This work was supported in part by the United States Department of Veterans Affairs Biomedical Laboratory Research and Development Program Merit Review Award I01-BX002336, the National Institute of Health grant R56-HL095498, as well as with the resources and the use of facilities at the VA Connecticut Healthcare System, West Haven, CT.

获得批准相应的机构动物护理和使用委员会。 1.麻醉和手术前的程序<ol><li>麻醉雄性C57BL / 6冰，8周龄，用汽化3％异氟醚和0.8升/分钟的氧气给予到丙烯酸感应腔室。 </li><li>确认缺乏反应脚趾捏足够的麻醉。通过连续吸入3％异氟烷 - 定位在动作表鼠标仰卧和定位硅氧烷掩模递送汽化2。 </li><li>腹部用化学脱毛霜去除颈部腹侧头发更低。 </li><li>之前AVF手术进行多普勒超声检查在感兴趣的领域，以记录动脉和静脉流动和血管直径的基线特征。1,2 </li><li>附加1ml注射器向地下25针和与所需药物加载注射器。弯针60度角约4从需要毫米乐一角。抓住一个弯针架在地下25针。 </li></ol> 2.手术过程<ol><li>准备切口部位有局部消毒剂和应用手术单。使用无菌手套和工具，以在整个手术保持无菌操作。 </li><li>使一个中线腹部切口用解剖刀从下肝脏边缘水平延伸到刚好耻骨之上。 </li><li>插入的牵开器以及从向右侧腹腔剔骨所有肠子。裹肠纱布用盐水浸湿。解剖连接复古腹膜和较低的结肠获得主动脉和下腔静脉的全视角膜。 </li><li>从周围组织解剖肾下主动脉和下腔静脉，对近端和远端夹紧准备。 </li><li>放置在这两个近端主动脉和在刚刚左肾静脉水平以下近端IVC单个显微剪辑。将第二显微剪辑跨两个为Distal主动脉和远侧IVC。 </li><li>把握结缔组织围绕主动脉和内侧旋转，使得主动脉的背表面稍微露出的动脉穿刺，如先前所述。1 </li><li>很快暴露穿刺部位。穿刺部位将在血管，从左肾静脉到主动脉分叉的距离的大约四分之三的尾方面。保持主动脉在一个旋转位置用左手，解剖主动脉的左侧缘，以便有充足的曝光，以允许用右手穿刺。要小心，不要主动脉和下腔静脉之间的解剖。 </li><li>保持在一个旋转位置的主动脉，并使用地下25针带有附加1ml注射器含有药物溶液通过刺穿主动脉进入下腔静脉。 （ 图1A） </li><li>注入药液 - 利用左手（100 200微升）。针可以通过扩张和薄可见下腔静脉壁当透明药液置换静脉血出下腔静脉（ 图1B，C）的。保持静止并保持在适当的位置的针15分钟。 </li><li>移除远侧显微剪辑脱夹具仅远端主动脉和远侧IVC。 </li><li>取出针再涂向上拉相邻复古腹膜组织主动脉的穿刺部位。 </li><li>取出近端显微剪辑去夹紧近端主动脉和近端IVC。在去夹紧，动脉血观察流入下腔静脉，而不是深静脉血流。保持覆盖所述穿刺孔进行1分钟。 </li><li>止血确认通过观察30秒，而没有压缩后，返回肠子到其自然的位置，并根据您批准的动物协议的连续缝合关闭的腹部。 </li></ol> 3.手术后的程序<ol><li>腹部闭合后，停止ANESThesia。应用术后护理包括镇痛和伤口护理按照机构动物护理和使用委员会建议的说明。镇痛我们使用丁丙诺啡在0.1毫克/千克intrasmuscularly以下的外科手术24小时，每12小时。 </li><li>在手术后的第一天，进行多普勒超声检查，以确认AVF通畅。此外，衡量其他船只和串行流量特性，比较从术前基线值的变化。1,2 </li></ol><img alt="图1" src="/files/ftp_upload/53905/53905fig1.jpg" /> 图1（a）执行照片AVF手术中显示管腔内交货。夹紧近端和远端主动脉，以及通过施加显微剪辑IVC。利用地下25针含附加注射器药液通过穿刺进入主动脉下腔静脉。（B）几点措施她电源图像输液前刺破下腔静脉（放大4倍）。针尖通过深色静脉血遮蔽。黄色箭头表示输液后刺破下腔静脉的墙到墙直径IVC。（C）更高的功率图（放大4倍）的。针尖（黑色箭头），可通过轻轻扩张和薄墙下腔静脉（黄色箭头）作为透明药材溶液中可以看出置换静脉血。

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The authors have nothing to disclose.

