Fig 1.
Schematic illustration of the study.
(A) Part 1: the mice were divided into three groups and trained for 1, 2, and 3 days. Next, the surgical operation took place on Day 1, and the behaviors of the subjects were observed on Days 1, 3, 5, 7, and 14. (B) Part 2: the surgical operation took place on Day 0. The subjects were euthanized 3 hours, 1, 3, 5, 7, or 14 days post-surgery. At each time point, the subjects’ brain tissues were harvested and the samples were stained with TTC for whole-brain imaging.
Table 1.
Fig 2.
Evaluation of motor dysfunctions after TBI induction with rotarod test (A), elevated body swing test (B), and cylinder test (C). (A) The rotarod test indicated motor disturbances in TBI-induced subjects, while the elevated body swing and cylinder tests indicated their asymmetric actions. (B-C) The plotted results of each behavioral test. After TBI induction, motor dysfunctions were observed in the subjects. The rotarod test showed a rapid decrease in motor functions for the TBI group, which failed to recover over time. Moreover, the TBI group showed increasing severity in their asymmetric action over time. *p < 0.05, **p < 0.01, ***p < 0.001 compared to the Con group; #p < 0.05, ##p < 0.01, ###p < 0.001 compared to the Sham group (n = 20).
Fig 3.
The transient changes in the brain after TBI induction.
(A) Whole-brain imaging was carried out 3 hours, 1, 3, 7, and 14 days post-injury. In contrast to the Sham group, the TBI group showed signs of hemorrhaging, which reached its peak on Day 1 and gradually decreased by Day 3. (B) In the TTC-stained images, the white line indicates the hemorrhage lesions and infarction areas. The empty sites are the hemorrhage lesions, while the white color sites are the infarction areas. The infarction areas gradually increased until Day 1, slightly decreased by 3 days, and virtually disappeared by Day 14. (C) The quantified infarction areas for different time points post-surgery. (D) The H&E stained images of each group. After the surgery, the cytosol showed signs of collapsing and aggregation, which were recovered by Day 7. Each experiment included three biologically independent mice per group. Significance levels: *p < 0.05, **p < 0.01, ***p < 0.001 compared to the Con group; #p < 0.05, ##p < 0.01, ###p < 0.001 compared to the Sham group.
Fig 4.
The change in the Bcl-2 family proteins’ levels in the brain after TBI induction.
(A-C) Western blotting analyses. (D-F) The quantified western blotting results. (G) The relative expresssion levels of Bax/Bcl-2 was calculated and quantified. All protein levels were normalized to that of β-actin and fold change was calculated relative to the Con groups. The western blotting analyses were carried out 3 hours, 1, 3, 7, and 14 days post-surgery. Each experiment included three biologically independent mice per group. Significance levels: *p < 0.05, **p < 0.01, ***p < 0.001 compared to the Con group; #p < 0.05, ##p < 0.01, ###p < 0.001 compared to the Sham group.
Fig 5.
The downstream factors of the apoptotic signaling pathway were stimulated after TBI induction.
(A-C) The western blotting analyses of cleaved caspase 3, pro-caspase 3, ROCK1, and p53, which are some of the downstream factors of the apoptotic signaling pathway. (D-G) The quantified western blotting analyses. All protein levels were divided by the β-actin level and normalized to that of the Con group. The protein levels increased until Day 3 and decreased by Day 14. Each experiment included three biologically independent mice per group. Significance levels: *p < 0.05, **p < 0.01, ***p < 0.001 compared to the Con group; #p < 0.05, ##p < 0.01, ###p < 0.001 compared to the Sham group.
Fig 6.
The expressions of Bax and Bcl-2 were also investigated by immunohistochemistry.
(A and C) The brown dot indicates Bax-positive cells. The Bax expression increased until Day 3 and gradually decreased for the remainder of the study. Each group was normalized to the Con group. (B and D) Bcl-2- positive cells increased until Day 3 and slightly decreased by Day 7. The brown areas and dots indicate the protein-positive areas. Each group was normalized to Con group. Each experiment included three biologically independent mice per group. Significance levels: *p < 0.05, **p < 0.01, ***p < 0.001 compared to the Con group; #p < 0.05, ##p < 0.01, ###p < 0.001 compared to the Sham group.
Fig 7.
There was a rapid loss of neurons and disruption in their shape after TBI induction.
(A and D) In the Con and Sham groups, the neuron nuclei showed a spherical shape. In contrast, from 3 hours to 1 day post-surgery, the TBI group’s neuron nuclei showed a sharp and long shape. By Day 7, such a shape disappeared in many nuclei. Each group was normalized to the Con group. (B and E) MAP2 expressed in dendrites, somas, and axons were stained and monitored for changes over time. The expression showed abrupt changes every day. Upon TBI induction, MAP2 was not detected initially. However, by Day 14, the protein’s expression recovered almost completely. Each group was normalized to Con group. (C and F) The changes in the level of NFH were also monitored over time. As shown in the image, NFH increased dramatically after TBI induction. Each experiment included three biologically independent mice per group. Significance levels: *p < 0.05, **p < 0.01, ***p < 0.001 compared to the Con group; #p < 0.05, ##p < 0.01, ###p < 0.001 compared to the Sham group.
Fig 8.
The changes in the levels of astrocyte and microglia markers after TBI induction.
After the apoptosis signaling was completed, astrocytes were activated. (A and C) GFAP (astrocyte marker) expression was increased on Day 7. (B and D) Iba1 (microglia marker) expression was gradually increased until Day 7 and decreased. Each experiment included three biologically independent mice per group. Significance levels: *p < 0.05, **p < 0.01, ***p < 0.001 compared to the Con group; #p < 0.05, ##p < 0.01, ###p < 0.001 compared to the Sham group.
Fig 9.
Experimental set-ups for the animal models of TBI and the results of the cellular levels.
We constructed an impact-acceleration model due to free falling weight. The animals showed a motor dysfunction in the behavioral tests. In addition, the levels of apoptotic signaling-related protein were highly expressed and neuronal cell loss was increased after TBI.