The deficiency of aquaporin-4 (AQP4) has been reported to alter release of neurotransmitters in the mouse brain. However, the functional relevance of AQP4 in mediating essential components of the general anaesthetic state is unknown. The aim of the present study was to investigate the role of AQP4 in general anaesthesia in mice lacking AQP4. The hypnotic effects of propofol, ketamine, and pentobarbital in AQP4 knockout (KO) and CD1 control mice were evaluated using the behavioural endpoint of loss of righting reflex (LORR). The effects of propofol on extracellular levels of amino acids in prefrontal cortex of freely moving mice were investigated using microdialysis coupled to high performance liquid chromatography with fluorescent detection. The result showed that, after receiving ketamine or pentobarbital, LORR occurred at earlier time in KO mice than that in control animals. Intraperitoneal injection of ketamine or pentobarbital increased the duration of LORR. After the administration of propofol, the duration of LORR was significantly reduced in KO mice compared with that in controls. Propofol increased the extracellular levels of aspartate, glutamate, and GABA, but not taurine, in prefrontal cortex. There were significant differences of increase patterns of the three kinds of neurotransmitters between KO and WT mice. Notably, the duration of GABA level increase correlated with the duration of LORR in two genotypes of mice. These results provide in vivo evidence of different responses in time-dependent release of excitatory and inhibitory neurotransmitters in prefrontal cortex of the two genotypes of mice. It is suggested that changes in anaesthetic reactions in mice with AQP4 loss may be related to neurotransmitter regulation, and that normal functioning of AQP4 plays an important role in the maintenance of anaesthetic hypnosis.
Anesthetics, Intravenous ; pharmacology ; Animals ; Aquaporin 4 ; deficiency ; genetics ; Hypnotics and Sedatives ; pharmacology ; Ketamine ; pharmacology ; Mice ; Mice, Knockout ; Neurotransmitter Agents ; metabolism ; Pentobarbital ; pharmacology ; Prefrontal Cortex ; drug effects ; metabolism ; Propofol ; pharmacology

OBJECTIVE: To investigate the effects of intravenous anesthetics on LPS-induced inflammatory responses of primary cultures of rat glial cells in vitro. METHODS: The primary cultures of rat glial cells were stimulated with lipopolysaccharide( LPS) to produce inflammatory responses. Glial cells were divided into 8 groups (n=4): blank control (Group C), LPS(Group L), 100micromol/L ketamine with LPS(Group K1), 1000micromol/L ketamine with LPS (Group K2), 30micromol/L propofol with LPS (Group P1), 300micromol/L propofol with LPS (Group P2), 3micromol/L midazolane with LPS (Group M1), and 30micromol/L midazolane with LPS (Group M2). TNF-alpha released into the culture media was measured by radioimmunity assay. RESULTS: Compared with the blank control Group C, LPS-induced TNF-alpha productions in Group L, K1, K2, P1, P2, M1 and M2 increased significantly. The levels of TNF-alpha in Group K1 and K2 were significantly lower than those in Group L (P<0.05), but TNF-alpha productions in Group P1, P2, M1 and M2 were not significantly different as compared with that in Group L. CONCLUSION Ketamine can reduce LPS-induced TNF-alpha production of glial cells, thereby inhabiting some of the inflammatory responses. Propofol and midazolam have no effect on the production of TNF-alpha from LPS-stimulated glial cells.
Anesthetics, Intravenous ; pharmacology ; Animals ; Cells, Cultured ; Female ; Glial Fibrillary Acidic Protein ; biosynthesis ; Immunohistochemistry ; Ketamine ; pharmacology ; Lipopolysaccharides ; pharmacology ; Neuroglia ; cytology ; drug effects ; metabolism ; Propofol ; pharmacology ; Rats ; Rats, Wistar ; Tumor Necrosis Factor-alpha ; biosynthesis

OBJECTIVETo investigate the expression of protein kinase C (PKC) in the spinal dorsal horn of rats with formalin-induced pain and the effect of intrathecal ketamine on PKC expression.

