We thank Amanda Fair, the CMI, and Kathy Sweet and the ORL at UM for assistance with &#181;CT imaging and analysis. Funding: BL Funded by 1K08GM109105-01 and Plastic Surgery Foundation National Endowment Award.

伦理声明：所有动物的程序进行，严格按照定义的指南实验动物的使用和注意事项中提供的准则良好做法的动物：第八版从学院实验动物研究（ILAR，2011）和人经密歇根大学的机构动物护理和使用委员会（PRO0001553）。 1.鼠标外科手术注意：使用8-10周龄C57BL / 6小鼠。其他年龄，背景，和小鼠的菌株也可用于测试不同的条件或遗传组成。对于该过程的切断术部，保持用面罩，吹风盖和无菌长袍，手套，和仪器无菌条件。手术部位应该用碘伏消毒，并进行立体裁剪无菌准备。通过使用兽医加热垫和升温复苏溶液至37℃，避免低温之前administra化。 <ol><li>麻醉，用2.5％的异氟烷​​吸入鼠标。用鼻锥离开背部和后肢肢体方便。调整异氟烷给药速度保持粘膜适当呼吸速率和着色，并确保麻醉通过检查肌肉张力，脚趾捏和角膜反射。应用平淡眼药膏鼠标的眼睛，以防止在手术过程中干眼的伤害。采用外科领域的下方兽医加热垫或温水循环保持足够的体温。 </li><li>辖丁丙诺啡，0.1毫克/千克，皮下之前立即手术镇痛。 </li><li>密切刮胡子使用指甲刀从脚跟到膝盖左侧后爪的手术部位。 </li><li>密切剃鼠标的背部，开始在脊柱和剃区域延伸到背中线的左边，结算的面积至少为2厘米×3cm至容纳烫伤。 </li><li>准备手术部位，包括后爪来使用3-交替碘伏磨砂膝盖。 </li><li>沿左跟腱的内侧面进行纵形切口。延长切口所以跟腱可以容易地可视化;约为0.5公分。 </li><li>在用锋利的剪刀组织的中点执行跟腱腱切断术与肌腱锐性剥离。插入所述组织剪刀的一个叶片在所述组织平面肌腱下方并沿平面解剖直到刀片是在肌腱中点。关闭剪刀片大幅割断肌腱。 注：确保所有列入筋后腓肠肌包括，比目鱼肌，跖肌腱。 </li><li>达到止血施加压力，用无菌纱布（应该有最小的出血）和5-0薇乔缝合关闭皮肤切口。 </li><li>与铝块重达35克与近似measuremen进行背Ⅱ度烧伤TS2厘米×2厘米×3厘米施加到小鼠的剃毛背部为17秒的水浴加热到60℃。 注：实现适当的烧伤深度由搁在块上的麻醉小鼠的顶部，确保该块的整个表面区域是在用鼠标接触，但是避免施加任何额外的压力来将挡， 即，允许重力将唯一力捧到位块。附连到块的一侧的薄塑料手柄便于操纵，稳定化，并从热水浴检索。这将创建在8-10周龄C57BL / 6小鼠的约30％体表面积烧​​伤。这种接触烧伤被选择了其他的方法（火焰或烫伤烧伤）因烧伤深度的均匀性伤口和动物之间的重现性。 </li><li>干烧伤部位用纱布和应用TEGADERM敷料。 </li><li>辖回暖复苏液：乳酸林格氏溶液1ml腹腔注射和0.5ml皮下注射。这一次复苏的管理是足够的距离烧伤和切断术恢复。 注意：不要单独在干净的笼子下监视返回麻醉小鼠住房笼与其他老鼠，房子，直到小鼠完全恢复。典型的恢复发生在1-6小时。烧伤部位一般在2-4周内自愈，很少通过伤口感染复杂，如果存放在干净的环境中的住房。 </li><li>辖丁丙诺啡0.1毫克/千克皮下注射，每12小时3天以下的步骤。何监督增长，串行μCT扫描在适当的间隔。异位骨的生长是第一明显约3周后运通过μCT。 注：大多数HO发展完成了9周后运。我们没有观察到何体积或反复μCT位置的差扫瞄每两周15周相比单终点μCT在15周扫描。在实验结束时，安乐死所有小鼠用CO 2吸入根据机构指南和10分钟后确认死亡颈椎脱位。 </li></ol> 2.μCT采集与分析<ol><li>确保麻醉鼠标放在扫描仪床上俯卧位。大盘后肢牢固地固定在床上，以防止呼吸运动伪影。包括空气，水，和羟基磷灰石含有小鼠用于图像校正的下方幻象。 </li><li>打开骨分析软件并定义感兴趣区域（ROI），它包括从髋关节都后肢近侧到后爪向远侧的末端的区域。使用以下参数获得图像：80千伏，500微安和1300毫秒曝光，48微米的体素尺寸14,15 </li><li>通过绘制的投资回报率在每三个幻影室，并输入到相应的字段的平均密度校准图像菲尔德单位（HU）该软件。 </li><li>使用在软件的“抓取器”工具，重新定向的图像，从而使左后肢的胫骨是沿Z轴平行，以允许原位皮质骨结构和HO划定清亮的解剖图。 </li><li>在膝盖开始，通过图像切片远端滚动，直到何遇到。使用手动样条曲线工具，使周围的异位骨的投资回报，每5 个片通过爪或直到何已持续超越的远端。使用外推工具来扩展和缝合的投资回报率连成一片的投资回报率是包含了所有的议员。 </li><li>做一个3D的投资回报率，然后选择菜单分析。通过设定下和上阈值的最佳显示骨窗计算骨体积。使用相同的固定阈值的所有扫描。 </li></ol>

