Name: in vivo multimodal imaging and analysis of mouse laser-induced choroidal neovascularization model
Uploaded: 2018-01-21T13:00:00
Description: Watch this Scientific Journal Video about In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model at JoVE.com

作者要感谢 Yuliya Naumchuk (芝加哥大学) 和 Agne žiniauskaitė (Experimentica Ltd.) 的优秀技术和录像支持。Dr. 卡洛特尼斯卡娅的研究项目是由 Dr. 的约翰 P 和苔蕾丝马尔卡希在美国芝加哥大学的眼科教授的支持。

所有的动物都是按照帕特声明, 为使用动物在眼科和视力研究和欧共体指令 86/609/EEC 动物实验, 使用的协议批准和监测的动物实验委员会芬兰。
1. 激光诱导小鼠 CNV 模型5
<ol><li>从宏观上检查动物的眼睛是否有异常。</li>
	<li>称量鼠标。</li>
	<li>计算和准备适当数量的麻醉剂使用, 根据动物的重量,例如medetomidine (1 毫克/千克), 氯胺酮 (75 毫克/千克) 和蒸馏...

<ol>
	<li>Dobi, E. T., Puliafito, C. A., Destro, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+model+of+experimental+choroidal+neovascularization+in+the+rat.">A new model of experimental choroidal neovascularization in the rat.</a> Arch. Ophthalmol. Chic. Ill 1960. 107, 264-269 (1989).</li><li>Tobe, T., 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=Evolution+of+neovascularization+in+mice+with+overexpression+of+vascular+endothelial+growth+factor+in+photoreceptors.">Evolution of neovascularization in mice with overexpression of vascular endothelial growth factor in photoreceptors.</a> Invest. Ophthalmol. Vis. Sci. 39, 180-188 (1998).</li><li>Seo, M. S., 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=Dramatic+inhibition+of+retinal+and+choroidal+neovascularization+by+oral+administration+of+a+kinase+inhibitor.">Dramatic inhibition of retinal and choroidal neovascularization by oral administration of a kinase inhibitor.</a> Am. J. Pathol. 154, 1743-1753 (1999).</li><li>Grossniklaus, H. E., Kang, S. J., Berglin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+choroidal+and+retinal+neovascularization.">Animal models of choroidal and retinal neovascularization.</a> Prog. Retin. Eye Res. 29, 500-519 (2010).</li><li>Shah, R. S., Soetikno, B. T., Lajko, M., Fawzi, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Mouse+Model+for+Laser-induced+Choroidal+Neovascularization.">A Mouse Model for Laser-induced Choroidal Neovascularization.</a> J Vis Exp. (106), e53502 (2015).</li><li>Giani, 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=In+vivo+evaluation+of+laser-induced+choroidal+neovascularization+using+spectral-domain+optical+coherence+tomography.">In vivo evaluation of laser-induced choroidal neovascularization using spectral-domain optical coherence tomography.</a> Invest. Ophthalmol. Vis. Sci. 52, 3880-3887 (2011).</li><li>Gong, Y., 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=Optimization+of+an+Image-Guided+Laser-Induced+Choroidal+Neovascularization+Model+in+Mice.">Optimization of an Image-Guided Laser-Induced Choroidal Neovascularization Model in Mice.</a> PloS One. 10, e0132643 (2015).</li><li>Sheets, K. G., 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=Neuroprotectin+D1+attenuates+laser-induced+choroidal+neovascularization+in+mouse.">Neuroprotectin D1 attenuates laser-induced choroidal neovascularization in mouse.</a> Mol. Vis. 16, 320-329 (2010).</li><li>Hoerster, 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=In-vivo+and+ex-vivo+characterization+of+laser-induced+choroidal+neovascularization+variability+in+mice.">In-vivo and ex-vivo characterization of laser-induced choroidal neovascularization variability in mice.</a> Graefes Arch. Clin. Exp. Ophthalmol. Albrecht Von Graefes Arch. Klin. Exp. Ophthalmol. 250, 1579-1586 (2012).</li><li>Sulaiman, R. S., 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=A+Simple+Optical+Coherence+Tomography+Quantification+Method+for+Choroidal+Neovascularization.">A Simple Optical Coherence Tomography Quantification Method for Choroidal Neovascularization.</a> J. Ocul. Pharmacol. Ther. Off. J. Assoc. Ocul. Pharmacol. Ther. Off. J. Assoc. Ocul. Pharmacol. 31, 447-454 (2015).</li></ol>

多模态成像为 CNV 病理评价提供了有价值的工具。在这里, 我们提出了一个成像协议, 包括 FA, SD OCT, 自动分割的快速, 重现性, 可靠的评价 CNV 病理。经激光管理后, 赫膜断裂。此外, 在这个阶段使用 SD OCT 也可以立即可视化可能的 intraretinal 和视网膜出血, 这可能混淆结果的解释。根据 FA 图像的荧光素信号对视网膜渗漏进行分级。使用 SD OCT 提供了更详细的描述 CNV 病理。此外, 纵向 SD OCT 分析在不同?...

