我们希望感谢帕蒂吉莱德曼在手术和苏珊. 波顿为 RPE 细胞准备的帮助。我们也承认 NYSTEM C028504 为这个项目提供资金。贾斯汀· d ·米勒得到 NIH 授予 F32EY025931 的支持。

所有涉及动物的程序都得到了纽约州立大学奥尔巴尼分校动物保育和使用委员会 (IACUC) 的批准。
1. 预注射制剂
<ol><li>
hRPESC 视网膜色素上皮细胞悬浮液的制备 注意: 所有以下步骤都在无菌组织培养罩中执行, 并且需要熟悉基本的无菌技术。<ol>
<li>从50-90 岁的人眼中分离出主要的 hRPE 细胞, 并在24井板中培养细胞12。冷冻的细胞在通道 1, 解冻的需要和文化通道 2 (P2) 细胞4-5 周 (图 1A) 注射。</li>
		<li>移除培养基12 , 用500µL 5ml 1X Dulbecco 磷酸盐缓冲盐水, 不含钙 & 镁 (1x DPBS-CMF), 用 1000 DPBS 吸管将 1X CMF µL 加入井中, 然后用真空吸尘器将其取出。</li>
		<li>每个井加300µL 胰蛋白酶/dnasei。孵育 hRPESC-RPE 细胞在胰蛋白酶/dnasei (4 kU dnasei 每1毫升0.25% 胰蛋白酶-EDTA) 4 分钟, 在37° c 离解细胞。</li>
		<li>检查显微镜下的细胞, 看它们是否已经围捕。继续孵育的细胞在胰蛋白酶/dnasei 的额外2分钟, 如果他们还没有四舍五入。</li>
		<li>一旦细胞被围捕, 使用1000µL 吸管研制分离的细胞从井和转移的胰蛋白酶/dnasei 包含分离细胞成一个15毫升锥形管与同等体积的5ml 培养基, 使胰蛋白酶/dnasei 灭活。</li>
		<li>用 5ml 1X DPBS CMF 的冲洗井, 通过轻轻移上下, 尤其是在井边;然后将这些细胞添加到前锥管中。</li>
		<li>离心锥形管在 286 x g 为 5 min 在4° c, 颗粒细胞。</li>
		<li>用1毫升培养基去除上清和重细胞。</li>
		<li>使用例计数单元格。</li>
		<li>离心机在 286 x g 为5分钟在4° c 的颗粒细胞。</li>
		<li>在5万细胞/µL 的无菌平衡盐溶液 (BSS) 中去除上清和重细胞 (在视网膜注射过程中, 在1µL 体积内提供5万细胞)。</li>
		<li>将最终的细胞悬浮液 (CS) 转化为1.7 毫升的离心管, 并将其放入冰水混合物中, 直至注射使用。</li>
	</ol>
</li>
	<li>
细胞注射器的制备
	<ol>
<li>插入一个无菌的33口径斜角针注入注射器和螺丝紧紧地组装注射器。</li>
		<li>冲洗注射器与100% 乙醇5-6 次。</li>
		<li>冲洗注射器与70% 乙醇5-6 次。</li>
		<li>用 BSS 冲洗注射器5-6 次。</li>
		<li>在解剖显微镜 (图 1B) 下, 用不育的黑色记号笔在600µm 的位置上标记注射器针。</li>
		<li>将注射器放在微上进行注射。</li>
	</ol>
</li>
</ol>2. 视网膜注射液
<ol><li>
手术区和动物制剂
	<ol>
<li>称一个4-5 周大的 RCS 大鼠 (60-100 克), 并麻醉它使用异氟醚蒸汽输送系统。 注意:诱导麻醉使异氟醚的流动率保持在5%。通过按压爪子来确认麻醉深度, 然后在手术过程中将麻醉维持的流速降低到2-3%。</li>
		<li>在解剖显微镜的舞台上放置一个不育的外科悬垂, 上面有一个加热垫, 以建立一个不育的手术区。</li>
		<li>将大鼠转移到手术区, 将大鼠置于与异氟醚系统相连的鼻锥中, 以维持麻醉。</li>
		<li>用纱布盖住老鼠的身体。捏住老鼠的脚趾以确认全身麻醉。</li>
	</ol>
</li>
	<li>
显微镜下反式巩膜视网膜注射液
	<ol>
<li>在老鼠的 unoperated 眼上涂抹一滴眼润滑剂。</li>
		<li>将老鼠的左眼朝向右侧, 将它的头部朝向外科医生的右手, 将它的背部朝向外科医生。</li>
		<li>修剪任何用小剪刀遮住眼睛的胡须。</li>
		<li>从左眼的颞侧滴下少量的眼部洗净, 用棉签将多余的部分收集在鼻侧, 冲洗眼睛。</li>