<ol>
	<li>Leblanc, E., 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=BMP-9-induced+muscle+heterotopic+ossification+requires+changes+to+the+skeletal+muscle+microenvironment.">BMP-9-induced muscle heterotopic ossification requires changes to the skeletal muscle microenvironment.</a> J Bone Miner Res. 26 (6), 1166-1177 (2011).</li><li>Shore, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoinductive+signals+and+heterotopic+ossification.">Osteoinductive signals and heterotopic ossification.</a> J Bone Miner Res. 26 (6), 1163-1165 (2011).</li><li>Wosczyna, M. N., Biswas, A. A., Cogswell, C. A., Goldhamer, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multipotent+progenitors+resident+in+the+skeletal+muscle+interstitium+exhibit+robust+BMP-dependent+osteogenic+activity+and+mediate+heterotopic+ossification.">Multipotent progenitors resident in the skeletal muscle interstitium exhibit robust BMP-dependent osteogenic activity and mediate heterotopic ossification.</a> J Bone Miner Res. 27 (5), 1004-1017 (2012).</li><li>Potter, B. K., 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=Heterotopic+ossification+following+combat-related+trauma.">Heterotopic ossification following combat-related trauma.</a> J Bone Joint Surg Am. 92, 74-89 (2010).</li><li>Van den Bossche, L., Vanderstraeten, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterotopic+ossification:+a+review.">Heterotopic ossification: a review.</a> J Rehabil Med. 37 (3), 129-136 (2005).</li><li>Chakkalakal, S. A., 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=An+Acvr1+R206H+knock-in+mouse+has+fibrodysplasia+ossificans+progressiva.">An Acvr1 R206H knock-in mouse has fibrodysplasia ossificans progressiva.</a> J Bone Miner Res. 27 (8), 1746-1756 (2012).</li><li>Yu, P. B., 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=BMP+type+I+receptor+inhibition+reduces+heterotopic+[corrected]+ossification.">BMP type I receptor inhibition reduces heterotopic [corrected] ossification.</a> Nat Med. 14 (12), 1363-1369 (2008).</li><li>Culbert, A. 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=Alk2+regulates+early+chondrogenic+fate+in+fibrodysplasia+ossificans+progressiva+heterotopic+endochondral+ossification.">Alk2 regulates early chondrogenic fate in fibrodysplasia ossificans progressiva heterotopic endochondral ossification.</a> Stem Cells. 32 (5), 1289-1300 (2014).</li><li>Dinther, 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=ALK2+R206H+mutation+linked+to+fibrodysplasia+ossificans+progressiva+confers+constitutive+activity+to+the+BMP+type+I+receptor+and+sensitizes+mesenchymal+cells+to+BMP-induced+osteoblast+differentiation+and+bone+formation.">ALK2 R206H mutation linked to fibrodysplasia ossificans progressiva confers constitutive activity to the BMP type I receptor and sensitizes mesenchymal cells to BMP-induced osteoblast differentiation and bone formation.</a> J Bone Miner Res. 25 (6), 1208-1215 (1359).</li><li>Peterson, J. 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=Burn+injury+enhances+bone+formation+in+heterotopic+ossification+model.">Burn injury enhances bone formation in heterotopic ossification model.</a> Ann Surg. 259 (5), 993-998 (2014).</li><li>Scott, M. A., 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=Brief+review+of+models+of+ectopic+bone+formation.">Brief review of models of ectopic bone formation.