Abstract:Objective To obtain zebrafish mutants with developmental defects in digestive organs from a forward genetic screening.Methods ENU mutagenesis and a classical F2 genetic screening were performed. RNA whole mount in-situ hybridization using lfabpas probe and BES-H₂O₂-Ac staining was applied to examine the liver, intestine and gall bladder phenotype in zebrafish embryos. Results We harvested 23 mutant lines with developmental defects in digestive organs (originated from 14 F2 families) after screening 128 mutagenized genomes. These mutants were classified into six groups according to their phenotypes. Conclusions The liver, intestine and gall bladder share and differ in their developmental molecular mechanisms in zebrafish.

Abstract:Objective To explore the role of ripply1 in zebrafish dorsal-ventral development. Methods Using zebrafish whole-mount in situ hybridization to examine the ripply1 expression pattern in early embryo development. To analyse the expression pattern changes of dorsal-ventral marker genes at shield stage and the morphological changes at 24 hpf (hours post-fertilization) after overexpression of ripply1 by injecting synthetic mRNA at 1-cell stage. Using Tol2 transposon technology to obtain a ripply1 promoter driven GFP transgenic fish and to identify promoter region that recapitulates endogenous ripply1 expression pattern. Results The in situ hybridization results revealed that ripply1 specifically expresses in the future dorsal region at shield stage. Overexpression of ripply1 caused an enhanced expression of dorsal marker genes and a reduction of ventral marker genes. Embryos overexpressing ripply1 also showed severely dorsalized phenotype, with enlarged head, reduced ventral yolk extension, and shortened posterior trunk and tail regions, and the formation of a secondary trunk axis. Transgenic fish revealed the maternal expression of ripply1 and suggested that a 1.2 kb promoter-driven GFP is able to recapitulate the endogenous gene expression pattern. Conclusions ripply1 may participate in the early development of dorsal-ventral axis in zebrafish embryo.

Abstract:Objective To better understand the mechanisms of cone opsin transport, we set to generate a transgenic zebrafish line with red fluorescence proteins expressing in the cone photoreceptors. Methods We used sws1 promoter to drive the expression of a chimerical protein, in which the last 44 amino acids of rhodopsin of Xenopus laevis were fused to the C-terminus of tdTomato to restrict its localization to the outer segment of photoreceptors. Results We successfully isolated such a transgenic zebrafish line and confirmed the localization of tdTomato by immunostaining analysis. Conclusions This transgenic zebrafish line will help us to better understand the transport mechanisms of cone opsins, especially the transport in live photoreceptors.

Abstract:Objective To investigate the functional role of a soluble secretion factor midkine-a in the process of zebrafish embryonic heart development. Methods Whole-mount in situ hybridization was performed to detect whether midkine-a is expressed in the embryonic heart. In the transgenic embryonic heart of Tg (pmidkine-a:EGFP), the expression of EGFP in the heart was monitored. Multiple heat shock treatments were applied to Tg (phsp:midkine-a:EGFP) embryos in order to overexpress midkine-a, and the phenotype of the heart was observed. The heterozygous Tg (phsp:midkine-a:EGFP) fish were crossed with homozygous Tg (pcmlc2:dsRed) fish, which specifically expresses RFP in the nucleoli of embryonic cardiomyocytes to facilitate myocyte number counting. To get Tg (phsp:midkine-a:EGFP/pcmlc2:dsRed) embryos, and the total number of cardiomyocytes in their heart was counted and compared with the controls when the heat shock induced overexpression of midkine-a. Morphonino knocked out of midkine-a was performed to observe the heart phenotype. Results Midkine-a was expressed in the zebrafish embryonic heart during development. Overexpression of midkine-a led to a smaller heart and reduced total number of cardiomyoctes in a single heart, which might be associated with the smaller heart phenotype. Morphonilo knocked out of midkine-a had no effect upon the heart development. Conclusions Midkine-a impedes zebrafish embryonic heart growth by limiting its cardiomyocyte pool.

