Cell Labeling and Injection in Developing Embryonic Mouse Hearts

Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect their in vivo potential. Preferably, these cells should be placed in a proper embryonic environment in order to acquire their definite phenotype. Furthermore, cell lineage tracing studies in the mouse after labeling cells with dyes or retroviral vectors has remained mostly limited to early stage mouse embryos with still poorly developed organs. To overcome these limitations, we designed standard and ultrasound-mediated microinjection protocols to inject various agents in targeted regions of the heart in mouse embryos at E9.5 and later stages of development.  Embryonic explant or embryos are then cultured or left to further develop in utero. These agents include fluorescent dyes, virus, shRNAs, or stem cell-derived progenitor cells. Our approaches allow for preservation of the function of the organ while monitoring migration and fate of labeled and/or injected cells. These technologies can be extended to other organs and will be very helpful to address key biological questions in biology of development.     

Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can be used both to investigate the biological responses to injury and to test potential therapies. Contusion or compression injury delivered to the surgically exposed spinal cord are the most widely used models of the pathology. In this report the experimental contusion is performed by using the Infinite Horizon (IH) Impactor device, which allows the creation of a reproducible injury animal model through definition of specific injury parameters. Stem cell transplantation is commonly considered a potentially useful strategy for curing this debilitating condition. Numerous studies have evaluated the effects of transplanting a variety of stem cells. Here we demonstrate an adapted method for spinal cord injury followed by tail vein injection of cells in CD1 mice. In short, we provide procedures for: i) cell labeling with a vital tracer, ii) pre-operative care of mice, iii) execution of a contusive spinal cord injury, and iv) intravenous administration of post mortem neural precursors. This contusion model can be utilized to evaluate the efficacy and safety of stem cell transplantation in a regenerative medicine approach.     

A Rapid and Efficient Method for Assessing Pathogenicity of Ustilago maydis on Maize and Teosinte Lines

Maize is a major cereal crop worldwide. However, susceptibility to biotrophic pathogens is the primary constraint to increasing productivity. U. maydis is a biotrophic fungal pathogen and the causal agent of corn smut on maize. This disease is responsible for significant yield losses of approximately $1.0 billion annually in the U.S.¹ Several methods including crop rotation, fungicide application and seed treatments are currently used to control corn smut². However, host resistance is the only practical method for managing corn smut. Identification of crop plants including maize, wheat, and rice that are resistant to various biotrophic pathogens has significantly decreased yield losses annually^3-5. Therefore, the use of a pathogen inoculation method that efficiently and reproducibly delivers the pathogen in between the plant leaves, would facilitate the rapid identification of maize lines that are resistant to U. maydis. As, a first step toward indentifying maize lines that are resistant to U. maydis, a needle injection inoculation method and a resistance reaction screening method was utilized to inoculate maize, teosinte, and maize x teosinte introgression lines with a U. maydis strain and to select resistant plants.Maize, teosinte and maize x teosinte introgression lines, consisting of about 700 plants, were planted, inoculated with a strain of U. maydis, and screened for resistance. The inoculation and screening methods successfully identified three teosinte lines resistant to U. maydis. Here a detailed needle injection inoculation and resistance reaction screening protocol for maize, teosinte, and maize x teosinte introgression lines is presented. This study demonstrates that needle injection inoculation is an invaluable tool in agriculture that can efficiently deliver U. maydis in between the plant leaves and has provided plant lines that are resistant to U. maydis that can now be combined and tested in breeding programs for improved disease resistance.     

Reverse Genetic Morpholino Approach Using Cardiac Ventricular Injection to Transfect Multiple Difficult-to-target Tissues in the Zebrafish Larva

The zebrafish is an important model to understand the cell and molecular biology of organ and appendage regeneration. However, molecular strategies to employ reverse genetics have not yet been adequately developed to assess gene function in regeneration or tissue homeostasis during larval stages after zebrafish embryogenesis, and several tissues within the zebrafish larva are difficult to target. Intraventricular injections of gene-specific morpholinos offer an alternative method for the current inability to genomically target zebrafish genes in a temporally controlled manner at these stages. This method allows for complete dispersion and subsequent incorporation of the morpholino into various tissues throughout the body, including structures that were formerly impossible to reach such as those in the larval caudal fin, a structure often used to noninvasively research tissue regeneration. Several genes activated during larval finfold regeneration are also present in regenerating adult vertebrate tissues, so the larva is a useful model to understand regeneration in adults. This morpholino dispersion method allows for the quick and easy identification of genes required for the regeneration of larval tissues as well as other physiological phenomena regulating tissue homeostasis after embryogenesis. Therefore, this delivery method provides a currently needed strategy for temporal control to the evaluation of gene function after embryogenesis.      