</a> Stem Cells Dev. 21 (5), 655-667 (2012).</li><li>Tannous, O., Griffith, C., O'Toole, R. V., Pellegrini, V. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterotopic+ossification+after+extremity+blast+amputation+in+a+Sprague-Dawley+rat+animal+model.">Heterotopic ossification after extremity blast amputation in a Sprague-Dawley rat animal model.</a> J Orthop Trauma. 25 (8), 506-510 (2011).</li><li>Tannous, O., 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=Heterotopic+bone+formation+about+the+hip+undergoes+endochondral+ossification:+a+rabbit+model.">Heterotopic bone formation about the hip undergoes endochondral ossification: a rabbit model.</a> Clin Orthop Relat Res. 471 (5), 1584-1592 (2013).</li><li>Peterson, J. 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=Treatment+of+heterotopic+ossification+through+remote+ATP+hydrolysis.">Treatment of heterotopic ossification through remote ATP hydrolysis.</a> Sci Transl Med. 6 (255), 255ra132 (2014).</li><li>Peterson, J. 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=Early+detection+of+burn+induced+heterotopic+ossification+using+transcutaneous+Raman+spectroscopy.">Early detection of burn induced heterotopic ossification using transcutaneous Raman spectroscopy.</a> Bone. 54 (1), 28-34 (2013).</li><li>Perosky, J. E., 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=Early+detection+of+heterotopic+ossification+using+near-infrared+optical+imaging+reveals+dynamic+turnover+and+progression+of+mineralization+following+Achilles+tenotomy+and+burn+injury.">Early detection of heterotopic ossification using near-infrared optical imaging reveals dynamic turnover and progression of mineralization following Achilles tenotomy and burn injury.</a> J Orthop Res. 32 (11), 1416-1423 (2014).</li><li>Peterson, J. 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=Effects+of+Aging+on+Osteogenic+Response+and+Heterotopic+Ossification+Following+Burn+Injury+in+Mice.">Effects of Aging on Osteogenic Response and Heterotopic Ossification Following Burn Injury in Mice.</a> Stem Cells Dev. , (2014).</li><li>Alfieri, K. A., Forsberg, J. A., Potter, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blast+injuries+and+heterotopic+ossification.">Blast injuries and heterotopic ossification.</a> Bone and Joint Research. 1 (8), 174-179 (2012).</li><li>Hunt, J. L., Arnoldo, B. D., Kowalske, K., Helm, P., Purdue, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterotopic+ossification+revisited:+a+21-year+surgical+experience.">Heterotopic ossification revisited: a 21-year surgical experience.</a> J Burn Care Res. 27 (4), 535-540 (2006).</li><li>Ring, D., Jupiter, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Operative+release+of+ankylosis+of+the+elbow+due+to+heterotopic+ossification.+Surgical+technique.">Operative release of ankylosis of the elbow due to heterotopic ossification. Surgical technique.</a> J Bone Joint Surg Am. 86-A, 2-10 (2004).</li><li>Crane, N. J., Polfer, E., Elster, E. A., Potter, B. K., Forsberg, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Raman+spectroscopic+analysis+of+combat-related+heterotopic+ossification.">Raman spectroscopic analysis of combat-related heterotopic ossification.</a> Bone. 57 (2), 335-342 (2013).</li></ol>

