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.

Cortical Acetylcholine Levels Correlate With Neurophysiologic Complexity During Subanesthetic Ketamine and Nitrous Oxide Exposure in Rats.

BACKGROUND: Neurophysiologic complexity has been shown to decrease during states characterized by a depressed level of consciousness, such as sleep or anesthesia. Conversely, neurophysiologic complexity is increased during exposure to serotonergic psychedelics or subanesthetic doses of dissociative anesthetics. However, the neurochemical substrates underlying changes in neurophysiologic complexity are poorly characterized. Cortical acetylcholine appears to relate to cortical activation and changes in states of consciousness, but the relationship between cortical acetylcholine and complexity has not been formally studied. We addressed this gap by analyzing simultaneous changes in cortical acetylcholine (prefrontal and parietal) and neurophysiologic complexity before, during, and after subanesthetic ketamine (10 mg/kg/h) or 50% nitrous oxide. METHODS: Under isoflurane anesthesia, adult Sprague Dawley rats (n = 24, 12 male and 12 female) were implanted with stainless-steel electrodes across the cortex to record monopolar electroencephalogram (0.5-175 Hz; 30 channels) and guide canulae in prefrontal and parietal cortices for local microdialysis quantification of acetylcholine levels. One subgroup of these rats was instrumented with a chronic catheter in jugular vein for ketamine infusion (n = 12, 6 male and 6 female). The electroencephalographic data were analyzed to determine subanesthetic ketamine or nitrous oxide-induced changes in Lempel-Ziv complexity and directed frontoparietal connectivity. Changes in complexity and connectivity were analyzed for correlation with concurrent changes in prefrontal and parietal acetylcholine. RESULTS: Subanesthetic ketamine produced sustained increases in normalized Lempel-Ziv complexity (0.5-175 Hz; P < .001) and high gamma frontoparietal connectivity (125-175 Hz; P < .001). This was accompanied by progressive increases in prefrontal (104%; P < .001) and parietal (159%; P < .001) acetylcholine levels that peaked after 50 minutes of infusion. Nitrous oxide induction produced a transient increase in complexity (P < .05) and high gamma connectivity (P < .001), which was accompanied by increases (P < .001) in prefrontal (56%) and parietal (43%) acetylcholine levels. In contrast, the final 50 minutes of nitrous oxide administration were characterized by a decrease in prefrontal (38%; P < .001) and parietal (45%; P < .001) acetylcholine levels, reduced complexity (P < .001), and comparatively weaker frontoparietal high gamma connectivity (P < .001). Cortical acetylcholine and complexity were correlated with both subanesthetic ketamine (prefrontal: cluster-weighted marginal correlation [CW r] [144] = 0.42, P < .001; parietal: CW r[144] = 0.42, P < .001) and nitrous oxide (prefrontal: CW r[156] = 0.46, P < .001; parietal: CW r[156] = 0.56, P < .001) cohorts. CONCLUSIONS: These data bridge changes in cortical acetylcholine with concurrent changes in neurophysiologic complexity, frontoparietal connectivity, and the level of consciousness.

Effect of ketamine on gene expression in zebrafish embryos.

Ketamine is an N-methyl-D-aspartate (NMDA) receptor antagonist. Used as an anesthetic, potential neurotoxic and cardiotoxic effects of ketamine in animal models have been reported. The underlying mechanisms of ketamine-induced toxicity are not clear. The zebrafish is an ideal model for toxicity assays because of its predictive capability in chemical testing, which compares well with that of mammalian models. To gain insight into potential mechanisms of ketamine effects, we performed real-time quantitative polymerase chain reaction-based gene expression array analyses. Gene expression analysis was conducted for multiple genes (a total of 84) related to 10 major signaling pathways including the transforming growth factor ß (TGFß), Wingless and Int-1 (Wnt), nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB), Janus kinase/signal transducers and activators of transcription (JAK/STAT), p53, Notch, Hedgehog, peroxisome proliferator-activated receptor (PPAR), oxidative stress, and hypoxia pathways. Our results show that ketamine altered the expression of specific genes related to hypoxia, p53, Wnt, Notch, TGFß, PPAR, and oxidative stress pathways. Thus, we can further focus on these specific pathways to elucidate the mechanisms by which ketamine elicits a toxic response.

