作者要感谢阿德里恩莫斯利和Libuse耶扎贝克（斯坦福大学），用于与手术技术援助。

所有的动物研究，根据加州大学洛杉矶分校的动物护理和使用委员会实验动物的护理和使用的指导方针和卫生指南的国家机构进行。下面描述的步骤的持续时间是从开始约45-60分钟来结束。 1。手术野的制备<ol><li>在一个干净的动物手术室执行程序。 </li><li>设备：异氟醚蒸发器，盖默牛逼泵用加热垫。 </li><li>无菌工具：两弯钳，细剪刀，持针器。 </li><li>无菌手套必须在整个手术过程中被使用。 </li></ol> 2。动物的准备<ol><li>将两个雄性或雌性小鼠相似的重量和大小，从相同的遗传背景，在同一个笼子和监测至少两周，以保证和谐的同居生活。雌性小鼠是首选，由于其较少攻击性的行为。 </li><li>由麻醉动物使用异氟醚蒸发器。这里的小鼠在一个压力平衡密封吸气室连接到异氟烷蒸发器（4-5％V / V）。一旦麻醉诱导，所述动物在整个过程通过鼻锥连接到异氟烷（1.5-2％V / V）转移到毛皮剃须区域和维持麻醉。用棉条，以防止眼睛干涩应用眼药膏。 </li><li>将动物的仰卧位。彻底剃鼠标的左侧放置在左侧和右侧的鼠标放置在右侧开始在大约1厘米以上的肘部到1厘米以下的膝盖。 </li><li>在无菌条件下彻底擦拭（2-3X）与聚维酮碘浸泡过的抹布其次是酒精擦拭准备剃领域。将小鼠在加热垫覆盖无菌垫。 </li><li>用于镇痛，分别管理卡洛芬和丁丙诺啡腹膜内或皮下，以10 mg / kg和0.1毫克/公斤的剂量。 </li><li>将动物就在自己身边，背靠背，与djacent朝上剃领域。避免了手术区域的任何污染，涵盖的小鼠用无菌披盖仅暴露操作区域。创建一个小的悬垂性开放进行手术时留下来的无菌。我们所做的悬垂窗口大偷拍时有更好的视觉。 </li></ol> 3。联体<ol><li>用锋利的剪刀，在0.5厘米肘以上进行纵向皮肤切口到每个动物的起始的剃边一路至0.5厘米以下的膝关节（ 图1）。继切口，轻轻按住皮肤与一双弯钳分离从皮下筋膜皮肤，分离筋膜与第二对创建0.5厘米免费皮肤。执行沿着整个切口这种分离。 </li><li>通过附加一个动物的左侧鹰嘴的其他的权利鹰嘴开始接合。既olecranons关节和膝关节都清晰可辨如下次e皮肤切口。为了方便加入，弯曲第一个鼠标的手肘并通过鹰嘴下的不可吸收缝线3-0的针。同样，第二个弯鼠标的手肘，并通过其下的相同的缝合。由双外科结连接接头紧密。 </li><li>连接膝关节按照相同的程序。 </li><li>以下关节的附件，一个连续吸收5-0薇乔缝合腹侧开始从肘部朝向膝盖连接两个动物的皮肤上。为了防止皮肤破裂和分离执行周围的肘部和膝盖的区域的皮肤紧缝合关闭。一旦腹侧皮肤附件已经完成时，执行双外科结。将小鼠俯卧位，并继续背侧缝合带双外科结结束。验证缝线的连续性，并确认没有开口。 </li><li>管理0.5毫升0.9％的NaCl皮下，每只小鼠以防止脱氢dration。 </li></ol> 4。术后恢复<ol><li>继续加热垫的动物，直至康复为止。 </li><li>以下恢复，提供止痛药卡洛芬和丁丙诺啡。重复腹膜内或皮下给药，每24和12小时，分别为48小时，在上述（步骤2.5）中描述的相同的剂量。监测动物的痛苦和困难的症状，如颤抖，嗜睡，咀嚼尾巴，每天弓背，缺乏疏导， 等了两个星期。 </li><li>预防，治疗小鼠磺胺甲恶唑/甲氧苄啶口服混悬液在他们的水2瓶磺胺毫克/毫升0.4毫克修剪/ ml的10天，以防止细菌感染。 </li><li>房子的每个连体对在一个干净的笼子与单片铺垫材料（ 如纸毛巾或吸水无菌垫），以防止铺垫材料的愿望。返回动物的床上用品填充笼的时候都能够保持胸骨斜卧与他们的头。为了尽量减少株深远的食物，而调整到连体的存在，放在笼地板上沾有食物颗粒。提供筑巢材料。在1-2周内连体小鼠有正常的走动配对手术前和后肢的能力。 </li><li>血液嵌合体发生10-14天以下的手术。 </li></ol> 5。确认和相反的步骤<ol><li>两周后抽血从尾静脉各小鼠的流式细胞分析来确认血液的嵌合体（ 图3）。 </li><li>血液处理流式细胞术分析：稀释2-3滴血液中加入500μl10mM的EDTA中。加入500μl2％葡聚糖（PBS稀释）中，充分混匀并在37℃下30分钟。将上清液转移到新管，旋于1200 rpm离心5分钟，重悬沉淀的细胞用PBS（或其它缓冲液）用于流式细胞仪分析。 </li><li>根据实验设计，连体一对可分开的吨稍后的时间点。根据我们的经验，我们一直保持着parabiosed对长达9个月，无任何并发症。 </li><li>在一个无菌手术表面执行相反的步骤。麻醉小鼠通过将它们放置在一个感应腔室连接到一个异氟烷蒸发器（4-5％）和维持麻醉用1.5-2％的异氟烷​​，上述（步骤2.2）所描述的。 </li><li>从周围的初始缝合的区域去除毛发和无菌制备如上所述的剃区域（步骤2.3，2.4）。 </li><li>用于镇痛，管理卡洛芬和丁丙诺啡腹膜内或皮下，以10 mg / kg和0.1毫克/公斤的剂量，分别为。 </li><li>覆盖手术区以上（步骤2.6）所述无菌的悬垂性。 </li><li>使用锋利的剪刀，通过沿横向缝合一纵行切口分离小鼠，轻轻地取下两只老鼠之间新形成的筋膜有一对弯曲的镊子。分开的接缝，切缝线的结连接它们。剪掉沿切口皮肤达到光滑的边缘和重新连接皮肤与可吸收连续涂5-0薇乔缝合。 </li><li>为了防止脱水，管理将0.5ml 0.9％的NaCl皮下，每只小鼠。继续加热垫的动物，直至康复为止。 </li><li>恢复后，重复镇痛药，卡洛芬（10毫克/千克），每24小时和丁丙诺啡（0.1毫克/千克），每12小时48小时。 </li><li>为防止细菌感染治疗小鼠磺胺甲恶唑/甲氧苄啶口服混悬液（2磺胺毫克/毫升0.4毫克修剪/毫升）在他们的水瓶10天。 </li></ol>

