Antinociceptive and Cardiorespiratory Effects of a Single Dose of Dexmedetomidine in Laboratory Mice Subjected to Craniotomy under General Anaesthesia with Isoflurane and Carprofen or Meloxicam.

Pain refinement represents an important aspect of animal welfare in laboratory animals. Refining analgesia regimens in mice undergoing craniotomy has been sparsely investigated. Here, we sought to investigate the effect of dexmedetomidine in combination with other analgesic drugs on intraoperative anti-nociceptive effects and cardiorespiratory stability. All mice were anaesthetised with isoflurane and received local lidocaine infiltration at the surgical site. Mice were randomised into treatment groups consisting of either carprofen 5 mg kg-1 or meloxicam 5 mg kg-1 with or without dexmedetomidine 0.1 mg kg-1 administered subcutaneously. Intra-anaesthetic heart rates, breathing rates, isoflurane requirements, and arterial oxygen saturations were continuously monitored. We found that administration of dexmedetomidine significantly improved heart and breathing rate stability during two of four noxious stimuli (skin incision and whisker stimulation) compared to non-dexmedetomidine-treated mice and lowered isoflurane requirements throughout anaesthesia by 5-6%. No significant differences were found between carprofen and meloxicam. These results demonstrate that dexmedetomidine reduces nociception and provides intra-anaesthetic haemodynamic and respiratory stability in mice. In conclusion, the addition of dexmedetomidine to anaesthetic regimes for craniotomy offers a refinement over current practice for laboratory mice.

Error-prone protein synthesis recapitulates early symptoms of Alzheimer disease in aging mice.

Age-related neurodegenerative diseases (NDDs) are associated with the aggregation and propagation of specific pathogenic protein species (e.g., Aß, α-synuclein). However, whether disruption of synaptic homeostasis results from protein misfolding per se rather than accumulation of a specific rogue protein is an unexplored question. Here, we show that error-prone translation, with its frequent outcome of random protein misfolding, is sufficient to recapitulate many early features of NDDs, including perturbed Ca2+ signaling, neuronal hyperexcitability, and mitochondrial dysfunction. Mice expressing the ribosomal ambiguity mutation Rps9 D95N exhibited disrupted synaptic homeostasis resulting in behavioral changes reminiscent of early Alzheimer disease (AD), such as learning and memory deficits, maladaptive emotional responses, epileptiform discharges, suppressed circadian rhythmicity, and sleep fragmentation, accompanied by hippocampal NPY expression and cerebral glucose hypometabolism. Collectively, our findings suggest that random protein misfolding may contribute to the pathogenesis of age-related NDDs, providing an alternative framework for understanding the initiation of AD.

Machine learning reveals interhemispheric somatosensory coherence as indicator of anesthetic depth.

The goal of this study was to identify features in mouse electrocorticogram recordings that indicate the depth of anesthesia as approximated by the administered anesthetic dosage. Anesthetic depth in laboratory animals must be precisely monitored and controlled. However, for the most common lab species (mice) few indicators useful for monitoring anesthetic depth have been established. We used electrocorticogram recordings in mice, coupled with peripheral stimulation, in order to identify features of brain activity modulated by isoflurane anesthesia and explored their usefulness in monitoring anesthetic depth through machine learning techniques. Using a gradient boosting regressor framework we identified interhemispheric somatosensory coherence as the most informative and reliable electrocorticogram feature for determining anesthetic depth, yielding good generalization and performance over many subjects. Knowing that interhemispheric somatosensory coherence indicates the effectively administered isoflurane concentration is an important step for establishing better anesthetic monitoring protocols and closed-loop systems for animal surgeries.

A role for spindles in the onset of rapid eye movement sleep.

Sleep spindle generation classically relies on an interplay between the thalamic reticular nucleus (TRN), thalamo-cortical (TC) relay cells and cortico-thalamic (CT) feedback during non-rapid eye movement (NREM) sleep. Spindles are hypothesized to stabilize sleep, gate sensory processing and consolidate memory. However, the contribution of non-sensory thalamic nuclei in spindle generation and the role of spindles in sleep-state regulation remain unclear. Using multisite thalamic and cortical LFP/unit recordings in freely behaving mice, we show that spike-field coupling within centromedial and anterodorsal (AD) thalamic nuclei is as strong as for TRN during detected spindles. We found that spindle rate significantly increases before the onset of rapid eye movement (REM) sleep, but not wakefulness. The latter observation is consistent with our finding that enhancing spontaneous activity of TRN cells or TRN-AD projections using optogenetics increase spindle rate and transitions to REM sleep. Together, our results extend the classical TRN-TC-CT spindle pathway to include non-sensory thalamic nuclei and implicate spindles in the onset of REM sleep.

REM sleep stabilizes hypothalamic representation of feeding behavior.

