Abstract:Objective To explore the effects of rapamycin on blood glucose level and insulin resistance in diet-induced obese mice. MethodsHealthy 4 week-old male C57BL/6 mice were fed with high fat diet (HF,n=18)or normal chow (normal control, NC, n=18) for 8 weeks. Both control and obese mice were treated with placebo, rapamycin [2 mg/kg,intraperitonealy (ip), Qod, n=6] and 2.37% leucine-containing drinking water (n=6) for 2 weeks, respectively. Oral glucose tolerance test (OGTT), insulin tolerance test (ITT) and morphological studies for pancreatic islets were performed. ResultsThe normal mice treated with rapamycin showed a significant increase of glucose level at 30 minutes after glucose challenge in comparison with the placebo- (P=0.038) and leucine-treated (P =0.035) groups. The rapamycin-treated obese mice showed a significantly elevated fasting glucose level compared with the placebo-treated obese mice (P =0.031), and a higher glucose level at 30 minutes after glucose challenge than that in the placebo-treated mice (P=0.013) and leucine-treated mice (P =0.041). The normal mice after rapamycin treatment had a decreased insulin sensitivity than those with placebo treatment (P =0.039). Rapamycin treatment significantly reduced the peritoneal white adipose mass in both normal (P<0.001) and obese mice (P =0.013)than in the placebo controls. ConclusionsRapamycin had significant effect on glucose metabolism in mice. The impairment of glucose metabolism in normal mice after rapamycin treatment is correlated with decreased glucose tolerance, while the impairment of glucose metabolism in obese mice is resulted from reduced insulin secretion and increased insulin resistance.

Abstract:ObjectiveThe purpose of this study was to investigate the mechanisms and characteristics of hyperglycemia-induced liver injury in streptozotocin-induced juvenile diabetic cynomolgus monkeys with poor glycemic control. MethodsA total of eight cynomolgus monkeys (3 years old) were used in this experiment. Four monkeys were induced insulin-dependent diabetes mellitus for four years. Other four age-matched healthy monkeys were used as the controls. Blood tests, periodic acid-Schiff (PAS) staining, Sudan III staining, Gordon-Sweet staining, Masson trichrome staining, conventional light microscopic and ultrastructural morphometry of the liver tissue were performed in both groups. ResultsTotal bile acid, blood urea nitrogen, alanine aminotransferase, aspartate aminotransferase, cholinesterase, lactate dehydrogenase, total cholesterol, triglyceride and low density lipoprotein cholesterol levels were significantly increased in the diabetic monkeys than in the normal monkeys. Histological examination showed swollen hepatocytes, increased number of positive PAS staining and Sudan III staining cells. Ultrastructural observation indicated that hepatocytes contained a drastic increase of glycogen granules and high electron-dense mitochondria, and hepatic stellate cells were also considerably more numerous in the diabetic livers. ConclusionsJuvenile diabetic cynomolgus monkeys with poorly controlled blood sugar showed specific pathologic changes including excess accumulation of glycogen in hepatocytes and increased number of hepatic stellate cells. These pathologic changes were distinct from nonalcoholic fatty liver disease, however, the mechanisms are unclear yet.

Abstract:Objective To explore the effects of telmisartan on the kidney function in diabetic rats and to investigate its possible mechanism. Methods SD rats were randomly divided into two groups:diabetic group (n=20) and control group (n=10). The diabetic group was injected with streptozotocin (STZ). Diabetic rats were randomly divided into two groups:telmisartan group (treated with 10 mg/kg/d telmisartan, n=8) and diabetic group (n=8). The fasting blood glucose, body weight, 24-hour urinary protein, creatinine clearance rate (Ccr), serum creatinine and blood urea nitrogen (BUN) were tested. Gene expression in the rat renal tissues was assyed with microarray analysis. ResultsTelmisartan significantly decreased 24-hour urinary albumin (P<0.01), serum creatinine (P<0.05), BUN (P<0.01), and increased Ccr (P<0.05), compared with those of the diabetic group. Microarray analysis showed that expressions of 1541 genes were significantly changed in the telmisartan group (554 increased, 987 decreased). Real-time PCR verified that ATP synthase beta subunit (ATP5b), cytochrome c oxidase subunit Vic (Cox6c), and NADH dehydrogenase (ubiquinone) Fe-S protein 3 (Ndufs3) were significantly down-regulated. Conclusion Telmisartan can improve kidney function in diabetic rats. Mitochondria oxidative phosphorylation and PPAR-γ pathway may be involved in the mechanism of action.

