Ketamine administration causes cognitive impairment by destroying the circulation function of the glymphatic system.

BACKGROUND: Ketamine, as a non-competitive antagonist of N-methyl-D-aspartate (NMDA) receptors, was originally used in general anesthesia. Epidemiological data show that ketamine has become one of the most commonly abused drugs in China. Ketamine administration might cause cognitive impairment; however, its molecular mechanism remains unclear. The glymphatic system is a lymphoid system that plays a key role in metabolic waste removal and cognitive regulation in the central nervous system. METHODS: Focusing on the glymphatic system, this study evaluated the behavioral performance and circulatory function of the glymphatic system by building a short-term ketamine administration model in mice, and detected the expression levels of the 5-HT2c receptor, ΔFosb, Pten, Akt, and Aqp4 in the hippocampus. Primary astrocytes were cultured to verify the regulatory relationships among related indexes using a 5-HT2c receptor antagonist, a 5-HT2c receptor short interfering RNA (siRNA), and a ΔFosb siRNA. RESULTS: Ketamine administration induced ΔFosb accumulation by increasing 5-HT2c receptor expression in mouse hippocampal astrocytes and primary astrocytes. ΔFosb acted as a transcription factor to recognize the AATGATTAAT bases in the 5' regulatory region of the Aqp4 gene (-1096 bp to -1087 bp), which inhibited Aqp4 expression, thus causing the circulatory dysfunction of the glymphatic system, leading to cognitive impairment. CONCLUSIONS: Although this regulatory mechanism does not involve the Pten/Akt pathway, this study revealed a new mechanism of ketamine-induced cognitive impairment in non-neuronal systems, and provided a theoretical basis for the safety of clinical treatment and the effectiveness of withdrawal.

Ketamine induces insomnia-like symptom of zebrafish at environmentally relevant concentrations by mediating GABAergic synapse.

Although the stimulative effects on the normal behaviors of fish posed by ketamine (KET) were well-studied, the adverse effects on the behavioral functions induced by KET at nighttime were unknown. Here, we used zebrafish larvae as a model exposed to KET (10, 50, 100, and 250 ng/L) at environmental levels for 21 days. The behavioral functions at nighttime, morphological changes during exposure stage, and alterations on the associated genes transcriptional levels of fish were determined. The difficultly initiating sleep was found in the fish exposed to KET, while the sleep duration of the animals was at the normal levels in exposure groups. The significant suppressions of the developmentally relevant genes, including bmp2, bmp4, and pth2ra were consistent with the developmental abnormalities of fish found in exposure groups. Moreover, the expression of γ-aminobutyric acid (GABA) receptor increased and melatonin (MTN) receptor decreased while the levels of GABA and MTN remained unchanged after exposure, by gene expression analysis and molecular docking. In addition, the transcriptional expression of apoptotic genes, including tp53, aifm1, and casp6, was significantly upregulated by KET. After a 7-day recovery, the insomnia-like behaviors (shorter sleep duration) were observed in zebrafish from the 250 ng/L-KET group. Accordingly, the adverse outcome pathway framework of KET was constructed by prognostic assessment of zebrafish larvae. This study suggested that the adverse outcomes of KET on the sleep health of organisms at environmentally relevant concentrations should be concerned.

Abdominal surgery under ketamine anesthesia during second trimester impairs hippocampal learning and memory of offspring by regulating dendrite spine remodeling in rats.

Recent evidence showed that general anesthesia produces long-term neurotoxicity and cognitive dysfunction. However, it remains unclear whether maternal non-obstetric surgery under ketamine anesthesia during second trimester causes cognitive impairment in offspring. The present study assigned pregnant rats into three groups: 1) normal control group receiving no anesthesia and no surgery, 2) ketamine group receiving ketamine anesthesia for 2 h on the 14th day of gestation but no surgery, and 3) surgery group receiving abdominal surgery under ketamine anesthesia on the 14th day of gestation. On postnatal day 1, the offspring rats in Ketamine group and surgery group were assigned to receive intra-peritoneal injection of Senegenin (15 mg/kg), once per day for consecutive 14 days. The offspring's spatial perception, anxiety-like behavior, and learning and memory were evaluated. Then the offspring's hippocampal tissues were collected. The offspring of the surgery group were impaired in the spatial perception in the cliff avoidance test and the spatial learning and memory in the Morris water maze test. Accordingly, the activity of histone deacetylases increased, the protein levels of NEDD9, BDNF, p-TrkB, Syn and PSD-95 decreased, and the density of dendritic spines reduced in the hippocampus of the offspring of the surgery group, and such effects were not seen in the offspring of the ketamine group, neither in the offspring of control group. Senegenin alleviated the learning and memory impairment, and increased the protein levels of NEDD9, BDNF, p-TrkB, Syn and PSD-95 and the density of dendritic spines in the offspring of the surgery group. ketamine anesthesia plus surgery during second trimester impairs hippocampus-dependent learning and memory, and the deficits could be rescued by treatment with Senegenin.

