*3.1. 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 our previous research, we demonstrated that the OT was the only damaged brain structure in the injured wild-type and TDP-43 transgenic mice already on the first day following the last head impact, lasting up to the end of the first week, suggesting early vulnerability of this structure to the investigated type of injury [27]. In the current study, we histologically detected neurodegeneration using Fluoro-Jade C staining and degenerating, argyrophylic, and swollen axons using neurosilver staining in the OT of the traumatized animals of both genotypes at 6 months after repetitive mTBI, indicating chronic axonal posttraumatic damage. Our results are in agreement with some previous studies in which the destruction of this structure in rodents was reported in different models by using various injury paradigms and time points of 1, 3, 7, or 60 days, 3 or 10 weeks, and 8 or 12 months following repeated mTBI [27,31,33,42,49–52]. Taking all these results together, including the fact that repetitive mTBI increases the sensitivity of the brain to each subsequent trauma, it is plausible that the time for the recovery between individual impacts was insufficient in the experimental models and protocols used in the mentioned investigations, resulting in neurodegeneration and axonal degeneration months after the final injury. Increased sensitivity and vulnerability of the OT to the damage induced by repeated mTBI may be a consequence of its position below the brain, as well as of its anatomic characteristics. Specifically, it consists of very long myelinated axons which are susceptible to compression in the optic canal during the direct injury, as well as to tension and torsion during acceleration and deceleration forces caused by the hits [53]. Moreover, the blood supply of the optic nerve arrives from pial arteries. Its swelling, induced by repeated head traumas, may cause localized ischemic injuries that may additionally contribute to the OT damage [31]. Moreover, neurodegenerative changes in the proximal part of the visual system, including the optic nerves and the optic chiasma, and decreased cellularity in the ganglion cell layer of the retina were previously described in mice at different time points following repetitive mTBI [40–42,50].

The current study is the first in which the neural and axonal degeneration in the OT of TDP-43 transgenic mice was investigated at a chronic time point following repetitive mTBI. Neurodegeneration was found in the injured TDP-43G348C animals compared with the related sham mice. Contrarily, significant differences in Fluoro-Jade C intensity were not observed between traumatized transgenic TDP-43 mice compared with wild-type animals, suggesting that human genetic TDP-43 background did not affect chronic damage of this structure.

In order to detect other chronic effects of repeated head impacts on the surviving axons of the OT, we investigated the levels of their myelination and the expression of the cytoskeleton NfL protein. Myelin preservation was determined by LFB and MBP stains and their quantification. Reduced LFB staining was evident in some parts of the OT in the injured mice of both genotypes. Furthermore, there was no significant difference in the staining density between the traumatized wild-type and transgenic TDP-43 mice compared with their related sham or between injured wild-type and TDP-43G348C animals in this brain structure at 6 months after head traumas. Previously, no changes in LFB-positive staining were observed in the brains of mice at 6 months following the first injury in the model of repetitive mTBI induced by electromagnetic controlled impact device [32], in which local areas of reduced myelination were described in the optic nerve at 3 and 13 weeks after the last head trauma [40,41]. To our knowledge, the level of myelination was not previously investigated in the OT in the models of repeated mTBI in TDP-43 transgenic animals.

In our experiments, no changes were observed in the MBP immunoreactivity and optical density in the OT of the injured wild-type and TDP-43 transgenic mice compared with related controls or in traumatized wild-type compared with TDP-43G348C animals. Taken together, the results of this study obtained by the LFB and MBP staining demonstrated that repetitive mTBI did not significantly affect myelination of the surviving axons of the retinal ganglion cells at 6 months after the final head impact and that the transgenic genotype did not influence it. In addition, Gangolli et al. [54] did not detect Myelin Black Gold staining in the OT 1 year following injury induced by CHIMERA in mice. In the same experimental model, MBP immunoreactivity was not altered in the OT 7 days after the final injury [51].

NfL chain is abundantly expressed in the long and large-caliber myelinated whitematter axons, and it is considered a promising candidate biomarker of axonal injury in different diseases of the central nervous system [55], including repetitive mTBI [56–58]. Increased exosomal and plasma levels of NfL chain have been detected in humans even years following repeated head traumas, suggesting chronic, long-term axonal dysregulation and degeneration induced by sustained brain injuries [57].