鼠AVF模型的这种修改包含在AVF创建时腔内药物递送到静脉内皮。一个AVF通过穿刺用地下25针的红外肾主动脉和延伸穿过相对的主动脉壁进入下腔静脉穿刺，随后的药物溶液注射通过同一针创建。该解决方案是维持内静脉， 即 ，在AVF的静脉肢体，直到去夹紧。什么区别于其他鼠类AVF模型17-19这种模式是缺乏缝合或胶水可能导致管腔狭窄，急性血栓形成，并与分子分析可能的干扰。使用该刺穿主动脉同一针和IVC创建AVF也药物递送到AVF管腔的能力允许以最小的容器操纵简单递送。 有一些关键步骤和点提高该过程的成功率和一致性。公关主动脉腔静脉和oximal夹具被放置在左肾静脉下方。如果有在帧内肾IVC的上部大腰静脉，然后将这些容器下夹紧站点的修改建议通过从腰静脉逆行回到出血，以允许无需稀释药物的有效培养。主动脉腔静脉和远侧夹具被放置在髂分叉的水平，以免可能与远侧主动脉和下腔静脉的穿刺干扰。保持与弯曲缝合针夹持器的地下25针使容器穿刺期间针的最佳方向。止血是通过覆盖与相邻的复古腹膜动脉入孔来实现的。特别注意的是，以提供小心压缩，同时避免AVF闭塞和血栓形成如先前所述。1 基因转移技术的发展提供了在分子水平上，以修改手术效果的潜力。报告描述traluminal基因递送到静脉主要集中在静脉移植失败的有针对性的治疗。相比之下，这种模式允许药物输送的AVF成熟的调制。这种模式的优点包括利用小鼠模型，允许不同的鼠标遗传品系的审查。此外，15分钟腔内照射协议是很容易转移到临床手术中的设置，并可能有助于评估围手术期干预措施，防止JUXTA，吻合口狭窄，人AVF手术一般复杂。从这些初步的结果，我们表明，该方法比传统的外部路由的内皮细胞中产生更多的位点特异性基因表达。 这项研究的潜在限制是对下腔静脉扩张可变压力的作用。下腔静脉段的隔离并不在此小鼠模型中， 即 ，侧分支（腰静脉）未结扎完美。因此，很难精确地MAINT艾因孵化过程中扩张的压力水平一致。虽然IVC轻轻手动输液胀得，胀痛较高的压力可能会导致内膜增生20,21和直接平滑肌细胞受损，11导致干扰分析。它也很难完全定量药物递送的效率，因为一些药物可以输送期间通过腰静脉丢失，以及后穿过近侧IVC夹具去除和潜在通过AVF进入主动脉。最后，该方法允许一些药物的钳除去后全身被释放;药物和站点可能防范系统性释放潮红，但药物洗脱程度的量化的撤离将需要得到证实。 总之，本技术修饰鼠AVF模型较为简单，重现性和使得能够靶向内皮药物递送到小鼠AVF。在腔内jection在AVF创建时在技术上是可行的，相比外膜递送可以成功地改善内皮病毒基因表达。此AVF的药物递送模式将不仅对调节静脉AVF肢成熟的机制解剖的有益形式，同时也为AVF成熟治疗性调节。

