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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Desipramine+attenuates+working+memoryimpairments+induced+by+partial+loss+of+catecholamines+in+the+rat+medial+prefrontal+cortex.">Desipramine attenuates working memoryimpairments induced by partial loss of catecholamines in the rat medial prefrontal cortex.</a> Psychopharmacology. 183, 404-412 (2006).</li><li>Cooley, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Stereotaxic surgery in the rat. , (1990).</li><li>Di Benedetto, M., Feliciani, D., D'Addario, C., Izenwasser, S., Candeletti, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Romualdi+P.Effects+of+the+selective+norepinephrine+uptake+inhibitor+nisoxetine+on+prodynorphin+geneexpression+in+rat+CNS.">Romualdi P.Effects of the selective norepinephrine uptake inhibitor nisoxetine on prodynorphin geneexpression in rat CNS.</a> Brain Res Mol Brain Res. 127, 115-120 (2004).</li><li>Ding, M., Haglid, K. G., Hamberger, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+immunochemistry+on+neuronal+loss,reactive+gliosis+and+BBB+damage+in+cortex/striatum+and+hippocampus/amygdala+aftersystemic+kainic+acid+administration.">Quantitative immunochemistry on neuronal loss,reactive gliosis and BBB damage in cortex/striatum and hippocampus/amygdala aftersystemic kainic acid administration.</a> Neurochemistry international. 36, 313-318 (2000).</li><li>Finsen, B. R., Sorensen, T., Castellano, B., Pedersen, E. B., Zimmer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte+infiltrationand+glial+reactions+in+xenografts+of+mouse+brain+tissue+undergoing+rejection+in+the+adultrat+brain.+A+light+and+electron+microscopical+immunocytochemical+study.">Leukocyte infiltrationand glial reactions in xenografts of mouse brain tissue undergoing rejection in the adultrat brain. A light and electron microscopical immunocytochemical study.</a> JNeuroimmunol. 32, 159-183 (1991).</li><li>Gliddon, C. M., Darlington, C. L., Smith, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+chronic+infusion+of+a+GABAA+receptoragonist+or+antagonist+into+the+vestibular+nuclear+complex+on+vestibular+compensation+inthe+guinea+pig.">Effects of chronic infusion of a GABAA receptoragonist or antagonist into the vestibular nuclear complex on vestibular compensation inthe guinea pig.</a> J Pharmacol Exp Ther. 313, 1126-1135 (2005).</li><li>Hauss-Wegrzyniak, B., Dobrzanski, P., Stoehr, J. D., Wenk, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+neuroinflammation+in+rats+reproduces+components+of+the+neurobiology+of+Alzheimer's+disease.">Chronic neuroinflammation in rats reproduces components of the neurobiology of Alzheimer's disease.</a> Brain Res. 780, 294-303 (1998).</li><li>Kim, H. S., Choi, H. S., Lee, S. Y., Oh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+of+GABA(A)+receptor+binding+and+subunitmRNA+level+in+rat+brain+by+infusion+of+subtoxic+dose+of+MK-801.">Changes of GABA(A) receptor binding and subunitmRNA level in rat brain by infusion of subtoxic dose of MK-801.</a> Brain Res. 880, 28-37 (2000).</li><li>Kim, S. Y., Chudapongse, N., Lee, S. M., Levin, M. C., Oh, J. T., Park, H. J., Ho, I. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomicanalysis+of+phosphotyrosyl+proteins+in+morphine-dependent+rat+brains.">Proteomicanalysis of phosphotyrosyl proteins in morphine-dependent rat brains.</a> Brain Res MolBrain Res. 133, 58-70 (2005).</li><li>Lockman, P. R., McAfee, G., Geldenhuys, W. J., Schyf, C. J., Abbruscato, T. J., Van Allen, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=der+Schyf+CJ,+Abbruscato+TJ,+Allen+DD.Brain+uptake+kinetics+of+nicotine+and+cotinine+after+chronic+nicotine+exposure.">der Schyf CJ, Abbruscato TJ, Allen DD.Brain uptake kinetics of nicotine and cotinine after chronic nicotine exposure.</a> J Pharmacol Exp Ther. 314, 636-642 (2005).