Impaired synaptic plasticity and decreased excitability of hippocampal glutamatergic neurons mediated by BDNF downregulation contribute to cognitive dysfunction in mice induced by repeated neonatal exposure to ketamine.

AIM: Repeated exposure to ketamine during the neonatal period in mice leads to cognitive impairments in adulthood. These impairments are likely caused by synaptic plasticity and excitability damage. We investigated the precise role of brain-derived neurotrophic factor (BDNF) in the cognitive impairments induced by repeated ketamine exposure during the neonatal period. METHODS: We evaluated the cognitive function of mice using the Morris water maze test and novel object recognition test. Western blotting and immunofluorescence were used to detect the protein levels of BDNF. Western blotting, Golgi-Cox staining, transmission electron microscopy, and long-term potentiation (LTP) recordings were used to assess synaptic plasticity in the hippocampus. The excitability of neurons was evaluated using c-Fos. In the intervention experiment, pAdeno-CaMKIIα-BDNF-mNeuronGreen was injected into the hippocampal CA1 region of mice to increase the level of BDNF. The excitability of neurons was enhanced using a chemogenetic approach. RESULTS: Our findings suggest that cognitive impairments in mice repeatedly exposed to ketamine during the neonatal period are associated with downregulated BDNF protein level, synaptic plasticity damage, and decreased excitability of glutamatergic neurons in the hippocampal CA1 region. Furthermore, the specific upregulation of BDNF in glutamatergic neurons of the hippocampal CA1 region and the enhancement of excitability can improve impaired synaptic plasticity and cognitive function in mice. CONCLUSION: BDNF downregulation mediates synaptic plasticity and excitability damage, leading to cognitive impairments in adulthood following repeated ketamine exposure during the neonatal period.

Phencyclidine-Like Abuse Liability and Psychosis-Like Neurocognitive Effects of Novel Arylcyclohexylamine Drugs of Abuse in Rodents.

Abuse of novel arylcyclohexylamines (ACX) poses risks for toxicities, including adverse neurocognitive effects. In vivo effects of ring-substituted analogs of phencyclidine (PCP), eticyclidine (PCE), and ketamine are understudied. Adult male National Institutes of Health Swiss mice were used to assess locomotor effects of PCP and its 3-OH, 3-MeO, 3-Cl, and 4-MeO analogs, PCE and its 3-OH and 3-MeO analogs, and ketamine and its deschloro and 2F-deschloro analogs, in comparison with those of methamphetamine (METH), 3,4-methylenedioxymethamphetamine (MDMA), and two benzofuran analogs of MDMA. PCP-like interoceptive effects for all of these ACXs were determined using a food-reinforced drug discrimination procedure in adult male Sprague Dawley rats. A novel operant assay of rule-governed behavior incorporating aspects of attentional set-shifting was used to profile psychosis-like neurocognitive effects of PCP and 3-Cl-PCP in rats, in comparison with cocaine and morphine. PCP-like ACXs were more effective locomotor stimulants than the amphetamines, PCE-like ACXs were as effective as the amphetamines, and ketamine-like ACXs were less effective than the amphetamines. Addition of -Cl, -OH, or -OMe at the 3-position on the aromatic ring did not impact locomotor effectiveness, but addition of -OMe at the 4-position reduced locomotor effectiveness. Lethal effects were induced by drugs with -OH at the 3-position or -OMe at the 3- or 4-position. All novel ACXs substituted at least partially for PCP, and PCP and 3-Cl-PCP elicited dose-dependent psychosis-like neurocognitive deficits in the rule-governed behavior task not observed with cocaine or morphine. Novel ACXs exhibit substantial abuse liability and toxicities not necessarily observed with their parent drugs. SIGNIFICANCE STATEMENT: Novel arylcyclohexylamine analogs of PCP, PCE, and ketamine are appearing on the illicit market, and abuse of these drugs poses risks for toxicities, including adverse neurocognitive effects. These studies demonstrate that the novel ACXs exhibit PCP-like abuse liability in the drug discrimination assay, elicit varied locomotor stimulant and lethal effects in mice, and induce psychosis-like neurocognitive effects in rats.

