关于潜在的金融关系, 作者发明是新加坡 I 船评估 (湿婆) 项目中使用的一份文件。

我们对新加坡国立大学计算机学院的技术支持表示感谢。

本协议描述了在视网膜体内量化神经元和血管变化的过程。由于视网膜与大脑有着相似的胚胎来源、解剖特征和生理特性, 这些视网膜的变化可能反映了大脑中血管和神经元结构的类似变化。
如图 10和表 1所示, 与健康主题相比, AD 主题显示的容器口径减少。据报道, 减少 CRVE 和 CRAE 是与阿尔茨海默氏病相关的46,</s...

由于预期寿命的增加, 痴呆已成为一个重大的医疗问题, 导致重大的社会和经济健康负担全球1,2,3,4,5。今天, 一个人在美国发展老年痴呆症 (AD), 最常见的形式的痴呆, 每六十六年代6。据估计, 到公元 2050, 1亿1500万人将受到 AD7的影响。
视网膜提供了一个独特的 "窗口", 以研究痴呆, 由于其类似的解剖和生理特性与大脑。在血管方面, 视网膜动脉和静脉, 测量直径100至300µm, 与脑小血管有相似的特征, 如无吻合的端动脉, 屏障功能, 和调节8,9. 在神经元结构方面, 视网膜节细胞 (视网膜) 与中枢神经系统 (CNS) 10 的神经元有着典型的特性。视网膜是突出的连接与大脑, 因为他们形成的视神经和项目的视觉信号从视网膜到外侧膝状核和优越的丘。视神经, 类似于中枢神经系统中的许多神经纤维, 由突髓, ensheathed 于脑膜层。值得注意的是, 对视神经的污辱会导致其他中枢神经系统轴突的类似反应, 如轴突逆行和顺变性、瘢痕形成、髓鞘破坏、继发变性和神经营养异常水平因素和神经递质11,12,13,14。一些 AD 患者的视觉症状的出现也可以通过视网膜和大脑之间的强关联来解释15,16。因此, 有人建议, 视网膜可能反映痴呆的病理过程中的大脑和视网膜成像可以用来研究痴呆症。
视网膜血管和神经元结构现在可以可视化性利用视网膜成像技术。例如, 视网膜眼底照片可以使用眼底照相机捕捉, 而视网膜血管的特征 (例如,血管口径、曲折和分形维数) 可以通过计算机辅助分析进行量化程序.此外, 还可以使用光学相干断层扫描 (OCT) 测量视网膜神经元结构 (如神经节细胞-内状层的厚度 [GC-光子) 和视网膜神经纤维层 [纤维]) 的参数, 并使用内置分析算法。
鉴于视网膜成像对研究痴呆的重要性, 本议定书的目的是描述一种成像和分析视网膜血管和神经元结构在体内使用视网膜成像技术的方法。该协议还提供了一些例子, 视网膜变化的主题与痴呆, 并讨论技术问题和目前的局限性, 视网膜成像。

<table><tbody><tr><td>Non-mydriatic Retinal Camera&amp;nbsp;</td><td>Topcon, Inc, Tokyo, Japan</td><td>TRC 50DX&amp;nbsp;</td><td>N/A</td></tr><tr><td>Singapore I Vessel Assessment Program</td><td>National University of Singapore</td><td>Version 4.0</td><td>N/A</td></tr><tr><td>CIRRUS HD-OCT&amp;nbsp;</td><td>Carl Zeiss Meditec, Inc, Dublin, CA</td><td>Model 4000</td><td>N/A</td></tr><tr><td>Mydriatic Agents&amp;nbsp;</td><td>N/A</td><td>N/A</td><td>Prepared from 1% tropicamide and 2.5% phenylephrine hydrochloride</td></tr></tbody></table>

using retinal imaging to study dementia

视网膜提供了一个独特的 "窗口" 来研究大脑中的老年痴呆的病理生理过程, 因为它是中枢神经系统 (CNS) 的延伸, 与大脑在胚胎起源、解剖特征和生理特性。 视网膜的血管和神经元结构现在可以很容易地被形象化和性使用视网膜成像技术, 包括眼底摄影和光学相干断层扫描 (OCT), 和量化半自动使用计算机辅助分析程序。研究视网膜和痴呆的血管和神经元变化之间的联系可以提高我们对痴呆症的认识, 并可能有助于诊断和风险评估。 本议定书的目的是描述一种量化和分析视网膜血管和神经元结构的方法, 这可能与痴呆有关。该协议还提供了一些例子, 视网膜变化的主题与痴呆, 并讨论技术问题和目前的局限性, 视网膜成像。

<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/56137/56137fig10.jpg"> 
图 10:一个例子, 以显示在正常的主题和一个广告主题之间的视网膜血管的差异.与正常人相比, AD 的眼底照片显示狭窄的血管口径 (CRAE 区 B、116.4 µm vs. 156.4 µm;CRVE 区 B, 186.9 µm vs. 207.5 µm;CRAE 区 C, 138.5 µm vs. 165.8 µm;CRVE 区 C, 206.6 µm vs. 232.2 µm), 更小...

