Name: 著者スポットライト:緑内障の診断と治療を改善するためのIn vivoおよびex vivo網膜イメージングの進歩
Uploaded: 2023-06-30T13:00:00
Description: Watch this Scientific Journal Video about Advancements in In Vivo and Ex Vivo Retinal Imaging for Improved Glaucoma Diagnosis and Treatment at JoVE.com

この研究は、緑内障研究財団シェイファー助成金、4-CA Cavalier Collaborative Award、R01EY029121、R01EY035088、およびテンプル騎士団眼科財団によってサポートされています。

すべての動物処置は、バージニア大学の動物実験委員会によって承認され、米国国立衛生研究所(NIH)の動物使用に関するガイドラインに準拠しています。このプロトコルで使用されるすべての材料、試薬、および機器に関連する詳細については、 材料表 を参照してください。
1. in vivo vis-OCTイメージング
<ol><li>vis-OCTシステム<ol><li>480 nm か...

マウス Retina OCT ファイバーグラムと共焦点画像によるアライメント

<ol>
	<li>Sernagor, E., Eglen, S. J., Wong, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+retinal+ganglion+cell+structure+and+function.">Development of retinal ganglion cell structure and function.</a> Progress in Retinal and Eye Research. 20 (2), 139-174 (2001).</li><li>Sanes, J. R., Masland, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+types+of+retinal+ganglion+cells:+current+status+and+implications+for+neuronal+classification.">The types of retinal ganglion cells: current status and implications for neuronal classification.</a> Annual Review of Neuroscience. 38, 221-246 (2015).</li><li>Seabrook, T. A., Burbridge, T. J., Crair, M. C., Huberman, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Architecture,+function,+and+assembly+of+the+mouse+visual+system.">Architecture, function, and assembly of the mouse visual system.</a> Annual Review of Neuroscience. 40, 499-538 (2017).</li><li>Cang, J., Savier, E., Barchini, J., Liu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+function,+organization,+and+development+of+the+mouse+superior+colliculus.">Visual function, organization, and development of the mouse superior colliculus.</a> Annual Review of Vision Science. 4, 239-262 (2018).</li><li>Quigley, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+glaucomatous+optic+neuropathy:+the+synergy+between+clinical+observation+and+investigation.">Understanding glaucomatous optic neuropathy: the synergy between clinical observation and investigation.</a> Annual Review of Vision Science. 2, 235-254 (2016).</li><li>Whitmore, A. V., Libby, R. T., John, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glaucoma:+thinking+in+new+ways-a+role+for+autonomous+axonal+self-destruction+and+other+compartmentalised+processes.">Glaucoma: thinking in new ways-a role for autonomous axonal self-destruction and other compartmentalised processes.</a> Progress in Retinal and Eye Research. 24 (6), 639-662 (2005).</li><li>Syc-Mazurek, S. B., Libby, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axon+injury+signaling+and+compartmentalized+injury+response+in+glaucoma.">Axon injury signaling and compartmentalized injury response in glaucoma.</a> Progress in Retinal and Eye Research. 73, 100769 (2019).</li><li>Puyang, Z., Chen, H., Liu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subtype-dependent+morphological+and+functional+degeneration+of+retinal+ganglion+cells+in+mouse+models+of+experimental+glaucoma.">Subtype-dependent morphological and functional degeneration of retinal ganglion cells in mouse models of experimental glaucoma.</a> Journal of Nature and Science. 1 (5), (2015).</li><li>Tatham, A. J., Medeiros, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+structural+progression+in+glaucoma+with+optical+coherence+tomography.">Detecting structural progression in glaucoma with optical coherence tomography.</a> Ophthalmology. 124, S57-S65 (2017).</li><li>Shu, X., Beckmann, L., Zhang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visible-light+optical+coherence+tomography:+a+review.">Visible-light optical coherence tomography: a review.</a> Journal of Biomedical Optics. 22 (12), 1-14 (2017).</li><li>Miller, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visible-light+optical+coherence+tomography+fibergraphy+for+quantitative+imaging+of+retinal+ganglion+cell+axon+bundles.">Visible-light optical coherence tomography fibergraphy for quantitative imaging of retinal ganglion cell axon bundles.