*4.2. Animals and Treatments*

The animal protocols were conducted in strict compliance with the guidelines established by the Kazan State Medical University Animal Care and Use Committee (approval No. 10, 2017). Experiments were performed on adult female Wistar rats (weight of 200–250 g) obtained from Pushchino Laboratory (Pushchino, Russia). Animals were housed one per cage according to approved procedures for the use of animals in laboratory experiments.

For preventive gene therapy, rats were deeply anesthetized intraperitoneally with Zolitil 100 (Virbac Laboratoires, France) 3 mg/kg and Xyla (Interchemie werken "De Adelaar" B.V., Netherlands) 4.8 mg/kg. A laminectomy was made over the L4–L5 vertebral level and gene-modified UCBC or adenoviral vectors were infused intrathecally. The rats were divided into five experimental groups according to the injected substances (Table 1).

Ischemic stroke in the rats was induced 3 days after intrathecal injection of UCB-MC+Ad5-GFP and UCB-MC+Ad5-VEGF-GDNF-NCAM or 4 days after intrathecal injection of 0.9% NaCl, Ad5-GFP and Ad5-VEGF-GDNF-NCAM. The ischemic stroke was induced by permanent MCAO, as described previously [25]. In brief, to reduce blood flow in the circle of Willis, the right common carotid artery was ligated with surgical silk. In the left side, a 4–5 mm hole was drilled in the temporal bone and MCAO was performed by thermocoagulation using an operating microscope.

To reduce suffering and distress, post-operative care included analgesic therapy with Ketamine (Dr. Reddy's Laboratories, Ltd., Hyderabad, Andhra Pradesh, India) intramuscularly (2.5 mg/kg) once-a-day and antibacterial therapy with Ceftriaxone (Sandoz, Austria) intramuscularly (50 mg/kg) once-a-day for 5 days. Based on our previous study [25] for evaluation of preventive gene therapy, experimental animals were sacrificed three weeks after MCAO.