</li><li>MA, K. C., Chang, Z. H., Shih, H., Zhu, J. H., Wu, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+compensatory+'rebound'+of+reactiveastrogliosis:+glial+fibrillary+acidic+protein+immunohistochemical+analysis+of+reactiveastrogliosis+after+a+puncture+wound+to+the+brain+of+rats+with+portocaval+anastomosis.">The compensatory 'rebound' of reactiveastrogliosis: glial fibrillary acidic protein immunohistochemical analysis of reactiveastrogliosis after a puncture wound to the brain of rats with portocaval anastomosis.</a> Acta neuropathologica. 82, 72-77 (1991).</li><li>Marchalant, Y., Rosi, S., Wenk, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-inflammatory+property+of+the+cannabinoid+agonistWIN-55212-2+in+a+rodent+model+of+chronic+brain+inflammation.">Anti-inflammatory property of the cannabinoid agonistWIN-55212-2 in a rodent model of chronic brain inflammation.</a> Neuroscience. 144, 1516-1522 (2007).</li><li>Naert, G., Ixart, G., Tapia-Arancibia, L., Givalois, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+i.c.v.+infusion+of+brainderivedneurotrophic+factor+modifies+hypothalamic-pituitary-adrenal+axis+activity,locomotor+activity+and+body+temperature+rhythms+in+adult+male+rats.">Continuous i.c.v. infusion of brainderivedneurotrophic factor modifies hypothalamic-pituitary-adrenal axis activity,locomotor activity and body temperature rhythms in adult male rats.</a> Neuroscience. 139, 779-789 (2006).</li><li>Nikkhah, G., Olsson, M., Eberhard, J., Bentlage, C., Cunningham, M. G., Bjorklund, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amicrotransplantation+approach+for+cell+suspension+grafting+in+the+rat+Parkinson+model:+adetailed+account+of+the+methodology.">Amicrotransplantation approach for cell suspension grafting in the rat Parkinson model: adetailed account of the methodology.</a> Neuroscience. 63, 57-72 (1994).</li><li>Perry, V. H., Andersson, P. B., Gordon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages+and+inflammation+in+the+centralnervous+system.">Macrophages and inflammation in the centralnervous system.</a> Trends Neurosci. 16, 268-273 (1993).</li><li>Radecki, D. T., Brown, L. M., Martinez, J., Teyler, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BDNF+protects+against+stress-inducedimpairments+in+spatial+learning+and+memory+and+LTP.">BDNF protects against stress-inducedimpairments in spatial learning and memory and LTP.</a> Hippocampus. 15, 246-253 (2005).</li><li>Rosi, S., Ramirez-Amaya, V., Hauss-Wegrzyniak, B., Wenk, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+brain+inflammationleads+to+a+decline+in+hippocampal+NMDA-R1+receptors.">Chronic brain inflammationleads to a decline in hippocampal NMDA-R1 receptors.</a> J Neuroinflammation. 1, 12-12 (2004).</li><li>Takahashi, M., Kakita, A., Futamura, T., Watanabe, Y., Mizuno, M., Sakimura, K., Castren, E., Nabeshima, T., Someya, T., Nawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustained+brain-derived+neurotrophic+factor+upregulationand+sensorimotor+gating+abnormality+induced+by+postnatal+exposure+tophencyclidine:+comparison+with+adult+treatment.">Sustained brain-derived neurotrophic factor upregulationand sensorimotor gating abnormality induced by postnatal exposure tophencyclidine: comparison with adult treatment.</a> J Neurochem. 99, 770-780 (2006).</li><li>Williams, L. R., Vahlsing, H. L., Lindamood, T., Varon, S., Gage, F. H., Manthorpe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+smallgaugecannula+device+for+continuous+infusion+of+exogenous+agents+into+the+brain.">A smallgaugecannula device for continuous infusion of exogenous agents into the brain.</a> Experimental neurology. 95, 743-754 (1987).</li><li>Zhang, X., Lee, T. H., Xiong, X., Chen, Q., Davidson, C., Wetsel, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ellinwood+EH.Methamphetamine+induces+long-term+changes+in+GABAA+receptor+alpha2+subunit+andGAD67+expression.">Ellinwood EH.Methamphetamine induces long-term changes in GABAA receptor alpha2 subunit andGAD67 expression.</a> Biochem Biophys Res Commun. 351, 300-305 (2006).</li></ol>

