我们要感谢我们的合作者，弗朗索瓦Delori，托马斯·伯克和Tobias Duncker。 研究支持: NIH / NEI R01 EY015520（RTS，JPG），并且从研究经费不受限制防盲（RTB）。

伦理学声明:就读于这些研究所有患者均按照医学在纽约大学法学院批准的机构审查委员会的监督，以便完成。 1.患者的选择和成像前期准备注意:需要以下物质:0.5％托眼用溶液，2.5％苯肾上腺素滴眼液，CSLO配备谱域光学相干断层扫描（SD-OCT）和内部荧光参考。 <ol><li>成像之前，根据制造商的说明适当的设置CSLO进行数据采集。 </li><li>安装在CSLO内部荧光参考。注意:内部荧光基准，从制造商购买的，被容纳在一个金属环，并直接放置在照相机镜头的后面。如果使用从其它制造商的CSLO，可能有不同的结构比其在所述此协议。 <ol><li>为了插入引用到CSLO，扭动镜头将其删除，旋开机器的金属环，并包含引用的新的金属环代替它。 注意:内部荧光基准是将QAF技术必不可少的，因为它允许校正在激光功率和检测器灵敏度/增益变化。 </li></ol></li><li>招募成像患者接受常规散瞳检查，并获得有关过去的眼部历史和可能与眼部表现的调查结果基本医疗条件的背景资料。 </li><li>扩张0.5％托和2.5％苯的学生。关键的一步:扩张小学生至少6毫米。注:这是光线通过不间断的本质，从而为眼底的最佳可视化和测量。 </li><li>成像前，正确定位患者的CSLO，用下巴搁在腮托，额头抵靠foreheað休息，外侧眼角与指标正确对齐。 </li></ol> 2.眼底病的基线成像<ol><li>首先，图像，以便集中相机在黄斑部，并获得大致对焦近红外反射光（IR）（820波长）眼底。 <ol><li>与患者适当地定位，开关控制面板红外成像模式的硬件环境，位置相机手动直到眼底处于全焦点，拍摄图像</li><li>调节控制盘为"IR-OCT"，即用谱域光学相干断层扫描（SD-OCT）结合红外成像以评估潜在的疾病黄斑上的设置。 </li><li>使用目前在成像窗口的导向正确定位华侨城眼底的红外图像。以实现最佳的SD-OCT质量，摄像机的位置，使得OCT图像是在它的成像窗口的顶部三分之一。获得至少一个水平线SC通过中央凹的并跨越整个摄像视野。 </li></ol></li></ol> 3.设置QAF成像<ol><li>使用"高速"图像采集。注:此设置可以实现更快速的图像采集，从而减少信号损失的风险，由于患者的运动和由虹膜或眼睑的光造成堵塞。 </li><li>使用"平均9"帧。注:此设置允许9的图像帧​​，随后可以快速，连续捕获"平均"，以减少噪声和假象（见下文）。 </li><li>使用"30×30度"字段。注:这是指度图像采集过程中捕捉的视网膜区域。 </li><li>此前成像，警告蓝光耐心，因为这可以在第一次是惊人。 </li><li>打开自动对焦模式和对准照相机轴线，使得屏幕最大是"充满"与眼底AF（两侧和图像的角部的最小变暗）。 <ol><li>如果患者^ h容忍明亮的蓝色光大街的困难，开始用相机成像进一步远离眼睛，然后把相机缓慢地向患者直到眼底是在众目睽睽之下。 关键的一步:如果光线进入，或从眼排出受阻，降低信号将导致。局部减小是作为图像边或角的不对称变黑可见。当眼球运动单独阻碍光线通道信号的广义损失看到。 </li></ol></li><li>将照相机使得AF信号在整个领域的最高水平。