鼠aortovenous瘘（AVF）的主动脉和下腔静脉（IVC）之间穿刺模型现在是一个既定的技术。1在该模型中，红外肾主动脉的两壁与地下25针被刺破，退出进毗邻肾下腔静脉;前主动脉入口孔被修复以简单的压缩，并且不需要缝合修复。串行随访检查采用高分辨率多普勒超声和病理分析表明，AVF有一个成熟阶段，再一个失败的阶段，扼要重述人类AVF的已知病理生理学2 探索了调节AVF成熟机制，需要用于输送治疗剂来熟化AVF内皮改进的方法。治疗剂递送至血管可以是通过血管内递送至内腔，或通过外部传递到外膜。外部递送的一个实例是玉米常用的聚醚凝胶的外膜应用。当加热到体温这种共聚物是热可逆的，并从液体到固体凝胶转化。当在体内局部施用药物在普卢兰尼克凝胶混合5,6-实现先前的研究已经表明持续的药物递送。3,4-病毒载体或siRNA与的Pluronic凝胶的外膜应用已报道是有效的血管周围递送系统，我们有还报告说，治疗由肽刺激外膜人外植隐静脉的导致内皮细胞受体蛋白的磷酸化。7 在另一方面，调查也同时使用病毒和非病毒载体腔内交付8-10犬和兔11,12车型静脉移植的。在这些报告中，静脉收获后进行基因转移体外 。伊斯拉米等报道的血管内的病毒基因递送y以颈动脉原位脉，而无需创建一个旁路13 Gloverman等。在大鼠股动脉，腹壁浅静脉瘘裸DNA的报道腔内和外膜交付14梅奥组报道在小鼠颈总动脉，颈内静脉瘘外膜的药物输送15,16然而，这些先前报告的模型需要缝合吻合创造在AVF。在这份报告中，腔内给药并同时创造AVF在小鼠中的描述，使用AVF创作的缝合线少的模型。通过使用本变形鼠AVF模型可以执行用于腔内药物输送到瘘管的静脉肢的简单方法。

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			<td height="69" style="height:69px;width:343px;">Vevo 770 ultrasound machine&#160;</td>
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</table>

intraluminal drug delivery to the mouse arteriovenous fistula endothelium

治疗剂的递送，以提高动静脉瘘（AVF）成熟可以通过腔内或外部途径给药。简单的鼠AVF模型用的药物溶液至静脉内皮同时作为瘘创建腔内施用相结合。这个模型的技术问题进行了讨论。在全身麻醉下腹部切口由和主动脉和下腔静脉（IVC）被曝光。在肾下主动脉和下腔静脉解剖夹紧。近端和远端夹紧后，穿刺部位进行曝光，将25g针头被用来穿刺主动脉并进入下腔静脉两壁。穿刺后，立即报告基因表达的病毒载体在IVC通过相同的针注入，随后温育15分钟。相比由外部给药途径腔内给药方法使能更健壮的病毒基因递送到静脉内皮。这诺夫递送l的方法将有利于研究，探索在AVF成熟内皮的作用，并能在外科手术的时间腔内给药。

在一系列33小鼠，在第一术后天存活率为97.0％; AVF通畅，如通过超声波来确定，为84.9％。 与传统的外部路由本血管内输送路线的基因转导效率进行比较。对于管腔内输送（ILD），在穿刺后，立即加入200μl腺病毒的GFP（广告-GFP）载体溶液（1×10 9 PFU / ml）的经穿刺针注入IVC，随后孵育15分钟时间。对照组小鼠用聚醚凝胶收到的Ad-GFP（1×10 9 PFU /毫升）下腔静脉的外膜交付（AD）。样品在暴露于腺病毒载体24小时后取出的。从用荧光显微镜，以及组织学检查内膜节用抗GFP进行了评估由烯面观察在IVC壁GFP和瘘管的表达抗体。 烯面观察显示在24小时（ 图2A）中的ILD治疗的小鼠与AD相比，下腔静脉壁的GFP表达更强，与周围的瘘特性增强。 GFP表达在两组永久在转染后72小时（数据未显示）。抗GFP抗体组织学分析，下腔静脉与ILD治疗的小鼠与AD相比，内膜显示更强大和特定地点的GFP表达。 （ 图2B）。为了证实这些结果，通过AD或ILD腺病毒-GFP治疗肾下IVCS而收获的印迹。仅ILD后的GFP蛋白质表达是足够的检测，在转染后24小时，这表明通过腔内路线优越病毒药物输送到下腔静脉壁。 <img alt="图2" src="/files/ftp_upload/53905/53905fig2.jpg" /> 图2.交货腺病毒GFP到AVF流明增强GFP表达。 （A）代表免疫表现在连接接收外膜交货（上）或腔内交付（底部）应用腺病毒GFP小鼠AVF内皮（24小时）的正视图。条表示25微米。（B）示出在接收由外膜输送（顶部）或管腔内输送（底部）施加腺-GFP小鼠AVF内皮（24小时）的部分代表性免疫荧光。 *表示下腔静脉管腔;条表示25微米。通过外膜或腔内航线应用于显示GFP在AVF腺病毒-GFP处理的检测（C）代表印迹。