Watch this Scientific Journal Video about Using Retinal Imaging to Study Dementia at JoVE.com

Using Retinal Imaging to Study Dementia

利用视网膜成像研究痴呆

视网膜与大脑有着显著的相似性, 因此它是研究脑性中血管和神经元结构的独特窗口。本协议描述了一种利用视网膜成像技术研究痴呆症的方法。这种方法对痴呆的诊断和风险评估有潜在的帮助。

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 ...

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

21.4K 次观看.  The Chinese University of Hong Kong. 视网膜与大脑有着显著的相似性, 因此它是研究脑性中血管和神经元结构的独特窗口。本协议描述了一种利用视网膜成像技术研究痴呆症的方法。这种方法对痴呆的诊断和风险评估有潜在的帮助。

视频: Using Retinal Imaging to Study Dementia

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with distinct functions and gene expression profiles. The roles of microglial activation in health, injury and disease remain incompletely understood due to their dynamic and complex regulation in response to changes in their microenvironment. Thus, it is critical to non-invasively monitor and analyze changes in microglial activation over time in the intact organism. In vivo studies of microglial activation have been delayed by technical limitations to tracking microglial behavior without altering the CNS environment. This has been particularly challenging during chronic neurodegeneration, where long-term changes must be tracked. The retina, a CNS organ amenable to non-invasive live imaging, offers a powerful system to visualize and characterize the dynamics of microglia activation during chronic disorders.
This protocol outlines methods for long-term, in vivo imaging of retinal microglia, using confocal ophthalmoscopy (cSLO) and CX3CR1GFP/+ reporter mice, to visualize microglia with cellular resolution. Also, we describe methods to quantify monthly changes in cell activation and density in large cell subsets (200-300 cells per retina). We confirm the use of somal area as a useful metric for live tracking of microglial activation in the retina by applying automated threshold-based morphometric analysis of in vivo images. We use these live image acquisition and analyses strategies to monitor the dynamic changes in microglial activation and microgliosis during early stages of retinal neurodegeneration in a mouse model of chronic glaucoma. This approach should be useful to investigate the contributions of microglia to neuronal and axonal decline in chronic CNS disorders that affect the retina and optic nerve.

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia activation and microgliosis are key responses to chronic neurodegeneration. Here, we present methods for in vivo, long-term visualization of retinal CX3CR1-GFP+ microglial cells by confocal ophthalmoscopy, and for threshold and morphometric analyses to identify and quantify their activation. We monitor microglial changes during early stages of age-related glaucoma.

视网膜胶质细胞活化的体内动力学在神经退行性疾病:共聚焦成像眼底及细胞形态计量小鼠青光眼

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with ...

科研

医学

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.

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.

定量自体荧光眼底视网膜疾病的评价

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

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular plasticity or damage occurring overtime and in relation to neuronal activity or blood flow. In vivo two-photon microscopy allows the study of the structural and functional plasticity of large cellular units in the living brain. In particular, the thinned-skull window preparation allows the visualization of cortical regions of interest (ROI) without inducing significant brain inflammation. Repetitive imaging sessions of cortical ROI are feasible, providing the characterization of disease hallmarks over time during the progression of numerous CNS diseases. This technique accessing the pial structures within 250 &#956;m of the brain relies on the detection of fluorescent probes encoded by genetic cellular markers and/or vital dyes. The latter (e.g., fluorescent dextrans) are used to map the luminal compartment of cerebrovascular structures. Germane to the protocol described herein is the use of an in vivo marker of amyloid deposits, Methoxy-O4, to assess Alzheimer&#39;s disease (AD) progression. We also describe the post-acquisition image processing used to track vascular changes and amyloid depositions. While focusing presently on a model of AD, the described protocol is relevant to other CNS disorders where pathological cerebrovascular changes occur.

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

This manuscript describes a procedure to track the remodeling of the cerebrovasculature during amyloid plaque accumulation in vivo using longitudinal two-photon microscopy. A thinned-skull preparation enables the visualization of fluorescent dyes to assess the progression of cerebrovascular damage in a mouse model of Alzheimer&#39;s disease.

