Delayed reduction in hippocampal postsynaptic density protein-95 expression temporally correlates with cognitive dysfunction following controlled cortical impact in mice

Abstract

Object: Traumatic brain injury (TBI) induces significant neurological damage, including deficits in learning and memory, which contribute to a poor clinical prognosis. Treatment options to limit cognitive decline and promote neurological recovery are lacking, in part due to a poor understanding of the secondary or delayed processes that contribute to brain injury. In the present study, the authors characterized the temporal and spatial changes in the expression of postsynaptic density protein-95 (PSD-95), a key scaffolding protein implicated in excitatory synaptic signaling, after controlled cortical impacts in mice. Neurological injury, as assessed by the open-field activity test and the novel object recognition test, was compared with changes in PSD-95 expression.

Methods: Adult male CD-1 mice were subjected to controlled cortical impacts to simulate moderate TBI in humans. The spatial and temporal expression of PSD-95 was analyzed in the cerebral cortex and hippocampus at various time points following injury and sham operations. Neurological assessments were performed to compare changes in PSD-95 with cognitive deficits.

Results: A significant decrease in PSD-95 expression was observed in the ipsilateral hippocampus beginning on Day 7 postinjury. The loss of PSD-95 corresponded with a concomitant reduction in immunoreactivity for NeuN (neuronal nuclei), a neuron-specific marker. Aside from the contused cortex, a significant loss of PSD-95 immunoreactivity was not observed in the cerebral cortex. The delayed loss of hippocampal PSD-95 directly correlated with the onset of behavioral deficits, suggesting a possible causative role for PSD-95 in behavioral abnormalities following head trauma.

Conclusions: A delayed loss of hippocampal synapses was observed following head trauma in mice. These data may suggest a cellular mechanism to explain the delayed learning and memory deficits in humans after TBI and provide a potential framework for further testing to implicate PSD-95 as a clinically relevant therapeutic target.

FIGURE 1. Delayed reduction in hippocampal PSD-95 expression following experimental TBI

(A) Representative data indicating a time-dependent reduction in the expression of PSD-95 within the whole hippocampus of mice following a moderate TBI. PSD-95 expression was normalized to β-actin, to control for equal protein loading. Brain sections stained with cresyl violet are provided to further document hippocampal injury by day 7 post-injury. (B) Quantification of Western blotting data in (A) by densitometry. Data are expressed as the ratio of PSD-95/β-actin and are presented as mean ± SEM. * p<0.05 vs. sham-operated control mice.

FIGURE 2. Spatial distribution of hippocampal PSD-95 following TBI

Immunoreactivity for NeuN (neuron-specific marker) and PSD-95 was performed within the hippocampus of sham or TBI (day 3 or 7 post-injury) mice. Immunoreactivity for both PSD-95 and NeuN was dramatically attenuated throughout the entire hippocampus at both time points following TBI, as compared to sham-operated control mice. Data are representative of 4–6 mice/group.

FIGURE 3. Activation of caspase-3 within the hippocampus at day 7 post-TBI

Representative data indicating an increase in the expression of cleaved caspase-3, a marker of apoptotic injury, within the whole hippocampus of mice following TBI. Cleaved caspase-3 expression was normalized to β-actin to control for equal protein loading.

FIGURE 4. Delayed neurological deficits following TBI

(A) Open-field activity testing was performed in sham-operated or TBI-induced mice at various time point following TBI. Activity, as measured by the average number of crosses per trial, was recorded. (B) Novel object recognition test of memory was performed in sham-operated or in mice at day 7 post-TBI, when PSD-95 expression was lowest. The average time spent exploring a novel object, a measure of recognition memory, was recorded and compared with sham-operated control mice. For all studies, n=6–8 mice/group and * was p<0.05 vs. sham.