Proteomic Characteristics of the Prefrontal Cortex and Hippocampus in Mice with Chronic Ketamine-Induced Anxiety and Cognitive Impairment.

Schizophrenia, a complex psychiatric disorder with diverse symptoms, has been linked to ketamine, known for its N-methyl-D-aspartate (NMDA) receptor antagonistic properties. Understanding the distinct roles and mechanisms of ketamine is crucial, especially regarding its induction of schizophrenia-like symptoms. Recent research highlights the impact of ketamine on key brain regions associated with schizophrenia, specifically the prefrontal cortex (PFC) and hippocampus (Hip). This study focused on these regions to explore proteomic changes related to anxiety and cognitive impairment in a chronic ketamine-induced mouse model of schizophrenia. After twelve consecutive days of ketamine administration, brain tissues from these regions were dissected and analyzed. Using tandem mass tag (TMT) labeling quantitative proteomics techniques, 34,797 and 46,740 peptides were identified in PFC and Hip, corresponding to 5,668 and 6,463 proteins, respectively. In the PFC, a total of 113 proteins showed differential expression, primarily associated with the immuno-inflammatory process, calmodulin, postsynaptic density protein, and mitochondrial function. In the Hip, 129 differentially expressed proteins were screened, mainly related to synaptic plasticity proteins and mitochondrial respiratory chain complex-associated proteins. Additionally, we investigated key proteins within the glutamatergic synapse pathway and observed decreased expression levels of phosphorylated CaMKII and CREB. Overall, the study unveiled a significant proteomic signature in the chronic ketamine-induced schizophrenia mouse model, characterized by anxiety and cognitive impairment in both the PFC and Hip, and this comprehensive proteomic dataset may not only enhance our understanding of the molecular mechanisms underlying ketamine-related mental disorders but also offer valuable insights for future disease treatments.

Curcumin-ZnO conjugated nanoparticles confer neuroprotection against ketamine-induced neurotoxicity.

BACKGROUND: Nanotechnology and its application to manipulate herbal compounds to design new neuroprotective agents to manage neurotoxicity has recently increased. Cur-ZnO conjugated nanoparticles were synthesized and used in an experimental model of ketamine-induced neurotoxicity. METHODS: Cur-ZnO conjugated nanoparticles were chemically characterized, and the average crystalline size was determined. Forty-nine adult mice were divided into seven groups of seven animals each. Normal saline was given to control mice (group 1). Ketamine (25 mg/kg) was given to a second group. A third group of mice was given ketamine (25 mg/kg) in combination with curcumin (40 mg/kg), while mice in groups 4, 5, and 6 received ketamine (25 mg/kg) plus Cur-ZnO nanoparticles (10, 20, and 40 mg/kg). Group 7 received only ZnO (5 mg/kg). All doses were ip for 14 days. Hippocampal mitochondrial quadruple complex enzymes, oxidative stress, inflammation, and apoptotic characteristics were assessed. RESULTS: Cur-ZnO nanoparticles and curcumin decreased lipid peroxidation, GSSG content, IL-1ß, TNF-α, and Bax levels while increasing GSH and antioxidant enzymes like GPx, GR, and SOD while increasing Bcl-2 level and mitochondrial quadruple complex enzymes in ketamine treatment groups. CONCLUSION: The neuroprotective properties of Cur-ZnO nanoparticles were efficient in preventing ketamine-induced neurotoxicity in the mouse brain. The nanoparticle form of curcumin (Cur-ZnO) required lower doses to produce neuroprotective effects against ketamine-induced toxicity than conventional curcumin.

