脑室周围组织的侧脑室及组织学特性对人类和鼠标3D建模
Summary
Using MRI scans (human), 3D imaging software, and immunohistological analysis, we document changes to the brain’s lateral ventricles. Longitudinal 3D mapping of lateral ventricle volume changes and characterization of periventricular cellular changes that occur in the human brain due to aging or disease are then modeled in mice.
Abstract
The ventricular system carries and circulates cerebral spinal fluid (CSF) and facilitates clearance of solutes and toxins from the brain. The functional units of the ventricles are ciliated epithelial cells termed ependymal cells, which line the ventricles and through ciliary action are capable of generating laminar flow of CSF at the ventricle surface. This monolayer of ependymal cells also provides barrier and filtration functions that promote exchange between brain interstitial fluids (ISF) and circulating CSF. Biochemical changes in the brain are thereby reflected in the composition of the CSF and destruction of the ependyma can disrupt the delicate balance of CSF and ISF exchange. In humans there is a strong correlation between lateral ventricle expansion and aging. Age-associated ventriculomegaly can occur even in the absence of dementia or obstruction of CSF flow. The exact cause and progression of ventriculomegaly is often unknown; however, enlarged ventricles can show regional and, often, extensive loss of ependymal cell coverage with ventricle surface astrogliosis and associated periventricular edema replacing the functional ependymal cell monolayer. Using MRI scans together with postmortem human brain tissue, we describe how to prepare, image and compile 3D renderings of lateral ventricle volumes, calculate lateral ventricle volumes, and characterize periventricular tissue through immunohistochemical analysis of en face lateral ventricle wall tissue preparations. Corresponding analyses of mouse brain tissue are also presented supporting the use of mouse models as a means to evaluate changes to the lateral ventricles and periventricular tissue found in human aging and disease. Together, these protocols allow investigations into the cause and effect of ventriculomegaly and highlight techniques to study ventricular system health and its important barrier and filtration functions within the brain.
Introduction
一个室管膜细胞单层线大脑提供的脑脊髓液（CSF）和间质液（ISF）的1-3之间的双向屏障和传输功能的脑室系统。这些功能有助于保持大脑毒物和无生理平衡2,3中。在人类中损失此衬里通过损伤或疾病的部分不会出现导致再生替换为在其他上皮衬里找到;而室管膜细胞覆盖的损失似乎导致脑室周围星形胶质细胞增生与星形胶质细胞覆盖在心室表面溶蚀室管膜细胞的区域的小梁。严重的影响到重要的CSF / ISF交换和通关机制，将预期从这个皮层1,2,4-7损失导致。
人类衰老的共同特征被放大侧脑室（脑室）和相关的脑室周围水肿作为observ由MRI和液体衰减反转恢复MRI（磁共振成像/ FLAIR）8-14编调查脑室和心室衬里的细胞组织之间的关系，死后人MRI序列进行匹配与侧脑室脑室周围组织的组织学制备。在脑室病例，胶质增生的重要方面开展了沿侧脑室壁室管膜代替小区覆盖。如果不是由基于MRI容积分析发现心室扩张，室管膜细胞衬里是完整，胶质细胞增生并没有沿着心室衬里6检测。这种组合的方式表示使用部分wholemount筹备或整个侧脑室壁和心室体积6三维建模侧脑室衬里细胞完整性的第一个全面的文档，详细的变化。几种疾病（阿尔茨海默氏症，精神分裂症）和损伤（创伤性脑损伤）显示脑室扩大作为早期神经病理学特征。的室管膜细胞衬里从而领域剥蚀将预期可干扰正常室管膜细胞功能和妥协CSF / ISF流体和溶质交换之间的稳态平衡。因此，改变的脑室系统，其细胞组合物，以及所产生的结果底层或邻近脑结构进行更彻底的检查将最终开始更多地揭示与脑室扩大相关的神经病理学。