Ketamine's schizophrenia-like effects are prevented by targeting PTP1B.

Subanesthetic doses of ketamine induce schizophrenia-like behaviors in mice including hyperlocomotion and deficits in working memory and sensorimotor gating. Here, we examined the effect of in vivo ketamine administration on neuronal properties and endocannabinoid (eCB)-dependent modulation of synaptic transmission onto layer 2/3 pyramidal neurons in brain slices of the prefrontal cortex, a region tied to the schizophrenia-like behavioral phenotypes of ketamine. Since deficits in working memory and sensorimotor gating are tied to activation of the tyrosine phosphatase PTP1B in glutamatergic neurons, we asked whether PTP1B contributes to these effects of ketamine. Ketamine increased membrane resistance and excitability of pyramidal neurons. Systemic pharmacological inhibition of PTP1B by Trodusquemine restored these neuronal properties and prevented each of the three main ketamine-induced behavior deficits. Ketamine also reduced mobilization of eCB by pyramidal neurons, while unexpectedly reducing their inhibitory inputs, and these effects of ketamine were blocked or occluded by PTP1B ablation in glutamatergic neurons. While ablation of PTP1B in glutamatergic neurons prevented ketamine-induced deficits in memory and sensorimotor gating, it failed to prevent hyperlocomotion (a psychosis-like phenotype). Taken together, these results suggest that PTP1B in glutamatergic neurons mediates ketamine-induced deficits in eCB mobilization, memory and sensorimotor gating whereas PTP1B in other cell types contributes to hyperlocomotion. Our study suggests that the PTP1B inhibitor Trodusquemine may represent a new class of fast-acting antipsychotic drugs to treat schizophrenia-like symptoms.

Protective Effect of GM1 Attenuates Hippocampus and Cortex Apoptosis After Ketamine Exposure in Neonatal Rat via PI3K/AKT/GSK3ß Pathway.

Ketamine is a widely used analgesic and anesthetic in obstetrics and pediatrics. Ketamine is known to promote neuronal death and cognitive dysfunction in the brains of humans and animals during development. Monosialotetrahexosyl ganglioside (GM1), a promoter of brain development, exerts neuroprotective effects in many neurological disease models. Here, we investigated the neuroprotective effect of GM1 and its potential underlying mechanism against ketamine-induced apoptosis of rats. Seven-day-old Sprague Dawley (SD) rats were randomly divided into the following four groups: (1) group C (control group: normal saline was injected intraperitoneally); (2) group K (ketamine); (3) group GM1 (GM1 was given before normal saline injection); and (4) GM1+K group (received GM1 30 min before continuous exposure to ketamine). Each group contained 15 rats, received six doses of ketamine (20 mg/kg), and was injected with saline every 90 min. The Morris water maze (MWM) test, the number of cortical and hippocampal cells, apoptosis, and AKT/GSK3ß pathway were analyzed. To determine whether GM1 exerted its effect via the PI3K/AKT/GSK3ß pathway, PC12 cells were incubated with LY294002, a PI3K inhibitor. We found that GM1 protected against ketamine-induced apoptosis in the hippocampus and cortex by reducing the expression of Bcl-2 and Caspase-3, and by increasing the expression of Bax. GM1 treatment increased the expression of p-AKT and p-GSK3ß. However, the anti-apoptotic effect of GM1 was eliminated after inhibiting the phosphorylation of AKT. We showed that GM1 lessens ketamine-induced apoptosis in the hippocampus and cortex of young rats by regulating the PI3K/AKT/GSK3ß pathway. Taken together, GM1 may be a potential preventive treatment for the neurotoxicity caused by continuous exposure to ketamine.

Integrin-dependent microgliosis mediates ketamine-induced neuronal apoptosis during postnatal rat retinal development.

