Department of Anesthesiology and Critical Care Medicine, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.
Neuroscience Graduate Group, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
Brain Pathol. 2023 Nov;33(6):e13163. doi: 10.1111/bpa.13163. Epub 2023 May 8.
Previous studies of human traumatic brain injury (TBI) have shown diffuse axonal injury as varicosities or spheroids in white matter (WM) bundles when using immunoperoxidase-ABC staining with 22C11, a mouse monoclonal antibody against amyloid precursor protein (APP). These findings have been interpreted as TBI-induced axonal pathology. In a mouse model of TBI however, when we used immunofluorescent staining with 22C11, as opposed to immunoperoxidase staining, we did not observe varicosities or spheroids. To explore this discrepancy, we performed immunofluorescent staining with Y188, an APP knockout-validated rabbit monoclonal that shows baseline immunoreactivity in neurons and oligodendrocytes of non-injured mice, with some arranged-like varicosities. In gray matter after injury, Y188 intensely stained axonal blebs. In WM, we encountered large patches of heavily stained puncta, heterogeneous in size. Scattered axonal blebs were also identified among these Y188-stained puncta. To assess the neuronal origin of Y188 staining after TBI we made use of transgenic mice with fluorescently labeled neurons and axons. A close correlation was observed between Y188-stained axonal blebs and fluorescently labeled neuronal cell bodies/axons. By contrast, no correlation was observed between Y188-stained puncta and fluorescent axons in WM, suggesting that these puncta in WM did not originate from axons, and casting further doubt on the nature of previous reports with 22C11. As such, we strongly recommend Y188 as a biomarker for detecting damaged neurons and axons after TBI. With Y188, stained axonal blebs likely represent acute axonal truncations that may lead to death of the parent neurons. Y188-stained puncta in WM may indicate damaged oligodendrocytes, whose death and clearance can result in secondary demyelination and Wallerian degeneration of axons. We also provide evidence suggesting that 22C11-stained varicosities or spheroids previously reported in TBI patients might be showing damaged oligodendrocytes, due to a cross-reaction between the ABC kit and upregulated endogenous biotin.
先前的人类创伤性脑损伤（TBI）研究表明，使用针对淀粉样前体蛋白（APP）的 22C11 小鼠单克隆抗体进行免疫过氧化物酶-ABC 染色时，白质（WM）束中的轴突呈现出空泡或球体。这些发现被解释为 TBI 诱导的轴突病变。然而，在 TBI 的小鼠模型中，当我们使用 22C11 进行免疫荧光染色而不是免疫过氧化物酶染色时，我们没有观察到空泡或球体。为了探讨这种差异，我们使用 Y188 进行免疫荧光染色，Y188 是一种 APP 敲除验证的兔单克隆抗体，在未受伤的小鼠的神经元和少突胶质细胞中显示出基线免疫反应性，并有一些排列的空泡。在损伤后的灰质中，Y188 强烈染色轴突泡。在 WM 中，我们遇到了大量大小不均的高度染色的点状结构。在这些 Y188 染色的点状结构中还发现了分散的轴突泡。为了评估 TBI 后 Y188 染色的神经元起源，我们使用了带有荧光标记神经元和轴突的转基因小鼠。观察到 Y188 染色的轴突泡与荧光标记的神经元细胞体/轴突之间存在密切相关性。相比之下，在 WM 中，Y188 染色的点状结构与荧光标记的轴突之间没有相关性，这表明这些 WM 中的点状结构不是来自轴突，这进一步怀疑以前使用 22C11 进行的报告的性质。因此，我们强烈推荐 Y188 作为检测 TBI 后受损神经元和轴突的生物标志物。使用 Y188，染色的轴突泡可能代表急性轴突截断，可能导致母神经元死亡。WM 中的 Y188 染色点状结构可能表明受损的少突胶质细胞，其死亡和清除可导致继发性脱髓鞘和轴突的沃勒变性。我们还提供了证据表明，先前在 TBI 患者中报道的 22C11 染色的空泡或球体可能显示受损的少突胶质细胞，这是由于 ABC 试剂盒与上调的内源性生物素之间的交叉反应。