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	<li>Bert, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exp&#233;riences+et+consid&#233;rations+sur+la+greffe+animale.">Exp&#233;riences et consid&#233;rations sur la greffe animale.</a> Journal de l'Anatomie et de la Physiologie. 1, 69-87 .</li><li>Bunster, E., Meyer, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+methods+of+parabiosis.">Improved methods of parabiosis.</a> Anat. Rec. 57, 339-380 (1933).</li><li>Waskow, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+parabiotic+mice+for+the+study+of+DC+and+DC+precursor+circulation.">Generation of parabiotic mice for the study of DC and DC precursor circulation.</a> Methods Mol. Biol. 595, 413-428 (2010).</li><li>Finerty, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parabiosis+in+physiological+studies.">Parabiosis in physiological studies.</a> Physiol. Rev. 32, 277-302 (1952).</li><li>Aicher, A., Heeschen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonbone+marrow-derived+endothelial+progenitor+cells:+what+is+their+exact+location.">Nonbone marrow-derived endothelial progenitor cells: what is their exact location.</a> Circ. Res. 101, e102 (2007).</li><li>Abe, S., Boyer, C., 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=Cells+derived+from+the+circulation+contribute+to+the+repair+of+lung+injury.">Cells derived from the circulation contribute to the repair of lung injury.</a> Am. J. Respir. Crit. Care Med. 170, 1158-1163 (2004).</li><li>Donskoy, E., Goldschneider, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thymocytopoiesis+is+maintained+by+blood-borne+precursors+throughout+postnatal+life.+A+study+in+parabiotic+mice.">Thymocytopoiesis is maintained by blood-borne precursors throughout postnatal life. A study in parabiotic mice.</a> J. Immunol. 148, 1604-1612 (1992).</li><li>Powell, A. E., Anderson, E. C., 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=Fusion+between+Intestinal+epithelial+cells+and+macrophages+in+a+cancer+context+results+in+nuclear+reprogramming.">Fusion between Intestinal epithelial cells and macrophages in a cancer context results in nuclear reprogramming.</a> Cancer Res. 71, 1497-1505 (2011).</li><li>Duyverman, A. M., Kohno, M., 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+transient+parabiosis+skin+transplantation+model+in+mice.">A transient parabiosis skin transplantation model in mice.</a> Nat. Protoc. 7, 763-770 (2012).</li><li>Ajami, B., Bennett, J. L., 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=Local+self-renewal+can+sustain+CNS+microglia+maintenance+and+function+throughout+adult+life.">Local self-renewal can sustain CNS microglia maintenance and function throughout adult life.</a> Nat. Neurosci. 10, 1538-1543 (2007).</li><li>Weissman, I. L., Jerabek, L., 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=Tolerance+and+the+H-Y+antigen:+Requirement+for+male+T+cells,+but+not+B+cells,+to+induce+tolerance+in+neonatal+female+mice.">Tolerance and the H-Y antigen: Requirement for male T cells, but not B cells, to induce tolerance in neonatal female mice.</a> Transplantation. 37, 3-6 (1984).</li></ol>