各种研究表明，减少交付脑内注射，组织创伤和侮辱血脑屏障套管的大小减少（佩里等人，1993年），炎症减少，免疫反应减弱（Finsen等人减少，1991），反应性胶质化（Nikkhah等，1994）。我们在座的一种neuroactive代理大脑内的慢性交付通过一个直径6倍，比常规使用的交付插管，减少microcannula方法。我们已经证明，该系统可靠地提供了一个有代表性的解决方案随着时间的推移和局部创伤和反应性胶质化水平显着降低...

A correction was made to: <a href="http://www.jove.com/video/716/">Construction and Implantation of a Microinfusion System for Sustained Delivery of Neuroactive Agents.</a> A key reference was excluded and a revised abstract was republished.
Additional Reference: 
22. Cunningham, M.G., Ames, H.M., Donalds, R.A., &amp; Benes, F.M. Construction and implantation of a microinfusion system for sustained delivery of neuroactive agents. Journal of Neuroscience Methods 167, 213-220, doi:10.1016/j.jneumeth.2007.08.016 (2008).
Revised Abstract:
Sustained delivery of neuroactive agents is widely used in neuroscience, but poses many technical challenges. It is necessary to deliver the agent with high precision while minimizing localized trauma and inflammation. Also, the ability to customize the system to accommodate animals of different species and sizes is desirable. This video presentation demonstrates the construction of an infusion system that can be fitted to any particular research animal. The delivery microcannula diameter is approximately 10-fold smaller than most infusion cannulas presently used. This translates into enhanced accuracy and reduced trauma to the brain region under study. The delivery cannula can also be sculpted to fit the contour of the surface of the animal's skull, thereby allowing closure of the scalp incision neatly over the infusion system, precluding the need for a skull-mounted pedestal, reducing risk of infection, and ensuring a greater level of comfort to the animal. The system is assembled in an air-free environment and requires the researcher to fashion glass micropipettes with a heat source. These construction methods require special skills that are best acquired, if not in person, using video instruction. (This article is based on work first reported in J Neurosci Methods. 2008 Jan 30;167(2):213-20. Epub 2007 Aug 28.).
Original Abstract:
Experimental protocols used for chronic infusion of neuroactive agents within regions of the brain often utilize a mini-osmotic pump system. Agents are commonly delivered via a stainless steel cannula with a diameter of 0.30 mm or greater. Systems utilizing a cannula of this caliber may impose trauma to the area of interest resulting in architectural damage, thereby compromising structural integrity and normal functioning. As neuroscience inquiry becomes more sophisticated, investigation of brain structures and circuitry requires improved levels of accuracy and higher resolution. We have developed a method for the preparation and implantation of a chronic infusion system within the brain utilizing a borosilicate microcannula with a tip diameter of 50 microns. This technique reduces damage to the local environment and diminishes reactive gliosis at the site of infusion. The configuration of the microinfusion system is also able to conform to the surface of the animal's skull, precluding the need for large cranial pedestals, thus facilitating closure of the scalp incision and reducing the risk of infection. We demonstrate reliable sustained delivery of a dye having a representative molecular weight using an in vitro model and in vivo studies in rats.

A correction was made to: <a href="http://www.jove.com/video/716/">Construction and Implantation of a Microinfusion System for Sustained Delivery of Neuroactive Agents.</a> A key reference was excluded and a revised abstract was republished.

Erratum: Construction and Implantation of a Microinfusion System for Sustained Delivery of Neuroactive Agents.

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<td>Mini-osmotic pumps (MOPs)</td>
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<td>Electrode puller</td>
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construction and implantation of a microinfusion system for sustained delivery of neuroactive agents.

