Using mice from different breeding sites fails to improve replicability of results from single-laboratory studies.

Theoretical and empirical evidence indicates that low external validity due to rigorous standardization of study populations is a cause of poor replicability in animal research. Here we report a multi-laboratory study aimed at investigating whether heterogenization of study populations by using animals from different breeding sites increases the replicability of results from single-laboratory studies. We used male C57BL/6J mice from six different breeding sites to test a standardized against a heterogenized (HET) study design in six independent replicate test laboratories. For the standardized design, each laboratory ordered mice from a single breeding site (each laboratory from a different one), while for the HET design, each laboratory ordered proportionate numbers of mice from the five remaining breeding sites. To test our hypothesis, we assessed 14 outcome variables, including body weight, behavioral measures obtained from a single session on an elevated plus maze, and clinical blood parameters. Both breeding site and test laboratory affected variation in outcome variables, but the effect of test laboratory was more pronounced for most outcome variables. Moreover, heterogenization of study populations by breeding site (HET) did not reduce variation in outcome variables between test laboratories, which was most likely due to the fact that breeding site had only little effect on variation in outcome variables, thereby limiting the scope for HET to reduce between-lab variation. We conclude that heterogenization of study populations by breeding site has limited capacity for improving the replicability of results from single-laboratory animal studies.

Development and in vivo validation of phospholipid-based depots for the sustained release of bupivacaine.

By direct deposition of the drug at the local site of action, injectable depot formulations - intended for treatment of a local disease or for local intervention - are designed to limit the immediate exposure of the active principle at a systemic level and to reduce the frequency of administration. To overcome known drawbacks in the production of some marketed phospholipid-based depots, here we propose to manufacture drug-loaded negatively charged liposomes through conventional technologies and to control their aggregation mixing a solution of divalent cations prior to administration. We identified phosphatidylglycerol (PG) as the most suitable phospholipid for controlled aggregation of the liposomes and to modulate the release of the anesthetic bupivacaine (BUP) from liposomal depots. In vivo imaging of the fluorescently-labelled liposomes showed a significantly higher retention of the PG liposomes at the injection site with respect to zwitterionic ones. In situ mixing of PG liposomes with calcium salts significantly extended the area under the curve of BUP in plasma compared to the non-depot system. Overall, controlling the aggregation of negatively charged liposomes with divalent cations not only modulated the particle clearance from the injection site but also the release in vivo of a small amphipathic drug such as BUP.

Anaesthetic and Perioperative Management of 14 Male New Zealand White Rabbits for Calvarial Bone Surgery.

Calvarial bone surgery on rabbits is frequently performed. This report aims to document a simple and practical anaesthetic and perioperative management for this procedure. Fourteen male New Zealand white rabbits were included in the study. Subcutaneous (SC) dexmedetomidine, ketamine and buprenorphine ± isoflurane vaporized in oxygen administered through a supraglottic airway device (V-gel®) provided clinically suitable anaesthesia. Supplemental oxygen was administered throughout recovery. Monitoring was clinical and instrumental (pulse-oximetry, capnography, invasive blood pressure, temperature, arterial blood gas analysis). Lidocaine was infiltrated at the surgical site and meloxicam was injected subcutaneously as perioperative analgesia. After surgery, pain was assessed five times daily (composite behavioural pain scale and grimace scale). Postoperative analgesia included SC meloxicam once daily for four days and buprenorphine every 8 h for three days (unless both pain scores were at the lowest possible levels). Rescue analgesia (buprenorphine) was administered in case of the score > 3/8 in the composite pain scale, >4/10 on the grimace scale or if determined necessary by the caregivers. Airway management with a V-gel® was possible but resulted in respiratory obstruction during the surgery in two cases. Hypoventilation was observed in all rabbits. All rabbits experienced pain after the procedure. Monitoring, pain assessments and administration of postoperative analgesia were recommended for 48 h.

Is xenon a suitable euthanasia agent for mice?