该作者有没有竞争的财务权益。

这里讨论的联体的方法呈现在低死亡率最小的技术难点和结果。膝关节和肘关节的附件是一个显著改善本斯特和迈耶技术。但是，该过程仍侵入从而维持无菌条件下整个手术是势在必行。以进一步防止外科手术部位的感染，这是很重要的parabiosed动物接受抗生素的组合，进行定期监测。为确保坚定的支持，防止疼痛，连接肘部和膝盖缝合应围绕关节，而不是通过组织。 作为替?...

联体，手术加盟的两种生物，于1864年首次描述了保罗·伯特，以此来建立一个模型来研究共享循环系统和由两只老鼠1的皮肤和肌肉壁的加盟。联体促进微血管的形成在3发炎的部位，并有多个应用程序在生理的研究，如脑下垂体和性腺与肾脏高血压4的作用之间的荷尔蒙沟通。它得到了进一步的来探讨祖细胞的招募和整合新生血管5，造血干细胞6的迁移和淋巴细胞贩运7，以及在肿瘤转移8,9循环炎性细胞或干细胞的作用和动力学，和神经退行性疾病10。 联体谎言中的一个显著优势在该合作的动物有着共同的循环抗原，使细胞迁移和血管新生，而不会触发免疫反应。重要的是，威斯曼等人已经显示雄性和雌性小鼠之间的联体不会导致形成抗HY的抗体11。 在由Paul伯特描述的原始协议的2只动物经皮肤和肌肉壁1的连接接合在一起。此方法然而，引起显著菌株对动物和由于伤口的感染导致高死亡率。自那时以来，联体技术已经修订了几组最最主要的是在1933年2提出的本斯特和迈耶的协议。他们的方法包括加入肩胛骨关节，体腔和皮肤，允许更好的支持和痛苦少的动物。与此同时，新的方法导致了最小的术后护理和显着地降低死亡率。本文所描述的协议是本斯特和迈耶技术，是微创的，并允许更牢固接合的变形。即，将小鼠通过肘和膝盖关节以及皮肤相连。皮肤的这种连接可以防止分机，因此导致更少的痛苦和并发症。在这里，我们描述了野生型（WT）成年小鼠的组成型表达GFP的小鼠的加盟。我们发现，手术后两周我们可以达到血液嵌合体的50％证明了这种外科手术的功效，以创建一个共享的循环系统。

