作者要感谢FORTE项目，由托斯卡纳大区（POR CREO FESR 2007-2013年，坂东的Unico R＆S 2012）资助，欧盟FP7 ECHORD ++的实验，部分支持的科研活动LA-玫瑰，和FP7 BiophotonicPlus工程“LITE”授托斯卡纳地区。

这项研究是与前瞻性审批医院的伦理委员会进行的;获得知情同意书。这项研究是在遵守赫尔辛基宣言的原则。 1.捐赠内皮准备<ol><li>使用角膜供体为在室温下编写的当地眼库。 </li><li>房中术，拉出捐献角膜从其输出容器分开设置的组织保存和营养是用于角膜运输解决方案。 </li><li>将供体角巩膜缘在一个人工前房，在其中一个可流动液体，并创建一个变量压力;盖与所述组织保持头部角膜。 </li><li>通过3路连接器连接的人工前房（AAC）来填充角膜保存和营养液的注射器。 </li><li>使用的压缩环，以牢固地保持在保持器中的地方。 </li><li>使用SYR英格连接到AAC维持前房压力：补前房与组织保存溶液，直到达到最优化的压力（见1.7）。关闭连接器。 </li><li>测试前房用手指内的压力。改变内压，直到达到（范围在60-70毫米汞柱）的右边的内部压力。关闭三路连接器。 </li><li>测量捐献角膜光学相干Thomography（OCT）的厚度。维持前房与角膜在文书光学前方的固定位置，然后取一个完整的厚度-OCT获取。 </li></ol>捐助内皮2.飞秒激光制备<ol><li>飞秒激光设备下进行捐献角膜压平的。使用激光femtosec切供体组织三种随后削减：后侧切，全板层和前侧切口。设置了三个切口以下参数。<ol><li>对于全片层切（光栅模式，开始时），设置相应的供体角膜（如1.8所述，OCT测量）的最薄点，始终减去95微米的切削深度。在范围0.8-0.9μJ（取决于工作深度）设定脉冲能量。在直径为8.7毫米。设置的切向点分离到2微米。设置径向点分离为2微米。 </li><li>对于前侧切口，设置后的深度比以前的全层状切割30微米深。设置脉冲能量至2.10μJ;直径8.6毫米;当场分离到3微米，而层分离到3微米。 </li><li>对于后侧切口，找到后侧的前深度削减30微米前方比全切薄片状。设置后深度900微米。设置脉冲能量至2.10μJ;直径为8.3毫米;当场分离到2微米;层分离到2微米。 </li></ol></l我&gt; </ol> 3.收件人准备眼<ol><li>准备病人进行手术。做一个4.00毫米角膜切口在12点有4.0毫米预校准刀片。做一个角膜缘穿刺术与1.2mm的预校准刀，放置2点。再拍一张缘穿刺术在6点钟位置有一个30°刺一刀。 </li><li>插入前房维持在患者的前房，通过2点钟探取。 </li><li>用专用钩子执行一个8.2毫米直径的圆形descemetorexis。 </li><li>剥开后弹力膜和内皮细胞的间质后，用弹力钩取出组织。 </li></ol> 4.准备生色<ol><li>把1毫克吲哚菁绿粉在1.5 ml离心管。 </li><li>加无菌水9毫克（ICG的水溶液是10％w / w的）。 </li><li>手动混合ICG粉末和水与金属搅拌器。 </l我&gt; </ol> 5.染色捐助内皮<ol><li>把供体内皮到BUSIN注射器的较宽部分，并在与喷射面接触的内侧。 </li><li>染色施主内皮与发色团溶液的内侧，在其周边部，用刮铲。的正确染色的组织具有均匀浅绿色。等待3分钟步开始之前5.3。 </li><li>拉动BUSIN注射器的前部的供体内皮，轧制。 </li></ol> 6.插入捐助内皮<ol><li>抓住和用无创钳同轴内皮微透镜插入折叠捐助内皮。 </li><li>取出前房维持。 </li><li>缝合角膜切口及穿刺术（步骤执行3.1）与尼龙单10.0施蒂希。 </li><li>注入气泡展开供体组织和其按压在受体角膜。气泡必须COMPletely填充前房空间。 </li></ol> 7.激光焊接<ol><li>将供体微透镜在接受者角膜的中心，从用钩子或移动的气泡从角膜的外部用刮刀内。 </li><li>使用二极管激光器发射在810纳米，配备有一个300微米的纤芯直径无菌光纤，以0.22作为数值孔径（NA）。 </li><li>保持眼球外的纤维笔尖，并提供激光朝向染内皮，透过透明的角膜组织。光纤头是在非接触式的配置。 </li><li>使用以下设置激光：单点发射模式，70-80毫秒脉冲持续时间，每个脉冲35-40兆焦耳。氦氖瞄准横梁上。 </li><li>提供单一的激光光斑在捐助内皮的外围染色。交付点顺序：最后的方面是在供体微透镜的周围斑点环。 2 ADJA之间的距离分点中心为点的直径的两倍。 </li><li>在患者眼睛应用一个接触透镜，连同0.3％妥布霉素和0.1％地塞米松的眼用悬浮液。 </li></ol>

