This research was supported by the Australian Research Council through the ARC Centre of Excellence in Vision Science (CE0561903), the National Health and Medical Research Council (1049990), and Progetto regionale speciale multiasse 'Reti per l'Alta Formazione' - PO FSE Abruzzo 2007-2013 - Azione 4.II.iii.

进行的所有实验均按照ARVO声明眼科及视觉研究用动物，并与阿奎拉动物伦理委员会的大学和澳大利亚国立大学动物实验伦理委员会的批准。成年大鼠（P100-200）用于在本研究中。 1，准备共聚树脂颗粒<ol><li>将20的Elvax 40W的小球进入在通风橱内的小玻璃烧杯中。 </li><li>填用100％乙醇确保覆盖所有粒料的烧杯中。 </li><li>用封口膜封住烧杯中，并将其浸泡在100％乙醇中的粒料在室温下搅拌7-10天。注：从这个步骤开始，不要用金属或塑料来处理或储存的树脂。 </li></ol> 2，准备好药液的加载<ol><li>溶解所选择的药物中的0.1％二甲亚砜（DMSO）中。使药液达到体积40微升。装入树脂用，将在单剂量给予的药物的三倍的浓度。 </li></ol> 3，准备和装入药物 - 树脂复合<ol><li>转移20洗涤粒料到另一个小玻璃烧杯中。溶解在4ml二氯甲烷中的丸粒约45分钟。用封口膜封住烧杯中，而颗粒溶解。 </li><li>通过在0.1％DMSO中溶解至0.001毫克/毫升的浓度制备坚牢绿FCF的溶液。 </li><li>取一吸管，并拟定40微升提前准备药液。加载另一个吸管40微升的快速绿色解决方案。 </li><li>同时添加两种溶液放入烧杯中。驴友迅速用玻璃搅拌棒，直到绿色染料在整个混合物均匀分布。 </li><li>将烧杯迅速转移到干冰10分钟快速冻结的树脂。 </li><li>设立“蒸发室”，以使溶剂蒸发。补较大的容器到三分之一以硫酸钙卵石。在卵石创建好，并小心地将烧杯中的井，使得卵石高达一半的烧杯中，以稳定腔室中的烧杯中。 </li><li>覆盖更大的外部容器的封口膜。 </li><li>蒸发室转移到冰柜。保持在-20℃下持续2-3周。 </li></ol> 4，准备手术植入的药物 - 树脂复合<ol><li>从冰箱中取出的蒸发室。固体药物树脂块转移至玻璃盘放在冰上。 </li><li>用显微镜或放大镜的帮助下，切下一块0.05mm左右的直径和0.1mm的长度从块使用环钻或冲压工具。 </li><li>包住块的剩余部分中防冻材料（ 如铝箔），并保持在-20℃直到需要。 </li></ol> 5，通过手术植入的药物 - 树脂复合成鼠眼</P&gt; <ol><li>准备动物进行手术。麻醉用腹膜内注射氯胺酮（100毫克/千克体重）和赛拉嗪（12毫克/公斤体重）的混合物。观察动物直到全身麻醉的实现。注：动物应该有动作（呼吸除外）的损失，以及角膜和脚趾捏反射损失。 </li><li>在完全麻醉的动物转移到手术台。使用mydriaticum（ 如硫酸阿托品）和局部麻醉剂（ 如盐酸丁卡因）滴在眼睛上（S）接受手术。应用人工泪液手术过程中，保持角膜湿润。使用一次性针头和消毒器械在整个手术。 </li><li>稳定使用双钝弯钳后瞳孔完全散大的眼睛。使用手术显微镜进行全面深入的穿刺伤口，以达到玻璃体，约2从角膜缘使用将25g针头毫米。 </li><li>将准备好的一件的T他的药物-树脂复合成使用一对细，尖镊子（ 例如镊子），其能够穿透穿刺部位的穿刺伤口。 </li><li>作为预防细菌感染，应用抗生素软膏至眼睛，以防止炎症以及防止角膜干燥而动物正在恢复。 </li><li>观察动物，直到完全康复。不要让动物无人看管，直到它恢复意识。不要将动物返回到其他动物的公司直至完全康复。如果动物显示压力或不适的迹象，镇痛药，应按照批准的职业道德协议中使用。 </li><li>按照通常的实验方法收集和组织的分析。 </li><li>在完成的试验中，实施安乐死的动物通过腹膜内注射戊巴比妥钠（60毫克/公斤体重）的2％利多卡因盐酸盐溶液。 </li></ol>注：盐酸利多卡因分钟imizes局部不适。