		<li>扩大瞳孔与1% 卡和2.5% 肾上腺素 (新鲜的10% 肾上腺素, 稀释它在不育0.9% 生理盐水的手术当天) 为一个后 OCT 考试通过应用一滴每一个。</li>
		<li>轻轻地将眼部周围的皮肤拉4-6 次以打开眼睑, 使眼睛稍微 proptosed, 以便更方便地进入角膜缘后部。</li>
		<li>应用一滴眼洗, 保持眼睛湿润。</li>
		<li>轻轻研制 cs (准备在步骤 1.1.12) 和负载的注射器与1.2 µL cs。额外的0.2 µL 用于补偿注入回流。 注意:根据我们的测量, 大约 5,000-8 000 细胞在回流中丢失, 注射了5万细胞/µL, 相当于大约10-16% 的细胞丢失, 另外还注射了0.2 µL CS 以补偿这种细胞损失。</li>
		<li>放置在一个微 (或有一个助理持有它) 垂直的注射器填充, 因为 RPE 细胞往往容易下沉悬浮。</li>
		<li>应用一滴0.5% 丙 (局部外用麻醉剂) 的眼睛和删除过剩的棉花涂抹剂。 注意:此步骤应抑制角膜反射, 防止眼睛在后续步骤中闪烁。</li>
		<li>用镊子夹住角膜缘后部的结膜, 旋转眼睛鼻, 抬起结膜作 "帐篷"。</li>
		<li>用剪刀剪下顶部的 "帐篷", 使一个小开口在结膜和暴露的基础巩膜。</li>
		<li>使用镊子握住角膜缘旁边剩余的结膜边缘的边缘, 并旋转眼睛鼻, 使瞳孔轴的角度相对于桌面 (图 1C) 大约30度。继续抓取的结膜边缘是需要提供一个反在针插入和保持眼睛的最佳角度。</li>
		<li>要做一个试验孔为细胞注射, 位置末端一个无菌的斜面31测量胰岛素针在 1,200-1, 500 µm 后到角膜缘与开头的尖朝向。</li>
		<li>调整胰岛素针的角度, 使它是10-15 度以上的巩膜 (切线到一个假想的平面在预期的注射地点)。慢慢穿透巩膜-脉络膜复合物到针深度约500µm。在色素大鼠, 针端将 ' 消失 ' 下色素脉络膜。在这里使用的胰岛素针的牌子, 从针尖到斜面的距离是500µm。</li>
		<li>小心地取出胰岛素针 (可以看到一个非常小的血液渗出)。</li>
		<li>如果注意到过多的出血, 可以使用眼枪清除孔, 如果需要。在矛应用后持续出血表明一艘船可能已经损坏。</li>
		<li>引导视网膜色素上皮细胞加载注射器针进入先导孔, 与开口朝下, 并在大约10-15 度的角度相对于巩膜的局部表面。</li>
		<li>轻轻地将注射器针插入先导孔, 深度约500µm, 进入视网膜空间。应该有大约100µm 的边缘之间的黑色钢笔标记和点的针是覆盖的有色脉络膜 (图 1C和1D)。</li>
		<li>请助手轻轻压低注射器的柱塞, 注入适当的细胞容积 (约1.2 µL)。准备提供一些反作为助手按下柱塞。 注意:事先与助手的模拟练习可以为外科医生和助手提供必要的经验。</li>
		<li>当视觉上专注于钢笔标记边缘, 按住注射器到位25-30 秒, 然后慢慢收回注射器。通常观察到少量的回流。 注意:如果没有观察到回流, 可能有一个玻璃注射。如果你看到通过密封或在巩膜下填充细胞的回流, 注射太浅。</li>
		<li>冲洗注射部位的细胞外排, 用无菌眼洗3次, 用棉签收集多余的。</li>
		<li>在操作眼睛上涂抹一滴眼部润滑剂, 将大鼠转移到 OCT 站, 检查移植细胞的位置和视网膜疱疹的大小。</li>
	</ol>
</li>
</ol>3. 注射后处理
<ol><li>
抗炎镇痛药治疗
	<ol>
<li>在生理盐水皮下注射0.1 毫克/千克体重的丁丙诺啡以减少疼痛。</li>
		<li>通过腹腔注射 (ip) 在生理盐水中注射地塞米松1.6 毫克/千克的体重以控制炎症。</li>
	</ol>
</li>
	<li>
动物康复
	<ol>
<li>将大鼠返回到热灯下的回收笼中, 以保持体温。</li>
		<li>观察手术眼出血的迹象。</li>
		<li>观察鼠, 确保它从麻醉中出来。</li>
		<li>将被恢复的动物带回一个新的笼子, 用手术卡将笼子标记起来, 每天监测任何遇险、眼部出血或角膜混浊的迹象。如果有问题, 立即通知兽医。</li>
	</ol>
</li>
</ol>

