Abstract:Objective To investigate the pathological characteristics of early lung adenocarcinoma in a mouse model with emphasis of the tumor growth patterns and tumor-bronch's relationship. Materials and Methods Early lung adenocarcinoma was induced in 10 mice by administering 0.2 mL N-methyl-N-nitro-N-nitrosoguanidine (MNNG, 2.0 mg/mL) once weekly for 4 consecutive weeks. The mice were sacrificed at the 100th day. The lung specimens were fixed in 10% formalin solution. One hundred tumors were randomly sampled, embedded in paraffin, cut into 3-μm sections, stained with hematoxylin and eosin (HE) and evaluated by histopathology. Pathological type, location, size, shape, margin, growth pattern and tumor-bronchus relationship of the tumors were assessed. Results One hundred and eighty-seven tumors were found macroscopically in the 10 mice. All of the randomly selected 100 tumors were adenocarcinomas of a size of 0.19 to 1.33 mm (mean size of 0.48 mm). The tumors demonstrated three types of growth pattern: lepidic (n=6), expansile (n=26), and mixed (n=68). Their mean size was 0.34 mm, 0.54 mm and 0.47 mm, respectively. Ninety-six out of the 100 tumors had direct relation with the bronchioles, of which 13% with the conductive bronchioles and 96% with the ventilating bronchioles. There were bronchioles at the center of tumor in 19% of the tumors, at the outer area of tumor in 49% of the tumors, and at the periphery of tumor in 96% of the tumors. Lobulation (33%) and spiculation (35%) of the tumors were formed by the block of bronchioles or by the invasive growth between or along bronchioles. Conclusions Analyzing the pathological growth patterns and tumor-bronchus relationships in early mouse lung adenocarcinomas will help us to better understand the corresponding CT manifestations of human early lung adenocarcinoma.

Abstract:Objective To establish a loop-mediated isothermal amplification (LAMP) method for detecting diarrhea pathogens (Shigella and Salmonella) in rhesus monkeys and evaluate the application of the LAMP method for detecting bacterial diseases in non-human primate laboratory animals. Materials and Methods A total of 205 fecal samples of rhesus monkeys were detected in this LAMP assay. The specificity and sensitivity of LAMP for Shigella and Salmonella were analyzed, and real-time polymerase chain reaction (REAL-TIME PCR) assay was employed as control. Results The LAMP method established here needed only 45 min to complete the reaction at 63℃. Its detection limit was 10 pg/μL and with a high specificity. The positive rate of Shigella and Salmonella was 1.5% and 6.3%, respectively. Conclusions Here we have established a fast and simple Shigella and Salmonella LAMP detection method that has strong specificity and high sensitivity and is suitable for rapid detection of bacterial disease in macaques. The development of this rapid detection kit is underway, and it will be helpful to the diarrhea detection.

Abstract:Objective To study the influence of androgen deprivation on prostate cancer bone metastasis in nude mouse models and to explore the biological characteristics of circulating tumor cells during tumor metastasis.Methods Forty male 6-7-week old nude mice were randomly divided into two groups:the castration group and untreated normal control group.All the nude mice were injected intracardially human prostate cancer PC3-Luc-GFPcells (labeled with GFP and Luc) in a dose of 1×10⁶/50 μL/mouse to establish the bone metastasis models. The resulted cancer bone metastasis was confirmed by detection of Luc signal using a small animal optical imaging system.Bone tissue samples were fixed with 4% paraformaldehyde and pathological examination using HE staining was performed. Circulating tumor cells (CTCs) were isolated from the periphery blood collected by heart puncture.The expression of metastasis-associated molecules TOPK and RANKL of CTCs was detected by Western blot assay. Results The nude mouse model of human prostate cancer bone metastasis was successfully established. The incidence rate of bone metastasis in the castration group was 2/13(15.38%), while that of the normal control group was 5/14=35.71%. The xenograft models were prepared twice and the results of the two experiments were pooled together, showing a bone metastasis incidence rate of 11.54%(3/26) in the castration group and 37.04%(10/27) in the normal control group (P<0.05). The expressions of TOPK and RANKL of CTCs were significantly higher than that in the original prostate cancer cells.Conclusions Castration can significantly reduce the incidence of prostate cancer bone metastases in nude mouse models. Metastatic potential of CTCs is significantly higher than that of the original prostate cancer cells.