<ol>
	<li>Tan, D. T., Dart, J. K., Holland, E. J., Kinoshita, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+transplantation.">Corneal transplantation.</a> Lancet. 379 (9827), 1749-1761 (2012).</li><li>El Husseiny, M. A., Manero, F., Gris, O., Elies, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Historical+Review+and+Update+of+Surgical+Treatment+for+Corneal+Endothelial+Diseases.">Historical Review and Update of Surgical Treatment for Corneal Endothelial Diseases.</a> Ophthalmol Ther. , (2014).</li><li>Rossi, F., 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=Laser+tissue+welding+in+ophthalmic+surgery.">Laser tissue welding in ophthalmic surgery.</a> J Biophotonics. 1 (4), 331-342 (2008).</li><li>Pini, 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=Combining+femtosecond+laser+ablation+and+diode+laser+welding+in+lamellar+and+endothelial+corneal+transplants.">Combining femtosecond laser ablation and diode laser welding in lamellar and endothelial corneal transplants.</a> Proc. SPIE Ophthalmic Technologies XVIII. 6 (844), 684411-1-684411-7 (2008).</li><li>Rossi, F., 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=All-laser'+endothelial+corneal+transplant+in+human+patients.">All-laser' endothelial corneal transplant in human patients.</a> Proc. SPIE Ophthalmic Technologies XXII. 8209, 82091O-1-82091O-3 (2012).</li><li>Rossi, F., Pini, R., Menabuoni, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+and+model+analysis+on+the+temperature+dynamics+during+diode+laser+welding+of+the+cornea.">Experimental and model analysis on the temperature dynamics during diode laser welding of the cornea.</a> J Biomed Opt. 12 (1), 014031-1-014031-7 (2007).</li><li>Menabuoni, 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=Laser-assisted+corneal+welding+in+cataract+surgery:+retrospective+study.">Laser-assisted corneal welding in cataract surgery: retrospective study.</a> J Cataract Refract Surg. 33 (9), 1608-1612 (2007).</li><li>Buzzonetti, 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=Laser+Welding+in+Penetrating+Keratoplasty+and+Cataract+Surgery+in+Pediatric+Patients.+Early+Results.">Laser Welding in Penetrating Keratoplasty and Cataract Surgery in Pediatric Patients. Early Results.</a> J Cataract Refract Surg. 39 (12), 1829-1834 (2013).</li><li>Menabuoni, 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=The+'anvil'+profile+in+femtosecond+laser-assistedpenetrating+keratoplasty.">The 'anvil' profile in femtosecond laser-assistedpenetrating keratoplasty.</a> Acta Ophthalmol. 91 (6), e494-e495 (2013).</li><li>Canovetti, 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=Laser-assisted+penetrating+keratoplasty:+one+year&#8217;s+results+in+patients,+using+a+laser-welded+&#8220;anvil&#8221;-profiled+graft.">Laser-assisted penetrating keratoplasty: one year&#8217;s results in patients, using a laser-welded &#8220;anvil&#8221;-profiled graft.</a> Am J Ophthalmol. 158 (4), 664-670 (2014).</li></ol>

The authors declare that they have no competing financial interests.

