TBI Impaired Cognitive Function of APP/PS1 Mouse Model.
To detect the cognitive function of AD mice after 4-week of TBI, we generated the TBI model with double transgenic AD mice, and novel object and position recognition test was performed to test the cognitive function in each group. The result showed that, in the acquisition phase, each group of mice spent nearly the same duration of time on exploring the two identical objects (C57BL/6, C57BL/6 TBI, APP/PS1, APP/PS1 TBI,P > 0.05)(Fig. 1A). In the testing phase after 1 h, mice in the C57BL/6, C57BL/6 TBI and APP/PS1 group preferred to spend more time on detecting the novel object and the novel position(C57BL/6 P > 0.05 vs. C57BL/6 TBI, P > 0.05 vs APP/PS1; C57BL/6, C57BL/6 TBI and APP/PS1 P < 0.05 vs. chance level 50%)(Fig. 1B,1D), however, APP/PS1 TBI mice showed no preference to the novel object and novel position relative to other groups (APP/PS1 TBI, P < 0.01 vs. C57BL/6; P < 0.01 vs. C57BL/6 TBI; P < 0.05 vs. APP/PS1).Of note, there is an interesting phenomenon that it is more often for the APP/PS1 TBI mice to climb on the object than any other group of mice during the test, which is never happened to the C57BL/6 mice. This result indicates that TBI impairs the hippocampal-dependent learning and memory function in APP/PS1 mice.
TBI Decreased Cell Density at TBI Lesion and CA3 Region of Hippocampus.
We performed TBI at the cortex of the brain, and the cell density decrease in the cortex is taken into granted (C57BL/6 TBI, P < 0.001 vs. C57BL/6; APP/PS1 TBI, P < 0.01 vs. APP/PS1)(Fig. 2). However, the hippocampus is not injured directly. It is well approved that hippocampus is a vital important grey matter area which is related to cognition, memory and emotion24. As cognitive function decline has been described above, we further detected the hippocampus pathological changes after TBI. HE stain was performed to determine the cell density in each region of hippocampus. By observing under the microscope, the neuron cells in the TBI brain exhibited loose and disordered in arrangement and the nucleus showed paramorphia and shrinkage. Our results revealed that neuron cell density in the region of CA3 was similar between C57BL/6 mice and APP/PS1 mice(APP/PS1, P > 0.05 vs. C57BL/6) (Fig. 2). After TBI, there is a trend of decrease, although not significantly, of cell density in the C57BL/6 TBI mice compared with C57BL/6 mice (C57BL/6 TBI, P = 0.18 vs. C57BL/6) (Fig. 2). Of note, the cell density in APP/PS1 TBI mice are significantly decrease compared with APP/PS1 mice (APP/PS1 TBI, P < 0.001 vs. APP/PS1) (Fig. 2). As for the cell density in the other region of hippocampus, it showed not significant difference between each group(Fig. 2). These results indicated that the cell density of APP/PS1 mice is no less than C57BL/6 mice, but TBI decrease the neuron cell in the cortex around the lesion and in CA3 region, which may be the reason of impairment of cognitive function in APP/PS1 mice post-TBI.
TBI Diminished Synaptic Function in the Cortex and the Specific Region of Hippocampus.
Synaptophysin(SYP) is the marker of synapse plasticity, which reflects synaptic function25. To further evaluate synaptic function in the brain of each group, immunofluorescence staining of SYP was performed to evaluate the expression in the cortex and in the hippocampus. In the cortex, C57BL/6 mice exhibited abundant of SYP, reflecting a normal stage of synaptic function, which is paralleled with its cognitive function(Fig. 3). C57BL/6 TBI mice exhibited down regulated of SYP compared with C57BL/6 mice (C57BL/6 TBI, P < 0.001 vs. C57BL/6), suggesting that TBI may impair synaptic function(Fig. 3). APP/PS1 mice expressed fewer SYP than C57BL/6 mice (APP/PS1 TBI, P < 0.001 vs. APP/PS1), indicating the impairment of synaptic function in its basic level(Fig. 3). As expected, compared with APP/PS1 mice, APP/PS1 TBI mice showed a significant decrease expression of SYP in the cortex (APP/PS1 TBI, P < 0.05 vs. APP/PS1), in paralleled with the cognitive function impairment of APP/PS1 TBI mice(Fig. 3). In the region of CA1 and CA3 of hippocampus, the expression of SYP in APP/PS1 TBI mice is also significantly decreased compared with APP/PS1 mice (APP/PS1 TBI, P < 0.05 vs. APP/PS1), suggesting that TBI aggravated the AD dementia via devastating synaptic function in AD mouse model(Fig. 3).
TBI Activated Microglia in the Brain and Sustained at Least for 3 Weeks.
