Actitrack system

After adaptation period at the beginning of the study basal behavioral profile was assessed in both males and females. In brightly lit room, rats were individually tested for locomotor activity using the Actitrack system (Panlab, Spain) as previously described (34 , 35 ). Each Plexiglas arena (45 cm × 45 cm × 30 cm) was equipped with 2 frames equipped with photocells located one above another 2 and 12 cm above the cage floor. Each animal was placed in the center of arena and the spontaneous behavior was tracked for 10 min. During the test, the horizontal locomotor activity (the trajectory as calculated by the system from beam interruptions that occurred in the horizontal sensors) and vertical activity (number of rearing episodes breaking the photocell beams of the upper frame) were recorded. At the end of the session, animals were returned to their home cage and arenas were wiped with 1% acetic acid to avoid olfactory cues.

Toxicity studies were conducted at Belgorod State University (Russia). The study included 30 male Wistar rats (21 days old). rAAV9-Hco-µUtrn-FLAG was tested at two doses: 2 × 10¹⁴ vg kg⁻¹ and 6 × 10¹⁴ vg kg⁻¹ after single injection via intravenous route (lateral tail vein). Euthanasia was performed by exsanguination with cardiac puncture under isoflurane inhalation. Animal behavior in the open field test was recorded daily using an ActiTrack system (Panlab, Spain). A modified Irwin test was performed daily for general assessment of the animals welfare. Terminal procedures, consisting of electrocardiography (ECG), blood pressure measurement, ophthalmoscopy, collection of whole blood, serum, and urine samples as well as necropsy were performed on the days 3 (½ animals) and 14 (½ animals). Collected urine was analyzed using test strips and a semi-automatic urine analyzer Urit 180 Vet (URIT Medical Electronic Group, China). Complete blood count analysis was performed with use of automatic hematology analyzer CELL-DYN 3700 (Abbott Diagnostics, USA). Biochemical blood analysis was performed on an A25 analyzer (Biosystems, Spain) using reagents and control materials from Hospitex Diagnostics. ECG was performed using the ECG-1003 DIXION machine (Dixon Technologies, India).

For the assessment of the explorative and general activity behavior, open field tests were performed. Mice were placed in a 50×50 cm arena with 50 cm high walls and their movement activity was recorded for 40 min using the Acti-Track System (Panlab, Barcelona, Spain). In the analysis, the first 10 min correspond to the exploratory behavior, the last 30 min to the general activity of the mice, the periphery of the arena a comfort and shelter zone and the center of the arena an insecure zone.