Assessment of thiamylal sodium as a euthanasia drug in mice.

Euthanasia agents should rapidly induce death and loss of consciousness without causing pain or distress. Various methods exist for the euthanasia of laboratory animals, and injectable anesthetics, particularly barbiturate derivatives, are widely used due to the rapid onset of unconsciousness induced by these agents. Moreover, pharmaceutical-grade drugs should be used to eliminate undesirable side effects as much as possible. However, in Japan, the sale of pharmaceutical-grade pentobarbital sodium (PB) ended in 2019, and that of secobarbital sodium (SB) ended in 2023, leading to a demand for new pharmaceutical-grade injectable euthanasia drugs. This study evaluates thiamylal sodium (TM), a barbiturate derivative that is available domestically, as a euthanasia agent for mice. The results showed that when administered at dosages of 200 mg/kg or more, TM exhibited effects equivalent to those of PB and SB. In addition, the impact of TM administration on hematological characteristics was examined. In female mice administered TM, decreased blood chloride and calcium levels and increased aspartate aminotransferase and alanine aminotransferase levels, which are markers of liver damage, were observed. These findings suggest that high concentrations of TM may affect renal and liver function. This study revealed that TM is effective as a euthanasia agent at dosages of 200 mg/kg or more. However, considering the potential risks of renal and liver damage due to TM administration, it may be preferable to use alternative euthanasia drugs when these risks could affect the objectives or outcomes of the research.

Isoflurane and Pentobarbital Anesthesia for Pulmonary Studies Requiring Prolonged Mechanical Ventilation in Mice.

Mechanical ventilation can be used in mice to support high-risk anesthesia or to create clinically relevant, intensive care models. However, the choice of anesthetic and inspired oxygen concentration for prolonged procedures may affect basic physiology and lung inflammation. To characterize the effects of anesthetics and oxygen concentration in mice experiencing mechanical ventilation, mice were anesthetized with either isoflurane or pentobarbital for tracheostomy followed by mechanical ventilation with either 100% or 21% oxygen. Body temperature, oxygen saturation, and pulse rate were monitored continuously. After 6 h, mice were euthanized for collection of blood and bronchoalveolar lavage fluid for evaluation of biomarkers of inflammation and lung injury, including cell counts and cytokine levels. Overall, both isoflurane and pentobarbital provided suitable anesthesia for 6 h of mechanical ventilation with either 21% or 100% oxygen. We found no differences in lung inflammation biomarkers attributable to either oxygen concentration or the anesthetic. However, the combination of pentobarbital and 100% oxygen resulted in a significantly higher concentration of a biomarker for lung epithelial cell injury. This study demonstrates that the combination of anesthetic agent, mechanical ventilation, and inspired oxygen concentrations can alter vital signs and lung injury biomarkers during prolonged procedures. Their combined impact may influence model development and the interpretation of research results, warranting the need for preliminary evaluation to establish the baseline effects.

Immunoassay testing for barbiturates using alternative matrices in postmortem tissues from cats and dogs.

The barbiturate drug pentobarbital is commonly used by veterinarians for the euthanasia of domestic animals. During the veterinary forensic autopsy, it is sometimes necessary to determine whether the animal was chemically euthanized with pentobarbital. The use of a human immunochromatographic test for barbiturate screening utilizing dog or cat urine has been previously validated; however, the use of alternative matrices for this purpose is yet to be explored when urine is not available. Postmortem heart, liver, spleen, skeletal muscle, blood and/or urine samples from 20 dogs and 26 cats with a reported chemical euthanasia status were processed using two different methods, bead homogenization and sonication, and screened for barbiturates using a human immunochromatographic test. There was 100% agreement of the immunochromatographic test results using the sonication method with the reported euthanasia status of both dogs and cats. Using the bead homogenization method, agreement with the reported euthanasia status was 93.3% and 96.7% for dogs and cats, respectively, due to invalid test results from four dog and two cat samples. A subset of liver samples (10 canine and 10 feline) was analyzed via gas chromatography-mass spectrometry, and there was 100% agreement between the immunochromatographic test results and gas chromatography-mass spectrometry results for both cats and dogs. Overall, our results support the use of a variety of alternative matrices for barbiturate screening in cats and dogs.