</a> Translational Vision Science and Technology. 9 (11), (2020).</li><li>Beckmann, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+the+inner+retinal+layer+structure+in+mice+after+eye-opening+using+visible-light+optical+coherence+tomography.">In vivo imaging of the inner retinal layer structure in mice after eye-opening using visible-light optical coherence tomography.</a> Experimental Eye Research. 211, 108756 (2021).</li><li>Grannonico, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+and+regional+damages+in+retinal+ganglion+cell+axon+bundles+monitored+non-invasively+by+visible-light+optical+coherence+tomography+fibergraphy.">Global and regional damages in retinal ganglion cell axon bundles monitored non-invasively by visible-light optical coherence tomography fibergraphy.</a> Journal of Neuroscience. 41 (49), 10179-10193 (2021).</li><li>Allen-Worthington, K. H., Brice, A. K., Marx, J. O., Hankenson, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraperitoneal+Injection+of+Ethanol+for+the+Euthanasia+of+Laboratory+Mice+(Mus+musculus)+and+Rats+(Rattus+norvegicus).">Intraperitoneal Injection of Ethanol for the Euthanasia of Laboratory Mice (Mus musculus) and Rats (Rattus norvegicus).</a> J Am Assoc Lab Anim Sci. 54 (6), 769-778 (2015).</li><li>Boivin, G. P., Bottomley, M. A., Schiml, P. A., Goss, L., Grobe, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiologic,+Behavioral,+and+Histologic+Responses+to+Various+Euthanasia+Methods+in+C57BL/6NTac+Male+Mice.">Physiologic, Behavioral, and Histologic Responses to Various Euthanasia Methods in C57BL/6NTac Male Mice.</a> J Am Assoc Lab Anim Sci. 56 (1), 69-78 (2017).</li><li>Chen, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+degeneration+of+retinal+and+superior+collicular+functions+in+mice+with+sustained+ocular+hypertension.">Progressive degeneration of retinal and superior collicular functions in mice with sustained ocular hypertension.</a> Investigative Ophthalmology and Visual Science. 56 (3), 1971-1984 (2015).</li><li>Feng, L., Chen, H., Suyeoka, G., Liu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+laser-induced+mouse+model+of+chronic+ocular+hypertension+to+characterize+visual+defects.">A laser-induced mouse model of chronic ocular hypertension to characterize visual defects.</a> Journal of Visualized Experiments: JoVE. 78 (78), (2013).</li><li>Gao, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+of+experimental+glaucoma+on+intrinsically+photosensitive+retinal+ganglion+cells+in+mice.">Differential effects of experimental glaucoma on intrinsically photosensitive retinal ganglion cells in mice.</a> Journal of Comparative Neurology. 530 (9), 1494-1506 (2022).</li><li>Thomson, B. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiopoietin-1+knockout+mice+as+a+genetic+model+of+open-angle+glaucoma.">Angiopoietin-1 knockout mice as a genetic model of open-angle glaucoma.</a> Translational Vision Science and Technology. 9 (4), (2020).</li><li>Feng, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustained+ocular+hypertension+induces+dendritic+degeneration+of+mouse+retinal+ganglion+cells+that+depends+on+cell+type+and+location.">Sustained ocular hypertension induces dendritic degeneration of mouse retinal ganglion cells that depends on cell type and location.</a> Investigative Ophthalmology and Visual Science. 54 (2), 1106-1117 (2013).</li><li>Grannonico, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longitudinal+analysis+of+retinal+ganglion+cell+damage+at+individual+axon+bundle+level+in+mice+using+visible-light+optical+coherence+tomography+fibergraphy.">Longitudinal analysis of retinal ganglion cell damage at individual axon bundle level in mice using visible-light optical coherence tomography fibergraphy.</a> Translational Vision Science and Technology. 12 (5), (2023).</li></ol>