The authors have nothing to disclose

Heterotopic ossification represents a major functional impairment faced by patients that sustain trauma, burns, and invasive musculoskeletal procedures. The most at-risk population are soldiers in modern conflicts with major blast injuries from mechanisms such as improvised explosive devices (IED).18 Improved body armor and forward positioned medical units allows for improved survival of major extremity injury. After initial stabilization and repair of their extremity injury, these patients are at high risk...

Heterotopic ossification (HO) is the formation of ectopic bone in which osteo-potent cells are aberrantly induced to form endochondral bone outside of the skeleton. While the details of the HO formation pathway are still largely unknown, an accepted paradigm includes three key factors: an inflammatory inciting incident, a permissive niche, and mesenchymal stem cells capable of forming bone.1-3 HO is a common comorbidity complicating over 60% of major burn injuries, 65% of combat-related injuries, and 10% of invasive orthopaedic surgery cases.4,5 However, it is often difficult to predict where HO will form because it can occur at sites of local injury or at distant locations that may be otherwise uninjured. This variability in location makes it difficult to intervene prophylactically to prevent reactive bone formation in a locally targeted manner. There are also congenital forms of HO such as fibrodysplasia ossificans progressiva (FOP) in which patients are prone to the development of robust HO in response to minor trauma or inflammatory insult. Powerful animal models using transgenic mice have reproduced this phenotype and provided insight to the molecular pathways that may also be important in trauma induced HO.6-9 Translational research into the pathogenesis of non-congenital HO has used a wide variety of constructs ranging from injury alone to the implantation of exogenous osteo-inductive materials and/or cells.10-13
In our prior work we have validated a simple and reliable model of HO formation in mice which does not require the administration of any exogenous material.14-17 This model created two key conditions to initiate HO: local trauma and global inflammation. This was achieved through the use of an Achilles tenotomy (local trauma) combined with a distant burn injury (global inflammation). Mice received both treatments concurrently and were found to develop a robust amount of HO that could be analyzed by histologic, radiologic, and molecular means. Interestingly, concurrent burn injury significantly increased the amount of HO that formed and accelerated its developmental time-course.14-16 HO developed at predictable sites around the calcaneus, ankle joint, and tibia/fibula of the limb that received the tenotomy. The reliability of HO development at a known location allowed for focused examination of molecular and histologic features in the early stages of ectopic ossification.14,17 To date, 100% of mice (over 50 animals) with a tenotomy and concurrent burn injury have developed HO. Additionally, longitudinal 2D and 3D imaging and spectroscopic analysis were conducted to examine the growth pattern and biochemical make-up of HO.15,16

