Ketamine is a noncompetitive antagonist of the N-methyl-D-aspartate (NMDA) receptors widely used as a dissociative anesthetic. In subanesthetic doses, ketamine is also currently used in pain management.1 Regardless of its generalized use, conflicting views regarding its neurotoxic or neuroprotective effects still persist.2 Currently, a large number of studies associate ketamine with mitochondrial dysfunction. In neural stem cells, ketamine decreased mitochondrial membrane potential, leading to cytochrome c release, reactive oxygen species production, and apoptosis.3 In primary cell cultures, ketamine was shown to be neurotoxic through increased expression of nitrotyrosine.4 Importantly, in hepatocytes and human lymphocytes, ketamine also led to mitochondrial dysfunction,5,6 indicating that its toxicity may be modulated through NMDA-independent pathways.
In vivo, the action of anesthetic doses of ketamine at the mitochondrial level is mostly unknown. Yet, it was recently reported that knockout mice with deficient mitochondrial complex I function displayed increased resistance to ketamine’s hypnotic action.7 Subanesthetic doses of ketamine led to transient generation of massive hydroxyl radical levels in mice8 and altered activity of the mitochondrial respiratory chain in several rat brain regions.9
Nitric oxide (NO) is an important brain messenger involved in anesthesia,10 as well as in regulating brain mitochondrial function.11 Importantly, ketamine-induced memory impairment was shown to be mediated by NO and could be antagonized by the nonselective NO synthase (NOS) inhibitor, N-nitro-L-arginine (L-NAME).12
We hypothesized that ketamine’s action at the mitochondrial level could be modulated through NO release. We administered escalating anesthetic doses of ketamine to adult rats and evaluated the ketamine-induced effects on the mitochondrial respiratory function and brain redox state. To reinforce our findings, we used L-NAME to inhibit the activity of mitochondrial NOS (mtNOS).
Animals and Reagents
All animal procedures were approved by the local ethical committee and by the Portuguese Agency for Animal Welfare, general board of Veterinary Medicine, reference 3053 of 2012/03/19. All efforts were made to ensure minimal animal suffering. Fifty-four adult male Wistar rats (90–110 days), obtained from Charles River (Barcelona, Spain), were randomly allocated into 5 groups using the Web site http://www.random.org. Rats were kept under controlled environmental conditions of 19 to 21°C and 45% to 55% humidity.
Substrates, enzymes, and standard chemicals reagents were of the highest grade commercially available and obtained from Sigma (Sigma-Aldrich, Steinheim, Germany). All solutions were prepared with ultrapure water purified by a Milli-Q Gradient system (Millipore, Bedford, MA).
Rats were injected intraperitoneally with a single isovolumetric dose (1.5 mL/kg) of ketamine (Imalgène1000®, Merial, Porto Salvo, Portugal, 100 mg/mL) in saline solution, at the following concentrations: 0 (K0), 50 (K50), 100 (K100), and 150 mg/kg (K150). The selected doses are regularly used in rodent models.13,14 Another group (L-K100) was treated with a dose of 100 mg/kg ketamine combined with L-NAME in a dose of 3 mg/kg, which was previously shown to be effective in antagonizing ketamine-induced memory impairment.12 Rats were allowed to breathe spontaneously with oxygen support during anesthesia. Continuous pulse oximetry (S&W 9040, Athena, Munich, Germany) at the hind limb was used to monitor heart rate and verify arterial oxygen saturation. Body temperature was maintained between 36 and 38°C by a homeothermic blanket connected to a rectal thermal probe (50-7061-F, Harvard Apparatus Ltd, Kent, UK).
The anesthetic level was continuously monitored; respiratory and heart rates were recorded every 5 minutes. Loss and recovery of righting reflex (consciousness), regain ability to walk/evaluation of ataxia, and recovery of normal gait were also assessed. The following stages were considered: (a) unconsciousness period—time frame between loss and recovery of righting reflex; (b) total time of behavioral ketamine effect—time frame between the onset of behavioral modifications and the recovery of normal gait. After anesthesia the animals recovered in their home cage. Rats were killed 6 hours after the administration time point by decapitation. Brains and blood samples were collected.
