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Background The Canadian Council on Animal Care and American Veterinary Medical Association classify intraperitoneal (IP) pentobarbital as an acceptable euthanasia method in rats. However, national guidelines do not exist for a recommended dose or volume and IP euthanasia has been described as unreliable, with misinjections leading to variable success in ensuring a timely death. The aims of this study were to assess and improve efficacy and consistency of IP euthanasia. In a randomized, blinded study, 51 adult female Sprague-Dawley rats (170–495 g) received one of four treatments: low-dose low-volume (LL) IP pentobarbital ( n = 13, 200 mg/kg pentobarbital), low-dose high-volume (LH) IP pentobarbital ( n = 14, 200 mg/kg diluted 1:3 with phosphate buffered saline), high-dose high-volume (HH, n = 14, 800 mg/kg pentobarbital), or saline. Times to loss of righting reflex (LORR) and cessation of heartbeat (CHB) were recorded. To identify misinjections, necropsy examinations were performed on all rats. Video recordings of LL and HH groups were analyzed for pain-associated behaviors. Between-group comparisons were performed with 1-way ANOVA and Games-Howell post hoc tests. Variability in CHB was assessed by calculating the coefficient of variation (CV). Results The fastest euthanasia method (CHB) was HH (283.7 ± 38.0 s), compared with LL (485.8 ± 140.7 s, p = 0.002) and LH (347.7 ± 72.0 s, p = 0.039). Values for CV were: HH, 13.4%; LH, 20.7%; LL, 29.0%. LORR time was longest in LL (139.5 ± 29.6 s), compared with HH (111.6 ± 19.7 s, p = 0.046) and LH (104.2 ± 19.3 s, p = 0.01). Misinjections occurred in 17.0% (7/41) of euthanasia attempts. Pain-associated behavior incidence ranged from 36% (4/11, LL) to 46% (5/11, HH). Conclusions These data illustrate refinement of the IP pentobarbital euthanasia technique. Both dose and volume contribute to speed of death, with a dose of 800 mg/kg (HH) being the most effective method. An increase in volume alone does not significantly reduce variability. The proportion of misinjections was similar to that of previous studies.

Electrophysiology (ERG) is widely used for retinal function assessment, but the effects of different anesthetics on ERG recordings, particularly in degenerated retinas, remain unclear. This study investigated the effects of different anesthetic drugs on ERG in wild-type (WT), Kv8.2 knockout (KO), and rd10 mice. Anesthetic drugs, avertin (300 mg/kg) and pentobarbital sodium (50 mg/kg) were administered intraperitoneally, isoflurane was given at 5% for induction and 1.5% for maintenance. Full-field flash electroretinography (ff-ERG) was recorded, including scotopic and photopic responses. Specifically, the amplitudes of a-wave, b-wave, oscillatory potentials (OPs), photopic negative response (PhNR), and c-wave were analyzed, respectively. Additionally, fundus imaging and optical coherence tomography (OCT) were performed to analyze retinal morphology. The three anesthetics had no obvious effect on retinal morphology. Pentobarbital sodium decreased scotopic OPs, increased scotopic and photopic b-wave amplitudes and decreased photopic a-wave amplitude in all groups of mice. Isoflurane resulted in larger scotopic OPs and photopic a-wave amplitudes in all groups, with a larger scotopic a-wave amplitude in KO mice. The PhNR amplitude was greater in WT mice anesthetized with avertin. The ERG amplitudes in mice showed significant differences among the three anesthetic conditions. Pentobarbital sodium markedly suppressed retinal OPs, suggesting it may not be suitable for assessing inner retinal function, particularly amacrine cells. Isoflurane enabled excellent recordings of various types of ERG, making it suitable for nearly all ERG recordings. Avertin might serve as a suitable alternative in the absence of isoflurane.