Effect of Repeated Application of Flos Daturae on Sedative and Hypnotic in Mice.

Insomnia is one of the most common sleep-related diseases. In traditional Chinese medicine, Flos daturae has been used as a traditional herbal totreatment of sizens of diseases. The research objective was to investigate the sedative and hypnotic effects of Flos Daturae. Kunming mice were divided into control group, Estazolam (positive drug, 0.0005 g/kg) group and Flos Daturae groups (0.01, 0.02, 0.04g/kg) with random, ig once a day for 7 days. The central sedative effect of flos Daturae on the spontaneous activity of mice was observed using the locomotive activity test, and the hypnotic effect of Flos Daturae was observed in mice using the direct sleep test and the sleep latency with synergistic supra-and sub-threshold doses of pentobarbital sodium. Flos Daturae (0.04g/kg) significantly inhibited mice locomotive activity (P<0.05) and had no direct sleeping effect (P>0.05), increased the number rate of sleep (P<0.05), and significantly shortening sleep latency (P<0.05), enhanced pentobarbital sodium-induced sleep. Flos Daturae possesses have sedative-hypnotic properties.

Pentobarbital coma for management of intracranial hypertension following traumatic brain injury: Lack of early response to treatment portends poor outcomes.

INTRODUCTION: 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. METHODS: 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. RESULTS: 25 patients were identified, and the majority were male (n â€‹= â€‹23, 92%) with an average age of 30.0 years â€‹± â€‹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 â€‹± â€‹11.5 and intensive care stay was 16.9 â€‹± â€‹10.8 days. 9 (36.0%) patients survived to hospital discharge. Mean follow-up time in months was 36.9 â€‹± â€‹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% â€‹± â€‹10% vs. GCS <15 or dead: 68% â€‹± â€‹22%, P â€‹= â€‹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. CONCLUSION: 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.

Survey of veterinarians who use pentobarbital for euthanasia suggests knowledge gaps regarding animal disposal.

OBJECTIVE: To assess (1) veterinarians' knowledge and practices regarding disposal of euthanized animals, (2) the extent to which veterinarians communicate with their clients about potential risks of rendering pentobarbital-euthanized animals, and (3) the extent to which veterinarians communicate potential relay toxicosis and environmental risks of pentobarbital-euthanized animals to clients. SAMPLE: A stratified random sample of AVMA members. METHODS: Over a 3-week period in early 2021, 16,831 of the AVMA's 99,500 members were surveyed, with 2,093 responses (a 12% response rate). Respondents were assigned to 1 of 3 categories on the basis of their answers: veterinarians euthanizing only food-producing species, veterinarians euthanizing only non-food-producing species, and veterinarians euthanizing both food-producing and non-food-producing species (ie, veterinarians euthanizing mixed species). RESULTS: Veterinarians responding to this survey appeared to be aware of the major methods of animal disposal, and about 89% reported communicating the method of euthanasia with clients to help ensure appropriate animal disposal. However, the need for additional education on local, state, and federal laws and rendering, as well as on risks of relay toxicosis including wildlife predation and environmental impacts, was reported. CLINICAL RELEVANCE: Survey results identified gaps in veterinarians' knowledge regarding animal disposal following pentobarbital euthanasia. Further education on this topic may be beneficial, particularly for early- and midcareer veterinarians who euthanize non-food-producing species and for veterinarians who euthanize mixed species in urban and suburban communities.

Securing lethal means for suicide: a focus group study exploring perceptions and barriers among practicing veterinarians.