<table><tbody><tr><td>Isoflurane-Phoenix</td><td>Clipper</td><td>NDC 57319-559-06</td><td></td></tr><tr><td>Posi-Seal Induction Chamber</td><td>Molecular Imaging Products</td><td>AS-01-0530-SM</td><td></td></tr><tr><td>Portable Anesthesia System</td><td>Molecular Imaging Products</td><td>AS-01-0007</td><td></td></tr><tr><td>Gaymer T Pump</td><td>Gaymar Industries, Inc</td><td>TP650</td><td></td></tr><tr><td>Warming Blanket (Heating pad)</td><td>Kent Scientific Corp</td><td>TP-22G</td><td></td></tr><tr><td>Curved forceps</td><td>Roboz</td><td>RS-5101</td><td></td></tr><tr><td>Scissors</td><td>Fine Science Tools (FST)</td><td>FST 14063-09</td><td></td></tr><tr><td>Needle holder</td><td>FST</td><td>FST 12501-13</td><td></td></tr><tr><td>Electrical shaver</td><td>Oster</td><td>Golden A5</td><td></td></tr></tbody></table>

parabiosis in mice: a detailed protocol

联体是两种生物体，允许共享的血液循环的外科结合。将两个动物的皮肤促进形成微脉管系统中的炎症部位。连体合作伙伴分享他们的循环抗原，因此是免费的不良免疫反应。首先由保罗·伯特在1864年1所述，联体手术是通过提炼和本斯特迈耶于1933年，以改善动物生存2。在当前的协议中，2只小鼠手术加入以下的本斯特和迈耶技术的一种改进。动物通过肘部和膝关节后面附着在皮肤允许，防止紧张的缝合皮肤坚定支持的连接。在此，我们详细描述了连体加入一个无处不在的绿色荧光蛋白表达小鼠与野生型（WT）小鼠。手术后两周，对被分离和GFP阳性细胞可以通过流式细胞术分析来检测血液中的circulat离子的WT鼠标。血液嵌合允许一个检查从在另一种动物的循环细胞的贡献。

的两个生物体联体的预期结果是每只动物的循环系统的一个共同的血液循环（ 图2）的相等的贡献。人们可以很容易验证parabiosed WT和GFP阳性小鼠经流式细胞仪分析血液的成功平衡。这里，从两个parabionts的尾部，得到在2周时，手术后静脉采血并进行分级分离为外周血细胞（排除红细胞）。分级分离的造血来源的细胞级分，随后用流式细胞仪对GFP阳性和WT细胞的存在进行分析。与以前的?...

Watch this Scientific Journal Video about Parabiosis in Mice: A Detailed Protocol at JoVE.com

Parabiosis in Mice: A Detailed Protocol

联体接合的两个生物体导致的共享循环系统的发展。在这个协议中，我们描述了手术步骤，以形成一个野生型小鼠和组成型表达GFP的小鼠之间的联体连接。

联体小鼠：一个详细的协议

Parabiosis is a surgical union of two organisms allowing sharing of the blood circulation. Attaching the skin of two animals promotes formation of ...

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

74.2K 次观看.  University of California, Los Angeles. 联体接合的两个生物体导致的共享循环系统的发展。在这个协议中，我们描述了手术步骤，以形成一个野生型小鼠和组成型表达GFP的小鼠之间的联体连接。 

视频: Parabiosis in Mice: A Detailed Protocol

Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae

Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for candidate genes of interest, migratory behaviors of cells, and secreted signals in distinct genetic settings. Because parabiotic animals share a common circulation, any blood or blood-borne factor from one animal will be exchanged with its partner and vice versa. Thus, cells and molecular factors derived from one genetic background can be studied in the context of a second genetic background. Parabiosis of adult mice has been &#160;used extensively to research aging, cancer, diabetes, obesity, and brain development. More recently, parabiosis of zebrafish embryos has been used to study the developmental biology of hematopoiesis. In contrast to mice, the transparent nature of zebrafish embryos permits the direct visualization of cells in the parabiotic context, making it a uniquely powerful method for investigating fundamental cellular and molecular mechanisms. The utility of this technique, however, is limited by a steep learning curve for generating the parabiotic zebrafish embryos. This protocol provides a step-by-step method on how to surgically fuse the blastulae of two zebrafish embryos of different genetic backgrounds to investigate the role of candidate genes of interest. In addition, the parabiotic zebrafish embryos are tolerant to heat shock, making temporal control of gene expression possible. This method does not require a sophisticated set-up and has broad applications for studying cell migration, fate specification, and differentiation in vivo during embryonic development.

This protocol provides step-by-step instruction on how to generate parabiotic zebrafish embryos of different genetic backgrounds. When combined with the unparalleled imaging capabilities of the zebrafish embryo, this method provides a uniquely powerful means to investigate cell-autonomous versus non-cell-autonomous functions for candidate genes of interest.