このプロトコルには、注意が必要な 2 つのステップがあります。まず、OCT画像診断の前に、動物が深い麻酔下にあり、目が完全に拡張していることを確認する必要があります。マウスに十分な麻酔がかけられていないと、呼吸が速くなり、 顔 画像の動きが不安定になり、ファイバーグラムの品質に悪影響を及ぼす可能性があります。さらに、拡張が不十分な場合、虹彩が光を遮り、?...

網膜神経節細胞(RGC)は、内網状層(IPL)の樹状突起樹を介してシナプス入力を受け取り、網膜神経線維層(RNFL)の軸索を介して脳に情報を伝達する視覚情報処理において重要な役割を果たします1,2,3,4。緑内障などの病態では、早期のRGC変性により、患者とげっ歯類モデルの両方で、RNFL、神経節細胞層(GCL)、IPL、および視神経に微妙な変化が生じる可能性があります5,6,7,8,9。したがって、RGCのこれらの形態学的変化の早期検出は、RGCと視力喪失を予防するためのタイムリーな介入に不可欠です。
最近では、可視光干渉断層撮影法(vis-OCT)と呼ばれる臨床可能な新しいイメージング技術を開発し、RGC損傷の生体内モニタリングのニーズに応えています。Vis-OCTは軸方向分解能を改善し、網膜10,11で1.3μmに達し、RNFLの個々のRGC軸索束の可視化を可能にしました。その後、マウスの単軸索束レベルでのRGC損傷を追跡および定量化するために、vis-OCTファイバーグラフィー(vis-OCTF)が確立されました11,12,13。ただし、ゴールドスタンダードと同じ網膜のex vivo共焦点イメージングは、in vivo所見を検証するために必要になることがよくあります。したがって、この研究では、vis-OCTFによって取得されたin vivo画像を、同じマウス網膜のex vivo共焦点画像と位置合わせする方法を示します。プロトコルはex vivoの共焦点イメージ投射によって生体内の調査結果を検証し、病気の状態のRGCの損傷の根底にある分子および細胞変化を検査するための基盤を確立することを向ける。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Halo 100</td>
			<td>Opticent Health, Evanston, IL</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss LSM800 microscope</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Drugs and antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4% paraformaldehyde (PFA)</td>
			<td>Santz Cruz Biotechnology, SC-281692</td>
			<td></td>
			<td>1-2 drops</td>
		</tr>
		<tr>
			<td>Bovine serum albumin powder</td>
			<td>Fisher Scientific, BP9706-100</td>
			<td></td>
			<td>1:10</td>
		</tr>
		<tr>
			<td>Donkey anti Mouse Alexa Fluor 488 dye</td>
			<td>Thermo Fisher Scientific, Cat# A-21202</td>
			<td></td>
			<td>1:1,000</td>
		</tr>
		<tr>
			<td>Donkey anti rat Alexa Fluor 594 dye</td>
			<td>Thermo Fisher Scientific, Cat# A-21209</td>
			<td></td>
			<td>1:1,000</td>
		</tr>
		<tr>
			<td>Euthasol (a mixture of pentobarbital sodium (390 mg/mL) and phenytoin sodium (50 mg/mL))</td>
			<td>Covetrus, NDC 11695-4860-1</td>
			<td></td>
			<td>15.6 mg/mL</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Covetrus, NADA043304</td>
			<td></td>
			<td>114 mg/kg</td>
		</tr>
		<tr>
			<td>Mouse anti-Tuj1</td>
			<td>A gift from Anthony J. Spano, University of Virginia</td>
			<td></td>
			<td>1:200</td>
		</tr>
		<tr>
			<td>Normal donkey serum(NDS)</td>
			<td>Millipore Sigma, S30-100 mL</td>
			<td></td>
			<td>1:100</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS, 10x), pH 7.4 
			(Contains 1370 mM NaCl, 27 mM KCl, 80 mM Na2HPO4, and 20 mM KH2PO4)</td>
			<td>Thermo Fisher Scientific, Cat# J62036.K3</td>
			<td></td>
			<td>1:10</td>
		</tr>
		<tr>
			<td>Rat anti-ICAM-2</td>
			<td>BD Pharmingen, Cat#553325</td>
			<td></td>
			<td>1:500</td>
		</tr>
		<tr>
			<td>Tropicamide drops&#160;</td>
			<td>Covetrus, NDC17478-102-12</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100 
			(Reagent Grade)</td>
			<td>VWR, CAS: 9002-93-1</td>
			<td></td>
			<td>1:20</td>
		</tr>
		<tr>
			<td>Vectashield mounting medium</td>
			<td>Vector Laboratories Inc. H2000-10</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Covetrus, NDC59399-110-20</td>
			<td></td>
			<td>17 mg/kg</td>
		</tr>
	</tbody>
</table>

alignment of visible-light optical coherence tomography fibergrams with confocal images of the same mouse retina

近年、眼疾患の神経損傷を客観的に評価するために、生体内網膜イメージングは、生体システムやプロセスに関する非侵襲的、 リアルタイム、縦 断的情報を提供することがますます適用されています。同じ網膜の Ex vivo 共焦点イメージングは、特に動物研究において 、in vivo 所見を検証するためにしばしば必要になります。本研究では、マウス網膜の ex vivo共焦点像と in vivo 像をアライメントする方法を実証した。可視光光干渉断層撮影ファイバーグラフィー(vis-OCTF)と呼ばれる臨床対応の新しいイメージング技術を適用し、マウス網膜の in vivo 画像を取得しました。次に、「ゴールドスタンダード」と同じ網膜の共焦点イメージングを行い、 in vivo vs-OCTF画像を検証しました。本研究は、分子・細胞機構の解明を可能とするだけでなく、 生体内の神経損傷を高感度かつ客観的に評価するための基盤を確立するものです。