Context: γ-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter and it is well established that activation of GABA A receptors favours sleep. l-Theanine, a naturally occurring amino acid first discovered in green tea, is a well-known anti-anxiety supplement with proven relaxation benefits. Objective: This study investigated the potential synergistic sleep enhancement effect of GABA/l-theanine mixture. Materials and methods: Pentobarbital-induced sleep test was applied to find proper concentration for sleep-promoting effect in ICR mice. Electroencephalogram (EEG) analysis was performed to investigate total sleeping time and sleep quality in normal SD rats and caffeine-induced awareness model. Real-time polymerase chain reaction (RT-PCR) was applied to investigate whether the sleep-promoting mechanism of GABA/l-theanine mixture involved transcriptional processes. Results: GABA/l-theanine mixture (100/20 mg/kg) showed a decrease in sleep latency (20.7 and 14.9%) and an increase in sleep duration (87.3 and 26.8%) compared to GABA or theanine alone. GABA/l-theanine mixture led to a significant increase in rapid eye movement (REM) (99.6%) and non-REM (NREM) (20.6%) compared to controls. The use of GABA/l-theanine mixture rather than GABA or l-theanine alone restored to normal levels sleep time and quality in the arousal animal model. The administration of GABA/l-theanine led to increased expression of GABA and the glutamate GluN1 receptor subunit. Conclusions: GABA/l-theanine mixture has a positive synergistic effect on sleep quality and duration as compared to the GABA or l-theanine alone. The increase in GABA receptor and GluN1 expression is attributed to the potential neuromodulatory properties of GABA/l-theanine combination, which seems to affect sleep behaviour.

Dried Chrysanthemum morifolium (Chry) flowers have been used in Korea as a traditional insomnia treatment. In this study, the sleep-promoting activity and improving sleep quality of Chry extract (ext) and its active substance linarin were analyzed by pentobarbital-induced sleep experiment in mice and electroencephalography (EEG), electromyogram (EMG) analysis in rats. In a dose-dependent manner, Chry ext and linarin promoted longer sleep duration in the pentobarbital-induced sleep test compared to pentobarbital-only groups at both hypnotic and subhypnotic doses. Chry ext administration also significantly improved sleep quality, as seen in the relative power of low-frequency (delta) waves when compared with the control group. Linarin increased Cl− uptake in the SH-SY5Y human cell line and chloride influx was reduced by bicuculline. After administration of Chry ext, the hippocampus, frontal cortex, and hypothalamus from rodents were collected and blotted for glutamic acid decarboxylase (GAD)65/67 and gamma-aminobutyric acid (GABA)A receptors subunit expression levels. The expression of α1-subunits, β2-subunits, and GAD65/67 of the GABAA receptor was modulated in the rodent brain. In conclusion, Chry ext augments pentobarbital-induced sleep duration and enhances sleep quality in EEG waves. These effects might be due to the activation of the Cl− channel.

Patients with prolonged seizures that do not respond to intravenous benzodiazepines and a second-line anticonvulsant suffer from refractory status epilepticus and those with seizures that do not respond to continuous intravenous anesthetic anticonvulsants suffer from super-refractory status epilepticus. Both conditions are associated with significant morbidity and mortality. A strict pharmacological treatment regimen is urgently required, but the level of evidence for the available drugs is very low. Refractory complex focal status epilepticus generally does not require anesthetics, but all intravenous non-anesthetizing anticonvulsants may be used. Most descriptive data are available for levetiracetam, phenytoin and valproate. Refractory generalized convulsive status epilepticus is a life-threatening emergency, and long-term clinical consequences are eminent. Administration of intravenous anesthetics is mandatory, and drugs acting at the inhibitory gamma-aminobutyric acid (GABA) A receptor such as midazolam, propofol and thiopental/pentobarbital are recommended without preference for one of those. One in five patients with anesthetic treatment does not respond and has super-refractory status epilepticus. With sustained seizure activity, excitatory N -methyl-d-aspartate (NMDA) receptors are increasingly expressed post-synaptically. Ketamine is an antagonist at this receptor and may prove efficient in some patients at later stages. Neurosteroids such as allopregnanolone increase sensitivity at GABA A receptors; a Phase 1/2 trial demonstrated safety and tolerability, but randomized controlled data failed to demonstrate efficacy. Adjunct ketogenic diet may contribute to termination of difficult-to-treat status epilepticus. Randomized controlled trials are needed to increase evidence for treatment of refractory and super-refractory status epilepticus, but there are multiple obstacles for realization. Hitherto, prospective multicenter registries for pharmacological treatment may help to improve our knowledge.