Abstract:Objective To establish a cisplatin-resistant 4T1 mouse model of triple negative breast cancer. Methods A drug resistant mice model was established with cisplatin (DDP) induction and in-vivo/in-vitro tumorigenic approach. Its resistance characteristics were identified by MTT assay. Changes of drug resistance gene (MDR1, BCRP, MMP7, GST-π) and protein (P-gp, BCRP, MMP7) expression, and phosphorate-Akt and total-Akt protein expression were evaluated by real-time PCR, immunohistochemistry and western blot method, respectively. Small animal live imaging technology was applied to detect tumor growth. Results Resistance fold (RF) of cisplatin-resistant 4T1 mouse model was 12.84. The expression of MDR1, BCRP, MMP 7, GST-π mRNA and P-gp, BCRP, MMP 7 proteins in the resistant mice were higher than that in the non-resistant mice. The result of western blot showed that a statistically higher expression of p-Akt in resistant mice than that in non-resistant mice at protein levels (P<0.01). No significant difference of tumor growth rate was observed between non-resistant and resistant mice(P>0.05). Given same dose of DDP, resistant mice showed lower sensitivity than non-resistant mice significantly (P<0.01). Conclusions We have successfully established a cisplatin-resistant triple negative breast cancer model in mice, which provides a new platform for further study on chemoresistant reversal strategy and individualized clinical treatment of this disease.

Abstract:Objective This study aims to investigate the effects of high-fat diet rich in perilla oil on the expression of key genes that regulate hepatic VLDL synthesis in obese rats. Methods Sixty healthy male 5-week old SD rats were randomly divided into 2 groups. The rats in the normal control group (NC, n=12) were given normal diet, and the rats in the high fat group (HF, n=48) were given a pure high fat diet in order to induce rat models of obesity. In the intervention period, the obesity model rats were randomly divided into 4 subgroups including consistent high fat group (CHF) and three intervention groups depending on perilla oil substitution rate of lard in CHF: 20%PO, 50%PO and 100%PO. The serum triglyceride (TG) of the rats was measured after 4 weeks. Real-time PCR was applied to measure microsomal triglyceride transfer protein (Mtp) and apolipoprotein B (Apob) mRNA, and western blot assay was used for detecting the expression of MTP and APOB in the liver. Results Compared with the NC group, the CHF rats exhibited significantly high fat deposition. The serum TG was markedly higher and the MTP and APOB were decreased at gene and protein levels in the CHF group compared with the NC group. After the intervention, PO remarkably reduced the level of serum TG and decreased hepatic fat deposition as it showed by pathological examination. At the gene and protein levels, MTP and APOB were upregulated by PO to different degrees. Conclusions All the three PO intervention can promote VLDL synthesis and secretion, and decrease the hepatic fat deposition in the obese rats. Furthermore, PO upregulates the expression of MTP at gene and protein levels in a dose-dependent manner.

Abstract:Objective The aim of the study was to establish a mouse model of endometriosis, and to observe the effects of endometriosis on the reproductive ability in the mice. Methods The mouse models of endometriosis was established by subcutaneous and intraperitoneal injection of heterogenous endometrium. The pregnancy rate and live embryo number in the experimental, sham operation and blank groups were observed and compared, and the effects of endometriosis on reproductive ability of the mice were evaluated. Results Two weeks after the establishment of the mouse model of endometriosis, there were lesions in the peritoneal cavity and subcutaneous tissue of the mice. Compared the pregnancy rates among the model group, sham operation group and blank group, the differences were statistically significant (P<0.05). Compared the live embryo number in the three groups, there were also significant differences (P<0.05). Conclusions A mouse model of endometriosis is successfully established, and it demonstrates that endometriosis may affect the reproductive ability of mice.

