**2. Results**

*2.1. Repetitive mTBI Induced Neurodegeneration, Axonal Injury, and Gliosis in the Optic Tract in Wild-Type and TDP-43G348C Mice at 6 Months Following the Last Head Impact*

In the first part of the study, we investigated whether repetitive mTBI causes the OT pathology 6 months after the last hit in wild-type and TDP-43 transgenic mice. We were especially focused on neuronal and axonal degeneration, demyelination, and glial activity in the mentioned brain structure after repeated brain impacts in the animals of both genotypes.

Fluoro-Jade C was used as the marker of neurodegeneration [34,35]. This dye was found to stain degenerating nerve cell bodies and distal dendrites, axons, and terminals [36]. Figure 1A shows representative microphotographs of Fluoro-Jade C-stained sections of the OT in the sham animals and the traumatized mice of both genotypes. It is evident that Fluoro-Jade C-positive staining was detectable in the OT of the traumatized mice, both wild-type and TDP-43 transgenic, but not in the sham animals. Quantitative analysis of Fluoro-Jade C staining intensity confirmed these observations (Figure 1B). It was demonstrated that Fluoro-Jade C intensity levels were significantly higher in traumatized wild-type and TDP-43G348C animals than in the sham animals of the corresponding control groups (*p* = 0.012; *p* = 0.012). No significant differences were observed between the levels of Fluoro-Jade C-positive staining in the OT of wild-type and transgenic TDP-43 injured mice (*p* = 0.676) (Figure 1B).

**Figure 1.** Neurodegeneration in the optic tract (OT) of wild-type (WT) and TDP-43G348C mice at 6 months after repetitive mild traumatic brain injury (rmTBI). (**A**) Representative microphotographs of the OT stained with the Fluoro-Jade C fluorescent dye. Arrows point to Fluoro-Jade C-positive staining. Scale bar: 100 μm. (**B**) The histogram shows the intensity levels of the fluorescent staining (AU/μm2) in the OT of WT and TDP-43G348C mice of the control groups (Sham) and animals with rmTBI. Results are expressed as means ± SEM (*N* = 5). \* *p* < 0.05, significantly different from the related Sham.

Silver staining is a method that has been used for visualization and localization of degenerating axons [37]. Representative microphotographs of silver-stained sections of the OT in mice of all experimental groups are shown in Figure 2. There was an increased silver uptake and staining in the OT of traumatized mice of both genotypes demonstrating evidence of axonal abnormalities compared with the sham-treated animals (Figure 2A). Furthermore, spheroids, a sign of axonal swelling, were observed in the axons of the OT in the injured wild-type and TDP-43 transgenic mice (Figure 2B). Results shown in Figure 2 suggest axonal injury in the OT at 6 months following repetitive mTBI in mice of both genotypes.

**Figure 2.** Axonal degeneration in the optic tract (OT) of wild-type (WT) and TDP-43G348C mice at 6 months after repetitive mild traumatic brain injury (rmTBI). (**A**) Representative microphotographs of the OT stained with the silver staining in the mice of the control group (Sham) and the animals with rmTBI. Scale bar: 200 μm. (**B**) Microphotographs are higher-magnification images of the areas in the boxes of the corresponding panels. Arrowheads point to the spheroids of degenerating axons. Scale bar: 50 μm.

In order to analyze the integrity of myelinated neuronal fibers in the OT, we used conventional histological methods, i.e., staining with luxol fast blue (LFB) and immunohistochemical staining with anti-myelin basic protein (MBP) antibody.

Representative photomicrographs of the OT sections stained with LFB in the traumatized wild-type and TDP-43G348C mice, as well as in the sham animals, are shown in Figure 3A. It is evident that myelin density and integrity of myelinated fibers were approximately equal in sham-treated wild-type and transgenic TDP-43 animals, while reduced myelin in some parts of the OT, characterized by porous and weaker LFB staining, was detectable in traumatized mice of both genotypes (Figure 3A). Quantitative analysis demonstrated that the staining densities in the OT of the injured wild-type and TDP-43G348C mice were slightly decreased in comparison to the levels in the related sham animals, but a statistically significant difference was not detected (*p* = 0.097). In addition, a significant difference in the levels of the LFB staining between the traumatized wild-type and TDP-43G348C transgenic mice was also not revealed (Figure 3B).

Representative photomicrographs of the coronal OT sections that were stained with anti-MBP antibody and the quantitative analysis of the MBP optical density for the mice of all experimental groups are shown in Figure 3C,D. There were no differences in the MBP immunoreactivity (Figure 3C) and optical density (Figure 3D) between the traumatized groups and their related control groups for both genotypes or between the injured wildtype and TDP-43 transgenic animals (*p* = 0.234) at 6 months after the last head trauma. The results obtained by LFB and MBP staining suggest that the myelination of surviving axons in the OT was mostly preserved at the investigated time point after the last brain trauma.