OBJECTIVE: To compare behavioural and electrophysiological variables of mice undergoing gas euthanasia with either xenon (Xe) or carbon dioxide (CO2). STUDY DESIGN: Single animals chronically instrumented for electroencephalography (EEG) recording were randomized to undergo euthanasia with either CO2 or Xe (n = 6 animals per group). ANIMALS: Twelve adult (>6 weeks old) male C57Bl6/n mice. METHODS: Mice were surgically instrumented with EEG and electromyogram electrodes. Following a 7-day recovery period, animals were placed individually in a sealed chamber and a 5-minute baseline recorded in 21% O2. Gas [100% Xe (n = 6) or 100% CO2 (n = 6)] was then added to the chamber at 30% chamber volume minute-1 (2.8 L minute-1) until cessation of breathing. EEG, behaviour (jumping and freezing) and locomotion speed were recorded throughout. RESULTS: Mice undergoing single gas euthanasia with Xe did not show jumping or freezing behaviours and had reduced locomotion speed compared to baseline, in contrast to CO2, which resulted in increases in these variables. EEG recordings revealed sedative effects from Xe but heightened arousal from CO2. CONCLUSIONS: Our data suggest that Xe may be less aversive than CO2 when using a 30% chamber volume minute-1 fill rate and could improve the welfare of mice undergoing gas euthanasia.

Nitrogen gas produces less behavioural and neurophysiological excitation than carbon dioxide in mice undergoing euthanasia.

Carbon dioxide (CO2) is one of the most commonly used gas euthanasia agents in mice, despite reports of aversion and nociception. Inert gases such as nitrogen (N2) may be a viable alternative to carbon dioxide. Here we compared behavioural and electrophysiological reactions to CO2 or N2 at either slow fill or rapid fill in C57Bl/6 mice undergoing gas euthanasia. We found that mice euthanised with CO2 increased locomotor activity compared to baseline, whereas mice exposed to N2 decreased locomotion. Furthermore, mice exposed to CO2 showed significantly more vertical jumps and freezing episodes than mice exposed to N2. We further found that CO2 exposure resulted in increased theta:delta of the EEG, a measure of excitation, whereas the N2 decreased theta:delta. Differences in responses were not oxygen-concentration dependent. Taken together, these results demonstrate that CO2 increases both behavioural and electrophysiological excitation as well as producing a fear response, whereas N2 reduces behavioural activity and central neurological depression and may be less aversive although still produces a fear response. Further studies are required to evaluate N2 as a suitable euthanasia agent for mice.

Epileptiform activity during inert gas euthanasia of mice.

Carbon dioxide (CO2) is one of the most commonly used euthanasia agents for mice, yet it is highly aversive and nociceptive. Inert gases are a possible alternative, however there are qualitative reports of seizures resulting from exposure. Here we evaluate epileptiform activity caused by inert gases (N2, He, Ar and Xe) and CO2 in mice chronically instrumented for EEG/EMG undergoing single-gas euthanasia. We found that N2, He and Ar caused epileptiform activity in all animals, CO2 in half of animals and no epileptiform activity produced by Xe. Atmospheric O2 concentrations at epileptiform activity onset were significantly higher for CO2 than for all other gases and occurred soon after loss of motion, whereas N2 and Ar epileptiform activity occurred at cessation of neocortical activity. Helium caused the longest epileptiform activity and these commenced significantly before isoelectric EEG. We did not detect any epileptiform activity during active behaviour. Taken together, these results demonstrate that whilst epileptiform activity from inert gases and particularly Ar and N2 are more prevalent than for CO2, their occurrence at the onset of an isoelectric EEG is unlikely to impact on the welfare of the animal. Epileptiform activity from these gases should not preclude them from further investigation as euthanasia agents. The genesis of epileptiform activity from CO2 is unlikely to result from hypoxia as with the inert gases. Helium caused epileptiform activity before cessation of neocortical activity and for a longer duration and is therefore less suitable as an alternative to CO2.