这项工作是支持由美国国立卫生研究院授予1R01EY022901，职业发展奖防盲研究（RPB），CMReeves＆MA里夫斯基金会，大肠杆菌明德齐格勒盲人基金会，圣殿骑士护眼基金，并无限制授予部眼科在犹他州立大学从RPB。 我们感谢Balamurali Ambati有关Spectralis多模态成像系统和张涛对稿件进行讨论和评论的技术援助。

1。仪器的研制<ol><li>在过程室中的动物设施中进行的过程。 </li><li>在开始实验前戴口罩，帽子的头发，手术衣，无菌脚罩和手套。 </li><li>耐热水的烧杯〜40℃的热板上。 </li><li>放置无菌蓝色垫加热垫，将在以后用于保持成像时的小鼠的体温的顶部。打开加热垫。 </li><li>准备成像系统: <ol><li>取下防尘罩，并打开激光。 </li><li>取出55°镜头，并将其安装到机器上。 </li><li>从电脑输入的鼠标成像的新病人的床单下（ 如基因型，年龄等 ）的信息，打开图像处理软件。在"设备类型"中，选择红外线（IR）模式。 </li></ol></li></ol> 注:据报道，使用OF予外部双非球面透镜可以改善图像质量17-20虽然我们也没有问题在由IV-ICGA获得高质量的图像，而无需使用外部透镜（见代表性的结果， 图1-4）。 2。 ICGA的尾静脉注射<ol><li>扩张鼠标的眼睛，用1％托吡卡胺滴眼液，并等待5分钟。 </li><li>权衡鼠标来确定麻醉（氯胺酮/甲苯​​噻嗪/乙酰丙嗪65-100/10-20/1-3毫克/千克）所需的量。 </li><li>检索无菌1毫升注射器以及一个32号针头。用麻醉剂（13毫克/毫升氯胺酮，2.6毫克/毫升的甲苯噻嗪，0.3毫克/毫升乙酰丙嗪在无菌PBS中）腹膜内注射小鼠。等到鼠标完全麻醉（〜5分钟）。 </li><li>将小鼠尾部成40℃的温水，以使静脉的血管舒张。 </li><li>检索1 ml注射器用32号针头。撤回集成电路所需量的G，通常为50微升的1毫克/毫升的ICG，其无菌过滤用0.2微米的针筒式过滤器进入无菌管中，对于一个25克鼠标（2毫克/千克）。注意不要引入任何空气进入注射器。 </li><li>擦拭尾巴用酒精棉签消毒被注入的区域。 </li><li>握住尾巴，用一只手使尾静脉是向上的。朝上针的斜面，以最小的角度注入针〜2毫米进入静脉。要小心，不要刺穿静脉。拉回注射器略微查找血流痕迹到针毂，这表明，在针被成功插入到静脉中。 </li><li>慢慢地注入ICG进入静脉。注射时应该有最小的阻力。取下针头，直接涂抹酒精棉球消毒注射部位为〜5-10秒停止任何出血。鼠标然后准备成像。为了赶早阶段（注射后0-4分钟），它是埃塞赶紧微分方程边值问题的图像鼠标。 </li></ol> 注:鼠标的眼睛可以很容易地得到干燥，可在麻醉下白内障的发展。它通过在手术过程中应用无菌PBS，以保持眼球湿润是很重要的。擦去多余的PBS与ICGA录制之前，无菌棉签。其他实验室已使用的隐形眼镜，以避免角膜17-20中脱水。 3。 ICG血管造影<ol><li>开始拍摄的影像注射ICG，允许脉络膜充盈的早期阶段被捕获后30-40秒，直到视网膜和脉络膜的环流是在最大亮度（0-4分钟）。视网膜血管是最擅长的焦点〜35-45屈光度和脉络膜血管可视化10-15屈光度可视化。 注:在动物模型中的第一次考试，建议从各个角度（鼻，颞，背，腹）捕捉图像，以确定所有可能的abnormalities在脉管系统。在早期阶段，无论是中型和大型脉络膜动脉和静脉都清晰可见。在本协议中使用的动物模型，脉络膜病变（ 如息肉状扩张术）可能开始出现1分钟到早期阶段。 </li><li>设置图像专注于血管。控制亮度，并采用集中控制模块和调焦旋钮分别。这些值是可调节的数字化，并且容易保持恒定。保持距离，从小鼠眼相机镜头恒定，以确保图像的质量是可重复使用下面的技术。 注:由于该设备只能像眼后的部分，我们尽量保持相机镜头和鼠标红眼常数之间的对焦，亮度和距离，因为我们的图像，整个眼后从不同的角度。这里的关键是使通过眼睛与争夺的领域由ICG发出的圆圈形发光瓦特的相机。这是通过使左到右的完成，向上和向下，并在和出调整相机的位置的，直到整个图像没有暗区。当视摄像机的发光和场一字排开，从眼睛到透镜的距离将是可重复用于下一组图像，以及在对成像质量的最佳距离。 </li><li>一旦血管是重点，按采集模块上的黑色圆形按钮捕捉图像帧。的圆形黑色按钮也可以用来降低或提高的ICG为最佳图像质量的信号。 </li><li>确定最佳的视角和聚焦深度图像脉络膜病变。它保持在眼睛的位置，聚焦深度，并固定在整个时间过程ICGA其他设备的设置是很重要的。该图像被保存按采集模块的触摸屏面板上获取按钮。 </li><li>在6-15分钟船尾收购中间相的图像呃注射。 注意:无论是脉络膜和视网膜血管变得不那么明显的。脉络膜血管表现为弥漫性的荧光。脉络膜病变表现出强荧光出现在对比周围正常背景荧光褪色。 </li><li>注射后收购的后期图像在17-25分钟。 注:强荧光消失。既脉络膜和视网膜血管不再可见。视神经乳头变黑。 Hyperfluorescent脉络膜病变具有与衰落背景最大对比度。 </li><li>完成收购图像后，将明确的润滑剂眼胶鼠标的眼睛，离开鼠标加热垫进行恢复。 </li><li>返回小鼠笼子和等候区。导出图像为TIFF或JPEG文件进行进一步的分析。 </li></ol> 注:各相的定时也不是绝对的。我们发现，EA的时机取决于ICG的注入量通道相可能会改变。更ICG趋于延长每个阶段。根据上面列出的每个阶段的主要特征来定义一个阶段的最佳方法是。