Watch this Scientific Journal Video about Intraluminal Drug Delivery to the Mouse Arteriovenous Fistula Endothelium at JoVE.com

Intraluminal Drug Delivery to the Mouse Arteriovenous Fistula Endothelium

After puncturing the aorta through the inferior vena cava (IVC) to create an aorto-caval fistula in the mouse, solution containing a drug is infused into the IVC via the same needle, followed by incubation. This method enables more robust drug delivery to the venous endothelium compared to the external route.

腔内药物输送到鼠标动静脉瘘内皮

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The ...

intraluminal-drug-delivery-to-mouse-arteriovenous-fistula

Nanyang Technological University

Research

JoVE Journal

Medicine

9.3K 次观看.  Yale University. After puncturing the aorta through the inferior vena cava (IVC) to create an aorto-caval fistula in the mouse, solution containing a drug is infused into the IVC via the same needle, followed by incubation. This method enables more robust drug delivery to the venous endothelium compared to the external route.

视频: Intraluminal Drug Delivery to the Mouse Arteriovenous Fistula Endothelium

Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to stimulate microcirculation. The choice of delivery method for these agents is important because the route of administration profoundly affects the bioactivity and efficacy of these agents1,2. In this article, we demonstrate how to locally administer a substance in ischemic hindlimbs by using a catheterized osmotic pump. This pump can deliver a fixed volume of aqueous solution continuously for an allotted period of time. We also present our mouse model of unilateral hindlimb ischemia induced by ligation of the common femoral artery proximal to the origin of profunda femoris and epigastrica arteries in the left hindlimb. Lastly, we describe the in vivo cannulation and ligation of the infrarenal abdominal aorta and perfusion of the hindlimb vasculature with Microfil, a silicone radiopaque casting agent. Microfil can perfuse and fill the entire vascular bed (arterial and venous), and because we have ligated the major vascular conduit for exit, the agent can be retained in the vasculature for future ex vivo imaging with the use of small specimen micro-CT3.

We show here the in vivo insertion of an osmotic pump for constant local drug delivery and the creation of hindlimb ischemia in a mouse model. Moreover, the hindlimb vasculature is perfused with Microfil, a silicone radiopaque agent, to prepare for micro-computed tomography (micro-CT) imaging.

药物输送到微型计算机断层扫描成像缺血后肢血管灌注与微滤渗透

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to ...

科研

医学

Technical Aspects of the Mouse Aortocaval Fistula

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta and inferior vena cava (IVC) are exposed. The proximal infrarenal aorta and the distal aorta are dissected for clamp placement and needle puncture, respectively. Special attention is paid to avoid dissection between the aorta and the IVC. After clamping the aorta, a 25 G needle is used to puncture both walls of the aorta into the IVC. The surrounding connective tissue is used for hemostatic compression. Successful creation of the AVF will show pulsatile arterial blood flow in the IVC. Further confirmation of successful AVF can be achieved by post-operative Doppler ultrasound.

The goal is to produce an arteriovenous fistula that is simple and reproducible. This method does not use sutures or glue adhesive. Therefore the samples can be used with the least amount of foreign materials for analysis.

在的鼠标Aortocaval瘘的技术方面

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta ...

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 ...

Slow-release Drug Delivery through Elvax 40W to the Rat Retina: Implications for the Treatment of Chronic Conditions

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these diseases. Chronic retinal diseases such as retinal oedema or neovascularization usually require multiple intraocular injections to effectively treat the condition. However, the risks associated with these injections increase with repeated delivery of the drug. Therefore, alternative delivery methods need to be established in order to minimize the risks of reinjection. Several other investigations have developed methods to deliver drugs over extended time, through materials capable of releasing chemicals slowly into the eye. In this investigation, we outline the use of Elvax 40W, a copolymer resin, to act as a vehicle for drug delivery to the adult rat retina. The resin is made and loaded with the drug. The drug-resin complex is then implanted into the vitreous cavity, where it will slowly release the drug over time. This method was tested using 2-amino-4-phosphonobutyrate (APB), a glutamate analogue that blocks the light response of the retina. It was demonstrated that the APB was slowly released from the resin, and was able to block the retinal response by 7 days after implantation. This indicates that slow-release drug delivery using this copolymer resin is effective for treating the retina, and could be used therapeutically with further testing.