联体斑马鱼胚胎一代发展囊胚的手术融合

Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for ...

科研

发育生物学

A Double Humanized BLT-mice Model Featuring a Stable Human-Like Gut Microbiome and Human Immune System

Humanized mice (hu-mice) that feature a functional human immune system have fundamentally changed the study of human pathogens and disease. They can be used to model diseases that are otherwise difficult or impossible to study in humans or other animal models. The gut microbiome can have a profound impact on human health and disease. However, the murine gut microbiome is very different than the one found in humans. &#160;There is a need for improved pre-clinical hu-mice models that have an engrafted human gut microbiome. Therefore, we created double hu-mice that feature both a human immune system and stable human-like gut microbiome. NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice are one of the best animals for humanization due to their high level of immunodeficiency. However, germ-free NSG mice, and various other important germ-free mice models are not currently commercially available. Further, many research settings do not have access to gnotobiotic facilities, and working under gnotobiotic conditions can often be expensive and time consuming. Importantly, germ-free mice have several immune deficiencies that exist even after the engraftment of microbes. Therefore, we developed a protocol that does not require germ-free animals or gnotobiotic facilities. To generate double hu-mice, NSG mice were treated with radiation prior to surgery to create bone-marrow, liver, thymus-humanized (hu-BLT) mice. The mice were then treated with broad spectrum antibiotics to deplete the pre-existing murine gut microbiome. After antibiotic treatment, the mice were given fecal transplants with healthy human donor samples via oral gavage. Double hu-BLT mice had unique 16S rRNA gene profiles based on the individual human donor sample that was transplanted. Importantly, the transplanted human-like microbiome was stable in the double hu-BLT mice for the duration of the study up to 14.5 weeks post-transplant.

We describe a novel method for generating double humanized BLT-mice that feature a functional human immune system and a stable engrafted human-like gut microbiome. This protocol can be followed without the need for germ-free mice or gnotobiotic facilities.

具有稳定的人类状古特微生物群落和人类免疫系统的双人性化BLT小鼠模型

Humanized mice (hu-mice) that feature a functional human immune system have fundamentally changed the study of human pathogens and disease. They can be ...

生物化学

Cardiopulmonary Bypass in a Mouse Model: A Novel Approach

As prolonged cardiopulmonary bypass becomes more essential during cardiac interventions, an increasing clinical demand arises for procedure optimization and for minimizing organ damage resulting from prolonged extracorporal circulation. The goal of this paper was to demonstrate a fully functional and clinically relevant model of cardiopulmonary bypass in a mouse. We report on the device design, perfusion circuit optimization, and microsurgical techniques. This model is an acute model, which is not compatible with survival due to the need for multiple blood drawings. Because of the range of tools available for mice (e.g., markers, knockouts, etc.), this model will facilitate investigation into the molecular mechanisms of organ damage and the effect of cardiopulmonary bypass in relation to other comorbidities.

This paper describes how to perform cardiopulmonary bypass in mice. This novel model will facilitate the investigation of the molecular mechanisms involved in organ damage.

小鼠体外循环模型: 一种新方法

As prolonged cardiopulmonary bypass becomes more essential during cardiac interventions, an increasing clinical demand arises for procedure optimization ...

医学

A Mouse Model of Orthopedic Surgery to Study Postoperative Cognitive Dysfunction and Tissue Regeneration

Surgery is commonly used to improve and maintain quality of life. Unfortunately, in vulnerable patients such as the elderly, complications may occur and significantly diminish the outcome. Indeed, after routine orthopedic surgery to repair a fracture, as many as 50% of elderly patients suffer from neurologic complications like delirium. Also, the capacity to heal and regenerate tissue after surgery decreases with age, and can impact the quality of fracture repair and even osseous integration of implants. Thus, a better understanding of mechanisms that drive these age-dependent changes could provide strategic targets to minimize risk for such complications and optimize outcomes. Here, we introduce a clinically relevant mouse model of tibial fracture. The postoperative changes in these mice mimic some of the cognitive impairments commonly observed after routine orthopedic surgery in humans. Briefly, an incision is performed in the right hind limb under strictly aseptic conditions. Muscles are disassociated, and a 0.38-mm stainless steel pin is inserted into the upper crest of the tibia, inside the intramedullary canal. Osteotomy is then performed, and the wound is stapled. We have used this model to investigate the effects of surgical trauma on postoperative neuroinflammation and behavioral changes. By applying this fracture model in combination with parabiosis, a surgical model in which 2 mice are anastomosed, we have studied cells and secreted factors that systemically rejuvenate organ function and tissue regeneration after injury. By following our step-by-step protocol, these models can be reproduced with high fidelity, and can be adapted to interrogate many biologic pathways that are altered by surgical trauma.