Abstract:Objective To develop an efficient, fast and stable establishment method of mouse model of hepatocellular carcinoma. Methods 128 healthy Kunming male mice were equally divided into eight groups: normal group supplied with routine drinking water and food; diethylnitrosamine (DEN)-water group received 16.5 mg/kg DEN in drinking water every day; high-dose DEN-oil groups received orally 16.5 mg/kg of DEN oil solution once, 3 times and 7 times per week, respectively; low-dose DEN-oil group and nano-DEN group received orally 8.25 mg/kg DEN in oil or in nano-vehicle every week, while nano-vehicle group received only an equal amount of nano-vehicle. After twenty weeks, all mice were sacrificed. The paraffin-embedded mouse liver specimens were examined by pathology using HE staining. Results (1) The mice treated with DEN solution showed dry and dull hair, slow movement and slow weight-gain to a varying degree. (2) At the twentieth week, both the DEN-water group and high-dose DEN-oil group showed tumors, and the mortality of high-dose DEN-oil group (once per week) was low. (3) There was no death of mice in the nano-DEN group, and the pathological examination revealed more serious liver injury in the nano-DEN group than in the low-dose DEN-oil group (once per week). Conclusions Oral administration of DEN-oil improved the dietary compliance of mice, and the mortality can be reduced significantly by long-term low frequency DEN administration. Microemulsion preparation of DEN dissolved in oil may enhance the DEN-induced liver damage.

Abstract:Objective To establish a method to evaluate the effect of paeoniflorin (PF)on free calcium (Ca²⁺)concentration in dorsal root ganglion (DRG)neurons in rats. Methods DRGs were obtained by microdissection, then were digested by trypsin and sieved.The dissociated neurons were alternately cultured and purified with DF-12 and anti-mitotic culture medium to obtain primary rat DRG neurons, and the purity of DRG neurons was measured by immunofluorescence technique. The fluorescence intensity of Ca²⁺ in DRGs was observed by laser confocal microscopy (LSCM)imaging, and the changes of fluorescence intensity were analyzed to explore the effects of PF on DRG intracellular free calcium concentration and capsaicin receptor. Results The purity of DRG cells isolated by the above mentioned method was up to 95%.Capsazepine(CAP)inhibited the increase of intracellular Ca²⁺ by blocking the capsaicin-activated transient receptor potential vanilloid 1 (TRPV1). PF exhibited the influx of extracellular Ca²⁺. Conclusions We would hypothesize that PF probably inhibits the significant increase of Ca²⁺ in DRG cells by acting on TRPV1 channel, and the method established in this study can be used to evaluate the effects of drugs on Ca²⁺ concentration in rat dorsal root ganflion neurons.

Abstract:Objective To explore the effect of compound rehmannia prescription on cannabinoid receptor 1 in the rats with wind syndrome due to deficiency of Yin in levodopa-induced dyskinesia (LID) in Parkinson's disease. Method A unilateral injection of 6-hydroxydopamine into the substantia nigra was performed to establish a Sprague-Dawley rat model of Parkinson's disease. The models were intraperitoneally injected with 50 mg/kg levodopa and 12.5 mg/kg benserazide for two weeks to prepare LID models with Wind syndrome due to deficiency of Yin. The models were randomly divided into LID group and compound rehmannia prescription groups. Moreover, the normal group and sham-operated group were set up, 6 rats in each group. Neurological behavior was tested after treatment for four weeks and six weeks, respectively. Then the rats were sacrificed, the brain was removed immediately and the corpus striatum was dissected out. The expression of cannabinoid receptor 1 was examined by Western blotting. Result With the prolonged time of levodopa treatment, the LID rats with wind syndrome due to deficiency of yin showed an increasing tendency of AIM scores (P<0.05), shortening of the rotation start time (P>0.05), a tendency of increased duration of continuous rotation time (P<0.01) and a reducing tendency of peak rotation rate (P>0.05). Compound rehmannia prescription improved the above mentioned neurological behavior. The expression of cannabinoid receptor 1 showed an increasing tendency in the LID groups compared with that in the normal groups. But with the prolonging time of levodopa treatment, the expression of cannabinoid receptor 1 in the LID groups showed a decreasing tendency (P<0.01). The expression of cannabinoid receptor 1 had an increasing tendency after the treatment with compound rehmannia prescription (P<0.01). Conclusions The expression of cannabinoid receptor 1 has an increasing tendency in LID rats with wind syndrome due to deficiency of yin. The changes can well reflect the severity of wind syndrome due to deficiency of yin. We would hypothesize that the treatment with compound rehmannia prescription may activate the cannabinoid receptor 1 in LID rats, inhibiting the release of excitatory amino acids, decreasing the excitability of neurons and inducing cell cascade, and finally reduce the excitatory toxicity of levodopa.

