*2.2. Experimental Procedure*

The rats were randomly allocated to three experimental groups: Group T for determination of the target serum concentration of medetomidine when administered with low dose isoflurane for rodent brain fMRI studies (*n* = 8); Groups IV and SC for determination of the SC bioavailability of medetomidine during isoflurane anaesthesia (*n* = 8 each).

On the days of the procedures, the rats were anaesthetised with isoflurane (Isothesia™, Henry Schein Animal Health, 2000, Australia) in an induction chamber (4% isoflurane in 100% medical oxygen, 2 L/min). Once adequately anaesthetised (recumbent, no response to toe pinch) the rats were transferred onto the experimental benchtop and positioned for delivery of isoflurane throughout the experiment (0.5–2% isoflurane vapouriser setting in 100% medical oxygen, 1.5 L/min, Darvall Zero Dead Space face mask circuit, Advanced Anaesthesia Specialists) under a heat lamp. Physiological monitoring included body temperature, respiratory rate, heart rate, electrocardiography (PC-SAM Small Animal Monitor, SA Instruments Inc., 1030 System), exhaled isoflurane and CO2 (data not shown) (ISATM Sidestream Gas Analyzer, Masimo Sweden AB and PHASEIN and Lightning Multi-Parameter Monitor Vetronic Services Ltd., Newton Abbot, UK) and blood glucose concentration (Accu-Chek Guide, Roche, Mannheim, Germany). These variables were recorded every 5 min. A single rat was studied at any one time, during the hours of 8 a.m. and 6 p.m.

Medetomidine (1 mg/mL, Ilium Medetomidine Injection, Troy Laboratories Pty. Limited, Glendenning, Australia) was administered according to the treatment group. In Group T, rats were administered an initial dose of medetomidine of 0.05 mg/kg SC over 1 s via a 29 G insulin syringe (BD Ultra-Fine Insulin Syringe, Becton Dickinson Pty Ltd., Macquarie University Research Park North Ryde, Australia), immediately followed by a continuous medetomidine infusion of 0.15 mg/kg/h SC, administered via a 25 G butterfly catheter connected to a single syringe infusion pump (Legato 100 Syringe Pump, KD Scientific Inc., Holliston, MA, USA). This protocol was developed empirically and used in our laboratory [10]. In the IV and SC groups, rats were manually administered a single dose of either IV (through a catheter placed in a lateral tail vein) or SC (under the skin over a flank) medetomidine at 0.05 mg/kg. The concentration of isoflurane was immediately reduced to 0.5% after administration of the initial dose of medetomidine and then subsequently altered to maintain an adequate depth of anaesthesia as assessed by response to toe pinch, heart rate and respiratory rate.

For serial blood sampling, a catheter was placed in the lateral tail vein (22 G, 1 IN, BD Angiocath IV Catheter, BD Australia, Seven Hills, NSW, Australia), secured with surgical tape and flushed with heparinised saline (5 IU/mL). In Group T, blood samples were collected 60 and 90 min after the initial dose of medetomidine. The conditions during anaesthesia were consistent with those observed in previous studies performed in this laboratory and were considered suitable for identification of the target concentration of medetomidine. In the IV group, blood was collected before medetomidine administration and 2, 5, 10, 20, 30, 60, 120 and 180 min afterwards. In the SC group, blood was collected before medetomidine administration and 10, 20, 30, 40, 50, 60, 120, 180 and 240 min afterwards. Following collection of the final sample, but before recovery from anaesthesia, the rats were euthanised via an intraperitoneal or IV injection of pentobarbitone (160 mg/kg, Lethabarb, Jurox, Rutherford, Australia).
