Name: ultrahigh resolution mouse optical coherence tomography to aid intraocular injection in retinal gene therapy research
Uploaded: 2018-11-02T12:30:00
Description: Watch this Scientific Journal Video about Ultrahigh Resolution Mouse Optical Coherence Tomography to Aid Intraocular Injection in Retinal Gene Therapy Research at JoVE.com

这些材料的部分依据是退伍军人事务部、退伍军人卫生管理局、研究与发展办公室 (生物医学实验室研究与开发) (退伍军人健康研究所) (退伍军人健康研究所赠款 1i01 bx000669) 所支持的工作。jms 部分由 va wny 聘为眼科工作人员医生-科学家;mcb 部分受雇于 va wny。这项研究是在纽约西部医疗系统 (纽约州布法罗) 进行的, 并得到了部分支持。内容不代表退伍军人事务部或美国政府的意见。还在很大程度上支持, NIH/NEI 授予 ey013433 (p:jms), nih变 nei r24 授予 ey016662 (ub 视觉基础设施中心, pi: m 屠宰, 目录-biophotonics 模块: jms), 向奥曼诺林大学系提供不受限制的赠款布法罗来自预防失明研究 (纽约州纽约), 以及 oishei 基金会 (纽约州布法罗) 提供的一笔赠款。我们承认 hc1 转基因rhop347s线的礼物和来自 janis lem 博士 (波士顿塔夫茨新英格兰医疗中心) 的 exon 1老鼠 rho淘汰赛的礼物, 以及 nrt-e 转基因模型在杂合状态下的天赋。鼠标外显子 2 rho 淘汰赛背景从g. 简·法拉尔博士和彼得亨弗里斯博士 (三一学院, 都柏林, 爱尔兰共和军)。

va wny hcs 机构动物护理和使用委员会和布法罗-suny 大学对动物规程进行了审查和批准。动物是根据视力和眼科研究协会 (arvo) 的规定和《赫尔辛基宣言》使用的。
1. 鼠标型号
<ol><li>确定要评估的鼠标模型, 包括控件。 请注意:对 C57BL/6(J)、hcenhcne/wtmwmwwwwwwwwwt 进行了成像, 这是一种部分人性化的小鼠视网膜变性模型纯合子, 用于人类突变体 rho p3477s hc1 等体对...

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hr-sd-oct 为人类疾病潜在动物模型的定性提供了一种简单的方法, 以确定它们在测试潜在疗法方面的有用性。快速可靠地描述人类疾病潜在动物模型的能力对于治疗药物发现的过程至关重要 (例如, 替代基因治疗、核酶或 shnna 敲除基因治疗、联合基因治疗)。hr-sd-oct 为评估视网膜健康提供了一种简单、快速和非侵入性的方法, 可用于描述和监测几乎所有小鼠模型中视网膜退化的进展。oct 图像可...