PURPOSE: Remodeling of the extracellular matrix (ECM) by matrix metalloproteinases (MMPs) plays a pivotal role for microglia in developing retina. We tested whether integrin-dependent microgliosis mediates ketamine-induced neuronal apoptosis in the developing rat retina. METHODS: We performed immunofluorescence assays to investigate the role of integrin receptors expressed in the microglia in ketamine-induced neuronal apoptosis. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA) were used to investigate the protein and mRNA levels of cytokines (TNF-α, IL-1ß) and/or chemokines (CCL2, CXCL6, CXCL10, and CXCL12). Experiments were performed using whole-mount retinas dissected from P7 Sprague-Dawley rats. RESULTS: Integrin receptors expressed in microglia were upregulated in ketamine-induced neuronal apoptosis in the early developing rat retina. Downregulating integrin receptors with RGD peptide ameliorated ketamine-induced microgliosis through: 1) ameliorating the change in microglia morphology from immature ramified microglia to an amoeboid state; 2) decreasing the number of microglia and intensity of activated microglia in the retinal ganglion cell layer (GCL); and 3) decreasing cytokine (TNF-α and IL-1ß) and chemokine (CCL2, CXCL10) levels in the retinal tissue. Inhibition of activated microglia with minocycline or the blockade of cytokines (TNF-α and IL-1ß) with a receptor antagonist (RA) attenuated neuronal apoptosis after exposure to ketamine. CONCLUSIONS: The upregulation of integrin ß1 receptors in the microglia acts as a signaling molecule, triggering microgliosis to aggravate ketamine-induced neuronal apoptosis via the release of TNF-α and IL-1ß in the early developing rat retina.

Ketamine inhibits neuronal differentiation by regulating brain-derived neurotrophic factor (BDNF) signaling.

Ketamine is widely used in pediatric anesthesia, perioperative sedation, and analgesia. Knowledge of anesthesia neurotoxicity in humans is currently limited by the difficulty of obtaining neurons and performing developmental toxicity studies in fetal and pediatric populations. However, mouse embryonic stem cells (mESCs) derived from embryos at the preimplantation stage demonstrate an unlimited ability to self-renew and generate different cell types and are a valuable tool for clinical research. Thus, in this study, a model was employed to investigate the mechanism by which ketamine (200 nM) influences the neuronal differentiation of mESCs. Mouse ESCs were treated with an anesthetic dose of ketamine, and neuronal differentiation was significantly inhibited on day 5. Downregulation of brain-derived neurotrophic factor (BDNF) by shRNA was found to have the same inhibitory effect. Furthermore, a rescue experiment indicated that BDNF overexpression markedly restored the neuronal differentiation inhibited by ketamine in the ketamine/BDNF group on day 5. Taken together, these data suggested that ketamine inhibited the neuronal differentiation of mESCs, possibly by interfering with BDNF. The results of the current study may provide novel ideas for preventing ketamine toxicity in the developing fetus.

Neonatal exposure to ketamine disrupts developmental synapse unsilencing and predisposes adult mice for stressor-evoked anxiety.

Accumulating evidence suggests long-lasting impairments in brain development and cognition caused by neonatal exposure to general anesthetics. To date, very little is known about potential abnormal psychiatric manifestations attributable to neonatal anesthesia. In this study, we used ketamine to induce anesthesia in neonatal mice. By applying mild stressors one day before behavioral tests, we found that adult mice exhibit significant anxiety-like behaviors that were indistinguishable at basal level. Recruitment of AMPA (a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) type glutamate receptors into silent synapses is a prominent cellular process during neonatal neurodevelopment. We found that exposure to ketamine significantly disrupted synapse unsilencing, and impaired the expression of unsilencing-mediated long-term potentiation (LTP). Pharmacologically enhancement of neural activities by AMPAkine drug CX546 [1-(1,4-benzodioxan-6-ylcarbonyl) piperidine] effectively rescued disrupted developmental synapse unsilencing and LTP at neonatal age, and prevented stressor-evoked anxiety-like behaviors in adult mice. Together, our results indicate that neonatal exposure to ketamine may predispose individuals for psychiatric conditions via disrupting synapse unsilencing, and potentiation of neural activities during the anesthesia-recovery period may be an effective approach to manage adverse effects on brain development. This article is part of the special issue on 'Stress, Addiction and Plasticity'.