瞄准最大的信号，而不是清晰的图像，但他们大致相关。通过移动CSLO手动或用检眼镜操纵杆重新调整相机调整相机焦距。 </li><li>调整灵敏度/增益，使得眼底AF是很容易看到，但要避免过度饱和。期间的图像采集，无论是在内部参考可见有色像素（位于的顶图像）或眼底表明过饱和并因此损失信号。 </li></ol> 4.图像采集注:关键的一步:在图像采集的目标应该是获得每个会话2高品质的9帧图像堆栈来控制一个会话内图像之间的差异。重新定位病人和相机后，获得两个图像的可变性一个第二次会议，评估和控制。所有图像将最终被校准到内部参考（以下描述）。 <ol><li>只有"漂白"（视网膜为AF的光的充分曝光）在感官视网膜9视紫红质，以尽量减少光的吸收的至少20秒后获得的图像。 <ol><li>利用这段时间来优化校准摄像机，对焦和感光度。 </li></ol></li><li>有一个新鲜的泪膜提高了信号质量，每个图像采集前闪烁的主题。 </li><li>避免在收购的平面上眼睑。 注意:它可能有助于手动打开眼皮更具挑战性的患者。笔者建议，助理执行此任务。 </li><li>每个图像采集之前优化对准，以确保光不被导致下降信号虹膜阻碍。 注:由于至少有轻微的运动是很常见的，作者建议每个图像采集前，根据需要精细调整。引导和鼓励患者，而成像有助于减少运动。作者还建议使用本机的脚踏板的"收购按钮"，以尽量减少操作者分心。 </li><li>通过计算"平均"的9帧堆栈，以增加信噪比进行图像后处理。计算使用CSLO软件，通过选择以计算平均值的期权的堆叠的平均值。 注:可能有堆栈，其中一些帧质量最优的不是（ 即有局限性或泛SI下降相对于最佳帧gnal），因此请仔细检查每个堆栈和平均计算之前取消选择次优的帧。 注意:具有可接受的信号 - 噪声比平均图像可以从至少3帧进行处理。这是正常的两侧和每个图像的四角比因CSLO本身的局限性中央20度较低的信号。 <ol><li>如果软件会询问经营者是否愿意正常化灰度级（ 即 ，舒展直方图）0和255之间，选择"否"。这保持了分析不变灰度级。 </li></ol></li></ol> 5.图像分析<ol><li>如以前使用的QAF技术开发的图像分析软件分析描述图像的自动对焦（IGOR; 9）。使用该软件的典型图像分析的简要说明包含下面。 <ol><li>加载从出口中IGOR的QAF程序，并导入图像（.bmp格式）CSLO软件的像素宽高比768 x 768（默认值输出参数）。 </li><li>选择下拉菜单中的影像，并开始分析。 </li><li>重新标定指标在图像上这样的"十字线"覆盖中心凹和"支架"紧靠视盘（这些指标被用于比例和位置的投资回报）​​。当正确定位时，使用软件提示然后填充周围中央凹圆周感兴趣区（ 见图1，2）的图像。 </li><li>选择在IGOR选项以每个图像的整体的灰度级校准中存在的内部荧光基准那些水平。注意:此步骤允许的机器有关的因素，包括电子零电平的各图像的各机器的内部参考校准因数，以及病人有关的因素，包括年龄，折射和角膜曲率校准。 注:分析软件然后显示固定ř所得到的图像和QAF值上egions论证每个区域内。 QAF值也自动输入到在单独的窗口的电子表格格式"。 </li><li>通过选择命令提示符中的选项生成QAF"热图"。所有的图像和数据可以从IGOR软件在Excel电子表格中选择相应的选项导出。 </li></ol></li></ol>