“全激光”内皮移植是一种原始的方法微创角膜移植。 所有在协议中所描述的程序进行，在手术室内进行，观察卫生和消毒过程是在手术常见的做法，如用无菌手套，长袍，面罩和帽。 ICG的溶液的制备在手术室，前不久其在染色施主内皮应用。 ICG的粉末，水和所有用于制备染色溶液的工具是无菌的，并且可商购的用于在人受试者中的使用。激光光纤灭菌并它被安装在一特?...

在这项工作中，我们提出了一种原始的方法角膜内皮移植术的基础上，使用飞秒激光，以制备供体组织和近红外二极管激光器将其焊接到收件人床。供体角膜术测量要正确设计供体组织尺寸。角膜内皮移植术已经提出在最近几年，以取代穿透性角膜移植术治疗内皮疾病1,2。该技术的主要优点是视力恢复快，相对于外科手术，移植物排斥的风险降低和眼完整性的保存期间穿透性角膜移植术，减少麻醉。的主要危险因素是术后施主微透镜错位。的标准技术是通过将供体的内皮在其最终位置，它是由喷射的气泡的保持进行：无缝合的使用，因为在机械，生物物理和尺寸恰拉的内皮cteristics。此外，视力恢复可以供体和受体组织之间的不匹配的，主要是因为由于一个厚的组织移植的限制。 在这里，我们提出在执行角膜内皮移植术，可以克服这些主要问题的过程。施主内皮可通过使用激光焊接技术来保证在其最终位置。这是一个控制和局部光热过程：它可诱发在捐献者/接受者接口。据研究，在过去十年，在穿透性角膜移植术和血管内皮3-5的移植方案。近红外光（波长：810纳米）通过低功率二极管激光器射出被传递向在伤口部位的生物组织。角膜是自然透明的该波长：为了使此组织吸收的激光光，有必要用一个生色留下污点。所提出的染料是一种无菌饱和吲哚菁绿（ICG）D水溶液。我们表明，当角膜组织的正常沾上这个ICG制剂，它显示了在810纳米6的吸收峰。此外，ICG被广泛用于临床诊断和其安全性已被证实在人受试者。染色角膜吸收二极管激光能量和主要产生的效果是一种受控的温度上升在焊接部位。无热效应引起的未染色的组织。温度增强诱导所述基质胶原可逆的热变性，与伤口壁的冷却时立即closuring。这种激光焊接效果首先表现在白内障手术7,8和穿透性角膜移植9,10。我们展现本文优化的方法已经研究了在角膜内皮移植术的应用。 在建议手术治疗，单个激光点（持续几毫秒）ARê输送到组织，导致点尺寸（几百微米的直径）内的局部的光热效应：诱导效应是硬激光​​焊接，由密闭在捐献者/主机接口胶原的光凝。胶原变性在焊接现场的结果是供体和宿主组织之间有很强的粘合性，从而提供了一种缝合效果是不可能获得使用标准技术（针）。所述组织恢复他自然外观在很短的跟进（1个月）和供体/宿主组织之间的粘合力是通过在愈合阶段的非常早的阶段提供的焊接改善。 为了避免角膜内皮移植术的另一主风险， 即厚的供体组织的移植，intrasurgical光学相干断层扫描（OCT）用于：商用装置测量供体角膜的厚度，以使得正确的切割形状可设计成具有该femtosec激光。所提出的“全激光”内皮移植这样看来，以改善这种微创手术的临床效果。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Indocyanine Green</td>
			<td>Pulsion Medical Systems, Germany</td>
			<td>ICG-PULSION (http://www.pulsion.com/international-english/perfusion/icg-pulsion/)</td>
			<td>Alternative product: IC-GREEN, Akorn&#160; Inc., Lake Forest, Illinois- US (http://www.icginjection.com/)</td>
		</tr>
		<tr>
			<td>Femtosecond Laser&#160;</td>
			<td>Abbott Medical Optics,&#160; Abbott Laboratories Inc. Abbott Park, Illinois, USA</td>
			<td>iFS150 (http://www.abbottmedicaloptics.com/products/refractive/ilasik/ifs-advanced-femtosecond-laser)</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical Coherence Tomography (OCT)</td>
			<td>Carl-Zeiss Meditec, Dublin, California- US (http://www.zeiss.com/meditec/en_de/home.html)</td>
			<td>Visante</td>
			<td></td>
		</tr>
		<tr>
			<td>Diode Laser</td>
			<td>E.l.En. Group s.pa., Calenzano-FI, Italy (http://www.elengroup.com/en/divisions/medical)</td>
			<td>Mod. WELD 800</td>
			<td></td>
		</tr>
		<tr>
			<td>Artificial Anterior Chamber</td>
			<td>CORONET, corneal graft products. Network Medical Products Ltd. Coronet House, Kearsley Road, Ripon, North Yorkshire, HG4 2SG, UK</td>
			<td>&#160;Artificial Anterior Chamber (A.A.C.) with large and small tissue-retaining heads. Code 51-935 (http://www.networkmedical.co.uk/ophthalmic_artificial_anterior_chamber.html)</td>
			<td></td>
		</tr>
		<tr>
			<td>Solution for tissue preservation and nutrition&#160;&#160;</td>
			<td>&#160;AL.CHI.MI.A. Srl, Viale Austria 14, 35020 - Ponte S. Nicol&#242; - PD 
			&#160;ITALY</td>
			<td>Carry-C media for corneal deturgescence and transport at room temperature - 12 x 50 ml (http://www.alchimiasrl.com/en/organ-culture-at-31&#176;-c-eb/carry-c-eb)</td>
			<td></td>
		</tr>
	</tbody>
</table>

