Name: an acute retinal model for evaluating blood retinal barrier breach and potential drugs for treatment
Uploaded: 2016-09-13T15:00:00.000+00:00
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Description: Watch this Scientific Journal Video about An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment at JoVE.com

Bringhurst Meats (Berlin, NJ) is acknowledged for their genuine help in providing the swine eyeballs.

所有协议均符合开展与机构动物护理和使用委员会在适用的政策。 1.准备<ol><li>用75％的制备稳定化的媒体的Dulbecco改良的Eagle培养基（DMEM），25％的Hanks&#39;平衡盐溶液（HBSS）中。在-20°C，直到使用拌匀，分装和储存。 </li><li>制备磷酸盐缓冲盐水（PBS）中，并通过高压灭菌消毒。在室温下储存的溶液。实验（5毫升，每孔）之前温PBS中至37℃。 </li><li>制备10％，20％，并在PBS中30％蔗糖。过滤器单独分开过0.22微米的过滤器进行消毒所有的解决方案。保存在4℃的溶液。 </li><li>制备在PBS中的组胺原液至90毫米的浓度。消毒通过过滤通过0.22微米过滤器的溶液。等分试样并保存在-80℃的溶液中。避免反复冷冻和解冻。 </li><li>试验前在PBS新鲜的4％多聚甲醛（PFA）。把它放在冰上。 注意:保持PFA在通风橱穿适当的防护设备。 </li><li>解冻稳定介质（20毫升，每视网膜）。青霉素 - 链霉素加入100单位/毫升的最终浓度。实验前的介质（每视网膜15毫升）中于37℃在培养箱中的温暖的一部分。留在冰介质的其余部分。 </li><li>放置的HBSS（10毫升，每视网膜）在冰上。 </li></ol> 2.视网膜器官文化<ol><li>转移样品到组织培养罩。通过镜头周围切开眼罩。使用刷子，不拉在视网膜上轻轻取出玻璃体。轻轻地从分离切割边缘视网膜用画笔暴露视盘。严重视神经用剃刀和释放视网膜。 </li><li>转移视网膜成培养皿并用冷HBSS仔细冲洗视网膜一次。放在HBSS样品上我CE。根据需要重复步骤2.1这一步解剖许多视网膜出来。 </li><li>切成两半视网膜对称用剃刀。轻轻样品转移至6孔板用3ml稳定介质的每孔。允许在37℃下30分钟的平衡在用含有5％的CO 2气氛的培养箱。 </li><li>在步骤2.3制备孵育期间下列试剂:稀释组胺原液在温暖介质所需浓度（450μM）。溶解LXA4（氩气下储存）到培养基中，以250纳克/毫升的最终浓度。 注:当测量LXA4的效果，从单一的视网膜的一半被用于单独组胺，而另一半被用于与LXA4组胺。 </li><li>小心吸稳定介质和制备的介质添加到每个孔中（3每孔毫升）中。孵育样品在37℃下1小时在用含有5％的CO 2气氛的培养箱。 </li><li>冲洗样品在温暖，无菌PBS一次。 </li></ol> 3.准备免疫染色的样品<ol><li>抽吸PBS从板上。添加4％PFA（每孔3ml）和孵育在室温下15分钟。 </li><li>迅速吸出PFA。冲洗一次使用PBS中的样品。转移样品至10％蔗糖（5毫升，每孔），并在4℃下孵育2至4小时。 </li><li>转移样品至30％的蔗糖（5毫升，每孔），并在4℃下孵育过夜。混合商用冷冻介质的一个部分，用20％的蔗糖的两部分，以使工作冷冻培养基。在4℃过夜或更长的时间离开它，直到气泡消失在空气中。 </li><li>转移样品到工作冷冻培养基，并允许平衡，在室温下5分钟。修剪每个视网膜成的矩形（大致为3毫米×5毫米）的。 </li><li>小心将样品转移到包含在一个筒状容器（约1cm半径×2cm的高度）dev的工作冷冻媒体从铝箔ISED。确保视网膜切片垂直（面向后切片，以提供在视网膜的一个横截面）并在液氮中冷冻。在-80°C，直到使用保存的块。 </li><li>第一个低温恒温器每块到14微米厚的片，并安装在载玻片上的章节。在-80°C，直到使用存储的幻灯片。 </li></ol> 4.免疫染色<ol><li>洗，用5％蔗糖磷酸缓冲液（SPB，在PBS中的5％蔗糖，pH值7.4）一旦部分。通过在封闭缓冲液（5％的SPB，用10％正常山羊血清和0.5％的Triton X-100）在室温下温育1小时的节阻断非特异性结合位点。 </li><li> （在封闭缓冲液500 GFAP或MAP2，在1稀释）1小时孵育适当的主要抗体的部分（上幻灯片）。吸出溶液。在5％的SPB洗涤三次。对于染色内源性抗体，跳过此步骤。 </li><li>孵育切片与相应塞康1小时:卡里抗体（800中的封闭缓冲液Cyanine3 / FITC缀合的驴抗小鼠/抗兔IgG，稀释为1）。 </li><li>洗部分彻底用5％的SPB三次。在安装中含有DAPI和盖玻片覆盖安装他们准备显微镜的样品。 </li><li>使用激光共聚焦显微镜，拍摄图像通过在相同的参数，如激光强度，增益值的20X镜头，停留时间等不同群体。设置的激发/发射波长为蓝色通道（检测DAPI）作为四百四十七分之四百零八纳米，对于红色信道（检测Cyanine3），为七百八十五分之五百六十一nm，对于绿色通道（检测FITC），为五百二十五分之四百八十八纳米。 </li></ol> 5.图像分析<ol><li>按照下列标准12量化渗漏血管的百分比:仅包含血管与计数部分的平面内的内皮细胞核;考虑血管渗漏，如果它显示了股份公司周围血管免疫染色radient。 </li><li>以确定表达水平，选择感兴趣的区域，并使用图像分析软件测量的平均灰度值。 </li><li>为了测量过程的宽度，选择在哪里的处理是不同的（外核层，ONL，例如）的部分的面积。然后，选择在哪里这样的过程是可见的，连续的光场。根据微观设置中设置的比例尺，得出整个过程线和执行测量。 </li><li>分析过程的连续性，选择感兴趣的区域，运用称为方差的滤波器，其由与它的邻域方差替换每个像素增强图像中的边缘，自动调节对比度和亮度，计算所述线和由正常化的计区。 </li><li>采用双尾学生t检验计算统计学意义。 *，P &lt;0.05; **，P &lt;0.01; *** P &lt;0.001。 P很多结果为平均±SEM。 </li></ol>