Binge and Subchronic Exposure to Ketamine Promote Memory Impairments and Damages in the Hippocampus and Peripheral Tissues in Rats: Gallic Acid Protective Effects.

Ketamine (KET) is a dissociative anesthetic for restrict medical use with high potential for abuse and neurotoxicity which does not prevent its recreational use. Gallic acid (GA) is a natural free radical "scavenger." We evaluated the GA protective role regarding binge or subchronic (SbChro) KET-induced toxicity in adolescent rats. In the binge protocol, animals were treated with GA (one dose of 13.5 mg/kg, p.o. every 2 h, totaling 3 doses) 12 h after KET exposure (one dose of 10 mg/kg, i.p., every 3 h, totaling 5 doses). In the SbChro, animals were treated with GA (one dose of 13.5 mg/kg/day, p.o., for 3 days) 48 h following KET exposure (one dose of 10 mg/kg/day, i.p) for 10 days. Our findings show that binge-KET impaired memory, increased pro-BDNF and TrkB levels in the hippocampus, and increased lipid peroxidation (LP) in the kidney and hippocampus, while SbChro-KET impaired memory, increased pro-BDNF, and decreased both BDNF and TrkB levels in the hippocampus, and increased LP in the kidney, liver, and hippocampus. GA treatment reversed the subchronically KET-induced harmful influences better. Interestingly, only memory impairment observed in the SbChro-KET protocol was reversed by GA. Memory impairments showed a positive correlation with hippocampal BDNF levels and negative with LP levels in the same brain area. This last hippocampal damage (LP) showed a negative correlation with BDNF levels in the hippocampus, indicating an interesting and close causal connection. Our outcomes show that the deleterious effects of SbChro-KET exposure can be attenuated or abolished with GA administration, a natural antioxidant that could be considered in KET abuse treatment.

Exogenous GM1 Ganglioside Attenuates Ketamine-Induced Neurocognitive Impairment in the Developing Rat Brain.

BACKGROUND: A prolonged exposure to ketamine triggers significant neurodegeneration and long-term neurocognitive deficits in the developing brain. Monosialotetrahexosylganglioside (GM1) can limit the neuronal damage from necrosis and apoptosis in neurodegenerative conditions. We aimed to assess whether GM1 can prevent ketamine-induced developmental neurotoxicity. METHODS: Postnatal day 7 (P7) rat pups received 5 doses of intraperitoneal ketamine (20 mg/kg per dose) at 90-minute intervals for 6 hours. Cognitive functions, determined by using Morris water maze (MWM) including escape latency (at P32-36) and platform crossing (at P37), were compared among the ketamine-exposed pups treated with or without exogenous GM1 (30 mg/kg; n = 12/group). The effect of GM1 on apoptosis in hippocampus was determined by terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling (TUNEL) staining and activated caspase 3 measurement. The hippocampal expression of brain-derived neurotrophic factor (BDNF), along with the phosphorylation of protein kinase B (AKT) and extracellular signal-related kinases 1 and 2 (ERK1/2), was detected by western blotting (n = 6/group). Anti-BDNF antibody (2 µg per rat) administered before GM1 treatment was applied to determine the neuroprotective mechanisms of GM1. RESULTS: The rats receiving ketamine exposure experinced cognitive impairment in MWM test compared to the control rats, indicated by prolonged escape latency at P34 (P = .006), P35 (P = .002), and P36 (P = .005). However, in GM1-pretreated rats, ketamine exposure did not induce prolonged escape latency. The exogenous GM1 increased the platform-crossing times at P37 (3.00 ± 2.22 times vs 5.40 ± 1.53 times, mean ± standard deviation; P = .041) and reduced the hippocampal TUNEL-positive cells and cleaved-caspase 3 expression in ketamine-exposed young rats. Ketamine decreased BDNF expression and phosphorylation of AKT and ERK in the hippocampus, whereas exogenous GM1 blocked these ketamine-caused effects. However, for the ketamine-exposed rat pups receiving exogenous GM1, compared to immunoglobulin Y (IgY) isotype control, the BDNF-neutralizing antibody treatment counteracted the exogenous GM1-induced improvement of the escape latency at P36 (41.32 ± 12.37 seconds vs 25.14 ± 8.97 seconds, mean ± standard deviation; P = .036), platform-crossing times at P37 (2.16 ± 1.12 times vs 3.92 ± 1.97 times, mean ± standard deviation; P < .036), apoptotic activity, as well as AKT and ERK1/2 phosphorylation in the hippocampus of ketamine-challenged young rats. CONCLUSIONS: Our data suggest that the exogenous GM1 acts on BDNF signaling pathway to ameliorate the cognitive impairment and hippocampal apoptosis induced by ketamine in young rats. Our study may indicate a potential use of GM1 in preventing the cognitive deficits induced by ketamine in the young per se.

