M&#252;ller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish

Federica M. Conedera; Petra Arendt; Carolyn Trepp; Markus Tschopp; Volker Enzmann

doi:10.3791/56249

JoVE Journal
Neuroscience

A subscription to JoVE is required to view this content. Sign in or start your free trial.

JoVE Journal Neuroscience

Müller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish

激光诱导的斑马鱼视网膜变性及再生模型中的 m ü ller 胶质细胞活化

Full Text

10,359 Views

06:27 min

October 27, 2017

DOI: 10.3791/56249-v

Federica M. Conedera^1,2,3, Petra Arendt¹, Carolyn Trepp^1,2,3, Markus Tschopp¹, Volker Enzmann^1,2

¹Department of Ophthalmology, University Hospital of Bern,University of Bern, ²Department of Clinical Research,University of Bern, ³Graduate School for Cellular and Biomedical Sciences,University of Bern

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

Overview

This study focuses on the use of zebrafish as a model to explore retinal degeneration and regeneration mechanisms. A protocol is described for inducing localized laser injury to the outer retina, monitoring subsequent cellular responses, particularly the involvement of Müller glia, throughout the recovery process.

Key Study Components

Area of Science

Neuroscience
Retinal biology
Regenerative medicine

Background

Zebrafish are recognized for their ability to regenerate retinal tissue.
Studying glial cell responses can provide insights into regenerative processes.
Localized injury models help minimize damage while focusing on specific cellular dynamics.

Purpose of Study

To establish a method for inducing focal retinal damage.
To observe morphological changes and cellular responses during regeneration.
To provide a framework for exploring repair mechanisms in other models.

Methods Used

The study utilized a zebrafish model to induce focal retinal damage using laser treatment.
Anesthetic procedures were detailed for the humane handling of zebrafish during experiments.
The protocol includes imaging techniques like optical coherence tomography (OCT) for monitoring changes.
Critical steps include preparing anesthetic solutions, applying laser treatment, and immediate imaging post-injury.

Main Results

Laser-induced injuries produced distinct hyper-reflective signals in the retina, indicating damage.
Müller glial responses were evident through immunohistochemistry, showing varying GFAP levels post-injury.
Recovery milestones were tracked, revealing a significant decrease in lesion size and restoration of retinal morphology over time.
The findings underline the dynamic cellular responses associated with retinal healing processes.

Conclusions

This study successfully demonstrates a method for inducing and monitoring retinal injury and regeneration in zebrafish.
The insights gained enhance understanding of regenerative mechanisms and their potential therapeutic implications for retinal diseases.

Frequently Asked Questions

What are the advantages of using zebrafish in retinal studies?

Zebrafish are advantageous due to their rapid retinal regeneration capabilities and the transparency of their embryos, allowing for easy in vivo imaging of cellular processes.

How is retinal damage induced in zebrafish?

Retinal damage is induced using a focused laser that targets specific regions of the outer retina, allowing for minimal impact on surrounding structures.

What types of data are obtained from this method?

Data obtained include morphological changes captured through imaging techniques like OCT, as well as immunohistochemical markers indicating cellular responses during regeneration.

How can this method be adapted for other models?

The protocol can be adapted to other vertebrate models by modifying anesthetic procedures and laser settings according to the specific anatomical and physiological needs of the species.

What are the critical considerations for anesthesia in this study?

Appropriate anesthesia is crucial for the welfare of zebrafish and to ensure successful operation; using freshly prepared anesthetic solutions is essential.