OBJECTIVE: Veterinarians are at higher risk for suicide than the general population, and 1 reason for this may be veterinarians' access to and knowledge of pentobarbital-a common suicide method in this population. One possible approach to reducing suicide risk is means safety. This study examined the acceptability and feasibility of means safety protocols in the veterinary workplace. SAMPLE: 43 veterinarians from a mix of specialty areas aged 26 to 53 years, currently practicing in the United States, recruited via social media and listservs. METHODS: Participants completed a 60- to 90-minute focus group with pre- and post-test surveys. Focus group content was qualitatively analyzed. RESULTS: Survey responses indicated that that 30% (n = 13) of veteriarians reported storing their pentobarbital unlocked at least part of the time. During focus group discussion, participants perceived work/life balance or being overwhelmed as the most common suicide risk factor in veterinarians, with normalizing mental health emerging as a primary way to improve mental health in veterinarians. Additionally, adding an extra lockbox for pentobarbital/firearms emerged as the most acceptable and feasible means safety method. Finally, at post-test, veterinarians increased in willingness to implement storage protocol changes (P = .02) and were more likely to endorse concern about a coworker's suicide risk than concern about their own suicide risk (P < .01) as a reason to change pentobarbital storage methods. CLINICAL RELEVANCE: Results from this study will inform public messaging campaigns and policy changes for pentobarbital storage and suicide prevention efforts in the veterinary workplace at the individual and organizational level.

Selecting goats for juniper consumption did not improve their liver Phase I detoxification.

A sodium pentobarbital-induced sleep time study was conducted on 15 adult intact male Boer × Spanish goats selected for high (J+, n = 7) or low (J-, n = 8) juniper consumption (estimated breeding values of 13.1 ± 1.0 and -14.3 ± 0.8, respectively; mean ± standard deviation). Pentobarbital sleep time is an in vivo assay of Phase I hepatic metabolism that can be induced by exposure to barbiturates and monoterpenes. Monoterpenes and pentobarbital are initially oxidized by this pathway; thus, we hypothesized that J+ goats would have shorter sleep times than J- goats. Time to the righting reflex after pentobarbital-induced sleep was measured in all goats following a minimum period of 21 d on three different diets: 1) grazing juniper-infested rangeland (JIR), 2) forage diet with no monoterpenes (M0), and 3) forage diet with 8 g/kg added monoterpenes from camphor, sabinene, and α-pinene in a w/w ratio of 5:4:1 (M+). Fecal samples from the JIR diet were analyzed with near-infrared spectroscopy for the percentage of juniper in the diet. Fecal samples from the JIR and M+ diets were analyzed for camphor and sabinene concentrations. The percentage of juniper in the diet of J+ goats grazing rangelands was greater (P = 0.001) than J- goats (31.1% and 18.6%, respectively). Sleep time did not differ between selection lines (P = 0.36). However, the sleep time of the goats fed M+ diet was 26 min shorter (P < 0.001) than JIR or M0 diets, which were equal. The concentration of camphor and sabinene in the feces was higher (P < 0.001) for goats on the M+ diet than on the JIR diet. There were no differences between selection lines in the serum enzymes indicative of liver disease (aspartate aminotransferase, bilirubin, gamma-glutamyl transferase, and glutamate dehydrogenase; P > 0.12), and all treatment means were within the reference interval. Selecting goats for juniper consumption did not affect the Phase I detoxification system, and several alternative hypotheses for differences in juniper consumption between J+ and J- goats are discussed.

Juniper is an encroaching woody plant with high levels of essential oils and condensed tannins that can limit its consumption by herbivores. Goats were divergently selected for 15 yr to increase or decrease their juniper consumption. This study was conducted to determine if a physiological pathway for metabolism of essential oils differed between high and low juniper-consuming goat lines. The metabolic pathway for the elimination of essential oils is similar to that of the barbiturate pentobarbital. A pentobarbital-induced sleep time was used to detect differences in detoxification rates between the divergent goat lines selectively bred for either a high or low percentage of juniper in their diet. We hypothesized that high juniper-consuming goats would have shorter sleep times, indicating their detoxification pathway was more active. However, there was no difference between these lines. Additionally, there were no differences between the selection lines in blood metabolites that indicate liver tissue damage or liver weights. Therefore, higher dietary juniper preference may be associated with other detoxification mechanisms, may not be limited by essential oils, or may be a socially facilitated learned behavior.