参麦注射液联合无创正压通气治疗慢性阻塞性肺疾病合并呼吸衰竭的临床观察

目的:观察参麦注射液联合无创正压通气治疗慢性阻塞性肺疾病(COPD)合并呼吸衰竭的疗效及其对患者血清脑钠肽(BNP)、纤维蛋白原和D-二聚体水平的影响。方法:选取2010年1月至2012年1月我院收治的COPD合并急性呼吸衰竭患者120例,随机分为观察组和对照组,每组各60例,对照组予以常规治疗,观察组在对照组的基础上予以无创正压通气和参麦注射液治疗,观察和比较两组治疗前后的pH值、动脉血氧分压(PaO2)、动脉血二氧化碳分压(PaCO2),、氧合指数(PaO2/FiO2)、纤维蛋白原、D-二聚体和BNP水平的变化。结果:治疗后,两组的pH、PaO2和PaO2/FiO2均较治疗前明显升高(P0.01),且观察组PaO2/FiO2水平较对照组升高更为明显(P0.05);两组PaCO2、纤维蛋白原、D-二聚体和BNP水平均较治疗前明显降低(P0.01),且观察组以上指标水平明显低于对照组(P0.01)。结论:参麦注射液联合无创正压通气治疗COPD合并呼吸衰竭的疗效显著,并可显著降低患者血浆BNP、纤维蛋白原和D-二聚体水平。     

氨溴索不同给药方式对机械通气大鼠肺内炎性因子水平及肺组织湿/干比值的影响

目的:探讨不同氨溴索给药方式对大鼠机械通气肺部炎性因子释放及肺组织湿/干比值的影响。方法:将45只健康SD雄性大鼠随机分成静脉组、雾化组和对照组,各15只。对照组:大鼠麻醉后行气管切开插管,并行单纯机械通气4h;静脉组:大鼠麻醉后从尾静脉泵入氨溴索,气管切开插管后行机械通气4h;雾化组:大鼠麻醉后行气管切开插管,通过自制装置氧气驱动雾化吸入氨溴索,并行机械通气4 h。各组机械通气后放血处死大鼠,取肺组织,计算肺湿/干重比(W/D)。收集支气管灌洗液(BALF)并检测TNF-α,IL-1β和IL-6浓度。结果:与对照组比较,静脉组和雾化组大鼠支气管灌洗液中TNF-α,IL-1β和IL-6的浓度明显降低(P0.05),但此两组间无显著性差异(P0.05);与对照组比较,静脉组和雾化组大鼠肺组织W/D比也明显降低(P0.05),但雾化组大鼠肺组织W/D比明显低于静脉组大鼠(P0.05)。结论:氨溴索静脉给予和雾化给予都能显著降低大鼠机械通气时肺内炎性因子水平和肺W/D比,而且雾化给予较静脉给予更能减轻大鼠肺组织的W/D比。     

Production of female bovine embryos with sex-sorted sperm using intracytoplasmic sperm injection: Efficiency and in vitro developmental competence

The production of embryos with a preselected sex sperm is important in the livestock industry. In this study, we examined the efficiency of producing female embryos by intracytoplasmic sperm injection (ICSI) with flow cytometry sorted (ssICSI) and unsorted (usICSI) bovine sperm, and their developmental competence in vitro. For comparison, bovine embryos were also produced by in vitro fertilization (IVF) with sorted (ssIVF) and unsorted (usIVF) bovine sperm. The semen used in this study was from a bull selected for its high fertility and blastocyst developmental competence among four bulls. We first examined and compared pronuclear (PN) formation and cleavage rates of the produced embryos among the treatment groups. Our results demonstrated that PN formation rates (judged by two pronucleus [2PN]) and cleavage rates in ssIVF group (23.1% and 43.6%) were lower than those in the usIVF (71.1% and 71.6%), usICSI (73.1% and 92.8%) and ssICSI (75% and 79.1%) groups, respectively (P < 0.05). Moreover, the blastocyst formation rate in the ssIVF group was less than those in the usIVF, usICSI, and ssICSI groups (2.7% vs. 30.2%, 28.7% and 24.7%, respectively; P < 0.05). Importantly, we reported that the blastocyst formation rate in the ssICSI group was similar to that in the usICSI group, which indicated that ICSI can rescue the damage introduced to sperm by flow cytometry–mediated sex-sorting. Of note, we achieved a blastocyst formation rate in the ssICSI group to be comparable with the usIVF group. We then examined embryo quality by counting the number of normal and apoptotic cells in blastocysts. It was found that, despite the fact that blastocyst formation rate in the ssIVF group was significantly lower than those in the usIVF, usICSI and ssICSI groups, there was no difference in total and apoptotic cell numbers among these groups (P > 0.05). Finally, karyotyping analysis demonstrated that the proportion of female embryos in the ssICSI and ssIVF groups was 100%, whereas it was 58.8% and 57.8% in the usIVF and usICSI groups, respectively. In conclusion, ICSI with flow cytometry sorted bovine sperm provides an alternative approach to produce embryos with predetermined sex.     

