The safety, efficacy and neuromotor effects of the neurosteroid anaesthetic alfaxalone in rats

Abstract

Rodents are routinely anaesthetised for husbandry and biomedical research purposes. All anaesthetics carry a degree of risk, complications and side effects. An ideal anaesthetic for rodents is safe, predictable, alleviates pain and distress and can be performed without specialist training. A survey was created to identify anaesthetic agents frequently administered to rodents currently utilised by the research community. Findings indicated injectable anaesthetics were very commonly used and complications were often associated with use, highlighting the lack of a safe, predictable laboratory rodent anaesthetic protocol.Alfaxalone is a neuroactive steroid injectable anaesthetic agent with a high margin of safety, and is commonly used in veterinary anaesthesia as the Alfaxan® formulation. I sought to establish whether alfaxalone could be used as an injectable anaesthetic agent for laboratory rodents. A plasma pharmacokinetics study was performed using IV and IP Alfaxan® in adult female Wistar rats (7-10 weeks). Mean T 1/2elim for 2 and 5 mg.kg -1 IV was short (~17 minutes), but could not be estimated for IP dosing due to sustained plasma levels for up to 60 minutes after injection. Cl p for IV injection was calculated at 24.5% and 23% of cardiac output respectively. The observed C max was 2.99 mg.L -1 for IP administration, and 2.2 ± 0.9 and 5.2 ± 1.3 mg.L -1 for 2 and 5 mg.kg -1 IV administration respectively. AUC 0-60 was 96 min.mg.L -1 for IP dosing. The relative bioavailability for IP dosing was 26% and 28%, compared to IV doses of 2 and 5mg.kg -1 respectively. Alfaxalone given IP caused longer sleep times than IV dosing, although anaesthesia was not induced in 30% of rats.Importantly, all rats exhibited muscle twitching after alfaxalone administration. I then sought to test whether combining IP injection of Alfaxalone with common premedication agents could improve anaesthetic induction rates and reduce muscle twitching, to provide a viable anaesthetic regimen for rats. Medetomidine (0.5 mg.kg -1 ), medetomidine-butorphanol (0.35 mg.kg -1 -0.2 mg.kg -1 ), Acetylpromazine (ACP) -methadone (3 mg.kg -1 -0.7 mg.kg -1 ) and ACP-xylazine (3 mg.kg -1 -3 mg.kg -1 )were administered IP 10 minutes prior to IP alfaxalone (20 mg.kg -1 ). ACP-xylazine, medetomidine and medetomidine-butorphanol all significantly reduced twitching. Medetomidine-butorphanol provided surgical anaesthesia without prolonged recovery and provided an adequate plane of anaesthesia for short painful surgical procedures.However, none of these premedication agents fully eliminated alfaxalone-induced muscle twitching. I therefore carried out electrophysiological studies of motor neurons (MN), to determine the mechanism causing neuromotor excitation in response to alfaxalone. Previous data has shown that alfaxalone can modulate strychnine-sensitive glycinergic receptors, which may, in part, contribute to the adverse neuromotor excitatory responses manifested as muscle twitching during alfaxalone anaesthesia. The 3 effects of alfaxalone on inhibito...

Improving the quality of neurosteroid induced anaesthesia in rodents

“…The lack of a suitable anaesthetic protocol was further emphasized when Lau and colleagues conducted a laboratory rodent anaesthetic survey, which identified inadequate depth of anaesthesia to be the most common anaesthetic complication followed by unexpected anaesthetic wake-up, sharp drop in body temperature and apnoea. The survey also found a significant rate of mortality in rodents after the use of popular anaesthetics (Lau, 2013). These findings, along with other reports (Zuurbier et al, 2014;Flecknell, 2016a), clearly show that, although we have come a long way from the earlier anaesthetics like chloroform and ether, modern agents of anaesthesia are not free of unwanted side effects.…”

“…In rats, severe facial and whole body twitches were reported during induction and recovery phases of anaesthesia (Lau, 2013). This phenomenon was also reported in mice earlier when older formulation, Saffan (alphaxalone and alphadalone) was used (File & Simmonds, 1988) and later when improved formulation (alfaxalone alone encased in hydroxypropyl-β-cyclodextrin) was used (Siriarchavatana et al, 2016…”

“…TRPM4 is a Ca 2+ activated nonselective cation channel activated by an increase of intracellular Ca 2+ and is regulated by several factors like temperature. Previous work has shown characteristic changes in inward current in hypoglossal motor neurons after application of increasing doses of alfaxalone where it induced an inward current, without a significant change in input resistance (Lau, 2013).…”

“…However, studies have reported that alfaxalone (as Alfaxan CD) has a major drawback for laboratory rodent anaesthesia, that of prominent neuromotor excitation (Lau, 2013;Siriarchavatana et al, 2016). Rats and mice show neuromotor excitation during the induction and recovery phase of anaesthesia.…”

“…Previous reports indicate that alfaxalone, particularly at high concentration potentiates the responses to glycine receptor activity, which could possibly contribute to the neuroexcitatory effects seen on alfaxalone anaesthesia. Lau and colleagues showed that inhibitory glycinergic neurotransmission to brainstem motor neurons is progressively suppressed by increasing alfaxalone concentration, potentially causing increased motor neuron excitability (Lau, 2013). However, to date, no studies have examined the effects of alfaxalone on excitatory synaptic transmission to brainstem motor neurons, which will be a key point of investigation in exploring the neuro-excitation seen with alfaxalone anaesthesia.…”