Abstract:Objective To construct the retroviral-mediated short hairpin RNA(shRNA) expression vectors of Noggin, and to analyze the silencing effect of the shRNA. Methods Four interference sequences were designed based onNoggin mRNA, and connected to Lenti-KD vectors. The recombinant plasmids were transiently transfected into HEK-293T cells, and finally obtained the recombinant virus.Then MC3T3-E1 cells were infected with the recombinant virus and screened by puromycin.The gene silencing effect of shRNA was evaluated using RT-PCR and Western blot. Results RT-PCR results showed that all the four interference sequences had silencing effect on the expression of Noggin, but only shNoggin-1(P<0.01) was different significantly. Western blot results showed that among the four interference sequences, only shNoggin-1(P<0.01) showed a significantly lowering effect on the expression of Noggin. Conclusions One interference sequence of Noggin gene is obtained, which can interfere the stability of mRNA of the Noggin gene, by affecting the protein expression. It is useful for the further study of unknown biological function of Noggin.

Abstract:Objective To establish a new model of distraction osteogenesis in rat tibia. Methods A customized external fixation device was attached to the right tibia of 40 male Sprague-Dawley rats. A 4-mm bone block was resected and the tibia was shortened. To investigate the bone healing during the distraction osteogenesis period and reconstruction period, and general observation, x-ray analysis, and histological examination were performed. Results The device was stable and effective without hindrance. The tibia length was recovered and bone union was achieved. Conclusions A novel Sprague-Dawley rat model of distraction osteogenesis in tibia is successfully established, and this is a reliable, practical and reproducible model.

Abstract:Objective To explore the effects of a Chinese medicine, Jiuxieling granules, on the expression of TLR4 and NF-κB p65 mRNA and proteins in ulcerative colitis model rats with spleen and kidney yang deficiency. Methods The rat model of ulcerative colitis with spleen and kidney yang deficiency was established by oral administration of rhubarb decoction, intramuscular injection of hydrocortisone and TNBS (2,4,6-trinitrobenzene sulfonic acid) combined with ethanol enema. All the 90 rats were randomly divided into blank control group, UC model of spleen kidney yang deficiency group, and Jiuxieling granules treatment groups for 7 days, 14 days and 21 days, respectively, and positive control group (SASP). After all the rats were killed and colon samples were collected, the expression of TLR4 and NF-κB p65 mRNA and proteins of colon tissues were detected by RT-qPCR and SP immunohistochemical. Results The results of immunohistochemiscal assay indicated that expressions of TLR4 and NF-κB p65 in the UC model group were significantly enhanced, when compared with that of the blank group (P<0.01). After treatment, the expressions of TLR4 and NF-κB p65 in all treatment groups were significantly decreased (P<0.01 or P<0.05). The RT-qPCR assay results indicated that the expressions of TLR4 and NF-κB p65 in the UC model group were significantly enhanced when compared with that of the blank group (P<0.01). After treatment, the expressions of TLR4 and NF-κB p65 in all treatment groups were reduced (P<0.01 or P<0.05). Conclusions Jiuxieling granules can alleviate the mucosal inflammation and facilitate the mucosal repair. The main reasons of which may be associated with reduction of TLR4 and NF-κB p65 gene transcription and protein expression in the ulcerative colitis model rats with spleen and kidney yang deficiency.