研究人员正在开发各种视网膜和视网膜退行性疾病的基因疗法, 希望将新的疗法转化为人类疾病的治疗方法1,2,3,4,5,6,7.,8,9,10,11. 时域或光谱域光学相干断层扫描 (sd-oct) 已被用来研究疾病 12、13、14的特定小鼠模型中视网膜外变性的各个方面.然而, hr-sd-oct 并没有被广泛用于优化小鼠模型的评价, 以确定视网膜变性的速度和空间一致性, 或者在基于基因的疗法的临床前评估中, 例如:评估救援、毒性或病媒传递的空间范围8、15、16。一旦小鼠模型被充分描述, hr-sd-oct 数据可以作为一个信息丰富和可靠的资源, 以衡量治疗方法的影响, 发挥救援或毒性的小鼠模型视网膜变性 17。许多群体使用视网膜下注射作为载体传递的方法, 因为它在转染光感受器和视网膜色素上皮 (rpe) 细胞的效率。然而, 这仍然是一个很难掌握的方法, 因为它通常是通过从角膜表面的自由手手术, 往往充满白内障, 出血, 和意外视网膜脱离发生仅仅是通过操纵后部玻璃体。许多群体仍然试图盲目地注射视网膜下注射, 并使用人工注射相对较大直径的不锈钢针头 (34g)8,17,18,19 提供病毒 20,21, 22,和一些使用光学相干断层扫描 (oct) 成像, 以确认适当的载体传递到视网膜8,17,20,22. 最近使用微机械手22驱动的微型针头描述了该方法的一些改进.
我们提出了一个综合的方法, 帮助在针的定位, 注射是由一个定制的定向立体眼镜仪设计在实验室专门为可视化内的小鼠17,23. 在使用拉玻璃微针头与立体定向微机械手相结合的情况下, 可以更好地控制针头的放置, 无需手术切除 (即通过结膜和结缔组织)。注射。使用压力调节的微注射器有助于提供一致的注射量, 并且与手持式注射器进行的手动注射相比, 注射的稳定性、精度和速度要慢得多, 从而降低了出现泡沫注射到眼睛。较小的针头有助于防止拔针后泄漏, 因为路径是自密封的。为了评估注射的程度, 许多研究小组依靠在实验结束时发现和评估视网膜中增强的绿色荧光蛋白 (egfp) 表达的区域范围 (由载体提供的表达结构)点 (安乐死) 确认注射成功11,19,20,24。这种方法 (不使用 oct) 来验证手术的成功, 浪费了大量的资源在外科手术时间和动物, 因为所有动物有 (未知) 手术失败需要保持, 其次是重复措施, 直到安乐死和眼睛收获 (当 egfp 被测量)。使用 hr-sd-oct 可以改善注射在视网膜中位置的确认, 以证明注射位于视网膜的正确层 (即视网膜下空间) 之间。hr-sd-oct 也可用于立即描述不成功的尝试 (手术失败), 以确定相关变量在实际手术时间, 以改进的方法。我们发现, HR-SD-OCT 通过允许快速定量评估视网膜外变性, 从而在临床前基因治疗研究中提供了许多优势, 从而能够识别不符合实验标准的研究动物 (例如,不正确的视网膜下注射), 并直接随访成像眼睛的区域, 其中矢量的交付 (在临床前的效果是最可能的), 以及控制区域, 其中矢量没有交付。自发展以来, sd-oct 的使用一直被眼科研究人员接受和使用, 现在被认为是小鼠或啮齿类动物模型 13,25视网膜科学研究中视网膜成像的标准。hr-sd-oct 及其软件能力以独特的综合方式被利用, 以促进小鼠模型在这一过程中的每一步成功进行定量基因治疗的目标, 包括动物模型的选择、选择中的退化特征疾病模型, 治疗传递, 病媒传递的映射, 和毒性/疗效评估。hr-sd-oct 的使用可以在整个过程的各个层面更有效地发现药物。在这里, 我们描述了这些方法, 用于我们的 rna 药物发现计划。

