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>

<ol>
	<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 border="1">
	<tbody>
		<tr>
			<td>Name of equipment</td>
			<td>Company</td>
			<td>Catalog number</td>
		</tr>
		<tr>
			<td>Isoflurane-Phoenix</td>
			<td>Clipper</td>
			<td>NDC 57319-559-06</td>
		</tr>
		<tr>
			<td>Posi-Seal Induction Chamber</td>
			<td>Molecular Imaging Products</td>
			<td>AS-01-0530-SM</td>
		</tr>
		<tr>
			<td>Portable Anesthesia System</td>
			<td>Molecular Imaging Products</td>
			<td>AS-01-0007</td>
		</tr>
		<tr>
			<td>Gaymer T Pump</td>
			<td>Gaymar Industries, Inc</td>
			<td>TP650</td>
		</tr>
		<tr>
			<td>Warming Blanket (Heating pad)</td>
			<td>Kent Scientific Corp</td>
			<td>TP-22G</td>
		</tr>
		<tr>
			<td>Curved forceps</td>
			<td>Roboz</td>
			<td>RS-5101</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Fine Science Tools (FST)</td>
			<td>FST 14063-09</td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td>FST</td>
			<td>FST 12501-13</td>
		</tr>
		<tr>
			<td>Electrical shaver</td>
			<td>Oster</td>
			<td>Golden A5</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 ...

Research

JoVE Journal

Medicine

73.6K 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

The Trier Social Stress Test Protocol for Inducing Psychological Stress

This article demonstrates a psychological stress protocol for use in a laboratory setting. Protocols that allow researchers to study the biological pathways of the stress response in health and disease are fundamental to the progress of research in stress and anxiety.1 Although numerous protocols exist for inducing stress response in the laboratory, many neglect to provide a naturalistic context or to incorporate aspects of social and psychological stress. Of psychological stress protocols, meta-analysis suggests that the Trier Social Stress Test (TSST) is the most useful and appropriate standardized protocol for studies of stress hormone reactivity.2 In the original description of the TSST, researchers sought to design and evaluate a procedure capable of inducing a reliable stress response in the majority of healthy volunteers.3 These researchers found elevations in heart rate, blood pressure and several endocrine stress markers in response to the TSST (a psychological stressor) compared to a saline injection (a physical stressor).3 Although the TSST has been modified to meet the needs of various research groups, it generally consists of a waiting period upon arrival, anticipatory speech preparation, speech performance, and verbal arithmetic performance periods, followed by one or more recovery periods. The TSST requires participants to prepare and deliver a speech, and verbally respond to a challenging arithmetic problem in the presence of a socially evaluative audience.3 Social evaluation and uncontrollability have been identified as key components of stress induction by the TSST.4 In use for over a decade, the goal of the TSST is to systematically induce a stress response in order to measure differences in reactivity, anxiety and activation of the hypothalamic-pituitary-adrenal (HPA) or sympathetic-adrenal-medullary (SAM) axis during the task.1 Researchers generally assess changes in self-reported anxiety, physiological measures (e.g. heart rate), and/or neuroendocrine indices (e.g. the stress hormone cortisol) in response to the TSST. Many investigators have adopted salivary sampling for stress markers such as cortisol and alpha-amylase (a marker of autonomic nervous system activation) as an alternative to blood sampling to reduce the confounding stress of blood-collection techniques. In addition to changes experienced by an individual completing the TSST, researchers can compare changes between different treatment groups (e.g. clinical versus healthy control samples) or the effectiveness of stress-reducing interventions.1

This article describes a protocol for inducing psychological stress in participants, which enables researchers to measure psychological, physiological and neuroendocrine responses to stress within single participants or between groups.

特里尔社会压力诱发心理应激的测试协议

This article demonstrates a psychological stress protocol for use in a laboratory setting.  Protocols that allow researchers to study the biological ...