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YF是两个正在申请的专利是有关在这项工作中使用AMD的小鼠模型的发明者。 SK，ZB，和ADJ什么都没有透露。

在这项研究中，我们演示了使用ICGA的图像脉络膜病变HTRA1转基因小鼠。的早，中，并在我们的小鼠模型ICGA后期阶段的特征在人类研究中1匹配的时间过程很好。这一点很重要，使人体病理学和动物的表型，这是无价的对相关的脉络，例如AMD的条件病理生理机制和治疗策略研究之间作出比较。 我们首先进行ICGA小鼠IP注射，发现的时间过程是从小鼠到小鼠高度可变的，这可能是由于在ICG染料从体腔中的腹部（ 图3）的可变的吸收。这使得难以用静脉注射进行人体试验比较。此外，第IV-ICGA不同阶段的血管造影特征是用于表征不同类型的脉络膜病变的动物的非常有用<html>模型。大多数人选择，以避免静脉注射（ 如 FA）对小鼠因进行尾静脉注射（小鼠尾静脉是微小的）的技术挑战。然而，这种努力是公略去考虑该技术的重复性的性质和信息获得的量。一旦我们掌握了尾静脉注射的技术，其他步骤都相当类似，IP-ICGA。这是值得一提的是一个人得到一切事前准备（ 如成像系统），以捕捉极短的早期阶段（0-4分钟）。我们比较了IP-ICGA与IV-ICGA学习各种HTRA1转基因小鼠。我们已经做了〜100老鼠每个方法。的结论是，IV-ICGA优于IP的ICGA在脉络膜病变的特征。时间进程IV-ICGA已经成为我们的标准实践检验AMD的小鼠模型。出于同样的原因，我们建议研究者应该考虑执行IV-FA对动物的研究。 T"&gt;除注射途径，我们注意到，色素的颜色也影响ICGA的质量。以前的研究也报告了这个"色素沉着的效果"18,25，但没有信息的毛色的不同的影响可ICGA的阶段。我们比较ICGA色素的小鼠（C57BL6）和白化小鼠（CD1）按时间进程IV-ICGA之间。大的脉络膜血管出现模糊和不清晰，而小血管都很难看到在C57BL6小鼠，这是锐相反，在CD1小鼠既大又小的脉络膜血管（ 图4）的更清晰的图像。最大的不同，观察在早期阶段，虽然中间阶段也受到影响。有后期阶段ICGA没有什么大的差异，由于在脉络膜血管ICG信号的衰落，显然，ICG荧光可部分阻断在色素小鼠脉络膜视网膜色素上皮和黑素细胞，作为一个建议，人们可能要考虑b穿筘他们的AMD机型到CD1背景，以获得高分辨率ICGA。 虽然FA是更广泛的基于AMD的动物模型中使用，ICGA是在脉络膜血管异常检测必不可少。观察小鼠脉络膜血管高分辨率实时能力可以极大地帮助研究人员在描述AMD的小鼠模型，并与组织病理学的数据关联。 ICGA，FA和华侨城的结合将是AMD的特征模型的表型作为AMD在人类患者的诊断非​​常有用。由于鼠标是目前使用最广泛的动物模型对于AMD的研究26-29，时间进程IV-ICGA可以在研究社会中发挥更大的作用。