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STA: no disclosures, JPG: no disclosures, RTS: consultant, Ocata Therapeutics.

异常RPE脂褐素分布，是否增加或减少，是视网膜疾病的敏感标记物和一般与感官视网膜功能的丧失相关。在这里，我们描述了RPE脂褐素的评价QAF的应用。内部荧光参考掺入纠正可变激光功率和探测器的灵敏度9与我们的标准化的成像技术使得AF水平的可靠的定量。这是我们的目标，该方法将在诊断和视网膜疾病的监测帮助，并最终在评估治疗性干预，如药物或基因疗法的功效。 QAF也可协助处于危险对于如AMD条件的个体的分层。 我们已经产生QAF数据的一个大的规范数据库被用作治疗视网膜病变13的解释的参考工具，并且已经在几个疾病STA还描述QAF工商业污水附加费，包括Stargardt病10，公牛的眼睛黄斑病变12和最佳的疾病11。在健康的视网膜上，有民族之间不同QAF水平，显著高于QAF白人比黑人和亚洲人，并表明女性QAF水平高于男性。也许最引人注目的是，QAF随着年龄的患者，如以前荧光分光光度计测量14随RPE脂褐素水平对应。虽然当前规范的数据只向上延伸到60岁，看来患者有70 14岁以后在RPE脂褐素测量下降。有趣的是，它不会出现在视网膜色素上皮细胞的数量的变化发生如患者年龄2等中观察到的下降AF可能是由于在RPE脂褐素3再分配或减少。这将是有趣的，以确定这些AF减少老年是否受损的视网膜色素上皮的功能和AMD的风险增加相关。 <p clasS ="jove_content"&gt;这个协议允许可靠的定量结果，可以纵向比，患者之间，并且CSLO机器之间。一个限制是，QAF不能准确在老年患者晶状体混浊，这限制了该技术在此高危人群的子集测量的AF。一个可能的解决方案是使用一个反射方法来单独地估计在媒体的光损失。幸运的是，老年患者谁是晶状体有明确的后路胶囊和已知的人工晶体传输特性是好的候选人QAF。然而，老年患者的学生和那些事先白内障手术经常不扩张到超过6mm，这限制了光通道，并防止准确的数据集合。为了图像这样的患者中，孔可以降低激光的直径插入CSLO由生产。这种修改已被成功用于在小鼠中执行QAF成像15。另一个限制是该技术是具有挑战性的执行，并且高度依赖于操作者。始终如一地得到高质量的图像，以确保自动对焦水平的精确测量，这是至关重要的。为了实现这一点，作者建议严格遵守成像协议以及适当的做法。有些患者虽然不好容忍AF成像所需的光的明亮闪烁，确保患者暴露水平远在安全范围内是有帮助1。以允许最佳成像，它是扩张瞳孔到至少6毫米，以获得具有通畅光传输的多个图像（见上文）的关键。实现最佳的对焦和避免像素的过饱和也是至关重要的。有益的做法包括与患者沟通，同时成像，如果眼睑需要如上所述被提升并使用脚踏触发摄像机，使用助手。 总之，联合国derstanding在视网膜变性的视网膜色素上皮的病理生理学仍然活跃的研究领域，并且潜在的重要治疗的影响。至于QAF允许纵向获得患者之间以及中心之间的图像自动对焦的水平直接比较，这是一个有价值的工具，可以促进这种理解，以及提供有用的临床信息。这里列出的详细的协议将有助于在获取可靠的数据QAF别人，那QAF的重要临床应用将鼓励研究中心和临床视网膜专家利用QAF技术。