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The authors have nothing to disclose.

In this report, we present a powerful ex vivo acute retinal model of BRB dysfunction using the swine retina. This model system does not require special instruments and can be easily adapted under most laboratory settings. However, to obtain a successful result, several steps require close attention. After obtaining the eyeballs from the source, they must be kept at 4 &#176;C or on ice and processed as soon as possible. When the effect of a treatment is being analyzed, two halves of the same retina must be used -...

越来越多的证据支持体血视网膜屏障（BRB）1-5和其相似的血脑屏障（BBB）6,7的存在。血脑屏障的折衷已经紧密连锁因果或作为诊断标志物的慢性神经变性疾病如阿尔茨海默氏病（AD）8,9和急性病症如谵妄10。机械分析上市这些病状和发现的潜在的药物靶标一般是由脑的有限访问性和网络复杂阻碍。替代品，如在体内成像11，脑器官型培养12，原代细胞培养13,14和共培养系统15已经产生。然而，大多数这些模型需要特殊的仪器，实验长周期或多个标记来识别细胞。血脑屏障和BRB之间的功能和结构相似性，以及镝之间的相关性两个sfunctions已经争论16-19。此外，更容易获得的，定义良好的细胞类型和层状结构已经允许充分表征视网膜作为窗口到大脑。血脑屏障和BRB的结构和功能的身份留在详细地进行比较。然而，视网膜病变，尤其是BRB违约，也被紧紧与各种疾病的进展，包括糖尿病18-19和AD 21,22相关联。因此，感兴趣的是建立一个BRB功能障碍系统不仅划定机制，但也以筛选​​潜在药物。在这份报告中，用一个简单的急性视网膜文化协议，实现BRB功能障碍是制定并提交。 增加血脑屏障通透性和AD样病理改变已经建立了与组 ​​胺，促炎症介质12孵育大脑器官文化。因此，在所提出的系统中，组胺是申请灭蝇灯对体外培养的视网膜诱导BRB功能障碍。从几个物种，如小家鼠和普通牛视网膜，进行了测试。由于它们的商业可用性和相似之处人体组织，新鲜的猪眼球，用来提供此报告的数据。组胺和/或其他药物孵育后，视网膜被免疫染色几种蛋白12，例如免疫球蛋白G（IgG）的，血液的主要成分之一处理进行评估;胶质纤维酸性蛋白（GFAP），神经胶质激活一个公知的标记;和微管相关蛋白2（MAP2），一个特定的神经元细胞骨架蛋白对微管组装至关重要。此外，视网膜的层状结构允许Müller细胞和神经节细胞的过程的详细分析，如在它们的宽度和连续性变化。因此几个附加参数可用来评估的后果BRB违反在早期阶段和评估的潜在治疗逆转的效果好。 血管渗漏（BVS），神经胶质细胞的活化和神经元细胞的损伤响应:在该协议中，筛选药物的潜在反转效果从三个方面进行评估。几个量化方法被利用​​，例如，通过免疫染色，处理并通过增强滤波器示出神经元过程的连续性的宽度测量的强度中所示的表达水平。为了更好地说明该方法，并帮助解释结果，脂氧素A4（LXA4），响应炎性损伤和衰减内皮功能障碍23内源性合成的化合物，已被选定为示范的目的。

<table><tbody><tr><td>DMEM</td><td>Life Technologies&amp;nbsp;</td><td>11965-092</td><td></td></tr><tr><td>HBSS</td><td>Life Technologies&amp;nbsp;</td><td>14170-112</td><td></td></tr><tr><td>Sucrose</td><td>J.T.Baker</td><td>4072-05</td><td></td></tr><tr><td>Histamine&amp;nbsp;</td><td>Sigma</td><td>H7125-1G</td><td></td></tr><tr><td>Penicillin-Streptomycin&amp;nbsp;</td><td>Invitrogen</td><td></td><td></td></tr><tr><td>PFA</td><td>Electron Microscopy Sciences</td><td>15710</td><td></td></tr><tr><td>Freezing Media&amp;nbsp;</td><td>Triangle Biomedical Sciences</td><td>TFM-5</td><td></td></tr><tr><td>Normal Goat Serum&amp;nbsp;</td><td>Rockland</td><td>D104-00-0050</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787</td><td></td></tr><tr><td>GFAP Antibody</td><td>Millipore</td><td>AB5804</td><td></td></tr><tr><td>MAP2 Antibody</td><td>EMD Millipore</td><td>MAB3418</td><td></td></tr><tr><td>FITC conjugated Donkey anti-rabbit IgG</td><td>Jackson ImmunoResearch Laboratories, Inc.</td><td>711-095-152</td><td></td></tr><tr><td>Cy3 conjugated Donkey anti-mouse IgG</td><td>Jackson ImmunoResearch Laboratories, Inc.</td><td>715-165-150</td><td></td></tr><tr><td>mounting medium containing DAPI</td><td>Vector Laboratories, Inc.</td><td>H-1200</td><td></td></tr><tr><td>Laser Confocal Microscope</td><td>Nikon</td><td>Eclipse Ti microscope</td><td></td></tr><tr><td>ImageJ</td><td>National Institutes of Health</td><td>1.45s</td><td></td></tr></tbody></table>

an acute retinal model for evaluating blood retinal barrier breach and potential drugs for treatment