Evaluation of Genotoxicity and Mutagenicity of Ketamine on Human Peripheral Blood Leukocytes and in Salmonella typhimurium.

Ketamine is a potent uncompetitive NMDA receptor antagonist that provides amnesia, analgesia, environmental dissociation and immobility, where it has its cytotoxic effect well described in the literature. However, the work on its genotoxic/mutagenic potentials are scarce and insufficient and does not allow a reasonable evaluation of its role. Thus, in the present work, we decided to evaluate the genotoxic and mutagenic effects of ketamine on human peripheral blood leukocytes (PBLs) and Salmonella typhimurium (TA98, TA97a, TA100, and TA102) through several well-established experimental protocols based on different parameters in the presence or not of exogenous metabolizing S9 fraction. Our data revealed that ketamine induces a weak cytotoxic effect on human PBLs after 24 h and is devoided of hemolytic effects. A small amount of DNA strand breaks levels were detected in the modified comet assay (employment of FPG enzyme) only at highest concentrations (500 and 700 µg/mL) of ketamine, highlighting our pro-oxidant data regarding ketamine. However, the oxidative DNA lesions were almost completely repaired which reflects in the lack of mutagenesis (micronuclei and chromosomal aberrations) on human PBLs and no increases in revertants numbers on S. typhimurium/microsome test (500 to 5000 µg/plate). In summary, ketamine is a weak oxidative DNA damaging agent and is devoid of mutagenic properties on eukaryotic and prokaryotic models.

Gallic acid prevents ketamine-induced oxidative damages in brain regions and liver of rats.

INTRODUCTION: Ketamine (KET) is an anesthetic agent widely used in human and veterinary medicine. According to studies, KET is associated to direct neutorotoxic damages due to its capacity to induce oxidative stress. Because of the free radical generation in the organism and its relation with diseases' development, there is a growing interest to study antioxidant molecules, such as gallic acid (GA), a natural phenolic compound. AIM: Evaluate the GA antioxidant potential for the prevention of oxidative damage in the brain and liver tissue of rats exposed to acute KET administration. MATERIAL AND METHODS: 32 Wistar male rats received GA (by gavage, 13.5 mg/kg) for three consecutive days, 24 h after the last GA dose, animals were anesthetized with KET (50 mg/kg, i.m.). All animals were euthanized by decapitation 60 min after KET administration. The liver, brain cortex and hippocampus were removed and homogenized for biochemical analysis. RESULTS: In brain cortex, KET increased reactive species (RS) generation, protein carbonyls (PC) levels and reduced non-protein thiols (NPSH) levels, while GA pre-treatment reduced PC and increased NPSH levels. KET increased PC and decreased NPSH levels in the hippocampus, and GA reduced PC and NPSH levels. In the liver, no difference was observed in the RS generation, while KET induced and increase of PC levels and decreased NPSH levels, while GA pre-treatment prevented it. CONCLUSION: GA administration can prevent oxidative damage caused by acute KET administration and minimize its noxious effects. Further studies are needed to evidence GA antioxidant properties regarding KET chronic use.

Protective effects of Spinacia oleracea seeds extract in an experimental model of schizophrenia: Possible behavior, biochemical, neurochemical and cellular alterations.

