Microphotographs of the sections were taken using the Olympus BX 51 microscope, equipped with the Olympus DP 70 digital camera (Olympus, Tokyo, Japan) and taken with cellSens software (Olympus, Tokyo, Japan). RAF1 Cresyl violet staining (Nissl staining) was used for identifying the basic neuronal structure and the brain injury extent. pathological changes of TDP-43 in the hippocampi of animals following LFPI and in mice exposed to repetitive moderate TBI (rmTBI) were studied. Changes in the hippocampal TDP-43 expression patterns at 14 d following different brain trauma procedures showed pathological alterations only after single moderate, but not following rmTBI. Hippocampal LFPI-induced TDP-43 pathology was not accompanied by the microglial reaction, contrary to the findings after rmTBI, suggesting that different types of brain trauma may cause diverse pathophysiological changes in the brain, specifically related to the TDP-43 protein as well as to the microglial reaction. Taken together, our findings may contribute to a better understanding of the pathophysiological events following brain trauma. (= 4C5 mice per group). Each triangle represents data from an individual mouse. * 0.05; significantly different from the related control group. # 0.05; significantly different from the LFPI D1 group. (D) Representative microphotographs of the ipsilateral cortices, double labeled with anti-TDP-43 antibody (green) and the markers of neurons (NeuN), microglia (Iba1) or astrocytes (GFAP) (all red), from the animal sacrificed 3 d following single moderate LFPI. Sections were counterstained with DAPI nuclear stain (blue). Arrows point to cells with prominent cytoplasmic, and arrowheads indicate cells with dominant nuclear TDP-43 expression. Scale bar: 100 m. In the ipsilateral cortices of injured animals, increased cytoplasmic TDP-43 immunostaining was evident at both day 1 and day 3 after the brain trauma compared to the control mouse, in which this protein was mostly situated in the cell nuclei (Physique 1A). Densitometric analyses of the Idasanutlin (RG7388) TDP-43 Western blots (Physique 1B,C) revealed a significant increase in its cytoplasmic content compared to the results from the cortical samples of the control animals at both 1 and 3 post-injury d [F(2,9) = 6.525; = 0.018]. A decrease in the nuclear expression of this protein was detected at day 3 following LFPI but only compared to the day 1 level [F(2,10) = 4.302; = 0.045]. Further, we wanted to determine whether these TDP-43 expression changes are cell type specific, i.e., if the increased cytoplasmic presence of this protein could be detected in the ipsilateral cortical neurons, microglia, or astrocytes after LFPI. Immunofluorescence analysis of the ipsilateral cortices of injured mice (Physique 1D) showed increased cytoplasmic TDP-43 staining in neurons and microglia; however, in our experiments, in the control and the injured animals, cytoplasmic expression of TDP-43 in the astrocytes was not detected. At this point, it was unclear whether the observed changes in the TDP-43 expression that we found in the ipsilateral cortices of injured animals was part of the physiological response to injury or if it was a sign of Idasanutlin (RG7388) pathological changes. Therefore, we wanted to determine whether LFPI is also associated with the formation of phosphorylated TDP-43 and its fragments in the mouse ipsilateral cortex at 1 and 3 d following trauma. As shown in the Physique S2, we did not detect any additional TDP-43 abnormalities in the context of single LFPI. Taken together, these results reveal the changes in the TDP-43 subcellular expression at the investigated time points following a single moderate TBI. This cytoplasmic TDP-43 mislocalization was not only restricted to neurons but was also found in microglial cells. 2.2. Single Moderate Brain Trauma Idasanutlin (RG7388) Triggers Changes in the Cortical Expression of p65 Subunit of NF-B That Are Area- and Cell Type-Dependent In the previous studies, it was shown that TDP-43 may act as a co-activator of the p65 subunit of NF-B in the models of ALS [23] and stroke [47], and that it is involved in the proinflammatory genes transcription as well as in the inflammation-mediated neurodegeneration [23]. Here, we first aimed to explore the NF-B expression changes in the ipsilateral cortices of injured mice acutely following a single moderate LFPI. Representative immunoblots and the densitometric analyses of the expression levels of the cytoplasmic p65 subunit of NF-B are shown in Physique 2A. Statistical analysis was conducted to examine the variances in the p65 expression levels between the experimental groups and no significant differences were found, even though a trend of a decrease in the p65 expression in mice with LFPI was apparent, especially at day 3 following LFPI [F(2,10) = 3.451; = 0.072]. Next, using the immunofluorescence method, we.