Rodents are widely used for animal research in Egypt. Pentobarbital is the most common anesthetic agent; however overdoses may affect the experimental outcomes and limit the use of tissues. To investigate the effects of sodium pentobarbital overdoses during exsanguination, three groups (6 rats/group) of male and female rats were injected i.p. with 50, 100 and 150 mg/kg of sodium pentobarbital, then carotid exsanguination was performed immediately after loss of consciousness. Hypoxia-inducible factor 1-alpha (Hif1a) and tumor necrosis factor-alpha (Tnfa) mRNA expressions in liver and kidney organs were evaluated. As well as, serum aminotransferase activities (AST&ALT), glucose, urea, creatinine, malondialdehyde (MDA), reduced glutathione (GSH) and catalase (CAT) levels were determined. The histological alterations in liver, kidney and spleen were studied. It was found that Hif1a and Tnfa were significantly overexpressed in the studied organs and serum AST, glucose, creatinine and urea levels were significantly increased after sodium pentobarbital overdoses (100 and 150 mg/kg) compared to 50 mg/kg dose. Similarly, significant increase in MDA and GSH levels of liver, kidney and spleen were noticed. Results showed gender difference where Hif1a and Tnfa levels were significantly overexpressed at high dose of sodium pentobarbital of liver and kidney organs in female more than male rats. Since euthanasia protocol may influence the physiological variables and affect genes’ expression, it is recommended to avoid sodium pentobarbital overdose during euthanasia as it may interfere with the biochemical, molecular and histological measurements.

Traumatic brain injury (TBI) results in the death of over 50,000 and the permanent disability of 80,000 individuals annually in the United States. Much of the permanent disability is the result of secondary brain injury from intracranial hypertension (ICH). Pentobarbital coma is often instituted following the failure of osmotic interventions and sedation to control intracranial pressure (ICP). The goal of this study was to evaluate the efficacy of pentobarbital coma with respect to ICP management and long-term functional outcome. Traumatic brain injury patients who underwent pentobarbital coma at a level 1 trauma center between 2014 and 2021 were identified. Patient demographics, injury characteristics, Glasgow Coma Scale (GCS) scores, intracranial pressures (ICPs), and outcomes were obtained from the trauma registry as well as inpatient and outpatient medical records. The proportion of ICPs below 20 for each hospitalized patient-day was calculated. The primary outcome measured was GCS score at the last follow-up visit. 25 patients were identified, and the majority were male (n ​= ​23, 92%) with an average age of 30.0 years ​± ​12.9 and median injury severity score of 30 (21.5–33.5). ICPs were monitored for all patients with a median of 464 (326–1034) measurements. The average hospital stay was 16.9 days ​± ​11.5 and intensive care stay was 16.9 ​± ​10.8 days. 9 (36.0%) patients survived to hospital discharge. Mean follow-up time in months was 36.9 ​± ​28.0 (min-max 3–80). 7 of the 9 surviving patients presented as GCS 15 on follow-up and the remaining were both GCS 9. Patients presenting at last follow-up with GCS 15 had a significantly higher proportion of controlled ICPs throughout their hospitalization compared to patients who expired or with follow-up GCS <15 (GCS 15: 88% ​± ​10% vs. GCS <15 or dead: 68% ​± ​22%, P ​= ​0.006). A comparison of the daily proportion of controlled ICPs by group revealed negligible differences prior to pentobarbital initiation. Groups diverged nearly immediately upon pentobarbital coma initiation with a higher proportion of controlled ICPs for patients with follow-up GCS of 15. Patients that do not have an immediate response to pentobarbital coma therapy for ICH universally had poor outcomes. Alternative therapy or earlier palliation should be considered for such patients. In contrast, patients whose ICPs responded quickly to pentobarbital had excellent long-term outcomes. •Persistent intracranial hypertension in TBI increases morbidity and mortality.•Pentobarbital can be used to decreased intracranial hypertension.•Patients with refractory intracranial hypertension after TBI may fully recover.