Isolation of Brain Mitochondria
Brain mitochondria were prepared at 4°C as previously described,15 modified as follows. The brain was homogenized in ice-cold isolation medium containing 225 mM mannitol, 75 mM sucrose, 5 mM HEPES, pH 7.4, 1 mM EGTA, and 1 mg/mL bovine serum albumin (BSA). The brain homogenates were first centrifuged at 2000g for 5 minutes. The supernatant was preserved and the pellet, including the synaptosomal layer, was resuspended in the isolation medium without BSA and containing 0.02% digitonin; after a 2-minute incubation, it was centrifuged at 4000g for 10 minutes. The new pellet, here referred to as postmitochondrial pellet, was quickly frozen in liquid nitrogen and stored at −80°C. Both resultant supernatants were mixed and further centrifuged at 12,000g for 10 minutes, twice, in isolation medium without BSA and EGTA. The supernatant of this centrifugation was also quickly frozen in liquid nitrogen and stored at −80°C. The end mitochondrial pellet was resuspended in 300 μL of the same medium and divided into 2 fractions. One was quickly frozen in liquid nitrogen and stored at −80°C. The other was used immediately for mitochondrial respiratory activity, mitochondrial membrane potential (ΔΨm), and hydrogen peroxide (H2O2) production evaluation. Mitochondrial protein concentration was determined by the Biuret assay and verified through citrate synthase activity to ensure that the amount of mitochondria per milligram of protein was similar within the different groups.
Mitochondrial Respiratory Activity
Oxygen consumption of isolated mitochondria was monitored polarographically with a Clark-type oxygen electrode, using Hansatech Oxygraph Measurement System (Hansatech, Norfolk, UK) maintained at 25°C. Experiments were conducted in respiration buffer containing 0.5 mg/mL mitochondrial protein.15 The effect of ketamine administration in mitochondrial respiration was measured by energizing the mitochondrial fraction with different subtracts: complex I substrates glutamate + malate, complex II substrate succinate, or complex IV substrate ascorbate + N,N,N′,N′-tetramethyl-p-phenylenediamine. Oxygen consumption was measured in the absence (state 4) or presence of 100 μM adenosine diphosphate (ADP, state 3). Respiratory control ratio (RCR = state 3/state 4) and ADP/O (number of ADP molecules added to the medium per oxygen atom consumed during phosphorylation) were calculated.
Mitochondrial Membrane Potential
The ΔΨm was assessed using the fluorescence of safranine O (5 μM), with excitation and emission wavelengths of 495 and 586 nm, respectively. ΔΨm was induced by the addition of glutamate + malate as substrate. Data obtained were calibrated using a K+ gradient. To this end, safranine O fluorescence was recorded in the presence of 2 nM valinomycin and stepwise increasing K+ concentrations (0.2–120 mM) which allowed calculation of ΔΨm by the Nernst equation assuming a matrix K+ = 150 mM.16
Mitochondrial Enzyme Assays
All enzymatic activities were evaluated at 30°C using a microplate spectrophotometer (Power Wave XS2, BioTek, Bad Friedrichshall, Germany). Mitochondrial samples stored at −80°C were freeze-thawed and shaken 3 times followed by sonication to break membranes.
Complex I Activity
The complex I (NADH-ubiquinone reductase) activity was quantified by spectrophotometric assay, as previously described.17
Mitochondrial Nitric Oxide Synthase Activity
The mtNOS activity was determined by spectrophotometric oxyhemoglobin assay, as previously described.18 Production of NO was calculated from the absorbance change at 401 to 420 nm (ε401–420 = 100 per mM/cm), inhibited by 2 mM NG-monomethyl-L-arginine.
Measurement of Hydrogen Peroxide Generation
The H2O2 production was assayed by measuring the increase fluorescence (530 nm excitation, 590 nm emission) due to the reaction of Ampliflu Red with H2O2 in the presence of horseradish peroxidise.19 The assays were performed by incubating fresh intact mitochondria in the presence of glutamate (5 mM) plus malate (2.5 mM) as complex I substrates and in the presence of succinate (5 mM) as complex II substrate. The rate of H2O2 generation was calculated using a standard curve of H2O2 stabilized solution.
Brain Redox State Assay
The first rejected supernatant and postmitochondrial pellet were used for brain redox state assay. The postmitochondrial pellet was homogenized (Ultra-Turrax T25 Basic, IKA, Germany) for 1 minute at 13,000 rpm in 4 volumes of ice-cold Tris-HCl (50 mM, pH 7.4) containing 0.5 mL/L Triton X-100. This was further centrifuged at 10,000g for 10 minutes to obtain the supernatant for determination of superoxide dismutase (SOD) and catalase activities, total antioxidant status, reduced and oxidized glutathione (GSH and GSSG). Protein was measured by the Bradford method.
Quantification of Lipid Peroxidation and Protein Carbonyls
Lipid peroxidation was determined measuring malondialdehyde equivalents, using the thiobarbituric acid assay, as previously described.20 Protein carbonyls were quantified through the spectrophotometric carbonyl assay using 2,4-dinitrophenylhydrazine.20
Quantification of Reduced and Oxidized Glutathione
The GSH and GSSG levels were evaluated by fluorescence detection using an emission of 420 nm and excitation at 350 nm in a Cary Eclipse fluorometer (Varian Analytical Instruments, Palo alto, CA).21 Their levels were compared with a series of fresh linear GSH and GSSG standard curves.