视网膜色素上皮细胞（RPE）支持感官视网膜通过众多方法1的功能。年龄相关性黄斑变性（AMD）是在工业化国家无法治疗失明的最重要的原因，其特征是在RPE中的变化，包括颜料的损失，功能和萎缩损失。在AMD和在正常的老化中，RPE积聚含有吞噬感光体的片段，称为脂褐素颗粒的荧光，溶酶体衍生的细胞器。 RPE脂褐素的累积已经认为表明氧化功能低下1，但最近的研究表明，视网膜色素上皮形态学老年眼睛保持正常高脂褐素水平2。然而，脂褐质分布异常模式，脂褐素的具体损失，都记录标志物AMD和AMD的进展，无论是组织学和临床3,4 缺陷PROCESRPE脂褐素唱也已显示在某些遗传性视网膜变性发生。患有斯特格病（STGD）遭受聚积在RPE脂褐素在年轻的时候，最终建立类似于AMD 5所示视力丧失。这些结果表明，脂褐质积聚本身可能是有毒的和驱动RPE功能障碍6,7。然而，受试者STGD随着时间的推移了详细的影像学研究并未证实焦点脂褐素的积累导致了随后的RPE损失8。因此，尽管脂褐质的异常是视网膜变性的标记，对于脂褐素的直接毒性作用尚未得到证实。 视网膜色素上皮是视网膜的最后的细胞层，但是产生从眼底的多数荧光信号。可使用共焦扫描激光眼底镜（CSLO），其允许六执行从RPE衍生产生和自体荧光（AF）的检测眼底AF的空间分布sualization。某些视网膜变性表明在这些条件下，诊断和监测眼底房颤独特的模式，和AF成像助剂。虽然标准的自动对焦成像是临床上重要的，定量的AF（QAF）已成为评估RPE健康的一个重要手段。我们和其他人开发了一种标准化的方法，能够可靠地确定在特定的视网膜部位9 QAF水平。 QAF在诊断和潜在的应用的视网膜条件监视，并且还可以具有在预后和风险分层效用。此外，QAF的诊断功能也已为某些视网膜疾病10-12所述。在这里，我们提供了对我们的技术伴随着它在健康和患病的眼睛的评价中的应用的可视化演示逐步细节。

<table border="0" cellpadding="0" cellspacing="0" width="671">
	<tbody>
		<tr height="19">
			<td height="19" style="height:19px;width:243px;">Spectralis HRA + OCT</td>
			<td style="width:143px;">Heidelberg Engineering</td>
			<td style="width:119px;">n/a</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">0.5% tropicamide ophthalmic solution</td>
			<td style="width:143px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td>Any brand can be used</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">2.5% phenylephrine ophthalmic solution</td>
			<td style="width:143px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td>Any brand can be used</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:243px;">Internal fluorescent reference</td>
			<td style="width:143px;">Heidelberg Engineering</td>
			<td style="width:119px;">n/a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">IGOR Pro software</td>
			<td style="width:143px;">WaveMetrics</td>
			<td style="width:119px;">n/a</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantitative fundus autofluorescence for the evaluation of retinal diseases

The retinal pigment epithelium (RPE) is juxtaposed to the overlying sensory retina, and supports the function of the visual system. Among the tasks performed by the RPE are phagocytosis and processing of outer photoreceptor segments through lysosome-derived organelles. These degradation products, stored and referred to as lipofuscin granules, are composed partially of bisretinoids, which have broad fluorescence absorption and emission spectra that can be detected clinically as fundus autofluorescence with confocal scanning laser ophthalmoscopy (cSLO). Lipofuscin accumulation is associated with increasing age, but is also found in various patterns in both acquired and inherited degenerative diseases of the retina. Thus, studying its pattern of accumulation and correlating such patterns with changes in the overlying sensory retina are essential to understanding the pathophysiology and progression of retinal disease. Here, we describe a technique employed by our lab and others that uses cSLO in order to quantify the level of RPE lipofuscin in both healthy and diseased eyes.

