Abstract:This is a systematic review of the history and progress of animal behavior experiment methods at home and abroad, and the conception associated with animal behavior experiments. In addition, the application of animal behavior experiment methods in the field of neuropsychology and military medicine is also reviewed, providing a reference for the scientific research based on animal behavior experiments.

Abstract:Objective To study the effects of different color light stimulus, duration of stimulus and day/ night training on the incentive conditioned reflex of rats, and to provide a data basis for the methodology of incentive conditional reflection experiments. Methods Three experiments were included in this study. During the experiment 1, a total of 30 male 8?week?old Wistar rats were divided into three groups: red light stimulation group, yellow light stimulation group and blue light stimulation group. During the experiment 2, a total of 32 male 8?week?old Wistar rats were divided into three groups: the group I (with a light stimulation duration of 10 s), the group II (with a light stimulation duration of 30 s), and the group III (with a light stimulation duration of 60 s). During the experiment 3, a total of 16 male 8?week?old Wistar rats were randomly divided into two groups, of which the first group was trained at 9:00 a. m. every day, and the second group was trained at 21:00 every day. All the laboratory animals were given reward conditioned reflex training, and indicators such as the number of correct nose pokes, the number of incorrect nose pokes, the ratio of correct nose pokes and so on were counted and calculated. Results During the experiment 1, there was no significant difference in the numbers of correct nose pokes and incorrect nose pokes, movement distances and average speeds of the rats among the three groups. During the experiment 2, compared with the group with a light duration of 10 s, the rats in the groups with a light duration of 30 s or 60 s had a higher number of correct nose pokes ( P < 0.05). Moreover, on the 13th and 14th days, the ratio of correct nose pokes of the group with a light duration of 30 s was significantly higher than that of the group with a light duration of 10 s ( P < 0.001). During the experiment 3, there were no significant differences in the number of correct nose pokes and the nose poke accuracy of the rats between the two groups. Conclusions In the reward conditioned reflex test, red, yellow and blue light stimuli have similar reward prediction effect. When stimulated with the same light, the time of stimulation has an impact on the learning ability of the rats, and an appropriate extension of stimulation is beneficial to the formation of conditioned reflex in them. The day/ night training has no effect on the learning and memory ability of the rats.

Abstract:Objective To explore the effects of high?frequency electromagnetic radiation exposure on the moodcognitive behaviors of mice, and to provide an experimental basis for the study on radiation damage mechanism and protection. Methods The electromagnetic reverberation chamber can produce continuous and uniform high-frequency electromagnetic waves. Different groups of ICR mice were exposed to high-frequency electromagnetic radiation in this chamber for 1 d, 3 d and 7 d, and then series of behavioral tests were conducted to observe the changes in locomotor activities, depressive-like and anxiety-like behaviors, and cognitive function of the mice. Results After exposed to the high-frequency electromagnetic radiation (3 GHz, SAR 4 W/ kg) 4 h/ d for 7 d, there was no obvious change in locomotor activity (times of grid crossing and standing up in the open field test) of the mice, while the times of entering the central region ( P < 0.05) and the time duration ( P < 0.05) were significantly decreased. Compared with the unexposed group, the percentage of the times of entering the open arm in the elevated-plus maze ( P < 0.05) and the time duration in the open arm ( P < 0.01) of the mice in the radiation group were significantly decreased. Meanwhile, the immobility time in the forced-swimming test ( P < 0.01) and the tail suspension test ( P < 0.05) of the mice exposed to electromagnetic radiation was significantly increased, and the discrimination index of new objects in the new object recognition test was significantly reduced ( P < 0.001). Conclusions Continual exposure to high-frequency electromagnetic radiation (3 GHz, SAR 4 W/ kg) can lead to anxious-depression behaviors and cognitive impairment of mice in a time-dependent manner, which provides an experimental basis for the relevant study of prevention and therapy techniques.

Abstract:Objective To develop a multi-mode, detailed and sensitive object recognition method for learning and memory research, including novel object recognition task, object location recognition task, temporal order memory task and object context recognition task in animals based on their spontaneous behavior. Methods Two strains of mice (KM and ICR) and four patterns were used to establish the object recognition method and several relative factors, such as time of test phase, delay between test phase and familiar phase were investigated. Results It was stable and reasonable to set the time of familiar phase as 5 min, the interval between test phase and familiar phase as 10 min, and the time of test phase as 3-5 min, under the precondition of habituation for three days and selecting the appropriate object pairs. Conclusions Object recognition test including four patterns has been systematically established as a stable, reliable and normative way for the research of animal learning and memory and drug development.