吲哚菁绿血管造影（ICGA）是一种诊断测试的眼睛相关的血管图像的问题。 ICG的吸收光谱从790-805纳米范围内，而发射光谱范围为770-880纳米，在835纳米1的峰值发射。这是从其他流行染料，荧光素钠，其光谱落在在可见光范围内的不同。红外光谱使ICG穿透视网​​膜色素上皮细胞（RPE），serosanguineous流体，及脂质渗出物，所有这些都通过钠 - 荧光素为基础的荧光素血管造影术（FA）可以很容易地阻挡可视化。 ICG是98％的蛋白质结合的从而减少渗出血管，使脉络膜血管与脉络膜病变1,2增强成像。 ICGA几乎是唯一的选择，以可视化的脉络膜血管，这是后到的RPE。 图1显示ICGA和FA在小鼠眼成像血管的比较。 FA可以bË用于图像的视网膜血管很好，但不是脉络膜血管。与此相反，ICGA可用于图像既视网膜和脉络膜血管系统。 ICGA与高分辨率数字成像系统或扫描激光检眼镜（SLO），连同红外感光摄像头，这是我们在本研究将使用执行。 在临床上，ICGA已在诊断多项的脉络膜视网膜疾病的累及脉络膜血管系统，包括息肉状脉络膜血管病变（PCV），视网膜血管瘤样增生（RAP），视网膜血管样纹，卵黄黄斑营养不良，中心性浆液性脉络膜视网膜病变，脉络膜血管瘤，出血性视网膜推荐动脉macroaneurysms，脉络膜肿瘤，及后的某些形式葡萄膜炎1,3。 ICGA与FA和光学相干断层扫描（OCT）的结合提供了强大的工具，在渗出性年龄相关性黄斑的诊断和管理的临床医生变性（AMD）4-10。 ICGA用于诊断涉及脉络膜情况特别有用。事实上，ICGA被认为是黄金标准诊断PCV，渗出型AMD 11-13的一个变种。 PCV的特点是分支血管与终端息肉状扩张术在脉络膜血管11-13网络。 PCV经常与视网膜色素上皮和视网膜有泄漏，并从息肉状组件11,14,15出血复发serosanguineous支队有关。我们最近通过转基因表达人HTRA1，多官能丝氨酸蛋白酶，在小鼠的视网膜色素上皮细胞（RPE）16报告第一PCV动物模型的生成。我们发现，增加HTRA1引起的肺炎球菌结合疫苗特征， 如息肉状病变。 在这里，我们展示了通过尾静脉注射使用我们的HTRA1小鼠模型研究的AMD使用时间过程ICGA的。我们的数据表明，IV-ICGA优于IP（或皮下（SC）），当前使用中的字段17,18为在脉络膜病变表征-ICGA。 在动物研究表动物实验时，按照批准的机构动物护理和使用委员会（IACUC）协议进行，并按照ARVO声明在眼科和视觉研究用动物被执行。