an &#8220;all-laser&#8221; endothelial transplant

The &#8220;all laser&#8221; assisted endothelial keratoplasty is a procedure that is performed with a femtosecond laser used to cut the donor tissue at an intended depth, and a near infrared diode laser to weld the corneal tissue. The proposed technique enables to reach the three main goals in endothelial keratoplasty: a precise control in the thickness of the donor tissue; its easy insertion in the recipient bed and a reduced risk of donor lenticule dislocation. The donor cornea thickness is measured in the surgery room with optical coherence tomography (OCT), in order to correctly design the donor tissue dimensions. A femtosecond laser is used to cut the donor cornea. The recipient eye is prepared by manual stripping of the descemetic membrane. The donor endothelium is inserted into a Busin-injector, the peripheral inner side is stained with a proper chromophore (a water solution of Indocyanine Green) and then it is pulled in the anterior chamber. The transplanted tissue is placed in the final and correct location and then diode laser welding is induced from outside the eyeball. The procedure has been performed on more than 15 patients evidencing an improvement in surgery performances, with a good recovery of visual acuity and a reduced donor lenticule dislocation event.

“全激光”手术建议进行微创角膜移植。该过程容易进行（参见图1）：相对于标准内皮移植测量角膜厚度，染色的供体组织，并提供激光被添加的唯一步骤。所取得的优势很大程度上弥补了几分钟增加手术时间。使用术OCT的测量供体角膜厚度和用于定制供体微透镜尺寸的飞秒激光使供血者/主机接口粘结的提高（参见图2）。在这样做时，手术的目的是以下的需求和单个?...

