[Show abstract][Hide abstract] ABSTRACT: Propofol is an important compound used for anaesthetic purposes in clinical practice. Nevertheless, in the recente years, the use of propofol has also been reported for recreational, abusive or even for suicidal and criminal purposes. So far, there is a lack of practical techniques validated for simultaneous quantification of propofol and its non-conjugated metabolites (2,6-diispropyl-1,4-quinol and 2,6-diispropyl-1,4-quinone) in plasma and organs, to optimize therapeutics, to prevent undesired effects, and for application in forensic settings.
A simple gas chromatography/ Ion trap – mass spectrometry method was optimized for the detection and quantification of propofol and its non-conjugated metabolites in plasma and organ (liver, heart, kidney and lungs) samples.
All compounds were simultaneously extracted from 0.5 mL of plasma and 0.2 g of each organ, following a straightforward and rapid procedure using thymol as internal standard. This method was validated according to international guidelines for analytical methods.
The standard curve ranged from 0.005 to 100 μg/mL for propofol and 0.005 to 50 μg/mL for the non-conjugated
metabolites. Intra and inter-assay variability for propofol and its metabolites was less than 15% and the average
recovery was greater than 90%. The proof of applicability of this methodology allowed the successful measurement of propofol and its non-conjugated metabolites in plasma and solid tissues from seven New Zealand White rabbits that were submitted to a long-term anaesthesia protocol with a continuous infusion of propofol ranging from 20 to 60 mg/kg/h.
This optimized and validated assay may also be suitable in the monitoring of sedated or anaesthetised animals
and humans with continuous infusions of propofol and for use in pharmacokinetic and toxicological studies.
Journal of Analytical & Bioanalytical Techniques. 08/2014;
[Show abstract][Hide abstract] ABSTRACT: 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 mitochondria 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 end. 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.
[Show abstract][Hide abstract] ABSTRACT: The ketamine/midazolam association of a dissociative with a sedative agent is used for the induction and maintenance of anaesthesia in laboratory animals. Anaesthesia may interfere with research results through side-effects on the nervous system, such as memory impairment. It is known that ketamine and midazolam affect cognition; however, their effects have not been clarified when used in a context of balanced anaesthesia. Thus, this study evaluated the effects of ketamine/midazolam on the acquisition of motor and of a spatial memory task in adult mice. Twenty-eight C57BL/6 adult male mice were divided into three groups: untreated control, treated with ketamine/midazolam (75 mg/kg / 10 mg/kg) and treated with midazolam (10 mg/kg) groups. Respiratory rate, heart rate and systolic pressure were measured every 5 min in the animals treated with ketamine/midazolam, as this was the only group that exhibited loss of the righting reflex. One day after treatment, animals were tested in the open field, rotarod and radial arm maze. There were no differences between treatments regarding open-field activity, rotarod performance or number of working and reference memory errors in the radial arm maze task. In conclusion, the learning process of spatial and motor tasks was not disrupted by the anaesthetic combination of ketamine/midazolam. These results suggest its safe use in adult mice in projects where acquisition of a spatial and motor task is necessary.
[Show abstract][Hide abstract] ABSTRACT: Objective measurements of physiological parameters controlled by the autonomic nervous system such as blood pressure, heart rate and respiration are easily obtained nowadays during anaesthesia by the use of monitors: oscillometers, pulseoximeters, electrocardiograms and capnographs are available for laboratory animals. However, the effect-site of hypnotic drugs that cause general anaesthesia is the central nervous system (the brain). In the present, the adjustment of hypnotic drugs in veterinary anaesthesia is performed according to subjective evaluation of clinical signs which are not direct reflexes of anaesthetic effects on the brain, making depth of anaesthesia (DoA) assessment a complicated task. The difficulties in assessing the real anaesthetic state of a laboratory animal may not only result in welfare-threatening situations, such as awareness and pain sensation during surgery, but also in a lack of standardization of experimental conditions, as it is not easy to keep all animals from an experiment in the same DoA without a measure of anaesthetic effect. A direct measure of this dose-effect relationship, although highly necessary, is still missing in the veterinary market. Meanwhile, research has been intense in this subject and methods based on the brain electrical activity (electroencephalogram) have been explored in laboratory animal species. The objective of this review is to explain the achievements made in this topic and clarify how far we are from an objective measure of DoA for animals.