Quantification of the Total Antioxidant Status
The 2,2′-azino-bis(3-ethylbenthiazoline-6-sulfonic acid) radical assay was used to measure the total antioxidant status according to procedures described elsewhere.22 The total antioxidant status in the samples was calculated with reference to the trolox standard curve.
The SOD activity was determined as described previously,23 in mitochondrial and postmitochondrial fractions. The determination of catalase activity was performed as described previously24 in postmitochondrial fractions.
Measurement of Nitric Oxide Levels in Plasma
As an index of NO levels in plasma, its oxidation products were measured. We used the Griess method after conversion of nitrate to nitrite by copperized cadmium granules25 in an autoanalysis segmented flow with a previous online separation step (dialysis) (Skalar® Sanplus, Breda, The Netherlands). The determinations were obtained by molecular absorption at 550 nm.
The number of animals used per group was based on previous related works,20,26 where we used mitochondrial whole brain extracts obtained from male Wistar rats to show that the administration of a neurotoxic dose of 3,4-methylenedioxymethamphetamine (“ecstasy”) resulted in increased lipid peroxidation, protein carbonylation, and mitochondrial DNA deletions, which could be prevented by the inhibition of monoamine oxidase B (MAO-B)26 or acetyl-L-carnitine.20 By running post hoc F tests power analyses (for α = 0.05, 4 experimental groups with n = 6), we obtained: for lipid peroxidation data a noncentrality parameter of λ = 34.19, a size effect f = 1.20 and a power of 1.00; for carbonilation a noncentrality parameter of λ = 13.99, a size effect f = 0.76 and a power of 0.82; and for changes in respiratory chain subunits a noncentrality parameter of λ = 35.80, a size effect f = 1.22 and a power of 1.00, with a critical F value F(3,20) = 3.10. Based on these results, we used 6 rats per experimental group. Because the K0, K50, K100, and K150 groups were repeated twice, for some evaluations (behavioral effect, state 3 and 4 respiration, NADH activity, mtNOS activity, and SOD activity), the sample size was n = 12 for those groups and n = 6 for the L-K100 group.
Comparisons among groups were made using 1-way analysis of variance followed by the Tukey multiple comparisons test. The normality of the residuals was verified using the D’Agostino-Pearson normality test for experimental groups with n = 12, and the Kolmogorov-Smirnov with Dallal-Wilkinson-Lilliefor P value for the L-K100 group (n = 6). Statistical significance was set at P < 0.05 or P < 0.01 when data failed to pass the Brown-Forsythe test to evaluate homogeneity of variance. P values for normality and homogeneity tests are reported in the figure legends. Data are expressed as mean values ± SD. All tests were conducted using the GraphPad-Prism software, version 6.00 for Mac Os X (GraphPad Software, Inc., San Diego, CA).
Ketamine Anesthetic Behavioral Variables
The respiratory and heart rates of anesthetized animals were not significantly different among groups and all rats maintained arterial oxygen saturation over 90% (data not shown). Within the K100, K150, and L-K100 groups, all animals lost the righting reflex after ketamine administration within 3 minutes. For the K50 group, this loss was only observed in 6 of 12 rats. Analysis of the unconsciousness period data revealed a significant effect of the ketamine dose (F(3,38) = 62.26, P < 0.001). Further testing showed that the unconsciousness period for the K50 group was lower than the K100 (P < 0.001) and the K150 (P < 0.001) groups. The unconsciousness period for the K100 group was also shorter than that in the K150 group (P < 0.001) (Fig. 1A). The total time observed for the ketamine behavioral effect was affected by different ketamine doses (F(3,38) = 55.74, P < 0.001). The behavioral effect of ketamine for the K150 group was higher than for the K100 (P < 0.001) and K50 (P < 0.001). The K100 group total time was also longer when compared with the K50 (P < 0.001) (Fig. 1B). L-NAME administration did not significantly affect the ketamine anesthetic variables (K100 versus L-K100, P = 0.38 for unconsciousness period; K100 versus L-K100, P = 1.00 for total behavioral time).