缺乏多模成像数据的，尤其是纵向的数据序列，用有限的访问相应的组织学组织样品一起使人类脑病理学的分析困难。在人体衰老或疾病发现的表型造型往往可以达到的小鼠模型和动物模型成为我们的最佳手段之一，以探讨有关人类疾病的发生和发展的问题。在一些研究中健康年轻小鼠所描述的侧脑室壁的细胞结构和底层干细胞小生境4,7-15。这些研究已经通过延长老化6,15包括脑室壁上的三维建模和细胞分析。无论是脑室周围胶质细胞增生，也不脑室老年小鼠中观察到的，而老鼠显示一个相对强劲的subventicular区（SVZ）干细胞小生境下层为一个完整的室管膜细胞衬里6,15。因此，在这两种一般的保养和侧脑室衬里的完整性存在老化6,15的过程中撞击物种特异性差异。因此，最好使用小鼠来询问在人类中发现的条件，需要进行表征，而在任何建模范例适当考虑这两个物种之间的差异。这里，我们目前的程序，以评估在人类和M纵向变化侧脑室和相关组织脑室周围乌斯河。我们的程序包括3D渲染和小鼠和人脑室体积测量，并利用脑室周围组织整装准备免疫组化分析，既细胞组织和结构进行了表征。连同这些程序提供表征在脑室系统的变化和相关的脑室周围组织的一种手段。
Protocol
Representative Results
Discussion
我们提出的工具和可用于在小鼠和人类中评估大脑脑室系统的完整性协议。这些工具，然而，也可以应用到进行，或在老化14,21,22的过程中，由于受伤，疾病改变其它脑结构或器官系统。策略提出的软件趁其允许剖面和纵向的MRI序列的比对，以产生特定区域或感兴趣结构的3D体积表示。纵向MRI序列允许的发生随着时间的推移，并且可以扩展到包括大脑总体积，为侧脑室总脑体积比，和/或其…
Disclosures
The authors have nothing to disclose.
Acknowledgements
An NINDS Grant NS05033 (JCC) supported this work. The University of Connecticut RAC, SURF and OUR programs provided additional support.
Materials
| Name of the Materal/Equipment | Company | Catalog Number | Comments/Description |
| Phosphate buffered saline (PBS) | Life Technologies | 21600-069 | |
| Paraformaldehyde (PFA) | Electron Microscopy Sciences | 19210 | Use at 4% in PBS, 4 °C |
| Normal Horse Serum | Life Technologies | 16050 | 10% in PBS-TX (v/v) |
| Normal Goat Serum | Life Technologies | 16210 | 10% in PBS-TX (v/v) |
| Triton X-100 (TX) | Sigma-Aldrich | T8787 | 0.1% in PBS (v/v) |
| Vibratome | Leica | VT1000S | |
| Fluorescence Microscope | Zeiss | Imager.M2 | |
| Camera | Hamamatsu | ORCA R2 | |
| Microscope Stage Controller | Ludl Electronic Products | MAC 6000 | |
| Stereology software | MBF Bioscience | Stereo Investigator 11 | |
| Stereology software | ImageJ/NIH | NIH freeware | |
| 3D Reconstruction software | MBF Bioscience | Neurolucida Explorer | |
| Confocal Microscope | Leica | TCS SP2 | |
| MRI Software | |||
| Freesurfer | https://surfer.nmr.mgh.harvard.edu/fswiki/DownloadAndInstall | Segmentation and Volume | |
| ITK-Snap | http://www.itksnap.org/pmwiki/pmwiki.php | Segmentation and Volume | |
| Multi-image Analysis GUI (Mango) | http://ric.uthscsa.edu/mango/ | Longitudinal overlay | |
| Whole Mount Equipment | |||
| 22.5° microsurgical straight stab knife | Fisher Scientific | NC9854830 | |
| parafilm | |||
| wax bottom dissecting dish | |||
| pins | |||
| fine forceps | |||
| aquapolymount | |||
| Dissecting Microscope | Leica | MZ95 | |
| Whole Mount Antibodies | |||
| mouse anti-b-catenin | BD Bioschiences, San Jose, CA, USA | 1:250 | |
| goat anti-GFAP | Santa Cruz Biotechnology | 1:250 | |
| rabbit anti-AQP4 (aquaporin-4) | Sigma-Aldrich | 1:400 | |
| Coronal Antibodies | |||
| Anti-S100β antibody | Sigma-Aldrich | 1:500 | |
| 4’,6-diamidino-2-phenylindole (DAPI) | Life Technologies | D-1306 | 10 µg/mL in PBS |
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