Mouse knockout of guanylyl cyclase C: Recognition memory deficits in the absence of activity changes

Guanylyl cyclase C (GC‐C) is found in brain regions where dopamine is expressed. We characterized a mouse in which GC‐C was knocked out (KO) that was reported to be a model of attention deficit hyperactivity disorder (ADHD). We re‐examined this model and controlled for litter effects, used 16 to 23 mice per genotype per sex and assessed an array of behavioral and neurochemical outcomes. GC‐C KO mice showed no phenotypic differences from wild‐type mice on most behavioral tests, or on striatal or hippocampal monoamines, and notably no evidence of an ADHD‐like phenotype. KO mice were impaired on novel object recognition, had decreased tactile startle but not acoustic startle, and females had increased latency on cued training trials in the Morris water maze, but not hidden platform spatial learning trials. Open‐field activity showed small differences in females but not males. The data indicate that the GC‐C KO mouse with proper controls and sample sizes has a moderate cognitive and startle phenotype but has no ADHD‐like phenotype.     

3,3′,4,4′,5-Pentachlorobiphenyl influences mitochondrial apoptosis pathway in granulosa cells

3,3′,4,4′,5-Polychlorinated biphenyl (PCB126) is a persistent organic environmental pollutant which can affect various biological activities of organisms, such as immunity, neurological function, and reproduction. In our study, we aimed to investigate the effects of PCB126 on granulosa cells (GCs). GCs were collected from ovaries in PMSG-treated mice, after 24 hours culture. GCs were then incubated with 10 pg/mL, 100 pg/mL, and 10 ng/mL of PCB126 for another 24 hours. Following these steps, exposed GCs were collected for further experimentation. Our data showed that the number of GCs in the 10 ng/mL PCB126 decreased. Meanwhile, pyknotic nuclei and condensed chromatin increased, while the apoptotic cells in the 10 ng/mL PCB126 group were significantly increased. Furthermore, the expression of the apoptotic executive protein caspase-3 increased after PCB126 treatment. The expression of Bax, Bcl-2, and Bim related to the mitochondrial apoptosis pathway were also influenced to different degrees. Thus, our data suggested that PCB126 affect the GCs apoptosis, and mitochondrial apoptosis pathway was involved in this process.     

Imaging the interaction of αv integrin-GFP in osteosarcoma cells with RFP-expressing host stromal cells and tumor-scaffold collagen in the primary and metastatic tumor microenvironment

Human osteosarcoma 143B cells were previously stably transfected with an α_v integrin green flourescent protein (GFP) vector. 143B cells expressing α_v integrin-GFP were transplanted orthotopically in the tibia of transgenic nude mice ubiquitously expressing red fluorescent protein (RFP). The primary tumors acquired RFP-expressing stroma and were passaged orthotopically in the tibia in noncolored nude mice, which maintained the RFP stroma. The interaction of α_v integrin-GFP expression in 143B cells with RFP-expressing host stromal cells was observed by confocal microscopy using the Olympus FV1000. Collagen fibers were imaged simultaneously in reflectance mode. The RFP-expressing stroma included cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs) which persisted even 3 weeks after passage to nontransgenic nude mice. CAFs expressing RFP were aligned between collagen fibers and cancer cells expressing α_v integrin-GFP. Six weeks after transplantation, pulmonary metastases expressing α_v integrin-GFP could be identified. TAMs expressing RFP accompanied metastasized osteosarcoma cells expressing α_v integrin-GFP in the lung. The current study demonstrates the importance of α_v integrin interaction with stromal elements in osteosarcoma.     

Quantitative assay of 5-HT1A receptor gene expression in the brain

5-HT_1A receptors are involved in the regulation of various behaviors and the mechanism of action of anxiolytics and antidepressants. It is rather difficult to study the expression of the 5-HT_1A receptor gene in the brain because of the low concentration of its mRNA. A method developed for quantitating the level of 5-HT_1A receptor gene expression in brain structures involves estimation of the copy number for contaminant genomic DNA, the cDNA of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene (a housekeeping gene), and the 5-HT_1A receptor gene cDNA in a cDNA preparation. To estimate the GAPDH and 5-HT_1A receptor cDNA copy numbers, the fluorescent intensity of the corresponding PCR products is calibrated using genomic DNA standards of known concentrations. The expression of the 5-HT_1A receptor gene is corrected for the content of contaminant genomic DNA and presented as a 5-HT_1A receptor cDNA copy number per 100 copies of the GAPDH cDNA. The method was used to demonstrate for the first time that expression of the 5-HT_1A receptor gene is increased in the frontal cortex and the amygdala of mice knocked-out in the monoamine oxidase A gene.     

