Transpupillary-Guided Trans-Scleral Transplantation of Subretinal Grafts in a Retinal Degeneration Mouse Model

Ying V. Liu; Kang V. Li; Zhuolin Li; Yuchen Lu; Minda M. McNally; Edward P. Esposito; Kanza Aziz; Mandeep S. Singh

doi:10.3791/65448

JoVE Journal
Neuroscience
Author Produced

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JoVE Journal Neuroscience

Transpupillary-Guided Trans-Scleral Transplantation of Subretinal Grafts in a Retinal Degeneration Mouse Model

经瞳孔引导下视网膜下移植物的视网膜下移植物经巩膜移植视网膜变性小鼠模型

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January 26, 2024

DOI: 10.3791/65448-v

Ying V. Liu¹, Kang V. Li¹, Zhuolin Li¹, Yuchen Lu¹, Minda M. McNally¹, Edward P. Esposito¹, Kanza Aziz¹, Mandeep S. Singh^1,2

¹Wilmer Eye Institute,Johns Hopkins University School of Medicine, ²Department of Genetic Medicine,Johns Hopkins University School of Medicine

Please note that some of the translations on this page are AI generated. Click here for the English version.

Overview

This study presents a transpupillary vision-guided trans-scleral approach for delivering subretinal cellular grafts in mouse models, achieving a low rate of surgical complications. The method utilizes a direct transpupillary guidance system to optimize the surgical precision of subretinal transplantation.

Key Study Components

Area of Science

Neuroscience
Ophthalmology
Cell transplantation

Background

Subretinal cell transplantation is crucial for treating retinal degeneration.
Current methods may pose risks and complications.
Transpupillary guidance offers a potential improvement in precision.
Cohorts of mice with retinal degeneration serve as experimental subjects.

Purpose of Study

To develop a surgical platform for safe subretinal cell delivery in mice.
To minimize complications during retinal grafting procedures.
To validate the effectiveness of grafts in restoring retinal function.

Methods Used

The platform involved transpupillary visualization for subretinal cell delivery.
Mice with retinal degeneration served as recipients for cellular grafts.
Key steps included isolating donor retinal cells, preparing the surgical site, and managing anesthesia.
Monitoring of the grafts was performed through imaging techniques post-surgery.

Main Results

Successful graft delivery was confirmed by the presence of blebs in the subretinal space.
Retinal sheets demonstrated survival and integration into the host retina.
Histological analysis revealed abundant photoreceptors in transplanted tissues.
Grafts reconstituted the outer nuclear layer of recipient retinas effectively.

Conclusions

The trans-scleral approach enables precise and safe subretinal cell delivery.
This method may facilitate advancements in therapeutic applications, including gene therapy.
Findings suggest potential for broader applications in treating retinal degenerative diseases.

Frequently Asked Questions

What are the advantages of the transpupillary approach?

This approach allows for greater surgical precision and minimizes the risk of complications during cell transplantation.

How is the cellular graft prepared for transplantation?

The donor retinas are isolated and cultured to create a cellular suspension or sheets, which are then delivered subretinally.

What types of imaging techniques were used to assess the grafts?

Multimodal confocal scanning laser ophthalmoscopy and spectral domain optical coherence tomography were utilized to evaluate graft survival and retinal layers.

What outcomes were observed post-transplantation?

Grafts showed signs of integration, with photoreceptors observed in histological samples, indicating functional recovery in recipient mice.

Can this method be adapted for other types of therapeutic agents?

Yes, the technique can be adapted to deliver different types of therapeutic agents, including gene therapy, along with retinal transplants.

What are some limitations of this study?

The study primarily focuses on mouse models, which may limit direct applicability to human conditions without further validation.