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作者没有什么可透露的。

本文所描述的视网膜注射技术是通过跨巩膜通路, 注射器针穿透眼壁的外层 (巩膜-脉络膜-RPE 复合体) 而不损害神经视网膜或扰乱玻璃腔。一个替代的反式 vitreal 方法有潜在的风险, 导致白内障的晶状体损伤, 因为啮齿目动物的镜头占据了大多数的玻璃腔。与这种方法相比, 我们的技术是低风险和造成最小的创伤, 因为注射器针不需要穿过整个玻璃腔, 以达到视网膜空间。事实上, 在我们的研究中, OCT ?...

人视网膜位于眼睛的后面功能作为一个轻的感觉组织并且在视觉知觉扮演一个关键的角色。因此视网膜细胞功能障碍或细胞死亡导致视力问题或永久性失明。在视网膜不同层次的细胞变性或功能紊乱的疾病被称为退行性视网膜疾病, 其中 AMD 是最常见的类型, 也是发达国家老年人不可逆转性失明的主要原因之一1,2。AMD 的病理过程与视网膜色素上皮层与底层赫膜之间的 "疣" 堆积有关, 从而损害了视网膜色素上皮支持的感光生理, 导致了神经系统的萎缩和视力丧失3, 4,5。到目前为止, 没有治疗高级干 (non-neovascular) AMD。干细胞治疗作为再生医学的一个新范式的出现, 带来了用干细胞衍生的健康细胞取代功能失调或死亡的 RPE 细胞的希望。事实上, 广泛的临床前研究的移植干细胞 (如, 人类胚胎干细胞) 衍生 rpe 细胞到 rpe-变性动物模型已经执行6,7, 其中一些已取得进展临床试验8,9 (NCT01344993, ClinicalTrials.gov)。最近, 我们的实验室发现了人类视网膜色素上皮细胞 (hRPESCs) 中的一种替代的干细胞来源, 目前正被用于 hRPESC 衍生-rpe 细胞 (hRPESC-rpe) 移植治疗 AMD 的前临床研究。10,11,12,13。
视网膜注射技术应用于上述多个群体, 包括本组的前临床研究。动物视网膜注射有两种一般的方法: 反 vitreal 和反式巩膜。vitreal 方法的优势是, 外科医生能够直接观察针端, 因为它穿透了前眼, 越过整个 vitreal 腔毗邻的镜头, 并穿透视网膜在后面的眼睛, 以达到视网膜空间14,15,16。然而, 它需要扰乱视网膜在两个位置 (前部和后部), 承担损害晶状体的风险, 并可能导致细胞回流到玻璃体时, 针被收回。相反, 跨巩膜的方法, 原则上, 避免介入视网膜和玻璃体, 和回流退出眼睛。在有色啮齿目动物中, 外科医生最初可以观察到巩膜的穿透, 但在进入色素脉络后, 针端不再可见。没有直接观察, 破坏视网膜是常见的, 并可能导致视网膜解剖和交付细胞和/或血液进入玻璃体。此外, 由于眼睛表面是弯曲的, 很难知道哪种针的角度和深度是最有效的反式巩膜注射。
在这一可视化的文章中, 我们介绍了一个跨巩膜视网膜注射方法, 通过使用后评价与光学相干断层扫描 (OCT), 这使得对注射场的详细检查。我们的反式巩膜注射技术利用定义的位置, 角度和深度注射针, 以产生非常低的外科创伤和高可靠性。在这里, 我们特别演示了 hRPESC-RPE 细胞注入视网膜空间的 RCS 大鼠, 一个临床前模型的人 AMD。采用这种注射方法, 成功、一致地将 hRPESC 视网膜色素上皮细胞导入到 RCS 大鼠眼的视网膜空间中, 成功率很高。注入的细胞以前发现, 导致保存 RCS 光至少2月后, 注入13。本程序在解剖显微镜下进行, 易于学习。它需要两个人 (一个外科医生和一个助理) 来执行注射和平均注射时间为每种动物少于5分钟。定义的角度和深度为注射针, 使之成为可能的实验室, 在那里 OCT 是无法获得成功的视网膜注射液。它允许高度可再生的视网膜通路, 不仅可以用于细胞移植, 也可用于药物传递和基因治疗。