Histological Alterations in the Internal Organs of Wistar Han Rats (Rattus norvegicus) Euthanized by Five Different Methods.

Selecting a method of euthanasia is an important step in designing research studies that use animals; euthanasia methods must be humane, cause minimal pain and suffering to the animal, and preserve the tissue architecture of the organs of interest. In this study, we evaluated the histomorphology of the internal organs (lung, spleen, heart, kidney, liver, brain, and adrenal gland) of rats submitted to five different methods of euthanasia, with the goal of determining which protocol caused the least alteration of histomorphology. Twenty adult Wistar Han rats (Rattus norvegicus) were divided into 5 groups of 4 rats each (2 females and 2 males) and were euthanized by CO2 or isoflurane inhalation, sodium thiopental or xylazine plus ketamine overdose, or decapitation. All euthanasia was performed in accordance with published guidelines and local legal require- ments. Necropsy was performed immediately after euthanasia. Specific internal organs were removed and placed in formalin and submitted for routine histologic processing. Histomorphological examination of hematoxylin and eosin-stained tissues revealed circulatory alterations in multiple organs, predominantly congestion in multiple tissues, pulmonary hemorrhage, and hepatic degeneration. The euthanasia methods that induced the most severe alterations were exposure to CO2 and anesthetic overdose with xylazine plus ketamine or sodium thiopental. Euthanasia by overexposure to isoflurane caused less damage, and the alterations were of minimal severity. Decapitation resulted in the lowest incidence of lesions in multiple organs but due its traumatic nature, it caused the highest incidence of pulmonary hemorrhage. In selecting a method of euthanasia, factors to consider are the species of animal, the purpose of the research, and the practical ability to perform the procedure to achieve maximal animal welfare without iatrogenic changes that could compromise the outcome and reproducibility of the study.

A Retrospective Comparison of Electrocardiographic Parameters in Ketamine and Tiletamine-Zolazepam Anesthetized Indian Rhesus Monkeys (Macaca mulatta).

Electrocardiographic evaluation is performed in rhesus monkeys to establish the cardiovascular safety of candidate molecules before progressing to clinical trials. These animals are usually immobilized chemically by ketamine (KTM) and tiletamine-zolazepam (TZ) to obtain a steady-state heart rate and to ensure adequate human safety. The present study aimed to evaluate the effect of these anesthetic regimens on different electrocardiographic parameters. Statistically significant lower HR and higher P-wave duration, RR, QRS, and QT intervals were observed in the KTM-anesthetized group in comparison to TZ-anesthetized animals. No significant changes were noticed in the PR interval and p-wave amplitude. Sex-based significance amongst these parameters was observed in male and female animals of TZ- and KTM-anesthetized groups. Regression analysis of four QTc formulas in TZ-anesthetized rhesus monkeys revealed that QTcNAK (Nakayama) better corrected the QT interval than QTcHAS (Hassimoto), QTcBZT (Bazett), and QTcFRD (Fridericia) formulas. QTcNAK exhibited the least correlation with the RR interval (slope closest to zero and r = .01) and displayed no statistical significance between male and female animals. These data will prove useful in the selection of anesthetic regimens for chemical restraint of rhesus monkeys in nonclinical safety evaluation studies.

Haloperidol alters neurotrophic factors and epigenetic parameters in an animal model of schizophrenia induced by ketamine.

This study aimed to evaluate Haloperidol's (Hal) effects on the behavioral, neurotrophic factors, and epigenetic parameters in an animal model of schizophrenia (SCZ) induced by ketamine (Ket). Injections of Ket or saline were administered intraperitoneal (once a day) between the 1st and 14th days of the experiment. Water or Hal was administered via gavage between the 8th and 14th experimental days. Thirty minutes after the last injection, the animals were subjected to behavioral analysis. The activity of DNA methyltransferase (DNMT), histone deacetylase (HDAC), and histone acetyltransferase and levels of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-3 (NT-3), and glial-derived neurotrophic factor (GDNF) were evaluated in the frontal cortex, hippocampus, and striatum. Ket increased the covered distance and time spent in the central area of the open field, and Hal did not reverse these behavioral alterations. Significant increases in the DNMT and HDAC activities were detected in the frontal cortex and striatum from rats that received Ket, Hal, or a combination thereof. Besides, Hal per se increased the activity of DNMT and HDAC in the hippocampus of rats. Hal per se or the association of Ket plus Hal decreased BDNF, NGF, NT-3, and GDNF, depending on the brain region and treatment regimen. The administration of Hal can alter the levels of neurotrophic factors and the activity of epigenetic enzymes, which can be a factor in the development of effect collateral in SCZ patients. However, the precise mechanisms involved in these alterations are still unclear.