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作者没有什么可披露。

本文证明了使用的药物 - 树脂复合物的药物缓释递送至视网膜。我们的目的是提出一种方法，它是相对便宜和容易在小动物模型适用。 鉴于APB的功能是作为一种谷氨酸类似物，它会阻止在眼睛的视网膜响应。结果表明，APB引起一个星期着床后的视网膜响应的堵塞。这表明，APB成功释放到玻璃体和视网膜，并且其作用仅限于眼睛的药物 - 树脂复合物注入。药物的全面影响变得?...

慢性疾病如糖尿病和高血压的治疗中提出了许多挑战，因为这些疾病通常需要治疗延长的时期，常常为生活。这叫做缓慢释放的药物递送系统，其减少需要频繁给药的发展。这些缓慢释放方法的有效性已经通过胰岛素泵的发展已经证明，减少治疗糖尿病需要胰岛素注射的次数。眼睛的慢性疾病，尤其是影响其内层，需要通过药物侵入性操作频繁给药。一个这样的慢性疾病，影响人的眼睛是年龄相关性黄斑变性（AMD）。它影响到中央视网膜，这是一个层位于负责发起视力的眼睛后面的神经组织。 AMD是导致失明的西方世界第一的主要原因。与治疗真是个特别的挑战tinal疾病是该药物是需要达到的眼睛，这经常需要递送的侵入性方法本深层。药物通常是给药于使用玻璃体内注射玻璃体腔和视网膜。然而，每次注射有一种postinjection并发症，包括眼内炎，视网膜脱离，白内障和玻璃体出血2的风险。这种风险被乘以与药物的每一个回注。 减少需要多次注射会在AMD的治疗的一个主要好处。治疗AMD，在新的血管生长是一个标志性的湿形式，既定的治疗策略是针对使用VEGF抑制剂3的血管内皮生长因子（VEGF）。目前，这是通过重复的玻璃体内注射递送。同样，在黄斑水肿，糖尿病性视网膜病变的常见并发症的治疗，皮质类固醇激素是通过反复注射交付<s达&gt; 4。通过缓慢释放的方式提供这些药物确实可以减少postinjection并发症的风险。 使用有机硅类橡胶车辆提供小分子进入动物组织5首次被描述缓慢释放的药物递送系统的概念。自那时以来，其他的缓慢释放的方法已被开发来提供更大的分子，其中一些在眼睛进行了测试。粒子的载体，如可生物降解的微球，聚交酯-共-乙交酯共聚物（PLGA）纳米颗粒和磷脂小泡（脂质体）可以作为运载工具6,7有用。 PLGA纳米颗粒和脂质体进行了比较， 在体外环境中为它们随着时间的推移7穿过巩膜递送抗癌剂的能力。这两款车均有效缓慢释放的药物。然而，该研究只在体外环境下进行。 Bochot 等人 （2002）8测试脂质体的效能，提供分子在体内的视网膜。他们已经证明，脂质体成功递送小的寡核苷酸对兔视网膜。作者认为脂质体可能是有益的视网膜疾病8的治疗。然而，这些小泡的性质在玻璃体漂浮装置，它们很可能会变得模糊或损害视力9。 冈部等人（2003）10中使用由33％的乙烯-乙酸乙烯酯的非生物可降解的聚合物的光盘适用的β-塞米松兔。它们植入光盘插入巩膜袋和演示的有效释放药物进入玻璃体和视网膜的长达一个月10。然而，在这种特定的协议中​​，植入物是相对大的和刚性的，并且需要更复杂的外科手术，包括一个大的巩膜切口和缝合。 早先的研究中调查了Tissu酒店ë响应于各种聚合物的车辆通过注入成兔眼的角膜，并发现，在醇洗涤的乙烯 - 乙酸乙烯酯共聚物并没有引起炎症或刺激。这些配合物显示出持续的递送较大的化合物的成动物组织为延长的时期，某些超过高达100天依赖于药物11。一个这种类型的共聚物树脂已开发工业中的Elvax 40W的形式（40％（重量）的乙烯 - 乙酸乙烯酯共聚单体含量用“W”酰胺添加剂以改善沉淀处理）。此共聚物树脂是一种惰性物质，其是稳定的，在室温和体温。它并没有被证明可导致在生物组织中的过敏或毒性。此树脂，有效地传递大量的各种药物在不同的实验模型，探讨各种系统，如乳腺导管系统12中，初级听觉皮层13,14 &lt;功能/ SUP&gt;，青蛙视觉系统15。这种树脂也被用于在眼睛将药物递送到显影龟16,17，鸡胚18,19，和成人鼬视网膜20。在大鼠中枢神经系统中，树脂仅被用于在大脑21-23，但其在大鼠眼用尚未记录。 使用该共聚物树脂慢慢将药物递送至视网膜方法比其他方法的优点是，它是一种不引起炎症或刺激的眼睛稳定的化合物。不同于粒子的载体，该药物 - 树脂复合物就不会在植入后损害视力的，因为它通常保持在输送，而不是浮在玻璃体的部位。这将只需要一个简单的植入过程进入玻璃体腔内靠近眼睛的角膜缘，并在植入后不会需要缝合。最近，出现了几种新颖的递送系统的出现，如封装的电池技术（ECT）24,25，水凝胶26和缩微胶片27。然而，在目前的研究中制备和输送的药物 - 树脂复合物所用的方法是既容易跟踪和便宜的，由此用于在基础研究环境中使用的更有利的。使用这种复合物能够提供长期的药物治疗的挑战是确定该药物，这将最大限度地具有较少的玻璃体内注射的治疗益处的最佳浓度。 本文旨在演示使用的药物 - 树脂复合物的长期治疗的成年大鼠视网膜。交付该模式的效果是用谷氨酸类似物2 - 氨基-4 - 膦酰（APB）作为药物测试。 APB块的ON双极细胞通过模仿谷氨酸，内源性神经递质在视网膜28的光响应。当建业竞争与谷氨酸受体，它会阻止二极管灯吨的回应。 APB已用于生理研究，以控制视网膜功能和使用电生理学方法，如电图（ERG）测量它的效果。在以前的研究中，APB已被用于短期29和长期治疗显影视网膜;后者所涉及的日常给予通过眼内注射单剂量为30天30。用APB的相同量（0.092毫克，无菌盐水，以50μM的浓度）为所有注射如以前的工作28,29,31建议。我们选择了APB以测试药物 - 树脂复合物作为缓释载体将药物递送到眼睛。在本研究中所述的方法类似的涉及药物-树脂复合物16,32的制备以前描述的方法;然而，我们也详细的使用特别是在成年大鼠的眼睛。后的APB-装载树脂进入眼睛的手术植入，ERG进行建立APB是否废除第retinal光响应，并且因此是否APB已经成功地释放到玻璃体和视网膜。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Elvax 40W Pellets</td>
