Abstract:Objective To establish a disease syndrome combined animal model, the miniature pig model of chronic myocardial ischemia of phlegm-blood stasis syndrome type, by high fat/cholesterol diet feeding and intravenous injection with VD₃ and isoproterenol. Methods Miniature pigs were randomly divided into the control (Ctr) group, high fat/cholesterol diet (HFC) group and chronic myocardial ischemia model of phlegm-blood stasis syndrome (CMI) group, 5 pigs in each group. The Ctr group was fed with normal regular chow diet, HFC group was fed with high fat/cholesterol diet, while the CMI group was fed with high fat/cholesterol diet and intravenous injection with VD₃ and isoproterenol. The experiment lasted for 24 weeks. The model establishment and its pathological process of phlegm-blood stasis syndrome were evaluated by examinations of body weight, electrocardiogram, activity, blood lipid, myocardial enzymes, hemorheology, inflammation, cardiac index(CI) and myocardial ischemia size (MIS). Results Compared with the Ctr group, the body weight, heart rate(HR), Total cholesterol(TC), low density lipoprotein-cholesterol (LDL-C), high density lipoprotein-cholesterol(HDL-C),atherosclerosis index(AI),low/middle/high shear rate of whole blood viscosity and reduced viscosity, erythrocyte electrophoresis time(EPT), high-sensitivity C-reactive protein (hs-CRP) and IL-6 levels in the HFC group were significantly increased (P<0.05, P<0.01), while the body weight, heart rate, total ST, ST_average, activity, TC, TG, LDL-C, HDL-C, AI, CK, LDH, cTn-1, low/middle/high shear rate of whole blood viscosity and reduced viscosity, EPT, Casson viscosity(CV), hs-CRP, IL-6, CI and MIS in the CMI group were significantly increased (P<0.05, P<0.01), and APN level in the CMI group was significantly decreased (P<0.05). Moreover, AI, CK, LDH, cTn-1, low/middle/high shear rate of whole blood viscosity, EPT, CI and MIS in the CMI group were significantly higher than those of HFC group (P<0.05, P<0.01), while APN in the CMI group was significantly lower than that of HFC group (P<0.05). Correlation analysis showed that MIS was closely correlated to TC, LDL-C, AI, CK, LDH, cTn-1, APN, high/middle/low shear rate of whole blood viscosity, EPT, CV, hs-CRP and IL-6 (P<0.05, P<0.01). The linear regression analysis also showed that phlegm-blood stasis was closely correlated to TC, LDL-C, AI, CK, LDH, cTn-1, APN, CV, EPT, hs-CRP, and IL-6 (P<0.01), and further linear stepwise regression analysis showed that the evolution of phlegm-blood stasis was closely related to TC, CK and IL-6. Conclusions Minipig model of chronic myocardial ischemia of phlegm-blood stasis syndrome type can be established by high fat/cholesterol diet feeding and intravenous injection with VD₃ and isoproterenol. Their blood lipid metabolism, hemorheology, myocardial enzymes and inflammatory indexes can be used as external biochemical basis of phlegm-blood stasis syndrome type, which may reflect related biological basis of the traditional Chinese medicine theory of "phlegm and stasis cementation, blood-stasis & toxin causing catastrophe".

Abstract:Objective To investigate the kidney yang deficiency constitution and hypothalamus-pituitary-adrenal (HPA) axis dysfunction in Lewis rats. Method Two kidney yang deficiency models were established by subcutaneous injection of hydrocortisone and by adrenalectomy to induce hypothalamic-pituitary-adrenal (HPA) axis dysfunction in Lewis rats. Wistar rats were used as control and compared with the two types of Lewis rat models of kidney yang deficiency. The degree of kidney yang deficiency of the Lewis rats was evaluated by examination and detection of the general signs, behavior, and neuroendocrine function, and so on. Result Compared with the normal Wistar rats, the body weight of Lewis rats was significantly higher than that of Wistar rats at the same age. The body temperature, urine volume and grip strength were significantly lower than those of Wistar rats (P<0.01 for all). The memory ability of the Lewis rats was slightly decreased. The liver, kidney and adrenal indexes were significantly decreased in the Lewis rats (P<0.01). The levels of serum ACTH, CRH, cGMP, Cort and urine 17OHCS were significantly decreased (P<0.01). Conclusion A very slight deficiency of adrenal cortex function of Lewis rats is caused by genetic inheritance, and Lewis rats have the constitution of congenital kidney yang deficiency (criticality or prophase of kidney yang deficiency).