Electroacupuncture attenuates ketamine-induced neuronal injury in the locus coeruleus of rats through modulation of the CAMK II/CREB pathway.

BACKGROUND: Ketamine, despite its efficacy in treating depression, raises concerns regarding safety due to potential abuse, cognitive impairment, and bladder toxicity. Ketamine can affect the locus coeruleus (LC) norepinephrine and attention networks. This study explored the protective effects of electroacupuncture (EA) on the LC of rats exposed to repeated administration of ketamine while investigating the potential role of the Calcium CaM-dependent protein kinase II (CAMK II)/ cAMP response element binding protein (CREB) pathway in mediating EA's impact on ketamine-induced neuronal injury in LC. METHODS: Rats were repeatedly injected intraperitoneally with ketamine hydrochloride (50 mg/kg) once daily for seven days. Subsequently, EA was performed at the acupoints "Zusanli" (ST36) and "Sanyinjiao" (SP-6) once daily following ketamine administration. The Morris water maze test was employed to assess behavioral changes in the rats. Neuronal injury was examined using Nissl staining, and the expression of CAMK II, CREB, and phospho-CREB (p-CREB) was evaluated through immunohistochemistry and western blotting. RESULTS: EA mitigated the cognitive and exploratory impairments and attenuated neuronal injury in the LC induced by repeated administration of ketamine. The expression of CAMK II and p-CREB proteins in the LC increased following 7 days of ketamine administration. However, EA treatment led to a downregulation of CAMK II and p-CREB expression. CONCLUSION: Repeated administration of ketamine in male rats can lead to neuronal injury and neurobehavioral dysfunction. However, EA was found to ameliorate neurodegeneration in the LC and enhance neurobehavioral symptoms. This therapeutic effect of EA may be attributed to its modulation of the CAMKII/CREB pathway, thereby mitigating the aforementioned adverse effects.

Resveratrol plays neuroprotective role on ketamine-induced schizophrenia-like behaviors and oxidative damage in mice.

This study aimed to determine effects of the resveratrol on ketamine-induced schizophrenia-like behaviors and oxidative damage in mice. Twenty-four male mice were allocated into four experimental groups as control, ketamine (20 mg/kg), resveratrol (80 mg/kg) and co-administration of the ketamine (20 mg/kg) + resveratrol (80 mg/kg). Mice were received resveratrol for 30 days and ketamine was used for an animal model of schizophrenia and was injected from days 16 to 30 of the study. After the drug administration was finished, schizophrenia-like behaviors were evaluated using object recognition test, tail suspension test, forced swimming test and open field test and brain malondialdehyde, glutathione peroxidase, superoxide dismutase and catalase levels were determined. According to the results, ketamine treatment significantly decreased body weight and pretreatment with resveratrol elevated body weight compared to ketamine group (P < 0.05). Ketamine treatment significantly decreased number of the cross in open field test and pretreatment with resveratrol improved i (P < 0.05). Immobility time in tail suspension and forced swimming tests increased in mice treated with ketamine (P < 0.05). Pretreatment with resveratrol diminished immobility time compared to ketamine group (P < 0.05). Ketamine significantly decreased memory deficits while pretreatment with resveratrol significantly reduced the memory deficits induced by ketamine (P < 0.05). Brain MDA increased in both cortical and sub-cortical area in ketamine treated mice while pretreatment with resveratrol decreased ketamine-induced elevation in MDA (P < 0.05). Ketamine significantly decreased brain SOD, GPx and CAT levels while pretreatment with resveratrol improved SOD, GPx and CAT levels (P < 0.05). Findings suggested resveratrol has neuroprotective effects against ketamine-induced behavioral deficits and oxidative damages.