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>C57BL/6 mice</td>
			<td>Jackson Laboratory</td>
			<td align="right">664</td>
			<td>8-10 weeks old</td>
		</tr>
		<tr>
			<td>Isoflurane &#8211; Fluriso</td>
			<td>VET one, Boise, ID</td>
			<td>V1 501017</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine &#8211; Buprenex</td>
			<td>Reckitt Benckiser Healthcare</td>
			<td>NDC 12496-0757-1</td>
			<td>0.3 mg/ml solution</td>
		</tr>
		<tr>
			<td>Betadine</td>
			<td>Owens and Minor, Mechanicsville, VA</td>
			<td>2047PVP202</td>
			<td></td>
		</tr>
		<tr>
			<td>5-0 Vicryl sutures</td>
			<td>Ethicon, Summerville, NJ</td>
			<td>J493</td>
			<td></td>
		</tr>
		<tr>
			<td>Tegaderm Film, 6cm x 7cm</td>
			<td>3M</td>
			<td>1624W</td>
			<td>Cut in half to properly cover burn site</td>
		</tr>
		<tr>
			<td>&#181;CT - GE eXplore Locus SP</td>
			<td>GE Healthcare Pre-Clinical Imaging, London, ON, Canada</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microview 2.2 Advanced Bone Analysis Application</td>
			<td>GE Healthcare Pre-Clinical Imaging, London, ON, Canada</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

direct mouse trauma/burn model of heterotopic ossification

Heterotopic ossification (HO) is the formation of bone outside of the skeleton which forms following major trauma, burn injuries, and orthopaedic surgical procedures. The majority of animal models used to study HO rely on the application of exogenous substances, such as bone morphogenetic protein (BMP), exogenous cell constructs, or genetic mutations in BMP signaling. While these models are useful they do not accurately reproduce the inflammatory states that cause the majority of cases of HO. Here we describe a burn/tenotomy model in mice that reliably produces focused HO. This protocol involves creating a 30% total body surface area partial thickness contact burn on the dorsal skin as well as division of the Achilles tendon at its midpoint. Relying solely on traumatic injury to induce HO at a predictable location allows for time-course study of endochondral heterotopic bone formation from intrinsic physiologic processes and environment only. This method could prove instrumental in understanding the inflammatory and osteogenic pathways involved in trauma-induced HO. Furthermore, because HO develops in a predictable location and time-course in this model, it allows for research to improve early imaging strategies and treatment modalities to prevent HO formation.

在目前的研究中，该协议被描述为创伤此前公布的小鼠模型/刻录诱导HO 14-17这涉及创建并发局部肌肉骨骼损伤与跟腱切断术和全球炎症侮辱与部分厚度烧伤。这导致在切断术部位可遵循与串行成像可靠的形成反应骨。迄今为止，所有的小鼠（50个以上）已持续两个跟腱切断术和烧伤已经开发HO可测量的量在切断术部位。量化异位骨的形成，一个校准图像的μCT协议用于在每个期望的时间点。...

Watch this Scientific Journal Video about Direct Mouse Trauma/Burn Model of Heterotopic Ossification at JoVE.com

Direct Mouse Trauma/Burn Model of Heterotopic Ossification

An Achilles tenotomy and burn injury model of heterotopic ossification allows for the reliable study of trauma induced ectopic bone formation without the application of exogenous factors.

异位骨化直接鼠标创伤/烧伤模型

Heterotopic ossification (HO) is the formation of bone outside of the skeleton which forms following major trauma, burn injuries, and orthopaedic surgical ...

direct-mouse-traumaburn-model-of-heterotopic-ossification

Research

JoVE Journal

Medicine

9.9K 次观看.  University of Michigan Medical School. An Achilles tenotomy and burn injury model of heterotopic ossification allows for the reliable study of trauma induced ectopic bone formation without the application of exogenous factors.