Abstract:Objective To clarify the protective action of a Chinese medicine, Jie-Du-Qu-Yu-Zi-Shen prescription, on the lupus nephritis in MRL/lpr mice through the TLR9-MyD88-NF-κB signaling pathway. Methods Thirty MRL/lpr mice were randomly divided into model group, prednisone group, and traditional Chinese medicine (TCM) group (n=10). Each treatment group received appropriate treatment or drug therapy for 8 weeks. The pathological changes of mouse renal tissues in the three groups were examined and compared using HE, PAS, and Masson staining. Real-time PCR was performed to compare the expression of TLR-9, MyD88, NF-κBm RNA mRNA in each group of the mice. Results Compared with the model group, the Chinese medicine Jie-Du-Qu-Yu-Zi-Shen prescription significantly improved the renal pathological tissue damages in the MRL/lpr mice. Real-time PCR assay showed that the expression levels of TLR-9, MyD88, NF-κB mRNA in the TCM group were significantly lowered (P<0.01 for all). The differences between the TCM group and prednisone group were statistically not significant (P>0.05). Conclusions Jie-Du-Qu-Yu-Zi-Shen prescription has protective effect on the kidney of MRL/lpr mice, alleviating its pathological changes, and delaying the disease course, probably, through inhibiting the TLR-9-MyD88-NF-κB signaling pathway in the kidneys of MRL/lpr mice.

Abstract:Objective To investigate the effect and mechanism of a Chinese traditional prescription, Yuebi Banxia decoction, on allergic asthma in mice by observing the changes of serum immunoglobulin E (IgE) and lung tissue superoxide dismutase (SOD) activity in the mouse model of allergic asthma. Methods Forty healthy Kunming mice were randomly divided into normal control group, asthma model group, dexamethasone intervention group and Chinese medicine preparation group. The mouse model of asthma was generated by peritoneal injection of ovalbumin (OVA) and suspension of aluminium hydroxide. The mice of traditional Chinese medicine group received Yuebi Banxia decoction in a dose of 1 mL/100 g, daily for 30 days. The mice of dexamethasone group were given intraperitoneal injection of dexamethasone 1 mg/kg. The normal control group and asthma model group were given gastric gavage of physiological saline. After 30 days administration, the serum levels of IgE and SOD activity of lung tissue were determined, and the pathological changes of lung tissue were observed using HE staining. Results The mice of traditional Chinese medicine preparation group showed significantly decreased serum IgE level, and significantly increased SOD activity than those of the asthma model group mice (P<0.05 for both). Conclusions Reduced serum IgE and increased SOD activity in the lung tissue of mice with allergic asthma, improving the antioxidant ability of lung tissue, may be involved in the anti-allergic asthma mechanism of the Chinese traditional prescription Yuebi Banxia decoction.