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			<td style="width:231px;">This is used to make 2.5% Phenylepherine</td>
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			<td height="44" style="height:44px;width:259px;">hRPESC-RPE cells</td>
			<td style="width:256px;">Not available commercially</td>
			<td style="width:119px;"></td>
			<td style="width:231px;">Please refer to &#34;Reference #12&#34; for cell isolation and mainteinance.</td>
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			<td height="21" style="height:21px;width:259px;">Nanofil syringe (10 ul)</td>
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			<td height="21" style="height:21px;width:259px;">Insulin syringe needles 31-gauge</td>
			<td style="width:256px;">Becton Dickinson</td>
			<td style="width:119px;">328418</td>
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			<td height="21" style="height:21px;width:259px;">Animal anesthesia system</td>
			<td style="width:256px;">World Precision Instruments</td>
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			<td height="21" style="height:21px;width:259px;">Stereo microscope</td>
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			<td style="width:119px;">R2210</td>
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			<td height="21" style="height:21px;width:259px;">Sterile black marker pen</td>
			<td style="width:256px;">Viscot Industries</td>
			<td style="width:119px;">1416S-100</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Miniature measuring scale</td>
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			<td style="width:119px;">13623</td>
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			<td height="63" style="height:63px;width:259px;">Infrared Basking Spot Lamp&#160;</td>
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			<td style="width:119px;">PT2144</td>
			<td style="width:231px;">This is used as a heating lamp for animals during the post-surgical recovery&#160; phase</td>
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development of a refined protocol for trans-scleral subretinal transplantation of human retinal pigment epithelial cells into rat eyes

退行性视网膜疾病, 如年龄相关的黄斑变性 (AMD) 是导致不可逆转的视力丧失全球。AMD 的特点是视网膜色素上皮 (RPE) 细胞的退化, 这是一个单层的细胞功能支持和解剖包裹周围的神经视网膜。目前的药理治疗 non-neovascular amd (干 amd) 只减缓疾病进展, 但不能恢复视力, 必须研究, 以确定新的治疗策略。用健康细胞取代退行性视网膜色素上皮细胞有望在未来治疗干性 AMD。广泛的临床前研究的干细胞替代疗法的 AMD 涉及移植干细胞衍生 RPE 细胞进入视网膜空间的动物模型, 其中视网膜注射技术的应用。在这些临床前动物研究中最常用的方法是通过经巩膜路线, 这是由于缺乏直接可视化的针端, 往往会导致视网膜损伤而变得困难。通过玻璃体的另一种方法可以直接观察针端位置 , 但是由于更多的眼部组织扰 , 它有很高的手术创伤风险。我们已经开发了一个低风险和可重复性的改良反式巩膜注射法, 使用定义的针角和深度, 成功地和一贯地提供 RPE 细胞进入大鼠视网膜空间, 并避免过多的视网膜损伤。以这种方式传送的细胞在皇家外科医生 (RCS) 大鼠中已经被证明是有效的, 至少2月。该技术不仅可用于细胞移植, 也可用于小分子或基因治疗的传递。

利用本文所描述的技术, 我们通过精确地控制插入到组织中的注射器针的位置、角度和深度, 将 hRPESC 视网膜色素上皮细胞送到 RCS 大鼠的视网膜空间中 (图 1B-D).在移植后立即进行 OCT 检查, 观察注射部位和移植细胞产生的视网膜疱疹。术后 OCT 评估是评估注射质量和视网膜损伤或出血监测的检查工具。在 OCT 扫描下, 可以清楚地看到视网膜疱疹 (<strong...