视频: Direct Mouse Trauma/Burn Model of Heterotopic Ossification

A Modified Method for Heterotopic Mouse Heart Transplantion

Mice are often used as heart transplant donors and recipients in studies of transplant immunology due to the wide range of transgenic mice and reagents available. A difficulty is presented due to the small size of the animal and the considerable technical challenges of the microsurgery involved in heart transplantation. In particular, a high rate of technical failure early after transplantation may result from recipient death and post-operative complications such as hind limb paralysis or a non-beating heart. Here, the complete technique for heterotopic mouse heart transplantation is demonstrated, involving harvesting the donor heart and its subsequent implantation into a recipient mouse. The donor heart is harvested immediately following in situ perfusion with cold heparinized saline and transection of the ascending aorta and pulmonary artery. The recipient operation involves preparation of the abdominal aorta and inferior vena cava (IVC), followed by end-to-side anastomosis of the donor aorta with the recipient aorta using a single running 10-0 microsuture and a similar anastomosis of the donor pulmonary artery with the recipient IVC. Following the operation the animal is injected with 0.6 ml normal saline subcutaneously and allowed to recover on a 37&#160;&#176;C heating pad. The results from 227 mouse heart transplants are summarized with a success rate at 48 hr of 86.8%. Of the 13.2% failures within 48 hr, 5 (2.2%) experienced hind limb paralysis, 10 (4.4%) had a non-beating heart due to graft ischemic injury and/or thrombosis, while 15 (6.6%) died within 48 hr.

A technique is demonstrated for the microsurgical procedure for heterotopic transplantation of hearts in mice, including simplified methods for donor harvesting and recipient vessel anastomosis.

对于异位小鼠心脏移植技术的一种改进方法

Mice are often used as heart transplant donors and recipients in studies of transplant immunology due to the wide range of transgenic mice and reagents ...

科研

医学

Heterotopic Mucosal Engrafting Procedure for Direct Drug Delivery to the Brain in Mice

Delivery of therapeutics into the brain is impeded by the presence of the blood-brain barrier (BBB) which restricts the passage of polar and high molecular weight compounds from the bloodstream and into brain tissue. Some direct delivery success in humans has been achieved via implantation of transcranial catheters; however this method is highly invasive and associated with numerous complications. A less invasive alternative would be to dose the brain through a surgically implanted, semipermeable membrane such as the nasal mucosa that is used to repair skull base defects following endoscopic transnasal tumor removal surgery in humans. Drug transfer though this membrane would effectively bypass the BBB and diffuse directly into the brain and cerebrospinal fluid. Inspired by this approach, a surgical approach in mice was developed that uses a donor septal mucosal membrane engrafted over an extracranial surgical BBB defect. This model has been shown to effectively allow the passage of high molecular weight compounds into the brain. Since numerous drug candidates are incapable of crossing the BBB, this model is valuable for performing preclinical testing of novel therapies for neurological and psychiatric diseases.

A mouse model of human endoscopic skull base reconstruction has been developed that creates a semipermeable interface between the brain and nose using nasal mucosal grafts. This method allows researchers to study delivery to the central nervous system of high molecular weight therapeutics which are otherwise excluded by the blood-brain barrier when administered systemically.

异位黏膜移植程序直接给药到小鼠的大脑

Delivery of therapeutics into the brain is impeded by the presence of the blood-brain barrier (BBB) which restricts the passage of polar and high ...

Murine Heterotopic Heart Transplant Technique

It is now over forty years since this technique was first reported by Corry, Wynn and Russell. Although it took some years for other labs to become proficient in and utilize this technique, it is now widely used by many laboratories around the world. A significant refinement to the original technique was developed and reported in 2001 by Niimi. Described here are the techniques that have evolved over more than a decade in the hands of three surgeons (Plenter, Grazia, Pietra) in our center. These techniques are now being passed on to a younger generation of surgeons and researchers.
Based largely on the Niimi experience, the procedures used have evolved in the fine details - details which we will endeavor to relate here in such a way that others may be able to use this very useful model. Like Niimi, we have found that a video aid to learning is a priceless resource for the beginner.

The manuscript describes the steps required to perform the heterotopic heart transplant in the mouse.

小鼠异位心脏移植技术

It is now over forty years since this technique was first reported by Corry, Wynn and Russell. Although it took some years for other labs to become ...

