Detection and verification of neurodegeneration after traumatic brain injury in the mouse: Immunohistochemical staining for amyloid precursor protein

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.