Abstract:Objective To select more sensitive animal model to evaluate the cardiotoxicity of drugs, we used the heart-specific CYP2E1 genetically modified mice (α-MHC CYP2E1 transgenic mice [Tg(+) mice] and α-MHC CYP2E1 silencing transgenic mice [sTg(+) mice] ) to evaluate the drug sibutramine-induced cardiotoxicity. Methods The 8-week old male Tg(+), sTg(+), and C57BL/6 mice (wild type, WT), 50 mice in each group, were randomly divided into 5 groups: the solvent control group (intragastric gavage of pure drinking water), and the four sibutramine (50 mg/kg, 100 mg/kg, 150 mg/kg and 300 mg/kg) treatment groups, respectively. The general condition of the mice was observed and the survival rate was determined during the drug treatment period. At 15 days after the sibutramine administration, the blood biochemical indicators of cardiotoxicity lactate dehydrogenase (LDH), creatine kinase (CK), and creatine kinase isoenzyme (CK-MB) were assessed, and then the mice were sacrificed and heart tissue samples were taken for pathological examination and immunohistochemical observation of the expression of connexin 43 (CX43). Results (1) The blood biochemical indicators in the sibutramine 50 mg/kg and 100 mg/kg Tg(+) treated mice were significantly higher than those in the WT mice (P<0.05 or P<0.01), and significantly higher in the 50 mg/kg, 100 mg/kg and 150 mg/kg sibutramine treated Tg(+) mice than in the sTg(+) mice (P<0.05 or P<0.01). However, when the sibutramine was given at a dose of 300 mg/kg, the values of those indicators in the Tg(+) mice were significantly lower than that in the WT and sTg(+) mice (P<0.05 or P<0.01), with the lowest level in the sTg(+) mice. (2) The pathological examination revealed cardiotoxic changes in the Tg(+) and WT mice. (3) The immunohistochemical analysis showed that alongside with the increasing drug dose, the expression of CX43 was decreased in the intercalated disks of cardiomyocytes in the Tg(+), sTg(+) and WT mice, and the color staining intensity was mostly decreased in an order of Tg(+)>WT>sTg(+) mice. Conclusions Tg(+) mice may have a higher sensitivity in the evaluation of potential cardiotoxicity than WT mice, and sTg(+) mouse is a good model of protection against cardiotoxicity.

Abstract:Objective To explore the mechanism of bone remodeling during the process of bone defect healing via topical application of exogenous β-NGF. Methods Thirty healthy male Sprague-Dawley rats were used in this study. Two 5-mm diameter bilateral bone defects were created on the parietal bone. The right side was given 10 μg β-NGF/PBS solution (experimental group) and the left side was given only PBS solution (control group) via an osmotic pump for 7 days. Expression levels of BMP-2 and tartrate-resistant alkalin phosphatase (TRAP) at postoperative 3, 7, 14, 21 and 28 days were detected using immunohistochemical staining and semi-quantitatively analyzed with integral optical density (IOD). Results Immunohistochemistry showed that the expression levels of BMP-2 (IOD values) in the experimental group were significantly higher (P<0.05) than that of the control group at postoperative day 14. Special TRAP staining results showed that the activities of osteoclasts in the experimental group was significantly decreased compared with that of the control group (P<0.05) at postoperative day 7, 21 and 28. Conclusions The results of this study indicate that topical application of exogenous β-NGF plays an important role in bone remodeling during the process of bone defect healing, which may be through increased production of BMP-2 and inhibition of osteoclast activity.