Schizophrenia is one of the psychotic mental disorders characterized by symptoms of thought, behavior, and social problems. Newer biomedicine and pharmacotherapy has been investigated for the treatment of various neuropsychiatric disorders in the past few decades. Spinacia oleracea is one of these, reported to have beneficial effect against several neurodegenerative disorders. The present study was carried to explore the protective effects of Spinacia oleracea seed extract (SOEE) in an experimental model of ketamine-induced schizophrenia in mice. Ketamine (50 mg/kg, i.p.) was used to induce stereotyped psychotic behavioural symptoms in mice. Behavioral studies (locomotor activity, stereotype behaviors, immobility duration and memory retention) were carried out to investigate the protective of SOEE on ketamine-induced psychotic symptoms, followed by biochemical, neurochemical and cellular alterations in the brain. Treatment with SOEE for 15 consecutive days significantly attenuated stereotyped behavioral symptoms in mice. Biochemical estimations revealed that SOEE reduced lipid peroxidation and restored total brain proteins. Furthermore, SOEE remarkably reduced dopamine levels, AChE activity & inflammatory surge (serum TNF-α) and increased the levels of GABA and reduced glutathione in mice. The outcomes of the study suggested that SOEE could ameliorate ketamine-induced psychotic symptoms in mice, indicating a protective effect in the treatment of schizophrenia.

Probable mechanisms involved in the antipsychotic-like activity of morin in mice.

Evidence derived from preliminary studies suggests that morin, a neuroactive flavonoid with proven antioxidant and antiinflammatory properties possess antipsychotic-like activity. The present study was designed to evaluate the probable mechanisms involve in the antipsychotic-like activity of morin in ketamine model of schizophrenia. The effects of morin, haloperidol and risperidone on neurobehavioral and anti-schizophrenia-like effects were evaluated in mice (n = 7) following intraperitoneal (i.p.) administration of morin (25-100 mg/kg), haloperidol (1 mg/kg) and risperidone (0.5 mg/kg) alone or in combination with ketamine (20 mg/kg, i.p.) for 10 days. Neurobehavioral and schizophrenia-like activities consisting of open-field (positive symptoms), Y-maze, novel-object recognition (cognitive symptoms), social interaction (negative symptoms) tests were assessed. Also, wood-block catalepsy and rota-rod tests were employed to evaluate extrapyramidal side effects of morin. Thereafter, brain levels of biomarkers of oxidative, nitrergic and acetylcholinesterase alterations as well as histomorphological changes in the striatum and prefrontal-cortex were determined. Administration of morin and risperidone alone but not haloperidol significantly (p > 0.05) prevented ketamine-induced hyperlocomotion, social withdrawal and cognitive impairments relative to controls, and were devoid of extrapyramidal side effects. Morin alone or in combination with ketamine significantly increased glutathione concentration, superoxide dismutase and catalase activities compared with saline- or ketamine-treated mice. Moreover, morin alone or in combination with ketamine also significantly decreased malondialdehyde, nitrite and acetylcholinesterase alterations in mice brains. Furthermore, morin prevented ketamine-induced brain neuronal alterations in the striatum and prefrontal-cortex. Together, our findings suggest that morin may demonstrate antipsychotic-like therapeutic effect via modulation of oxidative/nitrergic, cholinergic actions and neuroprotection.

N-acetylcysteine prevents ketamine-induced adverse effects on development, heart rate and monoaminergic neurons in zebrafish.

N-acetylcysteine, a precursor molecule of glutathione, is an antioxidant. Ketamine, a pediatric anesthetic, has been implicated in cardiotoxicity and neurotoxicity including modulation of monoaminergic systems in mammals and zebrafish. Here, we show that N-acetylcysteine prevents ketamine's adverse effects on development and monoaminergic neurons in zebrafish embryos. The effects of ketamine and N-acetylcysteine alone or in combination were measured on the heart rate, body length, brain serotonergic neurons and tyrosine hydroxylase-immunoreactive (TH-IR) neurons. In the absence of N-acetylcysteine, a concentration of ketamine that produces an internal embryo exposure level comparable to human anesthetic plasma concentrations significantly reduced heart rate and body length and those effects were prevented by N-acetylcysteine co-treatment. Ketamine also reduced the areas occupied by serotonergic neurons in the brain, whereas N-acetylcysteine co-exposure counteracted this effect. TH-IR neurons in the embryo brain and TH-IR cells in the trunk were significantly reduced with ketamine treatment, but not in the presence of N-acetylcysteine. In our continued search for compounds that can prevent ketamine toxicity, this study using specific endpoints of developmental toxicity, cardiotoxicity and neurotoxicity, demonstrates protective effects of N-acetylcysteine against ketamine's adverse effects. This is the first study that shows the protective effects of N-acetylcysteine on ketamine-induced developmental defects of monoaminergic neurons as observed in a whole organism.