采用此技术，研究QAF在健康13和疾病状态10-12。在健康的眼睛（ 图1），从RPE发射AF时相对均匀地分布在整个眼底（ 图1A）。减少强度被认为是在黄斑中心区域由于黄斑色素阻挡光，并在两侧和图像的角由于眼睛和照相机的光学系统。血管暗，并应在明确的重点公采集的图像。 图1B说明了从图1A的QAF水平的相应热图表示。冷色调对应强度较低的区域，同时温暖的颜色对应于高强度的领域。最大强度通常被在第二同心8段环（在图1B中所示）。这个区域也是成像相关variabil少受性比区域更接近图像边界，并在这里黄斑色素对QAF水平有很大的影响的中央区域的外侧。因此，可用于大部分数据这枚戒指的平均强度分析13。 图2给出与AMD展示的地理萎缩（GA），AMD的一种高级形式的眼睛有代表性的分析。 AMD结果的这种形式在RPE损失的局部区域​​，通过证明明显减少或缺失自动对焦，并导致进行性中心视力丧失。 <img alt="图1" src="/files/ftp_upload/53577/53577fig1.jpg" /> 图1.自体荧光在健康的眼睛。一个普通患者的右眼（A）自体荧光（AF）的形象。 OD:视盘，佛:凹，马:黄斑，参考:内部参考。 （B）后处理来自（A）AF形象QAF地图。暖色关联到更高的AF强度。固定章离子被示出，并且每个区域的QAF的值表示。在数据分析中使用8段perifoveal环由虚线表示。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53577/53577fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53577/53577fig2.jpg" /> 图2.由于AMD的自体荧光的眼睛显示地理萎缩。（A）的自发荧光（AF）与晚期AMD表示RPE（由虚线外接代表区域）的地图状萎缩（GA）的患者的左眼的图像。 OD:视盘，佛:凹，马:黄斑，参考:内部参考。注意对应于GA在黄斑区显着减少和缺席的AF。 （B）后处理来自（A）AF形象QAF地图。固定区域被示出，并且每个区域的QAF的值重新显示。在数据分析中使用8段perifoveal环由虚线表示。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53577/53577fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Quantitative Fundus Autofluorescence for the Evaluation of Retinal Diseases at JoVE.com

Quantitative Fundus Autofluorescence for the Evaluation of Retinal Diseases

定量自体荧光眼底视网膜疾病的评价

The retinal pigment epithelium (RPE) supports the sensory retina through recycling visual cycle byproducts, which accumulate as lipofuscin. These products are autofluorescent and can be qualitatively imaged in vivo. Here, we describe a method to quantitatively image RPE lipofuscin using confocal scanning laser ophthalmoscopy.

The retinal pigment epithelium (RPE) is juxtaposed to the overlying sensory retina, and supports the function of the visual system. Among the tasks ...

quantitative-fundus-autofluorescence-for-evaluation-retinal

Nanyang Technological University

Research

JoVE Journal

Medicine

11.3K 次观看.  NYU School of Medicine. The retinal pigment epithelium (RPE) supports the sensory retina through recycling visual cycle byproducts, which accumulate as lipofuscin. These products are autofluorescent and can be qualitatively imaged in vivo. Here, we describe a method to quantitatively image RPE lipofuscin using confocal scanning laser ophthalmoscopy. 

视频: Quantitative Fundus Autofluorescence for the Evaluation of Retinal Diseases

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

Parkinson's disease (PD) is the second most common movement disorder and affects 1% of people over the age of 60 1. Because ageing is the most important risk factor, cases of PD will increase during the next decades 2. Next to pathological protein folding and impaired protein degradation pathways, alterations of mitochondrial function and morphology were pointed out as further hallmark of neurodegeneration in PD 3-11.
After years of research in murine and human cancer cells as in vitro models to dissect molecular pathways of Parkinsonism, the use of human fibroblasts from patients and appropriate controls as ex vivo models has become a valuable research tool, if potential caveats are considered. Other than immortalized, rather artificial cell models, primary fibroblasts from patients carrying disease-associated mutations apparently reflect important pathological features of the human disease.
Here we delineate the procedure of taking skin biopsies, culturing human fibroblasts and using detailed protocols for essential microscopic techniques to define mitochondrial phenotypes. These were used to investigate different features associated with PD that are relevant to mitochondrial function and dynamics. Ex vivo, mitochondria can be analyzed in terms of their function, morphology, colocalization with lysosomes (the organelles degrading dysfunctional mitochondria) and degradation via the lysosomal pathway. These phenotypes are highly relevant for the identification of early signs of PD and may precede clinical motor symptoms in human disease-gene carriers. Hence, the assays presented here can be utilized as valuable tools to identify pathological features of neurodegeneration and help to define new therapeutic strategies in PD.

