Frigon Eve-Marie, Gérin-Lajoie Amy, Dadar Mahsa, Boire Denis, Maranzano Josefina
Department of Anatomy, University of Quebec in Trois-Rivieres, Trois-Rivieres, QC, Canada.
Department of Psychiatry, Douglas Research Center, McGill University, Montreal, QC, Canada.
Front Neuroanat. 2024 Apr 10;18:1372953. doi: 10.3389/fnana.2024.1372953. eCollection 2024.
Brain banks provide small tissue samples to researchers, while gross anatomy laboratories could provide larger samples, including complete brains to neuroscientists. However, they are preserved with solutions appropriate for gross-dissection, different from the classic neutral-buffered formalin (NBF) used in brain banks. Our previous work in mice showed that two gross-anatomy laboratory solutions, a saturated-salt-solution (SSS) and an alcohol-formaldehyde-solution (AFS), preserve antigenicity of the main cellular markers (neurons, astrocytes, microglia, and myelin). Our goal is now to compare the quality of histology and antigenicity preservation of human brains fixed with NBF by immersion (practice of brain banks) vs. those fixed with a SSS and an AFS by whole body perfusion, practice of gross-anatomy laboratories.
We used a convenience sample of 42 brains (31 males, 11 females; 25-90 years old) fixed with NBF (N = 12), SSS (N = 13), and AFS (N = 17). One cm tissue blocks were cut, cryoprotected, frozen and sliced into 40 μm sections. The four cell populations were labeled using immunohistochemistry (Neurons = neuronal-nuclei = NeuN, astrocytes = glial-fibrillary-acidic-protein = GFAP, microglia = ionized-calcium-binding-adaptor-molecule1 = Iba1 and oligodendrocytes = myelin-proteolipid-protein = PLP). We qualitatively assessed antigenicity and cell distribution, and compared the ease of manipulation of the sections, the microscopic tissue quality, and the quality of common histochemical stains (e.g., Cresyl violet, Luxol fast blue, etc.) across solutions.
Sections of SSS-fixed brains were more difficult to manipulate and showed poorer tissue quality than those from brains fixed with the other solutions. The four antigens were preserved, and cell labeling was more often homogeneous in AFS-fixed specimens. NeuN and GFAP were not always present in NBF and SSS samples. Some antigens were heterogeneously distributed in some specimens, independently of the fixative, but an antigen retrieval protocol successfully recovered them. Finally, the histochemical stains were of sufficient quality regardless of the fixative, although neurons were more often paler in SSS-fixed specimens.
Antigenicity was preserved in human brains fixed with solutions used in human gross-anatomy (albeit the poorer quality of SSS-fixed specimens). For some specific variables, histology quality was superior in AFS-fixed brains. Furthermore, we show the feasibility of frequently used histochemical stains. These results are promising for neuroscientists interested in using brain specimens from anatomy laboratories.
脑库为研究人员提供小的组织样本，而大体解剖实验室可以为神经科学家提供更大的样本，包括完整的大脑。然而，它们是用适合大体解剖的溶液保存的，这与脑库中使用的经典中性缓冲福尔马林（NBF）不同。我们之前在小鼠身上的研究表明，两种大体解剖实验室溶液，即饱和盐溶液（SSS）和酒精 - 甲醛溶液（AFS），能保留主要细胞标志物（神经元、星形胶质细胞、小胶质细胞和髓磷脂）的抗原性。我们现在的目标是比较通过浸泡（脑库的做法）用NBF固定的人脑与通过全身灌注（大体解剖实验室的做法）用SSS和AFS固定的人脑在组织学质量和抗原性保存方面的差异。
我们使用了一个便利样本，包含42个大脑（31名男性，11名女性；年龄在25 - 90岁之间），分别用NBF（N = 12）、SSS（N = 13）和AFS（N = 17）固定。将组织切成1厘米的块，进行冷冻保护、冷冻并切成40μm的切片。使用免疫组织化学对四种细胞群体进行标记（神经元 = 神经元细胞核 = NeuN，星形胶质细胞 = 胶质纤维酸性蛋白 = GFAP，小胶质细胞 = 离子钙结合衔接分子1 = Iba1，少突胶质细胞 = 髓磷脂蛋白脂蛋白 = PLP）。我们定性评估抗原性和细胞分布，并比较不同溶液处理的切片的操作难易程度、微观组织质量以及常见组织化学染色（如甲酚紫、Luxol固蓝等）的质量。
与用其他溶液固定的大脑切片相比，SSS固定的大脑切片更难操作，组织质量也更差。四种抗原均得以保存，在AFS固定的标本中细胞标记更常呈现均匀状态。NeuN和GFAP在NBF和SSS样本中并非总是存在。一些抗原在某些标本中呈异质性分布，与固定剂无关，但抗原修复方案成功恢复了它们。最后，无论使用何种固定剂，组织化学染色质量都足够，尽管在SSS固定的标本中神经元通常颜色更淡。
用人大体解剖中使用的溶液固定的人脑保留了抗原性（尽管SSS固定的标本质量较差）。对于某些特定变量，AFS固定的大脑在组织学质量上更优。此外，我们展示了常用组织化学染色的可行性。这些结果对于有兴趣使用解剖实验室脑标本的神经科学家来说很有前景。
