Controlled cortical impact results in an extensive loss of dendritic spines that is not mediated by injury-induced amyloid-beta accumulation

Abstract

The clinical manifestations that occur after traumatic brain injury (TBI) include a wide range of cognitive, emotional, and behavioral deficits. The loss of excitatory synapses could potentially explain why such diverse symptoms occur after TBI, and a recent preclinical study has demonstrated a loss of dendritic spines, the postsynaptic site of the excitatory synapse, after fluid percussion injury. The objective of this study was to determine if controlled cortical impact (CCI) also resulted in dendritic spine retraction and to probe the underlying mechanisms of this spine loss. We used a unilateral CCI and visualized neurons and dendtritic spines at 24 h post-injury using Golgi stain. We found that TBI caused a 32% reduction of dendritic spines in layer II/III of the ipsilateral cortex and a 20% reduction in the dendritic spines of the ipsilateral dentate gyrus. Spine loss was not restricted to the ipsilateral hemisphere, however, with similar reductions in spine numbers recorded in the contralateral cortex (25% reduction) and hippocampus (23% reduction). Amyloid-β (Aβ), a neurotoxic peptide commonly associated with Alzheimer disease, accumulates rapidly after TBI and is also known to cause synaptic loss. To determine if Aβ contributes to spine loss after brain injury, we administered a γ-secretase inhibitor LY450139 after TBI. We found that while LY450139 administration could attenuate the TBI-induced increase in Aβ, it had no effect on dendritic spine loss after TBI. We conclude that the acute, global loss of dendritic spines after TBI is independent of γ-secretase activity or TBI-induced Aβ accumulation.

FIG. 1.

Traumatic brain injury results in rapid loss of neurons in the cortical area surrounding the primary lesion site. (A) Representative image of a mouse brain coronal section with Golgi staining at 24 h post-injury. A neuronal “dead zone” exists around the lesion where no Golgi stain is taken up into neurons. This is especially evident at the rostral edge of the lesion, marked by an asterisk, where no Golgi stain has been taken up by neurons, but the lesion itself is not present. (B) Image of Golgi-stained neurons with dendrites that appear to be retracting from the dead zone surrounding the lesion. Arrows show dendrites on the left side of the neuron that have swelling and abnormalities associated with dendrite retraction. The dendrites on the side facing away from the dead zone appear to still have dendritic spines and do not show dendritic abnormalities. (C) High magnification image of a dendrite protruding into the dead zone, showing numerous swellings and abnormalities. (D) Representative Golgi-impregnated images of the contralateral and ipsilateral dentate gyrus (DG) of the same mouse. Although there appears to be less dense staining of granule cells in the ipsilateral DG compared with the contralateral DG, the stained neurons do not show overt signs of injury and have dendritic spines.

FIG. 2.

TBI causes dendritic spine loss in Layer II/III neurons at 24h post-injury. (A) Controlled cortical impact (CCI) causes a reduction in dendritic spines on the pyramidal neurons of cortical layers II/III in both the ipsilateral (ipsi) and contralateral (contra) cortex. (B) Representative Golgi-stained apical oblique (AO) dendrites of pyramidal neurons of layer II/III of the parietal cortex, and a graphic representation of the average spine number for each experimental condition. (C) Representative Golgi-stained basal shaft (BS) dendrites of pyramidal neurons of layer II/III of the parietal cortex, and a graphic representation of the average spine number for each experimental condition. Analysis of variance with Student-Newman-Keuls post-hoc test; *** p<0.001 vs. sham. TBI, traumatic brain injury.

FIG. 3.

TBI causes dendritic spine loss in the hippocampus and entorhinal cortex at 24 h post-injury. (A) Controlled cortical impact (CCI) reduces dendritic spine numbers in the dentate gyrus. Representative images of Golgi-stained dendrites from granule neurons of the dentate gyrus taken from the ipsilateral (ipsi) and contralateral (contra) hemispheres of a traumatic brain injury (TBI) brain, and the graph of the average spine count. (B–D) Spine counts from layer II/III neurons of the ipsilateral and contralateral entorhinal cortex at 24 h post-injury. Total (B), apical obliques (AO) (C), and basal shaft (BS) (D) dendritic spine counts are included. Analysis of variance with Student-Newman-Keuls post-hoc test; *** p<0.001 vs. sham.

FIG. 4.

Traumatic brain injury-induced Aβ₄₀ accumulation in the ipsilateral cortex is attenuated with the γ-secretase inhibitor LY450139. Controlled cortical impact (CCI) causes an increase in soluble Aβ₄₀ levels at 24 h post-injury that is significantly reduced by treatment with LY450139. Aβ₄₀ levels quantified by enzyme-linked immunosorbent assay. Analysis of variance with Student-Newman-Keuls post-hoc test; *** p<0.001 vs. sham.

FIG. 5.

Traumatic brain injury-induced dendritic spine loss is not attenuated by the γ-secretase inhibitor LY450139. (A) Total dendritic spine counts of layer II/III neurons in the parietal cortex of mice after controlled cortical impact (CCI) and treatment with LY450139. Spine counts on the apical oblique (AO) dendrites (B) and basal shaft (BS) dendrites (C) of layer II/III neurons of the parietal cortex after CCI and treatment with LY450139. (D) Dendritic spine counts from granule cells of the dentate after CCI and treatment with LY450139 treatment. Analysis of variance with Student-Newman-Keuls post-hoc test; ** p<0.01; *** p<0.001 vs .sham.