Abstract:Objective To measure and analyze the behavioral changes of Rncat congenital cataract mice. Methods Normal BALB/ c mice and KM mice were used as control group, and inbred and random mated Rncat congenital cataract mice were used as experimental group. Behavioral tests, including the open field test, coat-hanger test, forced swimming test, and tail suspension test, were conducted on the mice. Results Compared with the inbred Rncat congenital cataract mice, the residence time in the open field test, the immobility time in the forced swimming test and tail suspension test of the BALB/ c mice, randomly-mated Rncat congenital cataract mice and KM mice were significantly different. Conclusions There are certain differences in behavioral performance between the Rncat congenital cataract mice and the other mice. Our findings may provide a useful reference for future researchers.

Abstract:Objective To establish a rat model of post-traumatic stress disorder (PTSD) through single-prolonged stress (SPS) and to observe the effect of social isolation on the behavior of the SPS model rats. Methods A total of 36 rats were randomly divided into the normal group, SPS model group and SPS combined with social isolation group. The rats in both SPS model group and SPS combined with social isolation group were modeled by single-prolonged stress, and the rats in the SPS combined with social isolation group were raised separately after modelling. The weight gaining, the total movement distance in open field test, the frequency of grid crossing, the single maximum movement distance, and the freezing frequency and time durations in the freezing behavior test were measured after 7 days of modeling. Results Compared with the normal group, the weight gaining and the single maximum movement distance of the rats in the SPS model group were significantly decreased ( P < 0.01), as well as the total movement distance and the frequency of grid crossing ( P < 0.05), while the freezing frequency and time in the freezing behavior test were significantly increased ( P < 0.01). Compared with the normal group, the weight gaining and crossing times of the rats in the SPS combined with social isolation group was decreased ( P <0.05), and the freezing frequency and time durations in the freezing behavior test were increased ( P < 0.05). In addition, compared with the SPS model group, the total movement distance in the open field test, the frequency of grid crossing and the single maximum movement distance of the rats in the SPS combined with social isolation group were increased ( P < 0.05). Conclusions The rat model of post-traumatic stress disorder is successfully established by single-prolonged stress, and 7 days of social isolation may alleviate the anxiety state of SPS model rats.

Abstract:Objective To evaluate the effect of two polymer membranes, polyhydroxyalkanoates (PHA) and polylactic acid (PLA) during glaucoma filtration surgery (GFS), and to evaluate the morphology of membranous PHA after interlamellar implantation. Methods Twenty-eight New Zealand white rabbits were chosen and twenty-four of them were randomly divided into 6 groups (n =4): the PHA-low group, PHA-high group, PLA-low group, PLA-high group, positive control group (MMC group) and blank control group. The rabbits in each group received GFS. The corresponding polymer membranes were implanted under the scleral flap, while the MMC group was treated with 0.2 mg/ mL mitomycin C (MMC) for 3 minutes, and the blank control group was treated without extra drugs. The intraocular pressure (IOP) was examined at 0 d, 1 d, 3 d, 7 d, 14 d, 28 d and 84 d after GFS. The corneal layers of four rabbits were implanted with PHA membranes and the corneal morphological changes were observed after 84 d. Results The IOP of the PHA-low and PLA-high groups was lower than that of the blank control group at 84 d after GFS ( P < 0.05), and was similar with that of the MMC group ( P > 0.05). Morphological studies showed that there were no collagenous fibers filling in the duct, and the collagenous fibers around the membranes were generally arranged in parallel. There were no obvious changes in the peripheral collagen structure after implantation of PHA membranes between the corneal layers. Conclusions Application of PHA and PLA membranes during GFS in rabbits may maintain the level of IOP, and the effect is similar with MMC. The mechanism may be achieved through the mechanical blocking of fibrous tissue.