During rapid eye movement (REM) sleep, behavioral unresponsiveness contrasts strongly with intense brain-wide neural network dynamics. Yet, the physiological functions of this cellular activation remain unclear. Using in vivo calcium imaging in freely behaving mice, we found that inhibitory neurons in the lateral hypothalamus (LHvgat) show unique activity patterns during feeding that are reactivated during REM, but not non-REM, sleep. REM sleep-specific optogenetic silencing of LHvgat cells induced a reorganization of these activity patterns during subsequent feeding behaviors accompanied by decreased food intake. Our findings provide evidence for a role for REM sleep in the maintenance of cellular representations of feeding behavior.

Evaluation of Poultry Stunning with Low Atmospheric Pressure, Carbon Dioxide or Nitrogen Using a Single Aversion Testing Paradigm.

Low atmospheric pressure stunning (LAPS) has been suggested for use in poultry under 4 kg in the abattoir as a more humane alternative to carbon dioxide (CO2). However, there are currently no studies offering a direct comparison of the aversion between methods. Here, we trained adult female broiler breeders to relinquish a food reward by moving to another area of the gas chamber in response to aversive stimuli. They were then stunned and subsequently killed using single exposure to either CO2, N2, LAPS or medical air as a control. Birds exposed to CO2 relinquished the food reward the quickest and exhibited gasping and headshaking more than the other groups. LAPS resulted in the quickest time to loss of posture (LOP) and birds in the N2 group took the longest. Birds exposed to N2 displayed the longest duration of ataxia of any group; however, they did not show any wing-flapping prior to LOP, unlike the LAPS and CO2. Collectively these data demonstrate that both LAPS and N2 are less aversive to poultry than CO2 and may offer a significant welfare refinement for poultry killed for meat production.

Rapid fast-delta decay following prolonged wakefulness marks a phase of wake-inertia in NREM sleep.

Sleep-wake driven changes in non-rapid-eye-movement sleep (NREM) sleep (NREMS) EEG delta (δ-)power are widely used as proxy for a sleep homeostatic process. Here, we noted frequency increases in δ-waves in sleep-deprived mice, prompting us to re-evaluate how slow-wave characteristics relate to prior sleep-wake history. We identified two classes of δ-waves; one responding to sleep deprivation with high initial power and fast, discontinuous decay during recovery sleep (δ2) and another unrelated to time-spent-awake with slow, linear decay (δ1). Reanalysis of previously published datasets demonstrates that δ-band heterogeneity after sleep deprivation is also present in human subjects. Similar to sleep deprivation, silencing of centromedial thalamus neurons boosted subsequent δ2-waves, specifically. δ2-dynamics paralleled that of temperature, muscle tone, heart rate, and neuronal ON-/OFF-state lengths, all reverting to characteristic NREMS levels within the first recovery hour. Thus, prolonged waking seems to necessitate a physiological recalibration before typical NREMS can be reinstated.

Case report - azygos vein drainage into the left atrium in a dog with cor triatriatum dexter and a patent foramen ovale.

A 2-year-old Airedale terrier was presented with exercise intolerance since birth and newly developed chylous pleural effusion. Imaging procedures including echocardiography, cardiac magnetic resonance imaging, computed tomography, and selective angiography revealed an aberrant connection of the azygos vein and the left atrium, a membrane in the right atrium consistent with cor triatriatum dexter, and a patent foramen ovale with right-to-left shunt. Balloon dilation of the membrane in the right atrium seemed to result in transient improvement of exercise tolerance compared with the previous 2 years. When chylothorax relapsed after three months, the dog was euthanized. Necropsy confirmed the azygos vein to left atrial connection, the patent foramen ovale, and the cor triatriatum dexter.

Humanely Ending the Life of Animals: Research Priorities to Identify Alternatives to Carbon Dioxide.

: The use of carbon dioxide (CO2) for stunning and killing animals is considered to compromise welfare due to air hunger, anxiety, fear, and pain. Despite decades of research, no alternatives have so far been found that provide a safe and reliable way to induce unconsciousness in groups of animals, and also cause less distress than CO2. Here, we revisit the current and historical literature to identify key research questions that may lead to the identification and implementation of more humane alternatives to induce unconsciousness in mice, rats, poultry, and pigs. In addition to the evaluation of novel methods and agents, we identify the need to standardise the terminology and behavioural assays within the field. We further reason that more accurate measurements of consciousness state are needed and serve as a central component in the assessment of suffering. Therefore, we propose a roadmap toward improving animal welfare during end-of-life procedures.

Oscillating circuitries in the sleeping brain.