This paper details how Elvax 40W can be used as a slow-release method for drug delivery to the adult rat retina. The protocol for preparing, loading, and delivering the drug-resin complex to the eye is described.

通过40W的Elvax缓释给药的大鼠视网膜:对慢性疾病的治疗

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these ...

A Novel Murine Model of Arteriovenous Fistula Failure: The Surgical Procedure in Detail

The arteriovenous fistula (AVF) still suffers from a high number of failures caused by insufficient remodeling and intimal hyperplasia from which the exact pathophysiology remains unknown. In order to unravel the pathophysiology a murine model of AVF-failure was developed in which the configuration of the anastomosis resembles the preferred situation in the clinical setting. A model was described in which an AVF is created by connecting the venous end of the branch of the external jugular vein to the side of the common carotid artery using interrupted sutures. At a histological level, we observed progressive stenotic intimal lesions in the venous outflow tract that is also seen in failed human AVFs. Although this procedure can be technically challenging due to the small dimensions of the animal, we were able to achieve a surgical success rate of 97% after sufficient training. The key advantage of a murine model is the availability of transgenic animals. In view of the different proposed mechanisms that are responsible for AVF failure, disabling genes that might play a role in vascular remodeling can help us to unravel the complex pathophysiology of AVF failure.

Here we present a murine model of arteriovenous fistula (AVF) failure in which a clinically relevant anastomotic configuration is incorporated. This model can be used to study the pathophysiology and to test possible therapeutic interventions.

动静脉内瘘失败的新型小鼠模型:在详细介绍手术过程

The arteriovenous fistula (AVF) still suffers from a high number of failures caused by insufficient remodeling and intimal hyperplasia from which the ...

Pancreatic Duct Infusion: An Effective and Selective Method of Drug and Viral Delivery

The pancreas is a bifunctional organ with both endocrine and exocrine components. A number of pathologies can afflict the pancreas, including diabetes, pancreatitis, and pancreatic cancer. All three of these diseases mark active areas of study, not only to develop immediate therapy, but also to better understand their pathophysiology. There are few tools to further these areas of study. Pancreatic duct infusion is an important technique that can allow for lineage tracing, gene introduction, and cell line-specific targeting. The technique requires the intricate dissection of the second portion of the duodenum and ampulla, followed by the occlusion of the bile duct and the cannulation of the pancreatic duct. Although the technique is technically challenging at first, the applications are myriad. Ambiguity in the specifics of the procedure between groups highlighted the need for a standard protocol. This work describes the expression of a green fluorescent protein (GFP) within the pancreas after the pancreatic duct infusion of a viral vector expressing GFP versus a sham surgery. The infusion and therefore expression is specific to the pancreas, without expression present in any other tissue type.

Pancreatic duct infusion is an important technique that can allow for lineage tracing, gene introduction, and cell line-specific targeting. A pancreatic duct infusion technique for drug and viral delivery to pancreatic cells is presented here.

胰腺管输液:药物和病毒输送的有效和选择性方法

The pancreas is a bifunctional organ with both endocrine and exocrine components. A number of pathologies can afflict the pancreas, including diabetes, ...

Optimized LC-MS/MS Method for the High-throughput Analysis of Clinical Samples of Ivacaftor, Its Major Metabolites, and Lumacaftor in Biological Fluids of Cystic Fibrosis Patients