Transplantation of Pulmonary Valve Using a Mouse Model of Heterotopic Heart Transplantation

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed results. However, the cellular and molecular mechanisms of the neotissue development, valve thickening, and stenosis development are not researched extensively. To answer the above questions, we developed a murine heterotopic heart valve transplantation model. A heart valve was harvested from a valve donor mouse and transplanted to a heart donor mouse. The heart with a new valve was transplanted heterotopically to a recipient mouse. The transplanted heart showed its own heartbeat, independent of the recipient&#8217;s heartbeat. The blood flow was quantified using a high frequency ultrasound system with a pulsed wave Doppler. The flow through the implanted pulmonary valve showed forward flow with minimal regurgitation and the peak flow was close to 100 mm/sec. This murine model of heart valve transplantation is highly versatile, so it can be modified and adapted to provide different hemodynamic environments and/or can be used with various transgenic mice to study neotissue development in a tissue engineered heart valve.

In order to understand the cellular and molecular mechanisms underlying neotissue formation and stenosis development in tissue engineered heart valves, a murine model of heterotopic heart valve transplantation was developed. A pulmonary heart valve was transplanted to recipient using the heterotopic heart transplantation technique.

肺动脉瓣使用异位心脏移植小鼠模型移植

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed ...

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 ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Modified Heterotopic Hindlimb Osteomyocutaneous Flap Model in the Rat for Translational Vascularized Composite Allotransplantation Research

Vascularized composite allotransplantation (VCA) is a relatively new field in the reconstructive surgery. Clinical achievements in human VCA include hand and face transplants and, more recently, abdominal wall, uterus, and urogenital transplants. Functional outcomes have exceeded initial expectations, and most recipients enjoy an improved quality of life. However, as clinical experience accumulates, chronic rejection and complications from the immunosuppression must be addressed. In many cases where grafts have failed, the causative pathology has been ischemic vasculopathy. <span style="font-size: 11pt; line-height: 115%; font-family: Arial, sans-serif; color: rgb(41, 43, 49); background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">The biological mechanisms of the acute and chronic rejection associated with VCA, especially ischemic vasculopathy, are important areas of research. However, due to the very small number of VCA patients, the evaluation of proposed mechanisms is better addressed in an experimental model. Multiple groups have used animal models to address some of the relevant unsolved questions in VCA rejection and vasculopathy. Several model designs involving a variety of species are described in the literature. Here we present a reproducible model of VCA heterotopic hindlimb osteomyocutaneous flap in the rat that can be utilized for translational VCA research. This model allows for the serial evaluation of the graft, including biopsies and different imaging modalities, while maintaining a low level of morbidity.

Vascularized composite allograft offers life-altering benefits to transplant recipients, but the biological causes of graft rejection and vasculopathy remain poorly understood. The rodent surgical model presented here offers a reproducible, clinically relevant model of transplantation, allowing researchers to evaluate rejection events and potential therapeutic strategies to prevent their occurrence.

改良异位异体下肢骨皮瓣模型在大鼠转化血管化复合同种异体移植研究中的应用

Vascularized composite allotransplantation (VCA) is a relatively new field in the reconstructive surgery. Clinical achievements in human VCA include hand ...

A Mouse Model of Vascularized Heterotopic Spleen Transplantation for Studying Spleen Cell Biology and Transplant Immunity

The spleen is a unique lymphoid organ that plays a critical role in the homeostasis of the immune and hematopoietic systems. Patients that have undergone splenectomy regardless of precipitating causes are prone to develop an overwhelming post-splenectomy infection and experience increased risks of deep venous thrombosis and malignancies. Recently, epidemiological studies indicated that splenectomy might be associated with the occurrence of cardiovascular diseases, suggesting that physiological functions of the spleen have not yet been fully recognized. Here, we introduce a mouse model of vascularized heterotopic spleen transplantation, which not only can be utilized to study the function and behavioral activity of splenic immune cell subsets in different biologic processes, but also can be a powerful tool to test the therapeutic potential of spleen transplantation in certain diseases. The main surgical steps of this model include donor spleen harvest, the removal of recipient native spleen, and spleen graft revascularization. Using congenic mouse strains (e.g., mice with CD45.1/CD45.2 backgrounds), we observed that after syngeneic transplantation, both donor-derived splenic lymphocytes and myeloid cells migrated out of the graft as early as post-operative day 1, concomitant with the influx of multiple types of recipient cells, thus generating a unique chimera. &#160;Despite relatively challenging techniques, this procedure can be performed with &#62;90% success rate. This model allows tracking the fate, longevity, and function of splenocytes during steady state and in a disease setting following a spleen transplantation, thereby offering a great opportunity to discover the distinct role for spleen-derived immune cells in different disease processes.