Fibroblasts from patients carrying mutations in Parkinson's disease-causing genes represent an easily accessible ex vivo model to study disease-associated phenotypes. Live cell imaging gives the opportunity to study morphological and functional parameters in living cells. Here we describe the preparation of human fibroblasts and subsequent monitoring of mitochondrial phenotypes.

利用人类纤维原细胞线粒体表型的监测领域的帕金森氏病

Parkinson's disease (PD) is the second most  common movement disorder and affects 1% of people over the age of 60 1. Because ageing is  the most important ...

科研

医学

Techniques for Processing Eyes Implanted with a Retinal Prosthesis for Localized Histopathological Analysis: Part 2 Epiretinal Implants with Retinal Tacks

Retinal prostheses for the treatment of certain forms of blindness are gaining traction in clinical trials around the world with commercial devices currently entering the market. In order to evaluate the safety of these devices, in preclinical studies, reliable techniques are needed. However, the hard metal components utilised in some retinal implants are not compatible with traditional histological processes, particularly in consideration for the delicate nature of the surrounding tissue. Here we describe techniques for assessing the health of the eye directly adjacent to a retinal implant secured epiretinally with a metal tack.
Retinal prostheses feature electrode arrays in contact with eye tissue. The most commonly used location for implantation is the epiretinal location (posterior chamber of the eye), where the implant is secured to the retina with a metal tack that penetrates all the layers of the eye. Previous methods have not been able to assess the proximal ocular tissue with the tack in situ, due to the inability of traditional histological techniques to cut metal objects. Consequently, it has been difficult to assess localized damage, if present, caused by tack insertion.
Therefore, we developed a technique for visualizing the tissue around a retinal tack and implant. We have modified an established technique, used for processing and visualizing hard bony tissue around a cochlear implant, for the soft delicate tissues of the eye. We orientated and embedded the fixed eye tissue, including the implant and retinal tack, in epoxy resin, to stabilise and protect the structure of the sample. Embedded samples were then ground, polished, stained, and imaged under various magnifications at incremental depths through the sample. This technique allowed the reliable assessment of eye tissue integrity and cytoarchitecture adjacent to the metal tack.

Here we describe histological techniques for visualising ocular tissue directly adjacent to a metal epiretinal tack and retinal prosthesis.

为处理眼植入视网膜假体的局部病理组织学分析技术:第2部分视网膜前植入视网膜大头钉

Retinal prostheses for the treatment of certain forms of blindness are gaining traction in clinical trials around the world with commercial devices ...

Automated Radiochemical Synthesis of [18F]3F4AP: A Novel PET Tracer for Imaging Demyelinating Diseases

3-[18F]fluoro-4-aminopyridine, [18F]3F4AP, is a radiofluorinated analog of the FDA-approved drug for multiple sclerosis 4-aminopyridine (4AP). This compound is currently under investigation as a PET tracer for demyelination. We recently described a novel chemical reaction to produce metafluorinated pyridines consisting of direct fluorination of a pyridine N-oxide and the utilization of this reaction for the radiochemical synthesis of [18F]3F4AP. In this article, we demonstrate how to produce this tracer using an automated synthesizer and an in-house made flow hydrogenation reactor. We also show the standard quality control procedures performed before releasing the radiotracer for preclinical animal imaging studies. This semi-automated procedure may serve as the basis for future production of [18F]3F4AP for clinical studies.

Automated Radiochemical Synthesis of [18F]3F4AP: A Novel PET Tracer for Imaging Demyelinating Diseases

We demonstrate the semi-automated radiochemical synthesis of [18F]3F4AP and quality control procedures.

自动放射化学合成[<sup&gt; 18</sup&gt; F] 3F4AP:一种用于成像脱髓鞘疾病的新型PET示踪剂

3-[18F]fluoro-4-aminopyridine, [18F]3F4AP, is a radiofluorinated analog of the FDA-approved drug for multiple sclerosis 4-aminopyridine (4AP). This ...