Abstract:Objective To establish a rat model of atherosclerosis combined with syndrome of intermingled phlegm and blood stasis, to observe the inflammatory reactions and the treatment effect of prescription (Danlou tablet) on the rat model. Methods Thirty-two healthy male SD rats were randomly and equally divided into four groups, namely, normal control group, model control group, atorvastatin group (ATV group), and Danlou group (DLP group). The normal control group was given basic forage, while other three groups were given high fat forage plus intraperitoneal injection of vitamin D3 and balloon injury of the left common carotid artery to build rat atherosclerosis model combined with syndrome of intermingled phlegm and blood stasis, and then received intragastric administration of saline, atorvastatin suspension and Danlou tablets suspension for 4 weeks, respectively. After intervention, both serum lipid and hs-CRP, IL-6, TNF-α, and LP-PLA2 levels were determined by ELISA, pathological alterations in the thoracic aorta was analyzed using HE staining, the expressions of IL-6, TNF-α and LP-PLA2 mRNA in the thoracic aorta tissue were assessed by real-time fluorescent quantitative PCR technology. Results ① Compared with the normal control group, there were significant increases in serum TC, LDL-C, hs-CRP, IL-6, TNF-α, and LP-PLA2 levels (P<0.05 for all) and decrease of serum HDL-C levels in the model group (P<0.05). The expressions of IL-6, TNF-α, and LP-PLA2 mRNA in the rat aorta were significantly increased (P<0.05 for all). In the model control group, HE staining showed altered arrangement of aortic endothelial cells, irregular intimal thickening, broadened subendothelial space, and accumulation of foam cells and lipid deposition, forming typical atherosclerotic plaques. ② Compared with the model control group, the ATV and DLP groups showed significantly decreased serum TC, LDL-C, hs-CRP, IL-6, TNF-α, and LP-PLA2 levels (P<0.05 for all) as well as the expressions of IL-6, TNF-α, and LP-PLA2 mRNA in the thoracic aorta (P<0.05 for all). There were no significant differences between the ATV and DLP groups (P>0.05). There were also reduced aortic intimal hyperplasia, macrophage infiltration and plaque area compared with those of the model group. Conclusions Rat model of atherosclerosis combined with syndrome of intermingled phlegm and blood stasis can be established by high fat diet feeding combined with the intraperitoneal injection of vitamin D3 and balloon injury of carotid artery. The prescription (Danlou tablet) can inhibit the inflammatory reaction and ameliorate atherosclerotic changes in the rat models.

Abstract:Objective To explore the dynamic changes of oxidative stress and cytokines in Mongolian gerbils with nonalcoholic fatty liver disease (NAFLD) and their significance. Methods Forty-eight healthy male gerbils were randomly divided into normal group and model group, 24 in each group. Gerbils of the model group were fed with high fat diet while those of the normal group with normal diet. Eight gerbils in each group were killed at the end of 4 w, 8 w and 16 w, respectively. MDA content and SOD, GSH-PX and T-AOC activity in the liver tissue were detected by chemical method, and serum TNF-α, INF-γ and IL-10 levels were determined using liquid suspension chip. Results With the development of NAFLD, MDA content in liver increased gradually, and the MDA contents were all significantly higher than those of the normal group (P<0.01); T-AOC level slightly increased, and then decreased, the levels at 4 w and 16 w were markedly decreased compared with those of the normal group (P<0.05); SOD level was significantly increased and then markedly reduced, the level of the model group at 4 w was significantly increased (P<0.05), while that at 8 w and 16 w were significantly decreased (P<0.05, P<0.01). The level of GSH-PX was decreased gradually, the levels at 8 w and 16 w were significantly lower than those of the normal group (P<0.05). With the progression of NAFLD,serum TNF-α and IFN-γ were increased gradually, while the level of IL-10 decreased gradually, and the levels at 8 w and 16 w were significantly lower than those of the normal group (P<0.05, P<0.01). Conclusions The oxidative stress-related indicators and inflammatory cytokines in the gerbil NAFLD models induced by high fat diet are significantly changed as simple fatty liver develops into steatohepatitis, liver fibrosis and cirrhosis, and participate in the development and progression of NAFLD.