Impairment of Mitochondrial Respiration at the Complex I Level and NADH-Ubiquinone Reductase Activity
When using glutamate-malate as a substrate, state 3 respiration did not present significant differences among groups (Fig. 2A, F(4,39) = 1.46, P = 0.23). State 4 respiration, however, was affected by ketamine administration (F(4,35) = 4.23, P = 0.0068), increasing for K50 (P = 0.023), K100 (P = 0.012), and K150 (P = 0.015) versus the K0 group (Fig. 2B). L-NAME administration resulted in a value that was not significantly different from the control (L-K100 versus K0, P = 0.24, Fig. 2B). RCR and ADP/O are measures of integrity and efficiency of the mitochondrial phosphorylation system. Concerning the RCR values, analysis of variance displayed the following value F(4,33) = 2.16, P = 0.094; no further comparisons among groups were made (Fig. 2C). ADP/O values were decreased by ketamine (F(4,19) = 5.26, P = 0.0050), specifically, K50 (P =0.0033), K100 (P =0.014), and K150 (P =0.028) doses were decreased versus the K0 group (Fig. 2D). Again, L-NAME administration resulted in a value that was not significantly different from the control (L-K100 versus K0, P = 0.19, Fig. 2D). No significant differences were observed when measuring complex II (through succinate) and complex IV (through ascorbate + N,N,N′,N′-tetramethyl-p-phenylenediamine) respiration variables (data not shown, F(3,20) = 0.4086, P = 0.75; and F(3,16) = 0.23, P = 0.88, respectively). No significant differences were observed when measuring maximum membrane potential using glutamate-malate as a substrate (F(4,21) = 0.74, P = 0.58, Fig. 2E). When evaluating NADH-ubiquinone redutase activity (F(4,34) = 4.53, P = 0.0048), an inhibition was observed for the K50 (P =0.026), K100 (P =0.0041), and K150 (P =0.039) versus the K0 group but not when L-NAME was administered (L-K100 versus K0, P = 0.62, Fig. 2F).
Increased Brain mtNOS and Plasmatic NO
The activity of mtNOS is often associated with the respiratory chain complex I. Our results showed that brain mtNOS activity was affected by ketamine administration (F(4,32) = 15.77, P < 0.001) because both the K50 and the K100 groups had an increase when compared with the K0 group (P = 0.020 and P = 0.034, respectively) and the K150 group (P < 0.001 for both). As expected, L-NAME administration inhibited mtNOS activity (P < 0.001, L-K100 versus K100 and P =0.018, L-K100 versus K0) (Fig. 3A). In accordance, the NO plasma levels were also affected by ketamine (F(4,46) = 24.30, P < 0.001), with the K50 (P < 0.001) and K100 (P < 0.001) groups displaying increased NO versus the K0 group. These levels were also higher in the K50 group when compared with the K150 (P < 0.001) group. L-NAME administration prevented a ketamine-induced increase in NO levels (P<0.001, L-K100 versus K100) (Fig. 3B).
Brain Mitochondrial and Postmitochondrial Oxidative Stress
Ketamine treatment increased mitochondrial H2O2 generation when oxidizing complex I (glutamate + malate, F(2,13) = 19.14, P < 0.001) and complex II (succinate, F(2,15) = 20.49, P < 0.001) substrates. The K100 group had increased H2O2 levels (P <0.001, K100 versus K0, Fig. 4A), which were prevented by L-NAME administration (P < 0.001, L-K100 versus K100, Fig. 4A). However, when succinate was used as a substrate for complex II, the H2O2 levels were only partially reduced (P =0.030, L-K100 versus K100, Fig. 4B).
SOD activity was increased in ketamine-treated groups both in mitochondrial (F(2,27) = 22.60, P < 0.001) and in postmitochondrial fractions (F(2,27) = 20.99, P < 0.001). In both extracts, the K100 group was increased versus K0 (P < 0.001, in both cases, Fig. 4, C and D). However, in mitochondrial extracts, L-NAME administration did not prevent ketamine-induced SOD activity (P = 0.0064, L-K100 versus K0).
Ketamine administration did not affect lipid peroxidation, carbonyl formation, the GSH/GSSG ratio, or the catalase activity (data not shown, F(3,24) = 0.92, P = 0.44; F(3,24) = 0.16, P = 0.92; F(3,24) = 0.23, P = 0.87; and F(3,24) = 0.83, P = 0.49). In accordance, the total antioxidant capacity in these fractions remained unaltered (data not shown, F(3,24) = 0.14, P = 0.93).
The present work shows that in adult rats ketamine administration led to altered function of the mitochondrial respiratory chain complex I, which was concomitant with increased mtNOS and SOD activities, leading to H2O2 generation. Importantly, this could be prevented by the administration of L-NAME, a nonselective NOS inhibitor.
