**1. Introduction**

Repetitive mild traumatic brain injury (mTBI) represents a current and growing serious medical and economic problem worldwide. It is particularly common in athletes engaged in contact sports, such as soccer, ice hockey, American football, boxing, wrestling, and mixed martial arts [1,2], as well as in victims of domestic spousal violence or child abuse [3] and military personnel [4,5]. The true prevalence of repetitive mTBI is not known because the symptoms of a single mTBI or concussion frequently resolve without medical care, pass spontaneously, and stay unrecognized, unreported, or undiagnosed. For example, in most patients, especially adult athletes, some of the post-concussion symptoms, such as dizziness, disorientation, confusion, or headache, subside within 10 days [6] and, in some cases, within several months following the first head trauma without specific interventions [7]. Increasing evidence suggests that, in humans or experimental animals with prior mTBI history, the susceptibility to brain damage induced by a future TBI is

**Citation:** Pilipovi´c, K.; Rajiˇc Bumber, J.; Dolenec, P.; Gržeta, N.; Jankovi´c, T.; Križ, J.; Župan, G. Long-Term Effects of Repetitive Mild Traumatic Injury on the Visual System in Wild-Type and TDP-43 Transgenic Mice. *Int. J. Mol. Sci.* **2021**, *22*, 6584. https:// doi.org/10.3390/ijms22126584

Academic Editor: Anne Vejux

Received: 11 May 2021 Accepted: 17 June 2021 Published: 19 June 2021

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augmented and that repetitive injuries have cumulative effects, enhancing a risk for longterm and later-life cognitive, behavioral, and psychiatric disturbances, as well as the development of neurodegeneration [8–10].

Repetitive mTBI has long been recognized as a risk factor for chronic traumatic encephalopathy (CTE), a condition characterized by generalized cerebral atrophy associated with widespread deposits of phosphorylated tau protein occurring as neurofibrillary tangles, diffuse beta-amyloid deposits, neuroinflammation, axonal pathology through the brain, and, in the majority of cases, by transactivation response element (TAR) DNA/RNA-binding protein 43 (TDP-43) immunoreactive intraneuronal and intraglial inclusions [10–13].

TDP-43 is predominantly a nuclear protein with the primary amino-acid structure similar to the members of the heterogeneous ribonucleoprotein family that shuttles between nucleus and cytoplasm [14]. Its intracellular functions in physiological conditions are insufficiently characterized, but it is becoming increasingly evident that TDP-43 is involved in specific pre-mRNA splicing and transcription events, in the regulation of mRNA stability, transport, translation, and degradation, and in chromatin condensation [14–16].

Recent evidence suggests that TDP-43 proteinopathy has been identified not only in CTE, but also in most cases of amyotrophic lateral sclerosis (ALS) [17], in a subset of the frontotemporal lobar degeneration (FTLD) with tau-negative ubiquitin-positive TDP-43-positive inclusions [18,19], and in specific disorders such as Alzheimer's disease [20], Lewy body disease [21], hippocampal sclerosis [22], and corticobasal degeneration [20], suggesting its important role in the pathogenesis of neurodegeneration [23].

While TDP-43 dysregulation and accompanying neuropathological changes were documented in humans with previous history of repetitive mTBI [11], to our knowledge, they were investigated in only four published experimental studies. Elevated TDP-43 expression levels in the whole-cell lysates from the injured mouse cortical and hippocampal tissue [24,25], as well as the protein changes in the rat brain following blast TBI [26], were described. We detected transitory TDP-43 cytoplasmatic translocation and overexpression of the protein and its pathological forms in the frontal cortex within the first week following repetitive mTBI in mice [27]. Neurodegeneration and gliosis in the optic tracts (OT) of injured wild-type mice and animals with overexpression of human mutant TDP-43 protein (TDP-43G348C), a model of ALS/FTLD [28], were also demonstrated [27]. In addition, the level of damage in the OT was significantly increased in TDP-43 transgenic animals compared with wild-type mice at the end of the first week after the last injury [27]. TDP-43G348C mice used in the mentioned study were 9–11 week old animals at the beginning of the study and did not show any neurodegenerative and behavioral impairments before head traumas. Here, we expanded our previous research to investigate the changes in the OT, as well as in the lateral geniculate nucleus (LGN) of the thalamus and the superior colliculus (SC), the brain structures that receive input from axons traveling in the OT, in wild-type and TDP-43G348C animals at 6 months after the last brain trauma. We were interested in the level of neurodegeneration and glial activity in the OT and the mentioned nuclei, the presence of axonal injury and demyelination in the OT, and the possible synaptic changes included in visual information processing from retinal ganglion cells toward the nuclei. Chronic pathological changes in the OT, the LGN, and the SC of transgenic TDP-43 animals have not yet been studied. Furthermore, to the best of our knowledge, the preservation, glial responses, and synaptic organization in the mentioned nuclei of the visual pathway in wild-type mice in a model characterized with unconstrained head and body movements following the brain traumas have not been previously investigated. Moreover, because of different visual impairments detected in patients after mTBI [29], spatial learning and memory testing, which require preserved visual information processing, was conducted in mice of both genotypes. Such behavior in TDP-43 transgenic mice after repetitive mTBI has not been previously examined. Therefore, considering the results of our previous study and the fact that behavioral and the pathological brain changes are present for months after the initial injuries [12,27,30–33], this research hypothesized that the damage, gliosis, and synaptic reorganization in the OT and the investigated nuclei, as well as behavioral

impairments induced by repeated mTBI, would be detected 6 months after the last injury and that they would be more pronounced in TDP-43G348C mice.