纵<em&gt;在体内</em&gt;的Cerebrovasculature影像:相关性疾病CNS

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular ...

Microsurgical Dissection and Tissue Clearing for High Resolution Intact Whole Retina and Vitreous Imaging

Neuronal and vascular structures of the retina in physiologic and pathologic conditions can be better visualized and characterized by using intact whole retina imaging techniques compared to conventional retinal flat mount preparations and sections. However, immunofluorescent imaging of intact whole retina is hindered by the opaque coatings of the eyeball, i.e., sclera, choroid, and retinal pigment epithelium (RPE) and the light scattering properties of retinal layers that prevent full thickness high resolution optical imaging. Chemical bleaching of the pigmented layers and tissue clearing protocols have been described to address these obstacles; however, currently described methods are not suitable for imaging endogenous fluorescent molecules such as green fluorescent protein (GFP) in intact whole retina. Other approaches bypassed this limitation by surgical removal of pigmented layers and the anterior segment of the eyeball allowing intact eye imaging, though the peripheral retina and hyaloid structures were disrupted. Presented here is an intact whole retina and vitreous immunofluorescent imaging protocol that combines surgical dissection of the sclera/choroid/retina pigment epithelium (RPE) layers with a modified tissue clearing method and light sheet fluorescent microscopy (LSFM). The new approach offers an unprecedented view of&#160;unperturbed vascular and neuronal elements of the retina as well as the vitreous and hyaloid vascular system in pathologic conditions.

Presented here is a protocol for intact whole retina imaging in which the outer opaque/pigmented layers of the eyeball are surgically removed, and optical clearing is applied to render retina transparent enabling the visualization of the peripheral retina and hyaloid vasculature in intact retina using light sheet fluorescent microscopy.

Neuronal and vascular structures of the retina in physiologic and pathologic conditions can be better visualized and characterized by using intact whole ...

神经科学

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

小鼠视网膜的经瞳双光子体内成像

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

Time-Lapse Imaging of Neuronal Arborization using Sparse Adeno-Associated Virus Labeling of Genetically Targeted Retinal Cell Populations

Discovering mechanisms that pattern dendritic arbors requires methods to visualize, image, and analyze dendrites during development. The mouse retina is a powerful model system for the investigation of cell type-specific mechanisms of neuronal morphogenesis and connectivity. The organization and composition of retinal subtypes are well-defined, and genetic tools are available to access specific types during development. Many retinal cell types also constrain their dendrites and/or axons to narrow layers, which facilitates time-lapse imaging. Mouse retina explant cultures are well suited for live-cell imaging using confocal or multiphoton microscopy, but methods optimized for imaging dendrite dynamics with temporal and structural resolution are lacking. Presented here is a method to sparsely label and image the development of specific retinal populations marked by the Cre-Lox system. Commercially available adeno-associated viruses (AAVs) used here expressed membrane-targeted fluorescent proteins in a Cre-dependent manner. Intraocular delivery of AAVs in neonatal mice produces fluorescent labeling of targeted cell types by 4-5 days post-injection (dpi). The membrane fluorescent signals are detectable by confocal imaging and resolve fine branch structures and dynamics. High-quality videos spanning 2-4 h are acquired from imaging retinal flat-mounts perfused with oxygenated artificial cerebrospinal fluid (aCSF). Also provided is an image postprocessing pipeline for deconvolution and three-dimensional (3D) drift correction. This protocol can be used to capture several cellular behaviors in the intact retina and to identify novel factors controlling neurite morphogenesis. Many developmental strategies learned in the retina will be relevant for understanding the formation of neural circuits elsewhere in the central nervous system.

Here, we present a method for investigating neurite morphogenesis in postnatal mouse retinal explants by time-lapse confocal microscopy. We describe an approach for sparse labeling and acquisition of retinal cell types and their fine processes using recombinant adeno-associated virus vectors that express membrane-targeted fluorescent proteins in a Cre-dependent manner.

使用稀疏腺相关病毒标记遗传靶向视网膜细胞群的神经元树化延时成像

Discovering mechanisms that pattern dendritic arbors requires methods to visualize, image, and analyze dendrites during development. The mouse retina is a ...