The ketamine doses used in the present work led to a dose-dependent effect in the unconsciousness period and total ketamine behavioral effects that correspond to subanesthetic (K50), light anesthetic (K100), and deep anesthetic (K150) dose effects. This is in agreement with what was previously reported for the anesthetic depth under the same ketamine doses.13,14
At the mitochondrial level, ketamine administration led to increased oxygen consumption in state 4 and decreased oxidative phosphorylation efficiency when mitochondria were energized with the complex I substrate (glutamate/malate). When additional substrates were used, no other changes could be observed. Furthermore, the activity of the complex I (NADH-ubiquinone oxidoreductase) was inhibited by ketamine, which strengthens the possibility of a direct effect of ketamine over this complex. In agreement, it was recently reported that knockout mice with deficient complex I function displayed increased resistance to ketamine.7 Complex I activity plays a major role in the control of oxidative phosphorylation27; however, the excessive enzymatic capacity and the amount of intermediate substrates available, compared with what is in fact required for state 3 respiration,16 may mask the effect of ketamine in this state and explain the observed absence of effect. State 4 corresponds to an ADP-limited resting state where increased oxygen consumption can be explained by either increased proton leak or reversed electron transport.28 However, in the present case, proton leak can be excluded by the absence of effects on state 4 respiration when using succinate as substrate. Reversed electron transport, on the other side, is usually linked to increased superoxide production.29 There is also a functional association between complex I and mtNOS, since mtNOS activity is due to a reversed electron flow from complex I30 that leads to higher NO production in state 4.31 Thus, the reversed electron transport hypothesis seems to fit our results. Loss of complex I activity together with increased state 4 respiration can justify the decreased ADP/O observed for all ketamine doses. The present results are in accordance with those obtained in hepatic cell cultures, where exposure to ketamine reduced adenosine triphosphate synthesis due to downregulation of complex I activity.32 Of note, mitochondrial complex I function was unaltered when L-NAME was coadministered with ketamine. The present results are in accordance with a former report where ketamine-induced mitochondrial apoptotic events, in primary cell cultures, were attenuated using the neuronal NOS inhibitor, 7-nitroindazole.4
mtNOS is located in the inner membrane, and its activity is associated with the mitochondrial respiratory chain complex I.18,30 It was reported that mainly in state 4, electrons derived from oxidation of NADH to complex I instead of flowing through the respiratory chain can be reversed to mtNOS.30,31 Elevated levels of NO can signal through the guanylyl cyclase receptor and increase presynaptic glutamate release.33 Furthermore, increased production of NO can lead to peroxynitrite formation that was associated with complex I inhibition.11 Here, we observed a significant increase in mtNOS activity for both K50 and K100 groups but not for the K150 group. Adding to this, the NO plasma levels were also increased for the K50 and K100 groups. Increased NO both in plasma and in brain tissue has been observed after ketamine anesthesia.14,25 Acutely, ketamine, an NMDA receptor antagonist, blocks Ca2+ influx, decreasing glutamatergic activity and consequently NO production by NOS activity.10,34 However, after the ketamine washout in neuronal cultures, cytosolic Ca2+ levels were seen to suffer a 3-fold increase, this oscillation was associated with potential neurotoxic effects35 and may boost mtNOS activity.18 Importantly, L-NAME administration together with the K100 dose was efficient in preventing increased mtNOS activity and reduced plasmatic NO to the control levels. The unaltered activity of mtNOS after the K150 dose may result from a more prolonged inhibition of the NMDA receptors and a longer washout period, which could delay the activation of mtNOS in this group and explain our results. Moreover, contrary to what is observed for NMDA receptors, only high ketamine levels were reported to inhibit voltage-sensitive Ca2+ channels,36 which may also have contributed to the absence of mtNOS activation in the K150 group.
SOD is a crucial player in the antioxidant defense system, eliminating superoxide radical by converting it into H2O2 and oxygen and preventing the conversion of NO into peroxynitrite.11 Increased SOD activity may result from increased NO generation through the NO/Ras/extracellular-regulated kinase 1/2 pathway37 or from H2O2 upregulation of SOD2 transcription mediated by translocation of the nuclear factor κB into the nucleus38 (Fig. 5). Importantly, it was previously shown that in cortical neurons ketamine led to augmented translocation of nuclear factor κB to the nucleus.39 In the present work, increased H2O2 levels were observed as a result of ketamine administration both when glutamate/malate or succinate were used as substrates. Of note, L-NAME administration could only prevent the increased H2O2 generation when glutamate/malate was used. These results may indicate that the increase in H2O2 levels is only partially due to NO modulation. In accordance, we show that, in mitochondria, ketamine-induced SOD activity is not prevented by L-NAME administration. Importantly, although a positive correlation between SOD and mtNOS activities was previously established regarding the modulation of the brain redox status,40 SOD-increased activity can also be justified by complex I inhibition.41 Despite former reports showing that ketamine may induce hydroxyl radical generation8 and promote lipid peroxidation or carbonyl formation in different brain regions,42 and regardless of the increased H2O2 generation, in the present study ketamine did not affect the total antioxidant capacity, which probably results from SOD activation. Previous preconditioning studies performed with volatile anesthetics have shown that a transitory inhibition of complex I, with moderate increase in NO or superoxide radical and concomitant SOD-improved activity, was protective against later insults.43,44 At the neuronal level, the putative preconditioning effect of ketamine has not yet been fully explored. However, in vitro, the protective effect of a transient NMDA receptor inactivation was already demonstrated.45 This may be relevant at the clinical level and deserves further exploration.