METHODSThirty-two SD rats were randomly divided into 4 equal groups, namely the control group, intrathecal saline group (NS), 50 µg ketamine group (K1) and 100 µg ketamine group (K2). The rats were anesthetized with 10% chloral hydrate, and a microspinal catheter was inserted intrathecally into the lumbar region. Five days later, the rats in groups, K1 and K2 were subjected to intrathecal administration of 50 and 100 µg ketamine (10 µl), respectively, followed by 10 µl saline, and those in NS group received 20 µl saline only. Thirty minutes later, 5% formalin (50 µl) was subcutaneously injected into the left hindpaw. The pain intensity score (PIS) was utilized to assess antinociceptive behavior within 1 h after formalin injection. Twenty-four hours later, the left hindpaw thickness was measured and the expression of PKC in the spinal dorsal horn in the L5 segment was assayed using immunohistochemistry.

RESULTSCompared to group NS, groups K1 and K2 showed significantly decreased PIS (P<0.01) in the second phase of formalin-induced pain; 24 h later, the left hindpaw thickness of group NS increased obviously in comparison with that in the control group (P<0.01), whereas the thickness was significantly reduced in group K1 and K2 as compared to that in group NS (P<0.05). The number of immunoreactive cells and the immunohistochemical score of PKC in the spinal dorsal horn were significantly higher in group NS than in group C (P<0.01), but significantly lower in groups K1 and K2 than in group NS (P<0.05).

CONCLUSIONIntrathecal ketamine produces obvious antinociception against formalin-induced pain in rats and inhibits the enhanced PKC expression in the spinal dorsal horn in response to formalin-induced pain, suggesting the important role of PKC in nociceptive signal transmission and modulation in the spinal cord.

Animals ; Formaldehyde ; adverse effects ; Injections, Spinal ; Ketamine ; administration & dosage ; pharmacology ; Male ; Pain ; chemically induced ; metabolism ; Pain Measurement ; Posterior Horn Cells ; metabolism ; Protein Kinase C ; metabolism ; Rats ; Rats, Sprague-Dawley ; Spinal Cord ; drug effects ; metabolism

Animals ; Animals, Newborn ; Brain ; drug effects ; metabolism ; pathology ; Disease Models, Animal ; Excitatory Amino Acid Antagonists ; pharmacology ; therapeutic use ; Female ; Hypoxia-Ischemia, Brain ; drug therapy ; metabolism ; Ketamine ; pharmacology ; therapeutic use ; Male ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Treatment Outcome

OBJECTIVE: To study the effects of ketamine and alcohol on learning and memory in mice and its possible mechanism. METHODS: Forty mice were divided into 4 groups: normal control group, ketamine group, alcohol group, and alcohol plus ketamine group. Ketamine and alcohol were given by intraperitoneal injection and intragastric administration, respectively, 1 time per day, for 14 days. The ability of learning and memory in mice was tested by the method of step-down and Morris water maze. Acetylcholine (ACh) and 5-hydroxy tryptamine(5-HT) in mice brain tissue were analyzed for the possible mechanism. RESULTS: (1) Step-down: The treatment groups lessened the latency and added wrong times (P < 0.05). The number of errors in the combined treatment group significantly increased comparing with the single drug treatment group (P < 0.05). (2) Morris water-maze: The treatment groups prolonged the latency (P < 0.05), reduced the target quadrant activity time significantly (P < 0.05), and decreased the numbers of crossing the former platform significantly (P < 0.05). (3) Biochemical index determination: The concentrations of ACh and 5-HT in treatment groups decreased significantly (P < 0.05), showed a more decreasement comparing with the single drug treatment group. CONCLUSION Ketamine has a synergistic effect with alcohol on learning and memory impairment in mice, which may be related to the common inhibitive effect on the ACh and 5-HT.
Acetylcholine/metabolism* ; Alcohols/pharmacology* ; Animals ; Brain/physiopathology* ; Drug Synergism ; Ketamine/pharmacology* ; Male ; Maze Learning/drug effects* ; Memory/drug effects* ; Memory Disorders/physiopathology* ; Mice ; Mice, Inbred ICR ; Serotonin/metabolism* ; Spatial Behavior/drug effects*