Cellular consequences triggered by ketamine on exposure to human glioblastoma epithelial (LN-229) cells.

Ketamine is generally a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist that interrelates with various other receptors, contributing to a wide range of actions. They are mainly approved as a general anesthetic, but a low dose of ketamine is applied for pain management, depression, and as analgesics. However, there is a significant concern regarding the long-term usage as antidepressants and as an abused drug. The study mainly aims to exhibit the possible long-term side effects of ketamine as an antidepressant and in recreational users. The study explores the in vitro cytotoxicity revealed on LN-229 cells in a dose-dependent manner. According to the cell viability assays, there is a dose-dependent response toward ketamine. Morphological and nuclear integrity was changed on exposure and assessed using Giemsa, Rhodamine phalloidin, 4',6-diamidino-2-phenylindole (DAPI), and Acridine orange staining. The apoptotic cell death marked by nuclear condensation, Lactate dehydrogenase leakage, pro-inflammatory cytokine (interleukin [IL]-ß) release, and inhibition of cell migration was observed. The study highlights the importance of nonanesthetic usage of ketamine, which can lead to severe adverse side effects on long-term exposure rather than a single exposure as an anesthetic agent.

Low-Dose Ketamine-Induced Deficits in Arbitrary Visuomotor Mapping in Monkeys.

Ketamine, an NMDA antagonist, is widely used in clinical settings. Recently, low-dose ketamine has gained attention because of its promising role as a rapid antidepressant. However, the effects of low-dose ketamine on brain function, particularly higher cognitive functions of primate brains, are not fully understood. In this study, we used two macaques as subjects and found that acute low-dose ketamine administration significantly impaired the ability for arbitrary visuomotor mapping (AVM), a form of associative learning (AL) essential for flexible behaviors, including executions of learned stimuli-response contingency or learning of new contingencies. We conducted in-depth analyses and identified intrinsic characteristics of these ketamine-induced functional deficits, including lowered accuracy, prolonged time for planning and movement execution, increased tendency to make errors when visual cues are changed from trial to trial, and stronger impact on combining associative learning and another key higher cognitive function, working memory (WM). Our results shed new light on how associative learning relies on the NMDA-mediated synaptic transmission of the brain and contribute to a better understanding of the potential acute side effects of low-dose ketamine on cognition, which can help facilitate its safe usage in medical practice.

The effects of ketamine on viability, primary DNA damage, and oxidative stress parameters in HepG2 and SH-SY5Y cells.

Ketamine is a dissociative anaesthetic used to induce general anaesthesia in humans and laboratory animals. Due to its hallucinogenic and dissociative effects, it is also used as a recreational drug. Anaesthetic agents can cause toxic effects at the cellular level and affect cell survival, induce DNA damage, and cause oxidant/antioxidant imbalance. The aim of this study was to explore these possible adverse effects of ketamine on hepatocellular HepG2 and neuroblastoma SH-SY5Y cells after 24-hour exposure to a concentration range covering concentrations used in analgesia, drug abuse, and anaesthesia (0.39, 1.56, and 6.25 µmol/L, respectively). At these concentrations ketamine had relatively low toxic outcomes, as it lowered HepG2 and SH-SY5Y cell viability up to 30 %, and low, potentially repairable DNA damage. Interestingly, the levels of reactive oxygen species (ROS), malondialdehyde (MDA), and glutathione (GSH) remained unchanged in both cell lines. On the other hand, oxidative stress markers [superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT)] pointed to ketamine-induced oxidant/antioxidant imbalance.

NRX-101, a Rapid-Acting Anti-Depressant, Does Not Cause Neurotoxicity Following Ketamine Administration in Preclinical Models.