In summary, we propose that ketamine leads to a key interference in complex I activity, resulting in impaired oxidative phosphorylation efficiency and increased mtNOS activity due to reversed electron flow from complex I, which would lead to increased levels of superoxide and NO and trigger SOD activity. The triggering of SOD seems to preserve the overall redox status (Fig. 5). The present findings clarify the role of NO modulation in ketamine anesthesia, providing new data on a relevant clinical mechanism.
Name: Carlos Venâncio, DVM, PhD.
Contribution: This author participated in study design, data collection, the analysis reported in this manuscript, writing of the first draft of the paper, and revising the article.
Attestation: Carlos Venâncio approved the final manuscript. Carlos Venâncio attests to the integrity of the original data and the analysis reported in this manuscript. Carlos Venâncio is the archival author.
Name: Luís Félix, MSc.
Contribution: This author participated in data collection, data analysis, revising the article, and contributed to the final manuscript preparation.
Attestation: Luís Félix approved the final manuscript. Luís Félix attests to the integrity of the original data and the analysis reported in this manuscript.
Name: Vanessa Almeida, MSc.
Contribution: This author participated in data collection and revising the article.
Attestation: Vanessa Almeida approved the final manuscript.
Name: João Coutinho, PhD.
Contribution: This author participated in data collection, data analysis, and revising the article.
Attestation: João Coutinho approved the final manuscript.
Name: Luís Antunes, DVM, PhD.
Contribution: This author participated in study design and revised the analysis of the data and the article.
Attestation: Luís Antunes approved the final manuscript.
Name: Francisco Peixoto, PhD.
Contribution: This author conducted the study design, data collection, writing of the first draft of the paper, and contributed to the final manuscript preparation.
Attestation: Francisco Peixoto approved the final manuscript. Francisco Peixoto attests to the integrity of the original data and the analysis reported in this manuscript.
Name: Teresa Summavielle, PhD.
Contribution: This author conducted the study design, reviewed the analysis of the data, and the writing of the manuscript.
Attestation: Teresa Summavielle approved the final manuscript.
This manuscript was handled by: Marcel E. Durieux, MD, PhD.
1. Kohrs R, Durieux ME. Ketamine: teaching an old drug new tricks. Anesth Analg. 1998;87:1186–93
2. Himmelseher S, Durieux ME. Revising a dogma: ketamine for patients with neurological injury? Anesth Analg. 2005;101:524–34
3. Bai X, Yan Y, Canfield S, Muravyeva MY, Kikuchi C, Zaja I, Corbett JA, Bosnjak ZJ. Ketamine enhances human neural stem cell proliferation and induces neuronal apoptosis via reactive oxygen species-mediated mitochondrial pathway. Anesth Analg. 2013;116:869–80
4. Wang C, Sadovova N, Patterson TA, Zou X, Fu X, Hanig JP, Paule MG, Ali SF, Zhang X, Slikker W Jr. Protective effects of 7-nitroindazole on ketamine-induced neurotoxicity in rat forebrain culture. Neurotoxicology. 2008;29:613–20
5. Lee ST, Wu TT, Yu PY, Chen RM. Apoptotic insults to human HepG2 cells induced by S-(+)-ketamine occurs through activation of a Bax-mitochondria-caspase protease pathway. Br J Anaesth. 2009;102:80–9
6. Braun S, Gaza N, Werdehausen R, Hermanns H, Bauer I, Durieux ME, Hollmann MW, Stevens MF. Ketamine induces apoptosis via the mitochondrial pathway in human lymphocytes and neuronal cells. Br J Anaesth. 2010;105:347–54
7. Quintana A, Morgan PG, Kruse SE, Palmiter RD, Sedensky MM. Altered anesthetic sensitivity of mice lacking Ndufs4, a subunit of mitochondrial complex I. PLoS One. 2012;7:e42904