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An inflammatory factor, histamine, is demonstrated to compromise vessel integrity in the cultured retina through positive staining of IgG outside of the blood vessels. The effects of histamine itself and those of candidate drugs for potential treatments, such as lipoxin A4, are assessed using three parameters: blood vessel leakage via IgG immunostaining, activation of M&#252;ller cells via GFAP staining and change in neuronal dendrites through staining for MAP2. Furthermore, the layered organization of the retina allows a detailed analysis of the processes of M&#252;ller and ganglion cells, such as changes in width and continuity. While the data presented is with swine retinal culture, the system is applicable to multiple species. Thus, the model provides a reliable tool to investigate the early effects of compromised retinal vessel integrity on different cell types and also to evaluate potential drug candidates for treatment.

我们提出了一个低成本，省时高效且易于使用的系统，以评估可能防止组胺引起的BRB违约的潜在治疗。的IgG对照视网膜（ 图1A）的血管内的制约，但漏出的血管时组胺曝光（ 图1B），确认该模型建立成功的。 LXA4被选为演示药物对BRB破坏的系统呈现的筛选。 BRB功能障碍是在治疗与LXA4（ 图1D），<...

Watch this Scientific Journal Video about An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment at JoVE.com

An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment

一个低成本，易于使用和强大的制度的建立，以评估可能改善组胺引起血视网膜屏障破坏的潜在治疗。血管渗漏，穆勒细胞活化和神经过程的连续性被用来评估损伤反应及其逆转与潜在的药物，脂氧素A4。

急性视网膜模型评估血视网膜屏障违约和潜在的药物治疗

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An ...

an-acute-retinal-model-for-evaluating-blood-retinal-barrier-breach

Research

JoVE Journal

Medicine

7.1K 次观看.  Rowan University. 一个低成本，易于使用和强大的制度的建立，以评估可能改善组胺引起血视网膜屏障破坏的潜在治疗。血管渗漏，穆勒细胞活化和神经过程的连续性被用来评估损伤反应及其逆转与潜在的药物，脂氧素A4。 

视频: An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment

Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium (hfRPE) cultures with morphological, physiological and genetic characteristics of native human RPE. These hfRPE cell cultures exhibit heavy pigmentation, and electron microscopy show extensive apical membrane microvilli. The junctional complexes were identified with immunofluorescence labeling of various tight junction proteins. Epithelial polarity and function of these easily reproducible primary cultures closely resemble previously studied mammalian models of native RPE, including human. These results were extended by the development of therapeutic interventions in several animal models of human eye disease. We have focused on strategies for the removal of abnormal fluid accumulation in the retina or subretinal space. The extracellular subretinal space separates the photoreceptor outer segments and the apical membrane of the RPE and is critical for maintenance of retinal attachments and a whole host of RPE/retina interactions.

We provide a reproducible method for culturing confluent monolayers of human fetal retinal pigment epithelial cells (hfRPE) cells that exhibit morphology, physiology, polarity, and protein and gene expression patterns of adult native tissue. This work has been extended to an animal model of several eye diseases.

视网膜色素上皮的生理与病理生理学研究的实验模型

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium ...

