Carlos Venâncio

Performance of Anesthetic Depth Indexes in Rabbits under Propofol Anesthesia Prediction Probabilities and Concentration-effect Relations

Background:The permutation entropy, the approximate entropy, and the index of consciousness are some of the most recently studied electroencephalogram-derived indexes. In this work, a thorough comparison of these indexes was performed using propofol anesthesia in a rabbit model. Methods:Six rabbits were anesthetized with three propofol infusion rates: 70, 100, and 130 mg · kg −1 · h−1, each maintained for 30 min, in a random order for each animal. Data recording was performed in the awake animals 20, 25, and 30 min after each infusion rate was begun in the recovered animals and consisted of electroencephalogram recordings, evaluation of depth of anesthesia according to a clinical scale, and arterial blood samples for plasma propofol determination. Median and spectral edge frequencies were analyzed for single-scale permutation entropy and composite multiscale permutation entropy, approximate entropy, index of consciousness, and the spectral parameters. The spectral parameters and single-scale and multiscale permutation entropies were corrected for the presence of burst suppression. Performance of the indexes was compared by prediction probability and pharmacodynamic analysis. Results:The single-scale and composite multiscale permutation entropies with a burst suppression correction showed better prediction probabilities than did the other electroencephalogram-derived parameters but not better than the electromyographic activity. Conclusion:Single-scale and multiscale permutation entropies may be promising measures of propofol anesthetic depth when corrected for burst suppression. Additional studies should investigate the information measured by electromyography algorithms from commercial monitors of anesthetic depth. The rabbit may be a promising animal model for electroencephalographic studies because it provides a good-quality signal.

Impaired Spatial Memory after Ketamine Administration in Chronic Low Doses

Ketamine is a noncompetitive antagonist of the NMDA-receptors, used as a dissociative anesthetic, presently included in the category of the psychoactive substances known as “club drugs”. Ketamine administration was associated with impaired working memory and increased psychopathological symptoms, but there is a lack of information regarding the effects of chronic sub-anesthetic doses. Adult Wistar rats were administered ketamine, 5 and 10 mg/kg twice daily, subcutaneously for 14 days. One week later, rats were tested in an object recognition/object location task and in the open field arena. There was altered performance in both the object recognition/location and in the open field tests by the group chronically exposed to the lower dose of ketamine. These animals displayed a decreased discrimination index (p<0.05) in the object recognition task, were unable to recognize the displacement of a familiar object and displayed decreased activity across open filed sessions. Importantly, these alterations were not observed in animals administered a higher dose of ketamine. Collectively, these results consistently show that chronic administration of ketamine in sub-anesthetic doses may lead to decreased habituation and inability to update spatial representations.

Performance of electroencephalogram-derived parameters in prediction of depth of anaesthesia in a rabbit model

BACKGROUND The index of consciousness (IoC), the permutation entropy (PE), and the approximate entropy are recent EEG-derived indices of anaesthetic depth. In this study, a rabbit model under fentanyl and isoflurane anaesthesia was used to compare the performance of these indices and also the classic median and spectral edge frequency 95%. METHODS EEG recordings were obtained from six rabbits. Animals received fentanyl for premedication, followed by induction with propofol and maintenance with isoflurane. Anaesthetic depth was evaluated according to a clinical scale from 1 (awake) to 4 (surgical anaesthesia). Animals were submitted to surgical implantation of a small device in the lumbar muscles. A correction factor for the EEG suppression ratio was applied to the spectral parameters and to the PE. The correlation of the indices with the clinical scale of anaesthesia was analysed using prediction probability. Repeated-measures analysis of variance or its non-parametric equivalent was used to analyse the indices values at the study times and to compare their variability. RESULTS The IoC showed the best mean prediction probability value [0.94 (0.01)] followed by burst suppression-corrected PE [0.91(0.03)]. Both parameters also showed less variability than the others. CONCLUSIONS The IoC and PE are promising indices for anaesthetic depth monitoring. The PE might benefit from the application of a burst suppression correction at deeper stages of anaesthesia. The rabbit is useful as a translational research animal model for the validation of clinical indices.

Acute ketamine impairs mitochondrial function and promotes superoxide dismutase activity in the rat brain.

BACKGROUND:Ketamine is often associated with altered mitochondrial function and oxidative stress. Nevertheless, limited data are still available regarding the in vivo action of ketamine in mitochondrial bioenergetics and redox state. Accumulating evidence supports a role for nitric oxide (NO) as a possible modulator of ketamine’s side effects. In the present study, we investigated the role of NO modulation on ketamine anesthesia at the level of brain mitochondrial function and redox status. METHODS:Adult male rats received a single dose of ketamine (50, 100, or 150 mg/kg IP) or a combination of ketamine and N-nitro-L-arginine (3 mg/kg IP). Animals were killed 6 hours after treatment. Brain and blood samples were collected for plasma NO determination and mitochondria isolation. Several variables of brain mitochondrial function were evaluated. RESULTS:Ketamine interfered with complex I function, revealing increased oxygen consumption in state 4, impaired oxidative phosphorylation efficiency of glutamate-malate substrate, and decreased NADH-ubiquinone oxidoreductase activity. In addition, mitochondrial NO synthase (mtNOS) activity and NO plasma levels were increased for the 50 and 100 mg/kg doses. Ketamine administration increased hydrogen peroxide generation and triggered superoxide dismutase activity. All these effects could totally or partially be prevented by mtNOS inhibition through N-nitro-L-arginine. CONCLUSIONS:Acute ketamine administration impaired the function of mitochondrial complex I leading to increased mtNOS activity, increased generation of hydrogen peroxide and NO, resulting in superoxide dismutase triggering, and improved antioxidant activity. The present findings clarify the role of NO modulation in ketamine anesthesia, providing new data on a relevant clinical mechanism.