Agents that act at the N-methyl-D-aspartate receptor (NMDAR), such as ketamine, have gained increasing attention as rapid-acting antidepressants; however, their use has been limited by potential neurotoxicity. Recent FDA guidance requires a demonstration of safety on histologic parameters prior to the initiation of human studies. D-cycloserine (DCS) is a partial NMDA agonist that, along with lurasidone, is being investigated as a treatment for depression. The current study was designed to investigate the neurologic safety profile of DCS. To this end, female Sprague Dawley rats (n = 106) were randomly divided into 8 study groups. Ketamine was administered via tail vein infusion. DCS and lurasidone were administered via oral gavage in escalating doses to a maximum of 2000 mg/kg DCS. To ascertain toxicity, dose escalation with 3 different doses of D-cycloserine/lurasidone was given in combination with ketamine. MK-801, a known neurotoxic NMDA antagonist, was administered as a positive control. Brain tissue was sectioned and stained with H&E, silver, and Fluoro-Jade B stains. No fatalities were observed in any group. No microscopic abnormalities were found in the brain of animal subjects given ketamine, ketamine followed by DCS/lurasidone, or DCS/lurasidone alone. Neuronal necrosis, as expected, was seen in the MK-801 (positive control) group. We conclude that NRX-101, a fixed-dose combination of DCS/lurasidone, when administered with or without prior infusion of IV ketamine was tolerated and did not induce neurotoxicity, even at supratherapeutic doses of DCS.

Longitudinal effects of ketamine on cell proliferation and death in the CNS of zebrafish.

Zebrafish is known for its widespread neurogenesis and regenerative capacity, as well as several biological advantages, which turned it into a relevant animal model in several areas of research, namely in toxicological studies. Ketamine is a well-known anesthetic used both in human as well as veterinary medicine, due to its safety, short duration and unique mode of action. However, ketamine administration is associated with neurotoxic effects and neuronal death, which renders its use on pediatric medicine problematic. Thus, the evaluation of ketamine effects administration at early stages of neurogenesis is of pivotal importance. The 1-41-4 somites stage of zebrafish embryo development corresponds to the beginning of segmentation and formation of neural tube. In this species, as well as in other vertebrates, longitudinal studies are scarce, and the evaluation of ketamine long-term effects in adults is poorly understood. This study aimed to assess the effects of ketamine administration at the 1-4 somites stage, both in subanesthetic and anesthetic concentrations, in brain cellular proliferation, pluripotency and death mechanisms in place during early and adult neurogenesis. For that purpose, embryos at the 1-4 somites stage (10.5 h post fertilization - hpf) were distributed into study groups and exposed for 20 min to ketamine concentrations at 0.2/0.8 mg/mL. Animals were grown until defined check points, namely 50 hpf, 144 hpf and 7 months adults. The assessment of the expression and distribution patterns of proliferating cell nuclear antigen (PCNA), of sex-determining region Y-box 2 (Sox 2), apoptosis-inducing factor (AIF) and microtubule-associated protein 1 light chain 3 (LC3) was performed by Western-blot and immunohistochemistry. The results evidenced the main alterations in 144 hpf larvae, namely in autophagy and in cellular proliferation at the highest concentration of ketamine (0.8 mg/mL). Nonetheless, in adults no significant alterations were seen, pointing to a return to a homeostatic stage. This study allowed clarifying some of the aspects pertaining the longitudinal effects of ketamine administration regarding the CNS capacity to proliferate and activate the appropriate cell death and repair mechanisms leading to homeostasis in zebrafish. Moreover, the results indicate that ketamine administration at 1-4 somites stage in the subanesthetic and anesthetic concentrations despite some transitory detrimental effects at 144 hpf, is long-term safe for CNS, which are newly and promising results in this research field.

Prenatal Exposure to General Anesthesia Drug Esketamine Impaired Neurobehavior in Offspring.