Watch this Scientific Journal Video about An â€œAll-laserâ€ Endothelial Transplant at JoVE.com

An &amp;#8220;All-laser&amp;#8221; Endothelial Transplant

An all laser procedure is proposed in the endothelial transplant. The surgical technique is based on the use of a femtosecond laser to prepare the donor tissue. Laser welding technique is then used to secure the donor endothelium in the correct position.

“全激光”内皮移植

The &#8220;all laser&#8221; assisted endothelial keratoplasty is a procedure that is performed with a femtosecond laser used to cut the donor tissue at an ...

Research

JoVE Journal

Medicine

An &#8220;All-laser&#8221; Endothelial Transplant

9.3K 次观看.  Italian National Research Council. An all laser procedure is proposed in the endothelial transplant. The surgical technique is based on the use of a femtosecond laser to prepare the donor tissue. Laser welding technique is then used to secure the donor endothelium in the correct position.

视频: An &#8220;All-laser&#8221; Endothelial Transplant

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

采用实时定量PCR检测供体细胞植入一个具有竞争力的小鼠骨髓移植模型

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

科研

医学

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 ...

Technique of Porcine Liver Procurement and Orthotopic Transplantation using an Active Porto-Caval Shunt

The success of liver transplantation has resulted in a dramatic organ shortage. Each year, a considerable number of patients on the liver transplantation waiting list die without receiving an organ transplant or are delisted due to disease progression. Even after a successful transplantation, rejection and side effects of immunosuppression remain major concerns for graft survival and patient morbidity. 
Experimental animal research has been essential to the success of liver transplantation and still plays a pivotal role in the development of clinical transplantation practice. In particular, the porcine orthotopic liver transplantation model (OLTx) is optimal for clinically oriented research for its close resemblance to human size, anatomy, and physiology. 
Decompression of intestinal congestion during the anhepatic phase of porcine OLTx is important to guarantee reliable animal survival. The use of an active porto-caval-jugular shunt achieves excellent intestinal decompression. The system can be used for short-term as well as long-term survival experiments. The following protocol contains all technical information for a stable and reproducible liver transplantation model in pigs including post-operative animal care.

Experimental animal research plays a pivotal role in the development of clinical transplantation practice. The porcine orthotopic liver transplantation model (OLTx) closely resembles human conditions and is frequently used in clinically oriented research. The following protocol contains all information for a reliable porcine OLTx model using an active porto-caval-jugular shunt.

使用Active波尔图腔静脉分流术猪肝脏采购和原位移植技术

The success of liver transplantation has resulted in a dramatic organ shortage. Each year, a considerable number of patients on the liver transplantation ...

Mouse Model of Alloimmune-induced Vascular Rejection and Transplant Arteriosclerosis

Vascular rejection that leads to transplant arteriosclerosis (TA) is the leading representation of chronic heart transplant failure. In TA, the immune system of the recipient causes damage of the arterial wall and dysfunction of endothelial cells and smooth muscle cells. This triggers a pathological repair response that is characterized by intimal thickening and luminal occlusion. Understanding the mechanisms by which the immune system causes vasculature rejection and TA may inform the development of novel ways to manage graft failure. Here, we describe a mouse aortic interposition model that can be used to study the pathogenic mechanisms of vascular rejection and TA. The model involves grafting of an aortic segment from a donor animal into an allogeneic recipient. Rejection of the artery segment involves alloimmune reactions and results in arterial changes that resemble vascular rejection. The basic technical approach we describe can be used with different mouse strains and targeted interventions to answer specific questions related to vascular rejection and TA.

We describe a protocol for aortic interposition grafting in mice. The goal of the protocol is to provide a model with which to study pathological processes and therapeutic strategies relevant to alloimmune reactions in arteries and the resultant arterial changes that contribute to organ transplant failure.