Abstract:Objective To explore the establishment methods of rat models of diabetes mellitus by streptozotocin combined with different diets and to assess the learning and memory ability of the rat models, and to provide reliable models for diabetes research and related drug development. MethodsSeventy male Sprague-Dawley rats were randomly divided into 7 groups, (10 rats each):the normal group (I), high fat diet group (II), 0 week STZ (30 mg/kg) + high fat diet group (III), 0 week STZ (30 mg/kg) + standard diet group (IV), 6 weeks STZ (20 mg/kg) + high fat diet group (V), 6 weeks STZ (25 mg/kg) + high fat diet group (VI) and 6weeks STZ (30 mg/kg) + high fat diet group (VII). Diabetic models were established by intravenous injection of STZ, combined with different diets. The blood glucose (BG) levels were continuously monitored. Biochemical methods were employed to detect the serum lipids. The levels of serum insulin and glucagon were also determined. Morris water maze was used to test the changes of learning and memory ability in the model groups. ResultsCompared with the control, the blood glucose level in the rats of group III was significantly increased 72 h after STZ injection and reached to a peak 2 weeks after STZ injection (P<0.01). Then it decreased gradually, the BG remained at a level of 15 mmol/L 10 weeks after STZ injection (P<0.05). The BG value of the rats in the group IV was increased 72 hours after STZ injection, then it decreased rapidly, and dropped to a normal level 10 weeks after STZ injection. The BG values of rats in the groups V, VI and VII were elevated after the STZ injection and displayed a fluctuating profile in the following period. The range of BG changes depended to the dose of STZ. Compared with the control group, the values of insulin levels of the groups III, IV and V were increased after high fat feeding for 10 weeks. The values of glucagon in all experimental groups were increased except the group VI. The values of CHO, TG, LDL-c were increased in all high fat diet-fed rats. Morris water maze test results showed that the escape latency of group VII was significantly longer than that in the control group. ConclusionsOur findings indicate that intravenous injection of STZ (30 mg/kg) with high fat diet is a rapid and stable method for establishing a diabetic model in rats. Indeed, after 6 weeks of high fat diet and subsequent STZ (30 mg/kg) injection, the rats tend to develop type 1 diabetes since they exhibit a high level of blood glucose and low insulin level in the rat models.

Abstract:Objective To investigate the variation of serum amino acids in diabetic mice in order to explore the application of metabolic profiling analysis in research of diabetes mellitus in mice. Methods Healthy male adult SPF Kunming mice were fed with high-fat diet for 4 weeks and then injected with streptozotocin (STZ). Fasting blood glucose (FBG) was monitored weekly after STZ injection. Blood samples of normal mice and model mice were collected at 4 weeks after STZ injection. The metabolic profiles were analyzed using pre-column derivatization HPLC. ResultsCompared with the control mice,the FBG, TG, and FFA were significantly higher than those in the model mice. The metabolic profile of the model mice was significantly different from that of the control mice. ConclusionsPattern recognition method is applied and demonstrates that four amino acid markers (arginine, phenylalanine, lysine, tuarine) are different between the normal and model mice. Amino acid metabolism profiling combined with pattern recognition may reflect to a certain extent the pathological changes of type 2 diabetic mice.

