Abstract:Objective To examine the nonhuman primate (NHP) model of acute cerebral infarction thrombus?thrombolysis using multi?parameter high?field magnetic resonance imaging (MRI). Methods Altogether, 8 adult male rhesus monkeys aged 8.2 ( ± 1.2) years old and weighing 9.4 ( ± 1.0) kg were randomized into an infarction group (n =4) and thrombolysis group (n = 4). Middle cerebral artery occlusion (MCAO) was induced with a clot in the M1 segment. Monkeys in the thrombolysis group were treated with the recombinant tissue plasminogen activator, rt?PA, while those in the infarction group were treated with 0.9% NaCl only. T2 weighted imaging (T2WI), T2?weighted?fluid?attenuated inversion recovery (T2?FLAIR), time?of?flight magnetic resonance angiography (TOF?MRA), and diffusion?weighted imaging (DWI) were used to examine all monkeys at 4 and 24 h after onset of ischemia. Results The rhesus monkey thrombus?thrombolysis model was successfully established. MRA showed that the middle cerebral artery (MCA) was not recanalized in the infarction group, but was recanalized in the thrombolysis group. T2WI sequence showed an increase in infarction volume (12 027 ±5507 mm³ ) in the infarction group compared with the thrombolysis group (4910 ± 2764 mm³ ). DWI sequence showed an increase in infarction volume (9498 ± 5226 mm³ ) in the infarction group and thrombolysis group (4854 ± 1792 mm³ ). Both T2WI and DWI sequences showed no significant difference in infarction volume at 4 h between the two groups, while infarction volume in the thrombolysis group at 24 h was significantly lower compared with the infarction group. The increase in infarction volume was significantly lower in the thrombolysis group compared with the infarction group. Conclusions MRI sequences can be used to successfully evaluate recanalization.

Abstract:Objective To detect changes in expression of repressor element silencing transcription factor (REST) in rapid amygdala kindling epilepsy rats. Methods Adult Sprague-Dawley rats were randomly divided into a sham group (group S), epilepsy 2 h group (group EP-2 h), epilepsy 14 d group (group EP-14 d), and epilepsy 35 d group (group EP-35 d). An experimental epilepsy kindling model of the amygdala was established by electrical stimulation. After 2 h, 14 d, and 35 d, rats were perfused and their brains were fixed and frozen for coronal sections. Immunohistochemistry was used to detect expression of REST and the neuron specific nuclear protein, NeuN. Results Expression of REST was significantly increased in the CA1, CA3, and dentate gyrus regions in groups EP compared with group S ( P < 0.05), particularly in group EP-2 h. In contrast, NeuN staining was significantly decreased in groups EP compared with group S. Visible neuronal loss was observed in the hippocampus of the EP-35 d group. Conclusions Up-regulation of REST in rat hippocampus may be involved in epileptogenesis.

Abstract:Cells in two-dimensional (2D) cultures gradually lose their original traits as they are passaged in vitro. Existing animal models are expensive and animal experiments require too much work to conduct large-scale experiments. Three-dimensional (3D) cell culture models have attracted increasing attention as they can circumvent the limitations of these previous two models. In 2014, it was reported that the incidence of Alzheimer’s disease (AD) in people over 65 years old was 5.14%. In China, the number of AD patients reached 8 million in 2016, but the pathogenesis of the disease is not yet clear. Study of AD is a ‘hot’ yet complicated issue. This article will briefly introduce 2D and 3D cell cultures and the application of 3D cell culture to AD research in recent years.

Abstract:The Stroke Therapy Academic Industry Roundtable (STAIR) committee has suggested that nonhuman primates (NHPs) should be used for preclinical stroke studies owing to previous translational failures. Ischemia induced by endovascular method closely mimics thromboembolic or thrombotic cerebrovascular occlusion in patients. This method also shows potential for endovascular treatment. This review provides a detailed summary of NHP models using endovascular method , including advantages and disadvantages, and potential applications. Additionally, we also provide further analysis based on different kinds of emboli, infract size, and abnormal hemodynamics. Selection of the optimum model will pave the way for translational research.