<table border="0" cellpadding="0" cellspacing="0" style="width:775px;" width="774">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">C57BL6 (J)</td>
			<td style="width:245px;">Jackson Laboratories</td>
			<td style="width:215px;">664</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">N129R-</td>
			<td style="width:245px;">N/A</td>
			<td style="width:215px;">N/A</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">2HRho 1T/1T</td>
			<td style="width:245px;">N/A</td>
			<td style="width:215px;">N/A</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="78" style="height:78px;width:251px;">Envisu R-2200 Ocular Coherence Tomography Instrument (OCT)</td>
			<td style="width:245px;">Bioptigen</td>
			<td style="width:215px;">90-R2200-U1-120.&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Retinal Imaging System (RIS)</td>
			<td style="width:245px;">In-house</td>
			<td style="width:215px;">N/A</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Stemi 2000C Microscope</td>
			<td style="width:245px;">Zeiss</td>
			<td style="width:215px;">000000-1106-133</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">P-97 Flaming/Brown micropipette puller</td>
			<td style="width:245px;">Sutter Instrument Co</td>
			<td style="width:215px;">p-97</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">MMN-33 micro manipulator&#160;</td>
			<td style="width:245px;">Narishige USA</td>
			<td style="width:215px;">MMN-33</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">PLI-100&#160; micro injector</td>
			<td style="width:245px;">Harvard Apparatus</td>
			<td style="width:215px;">64-1736</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Micropipette Holder (Rotating)</td>
			<td style="width:245px;">In-house</td>
			<td style="width:215px;">N/A</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Micropipette Storage Receptacle</td>
			<td style="width:245px;">World Precision Instruments Inc.</td>
			<td style="width:215px;">E210</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">&#160;Borosilicate glass capillary tubes 1.5-1.8 X 100mm,&#160;</td>
			<td style="width:245px;">Harvard Apparatus</td>
			<td style="width:215px;">30-0053</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">2,2,2-Tribromoethanol</td>
			<td style="width:245px;">SIGMA Aldrich</td>
			<td style="width:215px;">T48402-25G</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Tert-amyl Alcohol</td>
			<td style="width:245px;">SIGMA Aldrich</td>
			<td style="width:215px;">240486-100ML</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Atropine Sulfate Ophthalmic Solution, USP 1%</td>
			<td style="width:245px;">Akorn Inc.</td>
			<td style="width:215px;">NDC 17478-215-05</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Goniovisc</td>
			<td style="width:245px;">BioVision Limited</td>
			<td style="width:215px;">NDC 17238-610-15</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Cyclopentolate Hydrochloride Ophthalmic Solution, USP&#160; 2%</td>
			<td style="width:245px;">Akorn Inc.</td>
			<td style="width:215px;">NDC 17478-097-10</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Gentamicin Sulfate Ointment USP, 0.1%</td>
			<td style="width:245px;">Perigo</td>
			<td style="width:215px;">NDC 45802-046-35</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Systane Ultra</td>
			<td style="width:245px;">Alcon Laboratories, Inc.</td>
			<td style="width:215px;">9006619-1013</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Tetracaine Hydrochloride</td>
			<td style="width:245px;">Bausch and Lomb</td>
			<td style="width:215px;">NDC 24208-920-64</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Ophthalmic Solution, USP 0.5%</td>
			<td style="width:245px;">Bausch and Lomb</td>
			<td style="width:215px;">NDC 24208-920-64</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">DPBS</td>
			<td style="width:245px;">Gibco Life Technologies</td>
			<td style="width:215px;">14190-136</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Virus Preparations</td>
			<td style="width:245px;">ViGene /UNC</td>
			<td style="width:215px;">N/A</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Gold nanorods</td>
			<td style="width:245px;">NANOPARTz</td>
			<td style="width:215px;">D12M-850-1.75</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Fluorescein Sulfate AK-FLUOR 25%</td>
			<td style="width:245px;">Akorn Inc.</td>
			<td style="width:215px;">NDC 17478-250-20</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Coverslips</td>
			<td style="width:245px;">Fisher Scientific</td>
			<td style="width:215px;">12-548-A</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Forceps</td>
			<td style="width:245px;">Milton</td>
			<td style="width:215px;">18-825</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Needles 30 guage</td>
			<td style="width:245px;">Beckton Dickenson&#160;&#160;</td>
			<td style="width:215px;">W11604</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Syringes</td>
			<td style="width:245px;">Beckton Dickenson&#160;&#160;</td>
			<td style="width:215px;">309659</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Bioptigen software Package</td>
			<td style="width:245px;">Bioptigen</td>
			<td style="width:215px;">N/A</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Proparacaine Hydrochloride Ophthalmic Solution, USP 0.5%</td>
			<td style="width:245px;">Akorn Inc.</td>
			<td style="width:215px;">NDC 17478-263-12</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Windows Excel</td>
			<td style="width:245px;">Microsoft</td>
			<td style="width:215px;">N/A</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Adobe Illustrator</td>
			<td style="width:245px;">Adobe&#160;</td>
			<td style="width:215px;"></td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Scale</td>
			<td style="width:245px;">Mettler</td>
			<td style="width:215px;"></td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Scissors</td>
			<td style="width:245px;">World Precision Instruments</td>
			<td style="width:215px;"></td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Ear punch</td>
			<td style="width:245px;">Nat&#8217;l band</td>
			<td style="width:215px;"></td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">CL 100 Light source</td>
			<td style="width:245px;">Welch Allyn</td>
			<td style="width:215px;">CL100</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Nitrogen Gas</td>
			<td style="width:245px;">Jackson Welding Supply</td>
			<td style="width:215px;">N/A</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Heated Water bath</td>
			<td style="width:245px;">Neslab</td>
			<td style="width:215px;">RTE-140</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Heating plate</td>
			<td style="width:245px;">In House</td>
			<td style="width:215px;">N/A</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Heating mat</td>
			<td style="width:245px;">Cincinnatti Sub Zero</td>
			<td style="width:215px;">273</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Clay mouse holder</td>
			<td style="width:245px;">Plast.i.clay American Art Clay Co.</td>
			<td style="width:215px;">N/A</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Betadine</td>
			<td style="width:245px;">MedLine&#160;</td>
			<td style="width:215px;">NDC53329-938-06</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Cotton Tip Applicators</td>
			<td style="width:245px;">American Health Service</td>
			<td style="width:215px;">Ctag</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">EtOH 70%</td>
			<td style="width:245px;">Fisher Scientific</td>
			<td style="width:215px;">BP2818-100</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Gloves Nitrile</td>
			<td style="width:245px;">VWR</td>
			<td style="width:215px;">89038-272</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Diagnosys ERG Color Dome instrument</td>
			<td style="width:245px;">Diagnosys Inc.</td>
			<td style="width:215px;">D125</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Contact lenses</td>
			<td style="width:245px;">In-house</td>
			<td style="width:215px;">N/A</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Diagnosys Software</td>
			<td style="width:245px;">Diagnosys Inc.</td>
			<td style="width:215px;">N/A</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Origin 6.1 software</td>
			<td style="width:245px;">OriginLab Corp.</td>
			<td style="width:215px;">N/A</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Reference electrodes</td>
			<td style="width:245px;">Ocuscience</td>
			<td style="width:215px;">F-Thread Electrode (DTL) 24&#8221;</td>
			<td></td>
		</tr>
	</tbody>
</table>