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening pulmonary manifestations. Ivacaftor (IVA) and ivacaftor-lumacaftor (LUMA) combination are two new breakthrough CF drugs that directly modulate the activity and trafficking of the defective CFTR-protein. However, there is still a dearth of understanding on pharmacokinetic/pharmacodynamic parameters and the pharmacology of ivacaftor and lumacaftor. The HPLC-MS technique for the simultaneous analysis of the concentrations of ivacaftor, hydroxymethyl-ivacaftor, ivacaftor-carboxylate, and lumacaftor in biological fluids in patients receiving standard ivacaftor or ivacaftor-lumacaftor combination therapy has previously been developed by our group and partially validated to FDA standards. However, to allow the high-throughput analysis of a larger number of patient samples, our group has optimized the reported method through the use of a smaller pore size reverse-phase chromatography column (2.6 &#181;m, C8 100 &#197;; 50 x 2.1 mm) and a gradient solvent system (0-1 min: 40% B; 1-2 min: 40-70% B; 2-2.7 min: held at 70% B; 2.7-2.8 min: 70-90% B; 2.8-4.0 min: 90% B washing; 4.0-4.1 min: 90-40% B; 4.1-6.0 min: held at 40% B) instead of an isocratic elution. The goal of this study was to reduce the HPLC-MS analysis time per sample dramatically from ~15 min to only 6 min per sample, which is essential for the analysis of a large amount of patient samples. This expedient method will be of considerable utility for studies into the exposure-response relationships of these breakthrough CF drugs.

Ivacaftor and ivacaftor-lumacaftor combination are two new CF drugs. However, there is still a dearth of understanding on their PK/PD and pharmacology. We present an optimized HPLC-MS technique for the simultaneous analysis of ivacaftor and its major metabolites, and lumacaftor.

经优化的 LC/ms 方法对囊性纤维化患者 Ivacaftor 及其主要代谢物和 Lumacaftor 的高通量分析

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening ...

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) ...

Trans-Tympanic Drug Delivery for the Treatment of Ototoxicity

The systemic administration of protective agents to treat drug-induced ototoxicity is limited by the possibility that these protective agents could interfere with the chemotherapeutic efficacy of the primary drugs. This is especially true for the drug cisplatin, whose anticancer actions are attenuated by antioxidants which provide adequate protection against hearing loss. Other current or potential otoprotective agents could pose a similar problem, if administered systemically. The application of various biologicals or protective agents directly to the cochlea would allow for high levels of these agents locally with limited systemic side effects. In this report, we demonstrate a trans-tympanic method of delivery of various drugs or biological reagents to the cochlea, which should enhance basic science research on the cochlea and provide a simple way of directing the use of otoprotective agents in the clinics. This report details a method of trans-tympanic drug delivery and provides examples of how this technique has been used successfully in experimental animals to treat cisplatin ototoxicity.

We present a technique for localized administration of drugs through the trans-tympanic route into the cochlea. Drug delivery through this route would not interfere with the anti-cancer efficacy of the chemotherapeutic drugs such as cisplatin.

经鼓膜药物治疗耳毒性的临床研究

The systemic administration of protective agents to treat drug-induced ototoxicity is limited by the possibility that these protective agents could ...

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves focal endothelial-selective transgenesis, thereby allowing an investigator to determine the biological roles of endothelial-expressed transgenes and to quantify the in vivo transcriptional activity of DNA sequences in large artery endothelial cells. The method uses surgical isolation of rabbit common carotid arteries and an arteriotomy to deliver a transgene-expressing viral vector into the arterial lumen. A short incubation period of the vector in the lumen, with subsequent aspiration of the lumen contents, is sufficient to achieve efficient and durable expression of the transgene in the endothelium, with no detectable transduction or expression outside of the isolated arterial segment. The method allows assessment of the biological activities of transgene products both in normal arteries and in models of human vascular disease, while avoiding systemic effects that could be caused either by targeting gene delivery to other sites (e.g. the liver) or by the alternative approach of delivering genetic constructs to the endothelium by germ line transgenesis. Application of the method is limited by the need for a skilled surgeon and anesthetist, a well-equipped operating room, the costs of purchasing and housing rabbits, and the need for expertise in gene-transfer vector construction and use. Results obtained with this method include: transgene-related alterations in arterial structure, cellularity, extracellular matrix, or vasomotor function; increases or reductions in arterial inflammation; alterations in vascular cell apoptosis; and progression, retardation, or regression of diseases such as intimal hyperplasia or atherosclerosis. The method also allows measurement of the ability of native and synthetic DNA regulatory sequences to alter transgene expression in endothelial cells, providing results that include: levels of transgene mRNA, levels of transgene protein, and levels of transgene enzymatic activity.