Abstract:Objective To explore the feasibility of establishing a tree shrew model of chronic gastrointestinal mucosal injury. Methods A total of 12 adult male tree shrews were randomly divided into 3 groups. The experimental groups 1 and 2 were administered with intraperitoneal injection of 2 mg/ (kg·d) and 1 mg/ (kg·d) of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) once every day for 56 days, respectively. The control group was given the same volume of sterile saline at the corresponding time points. Changes in the body weight of the tree shrews were observed. The contents of dopamine in the cerebrospinal fluid were detected. Gastrointestinal morphology was observed by stereoscope and histopathological changes of the gastrointestinal mucosa were examined by HE staining. Results The body weight and the contents of dopamine in the cerebrospinal fluid of the tree shrews in the model group were significantly decreased ( P < 0.05 for both). Pathological changes to some extent of the gastric antrum, the gastric body and the duodenum were observed, without obvious differences between the 2 mg/ kg group and the 1 mg/ kg group. No obvious changes were found in the control group. Conclusions Long-term intraperitoneal injection with a low dose of MPTP is a feasible method for the establishment of a tree shrew model of chronic gastrointestinal mucosal injury. The optimal dose is 2 mg/ (kg·d) every day for 56 days.

Abstract:Objective To investigate the effectiveness of the affinity adsorption material developed by our team for the specific removal of exogenous endotoxin in the blood circulation. Methods Fifteen beagle dogs were intravenously injected with endotoxin to establish a dog model of endotoxemia, and then they were randomly divided into the treatment group (n = 10) and the control group (n = 5). The treatment group received an extracorporeal perfusion to remove the endotoxin using the self-made disposable hemoperfusion device, while the control group using routine perfusion device. The levels of endotoxin, tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), interleukin 6 (IL-6) and interleukin 8 (IL-8) in the blood of the dogs were measured at the beginning and 120 min after hemoperfusion for 120 minutes. The vital signs of the dogs were monitored during the hemoperfusion. Results After successful establishment of the endotoxemia model, the level of endotoxin at the beginning of hemoperfusion in the treatment group and control group was 118.63 ±27.98 EU/ mL and 117.16 ±22.95 EU/ mL, respectively. After hemoperfusion for 120 min, it was 0.039 ±0.01 EU/ mL and 131.98 ± 7.01 EU/ mL, showing a significant difference (P ﹤ 0.05). The clearance rate of hemoperfusion in the treatment group was 94.07%. At the beginning of hemoperfusion, the levels of TNF-α, IL-1β, IL-6 and IL-8 in the treatment group were 1.53 ±0.27 ng/ mL, 12. 82 ± 1. 66 ng/ mL, 54.77 ± 3.98 ng/ mL and 0.25 ±0.32 ng/ mL, and the levels in the control group were 1.53 ±0.06 ng/ mL, 13.05 ±0.18 ng/ mL, 54.58 ±0.19 ng/ mL and 0.28 ±0.06 ng/ mL, respectively. After hemoperfusion for 120 min, the levels of TNF-α, IL-1β, IL-6 and IL-8 in the treatment group were 0.13 ±0.06 ng/ mL, 0.70 ± 0.36 ng/ mL, 1.62 ± 0.80 ng/ mL and 0.01 ± 0.00 ng/ mL, respectively, and as for the control group, the levels were 2.26 ±0.15 ng/ mL, 15.12 ±0.18 ng/ mL, 62.54 ±0.93 ng/ mL and 0.73 ±0.93 ng/ mL. There were significant differences between the beginning and after perfusion for 120 min in those two groups ( P < 0.05). Conclusions This affinity adsorption material can effectively remove endotoxin and the inflammatory mediators in the blood of experimental dogs, with a clearance rate of 94.07%.

Abstract:Objective To investigate the inhibitory effect of rosuvastatin on the migration and invasion of U87 glioma cells and its related mechanisms. Methods Cultured U87 cells were treated with 0, 5, 10 and 20 μmol/ L rosuvastatin for 24, 48 and 72 h. Cell viability was measured by CCK-8 assay. Migration ability of the cells was detected by scratch assay, and invasion ability of the cells was detected by Transwell assay. The expression of matrix metalloproteinase 2 (MMP2), MMP9 and the Wnt/ β-catenin signaling pathway-related proteins was detected by Western blot assay. Results Compared with the control group, the viability of U87 cells in the 5, 10 and 20 μmol/ L rosuvastatin groups was decreased ( P < 0.01) in a time and dose-dependent manner. Migration and invasion abilities of the cells were decreased ( P <0.01). In addition, the expression levels of MMP2, MMP9, Wnt1, Wnt3a, Wnt7a, and β-catenin were decreased as well ( P < 0.01). Conclusions Rosuvastatin can significantly inhibit the migration and invasion abilities of U87 glioma cells, probably related to the blocking of Wnt/ β-catenin signaling pathway.