To our knowledge, our study is the first in which NfL chain staining was investigated in the OT at a later time point following repetitive mTBI. We did not detect statistically significant differences in the levels of this protein's optical densities between the injured wild-type group and related sham 6 months after the final head trauma. Similar results were obtained by Cheng et al. [33] and Vonder Haar et al. [52], who also reported no differences in the Nf medium and heavy chains or the NfL chain staining in the OT between traumatized and sham mice at chronic post-injury time points in the CHIMERA model. Taking into account the results of our and other mentioned animal studies, it can be suggested that the changes in the NfL chain protein were not evident at later time points following repetitive mTBI. This could be due to previous death of the affected neurons and preserved cytoskeletal NfL architecture in surviving axons. Contrarily, axonal swellings and varicosities in the OT of the traumatized mice were observed 2 days after the injury induced by the CHIMERA method, but they disappeared by the seventh day following the last impact [59]. Taking all abovementioned results regarding the changes of the NfL following repetitive mTBI, it seems that this protein can be used as a brain marker of early axonal damage in animal models, in contrast to the human studies in which it has been detected in the blood 1 h to years after repeated head traumas [56–58].

Among other roles, under physiological conditions, TDP-43 binds and stabilizes NfL mRNA, regulating its transcription, metabolism, and axonal transport [60–62]. Contrarily, TDP-43 dysregulation, observed, e.g., in FTLD, is associated with NfL alterations and whitematter pathology [63]. In a recent study, Kumar et al. [64] found that cytoplasmic TDP-43 accumulation in mice expressing ALS-linked human TDP-43A315T mutant caused marked suppression of mRNA translation for NfL, Nf medium, and α-internexin, resulting in a decrease in the levels of these proteins at 12 months of their age when they exhibited TDP-43 proteinopathy in cortical neurons. In our research, we were interested if repetitive mTBI affects NfL chain staining intensity in the OT of mice presenting with cytoplasmic TDP-43 aggregates in the spinal cord starting at approximately 10 months of age and increased pathological TDP fragment in the brain and spinal cord at 10 months of age [28]. We did not detect any significant changes in NfL chain staining in the OT of injured TDP-43G348C mice compared to the related sham group or between traumatized wild-type and TDP-43 transgenic animals at 6 months following repeated head injury, suggesting that repetitive mTBI and the investigated genotype did not affect the structure and, consequently, function of this neuronal cytoskeletal protein in the used experimental conditions. Because the age of our experimental animals was approximately 8.5 months at the time of the experiments, it remains to be investigated whether there are age-dependent changes in NfL chain expression in mice of the tested TDP-43 genotype.

Neuroinflammation is one of the most important processes developing after head trauma that may have beneficial or detrimental effects in the acute TBI [65]; however, if it is chronic, it usually contributes to the brain damage [66–68]. In the current study, pronounced microglial and astrocytic response to the repetitive mTBI was found in the OT of the injured wild-type and TDP-43 transgenic mice related to their sham groups, suggesting chronic neuroinflammation of this structure as a result of synergistic exacerbating effects of repeated head traumas that provoked an increase in inflammatory responses during short time periods between each injury. In our previous study, using the same closed head weight drop method, significant microgliosis and astrocytosis were also demonstrated in traumatized wild-type and TDP-43G348C mice, in the acute and subacute posttraumatic periods [27]. Our previous and current results regarding gliosis in the OT following repetitive mTBI are in agreement with those obtained in animal studies in which the rodents were subjected to impacts induced by other methods and in which various head trauma protocols and different posttraumatic time points from 1 to 365 days after the injury were used [31,33,43,49–52,59,69,70]. Our study found no differences in the microglial and astrocytic hyperactivities between injured transgenic TDP-43 and wild-type mice, suggesting that the investigated genetic background did not affect inflammatory parameters used in this research. Taking all the abovementioned results together, it may be suggested that the OT is particularly susceptible and vulnerable to neuroinflammation induced by repetitive mTBI.