This protocol describes a mouse model of orthopedic surgery that has been used to study mechanisms of postoperative neuroinflammation and behavioral changes, and when combined with parabiosis, to study tissue regeneration during aging.

骨科手术的小鼠模型研究术后认知功能障碍与组织再生

Surgery is commonly used to improve and maintain quality of life. Unfortunately, in vulnerable patients such as the elderly, complications may occur and ...

Bone Marrow Transplantation Procedures in Mice to Study Clonal Hematopoiesis

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor cells (HSPCs). Mutations in driver genes, that confer cellular fitness, can lead to the development of expanding HSPC clones that increasingly give rise to progeny leukocytes harboring the somatic mutation. Because clonal hematopoiesis has been associated with heart disease, stroke, and mortality, the development of experimental systems that model these processes is key to understanding the mechanisms that underly this new risk factor. Bone marrow transplantation procedures involving myeloablative conditioning in mice, such as total-body irradiation (TBI), are commonly employed to study the role of immune cells in cardiovascular diseases. However, simultaneous damage to the bone marrow niche and other sites of interest, such as the heart and brain, is unavoidable with these procedures. Thus, our lab has developed two alternative methods to minimize or avoid possible side effects caused by TBI: 1) bone marrow transplantation with irradiation shielding and 2) adoptive BMT to non-conditioned mice. In shielded organs, the local environment is preserved allowing for the analysis of clonal hematopoiesis while the function of resident immune cells is unperturbed. In contrast, the adoptive BMT to non-conditioned mice has the additional advantage that both the local environments of the organs and the hematopoietic niche are preserved. Here, we compare three different hematopoietic cell reconstitution approaches and discuss their strengths and limitations for studies of clonal hematopoiesis in cardiovascular disease.

We describe three methods of bone marrow transplantation (BMT): BMT with total-body irradiation, BMT with shielded irradiation, and BMT method with no pre-conditioning (adoptive BMT) for the study of clonal hematopoiesis in mouse models.

小鼠骨髓移植程序研究克隆造血症

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor ...

免疫与感染

Mouse Heterotopic Cervical Cardiac Transplantation Utilizing Vascular Cuffs

Murine models of cardiac transplantation are frequently utilized to study ischemia-reperfusion injury, innate and adaptive immune responses after transplantation, and the impact of immunomodulatory therapies on graft rejection. Heterotopic cervical heart transplantation in mice was first described in 1991 using sutured anastomoses and subsequently modified to include cuff techniques. This modification allowed for improved success rates, and since then, there have been multiple reports that have proposed further technical improvements. However, translation into more widespread utilization remains limited due to the technical difficulty associated with graft anastomoses, which requires precision to achieve adequate length and caliber of the cuffs to avoid vascular anastomotic twisting or excessive tension, which can result in damage to the graft. The present protocol describes a modified technique for performing heterotopic cervical cardiac transplantation in mice which involves cuff placement on the recipient's common carotid artery and the donor's pulmonary artery in alignment with the direction of the blood flow.

Mouse cardiac transplantation models represent valuable research tools for studying transplantation immunology. The present protocol details mouse heterotopic cervical cardiac transplantation that involves the placement of cuffs on the recipient's common carotid artery and the donor's pulmonary artery trunk to allow for laminar blood flow.

利用血管袖带的小鼠异位宫颈心脏移植

Murine models of cardiac transplantation are frequently utilized to study ischemia-reperfusion injury, innate and adaptive immune responses after ...