该协议提出了一种经瞳孔视觉引导的经巩膜方法，可在有或没有视网膜变性的小鼠接受者中安全准确地输送视网膜下细胞移植物，手术并发症发生率低。

本研究的总体目标是开发一种具有直接经瞳孔引导的经巩膜移植平台，以促进小鼠受体中细胞的视网膜下递送。所有动物实验均根据美国国立卫生研究院指南进行。我们的动物使用声明，并经约翰霍普金斯大学动物护理和使用委员会批准。

采用出生后第3-6天年龄的EGFP小鼠作为视网膜细胞悬液的供体。采用出生后第 3 天年龄的 OPN1LW-EGFP/NRL 小鼠作为视网膜片供体。以免疫缺陷的成年视网膜变性Rd1/NS小鼠为受体。

用过量的二氧化碳对供体小鼠实施安乐死。为了隔离小鼠的眼球，使用微型剪刀小心地打开幼崽的眼睑并露出眼球。包裹视神经，用光滑的镊子拉出眼球。

隔离眼球后，使用 25 号针在角膜中心切开一个孔。将角膜切成两半。并扩大切口到巩膜和RPE。

然后取出巩膜和 RPE。接下来，使用微齿镊轻轻取出晶状体和玻璃体以隔离神经视网膜。为了收集供体视网膜悬浮液，将神经视网膜在 37 摄氏度的木瓜蛋白酶溶液中孵育 20 至 30 分钟，直到检测到无细胞团块。

按照木瓜蛋白酶试剂盒制造商的说明收集单细胞。为了准备视网膜片，将分离的视网膜放入含有PBS的培养皿中。然后用微型剪刀轻轻地将神经视网膜切成多片视网膜。

用腹膜内注射氯胺酮和甲苯噻嗪麻醉受体小鼠。确保达到的麻醉平面是动物失去眨眼和疼痛反射的手术平面，但呼吸和呼吸保持规律。通过捏尾或踏板退出反射来评估麻醉深度。

在手术过程中重新检查麻醉深度。将小鼠放在预热的手术台上以避免体温过低。手术前五分钟用托吡卡胺滴眼液散瞳。

散瞳的良好瞳孔可以促进手术显微镜下的透瞳孔可视化。将一滴盐酸丙美卡因滴在小鼠眼睛上以镇痛。用碘对手术小鼠眼睛和周围眼部组织进行消毒。

然后用无菌PBS清洁眼睛。穿透外周隧道进入内腔以降低眼内压。接下来，在角膜顶部滴一滴透明质酸钠和玻璃盖玻片。

小鼠眼底的经瞳孔可视化现在可以在手术镜下使用。用齿镊将眼壁推向经瞳孔视觉的中心，暴露注射位点。然后用显微注射针部分穿透巩膜，与眼壁成 90 度角。

视网膜浅表血管可作为解剖学参考，将针定位在视网膜下空间内。然后注射视网膜移植物。注射器中预装的小气泡有助于验证供体细胞的视网膜下位置。

如果角膜变得浑浊，请将针头保持在视网膜下间隙，直到角膜变得透明，以使眼压恢复正常。抓住注射孔的边缘，迅速拔出针头。成功递送细胞悬液的标准是视网膜下空间中恒定的气泡。

成功输送视网膜片，被可见的白片所说服。将移植的小鼠放在预热的干净恢复笼上，并仔细监测它们是否有任何痛苦迹象。如果发生这种情况，将一滴局部盐酸丙拉卡因放在手术眼上两到三次。

在小鼠完全警觉和移动后，将小鼠移回笼子。将少量食物颗粒放入笼子地板上的凝胶杯中。如果老鼠难以到达食物漏斗。

移植后两个月，进行多模态共聚焦扫描激光检眼镜检查视网膜移植物体内状态。光谱域光学相干断层扫描显示，视网膜移植物在视网膜下空间存活，并重建了所有受体小鼠的外核层。红外成像在所有移植小鼠中均未检测到明显的白内障。

其他手术并发症，包括出血，通过多色反射成像在移植小鼠中被严重检测到。组织学染色显示，移植视网膜片中大量视锥光感受器表达OPN1LW:EGFP和S-视蛋白。同样，移植视网膜细胞悬液在体内显示出很大比例的恢复阳性光感受器，包括许多成熟的EGFP阳性杆。

未移植的小鼠表现出外核层严重变性，残留的视锥形光感受器稀疏，表示恢复。然而，在未移植的小鼠中未检测到EGP信号。本研究为小鼠受者视网膜下移植提供了一个具有直接经瞳孔视觉引导的经巩膜手术平台。

该平台能够精确递送已知剂量的细胞。除了视网膜内或玻璃体内注射不同类型的治疗药物（包括基因治疗）外，学习和促进视网膜下递送相对容易。

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