abdominal distension, diarrhea, poor
activity, the weight decreased significantly ( P<0. 05) , swelling, gas accumulation and necrotic foci in
the intestinal tissue. The pathological damage of small intestinal tissue was serious, the injury score was
significantly increased (P<0. 05) , the number of Nissi bodies in brain tissue was significantly decreased
(P<0. 05) , and the cell apoptosis in the brain tissue was increased. Moreover, the intestinal tissue and
brain tissue injuries of 1-day-old NEC rats were the most serious. Conclusion There are differences in
intestinal and brain tissue injuries among NEC rats at different ages. The younger the age, the more
severe the injury, suggesting that the age of onset may be one of the risk factors for brain injury in NEC
rats.



Development of Reproductive System of Mongolian Gerbil#br# Before and After Sexual Maturity#br#

CHI Yafei , PENG Boya , MA Jun , LI Meng , CHEN Bai’ an , WU Yi , LU Jing

2024, 41(4):  22-28.  DOI: 10.3969/j.issn.1006-6179.2024.04.004

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To investigate in depth the development of the reproductive system during sexual

maturation in long-clawed gerbils, and to provide direct research evidence for the exact time of sexual

maturation. Method A total of 150 long-clawed gerbils were selected from 6 to 14 weeks of age and

divided into 5 groups of 15 males and 15 females according to the ages of 6, 8, 10, 12 and 14 weeks,

weighed and collected samples of ovaries, uterus, testes and epididymis, measured the wet weight of the samples and calculated the index of the organisms; the morphology of the ovaries was observed under the
microscope, and the spermatozoa were detected by a cytometer; 6 samples were randomly selected from
each group for HE staining to observe the development of the ovaries, testes and epididymis. Result In
female gerbils, ovarian wet weight and ovarian index increased significantly at 10 weeks of age, and there
was no significant change between 10 and 14 weeks of age; uterine wet weight and uterine index changed
in stages; under microscopic observation, the incidence of corpus luteum in the groups of 10 weeks of
age, 12 weeks of age and 14 weeks of age was 46. 7%, 60% and 73. 3%, respectively; the tissue
sections showed that corpus luteum could be seen clearly from 10 weeks of age. In male gerbils, the wet
weight of testes increased gradually with the increase of age from 6 weeks to 10 weeks, but there was no
significant change after 10 weeks; the testicular index increased significantly at 8 weeks and 12 weeks of
age, and decreased significantly at 14 weeks of age; both epididymal wet weight and epididymal index
increased gradually with weekly age from 6 weeks of age to 12 weeks of age, and there was no significant
change in epididymal wet weight at 14 weeks of age, and the epididymal index decreased significantly.
The result of the cell counter showed that the sperm count was zero at 6 weeks of age, the sperm count in
the epididymis increased significantly at 10 weeks of age compared to 8 weeks of age, and there was no
significant change between 10 and 14 weeks of age. Tissue sections showed that a small number of
spermatozoa began to appear in the testis and epididymis from 8 weeks of age and a large number of
spermatozoa were visible from 10 weeks of age. Conclusion As the gerbil’ s age increases, the ovaries of
female gerbils begin to develop and discharge follicles and eggs at about 10 weeks of age, and the gerbil
reaches sexual maturity and the ability to reproduce. Male gerbils began to produce sperm at around 8
weeks of age and entered the first estrus. At around 10 weeks of age, the testes developed stably and the
spermatozoa reached the maximum number, reaching sexual maturity, after which the epididymis
continued to develop, and its development stabilised at around 12 weeks of age.


Effects of Cinnamon Cortex on Endogenous Metabolism in SD Rats

WEI Lin, YAO Lihua, ZENG Jiacheng

2024, 41(4):  29-35.  DOI: 10.3969/j.issn.1006-6179.2024.04.005

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To investigate the effects of cinnamon cortex on the metabolomics of SD rats using
1H NMR combined with multivariate data analysis, to explore pharmacological mechanism of cinnamon
cortex. Method Twenty SD rats were randomly divided into cinnamon cortex-treated groups(CC) and the
control groups ( TCG) . The rats in the cinnamon cortex-treated groups ( CC ) were intragastric with
cinnamon cortex ( CC) decoction at the dose of 0. 5 g / kg, while the rats in the control groups were
intragastric with the same dose of distilled water once a day for 22 days. The dose of cinnamon cortex was
according to the Chinese Pharmacopoeia ( 5 g / person / day ) . It was determined that urine and fecal
samples were collected on day 0, day 5, 10 and 22 after cinnamon cortex administration. Result
Compared with the blank control group, the contents of 8 metabolites and 7 metabolites were significantly
up-regulated in the NMR map of urine samples of the cinnamon cortex administration group on the 22
day. The levels of cadaverine, carnitine, succinate, citric acid and hippuric acid were up-regulated,
while the levels of dimethylamine, creatinine, phenylalanine and glycine were down-regulated. In the
NMR pattern of fecal samples, the contents of 9 biometabolites were down-regulated and 3 up-regulated,
and the levels of adenine and uracil were significantly decreased, while the levels of lactic acid,
succinate and acetone were increased, with statistical significance between groups (P<0. 01,P<0. 05) .Conclusion Cinnamon cortex may exert its pharmacological effects by regulating aromatic amino acid
metabolism, choline degradation, short-chain fat synthesis and other metabolic pathways.