Apoptosis is a programmed, physiologic mode of cell death that plays an important role in tissue homeostasis. As for the central nervous system, ischemic insults can induce pathophysiologic cascade of apoptosis in neurophils. Impairment of astroctye functions during brain ischemia can critically influence neuron survival by neuronglia interactions. We aimed to elucidate the protective effect of ketamine on apoptosis by energy deprivation in astrocytes. Ischemic insults was induced with iodoacetate/ carbonylcyanide mchlorophenylhydrazone (IAA/CCCP) 1.5 mM/ 20 micrometer or 150 micrometer/2 micrometer for 1 hr in the HTB-15 and CRL-1690 astrocytoma cells. Then these cells were reperfused with normal media or ketamine (0.1 mM) containing media for 1 hr or 24 hr. FITC-annexin-V staining and propidium iodide binding were determined by using flow cytometry. Cell size and granularity were measured by forward and side light scattering properties of flow cytometry system, respectively. An addition of keta-mine during reperfusion increased the proportion of viable cells. Ketamine alleviated cell shrinkage and increased granularity during the early period, and ameliorated cell swelling during the late reperfusion period. Ketamine may have a valuable effect on amelioration of early and late apoptosis in the astrocytoma cells, even though the exact mechanism remains to be verified.
Anesthetics, Dissociative/*pharmacology ; Annexin A5/pharmacology ; Apoptosis ; Astrocytes/metabolism ; Astrocytoma/*drug therapy/pathology ; Brain/pathology ; Carbonyl Cyanide m-Chlorophenyl Hydrazone/pharmacology ; Cell Line, Tumor ; Cell Size ; Cell Survival ; Central Nervous System/drug effects/pathology ; Enzyme Inhibitors/pharmacology ; Flow Cytometry/*methods ; Humans ; Indicators and Reagents/pharmacology ; Iodoacetates/pharmacology ; Ischemia/pathology ; Ketamine/metabolism/*pharmacology ; Light ; Neurons/metabolism/pathology ; Neutrophils/metabolism ; Perfusion ; Propidium/pharmacology ; Scattering, Radiation ; Time Factors ; Uncoupling Agents/pharmacology

Ketamine (KTM), a N-methyl-D-aspartate (NMDA) receptor antagonist, was found to has an anti-inflammatory effect, but some patients suffered from exacerbated pro-inflammatory reactions after anesthesia with KTM. The present study was aimed to examine the underlying mechanism of pro-inflammatory effects of KTM. In this study, RAW264.7 cells were exposed to KTM and NMDA alone or combined for 30 min before lipopolysaccharide (LPS) stimulation. The expression levels of IL-6 and TNF-α were detected by RT-PCR and ELISA, and those of NMDA receptors by RT-PCR in RAW264.7 cells. Additionally, the TLR4 expression was determined by RT-PCR and flow cytometry, respectively. The results showed that in RAW264.7 cells, KTM alone promoted the TLR4 expression, but did not increase the expression of IL-6 or TNF-α. In the presence of LPS, KTM caused a significantly higher expression of IL-6 and TNF-α than LPS alone. NMDA could neither alter the IL-6 and TNF-α mRNA expression, nor reverse the enhanced expression of IL-6 and TNF-α mRNA by KTM in LPS-challenged cells. After TLR4-siRNA transfection, RAW264.7 cells pretreated with KTM no longer promoted the IL-6 and TNF-α expression in the presence of LPS. In conclusion, KTM accelerated LPS-induced inflammation in RAW264.7 cells by promoting TLR4 expression, independent of NMDA receptor.
Anesthetics, Dissociative ; pharmacology ; Animals ; Cell Survival ; drug effects ; Gene Expression Regulation ; Inflammation Mediators ; pharmacology ; Interleukin-6 ; genetics ; Ketamine ; pharmacology ; Lipopolysaccharides ; pharmacology ; Macrophages ; drug effects ; metabolism ; Male ; Mice ; N-Methylaspartate ; pharmacology ; RAW 264.7 Cells ; Signal Transduction ; drug effects ; Toll-Like Receptor 4 ; genetics ; metabolism ; Tumor Necrosis Factor-alpha ; genetics