8. Zuo DY, Wu YL, Yao WX, Cao Y, Wu CF, Tanaka M. Effect of MK-801 and ketamine on hydroxyl radical generation in the posterior cingulate and retrosplenial cortex of free-moving mice, as determined by in vivo microdialysis. Pharmacol Biochem Behav. 2007;86:1–7
9. de Oliveira L, Fraga DB, De Luca RD, Canever L, Ghedim FV, Matos MP, Streck EL, Quevedo J, Zugno AI. Behavioral changes and mitochondrial dysfunction in a rat model of schizophrenia induced by ketamine. Metab Brain Dis. 2011;26:69–77
10. Gonzales JM, Loeb AL, Reichard PS, Irvine S. Ketamine inhibits glutamate-, N-methyl-D-aspartate-, and quisqualate-stimulated cGMP production in cultured cerebral neurons. Anesthesiology. 1995;82:205–13
11. Riobó NA, Clementi E, Melani M, Boveris A, Cadenas E, Moncada S, Poderoso JJ. Nitric oxide inhibits mitochondrial NADH: ubiquinone reductase activity through peroxynitrite formation. Biochem J. 2001;359:139–45
12. Boultadakis A, Pitsikas N. Anesthetic ketamine impairs rats’ recall of previous information: the nitric oxide synthase inhibitor N-nitro-L-arginine methylester antagonizes this ketamine-induced recognition memory deficit. Anesthesiology. 2011;114:1345–53
13. Cui X, Li J, Li T, Ji F, Bu X, Zhang N, Zhang B. Propofol and ketamine-induced anesthetic depth-dependent decrease of CaMKII phosphorylation levels in rat hippocampus and cortex. J Neurosurg Anesthesiol. 2009;21:145–54
14. Wu J, Kikuchi T, Wang Y, Sato K, Okumura F. NOx- concentrations in the rat hippocampus and striatum have no direct relationship to anaesthesia induced by ketamine. Br J Anaesth. 2000;84:183–9
15. Vilela SM, Santos DJ, Félix L, Almeida JM, Antunes L, Peixoto F. Are fentanyl and remifentanil safe opioids for rat brain mitochondrial bioenergetics? Mitochondrion. 2009;9:247–53
16. Akerman KE, Wikström MK. Safranine as a probe of the mitochondrial membrane potential. FEBS Lett. 1976;68:191–7
17. Kiebish MA, Han X, Cheng H, Lunceford A, Clarke CF, Moon H, Chuang JH, Seyfried TN. Lipidomic analysis and electron transport chain activities in C57BL/6J mouse brain mitochondria. J Neurochem. 2008;106:299–312
18. Ghafourifar P, Asbury ML, Joshi SS, Kincaid ED. Determination of mitochondrial nitric oxide synthase activity. Methods Enzymol. 2005;396:424–44
19. Chen Q, Vazquez EJ, Moghaddas S, Hoppel CL, Lesnefsky EJ. Production of reactive oxygen species by mitochondria: central role of complex III. J Biol Chem. 2003;278:36027–31
20. Alves E, Binienda Z, Carvalho F, Alves CJ, Fernandes E, de Lourdes Bastos M, Tavares MA, Summavielle T. Acetyl-L-carnitine provides effective in vivo neuroprotection over 3,4-methylenedioximethamphetamine-induced mitochondrial neurotoxicity in the adolescent rat brain. Neuroscience. 2009;158:514–23
21. Hissin PJ, Hilf R. A fluorometric method for determination of oxidized and reduced glutathione in tissues. Anal Biochem. 1976;74:214–26
22. Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans C. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic Biol Med. 1999;26:1231–7
23. Payá M, Halliwell B, Hoult JR. Interactions of a series of coumarins with reactive oxygen species. Scavenging of superoxide, hypochlorous acid and hydroxyl radicals. Biochem Pharmacol. 1992;44:205–14
24. Aebi H. Catalase in vitro. Methods Enzymol. 1984;105:121–6
25. Alva N, Palomeque J, Carbonell T. Nitric oxide induced by ketamine/xylazine anesthesia maintains hepatic blood flow during hypothermia. Nitric Oxide. 2006;15:64–9
26. Alves E, Summavielle T, Alves CJ, Gomes-da-Silva J, Barata JC, Fernandes E, Bastos Mde L, Tavares MA, Carvalho F. Monoamine oxidase-B mediates ecstasy-induced neurotoxic effects to adolescent rat brain mitochondria. J Neurosci. 2007;27:10203–10
27. Venâncio C, Antunes L, Félix L, Rodrigues P, Summavielle T, Peixoto F. Chronic ketamine administration impairs mitochondrial complex I in the rat liver. Life Sci. 2013;93:464–70