慢性滴注neuroactive代理使用的大脑区域内的实验协议，通常利用一个微型渗透泵系统。代理通常是通过一个直径为0.30毫米或更大的不锈钢套管交付。利用这一口径套管的系统，可以并处创伤导致建筑损坏的利益方面，从而影响结构的完整性和正常运作。随着神经科学的查询变得更加复杂，调查的大脑结构和电路需要提高精度和更高的分辨率的水平。我们已经开发出一种利用与尖端直径为50微米的高硼硅microcannula了大脑内的慢性输液系统的准备和植入的方法。这种技术减少了对当地环境的破坏和减少输液部位的反应性胶质化。 microinfusion系统的配置也能够以符合动物的头骨表面，解除对大脑神经底座的需要，从而促进头皮切口封闭和减少感染的风险。我们表现​​出持续提供可靠的一个有代表性的分子量使用体外模型，并在大鼠体内研究的染料。

Watch this Scientific Journal Video about Construction and Implantation of a Microinfusion System for Sustained Delivery of Neuroactive Agents. at JoVE.com

Construction and Implantation of a Microinfusion System for Sustained Delivery of Neuroactive Agents.

随着神经科学的查询变得更加复杂，调查的大脑结构和电路需要提高精度和更高的分辨率的水平。我们已经开发出一种利用与尖端直径为50微米的高硼硅microcannula了大脑内的慢性输液系统的准备和植入的方法。

为持续给药Neuroactive代理Microinfusion系统的建设和植入。

Sustained delivery of neuroactive agents is widely used in neuroscience, but poses many technical challenges. It is necessary to deliver the agent with ...

construction-implantation-microinfusion-system-for-sustained-delivery

Research

JoVE Journal

Biology

15.4K 次观看.  Harvard Medical School. 随着神经科学的查询变得更加复杂，调查的大脑结构和电路需要提高精度和更高的分辨率的水平。我们已经开发出一种利用与尖端直径为50微米的高硼硅microcannula了大脑内的慢性输液系统的准备和植入的方法。

视频: Construction and Implantation of a Microinfusion System for Sustained Delivery of Neuroactive Agents.

Implantation of Engineered Tissue in the Rat Heart

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in common laboratory rats or mice and these quite frequently serve as an intermediate step between bench-top experiments and large animal testing or human trials. Given that rodents provide an established, cost-effective, and physiologically-relevant model system in which to test novel combinations of scaffolding materials and cells, they are particularly well-suited for cardiovascular tissue engineering studies. Presently, we describe an open-heart surgical procedure to implant engineered tissue containing myogenic progenitor cells in the atrioventricular (AV) groove of a rat heart. These implants are intended to create an electrical conduit between the right atrium and right ventricle with the ultimate goal of providing an alternative treatment to conventional pacemaker implantation in pediatric patients with complete heart block[1]. The engineered tissue is implanted in the AV-groove by means of a thoracotomy. For our purposes, Lewis rats are anesthetized and invasively ventilated to maintain positive airway pressure during the sterile surgical procedure. The approach to the heart is performed by a right thoracotomy through an antero-lateral incision at the 5th intercostal space. The tissue construct is fixed in the AV groove using a single 7-0 Prolene suture and positioned between the right ventricle and atrium at the ventral portion of the heart. The epicardium is partially removed to allow direct contact between the recipient myocardial cells and those contained in the engineered tissue. Following implantation, the chest wall is closed in layers, any pneumothorax is evacuated, and the animal is extubated and treated with analgesic.

Here, we describe a cardiac surgical procedure to implant engineered tissue in the atrioventricular (AV)-groove of an adult Lewis rat.

大鼠心脏中植入的工程组织

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in ...