			<td>Du Pont, DE, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich Co. LLC., MO, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2-amino-4-phosphonobutyric acid (APB)</td>
			<td>Sigma-Aldrich Co. LLC., MO, USA</td>
			<td>A1910</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichloromethane</td>
			<td>Sigma-Aldrich Co. LLC., MO, USA</td>
			<td>34856</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast Green FCF</td>
			<td>Sigma-Aldrich Co. LLC., MO, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Drierite, calcium sulfate</td>
			<td>Sigma-Aldrich Co. LLC., MO, USA</td>
			<td>238910</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine, Ilium Ketamil</td>
			<td>Troy Laboratories Pty. Ltd., NSW, Australia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine, Ilium Xylazil-20</td>
			<td>Troy Laboratories Pty. Ltd., NSW, Australia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Atropine Sulphate, Minims eye drops</td>
			<td>Bausch &#38; Lomb Pty. Ltd., NSW, Australia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tetracaine Hydrochloride, Minims eye drops</td>
			<td>Bausch &#38; Lomb Pty. Ltd., NSW, Australia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chloramphenicol, Chlorsig ointment</td>
			<td>Aspen Pharma Pty. Ltd., NSW, Australia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pentobarbital, Lethabarb</td>
			<td>Virbac Australia Pty. Ltd., NSW, Australia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lignocaine Hydrochloride, Ilium Lignocaine-20</td>
			<td>Troy Laboratories Pty. Ltd., NSW, Australia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Uni-Core Punch Tool</td>
			<td>World Precision Instruments Inc., FL, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Curved Forceps</td>
			<td>World Precision Instruments Inc., FL, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Operating Microscope, Zeiss OPMI 99</td>
			<td>Zeiss, West Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>25G Insulin Needle</td>
			<td>Terumo Corp., Tokyo, Japan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont Tweezers</td>
			<td>World Precision Instruments Inc., FL, USA</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