28. Brookes PS, Land JM, Clark JB, Heales SJ. Peroxynitrite and brain mitochondria: evidence for increased proton leak. J Neurochem. 1998;70:2195–202
29. Panov A, Kubalik N, Zinchenko N, Hemendinger R, Dikalov S, Bonkovsky HL. Respiration and ROS production in brain and spinal cord mitochondria of transgenic rats with mutant G93a Cu/Zn-superoxide dismutase gene. Neurobiol Dis. 2011;44:53–62
30. Parihar MS, Nazarewicz RR, Kincaid E, Bringold U, Ghafourifar P. Association of mitochondrial nitric oxide synthase activity with respiratory chain complex I. Biochem Biophys Res Commun. 2008;366:23–8
31. Boveris A, Valdez LB, Zaobornyj T, Bustamante J. Mitochondrial metabolic states regulate nitric oxide and hydrogen peroxide diffusion to the cytosol. Biochim Biophys Acta. 2006;1757:535–42
32. Chang HC, Chen TL, Chen RM. Cytoskeleton interruption in human hepatoma HepG2 cells induced by ketamine occurs possibly through suppression of calcium mobilization and mitochondrial function. Drug Metab Dispos. 2009;37:24–31
33. Neitz A, Mergia E, Eysel UT, Koesling D, Mittmann T. Presynaptic nitric oxide/cGMP facilitates glutamate release via hyperpolarization-activated cyclic nucleotide-gated channels in the hippocampus. Eur J Neurosci. 2011;33:1611–21
34. Sjakste N, Baumane L, Meirena D, Lauberte L, Dzintare M, Kalviņs I. Drastic increase in nitric oxide content in rat brain under halothane anesthesia revealed by EPR method. Biochem Pharmacol. 1999;58:1955–9
35. Sinner B, Friedrich O, Zink W, Zausig Y, Graf BM. The toxic effects of s(+)-ketamine on differentiating neurons in vitro as a consequence of suppressed neuronal Ca2+ oscillations. Anesth Analg. 2011;113:1161–9
36. Hirota K, Lambert DG. Ketamine: its mechanism(s) of action and unusual clinical uses. Br J Anaesth. 1996;77:441–4
37. Scorziello A, Santillo M, Adornetto A, Dell’aversano C, Sirabella R, Damiano S, Canzoniero LM, Renzo GF, Annunziato L. NO-induced neuroprotection in ischemic preconditioning stimulates mitochondrial Mn-SOD activity and expression via Ras/ERK1/2 pathway. J Neurochem. 2007;103:1472–80
38. Storz P, Döppler H, Toker A. Protein kinase D mediates mitochondrion-to-nucleus signaling and detoxification from mitochondrial reactive oxygen species. Mol Cell Biol. 2005;25:8520–30
39. Wang C, Sadovova N, Hotchkiss C, Fu X, Scallet AC, Patterson TA, Hanig J, Paule MG, Slikker W Jr. Blockade of N-methyl-D-aspartate receptors by ketamine produces loss of postnatal day 3 monkey frontal cortical neurons in culture. Toxicol Sci. 2006;91:192–201
40. Riobo NA, Melani M, Sanjuan N, Fiszman ML, Gravielle MC, Carreras MC, Cadenas E, Poderoso JJ. The modulation of mitochondrial nitric-oxide synthase activity in rat brain development. J Biol Chem. 2002;277:42447–55
41. Pitkanen S, Robinson BH. Mitochondrial complex I deficiency leads to increased production of superoxide radicals and induction of superoxide dismutase. J Clin Invest. 1996;98:345–51
42. de Oliveira L, Spiazzi CM, Bortolin T, Canever L, Petronilho F, Mina FG, Dal-Pizzol F, Quevedo J, Zugno AI. Different sub-anesthetic doses of ketamine increase oxidative stress in the brain of rats. Prog Neuropsychopharmacol Biol Psychiatry. 2009;33:1003–8
43. Chen CH, Liu K, Chan JY. Anesthetic preconditioning confers acute cardioprotection via up-regulation of manganese superoxide dismutase and preservation of mitochondrial respiratory enzyme activity. Shock. 2008;29:300–8
44. Zhao P, Peng L, Li L, Xu X, Zuo Z. Isoflurane preconditioning improves long-term neurologic outcome after hypoxic-ischemic brain injury in neonatal rats. Anesthesiology. 2007;107:963–70
45. Tremblay R, Chakravarthy B, Hewitt K, Tauskela J, Morley P, Atkinson T, Durkin JP. Transient NMDA receptor inactivation provides long-term protection to cultured cortical neurons from a variety of death signals. J Neurosci. 2000;20:7183–92