Abstract:Parkinson’s disease (PD) is a progressive neurodegenerative disorder, with an etiology that is now considered to be due to interaction between genetic and environmental factors. Typical PD features include loss of dopaminergic neurons in the nigrostriatal region, with typical motor traits of PD associated with dopamine deficiency. Animal models have contributed to determining PD etiology and pathogenesis, as well as testing new therapeutic schedules and novel drug research. Rodents, tree shrews, primates, and other animal models of PD have been established by different method . These models each have their own advantages and limitations, showing different clinical features and pathological mechanisms to those in humans. Therefore, the appropriate model for scientific research must be carefully considered. This article reviews the main neurotoxic and transgenic models of PD.

Abstract:Objective The aim of this study was to investigate the mechanism underlying pruritus by comparing the epidermal growth factor receptor inhibitor (EGFRI)-erlotinib mouse model with the substance P (SP)-induced pruritus mouse model. Methods Two randomized groups of mice were treated with erlotinib or SP to induce pruritus. Behavioral and skin manifestations were observed. Pathological images and neurokinin 1 receptor (NK-1R) expression of the skin were determined. Concentration of interleukin (IL)-31, IL-33, histamine, leukotriene B4, and SP was analyzed by enzyme-linked immunosorbent assay. Nitric oxide was analyzed by colorimetry. Results Transient pruritus induced by erlotinib appeared 2 to 5 days after treatment. In contrast, continuous pruritus was observed during the first hour, but was then gradually relieved. These two shared similar scratching behavior. Concentration of neurotransmitters showed similar trends in changes among the erlotinib group and SP group. Immunohistochemical expression was also consistent between the erlotinib group and SP group. Conclusions Erlotinib-associated pruritus is related to release of signaling factors through the SP/ NK-1R signaling pathway

Abstract:Objective To compare Masson staining, van Gieson staining, and Sirius red staining for evaluation of an intrauterine adhesion (IUA) model in rats. Methods In total, 24 female adult Sprague-Dawley rats were selected and bilateral uteri exposed. To establish an IUA rat model, the left uterus was cut and the endometrium scraped using a scalpel. The right uterus was used as a control. Fourteen days after surgery, all uteri were collected for histological evaluation of IUA by hematoxylin and eosin (HE) staining, Masson staining, van Gieson staining, and Sirius red staining. Results HE staining showed that the endometrial epithelial layer of the uterus was absent, with a smaller number of endometrial glands than the control uterus ( P < 0.01). Collagen fibers were clearly visible using all special staining method . The fibrotic area rate of uteri by Masson staining (using toluidine blue) was higher than Masson staining (using light green), van Gieson staining, and Sirius red staining ( P < 0.01). Under polarized light, type I and type III collagen fibers were clearly distinguished by Sirius red staining, but not using Masson and van Gieson staining. Conclusions Sirius red staining is a superior method than Masson and van Gieson staining for evaluation of fibrosis in IUA and can also differentiate collagen fiber types.

Abstract:Objective To examine the role of β-tubulin on the interaction between the cholecystokinin B receptor (CCKBR), dopamine D5 receptor (D5R), and water-sodium metabolism. Methods Normotensive and hypertensive renal proximal tubular cells (RPTC) were equally randomized into three separate groups: a gastrin group, fenoldopam group, and gastrin + nocodazole group. Immunofluorescence was used to determine localization of β-tubulin, CCKBR, and D5R. Western blotting was used to detect CCKBR, D5R, and Na⁺K⁺ -ATP expression. Results Gastrin stimulation in normotensive RPTC increased D5R expression ( P < 0.05) and decreased Na⁺K⁺ -ATP expression ( P < 0.05). These changes were blocked by a tubulin inhibitor ( P < 0.05). However, interaction between CCKBR, D5R, and Na⁺K⁺ -ATP expression was not significantly affected in hypertensive RPTC. Immunofluorescence showed that CCKBR and D5R can induce one another, followed by transport to the plasma membrane, which can prevented by a tubulin inhibitor. Further, tubulin is disordered in hypertensive RPTC, which cannot support intracellular CCKBR and D5R transport. Conclusions tubulin plays a key role in the interaction between CCKBR, D5R, and water-sodium metabolism by improving protein transfer from the cytoplasm to cell membrane.