Sleep-promoting activity of amylase-treated Ashwagandha (Withania somnifera L. Dunal) root extract via GABA receptors.

Ashwagandha (Withania somnifera L. Dunal), an Indian medicinal plant that has been used for centuries to treat insomnia, exhibits a variety of biological activities, such as improving cognitive function, immunity and anxiety. In this study, the effect of enzyme-treated Ashwagandha root extract (EA) and on sleep was evaluated using rodent models. Starch contained in the Ashwagandha root extract was removed by amylase treatment to prepare EA. To evaluate the sleep-promoting activity of EA, a pentobarbital-induced sleep test and electroencephalogram analysis were performed. In addition, the sleep-promoting mechanism of EA was elucidated by analyzing the expression of sleep-related receptors. In the pentobarbital-induced sleep test, EA dose-dependently increased sleep duration. Additionally, electroencephalogram analysis revealed that EA significantly increased δ-wave and non-rapid eye movement sleep times, which are involved in deep sleep, thereby improving sleep quality and quantity. EA also effectively relieved caffeine-induced insomnia symptoms. Furthermore, the γ-aminobutyric acid (GABA) content in the brain and mRNA and protein expression of GABAA, GABAB1, and serotonin receptors were significantly increased by EA compared to the normal group. In particular, EA showed sleep-promoting activity by binding to various GABAA receptor sites. Collectively, EA exhibited sleep-promoting activity through the GABAergic system and may be used as a functional material to improve sleep deprivation.

Pentobarbital intoxication as a potential underlying cause for electrocution in a bearded vulture.

A bearded vulture (Gypaetus barbatus) found dead in northern Spain presented external lesions consistent with electrocution as the cause of death. During forensic examination, macroscopic lesions suggested potential comorbidity, so samples were collected for molecular and toxicological analyses. Gastric content and liver were analysed for toxic substances, and pentobarbital (a common pharmaceutical used for euthanasia in domestic animals) was detected at a concentration of 37.3 and 0.05 µg/g, respectively. Other toxicological, viral and endoparasite analyses (avian malaria, avian influenza and flaviviruses) were negative. Thus, although the cause of death was electrocution, pentobarbital intoxication likely impaired the equilibrium and reflexes of the individual, possibly causing the bird to contact energized wires that it would not have otherwise. These results underline the importance of comprehensive analysis of forensic cases of wildlife deaths and reveal barbiturate poisoning as an additional threat for the conservation of the bearded vulture in Europe.

A Population Pharmacokinetic Model of Pentobarbital for Children with Status Epilepticus and Severe Traumatic Brain Injury.

BACKGROUND: Pentobarbital pharmacokinetics (PK) remain elusive and the therapeutic windows narrow. Administration is frequent in critically ill children with refractory status epilepticus (SE) and severe traumatic brain injury (sTBI). OBJECTIVES: To investigate pentobarbital PK in SE and sTBI patients admitted to the paediatric intensive care unit (PICU) with population-based PK (PopPK) modelling and dosing simulations. METHODS: Develop a PopPK model with non-linear mixed-effects modelling (NONMEM®) with retrospective data (n = 36; median age 1.3 years; median weight 10 kg; 178 blood samples) treated with continuous intravenous pentobarbital. An independent dataset was used for external validation (n = 9). Dosing simulations with the validated model evaluated dosing regimens. RESULTS: A one-compartment PK model with allometrically scaled weight on clearance (CL; 0.75) and volume of distribution (Vd; 1) captured data well. Typical CL and Vd values were 3.59 L/70 kg/h and 142 L/70 kg, respectively. Elevated creatinine and C-reactive protein (CRP) levels significantly correlated to decreased CL, explaining 84% of inter-patient variability, and were incorporated in the final model. External validation using stratified visual predictive checks showed good results. Simulations demonstrated patients with elevated serum creatinine and CRP failed to achieve steady state yet progressed to toxic levels with current dosing regimens. CONCLUSIONS: The one-compartment PK model of intravenous pentobarbital described data well whereby serum creatinine and CRP significantly correlated with pentobarbital CL. Dosing simulations formulated adjusted dosing advice in patients with elevated creatinine and/or CRP. Prospective PK studies with pharmacodynamic endpoints, are imperative to optimise pentobarbital dosing in terms of safety and clinical efficacy in critically ill children.