Differential Analysis of Gut Microbiota Between Guide Dogs and#br# Eliminated Dogs Based on Metagenomic Sequencing#br#

ZHANG Ying , ZHOU Zijuan , GUO Yijun , HOUZHENG Zitan , WANG Jingyu , DONG Jianyi

2024, 41(4):  36-40.  DOI: 10.3969/j.issn.1006-6179.2024.04.006

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Differential analysis of intestinal flora function in guide dogs and eliminated dogs
was performed using macro gene sequencing. Method Fifteen dogs were sequenced ( nine guide dogs
and six eliminated dogs) and analyzed by applying gene number and intestinal flora diversity. Result
There were 25 630 genes specific to the guide dog group and 10 527 genes specific to the elimination dog
group. There were 2 266 up-regulated genes and 1 043 down-regulated genes with significant differences
(P<0. 05) . The abundance of Clostridium purpureum was higher in the guide dog group than in the
elimination dog group; in addition, the abundance of rumenococci differed between the two groups of dog
samples. Conclusion The diversity of intestinal flora of both guide dogs and eliminated dogs was
different and had an effect on dog behavior.


Changes of Some Serum Inflammatory Cytokines in

Type 2 Diabetes Model of Minipigs

ZHAO Changqi, ZHAO Yuqiong, NIU Miaomiao, JIA Yunxiao

2024, 41(4):  41-48.  DOI: 10.3969/j.issn.1006-6179.2024.04.007

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To dynamically observe the levels of inflammatory cytokines in the serum of type 2
diabetes ( T2DM) model in miniature pigs, so as to provide reference data for the study of type 2
diabetes. Method Ten Bama minipigs were randomly divided into a Control group and a model group
(DM) . The Control group was fed with conventional diet, and the DM model in miniature pigs was
established by high-fat diet combinated with intravenous streptozotocin ( STZ) . The experiment lasted for
nine months, fasting blood glucose and insulin were measured monthly, and glucose tolerance was
measured every 3 months. Concentrations of 11 inflammatory cytokines in serum of animals at the 0th,
3rd, 6th, and 9th months were determined by ELISA kit. Result The fasting blood glucose was
significantly increased (P<0. 05) , severely impaired of glucose tolerance and insulin resistance occurred
in the DM group after high-fat diet for 3 months injected with STZ. The ELISA result showed that there was no significant change in the levels of 11 inflammatory cytokines of the DM group at the first 3 months,
and no significant difference compared to the Control group. The levels of platelet-derived growth factorBB ( PDGF-BB) and tumor necrosis factor-α ( TNF-α) were increased after the onset of T2DM and
significantly higher than those of in Control group ( PDGF-BB, P<0. 05 at the 6th month; PDGF-BB and
TNF-α, P<0. 05 at the 9th month) . The level of interleukin-6 ( IL-6) in the DM group was increased
gradually over time which the level at the 9th month was significantly higher than that at the 0 th and 3 r

d month (P < 0. 05) , but no significant difference compared with the Control group. Conclusion The

model of type 2 diabetes in minipigs was successfully established by using high-fat diet combined with
low-dose STZ. A simply high-fat diet was not obvious influence on the levels of inflammatory cytokines in
serum of mini-pigs, whereas the levels of PDGF-BB and TNF-α in serum of T2DM mini-pigs were
significantly increased and higher than those in normal animals.