科研

医学

Protocol for Long Duration Whole Body Hyperthermia in Mice

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body temperature that is observed during fever. The use of hyperthermia as an adjuvant has emerged as a promising procedure for tumor regression in the field of cancer biology. For this purpose, the most important requirement is to have reliable and uniform heating protocols. We have developed a protocol for hyperthermia (whole body) in mice. In this protocol, animals are exposed to cycles of hyperthermia for 90 min followed by a rest period of 15 min. During this period mice have easy access to food and water. High body temperature spikes in the mice during first few hyperthermia exposure cycles are prevented by immobilizing the animal. Additionally, normal saline is administered in first few cycles to minimize the effects of dehydration. This protocol can simulate fever like conditions in mice up to 12-24 hr. We have used 8-12 weeks old BALB/Cj female mice to demonstrate the protocol.

This paper describes a protocol for whole body hyperthermia in mice that can stimulate fever like conditions up to 12-24 hr.

持续时间较长的小鼠全身热疗协议

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body ...

Collection Protocol for Human Pancreas

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation of pancreas recovered from cadaveric organ donors. The pancreas is divided into 3 main regions (head, body, tail) followed by serial transverse sections throughout the medial to lateral axis. Alternating sections are used for fixed paraffin and fresh frozen blocks and remnant samples are minced for snap frozen sample preparations, either with or without RNAse inhibitors, for DNA, RNA, or protein isolation. The overall goal of the pancreas dissection procedure is to sample the entire pancreas while maintaining anatomical orientation.
Endocrine cell heterogeneity in terms of islet composition, size, and numbers is reported for human islets compared to rodent islets 1. The majority of human islets from the pancreas head, body and tail regions are composed of insulin-containing &beta;-cells followed by lower proportions of glucagon-containing &alpha;-cells and somatostatin-containing &delta;-cells. Pancreatic polypeptide-containing PP cells and ghrelin-containing epsilon cells are also present but in small numbers. In contrast, the uncinate region contains islets that are primarily composed of pancreatic polypeptide-containing PP cells 2. These regional islet variations arise from developmental differences. The pancreas develops from the ventral and dorsal pancreatic buds in the foregut and after rotation of the stomach and duodenum, the ventral lobe moves and fuses with the dorsal 3. The ventral lobe forms the posterior portion of the head including the uncinate process while the dorsal lobe gives rise to the rest of the organ. Regional pancreatic variation is also reported with the tail region having higher islet density compared to other regions and the dorsal lobe-derived components undergoing selective atrophy in type 1 diabetes4,5.
Additional organs and tissues are often recovered from the organ donors and include pancreatic lymph nodes, spleen and non-pancreatic lymph nodes. These samples are recovered with similar formats as for the pancreas with the addition of isolation of cryopreserved cells. When the proximal duodenum is included with the pancreas, duodenal mucosa may be collected for paraffin and frozen blocks and minced snap frozen preparations.

This video demonstrates a dissection procedure for processing human pancreas into multiple storage formats. Anatomical orientation is maintained throughout the pancreatic regions to allow definition of regional islet composition and density.

人体胰腺的收集协议

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

颈动脉输液的紫杉烷类化合物在小鼠体内的药代动力学和药效学分析

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

A Simple Critical-sized Femoral Defect Model in Mice

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all limb bone fractures fail to heal completely due to the extent of the trauma, disease, or age of the patient. Our ability to improve bone regenerative strategies is critically dependent on the ability to mimic serious bone trauma in test animals, but the generation and stabilization of large bone lesions is technically challenging. In most cases, serious long bone trauma is mimicked experimentally by establishing a defect that will not naturally heal. This is achieved by complete removal of a bone segment that is larger than 1.5 times the diameter of the bone cross-section. The bone is then stabilized with a metal implant to maintain proper orientation of the fracture edges and allow for mobility. 
Due to their small size and the fragility of their long bones, establishment of such lesions in mice are beyond the capabilities of most research groups. As such, long bone defect models are confined to rats and larger animals. Nevertheless, mice afford significant research advantages in that they can be genetically modified and bred as immune-compromised strains that do not reject human cells and tissue.
Herein, we demonstrate a technique that facilitates the generation of a segmental defect in mouse femora using standard laboratory and veterinary equipment. With practice, fabrication of the fixation device and surgical implantation is feasible for the majority of trained veterinarians and animal research personnel. Using example data, we also provide methodologies for the quantitative analysis of bone healing for the model.