Abstract:Objective Cisplatin (DDP) is widely used in the chemotherapy of lung cancer. However, cisplatin resistance represents a major obstacle in its effective treatment. Our preliminary work has demonstrated that inactivated Sendai virus (HVJ-E) shows that it induces apoptosis in murine melanoma cells (B16) and obviously inhibites the tumor growth in tumor-bearing BALB/ c nude mice. This study aims to investigate whether inactivated HVJ-E has an effect of inducing apoptosis in cisplatin-resistant A549/ DDP lung adenocarcinoma cells in vitro and in vivo. Methods HVJ-E and A549/ DDP cells were co-cultured in vitro, and the effect of HVJ-E on the apoptosis in A549/ DDP cells was detected by flow cytometry. In addition, HVJ-E was injected into the tumor in vivo, and its oncolytic effect was observed by TUNEL assay of tissue sections and measurement of tumor size. Results After co-cultured with HVJ-E for 12 h, 24 h and 36 h, the apoptosis rate of A549/ DDP cells in late stage detected by flow cytometry was 7.7%, 12.6% and 18.9%, respectively, showing a significant difference between 12 h and 24 h, and between 24 h and 36 h. TUNEL assay showed that there was more apoptosis in tumor cells in vivo in the experimental group than in the control group. Meanwhile, intratumoral injection of HVJ-E induced a significantly smaller tumor volume in the experimental group compared with the control group ( P ﹤ 0.05). Conclusions Our findings indicate that inactivated HVJ-E can induce apoptosis in A549/ DDP cells both in vitro and in vivo, and intratumoral injection of inactivated Sendai virus significantly reduces the tumor growth in vivo.

Abstract:Objective To establish a simple animal model of cough variant asthma (CVA) through sensitizing Brown-Norway (BN) rats with ovalbumin (OVA). Methods A total of 36 BN rats were randomly divided into three groups, including the normal control group, the model control group and the montelukast group. BN rats in the model group and the montelukast group were intraperitoneally administered with 2.0 mg of OVA and 100 mg of Al(OH) ₃ , and the same volume of sterile saline was given to the normal group by intraperitoneal injection. Boosting was carried out by intraperitoneal administration with 0.01 mg of OVA and 100 mg of Al(OH) ₃ 3 weeks later, and the rats in the normal group were injected with the same dose of physiological saline. Three weeks later, the actively sensitized BN rats were challenged with aerosolized OVA for 7 times on alternative days, and the rats in the normal group were treated with sterile saline instead of OVA. At the same time, the montelukast group was given 1.3 mg/ kg of montelukast 30 minutes before atomization by intragastric administration once a day for 2 weeks, and the normal group and the model group were given the same volume of water. The tests of cough sensitivity to capsaicin and bronchial responsiveness were performed 24 h after the last administration. Results Compared with the normal group, the times of coughing ( P < 0.01) and the lung resistance (RL ) ( P < 0.05) in the model group were significantly increased, while the lung compliance (Cdyn) was significantly decreased ( P < 0.05). There was a significant difference ( P < 0.05) in the times of coughing caused by capsaicin between the model group and the montelukast group. Compared with the model group, RL in the montelukast group was decreased significantly ( P < 0.05), and Cdyn was increased significantly ( P < 0.05). Conclusions This rat model of CVA is similar to a variety of clinical features of CVA and is easy to operate. Thus it can be used as an effective animal model of CVA.