第一次成功的尝试模仿人类 CNV 在啮齿目动物的病理证实了近三年前与氪激光在长埃文斯鼠1。此后, 氪激光被用来打破赫的细胞膜在最流行的鼠标应变, C57BL/6J2,3,4。用 FA 和组织学染色验证了 CNV 诱导的成功率。快速发展的无创成像模式, 如 OCT, 促进了啮齿动物的临床前模型领域的增长。在同一眼的多个时间点, 监测视网膜形态变化的能力大大降低了动物的使用, 提高了实验研究的效率。组织学评价 CNV 病变是相当直接的, 并要求标签的异常血管生长周围的激光管理, 图像采集, 和面积/体积估计使用图像分析软件。相比之下,在体内成像模式引入了对 CNV 病理及其解释的更复杂的分析。
在这里, 我们提出了一个简单的和相对快速的方法来评分的诱导, 进展, 和回归的 CNV 使用 FA, SD OCT, 和自动分割方法的小鼠激光诱导 CNV 模型。

<table border="0" cellpadding="0" cellspacing="0" width="917">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:275px;">Medetomidine (commercial name Domitor)</td>
			<td style="width:123px;">Orion</td>
			<td style="width:344px;">Vnr 01 56 02</td>
			<td style="width:177px;">Anesthesia</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Ketamine</td>
			<td>Intervet</td>
			<td>Vnr 51 14 85</td>
			<td>Anesthesia</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">0,9% NaCl</td>
			<td>B Braun</td>
			<td>357 0340</td>
			<td>Anesthesia</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Xylazine (commercial name Rompun vet)</td>
			<td>Bayer</td>
			<td>vnr 14 89 99</td>
			<td>Anesthesia</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tropicamide</td>
			<td>Santen</td>
			<td>Vnr 04 12 36</td>
			<td>Mydriatic agent</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Viscotears</td>
			<td>Alcon</td>
			<td>Vnr 44 54 81</td>
			<td>Lubricant</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Systane</td>
			<td>Alcon</td>
			<td>&#160;-</td>
			<td>Lubricant</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">5% Fluorescein sodium salt</td>
			<td>Sigma Aldrich</td>
			<td>F6377-100G</td>
			<td>Fluoresent agent</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Atipamezole (commercial name Antisedane)</td>
			<td>Orion</td>
			<td>Vnr 47 19 53</td>
			<td>Anesthesia</td>
		</tr>
	</tbody>
</table>

in vivo multimodal imaging and analysis of mouse laser-induced choroidal neovascularization model

激光诱导脉络膜新生血管 (CNV) 是模拟年龄相关性黄斑变性 (AMD) 的湿型的一个良好建立的模型。在本议定书中, 我们的目标是引导读者不只是通过技术考虑产生激光诱导的损害, 以触发新生血管的过程, 而是侧重于强大的信息, 可以获得从多式联运纵向在体内的整个随访期内的成像。
激光诱导的小鼠 CNV 模型是由二极管激光管理产生的。多模态在体内成像技术被用来监测 CNV 的诱导, 进展和回归。首先, 在激光后立即进行光谱域光学相干层析成像 (SD OCT) 来验证赫膜的断裂。随后使用荧光素血管造影 (FA) 进行的活体成像证实了赫膜从脉络膜水平获得的序列图像的成功损伤。CNV 增殖和回归的纵向后续在天 5, 10 和14以后使用了 SD OCT 和 FA 激光执行。提出了从 FA 图像中 CNV leasions 的简单可靠的分级方法。自动分割的测量总视网膜厚度, 结合手动口径应用测量视网膜厚度的 CNV 地点, 允许无偏见的评价存在水肿。最后, 利用 isolectin GS-IB4 染色对脉络膜 flatmounts 进行了 CNV 的组织学验证。染色是值, isolectin 阳性面积计算 ImageJ。
该协议特别适用于需要高通量样的 CNV 病理筛查的治疗研究, 因为它可以快速、多式、可靠地分类 CNV 病理和视网膜水肿。此外, 高分辨率 SD OCT 可以记录其他病理特征, 如视网膜或 intraretinal 液的积累。但是, 此方法不提供从 SD OCT 图像中自动进行 CNV 卷分析的可能性, 必须手动执行。

激光后立即出现气泡或视网膜出血并不总是可见的。因此, SD OCT 对于验证赫膜的损伤尤为重要。图 1显示了激光管理后在不同时间点的 OCT 成像的示例。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56173/56173fig1.jpg"> 图 1: 眼睛眼底的 OCT (VIP 图像) 显示在白色、绿色和红色圆圈?...

Watch this Scientific Journal Video about In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model at JoVE.com

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

在体内小鼠激光诱导脉络膜新生血管模型的多模态成像与分析

在这里, 我们介绍了纵向的在体内成像在激光诱导的小鼠脉络膜新生血管形态学变化中的作用。

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this ...

Research

JoVE Journal

Neuroscience

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

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在中央小鼠中枢神经系统的干细胞移植的多模态成像

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

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Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

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一个<em&gt;离体</em&gt;激光诱导脊髓损伤模型，以评估轴索变性的机制在实时

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A common technique for inducing stroke in experimental rodent models involves the transient (often denoted as MCAO-t) or permanent (designated as MCAO-p) ...

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Neurofilament protein polymers move along axons in the slow component of axonal transport at average speeds of ~0.35-3.5 mm/day. Until recently the study ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

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