Prenatal exposure to anesthetics has raised increasing attention about the neuronal development in offspring. Animal models are usually used for investigation. As a new drug, esketamine is the s-isoform of ketamine and is twice as potent as the racemic ketamine with less reported adverse effects. Esketamine is currently being used and become more favorable in clinical anesthesia work, including surgeries during pregnancy, yet the effect on the offspring is unknown. The present study aimed to elucidate the effects of gestational administration of esketamine on neuronal development in offspring, using a rat model. Gestational day 14.5 pregnant rats received intravenous injections of esketamine. The postnatal day 0 (P0) hippocampus was digested and cultured in vitro to display the neuronal growth morphology. On Day 4 the in vitro experiments revealed a shorter axon length and fewer dendrite branches in the esketamine group. The results from the EdU- imaging kit showed decreased proliferative capacity in the subventricular zone (SVZ) and dentate gyrus (DG) in both P0 and P30 offspring brains in the esketamine group. Moreover, neurogenesis, neuron maturity and spine density were impaired, resulting in attenuated long-term potentiation (LTP). Compromised hippocampal function accounted for the deficits in neuronal cognition, memory and emotion. The evidence obtained suggests that the neurobehavioral deficit due to prenatal exposure to esketamine may be related to the decrease phosphorylation of CREB and abnormalities in N-methyl-D-aspartic acid receptor subunits. Taken together, these results demonstrate the negative effect of prenatal esketamine exposure on neuronal development in offspring rats. G14.5 esketamine administration influenced the neurobehavior of the offspring in adolescence. Poorer neuronal growth and reduced brain proliferative capacity in late gestation and juvenile pups resulted in impaired P30 neuronal plasticity and synaptic spines as well as abnormalities in NMDAR subunits. Attenuated LTP reflected compromised hippocampal function, as confirmed by behavioral tests of cognition, memory and emotions. This figure was completed on the website of Figdraw.

Comparison of different QT correction methods for nonclinical safety assessment in ketamine-anesthetized Indian rhesus monkeys (Macaca mulatta).

Rhesus monkeys are a non-rodent species employed in the preclinical safety evaluation of pharmaceuticals and biologics. These nonhuman primate species have been increasingly used in biomedical research because of the similarity in their ionic mechanisms of repolarization with humans. Heart rate and QT interval are two primary endpoints in determining the pro-arrhythmic risk of drugs. As heart rate and QT interval have an inverse relationship, any change in heart rate causes a subsequent change in QT interval. This warrants for calculation of a corrected QT interval. This study aimed to identify an appropriate formula that best corrected QT for change in heart rate. We employed seven formulas based on source-species type, clinical relevance, and requirements of various international regulatory guidelines. Data showed that corrected QT interval values varied drastically for different correction formulas. Equations were compared on their slope values based on QTc versus RR plots. The rank order of the slope for different formulas was (closest to farthest from zero) QTcNAK, QTcHAS, QTcBZT, QTcFRD, QTcVDW, QTcHDG, and QTcFRM. QTcNAK emerged to be the best correcting formula in this study. It showed the least correlation with the RR interval (r = -0.01) and displayed no significant difference amongst the sexes. As there is no universally recognized formula for preclinical use, the authors recommend developing a best-case scenario model for specific study designs and individual organizations. The data from this research will be helpful in deciding an appropriate QT correction formula for the safety assessment of new pharmaceuticals and biologics.

Lipoxin A4 methyl ester attenuated ketamine-induced neurotoxicity in SH-SY5Y cells via regulating leptin pathway.

Ketamine, the widely used intravenous anesthetic, has been reported to cause neurotoxicity and disturbs normal neurogenesis. However, the efficacy of current treatment strategies targeting ketamine's neurotoxicity remains limited. Lipoxin A4 methyl ester (LXA4 ME) is relatively stable lipoxin analog, which serves an important role in protecting against early brain injury. The purpose of this study was to investigate the protective effect of LXA4 ME on ketamine-caused cytotoxicity in SH-SY5Y cells, as well as the underlying mechanisms. Cell viability, apoptosis and endoplasmic reticulum stress (ER stress) were detected by adopting experimental techniques including CCK-8 assay, flow cytometry, western blotting and transmission electron microscope. Furthermore, examining the expression of leptin and its receptor (LepRb), we also measured the levels of activation of the leptin signaling pathway. Our results showed that LXA4 ME intervention promoted the cell viability, inhibited cell apoptosis, and reduced the expression of ER stress related protein and morphological changes induced by ketamine. In addition, inhibition of leptin signaling pathway caused by ketamine could be reversed by LXA4 ME. However, as the specific inhibitor of leptin pathway, leptin antagonist triple mutant human recombinant (leptin tA) attenuated the cytoprotective effect of LXA4 ME against ketamine-induced neurotoxicity. In conclusion, our findings demonstrated LXA4 ME could exert a neuroprotective effect on ketamine-induced neuronal injury via activation of the leptin signaling pathway.