科研

神经科学

Rat Model of Blood-brain Barrier Disruption to Allow Targeted Neurovascular Therapeutics

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain barrier1. The barrier selectively excludes molecules from crossing between the blood and the brain based upon their size and charge. This function can impede the delivery of therapeutics for neurological disorders. A number of chemotherapeutic drugs, for example, will not effectively cross the blood-brain barrier to reach tumor cells2. Thus, improving the delivery of drugs across the blood-brain barrier is an area of interest.
The most prevalent methods for enhancing the delivery of drugs to the brain are direct cerebral infusion and blood-brain barrier disruption3. Direct intracerebral infusion guarantees that therapies reach the brain; however, this method has a limited ability to disperse the drug4. Blood-brain barrier disruption (BBBD) allows drugs to flow directly from the circulatory system into the brain and thus more effectively reach dispersed tumor cells. Three methods of barrier disruption include osmotic barrier disruption, pharmacological barrier disruption, and focused ultrasound with microbubbles. Osmotic disruption, pioneered by Neuwelt, uses a hypertonic solution of 25% mannitol that dehydrates the cells of the blood-brain barrier causing them to shrink and disrupt their tight junctions. Barrier disruption can also be accomplished pharmacologically with vasoactive compounds such as histamine5 and bradykinin6. This method, however, is selective primarily for the brain-tumor barrier7. Additionally, RMP-7, an analog of the peptide bradykinin, was found to be inferior when compared head-to-head with osmotic BBBD with 25% mannitol8. Another method, focused ultrasound (FUS) in conjunction with microbubble ultrasound contrast agents, has also been shown to reversibly open the blood-brain barrier9. In comparison to FUS, though, 25% mannitol has a longer history of safety in human patients that makes it a proven tool for translational research10-12.
In order to accomplish BBBD, mannitol must be delivered at a high rate directly into the brain's arterial circulation. In humans, an endovascular catheter is guided to the brain where rapid, direct flow can be accomplished. This protocol models human BBBD as closely as possible. Following a cut-down to the bifurcation of the common carotid artery, a catheter is inserted retrograde into the ECA and used to deliver mannitol directly into the internal carotid artery (ICA) circulation. Propofol and N2O anesthesia are used for their ability to maximize the effectiveness of barrier disruption13. If executed properly, this procedure has the ability to safely, effectively, and reversibly open the blood-brain barrier and improve the delivery of drugs that do not ordinarily reach the brain 8,13,14.

Blood-brain barrier disruption aids the delivery of certain drugs to the brain. Mannitol delivered intra-arterially shrinks cells surrounding blood vessels in order to physically disrupt the barrier.

允许有针对性的大鼠模型血 - 脑屏障破坏神经血管治疗

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain ...

医学

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes (ILM), and in conjunction with astrocytes, pericytes and endothelial cells they establish the blood-retinal barrier (BRB). BRB limits material transport between the bloodstream and the retina while the ILM acts as a basement membrane that defines histologically the border between the retina and the vitreous cavity. Labeling M&#252;ller cells is particularly relevant to study the physical state of the retinal barriers, as these cells are an integral part of the BRB and ILM. Both BRB and ILM are frequently altered in retinal disease and are responsible for disease symptoms.
There are several well-established methods to study the integrity of the BRB, such as the Evans blue assay or fluorescein angiography. However these methods do not provide information on the extent of BRB permeability to larger molecules, in nanometer range. Furthermore, they do not provide information on the state of other retinal barriers such as the ILM. To study BRB permeability alongside retinal ILM, we used an AAV based method that provides information on permeability of BRB to larger molecules while indicating the state of the ILM and extracellular matrix proteins in disease states. Two AAV variants are useful for such study: AAV5 and ShH10. AAV5&#160;has a natural tropism for photoreceptors but it cannot get across to the outer retina when administered into the vitreous when the ILM is intact (i.e., in wild-type retinas). ShH10 has a strong tropism towards glial cells and will selectively label M&#252;ller glia in both healthy and diseased retinas. ShH10 provides more efficient gene delivery in retinas where ILM is compromised. These viral tools coupled with immunohistochemistry and blood-DNA analysis shed light onto the state of retinal barriers in disease.

To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability.

使用腺相关病毒作为一种工具来研究疾病中的视网膜障碍

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes ...