Smartphone Fundus Photography

Smartphone fundus photography is a simple technique to obtain ocular fundus pictures using a smartphone camera and a conventional handheld indirect ophthalmoscopy lens. This technique is indispensable when picture documentation of optic nerve, retina, and retinal vessels is necessary but a fundus camera is not available. The main advantage of this technique is the widespread&#160;availability of smartphones that allows documentation of macula and optic nerve changes in many settings that was not previously possible. Following the well-defined steps detailed here, such as proper alignment of the phone camera, handheld lens, and the patient&#39;s pupil, is the key for obtaining a clear retina picture with no interfering light reflections and aberrations. In this paper, the optical principles of indirect ophthalmoscopy and fundus photography will be reviewed first. Then, the step-by-step method to record a good quality retinal image using a smartphone will be explained.

Fundus photography normally requires specialized fundus cameras that are not always available in all clinical settings.&#160;Here, a simple method to record ocular fundus images using a smartphone camera and a conventional high-plus handheld indirect ophthalmoscopy lens is described.

智能手机眼底摄影

Smartphone fundus photography is a simple technique to obtain ocular fundus pictures using a smartphone camera and a conventional handheld indirect ...

Using Retinal Imaging to Study Dementia

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central nervous system (CNS) and shares prominent similarities with the brain in terms of embryological origin, anatomical features and physiological properties.&#160; The vascular and neuronal structure in the retina can now be visualized easily and non-invasively using retinal imaging techniques, including fundus photography and optical coherence tomography (OCT), and quantified semi-automatically using computer-assisted analysis programs. Studying the associations between vascular and neuronal changes in the retina and dementia could improve our understanding of dementia and, potentially, aid in diagnosis and risk assessment.&#160; This protocol aims to describe a method of quantifying and analyzing retinal vasculature and neuronal structure, which are potentially associated with dementia. This protocol also provides examples of retinal changes in subjects with dementia, and discusses technical issues and current limitations of retinal imaging.

The retina shares prominent similarities with the brain and thus represents a unique window to study vasculature and neuronal structure in the brain non-invasively. This protocol describes a method to study dementia using retinal imaging techniques. This method can potentially aid in diagnosis and risk assessment of dementia.

利用视网膜成像研究痴呆

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central ...

Invasive Hemodynamic Assessment for the Right Ventricular System and Hypoxia-Induced Pulmonary Arterial Hypertension in Mice

Pulmonary arterial hypertension (PAH) is a chronic and severe cardiopulmonary disorder. Mice are a popular animal model used to mimic this disease. However, the evaluation of right ventricular pressure (RVP) and pulmonary artery pressure (PAP) remains technically challenging in mice. RVP and PAP are more difficult to measure than left ventricular pressure because of the anatomical differences between the left and right heart systems. In this paper, we describe a stable right heart hemodynamic measurement method and its validation using healthy and PAH mice. This method is based on open-chest surgery and mechanical ventilation support. It is a complicated procedure compared to closed chest procedures. While a well-trained surgeon is required for this surgery, the advantage of this procedure is that it can generate both RVP and PAP parameters at the same time, so it is a preferable procedure for the evaluation of PAH models.

Here, we present a protocol to perform an invasive hemodynamic assessment of the right ventricle and pulmonary artery in mice using an open-chest surgery approach.

小鼠右心室系统及缺氧诱发肺动脉高血压的侵入性血液动力学评估

Pulmonary arterial hypertension (PAH) is a chronic and severe cardiopulmonary disorder. Mice are a popular animal model used to mimic this disease. ...

Oxygen-Induced Retinopathy Model for Ischemic Retinal Diseases in Rodents

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR model induction and its readouts in both mice and rats. Retinal neovascularization is induced in OIR by exposing rodent pups either to hyperoxia (mice) or alternating levels of hyperoxia and hypoxia (rats). The primary readouts of these models are the size of neovascular (NV) and avascular (AVA) areas in the retina. This preclinical in vivo model can be used to evaluate the efficacy of potential anti-angiogenic drugs or to address the role of specific genes in the retinal angiogenesis by using genetically manipulated animals. The model has some strain and vendor specific variation in the OIR induction which should be taken into consideration when designing the experiments.