Retinal ganglion cells (RGCs) play a critical role in visual information processing, receiving synaptic inputs through their dendritic trees in the inner plexiform layer (IPL) and transmitting the information via their axons in the retinal nerve fiber layer (RNFL) to the brain1,2,3,4. In diseased conditions such as glaucoma, early RGC degeneration may result in subtle changes in the RNFL, the ganglion cell layer (GCL), the IPL, and the optic nerve in both patients and rodent models5,6,7,8,9. Early detection of these morphological changes in RGCs is thus essential for timely intervention to prevent RGC and vision loss.
We have recently developed a new clinical-ready imaging technology called visible-light optical coherence tomography (vis-OCT) to satisfy the need for in vivo monitoring of RGC damage. Vis-OCT improved the axial resolution, reaching 1.3 &#181;m in the retina10, 11, allowing for the visualization of individual RGC axon bundles in the RNFL. Subsequently, vis-OCT fibergraphy (vis-OCTF) was established to track and quantify RGC damage at the single axon bundle level in mice11,12,13. However, ex vivo confocal imaging of the same retina as the gold standard is often necessary to validate the in vivo findings. Therefore, this study will demonstrate how to align in vivo images acquired by vis-OCTF with ex vivo confocal images of the same mouse retina. The protocol aims to validate the in vivo findings by ex vivo confocal imaging and establish a foundation for examining the molecular and cellular changes underlying RGC damage in diseased conditions.

著者スポットライト:緑内障の診断と治療を改善するためのIn vivoおよびex vivo網膜イメージングの進歩 

可視光光干渉断層撮影ファイバーグラムと同一マウス網膜の共焦点像のアライメント

We present a protocol for aligning in vivo VIS-OCT images with ex vivo immunostained retinal nerve fibers to validate our novel imaging technology. VIS-OCT enables visualization of retinal nerve fibers in vivo, and this process confirms the accuracy of our findings. Our protocol combines in vivo and ex vivo imaging to provide a real time visualization of retina structure and confirm findings.The process is applicable to mouse models of eye diseases, provided the anterior eye portion allows for optical imaging. This includes diabetic retinopathy and retinal ischemia models. These studies lay the foundation for establishing an objective evaluation of neuro damage in humans, which can significantly improve glaucoma diagnosis and treatment in the future.

複合vis-OCTファイバーグラムを、RGC軸索のTuj-1で免疫染色したフラットマウント網膜の対応する共焦点画像と比較します(図1D、上パネル)。vis-OCTFによって画像化された軸索束は、共焦点画像上のTu-j1標識軸索束と一致することができます。血管は通常、ファイバーグラム画像では周囲の軸索束と比較して区別可能な分岐構造を示し、共焦点画像上のICAM-2標識血管と一致す?...

Watch this Scientific Journal Video about Advancements in In Vivo and Ex Vivo Retinal Imaging for Improved Glaucoma Diagnosis and Treatment at JoVE.com

本プロトコルは、in vivo画像で観察された網膜神経節細胞軸索束形態を検証する目的で、同じマウス網膜のex vivo共焦点画像とin vivo可視光光干渉断層撮影ファイバーグラフィー(vis-OCTF)画像を位置合わせするための手順を概説する。

In recent years, in vivo retinal imaging, which provides non-invasive, real-time, and longitudinal information about biological systems and processes, has ...

in vivo VIS-OCT画像をex vivo免疫染色網膜神経線維とアライメントするためのプロトコルを提示し、新しいイメージング技術を検証します。VIS-OCTは、生体内の網膜神経線維の可視化を可能にし、この過程は我々の知見の正確性を裏付けるものです。私たちのプロトコルは、in vivoイメージングとex vivoイメージングを組み合わせて、網膜構造のリアルタイム視覚化を提供し、所見を確認します。このプロセスは、眼球前部が光学イメージングを可能にする場合、眼疾患のマウスモデルに適用できます。これには、糖尿病性網膜症および網膜虚血モデルが含まれます。これらの研究は、ヒトの神経損傷の客観的な評価を確立するための基盤を築き、将来の緑内障の診断と治療を大幅に改善する可能性があります。

alignment-visible-light-optical-coherence-tomography-fibergrams-with

Research

JoVE Journal

Neuroscience

Alignment of Visible-Light Optical Coherence Tomography Fibergrams with Confocal Images of the Same Mouse Retina

In recent years, in vivo retinal imaging, which provides non-invasive, real-time, and longitudinal information about biological systems and processes, has been increasingly applied to obtain an objective assessment of neural damage in eye diseases. Ex vivo confocal imaging of the same retina is often necessary to validate the in vivo findings especially in animal research. In this study, we demonstrated a method for aligning an ex vivo confocal image of the mouse retina with its in vivo images. A new clinical-ready imaging technology called visible light optical coherence tomography fibergraphy (vis-OCTF) was applied to acquire in vivo images of the mouse retina. We then performed the confocal imaging of the same retina as the &#34;gold standard&#34; to validate the in vivo vis-OCTF images. This study not only enables further investigation of the molecular and cellular mechanisms but also establishes a foundation for a sensitive and objective evaluation of neural damage in vivo.