<table border="0" cellpadding="0" cellspacing="0" width="667">
	<tbody>
		<tr height="50">
			<td height="50" style="height:50px;width:216px;">Spectralis Multi-Modality Imaging System</td>
			<td style="width:152px;">Heidelberg Engineering, Germany</td>
			<td style="width:132px;">SPECTRALIS HRA+OCT</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tropicamide ophthalmic solution (1%)</td>
			<td style="width:152px;">Bausch &#38; Lomb</td>
			<td style="width:132px;">NDC 24208-585-64</td>
			<td>for dilation of pupils</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">GenTeal Gel</td>
			<td style="width:152px;">Genteal</td>
			<td style="width:132px;">NDC 58768-791-15</td>
			<td>&#160;clear lubricant eye gel&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ketamine</td>
			<td style="width:152px;">Vedco Inc</td>
			<td style="width:132px;">NDC 50989-996-06</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Xylazine</td>
			<td style="width:152px;">Lloyd Laboratories</td>
			<td style="width:132px;">NADA 139-236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Acepromazine</td>
			<td style="width:152px;">Vedco Inc</td>
			<td style="width:132px;">NDC 50989-160-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">32-G Needle</td>
			<td>Steriject</td>
			<td style="width:132px;">PRE-32013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1-ml syringe</td>
			<td style="width:152px;">BD</td>
			<td style="width:132px;">309659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Indocyanine Green</td>
			<td style="width:152px;">Pfaltz &#38; Bauer</td>
			<td style="width:132px;">I01250</td>
			<td></td>
		</tr>
	</tbody>
</table>

detecting abnormalities in choroidal vasculature in a mouse model of age-related macular degeneration by time-course indocyanine green angiography

吲哚菁绿血管造影术（或ICGA）是由眼科医生进行诊断脉络膜和各种眼病，如年龄相关性黄斑变性（AMD）的视网膜血管系统的异常的技术。 ICGA就是将图像特别有用的眼睛由于通过着色层，其红外光谱穿透它的能力后的脉络膜血管。 ICGA时间过程可分为早期，中期和晚期阶段。这三个阶段提供的眼部问题的病理有价值的信息。虽然时间过程ICGA通过静脉（IV）注射剂被广泛应用于临床为脉络膜问题的诊断和管理，ICGA腹腔注射（IP）是常用于动物研究。在这里，我们展示了技术，通过尾静脉注射和共焦激光扫描检眼镜在小鼠体内获取高分辨率ICGA时间过程的图像。我们使用这种技术对图像中的脉络膜乐本期开始的年龄相关性黄斑变性的小鼠模型。虽然这是很容易引入ICG鼠标脉管系统通过IP，我们的数据表明，这是难以通过IP ICGA获得可重复性ICGA时间过程的图像。与此相反，通过尾静脉注射ICGA提供高品质的ICGA时间过程的图像媲美人类的研究。此外，我们发现，ICGA对小白鼠进行使脉络膜血管比对色素的小鼠进行更清晰的图像。我们认为，时间进程IV-ICGA应该成为基于动物模型研究的AMD的标准做法。