Abstract:Objective To explore the role of Sestrin2 in pulmonary alveolar type II epithelial cell injury induced by cigarette smoking and its mechanism of action. Methods The cell injury model was induced by cigarette smoke extract (CSE) in the human pulmonary alveolar type II epithelial A549 cells. The generation of ROS was detected by DCFDA fluorescence probe. The levels of inflammatory factors TNF-α and IL-8 were determined by ELISA, and the expression of Sestrin2 and the peroxiredoxin, Prx-SO_2/3 H, was detected by Western blot. In addition, all the events were also measured in the A549 cells which were transfected with Sestrin2 siRNA and treated with azithromycin. Results After the CSE treatment, the expression of Sestrin2 in the A549 cells was decreased, the expression of Prx-SO_2/3 H was increased, the ROS production, secretion of cytokines TNF-α and IL-8 were increased ( P < 0.05). These changes were partly reduced by azithromycin, indicating that azithromycin significantly relieved CSE-induced oxidative stress and inflammatory injury. Silencing of Sestrin2 in the A549 cells result ed in an increase of Prx-SO_2/3 H expression, ROS production and the secretion of the cytokines TNF-α and IL-8. However, oxidative stress and inflammatory injury were not alleviated with the addition of azithromycin in the Sestrin2 siRNA silencing A549 cells. Conclusions Sestrin2 plays an protective role in the pulmonary alveolar type II epithelial cell injury induced by cigarette smoking through negatively regulating the level of intracellular ROS via catalyzing the reduction of the hyperoxidized peroxiredoxin Prx-SO_2/3 H.

Abstract:Objective To investigate the role and mechanism of PSGL-1 in development of salt-sensitive hypertension in mice. Methods PSGL-1 knockout (PSGL-1 ^{- / -} ) and wild type (PSGL-1 ^{+ / +} ) mice were fed a high salt (6% NaCl) or normal salt (0. 4% NaCl) diet for three months. Blood pressure was measured under anesthesia via the carotid artery. The status of tissue inflammation and kidney injury was tested by flow cytometry, immunohistochemistry, and western blotting. Results Compared with mice fed a normal salt diet, PSGL?1 ^{+ / +} mice fed a high salt diet for three months showed high blood pressure, increased inflammatory cell infiltration in the aorta and skin, and increased inflammatory cytokine expression (interleukin?6, interleukin?1β, and tumor necrosis factor?α) in the kidney, as well as elevated expression of the kidney injury marker, connective tissue growth factor. In contrast, inflammation and kidney injury were not found in PSGL?1 ^{- / -} mice fed a high?salt diet. Conclusions In mice, PSGL?1 via inflammation plays a key role in development of hypertension and kidney injury caused by high salt intake.

Abstract:Objective To investigate the effect of berberine on proliferation of neural stem cells (NSCs) induced by hydrogen peroxide (H₂ O₂ ). Methods NSCs from Sprague-Dawley rats were isolated and purified by suspension culture. Cells were divided into a control group, H₂ O₂ group (NSCs exposed to H2 O2 injury), berberine group (NSCs were incubated with berberine concentrations ranging from 0.5 to 20 μmol/ L and exposed to H₂ O₂ ), and DAPT (a blocker of the Notch signaling pathway) group. Cell viability was evaluated using the Cell Counting Kit-8 assay. Proliferation of NSCs was evaluated by a neurosphere formation assay and Ki67 protein expression. Expression of key proteins in the Notch signaling pathway (including notch1 and hes1) in response to berberine treatment or DAPT (a Notch inhibitor) was determined by Western blotting. Results Cell viability of NSCs was significantly increased by berberine compared with the H₂ O₂ group. The neurosphere growth assay showed that 5 or 10 μmol/ L berberine increased NSC proliferation. The ratio of Ki67 ⁺ / DAPI cells and notch1 and hes1 protein expression increased significantly compared with the H₂ O₂ group. Conclusions Berberine treatment upregulates Notch signaling in NSCs, whereas DAPT attenuates these effects. Berberine is a drug that promotes NSC proliferation and exerts a protective effect on NSCs via the Notch signaling pathway.