Abstract:Objective To analyze the changes and gender differences of organ coefficients, intestinal length and body size of SJ5-SPF chickens at different weeks of age, and to provide experimental data for the sex selection of laboratory chickens. Methods The body weight, the weight of 15 main organs, and length of 5 main segments of intestine and the 6 main parameters of body size of SJ5-SPF chickens at the age of 4, 20, 25 and 40 weeks were measured, respectively, and the organ coefficients were calculated. In addition, the gender differences in body weights, organ coefficients, intestinal lengths and body sizes between the female and male SJ5-SPF chickens at the four different weeks of age were compared, respectively. Results There were significant differences in body weights between the male and female SJ5-SPF chickens at each of the four different weeks of age ( P < 0.01). The organ coefficients varied between females and males to different extents at different weeks of age. As for the intestinal length, there were no significant differences in the length of jejunum and ileum as well as rectum between the male and female chickens, while the length of duodenum, left cecum and right cecum showed gender differences at some of the four different weeks of age. During the determination of body size, there were gender differences in body length, shank length, pelvic width, chest depth and chest width at two or three of the four different weeks of age. Conclusions The organ coefficients, intestinal length and body size of SJ5-SPF chickens are affected by both their age and gender.

Abstract:Objective To establish a quick and accurate method for detection of tree shrew adenovirus (TAV) using TaqMan real-time fluorescence quantitative PCR. Methods Based on the published TAV genome sequence, a 3^, conserved sequence was used to design specific probe primers. A standard curve was prepared using a recombinant plasmid containing the target gene fragment. A real-time fluorescence quantitative PCR method was established for detecting TAV based on TaqMan probe. Results The detection method was specific and was not cross-reactive with other common pathogens. The detection limit of the method was 3.7 copies/ μL, showing a high sensitivity. The correlation coefficient was 0.998, and the efficiency was 95.7%. The amplification result showed a fine linear relationship, and the repeatability test effect was good. Conclusions The TAV real-time quantitative PCR detection method based on TaqMan probe has been successfully established. It has high sensitivity and reproducibility and can be applied to early detection of TAV infection.

Abstract:Objective To establish a safe and effective method of rhesus monkey biopsy to take liver and kidney samples under B-mode ultrasound guidance. Methods A total of 4 adult monkeys (weight: 8 -12 kg; sex: male; age: 11 - 12 years old) were anaesthetized with 5 - 10 mg/ kg of ketamine hydrochloride for each through intramuscular injection. After successful anesthesia, abdominal shaving and iodophor disinfection, they were monitored from intercostal area of right upper quadrant or lateral waist subcostal abdomen portions to find liver or kidney organ by MyLab 30CV B-mode ultrasonography with 3.5 Hz transducer which was fixed with a guiding frame. Large vessels such as the portal vein and inferior vena cava were carefully avoided. The range of the biopsy gun was set to 15 mm. When the puncture target and the puncture needle were positioned in the guide line, the puncture target was perpendicular to the puncture needle, and then the trigger button of the puncture needle was pressed to obtain the liver or kidney tissue samples respectively. After puncture, the needle was pulled out quickly. The obtained liver and kidney tissues were used to extract RNA. Results About 13 mg of liver or kidney tissue was obtained by each puncture with volume convertion. This method was fast, reliable and safe, and the total RNA had high purity and integrity. There was no postoperative bleeding and infection. Conclusions This is a very important method for obtaining liver and kidney tissue samples of rhesus monkeys with the guidance of ultrasound. With this method , the research cost can be reduced, the life quality and animal welfare of laboratory nonhuman primates can be improved, and the accuracy of experimental result can be ensured.

Abstract:Objective To establish a micro volume blood sampling method via the saphenous vein for pharmacokinetic studies (PK studies) in mice, aiming at reducing animal use and alleviate animal pain in in vivo procedures. Methods CD-1 mice were intravenously or orally administered with model compounds A,B, C and D. Blood samples were collected by both the micro-sampling method and regular method at the same time points,and used for the measurement of plasma drug concentration. Pharmacokinetic parameters obtained from each method were compared. Results For each of the four compounds, plasma PK profiles generated by micro-sampling via saphenous vein were in good accordance with those by the regular sampling method via retro-orbital venous plexus. Conclusions Our newly developed micro-sampling blood collecting method can replace the regular blood collecting method used in pharmacokinetic studies in mice. It can significantly improve animal welfare by alleviating animal pain. The volume of blood withdrawn can be reduced by 80%, and the number of mice used for the pharmacokinetic studies can be reduced by 65%.

