Cryo-section Dissection of the Adult Subependymal Zone for Accurate and Deep Quantitative Proteome Analysis

Christian Friess; Magdalena G&#246;tz; Jacob Kjell

doi:10.3791/63047

JoVE Journal
Neuroscience

This content is Free Access.

JoVE Journal Neuroscience

Cryo-section Dissection of the Adult Subependymal Zone for Accurate and Deep Quantitative Proteome Analysis

冷冻切片解剖成人室管膜下区，用于准确和深入的定量蛋白质组分析

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06:24 min

October 7, 2021

DOI: 10.3791/63047-v

Christian Friess¹, Magdalena Götz^1,2,3, Jacob Kjell^1,2,4

¹Division of Physiological Genomics, Biomedical Center,Ludwig Maximilian University of Munich, ²Institute for Stem Cell Research,Helmholtz Zentrum München, ³SYNERGY, Excellence Cluster Systems Neurology,University of Munich, ⁴Department of Clinical Neuroscience,Karolinska Institutet

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Overview

This study presents a cryo-section-dissection method for the precise and efficient preparation of the murine ventricular neurogenic niche, enabling deep quantitative proteome analysis. By minimizing tissue perturbation, this technique is particularly suitable for exploring the molecular microenvironment across various biological contexts, such as health and disease.

Key Study Components

Area of Science

Neuroscience
Proteomics
Neurogenesis

Background

The ventricular neurogenic niche plays a critical role in neurogenesis in the murine brain.
Existing methods for isolating this niche can cause significant tissue damage.
Precise dissection is necessary for detailed molecular analysis.

Purpose of Study

To develop a method that enables accurate extraction of the ventricular neurogenic niche.
To facilitate quantitative analysis of proteins involved in neurogenesis.
To assess its applicability in different species and health conditions.

Methods Used

Cryo-section-dissection of the mouse brain was employed.
The study utilized male C57BL/6 mice, focusing on their ventricular neurogenic niche.
No multiomics workflow was mentioned, but proteomic analysis was highlighted.
Key steps included the removal of specific brain sections and subsequent freezing for analysis.
The technique requires dexterity in scalpel use during dissection.

Main Results

The method achieved robust identification and quantification of neurogenesis-related proteins.
Cryo-section-dissection yielded fewer contaminants compared to laser capture microdissection, improving data integrity.
Significantly, this approach revealed unique neurogenesis regulators.

Conclusions

This study demonstrates a novel approach for isolating neurogenic niches, enhancing the understanding of neurogenesis.
The technique enables deeper insights into protein profiles associated with neuroplasticity and regeneration.
Findings have implications for studying neurogenic regulation in diverse biological and pathological contexts.

Frequently Asked Questions

What are the advantages of the cryo-section-dissection method?

It provides precise isolation with minimal tissue damage, crucial for accurate proteomic analysis.

How is the ventricular neurogenic niche extracted?

The method involves precise scalpel cuts to remove specific brain structures, followed by freezing for sectioning.

What types of outcomes can this method produce?

The technique enables the identification of proteins involved in neurogenesis, allowing for deeper molecular insights.

Can this method be applied to other species?

Yes, while developed for mice, the technique is adaptable for use in various species.

Are there any key limitations of this method?

The technique requires skilled handling of instruments, particularly the use of a scalpel for accurate cuts.

What is the significance of the findings?

The study identifies novel regulators of neurogenesis, providing a foundation for future research in neuroplasticity.

How might this influence studies of neurological disorders?

By improving our understanding of neurogenic processes, this method could aid in developing therapies for cognitive deficits associated with neurological diseases.