Abstract:Objective To investigate the vasorelaxant effect and possible mechanism of niclosamide ethanolamine (NEN) on isolated rat coronary artery (RCA). Methods Wire myograph was used to record myogenic tone of vessels. The vasorelaxant effect of NEN was studied in RCA precontracted with either KCl or U46619. Study of related inhibitors was performed to investigate possible involvement of potassium channels and the mitogen-activated protein kinase (MAPK) signaling pathway in vasorelaxation. The effect of NEN on Ca^{2 +} mobilization was determined by observing vasoconstrictor-induced contractions in tissue solution deprived of Ca^{2 +} followed by Ca^{2 +} restoration. Results NEN (0.5 -3.0 μmol/ L) relaxed RCA precontracted with KCl or U46619 in a concentration-dependent manner. MAPK inhibitors (PD98059 and SB239063) reduced the relaxant effect of NEN, while the potassium-channel blockers (tetraethylamine, 4-aminopyridine, BaCl2 , and glibenclamide) did not significantly affect relaxation. NEN specifically inhibited the contraction component dependent on extracellular Ca^{2 +} influx in vessels stimulated with KCl and U46619, with a negligible effect on the component dependent on intracellular Ca^{2 +} release. Conclusions NEN exhibits vasodilator properties in RCA. Inhibition of extracellular Ca^{2 +} influx and activation of the MAPK signaling pathway may be involved in NEN-induced RCA vasorelaxation.

Abstract:Objective To examine hematological and biochemical parameters in 30 common marmosets, count the mean and standard deviations of each index, and analyze significant differences between female and male groups. Additionally, data from marmosets and macaque breeding were compared. Methods Blood was collected through the posterior limb vein while animals were awake. Hematology and serum biochemical indices were then measured with an automatic blood cell analyzer and blood biochemical analyzer, followed by statistical testing. Results No significant differences were measured in hematological indices between male and female groups. There was a significant difference between the female and male group in serum biochemical indices including high-density ipoprotein-cholesterol (HDL-C) and low-density lipoprotein-cholesterol (LDL-C) ( P < 0.05). Compared with the foreign marmoset group, HGB, MCHC, NEUT, ALT, AST, and GLOB were visibly increased in the group of marmosets fed by our institution, but in accordance with the data range in rhesus monkeys. Conclusions Hematological and serum biochemical indices of common marmosets have been detected in this study and compared with related data in macaques and marmosets. Our findings provide basic data not only for pharmacological and toxicological studies, but also diagnosis and treatment of diseases.

Abstract:Objective To investigate the changes of CD169 expression on the surface of peripheral blood monocytes and different subsets of monocytes in normal rhesus monkeys after SIVmac239 infection and the possible reasons. Methods Normal rhesus monkeys were infected with SIVmac239 through intravenous injection, and changes in the percentage of peripheral blood monocytes and the expression of CD169 before and after SIVmac239 infection were detected by flow cytometry. The peripheral blood CD14 ⁺ monocytes of normal rhesus monkeys sorted by flow cytometry were directly infected by SIVmac239 and stimulated by different cytokines, and changes in the expression of CD169 on the cell surface and the cytokine IFN-α were detected by flow cytometry. Results After SIVmac239 infection, the percentage of CD14 ⁺ monocytes of the normal rhesus monkeys was decreased and the expression of CD169 on their surface was increased. Meanwhile, the expression of CD169 on the surface of different subsets of peripheral blood monocytes was significantly increased, and the expression of CD169 on the CD14 ⁺ CD16 ^{+ +} monocytes was increased more obviously. CD169 was not expressed on the surface of peripheral blood CD14⁺ monocytes of the normal rhesus monkeys after stimulated by the cytokines M-CSF, IL-4 and IL-13. However, CD169 was highly expressed after the monocytes were stimulated by the cytokine IFN-α. The expression of CD169 on the surface of CD14 ⁺ monocytes and the intracellular cytokine IFN-α was not significantly changed after the monocytes were directly infected with SIVmac239. Conclusions SIVmac239 infection can lead to the increase of CD169 expression on the surface of peripheral blood monocytes in rhesus monkeys. Its expression is not associated with the direct infection of virus, but is related to the cytokine IFN-α secreted by other cells of the monkeys in vivo.