Chronic ketamine administration impairs mitochondrial complex I in the rat liver.

AIM Ketamine can induce hepatotoxicity which has been suggested to be dependent on mitochondrial impairment. This study investigated the long-term effects of chronic low-dose ketamine on liver mitochondrial function, oxidative stress parameters, liver histology and glycogen content. MAIN METHODS Adult rats were administered with saline or ketamine (5 or 10mg/kg) twice a day for a fourteen-day period in order to mimic chronic treatments. Effects between groups were compared ten days after the treatment had ended. Liver mitochondrial function was monitored in isolated mitochondrial extracts through evaluation of respiration parameters and activity of respiratory complexes, as well as oxidative stress, through lipid peroxidation, protein oxidation and superoxide dismutase activity. The hepatic histology and liver glycogen content were also evaluated. KEY FINDINGS Ketamine groups showed a decreased evolution in body weight gains during the treatment period. Ketamine had no effect either on serum liver enzymes or on the oxidative stress parameters of liver mitochondria. Ketamine decreased the hepatic glycogen content, inhibited mitochondrial complex I and oxygen consumption when glutamate-malate substrate was used. SIGNIFICANCE These findings reflect a long-term mitochondrial bioenergetic deterioration induced by ketamine, which may explain the increased susceptibility of some patients to its prolonged or repeated use.

HPV16 induces a wasting syndrome in transgenic mice: Amelioration by dietary polyphenols via NF-κB inhibition

&NA; Cancer patients often show a wasting syndrome for which there are little therapeutic options. Dietary polyphenols have been proposed for treating this syndrome, but their usefulness in cases associated with human papillomavirus (HPV)‐induced cancers is unknown. We characterized HPV16‐transgenic mice as a model of cancer cachexia and tested the efficacy of long‐term oral supplementation with polyphenols curcumin and rutin. Both compounds were orally administered to six weeks‐old HPV16‐transgenic mice showing characteristic multi‐step skin carcinogenesis, for 24 weeks. Skin lesions and blood, liver and spleen inflammatory changes were characterized histologically and hematologically. Hepatic oxidative stress, skeletal muscle mass and the levels of muscle pro‐inflammatory transcription factor NF‐&kgr;B were also assessed. Skin carcinogenesis was associated with progressive, severe, systemic inflammation (leukocytosis, hepatitis, splenitis), significant mortality and cachexia. Curcumin and rutin totally suppressed mortality while reducing white blood cells and the incidence of splenitis and hepatitis. Rutin prevented muscle wasting more effectively than curcumin. Preservation of muscle mass and reduced hepatic inflammation were associated with down‐regulation of the NF‐&kgr;B canonical pathway and with reduced oxidative stress, respectively. These results point out HPV16‐transgenic mice as a useful model for studying the wasting syndrome associated with HPV‐induced cancers. Dietary NF‐&kgr;B inhibitors may be useful resources for treating this syndrome.

The effect of high doses of remifentanil in brain near- infrared spectroscopy and in electroencephalographic parameters in pigs

OBJECTIVE To study the effects of a high remifentanil bolus dose on pigs electroencephalographic indices and on brain regional and global oxygenation. STUDY DESIGN Prospective experimental study. ANIMALS Twelve healthy Large-White male pigs, age 3 months and weight 26.2 ± 3.6 kg. METHODS Anaesthesia was induced with intravenous propofol 4 mg kg⁻¹, then maintained with constant rate infusions of propofol (15 mg kg⁻¹ hour⁻¹) and remifentanil (0.3 μg kg⁻¹ minute⁻¹). Following instrumentation, all pigs received a 5 μg kg⁻¹ remifentanil bolus. The responses of jugular venous oxygen saturation, cardiac output and cerebral oxygen saturation to the remifentanil bolus were studied. The Bispectral index, spectral edge frequency 95%, total power, approximate entropy and permutation entropy were also studied. Repeated measures anova and Pearson correlation were used to analyze the effect of remifentanil bolus on these variables until 5 minutes after the bolus. RESULTS Cardiac output and cerebral oxygen saturation decreased significantly after the remifentanil bolus from 4.6 ± 0.9 to 3.8 ± 1.0 L minute⁻¹ and from 65 ± 6 to 62 ± 1% (p < 0.05), respectively. No significant changes were observed in the jugular venous oxygen saturation (p > 0.05) nor in any of the electroencephalogram derived indices (p > 0.05). Correlation analysis revealed strong positive significant correlations between cerebral oxygen saturation and cardiac output (r = 0.82, p < 0.001) and between cerebral oxygen saturation and approximate entropy (r = 0.65, p < 0.001). CONCLUSIONS AND CLINICAL RELEVANCE The effect caused by the remifentanil bolus on the brain oxygenation seems to be better reflected by the cerebral oxygen saturation than the jugular venous oxygen saturation. The effect of remifentanil on the electroencephalogram may not be reflected in indices derived from the electroencephalogram, but the potential of the approximate entropy in reflecting changes caused by opioids on the electroencephalogram should be further investigated.