A Modified Murine Heterotopic Heart Transplant Protocol Matching Contemporary Standards of Aseptic Technique, Anesthesia, and Analgesia

The development of experimental models of cardiac transplantation in animals has contributed to many advances in the fields of immunology and solid organ transplantation. While the heterotopic vascularized murine cardiac transplantation model was initially utilized in studies of graft rejection using combinations of mismatched inbred mouse strains, access to genetically modified strains and therapeutic modalities can provide powerful new preclinical insights. Fundamentally, the surgical methodology for this technique has not changed since its development, especially with respect to important factors such as aseptic technique, anesthesia, and analgesia, which make material impacts on postsurgical morbidity and mortality. Additionally, improvements in perioperative management are expected to provide improvements in both animal welfare and experimental outcomes. This paper reports upon a protocol developed in collaboration with a subject matter expert in veterinary anesthesia and describes the surgical technique with an emphasis on perioperative management. Additionally, we discuss the implications of these refinements and provide details on troubleshooting critical surgical steps for this procedure.

The present paper describes a modified technique for heterotopic vascularized cardiac transplantation with updated aseptic technique, analgesia, and anesthesia.

符合当代无菌技术、麻醉和镇痛标准的改良小鼠异位心脏移植方案

The development of experimental models of cardiac transplantation in animals has contributed to many advances in the fields of immunology and solid organ ...

Blood Circuit Reconstruction in an Abdominal Mouse Heart Transplantation Model

The surgical technique of heterotopic abdominal heart transplantation in mice is a standard model for research in transplantation immunology. Here, the established technique for a modified blood circuit reconstruction in a heterotopic abdominal heart transplantation model is presented. This method uses the intrathoracic inferior vena cava (IIVC) instead of the pulmonary artery of the donor heart for the anastomosis to the inferior vena cava of the recipient. It is facilitating and improving success rates for abdominal heart transplantation in mice.

A novel technique for blood circuit reconstruction in a heterotopic abdominal mouse heart transplantation model is demonstrated.

腹部小鼠心脏移植模型中的血液回路重建

The surgical technique of heterotopic abdominal heart transplantation in mice is a standard model for research in transplantation immunology. Here, the ...

Isolation of Mouse Coronary Endothelial Cells

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new vascular formation. Endothelial cell dysfunction is implicated in the development and progression of many cardiovascular diseases including ischemic heart disease. To examine the function and characterization of coronary endothelial cells, cell isolation is the first step and it requires high purity and quantity to conduct subsequent experiments. This protocol describes an efficient method to isolate adult mouse coronary endothelial cells. The mouse heart is dissected and minced into small pieces. After the digestion of the heart using dispase and collagenase II, cells are washed&#160;and incubated with magnetic beads which are conjugated with anti-CD31 antibody. The beads with endothelial cells are washed several times and are ready to use in various applications, including imaging and molecular biological experiments. Efficient isolation yields approximately 104 cells per one heart with over 90% purity.

This protocol is prepared to share our method of isolating mouse coronary endothelial cells for the purpose of imaging or to conduct molecular biological experiments.

鼠标冠状动脉内皮细胞的分离

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new ...

生物学

小鼠肝移植和同种异体移植物通畅性分析

Successful Orthotopic Liver Transplantation in Mice Utilizing Microcomputed Tomography Angiography

Microcomputed tomography (microCT) angiography is an invaluable resource to researchers. New advances in this technology have allowed for high-quality images to be obtained of micro-vasculature and are high-fidelity tools in the field of organ transplantation. In this model of orthotopic liver transplantation (OLT) in mice, microCT affords the opportunity to evaluate allograft anastomosis in real time and has the added benefit of not having to sacrifice study animals. The choice of contrast, as well as image acquisition settings, create a high-definition image, which gives researchers invaluable information. This allows for evaluation of the technical aspects of the procedure as well as potentially evaluating different therapeutics over an extended duration of time. In this protocol, we detail an OLT model in mice in a stepwise fashion and finally describe a microCT protocol that can give high-quality images, which aid researchers in in-depth analysis of solid organ transplantation. We provide a step-by-step guide for liver transplantation in a mouse, as well as briefly discuss a protocol for evaluating the patency of the graft through microCT angiography.

作者聚焦:通过小鼠模型中的 MicroCT 血管造影增强移植研究 

In this protocol, we discuss the implementation of a model of successful orthotopic liver transplantation (OLT) in mice. Additionally, adjuvants to further analyze allograft patency after successful OLT in a mouse are discussed as well, specifically utilizing microcomputed tomography (microCT) scans.

利用显微计算机断层扫描血管造影成功对小鼠进行原位肝移植

Microcomputed tomography (microCT) angiography is an invaluable resource to researchers. New advances in this technology have allowed for high-quality ...