Abstract:ObjectiveTo explore the application value of combined detection of microalbumin, β₂-microglobulin, TRF, glycated hemoglobin and serum lipids in diabetic rhesus macaque models. Methods Streptozotocin in a dose of 30 mg/kg of body weight was intravenously injected to five rhesus macaque for consecutive 3 days. The dynamic changes of urine microalbumin, β₂-microglobulin, TRF, glycated hemoglobin were measured for 13 months. Resultsβ₂-microglobulin and transferrin were significantly higher than those before STZ injection (P<0.05). The metabolism of TC and HDL-C were abnormal 6 months after STZ injection. Abnormal TG and LDL-C metabolism occurred 9 months after STZ injection. Significantly positive correlations were observed between TRF and β₂-MG (F=0.2,0.153, P < 0. 05), mALB and TRF (F=0. 213, P< 0. 05), as well as HbAlc and β₂-MGTRF ( F=0. 3,0.097, P< 0.05). There was a positive correlation between urine mALB and β₂-MG (F=0.2,0.153, P < 0. 05), between TRF and β₂-MG (F=0.213, P< 0. 05), and between HbAlc and β₂-MG (F=0.3,0.097, P< 0. 05). Single factor linear regression analysis showed that TRF was a correlation factor of mALB and β₂-MG ( P < 0. 05). ConclusionsThe results show that the combined detection of urine microalbumin, β₂-microglobulin, glycated hemoglobin and serum lipids can be used in rhesus macaque models of diabetes mellitus, and are useful in their early monitoring, diagnosis, therapy and prognosis.

Abstract:ObjectiveIn order to provide scientific data for understanding the adaptive mechanism of Schizopygopsis pylzovi to seasonal cold environment at Qinghai-Tibetan Plateau, the obese gene (ob) of Schizopygopsis pylzovi was cloned and its encoding protein leptin was analyzed. Then the expression pattern of the ob mRNA in tissues and the relative expression level of the ob mRNA in hepatopancreas and white muscle of Schizopygopsis pylzovi from habitats of different temperatures were assessed. Methods The full-length cDNA of ob gene was cloned by RT-PCT, 5′-RACE and 3′-RACE methods. The expression pattern and relative expression level were determined by RT-PCR and real-time PCR. ResultsThe full-length cDNA of ob gene was 1 337 bp, including a 513 bp complete ORF encoding a 170 amino acid peptide. The ob gene was expressed in the nine examined organs, including heart, kidney, fat, intestine, spermary, hepatopancreas, gill, brain and muscle, while strongly expressed in heart and weakly expressed in muscle. The ob gene expression in the hepatopancreas and white muscle of Schizopygopsis pylzovi was suppressed by low temperature. ConclusionsThe tissue expression pattern of ob gene in Schizopygopsis pylzovi may be related to energy metabolism and biological functions in specific tissues. The encoding protein leptin plays an important role in the regulation of energy metabolism of Schizopygopsis pylzovi, as well as its adaptation to the seasonal cold environment at Qinghai-Tibetan Plateau.

Abstract:Objective To explore the electrophysiological characteristics of right atrial appendage (RAA) and left atrial appendage (LAA) in 48-h atrial fibrillation (AF) canine detected by microelectrode array (MEA) chip technique. Methods Twelve adult healthy mongrel canine were randomly divided into two groups:48-h pacing group and control group. The 48-h AF model was established by rapid pacing the right atrium. The RAA and LAA were quickly removed and immersed into Tyrode’s solution in MEA chip dish. The morphology, duration, voltage, discharge frequency and conduction of field action potential were recorded. ResultsIn the AF group, the rhythms of LAA and RAA tissue field potential were irregular. The frequency of LAA (185.22±25.62 beats/min) was increased by 15.67% (156.44±8.88 beats/min)(P<0.01), and the frequency of RAA (102.39±16 beats/min) was decreased by 34.62% than that in the control group (156.44±8.88 beats/min) (P<0.01). The voltages of LAA (458.33±26.73)μV and RAA (504.83±39.93)μV were decreased than those of the control group (740.55±18.93)μV and (840.56±18.93)μV, respectively, (P<0.01). The durations of field potential of LAA (45.28±8.59)ms and RAA (61.78±7.1)ms were significantly shortened than those of the control group (70.77±6.98)ms (P<0.01). The electric impulse presented anisotropic alteration. ConclusionsMEA is a sensitive, long-lasting and stable technique, and local tissue action potential can be used to assess the electrophysiological characteristics of field potential in animal heart slices using multiple-channel recording mapping system. The discharge frequency of left atrial appendage is increased and frequency irregularity occurs after 48 h atrial fibrillation, the voltage of field potential is decreased in the atrial appendage, and the field potential duration is prolonged.