Transmucosal pentobarbital is a successful euthanasia method in pond slider turtles (Trachemys scripta).

OBJECTIVE: To determine the ability of transmucosal euthanasia solution to induce euthanasia in pond slider turtles (Trachemys scripta). ANIMALS: 16 pond slider turtles (T. scripta). PROCEDURES: Pentobarbital 100 mg/kg was delivered through esophageal gavage (n = 8) or cloacal administration (8). The presence of voluntary movement, heart rate (HR), respiratory rate (RR), palpebral reflex, corneal reflex, and response to noxious stimuli were recorded until death, confirmed via the absence of reflexes, movement, heartbeat, and absence of cardiac electrical activity. RESULTS: No signs of irritation were observed in any turtles. Leakage after administration occurred in 75% (6/8) of the cloacal group, including 2 turtles with marked leakage or expulsion. Two of eight turtles in the cloacal group regained movement and required euthanasia by a standard method and 1 turtle in the oral group was excluded from further analysis due to a miscalculated dose. The remaining 13 turtles (7/8 oral, 6/8 cloacal) had cessation of a heartbeat at a median of 18 hours (range = 6 to 26 hours) with respiratory arrest occurring within 15 minutes. The median time to loss of corneal reflex was 45 minutes (range = 15 minutes to 4 hours). Time-to-loss of parameters was similar between oral and cloacal routes, respectively. CLINICAL RELEVANCE: Transmucosally administered pentobarbital via the oral and cloacal routes both result in euthanasia within approximately 24 hours. Given that 25% of the turtles in the cloacal group required a secondary method of euthanasia, the oral route is a preferred route to induce euthanasia in pond turtles.

Effect of pentobarbital as a euthanasia agent on equine in vitro embryo production.

Postmortem and pre-euthanasia oocyte retrieval provides the last opportunity to preserve the genetic material in mares. Pentobarbital (PB) is the most common euthanasia agent; however, its effect on the developmental competence of oocytes has not been determined. Here, we evaluated the concentration of PB in equine follicular fluid (FF) and investigated its effect on the developmental competence of oocytes using a bovine IVF model to overcome the low availability of equine oocytes. The concentration of PB was measured by gas-chromatography/mass-spectrometry in FF collected from mare ovaries immediately after euthanasia (n = 10), 24 h post-euthanasia (n = 10), and from the ovaries collected by ovariectomy (negative control; n = 10). The serum concentration of PB was also evaluated as a positive control. PB was detected in all FF samples with an average concentration of 56.5 µg/ml. Next, bovine cumulus-oocyte complexes (COC) were held in holding media with PB for 6 h at 60 µg/ml (H60, n = 196), 164 µg/ml (H164, n = 215) or without PB (control; n = 212). After holding, the oocytes were matured and fertilized in vitro, followed by in vitro culture to the blastocyst stage. The cumulus expansion grade, cleavage rate, blastocyst rate, embryo kinetic rate and the blastocyst cell numbers were compared among the experimental groups of bovine COC. Higher rates of Grade 1 cumulus expansion were found in controls (54%, 32-76%; median, min-max) in comparison to H60 and H164 (24%,11-33% and 13%, 8-44%; P < 0.001). The cleavage rate was higher in the controls than in H164 (64% vs. 44%; P < 0.01). Blastocyst rates (blastocyst/cleaved oocytes) and total cell number were not different among the groups (control 29%, H60 25%, and H164 24%). In a preliminary study, equine oocytes (n = 28) were exposed to PB in vitro for 6 h followed by intracytoplasmic sperm injection (ICSI) and in vitro embryo production. Exposed oocytes showed a numerically lower maturation rate (43% Vs 52%; P > 0.05) in comparison to the laboratory-established rate during the same timepoints. Overall, we showed that PB reaches the FF immediately after euthanasia, exposing oocytes to this drug. This exposure affected cumulus expansion and cleavage rates in a bovine model, suggesting initial damage caused by PB that may not completely impede the formation of embryos, although lower overall embryo numbers might be obtained.