Abstract:Objective To investigate the effects of decoction, ethanol extract, and formulated granules of different processed products of Polygonum multiflorum in conventional doses on kidney injury, renal cell apoptosis and expression of related protein in SD rats. Methods Half male and half female SD rats were randomly divided into 7 groups according to their body weight: normal control group, traditional water extract of raw Polygonum multiflorum group (SW group), traditional water extract of prepared Polygonum multiflorum group (ZW group), 70% alcohol extract of raw Polygonum multiflorum group (SA group), 70% alcohol extract of prepared Polygonum multiflorum group (ZA group), raw Polygonum multiflorum granules without water decocting extraction group ( SK group) and prepared Polygonum multiflorum granules without water decocting extraction group (ZK group). Among them, the rats in the normal group were intragastrically given distilled water, and the other groups were treated with the corresponding drug liquids [6 g/ (kg·d) of crude drug] for consecutive 30 days. At the end of the experiment, changes in the levels of blood urea nitrogen (BUN), creatinine (Crea), uric acid (UA) and β2 -microglobulin (β2 -MG) were measured by a semiautomatic biochemical analyzer. In addition, the histopathological changes of kidney tissues were examined, renal cell apoptosis was detected by TUNEL assay, and the expression of Bcl-2 and BAX was determined by immunohistochemical analysis. Results Compared with the normal control group, the levels of BUN in each drug administration group were significantly lower ( P <0.05, P < 0.01). The levels of Crea, UA, and β2 -MG in each drug administration group all showed an increasing tendency compared with the normal control group, especially, the level of β2 -MG in the ZA group was significantly increased ( P < 0.05). The result of TUNEL assay showed that the average optical density in each drug administration group was significantly higher than that in the normal control group ( P < 0.01). In addition, the expression level of BAX in the SA group was significantly increased ( P < 0.01), but expression of Bcl-2 showed no significant difference among the groups ( P > 0.05). Conclusions Our findings indicate that the long-term administration of Polygonum multiflorum with a daily dose of 6 g/ kg of crude drug can cause some damages to the kidneys, and the degrees of kidney injuries are ranked as alcohol extract > formulated granules > water extract. We would suggest that for patients with impaired renal fuction, Polygonum multiflorum should be used with caution, and do not used for patients with severely impaired renal function. For people long-term using Polygonum multiflorum for health care purposes, it is recommended only to use its water extract, and to control renal function, especially, β2 -microglobulin, at regular intervals, to avoid irreversible kidney injury.

Abstract:Objective To analyze the effect of a Chinese medicine Zhengqingfengtongning on the expression of aggrecanase-1 ( ADAMTs-4) and aggrecanase-2 ( ADAMTs-5) in cartilage tissue of knee joint in rabbit models of osteoarthritis, and to study the mechanism of action of Zhengqingfengtongning in treatment for osteoarthritis. Methods 32 New Zealand rabbits were randomly divided into the observation group and the model group, and 16 healthy New Zealandrabbits were chosen as the blank group. The blank group did not receive the intervention treatment, the model group received physiological saline in gastric gavage, the observation group was given the Zhengqingfengtongning by gastric gavage. The soft tissue samples of knee joint in the three groups were taken at 4 weeks after drug intervention. The joint morphology, joint fluid and articular cartilage were observed by histopathology. The ADAMTs-4 and ADAMTs-5 mRNA were detected by RT-PCR. The data were then statistically analyzed. Results The grades of articular cartilage and Mankin’s score of the model group were significantly higher than those of the observation group and blank group, the grades of articular cartilage and Mankin’s score of the observation group were significantly higher than those of the blank group, and the difference between the two groups was statistically significant ( P < 0.05). The ADAMTs-4 and ADAMTs-5 mRNA expressions of the model group were significantly higher than those of the observation group and blank group. The ADAMTs-4 and ADAMTs-5 mRNA expressions of the observation group were significantly higher than those of the blank group, and the difference between the two groups was statistically significant ( P < 0.05). Conclusions The Zhengqingfengtongning can improve the degree of joint lesions in osteoarthritis animal models, which may be related to inhibiting the expression of ADAMTs-4 and ADAMTs-5.