腺病毒介导hIL-24抑制裸鼠肝癌荷瘤生长及其机制研究

为了研究腺病毒hIL-24抑制SMMC-7721肝癌细胞裸鼠荷瘤的生长抑制及其作用机制,构建了重组腺病毒载体pAdeasy-1-pTrack-CMV-hIL-24(Ad-hIL-24),经PacI线性化后转染QBI-293A细胞,多轮感染后收获高效价腺病毒重组病毒子。用SMMC-7721肝癌细胞使16只裸鼠致瘤,然后随机NS分成干预组、5-Fu组、Ad组和Ad-hIL-24组,进行抗肿瘤试验。四组均使用瘤体内注射干预用药,100μL/只NS组、Ad组(107pfu)和Ad-hIL-24组(107pfu)隔日一次,共注射5次;5-Fu组20μg/kg,连续注射5d。用药前、用药后1周和2周分别测皮下瘤体的长径(L)、短径(W),计算出瘤体体积,治疗开始后第15天,将裸鼠脱颈处死,摘取瘤体称重,计算出抑瘤率。显微镜观察细胞生长形态、免疫组化法测caspase3蛋白表达、P53及P27核内抑癌基因表达、CD34染色标记测定的MVD及VEGF蛋白表达。与NS组比较,Ad-hIL-24组与5-Fu组的抑瘤率分别为68.52%(P<0.01)和65.64%(P<0.01);镜下Ad-hIL-24组肿瘤组织的caspase3蛋白表达细胞数较其它3组呈显著性升高(P<0.01),P27核内抑癌基因表达细胞数也较NS组明显增高(P<0.01),CD34染色标记测定的CD34及VEGF较NS组和Ad组明显减少(P<0.001),P53未见明显变化。结论Ad-hIL-24可以显著抑制裸鼠SMMC-7721荷瘤的生长,其机制可能通过激活caspase途径、上调P27抑癌基因表达、抑制血管形成等多途径发挥抑瘤作用。     

Faster nerve regeneration after sciatic nerve injury in mice over‐expressing basic fibroblast growth factor

Basic fibroblast growth factor (FGF‐2) is expressed in the peripheral nervous system and is up‐regulated after nerve lesion. It has been demonstrated that administration of FGF‐2 protects neurons from injury‐induced cell death and promotes axonal regrowth. Using transgenic mice over‐expressing FGF‐2 (TgFGF‐2), we addressed the importance of endogenously generated FGF‐2 on sensory neuron loss and sciatic nerve regeneration. After sciatic nerve transection, wild‐type and transgenic mice showed the same degree of cell death in L5 spinal ganglia. Also, the number of chromatolytic, eccentric, and pyknotic sensory neurons was not changed under elevated levels of FGF‐2. Morphometric evaluation of intact nerves from TgFGF‐2 mice revealed no difference in number and size of myelinated fibers compared to wild‐type mice. One week after crush injury, the number of regenerated axons was doubled and the myelin thickness was significantly smaller in transgenic mice. After 2 and 4 weeks, morphometric analysis and functional tests revealed no differences in recovery of sensory and motor nerve fibers. To study the role of FGF‐2 over‐expression on Schwann cell proliferation during the early regeneration process, we used BrdU‐labeling to mark dividing cells. In transgenic mice, the number of proliferating cells was significantly increased distal to the crush site compared to wild‐types. We propose that endogenously synthesized FGF‐2 influences early peripheral nerve regeneration by regulating Schwann cell proliferation, axonal regrowth, and remyelination. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

利用免疫抑制小鼠模型检测真菌病毒对黄曲霉菌致病力影响

【目的】构建用于比较黄曲霉（Aspergillus flavus,A. flavus）菌株之间致病力差异的小鼠感染模型,并利用该模型评价真菌病毒AfPV1对宿主A. flavus致病力的影响。【方法】用不同浓度环磷酰胺腹腔注射Institute of Cancer Research （ICR）小鼠,根据白细胞的数量判断小鼠免疫抑制程度;通过滴鼻和尾静脉两种感染方法接种不同浓度的A. flavus孢子量,统计14 d以内小鼠的死亡率,确定A. flavus最佳的孢子接种量;通过小鼠组织的菌落负荷量以及肺部组织的病理观察,确定A. flavus感染是否成功;最后利用该小鼠模型评价真菌病毒AfPV1对寄主A. flavus致病力的影响。【结果】腹腔注射环磷酰胺的浓度为250 mg/kg时,能够达到免疫抑制水平;小鼠组织真菌负荷和病理组织切片观察显示A. flavus成功感染接种的ICR小鼠组织;在滴鼻接种模型中,A. flavus的孢子接种量为40μL（1×10⁶CFU/mL）时比较合适评价A. flavus菌株之间的差异;在尾静脉接种的模型中,A. flavus的孢子...     

In vivo monitoring of thrombosis in mice by optical coherence tomography

The objective of this study is to establish a novel method for continuously monitoring thrombus progression with various outcome measures and to assess the efficacy of antithrombotic drugs in murine thrombosis model in mice. In the study, thrombus was induced in the femoral vein of mice by FeCl₃ and monitored over time by spectral‐domain optical coherence tomography (OCT). Three‐dimensional images of thrombi with or without heparin as an antithrombotic agent were obtained from OCT angiography. In addition, several parameters of thrombi were analyzed and compared between control and anticoagulant groups. By using OCT, we were able to trace thrombus generation in the same mouse in real time. We found that in our model heparin reduced thrombus size by ~60% and thrombus cross‐sectional area by 50%. OCT results also show that both time to thrombus size (>0.02mm³) and time to occlusion (>30%) were significantly reduced after heparin addition. This study demonstrates that OCT reliably monitors thrombus generation and progression from various aspects including thrombus size. This enables us to measure the kinetic of thrombosis more accurately, and effectively evaluate the efficacy and activities of antithrombotic drugs. This model may represent a useful tool in antithrombotic drug discoveries in preclinical studies.