Glycyrrhizae Radix suppresses lipopolysaccharide- and diazepam-induced nerve inflammation in the hippocampus, and contracts the duration of pentobarbital- induced loss of righting reflex in a mouse model.

Nerve inflammation is linked to the development of various neurological disorders. This study aimed to examine whether Glycyrrhizae Radix effectively influences the duration of the pentobarbital-induced loss of righting reflex, which may increase in a mouse model of lipopolysaccharide (LPS)-induced nerve inflammation and diazepam-induced γ-aminobutyric acid receptor hypersensitivity. Furthermore, we examined the anti-inflammatory effects of Glycyrrhizae Radix extract on LPS-stimulated BV2 microglial cells, in vitro. Treatment with Glycyrrhizae Radix significantly decreased the duration of pentobarbital-induced loss of righting reflex in the mouse model. Furthermore, treatment with Glycyrrhizae Radix significantly attenuated the LPS-induced increases in interleukin-1ß, interleukin-6, and tumor necrosis factor-alpha at the mRNA level, and it significantly reduced the number of ionized calcium-binding adapter molecule-1-positive cells in the hippocampal dentate gyrus 24 h after LPS treatment. Treatment with Glycyrrhizae Radix also suppressed the release of nitric oxide, interleukin-1ß, interleukin-6, and tumor necrosis factor protein in culture supernatants of LPS-stimulated BV2 cells. In addition, glycyrrhizic acid and liquiritin, active ingredients of Glycyrrhizae Radix extract, reduced the duration of pentobarbital-induced loss of righting reflex. These findings suggest that Glycyrrhizae Radix, as well as its active ingredients, glycyrrhizic acid and liquiritin, may be effective therapeutic agents for the treatment of nerve inflammation-induced neurological disorders.

Comparison of Low and High Doses of Pentobarbital or Thiopental for Euthanasia of Isoflurane-anesthetized Pigs.

Barbiturate overdose is a common method for euthanizing pigs. However, barbiturates can cause tissue damage and may affect experimental results, so the minimal dose should be used. The minimal dose of barbiturate for euthanasia in pigs under isoflurane anesthesia has not yet been determined. In this study, we compared the effect of low and high doses of 2 barbiturates (pentobarbital, 30 or 60 mg/kg; thiopental, 20 and 40 mg/kg) on hemodynamic parameters and time to cardiac arrest in female pigs maintained on isoflurane. Acute decreases in blood pressure and end-tidal CO2 occurred in all pigs shortly after administration of the barbiturate. However, these changes were not different between either of the high- and low dose groups. Cardiac arrest occurred significantly faster for high dose as compared with low dose thiopental groups, but this parameter was different between the 2 pentobarbital groups. The bispectral index fell immediately after dosing, in all pigs, but no significant differences were observed in the time needed to achieve 0 for the high or low-doses of either drug. In pigs maintained on isoflurane, a low dose of barbiturates is adequate for euthanasia and may result in less tissue damage.

Chrysanthemum morifolium and Its Bioactive Substance Enhanced the Sleep Quality in Rodent Models via Cl- Channel Activation.

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, ß2-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.