Abstract:Objective To investigate the role of eNOS/NO signaling pathway in peritubular capillary lesions of mouse renal interstitial fibrosis with unilateral ureteral obstruction (UUC) and the potential mechanism. Methods Sixty-four healthy male KM mice were randomly divided into sham operated group (n=32) and unilateral ureteral obstruction group (n=32). At each week, serum BUN, Scr and NO were determined and the percentages of CD133⁺/VEGFR⁺ endothelial progenitor cells in peripheral blood mononuclear cells were detected by flow cytometry. Morphological changes of the renal tissue were observed using Masson staining. The expression of CD34⁺ cells in the renal interstitium was analyzed by immunohistochemistry to calculate the peritubular capillary density. The expressions of eNos and VEGF mRNA were determined by real-time PCR. Results The expression of blood NO, the percentages of endothelial progenitor cells, peritubular capillaries, eNOS mRNA, and VEGF mRNA in the UUO group were significantly decreased compared with those of the sham group at 2, 3, and 4 weeks (P<0.05). NO was positively correlated with peritubular capillaries (r=0.715, P<0.05), eNOS mRNA was positively correlated with peritubular capillaries (r=0.624, P<0.05), endothelial progenitor cells (r=0.375, P<0.05), and VEGF mRNA (r=0.351, P<0.05). Conclusions eNOS/NO signaling pathway participates in regulation of peritubular capillary lesions in renal interstitial fibrosis of UUO mice. The mechanism may be partly related to the regulation of vasomotor reflex, the expression of VEGF mRNA and mobilization of endothelial progenitor cells.

Abstract:Objective To establish an appropriate animal model of brain metastases from lung cancer in nude mice by thoracic orthotopic implantation in the chest or left ventricular injection, and to serve further studies on the mechanisms of lung cancer brain metastasis. Methods PC-9 cells (1×10⁶/0.1 mL) in logarithmic phase were respectively injected into 18 nude mice by orthotopic implantation in the chest or left ventricular injection (n=9 each group). The statuses of nude mice were observed after implantation. Animals showing clear signs of dyscrasia were killed. At autopsy, the lung, brain, liver and kidney were removed and histological sections were stained with H/E to detect the presence of tumor cells. Results In the thoracic orthotopic implantation group, three weeks after implantation, the number 4, 6, 9 mice showed tumor nodules in the chest wall, they began to lose weight in the fourth to sixth week differently, showing signs of dyscrasia gradually, and were sacrificed at the fifth to seventh week. The thoracotomy revealed that the whole thorax was occupied by many large lung cancer masses, spreading into bilateral ribs, pleura and spinal vertebra, with scarce eroded, compressed, pale and distorted lung tissues left. Histological examination with HE staining showed the presence of neoplasms in their lung tissues but only the number 6 mouse showed metastatic lesions in the brain tissue. In the left ventricular injection group, the mice almost began to lose weight in the third week simultaneously and became moribund slowly, which were all sacrificed at the fourth week. After thoracotomy, the thoraxes were clear except the number 11 and 18 mice which appeared 2-3 tiny tumor foci in the chest wall, with normal lung tissues. Histological examination with HE staining showed the presence of brain metastases in all the nine mice. The rate of brain metastases from lung cancer in the left ventricular injection group was 100%, compared with 11.1% in the thoracic orthotopic implantation group. Conclusions The establishment method of mouse model by left ventricular injection shows significantly higher rate of lung cancer brain metastases than that by thoracic orthotopic implantation.

Abstract:Objective To determine the genotypic and phenotypic characteristics of alkaline sphingomyelinase (alk-SMase) knockout (KO) mice and to provide the foundation data of this animal model for further study of the role of alk-SMase in colon cancer. Methods DNA was extracted from the tail tissue of wild type, heterozygous, homozygous KO mice, respectively. PCR analysis was used for genotyping. HE staining, confocal microscopy, and mass spectrometry were used for the detection of morphology, alk-SMase expression and sphingolipid metabolites in liver and intestine of the mice. Results Compared with the wild type and heterozygous mice, the homozygous KO mice showed that no changes occurred in the appearance and body weight, there was only one band (247bp) appeared on the genotyping, the thickness in small intestinal mucosa was significantly increased with a lower expression level of alk-SMase, and the amount of sphingolipid metabolites in the intestine and liver was changed, i.e. increase of SM and S1P, and reduction of ceramide. Conclusions Our findings demonstrate that the homozygous KO mice have specific genotype and phenotype that do not affect their growth. These mice will provide an ideal animal model for further study of alk-SMase functions.