To evaluate the effects of different antagonists on the release of cytochrome c from mitochondria to cytosol and the expression of Bcl-2 in mitochondria in rat hippocampus after ischemia, we examined Bcl-2 and cytochrome c expression by immunoblotting using 4-vessel occlusion (4-VO) as brain ischemia model. The results showed that after 24 h ischemia/reperfusion (I/R) cytochrome c decreased markedly in mitochondria, which was correspondingly increased in the cytosolic fraction. Bcl-2 expression was time-dependent, reaching its peak level after 6 h I/R. In all those samples, there were no alterations in the subcellular distribution of cytochrome oxidase, a mitochondrial respiratory chain protein. The decreases in Bcl-2 and cytochrome c in mitochondria were restored by pretreatment with non-competitive NMDA receptor antagonist ketamine or L-type voltage-gated Ca(2+) channel (L-VGCC) antagonist nifedipine at 20 min prior to ischemia. The results demonstrate that the release of cytochrome c from mitochondria to cytosol and the up-regulation of Bcl-2 are possibly mediated by NMDA receptors or L-VGCC following brain ischemia. Cytochrome c release may be injurious while Bcl-2 up-regulation may be protective to ischemic hippocampus.
Animals ; Brain Ischemia ; metabolism ; Calcium Channel Blockers ; pharmacology ; Calcium Channels, L-Type ; drug effects ; Cytochromes c ; metabolism ; Cytosol ; Hippocampus ; metabolism ; Ketamine ; pharmacology ; Male ; Mitochondria ; metabolism ; Nifedipine ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; biosynthesis ; genetics ; Rats ; Rats, Sprague-Dawley ; Receptors, N-Methyl-D-Aspartate ; antagonists & inhibitors ; Up-Regulation

OBJECTIVETo investigate the protective effects of different concentrations of ketamine against acute lung injury induced by lipopolysaccharide (LPS) in rats and its mechanism.

METHODSForty-eight male Wistar rats were randomized into 4 equal groups, namely the control group, LPS group, ketamine group I (5 mg/kg), and ketamine group II (10 mg/kg). The neutrophil count, protein contents in the bronchoalveolar lavage fluid (BALF) and the wet/dry lung weight ratio were measured 4 h after LPS injection. TNF-alpha, IL-8, NO, iNOS and NF-kappaB were also measured in the lung tissues.

RESULTSIn LPS group, the neutrophil count, protein contents in BALF, the wet/dry lung weight ratio and the levels of tumor necrosis factor-alpha(TNF-alpha), interleukin-8 (IL-8), and NO were all significantly increased compared with the control group (P<0.01). The mRNA expression of iNOS and the protein expression of NF-kappaB were also increased in LPS groups. Ketamine treatment attenuated the increase in wet/dry lung weight ratio, neutrophil count, and protein contents in BALF in a dose-dependent manner. Ketamine also dose-dependently inhibited the production of TNF-alpha, IL-8 , and NO and lowered iNOS mRNA and NF-kappaB protein expression.

CONCLUSIONKetamine can offer protection against LPS-induced acute lung injury in rats by inhibiting the expression of NF-kappaB and attenuating the production of the inflammatory cytokines.

Acute Lung Injury ; chemically induced ; drug therapy ; Animals ; Bronchoalveolar Lavage Fluid ; Interleukin-8 ; metabolism ; Ketamine ; pharmacology ; Lipopolysaccharides ; Lung ; drug effects ; pathology ; Male ; NF-kappa B ; metabolism ; Neutrophils ; metabolism ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase Type II ; metabolism ; Rats ; Rats, Wistar ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVE: To explore the effect of ketamine on adrenal pheochromocytoma (PC12) cell proliferation inhibition and induction of apoptosis and its mechanism. METHODS: PC12 cells of rats were models for dopaminergic neuron. PC12 cells were cultured with ketamine at concentrations of 0.9, 1.2, 1.5, 1.8 and 2.1 mmol/L, respectively. The cell viability was measured by MTT method after incubation at 12, 24, 48 and 72h. Hoechst stain was used to observe the morphological changes of apoptosis. PC12 cells cultured after 48 h with different concentrations of ketamine were selected to detect apoptotic rate using flow cytometry and detect the expression of bax and bcl-2 proteins using Western blotting. RESULTS: For different concentrations of ketamine, vitality of PC12 cells significantly decreased with increase of the incubation time. Apoptosis was obviously observed using Hoechst staining. Flow cytometry showed that apoptosis rates significantly increased with increasing ketamine concentrations. CONCLUSION Ketamine can inhibit the proliferation of PC12 cell by inducing apoptosis of the PC12 cell in a concentrations-dependent manner. The underlying mechanism may be related to promoting the expression of bax and inhibiting the expression of bcl-2 in the cells.
Anesthetics, Dissociative/pharmacology* ; Animals ; Apoptosis/drug effects* ; Blotting, Western ; Cell Proliferation/drug effects* ; Dose-Response Relationship, Drug ; Flow Cytometry ; Gene Expression Regulation/drug effects* ; Ketamine/pharmacology* ; PC12 Cells ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Rats ; Time Factors ; bcl-2-Associated X Protein/metabolism*