Ketamine-induced neurotoxicity blocked by N-Methyl-d-aspartate is mediated through activation of PKC/ERK pathway in developing hippocampal neurons.

Ketamine, a non-competitive N-methyl d-aspartate (NMDA) receptor antagonist, is widely used in pediatric clinical practice. However, prolonged exposure to ketamine results in widespread anesthetic neurotoxicity and long-term neurocognitive deficits. The molecular mechanisms that underlie this important event are poorly understood. We investigated effects of anesthetic ketamine on neuroapoptosis and further explored role of NMDA receptors in ketamine-induced neurotoxicity. Here we demonstrate that ketamine induces activation of cell cycle entry, resulting in cycle-related neuronal apoptosis. On the other hand, ketamine administration alters early and late apoptosis of cultured hippocampus neurons by inhibiting PKC/ERK pathway, whereas excitatory NMDA receptor activation reverses these effects. Ketamine-induced neurotoxicity blocked by NMDA is mediated through activation of PKC/ERK pathway in developing hippocampal neurons.

Early Exposure to Ketamine Impairs Axonal Pruning in Developing Mouse Hippocampus.

Mounting evidence suggests that prolonged exposure to general anesthesia (GA) during brain synaptogenesis damages the immature neurons and results in long-term neurocognitive impairments. Importantly, synaptogenesis relies on timely axon pruning to select axons that participate in active neural circuit formation. This process is in part dependent on proper homeostasis of neurotrophic factors, in particular brain-derived neurotrophic factor (BDNF). We set out to examine how GA may modulate axon maintenance and pruning and focused on the role of BDNF. We exposed post-natal day (PND)7 mice to ketamine using a well-established dosing regimen known to induce significant developmental neurotoxicity. We performed morphometric analyses of the infrapyramidal bundle (IPB) since IPB is known to undergo intense developmental modeling and as such is commonly used as a well-established model of in vivo pruning in rodents. When IPB remodeling was followed from PND10 until PND65, we noted a delay in axonal pruning in ketamine-treated animals when compared to controls; this impairment coincided with ketamine-induced downregulation in BDNF protein expression and maturation suggesting two conclusions: a surge in BDNF protein expression "signals" intense IPB pruning in control animals and ketamine-induced downregulation of BDNF synthesis and maturation could contribute to impaired IPB pruning. We conclude that the combined effects on BDNF homeostasis and impaired axon pruning may in part explain ketamine-induced impairment of neuronal circuitry formation.

Apoptosis-related genes induced in response to ketamine during early life stages of zebrafish.

Increasing evidence supports that ketamine, a widely used anaesthetic, potentiates apoptosis during development through the mitochondrial pathway of apoptosis. Defects in the apoptotic machinery can cause or contribute to the developmental abnormalities previously described in ketamine-exposed zebrafish. The involvement of the apoptotic machinery in ketamine-induced teratogenicity was addressed by assessing the apoptotic signals at 8 and 24 hpf following 20min exposure to ketamine at three stages of early zebrafish embryo development (256 cell, 50% epiboly and 1-4 somites stages). Exposure at the 256-cell stage to ketamine induced an up-regulation of casp8 and pcna at 8 hpf while changes in pcna at the mRNA level were observed at 24 hpf. After the 50% epiboly stage exposure, the mRNA levels of casp9 were increased at 8 and 24 hpf while aifm1 was affected at 24 hpf. Both tp53 and pcna expressions were increased at 8 hpf. After exposure during the 1-4 somites stage, no meaningful changes on transcript levels were observed. The distribution of apoptotic cells and the caspase-like enzymatic activities of caspase-3 and -9 were not affected by ketamine exposure. It is proposed that ketamine exposure at the 256-cell stage induced a cooperative mechanism between proliferation and cellular death while following exposure at the 50% epiboly, a p53-dependent and -independent caspase activation may occur. Finally, at the 1-4 somites stage, the defence mechanisms are already fully in place to protect against ketamine-insult. Thus, ketamine teratogenicity seems to be dependent on the functional mechanisms present in each developmental stage.