Watch this Scientific Journal Video about Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes at JoVE.com

Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

视网膜注射液已广泛应用于干细胞置换治疗老年性黄斑变性的临床前研究。在这一可视化的文章中, 我们描述了一个低风险, 可重复和精确修改的视网膜注射技术, 通过巩膜的方法传递细胞到大鼠的眼睛。

人视网膜色素上皮细胞向大鼠眼巩膜视网膜移植精制方案的研制

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is ...

development-refined-protocol-for-trans-scleral-subretinal

Research

JoVE Journal

Neuroscience

9.4K 次观看.  Neural Stem Cell Institute. 视网膜注射液已广泛应用于干细胞置换治疗老年性黄斑变性的临床前研究。在这一可视化的文章中, 我们描述了一个低风险, 可重复和精确修改的视网膜注射技术, 通过巩膜的方法传递细胞到大鼠的眼睛。

视频: Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium (hfRPE) cultures with morphological, physiological and genetic characteristics of native human RPE. These hfRPE cell cultures exhibit heavy pigmentation, and electron microscopy show extensive apical membrane microvilli. The junctional complexes were identified with immunofluorescence labeling of various tight junction proteins. Epithelial polarity and function of these easily reproducible primary cultures closely resemble previously studied mammalian models of native RPE, including human. These results were extended by the development of therapeutic interventions in several animal models of human eye disease. We have focused on strategies for the removal of abnormal fluid accumulation in the retina or subretinal space. The extracellular subretinal space separates the photoreceptor outer segments and the apical membrane of the RPE and is critical for maintenance of retinal attachments and a whole host of RPE/retina interactions.

We provide a reproducible method for culturing confluent monolayers of human fetal retinal pigment epithelial cells (hfRPE) cells that exhibit morphology, physiology, polarity, and protein and gene expression patterns of adult native tissue. This work has been extended to an animal model of several eye diseases.

视网膜色素上皮的生理与病理生理学研究的实验模型

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium ...

科研

神经科学

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and cones). The RPE is a monolayer of pigmented cuboidal cells and associates closely with the neural retina just above it. This association makes the RPE of great interest to researchers studying retinal diseases. The RPE is also the site of an in vivo assay of homology-directed DNA repair, the pun assay. The mouse eye is particularly difficult to dissect due to its small size (about 3.5mm in diameter) and its spherical shape. This article demonstrates in detail a procedure for dissection of the eye resulting in a whole mount of the RPE. In this procedure, we show how to work with, rather than against, the spherical structure of the eye. Briefly, the connective tissue, muscle, and optic nerve are removed from the back of the eye. Then, the cornea and lens are removed. Next, strategic cuts are made that result in significant flattening of the remaining tissue. Finally, the neural retina is gently lifted off, revealing an intact RPE, which is still attached to the underlying choroid and sclera. This whole mount can be used to perform the pun assay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.

A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.

解剖鼠标眼的视网膜色素上皮的全山

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.

We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required.

大鼠胚胎神经细胞的分离和培养:一个快速议定书"

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in retinopathies using non-viral and viral gene delivery to the retina and retinal pigment epithelium (RPE). Subretinal injection is the best approach to deliver viral vectors directly to RPE cells. Before the clinical trial of a gene therapy, it is inevitable to validate the efficacy of the therapy in animal models of various retinopathies. Thus, subretinal injection in mice becomes a fundamental technique for an ocular gene therapy. In this protocol, we provide the easy and replicable technique for subretinal injection of viral vectors to experimental mice. This technique is modified from the intravitreal injection, which is widely used technique in ophthalmology clinics. The representative results of RPE/choroid/scleral complex flat-mount will help to understand the efficacy of this technique and adjust the volume and titer of viral vectors for the extent of gene transduction.

Subretinal injection is a surgical technique for effective gene delivery to retinal pigment epithelium in the mouse eye. Here we describe an easy and replicable method for subretinal injection of viral vectors to retinal pigment epithelium in experimental mice.

病毒载体的基因治疗的小鼠视网膜色素上皮缘方针，视网膜下注射

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in ...

