Medetomidine

Experiments were organized into two series. The first series utilized the A(H3N2) clinical isolate pair, and the second series utilized the A(H1N1pdm09) clinical isolate pair. Virus stocks for ferret inoculation were diluted to 10⁴ and 10⁵ TCID₅₀/500 μL in PBS for the A(H1N1pdm09) and A(H3N2) clinical isolate pairs, respectively. For the 100% WT pretreatment isolate and 100% posttreatment isolate, pure populations of virus were used for infection. In coinfected ferrets (20% variant:80% WT), the mixtures were prepared based on the TCID₅₀ titer. A total of 36 ferrets were used in each experiment (72 ferrets for the complete study) with 12 ferrets in each infection group: 100% WT, 100% variant, or 20% variant:80% WT. For viral inoculation, the ferrets were given a reversible anesthesia via an intramuscular injection using a mixture of ketamine (10 mg/kg; Troy Laboratories), midazolam (0.5 mg/kg; Troy Laboratories), and medetomidine (0.02 mg/kg; Troy Laboratories) that was antagonized following the procedure by atipamezole (0.01 mg/kg; Troy Laboratories). During anesthesia, the ferrets were inoculated via the intranasal route with 250 μL of virus suspension in each nostril.

For all mice, a coil support was attached to the skull to allow secure positioning of the coil for LI-rTMS or sham (coil switched off as no stimulation control; Fig. 1A). The coil support was constructed from a P20 pipette tip (Beckman-Coulter), trimmed 15 mm from the apex and fixed to a dental cement base (Paladur, Heraeus Kulzer).
To implant the coil supports, animals were deeply anaesthetized (75 mg/kg ketamine and 1 mg/kg medetomidine i.p.; Troy Laboratories), and an incision was made to expose the skull. Connective tissue on the skull surface was gently blunt-dissected. Cyanoacrylate (Uhu) was applied to the underside of the dental cement base to adhere it to the skull. The coil support fixation to the skull was then reinforced by applying further dental cement to the join between the (already set) dental cement base and the mouse skull. Excess glue and dental cement were removed, and the skin was sutured around the base of the pipette, leaving the tip accessible (Silkam, Aesculap). Pipette tips were trimmed to extend 10 mm from the surface of the skin. Anesthetic reversal (10 mg/kg atipamezole; Troy Laboratories) was injected subcutaneously.

OB was performed according to several previously established surgical protocols^{14 (link),19 (link)–21 (link)}. Briefly, mice were anesthetized with intraperitoneal injection of ketamine (75 mcg/kg; Troy Laboratories Pty Ltd, USA) and medetomidine (1 mg/kg; Troy Laboratories Pty Ltd, USA). A midline incision was made in the skin overlying the skull, and the skin was retracted to reveal the bregma and skull overlying the anterior cranial fossa. Bilateral burr holes 2 mm in diameter were drilled in the skull 4 mm rostral and 2 mm lateral to bregma. The olfactory bulbs were aspirated, and small pieces of hemostatic sponge were used to stop the bleeding before the skin was sutured. Animals receiving a sham operation were treated identically, but the olfactory bulbs were left intact. Following surgery, animals were returned to their home cage. All animals had a 2-week recovery before starting fluoxetine hydrochloride or rTMS treatment. During the second week, all mice were handled daily for 10 min to habituate the animals to the handling associated with rTMS treatment.