**1. Introduction**

Anaesthetic protocols using a combination of medetomidine and isoflurane, are increasingly being used in functional magnetic resonance imaging (fMRI) studies of the rodent brain [1–10]. The use of medetomidine for these studies was first reported in 2002 [9], whilst the use of low dose isoflurane (<0.5% vapouriser setting) in conjunction with medetomidine as an anaesthetic regime was first reported in 2012 [3,4,8].

Medetomidine is an <sup>α</sup>2-adrenoceptor agonist that causes sedation, hypertension, bradycardia, respiratory depression, hyperglycaemia, diuresis, muscle relaxation and analgesia [11–26]. The potency and receptor selectivity of medetomidine has led to its widespread use in veterinary anaesthesia, mostly in dogs and cats [27]. Medetomidine causes sedation through the activation of central <sup>α</sup>2-adrenoceptors in the locus coeruleus, which prevents excitatory neurotransmitter release in the central nervous system and thereby depresses cortical arousal [13–15]. Vascular side effects of medetomidine occur due to the activation of peripheral <sup>α</sup>2-adrenoceptors, which causes a transient and marked increase in systemic vascular resistance [17,18]. This vasoconstriction is followed by a decrease in vascular tone due to suppression of central nervous system-mediated sympathetic stimulation on blood vessels.

Isoflurane is a GABAergic fluorinated ether that causes anaesthesia, respiratory depression, bronchodilation, vasodilation, hypotension and muscle relaxation [28,29]. Isoflurane is commonly used for clinical and veterinary anaesthesia due to its rapid onset of action, short recovery time, safety and titratability [29–31]. The minimum alveolar concentration of isoflurane in adult Sprague-Dawley rats is 1.46 ± 0.06% [32].

The benefit of combining medetomidine with isoflurane specifically for fMRI studies has been described. When >0.1% isoflurane is administered with medetomidine, the epileptic activity caused by medetomidine is suppressed [4,21,22]. Furthermore, the drug combination allows for maintenance of a steady state of anaesthesia for >4 h, with consistent fMRI data [3]. In contrast, when medetomidine is administered alone via a constant rate infusion (subcutaneous (SC) or intravenous (IV)), it is not possible to maintain a steady state of sedation for >3 h. It has been reported that medetomidine administered alone can only be used in fMRI experiments >3 h if the initial infusion dose is increased three-fold after 90 min, or if medetomidine is specifically administered using an initial IV injection of at least 0.05 mg/kg medetomidine followed by a subsequent continuous SC or IV infusion of at least 0.1 mg/kg/h medetomidine, whereby the initial dose cannot be omitted, and the dose cannot be decreased [1,33].

Despite the increasing popularity of this combination of medetomidine and isoflurane, a standardised anaesthetic protocol for their combined use for rodent brain fMRI studies has not been established [4,34]. Various protocols are described with variable doses of both medetomidine and isoflurane, different routes of administration of medetomidine, and variation in the time of fMRI data collection relative to the time of medetomidine administration [3,4,6,7,34–36]. For example, in seven rodent fMRI studies employing medetomidine and isoflurane anaesthesia, the dose of isoflurane for maintenance of anaesthesia varied from 0.25–1.4% [3,4]. Furthermore, reported loading doses for medetomidine range from 0.03 to 0.15 mg/kg and the subsequent infusion doses range from 0.03 to 0.1 mg/kg/h [35,36]. In addition, the initial injection was administered via the intravenous (IV), intramuscular, intraperitoneal or subcutaneous (SC) routes and the infusion via the IV, intramuscular or SC routes. The time of fMRI data collection after the initial administration of medetomidine ranged

from 15 min to 90 min [3,7,36]. This variation in the use of medetomidine and isoflurane in rodent brain fMRI studies may be attributed to a lack of comprehensive data on the pharmacokinetics and pharmacodynamics of medetomidine in rodents. Importantly, the serum concentration of medetomidine when administered with low dose isoflurane for rodent brain fMRI studies is unknown. Thus, the rationale for the administration of medetomidine alongside isoflurane for rodent brain fMRI studies is largely derived empirically [1–9]. However, there is now evidence that both resting-state and evoked blood-oxygen-level-dependent (BOLD) fMRI signals are altered by the type of anaesthetic drug(s) used and their dose [37–44]. Thus, the many aforementioned inconsistencies in the use of medetomidine and isoflurane may be hindering the interpretation, generalisation, meta-analysis and reproducibility of rodent brain fMRI studies.

Medetomidine substantially reduces the dose of isoflurane required to achieve stable anaesthesia, therefore minimising anaesthetic-induced distortions of BOLD fMRI signals. When dogs are administered a dose of 0.03 mg/kg IV medetomidine, there is a reduction of the minimum alveolar concentration of isoflurane by 47.2% [45]. Furthermore, when rodents are anaesthetised with a combined medetomidine and isoflurane dose of 0.06 mg/kg/h IV and 0.5–0.6%, respectively, they exhibit levels of anaesthesia comparable to rodents treated either medetomidine 0.1 mg/kg/h IV or isoflurane 1.3% [34]. Reducing the dose requirement of each drug is beneficial, as high doses of each drug in isolation are associated with significant drug-specific distortions of BOLD fMRI signals [34]. This artefact occurs because BOLD fMRI studies rely on the coupling between local blood flow and local neuronal activity (known as neurovascular coupling) to infer and therefore measure neural activity [44,46,47]. BOLD signals in anaesthetised rodents are considered an accurate measure of neural activity when they produce an image reflective of brain activity in the awake rodent. Conversely, BOLD signals are considered inaccurate when they produce an image reflective of fMRI-induced-stress or anaesthetic-induced changes in the BOLD effect [34,48]. Recent evidence suggests that BOLD fMRI signals obtained during medetomidine and isoflurane anaesthesia can be used to accurately measure rodent brain activity [34]. This attribute can be partially explained by the synergistic effects of the drugs on preserving neurovascular coupling [3,35]. When administered alone, medetomidine alters the BOLD effect by causing cerebral vasoconstriction, bradycardia, decreased cerebral blood flow and altered astrocyte activity [4,35,49]. In contrast, when isoflurane is administered alone, it alters the BOLD effect by inducing vasodilation in cerebral vasculature [1,50]. Accordingly, when medetomidine and isoflurane are administered together, medetomidine appears to attenuate isoflurane-induced cerebral vasodilation, leading to better preservation of neurovascular coupling [51].

To better utilise medetomidine and isoflurane anaesthesia in rodent fMRI studies, their use should be standardised. To this end, the pharmacokinetic profile of medetomidine during combined medetomidine and isoflurane anaesthesia needs to be elucidated, and the serum concentration of medetomidine in this context needs to be identified. The aim of this study was to describe the pharmacokinetics of medetomidine during isoflurane anaesthesia and determine the serum concentration of medetomidine when administered with 0.5% (vapouriser setting) isoflurane, so that an evidence-based dosing regimen of medetomidine could be determined for rat brain fMRI studies.

### **2. Materials and Methods**

The study was approved by the University of Western Australia's Animal Ethics Committee (RA/3/100/1599) and conducted in accordance with the Australian code for the care and use of animals for scientific purposes, 8th edition [52]. The rats were housed in an AAALAC (Association for the Assessment and Accreditation of Laboratory Animal Care) facility.