我们进行ICGA时间过程中HTRA1转基因小鼠和野生型对照窝，这两者都在CD1背景。白化CD1背景被选中，以方便吲哚青绿血管造影（ICGA）成像（见讨论）。有些动脉瘤样扩张术开始出现在HTRA1小鼠的早期阶段（ 图2，红色箭头表示扩张在容器的顶端和一个红色的圆圈表示一个簇型息肉状病变）。脉络膜血管都清晰可见在WT和HTRA1老鼠在这个早期填充在ICG染料的阶段。在中间阶段，hyperfluorescent病变在早期阶段变得更清晰，更皮损开始出现，而脉络膜血管开始在HTRA1鼠标褪色（黄色圆圈表示的多病灶的外观）。在后期阶段，HTRA1鼠标脉络膜病变变得更加"独特的"，因为所有的船只消失在背景中。视神经<html>头呈暗在WT和HTRA1小鼠（绿色箭头）。这三个阶段的主要特点是相似的ICGA时间过程在人类的AMD患者（早期阶段，0-3分钟;中间阶段，5〜15分钟;后期阶段，18〜22分）1。 <img alt="图1" fo:content-width="5in" fo:src="/files/ftp_upload/51061/51061fig1highres.jpg" src="/files/ftp_upload/51061/51061fig1.jpg" /> 图1。 FA和ICGA的成像小鼠视网膜和脉络膜的血管比较。野生型CD1小鼠静脉-FA和IV-ICGA使用多模态成像系统进行成像。视网膜血管可以看出，在两个FA和ICGA。脉络膜血管只能看到ICGA。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51061/51061fig1highres.jpg" target="_blank">点击这里查看大图</a> 。 / 51061fig2highres.jpg"SRC ="/ files/ftp_upload/51061/51061fig2.jpg"/&gt; 图2。 HTRA1转基因小鼠中通过静脉注射ICGA时间过程。一个野生型的控制和HTRA1转基因小鼠进行成像由ICGA（带尾静脉注射）。一个红色箭头表示在容器顶端的扩张（单发息肉）和红色圆圈表示一个簇型息肉状病变，出现于早期阶段。黄色圆圈表示几种病变出现在中间阶段。绿色箭头指向黑暗的视神经头在WT和HTRA1小鼠。注意，息肉状病变出现在ICGA的早期阶段，变得更加明显，中间相在人类研究21-24。离散点的病变出现在中间阶段和明朗化后期阶段（ 如最大的黄色圆圈，表示3点病变）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51061/51061fig2highres.jpg" target="_blank">点击这里查看大图</a> 。 </P&gt; <img alt="图3" fo:content-width="5in" fo:src="/files/ftp_upload/51061/51061fig3highres.jpg" src="/files/ftp_upload/51061/51061fig3.jpg" /> HTRA1转基因小鼠通过IP注射的图3。ICGA时间过程。HTRA1转基因小鼠进行成像5，12，和IP注射ICG后20分钟。的两个面板的行的图像取自两个不同的HTRA1转基因小鼠。请注意，脉络膜血管大多是无形的，即使5分钟注射（12分钟为鼠标在较低的面板）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51061/51061fig3highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图4" fo:content-width="5in" fo:src="/files/ftp_upload/51061/51061fig4highres.jpg" src="/files/ftp_upload/51061/51061fig4.jpg" /> 图4。颜料的ICGA时间过程ED（C57BL6）和白化（CD1）小鼠静脉注射。请注意，在色素和小白鼠之间的脉络膜血管的清晰度的差别。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51061/51061fig4highres.jpg" target="_blank">点此查看大图</a> 。

Watch this Scientific Journal Video about Detecting Abnormalities in Choroidal Vasculature in a Mouse Model of Age-related Macular Degeneration by Time-course Indocyanine Green Angiography at JoVE.com

Detecting Abnormalities in Choroidal Vasculature in a Mouse Model of Age-related Macular Degeneration by Time-course Indocyanine Green Angiography

通过尾静脉注射进行吲哚青绿血管造影（ICGA或）提供高品质的ICGA时间过程的图像在小鼠脉络表征异常。

按时间当然吲哚菁绿血管造影检测脉络膜血管系统异常在年龄相关性黄斑变性的小鼠模型

Indocyanine Green&#160;Angiography (or ICGA) is a technique performed by ophthalmologists to diagnose abnormalities of the choroidal and retinal ...

detecting-abnormalities-choroidal-vasculature-mouse-model-age-related

Nanyang Technological University

Research

JoVE Journal

Medicine

13.6K 次观看.  University of Utah Health Sciences Center. 通过尾静脉注射进行吲哚青绿血管造影（ICGA或）提供高品质的ICGA时间过程的图像在小鼠脉络表征异常。 

视频: Detecting Abnormalities in Choroidal Vasculature in a Mouse Model of Age-related Macular Degeneration by Time-course Indocyanine Green Angiography

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

多光子成像在口腔鳞状细胞癌原位小鼠模型的肿瘤细胞的侵袭

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

科研

医学

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

在斑马鱼模型的胎儿酒精暴露评估致畸的变化

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

Noninvasive Assessment of Cardiac Abnormalities in Experimental Autoimmune Myocarditis by Magnetic Resonance Microscopy Imaging in the Mouse