Enrichment of Bruch's Membrane from Human Donor Eyes

Age-related macular degeneration (AMD) is a leading cause of visual impairment in the developed world. The disease manifests itself by the destruction of the center&#160;of the retina, called the macula, resulting in the loss of central vision. Early AMD is characterised by the presence of small, yellowish lesions called soft drusen that can progress onto late AMD such as geographic atrophy (dry AMD) or neovascularisation (wet AMD). Although the clinical changes are well described, and the understanding of genetic influences on conferring AMD risk are getting ever more detailed, one area lacking major progress is an understanding of the biochemical consequences of genetic risk. This is partly due to difficulties in understanding the biochemistry of Bruch&#8217;s membrane, a very thin extracellular matrix that acts as a biological filter of material from the blood supply and a scaffold on which the retinal pigment epithelial (RPE) cell monolayer resides. Drusen form within Bruch&#8217;s membrane and their presence disrupts nutrient flow to the RPE cells. Only by investigating the protein composition of Bruch&#8217;s membrane, and indeed how other proteins interact with it, can researchers hope to unravel the biochemical mechanisms underpinning drusen formation, development of AMD and subsequent vision loss. This paper details methodologies for enriching either whole Bruch&#8217;s membrane, or just from the macula region, so that it can be used for downstream biochemical analysis, and provide examples of how this is already changing the understanding of Bruch&#8217;s membrane biochemistry.

Identifying proteins specifically associated with Bruch&#8217;s membrane in human eyes is an important step in understanding the biochemical mechanisms behind eye diseases such as age-related macular degeneration. This protocol describes how to enrich this sheet of extracellular matrix for down-stream biochemical analysis.

从人类供体眼富集布鲁赫的膜

Age-related macular degeneration (AMD) is a leading cause of visual impairment in the developed world. The disease manifests itself by the destruction of ...

Single-cell RNA-Seq of Defined Subsets of Retinal Ganglion Cells

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for the improved understanding of neuronal diversity, it is important to identify genes whose expression defines various subpopulations of cells. The retina serves as an excellent model for the study of central nervous system diversity, as it is composed of multiple major cell types. The study of each major class of cells has yielded genetic markers that facilitate the identification of these populations. However, multiple subtypes of cells exist within each of these major retinal cell classes, and few of these subtypes have known genetic markers, although many have been characterized by morphology or function. A knowledge of genetic markers for individual retinal subtypes would allow for the study and mapping of brain targets related to specific visual functions and may also lend insight into the gene networks that maintain cellular diversity. Current avenues used to identify the genetic markers of subtypes possess drawbacks, such as the classification of cell types following sequencing. This presents a challenge for data analysis and requires rigorous validation methods to ensure that clusters contain cells of the same function. We propose a technique for identifying the morphology and functionality of a cell prior to isolation and sequencing, which will allow for the easier identification of subtype-specific markers. This technique may be extended to non-neuronal cell types, as well as to rare populations of cells with minor variations. This protocol yields excellent-quality data, as many of the libraries have provided read depths greater than 20 million reads for single cells. This methodology overcomes many of the hurdles presented by Single-cell RNA-Seq and may be suitable for researchers aiming to profile cell types in a straightforward and highly efficient manner.

Here, we present a combinatorial approach for classifying neuronal cell types prior to isolation and for the subsequent characterization of single-cell transcriptomes. This protocol optimizes the preparation of samples for successful RNA Sequencing (RNA-Seq) and describes a methodology designed specifically for the enhanced understanding of cellular diversity.

视网膜神经节细胞定义子集的单细胞RNA-Seq

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for ...

Homochronic Transplantation of Interneuron Precursors into Early Postnatal Mouse Brains

Neuronal fate determination and maturation requires an intricate interplay between genetic programs and environmental signals. However, disentangling the roles of intrinsic vs. extrinsic mechanisms that regulate this differentiation process is a conundrum for all developmental neurobiologists. This issue is magnified for GABAergic interneurons, an incredibly heterogeneous cell population that is born from transient embryonic structures and undergo a protracted migratory phase to disperse throughout the telencephalon. To explore how different brain environments affect interneuron fate and maturation, we developed a protocol for harvesting fluorescently labeled immature interneuron precursors from specific brain regions in newborn mice (P0-P2). At this age, interneuron migration is nearly complete and these cells are residing in their final resting environments with relatively little synaptic integration. Following collection of single cell solutions via flow cytometry, these interneuron precursors are transplanted into P0-P2 wildtype postnatal pups. By performing both homotopic (e.g., cortex-to-cortex) or heterotopic (e.g., cortex-to-hippocampus) transplantations, one can assess how challenging immature interneurons in new brain environments affects their fate, maturation, and circuit integration. Brains can be harvested in adult mice and assayed with a wide variety of posthoc analysis on grafted cells, including immunohistochemical, electrophysiological and transcriptional profiling. This general approach provides investigators with a strategy to assay how distinct brain environments can influence numerous aspects of neuron development and identify if specific neuronal characteristics are primarily driven by hardwired genetic programs or environmental cues.