Effects of acute bleeding followed by hydroxyethyl starch 130/0.4 or a crystalloid on propofol concentrations, cerebral oxygenation, and electroencephalographic and haemodynamic variables in pigs.

Bleeding changes the haemodynamics, compromising organ perfusion. In this study, the effects of bleeding followed by replacement with hydroxyethyl starch 130/0.4 (HES) or lactated Ringers (LR) on cerebral oxygenation and electroencephalogram-derived parameters were investigated. Twelve young pigs under propofol-remifentanil anaesthesia were bled 30 mL/kg and, after a 20-minute waiting period, volume replacement was performed with HES (GHES; N = 6) or LR (GRL; N = 6). Bleeding caused a decrease of more than 50% in mean arterial pressure (P < 0.01) and a decrease in cerebral oximetry (P = 0.039), bispectral index, and electroencephalogram total power (P = 0.04 and P < 0.01, resp.), while propofol plasma concentrations increased (P < 0.01). Both solutions restored the haemodynamics and cerebral oxygenation similarly and were accompanied by an increase in electroencephalogram total power. No differences between groups were found. However, one hour after the end of the volume replacement, the cardiac output (P = 0.03) and the cerebral oxygenation (P = 0.008) decreased in the GLR and were significantly lower than in GHES (P = 0.02). Volume replacement with HES 130/0.4 was capable of maintaining the cardiac output and cerebral oxygenation during a longer period than LR and caused a decrease in the propofol plasma concentrations.

Ketamine alone or combined with midazolam or dexmedetomidine does not affect anxiety-like behaviours and memory in adult Wistar rats

Ketamine administration has been associated with controversial behavioural impairments and psychotic episodes. Even though ketamine alone and in combination with midazolam or dexmedetomidine are frequently used in laboratory animals, the side-effects of such protocols are not well known. Therefore, our aim was to evaluate the effects of ketamine alone and in combination with midazolam or dexmedetomidine on emotional reactivity, as well as the effects on learning and memory in adult rats at least 48 h after anaesthesia. The evaluation of the potential influence of 100 mg/kg ketamine administered alone and in combination with midazolam (5 mg/kg), or dexmedetomidine (0.25 mg/kg) on spatial learning and recognition memory was studied in adult Wistar rats using the radial maze as well as object recognition and location tests. The influence of these combinations on emotional reactivity was investigated using the new exploration test and the elevated plus maze. Results showed that ketamine alone or in combination with midazolam or dexmedetomidine affected neither spatial and recognition memory, nor emotional reactivity. These results reinforce the safe clinical use of ketamine and its combinations in rats in a research context since the administration of these anaesthetic combinations did not produce significant changes with regard to spatial and recognition memory or emotional reactivity. Furthermore, these results indicate that the quality of scientific data produced in adult rat neurobehavioural research is not jeopardized by the use of these anaesthetic protocols.

Teratology Study Guidelines: An Overview

Developmental toxicology is a constantly evolving research field which needs to attend to a complex underlying regulatory network. Before entering the market new substances have to be tested for toxic effects on reproduction and development in order to ensure human health and environmental safety. Traditional in vivo mammalian models represent more adequately the intricacy of human development and provide an assessment of the interaction of chemicals on the reproductive system. However, in the last years, the main goal is to reduce the use of vertebrate animals, using those only as last resort. Consequently, the interest in the development and validation of a battery of alternative tests able to cover the various aspects of the reproductive cycle has increased. Reproductive toxicity is probably the most difficult endpoint to be replaced by alternative assays, since it should provide information on mechanisms interactions essential for female and male fertility, and also knowledge on the development of a new human being during its prenatal life. This complexity explains the slow progress in implementing alternatives for reproductive toxicity safety assessments. Alternative test methods may be based on in vitro systems and non-mammalian animal models. Many biological processes have been successfully implemented using in vitro models, opening the possibility to study the interference of teratogenic compounds using these models. Their validation and implementation have lagged behind, in part because of difficulties in establishing their predictability. Nevertheless, the advance toward the process of validation is crucial for a strategy aiming to replace and reduce the use of living animals. Based on the present state of the art, it is not probable that such testing strategies will completely replace the need to assess reproductive toxicity in vivo in the near future, but they contribute to reduce the animal testing and provide important information. In this chapter the approved guidelines for standard methods and alternative methods according to their regulatory and scientific status are enumerated and described.