科研

生物学

High Speed Droplet-based Delivery System for Passive Pumping in Microfluidic Devices

A novel microfluidic system has been developed that uses the phenomenon of passive pumping along with a user controlled droplet based fluid delivery system. Passive pumping is the phenomenon by which surface tension induced pressure differences drive fluid movement in closed channels. The automated fluid delivery system consists of a set of voltage controlled valves with micro-nozzles connected to a fluid reservoir and a control system. These voltage controlled valves offer a volumetrically precise way to deliver fluid droplets to the inlet of a microfluidic device in a high frequency manner. Based on the  dimensions demonstrated in the current study example, the system is capable of flowing 4 milliliters per minute (through a 2.2mm by 260um cross-sectional channel). Based on these same channel dimensions, fluid exchange of a point inside the channel can be achieved in as little as eight milliseconds. It is observed that there is interplay between momentum of the system (imparted by a combination of the droplets created by the valves and the fluid velocity in the channel), and the surface tension of the liquid. Where momentum provides velocity to the fluid flow (or vice-versa), equilibration of surface tension at the inlet provides a sudden stop to any flow. This sudden stop allows the user to control the flow characteristics of the channel and opens the door for a variety of biological applications, ranging anywhere from reagent delivery to drug-cell studies. It is also observed that when nozzles are aimed at the inlet at shallow angles, the droplet momentum can cause additional interesting fluid phenomena, such as mixing of multiple droplets in the inlet.

A novel microfluidic system has been developed using the phenomenon of passive pumping and a user controlled fluid delivery system. This microfluidic system has the potential to be used in a wide variety of biological applications given its low cost, ease of use, volumetric precision, high speed, repeatability and automation.

基于液滴在微流体装置的被动抽水高速输送系统

A novel microfluidic system has been developed that uses the phenomenon of passive pumping along with a user controlled droplet based fluid delivery ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

一个系统<em&gt;体外</em培养的胚胎胰腺

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Phosphopeptide Analysis of Rodent Epididymal Spermatozoa

Spermatozoa are quite unique amongst cell types. Although produced in the testis, both nuclear gene transcription and translation are switched off once the pre-cursor round cell begins to elongate and differentiate into what is morphologically recognized as a spermatozoon. However, the spermatozoon is very immature, having no ability for motility or egg recognition. Both of these events occur once the spermatozoa transit a secondary organ known as the epididymis. During the ~12 day passage that it takes for a sperm cell to pass through the epididymis, post-translational modifications of existing proteins play a pivotal role in the maturation of the cell. One major facet of such is protein phosphorylation. In order to characterize phosphorylation events taking place during sperm maturation, both pure sperm cell populations and pre-fractionation of phosphopeptides must be established. Using back flushing techniques, a method for the isolation of pure spermatozoa of high quality and yield from the distal or caudal epididymides is outlined. The steps for solubilization, digestion, and pre-fractionation of sperm phosphopeptides through TiO2 affinity chromatography are explained. Once isolated, phosphopeptides can be injected into MS to identify both protein phosphorylation events on specific amino acid residues and quantify the levels of phosphorylation taking place during the sperm maturation processes.

Proteomic analysis of any cell type is highly dependent on both purity and pre-fractionation of the starting material in order to de-complexify the sample prior to liquid chromatography mass spectrometry (MS). By using back-flushing techniques, pure spermatozoa can be obtained from rodents. Following digestion, phosphopeptides can be enriched using TiO2.

啮齿动物附睾精子磷酸分析

Spermatozoa are quite unique amongst cell types. Although produced in the testis, both nuclear gene transcription and translation are switched off once ...

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic mouse models using this method necessitates the construction of a &#8216;targeting&#8217; vector, which contains homologous DNA sequences to the target gene, and has for many years been a limiting step in the process. Vector construction can be performed in vivo in Escherichia coli cells using homologous recombination mediated by phage recombinases using a technique termed recombineering. Recombineering is the preferred technique to subclone the long homology sequences (>4kb) and various targeting elements including selection markers that are required to mediate efficient allelic exchange between a targeting vector and its cognate genomic locus. Typical recombineering protocols follow an iterative scheme of step-wise integration of the targeting elements and require intermediate purification and transformation steps. Here, we present a novel recombineering methodology of vector assembly using a multiplex approach. Plasmid gap repair is performed by the simultaneous capture of genomic sequence from mouse Bacterial Artificial Chromosome libraries and the insertion of dual bacterial and mammalian selection markers. This subcloning plus insertion method is highly efficient and yields a majority of correct recombinants. We present data for the construction of different types of conditional gene knockout, or knock-in, vectors and BAC reporter vectors that have been constructed using this method. SPI vector construction greatly extends the repertoire of the recombineering toolbox and provides a simple, rapid and cost-effective method of constructing these highly complex vectors.

Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting methodologies can be used to generate transgenic mice with knockout, knock-in and tagged alleles. Here, we describe an improved method of recombineering in E. coli, that we term &#8216;subcloning plus insertion&#8217;, which can be used to generate custom gene targeting vectors rapidly.

亚克隆加插入（SPI） - 一种新型重组工程方法基因打靶载体的快速构建

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic ...

Single Cell Collection of Trophoblast Cells in Peri-implantation Stage Human Embryos

Human implantation, the apposition and adhesion to the uterine surface epithelia and subsequent invasion of the blastocyst into the maternal decidua, is a critical yet enigmatic biological event that has been historically difficult to study due to technical and ethical limitations. Implantation is initiated by the development of the trophectoderm to early trophoblast and subsequent differentiation into distinct trophoblast sublineages. Aberrant early trophoblast differentiation may lead to implantation failure, placental pathologies, fetal abnormalities, and miscarriage. Recently, methods have been developed to allow human embryos to grow until day 13 post-fertilization in vitro in the absence of maternal tissues, a time-period that encompasses the implantation period in humans. This has given researchers the opportunity to investigate human implantation and recapitulate the dynamics of trophoblast differentiation during this critical period without confounding maternal influences and avoiding inherent obstacles to study early embryo differentiation events in vivo. To characterize different trophoblast sublineages during implantation, we have adopted existing two-dimensional (2D) extended culture methods and developed a procedure to enzymatically digest and isolate different types of trophoblast cells for downstream assays. Embryos cultured in 2D conditions have a relatively flattened morphology and may be suboptimal in modeling in vivo three-dimensional (3D) embryonic architectures. However, trophoblast differentiation seems to be less affected as demonstrated by anticipated morphology and gene expression changes over the course of extended culture. Different trophoblast sublineages, including cytotrophoblast, syncytiotrophoblast and migratory trophoblast can be separated by size, location, and temporal emergence, and used for further characterization or experimentation. Investigation of these early trophoblast cells may be instrumental in understanding human implantation, treating common placental pathologies, and mitigating the incidence of pregnancy loss.

Here, we describe a method for warming vitrified human blastocysts, culturing them through the implantation period in vitro, digesting them into single cells and collecting early trophoblast cells for further investigation.

近植入阶段人类胚胎中特罗福细胞的单细胞集合

Human implantation, the apposition and adhesion to the uterine surface epithelia and subsequent invasion of the blastocyst into the maternal decidua, is a ...

Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial Compounds

Staphylococcus aureus expresses virulence factors to trigger its internalization into eukaryote cells and to survive inside different subcellular compartments. This paper describes an enzyme protection assay to study the extent of S. aureus internalization and its intracellular survival in adherent non-professional phagocytic cells (NPPCs) as well as the intracellular efficacy of antimicrobial compounds. NPPCs are grown in a multi-well plate until they reach 100% confluence. S. aureus cultures are grown overnight in cell culture medium. The bacterial suspension is diluted according to the number of cells per well to inoculate the cells at a controlled multiplicity of infection. Inoculated cells are incubated for 2 h to allow the bacteria to be internalized by the NPPCs, following which lysostaphin is added to the culture medium to selectively kill extracellular bacteria. Lysostaphin is present in the culture medium for the rest of the experiment. 
At this point, the infected cells could be incubated with antimicrobial compounds to assess their intracellular activities against S. aureus. Next, the cells are washed three times to remove the drugs, and intracellular S. aureus load is then quantified by culturing on agar plates. Alternatively, for studying staphylococcal virulence factors involved in intracellular survival and cell toxicity, lysostaphin could be inactivated with proteinase K to eliminate the need for washing steps. This tip improves the reliability of the intracellular bacterial load quantification, especially if cells tend to detach from the culture plate when they become heavily infected because of the multiplication of intracellular S. aureus. These protocols can be used with virtually all types of adherent NPPCs and with 3D cell culture models such as organoids.

Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial Compounds

This protocol aims to describe how to study the extent of Staphylococcus aureus internalization and its ability to survive inside the human host cell, as well as the intracellular efficacy of antimicrobial compounds.

改进酶保护测定法研究 金黄色葡萄球菌 内化和抗菌化合物的细胞内效

Staphylococcus aureus expresses virulence factors to trigger its internalization into eukaryote cells and to survive inside different subcellular ...

Culture of Piglet Intestinal 3D Organoids from Cryopreserved Epithelial Crypts and Establishment of Cell Monolayers

Intestinal organoids are increasingly being used to study the gut epithelium for digestive disease modeling, or to investigate interactions with drugs, nutrients, metabolites, pathogens, and the microbiota. Methods to culture intestinal organoids are now available for multiple species, including pigs, which is a species of major interest both as a farm animal and as a translational model for humans, for example, to study zoonotic diseases. Here, we give an in-depth description of a procedure used to culture pig intestinal 3D organoids from frozen epithelial crypts. The protocol describes how to cryopreserve epithelial crypts from the pig intestine and the subsequent procedures to culture 3D intestinal organoids. The main advantages of this method are (i) the temporal dissociation of the isolation of crypts from the culture of 3D organoids, (ii) the preparation of large stocks of cryopreserved crypts derived from multiple intestinal segments and from several animals at once, and thus (iii) the reduction in the need to sample fresh tissues from living animals. We also detail a protocol to establish cell monolayers derived from 3D organoids to allow access to the apical side of epithelial cells, which is the site of interactions with nutrients, microbes, or drugs. Overall, the protocols described here is a useful resource for studying the pig intestinal epithelium in veterinary and biomedical research.

Here, we describe a protocol to culture pig intestinal 3D organoids from cryopreserved epithelial crypts. We also describe a method to establish cell monolayers derived from 3D organoids, allowing access to the apical side of epithelial cells.

冷冻保存的上皮隐窝仔猪肠道3D类器官的培养和细胞单层的建立

Intestinal organoids are increasingly being used to study the gut epithelium for digestive disease modeling, or to investigate interactions with drugs, ...

免疫组织化学染色的子宫内膜组织的图像分析

Platform for Quantitative Detection of Endometrial Immune Cells Based on Immunohistochemistry and Digital Image Analysis

To evaluate the endometrial immune microenvironment of patients with recurrent miscarriage (RM), a digital immunohistochemistry image analysis platform was developed and validated to quantitatively analyze endometrial immune cells during the mid-luteal phase. All endometrium samples were collected during the mid-luteal phase of the menstrual cycle. Paraffin-embedded endometrial tissues were sectioned into 4 &#181;m thick slides, and immunohistochemistry (IHC)staining was carried out for detecting endometrial immune cells, including CD56+ uNK cells, Foxp3+ Tregs, CD163+ M2 macrophages, CD1a+ DCs, and CD8+ T cells. The panoramic slides were scanned using a digital slide scanner and a commercial image analysis system was used for quantitative analysis. The percentage of endometrial immune cells was calculated by dividing the number of immune cells in the total endometrial cells. Using the commercial image analysis system, quantitative evaluation of endometrial immune cells, which are difficult or impossible to analyze with conventional image analysis, could be easily, and accurately analyzed. This methodology can be applied to quantitatively characterize the endometrium microenvironment, including interaction between immune cells, and its heterogeneity for different reproductive failure patients. The platform for quantitative evaluation of endometrial immune cells may be of important clinical significance for the diagnosis and treatment of RM patients.

作者聚焦：通过子宫内膜免疫细胞定量评估方案推进生殖免疫学 

Here, a digital immunohistochemistry image analysis platform was developed and validated to quantitatively analyze the endometrial immune cells of patients with recurrent miscarriages in the window of implantation.

基于免疫组化和数字图像分析的子宫内膜免疫细胞定量检测平台

To evaluate the endometrial immune microenvironment of patients with recurrent miscarriage (RM), a digital immunohistochemistry image analysis platform ...