Abstract:Objective To explore the behavioral differences of Sprague-Dawley (SD) and Wistar rats in Pavlovian conditioning and reward operant conditioning task, and provide a good reference for selection of experimental animals in establishing the animal models of conditional reflex for studying higher brain functions. Methods Healthy 8-week-old SD and Wistar rats were used in this study. Single operant task and continuous operation training task, and multiple operation training task were used to assess the learning and memory ability of the SD and Wistar rats. Results Significant difference was found in the Pavlovian conditioning. SD rats performed more nose pokes (NPs) (P<0.05) vs. Wistar rats. There were significant differences during a single operation task.Wistar rats preformed significantly more total lever presses (LPs) (P<0.05) and cLP/LP (P<0.05) vs. SD rats, and there was obvious increase in total nose pokes (NPs) (P<0.05) on the fifth day. The two indicators,the lever pressing rate and the latency of the first lever pressing could explain objectively the rat learning and memory abilities. The latency of the first lever pressing in the Wistar rats was significantly shorter than that of SD rats (P<0.05), while the lever pressing rate obviously higher (P<0.05). In the continuous operation training task, the Wistar rats showed significantly higher nose pokes (vs. SD rats, P<0.05); and also had higher lever presses, reward earned, and correct accuracy rate, but with no significant difference between them.Conclusions Wistar rats exhibit excellently in a single operation task and continuous operation training task, while SD rats show better performance in Pavlovian conditioning.

Abstract:Objective To analyze the genetic characteristics of seven frequently-used inbred mouse strains in core colonies from Shanghai suppliers. Methods 48 pairs of microsatellite primers with high polymorphism were screened out and combined into 11 sets of multiplex PCR primer mix labelled with two dyes (FAM and HEX). By these primer mix, the DNA samples of the seven strains were genotyped. The genotyping data were analyzed using statistical software. Results All the 48 microsatellite loci were homozygous for all inbred mice of the seven strains. On each microsatellite locus, the PCR results of the mice in the same population showed monomorphism, and that of the mice in different populations showed polymorphism, whether the populations were of the same strain or not. But the genetic distances between the populations of the same strain were all closer than that of different strains. The phylogenic tree showed that the populations of the same strain were first get into one cluster. The genetic relationship between the strains of C57BL/6 and other six strains were all far. Conclusions Genetic differences exist on 48 microsatellite loci between the core colonies of seven inbred mouse strains from two suppliers in Shanghai.

Abstract:Objective To explore the mechanism of ovariectomy on signal transduction of neuroendocrine signals in the rat brain. Methods Twenty-four healthy SPF 2-month-old female Sprague-Dawley rats were randomly divided into ovariectomized group and sham-operated group (n=12). Morphological changes of the brain neurons were examined by pathology using HE staining. The content of epinephrine (E), norepinephrine (NE), dopamine (DA), 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA) and homovanillic acid (HVA) in plasma and different brain regions were detected by high performance liquid chromatography (HPLC). Results Pathological examination showed obvious neuron degeneration in the brain of OVX rats. Compared with the sham-operated group, contents of NE and E in the OVX group were lower, especially in the hippocampus and serum. In the OVX rats, HVA level in the thalamus was higher than that in the sham-operated group, while DA content in the cortex was lower. 5-HT was higher in the hippocampus and cerebellum of OVX group when compared with the sham-operated group, and a lower level was observed in the cortex, thalamus and serum. Moreover, the changes of 5-HIAA was in contrary to that of 5-HT. Conclusions Declined secretion of estrogen after ovariectomy induces abnormal synthesis and secretion of main signal molecules (classic neurotransmitters and amino acids) in the brain tissue. Thus, being a model with abnormal neuroendocrine signaling, the OVX rat provides new ideas and methods for research of signal transduction in peri-menopausal syndrome.