Myocarditis is an inflammation of the myocardium, but only ~10% of those affected show clinical manifestations of the disease. To study the immune events of myocardial injuries, various mouse models of myocarditis have been widely used. This study involved experimental autoimmune myocarditis (EAM) induced with cardiac myosin heavy chain (Myhc)-&#945; 334-352 in A/J mice; the affected animals develop lymphocytic myocarditis but with no apparent clinical signs. In this model, the utility of magnetic resonance microscopy (MRM) as a non-invasive modality to determine the cardiac structural and functional changes in animals immunized with Myhc-&#945; 334-352 is shown. EAM and healthy mice were imaged using a 9.4 T (400 MHz) 89 mm vertical core bore scanner equipped with a 4 cm millipede radio-frequency imaging probe and 100 G/cm triple axis gradients. Cardiac images were acquired from anesthetized animals using a gradient-echo-based cine pulse sequence, and the animals were monitored by respiration and pulse oximetry. The analysis revealed an increase in the thickness of the ventricular wall in EAM mice, with a corresponding decrease in the interior diameter of ventricles, when compared with healthy mice. The data suggest that morphological and functional changes in the inflamed hearts can be non-invasively monitored by MRM in live animals. In conclusion, MRM offers an advantage of assessing the progression and regression of myocardial injuries in diseases caused by infectious agents, as well as response to therapies.

This study demonstrates the successful establishment of magnetic resonance microscopy imaging as a non-invasive tool to assess the cardiac abnormalities in mice affected with autoimmune myocarditis. The data indicate that the technique can be used to monitor the disease-progression in live animals.

心脏异常的实验性自身免疫性心肌炎的无创性评估磁共振成像显微镜在鼠标

Myocarditis is an inflammation of the myocardium, but only ~10% of those affected show clinical manifestations of the disease. To study the immune events ...

A Mouse Model for Laser-induced Choroidal Neovascularization

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) research. By administering targeted laser injury to the RPE and Bruch&#8217;s membrane, the procedure induces angiogenesis, modeling the hallmark pathology observed in neovascular AMD. 
First developed in non-human primates, the laser-induced CNV model has come to be implemented into many other species, the most recent of which being the mouse. Mouse experiments are advantageously more cost-effective, experiments can be executed on a much faster timeline, and they allow the use of various transgenic models. The miniature size of the mouse eye, however, poses a particular challenge when performing the procedure. Manipulation of the eye to visualize the retina requires practice of fine dexterity skills as well as simultaneous hand-eye-foot coordination to operate the laser. However, once mastered, the model can be applied to study many aspects of neovascular AMD such as molecular mechanisms, the effect of genetic manipulations, and drug treatment effects. 
The laser-induced CNV model, though useful, is not a perfect model of the disease. The wild-type mouse eye is otherwise healthy, and the chorio-retinal environment does not mimic the pathologic changes in human AMD. Furthermore, injury-induced angiogenesis does not reflect the same pathways as angiogenesis occurring in an age-related and chronic disease state as in AMD.
Despite its shortcomings, the laser-induced CNV model is one of the best methods currently available to study the debilitating pathology of neovascular AMD. Its implementation has led to a deeper understanding of the pathogenesis of AMD, as well as contributing to the development of many of the AMD therapies currently available.

Here, we present the mouse laser-induced choroidal neovascularization (CNV) protocol, an experimental model that re-creates the vascular hallmarks of neovascular age-related macular degeneration (AMD). Once mastered, it can reliably and effectively induce CNV as a model system to test various experimental measures.

小鼠模型的激光诱导脉络膜新生血管

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) ...