The present protocol outlines the steps for aligning in vivo visible-light optical coherence tomography fibergraphy (vis-OCTF) images with ex vivo confocal images of the same mouse retina for the purpose of verifying the observed retinal ganglion cell axon bundle morphology in the in vivo images.

神経科学

In Vivo Visible Light Optical Coherence Tomography (OCT) Mouse Retinal Imaging

Start by positioning the anesthetized mouse on the animal holder and securing it with two Velcro straps. Turn on the computer and open the software to activate the laser. Adjust the animal holder to center the laser in the mouse's eye.Visualize the posterior part through an on-face preview, the field of view of the superficial vascular plexus, a B scan, and the retina cross-section within the field of view. Acquire a visible light optical coherence tomography, or vis-OCT volume after making minor adjustments to the optical focus. Align the optical nerve head in each of the four corners of the field of view to cover different retinal areas.Use an intensity-based threshold method to detect the retina surface, and generate vis-OCT fiber grams from the volume. Crop the retinal nerve fiber layer, or RNFL, by selecting the first 16 micrometers depth. Next, calculate the mean intensity projection along the axial direction to generate the fiber gram image composed of retinal ganglion cell axon bundles and surrounding vasculature.Align the blood vessels using a graphics editor to montage the four images post-processing. The composite vis-OCT fiber gram is compared with the corresponding confocal image of flat-mounted retina immunostained with TUJ1 for RGC axons. Blood vessels exhibit distinguishable branching structures which can be matched with the ICAM II labeled blood vessels on the confocal image.Side-by-side comparison between ex vivo confocal microscopy and in vivo vis-OCT revealed identical RGC exon bundle networks and surrounding retinal vasculature.

In vivo可視光光干渉断層撮影法(OCT)マウス網膜イメージング

Ex Vivo Confocal Imaging of Mouse Retina

Enucleate the eyes of a euthanized mouse, marking the temporal side for orientation purposes. Remove the anterior chamber. Then remove the lens and vitreous and place eye cups in paraformaldehyde for 30 minutes.Wash eye cups with PBS for 30 minutes. Replacing the solution three times. Then, wash with 0.5%Triton X-100 for 30 minutes.Incubate eye cups in the blocking buffer for two hours at room temperature. After immunostaining, isolate the retinas from the eye cups using a microscope. Divide the retinas into four leaves and flat mount them with the retinal ganglion cell layer facing up.Ensure the mark on the temporal side remains attached for orientation. Cover the retinas with mounting medium. Place cover slips.And seal the slides using nail polish. Turn on the confocal microscope and under Locate mode, find the area of interest using the eye piece. Switch to Acquisition mode and set up tiles to cover the entire retina, along with Z-Stack slices for all layers of information.Image at least 25 tiles across the whole retina to achieve a total volume. Project the Z-Stack slices to generate two-dimensional on-face confocal microscopy images. The composite vis-OCT fibergram is compared with the corresponding confocal image of flat mounted retina immunostained with TuJ1 for RGC axons.Blood vessels exhibit distinguishable branching structures which can be matched with the ICAM-2 labeled blood vessels on the confocal image. Side-by-side comparison between ex vivo confocal microscopy and in vivo vis-OCT revealed identical RGC axon bundle networks and surrounding retinal vasculature.

Alignment of In Vivo and Ex Vivo Mouse Retinal Images

Start by creating a composite image using the four obtained images from each mouse. Utilize a graphics editor to align all the blood vessels, employ the optic nerve head and blood vessel patterns as landmarks for aligning the composite fiber gram overlap the composite images and the confocal images of the same retina stained with ICAM2, to achieve in vivo and ex vivo alignment. The composite vis-oct fiber gram is compared with the corresponding confocal image of flat mounted retina.Immuno stain with TUJ1 for RGC axons. Blood vessels exhibit distinguishable branching structures which can be matched with the ICAM2 labeled blood vessels on the confocal image. Side-by-side comparison between ex vivo confocal microscopy and in vivo vis-oct revealed identical RGC Axon bundle networks and surrounding retinal vasculature.