Microglia that resident in the brain and differentiate from peripheral phagocyte infiltrating into CNS through injured Blood-Brain-Barrier(BBB) can be activated soon after TBI. Also, the period that microglia activated by TBI will last for days or even months26. To further determine the situation of activated microglia, we performed immunofluorescence staining to detect the IBA-1 expressed on the surface of microglia at the 1st ,2nd ,3rd and 4th week of TBI. Also, to evade the disturbance to the result by the Aβ-plaques deposition which may also up-regulated the degree of activation of microglia, we just detect this marker from C57BL/6 TBI mice. The results showed that, compared with resting phenotype, in the each region of brain(cortex and hippocampus), microglia were significantly activated after the 1st week of TBI(Fig. 4), and the activation state is last at least for 3 weeks(C57BL/6 P < 0.05 vs. C57BL/6 TBI at the 1st week; C57BL/6 P < 0.01 vs. C57BL/6 TBI at the 2nd week; C57BL/6 P < 0.01 vs. C57BL/6 TBI at the 3rd week; C57BL/6 P > 0.05 vs. C57BL/6 TBI at the 4th week) (Fig. 4).
TBI Aggravated Aβ-plaques Deposition in the Specific Region of Hippocampus in APP/PS1 Mouse Model.
Aβ-plaques deposition is concerned to be the most distinctive and classic pathological change in AD brain. Generated from the cleavage of APP by β-secretase, Aβ molecules form into monomer, oligomer and fiber, which are both toxic to the CNS. As is reported previously, the up-regulated expression of APP mRNA and up-regulated activity of β-secretase has been observed in situ in the TBI mouse model, which provides abundant materials for the generation of Aβ-plaques via β-secretase27, 28. To further investigate whether TBI could aggravate Aβ-plaques deposition in the hippocampus in APP/PS1 mice brain, immunohistochemistry staining was performed. As expected, there were not any Aβ-plaques deposition in C57BL/6 and C57BL/6 TBI mice. By IHC staining, we found that the Aβ-plaques deposited mostly in the specific region in the hippocampus(Fig. 5 in the region of black curly line). It showed that only a few mounts of Aβ-plagues deposited in the brain of APP/PS1 mice. Whereas, after 4 weeks of TBI, APP/PS1 TBI mice exhibited significantly more Aβ-plaques deposition in these specific region of hippocampus than the APP/PS1 mice did at the same month of age (APP/PS1 TBI, P < 0.05, vs. APP/PS1) (Fig. 5), which infers that TBI might be the trigger of acceleration of Aβ-plaques deposition in the brain.
Aβ-plaques Aggravated Neuroinflammation in the Specific Region of Hippocampus post-TBI.
Microglia plays an important role in the progression of AD. It can be activated by the deposition of Aβ-plaques and the potent activation of microglia results in the persistent release of proinflammatory cytokines, which causes secondary injury to the CNS29. Neuroinflammation exists both in the progression of TBI and AD. In the acute stage of TBI, there is an activation of microglia in the CNS instantly, which can persist for a long time26. To further determine the degree of neuroinflammation in the hippocampus post TBI, immunofluorescence staining was performed to detect the expression of IBA-1 which is the marker of activated microglia. In our experiment, we found that the activation of microglia was significantly elevated in the region of CA2 and DG in APP/PS1 TBI mice(APP/PS1 TBI, P < 0.01 vs. APP/PS1(CA2 region); APP/PS1 TBI, P < 0.05 vs. APP/PS1(DG region)) (Fig. 6), but not the other regions of hippocampus(Fig. 6). Notably, the region of up-regulated activation of microglia is paralleled with the region of accumulation of Aβ-plaques deposition in the hippocampus, indicating that the possibility of correlation between potent activation of microglia and deteriorated AD pathology after TBI.
TBI Aggravate the Alzheimer’s-like Pathology by Altering the Phenotype of Microglia.
As is elucidated above, microglia are activated after TBI in the specific region of hippocampus. However, that which sort of phenotype of activated microglia is predominate in the damaged brain deserves further study. In that the biological behavior of microglia, doing good or harm to the CNS, depends on its phenotype. To further investigate the direction of microglia polarization post TBI, we performed western-blotting in the APP/PS1 TBI mice to detect the production of microglia(Arg1 and iNOS) for distinguishing the predominate phenotype. The results showed that in the normal circumstance, microglia seemed to exhibit an alternative phenotype which produced high level of Arg1(Fig. 7A,7B), however, it switched the phenotype much more to a classical phenotype with the feature of producing more iNOS than the normal state post TBI(Fig. 7A,7C). These results indicated that microglia switched the phenotype from an “M2-like” to an “M1-like” post TBI, a phenotype which was disadvantaged for the clearance of Alzheimer’s-like pathology.