Abstract:Objective To obtain imaging information of the cervical arteries and vertebral structure of rhesus macaques and provide useful reference data for medical research works using macaques as animal models. Methods Six adult macaques (3 males and 3 females) in age of 3-5 years, body weight from 3.5 to 5 kg, were used to examine the neck by 320 row spiral CT imaging under general anesthesia, and three-dimensional reconstruction was performed to observe the vertebral artery morphology, and to measure the diameter of vertebral artery and the size of transverse foramen of the macaques. Color Doppler ultrasound was combined with 320-CT imaging to assess the blood flow velocity in carotid arteries. After CT scan to make sure that the vertebral artery and cervical vertebra of macaques had no deformity, the size of transverse foramen and diameter of vertebral artery were measured in the cross-sectional images. Color Doppler ultrasound was used to analyze the peak systolic velocity (PSV), diastolic peak flow velocity (EDV) and the diameter (D) of common carotid artery (CCA), internal carotid artery (ICA), external carotid artery (ECA), and vertebral artery (VA) of the macaques. Results The 320-CT scan showed that in the monkeys, the vertebral artery enters the cervical spine through the intervertebral foramina at C6, and leaves the cervical spine at C2 level, and the cervical spine has Luschka joint, which is similar to the anatomic structure in humans. The diameter of left and right vertebral arteries was 1.89±0.44 mm and 1.72±0.39 mm, respectively, with no significant difference between them in the same segment (P>0.05), and the size of transverse foramen also had no significant difference in the same segment (P>0.05). Color Doppler ultrasound showed that the left and right side systolic peak velocity (PSV) and diastolic peak velocity (EDV) of CCA and ICA, the left and right side systolic peak velocity (PSV) of ECA and VA had statistically significant difference (P<0.05), and the left arteries were predominant in the macaques. Conclusions In this study we obtained some imaging information of vertebral artery morphology and artery blood flow of macaques and these information may provide some useful basic data for further studies using rhesus macaques as animal models.

Abstract:Objective To analyze the correlation between three evaluation methods of middle cerebral artery occlusion and focal cerebral ischemia, and to explore a new method and standard for the evaluation of the model. Methods Thirty healthy adult male SD rats were randomly divided into a sham-operated group (n=20) and model group. According to Zea-Longa procedure,we established the disease model to detect the changes in cerebral blood flow before and after surgery. The resulted cerebral infarction area was taken as gold standards. Then we analyzed the correlation between the standards and the changes in cerebral blood flow. Results The changes of infarction area ratio were positively correlated with the changes of cerebral blood flow in the model rats. Conclusions The changes of rat cerebral blood flow can be used to evaluate whether the cerebral ischemia model is successful or not.

Abstract:Objective To observe the influence of a Chinese traditional medicine, Shexiangbaoxinwan, on myocardial injury markers and C-reactive protein in exhaustively exercised rats. Methods A total of 52 male 11-12 week old Wistar rats were divided randomly into two groups: the experimental (n=26) and control groups (n=26).The two groups of rats took food and water freely. The experimental group of rats was given Shexiangbaoxinwan (2 capsules per day) for two weeks. Following a single bout of exhaustive swimming (≥2 hrs), the serum and myocardial CK, CK-MB, TnT, BNP and CRP in both groups were determined and compared. Results Compared with the control group, the exhaustive swimming time of the experimental group was significantly prolonged (P<0.01). The levels of serum and myocardial CK, CK-MB, TnT, BNP and CRP in the experimental group were significantly lower than those in the control group (P<0.05 for all). Conclusions The Chinese traditional medicine Shexiangbaoxinwan can significantly decrease the levels of rat serum and myocardial CK, CK-MB, TnT, BNP and CRP after a single bout of exhaustive swimming, and car alleviate myocardial injury.