ultrahigh resolution mouse optical coherence tomography to aid intraocular injection in retinal gene therapy research

hr-sd-oct 用于监测活小鼠模型中光感受器变性的进展, 评估治疗药物在视网膜下空间的传递, 并评估体内的毒性和有效性。hr-sd-oct 使用近红外光 (800-880 nm), 并具有专为鼠标眼睛独特光学器件设计的光学器件, 其轴向分辨率低于2微米。对转基因小鼠外视网膜 (光感受器) 变性和对照进行了成像, 以评估疾病进展情况。采用带状玻璃微针, 通过反层和反式脉络膜法,对腺病毒 (aav) 或纳米颗粒 (np) 进行视网膜下注射。在校准压力注射之前, 需要将针头仔细定位到视网膜下空间, 然后将液体输送到视网膜下空间。在视网膜成像系统 (ris) 上进行了实时视网膜下手术。hr-sd-oct 表现出由于毒性突变体人视紫红质 (p347s) (rhop347s) 转基因表达而逐渐变性视网膜变性。hr-sd-oct 允许对所有视网膜层进行严格的定量。外核层 (onl) 厚度和光感受器外段长度 (osl) 测量与光感受器的活力、退化或抢救相关。ris 分娩系统可实时显示新生儿 (~ p10-14) 或成年小鼠的视网膜下注射, hr-sd-oct 立即确定分娩的成功程度和地图面积。hr-sd-oct 是一个强大的工具, 可以评估小鼠视网膜下手术的成功, 以及测量光感受器在体内的活力。hr-sd-oct 还可用于识别统一的动物队列, 以评估视网膜退化的程度、毒性和治疗性抢救在临床前基因治疗研究中的应用。

模型外视网膜退化的存在性、速率和一致性评价 从 opl 到 elm 的测量记录, 使用仪器软件中提供的卡尺工具定义了 onl 的限制。目标是在部分人性化的 adrp 小鼠模型中绘制视网膜外变性的进展图。来自对照 C57BL/6(J) 小鼠和 hC1/hC1//mWT/mWT 小鼠模型的可比图像, 表达了突变的人棒蛋白 (rhop347s) 基因的两个副本, 显示出控制视网膜的发现和严重的和严?...

Watch this Scientific Journal Video about Ultrahigh Resolution Mouse Optical Coherence Tomography to Aid Intraocular Injection in Retinal Gene Therapy Research at JoVE.com

Ultrahigh Resolution Mouse Optical Coherence Tomography to Aid Intraocular Injection in Retinal Gene Therapy Research

在这里, 我们展示了一种新的方法, 使用高分辨率光谱域光学相干断层扫描 (hr-sd-oct), 以协助将基因治疗剂传递到视网膜下空间, 评估其区域覆盖, 并表征光感受器的活力。

超高分辨率小鼠光学相干层析成像辅助眼内注射在视网膜基因治疗中的应用

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the ...