的同种免疫引起的血管性排斥反应和移植物动脉硬化小鼠模型

Vascular rejection that leads to transplant arteriosclerosis (TA) is the leading representation of chronic heart transplant failure. In TA, the immune ...

Murine Kidney Transplant Technique

The first mouse kidney transplant technique was published in 19731 by the Russell laboratory. 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 using the donor aorta to form the arterial anastomosis instead of the renal artery was developed and reported in 1993 by Kalina and Mottram 2 with a further advancement coming from the same laboratory in 1999 3. While one can become proficient in this model, a search of the literature reveals that many labs still experience a high proportion of graft loss due to arterial thrombosis. We describe here a technique that was devised in our laboratory that vastly reduces the arterial thrombus reported by others 4,5. This is achieved by forming a heel-and-toe cuff of the donor infra-renal aorta that facilitates a larger anastomosis and straighter blood flow into the kidney.

The goal of this manuscript is to describe the steps required to perform a kidney transplant in a mouse, paying particular attention to the details of the arterial anastomosis.

小鼠肾脏移植技术

The first mouse kidney transplant technique was published in 19731 by the Russell laboratory. Although it took some years for other labs to become ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

一种基于猪脑内皮细胞的体外血脑屏障模型的改进方法

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

Optimized Griess Reaction for UV-Vis and Naked-eye Determination of Anti-malarial Primaquine

Primaquine (PMQ), an important anti-malarial drug, has been recommended by the World Health Organization (WHO) for the treatment of life-threatening infections caused by P. vivax and ovale. However, PMQ has unwanted adverse effects that lead&#160;to acute hemolysis in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency. There is a need to develop simple and reliable methods for PMQ determination with the purpose of dosage monitoring. In early 2019, we have reported an UV-Vis and naked-eye based approach for PMQ colorimetric quantification. The detection was based on a Griess-like reaction between PMQ and anilines, which can generate colored azo products. The detection limit for direct measurement of PMQ in synthetic urine is in the nanomolar range. Moreover, this method has shown great potential for PMQ quantification from human serum samples at clinically relevant concentrations. In this protocol, we will describe the technical details regarding the syntheses and characterization of colored azo products, the reagent preparation, and the procedures for PMQ determination.

This protocol describes a novel colorimetric method for antimalarial primaquine (PMQ) detection in synthetic urines and human serums.

抗疟药的紫外线-Vis和裸眼测定优化的格里斯反应

Primaquine (PMQ), an important anti-malarial drug, has been recommended by the World Health Organization (WHO) for the treatment of life-threatening ...

Organ Ischemia-Reperfusion Injury by Simulating Hemodynamic Changes in Rat Liver Transplant Model

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, including ischemia-reperfusion injury (IRI) of extrahepatic organs. This model requires numerous experiments and devices. The duration of anhepatic phase is closely related to the time to develop IRI after transplantation. In this experiment, we used hemodynamic changes to induce extrahepatic organ damage in rats and determined the maximum tolerance time. The time until the most severe organ injury varied for different organs. This method can easily be replicated and can also be used to study IRI of the extrahepatic organs after liver transplantation.

This paper provides a detailed description of how to build an animal model of the anhepatic phase (liver ischemia) in rats to facilitate basic research into ischemia-reperfusion injury after liver transplantation.

器官缺血-输液损伤模拟大鼠肝脏移植模型的血动力学变化

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, ...

