*4.1. Animals and Treatment*

This study was performed on wild-type C57BL/6J and transgenic TDP-43G348C male mice of C57BL/6J background. At the beginning of the experiments, the mice were 9–11 weeks old. Transgenic TDP-43G348C mice were obtained from the University Laval, Quebec, Canada, and the colony was raised in the Laboratory for Mice Breeding and Engineering Rijeka, Faculty of Medicine Rijeka, University of Rijeka, Croatia. All the experimental procedures were performed according to the Faculty's Ethical Committee approval and in accordance with the Croatian laws and rules (NN 135/06; NN 37/13; NN 125/13; NN 39/17), as well as the guidelines of the European Community Council Directive (86/609/EEC). Mice were maintained in the animal facility of the Faculty's Department of Basic and Clinical Pharmacology and Toxicology in temperature- and humidity-controlled holding rooms, with an alternating 12 hour light/dark cycle. Fresh water and standard rodent chew were available to animals ad libitum.

Mild brain traumas were induced using the closed head weight drop method previously described by Kane et al. [84]. In brief, mice were anesthetized with 3.5% isoflurane in a nitrous oxide/oxygen (2:1) mixture in an induction chamber and rapidly positioned on aluminum foil placed over a Plexiglas box, lined with a sponge. The box was situated beneath the vertical metal tube of the apparatus. A steel weight (1.2 cm diameter, mass 97 g), set above the mouse head and between the ears, was pulled rapidly upward to 1 m height and released. Following the impact, the mice fell down through the foil and onto the surface of the sponge, all while rotating their bodies by 180◦ horizontally. After each mild brain trauma, the mice were returned to their holding cages to recover. In our experiments, mice rapidly recovered and showed normal interactions with other animals

without demonstrating signs of pain or indisposition nor resistance to manipulations after mTBI. We did not observe respiratory arrest or seizures in any of the tested animals. For the control group, sham treated animals were only briefly anesthetized without receiving impacts. Sham procedures or mild brain traumas were repeated twice daily, in intervals of 6 h, for five consecutive days, i.e., a total of 10 impacts. Mice were euthanized at 6 months after the final impact or sham procedure.