此下视网膜注射程序的总体目标是以可实时观察到的微创和可重复的方式向视网膜提供载体。你好，该方法可用于回答基因治疗研究中有关载体传递疗效和治疗剂毒性的关键问题。该技术的一些优点包括对视网膜和其他眼睛结构的最小损伤，精确的注射部位映射，以及严格的定量体内测量，允许坚实的统计推理。对于眼内转基因注射，首先将麻醉小鼠置于视网膜成像系统显微镜的观察阶段，将无菌钝虹膜钳的尖端置于眼睑的七点和十点钟位置，同时将钳子打开和向下，以轻轻诱导促进性。使用钳子哄骗眼球下的眼睑，并引导针尖到角膜边缘以下约 1 至 1.5 毫米。接下来，将一滴无菌 PBS 溶液涂抹在眼睛上，用无菌盖滑盖住眼睛。小心地将针头通过结膜驱动到眼睛中，直到制造下性凹陷。使用微型操纵器向上旋转眼睛，直到眼部凹陷对焦，然后向前驱动针头，用针头在视网膜中形成一个尖锐的峰值。使用针架旋转针头，直到针头尖通过硬合体和RPE孔，使针尖中的面粉素在靠近下视网膜空间和视网膜色素的上皮细胞单层下的视网膜组织下可见。向下驱动针尖，使针尖与地球切线，然后用脚踏板开关激活喷射泵。在将0.5至1微升的转基因溶液送到亚中心空间后，取出针头并确认血液稳定，且液体不会从注射液部位泄漏出来。要确认注射成功，请记录一个矩形卷 OCT，并将图像与不同类型的潜在注射结果的图像进行比较。确认正确注塑放置后，使用该矩形 OCT 图像将 bleb 的范围映射到基金图像上。要映射下一个模糊的范围，请打开 bleb 的矩形体积 OCT 图像，以便图像中包含眼睛中的可识别地标。添加尽可能多的卡钳，以识别 B 扫描的左右边缘的位置，以及视神经头 ONH（如果存在）以及视网膜从 RPE 和 choroid 分离，然后保存数据的拐点。然后编译保存的文件并绘制数据，有效地将注射 bleb 的映射创建到 en face OCT 图像上。将绘制的数据叠加到包含注入站点的基金图像上，并保存合并的图像。使用此图像可查找后续 OCT 成像期间感兴趣的区域。要评估注射后视网膜退化，请首先打开记录的高分辨率光谱域 OCT 或 HRSD OCT 矩形体积图像，然后选择要测量的 B 扫描。放大所选图像，直到其填满屏幕并右键单击图像。单击卡钳以选择适当数量的卡钳，然后使用配置卡钳功能将卡钳指定为角度块列中的垂直卡钳，并打开显示卡钳位置，以便于在视网膜上均匀放置卡钳。单击应用并移动每个卡钳，在 B 扫描中相隔 0.1 至 0.2 毫米，小心在适当时将一个卡钳放入视神经头中心。放置所有卡钳后，单击每个卡钳并将其拖动到感兴趣区域的长度跨度，任意设置卡钳，不会将 B 扫描的可测量区域叠加到最大长度。要测量外层厚度，请将每个卡钳的顶部放在外部限制膜上，将每个卡钳的底部放在外层底部，在视网膜上以 0.1 到 0.2 毫米的增量。放置并调整所有卡钳大小后，右键单击图像并单击保存卡钳数据"，然后重复每十次 B 扫描所演示的测量值，使卡钳保持打开，并在 x 轴上的相同位置，并根据需要调整卡钳的长度，而无需在 x 方向上移动卡钳。测量所有扫描后，打开保存的数据文件并单击修改日期"，以便根据保存的时间排列文件。打开每个 B 扫描的所有文件，将每个 B 扫描的原始数据编译为从最低到最高帧数的单个文件，然后选择数据列，包括卡钳名称、长度和中心 X 列。确保对于处理的每个矩形卷 OCT 图像测量的所有 B 扫描中心 X 相同，并删除未用于记录测量的卡钳中的任何数据。将位于视神经头中心的卡钳设置为零，并绘制 X 数据集与多个 Y 数据集的数据，以获得外部核层或任何其他测量视网膜层的厚度的 3D 绘图函数。然后比较控制和实验动物与相应年龄之间的外部核层测量，以确定任何潜在的视网膜退化的速率和均匀性。对于视网膜测量的 3D 映射，请查看注射后 OCT 图像并记下任何可识别的地标，然后定位动物以从同一区域获取后续 SDO CT 图像，注意包括相同的可识别地标，并像刚刚演示的一样处理记录的图像。获得高质量的 OCT 图像（包括可识别的地标）非常重要，以方便重新成像以前注入的视网膜区域。为了测量操作系统长度的变化，卡钳从外部限制膜放置到布鲁克斯膜，从而允许可靠的测量，因为这些层在 OCT 图像中很容易识别。这些层之间不同鼠标线的测量值差异归因于较短的外部段长度。如果成功执行下重注入，则下一个空间的打开会产生一个 bleb，该 bleb 可以在 HRSD OCT 成像器的 en 面视图和 B 扫描图像中清晰可视化。叠加注射部位的边界图允许分割视网膜组织测量和卡钳位置数据集，以便随后对特定治疗剂对视网膜退化的影响进行统计测试。一旦掌握，这项技术可以在十分钟内完成，如果执行得当。在尝试这个程序时，重要的是要记住保持手术环境清洁和无菌。该技术开发后，为基因治疗领域的研究人员实时监控整个下体手术注射过程铺平了道路，并创造了一种立即确定体内手术成功的方法。按照此注射程序，可以执行 OCT 和其他方法，如电视网膜学，以回答有关转基因功能救援和/或毒性的其他问题。观看此视频后，您应该了解如何执行跨细胞和转义体下视网膜注射，如何从此图像将注射出血的范围映射到 OCT，并获得外部视网膜层的严格测量。不要忘记，使用基因治疗载体可能是危险的，并且在执行此程序时应始终使用预防措施，例如佩戴适当的个人防护设备。