The Monoiodoacetate Model of Osteoarthritis Pain in the Mouse

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The current treatments for OA pain such as NSAIDS or opiates are neither sufficiently effective nor devoid of detrimental side effects. Animal models of OA are being developed to improve our understanding of OA-related pain mechanisms and define novel pharmacological targets for therapy. Currently available models of OA in rodents include surgical and chemical interventions into one knee joint. The monoiodoacetate (MIA) model has become a standard for modelling joint disruption in OA in both rats and mice. The model, which is easier to perform in the rat, involves injection of MIA into a knee joint that induces rapid pain-like responses in the ipsilateral limb, the level of which can be controlled by injection of different doses. Intra-articular injection of MIA disrupts chondrocyte glycolysis by inhibiting glyceraldehyde-3-phosphatase dehydrogenase and results in chondrocyte death, neovascularization, subchondral bone necrosis and collapse, as well as inflammation. The morphological changes of the articular cartilage and bone disruption are reflective of some aspects of patient pathology. Along with joint damage, MIA injection induces referred mechanical sensitivity in the ipsilateral hind paw and weight bearing deficits that are measurable and quantifiable. These behavioral changes resemble some of the symptoms reported by the patient population, thereby validating the MIA injection in the knee as a useful and relevant pre-clinical model of OA pain.
The aim of this article is to describe the methodology of intra-articular injections of MIA and the behavioral recordings of the associated development of hypersensitivity with a mind to highlight the necessary steps to give consistent and reliable recordings.

Osteoarthritis (OA), or degenerative joint disease, is a debilitating condition associated with pain that remains only partially controlled by available analgesics. Animal models are being developed to improve our understanding of OA-related pain mechanisms. Here we describe the methodology for the monoiodoacetate model of OA pain in the mouse.

骨关节炎疼痛的鼠标Monoiodoacetate模式

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The ...

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Anastasis (Greek for &#8220;rising to life&#8221;) is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death processes that are generally assumed to be intrinsically irreversible. Promoting anastasis could in principle rescue or preserve injured cells that are difficult to replace such as cardiomyocytes or neurons, thereby facilitating tissue recovery. Conversely, suppressing anastasis in cancer cells, undergoing apoptosis after anti-cancer therapies, may ensure cancer cell death and reduce the chances of recurrence. However, these studies have been hampered by the lack of tools for tracking the fate of cells that undergo anastasis in live animals. The challenge is to identify the cells that have reversed the cell death process despite their morphologically normal appearance after recovery. To overcome this difficulty, we have developed Drosophila and mammalian CaspaseTracker biosensor systems that can identify and permanently track the anastatic cells in vitro or in vivo. Here, we present in vivo protocols for the generation and use of the CaspaseTracker dual biosensor system to detect and track anastasis in Drosophila melanogaster after transient exposure to cell death stimuli. While conventional biosensors and protocols can label cells actively undergoing apoptotic cell death, the CaspaseTracker biosensor can permanently label cells that have recovered after caspase activation - a hallmark of late-stage apoptosis, and simultaneously identify active apoptotic processes. This biosensor can also track the recovery of the cells that attempted other forms of cell death that directly or indirectly involved caspase activity. Therefore, this protocol enables us to continuously track the fate of these cells and their progeny, facilitating future studies of the biological functions, molecular mechanisms, physiological and pathological consequences, and therapeutic implications of anastasis. We also discuss the appropriate controls to distinguish cells that undergo anastasis from those that display non-apoptotic caspase activity in vivo.

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Anastasis is technically challenging to detect in vivo because the cells that have reversed the cell death process can be morphologically indistinguishable from normal healthy cells. Here we describe protocols for detecting and tracking cells that undergo anastasis in live animals by using our newly developed in vivo CaspaseTracker biosensor system.

用 CaspaseTracker 生物传感器检测 Anastasis体内

Anastasis (Greek for &#8220;rising to life&#8221;) is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death ...

Regenerative Therapy by Suprachoroidal Cell Autograft in Dry Age-related Macular Degeneration: Preliminary In Vivo Report

This study is aimed at examining whether a suprachoroidal graft of autologous cells can improve best corrected visual acuity (BCVA) and responses to microperimetry (MY) in eyes affected by dry Age-related Macular Degeneration (AMD) over time through the production and secretion of growth factors (GFs) on surrounding tissue. Patients were randomly assigned to each study group. All patients were diagnosed with dry AMD and with BCVA equal to or greater than 1 logarithm of the minimum angle of resolution (logMAR). A suprachoroidal autologous graft by Limoli Retinal Restoration Technique (LRRT) was carried out on group A, which included 11 eyes from 11 patients. The technique was performed by implanting adipocytes, adipose-derived stem cells obtained from the stromal vascular fraction, and platelets from platelet-rich plasma in the suprachoroidal space. Conversely, group B, including 14 eyes of 14 patients, was used as a control group. For each patient, diagnosis was verified by confocal scanning laser ophthalmoscope and spectral domain-optical coherence tomography (SD-OCT). In group A, BCVA improved by 0.581 to 0.504 at 90 days and to 0.376 logMAR at 180 days (+32.20%) postoperatively. Furthermore, MY test increased by 11.44 dB to 12.59 dB at 180 days. The different cell types grafted behind the choroid were able to ensure constant GF secretion in the choroidal flow. Consequently, the results indicate that visual acuity (VA) in the grafted group can increase more than in the control group after six months.