Abstract:Objective To study the pathological changes in some important brain areas of rats with post-traumatic stress disorder (PTSD) induced by combined stress.Methods Twenty healthy adult female SD rats were randomly divided into normal group and model group, with 10 rats in each group. The PTSD model was established by combined stress. After 4 weeks, the rat behaviors of the two groups were tested by elevated plus maze and Morris water maze. After the test, samples of the cerebral cortex, areas of CA1, CA2, CA3 and hippocampal dentate gyrus were taken for pathological examination using HE and Nissl staining. Results In the normal group, the cell morphology and distribution were regular and the cell nucleoli were clear in the cerebral cortex and hippocampus, cytoplasmic Nissl bodies were abundant, and there were no obvious neuronal degeneration and necrosis. In the model group, the cell morphology and distribution in the CA1 and CA3 areas were irregular, with increased cell gaps, and in addition, there were some pathological changes of physaliferous cells, especially in the CA3 area. The cell morphology and distribution in the CA2 area were regular. In the dentate gyrus, some cells arranged loosely, the cell gap was widened, and the amount of Nissl bodies was decreased. Conclusions There are some pathological changes in the hypocampal CA1, CA3 and dentate gyrus in rat models of post-traumatic stress disorder, and they will provide an experimental basis for studying the pathological mechanism of PTSD patients.

Abstract:Objective To compare the refractive development and axial eye growth between Zmu-1:DHP and DHP guinea pig strains after weaning. Methods After screening of the refractive status of two strains of guinea pigs at 3 weeks of age, twelve Zmu-1:DHP guinea pigs and eight DHP guinea pigs were chosen. Each group underwent a series of ocular measurements at five time-points (4, 6, 8, 10 and 12 weeks), including refraction, axial length, corneal curvature and other ocular parameters. Results The refraction of 3-week old Zmu-1:DHP strain(n=327)was-5.19±3.92 D with a myopic rate of 85.1%. While the refraction of DHP strain guinea pigs (n=117) was 2.13±4.40 D with a myopic rate of 22.6%. From 4 to 12 weeks of age, the guinea pigs of Zmu-1:DHP strain developed toward more myopic, their anisometropia was larger than 1.5 D. The DHP strain had a stable refractive state. The Zmu-1:DHP guinea pig strain had larger axial length and vitreous thickness than strain DHP (t-test: P<0.01), and the DHP guinea pig strain had better vision (P<0.01). Astigmatism of Zmu-1:DHP strain guinea pigs changed significantly from 4 to 12 weeks (ANOVA: P<0.01), while DHP strain did not (ANOVA: P =0.07). In the Zmu-1:DHP strain, the refractive status had significant correlation with axial length (r=-0.785, P<0.01) and vitreous chamber depth (r=-0.754, P<0.01). In the DHP strain, the correlation coefficients were-0.19(P =0.089)and-0.34 (P<0.01). Conclusions Zmu-1:DHP strain guinea pigs have a higher rate of spontaneous myopia than DHP strain. The emmetropization is disrupted. This myopia is highly correlated with axial length and vitreous chamber depth.

Abstract:A class of near infrared fluorescence (NIRF) heptamethine cyanine dyes, in their native form without chemical conjugation, can be taken up and accumulated specifically in cancer cells and used for tumors imaging. The different uptake of the dyes into cancer but not normal cells is possible due to the presence of specific isoforms of organic anion-transporting peptides (OATPs), and regulated by hypoxia. These advances have widely extended current application of NIRF imaging in cancer diagnosis.

Abstract:Heart failure (HF) is the final stage of various cardiac diseases. It's important to use appropriate biomarkers for identifying heart failure in the early stage, managing patients, and optimizing risk stratification in clinical setting. This review summarizes the important roles of markers related to cardiac stress, myocardial injury, extracellular matrix, micro RNA and inflammatory factors which were reported in literatures in recent years, and assessed their clinical value in the early diagnosis and prevention of heart failure.

Abstract:Purine receptors are divided into P1 and P2 receptors. P2 receptors are divided into the ligand gated ion channel receptor (P2X receptor) and G protein coupled receptor (P2Y receptor). The P2Y₆ receptor is a member of the P2Y family, P2Y₆ receptors are involved in cardiovascular diseases, endocrine diseases and neuropathy diseases, etc. With clopidogrel (P2Y₁₂ receptor antagonist) and other purine receptor blocker approved by the FDA for clinical application, and showed good efficacy,study on biological effect of P2Y₆ receptor also becomes one of the hot researches to new drug target for the P2Y receptor. Based on the development of molecular biological techniques, the research on biological effect of P2Y6 receptor achieved significant advances.