低温切片解剖允许新鲜，冷冻地制备小鼠大脑中最大的神经源性生态位，以进行深层定量蛋白质组分析。该方法精确，高效，并且引起最小的组织扰动。因此，它非常适合研究该生态位的分子微环境以及其他器官，区域和物种。

我们的解剖方法允许精确分离心室神经源性生态位，因此非常适合研究该生态位的分子微环境。这种解剖方法的主要优点是它精确，高效，并且引起最小的组织扰动，同时仍然与用于蛋白质组分析的质谱兼容。在该协议中，我们从小鼠大脑中提取心室的神经源性生态位，但这种方法也可以应用于其他物种以及处于各种健康和疾病状态。

该技术可能需要一些培训。特别是在暴露和提取心室神经源性龛位时使用手术刀切口。对8至10周龄的C57黑/6雄性小鼠实施安乐死后，通过手动解剖提取大脑并将其放入含有冰冷解剖培养基的培养皿中。

使用手术刀，通过在嗅球和皮层的内极之间做一个直的冠状切口来移除嗅球。接下来，通过进行冠状切口切除皮层的前极，确保外侧心室在冠状平面上可见。然后，使用剪刀，从顶部打开两个侧心室，从皮质表面到心室腔的矢状面开始。

在心室屈曲后以C形方式拉长该切口。接下来，连接左右矢状切口的尾端，采用额外的冠状切口。接下来，去除覆盖内侧心室壁的皮质和胼胝体。

如果组织附着在内侧心室壁上，请进行额外的切口或用剪刀抬起皮质和胼胝体以移走组织。然后，使用镊子去除覆盖外侧心室的皮质和胼胝体。使用镊子，小心地铺开心室壁并切除脉络丛。

然后，将大脑放在载玻片上，并将载玻片放在干冰的顶部，以将心室壁以开放配置冻结大脑。在切片之前，确保大脑连接到后脑的低温恒温器附着板上，并嵌入用于冷冻组织的培养基。此外，确保没有嵌入介质与前脑接触，特别是在心室。

然后，切开50至100微米厚的大脑冠状切片，直到侧脑室的末端，并将这些切片安装在载玻片上。将载玻片放在解剖显微镜下的干冰上。将切片从干冰上提起15至30秒，以实现短暂的，不完整的解冻，使纹状体的致密髓鞘可观察到致密的白点。

然后，使用预冷的手术刀，将室管膜下区域与相邻纹状体分开，并使用冷却手术刀的钝边将其作为一块或分成两到四个部分转移到微量离心管中。然后对内侧心室区做同样的事情。在全移植样品中鉴定出纹状体的髓鞘相关糖蛋白阳性内囊，但很少通过免疫组化在冷冻切片解剖样品中鉴定。

与躯体感觉皮层样品相比，通过在室管膜下区域富集髓鞘蛋白证实了整个样本中的纹状体污染。相比之下，冷冻切片解剖样品中这些髓鞘标志物蛋白的比较显示，室管膜下区和躯体感觉皮层样品没有显着差异。当比较冷冻切片解剖和激光捕获显微切割之间的质谱结果时，激光捕获显微切割产生的定量蛋白质数量约为其一半，尽管组织收集时间大约是其两倍。

主成分分析表明，用激光捕获显微切割（描绘为正方形）收集的样品之间的变异性大于用冷冻切片解剖收集的样品（描绘为圆形）的变异性。室管膜下和内侧室管膜下区域的冷冻切片解剖和激光捕获显微切割之间的2D注释富集测试显示，与细胞外空间相关的蛋白质在方法和区域中都有类似的富集。冷冻切片解剖可更可靠地鉴定和定量室管膜下区相关细胞外基质蛋白中的神经发生。

在tenascin-C的情况下，与内侧室管膜区相比，只有冷冻切片解剖在室管膜下区显示出富集。确保载玻片上的大脑部分不会完全解冻。总体而言，最好在解剖过程中练习这些步骤以获得一致的结果。

解剖方法还可以与其他蛋白质分析方法一起使用，这些方法可以很容易地检测出非常低丰度的蛋白质，例如生长因子和细胞因子。这种显微解剖方法使我们能够识别一种新的神经发生调节因子，我们相信它将允许其他人在各种情况下识别神经发生的其他调节因子。