Abstract:Objective To establish a rat model of increased blood flow-induced pulmonary arterial hypertension generated by anastomosis of the left common carotid artery to left external jugular vein. Methods 45 male SD rats were divided into three groups: the shunt group, the ligation group and the sham group. At twelve weeks after the procedure, the general status of the rats was observed. Heart conditions, cardiac output and shunt patency were measured by echocardiography. Right ventricular systolic pressure (RVSP) and Qp/Qs were checked by catheterization. Right ventricular hypertrophy index (RVHI) was calculated and lung tissues were examined by pathology using hematoxylin-eosin and elastin Van Gieson staining. All data were analyzed statistically by one-way ANOVA test using SPSS 16.0. Results There was no significant difference in body weight gains between the groups. The patency rate of shunt was 84.6%. The heart was enlarged in the group shunt. Cardiac output increased significantly in the shunt group than that in the other two groups [(309.8±33.1) mL/min·kg vs. (245.6±31.9) mL/min·kg, (240.8±30.9)mL/min·kg, respectively, P<0.05]. In the shunt group Qp/Qs was 2.16±0.38 and RVSP increased to (35.8±4.9) mmHg, RVHI was 0.3263±0.0342, significantly higher than that of the other groups. The pulmonary arteriolar wall was evidently thickened in contrast to that in the sham group [(22.3±1.7)% vs. (10.6±1.7)%, P<0.05). Conclusions Anastomosis of the left common carotid artery to left external jugular vein can successfully establish pulmonary arterial hypertension model induced by high blood flow in rats.

Abstract:Objective To explore the correlation of obesity and early puberty in immature female SD rats by comparison of puberty development-related indicators between the model group and control group after setting up SD immature rat models of nutritional obesity. Methods Ten female rats and 6 male rats were divided in two groups: the parent model group (5 females and 3males) and parent control group (5 females and 3 males). The model group was fed with high-fat diet for 8 weeks and control group was fed with normal diet. The rats of model group with body weight ≥20% higher than those of the control group were matched and their female offsprings were taken as model offspring group. The female offsprings of the parent control group were taken as control offspring group. The 21-days old immature rat model group was fed with high-fat diet for 5 weeks while the control group immature rats were fed with normal diet for the same time. Since 21-days old the immature rats were observed for the vaginal opening state daily, and after the vaginal opening they were examined by vaginal smear. The rats were weighed daily, and their abdominal circumference and body length were measured biweekly. The rats of both experimental groups were killed at the age of 56-days old, the serum triglyceride, cholesterol, estradiol, follicle stimulating hormone, and luteinizing hormone were measured, and the uterus, ovaries, pituitary gland were taken and weighed and the ovary index and uterus index were calculated. The uterus and ovaries were examined by pathology using HE staining, and the corpus luteum and mature follicles in the largest cross section of ovaries were counted. Results 1. The body weight and the abdominal circumference of the 56-days old immature rats in the model group were significantly higher than those of control group (P<0.001). 2. The values of serum TG and CH in the immature rats of model group were significantly higher than those in the control group (P<0.001). 3. The values of serum LH and E₂ in the immature rats of model group were significantly higher, and the value of serum FSH was significantly lower than those of the control group (P<0.001 for all). 4. The number of corpus luteum in the largest cross section of ovaries was significantly higher, and the number of mature follicles was significantly lower in the model group than those of control group (P<0.001 for both). 5. The Pearson correlation analysis showed that the body weight of immature rats was positively related to the serum LH level and quantity of corpus luteum, and negatively correlated with vaginal opening time and the first diestrus time; the abdominal perimeter of the immature rats was positively related to the quantity of corpus luteum, serum LH, and negatively correlated with vaginal opening time and the first diestrus time. Conclusions The early puberty of immature SD rats is positively related to obesity, i.e. the development of immature rats with obesity occurs earlier than in normal immature rats.