斑马鱼是研究脊椎动物视网膜变性/再生机制的流行动物模型。本议定书描述一种方法, 以诱导局部损伤破坏外视网膜, 对内视网膜的损害最小。随后, 我们监测在体内视网膜形态学和 m ü ller 胶质细胞反应在整个视网膜再生。

本视频的总体目标是展示如何监测斑马鱼局灶性激光诱导的视网膜损伤后体内的细胞变化。该模型可以帮助您回答视网膜再生的关键问题，例如形态变化、动力学以及所涉及的细胞类型。该技术的主要优点是，通过诱导局灶性损伤，可以直接在损伤部位研究生物过程。

虽然这种方法可以提供对斑马鱼视网膜再生的见解，但它也可以应用于研究不同动物模型中的修复机制。通过将 400 毫克三卡因粉末溶解在 97.9 毫米的水箱水和 2.1 毫米的一磨牙 TBS 中来制备麻醉剂储备溶液。在 pH 9 下用一颗磨牙 tris 调节至 pH 7.0。

为了制备工作溶液，将三卡因储备液在罐水中稀释 1 至 25 毫升，并将 50 毫升转移到培养皿中。然后，将斑马鱼放入麻醉溶液中 2 到 5 分钟，直到它们变得不动并且对外部刺激没有反应。用手将每条鱼转移到定制的硅胶针架上进行激光处理。

适当的麻醉对于动物的健康和手术的成功至关重要。因此，新鲜制备的三卡因溶液至关重要，出水时间不应超过 10 分钟。将 532 纳米拨盘激光器的输出功率设置为 70 毫瓦，脉冲持续时间设置为 100 毫秒，将天线直径设置为 50 微米。

然后，将一到两滴 2% 羟丙基甲基纤维素滴在眼睛上。将 methylcell 涂抹在眼睛上时，确保粘性溶液不会进入鳃。接下来，使用 2 毫米眼底激光透镜将激光瞄准光束聚焦在视网膜上。

在左眼的视神经周围放置四个激光点，并使用右眼未经治疗的眼睛作为内部对照。激光诱导后，立即将仍然麻醉的斑马鱼放在成像区域的定制硅胶针架上。为了获得最佳图像，通过打孔器切割市售的水凝胶隐形眼镜以适合斑马鱼的眼睛。

用甲基纤维素填充晶状体的凹面，然后将其放在角膜上。为光学相干断层扫描系统配备 78D 非接触式裂隙灯镜头。在 IR 加 OCT 模式下聚焦红外图像，以可视化眼底。

然后，通过单击获取按钮拍摄 IR 照片以定位视网膜上的激光点。然后，在 IR 加 OCT 模式下可视化视网膜层的三维切片，并通过单击获取按钮拍照。在这些图像中观察外核层损伤的严重程度。

要在治疗和成像后逆转麻醉，请将斑马鱼放入装有水箱水的容器中。为了支持恢复，通过在水中来回移动斑马鱼，在鳃上形成一股新鲜的水箱水流。激光治疗后，弥漫性高反射信号立即定位于视网膜外层。

它从视网膜色素上皮延伸到外丛状层。来自对照未损伤眼的视网膜中不存在弥漫性高反射信号。在受伤后的第一天检测到类似的弥漫性高反射信号。

第 3 天后，这种弥散信号变得更加有组织和密集。它始终出现在外核层，延伸到感光层。第一周后，平均病灶大小显著减少，仅检测到一个小的高反射信号。

从第 14 天开始直到研究的最后一个时间点，激光点在 IR 和 OCT 图像中不再可见，视网膜的形态与对照侧相当，如图所示。采用 H&E 染色研究视网膜变性和再生的程度和动力学。此图显示了控制部分。

这张图显示了受伤后 3 天的 H&E 染色，此时最大的光感受器损失很明显。进行免疫组化以可视化神经胶质细胞标志物，红色的谷氨酰胺合成酶和绿色的神经胶质纤维酸性蛋白。这张图片显示了一个对照视网膜，其中绿色荧光非常少，这表明 GFAP。

受伤后第 3 天，GFAP 信号上调，而红色谷氨酰胺合成酶信号保持不变。在此程序之后，可以执行其他方法，如双光子显微镜以及细胞和分子分析，以研究分子细胞参与内源性修复机制。看完这个视频，你应该对如何诱导斑马鱼视网膜的局灶性损伤，并在体内监测以下退行性和再生过程有一个很好的了解。

View the full transcript and gain access to thousands of scientific videos

Explore More Videos

神经生物学问题 128 细胞生物学斑马鱼激光治疗视网膜退化再生 m ü ller 细胞光学相干断层扫描