产生视网膜变性以研究非洲爪蟾的细胞修复策略

Generating Retinal Injury Models in Xenopus Tadpoles

Retinal neurodegenerative diseases are the leading causes of blindness. Among numerous therapeutic strategies being explored, stimulating self-repair recently emerged as particularly appealing. A cellular source of interest for retinal repair is the M&#252;ller glial cell, which harbors stem cell potential and an extraordinary regenerative capacity in anamniotes. This potential is, however, very limited in mammals. Studying the molecular mechanisms underlying retinal regeneration in animal models with regenerative capabilities should provide insights into how to unlock the latent ability of mammalian M&#252;ller cells to regenerate the retina. This is a key step for the development of therapeutic strategies in regenerative medicine. To this aim, we developed several retinal injury paradigms in Xenopus: a mechanical retinal injury, a transgenic line allowing for nitroreductase-mediated photoreceptor conditional ablation, a retinitis pigmentosa model based on CRISPR/Cas9-mediated rhodopsin knockout, and a cytotoxic model driven by intraocular CoCl2 injections. Highlighting their advantages and disadvantages, we describe here this series of protocols that generate various degenerative conditions and allow the study of retinal regeneration in Xenopus.

作者聚焦：探索非洲爪蟾的视网膜再生机制 

We have developed several protocols to induce retinal damage or retinal degeneration in Xenopus laevis tadpoles. These models offer the possibility of studying retinal regeneration mechanisms.

在 非洲爪蟾 蝌蚪中生成视网膜损伤模型

Retinal neurodegenerative diseases are the leading causes of blindness. Among numerous therapeutic strategies being explored, stimulating self-repair ...

发育生物学

A Mouse Model of Retinal Ischemia-Reperfusion Injury Through Elevation of Intraocular Pressure

Retinal ischemia-reperfusion (I/R) is a pathophysiological process contributing to cellular damage in multiple ocular conditions, including glaucoma, diabetic retinopathy, and retinal vascular occlusions. Rodent models of I/R injury are providing significant insights into mechanisms and treatment strategies for human I/R injury, especially with regard to neurodegenerative damage in the retinal neurovascular unit. Presented here is a protocol for inducing retinal I/R injury in mice through elevation of intraocular pressure (IOP). In this protocol, the ocular anterior chamber is cannulated with a needle, through which flows the drip of an elevated saline reservoir. Using this drip to raise IOP above systolic arterial blood pressure, a practitioner temporarily halts inner retinal blood flow (ischemia). When circulation is reinstated (reperfusion) by removal of the cannula, severe cellular damage ensues, resulting ultimately in retinal neurodegeneration. Recent studies demonstrate inflammation, vascular permeability, and capillary degeneration as additional elements of this model. Compared to alternative retinal I/R methodologies, such as retinal arterial ligation, retinal I/R injury by elevated IOP offers advantages in its&#160;anatomical specificity, experimental tractability, and technical accessibility, presenting itself as a valuable tool for examining neuronal pathogenesis and therapy in the retinal neurovascular unit.

This article describes a procedure for inducing retinal ischemia-reperfusion injury by elevated intraocular pressure in mice. Retinal ischemia-reperfusion injury by elevated intraocular pressure serves to model human pathologies characterized by compromised oxygen and nutrient delivery in the retina, enabling researchers to examine potential cellular mechanisms and treatments for human diseases of the retinal neurovascular unit.

视网膜缺血再灌注损伤的小鼠模型通过眼压升高

Retinal ischemia-reperfusion (I/R) is a pathophysiological process contributing to cellular damage in multiple ocular conditions, including glaucoma, ...

In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

Advancements in ophthalmic imaging tools offer an unprecedented level of access to researchers working with animal models of neurovascular injury. To properly leverage this greater translatability, there is a need to devise reproducible methods of drawing quantitative data from these images. Optical coherence tomography (OCT) imaging can resolve retinal histology at micrometer resolution and reveal functional differences in vascular blood flow. Here, we delineate noninvasive vascular readouts that we use to characterize pathological damage post vascular insult in an optimized mouse model of retinal vein occlusion (RVO). These readouts include live imaging analysis of retinal morphology, disorganization of retinal inner layers (DRIL) measure of capillary ischemia, and fluorescein angiography measures of retinal edema and vascular density. These techniques correspond directly to those used to examine patients with retinal disease in the clinic. Standardizing these methods enables direct and reproducible comparison of animal models with clinical phenotypes of ophthalmic disease, increasing the translational power of vascular injury models.