Abstract:Objective To study the characteristics of mice infected with egg drop syndrome virus (EDSV) strain NE4. Methods Kunming (KM) mice in age of 4[KG-*2]-[KG-*9]6 weeks were artificially infected with 10⁶ TCID₅₀ dose of EDSV strain NE₄, and the negative control mice were inoculated with normal allantoic liquid at the same time. FQ-PCR assay was used to detect the quantity of virus in different tissues. HE and immunohistochemical staining were used to observe the pathological changes and antigen localization. Results The virus inoculation affected somehow the ingestion of mice, but did not cause obvious clinical symptoms. The virus-infected mice produced specific antibody, reaching up to a titer of 2¹². The EDSV titer in the control mice was negative. EDSV was detected in the tissues of most organs such as liver, uterus, kidney, lungs of the mice by FQ-PCR assay, and at 7 days post infection, the mice began to release virus. Immunohistochemistry showed that EDSV was propagated in organs such as liver, uterus, kidney, and lungs of the mice, most notably, in the cytoplasm of uterine glandular epithelial cells and myocytes. No obvious pathological changes were found in tissues of infected mice. Conclusion EDSV strain NE₄ virus can infect KM mice.

Abstract:Objective To improve a model of hypoxic-ischmic brain injury (HIBI) in neonatal rats and observe the effects of hypoxic-ischemic brain injury on cerebral pathology and neurotrophic factor. Methods Thirty-two healthy 7-day-old neonatal Sprague-Dawley (SD) rats were used as research subjects and randomly divided into 3 groups:the control group (n=5), the sham operated group (n=8), and the HIBI group (n=19). The rats in the HIBI group received hypoxia immediately after operation without previous anaesthesia. The body weight, behavioral ability and neuropathological changes in each group were observed. β-NGF and human-NT3 of the brain tissue in the sham operated and HIBI groups were compared on d3 and d14. Results (1) The body weight gain of the HIBI group was significantly slower and lower than that in the control group and sham operated group (P<0.01). (2) Behavioral abnormalities were found in all rats of the HIBI group including unable to turnover (84%), muscle tremor and/or head tremble (63%) and mortality (21%). (3) HE staining showed neuronal damages and proliferation of neuroglial cells in the left hemisphere of the HIBI group. (4) The concentration of human-NT3 in the HIBI group was increased than that of sham operated group, but the concentration of β-NGF was not significantly different from that in the two groups on d3. ConclusionsA neonatal rat model of HIBI is successfully established and it is more closely resembling the course of human neonatal HIBI. The increase of neurotrophic factor plays an important role in the neuroprotection mechanism in the early phase of HIBI.

Abstract:ObjectiveTo investigate the changes of ocular biometric parameters during the recovery from form-deprivation myopia in guinea pigs. Methods Thirty guinea pigs, aged 2[KG-*2]-[KG-*9]3 weeks, were randomly divided into 2 groups:experimental group (n=20) and normal control group (n=10). The right eyes of guinea pigs in the experimental group were form-deprived with semi-transparent membranes for 4 weeks and subsequently allowed unrestricted vision for 3 weeks. The left eyes remained untreated as contralateral control eyes. The guinea pigs in the normal control group were raised with both eyes open for 7 weeks. Ocular biometric parameters of the guinea pigs were measured before and 4 weeks after form-deprivation, and 2,6, 0,4, and 21 days after re-exposure. Refraction was measured by retinoscopy after cycloplegia. Axial length, anterior chamber depth and lens thickness were measured by A-scan ultrasound, and the vitreous chamber depth was calculated. ResultsAfter 4 weeks of treatment, the right eyes of guinea pigs in the experimental group had myopic shift with refraction of (-2.88±2.30) D, and induced a relative myopia (-5.50±1.9) D. After re-exposure, the right eyes of guinea pigs in the experimental group experienced re-normalization, and the refraction was rapidly recovered in the initial 6 days. The refractive difference was (-0.18±0.26) D, vitreous chamber depth difference was (0.012 5±0.045 8) mm, and axial length difference was (0.018 3±0.035 9) mm between both eyes at 14 days after re-exposure, and were statistically non-significant (t=-2.049,P=0.080; t=0.610,P=0.575; t=1.443,P=0.192). Compared with the right eyes of guinea pigs in the normal control group, the refractive difference (-0.48±0.36) D was non-significant (t=-1.325,P=0.206) at 6 days after re-exposure, while the vitreous chamber depth difference was (0.0961±0.0630) mm and the axial length difference was (0.062 1±0.038 6) mm, statistically non-significant(t=1.652,P=0.14；t=1.607,P=0.125)at 2 days after re-exposure. ConclusionsGuinea pigs aged from 2 to 3 weeks with form-deprivation can experience re-normalization after re-exposure, along with a shortening of the vitreous chamber depth and axial length. The main recovery stage of ocular biometric parameters is in the initial 6 days after re-exposure.