Abstract:Objective To explore the stability of rat models of subdural hematoma prepared by subdural injection of different volumes of autologous blood. Methods The rats were randomly divided into sham group (36), 300 μL blood group, 500 μL blood group, and 700 μL blood group (each group 60 rats). The rats of model groups received subdural injection of 300 μL, 500 μL, or 700 μL autologous blood, respectively. At the postoperative 2nd, 4th, 6th, 8th, 10th, 14th days, blood samples were taken from the abnormal aorta, and the brains were taken out for gross examination and taking photographs, six rats were used for each time. Enzyme linked immunosorbent assay (ELISA) was performed to determine the content of serum NSE and S100B proteins in the rats in each group. Results Compared with the sham operation group, the serum NSE in the 300 μL group was significantly increased at the 2nd and 4th days (P<0.01), and then gradually reduced at the 6th and 8th days (P<0.05), indicating that the hematoma began to disappear, and at the 10th and 14th days returned to a similar level of the sham operation group (P>0.05). In the 500 μL and 700 μL blood groups, the NSE contents at 2nd, 6th, 8th, 10^th and 14th days were significantly increased (P<0.01), but not significantly increased at the 4th day (P>0.05). The content of S100B protein in the 300 μL blood group was significantly higher at the fourth day (P<0.01), lower at the 2nd and 6th days (P<0.05), and at 8th, 10th and 14th days was similar to that in the sham operation group (P>0.05 for all), indicating that the hematoma disappeared gradually, and the damages repaired. The S100B protein content of the 500 μL and 700 μL blood groups was constantly kept at a higher level (P<0.05). Conclusions Compared with the 300 μL ad 700 μL blood groups, the rat model of subdural hematoma developed by subdural injection of 500 μL autologous blood is the best, and can be used for studies of rat subdural hematoma.

Abstract:Objective The aim of this study was to provide a better positive control for allergic test by comparing the allergic effect of two kinds of positive materials, human albumin and ovalbumin, on active systemic anaphylaxis in guinea pig. Methods Guinea pigs were randomly divided into 14 groups, and were given human albumin, ovalbumin (2, 10, 100 mg/animal), or 0.9% sodium chloride injection as test substances, to assess the symptoms and incidence of systemic allergic responses induced by different sensitizing substances in different challenge doses and different challenge intervals.Results In the range of 2 to 100 mg/animal, the guinea pigs showed a 100% incidence rate of positive allergic reaction to human albumin and ovalbumin, the severity of anaphylactic symptoms was increasing along with the increase of sensitizing doses and challenge doses, and the allergic reaction was more strong induced by the same dose of ovalbumin than human albumin. Conclusions Our findings indicate that in the active systemic anaphylaxis test in guinea pigs, we recommend ovalbumin as the positive control in a dose of 2 mg/animal.

Abstract:Objective To compare the differences of two stocks of guinea pigs, the albino guinea pigs and pigment guinea pigs, in establishing dyslipidemic model, to evaluate their lipid-lowering action, and to compare their properties for development of hyperlipidemia. Methods Two stocks of the 5-week-old guinea pigs were randomly divided into two groups, normal group (NC) and model group (Model). For the NC group, 12 guinea pigs were fed with normal chew. For the model group, after fed with high-fat diet for four weeks, 24 male guinea pigs were randomly grouped and treated with vehicle (VC group) and pitavastatin (Pit group) calcium, respectively, by gavage as well as received high-fat diet. Before and after modeling and pitavastatin treatment, blood samples were collected and subjected to analysis of plasma TC, TG, HDL-C and LDL-C, respectively. Results In the normal group, the blood lipid levels of albino guinea pigs were more stable than that of the pigmented pigs with the increase of age. After fed with high-fat diet, the plasma lipid levels of TC, TG and LDL-C were significantly increased in the two strains of guinea pigs, while HDL-C showed a decrease to varying degrees. Interestingly, the lipid level in the albino guinea pigs was significantly higher than that of pigment guinea pigs. And also, after drug administration for four weeks, pitavastatin treatment significantly decreased the elevated lipid level of TC, TG and LDL-C in the albino guinea pigs compared with that in the pigment guinea pigs. Conclusions The albino guinea pigs and pigment guinea pigs demonstrate certain differences in establishing dyslipidemic model and evaluating lipid-lowering pharmacodynamics. However, compared with the pigment guinea pigs, the albino guinea pigs have obvious superiority because of easy establishment of hyperlipidemia model and are more sensitive to lipid-lowering drugs.