[Show abstract][Hide abstract] ABSTRACT: Ketamine is increasingly popular in clinical practice and its combination with α(2)-agonists can provide good anaesthetic stability. Little is known about the effects of this combination in the brain. Therefore, we investigated the effects of different concentrations of ketamine combined with medetomidine on cognition and its potential apoptotic neurodegenerative effect in adult mice.
Seventy-eight C57BL/6 adult mice were divided into six different groups (saline solution, 1 mg kg(-1) medetomidine, 25 mg kg(-1) ketamine+1 mg kg(-1) medetomidine, 75 mg kg(-1) ketamine+1 mg kg(-1) medetomidine, 25 mg kg(-1) ketamine, and 75 mg kg(-1) ketamine). Eight animals per group were tested in the T-maze, vertical pole, and open-field test. Five animals per group were used for histopathological [haematoxylin and eosin (HE) staining] and immunohistochemical analyses [caspase-3 activation and expression of neurotrophin brain-derived neurotrophic factor (BDNF)]. Cells showing clear HE staining and positive immunoreactions for caspase-3 and BDNF in the retrosplenial cortex, visual cortex, pyramidal cell layer of the cornu Ammonis 1 and cornu Ammonis 3 areas of the hippocampus, and in the granular layer of the dentate gyrus were counted.
There were no differences between groups regarding the number of dead cells and cells showing positive immunoreactions in the different areas of the brain studied. Similarly, no differences were detected in the number of trials to complete the T-maze task. Nevertheless, α(2)-agonist decreased hyperlocomotion caused by ketamine in the open field.
Neither apoptotic neurodegeneration nor alterations in spatial memory were observed with different concentrations of ketamine combined with medetomidine in adult mice.
BJA British Journal of Anaesthesia 02/2012; 108(5):807-14. · 4.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cerebral State Index (CSI) is a measure of depth of anesthesia (DoA) developed for humans, which is traditionally modeled with the Hill equation and the propofol effect-site concentration (Ce). The CSI has been studied in dogs and showed several limitations related to the interpretation of EEG data. Nevertheless, the CSI has a lot of potential for DoA monitoring in dogs, it just needs to be adjusted for this species. In this work, an adapted CSI model is presented for dogs considering a) both Ce and EMG as inputs and b) a fuzzy logic structure with parameters optimized using the ANFIS method. The new model is compared with traditional Hill model using data from dogs in routine surgery. The results showed no significant impact in the model performance with the change of model structure (Fuzzy instead of Hill). The residuals of the Hill model were significantly correlated with the EMG, indicating that the latter should be considered in the model. In fact, the EMG introduction in CSI model significantly decreased the modeling error: 11.8 [8.6; 15.2] (fuzzy logic) versus 20.9 [16.4; 29.0] (Hill). This work shows that CSI modeling in dogs can be improved using the current human anesthesia set-up, once the EMG signal is acquired simultaneously with the CSI index. However, it does not invalidate the search of new DoA indices more adjusted to use in dog's anesthesia.
Conference proceedings: ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Conference 08/2011; 2011:6593-6.
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
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.
[Show abstract][Hide abstract] ABSTRACT: 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.
DNA research: an international journal for rapid publication of reports on genes and genomes 03/2011; 9(1):251-5. · 1.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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%.
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.
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.
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.
BJA British Journal of Anaesthesia 02/2011; 106(4):540-7. · 4.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This paper address the problem of designing a system for automatic assessment of the Depth of Anesthesia (DoA) for general surgery in veterinary medicine. The aim is to develop a DoA sensor that coupled with an automatic control methodol-ogy may bring the benefits of automation to veterinary medicine, to relief the veterinary surgeon from routine activities due to the his/her dual role of surgeon and anaesthetist. At the core of an automatic DoA assessment system are mathematical methods used to extract features from the electroencephalogram (EEG) signal which are mapped into an index. These mathematical methods may use frequency/time domain analysis to evaluate the change in the EEG signal such as frequency shift and amplitude change. Other methods use a probabilistic approach to measure the change of complexity in the EEG signal, such as, Permutation Entropy (PE) and Shannon Entropy (ShEn). The method that is evaluated in based on the concept of computing the mean power of EEG frequency bands as in human anesthesia.