Abstract:Objective To establish a subcutaneous tumor model in nude mice by injection of human gastric carcinoma cell line BGC-823 cells in various concentrations and observe its biological characteristics．MethodsBGC-823 cell line was cultured and the cells were divided into four cell suspension groups:group1:5×10⁷cells/mL, n=10; group 2:1×10⁷cells/mL, n=10; group 3:1×10⁶cells/mL, n=10; group 4:1×10⁵cells/mL, n=10．Each of 40 mice was subcutaneously injected 0.2 ml BGC-823 cell suspension into the armpit. Food intake and activity status of tumor-bearing mice,tumor formation time,mental conditions, survival rate,tumor formation rate, growth and size of the tumors were observed．Immunohistochemical SP staining was adopted for assessment of the microvessel density (MVD). ResultsThere was no abnormity and death in the mice after injection of tumor cell suspension. The overall tumor formation rate was 100% in all groups except the group 4,and the average tumor appearing time was 3[KG-*2]-[KG-*9]7 days after injection of tumor cell suspension. The average tumor MVD was (123.26±31.57)/mm². ConclusionsThe subcutaneous nude mouse models of human gastric carcinoma and the lowest concentration of tumor cells needed for the modeling are preliminarily established. It lays a foundation for experimental studies of gastric carcinoma.

Abstract:ObjectiveTo establish a stable and reliable mouse model of systemic infection induced by methicillin-resistant Staphylococcus aureus strain 252 (MRSA-252), and to provide reference and experimental foundation for the studies on pathogenesis of MRSA infection and development of MRSA vaccine. MethodsBased on the establishment of MRSA-252 OD₆₀₀-CFU standard curve, BALB/c mice were infected with bacterial suspension via tail vein injection. Then we systematically evaluated the mouse model at different levels, including the selection of infection dose, the survival rate and change of body weight, bacterial number assay of blood samples and major organs, and the histopathological changes among different groups. ResultsThe lethal dose was 5.0×10⁹ CFU/mouse and sublethal dose was 1.0×10⁹ CFU/mouse, with a survival rate of 60%-70%. It showed a typical manifestation of systemic infection in the mice, including low survival rate, high bacterial load in blood and major organs, with a peak on the 3rd day after infection, and extensive histopathlogical changes including cell death and infiltration of inflammatory cells. ConclusionsA mouse model of systemic MRSA infection is successfully established. It provides a reliable technical support for efficacy study and safety evaluation of MRSA vaccines.

Abstract:Objective To improve the preparation of trachea spiral strips of guinea pigs by spiral cutting using surgical scissors and to establish a new method of spiral cutting using surgical blade.MethodsForty guinea pigs were enrolled in this experiment. By method of spiral cutting with surgical scissor or surgical blade to prepare isolated guinea pig trachea spiral strips, and incubate them in Kreb''s solution for 2 hours. Contraction of the trachea spiral strips were induced by histamine in concentration of 2.0×10-3 g/L or acetylcholine in concentration of 2.0×10-4 g/L. The changes of tension values were measured using a BL420 biological signal acquisition system and tension sensors. SPSS 11.5 software was used to analyze the data (t test, α=0.05). ResultsUnder 2 g tension, the contraction caused by histamine of the surgical blade-prepared trachea strips was 1.31-fold higher than that of surgical scissors-prepared tracheal strips, and the contraction caused by acetylcholine of the surgical blade-prepared trachea strips was 1.208-fold higher than that of surgical scissors-prepared tracheal strips, both with a significant difference (t test, P<0.05). The contraction amplitude caused by histamine of surgical blade-prepared specimens under 2 g tension was 1.48-fold higher than that caused under 1 g tension, and the contraction amplitude of that caused by acetylcholine under 2 g tension was 1.38-fold higher than that under 1 g tension, both with a significant difference (t test, P<0.05). When histamine and acetylcholine were washed out from the tracheal strips, their tension returned to baseline. The RSD values of contraction amplitude of surgical blade-prepared strips were 19.8% (by histamine) and 19.1% (by acetylcholine), and the RSD values of contraction amplitude of surgical scissors-prepared strips were 35.3% (by histamine) and 33.7%. ConclusionsComparing the surgical blade-prepared tracheal spiral strips with those of surgical scissors-prepared ones, the former is more sensitive to histamine and acetylcholine treatment, and it is better to be treated under 2 g tension. The contraction variation caused by recycling of surgical blade-prepared specimens is smaller than that of surgical scissors-prepared specimens.