Neurosteroids that positively modulate GABAA receptors are among a growing list of rapidly acting antidepressants, including ketamine and psychedelics. To develop increasingly specific treatments with fewer side effects, we explored the possibility of EEG signatures in mice, which could serve as a cross-species screening tool. There are few studies of the impact of non-sedative doses of rapid antidepressants on EEG in either rodents or humans. Here we hypothesize that EEG features may separate a rapid antidepressant neurosteroid, allopregnanolone, from other GABAA positive modulators, pentobarbital and diazepam. Further, we compared the actions GABA modulators with those of ketamine, an NMDA antagonist and prototype rapid antidepressant. We examined EEG spectra during active exploration at two cortical locations and examined cross-regional and cross-frequency interactions. We found that at comparable doses, the effects of allopregnanolone, despite purported selectivity for certain GABAAR subtypes, was indistinguishable from pentobarbital during active waking exploration. The actions of diazepam had recognizable common features with allopregnanolone and pentobarbital but was also distinct, consistent with subunit selectivity of benzodiazepines. Finally, ketamine exhibited no distinguishing overlap with allopregnanolone in the parameters examined. Our results suggest that rapid antidepressants with different molecular substrates may remain separated at the level of large-scale ensemble activity, but the studies leave open the possibility of commonalities in more discrete circuits and/or in the context of a dysfunctional brain.

Neuroinflammation is associated with the development of hypoactive delirium, which results in poor clinical outcomes. Drugs effective against hypoactive sur have not yet been established. Yokukansan has an anti-neuroinflammatory effect, making it potentially effective against hypoactive delirium. This study aimed to examine the effect of Yokukansan on the pentobarbital-induced loss of righting reflex duration extended with lipopolysaccharide (LPS)-induced neuroinflammation and diazepam-induced gamma-aminobutyric acid receptor stimulation in a mouse model. The active ingredients in Yokukansan and its anti-neuroinflammatory effect on the hippocampus were also investigated. Furthermore, we examined the in vitro anti-inflammatory effects of Yokukansan on LPS-stimulated BV2 cells, a murine microglial cell line. Findings revealed that treatment with Yokukansan significantly decreased the duration of pentobarbital-induced loss of righting reflex by attenuating the LPS-induced increase in interleukin-6 and tumor necrosis factor-alpha levels in the hippocampus. Moreover, treatment with Yokukansan significantly decreased the number of ionized calcium-binding adapter molecule-1-positive cells in the hippocampal dentate gyrus after 24 h of LPS administration. In addition, glycyrrhizic acid, an active ingredient in Yokukansan, partially decreased the duration of pentobarbital-induced loss of righting reflex. Treatment with Yokukansan also suppressed the expression of inducible nitric oxide, interleukin-6, and tumor necrosis factor mRNA in LPS-stimulated BV2 cells. Thus, these findings suggest that Yokukansan and glycyrrhizic acid may be effective therapeutic agents for treating neuroinflammation-induced hypoactive delirium. Graphical abstract

The ethical issues surrounding sacrifice methods in animal experimentation have garnered increasing attention, making immobilization and sedation an integral part of the sacrifice process for experimental animals. Currently, internationally recognized general sacrifice methods for commonly used laboratory animals have been established, but there remains significant controversy over the sacrifice methods for turtles. To explore the effectiveness of various immobilization methods and their impact on stress-related indicators in turtles, this study used red-eared sliders ( Trachemys scripta elegans ) as subjects. Our findings indicate that − 20 °C cryoimmobilization achieved immobilization after 45 min, with individual deaths occurring after 75 min or more. When the dose of pentobarbital sodium for intraperitoneal/intramuscular injection reaches 0.02 mg/g, an anesthetic effect can be achieved, but exceeding 0.03 mg/g may lead to the death of the individual. While for ether immobilization, 60 min can achieve the anesthetic effect, a duration of over 120 min may cause the risk of hypoxia. With the exception of the ether inhalation immobilization 90min group, the 0.03 mg/g pentobarbital sodium intraperitoneal injection group and − 20 °C cryoimmobilization 45 and 60 min groups, no significant differences in brain, heart, and liver function indicators were observed between the remaining groups and decapitation sacrifice group. The administration of pentobarbital sodium injection for immobilization is relatively simple, with a short induction period, a long immobilization duration, and good anesthetic effects. In summary, pentobarbital sodium injection immobilization is a worth considering method, with an effective injection dose of 0.02 mg/g. Intramuscular injection has a shorter induction period compared to intraperitoneal injection. The research results can provide a reference for the choice of sacrifice methods for turtles.