slow-release drug delivery through elvax 40w to the rat retina: implications for the treatment of chronic conditions

视网膜疾病是难以治疗的视网膜深藏在眼内。通常需要药物递送的侵入性的方法来治疗这些疾病。慢性视网膜疾病，如视网膜水肿或新血管形成通常需要多眼内注射，以有效治疗该病症。然而，与这些注射相关的风险增加具有重复输送药物。因此，替代交付方式需要建立以尽量减少回注的风险。几个其他研究已开发的方法来递送药物在延长的时间，通过能够释放的化学物质慢慢进入眼睛的材料。在本次调查中，我们概述了使用的Elvax 40W，共聚物树脂，以充当药物输送到成年大鼠视网膜的车辆。的树脂制成，并装入该药物。该药物 - 树脂复合物，然后注入到玻璃体腔，在那里它会慢慢地释放药物比TI我。使用2 - 氨基-4 - 膦酰（APB），谷氨酸类似物阻止视网膜的光响应这种方法进行了测试。由此表明了该APB慢慢从树脂释放，并且能够阻止在植入后7天的视网膜响应。这表明，使用缓释药物递送该共聚物树脂可有效治疗视网膜上，并且可以用于治疗与进一步的测试。

全视野视网膜电图（ERG）被用于检测APB的视网膜上的效果。有关如何在ERG进行详细说明，请参阅下面的研究33,34。简言之，麻醉动物后，金电极被放置在眼睛的角膜上，和参考电极放置在两眼之间的前头皮，以记录视网膜的电活动。使用电子闪光灯单元中产生的刺激和10的强度不等，以10 6个光异构化反应，其表示每根棒的活化光敏分子的数目。在这些实验中，ERG记录采取了在1和后...