Meta-analysis of the Effects of Intermittent Hypoxia on

Learning and Memory in Mammals

YAO Zhen, ZHANG Ziwei, WU Chunmiao, ZHANG Jingnan, LI Qinghua, WANG Zhenlong

2024, 41(4):  49-55.  DOI: 10.3969/j.issn.1006-6179.2024.04.008

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Objective Existing studies have shown that hypoxia affects mammalian brain function. In this
paper, Morris water maze experiment was employed to analyze the effects of intermittent hypoxia on
learning and memory ability of lactation. Method The retrieval time was limited to February 25, 2023
through the databases of CNKI, VIP database, WANFANG data, Web of Science, PubMed and
Elsevier. RevMan 5. 3 software was used for statistical analysis of the extracted data, Then sensitivity
analysis was used to evaluate the quality of the included studies. Result A total of 24 articles and 263
subjects were included in this study. In the Morris Water Maze experiments, the escape latency of rats/
mice was increased after treated with intermittent hypoxia for two weeks compared with the control group
[ SMD = 4. 17, 95%CI (3. 46, 4. 89) , P<0. 01] . The time of rats/ mice crossing the target quadrant was
decreased [ SMD = - 3. 07, 95% CI ( - 3. 56, - 2. 58 ) , P < 0. 01 ] . After treatment with intermittent
hypoxia for four weeks, the escape latency increased [ SMD = 8. 03, 95%CI (6. 21, 10. 40) , P<0. 01] ;
The time across the target quadrant shortened [ SMD = - 6. 79, 95% CI ( - 8. 60, - 4. 99) , P < 0. 01] .Conclusion Treated with intermittent hypoxia, the escape latency of rats/ mice in the water maze rises,
and the time of cross the target quadrant is shortened, indicating the learning and memory ability of mice /
rats is impaired.



Effect of In Vitro Fertilization Reagent on Fertilization Rate and Birth Rate of

Mice in Biological Purification

LI Site, TANG Yuling, CUI Jing, WANG Jin, WU Wenqing, QIU Yefeng

2024, 41(4):  56-60.  DOI: 10.3969/j.issn.1006-6179.2024.04.009

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To investigate the effects of IVF reagents on the fertility rate and birth rate of
genetically modified mice in biological purification. Method The experimental system was divided into
group A, group B and group C according to different manufacturers of IVF reagents. 5 - 11 genetically
modified mouse strains were selected for superovulation, IVF-ET experiments, and the number of eggs
collected, the number of 2-cell, the number of embryos transferred, and the number of piglets born were
recorded, and the fertilization rate and the birth rate were calculated. Result The fertilization rate of
group A was ( 51. 89 ± 13. 91 ) % and the puerperal rate was ( 14. 70 ± 10. 95 ) %. In group B, the
fertilization rate was ( 57. 71 ± 17. 12) % and the birth rate was ( 34. 96 ± 9. 92 ) %. In group C, the
fertilization rate was (54. 78±18. 56) % and the puerperal rate was (22. 01±9. 54) %. Fertilization rate:
B>C>A; Puerperal rate: B>C>A, but there was no statistical difference among all groups. The reagents
required by group A are difficult to prepare, complicated to use and have poor stability, while the
reagents of group B and group C are simple to use and have strong stability. However, compared with group A and group C, the unit price of reagents used in group B is high, so the experimental cost is
higher. Conclusion The reagents of group B and group C can be used as conventional reagents for IVF
of genetically modified mice, and the experimental can choose them according to the comprehensive
factors such as fertilization rate, puerperal rate, price and ease of use.



Establishment of a Multiplex Molecular Test Method for Three Types of

Conditional Pathogenic Bacteria in Laboratory Animals

CAI Lidong, TIAN Miaomiao, LI Naixin, WANG Xinyu, YANG Huixin

2024, 41(4):  61-67.  DOI: 10.3969/j.issn.1006-6179.2024.04.010

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A multiplex qPCR( Fluorescence quantitative PCR, qPCR) test method for three
conditional pathogens, Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa, was
established. Method The nuc gene of Staphylococcus aureus, the phoE gene of Klebsiella pneumoniae,
and the gyrB gene of Pseudomonas aeruginosa were selected to design specific primers and probes, and a
multiplex qPCR method was established. Its performance indicators such as specificity, sensitivity,
standard curve, and repeatability were tested. This method was used to test 30 clinical stool samples and
6 positive references. The test result were compared with the result of the culture method, and the
compliance rate was calculated. Result The multiplex qPCR method can stably test the genomic DNA of
standard strains of Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa; there is
no nonspecific amplification of pathogenic microorganisms such as Salmonella typhimurium, Pasteurella
pneumotropica, Corynebacterium kutscheri, and Bordetella bronchiseptica; the sensitivity of the genomic
DNA of standard strains of Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa is
1. 0×10^{- 5},1. 0×10^{- 4},and 1. 0×10^{- 5}ng / μL, respectively; the inter-batch and intra-batch differences are less than 2%, and the repeatability is good; the test result of clinical samples and positive references
show that the test result of the multiplex qPCR method are consistent with those of the isolation and
culture method. Conclusion This study successfully established a multiplex qPCR method for
simultaneous and rapid detection of Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas
aeruginosa.