Brain activity during sleep is characterized by circuit-specific oscillations, including slow waves, spindles and theta waves, which are nested in thalamocortical or hippocampal networks. A major challenge is to determine the relationships between these oscillatory activities and the identified networks of sleep-promoting and wake-promoting neurons distributed throughout the brain. Improved understanding of the neurobiological mechanisms that orchestrate sleep-related oscillatory activities, both in time and space, is expected to generate further insight into the delineation of sleep states and their functions.

Alternatives to Carbon Dioxide-Taking Responsibility for Humanely Ending the Life of Animals.

Carbon dioxide (CO2) is commonly used to kill rodents. However, a large body of research has now established that CO2 is aversive to them. A multidisciplinary symposium organized by the Swiss Federal Food Safety and Veterinary Office discussed the drawbacks and alternatives to CO2 in euthanasia protocols for laboratory animals. Dialogue was facilitated by brainstorming sessions in small groups and a "World Café". A conclusion from this process was that alternatives to CO2 were urgently required, including a program of research and extension to meet the needs for humane killing of these animals. The next step will involve gathering a group of international experts to formulate, draft, and publish a research strategy on alternatives to CO2.

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.

Influence of injection protocol and measurement technique on computed tomographic assessment of glomerular filtration rate in healthy Beagles.

OBJECTIVE To compare values of CT-derived glomerular filtration rate (GFR) determined by 3 contrast-medium injection protocols and 4 measurement techniques in healthy Beagles. ANIMALS 9 healthy Beagles (mean ± SD weight, 13.2 ± 1.6 kg). PROCEDURES Each dog underwent 3 iohexol-injection protocols (700 mg of iodine/kg administered at a constant rate over 20 seconds, 700 mg of iodine/kg administered following an exponentially decelerated injection over 20 seconds, and 350 mg of iodine/kg at a constant rate over 10 seconds) during dynamic, whole renal-volume CT in randomized order with an interval of ≥ 7 days between experiments. Values of GFR determined from Patlak plots derived by use of 4 measurement techniques (standard transverse section, optimized transverse section, dorsal reconstruction, and volume calculation techniques) were compared. RESULTS The measurement technique influenced the mean ± SD GFR results (standard transverse section technique, 2.49 ± 0.54 mL/kg/min; optimized transverse section technique, 2.72 ± 0.52 mL/kg/min; dorsal reconstruction technique, 3.00 ± 0.60 mL/kg/min, and volume calculation technique, 2.48 ± 0.51 mL/kg/min). The lower iodine dose resulted in a significantly higher GFR value (3.00 ± 0.65 mL/kg/min), compared with that achieved with either higher dose administration (constant rate injection, 2.54 ± 0.45 mL/kg/min and exponentially decelerated injection, 2.47 ± 0.48 mL/kg/min). CONCLUSIONS AND CLINICAL RELEVANCE In healthy Beagles, the CT-derived GFR measurements obtained after injection of a full dose of contrast medium were reduced, compared with measurements obtained after injection of a half dose. This finding is important with regard to potential nephrotoxicosis in dogs with impaired renal function and for GFR measurement with CT-contrast medium protocols.

Sleep-wake control and the thalamus.

Sleep is an essential component of animal behavior, controlled by both circadian and homeostatic processes. Typical brain oscillations for sleep and wake states are distinctive and reflect recurrent activity amongst neural circuits spanning localized to global brain regions. Since the original discovery of hypothalamic centers controlling both sleep and wakefulness, current views now implicate networks of neuronal and non-neuronal cells distributed brain-wide. Yet the mechanisms of sleep-wake control remain unclear. In light of recent studies, here we review experimental evidence from lesional, correlational, pharmacological and genetics studies, which support a role for the thalamus in several aspects of sleep-wake states. How these thalamo-cortical network mechanisms contribute to other executive functions such as memory consolidation and cognition, remains an open question with direct implications for neuro-psychiatric diseases and stands as a future challenge for basic science and healthcare research.

Thalamic dual control of sleep and wakefulness.

Slow waves (0.5-4 Hz) predominate in the cortical electroencephalogram during non-rapid eye movement (NREM) sleep in mammals. They reflect the synchronization of large neuronal ensembles alternating between active (UP) and quiescent (Down) states and propagating along the neocortex. The thalamic contribution to cortical UP states and sleep modulation remains unclear. Here we show that spontaneous firing of centromedial thalamus (CMT) neurons in mice is phase-advanced to global cortical UP states and NREM-wake transitions. Tonic optogenetic activation of CMT neurons induces NREM-wake transitions, whereas burst activation mimics UP states in the cingulate cortex and enhances brain-wide synchrony of cortical slow waves during sleep, through a relay in the anterodorsal thalamus. Finally, we demonstrate that CMT and anterodorsal thalamus relay neurons promote sleep recovery. These findings suggest that the tonic and/or burst firing pattern of CMT neurons can modulate brain-wide cortical activity during sleep and provides dual control of sleep-wake states.