Watch this Scientific Journal Video about Slow-release Drug Delivery through Elvax 40W to the Rat Retina: Implications for the Treatment of Chronic Conditions at JoVE.com

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

本文详细介绍了如何的Elvax 40W，可作为药物输送到成年大鼠视网膜缓释方法。该协议的制备，装载和输送的药物 - 树脂复合物对眼睛进行说明。

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 ...

slow-release-drug-delivery-through-elvax-40w-to-rat-retina

Research

JoVE Journal

Medicine

11.5K 次观看.  University of L'Aquila. 本文详细介绍了如何的Elvax 40W，可作为药物输送到成年大鼠视网膜缓释方法。该协议的制备，装载和输送的药物 - 树脂复合物对眼睛进行说明。 

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

The Measurement and Treatment of Suppression in Amblyopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current estimates put the prevalence of amblyopia at approximately 1-3%1-3, the majority of cases being monocular2. Amblyopia is most frequently caused by ocular misalignment (strabismus), blur induced by unequal refractive error (anisometropia), and in some cases by form deprivation.
Although amblyopia is initially caused by abnormal visual input in infancy, once established, the visual deficit often remains when normal visual input has been restored using surgery and/or refractive correction. This is because amblyopia is the result of abnormal visual cortex development rather than a problem with the amblyopic eye itself4,5 . Amblyopia is characterized by both monocular and binocular deficits6,7 which include impaired visual acuity and poor or absent stereopsis respectively. The visual dysfunction in amblyopia is often associated with a strong suppression of the inputs from the amblyopic eye under binocular viewing conditions8. Recent work has indicated that suppression may play a central role in both the monocular and binocular deficits associated with amblyopia9,10 .
Current clinical tests for suppression tend to verify the presence or absence of suppression rather than giving a quantitative measurement of the degree of suppression. Here we describe a technique for measuring amblyopic suppression with a compact, portable device11,12 . The device consists of a laptop computer connected to a pair of virtual reality goggles. The novelty of the technique lies in the way we present visual stimuli to measure suppression. Stimuli are shown to the amblyopic eye at high contrast while the contrast of the stimuli shown to the non-amblyopic eye are varied. Patients perform a simple signal/noise task that allows for a precise measurement of the strength of excitatory binocular interactions. The contrast offset at which neither eye has a performance advantage is a measure of the "balance point" and is a direct measure of suppression. This technique has been validated psychophysically both in control13,14 and patient6,9,11 populations.
In addition to measuring suppression this technique also forms the basis of a novel form of treatment to decrease suppression over time and improve binocular and often monocular function in adult patients with amblyopia12,15,16 . This new treatment approach can be deployed either on the goggle system described above or on a specially modified iPod touch device15.

Amblyopia is a developmental disorder of the visual cortex that is often accompanied by strong suppression of one eye. We present a new technique for measuring and treating interocular suppression in patients with amblyopia that can be deployed using virtual reality goggles or a portable iPod Touch device.

测量和制止在弱视治疗

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current ...

科研

医学

A Model of Chronic Nutrient Infusion in the Rat

Chronic exposure to excessive levels of nutrients is postulated to affect the function of several organs and tissues and to contribute to the development of the many complications associated with obesity and the metabolic syndrome, including type 2 diabetes. To study the mechanisms by which excessive levels of glucose and fatty acids affect the pancreatic beta-cell and the secretion of insulin, we have established a chronic nutrient infusion model in the rat. The procedure consists of catheterizing the right jugular vein and left carotid artery under general anesthesia; allowing a 7-day recuperation period; connecting the catheters to the pumps using a swivel and counterweight system that enables the animal to move freely in the cage; and infusing glucose and/or Intralipid (a soybean oil emulsion which generates a mixture of approximately 80% unsaturated/20% saturated fatty acids when infused with heparin) for 72 hr. This model offers several advantages, including the possibility to finely modulate the target levels of circulating glucose and fatty acids; the option to co-infuse pharmacological compounds; and the relatively short time frame as opposed to dietary models. It can be used to examine the mechanisms of nutrient-induced dysfunction in a variety of organs and to test the effectiveness of drugs in this context.

A protocol for chronic infusions of glucose and Intralipid in rats is described. This model can be used to study the impact of nutrient excess on organ function and physiological parameters.

在大鼠模型慢性营养输液

Chronic exposure to excessive levels of nutrients is postulated to  affect the function of several organs and tissues and to contribute to the  ...