TBase - an Integrated Electronic Health Record and Research Database for Kidney Transplant Recipients

TBase is an electronic health record (EHR) for kidney transplant recipients (KTR) combining automated data entry of key clinical data (e.g., laboratory values, medical reports, radiology and pathology data) via standardized interfaces with manual data entry during routine treatment (e.g., clinical notes, medication list, and transplantation data). By this means, a comprehensive database for KTR is created with benefits for routine clinical care and research. It enables both easy everyday clinical use and quick access for research questions with highest data quality. This is achieved by the concept of data validation in clinical routine in which clinical users and patients have to rely on correct data for treatment and medication plans and thereby validate and correct the clinical data in their daily practice. This EHR is tailored for the needs of transplant outpatient care and proved its clinical utility for more than 20 years at Charit&#233; - Universit&#228;tsmedizin Berlin. It facilitates efficient routine work with well-structured, comprehensive long-term data and allows their easy use for clinical research. To this point, its functionality covers automated transmission of routine data via standardized interfaces from different hospital information systems, availability of transplant-specific data, a medication list with an integrated check for drug-drug interactions, and semi-automated generation of medical reports among others. Key elements of the latest reengineering are a robust privacy-by-design concept, modularity, and hence portability into other clinical contexts as well as usability and platform independence enabled by HTML5 (Hypertext Markup Language) based responsive web design. This allows fast and easy scalability into other disease areas and other university hospitals. The comprehensive long-term datasets are the basis for the investigation of Machine Learning algorithms, and the modular structure allows to rapidly implement these into clinical care. Patient reported data and telemedicine services are integrated into TBase in order to meet future needs of the patients. These novel features aim to improve clinical care as well as to create new research options and therapeutic interventions.

TBase combines an electronic health record with an innovative research database for kidney transplant recipients. TBase is built upon an in-memory database platform, connected to different hospital systems, and used for regular outpatient care. It automatically integrates all relevant clinical data including transplantation-specific data creating a unique research database.

TBase - 肾移植受者的综合电子健康记录和研究数据库

TBase is an electronic health record (EHR) for kidney transplant recipients (KTR) combining automated data entry of key clinical data (e.g., laboratory ...

Endothelial Cell Transcytosis Assay as an In Vitro Model to Evaluate Inner Blood-Retinal Barrier Permeability

Dysfunction of the blood-retinal barrier (BRB) contributes to the pathophysiology of several vascular eye diseases, often resulting in retinal edema and subsequent vision loss. The inner blood-retinal barrier (iBRB) is mainly composed of retinal vascular endothelium with low permeability under physiological conditions. This feature of low permeability is tightly regulated and maintained by low rates of paracellular transport between adjacent retinal microvascular endothelial cells, as well as transcellular transport (transcytosis) through them. The assessment of retinal transcellular barrier permeability may provide fundamental insights into iBRB integrity in health and disease. In this study, we describe an endothelial cell (EC) transcytosis assay, as an in vitro model for evaluating iBRB permeability, using human retinal microvascular endothelial cells (HRMECs). This assay assesses the ability of HRMECs to transport transferrin and horseradish peroxidase (HRP) in receptor- and caveolae-mediated transcellular transport processes, respectively. Fully confluent HRMECs cultured on porous membrane were incubated with fluorescent-tagged transferrin (clathrin-dependent transcytosis) or HRP (caveolae-mediated transcytosis) to measure the levels of transferrin or HRP transferred to the bottom chamber, indicative of transcytosis levels across the EC monolayer. Wnt signaling, a known pathway regulating iBRB, was modulated to demonstrate the caveolae-mediated HRP-based transcytosis assay method. The EC transcytosis assay described here may provide a useful tool for investigating the molecular regulators of EC permeability and iBRB integrity in vascular pathologies and for screening drug delivery systems.

Endothelial Cell Transcytosis Assay as an In Vitro Model to Evaluate Inner Blood-Retinal Barrier Permeability

This protocol illustrates an in vitro endothelial cell transcytosis assay as a model to evaluate inner blood-retinal barrier permeability by measuring the ability of human retinal microvascular endothelial cells to transport horseradish peroxidase across cells in caveolae-mediated transcellular transport processes.

内皮细胞转吞测定作为 体外 模型评估内部血-视网膜屏障通透性

Dysfunction of the blood-retinal barrier (BRB) contributes to the pathophysiology of several vascular eye diseases, often resulting in retinal edema and ...