Effects of GABA-B receptor positive modulator on ketamine-induced psychosis-relevant behaviors and hippocampal electrical activity in freely moving rats.

RATIONALE: Decreased GABAB receptor function is proposed to mediate some symptoms of schizophrenia. OBJECTIVES: In this study, we tested the effect of CGP7930, a GABAB receptor positive allosteric modulator, on ketamine-induced psychosis-relevant behaviors and hippocampal electrical activity in behaving rats. METHODS: Electrodes were bilaterally implanted into the hippocampus, and cannulae were placed into the lateral ventricles of Long-Evans rats. CGP7930 or vehicle was injected intraperitoneally (i.p.) or intracerebroventricularly (i.c.v.), alone or 15 min prior to ketamine (3 mg/kg, subcutaneous) injection. Paired click auditory evoked potentials in the hippocampus (AEP), prepulse inhibition (PPI), and locomotor activity were recorded before and after drug injection. RESULTS: CGP7930 at doses of 1 mg/kg (i.p.) prevented ketamine-induced deficit of PPI. CGP7930 (1 mg/kg i.p.) also prevented the decrease in gating of hippocampal AEP and the increase in hippocampal gamma (65-100 Hz) waves induced by ketamine. Unilateral i.c.v. infusion of CGP7930 (0.3 mM/1 µL) also prevented the decrease in gating of hippocampal AEP induced by ketamine. Ketamine-induced behavioral hyperlocomotion was suppressed by 5 mg/kg i.p. CGP7930. CGP7930 alone, without ketamine, did not significantly affect integrated PPI, locomotion, gating of hippocampal AEP, or gamma waves. CGP7930 (1 mg/kg i.p.) increased heterosynaptically mediated paired pulse depression in the hippocampus, a measure of GABAB receptor function in vivo. CONCLUSIONS: CGP7930 reduces the behavioral and electrophysiological disruptions induced by ketamine in animals, and the hippocampus may be one of the neural targets where CGP7930 exerts its actions.

Ketamine-induced behavioural and brain oxidative changes in mice: an assessment of possible beneficial effects of zinc as mono- or adjunct therapy.

RATIONALE: We studied the influence of zinc, haloperidol or olanzapine on neurobehaviour (open-field, radial arm maze and elevated plus maze) and brain antioxidant status in vehicle- or ketamine-treated mice, with the aim of ascertaining the potentials of zinc in counteracting ketamine's effects. OBJECTIVES: Experiment 1 assessed the effects of zinc in healthy animals and the relative degrees of modulation of ketamine's effects by zinc, haloperidol or olanzapine, respectively. Experiment 2 assessed the modulation of ketamine's effects following co-administration of zinc with haloperidol or olanzapine. METHODS: Male mice weighing 18-20 g each were used. Animals were pretreated with ketamine (except vehicle, zinc, haloperidol and olanzapine controls) for 10 days before commencement of 14-day treatment (day 11-24) with vehicle, zinc, haloperidol or olanzapine (alone or in combination). Ketamine injection also continued alongside zinc and/or standard drugs in the ketamine-treated groups. Zinc, haloperidol and olanzapine were administered by gavage. Treatments were given daily and behaviours assessed on days 11 and 24. On day 24, animals were sacrificed and whole brain homogenates used for estimation of glutathione, nitric oxide and malondialdehyde (MDA) levels. RESULTS: Ketamine increased open-field behaviours, nitric oxide and MDA levels, while it decreased working memory, social interaction and glutathione. Administration of zinc alone or in combination with haloperidol or olanzapine was associated with variable degrees of reversal of these effects. CONCLUSION: Zinc may have the potential of a possible therapeutic agent and/or adjunct in the reversal of schizophrenia-like changes in behaviour and brain oxidative status.