Abstract:Objective To investigate the anatomical structure of arterial vessels in the head and neck of miniature pigs and the related application of vascular cast specimens, and the technology of three-dimensional model reconstruction by CT imaging. Methods A vascular cast specimen of a miniature pig head and neck was made by a 128-slice spiral CT scanning, and a three-dimensional model of the arterial vessels in the head and neck of the miniature pig was reconstructed. Results The cast specimen clearly showed the distribution and running characteristics of the arteries in the head and neck of a miniature pig. The three-dimensional digital model was realistic and stereoscopic, showing the running and distribution of arteries from multiple angles and layers. Conclusions The distribution and running characteristics of arterial vessels in the head and neck of a miniature pig have been investigated by the combination of cast specimen and three-dimensional digital model, providing a morphological reference for the establishment of pig cerebrovascular models in respects of both solid specimen and virtualized model.

Abstract:Objective To establish and preliminarily apply an effective PCR assay for detection of Tupaia (tree shrew) paramyxovirus (TPMV). Methods Using TPMV genomic DNA from NCBI GenBank, bases 8231 to 8720 were synthesized and inserted into a plasmid as a positive standard. One primer pair was designed based on this sequence. In total, 60 respiratory swabs and 12 lung tissues from the tree shrews were tested in this PCR assay. Results A PCR method for detection of TPMV was successfully established, with high specificity and sensitivity of 11.5 × 10 ^-5 μg/ mL. PCR result testing 60 respiratory swabs and 12 lung tissues were negative. Conclusions PCR for detecting TPMV has good specificity and high sensitivity and can be used for conventional tree shrew paramyxovirus detection.

Abstract:Objective Four different methods were examined to identify a safer and more reliable method for tail vein punctures in C57BL/6 mice. Methods In total, 320 mice were randomly divided into four groups: a blank group, incandescent lamp baking method group, three-line method group, and combined method group. Blood samples were taken from the left or right peripheral vein of puncture mice. Puncture success rate of each group was recorded. SPSS 13.0 software was used to compare statistical difference among groups. Results Compared with the blank group, success rates of the other three methods were significantly higher ( P < 0.001). Further, the three-line method was better than the incandescent lamp baking method ( P < 0.001). The success rate of the combined method was significantly higher than the three-line and incandescent lamp baking methods ( P < 0.001). Conclusions The combined method greatly improved the success rate of tail vein punctures in C57BL/6 mice. This method is more reliable and should be more widely used in the future.

Abstract:Chinese hamsters are a kind of valuable laboratory animal resources and play an important role in medical, genetics and pharmaceutical research. More and more biological characteristics of Chinese hamsters have been discovered with in-depth research, and many Chinese hamster models have been established so far. This paper is a brief overview of the development and research progress of Chinese hamsters and their application in taxonomy and medical research.

Abstract:Allergy contact dermatitis is a type IV delayed type hypersensitivity that is induced by exogenous compounds and involves many cell types. Traditional animal testing uses guinea pigs or mice as a model. With progress of the adverse outcome pathway (AOP) on skin sensitization, a concept for development of alternative methods based on a molecular initiating event and key events is provided. Dendritic cell (DC) activation plays a key role in the AOP. Many alternative methods have been developed, with several methods validated and accepted as guidance for assays. This paper examines DC screening, characteristics of test parameters, and limitations and applicability of DC-derived methods. Progress on interactions between DCs and other cells, co?culture systems, and the human body-on-a-chip will also be introduced. Altogether, this paper will provide information for optimization of in vitro alternative methods for sensitization detection.

Abstract:Laboratory animals are an important part of life sciences and medical researches, as well an important support for the science and technology innovation in our country. Laboratory animal science is of great significance to the protection of human health, food safety and biological safety. Laboratory animals are indispensable in the development of food safety, drugs, vaccines and biological products and the studies of human disease pathogenesis. In order to adapt to the requirements for overall development of the laboratory animal industry in China, our institute has independently developed the Network Training System for Laboratory Animal Managers. This system is an online education and training platform which integrates the practical operation and theoretical knowledge of laboratory animals, including seven knowledge modules such as animal welfare, animal breeding, animal surgery and so on. The training subjects of the system include managers, experiment operators, laboratory animal doctors and breeders, aimed at accelerating the personnel training and team building of laboratory animal sciences, and promoting the transformation and development of personnel training in laboratory animal industry.