In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

Here, we present three data analysis protocols for fluorescein angiography (FA) and optical coherence tomography (OCT) images in the study of Retinal Vein Occlusion (RVO).

体内 小鼠视网膜血管损伤读数以促进可重复性

Advancements in ophthalmic imaging tools offer an unprecedented level of access to researchers working with animal models of neurovascular injury. To ...

Retinal Pathophysiological Evaluation in a Rat Model

A posterior segment eye disease like diabetic retinopathy alters the physiology of the retina. Diabetic retinopathy is characterized by a retinal detachment, breakdown of the blood-retinal barrier (BRB), and retinal angiogenesis. An in vivo rat model is a valuable experimental tool to examine the changes in the structure and function of the retina. We propose three different experimental techniques in the rat model to identify morphological changes of retinal cells, retinal vasculature, and compromised BRB. Retinal histology is used to study the morphology of various retinal cells. Also, quantitative measurement is performed by retinal cell count and thickness measurement of different retinal layers. A BRB breakdown assay is used to determine the leakage of extraocular proteins from the plasma to vitreous tissue due to the breakdown of BRB. Fluorescence angiography is used to study angiogenesis and leakage of blood vessels by visualizing retinal vasculature using FITC-dextran dye.

Diabetic retinopathy is one of the leading causes of blindness. Histology, blood-retinal barrier breakdown assay, and fluorescence angiography are valuable techniques to understand the pathophysiology of the retina, which could further enhance the efficient drug screening against diabetic retinopathy.

大鼠模型中的视网膜病理生理学评估

A posterior segment eye disease like diabetic retinopathy alters the physiology of the retina. Diabetic retinopathy is characterized by a retinal ...

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 ...

急性视网膜模型评估血视网膜屏障违约和潜在的药物治疗

摘要

探索更多视频

此视频中的章节

视网膜色素上皮的生理与病理生理学研究的实验模型

允许有针对性的大鼠模型血 - 脑屏障破坏神经血管治疗

使用腺相关病毒作为一种工具来研究疾病中的视网膜障碍

在非洲爪蟾蝌蚪中生成视网膜损伤模型

在非洲爪蟾蝌蚪中生成视网膜损伤模型

在非洲爪蟾蝌蚪中生成视网膜损伤模型

视网膜缺血再灌注损伤的小鼠模型通过眼压升高

体内小鼠视网膜血管损伤读数以促进可重复性

大鼠模型中的视网膜病理生理学评估

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

急性视网膜模型评估血视网膜屏障违约和潜在的药物治疗

摘要

探索更多视频

此视频中的章节

视网膜色素上皮的生理与病理生理学研究的实验模型

允许有针对性的大鼠模型血 - 脑屏障破坏神经血管治疗

使用腺相关病毒作为一种工具来研究疾病中的视网膜障碍

在 非洲爪蟾 蝌蚪中生成视网膜损伤模型

在 非洲爪蟾 蝌蚪中生成视网膜损伤模型

在 非洲爪蟾 蝌蚪中生成视网膜损伤模型

视网膜缺血再灌注损伤的小鼠模型通过眼压升高

体内 小鼠视网膜血管损伤读数以促进可重复性

大鼠模型中的视网膜病理生理学评估

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

在非洲爪蟾蝌蚪中生成视网膜损伤模型

在非洲爪蟾蝌蚪中生成视网膜损伤模型

在非洲爪蟾蝌蚪中生成视网膜损伤模型

体内小鼠视网膜血管损伤读数以促进可重复性

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