The integrity of surviving axons of retinal ganglion cells following repetitive mTBI was also examined using immunohistochemistry for neurofilament light (NfL) chain that is a major constituent of the neuronal cytoskeleton. Approximately equal NfL-positive staining was revealed in the axons of the OT in the traumatized and the sham mice of both genotypes (Figure 4A), while a significant difference in the NfL optical densities between the experimental groups was not revealed (*p* = 0.254) (Figure 4B). These results suggest that the NfL expression in the axons of the OT was unchanged at 6 months following repetitive mTBI.

**Figure 3.** The integrity of myelinated neuronal fibers in the optic tract (OT) of wild-type (WT) and TDP-43G348C mice at 6 months after repetitive mild traumatic brain injury (rmTBI). (**A**) Representative microphotographs of the OT stained with luxol fast blue (LFB). Higher magnification of the boxed regions reveals an area with reduced myelin and characterized by porous and weaker LFB staining. Dashed lines indicate the OT. Scale bar: 200 μm. (**B**) The histogram shows the LFB staining density (AU) in WT and TDP-43G348C mice with rmTBI and related control groups (Sham). Results are expressed as means ± SEM (*N* = 4–6). (**C**) Representative microphotographs of the OT sections immunostained with anti-myelin basic protein (MBP). Dashed lines indicate the OT. Scale bar: 200 μm. (**D**) The histogram shows the MBP optical density (AU) in WT and TDP-43G348C mice with rmTBI and related control groups (Sham). Results are expressed as means <sup>±</sup> SEM (*<sup>N</sup>* = 4–5).

**Figure 4.** Neurofilament light chain (NfL) expression in the optic tract (OT) of wild-type (WT) and TDP-43G348C mice at 6 months after repetitive mild traumatic brain injury (rmTBI). (**A**) Representative microphotographs of the OT stained with anti-neurofilament light chain protein. Dashed lines indicate the OT. Scale bar: 200 μm (**B**) The histogram shows NfL optical density (AU) in the axons of the OT in WT and TDP-43G348C mice with rmTBI and related control groups (Sham). Results are expressed as means ± SEM (*N* = 3–5).

The activity of the glial cells in the OT was examined using microglial marker ionized calcium-binding adaptor molecule 1 (Iba1) and the astrocytic marker glial fibrillary acidic protein (GFAP) (Figure 5).

**Figure 5.** Gliosis at 6 months after repetitive mild traumatic brain injury (rmTBI) in the optic tract (OT) of wild-type (WT) and TDP-43G348C mice. (**A**) Representative microphotographs of the OT immunostained with anti-ionized calcium-binding adaptor molecule 1 (Iba1). Arrowheads point to Iba1-positive cells with "resting" microglial morphology, and arrows point to Iba1-positive cells with activated microglial morphology. Scale bar: 100 μm. (**B**) The histogram shows the Iba1-positive area (%) in the OT of WT and TDP-43G348C mice with rmTBI and related control groups (Sham). Results are expressed as means ± SEM (*N* = 5). \* *p* < 0.05, significantly different from the related Sham. (**C**) Representative microphotographs of the OT immunostained with anti-glial fibrillary acidic protein (GFAP). Arrows point to the GFAP-positive cells with hypertrophic morphology. Scale bar: 100 μm. (**D**) The histogram shows the GFAP-positive area (%) in the OT of WT and TDP-43G348C mice with rmTBI and related control groups (Sham). Results are expressed as means <sup>±</sup> SEM (*<sup>N</sup>* = 5). \* *<sup>p</sup>* < 0.05, significantly different from the related Sham.

Figure 5A shows representative microphotographs of the Iba1 immunostained OT sections in mice of all the experimental groups. "Resting" microglia, i.e., the microglial cells with thin Iba1-immunoreactive processes, were detected in the sham-treated mice of both control groups. Moreover, in all the traumatized animals, activated microglia, i.e., microglial cells with hypertrophic and large cell bodies, thick processes, and with amoeboid and migrating morphology, were noticed (Figure 5A). The higher magnifications images of "resting" and "activated" microglia are shown in Figure S1 (Supplementary Materials).

Quantitative analysis of the percentages of the Iba1 immunoreactive areas in the OT demonstrated statistically significant higher values in the injured wild-type and TDP-43G348C animals compared with related sham mice (*p* = 0.012; *p* = 0.012) (Figure 5B). However, a statistically significant difference in the percentages of the Iba1 immunoreactive

areas between the traumatized wild-type and TDP-43 transgenic mice was not found (*p* = 0.296) (Figure 5B).

Morphological changes that would suggest astroglial activation were not detected in the OT of the sham injured wild-type and TDP-43G348C mice at 6 months after the last head trauma (Figure 5C). Contrarily, the GFAP immunoreactivities were more pronounced in the OT of traumatized wild-type and TDP-43 transgenic mice than in sham-treated animals, suggesting astrocytic hypertrophy after repetitive mTBI (Figure 5C). Furthermore, the percentages of the OT areas covered by hypertrophic astrocytes in the traumatized animals of both genotypes were significantly higher compared with the values noticed in the noninjured mice (*p* = 0.012; *p* = 0.012) (Figure 5D). In addition, the values of GFAP-positive areas in the injured TDP-43G348C animals did not significantly differ from the values in wild-type mice (*p* = 0.210) (Figure 5D).