Animal models are frequently employed to mimic serious bone injury in biomedical research. Due to their small size, establishment of stabilized bone lesions in mice are beyond the capabilities of most research groups. Herein, we describe a simple method for establishing and analyzing experimental femoral defects in mice.

一个简单的临界尺寸股骨缺损模型小鼠

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all ...

Heterotopic Renal Autotransplantation in a Porcine Model: A Step-by-Step Protocol

Kidney transplantation is the treatment of choice for patients suffering from end-stage renal disease. It offers better life expectancy and higher quality of life when compared to dialysis. Although the last few decades have seen major improvements in patient outcomes following kidney transplantation, the increasing shortage of available organs represents a severe problem worldwide. To expand the donor pool, marginal kidney grafts recovered from extended criteria donors (ECD) or donated after circulatory death (DCD) are now accepted for transplantation. To further improve the postoperative outcome of these marginal grafts, research must focus on new therapeutic approaches such as alternative preservation techniques, immunomodulation, gene transfer, and stem cell administration.
Experimental studies in animal models are the final step before newly developed techniques can be translated into clinical practice. Porcine kidney transplantation is an excellent model of human transplantation and allows investigation of novel approaches. The major advantage of the porcine model is its anatomical and physiological similarity to the human body, which facilitates the rapid translation of new findings to clinical trials. This article offers a surgical step-by-step protocol for an autotransplantation model and highlights key factors to ensure experimental success. Adequate pre- and postoperative housing, attentive anesthesia, and consistent surgical techniques result in favorable postoperative outcomes. Resection of the contralateral native kidney provides the opportunity to assess post-transplant graft function. The placement of venous and urinary catheters and the use of metabolic cages allow further detailed evaluation. For long-term follow-up studies and investigation of alternative graft preservation techniques, autotransplantation models are superior to allotransplantation models, as they avoid the confounding bias posed by rejection and immunosuppressive medication.

Porcine models of organ transplantation provide an important platform to study mechanisms of organ preservation. This article describes a heterotopic porcine renal autotransplantation model, which allows investigating new approaches to improve the outcome of transplantation using marginal kidney grafts.

异位自体肾移植的猪模型：有步骤，分步协议

Kidney transplantation is the treatment of choice for patients suffering from end-stage renal disease. It offers better life expectancy and higher quality ...

A Step by Step Protocol for Subretinal Surgery in Rabbits

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in adults in industrially developed countries. RPE transplantation appears to be a promising therapy, as it may replace dysfunctional RPE, restore its function, and thereby vision.
Here we describe a method for transplanting a cultured RPE monolayer on a scaffold into the subretinal space (SRS) of rabbits. After vitrectomy xenotransplants were delivered into the SRS using a custom made shooter consisting of a 20-gauge metallic nozzle with a polytetrafluoroethylene (PTFE) coated plunger. The current technique evolved in over 150 rabbit surgeries over 6 years. Post-operative follow-up can be obtained using non-invasive and repetitive in vivo imaging such as spectral domain optical coherence tomography (SD-OCT) followed by perfusion-fixed histology.
The method has well-defined steps for easy learning and high success rate. Rabbits are considered a large eye animal model useful in preclinical studies for clinical translation. In this context rabbits are a cost-efficient and perhaps convenient alternative to other large eye animal models.

Retinal pigment epithelium (RPE) replacement strategies and gene-based therapy are considered for several retinal degenerative conditions. For clinical translation, large eye animal models are required to study surgical techniques applicable in patients. Here we present a rabbit model for subretinal surgery geared towards RPE transplantation, which is versatile and cost-efficient.

一种用于兔视网膜下手术分步协议

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in ...

