Background

Astrocytes play a critical role in CNS functions by providing physiological support to surrounding cells. These cells present a particularly unique challenge for in vitro immunohistochemical quantification due reactive gliosis after insult or injury, which is characterized by the extension of long processes.

New method

We present an optimized QuPath protocol that is scalable, fully automated, and capable of being applied to images generated by whole slide scanning technology using this open-source software.

Results

We induced mechanical injury in the rat brain and stained astrocytes using glial fibrillary acidic protein (GFAP) and 3,3-diaminobenzidine (DAB) chromogen detection. Slides were scanned using a whole slide scanner, Vectra Polaris. Using QuPath, we summarize and contrast three ways of quantifying astrocytes in uninjured (contralateral) and injured (ipsilateral) hemispheres: optical density, positive pixels and positive proportion.

Comparison with existing methods

Robust quantification of DAB stained astrocytes remains elusive. Previous methodologies have relied on software that is not compatible with whole slide scanner images. Use of such software can compromise the data integrity within the image and is limited by issues with scalability and lack of automation. Previous methods using manual histopathological scoring are also limited by the ability to quantify large numbers of astrocytes. Given these limitations, we were unable to directly compare our method with those using other software or manual histopathology.

Conclusions

Based on an analysis of our method, we conclude that positive proportion may be the most effective way to quantify astrocytic responses using GFAP and DAB immunohistochemistry in the brain.

中文翻译：

---

一种可扩展的全自动方法，用于大鼠脑中星形胶质细胞免疫组织化学染色的区域定量

背景

星形胶质细胞通过向周围细胞提供生理支持，在中枢神经系统功能中起关键作用。这些细胞由于损伤或损伤后反应性胶质增生而对体外免疫组织化学定量提出了特别独特的挑战，其特征是延长了过程。

新方法

我们提出了一种经过优化的QuPath协议，该协议具有可伸缩性，全自动性，并且能够使用此开源软件将其应用于通过整个幻灯片扫描技术生成的图像。

结果

我们使用胶质纤维酸性蛋白（GFAP）和3,3-二氨基联苯胺（DAB）色原检测技术诱导了大鼠大脑和星形胶质细胞的机械损伤。使用整个幻灯片扫描仪Vectra Polaris扫描幻灯片。使用QuPath，我们总结并对比了三种在未受伤（对侧）和受伤（同侧）半球中星形胶质细胞定量的方法：光密度，正像素和正比例。

与现有方法的比较

DAB染色星形胶质细胞的可靠定量仍然难以实现。先前的方法依赖于与整个幻灯片扫描仪图像不兼容的软件。使用此类软件可能会损害映像中的数据完整性，并受到可伸缩性和缺乏自动化问题的限制。先前使用手动组织病理学评分的方法也受到定量大量星形胶质细胞能力的限制。鉴于这些限制，我们无法直接将我们的方法与使用其他软件或手动组织病理学的方法进行比较。

结论

在对我们的方法进行分析的基础上，我们得出结论，正比例可能是使用GFAP和DAB免疫组化在大脑中量化星形细胞反应的最有效方法。