Abstract:Objective Comparing four groups of immunosuppressive mouse models established by cyclophosphamide administration in different doses and periods, we used partial least squares(PLS) analysis to evaluate the immunosuppressive mouse models comprehensively and select the appropriate way to establish this model. Methods In this experimental study, 58 male KM mice were randomly divided into five groups: normal group (10 mice) were given normal saline daily by i.p. injection, model group 1 (12 mice) was given 40 mg/kg CTX daily by i.p. injection for 10 days, model group 2 (12 mice) was given 80 mg/kg CTX daily by i.p. injection for 3 days, model group 3 (12 mice) was given 40 mg/kg CTX daily by i.p. injection twice for a week, model group 4 (12 mice) was given 50 mg/kg CTX daily by i.p. injection for 7 days. After the injection of cyclophosphamide, the daily metabolic activities were detected everyday such as body weight, water intake, and food intake, organ index and immunological indexes such as blood RT of the model mice were measured as well. Using partial least squares (PLS) analysis to the models and analyzing the final weight, final anal temperature, organ index, and blood routine examination in order to evaluate the immunosuppressive mouse models comprehensively. Results Compared with the normal group, different dosages of CTX reduced the weight and anal temperature of model mice (P<0.05), the food intake and water intake (P<0.01), and the spleen index and thymus index (P<0.01). Besides, the amount and percentage of basophilic granulocytes decreased (P<0.05), and the percentage of MCHC, macrophage went up, as well as the absolute value of WBC and lymphocytes were decreased in the model groups 1, 2, and 4 (P<0.05). According to the PLS analysis, there were significant differences among models 1, 2, and 4 when compared with the normal group, especially the most different in the model group 1. Conclusions After the PLS analysis of different indexes, the optimal way to establish immunosuppressive mouse models is the procedure with 40 mg/kg CTX daily injected i.p. for ten days. Our findings provide experimental evidence for the establishment of immunosuppressive mouse models.

Abstract:Objective To establish an ion chromatography (IC) method for detection of ammonia in the environmental air of laboratory animal facilities. Methods Dilute sulfuric acid was used as the absorption liquid to collect ammonia in the air. Ammonia was converted into ammonium ion in the absorption solution. With methylene sulfonic acid as the pouring liquid, the content of ammonium ion in the absorption solution was detected, and the concentration of ammonia in the air was calculated. Results The ammonium ion showed good linearity in the range of 0 to 1 μg/mL (r=0.9994), and high precision (RSD<6.38%). The lowest detected concentration was 0.012 mg/m³, and the average recovery was 98.8%. Conclusions This is a simple method, easy to operate, not interfered by other ions, and can be used to detect the ammonia in the environmental air of laboratory animal facilities.

Abstract:Schizophrenia is a common and serious mental disorder, which causes great harm to patients and their families. Because of the complicated pathogenesis of schizophrenia, there are still many problems in its drug treatment. Stable and reliable animal models are required for preclinical studies of new drugs. In this article, we reviewed the rodent models associated with positive symptoms, negative symptoms and cognitive impairment of schizophrenia and the behavioral evaluation on these models, hoping to provide useful references of animal models for the antipsychotic drug screening and preclinical studies.

Abstract:There are many ways to establish animal models of neonatal hyperbilirubinemia, such as intraperitoneal or intravenous injection, genetic defect animal models, the use of chemical drugs, and injection of bilirubin into cerebellomedullary cistern, and so on. In order to study the etiology, pathogenesis, and therapy of neonatal hyperbilirubinemia through animal models,we review the literature on rodent and primate models of neonatal hyperbilirubinemia,including establishment of models and their applications, in order to provide reference for the research of neonatal hyperbilirubinemia.

Abstract:Experimental animals are important basis for life science research and development. Along with the continuous development of science and technology, new technology and new ideas emerging, treatment and protection of animals during experiments are important condition to ensure the scientific results accurate and reliable, so scientists have paid more attention to the issues of animal welfare and protection. This article summarizes the animal treatment and protection measures during experiments based on both own work and experience and knowledge of other scientists.