Research

JoVE Journal

Medicine

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

在免疫缺陷小鼠的建立和传播人类视网膜母细胞瘤肿瘤

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

科研

医学

Production and Detection of Reactive Oxygen Species (ROS) in Cancers

Production and Detection of Reactive Oxygen Species ROS in Cancers

在癌症的生产和检测活性氧（ROS）的

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive ...

Doppler Optical Coherence Tomography of Retinal Circulation

多普勒光学相干断层扫描视网膜循环

Noncontact retinal blood flow measurements are performed with a Fourier domain optical coherence tomography (OCT) system using a circumpapillary double ...

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

一种新的手术方法气管内注射生物活性剂在胎鼠模型

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye

基因治疗的载体和干细胞视网膜下注射到老鼠的眼睛

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy ...

Near Infrared Optical Projection Tomography for Assessments of &beta;-cell Mass Distribution in Diabetes Research

Near Infrared Optical Projection Tomography for Assessments of ÃƒÅ½Ã‚Â²-cell Mass Distribution in Diabetes Research

近红外光学投影层析成像技术在糖尿病研究β细胞质量分布的评估

By adapting OPT to include the capability of imaging in the near infrared (NIR) spectrum, we here illustrate the possibility to image larger bodies of ...

Generation and 3-Dimensional Quantitation of Arterial Lesions in Mice Using Optical Projection Tomography

生成和动脉病变的小鼠的三维定量使用光学投影层析成像

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating ...

In Vivo, Percutaneous, Needle Based, Optical Coherence Tomography of Renal Masses

In Vivo, Percutaneous, Needle Based, Optical Coherence Tomography of Renal Masses

在体内 ，经皮，基于针，肾群众的光学相干断层扫描

Optical coherence tomography (OCT) is the optical equivalent of ultrasound imaging, based on the backscattering of near infrared light. OCT provides real ...

Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

通过重组腺相关病毒载体注射立体的α-突触核蛋白基于大鼠模型为帕金森氏病的发展

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on ...

A Mouse Model of Retinal Ischemia-Reperfusion Injury Through Elevation of Intraocular Pressure

视网膜缺血再灌注损伤的小鼠模型通过眼压升高

Retinal ischemia-reperfusion (I/R) is a pathophysiological process contributing to cellular damage in multiple ocular conditions, including glaucoma, ...