Creation of Abdominal Adhesions in Mice

Abdominal adhesions consist of fibrotic tissue that forms in the peritoneal space in response to an inflammatory insult, typically surgery or intraabdominal infection. The precise mechanisms underlying adhesion formation are poorly understood. Many compounds and physical barriers have been tested for their ability to prevent adhesions after surgery with varying levels of success. The mouse and rat are important models for the study of abdominal adhesions. Several different techniques for the creation of adhesions in the mouse and rat exist in the literature. Here we describe a protocol utilizing abrasion of the cecum with sandpaper and sutures placed in the right abdominal sidewall. The mouse is anesthetized and the abdomen is prepped. A midline laparotomy is created and the cecum is identified. Sandpaper is used to gently abrade the surface of the cecum. Next, several figure-of-eight sutures are placed into the peritoneum of the right abdominal sidewall. The abdominal cavity is irrigated, a small amount of starch is applied, and the incision is closed. We have found that this technique produces the most consistent adhesions with the lowest mortality rate.

Abdominal adhesions that form after surgery are a major cause of pain, infertility, and hospitalization and reoperation for small bowel obstruction. Our surgical procedure for creating abdominal adhesions in mice is a reliable tool to study the mechanisms underlying the formation of adhesions.

小鼠腹腔粘连的创建

Abdominal adhesions consist of fibrotic tissue that forms in the peritoneal space in response to an inflammatory insult, typically surgery or ...

A Detailed Protocol for Perspiration Monitoring Using a Novel, Small, Wireless Device

Perspiration monitoring can be utilized for the detection of certain diseases, such as thermoregulation and mental disorders, particularly when the patients are unaware of such disorders or are having difficulty expressing their symptoms. Until now, several devices for perspiration monitoring have been developed; however, such devices tend to have a relatively large exterior, considerable power consumption, and/or less sensitivity.
Recently, we developed a small, wireless device for perspiration monitoring. The device consists of a temperature/relative humidity (T/RH) sensor, battery-driven small data logger, and silica gel as a desiccant in a small cylindrical exterior. The T/RH sensor is placed between the detection windows (through which the water vapor from the skin enters) and the silica gel. The underlying principle of the perspiration monitoring device is based on Fick&#39;s law of diffusion, which means that water vapor flux from the skin to the silica gel (i.e.&#160;transepidermal water loss and perspiration) can be captured by change in humidity at the T/RH sensor. In addition, a baseline subtraction method was adopted to distinguish perspiration and transepidermal water loss.
As shown in the previous report, the developed device can monitor the perspiration at any sites of the body in an easy, wireless manner. However, detailed methods of how to use the device have not been disclosed yet. In this article, therefore, we would like to show the point-by-point tutorials of how to use the device for perspiration monitoring, by showing the sympathetic activity test with the sympathetic skin response monitoring as an example.

Recently, we developed a small wireless device for perspiration monitoring. In this article, we present detailed protocols on how to use the device for perspiration monitoring with an example of the sympathetic activity test.

详细的汗渍监控协议使用一种新的，小的，无线设备

Perspiration monitoring can be utilized for the detection of certain diseases, such as thermoregulation and mental disorders, particularly when the ...

A Detailed Protocol for Physiological Parameters Acquisition and Analysis in Neurosurgical Critical Patients

Intracranial pressure (ICP) monitoring is now widely used in neurosurgical critical patients. Besides mean ICP value, the ICP derived parameters such as ICP waveform, amplitude of pulse (AMP), the correlation of ICP amplitude and ICP mean (RAP), pressure reactivity index (PRx), ICP and arterial blood pressure (ABP) wave amplitude correlation (IAAC), and so on, can reflect intracranial status, predict prognosis, and can also be used as guidance of proper treatment. However, most of the clinicians focus only on the mean ICP value while ignoring these parameters because of the limitations of the current devices. We have recently developed a multimodality monitoring system to address these drawbacks. This portable, user-friendly system will use a data collecting and storing device to continuously acquire patients&#39; physiological parameters first, i.e., ABP, ICP, and oxygen saturation, and then analyze these physiological parameters. We hope that the multimodality monitoring system will be accepted as a key measure to monitor physiological parameters, to analyze the current clinical status, and to predict the prognosis of the neurosurgical critical patients.

Recently, we have developed a portable multimodality monitoring system for the monitoring of various physiological parameters in neurosurgical critical patients. Detailed protocols on how to use this multimodality monitoring system are presented here.

神经外科危重患者生理参数采集与分析的详细方案

Intracranial pressure (ICP) monitoring is now widely used in neurosurgical critical patients. Besides mean ICP value, the ICP derived parameters such as ...