Abstract:ObjectiveTo establish mouse models of ulcerative colitis (UC) by acetic acid (AA), dextran sodium sulfate (DSS), and Helicobacter pylori (Hp), and to determine the best UC mouse model assessed by histopathological comparison.MethodsForty male BALB/C mice were randomly divided into four groups:the experimental group I was induced by acetic acid, the group II was fed with 3.5% DSS solution, and the group III was infected with Hp. The control group received distilled water only. In all animals, the body weight, stool consistency and occult blood were observed daily, and the gross and histopathological changes of the colon were examined. ResultsBoth acetic acid and DSS could result in ulcerative colitis in the mice. ConclusionsHelicobacter pyroli is not suitable to generate UC mouse models. Acetic acid is not suitable for establishing UC mouse models for longer period observation. The mouse UC models induced by DSS is an ideal model, suitable for studies of pathogenesis and drug therapy of ulcerative colitis.

Abstract:ObjectiveTo establish an animal model of rheumatoid arthritis (RA) and isolate fibroblast-like synoviocytes (FLS) from inflammatory synovium. MethodsModified adjuvant was prepared using heat-killed Mycobacterium tuberculosis H37Ra with sterile mineral oil,and arthritis was induced in Lewis rats by injection of the adjuvant at base of tail. Synovial tissue specimens were taken from arthritic ankle joints followed by mincing adequately. FLS were isolated by collagenase digestion. ResultsArthritis was successfully induced in the rats. The incidence reached 100% with regular time of arthritis development, and the histological changls were similar to those of rheumatoid arthritis. FLS were successfully obtained,identified and characterized in vitro. ConclusionsAn animal model of RA has been successfully established in rats and FLS have been successfully obtained, providing a desirable in vivo animal model and in vitro cell model for exploration of RA pathogenesis and drug evaluation. 

Abstract:ObjectiveTo establish a simple, rapid, sensitive and accurate method for detection of Staphylococcus aureus (SA).MethodsAccording to SA thermostable nuclease nuc gene, a pair of universal primers and two specific probes were designed. 5′ end of the universal primers were biotinylated. The probes were fixed on the nitrocellulose,then the probes were hybridized with PCR products. ResultsSA DNA concentration of 2 ng/μL could be detected by reverse linear hybrid method. The specificity and accuracy were 100%.ConclusionThe reverse linear hybrid method has a high sensitivity and specificity. It can be used for rapid detection of SA.

Abstract:ObjectiveTo study the effects of heroin on Bax expression in the midbrain ventral tegmental area (VTA) of rat. MethodsHeroin was intramuscularly injected into rats and a rat model of addiction was generated. Bax expression in the midbrain ventral tegmental area (VTA) was detected by immunohistochemistry. ResultsThe rats had significant withdrawal symptoms after continuous injection of heroin for 7 days. The expression of Bax-positive cells in cerebellar cortex of rat was significantly increased than that in the control group (P<0.01).ConclusionHeroin has effect of inducing Bax gene expression and damage to brain cells.

Abstract:Blast-induced injury animal models are the premise and guarantee of simulation researches of primary blast injury. At present, most experiments simulating blast are conducted by two kinds of methods:explosion-induced blast and blast generators. In the early stage, high explosives were used to produce blast in blast-induced animal modeling. With the development of technology, blast generators instead of high explosives were applied to experimental researches. There are two common types of blast generators:the bio-shock tubes and laser blast generator. All kinds of bio-shock tubes have already developed and widely used, and laser blast generators shows also unique advantages. The development of blast generators has given an enormous impetus to the experimental researches of primary blast injury in the laboratory.