Abstract:Objective The aim of this study was to establish a mouse model of chronic Parkinson's disease induced by systemic administration of lipopolysaccharide plus 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP), and to study the changes of behavioral manifestation, numbers of dopaminergic neurons in the substantia nigra pars compacta. Methods Twenty C57BL mice were randomly divided into 2 groups: the saline control group and model group. The mice in the model group received three intraperitoneal (i.p.) injections of LPS (0.25 mg/kg), once daily for three consecutive days. Four hours following the final LPS injection, the mice received one subcutaneous injection of low-dose MPTP (25 mg/kg). The mice of control group were injected with the same volume of saline. Eight weeks later, the motor ability of the mice was evaluated by footprint test and rotarod test. The tyrosine hydroxylase (TH)-positive cells were observed by immunohistochemical analysis. Results Compared with the control group, the scores of behavioral test were significantly lower, numbers of TH immunoreactive cells were significantly less in the Parkinson's model group (P<0.05). Conclusions Behavioral manifestation,number of dopaminergic neurons in the substantia nigra are significantly changed in the mouse models of Parkinson's disease produced by repeated injection of LPS plus MPTP, suggesting that this chronic animal model can be used in the experimental study for pathogenesis and therapy of Parkinson's disease.

Abstract:Kinesin-2 family proteins, including KIF3A, KIF3B, KIF3C and KIF17, are members of the kinesin superfamily motor proteins, which transport various proteins and vesicles in the cell and play diverse biological functions. Recently, studies on members of kinesin-2 family proteins suggest that they play fundamental roles during ciliary transport, whose defects can lead to abnormal cilia development, the major cause of human ciliopathies. In this review, we will summarize the functions of this motor protein family during ciliogenesis and focus mainly on their roles in the development of model organisms.

Abstract:Zebrafish has been widely used as an important model system in research fields of genetic and developmental biology over the past 20 years. Similar to the mammalians and other vertebrate animals, zebrafish also has various tissue barriers. In recent years, more and more important progress of tissue barrier studies have been achieved using zebrafish as in vivo model, such as blood-brain barrier. These findings contribute to the understanding of the mechanisms of diseases caused by the disorders of physiological barriers. It also helps with the modulation of the permeability of tissue barriers for drug delivery. This review summaries recent progress of zebrafish applications in the study of tissue barriers, such as blood-brain barrier, blood-retina barrier, epidermal barrier, etc.

Abstract:Zebrafish, a new type of model animal, has been widely used in many fields of biological research because of its low cost, ability of external fertilization, high fecundity, allowance of embryo transplant, and ectogenesis. Recently, zebrafish and its embryos have been widely used in ecotoxicological studies and environmental monitoring. Furthermore, with the maturation of zebrafish transgenic techniques, a new era has come for environmental pollution monitoring.

Abstract:Experimental autoimmune encephalomyelitis (EAE) is a classic animal model for multiple sclerosis. After more than 2 centuries of development, EAE can be established in mice, monkeys and rats,etc. Some animals can be immunized by different immune antigens which have different features in sensibility and clinical characteristics. In this paper, we will review the development of EAE, discuss the characteristics of EAE models produced by immunization of different animals with different antigens. In summary, we explore the development of animal models of EAE.

Abstract:Laboratory animal is an important support condition in life science and biomedical research. With the rapid development of life science and biomedical industry, laboratory animals, and quantity and variety of animal model resource are rapidly increasing. According to the statistics, there are more than 200 kinds of a total of more than 26000 strains, including 2607 strains of conventional laboratory animals in the world. USA is a big country of laboratory animal resource in the world, with most of the laboratory animal breed resources and preservation organization. In this paper, we analyzed American laboratory animal resources, and provided references for the development of laboratory animal resource in China.

Abstract:Although the publication of peer-reviewed scientific papers is the major way in which scientists communicate with one another, these papers often lack essential information about the conditions under which the animals lived and how they were treated during the experiments. Without this information it may be impossible to evaluate the studies, replicate them in another laboratory, or use them to advance the 3Rs in other animal experiments. This paper gives some advice on how the reporting of animal research can be improved.