This protocol details the surgical steps of a mouse model of vascularized heterotopic spleen transplantation, a technically challenging model that can serve as a powerful tool in studying the fate and longevity of spleen cells, the mechanisms of distinct spleen cell populations in disease progression, and transplant immunity.

血管化异位脾移植小鼠模型在脾细胞生物学和移植免疫研究中的应用

The spleen is a unique lymphoid organ that plays a critical role in the homeostasis of the immune and hematopoietic systems. Patients that have undergone ...

A Simplified Model for Heterotopic Heart Valve Transplantation in Rodents

There is an urgent clinical need for heart valve replacements that can grow in children. Heart valve transplantation is proposed as a new type of transplant with the potential to deliver durable heart valves capable of somatic growth with no requirement for anticoagulation. However, the immunobiology of heart valve transplants remains unexplored, highlighting the need for animal models to study this new type of transplant. Previous rat models for heterotopic aortic valve transplantation into the abdominal aorta have been described, though they are technically challenging and costly. For addressing this challenge, a renal subcapsular transplant model was developed in rodents as a practical and more straightforward method for studying heart valve transplant immunobiology. In this model, a single aortic valve leaflet is harvested and inserted into the renal subcapsular space. The kidney is easily accessible, and the transplanted tissue is securely contained in a subcapsular space that is well vascularized and can accommodate a variety of tissue sizes. Furthermore, because a single rat can provide three donor aortic leaflets and a single kidney can provide multiple sites for transplanted tissue, fewer rats are required for a given study. Here, the transplantation technique is described, providing a significant step forward in studying the transplant immunology of heart valve transplantation.

This protocol describes a simple and efficient method for the transplantation of aortic valve leaflets under the renal capsule to allow for the study of alloreactivity of heart valves.

啮齿动物异位心脏瓣膜移植的简化模型

There is an urgent clinical need for heart valve replacements that can grow in children. Heart valve transplantation is proposed as a new type of ...

A Modified Cuff Technique for Mouse Cervical Heterotopic Heart Transplantation Model

Cardiac allograft rejection limits the long-term survival of patients after heart transplantation. A mouse heart transplantation model is ideal for investigating the mechanism of cardiac allograft rejection in preclinical studies because of their high homology with human genes. This understanding would help develop unique approaches to improving patients&#39; long-term survival treated with cardiac allografts. In a mouse model, abdominal donor heart implantation is commonly performed with an end-to-end&#160;anastomosis to the recipient&#39;s aorta and inferior vena cava using stitches. In this model, the donor&#39;s heart is implanted by end-to-end anastomosis to the recipient&#39;s carotid artery and jugular vein by the modified-Cuff technique. The transplantation surgery is performed without stitching and thus may increase the survival of the recipient since there is no interference with the blood supply and venous reflux of the lower body. This mouse model would help investigate the mechanisms underlying the immunological and pathological (acute/chronic) rejection of cardiac allografts.

In the present protocol, a mouse heart transplantation model is used for investigating the mechanism of cardiac allograft rejection. In this heterotopic heart transplantation model, operation efficiency is improved, and the survival of cardiac grafts is ensured by a cervical end-to-end anastomosis of heart implantation using a modified Cuff technique.

一种改进的袖带技术用于小鼠颈椎异位心脏移植模型

Cardiac allograft rejection limits the long-term survival of patients after heart transplantation. A mouse heart transplantation model is ideal for ...