Challenging young neurons in new brain regions can reveal important insights into how the environment sculpts neuronal fate and maturation. This protocol describes a procedure to harvest interneuron precursors from specific brain regions and transplant them either homotopically or heterotopically into the brain of postnatal pups.

Interneuron 前体 Homochronic 移植早期小鼠脑的研究

Neuronal fate determination and maturation requires an intricate interplay between genetic programs and environmental signals. However, disentangling the ...

Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is a complicated task, which is particularly difficult in regards to the development of &#947;-aminobutyric acid (GABA)ergic cortical interneurons (CIs). CIs are the main inhibitory neurons in the cerebral cortex, and they play key roles in neuronal networks, by regulating both the activity of individual pyramidal neurons, as well as the oscillatory behavior of neuronal ensembles. They are generated in transient embryonic structures (medial and caudal ganglionic eminences -&#160;MGE and CGE) that are very difficult to efficiently target using in utero electroporation approaches. Interneuron progenitors migrate long distances during normal embryonic development, before they integrate in the cortical circuit. This remarkable ability to disperse and integrate into a developing network can be hijacked by transplanting embryonic interneuron precursors into early post-natal host cortices. Here, we present a protocol that allows genetic modification of embryonic interneuron progenitors using focal ex vivo electroporation. These engineered interneuron precursors are then transplanted into early post-natal host cortices, where they will mature into easily identifiable CIs. This protocol allows the use of multiple genetically encoded tools, or the ability to regulate the expression of specific genes in interneuron progenitors, in order to investigate the impact of either genetic or environmental variables on the maturation and integration of CIs.

Here we present a protocol, designed to use chemogenetic tools to manipulate the activity of cortical interneuron progenitors transplanted into the cortex of early postnatal mice.

将化学遗传工程皮质内神经内生前生移植到早期产后小鼠大脑

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

坐骨神经粉碎损伤大鼠模型中功能评估的3D运动学分析

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...

Subretinal Transplantation of Human Embryonic Stem Cell-Derived Retinal Tissue in a Feline Large Animal Model

Retinal degenerative (RD) conditions associated with photoreceptor loss such as age-related macular degeneration (AMD), retinitis pigmentosa (RP) and Leber Congenital Amaurosis (LCA) cause progressive and debilitating vision loss. There is an unmet need for therapies that can restore vision once photoreceptors have been lost. Transplantation of human pluripotent stem cell (hPSC)-derived retinal tissue (organoids) into the subretinal space of an eye with advanced RD brings retinal tissue sheets with thousands of healthy mutation-free photoreceptors and has a potential to treat most/all blinding diseases associated with photoreceptor degeneration with one approved protocol. Transplantation of fetal retinal tissue into the subretinal space of animal models and people with advanced RD has been developed successfully but cannot be used as a routine therapy due to ethical concerns and limited tissue supply. Large eye inherited retinal degeneration (IRD) animal models are valuable for developing vision restoration therapies utilizing advanced surgical approaches to transplant retinal cells/tissue into the subretinal space. The similarities in globe size, and photoreceptor distribution (e.g., presence of macula-like region area centralis) and availability of IRD models closely recapitulating human IRD would facilitate rapid translation of a promising therapy to the clinic. Presented here is a surgical technique of transplanting hPSC-derived retinal tissue into the subretinal space of a large animal model allowing assessment of this promising approach in animal models.

Presented here is a surgical technique for transplanting human pluripotent stem cell (hPSC)-derived retinal tissue into the subretinal space of a large animal model.

猫大型动物模型中人类胚胎干细胞来源的视网膜组织的视网膜下移植

Retinal degenerative (RD) conditions associated with photoreceptor loss such as age-related macular degeneration (AMD), retinitis pigmentosa (RP) and ...