同一药物不同剂量足三里穴位注射对心气虚证的效应对比研究

目的 研究参芪扶正注射液足三里穴位注射对心气虚证的效应与用药剂量的关系.方法 通过负重游泳及灌服大剂量心得安法获得心气虚证大鼠模型,对各治疗组分别进行不同剂量参芪扶正注射液的足三里穴注治疗.连续治疗10 d后,观察并记录大鼠的一般状况和症状;通过ELISA法检测各组血清心钠素（ANP）及环磷酸腺苷（cAMP）含量;通过比色法检测各组血清超氧化物歧化酶（SOD）活性;通过HE染色法检测各组心肌组织病理改变.结果 和正常对照组相比,模型对照组出现疲软无力,舌质发紫,呼吸急促等明显的心气虚症状;血清ANP浓度升高,cAMP浓度降低,SOD活性降低,均具有极其显著性意义（P〈0.001）; 心肌组织病理示：炎细胞浸润明显,心肌细胞严重水肿,排列紊乱.和模型组相比,各治疗组症状缓解,血清ANP浓度降低,cAMP浓度升高、SOD活性增强,差异均具有显著性意义（P〈0.05或P〈0.01或P〈0.001）,其中参芪0.05 mL组变化最小（P〈0.05）,参芪0.20 mL组变化最大（P〈0.001）;心肌组织病理改变减轻,其中参芪0.20 mL组最接近正常.结论 参芪扶正注射液足三里穴位注射能有效治疗心气虚证,且其疗效与用药剂量在一定剂量范围（0.05～0.20）mL内成正相关.     

阿奇霉素连续与间歇注射给药治疗支原体肺炎的疗效与耐药性比较

为了比较阿奇霉素连续与间歇注射两种给药方式治疗支原体肺炎的临床疗效及耐药性,选取120例支原体肺炎患者,随机分为连续给药组(A组)与间歇给药组(B组),两组患者均连续治疗1个疗程(14d),比较临床疗效及耐药性情况。结果显示,两组患者在临床疗效、症状及体征消失时间方面均无明显差异(p>0.05),但治疗7d、14d后A组的最小抑菌浓度(MIC)值均明显低于B组(p<0.05)。结果表明,阿奇霉素的两种不同给药方式治疗支原体肺炎效果类似,但间歇给药法可减轻因连续给药带来的耐药性,对提高抗生素用药安全有一定价值。     

Elucidating the mechanisms of protein antigen adsorption to the CAF/NAF liposomal vaccine adjuvant systems: Effect of charge,fluidity and antigen-to-lipid ratio

The reverse vaccinology approach has recently resulted in the identification of promising protein antigens, which in combination with appropriate adjuvants can stimulate customized, protective immune responses. Although antigen adsorption to adjuvants influences vaccine efficacy and safety, little is generally known about how antigens and adjuvants interact at the molecular level. The aim of this study was to elucidate the mechanisms of interactions between the equally sized, but oppositely charged model protein antigens α-lactalbumin and lysozyme, and i) the clinically tested cationic liposomal adjuvant CAF01 composed of cationic dimethyldioctadecylammonium (DDA) bromide and trehalose-6,6′-dibehenate (TDB) or ii) the neutral adjuvant formulation NAF01, where DDA was replaced with zwitterionic distearoylphosphatidylcholine (DSPC). The effect of liposome charge, bilayer rigidity, isoelectric point and antigen-to-lipid ratio was investigated using dynamic light scattering, transmission electron microscopy, differential scanning calorimetry, intrinsic fluorescence and Langmuir monolayers. The net anionic α-lactalbumin adsorbed onto the cationic liposomes, while there was no measureable attractive interaction with the zwitterionic liposomes. In contrast, the net cationic lysozyme showed very little interaction with either types of liposome. Adsorption of α-lactalbumin altered its tertiary structure, affected lipid membrane packing below and above the phase transition temperature, and neutralized the liposomal surface charge, resulting in reduced colloidal stability and liposome aggregation. Langmuir studies revealed that α-lactalbumin was not squeezed out of DDA monolayers upon compression, which suggests additional hydrophobic interactions.