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.

Effects of contrast medium injection technique on attenuation values of adrenal glands in healthy dogs during contrast-enhanced computed tomography.

OBJECTIVE: To assess the effects of 3 contrast medium injection techniques on attenuation values for canine adrenal glands during contrast-enhanced CT. ANIMALS: 9 healthy Beagles. PROCEDURES: 3 protocols were evaluated in a randomized cross-over design study: 700 mg of iodine/kg at a constant injection rate over 20 seconds (full-dose constant rate), the same dose at a rate following an exponential decay curve over 20 seconds (full-dose decelerated rate), and 350 mg of iodine/kg at a constant injection rate over 10 seconds (half-dose constant rate). Multislice CT images were obtained before and at predetermined time points after the start of contrast medium injection. RESULTS: Median peak attenuation values were 129, 133, and 87 Hounsfield units with the full-dose constant rate, full-dose decelerated rate, and half-dose constant rate injection protocols, respectively. Peak attenuation differed significantly between the full-dose constant rate and half-dose constant rate injection protocols and between the full-dose decelerated rate and half-dose constant rate injection protocols. Median time to peak attenuation did not differ significantly among injection methods and was 30, 23, and 15 seconds for the full-dose constant rate, full-dose decelerated rate, and half-dose constant rate injections, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: The dose of contrast medium and the timing of postinjection CT scanning were main determinants of peak attenuation for adrenal glands in healthy dogs; effects of the 3 injection protocols on attenuation were minor. The exponentially decelerated injection method was subjectively complex. A constant injection protocol delivering 700 mg of iodine/kg over 20 seconds, with scans obtained approximately 30 seconds after starting contrast medium injection, provided images with maximum adrenal gland attenuation values.

Altered activity in the central medial thalamus precedes changes in the neocortex during transitions into both sleep and propofol anesthesia.

How general anesthetics cause loss of consciousness is unknown. Some evidence points toward effects on the neocortex causing "top-down" inhibition, whereas other findings suggest that these drugs act via subcortical mechanisms, possibly selectively stimulating networks promoting natural sleep. To determine whether some neuronal circuits are affected before others, we used Morlet wavelet analysis to obtain high temporal resolution in the time-varying power spectra of local field potentials recorded simultaneously in discrete brain regions at natural sleep onset and during anesthetic-induced loss of righting reflex in rats. Although we observed changes in the local field potentials that were anesthetic-specific, there were some common changes in high-frequency (20-40 Hz) oscillations (reductions in frequency and increases in power) that could be detected at, or before, sleep onset and anesthetic-induced loss of righting reflex. For propofol and natural sleep, these changes occur first in the thalamus before changes could be detected in the neocortex. With dexmedetomidine, the changes occurred simultaneously in the thalamus and neocortex. In addition, the phase relationships between the low-frequency (1-4 Hz) oscillations in thalamic nuclei and neocortical areas are essentially the same for natural sleep and following dexmedetomidine administration, but a sudden change in phase, attributable to an effect in the central medial thalamus, occurs at the point of dexmedetomidine loss of righting reflex. Our data are consistent with the central medial thalamus acting as a key hub through which general anesthesia and natural sleep are initiated.

Staying awake--a genetic region that hinders α2 adrenergic receptor agonist-induced sleep.

How external stimuli prevent the onset of sleep has been little studied. This is usually considered to be a non-specific type of phenomenon. However, the hypnotic drug dexmedetomidine, an agonist at α2 adrenergic receptors, has unusual properties that make it useful for investigating this question. Dexmedetomidine is considered to produce an 'arousable' sleep-like state, so that patients or animals given dexmedetomidine become alert following modest stimulation. We hypothesized that it might be more difficult to make mice unconscious with dexmedetomidine if there was a sufficient external stimulus. Employing a motorized rotating cylinder, which provided a continuous and controlled arousal stimulus, we quantitatively measured the ability of such a stimulus to prevent dexmedetomidine loss of righting reflex in two inbred strains of mice (C57BL/6 and 129X1). We found that whereas the C57BL/6 strain required a strong stimulus to prevent dexmedetomidine-induced hypnosis, the 129X1 strain stayed awake even with minimal stimuli. Remarkably, this could be calibrated as a simple threshold trait, i.e. a binary 'yes-no' response, which after crossing the two mouse strains behaved as a dominant-like trait. We carried out a genome-wide linkage analysis on the F2 progeny to determine if the ability of a stimulus to prevent dexmedetomidine hypnosis could be mapped to one or more chromosomal regions. We identified a locus on chromosome 4 with an associated Logarithm of Odds score exceeding the pre-established threshold level. These results show that complex traits, such as the ability of a stimulus to reverse drug-induced hypnosis, may have precise genetic determinants.