Investigation and Analysis of Experimental Animal Feeds

Quality of Different Manufacturers

DONG Qiaoyan, ZHANG Wensheng, SHANG Yanjiao, QIU Yefeng, CHEN Jingqing

2024, 41(4):  68-74.  DOI: 10.3969/j.issn.1006-6179.2024.04.011

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To understand the current situation of laboratory animal feed quality in China and
provide the basis for standardizing laboratory animal feed production. Method Five feed companies with
high market share and strong representativeness were selected to produce laboratory animal feeds, and the
conventional nutritional components of the feeds were determined and compared by the national standard
method. Result There were significant differences in the routine nutrient contents of rat breeding
material, monkey maintenance material, dog material, rabbit material and guinea pig material produced
by different companies, and the contents of feed moisture, crude protein, calcium and phosphorus met
the national standards. The rat breeding material, monkey maintenance material and rabbit material
produced by individual companies had ash, excessive crude fiber content and low crude fat content. In
addition, the detection rate of unqualified trace element content in the five feeds was high, mainly the
iron and iodine contents. Conclusion The conventional nutrient quality of different experimental animal
feed manufacturers can basically meet the national standard requirements, but lack of monitoring of trace
elements and other indicators, resulting in high product failure rate. Therefore, it is also necessary to
strengthen and improve the construction of feed standardization system and quality control supervision and other key links to ensure the standardization and standardization of laboratory animal feed.


Construction and Evaluation of Mouse Depression Models

WANG Jinhui, ZHANG Yong, DONG Weitao, ZHANG Quanwei

2024, 41(4):  75-79.  DOI: 10.3969/j.issn.1006-6179.2024.04.012

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Depression has received widespread attention from the medical community due to its increasing
incidence year by year. The factors of depression are extremely complex, so the construction of
depression models has become a key step when conducting related experiments. This paper mainly
introduces the construction method of common mouse depression models, such as chronic unpredictable
mild stimulus model, special drug-induced depression model, early life stress model, etc. , and related
behavioural experiments to assess the degree of depression, such as forced swimming experiment, sucrose
preference experiment, splashing experiment, etc. , which provide relevant researchers with experimental
ideas and method.


Research Progress of Abdominal Traumaanimal Model Making and Treatment

XIRENNAYI Selimu , LIU Jiangwei , SUN Yawei , XU Qin

2024, 41(4):  80-84.  DOI: 10.3969/j.issn.1006-6179.2024.04.013

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With the advancement of pathophysiology and treatment of the abdominal trauma research, the
establishment of standardized abdominal trauma animal models highlights its importance. This paper
reviewed and summarized the research progress of abdominal trauma-related animal models and damage
control surgery ( DCS ) treatment strategies according to relevant literature at home and abroad, and
compared and analyzed the advantages and disadvantages of the models, and that provided the reference
for the pathophysiology mechanism and treatment research of abdominal trauma.


Research Progress on Morphological Development of

Temporomandibular Joint During Embryonic Stage

XIANG Lei , WANG Xuewen , LIU Huawei , CHANG Ping , MU Xiaodan , HU Min

2024, 41(4):  85-91.  DOI: 10.3969/j.issn.1006-6179.2024.04.014

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Temporomandibular joint ( TMJ) is a synovial joint unique to mammals and its development
process is significantly different from that of long bone joints. The study of embryonic development in TMJ
has nearly one hundred years history. However, on the one hand, the continuity of human TMJ embryo
development research is limited. The initiation of TMJ development and the condyle ossification and
growth patterns have not yet been fully elucidated in human. On the other hand, there are some
differences between rodents as model animals for TMJ development research and human. Seeking model
animals similar to human to carry out TMJ embryo development research has never stopped. This article
reviews the morphological development of embryonic TMJ including human and animal models.


Principles and Methods for Behavioral Tests of Mental Disorder-like Rodents

WAN Yidian, LI Yuting, WANG Qi

2024, 41(4):  92-96.  DOI: 10.3969/j.issn.1006-6179.2024.04.015

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Mental disorders refer to the disorders in the mental activity ( affection, cognition, and
volition, etc) of patients, which are caused by the brain dysfunction. Implicit mental disorders are
usually accompanied by explicit abnormal behaviors. Building mental disorder-like animals is vital for
exploring the pathogenesis of mental disorders, and is meaningful for evaluating the validity of the
researches on mental disorders. Therefore, based on previous studies about mental disorder-like rodents,
we summarized the principles and method for behavioral tests of some mental disorders ( depression,
anxiety, bipolar disorder, autism spectrum disorder, attention deficit hyperactivity disorder, posttraumatic stress disorder, obsessive-compulsive disorder ) , expecting to provide a reference for the fundamental researches of psychiatry.