Regenerative Therapy by Suprachoroidal Cell Autograft in Dry Age-related Macular Degeneration: Preliminary In Vivo Report

The goal of this study is to assess whether the suprachoroidal graft of adipose-derived stem cells included in the stromal vascular fraction and platelets derived from platelet-rich plasma by the Limoli Retinal Restoration Technique can improve visual acuity and retinal sensitivity responses in eyes affected by dry age-related macular degeneration.

脉络细胞自体移植治疗老年性黄斑变性的再生疗法: 初步体内报告

This study is aimed at examining whether a suprachoroidal graft of autologous cells can improve best corrected visual acuity (BCVA) and responses to ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Evaluation of Capillary and Other Vessel Contribution to Macular Perfusion Density Measured with Optical Coherence Tomography Angiography

Parafoveal circulation of the superficial retinal capillary plexus is usually measured with vessel density, which determines the length of capillaries with circulation, and perfusion density, which calculates the percentage of the evaluated area that has circulation. Perfusion density also considers the circulation of vessels larger than capillaries, although the contribution of these vessels to the first is not usually evaluated. As both measurements are automatically generated by optical coherence tomography angiography devices, this paper proposes a method for estimating the contribution of vessels larger than capillaries by using a coefficient of determination between vessel and perfusion densities. This method can reveal a change in the proportion of perfusion density from vessels larger than capillaries, even when mean values do not differ. This change could reflect compensatory arterial vasodilatation as a response to capillary dropout in the initial stages of retinal vascular diseases before clinical retinopathy appears. The proposed method would allow the estimation of the changes in the composition of perfusion density without the need for other devices.

We describe the evaluation of a coefficient of determination between vessel and perfusion density of the parafoveal superficial capillary plexus to identify the contribution of vessels larger than capillaries to perfusion density.

评估毛细血管和其他血管对光学相干断层扫描血管造影测量的黄斑灌注密度的贡献

Parafoveal circulation of the superficial retinal capillary plexus is usually measured with vessel density, which determines the length of capillaries ...

A Neonatal BALB/c Mouse Model of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight infants, with high mortality and unclear pathogenesis. The cause of NEC may be related to inflammatory immune regulatory system abnormalities. An NEC animal model is an indispensable tool for NEC disease immune research. NEC animal models usually use C57BL/6J neonatal mice; BALB/c neonatal mice are rarely used. Related studies have shown that when mice are infected, Th2 cell differentiation is predominant in BALB/c mice compared to C57BL/6J mice. Studies have suggested that the occurrence and development of NEC are associated with an increase in T helper type 2 (Th2) cells and are generally accompanied by infection. Therefore, this study used neonatal BALB/c mice to induce an NEC model with similar clinical characteristics and intestinal pathological changes as those observed in children with NEC. Further study is warranted to determine whether this animal model could be used to study Th2 cell responses in NEC.

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight infants, with high mortality and unclear pathogenesis. The cause of NEC may be related to inflammatory immune regulatory system abnormalities. An NEC animal model is an indispensable tool for NEC disease immune research. NEC animal models usually use C57BL/6J neonatal mice; BALB/c neonatal mice are rarely used. Related studies have shown that when mice are infected, Th2 cell differentiation is predominant in BALB/c mice compared to C57BL/6J mice. Studies have suggested that the occurrence and development of NEC are associated with an increase in T helper type 2 (Th2) cells and are generally accompanied by infection. Therefore, this study used neonatal BALB/c mice to induce an NEC model with similar clinical characteristics and intestinal pathological changes as those observed in children with NEC. Further study is warranted to determine whether this animal model could be used to study Th2 cell responses in NEC.

一种新生儿BALB/c坏死性小肠结肠炎小鼠模型

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight ...