Mild traumatic brain injury results in extensive neuronal degeneration in the cerebral cortex

Abstract

Mild traumatic brain injury (mTBI) leads to long-term cognitive and emotional difficulties and behavioral disturbances, but the diagnosis and treatment of mTBI have historically been hampered by a lack of evidence-based correlates of these clinical manifestations. Unlike moderate and severe TBI, mTBI does not show significant tissue lesions or cavities in the cortex. Moreover, neuroimaging by magnetic resonance imaging or computed tomography is usually negative, suggesting that the damage is beyond the resolution of current structure-based scanning technologies. Therefore, we investigated the morphologies of spared neurons in the mouse cortex after mTBI in a controlled cortical impact injury model. Our results indicate that, although mTBI caused only a mild extent of cell death, it led to extensive dendrite degeneration and synapse reduction in the cortex in this model. This study sheds light on the neuropathologic consequences of mTBI in humans and suggests that neurodegeneration may be a novel target for developing diagnostic methods and therapeutic approaches for mTBI.

FIGURE 1

Mild traumatic brain injury (mTBI) in mice. (A) Gross examination of the brains 3 days post-TBI showed very limited tissue lesions without cavitation in the mTBI mouse brain (left) compared to the sham control (right). (B) Crystal violet staining shows that the tissue in the injured cortex is nearly intact without major disruption. (C) Quantitative measurement of the volume of the damaged brain region revealed that a reduction in the volume of the spared cortex was less than 5% and not significantly different versus the contralateral hemisphere (n = 10, p > 0.05).

FIGURE 2

Cell death in the neocortex of mice with mild traumatic brain injury (mTBI). (A) Fluoro-Jade B (FJB)–positive cells are green in the epicenter on the ipsilateral side of the neocortex. (B–F) Distribution pattern of dying cells around the epicenter. (G) Quantitative data show that the highest number of FJB-positive cells was observed in the epicenter and gradually decreased rostrally and caudally away from it (n = 12).

FIGURE 3

Cells lost in the neocortex of mice with mild traumatic brain injury (mTBI). (A–D) DAPI (blue) and NeuN (red) staining revealed the nuclei of cells and mature neurons at the injury site in the cortex (A and B sham control, C and D mTBI). (E) DAPI staining followed by counting of the stained cells showed that the total cell density in the epicenter was slightly reduced to 85.8% ± 9.5% compared to the sham control (n = 10; *, p < 0.05). (F) The density of NeuN-positive neurons was dramatically reduced to 57.2% ± 5.4% (n = 10; **, p < 0.005).

FIGURE 4

Mild traumatic brain injury (mTBI) causes extensive neural degeneration in the neocortex. (A–F) Golgi staining reveals individual neurons, their processes, and spines in a sham control (A, C1–C3, and D1–D3) and in a mouse with mTBI (B, E1–E3, and F1–F3). The density of the Golgi-stained neurons in the injury area on the ipsilateral side was decreased, and the dendrites and spine of the spared neurons in the same area were dramatically injured. (G) Quantitative data show that when degeneration is taken into account the spared cortex decreased dramatically to 82.5% compared with the sham control (n = 10; **, p < 0.005). (H) The volume of the neuronal degeneration region (marked by the red line in B) was 10.3 times higher than the tissue lesion volume in the injured cortex after mTBI.

FIGURE 5

Mild traumatic brain injury (mTBI) causes dendrite degeneration. (A, B) Neurolucida reconstruction of Golgi-stained layers II/III neurons in the cortex of sham (A) and mTBI mice (B). (C, D) The total length (C) and the number of branches (D) of the dendrites in cells from sham and mTBI mice. The total length of the dendrites in spared neurons decreased from 1905 ± 357 to 508 ± 55 µm; branches are dramatically decreased from 32 ± 5 to 10 ± 1 in the cortex after mTBI (n = 5; *, p < 0.05). (E) Sholl analysis–derived distribution of layers II/III neuron dendritic complexity based on the distance from the cell body. Mean number of intersections of dendrite branches with consecutive 10-µm-spaced concentric spheres. (F) There is a dramatic reduction of dendritic complexity in the spared neurons in mTBI mice versus sham controls (n = 5; *, p < 0.05).

FIGURE 6

Mild traumatic brain injury (mTBI) leads to dendritic spine degeneration. (A, B) High-resolution image of a single spine in sham control (A) and mTBI mice (B). (C–E) Mushroom-shaped (C), stubby-shaped (D), and filopodia-shaped (E) spines are shown. (E) Quantitative data show that the density of the dendritic spines is significantly reduced in spared neurons in the cortex of TBI mice (n = 5; **, p < 0.005). (G) The reduction is particularly apparent in mushroom-shaped and filopodia-shaped spines (n = 5; **, p < 0.005).

FIGURE 7

Mild traumatic brain injury (mTBI) decreases the number of synapses in the injury area. (A–D) Synaptophysin staining (red) reveals presynaptic puncta in layers II/III of the cortex in control (A, B) and mTBI mice (C, D). The synapse density decreased to 65.5% ± 8.1% of the control cortex in the cortex of injured mice (n = 10; *, p < 0.05).