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 ...

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 ...

Intraluminal Drug Delivery to the Mouse Arteriovenous Fistula Endothelium

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The simple murine AVF model was combined with intraluminal administration of drug solution to the venous endothelium at the same time as fistula creation. Technical aspects of this model are discussed. Under general anesthesia, an abdominal incision is made and the aorta and inferior vena cava (IVC) are exposed. The infra-renal aorta and IVC are dissected for clamping. After proximal and distal clamping, the puncture site is exposed and a 25 G needle is used to puncture both walls of the aorta and into the IVC. Immediately after the puncture, a reporter gene-expressing viral vector was infused in the IVC via the same needle, followed by 15 min of incubation. The intraluminal administration method enabled more robust viral gene delivery to the venous endothelium compared to administration by the external route. This novel method of delivery will facilitate studies that explore the role of the endothelium in AVF maturation and enable intraluminal drug delivery at the time of surgical operation.

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 ...

Intravascular Delivery of Biologics to the Rat Kidney

The renal microvascular compartment plays an important role in the progression of kidney disease and hypertension, leading to the development of End Stage Renal Disease with high risk of death for cardiovascular events. Moreover, recent clinical studies have shown that renovascular structure and function may have a great impact on functional renal recovery after surgery. Here, we describe a protocol for the delivery of drugs into the renal artery of rats. This procedure offers significant advantages over the frequently used systemic administration as it may allow a more localized therapeutic effect. In addition, the use of rodents in pharmacodynamic analysis of preclinical studies may be cost effective, paving the way for the design of translational experiments in larger animal models. Using this technique, infusion of rat recombinant Vascular Endothelial Growth Factor (VEGF) protein in rats has induced activation of VEGF signaling as shown by increased expression of FLK1, pAKT/AKT, pERK/ERK. In summary, we established a protocol for the intrarenal delivery of drugs in rats, which is simple and highly reproducible.

The administration of drugs for recovery of kidney function requires control of the localization and distribution of the therapeutic compound. Here, we describe in detail a simple technique for intrarenal delivery of drugs in rats. This procedure may be easily performed with no mortality and high reproducibility.

生物血管内送货到大鼠肾脏

The renal microvascular compartment plays an important role in the progression of kidney disease and hypertension, leading to the development of End Stage ...

Network Analysis of Foramen Ovale Electrode Recordings in Drug-resistant Temporal Lobe Epilepsy Patients

Approximately 30% of epilepsy patients are refractory to antiepileptic drugs. In these cases, surgery is the only alternative to eliminate/control seizures. However, a significant minority of patients continues to exhibit post-operative seizures, even in those cases in which the suspected source of seizures has been correctly localized and resected. The protocol presented here combines a clinical procedure routinely employed during the pre-operative evaluation of temporal lobe epilepsy (TLE) patients with a novel technique for network analysis. The method allows for the evaluation of the temporal evolution of mesial network parameters. The bilateral insertion of foramen ovale electrodes (FOE) into the ambient cistern simultaneously records electrocortical activity at several mesial areas in the temporal lobe. Furthermore, network methodology applied to the recorded time series tracks the temporal evolution of the mesial networks both interictally and during the seizures. In this way, the presented protocol offers a unique way to visualize and quantify measures that considers the relationships between several mesial areas instead of a single area.

This protocol describes a procedure to track the evolution of mesial network measures in temporal lobe epilepsy (TLE) patients. It is based on the combination of intracranial recordings with a novel numerical technique for data analysis. Specifically, we present a protocol for network analyses of foramen ovale recordings.

在耐药性颞叶癫痫患者卵圆孔电极记录的网络分析

Approximately 30% of epilepsy patients are refractory to antiepileptic drugs. In these cases, surgery is the only alternative to eliminate/control ...

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 ...

Methods Article

通过40W的Elvax缓释给药的大鼠视网膜：对慢性疾病的治疗