Abstract:Objective To introduce a single person operation procedure for tail vein injection in conscious tree shrews, and to improve the success rate of injection. Methods The tree shrew was fixed by a canvas glove and a clamp. The tail of the tree shrew was fixed by the operator’s left hand, and the drug was injected by the right hand with a 1 mL disposable syringe. Results This method had the advantages of simple operation, economy and practicality, good fixation effect, high matching degree of animals and high success rate of drug administration. Conclusions Compared with other methods, this method has obvious advantages such as single person operation, convenient, time-saving and labor-saving. The cost is low and the effect is good, thus significantly improving the success rate of injection.

Abstract:Objective To explore a new efficient extraction method of dorsal root ganglions (DRGs) of rats by exploring and extracting DRGs reversely along the nerve traveling pathway. Methods The DRGs were extracted by the traditional method of opening the intervertebral foramina and the new method , extracting DRGs reversely along the nerve traveling pathway, respectively. The time consuming and the number of intact DRGs obtained with these two method were compared. Results The number of intact DRGs (L3 -5 segments, both sides) extracted from each rat with the traditional method was (3.08 ±1.31), and the average time consuming of each DRG was (5.58 ±1.21) min. As for the new method , the number of intact DRGs extracted from each rat was (4.29 ± 1.08), and the average time consuming was (1.69 ±0.91) min, significantly better than that of the traditional method ( P < 0.05 for both). Conclusions The new method of exploring and extracting DRGs reversely along the nerve traveling pathway is more efficient for obtaining intact DRGs of rats, providing more useful tissue materials for subsequent culture and morphological studies of DRG cells.

Abstract:Optical molecular imaging is more and more widely used in the field of biomedical sciences due to its advantageous properties such as non-invasive, real-time and high resolution. As a kind of important optical molecular imaging probe, the near?infrared fluorescent (NIRF) dyes exhibit less tissue absorption and strong tissue penetration, and has been gradually applied to the early diagnosis of tumors. Researchers have developed a number of NIRF dyes with high potential for clinical application by conjugating tumor-targeting ligands, nano-modifications and multimodal NIRF imaging, and have significantly improved the specificity and sensitivity of these NIRF dyes in tumor diagnosis. In this paper we provide a review on the application of NIRF dyes in tumor diagnosis.

Abstract:Innate immune system rapidly detects and responds to viruses at the early stage of viral infection. However, the mechanisms by which the immune system recognizes and eliminates them have not been fully clarified so far. Studies have shown that receptors are the primary tool for cell recognition and detection of viruses, and cyclic GMP-AMP synthase (cGAS) is one of the newly found DNA recognition receptors. cGAS transmits the signal to the downstream protein called STING (stimulator of interferon genes) and mediates the production of type I interferon (IFN-I), thereby to initiates the antiviral immunity of cells. This review briefly introduces the mechanism of the cGAS-STING signaling pathway, in order to provide a theoretical basis for the research and development of new antiviral drugs.

Abstract:Angiogenesis is a process that new blood vessels generate on the basis of an original vascular plexus via sprouting or other forms during normal growth and development of an organism or under particular conditions such as wound repairing and ischemic hypoxia. In general, angiogenesis includes five stages: degradation of the vascular basement membrane, endothelial cell proliferation, vascular sprouting, lumen formation and vascular network turning stable and mature. Among ischemic heart diseases, acute myocardial infarction seriously threatens human health, its pathological characteristics include myocardial vascular stenosis and blockage, leading to myocardial infarction. Therefore, the promotion of angiogenesis has become one of the therapies for ischemic heart diseases. A variety of pro-angiogenic cytokines are involved in the process of angiogenesis, such as polypeptide growth factors and lipid mediators. In this paper we review the recent progress in research on the expression of pro-angiogenic factors in myocardium after ischemic myocardial infarction.

Abstract:The offspring generated by assisted reproductive technology (ART) are considered genetically identical with those generated by natural fertilization. However, studies have found that there are differences between them in some phenotypes to varying extents. In this review, we have summarized the effects of ART on the sex ratio, growth and development, behavior, life expectancy, metabolism of organs and tissues, and molecular phenotypes of mice, providing some guidance to the problems that may arise during the application of ART in the production of genetically modified mice, rapid propagation, purification, embryo cryopreservation, and so on.