Oxygen-induced retinopathy (OIR) can be used to model ischemic retinal diseases such as retinopathy of prematurity and proliferative diabetic retinopathy and to serve as a model for proof-of-concept studies in evaluating antiangiogenic drugs for neovascular diseases. OIR induces robust and reproducible neovascularization in the retina that can be quantified.

罗登斯缺血性视网膜疾病的氧诱导视网膜病变模型

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR ...

Retinal Pigment Epithelium Transplantation in a Non-human Primate Model for Degenerative Retinal Diseases

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These conditions include retinitis pigmentosa (RP) and advanced forms of age-related macular degeneration (AMD), such as geographic atrophy (GA). Together, these disorders represent a significant proportion of currently untreatable blindness globally. These unmet medical needs have generated heightened academic interest in developing methods of RPE replacement. Among the animal models commonly utilized for preclinical testing of therapeutics, the non-human primate (NHP) is the only animal model that has a macula. As it shares this anatomical similarity with the human eye, the NHP eye is an important and appropriate preclinical animal model for the development of advanced therapy medicinal products (ATMPs) such as RPE cell therapy.
This manuscript describes a method for the submacular transplantation of an RPE monolayer, cultured on a polyethylene terephthalate (PET) cell carrier, underneath the macula onto a surgically created RPE wound in immunosuppressed NHPs. The fovea-the central avascular portion of the macula-is the site of the greatest mechanical weakness during the transplantation. Foveal trauma will occur if the initial subretinal fluid injection generates an excessive force on the retina. Hence, slow injection under perfluorocarbon liquid (PFCL) vitreous tamponade is recommended with a dual-bore subretinal injection cannula at low intraocular pressure (IOP) settings to create a retinal bleb. 
Pretreatment with an intravitreal plasminogen injection to release parafoveal RPE-photoreceptor adhesions is also advised. These combined strategies can reduce the likelihood of foveal tears when compared to conventional techniques. The NHP is a key animal model in the preclinical phase of RPE cell therapy development. This protocol addresses the technical challenges associated with the delivery of RPE cellular therapy in the NHP eye.

The non-human primate (NHP) is an ideal model for studying human retinal cellular therapeutics due to the anatomical and genetic similarities. This manuscript describes a method for submacular transplantation of retinal pigment epithelial cells in the NHP eye and strategies to prevent intraoperative complications associated with macular manipulation.

非人灵长类动物模型中视网膜色素上皮移植治疗退行性视网膜疾病

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These ...

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 ...

Repetitive Blood Sampling from the Subclavian Vein of Conscious Rat

Rats are widely used in pharmacokinetics (PK) and toxicokinetic (TK) studies that need to collect a certain amount of blood at specific time points to detect drug exposure. The rat blood sampling method determines the quality of the plasma and further affects the precision of the test results. The subclavian vein blood collection method described in this protocol collects blood samples repeatedly in the conscious state of animals to meet the needs of PK and TK tests. The skills of restraint handling and appropriate procedure of needle incision ensure the success rate of blood collection. It is easy to operate while ensuring the quality of plasma and at the same time catering to animal welfare. However, this method requires skilled operation, and an improper one may cause animal weakness, pain, lameness, and even mortality. The current method has been used in the testing facility for a 4-week oral toxicity study in Sprague Dawley (SD) rats with TK. The maximum amount of blood collected within 24 h did not exceed 20% of the animal's total blood. The animals' body weight was more than 200 g for males and females. The data showed that the animals' bodyweight increased steadily every week, and the clinical observation was normal after repetitive sample collection.

The present protocol describes a simple and efficient method for collecting blood from the subclavian vein in rats. It enables rapid, timely, and easily identifiable sampling without anesthesia and obtains high-quality blood through repetitive sample collection.

意识大鼠锁骨下静脉的重复血液采样

Rats are widely used in pharmacokinetics (PK) and toxicokinetic (TK) studies that need to collect a certain amount of blood at specific time points to ...