Temporal and structural sensitivities of major biomarkers for detecting neuropathology after traumatic brain injury in the mouse.

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality, especially in teenagers to young adults. In recent decades, different biomarkers and/or staining protocols have been employed to evaluate the post-injury development of pathological structures, but they have produced many contradictory findings. Since correctly identifying the underlying neuroanatomical changes is critical to advancing TBI research, we compared three commonly used markers for their ability to detect TBI pathological structures: Fluoro-Jade C, the rabbit monoclonal antibody Y188 against amyloid precursor protein and the NeuroSilver kit were used to stain adjacent slices from naïve or injured mouse brains harvested at different time points from 30 min to 3 months after lateral fluid percussion injury. Although not all pathological structures were stained by all markers at all time points, we found damaged neurons and deformed dendrites in gray matter, punctate and perivascular structures in white matter, and axonal blebs and Wallerian degeneration in both gray and white matter. The present study demonstrates the temporal and structural sensitivities of the three biomarkers: each marker is highly effective for a set of pathological structures, each of which in turn emerges at a particular time point. Furthermore, the different biomarkers showed different abilities at detecting identical types of pathological structures. In contrast to previous studies that have used a single biomarker at a single time range, the present report strongly recommends that a combination of different biomarkers should be adopted and different time points need to be checked when assessing neuropathology after TBI.

Mild Traumatic Brain Injury Affects Orexin/Hypocretin Physiology Differently in Male and Female Mice.

Traumatic brain injury (TBI) is known to affect the physiology of neural circuits in several brain regions, which can contribute to behavioral changes after injury. Disordered sleep is a behavior that is often seen after TBI, but there is little research into how injury affects the circuitry that contributes to disrupted sleep regulation. Orexin/hypocretin neurons (hereafter referred to as orexin neurons) located in the lateral hypothalamus normally stabilize wakefulness in healthy animals and have been suggested as a source of dysregulated sleep behavior. Despite this, few studies have examined how TBI affects orexin neuron circuitry. Further, almost no animal studies of orexin neurons after TBI have included female animals. Here, we address these gaps by studying changes to orexin physiology using ex vivo acute brain slices and whole-cell patch clamp recording. We hypothesized that orexin neurons would have reduced afferent excitatory activity after injury. Ultimately, this hypothesis was supported but there were additional physiological changes that occurred that we did not originally hypothesize. We studied physiological properties in orexin neurons approximately 1 week after mild traumatic brain injury (mTBI) in 6-8-week-old male and female mice. mTBI was performed with a lateral fluid percussion injury between 1.4 and 1.6 atmospheres. Mild TBI increased the size of action potential afterhyperpolarization in orexin neurons from female mice, but not male mice and reduced the action potential threshold in male mice, but not in female mice. Mild TBI reduced afferent excitatory activity and increased afferent inhibitory activity onto orexin neurons. Alterations in afferent excitatory activity occurred in different parameters in male and female animals. The increased afferent inhibitory activity after injury is more pronounced in recordings from female animals. Our results indicate that mTBI changes the physiology of orexin neuron circuitry and that these changes are not the same in male and female animals.

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.