用于 AMD 研究的基于 OCT 的人类供体眼多模态成像

Ex Vivo OCT-Based Multimodal Imaging of Human Donor Eyes for Research into Age-Related Macular Degeneration

A progression sequence for age-related macular degeneration (AMD) learned from optical coherence tomography (OCT)-based multimodal (MMI) clinical imaging could add prognostic value to laboratory findings. In this work, ex vivo OCT and MMI were applied to human donor eyes prior to retinal tissue sectioning. The eyes were recovered from non-diabetic white donors aged &#8805;80 years old, with a death-to-preservation time (DtoP) of &#8804;6 h. The globes were recovered on-site, scored with an 18 mm trephine to facilitate cornea removal, and immersed in buffered 4% paraformaldehyde. Color fundus images were acquired after anterior segment removal with a dissecting scope and an SLR camera using trans-, epi-, and flash illumination at three magnifications. The globes were placed in a buffer within a custom-designed chamber with a 60 diopter lens. They were imaged with spectral domain OCT (30&#176; macula cube, 30 &#181;m spacing, averaging = 25), near-infrared reflectance, 488 nm autofluorescence, and 787 nm autofluorescence. The AMD eyes showed a change in the retinal pigment epithelium (RPE), with drusen or subretinal drusenoid deposits (SDDs), with or without neovascularization, and without evidence of other causes. Between June 2016 and September 2017, 94 right eyes and 90 left eyes were recovered (DtoP: 3.9 &plusmn; 1.0 h). Of the 184 eyes, 40.2% had AMD, including early intermediate (22.8%), atrophic (7.6%), and neovascular (9.8%) AMD, and 39.7% had unremarkable maculas. Drusen, SDDs, hyper-reflective foci, atrophy, and fibrovascular scars were identified using OCT. Artifacts included tissue opacification, detachments (bacillary, retinal, RPE, choroidal), foveal cystic change, an undulating RPE, and mechanical damage. To guide the cryo-sectioning, OCT volumes were used to find the fovea and optic nerve head landmarks and specific pathologies. The ex vivo volumes were registered with the in vivo volumes by selecting the reference function for eye tracking. The ex vivo visibility of the pathology seen in vivo depends on the preservation quality. Within 16 months, 75 rapid DtoP donor eyes at all stages of AMD were recovered and staged using clinical MMI methods.

作者聚焦:基于离体 OCT 的人类供体眼多模态成像，用于年龄相关性黄斑变性的研究  

Laboratory assays can leverage prognostic value from the longitudinal optical coherence tomography (OCT)-based multimodal imaging of age-related macular degeneration (AMD). Human donor eyes with and without AMD are imaged using OCT, color, near-infrared reflectance scanning laser ophthalmoscopy, and autofluorescence at two excitation wavelengths prior to tissue sectioning.

离体 基于OCT的人供体眼睛多模态成像用于研究年龄相关性黄斑变性

A progression sequence for age-related macular degeneration (AMD) learned from optical coherence tomography (OCT)-based multimodal (MMI) clinical imaging ...

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.

利用视网膜成像研究痴呆

摘要

探索更多视频

此视频中的章节

视网膜胶质细胞活化的体内动力学在神经退行性疾病:共聚焦成像眼底及细胞形态计量小鼠青光眼

定量自体荧光眼底视网膜疾病的评价

纵

Microsurgical Dissection and Tissue Clearing for High Resolution Intact Whole Retina and Vitreous Imaging

小鼠视网膜的经瞳双光子体内成像

使用稀疏腺相关病毒标记遗传靶向视网膜细胞群的神经元树化延时成像

离体基于OCT的人供体眼睛多模态成像用于研究年龄相关性黄斑变性

离体基于OCT的人供体眼睛多模态成像用于研究年龄相关性黄斑变性

离体基于OCT的人供体眼睛多模态成像用于研究年龄相关性黄斑变性

大鼠模型中的视网膜病理生理学评估

利用视网膜成像研究痴呆

摘要

探索更多视频

此视频中的章节

视网膜胶质细胞活化的体内动力学在神经退行性疾病:共聚焦成像眼底及细胞形态计量小鼠青光眼

定量自体荧光眼底视网膜疾病的评价

纵

Microsurgical Dissection and Tissue Clearing for High Resolution Intact Whole Retina and Vitreous Imaging

小鼠视网膜的经瞳双光子体内成像

使用稀疏腺相关病毒标记遗传靶向视网膜细胞群的神经元树化延时成像

离体 基于OCT的人供体眼睛多模态成像用于研究年龄相关性黄斑变性

离体 基于OCT的人供体眼睛多模态成像用于研究年龄相关性黄斑变性

离体 基于OCT的人供体眼睛多模态成像用于研究年龄相关性黄斑变性

大鼠模型中的视网膜病理生理学评估

离体基于OCT的人供体眼睛多模态成像用于研究年龄相关性黄斑变性

离体基于OCT的人供体眼睛多模态成像用于研究年龄相关性黄斑变性

离体基于OCT的人供体眼睛多模态成像用于研究年龄相关性黄斑变性